PLANT NEMATOLOGY NOTES

PLANT NEMATOLOGY WORKSHOPS

NORTH CAROLINA STATE COLLEGE

RALEIGH, N. C.

1954

ALABAMA POLYTECHNIC INSTITUTE

AUBURN, ALA.

1955

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Sponsored By The
SOUTHERN REGIONAL NEMATODE PROJECT

(S-19)

PLANT HEMATOLOGY NOTES

PLANT NEMATOLOGY WORKSHOPS

NORTH CAROLINA STATE COLLEGE

RALEIGH, N. C.

1954

ALABAMA POLYTECHNIC INSTITUTE

AUBURN, ALA.

1955

Sponsored By The
SOUTHERN REGIONAL NEMATODE PROJECT

(S-19)

FOREWORD TO 1955 EDITION

These lecture notes from the firct Plant Nematology Workshop, held at
Raleigh, North Carolina, Sept'ember 7 to 18, were prepared for the use
of the students at the Workshop and not for publication.

Instructors at the V/orkshop were: E. J. Cairns, iMabama Polytechn.ic
Institute^ J. N. Sasser, North Carolina State College; and A. L, Ta.^lor,
Section of Nematology, Cf.S.D.A.

This Workshop was a function of the Southern Regional Nematode Project
and was held to provide training in nematology for professional plant
pathologists.

Facilities were furnished by the Division of Plant Pathology, North
Carolina State College.

The services of Mr. A. L. Taylor were made possible through the co-
operation of Dr. G. Steiner of the Section of Nematology, U.S.D.A.

Drawings of nematodes for these notes were made by Dr. Hedwig Hirsch"iann,
North Carolina State College.

Reproduction of the notes was made possible through a grant from the
Rockefeller Foundation.

J. N. Sasser, Chairman
Technical Committee (S-19)

FOREWORD TO 1958 EDITION

Requests for copies of these Plant Nematology Notes soon depleted the
first and second printings of it. Therefore, a final printing has
been prepared and at the same time, extra sets of the new and revised
portions have been made for distribution to owners of the earlier
edition.

This manual was intended as a stopgap measure due to lack of up to
date and available texts j we hope it has served this purpose well.
Gratifying as the demand for the manual has been it is even better to
note that need for it should soon be over. A book dealing with the
plant-parasitic nematodes, the diseases they cause, and their control
is soon to be released by Dr. J. R. Christie. A text, which is to be
more taxonomic in its approach, is being written by Mr. G, Thorne.
The out of print book. Plant Parasitic Nematodes, by T. Goodey is to
revised by J. B. Goodey. Soil and Freshwater Nematodes, also by
T. Goodey and out of print, is to be reissued. In addition, the

number of recent workshops in nematology thnt hnve been held snd

the release of their notes have reduced the need for increasinf?

the scope or making an extensive revision of Plant Nematology Notes.

Appreciation is again acknowledged to Dr. Hedwig Hirschmann for her
work in preparing the new plates for the section on morphology.
Thanks are also due to the various authors from whose works we have
collected information for these Notes.

As before, reproduction of the Notes was made possible through a
grant from the Rockefeller Fovindation. The services rendered by
this Foundation to the southeastern regional plant-nematology program
have set a pattern of activities for all the regions. The result is
an arousal of interest and action in phytonematology, rewarding to
all who are interested in this important subject and who have so
generously supported it with money and active personal participation.

E. J. Cairns, Chairman
Technical Committee (S-19)

First and second editions copyrighted 1955 aJ^d 1958.

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TABLE OF CONTI'OTS

Forwards to 1955 and 1958 Editions

TECHNIQUF^

Tech. A:
B:
C:
D:
E:

Equipment and materials

Outline for processing samples

Isolation of nematodes from soil

Location and isolation of nematodes from plant tissue

Preparation of nemrtode slide moiints

F: Additional techniques and sources of information

FX3RPH0L0GY

Morph.A: General structure of nematodes

B: External characters

C: Digestive system

D: Reproductive system

E: Nervous system

. F: Excretory system

G: Plates I-IV

SYSTEI-IATICS

System. A

B

C

Nematode systematics

Key to the most common soil forms

Key to the females of the Tylenchoidea

PLANT-F/JIASITIC NEMATODES

Paras. A: Cyst-forming nematodes of the genus Heterodera

B: Root-knot nematodes

C: Meadow or root-lesion nematodes

D: Bud and leaf nematodes

E: Stem and bulb nematodes

F: Spiral nematodes

G: Sting nematodes

H: Ring nematodes

I: Stylet nematodes

J: Stubby root nematodes

K: Dagger nematodes

FREE-LIVING MEMA.TODES
Free-liv.A: Identification and biology

CONTROL
Control. A;
B;

Chemical
Other means

MISCELLANEOUS

riisc.A: Useful references

B: Scientific and common names

Tocl.aioue /. :1

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EQUIPMENT AND MATERIALS /fe" >'^'^* '^*'.. 'i*

I. Equipment and Materials for the Laboratory
A. Optical Equipment:

1. Binocular sterescopic microscope of a type possessing a pedestal
base or otherwise mounted so as to permit transmitted illumina-
tion of the specimens. A range of magnifications from low to
high powers is desirable.

2. Honobjective microscope equiped with a mechanical stage, sub-
stage condenser, and objectives ranging from low to high magni-
fications (oil immersion). A set of oculars with powers from
about $1 to l^X and interchangeable binocular body tube are
useful additions. Achromatic lenses are satisfactory for most
work, but an apochromatic oil immersion objective with compensat-
ing eyepieces is much to be preferred for critical examinations
requiring maximum resolution at high magnification.

3. Illumination froia an electric illuminator of a condenser type
capable of providing the Koehler system of illumination is
desirable, if detailed microscooic examinations of the nematodes
are to be" made .

U. Measurements of nematodes and their parts v/ill require an ocular
micrometer disc. A filiar micrometer eyeoiece is a useful acces-
sory for very accurate measurements, but it is not a necessity.
Calibration of the ocular disc or the filiar micrometer will
require access to a stage micrometer,

5. Camera lucida apparatus is also useful, if not necessary. Most
nematoiogists dispense with the small mirror provided and instead
use a large-sized, front- surfaced mirror. A source of these
mirrors, which are made to size and stocked just for this purpose,
is the Pancro Mirrors Co. Inc., 2958 Los Feliz Blvd., Los Angeles
39, California. The item is listed as camera lucida mirror, 6X9
inches, 1/8 inch thick mirror- quality glass, front-surfaced Pancro
coating. The mirror is mounted on a piece of wood, and a very
satisfactory assembly for the mirror can be made from standard
Flexiframe supports obtainable from scientific supply houses.

B. Slide Making Materials:

1. Microscope slides of the 3X1 inch size are most used. The best
quality, non-corrosive slides should be used for permanent slides.
Clinical grade or other lees highly finished or selected grades
are satisfactory for routine work. A special metal slide which
holds the nematode specimens moiinted between two coverglasses is
obtainable. This device permits study of both sides of the

Tech. A: 2

mounted specimen. Equipment for making these slides is available
on loan from various laboratories. Contact one of the authors
for more information.

2, Covfar-gLasses of best quality are to be preferred. They should be
of #0 thickness for routine work. Circles of 3/U inch diameter
are generally used. Square cover-slips may be satisfactory and
are less expensive,

3. Slide sealing materials are of various kinds. An ideal sealing
cement ZUT was devised by G, Thome (1935) and is widely used by
phytonematologists. It is used to seal water, formalin, T A F,
lactophenol, and glycerine m.ounts. It dries quickly and resists
action of solvents used for removing immersion oil from the
cover-slips. ZUT is obtainable in pint and quart amounts from;
Bennett's, 65 'iest First South Street, Salt Lake City 10, Utah.
The recommended thinner for ZUT is butyl acetate. Ethyl acetate
has been found to be a satisfactory suiDstitute, and acetone may
also be used, but the latter may produce small bubbles in the
applied ZUT seal.

Lactophenol gum is a cement used to seal lactophenol mounts. The
directions for its preparation (Davis, 192ii) are as follows:
Dissolve 38 grams of pure gum arable in 50 ml, of distilled water,
add 5 grams of glucose and 6 grams of lactophenol. The solution
is then filtered through glass-wool. Lactophenol consists of a
solution made by mixing 3 parts melted phenol, 1 part lactic acid,
2 parts glycerine, and 1 part water.

Other slide sealing cements include: Cleared, used to seal
lactophenol mounts, obtainable from H. W. Clark, 5Ul9 - 32nd
Street, N. W., Washington, D. C; par af fin- vaseline mixtiire)
50-50, for temporary water and formalin mounts; gold-size varnish
and bakelite resin varnish and other materials can be used, pro-
vided that they are not effected by the mounting medium or by the
solvents used to remove immersion oil.

A slide-ringing turntable is not necessary. When used for making
neat seals of round cover-glasses, extra precaution should be
taken to provide a seal of sufficient thickness, particularly
if the ringing compound is thinned for easier application. Seals
applied freehand are likely to be thicker, and round as well as
square cover-glasses can be safely sealed in this manner, even
if somewhat less neatly. Sources of turntables are the Will
Corp., Ho Chester 3, N. Y.j and the Southern Scientific Co., Inc.,
Atlanta 3, Georgia.

U. Cover-glass supports are necessary to prevent distortion of the
nematode specimens by the pressure of the cover-glass, liJhatever
the kind of support used, it is imperative that it be only
slightly thicker than the cross-sectional diameter of the nematodes.

Tech. A:3

This is necessary in orJor that tlie oil immersion objective,
which has a veiy limited working distance, may be focused on and
within the nematode without contacting the cover-glass. An
excellent means of supporting the cover-glass is by use of short
lengths of glass-wool fibers. Glass-wool is readily obtained
and will be found to have strands of various diameters, so that
supports of the optimum thickness can be selected. Thick sup- .
ports can be made from pieces of cover-glasses which come in
various thicknesses, pieces of slides, pieces of drawn glass rod
or tubing, plastic, or from numerous other materials.

One method of support is to apply a ring of ZUT or other inert
material to a slide, making a shallow well of desired depth.
This involves some practice in getting support of the correct
thickness. This method is worthy of consideration and refine-
ment to a more controlled degree, as it offers certain advantages,
particularly to the beginner, in the preparation of good slides.

C. Additional Special Laboratory Equipment:

1. Manipulation of nematodes, individually and without harm, can be
done in either~of two ways. They can be picked up on needles of
one kind or another or drawn up into a fine-tipped pipette, pro-
vided with a rubbir bulb or connected to a tube and mouthpeice
for oral operation. The use of a needle is usually preferred,
because liquid is unavoidably carried over with the nematodes,
when a pipette is used, which may not be desired.

Ordinary sewing needles of fine size can be used when inserted
in a suitable holder. They are improved by oxidizing in an open
flame which roughens the needle siurface. Cacti needles are
excellent, although not always readily obtained. Bristles, .
strands, or loops of hair mounted in a holder have also been used.

Long a favorite with nematologists has been the use of a needle
whittled from barnboo. Using a razor blade and observing progress
under the dissecting microscope, it is oossible to get a very
fine-tipped needle which is probably unsurpassed for picking such
tiny objects as nematode eggs out of water. Leaving the outer
hard covering of the bamboo piece as the actual needle end gives
added resilience and moisture resistance to the needle.

A very durable and satisfactory needle for manipulating larval
and adult nematodes has been found in the use of dental pulp
canal files. These spring metal, needle-like files can be fur-
ther improved by grinding or filing the tips to a fine point and
by slightly bending the^last eighth of an inch or so of the tip.
A local dentist may have such pulp canal files in stock or may
be able to procure them from his supplier. The manufacturer of
these files is the Kerr Manufacturing Cto., Detroit 3, i'lich. The
■•;. A. Lockvjood Dental Co., 1722 "I" Gt., I'J.W., V/ashington, U. C,

Tecb. A: It

is one source for this item which is designated as Kerr pulp
canal files, style D, No. 1, and are packaged one-half dozen
per box.

2. Slide making can be much easier if one has forceps having fine
pointed tips and weak spring tension. Such forceps are best for
handling the glass-wool strands used for cover-glass supports
and for handling the cover-slips. Low-cost forceps, having the
desired weak spring tension, are obtainable from scientific
supply houses as "analytical weight" forceps. Stainless steel
is preferable to brass. The tips of these forceps can be filed
or ground to the desired shape.

Surgical eye-knives are used for excising nematode heads and
tails for special slides. They are also of use in cutting the
body of root-knot females for identification to species by
perineal pattern study. These knives are made in Europe, and,
according to the manufacturer, they are now available in this
country, but only through medical supply houses.

The company will be glad to advise of surgical distributors for
any locality upon writing to the Kny-Scheerer Ctorp., 35 East 17th
St., New York 3, N. Y. One source which has been advised of the
special use of these knives and stocks the item is McKessen &
itobbins, Inc., Surgical Dept., 1706 First Ave., BiiT.iingham 3,
Ala. The description of the knife is No. 322U Wheeler's dis-
cission knife.

The general usefiiLness of this tool warrants purchasing two, one
of which should be used only for cutting nematodes. The cutting
edges can be renewed on a fine hone, and these knives, although
expensive, have a useful life of many years. Cutting nematodes
placed on a piece of celluloid or plastic will prolong the blade's
keen edge.

3. Observation dishes for working with the nematodes should be
stocked in good numbers. The most useful type of dish is the
Syracuse watch glass; a few dozen will be needed. These dishes
are ver'y convenient in that they can be stacked, thus preventing
evaporation of the water in which nematodes are almost invari-
ably maintained for observation, counting, and other purposes.

A similar type of dish, but of much smaller size, is the watch
glass, U. S. Bureau of Plant Industry Model, which was developed
for nematological work. A dozen or so of these will be found
useful. VJhen a small dish, having a flatter bottom surface, is
needed, as when counting nematodes, the 60mm. "Petri" dish may
be used. The Arthur H.* Thomas, Co., West Washington Square,
Philadelphia 5, Penn., is a source of the "B.P.I." watch glasses.
Syracuse watch glasses and small "Petri" dishes are obtainable
at all laboratory supply companies.

Tech. A:5

D. Sample Processin;^ Equipment:

Processing plant or soil samples to recover tivi nematodes pres-
ent involves separating the nematodes from the soil particles or
plant tissues, getting the nematodes into suspension in water,
and then concentrating the nematodes into a small voliune of v/ater
for examination and for other purposes. Various means of accom-
plishing these three steps have been devised and have been used
in different combinations. The equipment involved can be very
simple, as is described here, or it may take the form of machines,
usually utilising the saiiie principles, but requiring more descrip-
tion than space herein will allow.

1. A blendor, such as the Waring blender or its equivalent, is used
for facilitating the rapid recovery of nematodes from plant tis-
sues, which are reduced to small fragments by the blendor.

2. Pails or pans for containing soil and water are used for the
decatitation-sieving method. Galvanized pails which neat together
are more durable and space-saving than enameled pails. The 12
quart size is satisfactory. Four to srx containers should be
obtained with two as the minimum. Plastic pails are suitable.

3. Sieves in a series of meshes of 2$, 60, 200, and 270 should be
acquired. The addition of a 100 and a 325 mesh sieve may be
desirable, but it is not necessary. The eight-inch diameter, nest-
type sieve is stocked by all scientific supply companies. A
smaller five-inch type is available from some companies, but will
not be as generally useful as the larger size. A set of small
sieves of about two or three-inch diameter for use in rjpid re-
moval of nematodes from small samples is useful to have but is
not commercially stocked. Such a set is easily made by solder-
ing, fusing, or cementing sieve screening to small tin or plastic
dishes or suitable size and shape.

U. Sieve supports will eliminate the need for holding a sieve by
hand while pouring the nematode-soil suspension in water through
it. A simple, effective support can be made by shaping aluminum
clothesline wire into the form of a ring the size of the sieve,
with three or four lugs twisted outwards around the border. In
use, the sieve fits in the ring which is then placed over the
pail or pan and is supported there by the lugs. Similar sieve
holders can be made of wood or metal strips. Another possibility
is to make a stand to hold the sieve over the container.

5. Funnels are used for isolation of nematodes from soil and plant
materials; this m.uch used item, of equipment is referred to as the
Baerinann funnel. In its simplest form, the Daermann funnel con-
sists of a fimnel with a short piece of flexible tubing attached
to the stem and a clamp to close the tubing. The sample is
either supported on or enclosed in a porous material and immersed
in water with which the funnel is filled. Separation of the

Tech. A: 6

nematodes from the saimile occurs when the nematodes, moving
about in the water-soaked sample, eventually reach and pass
through the porous boundry which, however, holds back the soil
or plant particles. The nematode r; sink to the sides of the
funnel, and gradually move or drift to the apex of the funnel
and accumulate in the funnel stem and rubber tubing just above
the clamp. Releasing the clamp at intervals over a period of
hours or days permits collection of the nematodes, concentrated
in a relatively small voli:mie of water and reasonably free from
soil and plant debris.

There are many variations in actual practice, all utilizing the
principle of the Baermann funnel; there are more variations than
it is possible to describe here. However, a few generalizations
are offered which will be helpful in \inder standing the signifi-
cance of the results obtained and in devising improvements.

Most of the nematodes which come through the porous barrier do
so only because of their own activities. Nematodes which are
nonaally not very active or which are sluggish because of
insufficient oxygen, starvation, excessive bacterial contamina-
tion, or too cold a teraperatui^e are not likely to be recovered
in high percentages. Random movement is probably the rule rather
tha.'i tropistic movement, so the larger the surface area to vol-
ume ratio of the s;imple, the greater the chances of recovery of
nematodes. The shorter the distance from the sample to the
claraped-end of the furmel and the steeper the walls of the fun-
nel, the higher the collection rate. It is, of course, obvious
that the sample itself should be crumbled or cut into small frag-
ments to facilitate fr-eeing the nematodes.

In practice, funnels of about five to six inches in diameter are
satisfactory for small samples or for residues from the sieves
placed in the funnel for further cleaning. Funnels of about
eight inches in diameter are good for moderate sized soil or
plant samples which are placed directly in the funnel without
previous sieving. Verylarge funnels made of sheet metal can be
used to recover nematodes from large samples put to soak in the
funnel. Funnels may be of glass, metal, or plastics.

The steeper the angle of the conical section, the larger the
bore of the stem, and the shorter the stem, the better; both as
an aid to recovery of more nematodes and for ease of cleaning
after each use. Guard against using funnels designed for filter-
ing purposes which have a constriction in the bore of the stem
at the point of its union with the conical part of the funnel.
Cut the stem short, leaving support for attaching the tubing.
Spring-action hose clamps are preferable to the screw-type, in
order to have adequate control over the small amoimts of liquid
to be draim from the funnel.

Supports for the soil or plant samples, Xirhen placed in the funnel,

Tech. A:'

may be of vari'ius materials used in different Xirays. Unbleached
muslin or similar cloth serves the purpose vxell. Small piec-s
of cloth can be fastened in the top of the funnel by means of
clothespins, by lapping the covers over a ring of suitable
diameter, or by cutting and sewing into the shape of a skull-
cap with a ring sevm in its border. Cloth is re-usable after
thorough washing in hot water as a safeguard against carry-over
of nematodes from one sample to the next.

Aluminum clothesline Xirire is excellent for making supporting
rings, as it is easily formed and does not rust. Paper, when
properly supported, can also be used to hold the sample in the
Baerraann funnel. Toilet tissue, paper toweling, facial tissues,
or paper handkerchiefs (some of which are water resistant) can
be used. A disc cut from plastic or non-rusting metalic screen-
ing serves as a support for the paper, which, of course, is
discarded after each use.

6. Funnel racks are a necessity if a considerable number of Baennann
funnels are to be set up in the laboratory. A simple manner of
holding the funnels is to insert them into holes of about one and
one half inches diameter cut in pieces of wood shelving boards.

A slot should be cut from the edge of the board to each hole and
be wide enough to allow passage of the funnel stems. Tapering
the sides of the holes xTill help to hold the funnels more stable.
Use of a large sized Dipe reamer is satisfactory for this purpose.

The boards can be assembled or mounted in various ways as the
laboratory space permits. Sxamnles are: assemblies similar to
bookshelves, mountings such as racks over a xrark table, or
shelves along the wall. The one precaution to observe is that
there is no chance for water dripping or leaking from funnels
into others located below.

7. Soil processing sinks should be planned for, if one has the
opportunity to do so. Hovjever, any sink can serve the purpose,
if a few precautions are observed. The greatest hazard is the
accumulation of soil in the plumbing system leading from the sink
drain. Special traps are available for installation in place of
the usual sink drain traps. These special traps retain the larg-
er soil Particles and are easily cleaned out. In some sink
installations, the drainage can be piped directly to pits or
settling tanks outside the laboratory.

The sink itself should be large enough to acconmodate two pails,
and, if possible, have a swing-type faucet high enough to clear
the tops of the pails. A faucet which provides for mixing of
hot and cold water makes it possible to avoid use of water which
may be too cold. Stone laboratory sinks are rugged and serve
well for this soil processing ^^fork. Itegular porcelainod iron oi-
steel sinks can be used, if protected from abraaion by rubber

Tech. A-.r,

sinlc-mats or by wooden-slat racks in the bottom.

A useful addition is to have either a separate water outlet, to
which a piece of tubing can be fastened, or a spray-hose sink
attacliment. These are used in directing a strea/n or spray of
water on the sieves and for flushing soil out of the pails and
sink. It has been found that an Inexpensive aerorator device on
the faucet will result in stimulated activity of the nematodes
and is, therefore, highly recommended for installation at any
sink where samples are to be processed or where water for the
Baermann funnels is obtained.

8. Soil disposal is a matter requiring special consideration, if
there is a chance that nematodes which are new to the locality
may be brought in and become dispersed. Soil processing arrange-
ments having settling tanks, sumps, or pits can be treated peri-
odically with a poisonous chemical. This will reduce the hazard
of overflow or run-off water carrying viable nematodes. The
accumulated soil can be treated by chemicals, including soil
fumigants.

In laboratories where the processed sample residues are not passed
into special drains, these materials can be accumulated in pails
and carried out of the lab to special locations for contaminated
materials. Nematodes in these residues can b e killed by steam
sterilization, fumigation, or, in some cases, by retaining in such
a manner that plant-parasitic nematodes would be starved, des-
sicated, or subjected to lethal exposures of light, heat, or cold.

II. Equipment and Mat-?rial for the Field

A. A shovel is needed for digging plants v^xhen field examinations of the
root systems are necessary. The roots should be dug so that the
small roots remain intact. Remove the plant and its adhering soil
ball from the ground, and knock the soil from off the roots onto the
shovel blade. This assures getting the soil which was in closest
contact VJith the roots ard thus having the highest concentration of
nematodes of interest.

B. Soil sampling tubes and soil augers can be used to get samples from
various depths in the ground and are an excellent means of quiclcly
getting numerous, relatively small samples for consolidation into a
larger, single sample, such as may be desired for survey work. The
soil tube is preferable to the soil auger in most localities, as the
soil sample is retained in the tube and can be readily stripped out
of the slot in the side of ^ the tube and caught in the sample con-
tainer. Soil tubes which require tapping or a rami-od for removal of
the soil core are not as satisfactory as the above mentioned slotted-
type. Soil augers may have to be used where the soil is rocky or
very hard. The disadvantages of the auger are its slowness in use

Tcnh. A: 9

and the fact that dry or loose soils may fall off as the auj^er is
withdravm from the ground. Both soil tubes and soil auf^ers should
be obtained, however, in order to be able to obtain soil samples
from any type or condition of the soil to be encountered. One
source for soil sainple tubes and soil augers is the National Agri-
culture Supply Co.., Fort Atkinson, Wis.

It is important that, whenever possible, the soil samples should be
taken from the rhizosphere of the plant. Soil sampling tubes or
augers can serve to do this very well when it is not desired or
possible to uproot an entire plant because of its special value or
large size.

C. Sanple containers can be of various sorts, the only requirement
being that they are moisture resistant. The usual soil sample is
about one pint to one quart in size. Satisfactory containers
include: round ice- cream- type cartons; freezer-type boxes which
can be folded flat, a space-saving feature; moisture-resistant
lined paper ba5;;s; and plastic freezer- t^^De bags. This latter type
of container is ideal because of availability, low cost, durability,
and compactness when empty.

Plant specimens must be kept from drying out before examinations.
Roots can be left in the soil ball or replaced in the soil which
had been knocked off for field checking of the roots. Plastic bags
are particularly useful for packing plants, as they can be slipped
over the roots and soil mass and tie! securely about the plant's
stem.

All samples should be labeled when taken, and the use of vraterproof
pencils and heavy-stock paper is recommended. This is particularly
important, if there is to be an appreciable delay before processing
the sample, and in all cases, if the label is placed inside the con-
tainer. The moistiu'e from condensation which developes in the closed
contai.iers can quickly obliterate the pencil or ink writing and dis-
integrate ordinary papor.

Samples, when prevented from drying out, can be kept for several
days before being examined. If periods of extended delay before
processing can be conducted are necessary, the samples, perhaps, are
best stored under refrigeration, although this evidently is not tnie
for all kindi of the nematodes.

D. Miscellaneous. Another recommended item of field equipment is a
coarse brush whj ch is used to clean soil from sampling tools and
from the operator's shoes and pants cuffs. This is an important
safeguard when collecting eamplas in locations where the lilcelihood
of inadvertantly transporting and spreading a nematode oest must be
considered. Examples of this are: wliere an apparently new occur-
rence of a. nematode is being checked, where a quarantine is in
effect, and where there are separate planting sites which may not
have the same kinds of nematodes.

Tech. B:l

OUl'LIi^-E ?0K PROCBSoING SAMPLES

1. The complete and, therefore, the best sample consists of plants and
soil from the plant's rhizosphere.

2. The samples shoidd be reasonably representative of the condition
involved, whether it is only a part of a single plant, a row or
spot, or an extensive planting.

3. The samples should be maintained in such a manner that nematodes
present will not be dead from overheating or dessication upon
arrival at the laboratory or before processing.

Soil and Plant Samples

I. Soil with:'

Cyst forming nematodes

A. Dried

Recovery of "floaters"

B. Not dried
Decanting &. Sieving

Non-cyst forming nematodes

A. Baer-mann funnel

B. Decanting c^ Sieving

C. Combination of both

D. Direct observation

E. Seinhorst Slutriator apparatus

II. Plant with:

Endo- and/or Ectoparasitic nematodes

A. Direct examination

itoots and foliage for nematodes
and symptoms of nematode damage.

B. Isolation of nematodes

1. Teasing out

2. Soaking

3. Baermann funnel

U. Seinhorst extraction technique

5. Incubation method

6. Blendor and Sieving

C. Staining in situ

1. Osmic acid methods

*2. Lactophenol methods

3. Bromphenol or bromthymol method

U. Aceto-osmimn method

Tecli. G:l

ISOLATION OF NWATODES FROM SOIL

Cyst-f onuing nematodes.

A. Dry cysts will float at the surface of the water when added to the
soil sample. The material thus floating is poured, screened,
ladled off, or in some other manner removed, and then placed on

a 25 mesh sieve nested with a 60 mesh sieve. Both sieves are then
thoroughly washed with water. The residue from the 60 mesh sieve
is washed off and then distributed in shallow layers in Syracuse
watch-glasses prior to searching for cysts under the binocular
dissecting microscope.

The soil may be deliberately permitted to dry out or force dried in
order to get the cysts into the condition of being "floaters."

B. Non-dry cysts, whether they float or sink, may be recovered from
non-dried soil by roiling the soil in a pail of water, allowing it
to settle briefly, and then passing the decanted suspension through
25 and 60 mesh sieves. The process is repeated until only sand and
gravel remain in the pail. The residue from the 60 mesh sieve is
examined for the presence of nematode cysts.

It has been found that differential stain which colors the debris
and leaves the cysts unstained facilitates finding the cysts even in
lightly infested samples. (Taylor, A. L., J. Feldmesser, and
G. Fassuloitis, 1952) Stains siritable for this purpose are janus
green, brilliant green, malachite green, and gentian violet. The
time required for staining depends on the concentration of the
stain. Staining times of one hour are satisfactory with one gram
of janus green in h,000 ml. of water, one gram of brilliant green
or gentian violet in 32,000 ml. of water, or one gram of malachite
green in 6U,000 ml. of water. Staining overnight will require
solutions of one half these concentrations.

Non-cyst forming nematodes in the soil should never be permitted to
dry out, because some species are killed by dessication, and, in
any case, the number of forms recoverable from the sample will be
reduced by drying.

A. Baermann funnels will hold small volumes of soil from which the

nematodes can be extracted, largely as a result of their own efforts,
as explained previously. Small samples can, therefore, be put
directly on the funnels without any previous treatment.

Soil particles are most likely to settle into the funnel witliin the
first few minutes after adding the water and the soil. Per\nit these
particles to accumulate in the stem of the funnel, and after a short
period, draw off in a small volume of water and replace at the top
of the funnel. This practice will result in samples that are much

Tech. C:2

less obscured by debris, thus facilitating the examination for
nematodes in the Syracuse watch-glasses into which the nematode
suspensions have been drai«i off from the funnels.

Small volumes of the water from the funnels should be withdrawn
periodically over a period of 12 to 2U hours, or longer if desired.
The water in the funnel should be maintained at a level submerging
the sample.

Decanting and sieving is the method most used for samples ranging
up to a pint or, perhaps, a quart in size. The principle of decant-
ing involves washing the nematodes free from the soil particles by
roiling the sample in water, allowing the heavier soil particles to
settle momentarily, and then pouring off the supernatant liquid in
which the nematodes are suspended.

Sieves operate in two ways in removing the various kinds and sizes
of nematodes from the nematode suspension which is poured through
them. If the diameter of a nematode is greater than that of the
opening of the sieve, the nematode is retained by the sieve. How-
ever, if the diameter of the nematode is less than that of the
sieve openings, the nematode will pass through, unless it is caught
by hanging across the wires of the sieve. This latter condition
exists with mfiny of the typically eel-shaped plant-parasitic nema-
todes which have body diameters less than the hi^mm. diameter open-
ings of the finest, or 325 mesh screen. The following table illus-
trates the relationship of the sieve mesh number to the sieve's
value in processing samples for nematodes:

Mesh numbers: Effective for: Manner of operation:

25 Retains debris and large Too large to pass
soil particles through sieve

50-60 Retains cyst-forming Too large to pass
nematodes through sieve

100 Retains eel-shaped

nematodes of large size Most of the nema-
todes are caught

200 Retains nematodes of only \-ihen they hang
most sizes except the across the wire mesh,
smallest forms with the exception

of large nematodes

270 Retains all nematode which may not pass
sizes; s'ilted water tlirough the 270 or
flows through readily. 325 sieve openings,

325 Retains all nematode sizes; (as above)
sizes; silted water flows
flows through slowly.

Try;h. C:3

It will be apparent that more than one washing of the soil may be
required to get most of the nematodes of a sample into suspension.
Also, more than one passage through the finest mesh sieve used will
be necessary to catch the nematodes which are caught only when they
hang by chance over the wire mesh.

A good compromise between speed and efficiency in processing soil
samples consists of two or three washings of the sample and passage
of the supernatant suspension through the finest mesh sieve five
separate times, removing and saving the residues from this sieve
after each sieving. The choice of sieves will depend on the objec-
tive in mind, a good general series consists of: 2$ mesh to remove
large debris and soil particles; 60 mesh if cyst forms are being
sought, otherwise, it is eliminated from the series; and 270 mesh
for retention of all other nematodes. The 325 mesh clogs too
readily with most soils other than sandy types. Five repeated
sievings can usually be made with the 270 mesh sieve in the time
required to sieve only once or twice with the 325 mesh sieve. It
should be remembered that repeated sieving is necessary for higher
rates of recovery of small nematode species which are caught only
when they by chance hang over the fine mesh of the sieve. Thus,
the advantage lies with the finest mesh sieve which permits rapid
flow of silt laden water through it.

It may be worthwhile to note that sieves can be made of cloth, if
it is necessary to improvise. In fact, some workers use fine silk
bolting or flour miller's cloth for the fine-meshed series of sieves.

