Quantitative studies on spore production and host infection by Pythium spp.

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Quantitative studies on spore production and host infection by Pythium spp.
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vii, 81 leaves : ill. ; 28 cm.
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Sauve, Roger J
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Pythium   ( lcsh )
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Thesis--University of Florida.
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Includes bibliographical references (leaves 75-80).
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by Roger J. Sauve.
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Typescript.
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Vita.

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University of Florida
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QUANTITATIVE STUDIES ON SPORE PRODUCTION
AND HOST INFECTION BY PYTHIUM SPP.












BY

ROGER J. SAUVE


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY








UNIVERSITY OF FLORIDA


1978

















ACKNOWLEDGMENTS


The writer wishes to express his wholehearted appreciation to

Dr. David J. Mitchell, Chairman of the Supervisory Committee, for his

consideration, understanding, guidance and assistance throughout this

investigation and especially during the preparation of this manuscript.

Appreciation is also expressed to the other members of the Supervisory

Committee, to the entire staff and faculty of the Department of Plant

Pathology, and to various members of the Department of Botany of the

University of Florida for their courtesy and assistance.

Gratitude is expressed to the State of Florida for providing the

financial support that made this work possible.

Special gratitude is expressed to my wife, Deidre, for her limitless

patience during the three years that this manuscript was in preparation.

















TABLE OF CONTENTS


ACKNOWLEDGMENTS ................................................... ii

ABSTRACT ...................................... .................... v

PART 1. OOSPORE PRODUCTION BY PYTHIUM APHANIDERMATUM, P. DEBARYANUM,
P. MYRIOTYLUM, AND P. POLYMASTUM IN LIQUID MEDIA

Introduction .......................................... ....... 1

Materials and Methods ....................................... 3

Results ................................................ 7

Discussion .............. ....... ..................... .......... 17

PART 2. AN EVALUATION OF METHODS FOR OBTAINING MYCELIUM-FREE OOSPORES
OF PYTHIUM APHANIDERMATUM AND P. MYRIOTYLUM

Introduction .................................................. 20

Materials and Methods .................................... 21

Results ............................................. .. ..... 23

Discussion .............................................. ..... 29

PART 3. EFFECT OF SOME ENVIRONMENTAL AND CULTURAL FACTORS
ON INFECTION OF SEVERAL HOSTS BY PYTHIUM SPP.

Introduction .................................................. 31

Materials and Methods ...................................... 32

Results ........ ....................................... ........ 36

Discussion .................................................... 56
















PART 4. RELATIONSHIPS OF THE NUMBERS OF MOTILE AND ENCYSTED ZOOSPORES
OF PYTHIUM APHANIDERMATUM, P. MYRIOTYLUM, AND PHYTOPHTHORA
PALMIVORA TO INFECTION OF TOMATO

Introduction .................................................. 60

Materials and Methods ......................................... 62

Results ................................... .............. 66

Discussion .............................................. 72

LITERATURE CITED .............................................. ...... 75

BIOGRAPHICAL SKETCH ........... ...................... ............ 81

















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of
the Requirements for the Degree of
Doctor of Philosophy


QUANTITATIVE STUDIES ON SPORE PRODUCTION
AND HOST INFECTION BY PYTHIUM SPP.

by

Roger J. Sauve

December, 1978

Chairman: David J. Mitchell
Major Department: Plant Pathology

Oospore production by Pythium aphanidermatum, P. debaryanum, P.

myriotylum, and P. polymastum was evaluated on two natural media and

on two synthetic media. Abundant oospores were produced in the natural

media and in the synthetic medium that contained cholesterol. Cholesterol

was required by all isolates for oospore production in the synthetic

medium. The amount of cholesterol required for oospore production

differed with each isolate and ranged from 1 to 1,000 ug per ml of medium.

Oospore production was inhibited when the cholesterol concentration was

greater than the amount required for optimum production. Optimum

temperatures for oospore production ranged from 15 to 35 C and were 5

to 10 C lower than the optimum temperatures for vegetative growth.

Maximum oospore production occurred after 7 to 21 days of incubation in

the dark at 25 C. After 3 weeks of incubation under continuous darkness

or light, there were no differences in the number of oospores produced.










Oospore germination and host infection were not affected adversely

when either of the following methods were used to prepare oospore

suspensions that were free of viable mycelial fragments: filtering and

sonicating suspensions at 40 to 80% of maximum intensity of a Biosonic

III ultra-sonic system for up to 60 sec; or treating suspensions with

cellulase-hemicellulase solutions or with solutions of commercial snail

enzymes. Oospore germination and host infection were, however, reduced

by freezing. In vitro germination of oospores and host infection in soils

were greater with oospores of P. myriotylum that were fed to water snails

than with comparable numbers of untreated oospores; with P. aphanidermatum,

no increases in germination of oospores or host infection were observed

when oospores were fed to water snails.

When the four Pythium spp. were used in a host range test with

cabbage, corn, rye, and tomato, P. aphanidermatum and P. debaryanum

infected all plants tested but caused root weight reduction only in

tomato. Pythium myriotylum infected and caused root weight loss in all

plants tested, but P. polymastum infected and caused root weight loss

only in cabbage.

The relationships of the density of oospores in soil to the incidence

of infection were determined for P. aphanidermatum and P. debaryanum with

tomato, for P. myriotylum with rye, and for P. polymastum with cabbage.

Between 16 and 64 oospores per g of soil were required for 50% infection

of the host plants by the four Pythium spp. Percentages of infection of

tomato and rye by P. aphanidermatum and by P. myriotylum respectively,

were affected by the temperature, time, and the watering frequency while

the hosts were exposed to the inocula. Infection of rye by P. myriotylum

also was affected by isolates that differed in the ability to infect and










by the time that the isolate had been maintained in culture previous to

inoculation.

In addition, the relationships of numbers of motile or encysted

zoospores (cysts) of P. aphanidermatum, P. myriotylum, and Phytophthora

palmivora to infection of tomato were determined. Fewer numbers of

motile zoospores were required to obtain the same levels of infection

that occurred with cysts. The numbers of motile zoospores required

per plant for 50% infection of tomato seedlings were 275 for P.

aphanidermatum, 166 for P. myriotylum, and 1,505 for P. palmivora. The

numbers of cysts required per plant for 50% infection of tomato seedlings

were 23,419 for P. aphanidermatum, 10,988 for P. myriotylum, and 47,424

for P. palmivora. Optimum temperatures for infection of tomato by

motile zoospores of P. aphanidermatum, P. myriotylum, and P. palmivora

ranged from 20 to 30 C.

Percentages of infection of all hosts exposed to oospores or

zoospores increased with increasing inoculum levels, but the ratio of

infection to amount of inoculum decreased as the inoculum increased.

Slopes of regression lines of log010oge (1/l-y), where y equals the

proportion of infected plants, vs logl0 of inoculum level of oospores

or motile and encysted zoospores were between 0.62 and 0.69.
















PART 1


OOSPORE PRODUCTION BY PYTHIUM APHANIDERMATUM, P. DEBARYANUM,
P. MYRIOTYLUM, AND P. POLYMASTUM IN LIQUID MEDIA


Introduction


Various physical and nutritional factors influence oospore

production by Pythium spp. The specific numbers of oogonia and oospores

produced by several different species of Pythium in natural or synthetic

media under different cultural conditions rarely have been determined

precisely.

Although oospores are produced readily by most Pythium spp. in

natural media such as V-8 juice broth or hemp seed broth, synthetic media

may be desired for some studies which require a minimum of organic debris

or nutrients in oospore preparations, or which require reproducible

defined levels of medium components. Lenny and Klemmer (36) produced

mature oogonia of P. graminicola Subr. in a synthetic liquid medium,

but they did not indicate if oospores were formed. Yang and Mitchell

(67) observed the formation of mature oogonia of P. debaryanum Hesse,

P. irregulare Buis., and P. ultimum Trow in a chemically defined medium.

Adams (1) and Schmitthenner (56) produced oospores of P. aphanidermatum

(Edson) Fitz. in a synthetic medium for germination studies. Subsequently,

Schmitthenner's medium has been used for the production of oospores of

several other Pythium spp., including P. debaryanum, P. myriotylum Drechs.,

and P. ultimum (3, 44, 54, 55).










Several basic requirements must be met for oospore production in

synthetic media. For example, various carbon and nitrogen sources are

required by different species. The best carbon sources for oospore

production by P. debaryanum are lactose, glucose, and fructose, and the

best nitrogen sources are dl-isoleucine, 1-leucine and dl-asparagine (66).

Pythium butler Subram. produces oospores when amine asparagine is used,

but does not when isoleucine or leucine is used (35). The carbon to

nitrogen ratio may be more important for oospore production than the

source or concentration of these elements (34). Oospore production by

both Phytophthora and Pythium spp. is favored by a high carbon to nitrogen

ratio (34, 36, 55). Since Pythium spp. are incapable of synthesizing

sterols, cholesterol or some other suitable sterol must be incorporated

into synthetic media for oospore production (24, 25). Calcium is essen-

tial for the production of oogonia by P. debaryanum, P. graminicola, P.

irregulare, and P. ultimum (36, 67).

Optimum oospore production by Pythium spp. occurs near neutral pH;

oospores are produced typically at the same hydrogen ion concentrations

at which growth of these fungi occurs (1, 2). Al-Hassan & Fergus (2)

reported growth and oospore production by P. artotrogus (Mont.) deBary

over the pH range of 4.5 to 8.5. The optimum initial pH of the culture

medium ranged from 5.0 to 5.5 and the final pH ranged from 5.9 to 6.2.

Light may be an important environmental factor that affects oospore

formation by Pythium spp. High light intensities (10,760-13,988 Ix)

inhibit oospore production by P. ultimum (42).

The range of favorable temperatures for oospore production by

pythiaceous fungi is narrower than that for growth (2, 3, 28). In

general, temperatures lower than those for optimum vegetative growth









favor oospore formation, differentiation, and maturation by both

homothallic and heterothallic Pythium spp. The time required for ini-

tiation of antheridia and oogonia depends upon the temperature of

incubation. Gametangial initiation in some heterothallic Pythium spp.

begins after 3 days of incubation at 25 C (36). Germinable oospores

of P. aphanidermatum can be obtained after 5 days of incubation at 26

C (3).

Large quantities of oospores of P. aphanidermatum, P. debaryanum,

P. myriotylum, and P. polymastum produced under defined environmental

conditions were needed in our laboratory for studies on oospore ger-

mination, fungal survival in soil, and plant infection. The objective of

this study was to evaluate the cultural conditions in liquid media that

are most favorable for abundant production of viable oospores free of

other reproductive structures.


Materials and Methods


Pythium aphanidermatum and P. debaryanum were isolated from tomato

seedlings (Lycopersicon esculentum Mill.), P. myriotylum was isolated

from a peanut pod (Arachis hypogeae L.), and P. polymastum came from a

cabbage seedling (Brassica oleracea L.). Cultures of these fungi were

maintained on V-8 juice agar.

Hemp seed decoction broth was prepared as follows: 20 g of heat

sterilized hemp seeds (Cannabis sativa L.) were cracked by placing the

seeds in a Waring blender with 40 ml of distilled water (DW) and turning

the blender on and off at maximum speed ten times during a 10 sec period.

The cracked seeds with the water were then autoclaved at 121 C for 30 min.

After autoclaving, the mixture was filtered through four layers of cheese

cloth while still hot and the filtrate was adjusted to a 40-ml volume with










DW. Twenty milliliters of this solution were mixed with 980 ml of DW

and 15 ml of broth were added to each 250-mi Erlenmeyer flask.

Half strength V-8 juice broth was prepared by mixing 300 ml of V-8

juice (Campbell Soup Co., Camden, New Jersey) with 4.5 g of CaCO3 and

centrifuging the mixture at 5,860 X g for 5 min. One hundred milliliters

of supernatant were added to 900 ml of DW.

Yang's synthetic medium was prepared as described by Yang and

Mitchell (67). It was composed of 0.5 g of KH PO4, 0.5 g of K2SO 0.6 g

of MgC12-6 H20, 5.9 g of glucose, 1.3 g of dl-asparagine, and 1,000 ml

DW. The cultures were grown in 15 ml of this medium in 250-mi flasks for

7 days at 25 C in the dark, rinsed three times with sterile distilled

water (SDW) and then resuspended in 50 ml of replacement solution which

contained 0.001 M Ca+ in the form of a chloride salt (CaC12-2 H20).

The cultures were incubated for an additional 14 days.

