Population dynamics of Pythium aphanidermatum in field soil

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Authors Burr, Thomas James, 1949-

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/566316 POPULATION DYNAMICS OF FYTHIUM

APHANIDERMATUM IN FIELD SOIL

b y

Thomas James Burr

A Thesis Submitted to the Faculty of the

DEPARTMENT OF PLANT PATHOLOGY

In Partial Fulfillm ent of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 3 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillm ent of re­ quirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Deem of the Graduate College when in his judg­ ment the proposed use of the material is in the interests of scholar­ ship. In a ll other instances, however, permission must be obtained from the author.

Ty

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

ZMICHAEL E . STAgGHELLINI Assoc. Professor of Plant Pathology ACKNOWLEDGMENTS

The author expresses deep appreciation for the concern and guid­ ance of Dr. Michael E. Stanghellini, Associate Professor of Plant

Pathology, who made this study a rewarding educational experience.

Appreciation is also expressed to Dr. Richard B. Hine and Dr.

Homer E. Bloss who contributed constructive suggestions to this study.

The author is also grateful to Judith A. Bonsall for her unself­ ish contributions toward manuscript preparation.

i l l TABLE OF CONTENTS

Page

LIST OF TABLES...... v

LIST OF ILLUSTRATIONS...... v i

ABSTRACT...... v i i

1 . INTRODUCTION...... 1

2 . LITERATURE R E V IE W ...... 3

3 . MATERIALS AND M ETHODS...... 6

4 . R E S U L T S ...... 8

5 . DISCUSSION AND CONCLUSIONS...... 22

LITERATURE C IT E D ...... 29

i v LIST OF TABLES

T ab le Page

1. Origin of colonies of Pythium aphanidermatum from naturally infested soils on a species specific i s o l a t i o n m e d iu m ...... 11

2. Effect of cyclic wetting and drying, asparagine amendments and supersaturation on populations of Pythium aphanidermatum in a naturally in­ f e s t e d f i e l d s o i l ...... lU

3. Variability of oospore size and growth rate of 25 Pythium aphanidermatum isolates obtained from v a rio u s n a t u r a l l y in f e s t e d f i e l d s o i l s ...... 21

v LIST OF ILLUSTRATIONS

Figure Page

1. Relationship between Pythium aphaniderroatum popula­ tion and soil dilution ...... 9

2. Photographs of A) colony morphology and B) originat­ ing propagule (oospore) of Pythium aphanidermatum on a species specific isolation medium after 72 h o u rs in c u b a tio n a t 35 C ...... 10

3* The verticle distribution of oospores of Pythium aphanidermatum in soil cropped to alfalfa for th r e e c o n s e c u tiv e y e a rs ...... 15

4. Sequential stages of germinating zoospore cysts of P . aphaniderm atum in f i e l d s o i l ...... 18

v i ABSTRACT

Oospores were the sole survival structure of Pythium aphanider- matum in naturally infested soil. Population counts determined on a species-specific isolation medium from naturally infested soils of var­ ious types ranged from 10- 2$0 oospores/g soil. The highest concentra­ tion of oospores were found in the top 15 cm of soil "but could be detect­ ed t o 30 cm in depth.

No evidence was obtained for the presence of a constitutively dormant oospore population in naturally infested soils. Enzymatic deg­ radation of colonized organic matter apparently resulted in liberation of oospores which are exogenously dormant.

In naturally infested soils, the minimum inoculum density of P. aphanidermatum necessary for host colonization was two oospores/g soil.

Among 25 soil isolates, little or no variation was observed with respect to oospore size, growth rate, colony morphology, spore-producing ability or pathogenicity. All isolates caused 100$ post emergent damping off of sugar-beet seedlings after 48 hour incubation at 35 C.

v i i CHAPTER 1

INTRODUCTION

The genus Pythium vas first described by Pringsheim in 1858 ( 4 3 ) .

Since that time over 66 species have been identified (35)• The genus is worldwide in distribution, parasitizing aquatic as well as terrestrial plants (20). One member of the genus, Pythium aphanidennatum (Edson)

Fitzpatrick, which was first described causing a seedling disease on sugar beets in 1915 (ll), has subsequently been reported as a parasite of over 100 different genera of higher plants ( 63). Although the organ­ ism is usually reported as a pathogen of seedlings (6, 21, 24, 4$, 46,

53> 6l, 66), it is also known to cause rots of mature roots (l6, 23, 31*

32, 40, 4l, 44), blights (l8, 28), stem rots (12, 34, 42, 62), a n d s o f t r o t s (4 , 7 , 8, 30, $6). Disease incited by this fungus is associated with high soil moisture conditions and high soil temperatures, 27 C or g r e a t e r .