C. Combinations of Decanting and Sieving with the Baermann funnel tech-
nique. The value of combining the;, e two methods of processing a
soil sample (Christie and Perry, 1951) lies in the freedom from
debris and soil particles which results when the residues from the
sieves are placed on the Baermann funnel for 2\\ hours. A recent
modification of this method (Feder and Feldmesser, 195U) substi-
tutes a fritted glass Buchner funnel for the usual Baermann arrange-
ment. This permits use of vacuim filtration. This method is also
of general value in the handling of nematodes in suspension in
fixatives, stains, disinfectants, wash solutions, and toxicants.

D. Direct observation of a very small soil sample soaked in water in a
watch-glass is one manner of finding nematodes which may be of
value under certain conditions. The time consuming, tedious nature
of such a technique limits its general use. This method is, of
course, the most accurate way of recovering all the nematodes from
a sample, alive or dead. However, the very small size of the
sample leaves doubt as to its representative value.

E. Seinhorst Elutriator apparatus. A recently developed method for
quantitative extraction of nematodes was introduced into this
country by Dr. J. W. Seinhorst during his visit from the Netherlands.
A review of methods for determination of nematodes in soil samples

Tech. C:)4

and a complete d'^scription of the elutri?tor apparatus is available
(SeJn.horst, 1956).

The elutriator apparatus was developed for work involvinp removal
of a high percentage of nematodes from heavy clay soils; two of the
most troublesome aspects of soil processing. The elutriator appa-
ratus works well with all kinds of soil, resulting in a high rate
of removal, of the nematodes. The resulting samples will be found
to be much freer from debris and silty water. Another noteworthy
feature is that this can be a standardized technique, yj.elding
more comparable results between any laboratories equipped with the
elutriator apparatus.

The accompanying illustrations will assist in planning an installa-
tion of this apparatus in having the glassware and multiple sieves
fabricated. One source for obtaining the glass items and suitable
rubber stoppers is llr. D. E. Sampson, Scientific Supply Room, Uni-
versity of North Carolina, Chapel Hill, North Carolina. If no
special device is used for holding the rubber stopper at bottom of
the glass column, difficulty in keeping an ordinary rubber stopper
in place against the weight of the water and soil may result. A
special kind of stopper, stocked by Arthur H. Thomas Company, will
hold in place vrell. (Semi-solid type, Catalog No. 8806, HR-IO8,
size lOj.) The elutriator glassware parts diagrammed have to be
made by a glassworker but represent a design suitable for standard
glass materials available in this country.

The diagram of an installation consisting of a bank of five elu-
triators illustrates a convenient arrangement, utilizing readily
available materials and incorporating design features suggested by
Dr. Seinhorst. The photographs are used with the permission of
Dr. Seinhorst and illustrate two types of multiple sieves which vrill
be of use to anyone who uses sieving whether as a part of the Sein-
horst elutriator method or in the other methods described. Having
the sieves stacked in this manner obviously results in a great
saving of time and labor while retaining the desired feature of
several passages of the nematode suspension through the fine-mesh
final sieve. One person using the bank of five elutriators as
illustrated can routinely process at least forty-five samples per
work day.

It is advisable to purchase a duplicate set of the flasks and the
stems which fit the flasks so that a second lot of samples can be
prepared while one set is being processed in the elutriator. Also,
these portions of the apparatus are the most subject to breakage.

Another aspect of this method that should be noted by all who apply
the Baermann funnel method for a final cleaning and concentrating
step is the utilization of petri dishes in place of the funnels. A
simplification of the procedure discribed by Seinhorst that has been
tested and found very satisfactory is to pour the accumulated wash-
ings of the final sieves directly on to a double thickness of Scott

Tfcti. C:$

facinl tissues supported in a shallow wire dish formed frorr/ vindow
screen. The screen dish and its tissue paper filter are then placed
in a covered petri dish containing just enough water to keep the
tissue wet. Very few, if any, of the nematodes are poured through
the tissue, but once in the petri dish where conditions for aeration
and locomotion are ideal, the nemas work through the tissue and
screen to the water beneath. Most of the nematodes will be in water
of the petri dish by h hours, but routinely a 21; hour time is
allowed for convenience and for maximum removal of nematodes. Tests
have shown this procedure to be superior to use of the Baermann
funnel in cleanliness of the sample, numbers and viability of the
nematodes, and rapidity.

Tech. D:l

LOCATION AND ISOUTION OF NEt4AT0D'?S FRQ-l PLANT TISSU-^S

A. Direct examination of plant tissues is often an important part of
processing the plant sample in order to find nematodes and to dis-
cover evidence of their activities. The latter is frequently the
only clue to the presence of nematodes of the external-feeding or
ectoparasitic type, particularly when an inadequate soil sample
accompanies the specimen. The use of the binocular, steroscopic,
dissecting microscope aids in examination of the suspected plant
parts. Immersing the material in a shallow dish of water is
usually necessary when fine root structures are being examined or
if looking for nematodes at the plant's surface.

The typical eel-shaped nematode is usually seen as a glistening,
white or translucent form, often first noted because of its body
movement. Swollen, sedentary females and cysts are usually opal-
escent, but may range in colors from white or yellow to brown.
Some forms may be obscured by the matrix of their egg masses or by
aggregations of small soil particles adhering to them. Teasing the
plant tissues apart is usually necessary. Tissues in advance of
lesions may harbor more parasitic forms than in obviously decayed
areas.

Nematode disease symptoms are for the most part not to be considered
as having exact diagnostic value because other organisms and con-
ditions do cause similar plant responses. The best rule is to make
the diagnosis on the basis of the nematode forms recovered from the
host tissues or, in the case of root ectoparasites, on the basis of
neraatodes recovered from the rhizosphere. However, there is con-
siderable value in using plant symptoms as clues to the possibility
of plant-parasitic nematodes being involved. Roots are therefore
checked for galls, lesions, cortical sloughing, stunting, dead root-
tips, and distortions. Foliar parts are checked for lesions, dis-
colorations, and distortions.

B. Isolation of nematodes from pLant tissues can be done in a number of
ways, the choice depending upon the kind of nematode involved, the
type and size of the sample, and the quantity of nematodes required.

1. Teasing of suspected diseased plant parts in a watch-glass con-
taining water is usually the manner in which plant samples are
first examined and nematodes recovered for identification.

2. Prolonged soaking of small fragments of plant parts in water may
be necessary for the recovery of nematodes from light infections
or from tissues which have become dry. Breaking or cutting the
plant parts into small pieces expedites the release or movement
of nematodes from the tissues in all of the methods suggested
here. A harmless wetting agent such as Triton may also have value
when added to water used for washing or soaking of samples in any
of these methods.

Tech. D:2

3. The Bar-rmann funnel technique is applicable to plant tissues in
the sai'ne maiiner as with t;oil samples. The plant parts are reduced
to small pieces and are submerged in water in the funnel. As
decay of the tissues may quickly develop and is detrimental to
the nematodes, small portions of the liquid should be drawn fre-
quently from the funnel and clean aerated watar added to them.
If a prolonged retention of the plant parts in the funnel is
necessary, it is better to transfer the tissues to another funnel
containing clean water every day or two.

U. Seinhorst extraction technique. Seinhorst (1950) has devised an
extraction apparatus which overcomes the -ibove mentioned defect
of excessive bacterial cont£tmination and the resulting reduction
of oxygen in the water. Infested material is put in funnels in
the usual manner and sprayed with a mist of water from nozzles
located above. The excess water not entering the funnels is
caught on trays and floxiis away. The spray water slowly passing
around and through the tissues in the funnels gently flows out
the stem of the funnel into shallow trays below. The extracted
nematodes settle to the bottom of these collection trays and the
excess water passes through overflow outlets. The nematodes can
be collected and concentrated and are ready for use or examina-
tion.

5. The incubation method proposed by Youjig ( 19514 ) was found to be
more efficient than the Eaennann funnel technique for recovery
of burrowing and meadovr nematodes from avocado roots. In this
method, washed roots are collected in a covered container, such
as a Mason jar, with a small amount of water, to maintain a
humid atmosphere, and are incubated at room temperature.

Within a few hours, nematodes start accumulating with the small
amovint of wash vfater which drains from the roots to the bottom
of the container. These nonatodes are collected for examination
ii. Syracuse vratch-glasses. V/ater in the jar is maintained by
occasional additions from a wash bottle or pipette which, when
applied to the roots, washes down more nematodes. The roots can
be kept for periods up to several weeks, if care is taken to
keep the amount of standing water in the container small. If
cultures become overly contaminated, they may be flushed out
while Tn the jars with several changes of water. Nematodes can
be recovered from this wash water by use of the sleve-Baemiann
funnel combination process.

Some suggested refinements of this technique include splitting
the roots lengthwise, stripping back the cortex of larger roots,
and the use of antil)iotics or other fungal and bacterial inhibi-
tors, which are known fo be harmless to stylet-bearing nematodes.
Examples of theFO include penicillin, calciijn propionate, and
the conimon sulfa dru'S. These materials may also have value for
retarding contaminant growth in water held in the Baemiann funnel.

Tech. D:3

6. The blendor and sieving method developed by Taylor and Loegering
(1953) is essentially a means of freeing the nematodes from the
plant tissues so that they can be recovered by sieving. The
method is rapid because it does not depend upon the nematodes
moving out of the tissues by their own activity. The method
should be used with caution if infections are light, as some of
the specimens may be destroyed by the action of the blades or
may not be freed from the plant tissues. In practice, the plant
parts are cut into short pieces and placed in the blendor in a
small volume of water. The blendor is operated for about 10-20
seconds or until the plant pieces are reduced to small fragments.
The suspension is then washed simultaneously through a 2$ mesh
sieve to remove large debris particles, through a 6O mesh to
remove cyst-forming and female root-knot nematodes, and through
a 200 to 325 mesh sieve to remove other sizes and types of nema-
todes.

This method is also useful for getting pieces of the cuticle of
female root-knot nematodes free from internal contents for exami-
nation and photomicrographs of peiuneal patterns. The residue
from the 60 mesh sieve is searched for these fragments which
will be numerous and recognizable if heavily infected roots have
been used. The differential stains previously mentioned for use
with the cyst-nematode recovery techniques may also have value
in this process,

C. Staining in situ provides a means for detecting and studying nana-
todes within plant tissues without the necessity of making serially
sectioned slides v;ith the aid of a microtome. Plant parts treated
by the methods described may retain enough stain to be more or less
opaque if the tissues are too thick. The remedy for this is to
split the tissues.

Several different methods for in situ staining of root and foliar
parts for nematodes are presented.

Tech. D:ii

FIXATION TOR N^xIjVTIZR]} PXJ')TS

(Araberger technique; Flentrning Medium fixative; acetic acid-glycerine
clearing ; pure glycerine mounting . )

Section of Nematology, Eeltsville, Maryland

Flemming Medium fixative: 1% chromic acid solution 375 cc.

glacial acetic acid 25 cc.

distilled water 50 cc.

1 gram osmic acid (in sealed vial)
Mix the chromic acid solution, the glacial acetic acid, and the distilled
water in a graduate. Pour about 25 to 50 cc. of the solution into a
double- stoppered bottle that has been thoroughly cleaned. Avoid getting
any oil — even finger marks — where the fixative will touch. Remove the
labels from the outside of the osmic acid vial and clean its surface
with alcohol, again avoiding any finger marks. Drop vial into the
double- stoppered bottle, and break it by stamping down with a cleaned
heavy glass rod, thereby releasing the osmic acid. Immediately pour on
the rest of the chromic acid-acetic acid solution.

Store bottle in a dark container, away from light.

Place washed roots in the fixative, using only as much fixative as will
cover the roots, in a small, well-corked bottle. Allow to stand 1, 2,
or 3 days, depending on size of roots and color desired.

Wash in running water 1, 2, or 3 days.

Transfer by steps (20^, 30^, UO^i alcohol, several hours to 1 day each)
to S0% alcohol. For this a bottle or closely- covered glass dish pay be
used.

Transfer roots to an open Syracuse watch-glass or Petri dish, depending
on the quantity of roots in the lot.

Make up a solution of \ glacial acetic acid and \ pure glycerine; keep
in a drop-bottle.

Drop acetic-glycerine solution into dish containing roots gradually
during the period of a week or less, depending upon temperature and
humidity conditions, allowing the alcohol to evaporate at the same time
that the acetic-glycerine is penetrating the roots. For a small quantity
of roots, well covered by the S0% alcohol, in a Syracuse watch-glass,
the process may be begun with 5 to 7 drops of acetic- glycerine in the
morning, the same amount in the afternoon, then gradually increasing the
amount dropped during the next few days.

As each quantity of acetic-glycerine is added, agitate or stir the dish
to facilitate uniform mixing.

Tech. D:^

Keep dish uncovered during daytime to promote evaporation. Cover at
night or when preparation cannot be watched. Depending on atmospheric
conditions, adjust the cover to increase or decrease evaporation and
differentiation, so that after the U to 7 day period the roots lie in
a pure acetic-glycerine solution.

Roots may now be mounted in pure gylcerine. Nematodes within roots
should be black, root tissue pale yellow and clear.

Roots treated with Flemiriing strong fixative

Flamming Fixative, strong formula, consists of:

1% chromic acid 1$ parts

2% osmic acid k parts

Hoots are washed free of soil and immersed in water heated to 70-80° C
for 1-2 minutes in order to kill the nematodes within. The roots are
then transferred to the fixative for 10- 1^ minutes, the degree of
staining can be controlled by observation under the binocular stereo-
scopic microscope. Wash roots in running water for several hours or
overnight. Dehydrate by passage through a graded alcohol series to
absolute. Clear in clove oil and, if permanent slides are desired,
mount in Canada balsam.

Foliar parts treated with Flemming strong fixative

In order to get satisfactory in situ staining of tissues containing
chlorophyll it is necessary to pretreat before staining. Godfrey (1935)
recommended treatment with hot acetone prior to soaking in the Flemming
fixative.

Drop small pieces of the plant material into boiling acetone in a small
conical flask held in a water bath. Boil for a few minutes and leave
in the slowly cooling acetone until the green color is removed. Wash
in several changes of water and then transfer into the Flemming fixa-
tive. Observe the process under the microscope and remove from the
stain when the nematodes are sufficiently blackened. Wash in running
water several hours or overnight. Dehydrate, clear, and mount in balsam,
euparal, or diaphane, if permanent preparations are desired.

Osmic acid is quite expensive, and although it gives excellent results
in the methods cited, other techniques of in situ staining have been
developed.

Rapid method of staining for nematodes in roots
A modification of the original lactophenol plus cotton blue or acid

Tech. D:6

fuchsin method (Goodey 1937) was developed (McBeth, Taylor, and Smith
19hl) in order to eliminate the necessity for dehydrating the tissue.

Goodey' s formula for staining is used as follows:

Phenol, pure crystals 20 g.

Lactic acid 20 g.

Glycerine hO g.

Distilled water 20 ml.

Acid fuchsin or cotton blue ^ ml. (1 g. to 100 ml. water)

The washed roots are boiled in the staining solution for one minute.
Wash in tap water to remove excess stain and place in lactophenol solu-
tion vmtil cleared. The same lactophenol formula is used for clearing
that is used for staining, except that no stain is added. Clearing
requires from one to several days, depending upon the type of roots and
intensity of the stain. After clearing, the roots can be left in lac-
tophenol or in glycerine for examination. If permanent mounts are
desired, the material can be rtounted directly in glycerine.

In some cases, better results are obtained by using a weaker stain con-
centration, -g- ml. of stain solution in 100 ml. of clear lactophenol.
Boil the plant parts for two minutes in clear lactophenol, then for one
minute in the stain, or for two minutes in the stain.

Leaves stained in cotton blue lactophenol (Franklin 19U9)

Prepare a 0.1 per cent solution of cotton blue in lactophenol (stock
lactophenol for this purpose consists of phenol, 20 g.; lactic acid,
20 g.; glycerine, UO g.; and water, 20 g.).

Plunge leaves into boiling 0.1 per cent cotton blue lactophenol and
keep submerged in the gently boiling stain for 3-5 minutes. Leave in
the stain, which is permitted to cool. Remove tissues and wash in run-
ning water or in $0 per cent alcohol to eliminate excess stain. Clear
in lactophenol or in concentrated phenol. Concentrated phenol acts
more quickly and removes the chlorophyll. More or less permanent
mounts can be made by transferring the plant material through 50 and 70
per cent alcohol to iso-butyl alcohol and moimting in euparal. If
euparal gets cloudy before mounting is completed, warm the slide gently
until cloudiness disappears. The American equivalent for euparal is
diaphane. Mounting in this medium may be from either 9^ per cent or
absolute alcohol, but one should work quickly to avoid clouding.

Itoots of some plants, particularly of trees, are not satisfactorily
stained by the preceeding methods because of oils and other materials
present in the roots. These types of roots will reouire staining and
sectioning by standard methods. The procedures recommended for demon-
strating f\ingi and bacteria within plant tissues are also useful to
demonstrate nematodes. These methods are to be found i/i other refer-
ences (e.g. fiawlins 1936).

Toch. D:7

NZMTODE STAINING IN D2/iD LEAVES (MINDERMN 19^6)

A Ketiiod whereby nematodes even in opaque bro^^n leaves can be observed.
The leaves are bleached with hydrogen peroxide and made transparent by
immersion in lactophenol. Nematodes ?nd other organisms are stained
by appropriate stains dissolved in lactophenol.

Damp leaves or pieces of them are covered with the following bleaching
mixture :

Water 3 parts by volume

20/0 airononia solution 1 part " "

30^ hydrogen peroxide 1 " " "

Leave from 1 to 2U hours, depending upon the material to be bleached.
Wash in water. Stain by pouring a hot stain solution (± 65° C.) over
the leaves. After about 5 m:mutes, pour off dye and replace with pure
lactophenol until no appreciable ajnount of dye comes out of the leaves.
The specimens are mounted in lactophenol and sealed xd.th "Zut" if
permanent slides are desired. Acid Fuchsin and Cottonblue in 0.05^
solutions xjere found most suitable for nematodes from a wide selection
of stains tested for this purpose.

ACETO-OSMIUK IN SITU METHOD FOR ROOTS (TARJAN AND FORD 195?)

This method was developed for observing nematodes in citrus feeder
roots which do not stain satisfactorily using conventional methods
because of the presence of suberin and other unsaturated compounds.

The staining schedule found most effective was as follows: Insert
washed roots into the fixing and staining solution at 52° C. for 2
hours. (V/ater, 16 parts; 10% acetic acid, 10 parts; and 2% aqueous
osmium tetroxide, 2 parts.) Was.h stained roots for 1 hour in running
x-irater. Bleach in 10-30$ hydrogen peroxide at 32° C. until the dark-
ened tissues lighten perceptibly, then wash several times i-jlth water.
Pass through 70$, 90$, and 100$ ethanol at 52° C., 1/2 hour in each.
Immerse in methyl salicylate at 52° 0. until tissues clear, after
which the roots may be stored indefinitely in this liquid at room
temperatures. Although other clearing agents such as clove oil are
just as effective, methyl salicylate (synthetic oil of xidntergreen)
may be preferred due to its inoffensive odor.

Tissues that have been treated but still are opaque can be transferred
back through 95$ ethanol directly into the bleach and then passed up
through the alcohol series to the clearing agent again. Fxxcess expo-
sure to hydrogen peroxide will result in excessive destaining of the
.nematodes and, in some caset;, will cause disintegration of the roots.

(Nob included in ropyripht) Tech. D:8

BROMT'UFWI, OR BROITTHYMOL IN SITU STAIN FOR NEMATOTIFS
J. D. Kirkpq trick and W. F. Mai
Cornell University, Ithaca, New York

Clearing is by means of a bleach which makes possible the use of a
wide variety of stains. In the procedure described using pH indicator
dyes the color development and contrast obtained between plaiit and
nematode tissue apparently is due to the greater buffer capacity of
the nematode tissue. These maintain the absorbed dye in the pH range
in which it is most highly colored. The nematode tissues apparently
absorb considerable ajmounts of the dye. These two factors combined
result in good color contrast.

Bromphenol blue gives a deep blue to green color to the nematodes and
eggs contrasted with a very light yellow or clear background of plant
tissue. Orange or light green color contrasted with a very light
yellow background are obtained when using bromthymol blue or bromcresol
green.

Kill fresh material with F.P.A. (formalin, 5 ml.', propionic acid, 5 ml.;
and 5'0^ ethanol, 90 ml.). Rinse in ^0% ethanol and then place in water.
In the above steps higher alcohol concentrations dehydrate, twist, and
deform the nematodes; xylol destroys them. Drain water fiom the mate-
rial and then transfer to 1% sodium hypochlorite until "cleared." Drain,
rinse 3-h times with water to remove excess NaOCl2, rinse in ^0% ethanol
in which it can be stored. Transfer to 1% bromphenol blue in ^0% eth-
anol. and leave for at least h hours (0.0^-0.1^ in ^0% ethanol for 12-16
hours if more detail is required). Staining time is not critical and
can be extended; some destaining is possible by leaving material in S0%
ethanol for several days.

Drain off stain, rinse with ^0% ethanol until wash is faintly yellow,
drain briefly and transfer to a petri dish containing 0,2^ v/v acetic
acid in 50^ ethanol. (Too much acid will destroy the buffer capacity
of the tissues.) Observe the material for nematodes and eggs while
the material is in the petri dish. For semi -permanent mounts the
tissues can be transferred to water for several minutes and then
mounted in glycerol. For permanent mounts the tissues can be trans-
ferred through a series consisting of 50^ ethanol, 70/S ethanol, and
70% isobutanol (1-2 minutes in each) and mounted in diaphane (euparal).
If greater detail is desired of the plant cell walls, the tissue seg-
ments are placed in an aqueous solution of 1:50,000 safranin for 10
minutes, or until the desired degree of staining is obtained. Do this
after removal of the tissue from the acetic acid step and before pro-
cessing into semi -permanent or permanent mounts. Store all mounts away
from bright light.

Smaller rootlets may be ruined by the "clearing" times required for
the larger roots. The bleach appears to disintegrate nematodes that
were dead prior to sarm^ling and fixing. Prolonged immersion in the
bleach will dissolve swollen female root-knot and cyst nematodes, but
the eggs persist much longer. The stain, which is expensive, can be
used repeatedly when saved and filtered through cloth.

Tech. L:l

PhEPAhATION OF NWIATODE SLIDE MOUNTS

Accurate identification of plant and soil nematodes usually requires
the preparation of micrcscope slide mounts suitable for examination of
the specimens with the aid of the oil immersion objective lens. The
reader is referred to the section listinf^ laboratory equipment and
materials for details and discussions pertaining to manipulation of
nematodes and supplies needed for slide making.

Usually the nematodes recovered from the soil or plant samples are
collected in Syracuse watch-glasses, in water and reasonably free from
soil and plant debris. Processed materials preserved in % formalin
and kept in vials may be emptied into watch-glasses in preparation for
slide making. The nematode samples are then examined with the aid of
the binocular dissecting microscope and the desired nematodes selected,
removed, and made into slide mounts. It should be noted that adult
specimens are required for identification to species and that it is
always worthwhile to mount several specimens of each kind of nematode
present. Usually considerable n\imbers of individual nematodes can be
mounted on a single slide, although it may be necessary to mount nema-
todes with relatively thick bodies separately from thinner nematodes.

Nematodes may be examined while alive, but usually it is necessary to
inactivate them for detailed study. The very delicate complex struc-
ture of the nematodes requires appropriate means for killing, fixing,
and preserving; and the methods found satisfactory have been few. The
following table summarizes the types of slide preparations presented
in this manual.

Type of Mematode Mount Temporary Semi-permanent Permanent

Cyst forms (Heterodera) water
Root-knot (Meloidogyne)

lactophenol
S% formalin
TAF

lactophenol

Eel-shaoed forms

water

"^t formalin

TAF

glycerine

Head, tail and methyl-

cross-section mounts cellulose

glycerine-
jelly

Helaxin,^ the nematodes prior to killing and fixing them is usually
done to reduce th° likelihood of distortion of the nematode in the
fixative. Heat is most generally used for this purpose. Ihe nema-
todes can be placed in a drop of water on a slide or put into a
depression slide and gently heated until activity ceases. Some workers
maintain an incubator at 52° C. for standardizing this heat treatment.
Larger samples of nematodes can be heated by adding a small amount of
hot water or by placing the sample, concentrated by centrifugation, in
a hot water bath at 65° C. for at least two minutes.

Tec?). I<;:2

Fixation of the nematodes after relaxing is accomplished either by-
transferring the nematodes to the fixative or by removing the excess
water from about the nematodes and adding the fixative. Nematodes
concentrated in the bottom of a centrifuge tube or settled in a vial
of water are fixed by the addition of a volime of double strength
fixative equal to the volume of water present in the tube or vial.
Mijx the contents promptly by shaking.

Formxilae for various commonly used fixatives and mounting media are
given with notations on their use.

Formalin Fixative

5/0 formaldehyde is the concentration most frequently used and is pre-
pared from:

Stock formalin 5 ml. 133 ml.

(37.5^ formaldehyde) or

i"/ater 32.5 ml. to make one liter

The nematodes can be transferred directly to a drop of this fixative on
a slide, glass-wool supports added, and the coverglass sealed carefully.
This type of slide mount may last for as long as several months or
longer. Distortion of the cuticle and of the oesophagus may be noted
for some species when fixed and preserved in this material. This has
for many years been the fixative used for routine identification work,

TAP Fixative

(Courtney, Polley, and Miller 1955):

Water distilled 91 ml.

UO^ commercial formaldehyde solution 7 ml.
Triethanolamine 2 ml.

This new fixative, called TAF (triethanolamine formalin), has given
excellent fixation and preservation of specimens which have lasted for
more than two years. It is said to eliminate the objectionable features
of the ordinary S% formaldehyde solution.

Living nematodes may be placed directly in the cold fixative, or they
may be first relaxed. Although it is still too soon to know how long
permanent slides of nematodes fixed in TAF will last, good slides have
been obtained by the modified process of Baker, which is included in
this section of the manual.

Formalin - Aceto - Alcohol

Numerous variations of this popular type of fixative have been used
for fixation of nematodes, one recomra ended proportion is:

Tech. E:3

^$!. ethyl alcohol 15 or 20 ml.
Glacial acetic acid 1 ml.

Formalin (UO/^ f onnaldehyde) 6 ml.

Distilled water UO ml.

After relaxing by gentle heat, the nematodes are covered with the
fixative and left there for 2[i-U8 hours. A slight staining of the
nematodes can be obtained if a few drops of saturated picric acid are
added to the fixative.

Lacto-phenol Solution

Melted phenol 3 parts

Lactic acid 1 part

Glycerine 2 parts

Water 1 part

Cj'st-forms and female root-knot nematodes can be mounted directly from
water or formalin into lactophenol solution for examination of cuticu-
lar details. The cover-glass need not be sealed, as this material
evaporates at a very slow rate.

Permanent Slides

Slides of soil and plant nematodes capable of lasting many years can
be made but are usually, and perhaps wrongly, considered to involve
too much trouble for routine work. However, specimens of taxonomic
value should be made into permanent mounts. Two methods for making
such slides are presented. The first method requires a lengthy lapse
of time to allow dehydration of the nematodes to occur and is the
method developed by G. Thome of the Section of Nematology, Nematodes
on slides prepared in this manner over 30 years ago are still in an
excellent state of preservation. The second method is popular because
of being quicker, due to a different manner of dehydration.

Special slides include mounts made of nematodes for examining the en
face aspect of nematodes, cross-sectional views, and tail mounts, The
method of Buhrer given in this manual can be used for making permanent
mounts of cross-sections, nematode tail sections, as well as for head
mounts. The methylcellulose method is used only for rapid preparation
of mounts which are not permanent.

Miscellaneous Staining Methods

Intra-vital stains of various kinds are of value in water mount slides
of most of the nematodes, except those of the Tylenchoidea and Aphelen-
choidea groups. Anilin blue .WS (Syn: cotton blue, water blue, china
blue, Poirrier's blue) is a very useful stain which aids in the resolu-
tion of cuticular structures and markings and for locating amphidial
and phasmidial openin.';s. A small drop of saturated stain in vjater is
added directly to living or fixed specimens on the slide.

(Not included in cionyrnphfc.) Tech. E:h

DIRECTIONS TOR MAKING FACE VII'MS AND SECTIONS OF KH4AT0DES
E. M. Buhrer, Section of Nematology, Beltsville, Maryland

Satisfactory face views cannot be made from distorted, shrunken speci-
mens, especially spear-bearing species of Dorylamoidea, Tylenchoidea,
etc., in which spears are extruded if the specimens are not properly
killed.

1. Kill and relax specimens by placing them in a hollow ground slide
filled with water and hold over a small flame. Do not overheat and
cook them.

2. Fix in F.A.A. or dilute Flenmings: The latter gives a slight stain
which aids in locating the portions severed.

3. Place in 1^% glycerin in 30% alcohol and evaporate to pure glycerine
in a small desiccator, a process which requires at least two weeks.

U. Prepare slide with a drop of hard glycerine jelly. Do this by

placing a small piece of jelly on the slide and carefully warming it
into a drop over a very small flame. Do not heat too much or bubbles
will form. Spread drop as thin as possible with bamboo splinter.

5. Select the tip of a slender hair from a brush and imbed in jelly
drop while warm. This will act as a marker for the head and should
reach about half-way across the drop.

6. Place nematode in a drop of glycerine on a thin piece of celluloid
and decapitate with an "eye knife." This is done under the binocu-
lar with the hand held firmly on the hand rest.

7. Pick up head with a very fine bamboo splinter and hold in hand while
performing the next operation.

8. Insert head into glycerine jelly at end of pointer hair and stand on
end, holding it in place with the bamboo splinter just touching the
surface of the jelly above the face. The jelly sets in a few seconds,

9. Apply a very thin cover-glass to the drop, carefully centering it
before it touches.

10. Heat the dissecting needle and carefully touch the cover-glass, melt-
ing the jelly \anderneath \Kitil the cover- glass is lowered to near
the face. Do not use too much heat in this operation or the whole
drop of jelly will become warm and the head fall out of place and
migrate.

11. Place two small drops of candle wax just under the edge of the
cover-glass on opposite sides, lightly cementing it to the slide.

Tech. EiE"

12. Bring the head to perfect perpendicular by carefully pushing on the
edge of the cover-glass. This is first done under the binocular and
later, if necessary, perfected under the high power. The wax will
hold the cover-glass in place, yet allow for the necessary slight
movements necessary to bring the head into perpendicular. (Staining
the nematode before cutting off the head aids in locating and orient-
ing the face view. Acid Puchsin, either in Goodey's formula or in
fixative, may be used. The stain usually fades out withing 2k hours.)

Tech. E:6

r<APID PREPARATION OF TmPORAKY HEAD-MOUNTS

The desirability of a rapid method using a viscous mounting medium
into which a specimen could be transferred directly from fixative or
water led to the use of methylcellulose. (Cairns and Tarjan 1955).

Slides made by this method are of a temporary nature and are satis-
factory mainly for determining the surface structures and the internal
sclerotized parts of the nematodes' heads. These structures, unlike
the rest of the body, apparently are not distorted by this treatment
and are the anatomical elements usually considered of diagnostic value
in en face preparations.

The viscous, clear mounting medium is prepared by heating 50 ml. of
distilled water in a beaker to about 80 to 90° C. Enough methyl-
cellulose of one of the higher viscosity types (e.g. UOO-U,000 cps.)
is immersed in the water so that it appears well soaked. After 30
minutes the mixture is cooled by partial immersion of the beaker in
cold water. If the material fails to dissolve completely, the beaker
is ininersed in ice water. If the material becomes too viscous during
or after its preparation, a small quantity of distilled water is added.
The material becomes slightly more fluid after a few days and bubbles
made by stirring disappear.

The specimen is transferred into a drop of the medium on a glass slide,
its head is severed, and a cover-slip then placed directly on the
drop. Manipulation of the cover-slip will bring the nematode head into
the desired vertical position. The location of the head is indicated
in the usual manner by placing three dots on the cover-slip with India
ink in a triangular fashion aroiind the specimen. Application of molten
paraffin-petroleum jelly mixture to the edges of the cover-slip cements
it firmly in place so that shifting of the specimen will be greatly
reduced. The specimen is now ready for immediate examination.

