A STUDY OF THE CELERY EARLY
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A Study of the Celery Early Blight fungus,

Cercospora apii Fres.

Thesis Presented for the Degree of
Master of Science,

Michigan Agricultural College.

6 2
Le. J. Klotz,
1921.
BHesis
ACKNOWLEDGMENTS.

The writer is grateful to Dr. E. A, Bessey
and to Dr. G. He. Coons for advice and suggestions
given throughout this study and for the criticism
and correction of the manuscript.

To Dr. R. C. Huston the writer is indebted
for the directing of the chemical work and for aid
in preparing the report of the analyses.

Acknowledgment is made to Dr. G H. Coons
and Mr. R. Nelsm for photographs, and to Mr. Jd. E.
Kotila who did most of the photographic work.

4 O2335
| TABLE OF CONTENTS.
Introduction.
Name of Disease,
History and Distribution.
Economic Importance.
Hosts.

Signs of the Disease-
General.
On Leaves.
On Petioles.
On Fruit.

Etiology.
Proof of Pathogenicity.
Morphology.
Mycelium.
Sclerotium-like bodies.
Conidiophores.
Spores.
Name of Causal Organism.
Life History of Causal Organisn.

Relation of Parasite to Host.
Mode df Infection.
Relation to Light.
Water and Temperature Relations.
Pathological Histology.

Physiological Relations.

Effect of the Early Blight Fungus (Cercogpora
apii Fres.) on the Nitrogen Constants o
elery, (Apium graveolens L.)
Cultural Characteristics.
Light Relations, :
Growth on Muck and Celery Refuse.
Temperature Relations,
Vegetative Growth.
Spore Germination.
Thermal Death Point (Mycelium, Conidiophores, Spx
Relation to Reaction of Med ium.
Oxygen Relations.
Resistance to Desiccation.

Dissemination.
Varietal Resistance.
Control.

Summary.
Bibliography.
Description of Plates.
A Study of the Celery Early Blight Fungus,
Cercogpora apii, Fres.

INTRODUCTION.

fhe importance of celery as a truck crop in Michigan,
Florida, New York, California, Colorado, New Jersey, and
“several other states is well recognized. Fhe peat lands
of Orange County, California were set to over three
thousand acres of celery in 1910'7°) « fPfhroughout this
country the words "Kalamazoo" and "celery" have been closely
associated for the past twenty-five or thirty years. Ottawa,
Kalamazoo, Kent, and Muskegon are the counties in which
the industry is most developed in this state. Considerable
celery is also grown in truck gardens near Bay City, Detroit,
Newberry, and in smaller quantities by truck growers through-
out the state. The possibility of the furthe development
of the industry is indicated by the fact that there are
in Michigan alone some three million or more acres of
adaptable muck and other lands. The obtaining of seed from
many sources, domestic and foreign, in which the natural
enemies of the plant have long been known, together with
the long continued raising of celery year after year with-
out rotation, have led to the establishment in this state

of several serious fungous diseases to which celery is

subject. Among these is the disease known as Eerly Blight,

the importance of which, in hot seasons, makes desirable

its further investigation.
Name of Disease.

fhe disease has been called by several common names

among which are "celery rust" (56) | "celery blight™(1,36,54) |
(15)
9

{ 24)

"celery leaf blights! 4.78) “san blight of celery"

(19,20)

"early blight of celery » “celery leaf spot"

and “celery spot mou1a®!22) |
discarded due to the fact that at least two true rust fungi

have been recorded as attacking celery! ’?), The word "spot"

The name "rust" has long been

is not charactéristically descriptive because of the
spotting of celery leaves caused by several other fungi.
The disease would best be popularly known as the "Barly
Blight of Celery" because of its early appearance during
the growth period of the plant, that is, during the hot
months of July and August. It should never be designated
as simply “blight"™ due to the easy confusion with another
destructive celery disease known as "Late Blight" which

appears the later, cooler part of the growing season! 12,70,9,10)

Histo and Distribution.

fhe first report of the disease in the United States
which the writer has been able to discover was made in 1881
by Galloway!0) | in which he has said that practically all
of a lot of 10,000 plants were destroyed. At this time he
also observed that shaded plants escaped destruction. In
his 1888 report he stated that his observations were that
"the disease occasions greatest injury in sections where
~Ze

the summers are long, hot, and dry", and that the disease
was unknown where the soil remained cool and moist. He
recommended shading with lath screens and destroying the
diseased material in the autum. Ellis and Everhart (1885)
described the fungus from specimens collected in Micigan
by Dr. Beal! 22) , Soribner in his 1886 report described the
disease and its causal organism and stated that no satis-
factory fungicide had as yet been founa!78) , Brillieux
and Delacroix (1890) reporteé the disease as occurring

on celery in the experimental garden of the Agronomy Insti-
tute at Joinville le Pont, France. They stated that the
fungus had already been reported on various other Umbelli-
fers, particularly parsley. The appearance and cause of
the disease were described, as were the fungus spores.

Note was made of the fact that as the weather becomes cool
the disease becomes less virulent. The authors called
attention to the method of attachment of the spores by the
larger end, correcting the figure of Scribner which pictured
the spores attached by the small enas ©”). Halsted (1891)
found that the "fungus spores germinated with remrkable |
rapidity in water and in celery extract". Copper filings
placed in a drop of water with the spores inhibited
germination, whereas metallic zinc was apparently non-toxic,
not preventing the formation of germ tubes. In 8 small
spray experiment, in which he used ammoniacal copper oar-

bonate, he obtained almost complete control of the disease,
~~

the amount of edible product from the spayed celery being
twice that from the unsprayed plants! 36.47) |

McCarthy (1892) reiterated Galloway's observations
on the relation of Early Blight to soil, shade and moisture
and stated that celery cannot be successfully grown on
upland soils without irrigation. In his rules for combatting
the disease he advised making the seed bed in the shade,
planting some shading crop as Russian sunflower in the field,
using liver of sulfur spray or sulfur dust throughout the
growing season, and carefully destroying all diseased leaves.
Galloway (1892) as a result of a number of experiments found
that proper cultural methods more readily prevented leaf
blight than any or several of the various fungicides used.
He did not, in this report, state which fungicides were
employed, but judging from his 1888 report they were lime
and sulfur, Bordeaux mixture, liver of sulfw, ammoniacal
copper carbonate, sodium hypochlorate, and sulfur flour.
He emphasized an abundance of water, good drainage, and
@ heavy application of well-rotted manure!31), Stur gis
(1692, 1893, 1697) conducted control experiments in which
he fowmd sulfur dust superior to Bordeaux mixture, liver
of sulfur, ammoniacal copper carbonate and dilute solutions
of copper sulfate! 82,85,84,85) | Davis of the Michigan
station (1893) published Galloway's notes on the"Celery
Leaf Blight” which stated that complete control was realized
> ee eee ~
-5-

By flooding the ground sufficiently often to keep it always
soaked, whereas the plants which received only the water
falling naturally as rain blighted badly'15) °

Rolfs (1896-1898) found that crowded, poorly nourished
plants succumbed very readily to the disease and that the
blight spread with alarming rapidity during warm, moist,
foggy weather. As sprinkling the plants wets the foliage
and supplies the moisture necessary for spore germination
he advised flooding and trench irrigation. liver of sulfur
(1 oz. to 2 gale water) applied twice a week, thoroughly
wetting the plants, gave almost canplete control, as did
freshly prepared Bordeaux mixture applied at 10-day or
two-week intervals! 71,72 ), Duggar (1897) grew the fungus
in pure culture, using as media nutrient agar, celery petioles
and bean stema'19,20), tochnead (1900) found that intense
heat agcompanied by drought favored the disease. He, as
had Galloway, observed that celery on shaded, low, wet
ground escaped. Ammoniacal copper carbonate (6 oz. cuc0,
to 1 qt. ammonia water and 45 gal. water) sprayed on at
two week intervals was recommended! 54) . Hume (1899,1900)
by placing celery leaves in moist chamber, sowing the leaf
surfaces with cmidia, ahd then after 48 hours stripping
off portions of the epidermis and examining under the micro-
scope, demonstrated the entrance of the parasite through
the stamata. He recommended 4-4-40 Bordeaux mixture
applied twice weekly the first part of the season and later
~6=

at one week intervals. He preferred this to liver of
sulfur because of Bordeaux being the cheaper; he found
Bordeaux to have slightly greater fungicidal value than
ammoniacal copper carbonate (39),

Other contributions which do not add particularly
to the above, but which should be mentioned, are the
following:

Tracey (1864, 1885, 18686) noted "considerable damage"
to the celery crop at Columbia, Missouri(78) , Chester (1891)
told of his mistaking septoria blight lesions for those of

cercospora (9,10) Humphrey (1891) (40) and Lindau (1908) (53)

believed that septoria and cercospora blights of celery
genetically connected. Atkinson (1892) gave a gooé drawing
and description of the fungus and corrected the error of

(1,2),

Soribner’s figure Briosi and Cavara (1895 p.57)

accurately described the disease and its causal organien!”),
Hoack (1898) reported the presence of the disease in Brazi1!61) ,
Kellerman (1904) also errored in pieturing the spores as
attached by their small ends to the conidiophores!48) ,

Freeman (1905) very briefly described the effect of the fungus
on its host, saying that it was particularly effective against
young plants. He recommended Bordeaux mixture (27), Cooke
(1906) recorded the fungus under the common name of "celery
spot mould", stated its injurious extent in Germany, Austria

and the United States, and that it had not yet developed as
~7~

a pest in Englend?ll), giehahn (1906) mentioned the
presence of celery Early Blight in various localities of
Germany/ 50) , Fowmsend (1907) pointed out the seriousness
of "celery blight or rust” and claimed complete control by
the use of either Bordeaux mixture or ammoniacal copper
carbonate(88), wassee (1903,1910) described the disease
under the heading "Celery and Parsnip Leaf Blight" and
states that spores live through the winter, being capable of
infecting a crop the following season and inferred that rain
is the active agent of dissemination (56,57). Eriksson
(1913) after a discussion of the leaf spot of sugar beets
mentioned that similar spotting is caused by Cercospora

apii en carrots, parsnips, and other Umbellifers, (24) ,
Stevens (1913) described the diseased host and fungus '80) ,
Ferraris (1915) also described the parasite and morbid

host and advised the burning of the diseased Leaves! 25) |
Taubenhaus (1918) repeated a description and stated that the
"White Plume variety seemed to be resistant"!87) , Krout
(1919) compared the two celery blights and recommended
spraying with Bordeaux mixture for both, (51,52) ,

From the above contributions coming as they do from
England, Germany, Austria, France, Italy, Siberia, Canada,
South America, and many states of this comtry, it is reason-
able to infer that. the disease Celery Early Blight has been

present at some time, in mild or serious form, wherever
 

-~8=

celery is grown extensively. It is nearly always possible
to find few to many Early Blight grots on celery shipped
into Michigan from Florida and California. This general-
ization is corroborated by the following report.

Economie Importance.

Im April 1921, at the request of Dr. G H, Coons of
this station, Dr. & R. Lyman of the Burean of Plant In-
dustry, Plant Disease Survey, kindly compiled data as to the
occurrence of and losses fram the Early Blight of celery.
This report, a brief of whieh is here given, embraces
thirty-two different states.

California reported “heavy losses and disastrous
presence” in 1907 and 1908, "general occurrence but no
damage in 1910", "severe (10% loss) in 1919" and Blight
(2% loss) in 1920", Robbins of Coloradé stated that losses
from Early Blight were “very slight" in 1917 and 1918,
Clinton (1913) reported the disease as “common and bad” in
certain fields in the state of Connecticut, 2% loss from
Barly and Late Blights in 1917, common but not very serious
for the years 1918, 1919 and 1920. Jackson, Elliott and
Manns noted presence in Delaware from 1905 to 1919 but
stated that it was not severe. From Florida comes many and
important reports: Rolfs reportcd 20% injury in 1905 and
80% in 1906; Winters noted some damage in 1908 and 1910.

H. BE. Stevens (3912) recorded the disease as "very bad",
~-Q-

10% té 50% of the crop having been attacked, as "rather
severe"in 1913, as "common" in 1914 and 1915, "less preva-
lent” in 1916, that in 1917 the injury was up to 100%

locally. In 1918 Hesler noted whole fields “brown as a
result of the blight". In 1919 Link and Jagger observed

the effect of a "deluge of Early Blight upon celery", stating
that the plants as a result are “short, small, and of much
reduced market value". The economic importance of the
disease in the state of Florida is very evident from these
notations.