In Arizona P. aphanidennatum was found to be indigenous on na­ tive plant species and responsible for extensive crop losses during the summer months (23, $ 2 ).

The objectives of this thesis were to determine l) the nature of the persisting propagule of P. aphanidennatum in naturally infested soils, 2) the verticle distribution of the fungus in soil, 3 ) th e m in­ imum inoculum density necessary for colonization of a host, 4) environ­ mental and physical conditions which tend to alter natural soil populations

1 and, 5) pathogenic variation among soil isolates. Attainment of these objectives could provide knowledge for application of techniques for biological control of P. aphanidermatum. CHAPTER 2

LITERATURE REVIEW

Selective media allow for the isolation of a specific microflora from a complex environment such as soil. Before the advent of media con­ taining the polyene antibiotics (pimaricin, endomycin, or nystatin), se­ lective isolation of most Pythiacious fungi was virtually impossible.

The first selective medium for the isolation of Phytophthora and Pythlum from plant roots was developed by Eckert and Tsao in i 960 with the aid of the polyene antibiotic, pimaricin (10). Singh and M itchell in 1961

(47) were the first to report a highly selective pimaricin containing me­

dium specifically for the isolation of Pythium spp from soil. Pimaricin

inhibits the growth of nearly a ll common soil fungi except a small group

that includes the members of the Pythiaceae (10, 17, 47). L ittle empha­

sis has been previously placed on development of media for isolating in­

dividual species within the genus Pythium (60).

A wide range of natural populations of Pythium spp in soil have

been reported (19, 25, 51, 65). Where quantitative attempts have been

made for determining populations of a single species up to 38OO p ro p a -

gules/g of naturally infested soil have been reported (51)• Hine and

In n a in 1963 developed the first baiting technique for isolation of P.

aphanidermatum from soil (22). However, quantitative data and the na­

ture of the persisting propagule of P. aphanidermatum could not be de­

termined with this method. Indirect methods have implicated oospores as

3 4 the primary survival structure of P. aphanidermatum in field soil (2, 22,

52, 59)• Direct observation has shown that sporangia of P. aphaniderma­ tum are incapable of persisting in air dried soil for more than three days (49). Zoospore survival capabilities of P. aphanidermatum have not been fully elucidated, although zoospores have been recovered from soil up to seven days after artificial infestation (59)•

The verticle distribution of P. aphanidermatum in soil has been previously determined using semi-quantitative techniques (57, 39)• Re­ sults illustrated that the bulk of the population was present near the surface of the soil but some oospores could be found to a depth of 35 cm.

Similar results were obtained from studies of verticle distribution of other Pythium spp (29). However, in peach orchards where extensive root systems are present, Pythium has been found to a depth of 90 cm (20).

In nature, populations of Pythium are known to fluctuate depend­ ing upon substrate availability and environmental conditions (64).

Knowledge of factors which could change populations in naturally infested soils would be highly beneficial in understanding the behavior of the or­ ganism and is necessary for developing control measures. The oospores of P. aphanidermatum have been shown to germinate in the presence of ex­ ogenous nutrients in artificially infested soils, thereby leaving e i t h e r germlings or sporangia, neither of which can survive for more than three days in the absence of a colonizable substrate (49). Their germination could result in a decrease in inoculum density.

Once the quantity and verticle distribution of the propagule are established, as well as the factors influencing population changes in 5

soil, it is important to determine the minimum density of that inoculum

necessary for penetration and infection of a host. An understanding of the minimum inoculum density necessary for infection may be used to fore­

cast epiphytotics in the field. It may also be beneficial in determin­

ing the reduction in population of the propagule necessary for control of the disease. Ashworth showed populations of 3*5 m icrosclerotia of

Verticillium albo-atrum were sufficient to cause serious infection of

cotton (l), whereas Kendrich and Wilbur determined that $00 propagules/g

of Pythium irregulars in soil were necessary to infect lima bean seedlings

(26). Colonization of bentgrass by P. aphanidermatum was possible with

only a single zoospore, but several zoospores were required for infection

(20). Garrett (15) pointed out, however, that the values of inoculum

density necessary for infection is not only determined by the energy of

growth of the infecting propagule but also by environmental conditions

(3 ). Variability in pathogenicity between soil and plant isolates of

P. aphanidennatum has been reported (37). However, methods for assaying

these differences between isolates have not been carried out using inoc-

ula of known type, viability, or quantity ( 33, 37) • CHAPTER 3

MATERIALS AND METHODS

Litchfield sandy loam (LSL), Guest clay (GC), and Pomerene clay loam (PCL) soils were used in this investigation unless otherwise speci­ fied. Soils were collected from the Salt River Valley, Gila River Valley, and Cochise County in Arizona, respectively, and stored at 24 C in poly­ ethylene bags.