(Not included in copyright.) Tech. E:7

PEHMANENT MOUNTS IN GLYCEKDJE (THOWJE'S METHOD)

1. Heat specimens in water in a depression slide very gently until
they are killed. Frequent checking under the dissecting microscope
is required if heating is done over a small flame or on a slide
warming table. A better method is to put the specimens in a small
amount of water in a "B.P.I." watch-glass and place in an incuba-
tor at 110° F. for about 20 minutes or at 120° F. for about 5
minutes.

2. Drain off the water with a fine pipette, add formalin-aceto-alcohol
fixative. After a few minutes, draw off the liquid and add fresh
fixative. Leave for 2U to I48 hours, the longer time is preferred.

3. Transfer the nematodes to a solution of l\% glycerine in 30^ ethyl
alcohol contained in a "B.P.I." watch-glass.

U. Place the watch-glass in a small desiccator. The desiccant is kept
in a small, screw-capped vial with a hole of about one millimeter
in diameter bored in the cap. Two grams of desiccated calcium car-
bonate is the amount of desiccating agent used. Calcium chloride
is not used, because its use in this manner results in too rapid
uptake of the moisture in the small desiccator. Allow three weeks
for dehydration of the alcohol-glycerine solution; complete dehy-
dration in less time is not desired.

5. After the water has thus been removed, after the three or more weeks
required, transfer the watch-glass with the nematodes to a large
calcium chloride desiccator for three days. Transfer specimens to
pure glycerine, the supply of which is kept in a calcium chloride
desiccator.

6. Mouni. specimens in a drop of piire desiccated glycerine on a slide
clearied with acidulated alcohol. Aid glass-wool supports wMch
have been kept in dehydrated glycerine. Orient supports and speci-
mens and add a clean cover-glass that vras preheated slightly. The
cover-glasses used are selected for freedom from defects, #00 in
thickness, and cleaned in acidulated alcohol.

7. Seal the cover-glass with ZUT and label slides.

The advantage of using the special metal slides, by means of which
nematodes can be mounted between two cover-glasses for observation
of both sides, will be appreciated for permanent slides of scarce
specimens needed for taxonomic purposes.

(Not included in copyright.)

Tech. E:8

MODIFICATION OF RAPID METHOD FOR MOUNTING NH^IATODF^ IN GLYCERIN
Dr. A. D. Baker, Department of Agriculture, Ottawa, Canada

-jKas-

F 0

AM

U L A

_E

•JKHr

Seri

es

- I

II

III

IV

V

Glycerin

55

70

82

90.0

100.0

Lactic Acid

15

10

5

2.5

Phenol (crystals)

15

10

5

2.5

Distilled Water

15

10

5

2.5

Formol

0

0

3

2.5

With Lactophenol the proportions would be as follows:

Series - I II III IV
Lactophenol 75 50 25 12.5

Glycerin 25 50 72 85.0

Formol 0 0 3 2.5

V

From fixative to Lactophenol and then through the above series to" pure
Glycerin. About 10 minutes in each of these mixtures at a temperature
of 50 - 55° F.

In preparing these mixtures, it is convenient to have a stock solution
of equal parts of Lactic Acid and Phenol (crystals) available. This
should be kept in an amber bottle. Lactophenol and the above mixtures
should also be stored in amber bottles.

Stain may be included with the Lactophenol, as described for cotton
blue staining (0.01 per cent) by Franklin & Goodey in 19h9. In making
up these mixtures, add the stain to the distilled water used in making
up your Lactophenol, or dissolve the dry stain in Glycerin (with heat),
femember that most stains are soluble in Glycerin. Thus, if some fad-
ing of the color is not to occur, it is sometimes necessary to faintly
"tint" the mixtures, as well as the final mounting medi\im. It is also
important to note that all nematodes do not stain equally well with the
same stain. Cotton blue is a useful stain, but one has to be careful
not to overstain. Other stains that may be tested for particular needs

Tech. E:9

include Chlorazol Black, Fast Green, Bismark Brown, Purpurin K, and
B, Scarlet. Orange G is a good general stain. A stain that the
writer has found useful is Van Giesen's RLcro-Fuchsin. Some very
interesting differential staining may often be obtained with this
medium, with bright colors and very little danger of overstaining.

Controlling temperatures while using this mounting method may appear
to be a problem if constant temperature equipment is not available.
If possible, reduce the peak temperature of your hot plate below the
boiling point of water, (preferably around 60 - 70° C. ). Five drops
of one of the mixtures are about sufficient to fill the shallow de-
pression of a hanging-drop slide. Transfer the specimens to this slide.
Place the slide on the hot plate with the tip of your index finger on
top of the slide at one end. When the temperature is too hot for your
finger, take the slide off the plate.

There is no present indication that this method gives better results
than original Rapid Method (Baker 19^3 )> hut its main value may lie in
the fact that fewer transfersare necessary.

PERMNETJT NEllATODE ^DUNTS IN GLICERII\IE
Based on a procedure of Dr. J. W. Seinhorst

The following instructions are based on a procedure for making perma-
nent mounts as described by Dr. Seinhorst diiring his visit in 1957 to
the southeastern states as a function of the Southern Regional Nema-
tology Project (S-19). Complete details of this method are to be
published in the near future. The instructions given here are to be
considered only as preliminary and not necessarily according to
Dr. Seinhorst' s technique in all details. The method, or modifica-
tions of it, are in use in several laboratories in the South and
proving quite satisfactory for mounting most nematode species.

A. Relax and kill the nematodes with gentle heat (50-^2° C). The
progress of this step can be observed under the dissecting scope
by using a cavity slide containing a small quantity of vjater and
the specimens.

B. Transfer the nematodes to a Bureau of Plant Industry watch glass
or other suitable dish containing fixative. FA, FAA, Kahle's
solution, TAF, or 5^ formaldehyde can be used as fixatives. Cover
dish to retard evaporation.

1. If fixative used is at ^0-52° C, keep nematode in it for 12-21;
hours.

2. Or, if fixative used is at room temperature, keep nematodes for
2li-li8 hours or longer.

Tech« E: 10

PERMANENT NEMATODE MOUNTS IN GLYCERIN
Based on a procedure of Dr. J. W. Selnhorst

The following instructions are based on a procediare for making perma-
nent mounts as described by Dr.. Selnhorst during his visit in 1957 to
the southeastern states as a function of the Southern Regional Nema-
tology Pro;)ect (S-19) . Complete details of this method are to be
published in the near future. The instructions given here are to be
considered only as preliminary and not necessarily according to
Dr. Selnhorst 's technique in all details. The method, or modifica-
tions of it, are in use in several laboratories in the South and
proving quite satisfactory for mounting most nematode species,

A. Relax and kill the nematodes with gentle heat (50-52*^0.) The
progress of this step can be observed xmder the dissecting scope
by using a cavity slide containing a small quantity of water and
the specimens.

B. Transfer the nematodes to a Bureau of Plant Industry watch glass
or other suitable dish containing fixative. FA, FAA, Kahle's
solution, TAF, or S% formaldehyde can be used as fixatives. Cover
dish to retard evaporation.

1. If fixative used is at 50-52°C., keep nematode in it for 12-
2\x hours.

2. Or, if fixative used is at room temperature, keep nematodes
for 2U-U8 hours or longer,

C. Transfer fixed nematodes into methyl alcohol solution (glycerin
S% in methyl alcohol) in a Bureau of Plant Industry watch glass
or other container. Place on hot plate regulated to 60-6U°C,

D. Keep containers on a hot plate at 60-6U°C. until the methyl alcohol
has evaporated (about 30 minutes). Collapsing, if it occurs in
some specimens, can be corrected by leaving container on the hot
plate for a longer time.

E. Transfer containers with the specimens to a dessicator for at ]e ast
one day.

F. Mount specimens in anhydrous glycerin.

Toch. Fsl

ADDITIONAL TECI[NIQUF.S AND SOURCES OF INFORMATION

General Methods

Goodey, J. Basil, 1?37' Laboratory methods for work with plant and
soil nematodes. Tech. Bui. 2 Ministry of Agriculture, Fisheries and
Food. This third edition of a very useful manual is recommended to
all workers in phytonematology and particularly should be used by
those interested in the techniques of working id.th the cyst-forming
nematodes, a topic which is not dealt with jn any great detail in the
Plant Nematology Notes. The bulletin can be obtained in the U.S. from
the British Information Services, Ii5 Rockefeller Plaza, New York ?0,
New York, for $.70 (last quotation). In Canada, copies can be obt.Tined
from: United Kindgom In.formation Offices, 27^ Albert Street, Ottawn;
119 Adelaide Street, West, Toronto j 1111 Beaver Hall Hill, Montreal.

-,'c -;(■ -!!-

Additional useful information concerning survey procedures and process-
ing techniques for cyst-forming nematodes may be obtained upon request
in the form of a handbook prepared for its field and laboratory by the
Plant Pest Control Branch, Agricultural Research Service, U. S. Dept.
of Agriculture, Washington, D. C.

-ii- -»r -/<■

Kevan, D. Keith McE. (Fd.), 191??. Soil Zoology. Butterworth, London,
512 pp. This is an excellent reference source for anyone interested
in methods and ecology of soil fauna. The contents are presented as
papers by individual contributors and discussions which resulted at
the presentation of the papers at a symposium. A considerable portion
of the book deals with nematodes. Many other items of Interest to
those concerned with the soil as an environment for animal life.

Processing of Samples

Tar j an, A. C, VJ, A. Simanton, and E. E. Russell. 19^6. A labor
saving device for the collection of nematodes. Phytopathology U6 (12);
SlU-Skh- A workable and inexpensively constructed device for extrac-
tion of nematodes from soil and comminuted plant tissue. It uses
spray nozzles for washing and agitating the soil and a system of
inclined, graded sieves for selective trapping of different sized

nematodes,

-;(■ -;!• -a

Mller, Patrick M., 1957. Cheap, disposable filters for nematode
s-urveys. Plant Dis. Reptr. Ul:192.

Stoller, B. B., 1957. An improved test for nematodes in the soil.
Plant Dis. Reptr. Ul(6) :531-532.

Tlie above two methods, although described for use with small-sized
soil or plant samples, can be used for cleaning residues containing
nema.todes washed from sieves. Both methods feature use of expendable

Tech. F:2

tissue filter materials and final containers for the nematodes; the
first using paper cups, the second using tubes formed from polyethylene
films.

Caveness, Fields E. , and Harold d. Jensen, 1955. Modification of
centrifugal -flotation technique for isolation and concentration of
nematodes and their eggs from soil and plant tissues. Proc. Heljm.
Soc. Wash. 22(2): 87-89.

Miller, Patrick M., 1957. A method for the quick separation of nema-
todes from soil samples. Plant Dis. Reptr. I|1(3):19U. The centrifuga-
tion-flotation technique as described is applicable to rather small-
sized soil and plant samples but is of particular interest as a means
for recovery of nematode eggs. The further modification of the tech-
nique by Miller consists of screening the nematodes from the sepairating
sugar solution instead of alla^^ing them to settle, this reduces the
overall time of the process considerably.

Mijnderman, G., 1956. New techniques for counting and isolating free-
living nematodes from small soil samples and from oak forest litter.
Nematologica 1(3) :2l6-226. Describes two techniques for use In
investigating nematodes in small-sized soil samples. One involves
staining of the nematodes in samples where total counts of nemas are
desired. The other technique is a combination of centrifugation and
flotation of the nematodes using magnesium sulphate solution. This
method may have application for recovery of eggs from samples and a
way for mechanization of the processing is illustrated.

Manipulation of Nematodes

Ford, Harry W., 1957. A source of controlled vacuum for pipetting
nematodes. Plant Dis. Reptr. Ul(2):89-90. A simply constructed
device for applying a very weak suction to a pipette for removing
individual nematodes from a dish. One ingenious feature of the device
is that pipetting action will stop automatically when the tip is lifted
above the surface of the water because surface tension of the water at
the tip of the pipette is greater than the vacuum applied to the pi-
pette.

Pathogenicity Experimental Work

Mountain, W. B., 1955- A method of culturing plant parasitic nema-
todes under steril conditions. Proc. Helm. Soc. Wash. 22(1) :U9-52.
A technique for rearing plant-parasitic nematodes under aseptic con-
ditions on root cultures.

■K- -;;- •«•

Fenwick, D. W., 1956. The production of sterile viable larva? of the
potato root eelworm, Heterodera rostochiensis. Nematologica 1(h) :331-
336. A simrplified technique for obtaining eggs and Inrvae which may
be adaptable to Hcloidogyne and other plant-parasitic forms.

Tech. F:3

townsbei';^'', B. F., -^nd J-. W. Lownsbery, 1956. A procedure for testing
'the sterj.lity of large rt'tutibers of nematodes after treatment with vari-
ous sterilants. Plant Dis. Reptr. hO(ll) :9B9-990.

Holdeman, Q. L. , 195U. Value of greenhouse tests in evaluating the
host range of nematodes. Plant Dis. Reptr. Supplement 227:Bl-82.
Consideration of some of the factors in evaluating tests for the host
range of nematodes. Should be read by all who contemplate work of

this type.

-!!- -;<■ -Si-
Pitcher, R. S., 1957. A critical review of current techniques for the
study of migratory root nematodes as etiological agents. Nematologica
2 (Supplement) :Ul3-U23S. An excellent revievr paper which will be of
use to anyone planning work on pathogenicty of plant-parasitic nema-*
todes .

Miscellaneous

Korsten, L. H. J., J. W. Sieben, and L. Voskuyl, 1953. A colorimetric
determination of the number of eelworms in a suspension. Buphytica 2:
135-138. A quick method of determining the nematode concentration in
a suspension used for inoculation. The percentage absorption of a
quantity of nematodes in an aqueous solution of carboxymethlcellulose
is determined and, by comparison to a standard curve, the number of
nematodes per ml. is found. A suspension of a given concentration can
be made.

■5<- -il- -5'-

Feldmesser, J., and W. A. Feder, 195U. Maintaining and determining
viability of nematodes in vitro. Soil Sci. Soc. Fla. I5:l5i;-l56.
Discussion of a problem of obvious interest when viability of the
nematodes must be determined. Various techniques are discussed for
determining viability and for. maintaining in vitro populations of
nematodes under ideal conditions for examination.

■Yi- -;(■ it
Jensen, H. J., F. E. Caveness, and R. H. Mulvey, 1951i. A modification
of Thome's technique for examining soil diffusion patterlis of nema-
todes. Plant Dis. Reptr. 38(9):680-686. Detailed description of a
method using standard materials which can be applied to other situa-
tions in which nematode distribution or profiles are Involved.

MULTIPLE SIEVES

ELUTRIATOR INSTALLATION

SEINHORST ELUTRIATOR
Glassware parts, measureraents in mm.

IZ O.D,

C.

380

-J 3q|-*-

T

so

_i_

T

S"! O.D.

T

o|m

D-2

2 LITER

FLASK

ERLENMEYER

Prices 1958, Scientific Supply Room, U. of North Carolina,

Chapel Hill, Prices include the proper rubber stoppers. It

is necessary only to specify the parts as designated, as Mr.

D. E. Sampson at the above address knows the construction details.

A. B, C. D-1 D-2

$6,00 ea. $5.3l4 ea. $U.86 ea. $5.50 ea. $7.35 ea.

J'iorpholo;'y A, V.-.l

NlT^lATODE MORPHOLOGY

General Structiire of a Nematode

Nematodes are triploblastic, bilaterally symmetric, unsegmented, non-
coelomate animals. Their shape is more or less cylindrical, sometimes
fusiform, pear-shaped or otherwise modified, particularly in the adult
female. The mouth opening is generally anterior and is usually sur-
rounded by lips bearing sensory organs. The mouth is followed by a
mouth cavity or stoma, an esophagus, intestine, and a rectum terminating
in a ventral terminal or subterminal anus in females, or a cloacal
opening in males. The body is covered with cuticle. There are usually
no external appendages, but appendages do occur in rare forms. The
body wall is composed of a hypodermis or epithelium which is situated
beneath the cuticle, ^nd a single layer of muscles. Sexes are usually
separate. The male reproductive system opens directly into the rectum
forming a cloaca, while the reproductive system of the female has a
separate opening, the ventrally situated vulva. Excretory and nervous
systems are present, but there are no specialized organs of circulation
or respiration.

External Characters

The nonatodes, or roundworms, are generally vermiform animals of long
cylindrical shape, circular in cross section. There are two general
types of body form, the fusiform and the filiform, the latter being
less common. The fusiform is that of an elongated spindle, widest
through the middle and tapering toward the blunt or pointed ends; the
posterior end is generally more tapering and pointed than the anterior
end and in some species is very slender. Filiform is thread-like with
a uniform body diameter throughout. Other variations are the short,
plump, pyriforrii or oval shape assumed by females of Heterodera and
Meloidogyne. Most attain a length of 3 to U mm. Males are nearly always
smaller than the females (Plate I).

Nematodes in general lack coloration, being transparent or of a whitish
or yellowish tint conferred on them by the cuticle.

The body is not divisible into definite regions and lacks a distinct
head, although this term is sometimes applied to the anterior end. The
ventral surface of nematodes is identifiable by the presence in the
mid-ventral line of the excretory pore (Plate I, Fig. 1, G), the gono-
pore in the female, and the anus. In most forms, the excretory pore
is located about opposite the base of the esophagus. The vulva (Plate
I, Fig. 1, Q) is usually situated in the oosterior body half. The anus
or cloacal opening of the malQ (Plate I, Fig. 1 and 2, U) lies near the
posterior end. The body region behind the anus is commonly called the
tail.

The oral opening is surrounded by six lips (Plate I, Fig. 3, B) in

Korp?i. 13:2

many genera, but others show great modifications from this pattern.
There may be fusion of pairs of lips giving rise to three lips or
reduction of some and enlargement of others. >/hen there are six lips,
the submedial lips carry three papillae each, one apical and two sub-
lateral in position. The two lateral lips carry two papillae, but
along with the alteration or modification in lip structure there
may also go modification in the number and arrangement of the papillae.
The lips not infrequently bear, or are surrounded by, various cuticu-
lar protuberances. In certain terrestrial nematodes, such as Acrobeles,
peculiar structures termed probolae, three or six in number, project
fon-jard; they vary from simple rounded, conical, or forked eminences
to branched projections resembling antlers. VJhen six are present, they
are arranged in two circlets of three each.

In the region of each lateral lip, but behind the anterior face, there
is a pair of sense organs very characteristic of nematodes, the amphids
(Plate I, Hig. 3, A). The openings of the amphids are most conspicuous
and best developed in the Aphasmidia, but are also present as minute
pores in the Phasmidia. The amphidial openings of the Aphasmidia are
cuticular depressions of three general shapes: cyathiform (Plate II,
Fig. 1, A, B), spiral (Plate II, Fig. 1, D), and circular (Plate II,
Fig. 1, C), The amphids consist of a gland and nerve ending's and are
presumably chemo receptors. In most plant parasitic forms, the pore-
like opening of the amphid cannot be seen except by a study of en face
preparations. '

The -general body surface may be smooth, but very often is marked by a
regi.ilar series of transverse striations (Plate I, Fig. 1, 2, T). These
striations are often interrupted by the lateral fields (Plate I, Fig. 1,
K and Fig. k, A) which are quite prominent in some forms but are rather
inconspicuous in others. In addition to the transverse striae, there
may also be longitudinal striations (Plate I, Fig. U, B). In the tail
region of males of phasmidia, extentions of the cuticle often form lat-
eral alae (caudal bursa; Plate I, Fig. 2, Y) which are employed in
copulation and generally bear genital papillae (Plate IV,' Fig. 1, G).

Another type of cuticular marking is termed punctation. Punctations
are minute dots or ovals which nay occur in transverse or longitudinal
rox-Js and often are arranged in patterns.

Near the posterior end of many nematodes there occurs a pair of cuticu-
lar pouches resembling the amohids. These are called ohasmids (Plate I,
Fig. 1, 2, V) and are probably sensory in function. They are located
in the lateral fields, generally in the tail region or just above it.
Sach consists of a short duct opening on the surface of the cuticle,
and leading inward to a small unicellular gland. Often a surface pa-
pilla is associated with the phasmids. In some cas>=s the gland and
duct may degenerate, leaving ©nly the surface papillae as evidence of
theii" former existance.

Morr;h. C:l

Body V/all

The body wall of ueiaatodGs consists of cuticle, hypodorrtiis (epidermis,
subcuticle), and muscle layer. The cuticle (Plate II, Fig. 5j F) is
a non-cellular layer extended inward at the mouth, excretory pore,
vulva, and anus. It is intimately connected with and, undoubtedly, is
a product of the hypodermis. Histologically, it consists of several
layers ^^rhich are reducible to three kinds of material: the cortex,
the matrix, and the fiber layers . The cortical layer consists of a
dense material of the nature of a keratin and is resistant to solvents
and to digestion. The matrix layer, according to Chitwood (1936),
consists of, or contains, a fibroid named matricin, rich in sulphur.
The innermost part of the cuticle consists of tw^ or three fiber layers
of very dense connective tissue running: in different directions in
adjacent layers. These fiber layers consist chiefly of collagen.

The hypodermis (Plate II, Fig. 5, G) is a syncytial layer that bulges
into the pseudocoel at four places to form four longitudinal ridges
terraed the longitudinal chords (Plate II, Fig. $, A, D, I), middorsal,
midventral, and lateral in position. The nuclei of the hypodermis are
confined to the chords. The nerves and excretory canals (when present)
are in these chords. Some forms may have more than four chords.

The musculature of the body may be divided into two general types, the
somatic musculatuje and the specialized muscles. The somatic muscula-
ture is the general muscular layer of the body wall and is composed of
a single layer of obliquely arranged, more or less spindle-shaped cells
attached to the hypodermis throughout their lengths (Plate II, Fig. S,
H) . Specialized muscles, apparently of the sarae origin as the somatic
musculature, are limited to some particular part of the body, such as
labial muscles, somato-esophageal muscles, somato-intestinal muscles,
rectal muscles, and copulatory muscles.

The Digestive System

The mouth leads into the buccal capsule, very variable in size, shape,
and degree of differentiation in different nematodes. The buccal
capsule and its stiffenings often exhibit a triradiate arrangement,
corresponding with that of the esophagus. In some nematodes, espe-
cially rhabditoids, the buccal capsule is divisible into three sec-
tions: an anterior chamber enclosed by the lips, the vestibule pr
cheilostomj a middle and longest and most sclerotized portion, the
protostom,- and a small terminal chamber, the telostom (Plate II, I'ig.
2). The walls of the various parts of the stoma are called rhabdions
and are termed cheilorhabdions, protorhabdions, and telorhabdions,
respectively.

In certain groups of nematodes, the buccal capsule is armed with a
conspicuous protnasible spear or stylet, used to puncture plants and
animal prey. This may be forme.i, as in tylenchoids, by the coming

Morph . C : 2

together of the sclerotizations of the buccal capsule, so that it con-
stitutes a buccal stylet (Plate II, Fig. 3). This is necassarily
hollow and forras the uath of food intake. This structure is called a
stomatostylet. In other forms, as in dorylaimoids, the spear repre-
sents an enlarged tooth that originates in the esophagus wall. This
type is called ondontosbylet (Plate II, Fig. U).

The structur-e of the esophagus varies in different nematode groups
and is, therefore, an important taxonomic character (Plate III). It
is a tube lined by a thin cuticle and covered by a membrane. The
esophagus lumen is triradiate, being extended into three syrrmietrically
arranged longitudinal grooves that partially divide the esophagus wall
into three sectors, one dorsal and two ventrolateral (Plate II, Fig. 5,
B, j) . Three salivary glands are typically imbedded in the esophageal
wall, one dorsal and two ventrolateral. In most plant parasitic nema-
todes, the esophageal glands are single cells, often with conspicuous
nuclei (Plate III). The main cuticularized duct of each gland opens,
often by way of an ampulla, into the lumen of the esophagus. It is
usual for the dorsal gland to open much farther forward than the ven-
trolateral glands. In some tylenchoids, the glands protrude from the
esophagus wall into the pseudocoel (Plate III, Fig. 7,8).

The esophagus commonly has one or more muscular swellings known as
bulbs . Bulbs provided with a valvular apparatus are called true bulbs.
Those lacking such an apparatus are termed pseudobulbs. The true
bulbs are the cliief pumping and sucking structures of the nematode
esophagus. Bulbs may be situated near mid-length and spoken of as
median, or m.ay occur at the end of the esophagus and termed posterior,
cardiac, or end bulbs. VJjth regard to shape and the presence of bulbs,
esophagi of plant parasitic and soil nematodes are classifiable as
follows (Filipjev and Stekhoven, I9UI): cylindrical, when of nearly
the same diameter throughout (Plate III, Fig. 1); dotylaimoid, when
slender anteriorly and wider posteriorly (Plate llT, Fig. 2); bulboid
when provided with an end bulb (Plate III, l^lg. 3); rhabditoid, with
an anterior vjide region (corpus), usually leading into a mediam pseu-
dobulb, followed by a narrowed region (isthmus), succeeded by an end
bulb with valvular apparatur (Plate III, Fig. U) ; diplogasteroid,
with an anterior muscular region terminating in a median bulb, suc-
ceeded by a posterior glandular region forming a distinct bulb without
valvular apparatus (Plate III, Fig. 5); tylenchoid, having a very
narrow esophageal tube attached to the base of the stylet and enclosed
in a larger thin-walled tube. In most genera there is a muscular
median bulb with an ovoid valve (Plate III, Fig. 6, 7), but this bulb
is much reduced or absent in the neotylenchidae (Plate III, Fig. 8).
The posterior portion of the esophagus is glandular, and the glands may
form a distinct basal bulb (Plate III, Fig. 6) or a lobe ovcx'loyping the
anterior part of the intestine (Plate III, Fig. 7). The duct of the
dorsal esophageal gland empties into the esophageal tube well anterior
of the median bulb, usually very near the base of the stylet (Plate
III, Fig. 6, 7)5 aphelenchoid, similar to the tylenchoid, exceot that
the dorsal esophageal gland empties into the lumen of the esophagus in

Morph. 0:1

the median bulb (Plato III, J'lg. 9).

Esophagi may be quite diversified due to fe'Jdinf; habit. Certain thin.f^s,
however, arc constant: (1) triradiate lumen; (2) cuticular linint^, and
(3) slands.

The esophagus may be connected with the intestine through a short struc-
ture termed the esophago-intestinal valve^ which often projects some
distance into the Ivunen of the intestine. This functions as a valve
which irapedes the flowiiig back into the esophagus of food contained in
the intestine. Like the esophagus, it is lined with cuticle.

The intestine is composed of a single layer of epithelial cells (Plate
II, Fig. 6, a) . It is usually a straight tube in contrast to the
reproductive organs which may be reflexed or coiled.

Posteriorly, the intestine leads into the rectum, which is lined by an
invagination of the l^ody cuticle, and opens at the anus. The rectal
glands, generally three in number, open into the rectum, one dorsal
and two subventral. In the male, the spenri duct enters the ventral
wall of the rectum. The rectum is thus in whole, or in part, a cloaca
in male nematodes. In both sexes, the rectum empties posterdad on the
ventral surface of the body.

The itenroductive System

Nematodes, as a rule, are dioecious, existing as separate males and
females. Males are readily distinguished externally from females by
the presence of copulatory spicules (Plate I, Fig. 2, W). Other dis-
tinguishing features which may or may not be present are smaller size,
curvature of the posterior end, and presence of bursae, genital papillae,
arid other accessory copulatory structures. In marine and most parasitic
nematodes, the sexes occur in about equal proportions, but this is not
the case with terrestrial and fresh-water forms. In the terrestrial and
fresh-water forms, the female predominates. The scarcity or absence of
males indicates a tendency toward hermaphroditism or parthenogensis in
nematodes from these types of habitats. The hermaphroditism is usually
of the protandric type; the gonad first produces sperm that are stored
and later fertilize the eggs subsequently develooed by the same gonad.
The occurrence of parthenogenesis has been proved for several terres-
trial nematode , e.g. Mermis subnigrescens (Christie, 1929), and in the
root-knot nematode Heloidogyne sp. (Tyler, 1933), although males are
knox-m in both groups.

Intersexes are known in some nematode genera (Mermithid, Meloidogyne,
Ditylonchus) . An intersex is an individual which exhibits a blending
of male and female characters. In mo^t cases, the intersexes are
females which show secondary male characters. They may copulate vjith
ifiales ami lay viable eggs. The cause of intersex fonnation is not Quite
understood.

Morph. E:l

Nematode gonads are of tubular shape, varying greatly in length, and
may be straight, sinuous, reflexed, or coiled back and forth (Plate IV).
The male gonad consists of a single testis or paired testes. A single
testis is usually present, and this extends anteriorly (Plate IV, Fig.
1, A) . However, two testes occur in many nematodes, and these are
oppositely oriented, except in Heloidogyne, where they are parallel.
The terms diorchic and monorchic are convenient for referring to the
two-testes or one-testis condition, respectively. VJhen two testes are
present, one usually extends forward and the other backward in the
body cavity, but tliey join medially into the vas deferens, which runs
posteriorly ventral to the intestine and finally narrows down to an
ejaculatory duct which opens into the rectum to form a cloaca (Plate
IV, Fig. 1, C).

Male nematodes, with few exceptions (example, Trichinella), are pro-
vided with copulatory spicules lodged in and secreted by spicule pouches
(Plate IV, Fig. 1, D). Often the spicules are accompanied by an acces-
sory piece, the gubernaculum (Plate IV, Pig. 1, E), which is a sclero-
tization of the dorsal wall of the spicule pouch.

The female reproductive system, may be paired (Plate IV, Fig. 3, U, and
S) or single (Plate IV, Fig. 2), the organ lying in the body cavity
along-side the intestine being either outstretched (Plate IV, Fig, 2,
3) or reflexed (Plate IV, Fig. h, 5). The terms monodelphic and didel-
phic are convenient for indicating the single or double condition of
the female tract, respectively. Each gonad consists essentially of an
ovary, containing developing eggs, and a tubular portion which, in many
forms, is dividiV)le into an oviduct and a uterus. The uterus joins on to
the vagina vrhich opens on the ventral body surface at the vulva. Varia-
tions of the female reproductive system are shown in Plate IV.

>Jervous System

The most easily recognizable part of the nervous system is the nerve
ring which encircles the isthmus region of the esophagus (Plate III).
Associated with it are a number of ganglia, and, according to CJiitwood
and Chitwood (1937), there are six nerves which are directed anteriad
from the nerve ring. The four submedial have three chief distal branches.
The two lateral have only two branches. These branches innervate sen-
sory organs of the anterior extremity (sensory papillae or setae). The
amphids are innervated from the amphidial nerves which originate In the
lateral ganglia of the nerve ring. Thare are a dorsal, a ventral, four
submedian, and one, two or three pairs of lateral nerves situated in
the chords of the hypodermis, which proceed posteriorly from the nerve
ring. The paired postanal lateral sensory organs (phasmids), the ven-
tral supplementary organs of n\ales, and the genital papillae are all
innervated by branches from one or the other of the main nerves. The
nerves themselves and their branches cannot be seen xjithout special
methods for demonstrating thorn or by means of sections. In roiitine
microscopic examinations, the only part of the nervous system observed

Korph. F:l

is the nerve ring which surrounds the esopha;^us, and this is often
difficult to see.

The Excretory aystom

The excretory system presents a varied picture in the phylum as a
whole. It is simplest in the Class Aphasmidia where there is a sin^jle
ventral excretory cell, or renette, iijhich opens through an excretory
pore on the midventral line in the region of the esophagus by way of a
short to long duct. In the Class Phasmidia, there are two lateral
excretory canals imbedded in the lateral chords of the hypodermis
throughout most of the body length. They are connected anteriorly and
ventrally by a transverse canal, thus forming an H or U shape. A duct,
variable in length, connects the transverse duct with the excretory
pore. The terminal excretory duct is cuticularly lined in the Phas-
midia and can be observed in routine microscopic examinations. In the
Aphasmidia, the terminal excretory duct is not lined with cuticle (ex-
cept in some plectinae), thus making it difficult to see. There may
or may not be tvjo special cells (the renette) associated with the
transverse duct.

In a few genera, including Dorylaimus, no excretory system has been
fo\md. Considerable excretion through the digestive tract may occur
in all nematodes.

Circulatory and Respiratory Systems

Circulatory and respiratory systems are not knownj the movement of the
fluids of the body cavity apparently serving these purposes.

