DAMPING-OFF IN FOREST NURSERIES

A THESIS ACCEPTED IN PARTIAL SATISFACTION OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

AT THE UNIVERSITY OF CALIFORNIA

BY

CARL HARTLEY

DECEMBER, 1919

UNITED STATES DEPARTMENT OF AGRICULTURE
BULLETIN No. 934

Contribution from the Bureau of Plant Industry
~ WM. A. TAYLOR, Chief

Washington, D. C. PROFESSIONAL PAPER June 16, 1921

DAMPING-OFF IN FOREST NURSERIES

X

By

CARL HARTLEY, formerly Pathologist, Office of
Investigations in Forest Pathology

CONTENTS -

Damping-Off in General . . Aiadvars Density of Sowing
Damping-Off of Conifers Moisture and Temperature Factors . . .
Causal Fungi Chemical Factors
Relative Importance of the Damping-Off Biologic Factors
Fungi on Conifers Acknowledgments
Damping-Off Fungi as Causes of Root- Summary
Rot and Late Damping-Off Literature Cited
Relation of Environmental Factors to
Damping-Off

WASHINGTON
GOVERNMENT PRINTING OFFICE
1921

j

RECEIVED
“1921

SEP.8
BOOUMENTS DIVISION

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3
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De Caer ak hn hoi) soar ua ont) ol.
UNITED STATES DEPARTMENT OF AGRICULTURE

| BULLETIN No. 934

Contribution from the Bureau of Plant Industry
WM. A. TAYLOR, Chief

Washington, D. C. PROFESSIONAL PAPER June 16, 1921

DAMPING-OFF IN FOREST NURSERIES.

By Cart Harrrey, formerly Pathologist, Office of Investigations in Forest

Pathology.
CONTENTS.
Page. Page.
Damping-off in general________--~~_ 1 Damping-off fungi as causes of root-
Damping-off of conifers__________~ 7 rot and late damping-off________ 70
(OFDITSETW La Nata ya eb ae a 2 tvelation of environmental factors to
Connie PUM: 2 2G Gaim pin o- Ofte os Se i 73
Husariamispp® —. o-oo 34 je Density vor SOWwimNe == = ess are 74
Pythium debaryanum__—_____-_- 355 | Moisture and temperature factors—_ 7)
Rheosporangium aphanider- @hemicalisfactorns 225 ee eee 79
BRD USS Sse tn SW Pores ee) do.|) Biologic! factors. —=+2- 2-2 = > eee 82
Phytophthora spps se —— 2+ Se 59) | -Acknowledemen tes 22 62 86
Miscellaneous phycomycetes____ 61 ‘Summary eee Peet eas, Me 86
UW Eher sumo. see ee ae G2) | aterature cited =—---=— aan ae 91
Relative importance of the damping-
Bit Lune: OM cOniters = ss 5 te 65

DAMPING-OFF IN GENERAL.

Damping-off is the commonest English name for a symptomatic
croup of diseases affecting great numbers of plant species of widely
separated phylogenetic groups. It is commonly used for any disease
which results in the rapid decay of young succulent seedlings or soft
cuttings. Young shoots from underground rootstocks may also be
damped-off before they break through the soil (66).t| The same term
is even used for diseases affecting the prothallia of vascular crypto-
gams (2). The name apparently originated in the fact that the dis-
ease is usually most prevalent under excessively moist conditions.
In those cases in which the disease becomes serious without the pres-
ence of unusual amounts of moisture the term is a misnomer. It is,
however, so thoroughly established in practical use that it would be
impossible, even if desirable, to establish any other name.

1 The serial numbers in parentheses refer to ‘‘ Literature cited,’ at the end of this
bulletin.

19651°—Bull. 934—21——1

2 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

While the parasites reported as causing damping-off are probably
not as numerous as the host species which are subject to it, a con-
‘ siderable number are known. Two quite different types of damping-
off parasites may be recognized. In the first type we have fungi,
such as Pythium debaryanum Hesse and Corticium vagum B. and C..,
soil inhabiting and primarily saprophytic, which attack a great
variety of hosts, and are at least better known, if not more destruc-
tive, as damping-off organisms than as parasites on older plants.
They are specialized as to the type and age of tissues which they at-
tack rather than as to host. The second type includes fungi less
common as saprophytes and with a relatively limited, sometimes very
closely limited, host range. Phoma betae, the systemic parasite of
sugar beet (37), is an excellent example of the host-specialized para-
site, transmitted in the seed and capable of seriously injuring various
parts of the older plant at different stages of growth as well as at-
tacking seedlings.

Most damping-off parasites are intermediate in habit between the
extremes of these two types. Of those which are somewhat host
specialized, the following may be mentioned :

Phomopsis verans, the cause of foot-rot of eggplant, reported by Sherbakoff
(128) as a frequent cause of damping-off of this host and believed to be
carried on seed.

Gibberella saubinetii (Mont.) Sace. (29) and the imperfect fungi which kill
grain seedlings as well as cause diseases of the older plants (S80; 126,
p. 218). Species of Gloeosporium and Volutella named by Atkinson (2,
p. 269; 52) as able to kill seedlings er cuttings of particular host plants.

Glomerella (Colletotrichum) gossypii, described by Atkinson (1) and Barre
(4) as likely to cause damping-off of cotton (112).

Fusarium lini, the flax parasite, reported by Bolley (14) as destructive .to
young seediings.

Phoma lingam, the cause of black-leg of cabbage, at least under inoculation
conditions able to kill quickly seedlings of cabbage and other crucifers
Ci2ur

Peronospora parasitica (Pers.) De Bary, a downy mildew attacking cabbage
and various other crucifers, reported as killing thousands of very young
cabbage plants in Florida seed beds (41).

The entomophthoraceous Completoria complens, on fern prothallia (1; 87,
p. 203).

Bacillus malvacearum, a parasite of the leaves of cotton plants, which can
also cause damping-off of its favorite host (113) and the bacteria from
diseased cucumber plants with which Halsted (53) caused typical
damping-off of cucumbers.

Damping-off fungi with wider host ranges include Phytophthora
fagi, Aphanomyces levis (100), Rheosporangium aphanidermatus
(38, 39), Botrytis cinerea, and certain Fusaria. The so-called prop-
agation fungus, “ vermehrungspilz,” a sterile damping-off mycelium
which Sorauer (133, p. 821) believed related to Sclerotinia and for —
which Ruhland (115) has erected a new genus, considered by both

115 eae S

DAMPING-OFF IN FOREST NURSERIES. 3

authors the most serious enemy encountered in growing softwood
cuttings in Germany, if distinct would be a further addition to these
generalized parasites. However, it is now believed (34) to be identi-
‘al with Corticium vagum. Common generalized parasites of older
plants, such as Sclerotinia libertiana, Sclerotium rolfsti (129), and
Thielavia basicola (47), capable of attacking roots or other parts of
older plants of numerous species, may also be considered among the
damping-off fungi when they cause the death of small seedlings, as
occurs, for example, in attacks by Sclerotinia libertiana on lettuce
(20, p. 28) and celery (103, p. 536) in seed beds. Further study will
probably result in multiplying almost indefinitely the number of
more or less important damping-off parasites, both of the specialized
-and unspecialized groups, although the most important of the latter
type are probably already known.

Most of the references in literature to damping-off describe its
occurrence in truck crops and the losses caused in these crops. Ac-
cording to Haisted (53, p. 342), weed seedlings are also very com-
monly attacked. Duggar (33) names lettuce, celery, cotton, sugar
beet, cress, cucumber, and sunflower as especially susceptible to
injury by the two most important damping-off organisms. Except
for the plant species in which damping-off by seed-carried parasites
is common, it appears that the economic damage from damping-oft
is serious only with plants whose culture involves the raising of the
seedlings in crowded seed beds for subsequent transplanting. For
example, tomatoes do not ordinarily suffer from damping-off in the
field (70), but the growing of seedlings in flats for subsequent trans-
planting is sometimes seriously hampered as a result of the preva-
lence of damping-off. This same principle holds in general for trees.
Broad-leaved trees, which are usually not as crowded in the seedling
stage as are the conifers, seldom give rise to complaint on the score
of damping-off. The conifers, subject to serious losses in nursery
beds, are not believed to be greatly affected in this country by the
better known types of damping-off under forest conditions (68)
except in the less common cases in which seedlings come up in close
groups from squirrel hoards, artificial seed spots, or similar sources.

A considerable number of broad-leaved trees have been reported at
one time or another as injured by damping-off, though complaints
of commercially serious losses are not common. The cases which
have come to the writer’s attention are listed below:

Cause not determined:

Orange (438, 108).

Olive, in greenhouse at the University of California.

Russian wild olive (Hiaecagnus sp.), serious at an Iowa nursery; oral re-
port by Mr. C. R. Bechtle, formerly of the United States Forest Service ;
at another nursery in the same region this plant was reported as very
little subject to injury.

a BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

Cause not determined—Continued.

Magnolia (31), troublesome if the pulp is not washed off the seed before
planting.

Bucalyptus spp. (88, p. 45; 181), serious under moist conditions,

Betula spp. Communication by Dr. Perley Spaulding, of the Bureau of
Plant Industry ; found especially susceptible in a Pennsylvania nursery.

Carob, at United States Plant Introduction Garden, Chico, Calit. Dr.
Mel T. Cook states that damping-off is more serious in carob seedlings
if the seed is removed from the pod than if.pods and seeds are sown
together.

Robinia pseudacacia (138).

Apple, in greenhouse at the Michigan Agricultural College.

Sclerotinia sp. (Hurope) :

Betula (79), a disease of seed and germinating seedlings.

Phytophthora fagi (Europe) :

Fagus. Hartig (59) and many other writers; seriously affected, even
in forest.
Platanus (15).
Acer (15), A. platanoides and A. pseudoplatanus (86, 104).
Robinia (59, 73).
Fraxinus (73).
Acacia (59).
Cercospora acerina (Europe) :
Acer platanoides and A. pseudoplatanus (58).
Pythium debaryanumn:
Tilia europea and T. ulmifolia (187), serious.
Robinia (75, p. 18-14), killing germinating seed.
Catalpa (126).
Rhizoctonia:
Citrus seed beds (180) ; much loss.
Catalpa (126).
Botrytis cinerea:
Catalpa (126).
Fusarium sp.:
Citrus seed beds (1380) ; much loss.

The sugar beet is apparently the only plant whose damping-oft
diseases have been investigated with any degree of completeness
by modern methods. While there is a great mass of literature on
damping-off, it is mainly descriptive and on control measures. Most
of the reports of the causal relation between the different fungi and
the disease in the various host plants have been based on demon-
strations of the presence of the fungus in diseased seedlings. In
a great number of these cases identification has been doubtful.
Even when a fungus is known to belong to a parasitic species, it
is by no means certain that the mycelium found belongs to:a para-
sitic strain. It has been found, for example, that only part of
the strains of Corticium vagum occurring in sugar beets are able
to attack that host vigorously (38, p. 154). Similar data for pine
appear in figures 1 and 2. Furthermore, even parasitic strains of
several of the damping-off organisms are so widely distributed as

DAMPING-OFF IN FOREST NURSERIES. 5)

saprophytes that one of them might easily get into a killed seedling
after some other parasite had caused its death. Not only in the
‘ase of seedlings killed by fungi like Peronospora parasitica, but in

wosT \ PINUS BANKSIANA — PINUS RESINOSA

YEAR
expr| zea | 20a | a | oe | 7

LIVING SEEDLINGS PER 100 1M CONTROLS

Fig. 1.—Diagram showing the relative activity of different strains of Corticium vagum
in inoculations made at the time of sowing the seed. In experiments Nos. 36, 45, and
47 the values are plotted for the number of seedlings appearing above the soil. For the
other experiments the number of seedlings surviving at the close of the experiment have
been taken. Explanation of symbols: O=Strain 147, from spruce seedlings, Washington,
D. C., 1910; strain 50, from pine seedlings, Nebraska, 1909; (J/==strain 253, from
Elaeagnus sp., Kansas, 1913; [§=strain 230, from the same lesion as strain 233;
@ =strain 183, from bean, New York, 1910.

HOST PINGS BANKSIANA PINUS RESINOESA
YEAR| 1913 S13 191 F- (914- /H18
exrt| san | eee | # | 4 | ve | 7 | 7

A

lig. 2—Diagram showing the relative activity of different strains of Corticium vagum,
as indicated by the number of seedlings surviving in inoculated soil. Explanation of
symbols: @=—Strain 189, from sugar beet, Michigan, 1910 (light-brown mycelium with
few sclerotia) ; A=strain 211 and A= strain 212, from sugar beet, Colorado, 1910;
W=strain 186, from potato, Ohio, 1910; [J=strain 187, from potato, New York, 1910;
4+—=strain 205, from Douglas fir, Colorado, 1911; X==strain 192 and O=strain 206,
from pine, Nebraska, 1911.

sf)
°

SEEDLINGS SURVIVING PER 100/N CONT)
o
fo} fo)

cases of true damping-off produced by the rotting type of parasite,
much of the rapid decay of the seedling after death is brought
about by bacteria and fungi other than the one causing death.

6 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

Inoculation experiments are therefore probably even more neces-
sary in damping-off investigations than in studies of most other dis-
eases in order to demonstrate etiological relationships. Unfortu-
nately, most of the inoculation work with damping-off organisms
prior to 1900 was either crudely done by placing diseased seedlings
against healthy ones or consisted of experiments in which purity
of cultures and validity of controls did not receive sufficient con-
sideration. Recent investigations not primarily directed toward
damping-off, but which have decidedly increased our knowledge of
the relation between Corticium and the disease, are those of Peltier
(98) and Fred (43). The latter established a strong presumption
that the difficulty in securing stands of various field crops having
oily seeds in soil where green manures had been recently turned
under is due to the killing of the sprouting seed by damping-off
organisms.

In tobacco, sugar beet, and pine, whose damping-off has received
considerable attention, it has been found that the damping-off proper
is commonly preceded by the killing of many of the sprouting seeds
in the soil (88; 68, p. 522; 81, p. 5) and followed, after the plants
become too large to be killed by the damping-off organisms, by root
sickness and the death of small roots (88, p. 161; 64; 100). This latter
has been reported also as a serious matter in the case of Corticium
vagum for potato (51), a host on which damping-off is not important
because of the lack of commercial propagation from seed. Pythiuim
debaryanum further has been reported as continuing to work in the
cortical tissues and leaves of tobacco plants which have been in-
fected too late to result in death (81).

The fact that a number of the damping-off fungi are able to attack
young or soft tissues of so great a variety of plants and are much
less able to kill older plants suggests that resistance to damping-off
may be in part based on purely mechanical factors. Hawkins and
Harvey (71) recently have extended, to Pythium debaryanum the
idea, developed by Blackman and Welsford (12) and Brown (16) for
Botrytis cinerea, of the importance of mechanical penetration in the
fungous invasion of plant tissues. While for 2. cinerea mechanical
pressure was found to be the main factor only in cuticle penetration,
with P. debaryanum. the penetration of the cell walls of all parts of
the potato tuber was apparently largely dependent on mechanical
puncturing by the hyphe, only tubers with mechanically weak cell
walls being susceptible to decay by the fungus. The extreme sus-
ceptibility to P. debaryanum and Corticium vagum of soft, thin-
walled tissues and the resistance of older stems and root parts would
fit in well with such a theory as to the method of wall penetration,
as in the older tissues the thicker cell walls would obviously be a
serious bar to the extension of a fungus dependent partly or en-

DAMPING-OFF IN FOREST NURSERIES. if
tirely on mechanical puncturing for its progress from cell to cell.
Hartig (61, p. 147-150) shows a fungus which he does not name, but
which is evidently a species of Fusarium, dissolving the young un-
euticularized epidermis of pine seedlings; but he states that it can
not so dissolve older epidermis. The increased protective value of
the epidermis of older plants can only in part explain the immunity
most of them develop against serious attack by damping-off organ-
isms, as lesions already started or which may later develop from the
infection of young roots are unable to extend into the older parts
of the plants.

It may be mentioned here that the writer in a very preliminary
test. found strains of Corticitum vagum and Fusarium moniliforme
Sheldon which had been proved able to cause damping-off of pines
also apparently able to destroy filter paper in inorganic salt solu-
tion, while Pythiwm debaryanum seemed not so able. Ruhland
(116), on the other hand, found the strain of the “ vermehrungspilz ”
(Corticium vagum) which he tested to be very weak in cellulose-
destroying ability as compared with Botrytis cinerea.

DAMPING-OFF OF CONIFERS.
HISTORICAL.

While the losses from damping-off in seed beds of dicotyledonous
tree species are occasionally serious and in the case of beech in
Europe have required considerable study, they have been so far
overshadowed in this country by the losses in coniferous seed beds
that practically all the attention thus far, both as to etiology and
measures of prevention, has been devoted to the disease in conifers.

The literature on the damping-off of conifers is considerable.
A large part of it, because of the extensive early development of
plant pathology and forest planting in Germany, has been writ-
ten by Germans. A large portion of the German articles on it
was either by foresters or by botanists in the day when most patho-
logical work was of the reconnaissance type. Therefore, while the
work of one of the best known of the parasites on coniferous seed-
lings was noticed in Europe as early as the eighteenth century (21,
p. 252-253) most of the European data available are observational.
The only fungi which were at all definitely connected with the dis-
ease on conifers seem to have been Fusarium (/usoma spp.) and
Phytophthora fagi (P. omnivora De Bary in part). The dampine~
off Rhizoctonia was described in Germany in 1858 and Pythium de-
baryanum in 1874; the fact that neither of these, important in conif-
vrous seed beds in both the eastern and western United States, has
ever been reported from conifers in Europe is perhaps the best evi-

8 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

dence of the relatively small amount of actual investigation carried
on there on this disease in the nurseries. A number of references to
the damping-off of conifers in the English horticultural and botani-

‘al literature yield even less definite information as to the causal
fungi than do the German articles.

e With the awakening of interest in reforestation in the United
States between 15 and 20 years ago and the first efforts to grow
pimes in quantity for forestry purposes, attempts were made to de-
termine the cause of the disease in this country and to develop direct-
control methods. Duggar and Stewart (32) made what appears to
be the first report of Rhizoctonia in connection with the damping-off
of conifers. Spaulding (136, 137), in work begun in 1905, eon-
tributed much to our knowledge of the etiology of the damping-off
of pine in this country, especially in relation to Fusarium, and origi-
nated the sulphuric-acid method of control. The writer in 1910 re-
ported preliminary inoculations on conifers with both Rhizoctonia
and Pythium debaryanum (62). The work of Gifford (46) and
Hofmann (77) added to the information on the causal relation of
Fusarium spp. and P. debaryanum, respectively. Hartley, Merrill,
and Rhoads (68) have recently established the parasitism of a num-
ber of strains of the Corticium vagum type of Rhizoctonia on pine
seedlings under inoculation conditions, have confirmed Spaulding’s
conclusions as to the parasitism of Yusarium moniliforme Sheldon,
and have given preliminary data on other fungi. They consider ?.
debaryanum and C. vagum more important in pine seed beds than any
single Fusarium species. Hartley and Hahn (69) have announced
successful inoculations on pines with P. debaryanum and PRheospo-
rangium aphanidermatus Edson, with less satisfactory evidence of
the parasitism of Phytophthora sp. and a fungus tentatively referred
to Pythium artotrogus. Hartley and Pierce (67) report the finding
of P. debaryanum in Tsuga mertensiana and Pseudotsuga taxifolia
as well as in the pines. In damped-off pine seedlings they find /.
debaryanum more commonly than C. vagum, especially in beds which
have received disinfectant treatments. Other considerations, how-
ever, keep them from concluding that the former is necessarily the
more important of the two. Both of these latter papers and all of
the reports of Pythium with the exception of Hofmann’s are brief
notes, presenting no evidence in support of the statements mace.

DESCRIPTION.

The symptoms of damping-off in conifers have already been de-
scribed in some detail (68). In the paper cited, injury due to exces-
sive heat of the surface soil and injury caused by high wind, both of
which may easily be confused with damping-off, are described and
accompanied by colored illustrations both of different types of damp-

DAMPING-OFF IN FOREST NURSERIES. 9

ing-off and of these nonparasitic troubles. The detailed descriptions
will not be repeated here. A brief summary of the different types
of disease recognized as included in damping-off follows:

(1) Germination loss: The radicles are killed very soon after the seeds
sprout and before the seedlings can appear above ground. This is an important
type, which can be caused probably by any of the organisms commonly capable
of causing the better known types of trouble (61, 63, 68, 187).

(2) Normal damping-off (figs. 3, 4, and 5): The seedlings are killed by fungi
invading either the root or hypocotyl after the seedling has appeared above the
soil and while the stem is still dependent largely on the turgor of its cortical tis-
sues for support. In sandy soils root infection is more common than hypocotyl
infection, though the latter is the type most emphasized in the early horticul-
tural descriptions. Biittner (26) some time ago recognized the frequence of

Fic. 3.—Normal type of damping-off of Pinus: ponderosa. At the left is a damped-off
seedling or root sprout of the southwestern ragweed (Ambrosia psilostachya). (Photo-
graphed by 8S. C. Bruner.)

root infections. Damping-off in beds out of doors is primarily in most Cases a
root rot, either of this type or of the types preceding and following.

(3) Late damping-off includes cases of the root-rot type occurring only after
the seedling stems have started to become woody and the cortex has begun to
shrivel. The damping-off parasites, or at least part of them, continue to kill
seedlings by rotting their roots for some time after the stems become too woody
to be decayed. The seedlings affected do not fall over till a considerable time
after death. For convenience, all cases of this sort up to the purely arbitrary
age of two months are classed as damping-off. However, in weather permitting
of average speed of development the seedlings are usually able to resist attack
before they reach this age. Seedlings at the marginal age between suscepti-
bility and nonsusceptibility to killing infections are found often with the
younger parts of their roots killed, but with the older portions apparently able
to resist invasion by the fungus, recovery taking place by laterals. Dr.
R. D. Rands and the writer in 1911 established the ability of seedlings from
43-day-old beds of Pinus sylvestris, P: banksiana, P. nigra austriaca, and P.
nigra poiretiana to survive such infections, even when more than half of the
root system has been destroyed, by transplanting such root-sick seedlings and

10 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

observing their continued growth (fig. 6). An article recently found (25) shows
that Biittner had earlier made the same sort of demonstration of recovery of
root-sick conifers. Observations on olive seedlings in 1916 showed cases of
partially rotted roots which were recovering by sending out lateral root
branches.

(4) Top damping: The cotyledons or upper part of the stem are invaded by
the parasite, sometimes before the seedling breaks through the soil. The infec-
tion may or may not be fatal. A special case of this type, probably caused by ¢
different parasité from those most commonly active, is that which in a publica-
tion above referred to was described and figured as “black-top” (68). It is

Itc, 4.—The beginning of an epidemic in drill-sown Pinus banksiana. Black crosses (X)
indicate disease foci where the germinating seed were apparently killed and from which
the disease is now spreading to adjacent seedlings. (Photographed by Dr. J. YV.
Hofmann.)

distinguished from ordinary top damping by the very dark color of the invaded
tissues and its apparent dependence on some unusual set of climatic factors for
its progress in the seedling after infection.

The killing of dormant seed by fungi is a matter of some practical
interest in seed beds, and possibly still more so in forests, as it may
help to explain the failure of certain conifers to reproduce except on
mineral or certain other special soil types (68). With sugar beets
Pythium debaryanum (100) is said to attack dormant seed as well
as seeds which have sprouted. It is to be presumed that with conifers
some of the damping-off fungi will be found to attack dormant as
well as sprouting seed. This matter is now under investigation.

DAMPING-OFF IN FOREST NURSERIES. iT

Something is already known about the seed fungi of herbaceous
plants (76, 91), broad-leaved trees (79, 92), and juniper (95).

RELATIVE IMPORTANCE OF THE DIFFERENT TYPES.

Of the types of damping-off described in the foregoing pages the
first two are ordinarily the most important. Late damping-off is
rarely as serious as the normal type of damping-off. Top damping is
only of importance in cases of excessive and unusual atmospheric
moisture, so far as the writer’s experience indicates. In the Middle
West it has proved relatively insignificant. The three types which

Fic. 5.—Nearly complete destruction of the seedlings of Pinus banksiana at an unusually
early age, at Garden City, Kans. (Photographed by Dr. J. VY. Hofmann.)

occur after the seedlings appear above the soil surface can, of course,
be evaluated by frequent counts during the damping-off season. This
has apparently not yet been done by anyone. However, in experi-
ments on damping-off control by soil disinfection, data have been
obtained on comparative emergence (number of seedlings appearing
above the soil surface) in treated and untreated plats and on the total
parasitic losses after the seedlings appear which permit a certain
amount of analysis of the losses due to damping-off parasites. The

data from five nurseries bearing on this point are presented in
Table I.

12 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

TABLE I.—Relative importance of losses by damping-off before and after conifer
seedlings emerge from the soil.

Basis. Loss in control plats.
Number of Emerged ae
Nursery and species. Series. plats. (viable seeds). | nee
0.
Disinfectant. |- = Ta ema | 401 Ly
| to
Treat- Con- | Be- S AMinvng anil einer
ed. | trols. |'fore: After.|Total.| col. 7.
|
1 P4 3 4 5 6 7 8 9
Bessey (Nebraska sand hills): | | P.ct.| P.ct.| P.ct.|
Pinus banksiaha.........--. Average of 9....|Sulphurie acid} (a) | (») | 37.8 |e27.2 | 65.0) ¢1.39
z-Finus ponderosa.......-..- ASVeOT AGO Ol 2. aoa ek O2e oe ae 6 8] 28.1 |): 27.3}, 5504 1.03
SgeePinus resinosa.............- Average ona: -t\0c. doe 22220). 4 7} 29.4 | 54.1 | 83.5 . 54
. len City (southwestern
Syeeansas): |
PF einus austriaca. 2.3.5... Average of 2....| Copper — sul- 4 3 | 69.0 | 27.5 | 96.5 | 2.52
phate. }
ints banksianas..) ose os eee Cover sae Zine chlorid. .. 3 2) 8057 | 12.0: | 92°71) 6.72
Pinus ponderosa..........- Average of 7....| Copper — sul- 17 29 | 15.3 | 30.9 | 46.2 4
phate.
Cass Lake (northern Minne-
sota): ING LOD LTS iene Formaldehyde 6 3 | 3.8 | 37.2 | 41.0 10
INOSLO52 oes gee leneee Goes 6 3 | 25.7 | 36.2 | 61.9 71
See Bn tae No. 1058........| Zine chlorid. .. 4 4| 5.7 | 26,2 | 31.9 22
Pinus resinosa....-....----. NO HORE 5-225 dost 22502 4 3| 7.4] 43.6] 5.1 18
Nos. 1057 and | Heat.......- se 4 2/ 4.9] 16.9 | 21.8 | 29
1061.
Bese oh chigen): 107. Formaldehyde] * 6| 3)-5.9| 45.3 | 512| 13
Bihan Poneto abet cs Fores I\ (Lycee seeepiene tee Sulphuric acid 2 7 | 582] 18.0} 76.2) 3.25
|
Nos. 791 and | Formaldehyde | 7 8 | 12.6 | 36.1 | 48.7 35
Fort Bayard (New Mexico): 792.
>) linus ponderosae..-)).-- 2. Nos. 891 and | Sulphuricacid 8 6} 14.5.) 18:6") 33.2) .78
\{ 892.
a Area counted, 122 square feet. b Area counted, 78 square feet.

¢In Pinus banksiana av the Bessey Nursery, the loss after emergence is slightly low and the ratio slightly
high, because of the closing of counts on afew of the series before damping-off was erftirely over.