Determination of the P. aphanidennatum Edson (Fitz.) population

in naturally infested soils using existing selective media (26, 38; 4%) proved quantitatively unsatisfactory. A medium was therefore developed which contained Difco commeal agar (1 . 7%), pimaricin (Myprozine, poten­

cy 92.2^6, American Cyanamid) 100 jig/ml, streptomycin sulfate 200 jug/ml,

rose bengal 100 - 2$0 jig/ml (concentration variable depending upon source) and benomyl 5 Mg/ml. Each of the antibiotics was added after autoclav­

ing and cooling the commeal agar to 45 C. A ll ingredients were shown

to be necessary at the given concentrations for satisfactory results.

Each antibiotic was prepared as a 156 stock solution and stored at 10 C

for no longer than one month.

Soil dilutions were prepared at 1:5, 1:10, 1:20, and 1:40 soil/m l

with 0.3$ water agar. Dilutions were mixed on a Vortex stirrer for at

least 15 minutes and one ml aliquots were dispensed evenly across the

surface of the medium with the addition of 0.5 ml distilled water to

facilitate distribution of the soil. Media were then incubated at 35 C

6 7 for various time intervals. Subsequently, the soil was washed from the agar surface under a stream of tap water and colonies were observed.

Isolates of P. aphanidermatum, obtained from single oospore col­ onies originating on the selective medium, were maintained at 24 C on V8 juice agar in test tubes and transferred monthly. These isolates were utilized in subsequent experiments. CHAPTER 4

RESULTS

The following characteristics were observed using the selective medium described in this study: l) oospores, sporangia, zoospores, and mycelial fragments of P. aphanidermatvua were capable of greater than 90# germination and/or growth, 2 ) a p p ro x im a te ly 97$ of a known oospore pop­ ulation was recovered from artificially infested field soil, 3 ) t h a t th e medium is specific for growth of P. aphanidermatum with the elimination of growth of all other microorganisms from naturally infested field soils and, 4) that a linear relationship existed between the number of oospores of P. aphanidermatum/ml and the soil dilution (Fig. l).

Cultures prepared from soil dilutions were incubated at 35 C for

24 hours at which time colony size ranged from 3 - $ mm and could be traced easily to its origin using a light microscope. After 72 hour in­ cubation, P. aphanidermatum colonies were characterized by a distinct red ring visible on the medium (Fig. 2A). Longer incubation periods usu­ ally resulted in colony overlap and therefore made counting difficult.

In naturally infested soils tested over a nine-month period 96$ of the colonies examined originated from oospores (Table 1, Fig. 2B).

The remainder of the colonies could not be traced back to an originating propagule due possibly to dislodgement of the propagule from the agar surface during the washing phase. Direct microscopic examination veri­ fied that oospores were not found in association with organic matter

8 0 2 5 5 SOIL DILUTION (g/IOOml)

F ig u re 1 . Relationship between Pythium aphanidermaturn population and soil dilution.—The mean and standard error of 10 separate soil samples, each replicated 10 times, are plotted. 10

Figure 2. Photographs of A) colony morphology and B) originating propagule (oospore) of Pythium aphanidermatum on a species spe­ cific isolation medium after 72 h o u rs incubation at 35 C. Table 1. Origin of colonies of Pythium aphanidermatum from 1 1 naturally infested field soils on a species specific isolation medium.a

Experiment Total No. colonies Colonies traced $ colonies observed on 5 or to oospores tr a c e d to more dilution plates o o sp o re s

i ” 46 43 93-5 2» 50 48 96.0

3b 36 35 97-2 Ub 12 12 100.0

5° 131 130 99.2

6d 43 40 93.0

74 20 19 95.0

T o ta l 338 327 Avg. 96.3

aSoil dilutions of 1:10 (g soil/m l 0.3$ water agar) were incubated at 35C for 24 hours on the selective medium.