Morph. G:l

Plate I

Fig. 1-U. Tylenchorhynchus claytoni Steiner, 1937.

Fig. 1. Female

A - lip region

B - stylet

D - median bulb of esophagus

G - excretory pore

H - basal bulb of esophagus

I - cardia

K - lateral field

L - intestine

M - ovary

N - seminal receptacle

0 - uterus
P - vagina
Q - vulva
S - sperms

T - annulation of the cuticle
U - anus

V - phasmids

Fig. 2. Male

A - lip region

B - stylet

C - dorsal esophageal gland orifice

D - median bulb of esophagus

E - subventral esophageal gland orifice

F - nerve ring

G - excretory pore

H - basal bulb of esophagus

1 - cardia

L - intestine

R - testis

S - sperms

T - annulation of the cuticle

U - cloacal opening

V - phasmids
W - spicule

X - gubernaculum

V - caudal bursa

Fig. 3. Face view
A - arriphid
B - lips

Fig. U. Cuticle detail

A - lateral field with four incisures forming three ridges
B - annules subdivided by longitudinal striations

1

D

Uf

s

s

■ ■ ft

ro

Jk

1 1 1

jjIluj

U X Y

Fig.1

Plate I

Morph. G:2

Plate II

Fig. 1. Amphids

A - cyathiform type (Trilobus)
B - cyathiform type (Dorylaimus)
C - circular type (Monhystera)
D - spiral type (Choanolairaus)

Fig. 2-U. Variations of the buccal capsule (See also Plate TJi) ,

Fig. 2. Cylindrical buccal capsule (Rhabditis)

Fig. 3. Stomatostylet (Rotylenchus)

Fig. h. Odontostylet (Dorylaimus)

Fig. 5. Cross section through esophageal region
A - dorsal chord
B - dorsal sector of esophagus
G - esophagus lumen
D - lateral chord
E - lateral field
F - cuticle
G - hypodermis
H - muscle layer
I - ventral chord
J - the two ventrolateral sectors of esophagus

Fig. 6. Cross section through reproductive region of female
A - intestine
B - ovaiy with unfertilized egg (Oocyte)

B

Fig. 1

Fig. 2

Fig. 3

D
E

Fig. 5

Fig. 6

Plate 11

Morph. G:3

Plate III
Fig. 1-9. Different types of oesophagi,

Fis. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

A

CYLINDRICAL DORYLAIMOID BULBOID RHABDITOID DIPLOGASTEROID

(MONONCHUS)(OORYLAIMUS) (ETHMOLAIMUS) (RHABDITIS) (DIPLOGASTER)

Fig. 6

Fig. 7

Fig.8

Fig. 9

TYLENCHOID
(TYLENCHORHYNCHUS HELICOTYLENCHUS NEOTYLENCHUS)

APHELENCHOID
(APHELENCHUS)

Plate III

¥

Morph. G:U

Plate IV
Fig. 1-5. Reproductive Systems.

Fig. 1. Male reproductive system (Rhabditis)

A - single testis (monorchic)

B - intestine

C - cloaca

D - spiciile

E - gubernac ul\im

F - bursa

G - genital papillae

Fig. 2-5. Female Reproductive Systems.
Fig. 2. Single (monodelphic), outstretched (Ditylenchus)
Fig. 3. Paired (didelphic), outstretched (T;/lenchorhynchus)
Fig. h. Paired (didelphic), reflexed (Rhabditis)
Fig. 5. Paired (didelphic), reflexed (Meloidogyne)

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Plate IV

Sy stoma tics A:l

NH-IATODE SYST^lATIC.^i

According to Mayr et al. (1953)5 nematodes belong to the Kint^dom /.ni-
malia, Subkingdom Hetazoa, and Phyliirn Nematoda. Chitv70od and Chitwood
(1950) use "Nanatheln inthes" as the designation of the phylum. Ctoodey
(19$1) places the nematodes in the Subkingdom Vermes, Phylum Ascheliaintha,
and Class Nematoda,

Vifhether the nematodes are considered a class or a phylum, there is fairly
general agreement on the classification of Chitwood (1933). According to
this scheme, all nematodes are divided into two large groups (classes or
subclasses) called Phasmidia and Aphasmidia. The classes are divided
into orders with names derived from the type genus of the order and end-
ing in -ida (e.p., Rhabditida) . Names of suborders end in -ina (Rhab-
ditina); of superfamilies in -oidea (Rhabditoidea) ; of families in -idae
(Rhabditidae); and of subfamilies in -inae (Rliabditinae) . The subfami-
lies are composed of genera and the genera of species. In addition, a
few subspecies have been named.

As with plants and other animals, the various taxa are composed of forms
having a greater or lesser degree of resemblance and presumably have
evolved from cominon ancestry. Species in the same nematode genus may
differ from one another in only a few characters, and are usually easily
recognizable as belonging to the same genus. Genera in the same sub-
family differ to a larger degree, and so on through the higher categories.

Nematode names are subject to the International Rales of Zoological
Nomenclature, and some knowledL';e of these rules should form part of the
basic information of the nematologist. At present, the best available
explanation of the rules is to be found in the book, "Methods and Princi-
ples of Systematic Zoology," by Mayr, Linsley, and Usinger. These rules
provide for the changing of nan-es under certain circumstances, and this
is often done with nematode names. Consequently, desired infoimation on
a given species of nematode may be found in literature files under several
different names. As examples, the root-knot nematodes have been transfer-
red from the genus Heterodera to the genus Meloidogyne; many genera of the
Tylenchida were formerly grouped in the genus Tylenchus and later in the
genus Anf^uillulina; and the genus Hemicycliophora was formerly known as
Procriconema. The table of contents of Goodey's "Soil and Freshwater
Nematodes" is the best guide to general classification of the soil and
plant parasitic forms available at present. However, it will be found
lacking in several respects since several important changes have been made
since the book was prepared about 19U9.

It should also be remeinbered that, nematode taxonomy is in a fairly early
stage of development. New species are constantly being described and new
genera created, either through the discovery of new forms, the splitting
of old genera, or the correction of errors of the past. As concepts change.

Syt;b':ri. A:2

iit^w species are studied and better working methods are develooed, the
taxonomy must change.

In this course, emphasis has been placed on the most common soil and
plant parasitic nematodes. Two keys for identification of these forms
are presented. The first of these, the "Ke.v to the Most Common liematodes
of Agricultural Soils and Plants," permits identification to f airily or
subfarailv. Once this identification has been made, identification to
genus can usually be made by reference to the illustration and descrip-
tions in tne appropriate section of Goodey more quickly than by ttie use
of additional keys. The second "Key to the J.ature Females of the Tylen-
choidea" was designed to facilitate identification to genus of the great-
er part of the plant parasites. Both keys have been siraolified by omis-
sion of rare forms. While this simplifies construction and use of the
keys, it also in>plies that rare forms vjill not "key out."

In use of the keys, it should be kept in mind that keys are imperfect
tools at best and that verbal descriptions are very apt to be misleading.
Consequently, the use of the keys should be supolemented by frequent
reference to the illustrations in Goodey or other available publications.
Also, it is suggested that time and effort will be saved if no attempt is
made to identify larval forms (without sexual organs). VJhen possible,
several specimens should be studied. Visibility of the key characters
varies with specimens, and structures which are obscure on one may be
plainly visible on another. vJlien a tentative identification has been
made, available illustrations should be carefully checked.

£y G ten . B : 1

KEY TO THE MOST COMMON NBIATODSS OF AGKICULTURAL SOILS AND PLANTS

Oesophagus rhabditoid (with valve in basal bulb as in PI. Ill, h) ,
diplogasteroid (without valve in basal bulb as in PI. Ill, 5),
tylenchoid (PI. Ill, 6, 7, 8), or aphelenchoid (PI. Ill, 9). If
tylenchoid or aphelenchoid, always with stylet. Males of sorae
genera have a distinct bursa (PI. I, Fig, 2). Never with setae
or conspicuous amphids. Phasmids always present, but most often
difficult to locate. Mature females of some genera have a much
enlarged body, Phasmidia 2 .

Oesophagus bulboid (PI. Ill, 3), cylindrical (PI. Ill, 1) or
dorylaimoid (PI. Ill, 2). Amphids often conspicuous, appearing
as circles, spirals, stirrup forms, etc. (PI. II, A, B, C, D).
Often with setae. Males without bursa. Always with elongate,
vermiform body. Aphasmidia 6.

2. Stylet always present, usually, though not always, with well-devel-
oped, rounded knobs. Shapes and proportions of stylets vary
greatly with genera and species, but are usually recognizable as
variations of the form shown in PI. II, Fig. 3- Attached to the
stylet is a thin oesophageal tube which may be straight or coiled,
Tylenchida 3-

Stylet absent. Anterior portion of oesophagus muscular (striated)
(PI. Ill, h, 5). Rhabditida U.

3. Dorsal oesophageal gland orifice near base of stylet, or at most,
not more than one stylet length posterior to stylet knobs. The
oesophageal tube often has an abrupt bend at this point (PI. II,
Fig- 3). Stylet mostly with well-developed knobs. If stylet
knobs are absent, tail is lonp, and thin, Tylenchoidea

Dorsal oesophageal gland orifice in median bulb and difficult to
locate. Oesophageal tube witliout abrupt bends. Stylet knobs
small or absent. Median oesophageal bulb occupying nearly full
width of body (PI. Ill, 9). Tail rounded or conical.

Aphelenchoidea

h. Oesophagus rhabditoid, with valve in basal bulb (PI. Ill, U) 5.

Oesophagus diplogasteroid, without valve in basal bulb (PI. Ill,
5) Diplomas toridae

5. Stoma cylindrical, usually much longer than wide (Plato II, 2),

System. B:?.

Lios plain, except in one genus (Piploscapter) , which has hooked
projections on lips. Usually with two ovaries itfiabiiitidae

Stoma not cylindrical, or if nearly so, about as long as wide.
Lips of some genera have distinct projections ranging in shape
from rounded to elaborately ornamented. .Jith one ovary, this be-
ing reflexed so distal end is posterior to vulva — Cephalobidae

6. Oesophagus plectoid (with basal bulb, PI. Ill, 3). Tail tip with
a small projection (spinneret) Plectinae

Oesophagus cylindroid (PI. Ill, 1) or dorylaimoid (PI. Ill, 2)

7. Oesophagus cylindroid. Houth cavity large, subglobular, usually
with one or more large teeth (PI. Ill, 1) Monochidae

Oesophagus dorylaimoid (PI. Ill, 2). Stylet often as shown in
PI. II, U. Some with much longer stylet, others with a tooth
Dorylaimoidea

iys tera . C : 1

KEY TO thh: mature ptmalf.s of THR TYLENCHOIDEA

1. VJithout median oesophageal bulb, stylet length never more than 3

times width of lip region (PI. Ill, 8) Meotylenchidae

With median oesophageal bulb (PI. Ill, 6, 7) 2.

2. Body of mature female pear-shaped, lemon-shaped, or enlarged and
saccate. Found in roots of plants, either embedded or attached

by neck, some as cysts in soil 3«

Body of mature female vermiform (much longer than wide) 7.

3. Body of mature female pear- or lemon-shaped U.

Body of mature female saccate $•

h. Body of mature female pear-shaped, white. Found completely or
almost completely embedded in roots or other plant tissue, nearly
always in distinct knots or galls Meloidogyne

Body of mature female pear-shaped or lemon-shaped, white, yellow-
ish, or brown according to age, attached to root by neck only, or
found as a brown cyst in soil Heterodera

5. Mature female embedded in plant root, often in knot, body shape
ovoid to spheroid with elongated "tail," vulva nearly terminal,
Nacobbus

Mature female attached to root by neck, body more or less kidney-
shaped 6.

6. Vulva at 90^ of body length, parasites of citrus and olives

Tylenchulus

Vulva at 72% of body length, parasites of numerous plants,

mostly annuals Rotylenchulus

7. Tail long and thin, more than 6 times anal body diameter 8.

Tail not long and thin, but rounded, or conical and more or less
pointed 9.

Sys tern . C : 2

8. Tail tip frequently clavate, one or two ovaries, distance from
anterior end to center of median oesopha,";eal bulb equal to, or
greater than distance from center of bulb to base of ogsophafjus.
''-'silenchus

Tail tin usually pointed, often curved ventrally. Distance from
anterior end to middle of median oesophageal bulb less than dis-
tance from this point to base of oesophagus. One ovary Tylenchus

9. With one ovary 1*^-

With two ovaries IS-

10. Body more or less sausage-shaped, i.e., short and stout (a= 6-10),
length seldom more than about 0.6mm 11.

Body slender (a= 20 or more), length usually more than 0.6mm 13.

11. Body with prominent retrorse (directed posteriorlj^ annules,
usually longer than 0.3mm, a- 10 or more. Found in soil 12.

Body without prominent annules, very stout (a= 6-8). Length

about 0.3mjfii. Found iix roots Cacopaurus

12. Annules with prominent spines or scales Griconema

Annules of mature female without spines or scales Criconemoides

13. :Hature female more than 2mm, often 3-5mm long. Found in galls

in leaves, or in inflorescence of grains and grasses Anguina

Body length less than 2mm. Fcamd in soil, or in roots and

tubers; sometimes in bulbs, lesaves and stems lU.

lU. Stylet longer than 3 times width of lip region 15.

Stylet shorter or about twice width of lip region 16.

15. Posterior portion of body strongly curved ventually Paratylenchus

Body usually nearly straight, usually covered by loose cuticle

of fourth molt Hemicycliophora

System. C:3

16. Body long and slender (a= J4O or more), tail conical, pointed.
T^iidoparasitic in bulbs, stems, leaves and tubers, from mush-
room compost, or sometimes in soil Ditylenchus

Tail tip rounded 17.

17. Lip region distinctly set off from body; oesophageal glands
forming a lobe overlapping intestine; knobs of stylet closely
joined. A cormiion genus endoparasitic in roots and tubers, also
found in soil Pratylenchns

Lip region not distinctly set off from body; oesophageal gland
forming a distinct bulb; knobs of stylet separated like an in-
verted Y. An apparently very rare genus from soil Ghitinotylenchus

18. At least 2rain long, slim (a= U5 or more). Stylet very long, six

or more times as long as width of lip region 19.

Less than 1.5mm long, length stylet not more than 5 times width

of lip region 20.

19. Tail pointed, oesophagus with distinct basal bulb Dolichodorus

Tail rounded, base of oesophagus a lobe overlapping intestine

Belonolaimus

20. Lip region flattened, stylet length about twice width of lip

region Radopholus

Lip region convex conoid, stylet length 2 or more times width of

lip region 21.

21. Tail 2 or more times as long as anal body diameter, tapering 22.

Tail shorter than anal body diameter, rounded 23-

■^d.. Tail tip ro-mded Tylenchorhyrichus

Tail tip nearly pointed Tetylenchus

23. Lip region distinctly set -off from body, divided into minute

plates. Body at rest or fixed lying only slightly curved- Hoplolaimus

Annulated liu region continuous •l^rith body contour, body usually

Sy;;tora. C:U

lies in loose spiral when fixed or at rest 2U.

2I4. Dorsal gland orifice about one-third stylet length or more

posterior to stylet knobs Helicotylenchus

Dorsal gland orifice much less than one third stylet length
posterior to stylet knobs Itotylenchus

Parasitic Nematodes A:l

CYST NH'-UTODra (ifOTWODEHA SPP.)

The genus Heterodera was placed in the family Heteroderidae of the Tylen-
choidea by Thorne (19U9) with the genus Meloidogyne (root-knot nematodes).
Prior to 19U9j species of both these genera were placed in the genus Hete-
rodera. Prior to 19U0, there was a strong tendency to refer all of the
cyst-fonaing nematodes to a single s pecies, H. schachtii Schmidt, 1871,
though some effort was made to differentiate races, strains, or varieties
according to the host plants attached. However, little was done toward
study of morphology, and identification was difficult unless the host
plants were known. Some progress has been made in recent years, though
the situation is yet far from satisfactory.

Morphology

The adult females and cysts of Heterodera are the forms most cormonly
encountered. Adult females or cysts will be found on roots of various
plants if these are carefully removed from the soil and washed. The nema-
todes are attached to the roots by the neck only, with most of the body
outside the root. The females are white or yellowish in life, and the
cysts are light to dark brown. Average size is about 0.5mm by 0.75mm.
Some species are lemon-shaped, others are pear-shaped. Cysts are very
highly resistant to decay and may be found in soil in which infected
plants have grown, even many years afterward.

The males are slender worms shaped very much like Meloidogyne males. That
is, they are about 1.25 to 1.75itoti long, slender (a = 35-UO), taper slight-
ly anteriorly, and have a short rounded tail (Goodey, 1951, K.g. 70).
Males will be found in abundance at certain times of the year, but mey he
very scarce at other times. The larvae have an average length of about
0.5mj'i. They differ from root-knot nematode larvae in that the stylet is
20 to 30 microns long (Meloidogyne, 10-11 microns) and in the shape of the
anterior end.

Excellent drawings of the larvae and other stages of H. schachtii will be

found in "The Life History and Morphology of the Sugar-Beet Nematode,

Heterodera schachtii Schmidt," by D. J. Haski (Phytopath. U0(2): 135-152,

1950) . Larvae are seldom found free in soil, but can easily be obtained

from the cysts.

t

The cysts are important contaminants of imported plant material and also
are searched for in soil in connection with quarantine and rotation pro-
grams in various countries. Consequently, they have benn intensively
studied, and most of the present information on identification of species
is based on characters of the mature cysts and their contents.

A key to aid in identification of cysts is presented. Key characters will
be found on the mature cysts.

Par.'j„'3. A:2

Life History-
Larvae in the soil enter the roots of plants near the root tips and V}es^in
to feed on the developing tissues. Here they undergo three moults, break-
ing through the outer root tissue at the last one. Females remain attached
to the root by the neck. Males leave the larval cuticle and go in search
of females. Apparently all of the females of the H. schachtii and H. gflt-
tingiana groups deposit some eggs in a jelly-like substance, forming an
eg^ mass or "egg sac." However, these species also retain eggs in the body
so that by the end of the life of the female, the body is tightly packed
vfith eggs. Females of the H. rostochiensis and H. cacti groups do not de-
posit any eggs, retaining all in the body. The female finally dies and
her cuticle becomes transformed into a cyst filled with eggs. Eggs in the
cyst; develop to the first larval stage, then moult once, becoming second
stage larvae. Apparently hatching may then take place immediately, or the
larvae can remain in the eggs In the cyst indefinitely.

It has been shown that root excretions of various plants can stimulate
hatching. Most work on this subject has been done with H. rostochiensis.
l^en cysts of this species are placed in leachings from a growing potato
plant the rate of hatching of their eggs is enonnously increased. How-
ever, it is seldom that all the eg-'s in a cyst hatch even \inder the most
favorable conditions. In the absence of a host plant only a few eggs
hatch each year. Hatching after 17 years has been reported in the litera-
ture. Probably the maximum time under most conditions is less than half
of that. Once hatched, the larvae make their way to a host plant complet-
ing the life cycle.

Because of the limited host range of most of the Heterodera species, it is
usually easy to devise crop rotation methods for control. On the other
hand, delayed hatching of the eggs means that the rotations must be very
long; in heavily infested sugar beet fields, as long as 5 or 6 years must
be allowed between beet crops. Since it has been shown that larvae die
within 12 to 18 months after hatching if they do not reach a host plant,
efforts are being made to analyze the "hatching factor" in root leachings
in the hope that it can be synthesized and used in control. Some progress
has apparently been made.

The literature on Heterodera is voluminous, European workers having studied
the sugar beet nematodes and other species of this genus since around l06O.
A summary to about 1938 will be found in "A Manual of Agricultural Helmin-
thology," by I. N. Filipjev and J. H. Schuurmans Stekhoven (19Ul). A later
and shorter suini.iary, "The Cyst-forming species of Heterodera," by Mary T.
Franklin, was published by the Commonwealth Agricultural Bureaux, Farnham
Royal, Bucks, England, in 1951. "The Golden Nematode of Potatoes," by
B. G. Chitwood, (USDA circular No. 875) summarizes information to about
1951 on H. rostochiensis in this covmtry. There are several host lists,
but because of the confusion as- to identity of species, most are apt to be
misleading. Probably the best information on hosts is fovind in reports of
experiments by F. G. W. Jones, "Observations on the beet eelworm and other
cyst-forming species of Heterodera." (Annals of Applied Biology 37(3):

I' 0X0.0. A:3

U07-UU0), of 1950.

Available names of the genus Heterodera are listed in Table 1. Identi-
fication of some of these by characters of the cysts and contained egzs
and lai-vae is doubtful, and it is possible that some of the species are
not valid. Tlie final answer to this question must await careful study
and description of the males, females, and larval stages. It should also
be mentioned that while the writer is certain there are a considerable
number of undescribed species, very few cysts have been seen which could
not be placed in one of the species groups described below.

Species formerly referred to this genus are as follows:

Heterodera radicicola (Greef, 1872) Miller, I88U, was shown by Goodey
"0-932) to be a species of another genus and is now known as Ditylenchus
radicicola (Greef, 1872) Filipjev, 1936.

Heterodera vitis Phillipi, I88U, was shown by Giard (I89U) to be an
insect MaTgarodes vitivim (Phillipi, I88U) Giard, I89U.

Heterodera javanica Treub, 1885, is a root-knot nematode, Meloidogyne
javanicaTTreub, 188g) Chitwood, 19U9.

Heterodera exigua (Goldi, 1887) Loos and Foaden, 1902, is also a root-
knot nematode, Meloidogyne exigua Goldi, 1887. (The species name was
misspelled exigua by Loos and Foaden.)

Heterodera marioni (Comu, 1879) Goodey, 1932, is a root-knot nematode,
Meloidogyne marioni (Comu, 1879) Chitwood, 1952.

Heterodera lupuli Filipjev and Schuurmans-Stekhoven, I9UI, is an obvious
error, H. humuli having been intended.

Heterodera viale (Lavergne, 1901) Chitwood, 19ii9, is also an obvious error
of transcription for Anguillula viale.

Special attention should be called to the fact that several additional
names to be found in Cooper's paper published in 1955 have no nomencla-
torial standing, having been specifically designated a provisional name
by the author.

Table 1

The Species of Heterodera with Type Hosts and Localities
The ===»===

Species: Type Host: Type Locality

H. schachtii Schmidt, I87I Beta vulgaris Halle, Germany

H. gottingiana Liebscher, 1892 Pisum sativum Gottingen, Gennany

H. rostochiensis /Jollenweber, 1923 Solarium tuberosum Hostock, Germany

Species

H. punctata Thome, 1928
H. major (Schmidt, 1930)

Fi-anklin, 19UC»i-
H. trifolii (Goffart, 1932)

Oostenbrink, 19^9
H. humuli Filipjev, 1931;
H. galeopsidis (Goffart, 1936)

Pilipjev &L Schuurmans-

Stekhoven, 19^1
H. cacti Filipjev & Schuurmans-

Stekhoven, I9UI
H. cruciferae Franklin, 19h$
H. weissi Steiner, 19U9

H. carotae Jones, 1950

H, glycines Ichinohe, 1952

H, leptonepia Cobb and Taylor,
H. tabacum Lownsbery and
Lownsbery, 1952
H. fici - Kirianova, 195U

Type Host

Triticum vulgare
Avena sativa

Paras . kiU

Type Locality

Saskatchewan, Canada
Halle, fjermany

Trifolium pratense Schleswig-Holstein,

Germany
Humulus lupulus Kent, England
Galeopsis tetrahit Lauscha, Germany

Epiphyllum

ackermanni

Brassica oleracea

Polygonum pennsyl-
vanicum

Daucus carotae

Glycine max

1953 Unknown

Nicotiana tabacum

Picus sp.

Haartensdyk, Holland

England

Beltsville, Maryland,

U.S.A.
Isle of Ely, England
Tokachi Province

Hokkaido, Japan
Probably Peru
Hazardsville, Conn.

U.S.A.
U.S.S.fi

•«-/There is some confusion as to the proper name for this species. H. avenae
(Mortensen, Hostrup, and Kolpin Havn, I9O8) Filipjev, 193U, has been used
in some recent literature. However, as pointed out by Franklin (1957),
this name was never accompanied by an adequate "indication" as required by
the Eules of Zoological nomenclature, and is therefore invalid.

The Cysts

Cysts of Heterodera are of two general types which are easily distinguished
by examination of the lower end, l/This part of the cysts of H. rostochi-
ensis and H. punctata has a smooth romided contour (figs. 1 and 2T'. The
cysts of all other known species is shaped somewhat like the end of a
lemon, that is, the vulva is located on a definite protuberance. This is
shown in figures 3 to 6, the variety of forms illustrated being repre-
sentative not only of the species shown, but of the other known species as
well. The first type of cyst is conveniently referred to as "round" and
the second type as "lemon-shaped", or it might be said that the vulva does
not or does protrude. At the upper end of the cyst is a distinct neck

1/For convenience here and in the following parts of this paper, the cysts
will be described as viewed laterally with the vulva at the lowest point.
The "lower end" of the cyst would then refer to the region around the vul-
va, the vertical axis would extend from the viiLva along the center line of
the cysts, horizontal lines would be at right angles to the center line, etc.

Paras. A:^

which varies in length, shape, and position with reference to the
vertical axis of the cyst. Shape, size, and proportions of the cysts
are highly variable; and while it can be shown statistically that
averages of these dimensions or of the relations between them differ
between species, such averages are of little value for identification
of small lots of cysts.

As has been illustrated by Chitwood (1951) for H. rostochiensis, the
cysts of all species are made up of several distinct layers. iJhen the
cysts of some species are fresh, they may be covered or partly covered
by the "subcrystalline layer." This is a waxy, translucent substance
which apparently persists for only a short time in the soil.

The color of mature cysts is always some shade of brown. The cuticle
of living females may appear white, colorless, or yellow.

The outermost layer of cysts is marked by grooves and ridges which
form distinctive "patterns." These vary in detail with individuals,
but are sufficiently constant within groups of species to be of value
in identification. The pattern is visible on immature females and can
nearly always be seen on part or all of cysts even when these are very
old. In certain species, the basic element of the pattern at the mid-
dle of the cyst is a short zig-zag line which may appear as light on a
dark background or dark on a light background, according to the focus
of the microscope (figs. 7, 8, and 26). The segments of the line are
straight, and the angles between them are well defined. Usually these
lines near the middle of the cyst show no trace of regular arrangement.
Near the base of the neck and around the vulva there may be parallel
lines (fig. 9) or wavy lines (fig. 10). The size of the elements of
the pattern nay vary greatly, being small as shown in figure 8, rela-
tively large as shown in figure 26, or intermediate between these two.
A variation v;hich seems rare is the network pattern shown in figure 12.
An occasional cyst with partly zig-zag and partly netiTOrk pattern has
been seen. So far as is known at present, there is no constant dif-
ference in pattern between species in the H. schachtii group, though
it is possible that fine, coarse, or network patterns will be found
more frequently in some species than in others.

A second type of pattern is found in the group of species which in-
cludes H. weissi, H. cacti, and probably a number of undescribed
species. This pattern has as its basic element parallel lines running
around the cyst at right angles to the vertical axis (fig. 13). These
may be interrupted at intervals by short vertical or oblique lines
(figs, ill and 15). Sometimes this pattern may appear somewhat like
the zig-zag pattern but differs in that some trace of the parallel
lines always remains .

The cysts of H. rostochiensis and H, punctata have a third type of
pattern. Around the vulva it-is made up of wavy lines (fig. 19. On
the lower portion of the cyst, there are short, crooked lines, some-
times in horizontal rows (fig. 22). On the upper part of the cyst,
the lines tend toward the vertical, sometimes appearing as nearly
vertical striae (fig. 23).

Paras . A : 6

Some of the species with zi^-'^ag patterns have in the lower end a stri-
ated object shaped somewhat like a sheaf of grain, apparently the cuticu-
lar lining of the vagina (fig. 11). This is nearly always accompanied
by a number of dark bodies of irregular, though never angular shape.
These may be few or numerous. No constant number or arrangement has
been observed. These are absent in cysts of other species having zig-
zag patterns and can be used for separation of these cysts into two
groups.

Punctation is found on a layer of the cyst below that which carries the
pattern. According to Franklin (1939) punctation is minute pits in one
of the layers of the cyst. Under high magnification these appear as
round dots of uniform size, either light or dark, according to the focus
of the microscope.

Punctation is usually very prominent in H. rostochiensis and H. punctata,
with the dots often iDeing arrayed in distinct parallel horizontal rows
(figs. 20 and 2U) . In cysts of the H. schachtii group, punctation is of
several types. One of these is a prominent feature of most H. avenae
cysts, but also occurs on other species. The dots are about one-half
micron in diameter, and there is little or no trace of regular arrange-
ment (figs. 25 ana 26), This is called "coarse irregular" punctation.

Cysts of H. trifolii have dots of about the same size as those fo\ind on
cysts of H. avenae, but these are often arranged in parallel lines on
part of the cyst at least. This is sho;vn in figure 2? with the lines
running diagonally from lower left to upper right across the photograph,
but the rows are seldom as long as those shown.

In other species of the H. schachtii group, fine irregular punctation
occurs. The dots of fine irregular punctation are much smaller than those
of the coarse type, being difficult to see even with the oil immersion
objective of the microscope. Unfortiinately, punctation is a somewhat
variable character, being easy to see on some cysts and difficult or
impossible to find on others. Its presence is therefore a useful char-
acter, but its absence cannot be taken to indicate that a given specimen
does not belong to a species for which punctation is described.

Punctation has not been seen on H. weissi or H. cacti, though it may
occur on some of the undescribed species of this group. But cysts of
these species often have a grainy appearance (figs. 1? and 18) due to
the presence of dots of somewhat irregular size and shape on the outer
layer of the cyst.

The anus of H. cacti is shown in figure 17. All lemon-shaped cysts have
the anus located in about this same relationship to the vulva. The anus
of H. rostochiensis is shovm near the upper edge of figures 19 and 20.
The~pattern runs around the vi^l-va, but the anus is marked only by a
slight irregularity. The anus of H. punctata is located at a thin spot
on the cyst, which is about the same size as the vulvar opening, figure
No. 21 shows this clearly, though the cyst wall was split in the process

Paras. A:7

of preparing the slide for photographing. This difference, together with
the round shape of the cysts, perraits the identification of H. rostochi-
ensis and H. pvinctata from examination of the cysts alone.

The Larvae

Larval characters used in the key are average length, relation of body
length to breadth, shape of stylet knobs, location of the dorsal gland
orifice, and relation of the tail terminal to the stylet length.

Identification by average length of the larvae has distinct limitations
due to the fact that variation in length within a species might be as
much as 20^ of the total length. Relation of larval length to width is
useful for separation of only one species, H. leptonepia.

Stylet knobs are of two general types, concave anteriorly and convex
anteriorly. With most species, there is no doubt as to the type of
knobs, since the concavity or convexity is distinct, but some forms have
stylet knobs intermediate in type and difficult to distinguish.

Location of the dorsal gland orifice is used mostly to distinguish be-
tween H. trifolii and H. glycines. As was pointed out by Hirschmann
(1956), the dorsal gland orifice in larvae of H. glycines is located 3.0
to 5.2 microns posterior to the stylet knobs; in H. trifolii, the loca-
tion is 5.6 microns posterior to the stylet knobs.

The tail terminal is defined as the hyaline portion of the tail posterior
to the body cavity. This portion of the tail is usually clearly defined,
since the contents of the body cavity are more or less granular. In
poorly preserved specimens, the body contents may be shrunken, making the
tail terminal appear longer than it is in reality.

Key to The Mature Qysts of Species of Heterodera

Note: This key is designed to facilitate identification of the species

of Heterodera, using only characters of the mature cysts and
their contents, that is, eggs with second stage larvae. Certain char-
acters used in the key may not be visible on other than fully mature
cysts.

Measurements of larvae are from Fenwick and Franklin (195l) for most
species, from Jones (195l) for H. caret ae, from Ichinohe (1952) for H.
glycines, and from Kirianova for H. ficl.

1. Body of cyst ovoid to globular, that is with posterior portion

rounded and vulva not located on a distinct protuberance (figs. 1
and 2) Heterodera rostochiensis group k-

Body of cyst lemon-shaped, that is, with vulva located on a dis-
tinct protuberance (figs. 3 and 6) 2.