The procedure was to average the number of seedlings which
emerged in the control plats in each series and subtract this number
from the average number emerging (that is, appearing above the
soil surface) in the treated plats in the same series. The treated
plats chosen were the ones which allowed the averaging of the
greatest number of plats treated with the same disinfectant. Only
those plats were taken in which there was no evidence of injury to
the seed or seedlings by the disinfectant and in which the amount
of normal damping-off during the first few days after emergence
was so slight as to indicate satisfactory initial control of the parasites
by the treatment. In such plats it was assumed that the germination
loss was unimportant, and the average number of seedlings appear-
ing on them was taken as representing the number of viable seeds per
plat. The difference between this emergence figure and the average
emergence in the controls was taken as indicating the extent of para-
sitic loss before the seedlings appeared, including any destruction
of dormant seed by parasites which may have occurred as well as
the killing of germinating seed. Both this figure and the number

DAMPING-OFF IN FOREST NURSERIES. 13

of seedlings which succumbed to damping-off after emergence were
reduced to a percentage based on the indicated number of viable seeds,
and they are directly compared in columns 6 and 7 of Table | ee
three of the nurseries the data of the same species of pine and with
the same treatment were averaged.

The data in Table I do not indicate any regularity either in the
extent of loss before emergence, the loss after emergence, or in the

ratio between these RNs 2 ONOR

two values. For ob- SS ZX oP VAN Nels ret
vious reasons, no reg- ¥ SW gr Wy y
ularity is to be ex-
pected in any of these
items. The table is
of some interest,
however, in confirm-
ing the evidence of
the inoculation ex-
periments, of obser-
vation of sprouting
seed dug up in the
beds, and of the par-
tial or complete fail-
ure of emergence at
the centers of large
damping-off foci
(figs. 4,7, and 8) that
the work of parasites
before the seedlings
appear may in some
cases be of consider-
able importance. ‘It
is obviously impos-
sible to make any
general quantitative
statement of the se-
riousness of such loss,

5 ; ‘ ; Ic. 6.—Root sickness in Pinus nigra poiretiana. The two
in view of the yvarla- seedlings at the right are healthy. The three at the left

Mone in: its extent at or tye call quctecs, All axe putting out lateral roots trom
different times and the lowermost sound point. Similarly injured seedlings
places and of the in- when transplanted lived and made satisfactory growth.

accuracy of any computations based on the relative emergence
of hosts as irregular in their germination as the conifers are
known to be. The case is complicated in addition by the fact

that, despite careful avoidance of treated plats known to have
suffered chemical injury, it is probable that a few seedlings were
killed before emergence by the disinfectants used in some of the

14 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

cases. It may furthermore be that in other cases the disinfectant had
a stimulating effect, resulting in better germination in the treated
plats, entirely aside from that resulting from parasite control. The
number of disinfectant methods which concurred in giving apparent
increases in germination, however, makes it seem reasonably cer-
tain that no great part of the increase was due to this stimulation.

In addition to the different disinfectants shown in the table, mer-
curic chlorid, heat, hydrochloric acid, nitric acid, and ammonia all
apparently resulted in approximately the same increases of germina-
tion in tests at the Bessey Nursery as the sulphurie acid which was
used as the standard for comparison in most of the series. Relative
emergence in treated and untreated plats, as well as damping-off

ig. 7.—A clean-killed area in a bed of Pinus ponderosa, caused by Corticium vagum.
Inside a 12-inch circle at the center of this ‘‘ patch”? no seedlings appeared. It will
be noted that the weeds as well as the pines have been killed with the exception of
Salsola tragus.

loss after the seedlings appeared, was determined at two nurseries
in addition to those given in the table. The results at these nurseries
in general confirmed those at the five nurseries covered by the
table in showing lower emergence in the controls. Although it is
impossible to draw positive conclusions, some idea of the seriousness
of losses before the appearance of the seedlings above ground can be
obtained by studying the data in Table I. The fact that such losses
appear considerable, sometimes exceeding the losses from the damp-
ing-off that occtirs after emergence, is believed to explain the com-
mon failure to secure satisfactory results from control measures
taken after the seedlings have come up and the disease has become
noticeable. It is somewhat interesting to note that the data in the

DAMPING-OFF IN FOREST NURSERIES. 15

table tend to confirm field observations that, as compared with other
species, Pinus resinosa is more susceptible to the later forms of
damping-off than to germination loss.

Further indication that the killing of germinating seed before
emergence may be important enough to help explain cases of poor
germination is obtained by an entirely different method, as follows:
At the Wind River Experiment Station of the United States Forest
Service counts of the seedlings emerging and of those which later
died were made on a number of untreated plats by forest officers,
who kindly permitted the writer to use the data obtained. The
counts were made separately on 10 plats each of noble fir (dies
nobilis) and silver fir (Abées concolor). The plats of each species
had been sown with equal quantities of seed. It appeared on in-
spection of the figures that the plats which showed the poorest emer-

Fic. 8.—The area shown in figure 7 after the bed had been weeded and damping-off had
practically ceased. (Photographed by S. C. Bruner.)

gence were also the ones which suffered the most subsequent loss. The
coefficient of correlation between the number of seedlings emerging
and the percentage of subsequent loss in the same plats was found
to be —0.49+0.16 for the noble fir, and —0.50+0.16 for the silver
fir, an average of —0.49+0.11 for the two species, confirming the
conclusion drawn from inspection of the figures. In other words,
poor emergence and heavy subsequent loss were in general associated.
The simplest explanation of this association appears to be to suppose
that both poor emergence and subsequent loss were largely due to
the same cause, namely, the damping-off parasites. Another possible
explanation of the correlation would be to neglect parasites as im-
portant causes of the poor emergence in certain plats and to suppose
that the higher subsequent loss in such plats was due to heat injury,
the less dense stands affording less shade to the bases of the seedlings
composing it. As damping-off is in general so much more important
than heat injury as a cause of death after emergence and the dif-
ference in the degrees of shade between the plats with the denser

16 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

and the plats with the thinner stands must have been very slight, this
latter explanation has not much to support it. The data are believed
to constitute further evidence of the importance of parasites in de-
creasing the percentage of emergence in coniferous seed beds. That
the effect of parasites on emergence should have been large enough
in this case to make itself apparent on the face of the figures, despite
the variations due to other sources, is especially interesting in view
of the fact that the losses after emergence in these plats were not

high
=e
ECONOMIC IMPORTANCE OF DAMPING-OFF.

The importance of damping-off in coniferous nurseries in Europe
is indicated by frequent reference to the disease in the literature.
Biittner (25, 26) states that whole beds are frequently destroyed by
it. Baudisch (9) speaks of the death of entire stands in many
nurseries as the result of damping-off. In the United States Spauld-
ing (137) considers damping-off a serious obstacle in forestation
operations. Clinton (28, p. 348-349) reports serious damage to
conifers in New England nurseries. The writer has found the dis-
ease especially prevalent in nurseries in Nebraska and Kansas, a some-
what unexpected situation in view of the relatively dry conditions
prevailing there. A correspondent has reported heavy loss in seed
beds in Texas.

The economic importance of the disease in conifers is due in part
to the rather heavy average losses experienced at many nurseries
and in part to the irregular character of the losses. In one season
losses may be negligible, while the next season the beds of certain
species may be practically wiped out. Even without this element of
uncertainty the losses experienced are expensive, because of the high
cost of coniferous seed. The seed of some species costs from $3 to
$5 a pound and seldom shows a germination of more than 60 per
cent under nursery conditions. <A loss of half of this 60 per cent from
parasites, both before and after the seedlings break through the soil,
is therefore a matter which deserves attention. The figures in
column 8 of Table I, obtained by adding together those in columns
6 and 7, show that the loss is frequently higher than this. At
the nurseries at which control experiments have been conducted, the
percentage of the seedlings in untreated beds which have been found
by actual count damped-off after emergence is frequently more than
50 per cent, in addition to the considerable but less accurately de-
terminable loss indicated by the foregoing data as being caused by
the parasites before the seedlings appear.

It has been suggested by foresters and others that the net economic
damage from damping-off is not as great as is indicated by the loss
of seed and seedlings which it may cause. The argument is ad-

SEEDLINGS PER SQUARE FOOT OF BED

DAMPING-OFF IN FOREST NURSERIES.

~onseoorern

yo a 2 Rae

SULPHURIC AC/D

Poe aie

8

FORMALDEHYDE

SULPHURIC AC/D

SULPHURIC ACID “ SULPHURIC ACID

70 20 30 oO 5O T7aErG0i GO.) FO! GO
QaYS SINCE GERMINATION

Fic. 9.—Diagram showing the progress of damping-off in treated plats

(solid line) as compared with untreated plats (broken line). Graphs
1 to 4 represent Pinus ponderosa; graphs 5 to 8, P. resinosa; graph 9,
P. banksiana; and graph 10, plats each of which was half P. nigra
poiretiana and half P. sylvestris. Nurseries in Kansas, Nebraska, Minne-
sota, and Michigan are represented. The relatively high total number
damped-off in the treated plats shown in two of the graphs is due to the
fact that a large proportion of the seedlings in the untreated plats had
been killed before they appeared above the soil surface. In both the
cases in which the absolute number of seedlings killed was as great
in the treated plats as in the controls, the percentage of the seedlings
killed was nevertheless lower and the survival more than twice as good
as in the controls.

19651°—Bull. 934—21——2

iy

18 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

vanced that damping-off may be a valuable selective agent in nursery-
grown stock for forest planting, eliminating the weaker individuals
and thus insuring the vigor of the trees which go into the forest
plantation. This is a possibility which must be considered. It is by
no means certain, however, that escape from damping-off is correlated
with permanently superior vigor. It is believed that temperature,
moisture, and other environmental factors, which as yet are very im-
perfectly analyzed, together with the age of the seedlings and the
presence or absence of virulent strains of the parasites, are much
more important factors than inherent differences in individual re-
sistance in determining whether or not seedlings are destroyed. Evi-
dence from inoculation experiments and from field observation, sup-
ported by data of the sort presented in Table I, indicate that damp-
ing-off ordinarily does a considerable part of its damage by killing
the sprouting seed before emergence from the soil, while the graphs
in figure 9 show that of the loss which occurs after emergence in un-
treated beds a large part occurs very early in the life of the seedlings.
Observation of the clean sweeps which the disease commonly makes
in the immediate neighborhood of infection foci (figs. 3, 4, and 5)
indicates that either before or just after the seedlings break through
the soil none of them have any considerable resistance to the really
virulent strains of the parasites, which are believed to be the ones
responsible for the major share of the damping-off.

‘ven if there should be found to be an appreciable selective value
in damping-off, this would not be a valid argument against control
by seed-bed disinfection for the following reason: The graphs show-
ing the course of damping-off in treated plats in figure 9, together
with the decided differences in germination between treated and un-
treated plats, indicate that the very early damping-off is more com-
pletely controlled by disinfectants than the later damping-off. This
early damping-off which the treatments so largely prevent is the
part of the loss which has the least possibility of selective value.
The later damping-off is rarely controlled at all thoroughly by disin-
fectants. As shown by the graphs, it is often even heavier on the
treated than on the untreated soil. It is the part of the loss which
is most likely to have selective effect. At this stage beds are not
taken clean, as earlier; only seedlings which are below normal re-
sistance succumb. The damping-off in disinfected beds seems there-
fore at least as likely to have true selective value as that which
occurs in untreated beds.

The only way in which the effect of damping-off as a selective
agent can be positively determined will be to compare through sev-
eral subsequent years the growth rate, or survival after transplant-
ing, of trees from beds which suffer seriously from damping-off
with the growth of trees from the same lot of seed in seed beds in

DAMPING-OFF IN FOREST NURSERIES. 19

which the disease is either accidentally absent or is artifically con-
trolled. Such an experiment is within the silvical rather than the
pathological investigative field. If it be found that there is some
selective value in the action of the disease and that it is greater in
untreated than in treated beds, it would still seem that a much more
desirable and dependable selection could be obtained by discarding
weak plants at the time of transplanting than by letting damping-
off run uncontrolled in the seed beds. Damping-off is sometimes
negligible and sometimes destroys practically all the seedlings in a
given area, in neither of which cases can it have any material selective
value.

: RELATIVE SUSCEPTIBILITY OF DIFFERENT CONIFERS.

Biittner (25) writing of European conditions, states that exotic
conifers are especially subject to damping-off. He includes fir,
spruce, pines, larches, and cypress in this statement. He mentions
the same subject in a later paper (26). Neger and Biittner (94) give
a long list of different species of conifers from various parts of the
world with statements as to their susceptibility to damping-off.
Beissner (11, p. 656-657), Neger (93), Clinton (28), Bates and
Pierce (7), Boerker (13), and Tillotson (139, p. 69) have all given
information on the susceptibility of different conifers. The data re-
ported by Tillotson are drawn from reports by various officers of the
United States Forest Service which he has compiled. While it is
probable that the nurserymen who are responsible for most of his
records have not observed the disease as closely as Neger and Biittner,
the fact that their observations are mostly based on repeated seasons’
work with large-scale seed beds of the species they report on makes
their observations in some respects more reliable than the other pub-
lished data. Neger and Biittner presumably worked in most cases
with small beds of the various conifers on which they report, and the
variations which they attribute to differences in specific resistance
might easily in such case be largely due to accidental variation. The
error which nurserymen are most likely to make in their notes on
susceptibility is to underestimate the loss, especially for the small-
seeded species. The seedlings of small-seeded conifers decay and
shrivel so quickly after they fall that in taking notes at any one time
only a small proportion of the total loss is visible. Frequent counts
of dead seedlings are the only way by which the loss after germina-
tion in such species can be properly appreciated.

The data given by the authors mentioned in the foregoing para-
graph, together with unpublished data obtained by personal observa-
tion or from commercial and other nurserymen in the United States,
are summarized in Table II, the source of each report being shown
by letters signifying the authority. The unpublished data on two

20 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

nurseries in Illinois and Minnesota were obtained from the nursery-
men by Mr. R. G. Pierce and are indicated by the initial “ P.” In-
formation obtained directly from the nurserymen by the writer is in-
dicated by “ N.” For nurseries where the statements are based on
the writer’s personal observation rather than on the authority of the
nurserymen, his own initial (“H™”) is given. Most of the writer’s
own estimates of relative susceptibility are based on a comparison
of detailed counts of the damped-off seedlings in a large number of
untreated plats at different times, as well as on observation. The
nurseries on which Tillotson reported were all west of the Missouri
River, most of them being in the mountain region. The reports in-
dicated by “H” and “N” were mostly from nurseries east of the
Rocky Mountains. In cases in which the data permit it, the species
are classified as most susceptible, intermediate, least susceptible, or
immune. Ina number of cases, however, it is only possible to classify
-them as “more” or “less” susceptible.

TasLe I1.—Relative susceptibility to damping-off of different conifer species.

[Figuresin parentheses in this table indicate the number of nurseries from which the susceptibility noted
has been reported by the observer to whom the preceding letter refers. ]

Reports of relative susceptibility.

Host species. Not | Least Li More sus- | :
sus- | sus- Ss Eee ceptible | oer
wales cep- | average. | ate. than the tible.

tible. | tible. : average. |

Pinacee (Abietoidez): | |
A DIES SP P32 Seek 2 han, coe c oe nes eee eee eames Ce) PC es eee eos Seen |S ces wane
“Abies balsamene -o.e22 22 - av edae eee eee ee INDs2a ses ees ee aeons
‘Aibiesicephialonica S38 6 2 oh ae See | Seen | Sees IN‘ DS ea) i sateen
Abies concolor'2s2 5-325 Gee c= been ee Peace oe [See aes eee ene eae Seen eso Ee Bu, Nb.
JASDIES MODIS: Are Vets eee ays ae ate ae ee |e eae eee il Nati te [ie re no Ls eee eel pe Nb.
AibicsMordmamn arias oe Les Sas oS Sea ee aie | pee loon eee Nf oma sao cee
sAibies ‘picear( A: pechiniata)sss-- -. asa e te eatas cee eos see eae eee IND eae se heen
‘Abies sacchalkinensis; (2) 2 =< 2 asso [saan ots |eo ee seOe ce ences I Opel ee See nc ce |
ADIeS SibINICaAss «Peas ae eae ee IN Diese) ya see eee | seen 2 sacar nets
(MbIGS VEltGhil sees ase ce eee eee eee V Aoete be al. ae Be ral aes A me ae | a ae ee Nb.
Cedrus deodana- 855-455 he a oe ree tral leah Scat Dp ee eS ee See |
bari gieurop ceases eee ee eee eae cee hpi el pe RE ae INiDio wel sia (22 seam
Darix leptolepisn. 2. os es eee es ee eer | oc ese eee re al Ei en een a | Nb.
Marix occidentalis-a. <2. S3te sree. 2 hoes aoe Ob Deere TY 2 alee eee] aE ees
Picea, ajanensises se ks ike eee eS el es I eee ING eee INDE Ss ares 2
Picea: canadensisiaa. 55-5 -fhaeas meee eee ee oe aH ose ae ee or ene Pia ONS rss
Picea engelmannis. 09. on) ss carcass atl | eee mee Di(2) S55. esac H, N, Ne, | Nb.
Piceaexcelsars a: oe chee ce ose eee eee Pree eee Nb 12 ba Sees Isaeee aes Nes Nek =
Picea omorika -- oy. oe ts ese eee | eee eee INO) ospeee INDD Ses] sac eeye eae
IPICHAOLION PALS: «aes hic omcn aan eee ae ere Pe ener NOSE Sees dn OF ai [eae eee A
Picea pungens:<.. 5.2: 3 te ee eee eee IN (2) 22ers | PEE Pes |VING See, cere ee
PiCes SItCHeNSIS<, .<.. 2 seen See ore oc ee | ee | Se eee eee ING. Nb.
Pins aristata.as--2s2 eee ee at 500 Ae oe IND ees ee ene ce ale eee es | eee cece
PinUSAtpenuatsss 2. see ee ee es | eee | ero reer se Deere tie eke ING Use a eee.
Pinus banksiana....-- SE rae Se ea | ee ee Nibesee | Nees eco 1 B, ae Bere
(2)

Pins CONtOnta....--4-- eee ee eee 4 Nb ec rath a (Pee ete a tos aa ane
PINS 6 GUS es) | eee eee TS ee | ee See Ls we aoe | Cea | See Pee eee

6 Symbols signifying the authority for the report: B= Boerker (13), Bu= Bittner (25, 26), Bp= Bates
and Pierce (7), C=Clinton (28), H= Writer’s estimate, N= Nurserymen’s estimate (obtained by the writer),
Nb=Neger and Biittner (94), Ne= Neger (93), P= Nurserymen’s estimate (obtained by Pierce), T= Forest
officers’ estimate (compiled by Tillotson, 139).

¢ Susceptibility to Phytophthora fagi. =

DAMPING-OFF IN FOREST NURSERIES.

21

TABLE IIl.—Relative susceptibility to danvping-o[f of different conifer

Host species.

specics—Continued.

Reports of relative susceptibility.

Least More sus-

Pinacew (A bietoides )—Continued.
Meta S OX COIS see ate ce sine a sini a setae oye bars
nS MOXBISS 2 =e Geosocs seeee es eese
Pinus jefireyi
Pinus lambertiana
PMgsmontanarmMucnus: =. G..2cjes- ee see
1Fitey boUsaa(ol elo) (2 ea) en ay a aes ee
Pinus nigra austriaca (Austrian pine)... -
Pinus nigra poiretiana (Corsican pine)... -
PAIFAVISHPRIUISUIIS fcc cise cei alee was isin nie wine
PINS POU Chsk ae cen ce oe cc omeeinc ake ace
Pinus ponderosa (type not specified)

Pinus ponderosa (Eastern Rocky Moun-
tain type)
Pinus ponderosa (Pacific coast type). --- -
PTUIS MOSINOSaceet oe lajela aici s cba wale soca
TENE SORIA ee Soe ee ea ae sae ten wtnjae <.cistoie
Pinus strobus
HEMMIUSISVIMEStIIS= a2 - 2a cence See c owen
TENE) FAG 6 8 2 a ee SOR ne CORE ee ence
Pinus thunbergii
Pseudotsuga taxifolia (type not specified ).
Pseudotsuga taxifolia (Colorado type).. --
Pseudotsuga taxifolia (Northwestern
type)
Tsuga canadensis....-.--.-- eee see caine

Number of reports
Percentage of total

Sciadopitoider: -
Sciadopitys verticillata.................--

Cupressaceze (Cupressoide ):
Chamaecyparis lawsoniana...........----
Chamaecyparis pisifera
Cryptomeria japonica
Cupressus spp.. ----
Cupressus arizonica
Juniperus communis
Juniperus monosperma
Juniperus pachyphloea
Juniperus Virginiana
Libocedrus decurrens

PAO StiChuN 66-2 oo 93 sos 525-2

Thuja occidentalis......-.--:---.---------
aia Onientaliss =f. a cece: nets ee ce

aS PUCAtS Scci oewe as -5 -S eee Aeon

MINT Cr OTC DONS semis = che ge ato we loe ain
Percentage of total

Sequoidexe:

Sequoia spp......-- ere See se pt et eae |
Sequoia washingtoniana......-.-..- mane

Taxacer:
Taxus cuspidata

Saw ne Less Inter- sere Most
or Hae than medi- eepee suscep-
tiple tible. | 2VeTage- ate. average. tible.
See Ios Be eeeee WG) senor eaeeccise) ple ope els Aiea tele ye tel
ees eos cee Rae eine nce ames Ni cas aseee
de eet lees tee SEIN ie Nicaes,. C,N, T-
weer got AY (Q)nc. 4. Hs Po.) Bp, EG)--| Ne
FIR IO OS KS NG Tike eee PS SN
BAN pe Sl seen N (3) T(3)| H.....| N, T (5)
te ese ye Beas Sbres- es cee NDS ara: sci qeeeerex
Bs, edema pease hens ese IND iba Bieeeeese

9 17 51 23 48 15

6 10 31 14 29 9
We) Elias Feavealliian oe. dea aloes (ribs: Siete Nb.
DN Se Nb UN ees cate | okey cse el ipl CHU ee

Nb

The fact most evident in Table IT is the extreme variation between
reports, not only on closely related species but even on the same

species.

While it is, of course, possible that the obvious lack of a

definite basis and method of comparison in most of the reports is
responsible for most of this variation, it seems to the writer more
probable that different species do actually vary in their relative sus-
ceptibility to damping-off in different localities. In the first place,

29 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

the conditions which might increase resistance of one host might
very easily decrease its resistance for a host with different environ-
mental requirements. ‘To illustrate by an extreme example, the
pinon (Pinus edulis) of the arid or semiarid region might remain
resistant in soils in which Picea engelmanni of the high mountain-
stream bottoms or Picea mariana of the northern swamps might be
low in vigor and easily attacked. In the second place, it is to be
expected that species with a certain order of relative susceptibility to
the parasites which predominate at one nursery may exhibit a very
different order of susceptibility to the different combination of para-
sites which might be prevalent in another locality.

The only individual species on which there are a sufficient number,
of reports and a sufficient agreement between the reports are the two
common western spruces, Picea pungens and P. engelmanni, which
(at least as compared with Picea excelsa) seem rather susceptible,
and Pinus ponderosa, which (as compared with most of the other
species of the Abietoidez) is to be regarded as generally more re-
sistant than the average. Within each of the larger genera of this
group it seems evident that susceptibility is extremely varied and
that no statement as to the relative susceptibility of the genera them-
selves can therefore be made. The only group generalization that
is perhaps permissible is derived from the consideration of the
Cupressoidee. In this group, out of 23 reports, 16 are in the “ not
susceptible” or “least susceptible” columns and only one indicates
more than intermediate susceptibility. Of 163 reports pertaining
to the Abietoidex, only 26 place them in the “not susceptible” or
“least susceptible” columns and 63 in the classes of more than inter-
mediate susceptibility. The general feeling among nurserymen
seems to be that serious damping-off need not be feared among the
cedars and their relatives. The data at hand tend to justify this
confidence.

CONTROL OF DAMPING-OFF.

Early efforts to prevent damping-off were chiefly directed to the
avoidance of excessive moisture in either the air or soil. A means
to this end, which has been observed more or less by nurserymen for
many years, both in the United States and elsewhere, is the applica-
tion of small quantities of dry sand to the seed beds after the disease
becomes noticeable (18, p. 166; 83). This is sometimes applied hot
(101, p. 48-44; 145), though even this procedure does not result in
very great advantage. Surfacing with hot sand can not always be
counted on to give any measurable advantage over untreated beds (67,
p. 8). The use of sand (25) or sterile subsoil (101) instead of ordi-
nary soil in covering seed at the time of sowing has been advised.
Johnson (82) did not secure satisfactory results with sand in tobacco

DAMPING-OFF IN FOREST NURSERIES. os

beds. Making the upper part of the bed to'a depth of several inches of
recently dug subsoil appeared effective in a single test by Spaulding
(137) and at four nurseries by cooperators of the writer in later
tests, the results of which will be published elsewhere. The procedure
is unfortunately rather expensive in large-scale work and under some
conditions at least undesirable because of the poor subsequent growth
on such soil. Excessive vegetable matter (45), imperfectly rotted
manure (67), or green manures recently plowed under (43) have all
been advised against as likely to favor the disease. The experience
reported with conifers (67, 139) indicates that damping-off can be
to a certain extent decreased by broadcast sowing as compared with
sowing in drills. The usual recommendation of thin sowing to avoid
the seed-bed disease of other plants has also been made for conifers
(67). Transplanting healthy seedlings from infected beds into new
soil is recommended as a means of saving them from attack (11, 145).
The writer’s tests of transplanting at a Nebraska nursery gave no
promise of economic value as a control method, although he is in-
formed that it was successfully employed in a nursery in New Mex-
ico. The time of sowing appears to have a relation to the amount of
disease at some nurseries, but conditions in this regard evidently
differ in different localities, so that the best time to sow from the
standpoint of avoiding damping-off must be determined separately
by repeated tests at each nursery. For example, observations both
by the nurserymen and the writer during several seasons at the
Bessey Nursery, in Nebraska, indicate that fall sowing is an ex-
cellent means of decreasing loss from damping-off in at least one
pine species, and Retan (110) reports the same thing for a nursery in
Pennsylvania, while at two Kansas nurseries and at nurseries men-
tioned by Tillotson (139) fall-sown beds suffer more than those
sown in the spring.

Treatment of the seed with mercuric chlorid (25) or with copper
sulphate (122) has been recommended. While it has been demon-
strated (38) that a proper heat treatment of the seed will greatly
decrease the damping-off in sugar-beet seedlings, this is explained
by the fact that one of the most important parasites of the sugar
beet is systemic and often present in the seed. There is no reason
to believe that seed-carried infection is of any importance in conif-
erous seed beds. The only advantage that could reasonably be
expected from a seed treatment of conifers would be that which
would come from the prevention of seed decay in the soil before
germination starts, and this protection could be expected to be ef-
fective only if a relatively insoluble disinfectant, such “as Bordeaux
mixture, was used.

Soil treatment is the most direct and probably the most profitable
method of attack on the disease. It is especially easy, for tobacco

24 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

seedlings (82) as well as for pines, to prevent by soil disinfection
losses before the seedlings appear above the ground. Heat disinfec-
tion of seed beds has boen frequently mentioned. Burning wood
or litter on the surface of the beds before sowing, said by Gilbert (47,
p. 36) to be a common procedure in preparing tobacco seed beds both
in Italy and in parts of this country, has been recommended for
coniferous seed beds by Biittner (25). The disadvantageous results
sometimes noticed following the application of wood ashes to pine
seed beds may prove an objection to this type of treatment in some
of the nurseries. Ata Nebraska nursery (67) moist heat proved only
partly satisfactory, unavoidable reinfection having serious results.
Steam disinfection, using the inverted-pan method commonly advo-
cated for tobacco seedlings (10, 47, 81), has been reported by Scott
(123) as successful at a nursery in Kansas. Gifford (46) found
steaming with the inverted pan only partly satisfactory. It is not
believed that it is likely to pay to install the necessary apparatus for
steam disinfection at nurseries in nonagricultural districts where
steam tractors are not available for temporary use. The hot-water
soil treatment as used by Byars and Gilbert (27) is probably worth
a trial at any nursery where damping-off is serious and fuel cheap.
It may be that in some localities where steam or hot-water treatment
of the soil is not sufficiently effective, its efficiency can be increased
by reinoculating the soil immediately after treatment with sapro-
phytic molds and bacteria to provide maximum competition for
parasites which come in from the outside. Tests of this procedure
will be described later in the present bulletin. The value of char-
coal has been emphasized by Retan (109, 110).

