PHOMA ROOT ROT OF CELERY

 

THESIS FOR DEGREE OF M. §S,
C. W. BENNETT

1920

 

 
 

 
PHOMA ROOT ROT OF CELERY

Thesis for Degree of Master of Science

Michigan Agricultural College

Cc. W. Bennett

1920
Table of Contents

Tntroduction
Name of the Disease
History and Distribution
Economic Importance
Hosts
Signs of the Disease
General
On Leaves

On Crpwn and Roots

Etiology
Morphology
Mycelium
Pycnidia
Spores

Proof of Pathogenicity
Name of Causal Organism
Relation of Parasite to Host
Method of Infection
Pathological Histology
Physiological Relations
Cultural Characteristics
Temperature Relations
Vegetative Growth
Pycnidium Formation
Germination of Spores

Thermal Death-Point

93898
Light Relations
Oxygen Relations
Relation to Dessication
Relation to Reaction of Medium
Dissemination
Varietal Resistance
Life History of the “ausal Organism
Control

Summary
INTRODUCTION

Muck lands of Michigan equal five million acres and
constitute an appreciable percentage of the total farming area
of the State. Much of this is highly improved and forms some
of the most valuable land to be found in any of the great
agricultural sections of the country. This type of soil,
on account of its hich organie content and physical properties,
is peculiaray adapted to the growing of truck crops. Celery,
cabbage, lettuce, and onions, in particular, are crops which
thrive well and which yield large returns. The growing of
these crops for the markets of Chicago, Detroit, and other
large cities of the north central and middle west, constitutes
an important part of Michigan agriculture.

On account of the liberal returns per acre and the
brisk market demand, celery has become the leading crop on
mach of the best mck land. The high organic content of
mick soil is conducive te rapid vegetative growth which pro-
duces the very best quality in celery and also enables the
grower to put his product on the market early in the season.
It is eften the practice to grow two or three crops on the
same soil each year. In such cases the second and third
crop are put in between rows just before the preceding crop
goes in boards for bleaching. Thus, the land is in celery
the entire growing season. Along with this intensive
culture, the value of the crop has led to an almast complete
absence of rotation in many of the chief celery growing
districts. Not infreqently do we find fields which have
 

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grown celery continuously for a period of more than thirty
years, the fertility of the soil not only being maintained
but greatly improved by liberal application of manure.

Phe absence of rotation, the interchange of plants,
ana the procuring of seeds from a great many sources has
resulted in the introduction of practically every disease
known to the celery plant. One of the most recent of these
invaders is a Phoma disease, which, in Michigan, was first
discovered at Kalamazoo in the spring of 1914 by Dr. G.

H. Coons of the Michigan Agricultural College. The disease,
since its introduction has yot spread rapidly in an
epiphylotic form, but in several cases it has been very
destructive en small acreages. These destructive outbreaks
have seemed serious enough to warrant a systematic study of
the disease with a view to adding something to the knowledge
of its relation to weather and soil conditions and to dus
methods of coping with the problem which this disease
presents to celery growers.

NAME OF THE DISEASB.

Phis disease, like many others, is known by a number
of common names. In Germany it is called "Schorfkrankheit"
and the name "Scab" has been applied in thie esountry. ‘Root
Rot” is a term which is commonly used but sometime confused
with rot due to Sclerotinia and other causes. In choosing a
common name for a disease one should be selected which will
not be confused with other troubles and one which is as
descriptive as the nature of the thing permits. The diseased
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area is not typically scabby, but simply composed of dead
tissue. Since this is true the term "Scab" seems inappropriate
for the trouble as it occurs in America; on turnip .. rooted
celery the terms may be more descriptive. The roots constitute
one of the chief points of attack and it seems that no term

is more fitting than Root Rot. Po avoid confusion with other
Reot rots of celery we suggest that the disease be called
Phoma Root Rot, the causal organism being, as will be shuwn

later, a species of Phoma.

HISTORY AND DISTRIBUTION.

Phere is considerable difficulty in determining the
approximate time at which Phoma Root Rot first mde its
appearance as a disease of celery, and in tracing the history
of the trouble. While the same disease has been described
by certain Buropean investigators on celeriac in Germany,
Holland, and France, little has been reported regarding its
ocourrence in this country. It is hardly probable that this
is due to a recent introduction of the pathogene, or to a
limited distribution of the disease. WNumerous references
in literature te root rots of celery, all presenting the same
pathological aspects, make it more plausible to believe that
the disease has been present in certain celery districts for
a number of years, od that it is generally distributed
through the celery growing district or east and east central
parts of the United States. Working as it does beneath the
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surface of the soil, the true cause of the disease has
probably escaped observers because of its obscure nature
and because of confusion withother diseases.

&atee from a Phyllosticta leaf spot reported by
Halsted (1891), ne description of anything similar to this
disease seems to have been made unéil Van Hook (1907)
discovered and described a root rot of celery from the
celery districts of Ohio. He did not identify the organism
causing the trouble, but found Rhizoctonia in the diseased
tissue. However, he states that he does not believe that
Rhisoctonia alone was responsible for the é@isease. Neither
can the writer believe that any species of Rhisoctonia had
a part other than as a saprophyte coming in after the disease
had been initiated by another organism. In our work with
diseased plants, Rhizoctonia has quite frequently been
associated with diseased condition, but inoculation from
several isolatiens, and with Rhizoctonia solani and
Rhizoctonia from milkweed have given no results which would
indicate that any of these forms are pathogenic on celery.

Van Hook's descriptions, his photographs of diseased
Plants, and the environmental conditions under which the

disease became destructive, furnish evidence conolusive
enought to justify the assumption that he was dealing with
the Phoma Root Rot which has Been reported from other celery

growing regions.
on
es

v
-5-

Van Hook also reported that he had observed t§je samp
or e similar disease on celery in New York State as early
as 1903.

in 19192 the disease was reported from Ohio and
specifically attributed to Phoma apiicola.

Klebahn (1910) described the disease on celeriac
in Hamburg Lowlands. It seems to have a general dis-
tribution over Holland and southern Germany where it has
caused serious loss to the growers of turnip rooted celery.

Quanjer and Slagter (1914) described the disease
from Holland, and state that it isvery generally dis-
tributed in that country.

Dye and Whetzel” in 1918 reported the disease as
present in New York State.

This disease in Michigan was first found at Kalamazoo
where it appeared in a very virulent form from a seed bed
infection. Since that time it has been found at Byron
Center, North liuskegon, and Portage. It is extremely
likely that it hes been generally present throughout the
celery growing districta of the State.

 

1. Plant Disease: Bulletin. p. 109. 1919.

2. Verbal statement to Dre G H. Coons to whom the author
is indebted for this information.
-6<
- BCONOMIC IMPORTANCE.

The sporadic occurrence of this disease makes it

impossible to give even an approximate estimate of annual
loss. The seriousness of the attack after infection depends
entirely on environmental conditions. In wet cool seasons,
the loss may reach 75% of an infested district; under other
conditions, the loss may be negligible.

The disease in America has never been so serious as
in Europe. Klebahn and Quanjer and Slagter report considerable
damage to eeleriac occurring at more frequent intervals than

has been noted in America. Many plants are rotted off at

the base, while the damage done by stunting and pruning
away of the outer leaves, calls for serious consideration,
reducing as it does the market value of stalks so greatly.
Several rather severe outbreaks of this disease
have been recorded in America in certain localities. Van
Hook reports that in the celery districts of Ohio in 1902
one grower suffered a loss of 75% of his crop; minor
Losses were reported from other sections. At Kalamazoo,
Michigan, in 1914, and again in 1915/the first crop on an
area of three acres was a complete loss. At Kalamazoo and

@
Byron Center Coons (1918) states that the disease was very

severe in the spring of 1917. In the past two seasons the

 

1. Verbal report to the author from Dr. G. H. Coons.
_

disease has been present in a number of celery fields which
have been inspected, but diseased plants have shown nothing
more than black rings around the base. These seasons have
been rather dry and warm in the early part of the summer;
this no doubt accounts for the scarcity of the disease during
these years. Where the disease producing organism is known
to be present growers in cool wet seasons should expect to
enoounter léss if due precaution for protections are not
taken.

HOSTS.

So far as we have been able to discover by a careful
survey of the literature on the subjeot, this disease has
been reported only on celery and celeriac (Apium graveolens L.).
Other Phomas have been reported on species of the Family
Umbelliferae, but in many cases the available descriptions
are too meager to admit of satisfactory comparison with the
one on celery. It is, of course, entirely possible that this
organism may have been reported on other species of plants.
fhe genus Phoma embraces so many. little-known forms, with
present descriptions so inadequate, that, until & more
comprehensive study of the genus is made, it will be
impossible to determine the validity and host range of many
of the parasitic species.

