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| CULTURE EXPERMENTS
WITH RUSTS

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| THESIS FOR DEGREE OF W. S.
GEORGE R. GAGE

| 1915

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CULTURN BXPERDIVUSNTS (/ITH RUSTS’

thesis for Degree of Master of Science

Michigan Agricultural College |

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George RK, Gage
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1915,
THESIS

 
Culture Experiments with Rusts

The Uredinales, also spoken of as the Uredineae
or rusts, form a group of fungi which insofar as we un-
derstand them represent the extreme of obligate para-
sitism,

the purpose of this work has been to find out
if it is possible to grow these rusts in artificial cul-
ture, that is to say, apart from their hosts, Little
need be said of the importance of any successful efforts,

Incidentally several experiments have been made
on the germination of rust spores, the procedure and

results of which have been included in this paper,

Previous ‘/ork
In some of the later text books on fungi the
following statement, or one similarly worded, has been
found:- "In no case has it been possible to grow these
organisms upon artificial media, or apart from their
hosts beyond the stare of mere germination or promycelial
production,” (1). Nowhere in the literature on rusts,
however, can be found any work the results of which
would warrant such a conclusion, Furthermore only one

publication whatsoever on artificial rust culture can

(1). Duggar, B. li, Fungus Diseases of Plants, 384, 1909,

96345

-|-
be found, Ray (2) makes the following statements: "Sur
trente especes d'Uredinees on d' Ustilaginees recueilles,
nous avons pu obtenir des cultures pures pour neuf d'entre
elles; Charbons du Ble, de l'Avoine, du lvais, de la
Saponaire, du Lychnis; Rouilles du Ble, de la Clematite,
du Xosier, du Fusain, Dans tous ces cas, il s'est
manifeste un developpement vegetatif abondant; les
Charbons ont donne une forme levure, seule au debut
accompagnee plus tard de mycelium: les Rouilles ont
immediatement produit un myceliun, et le Rouille du
nosier a forme une quantite considerable de spores
noires cloisonnees," As for media and methods employed:-
"Le milieu de culture employe le premier est un bouillon
prepare avec des echantillons sains des divers hotes et
additionne de gelose, l.ais sur d'autres milieux, tels
que la carotte cuite a 115°, le lait sterilise, il est
possible, pour les charbons surtout, d'obtenir des
cultures tres developpees, sinon physiologiquement

identiques aux premiers,”

Summary of Results
1, A duplication of the methods of Julian Nay,
who claims to have succeeded in growing rusts in arti-
ficial culture, insofar as they could be carried out
from his description, failed to produce rust development
(2), Rey, J, MM. Etude biologique sur le parasitisme,
vomptes Kendus De L'Academie Des Sciences,

136:568, 1903,

=).
beyond spore germination, Page 5,

2, The mycelial development of Puccinia malvace-
arum could not, at ordinary temperatures, be produced
upon sterilized and sterile cut plugs of vegetables
usually used for such purposes, Page 7,

3, The mycelial development of Puccinia malvace-
arum could not be produced at ordinary temperatures,
upon media made from decoctions of ordinary vegetables,
Page 8,

4, Mycelial development of Puccinia malvacearum
could not be produced, at ordinary temperatures, upon
substrata of filter paper, elder pith, and mallow stens,
supplied with a nutrient of sterile (filtered) I'alva
rotundifolia juice, Page 19.

dD, Mycelial development of Puccinia malvacearum
could not be produced, at ordinary temperatures, upon
mallow stems rendered sterile by removal of the epidermis,
the stems being supplied with nutrients of sterilézed
mallow juice, (autoclaved), sterile filtered mallow juice
(Chamberlain filter), and sterile distilled water, Page 11,

6, Mycelial development of Puccinia valvacearum
could not be produced, at ordinary temperatures, upon
the following media:- Plain agar, glucose agar, nutrient
agar, Ashby's synthetic agar, and nutrient gelatin, Page 12,