Chemical disinfection of the soil has also been employed. Sulphur
has long been in use as a soil treatment against the damping-off of
various plants (45, 111) in addition to its use in combating potato
scab and onion smut. It was tested on conifers by Spaulding (136,
137) in the form of light surface applications to the beds after ger-
mination, but without decisive result. In later cooperative tests pow-
dered sulphur raked into the soil before the sowing of the seed failed
to indicate any large measure of value. Very finely divided forms
of sulphur in various amounts and. times of application are prob-
ably worth some further test.

Moller (90) and Sherbakoff (128) have reported the wicceeten use
of copper sulphate in combating attacks of Corticium on dicotyle-
dons. In Johnson’s experiments on tobacco seedlings (82, table
3) copper salts and Bordeaux mixture were the only chemicals for
which any value was indicated. Sherbakoff apparently used copper
sulphate and other strong disinfectants chiefly to stop the extension
of vigorously spreading damping-off foci by local treatment rather
than as a general treatment for use over the beds. Such treatment

DAMPING-OFF IN FOREST NURSERIES. 25

would presumably kill all seedlings on the area treated, but would,
of course, be of considerable value in stopping at the outset such
mycelia as those which caused the damped-off area in figure 7. The
procedure would be of practical value only in cases in which damp-
ing-off was chiefly limited to a few large patches of this sort, a rather
rare condition in conifers.

Copper sulphate solutions have been used on pine seed beds at the
time of sowing with considerable success at some nurseries (65, 67).
Except in a nursery in which the soil contained carbonates, it has
proved rather difficult to prevent injury to the pines. The trial of
some ‘such combination of copper sulphate and lime as was used
by Spaulding (136) on the surface of pine beds before sowing, which
apparently prevented the damping-off of lettuce in some unpub-
lished pot experiments of Mr. J. F. Breazeale, is considered desir-
able. Treating seed beds with ordinary Bordeaux mixture has also
been recommended. Horne (78) secured especially good results
against Corticium vagum in tobacco seed beds by heavy applications
of Bordeaux mixture, and Schramm (122) and Clinton (28) have
obtained indications of its value as a spray in preventing the damp-
ing-off of conifers. It is probably worth further tests in various
amounts of application. In tests conducted by the writer in 1912
and still unpublished, some advantage was indicated for Bordeaux
mixture as a surface treatment after soil disinfection with acid.
Zine chlorid as a soil disinfectant has also been found valuable in a
number of cases (65, 67), but it is more expensive and apparently less
dependable than copper sulphate.

Formaldehyde and sulphuric acid have been tested more fre-
quently than other disinfectants. The use of sulphuric acid on
coniferous seed beds was originated by Spaulding (136). The first
intensive experiments with this acid were reported by the writer (63).
The first experiments with formaldehyde on conifers seem to have
been in the early greenhouse tests of Spaulding (137), repeated in
forest nurseries in 1907 by Jones (83) and Spaulding (136). Most
of the experiments with these two substances have already been sum-
marized (67). A report not mentioned in this summary is that of
Schaaf (119, p. 88), who obtained favorable results with the acid.
The great trouble with formaldehyde is its tendency to kill dormant
seed. The length of time which must be allowed to elapse between
treatment and sowing in order to avoid this killing varies with con-
ditions. Formaldehyde is more expensive than acid and seems on
the whole to have been less effective in disease control. Acid, on the
other hand (applied just after the seed is sown, which is found to be
the best time), on a few soils has caused injury to radicles, which it was
at first thought could be prevented only by very frequent watering
during the germination period; while in a few cases, when cold

96 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

weather resulted in a long germination period, it has killed or in-
hibited the germination of some of the dormant seed. All injury
can be prevented by treatment a few days before sowing, followed
by the addition of lime just before sowing, but lime used in this
way has apparently destroyed a considerable part of the value of the
acid treatment against the disease. Further consideration of the
data on which earlier papers (63, 67) were based indicates that the
apparent need for frequent watering during the germination period,
which was required at a few of the nurseries where the fifst tests of
acid treatment were made, as well as practically all of the germina-
tion reduction, was due to the use of unnecessarily large applications
of acid and that the trouble can be eliminated by determining by
test the minimum quantity of acid which will be reasonably effective
in each locality. If this can be done it should establish the acid
treatment as the most profitable for general use of any of the
methods of damping-off control which have so far been extensively
tested.

In view of the various parasites which may cause damping-off at
different times and places and which vary greatly both in their
means of dissemination and in their physiological qualities, it is not
believed that any single disinfectant will be found entirely satisfac-
tory at all nurseries. It is also unfortunately true that no one
strength of treatment can be recommended for all nurseries. The
quantity of acid to be used at any specified nursery will have to be
determined by test at that nursery. <A single test, no matter how
well conducted, is not sufficient to serve as a basis for conclusions.
However, a number of small-scale tests, made at different times and
in different parts of the seed-bed area, should determine the best
treatment for any particular nursery with a reasonable degree of
certainty and with very little work. If the plats are equal in size
and receive equal quantities of seed, all the nurseryman needs to do
to determine the value of the treatments is to count the number of
living seedlings on the different plats at the end of the season. The
decrease in the number of weeds as a result of the use of acid is
itself sufficient at a number of nurseries to pay the entire cost of the
treatment. Detailed methods of application have already been pub-
lished (67). The differing proportions of acid between which the
best treatment will ordinarily be found to he are 2 and 7 ¢. ¢. (one-
sixteenth and one-fourth of a fluid ounce) of the concentrated com-
mercial acid per square foot of seed bed, applied just after the seed is
sown and covered. It should be dissolved in 500 to 1,000 ¢. ec. (1 to 2
pints) of water per square foot of bed before applying. The drier
the soil before treatment, the more water should be used in dissolving
the acid.

No treatment applied after germination begins can have the maxi-
mum value in controlling the disease, because the damping-off para-

DAMPING-OFF IN FOREST NURSERIES. rd

Sites frequently, if not usually, do part of their work before the seed-
lings appear above the soil. Furthermore, any treatment at all effect-
ive against the disease is almost certain to hurt the seedlings if applied
after the seed starts to sprout.

Both the acid and copper-sulphate treatments which have been
found useful in pine seed beds are of very doubtful value for most
hosts other than conifers, as the angiosperms on which observations
have so far been made are too easily injured by the disinfectants.
The weeds in the nurseries have been injured or entirely kept from
appearing by treatments which caused no injury to the pines.

CAUSAL FUNGI.

CORTICIUM VAGUM.

Occurrence and parasitism.—tIn a recent publication (68) Corti-
cium vagum B. and C. (C. vagum solani Burt, Typochnus solani
Pril. et Del., the common damping-off Rhizoctonia) has been reported
on a number of conifers. Inoculation, reisolation, and reinoculation
on pine have established its parisitism on this host beyond a reason-
able doubt, though in these inoculations, as in most, if not all, the
work which has been done with the fungus on angiospermous hosts,
the cultures employed have been from plantings of diseased tissue
instead of from single spores. The inoculation experiments have
confirmed the field observations indicating that this fungus is fully
as able to cause loss by destroying germinating seed below the soil
surface as to cause damping-off of the better known type after the
seedlings appear above the soil surface.

An extensive list of angiosperms on which the fungus has been
reported is given by Peltier (98). Cross-inoculations between the
pines (68), on the one hand, and potato (40) and sugar beet (38)
have shown the same strains to be parasitic on both conifers and
angiosperms and established the physiological as well as the morpho-
logical identity of the fungus attacking pines with the common
Corticium vagum. Now that Duggar (34) has offered strong,
though not yet entirely conclusive, evidence of the identity of C.
vagum with the European “ vermehrungspilz” (the J/oniliopsis
aderholdii of Ruhland; 115) it is to be presumed that it is
a cause of damping-off of conifers in Europe as well as in Amer-
ica, though no reports of it on conifers have been so far en-
countered in European literature. The Rhizoctonia reported by
Somerville (132) on Pinus sylvestris and the Rhizoctonia strobi de-
scribed by Scholz (121) as killing young Pinus strobus were both
on trees more than 4 years old, so that they had no relation to damp-
ing-off. Furthermore, the first of these was apparently the old
Rhizoctonia violacea, now known as 2. crocorum (R. medacaginis) ,
a fungus entirely distinct from Corticium vagum, probably belong-

28 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

ing to an altogether different group of fungi and not known to cause
damping-off of any host. Rhizoctonia strobi is not sufficiently de-
scribed to allow determination of its identity.

Variations in virulence—In the inoculations earlier reported on
conifers, different strains of Corticium vagum were said to vary
greatly in virulence (68). Further examination of the data on
which this statement. was based yields confirmatory evidence. Part
of this evidence is shown eraphically in figures 1, 2, and 10. The
experiments on which these graphs were Hines involved at the time of
seed. sowing the addition to the soil of apparently pure cultures of
C. vagum. Throughout each experiment the different units received

FINUS BANKSIANA PINUS RES(NOSA
1918
ere ee Ze

8

[PN

SEFOLINGS GERMINATED PER 100IN CONTROLS
°

Fic. 10.—Diagram showing the relative activity of different strains of Corticium vagum,
as indicated by the number of seedlings appearing in inoculated pots. Explanation of
symbols: Q —Strain 147, from spruce seedlings, Washington, D. C., 1910; V =strain
213, from sugar beet seedlings, Washington, D. C., 1911; —J—strain 230, from Hlaeagnus
sp., Kansas, 1913; 0 =strain 233, from the same lesion as strain 230. Strains re-
isolated from these, the results of which appear in experiments Nos. 71 and 72, are
indicated by the same signs as the original strains used in the inoculations from which
they were taken. The original strains in experiments Nos. 71 and 72 are indicated
by arrows.

equal quantities of seed, and the culture substratum used in inoculat-
ing was the same for all strains. Experiments 36, 45, 47, 49, and 51
were conducted on plats in out-of-door drill-sown beds, experiment
36 on an alkaline soil, all of which had been heated in a moist con-
dition at a temperature of not less than 80° C. for not less than 10
minutes,? and experiments 45, 47, 49, and 51 on a sand which had

2 This temperature is probably high enough to eliminate damping-off organisms. Tests
by Dr. Theodore C. Merrill indicate that the three most virulent parasites so far
worked with are killed by placing agar tube cultures for 10-minute periods in water at
the following temperatures: Pythium debaryanum, 65° C.; Corticium vagum, 50° C. for
mycelium and 60° C. for sclerotia; Fusarium moniliforme, 70° C. Both the Pythium
and Fusarium cultures contained spores. The possibility of the survival of oospores
which would not be capable of germination for several months was apparently eliminated
by the writer, who retained Dr. Merrill’s heated Pythium tubes and made final transfers
from them 73 months after heating, still without securing growth. Plenty of typical
oospores* were present in the part of the heated culture from which transfers were
made.

DAMPING-OFF IN FOREST NURSERIES. 29

been treated with sulphuric acid followed later by lime. ‘The other
experiments included in the graphs were on autoclaved sandy loams
in pots in the greenhouse. In these graphs are included all of the
results in which the same groups of strains were used repeatedly in
different experiments. In figure 1, the values plotted for experi-
ments 36, 49, and 51 are for the number of seedlings which appeared
above ground, the heavy inoculations and favorable conditions for
damping-off in these experiments being such that even weak strains
caused heavy losses and the survivals therefore do not give differ-
ential results. Comparison of the survival data in the other experi-
ments in figure 1 with the emergence data for the same strains in
that figure and in figure 10 indicates that the strains best able to
reduce survival are also the ones best able to reduce emergence.
While the data presented in the graphs are not entirely consistent,
it is very evident from them that strains 147, 213, and in a lesser
degree 206 were regularly more virulent than most of the strains in
‘tests conducted several years apart on different species of Pinus.
It is also evident that certain strains of 186 and 189 which appear
in figure 2 are quite regularly of low or doubtful virulence. Strains 50,
183, 192, 211, 212, 230, and 233, whose virulence is apparently inter-
mediate, show a greater variability. In experiments 36, 45, 47, 49,
and 51, in which conditions especially favor parasitism, they may
cause practically as serious loss as the regularly virulent strains, the
best differential results being shown in experiments in which the
disease is less favored. The apparent variation in the relative viru-
lence of such strains in different experiments may, of course, mean that
their virulence is differently affected by different conditions. It
seems rather more probable that the variation in relative activity is
to be classed as accidental variation, necessarily great with small
units which are subject to numerous uncontrollable variables. It
seems entirely possible, however, that part of the observed differences
in relative activity may be due to differences, not in virulence, but in
the ability of the different strains to maintain themselves saprophyt-
ically in different soils during the period between inoculation and
the commencement of germination. For example, strains 230 and
233 came from a nursery in southwestern Kansas in which the soil-
acidity exponent, as determined by Dr. L. J. Gillespie, of the United
States Bureau of Plant Industry, is 8.4. It seems entirely possible
that these strains, rather strongly parasitic in some of the experi-
ments, including an experiment on the soil from which they were
taken, might prove less able than strains from some other habitats
to maintain themselves on some of the eastern soils used in the green-
house tests. The source of strains 230 and 233 was furthermore a
locality where high soil temperatures are to be expected. The fact

30 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

that experiments 71 and 72, in which they showed the least virulence,
were conducted in a colder greenhouse than any of the other tests
may have had something to do with the lower activity indicated for
these strains. Variation in the temperature requirements of different
strains in accordance with the temperature of the source locality has
already been demonstrated by Edgerton (35) for one of the anthrac-
noses. It is hoped later to determine the temperature and acidity
preferences of these two strains as compared with the others used.
It should be noted that the consistently weak strain No. 189 (fig. 2),
was abnormal in habit, lighter brown, and produced fewer sclerotia
than typical strains. The other strains appearing in the graphs
showed no conspicuous morphological or cultural differences that
were identical. The only other strain which was noticeably abnormal
in culture was one from pine seedlings in Kansas, intermediate in
habit and color between No. 189 and the typical strains and indicat-
ing little more virulence than No. 189 in the few experiments in
which it was used. It does not appear in figures 1 and 2, but was
included in the experiments reported in the following paragraph.
Peltier (99) believes low sclerotium-forming capacity to be a sign
of degeneration and low virulence; the writer’s experience agrees
with his as to virulence, but these two strains showed no other evi-
dence of lack of vigor.

As a further check on the reality of the apparent differences in
virulence between different strains, all of the original strains avail-
able at the time, a total of 29, were used in the practically duplicate
experiments 71 and 72 and the relative survivals of the same strains
in the two series mathematically and graphically compared (fig. 11).
The very decided correlation between the two experiments indicated
by the graph has a coefficient * of 0.813+-0.042, nineteen times its

’The correlation coefficient, a very useful thing for many kinds of biological work,
which unfortunately has received little attention from plant pathologists, is explained
by Secrist (124, p. 43 et seq.) and the process of computation described (124, p. 453-467).
A shorter method of computation is given by E. Davenport (30, p. 465-467); the
example he gives is of a series with a large number of varieties, in which the correlation
table is employed. Davenport's method is, however, just as useful in such a case as
this, in which the number of varieties is too small to make the formal table advantageous.
In such a case the computation should be arranged as by Secrist (124, p. 460-461), but
the guessed rather than the true mean used and Davenport’s formula employed. If the
coefficient is +1, the correlation is perfect; if it is 0 there is no correlation,. and if
—1, perfect negative correlation. The significance of a coefficient less than 1 is judged
from its relation to its probable error. Wing (84), in an excellent discussion of cor-
relation, gives rules for judging the degree of significance of the coefficient. The
correlation coefficient has its greatest potential usefulness in examining apparent causal
relations. It is so used in connection with the relation between the hydrogen-ion exponent
and damping-off in considering figure 12 of. the present bulletin. Interexperimental,
or, as Harris calls them, ‘‘ interannual’ correlation coefficients of the sort used for
these Corticium strains have been used by Norton (96, p. 51) in measuring the constancy
of rust resistance of asparagus strains, by Harris (54) in demonstrating the constancy
of differences in various characters between strains or individuals, and they appear. to
be usefuj for this purpose in the present case.

DAMPING-OFF IN FOREST NURSERIES. 81

probable error. Peltier’s results permit similar correlations for the
18 strains common to his experiments 1 and 1A on carnation cuttings
and for the 22 strains common to experiment 1 on cuttings and ex-
periment 2 on seedlings. The coefficients found are decidedly lower
than those obtained from the experiments on pine, 0.510.117 for
the experiments on cuttings and 0.360.124 for the comparison
of the results on cuttings with the results on seedlings, but neverthe-
less indicate some interexperimental correlation for the same strains
and therefore inherent differences in parasitic ability between the dif-
ferent strains.

gd
3.3

g

SEEDLINGS SURVIVING PER 2P0T UNIT
38

Lo)

SRR EERE SE EEE EERE CRLELEE

STRAINS OF CORTICIUM LAGU

Fic. 11—Diagram showing the comparative virulence of 29 strains of Corticium vagum
in two successive inoculation experiments on Pinus resinosa. The results in experiment
No. 71 are shown by the solid line, the strains being arranged from left to right in the
order of descending virulence indicated by the number of seedlings surviving in this
experiment. The results from the use of the same strains in experiment No. 72 are
shown by the broken line. The obvious correlation between the two curves (coefficient
0.81+0.04) indicates a real difference in virulence between the different strains. The
strains indicated by the underscored numbers are original strains and those not under-
scored reisolations from the original strains in earlier inoculation experiments on pine
seedlings.

In the work on pine seedlings, with the possible exception of
strains 230 and 233, there was no evidence of attenuation in arti-
ficial culture. Strains 147 and 213, isolated in 1910 and 1911, re-
spectively, seemed as strongly “erierte in experiments 71 and 72
(1917 and 1918) as any of the five strains isolated in 1916 or the fe
strains isolated in 1915.

Of the 20 strains above mentioned, three pairs were isolated under
such conditions and. showed such later agreement in performance as to
indicate their individual identity. For the purposes of considera-
tion in the following paragraph, the one of these probably duplicate
strains which happens to have the higher number was eliminated from
each pair. The survival figures in pots inoculated with the 17 strains

82 BULLETIN 934; U. S. DEPARTMENT OF AGRICULTURE.

remaining, giving the mean of the results in experiments 71 and 72,
are shown graphically in figure 13, together with the results of some
of Peltier’s experiments in which other strains were used. Per-
centages of seedlings damped-off after germination are not included
in these and most of the other data on pines because the most viru-
lent strains often entirely prevent germination, and no value for sub-
sequent loss is obtainable. The grouping of most of the writer’s
strains at the least virulent end of the register (that is, the one with
the highest number of living seedlings) is of some interest. The
distributions based on the two experiments considered separately

10 93
“t
ee be
| §
be
ab

6 7
S| 8
3 ?
ar

4-6
v1 8
idl BS
cS |
%

u')
14]

DITION Hie Nea oe FOO) (i SEP S 10 123 TF 16 SIE

Iic. 12.—Diagram showing the relation between damping-off of conifers (broken line)
and soil acidity (solid line). The acidity of soil samples from the different. nurseries,
determined by Dr. L. J. Gillespie, is reported as Pu7, indicating approximate neutrality
while Px6 indicates ten, and Px5 one hundred times as great a hydrogen-ion concentra-
tion as Pu7 ; therefore the lower the hydrogen-ion exponent line, the greater the acidity.
The seriousness of damping-off at each nursery is on an arbitrary scale in which nurseries
with negligible loss are rated as 1, and the nursery which suffered most is rated as 10.
These values are estimates, though for some of the nurseries extensive counts were
available on which the estimates were based.

agreed very well in this grouping. The minor group at the end of
extreme virulence is not taken to indicate an actual grouping but,
‘ather, an artificial one, due to the fact that both the strongest
strains and some less strong were thrown into the same group by
the lack of additional seedlings for the stronger strains to kill. This
lack of additional seedlings constituted a limiting factor. In other
words, conditions favored damping-off even in these two experiments
too much to permit completely differential results for the more viru-
lent strains. Despite this artificial limit preventing the full vari-
ability becoming evident, the coefficient of variability of the survivals

DAMPING-OFF IN FOREST NURSERIES. 3

had the high value of 63+9.7 per cent. The graph indicates also a
decided, though less extreme, degree of variability for Peltier’s
strains on carnations; the survivals for the 18 strains which he used
in both of his experiments on cuttings have a variability coefficient
of 29+3.5 per cent and the 23 strains in experiment 2 on seedlings
55+6.9 per cent.

PYTHIUM DEBARYANYM
® ccoh Sede bos 38 ® 0O

CORTICIUM LAAGUM
WAITERS STRAINS ON FINE

oO
oo Oo ao ooo

Aa PELTIERS STRAINS ON CARNATION CUTTINGS

ee | O
Been ab aoa. oO oo O

PELTIER’S STRAINS ON CARNATION SEEQLINGS
O

Db .oO oO Oo ©.
OO Bb” S'S) ee

O 20 40 60 80 ‘/00
PLANTS SURVILING PEP 10Q/N CONTROLS

lic. 13.—Diagram showing the results of inoculations with 17 strains of Corticium vagum
and 35 strains of Pythium debaryanum, arranged in decreasing order of virulence from
left to right, as indicated by the survivals in pots of pine seedlings artificially inoculated
with them. The Pythium results represent the mean survivals in 5 pots inoculated with
each strain in each of experiments Nos. 66, 67, and 68. Each point located is therefore
based on the results in 15 pots, 10 of Pinus banksiana and 5 of P. resinosa. The
Corticium results on pine represent 5 or 6 pots each, in two experiments (Nos. 71 and 72)
on P. resinosa. The outline circles represent P. debaryanum strains from Hast Tawas,
Mich.; the solid circles represent strains from other localities. The second row of
squares shows the sum of the results in Peltier’s experiments Nos. 1 and la (99, his
table 8). The lowest row of squares shows his results in experiment No, 2 (his
table 4).

The strains represented in figure 13, as used on pine, include 1
from bean, 2 from potato, 1 from sugar beet, 1 from Elaeagnus, 2
from Picea engelmanni, and 10 from Pinus resinosa, P. ponderosa,
and P. banksiana. Two were from Washington, D. C., 2 from New
York, 1 from Ohio, 4 from Michigan, 4 from Minnesota, 1 from Ne-
braska, 2 from Kansas, and 1 from California. The sources of these
strains are widely distributed both as to host and locality; they are

19651°—Bull. 934—21 3

34 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

rather more representative of the country as a whole geographically
than the strains in the larger of Peltier’s experiments, though less
representative as to host sources. The number is too small to justify
conclusions as to the proportion of Corticium vagum strains which
can be expected to prove strongly virulent on pine. The data are
offered merely as a beginning, to which it is hoped experimenters
with other strains of C. vagwm will make additions.

In addition to the strains used in these two experiments, several
others which had been lost or for other reasons could not be included
in both the final experiments had been previously tested on pines in
sarlier experiments. Of these, 6 strains, 1 each from alfalfa, sugar
beet, Pseudotsuga taxifolia, Pinus banksiana, P. resinosa, and P.
strobus, gave indications of low virulence on the pines; 3 strains, 1
each from sugar beet, Pinus sylvestris, and P. ponderosa, gave indi-
cations of rather high virulence; while another strain from P. ponde-
rosa indicated an intermediate ability to attack pine. Combining
these strains with those represented in figure 13, there are data on 27
original strains, of which 8 are roughly classed as strongly virulent
on pine seedlings, 14 as weak, and 5 as intermediate.

Edson and Shapovaloy (40) have conducted inoculation experi-
ments on potato stems with 6 of the Corticium strains which had
been used on pine, including the 5 strains mentioned by them on page
218 and their strains R. XV (the writer’s strain 192 of fig. 2) on page
215. Strains 147 and 724, which had proved the most destructive
in the inoculations on pine, appeared also rather strongly virulent on
potato. Strain No. 186, originally from potato, which had given no
definite evidence of parasitism on pine, also proved unable to cause
lesions on the potato stems. The remaining 3 strains, all of inter-
mediate virulence on pine, gave results on potato which were less in-
dicative of agreement with the order of virulence on pine. The data
suggest that strains strongly parasitic on potato are hkely to be
strongly parasitic on pine, and vice versa, but the agreement between
their results and the writer’s is not sufficiently complete to establish

the point.
FUSARIUM SPP.

Fusarium is often found on or in damped-off seedlings (24, 46, 60,
94, 120, 187, 141, 142). The early inoculation experiments, conducted
in the main with strains not sufficiently described to allow their iden-
tification, have been recently summarized (68, p. 537), together with
descriptions of inoculation experiments on pine seedlings with four
commonly recognized species of Fusarium. These, though not fol-
lowed by reisolation, gave rather definite evidence that /usariwm
moniliforme Sheldon was decidedly parasitic and /. solani less
strongly so. Fusarium ventricosum Appel and Wollenw. was indi-

DAMPING-OFF IN FOREST NURSERIES. 35

cated as more strongly parasitic than /’. solani, but in a single test
only and with a culture of doubtful purity. /usariwm acuminatum
KE. and FE. gave no evidence of parasitism. These results agreed with
those of Spaulding (137) in indicating that the ability to attack
seedling conifers is not limited to a single species of Fusarium and
that /. moniliforme is one of the more virulent. The statement by
Hartig (61, p. 147-150) that a Fusariumlike fungus was able to cor-
rode the young epidermis of pine seedlings has already been men-
tioned.
PYTHIUM DEBARYANUM.

Pythium debaryanum Wesse (Artotrogus debaryanum Atkinson,
Lucidium pythioides Lohde) has been known since 1874 (74, 86) as
a common cause of damping-off of various angiosperms. ‘The first
known observation was made by De Bary about 1864 (74). Despite
the large number of hosts on which it has been listed, its parasitism
has been definitely established on few. Peters (100) has successfully
inoculated sugar beets with pure cultures; at least part of his strains,
including presumably part or all of those he used in inoculation tests,
were obtained from single spores. Edson (38) working with the
same host, reisolated the fungus from inoculated seedlings, and made
reinoculations with it. Both find it able to cause root sickness of
plants not attacked early enough to be killed outright. Johnson
(82) and Knechtel (85) have caused damping-off of tobacco seed-
lings with it, and the former reported it also able to persist in the
cortex and kill the lower leaves of tobacco plants which survived
attack. The fungus has long been reputed parasitic on potato tubers
and has now been found by Hawkins (70) to be the chief cause of
the rot known as “leak” in California. Peters (99) made success-
ful inoculations with pure cultures on cuttings of Pelargonium.
Most of the reports of parasitism, however, have been based on
microscopic examination or more or less crude inoculation experi-
ments. Noteworthy among the latter are those reported by Hesse
(74) on Camelina sativa in the original description of the fungus.
These were made before pure-culture technique had come into use
with fungi, but were so thoroughly checked by microscopic obser-
vations at every step in the process that they must be admitted as
very good evidence of the parasitism of the fungus. A number of
reported angiospermous hosts are listed by Butler (23), Voglino
(143), and Johnson (82, p. 34, footnote, and p. 35). Reinking (107)
recently reported Canica papaya as attacked. A host which the
writer has not found in the literature is rice, found by Dr. Haven
Metcalf seriously attacked in the seedling stage in a field in South
Carolina. A second apparently new host for the fungus is fenu-
ereek (Trigonella foenum-graecum). The writer found oospores
typical of Pythium debaryanum in the tissues of damped-off seed-

36 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

lings received by Prof. William T. Horne, from Sonoma County,
Calif., with the statement that the disease was seriously affecting
the stand. The fungus was easily isolated, and the results of suc-
cessful inoculations on pine with the cultures obtained are included
in Table V (p. 47). A fungus resembling P. debaryanum was
also found in damped-off cowpea (Vigna sp.) seedlings grown in
rotation with pines at a Nebraska nursery.

Pythium equiseti Sadebeck, reported as parastic on the prothallia
of E'quisetum arvense, was successfully used by Sadebeck (117) in
crude cross-inoculations direct from £’. arvense to potato tubers.
De Bary (5) reversed the direction of the experiment between
cryptogamous and phanerogamous hosts by successfully inoculating
prothallia of Hquisetum arvense with Pythium debaryanum directly
from diseased Lepidium seedlings. He also secured positive results
on prothallia of the fern 7odea africana by the same method. The
Equisetum prothallia he found to be especially favorable media on
which to develop Pythium debaryanum. Fischer considers the
fungus found by Bruchmann (17) and Goebel (49) on prothallia of
Lycopodium sp. as probably identical with P. debaryanum. <A care-
ful reading of the original articles is sufficient to show that the sym-
biotic fungus which they described was an entirely different or-
ganism. Saprolegnia schachtii and Sporodospora jungermanniae, re-
ported on two of the Hepatice, are of doubtful position (42, p. 403),
though Butler (23, p. 89), after a survey of the literature, apparently
favors the view that the former is distinct from the damping-off
fungus. De Bary (5) reported Vaucheria and Spirogyra apparently
immune against ?. debaryanum.