Inoculation experiments leave no doubt as to the

ability of the fungus to attack plants other than celery.
~B=

Its potentialities along this lines have been tested by
inoculation of a number of species of Umbellifers and several
species of Crucifers.

Plants, nearly related to celery and of economic
importance, were inoculated with pure cocultures of the
pathogene, under conditions very favorable to the production
pf the disease on celery. Inoculation of carrot (Daucus
carota L.) produced black sunken spots on the roots and a
killing of the outer leaves. Pycnidis were found scattered
over the diseased parts.

Parsnip (Pastinaca s@biva L.), while it is by no
means immune, seems to be less susceptible than carrot.
fhe region of invasion was largely confined to the upper
part of the root and to the base of the leaf stalks, The
diseased parts were typically dark brown er black. On the
root the epidermis was broken and the underlying tissue
invaded, which resulted in a “cankered" area. Invasion was
very slow and the damage slight, except as the diseased
spots disfigured the roots.

Under greenhouse conditions Moss Curled parsley
(Carum petroselinum B. & H.) and celery seem to be equally
susceptible to attack of the fungus. Young plants were
killed, and older ones were severely "scabbed" around the

base. Pyonidia of the pathogene werd produced in all
=D =

diseased parts. Although it has not been reported, it

would not be surprising to find this disease éehhg, damage
te parsley under field conditions, particularily in
trucking districts where celery and parsley are grown
extensively.

The attacks of the fungus on caraway (Carum carvi L.)
were very weak. A few leaves have been killed by basal
invasion but the region of attack is usually limited.

Peisen hemleck (Conium maculatum IL.) and dill (Anethum
graveclens L.) have been free from all signs of disease

after inoculation with heavy doses of mycelium and spores.

SIGNS OF THE DISEASE.

General.
Cases of mild infection are often impossible to detect,

unless the plants are removed from the soil and examined
for dark diseviemations. <Above ground, the first indication
of this disease is the dying of a few outer leaves. Often
one or two withered leaves lying on the ground, but still
attached to the plant, betray the presence of the disease,
while the remainder of the plant has all the appearance of
@ normal, undiseased individual. As invasion progresses,
more leaves may die and fall to the ground. The plant takes
on a general unthrifty appearahce, and, quite often, remains
stunted throughout its period of grewth. In the case of

severe attacks, plants topple over, so completely are the
-10-

crowns and roots destroyed.
On Leaves.

Under Michigan conditions, natural infection of the
leaves has not been noted, and if it does take place it is
probably eof rare occurrence. Wherever the disease has
appeared in America, it seems to have been restricted to
underground parts. In literature and references pertaining
te celery diseases, there are only two citations of an
eceurrence of an organism of a Phoma or Phyliosticta type
on leaves of celery. Halsted (1891) has described a
Phyllosticta on celery leaves, which closely resembles the
organism causing Phoma Root Rot, and which produced a leaf-
spot such as can be produced by inoculating, under the
proper environmental conditions, with the root rot erganisn.
Recently a Phyllosticta leaf spot has been reported from
Porte Rica (1918). <A complete description ef this fungus
has not been obtained and it is not known whether or not
it is a distinct organisn.

In Holland, Klebahn found the disease on celeriac
leaves and on the flowering pasts, and alse discovered
pycnidia of the pathogene on the seeds.

Given the proper conditions, the fungus is capable
of attacking the deaves. Spraying with spores outside and
in the greenkeuse has not resulted in leaf infection. Only
@ small amount of infection has been produced by spraying

the plants with a spore suspension and placing them under
ere

 
battery jars. Much more uniform infection has been obtained
by first germinating the spores, then placing them on the
leaves under small bits of wet cotton. On leaves inoculated
in this manner, signs of the disease began to appear after
two days. The results of infection may be of several kinds,
Gepending on conditions. If there is not too great a
quantity of moisture present there is first produced a light
colored spot which later turns inte an irregular red blotch.
Given more moisture, the fungus, possibly by the aid of other
organisms, produces soft, water-soaked areas, which are often
studded with pyonidia. In the first type of spot, fruiting
bodies begin to be formed in the mesophyll of the leaf, but
seldom: reach maturity unless the leaves are subjected to
exceptionally moist conditions. The progress of the disease
has been slow under all conditions mainéeained, and
individual spots cease to spread when the plant is taken eut
of a moist atmosphere, and more or less normal relations re-
stored. Leaves that have been killed by inwasion at the base,
often show pyonidia on the petioles and blades, after remain-
ing on damp soil for a time, which indicates that the fungus
is not selective as to the part of the plant attacked, but
is governed chiefly by environmental factors.

Inoculatiem experiments, and ebservations on the
disease in the field, establish clearly that climatic

relations in Michigan are not favorable to the development
-lZ=-

of the leaf-spot phase. In no place, either in America or
Kurope, has leaf infection been reported as common. The
pathological significance lies in the bearing, which leaf
and flower infection may have on the dissemination of the

disease producing fungus.

On Crown and Roots of Plants.

The chief loss to the celery crop from this disease
comes from attacks on the roots and crown of the celery
plant. In the incipient stages of the disease, there is
usually a bluish-green discoloration of diseased parts;
which, as invasion continues, gives rise to a black sourfy
surface. A bluish-green border, more marked on the leaf
Stalks than on any other part, is usually found around these
"scabby” areas. The fungus may confine its ravages to the
outer part of the crown and produces a black ring of diseased
tissue. This kind of an attack, by killing the epidermal
and neighboring cells, often leads to large cracks in the
crown and bases of the leaf petiole, as the plant continues
to grow and expand from within. The course of the disease
after infection depends entirely on environmental conditions.
The plant may attein normal size and reach maturity, with
only a black ring around the crown to indicate the presence
of the disease. In other cases, the plant may rot off at
the orown or the roots may be attacked and destroyed,
leaving the plant to fall over, or to maintain itself

by means of new roots which may be shot out from the diseased
-13-

base. Plants of this latter class are usually stunted and
are easily pulled out of the soil. They exhibit a black,
ragged, con-shaped butt which is invariably studded with
black fruiting bodies of the pathogene.

In some cases, when the plants are deeply set, the
attack may be confined to the leaf stalks in the region where
they come in oontact with the first inch or two of surface
soil. In such instanoes, a killing of the outer leaves
usually oceurse In most cases, the disease spreads to the
crown and roots, where the work of destruction is continued.

Root attacks are most severe in close proximity
to the crown. There is rarely a general attack on the entire
root system. Typically, infection tekes place at some point
Near the base of the leaves, and spreads dow and around the
basal portion of the plant, involving the roots as it
progresses. Roots are usually rotted from the plant before
they become disoused: Those roots, which are near the surface
are more subject to attacks along their entire length than
ere those which extend down deep into the soil. The disease
on the roots is characterized by a brownish discoloration.
The black surface, so typical of the disease on other parts
of the plant, is also noted on the larger roots; small
roots usually disintegrate before this color appears. fhis
disease will be shown subsequently is due to the pathogene,

Phoma apiicola. The following account may be given of its
Morphology and life history.
-14-
ZTIOLOGY.
Morphology.
iycelium.

Within the tissue of the host plant, the mycelium is
composed of septate threads, sparingly branched, and
comparatively small; averaging between three and four microns
in diameter. In young hyphae, cross walls are rare and the
threads are very uniform throughout. With age, the hyphae
sometimes inorease in diameter, and the cells may bulge
Slightly as the cell walls thicken, and as the septa become
more numerous. Very young hyphae are hyaline and the aell
contents finely granular; older threads are vaculate
and often densely granular within.

In pure culture, the organism is subjeot to great
variation in size of threads,in thickness and color of cell
walls, and in structure of oell contents. New hyphae vary
from two to four microns in diameter. These become very
wlosely septate, and the cells bulge and inorease in size,
until they are large spherical bodies, twenty or more microns
in thickness. The cell walls are rather thick and have a
Gark-brown color. When the mycelium comes in contact with
glass, it forms e black mass of thick walled cells which
clogely adheres to the smooth surface; in liquid cultures it
forms a black ring immediately above the surface of the
culture medium. Old cultures, on favorable media, form

black compact masses of densely interwoven threads approach-
-15-

ing a pseudoparenchymatous tissue. On dextrose agar,
sclerotium-like bodies, several millimeters in diameter,
are sometimes produced in the mass and near the surface

of mycelial growth. These very much resemble sclerotia

of the genus Sclerotinia. Sectioning, however, reveals

the same context throughout; the entire strueture is
composed of dark brown cells closely compacted and cemented
together. Kilebahn, in addition to find these bodies in

pure culture, has noted them on diseased plants.

Pyonidia.