Schmitthenner's synthetic liquid medium consisted of 0.15 g of

KH2PO4, 0.15 g of K2HPO4, 0.1 g of MgSO4-7 H20, 4.4 mg of ZnS047 H20,

1.0 mg of FeSO4*7 H20, 0.07 mg of MnC12*4 H20, 2 mg of thiamine HC1, 10

mg ascorbic acid, 30 mg of CaC12, 2.4 g of sucrose, 0.27 g of asparagine,

30 mg of cholesterol, and 1,000 ml of DW (56). Cholesterol was added to

15 ml of medium in each 250-mi flask as 0.45 mg of cholesterol in 1 ml

of dichloromethane after the addition of the medium and before autoclaving.

Cultures were initiated for each fungus by adding a 6-mm agar disc

cut from the perimeter of a 24 to 48-hr-old culture to each of four 250-ml

flasks containing 15 ml of a liquid medium. The agar medium used for

inoculum production was the same as the respective liquid medium used

in each test except for the addition of 17 g of Difco agar per liter of

medium. All media were adjusted to pH 6.5 with 0.1 N NaOH or 0.1 N HC1








5

before autoclaving. After inoculation, the cultures routinely were

maintained at 25 C for 21 days under continuous darkness unless

otherwise treated.

After 3 weeks of incubation, each culture was washed on a nylon

screen with SDW, resuspended in SDW, and homogenized with a glass tissue

grinder. The final homogenate from each culture was adjusted to a 50-ml

volume with SDW. This suspension then was subjected to 40% of maximum

intensity of a Biosonic III ultrasonic system for 40 sec to disrupt

mycelial fragments and to disperse the spores more evenly in water. The

numbers of oogonia and oospores within oogonia in the suspension were

determined immediately after sonication by counting four fields for each

of ten samples from each culture on a standard hemacytometer. Aborted

oospores and oospores with excessive vacuoles were not included in the

oospore counts.

The ability of the Pythium spp. to grow on different media was

observed on agar plates. Cultures for radial growth determination were

initiated by placing a 6-mm inoculum disc obtained from the margin of a

24- to 48-hr-old culture grown on the same medium as that used in the

test. Each petri dish contained 15 ml of medium. Four replicates of each

isolate were grown on each test medium. After 24 or 48 hrs of incubation,

four measurements of each colony diameter were taken and measurements for

each isolate on a particular medium were averaged.

The effects of cholesterol on colony diameter and on oogonium and

oospore production by each Pythium spp. were determined in Schmitthenner's

medium with or without agar. Concentrations of cholesterol ranging from

0.1 to 1,000 ug per ml of substrate were tested.

The time required for initiation of oogonia was determined at six

temperatures. Individual cultures were examined at 24-hr intervals and










the numbers of oogonia were recorded.

The effect of time on the production of oogonia and oospores was

determined by harvesting cultures after 1, 2, 3, 6, or 12 weeks of

incubation and counting the spores.

The effect of light was studied in a growth chamber illuminated with

fluorescent lamps producing 10,760 Ix at the level of the cultures. One

set of cultures was illuminated continuously and one set was wrapped in

aluminum foil.

The standard medium and incubation conditions (unless otherwise

indicated) were as follows: Schmitthenner's liquid medium was used for

oogonium and oospore production; Schmitthenner's agar was used for growth

determination; cultures were maintained in the dark at 251 C: cultures

in liquid medium were incubated for 21 days and those on agar for 24 or

48 hrs. Each experiment was repeated, and each treatment was replicated

four times. The results in tables represent data from one of the two

experiments since the results were similar. Duncan's multiple-range

test was used to determine significant differences between treatments.

Analyses of data in the form of percentages were performed with the data

transformed to arcsin degrees.










Results


Generally, the natural media were the best substrates for oospore

production by the Pythium spp. studied (Table 1). More oospores of

P. aphanidermatum and P. polymastum were produced in V-8 juice broth

than in the other media. The best medium for oospore production by

P. debaryanum was hemp seed broth. Schmitthenner's medium supported

good oospore production by all isolates. There were no significant

differences (P=0.05) in the number of oospores produced by P. debaryanum

and P. myriotylum in Schmitthenner's medium or in V-8 juice broth. No

oospores of P. myriotylum or P. polymastum and very few oospores of P.

aphanidermatum and P. debaryanum were produced in Yang's replacement medium.

Pythium aphanidermatum consistently produced the highest percentage

(70%) of oogonia with oospores that appeared normal, P. debaryanum (43%)

and P. myriotylum (37%) were intermediate and P. polymastum produced the

lowest percentage of oogonia with oospores (24%). When P. aphanidermatum,

P. myriotylum, or P. polymastum were cultured in hemp seed broth, V-8

juice broth, or Schmitthenner's medium, there were no significant

differences in the percentages of oogonia with oospores. However, the

percentages of oogonia with oospores of P. debaryanum differed with each

medium used. Pythium debaryanum produced the highest percentage of oogonia

with oospores in hemp seed broth (66%) and the lowest percentage in Yang's

medium (3%). Forty percent of the oogonia of P. debaryanum that were

produced in V-8 juice broth contained oospores and 20% of those produced

in Schmitthenner's medium contained oospores.

A significant increase in oospore production by all isolates was

observed when cholesterol was added to Schmitthenner's basal medium at

concentrations greater than 1.0 ug per ml (Table 2). Pythium aphanidermatum










and P. debaryanum produced the most oospores at 200-300 and 10-1,000

ug of cholesterol per ml of medium, respectively. Maximum production of

oospores by P. myriotylum and P. polymastum occurred at 100-300 and 10

ug per ml of medium, respectively.

At concentrations of cholesterol greater than that required for

optimum oospore production, oospore production by most isolates was

inhibited. Only P. polymastum did not produce oospores at high concen-

trations of cholesterol. No inhibition was observed when P. debaryanum

was cultured in media containing up to 1,000 ug of cholesterol per ml of

medium. The proportion of aborted oospores of P. aphanidermatum and P.

polymastum increased when the concentration of cholesterol was greater

than the concentration required for optimum oospore production. The

percentages of oogonia of P. debaryanum and P. myriotylum with oospores

that appeared normal were not significantly greater at 1,000 ug of

cholesterol than at 10 ug per ml of medium (Table 2).

Vegetative growth by P. debaryanum, P. myriotylum, and P. polymastum

appears to be stimulated slightly when the medium contained concentrations

of cholesterol ranging from 0.1 to 10 ug per ml of medium. No stimulation

of vegetative growth of P. aphanidermatum was observed when cholesterol

was added (Table 2).

For two isolates the range of temperatures for optimum oospore

production was broader than the range of temperatures for optimum

growth. The optimum temperatures for growth were 35 C for P. aphanidermatum

and P. myriotylum, 25 C for P. debaryanum, and 30 C for P. polymastum

(Table 3). Temperatures for optimum oospore production were 20-30 C for

P. aphanidermatum, 15 C for P. debaryanum, 25 C for P. myriotylum, and

15-25 C for P. polymastum.










The earliest production of oogonia by all isolates in Schmitthenner's

medium occurred at 25 C (Table 4). Pythium aphanidermatum produced

oogonia after 2 days at 25, 30, and 35 C; P. debaryanum, P. myriotylum,

and P. polymastum required an additional 2 to 3 days at 20 or 30 C.

Only P. aphanidermatum and P. myriotylum produced oogonia at 35 C. All

isolates produced oogonia at 15 C, but longer incubation times were

required.

Pythium aphanidermatum and P. myriotylum produced maximum numbers

of oospores after 7 days of incubation at 25 C. Pythium debaryanum

and P. polymastum required 14 to 21 days of incubation, respectively, for

maximum oospore production (Table 5). The number of oogonia per culture

remained the same after the first and second week of incubation. With

the exception of P. myriotylum, the number of oospores per culture and

the percentage of oogonia with oospores that appeared normal remained

relatively constant or declined slightly during the 12 week study.

Aborted oospores were observed in all cultures after the first week of

incubation. As time progressed the number of aborted oospores of P.

myriotylum steadily increased.

Cultures of Pythium spp. incubated under continuous darkness

produced 4 to 25% more oospores than those incubated under continuous

light; however, the increased production was not significant (P=0.05).

There were no differences in the number of oospores produced when the

four isolates were exposed to the following light regimes: 3, 7, and

14 days of continuous light followed with 18, 14, and 7 days of darkness,

respectively, or 3, 7, or 14 days of darkness followed with 18, 14, and

7 days of continuous light. When cultures of the four isolates were

exposed to strong light (10,760 Ix), growth was inhibited slightly.







10

None of the four Pythium spp. grew or produced oospores in Schmitt-

henner's medium adjusted to pH 4.0, but all isolates grew and produced

abundant oospores in media with initial hydrogen ion concentrations

ranging from pH 4.5 to 8.5. The final pH of the filtrates obtained from

cultures that produced oospores were all near neutrality, and the number

of oospores produced by each isolate in cultures with different initial

hydrogen ion concentrations did not differ.










Table 1. The influence of media on colony diameter and production of
oogonia and oospores by Pythium spp. after 21 days in the dark at 25 C.




Medium Colonyx Number of Number of % oogonia
diameter oogonia oospores with
(mm) per culturey per culture oospores


Pythium aphanidermatum
Hemp seed 67 bz 1.2 X 100 b 0.8 X 106 c 70 a
V-8 juice 73 a 3.1 C 106 a 2.3 X 106 a 74 a
Schmitthenner's 61 c 2.6 X 106 a 1.7 X 106 b 65 a
Yang's 55 d 0.4 X 106 c 0.1 X 105 d 2 b

Pythium debaryanum
Hemp seed 61 b 1.1 X 10b a 0.7 X 106 a 66 a
V-8 juice 70 a 0.5 X 106 c 0.2 X 106 b 40 b
Schmitthenner's 61 b 0.9 X 106 b 0.2 X 106 b 20 c
Yang's 62 b 0.2 X 106 d 0.1 X 105 c 3 d

Pythium mvriotylum
Hemp seed 72 b 1.0 X 106 b 0.3 X 106 b 32 a
V-8 juice 81 a 1.9 X 106 a 0.7 X 106 a 40 a
Schmitthenner's 61 c 2.1 X 106 a 0.8 X 106 a 39 a
Yang's 55 d 0 c 0 c 0 b

Pythium polymastum
Hemp seed 40 a 1.4 X 105 b 0.5 X 105 b 27 a
V-8 juice 29 b 5.6 X 105 a 1.1 X 105 a 19 a
Schmitthenner's 21 c 0.7 X 105 bc 0.3 X 105 b 27 a
Yang's 16 d 0.1 X 105 c 0 c 0 c


x
Growth after 24 hrs (48 hrs for P. polymastum) of incubation on 1.7%
agar incorporated into the respective liquid media (described in text).

YThe cultures were incubated in 250-ml Erlenmeyer flasks containing 15
ml of liquid medium; all media were adjusted to pH 6.5 before autoclaving.

ZMeans within a column not followed by the same letter are significantly
different (P=0.05) as determined by Duncan's multiple-range test; all
percentages were converted to arcsin degrees before analyses.











Table 2. The influence of cholesterol on colony diameter and production
of oogonia and oospores by Pythium spp. in Schmitthenner's medium in the
dark at 25 C.


Cholesterol Colonyx Number of Number of % oogonia
(ug/ml) diameter oogonia oospores with
(mm) per culturey per culture oospores


Pythium aphanidermatum
59 az 0.4 X 105 d
59 a 0.4 X 105 d 0.2
60 a 0.5 X 105 c 0.3
59 a 2.4 X 106 a 1.5
59 a 2.6 X 10 a 1.7
61 a 2.5 X 10 a 1.8
60 a 2.6 X 10 a 2.0
59 a 2.8 X 106 a 2.3
59 a 1.4 X 106 b 0.7
61 a 1.4 X 106 b 0.6


55 b


49 b


f
cde
bcde
bcd
bcd
abc
ab
a
de
e



b
b
b
a
a
a
a
a
a
a



c
c
b
a
a
a
a
a
a
a


Pythium debaryanum
0.9 X 10' c
7.1 X 105 b
7.8 X 105 ab
7.9 X 105 ab
8.5 X 105 a
8.3 X 105 a
8.6 X 105 a
8.5 X 105 a
8.5 X 105 a
8.3 X 105 a

Pythium myriotylum
1.7 X 10 c
2.0 X 106 be
2.0 X 106 be
2.0 X 106 be
2.1 X 106 be
2.4 X 106 ab l
2.5 X 106 a
2.8 X 106 a
2.5 X 106 a
2.6 06 a
2.6 X 10 a


0.1
1.0
10.0
30.0
100.0
200.0
300.0
500.0
1000.0


0.1
1.0
10.0
30.0
100.0
200.0
300.0
500.0
1000.0



0.0
0.1
1.0
10.0
30.0
100.0
200.0
300.0
500.0
1000.0










Table 2 continued.