Early references to Pythium debaryanum in connection with
gynosperms seem to have been based on the probability that it
would be found to be the cause of damping-off in conifers (6; 97; 134,
p- 27). The first actual finding of the fungus in any gymnosperms
of which the writer is aware is indicated by a label marked Pythium
debaryanum in the handwriting of Mrs. Flora W. Patterson on a
package of coniferous seedlings from a New York nursery collected
in 1904.4 The seedlings, judging from the several rather long cotyle-
dons and the fact that both cotyledons and primary leaves are denticu-
late, are probably of one of the species of Pinus having medium-sized
seed. In 1908 Dr. R. J. Pool, of the University of Nebraska, and his
student, Mr. H. S. Stevenson, obtained in culture from damped-off
coniferous seedlings a nonseptate fungus which was _ probably
Pythium debaryanum, but which formed no distinctive spores on the
media on which it was grown. <A year later the writer obtained the
fungus from pine seedlings at the same nursery and reported it as

*In the Office of Pathological Collections, United States Bureau of Plant Industry.

DAMPING-OFF IN FOREST NURSERIES. 37

parasitic on pines in preliminary inoculation experiments (62). In
1910 Spaulding (137) found it on spruce in New York, and Hof-
mann (76) later made successful inoculations on both pine and spruce
seedlings, using ?. debaryanum cultures both from aerial trap plates
and from recently damped-off seedlings of cabbage, radish, and
Russian thistle (Salsola tragus). Hofmann’s work, detailed notes of
which the writer has been permitted to examine, was done with cul-
tures which were contaminated by molds, but was checked up by
microscopic examination of the lesions resulting, which showed the
affected tissues filled with nonseptate hyphe. His results are taken as
a rather strong indication that P. debaryanum attacks spruce as well
as pine and that the fungus attacking conifers is physiologically as
well as morphologically identical with that causing the damping-off
of angiosperms.

There thus appears from the literature to be reason to believe that
Pythium debaryanum is parasitic on representatives of two groups
of the Pteridophyta and on gymnosperms, as well as on various
monocotyledons and dicotyledons, a range of hosts not only remark-
able but perhaps unequaled in our present knowledge of plant
parasites. Final published proof of parasitism seems to be available
for three or four species of dicotyledons only. Additional imocula-
tions on conifers with strains isolated from various other hosts are
reported in the present bulletin. Some of the detailed evidence neces-
sary for complete proof of the parasitism of the Pythium on conifers,
lacking in experiments previously reported because of the doubtful
purity of the cultures used and failure to reisolate and reinoculate
with the organism, is also given here, together with evidence of the
ability of the parasite to cause root sickness of pines too old to suffer
from damping-off.

Descriptive data of interest on Pythium debaryanuwm have been
supplied by Hesse (74),.De Bary (5), Ward (144), Miyake (89),
Butler (23), and Butler and Kulkarni (24). An important contri-
bution to the physiology of the fungus and the factors controlling its
passage through the tissues of one of its hosts has recently been made
in the previously mentioned paper of Hawkins and Harvey (71).

IDENTITY AND ISOLATION,

The fungus in the writer’s cultures referred to Pythium debary-
anum Hesse has been so called for the following reasons :

(1) The morphological characters agree with those described and figured for
gas debaryanum by other workers and with those of strains obtained from
. H. A. Edson under this name.
ati The absence of zoospores in the writer’s cultures agrees with the experi-
ence of others with Pythium debaryanum (2, 5, 28, 24, 38, 100), all workers
with pure cultures haying obtained zoospores infrequently, if at all. The
earliest work by Hesse (74) in which zoospores were apparently produced

38 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

readily at certain times of the year was done before the development of pure-
culture methods. Water cultures kept in the dark and in the light, at constant
and at varying temperatures, with nutrient substrata consisting of steamed
or outoclaved fragments of potato, carrot, sweet potato, turnip, sugar beet, corn
meal or rice, nutrient agar, sugar-beet seedlings, and insects have all produced
only sexual fruiting bodies and chlamydospores (the so-called conidia ).

(8) The successful cross-inoculations, those’ which Edson (88) used on sugar-
beet strains and the writer had found parasitic on pine and had used on pine,

strains which Hawkins had found parasitic on potato tubers and Edson on’

sugar beets, confirm the work of Hofmann (77) in indicating that the Pythium
which causes the damping-off pine is a parasite on entirely unrelated species
of host plants, a commonly recognized characteristic of Pythiwm debaryanum.

The organism is easily isolated from recently damped-off conifer-
ous seedlings or from soil direct by placing the seedlings or a lump
of soil at the edge of a Petri dish of solidified prune agar and transfer-
ring to tubes mycelium from the advancing edge of the resulting
growth. It has been found in or obtained from damped-off conifers in
California, Kansas, Nebraska, Minnesota, and the District of Columbia,
as well as in cultures made by Mr. Glenn G. Hahn in Michigan. Picea
engelmanni, P. sitchensis, Tsuga mertensiana, Pinus nigra austriaca,
and Pseudotsuga taxifolia are the coniferous hosts from which cul-
tures of Pythium debaryanum have been obtained. It has been iso-
lated directly from soil not only in coniferous seed beds but from
open grassland in California not adjacent to any seed bed or culti-
vated crop. Unless Mucor is abundant, Pythium is commonly ob-
tained in apparently pure condition on the first transfer from the
plate, as prune agar appears unfavorable for most bacterial growth
while allowing rapid spread of the Pythium. On media made from
prunes which taste sweet and with a total gross weight of not more
than 40 or 50 grams per liter of medium, the Pythium will make a
rapid growth, often extending radially 1 mm. per hour at tempera-
tures in the neighborhood of 22° C. and produce both chlamydospores
and oospores. A less valuable medium for isolation work, but more
convenient for subcultures than any other which has been tested, is
autoclaved corn-meal agar. The growth is not luxuriant, but spores
are always formed and the cultures seem to be as long lived as those
on any other medium, retransfer being rarely necessary more often
than twice a year. Much stronger growth and more abundant fruit-
ing is obtained on such media as sugar-beet or rice-stem agar, but
the leathery surface of the culture on ‘such media makes transferrie
difficult. On rice grains, corn-meal mush, beef agar, and on corn-
meal agar plus 2 per cent dextose or sucrose no spores are formed
and the cultures are short lived, though growth is heavy and on
the last-named medium extremely rapid. On agar containing the
juice from sour prunes or on corn-meal agar prepared without sub-
jecting it to the high temperature of the autoclave, both growth and
fruiting have been very poor or even lacking.

DAMPING-OFF IN FOREST NURSERIES. 39

In both artificial cultures and in the tissues of coniferous and
dicotyledonous hosts the numerous strains observed showed no con-
spicuous differences in the size or other characters of the spores pro-
duced, though noticeable and constant abnormality was found in one
strain in the readiness with which spores were produced and in two
strains in the ratio between chlamydospores and oospores in agar
cultures. In the first-mentioned strain, obtained from pine in
Kansas, and in cultures reisolated from seedlings inoculated with it,
both chlamydospores and oospores are produced tardily and so
scantily that it is often difficult to find them. In most strains, on
the other hand, almost the entire contents of the mycelium are
promptly emptied into the spores as soon as the limits of rapid vege-
tative growth are reached. In another abnormal strain from pine
from the same locality, and in still another furnished by Hawkins
from a California potato, chlamydospores are produced in large
numbers, but oospores are few. In many other strains, including
several from California, the opposite condition obtains, oospores in
plate cultures being decidedly more numerous than chlamydospores.
These peculiarities of particular strains seem to be fairly constant
characters, the first abnormal strain mentioned having been under
observation for more than three years without any change in its tend-
ency to scanty fruiting, and the low ratio of oospores to chlamydo-
spores having been constant during the shorter periods over which
the observation of the other strains extended. In view of the small
variation between different strains in the matter of speed of growth,
a purely vegetative character, this variation in reproductive habit
is somewhat surprising. The strain which produced spores infre-
quently was unquestionably parasitic, though it killed fewer seed-
lings than the average Pythium debaryanum strains. The strains
with the high ratio of chlamydospores were both of at least average
virulence on pine.

Oospores in the strains the writer has had in culture, whether ex-
amined in agar, in water cultures, or in root tissues, have ordinarily
been somewhat larger than the diameter of 14 or 15 to 18 uw given in
a number of the descriptions. The maximum range has been 12.8
to 20.6 pw, the same strain sometimes being well down within the usual
size range and sometimes ranging from 17 to 20 wu. The largest
oogones observed were 26°y in diameter. Various stages of fertili-
zation are shown in Plate I, figures 2 to 4. Chlamydospores attain
a maximum diameter in the case of the hmoniform intercalary forms
of 32 », and spherical chlamydospores sometimes reach a diameter
of 28 y. There is no lower limit for these bodies, as under unfavor-
able conditions—e. g., in sour-prune agar—they are sometimes all less
than 15 yw in diameter, and the smaller ones are little larger than the

AQ BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

hyphe which bear them. Both oogones and chlamydospores may be
either terminal or intercalary.

The normal hyphe are large, varying from 3 to 7 » and sometimes
more in diameter. ‘Typical hyphe, showing the false septa developed
at the boundary of the protoplasm and the portions of the hyphee
which have been evacuated in the extension of the younger parts, are
shown in Plate I, figure 1. At points at which the ends of hyphe come
in contact with the glass of the culture dish, peculiar contact swell-
ings are produced (PI. I, figs. 5 to 7), much the shape and size of
antheridia, but not walled off from the adjacent hyphe and having
no apparent significance in the life history of the fungus. These are
not always terminal (PI. I, fig. 8). It is noteworthy that Hesse de-
scribed contact swellings at the tips of the hyphe just before pene-
trating the epidermis bf Camelina sativa.

The : asexual nonsporangial fruiting bodies of Pythium debaryanum
are referred to as chlamydospores ites than as conidia, though in
most of the previous literature the latter term has been used for
them. Hesse called the terminal bodies conidia and the intercalary,
gemme (74). It is believed that the best terminology and the one
which should be followed for all fungi, as it now is for most, is that
which limits the term conidium to a spore which is adapted primarily
for aerial distribution or which is at least readily separated as soon
as it is mature from the parent hypha from which it arises. The
most typical conidium, in fact, is a spore which is abstricted by the
parent hypha at maturity. The asexual spores of this Pythium re-
main attached to the parent hyphe indefinitely even after the hyphe
are dead and empty. It is a common thing to find numbers of these
bodies in water cultures, still attached to hyphez which are so com-
pletely empty that it is only with favorable hghting that their thin
colorless walls can be seen. So firm is the attachment that vigorous
shaking is required to release any considerable proportion of the
spores. It seems probable that in nature the spores are released
chiefly as a result of the destruction of the hyphe walls by bacteria.
While there is reason to think that Pythiwm debaryanum is some-
times disseminated by wind, it is by no means certain that it is
through the medium of these spores. It is true that these bodies have
thinner walls than are commonly found in chlamydospores of some
other fungi, but they have somewhat thickened walls as compared
with the vegetative hyphe, and they are commonly intercalary.
These facts, and the indications that they are better able to with-
stand unfavorable conditions than are the hyphae, all tend to entitle
them to rank as chlamydospores. De Bary (5) speaks of them as
“dauerconidia.” Their ability to stand drying is not entirely demon-
strated, but is indicated by the relative longevity of the fungus on
different media. On-beef agar and on rice, on which no spores are

Bul, 934, U. S. Dept. of Agriculture. PLATE |

PYTHIUM DEBARYANUM FROM ARTIFICIAL CULTURES.

Fic. 1.—Hyphe, showing old portions of hyphz and false septa separating them from the portions still
containing protoplasm. Fias.2 to 4.—Various stages of oospore formation. Fras. 5 to 8.—Hyphal
swellings at points of contact with glass. From camera-lucida drawings.

DAMPING-OFF IN FOREST NURSERIES. Al

formed, a few tests indicate that the fungus is very short lived,
sometimes dying in a month. On media on which spores are pro-
duced, transfers any time before the sixth month, and often as late
as the tenth month, start immediate growth on fresh media. This
is true even for strains which produce few or no oospores. The im-
mediate commencement of growth from cultures 3 or 4 months old
is taken as an indication that the new growth results from the
asexual spores, as oospores are commonly believed to require a rest-
ing period of five or more months before they are able to ger-
minate (5,38).
INOCULATION ON STERILIZED SOIL.

Pythium debaryanum has been used in inoculation in pots of
recently autoclaved soil in 16 different series of tests. In 10 of these,
fragments of agar cultures were scattered over about one-fourth
of the area at the side of each pot when seed was sown; in 2 of these
10 and also in 2 other tests some pots were inoculated over their
entire surface. In every one of these 12 heavily inoculated series
positive results were indicated by smaller emergence and where any
considerable number of sprouting seeds escaped the fungus by heavier
damping-off loss in the inoculated pots than in the controls. In
some cases the fungus killed all or practically all of the seed or
seedlings in the inoculated pots before they emerged from the soil.

In a total of 7 series, part or all of the pots received lighter in-
oculations, consisting of one or two small fragments of an agar
culture placed just below the surface of the soil at the edge of each
pot. In 5 of these success was indicated. In the sixth and seventh
also of these lightly inoculated sets, there was more damping-off in
the inoculated pots than in the controls, but the difference was neg-
ligible. The damping-off caused by light inoculations was in general
distinctly less than that resulting from broadcast inoculations. To
sum up the evidence: Sixteen separate experiments were conducted
with Pythium debaryanum on pine seedlings in autoclaved soil, and
in every one fewer seedlings survived in the inoculated pots than in
the checks; the difference in most of the experiments was large.

Of the successful inoculation experiments—that is, those in which
the difference between the inoculated pots and the checks seemed
significant—9 series included jack pine (Pinus banksiana), 7 series
western yellow pine (?. ponderosa, Colorado and New Mexico seed),
and 3 series red pine (P. rvesinosa). In addition to the pines, Doug-
las fir (Pseudotsuga taxifolia, Colorado seed) was grown in two large
plats in one of the earlier series, one being inoculated over its entire
surface with Pythium debaryanum. Because of the poor quality
of the seed in the test on Douglas fir, too few seedlings were obtained
to furnish a decisive test, but the difference in the emergence in the
inoculated plat and the control affords preliminary evidence that

42 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

Pythium can cause the “ germination-loss” type of damping-off in
Douglas fir as well as in species of Pinus. Of the seeds sown in the
control plat 48 produced seedlings which appeared above the soil,
while only two seedlings appeared from an equal number of seeds
sown in the inoculated plat.

Altogether, 38 strains, excluding reisolations, have been tested on
one or more of the 3 pine species. Strains from both the Pacific
coast and the eastern United States and from a number of hosts
other than pines were among those used. With the exception of two
or three strains from a pine nursery in Michigan, the use of which
was. followed by so little damping-off as to leave their parasitism
uncertain, all of the strains proved parasitic under favorable condi-
tions, though some were more virulent than others. The positive re-
sults in the 14 successful experiments are based on the comparison
of a total of approximately 1,160 inoculated pots with 195 control
pots.

Tasie IIT.—Inoculation experiments with Pythium debaryanum in pots of steri-
lized soil.

Pythium strain. Results.
: | Num- Damp-
Series, experiment number, ber | Inoculation tap on Sur-
and host. Tnitial strain of method. Emerged| after | vival
No. from which it | pots. (per 5-pot| germi- | (per 5-
wasreisolated. unit). | nation} pot
| (per | unit).
| cent).
Series A.—Initial inocula- ;
tions: |
1 1b ae ae ee ee eee 5 | Agarcultures up 100 0
| broadcast
No. 58, Pinus banksiana: .| a mage
Controls: eee s-e.n sees ee 5 |Noinoculum 74 0 74
SGOS eta eaten ee eens Sul eaee doves: 82 0 82
Cus aes ee ee Bee eee 5 | Agarcultures 41 28 30
: at single
No. 58, Pinus ponderosa... point in
each pot.
Controls. tes se sek Rt 5 | Noinoculum 55 0 55
DURE ALE | Woon k eee See 5 | Agarcultures 16 81 3
broadcast
at oneside
of pot.
at Es Shs eae 3-6 oe 5 ens GOs see 18 89 2
fo ak (0) Cy Re ee Cees 5 | Noinoculum 90 0 90
S400: Hoz|tee . -LSSe 2 the UW Sade O5:225=4 82 0 82
No. 62, Pinus banksiana.«|{ | d0..2.2). 2-22. coc cn ne oe Gries doi 82 0 82
BEC Ee Pe SBR em SeGo ane 5 | Agarcultures 14 72 4
° broadcast
at one side
of pot.
OAT Cee Plc eae cea eee Bile ea OO: ate4s 3 67 1
S48 Goon )d sans nscaismenste Dilecce dos 52 13 67 4
Controla!|ie- See eee 5 | Noinoculum 78 0 78
SERIES B.—Reisolatedstrains:
S88 565-53 No. 295 (in P. 5 (b) 14 72 4
No. 62, Pinus banksiana. . er
Controls!|oocccn-lnceeaaee (Fl |e eset el 5 A 78 0 78

2A different soil used in these pots from that used with strain 258 and the first three control units.

>All inoculations with fragments of agar cultures scattered broadcast at one side of the pot, including
about one-fourth ofits area. Nothing was added to the controls in experiment 62, but sterile agar was
added to the controls in experiments 66, 67, and 68.

DAMPING-OFF IN FOREST NURSERIES. 43

TABLE III.—/noculation experiments with Pythium debaryanum in pots of steri-
lized soil—Continued.

| Pythium strain. Results.
Damp-
Num- ;
: : : ing-off | Sur-
Series, pepe nent number, Initialstrain px jhe aR Emerged| after | vival
5 | No. from which it at * |\(per 5-pot| germi- | (per 5-
| wasreisolated. | POS unit). | nation} pot
(per | unit).
cent).
Series B.— Reisolated strains—
Continued.
OOOtes e- No. 295 (in P. 5 (a) 9 67 3
ponderosa,
expt. 58).
345...... No. 218 (in P. 5 (a) 15 33 10
banksiana, E
expt. 58).
Palace 2A No. 258 (in P. 5 (a) 36 25 27
banksiana
No. 66, Pinus banksiana. . F expt. 62, 2
| unit).
CG ae eee dote Aen. 5 (a) 59 10 54
cit Sea e No. 348 (in P. 5 (a) 25 100 0
banksiana,
expt. 62).
ABOE: S5e8 No. 347 (in P. Sup (a) 41 72 11
banksiana,
expt. 62).
Controls. |-5..-c8teonctho = 71) BARA See Bee 75 14 64
(eas. J252 No. 295 (in P. 5 (a) 7 57 3
ponderosa,
expt. 58).
Wa5be ao. No, 218 (in P. 5 (a) 24 37 15
banksiana,
( expt. 58).
BUA eo No. 258 (in P. 5 (a) 57 24 44
banksiana,
expt. 62, 2d
unit).
No. 67, Pinus banksiana.. (415...... No. 258 (in P. 5 (a) 30 37 19
banksiana,
expt. 62, Ist
unit).
\|419...... No. 348 (in P. 5 (a) 62 65 22
banksiana,
| expt. 62).
(450: aa52 3 No. 347 (in P. 5 (a) 53 27 | 39
banksiana,
expt. 62). |
@orrtrolsile seen. 93 Resta eameatrs 87 5 | 83
Doorn ae ws No. 295 (in P. 5 (a) 85 26 | 63
; ponderosa,
expt. 58).
Bane No. 218 (in P. 5 (2) 76 24 58
banksiana,
expt. 58).
HALAS ees No. 258 (in P. a (a) 98 14 84
banksiana,
expt. 62, 2d
unit).
No. 68, Pinus resinosa. -. ..|{415...... No. 258 (in P. 5 (a) 92 11 82
banksiana,
expt. 62, Ist
unit).
419 eos No. 348 (in P. 5 (a) 95 45 52
banksiana,
expt. 62).
4505 = 52 No. 347 (in P. 5 (a) 84 40 51
banksiana,
expt. 62).
(Ontrols)| sete aes f. TS. eee eee 104 0 104

@ Allinoculations with fragments of agar cultures scattered broadcast at one side of the pot, including
about one-fourth of its area. Nothing was added to the controls in experiment 62, but sterile agar was
added to the controls in experiments 66, 67, and 68.

As has been stated, securing positive results did not always mean
that the control pots remain uninfected. Even with the most care-
ful treatment and the use of boiled water throughout the experiment

44 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

it proved difficult to keep the control pots entirely free from damp-
ing-off. Cultures from seedlings which damped-off spontaneously
in control pots indicated that Pythium as well as Fusarium may be
introduced by accident, even when insects, birds, and rodents are ex-
cluded. This agrees with the evidence of Hofmann (77) that
Pythium debaryanum is sometimes disseminated by wind, despite
its apparent lack of adaptation to wind distribution. It is also in-
dicated, however, that unheated tap water increases damping-off
when used on control pots and probably carries this semiaquatic
fungus. Notwithstanding infections in the controls of a number of
the experiments, it is believed that the large number of pots whose
results have been considered in drawing conclusions, the fact that the
Pythium pots lost more heavily than the controls in every one of the
16 experiments, and the magnitude of the differences between both the
emergence and subsequent damping-off figures for the inoculated pots
and the controls in most of the experiments establish the parasitism
of the fungus in inoculation on autoclaved soil without it being neces-
sary to present all the evidence in detail. The pot series which in-
volved reisolation and reinoculation (Table IIT), together with the
results given for other purposes in Tables V and VI, seem sufficient
by themselves to establish a parasitic relationship.

REISOLATION AND REINOCULATION.

In a number of the experiments dead seedlings in the inoculated
pots were examined and typical Pythium hyphe and spores were
found. In three of the experiments in which the controls remained
entirely free from disease up to the time the experiment was closed,
reisolations: and reinoculations were made in accordance with the
usual rules of proof. The results are given in Table III.

From Table IIT it will be seen that five strains reisolated from
Pinus banksiana and one strain reisolated from P. ponderosa gave
positive results in pots of P. banksiana and P. resinosa. In addition
to the reinoculations shown in the table, the strain reisolated from
Pinus ponderosa (No. 338) was again reisolated in duplicate from
P. banksiana in experiment 62, and both these secondary reisolations
gave cultures which were parasitic on P. banksiana and P. resinosa
in subsequent inoculations.

That the organisms reisolated were actually the same as those used
in the initial inoculation is indicated not only by the absence of dis-
ease in the control pots of experiments 58 and 62, but by the distine-
tive characters of some of the strains. In general, cultures reisolated
from strongly parasitic initial strains were themselves strongly
parasitic and vice versa. This is shown by comparing the figures for
the initial and reisolated strains, as shown in Table IV. Each figure
represents the average results in 10 pots of jack pine and 5 of red

DAMPING-OFF IN FOREST NURSERIES. 45

pine in experiments 66, 67, and 68 combined. The figures are rel-
ative, the mean survival of 47 different strains used in all three ex-
periments being taken as 10. A survival figure above 10 therefore
means that the strain was less destructive than the average Pythium,
and a figure below 10 indicates more than average virulence. As
strain 218 was not used in these three experiments, strain 345 can not
be compared.

Tas_e 1V.—Comparative virulence of original cultures and reisolated strains of
Pythium debaryanum in experiments 66, 67, and G68.

a =

Rela- Rela-
Pythium strain. Description. Be | Pythium strain. Description. ae

vival. | vival.
IN@nouscisen se .aca Original culture......... UGH |WIN} 4005s oe ee Reisolated from 338. ..-- 9
HAS Seon aon Reisolated from 258... .. UZ} HW ENO34 fee ose cas Original culture......... 4
INGHAT DE. osc oseet Se ccs Oe ose Sseeee DN | Nowe 50 meee eee Reisolated from 347... .. 7
Nos2d0e-2.-2--.2|. Original culture. ....2..2 AvIMNOy S48ee creas ee Original culture......... 10
INCHGK eee Reisolated from 295. .... AN Ov4O es tee ee Reisolated from 348. .... 4
NMOe408S2-320--52 Reisolated from 338. ...- 6

These figures are not absolutely consistent, but are to be viewed
as contributing to the evidence furnished by the absence of damping-
off in the control of experiments 58 and 62 that the cultures reiso-
lated in those experiments were actually identical with the original
strains. A further proof of this identity is in the fruiting tendencies
of the strains. Both Nos. 414 and 415, the strains neolited from
original strain 258, exhibited the peculiarly sparse spore production
which has been characteristic of strain 258 for the entire period dur-
ing which it has been in culture. The other reisolated strains, taken
from pots inoculated with normally fruiting strains, all showed
normal spore production.

: PURITY OF CULTURES.

A slight deficiency in the evidence as to the parasitism of Pythium
debaryanum both in the writer’s work and apparently in all previous
investigations except those of Peters (100) and possibly Knechtel °
is the lack of single-spore cultures. The large number of strains
which have remained apparently pure through numerous subcultures
and have retained their individual characteristics as to virulence and
fruiting tendencies (one strain having been carried on artificial
media continuously for eight years without material change) give
very strong justification for believing that the cultures used were
pure. In three early inoculation tests the cultures used were after-
wards found to have been contaminated by bacteria carried by mites;
the positive results obtained in these three were the basis of the ear-

5 Knechtel’s work in Rumanian has been available to the writer only in the German
abstract, which makes an ambiguous statement on this point.

46 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

liest report of pathogenicity (62), but have not been used as evidence
in the present bulletin, though the contaminating bacteria in one of
them, when tested independently, showed no evidence of parasitism.
In all the experiments mentioned in the foregoing as giving positive
results with Pythium the cultures used were apparently pure.

Cultures from single chlamydospores should be reasonably easy
to secure, part of the chlamydospores in water cultures being separa-
ble from the mycelium by vigorous shaking, and further inoculation
tests with cultures so obtained are probably desirable. The experi-
ments so far conducted are believed to be sufficiently conclusive, how-
ever, for all practical purposes. For isolation of absolutely pure
lines of this or any other coenocytic fungus, it is evident, as pointed
out by Dr. W. H. Weston (146), that isolations should be made from
the uninucleate swarm spores. For the determination of the bare
fact of pathogenicity such a refinement would be superfluous.

CROSS-INOCULATIONS.

The physiological identity of the Pythium attacking coniferous
seedlings with the one which attacks dicotyledons is indicated by
the results of several inoculation experiments. The last two experi-
ments, one with jack pine and one with red pine for the host, are
the most comprehensive and give results sufficiently decisive so that
quotation of the corroborative evidence from earlier experiments
is considered unnecessary. The results appear in Table V. Each
unit consisted of five 3-inch pots except in the controls, in which
23 pots were used in the jack-pine experiment and 18 in that with
red pine. In the second experiment, separate records were kept of
the survival in each pot, and the probable error calculated from the
controls was less than two seedlings per pot for a single pot, less
than 0.9 for a mean of 5 pots, and less than 0.5 for the mean of the
18 control pots. While the number of controls was, of course, in-
sufficient to furnish an exact basis for such a calculation, the small
value found tends to confirm the impression gained from inspection
of the table that considerable confidence can be placed in the results.

The difference appearing in Table V between jack pine and red
pine in point of susceptibility to germination loss from Pythium
agrees with field observations in Nebraska, the red pine at the Bessey
Nursery, though on the whole more susceptible than jack pine to
damping-off losses, having given indication of more resistance to the
disease Tor the first week or two. Inoculations in other experiments
on western yellow pine indicate that the strains which attack it are
identical with those attacking jack pine and red pine.

The conclusion reached from the cross-inoculation results is that
the Pythium causing damping-off of the three species of pine men-

DAMPING-OFF IN FOREST NURSERIES. 47

tioned is identical with Pythiwm debaryanum, causing leak of potato
tubers and the damping-off of seedlings of two dicotyledonous
families.

TABLE V.—Results pf inoculations on jack pine and red pine with Pythium
debaryanum from various hosts.

