Marine Biological Laboratory

RprpivpH June 25, 1947

Accession No. 60980

Given By ^e derail Ian Co,
Place New York City

0

0

)

TRACE ELEMENTS

IN

PLANTS AND ANIMALS

TRACE ELEMENTS

IN

PLANTS AND ANIMALS

by
WALTER STILES

M.A., Sc.D., F.L.S., F.B.S.

Mason Professor of Botany in the
University of Birmingham

vl

CAMBRIDGE: AT THE UNIVERSITY PRESS
NEW YORK: THE MACMILLAN COMPANY

1946

Copyright, 1946, by
THE MAC MIL LAN COMPANY

All rights reserved — no part of this hook
may be reproduced in any form without
permission in writing from the publisher,
except by a reviewer who wishes to quote brief
passages in connection with a review written
for inclusion in magazine or newspaper.

FIRST PRINTING

LITHOGRAPHED IN THE UNITED STATES OF AMERICA

To

EDWARD JAMES SALISBURY

Director of the Royal Botanic Gardens, Kew

Chairman of the Mineral Deficiencies Conference of the

Agricultural Research Council

IN FRIENDSHIP

I

CONTENTS

Preface page ix

List of Plates xi

Chapter I. Historical Introduction 1

II. Methods of Investigating Micro-
Nutrient Problems 21

1. The Purification of Materials used in
Culture Experiments 21

2. The Estimation of Micro-nutrient Ele-
ments in Plant Material 26

3. The Diagnosis of Mineral Deficiencies of
Plants 51

III. Trace-Element Deficiency Diseases of

Plants 57

1. Diseases Attributable to a Deficiency of
Manganese 57

2. Diseases Attributable to a Deficiency of

Zinc 68

3. Diseases Attributable to a Deficiency of
Boron % 80

4. Diseases Attributable to a Deficiency of
Copper 90

5. The Symptoms of Molybdenum Deficiency 96

IV. The Functions of Trace Elements in

Plants 97

60980

Vlii CONTENTS

Chapter V. Trace Elements in Animals page 124

1. Diseases due to Trace-Element Excess 127

2. Trace-Element Deficiency Diseases of
Animals 136

3. The Functions of Trace Elements in
Animals 150

VI. Concluding Remarks I52

List of Literature 156

Index 179

IX

PREFACE

Trace elements, micro-nutrients and minor elements are terms
applied to a number of chemical elements which are essential
for the lives of plants and animals, but which are required in
extremely small quantity. The development of our knowledge
of the part played by the trace elements in the life of plants and
animals is very recent, nearly all our present information on the
subject having been acquired during little more than the last
twenty years. Nevertheless, in the course of that time a great
many observations have been made in the field, and much
experimental work carried out in both field and laboratory,
while many hundreds of publications, some of slight value,
others of considerable scientific and economic importance,
dealing with trace elements in living plants and animals have
appeared in the scientific press. The time thus seems ripe for the
presentation of a digest of this information, so that the salient
facts may be available in a convenient form, and the present
position of our knowledge of trace elements in living organisms
made plain. It is with this intention that the present book has
been written.

The role of the trace elements in organisms is, in the first
place, a matter for the plant or animal physiologist, but as
deficiency or excess in the supply of the various trace elements
may lead to a diseased condition of the plant or animal with
serious economic consequences, the trace elements are also of
interest to the plant pathologist, the veterinary surgeon, the
agriculturist and horticulturist. Indeed, as regards plants at
any rate, much more is definitely known of the plant patho-
logical aspect of the trace elements than of their physiological
functions.

I would take this opportunity of expressing my thanks to
Mr W. Morley Da vies of Harper Adams Agricultural College,
who first introduced to me the effects of trace-element defi-
ciencies in the field, and who has generously and constantly put
at my disposal both his knowledge of the pathological effects of
trace-element deficiency, and the pathological material itself.

X PREFACE

I also have pleasure in thanking Dr C. S. Piper of the Waite
Agricultural Research Institute, University of Adelaide, for
permission to use the photographs reproduced in Figs. 4 and 12,
and my colleagues Dr K. W. Dent and Mr A. E. Roberts for the
preparation of the rest of the photographs in the book.

Throughout the book the following contractions have been
used:

ml. (millilitre) = one -thousandth of a litre.

//g. (microgram) = one-millionth of a gram.

A. (Angstrom unit) = one ten-thousandth of a millimetre.

p. p.m. = parts per million.

WALTER STILES

Birmingham
28 October 1944

XI

PLATES

Fig. 1. Oat seedling affected with grey speck facing page 60

Fig. 2. Oat leaf exhibiting the characteristic symptoms

of grey speck 60

Fig. 3. Sugar beet suffering from manganese deficiency 60

Fig. 4. Peas suffering from marsh spot 61

Fig. 5. Sugar beet suffering from boron deficiency 84

Fig. 6. Mangold suffering from boron deficiency 84

Fig. 7. Transverse section through swedes showing

characteristic appearance of brown heart 85

Fig. 8. Longitudinal section through swede affected with

brown heart 85

Fig. 9. Late stage in boron-deficient swede 86

Fig. 10. Boron-deficient cauliflower 86

Fig. 1 1 . Stem of cauliflower suffering from boron de-
ficiency and showing very typical breakdown of
central tissue 87

Fig. 12. Effect of concentration of copper on the growth

of oats 9 2

CHAPTER I

HISTORICAL INTRODUCTION

In the year 1699 Woodward published the results of experiments
in which cuttings of plants were grown, not in soil, but in rain
water. More than a century and a half later Julius Sachs (1860)
and W. Knop (1860) independently developed this method of
water culture by growing plants of several different species in a
dilute aqueous solution of various salts. In this way the materials
available for absorption by the roots of the growing plants were
controlled, and Sachs and Knop concluded from their experi-
ments that so long as the culture solution contained salts in-
volving the elements nitrogen, sulphur, phosphorus, potassium,
calcium, magnesium and iron, a perfectly healthy plant would
result. The elements occur in soil as constituents of compounds
present in, or derived from, the minerals of the underlying rock,
and are therefore generally known as mineral nutrients. Ex-
periments of this kind have been repeatedly carried out by
subsequent investigators and many formulae for water-culture
solutions have been used and recommended. The water-culture
method has also been extensively used for research in plant
nutrition. Some of the best known and most widely used water-
culture solutions are those of Sachs and Knop themselves and
the later ones of Pfeffer and Von der Crone. Plants of a great
number of species have been successfully grown in water culture,
and in recent years the method has been advocated, perhaps too
optimistically (cf. Hoagland and Arnon, 1938), as a means of
cultivating certain crop plants on a commercial or semi-
commercial scale. The compositions of some of the best known
and most widely used water cultures are given below.

Sachs's nutrient solution

Potassium nitrate l'Og.

Sodium chloride 0-5 g.

Calcium sulphate 0-5 g.

Magnesium sulphate 0-5 g.

Calcium phosphate 0-5 g.

Water 1 1.

A few drops of a solution of ferric chloride or ferrous sulphate.

2 HISTORICAL INTRODUCTION

Pfeffer's stronger nutrient solution

Calcium nitrate 40 g.

Potassium nitrate 1'Og.

Potassium dihydrogen phosphate 1-0 g.

Potassium chloride 0-5 g.

Water 3 1.

Ferric chloride medicinal solution 3 to 6 drops

Pfeffer's weaker solution is similar, except that the same quantity of these
salts is dissolved in 7 1. of water.

Von der Crone's nutrient solution

Potassium nitrate 10 g.

Calcium sulphate 0-5 g.

Magnesium sulphate 0-5 g.

Calcium phosphate 0-25 g.

Ferrous phosphate 0-25 g.

Water 1 1.

It will be noticed that, in addition to the elements given above,
Pfeffer's solution also contains chlorine, while Sachs's solution
contains not only chlorine, but sodium. While the general
opinion of plant physiologists for many years was that most
plants required neither of these elements, it was rather vaguely
held that some plants required sodium or chlorine in addition to
those regarded as always necessary. Thus Pfeffer (1900) re-
marked that probably no plant had been grown in complete
absence of chlorine, and he considered it uncertain whether a
minimum amount was essential or not. Miller (1931) considered
that experimental results had shown that under certain con-
ditions sodium had a definite effect on the growth of plants of
some species.

So opinion on the mineral nutrition of plants remained during
the latter half of the nineteenth century. It was well established
that many elements, apart from those regarded as necessary,
were present in plants, but their presence was regarded as
incidental, due to their presence in the soil in which the plants
grew. But in 1897 G. Bertrand claimed that manganese was
constantly associated in the plant with an oxidizing enzyme,
laccase, and he came to regard manganese as an essential con-
stituent of the oxidase system. In 1905 he claimed that an
insufficient supply of manganese to plants brought about
diminution or cessation of growth, and that manganese was to
be regarded as an essential element. Much subsequent experi-

HISTORICAL INTRODUCTION 3

mental work and many field observations have gone to confirm
Bertrand's view.

In 1914, by the use of water-culture experiments with care-
fully prepared culture vessels and nutrient salts, Maze showed
that not only manganese but zinc also was necessary for the
growth of maize. As a result of later work on similar lines he
claimed that aluminium, boron, chlorine and silicon were all
essential in small amounts for the healthy growth of plants. Of
these various elements, boron had already been found by
Agulhon (1910) to induce an increased production of dry matter
in wheat, oats and radish grown in sand cultures and in maize,
colza and turnip grown in field-plot experiments. It is, however,
scarcely correct, as has sometimes been done, to quote Agulhon
as the first worker to point out the essential nature of boron for
higher plants. As Dennis and O'Brien (1937) have pointed out,
Agulhon provided no proof that normal growth was not possible
without boron, and the credit for first calling attention to the
necessity of this element for any plant is due to Maze. The
importance of boron as an essential plant nutrient was brought
into prominence in 1923 by Miss Warington, who showed by
means of water cultures that while a concentration of boric acid
as low as 1 in 12-5 xlO6 was sufficient to allow the normal
growth of the broad bean (Vicia faba), in complete absence of
boron death of the plant supervened after the development of
quite characteristic pathological symptoms. A few years later
Sommer and Lipman (1926) added cotton, castor oil, buckwheat,
flax, mustard and barley to the list of plants for the growth of
which a supply of boron is essential. Since then the list of such
species has been further extended.

Confirmation of Maze's finding that zinc is necessary for the
healthy development of maize has been provided by more
recent observations of Barnette and Warner (1935), Mowry and
Camp (1934) and others, who have shown that the curious
chlorotic condition of this plant known as ' white-bud ' can be
cured by application of zinc sulphate. Sommer and Lipman
(1926) also found zinc essential for the growth of sunflower and
barley, and later Sommer (1928) reported that buckwheat and
beans could only undergo normal development in presence of
zinc. In beans, for example, abscission of leaves and flower buds

4 HISTORICAL INTRODUCTION

occurred in cultures deprived of zinc, whereas in controls
adequately supplied with this element flowering took place and
seeds were produced. A number of trees have been shown to
require a supply of this element.

As regards the remaining elements that Maze concluded were
necessary for the growth of maize, reference has already been
made to the doubt which has existed for many years about the
necessity of chlorine for plant development. As long ago as 1862,
Nobbe and Siegert concluded that chlorine was essential for the
normal development of buckwheat, but many subsequent
experiments carried out to check this conclusion yielded con-
flicting results. But in 1938 the much-debated question of the
effect of chlorine on the growth of buckwheat was examined by
Lipman, and it was found that plants grown with 5 p. p.m. of
chlorine added as potassium chloride produced markedly more
dry matter and seeds than plants grown without added chlorine.
Moreover, the seeds from plants grown with added chlorine
gave a higher percentage of germination than the seeds from
plants without added chlorine, and of the seedlings produced
from the latter little more than half developed to any extent.
A second series of cultures was made from the seed obtained in
the first series, the seedlings from seeds obtained from the
cultures with added chlorine being again supplied with this
element, and those from seeds grown in cultures without added
chlorine being again deprived of it. The superiority of the plants
supplied with chlorine was emphasized, and Lipman concluded
that if chlorine is not absolutely essential to buckwheat it
certainly greatly influences its growth and seed production.
A like conclusion was drawn from water-culture experiments
with peas.

As regards aluminium, Stoklasa in 1922 grew a number of
hydrophytes in water cultures and silica gel cultures with and
without aluminium, and found growth was considerably improved
in presence of aluminium. Indeed, silica gel cultures of Glyceria
aquatica without aluminium died in 22 days and aluminium-free
water cultures of J uncus ejfusus died in from 56 to 69 days.
Addition of aluminium also improved the growth of wheat,
barley and oats, but no beneficial effect of this element was
observed with plants of a number of other species, including

HISTORICAL INTRODUCTION 5

Polygonatum officinale and Iris bohemica. More critical water-
culture experiments to test the essentiality of aluminium were
carried out by Sommer (1926) with peas and millet. Specially
purified nutrient salts were used in making up the culture
solutions, and in those containing aluminium this element was
present to the extent of 5-5 or 1 p.p.m. of the solution. The
presence of 1 p.p.m. brought about a slight increase in the
dry weight of peas, but with millet the presence of aluminium
brought about a definite increase in dry weight and a very pro-
nounced increase in the weight of seeds produced, the respective
weight of seeds produced with and without aluminium being
0-23 and 4-98 g.

In 1938, Lipman obtained similar results with sunflowers and
maize, the effect being particularly noticeable with the latter,
where the addition of aluminium sulphate to the extent of
1 p.p.m. of aluminium (renewed from time to time) resulted in
an increase in dry weight of the vegetative parts of about 20 per
cent and an increase in the dry weight of the ears of about
155 per cent.

Sommer and Lipman also investigated the essentiality of
silicon. Sommer in 1926 found that the presence of silicon,
added as silica gel, increased the dry weight of rice plants from
4-4 to 7 g., and she concluded that this increase was big enough
to indicate that silicon is essential for the growth of rice. Marked
increases in seed production as a result of the presence of silicon
in the culture solution were observed with millet. Lipman in
1938 found that sunflowers and barley grown in water culture
produced more dry matter in a given time with colloidal
silica added to the solution than without such addition. With
barley the addition of silica also resulted in a greater yield of
grain.

Since Lipman could not be sure that in his control cultures
without added silicon, aluminium or chlorine respectively there
was not a small quantity of the particular element present, he
did not go further than emphasize the importance of silicon,
aluminium and chlorine for the plants used and probably for
higher plants in general. However, he regarded the probability
of the indispensability of these elements as very great indeed.

In recent years evidence has been accumulating that copper

6 HISTORICAL INTRODUCTION

is essential for the normal growth of a number of plants. Thus
in 1931 Sommer found that the addition of small quantities of
copper induced a considerable increase in growth of sunflowers,
flax and tomatoes, while in the same year Lipman and Mac-
kinney found that flax and barley grown in water culture failed
to produce seed in complete absence of copper. Evidence has
also been presented by Anderssen (1932) in South Africa and by
Oserkowsky and Thomas (1933) in America indicating that
chlorosis and dying back of the branches of various fruit trees
result from a deficiency of copper. Further, a pathological
condition of cereals and some other plants known as ' reclama-
tion disease ', occurring in plants growing on reclaimed heath land
in Holland and elsewhere, was attributed by Sjollema in 1933 to
copper deficiency. In cereals exhibiting this condition the tips
of the leaves turn yellow or white while seed fails to form.

In 1939, Arnon and Stout obtained evidence that molyb-
denum may be an essential element for higher plants. They
found that tomato plants exhibited pathological symptoms when
grown in a culture solution containing all the ordinary nutrient
elements along with manganese, boron, zinc and copper but
which was freed from all trace of molybdenum. The lower leaves
of the plants first developed a mottling; then dying of the.
marginal cells followed while the flowers fell without setting
fruit. The condition was prevented by the addition of one part
of molybdenum as molybdic acid in 100,000,000 to the culture
solution, while the pathological symptoms were removed in
the molybdenum-deficient plants by spraying the leaves with
a very dilute solution of molybdic acid. More recently, Stein-
berg (1941) concluded that both molybdenum and gallium are
essential for the growth of Lemna.

So far only higher plants have been considered, but it is quite
clear that the same trace elements may be equally essential for
the growth of lower plants, at any rate of fungi. Indeed, the
necessity of an element for the growth of a fungus has in more
than one instance been demonstrated before its essentiality for
a higher plant has been claimed.

The favourite species among the fungi for studies on mineral
nutrition has been Aspergillus niger. In addition to the
generally acknowledged mineral nutritive elements, with the

HISTORICAL INTRODUCTION 7

exception of calcium,1 but definitely including iron,2 it has been
shown that this fungus definitely requires zinc, manganese,
copper, molybdenum and gallium. Indeed, as long ago as
1869, Raulin showed that for A. higer zinc is necessary, although
the significance of this finding was rather obscured subsequently
by the attempt to explain the increased growth resulting from
the presence of a small quantity of zinc as a stimulation effect
on normal development due to the action of a poison in low
concentration. While the possibility of such a stimulation effect
in general is not to be ruled out without proof, such proof is
forthcoming when it has been shown that with rigid exclusion
of any substance normal development is prevented. For zinc
this proof was definitely provided by Steinberg (1919), who,
by means of a special technique, succeeded in removing all
but the minutest traces of zinc from the culture medium, and
then showed that the cultures of A. niger provided with zinc
produced more than 2000 times as much dry matter as the
controls deprived of all but the last traces of that element.
Steinberg's result was later confirmed by Bortels (1927), Roberg
(1928, 1931) and Gollmick (1936).

The need for manganese for the normal development of
A. niger was claimed by Bertrand and Javillier (1911a), and this
was confirmed by Steinberg (1935a). That copper is essential
for the growth of this fungus was shown by Bortels (1927) and
by Wolff and Emmerie (1930). In 1928, Davis, Marloth and
Bishop reported that the yield of a species of Dothiorella grown
on an artificial medium was reduced to half by the removal of
traces of boron from the nutrient salts used, while in 1933
Lockwood found that the growth of Penicillium Javanicum was
increased by additions of columbium, molybdenum and tung-
sten. The rigid proof of the need of molybdenum for the growth

1 There exists considerable doubt about the necessity or otherwise of
calcium for the growth of fungi. The general opinion at present appears
to be that this element is necessary for the growth of some species but
not of others. Much experimental work is necessary to place our know-
ledge of this question on a reliable basis. According to Davis, Marloth
and Bishop (1928) calcium is necessary for the development of A. niger.
Mann (1932), on the other hand, came to the opposite conclusion.

2 Iron is placed in the category of micro -nutrients by some workers
on fungus nutrition, e.g. Steinberg (1939) and Foster (1939).

8 HISTORICAL INTRODUCTION

of Aspergillus niger was given by Steinberg in 1937, while in the
following year the same worker added gallium to the list of
elements essential for this fungus.

While the mineral nutrition of no other fungus has received
so much attention as that of Aspergillus niger, sufficient in-
formation has now accumulated to justify the conclusion that
for the fungi in general a supply of trace elements is necessary.
Thus McHargue and Calfee (1931) concluded that zinc, man-
ganese and copper are necessary for the growth of A . flavus and
Rhizopus nigricans, and the same three elements were con-
sidered necessary for Ceratostomella Ulmi by Ledeboer (1934),
for Trichophyton interdigitale by Mosher, Saunders, Kingery and
Williams (1936), and for Phymatotrichum omnirorum by Rogers
(1938). Foster and Waksman (1939) reported that Rhizopus
nigricans fails to produce zygospores in absence of zinc, while the
favourable effect on development produced by zinc and copper
on a number of fungi belonging to different families has been
reported by Metz (1930) and for a number of species of Asper-
gillus by Roberg (1928, 1931).

Thus, as far as our information goes at present, both higher
plants and fungi, or some of them, require a supply of man-
ganese, zinc, copper and molybdenum. Many higher plants have
been shown to need boron, and it is rather surprising that this
element, one of the most definitely established micro -nutrients
of higher plants, has received scarcely any attention from
workers on fungus nutrition. On the other hand, the need for
columbium and tungsten has so far not been claimed for any
higher plant.

Very little information is available about the need for trace
elements of plants other than angiosperms and fungi, although
a few observations dealing with algae are on record. Thus man-
ganese has been shown to be essential for the unicellular green
alga Chlorella by Hopkins (1930, 1934) and for the diatom
Ditylum brightwelli by Harvey (1939). Roberg (1932) has
reported increased growth of two unicellular green algae
Coccomyxa simplex and Chlorella vulgaris as a result of small
additions of salts of iron, zinc and copper to the normal nutrient
solution. From his work, however, it is not made clear that
zinc and copper are actually essential for the growth of these

HISTORICAL INTRODUCTION 9

plants, and Roberg himself says that these elements are to be
considered as acting as stimulants. However, having regard to
work with plants of other groups, it may well be that these
elements v/ill prove to be actually essential for the growth of
these and other algae.

From what has so far been discovered regarding the essenti-
ality of trace elements, two questions arise which only further
research can answer. The first is how far the necessity for these
elements is general throughout the plant kingdom, and the
second is whether we now know the complete list of these
elements. As regards the first question, it would seem probable
rather than merely possible that when plants differing as widely,
both taxonomically and anatomically, as well as physiologically,
as angiosperms and fungi, both exhibit these requirements, we
are dealing with something very fundamental in plant nutrition,
and we are justified in concluding that the best established of the
trace elements, manganese, boron, zinc and copper, are likely
to be found essential for the nutrition of plants in general. In
regard to the second question, Steinberg (1938 c) has contributed
an interesting discussion on the relations between essentiality
of elements and their atomic structure, and he draws the con-
clusion from such considerations that scandium may be an
essential element for plant nutrition. In this connexion some
experiments carried out by Arnon are of interest. In 1937 this
worker described the results of water-culture experiments in
which the growth of barley plants was improved by the addition
of small quantities of molybdenum, chromium and nickel. In
the following year further experiments were described in which
plants of asparagus and lettuce were grown in four different
culture solutions. The first of these contained the ordinary
nutrient elements. The second contained these together with the
four well-established micro-nutrients, manganese, boron, zinc
and copper (designated A 4) and some chlorine. The third con-
tained all the elements present in the second solution together
with another seven (designated B7); these were molybdenum,
titanium, vanadium, chromium, tungsten, cobalt and nickel.
The fourth solution contained all the elements present in the
third solution together with thirteen other elements, namely,
aluminium, arsenic, cadmium, strontium, mercury, lead, lithium,

10 HISTORICAL INTRODUCTION

rubidium, bromine, iodine, fluorine, selenium and beryllium.
This group was designated C13. The fourth solution also con-
tained sodium. All the elements of the A 4, B7 and C13 groups
were present in very low concentration, that is, as traces. The
four solutions, which may be denoted by I, II, III and IV thus
contained respectively the following mineral elements :

I: N, S, P, K, Ca, Mg, Fe.
II: elements of I + B, Zn, Mn, Cu, +C1.
Ill: elements of 11 + Mo, Ti, V, Cr, W, Co, Ni.
IV: elements of TII + A1, As, Cd, Sr, Hg, Pb, Li, Rb, Br, I, F, Se, Be,

+ Na.

The fresh weights in grams of the plants grown in these
different solutions are shown in Table I. Thus the great effect
of the four well-established micro-nutrients is well demon-
strated, but the seven additional elements of solution III
produced a further increase in growth of asparagus, while for
lettuce their effect was most striking, the yield being increased
about ten times as a result of their addition. The subsequent
demonstration by Arnon and Stout that molybdenum is neces-
sary for the tomato has been mentioned earlier, and this leaves

Table I. Growth of plants of asparagus and lettuce
in four different culture solutions. (Data from Arnon)

Fresh

weight in g.

A

f^nltjirfi

Asparagus

A.

Lettuce

A

^

solution

I

II
III
IV

1

Shoots

16-2

88-2

1181

121-7

Roots

12-8
38-2
81-3
74-5

t

Shoots

71-4

105-7

1068-3

984-4

Roots

14-5
220

188-6
196-2

in doubt whether the effectiveness of the seven elements of
solution III not included in solution II is due solely to the
molybdenum or whether some or all of the others are also in part
responsible for this. That no further increase in yield results
from the addition of the extra thirteen elements of solution IV
at first sight suggests that none of these is essential for the
growth of asparagus and lettuce, but it may be that one or more
of these is actually necessary but exists in sufficient quantity
as impurity of one of the salts used in making up solution III.

HISTORICAL INTRODUCTION 11

Using Arnon's technique, Twyman (1943) obtained a similar
result with oats. Four weeks after germination of the grains
the average dry weights of plants supplied with the A 4, B7
and A4 + B7 groups of trace elements were respectively 0-101,
0-130 and 0-238 g. This again shows the necessity of one or more
of the elements of Arnon's group B7.

The question arises why for so many years the necessity for
the trace elements in plant nutrition was not recognized. The
answer given to this question by Maze was no doubt the correct
one. Maze considered that the importance of the micro-
nutrients had been overlooked because in water-culture experi-
ments a sufficient quantity of these was introduced into the
cultures from (1) the seeds used, (2) impurities in the salts used
in preparing the culture solutions, and (3) solution from the
vessels containing the culture solutions. Indeed, knowledge of
the existence of the various trace elements has only been
obtained through the purification of the water and nutrient
salts used, and the choice of suitable culture vessels.

The securing of an adequate degree of purity of the materials
used is of the utmost importance in experimental work designed
to examine the indispensability or otherwise of particular
substances. The methods that have been developed to this end,
along with other experimental methods of value in work on
micro -nutrients, form the subject of the next chapter.

Although addition of a compound of a particular element to
the nutrient medium in which plants are growing may bring
about increase in rate of growth of the plants, it does not follow
that the element is essential for the growth of plants. Indeed,
increases in growth rate as a result of the addition of com-
pounds of a number of different elements have been recorded
from time to time. Among recent observations of this kind
particular mention may be made of those of R. S. Young (1935),
who examined the effect On the growth of timothy (Phleum
pratense) of thirty -five of the rarer elements when added in
five different concentrations (2000, 500, 100, 10 and 0-1 p. p.m.)
to a sandy loam. Beneficial effects were observed with molyb-
denum, supplied in 2000 p.p.m., and with antimony, barium,
bismuth, bromine, cerium, manganese, strontium, tungsten,
uranium and yttrium, supplied at the rate of 500 p.p.m.

12 HISTORICAL INTRODUCTION

Aluminium, cadmium, copper, fluorine, lanthanum, lead, mer-
cury, tin and zinc gave an increased growth when supplied at
the rate of 100 p. p.m., while with 10 p.p.m. arsenic, beryllium,
chromium, iodine, lithium, selenium, thorium, titanium, vana-
dium and zirconium were beneficial. At a concentration of
0-1 p.p.m., boron, nickel and thallium brought about an increase
in growth. Of the thirty-five elements, the effects of which were
tested, only cobalt appeared to be slightly toxic at this lowest
concentration employed, while silver at this concentration
appeared to have no effect. At higher concentrations than those
stated for the respective elements the action of these was
depressing on growth.

Experiments with cultures of two green algae, species of
Chlorella and Crucigina respectively, led Young to conclude that
on the whole any element will stimulate the growth of algae at
a definite concentration which depends on the element and the
species.

We certainly cannot conclude that an element which stimu-
lates growth is necessarily essential for growth, although if
increased growth is observed to result from the presence of a
particular element there is always the possibility that that
element may be an essential one. Whether it is so or not can
only be proved by growing the plant in carefully controlled
cultures, in which every care is taken rigidly to exclude from the
culture medium the element under examination.

To conclude this introduction to our subject here is appended
a list of those elements which, it has been claimed, constitute
micro-nutrients of plants. Under the name of each of these
elements are listed those species for which it has been claimed
that the element either is essential or induces an increase in rate
of growth or in yield. In the latter circumstance it does not
necessarily follow that the element is essential although the
evidence of increased growth indicates that this may be so.

In the following list the name of the first worker to call
definite attention to the favourable effect of each particular
element on the growth of the species concerned is given, whether
that worker regarded the element as essential for the species
or not. Thus Nakamura in 1903 reported increased growth of
peas and spinach as a result of adding boron to the soil, but it

HISTORICAL INTRODUCTION 13

was not until 1915 that Maze claimed the essential quality of
boron for plant growth. For a few the claim is very tenta-
tive and in others the justification for the claim rather tenuous,
but for most the evidence is definite enough to be accepted.

Manganese

Rumex Acetosa (sorrel) Boullanger, 1912
Fagopyrum esculentum (buckwheat) Meyer, 1931
Beta marilima (mangold) Gilbert and McLean, 1928

B. maritima (beet) Gregoire, Hendrick and Carpiaux, 1907
Spinacia oleracea (spinach) Tacheuchi, 1909
Bougainvillea sp. Dickey and Reuther, 1938

Cochlearia Armoracia (horse -radish) Picado and Vicente, 1923

Sinapis alba (white mustard) Clausen, 1913

Brassica campestris (turnip) Chittenden, 1915

Raphanus sativus (radish) McHargue, 1923

Fragaria vesca (strawberry) Hoagland and Synder, 1933

Prunus Persica (peach) Weinberger and Cullinan, 1937

Lupinus spp. (lupins) Montemartini, 1911; blue lupin, Scholz, 1934

Medicago saliva (lucerne) D'Ippolito, 1914

M. denticulata (toothed bur clover) Samuel and Piper, 1929

Trifolium subterraneum (subterranean clover) Samuel and Piper, 1929

Vicia Faba (broad bean) Bonomi, 1908; Tacheuchi, 1909; Monte-
martini, 1911

Pisum arvense (field pea) McHargue, 1923

P. sativum (garden pea) Kakahi and Baba, 1907

Lathyrus tuberosus (earth-nut pea) Zlartarov, 1934

Cicer arietinum (chick pea) Zlartarov, 1934

Phaseolus communis (kidney bean) Andouard and Andouard 1911;
Stoklasa, 1911

Glycine hispida (soya bean) McHargue, 1923

Vigna sinensis (cow pea) McHargue, 1923

Linum usitatissimum (flax) Takeuchi, 1909

Citrus sinensis (orange) Haas, 1932

C. limonia (lemon) Haas, 1932

C. limonia (rough lemon) Haas, 1932

Aleurites fordii (tung-oil tree) Reuther and Dickey, 1937

A. montana (mu-oil tree) Reuther and Dickey, 1937

Vitis vinij 'era (vine) Montemartini, 1911

Lagerstroemia indica (crape myrtle) Dickey and Reuther, 1938

Psidium cattleianum (cattley guava) Dickey and Reuther, 1938

Apium graveolens (celery) Boullanger, 1912

Daucus Carota (carrot) Boullanger, 1912

Vaccinium corymbosum (blueberry) Shive. 1933

Allamanda cathartica Dickey and Reuther, 1938

Solarium tuberosum (potato) Gregoire, Hendrick and Carpiaux, 1907

Lycopersicum esculentum (tomato) Schreiner and Dawson, 1927

14 HISTORICAL INTRODUCTION

Bignonia venusta (flame vine) Dickey and Reuther, 1938
Thunbergia grandiflora Dickey and Reuther, 1938
Lactuca sativa (lettuce) Gilbert and McLean, 1928

Zea mais (maize) Suthert and Ingle, 1908

Saccharum officinarum (sugar cane) Lee and McHargue, 1928

Oryza sativa (rice) Aso, 1904

Phalaris bulbosa (Toowoomba canary grass) Samuel and Piper, 1929

Phleum pratense (timothy) Gram, 1936

Avena sativa (oat) Bertrand, 1905

Arrhenatherum avenaceum (tall oat grass) Gram, 1936

Danthonia penicillata (slender Wallaby grass) Samuel and Piper, 1929

Bromus uniloides (prairie grass) Samuel and Piper, 1929

Triticum vulgare (wheat) Nazari, 1910; Andouard and Andouard, 1911

Secale cereale (rye) Scharrer and Schropp, 1934

Hordeum distichum (barley) Katayama, 1906

Lolium subulatum (Wimmera rye-grass) Samuel and Piper, 1929

Lemna minor (duckweed) Hopkins, 1931

Allium Cepa (onion) Gilbert and McLean, 1928

Chlorella Hopkins, 1930
Ditylum brightwelli Harvey, 1939

Rhizopus nigricans McHargue and Calfee, 1931

Saccharomyces cerevisiae (yeast) McHargue and Calfee, 1931

S. apiculatus (yeast) Seiss, 1908

S. ellipsoideus (yeast) Seiss, 1908

Aspergillus niger Bertrand and Javillier, 1911

A. flavus McHargue and Calfee, 1931

Ceratostomella Ulmi Ledeboer, 1934

Trichophyton interdigitale Mosher, Saunders, Kingery" and Williams,

1936
Phymatotrichum omnivorum Rogers, 1938

Streptococcus lactis Zlataroff and Kaltschewa, 1936

Lactobacillus casei Woolley, 1941 (increased rate, but not extent, of
growth)

Zinc

Carya olivaejormis (pecan) Alben, Cole and Lewis, 1932

Juglans regia (walnut) Chandler, Hoagland and Hibbard, 1932

J. Hindsii (black walnut) Chandler, Hoagland and Hibbard, 1935

Populus sp. (Carolina poplar) Chandler, Hoagland and Hibbard, 1935

Ficus Carica (fig) Chandler, Hoagland and Hibbard, 1935

Fagopyrum esculentum (buckwheat) Sommer, 1928

Sinapis nigra (black mustard) Hoagland, Chandler and Hibbard, 1936

HISTORICAL INTRODUCTION 15

Pyrus Malus (apple) Chandler, Hoagland and Hibbard, 1932

P. communis (pear) Chandler, Hoagland and Hibbard, 1932

Fragaria vesca (strawberry) Hoagland and Snyder, 1933

Prunus Armeniaca (apricot) Chandler, Hoagland and Hibbard, 1934

P. domestica (plum) Chandler, Hoagland and Hibbard, 1932

P. cerasus (cherry) Chandler, Hoagland and Hibbard, 1932

P. Persica (peach) Chandler, Hoagland and Hibbard, 1932

Vicia Faba (broad bean) Sommer, 1928

Pisum sativum (garden pea) Scharrer and Schropp, 1934

Phaseolus vulgaris (red kidney bean) Sommer, 1928

Citrus sinensis (orange) Johnston, 1933

C. limonia (lemon) Haas, 1932

G. grandis (grape-fruit) Parker, 1936

Melia sp. Chandler, Hoagland and Hibbard, 1935

Aleurites fordii (tung-oil tree) Mowry and Camp, 1934

A. montana (mu-oil tree) Mowry and Camp, 1934

Vitis vinifera (grape) Chandler, Hoagland and Hibbard, 1932

Gossypium sp. (cotton) Hoagland, Chandler and Hibbard, 1936

Ligustrum sp. Chandler, Hoagland and Hibbard, 1935

Nicotiana tabacum (tobacco) Hoagland, Chandler and Hibbard,

1936
Lycopersicum esculentum (tomato) Hoagland, Chandler and Hibbard,

1936
Cucurbita maxima (squash) Hoagland, Chandler and Hibbard, 1936
Heliantkus annuus, (sunflower) Sommer and Lipman, 1926
Zea mais (maize) Maze, 1914

Triticum vulgare (wheat) Scharrer and Schropp, 1934
Secale cereale (rye) Scharrer and Schropp, 1934
Hordeum distichum (barley) Sommer and Lipman, 1926
Lemna minor (duckweed) Steinberg, 1941
Pinus radiata Kessell and Stoate, 1938

Coccomyxa simplex Roberg, 1932
Chlorella vulgaris Roberg, 1932

Rhizopus nigricans McHargue and Calfee, 1931

R. suinus Nielsen and Hartelius, 1933

Aspergillus niger Raulin, 1869

A.flavus Metz, 1930

A. fumigatus Roberg, 1928

A. oryzae Roberg, 1928

A. ficuum Roberg, 1928

A. cinnamomeus Roberg, 1928

A.fuscus Roberg, 1928

Ceratostomella Ulmi Ledeboer, 1934

Trichophyton interdigitale Mosher, Saunders, Kingery and Williams

1936
Phymatotrichum omnivorum Rogers, 1938
Fusarium oxysporum Niethammer, 1938

16 HISTORICAL INTRODUCTION

Acaulium nigrum Niethammer, 1938
Penicillium sulfureum Metz, 1930
P. luteum Metz, 1930
Macrosporium sp. Metz, 1930
Phonia betae Metz, 1930
Ovularia sp. Metz, 1930
Botrytis cinerea Metz, 1930

Boron1

Carya olivaeformis (pecan) Blackmon and Camp, 1932

Cannabis sativa (hemp) Skolnik, 1935

Fagopyrum esculentum (buckwheat) Sommer and Lipman, 1926

Beta vulgaris (sugar beet) Brandenburg, 1921

Spinacia oleracea (spinach) Nakamura, 1903

Persea gratissima (avocado) Haas, 1939

Papaver officinale (poppy) Brandenburg, 1942

Iberis umbellata (candytuft) Lohnis, 1937

Sinapis alba (white mustard) Sommer and Lipman, 1926

£. nigra (black mustard) Chandler, 1941

Brassica campestris (turnip) Scharrer and Schropp, 1934

B. campestris (swede) Jamalainen, 1935

B. campestris (rutabaga) Muhr, 1942

B. oleracea (cabbage) Walker, Jolivette and McLean, 1939

B. oleracea var. gemmifora (Brussels sprout) Chandler, 1940

B. oleracea (kale) Chandler, Chucka and Mason, 1938

B. oleracea var. italica (sprouting broccoli) Chandler, 1940

B. oleracea (cauliflower) Ferguson, 1938

B. pekinensis (Chinese cabbage) Chandler, 1940

B. caulorupa (kohlrabi) Chandler, Chucka and Mason, 1938

B. Napus (rape) Schropp and Areuz, 1938

Raphanus sativus (radish) Agulhon, 1910

Camelina sativa (gold of pleasure) Schropp and Arenz, 1938

Ribes rubrum (red currant) Lohnis, 1937

Pyrus Malus (apple) McLarty, 1928

Fragaria vesca (strawberry) Hoagland and Snyder, 1933

Rosa sp. (rose) Davidson and Biekert, 1939

Prunus Persica (peach) Weinberger and Cullinor, 1937

P. Armeniaca (apricot) Hoagland, Chandler and Hibbard, 1936

Lupinus albus (white lupin) van Gennep, 1936

L. hispidus (blue sweet lupin) Schropp and Arenz. 1942

L. luteus (yellow lupin) van Gennep, 1936

Medicago sativa (lucerne) Brenchley and Warington, 1927

Melilotus sp. (melilot) Cook, 1938

Trifolium incarnatum (scarlet clover) Warington, 1923

T. pratense (red clover) Brenchley and Warington, 1927

1 A number of the later entries under this head are given on the
authority of Dennis and Dennis (1941, 1943).

HISTORICAL INTRODUCTION 17

Trijolium repens (white clover) Brenchley and Warington, 1927

T. minus (lesser clover) Brenchley and Warington, 1927

T. hybridum (Alsike clover) Cook, 1938

Onobrychis sativa (sainfoin) Mevius, 1928

Ornithopus sativus (serradella) Kedrov-Sichman and Dankova-Ano-

china, 1940.
Arachis hypogea (pea-nut) Burkhart and Collins, 1942
Vicia Faba (broad bean) Warington, 1923
V. sativa (common vetch) Sommer, 1927
V. villosa (hairy vetch) Lohnis, 1937
Pisum sativum (garden pea) Nakamura, 1903
Lathy rus odoratus (sweet pea) Laurie and Wagner, 1940
Lens esculenta (lentil) Kalantyr, 1939
Phaseolus multijlorus (scarlet runner) Warington, 1923
P. vulgaris (dwarf bean) Brenchley and Warington, 1927
Glycine hispida (soya bean) Brenchley and Warington, 1927
Linum usitatissimum (flax) Sommer and Lipman, 1926
Citrus sinensis (orange) Haas, 1929
C. limonia (lemon) Haas, 1929

Ricinus communis (castor oil) Sommer and Lipman, 1926
Euphorbia pulcherrima (poinsettia) Laurie and WTagner, 1940
Impatiens balsamina (balsam) Rehm, 1937
Vitis vinifera (vine) Maier, 1937

Malva verticillata (forage mallow) Schropp and Arenz, 1940
Gossypium s-p. (cotton) Sommer and Lipman, 1926
Begonia semperflorens Laurie and Wagner, 1940
Apium graveolens (celery) Purvis and Ruprecht, 1935
Daucus Carota (carrot) Warington, 1940
Vaccinium corymbosum (blueberry) Shive, 1935
Ipomoea Batatas (sweet potato) Cooper, 1938
/. purpurea (morning glory) Ark and Thomas, 1940
Phacelia sp. Schropp, 1941
Salvia occidentalis s' Jacob, 1936
Perilla ocymoides Skolnik, 1935
Solanum tuberosum (potato) Breckenridge, 1921
Lycopersicum esculentum (tomato) Johnson and Dore, 1928
Nicotiana tabacum (tobacco) Swanback, 1927
Calceolaria herbeohybrida (calceolaria) Ark and Tompkins, 1941
Sinningia speciosa (gloxinia) Ark and Tompkins, 1941
Gardenia Veitchii Laurie and Wagner, 1940
Coffea arabica (coffee) s' Jacob, 1936
Cucumis Melo (melon) Brenchley, 1926
Cucurbita Pepo (pumpkin) Sommer, 1927
C. maxima (squash) Purvis and Hanna, 1940
C. Melo var. cantalupensis (cantaloupe) Stier, 1942
Helianthus annuus (sunflower) Sommer and Lipman, 1926
Dahlia variabilis Sommer, 1927

Chrysanthemum indicum Ferguson and Wright, 1940
Carthamnus tinctorius (safliower) Schropp, 1941

18 HISTORICAL INTRODUCTION

Crepis biennis (hawk's beard) Herzinger, 1940

Lactuca sativa (lettuce) McHargue and Calfee, 1933

Sonchus oleraceits (sow thistle) Herzinger, 1940

Cichorium Intybus (chicory) Muhr, 1942

Taraxacum dens-leonis (dandelion) Muhr, 1942

Zea mais (maize) Maze, 1919

Saccharum officinarum (sugar cane) Martin, 1924

Sorghum vulgare Sommer, 1927

S. sudanense Skolnik, 1935

Panicum miliaceum (millet) Sommer, 1927

Phalaris canariensis (canary grass) Schropp, 1940

Avena sativa (oat) Agulhon, 1910

Triticum vulgare (wheat) Agulhon, 1910

Secale cereale (rye) Lohnis, 1937

Hordeum distichum (barley) Sommer and Lipman, 1926

Lemna minor (duckweed) Steinberg, 1941

L. polyrrhiza (small duckweed) Geigel, 1935

Tradescantia albiflora Meier, 1938

Allium Cepa (onion) Lohnis, 1937

Chlorella sp. Geigel, 1935
Ulothrix tenerrima Herzinger, 1940

Dothiorella sp. Davis, Marloth and Bishop, 1928
Azotobacter chroococcum Herzinger, 1940

Silicon

Beta vulgaris (red beet) Raleigh, 1939
Helianthus annuus (sunflower) Lipman, 1938
Zea mais (maize) Maze, 1919
Oryza sativa (rice) Ishibashi, 1937
Hordeum distichum (barley) Lipman, 1938

Aluminium

Pisum sativum (garden pea) Sommer, 1926

Helianthus annuus (sunflower) Lipman, 1933

Zea mais (maize) Maze, 1919

Panicum miliaceum (?) (millet) Sommer, 1926

Avena sativa (oat) Stoklasa, 1922

Glyceria aquatica (reed meadow grass) Stoklasa. 1922

Triticum vulgare (wheat) Stoklasa, 1922

Hordeum distichum (barley) Stoklasa, 1922

J uncus effusus (rush) Stoklasa, 1922

Chlorine

m

Fagopyrum esculentum (buckwheat) Nobbe and Siegert, 1862
Pisum sativum (garden pea) Lipman, 1938
Zea mais (maize) Maze. 1919

HISTORICAL INTRODUCTION 19

Copper

Brassica oleracea (cabbage) Russell and Manns, 1933

Linum usitatissimum (flax) Sommer, 1931; Lipman and Mackinney,

1931
Pyrus Mains (apple) Anderssen, 1932
P. communis (pear) Anderssen, 1932
Prunus domestica (plum) Anderssen, 1932
P. Persica (peach) Anderssen, 1932
P. Armeniaca (apricot) Anderssen, 1932
Trifolium subterraneum (subterranean clover) Piper, 1942
Medicago sativa (lucerne) Piper, 1942
Pisum sativum (garden pea) Piper, 1942
Vicia Faba (broad bean) Russell and Manns, 1933
Glycine hispida (soya bean) Russell and Manns, 1933
Ipomoea Batatas (sweet potato) Russell and Manns, 1933
Solarium tuberosum (potato) Russell and Manns, 1933
Lycopersicum esculentum (tomato) Skinner and Ruprecht, 1930
Helianthus annuus (sunflower) Sommer, 1931
Zea mais (maize) Russell and Manns, 1933
Saccharum officinale (sugar cane) Allison, 1930
Oryza sativa (rice) Harrison and Subrahmanya Azya, 1917
Phalaris tuberosa (a canary grass) Piper, 1942
A vena sativa (oat) McHargue and Shedd, 1930
Triticum vulgare (wheat) Russell and Manns, 1933
Hordeum distichum (barley) Lipman and Mackinney. 1931
Lolium subulatum (Wimmera rye-grass) Piper, 1942

Rhizopus nigricans McHargue and Calfee, 1931

Aspergillus niger Bortels, 1927

A. fumigatus Roberg, 1928

A. oryzae Roberg, 1928

A. ficuum Roberg, 1928

A. cinnamomeus Roberg, 1928

A. fuscus Roberg, 1928

A.flavus McHargue and Calfee, 1931

Ceratostomella Ulmi Ledeboer, 1934

Trichophyton interdigitale Mosher, Saunders, Kingerv and Williams,

1936
Phymatotrichum omnivorum Rogers, 1938

Bacillus fluoresceins Russell and Manns, 1933

Columbium

Penicillium Javanicum Lockwood, 1933

Molybdenum

Prunus cerasifolia (myrobalan plum) Hoagland, 1941
Lycopersicum esculentum (tomato) Arnon and Stout, 1939

20 HISTORICAL INTRODUCTION

Avena sativa (oat) Piper, 1940
Hordeum distichum (barley) Arnon, 1937
Lemna minor (duckweed) Steinberg, 1941

Penicillium Javanicum Lockwood, 1933
Aspergillus niger Steinberg, 1936

Tungsten

Penicillium Javanicum Lockwood, 1933

Gallium

Lemna minor (duckweed) Steinberg, 1941

Aspergillus niger Steinberg, 1938

In addition to the elements included in the above list, claims
have been made that others, for example, tin and uranium,
'stimulate' plant growth. Until further information accumu-
lates it would be wiser to defer judgement on the significance of
these claims. It should also be noted that some of the elements
in the list are scarcely generally accepted at present as micro -
nutrients. In this category are columbium and tungsten. The
elements most definitely accepted as micro -nutrients are man-
ganese, zinc, boron, copper and molybdenum, but the evidence
for aluminium, silicon, chlorine and gallium for the plants cited
in the list is very strong.