Cholesterol Colonyx Number of Number of % oogonia
(ug/ml) diameter oogonia oospores with
(mm) per culture per culture oospores


Pythium polymastum
0.0 25 bz 0 c 0 d 0 d
0.1 25 b 0 c 0 d 0 d
1.0 26 b 0.2 X 10 c 0 d 0 d
10.0 30 a 5.4 X 105 a 7.2 X 104 a 13 b
30.0 31 a 1.0 X 105 b 2.6 X 104 b 28 a
100.0 29 a 0.8 X 105 bc 0.8 X 10 c 9 c
200.0 31 a 0.7 X 105 bc 0.6 X 10 c 7 c
300.0 30 a 0.2 X 104 c 0 d 0 d
500.0 31 a 0 c 0 d 0 d
1000.0 32 a 0 c 0 d 0 d


XGrowth after 24 hrs (P. polymastum 48 hrs) of incubation in the dark
at 25 C on 1.7% agar Incorporated into the respective liquid media
(described in text).

YThe cultures were incubated for 21 days in 250-ml Erlenmeyer flasks
containing 15 ml of liquid medium; all media were adjusted to pH 6.5
before autoclaving.

ZMeans within a column not followed by the same letter (s) are signif-
icantly different (P=0.05) as determined by Duncan's multiple-range
test; percentages were converted to arcsin degrees before analyses.










Table 3. The influence of temperature on colony diameter and production
of oogonia and oospores by Pythium spp. in Schmitthenner's medium.




Temperature Colonyx Number of Number of % oogonia
(C) diameter oogonia oospores with
(mm) per culture per culture oospores


Pythium aphanidermatum
15 21 fz 1.2 X 100 c 5.0 X 105 b 41 b
20 37 e 2.7 X 106 a 1.7 X 106 a 63 a
25 61 c 2.6 X 106 a 1.6 X 106 a 62 a
30 70 b 2.5 X 10 a 1.6 X 10 a 67 a
35 77 a 1.7 X 106 b 5.0 X 105 b 28 c
40 52 d 0 d 0 c 0 d

Pythium debaryanum
15 33 d 1.1 X 100 a 6.6 X 105 a 59 a
20 51 c 7.4 X 105 b 4.7 X 105 b 63 a
25 61 a 8.5 X 105 b 1.7 X 105 c 20 b
30 56 b 4.5 X 105 c 2.4 X 10 d 5 c
35 11 e 0 d 0 d 0 c
40 6 f 0 d 0 d 0 c

Pythium myriotylum
15 20 f 1.6 X 10 b .1 X 105 d 7 d
20 31 e 2.2 X 106 a 5.3 X 105 b 24 c
25 61 c 2.1 X 106 a 8.1 X 105 a 39 a
30 75 b 1.5 X 106 bc 4.8 X 105 b 31 b
35 87 a 1.2 X 106 c 3.3 X 105 c 28 bc
40 54 d 1.2 C 10 c 0 d 0 d

Pythium pplymastum 4
15 24 d 7.0 X 10 a 2.2 X 10 a 33 a
20 26 c 9.8 X 104 a 3.1 X 10 a 30 a
25 31 b 9.5 X 104 a 2.6 X 104 a 27 a
30 39 a 9.2 X 10 a 1.4 X 10 b 16 b
35 6 e 0 b 0 c 0 c
40 6 e 0 b 0 c 0 c


Growth after 24trs(48hrsfor P. polymastum) on 1.7% agar incorporated into
the respective liquid media.

YThe cultures were incubated for 21 days in the dark in 250-ml Erlenmeyer
flasks containing 15 ml of liquid medium; a 1 1 media were adjusted to pH
6.5 before autoclaving.

ZMeans within a column not followed by the same letter are significantly
different (P=0.05) as determined by Duncan's multiple-range test; all
percentages were converted to arcsin degrees before analyses.










Table 4. Time required for initiation of oogonium production by four
Pythium spp. at six temperatures.





Temperature (C)x
Pythium spp. 15 20 25 30 35 40
Days after inoculation


y
P. aphanidermatum 8 3 2 2 2 --

P. debaryanum 7 5 3 4 --

P. myriotylum 7 5 2 3 3

P. polymastum 6 4 3 4 --


in the dark.


XThe cultures were maintained in Schmitthenner's liquid medium

YNo oogonia were produced during 21 days of incubation.










Table 5. The effect of time of incubation at 25 C on oogonium and
oospore production by Pythium spp. in Schmitthenner's liquid medium.




Days after Number of Number of % oogonia
inoculation oogonia oospores with
per culturey per culture oospores


Pythium aphanidermatum
7 2.5 X 10b a 1.9 X 106 a 76 a
14 2.4 X 106 a 1.7 X 10 ab 70 ab
21 2.6 X 106 a 1.7 X 106 ab 65 ab
42 2.5 X 106 a 1.6 X 106 b 63 b
84 2.5 X 106 a 1.5 X 106 b 62 b

Pythium debaryanum
7 7.2 X 105 b 1.0 X 105 b 14 b
14 8.4 X 10 a 2.0 X 10 a 24 a
21 8.5 X 105 a 1.7 X 105 a 20 a
42 8.8 X 105 a 1.7 X 10 a 19 ab
84 8.9 X 10 a 1.7 X 10 a 19 ab

Pythium myriotylum
7 2.3 X 106 a 1.0 X 10' a 44 a
14 2.2 X 106 a 9.3 X 105 ab 42 a
21 2.1 X 106 a 8.1 X 105 b 39 a
42 2.2 X 10 a 3.5 X 10 c 16 b
84 2.2 X 106 a 2.9 X 105 c 13 b

Pythium polymastum
7 5.2 X 10 b 1.3 X 104 b 25 a
14 5.4 X 10 b 1.6 X 10 b 28 a
21 9.5 X 104 a 2.6 X 10 a 27 a
42 9.9 X 10 a 2.2 X 10 ab 22 a
5 4
84 1.0 X 10 a 1.8 X 10 ab 18 a


YThe cultures were incubated in 250-ml Erlenmeyer flasks containing 15 ml
of liquid medium, the medium was adjusted to pH 6.5 before autoclaving.

ZMeans within a column not followed by the same letter (s) are significant-
lv different (P=0.05) as determined by Duncan's multiple-range test;
percentages were converted to arcsin degrees before analyses.










Discussion


The natural media used most often for culturing pythiaceous fungi

are: cornmeal, hempseed, lima bean, oatmeal, pea, potato-carrot, soybean

or V-8 juice (2, 22, 27, 28, 42, 50, 52). Although these media provide

the nutritional requirements for growth and sporulation of pythiaceous fungi

they are defined poorly and are subject to change with locality, source

of ingredients, manufacturing, and consumer trends. Agar contains calcium,

nitrogen and other contaminants. Unless purified, it is unreliable for

use in studies with fungi that have exacting nutritional requirements.

Thus, chemically-defined liquid media are preferred over natural liquid

or solid media for nutritional or physiological studies. Synthetic liquid

media provide a homogenous environment for fungi, can be duplicated, and

can be separated more easily from the organism (27).

Optimum oospore production by the species of Pythium tested in this

study varied with the media used. The natural media were better than the

synthetic media for oospore production by most species. The replacement

medium of Yang and Mitchell (67) supported production of very few oospores

but the synthetic medium developed by Schmitthenner (56) sustained

abundant production by all species.

The percentage of oogonia with oospores changed when the cholesterol

concentration in Schmitthenner's medium was altered. No oospores were

produced when the cholesterol was omitted, but most isolates produced

oogonia. Ayers and Lumsden (3) obtained mature oospores of P. aphanider-

matum and P. ultimum in this medium when it lacked cholesterol; however,

their production was much greater when they added the sterol.

The results obtained by Ayers and Lumsden (3) on oospore production

in V-8 juice broth with P. aphanidermatum were similar to ours; 71% of










the oogonia in their study and 74% in this study contained mature

oospores. Similar numbers of oospores of P. myriotylum were formed in

the two studies; however, only 15% of the oogonia in their study

compared to 40% of the oogonia in our experiments produced mature oospores.

The differences that exist between the two studies are understand-

able since the isolates (20, 40) and the methods used were different.

All cultures used by Ayers and Lumsden (3) were grown in 25 ml of media

in petri dishes for only 14 days, while the fungi in this study were

grown in 15 ml of media in 250-ml Erlenmeyer flasks for 21 days. In

some of their tests, cultures were comminuted in a Tekmar Tissuemizer

before oospores were counted in a hemacytometer; in other tests oospores

were counted directly from mats. All cultures used in our tests were

comminuted with a glass tissue grinder and sonicated before counts were

made. This procedure eliminated the variability due to non-uniform

distribution of oospores on mats encountered by Ayers and Lumsden (3),

but the use of a tissue grinder for comminuting fungal mats probably

destroyed some oospores (55). Loss of oospores due to grinding appears

to be uniform since counts among replicates of a treatment were not

significantly different.

Of the physical environmental factors studied, temperature had the

greatest influence on oospore production and growth. For two isolates

the ranges of temperatures for optimum oospore production were broader

than for growth. Temperature ranges for optimum growth were higher than

temperatures required for optimum oospore production.

The use of oosDores obtained from cultures older than 2 weeks for

studies on oospore germination in vitro or on inoculum density in soils

reduces the possibility of introducing immature oospores or unfertilized

oogonia, which may lead to quantitatively inaccurate results. Oogonium










germination or growth from antheridia not yet moribund has been

reported to occur in vitro with several pythiaceous fungi (3, 9, 52).

Furthermore, oospores older than 2 weeks have been reported to germinate

at a higher rate than those obtained from younger cultures (1, 3).

Pythiaceous fungi have the ability to alter the hydrogen ion concen-

tration of the medium in which they are cultured (2, 31, 32, 39). All

Pythium spp. grown in Schmitthenner's medium neutralized the hydrogen

ion concentration. Since the phosphate buffer systems used were not

adequate to maintain the initial hydrogen ion concentration, better

buffer systems (12) should be evaluated in the future.

The presence or absence of light greatly influences oospore produc-

tion by Phytophthora spp. (10, 24, 27), but it does not appear to have a

significant effect on oospore production by the Pythium spp. tested in

this study.

Various physical and nutritional factors influence oospore produc-

tion by Pythium spp. Schmitthenner's liquid medium is a good medium for

oospore production by all of the Pythium spp. used in this study, but for

optimum oospore production the cholesterol level and the incubation tem-

perature should be adjusted to suit the individual isolate.

















PART 2


AN EVALUATION OF METHODS FOR OBTAINING MYCELIUM-FREE OOSPORES OF
PYTHIUM APHANIDERMATUM AND P. MYRIOTYLUM


Introduction


Techniques for obtaining oospoms free of mycelial fragments and

other viable propagules are useful for studies on oospore germination,

population dynamics, and the relationship of inoculum densities of

various species of Phytophthora and Pythium to infection and disease

incidence.

Some of the methods that have been used by other workers to

separate oospores of various oomycetes from mycelial fragments are

centrifugirg an oospore suspension in a sucrose density gradient after

freezing the culture to kill the mycelium (48); forcing cultures through

a 50-u sieve and separating oospores from mycelial fragments on a column

of water (14); coupling sonication and isopycnic centrifugation of an

oospore suspension (66); grinding mats in a tissue grinder and sonicating

the suspension to disrupt mycelial fragments (44, 54); grinding mats and

treating the suspension with commercial snail enzymes or combinations of

cellulase and hemicellulase (58, 60); and feeding the mycelial mats to

snails such as species of Helisoma, Helix, Planorbarius, and Planorbis

(7, 19, 57, 60).

The objectives of this study were to evaluate several methods for

producing oospore inocula free of viable mycelial fragments and to evalu-

ate the effect of the inocula on root infection.

20










Materials and Methods


The isolate of Pythium aphanidermatum (Edson) Fitz. used in this

study was isolated from a tomato seedling (Lycopersicon esculentum Mill.)

and the isolate of P. myriotylum Drechs. was isolated from a peanut pod

(Arachis hypogeae L.). Single oospore isolates of these fungi that had

been isolated 11 to 13 months before they wereused in this study were

maintained on V-8 juice agar (200 ml of clarified V-8 juice, 800 ml of

H20, 17 g of Difco agar) in the dark at 20 C.

Oospores were produced in 250-ml Erlenmeyer flasks containing 15

ml of Schmitthenner's liquid medium (56). Mycelial mats free of oospores

were produced either in Difco nutrient broth or in Schmitthenner's

liquid medium without cholesterol. All cultures were incubated for

three weeks in the dark at 25 C.