Dean of Georgia reported “aomplete destruction" of
certain celery crops in Stewart County in 1904 and 1905:
that it was negligible in 1906. femple of the Jdaho station
recorded it as very prevalent in 1913, the injury being
1 to 15%. Barrett (1907) stated the disease as having been
very bad in one Illinois locality and (1911) as having dme
76% ingury to the crop in Champaign County. In 1913 the
presence of the pest was reported in Elkhart County, Indiana.
Jacksm of the same state reported 25% injury in Marshall
County (1916) and as common in Elkhart, Tippecanoe and
Madison Counties in 1917. Gardner noted 10% injury to
Marion and Elkhart Counties’ crop in 1919 and similar loss
in 1920. Kentucky reported the disease as very prevalent
about Lexington, injury being 10%. EdAgerton of Louisiana
recorded the presence of Early Blight from 1908 to 1917,

reporting losses and injury ranging from "slight" to "con-
 

-10-

siderable". Morse (1988) noted its presence in Andros-
coggin County, Maine and Shapovaloyv recorded a "bad out-
break" in Penobscot County. Phe reports from Maryland
(1903-1917) indicate but slight loss, although severe in
some localities. Massachusetts reported considerable loss
in 1906, as "present but not serious" 1907 to 1912, as
"doing large injury” in 1915, and that the losses ranged
(1917-1920) from very slight to 4% (Davis of the Michigan
station (1893) stated that "it took nearly the wiole crop
at Kalamazoo several years ago".)* The same station
reported the common presence of thedisease (1903-1918) and
stated (1914) that the "damage done was less than from
Septoria". Minnesota (1908-1918) reported very little
injury by Cercospora and Septoria. Reed of Missouri recorded
the disease as being very serious in St. Louis Cowmty in 1911.
Hebrasks noted its presence in Lancaster, Gage, and Buf falo
Counties (1905-1917). The New Hampshire station reported
20% injury in 1906, 5% in 1908, "not seen” (1909), 15% injury
in 1910, and "practically none" in 1911. New Jersey noted
the prevalence of Early Blight (1906-1909), recorded as
serious in South Jersey in 1912, as “common" and "abundant"
in 1919 and 1920. |
Jagger of the New York station stated that "losses from
celery blights were at least 5 to 10% in 1917. Other reports

of losses from this state range from "no reduction in yield"

 

“Hot in Pathologist's Report.
-lle

to "common but not severe". Stevens and Pulton of North
Carolina notéd its presence (1904-1916), recording "much
injury" to crop in 1913. North Dakota reoorded Early
Blight as "occurring" in 1906, "rare" in 1909, "commén"
in 1916, and “uncommon” in 1917. Ohio in 1905 reported
10% injury due to the Cercospora blight, 5% to 10% in 1906
due to both blights, as negligible fran 1907 to 1911, both
blights as causing "considerable injury" (1912) and as
"less" (1913-1920). From Roseburg, Oregon, N.D. McCall
wrote that the celery disease due to Cerocospora apii
caused widespread injury in 1908. Pennsylvania reported
less than 5% injury in 1903, as "slight" (1906-1912), as
"present" and causing but "slight loss" 1915-1919, and

as very bad, “causing 50% loss in some fields" in 1920.

Stene of Rhode Island reported 5% loss in 1903 and quite

 

commen in 1907. The presence of the disease is noted

as common in South Carolina for 1909-1919, causing 5%

‘loss in 1917. Tennessee noted the presence of Early
Blight in 1915. Utah reported (1918) "no measurable loss".
Lutman of Vermont (1915-1920) recorded the disease as
being "very common but not serious” and as causing injury
ranging from 5 to 75% and losses from "negligible" to 5%,
Virginia (1911-1916) reported it as "not common" and
causing not more than 2% injury. West Virginia wrote

that it had occurred in small amounts only. Jones of

Wisconsin recorded it as being "bad in some localities and
~1l2=

entirely absent in others" in 1910, and that it caused a
90% loss to some Milwaukee celeriac growers in 1909.

These reports, particularly the one from Florida,
prove that the Celery Early Blight dimease is of economic
importance. It is evident that at times the fungus can
cause slight or serious injury to its host wherever celery
is grown. Given an abundance of plant food and plenty of
rain celery makes a good vigorous growth in spite of much
Early Blight spotting. Under these conditions the principal
injury is due to the disfiguration of the leaves which
necessitates excessive trimming in the preparation of the
plant for market. The spotted leaves, moreover, dry out
much more rapidly than sound ones, thus causing greater
deterioration in shipment. Under less favorable conditions
the Barly Blight disease supplements the other factors
in rendering the crop of little or no market values.

Hosts.

Although in the above citations there are frequent
references which indicate that the same specific fmgus is
by many investigators considered responsible for the production
of similar diseases in other umbelliferous hosts, there are

no records of the results of work by weich cross- infection
was demonstrated. Thus far, I have been able to secure

infection by Cercospora apii Fres. on only two hosts, namely

celery (Apium graveolens L.) and Celeriac (Apium graveolens lL.
var. rapaceum D.C.). Repeated inoculations with pure
cultures of this fungus and long continued exposure to
very badly blighted celery of carrot,(Daucus carota L.),
parsnip (Pastinaca sativa L.), parsley (Petroselinum
hortense Hoffm.), dill (Anethum graveolens L.), carroway
(Carum carvi L.), Pimpinella (Pimpinella saxifragia L.),
coriander (Coriandrum sativum L.), and fennel (Foeniculum
vulgare Hill.) gave negative results. Likewise I have been
unable thus far to secure infection on celery using the
fungus isolated from carrot. It thus appears that the
Cercospora apii Fres. of celery is a strain or variety
distinct from those forms causing similar spotting on

other related hosts.

Signa of the Disease.

General.
The common term "blight" at once brings to mind the

picture of an afflicted plant, one whose outer foliage at
least hag the appearance of being scorched as if by the hot
sm. In fact, this is the sight presented by celery plants
under the worst conditions of Farly Blight (Plate III).

With the coming of droughty weather, diseased plants appear
to wilt and droop more readily than healthy ones. The older
stems soon fall and, coming in contact with the moist earth,
become rapidly filled with the fungous threads which spread
throughout the tissue in part saprophytically. These fallen
——_—e ee ae ce eee

~1l4=

leaves and stalks are virtually so much living fungus and
this material soon becomes so thoroughly cowered by the
large numbers of fruiting structures as to have a dark
grayish, velvety a@pearance, It may be said here that
these fallen stems and leaves are a very important source

of further infection of the crop.

On the Leaves.

The first macroscopic &ndication of the fungus within
the leaf is revealed by the presence of from few to many
small slightly yellowish or pale greenish spots a millimeter
or more in diameter. These may be noted in from 5 to 8 days
after inoculation, when conditions best favor the fungus.
These small spots enlarge and coalesce into a rather indefinite
yellow spot, the size of which depends upon the number of }
initial entrances of germtubes, the time elapsed since
infection, the climatic conditions omourring during the growth
ef the fungus within the leaf tissue, and the condition of the
host plant itself. Under the conditions described as best
for the fungus, lesions have been found to attain the diameter
of from one to two centimeters within ten days after inocn-
lation. These spots are in general approximately elliptical
in shape, and cause a wrinkling or slight folding of the
healthy tissue immediately surrounding (Plate I, II.).

The border of the lesion is frequently, but not always,
slightly raised, particularly where the edge of the involvment
meets the larger veins of the leaves, and at times there is

a yellowing of the tissue extending two millimeters or more
-15=
beyond th border of the brown spot. More spots are found
beginning at the leaf margins than in from the edges. As
the nutriment of the affected area is exhausted the color
of the spot changes, progressively, to a pale brown, a
slightly reddish brown, and ultimately to a dull slate or
ashen gray as the fruiting structures appear. As indicated
under general signs of the disesse, the presence of high
temperature and moisture and a cutting short of the food
supply are instrumental in bringing about abundant fructi-
fication of the fungus. Conidiophores under these conditions
are capable of producing a crop of spores in from 6 to 10
hours. In one experiment, at 12:00 P.M. a portion of a fruiting
surface was found to have conidiophores which bore no spores,
but when examined at 6:00 P.M. the following dgy was fowmd
to have produced an entire new crop of typical conidia,

This was observed microscopically by using the procedure
described later under the heading "Dissemination".

On the Petioles.

Mention has been made of the presence of the fungus on
the fallen stems. Occasionally the active, lfving petioles
are attacked, elliptical spots having a water-soaked appear-
ance being produced. The greatest diameter of the ellipse
extends in the direction of the length of the stem. Phe
stems frequently break ove® at these points of infection
leaving the foliage beyond to droop and die (Plate III}.
~l6=
On the Fruit.

I have not, as yet, had the opportunity to examine
celery flowers for the presence of the fungus. Repeated
examination with a hand lens and with the microscope of the
seeds kept in stock at this laboratory has failed to reveal
the presence of the Early Blight organism or any of its
associated effects. Examination was made microscopically
of the sediment obtained by centrifuging water in which
celery seeds were suspended. What may have been conidio-
phores of the parasite were seen on several occasions, but
the parts bearing the characteristic scars were missing.
Because of the wide-spread occurrence of the disease and
because of the probable specific nature of the causal
organism it is reasonable to believe the parasite is seed-
borne. Undoubtedly bits of blighted material containing
viable fungus find their way, in the course of seed collection,
into stocks of seeds and are thus distributed. Further

search should be made using material from other sources.

Etiology.
Proof of Pathogenicity.

Single spore cultures of the organism (Plate XI), were
obtained by the use of the ordinary poured plate procedure,
spores being obtained at times from diseased leaves and at
Other times from artificial cultures. The petri dish was
inverted and by means of the low power of the microscope single

isolated spores of the fungus were located and marked by a
-17<

small ring of ink. These were transferred immediately to
a cornmeal agar slant and others allowed to grow for two
to four days and then transferred. The cultures thus
obtained were used in many subsequent successful infection
experiments. A typical infection experiment is here described.

Celery trash that had overwintered near the end of the
Botany Byilding was on April 15th, 1920, shaken over two
large healthy plants in the rain chamber, some of the refuse
being left in contact with the sowmd leaves. Seven days
after (April 22nd) spots were noted on the leaves (Plate II).
These gradually assumed the pale brownish color of typical
Cercospora lesions and by April 28th had attained an ashen
gray appearance. Characteristic spores were first noted.on
May 2rd. Using spores from the plants thua infected, plates
were poured May 4th, 1920 and the organism isolated on
May 1LOth, 1920. Jue 12th, 18 celery plants and 4 celeriac
plants were inoculated using the pure cultures obtained from
this isolation. Thirteen days later, June 25th, all inoculated
plants showed typical infection. The checks two weeks later
also showed infection which was probably due to the distri-
bution of the spores to them by air currents created by
opening and closing the doorsg.of the rain chamber. This
experiment has been repeated several times with similar
results; the checks, however, being better isolated, showed
no infection by the Barly Blight fungus. In one infection
experiment the organism was reisolated and the single spore

culture thus obtained was used with success in two subsequent
-18—=

inoculations.

Morphologye
(Mycelium)--The mycelium of the fungus eas it occurs

within the host tissue is composed of irregularly septate,
fairly heavily walled threads (Plate VI). These vary in
width from 2 to 3.5 microns, attaining a diameter of 4 to 5.5
microns in the case of the threads that make up the stroma-
like masses from which the fruiting structures arise. The
young hyphae are small tubes nearly hyaline, having rela-
tively thin walls with septa only at long intervals and having
a finely granular, homogenous appearing protoplasm As they
grow older they become gradually pale browm in color, gnarly,
tortuous and very irregular in shape, form septa at shorter
intervals, build heavier walls, md their protoplasm becomes
more coarsely granular with a few large (.5 to 1.0"), highly
refractive, globular bodies (Fig. 4-5, Plate VI). ‘The
mycelium formed in culture varies greatly according to the food
constituents and character of the medium. Figure 5, Plate VI
shows its growth on Melilotus stems and Figures 3 and 6

in cornmeal broth of Py 5 and 8.

Sclerotium-like Bodies)--Dark brown, sclerotium-like
masses of threads, having a diameter of 20 to S0n, are fommed
in the substomatal spaces (Plate VI, Fig.6). From these
through the stomatal pore are thrust the conidiophores which

wedge apart and soon obliterate the guard cells.
-19-

(Conidiophores)--The conidiophores are fused together
in the region where they emerge from the leaf, forming a
distinct fascicle (Plates VII, VIII). fThey are a decided
brown in color, usually with 1 to 2 rather indistinct septa
near the base but not infrequently having cross walls at
10 to 15p intervals throughout their length. They vary
from 40 to 180 in length, the average being 40 to 601; their
width varies from 53-5 to 5. bh. At the end of a mature
conidiophore is seen a dist inet scar, circular in outline and
with a small circular dot occupying the center which indicates
the point of attachment of a spore previously borne (Plate
VIII, Figs. 2,3,4). Older conidiophores have along their
surface from 2 to 6 of these scars, with a bend or genicu-
lation at each scar (Plate VIII, Figs. 8, 12), The expla-
nation for the presence of these geniculations is evident.

A conidiophore having borne a spore, on being brought into
moist surroundings sends out from just beneath the scar a
new growth. From thés new growth a new conidium is
abscissed, &4t a distance of from 10 to 30m from the first
scar. This sympodial branching may be repeated several
times, leaving as many distinct knees. As many as sig geni-
culations have been seen (Plate VIII, Fig.8.)

(Spores)--The spores or conidia which are borne at the
apices of the conidiophores are much elongated and slender
(Plate IX). They are spoken of as inversely clavate or
obclavate in form; thet is, the part next the point of attach-

ment is greater in diameter than the free ends. (Several
-20-
writers have made the error of considering the spores as
attached by their small ends !78,48); 4 siignt tendency
toward chain formation was also noted (Plate IX, Fig.
11j,K). Over 200 spores from 10 sources were measured,

the dimensions being as follows:

Minimum Maximum Average
Length (microns) 22.0 290.0 55.-100.
Width at base " 318 4.5 4.0-4.5
Number of cells Ze 30. 4.-12.