Pycnidia are produced singly,either scattered or
clustered on the surface of the host. lhiycelial development
around the pycnidium is sparce; nothing approaching a stroma
has been observed. Young pycnidia are hyaline; older ones
vary from brown to black. <A few pycnidis are more or less
Spherical, but more commonly, they are considerably
depressed; all are very symmetrical. In size they measure
80--100 x 100~-250 microns. The pycnidial walls vary in
thickness from 6 to 8 microns. The neck is oylindrical
or expanded at the base and top. Pyonidia usually originate
immediately below the epidermis; as they increase in size,
they may push out-eward, until, at maturity, they are firmly
seated on the surface with only the base imbedded in the
host tissue; or they may remain imbedded in the host tissue

with only the neck exposed.
-l6=

The pyonidium has its beginning in a tangled weft
of hyphae which rounds out into a globose, fruiting body.
The first part to darken is the neck of the ostiole which is
conspicuous as a black ring, even before any part of the
pycnidium becomes superficial. The remainder of the pycnidial
wall is hyaline until after the spores are formed. fThen,
there is a deposition of brown-coloring matter in the outer
layer of cells which produces the dark colored pycnidiun.
The greater part of the black outer portion of the pycnidial
wall consists of a layer of large, rather thick walled cells.
This is rarely more than one cell in thickness. Near the
base of the ostiole, the wall begins to inorease in thickness,
until the well of the neck itself is two to three cells thick.
To this greater number of thick walled cells, is partly due
the darker color of the pyenidial neck. Within, covering the
internal surface and extending well up into the neck of the
pycnidium, is a layer of thin-walled cells filled with
dense masses of protoplasm. These give rise to minute
conidiophores which, in turn, give rise to spores by

constriction and formation of cross walls near their tips.

Spores.
Spores of Phoma apiicola are small rod-shaped, thin-
walled, hyaline bodies, measuring 3-3.8 x 1-1.6 microns.

Staining discloses the presence of vacuoles which are

Surrounded by eytoplaem having a finely granular structure.
-L7-

The size of spores on the host plants does not vary to
any considerable degree. This uniformity apparently holds
true with the organism very generally, as the following
table of measurements by different investigators will show.

Spore and Pygnidium Measureitent.

Spore. Pycnidium.
Klebahn  « Be4 x 168--168 microns 90--240 microns
Quanjer & Slagter 4 x 1.5 microns 98--210 "
Author 3-3-8 x 1-1.6 microns 80--100 x 100--

250 mierons
The pycnidia, as shown by the above table, are subject
to great variation in size. Quanjer and Slagter found that
spores produced in pure culture were smaller than those on
the host, a thing which has not been noted in any culture

used in this work.

PROOF OF PATHOGENICITY.

The organism causing "Scab" of celeriac has been
isolated by Klebahn ana its pathogenicity proved,by
inoculation experiments Coons (1917) isolated a Phoma
from diseased celery plants. Regarding this organism he
says: “This fungus was obtained in pure culture and
typical lesions were obtained in inoculation experiments.
The etiological relation of the Phoma thus obtained has
been fully established. "

The author has obtained a Phoma from celery from a
-18-
number of sources. Single spore isolations have been made
from digeased plants from Kalamazoo, Portage, and North
Muskegon, as well as from a number of diseased plants
which were found in the greenhouse at the Michigan Agri-
cultural College. MInooulations with these cultures have
given oharacteristic diseased plants in a large percentage
of cases. The organism has been reisolated from celery,
parsnip, and parsley and the identity of the different cul-
tures proved by comparison of cultural characteristics and
by inoculation followed by typical signs of disease.
Repeated inoculations and isolationslaave no doubt as to
the pathogenicity of the organism and its causal relation-
Ship to the disease.
NAME OF ORGANISL.

Huropean writers have been unanimous in ascribing
the cause of "Scab" of celeriac to Phoma apiicola Speg.
A culture of this organism was obtained from hiss
Johama Westerdijk of the International Station for
Fungous Cultures by Dr. G. H. Coons who compared this
culture with the Phoma which he isolated from oélery.
As a result of this comparison he states that, "The
fungus (the one isolated in Michigan) in culture and
pathological habit greatly resembles rhoma apiicola
described by Klebahn as producing the "Scab" of
Celeriac."

The author has further compared this Suropean
culture with the ones which have been found in Michigan

and in all oases responsible for the Phoma disease
-19-

of celery in America.

This organism is a typical Phome and in the light
of most recent taxonomic work by Diedicke on this genus,
cannot be classified otherwise. The walls of the mature
pycnidia are typically black amd parenchymatous, the
spores are small and the conidiophores are neither branched
nor hooked. This organism has none of the structural
characteristics of which Diedieke took advantage in his
work with the species formerly embraced in the genus Phoma,

and to which he attached generic value.

RELATION OF PARASIT# TO HOST.
hLethod of Infection.

The factors involved in the physiology of parasitism
have been baffling problems which investigators have
attacked with varying degrees of success every since the
study of plant diseases has attracted scientific research.
liuch of the work done has hinged sround the method by
which the parasite entsrs the host and around relations
of the pathogene to the internal tissues of the host.

It is to be hoped that a thorough knowledge of these
phases will aid in the discovery of at least some of

the fundamental factors which oonstitutethe difference
between parasites and saprophytes. Aside from the
Scientific value connected with this kind of investigation,
there is often considerable practical significanee.

attached to the method of host penetration and invasion.
 

 

 

 

-2Q0-

Parasites, of course,employ a number of different
kinds of methods in entering host tissue. Germ tubes may
enter through stomata, as Gregory* has shown for
Plasmopara viticola, and as Pool and Liokay# have shown
Phoma betae. Other natural openings are often very
important. Wounds are recognized as weak places in any
plants defensive equipment and, with many parasites,
are important points of infection. Still other organisms
enter by boring directly through the epidermal cell walls.
In the case of the Botrytis of lily Waratt very early
believed that the fungus gained entrance by dissolving the
cell walls by means of a “ferment substance", an
observation which has been confirmed repeatedly and which
has been found to be true for a great number of other
plant pathogenes.

Whether or not all organisms which penetrate cell
walls employ some kind of dissolving substance as they
come in contact with the host, is not definitely known.
it is possible that the substances are not necessary for
&ll parasites.

Hewkins and Harvey! think that in Pythium de
Baryanum they have an organism capable of exerting

enough mechanical pressure to enable the threads to

 

“Gregory, C. T. Phytopath. 2: 233-247. 1912.
# Pool, Venus W. and kh. B. MioKay. Jour.AgreRes. 5:1011-1036.1916
H# Ward, H. Me Anne Bote 2: 319-382. 1888.

1 Hawkine, Lon A. and Harvey, A. B. Hour.Agr.fes.
18: 275--298. 1919.
-21-

push their way through the cell wall.
With a view to determining the relation of the Phoma

Root Rot organism to its host in the early stages of
infection a number of inoculations and microscopic studies
have been conducted. Virulent strains of this parasite
uniformly give infection regardless of whether spores or
mycelium are used as the inoculum, but on account of the
greater uniformity of time required for penetration sporeshave
been used for studying methods of infection.

Leaf and petiole inseutations have been made to avoid
the difficulty of making observations on crowns and roots.
The spores were first germinated in water, then bits of
filter paper were soaked in this spore suspension and
Placed on the plant under flecks of wet ootten. he spots
inoculated were examined at different times by stripping

off pieces of epidermis, or by placing bits of leaves under
the microscope. In this way, it was possible to study
relation of host and parasite under high power without
resorting to stains.