Before use, all of the mycelial mats were washed three times in

sterile deionized water and homogenized with a glass tissue grinder.

Sterile deionized water was added to the homogenate to provide a final

concentration of 10,000 oospores per ml of suspension. The suspension

was divided then into six 50-ml aliquots and each aliquot was subjected

to one of the treatments listed in Table 6.

At the termination of each treatment, the number of oospores

remaining in each suspension was determined by counting six fields for

each of 10 samples in a standard hemacytometer. The final concentration

was adjusted to 1,000 oospores per ml and 10 plates containing a

selective medium (PV) modified from that of Tsao and Ocana (62) were

each inoculated with 1 ml of the suspension. The medium contained 5 mg

of pimaricin (Delvocid, Gist-Brocades, Delft, Holland), 300 mg of

vancomycin hydrochloride (Vancomycin, Eli Lilly & Co.), and 17 g of









of Difco cornmeal agar in 1 liter of sterile deionized water. After

24 and 36 hrs of incubation at 30 C in the dark, the plates were examined

microscopically for growth from mycelial fragments or for oospore

germination.

Autoclaved Arredondo fine sand (pH 6.5, measurement obtained from

a 1:2 suspension of soil in 0.01 M CaC12) was infested with

oospores to give a final density of 25 oospores per g of soil for each

test using P. aphanidermatum inocula and 50 for those using P. myriotylum

inocula. Before the inocula were introduced into the soil, the water

content of the soil was adjusted to 5% (w/w).

The system developed by Mitchell (44) was used to expose noninjured

roots of tomato ('Bonny Best') and rye (Secale cereale L. 'Wesser')

seedlings to treated inocula of P. aphanidermatum and P. myriotylum.

respectively. Tomato seedlings were maintained for 7 days and rye

seedlings for 5 days in growth chambers at 30 C under 12 hrs of daylight

(10,760 Ix at the level of the plants). Fifty plants were used in each

treatment.

At the termination of each experiment, seedlings were harvested

by gently washing the soil away from the roots with a steady stream of

tap water. After the shoots were cut off, the roots were submerged in

70% ethyl alcohol for 5 sec and rinsed three times in sterile deionized

water. The roots were blotted dry on sterile paper towels and

plated on the PV medium. The plates were incubated in the dark at 30 C

and examined for growth of Pythium after 24 and 36 hrs.

All experiments were repeated at least twice. Those dealing with

root infection were repeated three times. The data presented on the

following tables are means of the experiments.











Results


Sonication of oospore suspensions at 20% of maximum intensity for

periods in excess of 100 sec, or at 40, 60, or 80% of maximum intensity

for 20 sec or longer, resulted in suspensions that contained only

oospores as viable propagules (Table 7). At the lower sonication

intensities or at shorter time intervals, some hyphal fragments gave

rise to new growth. All growth, at the higher intensities or at longer

sonication time intervals, was traced to germinated oospores. Pythium

myriotylum oospores were slightly less resistant to high sonication

intensities than were those of P. aphanidermatum. Oospores of P.

aphanidermatum, but not those of P. myriotylum, germinated after being

subjected to intensities as high as 80% of maximum for up to 100 sec.

No oospores or hyphal fragments of P. myriotylum produced new

growth when the suspension of the culture was frozen, but some growth

was observed from hyphal fragments and oospores of P. aphanidermatum

(Tables 8, 9). No infection of rye seedlings occurred with inoculum of

P. myriotylum that had been frozen (Table 9). Only 7% of the tomato

seedlings were infected after exposure to soil infested at 25 oospores

per g of soil with inoculum of P. aphanidermatum that had been frozen,

but 70-80% of the seedlings were infected after growth in soil infested

at the same inoculum density with oospores that had been exposed to the

other treatments (Table 8).

Although hyphal fragments from fresh mats produced new growth on

PV, seedlings exposed to soil infested with only hyphal fragments remained

healthy and were not infected (Tables 8, 9).

When oospores of P. myriotylum were recovered from pond snails and

used to infest soil, a significant increase in host infection occurred








24

(Table 9). Oospores of P. myriotylum that were treated with the enzymes

or fed to the pond snails germinated better in vitro than those that were

treated by the other methods (Table 9). No increase in oospore

germination or host infection was observed with P. aphanidermatum

inoculum that was treated with enzymes or fed to pond snails (Table 8).










Table 6. Treatments used for the preparation of oospore inocula.


Description


Untreated suspension


Filtered suspension



Frozen suspension


Sonication




Cellulase & hemicellulase








Snail enzymes








Snail ingested


The suspension was used immediately after
grinding in a glass tissue grinder.

The suspension was filtered twice through
4 layers of cheese cloth to remove mycelial
fragments.

The suspension was frozen at -5 C for 48 hrs
to kill mycelial fragments.

The suspension was subjected to 40% maximum
intensity of a Biosonic III ultrasonic
system for 40 sec at 20-30 C.

The suspension was centrifuged (6,000 g for
2 min) and resuspended in 20 ml of a 2.0%
buffered solution (0.1 M phosphate buffer
at pH 6.0) of cellulasea and hemicellulaseb.
After 48 hrs of incubation at 30 C in the
dark, the oospores were washed three times
in sterile distilled water.

The suspension was centrifuged (6,000 g for
2 min) and resuspended in 20 ml of a 5.0%
buffered solution of snail intestinal fluid
(0.1 M acetate buffer at pH 5.0) obtained
from Helix pomatiac. After 48 hrs of incubation
at 30 C in the dark, the oospores were washed
three times in sterile distilled water.

Intact mats containing oospores were fed to
small pondsnails that had been starved
overnight. After 48 hrs, the fecal pellets
were collected and the oospores were washed
four times in sterile distilled water that
contained 200 ppm Vancomycin hvdrochloride.


a"Onuzuka R-10", Calbiochem, P. O. Box 12087, San Diego 92112.

b"Rhozyme Hp-150", Rohm & Haas Co., Philadelphia 19105.

C"Glusulase", Endo Laboratory, 1000 Stewart Street, Garden City, N. J.


Treatment





















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Table 8. The effect of various treatments on hyphal growth, oospore
germination and infection of tomato seedlings by Pythium aphanidermatum.


Hyphal Oospore
growthw germinationx
(%)


Frequency of
infectionv
(%)


Nontreated mycelial fragments

Frozen suspension

Nontreated suspension

Filtered suspension

Sonicated suspension

Cellulase & hemicellulase

Snail enzymes

Snail ingested



WGrowth from hyphal fragments
the dark at 30 C (growth= +;

XOospore germination recorded
dark at 30 C.


4 bz


82 a

86 a

84 a

86 a

94 a

91 a


recorded after
no growth= -).


77 a

73 a

80 a

73 a

80 a

70 a


24 hrs of incubation in


after 24 to 36 hrs of incubation in the


V
Percent infection of tomato seedlings ((number of infected seedlings/
50 plants) X 100) after 7 days of incubation in soil infested with
25 oospores per g of soil at 30 C in a growth chamber with a 12-hr
day length.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.


Treatment











Table 9. The effect of various treatments on hyphal growth, oospore
germination and infection of rye seedlings by Pythium myriotylum.


Treatment


Hyphal Oospore
growth germinationx
(%)


Nontreated mycelial fragments

Frozen suspension

Nontreated suspension

Filtered suspension

Sonicated suspension

Cellulase & hemicellulase

Snail enzymes

Snail ingested


Frequency of
infectionY
(%)


0 bz


4b

6 b

4 b


14 ab

12 ab

21 a


40 b

43 b

47 b

43 b

40 b

67 a


WGrowth from hyphal fragments
the dark at 30 C (growth= +;
x
Oospore germination recorded
dark at 30 C.


recorded after
no growth= -).


24 hrs of incubation in


after 24 to 36 hrs of incubation in the


YPercent infection of rye seedlings ((number of infected seedlings/
50 plants) X 100) after 5 days of incubation in soil infested with
50 oospores per g of soil at 30 C in a growth chamber with a 12-hr
day length.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.









Discussion


It is important to have oospore suspensions free of viable

hyphal fragments for in vitro studies on oospore germination so that

slow germinating oospores are not overgrown, and for inoculum density

studies to ensure that propagule counts are as accurate as possible.

The presence of mycelial fragments from cultures 21-day-old or older

in non-amended soil, however, does not appear to influence the infection

rates at defined oospore densities in soil. Laviolette and Athow (33)

also noted that mycelia obtained from older cultures of Pvthium spp. are

not infective.

In our experiments P. aphanidermatum oospores were not affected

by any of the treatments and germinated readily in vitro. In contrast

to P. aphanidermatum oospores, P. myriotylum oospores germinated poorly

and were not affected greatly by any of the treatments. This difficulty

in germinating P. myriotylum oospores in vitro has been reported

previously (3, 44).

Although the percentages of infection of rye seedlings exposed to

P. myriotylum oospores from most treatments used in this study at 50

oospores per g of soil were low compared to the 713% infection observed

by Mitchell (44, 45) at the same oospore density of this fungus, the latter

value was close to the 67% infection obtained with oospores ingested by

pond snails. Oospores of two isolates of P. myriotylum (Part 3 of this

dissertation) gradually lost the ability to infect rye roots after

varying times (3 to 18 months) in culture or in soil in pots under

greenhouse conditions, but no changes were observed in the oospores

after inoculum was produced as described above. Thus, the higher

infection of rye seedlings with oospores that had been passed through










pond snails may be due, in part, to a screening effect produced by

the snails wherein damage or aborted oospores that are not discerned

easily visually from healthy oospores are destroyed and those retrieved

from fecal pellets are mostly germinable oospores. Counts of total

oospores recovered from fecal pellets were about 40% less than those

from mats that only had been homogenized. No increase in infectivity

was observed when oospores of an isolate of P. aphanidermatum that had

not been observed to lose its ability to infect tomato seedlings were

ingested by snails.

This study shows that sonication provides a rapid reliable method

for destruction of mycelial fragments for work requiring suspensions of

oospores of P. aphanidermatum and P. myriotylum. Sonication has been

used to prepare inocula of P. debaryanum Hesse, P. irregulare Buisman,

and P. polymastum Drechs. which contained only oospores as viable

propagule (44, 45, 54). This method has also been used to obtain

chlamydospore suspensions free of hyphal fragments of several

Phytophthora spp. (45, 47, 49, 51).

















PART 3

EFFECT OF SOME ENVIRONMENTAL AND CULTURAL FACTORS
ON INFECTION OF SEVERAL HOSTS BY PYTHIUM SPP.


Introduction


Under environmentally favorable conditions for host infection in

growth chambers, populations or Pythium spp. of 15 to 50 oospores per

g of soil were required for 50% infection of several hosts (43, 44, 53).

Disease under natural conditions, however, may not occur in soils that

contain abnormally high populations of Pvthium spp. propagules if the

environmental conditions are not favorable (23). Although information

is available on variability in host susceptibility to Pythium spp.

(23, 40), on intraspecific variability in pathogenicity of the fungi

(23, 40), on the number of oospores required for infection of plants

in soils (44, 45), and on certain environmental conditions that

contribute to infection of host plants during exposure to oospores

(23, 37, 44, 55), little or no information is available on the influence

of environmental conditions during oospore production on subsequent

infection of host roots.

The objectives of this study were: (i) to evaluate the effect of

four Pythium spp. at two inoculum levels on infection and disease

development in four plant species; (ii) to determine quantitatively

the relationship between numbers of oospores of P. aphanidermatum,

P. debaryanum, P. myriotylum, and P. polymastum to infection

31










of tomato, rye, and cabbage; and (iii) to evaluate the influence of

oospore age, different isolates of a species, media used for oospore

production, temperature used for oospore production,and of certain

environmental factors during exposure of oospores to host plants on

infection.


Materials and Methods


Pythium aphanidermatum (Edson) Fitz. and P. debaryanum Hesse were

isolated from tomato (Lycopersicon esculentum Mill.), P. myriotvlum

Drechs.was isolated from a peanut pod (Arachis hypogeae L.) and P.

polymastum Drechs. was isolated from a cabbage seedling (Brassica

oleracea L.). Hyphal-tipped cultures of these fungi were maintained

at 20 C on V-8 juice agar and transferred at monthly intervals.

The media and methods used for production and preparation of

oospores for soil infestation are described in Parts 1 and 2 of this

dissertation.

Arredondo fine sand (pH 6.5, measurement obtained from a 1:2

suspension of soil in 0.01 M CaC12) that had been sieved through a

20 mesh sieve and autoclaved at 15 psi and 120 C twice for 4 hrs at

24-hrs intervals was used throughout this study. Before inocula was

introduced, the water content of the soil was adjusted to 5% (w/w) by

adding sterile deionized water. The inocula were suspended in various

volumes of sterile deionized water and added to moist soils; the final

water content of the soil was 10%.