^Litchfield sand loam soil.

cPomerene clay loam soil.

^Safford clay loam soil. 1 2

either on dilution plates or in organic matter separated from soil prior to dilution studies. These observations were made using Arizona soils which had been fallow from 4 -6 weeks.

Determinations of propagule densities of F. aphanidermatum in

cultivated soils, which had a known disease history, showed that LSL which was cropped to potatoes one month prior to sampling had a popula­

tion of 60-80 oospores/g soil, whereas GC which had been continuously

cropped to sugar beets for three consecutive years contained populations

up to 250 oospores/g soil. PCL which was cropped to alfalfa contained

40 - 50 oospores/g soil.

In addition to cultivated soils of Arizona, species specificity

of the selective medium was also demonstrated in non-cultivated Gila

River sand as well as cultivated soils from Obregon, Sonora and Culiacan,

Sinaloa, Mexico where populations ranged from 10-27 oospores/g soil.

No change in the oospore population was observed in naturally

infested soils stored over a nine-month period at 24 C. When various

moisture and nutritional conditions were experimentally imposed on these

soils, however, population changes were observed. Twenty grams of LSL

soil which contained a natural population of 20 -2 3 oospores/g soil was

added t o $0 mm diam petri dishes and treated as follows: l) amended

with tap water to saturation and allowed to air-dry for 24 hours, 2) re-

moistened to saturation after a 24 hour air-drying period and allowed to

dry for an additional 24 hours, 3) supersatured (5 mm standing water

above the soil surface) for 24 and 48 hours and, 4) saturated with a 1^

solution of L-asparagine (ca. 25 mg) for 24 and 48 hours. P. aphanidermatum 1 3 populations were determined as previously described after the various

treatments. Soil samples were incubated at 2b C, replicated 10 times

and repeated twice. Results are given in Table 2. Microscopic examina­

tion (X100) of colony origin of P. aphanidermatum colonies on dilution

plates made from the supersaturated soil samples showed that colonies

originated from zoospores and oospores where only the latter was the

originating propagule from the other soil treatments.

Verticle distribution of P. aphanidermatum was determined in sev­

eral agricultural soils. Erratic results were obtained in soils which

had been recently plowed. Pockets of oospores were found at various

levels within the plow level. However, when a clay loam soil was sam­

pled from a three-year-old unplowed alfalfa field, consistent data were

obtained. Samples were collected by digging pits approximately 50 X 90 cm

and 90 cm in depth. Samples were collected at 7 cm i n t e r v a l s an d s to r e d

separately in polyethylene bags at 24 C. Soil samples were passed through

a 7 nm sieve prior to making dilutions for population determinations,

which were calculated as described previously. Results are given in

F ig . 3-

The minimum inoculum density necessary for host colonization by

P. aphanidermatum was determined by diluting soils of known density with

sterile soil of the same type. Sterilization was accomplished by auto­

claving the soil for three hours at 248 C and 15 psi pressure. P. aphan­

idermatum could not be recovered from the sterilized soil used as the

d il u e n t Ik

Table 2. Effect of cyclic wetting and drying, asparagine amendments and supersaturation on populations of Pythium aphanidermatum in a naturally infested field soil.

Soil Treatments

Soil Samples* 6 C o n tro l 24 h r Supersaturated Supersaturated W et-dry 48 h r^ Wet-dry 24Wet-dry hr 24 hre Amended 24 hr® Amended 48 hr® Asparagine Asparagine

1 22 9 4 27 30 2 1

2 22 20 5 4 l 31 5 4

3 42 22 26 22 25 12 9

aSoil dilutions of 1:10 (g soil/m l 0.3$ water agar) were incubated a t 35C f o r 36 hours on previously described selective medium.

bSoil samples were obtained in July 1972 from one month fallow fields which had a disease history caused by P. aphanidermatum.

^Control samples were maintained at field capacity.

^Samples wetted to saturation and then allowed to dry 2k h o u rs before population determinations made or,

eRewetted and dried an additional 24 hours before analysis.