Forty-eight hours after inoculation the germ tubes
were found to have entered the host. The method of entrance
was a direct penetration of the epidermal cell walls by the
fungus hyphae. Wo cases of penetration between cells, by

wedging apart of cell walls or by dissolving the cementing
=22-
substances, were discovered. A few cases of stomatal entrance
were noted but these were not numerous enough to denote any
special attraction on the part of the stomata for the fungus.
Those tubes which entered in this manner probably did so by
chance rather than as an easy method of gaining entrance to
the host.tissue. As normally the fungus attacks the under-

ground parts where stomata are scarce or lacking, it is
obvious that methods of entrance mst be confined to other
natural openings, to wounds, or to direct penetration. These
observations establish the fact that the fungus is capable
of making its own openings in the leaf-blade and petiole.
The same is doubtless true in the case of roots and crown.
With young plants, 100% infeotion is quite frequently
obtained; older plants, when inoculated, often fail to take
the disease. This observation has been made repeatedly with
greenhouse plants. Young seedlings are subject to rapid
infection. As the plants increase in age, indicatiens of
extreme susceptibility become less marked, though age by no
means renders them i:mmne. Pull grown plants can be made
to taJe the disease under certain conditions. As the plants
become older the tissues become tougher and no doubt offer more
resistance to invasions of this fungus. Breaks in the
epidermis under these conditions wowld offer opportunity for

the pathogene to gain a foothold.
Peo determine the effect which wounding would have on
=23-
the percentage of infection in the case of older individuals,
50 plants were selected and wounded in the region of the
ecrewn by scratching into the tissue with sharp needles.
Bits of mycelium were placed in direct contact with these
breaks in the epidermis. At the same time, an equal number
of checks was inoculated by placing mycelium in contact
with unbroken tissue. The plants were kept under conditiens
favorable for the progremof the disease. At the end of
two months, 48 of the 50 wounded plants had developed black
rings around the crowns, and a large number of roots had
been lost. Of the check plants, 32 of the total 50 showed
typical signs of the disease, but the invasions were more
restricted and much less serious in their affect on the

normal functioning of the host plants.
Under average conditions in the field, wounds due to

insect bites, fungus invasion, mechanical injuries, etc.,
are common. Such injuries in all probability are an aid
in admitting the root rot fungus, but by far the greater
percentage of infection must take place through the
unbroken epidermis. Especially is this true in the case of

young plants which may suffer a high percentage of
infections. Older plants which offer more resistance to
invasions are probably less susceptible when they present

an unbroken surface.
~% -
PATHOLOGICAL HISTOLOGY.

Previous investigation by Klebahn has shown that the
Phoma Root Rot organism is both an intercellular and intra-
cellular fungus. This point was confirmed early in the
course of the present work, and an attempt was made to
determine something of the effect of the pathogene on the
individual host cells. “‘Yigquid caltures in which the fungus
had grown, extracts from mycelium, and alcoholic
precipitates from both of these types of substances have
produced no perceptible effect on raw sterile celery when
pieces were immersed in such solutions. It is very certain
that enzymes which will react on cellulose or pectin and
allow the fungus to make progree between cells in the
middle lamella or which would cause a falling apart of the
cells of pathological tissue, if produced by the fungus,
are not present in sufficient quantities to cause noticeable
results in experiments of this kind. Apparently hyphae are
capable of penetrating cell walls at any point at whidh
they come in contact and of running through cell walls,
protoplasm or intercellular spaces as these are encountered.
Sections of diseased plants show the hyphae ramifying
through all parts of the tissue, forming a perfect network
of threads through the cell walls and protoplasm. Hyphae
have been found in all the various tissues of the celery
plant. In the later stages of disease mycelium sometimes
=-25-
even produces extensive growth in the vascular systen.
Both stained and unstained sedtions reveal no injury
preceding invasion as is noted with many rot producing
organisms. Even after penetration the cell is net
fimmediately destroyed; many invaded cells, but for the pre-
gence of mycelium, could not be distinguished from cells
of normal unattacked tissue. The first indication of
change in the invaded cells comes about through a disinteg-
ration of protoplasm, follewed by production of brown
coloring matter in the cell walls preceding a general break
down and collapse of diseased tissue. Many soil organisms
no doubt come in at this point and continue the work of
destruction. It is to these last organisms that we attribute
the true rotting found in diseased plants as the primal
organism concerned, while effective in killing tissue, is
incapable of producing a typical rot such as is usually
encountered in diseased specimens, which are found in the
field.

PHYSIOLOGICAL RELATIONS.

Cultural Characteristics.
General.
The fungus grows readily on all of the common
laboratory media and on many other media which have been
prepared and used in cultural studies. It is easily

from spores by the dilution method. Single spore colonies
 
appear as thin feathery growths, so characteristic of
many of the Fungi Imperfecti. The colonies are at first
white; after four to eight days, on certain media, the
superficial growth is bluish or bluish-green. The sub-
merged threads are hyaline to black. Acidity, high
temperature, and carbohydrates seem to favor color
production in the mycelium. Proteins, low temperature,
and alkalinity tend to cause little color production.

A protein medium also tends to cause permanent loss of
potentialities for color production, and for pycnidiun
production, as well as loss of virulence. Ppyonidiun
production is sparse on all media solidified with agar
and on sterile vegetables, with the exception of celery
plugs. Ten to twenty days are required for pycnidia to
develop. Characteristics of growth on different media
are given briefly in a tabular form in the following out-
line.
-27 =
TABLE l.

Vegetative growth.

Characteristics on Different Media.

Standard nutrient
agar

Dextrose agar

Celery agar

Corn meal agar.

Oat meal agar.

Prune juice agar.

Potato plugs.

Carrot plugs.

Celery plugs.

Growth good, largely super-
ficial, slight mass colored
tinge

Growth abundant, fluffy,
largely superficial; tough
masses of dark mycelium
formed over surface with
black sclerotial-like

bodies formed in the surface
mycelium.

Growth good, considerable

submerged mycelium; aerial

eycelium Slightly tinged with
lue.

Considerable growth below
the surface ef the substrate;

peers mycelium very faint
luee

Mycelium abundant, sometimes
filling entire slant in test-
tube; black masses of
mycelium formed at the edges
of cultures.

Growth moderate, largely
aerial, fluffy, white.

Mycelium abundant, compact,
white, old cultures form
tough black mats.

Very much the same as with
potato plugs; mycelium hot
so compact and slightly
bluish.

Growth moderate, fluffy and
white at first, later turns
ine

Mycelium very abundant,
white or slightly tinged
with blue.

Few
pycnidia.

Pycnidia
scarce.

Pycnidia
Scarcee

Pycnidia some-

times numerous

in old
cultures.

Pycnidia have
been found in
old cultures.

No pycnidia
were found.

Fruiting not
abundant.

Few pycnidia.

Pycnidia found
in great
numbers, seatéda
on the surface
and visible to
the unaided ey

No pycnidia
observed.
Corn

Coons' synthetic
(on filter paper)

Richard's solution

-28-
PABLE 1 continued.

Vegetative growth

Color of rice changed
from white to orange
color; mycelium varying
from light blue to black
Growth abundant.

Growth moderate, color
mycelial mass light
bluee

Mycelial growth sparse;
at first white, later
turning dark.

At first submerged
coming to the surface;

very dense masses formed;

Color varying from white
to black.

Pycnidia
produced in
old cultures.

Pycnidia
produced in
old cultures.

Pycnidia very
abundant on
both side of
filter paper
cone.

No pycnidia
observed.
=29-
Temperature Relations.

Vegetative growth.

The seaspns of the year, spring and fall, in which this
disease is prevalent, suggest a sharp susceptibility on
the part of the fungus to high temperatures. This is force-
fully brought to ones attention when he attempts to isolate
the organism during a hot period, or attempts to grow it
in pure culture at a temperature higher than 27°C. During
the summer months it has invariably refused to grow in the
laboratory, # fact which-has previously been noted by Coons.
Nearly all the cultural work at such times has been done in
a basement where the temperature ranged around 21°C. Where
more rapid growth was desired an ice box has been used.
This gave a temperature of about 16°C, which is very favor-
able for vegetative growth.

In the more exact temperature studies with the fungus,
a differential thermostat, modeled after that of Ganong's,
has been employed. This consists, at one end, of a cubical
container which is filled with ice, at the opposite end, of
a compartment for water which is heated with a hot-point;
a galvanized sheet iron connection is between the two.
Between is the boiling water at one end, and the ice at the
other, there are nine compartments, separated by cardboard
partitions. A glass covering is provided ge that light is
admitted. This gives a range of temperatures which is
fairly constant in the middle compartments, but which admits
oN
~30=
of considerable variation near the two extremes.

Using this equipment, preliminary experiments were
conducted to determine the effect of temperature on quantity
of vegetative growth. Test-tube culture on agar were first
used, then, later, in order to get a giant colony effect for
more exact comparison, these were replaced with Ehrlenmeyer
flasks. Twenty c.c. of celery agar were placed in each
flask; bits of mycelium of Phoma apiicola were placed in the
Genter of the medium and subjected to different temperatures.
The experiment was run for 30 days in duplicate. The
colonies progressed slowly in uniform circles and were
actively growing at the time the experiment was concluded.
Results in the following table are recorded in terms of
average diameter of the colonies. Temperature variations
are given as well as the average of twenty readings made

during the experiment, values being recorded in terms of the

nearest degree.
-3l-
TABLE 2.
The Effect of Temperature on Growth of Mycelium.

femp. Variation. Average Temp. Average diameter
of colonies.

4--6°C 2° 5 om.
8--128 g@ 1.6
13--15° 13° 2.5
15 --16° 16° 31

180 18° 3

200 20° 2.8
21--23° 22° 2.6
25--27° 26° 1.7
27 --30° 28° 3
34--400 36° no growth.

fhese results show that minimum temperature for
growth is close to the freezing point of water; maximm
temperature is around 28 degrees. Optimum temperature is
somewhere in the range of 16 to 20 degrees, though very
good growth is produced with a few degrees variation either
Wwaye
Pyonidium Production.