The conidia attained their maximum length in the warm,
moist atmosphere of the rain chamber where the humidity was
high and prolonged. The spore contents are non-vacuolate,
finely granular and subhyaline, the ones formed in pure
culture being slightly more transparent than ones collected
in the field. They are commonly constricted for the length
of a cell, this portion appearing devoid of protoplasm
(Plate IX, Fig. llb). One culture media a few typical
obclavate spores are formed and abjointed at first, but later
the new conidiophore growth becomes longer and remains attached
as hypha-like branch. These when mounted appear as long,
slender filaments which may be considered aty»ical spores,

(Plate IX, Fig. 1lh).

Name of Causal Organism.

The causal organism was first described by Fresenius

in 1863 under the name Cercospora apii' ®°) Through error

the credit for this was assigned by Soribner and some

others following him to Fries. To avoid mistaking the
@2le

authority for the name the abbreviation should be
written Fres. or Fresen, not Fr., thus--Cercospora
apii Fres. (or Fresen.)

Life History of Causal Organism.
The writer has twice proved that the fmgus will

overwinter on celery refuse. Microscopical examination

of this refuse reveals the presence of the typical
conidiophore fascicles, and leaves containing the over-
wintered parasite when placed in a moist chamber in the
warm leboratory are soon covered by a growth of typical
Cercospora spores. Early this spr ing spores which had
overwintered on trash were found. They were so very
hyaline as to be difficult to see, were much swollen--

5.5 to 6.58 at the base--and failed to germinate. This
tends to discredit Massee's atatement! 55,57) that spores
live through the winter ani are capable ef infecting a
crop the following season. The fact that the fungus makes
@ good growth on sterile muck (Plate V) may indicate
another means of overwinter ing and a source of infection
the following season. (Growth on muck is described under
"Cultural Cheracteristics".) The spore, under the suit-
able climatic conditions described, on coming in camtact
with a celery leaf germinates, sending its delicate thread-
like germ tubes into the leaf tissue. (See under "Infection
Phenomena".) Here it grows absorbing nutriment from the
leaf cells and producing a well-defined spot. The nutri-
-22—

ment at the center of the lesion being first exhausted
the fuwgus here sends out fruiting hyphae which absciss
spores capable of immediately infecting the crop. This
course continues throughout the hot, humid summer months
until the coming of the cooler, rainy fall weather. Then
the fungus within the fallen leaves and petioles assumes
a resting stage in the form of the stroma-like masses of
heavy-walled mycelial threads described. It thus con-
tinues its existence until the coming of hot humid weather
again. |

Relation of Parasite to Host.

Mode of Infection.

To demonstrate the mode of entrance of the organism
healthy celery leaves were placed in a moist chamber and
inoculated with spores and mycelium from diseased material.
The points of inoculation were marked by means of small
pieces of moist filter paper, each having a 5 mm. cir-
cular hole in its center. From time to time bits of the
leaf epidermis were stripped off and examined. A mowmt
made after 25 hours showed two distinct germ tubes from
@ piece of mycelium entering a stroma (Plate VI, Fig.l.)
Spore germ tubes were also seen entering the leaf tissue
in the same manner, but the view was too indistinct to be

traced by means of the camera lucida. The spore shown
~25=

in Plate VI, Fig. 1, was not seen in the mount illus-
trated but was placed there for the purpose of size
comparison. Hume (1900) demonstrated stomatal entrance
in a similar manner. Although repeatedly tried, I have
as yet not been fortunate enough to secure pieces of
epidermis from young heart leaves which show this entrance;
nor have I observed entrance through wounds. Although it
is reasonable to believe that the fungus may enter through
Wounds. Experiments show no increase in percent of
infection where the leaves have been wounded, That the
parasite can gain entrance to the interior tissue of the
leaf when placed on either the upper or lower leaf sur-
faces was demonstrated in infection experiments. This
is to be expected, the stomata being present in both
upper and lower epidermis.

Making use of the method employed by Makemson
(1918) (55) , germ tubes were observed to enter leaves that
were not detached from the plant. In this method the
leaf is bent over onto a slide on the microscope stage
and held in place by means of rubber bands. fo supply
moisture to the leaf surface a narrow ribbon of filter
paper is employed. The end of this paper, which is placed
over the leaf, has a small ciroular opening which marks
the place of inoculation. The other end dips into a
beaker of water set near the stem of the potted plant.
~2h=

Spores of the fungus are placed in the small exposed
leaf area and ower this is placed a cover slip. Using
low power direct microscopic observation is possible.

Hot finding sufficient moisture to be supplied by the
filter paper, a modification was made in the form of
capillary glass siphons. These supplied water to a small
piece of filter paper strip kept on the leaf, at any rate
desired, depending upon the diameter of the tube and
height of the source of the water. In same experiments,
for the purpose of better viewing the spores and germ
tubes, the cover slip was removed after sufficient time
had elapsed for entrance, and the fungus stained a minute
with a 1% aqueous solution of eosin! 24} - Phe stain ws
removed from the leaf surface with filter paper and water,
the paper strip removed, the cover slip replaced ove r
the area of inoculation, and direct mieroscopic obser-
vation with both high and low power was possible.

It was noted that the fungus most readily enters the
leaf when conditions of high humidity are present in the
purr ounding air. However, where the water forms a visible
film over the leaf surface, the germ tubes do not pene-
trate the stomata. It was noted with spores germinating

upon a glass surface that where a moisture film enveloped

the germ tube by capillary action this film was pulled
along with the extending tube. This small film enveloping

the spores may be significant in the infection process.
It is likely that the tube extends the enveloping water
film until this film is brought in cmtact with a stomatal
opening into whieh the water is quickly drawn by capillary
action. Thus the germ tubes would build continuous
Water passageways fram the spore to the leaf interior.
The tubes, naturally hydrotropic, continue down along the
water paths entering into the minute capillary-sized
passageways of the parenchyma. The factors involved in
the establishment of the parasite within the host after
invasion have not been determined, Entrance may be
effected in from 6 hours upwards, depending upon the
rapidity of germination and the proximity of the stomata.
Although I have not as yet observed entrance of the
fungus into young heart leaves, that such does take place
is undoubtedly true. Poole and McKay attempt to correlate
age of the leaf and stomatal activity with the suscepti-
bility of beet leaves of different maturity to the leaf

(64,65)
spot fungus, Cercospora beticola Sacc. - The stomata

of immature celery leaves being less frequently open and
having smaller slits than those of mature leaves are
facts which mst be taken into account when we consider
that heart leaves infrequently show evidences of blight.
To note accurately the relative susceptibility of young
and old leaves, the heart leaves of four healthy plants
were tagged March 3lst, last; the plants were placed in
the rain chamber and inoculated with spores from blighted
leaves. fen days later pale yellowish spots 1-3 mm. in
diameter sppeared at the margins of sane of the heart
leaves. The spots not appearing to enlarge, some of
the heart leaves bearing them were placed in moist
chamber on April 16th. April 19th the lesions had
enlarged toe sreas measuring about 5 mm. by 8 mm... Exami-
nation April 21st revealed them bearing typical conidio-
phores and spores. Sy April 23rd the diseased heart
leaves remaining in the rain chamber had borne spores.
From this it appears that the youmg immature leaves of
celery are capable of being entered by the organism which,
however, is unable to cause a definite, easily recogniz-
able Cercospora lesion within twelve to sixteen days.
It seems that after the parasite has gained entrance it
makes but little apparent progress while the leaf is
actively growing. There may be same correlation between
the hydrogen ion cmecentration of the young tissue and
its seeming resistance, while young, to the fungus attack.
The Pp of immature foliage has not been determined. It
is evident from innumerable observations that the fungus
makes its most rapid progress in the old retrograding
leaves. On mature leaves spots are produced in from
five to eight days, whereas it takes at least 10 to 14
days on the immature heart leaves.

On April 15th the heart leaves of four plants were
inoculated with diseased material and tagged for later
-27 =

identification. Eighteen days later these leaves had
attained almost their final mature size anda bore the
characteristic fruiting bodies of the fungus.

Relation to Light.

To determine whether darkness has any influence on
the infective powers of the fungus two potted plants
were placed in a dark constant temperature apartment
and inoculated with diseased material. One plant was
kept moist by inverting over it a battery jar in the
bottom of which and down for about half the height of the
jar was fastened moist filter paper. Some leaves of the
other plant were kept moist at the points of inoculation
by means of wet absorbent cottem wisps, while others
were irrigated with capillary siphons after the manner
described in the study of the mode of infection (pp.22
and 23). The wminoculated leaves were considered as
checks. Plants were similarly treated in the light of
the laboratory. Six days later same of the inoculated
leaves began to show signs of infection. Spotted leaves
were placed in moist chamber and four days later were
observed to have produced typical cmmidiophores and spores,

These results are in contrast to the observations by Pool

and McKay!64,55) 4, workea with the Cercospora leaf
spot of sugar beet. They reported that infection probably
takes place only in the day time and that immature leaves

are not susceptible.
Water and Temperature Relations.
Once established within the host leaves the fungus

 

makes rapid progress during hot, dry weather when the
plant is least resistant. The factors which most favor
the fungus, as determined by field observations and by
controlled conditions in the greenhouse, are high temper-
atures (80 to 90°F.) coupled with a droughty condition
of the soil when the activities and resistance of the
celery plant are at low ebb.

When we consider dissemination of the causal organism
we must add to these factors the necessity for air currents
ef sufficient strength to distribute the fungus conidia
and of an adequate amount of moisture being present on the
celery leaves to permit of the germination of the spores
when they arrive. Dews, a high hugidity, and light raina,
while not being of much value to the plant, supply this
moisture requirement of the fungus. One notes a most
rapid spread of the disease and fruiting of the fungus in
hot, muggy times following a period of drought. To demon-
strate the germination of spores in condensation water
the method of Duggar!20) (page 59) was employed. A
van Tieghem ring was fastened to a slide with vaseline
and paraffin, a few drops of water placed in the ring, and
over this was inverted a cover slip on the top of which
had been placed a number of conidia. The spores readily

germinated in the moisture which condensed on the glass,
~29—

germ tubes appearing in from 2 to 5 hours at a room
temperature of 28°C. (Plate IX, Figs. 3,4,5.)

In the greenhouse proper conditions for a thorough
infection of healthy plants were secured in the rain
chamber which is a plate glass cage (Wardian case) approx-
imately cubical, having a capacity of 12 cubic feet. lIEnter-
ing this through a hole in one corner of the bottam and
extending upward until it nears the center of the glass
top is a half-inch water pipe terminating in a small
opening fran which water emerges forcibly. By means of
this watering system is directed forcibly against the
top of the small chamber a small jet of water whioh,
breaking into a fine spray, thoroughly wets the leaves
of plants placed therein but does not supply sufficient
water to the soil to keep the plants vigorous. Healthy
potted plants were placed in this apparatus, the mist
turned on for a half minute, and the plants "peppered"
with spores by shaking diseased material over then. The
leaves were kept moist for a period of 8 to 10 hours by
turning on the mist for a few seconds at intervals of
about two hours. The chamber was maintained at a high
temperature by means of two 16 candle-power carbon filament
lights. Making use of this apparatus, conditions were
obtained which best simulate those in nature when Early
Blight is most prevalent and destructive. Knowing these
 
-30-
conditions we are thus better able to understand and explain

the severe losses experienced in Florida and other localities.

Pathological Histology.
The character of the mycelium as it ocours within the

leaf was described under "Morphology" (page 18). Mention
was not made as to the means by which the parasite obtains
its nourishment. The mycelium viewed in mounts of small
portions of diseased leaves can,-by careful focussing,

be seen to possess small knob-like structures 2 to 3.5 in
width (Plate VI, Fig.4). These outgrowths or haustoria
of the fungus mycelium extend through the cell wall and
lie in close contact with the cell contents therefrom
absorbing nutriment for the organism. The hyphae as seen
in stained paraffin sections of blighted leaves and stems
are exclusively intercellular, although in the very old
disintegrated tissue they appear to pass right through,
ramfying seemingly unobstructed as in the growth on
artificial culture medium. An abundance of hyphae shows
evidence of a rapid growth throughout the sponge paren-
chyma and palisade tissue, here readily wedging the cells
apart. Distortion of the host tissue is most evident in
the regions where the coarse, heavy-walled threads collect
to form spherical, stroma-like masses. Guard cells of the
stomata are forced apart as the conidiophore bundles are
protruded, are overshadowed by the short immature stalks

of the bundle, and soon lose their identity. Plate VI,
-3l1-
Fig. 2, illustrates the beginning of this procedure, and
Plate VII and Fig. 1 of Plate VIII the ultimate effect.
The bases of the fascicles were found to measure as wide
as 50% whereas the slits of mature stomata are only

1LOu to 13u in length.

Physiological Relations.

Effect of the Early Blight Fungus (Cercospora apii Fres.)
on the Nitrogen Constants of Celery (Apium graveolens).

To discover what changes in the nitrogenous compounds
of celery are brought about under the pathological work-
ings of the Early Blight fungus, and to note the similar-
ities and differences in the action of this fungus and
that of the so-called virus or mosaic diseases! 44) | the
following chemical studies were undertaken during the
fall of 1920. The methods used were, in the main, those
employed by Jodidi and his collaborators in their spinach
and cabbage mosaie investigations! 46>47) , Minor modifi-
cations and deviations are described in the following.