**Soil samples supersaturated (5 - 6mm standing water above soil line) and incubated 24 or 48 hours, respectively.

^Samples amended with a 1$ asparagine solution to saturation (ca. 25 mg L-asparagine/10 g soil) and incubated 24- 48 hours respectively. 1 5

3 0 3 0 3 0 LOCATION I LOCATION 2 LOCATION 3 SOIL DEPTH (cm)

F ig u re 3 . The verticle distribution of oospores of Pythlum aphanidermatum in soil cropped to alfalfa for three consecutive years. 16 In pre-emergence colonization experiments, non-treated alfalfa seed (Medicago sativa L.) was used as the host. Twenty seeds were added to 20 g soil samples in 50 mm diam petri dishes containing decreasing amounts of infested soil, saturated with tap water and incubated for 24 hours at 35 C. Seeds were then recovered from the soil and examined mi­ croscopically for the presence of coenocytic mycelium within host tissue.

Seeds were also plated onto water agar and incubated at 35 C for 24 hours for the isolation of P. aphanidermatum. It was found that populations as low as two oospores/g soil were effective in colonization of the alfalfa seeds and coenocytic mycelium was also observed in colonized host tissue.

Each dilution was replicated three times and repeated twice. No attempt was made to record the actual percentage of alfalfa seeds colonized by

P. aphanidermatum at the various inoculum levels since the possibility of secondary spread subsequent to primary colonization of a single seed could not be ruled out.

Post-emergence colonization was assayed by adding 15 untreated

sugar beet seeds (Beta vulgaris L.) to pots containing 50 g samples of

diluted soil of known inoculum density. Pots were incubated at 65 C f o r

one week until seedlings had emerged. Pots were then incubated at 100 C,

the optimum temperature for host colonization by P. aphanidermatum, for

48 hours. Roots were examined at the end of the incubation period for

the presence of coenocytic mycelium and oospores within root tissue and

plated out on water agar at 35 C for 24 hours for the isolation of P.

aphanidermatum. Each inoculum level was replicated three times and re­

peated twice. Populations as low as two oospores/g soil were effective 1 7 in causing infection of the sugar beet seedlings. Again no attempt was made to record the percentage of infection.

The survival ability of zoospore cysts of P. aphaniderroatum in soil was analyzed under various moisture regimes. Zoospores were produced by incubating three, 2 mm diam plugs of P. aphanidermatum in 1$ ml dis­ tilled water contained in a $0 mm diam petri dish at 24 C for 18 h o u r s .

Mycelia taken from five-day-old cultures of P. aphanidermatum were grown, on V8 juice agar at 24 C. Zoospores were encysted by agitating following the method of Tokunaga and Bartnicki-Garcia ($8). Zoospore cysts were collected on a .47 n millipore filte r and re-suspended in a small volume of distilled water. Approximately 200 cysts were added to two g samples of soil contained in Model 96U-CS-Clear Disposo-Trays (Linbro of the

Pacific). Soil samples were either supersaturated, leaving 5 nm stand­ ing water above the soil line and covered to prevent evaporation, or brought to saturation and left uncovered to air-dry. Behavior of zoospore cysts in soil were examined microscopically at X100 for germ tube length, production of spore structures and presence of lysing mycelium over a 48 hour period at 24 C. In both supersaturated and drying soils, cyst ger­ mination occurred in two hours and produced germ tubes of ca. 95 J1 and

345 P- after six and 12 hour incubation respectively (Fig. 4a - D ). A t these times the drying soils were. at 6 and 4$ of their moisture holding capacities, respectively. After soils were air-dried to 1$ of their holding capacity, zoospore germlings were not observed to persist for more than 12 hours. This observation was confirmed by plating these soils onto the selective medium previously described and observing that 18

Figure 4. Sequential stages of germinating zoospore cysts of P. aphanidermaturn in field soil.-- A) Zoospore cyst in soil after two hour in­ cubation at 24 C; B - C) Cyst germlings after 6-24 hour incubation at 24 C with lengths of 95 M and 345 ji respectively; D) Produc­ tion of terminal sporangia by cyst germlings after 48 hour incubation at 24 C in super­ saturated soil. 1 9 colonies of P. apbanidermatum did not arise from zoospores. However, in soils maintained in the supersaturated state for 48 hours, germlings were in a state of lysis and produced terminal sporangia (Fig. 4d ). Enzyme- treated oospores (54), added to the same soil used above, did not germi­ nate illustrating that they, unlike the zoospore cysts, were under the influence of soil fungistasis.