Quite often pycnidia are not produced es a solid

medium and when they do make their appearance it is

usually in old cultures where there is great difficulty
in making estimates for comparison of response to different

treatments.
 
“52 —

Cultures of the organism on filter paper, prepared as
described py Coons are more satisfactory for determining

the value of various factors, in pycnidium production.
Fruiting bodies have been found to be formed in greater
numbers and results are more easily and accurately estimated.

This method consists, esentially, in placing filter paper
cones in a deep culture dish with the addition of s liquid
medium. The nutrient solution used has been either corn-

meal broth or Coons' synthetic solution. Either is very

satisfactory although the latter gives a greater mycelial
development and a larger number of pycnidia.

Four deep culture dishes, prepared as outlined above,
were inoculated with mycelium of Phoma apiicola and placed
in each compartment of the differential thermostat. They
were allowed to remain thirty days. During this period, the
temperature in the basement in which the test was conducted,

was fortunately very constant. Therefore, the temperature in

the different compartments varied little, after the first
two days. The temperatures given below are averages for
the period: except in the case of the last two figures the

variation was not more than one degree in either direction.

 

1. Coons, G. H. Jour. Agr. Res. 5: 752. 1916.
~5S-

TABLE Ss.

fhe Effect of Temperature on Pycnidium Formation.

Average Temp. Percent Mycelial Pycnidia.
Growth.

4C 75 -
10 80 -
13 90 +
16 100 *
18 100 .
20 80 ’
23 40

+
26 10
35 0 ~

The quantity of mycelium produced at different temper-
atures is in harmony with previous experiments. Color
reactions on different media have been noted before, but they
came in here in a very pronounced form in response to heat.
At 4 degrees and 10 degrees, the mycelium was almostwhite.
As the temperature increased, the color became more intense.
At 26 degrees there was very little vegetative growth,
but that which was produced had a very intense dark bluish
cast.

Pycnidia begin to appear at the end of 14 days.

Larger number of fruiting bodies were produced at 15°,
16° and 18°, though, per unit area of mycelium, there
were as many produced at 25° as at any other temperature.
~34-

At 4°and 10° while there was a very good mycelial growth,

there were no pycnidia; however, after these cultures were
removed and placed at 21° the characteristic bluish color

came in and pycnidia were produced as abundantly as in any
of the; other cultures.

High temperature seems to lessen pycnidium production
only as it lessens mycelial growth. Low temperature,
1.e. below 10°, represegspycnidium production, but a

short exposure does not destroy the potentialities for

fratting.
Germination of Spores.

In moist chambers and liquid culture, the spores of
Phoma apiicola have been very slow in germinating. From
36 to 48 hours have been required for the first indications

of germination at room temperature. After the mycelium had
shown such a marked response to relatively high temperature,
it was suspected that spores were being kept at too high
a temperature for the mist rapid germination.

Pycnidia from deep culture dishes were crushed and the
spores placed in small test-tubes containing sterile
tap water. These were incubated, four tubes being placed
at each temperature recorded below. Loops from these tubes
were examined every 12 heures for germination of spores.
Results are given in the following table.
-35 -
TABIE 4.
Effect of Temperature on Germination of Spores.

Temperatures. Stage of Germination.
ga hye.) 48 bra. =O ars. =~ 84 hrs.
ae 8 = - —s Swelling = © budding
10° - - Swelling germ tube
13° - budding == germtube germ tube
16° gwelling germ tube germ tube germ tube
18° swelling germtube germ tube germ tube
20° - swelling germtube germ tube
23° - swelling budding germ tube
26° - - - swelling

At least 24 hours are required for germination at
any temperature. The most favorable temperature for
vegetative growth, 16°to 18°, is also conducive to most
rapid spore germination. As indicated by weak germination
at 4°, the minimum is very little below that temperature.
Maximum temperature is close to 26°.

The most outstanding feature is the length of time
required for germination even at the most favorable
temperature. In this, we probably have on of the minor
factors for making restriction of the fungus to the
underground parts of the host plant under field conditions.
The spores, as indicated by inoculation and germination

experiments, require considerable moisture for germination.
~36=
A fungus restricted by this moisture requirement, in order
to become am important leaf disease producing organisn,

mst necessarily have spore which will germinate rapidly
and produce infection before drying out takes place.

Thermal Death-Point.

The ability of the fungus mycelium to withstand low
temperatures has been tested by placing flask cultures out-
side of a window when the temperature ranged from-18° to
- 28°¢, fransfers proved that the mycelium was not killed
after several days exposure.

In the higher range of temperatures the mycelium is
killed by subjecting it to 35° for a period of 72 hours.

A ten day exposure at 32° stopped growth but was not
sufficient to produce death. Shorter exposures at lower
temperatures indicate that the thermal death-point of the
mycelium is very close to that of the spores which is
very close to 49°.

In determining the thermal death-point of spores the
capillary tube method, as described by Novy, has been used.
Capillary tubes, five inches in length, and about 2 m.
in cross section, were drawn out from a piece of this walled
glass tubing. These were inserted in a spore suspension
until there were properly filled, then sealed at each end.
The tubes were immersed in water baths held at constant
temperatures. At the end of the time of treatment they

were co@led in water at a temperature of about 21°C.
-37-

fhe tip of one end was then broken off, using aseptic
precautions, and the contents discharged into melted agar
by applying heat to the unbroken end. The experiment was
run twice in duplicate, first with an interval of 5
degrees and later with an interval of 1 degree. Results
are combined in the following table:

TABLE 5.

fhermal Death-Point of Spores.

Temperature Degrees Germination of spores.
Centigrade Time of Expostre in Minutes.
ge yo a go

40° + + + +
45° + + + -

46° +
47° +
48° +
49° -
50° - - - -

The thermal death-point of spores, at an exposure of
10 minutes, lies between 48 and 49 degrees. Only a few spores
germinated at 48 degrees, indicating that there was some slight
variation in the treatment which thespores in any one tube
received or that some of the spores had slightly greater
potentialities for withstanding heat than did others.
-~38-
Light.

Physiological investigation in this field indicated that,
with many species of higher fungi, light. is of little import-
ance either as a stim lamb or as a repressant of vegetative
growth. With many of these same organisms, however, light
is as important and often an essential factor in spore
production. Levin’, working with a number of species of
Sphaeropsidales, has found that, while mycelial geowth was
just as abundant, fruiting was considerably lessened and,
in several species, completely suppressed by absence of light.
Species of Coniotherium, Phyllosticta and Cytospora showed
considerable pycnidium production im the dark but at that
suffered a falling off of more than 55%, when light was
excluded. Species of Ascochyte, Phoma, Sphaeropsis, and
Pusicoccum produced no pycnidia in the dark. S@eene’ has
found that, in the case of Plenodoms fuscomaculans,
darkness has little or no effect on vegetative growth, but
is prohibitive to pycnidium production.

With these results in mind, experiments were planned

to determine the effect of light and darkness in vegetative

growth and pycnidium production in the case of Phoma apiicola.
To provide for darkness and at the same time allow for the

circulation of air, a method modeled after that of Coons was

used. To exclude light, two battery jars were wrapped in

 

1. Levin, Ezra, Report Mich. Acad. Sci. p.134-1355. 1915.
Ze Coons, Ge. He JOUXS. -- Agre Rese 5: 720--725. 1916.
~39@
heavy black paper. The larger was placed bottom end up

over the smaller one and allowed to rest on block of wood,
thus providing for a passage of air at the base and over

the top of the jar within. A similar arrangement was
prepared, using heavy wrapping paper, for obtaining diffused
light. Two jars without any covering were used in the

series exposed to light. Filter paper comes were placed

in Soyka dishes coygjtaining 5 c.c. of corn meal broth as
the nutrient medium. Inoculations were made directly from
mycelium. Eight dishes were placed in each jar. Results
are tabulated below.
TABLE 4.
Bffect of Light on Growth and Pyonidium Production.

Treatment Percentage Development, based on best
“eee er eer ee eer er er ree ee Ree er ewe eS culture.
Pycnidia Mycelium
Light 100 90
Diffused Light 80 100
Dark 80 90

Pycnidia were produced in greater numbers in light

than under any other conditions. However, it is conclusively
dempnstrated that light is not an essential factor in the

fruiting of this organism. Light seems to stimlate fruit

bedy production, probably due to the lessening of vegetative

growth er to some other indirect action.
-40-
Greatest mycelial development was obtained in diffused
light, here also the hyphae were more upright producing a

leoser and more fluffy mass. In darkness there was

apparently as much growth as in strong diffused light. Under
normal conditions on the host the organism is not subjected
to strong light when it grows around the base of the plants
just beneath the surface of the soil. Long contimed growth
under such conditions may have produced a physiological
“adaptation to environmental conditions. At any rate it is

a signficant fact that the light relations which are most

favorable for development in culture are those to which the

fungus is subjected when functioning as apathogenic organism,

Dessication.