All the determinations, except water content, were
made with diseased and healthy celery of one variety,
Easy Bleaching, which was collected from plants of the
same age and growing in the same garden plot. Due to the
ebundance of relatively uniform leaf infection and to the
small amount of stem infection the work was done entire-
ly on leaves. As far as practicable, mature leaves of

apparently the same age were selected. The lesions on
~32—
the blighted leaves involwed approximately from one-
sixth to one-fourth of the leaf area. The leaves were
spread in thin layers on cheese cloth and dried at room
temperature (20-25°C.) for three days, and then placed
for three days in an eleotric oven which maintained a
temperature of 49-54°C, The dried materials were next
rubbed through a 40-mesh sieve, then mixed to assure
more uniform sampling, and finally put into mason jars
and sealed. The dried, powdered leaves thus made ready
for use were about the fineness of table pepper. The
color of the powdered blighted material was an ashen
grayish green, while that of the healthy material was
distinctly chlorophyll green.

The TOTAL NITROGEN was determined by the Kjeldahl
and Kjeldahl-Gunning methods, 2g. samples of dried, un-
powdered leaves being used in the first method, and 2g.
Samples of the more representative powdered materials
in the second. It may be said here that in all ammonia
distillations 4% boric acid solution was used as the
receiving liquid, brom-phenol blue indicator being used
in the titration’ 7° °

NITRIC NITROGEN was estimated by two different
methods, F,. M. Scales® zinc-copper couple reduotion

(75)''

method and the Schulze-Tiemann nitric oxid gas
H

method . According to Jodidi's procedure, 12g. each
of healthy and blighted celery were repeatedly extracted
and thoroughly washed with 85% alcohol. Milk of lime

at a I ‘
‘ - ‘ a lf
. - . f. \ i Are row

i“ ”

ry ; | , ;
» 4 ‘ ‘ e-~%
wr C Oo oY v Lo 9
-33-

was then added to the combined extracts and washings,
which were then evaporated to dryness in a vacuum oven
at low temperature. Phe residue was then taken up with
hot water, lead acetate solution added, and the whole
filtered, washed, and the combined filtrate and washings
made up to 2 liters. In the Scales" method 250 cc,
Samples of the extract were placed in 500 cco. Kjeldahl
flasks containing 80g. of the Zn-Cu couple coils. Five
grams of c.p. sodium chloride and lg. of cep. magnesium
oxid were added and the ammonia from the nitrates thus
reduced distilled into boric acid. In the gas method 250
cc. aliquot portions of the same extract were used and
the work carried out exactly as desoribea (23,

Distillation with Magnesia not being found to give
dependable results in the estimation of AMMONIA NITROGEN,
a modification of Grafe's methoa ‘°™ of distillation
in vacuo at low temperature was employed. Ten gram
samples were placed in one liter flasks, treated with
25cco. of concentrated sodium chloride solution, 35cc.
of water, and 15cc. of alcohol. Then the apparatus was
carefully made tight, l5co. of saturated sodium carbonate
added by means of the separatory funnel, and the water

pump turned on. The temperature of the water bath was

then raised to 25-29°C., at which temperature it was held
for three hours and then raised to 40-435°C. The water

pump reduced the pressure to 21-52 mm. mercury, the
~54=

average being 28 mm. Fifty cc. portions of a 4% boric
acid solution were used in the Peligot tubes to receive
the ammonia. The Peligot tubes were placed in the same
ice bath and attached by a Y to the same water pump.
Near the end of the 6-7 hour distillation period l5co.
more of alcohol were cautiously added through the sepa-
ratory funnels. The distillation was continued 20 to
50 minutes longer, during which time the particles of
moisture that collect in the necks of the flasks and the
cenducting tubes and which contain sane ammonia were
removed by squirting a stream of boiling hot water against
the exterior of the glass. The contents of the Peligot
tubes were transferred to Erlenmeyers and titrated against
standard sulphurie acid, using brom-phenol blue as an
indicator. |

NITROGEN of NITRITES was tested for only qualitatively.
Portions of the extract used in the Hitric Nitrogen deter-
mination were made neutral and to them were added 1 co.
quantities of Sulfanilic acid mixture (69) * A red color
in the extract from the diseased leaves indicated the
presence of nitrites. A faint pink appearing in the
extract from the healthy material indicated a trace of
Nitrite nitrogen.

Total HYDROLYZABLE NITROGEN and NITROGEN DISTRIBUTION
in the hydrolyzed portions were determined as follows:
Sg. samples with 400cc. 20% hydrochloric acid were boiled
-35@

for 9 hours under a reflux condenser. The contents of
the flasks were then filtered and washed with ammonia-
free water until free from chlorine. The filtrate and
Washings of each 8g. sample were thereupon made to 2
liters. Total nitrogen was determined at 500 co. aliquots
by the Kjeldahl-Gunning method. Other 500 cc. portions
were evaporated to dryness on the water bath and the
ACID AMIDE, HUMIN, and DIAMINO ACID NITROGEMS estimated
according to Hausmann’s nitrogen distribution methoa! 58,45) .
The results for MONOAMINO ACID NITROGEN were arrived at
by difference. Because of limited time and because of
the doubtful nature of the nitrogen extracted with
boiling hot water (28) , nitrogen distribution by Hausmann’ s
method was not estimated on the extract containing the .
so-called non-protein nitrogen. |

PROTEIN NITROGEN was determined by Stutser’s methoa 2)
a development of the work of Rit thausen! 9) , In this
method, lg. samples of the dry celery powder were treated
in the beaker with water (100 cc.) heated to boiling and
kept on the water bath for 10-15 minutes. Then 2ccc. of
concentrated potassium-alum solution and lScc. (.45 g.
Cu(OH),) of Stutzer's solution were added and the mixture
stirred thoroghly. On cooling, it was filtered and
thor oughly washed with water. The residue and filter
paper were transferred to a Kjeldahl flask and the "protein
nitrogen" exidized by the Kjeldahl-Gunning method.

The nitrogen in the filtrate and washings from the
 

 

 

~35 Ge

Cu(OH), residue was determined by the Kjeldahl-Gunning
method and called NON-PROTEIN NITROGEN. The results
for this analysis are nearly the same as those arrived
at by subtracting the figures for protein HN from those
for total N; they are at least within the error due
to sampling which must be considered in all the esti-
mations.

MOISTURE CONTENT was found by immediately weighing
freshly gathered leaves, drying to a constant weight
in an electric oven (temp. 99-103°C.) and reweighing.
Being more conveniently located than garden material,
greenhouse material was chiefly used in this determination.

A tabulation and brief discussion of results follow:
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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From the above data it is seen that the blighted
leaves of celery plants have approximately only 3/5
as much total nitrogen and about 1/3 as much nitrate
nitrogen as normal leaves. Further examination shows
that the protein nitrogen content of the diseased material
is 2/3 that of the healthy, whereas the non-protein and
total hydrolyzable nitrogen are slightly over 1/2 as
much. The hydrolyzable portion of the unaffected leaves
revealed on analysis considerably more acid amide,
diamino acid, and monoamino acid nitrogen than the corres-
ponding extract of blighted material. "Humin™ nitrogen
was found to be about the same in both normal and diseased
leaves. On the other hand, blighted material in every
instance showed more ammonia nitrogen and a much stronger
qualitative test for nitrites than the sound material.
The slight trace of nitrite nitrogen in “healthy” leaves
may be due to the fact that some of them were possibly
not free from the disease, although at the time of
collection they showed no outward evidence of infection.
(The incubation period was found to be 5-7 days.) One
may concede the possibility that part of this denitri-
fication may be due to the action of reducing bacteria
coming in as secondary infection! §),

The results of these analyses make it evident that
the action of the celery early blight organism on the

nitrogen constituents of celery is quite similar to the
~39-
workings of the so-called mosaic diseases on the
various nitrogen compounds of cabbage and spinach, dAgs
in the mosaic diseases, the fungus brings about a process
of denitrification in the plant tissues whereby the
nitrates are reduced. This accounts for less nitrate
nitrogen and the presence of nitrites in the diseased
tissue.

The nitrites formed in the denitrification process
probably react with the amino acids of the celery tissue,
nitrogen being eliminated. For example, note the possible
reaction with tyrosin, asparagin or glutamin, all three
of which haye been found in celery (89) ,

HOCgH«CHecHwH,coon —HOOM, HocgHacHsCHOHCOOH + Not + Hp0

Tyrosine Hydroxy Acid Elementary
Nitrogen.
We should, therefore, expect less mono- and diamino acid
nitrogen.
(86)
It also has been shown that under certain biological
conditions the nitrogen of the amide groups is readily split

off as ammonia.

RCONH, + HOH => RCOOH + NHg

Hence, we find less acid amide nitrogen in the diseased
tissue. The anmonia formed in this hydrolysis may indi-
cate the reasm, entirely or in part, why more nitrogen

of ammonia was found in blighted leavese
From the above it is evident that the total nitrogen
~40-
is reduced through the loss of the nitrogen of proteins,
nitrates, amino acids, and acid amides, nitrogen being
evolved for the most part as elementary nitrogen and
ammonia. Closer examination of the data reveals the

fact that based on the total nitrogen there is 6% more
protein nitrogen in the diséased than in healthy material,

but that when calculated to the weight of the oven dried
leaves there is only 67% as much protein nitrogen in
the blighted as in the sound celery leaves. In account-
ing for the figures based on total nitrogen, it is known
that proteins as such are but slightly attacked by nitrous
acid and therefore directly contribute very little to this
loss of total nitrogen. Considering the result based on
the actual weight of the oven-dried leaves it is reason-
able to believe that the proteins of the tissue under
the immediate influence of pathological conditions are
in part hydrolyzed to peptids and amino acids which are
readily attacked by nitrous acid. This would explain the
presence of less protein nitrogen in the diseased leaves.
The effect of the fungus on the nitrogen metabolism
seems to be primarily denitrification. This causes
deaminization of the amino acids and possibly to a small
extent, proteins. The loss in total nitrogen appears to
be the result of the formation of free nitrogen and ammonia.

Accompanying and following the death of the tissue
and progress of the fungus there is, naturally, a drying
-4l=
of the affected parts. Moisture determinations showed
that blighted leaves contained 8% less water than sound
material.

In brief, the parasitic organism at the expense of
and in competition with its hosts selects and utilizes
the forms of food suited to its energy and tissue require-
ments. The fact that this fungus, in common with the
"Mosaic™ trouble of other plants, is responsible for a
smaller percentage of total protein, nitrate, acid amide,
monoamino and diamino nitrogen, for a higher percentage
of ammonia, and for the presence of nitrites in its
host, may give a sort of picture of the chemical changes

that accompany disease in plants.

Cultural Characteristics.
In pure culture the fungus was foud to grow well

on all media employed. Plantings from cornmeal agar
cultures were made on the various media shown in the
following record, and the growth observed from day to

day. The effects of light and darkness on growth and
sporulation were also studied at the same time. For
growth in darkmess, an Arnold sterilizer was used. The
diffused light of the laboratory supplied a second lighting
effect, and a 50-watt Mazda bulb in the dark room of the
laboratory gave a third condition of illumination. In

the second and third cases the cultures were placed in a

single row to give the full effect of the light to each
~42-
tube. The character of the growth was observed fre-
quently but the results here given are those recorded
at the end of the experiment. Not considering the
amount of growth, the macroscopic appearance of the
fungus varied but little from time to time. The color of
the small mycelial threads as they first invade the
fresh medium is a translucent gray. Growth progressing,
the hyphae as they become older manufacture a pigment
which varies in color with the character of the medium.
On same media it is a dark gray to black, on others
shades of brown, but it is generally a deep chromium or
mineral green. The dark green pigment so pronounced on
cornmeal agar was found by the microscopic examination of
sections to be entirely within the mycelial threads
and not in the medium. The mycelium penetrates the agar
to a depth of .5 to 1.5cm., the color not being present
in the yowg branches which make the borders of the
growth (Plate IV.)

Light Relations.

The following record was made after ten days’ growth
on the various media. The growth in darkness and diffused
light took place between 20°C. and 24°C; that in electric
light was necessarily a little higher (23°-26°C.)
l. FPurnip plug:

2. Rice:

3- Potato plug:

4. Bean pods:

5. Corn Meal:

-43=
Dariness~ The superficial growth covered
about 1/3 the surface of medium
and was a dull mouse gray in color.
Prominent folds and bunches of a
deep green pigmented growth in the

medium. This extended to a depth
of .5 cm. No typical spores found.

Diffused light- As in darkness. No typ-
ical spores.

Electric light- As in darkness. No typ-
ical spores found.

Darkness- Mouse gray as in (1). In
medium a chromium green pigment
2mm. deep. No typical spores
found.

Diffused light- Similar to that in dark-
ness but aerial growth slightly
lighter colored. Typical spores
not found.

Electric light= As above. A few typical
spores.

Darkmess= Aerial growth not distinct.
Considerable dull, dirty, yew-green
growth covering 1/4 of surface of
plug. Less elevated than (1)
and (2). Spores-

Diffused light- Slightly more elevated
and folded.
Electric light- As in diffused light.

Darkness- Considerable dull, intensely
black growth with short velvety
mouse-grayish aerial growth on
the many folds and bunches. No
typical spores.

Diffused light- Exactly similar to that
in dark.

Electric light- Exactly similar.

Darkness- Considerable, the greater por-
tion being submerged and having a
dark chromium green pigment which
is most intense near center of growth.
Corn Meal cont'd:

6.