V ariability among 25 soil isolates of P. aphanidermatum was ana­ lyzed based on five parameters and compared with data obtained from iso­ lates from infected plants (Stanghellini, unpublished data). No differ­ ences were observed based on colony morphology; sporangia, zoospore, or oospore producing ability; or on pathogenicity of the isolates to sugar beet seedlings. Colony morphology of isolates was compared by growing cultures on V8 juice agar in 100 mm petri dishes incubated at 35 C for three days. All isolates were characterized by having arachnoid-type growth characteristics of P. aphanidermatum (35)• Microscopic examina­ tion of all colonies revealed abundant oospores and sporangia by a ll iso­ lates after 72 hour incubation at 24 C.

Abundant zoospores were produced by each isolate after incubating mycelial mats in distilled water at 24 C for 4 -6 hours. Mats were re­ moved from six-day-old cultures grown on V8 juice agar with the aid of a rubber policeman. Mats were rinsed twice in distilled water before they were placed into petri dishes containing 15 ml distilled water.

Variation in virulence was determined by dispensing 25 ml of a zoospore suspension which contained approximately 1000 zoospores/ml over 15, o n e - week-old sugar beet seedlings growing in 4" plastic pots. Seedlings and 2 0 inocula were incubated at 100 C air temperature for 36 hours. In all cases 100% damping-off of the sugar beet seedlings occurred.

Variation in oospore size between and among isolates was deter­ mined by microscopic measurement of oospores at X hgO. Oospores were washed from mycelial mats of one-week-old P. aphanidennatum colonies grown on V8 j u i c e a g a r a t 2b C. Oospore diameter of the 25 isolates ranged from l4 - 30 m • The range and average diameters of each isolate along with their standard deviations are given in Table 3*

The growth rate of a ll isolates was calculated by growing iso­ lates on V8 juice agar in 100 mm diam petri dishes at 35 C, the optimum growth temperature of P. aphanidermatum. A 5 am diam plug from a four- day-old culture was placed in the center of a 100 mm diam petri dish con­ taining 20 ml V8 juice agar which had been pre-incubated at 35 C for two hours. Measurements of diameters of colonies were made after 15, 18, and

21 hour incubation. Results are presented in Table 3*

It was observed that three of the isolates of P. aphanidennatum produced an unusually high percentage of aborted oospores (70 - 90%).

This abortive condition did not change with continued culturing but re­ duction in one isolate, P-3, from 72% oospores aborted to 16% a b o r te d was noticed when the isolate was grown at 35 C and 2b C, respectively.

The other isolates (L-l and L-2) did not differ in percent oospore abor­ tion when grown at variable temperatures. 2 1 T ab le 3 . Variability of oospore size and growth rate of 25 Fythium aphanidermatum isolates obtained from various naturally— infested field soils.a

P. aphanidermatum Range o f A verage Growth rate i s o l a t e oospore sizefai oospore size(u) (mm/3 h r ) c

L i tc h f ie ld , i6.10-27.37 23.44 + 2.28 11. 2+ 1.1 2 19.32 - 27.37 24.15+1.84 1 2 .3 + 1 .5 l 22.9 4 - 27.3^ 23.73+1.55 i 24.15-32.20 1 2 .8 ± 1 .4 27.43+2.11 9 . 7 + 0 .8 c 20.93 - 27.37 23.79+1.70 10.5 + 1.0 26. 27± 2 .4 6 9 .7 + 1 .2 22.70±1.95 24.92 + 2.24 9 .3 + 1 .0 23.79+2.48 9 . 5 ± 1 .5 1 0 .0 3 :0 .9 10 19.32 - 25.76 2 3 .3 4 ± 2.32 12.2± 0.8 11 16.10-28.93 23.34+2.33 S a ffo rd 1 19.32-28.98 26.40 + 2.41 13. 3 ± 1 .0 2 19.32 - 32.22 8 . 7 ± 0 . 8 27.75+2.29 10 2 1.2 3 16.10 -27.37 23. 6 9 + 2.51 . + 10.2 ± 1.6 4 19.32 - 27.37 23.34+1.78 1 3 .2 ± 0 .8 5 22.5k-28.98 24.60+1.89 6 20.93 - 28.93 24.53+2.12 9 . 8 + 1 .7 7 19-32-25.76 2 3 .2 8 + 3 .6 6 9 - 5 + 1 .9 11.2 +2.3 8 19.32-27.37 23.86 ± 1.83 G ila R iv e r 1 17.71-27.37 23. 8 9 + 2.68 12.5 ± 0 .8 9 .7 ± 1 .4 Campbell Farm 1 20.93 - 27.37 2 4 .0 8 ± 1.87 A rkansas 1 17.71-28.98 2 3 .1 5 + 2.82 9 .5 ± 1 .5 ± C u lia c a n 1 14.49-27.37 23.44 12.21 8 .3 0 .8 9 .5 ± 1 -5 2 14.49-27.37 22.73 +2.57 Obregon 1 l 6 . l O - 3O.59 25.43 ±3.28 9 . 0 ± 2 .3 9 .0 ± 0 .6

aSingle oospore isolates were obtained from soil specific medium for P. aphanidermatum. x using the species