CHAPTER II

METHODS OF INVESTIGATING MICRO-
NUTRIENT PROBLEMS

1. The Purification of Materials used
in Culture Experiments

The existence of the micro -nutrients raises a number of pro-
blems which can only be solved after the development of
methods designed specially to deal with them. In the first
place the question inevitably arises as to how we can be certain
that any particular element is really essential or not. This is
obviously a problem of mineral nutrition requiring immediate
solution; it is clearly an essential preliminary for all investiga-
tions on the micro -nutrients that we should know what these are.

It has already been pointed out that the essentiality of the
micro -nutrients for plant development was overlooked for half
a century simply because an adequate supply of them was
introduced into the culture (1) from the seed from which the
plant developed, (2) from impurities present in the water and
salts used in preparing the culture solutions, and (3) by solution
from the vessels used to hold the culture solutions. Hence the
problem of determining the micro -nutrients clearly resolves
itself into devising means for preventing the introduction of
micro -nutrients from these three sources. If this can be done
the effect of the absence or presence of any particular element
in the culture medium can then be determined for a wide range
of plant species.

The prevention of the introduction of any particular elements
from the seed used for the cultures is perhaps scarcely possible.
But when the seed is small the amount of any micro -nutrient
present in it is likely to be negligible, and when the seed is large
the amount introduced can often be considerably reduced by
removal of the cotyledons from the seedling as soon as the latter
is established. Reference has already been made to Lipman's
method of meeting the difficulty in his experiments with buck-
wheat, in which seed was used from plants grown in culture

22 METHODS OF INVESTIGATING

solutions devoid of chlorine. It appears clear that by this
procedure seed was obtained containing a negligibly small
quantity of this element.

The problem of obtaining water and salts free from the trace
elements for work on the nutrition of fungi has been dealt with
by a number of workers, notably Steinberg (1919, 1935 6). It
may be stated at once that pure salts sold as analytical reagents
may contain a sufficiency of trace elements present as impurities
to allow the growth of plants, nor does recrystallization neces-
sarily afford an adequate means for the removal of these
contaminants.

Steinberg's original precedure for obtaining a nutrient solu-
tion free from so-called heavy metal contaminants consisted in
heating the complete nutrient solution with pure calcium
carbonate under pressure. The nutrient solution used was one
due to Pfeffer and was made up as follows :

Sucrose 50 g.

Ammonium nitrate ] 0 g.

Potassium dihydrogen phosphate 5 g.

Magnesium sulphate 2-5 g.

Ferrous sulphate Trace

Since Steinberg's treatment of the solution leads to the
removal of iron it is obvious that the trace of ferrous sulphate
may be omitted.

To a litre of this solution 15 g. of pure calcium carbonate were
added and the mixture heated in an autoclave for 20 min. under
a pressure so as to give a temperature of 120-5° C. The mixture
was then allowed to stand overnight and the clear solution
then decanted from the sediment. Subsequently, Steinberg
recommended filtering the solution from the sediment im-
mediately after autoclaving.

The principle involved in this method of purification consists
in increasing the alkalinity of the solution so that the traces of
heavy metals are precipitated as carbonates, hydroxides and
phosphates which are adsorbed on calcium salts precipitated in
some bulk. In this way traces of iron, manganese, zinc and
copper are removed from the solution.

Various modifications of this procedure have been proposed.
Steinberg himself points out that the substitution of basic
magnesium carbonate for calcium carbonate has certain advan-

MICRO-NUTRIENT PROBLEMS 23

tages where work on fungi is concerned. It does not involve the
introduction into the solution of calcium, an element of which the
necessity for fungus nutrition is, as we have already seen,
doubtful. Indeed, it appears to effect a pretty complete re-
moval of any calcium present as impurity in the nutrient solu-
tion. Also the use of an autoclave is unnecessary, heating for
20 min. at 100° C. being sufficient to precipitate the heavy
metals. Care has, however, to be taken to avoid excess of the
basic magnesium carbonate, as otherwise more or less complete
removal of phosphate may result.

Bortels (1927) similarly purified the nutrient solution by pre-
cipitating the traces of the heavy metal contaminants with a
small quantity of ammonium sulphide and adsorbing the
precipitate on charcoal. Actually the ammonium sulphide
appears to be unnecessary, according to Roberg (1928), who also
purified the charcoal from mineral ash constituents by a pre-
liminary treatment with acid. However, Steinberg pointed out
that removal of the ash constituents appears to reduce the
adsorbing power of charcoal, and he concluded that the use of
charcoal is only advisable when for some reason it is essential
to avoid the use of an alkaline earth compound.

In 1927, Hopkins and Wann made use of the adsorptive pro-
perty of calcium phosphate for the removal of iron from culture
solutions for the green alga Chlorella and later, for work with
green algae and Lemna, Hopkins (1934) again employed calcium
phosphate as an adsorbent for removal of traces of manganese
from the nutrient solution. Sakamura, who had previously
(1933, 1934) used charcoal for removal of traces of heavy metals
from the nutrient medium of Aspergillus spp., concluded (1936),
by polarographic examination1 of nutrient solutions after treat-
ment with charcoal and calcium phosphate respectively, that
the latter effected a much more complete removal of the heavy
metal contaminants, a conclusion which was confirmed by growth
experiments. His procedure was as follows. Calcium phosphate
was first purified by washing with water distilled in a glass still,
a suspension of 50 g. calcium phosphate in a litre of distilled
water being shaken for 5 hr., during which time the water was

1 See this chapter, p. 27.

24 METHODS OF INVESTIGATING

changed four times. The calcium phosphate was then filtered off
with the use of ash -free filter paper and dried. Calcium phos-
phate so purified was then added to the culture solution so that
it was contained in the latter to the extent of 0-5 per cent and
the whole was brought to a pH of 5-5 by means of sodium
hydroxide. The solution was then shaken for 2 hr. and twice
filtered. The final filtrate comprised the working culture solution.

The preparation of culture solutions free from trace elements
for work with higher plants presents a somewhat different
problem from the preparation of nutrient solutions for the growth
of fungi. It will be appreciated that it is impracticable to purify
the complete culture solution for higher plants owing to the
large quantities of solution required, and it is thus necessary to
remove the trace elements from the water and nutrient salts
separately. The means by which this may be done have been
described in detail by Stout and Arnon (1939). As regards the
water used, it is necessary to avoid the use of distillation
apparatus made of or containing copper, silver, tin or other
metal. Although the actual content of contaminants in distilled
water from a metal still may be very small, yet the quantity of
water used, particularly in the culture of higher plants, is very
considerable, so that the absolute amount of contaminant
presented to the plant may be far from negligible. Stout and
Arnon found that ordinary distilled water contained from 0- 1 to
0-01 p. p.m. of metal contaminants. They recommended, there-
fore, that water should be redistilled, using a trap and con-
denser of pyrex glass and distilling at a rate slow enough to give
a cool distillate. Water is obtained in this way free from metal
impurities. It should be noted that the use of Jena glass is to
be avoided, since this contains zinc which may appear in the
distilled water.

The mineral salts used by Stout and Arnon were calcium
nitrate, potassium nitrate, magnesium sulphate, diammonium
phosphate, dipotassium phosphate and ammonium sulphate.
Molar solutions of each of these salts were prepared and purified
separately, 5 1. at a time, in 6 1. pyrex flasks provided with a
plug of cotton-wool. The principle involved in the purification
of the solutions was the same as that employed by Steinberg,
but the details of the purification varied somewhat for different

MICRO-NUTRIENT PROBLEMS 25

salts. In all cases 65 g. calcium carbonate and a small quantity
of a solution of some other salt were added to 5 1. of the solu-
tion to be purified. For calcium nitrate and potassium nitrate
the solution added along with the calcium carbonate was
50 ml. of molar dipotassium phosphate; with dipotassium phos-
phate and diammonium phosphate the added solution con-
sisted of 25 ml. of molar calcium nitrate, and with magnesium
sulphate and ammonium sulphate the added solution was 50 ml.
of molar calcium nitrate +50 ml. of molar dipotassium phos-
phate. The purification of all the solutions except that of
ammonium sulphate was then effected by autoclaving the mix-
ture for an hour at 20 lb. pressure, allowing the solution to stand
overnight and then filtering. The ammonium sulphate was
treated similarly except that the solution was heated for 45 min.
in a steamer instead of in an autoclave. The final filtrates of the
dipotassium and diammonium phosphate were acidified to
pH 5-5 with pure sulphuric or nitric acid.

For testing the purity of the solutions so prepared Stout and
Arnon made use of dithizone (diphenylthiocarbazone). The
testing reagent is prepared by dissolving 0-1 g. of purified
dithizone in 100 ml. of redistilled chloroform. This reagent gives
a red or purple colour in the chloroform layer when it is added
to a solution containing zinc, copper, lead, nickel, cobalt,
cadmium, thallium, mercury or bismuth. By comparing the
colour produced by standard solutions with that produced by
the purified nutrient solutions it was found that the latter
usually contained less than one part of metal contaminants in
108 parts of solution, a degree of purity which was deemed
sufficient for culture work on micro -nutrients. Although man-
ganese does not give the colour reaction with dithizone it was
concluded that if the other metal contaminants which do
produce the colour with dithizone are removed, the manganese
will have been removed also.

The salts so purified did not include iron. This was provided
as a solution containing 0-5 per cent ferrous sulphate +0-5 per
cent tartaric acid which was added twice weekly to the extent
of 0-5 ml. per litre of culture solution.

That the method of purification used by Stout and Arnon was
justified is clear from the fact that they obtained definite effects

26 METHODS OF INVESTIGATING

which were reproducible in the growth of plants by adding
1 part of zinc in 2 x 108 parts of culture solution.

There remains the question of the vessels used to contain the
culture solutions. There appears to be a general agreement that
containers made of pyrex glass form suitable culture vessels for
work on micro-nutrients.

2. The Estimation of Micro-nutrient
Elements in Plant Material

In order to investigate the part played by micro -nutrients in
plants it is a prerequisite that methods should be available for
the quantitative determination of each of them in plant tissues,
for without quantitative data little advance in knowledge of any
value is likely to accrue. In general, however, the quantities of
these elements present in the tissues are so small that the
ordinary methods of quantitative chemical analysis are useless
for the purpose, and methods have to be found by which very
small quantities of the elements concerned can be determined
with a reasonable degree of accuracy. Indeed, the advance of
plant physiology in general has been retarded very considerably
by the lack of methods for measuring many substances in very
small quantity, and it is certain that increase of knowledge of
the physiology of plants waits in large measure on the develop-
ment of such micro-methods.

During the last decade a number of physical instruments
have been developed which can be employed by the plant
physiologist for the measurement of small quantities of material,
and it is now possible with their aid to determine with sufficient
accuracy all the known micro -nutrients in plants. These instru-
ments are the absorptiometer, the polarograph and the spectro-
graph. The absorptiometer is an adaptation of the colorimeter
in which the depth of colour of a solution is measured by
matching it against that of a standard solution, the matching
being made, not by the eye, but by a photoelectric cell. The use
of this instrument for the determination of small quantities of
phosphorus has been described by Berenblum and Chain (1938),
who have shown that quantities as small as 0-1/Ag. can be
measured with it. Not only have A. D. Skelding and I used the

MICRO-NUTRIENT PROBLEMS 27

instrument for this purpose, but it has also been used success-
fully in my laboratory to determine small quantities of mag-
nesium, iron, aluminium and manganese in the course of work
not yet published . The smallest quantities of each of these elements
which have so far been determined in this manner are noted later.1

The polarograph is an instrument in which a solution of an
electrolyte in presence of another electrolyte in much higher
concentration (known as the ground substance or supporting
electrolyte) is subjected to a gradually increasing difference of
potential between two electrodes, one of which consists of a series
of small drops of mercury delivered from the end of a capillary
tube, while the other consists of a still mass of mercury with a
comparatively large surface. In these circumstances, when cer-
tain experimental conditions are fulfilled a current (the so-called
"wave") flows through the solution when the potential difference
reaches a certain value determined by the nature of the cation,
or in certain circumstances by the anion, present, while the
magnitude of the current is determined by the concentration of
these cations (or anions). By means of this instrument it is
possible to measure quantities of a number of cations and anions
of the order of Ifxg. or less. Among the ions which have so far
been determined in this way in my laboratory in the course of
the last five years are potassium, copper, manganese, aluminium,
iron, zinc, barium, chloride, sulphate and nitrate, though in
general it should be noted that it is not possible with the polaro-
graph to determine one alkali metal in presence of another.

The polarograph was developed by Jaroslav Heyrovsky and
appears to have been first described by Heyrovsky and Shikata
in 1925, but although much of the pioneer work with the instru-
ment was described in English in the Collection of Czechoslovak
Chemical Communications, it appears until recently to have been
little used in this country. There can, however, be no doubt
whatever that this instrument is a most valuable tool for the
student of plant nutrition, and the recent publication in English
of a book on polarographic analysis by Kolthoff and Lingane
(1941) may render an appreciation of the value of the polaro-
graph more widespread.

1 Since this was written there has appeared an authoritative work
on the absorptiometer by F. W. Haywood and A. A. Wood (1944).

28 METHODS OF INVESTIGATING

Undoubtedly the use of the spectrograph affords the most
sensitive method of measuring small quantities of a large number
of elements.

Under certain conditions a substance can be made to emit
radiation, this radiation being limited to certain wave-lengths
which are characteristic of the elements in the substance and
of the conditions used to excite the radiation. If the radiation
passes through a prism or diffraction grating the radiations of
different wave-lengths are separated and a spectrum results, the
radiation possessing the longest wave-lengths being at one end
of the spectrum and that possessing the shortest wave-lengths
at the other. In the spectrograph such a spectrum is made to
fall on a photographic plate, and the plate, on development,
constitutes a photographic negative of the spectrum. This
usually consists of a number of lines, each line corresponding to
radiation of a definite wave-length and possessing an intensity
of blackness depending, within the limits of under-exposure
and over-exposure of the plate, on the intensity of radiation of
that particular wave-length. Determination of the intensity of
blackening of a line characteristic of a particular element should
therefore afford a means of estimating the quantity of that
element in a sample of material, for example, plant ash, which
has been subjected to the necessary conditions for inducing an
emission of radiation from it.

Of the various ways in which radiation suitable for spectro-
graph^ examination may be produced three have been developed
to a considerable extent; these are by means of a flame, by the
use of the electric arc and by the use of an electric spark. The
spectra produced in these ways are known as flame, arc and
spark spectra respectively. Each method has its own particular
advantages and disadvantages. Perhaps with workers on plant
material the flame method has so far proved the most popular,
but this is by no means so with workers in other scientific
fields.

Flame spectra, as the name suggests, are produced when a
substance is heated in a flame. Although spectra are produced
when the flame is that of a Bunsen burner, for analytical pur-
poses a flame much hotter than this is generally required, and
air-acetylene, oxy-coal gas, oxy-hydrogen and oxy-acetylene

MICRO-NUTRIENT PROBLEMS 29

flames have all been employed with more or less success. Two
distinct procedures have been employed for introducing the
substance into the flame. In one a small quantity of the sub-
stance is contained on or in a piece of filter paper and the latter
then introduced into the flame. In the other a solution of the
substance is sprayed into the flame through a very fine nozzle.
These two methods are largely associated with the names of
Ramage and Lundegardh respectively, but they have both been
used, with a variety of modifications in detail, by other workers
as well. By maintaining the conditions of experimentation
constant the same density of spectral line can be obtained from
the same quantity of material, so if calibration is made by the
use of a number of samples of known composition it is possible
to determine the amount of the element in a sample under
examination by measurement of the density of the line and
reference to a calibration graph.

In exciting spectra by means of the arc, electrodes in the form
of rods, usually of graphite, but sometimes of copper, nickel, iron
or other metal, and of as great a purity as possible, are used. The
electrodes are in a vertical line and the lower contains at its
upper end a cavity in which the substance under examination
is held. After bringing the electrodes into contact they are
separated to a standard distance apart so that the conditions of
the arc are kept as constant as possible. Even so the arc is so
variable that it has so far been found impossible to devise an
arrangement such that the same amount of material subjected
to excitation will produce the same intensity of spectral lines.
Hence in quantitative estimation of any element by means of
arc spectra it is necessary to have recourse to the device of the
'internal standard'. This is achieved by introducing along with
the substance to be examined a known amount of some other
substance involving an element which yields a spectral fine in
the near neighbourhood in the spectrum of the fine to be
measured. This same consideration holds when the spark dis-
charge is used for exciting spectra. Thus Foster and Horton, in
determining boron in plant material by the spark method, added
a known quantity of a gold salt to the material they were
examining. Within limits the ratio of the intensities of the fines
of the element to be measured and of the internal standard is

30 METHODS OF INVESTIGATING

proportional to the ratio of the quantities of the two elements
present, so that by measuring the intensities of both lines and
reference to a calibration graph obtained by the use of known
mixtures in which the quantities of the two elements are varied
the desired determination can be made.

For measuring the intensities of the spectral lines various
methods have been devised, but this is now generally effected
by means of the microphotometer. In this instrument a narrow
beam of light passes through the photographic negative of the
spectrum, and then falls on a photoelectric cell, with the result that
a current is induced which is measured by a galvanometer. By
means of a rack and pinion the plate is moved very slowly over
the beam of light so that this passes in turn through the clear
plate and the spectral line. The difference in the galvanometer
deflexion obtained for the clear plate and the spectral line gives
a measure of the intensity of the line and hence of the amount
of material. The principles of spectrographic analysis have, of
course, been given here in the broadest and simplest terms.
Actually such analysis is full of difficulties and many pre-
cautions have to be taken to ensure reliable results. A descrip-
tion of these details is outside the scope of this book, and those
interested should consult works on spectrographic analysis,
particularly the publications by F. Twyman (1935, 1938a,
19386), published by Adam Hilger, Ltd., which aim at keeping
information on this subject up to date, and the four major works
by Lundegardh(1929, 1934, 1936, 1938), which are of particular
value to workers with, biological material. The same remarks
apply to the use of the microphotometer and the method of
calculating results.

It is to be noted that in the method developed by Lundegardh
solutions are analysed, whereas in most other procedures solid
samples are used.

It has already been stated that the spectrograph affords the
most sensitive method of measuring small quantities of many
elements. This is undoubtedly the case when an arc or spark is
used, but results obtained with the flame method indicate that
the latter, as used up to now, is capable, broadly speaking, of
yielding about the same degree of sensitivity as the polarograph
and absorptiometer.

MICRO-NUTRIENT PROBLEMS 31

But from Lundegardh's experience (1929, 1934) it would
appear that the flame method is sufficiently sensitive for the
quantitative determination of only three of the micro-nutrient
elements listed in Chapter i, namely, manganese, copper
and gallium. The most dilute solutions of manganese and
copper that can be used are those of a concentration of
5x 10~6 M . As a quantity of 5-15 ml. of solution is required
this would mean that the smallest quantity of these elements
determinable by the flame method as Lundegardh used it would
be of the order of 1-4-4/zg. With a procedure more recently
described by Griggs, Johnstin and Elledge (1941), the minimum
concentration of manganese usable is given as 1-125 x 10~5 M ,
while the use of .as little as 2 ml. of solution is possible. This
would mean that the smallest quantity of manganese measur-
able would be about 1-37/xg.

Data for gallium are not at present available, but a flame
spectrum of this element reproduced by Lundegardh suggests
that the sensitivity is certainly high for this metal.

Using the arc the author has found it possible to measure
quantities of manganese as small as 0-05^g., and no doubt even
smaller quantities than this could be measured with a suitable
choice of experimental conditions.

It must be borne in mind that for the determination of any
particular element one or other of the methods that have been
here indicated may be inapplicable. Thus so far it has not been
found possible to determine either boron or magnesium, both
important plant nutrients, by means of the polarograph. The
presence of magnesium as an impurity in graphite may render
the use of a graphite arc impracticable for the determination of
that element spectrographically. A method, while usable for the
determination of larger quantities, may not be sufficiently sen-
sitive for measuring the small amounts of micro -nutrients
present in available samples of plant material. Many pre-
liminary trials of the different methods may thus be necessary
before the investigator can decide what method to use for the
estimation of any particular micro -nutrient.

The degree of accuracy generally obtainable by what may be
called micro-methods is much less than that obtainable in most
macro -chemical determinations, but is generally sufficient for

32 METHODS OF INVESTIGATING

the kind of problem which faces the investigator of plant
nutritional and pathological problems. Broadly speaking it may
be said that the results of a single determination obtained by
the methods that have been outlined here are correct within
about 5-10 per cent. A higher degree of accuracy is no doubt
often possible, and with the absorptiometer, polarograph and
spectrograph it has been claimed in certain circumstances that
the error of a single determination does not exceed 2 per cent.
By making a number of replicate determinations and taking the
mean value a considerable increase in the accuracy of an estima-
tion may be achieved, but the limited amount of material
available may often render this procedure impossible.

We may next turn to a consideration of the methods suit-
able for the estimation of the individual micro -nutrients in
plant material. They are usually determined in plant ash. This
should be prepared by first drying the material in an oven at
70-100° C, grinding it to a powder and then incinerating it in a
furnace at a temperature of from 450 to 600° C. Burning in a
crucible over a Bunsen burner or blowpipe is not to be recom-
mended, as this leads to an intense local heat causing partial
volatilization of some of the mineral matter. The ash, after
cooling, is dissolved in a small quantity of mineral acid and the
resulting solution evaporated to dryness on a water-bath. The
residue is then dissolved in a definite volume of water or dilute
acid. Insoluble silica may be removed by filtering.

The preparation of ash in this way is not always to be recom-
mended. For example, Reed and Cummings (1941) found that
there was a considerable loss of copper, amounting to 50 per
cent or more, involved in this method of ashing, even when the
temperature of ignition was as low as 450° C. For the estimation
of copper in plant material they therefore recommend a pro-
cedure in which from 0-5 to 2 g. of the plant material are heated
with 5 ml. of concentrated nitric acid until brown fumes are
evolved, when 1 ml. of concentrated sulphuric acid is added and
heating continued until charring begins and all nitric acid is given
off. After addition of 1-2 ml. of 60 per cent perchloric acid, heating
is resumed until the solution is colourless or pale yellow and ex-
cess of perchloric acid also given off. The resulting solution should
then contain all the copper originally present in the sample used.

MICRO-NUTRIENT PROBLEMS 33

It is also possible that such a 'wet ashing' method may be
preferable to dry ashing when micro-nutrients other than copper
are to be estimated. In this connexion it may be mentioned that
Griggs, Johnstin and Elledge (1941) found that dry ashing at
400° C. resulted in a loss of 30 per cent of the total potassium
and 10 per cent of the total calcium. They themselves recommend
the extraction of the mineral elements with nitric acid and
perhydrol. Nitric acid is added to a weighed quantity of the
plant material in a 70 ml. pyrex test-tube and the mixture
heated on a sulphuric acid bath at a temperature between 120
and 140° C. until the solution is clear. Perhydrol is then added
and the tube carefully heated over a micro-burner. If necessary,
small additions of nitric acid and perhydrol may be made to
effect complete decoloration of the solution.

The best established of the trace elements are, as we have seen,
manganese, zinc, copper and boron. The determination of each
of these in plant material will now be considered.

Manganese. A considerable number of methods of reasonable
accuracy are available for the determination of manganese in
small quantities. The spectrograph, polarograph and absorptio-
meter can all be employed successfully for this purpose. With
regard to the spectrograph, the flame, arc and spark methods can
all be used, but, as has already been indicated, the sensitivity of
the flame method is less than that of the arc. For the deter-
mination of manganese in plant material the flame method has
been used and described by Lundegardh (1929, 1934) and more
recently by Griggs, Johnstin and Elledge (1941). As already
mentioned, by its means about 1-4/xg. of manganese can be
determined. Lundegardh claims that the probable error of a
single determination made by the flame method is about 1-2 per
cent, but the accuracy is no doubt less than this as the amounts
determined approach the lower limit of measurable quantities.
The line used for the measurement is 4031 A. (Lundegardh,
1929). Considerably smaller quantities of manganese can be
determined by the arc and spark, but, although they have been
used quite extensively in metallurgical work, they have received
relatively little attention from the point of view of the deter-
mination of manganese in plants.

However, Melvin and O'Connor (1941) have used the arc

34 METHODS OF INVESTIGATING

method for the simultaneous determination of manganese,
boron and copper in fertilizers, and their method would appear
to be suitable for the determination of these same trace elements
in plant material. The lines used were the manganese 2605-7 A.,
boron 2497-7A. and copper 3247-5A., with the beryllium line
3130 A. as internal standard. An accuracy of about ± 5 per cent
is claimed for the estimations. Analyses published by the
advocates of the method indicate that quantities as small as, or
smaller than, 0-1 fig. can be determined by means of their pro-
cedure.

Manganese is readily determined with the polarograph, a
good, well-defined wave being obtained when a chloride of an
alkali or alkaline earth is present in considerable excess. It is
usual to employ a few ml. of solution, and as concentrations of
from if/250,000 to if/300,000 are measurable, with the use of,
say, 5 ml. of solution l/u,g. of manganese is determinable. With
the use of special micro -cells taking smaller quantities of solu-
tion, very much smaller quantities of manganese can be mea-
sured.

But although the polarograph would at first sight appear to
offer an ideal way for determining manganese in plants, actually
the polarographic determination of manganese in plant ash is
not straightforward, and in general is not to be recommended.
This is particularly so where ash or extract contains a con-
siderable quantity of phosphate. Plant ash consists chiefly of
oxides, phosphates and sulphates of potassium, calcium, mag-
nesium and other metals and is only soluble in acid. Since
hydrogen-ion gives a wave in solutions of alkali or alkaline earth
chlorides very near to that of manganese, an ash solution cannot
be polarographed for manganese directly because the waves for
manganese and hydrogen tend to coalesce. On neutralizing the
solution the manganese precipitates as phosphate, and in con-
sequence no wave for manganese is then given. To deal with this
situation the following procedure has been found by the writer
to give in some cases fairly reasonable results. After removal of
sulphate by barium chloride the phosphate is removed from the
ash solution by the addition of barium carbonate in excess and
filtering. This also removes ferric iron and aluminium, the wave
for the latter of which is sufficiently close to that of manganese

MICRO-NUTRIENT PROBLEMS 35

to render the end-point of the wave for the latter rather indeter-
minate, even if the two waves do not coalesce. The large quantity
of potassium, calcium and magnesium chlorides present in the
solution acts as ground substance and the filtered solution can
be polarographed directly. Where the quantities of sulphate and
phosphate in the ash are relatively small, results obtained by this
treatment have been in reasonable agreement with results ob-
tained by other methods, but where much sulphate and phosphate
are present results are not reliable, owing probably to adsorp-
tion of manganese by barium sulphate or phosphate. For each
determination two solutions are taken, one consisting of ash
solution, the other consisting of ash solution containing the same
concentration of ash but with a known amount of added man-
ganese. The difference in the heights of wave given by the two
solutions is then attributable to the added manganese, to which
the height of the wave given by the pure ash solution is referred.

Since a number of metals are reduced at more positive
potentials than manganese there is the possibility that they might
interfere with the wave for this ion. The principal elements
concerned are copper, cadmium, lead, chromium, molybdenum,
cobalt, nickel, iron and zinc. It is extremely unlikely that any of
these, with the exception of the last two, are likely to occur in
sufficient quantity in plant material to disturb the polaro-
graphic determination of manganese. As regards iron this is
practically all removed by the treatment of the ash solution
outlined above, and, as a matter of fact, no wave for iron appears
in the polarograms of solutions so treated. The possible influence
of zinc on the manganese wave was examined by the writer, who
found that no effect was produced on the wave of Mj 10,000
manganese by zinc in concentrations up to five times that
amount. No ash examined by the writer has been found to
contain a proportion of zinc to manganese approaching that
value.

A number of methods for determining manganese with the
use of the absorptiometer are available. Most of these depend
on the oxidation of the manganese salt with the production of
permanganate, the intensity of the colour of which is determined
with the absorptiometer. The methods are known as the per-
iodate, persulphate and bismuthate methods, according to the

36 METHODS OF INVESTIGATING

reagent used for effecting the oxidation. Trials carried out by
my colleagues, Dr K. W. Dent and Dr E. S. Twyman, indicate
that the periodate method is the most satisfactory of these, as
regards both simplicity of procedure and accuracy of the results
obtained. This experience appears to be fairly general if we are
to judge from the fact that this method is the one most com-
monly used by recent workers. A number of accounts of the
procedure employed in using the method have been published;
two of the more recent are those by Coleman and Gilbert (1939)
and Cook (1941). Coleman and Gilbert found that the same
values for manganese content of tea and coffee were obtained
with a wet ashing process and with incineration in a muffle, so
it is concluded that no loss of manganese occurs with either
ashing process. When dry ashing is employed 1 g. of material is
moistened with 1-2 ml. of concentrated sulphuric acid and ashed
at a dull red heat in a muffle. The ash is dissolved in 15 per cent
sulphuric acid and filtered. The filter paper is ignited and any
residue from this dissolved in about 10 ml. of dilute sulphuric
acid and filtered into the main filtrate. To the combined filtrate
0-1 g. of potassium iodate is added and the mixture boiled for a
few minutes and then kept hot for 30 min. for the development
of the pink colour. The solution is made up to standard volume
and the intensity of the colour determined.

Amounts of manganese down to about 12/xg. can be estimated
in this way.

For a description of the bismuthate method, which is par-
ticularly recommended for the determination of manganese in
soils, reference may be made to a paper by Dean and Truog
(1935) and for the persulphate method to papers by Majdel
(1930) and Olsen (1934).

A method for the estimation of small quantities of man-
ganese which has been described by Sideris (1940) depends on
the colour produced when formaldoxime is added to a solution
of a manganese salt. The formaldoxime reagent is prepared by
dissolving (by boiling) 20 g. of trioxymethylene + 47 g. of hydro-
xylamine sulphate in 100 ml. of distilled water. Ten ml. of the
hydrochloric acid solution of the ash are neutralized with sodium
hydroxide and then acidified with 2 ml. of a 20 per cent solution
of acetic acid. Excess phosphate is then removed by adding

MICRO-NUTRIENT PROBLEMS 37

0-5 ml. of a 5 per cent solution of lead acetate, the mixture
shaken, allowed to stand for 10 min. and then treated with 1 ml.
of a 20 per cent solution of sodium sulphate to remove excess
lead. After 30 min. the precipitate is removed by filtration or
centrifuging and the clear solution neutralized with 40 per cent
sodium hydroxide. Three or four drops of formaldoxime reagent
are added to the liquid and then more 40 per cent sodium hydro-
xide until a wine-red colour develops, the intensity of which is
said to be directly proportional to the concentration of man-
ganese. The liquid is made up to a standard volume and the
intensity of the colour determined. According to Sideris it would
appear that quantities of manganese of the order of 5/xg. can be
determined in the presence of 10-lOO^tg. of phosphate with an
error not exceeding 4 per cent. Under more favourable con-
ditions quantities of manganese down to 0-25/xg. would appear
to be determinable.

Yet another method for the determination of small amounts
of manganese has been described by Wiese and Johnson (1939).
The nitric acid solution of the ash containing from 1 to 10/xg. of
manganese is first rendered free from chlorides by three times
evaporating it to dryness and redissolving in nitric acid +10 ml.
of distilled water. About 0-2 g. of sodium bismuthate is added
and the mixture boiled for 2-3 min. On cooling to below 30° C.
0-2-0-3 g. of sodium bismuthate is added, and after mixing the
sample thoroughly and allowing it to stand for a few minutes
the excess of sodium bismuthate is filtered off through a Gooch
crucible. The solution filters directly into the absorptiometer cell
containing two drops of a solution of benzidine (i per cent in
5 per cent acetic acid) in 3 ml. of distilled water. The solution is
made up to standard volume and the intensity of the yellow-
green colour which develops is estimated after 5 minutes.

Zinc. For the determination of zinc in plant material by
means of the spectrograph, the flame method is not sufficiently
sensitive. Several workers, however, have described procedures
for the spectrographic determination of zinc in such material
by using the arc. Thus Rogers (1935) advocated using the zinc
line 2 138- 5 A. but found it was necessary to sensitize the
photographic plate by spreading mineral oil over the emulsion,
or, alternatively, using a special plate (Eastman spectroscopic

38 METHODS OF INVESTIGATING

plate type III-O) with ultra-violet sensitization. The tellurium
line 2143-OA. was used as internal standard. Vanselow and
Laurance (1936), on the other hand, recommended the use of
the zinc line 3345-OA. with cadmium line 3252-5A. as internal
standard. To a hydrochloric-acid solution of plant ash a known
amount of cadmium sulphate was added and the zinc and
cadmium then precipitated as sulphides by a special technique ;
the sulphides were then spectrographed. Rogers and Gall (1937)
reported unfavourably on the procedure of Vanselow and Lau-
rance and suggested that zinc in plant ash is not completely
extracted by hydrochloric acid. O'Connor (1941), in developing
spectrochemical methods for the determination of trace elements
in fertilizers, like Rogers used the zinc line 2 138-5 A. for the
determination of this element, but employed the beryllium line
2348-6 A. as internal standard. The spectrograms were obtained
on photographic plates with ultra-violet sensitization (Eastman
1-0 spectroscopic ultra-violet sensitive plate). O'Connor claims
that in this way zinc can be determined within the limits of
2 p. p.m. to 1 per cent with an accuracy within + 5 per cent. As
a 20 mg. sample is used for a determination this means that a
quantity of zinc as small as 0-04/xg. can be determined.

Lundegardh (1934) recommends the use of the line 3345-OA.
for the determination of zinc by the spark method.

Methods for the polarographic determination of zinc in plant
materials have been elaborated by Stout, Levy and Williams
(1938), by Reed and Cummings (1940) and by Walkley (1942).

In the method of Stout, Levy and Williams the hydrochloric-
acid ash solution (about 100 ml.) from 1 to 2 g. of dried plant
material is treated with 5 ml. of N ammonium citrate and then
rendered slightly alkaline by the addition of ammonium hydro-
xide. The resulting solution is then shaken with 10 ml. of a
solution of 1-3 mg. of dithizone in chloroform. The resulting
chloroform layer, which then contains the zinc, nickel, cadmium,
lead and copper, is separated from the aqueous layer containing
iron and manganese. The zinc, and accompanying metals, are
then removed from the chloroform by two extractions with
10 ml. of 0-5 N hydrochloric acid. The hydrochloric-acid extracts
are evaporated to dryness and the residue dissolved in a solution
of 0-1 N ammonium acetate + 0025 AT potassium thiocyanate.

MICRO-NUTRIENT PROBLEMS 39

On polarographing this solution well-defined and well-separated
waves of lead, cadmium, nickel and zinc are obtained.

Reed and Cummings experienced some trouble with the method
of Stout, Levy and Williams, particularly when large quantities
of zinc or of cadmium, lead, copper, nickel or cobalt were
present, and they found that to effect a quantitative separation
of the zinc from aluminium, iron and alkali metals five or six
extractions, instead of only one, were necessary. They therefore
devised a different procedure in which the ash solution in hydro-
chloric acid was brought to a pH of between 4 and 5 by addition
of dilute ammonium hydroxide. This precipitates practically all
the aluminium and ferric iron which are filtered off. The filtrate
is evaporated to dryness on a steam bath and the residue dis-
solved in a solution of 0-1 N ammonium acetate of pH 4-6 and
containing also potassium thiocyanate of concentration 0-025 N,
and the resulting solution polarographed. It is stated that no
interference with the height of the zinc wave then results from
the presence of chloride, sulphate, phosphate, carbonate, sodium,
potassium, calcium, magnesium or manganese, and no inter-
ference results from lead, cadmium or nickel up to concentra-
tions ten times that of the zinc. Copper interferes if it is present
in concentrations ten times or more that of zinc, while the
cobalt and zinc waves tend to coalesce if the former is present
in relatively high concentration. Actually Reed and Cummings
found that if cobalt occurs in concentrations greater than
1 x 10-5 g. per ml. it only interferes with determinations of zinc
when the ratio of cobalt to zinc exceeds 2. It would, as a matter
of fact, be a very exceptional plant ash in which any of the ions
noted occurred in sufficient amount to interfere with the polaro-
graphic determination of zinc in this way.

The lowest measurable concentration of zinc is stated by Reed
and Cummings to be 0-2/xg. of Zn per ml., that is, about
Jf/300,000, so that, using 5 ml. of solution, 1/xg. of zinc should
be determinable.

In Walkley's procedure the dried plant material is first sub-
jected to wet ashing, about a gram of the material being digested
with a mixture of 10 ml. of nitric acid, 1 ml. of sulphuric acid
and 1 ml. of perchloric acid. Frothing is prevented by addition
of a drop of kerosene. The cooled digest is taken up in 15 ml. of

40 METHODS OF INVESTIGATING

water and boiled, and on cooling 25 ml. of an ammonium citrate
buffer are added. This buffer is prepared by dissolving 5 g. of
citric acid in 50 ml. of water and 200 ml. of 4 jV ammonium
hydroxide, and then extracting impurities by three successive
shakings with 10 ml. of a solution of dithizone in chloroform
(1 g. of dithizone in 100 ml. of chloroform), and running off the
chloroform layers. The final purified buffer solution contains
dithizone in solution.

The zinc is separated from the digest after treatment with the
ammonium citrate buffer by three extractions each with 5 ml.
of chloroform. The chloroform extracts are evaporated to dry-
ness, then treated with a mixture of 2-5 ml. of nitric acid, 0-5 ml.
of perchloric acid, and two drops of sulphuric acid and again
evaporated to dryness, boiling being avoided. The residue is
then dissolved in 1 ml. of a ground liquid of 0-1 N ammonium
chloride -I- 0-02 N potassium thiocyanate containing 0-0002 per
cent of methyl red and polarographed in a small electrolysis
vessel. Practical details for carrying out Walkley's method will
be found in Piper's book on Soil and Plant Analysis (1942).

For the absorptiometric determination of zinc the most
promising method appears to be that based on the coloration
given by zinc salts with dithizone (diphenylthiocarbazone).
Reference has already been made in this chapter to the fact that
this reagent gives a red or purple colour when added to solu-
tions of compounds of a number of metals, including zinc (see
p. 25). The resulting coloured compound is quantitatively ex-
tracted with chloroform or carbon tetrachloride, and according
to R. H. Caughley (see Holland and Ritchie, 1939) sodium
diethyldithiocarbamate in 0-02 N ammonium hydroxide solu-
tion inhibits the reaction of dithizone with all metals except zinc.
Cowling and Miller (1941) have made use of this very useful fact
to work out an absorptiometric method for the quantitative
estimation of zinc in plant materials. Unfortunately, the
addition of the sodium diethyldithiocarbamate (generally
denoted by 'carbamate' for the sake of brevity) renders the
extraction of zinc by dithizone incomplete. By carefully stan-
dardizing the technique, however, Cowling and Miller claim that
this drawback can be overcome and the quantity of zinc in
plant ash determined with reasonable accuracy.

MICRO-NUTRIENT PROBLEMS

41

After ashing a 5 g. sample of the dried plant material at 500-
550° C, the ash is dissolved in hydrochloric acid, insoluble matter
being removed by nitration. The metals which form complexes
with dithizone are then extracted from the solution by repeated
treatment with excess of a solution of dithizone in carbon
tetrachloride at a pH of 8-5-9 in presence of ammonium citrate ;
the latter prevents the precipitation of iron and aluminium. The
dithizone extract is then treated with 0-02 N hydrochloric acid;
copper and the excess of dithizone remain in the carbon tetra-
chloride phase while the zinc and other metals pass into the
aqueous phase. The pH of the latter is then adjusted to between
8-5 and 9 by the addition of ammonia-ammonium citrate buffer
containing carbamate and the zinc extracted with dithizone in
carbon tetrachloride. The resulting extract is used for the deter-
mination of the zinc, the reading obtained being compared with
those given by solutions of known zinc content which are plotted
to give a standard curve. It is essential that the same conditions
should be rigidly adhered to both in obtaining the standard
curve and in the analysis of samples of plant material; that is,
the same pK should be used in the extraction, the volumes of
phases, the amount of dithizone and the amount of carbamate
used should be the same. Tests made by the authors show that
if this is done the method is highly reliable. The presence of
other metals does not interfere significantly with the deter-
mination, a good degree of accuracy was obtained in the
reovery of zinc added to plant material, while good agreement
was obtained between determinations of zinc in duplicate samples
of the same material. The method would appear to be capable
of determining quantities of zinc as small as 2/xg. or even less.
Copper. The spectrograph^ determination of copper in plant
material can be carried out by both the flame and arc methods.
With the use of the Lundegardh flame method the sensitivity,
according to the recent experience of Griggs, Johnstin and
Elledge (1941), is about half that found for manganese, these
workers giving the minimum concentration of copper usable as
0-000025 M . As the minimum quantity of solution which can be
employed in their arrangement is 2 ml. this would give the lowest
measurable amount of copper to be 3-15/xg. The line used for the
measurements is 3247- 5 A. (Lundegardh, 1929).

42 METHODS OF INVESTIGATING

It has already been mentioned (p. 33) that with the use of the
arc Melvin and O'Connor (1941) have achieved the simultaneous
determination of boron, manganese and copper. The copper line
used for the determinations was 3248 A., and the beryllium line
3 130 A. was used to provide the internal standard. From the
analyses published by Melvin and O'Connor it would appear that
the copper forming 0-001 per cent of a 10 mg. sample of material
can be measured, from which it would seem that quantities
of copper of the order of 0-1 fxg. can be estimated.

For the spectrograph^ determination of copper in grasses
Rusoff, Rogers and Gaddum (1937), employing the arc, used
cadmium as internal standard.

A procedure for the estimation of copper in plant materials
by means of the polarograph has been described by Reed and
Cummings (1941). The solution obtained by wet ashing of from
0-5 to 2 g. of plant material (see p. 32) is diluted to 15-20 ml.,
heated to boiling and then rendered alkaline by the addition of
a slight excess of concentrated ammonium hydroxide. The
solution is then boiled for a minute and filtered, the residue being
washed with slightly ammoniacal water and the washings added
to the nitrate. The latter is evaporated to dryness and the
resulting residue dissolved in 10 ml. of a ground liquid consisting
of 4-5 ml. of 0-5 M sodium hydroxide +4-5 ml. of 0-5 M citric
acid + 1 ml. of 0-05 per cent of acid fuchsin. This liquid, when
polarographed in absence of oxygen, gives a well-defined wave
for copper. In this way, according to Reed and Cummings, con-
centrations of copper down to about 3 x 10~6 M can be deter-
mined, corresponding to about 2 p. p.m. of copper in the plant.
Published data indicate that in normal plants the amount of
copper is usually well above this value, but in plants showing
symptoms of copper deficiency it may be lower than this. In
such cases it would be necessary either to use larger quantities
of plant material for the determination or to use a method other
than a polarographic one.

The absorptiometric method generally used for the deter-
mination of copper in plant material depends on the intense
colour produced by copper salts with diethyldithiocarbamate
(Callan and Henderson, 1929), the coloured complex being
extracted with amyl alcohol. Procedures particularly applicable

MICRO-NUTRIENT PROBLEMS 43

to biological material have been described by Eisler, Rosdahl and
Theorell (1936), by Eden and Green (1940), and by Piper (1942).
In the procedure of the first group of workers the dried material
is heated on a sand-bath with sulphuric acid and perchloric acid
until the mixture is colourless or pale yellow. The resulting liquid
is cooled under the tap and then rendered slightly alkaline to
litmus by the addition of 8-9 per cent ammonium hydroxide.
Iron is precipitated by treating the alkaline solution with a few
ml. of a 4 per cent solution of sodium pyrophosphate, then
heating at 80° C. for 30 min. and cooling to room temperature.
If a crystalline precipitate is present at this stage it must be dis-
solved by the addition of water. Now 2 ml. of water and 5 ml. of
amyl alcohol are added and then immediately 0-5 ml. of a 2 per
cent solution of sodium diethyldithiocarbamate.

The mixture is now strongly shaken and centrifuged. The
amyl alcohol layer is separated and its light absorption in light
of wave-length 4400 A. measured. The depth of colour of the
amyl alcohol layer is dependent not only on the concentration
of copper, but also on the salt concentration of the aqueous
phase from which the amyl alcohol layer is separated, the salt
concentration depending on the amount of sulphuric acid used.
Hence it is necessary to use a constant technique in order to
obtain reliable results.

Eden and Green also used a wet ashing method, the material
e.g. 5 ml. of blood, 1-5 g. of fresh tissue or 1 g. of dried material,
being digested with a mixture of sulphuric and perchloric acids
to which nitric acid is added later. After dilution with water the
digest is treated with 2 ml. of 50 per cent ammonium citrate
and 5 ml. of ammonia (sp. gr. 0-880). The ammonium citrate
effects the deionization of the iron and prevents the precipitation
of calcium phosphate. The solution is then made up to 25 ml.
with water. If the material is relatively low in calcium and
phosphorus but high in iron it is preferable to use 10 ml. of 4 per
cent hydrated sodium pyrophosphate instead of the ammonium
citrate, as the resulting solution is colourless, whereas with
citrate the solution is slightly coloured.

Two ml. of a recently filtered 0-5 per cent solution of sodium
diethyldithiocarbamate are now added to the solution, kept
constantly shaken, then 5 ml. of amyl alcohol added, and the

44 METHODS OF INVESTIGATING

mixture vigorously shaken for 30 sec. The amyl alcohol extract,
which contains the coloured copper diethyldithiocarbamate, is
centrifuged or filtered through acid-extracted filter paper to
remove any water in suspension and the depth of colour deter-
mined with a colorimeter or absorptiometer by comparison with
a standard solution of copper diethyldithiocarbamate prepared
in the same way as that from the tissue digests. With adequate
precautions it would appear that quantities of copper as small
as 0-3 g. are determinable by the procedure of Eden and Green.