7, Wycelial development of Puccinia coronata could
not be produced, at ordinary temperatures, upon oat seed-

lings, sterilized in the autoclave and also by exposing

~%-
to 60° for 1 hour on 5 consecutive days, Page 14,

8 and 9, liycelial development of Puccinia
malvacearum could not be produced, at ordinary temper-
atures, upon various agar media, constantly washed with
water (distilled) to carry off by-products of the fungus
wnich might be injurious to itself, Page 15,16,

10, iiycelial development of Uromyces caryophyllinus
could not be produced, at ordinary temperatures, upon
@ porous cup atmometer constantly supplied with a nu-
trient solution and in a slightly greater concentration

of Carbon Dioxide than in ordinary air, Page 17,

Germination exveriments:-

1, Carnation rust urediniospores exhibited no
heliotropic curvatures when subjected to unilateral
diffused daylight, Equilateral light, unilateral light,
and total darkness, seemed to have no effect upon the
percentage of germination or upon the incubation period,
Page 18,

2, Carnation rust urediniospores exhibited no
thermotropic curvatures when subjected to unilateral low
or high temperatures, Page 19,

3. Puccinia coronata urediniospores exposed to a
variation of temperatures act as follows:- Spores at
all temperatures from 16° to 36° begin to swell at the

germ pores at the same time, The germ tube emerged also

~4-
 

 

at the same time, At a temperature of 18°, however,

not only did a higner percentage continue their growth,

but the rate of growth was somewhat increased, Page 20,
4, Alternate wetting and drying urediniospores of

Puccinia coronata had no effect upon the incubation

period, ‘The incubation period was the same for spores

in a strong glucose solution as in double distilled

water, likewise the emergence of the germ tube, but the

rate of growth of the tyvhes was somewhat increased in the

sugar solution, Page LR,

Procedure

1. Ray's methods were duplicated insofar as they
could be ascertained from his description,

Stems and leaves of lialva rotundifolia were
collected from non-infected parts of susceptible plants,
these were ground up and the juice strained through
cheese cloth, Half of the juice was then filtered
through filter paver andi tne other half through a
Chamberlain filter, To the former distilled water and
agar were added in the proportions: 500 cc, mallow
juice, 500 cc, water, 20 grams of shredded afar, ‘This
was steamed for two hours, filtered through cotton,
tubed and sterilized in the autoclave 20 minutes at

£Q pounds pressure, ‘To the second lot, water and acar
were added 3s foilows:- 500 cc, water and <O grems of
agar were steaned, filtered throufn cotton, and steril-
ized twenty minutes at £0 pounds, “his was cooled to
45° and mixed witn the mallow juice which hed been
warmed to 45°, ‘he mixing was done in a culture room,
all precautions against contamination being taken,
“nus two differant meiia were obtained, the

first with the mallow juice subjected to heat and the
second with it in its natural condition as far as temper-
ature was concerned,

cSeliospores of Puccinia malvaceerum were brougnt
in directly from the field and used in mekxine both pauref
and sprayed plates with each arar, ‘ubes of each agar
were elso inoculated with spores and with the rust mycelium
itself, ‘The mycelium was obtained from stems on which the
flecks were just eaevpearing as yellow spots, These were
washed in tep water, immersed in l’gCl, (1-1000) and rinsed
in sterile distilled water, The mycelium was picked fron
the flecxs with a sterile needle,

the plates and tubes were exemined for a month
or so, but nothinse more tian mere germination of the
spores resulted, Ina few tubes and plates there was
present contamination which proved to be either a *usariun,
Alternaria, Iiucor, or Panicilliun,

Since Ray's description of his methods is so

brief, we can only assume that it has been duplicated,
However, it is rather difficult to refrain from drawing
the conclusion that his "developpement vegetatif
abondant” and "quantite considerable des spores noires
cloisonnees" were rather illusionary or, better, the
mycelium and spores of some other fungus than rust,