^Growth rates were measured after 18, 21 and Pk u V8 juice agar at 35 C. Each isolate was replicated ^ubation on peated once. aree times and re- CHAPTER 5

DISCUSSION AND CONCLUSIONS

The importance of P. aphanidermatum as an economic pathogen throughout the world is well documented (20). Control of diseases caused

"by this fungus, however, has not in most cases been effectively accom­ plished. Knowledge of the biology and epidemiology of P. aphanidermatum in soil is essential if control methods are to be developed. Develop­ ment of rational control measures is dependent upon knowledge of: l) the nature of the persisting propagule in soil, 2) their distribution in soil, 3) the propagule density necessary for successful host coloni­ zation and, U) the existing pathogenic variation among isolates of the fu n g u s .

Oospores, as previously postulated (2, 25, 52, 59), were shown by direct observation to be the major if not the sole survival propagule of

P. aphanidermatum in naturally infested field soil. Knowledge of the type of oospore dormancy whether constitutive or exogenous (55), is fun­ damental to an understanding of the life and disease cycle of soil-borne f u n g i.

Constitutive dormancy of oospores has been postulated, based on on cultural studies, as a long-term survival mechanism of various fungi in soil, thereby preventing spontaneous germination of the entire popu­ lation when suitable environmental and nutritional conditions are present

(15) • Observations made in this study indicate a population of exogenously

2 2 2 3 dormant oospores of P. aphanidermatum in soil which are under the influ­ ence of soil fungistasis and which w ill germinate maximally when subjec­ ted to appropriate environmental conditions. Aging of culturally produced oospores over a three-month period has been shown to break some constitu­ tive dormancy ( 9 )• If this phenomenon were operative for oospores in a naturally infested soil, a fluctuation in population over the nine-month • storage period used in this study would have occurred. Although oospores are apparently constitutively dormant while embedded in colonized host tissues, their subsequent release through bio-degradation (48, $4) could alter oospore wall permeability thereby establishing a population having only an exogenous nutrient requirement for germination. Oospores of P. aphanidermatum were not found embedded in organic matter; they exist as free propagules. Biodegradation of organic matter in Arizona desert

soils occurs rapidly and soils usually have an organic matter content between 0.2 and 0.8$. This particular condition accounts for the appar­ ent lack of a constitutively dormant oospore population in Arizona soils.

In more temperate clim atic regions, however, where Pythium spp have been reported as pathogens on perennial crops ( 36), many oospores are embedded

in host residues during inter-seasonal periods, thus contributing to a constitutively dormant population. However, these oospores would not be considered as inoculum for current season infection.

Interpretation of the significance of soil fungal populations frequently reported as propagules/g soil is difficult unless the primary propagule of survival is known ( 5, 13, l4 ). Soil samples may contain oospores, sporangia, zoospores, and mycelial fragments a ll of which could 2 4

germinate and/or grow to give rise to colonies. They may, however, have

no significance as primary inoculum unless they can be shown to survive

intersubstrate periods. Sporangia of P. aphanidermatum have been shown

to survive in air-dried soil at 24 C for no longer than ?2 hours (49).

Zoospores of P. aphanidermatum, also under air-dried soil conditions, were shown to survive no longer than 12 hours and apparently are not un­

der the influence of soil fungistasis. Neither zoospores nor sporangia

play a role in the long-term survival of P. aphanidermatum in field soils

having fluctuating moisture conditions. However, their significance may be in facilitating secondary dissemination of the pathogen and subsequent­

ly increased severity of disease during periods of excess moisture ( 50)*

As shown in this study, high populations of exogenously dormant oospores

of P. aphanidermatum in soil are capable of surviving inter-substrate

periods and function as primary inocula.

Previous studies have shown a fluctuation in soil populations of

various Pythium spp (64). Since fluctuations were not observed in natu­

rally-infested stored soils, various moisture and nutrient conditions were imposed upon soils to determine whether such fluctuations could be

induced in the laboratory. In two of the three soils tested, wetting

followed by drying of the soil reduced the oospore population. This re­

duction, even to a greater extent, was observed in soils that were amend­

ed with asparagine for various time intervals and confirmed the results

of Stanghellini and Burr (49), who used artificially infested soils.