In the method as used by Piper a wet ashing process is also
used, and after diluting the digest with water and treating it
with ammonium citrate solution to dissolve any hydrolysed
manganese and to prevent precipitation of phosphates, the
resulting solution is brought to pH 3 and the copper extracted
with dithizone in carbon tetrachloride. The carbon tetrachloride
is then removed by evaporation and the dithizone by heating
with a little sulphuric acid containing a drop or two of per-
chloric acid. After dilution with water and rendering the solu-
tion alkaline with ammonium hydroxide, the copper is pre-
cipitated by the addition of a few drops of a 3 per cent aqueous
solution of sodium diethyldithiocarbamate. The copper diethyl-
dithiocarbamate is then extracted with amyl alcohol and the
intensity of colour of the extract compared with that of a
standard. For full details of the experimental procedure refer-
ence should be made to Piper's book.

Boron. As regards the determination of boron by means of
the spectrograph, the usual flame method is useless. Even with
a 0-1 M solution of boric acid no boron line was obtained by
Lundegardh in a flame spectrum. Boron in plant material has,
however, been determined by means of the spark method by
Foster and Horton (1937). The plant material was used fresh,
being neither dried nor ashed, but crushed and disintegrated
into a fine pulp in a small copper mortar. The samples used
consisted of 100 mg. of fresh material. Measurements were
made on the boron line 2497-7 A., and the gold line 2427-9 A. was
used as internal standard. Six replicate determinations of the
boron in turnip leaves gave values varying from 3-7 to 4-3/zg. of
boron per g. of fresh tissue, with an average value of 4-lyu,g.
Foster and Horton conclude that the error of a single deter-

MICRO-NUTRIENT PROBLEMS 45

mination is not in excess of 10 per cent. The limit of sensitivity
of the method is not clearly stated, but it would seem probable
that quantities of boron as small as 0-1/xg. might be measurable
in this way. Lundegardh (1929) suggested the use of the cad-
mium line 2573 A. as internal standard in the determination of
boron by the spark method.

As mentioned earlier (see p. 33), Melvin and O'Connor (1941)
have used the arc method for the simultaneous determination
of boron, manganese and copper in fertilizers, using beryllium
as internal standard. The method would seem to be applicable
to the determination of boron in plant material, and the
accuracy and sensitivity would appear to be of the same order
as in the spark method of Foster and Horton.

Although the flame method is not suitable for the speetro-
graphic determination of boron, McHargue and Calfee (1932)
have successfully made use of flame spectra for the optical
spectroscopic determination of boron. In their earlier procedure
the boron was first converted into methyl borate, then vola-
tilized with methyl alcohol and burnt in an atmosphere of
oxygen in front of a cell containing a solution of potassium
permanganate. The concentration of this solution was adjusted
so that the bright lines of the boron spectrum were just obscured
by it. By previous standardization of solutions of potassium
permanganate against standard boron solutions the concentration
of the experimental boron solution was obtained.

Later (Calfee and McHargue, 1937) a different procedure was
devised. The spectrum was excited in an oxygen-methane flame,
methane saturated with a solution of methyl borate in methyl
alcohol being ignited in an oxygen blast. The light emitted from
this was polarized and by an optical arrangement the spectrum
was brought into juxtaposition with a second spectrum similarly
produced by the burning of a standard boron solution. By rota-
tion of an analysing plate the intensity of the spectrum of the
standard boron solution could be varied and so matched with
that of the solution the boron concentration of which it was
desired to measure. For a quantitative determination in this
way the boron content of a sample should lie between 25 and
50/xg. Agreement between the two procedures was good, but
the second method was found to be more exact.

46 METHODS OF INVESTIGATING

So far it has not been found possible to estimate boron

polarographically .

A colorimetric method for the determination of small quantities
of boron which has been applied to the estimation of boron in
plant material depends on the colour change of quinalizarin
effected by boric acid (see e.g. Smith, 1935). To 1 ml. of solution
containing from 1 to 40/xg. of boric acid 9 ml. of concentrated
sulphuric acid are added followed by 0-5 ml.. of a 0-01 per cent
solution of quinalizarin in 93 per cent sulphuric acid. A colour
change from reddish violet to blue results, the complete pro-
cess taking about 5 min. Nitrate, dichromate and fluoride
must not be present, but the common metals do not interfere
with the reaction.

While this method appears to be quite satisfactory for the
estimation of boron in plant material, the necessity of using
concentrated sulphuric acid is something of a drawback. Another
colorimetric method for the determination of small quantities
of boron, which does not involve the use of this reagent, has
more recently been described by Naftel (1939). This method
depends on the colour produced when a solution of boric acid
is treated with oxalic acid and either curcumin or an extract of
turmeric and the mixture evaporated to dryness. According to
the procedure recommended by Naftel, the soil or plant-ash
extract containing from 0-5 to 8/xg. of boron is first rendered
alkaline by the addition of 5 ml. or more of 0-1 N calcium
hydroxide and then evaporated to dryness on a water-bath.
After cooling there are added to the residue 1 ml. of a freshly
prepared solution of oxalic and hydrochloric acids (made by
adding 80 ml. of a saturated solution of oxalic acid to 20 ml. of
concentrated hydrochloric acid) and 2 ml. of a 0-1 per cent
solution of curcumin or a 1 per cent freshly prepared extract of
turmeric in 95 per cent ethyl alcohol. The mixture is evaporated
to dryness on a water-bath at 55° C, heated for a further
30 min. at this temperature and then cooled, extracted with
95 per cent ethyl alcohol and the colour of the clear solution
obtained after filtering or centrifuging compared with that of
standard solutions prepared in the same manner. Quantities of
boron down to 0-5ju.g. can be determined by this method. In
soils other elements present do not appear to interfere with the

MICRO-NUTRIENT PROBLEMS 47

sstimation of boron, but if large quantities of other substances
present are found to interfere with the determination of boron,
:he latter may first be separated by volatilization with methyl
ilcohol.

An electrometric titration method has been specially devised
by Wilcox (1940) for the determination of boron in plant
material. A quantity of the dried and powdered plant material
(say, from 5 to 25 g.) containing not more than 2 mg. of boron
is mixed with one-tenth of its weight of calcium oxide and
ignited in a furnace at a low red heat. The resulting ash after
cooling is moistened with water and taken up in 15-20 ml. of
6 N hydrochloric acid and then heated on a steam-bath for
30 min. Phosphate is removed from the resulting solution by
the addition of N lead nitrate solution to the extent of 1 ml. for
each gram of plant material used, followed by sodium bicar-
bonate until a precipitate is produced, when the mixture is
heated on a steam -bath and more sodium bicarbonate added
until the solution is neutral to brom-thymol-blue (about pR 7).
The mixture is then made up to 250 ml. and filtered through a
dry filter paper. Carbon dioxide is removed by acidifying with
6 N hydrochloric acid and heating to boiling, then making
alkaline with 0-5 N sodium hydroxide and reacidifying with 2 N
hydrochloric acid until 5-10 drops in excess have been added.
On making up to 300 ml. the solution is boiled for a few minutes.
It is then ready for electrometric titration. For this the quin-
hy drone electrode may be used in conjunction with a 0-7AT
calomel electrode, these giving a null point at approximately
pB. 7. The electrodes having been introduced into the solution,
0-5 JV sodium hydroxide is added until the solution is neutral
to brom-thymol-blue, when the galvanometer should register
approximately zero ; if it does not the null point is obtained by
the addition to the solution of either 0-0231 N sodium hydroxide
or hydrochloric acid. Five grams of mannitol are then added,
and if boric acid is present a galvanometer deflexion results.
Standard 0-0231 N sodium hydroxide is then added until the
null point is again reached. The volume of sodium hydroxide
gives a measure of the amount of boron present, 1 ml. 0-0231 N
sodium hydroxide being equivalent to 0-25 mg. boron. This
method is claimed by Wilcox to be specially suitable for deter-

48 METHODS OF INVESTIGATING

mination of boron in tissues where this element is present in
quantity less than 50 p. p.m.

Molybdenum. Molybdenum in plant material is generally
determined with the colorimeter or absorptiometer as molyb-
denum thiocyanate by the method described by Marmoy (1939).
A 50 ml. sample of the hydrochloric acid solution of the ash
containing not more than 20jug. of molybdenum and having an
acid concentration of 1 4 per cent by volume is treated first with
3 ml. of potassium thiocyanate and then with 3 ml. of stannous
chloride. The molybdenum thiocyanate produced is then ex-
tracted with ether, the extractions being repeated until the
ether layer is colourless, and the depth of colour of the com-
bined ether extract is compared with that of a standard solution
in ether of molybdenum thiocyanate prepared from ammonium
molybdate in the same way as that from the ash.

Aluminium. Aluminium is present in quantity in many soils
and is generally present in plants, but so far its essentiality has
been indicated for only a few species. Perhaps for this reason
not so much attention has been given to the determination of
aluminium in plant material during recent years as to the better
established micro -nutrients.

For the measurement of small quantities of aluminium, such
as might be expected in samples of plant material, direct measure-
ment of the intensity of the aluminium line in the air-acetylene
flame spectrum is not suitable, for Lundegardh (1929) found that
no line was obtained with even a 0- 1 M solution of aluminium
chloride. Mitchell and Robertson (1936) have, however,
described a means by which aluminium in concentrations
ranging from about 2 to 10 mg. per litre can be determined by
the Lundegardh method. It depends on the fact that the
presence of aluminium brings about a lessening of the intensity
of the calcium and strontium lines of the flame spectrum, the
decrease in intensity varying with the amount of aluminium
present and also with the relative, amounts of calcium and
strontium present. Hence with careful control of the conditions
the depression in the intensity of these lines can be used to
determine the aluminium content of solutions of the concentra-
tions indicated above. Using 15 ml. of solution for a determina
tion, this means that quantities down to 30/xg. of aluminium
can be measured in this way.

MICRO-NUTRIENT PROBLEMS 49

With the spark the best aluminium line for measurement is,
according to Lundegardh (1934), 3961-5 A., but if the sample
under examination has a high calcium content, which may
frequently be so with plant material, the calcium line 3968-5 A.
may interfere with the aluminium line.

Aluminium may be determined polarographically with the
use of lithium chloride, barium chloride or magnesium chloride
as supporting electrolyte, but, owing to the fact that the
aluminium wave occurs at a rather high negative potential as
well as to difficulties resulting from the presence of phosphates,
it is unlikely that the polarograph will afford a simple means
for the determination of aluminium in plant material.

Two colorimetric methods, suitable for use with the absorptio-
meter, depend on the formation of lakes, fairly stable in the
presence of acetic acid, when alizarin and the ammonium salt
of aurin tricarboxylic acid, respectively, are added to a solution
of an aluminium salt. Both appear to be adaptable to the
determination of aluminium in plant tissues. The first method
appears to have been described first by Atack (1915). The
modification of it described by Underhill and Peterman (1929)
has been used in my laboratory on fairly pure solutions with
marked success. The second method was described by Hammett
and Sottery (1925) and was adapted for the determination of
aluminium in animal tissues by Myers, Mull and Morrison (1928).
Amounts of aluminium of the order of 5/xg. can be measured by
both methods.

Cobalt and Nickel. Although evidence has occasionally been
adduced to indicate that small quantities of cobalt and nickel
may bring about an increase in the rate of growth of plants,
there has up to now been no definite proof provided that
either of these elements is essential for the growth of any plant.
There is, however, very definite evidence that cobalt is essential
for sheep and cattle, and as the deficiency of this element in the
animal must arise from the low content of cobalt in the plants
on which the animal feeds, the determination of small quantities
of cobalt, at any rate, in plants may be necessary for investiga-
tions on cobalt deficiency in animals. There does not seem so far
to be any very definite indication that nickel is essential either
for any plant or any animal, but since the determination of

50 METHODS OF INVESTIGATING

nickel can be made in the same way as that of cobalt it is as
convenient to consider the two elements as cobalt only.

Although these elements can be determined spectrographic-
ally, the flame method scarcely has sufficient sensitivity for
their ready estimation in plant material, for it would appear from
Lundegardh's data that the smallest amount of either metal
measurable in this way is of the order of 100/xg., which means
that decidedly large samples of material would generally have
to be used.

For the semi-quantitative determination of cobalt in soils,
Mitchell (1940), who has made a special study of the estimation
of trace elements in soils, recommends the use of the cathode
layer arc. The method is based on the fact that in the region of
the arc adjoining the cathode (the cathode layer), the emission
of spectral energy from cations may be up to 100 times as
intense as that from the column of the arc. By means of a
spherocylindrical quartz lens the image of the arc is focused
sharply on the slit, so that the image of the cathode appears as
a horizontal line across the top of the slit while the image of the
anode falls well below the bottom of the slit. This arrangement
results in greatly increased sensitivity as compared with the
ordinary arc method, and 2 p. p.m. of cobalt and 1 p. p.m. of
nickel can be detected by its use. The spectral lines used for the
determination of these two elements are 3453-5 and 34 14- 8 A.
respectively.

Cobalt and nickel can be readily determined simultaneously
with the polarograph by the procedure described by Lingane
and Kerlinger (1941), the essential feature of which is the use of
pyridine as supporting electrolyte. Using a normal solution of
potassium chloride containing from 0-05 M to M pyridine and
0-05 per cent gelatin the waves of nickel and cobalt are well
separated and defined, while manganese does not interfere with
them. Ferric iron, if present in large excess, interferes with the
determination of nickel and cobalt, but its effect can be elimi-
nated by the use of a supporting electrolyte with pH of about
5-4 containing equal concentrations of pyridine and pyridium
chloride in which the ferric iron is precipitated as hydrous ferric
oxide. Small amounts of copper do not interfere with the deter-
mination of nickel and cobalt, but if present in considerable

MICRO-NUTRIENT PROBLEMS 51

excess the bulk of it must be removed before polarographing for
nickel and cobalt. The author is not aware of the polarographic
method having been used for the determination of nickel or
cobalt in plant material, and Piper (1942) points out that the
nearness of the deposition potential of zinc to that of cobalt, and
the small amount of the latter relative to that of zinc, usually
present in plant material, renders the polarographic determina-
tion of cobalt in plant ash uncertain.

The estimation of cobalt in plant material and in soil is usually
effected by colorimetric or absorptiometric means depending on
the intense coloration produced by cobalt compounds on treat-
ment with the sodium salt of l-nitroso-2-naphthol-3 : 6-disulphonic
acid, generally known as nitroso-i?-salt. Procedures have been
described by Kidson, Askew and Dixon (1936) and by Davidson
and Mitchell (1940) for the determination of cobalt in soils in
this way and by McNaught (1938), Kidson and Askew (1940)
and Marston and Dewey (1940) for the similar estimation of
this element in plant material. It would appear that quantities
of cobalt down to about 0-5^,g. are determinable in this way.

3. The Diagnosis of Mineral
Deficiencies of Plants

It is obvious that a ready means of diagnosing deficiencies of
the various mineral constituents of plants is likely to have great
economic value, particularly where crop plants are concerned.
Where the deficiency of a particular element is great the plant
generally displays symptoms which are readily recognizable by
an observer with experience of the effects on the species in
question of deficiency in that element. These symptoms are
often so definite that the resulting condition has a descrip-
tive name, and a number of well-defined deficiency diseases
of crop plants are known to agriculturists and horticulturists :
the most important of these are described in a later chapter.1

1 See particularly Wallace (1943, 1944) for diagnoses and coloured
illustrations of crop plants of Britain affected by deficiency diseases, and
Hunger Signs in Crops by a number of authors (Washington, 1941) for
an account, with coloured illustrations, of deficiency diseases of crop
plants in the United States.

52 METHODS OF INVESTIGATING

But by the time unmistakable symptoms of deficiency have
shown themselves it may be late, and, perhaps, too late, to
effect a cure of the condition by the application of the deficient
mineral; this is likely to be so with annuals such as cereals
and leguminous crop plants with a short life period rather than
with perennials such as fruit and other trees where the longer life
of the plant may provide adequate time for recovery. But
even for the latter early diagnosis is obviously desirable in
order to avoid a period of feeble growth or poor fruit yield. Also
it may be possible that the deficiency of a particular element is
insufficient for actual symptoms of a' deficiency disease to
develop and yet sufficient to bring about a reduction in the
rate of growth and finally in crop yield.

A second way of determining micro-nutrient deficiency is
provided by analysis of plant material by the methods described
earlier in this chapter. This might provide very definite evidence
of the adequacy or otherwise of the quantity of the various
nutrients in the plant, although it would be necessary first to
establish the minimum quantities of the respective mineral
elements which must be expected in the different organs of the
plants of each species at different stages of development. While
a certain amount of such information is available it must be
admitted that it is far from complete for any one species. The
acquisition of the requisite data takes time, but there can be
no doubt that the necessary information will ultimately be
obtained.

A third method of diagnosing mineral deficiencies, and one
which should enable this to be made early, consists in intro-
ducing a solution of the salt of the element in question, or even
the solid salt, into the plant and observing the reaction. The
introduction of the salt into the plant is generally spoken of as
'injection'. The generally accepted meaning of this word is the
forcing of material into the organism under pressure, whereas
in practice the plant is generally allowed to absorb the solution
through a cut surface, or even through an intact leaf, without
the application of pressure. However, there is no other simple
term to denote this process, and we may follow Roach, who has
developed this method for diagnosis of trace-element deficiencies,
in extending the use of the term 'injection' to include 'the

MICRO-NUTRIENT PROBLEMS 53

introduction by various methods of liquids and solutions into
plant organs, whether under pressure or not, and their spread
therein'.

In a long discussion on injection of plants as a physiological
method, Roach (1939) describes no less than ten ways in which
injection can be carried out ; each of these has its own particular
value. The ten injection methods are these:

1. Intervenal leaf injection.

2. Leaf- tip injection.

3. Lestf immersion (Anderssen).

4. Leaf-stalk injection.

5. Shoot-tip injection.

6. Branch-tip injection.

7. Shoot injection (Leach) and branch injection (Collison,

Harlan and Sweeney).

8. Injection of individual branches.

9. Injection of individual branches together with their

roots.
10. Injection of whole trees.

Not all these methods of introducing material into plants
have been designed for the purpose of diagnosing mineral
deficiencies, nor are all of them equally valuable for this purpose,
although any one of them could no doubt serve to demonstrate
the existence of such deficiency. But on the whole the methods
in which leaves or young shoots are injected are those which are
most useful for diagnostic purposes, while those in which larger
branches or a whole tree are used are more generally useful for
some other purpose, as, for example, the cure of a deficiency.

The principle underlying injection methods of diagnosis is
that the introduction into a leaf of a salt of an element in which
the plant is deficient will produce a definite response which is in
the direction of a cure of the deficiency. The most usual response
is a colour change in the leaf which generally becomes greener ;
sometimes increased rate of growth of a leaf occurs. These
responses are best observed when leaf areas permeated by the
nutrient are in close juxtaposition to control non-permeated
areas; a difference in colour between permeated and control

54 METHODS OF INVESTIGATING

areas is then most easily recognizable, while if one simple leaf
or leaflet contains both permeated and control areas a difference
in the rate of growth of the two parts of the leaf will result in
a puckering of the leaf which is readily observed.1

These conditions are fulfilled when leaves of certain species,
as, for example, apple, pear, plum, strawberry and broad bean,
are subjected to inter venal injection. For this treatment a small
incision is made near the midrib of the leaf between two major
secondary veins. A dilute solution of the salt of the element of
which a deficiency is suspected is contained in* a small tube,
and a wick made of filter paper, or, for small leaves, of
darning cotton, passes from the solution through the incision
into the leaf. By the use of dyes it is shown that the solute
diffuses through the whole area between the two secondary veins
and the leaf margin before diffusing into neighbouring inter-
venal areas. The length of time for which injection is allowed to
proceed should be such as to give a long boundary between the
injected and neighbouring control area, but not so long that the
solute diffuses into neighbouring areas. The best time must be
found by preliminary trials, for it varies with the species and
climatic conditions. However, for apple, pear, strawberry and
Shasta daisy, Roach suggests a period of from 7 to 12 hr. The
leaves which give the best response are those about half -grown.
The maximum response is generally given in about 10 days, but
a response has been observed in as short a time as 2 days. This
was recorded by Roach as having been observed by Lai as a
result of intervenal injection of soya-bean leaves with a 0*025
per cent solution of ferrous sulphate.

In leaf -tip injection the tip of a leaf or leaflet is cut off at right
angles to the midrib and the cut edge of the leaf immersed in a
solution *of the substance to be injected. This method can be
used for any type of leaf but is particularly suitable for long
narrow leaves. The greater the proportion of the leaf removed,
the greater is the penetration of the solute into the rest of the
leaf. For example, it was found that if the removed tip con-

1 It should, perhaps, be pointed out that these responses should only
be regarded as indicative of deficiencies when they have been correlated
with successful curative treatment, since an improved appearance of
the leaf might result from injection without there being a deficiency.

MICRO-NUTRIENT PROBLEMS 55

tained one-tenth of the midrib, half the rest of the leaf was per-
meated, but if more than one-fifth of the midrib was contained
in the part removed, the whole of the remainder of the leaf and
parts of neighbouring ones became permeated. This should be
avoided since neighbouring leaves can serve as a control. With
compound leaves, such as those of the strawberry, injection can
be so contrived that one leaflet becomes permeated and another
unaffected, while the third is partially permeated. Roach found
that with leaves of apple and pear injection should proceed for
about 10 hr. ; a response is apparent in from 7 to 10 days.

With leaf -stalk injection the whole, instead of part only, of
the lamina is removed, and the leaf stalk left attached to the
plant is connected with narrow rubber tubing (such as tyre
valve tubing) to a reservoir of the solution. As a result certain
leaves of the plant become completely permeated, others par-
tially and yet others not at all; the greater the angular distance
of any leaf from the injected leaf stalk the less the permeation.
In partially permeated leaves the permeated and non-permeated
areas are, at any rate in the case of apple, sharply delimited, and
such leaves are considered by Roach to be almost ideal for
showing differences in colour and rate of growth of affected and
control areas. The method would appear to be applicable to a
wide range of species.

For shoot-tip injection the tip of a shoot is removed and either
a small glass tube of solution is attached to the cut end of the
shoot with fine rubber tubing if the shoot is rigid enough to
support it, or the cut end of the shoot is bent over into a reservoir
of the solution. As a result one or more of the leaves on the shoot
become permeated.

The remaining methods of injection listed by Roach are of
less interest from the point of view of their value for diagnostic
purposes, but reference may be made to the methods used by
Anderssen and by Storey and Leach, since these were both devised
in connexion with work on mineral deficiency of plants. Anders-
sen's method consisted in bending over the leaf and immersing
it in a weak solution of copper sulphate containing 0-3 p. p.m.
of copper. By this treatment chlorotic leaves of plum recovered
their normal green colour in 2 weeks, a result which afforded
confirmatory evidence that the pathological condition of the

56 MICRO-NUTRIENT PROBLEMS

trees bearing the leaves was due to a deficiency of copper.
Anderssen's work is referred to in more detail in the next
chapter.

Storey and Leach (1933) were interested in a disease of the
tea plant known as 'yellows', which, as the name implies, in-
volves a chlorosis of the leaves. They traced this to a deficiency
of sulphur. Among other pieces of evidence which led them to
this conclusion was the effect of introducing various salts into
plants growing in the field. The injection of any particular salt
was effected by cutting a small side shoot under water and
immersing the cut end of the shoot in a solution of the salt. The
quantity of solution was maintained by daily additions, and
every fourth day the immersed shoots were cut farther back to
give a fresh absorbing surface of unchoked wood. It was found
that when a 0-5 per cent solution of sodium sulphate, potassium
sulphate or magnesium sulphate was used the normal green
colour was regained by the leaves on the branch beyond the cut
shoot, but that no such recovery resulted when other salts, such
as chloride or nitrate, of these metals were used.

CHAPTER III

TRACE ELEMENT DEFICIENCY
DISEASES OF PLANTS

At the end of Chapter I a list was given of species which have
been shown to be dependent for growth on one or other of the
micro -nutrients, or which at least have benefited by treatment
with a micro-nutrient. It has been established that certain
well-recognized pathological conditions met with in the field
are associated with deficiency of a micro -nutrient, and some
of these are so widespread or of sufficient economic importance
to be designated by common names. Such, for example, are
the grey speck disease of oats, and heart-rot of sugar beet. In
this country diseases due to a deficiency of manganese and
boron are both widespread and of economic importance; else-
where shortages of zinc and copper have been shown to be
responsible for diseases causing considerable damage to fruit
crops. So far no well-defined diseases attributable to lack of
molybdenum or other trace elements have been recognized.

The more important of the deficiency diseases attributable to
shortage of trace elements are described in this chapter.

1. Diseases Attributable to a
Deficiency of Manganese

The most general effect of manganese deficiency appears to be
in the first place the development of small chlorotic patches
localized in intervenal areas of the leaves. The form these patches
take in different species is no doubt largely dependent on the
anatomy of the leaf, so that in grasses with their parallel
venation they tend to take an elongated form, producing
'stripes' or 'streaks', while in reticulate-veined dicotyledons
they produce a spotted, speckled or mottled effect, as in potatoes
and sugar beet. Other symptoms may follow, including reduc-
tion or cessation of growth and the development of necrotic
areas which may not be limited to the affected regions of the leaf,

58 TRACE ELEMENT DEFICIENCY

but which may even affect the seeds, as in the case of the
garden pea.

Grey Speck of Oats. The disease of oats most usually known
as grey speck, but also sometimes called grey stripe, grey spot,
or dry spot, is characterized by the appearance in the leaves of
spots of a greyish colour, small chlorotic areas, chiefly in the
lower half of the leaf, which tend to coalesce and form elongated
streaks which finally turn brown. The first sign of the disease
often occurs in young plants in the third or fourth leaf. Very
characteristically a line of withering and weakness develops
transversely across the leaf blade so that the distal portion of
the leaf hangs down (see Figs. 1,2). In the young leaves this
line of weakness is often about 1 or 2 in. from the base of the
leaf lamina, but correspondingly higher up in older and longer
leaves. The leaves may eventually turn completely brown and
die. Colour photographs of oats badly affected by grey speck are
given by Wallace (1943, p. 95, pi. 77; 1944, p. 37, pi. 188).

Badly affected plants may be stunted and die early; in less
severe cases flowers may be produced but little grain is formed.
Root development tends to be poor, so that affected plants are
much more readily pulled out of the soil than healthy ones.

Grey speck appears to be widely distributed. It occurs in
different parts of Europe, including Britain, and in America and
Australia. It appears to be most liable to occur on certain soils
with an alkaline reaction, especially if they contain much humus,
and in such conditions the disease may be so serious as to lead
to the complete failure of the crop.

It has been recognized for many years that grey speck disease
could be controlled by treatment with a soluble manganese salt,
either as a soil dressing or by spraying the foliage, but the proof
that grey speck was actually related to manganese deficiency was
provided by Samuel and Piper (1928, 1929). They grew Algerian
oats in carefully controlled water cultures, using carefully
purified materials, and with various amounts of manganese
sulphate added to the culture solutions containing the usual
major nutrients. The initial concentrations of manganese in the
different cultures were 0, 1 in 50 x 106, 1 in 10 x 106, 1 in 5 x 106
and 1 in 1 x 106. Cultures grown in solutions free from man-
ganese developed the symptoms of grey speck in about 4 weeks,

DISEASES OF PLANTS 59

and this occurred whether the solutions also contained some
other trace element such as boron, zinc, cobalt, copper, etc., or
not. With culture solutions containing 1 part of manganese in
50 x 106, the symptoms developed suddenly in about 8 weeks.
On renewing the culture solution, including the manganese
supply, new healthy growth took place, but the symptoms of
deficiency again appeared after about 4 weeks. Recovery again
took place after a second renewal of the solution. With culture
solutions containing 1 part or more of manganese in 10 x 106
no symptoms of grey speck appeared, the solutions being
renewed after 10 weeks.

Reference has been made above to the fact that grey speck
tends to occur on plants growing on alkaline soils containing much
humus. This has led to suggestions that the disease might be
associated with excess of calcium ions or with certain organic
compounds in the soil. Water-culture experiments carried out
by Samuel and Piper to test these possibilities yielded no sup-
port for such views. Culture solutions containing calcium ions
in various degrees of excess, or various organic substances
(humus, sucrose, glucose, starch, cellulose), in no case induced
symptoms of manganese deficiency in oats growing in them
provided manganese sulphate had been added to the solutions.

That grey speck disease is the direct effect of manganese
deficiency has been disputed by Gerretsen (1937). He points
out that Lundegardh (1932) had recorded that the manganese
content of affected plants might be higher than that of healthy
ones; indeed, he gave values up to 420 p. p.m. of manganese in
affected plants and down to 1 p.p.m. in healthy plants. This is
certainly contrary to general experience, and Samuel and Piper
found that about 14 p.p.m. was the minimum amount of
manganese likely to be present in healthy Algerian oats at the
flowering stage.

Gerretsen states that when a soil which had borne a crop
showing symptoms of grey speck was sterilized with formalin, oat
plants subsequently grown on it were free from grey speck,
although the manganese content was the same as before and there
had been no increase in either water-soluble or exchangeable
manganese. On reinfecting such sterilized soil with 10 per cent
of the original soil grey speck again appeared on oats grown on

60 TRACE ELEMENT DEFICIENCY

it and the dry weight of the plants was reduced to 59 per cent
of that of plants grown on the sterilized* soil, while the man-
ganese content of the affected plants was reduced from 51-5 to
19-3 p. p.m. Some very striking results were given by sand-
culture experiments. Plants were grown in sterile sand and in
the same sand infected with 5 per cent of so-called 'diseased'
soil. The plants in the sterile sand were all healthy, had a mean
dry weight of 436 mg. and a manganese content of 15-0 p. p.m.,
whereas those in the infected sand all showed typical symptoms
of grey speck, a mean dry weight of only 216 mg., but a man-
ganese content of 26-6 p. p.m.

Oats grown in sterile water-culture solutions containing very
small quantities of manganese showed no symptoms of grey
speck, although the plants were stunted and might contain less
than 10 p. p.m. of manganese. But when the solutions were
inoculated with a root tip from an affected plant or with bacteria
isolated from affected roots, the symptoms of grey speck deve-
loped strongly. Again, the addition of 0-001-0-002 per cent of
Germisan, a germicide, to the culture solution of non-sterile
plants kept the plants healthy even when the manganese con-
tent was low, whereas without the addition of the Germisan the
plants were badly affected.

These facts are held to indicate that grey speck is related to
the presence of micro-organisms. According to Gerretsen the
roots of affected plants always show signs of microbiological
disintegration. It is suggested that alkaline products are pro-
duced in the roots by the infecting micro-organisms and that
these products are carried in the transpiration stream to the
leaves, where they produce the grey spots.

Gerretsen therefore concludes that it is necessary to dis-
tinguish between the direct physiological effect of manganese
deficiency which is a retardation of growth, and the symptoms
of grey speck disease which are related to the infection of the
roots by micro-organisms. The capacity of the root to resist
parasitic attack by micro-organisms is indeed held to depend
on the manganese content, but if the roots are maintained
sterile healthy plants are produced in presence of a very small
supply of manganese so that the manganese in the plant is only
from 5 to 35 p. p.m.

Fig. 1. Oat seedling affected with
grey speck. Note the grey areas
and the characteristic line of
weakness in affected leaves.

Fig. 2. Oat leaf exhibiting the
characteristic symptoms of grey
speck.

Fig. 3. Sugar beet suffering from manganese deficiency. Note the speckling of
the leaves, appearing in the photograph as a mottling.

By courtesy of Dr. C. S. Piper

Fig. 4. Peas suffering from marsh spot.

DISEASES OF PLANTS 61

It must be admitted that Gerretsen has made a strong case
for the view that grey speck disease is not the result of man-
ganese deficiency only. The problem is clearly deserving of
further study.

In wheat, Gallagher and Walsh (1943) observed that the first
sign of manganese deficiency usually appears with the develop-
ment of the third or fourth leaf. Frequently there is a general
similarity with grey speck of oats, but the transverse line of
withering tends to occur nearer the tip of the leaf than in
oats, later extending to the lower regions of the leaf. The
withering may develop as in oats by the coalescence of small
grey elongated areas or it may begin at the leaf margin. As
soon as the line of withering reaches right across the leaf the
upper part of the leaf soon loses its green colour. Sometimes,
instead of the development of the line of withering across the
leaf as in oats, small grey oblong areas appear scattered parallel
with the veins. The whole plant becomes chlorotic and the leaves
wither, beginning at the tip.

In barley, Gallagher and Walsh record the first symptom of
manganese deficiency to be a localized paling of the leaf, followed
by the development in a few days of small grey oblong spots with
brownish margins. These enlarge and coalesce to form stripes
parallel with the veins. Sometimes the spotting is most marked
near the tip, which finally withers. The plant as a whole becomes
somewhat chlorotic.

Rye appears to be affected by manganese deficiency in much
the same way as oats, the spots that develop on the leaves being
described by Gallagher and Walsh as whitish. The leaves bend
over in the same manner as those of oats affected by grey speck.
Neither barley nor rye, however, appears to be so badly affected
by manganese deficiency as oats.

From the work of Pettinger, Henderson and Wingard (1932),
manganese deficiency appears to produce a chlorotic condition
in maize very similar to grey stripe. In sand-culture experi-
ments with maize they met with three types of chlorosis which
they attributed respectively to deficiency of magnesium
(type A), excess of sodium (type B) and deficiency of manganese
(type C). In type A the chlorotic areas take the form of long
narrow streaks more or less continuous from the base to the

62 TRACE ELEMENT DEFICIENCY

apex of the leaves, but with very irregular margins. In type B
the streaks are also continuous throughout the leaf but have very
regular margins and occupy the whole intervenal region. In
the type attributable to manganese deficiency, on the other
hand, the chlorotic areas form discontinuous spots or stripes.
White or chlorotic spots first appeared in the cultures when
these were about 3 weeks old. As the leaves grew the spots also
increased in area, and as the affection became more severe the
spots tended to coalesce into elongated chlorotic streaks. The
tissue in the middle of the chlorotic areas then turned brown,
broke down and was dead. Sometimes the dead tissue fell out
of the leaf, leaving a number of holes. The similarity of the con-
dition to grey stripe of oats is striking, and a photograph of
maize leaves affected by their type C chlorosis published by
Pettinger, Henderson and Wingard bears a close resemblance
to that of oat leaves affected by grey stripe reproduced in Fig. 2.

Pahala Blight of Sugar Cane. The disease of sugar cane
named Pahala blight, after a small town in Hawaii where it was
first observed, is characterized by a partial chlorosis of the
leaves, the chlorotic areas taking the form of long white streaks.
These are limited to the leaf blades and do not occur on the leaf
sheaths. The third, fourth and fifth youngest leaves are generally
those most affected. As the chlorotic cells die red spots appear,
and as these increase in number neighbouring spots may coalesce
so that continuous red streaks result, and there may then follow
splitting of the leaf along the line of the streak. By the time
red spots appear the plant is generally very much stunted.
A fungus, Mycosphaerella striatiformans, frequently appears on
the red spots, and when the disease was first described in 1906
it was attributed to the attack of this fungus, but in 1928 Lee
and McHargue produced evidence that the Pahala blight results
from a deficiency of manganese, the fungal attack being secon-
dary to this. This conclusion is based on three lines of evidence
derived respectively from the results of the application of
solutions, and particularly powders, containing manganese
sulphate to the leaves, from chemical analyses of normal and
affected leaves, and from sand-culture experiments.

As regards the effect of applying manganese sulphate to the
leaves it was found that this salt, generally applied as a dust with

DISEASES OF PLANTS 63

dusting sulphur as a carrier, brought about good recovery of
affected plants so that new leaves were healthy and of a dark
green colour. No recovery resulted from similar treatment with
ferrous sulphate, which indeed had the effect of 'burning' the
leaves.

Chemical analyses of normal, semi-chlorotic and chlorotic
leaves showed no very marked difference in the content of any
of the mineral constituents except manganese. The difference
in this element, however, between normal and affected leaves
is marked, the percentages of manganese in normal, semi-
chlorotic and chlorotic leaves being respectively 0-003, 0-0005
and a trace,

The sand-culture experiments were carried out with cuttings
of a variety of sugar cane very susceptible to Pahala blight.
Manganese -free sand was used. Ten cultures were supplied with
a culture solution free from manganese while another ten, sup-
plied with a solution containing manganese, served as controls.
These latter plants grew normally, but the plants grown without
manganese gradually developed Pahala blight, and by the end
of six months the affection was quite severe.

It may be noted that Lee and McHargue report that Pahala
blight only appears to occur on plants growing in alkaline or
neutral soils. Addition of substances such as sulphur and super-
phosphates which increase the hydrogen-ion concentration of
the soil and so make the manganese in the soil more readily
available for absorptionvwill tend to diminish the incidence of
Pahala blight.

Speckled Yellows of Sugar Beet. The disease of sugar beet
known as speckled yellows also involves an intervenal chlorosis
in the leaves, the general appearance of the plant resulting from
the presence of the yellow chlorotic areas being indicated by its
name. As the disease progresses the margins of the affected
leaves curl upwards and over the upper surfaces of the leaves.
Other cultivated varieties of Beta maritima, namely, mangold,
red beet, and spinach beet, may also show the same condition,
although in red beet the characteristic speckled yellow effect
is masked by the red pigment present in the sap of the leaf
cells. Spinach (Spinacia oleracea), which belongs to the same
family as beet (Chenopodiaceae), may be similarly affected.

64

TRACE ELEMENT DEFICIENCY

Some good colour photographs of both sugar beet and red
beet affected with the disease are given by Wallace (1943,
pp. 97-9, plates 81-5).

The attribution of speckled yellows to a deficiency of man-
ganese is chiefly based on the fact that the disease is cured by
applications of a soluble manganese salt. Analyses carried out
by the writer and Dr K. W. Dent, however, show a very
striking difference in the manganese content in normal and
affected plants of sugar beet. The affected plants examined were
grown by Mr W. Morley Da vies on soil which had been heavily
limed in order to induce manganese deficiency. The normal
plants were grown on an adjoining plot not subjected to heavy
liming. The differences in manganese content of healthy and
affected plants are clearly shown by the data in Table II. The
values marked £> were obtained by the polarograph, those marked
a by the absorptiometer.

Table II. Manganese content of normal
and speckled sugar beet

Manganese content in p. p.m. dry matter

Date of

collection

of material

Plant
organ

r

Normal

Speckled

A

<
P

a

r

V

a

10 July 1942

Leaf
Petiole

Root

181

183

7

6

13

10

Not
recognizable

Not
recognizable

lit August 1942

Leaf
Root

551
32

36

51

18

54
14-5

Although the determinations by the two methods show in
some cases a little divergence, they make it clear that the
plants affected with speckled yellows contain considerably
less manganese than normal plants. This is particularly so
in the leaves, where the manganese content of the speckled
plants is of the order of one-tenth that of healthy plants. The
data afford supporting evidence for the view that speckled
yellows is a manganese deficiency disease.

Marsh Spot of Peas. The symptom of the disease of peas
known as marsh spot is the occurrence on the seeds in the pod
of brown or black spots or cavities on the internal surface of the

DISEASES OF PLANTS 65

cotyledons. These necrotic spots generally only affect coty-
ledonary tissue, although occasionally the plumule may be
affected. Pods containing exclusively healthy seed, and
pods containing only diseased seed may occur on the same
plant, and pods are even found containing both healthy and
diseased seed. Externally the plant may appear quite normal,
although sometimes mild chlorosis or mottling of the younger
leaves may be present. In this country it occurs particularly
in Romney Marsh where it appears to be limited to alkaline
soils (Heintze, 1938). In Holland, Ovinge (1935) generally found
it on alkaline and relatively new polder soils.

The fact that he found peas affected with marsh spot growing
near oats wThich had developed grey speck led Pethybridge
(1936) to suspect that marsh spot might be attributable to the
same condition as grey speck, that is, to manganese deficiency.
This view was supported by the finding of Lohnis (1936) that
peas affected by marsh spot contained somewhat less man-
ganese than healthy peas, and by the experiences of Ovinge
(1938) in Holland and of Lewis (1939) in this country who found
that the application of soluble manganese salt either as a soil
dressing or a spray was effective in reducing the incidence of
marsh spot. Also Heintze found that among the Romney Marsh
soils those on which marsh spot occurred contained less salt-
soluble manganese (manganese extracted by a normal solution
of magnesium nitrate or calcium nitrate) than those on which
peas were free from the disease, the difference in the extractable
manganese being related not to the total manganese content,
but to the acidity or alkalinity of the soil.

Determinations of the manganese content of different parts of
healthy peas and of peas affected by marsh spot made by Glass-
cock and Wain (1940) show a considerably lower manganese
content in the diseased seed. The peas examined were of the
variety Harrison's Glory, the diseased sample having been
obtained from Romney Marsh and the healthy peas from
Folkingham in Lincolnshire. The results are summarized in
Table III.

66 TRACE ELEMENT DEFICIENCY

Table III. Manganese content of healthy and marsh
spotted peas. (Data from Glasscock and Wain)

Manganese content in p. p.m.

^i

Part of seed Healthy seed Diseased seed

Germ 15 3

Cotyledon outer tissue 11 5

Cotyledon centre tissue 6 < 2

Seed coat 4 2

The relationship of marsh spot to manganese deficiency was
definitely established by Piper (1941) by means of carefully
controlled water-culture experiments in which specially purified
media and carefully regulated amounts of manganese were used.
In addition to the ordinary major mineral nutrients the culture
solution contained small amounts of boron, copper, zinc and
molybdenum as well as sodium chloride. One series of peas was
grown in this culture solution without manganese, to four other
series 5, 10, 20 and 500/xg. manganese per litre were respec-
tively added. For the first 39 days after the seeds were put to
germinate no differences were observable in the various cultures,
but then in the manganese -free cultures there appeared mottling
of the younger leaves and brown lesions on the internodes and
tendrils. In a further 2-3 weeks growth stopped.

In the cultures supplied with 5/xg. of manganese per litre
these same symptoms appeared, but not until 8 weeks from the
beginning of germination. On renewal of the solution, including
the manganese, healthy new growth was resumed, but in a
fortnight the same pathological symptoms again appeared. The
cultures produced a few flowers, but no fruits formed.

The cultures supplied with 10/xg. of manganese per litre
showed no unfavourable symptoms after 8 weeks, apart from
slight mottling of the upper leaves, and after renewal of the
culture solution growth was vigorous and moderate flowering
took place. After another 3-4 weeks, however, the symptoms
of manganese deficiency appeared, and although some fruits
formed only a few ripened and these were small and imperfectly
developed, while the seeds they contained were all badly
affected with marsh spot.

The cultures supplied with 20/xg. of manganese per litre grew
normally, flowered freely and produced numerous fruits with a

DISEASES OF PLANTS

67

good yield of ripe seeds. But although the vegetative parts of
the plant were free from symptoms of manganese deficiency,
33 per cent of the seeds were severely affected with marsh spot,
24 per cent were slightly affected, while 43 per cent were normal.

The cultures supplied with 500/u,g. of manganese per litre grew
normally and vigorously and showed no symptoms of marsh spot.

The necessity for manganese was also shown by the yield of
the cultures; Piper's results are given in Table IV.

Table IV. Yield of peas in water cultures supplied with
different quantities of manganese. (Data from Piper)

Cone, of

Mn in

^g. per

litre

0

5

10

20

500

Yield in g. per plant

Shoots

4-3
12-5

18-2
21-9
30-7

Roots

1-3
4-0
3-7
3-2
3-5

Seeds

0

0

0-4
10-2
13-5

Total

5-6
16-5
22-3
35-3

47-7

No. of

seeds

per

plant

0
0
6

68
88

Incidence
of marsh

spot
per cent

100

57

0

These results demonstrate very clearly that marsh spot arises
from a partial deficiency of manganese.1

Frenching of Tung Trees. Aleurites, a genus of the Euphor-
biaceae, contains five species which are of economic importance
on account of the oil yielded by their fruits. They are all trees
growing to a height of 25 to 40 ft. The species A. fordii, the
tung tree or tung-oil tree is the source of tung oil, a drying oil
used in the manufacture of paints, varnishes and linoleum and
for waterproofing.

In plantations of tung trees in Florida, Reuther and Dickey
(1937) reported a rather widely distributed affection which they
described as 'frenching'. It is possible that this disease was
previously unnoticeo because it was masked by another, known
as bronzing, attributed to a deficiency of zinc and described
later. In trees affected with frenching, chlorotic areas develop
between the veins of the leaves, and as the disease advances the
tissue in the chlorotic areas dies and necrotic spots arise.
Premature abscission of leaves may follow.

1 After this was written E. J. Hewitt (1945) described experiments
in which he succeeded in inducing symptoms of marsh spot in broad
beans ( Vicia Faba) and runner beans (Phaseolus multiflorus) grown in
manganese -deficient sand cultures.

68 TRACE ELEMENT DEFICIENCY

Frenching may also occur in another species of Aleurites,
A . montana, the mu-oil tree.

It was found that frenching was not limited to alkaline soils but
was related to a low value of exchangeable manganese in the soil.

In the earlier stages of chlorosis recovery could be brought
about in from 3 to 6 weeks by dipping the shoots in a 1 per cent
solution of manganese sulphate containing 1 per cent calcium
hydroxide and 1 per cent calcium caseinate spreader.

Determinations by Reuther and Burrows (1942) of the photo-
synthetic activity of affected leaves and leaves which had
regained their normal colour by treatment with manganese
sulphate did not indicate any very significant increase in photo -
synthetic activity as a result of treatment, and it is suggested
that an environmental condition such as high leaf temperature,
solarization or stomatal closure might limit the rate of photo-
synthesis under field conditions in Florida. Reuther and Bur-
rows also point out that trees severely affected by frenching tend
to produce small leaves, so that the total photosynthetic
activity of the tree and consequently its production of new
material may be reduced by frenching.

2. Diseases Attributable to a
Deficiency of Zinc

So far there have been no records from Britain of plant diseases
attributable to a deficiency of zinc, but in America such diseases
may be serious. They largely affect fruit trees, but have also been
recorded as occurring in maize and some other herbaceous plants.
As with manganese deficiency, the first sign of zinc deficiency
is usually an intervenal chlorosis, but in trees this is generally
followed by very characteristic symptoms of abnormal growth
known as rosetting. In spring, instead of the development of
elongated shoots with normal-sized leaves distributed along the
length of the shoot, there develops a rosette of small stiff leaves.
According to the species affected, the disease is variously known
as rosette, little-leaf, mottle-leaf or yellows. Chandler (1937)
prefers to class all these conditions, including those of zinc
deficiency in maize, as one disease, which he calls zinc-deficiency
disease, although he points out that the evidence may not yet

DISEASES OF PLANTS 69

be sufficient to justify the conclusion that the disease is due only
to a shortage of zinc. This attitude is no doubt logically sound,
but as the symptoms in different species may vary, and as the
investigations of the diseases in the various species or groups of
species have been to a large extent carried out independently,
it has been considered more satisfactory here to describe the
disease in these various groups separately.