Cabbage ('Early Jersey Wakefield'), corn (Zea mays L. 'Fla 200 N'),

rye (Secale cereale L. 'Wesser') or tomato ('Bonny Best') seeds that

were surface-disinfested with 1% sodium hypochloride for 30 sec and










rinsed three times with sterile deionized water were placed on a

layer of sterile paper towels, moistened, wrapped in aluminum foil and

incubated at 30 C for 24 hrs.

The following methods were used for greenhouse experiments where

cabbage, corn, rye, and tomato were exposed to oospores of P. aphanider-

matum, P. debaryanum, P. myriotylum, or P. polymastum. Fifty grams

of soil infested with 100 or 1,000 oospores per g of soilwere layered

over 200 g of autoclaved soil packed in the bottom of a 10-cm (diameter)

clay pot. A 200 g layer of autoclaved soil was distributed evenly and

packed over the infested soil, and five germinated seeds were placed

on the surface of the layer. The seeds were covered with 50 g of

autoclaved soil. Ten pots were planted at each inoculum level for each

treatment. The pots were randomized, placed in saucers, and maintained

in the greenhouse for 3 weeks at temperatures which fluctuated between

28 and 37 C. Every 48 hrs the saucers were filled with tap water and

after 15 min the excess water was removed.

For experiments on the relationship of inoculum density to

infection of cabbage, rye and tomato or on recovery of oospores from

soil, the infested-soil layer technique developed by Mitchell (44) was

used. Fifteen grams of infested soil were layered over 100 g of

autoclaved coarse-builder's sand packed in the bottom of 100-ml

polypropylene beakers that each had 3 small holes at the base for water

movement. Five germinated seeds were placed on a 15 g layer of autoclaved

soil over the infested soil. The seeds were covered with 5 g of vermic-

ulite and the beakers were placed in a nylon pan. Ten beakers were used

for each treatment; an additional two beakers were used to follow soil

populations of Pythium spp. These beakers contained 30 g of infested soil









layered over the builder's sand and no autoclaved soil was used to

separate the seeds from the infested soil. No seeds were planted in

one of the two beakers used for soil population studies. The pans

containing 12 beakers each were maintained in growth chambers with 12 hrs

of light (10,760 Ix) at 30 C except for those containing cabbage seedlings

which were held at 25 C. The plants were kept in the growth chambers

for 7 days except for rye seedlings which were maintained for 5 days.

The plants were watered every 48 hrs by filling the pans with tap

water. After 15 min, the water was drained and the pans were returned

to the growth chambers. Unless otherwise indicated, these were the

standard conditions for all tests.

At the termination of each experiment, plants were harvested by

gently washing the soil and sand away from the root systems with a

stream of tap water. Root systems that were visibly free of adhering

soil particles were surface disinfested by dipping the roots for 5 sec

in 70% ethanol, rinsing three times in sterile deionized water, and

blotting on paper towels to remove excess water. Two root systems were

plated on each plate that contained a selective medium (PV) that was

modified from that of Tsao and Ocana (67). This medium contained 17 g

of Difco corn meal agar, 5 mg of Pimaricin (Delvocid, Delft, Holland)

and 300 mg of vancomycin hydrochloride (Vancocin, Eli Lilly & Co.) in

a liter of sterile deionized water. The inoculated plates were incubated

in the dark at 30 C and examined for growth of Pythium spp. after 24 and

36 hrs.

Populations of Pythium spp. in soil were evaluated by a soil

dilution method using dilutions of 1:10 and 1:20. Samples taken from

soils with or without plants were mixed with 200 ml of 0.3% water agar

that contained 300 mg vancomycin hydrochloride and 3.68 g of CaC12"2 H20









in a liter of deionized water. For each dilution a 1-ml aliquot was

spread onto each of 10 PV plates with a bent glass rod, and the plates

were incubated in the dark. After 36 hrs at 30 C (unless otherwise

indicated), the soil-agar mixture was washed from the surface of the

plates under a slow stream of tap water and the colonies were counted.

To evaluate the influence of the hydrogen ion concentration of

the dilution medium on recovery of propagules of Pythium spp. from

soil, the 0.3% water agar dilution was adjusted to hydrogen ion

concentrations ranging from pH 2.5 to 11.5 with 0.1 N HC1 or 0.1 N

NaOH.

All data presented in this study are means of at least two

experiments. The data presented for inoculum density studies at

various levels were repeated from three to eight times.










Results


The four plant species tested in this study differed greatly in

susceptibility to P. aphanidermatum, P. debaryanum, P. myriotylum, and

P. polymastum (Table 10). Although cabbage, corn, rye or tomato

seedlings were infected when grown in soils infested with P. aphanidermatum

or P. debaryanum at 100 oospores per g of soil, root weight loss and

mortality occurred only in tomato. All plants grown in soils infested

with oospores of P. myriotylum were infected and developed disease

symptoms. Although no dead corn seedlings were observed in this study,

root weight loss occurred when they were grown in soil infested with

1,000 oospores per g of soil of P. myriotylum. Pythium polymastum

infected and caused root weight loss only in cabbage.

Percentages of root infection of tomato, rye, and cabbage grown

in soils infested with 10 to 150 oospores per g of soil of P. aphanider-

matum or P. debaryanum, P. myriotylum, and P. polymastum, respectively,

increased with increasing inoculum levels (Fig. 1). However, the propor-

tion of infection to inoculum density decreased with increasing inoculum

densities (Fig. 1, and Table 11).

Points of proportion of infection (y) transformed to log10 loge /l-y

plotted against log10 of the inoculum density lie in a straight line

between 10 and 150 oospores per g of soil for all four host-pathogen

combinations (Fig. 2). The slopes of the data calculated by linear

regression analyses were between 0.66 and 0.68 (Fig. 2). Interpolated

inoculum levels required for 50% infection of the host plants (ID50)

were 16, 64, 22, and 51 for P. aphanidermatum, P. debaryanum, P. myriotylum,

respectively.










The isolate of P. polymastum used in this study rapidly lost the

ability to infect cabbage seedlings. When the experiment on the

relationship of the number of oospores of P. polymastum per g of soil

to infection of cabbage seedlings was repeated, 23 times more oospores

were required to infect 50% of the seedlings than were required previously.

At that time, the isolate of P. polymastum had been in culture for 4

months. The experiment was repeated a third time but no infection of

cabbage occurred. The data presented for P. polymastum on cabbage in

Table 11 is from the first experiment.

The effects of maintaining P. aphanidermatum and P. myriotylum in

culture for 1, 2, 4, and 8 months on infection of their respective hosts

were evaluated (Table 12). During the study, no noticeable decrease

in the infection of tomato by P. aphanidermatum at 10 or 100 oospores

per g of soil was observed, but a considerable decrease in the percentages

of infection of rye by P. myriotylum at 25 and 100 oospores per g of soil

occurred. After 6 months in culture, approximately three times more

oospores of P. myriotylum were required to produce the same percentages

of infection that were obtained when the isolate was freshly isolated.

When a 2-year-old culture of P. myriotylum (Pm-l)that had been

observed to lose its ability to infect rye was compared to six cultures

that were freshly isolated from peanut pods, considerable variations

in their ability to infect rye were observed (Table 13). Only Pm-15

behaved similarly to the original isolate Pm-l before it lost the

ability to infect rye. Isolates Pm-10 and Pm-12 did not differ from

Pm-1, after it had lost the ability to infect rye, in their ability to

infect rye. Isolates Pm-11, Pm-13, and Pm-14 were relatively non-infective.










In an experiment to determine the influence of time of exposure

of host roots to inoculum on incidence of infection, maximum infection

ot tomato by P. aphanidermatum occurred after 7 days, and maximum

infection of rye occurred after 4 days (Table 14). No increases in

infection of tomato or rye seedlings were observed when the seedlings

were exposed to infested soil for longer times.

Maximum infection of tomato by P. aphanidermatum or rye by P.

myriotylum occurred when the plants were incubated at 35 C (Table 15).

Lower infection rates were observed when the plants were incubated at

lower temperatures,

When weight of roots obtained from rye seedlings that were grown

in soils infested with P. myriotylum at 25 or 150 oospores per g of soil

and maintained at temperatures ranging from 20 to 35 C were compared, the

largest loss in root weight was from seedlings that were grown in soil

infested with 150 oospores per g of soil and maintained at 35 C. The

optimum temperature for root growth was at 30 C.

Tomato and rye seedlings that were exposed to infested soils that

were watered every 24 hrs had higher levels of infection than those

that were watered every 48 or 72 hrs (Table 16). The moisture content

of the soil 30 min after watering was 23.6% (w/w). After 24, 48, 72, and

96 hrs of incubation at 30 C in the growth chamber, the moisture contents

were 21.1, 20.1, 17.2, and 16.4%, respectively.

The percentages of tomato seedlings infected when grown in soils

infested with oospores of P. aphanidermatum that were produced in hemp

seed broth, V-8 juice broth, or Schmitthenner's liquid medium did not

differ. But, when rye seedlings were grown in soils that were infested

with oospores of P. myriotylum that were produced in hemp seed broth or










V-8 juice broth, slightly greater infection occurred than in soils

infested with oospores that were produced in Schmitthenner's medium

(Table 17).

Slightly higher infection rates occurred when tomato or rye

seedlings were grown in soils infested with oospores of P. aphanidermatum

or P. myriotylum, respectively, produced at 20 to 25 C than when they were

grown in soils infested with oospores produced at 30 or 35 C (Table 18).

The rate of recovery of P. aphanidermatum from soils infested with

oospores produced in hemp seed decoction broth was lower than from soils

infested with oospores produced in V-8 juice broth or Schmitthenner's

medium (Table 17). No differences in recovery rates of P. myriotylum

from soils infested with oospores produced in hemp seed decoction

broth, V-8 juice broth, or Schmitthenner's liquid medium were observed.

When soils infested with oospores of P. aphanidermatum, or P.

myriotylum were incubated at 30 C in a growth chamber with a 12-hr

day length and plated at 24-hr intervals, maximum recovery of P.

aphanidermatum from infested soils occurred after 5 days of incubation

and maximum recovery of P. myriotylum occurred after 4 days (Table 14).

A higher population of P. aphanidermatum occurred in infested

soils that were maintained at 30 C than at 20, 25, or 35 C (Table 15).

However, the highest populations of P. myriotylum occurred in soils

that were maintained at 35 C.

Lower rates of recovery of P. aphanidermatum or P, myriotylum

were observed from soils that contained plants than from soils that

did not contain plants (Table 19). But, maximum recovery of propagules

from soils that contained plants occurred earlier than from soils that

were without plants.










Optimum recovery rates were observed when the pH of the medium

used for making soil dilutions ranged from 3.5 to 10.5. At pH 2.5

or 10.5 reductions in the rates of recovery occurred (Table 20).

Incubation temperature and the length of time that oospores in

soil suspensions were maintained on the selective medium affected the

percentage of propagules recovered from soil. Germinated oospores of

P. aphanidermatum and P. myriotylum were observed on PV medium 6 hrs

after plating (Table 21). The percentages of germinated oospores

increased with time of incubation at 30 C in the dark. Maximum

germination percentages occurred after 30-36 hrs of incubation, after

which no additional germinated oospores were observed. Better recovery

of P. aphanidermatum occurred when the selective medium was maintained

at 30 C than at 20, 25, or 35 C (Table 22). Maximum recovery of P.

myriotylum occurred at 30 C but the rate of recovery at that temperature

was not significantly different to recovery rates at 25 or 35 C (Table 22).






























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Table 11. The effect of inoculum density on percentage root infection.




Infection


Pythium spp. Plant Inoculum density (oospores/g soil)


10 25 50 100 150
(%) (%) (%) (%) (%)


Pythium aphanidermatum Tomato 463 653 754 846 966

Pythium debaryanum Tomato 174 332 432 643 695

Pythium myriotylum Rye 343 561 713 805 100

Pythium polymastumx Cabbage 24 30 50 85 ---y


XData from one experiment.


YNo data.









Table 12. The effect of culture age of Pythium aphanidermatum on
infection of tomato seedlings and of P. myriotylum on infection of
rye seedlings.




Infectionx


Age of culturey Pythium aphanidermatum Pythium myriotylum
(months) oospores/g of soil oospores/g of soil
10 100 25 100
(%) (%) (%) (%)


1 44 az 90 a 38 a 86 a

2 41 a 84 a 29 a 74 a

4 49 a 87 a 6 b 44 b

8 38 a 89 a 0 b 53 b


xTomato and rye seedlings were harvested after 7 and 5 days, respectively,
of incubation in a growth chamber at 30 C with a 12-hr day length.