Inoculation results.
On jack pine. On red pine.
Strain. Host from which isolated. a
; | Sur- " -
Emerged Damp-| vival Emerged Damp Sur
ing-off 2 ing-off | vival
(per 5-pot (per (per |(per 5-pot (per_ | (per
unit). cent) 5-pot]| unit). coat a5
eee on |pULULD) = Sea eees
| Dicotyledons: |
No. 131 4 POLAUOTOIIDOD. Seci--a2 noc acmica coe cee 45 36 30 101 23 15.6
No. 810) DO or daic.s tess adowscie ce ceseemicisee 45 34 | 30 62 41 7.4
Average potato. ............--- 45 ah} 30 82 32] 11.6
No. 294 ¢ Sugar-beet seedlings. ..........-.-.-- 50 8 | 46 79 36] 10.2
No. 295 ¢ Originally potato strain 131, but
twice inoculated on and reisolated
from sugar-beet seedlings by Ed-
SOM sce cinas oceans sowles Seas we 28 | 32 19 62 48 6.6
No. 296d Sugar-beet seedlings. ...............-- 19 | 32 13 68 58 5.8
Average, sugar beet..........- 32 | 24 26 70 47 7.4
No. 529 Fenugreek seedlingsd...............- 36 31 25 102 26] 15.0
No. 530: DOS sere see oe ten ee cca eee oeeee | 60 49 3 108 22 16.8
Average, fenugreek......-.,... 48 40 28 105 24) 16.0
Conifers:
No. 258 Western yellow-pine seedlings.---...- 58 | 9 53 109 Lal eeLSe10
No. 550 Sitka spruce seedlings.............-- 15 80 3 39° 98 2
No. 555 Engelmann spruce seedlings........- 42 29 30 45 70 5.0
Average, Spruces.......-------| 29 55 17 42 84 2.6
Raa cartinale es ot ay ti) ats 87 Ble 183 104 0| 20.9

a Furnished by Mrs. C. R. Tillotson: has been used » Furnished by Dr. L. A. Hawkins: cause of leak.
successfully on sugar-beet seedlings by Dr. H. A. ¢ Furnished by Dr. H. A. Edson.
Edson. : d Viseased material furnished by Prof. W. T. Horne.

VARIATIONS IN VIRULENCE OF PYTHIUM STRAINS ON PINE.

In Pythium debaryanum strains, as in the case of Corticium
vagum, there appeared to be a considerable difference in the parasitic
activity of different strains used in the same experiment. Figures
14, 15, and 16 show graphically the results from inoculations with
different strains of P. debaryanum in all the experiments in which
it was possible to compare directly the activity of different strains.
All the inoculations involved at the time of sowing the addition to
the soil of cultures on nutrient media in recently autoclaved 3-inch
pots. In experiment 31C the inoculum fragments were scattered over
the whole pot, in 31D at only one point in each pot, and in the others
were distributed over about one-fourth of the pot’s area. As noted
elsewhere, the variations observed in the results may have been due
in part to differences in the ability of the different strains to main-

48 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

tain themselves saprophytically in the soil used rather than entirely
due to difference in virulence.

The data shown in figures 15 and 16 indicate in the first place
‘rather more accidental variations in the results with Pythium than
with Corticium (see figs. 1, 2,10, and 11). The agreement between
original and reisolated strains from the same original source is de-
cidedly less good than in the case of Corticium (see experiments 71
and 72, figs. 10 and 11). In general, there are only two strains of

HOST PINUS BANASIANA
YEAR IAB

EXPT.

gS"

o 8

LIVING SEEDLINGS
FERIOOIN CONT)

Fig. 14.—Diagram showing variations in virulence as indicated by the living seedlings
in pots of autoclaved soil inoculated with different strains of Pythium debaryanum. For
experiments Nos. 31 and 66 to 72, inclusive, the surviving seedlings at the end of two
or three weeks after germination are shown. For the other experiments damping-off
was so heayy in the inoculated pots that the survivals did not give differential results
for the different strains, and the germinations are therefore shown. The reports are
based for experiments Nos. 71 and 72 on 2 or 3 pots for each strain in each experiment,
and for the other experiments on not less than 5 pots for each strain. In experiments
Nos. 66, 67, and 68 the number of pots in each experiment for the strains whose
reisolations were also used varied from 10 to 40 for each strain, the results of the
separate 5-pot units being shown in figure 15. The strains indicated by the different
symbols are as follows: From potato: @—Strain 131, isolated in 1909, California,
Furnished by Mrs. C. R. Tillotson. From sugar beet: O=Strain 295 and its reisola-
tions from pine. No. 295 was furnished by Dr. H. A. Edson as a reisolation of strain
131, after having been passed by him through two generations of sugar-beet seedlings.
A =Strain 294, isolated in 1912. Furnished by Dr. Edson. A=Strain 296, isolated
in 1912, Wisconsin. Furnished.by Dr. Edson. X=Strain 297, originally from pine,
Nebraska, 1911. Passed through two generations of sugar-beet seedlings by Dr. Edson.
From pine seedlings: +—=Strain 255, Kansas, 1913. Chlamydospores numerous ; oospores
rare. Strain 258 and its reisolations, Kansas, 1913. A sparsely fruiting strain.

=Strain 218 and its reisolation, Kansas, 1912. ©—Strain 347 and its reisolation,

Washington, D. C., 1915. -J—Strain 348 and its reisolation, Washington, D. C.,

1915. A=Strain 349, Washington, D. C., 1915. H]—Strain 354, Minnesota, 1915.

Pythium which can be said to have definitely shown difference in
activity continuing through several years and on different species of
pine. These are strains 295 and 258. As No. 258, the weak strain,
has also been found abnormal in its fruiting tendencies, the evidence
in these graphs does not indicate a decided difference in virulence
between different typical strains of Pythiwm debaryanum. The
other strain, which seems rather uniformly weaker than No. 295, is

No. 131, which according to Dr. Edson’s records was originally the

DAMPING-OFF IN FOREST NURSERISS. 49

same strain, No. 131 having been twice used in his inoculation ex-
periments on sugar beets and strain 295 recovered from the second
experiment. The apparent difference between this original strain
and its supposed reisolation may possibly be due to the treatment
given strain 131. Before it was used in any of the experiments
shown but after it had been used by Dr. Edson, it was allowed to
get very dry and was revived with great difficulty, growth being
very slow. While it apparently recovered all of its normal growth
qualities after one or two transfers, it 1s thought that this may

possibly explain the
apparently decreased
6a ema

virulence in the later yggp

experiments. EXPT.
The failure to se-
cure as definite indi-

cations of constant OX”
virulence differences Se
as were obtained for NES
several of the Cor- N80
ticilum strains is be- =
lieved to be in part }Q20
due to a smaller S&
actual difference be- Nit i)
tween the different q

0 CEES Sa onermpeguriep tenn ty amr
pearing inthe graphs figure 14, giving the results for original and reisolated
ea coon oni ho ae) rts Latent, Taek meh Bet Des
larger accidental va- give an idea of the degree of variability in the success of
riation between re- ite harass fn testad at ee in symbols used will
sults in pots inocu-

lated with the same strain. The growth of Pythium on agar media is
much more affected by variations in the substratum than is the growth
of Corticium, and it is rather natural to expect greater variations when
the two fungi are added to autoclaved soil. In experiments 66, 67, and
68 a number of strains not used in the earlier experiments were tested,
in addition to the strains previously used. The survival results for
all the different strains, both original and reisolated, 47 in all, are
shown graphically in figure 16. The results in experiments 66 and
67, both on Pinus banksiana, are averaged and taken as the subject,
while the results with the same strains in experiment 68 are made
relative and shown by the broken line. . The correlation between the
performance of the same strains on the two species of pine is by
no means as clear in the graph as it was in the case of the Cor-
ticium strains (fig. 11). The areas bounded by the broken line and

19651°—Bull. 984—21——_4

50 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

»

the horizontal line showing the location of the mean for experiment
68 are much larger below "ihe means than above it in the left-hand
portion of the graph, while the reverse is true in the right-hand
portion. To this extent the relative activity of the strains in this
experiment agrees with the performance of the same strains in the
two jack-pine experiments, as shown by the solid line. It can not
be decided from an inspection of the graph whether there is a real
agreement, in view of the large accidental variation present. How-
ever, the correlation coefficient, 0.446-+0.079, five and one-half times
its probable error, indicates a considerable correlation, not as good
as was found for the Corticium strains, but sufficient to establish a
strong presumption that observed differences in activity of the dif-

®
is}

p
8

8

SEEDLINGS SURVIVING PER S-POT UNIT

6

SCORE ERE GEE EEE CE EL OCECR OPC EGKEE ERR ORSE CLES EE

STRAINS OF PYTH/IUM poh Bs NUM

Fig. 16.—Diagram showing the comparative virulence of 47 strains of Pythiwm debaryanum
in successive inoculation experiments on species of Pinus. The results in experiments
Nos. 66 and 67 (on Pinus banksiana) are shown by the solid line, the strains being
arranged from left to right in the order of descending virulence indicated by the number
of seedlings surviving in those experiments. The results from the use of the same
strains in experiment No. 68 (on Pinus resinosa) are shown by the broken line. Such
correlation as there is between the two curves (coefficient 0.45+0.08) goes.to indicate
a real difference in virulence between the different strains. The strains indicated by
the underscored numbers are original strains,-and those not underscored are reisolations
from the original strains in earlier inoculation experiments on pine seedlings. ~

ferent strains in these inoculation experiments were in part actually |
due to differences in the capacity of the strains.

It has been suggested in the foregoing that the difficulty in demon-
strating constancy in the difference in virulence between the various |
strains of Pythium debaryanum is due in part to the lack of such
extreme differences as were observed between the various Corticium
strains. Figure 13 shows the distribution of the different original
Pythium strains according to the virulence indicated in the three
inoculation experiments of figure 16 (application to autoclaved soil
at the time of sowing). Each value plotted is based on the average
results in 15 ge? Of the strains used, 21 were from species of pine,
1 from spruce, 2 from potato tubers, 2 fore fenugreek, 3 from sugar
beet, and 6 from soil direct. Despite the considerable paeee of 4}

DAMPING-OFF IN FOREST NURSERIES. 51

strains, they are not much more representative than the smaller num-
ber of Corticium strains experimented with. All of the strains from
soil direct and 11 of the strains from pine were taken at approxi-
mately the same time from the same nursery in Michigan by Mr.
Glenn G. Hahn; despite the fact that these were the most recently
isolated of the strains used, nearly all of them proved weak in the
inoculations. Of the 17 strains which proved the weakest (out of
35), all but 3 were from this Michigan nursery. The 18 strains from
other sources (5 from California, 2 from Minnesota, 2 from Kansas,
1 from Wisconsin, 2 from an unknown locality, and 6 from Washing-
ton, D. C., representing two coniferous and three dicotyledonous host
genera), as shown by solid circles in figure 13, for the most part were
rather closely grouped within the more virulent portion of the range.
The coefficient of variability in the survivals allowed by the 35
Pythium debaryanum strains is 39+38.6 per cent, while for the
smaller number of original strains of Corticitum vagum in experi-
ments 71 and 72 it is 63+9.7 per cent. It is evident from figure 13
that if there had not been a disproportionately larger number of
strains from the Michigan nursery the variability of the P. de-
baryanum strains would have been much less than 39 per cent.
The number of strains was, of course, altogether insufficient for either
fungus to represent adequately a population as immense as the total
number of strains of either of these omnipresent species. The above
data, however, contain the only available information of which the
writer is aware on variation in the virulence of different strains of
P. debaryanum. .

The evidence as a whole, both from the results shown in figure 13
and the experience with 6 other strains which were not used in the
experiments on which figure 13 was based, lead the writer to believe
that most strains of Pythium debaryanum taken from lesions in
plants are ordinarily likely to prove rather virulent parasites on pine
seedlings. It further appears that the variation in virulence between
the different strains of P?. debaryanwm on pine seedlings is less than
the variation in strains of Corticium vagum.

PYTHIUM INOCULATIONS ON UNHEATED SOIL.

Inoculations with Pythium debaryanum were made in western
Kansas on a fine sand containing little humus after treating the soil
with acid followed by lime. Commercial sulphuric acid was applied at
the rate of 14.8 c. c. per square foot of bed, followed two days later
by 25.5 grams of air-slaked lime raked into the soil (0.16 liter of
acid and 0.274 kg. of lime per square meter). The acid was diluted
before applying with 256 volumes of water. The seeds were sown
in drills, and inoculum was placed in the drills at the time of sowing.
Each unit involved approximately 11 linear inches of drill, and all

59 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

received equal quantities of seed. Three strongly parasitic strains
of Pythium were used, and a total of 12 units of jack pine and an
equal number of western yellow pine was inoculated with 12 inter-
spersed units of each species as controls. The mean results are as
follows:

Pinus banksiana.—Inoculated plats: Emerged, 64.24.9; died during the next
17 days. 25 per cent. Control plats: Emerged, 85.53.6; died during
the next 17 days, 13 per cent.

Pinus ponderosa.—Inoculated plats: Emerged, 34.6£1.8; died during the next
9 days, 39 per cent. Control plats: Emerged, 45.4+1.3; died during the
next 9 days, 25 per cent.

The difference in emergence apparently due to the inoculation is
for the first species three and one-half and for the second nearly five
times its probable error. While, of course, 12-unit means are in-
sufficient to allow the calculation of entirely reliable probable errors,
they give some idea of the amount of variability of the results and
the confidence which can be given them. It is impossible to give any
such expression applying directly to the damping-off percentages and
their differences, for the reason that averages for this item have
been made in the writer’s work not by averaging the percentages for
the individual units but by totaling all the seedlings and the dead
seedlings on the plats to be averaged and recalculating the percent-
age from these figures. This seems the only safe method, as other-
wise units in which germination is low by accident or by the action
of parasites will be given an influence on the resultant mean entirely
disproportionate to the number of seedlings which they contain.
Average values for damping-off obtained by this method and by the
method of averaging the percentages of the individual plats or pots
are often very different; it not uncommonly happens that the units
in which germination is lower than the average also have especially
high damping-off percentages, both phenomena being caused by an
unusual activity of parasites. In such case to average the percentages
themselves usually gives a higher damping-off figure than to total
the seedlings for the different units and redetermine the percentage,
and the latter practice is considered the better. In the present case
the differences in the damping-off percentages obtained by the two

methods are not great. The figures obtained by averaging the per-

centages of the ultimate units are as follows:
Pinus banksiana.—Inoculated, loss 30.95.0 per cent; controls, loss 13.2+2.8
per cent.
Pinus ponderosa.—tInoculated, loss 40.05.1 per cent; controls, loss 24.1-43.3
per cent.
The differences between the inoculated and control plats in damp-
ing-off percentage were for the first species a little over and for the
second a little under three times their indicated probable errors.

DAMPING-OFF IN FOREST NURSERIES. 53

The results in general make it appear that the Pythium was able to
kill some pines both before and after their appearance above the soil
surface on the soil treated with the acid and lime. The control in
this experiment did not receive the nutrient substratum added with
the Pythium inoculum, but an experiment run under the same con-
ditions at nearly the same time, in which seven strains of hyphomy-
cetes with the same substrata entirely failed to decrease survival,
indicates that the rice subtratum was not in itself the cause of the
observed result. The rather weak action of the Pythium in these
experiments stands out in sharp contrast to the results with Corticium
vagum in the same experiments, practically all emergence being pre-
vented by most of the Corticium strains used, some of which had
proved less active than Pythium in tests on autoclaved soil.

In a soil in Nebraska, somewhat similar but with more humus, 5.5
ec. c. (three-sixteenths fluid ounce) of sulphuric acid per square foot
applied in solution at the time of sowing had been found greatly to
decrease damping-off. In different parts of beds treated with acid
from 10 to 17 days earlier, 96 plats, each 3 inches square, were laid
out, and each plat was inoculated at the center. Interspersed with
these were 96 plats set apart as controls. Emergence had already
begun at the time of inoculation. Jack pine, red pine, and Corsican
pine were the hosts, and three Pythium strains of known parasitism,
growing in pieces of prune agar the size of peas, constituted the
inoculum. The damping-off after emergence was less than 1 per
cent higher for the inoculated plats than for the controls. Even such
a light inoculation would probably have given some results in auto-
claved soil, so the experiment indicates, as would be expected, that
this acid-treated soil was less favorable for Pythiwm debaryanum
than steamed soil.

On pots containing entirely untreated soil the following series of
inoculations were made at the time of sowing the seed :

Inoculation at one point in each pot:

Experiment 25. Jack and western yellow pine, 1 pot of each inoculated ;
survival 18 days after emergence slightly greater in both than in the
six controls.

Experiment 27. Jack pine, 73 pots, 27 controls; average emergence, 59
in inoculated pots and 56 in controls; damping-off, 39 per cent in
inoculated pots and 87 per cent in controls.

Experiment 29. Jack, Corsican, and western yellow pine, 112 plats in-
oculated just as emergence commenced instead of at seed sowing,
as in other cases, and 112 controls alternating with them; damping-
off was less in the inoculated plats than in the controls.

Experiment 31. Jack pine, 8 pots inoculated, 8 controls; inoculated,
emergence 33 per cent, damping-off 18 per cent, survival 198; con-
trols, emergence 88 per cent, damping-off 26 per cent, survival 196.

Experiment 58A. Jack pine, 5 pots inoculated, 5 controls; inoculated,
emergence 59 per cent, damping-off 32 per cent, survival 40; controls,
emergence 51 per cent, damping-off 12 per cent, survival 45.

54 BULLETIN 934, U. S: DEPARTMENT OF AGRICULTURE.

Inoculations at two points in each pot:

Experiment 26A. Jack pine, 8 pots inoculated, 4 controls; inoculated,
emergence 29 per cent, as compared with 89 per cent in the controls ;
subsequent damping-off the same in both.

Inoculations at four points in each pot:

Experiment 58B. Jack pine, 5 pots inoculated, 5 controls; inoculated,
emergence 51 per cent, damping-off 10 per cent, Survival 46; controls,
emergence 438 per cent, damping-off 22 per cent, survival 34.

Hxperiment 59A. Jack pine, 5 pots inoculated, 5 controls; inoculated,
emergence 55 per cent, damping-off 2 per cent, survival 54; controls,
emergence 50 per cent, damping-off 8 per cent, survival 46.

Of these experiments, No. 29 was in the original fine sandy soil
of a nursery in Nebraska in which Pythium is commonly found
native and damping-off losses are usually heavy. Experiments 58A
and 59 were conducted on soil from the same source which had, been
kept dry in the laboratory for five years; experiments 25, 27, 31,
and 58A were on greenhouse mixtures of sand and soil. In experi-
ments 31, 58A, 58B, and 59 parallel inoculations were made on auto-
claved portions of the same soil, with definitely positive results in
three of the four cases. In the heated soil the results were positive,
not only because of smaller losses in the controls but because the losses
in the inoculated pots were actually heavier in the sterilized soil than
in that untreated.

Tnoculations broadcast :

Experiment 31. Jack pine, 8 pots inoculated, 8 controls; inoculated,
emergence 31 per cent, damping-off 89 per cent, survival 129; con-
trols, emergence 388 per cent, damping-off 26 per cent, survival 196.

Experiment 59. Jack pine, 5 pots inoculated, 5 controls; inoculated,
emergence 58 per cent, damping-off 22 per cent, survival 45; controls,
emergence 44 per cent, damping-off 2 per cent, survival 48.

Even with these broadcast inoculations the results on untreated
soil were too indefinite to allow the drawing of positive conclusions.
In both experiments much heavier losses than these resulted from
inoculations on steamed soil. It is evident that experiments on steril-
ized soil do not always show what can be expected on ordinary soil.
The same thing is indicated by the results of Edgerton with tomato
wilt (36).

CONCLUSIONS AS TO THE PARASITISM OF -PYTHIUM DEBARYANUM.

Pythium debaryanum has been found in low-altitude nurseries in
all the species of conifers from which a serious effort has been made
to obtain it, and its parasitism has been indicated in autoclaved soil
on all of the conifers on which inoculation has been attempted.
Therefore, although the work reported has been limited to a relatively
small number of hosts, it seems likely that it will be found able to
cause damping-off in most of the species of the Abietoideze which
suffer seriously from the disease. Just how active as a parasite it is

DAMPING-OFF IN FOREST NURSERIES. 55

under ordinary nursery conditions is yet to be proved. The results
in inoculations on disinfected soil, together with the frequency with
which the fungus has been isolated from seedlings in the nurseries,
lead the writer to believe that it is an important cause of disease in
the seed beds. Further experiments on unheated soil, however, are
considered desirable.

RHEOSPORANGIUM APHANIDERMATUS.

; CULTURAL STRAINS.

A culture of a parasite on radishes and sugar beets, described by
Edson (39) under the above name, was obtained from him, and an-
other strain, shown by Edson’s records to be a subculture from the
same original strain, was furnished by the department of plant pathol-
ogy of the University of Wisconsin. In parallel cultures on solid
media this fungus proved in many ways remarkably like Pythiwm
debaryanum, reacting in practically the same way to the different
media on which it was grown both in relative growth rate and in
spore production. Mycelium, chlamydospores, oogones, antheridia,
and oospores are not recognizably different from those of Pythiwm
debaryanum. The oospores have seemed on the whole slightly larger
-and the mycelium a little more inclined to aerial growth than most
of the Pythium debaryanum strains, but neither difference was suffi-
cient to have diagnostic value. Swellings of the hyphe occurred at
points in contact with glass, just as with Pythiwm debaryanum (P1. I,
figs. 5 to 7).

In liquid cultures the Rheosporangium was readily distinguished
from Pythium by the formation of the presporangia described by
Edson. Autoclaved cylinders of turnip, 15 to 20 mm. long, cut with
a 5-mm. cork borer, proved convenient bases for growth of both
Rheosporangium and Pythium in water culture and quite as satis-
factory as sterilized beet seedlings. Presporangia were also pro-
duced in autoclaved soil, and in a single lot of corn-meal agar they
were formed abundantly in the agar in Petri dish cultures. In none
of the writer’s cultures, either with flies, sugar-beet seedlings, or
turnip cylinders as nutrient bases, were mature escaped sporangia
or swarm spores commonly produced.

The Rheosporangium was not obtained in any of the numerous
cultures made from coniferous seedlings or from seed-bed soil.

INOCULATION EXPERIMENTS.

The Rheosporangium cultures above referred to, strain 229 fur-
nished by Dr. Edson and strain 351 received from the University of
Wisconsin, were tested on pine and red-beet seedlings, with parallel

inoculations with Pythium debaryanum. The results appear in
Table VI.

56 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

Ls
TaBLE VI.—Results of parallel inoculation with Rheosporangium aphanidermatus
and Pythium debaryanum on pine seedlings in autoclaved soil.

—————————e

Results,
Experiment number, host, and inoculating fungus.« Phpetns Emerged |Damping-| Survival
(per cent | off (per | (per cent
ofseed). | cent). | ofseed),
No, 30, Pinus ponderosa: : Per pot. | Per pot. | Per pot.
Rheosporangium, strain 229)... -...-:-....22.-. ce sesee 5 23 3 22
DAVIN ANSEL ORNS = so eee ee een eee en eee 5 M4 38 9
Can trp ISHRaee sae ee ce cater teehee et ee ne 5 20 22 16
No. 31, Pinus banksiana:
Rheosporanpium, Strain 220... soe cee as ease ec ce ence b) 45 13 40 °
PV ihiome strainieen wu ek ee eee 10 12 44 7
CONTLOIS® sane dow ae sce he mate fae tee aenoetae cats ee ee ane 25 43 5 41
Per 5-pot Per 5-pot
No. 58, Pinus banksiana: unit, unit.
Reosporaneium’ Strain 220.225 Sa. eee ee ee aa 5 46 12 45
PAV UHI MI AS SCE AIS Sees 2 Se hse rhe ech ott ee eg a 40 2.6 50 123
ConthOlsee peepee eee se een eee a Oe rae 10 7 0 78
No. 59, Pinus banksiana:
Rheosporanpium strain 2200220 -2- 22. ken ee. ee eee ce 5 46 11 41
VRS SUPAINS 8b eae kes eras Rae et pew ce be ea Re eee 40 15 35 10
DiS TNT OH Ra Seat afog SARE MEE ah Salt cd pet AD el hPa ee 10 64 0 64
No. 61, Pinus banksiana:
RUNSOSPOrANPiumMs StraiNisol 2. ss ae ecS-s a naese ooee noses 20 16 36 10
VEDI a SCPAMNS 2. cole soca aaass abo eee ese yeas 20 2 57 1
CGT RO See ee ce Tey eee cee ne care e Beaeree a eet 20 60 2 59
No. 61, Pinus banksiana and beets in same pots:
Beets} Rheosporangium, SUSI Sole ee oer cena ee eet son 5 { os c) 88 (>) 8
No. 61, beets alone:
heosporanriumy strain ool’ ..2) ba. -aase Saeco ean ee 5 43 72, 12
No. 62A, beets: x re aa , 5
A SERBIA 2O tone ena ea ree ees 5 ¢ ( 8s
Rheosporangium{* 7a DOW espace et ae ee noe eck er Re ae 4 42 35 27
PD ULI eaS GPS atte ese nephews | eee Be ahs afar 20 12 83 2
Goniroissee ere sen oa eee ae es Bee dancin 3 gee etn Mp ee 11 86 0 86
No. 62A, beets: 6 5 Me v 4
; . SULA 220 cree oe cee aero ie eee E 4
Rheosporangium eteaiyt rt lads ere ade Cie Pe ia 5 © 27 78 c6
CONT TOIS ee tees ae ech ce tae ene ee eee ee eee 5 c77 1 c 76
No. 62A, beets and Pinus banksiana in same pots: E
Boots} Rheosporangium, Strain/220.- So cceegs c+ os oe 5 { re os “e
Beek ;Rheosporangium, Strainigole eon sass een ee Sats 5 { ae ae -
Pines ‘ $1 0 81
Boots yoontrols Stee craigs cistela betes be oases aes case eee eee 5 { 67 0 57
No. 62B, Jack pine: @ 35 ¥ = ” ©
‘ F Strain 220). co n8,: seen eec cue hace ee meet oe 2
Rheosporangium( rain Sole ene cree eet pe ueareee 2 53 10 48
Py EITM, Strain: 258 a2 ganache a. ac ese seenes ie eaa ses 2 10 100
COnmtimolseers he ee ee eet. ne plene es ee ce inem 3 82 0 82
No. 66, Jack pine: oe : : 5 “ 5 te
: SULAUM: 229 cies comic cae on tn oe sete ae a :
Rheosporangium froin OR Waa een wee bee iat Bae TY 5 80 52 39
Pythiam, 47'strains and substrains.....//522.--..-..----.-e 235 43 33 29
Controlsi2eee Se aaa goo. Re Sateen eerie et ras epee 25 75 14 64
No. 67, Jack pine: Se 8 ae - see
ALATIG strain 229........ Raiarete Saale seein. ERS
Rheosporangium{ strain 391 220.2 5 88 6 83
Pythium, 47 strains and substrains..............-. ae 235 51 26 38
Controls shee see nc Sa, ee eee ate ee ea 23 87 5 83
No. 68, Red pine: t a ie
ae ee Strain) 229" < cee n cee omeren eens Sea re 5 5
Rheosporangium) strain $51... ss lecceclseee heen tL 5 124 0 124
Pythium, 47 strains and substrains.-....- bs SARS occ Reo 235 86 7 63
Controlssze se a5 oP es ee er ce eee a ee eee 18 104 0 104

2 Location of the inoculum: In experiment 30, at one point at the edge of each pot; in experiment 31,
over the entire pot; in all other experiments, over one-quarter the area of each pot.

> The breakage of the one seedling not killed while sprouting prevented the determination of results.

c Double seed density in these pots; emergence and survival figures halved to allow direct comparison
with otherunits. This high seed density may explain in part the higher loss in strain 351 than in strain 229.

@ Experiment 62B was conducted at the same time as 62A, but with a different soil.