Mycelium of this organism will live for a period of two
er three months when grown on artifical medium and allowed to
dry. Longevity waries with the character of the growth on
the different media; rice, which produces thiek tough mats
of growth and hyphae with thick walled cells, is conducive to
great resistance; soil and certain synthetic media produce a
more delicate type of growth which succumbs more readily to
dessication. The organism was grown on sterile muck until
the soil was thoroughly filled with a loose growth of
mycelium. Samples of this were removed, placed in sterile
filter paper and subjected to drying at room temperature.
Plating brought out the fact that the mycelium under these
 
-41-
conditions was not living at the end of 35 days but was
still capable of producing growth at the end of 28 days,

With many organisms, it is unquestionably truethat
longevity of spores subjected to drying on glass surfaces
furnishes noreliable index as to their persistance in
nature. The longevity of fungus spores as well as bacteria
depends largely on the medium upon which they are subjected
to drying and also on whether or not they are imbedded ih a
gelatinous matrix. Hansen has found that Pseudomonas
radicicola dried on filter paper or cover-glasses died within
14 hours, while on seeds it lived 14 days, and, in the dried
nodules of certain legumes, it was able to grow after two
years of dessication. Yeast cells shew a similar but less
striking response and the same is true of many fungi.

Spores of Phoma apiicola were tested in the usual
manner by drying them on coverglasses, and then germinating
them in broth. Under these conditiens, 3 days were
sufficient to eliminate all signs of life. Other methods
have been employed to more nearly approximate some of the

conditions to which spores might be subjected in nature.
Celery seeds were treated with mercuric bichloride,
thoroughly washed, inoculated with spores and subjected to
drying on sterile filter paper. The seeds were placed

in broth at regular intervals. Speres germinated after 38
=42-
days Gessication; above this, the limit of resistance seens
to be reached and ne more germination is obtained. To
eliminate any inhibiting or germicidal effect, which might
be produced by traces of mercuric bichloride untreated seeds
were inoculated with spores, divided into two lots and sub-
jected to drying. Platings were made from the first lot
while seeds from the second lot were planted in pots at the
end of every seventh day. Platings gave, substantially, the
same results as obtained in previous experiments. In the
first three plantings of seeds, infection was produced in
the seedlings; the fourth planting gave negative results,
and subsequent plantings failed to produce infection in the
seedlings.

Phese results merely indicate that free spores
exposed on the surface of celery seeds, are not able to
survive dessication at room temperature for a period longer
than 30 days, because of the many varying factors, they
cannot be taken as an accurate index as to the time which
must elapse, before infected seeds may be considered safe.
Spores used in the above experiments were produced in pure
culture. Under such conditions, there is always the
possibility of the spores being less resistant than those
produced on the host plant. Under natural conditions, the
spores discharged are imbedded in a gelatinous matrix which

would serve to make them more resistant to adverse conditions
-435-

of environment. When we consider these differences and

the fact that pycnidia may be produced on the seeds,
possibly existing there as immature fruiting bodies capable
of later producing spores, it seems extremely likely that,
while ordinary conditions of dessication may be important
in killing large numbers of superficial spores, there is not
enough evidence to conclude that dessication is effective
in entirely freeing seeds from the living organisn.

Oxygen Relations.

The seat of this disease is near the surface of the
soil regardless of whether leaf stalks, crowns, or roots are
located at that place. Light is evidently not an important
factor in this restriction. Experiments early indicated that

1 H- tubes

the organism is an aerobe. Inoculation to Giltner
produced he growth and broth cultures covered with paraffin
oil showed the same results. Very little growth was produced
on oatmeal agar in test-tubes when the tops were sealed with
paraffin though the volume of air, in such cases, was many
times that of the fungus. In tubes of sterile muck, the

depth to which the fungus penetrates can be regulated by
changing the compactness of the soil. In very loose muck,

the fungus seldom penetrates deeper than an inch and a half; mn
more compact or water. logged soil the growth is largely
superficial. "Phe same observation has been made in flasks
where, in order to get thorough penetration, it is necessary

te have the proper amount of moisture and to aerate the soil

by shaking the flasks at frequent intervals.

 

1. Giltner, We. Microbiology, p.- 160--162. 1916.
Tat.
~44<
To determine the effect of introducing large supplies
of oxygen, an experiment was planmdd to be set up in the
following manner: Potted plants, having a well established
root system, were allowed to dry to the wilting point. They
were then taken from the pots, none of the soil around the
roots being disturbed, and soaked in water having a heavy
charge of spores. After being placed in 6-inch battery jars
and well tamped in with more soil, the entire mass was then
drenched with a spore spgspension. Glass tubes, bent in such
a manner as to deliver air currents upward, were placed in the
bottom of the jars and connected with an air line. The amount
ef oxygen was regulated by allowing the air to bubble through
water tn a filtering flask. An average of about 120 bubbles
per minute was introduced for a period of two months. Checks
were run where no oxygen was supplied and also where ne
{inoculation was made. When a difference in the size of tops,
between the plants supplied with oxygen and the checks,
became noticeable they were removed, washed and examined for
signs of disease. Plate VI shows a picture of a typical
Plant supplied with oxygen. The roots were attasked at many
different points and many of these rotted off two or three
inches from the crown; smaller roots had suffered severely
and the whole root system was in an advanced stage of decay.
The presence of hundreds of pycnidia scattered over the root
system furnishes proof of the organism causing the destruction.

On the inoculated check plants, the disease was produced only
~45<

around the crown; the roots were as thrifty and as free from
disease as in the case of the uninoculated plants.
This air relation, We believe, largely explains why

reets a short distance from the crown are usually free from

disease. Excessively high moisture content in the soil, since
it drives out the air and makes less oxygen available, would
tend to still further restrict the susceptible area, though
relatively wet conditions are requisite for mycelial

proliferation. Lack of moisture, in nearly all of the celery

growing distriots where the fungus is a pest, drives it from
the aerial parts. Thus, the physiological relation of the
fungus to these two environmental factors, namely water and
air, determines the type of disease which is produced, and the
part of the host plant which is dangerously subject to attack.
Relation to the Reaction of the Mediun.
Several different media have been used in determining the

relation of acidity and alkalinity to the growth of Phoma

apiicola. Celery agar, nutrient broth, and Coons! synthetic
solutions have been employed in several experiments.

With the solid media used, a neutral or slightly acid
reaction is most favorable for growth. The same is true of
the liquid media used but the initial reaction for production
of most growth varies with the chemical composition of the
medium. This can be brought out more clearly by a description

of a typical experiment. Flasks of nutrient broth were prepared
-46-
to range in reaction from -30 degrees to +30 degrees

Fuller's scale. Each flask wasinoculated with a small bit

ef mycelium and inoubated at room temperature. In the
following table, results are given for different periods
of time on a percentage basis, reckoning the flask showing
best growth as 100%.

TABLE 5.
Effect of Acidity and Alkalinity on Mycelial Growth.
Reaction of Medium Percentage Development Based on
Degrees Fuller's Flask Showing Largest Myeelial
Scale Mass.
2 weeks 4 weeks 8 weeks.
-30 5 5 5
-20 10 5 5
-10 25 50 50
0 100 90 . 90
+10 90 100 95
+20 50 70 100
+30 5 5 5

At the beginning, there was a much more rapid growth at
the neutral point. <As development continued, those flasks
having an initial reaction of +10 and +20, respectively
replaced the neutral flasks in quantity of mycelium produced.
This came about, both through a gratiual accektration of rate
of growth in the acid and a slowing up in the alkaline media.
-47~-

Flasks, neutral ani alkaline had practically ceased growth
at the end of four weeks. In those having an acid reaction,
the fungus continued a fair rate of growth up to the end of the
eighth week. The slowness of gpewth in the acid flasks at the
beginning was no doubt due to an unfavorable reaction, the
longer period of growth being made possible through the
breaking down of protein molecules into alkaline compounds.
This decomposition, brought about through the activities of
the fungus, had the effect of making the reaction of the
medium increasingly more favorable for growth up to the
point et which the alkaline compounds began to exert a
deleterious influence, and then, of slowing up growth until
the medium became too toxic for further development. Thus,
in a Single flask, the organism was able to pass from the
limits of acid tolerance on the one hand to an alkaline
solution on the other. Various toxic substances thrown off
by the fungus were no doubt important in stopping growth
before the outer limits of alkaline tolerance were reached,
since, titrations, at the end of the experiment, proved
that the flasks having an acid reaction at the start were
only slightly alkaline. Inoculated flasks of broth, acid to
#20 degrees, upon being titrated every seventh day, showed
@ gradual change in reaction up until the end of the eighth
week, when they were 6 degrees alkaline.