YHyphal tipped isolates of P. aphanidermatum and P. myriotylum were
maintained on V-8 juice agar at 20 C and transferred at monthly
intervals to fresh agar slants.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.









Table 13. The effect of different isolates of Pythium myriotylum on
infection of rye seedlings.





Infectionx


Isolate (oospores/g soil)
25 150
(%) (%)


Pm-1 2 bz 16 b

Pm-10 0 b 19 b

Pm-11 0 b 3 c

Pm-12 2 b 23 b

Pm-13 0 b 0 c

Pm-15 54 a 98 a


XSeedlings were
at 30 C with a


harvested after 5
12-hr day length.


days of incubation in a growth chamber


All isolates were obtained from different peanut pods collected from a
field in Levy County, Florida, and were maintained on V-8 juice agar
at 20 C.
z
Within each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.










Table 14. The effect of time of exposure of tomato seedlings to soil
infested with Pythium aphanidermatum and of rye seedlings to soil
infested with P. myriotylum on percentages of infection.


Frequency of isolationw


Pythium aphanidermatum


Pythium myriotylum


Soil


100 opg 100 opg 25 opg
(%) (%) (%)


Soil


100 opg 100 opg
(%) (%)


84 a

17 b


14 cd 14 b


25 c

67 b


16 b

20 b


77 ab 86 a

74 ab 81 a


84 a

84 a

81 a


89 a

83 a

89 a


0 c

7 c

9 c

25 b

43 a

56 a

54 a

49 a

45 a

56 a


20 c

36 c

69 b

82 a

80 a


4 c

8 c

28 b

41 a

44 a


78 ab 57 a


82 a

79 a

93 a


39 ab

42 a

42 a


Seedlings


were incubated in beakers in growth chambers at 30 C with a


12-hr day length and watered every 48 hrs.


XAfter 1 to 21 days, 50 seedlings were harvested and the root systems
were plated on a selective medium (PV); soil samples were assayed for
populations after 0 to 21 days, only soils that did not contain plants
were sampled.

Yopg= oospores per g of soil.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.


Time
(days)


Tomato


10 opgy
(%)


0 bz


14 b

39 a

39 a

44 a

46 a

41 a

45 a









Table 15. The effect of temperature on percent infection of tomato
by Pythium aphanidermatum and of rye by P. myriotylum.


Frequency of isolation


Pythium aphanidermatum


Pythium myriotylum


Temperaturev
(C)


Tomato


10 opgy
(%)


Soil


100 opg 100 opg
(%) (%)


Rye


Soil


25 opg 100 opg 100 opg
(%) (%) (%)


20 26 bz 49 c 62 b 0 c 17 c 16 c

25 39 ab 69 b 74 ab 29 b 47 b 21 c

30 46 a 84 ab 89 a 56 a 80 a 46 b

35 44 a 91 a 52 b 61 a 84 a 62 a



Seedlings were incubated in beakers in growth chambers with a 12-hr
day length at temperatures ranging from 20 to 35 C.


WTomato seedlings and infested soil without plants were
7 days.


maintained for


Rye seedlings and infested soil without plants were maintained for 5
days.

YOospores per g of soil.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.









Table 16. The effect of watering frequency on percent infection of
tomato by Pythium aphanidermatum or of rye by P. myriotylum.





Infection


WateringY Pythium aphanidermatum Pythium myriotylum
frequency oospores/g of soil
(hrs)
10 100 25 100
(%) (%) (%) (%)


24 49 a 100 a 73 a 94 a

48 44 a 74 b 54 b 78 b

72 41 a 79 b 54 b 72 b


YAfter 24, 48, or
were taken out of
after 15 min, the
growth chamber at


72 hrs, the pans containing tomato or rye seedlings
the growth chamber and were filled with tap water;
water was drained and the pans were returned to the
30 C with a 12-hr day length for 6 days.


Within each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.









Table 17. The effect of the media used for oospore production on percent
infection of tomato by Pythium aphanidermatum or of rye by P. myriotylum.


Frequency of isolation


Pythium aphanidermatumw
Pythium aphanidermatum


x
Pythium myriotylum


Tomato


10 opgY
(%)


Soil


Soil


100 opg 100 opg 25 opg 100 opg 100 opg
(%) (%) (%) (%) (%)


Hemp seed 41 az 78 a 56 b 47 a 94 a 51 a

V-8 juice 43 a 84 a 86 a 41 a 88 a 55 a

Schmitthenner's 46 a 84 a 89 a 35 a 80 a 46 a


VCultures of P. aphanidermatum and P. myriotylum
dark at 25 C for 21 days.


were incubated in the


WTomato seedlings and infested soil without plants were maintained at
30 C for 7 days in a growth chamber with a 12-hr day length.
x
Rye seedlings and infested soil without plants were maintained at 30 C
for 5 days in a growth chamber with a 12-hr day length.

YOospores per g of soil.
z
Within each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.


Medium








Table 18. The effect of the temperature at which cultures were maintained
for oospore production on percent infection of tomato by Pythium
aphanidermatum or of rye by P. myriotylum.


Frequency of isolation


Pythium aphanidermatumw


Pythium myriotylumx


Temperatures
(C)


Tomato


10 opgy
(%)


Soil


Rye


100 opg 100 opg 25 opg 100 opg 100 opg
((%) (% (%) (%) (%)


20 52 az 89 a 84 a 41 a 92 a 34 b

25 46 a 84 a 89 a 34 ab 80 ab 46 a

30 37 ab 66 b 89 a 28 ab 74 b 38 ab

35 28 b 41 c 81 a 24 b 65 b 17 c



Oospores of P. aphanidermatum and P. myriotylum were produced in
Schmitthenner's liquid medium; the medium was incubated in the dark
at temperatures ranging from 20 to 35 C for 21 days.

Tomato seedlings and infested soil without plants were maintained at
30 C for 7 days in a growth chamber with a 12-hr day length.

XRye seedlings and infested soil without plants were maintained at 30 C
for 5 days in a growth chamber with a 12-hr day length.

YOospores per g of soil.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.


Soil









Table 19. The influence of the presence of host plants on recovery of
Pythium aphanidermatum or P. myriotylum from artificially infested soils.





Percent recovery
x
Plant
Pythium aphanidermatum Pythium myriotylum

(%) (%)


+ 39 bz 17 b

86 a 46 a



XPopulations of P. aphanidermatum or P. myriotylum were assayed from
soils that contained plants and from soils that were without plants
(Plants= +, no plants= -); initial inoculum was 100 oospores per g of
soil.

YSoil infested with P. aphanidermatum or P. myriotylum was maintained
at 30 C in a growth chamber with a 12-hr day length for 7 or 5 days,
respectively.
z
Within each column, entries without a common letter are significantly
different (p= 0.05) as determined by "T" test; analyses were performed
with data transformed to arcsin degrees.









Table 20. The effect of the hydrogen ion concentration of the suspension
used for making soil dilutions on recovery of Pythium aphanidermatum or
P. myriotylum from artificially infested soils.





Percent recovery


Pythium aphanidermatum Pythium myriotylum

pH (%) (%)


2.5 55 cz 15 b

3.5 96 a 36 a

4.5 100 a 42 a

5.5 100 a 47 a

6.5 89 ab 46 a

7.5 86 ab 46 a

8.5 83 ab 42 a

9.5 80 b 34 a

10.5 77 b 32 a

11.5 58 c 19 b



oospores of Pythium aphanidermatum and of P. myriotylum were incorporated
in Arredondo fine sand (pH 6.5) and maintained in a growth chamber at
30 C for 7 days; no plants were used in this test; soil samples containing
100 oospores per g of soil were plated on a selective medium (PV) using
water agar solution with a pH ranging from 2.5 to 11.5.

ZWithin each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.










Table 21. The effect of time on the selective medium on recovery of
oospores of Pythium aphanidermatum or P. myriotylum from artificially
infested soils.




Percent recoveryy


Time Pythium aphanidermatum Pythium myriotylum

(hrs) (%) (%)


6 3 c 1 b

12 12 c 4 b

18 52 b 10 b

24 77 a 31 a

30 89 a 43 a

36 89 a 46 a



Sixty petri dishes that each contained 15 ml of a selective medium (PV)
were inoculated with a suspension of soil infested with 100 oospores
of Pythium aphanidermatum'or P. myriotylum per g of soil and incubated
in the dark at 30 C; after 6, 12, 18, 24, 30, and 36 hrs of incubation,
10 plates were removed from the incubator and examined under magnification
(200 X) for germinated oospores.

Prior to plating the soil on a selective medium, the cups that
contained the infested soil were maintained at 30 C in a growth
chamber with a 12-hr day length for 7 days for P. aphanidermatum and
for 5 days for P. myriotylum; only soil from cups that did not contain
plants was sampled.

Within each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.









Table 22. The effect of temperature during the incubation of fungi
on the selective medium on percent recovery of Pythium aphanidermatum
or P. myriotylum from artificially infested soils.





Percent recoveryy
x
Temperature Pythium aphanidermatum Pythium myriotylum

(C) (%) (%)

z
20 62 b 24 b

25 74 b 42 a

30 89 a 46 a

35 44 c 39 ab



XInoculated petri dishes that contained a selective medium were incubated
at temperatures ranging from 20 to 35 C in the dark for 36 hrs.

YSoil infested with P. aphanidermatum or P. myriotylum without plants
was maintained at 30 C for 7 or 5 days, respectively, prior to plating.
z
Within each column, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.










100






80 .."""


_UJ~ ~ *' ^.









S.............. polymastum on cabbage
20 -













0 10 50 100 150
INOCULUM DENSITY (OOSPORES/g SOIL)

Fig. 40 The relationship of incidence of infection (arithmetic) of several
hosts t densities of oospores deb(arithmetic) of several Pythium spp.tomato
.-.-....-- P myriotylum on rye
.............. P polymastum on cabbage



20







10 50 100 150
INOCULUM DENSITY (OOSPORES/g SOIL)

Fig. 1. The relationship of incidence of infection (arithmetic) of several
hosts to dernsities of oospores (arithmetic) of several Pythium spp.













3.0 *


....... P aphanldermatum on tomato
P debaryanum on tomato
P myriotylum on rye ,
.............. R polymastum on cabbage


1.0 ... .




O *
I .' o
z
o ...


.LL








.1 I.
1 10 100 200
INOCULUM DENSITY (OOSPORES/g SOIL)

Fig. 2. The relationships of percentages of infection adjusted for
multiple infections (logarithmic) to densities of oospores of several
Pythium spp. (logarithmic); the linear correlation coefficient was
significant at P= 0.05 (*) or P= 0.01 (**).










Discussion


In this study reductions in the rates of infection occurred

when factors that affect the susceptibility of the host, the pathoge-

nicity of the pathogen or the favorability of the environment were

adversely altered. The curvilinear arithmetic and the log-log plots

drawn from data obtained from experiments with the four host-pathogen

systems studied were similar to the results obtained by other workers

for host-pathogen systems involving non-motile inoculum and motile

infection courts (4, 44, 45). The slope values of the log-log

transformations were between 0.66 and 0.68. Baker (4) predicted that in

systems where pathogen response to the host operates in the rhizoplane

the slope of the log-log transformation of the inoculum density-disease

curve should be near 0.67.

The pathogen response to the host in the four host-pathogen system

studied may operate only in the rhizoplane as postulated by Baker (4),

but, as pointed out by Mitchell (45), a rhizosphere effect modified by

various factors that adversely affect host infection could result in

similar slopes. In an ideal system each propagule that is encountered

by a growing root will germinate and infect that root and a slope

of 1.0 will be attained (63). Since 100% efficiency is rare in nature,

a slope of less than 1.0 is expected.

When the interpolated ID50's of the four host-pathogen systems were

compared with those obtained by Mitchell (45), our values were in the

same range. Mitchell (45) reported ID50's ranging from 15 to 50

oospores per g of soil with Pythium spp.

Variability in pathogenicity of the different Pythium spp. to

to different plant species was observed when the four plant species were










inoculated at two inoculum levels with oospores produced by the four

Pythium spp. Variability among species of Pythium is not unusual even

though many have wide host ranges (23). McCarter and Littrell (40)

observed basic differences in pathogenicity of P. aphanidermatum and

P. myriotylum to different crops and found large intraspecific variability

in the pathogenicity of these fungi.