£. Vegetables used as media:-

Carrots, white potatoes, sweet potatoes, pumpkins,
celery, sugar beet, turnivs, cabbage stalks, bean stems,
bean pods, and mallow stems were prepared in two differ-
ent wavs, as follows:-

First set, The vegetables were scrubbed in tap
water, placed in EgCl, (1-1000) for 15 minutes, and
rinsed in distilled water, Plugs were then cut with a
sterile (flamed) knife and placed in tubes containing
a little moist cotton, These were sterilized for 20
minutes on each of three consecutive days in the arnold
steamer,

Second set, The vegetables were scrubbed in tap
water, placed in Hg0lp (1-1000) for 15 minutes, and
rinsed in sterile distilled water, The plugs were cut
in a culture chamber with a sterile (flamed) knife, the
knife being sterilized after each direct cut, These were
then placed in sterile tubes containing moist cotton,
they were not steamed,

Tubes of both kind of plugs, sterilized and sterile
cut, were inoculated with Puccinia malvacearun telio-
spores and myceliam from young flecks of the same rust,

she tubes were examined for a month or more but
nothing developed which gave any indication of rust,
Kleven of the sixty-six tubes inoculated were contamin-
ated with either Fusarium or kucorg, All the plugs
were finally macerated and examined microscopically,
but no internal mycelial development of rust was found,
3, Plant decoctions used as media:-

Prune, oatmeal, cornmeal, potato, cucumber, bean,
celery, carrot, pumpkin, beet, cabbare, and turnip were
used, ‘hey were prepared as follows:

Prune:=- 120 grams dried prunes

1000cce double distilled water

Steamed in Arnold for 2 hours

Filtered through cheese cloth

Restored to original volume

12 grams shredicd agar added

Steamed in Arnold for 2 hours

Cleared with egg white

Filtered and adjusted to +12 (Fuller's scale)
Tubed and autoclaved 20 minutes

at 20 pounds,
Ontmoal:- £0 prra.ss oatmeal
200 co double distilled water
~vteaned in Arnold @ houre
riltered through cheese cloth
10 rrams of shredded apar added
Double distilled water added to
nake DOO «ce,
vteaucd in &Srnold e& hours
Milterod and adjusted to +12

Tubed and nutoclaved 20 minutes

at 2©O pounds,

Cornmenl:-lLO0O0ce double distilled water
3 tablespoons of cornmeal
Steaned in Arnold for & hours
Decanted and restored to orifrinal
volune
lo grans shredded arar added
Steniod in Arnold for 2 hours
Filtered and adjusted to +12
Tubed and autoclaved 20 minutes

at 20 pounds,

Cucumber, bean, celery, oarrot, pumpkin,
beet, turnip, and potato:-

£250 grans of rround up veretuhle

 
1000 ce double distilled water

Steamed in Arnoid for 2 nours

Yecanted and restored to original

volume

20 erans of glucose added (except beets)
+735 grams of shredded agar added

Steamed in Arnold 2 hours

Filtered and cleared with egg white

Adjusted to +1le2

Tubed and autoclaved 20 minutes

at £0 pounds,

Plates were made by the dilution method and also
by spraying, Tubes were inoculated directly, Both
spores and mycelium of Puccinia malvacearum were used
in each case, the infected plants having been brought
into the greennouse,

The plates and tubes were examined daily for a
month or so, but no sign of rust develonment appeared,
A contamination of liucors was present in a few cases,

especially in tnose tubes inoculated witn spores,

4, Special decoction of mallow leaves:-
A special decoction of mallow leaves was inade as

follows:-

Enough leaves were ground up and pressed

-10-
to give 20 cc, of juice, This was diluted 5 times and
filtered through a Chamberlain filter, ‘Zubes contain-
ing as substrata filter paper, elder pith, and mallow
stems and leaves, were sterilized and 3 ce, of the
decoction poured into each,