Paras. A:8

Basic element of pattern of outer layer of cyst wall at middle
portion of cyst short zig-zag lines with little or no trace of
regular transverse arrangement (figs. 7 and 0) sometimes modi-
fied to appear as network (fig. 12) 3,

Basic element of pattern at outer layer of cyst wall at middle
portion of cyst straight or wavy lines (figs. lU and 16) lines
at right angles to axis of cyst; sometimes broken by short ob-
lique or vertical lines; outer layer of cyst may have grainy
appearance (fig. 18) Heterodera cacti group ?■

Mature cysts with dark bodies (brown knobs) and often sheaf -
shaped object (lining of vagina) at posterior end (fig. 11).
On immature cysts, these seldom visible, and then do not appear
dark Heterodera schachtii group 8.

Matiore cysts without brown knobs or sheaf-shaped object at
posterior end Heterodera gtittingiana group 11.

I4. H. rostochiensis group. Cyst often ovoid, anus located at a
transparent spot on cyst so that anal and vulvar openings
appear to be about the same size when seen by transmitted
light (fig. 21). Hyaline portion of larval tail much longer
than stylet Heterodera punctata

Cyst ovoid to globular; anal opening appears much smaller than

vulva opening (figs. 19 and 20). Ify"aline portion of larval

tail about the same length as stylet 5-

5. Laivae very slender; length about 39 times greatest width;
orifice of dorsal oesophageal gland about two-thirds stylet
length posterior to stylet knobs — Heterodera leptonepia

Length of laarvae about 22 times greatest width; orifice of

dorsal oesophageal gland about one-fourth stylet length

posterior to stylet 6 .

6. Distance between vulva and anus about one and one- half times
diameter of vulva Heterodera rostochiensis

Distance between anus and vulva about two and one-half times
diameter of vulva Heterodera tabacum

7. H. cacti gioup. Hyaline portion of larval tail about as long

as stylet; stylet knobs concave anteriorly

Heterodera weissi

Hyaline portion of larval tail usually shorter than stylet;
stylet knobs convex anteriorly — Heterodera cacti

8. H, schachtii group. Cyst always with distinct punctation
consisting of dots of uniforra size but not in rows (fig. 25);

Paras . A : 9

brown Icnobs closely clustered around vulva. Hyaline portion
of larval tail at least one and one-half times longer than
stylet Heterodera major

Cyst with or without punctation, mostly in rows if present;

brown knobs not closely clustered around vulva. Hyaline

portion of larval tail about as long as stylet 9.

9. Average length of lai'vae I4.8O p or more 10.

Average length of larvae about U6O p Heterodera schachtii

10. Average length of larvae U8U }i Heterodera glycines

Average length of larvae 502 ji Heterodera trifolii

Average length of larvae 5l8 }i Heterodera schachtii

galeopsidis

11. H. gOttingiana group.

Average length of larvae UlU p. Heterodera cruciferae

Average length of larvae kSh p. Heterodera caret ae

Average length of larvae UTU p Heterodera g5ttingiana

Average length of larvae UO^ p Heterodera humuli

Average length of larvae li06 p Heterodera fici

Host Plants

Many lists of host plants of Heterodera species have been published, but
it seems probable that many of these are inaccurate in that they include
plants which are not hosts of the species discussed. This is especially
true of the older lists and of those based on Information compiled from
the literature rather than from host tests. In order to avoid this par-
ticular error, the following list of hosts includes only the type host, of
each species and an indication of the other plants which it attacks so
far as there is general agreement or information on host tests available.
That is, the list is not intended to be complete, but is believed to be
accurate so far as what is included is concerned.

The species of Heterodera and their principal hosts are:

H. schachtii. T.'ype host, sugar beet (Beta vulgaris L.). Also other
Chenopodiaceae, many species of Cruciferae (Oostenbrink, 1950 and Jones,
1951) and various species of other plant families (Thome 1932). It seems
possible that H. schachtii attacks a wider variety of plants than any

Paraa. A: 10

other kiiovm species of Heterodeia.

H. gtfttingiana. Type host, garden peas (Pisum sativum L.)- Also other
Leguminosae, but according to Oostenbrink (195l)j this species does not
attack beans (Phaseolus vulgaris L.)j clover (Trifolium spp.)j alfalfa
(I'ledicago sativa L.), or soybeans (:"')oya max Piper) .

R. trifolii. Type nost, red clover (Trifolium pratense L.)- Also other
Leguminosae, including beans (Phaseolus vulgaris L.), but not peas (Pisum
sativum L.), alfalfa, or soybenas. (Oostenbrink, 19^1) •

H. glycines. Type host, soybean (Glycine max L.). Also snap bean (Phas-
eolus vulgaris L.)j Adzuki Bean (P. annularis), vetch (Vicia sp.), Annual
lespedeza (Lespedeza stipulacea Maxim. ), Henbit (Lamium sp. ) .

H. major. Type host, oats (Avena sativa L.). Also other Gramineae
^Oostenbrink, 1950) .

H. cruciferae. Type host, cabbage (Brassica oleracea L.). Also other
Gruciferae.

H. carotae. Type host, carrot (Daucus carotae L.). Wild carrot (Daucus
carotae) is the only other kno\im host (Jones, 1950b).

H. humuli. Type host, hops ( Humulus lupulus L.). Also other Urticaceae.

H. galeopsidis. Type host, hemp nettle (Galeopsis tetrahit L.). Also
other Labiatae and some species of Chenopodiaceae and Carophyllaceae
(Jones, 1950b).

H. fici. Type host, mbber plant (Ficus sp.)

H. weissi. Type host, knotweed (Polygonum pensylvanicum L.). No other
hosts kno^-m.

H. cacti. Type host, Phyllocactus (Epiphyllum ackermanni). Also other
Cactaceae.

H. rostochiensis. Type host, potato (Solanum tuberostim L.). Also tomato
^ycopersicon esculentum Mill.) and a few other species of Solanaceae
(Oostenbrink, 1950), but not tobacco (Nicotiana tabacum L.) (Taylor, 1952)

H. tabacum. Type host, tobacco (Nicotiana tabacum L.) and tomato.

H. punctata. Type host, wheat (Triticum vulgare Vill.). Also other
Gramineae.

Species of other plant families than those mentioned above have been
reported as infected by nematodes of the genus Heterodera. Some of these
may be attacked by known species, but it is highly probable that others
are attacked by species as yet undescribcd. Among these should be men-
tioned the species found attacking sea marram grass (Ananophila arenaria

Paras. A: 11

(L.) Link) by Triffitt (1929), one found by Thorne on Shadscale (Atri-
plex confertifolia (Torr. and Frem.) S. VJats.)} one found by Chitwood
(19U9) in soil from North Dakota, and several found by Oostenbrink (1950)
attacking plants of various species.

Captions for Illustrations

Plate 1.

Shapes of cysts of Heterodera species. Fig. 1.
H. rostochiwnsis* Fig. 2. H. punctata. Fig, 3.
H. schachtii. Fig. U. H. avenae . Fig. 3. H. weissi,
FIp. 6. H. cacti.

Plate II.

Cyst patterns of Heterodera schachtii and related
species. Figs. 7 and b. Zig-zag line pattern near
middle of cysts of H. trifolii and H. schachtii
respectively. Fig. 9« Pattern at junction of neck and
body of cysts of H. schachtii. Fig. 10. Pattern near
vxilva of cyst of H. gottingiana. Fig. 11. Sheaf-
shaped object and dark bodies at lower end of cyst
of H. schachtii. Fig. 12. Network pattern, a
varTation of that shown in Figs. 7 and 8. Magnifi-
cation of figure 11 is about 200X, all other about

iaox.

Plate III.

Cyst markings of Heterodera weissi and H. cacti.
Fig. 13. Lower part of cyst of H. cacti. Fig. lU.
Pattern near middle of cyst of H. weissi. Fig. 15 •
Pattern at junction of body and neck of H. weissi.
Fig. 16. Pattern near middle of cysts o7 H. cacti.
Fig. 17. Lower end of cysts of H. cacti showing
anus. Fig. I8. Grainy appearance of cyst of H,
cacti. Ail about UIOa.

PUte IV.

Cyst patterns and punctation of Heterodera rostochiensis
and H. punctata. Fig, 19. Pattern at vulva and anus
of H. rostochiensis. Fig. 20. Same as Fig. 19, but
with deeper focus to show ptinctation. Pig.
and vulvar openings of cyst of H, punctata.
split in process of preparationj. Fig. 22.

21. Anal
(Cyst
Pattern

. Fig. 23.

at about middle of cyst of H. rostochiensis,

Pattern of upper part of cyst of H. punctata. Fig. 2U.

Punctation of H. rostochiensis. All about UlOX.

Plate V.

Cysts and eggs of Heterodera species. Fig. 25,
Pionctation of cyst of H. avenae. Fig. 26. Puncta-
tion and pattern of cyst of H, humuli . Fig. 27.
Punctation of cyst of H. triTolii. Fig. 28. Puncta-
tion of egg shell of H. cacti.

h

Fig. 2

Fig. 3 ^^

Fig. 4

in

Fig. 5

Fig. 6

■MP

?f1IBM

*

^aJSSt-

inr>*S;

ilflii*^^^.

'^"^h^-

m^^

'^4>?':i

, fc'

Paras. B:l

ROOT-KNOT NEMATODES (MF.LOIDOGYME SPP.)

The genus Ileloidogyne was placed in the family Heteroderidae of the
Tylenchoidea by Thorne (19U9) with the genus Heterodera (cyst-forming
nematodes). Prior to 19h9, all the root-knot nematodes were included
in Heterodera and considered as one species, Heterodera marioni .
Chitwood (19U9), after making a morphological study of the root-knot
nematodes, removed them from the genus Heterodera and reassigned them
to Meloidogyne , since this was the earliest valid generic name for this
group, having been used by Goeldi in 188? for a nematode causing root
galling of coffee trees in Brazil. Five species and one subspecies
were described by Chitwood at this time and later (1952) he described
another subspecies.

The root-knot nematodes differ in many respects from the cyst-forming
group and the following comparisons might be made: Meloidogyne females
disintegrate soon after death; the Heterodera female body turns into a
tough, durable cyst which may remain in the soil for several years.
The Meloidogyne female never retains eggs but instead deposits them in
a mucoid fibrous mass outside the body; the Heterodera female always
retains eggs in the body which acts as a protective cyst. Meloidogyne
males possess two lateral cheeks on the lip region and sometimes have
two testes; Heterodera males have no cheeks but the lip region has
ridges dividing the labial region into six sectors, and only one testis
is present. The lip region of Meloidogyne larvae is not definitely
set off from the body, and the stylet is 10 to 15 microns long; the
lip region of Heterodera larvae is set off from body by a definite
constriction and the stylet is 20-33 microns long. Meloidogyne species
characteristically cause root-swellings or knots on siu. table hosts with
females tending to remain inside roots at maturity; Heterodera species
usually do not cause gall formation and females tend to be located on
the external surface of root at maturity. Meloidogyne species have a
rather wide host range while Heterodera species are usually rather
restricted in host range. Other differences between these two large
and important groups are known but those listed above are the easiest
to recognize.

At the present time, eight species and three subspecies are known and
no doubt more will be described. A listing of these will be of use
because some of the original articles are not likely to be encountered
without library facilities.

Meloidogyne exigua Goeldi, 188?

M. javanica (Treub, 1885) Chitwood, 19U9

M. javanica bauruensis Lordello, 1956

M. hapla Chitwood, 19ii9

M. incognita (Kofoid and White, 1919) Chitwood, 19U9

Paras. B:2

M. incognita acrita Chitwood, 19l49

M. arenaria (Neal, I889) Chitwood, I9I49

M. arenaria thamesi Chitwood, 19^2

M. inornata Lordello, 19^6

M. brevicanda Loos, 1953

M. acronea Coetzee, 1956

At the present time, the following species and subspecies are known to
occur in the United States: Meloidogyne incognita, M. incognita acrita,
M. hapla, M. arenaria, M. arenaria thamesi, and M. javanica» Detailed
descriptions of these forms are given by Chitwood (19U9) . Sasser (195iU)
studied the host-parasite relationships of certain of the root-lcnot
nematode species and proposed a method of identification by host reac-
tion. Taylor, Dropkin, and Martin (1955) discuss in detail the identi-
fication of the various known species. This paper is very useful
because of its illustrations and a key to the species reported up to
1955 (which includes the knoxm species in the United States up to the
present) .

The reader is referred to a recent review of the genus Meloidogyne by
Franklin (1957) which deals with some aspects of the taxononry of these
nematodes. The taxonomy of these forms is not easy and it involves an
appreciation of the fact that one is not dealing with identical speci-
mens, but rather with specimens which fall within relatively consistent
morphological and host ranges. Separation of the species is of prac-
tical importance because the host ranges of the different species .are
not identical and thus offer a means of control.

It is of Interest to note that a new genus, Meloidodera, has been re-
ported (Cidtwood et al, 1956). As its name suggests, it has features
of both Meloidogyne and Heterodera. It is intermediate between them
in that, unlike root-knot nematodes, no gall is formed in the plant,
the female body wall is tough, and there is retention of eggs within
the body. It differs from the cyst-forming nematodes in that no dis-
tinct cyst stage exists and there is a distinct pattern form of the
anniiles. There are other defijiite morphological distinctions which
clearly set the Meloidodera apart. The original description should
be studied, because this nem-atode is already being found in various
places in the United States.

Paras. B:3

LITERATURE CITED

1. Chitwood, B. G. 19h9 . Root-knot nematodes — Part I. A revision
of the genus >1eloidogyne Goeldi 188? . Proc. Helm. Soc. Wash. 16:
90-lOh.

2. Chitwood, B. G., A. W. Specht, and Leon Havis. 1952. Root-knot
nematodes. III. Effects of Meloidogyne incognita and M. javanica
on some peach rootstocks. Plant and Soil U(l) : 77-95-

3. Cliitwood, B. G., C. I. Hannon, R. P. Esser. 1956. A new nematode
genus, Meloidodera, linking the genera Heterodera and Meloidogyne .
Phytopathology 115(5) :26i;-266.

U. Coetzee, V. 1956. Meloidogyne acronea, a new species of root-knot
nematode. Nature, London 177 (i;515) : 899-900.

5. Franklin, M. T. 1957. Review of the genus Meloidogyne. Nema-
tologica II (Supplement) :387-397S.

6. Goeldi, E. A. 1887. Relatoria sobre a molestia do cafeiro na
provincia da Rio de Janeiro. Apparently an advance separate of:
Arch. Mus. nac. Rio de J. 8:7-121, 1892.

7. Loos, G. A. 1953. Meloidogyne brevicauda, n. sp. a cause of root-
knot of mature tea in Ceylon. Proc. Helm. Soc. Wash. 20(2):83-91.

6. Lordello, L. G. E. 1956. Nematoids que parasitam a soja na regiao
de Bauru. Bragantia 15(6):55-6U.

9. Lordello, L. G. E. 1956. nMeloidogyne inornata" sp. n., a serious
pest of soybean in the State of Sao Paulo, Brazil, (Nematoda,
Heteroderidae) . Rev. bras. Biol. l6(l): 65-70.

10.. Sasser, J. N. 195U. Identification and Host-Parasite relationships
of certain root-knot nematodes (Meloidogyne spp.). Univ. of Md.
Agr. Piq5. Sta. Tech. Bui. A-77, 30 p.

11. Taylor, A. L., V. H. Dropkin, and G. C. Martin. 1955. Perineal
patterns of root-knot nematodes. Phytopathology i;5:26-3U.

12. Thorne, Gerald. 19ii9. On the classification of the Tylenchida,
new order (Nematoda, Phasmidia). Proc. Helm. Soc. Wash. l6(2):
37-73.

Paras . C : 1

MEADOW OR ROOT-LFSION NEMATODES (PRATYLENCHUS SPP.)

The genus Pratylenchus is made up of several species, all of which are
known plant parasites. As a group, these nematodes are widely dis-
tributed, have an extensive host range, and rank as one of the most
important plant-parasitic genera. All old host lists should be used
with caution because considerable changes have been taking place in
the taxonon^r of this group. A monograph (Sher and Allen, 1953) revis-
ing the genus and adding three new species to it has marked the begin-
ning of more attention being paid to the taxonomy of these important
parasites in various parts of the world. Additional new species were
described along with a study of morphological variation within certain
of the species (Taylor and Jenkins, 1957) in a paper. It also contains
some very worthwhile suggestions regarding consideration of morpho-
logical variation and their expression which ought to be considered by
anyone doing taxonomic work, particularly with economically important
genera which will be intensively studied for many years to come.

The useful, but now outdated, key to Pratylenchus species from Sher
and Allen (1953) is furnished in these Notes. In addition there is
given a listing of the Pratylenchus spp. to date with citations where
information can be obtained. It is suggested that forms which may not
be identified readily be sent to workers who are specialize ng in the
taxonomy of this genus.

The Pratylenchs are primarily root-parasites and may be endo- or ec to-
parasitic in habit. Infections may give rise to disease symptoms of
various kinds according to the hosts involved. In general, the damage
resulting is what would be expected of a plant debilitated ty increas-
ing amounts of root injury and loss. In boxwoods, a characteristic
bronze coloration of the foliage has been noted, and foliar discolora-
tions in other kinds of plants may also suggest the below-ground pres-
ence of these nematodes.

Meadow nematodes in various stages of their life cycle are to be found
in soil samples. These nematodes are known to evacuate root tissues
which are decaying, and thus may be overlooked if root examination
alone is relied upon. Infected roots may be stunted and show spots,
streaks, or encircling bands of discolored necrotic tissue. These
symptoms are not exclusive for Pratylenchus, although they are usually
found when these nematodes are the parasite involved. The nematodes
can enter into the root tissues, migrate within the root, and complete
their life cycle. The external feeding sites, places of entry, and
other damaged areas of the root may serve as portals of entry for other
infective soil microorganisms.

It is important to remember that these nematodes usually leave roots
when decay sets in and that most of the species are reported to be
unable to withstand drying. Both of these factors can effect chances
of finding the nematode in older infections or in samples of soil and
plants which become dry before being processed.

Paras. C:2

LISTING OF SPECIES OF FRATYLENCHUS

Sher, S. A., and M. W. Allen. 1953. Revision of the genus Pratylen-
chus (Nematoda: Tylenchidae ) . Univ. Calif. Public, in Zool.
W(B):hla-h70.

P. brachyurus (Godfrey); Syn. P. leiocephalus Steiner

P. coffeae (Zimmerman); Syn. P. mahogani, P. musicola

P. goodeyi n. sp.

P. minyus n. sp.

P. penetrans (Cobb)

P. pratensis (DeMan); (Tylenchus gulosus, Aphelenchus neglecbus

P. scribneri (Steiner)

P. thornei n. sp.

P. vulnus Allen & Jensen

P. zeae Graham (P. zeae Steiner, nomen nudum)

Species Inquirendae

P. sac char i (Soltivedel)
Dolichodorus heterocercus Kreis

Taylor, D. P., and ¥. R. Jenkins. 1957. Variation within the nema-
tode genus Pr a tylenchus, with the descriptions of P. hexincisus,
n. sp. and P. subpenetrans, n. sp.

P. hexincisus n. sp.

P. subpenetrans n. sp.

Also information on morphology of:

P. zeae

P. penetrans

Merzheyevskaya, 0. I. 1951. New species of nematodes. (In Russian).
Akademiia Nauk Belaruskaia, Minsk. Instytut Biialogii. Sbornik

Paras. C:3

Nachnykh Trudovft. 2:112-120. Fig. 1-6.
P. tumldiceps n. sp.

Meyl, A. H. 1953. Beitrage zur Kenntnis d6r Nematoden fauna vulkanisch
erhitzter Biotope I. Die terrikolen Nematoden in Bereich von
Fumarolen a\if der Insel Ischia. Z. Morph. Okol. Tiere. U2:67-ll6.

P. pratensis var. tenxxistriatus n. sp.

Meyl, A. H. 19$3. Die bisher in Italien gefundenen frei lebenden Erd-
und Sllsswasser-Nematoden. Arch. zool. Italiano 39 :l6l-26U.

P. pratensis var. bicaudatus n. sp.

Lordello, L. G. E. 1956. Sobre \m nematodeo do genero Pratylenchus ,
parasite das raizes de Allium cepa. Revista de Agricultura 31(3):
181-188.

P. coffeae brasiliensis n. subsp.

Loof , r. A. A. 1957. Was ist Aphelenchus neglec tus Rensch? Nemato-
lofTica 2 (Supplement) :3li8S.

P. neglec tus (Rensch, 1921;) n. comb.

Paras, C:U

KEY TO THE SPECIES OF PRATYLENQIUS
Sher-Allen, 1953

1. Lip region with 2 annules (1 striation) 2

Lip region with 3 or U annules (2 or 3 striations) 5

2. Lateral margin of lips angular brachyurus (Godfrey)

Lateral margin of lips rounded 3

3. Body long and slender (L=0,U-0.7, A=25-UO), males numerous

coffeae (Zimmennan)
Body short and stout (L=0.3-0.67, A=l8-26), males rare U

U. Vulva (=V) at 75-80 per cent scribneri Steiner

Vulva at 80-88 per cent minyus n. sp.

5. Striations around terminus of tail pratensis (de Man)

No striations around terminus of tail ~ — — — » 6

6. Outer margins of cephalic framework prominent, extending

posteriorly about two body annules, tail bluntly rounded

thomei n. sp.

Outer margins of cephalic framework nonmal 7

7. Vulva 68-76 per cent, tail tapering to narrow rovinded

terminus 8

Vulva 78-8U per cent 9

8. Three annules on lip region, males absent — zeae Graham

Four annules on lip region, males numerous goodeyi n, sp.

9. Postuterine branch short, length about equal to body width

at vulva, tail broadly rounded penetrans (Cobb)

Postuterine branch long, two or three times body width at
vulva, tail tapering to narrow roxinded terminus

vulnus Allen and Jensen

J'ary;;. . D:l

RID AND LFAF Nl'llATODES (APIIELKf-ICtlOIDTsS SPP.)

The genus Aphelenchoidos consists of numeroiis species, is widespread,
and specimens are frequently found in soil and plant samples. This
genus has a diversity of biological associations. There are species
that are parasitic on and in plant roots, buds, and leaves. Some
species feed on fungi and possibly on soil algae, others are parasites
or associates of insects, and still others are predators of nematodes
and other soil microorganisms.

As the common name of this genus suggests, the plant parasitic species
of this group are probably best known because of the foliar diseases
they cause. Symptoms of disease vary, depending upon the host plant
and the nematode species Involved. Plants may show varying degrees of
deformation of stems, leaves, flowers, and buds may remain rudimentary.
"Dwarf" or "crimp" are descriptive terms applied to such conditions as,
for example, in strawberries. In chrysanthemums, dahlias, ferns, and
other plants leaves invaded by the nematodes show discolorations and
eventually necrotic areas appear, these spots are often bounded by the
larger leaf veins and appear as distinctly angular in outline. Aphelen-
choides may lodge in the leaf sheathes of grasses or between the devel-
oping leaves or petals in the buds. These nematodes feeding as ecto-
parasites, as they are not within the plant tissues, can cause symptoms
of disease in the parts noted, such as abnormal foliar color, distortion
of the leaves, and in some cases lead to decay of the structures.

An important example of a disease caused by a species of Aphelenchoides,
in which the stem of the host is affected, is the red ring disease of
coconuts. The nematodes may initiate invasion of the host by penetrat-
ing at the terminal growing point, being carried there in some cases
by an insect. Eventually the entire parenchyma of the host is pene-
trated by the migrating nematodes, and a red-collored ring which char-
acterizes the disease is found in the parenchyma of the tree's trunk.

It is not possible to assign a definite role to all of the Aphelen-
choides species that are recovered from about plants. The fact that
some of the plant-parasitic species can thrive on cultures of fungi
suggests parasitism on the higher plants may be only incidental or of
a facultative nature. It is possible that incidental feeding on the
root surfaces by some of the often quite numerous Aphelenchoides and
similar forms could be of importance. Numerous minute wounds in the
presence of other soil microorganisms may be related to establishment
of disease complexes. The ability of Aphenlenchoides to survive on
fungi and perhaps algae, plus an ability to withstand periods of inac-
tivation in conditions of adversity, must be considered when developing
control practices for this genus.

The taxonomy of the genus is difficult, not because of a lack of reports
in the literature, but rather because of so many accounts without

P.'ira3. D:?

adeqiiato descriptions and which are occasionally in conflict. Differ-
ences in hosts, rather than morphological differencen of the nematodes,
were sometimes the criteria used for setting up a species. The text-
book by Pilipjev and Stekhoven (I9UI) is a good reference for this
genus and is provided with a taxonomic key, descriptions, and bionomics
for many of the species. A recent paper (Allen 1952) presents a recon-
sideration of important foliar species which is based entirely upon
the comparative morphology of the nematodes. The result is a synony-
mizing of numerous species previously regarded as distinct on the basis
of the host involved.

Literature Cited

1. Allen, M. W. 19^2. Taxonomic status of the bud and leaf nematoc3'2S
related to Aphelenchoides fragariae (Ritzema Bos L891) . Proc.
Helminth. Soc. Washington 19(2): 108-120.

Faraa. D:3

KKY TO THE APHI'lLMCHIDAK

The superfamily, Aphelenchoidea, has a single family, Aphelenchidae,
which is divided into two subfamilies, Aphelenchinae and Paraphelen-
chinae. A key is presented for distinguishing genera of these sub-
families which are found in. the soil in association with plant life.

1. Posterior portion of esophagus not a distinct glandular bulb,
esophageal glands considerably overlapping the beginning of
the intestine. Aphelenchinae 2.

Posterior portion of esophagus a distinct structiire contain-
ing the esophageal glands and not overlapping the intestine.
Paraphelenchinae U.

2. Head possessing a shallow, sclerotized frontal-disc. (Lat-
eral field with 3 incisures, male unknown.) Anomyctus

Head not possessing a shallow, sclerotized frontal-disc> 3»

3. Isthmus leading to a more or less distinct posterior portion
region about 1/2 to 1/3 length of pre corpus and blending
with the beginning of the intestine. Lateral field vrLth
10-12 incisures, spicules slender, gubernaculum present,
bursa present. Spear without basal knobs. Aphelenchus

Isthmus comparatively short and blending with beginning of
intestine. Longitudinal striae present, characteristic
rose-thorn shaped spicules, no gubernaculum, bursa absent.
Spear with or without basal knobs. Aphelenchoides

U, Post-bulbar region of esophagus set-off from intestine by
a constriction. Absence of longitudinal striae in lateral
fields . Paraphelenchus

Post-bulbar region of esophagus usually not set-off from
intestine by a constriction. Lateral field with 10-12

incisures with a bulge ventrally on tail region.

Metaphelenchus

Vnv.'r.. Dih

KKY TO •nil': srEcu:,' ok dud mu lI'IAI'' iiLMi^Di'irj

(From Allen, 195?)

1. llcnd swollen, wider thnn iiecl; . )i liner, in win," ere^').

Head not swollen, 2 lines In m.n;' nrea . 1^ . fr-ij-;nriae

2. Length of posterior-uterine branch 5 or more tim.es body widthx

Lcnpth of posterier-uterine brnneh less than U times body -width.

A. Besseyi

3. Tail bluntly rounded, armed by a single ventral spine. -A. subtenuis
Tail terminus peg-like, -rmcd with h sm^ll mucrons.-A. ritzema-bosi

Aphelenchoides fragariae (Ritzema Bos, I89I) Christie, 1932
Synonyms :

Aphelenchoides olesistus, Ritzema Bos, 1893
Aphelenchoides olesistus, oteiner, 1932

Aphelenchoides olesistus var. longicollis, Schwartz, 1911
Aphelenchoides olesistus var. longicollis, Goodey, 1933
Aphelenchoides longicollis, Filip. and Stek., 19^1
Aphelenchus psuedolesistus, Goodey, 1928
Aphelenchoides psuedolesistus, Goodey, 1933
Aphelenchus omerodis, Ritzema Pos, 1891, in part.

Aphelenchoides besseyi, Christie 19li2
Synonym :
Aphelenchoides oryzae, Yokoo, 19i|8

/nhelenchoides subtenuis (Cobb, 1926) Steiner and Buhrer l'^32
Synonym :

Porn.". D:5

AphclorichoidrG hodsoni, Goodeyj 193^

Aphelenchoides ritzema-bosi (Schwartz, 1912) Steinrr, 1932
Zynoixfnis:

T^rlenchus ribes, Taylor, 191?
Aphelenchus ribes, Goodey, 1932
Aphelenchoides ribes, Goodey, 1933
Ap^'elenchns phyllophagus, Stewart, 1921

Paras. Eil

STM AND BULB NWIATODE (DITYLENCHUS SPP. )

This genus represents one of the major plant-parasitic groups and is
reported in association with many kinds of host plants and from many
countries. There are about 30 si'ecies described, the best known be-
ing Ditylenchus dipsaci. As perhaps is to be expected from a genus
with many species and having an extensive host range and wide geo-
graphic distribution, there exists difficiilty in the exact taxonomy
of the group. There has long been recognition of "races" and applica-
tion of existing host lists must take into consideration this matter
of our not presently being able to accurately distinguish between
certain closely related species on the basis of morphology. Mixed
infections are also known to occur and must be guarded against in host
range studies. The book by Filipjev and Stekhoven (I9UI) is a good
reference for this genus and gives a taxonomic key, descriptions of
the species, and considerable information regarding the biology and
control of the more important species.

Symptoms of plant disease caused by these nematodes vary widely, depend-
ing upon the plant and nematode species involved. The diseased parts
of the plants may be the roots, leaves, stems, flowers, and seeds. In
the case of the teasle, the entire plant may be invaded by the nematode,
Ditylenchus dipsaci. More usually, only particular parts of the host
plants are involved, for example, the foliar parts of alfalfa or the
tubers of potatoes. Almost all row crop plants, forage plants, grains,
and many ornamentals and weeds have been reported as hosts for nema^
todes of this genus. Some of the forms parasitic on higher plants can
be cultijred on fungi, and it is possible that all the species may be
incidental feeders on the surface of roots, whether or not they ever
become endoparasitic in habit. Members of the genus which are regarded
as parasites of known importance, for the most part, are foxind within
the diseased host tissues.

Plants exhibiting irregularity of growth form and distortions of foliar
parts should be checked for the presence of Ditylenchus . Lesions of
stems and iinderground parts of plants showing a loose, granular, brown-
colored condition of the cells should also be checked carefully for
these nematodes which are likely to be located in advance of the lesions.
A galled condition of the roots similar to root-knot is also known to
occur on some plants as a result of invasion by Ditylehchus. The damage
caused by these nematodes is by chemical in addition to mechnaical
action, as evidenced by what appears to be dissolution of the middle
lamella of the hosts' cells and abnormal hypertrophy of cells and tis-
sues in the vicinity of the nematodes.

Some members of this genus have the ability to withstand adverse envi-
ronmental conditions by going into a state of dormancy called anabiosis.
This capacity for survival under a wide range of circumstances is a
matter of significance in dissemination and control of such nematodes.

Vrrrjs. V-J

SPIRAL NEMATODES (TIELICOTYLENCTUJS SPP. AND ROTYLENCHIJS SPP.)

These organisms are commonly called spiral nematodes because of the orien-
tation of the body into a spiral when the animals are inactive or dead.
As a group, these nematodes are found associated with the roots of numer-
ous kinds of plants from many countries. They may be found with heads
embedded to shallow depths in -lesions of roots and other underground
plant parts or, in some cases, penetration may be deeper. In most
instances of lesions other microorganisms are present. There is no report
of successful cultivation of these nematodes in cultures with fungi, so
it is likely that they are obligate parasites of the higher plants. The
spiral nematodes do not appear to be as harmful to their hosts as other
parasitic nematodes such as root-knot or meadow nematodes. Recent
experimental work (Sledge, 1955) with Helicotylenchus nannus Steiner, IS^USj
indicates that these nematodes, although obligate parasites, are highly
successful parasites in that their hosts are not quickly rendered unsuit-
able as sources of food and sites for nematode reproduction. Such nema-
todes, however, may have significance as wounding agents providing portals
of entry for other soil microorganisms, some of which may be harmful in
their effects. It is too soon to do much generalizing about the sprial
nematodes, because intensive consideration of them as plan -parasites has
only recently been started.