They showed that asparagine effectively stimulated maximum oospore ger­

mination in soil and that in the absence of a colonizable substrate 2 5 capable of supporting reproduction, lysis of oospore gennlings occurred with a resultant decrease in the oospore population. In the presence of a colonizable substrate it has been demonstrated that P. aphanidennatum is capable of abundant oospore formation (52, 59)• These behavioral characteristics offer an interesting approach to a biological control method in climate regions where colonizable residues in soil are at a minimum as in Arizona so ils.

Studies on the verticle distribution of Pythium spp in soil have, for the most part, been qualitative or semi-quantitative, using baiting techniques for verticle assay (27, 57)♦ Such methods do not reveal lo­ calities of the most densely populated sites within a soil profile. High concentration of oospores of P. aphanidermaturn in undisturbed soils were found in the upper 15 cm and rarely found below 30 cm. Plowing of recent­ ly infected crops, however, resulted in pockets within the plow depth that contained many oospores. This distribution of inoculum can be sig­ nificant in the choice of method and timing of control practices.

Before biological or chemical control measures can be devised, it is beneficial to know the minimum inoculum density of P. aphanidermatum necessary to cause infection, i.e ., the level to which inoculum must be reduced before effective control is accomplished. In contrast to previ­ ous findings using related fungi (26, 31) it was observed in this study that two oospores/g soil were effective in causing both pre- and post­ emergence damping off. Since inoculum density is one of the factors re­ lating to inoculum potential, the quantity of effective inoculum may vary under different field conditions. Substrate availability, 26 environmental conditions, as well as location of inoculum with respect to the host may all influence the quantity of inoculum necessary for in­ f e c t io n (15).

Knowledge of the existing range of pathogenic variability of P. aphanidermatum in soil is necessary for establishing the significance of soil populations. Previous reports have indicated variability in patho­ genicity of isolates of P. aphanidermatum using various hosts (33) and comparing plant vs. soil isolates (37)• No differences, however, were observed in pathogenicity of isolates from Arizona and Mexico soils in this study. Similar results were observed by Stanghellini using isolates from infected plant tissues (52). Degrees of virulence reported in pre­ vious studies are difficult to interpret since propagule nature, quantity and viability were not established (33, 37)• Inoculation procedures may vary among workers and may also contribute to what appears to be differ­ ence in virulence of isolates. In virulence experiments the chance of secondary spread may contribute to infection caused by P. aphanidermatum and thus may cause fluctuations in percentage of infection with time.

In addition, the genetic variation within the host species may contribute to difference in percentage of infection. Isolates obtained in this study that exemplify a high percentage of oospores aborted may also ac­ count for variance in pathogenicity reported by previous researchers

(3 3 , 3 7 ).

Since the nature of the persisting propagule, verticle distribu­ tion, minimum inoculum density necessary for infection, and behavioral characteristics in soil have been elucidated in this study, possible 27 biological control methods can now be utilized to lower populations of

P. aphanidermatum in the field to effect control. Using the selective medium developed in this study along with knowledge of inoculum density necessary for infection it may also be possible to forecast potential dis­ ease incidence in the field.

Media for the selective isolation of Pythium spp have previously . been developed with little emphasis placed on species specificity (60) .

Therefore, to determine soil populations of Pythium spp using these me­ dia it is not only necessary to incubate cultures over a range of temper­ atures from 25 - 37 C, since the optimum temperature for growth is species variable, but also necessary to isolate and identify each colony growing on the medium. Even then high densities of Pythium spp in general may little influence disease incidence. Pathogenic species involved may not be increasing in population at the time of soil sampling and/or may re­ quire low levels of inoculum to incite disease as has been shown for P. aphan 1 dermatum in this study. The above study was facilitated by the use of a selective isolation medium which had the following desirable char­ acteristics: l) species specificity eliminated the need for isolation and identification of all colonies on the medium, a persistent problem with other less selective media, 2) allowed identification of the orig­ inating colony propagule and subsequent isolation of pure single oospore i s o l a t e s , 3 ) permitted demonstration of the linear relationship between oospores/g soil and soil dilutions eliminating preparation of numerous soil dilutions when dealing with soils containing populations of P. aphan- idermatum of unknown. The linear relationship, along with species 2 8

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