the tubes were then inoculated with Puccinia
malvacearum snores and mycelium as in the previous
experiment, “hese were examined externally for a month
but no trace of rust development appeared, At the end
of the month the substrata were macerated and examined,

but no internal development could be found,

5, Special substratum of mallow stems:-

Mallow stems were washed in tap water and placed
for 15 minutes in HgCl, (1-1000), After rinsing in double
distilled water the epidermis of each was carefully
Skinned off, “nis process was carried on in a culture
chamber and with sterile (flamed) instruments, A decoc-
tion of mallow juice was prepared as in the previous
experiment, <A similar decoction but autoclaved instead
of filtered was also prepared, Using the stems as plugs
and the two decoctions and sterile distilled water as
nutrients, tubes of each were inoculated with spores
and mvcelium of the same rust, Puccinia malvacearum,

At the end of a month, no external development

-ll-
of rust appearing, the stems were macerated and exan-

ined, whese, however, were also free of internal

mycelial develovnent,

6, Special agar media used:-

Plain agar:-

Glucose agar:

1000cc double distilled water

15 grans of agar

oO grams of beef extract

10 grams of peptone

Titreted to #12 (Suller's Scale)
Cleared with egg white

“iltered and tubed

Autoclaved 20 minutes

at 2<O pounis

1000ce double distilled water
15 grams of shredded agar

5 grams of beef extract

10 grams of peptone

cO grams of glucose

titrated to +12

Vleared with egg white
Filtered and tubed
Autoclaved for 20 minutes

at 20 pounds,

-l|e-
Nutrient glucose agar:-

Ashby's

1000cce double distilled water
40 grams of glucose

3 grans of lad

£0 graus of peptone

5 grams of beef extract

40 grams of shredded agar
Cleared with erg white
“iltered and tubed

Autoclaved for c2O minutes

at 2O pounds,

Synthetic agar:-

1000ce double distilled water
cO frans of mannite

0.2 grams K,#PO,

0,2 gtams I'gs0,

O.2 grams NaCl

O.,1 gram Caso,

5,0 grams CalOs

15 grans powdered agar

Tubed and autoclaved for

15 minutes at 15 pounds,

~13-
Nutrient gelatin: -
1L000ce of bouilion
110 grams of gelatin.
Titrated to +10 (fuller's scale)
~ubed and steamed 3O min,
on each of three consecutive

davs,

Tubes of eacn of the above media were inoculated
with spores and mycelium of Puccinia malvacearun as
before, rlates were also poured and sprayed with spores
and mycelium, Again no rust development took place, A

contamination of “usarium was present in several tubes,

7. Oat seedlings as media:-

Oat seedlings were grown and when they head reached
the heignt ot five or six inches were placed in tubes
kept moist with wet cotton and sealed with wax, kHEelf
of the tubes were sterilized in the autoclave at 2O pounds
for 2<O minutes, ‘he otner half were placed in & water
bath at 60° for one hour on each of five consecutive days,
shese tubes were then unsealed and inoculated with uredinio-
sovores and mvcelium of Puccinia coronata, infected oat
plants havdng been introduced into tre freenhcuse,

‘These tubes were kent for two months and very care-

fully examined from time to time, No signs or rust

=-j|4-—
appeared externally and when the seedlines were mazer-
ated nothing was found to have developed internally,
In some of the checks aad also in a few inoculated

tubes a sporotrichum was found to have developed,

8, Constantly washed media:-

On the assumption that the rust mycelium pro-
duces a by-product injurious to itself but carried off
by the susceptible host the followinre experiment was
made:

peveral of the various media used previously
were put on glass slides in drops, ‘Zhe: were inoculated
with Puccinia malvacesrumn spores ani mycelium, Zech
slide was then clamped between two strivs of filter
paper and the whole used as a Siphon in double distilled
water, -hus the media were kept constantly moist and
any by-produce of the fungus was washed away,