Table VI shows that in experiments 30 and 67 the loss was less in
the Rheosporangium pots than in the controls and that in experiment
68 the results were entirely negative, while in the remaining seven

DAMPING-OFF IN FOREST NURSERIES. 57

experiments the losses were heavier in the Rheosporangium pots.
Especially in experiments 61 and 62A the evidence indicates very
strongly that both germination loss and subsequent damping-off of
the seedlings which come up can be caused by inoculation with Rheo-
sporangium on jack pine under favorable inoculation conditions. It
is, however, obvious that in all of the experiments the parallel in-
oculations with Pythiwm debaryanum gave much more positive re-
sults. The Pythium was active under conditions in which the Rheo-
sporangium gave no evidence whatever of parasitic capacity. It
furthermore appears that the two strains of Rheosporangium, though
probably identical originally, differed in virulence at the time of
their comparison in these experiments. The greater virulence of
strain 351 was quite distinct in most of the comparative tests on beets
as well as on pines. The possibility that the original culture was
really a composite of two or more strains, of which different ones
survived in the subcultures kept at Washington and Madison, re-
spectively, seems worth considering. Such an accident might also
have been responsible for the divergence of Pythium strains 131 and
295 referred to in another section.

Further evidence of the parasitism of Rheosporangium was ob-
tained in inoculations with cultures reisolated from seedlings killed
by the original strains in experiment 62. Typical Rheosporangium,
identified by presporangium formation, was easily recovered from
the damped-off seedlings in pots of pines only, those of beets only,
and the pots in which both hosts were sown. The recovery of a
virulent Pythium strain from a single one of the pots inoculated with
the weaker Rheosporangium shows that despite the absence of dis-
ease in the controls a shght amount of contamination did occur.
However, the comparative ease with which the Rheosporangium was
isolated from seedlings in other pots inoculated with it and the fact
that it has never been obtained in the numerous cultures made from
controls and from pots inoculated with other organisms leave little
room for doubt that the strains isolated were really recoveries of the
Rheosporangium used in the original inoculations. The results of
reinoculation with these strains are shown in Table VIT.

From Table VII and by comparison with Table VI it appears—

(1) That in one experiment each on jack pine and red pine the reisolated
Rheosporangium strains gave positive results. In a second experiment on jack
pine (No. 67) the difference between the Rheosporangium pots and the con-
trols was not significant.

(2) That, as in Table VI, the Pythium strains used proved on the- whole
decidedly more parasitic than the Rheosporangium strains. In experiment 66
this is not shown by the percentage of seedlings damped-off, but is sufficiently

evident when the germination loss as well as the subsequent damping-off per-
centage is considered, the survival being here, as in most other cases in which

58 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.
‘

either of the groups of pots compared is seriously affected by parasites, the
most convenient index of the comparative activity of the fungi used. In such
a comparison as that between the Rheosporangium pots and the controls in
experiment 68 (Table VII), accidental variations in emergence, of course, over-
shadow the slight effect of the fungus, and the definitely determinable per-
centage of loss after emergence is the only value which can serve as a basis for
any definite conclusion,

Taste VII.—Results of inoculations on pine seedlings with initial and reisolated
strains of Rheosporangium aphanidermatus compared with parallel inocula-
tions with Pythium debaryaniumn,

Results
- ‘ Num-| Num-
Experiment number, host, and ate . .
inoculating fungus. Reisolation source. borat ey ot Emerged Dara Survival
pots. "|(per 5-pot ie cr |(per 5-pot
unit) cent) unit).
No. 66, Pinus banksiana:
Rheosporangium— |
LAND eae aca ce coe ener Edson from beet...-...-- 5 1 63 9 58
URAL Sole seo es tek scmste se aac Olnss ccc chesnce cmos 5 1 80 52 39
Strains 403 and 404.....-.-- Strain 229 from beet. -.-- 10 2 78 43 44
Strains 405, 406, and 407...| Strain 351 from beet... - 15 3 63 48 36
Strains 417, 430, and 433...| Strain 351 from pine..... 15 3 72 42 41
AIM OLAL OE soe acess scan shee Sensis. cece tenses <2 50 10} 70+2.7 40 4243. 8
GontroOlsieese= seers cscs cess tin |s=ecnaeee dan tececs ss.caceige hy aeeeade 75 15 64
IPVUNWIM NW AVerar Ose chen t acted Co ceecar soa eccwae need ane 235 47 43 33 29
No. 67, Pinus banksiana:
Rheosporangium—
Strain@2on eet 22 eek 5 1 107 0 107
Sursimispleas sees scene d 5 1 88 6 83
Strains 403 and 404 10 2 91 8 84
Strains 405, 406, and 407...) Strain 351 from beet... -- 15 3 92 4 88
Strains 417, 430, and 433...| Strain 351 from pine...-- 15 3 83 3 80
PN METARO MS metres ae cee so ctmte sae mee ee aaa ets ate e 50 10} 90+2.4 4 8642.9
@ontrolsteie Sete Hee e222] cee ease Sess oe sels. a5 Eee 87 5 83
IPYUHINMFaVverage:.< S.cncocea|socn ec tecccasben nesescece 235 47 51 26 38
No. 68, Pinus resinosa:
Rheosporangium—
Seren 220 ee caenieesecs « Edson from beet......-- 5 1 105 0 105
Siren sole. ee sig Se OLS. eee eo 5 1 124 0 124
Strains 403 and 404.-.....-.- Strain 229 from beet... -. 10 2 102 1 101
Strains 405, 406, and 407...) Strain 351 from beet.....| 15 3 105 5 100
Strains 417, 430, and 433...) Strain 351 from pine...--| 15 3 100 3 97
JAY BERS GSE Gn Saga) Ga NOUS EE BE Osos SAH SoBe Sac 50 10 | 105+2.3 2 102+2.5
Controls: eee semdut cle ac ca wteetee bh coos suas sk ceempeuacmesees US ies acer 104 0 104
Pythium) averace:.-. csc eesc | Sees seae cons eeaeees Orie) 235 47 86 27 63.

A frequency graph based on the survivals of the 50 individual pots
inoculated with Rheosporangium in experiment 68 yields a rather
interesting asymmetrical curve (fig. 17). The shape of the curve
is taken as indicating that in a large number of the pots the inocula-
tion produced no effect, while in the smaller number of pots in which
the inoculation apparently “took,” the loss was rather heavy. This
is a rather common phenomenon in inoculations which are only
partly successful, part of the pots being free or practically free from
loss, while others are nearly cleaned out. It will be seen again in

NN eeeeeeeeeeeeeeeeeeeeEEEEEEEeEeEeEeEeEeEeEeEeEeEeEeEeeeeEeEeEeEeE

DAMPING-OFF IN FOREST NURSERIES. 59

figure 18. This suggests, further, that part of the lack of activity
was due to the failure of the fungus to maintain itself vigorously
in the soil till the pines reached a stage of sprouting in which they
could be readily attacked. Direct inoculations after the seed starts
to sprout are therefore desirable to supplement the experiments
reported. The survivals in the controls did not show any such
asymmetrical distribution.

While the Rheosporangium has given rather definite evidence of
parasitism. on Pinus banksiana under favorable conditions, the
activity of the strains available has been much less than that of the
Pythium debaryanum strains. In view of the fact that the fungus
has not so far been isolated from pine it can be concluded to have no
general importance
in pine seed beds. Its
very rapid growth on
prune agar makes it
very easy to isolate
when present.

&
°o

LY
°

PHYTOPHTHORA SPP.

~
Ss

Phytophthora fagi
R. Hartig has been
commonly reported

FERCENTAGE OF POTS

10 13 16 19 22 25 28 3I SF
as the cause of death SEEDLINGS SURVIVING PER POT
of seedlings of va- Fic. 17.—Diagram showing the results of inoculation of
rious plants in Eu- Pinus resinosa seedlings with Rheosporangium aphanider-
a = matus, as indicated by the number of seedlings surviving
rope, including con- in inoculated pots (solid line) and control pots (broken

ifers and herbaceous line). The shape of the curve for the inoculated pots is
] t 1] taken as indicating that a large proportion of them
plants as we as were entirely unaffected by inoculation, while those which
beech iD, 8, 15. 55, ‘were at all affected suffered considerably. This is a

= F frequent result in inoculations with weak parasites added
56, a1, 59, 73, 104.) at the time of sowing the seed.

It has been grouped

with the rather indefinite Phytophthora omnivora and with P.
cactorum, the enemy of cactus, ginseng, and other plants. Wil-
son (147, p. 54) considered it distinct, but Rosenbaum (114).
in his biometric comparison of Phytophthora cactorwum and a
single strain of P. fagi, failed to find ‘significant morphological
differences. If P. fagi is even physiologically different from
the American strains of P. cactorum, its introduction into the
United States is to be guarded against. There is certainly no
fungus in the United States causing the damage to coniferous
seedlings which European reports have attributed to P. fagi there.
As P. fagi attacks roots, it presumably can be carried in soil as well
as on plant parts.

60 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

A test made on jack pine with a culture of Phytophthora cactorum,
furnished by the department of plant pathology of Cornell Univer-
sity, resulted negatively. At the time of sowing the seed three pots
were inoculated with cultures on nutrient agar inserted at several
points in each pot. After emergence additional fragments of prune-
agar cultures were placed in contact with the seedlings, and they were

POTS WW/7H
PYTHIL/T

n 40

Q

9 DO

S

4

X40 POTS WITHOUT
: PYTHIUM

9

&

30

8
O-VF- 1S-24 3O-F4 FS-SI9 60-74 75-89 90-10F-
SLLQOLINGS SURVIVING PER POT

Fig. 18.—Frequency of pots with different numbers of surviving seedlings of Pinus
banksiana, inoculation experiment No. 81. The solid lines represent pots to which
cultures of saprophytic organisms were added. The broken lines are based on pots to
which no saprophytes had been added. The solid lines are based on 78 pots in the
upper graph and 80 pots in the lower one; the broken lines on 33 pots in the upper
and 25 pots in the lower graph. Pythiwm debaryanum was added just after sowing the
seed at a single point in each pot represented by the two upper lines. Cultures of
Saprophytes were applied broadeast two days before the Pythium inoculations were made.

sprayed with a spore suspension. The pots were covered with glass
to increase atmospheric moisture, and the seedlings were occasionally
sprayed with an atomizer. The soil was an autoclaved mixture in
which simultaneous inoculations in a different room with Pythium
and Corticium proved successful. The failure of the Phytophthorz
may possibly have been due to the lower temperature at which the
pots incculated with it were kept (15° to 20° C.).

DAMPING-OFF IN FOREST NURSERIES. 61

A species of Phytophthora was isolated by Mr. R. G. Pierce from
damped-off Pinus resinosa in Minnesota and used in four inocula-
tion experiments, the results of which appear in Table VIII. In the
first of these experiments unboiled water was used on the pots, and
mice obtained access to the pots of the second test. Probably as a
result of these things infection occurred in the controls in both cases,
and the results were inconclusive; in the later experiments these
two sources of infection were eliminated, and in experiments 68 and
72B the controls were free from disease. Parasitic activity was in-
dicated rather strongly in experiments 68 and 72 (on P. resinosa and
P. banksiana) and to a certain extent in experiment 66. In experi-
ment 67 it was evident that the Phytophthora was nearly or entirely
inactive. Comparison of the results in experiments 66 and 68 with
the results from inoculations with Rheosporangium aphanidermatus
in the same experiments (Table VII) suggests that the Phytoph-
thora may be better able to attack the pine from which it was isolated
than the Rheosporangium, while the latter fungus caused consider-
ably more destruction to Pinus banksiana than the Phytophthora.
Comparison of the results in the pots inoculated with Phytophthora
and those inoculated with Pythium debaryanum in all the experi-
ments indicates that the Phytophthora strains used were less virulent
than most of the strains of P. debaryanum and very certainly less
destructive than the most dctive strains of either P. debaryanum or
Corticium vagum. ‘This species of Phytophthora has been reisolated
from damped-off Pinus ponderosa in experiment 72.

Direct inoculations of the stems of seedlings of Pinus resinosa soon
after they emerge from the soil have so far confirmed the lack of
parasitism of Phytophthora cactorum and of the cultures of Phy-
tophthora sp. grown by the writer. The identity of this species has
not yet been determined. It is able to grow only about one-fourth as
rapidly as Pythiwm debaryanum on the medium which has been
used for isolation and may therefore be more common in the seed
beds than the small number of isolations by the planted-plate method
would indicate. However, its oospores, larger and darker than those
of Pythium debaryanum (usually over 20 yin diameter), should have
been recognized in the routine microscopic examination of planted-
plate cultures had this species been frequently present, even if it
had not grown fast enough to get out ahead of the other organism
and allow isolation. It is not believed that it is common enough in
pine seed beds to be of importance, even if other strains should be
found more virulent than those which have been available.

MISCELLANEOUS PHYCOMYCETES.

A fungus, apparently referable to the somewhat indefinite Pythium
artotrogus (Mont.) De Bary, was isolated by Mr. Glenn G. Hahn
from Pinus resinosa in Michigan and from damped-off Pinus bank-

62 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

siana in pots of autoclaved soil which had received tap water at
Washington, D. C. It agreed both in the appearance and measure-
ments of its spiny oogones and smooth oospores with Pythiwm arto-
trogus (P. hydnosporus) as described and figured by Butler (23).
In addition to the spores which Butler describes, there appeared in
apparently pure prune-agar cultures of different strains bodies with
smooth walls, of camerliat irregular ovoid outline, and mostly larger
than either oospores or oogones. They are very much less abundant
than the sexual spore forms. Their greatest diameter varied from
11» to over 40 y. The germination of these bodies was not observed.
Efforts to induce the fungus to produce swarm spores by growing
them in liquid nutrient media and transferring them to pure water
were unsuccessful. This failure to produce zoospores is further in-
dication of the identity of the fungus with that described by Butler,
who says that asexual reproduction is unknown.

The strain from Michigan was a rather weak growing organism,
difficult to maintain in tube cultures without rather frequent trans-
fers. Its parasitic activity in the experiments reported in Table VIII
is nil or negligible. Because of the poor seed and small number of
seedlings involved in experiment 72B, the percentage of damping-
off there given means only a single Seolinie dead. The Washington
strains, on the other hand, though evidently not strong parasites, did
apparently cause the death of a number of seedlings. The best evi-
dence of this is in experiment 68, in which there was damping-oft
in each of the five 5-pot units containing the Washington strains and
none in any of the 18 control pots. The available strains were less
active not only than Pythiwm debaryanum, but less than the Rheo-
sporangium and Phytophthora strains used. The fungus is be-
lieved to be a potential parasite on pine seedlings, but not one of any
general importance. What is probably the same fungus had ap-
peared in the writer’s cultures from western nurseries in conjunc-
tion with P. debaryanum, but not commonly, and it had not been
isolated. While its growth rate is only about half that of P. debary-
anum on prune agar, it is nevertheless so much faster than that of
many fungi that it Spaultl have been more often obtained in culture
were it at A common in damped-off seedlings.

Another fungus, presumably an oomycete but producing only
chlamydospores in the writer’s cultures, was obtained from damped-
off olive seedlings furnished by Prof. W. T. Horne and from soil
direct, both at Berkeley, Calif. The fungus is apparently the same
as one which has been occasionally seen in cultures from pine seed-
lings in the Middle West, but had not before been isolated. The
hyphe are ordinarily nonseptate, and the growth on corn-meal agar
is superficially much like that of Pythiwm debaryanum, but with
greater tendency toward local zonation and aerial growth and less

DAMPING-OFF IN FOREST NURSERIES. 68

than half as rapid. Chlamydospores are mostly intercalary, at first
subspherical, soon becoming polygonal, and after a few days they
shrivel and exhibit thick, angular walls. In size the unshrunken
spores usually he between 8 and 12 y in diameter, but bodies as
large as 20 p occasionally occur. Antheridia have not been observed,
and the shriveled bodies are not believed to be oospores, though the
observations made have not been sufficient to exclude such a possi-
bility. No other spore form was obtained in water culture, using
various nutrient substrata. In inoculation the strain from olive (the
“undetermined Phycomycete” included in Table VIII) has given
negative or nearly negative results in three inoculation tests in which
other fungi gave positive results. In a test not included in the table,
in which Pinus ponderosa was the trial host, damping-off was slightly
higher in the inoculated pots than in the controls, but the difference
was apparently due to accidental infection with Botrytis and
Pythium debaryanum. As all the seedlings in pots inoculated with
P. debaryanum in this additional experiment were killed, the rela-
tive unimportance of this strain of the small-spored fungus was
further indicated. An additional test of both the olive strain and
the strain from soil was made by inoculating seedlings of Pinus
banksiana and P. ponderosa growing on filter paper in Petri dishes.
Some of these were kept wet with water, some with an inorganic
culture solution, and some with the inorganic solution plus peptone
and dextrose. Agar cultures were applied directly to the seedlings.
The seedlings inoculated with the small-spored fungus remained
alive as long as the control seedlings, while parallel inoculations with
Pythium debaryanum resulted in the early decay of the seedlings.

TasLe VIII.—Results of inoculations with miscellaneous oomycetes on pines in
autoclaved soil at the time of sowing.
{In all the experiments included in this table, the inoculum consisted of fragments of agar cultures

distributed with the seed at one side of each pot over about one-fourth of the pot area. The controls
received sterile agar in the same way.]

Results.
Num- es ‘h pel
Experiment number, host, and inoculating fungus. ber of
pots. |Emerged. ee Survival.
No. 66, Pinus banksiana: Per 5-pot Per 5-pot
Phytophthora sp.— unit. |Percent.| unit.
BURST oer nate eos a arr e cman criss oie osiotiaae ten emae Ne aie. sit 5 70 30 49
PPT T Nis ect, COA oat dat Je ka Sak Sab ahha aeecteeiaere gates 5 75 28 54
(SUID G TSU ENB YA | mata Se a SS AR Se ee eee me bce ek 5 94 16 79
Pythium artotrogus (?), Michigan strain. ...............-....-- 4 115 14 99
Wndetermingd Piycomycete: 922-6 -- ooce- -secccen se ceceseereee 5 83 9 76
MELT OSS sereag he SSO ey, Sapte 2 BA SE RL EC) St as habiins 25 75 14 64
No. 67, Pinus banksiana:
Phytophthora sp.— | [
ming CCS te eae = a ie aa Na ag ea A ie oa wen ah er | 5 96 | 1 95
BirAInt ye? Nace eerie se. co ee ee Bevis: | aay aes ae seeeS. 5 88 3 85°
SLRs Te Teena ts ee oe on cee aid GT EE oS cwie as aa oe bin sire pele Gace 5 99 0 99
Pythium artotrogus (?), Michigan strain. ...............------- 5 102 ny) 100
WmnderenntinsavenyeCOmMycetess. 2... os = ccna csewewensawisinwser ens 5 98 0 98
Gtr Olena soe eet eke yatsmenemecelive ys aap asiweaicite ves 23 87 5 83

64 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.
TasBLE VIII.—Results of inoculations with miscellaneous oomycetes on pines in
autoclaved soil at the time of sowing—Continued.

Results.
INGE |e ee
Exporiment number, host, and inoculating fungus. ber of | cabal
pots. Emerged. ae Survival.
No. 68, Pinus resinosa: : Per 5-pot | Per 5-pot
Phytophthora sp.— unit. |Per cent.| wnit.
SUN EEL GTI a 8, a ef lio Nest i ea pe 5 104
DUPAIN Na eater cee aeiaton sche of ators chs Aad Seta ooh ee ka 5 109 18 , 89
IGUAL ot Orme he cee ete ean eae tec Cee not ote ceva 5 98 5 93
Pythium artotrogus (?), Michigan strain................-2.26+- 5 121 0 121
Pythium artotrogus (?), Washington, D. C.—
SUVA R 2a ee ten Oe eh oa (oan at rate ha heel 5 122 1 121
SUSE NGPA PS el ie Sk ah Bi iy AR ee eh Zi alana re 5 120 9 109
SU ha hts 55 Ss Sea ee OR Se ee eee ee eee See ar eae eer Pe 5 96 5 91
SUPA Odea see a ae ahois reas cone amie cine caches ween fee 5 110 6 103
SUPAITN ROS ote les ect em aamactsloaeineniae doe soe oc emee eoeaeten 5 94 uf 93
Undetermined’ Phycomycete.-.. ie oc ee 5 84 Z 82
Goniralses sane (Eee on oldie ac Rats aioe neck ae acee caa ges beea Pee 18 104 0 104
No. 72A, Pinus resinosa: Per 3-pot Per 3-pot
Phytophthora sp.— wnit. unit.
PUUBEALENS (id Oi a, NOR ine cedars ean cere A eae oe a Se ae ra 3 20 35 13
Pythium artotrogus (?), Michigan strain. .....................- 3 62 0 62
Pythium artotrogus (?), Washington, D.C.—
SS GRETA OL op eee cae ee ee ee anc oe Mc eee aes 2 45 i 42
SSUggo0t PUES ARIES Se Seale nes 4 ent ee ene ae Ge eee eee | 3 37 0 37
OYA CUAOURSS SE LL w, SAV ARSE MEE EM ad tas Se ins banat NE Ue Bae in ame Se 3 40 25 30
@ontrolss2-p- sass Bais Site d ciate see eee ania ae & plow ane reede 16 35 5 33
No. 72B, Pinus ponderosa:
lel ayyucejovakilaleyes Ws ob RAs oe ogee Set Se eee ia eee eee 3 8 50 4
Pythium artotrogus (?), Michigan strain. ..................-..- | 3 13 8 paz
Pythium artotrogus (?), Washington, D.C.— :
BTCA eee Nee ne ee te oe Se RE on eee ees 3 11 0 il
Siren bay SVS ROME 8 SS RUE Ne ere Pa I eR ee ee Ty eae Wi 2 29 6 27
DIEERIDESGO Re Soe see te dace ear eion Rinne oe ac eeeee eens onan 2 6 0 6
Gontrolss--. - ste- oc on « Be SEES HE COC E Een TS cc On Sener 14 9 0 9

OTHER FUNGI.

Data on the possible relation between various other fungi and the
damping-off of conifers have been already summarized by Hartley,
Merrill, and Rhoads (68, p. 546-550). Pestalozzia funerea on the
basis of the experiments of Spaulding (135), Botrytis cinerea on the
basis of observation and very preliminary inoculations, and 7'richo-
derma koningi on cultural evidence only are all believed to be po-
tential causes of damping-off, though not ordinarily important. A/-
ternaria sp. is under a certain amount of suspicion on account of its
frequent association with the damping-off of conifers, but it has
never been used in experiments. Rhizopus nigricans (incorrectly re-
ported as Mucor), 7'richothecium roseum, Rosellinia sp. from nursery
soil, Chaetomium sp. from maple roots, strains of Penicillium and
Aspergillus, Phoma betae, and Phoma spp. are all reported to have
been used in inoculations with negative results. Pay

Since the publication of the above summary a preliminary success-
ful inoculation experiment with Botrytis cinerea on recently emerged
Pseudotsuga taxifolia has been found briefly mentioned in an article
by Tubeuf (140) on another disease. Further experiments with va-

DAMPING-OFF IN FOREST NURSERIES. 65

rious strains of Botrytis, both from conifers and from other hosts
(the latter supplied by the departments of plant pathology of the
California and New York (Cornell) Agricultural Experiment Sta-
tions), have already yielded confirmatory evidence of the parasitism
of B. cinerea.

While a considerable number of fungi have been considered in the
_ foregoing, it is entirely possible that there are still parasites which
have received no consideration and that some of them may perhaps
be important. The moist-chamber method of culturing parasites
for isolation yields only those which produce spores readily; the
planted-plate method is not well adapted to the isolation of slow-
growing fungi or bacteria. It is suggested that in further culture
work with damped-off conifers an attempt be made to secure slow-
growing organisms by dilution plates of teased-up fragments of
recent lesions.

RELATIVE IMPORTANCE OF THE DAMPING-OFF FUNGI ON
CONIFERS.

The relative importance of the different damping-off parasites is
something that has not been thoroughly investigated for any host.
The most information on this point is that given by Busse, Peters,
and Ulrich (22) for sugar beet. In this case they find the special-
ized Phoma betoe distinctly the most important, with Pythiwm de-
baryanum second and Aphanomyces levis third.

Peters (100) apparently considered Rhizoctonia unimportant as
a cause of beet damping-off. The opposite was indicated by a small
number of cultures by Edson (38) from beet seedlings on Kansas
and Colorado soil. These yielded more Corticium vagum than any
other parasite and no Pythium at all. Johnson (81) states that
most of the damping-off of tobacco seedlings is due to Pythiuwm
debaryanum and Corticium vagum. Atkinson (1), speaking for
cotton in Alabama, and Sherbakoff (127, p: xev; 128; 129), speak-
ing for truck crops in Florida, make Corticium vagum the impor-
tant damping-off parasite, with P. debaryanum negligible. Horne
(oral communication) found the same situation in tobacco seed
beds in Cuba. Atkinson (3), in an article on trees, held that many
of the cases of damping-off attributed to P. debaryanum are in real-
ity due ta C. vagum. Peltier (98, pp. 336-337) has reported P/i-
zoctonia solani as the cause of damping-off of a large number of
plants, recording his observation of the damping-off of seedlings of
nearly 50 species of miscellaneous genera and cuttings of 15 different
species, all of which he attributes to the Rhizoctonia. He does not
state whether in this case he used diagnostic methods likely to de-
tect Pythium debaryanum if it had been present.

19651°—Bull. 934—21

4
wv

66 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

For the conifers, no very reliable data on relative importance have
been published. Numerous European reports emphasize the damage
due to Fusarium spp., while a smaller number attribute loss to
Phytophthora fagi or to both. There seems to have been little effort
to determine the presence or absence of Corticium or Pythium, so
these reports can not be given great weight. Spaulding’s evident
belief (136) in the importance of Fusarium has more weight, as he
was on the lookout for the other fungi; the moist-chamber diagnostic
method employed in most of this work was, however, not well
adapted to the detection of either one. The same is true of the work
of Rathbun (106), in which dilution plates of seed-bed soil were
employed. Rankin (105) attributes to /usariwm spp. the greatest
importance in tree seed beds in this country, with Pythiwm debary-
anum and Rhizoctonia spp. important in certain cases. Gifford (46)
emphasizes the importance of Fusarium, while Clinton (28) appar-
ently found Rhizoctonia (Cortictum vagum) especially prevalent
in the examinations he made.

On the basis of the data presented or summarized in this bulletin,
it is believed that of the various organisms which have been con-
nected with damping-off in coniferous seed beds Pythium debary-
anum, Corticium vagum, and Fusarium spp. include all of impor-
tance. The others, either because of low indicated virulence or
infrequent occurrence, and in most cases both, do not seem to merit
extensive consideration.

In order to form an idea of the relative frequency of the parasites
named above as important, there have been brought together in -
Table IX the results of the examination of 438 damping-off foci in
untreated beds and 304 foci which have appeared in beds which had
received various disinfectant treatments. The data are presented
by foci rather than by individual seedlings, as was done in the census
reported by Busse and his coworkers. Most of the diagnoses were
made by planting recently diseased seedlings in plates of solidified
prune agar, all the seedlings taken from the same focus, or “ patch,”
of damped-off seedlings being put into the same Petri dish. The
resulting growth was in some cases transferred to a tube for later
examination, but was usually examined directly in the plate. In a
smaller number of foci the seedlings were macerated and examined
directly without recourse to culture methods. As Pythium debary-
anum does not commonly fruit in diseased seedlings of pine or of
tobacco (81) and its hyphe are both difficult to find and not in
themselves considered a sufficient diagnostic character, this latter
method of examination is not so satisfactory for the determination of
Pythium as it is for Corticium, which is easily recognized by its

DAMPING-OFF IN FOREST NURSERIES. 67

thick-walled truncate-tipped hyphe and characteristic branching. A
further difficulty in the direct-examination method, unless the seed-
lings are sectioned, is in distinguishing between Corticium hyphe
which are in the tissues and those outside. The well-known habit
of the Corticium of sending hyphe superficially over the surface of
plants which it is not appreciably injuring makes it evident that only
hyphe actually found in the tissues have diagnostic value. Direct
microscopic examination is, furthermore, very likely to fail to detect
Fusarium. The planted-plate method therefore appears the better of
the two, and the results of the culture diagnoses appearing in the
lowest two lines of Table IX deserve probably more attention than
the total occurring a few lines above, in which the results of direct
examination of the seedlings are also included. The high proportion
of Corticium reported from the Michigan and Minnesota nurseries
is probably due in part to the fact that most of the examinations
made there were of the direct microscopic type.