Te

8.

9.

Oat Meal Agar:

Nutrient Broth:

Carrot plug:

Nutrient Dex-
trose Agar: —

-44=
Diffused light- As in darkness. A
few typical spores found.

Electric light- As in darkness.

Darkness- An abundance of dull black
growth, citrm green at the
border. <A few clumps of gray
aerial mycelium. No typical
spores found.

Diffused light- Similar.
Electric light- Similar.

Darkness- Characteristic growth of
(5) at the surface of the liquid
and on the glass. Many small
rainette green submerged colonies
against the glass. No spores.

Diffused light- Same.
Electric light- Same,

Darkness- Tomentose mouse-gray aerial
growth and a tough folded black
growth in substrata. Spores
abundant.

Diffused light=- More abundant growth
and spores.

Electric light- As in diffused light.

Darkness- Black in substrata, mouse-
gray to a faint pinkish gray to
Slmost white aerial growth. Ex-
uding amber colored drops of
Liquid over white serial myceliun.
Spores +.

Diffused light=- As in darkness, but
| with more numerous amber and
colorless moisture droplets.
Spores +¢.

Electric light- Similar.

10. Potate broth: Growth very similar to that in

Nutrient broth. Submerged
growths more fluffy.
oS | -45
ll. Celery Agar: Darkness- Moderate amount (1/2 of
surface). Mouse gray to white
aerial mycelium in clumps simu-
lating conidiophore fascicles.
A black-green pigmentation.
No spores.

Diffused light- Same as in dark.
Electric light- Same as in dark.

12. Prune juice Darkness- Abundant mineral green to
Agar; black growth with small mouse-
brow oottony clumps, of serial

mycelium. Wo spores found.

Diffused light- Same. Aerial mycelium
green near the point of planting.

Electric light- Same as in diffused light.

1S. Corn Meal Darkness- As in (11) but with fewer,
Agar: more scattering cmidiophorerlike
Glumps. Spores +¢+¢.

@ - oe
Electric light- Same.

14. Nutrient Darkness- Abumdant, deeply submerged
Agar: (5 om.) black growth with mouse-
gray to white aerial clumps.
No spores found.

Diffused light- Same but with less
white aerial mycelium.

Electric hight- Same as in diffused light.
15. Nutrient Sucrose Agar:

Darkmess=~ As in nutrient glucose agar,
with amber droplets exuding fran
white aerial mycelium. Spores ++.

Diffused light- Similar, but no moisture
droplets. Spores ++.

Electric light=- Aerial growth shows a
slightly pinkish coloration.
Spores ++.
It ig evident from the above record that Light

effects are not very marked. Corn meal agar cultures in
preparation dishes were later similarly exposed to
light and darkness with similar results.

To repeat the above observations on the effect
of the absence of light on the growth of the fwmegus in
pure culture, five corn meal agar slants were planted
with mycelium and placed in a black wooden chalk box.
This Was enclosed in a pasteboard box and the whole set
near a north window of the laboratory. Three similar
plantings were placed in a wire basket and set along-
side of the boxed tubes. Two weeks later the cultures
were examined. There was no apparent difference in the
growth of the fungus except that the aerial mycelium
of the tubes kept in the darkness was slightly darker
gray. Typical spores were produced in both groups of

tubes.

Growth on Muck and Celery Refuse.

The record given above shows that several media
which supply the food necessary for a good growth of the
fungus in pure culture may be employed. Particularly
may be mentioned carrots, the nutrient sugar agars, rice,
and corn meal agar. Cultures on carrot ani rice are
rather difficult to transfer rapidly, due to the tough
resistant grovth. The sugar agars, while being conven-
ient to prepare, are good media for bacterial as well
as fungus life. They could not be used to good
advantage in isolation work. Because of its convenience

in handling and because of its apparent suitability
47 =
as a food for the isolation of the organism, corn meal
agar was the medium most employed in this work (Plate IV).
Good growth of the fungus was secured also on sterile
lettuce, celery refuse, beets, orange and lemon peelings,
Conns* cheap synthetic media, Richards" synthetic, and
muck.

Po determine whether or not the organism would grow
on sterilized muck and celery refuse, and to note the
cha racter of the possible growth as influenced by three
different treatments of these natural media, the follow-
ing was performed:

Five separate portions of quantities of muck,
muck and refuse in about equal volumes, and refuse alone
were treated respectively as follows: One set of the
materials was placed in preparation dishes and distilled
water added until it was within .5 om. of the surface
of the medium. This set was called "saturated". Another
group was treated with just sufficient water to give the
soil a nice friable condition, the material put in pre-
paration dishes and labelled "Optimum water content".

A third series was treated as the second, using filtered
lime water ani marked "Lime water". A fourth was

saturated with water conta ining sulfuric acid in the
proportion of 250 of He0 to 1 of HeS804; the preparation
dishes containing the media thus treated were called "acid".
The fifth set, used as a check, was left untreated. All

were autoclaved at 20 pounds pressure for forty-five
 
-48-
Minutes and planted with mycelium from a corn meal
agar culture. The results are shown tn the follow-

ing table, page 49.

This experiment simply shows further the sapro-
phytic nature of the organism and suggests the possi-
bility of the fungus’ overwintering, if not persisting
longer, in the soil and being capable of infecting a
celery crop the following summer (Plate V).

Temperature Relations.

The season in which the disease appears in the
field together with its reactions to controlled con-
ditions in the greenhouse suggest a rather sharp sus-
ceptibility on the part of the parasite to heat. To
obtain further knowledge of the temperature relations
of the fungus use was made of a model of a Ganong
differential thermostat. This consists of two galva-

nized iron chambers, one for a freezing mixture and the

other for boiling viater, which are connected by a square
trough of the same material. This trough is divided .
into nine small compartments by means of cardboard
partitions and is covered by two pieces of glass, making
@ gable-like effect. Each division is equipped with

@ small immersion thermometer. Ice was renewed each
day in the one chamber, and the water in the other,

kept boiling by means of an electric immersion heater,

was automatically supplied from an aspirstor bottle.
-49—

Table showing Growth on Muck and Celery Refuse.

 

MEDIUM

TREATMENT.

GROWTH

SPORES

 

 

Optimum

Water Content

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1. MUCK Medium 3/4 covered with None found.
mouse-gray and white growth
the white immediately
around the point of inocu-
lation and amobinting to 1/3
the viable fungus.
2. REFUSE Entire medium covered by a | Many.
brownish-gray plushy growth.
3. MUCK & Surface 3/4 covered. Char- | Foud on
REFUSE acter of growth as in (1) refuse.
except less white area.
Water
Saturated
4. MUCK Surface 1/4 covered. Char- “=
acter as in (1).
5. REFUSE As (2) where medium is Abundant,
out of water. gome ger-
_ minating.
6. MUCK & Nearly entire surface Few spores
REFUSE covered as in (1). found on
_ refuse.
Lime
Water. Few atyp-
7. MUCK As in (1). ical spores
found.
8. REFUSE As in (2). Aw in (5).
9. MUCK & As in (3) with a little As in (3)
REFUSE more white growth. but fewer.
-Acidified
10. MUCK Character of growth as
in (1), 1/3 surface Few.
coverede
11. REFUSE 1/2 surface covered as Many.
in (2).
12. MUCK & As in (9), 1/2 of sur- Few.
REFUSE face covered.

 

 

 

 
Table continued.

 

 

 

 

 

MED I UM
& GROWTH SPORES
TREATMEN®.
Untreeted.
13. MUCK Appearance as in (1), Few.
1/2 covered.
14. REFUSE As in (2). Many.
15. MUCK & Character of (3), Found on
REFUSE more growth being on refuse.
refuse than soil.

 

 

eS
-51-

(Vegetative Growth) --Making use of the apparatus
described above the series of temperatures as described
above the series of temeratures as recorded in the follow-
ing tabulation was obtained. Plantings were made on
corn meal agar in preparation dishes and four cultures
placed in each compartment. The temperatures of the
several compartments were recorded and the growth of
the cultures observed each day for 16 days. The dishes

were then removed and measurements and observations

 

 

 

 

 

 

 

 

 

recorded.
T
No. of |Temp. range Diameter of| Growth. Spores.
compart-|of Compart- growth,
ment. iment, ( °C.) mm.
] | Max. y Min. | }
1. | 41-43 --- --- | Death ---
Re 30-30 Ted 4.0 | Poorest, 2
Heavy,
resistant
mycelial
threads.
Se | $0-31 728.0 25.0 Next +, Lew
best.
re 27-29  O4.0 Sle Optimum. +
+ Ba 24-25 23. 2de0 Good. + Lew
6. 22-23 27. 22. Good. +, 1eW
short
ae = spores.
7. | gO=-21 18. 18. Declin- ++, many
ing. more.
«Be | Jdo-L7 L7. ‘ll. Not so
good as % +++ Abun-
dant.

 

9. 4-8 66 5.5 Poor. + few.
-52-

These data indicate that the fungus grows well
between 23 and 30°C., temperature above or below this
range being distinctly opposed to the best progress of
the organism Most abundant sporulation took place
between 15 and 21°C. Im several attempted repetitions
of this experiment upon carrot disks, Coons’ synthetic
medium and corn meai agar, the cultures were contami-
nated by mites in spite of @ thorough dusting of the
apparatus with Pyrethrum powder and spraying with
nicotine selution. Observations of test tube cultures
confirm the above results except that the greatest
numbers of spores were found at 20-21°C. Work with
the living host was impossible because potted celery
plants small enough for the compartments did not

survive there long.

(Spore Germination)--Spores and comidiophores
were removed from diseased celery, placed in tap water
on a cover slip and inverted over van Tieghem rings
as already describea!®°) , They were then exposed for
14 hours to the temperatures shown in the table and

observations made and recorded.
 
~5 50

 

 

 

 

 

 

 

Temp. New Conidiophore Spore Germination.
(°Cc,) Growth.
42-43 oe Death.
35-36 About half the number 60% germinated. Tubes
germinated. Growth 30-85" long.
16-100u in length; ,
_one was 150}.
27-29 Many (50%) germinated; 100%. Practically
170-190» long. every cell showed
& perm tube.
22-24 (50%) 60-120n long. As at 27-29%, but
fewer cells germinated.
19 (50%) Growth 30-40n 100% tubes 100-170p

long. '

long, fewer cells »
germinated than at
22-24°.

 

 

 

16 Few (10%) tubes 16-50n Many (60%) tubes 38-
' 70u, some attaining
85 in length.
11 --= Small germ tubes on
_many (60%). 14-20p

 

 

This indicates that new growth is rapidly made in

temperatures ranging from 22 to 29°C.

At 27° to 29° the

greatest quantity of growth was put out and at this ten-

perature cmidia, moisture conditions being optimum, are

no doubt most efficient in infecting the host.
-54-
(Thermal Death Point)--The thermal death point of
myce lium as grown in corn meal agar was determined by
means of Novy’s capillary tube ne thoa! ®?) , By crushing
the agar of a culture with a sterile scatpel the mycelium
was broken into relatively small bits. Five cc. of
sterile distilled water were then added and the test-
tube vigorously shaken making a suspension of the
broken hyphae. Sterile capillary tubes, 4 inches in
length, were @ipped into this suspension which was per-
mitted to rise 4-5 centimeters. The capillary thus
charged was sealed at both ends in the fiame of a micro-
burner. Using heavy iron water baths, the temperatures
of which were carefully regulated by the size of the
flame below and by having the room as nearly devoid of
air currents as possible, the capillaries were exposed
as shows and then discharged onto corn meal agar in

petri di shes.

 

Pemp. : Time of Exposure.

 

 

°C. - id min. 2 min. 5 min. LO min. 20 min.
40 + + + + +
45 + + + + +
50 + + + - ~
55 - Contaminated. - - -
60 - Contaminated. - Contaminated. -
65 - ~ - - -
70 ~ - - - -

 

Using aerial mycelium and spores, scraped from

cornmeal agar slant cultures, the above was repeated.
5 5=
In this instance the temperature intervals were smaller

and the capillaries exposed for 10 minutes in all cases.

 

 

 

Capillary : TEMPERATURES.
Noe = "4g. 43.5 45. 48 50 51 52 54
Le + + + + + + - -
Re + + + + + - =
+ + + = © = @

Se +

 

For an exposure of ten minutes the therm] death
point of the fungus mycelium, therefore, dies between
50 and 52°C. Two of the three ten-minute exposures at
51°C. were lethal.

It being found impossible to obtain a sufficient
number of typical spores and conidiophores from pure
culture to carry out the above work satisfactorily,
these fruiting bodies were obtained from diseased leaves.
They were removed with small forceps and placed in 2cc.
of water in a preparation dish cover. The suspension thus
made was taken up with capillary tubes subjected to
temperatures as above described and discharged on cover
slips and inverted over van Tiewhem rings. The hanging
drops were examined at intervals for three successive

days and germination or its absence noted.
~56<

 

 

 

 

 

Conidiophores: TEMPERATURES °C.

Tube No. ; 42 43.5 45. #48. #50 #51 58 #52”
l. + + + + + - - -
Ze + + + + + - - -

Conidia

Tube No.
l. + + + + - - - -
Le + + + +? - =” - ~
3 - - - - -
4. - - - - -

 

Spores of the two of the fow oapillaries exposed
to 46°C. for 10 minutes failed to germinate; conidiophores
put out no new growth after the 51°C. exposure. While
this experiment is faulty, a pure culture not being
employed, it at least indicates something of the relative re-
action to high temperatures of spores and the heavier walled

mycelium.