Knowledge of the internal symptoms of zinc deficiency is due
to the work of Reed and his co-workers. In 1935, Reed and
Dufrenoy described the result of a microscopical examination
of mottled leaves of Citrus, which, as we shall see later, may suffer
from a deficiency of zinc. In such leaves the palisade cells are
broader than in normal leaves, being often transversely divided
so that the cells are rhomboidal rather than columnar in shape,
while the contents show various abnormalities. Thus chloro-
plasts are few, their stromata are often rich in fat, and the
starch grains within them are generally thin and elongated. The
vacuoles of the cell contain phenolic material and little spheres
of phytosterol or lecithin. These substances are absent from
normal leaves and tend to disappear when zinc is applied to
plants affected by mottle leaf.

Later the cytology of the leaves of a number of other species
suffering from zinc deficiency was examined by Reed (1938).
These included apricot, peach, tomato, maize, squash, mustard
and buckwheat. The general effect of zinc deficiency on the
growth of the leaves appears to be retarded differentiation, the
palisade cells appearing rhomboidal in shape rather than colum-
nar, while the mesophyll is markedly compact, owing to a
great reduction in intercellular spaces. Hypertrophy of cells
may also occur, and it may be said that zinc deficiency promotes
enlargement of the palisade cells rather than their multiplication
and differentiation, while in tomato actual atrophy of mesophyll
was observed.

In very young apricot and peach leaves the protoplast shows
an abnormally great affinity for dyes. This character disappears
later, at any rate in apricot, but proteolysis of the cytoplasm
may reduce this to an almost invisible layer.

The chloroplasts are particularly affected by zinc deficiency,
for this may result in inhibition of their development or in

70 TRACE ELEMENT DEFICIENCY

their destruction, injury being greatest in cells receiving the
strongest illumination. Plastid injury may be very localized,
affected and normal cells being found in juxtaposition.

The phenolic substances, which were noted b}^ Reed and
Dufrenoy as occurring in zinc -deficient Citrus leaves, were also
observed in zinc-deficient leaves of apricot, peach and buck-
wheat, but were absent from similarly affected leaves of mustard
and maize. Since some phenolic material is present in the
normal healthy leaves of some species such as apricot, Reed
concludes that the differences in the content of phenolic sub-
stances in healthy and affected leaves may be one of degree.
No toxic effect appears to be involved.

Reed (1939) also examined the structure of zinc-deficient
leaves of tomato grown in water culture. Such leaves ex-
hibited dwarfing, paleness, downward curvature of the leaflets,
incurved laminae and necrotic spots on the midrib and
laminae. The palisade cells were longer and the spongy tissue
more compact than in normal leaves. The chloroplasts of the
palisade cells of affected leaves were small and tended to aggre-
gate at the lower end of the cell and, owing to degeneration of
some of them, the number of plastids was abnormally low.
Degeneration was even more conspicuous in the spongy tissue,
the signs of it being increase in the amount of calcium oxalate,
shrinkage, the formation of a melanotic substance, and reduc-
tion in size and number of plastids.

Reed has also examined the cytological effects of zinc defi-
ciency in the apical buds of apricot and peach trees suffering
from little leaf. In apricot some of the meristematic cells in
such buds exhibit strong staining with haematoxylin and
methyl green; this is followed by premature vacuolization
and polarization. A similar state of affairs was observed in zinc-
deficient peach buds except that the strong affinity for dyes was
not evident. In the apricot nuclei may become masked by
densely stained masses of cytoplasm, and phenolic materials
arise from altered cell constituents. These changes were observed
while the buds were still in the resting stage. During the early
spring tannins, which are present in normal cells, become
replaced by phloroglucinol in affected cells, especially in the
more active of these. As growth and differentiation proceed

DISEASES OF PLANTS 71

tannin compounds reappear, their accumulation being associated
with enlargement of the cells and inhibition of cell division. At
the same time the amount of phenolic compounds diminishes,
reaching a minimum in early summer, after which their quan-
tity increases until it reaches a maximum at the onset of the
resting stage. The accumulation of these phenolic compounds
in the vacuoles results in an increase of cell size but does not
appear to be connected with necrosis of the cells.

Reference has already been made (p. 3) to the observation
of Sommer on the necessity of zinc for the completion of the
normal life cycle of beans and buckwheat. More recently, by
means of carefully controlled water cultures of garden pea, wax
bean and milo (Andropogon sorghum), Reed (1942) has shown
that a supply of zinc is necessary for seed production in these
plants. Zinc was supplied in a range of concentrations, namely,
0-0, 0-005, 0-02, 0-10 and 0-20 p.p.m. In garden peas no
seed was produced when the concentration of zinc was 0-005
p.p.m. or less, but with zinc concentrations of 0-02, 0-10 and
0-20 p.p.m. seeds were produced, the numbers forming increasing
with the concentration of zinc supplied. Results with beans and
milo were similar except that in these the minimum concentra-
tion necessary for seed formation was 0-10 p.p.m.

Pecan Rosette. The pecan (Carya olivaeformis) , a member of
the Juglandaceae, is not cultivated in Britain, but its fruit,
resembling a small walnut, was becoming familiar to people in
this country in the years immediately before 1939. The tree is
largely cultivated in the United States where the disease known
as pecan rosette is widely spread, and where, according to
Finch and Kinnison (1933), it was recognized by growers as long
ago as 1900.

The first symptom of the disease is a yellow mottling of the
leaves at the tip of a branch, the chlorosis being often evident
as the leaves unfold. The leaves at the top of a tree are generally
those first affected. As the disease proceeds the affected leaves
remain small and are usually crinkled, brittle and misshapen,
while the veins tend to stand out prominently. The chlorotic
areas of the leaves are abnormally thin and frequently become
dark reddish brown in colour and die. Sometimes the intervenal
tissue fails to develop at all, with the result that smooth-

72 TRACE ELEMENT DEFICIENCY

margined holes are scattered over the leaf. Internode develop-
ment is poor and finally the branches die back. The development
of lateral buds below the dead region results in the rosette
appearance from which the disease takes its name. A morpho-
logical examination by Finch and Kinnison of the roots of
affected trees revealed no indication of an abnormal condition,
either externally or internally.

Death of the tree rarely, if ever, results from rosette, but fruit
production may be so poor that the cultivation of the trees
becomes unprofitable, and in 1932 Alben, Cole and Lewis stated
that in some south-eastern states hundreds of acres of pecan
orchards had been abandoned on account of rosette, while in
south-western states where plantations were more recent, as
many as 95 per cent of the trees were rosetoing in some places.
There can thus be no doubt of the economic importance of
pecan rosette.

Researches by Orton and Rand (1914) showed fairly con-
clusively that the disease is not due to the attack of any micro-
organism, nor did it appear to be limited to any type of soil.

At first it appeared that the disease might be related to iron
deficiency, for Alben, Cole and Lewis (1932a) found that some
improvement in rosetted leaves was brought about by dipping
them in, or spraying them with, a 0-6-1 per cent solution of
ferric chloride or ferric sulphate.

A little later, however (19326), they found that favourable
results by such treatment were obtained only when galvanized
iron containers were used for the solutions. This suggested the
possibility that the effect at first attributed to iron might be
due to zinc salts present as impurities in the iron salts used,
and accordingly treatment with solutions of zinc chloride and
zinc sulphate was tried. It was found that an immersion of the
terminal branches of trees exhibiting rosette in a solution of an
iron salt produced no improvement in the condition of the
leaves, but that with a solution of a zinc salt young leaves were
restored to their normal condition. Similar favourable results
were obtained by the use of zinc-lime and zinc-sulphate sprays.
Alben, Cole and Lewis concluded from their experiments that
zinc is essential for the healthy growth of the pecan tree.

Similar conclusions with regard to the cause of pecan rosette

DISEASES OF PLANTS

73

were reached by Finch and Kinnison (1933). Among other
aspects of the problem they examined soils on which rosette
appeared, but could find no relation between any soil factor and
the incidence of rosette. The effects of a number of substances
on affected trees were examined. These substances included
salts of iron, magnesium, manganese and zinc. Three treatments
were employed: (1) placing the dry material in holes bored
in the trunk of the tree, (2) spraying leaves with solutions
of the substances or dipping the leaves in the solutions, and
(3) injecting the solutions into the tree trunks. With zinc salts
a great improvement in the condition of the trees was effected
by all three methods of treatment. In some cases, but by no
means in all, some improvement was observed with the use of
iron salts, a result which could be attributed to the presence of
zinc as an impurity in the iron salts, especially as no improve-
ment resulted with the use of purer iron salts. No benefit
occurred as the result of treatment with either magnesium or
manganese salts.

Determinations of the zinc content in different parts of
the terminal 6 in. of some shoots taken from the top of pecan
trees were made; the results are summarized in Table V. They
show that the shoots of the tree affected with rosette contained
very much less zinc than those of healthy trees, while in a rosetted
tree treated with zinc chloride by solid injection and in which
recovery from rosetting had taken place there was already after
8 weeks a very considerable increase in zinc content.

Table V. Zinc content of leaflets, petioles and stems of pecan
(Carya olivaeformis) . The quantities are given as p. p.m. of
dry matter. (Data from Finch and Kinnison)

Condition of tree

Healthy

Healthy

Rosetted

Rosetted ; then treated with 6 g.

zinc chloride injected in trunk

(55 days after treatment)

Leaflets

Petioles

Stems

16-7

110

7-9

100

Trace

10-3

3-5

Trace

Trace

3-9

8-6

15-3

Finch and Kinnison also published data of the zinc content
of irrigation waters used in different districts of Arizona for
supplying pecan plantations. Five out of six of these waters

74 TRACE ELEMENT DEFICIENCY

contained no measurable amount of zinc, and in all cases rosette
was severe or common in the districts supplied. In the sixth
case the water contained a measurable amount of zinc (0-14
p. p.m.) and the district was essentially free from rosette.

The evidence presented by Finch and Kinnison thus supports
the view that zinc is an essential element for the growth of the
pecan, and that when there is a deficiency of it the condition
known as rosette results. The favourable effect of zinc in con-
trolling pecan rosette was also recorded by Demaree, Fowler
and Crane (1933) working in Georgia.

Another member of the Juglandaceae, the walnut, may also
exhibit the effects of zinc deficiency, but according to Chandler
(1937) these do not include rosetting, although the leaves are
mottled, crinkled and rather small.

Little Leaf or Rosette of Deciduous Fruit Trees. Similar
to pecan rosette is the disease of deciduous fruit trees known in
California as little leaf. The most characteristic symptom is the
development in the spring of rosettes of very small leaves
which, according to Chandler, Hoagland and Hibbard (1932),
who have made a special study of the disease, generally possess
less than 5 per cent of the area of normal leaves. The affected
leaves generally exhibit a chlorotic mottling. Shoots bearing
normal leaves may develop later in the season below the little -
leaf rosettes, but as the season proceeds the new leaves are pro-
gressively smaller and mottled and may be abnormal in shape.
Sometimes after one or two years, in other trees after a much
longer period, the branches begin to die back. Fruit generally
fails to set on badly affected branches and any which does is
small and malformed. Stone fruits tend to have brown areas in
the flesh (Chandler, 1937). Chandler, Hoagland and Hibbard
mention apple, pear, plum, cherry, peach, apricot, almond and
grape as all liable to the affection.

Concluding that little leaf was not the result of attack by
micro-organisms, Chandler, Hoagland and Hibbard sought for
its cause in the soil. As a result of various fertilizer trials they
found that affected trees responded to the application of ferrous
sulphate, but only when this contained zinc as an impurity.
Further trials showed that it was the zinc that was actually
responsible for the improvement. Solid injection of zinc sulphate

DISEASES OF PLANTS 75

into the trunks of the trees appears to be the most effective
treatment, but favourable results are also obtained by the use of
zinc sulphate as a soil dressing or as a spray in winter. No
improvement could be detected as a result of treatment with
salts of silver, nickel, cobalt, tin, cadmium,- mercury, iron,
copper, chromium, manganese, aluminium, molybdenum, sele-
nium, zirconium, uranium, strontium, tungsten and titanium
(Chandler, Hoagland and Hibbard, 1934, 1935). A number of
organic compounds gave equally negative results.

Determinations of the zinc content of stems and leaves of
various fruit trees affected by, and free from, little leaf published
by Chandler, Hoagland and Hibbard suggest that the zinc
content of shoots from trees in orchards free from little leaf is
on the whole higher than in those of trees affected by little leaf.
Hoagland, Chandler and Hibbard (1936) have been able to
induce the symptoms of little leaf in young apricot trees grown
in water cultures from which care was taken to exclude zinc,
as far as practicable.

Chandler, Hoagland and Hibbard were at first very reluctant
to attribute little leaf to actual zinc deficiencv. Their reasons
for this reluctance were the suddenness with which healthy trees
might begin to die from little leaf, the recovery of some trees
without any obvious improvement in the zinc supply, and the
fact that, whereas trees are susceptible to little leaf, annual
plants growing on the same soils are apparently free from any
symptoms of zinc deficiency.

That simple zinc deficiency alone may not afford a complete
explanation of the cause of little leaf is suggested by the work
of Ark (1937). This investigator sterilized, by means of steam,
soil from orchards showing little leaf and found this treatment
very beneficial to maize and tomato. Also sand cultures of
maize were treated respectively with little -leaf soil and sterilized
soil. In the former the plants soon showed symptoms of zinc
deficiency (white bud, see p. 79), while the plants receiving
sterilized soil, or in sand without any soil, remained normal.
Further, from little-leaf soils he isolated two strains of bacteria
which when added to the artificial culture medium in which
maize seedlings were growing induced symptoms of white bud
which were removed by raising the content of zinc in the

76 TRACE ELEMENT DEFICIENCY

medium or by the injection of a zinc salt into the stems. Addition
of one of the bacteria to cultures of peach and walnut also
resulted in symptoms very similar to little leaf, symptoms which
were prevented by the presence of zinc. Altogether Ark's
observations are verv reminiscent of those of Gerretsen on the
relation of micro-organisms to the grey-speck disease of oats.

Chandler (1937) suggests that if little leaf is the result of a
simple zinc deficiency this might be brought about by the pro-
duction in certain soils of a flourishing micro-flora that absorbs
zinc in large quantities. The effect of sterilization, by killing
this flora, would thus be to leave more zinc available for higher
plants rooted in the soil. Alternatively, Chandler suggests that
soil organisms might excrete, or yield on dying, substances
which combine with zinc to form insoluble zinc compounds
and so render it non-available. In these circumstances the effect
of the sterilization treatment might be to break up the insoluble
zinc compounds and release the zinc in a soluble form.

Mottle Leaf (Little Leaf Type) or Frenching of Citrus.
The disease of Citrus trees known as mottle leaf in California has
been described in detail by Johnston (1933). The rfame is derived
from the fact that yellow areas arise between the veins of the
leaves giving these a mottled appearance. These chlorotic areas
enlarge, and as fresh leaves develop these are progressively
smaller until in severe cases they may be only an inch long, with
chlorophyll developing only at the basal end of the midrib. In
extreme cases dieback may follow, ultimately resulting in the
death of the tree. The root system is also affected, the smaller
rootlets first and the larger ones later, until in severe examples
only large roots with a few rootlets remain functional. All
species and varieties of Citrus can be affected by the disease.
The severity of the affection is increased by extremes of high or
low temperature.

Since mottle leaf of Citrus and little leaf of deciduous trees
often occur in the same orchard, and since Chandler, Hoagland
and Hibbard (1932. 1933) had found that application of zinc
sulphate was a cure for little leaf, Johnston tried the same treat-
ment for mottle leaf of Citrus and found that this resulted in
restoring the normal green condition to mottled trees. The zinc-
sulphate was applied as a circle of salt on the soil round the tree,

DISEASES OF PLANTS 77

by injection of crystals of the salt in holes bored in the trunk
and then sealed, and by spraying the foliage with various sprays
containing zinc sulphate. In all cases a favourable result was
obtained. Johnston is of opinion that there are probably several
kinds of mottle leaf affecting Citrus which are due to different
causes, and he proposes to designate the type which responds
to treatment with zinc as little leaf, thus bringing the terminology
into line with that used for the similar condition found in
deciduous trees.1

In Florida the chlorotic condition of Citrus trees is known as
frenching. This is presumably the same disease as the mottle
leaf described by Johnston, and affected trees respond favour-
ably to the application of zinc sulphate. Thus Satsuma orange
trees showing symptoms of frenching were treated by Mowry
and Camp (1934) with a soil dressing of zinc sulphate, and as a
result showed signs of complete recovery.

Whether mottle leaf or frenching is a zinc-deficiency disease
in the strict sense is not clear. Johnston, indeed, stated that
mottle leaf does not appear to be a case of soil deficiency and
suggests that the zinc may act as an antitoxin.

Bronzing of Tung Trees. The condition of tung trees (see
p. 67) known as bronzing was first noted in 1930 by Newell,
Mowry and Barnette as occurring in trees in Florida growing on
soils containing large amounts of phosphate. Later observation
has shown that bronzing is not confined to such soils.

The disease usually appears first in the late spring or early
summer or even later. The first symptoms are the appearance
of a bronze colour in a number of leaves, together with a defor-
mation of the terminal leaves of the shoots. With the develop-
ment of the dark bronze colour in the leaves necrotic spots
develop and parts of the leaves die so that these appear ragged.
Ultimately, a twig may lose many or all of its leaves. After the
first appearance of the disease in a tree the severity of attack
generally increases rapidly. New loaves are successively smaller
and become more deformed, the internodes fail to develop

1 Already in the earlier of the papers by Chandler, Hoagland and
Hibbard cited above they had included (JUrus fruits and walnut along
with deciduous fruit trees as liable to little leaf. They also showed (1934)
the favourable effect of zinc on orange trees affected in this way.

78 TRACE ELEMENT DEFICIENCY

normally, resulting in a bunched appearance of the foliage, the
twigs remain thin and adventitious buds sprout on the older
wood. Although the affection may be at first local, finally the
whole of the tree may be involved. Affected trees are unduly
subject to injury by the low temperatures of winter, and a
bronzed tree may come into activity in the following spring
smaller in size. By the third season after the first appearance
of bronzing a tree may be reduced greatly in size and, with its
almost bare branches, appear half dead.

Mowry and Camp (1934) found that bronzing could be cured
by the application of about 1/4-1/2 lb. of zinc sulphate per tree
to the soil or by spraying the foliage with a spray containing
6 lb. of hydra ted lime and 3 lb. of 89 per cent zinc sulphate in
50 gal. of water containing some calcium caseinate spreader.
While not definitely committing themselves to the view that
bronzing of tung trees is a deficiency disease they conclude that
it should be considered the result of zinc deficiency until proved
otherwise. As a result of this work by Mowry and Camp on the
control of bronzing by the application of zinc sulphate Reuther
and Dickey were able to report in 1937 that comparatively little
bronzing was then to be found in properly conducted tung
plantations.

Pinus radiata Rosette. Trees of Pinus radiata growing in
plantations on poor soil in Western Australia are liable to
exhibit a condition of rosetting reminiscent of that occurring on
so many species of trees in America already described. Field
trials of the effect of spraying with zinc chloride or zinc sulphate
described by Kessell and Stoate (1936, 1938) showed that, as
with other species affected by rosetting, treatment with zinc
was a cure for the disease. The presumption that rosette of
P. radiata is brought about by a deficiency of zinc was shown
to be correct by carefully controlled water cultures carried out
by Smith and Bayliss (1942). Removal of zinc, copper and lead
from the stock solutions was effected by the use of dithio-
carbazone. Boron, manganese and copper were added to
the culture solutions containing the usual major nutrients.
Zinc was added to the controls only. The method of purifying
the solutions did not remove molybdenum and it was not
necessary to add it.

DISEASES OF PLANTS 79

The first symptom of zinc deficiency was a decreased rate of
growth which began to be noticeable after about 3 months.
This was soon followed by the shoot apices acquiring a flat-
topped appearance due to the lower activity of the apical
meristem. The apical buds appeared bunched together while
secondary needles stopped growing and so appeared short, thick
and stiff and the bundles did not spread open. Chlorotic symp-
toms did not appear until the lapse of another 5 weeks. Then
small yellow dots appeared near the tip of the needles followed

*

by browning, soon giving the tops of the branches a bronzed
appearance. With the appearance of the first signs of chlorosis
in the leaves conical swellings sometimes arose on the root
apices. As far at least as the shoots are concerned the effect of
zinc deficiency on P. radiata thus resembles the effects produced
on other trees.

It should be noted that the cultures grown by Smith and
Bayliss were non-mycorrhizal.

White Bud of Maize. As stated early in this book (p. 3),
as long ago as 1914 Maze demonstrated the essentiality of zinc
for the growth of Zea mais. Maze reported that maize plants
grown in water culture deficient in zinc at first developed
normally, but that quite suddenly the leaves darkened and
developed a metallic sheen while nocturnal exudation became
very abundant, a deposit of soluble salts being left on the
leaves. Death of the plants followed in 3-5 days.

According to the investigations of Barnette and Warner
(1935) it would appear that the disease of maize known in
Florida as white bud is due to zinc deficiency. They report the
disease as occurring chiefly on land which has been under
cultivation for a number of years and also on poorer land
recently brought into cultivation. The disease may sometimes
be serious enough for the crop to fail completely.

The first symptoms of the disease may appear within a week
of the emergence of the seedlings from the soil. The first sign of
the trouble is the appearance of light yellow streaks between the
veins of the older leaves followed by the rapid development of
white necrotic spots. As the newer leaves unfold they are often
pale yellow to white in colour, a symptom which gives its name
to the disease. The older leaves develop light slate to dark brown

80 TRACE ELEMENT DEFICIENCY

necrotic areas which increase in size and merge with one
another until the whole leaf is dead. Meanwhile the younger
chlorotic leaves continue to unroll and as they expand develop
typical yellow intervenal striping. Internodes fail to develop
properly so that the whole plant is stunted. The root system,
however, appears to be normal.

The effect of various fertilizer treatments on maize plants
affected with white bud was examined by Barnette and Warner.
They found that a dressing which included 20 lb. of zinc sulphate
per acre induced a very much higher yield of grain than any
inorganic dressing which did not include zinc. With the applica-
tion of zinc sulphate the chlorotic plants recovered a normal
green colour, made healthy growth and produced grain. The same
effect was produced by the application of stable manure or leaf
mould, while chicken manure and alkaline peat produced some
improvement in the condition of affected plants. Spectroscopic
examination of the ash of these various organic manures
revealed the presence of zinc in all of them. The work of Barnette
and Warner, although not definitely establishing white bud of
maize as a disease due to deficiency of zinc, indicates the prob-
ability of this being the case.

The possible relationship of bacteria to white bud has already
been mentioned in the discussion of little leaf of deciduous fruit
trees (p. 75).

3. Diseases Attributable to a
Deficiency of Boron

The effects of boron deficiency in plants have been well dealt
with by Brenchley and Warington (1927) and more recently by
R. W. G. and A. C. Dennis (1937, 1939, 1941, 1942). From the
accounts of these and other authors it appears that the first
external symptom of boron deficiency is generally the death of
the apical growing point of the main stem. This is followed by
growth of lateral buds into side shoots, the apices of which
then die. Further symptoms are slight thickening of the leaves,
a tendency for these to curl, and sometimes a slight chlorosis.
The petioles, and even the leaves, often become brittle, flowers
may not form, or if they do fruit may not set. Stunted root
growth is general.

DISEASES OF PLANTS 81

Quite a number of investigations have been made on the
histology of boron-deficient plants. These include broad bean
(Warington, 1926), tomato (Johnston and Dore, 1929; Fisher,
1935; Van Schreven, 1935), Citrus (Haas and Klotz, 1931),
tobacco (Van Schreven, 1934), sugar beet (Jamalainen, 1935),
maize (Eltinge, 1936), potato (Van Schreven, 1939), apple
(Mac Arthur, 1940). carrot (Warington, 1940), Brassica spp.
(Chandler, 1941), radish (Skok, 1941), squash (Alexander, 1942),
sunflower (Lowenhaupt, 1942), garden beet and cabbage
(Jolivette and Walker, 1943).

In Vicia Faba Warington found that the chief internal symp-
toms of boron deficiency were frequent hypertrophy of the cells
of the cambium and subsequent discoloration and degeneration
of the cells ; disintegration of the cells occurred, however, whether
there was previous enlargement or not. Disintegration of
phloem and ground tissue was also frequent, while xylem de-
velopment was poor and the cells might also ultimately dis-
integrate. Brenchley and Thornton (1925) had previously found
that development of the xylem of the root nodules either failed
altogether or was very poor.

In sugar beet and Citrus hypertrophy of the cambium, fol-
lowed by its disintegration and that of the phloem, is also symp-
tomatic of boron deficiency. In Citrus boron was found to be
essential for meristematic and cambial activity. In tobacco Van
Schreven found that the first symptoms appeared in the cells of
the root apex and later in the stem apex. In these regions cells
became brown and degenerated. The symptoms then extended
backwards from the apices, the vascular tissue being par-
ticularly affected. Proliferation of the cells of the phloem
occurred resulting in the individual elements being compressed
and distorted, while the xylem development was poor. Cells of
the ground tissue might also undergo disintegration. The same
worker recorded similar effects in tomato where thin-walled
cells, such as those of cambium, phloem and ground tissue,
undergo degeneration. The degeneration of the phloem in
tomato was earlier recorded by Johnston and Dore and at about
the same time by Fisher.

A number of species of Brassica were examined by Chandler.
In broccoli and Brussels sprouts boron deficiency was found to

82 TRACE ELEMENT DEFICIENCY

bring about cessation of cell division in the root apex and
degeneration of the root cap. In rutabaga the thin- walled cells
of the meristematic tissue of the stem apex and of the root
cortex became crushed, while cells near the cambium became
elongated and the cork cambium failed to produce cork. In
cabbage Jolivette and Walker recorded considerable prolifera-
tion of cells in the cambial region resulting in a zone of meri-
stematic tissue between xylem and phloem several times the
usual width of this band, while there was a corresponding reduc-
tion in the amount of differentiated xylem and phloem. In
radish Skok found that vascular tissue near the axis which
might have been produced while boron from the seed was still
available, or from boron present as impurity in the substrate or
chemicals used, was usual, but that there was a complete absence
of normally developed and lignified xylem vessels in the region
between this inner tissue and the cambium, while the later
formed phloem disintegrated. Xylem parenchyma cells, al-
though smaller than normal, appeared uninjured. Owing to the
failure of the development of normal vascular tissue the roots
cracked and then the cambium and phloem mostly disintegrated.
In swede, according to Jamalainen, the first symptom of boron
deficiency is enlargement of xylem parenchyma.

In the squash Alexander noticed that boron deficiency
brought about hypertrophy and collapse of the meristematic
cells and of more mature cortical cells of the apical region of the
stem, while, towards the apex of the root, cells of the central
cylinder were similarly affected. In older regions of the stem
parenchyma of the ground tissue, xylem, and the region be-
tween xylem and internal phloem showed abnormal enlargement.
Cells of the cambium were also enlarged. Enlargement of thin-
walled cells also occurred in the leaves.

In garden beet the first internal symptom, according to
Jolivette and Walker, appears in the phloem where certain cells
resembling companion cells become filled with a densely staining
substance. Occasional hypertrophy of cambial cells was also
noted. Later degeneration of xylem tissue occurs.

The first internal deficiency symptom of maize was found
by Eltinge to be a disintegration of some of the leaf cells.
Later, entire cross-sections of a leaf might collapse, and in other

DISEASES OF PLANTS 83

parts of such leaves cells might fail to differentiate, and in yet
other parts hypertrophy of cells, particularly those of the lower
epidermis, might occur. Later, disintegration of cells of the
stem apex took place:

Thus, although the internal symptoms vary somewhat from
species to species, it may be said that in general boron deficiency
leads to degeneration of the meristematic tissues, including the
cambium, to breakdown of the walls of parenchyma cells and
to feeble development of the vascular tissues. Of these the
phloem appears to be most affected, but imperfect development
of xylem is also a common feature. Hypertrophy of thin-walled
cells and then discoloration are frequent precursors of their dis-
integration. Sometimes this latter is preceded by abnormally
active cell division.

Heart Rot of Sugar Beet and Mangold. The disease of
sugar beet and mangold known as heart rot, crown rot or dry
rot, is widely distributed through Britain, Europe and America.
It is generally most severe on alkaline soils and in dry years. As
the names given to it imply, the most prominent symptom is a
necrosis of tissues of the crown and interior of the root. The
first symptoms of the disease, however, appear in the youngest
(inner) leaves (cf. Fig. 5). These are stunted, become markedly,
curled, and the petioles develop a brown to black colour which
may extend into the veins of the lower part of the laminae. As the
plants grow older the leaves become affected, the veins becoming
yellowish and the petioles brittle. Next, the inner leaves become
brown or black and die, the main growing point dies and the outer
leaves turn yellow, wilt, wither and finally also die. New shoots
now develop in the axils of the dead leaves (cf. Fig. 6), but these
become affected in the same way as the earlier formed leaves.

With the death of the first crop of leaves the tissue of the
crown begins to rot. First necrosis occurs at a number of spots
on the crown and these develop inwards into the root, in-
creasing in size until in severe cases the greater part of the
tissues of the root may be destroyed. Invasion of the affected
parts by Phoma betae usually follows. The percentage of sugar
in even the healthy parts of affected beets is less than that of
healthy roots, so that from an economic point of view heart rot
can be of very serious consequence.

84 TRACE ELEMENT DEFICIENCY

The connexion of heart rot with boron deficiency was shown
by Brandenburg by means of controlled water cultures, first of
mangolds (1931) and later of sugar beet (1932). Similar results
were obtained by Bobko and Belvoussov (1933) and by Rowe
(1936). In the experiments of Bobko and Belvoussov, seedlings
provided with no boron developed symptoms of heart rot after
about a week. Renewed root development of boron-starved
cultures resulted on the addition of boric acid to the culture
solutions. Too high a concentration proved toxic, the most
satisfactory range of boron concentrations being from 0-5 to
5 mg. of boric acid per litre. These findings were confirmed with
sand cultures and field trials carried out by Brandenburg and
others.

Canker and Internal Black Spot of Red or Garden Beet.
Pathological conditions of garden beet attributed to boron
deficiency have been described both here and in the United
States. A full description of the disease as it occurs in the
United States has been given by Walker (1939), who terms it
internal black spot in reference to its most prominent symptom,
the presence of internal hard black necrotic masses which render
the beet unfit for canning. These masses, irregular in size and
shape, are not characteristic of any particular part of the root,
for although they may be confined either to the central region
or the peripheral region, they may also be scattered throughout
the root. When the black spot occurs near the periphery a rift
in the surface tissues may occur, soil micro-organisms may enter
and attack the root and a surface canker may result. But when
the necrotic areas are well inside the root there may be no
external symptom of the disease.

In this country the lesions on boron-deficient garden beet
appear always to be superficial and are known as canker. Thus
Wallace (1943) states that 'rotting occurs on the sides of the
roots and may not penetrate into the more central tissues', and
he gives some good colour photographs of cankered garden
beet (op. cit. plates 106, 107, pp. 109, 110). The absence of the
internal necrosis in red beet grown in this country is not ex
plained, but it may be related in some way to varietal differences
in growth. Walker states that the necrotic areas are most
obvious between the prominent rings marked by the thick-

*4L 0|

.

Fig. 5. Sugar beet suffering from boron deficiency. Note the stunted and
curled appearance of the young inner leaves.

Fig. 6.

Mangold suffering from boron deficiency. Note the dying or dead inner
leaves and the small fresh leaves which are developing on the crown.

Fig. 7. Transverse section through swedes showing
characteristic appearance of brown heart.

Fig. 8. Longitudinal section through swede affected with brown heart.

DISEASES OF PLANTS 85

walled vessels formed by the activity of the secondary cambium
zones in the pericycle.

The symptoms of boron deficiency exhibited by the shoot of
garden beet are similar to those of sugar beet and mangold but
generally less conspicuous. The older leaves are generally nor-
mal, but the younger leaves towards the middle of the crown
become malformed, abnormally small, and rich in anthocyanin.
The malformed leaves tend to die early and a rosette of dead
stunted leaves may result. This condition is generally followed
by the sprouting of dormant buds at the base of the dead leaves
and the consequent development of new leaves, which, however,
generally develop the characteristic symptoms indicative of
boron deficiency.

Brown Heart or Raan of Swede and Turnip. The disease
of swedes generally known as brown heart in England and raan
in Scotland has a wide distribution, having been recorded in all
parts of Britain, different countries of northern Europe, Iceland,
Canada, Newfoundland and the United States, Australia and
New Zealand. A good account of it has been published by
Dennis and O'Brien (1937).

No external symptoms of brown heart appear during the
growing season, and the root appears normal externally. On
cutting an affected root across, however, the middle region
presents a mottled appearance owing to a discoloration of
patches of tissue within the xylem (see Figs. 7, 8). According
to Dennis and O'Brien the affected tissue is limited to that
within the cambium. Usually it is the outer region of the xylem
which is affected so that the middle of the root has a normal
healthy appearance, but in severe cases the whole of the region
within the cambium may exhibit mottling, and in very severe
cases the central tissue may break down and the root become
hollow (Wallace, 1943, and see Fig. 9). The colour of the affected
patches shows some variation. Sometimes they have a greyish,
slightly brownish or 'water-soaked' appearance (Dennis and
O'Brien, 1937; Wallace, 1943), the coloration being partly due
to reduction in the intercellular spaces owing to slight swelling
of the affected cells, partly to the development of a brown pig-
ment apparently connected with slight swelling of the cell walls.
Dennis and O'Brien state that bacteria can always be isolated

86 TRACE ELEMENT DEFICIENCY

from the affected tissue, and they suggest that the coloration
may be partly related to increased activity of bacteria normally
present in the intercellular spaces.

Swedes affected with brown heart are unfit for human con-
sumption, as the discoloured parts remain hard when the root is
cooked, while affected roots contain less sugar than healthy roots
and have a bitter taste.

In 1934, Giissow reported that application of boron as sodium
tetraborate resulted in a large measure of control over brown
heart, whereas other elements were not effective, and in the
following year a number of investigators working independently
in different countries published the results of field experiments
on the control of brown heart by the application of borax or boric
acid. These workers included O'Brien and Dennis in Scotland,
Whitehead in Wales, Jamalainen in Finland and Hurst and
Macleod in Canada.

The proof of the essentiality of boron for swedes was, however,
provided by the sand-culture experiments of Hill and Grant
(1935) and Jamalainen (1935) and the water-culture experi-
ments of Dennis and O'Brien (1937), which clearly indicate that
without a supply of boron swedes fail to grow beyond a very
young stage.

The effect of boron deficiency in turnips is, as might be
expected, similar to that in swedes.

Browning of Cauliflower. The most noticeable symptom
of boron deficiency in cauliflower is the formation of brown
patches in the heads. The disease has been investigated by
Dearborn et at. (1936, 1937, 1942) by means of field trials, pot
cultures and histological examination. The first external sign of
the disease is the appearance of ' water-soaked ' patches on the
developing head (see Fig. 10). These patches soon turn brown
and hard and in wet weather may rot. A certain amount of
chlorosis may become apparent in the leaves, particularly at
the apices of the older ones, which may become thicker, brittle
and liable to curl downwards. Blistering may occur on the
petiole and along the midrib. The root system is poorly de-
veloped. The internal symptoms resemble those found in other
cases of boron deficiency (see p. 81). Thin walled parenchyma
cells of pith and cortex appear to be the first affected, individual

Fig. 9. Late stage in boron-deficient swede.

Fig. 10. Boron-deficient cauliflower. Note patches of discoloration of head.

Fig. 11. Stem of cauliflower suffering from boron deficiency
and showing very typical breakdown of central tissue.

DISEASES OF PLANTS 87

cells undergoing enlargement while the intercellular spaces
appear to become filled with mucilage, giving the tissues first a
water-soaked appearance, followed by a gradual darkening to a
deep brown colour. The brown patches on the head appear to
arise in the same way. Very characteristic is the breakdown of
the pith to form an elongated cavity (Fig. 11).

In field experiments it was found that browning of cauli-
flower was completely eliminated by a dressing of 10 lb. of
borax per acre.

Cracked Stem of Celery. A disease of celery, known as
cracked stem, is widely distributed in the United States and
Canada. It was first recorded in Florida and described by
Purvis and Ruprecht (1935, 1937). The first external symptoms
of the disease are a brownish mottling of the leaf, first in the
marginal region, and brittleness of the stem. Next, cracks
develop transversely across the leaf stalks which then curl
outwards and turn brown. The roots suffer the same discolora-
tion and finally die. In extreme cases the terminal bud may
suffer the same fate. Where the disease is prevalent it may be
very serious, and bring about a loss of half the crop in bad cases.
Investigations by Purvis and Ruprecht, including the use of
water cultures and sand cultures, as well as field trials, indicate
that cracked stem results from boron deficiency and can be
controlled in the field by the application of borax either as a
soil dressing or as a spray.

As far as I am aware cracked stem of celery has not been
recorded in Britain, although I have seen examples of it induced
artificially by Mr Morley Davies through heavy liming of the soil.

Lucerne Yellows or Yellow Top. The effects of boron
deficiency on lucerne (alfalfa) were described by McLarty,
Wilcox and Woodbridge (1937) as- a uniform yellowing of the
terminal leaves, or a bronzing of the intervenal areas, poor
development of internodes and death of the growing points. It
appears that the symptoms may be confused with those resulting
from attacks by the potato leaf hopper (Empoasca fabae), and
recently Colwell and Lincoln (1942) have published a compara-
tive account of the symptoms produced in lucerne by boron
deficiency and by the leafhopper, their account being the result
of work carried out both in the greenhouse and under field

88 TRACE ELEMENT DEFICIENCY

conditions. J^rorn this it would appear that when due to boron
deficiency yellowing or reddening is always confined to the
terminal leaves, including those of lateral branches, whereas
when due to the leafhopper, yellowing or reddening may
occur at various levels on the shoots. In boron-deficient
plants the terminal internode is always abnormally short,
whereas with leafhopper injury this is not so, although the
plant may be generally stunted. With boron deficiency the
terminal bud is always abnormal, and death of the growing
points results. Colour photographs of boron-deficient lucerne are
published in the paper by Colwell and Lincoln referred to above.
Several workers have reported increased growth and flowering,
and increased yield of seed of lucerne as a result of application
of boron to the soil (e.g. Grizzard and Mathews, 1942), although
apparently the non-treated plants did not suffer from yellowing.
This suggests that boron deficiency may be met with which is
insufficient to induce yellowing but which is yet sufficient to
bring about a decrease in growth, flowering and fruiting.

Top Sickness of Tobacco. The effect of boron deficiency on
tobacco growing in the field in Sumatra was described by Kuy-
per (1930) who called the disease 'top sickness'. In America the
effect of boron deficiency on tobacco growing in water cultures,
pot cultures and on field plots has been described by McMurtrey
(1929, 1933, 1935). The first external symptoms appear in the
terminal bud, the leaves of which have a pale green colour, the
bases of the leaves being paler than the tips. At the same time
the leaves have stopped growing. The tissue at the base of the
young leaves of the bud now breaks down and the bud dies.
Next the older leaves become thicker and increase in area and
later become brittle. The midrib may break and the vascular
tissue then becomes discoloured. The upper leaves tend to take
a drooping position. If the disease is not too severe and lateral
buds develop in the axils of the leaves, they generally degenerate
in the same way as the terminal bud. If there is partial recovery
from the disease the younger leaves and the upper part of the
stem as they develop may appear twisted to one side owing to
growth round the damaged tissue.

Internal Cork of Apples. Disease of apples as a result of
boron deficiency occurs in Europe, Canada, the United States,

DISEASES OF PLANTS 89

Australia and New Zealand. According to Dennis (1937) the
disease was recorded in Australia as long ago as 1892, when it
was, however, confused with bitter-pit. The symptoms of the
disease are rather varied, and partly as a result of this a number
of names have been given to it including internal cork, drought
or drouth spot, corky pit, corky core, brown heart, poverty
pit, die -back and rosette. Internal cork refers to lesions which
first appear as clear or slightly greenish rounded regions, gener-
ally not exceeding 1 cm. in di° .ieter, and which may arise any-
where in the flesh of the f«.rtt. The patches become dry and
darken, and finally are dark brown in colour and of a corky or
spongy consistency according as the lesions appear earlier or later
in the developing fruit (Carne and Martin, 1937; Burrell, 1937).
In apples affected with drought or drouth spot the lesions are
in the form of large superficial patches, while in the case of
corky core it is the middle region including the core which is
affected. According to Burrell a greater deficiency of boron is
required to produce rosetting and die-back than to induce
internal cork in the fruit. Many investigators have demon-
strated that this disease can be controlled b}^ the application of
borax or boric acid to the soil (see, e.g., Askew, Chittenden and
Thomson, 1936). Internal cork does not appear so far to have
been recorded in Britain.

There is evidence that boron deficiency may also induce the
formation of internal cork in the fruit and die -back of the shoots
in pears.

Brown -spotting of Apricots. On certain light-textured
soils in New Zealand apricots tend to develop brown spots in
the flesh, especially near the stem end, and a dry, spongy con-
dition round the stone. This condition is attributed by Askew
and Williams (1939) to boron deficiency. The condition is con-
trolled by the application of \ lb. of hydra ted borax per tree to
the soil, or as a 0-1 per cent spray. The increase in the boron
content of the leaves and fruit resulting from these treatments
was accompanied by freedom from brown-spotting.

90 trace element deficiency

4. Diseases Attributable to a
Deficiency of Copper

Although copper has now been shown to be essential for the
growth of a number of plants, diseases attributable to a shortage
of this element are rarely met with in the field and none are
known to occur in Britain. The two well-recognized diseases
which appear to be related to copper shortage are an affection
of fruit trees known as exanthema, die-back or chlorosis, and a
disease of various herbaceous plants known as reclamation
disease. These are described later.

The general effects of copper deficiency on the leaves of
tomato plants have been described by Reed (1939). The leaves
exhibit restricted growth, but although they are considerably
smaller than normal leaves the number of leaflets and lobes of
the leaflets is not reduced. The laminae of the leaflets are in-
curved and they develop a bluish green colour with a distinct
sheen. Later necrotic areas appear.

Microscopic examination showed that in the early stages of
development the palisade cells of affected leaves contained
many large hyper chromatic plastids. These plastids ulti-
mately degenerate, at the same time tending to form aggrega-
tions at one end of the cell. Cavities tend to form below stomata
by the separation of the upper ends of adjacent palisade cells.
The cavity may lengthen so that ultimately it extends the
whole length of the cells and at the same time broadens owing
to the shrinkage of the cells, this being followed by a disappear-
ance of the cells owing to lysis of their contents. These processes
appear to lead to the production of the necrotic areas already
mentioned.

Exanthema or Die -back of Fruit Trees. A pathological
condition known as exanthema affects various fruit trees in-
cluding both Citrus species and rosaceous trees as well as the
olive. The disease was recorded in Citrus in Florida in 1875, but
it was not described for other trees until 1928, when Smith and
Thomas recorded that for a long time it had been known in
California as affecting other fruit trees. They reported it as
occurring in French prune, Japanese plum, apple, pear and
olive trees.

DISEASES OF PLANTS 91

Exanthema in Citrus is of widespread occurrence throughout
the world. The symptoms of it in Citrus trees in Western Aus-
tralia have been described by Pittman (1936) as follows. In the
orange strong water shoots tend to bear abnormally large leaves
while the shoots themselves, instead of growing straight, form
an S-shaped curve. Small blister-like swellings containing gum
develop on the young shoots. Later these swellings develop into
longitudinal ruptures bordered with brown or reddish brown
ridges from within which the yellow or reddish gum exudes in
wet weather. Shoots so affected lose their leaves and die back,
and lateral shoots developing at the base of affected twigs pro-
duce a typically bunchy appearance. A condition of 'multiple-
bud' development, resulting from the development of a cluster
of buds instead of two, is frequent, thus emphasizing the bunchy
habit of the tree. Die -back is typical of badly affected trees.
The fruit is small and frequently marked with irregular-shaped
brown spots or blotches where finally the skin, after becoming
dry, splits open.

In the lemon, gum formation on the branches is rare, but gum
pockets may develop on the skin of the fruit.

Haas and Quayle (1935) state that leaves of Citrus in the
early stages of exanthema may be unusually green, but may
become mottled or chlorotic in later stages of the disease.

According to Smith and Thomas (1928), in French prune trees
(Prunus domestica) affected with exanthema there is vigorous
growth of new shoots each spring, but in June the terminal buds
wither and the terminal leaves develop a chlorosis. There is a
similar development of, lateral shoots and of multiple buds as
in Citrus as well as the production of eruptions of the bark.
Apples, pears and Japanese plums can develop similar symptoms.
Pittman describes exanthema of Japanese plums in Western
Australia as involving the development of cracks in the bark
which in some varieties may reach the cambium. Later, exuda-
tion of gum takes place through the ruptures. Die-back is here
also very characteristic of the condition.

As long ago as 1917 Floyd reported the beneficial effect of
copper sulphate on Citrus trees in Florida affected with this
disease, and confirmation of this was forthcoming from Wickens
(1925) in the treatment of affected orange trees in Western

92 TRACE ELEMENT DEFICIENCY

Australia, from Smith and Thomas (1928) in regard to plum,
apple, pear and olive in California and from Pittman (1936) for
Citrus, plum and apple in Western Australia.

Oserkowsky and Thomas (1933) showed that the condition of
the leaves of Bartlett pear trees in relation to this disease corre-
sponded to their copper content. Thus, leaves affected with
exanthema contained 3-1-5-1 p. p.m. of the dry weight, while
normal leaves from localities free from the disease contained
11-20 p. p.m. of the dry weight. While these workers considered
that these analyses afforded strong evidence that exanthema
resulted from a deficiency of copper, they pointed out that there
was no evidence to decide whether the disease was due directly
to copper deficiency or whether the effect was an indirect one
such as might be brought about, for example, if the action of
copper resulted from its neutralizing the effect of toxins absorbed
by the plant from the soil.

More definite evidence that exanthema in Citrus is due to a
deficiency of copper was provided by a culture experiment
described by Haas and Quayle (1935). Valencia orange trees
grafted on sour-orange stocks were grown in twelve large tanks
containing pure sand. A nutrient solution containing all the
known mineral nutrients except copper was supplied to the
trees. Although for some years the trees grew vigorously, after
seven years they displayed typical and pronounced symptoms
of exanthema including the S-shaped growing shoots, exudation
of gum, blisters on the surface of the shoot and dying back of
many shoots. The leaves became covered on the ventral side by
a resinous stain and many leaves developed chlorosis.

