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THESIS

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This is to certify that the

dissertation entitled
HYPERPARASITES OF OOSPORES 0F PHYTOPHTHORA MEGASPERMA VAR. SOJAE:
HOST RANGE, ENVIRONMENTAL PARAMETERS, AND BIOLOGICAL CONTROL

 

presented by

ELIZABETH DIAN SUTHERLAND

has been accepted towards fulfillment
of the requirements for

fiL—degreein P/dn 7‘ KLf/wlq7

/ Major professor

Date ~ V /¢?2

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HYPERPARASITES OF OOSPORES OF PHYTOPHTHORA MEGASPERMA VAR. SOJAE:
HOST RANGE, ENVIRONMENTAL PARAMETERS, AND BIOLOGICAL CONTROL

 

by

Elizabeth Sutherland

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1981

b/ 1’

ABSTRACT

HYPERPARASITES 0F OOSPORES OF PHYTOPHTHORA MEGASPERMA VAR. SOJAE:
HOST RANGE, ENVIRONMENTAL PARAMETERS, AND BIOLOGICAL CONTROL

 

by
Elizabeth Sutherland

Oospore hyperparasites were investigated as potential biocontrol
agents of Phytophthora root rot of soybean. Hyphochytrium catenoides,

Pythium monospermum and Humicola fuscoatra applied to soil 7 days before

 

seeding reduced root rot severity by 38-66% in natural soil in various
laboratory experiments.

Seed treatments with Actinoplanes missouriensis or R. fuscoatra
increased plant stand 30-80% in soil artificially infested with
Phytophthora megasperma var. sojae (Pms) in greenhouse tests.

Disease incidence in field plots was very low during three
consecutive years, and biological control could not be adequately
assessed. A soybean seedling assay of soil taken from treated rows,
showed no reduction in disease incidence in soil to which A.
missouriensis or E. fuscoatra were applied prior to planting in the

spring, or to which A. missouriensis, fl. fuscoatra, or E. catenoides

 

were applied at seeding.

The host range of the Pms oospore parasites A. missouriensis,

 

Diheterospora chlamydosporia, fl. catenoides, and E. monospermum was

 

 

extended to include Aphanomyces cochlioides, A. euteiches, E.

 

citrophthora, Pythium aphanidermatum, and E. ultimum, except that H,
catenoides did not infection 3, ultimum oospores. ‘fim§_oospores were

parasitized by two other Actinoplanes species, A. philippinensis and A,

 

 

utahensis; Amorphosporangjum auranticolor, Ampullariella regularis, and

Elizabeth Sutherland

Spirillospora albida, all members of the Actinoplanacae; Micromonospora
Sp. of the Micromonosporaceae. 13mg oospores of races 1, 3, 4, and 7
were equally susceptible (>95%) to infection by A. missouriensis and fl.
fuscoatra. The age of host and parasite cultures had no effect on

oospore infection by A. missouriensis and fl. fuscoatra.

 

Using transmission electron microscopy, hyphae of A. missouriensis
and fl. fuscoatra were observed to directly penetrate the oogonial and
oospore walls, and infect the oospore cytoplasm of fig; within 4 days.

No specialized infection structures were formed by either hyperparasite,
and no ultrastructural change in response to infection was observed in
the host.

Conditions highly conducive to parasitism of Em§_oospores in

autoclaved soil by A, missouriensis were 0 bars matric potential, soil

 

temperature 15-30 C, and soil pH at 5.5 or between 6.6 and 8.0. Optimum
conditions for parasitism of Em§_oospores by E. fuscoatra were -0.3 bars
matric potential, soil temperatures of 20-30 C, and soil pH between 6.6

and 8.0.

TABLE OF CONTENTS
Page
LIST OF TABLES ..................................................... iv
LIST OF FIGURES .......... ......... ................................. vi
INTRODUCTION ....................................................... 1
LITERATURE REVIEW .................................................. 3