Relation to Reaction of Medium.

Germination or noh<-germination of spores in several
solutions which have a range of Hydrozen Ion concentration
values give an indication of the relation of the psrasite
to the reaction of the medium.

As a preliminary tect co-ridi2 were pleced uson corn
mesk agor, distillc2 water ond citr water and the
rapidity of germination noted by measuring the length
of the germ tubes from time to time. The results are
recorded on Plate X. In the same period of time the germ

tube of the spores on corn meal agar attained greater
~57~
growth than those in tap weter, those in tap water
in turn growing faster than the ones in distilled water.
The P, of the agar was 6.7, that of the tap water 7.6,
while the distilled water wes Ps 5.7. This small
preliminary test may give a slight indication of the
effect of Hydrogen Ion Coneentration and possibly of
the food and character of the medium on germination.

Corn meal broth solutions were next adjusted to
different hydrogen ion concentrations using hydrochloric
acid and sodium hydroxide solutions, spores mounted there-
in and germination observed. The range as determined
by Gillespie* colorometric method was Py 4. to Py 8.
Solutions having a higher H* concentration than Py 5.
inhibited germination.

To eliminate, more or less, the food and other
factors, spores were placed in various buffer solutions
of known hydrogen ion concentration and observed from

time to time. The buffersgused which were made according

to the formulae of Clark** are the following:

 

*Gillespie, Le J. The reaction of soil and measure-
ments of hydrogen ion concentration. Jour. Wash.
Acad. Sci. 6:7-16, Figs. 2. 1916.

Gillespie, L. J. and Hurst Le A. Hydrogen ion Con-
centration measurements of soil of two types: Carbon
loam and Washburn loam. Soil Sci. 4:313-319. 1917.

**Clark, We Me. The determination of hydrogen ions
1-317, Figs. 1-38. 1920.
-58=

lL. Primary potassium phosphate - Sodium hydroxide
solution. Spores germinated in the entire range of Py
values given by this solution (P, 5.8-8.0), the germ
tubes attaining greatest length at Py 7

2. Acetic Acid - Sodium Acetate mixture Py 3.6-5.6.
No germination took place at any value. It was reagmed
that the H.1.C. was so high as to be detrimental to germi-
nation, the salts themselves toxic, or both factors
combined may be instrumental in preventing growth. That
the high H.I.C. alone was not responsible in some
mounts is shown by the third buffer used.

5. Potassium Acid Phthalate - Sodium hydroxide
(Py 4,-5.6). Gonidia did not germinate below Py 4.6 but
did from Py 4.6-5.4.

The conclusions to be drawn are evident. Many more
buffer combinations must be tried to reduce the para-
doxes due to the toxicity of the soil itself to a minimum.
Quantities of corn meal broth or some other medium that
proves satisfactory should be treated with various amounts
of appropriate acid and alkali or suitable buffer, and
the Pa values determined fram time to time by the
accurate electrometric method. Having thus obtained
solutions of media having a long range of H.I.C. values,
the organism should be placed in them and observed as
to germination and growth. There are probably so many
and complex factors to be taken into account to attain

these hypothetical media as to make them impossibilities.
-59~
Trust could not be placed in such an unknown quantity as
corn meal extract. Its reaction varies in solution
with the progressive hydrolysis of its various components.
Nevertheless, a procedure very roughly simulating the
above was carried out.

To definite portions Of sterile corn meal broth
(Py.6.7) were added varying quantities of autoclaved
hydrochloric acid of known strength, the hydrogen ion
concentration determined colorimetrically, making use of
Medalia' s* short-cut system This was repeated adding
aseptically to proportional quantities of the broth,
corresponding amounts of the acid, giving stock quantities
of the medium whose reactions were found to be fram
Py 6.7-1.9. To obtain the alkaline range the process
was repeated using sterilized N/4.0 NaOH. Five co.
portions of the stock solutions were added aseptically
to sterile test tubes containing a small filter paper
cone. Two transfers were then made for each Py value,
planting a small piece of fungus mycelium near the apex
of each cone. The growth was observed from time to time

and was begun February 1, 1921.

 

* Medalia, L, S. Color Standards for the Colorimetric
Measurement of H-ion Concentration Py 1.2 to P, 9.8.
Jour. Bacter. 5:441-468. Figs. 1-4. 1920.
-60@=

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pa Vaiues Growth | Growth Growth
(1 week) | Spores.
g/8/2. | 2/14/21 2/24/21
T
21 [- - - -i[- -
205 - - [= - - -
2.5 - = =~ = - =
520 - - 7 - - + +
| Very
. eth, No nor-
4 ° mal spores
~ ced - - - + + \. found; a
Sit. > few
| : atypical.
~ £20 - = | + + + + v
Slt. 60m. "».
Zed + + | + + + +
e7om.e «7Ccmy lom.
i ' Sclerotium-like Mycel-
5.0 . ial Mats.
+ + + + + +
Lome 1LeCMe |
5.5 + + + + + ,
6.9 + + + + +
6.38 + + + + !
1.5@n.
6.7 + + + + + +
1l.e2an to
6.9  i$| ee-cecee Contaminated----------- \©6©Spores.
762 + + | + + + +
1.2cm = | 1.5amn.
76 + + | + + + +
| | Sclerotium-like Mycel--
| ial Mats.
8.0 + + + + + + Lar gest
1 cm. number
typical
8.4 + + + + + + spores.
9.0 + + + + + + i

 

 

 

 

 
-6l=

The inability of the fungus to grow at Po 4, 305
and P; 3 at first and the ability later to make
appreciable growth at these concentrations of the
hydrion show the varying character of the medium. fThis
Was due either to the effect of the fungus or the medium
being unstable itself, or both. However, titration
of the stock quantities at the end of the experiment
showed that it had varied greatly. That which tested
Fy 1.9 at the initial determination showed Py, 4 at the
final, Py 8.4 media had fallen to Py 7.6, 2&5 had
gained 2, and so on. Only those solutions near neu-
trality showed any constancy. We may say that the
figures only in a general way indicate a tolerance of
the organism toward the acidity and alkalinity of the
medium in which it was grown. It is obvious that
exact descriptive statements and conclusions are here
impossible, due primarily to the varying of the very
factor under consideration.

To observe something as to the ability of the
fungus to change the reaction of the medium there were
added to 5 cc. portions of corn meal broth (P,, 6.7) the
necessary quantities of the various indicators and the
organism planted therein. One week later the colors
showed the H+ concentration of the broth to have
increased to Py 6.5, and two weeks after planting to Py 6.1.
Observations one and two weexs from the date of the last
record showed Py 6.2, and five weeks after planting 6,5.

The last record, made 10 weeks after the initial
~62=
was Py 6.6.

Hydrogen ion concentration values for the juice
expressed from celery leaves and carrot leaves and for
water extracts of the celery powder used in the chemical
determinations were determined electrometrically. The
writer is indebted to Dr. BE. C. Robinson of the Chen-
ical Division of the Experiment Station for these data.
These represent values for material from only two
sources and are not in any way to be considered compre-
hensive. The leaves were removed from the plants, washed
in distilled wseter, and then subjected to 250 pounds
pressure in a hydraulic press. The dry celery power
was moistened with an equal volume of doubly distilled
water and before extraction placed in an ice box at
a temperature of 12°C. for 16 hours. Two pressings
of the material were made, the extract obtained in the

second being labelled "residue" in the following:

 

 

Material Expressed 3 Pu 33 Material Expressed ; Pa
Juice from: 3 33 Water extract: ;
: 33 :
Carrot leaves >; 6.36 :: Healthy powder : 5.33
Celery leaves ; 6.42 :; Blighted powder : 6.08
Carrot residue ; 5.68 :: Blighted powder ; 5.00
Celery residue ; 5.66 3: residue :

 

The H.I.C. of this material was considerably
higher than that at which the fungus made its best
growth in culture. It is believed that the figures for

"residue" more nearly represent the correct values for
 
-63~
the true sap of the plant leaves, the liquid from the
first being diluted by the wash water. More determi-
nations should be made as more material becomes avail-
able, dry leaves from living plants that for several
days previous have been washed with distilled water
being used. Heart leaves and mature leaves should be

determined separately.

Oxygen Relations.

To study the relation of the amount of air to the
quantity of growth on corn meal agar slants, test tubes
of various lengths and the same diameter were used.

The tubes were closed with rubber stoppers and sealed
with paraffin. Comparing the longest and shortest tubes
a gemall difference in the amount of growth was noted.
There was, however, no appreciable difference between
the other tubes and checks all compared.

Plantings were then made on various media and
covered with paraffin oil. The fungus was apparently
unchecked, it making a white cottony growth, most
abundant at the plane of contact of the medium with
the oil. In this plane the most mycelium was found on the
glass of the enclosing test tube.

Much further investigation was evidently necessary.

Making use of the H-tube method* the oxygen of the air

 

*Giltner, W. A laboratory mamal in Gereral Micro-
biology i-xiv, 1-422, Figs. 1-74. 1916.
-64=
above corn meal agar slants and carrot plugs was
absorbed by means of alkaline pyrogallol, the tubes
being quickly and carefully closed by rubber stoppers
and paraffin. On the corn meal agar and all but one
of the carrot plugs absolutely no growth took place.

One carrot culture showed a very slight growth at
first but this was soon checked. In the cmmtrol tubes,
which were treated the same as the others except that
the chemicals were not permitted to came in contact
with each other, the cultures made an abundant growth
completely covering the medium. The tubes were ob-
served at intervals for several months. The stoppers
were then removed and the mycelium plantings broken into
pieces and placed upon freshly prepared corn meal agar
slants. These were observed for two weeks, no growth
taking place. The mycelium was evidently lifeless.

It is known that pyrogallic acid under the
conditions of this experiment gives off a trace of carbon
monoxide gas, which in sufficient quantity is detrimental
and in larger proportions even lethal to many forms of
life. To obviate this possible objection test tube
cultures were placed in atmospheres made of mixtures of
carbon monoxide, carbon dioxide and air as shown in
the tabulation. These atmospheres were obtained by
displacing weter in liter bottles with CO and CO, gases

prepared by heating together oxalic and sulphuric acids.
~65=€
Where the COo was not wanted, the mixture of the two
gases was bubbled through stron~ sodium hydroxide sol-
ution. Three corn meal agar slant cultures were first
placed in the inverted bottle, the bottoms of the test-
tubes restins on the flange of the bottle and the cotton
plugs pressing tightly against the bottom. The mouth
of the bottle was then put into a battery jar of water
and the water made to rise to the mouths of the test
tubes by removal of the air. This was accomplished by
sucking out the air with a U-shaped glass tube, one
arm of which extended upward to the bottam of the inverted
bottle. In this wey practically all the air, except
that remaining in the test tubes, was removed and the
water ready to be displaced by the gas desired. The
desired atmospheres obteined, the bottles were closed
under water by means of rubber stoppers. The tubes of
each bottle were then shaken off the flange onto the
bottom of the stopper, thus permitting free diffusion
of the air of the tubes with the surrounding gases.

 

Growth after

6 days’ growth

 

 

 

 

Atmosphere : : removal.
; Tubes : Tubes
a > tl. 2 Se 3: le Le Be
L/20C0-1/2005 ; - :° +
1/3C0-1/3COo-1/3Air :Diam. > + + +
;.Scm. -Som. eSom.:Pink color de-
° _ velops.
1/2C00-1/2 Air + + + ¢ i+ + +
$ . gPink color de-
; ; velops.
1/3C0-2/3 Air :Diam. :
>2.e0cm. + + 3
:Pink. Pink. Pink.:7---PINK-----

 
-66=

All the plantings grew except those in an atmos-
phere of half CO and half COo, (minus, of course, the
30-40 cc. of air contributed by the volume of each of
the test tubes). The purpose of the experiment was thus
accomplished. Carbon monoxide in relatively high concen-
trations is not inhibitive to the growth of the fungus.

It was noted in the above that a pink coloration
appeared in the agar immediately below the fungus growth.
This color extended into the agar about a centimeter.
It gradually disappeared as the fungus made further
growth although same remained as long as the cultures
were kept in atmospheres cmtaining CO. Microscopic
examination of sections of the pink agar did not reveal
the presence of any fungus hyphae. This pigment had on
several previous occasions been noted under young
cultures which to all knowledge had not been exposed to
any gas. It was thought, however, that possibly some
illuminating gas may have entered the tube during
flaming. Accordingly an experiment which had the dual
purpose of revealing something of the relation of the
fungus to oxygen and oxygen diluting gases, and of the
production, if possible, of the pink coloration by means
of illuminating gas was carried out. The method used
was exectly as described under the CO experiment. The
gases used and their proportions were as indicated in
the following table. The work was begun March 6, 1921

and results recorded on the three dates show.
~67<

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GROWTH —
ATMOSIHERE 3/9/21 3/13/21 3/18/21
All CO» Slight. No | Growth very | As 3/13/21.
pink color. | slight. Con- | Shallow.
centric dark-
ened rings. _
3/4 COo, 1/4 of ched| As 3/9/21. do.
1/4 Air. No pink.
1/2 COs, do. do. do.
y : |
1/4 COs, 1/2 check. | do. | do.
3/4 Air. No pink. !
All Np * Good growth | Thin spread- | 1/2 check.
'No pink. ing growth. | Many dark brown
: No pink. sclerotium-like
| mats on surface.
3/4 No, As check. | Check. | Check exactly.
° T a ne
1/2 Ne, Do. Check. | 2/3 check.
1/2 Oo. No pink.
1/4 No, Good growth | Check. Check size. 1 cm.
3/4 Oo. Slightly in med. Intense
pink. brown color at
inner surface
| __. of crowth.
All Oo * Good As check 3/4 check. As
No pink. but slight- above. No pink.
Ly pink.
All Gas* No growth. No growth. 1/4 check.
(Lab.Illum.) | No pink. Very pink.
3/4 Gas, +2/3 check | 2/3 check. 2/3 check.
1/4 Air. Slt. pink. | Pink. Pink.
1/2 Gas, 1/2 check. 1/2 check. 3/4 check.
1/2 Air. Very pink. | Pink. | Pink.
1/4 Gas, 3/4 check. | As check. As check.
3/4 Air. Very pink. | Pink. Pink.
Cheok- °7om. 1.5 om. 7/8 surface
All Air. i} covered.