Again negative results only were obtsined,
Contamination of course wes very great, so a Similar
experiment in wnich contamination would be prevented

was planned,
9, Tubes of agar made from decoctions of oat leaves

and mallow leaves were used as shown in the photograph

below,

 

 

The tubes were sterilized and sterile water was al-
lowed to drip constantly over the inoculated surface
of the agar, No developi.ent of rust resulted, nor

was there any contamination,
 
10, Medium of COs increased:-

Thinking that slightly greater concentra-
tion of COs in the intercellular leaf spaces may have
some influence upon fungus development, this peaten
was introduced as follows:

| A porous cup atmometer was placed in a glass
cylinder and connected to a feed tube through the bot-
tom of the same, Cotton was used in Both ends of the
cylinder to prevent eontamination, Through the upper
plug two small glass tubes were fitted, one for the

entrance of COp from a Kipp generator and the other

tube for an exhaust, See photograph, The feed tube

 

 
of the porous cun was connected to a large flasx con-
taining the followinz solution: 2000 ce, double dis-
tilled water, 1 fram glucose, and 1 frem of asparagin,
she wnole was then sterilized in the autoclave, ‘ne

COs was allowed to flow very slowly into the cylinder

a)

and a circulation was accomplisned by connecting the
exnaust tube to a suction pumo, Urediniosvores of
Uromyces caryophyllinus were spread freely upon the
surrace of the atmometer and the apparatus was then

run for 18 days, At the end of that time the atmometer
was examined but no rust develonvment was noticeable, A

contamination of Penicillium, however, was present,

It is the intention of the writer to garry on
a series of similar exneriments with different nutrient

solutions when time vernuits,
Germination ixperiments

1, Light effects:-

A search of the literature brought forth
three instances in which a phototropic reaction was
noted in germination of rust spores, ard (1) states

that in some of his work the germ tube of urediniospores

(1), Ward, H, M, Ann, Bot, 16:267, 1902,

-18-
of Puccinia dispersa exhibited phototropic curvatures,
Robinson (1) has shown recently that the sporidial

germ tubes of Puccinia malvacearum react negatively to
daylight. Fromme (2), working with urediniospores of
Puccinia xhamni, has likewise shown that the cerm tubes
of these spores exhibit negative phototropism, ‘he
writer using methods similar to those of *romme has
tested out urediniospores of Uromyces caryophyllinus,
Spores were dusted over the surface of plain gelatin

in plates containing moist filter papers, Checks were
maintained in total darkness and equilateral diffused
daylight, ‘Those exposed to unilateral daylignt were
placed on a window ledge in dark boxes with an aperture
of 1 cm, in diameter toward the window, ‘This experiment
was repeated at least 12 times, but in no case did those
spores subjected to unilateral light show any pnototropic
curvatures whatsoever, It was noted also that the time
for germination and percentage of same was practically the
same in total darkness, unilateral light, and equilateral

light,

z, Thermotropic influences:-=

In view of the fact that evavoration from a

(1). Robinson, ‘J, Ann, Bot, 28:331-340, 1914,

(2), Fromme, F, D, Journal Bot, £:82-85, 1915.

-19-
leaf is greater at the stomata, it was thought that the
lower temperature due to this greater evaporation may
have some effect upon the direction of germ tube growth,
The surface of plain gelatin in plates with moist fil-
ter paper was dusted with urediniospores of Uromyces
caryophyllinus, These plates were kept in boxes with
bags of cracked ice at one end and with the other end
in contact with an incubator of 37°, lo thermotropic

curvatures, however, resulted,

3, Temperature effects:-
To test out accurately the effects of temper-
ature without varying other factors a piece of apparatus