The effect of copper deficiency on deciduous fruit trees in
South Africa has been described by Anderssen (1932). Apple,
pear, plum, peach and apricot trees are all affected to different
degrees. Thus plums, peaches and apricots exhibit a very
decided chlorosis in the areas of the leaves between the veins.
This is accompanied by very marked rosetting of the leaves,
cessation of apical growth followed by the dying back of the
branches from their apices. Apples, on the other hand, exhibit
chlorosis much less frequently, but rosetting is severe and the
long shoots die back. Pears similarly do not show chlorosis very
often, but unlike apples they do not develop rosetting; however,

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DISEASES OF PLANTS 93

the shoot apices and the youngest leaves become browned and
finally die back.

Anderssen makes no reference to the surface eruptions on the
shoot which are so characteristic of copper-deficient Citrus trees
as to give the name exanthema to the disease, and which are
recorded both by Thomas and his collaborators and by Pittman
as occurring in deciduous fruit trees.

Analyses of leaves of affected and normal plum trees showed
that of the mineral constituents determined (CI, N, S04, P04,
Ca, Mg, K, Fe, Mn, Cu) the only ones which were present in
lower amount in chlorotic than in normal plants were manganese
and copper. Some of Anderssen's determinations are shown in
Table VI. No effect was, however, produced by applying
manganese to the chlorotic trees, but dipping chlorotic leaves
in a solution of copper sulphate (0-3 p. p.m.) cured the chlorosis,
and treatment of the soil with copper sulphate to the extent of
0-25-2 lb. of copper sulphate per tree brought about recovery
from the diseased condition. Isaac (1934) also reported the cure
of chlorosis in young peach trees in South Africa by the applica-
tion of copper sulphate to the soil, while manganese sulphate
effected no improvement.

Table VI. Ash, manganese and copper content of leaves from
chlorotic and normal Kelsey plum trees. (Data from
Anderssen)

Ash

Manganese

Copper

per cent of

p. p.m.

p. p.m.

Sample

dry weight

dry weight

dry weight

Top leaves chlorotic

10-56

100

3-2

Top leaves normal

8-85

171

7-3

Middle leaves chlorotic

12-34

14-7

2-9

Middle leaves normal

915

16-6

4-6

Lower leaves chlorotic

14-28

25-5

3-5

Lower leaves normal

10-83

19-5

6-9

Die-back of apple trees ('wither tip' or 'summer die-back') as
a result of copper deficiency has also been reported as occurring
in Western Australia by Dunne (1938), but the earlier sympt^ „
of the disease do not seem to be quite the same as those described
by Anderssen. Thus Dunne makes no reference to rosetting, but
states that first brown spots and then small necrotic areas appear
on the terminal leaves. Eventually the leaves wither and fall,
and then follows the dying back of the shoot. Application of

94 TRACE ELEMENT DEFICIENCY

copper sulphate, either to the soil or by injection, was effective
in bringing about arrest of the disease and recovery of the trees.
The copper content of leaves from healthy trees varied from 5-5
to 12 p. p.m. of the dry weight, whereas in leaves from affected
shoots the content varied from 1 to 3-6 p. p.m. of the dry weight.
These values agree well with those found by Anderssen for the
leaves of Kelsey plum trees, and by Oserkowsky and Thomas
for the leaves of Bartlett pear.

However, Haas and Quayle made many determinations of the
copper content of orange and lemon leaves and fruits, including
samples from normal and exanthematic trees and from affected
trees that had recovered as a result of the application of copper
sulphate, and concluded that so much variation exists in the
copper content of the leaves and fruits of trees from different
localities and sites that it is not possible to decide from a
knowledge of the copper content whether trees are suffering from
a deficiency of that element or not. It may be said, however,
that the values they obtained are of the same order as those
already noted here. Thus the copper content of mature healthy
orange leaves from untreated trees varied from about 7 to
15 p. p.m. of the dry weight, the corresponding values for the
leaves of lemon being between 4 and 13 p.p.m. For two grape-
fruit leaves the values were 6-4 and 7-38 p.p.m. The fruit appears
to contain much less copper calculated on a dry -weight basis, the
content of the element in oranges and lemons ranging respec-
tively from roughly 2 to 4 and 3 to 5 p.p.m. in these fruits.

Reclamation Disease. A disease attributed to copper
deficiency, which affects oats and other cereals, beet and legu-
minous crop plants, occurs on reclaimed heath and moorland
soils in Denmark, Holland and other parts of Europe. In affected
plants the tips of the leaves become chlorotic, and in cereals
this is followed by a failure of the plants to set seed. This disease,
known as reclamation disease or yellow -tip, was originally
attributed to the toxic action of a constituent of peat, but
Sjollema (1933) in Holland showed that the disease could be
cured by the addition of copper sulphate to the soil, and that the
content of copper in wheat, rye and hay grasses was raised by
this soil treatment. Later, Gram (1936) obtained similar results
with barley and oats in Denmark, and Undenas (1937) with oats
in Sweden, while in 1938 Piper found the disease affecting

DISEASES OF PLANTS 95

cereals in South Australia and also found that it could be con-
trolled by the application of copper sulphate to the soil.

Oats were grown by Brandenberg (1933, 1934) in water
culture in which the amount of copper present in the culture
solution was carefully controlled. When copper was excluded,
growth was poor and the plants developed the symptoms of
reclamation disease. With small amounts of copper added to
the solution vegetative growth was more normal, but fruiting
was not unless the concentration of copper in the solution was
at least 0-5 mg. per litre. More recently, Piper (1942) has carried
out water-culture experiments with specially purified reagents
in which the initial copper concentration of the culture solutions
varied from 0 to 3 mg. per litre. In addition to the ordinary
major mineral nutrients the solutions contained small quantities
of boron, zinc, manganese and molybdenum as well as sodium
chloride. The main results were as follows. In the cultures
without copper, 27 days after setting the seeds to germinate,
growth and tillering were noticeably less than in cultures pro-
vided with copper. During the next fortnight these symptoms
became more pronounced, the leaves were a paler green, the tips
of the younger leaves appearing definitely chlorotic, withering
and dying without unrolling, while the base of the leaf con-
tinued to emerge and grow. In subsequently emerging leaves
these symptoms were more pronounced until growth of the
tiller ceased and its death ensued.

In cultures provided with 3/tzg. of copper per litre definite
symptoms of copper deficiency were not observed until 7 1 days
after setting the seeds to germinate, although the plants made
less growth than tho&e with more copper. Slightly older leaves
developed a marginal chlorosis. Ultimately the main tillers
ceased growth and died, and although secondary tillers were
produced these also developed the symptoms already described
and ultimately stopped growing.

When cultures were initially provided with 6^g. of copper per
litre more growth took place before the symptoms of deficiency
developed in the youngest leaves. In these cultures the slightly
older leaves exhibited a loss of turgor so that they appeared limp
and drooping. Secondary tillers developed as before, but all
stopped growing before ear production.

The plants in a culture solution containing initially 10/i,g. of

96 TRACE ELEMENT DEFICIENCY

copper per litre grew normally for 19 weeks before the symptoms
of copper deficiency were evident. Flowering took place but
grain was not formed. When the concentration of copper in the
culture solution was 20 fig. per litre some grain was produced but
a proportion of the spikelets were sterile. In cultures grown in
solutions containing from 50 to 500/xg. of copper per litre growth
was normal. If the concentration of copper was higher than this,
a toxic effect was observed, growth and tillering being depressed
while the leaves became stiff and erect. Although the leaves
were first of a particularly deep green colour the younger leaves
developed a strong chlorosis, which was more marked the higher
the copper concentration of the culture solution.

The effect of copper concentration on the growth of oats is
very clearly demonstrated by Piper's photograph of his cultures
reproduced in Fig. 12.

Piper's work has confirmed the view that reclamation disease
in oats is the result of copper deficiency. Similar experiments by
Piper on other members of the Gramineae, namely, wheat,
Lolium subulatum and Phalaris tuber osa, showed that these also
are affected in the same way by shortage of copper.

5. The Symptoms of Molybdenum Deficiency

The symptoms ascribable to molybdenum deficiency displayed
by tomato plants in water culture as described by Arnon and
Stout (1939) are these. First the lower leaves develop a very
characteristic mottling. This is followed later by necrosis at the
leaf margins along with a characteristic curving ever of the
marginal regions of the leaf. Abscission of flowers takes place so
that no fruit is produced.

The symptoms of molybdenum deficiency of oats grown in
water culture are described by Piper (1940). About the time of
emergence of the panicles necrotic areas appear about midway
along the lamina of the upper leaves, and these areas frequently
extend right across the leaf. With a line of weakness so produced
the leaf bends back sharply; the band is first smooth, but
finally a kink develops and the middle necrotic region of the
leaf dries out a light reddish brown. Although the inflorescence
develops normally the grain consists entirely of empty husks.

So far there is no record of molybdenum deficiency occurring
in plants in the field.

CHAPTER IV

THE FUNCTIONS OF TRACE ELEMENTS

IN PLANTS

Some years ago R. W. Thatcher (1934) published a short
paper in which he put forward a classification of the chemical
elements based on their functions in plant nutrition. In the
first place he pointed out that nearly all the elements which are
known or have been suggested to have a function in plant
nutrition are included in the first four periods or orbits of the
periodic classification. These first four periods, with the atomic
numbers1 of the elements, are shown in Table VII. Of the

Table VII. The first four periods of the periodic classification

Group

Period

I

II

III

IV

V

VI

VII

VIII

0

1

H
1

—

He
2

2

Li

Be

B

C

N

0

F

—

Xe

3

4

o

6

7

8

9

10

3

Na

Mg

Al

Si

P

S

CI

—

A

11

12

13

14

15

16

17

18

4

K

Ca

Sc

Ti

V

Cr

Mn

Fe

Co

Ni

19

20

21

22

23

24

25

26

27

28

Cu

Zn

Ga

Ge

As

Se

Br

—

—

Kr

29

30

31

32

33

34

35

36

elements shown in this table we may dismiss the inert gases of
group O. In the other groups there are a number of elements
which so far have not been found essential for plants, but all
the elements known to be necessary for plants are included in
these first four periods with the one exception of molybdenum
(period 5. group VI, atomic number 42). The heavier metals do
not figure among elements necessary for plants.

Thatcher's classification of the elements in relation to their
functions in the plant was based on the conception that "green
plants are the energy-absorbing and energy-storing agents of

1 The atomic number is the number of free positive charges in the
nucleus of the atom. Except for hydrogen it is very roughly half the
atomic weight.

98

THE FUNCTIONS OF

the cycle of life'. From this point of view he divided the
elements into eight groups as shown in Table VIII.

Table VIII. Thatcher's classification of elements in plants

Group

Type

Function

Elements

I

Energy exchange

H, 0

II

Anion (or acid) group
formers

Energy storers

C, N, S, P

III

Cation (or base) formers
with fixed valency

Translocation
regulators

Na, K, Ca, Mg

IV

Cation (or base) formers
with varying valency

Oxidation-reduction
regulators

Mn, Fe, (Co,
Ni), Cu, Zn

V

Ampholytes with vary-
ing valency

Functions unknown

B, Al, Si, As, Se

VI

Anion (or acid) formers
with fixed valency

Functions unknown

CI, F, (Br, I)

VII

Cation (or base) formers
with varying valency

Perhaps those of
group IV

Co, Ni

VIII

Ampholytes

Functions unknown

Ge, Ga and
other rare
elements

It is not within the scope of the present discussion to con-
sider the value of this scheme of classification, but we may note
that three of the well-established trace elements, manganese,
copper and zinc, are placed, along with iron, itself often classed
as a trace element, in one group, that of oxidation-reduction
regulators. The fourth, boron, is placed in another group, one
with unknown function, that of ampholytes of varying valency.
At the time of publication of Thatcher's paper molybdenum had
not been recognized as a plant trace element; had it been, it
might have been included in the same group as boron. Further,
it is not clear why the ampholytes of Thatcher's group VIII.
which included gallium, were separated from those of his
group V, since in both the functions are listed as unknown.
However this may be, Thatcher's classification divides the
trace elements into two groups, the oxidation -reduction regu-
lators including manganese, copper and zinc, and also iron, the
other including boron of unknown function. With regard to the
oxidation-reduction regulators Thatcher pointed out that if
nickel and cobalt are included the group contains six elements

TRACE ELEMENTS IN PLANTS 99

with consecutive atomic numbers, namely, 25-30. It is inter-
esting to note that gallium, only recently shown to be essential
for any plant, is the next element in the series with an atomic
number of 31. This means that each successive member of the
series differs as regards atomic structure from the element before
it only by the addition of one electron to the proton nucleus.
Whether this close connexion between these elements really has
any significance in respect of their physiological function it
would be premature to say, but it is a fact that several of them
have rather similar chemical properties. Thatcher expressed the
opinion that there was sufficient evidence to justify the opinion
that manganese and iron on the one hand, and copper and zinc
on the other, were pairs of ' mutually co-ordinating catalysts for
oxidation-reduction reactions', the former pair for reactions in
which the addition or removal of oxygen is involved, the latter
for reactions which concern the transference of hydrogen. As we
shall see in the sequel there is a certain amount of experimental
evidence in favour of at least one of these hypotheses.

Frey-Wyssling (1935) has also attempted to find a relation
between essentiality and position of elements in the periodic
table. He uses a table in which group O appears both on the
extreme left and the extreme right, but with each element of the
group one period higher in the right than in the left column. If
a line is then drawn through the table from argon on the left
to carbon and then on to argon on the right, this line passes
through or near the positions in the table of all the essential
elements with the exception of hydrogen. This line Frey-
Wyssling calls the nutrient -line. This relationship expresses with
greater precision the fact pointed out by Thatcher, that all the
essential elements occur in the first four periods of the periodic
table. The further the position of an element is from the nutrient-
line the more toxic it is in general. It will be observed that
essential elements occur in all groups I-VIII.

A very interesting discussion on the relation between the
biological essentiality of the elements and their atomic structure
has been contributed by Steinberg (1938c). When considered
from the point of view of their position in the periodic table
Steinberg concludes that three and no more than three essential
elements are to be found in each group. Where less than three

100 THE FUNCTIONS OF

essential elements are known to exist in a group there is a pre-
sumption that the missing ones are yet to be found. However,
Steinberg considers that correlations between essentiality and
atomic structure can best be shown by tabulating the elements
on the basis of their transition subshell, those in which the
electron numbers have undergone a regular change in the
formation of the elements and which largely determine the
chemical properties of the latter. From such considerations
Steinberg makes certain deductions regarding the relationship
between essentiality and atomic structure and of the possible
essential elements not yet recognized as such. Thus the non-
essentiality of silicon is indicated, while the possibility of
columbium as an essential element is suggested. Steinberg's
conclusions only claim to be very tentative.

That the trace elements are required by plants in such small
quantities strongly suggests that they all function as catalysts
or are at least closely linked up with some catalytic process.
Miss Warington (1923) thought that the function of boron in
the broad bean was nutritive rather than catalytic, and it is
certainly true that the results of boron deficiency are generally
of a distinctly different kind from those resulting from de-
ficiency of manganese, zinc and copper. With boron deficiency,
as we have seen, the most characteristic effect is a breakdown
of thin- walled tissues, especially those of the meristematic
regions, followed by degeneration of the vascular tissues, whereas
with the other three well-established trace elements early
external symptoms are generally localized chloroses, although
these may precede more serious disturbances in growth such as
those which lead, in the case of zinc and copper, to die-back of
the terminal buds of the shoots. Nevertheless, chlorosis may
also be a symptom of boron deficiency, as, for example, in
tobacco (Van Schreven, 1934) and in sugar cane (Martin, 1934),
while plants do not appear to require more boron than man-
ganese. It is not very clear why a catalyst essential for the
maintenance of normal metabolism and growth should not be
regarded as fulfilling a nutritive function.

Manganese, zinc and copper are generally regarded, like iron,
as playing a part in vital oxidations and reductions, and some
writers consider boron to play a similar role. It must be

TRACE ELEMENTS IN PLANTS 101

emphasized, however, that if the parts the various elements play
are similar, they are not identical, for they cannot replace one
another in the organism. Reference has already been made to
Thatcher's view that manganese and iron form a pair of mutually
coordinating catalysts for oxidation-reduction processes in-
volving the addition and removal of oxygen, while zinc and
copper form another pair concerned in similar processes in-
volving the addition and removal of hydrogen.

Since deficiencies of manganese, zinc and copper character-
istically induce chlorosis, it is understandable that they have
been held to be concerned in chlorophyll formation (cf. McHargue.
1923, 1926 a; Bishop, 1928). They have also been thought to act
as catalytic agents in photosynthesis (see e.g. Stoklasa, 1911;
McHargue, 1923; Dufrenoy and Reed, 1934). Recent support
for this view is forthcoming from work by Emerson and Lewis
(1939), who found that when the trace elements of Arnon's
groups A 4 and B7 (see p. 9) were added to the culture medium
of Chlorella pyrenoidosa not only was the rate of growth of the
alga increased but the amount of photosynthesis per unit
quantity of light absorbed was also increased. The trace elements
were added in two groups, those of Arnon's A 4 group + molyb-
denum, now denoted by the symbol A 5, and those of Arnon's
B7 group without molybdenum, this group now being denoted
B6. The addition of the A 5 group was more effective than the
B6 group, and the addition of A5 + B6 more effective than
either alone. It would thus appear that the trace elements play
some part in the photosynthetic process, but what this is can at
present be only a matter of conjecture.

Some observations of Steinberg (1942) may be mentioned here.
Starting from the observation that when nitrogen is supplied
only as nitrate the growth of the fungus Aspergillus niger is
lessened in air deprived of carbon dioxide, he examined the
effect of removal of carbon dioxide on the growth of this fungus
in absence of various micro-nutrients. Under these conditions
omission of zinc, copper or manganese from the culture medium
reduced the growth of the fungus proportionately more when
carbon dioxide was absent than when it was present. Steinberg
concludes that the trace elements probably play a specific part
in the utilization of carbon dioxide by A. niger, and compares

102 THE FUNCTIONS OF

this with the conclusion of Emerson and Lewis on the part they
play in the utilization of carbon dioxide in green plants. In-
cidentally, Steinberg calls attention to the significance of this
similarity in regard to the question of the validity of the sugges-
tion of Ruben and Kamen that carbon dioxide utilization by
micro-organisms is essentially the same process as the dark or
Blackman reaction in photosynthesis.

So far the functions of the trace elements in general have been
considered. With regard to the specific functions of the in-
dividual trace elements little, if anything, of any definite value
can be said regarding copper, molybdenum and the less well-
established trace elements, but contributions of considerable
interest have been made regarding the functions of manganese,
zinc and boron. A consideration of these follows.

Manganese. The view has been very generally held that

manganese is related to oxidation in the plant. Bertrand (1897),

as we have seen, first called attention to the importance of

manganese when he found it essential for the action of the

oxidizing enzyme laccase. Later he maintained that it was

essential for the action of oxidizing enzymes in general. In

recent years Lundegardh (1939) has produced more direct

evidence that manganese is concerned in respiration. Thus he

found that the oxygen intake by wheat roots was increased by

155 to 470 per cent by the addition of 0-00005 M manganese

chloride. Contrasted with this, addition of ferric chloride or

ferric citrate generally brought about a decrease in oxygen

intake, the average reduction with 0-00005 M ferric citrate being

about 21 per cent. From these observations it is concluded that

manganese, but not iron, catalyses aerobic respiration.

About the same time Burstrom (1939) examined the assimila-
tion of nitrate both by whole wheat roots and by wheat-root
pulp in presence of small quantities of iron and manganese, and
came to the conclusion that, whereas without iron and man-
ganese nitrogen assimilation does not take place, in presence of
a small concentration of manganese assimilation takes place
both with whole roots and root pulp, the optimum effect being
produced with about 4 nig. manganese per litre with whole
roots and about 12-3 mg. per litre with pulp. Without added
manganese the addition of iron brought about feeble nitrogen

TRACE ELEMENTS IN PLANTS 103

assimilation of whole roots but not of pulp, and even this
assimilation by whole roots is ascribed by Burstrom to its effect
on respiration and ion uptake. His general conclusion is there-
fore that manganese, and not iron, directly catalyses nitrate
assimilation.

It will be observed that the observations of both Lundegardh
and Burstrom emphasize the contrasting effects of manganese
and iron. A number of other workers have called attention to
this, and indeed there appears to be considerable reason to
suppose that the function of manganese is to be found in its
relation to the oxidation and reduction effected by iron salts.
Thus Hopkins (1930), from observations on the growth of the
unicellular green alga Chlorella, held that manganese brings
about the reoxidation of iron after its reduction in the plant to
the ferrous state ; if the amount of manganese in the plant is
deficient there results too high a proportion of ferrous iron,
while if manganese is present in excess the reduction of ferric
iron is prevented and the concentration of ferric iron is too high.
In either condition the oxidation-reduction processes of the cell
involving iron are disturbed.

On this view it is to be expected that the ratio of manganese
to iron in the plant is of more importance than the absolute con-
centration of manganese. In this connexion it may be noted that
Bertrand (1912 b, c) found the ratio of manganese to iron + zinc
determined the development of conidia in Aspergillus niger, the
capacity to produce these reproductive bodies being inhibited
if the ratio Mn/Fe + Zn was too low. With higher plants the im-
portance of the ratio of manganese to iron has been emphasized
by a number of workers. Thus Pugliese (1913) and also Totting -
ham and Beck (1916) write of an antagonism between iron and
manganese in the growth of wheat, and the former gives the
optimum ratio of the two elements in the culture solution as
1/2-5. Scharrer and Schropp (1934) found that with maize in
water culture the growth of the roots was at a maximum when
the ratio of Mn/Fe in the culture solution was 1/7. That chlorosis
might be induced by manganese when iron is deficient was
reported by Gile in 1916 in the pineapple and again by Scholz
in 1934 in the blue lupin.

The relation between manganese and iron in the plant has

104 THE FUNCTIONS OF

more recently been dealt with by Shive (1941). This writer calls
attention to two important points in this connexion, the first
being that the active functional iron in the tissues is in the
ferrous state, the second that the oxidizing potential of man-
ganese is higher than that of iron. Shive holds that if iron is
absorbed in the ferric state much of it is reduced in the plant by
powerful reducing systems unless this is prevented by a counter-
reactant. The manganese functions as such a counter-reactant,
oxidizing ferrous to ferric iron which is precipitated, probably
in organic complexes. Hence, if manganese is deficient in the
plant, there will be an excess of active ferrous iron which induces
chlorosis, a chlorosis due to iron toxicity. On the other hand,
if the concentration of manganese is high the concentration of
active ferrous iron is low, and if too low a chlorosis due to
iron shortage will result. Thus it is necessary for healthy growth
that the proportion of iron to manganese should lie within
certain limits, and Shive concluded that, for the species in-
vestigated by him, the ratio of active iron to active (soluble)
manganese in the plant should lie between 1-5 and 2-5. This
conclusion was derived from the results of a series of culture
experiments with soya bean described by Somers and Shive
(1942) in which the quantities of both iron and manganese in
the culture solutions were varied. They used in all eighteen
different combinations of iron and manganese in their culture
solutions. The iron content varied from 0-005 to 3-00 p. p.m. and
the manganese content from 0 to 5-00 p. p.m.; the various
combinations are shown in Table IX. Actually complete absence
of manganese is never attained in the culture itself since there
will be some present in the seed used and no doubt a little will
also be introduced as impurity either from culture vessels or
other nutrient salts used. The culture solutions also contained
the usual major nutrients and boron. The concentrations of iron
and manganese and a pK of 4-6-4-8 (to prevent precipitation of
iron) were maintained approximately constant by the use of a
technique in which a continuous flow of solution passed through
the culture vessels, and the solutions were completely changed
every other day.

Approximate determinations of both soluble and insoluble
iron and manganese in the tissues were also made. To separate

TRACE ELEMENTS IN PLANTS 105

the soluble and insoluble fractions the fresh material was frozen
by means of an ice-salt mixture and from this, in thawing, the
juice was expressed under a pressure of 1600 lb. per sq. in. applied
for 2J min. The expressed juice together with washings of the
press cake and muslin containing it were taken as containing
the soluble iron and manganese, the press cake the insoluble
fraction.

A study of Table IX shows at once how the dry weight and
condition of the plants is related to the Fe/Mn ratio and not to
the absolute concentrations of these nutrients. Thus plants
growing in solutions containing 0-002, 0-250 and 2-00 p.p.m.
manganese respectively were all normal and possessed about the
same dry weight provided that in each case the ratio of soluble
iron to soluble manganese in the leaves was within the range
1-5-2-5. Whenever the ratio was outside this range pathological
symptoms tended to develop.

If the ratio were above 2-5 the symptoms were of one kind, if
the ratio were below 1-5 the symptoms were of a different kind.
The former were thus those of a too high Fe/Mn ratio, the latter
of a too low Fe/Mn ratio. The first could be described either as
manganese deficiency or iron excess, the second either as iron
deficiency or manganese excess. The first sign of a too high
Fe/Mn ratio was a fading of the green colour of the lower leaves
which later developed into an intervenal yellowing on the basal
part of the leaves. Next the upper leaves showed a fading of the
green colour in the intervenal areas followed by the development
of small brown necrotic spots. Finally, the new leaves opened
with the necrotic areas already present, and these leaves might
fail to develop and fall, also stem apices might die. Roots showed
no visible symptoms apart from being smaller than those of
normal plants.

The symptoms of a too low Fe/Mn ratio (iron deficiency,
manganese excess) were quite distinct from those just described.
The first sign was a slight brown discoloration of the roots
followed by yellowing and slight curling of the upper leaves.
As the condition developed the discoloration of the roots and
chlorosis of the upper leaves continued until the newer leaves
were almost white. The leaves curled downwards and sometimes
the midribs darkened and their tissue broke down. Large

106

THE FUNCTIONS OF

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TRACE ELEMENTS IN PLANTS 107

necrotic areas developed in the most chlorotic leaves, this being
accompanied by death of the stem apices.

In both it was possible to correct the pathological symptoms
by altering either the iron or manganese concentration in such
a way as to bring the Fe/Mn ratio to a value between 1-5
and 2-5. A further indication of the importance of the Fe/Mn
ratio was obtained by Somers, Gilbert and Shive (1942) in the
respiration rate of soya beans in water cultures supplied with
different proportions of the two nutrients. Respiration rates
were always definitely lower when the Fe/Mn ratio was outside
the range 1-5-2-5 than when it was within it.

In further support of the oxidation-reduction hypothesis out-
lined above, Somers and Shive mention a series of tests carried
out with maize seedlings in which cobalt was substituted for
manganese. The oxidation potential of cobalt is higher than
that of manganese, so it should, on the hypothesis, have a
greater tendency than manganese to lessen the metabolic
efficiency of iron by effecting the oxidation of the latter to
the insoluble ferric state, and this, Somers and Shive state,
was so.

The work of Shive and his associates constitutes a very strong
argument in favour of the hypothesis they present regarding
the connexion between iron and manganese. An extension of
such experiments to other species is clearly very desirable. If
the connexion, which means that manganese deficiency is the
same thing as iron excess and vice versa, should prove to be
general, it must be admitted that it has not so far been realized
by those workers who have had much experience with the effect
of mineral deficiencies in the field. It would, however, afford an
explanation of why a characteristic manganese deficiency has
been observed in plants containing much manganese, while
in others the symptoms of this deficiency have not appeared
when the manganese content has been exceptionally low
(cf. p. 59).

In a study of cation absorption by tobacco, Swanback (1939)
made some observations on the effect of manganese on the
absorption of potassium and calcium. The plants were grown in
nutrient solutions containing calcium in three different con-
centrations. In the absence of manganese the symptoms of

108 THE FUNCTIONS OF

deficiency of this element were most pronounced in the plants
grown in the solutions containing the highest concentration of
calcium (0-403 g. per litre), were less with a medium calcium
concentration (0-143 g. per litre) and were not observed with low
calcium content (0-042 g. per litre). The effects with potassium
were in the reverse order, the symptoms of manganese deficiency
being most pronounced with low potassium supply (0-026 g. per
litre), less with medium potassium supply (0-082 g. per litre),
and not noticeable in the plants supplied with the highest con-
centration of potassium (0-26 g. per litre). These results are
interpreted as suggesting that there is an antagonism between
calcium and manganese in their absorption, while there is none
between potassium and manganese. Other observations by
Swanback support this suggestion. Thus with a low supply of
calcium the dry matter produced by the tobacco plant cultures
was 6 times as much when manganese was not supplied as when
0-0054 millimol. per litre of this element was supplied to the
culture solution. With a high supply of calcium the reverse
resulted, the dry matter of the plants provided with manganese
being 3-5 times that of the plants not supplied with it. These
results are explained on the view that with low calcium supply
the manganese retards the absorption and utilization of calcium,
shortage of which results in the small amount of dry matter
produced, while with high calcium supply the antagonistic effect
of the manganese is insufficient to reduce the absorption and
utilization of the calcium to a low level while the favourable
effect of the manganese itself brings about an increase in growth
and so of dry matter produced.

That calcium antagonizes the absorption and utilization of
manganese while potassium is indifferent is shown by deter-
minations of the manganese content of shoots and roots of
tobacco plants grown in the solutions containing the various
concentrations of calcium and potassium already mentioned and
0-0054 millimol. of manganese per litre. The- results are shown in
the following table. The values in the fourth and fifth columns
of the table are of what Swanback calls the 'translocation
quotient'. This is the ratio of the manganese content of the
shoot to that of the root and is supposed to give a measure of the
mobility or relative translocation of the manganese. They show

TRACE ELEMENTS IN PLANTS

109

clearly the effect of calcium in reducing both the intake of man-
ganese and its translocation to the leaves, while no such effect
is suggested as regards potassium.

Table X. Effect of varying concentrations of calcium and
potassium on the absorption of manganese by tobacco
plants in water culture. (From Swanback)

Plant

Manganese content

in millimol. x 10-3

per g. after

Manganese
translocation
quotient after

Treatment

t

45 days

60 days

45 days

60 days

Low calcium

Shoot
Root

0-74

0-82

0-75
0-60

0-91

1-25

Medium calcium

Shoot
Root

0-25
0-36

0-36
5-00

0-70

007

High calcium

Shoot
Root

016
0-26

0-33
5-60

0-61

0-06

Low potassium

Shoot
Root

0-22
0-51

0-20
210

0-43

010

Medium potassium

Shoot
Root

0-25
0-36

0-36
5-00

0-70

0-07

High potassium

Shoot
Root

0-30
0-49

0-28
3-20

0-61

009

Zinc. It has already been mentioned (p. 99) that Thatcher
thought that zinc and copper were a pair of catalysts concerned
together in oxidation-reduction reactions. That zinc, at any
rate, is concerned in such reactions is the conclusion reached by
Reed and Dufrenoy (1935) mainly as a result of their micro-
scopical examination of mottled leaves of Citrus. They conclude
that zinc is concerned with the functioning of sulphydryl com-
pounds such as cysteine in their regulation of the oxidation-
reduction potential within the cells. We have already noted that
they found that the stromata of the chloroplasts in the palisade
cells of such leaves are often rich in fat, while the vacuoles con-
tain phenolic material and phytosterol or lecithin, which are
absent from normal leaves. Reed and Dufrenoy interpret these
substances as suboxidized products of carbohydrates and
proteins, and their presence suggests a disturbance in the
oxidation-reduction potential within the leaf cells.

For the view that the maintenance of the oxidation-reduction
potential at its normal level depends on sulphydryl compounds

110 THE FUNCTIONS OF

the following arguments are advanced. First, Hopkins and
others have shown that such compounds appear to be present in
all living cells and may control oxidation and reduction processes.
Secondly, the oxidation of cysteine to cystine is catalysed by
metals.1 Thirdly, Giroud and Bulliard have shown that zinc
has a specific effect in stabilizing the nitroprusside colour
reaction of the sulphydryl (SH) group.2 Reed and Dufrenoy
also refer to Maze's finding that roots of maize grown in a culture
solution deficient in, though not completely free from, zinc,
contain sulphides in the ash, indicating that the sulphur meta-
bolism of the plant was adversely affected.

Chandler (1937) has pointed out that zinc deficiency has its
most serious effects in plants where carbohydrates have accumu-
lated, and he suggests that zinc deficiency brings about in-
hibition of some process of carbohydrate transformation. This,
as Reed (1938) suggests, may mean that zinc catalyses oxidation
processes which in its absence may run the other way.

Following on their earlier work, Reed and Dufrenoy (1942)
have made a cytological study of catechol aggregates which
arise in the vacuoles as a result of zinc deficiency. They consider
that they form by a process which they call coacervation, in
which disperse phase particles of the colloidal system con-
stituting the vacuole become aggregated into spherical masses.
This process is regarded as something more than a separation
of phases, as the aggregates become surrounded by a precipita-
tion membrane composed of orientated molecules of a phospho-
lipoid. Tests for oxidase show (Dufrenoy and Reed, 1942) that
these aggregates are not only centres of catechol derivatives,
but also for catechol oxidase activity.

1 It is stated, however (cf. Meldrum, 1934), that the most active
metals are iron, copper and manganese.

2 This test, due to Morner, consists in adding a 5 or 10 per cent solu-
tion of sodium nitroprusside rendered alkaline with ammonia to the
liquid to be tested, and shaking. A violet colour, which soon fades, is
produced if a cysteine peptide such as glutathione is present. Some other
substances, including creatinine and acetone, give somewhat similar
colours. According to Giroud and Bulliard the addition of salts of zinc
gives a red colour much more stable than the violet colour of the reaction
as usually produced. It appears to be quite specific for the sulphydryl
group and is not given by either creatinine or acetone.

TRACE ELEMENTS IN PLANTS 111

Reed and Dufrenoy conclude that the vacuolar sap contains
both oxidizable phenolic compounds and catechol oxidase cap-
able of catalysing the oxidation of these compounds. Normally
this oxidation is prevented owing to the presence of hydrogen
donators which may include the ascorbic-dehydroascorbic acid
system, dihydroxymaleic acid, cysteine and glutathione. During
the earlier part of the growing season the relatively high con-
centration of hydrogen donators in the cell protects the catechol
compounds from oxidation and they remain dispersed through-
out the vacuole. With the approach of senescence, or with
nutrient deficiency such as a shortage of zinc, the oxidation-
reduction equilibrium is disturbed and coacervation results, the
process, according to Reed and Dufrenoy, being a 'simple
consequence of a gradient in the distribution of cations and
correlative distribution of polyphenol oxidase'. They further
suppose that a difference in electrical potentials will exist
between the aggregations and the surrounding medium, since
the former are foci for catechol oxidase, and that there will
therefore be a tendency for cations to move into the coacervate,
and this in turn will greatly influence the intake of dissolved
material by the cell and consequent derangement of meta-
bolism.

A somewhat more precise suggestion of the way in which zinc,
through its effect on oxidation-reduction systems, may affect
growth, has been put forward by Skoog (1940). Experiments
made and described by this worker on tomatoes and sunflower
grown in zinc-deficient culture solutions indicate a connexion
between zinc and the growth-promoting substance auxin.
Terminal buds and stems of such zinc-deficient plants appeared
to contain no auxin or only a trace of it. Appreciable amounts
were, however, found to be present in the leaves, although less
than in leaves of control plants provided with an adequate supply
of zinc. The visible symptoms of zinc deficiency only appear
after the decrease in auxin content, and if plants in an extreme
state of zinc deficiency are supplied with zinc, the auxin content
of these plants increases considerably in one to a few days, while
growth is resumed after the passage of several more days. These
observations suggest that zinc is necessary for the maintenance
of a normal auxin content.

,

112 THE FUNCTIONS OF

Skoog also placed sections of stems from zinc-deficient and
control plants, from which auxin had previously been removed,
on agar blocks containing a known concentration of indole-3-
acetic acid, and found that always more of this growth hormone
was inactivated in the blocks in contact with tissue from zinc-
deficient plants than in the blocks in contact with control tissue.
This suggests that deficiency of zinc brings about excessive
destruction of auxin, an effect attributed to an increased oxida-
tive activity of the tissues, since these displayed increased
capacity to oxidize benzidine in presence of hydrogen peroxide.
That zinc functions as a catalyst in relation to oxidation-
reduction processes in the cell is thus again indicated, while its
relationship to auxin maintenance suggests why zinc deficiency
may lead to retardation or cessation of growth.

As will be mentioned later, zinc enters into the composition
of the molecule of the enzyme carbonic anhydrase, which cat-
alyses the action H2C03^C02 + H20. As far as the writer is
aware this enzyme has so far only been found in animals, but
if it should occur in plants, it would suggest that zinc might be
concerned in the excretion of carbon dioxide and possibly in
other processes about which, in the absence of any information,
it would be idle to speculate. It may, however, be pointed out
that deficiency of zinc would then mean a shortage of the enzyme
catalysing the release of carbon dioxide so that this end product
of the oxidation of carbohydrate would accumulate as carbonic
acid in the tissues. This would lead to a slowing down of the
oxidation process and conceivably to just such an interference
in the normal oxidation mechanism in the tissues as Reed and
Dufrenoy hypothesize.

Copper. Precise information regarding the functions of
copper in the plant is forthcoming on one point: it has been
found that this metal enters into the composition of the oxidizing
enzyme or enzymes known as catechol oxidase or polyphenol
oxidase. This was shown by Kubowitz (1937) for the oxidase
present in potato tuber and by Keilin and Mann (1938) for the
similar oxidase present in the cultivated mushroom (Agaricvs
campestris). The enzyme, in fact, appears to be a copper-
protein compound containing not less than 0-30 per cent of
copper.

TRACE ELEMENTS IN PLANTS 113

Catechol or polyphenol oxidase catalyses the oxidation of
compounds with the orthodihydroxyphenol grouping such as
catechol and pyrogallol. In the presence of the enzyme and a
low concentration of catechol a number of other substances such
as guaiacum, benzidine and ascorbic acid are also oxidized
which are not oxidized by the pure enzyme alone. These further
oxidations may require the presence of some substance such as
orthoquinone, produced by the oxidation of catechol in the
primary reaction, so the presence in plant tissues of a substance
either of the catechol or orthoquinone type along with the
oxidase would make possible the oxidation of a wide range of
phenolic compounds.

Enzymes of this type are widely distributed in plants, but
whether they are all copper compounds it is not yet possible to
say. That the enzymes from plants so different from one another
as the potato and mushroom both contain copper suggests that
this is a possibility, and if this should prove to be so, the func-
tion of copper as a catalyst in vital oxidations can be regarded
as established.

Reference has already been made to Thatcher's opinion that
zinc and copper form a pair of mutually co-ordinating catalysts
for oxidation-reduction reactions. It is therefore interesting to.
find that Reed and Dufrenoy connect the action of zinc in plants
with the distribution of the copper-containing polyphenol
oxidase.

Boron. Various functions have been ascribed to boron in
higher plants. The effects attributed to the action of this element
include an influence on the water relations of the protoplasm,
a favourable influence on the absorption of cations and a
retarding influence on the absorption of anions, a favourable
influence on the absorption of calcium, a part in the formation
of pectic substances in the cell wall, and an essential part in
carbohydrate and nitrogen metabolism.

As regards the supposed effect of boron on the water relations
of the protoplasm, it has already been pointed out that a very
general symptom of boron deficiency, and often one of the
earliest internal symptoms, is an enlargement of thin-walled
cells. In this connexion some observations made by Schmucker
(1933. 1935) on the germination of the pollen grains of tropical

114 THE FUNCTIONS OF

water lilies and other species in presence and absence of boron
are of interest. He found that when the pollen was placed in a
drop of nectar from the flower, germination was normal, but
when placed in a drop of sugar solution of the same concentra-
tion, the grains either failed to germinate or the pollen tubes
produced quickly burst. But the pollen tube remained intact
and its growth continued if the sugar solution contained 0-001
or 001 per cent of boric acid. From such observations Schmucker
not only concluded that boron influenced the absorption of
water by the protoplasm but that along with sugar it played some
part in the formation of pectin in the cell wall. With regard to
the latter supposition we have already noted that one of the
most general features of boron deficiency is the breakdown of
parenchymatous and other thin cell walls, especially those of
the apical meristems in which pectic substances are, by some,
supposed to predominate owing to the relative importance of
the middle lamella in such cell walls. Other features of boron
deficiency, such as discoloration of cell walls and the brittleness
of petioles and leaf laminae, might also be held to support this
view. On the other hand, Dennis (1937) has pointed out that
discoloration of the cell wall is not confined to cases of boron
deficiency, and he suggests that the effect of boron deficiency
on the cell wall may be part of a more far-reaching effect of this
deficiency on carbohydrate metabolism.

The view that boron as boric acid increases the intake of
cations and decreases that of anions was put forward by Rehm
(1937) as a result of experiments with Impatiens balsamina
grown in water culture. The cultures were provided either with
single salts or complete nutrient solutions with and without
boron. The intake of the different ions was determined by
analysis of the solution with the results shown in Tables XI
and XII.

These data certainly indicate an increase of cation absorption
and a relative or absolute decrease of anion absorption as a
result of the presence of boron. At present it would seem to be
extremely doubtful whether such an effect of boron can be
accepted as a general one.

There is, however, much more evidence of a relation between
boron and calcium. In a series of water-culture experiments

TRACE ELEMENTS IN PLANTS

115

with Vicia Faba, Brenchley and Warington (1927) obtained
results that suggested an association of boron with the absorp-
tion or utilization of calcium. Cultures without boron and with
a very small supply of calcium as calcium sulphate (0-025 g. per
litre) made poor growth and developed very small blackened

Table XI. Absorption by Impatiens balsamina of various ions
from solutions of single salts in presence and absence of
boron. (Data from Rehm)

Concentra-

tion of

Ratio of uptake of

boron, when

ions in

presence and

present, in

in absence of boron

mg. per

t

A y

Salt

Concentration

litre

Cation

Anion

K2S04

0001 N

0-5

1-378

105

KC1

0001 N

0-5

1-718

107

KH2P04

0002 N

0-5

20-82

14-56

KH2P04

0002iV

10

95-00

2710

CaCl2

0-002iV

0-5

1013

0-789

MgS04

0-002 N

0-5

3022

1-244

MgS04

0002 N

10

3055

1-065

NH4N03

0002 N

1

1-038

0-872

Table XII. Absorption by Impatiens balsamina of various
ions from solutions containing the major plant nutrients
with and without boron. (Data from Rehm)

Ratio of absorption of ion with

boron present (0-5. mg. litre)
to absorption with boron absent

<

N supplied

N

supplied

Ion

as Ca(NO)2

as

NH4N03

NH4

—

1-61

K

1-215

4-46

Ca

1-24

109

Mg

1045

103

N03

1-834

105

CI

0-788

1127

so4

0-721

1002

H.PO,

0-74 -

01 7

leaves, finally turning black at the stem apices and withering
backwards from these. It will be noted that these are the
symptoms of calcium shortage and not of boron deficiency (see
p. 80). The blackening is attributed to the toxic effect of other
nutrient salts, a toxicity which is normally counteracted by
calcium, and the plants probably died before even the calcium
in the seed was used up. In cultures containing the same supply

116 THE FUNCTIONS OF

of calcium, but also containing boric acid, development was
very much better, and, although there was some blackening of
the leaves, the plants grew fairly tall, the stems did not blacken
and normal flower buds developed. Brenchley and Warington
interpreted these results as indicating that without boron the
plants were unable to absorb or utilize sufficient calcium to
prevent poisoning by the other nutrient salts, while when boron
is present the latter enables the plant either to absorb calcium
more rapidly or to utilize it more readily so that the toxic
effect of the other nutrients is antagonized. It may be noted
that in cultures without boron, as the supply of calcium is in-
creased the symptoms of calcium shortage become less marked
until with 0-1 g. of calcium sulphate per litre they disappear
and the plants show typical symptoms of boron deficiency.

Later, Warington (1934), by actual determination of the
amount of calcium absorbed by plants growing in culture solu-
tions, showed that the presence of boron does indeed result in
a very considerable increase in the amount of calcium absorbed
by plants of Vicia Faba. The actual values she obtained are
summarized in Table XIII.

Results comparable with those of Miss Warington have been
obtained with soya bean by Minarik and Shive (1939). Plants
were grown in sand cultures supplied with the usual major
nutrients and manganese. Boron was supplied to the different
cultures in concentrations varying from 0 to 10 p. p.m. As the
results summarized in Table XIV show, the boron supply
definitely influenced the amount of calcium which accumulated
in the leaves, and that indeed the effect of boron on the growth
of the plants was parallel with its effect on calcium uptake.

But this effect of boron in influencing calcium intake does not
appear to be general. Indeed, Holley and Dulin (1937), working
with cotton, could find no indication of a relationship between
boron and calcium, and Morris (1938) found no difference in
calcium content in normal and boron-deficient oranges, while
Talibli (1935) actually found that addition of boron to the
medium on which flax was growing brought about a reduction
in the calcium content of the flax straw.

Work by Marsh and Shive (1941) on maize appeared to throw
some light on the apparent divergence between the results

TRACE ELEMENTS IN PLANTS 117

Table XIII. Absorption of calcium by Vicia Faba from
nutrient solutions with and without boron. (Data from
Warington)

Age of Total calcium absorbed mg. per plant

plant in <— A ■ s

weeks With boron Without boron

1930 series: Solutions not renewed

2 2-2 2-6

3 8-3 51

4 151 71

5 251 6-6

1931 series: Solutions not renewed

2 2-2 2-2

3 6-2 3-5

4 13-8 5-5

5 171 GO

Solutions renewed fortnightly

5 17-8 101

9 50-9 15-6

Solution renewed weekly
9 54-83 26-65

Table XIV. Absorption of calcium by Glycine hispida from
media containing various amounts of boron. (Data from
Minarik and Shive)

Average fresh Calcium in

Concentration of

weight of leaves

leaves m

boron in medium

per plant

per g. of fi

p.p.m.

g-

weight

0-0

4-7

2-6

0-001

110

2-5

0-0025

15-8

2-6

0-005

16-2

2-9

0-010

240

30

0-025

32-8

3-5

005

38-7

4-5

0-1

32-6

40

0-25

28-4

3-9

0-5

28-1

3-4

10

31-5

3-5

2-5

13-6

4-2

5-0

12-8

3-6

10-0

0-8

20

obtained up to that time with these various species. Plants were
grown in sand cultures and for the first week were supplied with
a nutrient solution containing all necessary elements except
boron, this being omitted so that any in the seed or in external
sources should be exhausted. During the second week all the

118 THE FUNCTIONS OF

cultures were supplied with a complete culture solution which
included 0-25 p. p.m. of boron as boric acid. From the beginning
of the third week, two series of cultures receiving no calcium and
four supplied with a nutrient solution containing 170 p. p.m. of
calcium, received different amounts of boron. These treatments
were continued for 10 days, at the end of which time the dry
weights of the shoots, the total calcium and boron content and
the contents of soluble calcium and soluble boron of the shoots
were determined. The various treatments and the results obtained
from them are indicated in Table XV.