TABLE IX.—Results of the examination of damping-off foci in coniferous seed
beds for Pythium debaryanum, Corticium vagum, and Fusarium spp.

|
= Beds ofheated Beds treated with
Untreated beds. soil. strong acids.
|
Niuuibet Number Number
: showing— - | showing— - | showing—
Grouping. : 3 se] ore z Howang
AS ‘a5! A
BAU Sei Sel. Tree Secteee eialereitenet 1s iinet
S/E |S /Eleisiels| aisles
o bas | o Lod ) om Oo i] oO wo. ~
“ef ee | ered ee heme) Diced Slee
iS) Balice 3 olBBIS | 6 |B) es
cS mB iS) & BR 1} |/O|m& | & aA O | &
By locality:
IBERKOIE Ye CAMion sess sccioao--s---5-55= <= 4 3 1 PAN oeece| acc aaa soc 5| 5| 0 2
MAM TOM COLON Sees: os 2h l Scenes s—5- sce sb 22; 0}. 19 (A Seu ec eee sume leecoc beac REE acs
MOMINNENTMCOLO sen as 2252565 20-2 225e24sce5 3 Ls e000 i 5 ee ee eos eed scoed pecs seosibere
Garden City, Kans.—
Garden City, Nurseries.-...-.-..22----.22 18 4 0 OU Pa SO) | i) | 28)). 852 9
as Nurseries (sand)-.-........-.----. 20 4 9 i LD) ee ae ONE Gr el Ones 9) ae? 8
LB EDISTO) sta pang a 224 | 124} 45) 155] 28/10] 0} 17) 99] 34) 2) 61
OsSS US vee cI Wi gh A 15: | 22 Dil seas pee /eeee| see dt FOnteAO! 0
a Circ.) Le ee ae i ie eae a 7a) PTE Pn) nC In AS | Sr a a a sees tees
East Tawas, Mich.—
* Beal Nurseries (sand)......-:.-.-.---.--- 45| 14| 33 2 (enone ee fe ee HE i 0
Basti lawasi Nurseries... ..5-22---5.--<26- ie) Sot 7 1 ee eee oe | eee 13} 7) 6 2
Washington ereenhouse.....----.----------- 12 3 3 9 Ualiy CN UN ES ee ee Boe Seco
Total: :
INAIDRT see Sie eee do cece ous eons 438 | 184 | 162 | 204 | 64/19; O| 33] 163] 64; 12] 82
REECE TAeCraae oceise ms cee 2 Reet ace 100} 42) 37] 47) 100} 30} 0} 52] 100) 39} 5] 50
By diagnostic methods:
Direct examination— F
LN VET 1 Gel ee Sy lS a oe 156 |}. 39 | 108} 25 Wi eae! eee sae 16| 6] 8 4
BOICON par Gra tet foe te one a ruses see LOD T9257 69! | eetG, eaten eee oleae 100 | 38] 50] 25
Planted-plate cultures—
METH DORs ac. shes CMe eee sete 282 | 145}; 54)179| 64/19] 0} 33] 147] 58) 4

78
IR OECOLLG ARO sar serie aiaa desee aia pieces Bae 100! 517 19{ 631100] 30} 0, 5211001 39| 3] 53

68 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

TABLE IX.—Results of the examination of damping-off foci in coniferous seed
beds for Pythium debaryanum, Corticium vagum, and Fusarium spp.—Con.

Beds treated with| Beds treated with |}Beds treated with

formaldehyde. | copper sulphate. zine chlorid. All treated beds.
Number Number Number Number

, : | showing— . | showing— ; | showing— : showing—
Grouping. Z Z ‘ 3
iS a. g

Eleldleldlelals|dlelealalaleldla

P/GTe Ele ISIS e| eSB) gaat

o bd as | oO mH oa a! oO we o —“ oO i] oO = oO a

~ |/SIBl Sila |/Sl/SB]_ slau lSl/s]/si[s1]/ 8 l1sis

o = mr Q o = me Q o Bc a 2) [S) 5 ed a n

°o ml oO =) °o m1 oO =) fo) inl a) = fo) ca) >) =]

- alos ey A lO] Ba A }O|e& PY Oe

By locality:

CGO yy Calif ment- eel (ten Baa boos |oedel Beare laans BE calsodb| |ncses| ass cdas scot! cae HX) 2
Manito Golot se eee. slp aceceeee Pes heer piace BS BES FSA roca roe Meee Shea lSoane ciccelc< sills on
Monument; Colosssssces--|-oec= COE Rebs eee Sere <vec|Saeclectea|Seon| ooanlooce | teacleeece |p eeer Sees eee
Garden City, Kans.—

Garden City Nurseries.--| 6] 0] 6| 3 La 10.) Oa BD ON 35] 5255 Ta sales

Kansas Nurseries (sand)|..... Bee eas es eres Sane ae Bane pees Foes |e seeloee-|| Sola Ls eee
FAISGy NODE acne esses Sa HEZOU GL 1) 24.) Gaesals O|) 8) 0} Ol). 5.) 275s) S67 esa eae
Cassiitalke: Minin 4 5322222) eens JeBetl toe s|ceca| Seeet SSaelsece|-saelsccee BOA) beGellocee| meth 0; 0 0
Pm dec sn gh... slecuee ps Sd Ka Pac HE | Ge [Ga Pas oebelSie|2osa[se5.¢]-.ce eae
East Tawas, Mich.—

Beal Nurseries (sand). -.|-.--- Bees Seng eee) Basa oe ole | eters see = See ass) Meas [ol 11230 0

East Tawas Nurseries.--| 8] 5| 4] 0 Gil Salona 5/53) | 10)" 2) | 325) 20n as i
Washington greenhouse ...!.__-- Hes -5el Saat|soctlecc SA one (noe octet seer |----]----|---2! Ca Wee er) 3

Total: Bad

INtMDer Ss Secs ssee 48 | 25 11 | 27 13 8 3 9 16] 4 0 | 10 | 304 | 120} 26 | 161
Percentage. -.<.---<2-< 100 | 52 | 23 |.56 | 100 | 62 | 23 | 69 | 100 | 25} O|} 63) 100] 39 9 53
By diagnostic methods:

irect examination—

Numbers... sces8s 2 8: ebal\ "74 0 5| 4 2| 2 Die tal ON eee 34 18 | 14 8

Percentage.<..-. 2. ste< 100 | 638 | 50 0 | 100} 80 | 40 | 40 | 100 | 60 0 | 40 | 100 53 | 41 24
Planted-plate cultures—

Numberste.. fess. occa 40 | 20 LON PA 8 4 i 7 ll 0 8 | 270.) 102 | 12 } 153

Percentages: -ndems o=.2 100 | 50 | 18 | 68 | 100 | 50} 13 | 88} 100 9 0| 73] 100) 38 4 57

The data on the different nurseries do not allow any generalizing
on the basis of locality except to say that all of the fungi seem quite
generally distributed in the Lake States and Great Plains region.
In general, it appears that the Fusaria as a group are more common
than either of the other fungi; as they grow more slowly than either
the Pythium or the Corticium, they were probably rather more
common relatively than even the plate-culture method indicated.
It also appears that the Pythium occurred in more foci than the
Corticium in the beds examined. Further culture work, perhaps
by the method of dilution plates of fragments of lesions, seems de-
sirable, especially in the East and the Northwest, regions in which
there are large coniferous nurseries and in which nothing like a
parasite census has been attempted. Observations on the type of
focus occurring in most of the nurseries in the Rocky Mountains
leads the writer to believe that Corticium will be found especially
important there.

While the data on the fungi in foci in disinfected beds are insuffi-
cient to serve as a basis for much in the way of conclusions for any
individual treatment, they in general agree with the assumption,
which knowledge of the fungi would favor, that Corticium is the

DAMPING-OFF IN FOREST NURSERIES, 69

most easily controlled by soil disinfection (see the bottom line in
the last four columns of Table IX). Its poor adaptation for aerial
dissemination would lead one to expect to find it seldom in beds
treated with efficient disinfectants. The entire absence of Corticium
in heated soil therefore seems somewhat significant. The rather
high Corticium yield in the formaldehyde plats is of some interest
in view of the reported inefficiency of formaldehyde in destroying
Corticium vagum on potato tubers (48, 50). As will be noted from
the data given, more than one suspected parasite was often found in
what appeared to be a single focus. This was probably in some
cases due to independent foci being nearly concentric; it also in
some cases undoubtedly means that one of the organisms found was
only secondary. In the beet-seedling cultures by Busse and _ his
associates, individual seedlings yielded two or more potential para-
sites in 100 of their nearly 1,300 examinations. It not infrequently
happened in the work on pine seedlings that no fungus recognized
as a likely parasite could be isolated. This was especially true
in plate cultures when Rhizopus or Trichoderma happened to be
abundant, as both are very fast growing and often suppress para-
sites. This is an additional reason for the development of some
method as a dilution plate of lesion fragments for diagnosing damp-
ing-off.

Even an accurate and complete census of the organisms present in
the different foci could not be directly interpreted in terms of rela-
tive importance. None of the parasites so far used in inoculation
have been vigorously parasitic under all conditions. Of both Corti-
cium vagum and Pythium debaryanum some strains, microscopically
indistinguishable from the others, are very weak as parasites. Only
part of the Fusarium species are parasitic on pine, and data showing
which are and which are not parasitic are known for only a very
few. There is therefore no fungus which can be said positively to
be the cause of any particular damping-off “ patch” simply because
it was found in some of the dead seedlings in the patch. In an occa-
sional exceptional case, such as the large Corticium patch in figures
7 and 8, there is such a vigorous growth of the fungus that its pre-
dominance is undoubted, but such cases are rather rare. A census
throws light on the importance of the different fungi, but can be
interpreted only in the light of inoculation results.

For Pythium and Corticium the inoculation data do not permit
any simple comparison between the two, for the reason that neither
is uniform. Each has strains of high’ virulence and strains having
practically no effect on pines. In the inoculations in autoclaved soil at
sowing time the strongest strains of Corticium vagum have on the
whole caused more damage than any of the Pythium strains, but, en
the other hand, there has seemed to be a higher proportion of very

70 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

h

weak strains of C. vagum than in the case of Pythium. In inocula-
tions on Pinus banksiana and P. ponderosa in Kansas sand treated
with acid followed by lime, the average Corticium was very much
more destructive than even the strongest Pythium strains, allowing
practically no germination in most cases. On the other hand, in ex-
periments in which the inoculum was applied directly to Pinus
resinosa and P. ponderosa seedlings, either immediately after germ-
ination or after the older parts had become resistant, the Pythium
has been the more effective. The inoculation evidence so far avail-
able justifies so nearly equal emphasis on the two that it can prac-
tically be eliminated from the calculations. It is the writer’s opinion
that the Corticium strains are probably rather less virulent on the
average than the Pythium strains, but perhaps better able to main-
tain themselves and spread from one seedling to another in most
soils. The evidence of Table IX that the Corticium seemed less fre-
quent in the damping-off foci is more or less counterbalanced by the
apparent larger size of many of the disease patches which it seems
to cause in the seed beds. Nearly all the large clean areas such as
are shown in figures 7 and 8 have been found to contain abundant
Corticium hyphex. The evidence on the whole seems to indicate a
very nearly equal importance for the two fungi. The Pythium is
probably somewhat the more important for the stations at which
most of the cultures in Table IX were made, but the Corticium has
received more emphasis from other observers in this country and is
indicated by the writer’s observations to be more important in the
western mountains than any other damping-off fungus.

The inoculation evidence for /usarium spp., though less complete
than for Corticium and Pythium, is nevertheless rather helpful in
indicating their importance rating. None of those so far tested in
inoculations at sowing have shown the destructiveness of the aver-
age strains of Pythium or of the stronger strains of Corticium; while
this is only in part a test of virulence and in part a test of the
ability of the fungus to grow saprophytically in the soils used, the
indication is that no one Fusarium species is the equal in destructive
capacity of either Corticium vagum or Pythium debaryanum. How-
ever, when all of the Fusarium species which occur in the seed beds
are considered, the group as a whole may prove quite as important
or even more important than either of the other two fungi. The data
already at hand rather definitely indicate considerable importance for
all three.

DAMPING-OFF FUNGI AS CAUSES OF ROOT-ROT AND LATE
DAMPING-OFF.

As has been already stated, root-rot, often with frequent recovery,
has been commonly observed in seedlings several weeks old. It has
been especially common in the vicinity of old damping-off foci in

DAMPING-OFF IN FOREST NURSERIES. ad

which Corticitum vagum appeared to be the active parasite, but
beyond this indication of the causal relation of C. vagum it was not
known which of the damping-off fungi were able to attack the roots
of seedlings too old to be killed by damping-off. To throw light on
this point, seedlings of Pinus ponderosa and P. resinosa grown in
autoclaved soil in the greenhouse and approximately 14 months old
were inoculated with different fungi. There had been a certain
degree of early damping-off in these pots, but it had apparently
ceased before the inoculations were made. The inoculum used con-
sisted of cultures on rice introduced through the drainage holes at
the bottoms of the pots. The strains of Pythiwm debaryanum and
Corticium vagum used were the ones which had given maximum
results in earlier inoculation experiments at the time of sowing. The
strain of Fusarium ventricosum was the only one available, and the
Fusarium moniliforme strains were all of approximately equal viru-
lence, the three used having given as much evidence of parasitism
as any of the strains of this species in the earlier damping-off experi-
ments. Three pots of each pine were inoculated with each strain.
Two 3-pot units of each pine were set aside as controls and inoculated
with sterile rice. In addition, three pots of each pine were kept in
the same bench without the addition of any inoculum, for comparison
with the controls with rice. The results of this experiment, taken
a month after the inoculations were made, with the seedlings averag-
ing 24 months old, appear in Table X. The roots of the living seed-
lings were washed out carefully with water to permit examination.
The results in so far as they indicate root-rot of the oldest seedlings
are best shown by the figures in columns 4 and 5. These seedlings
were so far advanced that the fungi had not been able to kill them,
and nearly all would probably have recovered if they had not been
dug up. It will be noted from column 4 of Table X that a consider-
able portion of the Pinus ponderosa seedlings with root-rot had al-
ready made their recovery apparent by pushing out adventitious
roots above the decayed portion at the time they were examined.
For Fusarium ventricosum there was only the merest indication of
ability to attack pine roots at this stage. For 2’. moniliforme the
evidence is somewhat better, more pots being included and the dif-
ference in healthy-topped seedlings with injured roots between the
inoculated pots and the controls being approximately twice its indi-
cated probable error for each species. The percentage of root-injured
seedlings in the Pythium debaryanum pots exceeded that in the con-
trols in each species by between three and four times the probable
error of the difference, while the difference in percentage between
the Corticium vagum pots and the controls is approximately four
times its probable error in the case of Pinus ponderosa and five and
one-half times its probable error in the Pinus resinosa pots. The

72 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

weak point in the results is, of course, the insufficiency of the 6-pot
and 9-pot groups as bases for probable-error determination. The
indicated relative ability of these different fungi to cause root-rot
is about the same as their relative ability to cause the damping-off of
sprouting seed and young seedlings, as indicated by the results of the
earlier experiments in which inoculations were made at the time of
sowing. The fact that only the very strongest available strains were
used and that the pots were rather heavily inoculated is to be kept
in mind in considering these results. As in the seedlings examined
in the nursery beds, when a root system was partly rotted it was
only the younger portions of the roots that were affected. The evi-
dence obtained from this experiment needs to be amplified by experi-
ments with other coniferous hosts, other strains of the fungi, and
under other conditions. The experiment just described furnishes
the only evidence available on the relation of the important fungi
Pythium debaryanum and Corticitum vagum to the root-rot of conifers
and is therefore presented as a preliminary contribution.

TaBLe X.—Results of root inoculations of older pine scedlings with damping-off
fungi.

Seedlings which developed root-rot

Number of— (per cent).

Tops still healthy.
Host and inoculating fungus. co
Pots.| Seed- | Rootrecovery. | 4 verage | Killed.| Total.

lings. Biine
- dividual
pots.
Started. Started: Acct
1 2 3 4 5 6 7 8
Pinus ponderosa:
Pythium debaryanum, strains 295, 550, 9 71 27 25 | 5344.5 4 56
and 810.¢
Corticium vagum, strains 147, 213, and 9 56 16 34] 5143.5 4 54
47.a
Fusarium moniliforme, strains 249, 251, 9 64 19 27 | 4246.2 0 45
and 260.0
PVISATUMLM VENEDICOSUM soe cce oem cree eee 3 18 17 39} 50 0 56
Controls Seese--2 5-8 oe Ae oul See Saeiastes 6 41 2 15 | 2246.5 0 17
Controls*withoutnice <2. 5-c.cs-seeessse- 3 18 0 7ia|pezs: 0 17
Pinus resinosa:
Pythiumideparyanui. 3. -.- cece issc-= 9 140 3 16] 1844.0 12 31
Corticitim wapimicse: o-ceses-seereree ee. - 9 146 3 16] 2142.4 13 33
Fusarium moniliforme................... 8 128 0 11 1243.7 2 13
Fusarium ventricosum..................- 3 39 0 5 4 0 5
Controlst 2025 cor cones eee 6 115 0 33 442.0 6 10
Controls withoutrice... 2.22. 2222-.----.- 3 51 0 0 0 2 2

2 Forrelative virulence of these strains on younger seedlings as compared with other strains of the same
species, note their position in figures 11 and 14.
; ri aor performance of these strains in inoculations at time of sowing, see an earlier publication (68,
able 2).
The figures in column 7 give information as to the percentage of
late damping-off resulting from the inoculations. A certain per-

centage of the early type of damping-off appeared in some of the |

DAMPING-OFF IN FOREST NURSERIES, 73

pots, as there were still present a number of soft-stemmed seedlings
from seeds which were slow in germinating. These younger seed-
lings were excluded in counting the dead, the rule being to include
only plants which had developed a sufficiently rigid stem to remain
upright after death. Comparison of the percentage of killed with
the total percentage attacked for the two pines is rather interesting.
As has already been pointed out, while Pinus resinosa suffers very
heavy damping-off losses at a number of nurseries it seems to be
less susceptible than some other species to parasitic injury during
the sprouting period, before the seedlings appear above the soil sur-
face. Observation of beds of this species during different seasons
has indicated that it has not a greater susceptibility, but rather the
fact that its susceptibility lasts longer, which causes it to suffer as
seriously as it does at certain nurseries. It is indicated in Table X
that the succulent root tips of Pinus ponderosa are just as easily
attacked by damping-off parasites as those of P. resinosa—in fact,
considerably more easily attacked, as indicated by the figures in col-
umn 8. With the P. ponderosa seedlings, however, the older parts
of the roots had become resistant at this age in nearly all cases, while
of the affected P. resinosa seedlings more than one-third were still
unable to limit the lesions, and death resulted.

In general, this experiment indicates that Corticium vagum and
Pythium debaryanum are able to cause the death of some pine seed-
lings which have developed rigid stems and that both are also able,
as has been found by other workers in the case of sugar beets, to
cause “ root sickness,” the rot of the younger portions of the root
systems, in seedlings which have developed too much resistance to
be killed. The evidence for the parasitism of the two Fusarium
species on these older root systems is not so good; as in the experi-
ments on younger seedlings, their ability to attack the pines is prob-—
ably less than that of the other two fungi. Further inoculation ex-
periments are desirable both with these fungi and with others on the
roots of seedlings too old to succumb to the more ordinary types of
damping-off.

RELATION OF ENVIRONMENTAL FACTORS TO DAMPING-OFF.

In the earlier section dealing with disease control, mention was
made of the general belief on the part of men who have had experi-
ence with seedling diseases that damping-off is favored by thick seed-
ing, by much organic matter, especially by poorly rotted manure in
the soil, and by excessive moisture in the air and soil. It is also
commonly stated that high temperature favors the disease; on this
point there is perhaps a less general agreement. Practically all the
evidence on these points is observational.

74 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

DENSITY OF SOWING.

The relation between the disease and thick sowing was strikingly
indicated for tobacco seedlings in a single experiment by Johnson
(82). For pines the only available information is from four experi-
ments on Pinus banksiana. The results of the first two appear in
figure 19. In both experiments there is an indication of an increase
in the percentage of diseased plants as the seed density is increased.
There is, however, no such marked relation as in Johnson’s work.
As the pines were sown in drills, they were so close together even in
the less dense plats that no very great increase in the ease of spread
of the disease was to
be expected from in-
creasing the density.
Greater differences
should be expected
in broadcast beds.
That heavier losses
have been found in
drill-sown beds than
in those sewn broad-
cast (69, 139) is pre-

8

8

®
9S

t+.)
ie}

8

PERCENTAGE OF SEEOLINGS OAMPED-OF F
8 SIFTER GERMINATION.

600 4200

4800
NUMBER OF SEEDS SOMWN PEI A SQUARE FOOT OF BED

2400 3000

sumably explained
by the fact that with
equal numbers of

seed per square foot

of seed bed the seed-
_lings are much closer
together in drills
than in broadcast beds, and thus the spread of the mycelium of para-
sites from one seedling to another is facilitated.

Two tests of different seed densities were also made in 3-inch pots
of autoclaved soil in the greenhouse. Each regular pot was sown
with 28 seeds (equivalent to 600 per square foot). The pots were
inoculated by adding to each a single small fragment of an agar
culture of Pythium debaryanum. Uninoculated pots showed an emer-
gence of approximately 50 per cent of the seed and were entirely free
from subsequent damping-off in both experiments. The results ap-
pear in Table XI.

In this case not only the damping-off after emergence but the loss
before the seedlings appeared bore an apparent relation to sowing
density. In the field experiments there was no evidence that the loss
before the seedlings appeared was affected by seed density.

Fig. 19.—Diagram showing the extent of damping-off in
drill-sown Pinus banksiana in plats with different seed
densities. The regular seed density at this nursery was
600 seeds per square foot.

DAMPING-OFF IN FOREST NURSERIES. 75

Taste XI.—Results of inoculation, at the time of sowing, with Pythiwn

debaryanum on Pinus banksiana in different sowing densities in pots of
autoclaved sou.

[The percentages of ‘‘ Damping-off,”’ columns 4 and 7, are based on the number of seedlings; the percentages
y given in columns 3, 5,6, and 8 are based on the number of seeds.]
Num- Results (per cent).
ber —
of 2
T nari FQ aX ej] y (
Density of seed sowing. ner Experiment 58. Experiment 59.
ex- ee
perl- |, é Damp-| Sur- }y7..,-,,,, |Damp-| Sur-
ment. |Emerged. ing-ofl.| vival. Emerged. ing-oll.} vival.
1 2 3 4 5 6 7 8
FSR PUR oo Sos eee tect Shae reel Laks 10 15 43 10 26 13 23
1DYa(0) 2) CYQC Ss Bee ae ee ee 5 8 65 3 8 91 1
ALES VO) A pee ene Ae 5 1 100 0 11 34 7
Regular, but 10 additional seeds near -
MOM PONIMOCWALION Ss 2-1. 06 ho — ees es occ 5 6 33 4 17 37 11

MOISTURE AND TEMPERATURE FACTORS.

The relation of damping-off to moisture and temperature are sub-
jects less easily studied. In 1907 and 1908 Mr. W. H. Mast, then
supervisor of the Nebraska National Forest, conducted daily counts
of the number of seedlings damped-off and compared these records
with temperature and rainfall records. The writer in 1909 repeated
his work, maintaining parallel records of damping-off, air and soil
temperatures, soi] moisture, atmospheric humidity, wind movements,

* and evaporation. The 1909 records of damping-off, temperature,
soil moisture, and evaporation appear in figure 20. The damped-off
seedlings were counted and removed in the morning and evening, the
day period thus being in most cases 10 to 11 hours and the night
period 13 to 14 hours. Because the period was-not always the same
length, the data are reduced to a per hour basis. Air temperature
was recorded by a sheltered thermograph 3 feet above the soil sur-
face. The evaporation graph represents the mean loss per hour
from two porous cup atometers of the writer’s own design, in which
the rather long and slender Chamberlain filter bougie was used and
supported in a horizontal position just above the soil surface so as
to be under as nearly as possible the same atmospheric conditions as
the seedlings. The two bougies were placed at right angles to each
other in order to eliminate as far as possible the effect of change of
wind direction on their mean loss. While the rain-correction mount-
ing had not at that time come into use, the error due to rain absorp-
tion appeared negligible; atometers: filled shortly before rainfall
were read immediately after without any gain being found in the
water in the reservoir. The psychrograph and wind-movement rec-
ords are not presented, as the evaporation values are more easily inter-
preted. Soil moisture was periodically determined in the soil of the

JULTURE.

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76

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DAMPING-OFF IN FOREST NURSERIES. 77

plats on which the seedling counts were conducted, each determina-
tion representing two, and in some cases four, points. The deter-
minations for the upper one-fourth inch of soil, made more frequently
than for lower levels, are connected in figure 20 by a dotted line,
which gives some idea of the amount of moisture in the surface soil
during the periods between determinations. The determinations were
too infrequent to permit anything more than an estimate of the
moisture conditions between determinations, but the writer, having
before him the records of the times and amounts of rainfall and
artificial watering as well as the evaporation and soil-moisture de-
terminations, is in a better position to make such an estimate than
the reader. The dotted line which gives this estimate should not be
depended on to show what the percentage of moisture was at any one
time, but is believed reasonably reliable as showing whether in gen-
eral the soil was wet or dry. In interpreting the soil-moisture rec-
ords, it should be kept in mind that the soil was very sandy, the
wilting coefficient of composite samples from various parts of the
nursery, as determined by the indirect method of Briggs and Shantz
in the Laboratory of Biophysical Investigations of the Bureau of
Plant Industry, being only 3.4 per cent. The hygroscopic moisture
in dry air for the soil of the plats actually under consideration was
indicated by repeated determinations for the surface soil on dry days
to be in the neighborhood of or slightly below 2 per cent. The
nursery is located in a region of large temperature fluctuations, where
the air during the day is generally dry, and consequently the dew
is heavy at night.

The first result of interest is the difference between the damping-
off for the day and the night periods. In the records of every
day but two, more seedlings went down during the day period than
during the night, the differences in most cases being large. As the
evaporation and temperature showed similar day and night fluctua-
tions, it is difficult to say whether temperature or moisture condi-
tions were responsible. The other interesting result brought out by
the graphs is the sudden drop in the general level of the damping-
off graph following the rains of June 15, June 19-20, and July 3.
In each of these three cases the damping-off came up again only
after the soil moisture came down.

The fact that in the daily fluctuations the damping-off varied
directly with the evaporation rather than inversely is an apparent
contradiction of the generally accepted doctrine that moisture favors
the disease. This contradiction is, however, only apparent. Dur-
ing the first part’of the damping-off period, when the seedlings are
still soft, the recognition of damping-off depends on the decay of
that part of the stem just above the soil surface which allows the
seedling to fall over. This usually takes place at this nursery as

78 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

a result of the extension upward of lesions which have started on
the parts a little below the soil surface. It is supposed that such
decay takes place most rapidly at high temperatures and that it is
the temperature rather than the evaporation graph which the damp-
ing-off is following in these early day and night fluctuations. Dur-
ing the latter part of the damping-off period a dying seedling shows
its first signs of distress in the drying up of its leaves, the stem being
too stiff to go down until after the infection has gone far enough
in the roots to cut off most of the water supply. It is, of course,
under dry conditions that such a sign of distress will be most in
evidence. During the latter part of the damping-off period it is
therefore altogether likely that the day and night fluctuations are
caused, at least in part, by the higher evaporation rate which ob-
tains during the day. This is a relation not to the rate of progress
of the disease, but rather to the rate at which the symptoms of dis-
ease appear in plants already seriously affected.