During the course of this study, a gradual loss in the ability of

P. myriotylum and P. polymastum to infect rye and cabbage, respectively,

was observed. Although loss of pathogenicity among pythiaceous fungi

in culture is of common occurence, it rarely has been documented (55, 64).

No loss in the ability of P. aphanidermatum or P. debarvanum to infect

tomato occurred during this study. However, Mitchell (personal

communication) observed loss of the ability to infect host plants in

several isolates of P. aphanidermatum that were 'ept in culture for a

long time. When Laviolette and Athow (53) inoculated soybean seedlings

with pieces of mycelium produced by several isolates of P. ultimum that

had been maintained in cultures for several years, they did not observe

any loss of pathogenicity.

Loss of pathogenicity by an isolate sustained while it is maintained

in culture sometimes may be regained by inoculating susceptible plants

and reisolating from affected plants (64). When this procedure was tried

with P. myriotylum, it did not recover the ability to infect rye. Loss

of pathogenicity by P. myriotylum and by P. polymastum may be due to a

deficiency of a substance that is required in minute quantities; thus,

when this substance is exhausted, oospores that are produced subsequently

may be unable to germinate and infect host plants. Progressive changes

in pathogenicity may result through genetic recombination in the same










way that an avirulent strain of Phytophthora infestans can change to

a virulent strain (64). The loss of the ability of P. myriotylum to infect

rye may be attributed to a loss in the ability of this fungi to produce

germinable oospores. Perhaps if rye seedlings were inoculated by inserting

a piece of mycelium into an incision in the hypocotyl of the plant, as was

done by Laviolette and Athow (33) with soybean, no loss in the ability

of P. myriotylum to infect rye would have been detected.

Hendrix and Campbell (23) regarded soil temperature and moisture

as the two most important factors that influence infection. Maximum

infection of tomato by P. aphanidermatum and of rye by P. myriotylum

occurred when the seedlings were incubated at 35 C. These results are

in agreement with those reported by Littrell and McCarter (38) for infec-

tion of rye and tomato by P. aphanidermatum and by P. myriotylum, respec-

tively, and with those reported by Mitchell (44) for infection of rye

by P. myriotylum. Although disease caused by Pythium spp. usually is

low in soils maintained near water saturation (23), disease caused by

Pythium spp. increases as the percent moisture holding capacity (MHC) of

the soil is increased (5, 53). Bateman (5) observed that the development

of poinsettia root rot increased when the percent MHC of a soil that

held 56% moisture expressed on a dry weight basis was increased to

70% MHC. When the percent MHC of a soil was increased from 15 to

100%, damping-off of red pine seedlings increased linearly (52). In

this study, maximum infection of tomato by P. aphanidermatum and or rye

by P. myriotylum occurred when the soil was maintained at 89-100 MHC

(soil watered every 24 hrs). When the soil was maintained at 85-89 MHC

or at 73-85% MHC (soil watered every 48 or every 72 hrs, respectively)

the percentages of infection were lower.






59

Temperatures and media that favored oospore production resulted in

oospores that were more infective. Although there were no great differ-

ences in the rates of infection of tomato or rye when grown in soils

infested with oospores that were produced in the various media, slightly

higher rates of infection occurred when the seedlings were grown in soils

infested with oospores that were produced at 20-25 C than at 30-35 C.

The media used for producing inocula have been reported to be important

when the inocula consist of blended fungal mats. Mildenhall et al (43)

obtained marked differences in disease incidence and severity of carrot

with identical clones of any of three Pythium spp. that were cultured

in two different natural media.

The reductions in inoculum densities that occurred in soils which

contained plants may have been due to microbial lysis of oospores that

had been stimulated to germinate by root exudates. Stanghellini and Burr

(59) observed complete lysis of hyphae and/or germ tubes that originated

from oospores of P. aphanidermatum 96 hrs after amending artificially

infested soils with either bean seed exudate or nutrient solutions. Reduc-

tions in inoculum densities of P. myriotylum in field soils was observed

also by Frank (15) following cyclic wetting and drying of naturally infested

field soils. Similarly, Burr (11) found a significant decrease in the

populations of oospores of P. aphanidermatum in naturally infested soils

after cyclic wetting and drying, or after amending soils with asparagine.

In this study, we have shown that many cultural and environmental

factors can alter the rates of infection by Pythium spp. Since these

factors can alter the results of an experiment, they should be considered

when establishing tests with defined inoculum levels to evaluate certain

chemicals for disease control or when evaluating host resistance or

tolerance to these fungi.

















PART 4


RELATIONSHIPS OF THE NUMBERS OF MOTILE AND ENCYSTED ZOOSPORES
OF PYTHIUM APHANIDERMATUM, P. MYRIOTYLUM, AND PHYTOPHTHORA
PALMIVORA TO INFECTION OF TOMATO


Introduction


In recent studies where care was taken to prevent or reduce the

rate of encystment of zoospores during inoculation procedures, a lower

number of zoospores was required for infection of plants by pythiaceous

fungi than was expected from previous studies (45,46). Less than

300 zoospores per plant were required for 50% infection of cotton,

tomato, or watercress by Pythium ostracodes Drechs., P. aphanidermatum

(Edson) Fitz., or Phytophthora cryptogea Pethyb. and Laff., respectively

(45). Formerly, a minimum of 1.25 X 10 zoospores per plant were

required for 50% infection of papaya (50). The large difference

between the studies may be due to the method used for inoculation.

In the former study, zoospores were introduced to the plants while

they were under flooded conditions as opposed to injection of the

inoculum directly into nonflooded soil.

Most studies involving specific numbers of zoospores as inocula

have had results reported as percentages of disease incidence or

mortality of host plants rather than percentages of infection (6, 16,

17, 29, 30, 31). However, when percentages of infection were compared

with percentages of mortality, at least ten times more zoospores were

required to kill seedlings than were required for infection (45, 46, 49).

60








61

Fewer motile zoospores than encysted zoospores are required to cause

specific levels of infection or disease incidence. For example, 98%

of the papaya seedlings inoculated with 6 X 104 motile zoospores of

P. palmivora Butler were killed, but only 11% of the seedlings were

killed when exposed to a similar number of encysted spores (29).

Among the many factors that can cause zoospoms to encyst during

inoculation procedures are changes in hydrogen concentration, ion or

nutrient concentration, water current, and temperature (8, 16, 17, 21,

23, 41, 46, 61). Temperature may be one of the most important factors

that causes immobilization of zoospores. For example, no zoospores of

P. aphanidermatum or P. myriotylum Drechs. were motile after 1 hr of

incubation at 37 C while 70% of the zoospores were motile after 18 hrs

of incubation at 19 C (40). Immobilization of P. palmivora occurred after

0.5 hr at 33 C, 2.5 hrs at 28 C, or 6 hrs at 8 or 12 C (8, 29).

The objectives of this study were to determine the relationships of

the numbers of motile and encysted zoospores of P. aphanidermatum, P.

myriotylum, and P. palmivora to percentages of infection of tomato

(Lycopersicon esculentum Mill.) seedlings, and to evaluate the effects

of temperature on root infection and encystment of zoospores.










Materials and Methods


The isolates of Pythium spp. used in this study were obtained from

diseased plants found in Florida. Pythium aphanidermatum was isolated

from pigeon pea (Cajanus cajan (L.) Mill.) and P. myriotylum was

isolated from a peanut pod (Arachis hypoceae L.). The strain of

Phytophthora palmivora (P-455) examined as a comparison to Pythium spp.

was isolated from papaya (Carica papaya L.), and was obtained through

the courtesy of G. A. Zentmeyer (University of California). All fungal

isolates were maintained on V-8 juice agar in the dark at 20 C; the

cultures were transferred monthly to fresh agar slants.

Zoospores of P. aphanidermatum and P. myriotylum were produced by

a method described by McCarter and Littrell (41). Mycelium-bearing V-8

juice agar discs (15 mm in diameter) were cut from the periphery of 24-hr-

old colonies (grown at 30 C in the dark) with a sterilized cork borer,

and four discs were placed aseptically in an inverted position in a

sterile petri dish containing 10 ml of sterile deionized water. The

dishes were incubated for 15 hrs at 30 C in the dark, removed from

the incubator, the water was replaced with fresh sterile deionized water,

and the cultures were incubated for an additional 3 hrs at room temperature.

Zoospores of P. palmivora were produced from cultures prepared by

a method used by Ramirez and Mitchell (49). Each culture was initiated

by adding three 15 mm discs cut from the margin of a three-day-old

culture of P. palmivora on V-8 juice agar to a 250-ml Erlenmeyer flask

containing 15 ml of V-8 juice broth. After incubation at 30 C in the

dark for 48 hrs, the medium was drained from the flask and the mycelia

were washed three times with 50-ml aliquots of sterile deionized water

and incubated for an additional 48 hrs in a growth chamber at 25 C










under continuous illumination (10,760 Ix at the level of the cultures).

The water in the flasks was removed and the mycelia were resuspended in

15 ml of fresh sterile deionized water, chilled at 9 C for 30 min and

returned to the growth chamber for 1 hr.

Four 1.0-milliliter samples were removed from suspensions containing

motile zoospores for counting. The samples were placed in test tubes

and immersed for one min in a water bath at 46 C to induce rapid

encystment. The concentrations were estimated by counting Five fields

of four samples from each of the four tubes on a standard hemacytometer

For comparisons of motile and encysted spores, zoospores were

induced to encyst by a method used by Tokunaga and Bartnicki-Garcia (61).

Samples of each fungus were agitated in test tubes for 60 sec on a Vortex

mixer set at maximum speed.

Immediately after the counts were made, dilutions were prepared

with sterile deionized water at ambient temperature and inoculation

were performed.

Tomato seeds were soaked in tap-water for 30 min, surface-disin-

fested by soaking the seeds for 3 min in 0.5% (w/w) sodium hypochlorite.

rinsed in sterile deionized water, and incubated at 30 C on moist paper

towels for 2 days. One 2-day-old seedling was planted in 75 g of auto-

claved coarse builder's sand in a 50-ml polypropylene beaker that had

three small holes at the base for water movement. After planting, 4 g

of vermiculite were layered around the base of the seedling to prevent

rapid dying, and the beakers were placed in a nylon pan (30 X 18 X 16 cm).

The pan was placed in a growth chamber at 30 C with a 12-hr-day-light

(10,760 Ix at the level of the plants) and maintained for 5 days. All

plants were watered daily by filling the pan with sterile deionizedwater,










maintaining the flooded condition for 10 min, and draining the pan.

Fifteen beakers were placed into each pan and one pan was used for

each treatment.

Before inoculation, the pans were removed for the growth chambers,

the layer of vermiculite was removed and the cups were flooded by filling

the pan with sterile deionized water. One milliter of a known concentration

of zoospores was pipetted onto the surface of the standing water (16.6 cm2

X 0.5 cm deep) approximately 2 cm away from the stem of the tomato seedlings.

The zoospores were allowed to disperse in the sterile deionized water for

5 min, the water was drained off and the pans were returned to the growth

chambers. Plants that were inoculated with zoospores of P. aDhanidermatum

or P. myriotylum were maintained in the growth chamber for 2 days while

those inoculated with zoospores of P. palmivora were maintained for 3

days. In tests conducted to determine the effects of temperature on

infection, the pans were maintained in growth chambers at 15, 20. 25, 30,

35, and 40 C.

Tomato seedlings were harvested by washing the sand from the roots

under running tap water. Each stem and root system was surface-disinfested

by dipping in 70% ethyl alcohol for 5 sec, rinsing in sterile deionized

water, and blotting on paper towels to remove excess water. The root

systems were plated then on a selective medium (PV) which was modified

from that of Tsao and Ocana (62) and contained 5 mg pimaricin (Delvocid,

Gist-Brocades, Delf, Holland), 300 mg vancomycin hydrochloride (Vancocin,

Eli Lilly & Co.) and 17 g of Difco cornmeal agar in 1 liter of deionized

water. Plates were observed for growth of P. aphanidermatum or P.

myriotylum after 1 to 2 days of incubation at 30 C, or for growth of P.

palmivora after 3 to 4 days of incubation at 30 C.










The effects of time and temperature on encystment of zoospores were

evaluated after 1, 3, 6, 12, 18, 24, and 48 hrs of incubation in the

dark at 10, 15, 20, 25, 30, 35, and 40 C. Samples (2.5 ml) of a zoospore

suspension that contained 1 X 105 zoospores per ml were placed into

each of four stender dishes (37 X 25 mm) for each treatment and

incubated at the appropriate temperature and time intervals: the percent-

age of motile zoospores was estimated under magnification (200 X). The

stender dishes were returned to the incubator for additional incubation

time and the samples were estimated again for zoospore motility.