Table XV. Calcium and boron content of maize supplied with
different amounts of boron. (Data from Marsh and Shive)

Concen-
tration Dry weight Total Ca Soluble Ca Total B Soluble B
of boron of shoot per g. of per g. of per g. of per g. of
supplied per plant dry matter dry matter dry matter dry matter
p.p.m. g. mg. mg. mg. mg.

No calcium supplied in nutrient solution
0-0 1-50 30 0-3 0-001 00005

0-25 2-60 30 1-0 0-008 00069

170 p.p.m. calcium supplied in nutrient solution

00 2-85 7-6 2-1 0-002 00015

0-1 5-40 7-7 2-4 0-005 0-0042

0-25 5-30 80 2-8 0-008 0-0070

50 4-37 7-7 4-2 0-025 00232

Inspection of these results shows that the total calcium content
of the shoots is independent of the amount of boron supplied ;
on the other hand, the soluble calcium content runs parallel
with both the soluble boron content and the total boron content
of the plant and also with the boron content of the medium. It
is therefore concluded that the soluble calcium content is deter-
mined by the boron content, a large proportion of which is in
a soluble form, and which is itself determined by the boron
content of the medium.

There is thus, according to Shive (1941), a difference between
dicotyledons as exemplified by Vicia Faba (and presumably
Glycine hispid a) and monocotyledons as exemplified by Zea mais,
in that in the former the calcium and boron contents are gener-
ally much higher than in the latter, but in the dicotyledons
studied only a small fraction of the boron is soluble, whereas in

TRACE ELEMENTS IN PLANTS 119

monocotyledons practically all the boron remains in solution.
In both groups the soluble calcium is directly related to the
soluble boron which is itself determined by the total boron,
which in its turn is determined by the concentration of the boron
in the medium. This much smaller proportion of soluble boron
in dicotyledons explains why the boron requirement of these
plants is so much higher (5-10 times) than that of mono-
cotyledons.

That this explanation is not of general applicability, however,
appears from recent work on tomato by Reeve and Shive (1944),
who investigated the relations of boron to potassium and calcium
in this species. In their experiments on the relation of boron to
potassium twenty cultures, each containing three plants, were
grown in sand cultures which received a nutrient solution
supplied in a continuous flow. Five different potassium con-
centrations were used, namely, 10, 50, 89, 250 and 500 p.p.m.,
there being thus four cultures receiving potassium in each one
of these concentrations. The four cultures at each potassium
level respectively received boron in the concentrations 0-001,
0-1, 0-5 and 5-0 p.p.m. The nutrient solution contained iron,
manganese and zinc in addition to the major nutrients.

It was found that symptoms of boron deficiency appeared
first in the culture supplied with 0-001 p.p.m. of boron and
500 p.p.m. of potassium, and last in the culture supplied with
0-001 p.p.m. of boron and 10 p.p.m. of potassium and that,
in general, the severity of the symptoms of boron deficiency
increased with increase in the potassium concentration. No
symptoms, either of boron deficiency or excess, were observed
in any of the cultures receiving the intermediate concentrations
of boron (0-1 and 0-5 p.p.m.), butull cultures receiving 5 p.p.m.
of boron developed symptoms of boron toxicity. With these, the
severity of the symptoms increased with increase in potassium
concentration. Thus, increasing potassium concentration brings
about a progressive increase in the severity of the symptoms of
boron deficiency in low boron concentrations and of the
symptoms of boron excess in high boron concentrations.

Analysis of the plants showed that at each level of supplied
boron the amount of both soluble and total boron in the
tissues increases with increase in the potassium concentration.

120 THE FUNCTIONS OF

This explains the accentuation at the high boron level of
the symptoms of boron toxicity with increasing potassium
concentration, but does not explain why the boron deficiency
symptoms at the low boron level should be accentuated with
increase in the potassium supply.

In their experiments on the relation of calcium to boron thirty
cultures were used in which six levels of calcium supply (5, 10,
50, 100, 250 and 500 p.p.m.) and five of boron (0-001, 0-01, 0-5,
5-0 and 10-0 p.p.m.) were employed. The results show that
calcium acts similarly to potassium in that with the lowest
boron concentration (0-001 p.p.m.) the severity of the symptoms
of boron deficiency increases with increase of the supply of
calcium, the effect of the latter in this respect being, indeed,
greater than that of potassium. In its influence on boron toxi-
city produced by the highest boron concentration (10 p.p.m.),
however, calcium acts in exactly the opposite way to potassium,
for with progressively increasing calcium supply the severity of
the symptoms of boron toxicity becomes less. Chemical analyses
of the plants show that with the lower concentrations of boron
in the nutrient solution (0-001, 0-01 and 0-5 p.p.m.) the boron
content (both total and soluble) is independent of the calcium
concentration of the nutrient solution, but with the higher con-
centrations of boron in the nutrient solution (5-0 and 10-0 p.p.m.)
increase in the concentration of calcium brought about a decrease
of both total and soluble boron in the plants. This accounts
for the effect of increasing concentration of calcium in reducing
the toxicity of boron when supplied in high concentration. It
may be noted that, in contrast to the earlier findings of Miss
Warington with broad bean and of Minarik and Shive with soya
bean, boron appears to have no effect on the absorption of
calcium by tomato, and there does not appear to be any relation
even between soluble boron and soluble calcium such as Marsh
and Shive found in maize. However, the ratio of calcium to
boron in the plant is influenced by the supply of potassium,
increase of this cation in the nutrient solution bringing about a
lowering of the calcium/boron ratio. Calcium appears to have
no significant influence on the potassium/boron ratio.

The results obtained by Reeve and Shive are in harmony with
the well-known fact that heavy liming of certain soils will

TRACE ELEMENTS IN PLANTS 121

induce boron deficiency in a number of crop plants such as beet
and swede. It will also be observed that if a soil contains
sufficient boron to induce toxicity symptoms, heavy liming
should reduce the severity of these. This was found to be so
with oats by Jones and Scarseth (1944). These workers grew
lucerne, oats and tobacco in pots of limed and unlimed soils
to which various quantities of borax were added. The calcium
and boron in the plants were determined. As a result the con-
clusion was drawn that a plant will only make normal growth
when there is a certain balance between the intake of calcium
and that of boron. From a consideration of their own data and
those obtained by Cook and Miller (1939) for sugar beet, Muhr
(1940) for soya bean and Drake, Sieling and Scarseth (1941) for
tobacco, they come to the conclusion that the ideal balance for
these various plants is attained when the ratios (in equivalents)
of calcium to boron in the respective plants are 100 for sugar
beet, 500 for soya bean and 1200 for tobacco.

While, then, there is a quite considerable amount of evidence
of a relationship between boron and calcium, and also between
boron and potassium, in plant nutrition, the results obtained
with different species are so different that no generalization as
to the nature of this relationship appears possible at present.

Microchemical tests made by Marsh and Shive on the apical
meristem of maize plants supplied with different quantities of
boron suggested a relation between boron and the pectin and
fat contents of these tissues. Thus with a supply of 170 p.p.m.
of calcium without boron tests for pectin in the cytoplasm were
positive and for fats negative, but with 5 p.p.m. of boron tests
for pectin in the cytoplasm were negative and for fats positive.
With an intermediate supply of boron (01 and 0-25 p.p.m.)
which was found optimal for growth (cf. Table XV) tests for
both pectin and fat were positive. It is suggested therefore that
boron plays some part in carbohydrate and fat metabolism.

Swanback (1939), to whose work on absorption of cations by
tobacco reference has already been made, concluded from
analyses of tobacco plants supplied with calcium and potassium
at different levels with and without boron, that the latter
element aids the absorption and utilization of calcium.

That boron is connected with carbohydrate metabolism was

122 THE FUNCTIONS OF

indicated in the work of Johnston and Dore (1929), who found
that in tomato plants suffering from boron deficiency there was
a marked accumulation of sugars in the leaves and a corre-
sponding reduction of the sugar content of the stems, indicating
some considerable reduction below normal in the translocation
of carbohydrates.

The work of Wadleigh and Shive (1939) on cotton seedlings
is also important in this connexion. They examined the effects
of boron deficiency in the seedlings by means of microchemical
tests. For this purpose seedlings were grown in water cultures
with and without a supply of boron. The first internal symptom
observed was the increased acidity of a few cells scattered
through the pith and cortex, the pH of these cells being from 3-8
to 4-4 as compared with the normal value of 5-8 to 6-4. As boron
deficiency increased so did the number of these abnormally acid
cells, which then also appeared in the pericycle and the older
xylem parenchyma. When the majority of the cells of the pith
had become very acid their cell walls began to break down, at
the same time developing a deep brown colour. Next, some of
the cells of the phloem and younger xylem parenchyma developed
high acidity and ultimately a breakdown of cells occurred in
these regions also.

While these changes were proceeding accumulation of sugars
was observed in the boron-deficient plants, while starch was
abnormally abundant in the endodermis. In the cells of the
stem apices the nitrate-nitrogen content was much lower in
boron-deficient than in normal plants. This was attributed to
failure of nitrate absorption owing to death of the root apices.
There was a very marked accumulation of ammonia nitrogen
in the cells which develop high acidity. Wadleigh and Shive
conclude from the fact that both sugars and ammonia nitrogen
accumulate in boron-deficient plants that boron deficiency
brings about a decreased rate of oxidation of sugars, and of
amination of carbohydrate derivatives so that protein sub-
stances necessary for maintenance of protoplasm are not formed.
Microchemical tests for proteins supported this view, for Millon's
reagent, the xanthoproteic test and the biuret test all gave
immediate results with the abnormally acid cells of boron-
deficient plants, whereas in normal plants pre- treatment with

TRACE ELEMENTS IN PLANTS 123

ether and alcohol was necessary to denature the proteins before
these tests gave a positive result, thus indicating the degeneration
of the proteins of the cells with high acidity and ammonia
nitrogen content. The disturbance in the carbohydrate and
nitrogen metabolism of the boron-deficient plants may be
attributed to a disturbance of the normal oxidation-reduction
relations of the cells, and it has been pointed out by Johnston
and Dore (1929) and Eaton (1935) that boron has considerable
affinity for the hydroxy groups of polyhydric alcohols.

Heggeness (1942) has recently suggested that boron may play
a considerable part in protecting flax from attack by the rust
Melampsora Lini. Borax was applied to the soil at the rate of
60 lb. per acre, 2 weeks after germination of the flax, which was
sown on 9 May 1941. Some 10 weeks later plants on control
plots which did not receive boron showed 100 per cent infection,
while plants on the boron-treated plots showed very little
infection in the field. When leaves from boron- treated plants
were cleared with 80 per cent alcohol many points of infection
could indeed be seen, but they mostly failed to develop. With a
sowing later in the year, made on 16 June, the boron-treated
plants were not so free from rust, but were yet very much freer
from rust than the controls.

CHAPTER V

TRACE ELEMENTS IN ANIMALS

The study of trace elements in animals has not proceeded so
far as with plants, but analysis has shown that animals, no less
than plants, may contain small amounts of many elements not
generally regarded as essential. The well-established indispen-
sable elements are carbon, hydrogen, oxygen, nitrogen, sulphur,
phosphorus, sodium, potassium, magnesium, calcium, iron and
chlorine. Fox and Ramage (193 1),1 by spectrographic examina-
tion of forty -three whole animals, separated tissues of eighteen
others and blood of three more, found that in addition to the
above-named essential elements copper was universally present.
Manganese was also found to be widely distributed. Cobalt and
nickel were also found to be present in a number of species,
nickel being met with more frequently than cobalt, but the
incidence of both appeared to be irregular. Lithium and stron-
tium were also found in many of the species examined, while
lead, silver, and rubidium were found in several. Cadmium and
calcium fluoride were each found in one instance only.

The flame method of spectrographic analysis used by Fox and
Ramage (cf. p. 28) is not applicable for the detection, and still
less for the estimation, of a number of elements, including
aluminium, arsenic, boron, antimony, bismuth, gold, molyb-
denum, tin, titanium, uranium, vanadium and zinc, all of which
had earlier been recorded as occurring in at least one animal.
In a later spectrographic examination of marine invertebrates
made by Webb (1937) the silver and carbon arcs were used, by
means of which these elements are detectable in small amounts.
Twenty-one marine animals, as well as three algae, were ex-
amined ; the animals included representatives of Gastropoda,
Lamellibranchiata, Echinodermata, Pisces, Urochordata, Crus-
tacea, Nemertea and Polychaeta.

As far as those elements which are also detectable by the flame

1 References to earlier literature on trace elements in animals are to
be found in this paper, and in that by Webb referred to below.

TRACE ELEMENTS IN ANIMALS - 125

method are concerned Webb's results are in general agreement
with those of Fox and Ramage. Copper and strontium were
found in every animal examined, while manganese was detected
in all except two of the species examined. Lithium was found
to be widely distributed, although Webb was of the opinion
that the concentration of this in the tissue fluids is never higher
than it is in sea water. Cobalt was found in only one species,
the gastropod Pleurobranchus plumula, while nickel was definitely
found only in this and one other species, also a gastropod. It
should be pointed out that neither cobalt nor nickel has been
detected in sea water so that the concentration of these ele-
ments in the environment must be exceedingly low. Silver,
lead and cadmium were found to be fairly widely distributed
and the same was found for barium, which Fox and Ramage
had been unable to detect in any of the specimens they exam-
ined. This, and the greater incidence of cadmium as established
by Webb, may be due to the greater sensitivity of the arc
method employed by the latter as compared with the flame
method of Ramage.

As regards elements not detectable in small quantities by the
flame method a measurable amount of boron was found in every
species except two, while aluminium was detected in nine out of
twenty species examined. Silicon was found in a number of
species, but Webb concluded that apart from those in which
there is a siliceous skeleton there is little tendency for the
accumulation of this element. Chromium was found in four
species, but unfortunately the data obtained for molybdenum by
Webb are regarded as of uncertain value on account of the pos-
sibility of its presence in the electrodes used. Zinc in com-
paratively large quantities was found in some species, though in
others this element was not detectable, while tin was found in
small amount in several of the species examined. Fluorine in
quantity was found in two gastropods. Vanadium is of interest
because it is known to occur as a pigment in the blood of the
ascidians (Henze, 1911). It appears, however, to be generally
absent in recognizable amount from animals of other groups, the
only example of an animal other than an ascidian in which Webb
found it being the mollusc Pleurobranchus plumula. Finally,
there is a group of elements for which Webb searched but which

126 TRACE ELEMENTS IN ANIMALS

were never found; these were antimony, arsenic, beryllium,
germanium, gold, mercury and titanium. It will be observed
that the spectrographic methods will not allow the detection of
chlorine, bromine and iodine.

It thus appears that in addition to the generally recognized
indispensable elements a large number of others have been
found in animals. Of these copper would appear to be a constant
constituent of animals while almost all animals examined con-
tain manganese in significant amount. There is no indication
that any other of the spectrographically recognizable trace
elements are universally present although some of them may
occur in relatively considerable amounts in a limited number of
species. Of the few elements not recognizable by the spectro-
graph iodine is known to be widely distributed. Keilin and
Mann (1940) state that 'it is now well established that zinc is a
true and general microconstituent of living organisms ' . In view
of Webb's work, this statement would appear to be somewhat
premature, though it may well be that the view of the universal
presence of zinc in both plants and animals will ultimately prove
to be correct, and the essentiality of zinc for mammals appears
to be well established.

But as with plants, so with animals, the presence of an
element in the organism is no evidence of its necessity. Thus
although Webb found that lithium, boron, strontium, aluminium
and silver were frequently present, he emphasized that there
never appeared to be any appreciable accumulation of these
elements from the environment. Even accumulation is no
evidence of essentiality, which can only be proved by observa-
tion of the deleterious effects produced by the exclusion of the
element from the animal's diet and the recovery of the animal
from the ill-effects of deficiency when a supply of the element
is restored.

Although the investigation of trace-element effects in animals
has not been carried nearly as far as with plants, there are
nevertheless a few trace elements which are well-established
as essential for certain groups of animals. The best known of
these are perhaps copper and iodine, the former of which is
known to be needed for the utilization of iron in haemoglobin
formation, while iodine is essential for the functioning of the

TRACE ELEMENTS IN ANIMALS 127

thyroid gland in higher animals. Cobalt is now known to be
essential for sheep and cattle, for without a sufficiency of this
element a serious disease which may prove fatal develops. Its
wide distribution in animals suggests that manganese may be
an essential element for animals as well as for plants, and there
is evidence that a deficiency of it is associated with a disease of
fowls known as perosis characterized by deformity of the leg
bones. The essentiality of vanadium for the blood of ascidians
has already been mentioned, but of the other elements, although
a number of them may be constantly present in the bodies of
man and the higher animals, nothing really definite is known
regarding their indispensability.

Practically any trace element may produce poisoning if
administered or presented to the animal in too great excess. Two
diseases of animals, the causes of which have for long been
obscure, have in recent years been tracked down to the effects
of excess of two unexpected sources. One of these is the so-called
' alkali disease ' affecting livestock in a number of the northern
United States, the other is the scouring of cattle, and to a less
degree of sheep, which occurs in England on certain land known
as 'teart' in Somerset and a small area of Warwickshire and
Worcestershire. The former disease has now been shown to be
due to an excess of selenium, the latter to an excess of molyb-
denum, in the pasture plants growing, and consumed by the
animals grazing, on the lands in which the two diseases are
respectively incident.

In the following pages accounts will first be given of these
two diseases due to trace-element excess, after which the trace-
element deficiency diseases will be considered.

1. Diseases due to Trace-Element Excess

Alkali Disease (Selenium Poisoning). For many years a
disease affecting horses, cattle, pigs and poultry has been
known to occur in certain areas of the great plains of the Middle
Western United States. The symptoms of the disease include
loss of hair from the mane and tail of horses, from the switch of
cattle and from the body of pigs, and a marked change in the
growth of the horn of the hoof in all these animals which , when

128 TRACE ELEMENTS IN ANIMALS

severe, results in the sloughing of the hoof. General loss of
condition involving emaciation and listlessness may follow, and
in the worst cases the animals may die. There may be lesions
in various internal organs, including the heart, liver, spleen and
kidneys. With affected poultry eggs do not hatch. Further, the
symptoms can develop in animals in non-affected areas when fed
with hay or grain produced in the affected areas. Because of the
popular opinion that these effects were due to alkali in the water
drunk by the animals, the disease was generally known as
'alkali disease', but more than 30 years ago careful work by
Larsen, White and Bailey (1912, 1913) showed that this con-
clusion was false and the cause of the disease must be sought
elsewhere. That this was to be found in poisoning by selenium
appears to have been first suggested in 1931 (see Byers, 1934).
First investigations showed that selenium was present in the
soils of the affected areas and that wheat grain from such areas
might contain from 10 to 12 p.p.m. of selenium (Robinson,
1933). Nelson, Hurd-Karrer and Robinson (1933) found that
wheat appeared to grow normally on soil to which 1 p.p.m. of
selenium had been added in the form of sodium selenate, but the
grain from this wheat was toxic to white rats fed with it, re-
tardation of growth, and finally death, resulting. Schoening
(1936) developed the typical symptoms of alkali disease in hogs
by feeding them with maize grown in the affected area. Two lots
of maize were used, one containing 5 p.p.m. the other 10 p.p.m.
of selenium.

Further experiments on dosing animals with selenium leave
no doubt that this element is responsible for producing the
symptoms of alkali disease, and that this is, in fact, selenium
poisoning. The animals used include rats (Franke and Moxon,
1936), rabbits (Smith, Stohlman and Lillie, 1937), swine (Miller
and Schoening, 1938; Miller and Williams, 1940a), cattle, horses
and mules (Miller and Williams, 1940a). As an example of the
type of experiment employed may be cited one by Miller and
Schoening on swine. In this eight pigs about 4 months old were
separated into four pairs. All received a sufficient ration of
grain, but the four groups received different amounts of selenium,
the proportions of this, as sodium selenite in the ration, being
respectively 392, 196, 49 and 24-5 p.p.m. Four of the animals

TRACE ELEMENTS IN ANIMALS 129

developed typical symptoms of alkali disease, namely, loss of
hair and disturbance of the growth of the hoof, and all died in
from 10 to 99 days. Post-mortem examination revealed lesions
of internal organs, particularly of the liver, while kidneys, heart
and spleen were also affected in some cases. Two control animals
fed with similar grain without added selenium developed nor-
mally.

The minimum lethal dose of selenium for rats, rabbits, horses
and mules appears to be of the order of 1-5 or 2 mg. per lb. of
body weight, for cattle 4-5-5 mg. per lb. of body weight, and for
pigs between 6 and 8 mg. per lb. of body weight.

By the continued administration of small doses of selenium
to horses Miller and Williams (19406) produced symptoms
similar to, though not so striking as, those observed in chronic
alkali disease under natural conditions. These symptoms included
listlessness, loosening of hair in the mane and tail, softening of
the horny wall of the hoof, and lesions in the liver, heart,
kidneys and spleen.

It being thus clear that ' alkali disease ' results from poisoning
by grain or forage containing selenium, it is important to know
whether there are differences in the selenium-absorbing capacity
of different plants. There is no doubt that this is very much
the case. Byers (1935) found notable differences in the selenium
content of plants occurring naturally on seleniferous soils, while
Hurd-Karrer (1935) also found marked differences in the
selenium content of crop plants grown on artificially selenized
soils. Thus Miller and Byers (1937) record a selenium content of
1110 p.p.m. in Astragalus bisulcatus and one of only 45 p.p.m.
in wheat grown in the same area. In another area they found
1250 p.p.m. of selenium in A. bisulcatus, but only 3 p.p.m. in
A. missouriensis . Byers had earlier found as much as 4300
p.p.m. of selenium in A. bisulcatus, while relatively enormous
quantities of selenium have also been found in other species of
Astragalus, as, for example, 5560 p.p.m. in A. ramosus and
1750 p.p.m. in A . pectinatus . On the other hand, A. missourien-
sis, A . mollissimus and A . drummondii absorb only little selenium.
Prairie grasses in general have a very low capacity for absorbing
selenium ; thus Andropogon scoparius (little blue-stem) was found
to contain only 0-8 p.p.m. of selenium.

130 TRACE ELEMENTS IN ANIMALS

Miller and Byers (1937) distinguish three classes of plants in
regard to their capacity for absorbing selenium: (1) highly
seleniferous plants which absorb selenium readily and which are
either absent from or rare in neighbouring non-seleniferous areas .
Plants of this group include Astragalus bisulcatus, A. racemosus,
A. pectinatus, A. carolineanus, Stanleya pinnata, S. bipinnata,
Applopappus fremonti, Xylorrhiza parryi; (2) plants capable of
absorbing selenium, even in considerable amount, without
severe injury, but which are widely distributed on both seleni-
ferous and non-seleniferous soils. This group includes Aster
ericoides (white wreath aster), A.fendleri (blue aster), Gutierrezia
sarothrae (turpentine weed), Helianthus annuus (sunflower), Ag-
ropyron smithii (western wheat -grass) and the common cereals,
wheat, rye, barley and maize; (3) plants with a low tolerance for
selenium, which make poor growth at best on seleniferous soils
and which absorb only small amounts of selenium. Plants in
this group include Bouteloua gracilis (buffalo grass) and B. curti-
pendula (grama grass).

For the control of selenium poisoning the relation of selenium
absorption to sulphur absorption by plants may be of great
significance. Hurd-Karrer (1934, 1935) showed that increasing
the sulphur content of the soil brought about a reduction in the
quantity of selenium absorbed by plants. Similarly, with plants
grown in water culture selenium uptake was reduced by in-
creasing the concentration of sulphate in the nutrient solution.
Hurd-Karrer and Kennedy (1936) found that grain from wheat
grown on soil containing 2 p. p.m. of selenium was toxic to white
rats, whereas grain from wheat grown on similar selenized soil
treated with flowers of sulphur or gypsum was not toxic, the
selenium content of the grain being reduced by such treatment
from about 12 p. p.m. to about 4 p. p.m.

In 1937 Hurd-Karrer reported the results of experiments on
the uptake of sulphur and selenium by some fifteen different
crop plants grown in the greenhouse on soil artificially selenized
by the addition of 4 p. p.m. of selenium as sodium selenate. The
plants were known to differ widely with regard to their capacity
for absorbing sulphur. The shoots were cut when 1, 2 and 3
months old and the sulphur and selenium determined. The
results obtained after the first month are shown in Table XVI.

TRACE ELEMENTS IN ANIMALS

131

Sulphur

Selenium

per cent of

per cent of

dry weight

dry weight

3-37

00793

2-88

0-0710

2-78

0-0744

2-39

0-0780

2-24

0-0615

1-28

00474

1-32

00310

1-21

00315

0-92

00280

0-99

00195

0-87

00240

0-80

0-0280

0-59

00130

0-42

00140

0-48

00130

Inspection of this shows that although the species used exhibit
a wide range in sulphur and selenium uptake, the ratio of
sulphur to selenium absorbed is roughly the same in all.

Table XVI. Content of sulphur and selenium of a number of
crop plants one month old. (From Hurd-Karrer)

Species

Brassica oleracea capitata (cabbage)

B. oleracea botrytis (cauliflower)

B. nigra (black mustard)

B. napus (rape)

B. oleracea acephala (kale)

Linum usitatissimum (flax)

Helianthus annuus (sunflower)

Trifolium pratense (red clover)

Vicia villosa (vetch)

Lactuca sativa (lettuce)

Triticum sativum (wheat)

Secale cereale (rye)

Glycine hispida (Soya bean)

Zea mais (maize)

Sorghum vulgare

Hurd-Karrer also found that the sulphur /selenium ratio was
practically the same for stems and leaves and also that the ratio
depended on the relative amounts of sulphur and selenium in
the soil. If the former remained the same while the selenium
content was varied, the sulphur/selenium ratio in the plant rose
with a fall in the selenium content of the soil. Thus, with a
sulphur content of the soil of 0-06 per cent, the average ratio
of sulphur to selenium in the plants was found to be 114 when
the selenium in the soil was 3 p. p.m., but when this latter was
increased to 5 p. p.m. the sulphur/selenium ratio in the plants
was only 22.

These results are readily understandable in view of the
chemical similarity of sulphur and selenium. They indicate very
definitely a way of reducing the selenium content of plants
growing on seleniferous soils.

For a good detailed account of the earlier work on selenium
poisoning reference may be made to a review by Trelease and
Martin (1936).

The Scouring of Cattle on the Teart Lands of Somerset.
On certain soils derived from the Lower Lias, where this is

132 TRACE ELEMENTS IN ANIMALS

directly exposed and not covered by drift or boulder clay, there
are pastures which bring about the complaint of cattle known as
scouring. Cattle, on being turned into these pastures, develop
the symptoms of scouring within a few days. The symptoms are
described by Ferguson, Lewis and Watson (1943) as follows:
; The dung becomes extremely loose and watery, yellowish green
in colour, bubbly, and has a foul smell. The animals become
filthy, their coats stare and they lose condition rapidly. Red
Devon cattle turn a dirty yellow and black beasts go rusty in
colour. Milk yields drop considerably.' Sheep are not affected to
the same extent, but the dung is very soft and the fleeces some-
times become stained.

The principal area in which teart soils occur is in central
Somerset, but smaller areas occur in Warwickshire and Glouces-
tershire. It is to be noted that an area in Glamorgan in which
the soil is also derived from the Lower Lias does not exhibit
teartness.

The effect of teart land on cattle has been known, according
to Ferguson, Lewis and Watson, for over a hundred years, and
has been attributed to a number of factors, including bacteria,
parasites, particular species in the herbage, water supply, poor
drainage, soil texture, a high proportion of non-protein nitrogen
in the herbage, and some particular chemical constituent
present in the herbage (Muir, 1936). Bacteria cannot be the
primary cause of scouring because the symptoms cease directly
the cattle are removed from teart pastures, while as regards
parasites there is no abnormally high number of these in affected
animals. Nor do the water supply, drainage and soil texture of
teart pastures show constant and characteristic differences from
non -teart areas. Ferguson, Lewis and Watson determined the
nitrogen fractions in herbage from teart and non-teart areas but
found no difference in the non-protein nitrogen of the two.

These workers were thus led to investigate the last of the
suggestions listed above and made a spectrograpliic examina-
tion of a number of samples of herbage from different sources.
As a result they found one constant difference between the
samples from teart and from non-teart pastures, namely, in the
content of molybdenum, which was considerably higher in the
case of the teart herbage. Thus the mean molybdenum content

TRACE ELEMENTS IN ANIMALS 133

of the herbage from twelve teart localities was 33 p.p.m. of dry
matter in 1937 and 38 p.p.m. of dry matter in 1&38; the corre-
sponding mean values for eleven non-teart localities were 5 and
4 p.p.m. for the respective years. This finding strongly suggested
that molybdenum might be the cause of scouring, and this was
confirmed by administering doses of ammonium or sodium
molybdate to cattle when the symptoms of scouring were pro-
duced in a number of cases, although there was considerable
variation in the degree to which different individual animals
reacted to molybdenum. Scouring was also produced in cattle
and sheep turned into non-teart pasture dressed with sodium
molybdate.

Hay and frosted herbage do not have the same effect as the
fresh material in inducing scouring. This appears to be due to
the fact that fresh herbage contains a much higher proportion
of the molybdenum in soluble form than does hay or frosted
material, and it would thus appear that it is the soluble fraction
of the molybdenum that is responsible for the effect on cattle.

It has been mentioned that the teart soils occur on the Lower
Lias where this is directly exposed, but that such an area in
Glamorgan does not exhibit the properties of teart land. Lewis
(1943 a) has examined a number of Lower Lias soils in their
relation to teartness and finds that the teart soils contain about
20-100 p.p.m. of molybdenum in the surface horizon and are
neutral or alkaline in reaction. The soils of the Glamorgan area,
however, were found to contain only about 2-4 p.p.m. of
molybdenum, which explains why they are not teart in
character.

Very interesting are the results of experiments made by Lewis
(19436) to examine the uptake of molybdenum from teart soil
by plants of a number of different pasture species. Ten pasture
grasses and two species of clover were used in these experiments,
the seeds being sown in pots of teart soil, four pots of each species
being used. The herbage was cut when it was about 3-4 in. high
and the molybdenum content determined. After a further period
of growth it was again cut and the molybdenum content in the
new sample determined. This procedure was repeated so that
four samples were obtained in all for each species. The results
are summarized in Table XVII.

134 TRACE ELEMENTS IN ANIMALS

Table XVII. Uptake of molybdenum by pasture
grasses and clovers. (Data from Lewis)

Molybdenum content
p. p.m. dry matter

A

in

r

May-
June
1939

July-

Aug. Sept.

1939 1939

May-
June
1940

36

83 61

42

14

10 10

9

8

8 4

6

15

10 6

9

13

17 15

9

12

18 15

11

12

13

11

10

93

109

69

57

87

103

62

59

Species

Holcus lanatus (Yorkshire fog)
Agrostis alba (fiorin)
Phleum pratense (timothy)
Festuca pratensis (meadow fescue)
Dactylis glomerata (cocksfoot)
Poa trivialis (rough-stalked

meadow grass)
P. pratensis (smooth -stalked 8 6 6 8

meadow grass)
Cynosurus cristatus (crested dogs- 10 8 5 9

tail)
Lolium perenne (indigenous peren- 11 11 11 11

nial ryegrass)
L. italicum (Italian ryegrass)
Trifolium repens (wild white clover)
T. pratense (wild red clover)

These results show very clearly that the clovers and Holcus
lanatus contain a much higher proportion of molybdenum than
the other pasture plants examined. Teart pastures generally
contain very little Holcus, but often a good deal of clover.
Lewis is of the opinion that it cannot be assumed that the clovers
are the only cause of teartness because analyses made of a
number of grasses other than Holcus growing on teart land show
that these can contain a much higher proportion of molybdenum
than was found in the plants grown in pots (cf. Table XVIII).

Table XVIII. Molybdenum content in p. p.m. of dry matter
of pasture plants growing on teart land. (Data from Lewis)

Soil type

, *■

Ham- I
Species

Dactylis glomerata (cocksfoot)

Poa pratensis (smooth-stalked meadow grass)

Lolium perenne (perennial ryegrass)

Cynosurus cristatus (crested dogstail)

Bromus mollis (soft brome)

Agrostis spp. (bent)

Phleum pratense (timothy)

Festuca spp. (fescues)

Arrhenatherum avenaceum (tall oat grass)

Trifolium spp. (clovers)

Ham-

Eves-

\

bridge

ham

Haselor

70

42

13

54

39

18

54

36

11

60

36

—

60

11

18

54

41

—

—

30

8

—

—

20

90

156

12

28

TRACE ELEMENTS IN ANIMALS 135

Ferguson, Lewis and Watson found that the scouring of
cattle and sheep resulting from the absorption of molybdenum
can be prevented and cured by dosing the animals with copper
sulphate. On very teart land a daily dose of 2 g. of this salt for
cows and 1 g. for young stock was found to be adequate for the
purpose, while on mildly teart land a smaller dose would probably
be sufficient. The mode of action of the copper is not evident.

Experimenting with six dairy cows, Ferguson (1943) found
that giving a daily dose of 2 g. of copper sulphate to each cow
for from 10 to 18 weeks produced no ill effects, the cows re-
maining in perfect health and calving normally. Having regard
to the facts that ( 1 ) on most farms in teart areas there is a pro-
portion of non- teart land, so that cattle would graze on the
teart land for only a portion of their time, and (2) the scouring
tendency of the herbage varies with the season, being greatest
in early spring and September and less at other times, Ferguson
considers that continuous daily dosing with copper sulphate
would probably not be necessary for more than about 6 weeks,
so that the cows used in the experiment actually received con-
siderably more copper sulphate than would usually be necessary
to prevent scouring.

Ways to reduce the teartness of pastures were also examined
by Lewis. He found that application of acidic nitrogenous
fertilizers containing ammonium sulphate reduced the propor-
tion of molybdenum in the herbage. This was largely due to a
reduction in the proportion of clover in the herbage, but the
actual proportion of molybdenum in the grasses was also reduced.
This might have been due to the ammonium sulphate bringing
about an increase in the yield of the grasses without increasing
the weight of molybdenum taken up. Also it has been noted that
teart soils are neutral or alkaline. Rendering the soil more
acidic will reduce the availability of the molybdenum and so
induce a lowering in molybdenum absorption by the plants.

Another point observed by Lewis was that the molybdenum
content of newly sown grasses is low, but increases with age,
although clovers have a high molybdenum content from the
beginning. A system of ley farming with short leys and with only
a small percentage of clover would thus appear to be very
suitable for teart land.

136 trace elements in animals

2. Trace-Element Deficiency
Diseases of Animals

The Effects of Copper Deficiency. In certain Crustacea,
Arachnida and Mollusca copper enters into the composition of a
pigment haemocyanine which is concerned in the respiration of
these animals.

Mann and Keilin (1938) have isolated two copper protein
compounds, one from the blood, the other from the liver of
mammals. The former of these, haemocuprein, is a blue com-
pound present in both the red corpuscles and serum, and appears
to account for all the copper in the corpuscles. The other copper-
protein compound discovered by Mann and Keilin they obtained
from ox liver and named hepatocuprein. This has several pro-
perties in common with haemocuprein, but is almost colourless.
Both these copper-protein compounds contain 0-34 per cent of
copper. It may be that these two compounds are intimately
connected, but their precise function is not yet known.

The work of Hart, Steenbock, Waddell and Elvehjem (1928)
and McHargue, Healy and Hill (1928) first showed that in
mammals copper is necessary for the utilization of iron in the
formation of haemoglobin. Later work by Waddell and Elveh-
jem and their collaborators, Cunningham (1931), Keil and
Nelson (1931), Josephs (1932) and others has confirmed without
doubt the essentiality of copper for haemoglobin formation,
although the copper itself does not form part of the haemoglobin
molecule as it does that of haemocyanine. Consequently de-
ficiency of copper in mammals may lead to anaemia, and the
cure of anaemia traceable to this cause can be effected by dosing
with copper sulphate. For a general review of this question a
paper by Elvehjem (1935) may be consulted.

A disease of cattle known as 'licking sickness', apparently
attributable to copper shortage, has been described by Sjollema
(1933) as occurring on farms in Holland where reclamation
disease of cereals occurs. Reclamation disease, as we have seen
(p. 95), is traceable to copper deficiency, and it was the occur-
rence of licking sickness of cattle along with reclamation disease
that led Sjollema to connect the two.

The external symptoms of lickmg sickness are anaemia and

TRACE ELEMENTS IN ANIMALS 137

loss of appetite with a general degeneration in the condition of
the animal. Internally, the proportion of dry matter in the
blood is low, being only 13-14 per cent instead of the normal
18-20 per cent, while both the iron content and haemoglobin
content are generally very low. From what is now known of the
part played by copper in haemoglobin formation these are the
symptoms which might be expected to result from a shortage
of copper.

That a particular substance is concerned in the disease is
indicated by the fact that moving affected animals to land on
which reclamation disease does not occur brings about rapid
improvement in the animals. That the shortage is not due to
iron or manganese, but probably to copper, is indicated by
analyses of the hay from farms in which the disease occurs.
Such analyses show that there is no constant shortage of either
iron or manganese, but that there is consistently an abnormally
low content of copper, namely 2-3 p.p.m. and sometimes even
less than 1 p.p.m., whereas hay from normal farms contains
from 6 to 12 p.p.m.

Support for the attribution of licking sickness to copper
deficiency was obtained by Sjollema by dosing affected animals
with copper sulphate.

Later Sjollema (1938) described another disease of animals
which he also attributed to copper deficiency, both cattle and
goats being affected. The chief symptoms of this disease, which
occurs only on fine dry sandy soils, are diarrhoea, wasting, and in
black cattle, loss of colour in the coat which becomes brown-grey.
The disease is apparently not the same as licking sickness.

The evidence adduced by Sjollema in support of copper
deficiency as the cause of this second disease is similar to that
respecting licking sickness. Thus the copper content of the
blood, fiver and milk of affected cows and goats was abnormally
low, ana so was the hair of affected cows. The grass and hay
from farms on which the disease occurred were low in their
content of both copper and manganese, but the quantity of the
latter appeared more than adequate for the needs of the animals.
The hay was also poor in zinc, but the blood of affected animals
contained a normal amount of this element and the liver a
higher content than the normal. As regards iron the content of

138 TRACE ELEMENTS IN ANIMALS

this element in diseased animals was abnormally high. Finally,
dosing with copper sulphate brought about recovery of the
affected animals.

If this latter disease is indeed different from licking sickness,
and Sjollema's accounts appear to indicate this, it would appear
quite unlikely that deficiency of copper alone could produce two
distinct diseases in the same species. The fact that administra-
tion of copper sulphate can cure the disease cannot be regarded
as sufficient evidence in itself that the disease has its origin in
copper deficiency, for, as we have already seen, molybdenum
poisoning of stock can be cured by dosing with copper sulphate.
Indeed, the symptoms of the second disease described by Sjol-
lema are rather reminiscent of those characteristic of the
scouring of cattle, but the soils on which the disease described
by Sjollema occurs suggest a deficiency, rather than excess, of
some element as the cause. The possibility that the curative
effect of copper sulphate might be due to traces of some other
essential element present as an impurity in the copper sulphate
administered is not to be ruled out. Altogether, the attribution
of both licking sickness and the diarrhoea and wasting disease
to copper deficiency should perhaps be treated with some
reserve until confirmatory evidence is forthcoming and other
possibilities eliminated.

A disease of cattle known as 'salt sick' has been known for
many years in Florida. This disease, according to Becker, Neal
and Shealy (1931), is a nutritional anaemia resulting from an
insufficiency of copper and iron in the diet. Bryan and Becker
(1935) reported that the disease occurs on certain sandy soils,
the surface layers of which possess roughly only one-tenth of the
iron content and one-half of the copper content of healthy soils
of Florida. They found that cattle become salt sick on soils
containing 0-036 per cent of iron and 3-85 p. p.m. of copper, and
remain healthy on soils containing 0-42 per cent of iron and
8 p.p.m. of copper. The similarity of the disease and its method
of cure with the licking sickness dealt with by Sjollema strongly
suggests that the two diseases are the same. Whether, as sug-
gested by Aston (1931) and Greig, Dryerre, Godden, Crichton
and Ogg (1933), it is also identical with the disease known as
bush sickness in New Zealand (see e.g. Askew and Rigg, 1932),

TRACE ELEMENTS IN ANIMALS 139

that known as Nakuruitis in Kenya (Orr and Holmes, 1931)
and that called pining in Scotland (Greig et al. 1933) is
another matter. If this should indeed be so it would mean
that salt sick results, not from deficiency of copper, but from
deficiency of cobalt. The United States Department of Agriculture
Yearbook for 1939 records that although surveys were not then
complete, observations of cattle indicate a few copper-deficient
areas and a considerable cobalt- deficient area in Florida. Thus
it may be that both copper deficiency and cobalt deficiency may
occur in cattle in that State. The work of Rusoff, Rogers and
Gaddum (1937) rather suggests that all the cases of salt sickness
may not be due to the same cause. Thus they state that workers
at the Florida Agricultural Experiment Station have found that
cobalt is a limitixig factor in a type of salt sick known as 'hill
sick ' . It would therefore appear likely that this is identical with
pining or bush sickness. Rusoff, Rogers and Gaddum, however,
were unable to detect cobalt spectrographically in the grasses of
either salt-sick or healthy areas, but it may be, as they point out,
that this element, as well as others, may be present in quantities
too small to be determined by the method used. But as regards
copper, they found the same content of this element in the
grasses from salt-sick and normal areas, so that their analyses,
lend no support to the view that salt sick results from shortage
of copper. But, as they point out, it may be that the ratio of
various elements is the important factor and that the disease
might occur where the iron content of the grass is high and the
copper content low, or vice versa, or the ratio of iron or copper
to some other element might be the determining condition.

A disease of lambs which may result from shortage of copper
occurs widely in Great Britain, but appears to be most trouble-
some in an area of north Derbyshire, in part of the Cheviot Hills,
in certain districts of Yorkshire, and in an area of Gloucester-
shire and Somersetshire where the Mendip Hills appear to
provide the worst cases. The disease also occurs in Australia,
while the same or a similar disease has been described as
occurring in Sweden, in India and in Peru and other parts of
South America. In this country it is known as sway back,
swingback or swingleback, and in Australia as enzootic ataxia.
In this country it has probably been known to farmers for a very

140 TRACE ELEMENTS IN ANIMALS

long time, but the first scientific description of it appears to have
been recorded only some 14 years ago (Stewart, 1932). It was
recorded for Australia at about the same time (Bennetts, 1932).
Dunlop, who has specially studied the disease, thus (1939) de-
scribed the symptoms : ' In most cases the symptoms are noticed
in the lambs at birth. Some lie recumbent swaying their heads
and make spasmodic efforts to rise and obtain milk from their
dams. Such attempts frequently result in the lamb rolling over on
its side, kicking vigorously with the hind legs. Others may be able
to stand, but often the hindquarters swing about and eventually
fall over drawing the rest of the body to the ground. Attempts
to walk are characterized by incoordination of movement, a
swaying gait, staggering and finally collapse.' Blindness may
sometimes occur. In some cases the symptoms at birth may be
slight and develop later. Dunlop states that the mortality of
affected lambs is almost 100 per cent, so that adult animals
showing symptoms of swayback are rare. However, according
to Innes and Shearer (1940) mildly affected animals may survive
and when later used for breeding may give birth to healthy
lambs. Post-mortem examination of affected animals shows that
lesions of the brain occur, the white matter disintegrating leaving
cavities filled with a clear fluid or jelly. The degeneration may
extend down the spinal cord. These lesions arise while the lamb
is still in utero.

Investigations show that the disease is not hereditary, nor
could any evidence be obtained to suggest that it was due to
infection by micro-organisms. For example, no transmission of
the disease results from inoculating new-born healthy lambs
with extracts from affected tissues. All the facts, including the
localization of the disease to certain limited areas, point to the
disease being nutritional in origin. The coincidence of the disease
in Derbyshire with soils containing lead might suggest that the
disease results from lead poisoning, but this opinion has no
experimental evidence to support it and is by no means generally
held.

Dunlop describes a large-scale experiment carried out in
Derbyshire designed to test the theory that swayback is a
nutritional disease. In this experiment 1800 ewes distributed
over fifty farms were used. Four groups of 300 ewes each, the

TRACE ELEMENTS IN ANIMALS 141

groups as equal as possible, were fed with mineral mixtures,
made up into licks, the four mixtures containing respectively
copper, cobalt, manganese or boron in the same amount. These
were made available to the ewes on the various farms early in
December, and by lambing time about the beginning of April
practically all the licks had been consumed. As a result it was
found that of the lambs born from ewes which received minerals
with copper 1-34 per cent were affected with swayback as
compared with 13-1 per cent of the lambs from ewes which
received minerals without copper and 15-2 per cent where no
minerals were supplied. While this experiment indicates that
the addition of a small amount of copper to the diet of the ewes
is effective in preventing swayback, it does not follow that the
vegetation on which the ewes normally feed in swayback areas
is necessarily deficient in copper. As Dunlop points out, the
presence of excess of lead or other minerals may render the
copper unavailable and it may not be absorbed in adequate
quantity from the gut.

The favourable effect of feeding copper to pregnant ewes had
also been demonstrated by Bennetts and Chapman (1937) in
Australia and was confirmed in further work by Dunlop, Innes,
Shearer and Wells (1939), but although it seems clear that the
addition of copper to the diet is an effective preventive of sway-
back, it would be premature to assume that the disease is
actually due to copper deficiency. It has already been pointed
out that copper similarly prevents scouring of cattle, which, as
we have seen, is due to poisoning by excess of molybdenum. At
present therefore we cannot rule out the possibility that sway-
back may be due to poisoning by lead or some other metal, and
that copper has a similar action in preventing the disease as it
has in preventing molybdenum poisoning of cattle on teart lands.
It may be significant that at least two of the areas in which
swayback occurs, north Derbyshire and the Mendips, are areas
in which lead occurs in sufficient quantity to be mined. It
should also be observed that the symptoms in goats supposed by
Sjollema to be suffering from copper deficiency are quite different
from those of swayback. One might expect the symptoms pro-
duced in two such nearly related species as sheep and goats by
deficiency of the same element to be similar.