Despite the present unsettled state of the taxonomy of the genera of the
Hoplolaiminae, which includes the spiral nematodes, the worker is advised
to make careful observation of these forms because of the increasing
appreciation of their distribution and potential importance as plant
pests. As should be done with the other nematode species, when reporting
work with these nematodes, cite the complete scientific name with the
authors and revisers of it. This is particularly important with the
spiral nematodes because of the state of flux of the taxonony. Consid-
erable experimental work may be reduced to lesser value, if in the future
it is uncertain as to which spiral nematode the results pertain. The
complete listing of synonyms as taken from Andrassy (1958) is presented
in the notes to assist in applying exact names.

Two important papers dealing with the taxonomy of the group are available
(Golden, 1956, and Andrassy, 1958). The first paper also includes a
study of the developmental stages and host-parasite relationships of
Rotylenchus buxophilus Golden, 1956. Taxonomic keys are copied from
both of these papers as that of Andrassy gives a synopsis of the sub-
family Hoplolaiminae and makes certain transfers of species and sets up
two new genera. The paper by Golden gives a key to the genera and
species of spiral nematodes (Helicotylenchus spp. and Rotylenchus spp.)
Both papers have excellent literature reviews and should also be referred
to for the illustrations. The book by Filipjer and Stekhover (19l;l)
gives a key to the Rotylenchus, but will be more useful now because of
the illustrations and descriptions. If one follows the taxonony as set
forth by Andrassy, the key to species in the paper by Golden will also
be found useful.

Parac. K;2

Literature Cited

Andrassy, I. 1958. Hoplolalmus tylenchlformls Daday, 190p (Syn. H. coro-
na tus Cobb, 1923) und die Gattungen der unterfamille Hoplolaiminae
Filipjev, 1936. Nematologica 3(l):i4U-U6.

Fillpjev, I. N. and J. H. Schuurmans Stekhoven, Jr. I9UI. A manual of
agricultural helminthology. E. J. Brill, Leiden, Holland, pp. 213-
229.

Golden, A. M. 1956. Taxonomy of the spiral nematodes (Rotylenchus and
Helicotylenchus ) and the developmental stages and host-parasite
relationships of R. buxophilus, n.sp., attacking boxwood. Univ. of
Md. Bui. A-e5. 2F pp.

Sledge, E. B. 1955- Pathogenicity of the spiral nematode, Helicotylen-
chus nannus Steiner, 19U5> in relation to selected varieties of
corn. Thesis. Ala. Poly tech. Inst., Alabama. 60 pp.

Paras. Fi3

A KFY TO THE GENERA AND SPECIES OF SPIRllL NEMATODES
(From Golden, 1955)

1. Opening of dorsal esophageal gland usually less than 1/3 of
stylet length from base of styletj phasmids either small,
dot-like or large, round -or oval structures (scutellum).

Rotylenchus Filipjev, 193h h

Opening of aorsal esophageal gland 1/3 or more of stylet
length from base of styletj phasmids always small, dot-like
structures. Helico tylenchus Steiner_, 19U5 2

2. Tail generally rounded; stylet approximately 22 y.

H. multicinctus (Cobb, 1893) n. comb.

Tail not rounded, with most of the curvature being on the

dorsal side; stylet about 25-28 yi. 3

3. Tail usually with long ventral terminal process; well-developed
spherical spermatheca present.

H. erythrinae (Zinmermann, 190li) n. comb.

Tail with or without short, bl\int ventral terminal process;
well-developed spherical spermatheca absent.

H. nannus Steiner, 19U5

U. Phasmids large, round or oval structures ( scutelltan) . 7

Phasmids small, dot-like structures. 5

5. Tail with distinct ventral terminal process; stylet 23-25 y.

R. melancholicus Lordello, 1955

Tail without a ventral terminal process; stylet 28 y or over. 6

6. Phasmids at about latitude of anus; stylet approximately 28 y.

R. robustus (de Man, 1880) Filipjev, 19i;5

Phasmids well forward of anus; stylet about 33 V--

R. buxophilus n.sp.

7. Phasmids not opposite each other, location of anterior one being
at average of 79^ of body length and posterior one at an average
of 86^; lips of vulva protruding.

R. christiei Golden and Taylor, 1956

Paras. F1I4

Phasmids opposite esch other and located on tail or in vicinity

of anus| lips of vulva not protuding. --.-.— ~ 8

8. Tail as long as or longer than anal body diameter j head with

■5even or more annules. — 10

Tail much shorter than anal body diameter; head with five

annules or less . 9

9. Lateral field aerolated""' in esophageal region; five annules
on head. R. brachyurus Steiner, 1938

Lateral field not aerolated in esophageal region; three
annules on head, rarely four. R. coheni Goodey, 19^2

10. Stylet knobs anteriorly flattened or slightly concave.

R. bradys (Steiner and LeHew, 1939) Filpjev, 1936

Stylet knobs ovoid, anteriorly convex.

R. blaberus Steiner, 1937

Species of undetermined status:

Rotylenchus pararobustus (Schuurmans-Stekhoven and Texmissen, 1938 j

Filipjev and Schuurmans-Stekhoven, 19Ul.
Rotylenchus africanus (Micoletzky, 19l5) Filipjev and Schuurmans-

S tekhoven, 19U1.

■*\ransverse body striae extending into or across the lateral field.

A KEY TO THE GENERA OF HOPLOLAIMINAE
(From Andrassy, 193B)

1. Phasmids normal, small, pore-like: Rotylenchus genus - group 2
Phasmids extremely large, scutellum-like: Hoplolaimus genus - group h

2. Opening of dorsal esophageal gland about 1/2 spear length from base
of knobs of spear: Helicotylenchus Steiner

Opening of dorsal esophageal gland not more than l/3 spear length
from base of knobs of spear: 3

3. Lip region marked by annules divided into numerous small sections;
gubernaculum bearing titillae: Ro tylenchus Filipjev

Lip region with transverse annules onlyj gubernaculum without
titillae: Gottholdsteineria n. gen.

h. All three fields of lateral field aerolated by transverse striae j
spear knobs anteriorly furcate and pointed: Hoplolaimus Daday

Center field of lateral field smooth, without transverse striaej,
spear knobs rounded: Scutellonema n. gen.

I. Rotylenchus genus-group

A. Ro tylenchus Filipjev, 1936

R. robustus (de Man, I876) Filipjev, 1936 (Type for the genus)
syn: Tylenchus robustus de Man, I876

Tylenchorhynchus robus tus (de Man, I876) Micqletzky,

1921 partim
Hoplolaimus uniformis Thorne, I9I4.9

B. Helico tylenchus Steiner, 19U5

H. africanus Micoletzky n. comb.

syn: Tylenchus africanus Micoletzky, 1915

Tylenchorhynchus africanus (Micoletzky, 1915) Schuvirmans-

Stekhoven and Teunissen, 1938
Rotylenchus africanus (Micoletzky, 1915) Filipjev and
Schuiirmans-Stekhoven, 19Ul
H. erythrinae (Zimmermaan, 190ij) Golden, 1956
syn: Tylenchus erythrinae Zimmermann, 190U
Tylenchus psuedorobus tus Steiner, 191)|
Aphelenchus dubius var. peruensis Steiner, 1920
Tylenchus spiralis Cassidy, 1930

Paras. F:6

Tylenchorhynchus robustus var. erythrlnae (Zitnmermann,

IpoHTBally and Reydon, 1931
Anguillulina multlclncta in Goodey, 1932 nee Cobb, 1893
Tylenchorhynchus multiclnctus in Schuiirmans-Stekhoven and

Teunissen, 1936 nee Cobb, 1893
Anguillulina erythrinae (Ziiranermann, 190U) Goodey, 19U0
Rotylenchus erythrinae (Zimmermann, 1901;) Goodey, 1951
partim
H. iperoiguensis (Carvalho, 1956) n, comb.
" syn: Rotylenchus iperoiguensis Carvalho, 1956
H. melancholic us (Lordello, 1955) n. comb.

syn: Rotylenchus melancholicus Lordello, 1955
H. multiclnctus (Cobb, 1893) Golden, 1956
syn: Tylenchus mul ticinc tus Cobb, 1893

fiotylenchus multiclnctus (Cobb, 1893) Filipjev, 1936
Anguillulina mul tic in ta (Cobb, 1893) Goodey, 19kO
H. nannus Steiner, 19ii5 (Type for the genus)

C. Gottholdsteineria n. gen.

G. buxophila {Golden, 1956) n. comb.

syn: Rotylenchus buxophilus Golden, 1956
G. goodeyi (Loos and Oostenbrink, 1958) n. comb. (Type for the
genus)

syn: Tyl enchorhyachus robustus in Micoletzky, 1921 partim nee
de Man, 1876
Rotylenchus robustus in Filipjev and Schuurraans-Stekhoven,

19U1 and in Thorne, 19U9 nee de Man, I876
Rotylenchus goodeyi Loof and Oostenbrink, 1958
G. pararobusta (Schuurmans-Stekhoven and Teunissen, 1938) n.
comb.

syn: Tylenchorhynchus robustus in Schuvirmans-Stekhoven, 1936
nee de Man, 1875
Tylenchorhynchus pararobustus Schuurraans-Stekhoven and

Teunnisen, 1938
Rotylenchus pararobustus (Schuurmans-Stekhoven and

Teunnisen, 1938) Filipjev and Schuurmans-Stekhoven,
19U1
G. quarta Andrassy, 1958

II. Hoplolaimus genus -group

A. Hoplolaimus Daday, 1905

H. proporicus J. B. Goodey, 195?

H. tylenchiformis Daday, 1905 (Type for the genus)
syn: Hoplolaimus coronatus Cobb, 1923

B. Scutellonema n. gen.

S. blaberum (Steiner, 1937) n. comb. (Type for the genus)

syn: Rotylenchus blaberus Steiner, 1937
S. booeki (Lordello, 1957) n. comb.

syn: Rotylenchus booeki Lordello, 1957

Paras. F:7

S. brachyiirum (Steiner, 1938) n. comb.

syn: RotyLenchus brachyurus Steiner, 1938
S. bradys (Steiner and LeHew, 1933) n« comb.

syn: Hoplolaimus bradys Steiner and LeHew, 1933

Anpulllulina bradys (Steiner and LeHew, 1933) Filipjev,
193^
S. christiei (Golden and Taylor, 1956) n. comb.

syn: Rotylenchus christiei Golden and Taylor, 1956
S. coheni (J. B. Goodey, 1952) n. comb«

syn: Rotylenchus coheni J. B. Goodey, 1952

Paras. G:l

STING NEMATODES (BEL0N0LAIMU3 SPP.)

The sting nematode, Belonolamus gracilis, was described by Steiner
in I9U9 ("Plant Nematodes the Grower Should Know." Soil Science
Society of Florida Proceedings IV-B pp. 72-117). This genus was not
described in time to be included in either Goodey's 1951 "Soil and
Fresh-water Nematodes" or Thome's "On the Classification of the Ty-
lenchida" (Proc. Helm. Soc. Wash. 16(2): 37-73, of 19U9). However,
Steiner has an excellent illustration and description. The fairly
large size (about 2mm.), the very long stylet, and the other distinc-
tive features illustrated make identification easy. It should be
pointed out that only one species of the genus has been described, and
it is quite probable that other species exist. If so, these will differ
in details from B. gracilis.

Belonolaimus is an ectoparasite and will, therefore, be found in soil
collections, though occasional specimens, usually young individuals,
may be found in the root tissues. (Christie, J. R., A. N. Brooks and
V. G. Perry, 1952. Phytopath. U2(U): 173-176).

Christie et al. (loc. cit.) gives an excellent discussion of the sting
nematode and its effects. The following excerpts are taken from this
paper:

"The principal above ground symptom of attack by sting nematodes is
poor growth of the crops, with severe stunting in patches in the field.
As with other nematodes, plants will show symptoms of nutrient deficien-
cies. Hoots of attacked plants may be injured at the tips with conse-
quent development of short, stubby branches. Necrotic lesions may be
produced along the sides of the roots. On celery growing in soil heavily
infested with Belonolaimus, most of the roots are apt to be in the upper
two inches of soil where the nematodes are least numerous. Final diag-
nosis of the trouble depends on finding the nematodes in the soil. It
should be kept in mind that Belonolaimus, like most other plant para-
sitic nematodes, is an obligate parasite. That is, it feeds only on
living roots. For this reason, it is often difficult to establish a
correlation between apparent damage to the plant and the number of nema-
todes which can be found in the adjacent soil. In fact. Just the oppo-
site might be found. There may be more nematodes around a root system
which is still healthy than around one which has already been mostly
destroyed by the nematodes."

Christie et al. reported a series of experiments which demonstrated
injury to strawberry, celery, corn, and other plants. In some of these
experiments they used a system which is well worth considering for
similar work. This system was devised to meet the objection that damage
observed on plants exposed to nematodes extracted from the soil might be
due to associated organisms rather than to the nematodes. They say:
"While it may be impossible to obtain nematodes free from contaminating

Paras. Qt2

organisms, it is neither impossible nor especially difficult to obtain
these same organisms free from nematodes. One can remove the nematodes
from the water in which they are suspended when drawn from the fiinnels,
and this fluid can be placed around the roots of control plants or
around the roots of a third set of plants provided for the purpose."
If a third set of plants is provided, comparisons can be made between
the three sets of plants: (1) Controls in sterilized soil; (2) In
sterilized soil inoculated with nematodes and associated organisms; and
(3) In sterilized soil inoculated with associated organisms without
nematodes. If growth in (1) and (3) is equal, while growth in (2) is
depressed, the logical assumption is that the decrease in grcr^th is
either due to the nematodes alone or to the combination of nematodes
and associated organisms. For practical purposes, it makes little
difference as it has been shown that the nematodes are an essential
member of the complex, and control might be expected to be obtained by
their elimination.

The fact that sting nematode may be an important factor in cotton wilt
was shown by Holdeman and Graham (Phytopath. U2(5): 283-28U, of 1952).
They collected infested field soil and eliminated sting nematodes from
a portion of it by air drying, while a second portion was kept moist.
Cotton Fusarium inoculum was added to each lot of soil and wilt-resist-
ant and wilt- susceptible cotton varieties were planted. With the sting
nematodes, the resistant variety averaged S'^% wilt, compared with no
wilt where the nematode was absent. The susceptible variety had an
average of 88^ wilt with the sting nematode and only 10^ without it.

Information on hosts of Belonolaimus can be obtained from Christie ©t
at. (loc. cit.) and from Holdeman and Graham (Plant Dis. Reporter 3T(lO):
II^T-S'OO, of 1953). Apparently, sting nematodes are capable of feeding
on a large variety of plant species, including both crop plants and weeds.
As would be expected, certain other species appear to be unsuitable hosts,
among them being tobacco, watermelon, and crotalaria. However, establish-
ment of a host list for sting nematodes is still in the early stages, and
it should be kept in mind that there may be species, subspecies, popula-
tions, or races with varying host habits, as have been found with other
plant-parasitic nematodes.

Control of sting nematodes by crop rotation or by soil fiimigation appears
to present no special problems. Miller (Phytopath. U2(9): U70, of 1952)
reports excellent control with ethylene dibromide at moderate rates of
application. VJhen adequate host lists are available, control rotations
can be devised.

ParaH. H:l

RINQ NEMATODES (CRICONEMOIDES SPP.)

Rinf); nematodes are fairly common in agricultural soils and sometimes
occur in great numbers. However, because of their rather odd shape and
slup;gish movements, they may sometimes be overlooked. Shape is typi-
cally plump, since they are usually only about ten to fifteen times as
long as wide. Often the body is strongly curved, as shown in Fig. 7^
of Goodey's "Soil and Freshwater Nematodes." Once found, identification
to genus presents no special difficulty. The cuticle is strongly annu-
lated, and the annules are retrorse (directed backward). Apparently
this feature is connected with their locomotion, which is of a somewhat
different type than the serpentine motion of longer and slimmer nematodes.
Ring nematodes move through the soil by alternate contraction and expan-
sion of the body, the retrorse annules acting somewhat in the fashion of
a ratchet, catching on soil particles when the body is extended, and
slipping past when the body is contracted. The common rame, "ring nema-
todes," is applied to two genera, Criconema and Criconemoides . These
are closely related, differing mostly in that the annules of the latter
are smooth, while those of the former are fringed with scale-like pro-
jections, the shape and arrangement of which varies with species (Fig. 76,
Goodey) . Criconema appears to be rare in agricultural soils, but is
often found in forest soils.

Ring nematodes are usually about one-half millimeter long. Tliey have
stylets which are often-one-tenth of the body length and may be as long
as one-third of the body length. The stylet knobs are well developed
and are to be seen in the anterior part of the median oesophageal bulb
when the stylet is retracted.

An aid to identification to species is "On the Morphology of Criconeraoidesj
etc.," by Dewey J. Raski (Proc. Helmin. Soc . Wash. 19(2): 8^-99 of 1952).
This paper has a key to the species and is copied for inclusion in these
Notes.

Very little information, either on the biology or pathogenicity of ring
nematodes, is available. It is quite certain that they are plant-para-
sites. Steiner (Plant Nematodes the Grower Should Know) presents a
photograph of a ring nematode partially embedded in a root, though it
.is probable that most feeding is done from the outside of the root. It
is a fair assumption that this feeding kills or injures cells and that
the dead tissue might provide an entrance for pathogenic bacteria or
fungi which could not otherwise attack the plant.

Present evidence that ring nematodes may be a cause of important losses
in crops is mostly limited to observations of great numbers of ring
nematodes associated with roots of plants which were growing poorly.

Paras, u-.o

KEY TO THE SPECIES OF CRIC0NEMDIDE3
(From Raski, 19^f)

1. Spear length 100 v» or more 2

Spear length 90 ji or less ^

2. Total body annules 9$ or more »_3

Total body annules 58-61 annulifer (de Man)

3. Length 0.U50 mm. or morej spear not very long and thin (less

than 1/3 of body length) 1^

Length 0.270-0.300 mm.^ spear very long and thin (more than

1/3 of body length) macrodorum Taylor

U. Spear 105 ^j total body annules lUO; length 0.880-1.000 mm.

annulatum Taylor

Spear 122 )ij total body annules 95-103} length O.U56 mm.

sphagni (Micoletzky)

5 . Tail pointed ^

Tail rovmded jj^

6. Total body annules 110 or more 7

Total body annules less than 80 8

7. Length 0.700 mm.j vulva on 16-I7th annule from terminus

komabaensis (Imamura)

Length 0.550-0.590 mm.} vulva on 8th annule from terminus

morgense (Hofmanner and Menzel)

8. Total body annules 70 or more o

Total body annules 65 -heideri (Stefanski)

9. Vulva located on 12-15 th annule from terminus; total body

annules 70-76 2.0

Paras. Hi3

Vtilva located on 7th annnle from ternilnusj total body ajinules 79

peruense (Steiner)

10. Lenpth 0.700 mm.; first ajintile larger than second annule

crotaloldes (Cobb)

Length O.hOO-O.laS^ mm.; f-irst annule smaller than second annule

demaiil (Micoletzky)

11. Joints on lateral line except on anterior end of body 12

No joints on lateral line, annules unbroken except occasional
anastomosis 13

12. Lateral line zig-zag; spear 57 V sphaerocephalum Taylor

Lateral line with simple breaks; spear 50 yi citri Steiner

13. Total body annules 115 or less; spear U8 ya or more lU

Total body annules l[(2 or more; spear 38-Ul \i parvxun Raski

lU. Total body annules more than 73 *—!$

Total body annules 60-65 informe (Micoletzky)

15. Spear length 70-86 y 16

Spear length U8-67 p 18

16. Sublateral lobes absent 17

Sublateral lobes present xenoplax Raski

17. Total body annules 106-113; length 0.338-0.1^20 mm. teres Raski

Total body annules 73i length 0.532 mm.

congolense (Stekhoven and Teunissen)

18. Sublateral lobes not prominent or flattened anteriorly 19

Sublateral lobes prominent, flattened anteriorly presenting a
truncated head lobatujn Raski

Paras, tiik

19. First anntde not well set off j cuticle of larvae smooth or vrlth

delicate fringe 20

First annule well set offj cuticle of larvae provided with rows
of spines mutabile Taylor

20. Length 0. 303-0. U52 mm.; head and tail not blunt — truncate (tail

of cylindricum somewhat truncate) 21

Length 0.600 mm.; head and tail both blunt — truncate

rusticum (Micoletzky)

21. Anterior flap of vulva forming 2 definite points; larvae with

longitudinal culticular fringes cylindricum Raski

Anterior flap of vulva bilobed, rounded; larvae without
cuticular markings curvatum Raski

Paras. iiX

STTT^ET- NEMATODES (TYLENCHORHYNCHUS SPP. )

The genus Tylenchorhynchug Cobb, 1913, is comprised of many species,
some of which are important plant pathogens, Thorne (19U9) places this
genus in the subfamily Tylenchinae, of the family Tylenchidae . Nematodes
of this genus are medium in-size ranging from .6 to 1.7 mm in length.
The cuticle is coarsely annulated and in at least one species, T. claytoni,
longitudinally subdivided (Morphology section, Plate I, Fig. U). Lateral
fields are marked by four to six incisures. The esophagus is of the
tylenchoid type with a well developed basal bulb connected with the
intestine by a large cardia. Females have double ovaries, outstretched,
with the vulva near the middle of the body. Female tail blunt, rounded.
Male tail slightly arcuate and enveloped by the bursa.

At least two species, and undoubtedly there are others, have been shown
to cause considerable damage to crop plants. Reynolds _et. al. (1953)
have demonstrated that T. dubius can cause moderate stunting of cotton
under both greenhouse and field conditions. Steiner (1937) described
T. clay to ni from tobacco in South Carolina and at the time considered
it to be an apparently rare parasite of tobacco. Subsequent to that
time, Graham (195U) reports that T. claytoni was present in 67 per cent
of 175 soil samples collected from fields where tobacco jas stunted
(1951 through 1953). It was also found in cotton and corii samples. He
demonstrated its pathogencity on tobacco in both greenhouse and field
trials. Effects on tobacco are stunted top growth and a much retarded
root system. Graham fvirther reports (195U) that root lesions are absent
and root decay does not occur, although the roots are seriously retarded
in their growth and do not elongate normally.

In North Carolina, T. claytoni is one of the most serious nematode para-
sites to tobacco and is widespread in the state, having been identified
from 27 counties. It apparently has a wide host range, although few
studies have been made to date to determine this. Aside from tobacco,
high populations have been found in soil samples from cotton, corn, mile,
alfalfa, strawberry, oats, soybeans, peanuts, and various ornamentals.
It appears to be primarily an ectoparasite.

A monograph dealing with the taxonomy of the genus has been prepared by
Allen (1955). The key to the species known to that date is copied from
the monograph for inclusion with these Notes. At the present time, two
additional species have been described and are not included in the mono-
graph or key by Allen. These are as follows:

Tylenchorhynchus martini Fielding, 1956. Resembles T. claytoni
Steiner, 1937, but differs in having simple body annulationsj
female tail blunt with distinctive, finger-like shape, that is,
having parallel outline instead of being tapered j and presence
of a slight constriction setting off the lip region. Males
absent .

Paras. 1:2

Tylenchorhynchus lenorus Brown, 1956. T. lenorua keys to
t". ornntus, according to the key (Allen, 1955 )» but differs from
it in having a set-off lip region, a conoidobtuse tail, and
fewer longitudinal striae. T. lenorus differs from T. quadri -
fer in having fewer longitudinal striae and from T. tessalatus
in being smaller in size and having no axmulations around
terminus of tail.

Three other new species' descriptions have been submitted to Nematologica
by Bruce E. Hopper. It is likely that additional taxonomic work will be
done by others as this plant-parasitic group is investigated more fully.

Literature Cited

1. Allen, M. W. 1955. A review of the genus Tylenchorhynchus . Univ.

of Calif. Public, in Zoology 61(3) :129'^^lSS^

2. Brown, G. L. 1956. Tylenchorhynchus lenorus, n. sp. (Nematoda:

Tvlenchida), associated with the roots of wheat. Proc.
Heiminthoi. Soc. V/asn. ^jju) :152-153.

3. Fielding, M. J. 1956. Tylenchorhynchus martini, a new nematode

species found in the sugar cane and rice fields of Louisiana
and texas. Proc. Heiminthoi. Soc. Wash. 23(l):U7-U8.

h. Grahajn, T. W. 195U. The tobacco stunt nematode in South Carolina,
(Abs.) Phytopathology hi;: 332.

5. Graham, T. W. 195h. Plant abnormalities caused by parasitic nema-

todes. U. S. Dept. Agr. Plant Dis. Rptr. Supplement 227, p. 83.

6. Reynolds, Harold W. and Milton fl. Evans. 1953. The stylet nematode,

Tylenchorhynchus dubius, a root parasite of economic importance
in the southwest. U. S. Dept. Agr. Plant Dis. Rptr. 37:5UO-5Uh.

7. Steiner, G. 1937. Tylenchorhynchus claytoni, an apparently rare

nemic parasite of the tobacco plant. Proc. Heiminthoi. Soc.
Wash. J4(1):33-3U.

8. Thorne, Gerald. 19U9. On the classification of the Tylenchida, a

new order (Nematoda, Phasmidia). Proc. Helminthpl. Soc. Wash.
16(20) :37-73.

Vrr;-:s. J:h

KEY TO THE SPECIPS OF TYLENCHOlimNCHUS
(From Allen, 1955)

Females

1. Cuticle marked by longitudinal striae 2

Cuticle not marked by longitudinal striae 6

2. Lateral field marked by k lines 3

Lateral field marked by 6 lines h

3. Lip region bearing 3 or I4 annules claytoni Steiner

Lip region bearing 6 or 7 annules lamelliferus (de Man)

h' Annules extending around terminus of tail

tessellatus J. B. Goodey

Annules not extending around terminus of tail 5

5. About 32 longitudinal striae at middle of body ornatus n. sp.

About 60 longitudinal striae at middle of bo dy-quadrif er Andrassy

6. Lateral field marked by h lines 7

Lateral field marked by $ lines 19

Lateral field marked by 6 lines 20

7. Annules extending around terminus of tail 8

Annules not extending around terminus of tail 12

B. Lip region set off by constriction dubius (Butschli)

Lip region continuous with body contour* 9

9. Lip region bearing U or 5 annul csj tail tapering conoid

eremicolus n. sp.

Paras. 1:5

Lip region bearing 6 or 7 annulesj tail cylindrical, bluntly
rounded 10

10. Lip sclerotization heavy, conspicuous magnicauda (Thome)

Lip sclerotization faint, inconspicuous 11

11. Tail less th^in 3 x anal body diameter parvus n. sj>

Tail more than 3 x anal body diameter maximus n. spi

12. Lip region continuous with body contour 13

Lip region set off by constriction or depression 18

13. Lip region bearing 2 aimules nudus n. sp

Lip bearing more than 2 annules 111

Ik. Lip sclerotization faint, inconspicuous 15

Lip sclerotization strong, conspicuous 16

15. Tail bearing 10 to 15 annules clarus n. sp.

Tail bearing 20 to 2? annules striatus n. sp.

16. Spear less than 20 ji long manubriatus Litvinova

Spear more than 20 yi long . 17

17. Spear not more than 31 ii long, tail bearing SO annules

kegenicus Litvinova

Spear more than 31 V long, tail bearing kS annules

galeatus Litvinova

18. Spear more than 20 ji long, lips rounded, sclerotization con-
spicuous cyllndricus Cobb

Spear less than 20 \l long, lips broadly rounded, sclerotization
faint latus n. sp.

Paras. 1:6

19. Tall about 2 x anal body diameter, bearing less than 2^ annulns

acutun n. sp.

Tail about 3 x anal body diameter, bearing more than 2^ annules

capitatua n. sp.

20. Annules extending around, terminus of tail 21

Annules not extending around terminus of tail 25

21. Lip region set off by constriction or depression leptus n. sp.

Lip region not set off by constriction or depression 22

22. Lip sclerotization well developed, conspicuous — macrurus (Goodey)
Lip sclerotization faint, inconspicuous 23

23. Spear more than 23 y long obscurus n. sp.

Spear less than 22 p long 2U

2I;. Spear more than 15 yi long, tail less than 3 x anal body diameter*

nothus n. sp.

Spear not more than 1$ u long, tail more than 3 x anal body
diameter nanus n. sp.

25. Lip region continuous with body contour 26

Lip region set off by constriction or depression — 28

26. Tail short, conoid, terminus pointed brachyc ephalus Litvinova

Tail subcylindrical, bluntly rounded 27

27. Spear less than 20 ji long brevidens n.sp.

Spear more than 20 ji long af finis n. sp.

28. Lip sclerotization heavy, conspicuous 29

Lip sclerotization faint, inconspicuous 31

Parns. 1:7

29. Tail more than 3 x anal body diameter alpinus n. sp.

Tail less than 3 x anal body diameter 30

30.. Spear more than 35 p long macrodens n. sp.

Spear less than 35 >i lon| grandis n. sp.

31. Sp6ar less than 35 ,ii long lineatus n. sp.

Speai* more than 35 Vi long 32

32. Spear more than S^ ji long, tail less than 2 x anal diameter

superbus n. sp.

Spear less than 55 Ji long, tail more than 2 x anal diameter

conicus n. sp.

Pnr.-;;;;

STUT3r3Y-RnOT NI-mTODES (TRICHODORUS SPi'.)

"8fcubby-root" and "stubby-root nematode" have been £;u,f;f;eGtcd as coTnmon
names for the disease and the causal organism, respectively, by Christie
and Perry (1951). Since this report of Trichodorus being the causative
agent of diseases of various crop plants in Florida, considerable atten-
tion has been given to the genus. Now that a monograph of the genus has
been prepared by Allen (1957) progress can be expected in the study of
this interesting plant-parasitic genus. The Taxonomic key from the
monograph is included for use in these Notes.

These nematodes are not in the Class Phasmidia as have been the previ-
ously mentioned generaj rather, they are in the Class Aphasmida, Dory-
laimoidea. The species are small (0.5 to 1.5 mm. long), thick-bodied,
cylindrical nematodes, tapering at the anterior end. Fixed specimens
often appear as though they had a swollen condition of the cuticle or
as though they had retained the last molted cuticle. Allen (1957), who
made studies of cross sections of these nematodes, points out that the
spear or stylet should be referred to as a dorsally located tooth. This
onchiostyle is hollow but not throughout its entire lengthy axid, although
it may be used for puncturing cells, the actual feeding is by a somewhat
different mechanism than in nematodes having a hollow axial stylet.

These nematodes have been found associated with the roots of a diversity
of plants and from many different localities. Perhaps, primarily exter-
nal feeders, thorough examination of the soil rather than of the roots
may be necessary for finding these nematodes. Allen (1957) suggests
that it may be preferable to confine the use of the name "stubby-root
nematode" to Trichodorus christiei, since no other species of the genus
is known to produce the type of symptoms that are associated with the
feeding of this species. T. christiei appears at the present to be the
most tcidely distributed species of this genus in the United States and
is reported from the root zones of quite a number of economic and orna-
mental plants. Old host listings or reports of T. primitivus prior to
1957 should be used with caution because, until that time, only this one
species had been described.

Literature Cited

Allen, M. W. 1957- A review of the nematode genus Trichodorus with
descriptions of ten new species. Nematologica 2(1) :32-52.

Christie, J. R. and V. G. Perry. 1951. A root disease of plants

caused by a nematode of the genus Trichodorus. Science 113:1491-
U93.

Paras. J:?.

KEY TO THE SPECIES OF TRICHODORUS
(From Allen, Ip^^D

Males and Females

1. Females with one ovary (monodelphic ) monohys bera n. sp.

Females with two ovaries (amphidelphic) 2

2. Spear more than 130 microns long elegans n. sp.

Spear less than 110 microns long 3

3. Excretory pore posterior to base of the esophagus, spear less
than 30 microns long, males not known nanus n. sp.

Excretory pore anterior to end of esophagus, spear more than

30 microns long, males known U

li. Females with ventro-median or ventro-submedian pores near vulva,
males with 2 or 3 supplementary papillae — 5

Females without ventro-median or ventro-submedian pores near

vulva, males with one or 3 supplementary papillae 6

5. Females with ventro-median pores, 2 anterior and 2 posterior to
the v\ilva, male with caudal alae, 2 supplementary papillae

porosus n. sp.