All experiments were repeated twice except those on the relationship

of the number of zoospores per plant to root infection which were

repeated at least three times. The data presented in this paper are

means of the experiments.








Results


Higher percentages of root infection were obtained with motile

than encysted zoospores (Fig. 3). From 32 to 78 times more encysted

zoospores than motile zoospores were required to produce the same

level of root infection. The number of zoospores required to infect

50% of the plants with motile and encysted zoospores of P. aphanidermatum,

P. myriotylum, or P. palmivora were 275 and 23,419, 166 and 10,988, or

1,505 and 47,424, respectively. Percentages of seedling infection

increased with increasing numbers of zoospores per plant (Fig. 3-A,

3-B). Percentages of infection of tomato seedlings inoculated with

50, 100, 250, 500, 1,000, and 10,000 motile zoospores of P. aphanidermatum

were 186, 30+8, 519, 649, 789, 983,-and 98+3%; percentages of

infection with P. myriotylum at the same inoculum levels were 268,

4215, 5911, 735, 925, 98+3, and 100%; and the percentages of

infection with P. palmivora were 0, 13, 2014, 3111, 4213, 768, and

898%. Tomato seedlings inoculated with 1, 5, 10, 50, 100, and 500

(X 103) encysted zoospores per plant had percentages of infection of

23, 206, 345, 667, 875, and 983% with zoospores of P. aphanidermatum;

and 145, 335, 487, 875, 93+5, and 100% with zoospores of P. myriotylum.

Percentages of infection of tomato seedlings inoculated with 10, 50, 100,

and 500 (X 103) encysted zoospores of P. palmivora were 2016, 536, 736,

and 953.

Slopes determined by linear regression anaylsis of log10loge

(1/l-y), where y equals the proportion of infected plants, vs logl0

of the number of motile or encysted zoospores of P. aphanidermatum,

P. myriotylum, or P. palmivora per plant were 0.68 and 0.69, 0.67 and

0.64, or 0.62 and 0.67, respectively (Fig. 3-C, 3-D).










Optimum temperatures for root infection by zoospores of P.

aphanidermatum, P. myriotylum, and P. palmivora were 25, 30, and

20-25 C, respectively (Table 23). No recovery of P. aphanidermatum,

P. myriotylum or P. palmivora was observed from inoculated tomato

seedlings that were maintained at temperatures below 20 or above 35 C

after inoculation.

In a test in which rye (Secale cereale L.) seedlings were used

instead of tomato seedlings, the slope of the regression line and the

number of motile zoospores of P. myriotylum required to infect 50%

of the seedlings approximated the results obtained for tomato. The

percentages of infection of 7 day-old rye seedlings inoculated with

31, 62, 125, 250, or 500 motile zoospores of P. myriotylum per plant

were 20, 20, 33, 67, or 88%,respectively. The slope of the regression

line for the log-log transformation was 0.69 and the number of zoospores

per plant required to infect 50% of the seedlings was interpolated to

be 209.

When the root systems of plants that were inoculated by adding the

inoculum to seedlings that were flooded and left for 5 min before

draining were plated on PV, the fungi were recovered from random locations

on the root systems. Increasing the time of flooding after inoculation

resulted in a slightly lower infection rate.

Zoospores produced by P. aphanidermatum, P. myriotylum, or P.

palmivora retained motility longer at 20 C than at other temperatures

(Fig. 4-A, 4-B, 4-C). Twenty percent of the zoospores produced by

P. aphanidermatum and 2% of those produced by P. palmivora were motile

after 48 hrs of incubation at 20 C. At temperatures above or below 20 C,

the rate of encystment increased with rise or fall in temperature. Very








68

few slow moving zoospores were observed after 1 hr of incubation at 40

or 10 C. At 47 C, motility was terminated within 40 sec (Fig. 4-D).

Eighty-eight to 97% of the cysts germinated after 48 hrs of incubation at

temperatures ranging from 20 to 35 C. No germinated cysts were observed

at 10 or 40 C after 48 hrs of incubation in the dark. At 15 C, few

germinated cysts of P. aphanidermatum and of P. palmivora were observed

after 48 hrs of incubation; but no germinated cysts of P. myriotylum

were observed.









Table 23. The effect of temperature on infection of tomato seedlings
by zoospores of Pythium aphanidermatum, P. myriotylum, or Phytophthora
palmivora.






Temperature (C)


FungusX Zoospores 20 25 30 35
per plant Infectiony


(%) (%) (%) (%)


Pythium aphanidermatum 100 25 az 34 a 29 a 29 a

Pythium myriotylum 100 38 ab 42 ab 50 a 36 b

Phytophthora palmivora 400 27 a 27 a 22 a 7 b



XSeedlings were inoculated with motile zoospores of Pythium spp. and of
Phytophthora palmivora while they were flooded; seedlings that were
inoculated with zoospores of Pythium spp. were maintained for 48 hrs
in the growth chambers and those inoculated with P. palmivora were
maintained for 72 hrs.

YMean of two experiments, each with 15 plants per treatment.

ZWithin each row, entries without a common letter are significantly
different (P= 0.05) as determined by Duncan's multiple-range test;
analyses were performed with data transformed to arcsin degrees.






















































100 1000 10000 0uuu 0UUUU 100000
NUMBER OF ZOOSPORES/PLANT NUMBER OF ENCYSTED ZOOSPORES/PLANT

Fig. 3-(A to D). The relationship of the number of zoospores of Pythium!
aphanidermatum (-. ), P. myriotylum ("'), and Phytophthora palmivora
(----) to infection of tomato seedlings. A) Percentage infection
(arithmetic) and number of motile zoospores per plant (arithmetic). B)
Percentage infection (arithmetic) and number of encysted zoospores per
plant (arithmetic). C) Percentage infection (logarithmic) and number of
motile zoospores (logarithmic). D) Percentage infection (logarithmic)
and number of encysted zoospores (logarithmic). C & D) The linear
coefficient was significant at P= 0.05 (*) or P= 0.01 (**).


___


_^I^^


































10000 .....

S'c 'p r D


SP.plmIvora 0 '


\ \ \ ,, ....
I60 \



040 40 -
25 P. aphanidermatum \
S\P. myriotyl..um ..
S\ P. p lmvor -- \
20 \ 20


.-I5.. .
1 3 6 12 18 24 30 48 10 15 20 25 30 35 40
TIME IN HOURS TIME IN SECONDS

Fig. 4-(A to C) The effect of temperature on motility of zoospores of
(A) Pythium aphanidermatum, (B) P. myriotylum, (C) Phytophthora palmivora.
(D) The time in seconds for encystment of zoospores of P. aphanidermatum,
P. myriotylum, and of P. palmivora at 47 C.








Discussion


Fewer motile zoospores generally are required to produce the same

levels of infection obtained with encysted zoospores. From 32 to 85

times more encysted zoospores than motile zoospores were required to

infect 50% of the tomato seedlings in this study. Kliejunas and Ko (29)

reported that ten times more encysted than motile zoospores of P.

palmivora were required to kill the same percentage of papava seedlings,

but they inoculated 25 plants per container instead of individual plants.

The percentages of infected seedlings increased as the numbers of

zoospores per plant were increased but the ratio of infection to number

of zoospores decreased as the number of zoospores increased. The

arithmetic and the log-log plots obtained from our data were similar

to those reported by other workers for host-pathogen systems involving

motile inocula (4, 45, 46). The slopes of data in the log-log

transformations were between 0.62 and 0.69. These values support the

predictions of Baker (4) since they are near the expected value of 0.67.

The results of this study support previous work with zoospores of

several Pythium and Phytophthora spp. which indicates that fewer than

300 zoospores per plant are required for 50% infection of various hosts

(45, 46). There were no indications of quantitative differences in

infection of different hosts by a Pythium sp. or in infection of the

same host by two different Pythium spp. The number of zoospores of P.

myriotylum required for 50% infection of rye was similar to the number

of zoospores required for 50% infection of tomato seedlings. Similar

numbers of zoospores of P. aphanidermatum and P. myriotvlum were required

for comparable levels of infection of tomato.









The relativelygreat number of zoospores required by an isolate

of P. palmivora that had been maintained in culture for several years

for 50% infection of tomato seedlings may be due to a loss of pathoge-

nicity or a loss of the ability to infect host plants since approximately

five times more zoospores were required for 50% infection of this plant

than were required with zoospores of other Phytophthora or Pythium spp.

on various hosts (45, 46). Less than 300 zoospores per plant were

required for 50% infection of watercress by Phytophthora cryptogea (45),

milkweed vine by P. citrophthora (R. E. Sm. and E. H. Sm.) Leonian

(Mitchell and Ridings, unpublished) or tobacco by P. parasitica var

nicotiana (Dastur ) var. nicotianae (Breda de Haan.) Tucker (Kannwischer

and Mitchell, unpublished).

Low or high temperatures were unfavorable for root infection of

tomato by P. aphanidermatum, P. myriotylum, or P. palmivora. The

reduction in the percentages of infection of tomato by these fungi

may be related to a rapid encystment of zoospores at low or high

temperatures. At 20 C, zoospores of P. aphanidermatum, P. myriotylum,

or P. palmivora were motile for a longer time in vitro than at other

temperatures used in the tests. McCarter and Littrell (41) reported that

at 19 C, zoospores of P. aphanidermatum and P. myriotylum were motile

longer than at other temperatures. They also noted that the time of

motility decreased with fluctuations in temperatures. Zoospores of

Pythium iwayamai S. Ito remained motile for 16 days in unfrozen water

at 0 C (37). In a 1% dextrose solution, zoospores of P. parasitica

were motile for more than 7 hrs at 20 C (18). Bimpong and Clerk (8)

reported that zoospores of P. palmivora were motile for 84 hrs in

distilled water at 17 C.









In soils, zoospores encyst very rapidly (26). The period of

motility of zoospores of P. palmivora, however, has been reported

to be as long as 4.5 hrs at 25 C (29). Although the movement of

zoospores through soils may not be as important as the passive

transport of zoospores in soil solutions, the distance that they ac-

tively move toward plant roots, even if it is only 25-35 mm, can

be important since it creates the effect of increasing the inoculum

level (13, 26, 29, 65).

If zoospores are to be used in studies dealing with host infection,

care must be taken to insure that they are motile during and after

inoculation since loss of motility can create the effect of a 10 to

90 fold decrease in inoculum (26, 29). Forcing zoospore suspensions

through a cannula or a hypodermic needle into soils could cause the

zoospores to encyst rapidly since they can be induced to encyst by

any rapid change in their environment (8, 16, 18, 26, 29, 41, 46, 61).

To avoid the problem of excessive encystment during inoculation and to

simulate conditions for the dispersal of zoospores in nature, zoospores

can be added to seedlings flooded with water of similar qualities to

the water used for preparing zoospore inoculum.
















LITERATURE CITED


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in zoospores of Phytophthora palmivora (Butl.) Butl. Ann. Bot.
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12. Child, J. J., and C. Knapp. 1973. Improved pH control of fungal
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80





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BIOGRAPHICAL SKETCH


Roger J. Sauve is the head of the Plant Pathology Section and of

the Plant Disease Diagnostic Clinic, Division of Plant Industries, Tennes-

see Department of Agriculture at Nashville, Tennessee. Born in Montreal,

Quebec, he was awarded a B.S. in Biology from the University of Florida

in 1970.

During the Fall of 1970, he began studies toward the degree of

Doctor of Philosophy at the University of Florida. Shortly afterward,

he accompanied Dr. Howard N. Miller to Jamaica, West Indies, where they

made a survey of some plant diseases and taught a course in Mycology-

Plant Pathology at the Jamaica School of Agriculture from March to

September of 1971. Upon return to the U. S., he resumed his studies at

the University of Florida. In 1975, he assumed his present position with

the Tennessee Department of Agriculture and married the former Deidre D.

Tobin of Princeton, New Jersey. Recently, he initiated and implemented

the Tennessee Fruit Tree Improvement Program.

He is a member of the American Phytopathological Society, the

Southern Association of Agricultural Scientists, the Mycological Society

of America, the Tennessee Entomological Society and the International

Society of Arboriculture. In addition, he is a member of Gamma Sigma

Delta, Alpha Zeta, the National Honor Society, the National Audubon

Society, and the Boy Scouts of America.













I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



David J. Mitchell
Chairman
Associate Professor of Plant
Pathology

I certify that I have read this study and that in mv opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


Howard N. Miller
Professor of Plant Pathology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


orman C. Schenck
Professor of Plant


Pathology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Jmes W. Kimbrough
professor of Botany

This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

December, 1978 /1


Dean, Graduate School


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UNIVERSITY OF FLORIDA


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