142 TRACE ELEMENTS IN ANIMALS

Again, determinations of the copper content of the blood do
not reveal any correlation between copper content of blood and
incidence of sway back. Certainly Bennetts and Chapman (1937)
in Australia found particularly low values for the copper content
of the blood of four ewes that gave birth to affected lambs (less
than 0-01 mg. per cent). On the other hand, Innes and Shearer
(1940) found that in Derbyshire the average copper content of
the blood of ewes giving birth to swayback lambs was 0-058 mg.
per cent, while the value with ewes on swayback farms bearing
normal lambs was actually somewhat lower, namely 0-045 mg.
per cent, but in both groups there was quite a wide range of
values, 0-037-0-070 mg.# per cent in the swayback group and
0-034-0-061 mg. per cent with the ewes bearing normal lambs.
Higher mean values were found for the copper content of the
blood of ewes in non-sway back areas, but the range was even
wider than in affected areas. There was thus no indication of
any close relationship between copper content of the blood of
the ewes and the incidence of swayback. This conclusion is
supported by the results of the examination by Eden (1939,
1941) of the blood of more than 300 sheep in an area of Northum-
berland where swayback is unknown. Here very wide variations
in copper content were found, the mean value for ninety-four
sheep examined in 1940 being 0-080 mg. per cent with a range
of 0-013-0-210 mg. per cent. Eden also points out that the
pastures in this Northumbrian area have an even lower copper
content (6-10 p.p.m. of the dry weight) than those of the sway-
back area of Derbyshire. It may be concluded that much work
requires to be done to put our knowledge of the effects of copper
deficiency in animals on a firm basis.1

The Effect of Iodine Deficiency. Iodine deficiency is gener-
ally regarded as the cause of goitre in man and other mammals.
Goitre results from enlargement of the thyroid gland which
contains the characteristic protein thyroglobulin, one of the
constituent amino-acids of which, namely thyroxine, contains

1 In a paper which appeared since the above was written Shearer
and McDougall (1944) express the opinion that the Australian disease
is due to an uncomplicated deficiency of copper in soil and herbage
but that in this country this is not so although affected animals suffer
from copper deficiency.

TRACE ELEMENTS IN ANIMALS 143

iodine in its molecule. In various parts of the world where
iodine is deficient in the soil and where, in consequence of this,
very little of this element is absorbed by pasture plants, farm
animals may be liable to goitre, as, for example, in parts of Cali-
fornia and the Middle Western United States. Affected animals
are born with necks swollen as the result of enlargement of the
thyroid gland and are generally weak or dead. According to
Maynard (1937) iodine deficiency in dairy cows can be cured by
adding 0-02 per cent to the food given to the animals.

Actually man appears to be more liable to goitre than farm
animals, and in this country, where animals are rarely affected,
there are areas, particularly in Derbyshire, where the incidence
of goitre among the human population is quite high and where
special measures in the way of administering iodine have to be
taken to combat it.

It should be noted that although there is no doubt of the
beneficial effects of iodine in the treatment of goitre doubts
have been expressed as to whether iodine deficiency is the sole
factor responsible for this condition. It has, for example, been
quite widely held that the presence of radioactive material in
the soil is also a factor responsible in part for goitre.

Manganese Deficiency in Chicks. Perosis. The very wide
distribution of manganese throughout the animal kingdom
suggests that this element may be as generally essential to
animals as to plants. It is thought that manganese in small
quantity is necessary for man although its function in the
human body is not known. As a result of experiments with rats
it has been suggested that manganese may be an essential
catalyst in the utilization of vitamin Bl3 since .deficiency of
manganese in the diet brought about similar changes, for
example, loss of maternal instinct and progressive loss of
fertility, as excess of vitamin Bx (Perla, 1939).

The effect of manganese deficiency in bringing about a disease
of young chickens known as perosis, hock disease or slipped
tendon was first shown by Wilgus, Norris and Heuser in 1936.
This disease is characterized by a deformity of the leg bones,
and to some extent of the wing bones, of the chick, particularly
of the tibio-metatarsal joint. The bones of the leg and wing are
shorter than the normal. The relationship of manganese deficiency

144 TRACE ELEMENTS IN ANIMALS

to perosis was soon confirmed by Gallup and Norris (1937) and
several other groups of workers. Thus Heller and Penquite
(1937) found that addition of an aqueous extract of rice bran
or of traces of manganese to perosis-induoing diet practically
prevents the occurrence of the disease. Insko, Lyons and
Martin (1938 a) also showed that manganese exercised a protec-
tive action against perosis, whereas no such action resulted with
aluminium or zinc, but rather the reverse. The same workers
found (19386) that a minimum quantity of 30 p.p.m. of man-
ganese as manganese sulphate had to be added to an all -mash
basal ration containing 6 or 7 p.p.m. of manganese, if growth was
to be normal and perosis prevented. Amounts of manganese up
to 646 p.p.m. were not toxic. With low contents of manganese
they found that up to about 30 p.p.m. of manganese the degree
of bowing of the legs of their experimental chicks was inversely
proportional to the manganese content of the diet.

Wiese, Elvehjem and Hart (1938) also found both rice bran
(15 per cent of the ration) and 0-0025 or 0-005 per cent of man-
ganese as manganese sulphate gave protection against perosis.
They found, however, that autoclaving the rice bran removed its
protective property, from which they concluded that some ther-
molabile substance must be involved, perhaps in association with
manganese, in preventing perosis. This finding would suggest
then that perosis may not be an outcome of simple manganese
deficiency, but that some other substance is involved as well.

Gallup and Norris (1938) also showed that a large proportion
of the chicks hatched from eggs with a low manganese content
have short leg bones, and Lyons and Insko (1937) showed that
the embryos which were produced in eggs of hens fed on a
manganese-deficient diet were characterized by abnormally
short and thick legs and short wings. They found the deformities
could be prevented either by adding manganese to the diet of
the hen or by injecting manganese into the egg before incubation.

Caskey and Norris (1939) found that excess of calcium and
phosphorus in the diet greatly reduced the availability of man-
ganese, and they found that 15-5 mg. of manganese supplied
orally over a period of 6 weeks with diets containing 1 per cent
of calcium and 0-5 per cent of phosphorus was as effective in
reducing perosis as 142 mg. of manganese supplied with 3 'per

TRACE ELEMENTS IN ANIMALS 145

cent of calcium and 1-5 per cent of phosphorus. With neither diet,
however, was perosis completely prevented, but when the man-
ganese was supplied by intraperitoneal injection it was found
that 10 mg. completely prevented perosis even at the higher
levels of calcium and phosphorus supply.

Gallup and Norris (1938) have recorded the results of X-ray
examination of the leg and wing bones of chicks fed on a diet
low in manganese (10 p.p.m.), the X-ray photographs being
taken at intervals from the time of hatching until the chicks
were 7 weeks old. These were compared with similar photographs
of chicks fed on a diet containing an adequate supply of man-
ganese (100 p.p.m.). This X-ray examination confirmed the
effect of manganese deficiency in producing shortened leg bones.
Caskey, Gallup and Norris (1939) found that the ash content of the
bones of chicks fed on a diet low in manganese was significantly
below the normal. This was not due to a rachitic condition,
since the chicks were supplied with ample vitamin D, while
X-ray examination and staining with silver nitrate showed that
calcification was normal.

Gallup and Norris point out that other investigators have
reported measurable amounts of manganese in the bones of
rabbits and rats, and they suggest that a small amount of man-
ganese may not only be an essential constituent of bone in the
chick, but that it may be essential for the development of bone
in general.

Pining, Enzootic Marasmus, Bush Sickness or Morton
Mains Disease. In countries as far apart as Scotland and
Australia and New Zealand there has occurred for many years a
disease of sheep characterized by the rapid deterioration of the
animal. In Scotland, where the disease occurs in many parts of
the country, it is known as pine or pining, vinquish and daising,
and young cattle are also affected. Here the disease was re-
corded by Hogg, the Ettrick Shepherd, as long ago as 1831. The
symptoms as they appear in the island of Tiree, Inner Hebrides,
have been described by Greig, Dryerre, Godden, Crichton and
Ogg (1933) as 'those of a progressive debility, accompanied by
anaemia and emaciation. The onset is frequently insidious. The
affected animal is dull, and the coat, in the case of cattle, is
rough and staring, the visible mucous membranes, especially

146 TRACE ELEMENTS IN ANIMALS

the conjunctiva, become pale: physical condition is gradually
lost, the eyeball becomes sunken, and there is commonly a
serous discharge from the inner canthus. In young animals
growth is markedly retarded, and they soon present a stunted,
unthrifty appearance. Thereafter the anaemia and emaciation
progress to the condition of cachexia, and, finally, as the result
of extreme weakness, the animal is unable to rise. In severe
cases the gait is stilted and somewhat incoordinated.' The
disease usually ends fatally.

The disease of sheep in North Island, New Zealand, known as
bush sickness, that in Southland, New Zealand, known as
Morton Mains disease, and that in Australia called enzootic
marasmus, all appear to be the same as pine in Scotland. Morton
Mains disease in a bad year and in a bad locality was described
by Wunsh (1937) thus: 'About midsummer a majority of the
season's crop of lambs would fall off in condition. Their wool
would become "chalky" and harsh, their eyes would water, and
they would lose their activity. A large number — 30 per cent or
more — would gradually lose weight, become more and more
helpless and finally die.'

A consideration of the facts known about Morton Mains
disease in New Zealand suggests that it is a deficiency disease.
Thus the disease does not appear on newly broken land, but only
after several years of sheep farming on the same land. Also deep
ploughing lessens the disease for a few years. Both these facts
suggest a deficiency, and certainly not an excess, of some sub-
stance as the cause of the disease. That the disease is not due to
bacteria or other parasites is indicated by the fact that a sick
lamb transferred to healthy country recovers rapidly. Since the
climate, physical conditions and type of herbage of healthy and
affected farms might show no appreciable differences, there is
a strong suggestion that some substance in very small amount,
such as a trace element, is involved.

In 1935 it was reported by Underwood and Filmer that
enzootic marasmus of sheep in Western Australia could be cured
by administering a dose of 0-1-2 mg. of cobalt nitrate each day,
and 2 years later it was reported that affected cattle could be
cured in the same way (Filmer and Underwood, 1937). In the
meantime Askew and Dixon (1936) had reported a similar

TRACE ELEMENTS IN ANIMALS 147

favourable effect of cobalt on sheep suffering from Morton Mains
disease in New Zealand. This clearly suggested that deficiency of
cobalt might be the cause of the disease, and this view was
supported by the results of analyses of some New Zealand soils
made by Ramage by means of his spectrographic technique (see
p. 29), and which showed that two healthy soils each con-
tained 7 p.p.m. of cobalt while a sick Morton Mains soil con-
tained none of this element. Subsequent treatment of affected
lambs in New Zealand with weekly amounts of 7 mg. of cobalt
brought about a remarkable improvement after a fortnight.

In 1938, Underwood and Harvey showed that both soil and
herbage of affected areas in Australia have a lower cobalt con-
tent than those of neighbouring healthy areas, but that the
cobalt content of the herbage is considerably increased by
dressing the soil with a little cobalt acetate. Similar results
have been obtained in New Zealand (cf. Kidson and Maunsell
1939), although it would appear that there is not always a
direct relation between cobalt contents of soil and herbage.

Experiments in Scotland on pining have similarly shown the
relationship of this disease to a deficiency of cobalt. In south-
east Scotland Corner and Smith (1938) have shown that pining
could be cured and its reappearance prevented for 6 months by
a daily dose of 1 mg. of cobalt for 14 days.

An examination of a number of soils in the north of Scotland,
an area where pining occurs, made by Stewart, Mitchell and
Stewart (1941), showed that the cobalt content of the soils
varied from 1 to 300 p.p.m., and that most of the soils on which
pining occurs have a cobalt content of less than 5 p.p.m. while
some contain as little as 1 p.p.m. The same workers also carried
out an experiment on the treatment of affected lambs with
cobalt. In this experiment, which was started towards the end
of June 1939, two sets of lambs were used, one comprising forty
individuals which were treated with cobalt, the other of twenty-
five which served as controls, and which at the beginning of the
experiment were the best in the flock. Both groups ran together
on bad pining land in which the cobalt content of the soil was
only 1-2 p.p.m. The lambs of the experimental group were each
given 10 mg. of cobalt as cobalt chloride each week for 10 weeks ;
the control- animals received no cobalt. Apart from one lamb

148

TRACE ELEMENTS IN ANIMALS

which died from an unknown cause 3 days after the beginning
of the experiment and which was immediately replaced by one
from the control group, all the treated animals improved in
condition and growth and were stated by the farmer to be the
best set of lambs he had seen on the farm where the experiment
was carried out. The control animals, on the other hand, rapidly
lost condition, four died from pining, and by 4 September many
others were in the last stage of the disease.

Stewart, Mitchell and Stewart also examined on a set of plots,
the effect on the cobalt content of soil and herbage which
resulted from the application of various quantities of cobalt
chloride to the soil, the amounts ranging from \ to 80 lb. of
cobalt chloride (CoCl2.6H20) per acre, superphosphate to the
extent of 2 \ cwt. per acre being used as filling material for the
small quantity of cobalt used. Ten weeks after the application
of the dressings the total cobalt content of the soil and the
amount soluble in dilute acetic acid were determined on each
plot as well as the cobalt content of the herbage. The results,
given in Table XIX, show how in this comparatively short time
the application of cobalt to the soil affects the cobalt content
of the herbage.

Table XIX. Effect of cobalt manuring on the cobalt content
of herbage. (Data from Stewart, Mitchell and Stewart)

Cobalt

applied

to soil

p.p.m.

Cobalt in soil in p.p.m.

r

Total

>! ^\JKJ**1.V 111

Readily herbage
soluble p.p.m.

0

2-9

0177

011

•

2-9

[2-8

0196
0-200

012
0-20

0-6

—

—

0-23
0-35

0-125

•

2-9

2-9

0-215
0-230

0-34
0-41

0-25

13-25

13-2

0-255
0-236

0-51

0-83

1-25

■

40
4-2

0-479

1-63

2-28

500

5-8

1-75

3-94

1000

9-6

1-96

817

The favourable effect on lambs produced by such manuring
was shown by another experiment carried out by Stewart,

TRACE ELEMENTS IN ANIMALS 149

Mitchell and Stewart in which a field with a low cobalt content
of the soil (about 1-8 p.p.m.) was divided into two halves, one
of which was manured with 2 lb. cobalt chloride + 3 cwt. super-
phosphate per acre, the other with 3 cwt. superphosphate only.
Four to five weeks after this manuring fifteen lambs were
allowed to run on each half of the field, the two groups being of
equal value at the start. After 6 weeks the difference in the
appearance of the lambs of the two groups was striking, all
those on the half of the field with added cobalt being in fine
condition, whereas eight of the fifteen lambs on the other half
of the field showed severe pining while the remaining seven
animals were in poor condition.

While the effectiveness of cobalt manuring in increasing the
cobalt content of the herbage of ' pining ' lands can be regarded
as established, work by Mitchell, Scott, Stewart and Stewart
(1941) indicates that such manuring requires care. They find by
spectrographic analysis that the herbage growing on cobalt -
treated soil may take up an abnormally large amount of molyb-
denum. Where the herbage without cobalt treatment already
had a fairly high molybdenum content, this latter may be so
increased by cobalt manuring that it approaches the molyb-
denum content of the herbage of teart land, so that danger of
scouring may ensue. Analyses of the herbage from two soils, one
with a low, the other with a high molybdenum content, cut 15
months after various degrees of cobalt manuring, are shown in
Table XX. Since Lewis has shown that molybdenum uptake is
reduced by the use of acidic nitrogenous fertilizers (see p. 135),
and since teartness occurs on neutral and alkaline soils, it would
seem inadvisable to apply cobalt in the form of a cobalt-rich lime
or in conjunction with fertilizers having an alkaline reaction.

Table XX. Cobalt and molybdenum contents of herbage from
soils treated with cobalt chloride. (Data from Mitchell,
Scott, Stewart and Stewart)

CoCl2.6H20
added

Herbage

from soil A

Herbage

-*-

from soil B

Co

Mo

Co

Mo

lb. per acre

p.p.m.

p.p.m.

p.p.m.

p.p.m.

0

0-08

1-7

0-07

6-3

2

0-22

22

0-20

9-2

10

0-63

24

0-89

100

80

3-20

7-5

2-75

14-2

150 TRACE ELEMENTS IN ANIMALS

The possibility that cobalt deficiency may also be met with
in cattle in Kenya and in Florida has already been mentioned.

It is now recognized that pining in sheep occurs in various
parts of England, notably in parts of Northumberland and
Cumberland and on Dartmoor and Exmoor in Devonshire.

3. The Functions of Trace Elements

in Animals

The trace elements which have been shown to be essential for
animals of one or more species include iodine, manganese,
copper, zinc and cobalt. It has already been mentioned that
iodine enters into the composition of thyroxine, one of the
amino-acids of thyroglobulin, the protein of the thyroid gland.
This appears to be the only function of iodine in mammals, as
far as is known at present. Copper has long been known to form
part of the molecule of haemocyanine, a pigment concerned in
the respiration of certain Crustacea and some other lower
animals, while Mann and Keilin have shown that copper protein
compounds, haemocuprein and hepatocuprein, occur in the
blood and liver respectively of mammals. Although the actual
part played by these compounds in the physiology of the animal
is not clearly understood, it would appear to be established that
the functions of copper, wholly or in part, are to be found in
the roles of these copper-protein compounds.

Although diseases are now definitely associated with defi-
ciencies of manganese and cobalt nothing precise is known of the
functions of these elements nor in what form they are present
in animals. With zinc, on the other hand, the reverse is the case,
for whereas no condition has been associated with deficiency of
this element, recent work by Keilin and Mann (1940) has thrown
considerable light on the function of zinc in animals.

In 1933, Meldrum and Roughton showed that in the erythro-
cytes (red blood corpuscles) of mammals there occurs an en-
zyme which catalyses the reaction H2C03 = C02-(-H20. To this
enzyme they gave the name carbonic anhydrase. The enzyme
was shown by Davenport in 1939 to occur in relatively high
concentration in the gastric mucosa of mammals. It has now
been found by Keilin and Mann (1940) that preparations of this

TRACE ELEMENTS IN ANIMALS 151

enzyme, which they regard as practically pure, obtained from
the erythrocytes of ox and sheep, contain constantly about
0-33 per cent of zinc, but no iron, copper, manganese, mag-
nesium or lead. In preparations of the enzyme of varying degrees
of purity the enzyme activity is directly proportional to the zinc
content, which indicates that the zinc forms part of the enzyme
molecule. The enzyme, in fact, appears to be a zinc-protein
compound in which the protein in each molecule is combined
Avith two atoms of zinc. It may be noted that no other zinc
compound is known which has catalytic properties. Keilin and
Mann found the concentration of carbonic anhydrase to be
about 0-21 g. per 100 ml. of erythrocytes.

The function, or at any rate one function, of zinc in mammals,
is to be found therefore in the action of carbonic anhydrase. In
the blood this action accelerates the dissociation of carbonic
acid into water and carbon dioxide and so furthers the escape
of the latter from the blood into the alveolar spaces. As well as
this function in carbon dioxide excretion from the blood, the
presence of the enzyme in the parietal cells of the gastric
mucosa suggests another function in the stomach, probably in
connexion with the formation of hydrochloric acid.

CHAPTER VI

CONCLUDING REMARKS

I t will be clear from the preceding review of the present position
of our knowledge of trace elements in plants and animals that
these elements present the biologist with two sets of problems,
the one pathological, the other physiological. The pathologist is
concerned with the abnormal conditions resulting in plants and
animals from deficiency, and in a few cases from excess, of the
various trace elements, and of the means by which the deficiency
or excess can be removed. The problems of the physiologist are
more subtle, for it is his business to discover the functions in
the life of the organism of these various elements which are
present in only minute amounts. The two sets of problems are
of course interdependent, for the work of the pathologist in
discovering the effects of deficiency greatly aids the physiologist,
while knowledge of the part played by the trace elements in the
life of the organism must necessarily help the pathologist in the
diagnosis and treatment of deficiency diseases. But on the
whole, knowledge of the pathology of trace elements, particu-
larly in plants, is much more advanced than our knowledge of
their physiological functions. A considerable number of well-
recognized and defined plant diseases are now correctly attri-
buted to various trace-element deficiencies, and means of con-
trolling these diseases have been determined with considerable
precision. Future work on the pathological side will no doubt
extend the number of such known deficiency diseases. In
addition to such work it would appear that two lines of research
in the field of pathology would well repay attention. The first of
these is the development of rapid means of early diagnosis. The
usual method of observing deficiency symptoms, employed with
outstanding success by Wallace, is naturally only applicable
after visible external symptoms have developed. The injection
methods elaborated by Roach appear to be particularly useful
for trees, but although certainly usable for annual crops, and
affording a means of early diagnosis, require some degree of
technical skill for their employment with small herbaceous plants.

CONCLUDING REMARKS 153

The second field of research which should afford valuable
results in the control of deficiency diseases is the investigation of
the relationship of soil and climatic factors to the incidence of
these diseases. It is, for example, well known that the man-
ganese available in the soil for absorption by plants may be only
a fraction of the total manganese present, and that one of the
factors determining the degree of availability of this element is
the acidity (pR) of the soil, the availability in general de-
creasing with decreasing acidity (increasing pH). Hence liming
the soil, by bringing about an increase in the pR value of the soil,
may seriously affect the availability of manganese. This appears
to be particularly so when the soil contains much humus,
though the relations between pH, humus content and con-
centration of available manganese are at present far from being
completely understood. Liming may also adversely affect the
absorption of boron by plants, perhaps again because of its
effect in lowering the availability of this element, perhaps partly
because of the relations between calcium and boron. Again, it is
well established that the severity of deficiency diseases may vary
in different years, a variation which must be attributed to the
differences in climate experienced in different seasons; the
symptoms of boron deficiency, for example, are generally much
more severe in periods of drought than in wetter seasons. It is
to be presumed that the different water contents of the soil in
the different seasons must affect the availability of the trace
elements concerned.

Nor must the micro-organisms of the soil be forgotten. These
may themselves play a part in determining the availability of
various elements by bringing about oxidations and reductions of
compounds of the different trace elements and so affecting their
solubility, or they may play a more direct part in the deficiency
diseases themselves as suggested by the work of Gerretsen and
Ark referred to in an earlier chapter. There is here a wide field
of investigation which at present has scarcely been entered.

In the physiological field the outstanding fact is, of course,
that the trace elements as their designation implies are needed
in very minute amount. This does not make the solution of the
problems of their functions any easier. Because of the small
quantities of them involved in the organism it has been usual to

154 CONCLUDING REMARKS

assume that they play the part of catalysts, although there is
quite a definite suggestion on the part of some workers that the
function of boron is of a different kind from those of man-
ganese, zinc and copper.

Two lines of attack on the role of the trace elements which
have developed during recent years appear to give promise of
fruitful results. The first of these is that displayed in particular
by the work of Keilin and Mann in which compounds involving
the trace elements in their molecules have actually been isolated
and the chemical properties of these compounds determined. It
has been mentioned earlier that several copper compounds and
one zinc compound have been isolated in this way. They all
appear to be protein compounds of the metal concerned, and
two of them, the catechol oxidase of plants and carbonic
anhydrase of animals, are, indeed, enzymes which had been
previously known, and the properties of which were well
established. As far as these results go they thus confirm the view
of the catalytic nature of the trace elements in organisms, and
suggest the possibility that manganese and molybdenum also
will turn out to be essential constituents of enzymes. The further
view that the trace elements are concerned in vital oxidations
and reductions is also supported by our knowledge of the nature
of at least two of these compounds, for catechol oxidase is
definitely an oxidizing enzyme, while carbonic anhydrase is con-
cerned in the release of an end-product of oxidation, carbon
dioxide. The condition of boron is more problematic.

The second promising line of attack on the physiological
aspects of trace elements lies in growing plants under experi-
mental conditions in which the supply of various mineral
nutrients is controlled and determining the resultant effects on
growth and, as far as possible, the fate in the plant of the mineral
elements concerned . The recent work of Shive and his associates
along these lines, which has been dealt with in an earlier chapter
of this book, has already produced very promising results with
regard to the functions of manganese and boron in plants, and
it would seem probable that a development of work along these
lines will serve greatly to increase our understanding of the role
of the trace elements in plants.

The realization of the importance of trace elements in plants

CONCLUDING REMARKS 155

and animals is actually very recent, for, apart from a few pioneer
observations, our present not inconsiderable knowledge of the
subject is the result of work done during the last 25 years, and,
indeed, for the most part, during the last decade. Of late, more
and more workers have been attracted to the investigation of
the problems these elements present to the biologist, and with
the excellent research on these problems now being carried out
in various parts of the world, we may or may not find the list of
essential trace elements has to be increased, but we shall cer-
tainly see a rapid expansion in our knowledge of the physiology
and pathology of plants and animals in relation to the trace
elements.

LIST OF LITERATURE

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Alexander, T. R. (1942). Anatomical and physiological responses of
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Anderssen, F. G. (1932). Chlorosis of deciduous fruit trees due to a
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Ark, P. A. and Thomas, H. E. (1940). Apple die-back in California.
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Arnon, D. I. (1937). Ammonium and nitrate nitrogen nutrition of
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Arnon, D. I. (1938). Microelements in culture -solution experiments
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Arnon, D. I. and Stout, P. R. (1939 a). The essentiality of certain
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Arnon, D. I. and Stout, P. R. (1939 6). Molybdenum as an essential
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Askew, H. O., Chittenden, E. and Thomson, R. H. K. (1936). The use
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Askew, H. O. and Dixon, J. K. (1936). The importance of cobalt in the
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Askew, H. O. and Rigg, T. (1932). Bush sickness. Investigations con-
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Askew, H. O. and Williams, W. R. L. (1939). Brown -spotting of
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Aston, B. C. (1929). Cure of iron starvation (bush sickness) in stock.
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Atack, F. W. (1915). A new reagent for the detection and colorimetric
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LIST OF LITERATURE 157

Barnette, R. M. and Warner, J. D. (1935). A response of chlorotic
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Beath, 0. A., Draize, J. H., Eppson, H. F., Gilbert. C. S. and Mc-
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Beath, O. A., Eppson, H. F. and Gilbert, C. S. (1935). Selenium and
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Bennetts, H. W. and Chapman, F. E. (1937). Copper deficiency in
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158 LIST OF LITERATURE

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Bryan, O. C. and Becker, R. B. (1935). The mineral content of soil
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LIST OF LITERATURE 159

Burstrom, H. (1939). Uber die Schwermetallkatalyse der Nitrat-

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Cahill, V. (1929). Experiments for the control of exanthema in

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Little-leaf or rosette of fruit trees. II. Proc. Amer. Soc. Hort. Sci.

1932, 29, 255-63.

Chandler, W. H., Hoagland, D. R. and Hibbard, P. L. (1934).
Little-leaf or rosette of fruit trees. III. Proc. Amer. Soc. Hort. Sci.

1933, 30, 70-86.

Chandler, W. H., Hoagland, D. R. and Hibbard, P. L. (1935).

Little-leaf or rosette of fruit trees. Proc. Amer. Soc. Hort. Sci. 1934,

32, 11-19.
Coleman, D. R. K. and Gilbert, F. C. (1939). Manganese and caffeine

content of some teas and coffees. Analyst, 64, 726-30.

160 LIST OF LITERATURE

Colwell, W. E. and Lincoln, C. (1942). A comparison of boron defi-
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Cook, J. W. (1941). Rapid method for determination of manganese in
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Cook, R. L. and Miller, C. E. (1939). Some soil factors affecting boron
availability. Proc. Soil Sci. Soc. Amer. 4, 297-301.

Corner, H. H. and Smith, A. M. (1938). The influence of cobalt on
pine disease in sheep. Biochem. J . 32, 1800-5.

Cowling, H. and Miller, E. J. (1941). Determination of small amounts
of zinc in plant materials. Industr. Engng Chem. (Anal, ed.), 13, 145-9.

Cunningham, I. J. (1931). Some biochemical and physiological aspects
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Davidson, Annie M. M. and Mitchell, R. L. (1940). The determination

of cobalt and chromium in soils. J. Soc. Chem. Ind.,Lond., 59, 232—5.
Davis, A. R., Marloth, R. H. and Bishop, C. J. (1928). The inorganic

nutrition of the fungi. I. The relation of calcium and boron to

growth and spore formation. Phytopathology, 18, 949.
Dean, L. A. and Trtjog, E. (1935). Determination of manganese and

magnesium in soils and silicate rocks. Industr. Engng Chem. (Anal.

ed.), 7, 383-5.
Dearborn, C. H. (1942). Boron nutrition of cauliflower in relation to

browning. Bull. Cornell Univ. Agric. Exp. Sta. no. 778.
Dearborn, C. H. and Raleigh, G. J. (1936). A preliminary note on the

control of internal browning of cauliflower by the use of boron.

Proc. Amer. Soc. Hort. Sci. 1935, 33, 622-3.
Dearborn, C. H., Thompson, *H. C. and Raleigh, G. J. (1937). Cauli-
flower browning resulting from a deficiency of boron. Proc. Amer.

Soc. Hort. Sci. 1936, 34, 483-7.
Demaree, J. B., Fowler, E. D. and Crane, H. L. (1933). Report of

progress on experiments to control pecan rosette. Nation. Pecan

Assoc. Bull. Proc. Ann. Conv. 32, 90-9. (Summary in Biol. Abstr. 9,

1225, 1935.)
Dennis, A. C and Dennis, R. W. G. (1939). Boron and plant life. III.

Developments in agriculture and horticulture. Fertil. Feed. St. J.,

19 pp. Feb., Mar. and Apr.
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Developments in agriculture and horticulture, 1939-40. Fertil.

Feed. St. J. 24 pp. Nov. 1940-Feb. 1941.
Dennis, A. C. and Dennis, R. W. G. (1943). Boron and plant life. V.

Developments in agriculture and horticulture, 1940-42. Fertil.

Feed. St. J. 38 pp. in reprint Mar. ; Apr. ; May.
Dennis, R. W. G. (1937). The relation of boron to plant growth. Sci.

Prog. 32, 58-69.
Dennis, R. W. G. ( 1937). Boron and plant life. II. Recent developments

in agriculture and horticulture. Fertil. Feed. St. J. 15 pp., Sept.-Oct.
Dennis, R. W. G. and O'Brien, D. G. (1937). Boron in Agriculture.

Res. Bull. W. Scot. Agric. Coll. no. 5.

LIST OF LITERATURE 161

De Rose, H. R., Eisenmenger, W. S. and Ritchie, W. S. (1938). The
comparative nutritive effects of copper, zinc, chromium and molyb-
denum. Bull. Mass. Agric. Exp. Sta. 1937, no. 347, pp. 18-19.

Draize, J. H. and Beath, O. A. (1935). Observations on the pathology
of blind staggers and alkali disease. Amer. Vet. Med. Ass. J. 86
(N.S. 39), 753-63.

Drake, M., Sieling, D. H. and Scarseth, G. D. (1941). Calcium-boron
ratio as an important factor in controlling boron starvation.
J. Amer. Soc. Agron. 33, 454-62.

Dregne, H. E. and Powers, W. L. (1942). Boron fertilization of alfalfa
and other legumes in Oregon. J. Amer Soc. Agron. 34, 902-12.

Dufrenoy, J. and Reed, H. S. (1934). Pathological effects of the
deficiency or excess of certain ions on the leaves of Citrus plants.
Ann. Agron. N.S. 4, 637-53.

Dufrenoy, J. and Reed, H. S. (1942). Coacervates in physical and bio-
logical systems. Phytopathology, 32, 568—79.

Dunlop, G. (1939). Mineral Deficiencies in Live Stock on British Pastures.
Glasgow: The Scottish Agric. Publ. Co.

Dunlop, G., Innes, J. R. M., Shearer, G. D. and Wells, H. (1939).
'Swayback' studies in North Derbyshire. I. The feeding of copper
to pregnant ewes in the control of 'Swayback'. J. Comp. Path.
52, 259-65.

Dunlop, G. and Wells, H. E. (1938). 'Warfa' ('Swayback') in lambs
in North Derbyshire and its prevention by adding copper supple-
ments to the diet of the ewes during gestation. Vet. Pec. 50,
1175-82.

Dunne, T. C. (1938). 'Wither-tip' or 'Summer Dieback'. J. Agric. W.
Aust. 15 (2nd Ser.), 120-6.

Eaton, F. M. (1935). Boron in soils and irrigation waters and its effect
on plants. Tech. Bull. U.S. Dep. Agric. no. 448.

Eaton, S. V. (1940). Effects of boron deficiency and excess on plants.
Plant Physiol. 15, 95-107.

Eden, A. (1939). The influence of varying copper intake on normal
blood copper of Northumbrian sheep. J. Comp. Path. 52, 249-57.

Eden, A. (1941). Further observations on the blood copper of Northum-
brian sheep. J. Agric. Sci. 31, 186—93.

Eden, A. and Green, H. H. (1940). Micro -determination of copper in
biological material. Biochem. J ..34, 1202-8.

Eisler, B., Rosdahl, K. G. and Theorell, H. (1936). Uber die Mikro-
bestimmung des Kupfers mit Hilfe der lichtelektrischen Photo-
metric. Biochem. Z. 285, 76-7.

Eltinge, E. T. (1936). Effect of boron deficiency upon the structure of
Zea mays. Plant Physiol. 11, 765-78.

Eltinge, E. T. and Reed, H. S. (1940). The effect of zinc deficiency
upon the root of Lycopersicum esculentum. Amer. J. Bot. 27,
331-5.

Elvehjem, C. A. (1935). The biological significance of copper and its
relation to iron metabolism. Physiol. Rev. 15, 471-507.

162 LIST OF LITERATURE

Emerson, R. and Lewis, C. M. (1939). Factors influencing the efficiency

of photosynthesis. Amer. J. Bot. 26, 808-22.
Ferguson, W. S. (1943). The teart pastures of Somerset. IV. The

effect of continuous administration of copper sulphate to dairy

cows. J. Agric. Sci. 33, 116-18.
Ferguson, W. S., Lewis, A. H. and Watson, S. J. (1943). The teart

pastures of Somerset. I. The cause and cure of teartness. J. Agric.

Sci. 33, 44-51.
Filmer, J. F. and Underwood, E. J. (1937), Enzootic marasmus.

Further data concerning the potency of cobalt as a curative and

prophylactic agent. Aust. Vet. J. 13, 57-64.
Finch, A. H. (1933). Pecan rosette, a physiological disease apparently

susceptible to treatment with zinc. Proc. Amer. Hort. Sci. 1932,

29, 264-6.
Finch, A. H. and Kinnison, A. F. (1933). Pecan rosette: soil, chemical

and physiological studies. Tech. Bull. Arizona Agric. Exp. Sta.

no. 47, pp. 407-42.
Fisher, P. L. (1935). Responses of the tomato in solution cultures with

deficiencies and excesses of certain essential elements. Bull. Md

Agric. Exp. Sta. no. 375, pp. 282-98.
Floyd, B. F. (1917). Dieback, or exanthema of Citrus trees. Bull.

Fa Agric. Exp. Sta. no. 140, 31 pp.
Foster, J. S. and Horton, C. A. (1937). Quantitative spectrographs

analysis of biological material. II. Proc. Roy. Soc. B, 123,

422-30.
Foster, J. W. (1939). The heavy metal nutrition of fungi. Bot. Rev.

5, 207-39.
Foster, J. W. and vVaksman, S. A. (1939). The specific effect of zinc

and other heavy metals on growth and fumaric-acid production by

Rhizopus. J. Bact. 37, 599-617.
Fox, H. M. and Ramage, H. (1931). A spectroscopic analysis of animal

tissues. Proc. Roy. Soc. B, 108, 157-73.
Franke, K. W. and Moxon, A. L. (1936). A comparison of the minimum
fatal dose of selenium, tellurium, arsenic and vanadium. J. Phar-
macol. 58, 454-9.
Franke, K. W., Rice, T. D., Johnson, A. G. and Schoening, H. W.
(1934). Report on a preliminary field survey of the so-called
'alkali disease' of livestock. Circ. U.S. Dep. Agric. no. 320, 10 pp.
Frey-Wyssling, A. ( 1935). Die unentbehrlichen Elemente der Pflanzen-
nahrung. Naturwissenschaften, 23, 767-9.

Gallagher, P. H. and Walsh, T. (1943). The susceptibility of cereal
varieties to manganese deficiency. J . Agric. Sci. 33, 197-203.

Gallup, W. D. and Norris, L. C. (1937). Studies on the importance of
manganese in the nutrition of poultry. Poult. Sci. 16, 351-2.

Gallup, W. D. and Norris, L. C. (1938). The essentialness of man-
ganese for the normal development of bone. Science, 87, 18-19.

Gerretsen, F. C. (1937). Manganese deficiency of oats and its relation
to soil bacteria. Ann. Bot., Lond., N.S. 1, 207-30.

LIST OF LITERATURE 163

Gilbert, B. E. and McLean, F. T. (1928). A 'deficiency disease': The

lack of available manganese in a lime-induced chlorosis. Soil Sci.

26, 27-31.
Gile, P. L. (1916). Chlorosis of pineapples induced by manganese and

carbonate of lime. Science, 44, 855-7.
Glasscock, H. H. and Wain, R. L. (1940). Distribution of manganese

in the pea seed in relation to marsh spot. J. Agric. Sci. 30,

132-40.
Gollmick, F. (1936). Der Einfluss von Zink, Eisen und Kupfer und

deren Kombination auf das Wachstum von Aspergillus . niger.

Z. Bakt. ii, 93, 421-42.
Gram, E. (1936). Bormangel og nogle andre mangelsygdomme. Tidsskr.

Planteavl. 41, 401-49.
Greig, J. R., Dryerre, H., Godden, W., Crichton, A. and Ogg, W. G.

(1933). Pine: a disease affecting sheep and young cattle. Vet. J.

89, 99-110.
Griggs Mary A., Johnstin, Ruth and Elledge, Bonnie E. (1941).

Mineral analysis of biological materials. Industr. Engng Chem.

(Anal, ed.), 13, 99-101.
Grizzard, A. L. and Mathews, E. M. (1942). The effect of boron on

seed production of alfalfa. J. Amer. Soc. Agron. 34, 365-8.
Gussow, H. T. (1934). Brown-heart of swede turnips. Rep. Third Imp.

Mycol. Conference, p. 26.

Haas, A. R. C. (1932). Some nutritional aspects in mottle -leaf and other
physiological diseases of Citrus. Hilgardia, 6, 484-559.

Haas, A. R. C. (1937). Zinc relation in mottle-leaf of Citrus. Bot. Gaz.
98, 65-86.

Haas, A. R. C. and Klotz, L. J. (1931). Some anatomical and physio-
logical changes in Citrus produced by boron deficiency. Hilgardia,
5, 175-97.

Haas, A. R. C. and Quale, H. J. (1935). Copper content of Citrus leaves
and fruit in relation to exanthema and fumigation injury. Hilgardia,
9, 143-77.

Hammett, L. P. and Sottery, C. T. (1925). A new reagent for aluminium.
J. Amer. Chem. Soc. 47, 142-3.

Hammond, W. H. (1928). A rapid method for the detection of zinc in
the presence of iron. Chem. Anal. 17, 14.

Hart, E. B., Steenbock, H., Waddell, J. and Elvehjem, C. A. (1928).
Iron in nutrition. VII. Copper as a supplement to iron for hemo-
globin building in the rat. J. Biol. Chem. 77, 797-812.

Harvey, H. W. (1939). Substances controlling the growth of a diatom.
J. Mar. Biol. Ass. U.K. 23, 499-520.

Haselhoff, E. (1913). Uber die Einwirkung von Borverbindungen auf
das Pflanzen wachstum. Landw. Versuchs-Stat. 79/80, 399-429.

Haywood, F. W. and Wood, A. A. R. (1944). Metallurgical Analysis
by means of the Spekker Photo-electric Absorptiometer . London.

Heggeness, H. G. (1942). Effect of boron applications on the incidence
of rust on flax. Plant Physiol. 17, 143-4.

164 LIST OF LITERATURE

Heinicke, A. J., Reuther, W. and Cain, J. C. (1942). Influence of

boron application on preharvest drop of Mcintosh apples. Proc.
Amer. Soc. Hort. Sci. 40, 31-4.
Heintze, S. G. (1938). Readily soluble manganese of soils and marsh

spot of peas. J. Agric. Sci. 28, 175-86.
Heller, V. G.jand Penquite, R. (1937). Factors producing and pre-
venting perosis in chickens. Poult. Sci. 16, 243—6.
Henze, M. (1911). Untersuchungen iiber dans Blut der Ascidien. I.

Mitteilung. Die Vanadiumverbindung der Blutkorperchen. Hoppe-

Seyl.'Z. 72, 494-501.
Hewitt, E. J. (1945). 'Marsh spot' in beans. Nature, 155, 22-3.
Hill, H. and Grant, E. P. (1935). The growth of turnips in artificial

culture. Sci. Agric. 15, 652-9.
Hoagland, D. R. (1941). Water culture experiments on molybdenum

and copper deficiencies of fruit trees. Proc. Amer. Soc. Hort. Sci.

38, 8-12.
Hoagland, D. R. and Arnon, D. I. (1938). The water-culture method for

growing plants without soil. Circ. Univ. Calif. Coll. Agric. no.

347.
Hoagland, D. R., Chandler, W. H. and Hibbard, P. L. (1936).

Little-leaf or rosette of fruit trees. V. Effect of zinc on the growth

of plants of various types in controlled soil and water culture

experiments. Proc. Amer. Soc. Hort. Sci. 1935, 33, 131-41.
Hoagland, D. R., Chandler, W. H. and Stout, P. R. (1937). Little-
leaf or rosette of fruit trees. VI. Further experiments bearing on

the cause of the disease. Proc. Amer. Soc. Hort. Sci. 1936, 34, 210-12.
Hogg, J. (The Ettrick Shepherd) (1831). Remarks on certain diseases of

sheep. Quart. J. Agric. 2, 697-706 (and note by the Editor, 706-12).
Holland, E. B. and Ritchie, W. S. (1939). Report on zinc. J. Ass.

Off. Agric. Chem. 22, 333-8.
Holley, K. T. and Dulin, T. G. (1937). A study of ammonia and

nitrate nitrogen for cotton. IV. Influence of boron concentration.

Bull. Ga Agric. Exp. Sta. no. 197.
Hopkins, E. F. (1930). The necessity and function of manganese in

the growth of Chlorella sp. Science, 72, 609-10.
Hopkins, E. F. (1930). Manganese an essential element for a green alga.

Amer. J. Bot. 17, 1047.
Hopkins, E. F. (1934). Manganese an essential element for green plants.

Mem. Cornell Agric. Exp. Sta. no. 151.
Hopkins, E. F. and Wann, F. B. (1927). Iron requirement for Chlorella.

Bot. Gaz. 84, 407-27.
Hove, E., Elvehjem, C. A. and Hart, E. B. (1937). The physiology of

zinc in the nutrition of the rat. Amer. J. Physiol. 119, 768-75.
Hurd-Karrer, Annie M. (1934). Selenium injury to wheat plants and

its inhibition by sulphur. J. Agric. Res. 49, 343-57.
Hurd-Karrer, Annie M. (1935). Factors affecting the absorption of

selenium from soils by plants. J. Agric. Res. 50, 413-27.
Hurd-Karrer, Annie M. (1937). Selenium absorption by crops as

related to their sulphur requirement. J. Agric. Res. 54, 601-8.

LIST OF LITERATURE 165

Hurd-Karrer, Annie M. and Kennedy, Mary H. (1936). Inhibiting
effect of sulphur in selenized soil on toxicity of wheat to rats.
J. Agric. Res. 52, 933-42.

Hurst, R. R. and MacLeod, D. J. (1936). Turnip brown heart. Sci.
Agric. 17, 209-14.

Innes, J. R. M. and Shearer, G. D. (1940). 'Swayback': A demyelin-

ating disease of lambs with affinities to Schilder's encephalitis in

man. J. Comp. Path. 52, 249-57.
Insko, W. M., Lyons, M. and Martin, J. H. (1938a). The effect of

manganese, zinc, aluminium and iron salts on the incidence of

perosis in chicks. Poult. Sci. 17, 264-9.
Insko, W. M., Lyons, M. and Martin, J. H. (19386). The quantitative

requirement of the growing chick for manganese. J. Nutrit. 15,

621-7.
Isaac, W. E. (1934). Researches on the chlorosis of deciduous fruit trees.

II. Experiments on chlorosis of peach trees. Trans. Roy. Soc. S.

Afr. 22, 187-204.

Jacks, G. V. and Scherbatoff, H. (1934). Soil deficiencies and plant

diseases. Tech. Comm. Imp. Bur. Soil Sci. 31.
Jamalainen, E. A. (1935 a). Tutkimuksia lantun ruskotandista. Valtion

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summary). Cited from Dennis and O'Brien (1937).
Jamalainen, E. A. (1935 6). Der Einfluss steigender Borsauremengen

auf die Kohlrubenernte. J. Agric. Soc. Finland, 7, 182-6. Cited from

Dennis and O'Brien (1937).
Johnston, E. S. and Dore, W. H. (1929). The influence of boron on the

chemical composition and growth of the tomato plant. Plant

Physiol. 4, 31-62.
Johnston, J. C. (1933). Zinc sulfate promising new treatment for mottle

leaf. Calif. Citrograph, 18, 107, 116-18.
Joltvette, J. P. and Walker, J. C. (1943). Effect of boron deficiency

on the histology of garden beet and cabbage. J. Agric. Res. 66,

167-82.
Jones, H. E. and Scarseth, G. D. (1944). The calcium-boron balance

in plants as related to boron needs. Soil Sci. 56, 15—24.
Josephs, H. W. (1932). Studies on iron metabolism and the influence

of copper. J. Biol. Chem. 96, 559-71.

Keil, H. L. and Nelson, V. E. (1931). The role of copper in hemoglobin

regeneration and in reproduction. J. Biol. Chem. 93, 49-57.
Keilin, D. and Mann, T. (1938). Polyphenol oxidase. Purification,

nature and properties. Proc. Roy. Soc. B, 125, 187-204.
Keilin, D. and Mann, T. (1940). Carbonic anhydrase. Purification and

nature of the enzyme. Biochem. J. 34, 1163—76.
Kessell, S. L. and Stoate, T. N. (1936). Plant nutrients and pine

growth. Aust. For. 1, 4—13.
Kessell, S. L. and Stoate, T. N. (1938). Pine nutrition. Bull. W. Aust.