A series of deep culture dishes, with Coons’ synthetic

solution as the mutrient medium, were inoculated to determine
 

 

~48-

the effect of reaction of the medium on pycnidium production.

 

TABLE 6.
Reaction of Medium Comparative Development Based on
Degrees Fuller's Greatest Number of Pycnidia and
Scale largest Mass of Mycelium.
BS Pyonidia SO Mycelium ;
~20 5 5
-10 50 75
0 90 100
+10 100 85
+20 0 slight
+350 0 slight

fhe limits of fruiting seem to be as wide as those
for mycelial development. The neutral medium again
markedly favored vegetative growth, while solutions slightly
acid brought forth the largest number of pycnidia. Phe
salient features io these results is the failure of the
solutions acid to #20 to produce growth in measurable
quantities. <At first glance, this seems contradictory to
results obtained in broth cuitures. However, the solution
used in this experiment has more carbohydrate and less
protein in its composition than has beef broth. The
probability is, that, on account of the nature of the mediun,
the fungus was unable to adjust the reaction in its favor
and that +20 is too acid for growth.

fhe cultures at differant reactions produced a noticable

series of color changes; though there is not such a wide
 
~49-
variation as is produced in the case of extreme temperatures.
In the alkaline solutions, the fungus produced a pink dis-
coloration, which is never observed in neutral or acid
solutions, and the color of the mycelium varied from an
almost white to a light blue color. The acid solution

produced a bluish or bluish-green myceliun.
DESSEMINATION.

The restriction of the pychidia of the causal organism
to the roots and crown of the host plant makes air currents
of little importance in the dissemination of the disease.
The gelatinous matrix in which the spores are imbedded
necessitates a water distribution. When pycnidia are mature
and the spores pushed out in kong tendril-like threads, the
jelly-like matrix is dissolved away leaving the spores free
to be washed through the soil.

The importance of this method of dissemination will
depend largely on the frequency of heavy rains and on the
amount of surface drainage. When the disease is present
in the seed bed spores are disseminated by handling plants
at transplanting time. It is quite a common practice to
take plants fram the seed bed and place the roots in water
in a shallow pan until they can be placed in rows in the
field. Under such conditions a single diseased plant,
bearing pycnidia of the pathogene, would be capable of
infecting an enormous number of sound individuals.

With an organism capable of living and growing for a

period in soil, the question of lateral dissemination by
@50=

means of the growth of mycelium through the soil must be
considered. To gather experimental evidence on this point
plants in one side in each of five average size flats were
inoculated in the greenhouse and checks placed in the same
flats at a distance of 4 to 12 inches fram inoculated
plants. Watering was done with a sprinkler and the soil
was kept moist enougn for a good growth of celery. After
three months the plants were pulled up and washed free fra
soil. Of the inoculated plants 96% were diseased; 2% of the
plants 4 inches fram inoculated plants were attacked, and all
other uninoculated plants were free fram disease. Pot
experiments and observations on checks used in the greater
part of greenhouse work have confirmed these results. It

might be pointed out that in the above experiments con-

ditions were wery favorable for spread of mycelium as

the soil was kept damp at all times. Due to its oxygen
relation the fungus can penetrate soil only to a short depth,
and since in the field surface soil is often dry the spread
from plant to plant by means of mycelium will assume even
less importance than in the greenhouse.
VARIETAL_SUSCEPTIBILITY.

The whole question of varietal susceptibility is
one which will require more searching investigation and critical
observation to solve the problems here presented. Van Hook
reports that Giant Pascal and Evan's Triumph are much freer
from the disease than is Golden Self Blanching. The most

destructive outbreaks of the disease in Michigan have been

on Golden Self Blanching celery, but unfortunately in
-5l-
these cases, not other varieties have been present in
the infested districts on which to make observations,
or with which to make comparisons. White Plume and Easy
Bleaching are knownto be subject to attack under field

conditions; other varieties have such a limited use that

it is unwise to draw any conclusions regarding their pre-
disposition to the disease, other than from results obtained
from the inoculation of a limited number of plants.

A variety test was conduced using Perle La Grand,
Smallage, Schumacker, Dreer's Mammoth, Perfection's
Heartwell, Dwarf Golden Heart, Winter Queen, Rose Ribbed
Patie, Golden Self Blanching, Boston Market, Columbia and

Golden Heart. The method adapted for testing these

varieties was to place 25 plants of each variety in a
separate flat and thoroughly spray them with spores.

Indications of infection first became apparent on
Golden Self Blanching and Golden Heart plants through

a dying of the lower leaves and a general checking of
growth. In time, the same symptoms were to be noted on
all other varieties except White Plume and Giant Pascal.
All inoculated plants bf these two varieties, upon
examination, proved to be diseased; but the characteristic
symptoms were not to be seen on the parts of the plants
above ground: <At the base a thin ring of black diseased
tissue showed that the fungus was active but was not
-52-
seriously interfering with growth. In the case of the
other varieties there seemed to be no great difference in
susceptibility.

As a result of these tests we feel justified in
saying that though none of the common varieties are immune
to this disease, some give indication of being more
resistant than others. Golden Heart, Golden Self Blanching
and Dwarf Golden Heart, seem to be the three varieties which
suffer most from the disease. White Plume, Giant Pascal,
and Easy Bleaching give indication of being more resistant.
These last three varieties are large, rapid growing plants
of a tougher texture. Herein probably lies the secret of
resistance. Succulent plants of any variety are more
susceptible than plants of a tougher texture. Similarly

different species of Umbellifers seem to correlate resistance

with woodinesg.
gee =D aD Gp oS GP swe =e eu a SPP lel || lel eS ee eee

In tracing the detailed hife history of the parasite
in relation to disease production, it is well first to
consider the periodical development of the disease in the
greenhouse and under field conditions. Phe first plant
infection quite often came in the greenhouse or in the
out-door seed bed in the early spring. Clean plants of
the first crop may be attacked after they are transplanted
to the field. The second crop of celery growing during
midsummer is from serious attacks. As cool days offall
come on the Phoma root rot fungus again becomes active and

may produce serious damage in the last crop.

There are four possible ways in which the causal

organism may pass the winter: (1) in soil or trash of the
greenhouse or in the cold frame; (2) the refuse of the
previous year's crop in the field; (3) as a saprophyte in
the soil; (4) on seeds, and (5) of course, there is elways
@ possibility, with an organism of this type, that there

is an undiscovered perfect stage produced. As to the first
possibility the fungus is know to persist through one
season under such conditions. In case of the most serious
outbreaks of the disease at Kalamazoo, Michigan, the green-
house was the location where infection took place in two
successive years and the point from which the disease was

carried to the field. The source of the organism for the

initial infection is unknown, but the appearance of the
 

disease in a very virulent form the record year points
strongly to the fungus having lived over in the soil or
trash of the seed bed. In the greenhouse at Michigan
Agricultural College, plants became diseased when placed
in soil from around diseased plants, after this soil had
been kept in flats through the winter up to February.

There has been some difference of opinion fagarding
the importance of trash in harboring the organism. Klebahn
is inclimed to the belief that seeds are very important in
the distribution of this disease, while Quanjer and
Slagter minimize seed carriage and state that the chief
source of infection is to be found in the trash, manure, etc.
fo determine something of the importance of trash and also
to determine whether or not the organism lived as a sapro-
phyte in the 3011 through the winter an experiment was started
September 1919, on the following basis. Seven large flats
were placed outside to go through the winter. MTwo of
these contained gwowing plants which had been inoculated in
midsummer and which were know to be diseased, in two othars
were diseased plants pulled from the soil and left on
the surface; two contained mck soil, mixed with sterile
muck on which the fungus was growing. In one flat diseased
bases of plants were placed between layers of thick filter
paper and covered with soil to depths varying from 1 to 6
inches. April 1, 1920, the first six flats were taken
into the greenhouse and planted to celery. By May 1, the
plants in the four flats containing trash were showing
 

 

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signs of disease. The two flats containing soil inoculated
with a pure culture of the pathogene did not produce the
disease indicating that a long?’ “Si stance under the
above condition at least is not common. The diseased parts
in the remaining flat were examined for pycnidia and

for a possible perfect stage. The latter quest was wholly
unsuccessful, bgt pycnidia centaining an abundance of spores
were found on nearly all diseased parts. These spores
germinated readily in tap water and indeed many of them

had apparently germinated between the layers of filter
paper as masses of bluish mycelium were everywhere present.
Phis mycelium after isolationcorresponded to that of Phoma

and
apiicola produced disease when placed in contact with celery

plants.