 

 

 

 

All cultures

had a few atypical spores.

*except the air conteined in the test tubes.
-68~

The presence of the pink in the culture agar
remained as long as the tubes were kept in the illumin-
ating gas. The slight pink which was seen at first in
other atmospheres disappeared while the tubes still
remained in those gases. The fungus may have sim ly
overshadowed it, used it in its metabolism, or excreted
gzomething which changed it into a leuco-form. Judging
from this and the CO experiment it seems there is a
possibility that the CO gas is responsible for the
production of the color by the fungus. The CO content
of the illumination gas used was 4.4%. The poison gas
even a short exposure to it, probably so influences the
organism that it excretes the something which is either
colored or which reacts with the constituents of the
nutrient producing the pigment. Time did not permit
of more investigation of this interesting phenomena.

Another experiment was performed to learn more
coneerning the relation of the fungus to atmospheric
oxygen. Four corn meal agar slant cultures were placed
into each of several chancel flasks and the air exhausted
by means of a May-Nelson vacuum pump. fhis quickly
lowered the pressure to 3 mm. mercury, at which point
it was held for 10 minutes. The glass cock was then
closed and the pump stopped. Two cc. of water were
placed in the bottom of each flask before evacuation to
prevent excessive drying of the agar. This gives

practically an atmosphere of water vapor. The plantings
=69-
were examined from time to time and no growth was
observed. The checks showed normal growth, being l.cm.
in dismeter 4 days after the planting and two weeks
later completely covering the agar surface. Ten days
after the beginning of the experiment one of the flasks
was tested with a mercury manometer and was found to
have maintained its vacuum, the reading being 20 mm.
or about the vapor pressure of water at the existing
temperature. Air was admitted to this flask and its
plantings examined 3 days later. A good growth was
evident in all three tubes. For ten days the fungus
head either been able to remain alive on the very small
quantity of oxygen not removed from the agar and
surroundings, or it is able to respire anerobically for
some time, or it can remain dormant for this periods
The air was again exhausted from the flask and growth
stopped. It was reopened two weeks after the second
evacuation. At the same time another flask, whose
vacuum had been maintained continucusly for 26 days,
was tested and opened. Growth was not resumed in either
flask, showing that two weeks exposure to these con-
ditions is fatal. The last flask was tested after
a month's time and although it had leaked until the.
pressure was only 20 mm. lower than atmospheric
pressure, the fungus in no one of the tubes had grown.
How long it had maintained a good vacuum it is im-

possible to say, but it probably leaked very gradually.
-70=
Further experiments on the effects of air exhaustion
and reduced pressure, more exact and conclusive, were
not carried out because of lack of time.

These experiments demonstrate that the fungus,
Cercospora apii , while able to maintain apparently
normal growth in small quantities of free oxygen, is
killed by the almost complete absence of this gas.
Tolerance and reaction to the poisonous carbon mon-

oxide gas was also noted.

Resistance to Desiccation.

To determine the effect of drying on the life of
the fungus a spore mycelium suspension was made as
described under the Thermal Death-point experiment.
Using aseptic precautions, small, loose wisps of
absorbent cotton were dipped into the suspension and
then plac . in sterile petri disres. Several one-
inch cover slips were covered with the suspension and
placed in another petri dish. Both petri dishes were
placed in a desiccator where they were allowed to
remain until just the last trace of visible moisture
vanished. The dishes were then removed and each,
for protection against contamination, was placed in a
cover of a deep culture dish which had been lined with
cotton. The covers of the petri dishes were propped
up on one side by means of sterile pieces of wire.

Desiccation was allowed to proceed at room temperature.
 

 
-7l-
At the time intervals shown in the following table bits

of the glass circles and cotton wisps were planted on

 

cornmeal agar slants.

 

 

 

 

 

 

 

 

 

 

‘ime of Drying - Déys

Glass

a 4; 8 |16 22| 32) 38; 68
St + + + + + + +
No. l

2 + + + + + + +
Cotton
Wisps 8 | 28 | 30: 62
Set
Noe l + + + +

2 + | + + | + |

 

 

 

 

 

 

 

 

 

 

 

+ indicates growth.

It is worthy of note that successful plantings were
made from cultures which had been permitted to dry for
fourteen months. The above results show simply the com-

parative tolerance of the fungus to desiccation when less

protected.

To test the effect of drying on the conidia several
blighted leaves were put in a desiccation for 36 hours and

then removed to a petri dish where they were permitted to

dry at room temperature. One edge of the petri dish cover

was raised. At intervals of time shown below spores were

removed with forceps and placed in tap water on cover slips
and germination observed as in the Thermal Death Point

experiment. On the 15th day spores and conidiophores were

mounted also from garden material that had been pressed last
fall, placed in a pasteboard box and set in the specimen
room. Both conidia and conidiophores germinated very

readily. The material in the petri dish was consequently
 

-72-
abondoned. Fruiting bodies were then taken from old
herbarium material, dating from 1889 to 1912, and germin-

ation attempted.

 

 

 

 

 

Time of Drying - Days Mounts of
Mount Material
1-5; 3 4 6 7 9 | 134170 1889-1912
I i
Noe 1 Germination + + + + + + + + -- --
Woe 2 Gernination + + + + + + + + | -= ~—

 

 

 

 

 

 

 

 

 

 

 

 

 

These results indicate great tolerance of the conicia
to desiccation and the practical application of this to
dissemination and infection is obvious. There were no
indications whatsoever of living fungus in the mounts from
the old herbaruim specimens anc, altho allowed to remain
several days in the moist chumbers no conidionhore or spore
produced a germ tube.

Dissemination.

That air currents are to be considered as the main
agents in the dissemination of Cercosporae and other fungi
of the moniales type is indicated in various writings.” with
a view toward determining the presence or absence of forcible
expulsion of Cercospora spores from conidiophores the follow-
ing experiment was made. Using paraffin, corxs were fastened
to the lower surface of the tops of deep culture dishes. To
the corks, about 5 mm from the inner glass surface were
pinned small pieces of diseased leuves bearing an abundance
of conidiophores and Spores. Melted cornmeal agar was then
poured into the bottoms of the dishes and the toos replaced.

Examined with the microscope the dry fruiting structures

present the appearance of irregularly twisted, collapsed
m7 Zu

(16) Absorbing the moisture given off by the.

rubber tubes
agar they ravidly become turgid and appear as when mounted in
water. On being removed to the dry air again they very
readily resume their original shrivelled avpearance. Two
microscopes were used in the observations. Examinations were
made at varying intervals of time for a period of 36 hours.
Spores were not observed to leave the conidiophores. The
dishes were then set aside and examined a week later to see
if any cercospora colonies formed on the agar below. One
colony had formed immediately below the leaf in one dish. The
others, however, showed no growth. <Apparently this fungus is
provided with no means for the forcible expulsion of its
conidia.

Wind distribution was demonstrated as follows: Pieces
of blighted leaves were fastened to the inner top surface
of a deep culture dish as described above. The upper
surface of the glass immediately over the conidia was cooled
by other evaporation. Air wax. then blown onto the leaf ty
using a bent glass tube which passed down around the bottom
of the microscope stage and up into the dish where it ended
in a capillary opening using low power of the inicroscope and
getting the lower surface of the glass in focus, wpores
were observed to appear and stick in the condensation
moisture which collected on the cool glass.

It should here be repeated that fungus conidia which
had over..intered in the garden were found incapable of
rermination the following spring. They are probably

very rarely able to withstand the vicissitudes of winter

conditions, being designed for distribution during the
a 7
season in which they were produced.
Varietal Resistance.

The literature shows evidence of some little
observation on the resistance or susceptibility to the
fungus of a few celery varieties. Rolfs (1896) wrote
that "self blanching varieties suffer worse than other
kinds." (72) Davis (1893) recorded that "Shite Plume and
Golden Self Blanching suffered more than other varieties.
Hendersons New Rose has been entirely free from pricnt 2h
Taubenhaus (1918) stated that "Boston Marxet and Gold Heart
should be avoided because of their susceptibility to the

(87),

disease. The white Flume seems to be resistance. Jones

(1910) reported a Milwaukee celeriac grower as losing 90%

6
of his crop in 1909! i,

Although observations, both in the field and the
preen house, were numerous and records were made, they fail-
ed to revesl any marxed resistance of any variety to the
disease. Twelve different varieties of celery were used
in these tests anc there could be noted in the susceptibility
to Early Blight no difference between the green and self
blanching Kinds or between celery of any one or several

varieties.

Control

Being a leaf spot disease it would seem that Farly
Blight would be amenable to control br & srraying of the
plant foliage with some suitable fungicide. The published
results of early trials are somewhat confusing. Halsted (1891)

26
obtained good results with ammoniacal copper carbonate (”
~75=

Scribner (1886) found lime and sulfur of no value. He

wn

stated that "liver of sulfur msy arrest its wrogress,"
but that he hesitatec to recommend copper calts for

(78)
"hygienic reasons". Sturgis fourd culfur dust superior

to liver of sulfur solutions end the copper salt sprays (82-85)
Atkinson (1893) recommended the use of Anmoniacal copper
carbonate. 7! MeCarthy (1895) in one of his six rules for
combatting the Blight wrote that "liver of sulfur or sulfur
flour may be applied in the seed bed after transplanting." 2
Rolfs (1898) stated that the plants being "drenched tv.ice
aveek with liver of sulfur solution gave complete control,"
as did the copper fungicides which had the disadvantage of
coloring the foliage! =) Hume (1900) preferred Bordeaux to
potassium sultide and ammoniacil copper carbonate eotat ton?
Gallov.ay (1893) wrote Davis of the Michigan Ctation that
celery on upland soil blighted badly in spite of the fact
that the lesves vere kept covered with Bordeaux, amnoniacal
copper carbonate anc other fungicides, cut that celery co
treated was less attacxed than the checks $15) Locknead (1900)
recommended svraying st two week intervals with amnontacal

(54)
copper carbonste.e

Later investigators, as incicated in tne patnolorists
reports, have come to the opinion that Bordeaux is tre most
convenient and effective fungicide to be used in covbattine
this disesse. The reports are varied, "ordesux unsaticfuctory",
"Borde.ux and amnoriads«1 copper curbonate not very effective,"
"Borfeaux gives partisl cortrol,”™ "Borcewux good,” ana
“Bordeaux gives excellent results," beine sone of the statenents.

The fact that Cslifornia end Florida celery receivei here
~76-

generally snow Early Blight cpots in snite of the lez:vres
being coated by Bordceszux, indicates tnat the sirsey as enrlied
coes rot five complete control.

oprey exverinents ere run in tne greenhouce vith
the object of determinine the relative vulne of several
Tunricides in crevertire ~erly Blisht spotting. Frelininury
to tnese ex.eriments ccores vere placed in a larfre vuriety
of cheniculs (25 of them) arc observed for gernination.
Copner filines placed in cistilled water, 44-59 Bordesux
mixture, munoniacal copper curborete (5 oz. CaCo; and 5 pints
26° Beaund t:rconia to 50 callons of vater mercury crsnice end
bichloride (1-1000), a number of other salts whose very
poisonous pilopertics or ready soluvilitr nade then ivpocvitle
for use as snrays, end copper sulpnate in Cilution 28 hisn

as Ll in 128,900 of veter were inhititive to the production of

~

: . ~ ~,0 -«, 4 - - _ a .
rern tubes. Lime cul ur (84° Buune made 1-45) erc Liver 3f

sulfur (4 02 to 10 gal. veter) cic not per'it rercinution

wniile the crores vere in contact vith tne frech envlution, tnt
on being sillowea to cry arc then vet sr-in cid not prevent wr ¢

coricia fron pearninstire. Talo .nvcy purticlly exil:in tue epry-
ing results obtnined with vrotacciun sulfide solution. vulfur
aust died not erroeir to innibit serminatio. It is adifficrls

to uncersterd the gooc results oodt:.ined by Sturgis vith

sulfur -lour. (82-85)

Limesculfur (1-40 ard 1-45) “as found injurious

to the gelery le: ves caoisine cevere burnirne. It has,

moreover only a tenporary effect in inhibiting conidial
rrovthe

OF the ceversel cusll epry ex eriznents run in the
 

77.

greenhouse the follovwirs is en exanple. ‘ets of six vlants
eecn were tnorournly crrsyed, top ard bottom of the foliare,
with the resvective furnsicices as vhovn in the teble, sllowe
to ary, and vlacec in the rain chember. They were then crrey-
ed with a spore-mnycclium sucpension sede fron 8 cornmeal srer
Slant cultures ena moistened frou tine to time vith the ~iict.

seventeen duys later tna results exrre.sed in the nucbver of

cercospora srots ver plant, vere recorded. TT:

4 y .. n 1 -. 3 -. Lege Ue ~ Se ry 7 - +: won 4 we... .
sboout helf of the lesions vias corvfirmed hy -ierodse2.1e@ eweeuine-

e/

tion.