For. Dep. no. 50.

166 LIST OF LITERATURE

Kidson, E. B. and Askew, H. O. (1940). A critical examination of the

nitroso-R-salt method for the determination of cobalt in pastures.

N.Z. J. Sci. Tech. 21 B, 178b-189b.
Kidson, E. B., Askew, H. O. and Dixon, J. K. (1936). Colorimetric

determination of cobalt in soils and animal organs. N.Z. J. Sci.

Tech. 18, 601-7.
Kidson, E. B. and Maunsell, P. W. (1939). The effect of cobalt com-
pounds on the cobalt content of supplementary fodder crops.

N.Z. J. Sci. Tech. 21 A, 125a-128a.
Knight, H. G. (1935). The selenium problem. J. Ass. Off. Agric. Chem.

18, 103-8.
Knop, W. (1860). Ueber die Ernahrung der Pflanzen durch wasserige

Losungen bei Ausschluss des Bodens. Landw. Versuchsst. 2, 65-99,

270-93.
Kolthoff, I. M. and Lingane, J. J. (1941). Polarography . New York.
Kubowitz, F. (1937). Uber die chemische Zusammensetzung der

Kartoffeloxydase. Biochem. Z. 292, 221-9.
Kuyper, J. (1930). Boorzuur tegen de topziekte van de tabak. Deli

Proefstat. te Medan, Sumatra, Vlugschr. 50, 7 pp.

Larsen, C. and Bailey, D. E. (1913). Effect of alkali water on dairy

cows. Bull. S. Dakota Agric. Exp. Sta. no. 147, 300-25.
Larsen, C, White, W. and Bailey, D.E. (1912). Effect of alkali water on

dairy products. Bull. S. Dakota Agric. Exp. Sta. no. 132, pp. 220-

54.
Ledeboer, M. S. J. (1934). Physiologische onderzoekingen over Cerato-

8tomella ulmi (Schwarz) Buisman. Diss. Utrecht.
Lee, H. A. and McHargtje, J. S. (1928). The effect of a manganese

deficiency of the sugar cane plant and its relationship to Pahala

blight of sugar cane. Phytopathology, 18, 775-86.
Lewis, A. H. (1939). Manganese deficiencies in crops. I. Spraying pea

crops with solutions of manganese salts to eliminate marsh spot.

Emp. J. Exp. Agric. 7, 150-4.
Lewis, A. H. (1943a). The teart pastures of Somerset. II. Relation

between soil and teartness. J. Agric. Sci. 33, 52-7.
Lewis, A. H. (19436). The teart pastures of Somerset. III. Reducing the

teartness of pasture herbage. J. Agric. Sci. 33, 58-63.
Lewis, J. C. (1942). The influence of copper and iodine on the growth

of Azotobacter agile. Amer. J. Bot. 29, 207-10.
Lewis, J. C. and Powers, W. L. (1941). Iodine in relation to plant

nutrition. J . Agric. Res. 63, 623-37.
Liebig, G. F., Vanselow, A. P. and Chapman, H. D. (1943). Effects of

gallium and indium on the growth of Citrus plants in solution cul-
tures. Soil Sci. 56, 173-85.
Lingane, J. J. and Kerlinger, H. (1941). Polargraphic determination

of nickel and cobalt. Simultaneous determination in presence of

iron, copper, chromium, and manganese, and determination of

small amounts of nickel in cobalt compounds. Industr. Engng Chem.

(Anal, ed.), 13, 77-80.

LIST OF LITERATURE 167

Lipman, C. B. (1938). Importance of silicon, aluminium and chlorine

for higher plants. Soil Sci. 45, 189-98.
Lipman, C. B. and Mackinney, G. (1932). Proof of the essential nature

of copper deficiency. J. Pomol. 10, 130-46.
Lockwood, L. B. (1933). A study of the physiology of Penicillium

Javanicum Van Beikma with special reference to the production of

fat. Catholic Univ. Amer. Biol. Ser. 13.
Lohnis, M. (1936). Wat Veroorzaakt Kwade Harten in Erwten?

Tijdschr. PIZiekt. 42, 159-67 (with English summary).
Lowenhaupt, B. (1942). Nutritional effects of boron on growth and

development of the sunflower. Bot. Gaz. 104, 316-22.
Lundegaedh, H. (1929). Die Quantitative Spektralanalyse der Elemente.

Jena.
Lundegardh, H. (1932). Die Nahrstoffaufnahme der Pflanze. Jena.
Lundegardh, H. (1934). Die Quantitative Spektralanalyse der Elemente.

Zweite Teil. Jena.
Lundegardh, H. (1936). On spectral analysis of inorganic elements

Landboukhogskolano Ann. (Ann. Agric. Coll. Sweden), 3, 49-97.
Lundegardh, H. (1939). Mangan als Katalysator der Pflanzenatmung.

Planta, 29, 419-26.
Lundegardh, H. and Phdlipson, T. (1938). The spark-in-flame method

for spectral analysis. Landboukhogskolano Ann. (Ann. Agric. Coll.

Sweden), 5, 249-60.
Lyons, M. and Insko, W. M. (1937). Chondrodystrophy in the chick

embryo produced by manganese deficiency in the diet of the hen.

Bull. Ky Agric. Exp. Sta. no. 371, pp. 61-75.
Lyons, M., Insko, W. M. and Martin, J. H. (1938). The effect of

intra-peritoneal injections of manganese, zinc, aluminium, iron

salts on the occurrence of slipped tendon in chicks. Poult. Sci. 17,

12-16.

MacArthur, M. (1940). Histology of some physiological disorders of
the apple fruit. Canad. J. Res. Sect. C, 18, 26-34.

McClelland, J. A. C. and Whalley, H. K. (1941). The Lundegardh
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McHargue, J. S. (1922). The role of manganese in plants. J. Amer.
Chem. Soc. 44, 1592-8.

McHargue, J. S. (1923). Effect of different concentrations of man-
ganese sulphate on the growth of plants in acid and neutral soils
and the necessity of manganese as a plant nutrient. J. Agric. Res.
24, 781-94.

McHargue, J. S. (1926a). Manganese and plant growth. J. Industr.
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McHargue, J. S. (19266). Further evidence that small quantities of
copper, manganese and zinc are factors in the metabolism of
animals. Amer. J. Physiol. 77, 245-55.

McHargue, J. S. and Calfee, R. K. (1931 a). Effect of Mn, Cu and Zn
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168 LIST OF LITERATURE

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170 LIST OF LITERATURE

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172 LIST OF LITERATURE

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174 LIST OF LITERATURE

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Underwood, E. J. and Filmer, J. F. (1935). Enzootic marasmus. The
determination of the biologically potent element (cobalt) in limonite.
Aust. Vet. J., 11, 84-91.

Underwood, E. J. and Harvey, R. J. (1938). Enzootic marasmus:
the cobalt content of soils, pastures and animal organs. Aust. Vet.
J. 14, 183-9.

Van Schreven, D. A. (1934). Uitwendige en inwendige symptomen van
boriumgebrek bij tabak. Tijdschr. PIZiekt. 40, 98-129 (with English
summary).

Van Schreven, D. A. (1935). Uitwendige en inwendige symptomen van
boriumgebrek bij tomaat. Tijdschr. PIZiekt. 41 , 1-26 (with English
summary).

Van Schreven, D. A. (1939). De gezondheidstoestand van de aardap-
pelplant onder den invloed van twaalfelementen. Meded. Inst.
Phytopath. Wageningen, 43, 166 pp. (with English summary).

Vanselow, A. P. and Laurance, B. M. (1936). Spectrographic micro-
determination of zinc. Industr. Engng Chem. (Anal, ed.), 8, 240-2.

Vinogradov, A. P. (1934). Distribution of vanadium in organisms.
G.R. Acad. Sci. U.R.S.S. pp. 454-9 (with English summary).

Waddell, J., Steenbock, H., Elvehjem, C. A. and Hart, E. B. (1929).

Iron in nutrition. IX. Further proof that the anaemia produced on

diets of whole milk and iron is due to a deficiency of copper. J. Biol.

Chem. 83, 251-60.
Waddell, J., Steenbock, H. and Hart, E. B. (1929). Iron in nutrition.

VIII. The ineffectiveness of high doses of iron in curing anaemia in

the rat. J. Biol. Chem. 83, 243-50.
Wadleigh, C. H. and Shive, J. W. (1939). A microchemical study of

the effect of boron deficiency in cotton seedlings. Soil Sci. 47, 33-6.
Walker, J. C. (1939). Internal black spot of garden beet. Phyto-
pathology, 29, 120-8.
Walker, J. C, Jolivette, J. P. and McLean, J. C. (1943). Boron

deficiency in garden and sugar beet. J. Agric. Res. 66, 97-123.
Walker, J. C, McLean, J. G. and Jolivette, J. B. (1541). The boron

deficiency disease in cabbage. J. Agric. Res. 62, 573-87.
Walkley, A. (1942). The determination of zinc in plant materials.

Aust. J. Exp. Biol. Med. Sci. 20, 139-47.

LIST OF LITERATURE 177

Wallace, T. (1943). The Diagnosis of Mineral Deficiencies in Plants.

London. [Supplement, 1944.]
Warington, K. (1923). The effect of boric acid and borax on the

broad bean and certain other plants. Ann. Bot., Lond., 37, 629-72.
Warington, K. (1926). The changes induced in the anatomical structure

of Vicia Faba by the absence of boron from the nutrient solution.

Ann. Bot., Lond., 40, 27-42.
Warington, K. (1934). Studies in the absorption of calcium from

nutrient solutions with special reference to the presence or absence

of boron. Ann. Bot., Lond., 48, 743-76.
Warington, K. (1940). The growth and anatomical structure of the

carrot (Daucus Carota) as affected by boron deficiency. Ann. Appl.

Biol. 27, 176-83.
Webb, D. A. (1937). Studies on the ultimate composition of biological

material. Part II. Spectroscopic analyses of marine invertebrates,

with special reference to the chemical composition of their environ-
ment. Sci. Proc. Roy. Dublin Soc. N.S. 21, 505-39.
Webb, D. A. and Fearon, W. R. (1937). Studies on the ultimate com-
position of biological material. Part I. Aims, scope and methods.

Sci. Proc. Roy. Dublin Soc. N.S. 21, 487-504.
Weinberger, J. H. and Cullinan, F. P. (1937). Symptoms of some

mineral deficiencies in one-year Elberta peach trees. Proc. Amer.

Soc. Hort. Sci. 1936, 34, 249-54.
Whitehead, T. (1935). A note on 'brown -heart', a new disease of

swedes and its control. Welsh J. Agric. 11, 235-6.
Wtickens, G. W. (1925). Exanthema of Citrus trees. Rep. Proc. Imp.

Bot. Conference, London, 1924, pp. 353-7. Cambridge.
Wiese, A. C, Elvehjem, A. C. and Hart, E. B. (1938). Studies on the

prevention of perosis in ttie cluck. Poult. Sci. 17, 33-7.
Wiese, A. C. and Johnson, B. C. (1939). Microdetermination of man-
ganese. J. Biol. Chem. 127 ', 203-9.
Wilcox, L. V. (1940). Determination of boron in plant material. An

ignition-electrometric titration method. Industr. Engng Chem.

(Anal, ed.), 12, 341-3.
Wilgus, H. S., Norris, L. C. and Heuser, G. F. (1936). The role of

certain inorganic elements in the cause and prevention of perosis.

Science, 84, 252-3.
Wilgus, H. S., Norris, L. C. and Heuser, G. F. (1937). The effect of

various calcium and phosphorus, salts on the severity of perosis.

Poult. Sci. 16, 232-7.
Wolff, L. K. and Emmerie, A. (1930). Uber das Wachstum des

Aspergillus niger und den Kupfergehalt des Nahrbodens. Biochem.

Z. 228, 441-50.
Woodward, J. (1699). Some thoughts and experiments concerning

vegetation. Philos. Trans. 21, 193-227.
Wooley, D. W. (1941). Manganese and the growth of lactic acid

bacteria. J. Biol. Chem. 140, 311-12.
Wunsh, D. S. (1937). Tracking down a deficiency disease. Chem. and

Ind. 15, 855-9.

178 LIST OF LITERATURE

Yakwood, C. E. (1942). Stimulatory and toxic effects of copper sprays

on powdery mildews. Amer. J. Bot. 29, 132-5.
Young, R. S. (1935). Certain rarer elements in soils and fertilizers and

their role in plant growth. Mem. Cornell Agric. Exp. Sta. no. 174,

70 pp.

Zlataroff, A. and Kaltschewa, D. (1936). Der Einfluss einiger
Metallsalze auf die Milchsauregarung. Biochem. Z. 284, 12-23.

INDEX

Absorptiometer, 26-7
Absorptiometry determination of alu-
minium, 49

of cobalt, 51

of copper, 42-4

of manganese, 35—7

of molybdenum, 48

of zinc, 40—1
Absorption of manganese retarded by
calcium, 108-9

of selenium by plants, 129-31
Agaricus campestris, see Mushroom
Agulhon, H., 3
Alben, A. O., 72
Aleurites fordii, see Tung
Alewites montana, see Mu-oil tree
Alexander, T. R., 81-2
Alfalfa, deficiency of boron in, 87—8
Algae, trace elements necessary for, 8
Alkali disease, 127-31
Almond, zinc deficiency in, 74
Aluminium, determination of, in plant
material, 48-9

species requiring, 18

as micro-nutrient, 3, 4

in animals, 124-6
Anderssen, F. G., 6, 53, 55-6, 92-4
Andropogon scoparius, selenium con-
tent of, 129
Andropogon sorghum, zinc necessary

for, 71
Anions, effect of boron on absorption

of, 114-15
Antagonism between manganese and

calcium, 107—9
Apical buds, effect of zinc deficiency

on, 70-1
Apple, boron deficiency in, 81, 88-9

copper deficiency in, 90, 92-4

intervenal injection of, 54

leaf stalk injection of, 55

zinc deficiency in, 74

leaves, copper content of, 94
Apricot, boron deficiency in, 89

copper deficiency in, 92

zinc deficiency in, 69, 70-1, 74-5
Arachnida, copper in, 136
Arc spectra, 29-30
Ark, P. A., 75-6, 153

Arnon, D. I., 1, 6, 9, 10, 11, 24-5, 96,
101

Ascidians, vanadium in blood of, 125

Ash, preparation of, 32

Askew, H. O., 51, 89, 138, 146

Asparagus, effect of trace elements on
growth of, 9

Aspergillus flavus, micro-nutrients for, 8

Aspergillus niger, micro-nutrients for,
6-8
trace elements and conidia forma-
tion in, 103

Aston, B. C, 138

Astragalus spp., selenium content of,
129-30

Atack, F. W., 49

Ataxia, enzootic, 139-42

Atomic structure and biological essen-
tiality, 97-100

Auxin, relation of zinc to, 111-12

Avena saliva, see Oat

Bacteria associated with little leaf and

white bud, 75-6
Bailey, D. E., 128
Barium in animals, 125
Barley, boron necessary for, 3
copper deficiency in, 94
copper necessary for, 6
manganese deficiency in, 61
silicon necessary for, 5
zinc necessary for, 3
Barnette, R. M., 3, 77, 79-80
Bayliss, N. S., 78-9
Bean, broad, boron necessary for, 3
broad, effect of boron deficiency on,
81
effect of boron on absorption of
calcium by, 114—17
zinc necessary for, 3
soya, calcium- boron balance in, 116-
17, 121
effect of boron on absorption of

calcium by, 116-17
effect of iron-manganese ratio on
growth of, 104—7
wax, zinc necessary for, 71
Beck, A. J., 103
Becker, R. B., 138

180

INDEX

Beet, boron deficiency in, 82
copper deficiency in, 94
heart-rot of, 57, 83-4
speckled yellows of, 63-4
sugar, calcium-boron balance in, 121
leaves and roots, manganese content
of, 64
Belvoussov, M. A., 84
Bennetts, H. W., 140-2
Berenblum, I., 26
Bertrand, G., 2, 7, 102-3
Beta maritima, see Garden or red beet,

Sugar beet, Mangold
Bishop, C. J., 7
Bishop, W. B. S., 101
Black spot, internal, of red beet, 84-5
Blood of ewes, copper content of, 142
Bobko, E. V., 84
Boron deficiency diseases, 80-9

determination of, in plant material,

44-8
function of, in plants, 98, 100, 113-23
relation of, to carbohydrate and fat

metabolism, 121
species requiring, 16-18
as micro-nutrient, 3
content of plants, 44
in animals, 125
Boron-calcium ratio in plants, 120-1
Bortels, H., 7, 23
Brandenburg, E., 84, 95
Brassica spp., effect of boron deficiency

on, 81-2
Brenchley, W. E., 80-1, 115-16
Broccoli, effect of boron deficiency on,

81-2
Bronzing of tung, 77-8
Brown heart of apples, 89

of swede and turnip, 85-6
Brown-spotting of apricots, 89
Browning of cauliflower, 86-7
Brussels sprouts, effect of boron de-
ficiency on, 81-2
Bryan, O. C, 138
Buckwheat, boron necessary for, 3
chlorine necessary for, 4
zinc necessary for, 3
Buds, apical, effect of zinc deficiency

on, 70
Burrell, A. B., 89
Burrows, F. W., 68
Burstrom, H., 102-3
Bush sickness, 139, 145-50
Byers, H. G., 128-30

Cabbage, boron deficiency in, 82
Cadmium in animals, 124-5
Calcium, effect of, on absorption of
boron, 119-20
effect of, on absorption of man-
ganese, 107—9
effect of boron on absorption of,

114-21
effect of manganese on absorption
of, 107-9
Calcium-boron ratio in plants, 120-1
Calfee, R. K., 8, 45
Callan, R., 42
Camp, A. F., 3, 77-8
Canker of red beet, 84-5
Carbohydrate metabolism, relation of
boron to, 121-3
relation of zinc to, 109-10
Carbonic anhydrase, 112, 150-1, 154
Came, W. M., 89
Carya olivaeformis, see Pecan
Caskey, CD., 144-5
Castor-oil plant, boron necessary for, 3
Catalysts for oxidation-reduction re-
actions, 99, 109-13
Catalytic function of trace elements,

98-101, 112-13, 153-4
Catechol oxidase, 110-13, 154
Cations, effect of boron on absorption
of, 114-21
effect of manganese on absorption of,
107-9
Cattle, cobalt deficiency in, 145-50
cobalt essential for, 127
copper deficiency in, 136-9
scouring of, 127
selenium poisoning of, 127-9
Caughley, R. H., 40
Cauliflower, boron deficiency in, 86-

7
Celery, boron deficiency in, 87
Ceratostomella Ulmi, micro-nutrients

for, 8
Cereals, copper deficiency in, 94-6
Chain, E., 26
Chandler, F. B., 81
Chandler, W. H., 68, 74-7, 110
Chapman, F. E., 141-2
Cherry, effect of zinc deficiency on, 74
Chick, manganese deficiency in, 143-5

manganese essential for, 127
Chittenden, E., 89

Chlorella, effect of trace elements on,
8, 12, 101

INDEX

181

Chlorella, function of manganese in,
103

manganese necessary for, 8
Chlorine, species requiring, 18

as micro-nutrient, 2-4
Chlorophyll formation and trace ele-
ments, 101
Chlorosis of apricot, 92

of French prune, 91

of fruit trees, 6, 55, 74-7

of maize, 61-2, 79-80

of oat, 94-6

of orange, 92

of peach, 92—3

of pecan, 71

of Pinus radiata, 79

of plum, 92-3

of sugar beet, 63—4

of sugar cane, 62-3

of tung, 67-8
Chromium in animals, 125
Citrus, copper deficiency in, 90-4

cytology of mottled leaves of, 69-
70

effect of boron deficiency on, 8 1

zinc deficiency in, 76—7, 109
Coacervation, 110-11
Cobalt, determination of, in soil and
plant material, 49-51

content of soils, 147-9

deficiency in sheep and cattle, 139,
145-50

essential for sheep, 127

in animals, 124—5, 127
Coccomyxa simplex, effect of iron, zinc

and copper on, 8
Cole, J. R., 72

Colorimetric determination of alu-
minium, 49

of boron, 46-7

of cobalt, 51

of molybdenum, 48
Columbium, effect of, on growth of

Penicillium Javanicum, 7, 19
Coleman, D. R. K., 36
Colwell, W. E., 87-8
Cook, J. W., 36
Cook, R. L., 121
Copper, determination of, in ash, 41-4

effect of, on scouring, 135

function of, in animals, 150
in plants, 98-101, 112-13

species requiring, 19

as micro-nutrient, 5-6, 7, 8

Copper content of blood of ewes, 142
content of plants, 92-4, 137
deficiency diseases, 90-6
deficiency in animals, 136-42
essential for haemoglobin formation,

126
in animals, 124-6

Cork, internal, of apples, 88-9

Corky core, 89

Corky pit, 89

Corner, H. H., 147

Cotton, boron necessary for, 3

effect of boron on metabolism of,
122-3

Cowling, H., 40

Cracked stem of celery, 87

Crane, H. L., 74

Crichton, A., 138, 145

Crone, Von der, 1, 2

Crucigina, effect of trace elements on,
12

Crustacea, copper in, 136

elements present in, 124-5, 150

Culture solutions, purification of, 22-6

Cummings, R. W., 32, 38-9, 42

Cunningham, I. J., 136

Cysteine, 109-11

Cystine, 110

Cytology of zinc -deficient leaves, 69-70,
109

Daising, 145-50

Davenport, H. W., 150

Davidson, A. M. M., 51

Davies, W. Morley, 64, 87

Davis, A. R., 7

Dean, L. A., 36, 64

Dearborn, C. H., 86

Deciduous fruit trees, little leaf or

rosette of, 74-6
Deficiencies, diagnosis of mineral, 5 1-6
Deficiency diseases of animals, 136—50

of plants, 57-96
Deficiency of boron, symptoms of, in
plants, 80-3
of boron in plants, 80-9
of copper, symptoms of, in plants, 90
of copper in animals, 136-42

in plants, 90-6
of manganese, symptoms of, in

plants, 57-8
of manganese in animals, 127, 143-5

in plants, 57-68
of molybdenum in plants, 96

182

INDEX

Deficiency of sulphur, 56

of zinc, symptoms of, in plants, 68-71

of zinc in plants, 68-80
Demaree, J. B., 74
Dennis, A. C, 16, 80
Dennis, R. G. W., 3, 16, 80. 85, 89,

114
Dent, K. W., 36

Determination of aluminium in bio-
logical material, 48-9

of cobalt in soil and plant material,
49-51

of copper in ash, 41-4

of manganese in ash, 33-7

of molybdenum in ash, 48

of nickel in soil and plant material,
49-51

of zinc in ash, 37-41
Dewey, D. W., 51
Diagnosis of mineral deficiencies in

plants, 51-6
Dickey, R. D., 67, 78
Die-back of apple trees, 89

of fruit trees, 6, 74, 76, 90-4
Ditylum brightwelli, manganese neces-
sary for, 8
Dixon, J. K., 51, 146
Dore, W. H., 81, 122-3
Dothiorella, boron necessary for, 7
Drake, M., 121
Drought spot, 89
Dry ashing, 32
Dry spot, see Grey speck
Dryerre, H., 138, 145
Dufrenoy, J., 69-70, 101, 109-12
Dulin, T. G., 116
Dunlop, G., 140-1
Dunne, T. C, 93

Eaton, F. M., 123

Echinodermata, elements present in,

124-5
Eden, A., 43-4, 142
Eisler, B., 43

Electrometric titration of boron, 47—8
Ellidge, B. E., 31, 33, 41
Eltinge, E. T., 81-2
Elvehjem, C. A., 136, 144
Emerson, R., 101-2
Emmerie, A., 7
Enzootic ataxia, 139-42
Enzootic marasmus, 145-50
Estimation of aluminium in biological

material, 48—9

Estimation of cobalt in soil and plant
material, 29-51

of copper in ash, 41-4

of manganese in ash, 33-7

of molybdenum in ash, 48

of nickel, 49-51

of zinc in ash, 37-41
Ettrick Shepherd, 145
Ewes, copper content of blood of, 142
Exanthema of fruit trees, 90-4

Fats, relation of boron to, 121
Ferguson, W. S., 132, 135
Filmer, J. F., 146-7
Finch, A. H., 71-4
Fisher, P. L., 81
Flame spectra, 28-9
Flax, boron necessary for, 3
copper necessary for, 6
protective action of boron against
rust of, 123
Floyd, B. F., 91
Fluorine in animals, 124-5
Foster, J. S., 29, 44-5
Foster, J. W., 7, 8
Fowler, E. D., 74

Fowls, manganese deficiency in, 143-5
manganese essential for, 127
selenium poisoning of, 127-8
Fox, H. M., 124-5
Franke, K. W., 128
French prune, copper deficiency in,

90-1
Frenching of Citrus, 76-7
" of mu-oil tree, 67

of tung, 67-8
Frey-Wyssling, A., 99
Fruit trees, deciduous, little leaf or

rosette of, 74-6
Function of boron in plants, 98, 100,
113-23
of copper in animals, 150

in plants, 99
of manganese in plants, 99
of zinc in animals, 150—1
in plants, 99
Functions of trace elements in animals,
150-1
in plants, 97-123

Gaddum, L. W., 42, 139
Gall, O. E., 38
Gallagher, P. H., 61

INDEX

183

Gallium, spectrograph^ estimation of,

31
as micro-nutrient, 6, 8, 20
Gallup, W. D., 144-5
Garden beet, manganese deficiency in,

63-4
Gastropoda, elements present in, 124-5
Gerretsen, F. C, 59-61, 76, 153
Gilbert, F. C, 36
Gilbert, S. G., 107
Gile, P. L., 103
Glasscock, H. H., 65-6
Olyceria aquatica, aluminium necessary

for, 4
Glycine hispida, see Bean, soya
Goats, copper deficiency in, 137
Godden, W., 138, 145
Goitre, 142-3
Gollmick, F„ 7
Gram, E., 94
Grant, E. P., 86

Grape, effect of zinc deficiency on, 74
Grape-fruit leaves, copper content of,

94
Green, H. H., 43-4
Greig, J. R., 138-9
Grey speck, 57, 58-62
Grey spot, see Grey speck
Grey stripe, see Grey speck
Griggs, M. A., 31, 33, 41
Grizzard, A. L., 88
Giissow, H. T., 86

Haas, A. R. C, 81, 91-2, 94
Haemocuprein, 136, 150
Haemocyanine, 136, 150
Haemoglobin, copper essential for

formation of, 126
Hammett, L. P., 49
Hart, E. B., 136, 144
Harvey, H. W., 8
Harvey, R. J., 147
Haywood, F. W., 27
Healy, D. J., 136
Heart-rot of sugar beet and mangold,

57, 83-4
Heggeness, H. G., 123
Heintze, S. G., 65
Heller, V. G., 144
Henderson, J. A. R., 42
Henderson, R. G., 61-2
Henze, M., 125
Hepatocuprein, 136> 150
Heuser, G. F., 143

Heyrovsky, J., 27

Hibbard, F. L., 74-7

Hill, E. S., 136

Hill, H., 86

Hoagland, R. D., 1, 74-7

Hock disease of chicks, 143-5

Hogg, J., 145

Holland, E. B., 40

Holley, K. T., 116

Holmes, A., 139

Hopkins, E. F., 8, 23, 103

Hopkins, F. G., 110

Horses, selenium poisoning of, 127-9

Horton, C. A., 29, 44-5

Humus, effect of, on availability of

manganese 153
Hurd-Karrer, A. M., 128-31

Immersion, leaf, 55

Impatiens balsamina, effect of boron
on absorption by, 114-15

Injection method for diagnosing mineral
deficiencies, 52—6

Innes, J. R. M., 140-2

Insko, W. M., 144

Intensity of spectral lines, measure-
ment of, 30

Internal black spot of red beet, 84-5
cork, 88-9
standard, 29

Intervenal leaf injection, 53-4

Iodine essential for functioning of
thyroid gland, 126-7, 142, 150
in animals, 126, 142-3, 150

Iron-manganese relations in plants, 99,
101-7

Isaac, W. E., 93

Jamalainen, E. A., 81-2, 86
Javillier, M., 7
Johnson, B. C, 37
Johnstin, R., 31, 33, 41
Johnston, E. S., 81, 122
Johnston, J. C, 76-7
Jolivette, J. P., 81-2
Jones, H. E., 121
Josephs, H. W., 136
J uncus effusus, aluminium necessary
for, 4

Keil, H. L., 136

Keilin, D., 112, 126, 136, 150-1, 154

Kennedy, M. H., 130

Kerlinger, H., 50

184

INDEX

Kessell, S. L., 78
Kidson, E. B., 51, 147
Kingery, L. K., 8
Kinnison, A. F., 71-4
Klotz, L. J., 81
Knop, W., 1
Kolthoff, I. M., 27
Kubowitz, F., 112
Kuyper, J., 88

Laccase, 2, 102

Lactuca sativa, see Lettuce

Lambs, swayback in, 139—42

Lamellibranchiata, elements present

in, 124-5
Larsen, C, 128
Laurance, B. M., 38
Leach, R., 53, 55-6
Lead and incidence of swayback, 140—1

in animals, 124—5
Leaf immersion, 53, 55-6

injection, 53-4
Leaf -stalk injection, 55
Leaf-tip injection, 54—5
Ledeboer, M. S. J., 8
Lee, H. A., 62-3
Lemna, gallium necessary for, 6
molybdenum necessary for, 6
Lemon, copper content of, 94
Lettuce, effect of trace elements on

growth of, 9
Levy, J., 38-9
Lewis, A. H., 65, 132-5
Lewis, C. M., 101-2
Lewis, R. D., 72
Licking sickness, 136
Lillie, R. D., 128

Liming, effect of, on availability of
manganese, 153
effect of, on boron deficiency and
toxicity, 120-1
Lincoln, C, 87-8
Lingane, J. J., 27, 50
Lipman, C. B., 3, 5, 21
Lithium in animals, 124—5
Little leaf, 74-7
Lockwood, L. B., 7
Lohnis, M., 65
Lolium subulatum, copper deficiency in,

96
Lowenhaupt, B., 81
Lucerne, deficiency of boron in, 87-
8
yellows, 87-8

Lundegardh, .H., 29, 31, 33, 38, 41,
44-5, 48, 50, 59, 102-3

Lupin, chlorosis induced in, by man-
ganese, 103

Lyons, M., 144

Mac Arthur, M., 81
McDougall, E. I., 142
McHargue, J. S., 8, 45, 62-3, 101, 136
Mackinney, G., 6
McLarty, H. R., 87
McMurtrey, J. E., 88
McNaught, K. J., 51
Maize, aluminium necessary for, 5
chlorosis of, 61-2
effect of boron on absorption of

calcium by, 116-19
manganese deficiency in, 61—2
relation of boron to metabolism of,

121
white bud of, 3, 75, 79-80
zinc deficiency in, 69, 75, 79-80
zinc necessary for, 3, 79-80
roots, effect of iron-manganese ratio
on growth of, 103
Majdel, J., 36

Manganese, determination of, in plant
ash, 33-7
function of, in animals, 150

in plants, 98-109
species requiring, 13-14
as micro -nutrient, 2, 7-8
content of plants, 59, 64, 93
deficiency diseases, 57-68
in barley, 61
in beans, 67
in beet, 63-4
in chicks, 127, 143-5
in maize, 61-2
in mangold, 63
in mu-oil tree, 68
in oat, 57, 58-61
in peas, 64-7
in rye, 61
in spinach, 63
in sugar beet, 57
in sugar cane, 62-3
in tung, 67-8
in wheat, 61
essential for chicks, 127, 143-5
in animals, 124—7
Manganese -iron relations in plants, 99,

101-7
Mangold, heart rot of, 83-4

INDEX

185

Mann, M., 7

Mann, T., 112, 126, 136, 150-1, 154

Marasmus, enzootic, 145-50

Marloth, R. H., 7

Marsh, R. P., 116-18, 120-1

Marsh spot, 64-7

Marston, H. R., 51

Martin, A. L., 131

Martin, D., 89, 100

Martin, J. H., 144

Mathews, E. M., 88

Maunsell, P. W., 147

Maynard, L. A., 143

Maze, P., 3, 11, 13, 110

Melampsora Lini, protective effect of

boron against, 123
Meldrum, N. U., 110, 150
Melvin, E. H., 33, 42, 45
Metz, O., 8

Micro-organisms associated with grey-
speck, 59-61

little leaf and white bud, 75-6
Microphotometer, 30
Miller, C. E., 121
Miller, E. C, 2
Miller, E. J., 40
Miller, J. T., 129
Miller, W. T., 128-9
Millet, aluminium necessary for, 5

silicon necessary for, 5
Milo, zinc necessary for, 71
Minarik, C. E., 116-17, 120
Mineral deficiencies in plants, diagnosis

of, 51-6
Mitchell, R. L., 48, 50-1, 147-9
Mollusca, copper in, 136
Molybdenum, determination of, in
plant material, 48

species requiring, 20

symptoms of deficiency of, 6

as micro -nutrient, 6-7

content of pasture plants, 132-5
of soils, 133, 149

deficiency, symptoms of, 96

poisoning, control of, 135

of cattle and sheep, 127, 132-5
Morner's test, 110
Morris, A. A., 116
Morrison, D. B., 49
Morton Mains disease, 145-50
Mosher, W. A., 8
Mottle leaf of Citrus; 76-7
Mottling of Citrus, 76-7

of deciduous fruit trees, 74

Mowry, H., 3. 77-8

Moxon, A. L., 128

Muhr, G. R., 121

Muir, W. R., 132

Mules, selenium poisoning in, 128-9

Mull, J. W., 49

Mu-oil tree, frenching of, 68

Mushroom, catechol oxidase of, 112

Mustard, boron necessary for, 3

Mycosphaerella striatiformans, 62

Myers, V. C, 49

Naftel, J. A., 46
Nakamura, M., 12
Nakuruitis, 139
Neal, W. M., 138
Nelson, E. M., 128
Nelson, V. E., 136

Nemertea, elements present in, 124-5
Newell, W., 77

Nickel, determination of, in soil and
plant material, 49-51

in animals, 124—5
Nitrogen assimilation, effect of man-
ganese on, 102-3

metabolism, effect of boron on, 122-3
Nobbe, F., 4
Norris, L. C, 143-5
Nutrient solutions, purification of, 22-6

Oat, copper deficiency in, 94-6

effect of trace elements on growth of, 1 1

grey speck disease of, 57, 58—61

manganese content of, 59
deficiency in, 57, 58-61

molybdenum deficiency in, 96
O'Brien, D. G., 3, 85
O'Connor, R. T., 33, 38, 42, 45
Ogg, W. G., 138, 145
Olive, copper deficiency in, 90, 92
Olsen, C, 36
Optical spectroscopic determination of

boron, 45
Orange, copper content of, 94

copper deficiency in, 92

zinc deficiency in, 7
Orr, J. B., 139
Orton, W. A., 72
Oserkowsky, J., 6, 92
Ovinge, A., 65
Oxidase, 110-11, 154
Oxidation-reduction catalysts, 99, 109-
13, 154

regulators, 98-9, 103-7, 109-11

186

INDEX

Oxidations, vital, 98-107, 109-13, 154

Pahala blight, 62

Pea, garden, aluminium as micro-
nutrient for, 5

garden, chlorine as micro -nutrient
for, 4

garden, necessity of zinc for, 11
Peach, copper deficiency in, 92

zinc deficiency in, 69, 70-1, 74
Pear, boron deficiency in, 89

copper deficiency in, 90, 92, 94

effect of zinc deficiency on, 74

intervenal injection of, 54

leaves, copper content of, 92
Pecan, zinc content of, 73

rosette, 71-4
Pectin, relation of boron to, 121
Penicillium Javanicum, effect of trace

elements on growth of, 7, 20
Penquite, R., 144
Perla, D., 143
Porosis, 127, 143-5
Peterman, F. I., 49
Pethybridge, G. H., 65
Pettinger, N. A., 61-2
Pfeffer, W., 1, 2
Phalaris tuberosa, copper deficiency in,

96
Phleum pratense, see Timothy
Photosynthesis and trace elements, 101
Phymatotrichum omnivorum, micro -

nutrients for, 8
Pigs, selenium poisoning of, 127-9
Pineapple, chlorosis induced in, by

manganese, 103
Pining, 145-50
Pinus radiata rosette, 78-9
Piper, C. S., 40, 43-4, 51, 59, 66-7, 94-6
Pisces, elements present in, 124-5
Pisum sativum, see Pea, garden
Pittman, H. A., 91-3
Pleurobranchus plumula, cobalt and

vanadium present in, 125
Plum, copper deficiency in, 90-4

chlorosis of, 55-6

effect of zinc deficiency on, 74

intervenal injection of, 54

leaves, copper content of, 93
Poisoning by selenium, 127-31

by molybdenum, 127
Polarograph, 27

Polarographic determination of alu-
minium, 49

Polarographic determination of cobalt,
50

of copper, 42

of manganese, 34-5

of nickel, 50-1

of zinc, 38-40
Pollen, effect of boron on germination

of, 113-14
Polychaeta, elements present in, 124-5
Polyphenol oxidase, see Catechol oxidase
Potassium, effect of, on absorption of
boron, 119-20

effect of manganese on absorption
of, 107-9
Potato, catechol oxidase of, 112
Poultry, manganese essential for, 127

selenium poisoning of, 127-8
Poverty pit, 89
Pugliese, A., 103

Purification of nutrient media, 22-6
Purvis, E. R., 87

Quale, H. J., 91-2, 94

Raan, 85-6

Rabbits, selenium poisoning of, 128-9

Radish, boron deficiency in, 82

Ramage, H., 29, 124-5, 147

Rand, F. V., 72

Rats, selenium poisoning of, 128-9

Raulin, J., 7

Reclamation disease, 6, 94—6, 136-7

Red beet, manganese deficiency in, 63-4

Reductions, vital, 98-107, 109-13, 154

Reed, H. S., 69-71, 90, 101, 109-12

Reed, J. F., 32, 38-9, 42

Reeve, E., 119-20

Rehm, S., 114-15

Respiration and manganese, 102

Reuther, W., 67-8, 78

Rhizopus nigricans, micro-nutrients for,

8
Rice, silicon necessary for, 5
Rigg, T., 138
Ritchie, W. S., 40
Roach, W. A., 52-5, 152
Roberg, M., 7, 8, 23
Robertson, I. M., 48
Robinson, W. O., 128
Rogers, C. H., 8
Rogers, L. H. 37-8, 42, 139
Rosdahl, K. G., 43
Rosette of apple tree, 89
of pecan, 71-4

INDEX

187

Rosette of Pinus radiata, 78-9
Rosetting, 68

of deciduous fruit trees, 74-6, 92-3
Rubidium in animals, 124
Ruprecht, R. W., 87
Rusoff, L. L., 42, 139
Rutabaga, boron deficiency in, 82
Rye, manganese deficiency in, 61

Sachs, J., 1

Sakamura, T., 23

Salt sick, 138-9

Samuel, G., 59

Saunders, D. H., 8

Scarseth, G. D., 121

Scharrer, K., 103

Schmucker, T., 113

Schoening, H. W., 128

Scholz, W., 103

Schropp, W., 103

Scott, R. O., 149

Scouring of cattle and sheep, 127, 131—5

Seed production, necessity of zinc for,

3-4, 71
Selenium content of plants, 128-31

poisoning, control of, 130-1
of animals, 127-31
Shasta daisy, intervenal injection of, 54
Shealy, A. L., 138
Shearer, G. D., 140-2
Sheep, cobalt deficiency in, 145-50

cobalt essential for, 127

scouring of, 127
Shikata, M., 27

Shive, J. W., 104-7, 116-22, 154
Shoot injection, 53, 56
Shoot-tip injection, 55
Sideris, C. P., 36
Siegert, T., 4
Sieling, D. H., 121
Silicon, species requiring, 18

as micro-nutrient, 3, 5

in animals, 125
Silver in animals, 124-6
Sjollema, B., 6, 94, 136-8, 141
Skelding, A. D., 26
Skok, J., 81-2
Skoog, F., 111-12
Slipped tendon of chicks, 143-5
Smith, A. M., 147
Smith, G. S., 46
Smith, M. E., 78-9
Smith, M. I., 128
Smith, R. E., 90-2

Sodium as micro-nutrient, 2
Soils, cobalt content of, 147-9

effect of liming on boron availability
of, 120-1

effect of sulphur on selenium absorp-
tion from, 130-1

molybdenum content of, 133
Somers, I. I., 104-7
Sommer, A. L., 3, 5-6
Sottery, C. T., 49
Soya bean, effect of iron-manganese

ratio on growth of, 104-7
Spark spectra, 29
Speck, grey, 57, 58-62
Speckled yellows, 63-4
Spectral lines, measurement of in-
tensity of, 30
Spectrograph, 28-32
Spectrographs determination of alu-
minium, 48-9

of boron, 44-5

of cobalt, 50

of copper, 41-2

of manganese, 33-4

of nickel, 50

of zinc, 37-8
Spinach, manganese deficiency in, 63

beet, manganese deficiency in, 63-4
Squash, boron deficiency in, 82
Steenbock, H., 136

Steinberg, R. A., 6-9, 11, 22-3, 99-102
Stewart, A. B., 147-9
Stewart, J., 147-9
Stewart, W. L., 140
Stimulation of growth by trace ele-
ments, 11-12, 20
Stoate, T. N., 78
Stohlman, E. F., 128
Stoklasa, J., 4, 101
Storey, H. H., 55-6
Stout, P. R., 6, 24-5, 38-9, 96
Strawberry, intervenal injection of, 54
Strontium in animals, 124, 126
Sugar beet, heart rot of, 57

speckled yellows of, 63-4
Sugar cane, Pahala blight of, 62
Sulphur, relation of, to absorption of
selenium by plants, 130-1

deficiency of tea, 56
Sulphydryl compounds, 109-10
Summer die-back, 93
Sunflower, aluminium ner.essary for, 5

copper necessary for, 6

silicon necessary for, 5

188

INDEX

Sunflower, zinc necessary for, 3
Swanback, T. R., 107-9, 121
Swayback, 139-42
Swede, boron deficiency in, 82, 85-6
Swine, selenium poisoning in, 127-9

Talibli, G. A., 116

Teart lands, 127, 131-5

Tea-plant yellows, 56

Thatcher, R. W., 97-9, 101, 109, 113

Theorell, H„ 43

Thomas, H. E., 6, 90-3

Thomson, R. H. K., 89

Thornton, H. G., 81

Thyreoglobulin, 142, 150

Thyroid gland, 142-3

Thyroxine, 142, 150

Timothy, effect of trace elements on

growth of, 11
Tin in animals, 125

Titration, electrometric, of boron, 47-8
Tobacco, boron deficiency in, 81, 88
calcium -boron balance in, 121
effect of boron on absorption of

calcium by, 121
effect of manganese on absorption of
potassium and calcium by, 107-9
Tomato, boron deficiency in, 81
copper necessary for, 6
effect of boron on metabolism of,

121-2
effect of calcium and potassium on
absorption of boron by, 119-20
molybdenum deficiency in, 96

necessary for, 6
zinc deficiency in, 69-70
Top sickness of tobacco, 88
Tottingham, W. E., 103
Translocation quotient, 108
Trelease, S. F., 131

Trichophyton inter digitate, micro-nu-
trients for, 8
Truog, E., 36
Tung, bronzing of, 77-8

frenching of, 67-8
Tungsten, effect of, on growth of

Penicillium Javanicum, 7, 20
Turnip, boron deficiency in, 85—6
Twyman, E. S., 11, 36
Twyman, F., 30

Undenas, S., 94
Underhill, F. P., 49
Underwood, E. J., 146-7

Urochordata, elements present in, 124-5

Vanadium in animals, 125
Van Schreven, D. A., 81, 100
Vanselow, A. P., 38
Vicia Faba, see Bean, broad
Vinquish, 145-50
Vitamin B and manganese, 143

Waddell, J., 136

Wadleigh, C. H., 122

Wain, R. L., 65-6

Waksman, S. A., 8

Walker, J. C, 81-2, 84

Walkley, A., 38, 40

Wallace, T., 51, 58, 64, 84-5, 152

Walnut, zinc deficiency in, 74, 77

Walsh, T., 61

Wann, F. B., 23

Warington, K.,3, 80-1, 100, 115-17, 120

Warner, J. D., 3, 79-80

Water culture, 1

Water-relations of protoplasm, effect

of boron on, 113-14
Watson, S. J., 132 135
Webb, D. A., 124-6
Wells, H., 141
Wet ashing, 32
Wheat, copper deficiency in, 96

effect of iron-manganese ratio on
growth of, 103

manganese deficiency in, 61

selenium content of, 128

roots, effect of manganese on nitrogen
assimilation of, 102-3
effect of manganese on respiration
of, 102
White, W., 128

White-bud of maize, 3, 75, 79-80
Wickens, G. W., 91
Wiese, A. C, 37, 144
Wilcox, J. C, 87
Wilcox, L. V., 47
Wilgus, H. S., 143
Williams, K. T., 128-9
Williams, L. C, 38-9
Williams, R. J., 8
Williams, W. R. L., 89
Wingard, A., 61-2
Wither tip, 93
Wolff, L. K., 7
Wood, A. A., 27
Woodbridge, C. G., 87
Woodward, J., 1

INDEX

189

Wunsh, D. S., 146

Yellows of lucerne, 87-8

of tea plant, 56
Yellow-tip, 94
Yellow-top, 87-8
Young, R. S., 11

Zea mais, see Maize

Zinc, determination of, in plant ash,
37-41
function of, in animals, 150-1

in plants, 98-101, 109-12
species requiring, 14-16
as micro-nutrient, 3, 7—8
content of plants, 73
deficiency diseases, 68-80
in almond, 74

Zinc, deficiency diseases, in apple, 74

in apricot, 69-71, 74-5

in Citrus, 69-70, 76-7

in garden pea, 71

in grape, 74

in maize, 69, 75-6. 79-80

in milo, 71

in peach, 69-71, 74

in pear, 74

in pecan, 71-4

in Pinus radiata, 78-9

in plum, 74

in tomato, 69-70

in tung, 77-8

in walnut, 74, 77

in wax bean, 71
in animals, 125-6
Zinc-deficient leaves, cytology of, 69-70