Before the late "ara great pertion of the celery
seeds used in the United States are grown in Holland and
France, where this disease is most destructive. Since
this is apt to continue to be one of our chief sources of

seeds, it is a question of first importance to determine

whether or not the causal organism is seed borne.

Klebahn has found pycnidia of Phoma apgicola on the
seeds and suggests that seed distribution is important.
Quanjer and Slagter as stated before incline to the
belief that infection from the seeds is exceptionable.

This latter view seems to fit in well with the facts

as observed in Michigan. The disease has, no doubt, been
~56=
introduced from Europe on the seed or on trash present

with the seeds, but the occurrence of the fungus on seeds

is probably rare. Examination of French, Emglish, and

Dutch grown seeds has not shown pycnidia to be present.

The infrequency with which the disease is found in the
seed bed indicates that in the case of seeds shipped to
America from infested European countries seed distribution
does not commonly occur. While not qften carried on the
seeds, sporadic outbreaks of the disease in one or two
instances indicate that the seeds in some cases may be
heavily inoculated. It is probable that seed carriage

is important in introducing the disease into new
territories and in occasionally being the source of bad

seedling infections, but as a means of annual distribution
of the disease, it is probably of minor importance.
 

 

~57~

CONTROL.
Because of the fact that plants are attacked at a

point Bocated below the surface of the soil, none of our
common sprays which are used to fight other diseases of the

celery plant are effective against this disease. Control
measures must be based largely on certain relations of the
disease to environmental conditions amd on the application
of sanitary principles. In applying such measures it is
well to keep the following points in mind: (1) While some
varieties of celery are more or less resistant, none are

known to be wholly immune; (2) fhe disease may occasionally
be seed borne; (3) low temperature and high moisture

content in the soil favor the production of disease;
(&) attacks are most severe in the spring and fall; (5)
greatest injury comes from attacks on small plants;
(6) older plants are not so seriously affected; (7) trash
of the greenhouse and field are important sources of
infectious material.

It is out of the question to rely on any of our common
varieties to resist the attacks of the fungus. The
varieties of best quality seem to be most susceptible to

the disease, and some of the poorest quality seem to be
 

-58<

most resistant, but not enough difference has been foynd
to justify the grower in sacrificing quality for resistance.
The possibility of the disease being seed borne leads
to the. question of seed treatment and sources of contaminated
seeds. Phoma root rot has not been reported from any seed
producing section of the United States. If it is present
in any of these districts it is evidently causing little
damage and chances of seed infection would be very small.
European grown seeds sare more apt to carry infection and
treatment of such seeds may prove to be a good insurance
measure against introduction of the disease into the seéd-
bed.
A low temperature, combined with a high moisture content
in the soil, is necessary if the disease is to become of

economic importance. lin seed beds where conditions can

be controlled as in the greenhouse, we believe that it

would be possible to check the disease and in many cases pre-
vent infection by limiting the water supply and keeping the
temperature relatively high; but, since the seedling stage

is the critical one for infection, it is dangerous to rely
on this treatment. If seedlings of the first crop are kept
free from infection until they are transplanted to the field,

it is believed that in an average year the disease, cawed by
 

-59-
infection from field sources, is not capable of producing
@ great amount of destruction before its pragress is

arrested by the warm weather of su:mer. It is far safer

to keep watch of the seed bed and if the disease appears
change or sterilise the soil. In the greenhouse, if the

soil is changed, the benches should be drenched with a
formaldehyde solution (1-40f. This strength of solution

be
has been found to wery effective in killing spores and

mycelium of the causal organism when this fungus was grown
in sterile muck. Drenching diseased soil with the same
strength of solution would no doubt prove effective in
destroying the funzus. Where facilities are available,
steaming soil will pay. The organism is very sensitive to
high temperatures and this method of freeing the soil from
its presence should prove to .be very satisfactory.

In the case of the fall orop, field infection may be
a source of considerable loss. In dealing with this, it may
be necessary to practice rotation and to remove all diseased
trash from the crops of celery which are grown in the
rotation. Under field conditions it is not know how long
the causal organism may persist in the soil and trash, but
removal of diseased parts from the field should decrease
considerably the amount of infection. Orep rotation would
seem to be a very effective method of control in sections

where land is available and celery can be replaced by crops
 
wn

50 —

equally valuable. In districts where rotation is not practiced
it is believed that the removal of diseased trash and the use
of disease free plants as mentioned above, will hold the
disease in check and insure the grower a crop comparatively

free from this type of root rot.

SUMMARY
Phoma Root Rot is a disease of celery and celeriac
known both in Burope and America and is caused by the same
fungus (Phoma apiicola) in both cases.
The fungus also attacks parsley (Carum petroselinum) ,
parsnip (Pastinaca sativa), carrot (Daucus carota), and
caraway (Carum carvi).

The disease comes in on the crown of plants and causes

dead leaves at the base, "stunting", and a pinching off near
the surface of the soil.

The causal organism requires a relatively low temperature,
(optimum about 18°C) and abundance of moisture and a large
supply of oxygen for best growth.

The disease reaches its maximum of destructiveness in
the spring and fall, the hot weather of midsummer checks
the advance of the fungus and gives a clean crop so far as
this trouble is concerned.

Overwintering is knowm to take place in the trash of
the greenhouse and in the trash of the field.

Control measures recommended, the use of disease free
plants and the destruction of trash, which harbors the pathogene

or when the disease is severe the rotation of crops.
 

BIBLIOGRAPHY. *

Coons, G.- He Annual Report State Board Agr. p. 28, 1917.
Coons, G. H- A Phoma Disease of Celery. Report Mich.
Acad. Sci. p.- 444. 1918.
Halsted, Be. De Ann. Rept. Ne Je Agr. Exp. Sta. p.e 253, 1891.
Klebahn, Yon H. Krakheiten des Selleries.
Zeitschr. fir Pflansenkrankheiten. 20: 17-39. 1910.
Quanjer, He M. and N. Slagter. De Roest of Schurftziekte
wan de Selderie knol en Enkele Opmnerkingen . over
Andere Selderieziekten. Tijdschr. over Plantenszeikten.
20: 13-27. 1914.
Van Hook, J. M. Celery Root Rot. Ohio Agr. Exp. Sta. Cir. 72.
1907.

*®his bibliography contains only those references which
bear directly on the disease in question, others are given

simply as footnotes.
 

Platss [L-XII

 

 
Plate I.

 

 

 

Fig. 1. Golden Heart Celery inoculated

With Phoma apttcola from Holland and

Michigan.
Plate IL.

 

 

 

Fig. 2. Diseased celery plants.

Inoculated artificially with mycelium
PLATE III.

 

 

 

 

 

SF / . 7
4 i
|
{
|}
|
| ‘
| |
| |
oe
l i . y
| ; 4 4
-
vy {
; | :
e Se and undiseased Fig. 4. Diseased plant
Fig. 3. Diseased (left) eee atta lc

(right) plants of the Same age
(natural size).
PLATE IV.

 

 

 

Fig. 5. Diseased spots on Celery leaves. These leaves were inoculated
by spraying with germinated spores. Infection took place
under a bell-jar.
 

 

Pigs 6. Diseased Crown (Enlarged 6 times).

Pycnidia are shown on the base of one
of the leaf petioles.
 

SE

Pige 7. Effect of Oxygen on root attacks.

The plant at the left was not inocud&ated; the
one in the middle was inoculated with spores and
supplied no oxygen; the plant on the right was
inoculated with spores and supplied oxygen from
below. Note the scarcity of small roots and the
diseased condition of the larger ones.
 

PLATE VII.

 

 

 

Fig. 8 Parsley artificially inoculated. Fig. 9. Carrots (left) and

parsnips (right
artificially inocula ted
PLATE VIII.

 

 

Fig 10. Showing type of lesions on parsnip caused by
artifical inoculation.
PLATE IX.

 

 

 

Fig. ll. Group of pycnidia from leaf petiole
(magnified 150 times).

 

Fig 12. Pycnidium imbedded in host tissue.
(maenified 200 times).
PLATE X.

 
 

Fige 13. Pycnidia on filter paper with Fig. 14. Growth on rice.
corn meal broth (above) and Coons'
synthetic (below).
PLATE XI.

 

Fig. 15. Mycelium on sterile soil.

 

Fig. 16. Showing growth on celery agar at
different temperatures. (The ranze of
temperatures is given on page 3i.
PLATE XII.

 

Fige 17. Colonies on agar in Petri dish.
(Note rings of pycnidia).
 
 

 

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