Females with 2 ventro-submediaxi pores posterior to the vulva,
males with caudal alae, 3 supplementary papillae

atlanticus n. sp.

6. Females without caudal pores or lateral hypodermal pores, males
with a single supplementary papilla, caudal alae present

christiei n. sp.

Females with caudal pores and lateral hypodermal pores, males
with or without caudal alae, more than one ventral supplement

7. Females with three lateral hypodermal pores posterior to vulva,
males with caudal alae and three ventral supplements

pachydermus Seinhorst

Females with one lateral hypodermal pore posterior to vulva,

males without caudal alae 8

Par;iH. J: 3

8. Females with three lateral hypodermal pores, 2 anterior and one
post'-^rior to vulva, males with 3 or h ventro-median papillae
anterior to excretory pore primitivus (de Mnn)

Females with not more than two lateral hypodermal pores, males

with 0-2 ventro-median papillae anterior to excretory pore 9

9. Females with one lateral hypodermal pore posterior and one
anterior to vulva, 1st supplement in male about opposite
proximal end of spicules 10

Females with one lateral hypodermal pore at level of vulva,

males with 1st supplement near distal end of spicules 11

10. Excretory pore in female slightly posterior to middle of
esophagus, males with 2 ventro-median esophageal papillae
anterior to the excretory pore aequalis n. sp.

Excretory pore in female near base of esophagus, male with
one ventro-median papilla anterior to excretory pore

proximus n. sp.

11. Male with one ventro-median papilla anterior to excretory

pore californicus n. sp.

Male without ventro-median esophageal papilla obscurus n. sp.

I','irn;j. K:l

DAHGER NEMTODES (XIPHINEM SPP.)

The penus Xiphlnema Cobb, 1913j is comprised of several species and is
a member of the subfamily Longidorinae and family Dorylaimidae of the
Class Aphasmidia.

The status of this genus as a plant-parasitic group has been recently
reviewed by Schindler (1957 s.) , who has done work demonstrating the
pathogenicity of X. diver sic auda turn (Micoletzky, 192?) Thorne, 1939,
on various hosts TSchindler, 195^, 1957j Schindler and Braun, 195?) •
X. americanum is a form commonly encountered in the southeastern United
States. It is found, sometimes in large numbers, in soil from such
crops as tobacco, cotton, oats, vetch, peach, boxwood, and azaleas.
This species is believed to also be an ectoparasite of roots and to
cause damage under certain conditions. Although not yet demonstrated
under controlled conditions to be pathogenic, various species have been
found under circumstances strongly indicating the likelihood of a patho-
genic role. Biennial and perennial plants would appear to be hosts most
likely to show eventual damage because of large numbers of the nematodes
are required to produce appreciable damage.

Galling of rose, peanut, and strawberry root tips has been reported
(Schindler, 195U) for X. diver s ic auda turn and is characteristic enough
on these hosts to be given the name of "c\irly-tip." Characteristically,
an enlargement of the tip and a curling of the end of the root is pro-
duced with apparent necrosis and shriveling of the proximal portion*
These galls are somewhat similar to those caused by root-knot nematodes
but differ in that they almost invariably involve the distal portion of
the root. There are some other aspects concerning the pathogenicity of
X. diversicaudatum which Schindler, 1957, points out as being worthy of
further investigation.

A steadily growing list of species exists for this genus; forms being
reported from various countries and in association with various kinds of
plants. There are two recent taxonomic studies of the group (Lordello,
1955, and Luc, 1958). Keys from both of these papers are taken for in-
clusion in the Notes because the later paper by Luc does not contain all
the forms contained in the paper by Lordello. Another important earlier
paper containing illustrations of species of Xiphinema known up to that
date is the monograph by Thorne (1939).

Literature Cited

Lordello, L. G. E. 1955. Xiphinema krugi n. sp. (Neraatoda, Dorylai-
itiidae) from Brazil with a key to the species of Xiphinema . Proc.
Helminth. Soc. Wash. 22(l):l6-21.

Luc, M. 1958. Xiphinema de I'ouest Africain: Description de cinq
nouvelles especes (Nematoda:Dorylaimidae) . Nematologica 3(1)5
57-72.

Schindler, A. F. 195U. Root galling associated with the dagger nema-
tode, Xiphinema diversicaudatimi (Micoletzky, 1927) Thorne, 1939.
(Abs.) Phytopathology hh:3ti9.

Schindler, A. F. 1957. Parasitism and pathogenicity of Xiphinema
diversicaudatum, an ectoparasitic nematode. Nematologic a 2(1):
25-31.

Schindler, A. F. and A. J. Braun. 1957. Pathogenicity of an ectopara-
sitic nematode, Xiphinema diversicaudatxim, on strawberries. Nema-
tologica 2(1): 91-93.

Thorne, G. 1939. A monograph of the nematodes of the superfamily
Dorylainoidea. Capita Zool. Vol. 8, Part 5j 261 pp.

lat-rio. K:3

KKY TO TllF, .Sri'.GIK;". 01'' TlfF, GKMUr. XJIIIl Ml'.HA COHD, 1913
(Krom T,(irdpllo, 19Ip!?1

As several ne\< forms of "dagr^'V nematodes" have been described since the
publication of Thome's mono):raph (1939), the writer organized the follow-
ing key for the separation -of the species known to date. Only female
characters were used in the key, as the Xiphinema males are frequently
rare and unknown in some species. Unfortunately, it was not possible to
place in the key the species described by Schuurmans Stekhoven in 1931
(X. brevicaudat\im, X. effilatum, and X. digiticaudatum) as they were
based on larvae.

In previous publications, the writer emended the names of three species
of the genus, because they were established either by a printing error
or a lapsus calami. The writer's procedure was based on the 19th article
of the International Rules of Zoological Nomenclature (apud Amaral, 1950).
These three forms are: X. grande Steiner, 19lUj X. pratense Loos, 19h9j
and X. insigne Loos, 19U9; formerly referred to as X. grandis, X. pra-
tensis, and J^. insignis . )

1. Ovary one 2

Ovaries two 5

2. Tail rounded ajid short X. ensiculif erum (Cobb, 1893) Thorne, 1939

Tail elongated, conoid or digitate 3

3. Two pairs of caudal papillae present h

Three pairs of caudal papillae present X. radicicola Goodey, 1936

II.. Tail elongate, amphids narrow and long X. chambers! Thorne, 1939

Tail distinctly digitate, amphids iiride and short

X. brasiliense Lordello, 1951

5. Tail short and rounded 6

Tail elongate, conoid, subconoid or digitate 9

6. Lip region set off by constriction 7

Iiip rcf^lon continuous o

Pnr.-i;-. . K:)i

7. Sninll species (I.6I6 mm.)j V = iiO.O;^, oesophagus short (b = 6)

X. grande Steiner, 19llt.

Lar^^P species (h.O mm.), V =^ GO.0%, oesophagus very long (b = 2)

X. maJvrodorum (Vanha, 1893) Thorno, 1939

3. Small species (0.8 iran.) X. obtusujn Cobb, 1939

Large species (i.|.l mm.)

X. rotunda turn Sch. Stekhoven & Teunissen, 1938

9. Lip region expanded -X. lineum (Grube, 18[|.9) Thorne, 1937

Lip region not expanded 10

10. Head truncate X. truncatum Thorne, 1939

Head more or less rounded, not truncate 11

11. Length 3'h-h'O mm., or much larger (8.9U mm.) 12

Length 2.60 mm. or less lU

12. V = 38.9^ ^X. index Thorne & Allen, 1950

V = ii7.8-ij8.0^ 13

13. Length 8.9I; mm.

X. cylindricauda turn Sch. Stekhoven & TeTinissen, 1938

Length .UO rm. }C diversicaudatmnQlicoletzky, 192?) Thorne, 1939

lit. V = Sl.O-^k.0% ^X. gjnericanum Cobb, 1913

V = 29.8-147.0^ 15

15. Tail subconoid or digitate, not ventrally arcuate 16

Tail distinctly elongated, more or less deeply ventrally arcuated-17

16. Tail distinctly digitate (55.5 V- long), V = 39. li^

X. mammdllatum Sch. Stekhoven & Teunissen, 1938

Paras. K:p

Tail not digitate, subconoid (32 u long), V = 33»k-3h,2%

X. krugi n. sp.

17. Four pairs of caudal papillae present — X. campinense Lordello, 1951
More than h pairs of caudal papillae present 18

18. Combined length of spear and spear extension shorter (123. U Ji)

X. italiae Meyl, 1953

Combined length of spear and spear extension longer

(li.2. 0-158.0 }i) 19

19. Si::c pairs of caudal papillae present, spear apparently con-
sisting of two parts — X.elongatun Sch . Stekhoven & Teunis sen, 1938

Seven pairs of caudal papillae present, spear consisting of

only one part, as is usual 20

20. Tail long (77.0-102.0 ji), V = 29.8-31.6^, with a cuticular
triangular-shaped structure in the anterior slender part of

the oesophagus ^X. insigne Loos, 19^9

Tail short (1^8.0-57.0 ^), V = 39.0-ii2.0^, without such a
triangular-shaped structure in the anterior portion of the
oesophagus X. pratense Loos, 19U9

Pnrns. K:6

KKf TO THE SPECIES OF XIFFIINIJiriA COBB, 1913
(After LuFTTJbW

Females

1. One ova.ry « 2

Two ovaries (the anterior one reduced and obscure in X. krugi) 5

2. Tail long (more than four times body width at anus)

-' chainbersi Thorne, 1939

Tail short (less than fotir times body width at anus) 3

3. Tail hemispherical ^X. ensiculiferum (Cobb, 1893) Thorne, 1939

Tail mammillate (digitate) 1|,

)4. T;to pairs of caudal papillae ^X. brasiliense Lordello, 1951

Three pairs of caudal papillae ^X. radicicola Goodey, 1936

5. Tail long (mere than four times body width ar anus) 6

Tail short (less than fom- times body width at anus) 7

6. V ^ 29-32^; seven pairs of caudal papillae—X. insigne Loos, 19[t9
V = h7%', three to five pairs of caudal papillae~X. hallei n. sp.

7. Tail hemispherical or mfjiL-nillate (digitate) 8

Tail conical 13

8. Tail hemispherical 9

Tail mamnillate (digitate) 10

9. L = 0.8 rai,: lip region not. set off by constriction, i. e.

contimiouG ^X. obtusum Thorne, 1939

L = 3 r-'-i.j lip region set off by distinct constriction

X. yaooense n. sp.

Par.-in.

.10. Lips diGtcndcri. , si^parabcd one from mioUior

X. niamillntum Schuurmnns Sfcekhoven & TeunlG.'^on, 193^

Lips smooth, united 11

11. Lips flattened; lip region not set off by a contriction

X. index Thornc & Allen, 19^0

Lips rounded; lip region set off by distinct constriction 12

12. L = 2 mm.; "orgaji Z"""' in the females ^X. ebriense n. sp.

L = 3-k ran.; without "organ Z" in the females

X. diversicaudatum (Micholetzky, 192?) Thorne, 1939

13. L = at least 8 mm.

X. cylindricaudatujn Schuurmans Steckhoven & Teunissen, 1958

L = at most 3 iron. lit

lit. V = 5W ^X. americanum Cobb, 1913

V = at most h7^ 15

15. Length of tail one to one and one-half body with at anus;

anterior ovary reduced ^X. krugi Lordello, 1955

Length of tail longer than twice body width at anus; anterior

ovary normal -I6

16. Head truncate X- truncatum Thorne, 1939

Head rounded 17

""Organ Z" in the females has reference to a structure seen in the
genital tract of X. ebriense. This organ lies between the spermatheca
and the uterus. It is golbular in shape; very muscular; the lumen,
which opens wider in the center of the bulb, is bounded by a cuticular-
ized lining and possesses two to four moveable cuticularzied "teeth."
Generally, three "teeth" are seen, but it is difficult to distinguish
the exact number because of the thickness of the musculatu.re of the
orgp.n. Because of the unknown function of this organ M. Luc proposed
the nojne "organ Z."

Pnrnr,. K:8

17. Two-three pairs of caudal papillae 18

At least four pairs of caudal papillae 20

18. Two pairs of caudal papillae 19

Three pairs of caudal papillae X. pra tense Loos, 19h9

19. A = 50-55; ventral pores X. setariae n. sp.

A = 70; without ventral pores X. parasetariae n. sp.

20. Four pairs of caudal papillae X. c ampin ens e Lordello, 1951

More than four pairs of caudal papillae 21

21. V = UO-li?^; six pairs of caudal papillae

X. elongatum Schuurmans Steckhoven & Teunissen, 1938

V = ii3-ii7^ ^X. italie Meyl, 1953

''reti-Llv-.l

njf'I^TIKICATION AND BTOLO(]Y 01'^ FJfKF,- LIVING NKHATODffi

Free-living nematoder;, as the term vrill be used here, are defined as
nanatodes found in soil, in decayinf'; plant tissue, and in similar
habitats, but which do not feed on living plants. Discussion is limited
to the most common genera.

Free-living nematodes are found in all agricultural soils without
exception, and also are found in other locations where food is available,
as in manure piles, compost heaps, and other places where access can be
had to dead plant or animal material. \iJhile not quite so ubicuitous as
bacteria or fungi, they can still be found in many odd places where any
sort of food has been present for a considerable time. This includes
some very strange habitats indeed; one of the oddest being in the felt
mats which were used in German taverns for parking beer steins between
drinks. Another form is found in paperhangers ' paste; another in
pitchers of pitcher plants; and another in vinegar, etc. Occasionally
they may be something of a pest. In a shipment originating in Argen-
tina, Dr. Steiner found nematode bodies in great abundance in canned
tomato paste. Undoubtedly, they also occur in other food processing
operations where the sanitation is not all that might be desired.

In agricultural soils, free-living nematodes are often very niimerous,
counts of from several to hundreds per gram of soil not being unusual.
For the most part, little is known of food habits. However, it is a
fair assumption, with some evidence to support it, that many live on
bacteria. Others are predators, feeding by killing other nematodes.
Possibly others may feed on dead plants directly, though the evidence
for this is scanty.

The nematologist working on agricultural problems will encounter free-
living forms in the soil, in decaying plant tissues, and at times in
apparently healthy plant tissues. Nearly every sample of alfalfa stems
and leaves collected on a field trip in Virginia and examined for stem
nematodes had a number of specimens of Panagrellus.

With practice, it is easy enough to identify free-living nematodes to
genus, but identification to species is difficult or often impossible.

For the Cephalobidae, an aid to identification is Thome's paper, "A
Hevision of the Nematode Family Cephalobidae, Chitwood and Chitwood,
I93U." (Proc. HeLninthological Soc. Washington. U(l): I-I6 of 1937).

Genera of the Aphasmidia most comnon in soil samples are Flectus,
Fononchus (and subgenera) and various Dorj^laimina. The Dorylainina
include a large nuifiber of genera which are best separated by the use
of "A Monograph of the Nematodes of the Superfamily Dorylaim.oidea, " by
G. Thorne (Capita Zoologica 8(5): 1-261 of 1939). "A Monograph of the
Nematode Genera Dor:/laimns Du.jardin, Aporcelaimus n.g., Dorylain'oides

''r<x,-J..i v:

n.p., ai)d Punrentiis n.f:." (ibid, 6(l4):l-223 of 1936) is indj.spensible
for IdoiiMfirMtJon of Dorylaims to species. Forhunately, fchcso two
lr>r[-;c workr which had been out of print can now be obtained nnain.
(Ilnrt.inijs Ni.ihoff, Publisher, Post Office Box //269, The Hafjue, Nether-
lands, or through other booksellers. Price at last listing (N^y, 1959)
was 3P guilders each (approx. $10.10).)

Though Dorylaims are often found in large numbers around the roots of
plants and many have stylets vxhich would seem to be well adapted for
feeding on plants, there is little or no evidence that they are ever
serious plant parasites, with the exceptions- of species of the genera
Xiphinema , Trichodorus, and Longldorus . Except for these genera, one
seldom finds large numbers of a single species of Dorylaims around the
roots of plants, as would be expected if they are dangerous plant para-
sites. Some are believed to be predators, feeding on nematodes and
other small soil animals.

Kononchus species are easy to recognize, since they have a cylindrical
oesophagus and a somewhat globiilar mouth cavity, usually with one or
more teeth. Mononchus species are predatory.

(fe- ^o'-' "••"< V\

•Vv.-,^.

Control A:l

CHEKIGAL CONTROL OF NEMATODES

Historical

Almost as soon as it was realized that nematodes in the soil were
capable of damaging crop plants, attempts were made to kill them by
means of chemicals. The literature contains a long list of substances
which have been tested for this purpose. As early as I88I, Ktoin
attempted to kill nematodes with carbon bisulphide, though without
conspicuous success. Carbon bisulphide was also recommended by Bessey
(1911) for killing nematodes, his methods being copied from those used
for control of Phylloxera, an insect pest of grapes, in France. The
method was essentially that used today; application of measured amounts
in holes punched at intervals in the soil. Shortly after the first
VJorld War, chloropicrin was tested for nematode control in England witb
good results. About ten years later, Johnson and Godfrey again tested
chloropicrin and developed methods and machinery for field application
in pineapple fields in Hawaii. By 1937, this work was sufficiently
advanced so that Innis, Speiden & Co. started commercial development
of chloropicrin, finding most of their customers among greenhouse
owners. About I9U0, methyl bromide was found to be a good nematocide
and shortly thereafter, Dow Chemical Company entered the nematocide
business, again finding most of their customers among greenhouse
owners .

During the World War II years, the value of a mixture of dichloropro-
pane (D-D) was tested by Carter in Hawaii and ethylene dibrornide (EDB)
was tested by the Dow Chemical Company, with good success in both
cases. These being much less expensive than chloropicrin and methyl
bromide, they could be used on a large scale. Intensive efforts to
develop a market for them started in 19h^. In 1950, Shell Chemical
Corporation started development of Chlorobromopropene (CBP). This
company soon folloiired this irith the development of another chemical
first referred to as OSI897, novi trademarked Nemagon, and chemically
designated as l,2-dibromo-3-chloropropane.

The Virginia-Carolina Chemical Corporation, at about this same time,
released a commercially available chemical called V-C13 Nemacide, the
active ingredient of which is 0-2-l4.-dichlorophenyl O-O-diethyl phos-
phorothioate. This was sold as a water emulsifiable form to be applied
to the soil as a pre- or post-planting treatment and was one of the
first of the new nematocides to be recommended for treatment of soi]
around living plants.

About 195?, an experimental soil fumigant, formerly called compound
N-869, was released for comjnercial use as Vapam, a liquid manufactured
by the Sta\iffer Chemical Company. Its active ingredient, sodium methyl
dithiocarbamate, is lethal not only to nematodes but to various soil
fungi and insects and x^'eeds. Thus, it is considered as a temporary
soil sterilant.

Gojilrol /:?

Another recent nemotocide, also in the class of teTrmoivsry soil sterilsnts
nnd commercially available, is ^tylnne Q^V under the Crr^.r brand of the
Carbide and Carbon Chemicals Company, a Division of the Union Carbide
ajid Carbon Corporation. Mylnne is marketed as a dust or as a wettable
powder and is identified chemicallv as 3i'?-dimethyltetrahydro-l,3-3,2H
thiadiazine-2-thione.

Some other recent developments have been initiated by the Dow Chemical
Comppny. The introduction to commercial use in 1957 of Telone, the
trademark name for a liquid fumigant composed of undiluted technical
dichlcropropenes and containing no bromine materials. Also, tliere vras
commercial use at this same time of Dorlone, the trademark name for a
mixture of two nematocides (Telone 75.2^ and Dowfume W-85 (EDB) 2U.8^
by weight). This was in response to the observations that tinder various
soil and environmental conditions the present day soil fumigants exhibit
varying degrees of effectiveness in controlling different species of
plsjit-parasitic nematodes. At the present time, several other promising
chemicals are under test, but have not been placed on the market yet.

Thus, commercial soil fumigation in the United States began in 1937, ".nd
the greater part of the development to its present status as a multi-
million dollor business has taken place since 19h5' The gradual develop-
ment of new materials, permitting new ways of application, broader
lethalness for use as temporary soil sterilants or limited phy to toxicity
for use on and around living plants, is greatly expanding the usefulness
of chemicals for nematode control.

Basic Principles

Most nematocides are used for control of nematodes in soil before
planting, and this situation ifri.ll be discussed first. Since plant-
parasitic nematodes feed only on living plants, they must be killed
by "contact" rather than "stomach" poisons. Also, movements of nema-
todes in soil are slow and limited in extent, which means that the
poison must be distributed throughout the soil. ■.iJhere annual crops
have been grown, the nematodes will be in the upper 15 to 18 inches of
soil, 1-ri.th the grr;ater part of the population in the upper 12 inches,
nematodes in the soil may be in the form of eggs, larvae, or adults;
but, in any case, will be surroimded by a thin film of water. In
certain cases, they may be further protected as cysts ( Heterodera ) , or
the gelatinous mediurii of egg masses (Meloidogyne ) > or m?.y be in more or
less decomposed plant material.

Nematodes have a cuticle which is of a lipoidal nature and impermeable
to many substances.

\-ihen. these various fncts are considered, it is evident that the require-
ments for a soil nemntoeide are: 1) It must thoroughly permeate or be
mixed with the roil at least as deep as 12 inches and perhrps as deep
as 18 inches. 2) It must be able to penetrate the various barriers
fjurroundinp the ncmato'lns, eysts, pl-uit material, or at the veiy least.

Gontrol A:3

the film of vrater and the nematode cuticle. 3) It must kill or at
least disable the nematodes, h) Since the ultimate object of treating
the soil is the growing of crop plants, no residue toxic to plants must
be left in the soil after a reasonable waiting period.

The first requirement is best met by gases. These are difficult to
confine in large scale work, though this can be done on a small scale.
The next most favorable material is vapors or fumes of volatile liquids.
If very large amounts of water are available, it is possible to dis-
tribute a water soluble chemical through the upper layers of the soil.
Thorough mixing of a solid (powder or granule) with the soil is possible
only on a small scale or to a depth of not more than 6 or 8 inches id-th
ordinary farm machinery.

The water film around the nematode will be penetrated by any substance
even slightly soluble in water. The lipoidal cuticle is best penetrated
by fat solvents.

The fourth requirement (lack of phytotoxicity) may be inherent in the
nematocide used. However, if the substaiice is at all toxic, it must
either: 1) be dissipated into the atmosphere] 2) decoii5)ose, forming
harmless compounds^ 3) be leached out of the soil.

Materials which are gases at ordinary temperatures must be confined by
an impervious cover over the soil. Methyl bromide (boiling point k° C)
is used in this way for seedbeds and other comparatively small areas.

The most successful nematocides are volatile materials. They may be
applied as solutions, emulsions, powders, or granules, but the active
ingredient spreads in the soil as a volatile agent. Distributed at
intervals through the soil, the chemicals diffuse evenly in all
directions — downward, laterally, and upward. As they diffuse, the
vapors pass over soil particles and soil organisms, each surrounded
by a thin film of water. As conditions are ideal for solution of the
chemical vapors in the water, they make a nearly saturated solution
until equilibrium is reached.

It is evident that this simple and automatic process is superior to
anything that can be done with a non-volstile nematocide. It would be
difficult to get as thorough a distribution of such a chemical even if
it were water soluble. Obviously, the mixing of a non-volatile solid
nematocide as thoroughly in the soil would be far more laborious, even
if machinery capable of doing such a job were available.

Procedure in application of liquid and solid-carrier soil fumigsnts is
designed to make the best possible use of the fumes. For large scale
wor'-, tractor applicators which deliver the fumigant in continuous
streaias spaced at intervals in the soil are used. Generallj'-, linos of
fujninant are f^paced at about 12-inch intervals, thougli 10-inch inter-
vals ore usnd in some soils.

In e:>"norimontal work, spacin;- and application rates are varied Kyste-

Control Aik

matically to determine the most favorable combination. In ordinary farm
practice, manufacturers' directions are followed.

To date, no one has devised a method which is demonstrably better than
the one described. There is, for example, no evidence that distributing
the fumigant over a plane several inches wide (spraying over the whole
width of a flow furrow, for example) is any better than simply ciribbling
the fumigant in lines. And most certainly, emulsions, powders, and
granules applied to the soil surface are wasteful, since some fumes are
lost into the atmosphere. In addition, the great bulk of the chemical
is likely to be retained in the upper few inches of the soil whore the
fumes are too readily dissipated. Instructions regarding application
of sxifficient quantities of water to obtain adequate leaching of the
chemicals, and recommendations to working solid forms into the soil must
be carefully followed.

At best, soil fumigants are still expensive and the usual reason for
the commercial grower to use them is to increase profits from the
projected crop. The profits may come as a result of increased yield,
better quality, in savings on some other operation, or from a combina-
tion of all three. The tobacco farmer gets more pounds per acre and is
content if quality is not appreciably reduced. The sweet potato grower
may increase both yield and quality. The carrot grower eliminates culls.
The celery grower who fumigates his seedbeds with methyl bromide saves
expensive hand weeding. But whatever the advantage, it can be reduced
to a dollars and cents basis so that the cost of fumigating an acre ran
be balanced against returns due to fumigation.

It is this comparison which is of interest to the practical farmer,
rather than a comparison of nematode control in treated and untreated
areas, or one based on general appearance of the field. Thus, the
optimum application of soil fumigant is that which produces the greatest
cadireturn for each dollar expended for fumigation. Considered from the
purely monetary angle, row treatments may be better than solid treatment
and a light application better than a heavy one under certain coniiitions.
A fact which has become apparent as soil fumigation has progressed is
that it is not necessary to kill all of the nematodes to make a ver^
great difference in the crop. In many cases, all that is necessary is
to kill enough nematodes so that the plants are not subjected to an
overwhelming attack in the early stages of growth when they are most
vulnerable.

It should be recognized that the considerations and techniques may be
different for certain special commercial growing enterprises, such as
where biennial and perennial plants are concerned and for the home
gardener determined to grow good home garden and ornamental plants.
The point is, due consideration should be given to the intentit)]i;; and
needs of the grower, particiilarly when the cost factor is invlvid.

In addition to control of nematodes in soil before the crop in ii:nled,
there are several other situations where nematocides am or luiclit he
used.

Control At 5

One of the most important is for control of ectoparasites or endopara-
sites in or around the root.s of living plants, particularly perennials,
such as orchard trees and ornamentals. Two of the presently available
nematocidps may be generally recommended for this purpose (1,2-dibromo-
3-chloropropane and V-C13 Nemacide). Most of the nenatocides currently
available are more or less toxic to plants and might do more harm than
good. There are some recent experiments which point out that even with
a phytoxic material (D-D) a dosage applied to one side of a tree one
year end the other side the following year can be an overall beneficial
treatment. However, a degree of balance between plant injury and nema-
tode control must be obtained. This kind of application is rather an
exception, not the general rule. An intensive search is being made for
nematocides which are less toxic to plants.

Tliere is also a specialized but important need for nematocides which
will kill nematodes on bare-rooted plants to be transplanted, but at
this time no material is being generally recommended for this purpose.
Search is also being made for suitable chemicals for this purpose,
because of the hazard of nematode transport in infected plant stocks.

It has been shown that foliar nematodes in chrysanthemums and certain
other plants can be killed by nematocides which apparently have a
systemic action. One of these is sodium selenate, which is applied to
the soil. However, it should be remembered that selenivim taken up by
edible plants can be very poisonous to man and to domestic animals,
IfJhere selenium occurs naturally in soils, serious trouble with live-
stock has been encountered. Selenium, once added to the soil, might
remain for a long time, so its use is discouraged. It has also been
shown that repeated spraying with Parathion will control foliar nema-
todes on chrysanthemums and other plants. Amounts used are 0.2^ to
0.50 lbs. active ingredient (as wettable powder) to 100 gallons of
water, and control is obtained by h or more applications by spraying
at weekly intervals,

V.Tiite tip of rice is caused by Aphelenchoides oryzae carried on the
seed. Control can be obtained by seed treatment with several compounds,
the most satisfactory of which seems to be 3-p-chlorophenyl-5-inethyl
rhodanine (N-2iiij., made by Stauffer Chemical Company) .

Space fumigation is also mentioned because it has been used for many
years generally for disinfecting non-living materials. The generally
used chemical is methyl bromide which, although highly phytoxic, can
be safely used for a few special problems involving nematodes on living
plant materials. Examples are fumigation of onion and clover seed
infested with the stem or bulb nematode.

Materials and Methods

Selection of a nematocide for any particular purpose involves considera-
tion of the area to be fumigated, the crop to be grown, and other factors,
including economic ones.

Control AsS

In general, D-D, Telone, EDB, or l,2-dibromo-3-chloropropane will be
used for large scale work on soils to be planted to crops of fairly
high value. Seedbeds or other small areas will be fumigated with
methyl bromide, chloropicrin, Vapam, or >tylone where the broader lethal
spectrum of a soil sterilant is desired. In greenhouses, methylbromide
or chloropicrin will be used, though use of the latter should be avoided
in greenhouses where plants are growing, since small concentrations in
the air are highly poisonous to plants.

In case of doubt, it is well to inquire of the manufacturer before using
nematocides. If informed of the exact circumstances possible hazards
to avoid will be pointed out. Exact descriptions of methods are also
best obtained from the literature of manufacturers of soil fiamigants and
applicators. Discussion here will be confined to generalities.

Improvised equipment can be used for small jobs. Perhaps the easiest
way is to use a fruit jar with two nail holes punched in the lid, one
for the fumigant to run out, the other to admit air. The fumigant is
poured from the jar into an open furrow which is promptly covered.
Dosage is adjusted by trial and error, changing size of nail hole until
the proper amount is delivered at a steady walking pace. The availa-
bility now of water soluble materials, emulsifiable formulations, wet-
table powders, and impregnated granular carriers open up a wide range
of simple methods of application of the chemicals. The chart on the
following page illustrates various methods s\Ji table for treating small
areas of soil.

If commercial fumigation of small areas is a job which has to be done
often, as in a greenhouse or nursery, a hand applicator or injector may
be a good investment. Where topical applications may be preferred, a
rotory tillage device is recommended and can be rigged for distribution
of the chemicals at the time of tilling them thoroughly into the soil.

For work involving greater area, it is usually best to use some sort
of continuous flow applicator. These can be made in any size desired,
from a single shank on a garden tractor to six br eight shanks on a
regular farm tractor. Kits of parts to make a machine for delivery of
liquids are readily available, and regular distributor devices for
granular insecticides and fertilizers are available, or such equipment
on hand may be adapted. No matter what the size of the machine, it
must be capable of delivering accurately measured amounts of the chemi-
cal and placing them usually at a depth of at least six to eight inches.

In all fumigation jobs, except for experimental work, the manufacturers'
directions should be followed exactly. Read the label and any circular
you can get. Observe all safety precautions. It is also an excellent
idea to observe procedures used by others in your area.

Control A: 7

Effect of Soil Nematocldes

The effect of soil nematocides depends on the kind and amoimt of chemical
used. There are now available materials with different ranges of toxic-
ity to living organisms. Materials with a broad spectnm of toxicity are
conveniently termed soil sterilants. For example, methyl bromide at one
pound or more per 100 square feet does a fairly effective sterilization
job in the upper 12 inches or so of soilj killing weed seeds, many bac-
teria, and fungi as well as the nematodes and soil insects. Chloropicrin
at high rates (hOO-^OO pounds per acre) has a similar effect. Vapam and
l^lone are sold as being able to control weeds and fungi in addition to
nematodes. The other nematocides are intended principally for control
of nematodes and do not have as wide a spectrum of toxicity as the soil
sterilants, and these materials fall into two groups depending on whether
or not the chemical is toxic to plants. This is a very important dis-
tinction because phytotoxic nematocides require a waiting period before
planting to permit the chemical to be lost from the soil or to be changed
to a non-phy to toxic form.

Two of the most widely-used nematocides are fumigants requiring a wait-
ing period before planting. These are ethylene dibromide (EDB) and some
form of dichloropropane-dichloropropene (D-D and Telone) or a mixture of
both (Dorlone). With the standard application of D-D (20 gallons per
acre) or EDB (5U pounds of active ingredient per acre), the usual result
is the killing of 80 to 95^ of the nematodes. Effect on weed seeds,
bacteria, and fungi seems to be small, though not entirely absent. That
there is some effect on bacteria is indicated by reports that the propor-
tion of ammonia nitrogen to nitrate nitrogen remains high for some weeks
after fumigation, presumably due to destruction of some bacteria.