as shown in the photograph was constructed and used,

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This consists of a water-tight tube the ends of which
are enclosed in two large tanks, One tank is kept con-
stantly filled with ice and the other with hot water,
heated with an electric imarsion boiler, The tube is
fitted with thermometers at regular intervals, Alone
the top and alternating with the thermometers are siall
holes just large enough to admit Van Tiegnem cells,
These cells are held in place by means of rubber washers,
Under each cell there is fitted in the tube a glass win-
dow to admit light in order that microscopés can be used
in watching germination, ‘Yhen in operation a quite reg-
ular variation in temperature is obtained along the tube,
This is not as constant as is preferred but with careful
manipulation the change is never more than one degree,
Urediniospores of Puccinia coronata were used,
Samples were always taxen from tne same fleck in order
to get them of the same age and produced under the same
conditions, The temperature range used was 16, 18, <0,
£2, 24, 26, 28, 30, 32, 34, 36 degrees C, At all temper-
atures the swelling of the germ pores seemed to take
place at about the same time and with about an equal
percentage, ‘the germ tubes emerged also at the same
time at all temperatures but it soon became noticeable
that at a temperature around 18° not only did a higher
percentage continue their growth but that the rate of

growth was also increased,

-2l|-
4, Alternate wetting and drying of rust spores:-

she purpose of this experiment was to note
any effect of alternate wetting and drying upon the
incubation period of rust snores, ‘The actual wetting
and drying process is hardly possible without changing
other factors so another method had to be employed,
Since svores contain some moisture in their natural con-
dition, it was therefore concluded that wetting and dry-
ing is no more or less than changing the concentration
of the spore content, ‘aking advantage, then, of osmotic
forces, the spores were subjected alternately to double
distilled water and a strong sugar (glucose) solution,
the former being tne wetter, the latter the dryer, ‘The
following method was used:-

Gooch crucibles were fitted with filter paper
discs which had been cut out with a cork drill, Uredin-
iospores of Succinia coronata taken from one fleck were
divided into three lots, ‘The first was placed immediately
in Van Tieghem cells in drops of double distilled water,
The second was placed in Van Bieghem cells in drovs of
the strong sugar solution, The third lot was placed in
one of the crucibles and was subjected alternately to
distilled water and sugar solution by means of wash

bottles, The solution was changed every minute, quick

~LPea
changes being accomplished by blowing out the solu-
tion through tne filter paper discs, ‘The spores were
subjected 25 times to each solution in as many minutes,
These were then also placed in Van Tieghem cells in
drops of double distilled water,

the three sets were carefully watched throuph
a microscope with the following results: ‘Zhe germ pores
in all cases commenced swelling at tne same time; the
germ tubes emerged at the same time; and the percentage
of germination was equal, However, it was noted that
the rate of growth was greatest from spores which had
been subjected only to the sugar solution, next from
the spores which had been wetted and dried, and least
from the sppres which hed been subjected only to double
distilled water,

The experiment was reveated several times with
Similar results, It was concluded, therefore, that wet-
tinge and drying, or at least, changing the concentration
of spore contents has no effect upon the incubation period,
but that the concentration of the media has some effect

upon the rate of growth of the germ tube,

-23-
Conclusions

Although time permitted the testing of little
more than ordinary methods of artificial culture, we
can at least conclude that rusts are extremely para-
Sitic, tnat is, restricted to a very hifh derree to
living substvata, “ne fact trat in nearly all methods
used, contamination was present, substantiates the above
conclusion, in that the containination indicated favora-
ble conditions for the growth of various other forms
of fungi, ‘ne work was hardly extensive enough to f0
so far as to state tat rusts cennot be grown in arti-
ficial culture, but it nevertneless leads one to believe
that in the present state of our knowledge of the sym-
biotic neture of rusts end hosts the former cannot be
grown separately.

ene results of the experiments on spore geriina-
tion, esvecially tnose on the effect of temperature and
concentration of medium, bring forth a new es-ect of the
problem, cince the mere emergence of the germ tube must
be called germination, then the above factors have, with-
in certain li.its, no effect uvon tne ability of the
spores to germinate, On the other hand, however, the
results plainly snow that trese factors do influence the
germ tube after its emergence by restricting it entirely

or by regulating its rate of growth,
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