The drop in damping-off following the increased soil moisture of
June 15, 19-20, and July 3 also apparently contradicts established
doctrine. While it is ordinarily true that a wet soil is a cold soil
and that in the rainy weather which causes wet soil the evaporation
is usually low, it does not seem possible on inspection of the graphs
for these items to attribute entirely the reduction of damping-off
during these periods of wet soil either to low temperature or to low
evaporating power of the air. Lowered soil temperature probably
had something to do with the reduced loss following the rains. It —
is also suggested that a sudden change in moisture content may tem-
porarily hinder a soil fungus by decreasing its air supply. In this
sandy soil the fungi can work at very considerable ‘depths during
dry periods. Initial lesions have been found as much as 12 inches
below the surface. If this soil is as completely changed in its aera-
tion qualities by wetting as the sandy soil with which Buckingham
(19) worked, a rain might result in a rather sudden change in the level
at which the fungus is able to operate.

On the whole, the graphs tend to confirm the common statement
that high temperature favors damping-off. It must, however, be
borne in mind that in uncontrolled field plats several factors vary
simultaneously, and it is impossible to definitely attribute any ob-
served phenomenon to any one of them. Furthermore, it is not
possible to say for the seedlings at different ages just how long it will
take a factor to exert an effect on the damping-off curve. An addi-
tional consideration is that a method of investigation which gives
entirely reliable information on the speed with which the disease
develops does not necessarily throw light on the conditions under
which the greatest total amount of disease can be expected before
the seedlings become old enough to resist attack. High temperatures,

DAMPING-OFF IN FOREST NURSERIES. 79

within reasonable limits, are expected to increase the speed with
which the disease works, but these should also hasten the develop-
ment of the host to a point at which infections are unable to cause
death. It is the total amount of damage in the beds rather than the
damage per unit of time which is of practical importance. For a
number of reasons, then, the method followed in obtaining the data
for these graphs can not give information of maximum value. While
data of the sort mentioned are of undoubted interest and would be
of still more value if the records had been commenced when the first
seedlings appeared instead of a few days later, the relation of any
specific factor to the total extent of the disease can be better deter-
mined by comparing plats in series in which the factors are as far
as possible controlled and varied one at a time. To vary soil moisture
and soil temperature independently will prove somewhat difficult,
but it can be done with the proper facilities. Some work with en-
vironmental factors should be done under conditions of artificial in-
oculation in the greenhouse, in which the different damping-off
parasites can be experimented with separately, as it is obvious that
the factors which favor the activity of one may not be favorable for

another.
CHEMICAL FACTORS.

Chemical factors are presumably also important, as the soil is
in most cases the culture medium for both the parasite and the host.
The much greater activity of Pythiwn debaryanum in autoclaved
soil than in untreated soil may be due to the larger quantity of
soluble organic matter commonly present in autoclaved soil. Pythium
debaryanum has been found more sensitive to unfavorable substrata
in artificial culture than Corticitum vaqgum and is apparently more
dependent on soil organic matter in the nurseries than is C. vagwm.
For example, in the normal humus-containing surface sand in the
beds at Cass Lake, Minn., both Pythium and Corticium occurred
frequently in the damped-off seedlings, while in beds a few feet
distant, from which enough of the surface soil had been removed to
leave no humus, nearly all the damping-off foci contained abundant
Corticium, and no Pythium could be found. With both fungi and,
in addition, with two species of Fusarium (68) heavy inoculation has
been more successful in experiments at the time of sowing than
light inoculation. This has been thought possibly due in part to the
larger amount of nutrient substratum added in the heavy inocula-
tions, allowing better saprophytic development of the fungus in the
soil. In each of the two experiments with Pythium reported in
Table XI, a 5-pot unit was treated with corn-meal infusion and
another with prune infusion at the time of inoculation. In both
experiments germination was lower, damping-off after germination
higher, and the survival less than half as great in the pots with

80 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

infusion as in the inoculated pots not so treated. In the first experi-
ment 5-pot units of unheated soil were also inoculated in the same
way. In these also both the units which received infusions showed
less germination and more loss after germination than the unit which
received no infusion, though the differences were smaller than in the
autoclaved soil. In the second experiment the ight inoculation used
failed to cause material loss in the unheated soil units, even though
two of them were treated with the infusion as in the previous test
and two others received triple portions of the infusion.

The experience in the nurseries, in which heavy applications of
manure, and especially poorly rotted manure, in a number of cases
have apparently resulted in increased disease, and the finding of
Fred (48) that green manures recently plowed under favored the
work of Corticium have already been mentioned.. The addition of
dried blood at two nurseries in Kansas was in both cases followed
by very much heavier loss than in the controlled plats. The only
instances known to the writer in which the addition of organic
matter to the soil has shown any indication of materially decreasing
damping-off (with the exception, of course, of the organic disin-
fectants) are the result reported by Gifford (46) with tankage, a
single case in the writer’s experience with bone meal, and the cases
in which cane sugar has seemed to decrease losses somewhat (67).
It is of some interest to note that the experience available also indi-
cates increased disease as a result of the addition of inorganic nitrog-
enous substances. Sodium nitrate and sodium nitrite have both
given some indication of increasing damping-off. Ammonium sul-
phate in six separate series has in every case resulted in decreased
stands, though unfortunately in experiments in which the damped-
off seedlings were not counted. Ammonium hydroxid, though ap-
parently having some initial value as a disinfectant, as indicated by
early damping-off losses, in a number. of cases has been followed by
very heavy total losses. This experience is of some interest in view
of the apparently rather general belief that plants on a soil rich
in nitrogen are especially susceptible to disease.

The chemical factor for which there is perhaps the most evidence
of a relation to damping-off of conifers is acidity. The fact that
sulphuric-acid soil treatment has been found to be one of the most
effective means of controlling the disease, that its value is mainly
lost if lime is later added to the soil, that soil treatment with sulphur
in a number of cases has seemed to decrease the disease, and that
lime alone and wood ashes have had either no effect or have appar-
ently increased the damping-off whenever they have been tried, all
suggest that soil acidity is not favorable to the disease. Additional
indication of this appears in figure 12. The acidity determinations
serving as the basis for the graph were made by Dr. L. J. Gillespie,

DAMPING-OFF IN FOREST NURSERIES. 81

of the Bureau of Plant Industry. The estimates of the relative se-
riousness of damping-off are very approximate, based in part on
observation only. The stations at which damping-off is rated as 1
are places at which it has been reported by nurserymen or foresters
as negligible or absent. The estimates for stations 10, 11, 14, and 15
are based entirely on the reports of others, and for station 5 on the
basis of counts of damped-off seedlings made by Mr. R. G. Pierce
and Mr. Glenn G. Hahn. The writer personally has made the esti-
mates or checked the estimates of the nurserymen at the other sta-
tions. A considerable degree of correlation between the hydrogen-
ion exponent and the amount of damping-off appears on the face
of the graphs, the coefficient being 0.75+-0.07. If the correlation is
calculated with the H* concentration itself instead of its negative
exponent, the coefficient, in this case itself negative, is not so high
(—0.58+0.11). AI] of the above data on acidity relation have been
picked up incidentally in connection with other work and are merely
suggestive. The suggestion, however, seems sufficiently strong to
warrant further experimental work directed specifically at the rela-
tion between soil acidity and the disease.

‘The indication in the graph that damping-off is not serious in
soils in which the hydrogen-ion exponent (P,,)° is less than 6 is of
particular interest, in view of the experience of Hawkins and Har-
vey (71) with cultures of Pythium debaryanum on potato juice.
They obtained good growth through a range of acidity from Py 3.4
to 5.8, with no growth or practically none at 3.06 or 8.4. If this
represents the acid tolerance of the fungus in the soil solution, it is
evident that ordinarily acid soils can not be expected to remain free
from damping-off because of inhibition of this particular fungus.
This suggests that the apparently salutary influence of soil acidity
in decreasing the damping-off of some of the conifers may be ex-
erted in the direction of increasing the resistance of the host rather
than of inhibiting the parasites. In any case, it must be kept in
mind that as the numerous conifer hosts commonly grown in nurseries
have many different habitat preferences and many very different
parasites of potential importance, it is not to be expected that there
will be found any such constant relation between any factor and the
amount of disease as would be expected in a disease in which only a
single parasite and a single host are involved. .

6Py 6 is equivalent to a hydrogen-ion concentration, expressed in mols per liter, of
1<10-* or 0.000001. The higher the exponent, the smaller the hydrogen-ion concentra-
tion. An exponent of 7 means approximate neutrality. In dealing with this exponen-
tial form of expression, it should be kept in mind that Pu6 means ten times and Px5
one hundred times the hydrogen-iron concentration indicated by Pxu7. Conversely, the

-

concentration of hydroxyl-ions at Pu7 is one hundred times as great as at Psd.

19651°—Bull. 984—21——-6

82 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

BIOLOGIC FACTORS.

Mention has already been made of two strictly biologic factors
which may influence the amount of damping-off. Taylor (138) and
Rathbun (106) have found Fusarium not only at considerable depths
in the soil of pine seed beds, but viable Fusariuin spores without
hyphe in the alimentary canals of earthworms and insect larve in
the soil, and they attribute to the migrations of these and to the
tunnels which various animal forms make in the soil a possible im-
portance in the distribution of damping-off Fusaria. A likely rela-
tion between Cortictum vagum epidemics in pine seed beds and the
character of the weed flora has also been considered (66).

The relation between the damping-off parasites and other micro-
organisms in the soil is also a matter of some interest. The effect of
the microfauna of the soil on the microflora in general has been
considered by Russell and others in a number of papers. The effect
of soil disinfection by heat in favoring the work of artificially intro-
duced soil-inhabiting fungous parasites, apparently a rather frequent
phenomenon and quite evident in the inoculation experiments with
Pythium debaryanum reported in the present bulletin, has been in
other cases attributed to the removal of bacteria and other fungi
which might compete with the parasites (36, 80). Heating soil is
known to produce physical changes and also very considerable chemi-
cal changes both in organic and inorganic substances. These must not
be ignored in considering the effect of previous soil heating on para-
site activity. With a view to determining whether all the difference
noted in the behavior of P. debaryanum in heated soil is due to the
direct effects of the heating or in part to the elimination of com-
peting microorganisms, an experiment was conducted in 3-inch pots
of autoclaved soil in which 111 of them were inoculated with agar
cultures of the Pythium at one point in each pot shortly after seed
sowing. The seeds sown in each pot approximated 136, considerably
more than are used on equal areas of nursery seed bed. Of these,
fifteen 5-pot units and one 3-pot unit had been inoculated broadcast
with rice or nutrient agar cultures of various organisms supposed to
be saprophytic on pines. These included Phoma betae, Phoma sp.,
Chaetomium sp. (from a maple root), Rhizopus nigricans, Tricho-
thecium roseum, Trichoderma koningi, Aspergillus spp. (including
one with black and one with bright-colored spore heads), Rosellinia
sp. (from soil), Penzcillium sp., an undetermined bacterium, and three
undetermined higher fungi. The whole 78 pots inoculated with P.
debaryanum and saprophytes, the percentages being based on the
total number of seeds in the case of emergence and survival and on
the number of seedlings which appeared above ground for damping-
off loss, as compared with those which had received the parasite only,
gave results as follows:

DAMPING-OFF IN FOREST NURSERIES. 83

Pots with saprophytes: Emergence, 47.4£0.86 per cent; damping-off, 9.1 per

cent; survival, 41.01.23 per cent.

Pots without saprophytes: Emergence, 35.7 per cent; damping-off, 14.3 per

cent; survival, 29.2 per cent.

It has been noted in the attempts to diagnose damping-off by
planted-plate cultures that when Rhizopus appears Pythium debary-
anum is not frequently obtained. It is therefore of some interest to
note that in this case, in the two 5-pot units which received Rhizopus
in addition, the parasite killed only 1.2 per cent and 3.3 per cent,
respectively, of the seedlings which appeared above the soil.

At the same time pots not inoculated with parasites were sown, 16
other 5-pot units were inoculated with the same saprophytes as those
used in the Pythium inoculated pots, while 25 pots were left entirely
without inoculation. A certain amount of damping-off occurred in
these pots also as a result of accidental infection. The results were
as follows:

Pots with saprophytes: Hmergence, 47.8+0.8 per cent; damping-off, 3.9 per

cent; survival, 43.70.95 per cent.

Pots without saprophytes: Emergence, 43.0 per cent; damping-off, 5.2 per

cent ; survival, 38.4 per cent.

It is of some interest to note that in these pots also the 5-pot units
inoculated with Rhizopus suffered less from damping-off than the
average of the saprophyte-inoculated pots.

The probable-error values given above are based on the variability
of the emergence and survival figures of the different 5-pot units.
No individual figures are available to serve as a basis for the deter-
mination of the variability of the pots without saprophytes. The
16 units which support the error determinations are, of course, not a
sufficient number to give an entirely reliable index of variability,
though these 16 units are respectively derived from the combination
of a total of 78 and 80 ultimate units. The distribution of the data
appears to be such as to justify the use of probability methods. Of
the 64 items which went into the germination and survival calcula-
tions, 34 showed a deviation equal to E, (probable error of a single
unit), 9 to a deviation equal to 2E,, and only one a deviation equal
to 3E,.

All of the above figures are based on the results at the end of 10
days after average emergence in the pots. The pots were kept on the
benches till practically all damping-off had ceased, 36 days after
emergence. As additional accidental infection with saprophytes
certainly, and probably with parasites, occurred during this period,
the results at the end of the tenth day are considered to give a better
indication of the effect of the original inoculations. It is of some
interest, however, to note that during the period from the tenth to
the thirty-sixth day the difference between the pots to which sapro-

84 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

phytes had been added and those which received no saprophytes
showed a slight increase, proportionally as well as in the absolute
figures. At the end of the 36 days the survivals on all the pots were
counted separately. The average number of seedlings per pot were
as follows:

Without Pythium :

Without saprophytes, 42.82.38; with saprophytes, 52.1+1.1

With Pythium:

Without saprophytes, 30.1-=2.4; with saprophytes, 48.11.4.

The difference in the survivals in favor of the pots with sapro-
phytes in the first case is 9.82.5, three and two-thirds times its
probable error. In the second case it is 18.0++2.8, more than six times
its probable error.

In general, it appears that in this experiment the inoculation of ster-
ilized soil with saprophytes gave the seedlings some protection both
against damping-off due to accidental infection with unidentified
parasites and from the additional loss caused by light inoculation
with Pythium debaryanum. The indication is, as would be expected,
that only part of the favoring influence of heat sterilization of soil
on introduced P. debaryanum is immediately due to the elimination
of competition with other fungi. If a mixture of different bacteria
and fungi had been added to each of the pots instead of but one or
two organisms to each 5-pot unit, the effect might have been more
marked.

Tt will be noted that for all the groups (fig. 18), whether with or
without Pythium inoculation and with or without added parasites,
the frequency polygon is asymmetrical, indicating by its shape, as
did the frequency polygon of survivals in pots inoculated with Rheo-
sporangium (fig. 17), that with infections which do not kill all of
the seedlings the selection tends to be by pots rather than by seed-
lings. In other words, in pots in which the parasites succeed in kill-
ing any of the seedlings, they usually kill a considerable number.
The tendency is illustrated not only by inspection of the graphs, but
by the variability of the different groups. The greater variability
in survivals between different pots was in both cases in the groups in
which both the damping-off after emergence and the survival per-
centages indicated the largest loss. The percentages of seedlings
damped-off during the entire 36 days following emergence and the
coefficients of variability of the survivals of the individual pots at
the end of that time are as follows:

Without Pythium:

Without saprophytes, 15.5 per cent damped-off; coefficient of variability,
39+4.2 per cent.

With saprophytes, 11.1 per cent damped-off; coefficient of variability,
28+1.6 per cent.

DAMPING-OFF IN FOREST NURSERIES. 85

With Pythium:

Without saprophytes, 27.5 per cent damped-off; coefficient of variability,
67+7.8 per cent.

With saprophytes, 16.9 per cent damped-off; coefficient of variability,
39+2.4 per cent.

This tendency has been frequently observed in experiments in
which inoculum is applied to the soil at the time of sowing. Even
in experiments in which a relatively small proportion of the seed-
lings are killed, some of the pots are nearly or entirely cleaned out.
It is taken as an indication that failure of inoculation to give results
is often due to the inability of the fungus to maintain itself in a
vigorous condition till the germinating seed is far enough along to
allow easy infection. It may also be in part due to lack of uni-
formity of the soil in different pots affecting virulence of parasites
or resistance of hosts.

In addition to this experiment on autoclaved soil, a somewhat
similar experiment was conducted in a nursery in the Kansas sand
hills on soil which had been treated with sulphuric acid, followed
by lime raked into the soil. Saprophytes, for the most part the
same strains that had been used in the experiment in the greenhouse,
were added to 24 plats, each of one-half square foot, of Pinus bank-
siana and 24 of P. ponderosa, with 16 interspersed plats of each
species serving as controls. The saprophytes were growing on rice,
part of which was added to the plat with the inoculum in addition
to the fungous mycelium. Damping-off was rather heavy in this soil
from accidental infection or from parasites which survived the
initial acid treatment, no parasites having been artificially intro-
duced. The loss was probably due to Corticium vagum or Fusarium
spp. rather than to Pythium debaryanum in this case. In both pines,
emergence was slightly better in the control plats than in those to
which the saprophytes had been added, the difference for Pinus bank-
siana being less than half its probable error and for P. ponderosa
slightly more than its probable error. Damping-off for the first few
days after emergence was somewhat less in the controls in one species,
but higher in the saprophyte-inoculated pots in the other. The
saprophytes therefore gave no evidence of effective competition with
the parasites on this acid-lime treated sand.

While the competition for water which seems to be the form of
competition most common among green vascular plants is not likely
to be of significance between fungi such as those which cause damping-
off, a very little observation of the growth of mixed cultures of the
parasites and other organisms in Petri dishes is sufficient to make
one realize that the latter may very considerably decrease the activity
of certain of the parasites. In nutrient agar most of the fungi and
bacteria introduced from the soil in attempting parasite isolations,

86 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE,

as well as to a less extent the paramecia, nematodes, and amcebe
which develop in such plates, exert a very considerable limiting in-
fluence on the growth of most of the damping-off fungi. That they
should also limit the growth of parasites in soil, whether by the
production of toxic compounds, the exhaustion of food materials, or
in other ways, seems entirely reasonable.’ The results in the writer’s
experiments on heated soil warrant the suggestion that further trials
should be made of the introduction of vigorously growing bacteria
or molds, preferably mixed cultures containing a number of different
organisms, on seed beds which have been disinfected by some such
method as steam or hot water, which leaves the soil in a favorable
condition for the development of accidentally reintroduced parasites.
Tf such treatment should be successful in improving the rather dis-
appointing results with soil heating at some nurseries, it might
easily become of practical value, as the cultivation of certain of the
more easily grown saprophytes on a scale large enough to yield con-
siderable quantities of bacterial or spore suspensions should be fairly
easy and entirely practicable in an operation as intensive as that of
raising coniferous seedlings.

ACKNOWLEDGMENTS.

The writer wishes to express his obligations to Dr. H. A. Edson,
of the Bureau of Plant Industry, United States Department of
Agriculture, and to Prof. W. T. Horne, of the University of Cali-
fornia, for suggestions during the progress of this work; to Mr. Roy
G. Pierce and Mr. Glenn G. Hahn, of the Bureau of Plant Industry,
for assistance in a number of the inoculation experiments; and to
other members of ‘the staff of the Office of Forest Pathology for
assistance and suggestions at various times.

SUMMARY.

(1) Damping-off in nurseries is caused mainly by seedling para-
sites which are not specialized as to host; Pythium debaryanum and
Corticium vagum are probably the most important of these. Damp-
ing-off of various herbaceous hosts, including ferns, is often caused
by specialized parasites which are limited to a particular host or
group of hosts. Phoma betae is a rather extreme example of such
specialization. For the conifers all the damping-off appears to be
due to parasites of the generalized type.

(2) Damping-off of trees, as of herbaceous plants (with the ex-
ception of the cases caused by specialized seed-carried parasites), is
ordinarily serious only in seed beds or cutting beds in which large
numbers of plants are crowded together in a small space. In most of
the forest nurseries it is a much more serious matter in conifers than
in dicotyledonous seedlings.

DAMPING-OFF IN FOREST NURSERIES. 87

(3) The most serious losses in conifers are ordinarily from the
root-rot type of damping-off, occurring soon after the seedlings
appear above ground and while the hypocotyls are still soft. Losses
due to the killing of dormant or sprouting seed by parasites before
the seedlings appear above the soil are also frequently serious, some-
times necessarily more so than the later types, as in extreme cases
more than half of the seed or young seedlings are destroyed in this
way. Damping-off due to infections of parts above the soil surface
is serious only under extremely moist atmospheric conditions. The
late type of damping-off, in which the roots are rotted after the
stem becomes too rigid to be easily decayed, is ordinarily less im-
portant than the early types. Seedlings more than 2 months old are
ordinarily able to recover from infections by the damping-off fungi.
Even after the first month, seedlings with part of their root system
killed often recover.

(4) It is possible that damping-off has a certain value as a selec-
tive agent by eliminating weak individuals in the seed-bed stage and
allowing only the best trees to go into forest plantations. This

value, owerea is believed to be shane. Disinfectant treatments of
seed beds, even when controlling early parasitic losses very well,
allow a considerable percentage of disease during the last part of
the damping-off period, often as much as occurs at the same stage of
development in untreated beds. As it is only this late damping-off
in which differences in individual resistance of the seedlings seem to
be of importance in determining whether or not they ‘succumb, it is
believed that whatever selective value the disease may have will
appear in a larger proportion of the damping-off in the treated than
in the untre ated beds.

(5) Of the different conifers, reports are available as to the sus-
ceptibility of 63 species. Se which are especially susceptible
at somé nurseries may prove more resistant than the average at
others. Pinus resinosa, which is especially subject to loss at some
nurseries, is believed to be so because its growth at these nurseries is
slow and its period of susceptibility is therefore especially long.
In its-early stages it does not seem especially susceptible. Repre-
sentatives of all the commonly grown genera of the Abietoidez have
been reported by one observer or another as decidedly susceptible.
The reports on junipers and other members of the Cupressoidex, on
the other hand, have indicated a considerable amount of group re-
sistance to penis off.

(6) The best control method appears to be the disinfectant. treat-
ment of the seed-bed soil before or immediately after seed is sown.
Sulphuric acid has been found very useful for conifers, as they are
apparently especially tolerant of acid treatment. No method has yet
been worked out to a point at which all of its details are entirely

S8 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

satisfactory, though the acid treatment has now been in successful
use for several years at some nurseries. At most nurseries, if the
minimum effective quantity of acid is used, there is no need of any
special precautions to prevent injury to the seedlings. It is not
expected that any single treatment can be found that can be uni-
versally applied without change in details irrespective of differences
in soil characters and in fungous flora.

(7) Cortictum vagum and Fusarium spp. have been previously
shown to be parasitic on pine seedlings. Different strains of C.
vagum are found to vary considerably in their ability to cause damp-
ing-off, certain strains being consistently destructive and others
much less active in tests conducted on different species of pine and
several years apart. The differences in activity between strains
were greater, and apparently rather more constant from one ex-
periment to the next, than with Peltier’s strains in his carnation ex-
periments. Comparison of the results on pine with those of Edson
and Shapovaloy on potato gives some indication that strains vigor-
ously parasitic on one of these hosts are likely to be parasitic on the
other also.

(8) Pythium debaryanum, reported on many hosts and proved to
be parasitic on few, is shown by repeated inoculation, reisolation,
and reinoculation to be capable of causing the damping-off of seed-
lings of pine species. The identity of the fungus causing the damp-
ing-off of conifers with that attacking dicotyledons has been estab-
lished by cross-inoculations as well as by morphological comparison.
Inoculations on unheated soil are much less destructive than on
heated soil. Pythium debaryanum has been obtained in culture from
Picea engelmanni, P. sitchensis, Tsuga mertensiana, Pinus banksiana,
P. nigra austriaca, P. ponderosa, P. resinosa, and Pseudotsuga taxi-
folia. In addition, fenugreek (7'rigonella foenum-graecum) , cowpea
(Vigna sp.), and rice (Oryza sativa) are reported as apparently new
hosts among the dicotyledons. In inoculations the fungus has been
successfully used on Pinus banksiana, P. ponderosa, P. resinosa, and
in a preliminary experiment on Pseudotsuga taxifolia. It had
already been successfully used in preliminary inoculations on Picea
canadensis by Hofmann (77).

Differences in parasitic activity on pine are found between differ-
ent strains of Pythiwm debaryanum. These differences are not as
large and partly for this reason their constancy is not quite as con-
clusively demonstrated as in the case of the strains of Cortictum
vagum.

(9) Rheosporangium aphanidermatus Edson, a parasite of radish
and sugar beet, in many ways closely resembling Pythiuwm debary-
anum, has killed seedlings of Pinus bankstana and P. resinosa in
certain experiments, and reisolations and reinoculations have been

DAMPING-OFF IN FOREST NURSERIES. 89

made. The strain available is much less destructive to the pines
than most of the P. debaryanum strains used, and as the fungus has
never been isolated from coniferous seed beds it is not believed to
be of any great importance in forest nurseries.

(10) Phytophthora sp. from Pinus resinosa seedlings has been suc-
cessfully used in inoculation on Pinus resinosa and in a preliminary
test on P. ponderosa. The strains available have been less destruc-
tive to the pines than Pythium debaryanum and the stronger strains
of Corticium vagum. It is not common. Its relation to Phytoph-
thora fagi, tne European damping-off parasite of both conifers and
dicotyledons, which has not been reported in this country, is being
investigated.

(11) A fungus referred to Pythium artotrogus, also obtained from
damped-off Pinus resinosa, has indicated a very low degree of par-
asitism on this host, even less than that shown by the Rheospor-
angium and Phytophthora strains. An addition is made to the
statements in a previous paper concerning the ability of Botrytis
cinerea to cause damping-off in conifers.

(12) The results of the cultural or direct examination of 742 dis-
ease foci in seed beds of various conifers are reported. Pythiwm
debaryanum in the plate-culture examination method, considered
more reliable than direct examination, appeared in 51 per cent of
the foci from untreated beds, while Cortictum vagum was found in
19 per cent. In foci in beds treated with various disinfectants, P.
debaryanum was identified in 38 per cent of the foci and C. vagum
in only 4 per cent. When direct microscopic examination was sub-
stituted for isolation, C. vagum was found on a larger proportion
of the seedlings. It was not found at all in soil. which had been
heated. The relative ease with which it appears to be controlled by
soil disinfection is in agreement with its poor adaptation for aerial
distribution. It was found more commonly in cases in which’ the
seedlings were directly examined than when cultures were made.
In view of the fact that at least some of the Corticium foci extend
rapidly and include very large numbers of seedlings, it seems that
the Corticium may be as important as P. debaryanum in causing the
damping-off of pines.

(13) Fusarium spp. have occurred more commonly in plate cul-
tures than either of the above-mentioned fungi. Because little is
known as to the parasitism of different species of this group on
conifers, it is not possible to make any statement regarding the im-
portance of the individual species. The evidence as a whole indi-
cates so much importance for Pythiuwm debaryanum, Corticium
vagum, and for the /usarium spp. considered as a group that no one
of the three can be safely said to be more important than the others.
None of:the other fungi considered appear to be of real economic
rank in the United States.

90 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

(14) In an inoculation experiment on the roots of pines 14 months
old, Cortictum.vagum and Pythium debaryanum were found able
to cause the death of seedlings which had already developed rigid
stems and to destroy the younger parts of the roots of seedlings which
they were unable to kill. Indications were also obtained of similar
but less vigorous action by Fusarium moniliforme and F. ven-
tricosum.

(15) Data are given confirming the general belief that thick sow-
ing favors the disease and indicating that soil acidity is, in general,
unfavorable. Preliminary data on the relation of temperature and
moisture to the disease are also presented. The parasitic activity of
Pythium debaryanum in steamed soil was in one extensive test con-.
siderably decreased, following the inoculation of the soil with various
saprophytes; this indicates both that competition of different fungi
is a factor to be considered and that the inoculation of treated soil
with saprophytes may sometimes prove of value in increasing the
efficiency of heat disinfection. It is pointed out that with such a com-
plex of parasites capable of producing identical symptoms on a num-
ber of different hosts, no relationship between environmental factors
and the disease can be expected to hold in all cases.

(1)
(2)
(3)

(4

—

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13

~—

(14)

(15)

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94 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

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96 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE.

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