Less is known as yet about the toxicity spectrum of the two generally
available nematocides reported to be non-toxic to some plants and having
low or moderate toxicity to many others. These chemicals are 1,2-dibro-
mo-3-chloropropane (Nemagon, Fumazone and V-C13 Nemacide).

If the soil was heavily infested with nematodes, elimination of a large
proportion of the population permits increased root growth with a con-
sequent improvement in vigor of the plants. There is often a noticeable
increase in uniformity of growth over the field. Yield increases of 2S%
to S0% or more, often with improvement in quality, are obtained.

However, the increased root growth of the plants provides nearly ideal
conditions for increase of the remaining nematodes, and the final result
may be that there are more nematodes at the end of the season than there
were at the start. Thus the effect of soil fumigation lasts only for
one season, as a rule, though if conditions are particularly favorable,
some effect may be seen the next season.

Gont.j-Ml A: 8

The Economics of Soil Fumigation

At present prices, cost of f-umigating an acre of soil with the usual
amount of D-D or EDB is about 35 dollars, including an allowance for
application. The prudent farmer sees little point in making this
expenditure unJ-ess he has a fair prospect of returns which will amount
to about four times the investment. In other words, the value of the
crop, over and above that which would have been obtained without fumi-
gation, must be four times 35 dollars, or about lIiO dollars. This is
not an excessive demand, but about the same sort of return the frrmcr
expects from fertilizer, etc.

For example, the tobacco farmer producing 1,200 lbs. of tobacco per acre
without fumigation will sell his crop for an average of 55 cents per
poiuad, or a total of 660 dollars. By fumigating his soil he can increase
his crop 2S% to a total of 1,500 lbs., and sell it for 825 dollars, an
increase of 165 dollars, which is more than four times his investment in
fumigation.

The peanut farmpt- who grows 1,200 lbs. of peanuts per acre and sells
them for 12 cents a pound, or ll4i; dollars, would need to increase his
crop nearly 100^ to realize four times his investment. Fumigation of
peanut soil with prospects of no more than 25^ increase would be a very-
poor investment.

For purposes of rough calculation, the prospective selling price of the
crop from fumigated soil should be about 16 times the cost of fumigation
if an average increase of 25^ in crop value can be expected. ¥ith fumi-
gation at 35 dollars per acre, this works out to a total crop value of
560 dollars per acre.

In calculating total crop values, an allowance can sometimes be made for
an increase in average quality as well as for increase in yield.

About the sajne ratio between fumigation cost and total value of the crop
applies equally to the less expensive row fumigation. If row fumigation
can be done for 10 dollars per acre, it will be a good investment on
cx'ops selling for 160 dollars per acre .

Another cost cutting possibility being explored is to spread the cost of
a single soil treatment by getting the benefits of nematode control
extended to two different crops planted in succession on the treated
land. Experiments indicate this is feasible.

The experiment station worker or soil fumigant salesman is often asked
V7hether or not a particular field should be fumigated. In answering this
question, the first consideration should be the value of the crop to be
planted. Knowing approximate yields and selling prices for the crop, it
is possible to calculate whether or not there is any prospect that fumi-
gation will be a profitable investment. If the crop is not of sufficient
value, the farmer should be advised against the use of fumigants.

Contro] Aj9

Where a high value crop is planted, prospective retiirns fjrom the fumiga-
tion must be considered* Since this depends on the nematode population
of the field, Information on this point is desirable, but often difficult
to obtain.

If the nematodes are one of the root-knot species and a susceptible crop
is growing in the field, it is easy to get a fairly accurate idea of the
size of the population by examination of the roots of the plants. But
if syii5)toms of attack are not so readily recognized, or no crop is grow-
ing on the field, the task is more difficult.

Theoretically, it should be possible to estimate the nematode population
of a field by examination of soil samples. In practice, attempts to
apply this method reveal a number of difficulties. The first of these
is obtaining and examining an adequate sample of the field. Examination
of a sufficient number of soil samples simply requires more time than is
usually available. And, even if an accurate estimation of the nematode
population can be made, the background information necessary to correlate
such findings with subsequent crop growth is largely lacking.

In case of doubt, the grower should be encouraged to experiment on his
own farm. That is, he shoixld be advised to use soil fumlgants on a
trial basis, in such a way that easy comparisons between fumigated and
vuifumigated soil are possible.

Control B:l

METHOD OF CONTROL OTHER THAN CffilllCAL

The pxirpose of this section is to stress that in phytonematology the
general principles of plant disease control are applicable and should
not be overlooked.

I. Exclusion. Exclusion of any parasitic nematode from the farm,

nursery, greenhouse, or garden is probably the least
expensive and is certainly the most effective control available to the
plant grower. Prohibition of the introduction into an area of para-
sitic nematodes and the additional safeguard of Interception are methods
of nematode control being conducted on international and interstate
levels. The grower shoiild, whenever possible, take advantage of the
benefits of these government services, as for example, purchasing in-
spected and certified nematode-free plants and propagules. Elimination
of nematodes from infected plants or from infested shipping and packing
materials is conducted in a number of ways, depending upon the nema-
todes, plants, and carriers involved. Some examples are sorting, fumi-
gation, hot water treatments, and disinfesting dips or washes. It
should be remembered that every new planting, large or small, offers
the chance to apply the common-sense methods which can prevent a nema-
tode problem from developing. Simply try to keep parasitic nematodes
from being brought in on plants, propagules, and with their soil, pack-
ing, or containers.

II. Eradication. The phytonematologist and the plant pathologist are

usually sought for only' after a nematode problem has
been found to exist, and getting rid of the troublesome little animals
then becomes the aim of control.

Item oval of the nematodes in one way or another is a means of eradica-
tion which is applicable in a surprisingly large number of instances
and usually at no greater cost than that of the plants involved. Re-
moval of nematized individual plants is practiced in plantings of all
sizes and of almost all kinds. Removal of nematized plant parts may
be all that is necessary in some cases, for example, removal of the
diseased lower leaves of chrysanthemums, or sorting out of infected
seed. Removal of nematode infested soil is feasible and effective for
greenhouse operations, flower beds, and potted plants.

Destruction of the nematodes in place is the objective of most control
efforts. liJhether one is dealing with nematodes on or in viable hosts
or carriers, or else dealing with nematodes in the non-living environ-
ment determines how drastic the control treatment can be made. Nema-
todes on or very near to the outer surfaces of plant parts can, in some
cases, be removed by washing or killed by toxic wash or dip solutions,
fumigation, and other chemical treatments. Examples of non-chemical
controls are heat treatments of one kind or another, depending upon the
thermal tolerance of the plant part and whether or not surface or deep

Control B!2

penetration of the heat is required. Other, as yet, little investigated
possibilities include the use of ultrasonics, high frequency heating,
and irradiation treatments. Destruction of nematodes in the environ-
ment, whether this be the soil, the greenhouse bench, the flat or flower
oot, is done by various techniques usually based on one of the follow-
ing principles: a. Heat - hot water drenches or soaks, steam, flame,
electrical resistance, heated inclosures such as ovens and pressure
containers^ b. Exposiire - exposure to the effects of sunlight and desic-
cation; c. Electricity - probably effective only by production of heat,
rather than by the electrical effects alone; and d. Chemical - as is
covered in the section dealing with chemical control of nematodes.

Attrition is a form of eradication which has taken many forms in actual
practice, but which has as its goal "making life too tough" for the
nematodes to thrive. The principles involved include inactivation,
starvation, and antibiosis. Removal of carry-over and weed hosts,
falloirring, flooding, tillage, and crop rotations are possibilities used
vjhen feasible. Antibiosis has been used in some instances as a nema-
tode control in commercial plant production and is a fertile field for
further research. Antibiosis involves the application and manipulation
of the biological factors of the environment in such ways as to be
detrimental to the plant-parasitic nematodes present. . Examples are the
use of trap crop plants such as Crotolaria, and green manure to promote
development of nematode- trapping fungi. Other possibilities include
the use of antibiotics, predacious mites and nematodes, and introduction
of the various bacterial, fungal, and protozoan pathogens causing dis-
eases of nematodes. Isolation, identification, and synthesis of nema-
tode effecting root exudates is now being done and may be effective for
some kinds of nematodes.

III. Protection. Protection is a low-cost form of nematode control

which is applied in situations where parasitic nema-
todes are thought to be present. In its simplest forms it may be
nothing more than manipulation of the environment in such a way that
nematodes are not blown, washed, splashed, or transported from infested
to non-infested sites. Control of nematode dissemination may also be
had by choice of planting sites, plant spacing, and planting dates.
The use of protectant materials has not been very much explored, and
although this is in the realm of chemical control, the concept bears
mentioning here. One should keep in mind that for many of the annual
plants, a satisfactory economical control is attained if the plants
can be protected from the nematodes long enough to get a vigorous root
system well started. After that, the plant may be able to support the
nematodes which attack it and yet prove to be a worthwhile plant.

rv. Resistance. This is probably the ultimate goal in the development

of control for all plant diseases. There are nematode
resistant varieties of some crop plants, and others are being developed.
The problems in producing nematode resistant plants are difficult be-
cause of the numerous kinds of plant-parasitic nematodes, the fact that
usually more than one species is present, and the interrelationship of
nematodes with other pathogenic organisms in disease complexes.

Control B:3

Resistance, due to heredity, and its development, by means of selec-
tion and liybridization, is the task of the plant breeder; and althoiigh
tlid results may be lasting, their attainment is necessarily a slow
process, liesistance or tolerance to nematodes due to non-hereditary
factors is becoming of interest with the present-day development of so
many new chemicals and the extensive screening programs to test for
possible applications. Non- hereditary resistance or increased tolerance
to plant nematodes, perhaps, could result from nutritional therapy to
compensate for the losses due to the activities of these parasites, or
from the application of counteractants or inhibitors of the enzymatic
secretions, and to the excretory products of the nematodes. Instances
of immunity, tolerance, and inhibition of plant nematodes are known in
nature; therefore, development of non-hereditary resistance is con-
sidered possible. It is already almost axiomatic to recommend providing
ample water and nutrients to valuable nematized plants to compensate for
root damage sustained.

The use of resistance as a practical control measure always calls for
careful consideration of the nematode situation in which the resistant
plants are to be used, because of the diversity of the nematodes and
their relatioAships as mentioned above.

CHEMICAL TREATMENT ON SMALL SCALE

NEMATOCIDES

LIQUID

CAPSULES

/ \

APPLICATION
OF LIQUIDS

MEASURED
'AMOUNTS OR
CAPSUl-ES PUT IN HOLES

iJ^^/measuring
into furrow from
jar with punched lid

>i

GRAVITY- FLOW KIT
ON PLOW OR HARROW

SOLIDS

EMULSIFIABLE 8
WETTABLE FORMS
MIXED IN WATER

APPLICATION OF
DRY GRANULES

MEASURED INTO HOLES
OR FURROWS WITH
SPOON, SCOOP, OR
POUR-SPOUT B0>

GASES

PRESSURE CANS

• ^

//APPLICATOR

GAS RELEASED UNDER
CONFimM& COVERS

FUMIGATING A SOIL PLOT

OVERALL SURFACE APPLICATIONS MUST BE
FOLLOWED PROMPTLY BY R0T0TILLA6E ft/OR
WASHING INTO SOIL WITH AMPLE WATER

FUMIGATING A PILE
OF SOIL , POTS , OR FLATS

ICE PICK OR

AWL FOR HANDLING AND
PERFORATING CAPSULES

TIGHTLY COVERED
CONTAINERS FOR
TREATING SMALL

LOTS OF SOIL OR

POTS

MEASURES

USEFUL ITEMS FOR FUMIGATION

Misc. K'J

USEFUL REFERENCES

Joiirnals

In the United States papers dealing with plant-parasitic nematodes are
published most often in:

Froceedings of tne Helminthological Society of Washington

Phytopathology

The Plant Disease Reporter

In England:

Annals of Applied Biology

Nature

Journal of Helminthology

Plant Pathology

Euphytica

Empire Jo\irnal of Experimental Agriculture

International:

Nematologica

Books and Bulletins

Chitwood, B. G. and W. Birchfield. 1956. Nematodes, their kinds and
characteristics. Vol. 2. Bui. 9. State Plant Board of Florida.
Liberally illustrated and brief descriptions of the principal
plant-parasitic nematodes with a useful outline of control measures
with specific recommendations.

Chitwood, B, G. ajid M. B. Chitwood. 1950. An introduction to nema-

tology. Section 1. Anatory. Revised ed. The most comprehensive
source for detailed information on the anatomy and morphology of
nematodes of all kinds,

Dollfus, Robert Ph. 19U6. Parasites (animaux et vegetaux) des hel-
minthes. Ehcyclopedie Biologique. Paul Lechevalier, Editeior.
Paris. U82 pp. Probably the most complete compilation of work
on the parasites of the helminths, including nematodes. Informa-
tion and illustrations of the bacterial, fungal, protozoan, and
metazoan parasites of nematodes. Nany illustrations.

Duddington, C. L. 1957. The friendly fungi. A new approach to the
eelworm problem. Faber and Faber, London. 188 pp. An interest-
ingly written and informative book concerning the role and appli-
cation of fungi in the control of plant-parasitic nematodes.
Information on how to collect, culture, and observe predacious
fungi. Illustrated.

Filipjev, I. N. and J. H. Schuurmans Stekhoven. 19Ul. A manual of
agricultural helminthology. E. J. Brill Co., Leiden, Holland.

Misc. A:2

878 pp. A comprehensive source of infonnation concerning diseases
caused by nematodes and nematode taxonomy as reported in the
extensive literature up to about 1939.

Franklin, M. T. 19^1. The cyst-forming species of Heterodera. Common-
wealth Agricultural Biireaux, England, lli7 pp. An extensive review
of this group of nematodes, their history, the diseases they cause,
control, and taxonomy. Illustrated.

Goffart, H. 1951. Nematodes der kulturpflanzen Europas. Paul Parey,
Berlin. lUU pp. Information on diagnosis of the nematode para-
sites of various crops and plants, primarily those of Europe.
Numerous illustrations.

Goodey, J. Basil. 1957. Laboratory methods for work with plant and
soil nematodes. Tech. Bui. 2. Ministry of Agric, Fisheries and
Food, London. An excellent source for methods, particularly for
work with the cyst-forming nematodes. Illustrated.

Ck)odey, T. 1933. Plant parasitic nematodes and diseases they cause.
E. P. Button and Co., New York. 306 pp. Considers each parasite
according to historical, morphology, life-history, pathology,
hosts, and control topics. Illustrated.

Goodey, T. I9U0. (Revised 1956 by J. B. Goodey and M. T. Franlclin)

The nematode parasites of plants cataloged under their hosts. Imp.
Bur. Agric. Parasit., St. Albans. A very useful publication for
host range considerations with an extensive bibliography.

Goodey, T. 1951. Soil and freshwater nematodes. Methuen and Co., Ltd.,
London. 390 pp. The latest compilation of nematodes of these
kinds with descriptions down through the various taxonomic levels
to genotype. Illustrated.

Hyman, Libbie Henrietta. 1951. The invertebrates. Vol. 3. McGraw-
Hill Book Co., New York. 572 pp. A good consideration of the
entire Nematoda as a group of the invertebrates. Covers such
topics as anatomy, embryology, ecology, physiolbgy, host-parasite
relations (plant and animal hosts), and presents general taxonomic
and life history information for the various genera within the
nematode orders. Illustrated.

Kevan, D. K. McE. (Editor) 1955. Soil Zoology. Butterworth' s Scien-
tific Publications, London. 5l2 pp. Collection of a diversity of
papers dealing with the soil animals, including neariatodes. Highly
recommended for broadening ones concept of the soil as an environ-
ment. A source for techniques for study of the soil and the soil
animals. Contains a useful key to the orders and suborders of
soil and litter inhabiting animals. Illustrated.

Pennak, R. W. 1953. Fresh-water invertebrates of the United States.
Ronald Press Co., New York. 769 pp. The chapter dealing with

Misc. A:3

nematodes has an excellent key supplemented with illustrations of
representatives of the common genera encountered in soils and
moist habitats.

Steiner, 0. 19U2. Plant nematodes the grower should know. Proc. Soil
Sci. Soc. Fla. h-B: 72-11? (Issued 19^9) Well illustrated ajid
informative description of the major types of plant-parasitic
nematodes, the diseases they cause, and host-parasite relations.

Steinhsus, Edward A. 19U9. Principles of insect pathology. McGraw-
Hill Co., New York. p. 633-66U. A general introduction to the
nematodes parasitic on insects; life histories, host-parasite
relations, and applications. Illustrated.

Stewart, M. A. 19U5. A laboratory manual of helminthology . U. of
Calif. Syllabus Series ZN, Univ. of Calif. Press, Berkeley.
19h pp. (Third printing 195U) A useful compilation of methods
and taxonomic keys for the study of Platyhelminthes, Annelida
(Hirudinea), Acanthocephala, Nematoda, Nematomorpha (Gordiacea)
of importance as human and animal parasites.

Thorne, Gerald. 1939. A monograph of the nematodes of the superfamily
Dorylaimoidea. Capita Zoologica 8 (Part 5), Martinus Nijhoff, The
Hague, Netherlands. 26l pp. A basic reference in taxonomy pro-
vided with descriptions, drawings, and keys. Recently reprinted
with the addition of a listing of the new species in the Dorlyai-
moidea for the interval between the first printing and about 1955.

Thorne, Gerald and Helen Swanger. 1936. A monograph of the nematode
genera Dorylaimus Dujardin, Aporcelaimus, n. g., Dorylaimoides
n. g., and Pungentus n. g. Capita Zoologica 6 (Part h) , Hsxtinus
Nijhoff, The Hague, Netherlands. 223 pp. A basic reference book
provided with descriptions, drawings, and keys. Recently reprinted
■tfith the addition of the new species reported for the interval
between the first printing and about 1955.

Chemical Control Recommendations

A. Dow Chemical Company, Midland, Michigan.

ACD Information Bulletin No. 110, Nov. 29, 1957.

Factors Influencing diffusion and nematode control by soil

fumigants. Cleve A. I. Goring. 57 pp.
ACD Information Bulletin No. 112, Feb. 1958.

Dow soil fumigants. 15 pp.

B. Shell Chemical Corp., Agricultural Chemical Sales, New York 22, N. Y.

Specimen labels for Shell agricultural products. (A plastic bound
book permitting revisions as needed.)

Msc. B:l

SCIENTIFIC AND COMMON NAMES FOR PLANT-PARASITIC NEMAS"^

This cross-reference listing of the scientific and common names is
based on the largest compilations of common names as yet available.
The first reference (Buhrer, E. M. 195U. Common names of some
important plant pathogenic nematodes. PI. Dis. Reptr. 38(8) :535-5Ul. )
shoiild be carefully read by all who are concerned with this useful but
sometimes controversial aspect of phytonematology. The second refer-
ence is a chart issued by the Florida State Plant Board which, for the
most part, follows the listing of the reference just mentioned but, in
addition, lists a number of new common names to species for which no
common names have been designated. Perhaps the compilation presented
in these Notes will further promote the use of reasonably standardized
common names.

The words "nematode(s)" and "nema(s)" have been used interchangeably
in the text of these Notes. For a review of the origins of these words,
derivations from them, and suggestions concerning future usage, see:
Chitwood, B. G. 1957. The English word "Nema" revised. Systematic
Zoology 6(U):18U-186.

I. SCIENTIFIC NAMES

Misc. B:2
(Revised)

Common

Common

No,

Nematode Name No.

No.

Nematode

Name No.

1

Anguina

152

37

Hemicriconeraoides

160

2

A. agrostis

83

38

H. biformis

161

3

A. tritici

212

39

H. floridensis

162

UO

H. wessoni

163

h

Aphelenchoides lU

, 80

S

A. besseyi 131,196

Ul

Hemicycliophora

157

6

A. cocophilus

Uo

U2

H. similis

158

7

A. fragariae

180

U3

H. parvana

159

8

A« olesistus 8

, 77

9

A. ritzana-bosi

29

Ut

Heterodera

U8

U5

H. cacti

50

10

Belonolaimus

185

U6

H. carotae

52

11

B. gracilis

12U

U7

H. cniciferae

U9, 51

U8

H. glycines

62

12

Cacopaurus

15U

1x9

H. goettingiana

60

13

C. epacris

155

50

H. humuli

57

lU

C, pestis

156

51

H. major

53, 59

52

H. punctata

56

15

Criconema

166

53

H. rostochiensis

BS

16

C. civellae

167

5U

H. schachtii

63

17

C, decalineattnn

168

55

H. tobacxm

6U

18

C. spinalineatum

169

56

H. trifolii

5U

57

H. weissi

58, 61

19

Criconemoides

132

20

C. citri

133

58

Hoplolaimus

93

21

C. simile

13U

59

H. coronatus

9U

60

H, Tiniformis

95

22

Ditylenchus

15

23

D. angustus

129

61

Longidoms

109

2U

D. dipsaci

199

62

L. sylphus

110

25

D. destructor

126

26

D. myceliop>iagu3

108

63

Meloidodera

65

6U

M. floridensis

66

27

Dolichodoras

3

28

D. hetrrocephalous U

65

Meloidogyne

136

66

M. arenaria

lUli

29

Doryl'.iinus

165

67

M. arenaria tharaesi

lli7

68

M. brevi Cauda

Ihl

30

Gottholdsteineria

170

69

M. exigua

137

31

G. b\ixophila

172

70

M. hapla

1U3

71

M. incognita

1U5

32

Helicotylenchus (?)170

72

M. incognita acrita

lUo

33

H. africanus***-

171

73

M. javanica

lli2

3U

H. erythrinae

179

35

H. multicinctus

176

7U

Hacobbus

75

36

H. nannus

177

75

N, batatifortnis

76

;

Misc. B:3

\

Common

(Revised)
Common

No.

Nematode

Name No.

No.

Nematode

Name No,

76

Paratylenchus

118

lOU

Scutelloneroa

(?)170

77

P. elachistus

121

105

S. blaberum**

178

78

P. hamatus

120

106

S. brachyurum

17U

79

P, dianthus

119

107

S. christiei

175

108

S. coheni

173

80

Pratylenchus

96

81

P, brachyurus

103

109

Trichodorus

186

82

P. coffeae

100

110

T. christiei

187

83

P. leiocephalu£

i 105

111

T. obtusus

188

8U

P, minyus

98

112

T. pachydermis

189

8$

P. musicola

97

86

P. penetrans

99

113

Turbatrix

87

P. pratensis

102

nU

T. aceti

208

88

P. scribneri

lOU

89

P. thomei

106

115

Tylenchorhynchus

191

90

P, Tulnus

107

116

T. claytoni

190, I9U

91

P, zeae

101

117

T. martini

192

92

Radopholus

118

Tylenchulus

93

R. oryizea

130

119

T. semipenetrans

30

9U

R. similis

16

120

Xiphinema

67

95

Rotylenchulns

121

X. americanum

68

96

R, renifonnis

128

122

X. chambersi

70

123

X, diversicaudatum

71

97

Rotylenchus (see

also:

12U

X. index

69

Gottholdsteineria &

125

X. radicicola

72

Scut ell onema)

170

98

R, blaberus**

178

99

R. brachyvirus

17U

ICXD

R. buxophilus

172

101

R. Christie!

175

102

R, coheni

173

103

R, robustus

95

■JHt

It is suggested that Helicotylenchus africanus be called the "African
spiral nema" rather than Scutellonema blaberum syn, RotyleAchus blaberus.
As the latter was discovered in West Africa, the ccmimon name "Wesi
AfiT-can spiral nematode" seems appropriate.

Misc.

B:U

(Revised)

Scientific

Scientific

lo^

Nematode Name No.
African spiral neraa*» 98

No.

Uo

Nematode Name No.

1

Coconut paLn nema

6

2

American dagger nema

121

Coconut nema

3

Awl nemas

27

Cocopalm nema

h

Cobb's awl nema

28

la

Coffee meadow nema

82

U2

Coffee root-knot nema

69

5

Banana nema

85

U3

Confused root-knot nema

72

6

Banana meadow nema

85

hh

Com meadow nema

91

7

Beet nema

5U

hS

Cotton root-knot nema

72

8

Begonia leaf nema

8

he

Crimp nema

7

9

Boxwood spiral nema 31j

100

U7

Currant nema

9

10

Brassica-root nema

U7

U8

Cyst nemas

hk

11

Brazilian root-knot nema

69

U9

Brassica-root nema

U7

12

British spiral nema

102

50

Cactus cyst nema

U5

13

Bud nemas

h

51

Cabbage cyst nema

U7

Bud nema

7

52

Carrot cyst nema

U6

11+

Bud and leaf nemas

U

53

Cereal-root nema

51

15

Bulb or stem nema

22

5U

Clover cyst nema

56

16

Burrowing nema

9U

55

Golden nema

53

56

Grass cyst nema

52

17

Cabbage cyst nema

U7

57

Hops cyst nema

50

Cabbage-root nema

58

Knotweed cyst nema

57

18

Cactus cyst nema

U5

59

Oat cyst nema

51

19

California dagger nema

12U

60

Pea cyst nema

U9

20

California meadow nema

8U

61

Polygonum cyst nema

57

21

California sessile nema

13

62

Soybean cyst nema

U8

22

Carnation pin nema

79

63

Sugar beet nema

5U

23

Carolina spiral nema

99

6U

Tobacco cyst nema

^^

2li

Carrot cyst nema

U6

65

Cystoid nemas

63

Carrot-root nema

66

Pine cystoid nema

61;

25

Cereal-root nema

51

26

Chamber's dagger nema

122

67

Dagger nemas

120

27

Christie's spiral nema

101

68

American dagger nema

121

28

Christie's stubby root nemallO

69

California dagger nema

12U

29

Chrysanthemum nema

9

70

Chamber's dagger nema

122

Chrysanthemum leaf nema

71

European dagger nema

123

30

Citrus nema

119

72

Pacific dagger nema

125

Citnis-root nema

73

De Man's meadow nema

87

31

Citmis ring nema

20

32

Citrus spine nema

16

7U

European dagger nema

123

33

Clover cyst nema

56

75

False root-knot nemas

7U

3U

Cobb's awl nema

28

76

False root-knot nemas

35

Cobb's lance nema

59

of sugar beets

75

36

Cobb's meadow n^ma

86

77

Fern nemas

8

37

Cobb's ring nema

21

78

Fig pin nema

78

38

Cobb's spiral nema

35

79

Fig spine nema

17

39

Cobb's stubby root nema

111

80

Foliar nemas

k

No, Nematode

Scientific

Name No. No,

81 Gallworni

82 Garden nema

83 Grass nema

8U Grass cyst nema

8$ Godfrey's meadow nema

86 Golden nema of potato

87 Grass sheath nema

88 Hops cyst noma

Hops-root nema

89 Indian root-knot nana

90 Javanese root-knot nema

91 Kidney-shaped nema

92 Knotweed cyst nema

93 . Lance nemas

9U Cobb's lance nema

95 Thome's lance nema

96 Meadow nemas (see;

Root-lesion nemas)

97 Banana meadow nema

98 California meadow nema

99 Cobb's meadow nema

100 Coffee meadow nema

101 Com meadow nema

102 De Man's meadow nema

103 Godfrey's meadow nema
lOli Scribner's meadow nema

105 Smooth-headed "nema

106 Thome's meadow nem^j

107 Walnut meadow nema

108 Mushroom spawn nemr.

109 Needle nemas

110 Thome's nepile nema

111 Northern roo1/-knot nema

112 Oak sheatTioid nema

113 Oat cyst nema

Oat nema
Oat-root nema

nil Pacific dagger nema
11$ Pea cyst nema

Pea-root nema
116 Peanut root-knot nema

65
65
2
52
81

53
U2

50

68
73

96
57

58

59
60, 103

80
85
Qk
86
82
91
87
81
88
83
89
90
26

61
62
70

38
51

125
U9

66

117
118
119
120
121
122
123
12U
125
126

127
128
129
130
131
132
133
13U
135

136
137
138
139
lUO
lUl
1U2
II43
lUh
1U5
1U6

1U7
1U8

lh9
150

151
152
153
15U
155
156

157
158
159

160

Misc!

: B:5

(Revised)

Scientific

Nematode Nane

No.

Persian sessile nema

lU

Pin nemas

76

Carnation pin nema

79

Fig pin nema

78

Ramie pin nema

77

Pine cystoid nema

6U

Pine sheathoid nema

39

Pine sting nema

11

Polygonran cyst nema

57

Potato rot nema

25

Potato tuber nema

Ramie pin nana

77

Reniform nema

96

Rice nema

23

Rice root nema

93

Rice white tip nema

5

Ring nemas

19

Citrus ring nema

20

Cobb's ring nema

21

Root-gall nema (seer

Root-knot nemas)

65

Root-knot nemas

65

Brazilian root-knot nema

69

Confused root-knot nema

72

Coffee root-knot nema

69

Cotton root-knot nema

72

Indian root-knot nema

68

Javanese root-knot nema

73

Northern root-knot nema

70

Peanut root-knot nema

66

Southern root-knot nema

.71

Tea root-knot nema

68

Thames' root-knot nema

67

Root-lesion nemas (see:

Meadow nemas)

80

Root nemas

80

Root rot nemas

80

Scribner's meadow nema

88

Seed gall nemas

1

Seinhorst stubby root nema

112

Sessile nemas

12

California sessile nema

13

Persian sessile nema

Hi

Sheath nemas

Ul

Grass sheath nema

U2

Tarjan's sheath nema

U3

Sheathoid nemas

37

MJlsc. B:6
(Revised)

No, Nematode

Scientific
Name No.

161 Oak sheathoid nema 38

162 Pine sheathoid nema 39

163 Wesson's sheathoid nema UO
l61i Soybean cyst nema U8

165 Spear nemas 29

166 Spine nemas - 15

167 Citrus spine nema 16

168 Fig spine nema 17

169 Zoysia spine nema 18

170 Spiral nemas (?)30, 32,

97, (?) lOU

171 African spiral nema«* 33,

98, 105

172 Boxwood spiral nema 31, 100

173 British spiral nema 102, 108
I7U Carolina spiral nema 99, 106

175 Christie's spiral " 101, 107

176 Cobb's spiral nema 35

177 Steiner's spiral nema 36

178 West African spiral

nema** 98, 105

179 Zimmermann's spiral nema 3U

180 Spring dwarf nema 7

181 Steiner's spiral nema 36

182 Stem or bulb nemas (see:

Bulb or Stem nemas)

183 Strawberry bud nemas 5, 7
18U Strawberry dwarf nemas 5, 7

185 Sting nemas 10

186 Stubby root nemas 109

187 Christie's stubby root

nema 110

188 Cobb's stubby root nana 111

189 Seinhorst's stubby root

nema 112

No.
190

191
192

193
I9U
195
196

197
198
199
200
201
202
203
20U
205
206
207
208

209
210
211
212

213
211i

Nematode

Scientific

Name No*

Stunt nemas ll5

Tobacco stunt nema 116

Stylet nemas 115
Sugar cane stylet nema , 117

Tesselate stylet nema II6

Tobacco stylet nema II6

Sugar beet nema 5U

Summer dwarf nema $

Tarjan's sheath nema U3

Tea root-knot nema 68

Teasel nema 2k

Tesselate stylet nema II6

Thames' root-knot nema 67
Thome's lance nema 60, IO3

Thome's meadow nema 89

Thome's needle nema 62

Tobacco cyst nema 55

Tobacco stunt nema II6

Tobacco stylet nema II6

Vinegar eelworm 113
Vinegar eels

Walnut meadow nema 90

Wesson's sheathoid nema UO

West African spiral nema»» 33

Wheat nema 3

Wheat gall nema

Wheat eelworm

Zimmermann's spiral nema 3U

Zoysia spine nema Id

»» It is suggested that Helicotylenchus afriCanus be called the "African

spiral nema" rather than Scutellonema blaberum syn. Rotylenehus blabeinis .
As the latter was discovered in West Africa, the common name •HflTest
Afidcan spirl nema" seems appropriate.

"^