       

QQ

Cn

r

  
 
 

Bordenux

  
  
 

   
 

MNOe .vots

  

sm Cu CO%

   
  
 

 
  

   

oN

spots 12

       
  
  

  

Tod
PP

17
16

    

   

     
  
   

Oe Spots

 

    
  

  
  

£5
LO

Lo
}

Lime Sulfur

 
 

      

 
       

le

roe Srots

     
 

 

  

9 : . nv ‘ OY am S ' NO . xs
4 Cheerce no.ed 40 of pots exche

 

a / A ° - -~-— “as . ££ SL ~~ ° 3 n we ° 7. e rn
Tne relative value of te four funsicices, in tris
oimall ervcooriment at lesct, is et orce ceaen. The ti.o copper
serays have a decided edventrrse eve Liver of Culfur ere aine-
’ . Ad wv, ClItG L 47>. “A tot ase -% *- a a
sulfur solutions. Because of toc vrovth of the celery,
BAe Pane ae e touched by oo wo one <nonid rot ex ect
EeXNOS INE New SPreas UnNnvoucned 5 re an Hebe mS
% ~ a ‘ Soe rue , rad ’ 4 tt -s yy ~~
avpolute freedom fiona the disear>. vovever, it ic avident

thet benefits ere cerived thri 2 thoronen cpplicaution of an

efficient fungicide. In ragione weeare thi. dietase 1s snaun
~75-

4. a4, . a9 ° r ‘ soe aed ywry * “
vo be ted una curine sesesors when the climnetie eorditions,
oN _ s wo f me ~w—Aa we wv we: ‘
S aesceribed cre unfevor:. ble for celery, vcoreyine with 4-4-50

ordesux mixture is advisahle. The apmlicentions chold
erin esrly in tno cue ver, Civ be very thoroeh, covering

B
dD
both lesf surfeece, ard et intervels of a veer or rot less tren

~ y ry s ae: 5 qn -y NG *. “7 . (a - $ 7 wo * yy 7
ten devs. Mis curries Gouule protection es tre ce

e-

Dlirht clso is thus not :ersitted to ortein « footnold.

tae observation of meny is tnut Larly Blireht is very
uncommon on celery grovn in rich soils thet heve plenty of
Water. Celery, grovn for 2 deny yeirs on the cooleotct

uv

wolanazoo muck hes rarely been bedly attscxeu by tits Tursuse

tome . (<9 ol 19
Cellovesy's exreriiwrents ure very indicative. oF Tne celery
J .

erovn on &@ cooperstive ex:erimental plot ct Jisson, Vateh vas
unucus.lly large ard tarifty lect yeur, nea been vatered oy flood-
ing. It snowed no evidence of Ecrly plight. Tnst raiced «t

Esot Lensinre whicn :es vatered ty the overhead uysten, produced

a

large ecible stalxs in spite of the lesves beire badly spotted.
E ft

e It aproers tht, eiven en adertanvle, fertile

wD

by tne dise.us
soil, water is a very imnrortant factor in controllins Turly
Blight. “rnere it is roesible to irrisete thorouwhly or flood the
lend durire the hot, ary perts of cuinver, corayine oy be wun-
nececs:ry or not of sufficiert velue to be econd ically ivvortunt
Novwever, as the cesisn prorrecees tauura the tia of rervect,
e°rnyines for the purroee of procucins unspottea, more attructive
Folissee, ard nore inportart, as incurence apainct the cectructive
late blight, is sdvisable. fo produce 2 cleerer, “ore murzet-
able prodict, anuoniacal copnver czrbonate (fornule 4 o2z Cubo.
and 3 pints of 269 Baumé ammonia to 50 gal. of water.) mey te
substitutec for the Borcennx mixture in the last tyvo erruyirnrs.

after harvest, to remove tne dig source of infeution
-79—
to the followirs crop, the field should be clenned of £11 refuse.
Infectea meterizl voild best be burned.

sunnary

Colery fearly Blight is a disease of celery ord celorinze
«novn nractically wrerever tiece vlents are provn, in Europe end
america. It is caused by a srecific fungus, Cereoypore spii, via
was first described by Fresenius in 1863. The disense anpears
as suborbicular gnots on the lewves arc also, but not comiuonly,
on the stemse In tires of hieh humidity the losiors are covered
vith the frviting bodies which give a velvety &ppearunce.

Given high tenreratures suvplemented by heavy
evening dews, & arougnty coil and corscecuently plants of low
vitality, the dicesse may becove of great ecovo ic tinortauncs.
Injury as high as 100% and losses up to 20% of the edible
product due to the nececvoary excessive trimming, may result.

The fungus enters its hoct thru open stomuta by means
of conidiel ferm tuboes. It grows intercellulsrly, absorbing
nutriment from the surrounding tissue by means of heustoriae As
the food components of a lesion become exhausted, the fungus
thrusts thru the stomata in fescicles long fruiting hyphae, each
of which bears a long obclavete spore at its end. when mature
these spores are easily detached and distributed by air currents.
Wind dictritution was denons trated.

Chemiecsl analysis of the nitrogen constituents of the
sound and blighted leaves revealed lerge differences which may
indicate somethin: of the metabolism of the fungus and the effect
on the invaded host.

The organism grew well on all media employed,
 
~60-

inclucing sterile muck ard refuse. It grew in the presence

of a hydrogen ion concentration es high as Py 3.5 ard us low

as P, 9. Spores were capable of germination in the renee

Py 406 to Py 8. in sone buffer solutions.

study of tre tenperature relation. of the funrus shovs
that the psresite maces its largest veretutive frovth betveen

259 C and 30° C, thst an exposure of 489 C for 10 minutes ig

fatal to the conidia and 51° C to the conidio:hores and mycelium.
Spores frerminated best at 27-299 CG. There vas no parnination
after 12 hours exposure at. 42° a.

Fourteen months desiccation vas not lethal to mycelium
and spores were found carable of germination after cix months
drying at room tenperature.

Though requiring some free oxygen for life, the
fungus is very tolerant in this reparc.

A study of infection on 12 different varieties
of celery showed no varietsl resistance to Fsurly Blight .

Control is a matter of xeeping the plants vigoroucly
grovine; that is, on soil of high fertility ard good drainage
and having an abundance of moisture. Th's shold be supplemented

by spreying with Bordeaux mixture «ss indicated.
l.

Re
Se
4.

5.

6.

8.
9.

LO.
ll.
L2.

13.

14.

-81-

Bibliography.

Atkinson, G F, Note on the Cercospora of celery
blight. Cornell Agr. Exp. Sta.
Bul. 49:314-316, Fig. 5, 1892.

oe een neem enne On the fungus of celery blight. Am.
Monthly Micr. Jour. 14:115, 1893.

Bailey, L. H. The Standard Cyclopedia of Horticulture
2:3-1200, Figs. 701-1470, 1914.

Beattie, W. R. Leaf blight of celery. U,S.D.A.-
Farmers’ Bul. 148:17-18, 1902.

Bessey, E. A. Celary. Rept. of the Botanist. Reprint
from 30th Ann. Rept. State Board of
Agric. 1-28, 1917.

Bonequet, P. Ae Presence of Nitrites and Ammonia in
Diseased Plants. Jouwr. Amer. Chem.
Soce 38:2572—2576, 1916.

Briose e Cavara. I funghi parassite delle piante colti-
vate od utili. No. 268 Cerocospora
apii Fres. Fi g. 1-3, 1895,

Buller, Ae He BR. Researches on Fungi, 1-287, 1909.

Chester, F. D. A leaf spot of celery (Notes on three
new or noteworthy diseases of plants).

ieee teteteteteteteater wm- A noteworthy disease of celary.
Rept. Del. Agr. Exp. Sta. 1891:63.

Cooke, M. C. Fungoid pests of cultivated plants I-XYV,
1-278, Pls. 1-15, Figs. 1-23, 1906.

Coons, G. H. and Levin, E. The Septoria leaf spot
disease or célery blight. Mich. Agr.
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Figs. 191-233. 1915.
15.

16.

L7.

18,

19.

21.

Zee

235-6

26.

27 6

28

29.

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Davis, G. C, Celery Insects. Mich. Agr. Exp. Sta.
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De Bary, A. Comparative Morphology and Biology of
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Delacroix, G,. et Maublanc, A. Maiadies Plantes Cultivees-
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Dorsett, Pe H. Treatment of celery leaf blight. Amer.
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we ee nen eee Fungous diseases of plants V-XII, 1-508,
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Durand, BE. J. Differential staining of intercellular
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Ellis, J. B. and Everhart, B. M. Enumeration of the
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Merrauris, Teodoro. I parassite vegetali delle piante
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Freeman, HE, M. Minnesota Plant Diseases. vii-xxiii,
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250-259.

oe ep ee me mm moe ~ Some fungous diseases of the celery.
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e
° a
o <
* e °
e a
oe -_ — ~
-
*
e - «
° o
— i _

 
44,

45.

46.

49.

50.

51.

52.6

53.

55.

84 oo
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— 65-6

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---------------- - Celery Blight. Conn. (New Haven)
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87 =

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L911.

90. Whipple, O. B, Celery culture in Montana. Montana
Agr. Exp. Sta. Circ. 26. 1913.
Plate

Plate

-~88-

 

Description of Plates.

I. Stem bearing naturally infected leaves.

Photo by Dr. G. H. Coons.

II, Plants diseased as a result of shaking

over-wintered diseased material
over them. Photo by Mr. Re Nelson.

Plate II1l. Naturally infected plant showing broken

Plate

Plate

Plate

diseased outer stems. Photo by Dr.
G. He Coons.

IV. Corn meal agar cultures of the Celery

Barly Blight organism, Cercospora
apii Fres. Photo by Mr. J. ee XYotila.

VV. Cultures on sterilized muck and refuse.

Photo by Mr. R. Nelsen.

VI. Camera lucida drawings of-

Fig. 1.-Germ tubes from mycelium
entering stoma of celery leaf.

Fig. 2.-Conidiophores of the fungus
rotruding through a stoma
tyoung stage).

Fig. 3.-Mycelium from corn meal broth
culture having H-ion concen-
trat ion 8.

Fig. 4,-Mycelium from diseased leaf
showing knob<-like haustoria.

Fig. 5.-Gnarly, tortuous mycelium from
culture on Meliotus stems.

Fig. 6.“Sclerotium-like bunches of
mycelium formed in corn meal
broth Pp .5 and Py 8.

(Photo by Mr. Jk. Kotila).

Plate VII, Camera lucida tracings of conidiophore

fascicles (advanced stage) showing
spore (Sp.) germinating in situ, and
new conidiophore growths from each of
which another spore would have been
abscissed.

N.C.G. = New Conidiophore Growth.
GT. = Germ Tube.

Small letters refer to time showing
rate of growth. The mount was made
at 9:45 A.M.
Plate VII, cont'd.

 5’0Q Hh ® RO o' ®

~B9~

11:00
12:00

1:00
2:00
2:

4:15
43:30
8300
5:00

A.M.
M.

P.M.
PeMe
P.M.
PeM.
PoMe
PM.
AM,

(next day).

Mr.
Plate VIII. Camera lucida drawings of-

(Photo by Je E. Kotila).

Fig. 1.-Base of conidiophore fas-
cicle (Oil immersi on).

Fig. 2.-Tracings of conidiophore
tips showing characteristic
scars and new growth (0il
immersion).

Figs. 3, 4, 5.-Showing attachment
of conidium to conidiophore
(Oil immersion).

Figs. 6-12.-Tracings of permanent
mounts (paraffin method),
showing conidiophores, spores,
scars and geniculations.

(Photo by Mr. J. E. Kotila.)

Plate IX. Camera lucida drawings (high power) of-

Figs. 1-5, 8,9,10.-Germinating cmidia.

Figs. 6,7.-Germinating c midiophores.

Fig. 11 (a to g, i) Forms of conidia.

Fig. llh.-An atypical spore from pure
culture.

Fig. 11j,k.-Two attached conidia show-
ing tendency toward chain
formation. .

(Photo by Mr. J. E. Kotila.)

Plate X. Camera lucida drawings (high power) of germi-
nating spores, showing relative rate of
growth of germ tubes in tap water, dis-
tilled water, and corn meal agar. The
experiment was begun at 9:00 P.M. and
the temperature was 28-30°C. The small
letters refer to time:
> b'0e +) @ CO OP

~90-

10:30 P.M.
12:00 PeMe
2:00 AeMe
5:00 AM.
7:00 AM.
7:30 AM.
9:00 A.Me
9:45 A.M.
11:00 A.M.

(the following day)
 

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PLATE V.

Water
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PLATE VII.

     
   

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PLATE VIII.

 

 

 

 

 
PLATE IX.

 

 

 
PLATE X.

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