Parasitism of microorganisms causing rots of fruits and
vegetables .................................................. 5
Parasitism of organisms infecting aerial plant parts .......... IO
Peronosporaceae .......................................... 10
Ascomycetes and fungi imperfecti ......................... 11
Powdery mildews .......................................... 14
Rusts .................................................... 15
Pathogens of woody plant parts ........................... 18
Parasitism of organisms causing root diseases ................. 21
Chytrids ................................................. 21
Oomycetes parasitized by slime molds ..................... 21
Oomycetes and their chytrid parasites .................... 22
Oomycetous hyperparasites of other Oomycetes ............. 26
Fungi Imperfecti as hyperparasites of Oomycetes ............... 28
Hyperparasitism of some of the lower fungi Rhizoctonia ... 31
Ascomycetes and Fungi Imperfecti ......................... 32
Sclerotinia .............................................. 34
Rhizoctonia .............................................. 41
Basidiomycetes ........................................... 51
Parasitism of beneficial root-infecting fUngi, the
endomycorrhizae ............................................. 52
Potential biological control agents ........................... 53

 

 

MATERIALS AND METHODS ....OOOOOOOOOOOOOOOOOOOOO.......0.0.0.0000...O 55

Host fungi .................................................... 55
Maintenance of potential hyperparasites ....................... 57
Production of potential hyperparasites for laboratory use ..... 58
Evaluation of hyperparasitism in vitro ........................ 59
Evaluation of host Spore viability using tetrazolium

bromide ..................................................... 60
Environmental factors affecting hyperparasitism of E.

megasperma var. sojae oospores by A. missouriensis

and H, fuscoatra ........................................... 61
Electron microscope studies of oospore penetration ............ 63

 

 

ii

Page

Biological control experiments in vitro ....................... 63
Production of inoculum for greenhouse and field
experiments ................................................. 64
Carriers for biological control agents ........................ 66
Greenhouse experiments ........................................ 67
Field experiments: Biological control with hyperparasites .... 68
1979 ..................................................... 69
1980 ..................................................... 70

1981 O0......00.0.00.........OCOOOO.........OOOOOOOOOOOOOO 73

StatiStica] analyses ......OOOOOOOOOOOO......OOIOOOOOOOOOOOOOOO 74
RESULTS ......OOOOOOOOOOO......OOOOOOOOOOOOOOOOO.......OOOOOOOOOOOOO 75

Tetrazolium bromide as an oospore stain ....................... 75
Host range experiments ........................................ 79
>Invasion of living vs. killed oospores ........................ 84
Effect of Oomycete culture age on susceptibility of
oospores to hyperparasites .................................. 86
Effect of hyperparasite culture age on hyperparasitism ........ 86
The influence of soil pH, moisture, and temperature on
hyperparasitism of P. megasperma var. sojae oospores
by A. missouriensis and H. fuscoatra ........................ 90
Electron microsc0pic studies of the host- -parasite
interaction ................................................. 94
Biological control experiments using the soybean seedling
assay ....................................................... 100
Attempts to enhance indigenous hyperparasites through
soil treatment with oospores ................................ 100
Inoculum substrates for test organisms ........................ 102
Greenhouse experiments ........................................ 104
Effect of potting medium on disease ...................... 104
Effect of culture medium on infectivity of A.
megasperma var. so ae oospores ......................... 105
Effect of inoculum ensity on disease using two
different potting media ................................ 105
Effect of length of time of flooding on disease .......... 108
Biological control in the greenhouse by A.
missouriensis applied as an aqueous suspension ......... 108
Biological control with A. missouriensis and A,
fuscoatra as seed treatments ........................... 108
Additional biological control experiments ................ 111
Effect of viable Pms mycelium on disease incidence and
biological contFET .......................................... 113
Field experiments ............................................. 113

 

 

 

DISCUSSION 0.00.00...00.........OOOOOOOOOOOOOOOOOO0.0......00.0.0...116
BIBLIOGRAPHY ...00.0.........OOOOOOOOOOO.......OOOOOOOOOOOOOO0......124

Table

03

10

LIST OF TABLES
Page

Stain reactions of several Oomycetous fungi with
tetraZOTTUm bromide (MT-r) OOOOOOOOOOOOOOOIOOOOOOO0.0.0000.000 76

Parasitism of oospores of six plant pathogenic fungi
by five hyperparasites after 14 days of incubation
on water agar CO0.0...0.0.000...OOOOOOOOOOOOOOO0.0.0.0.000... 80

Parasitism of oospores of four Phytophthora species
by Actinoplanes missouriensis or Humicola fuscoatra
after 14 days of incubation on water agar ................... 82

Comparative susceptibilities of oospores of four races of
EAytophthora megasperma var. sojae to the hyperparasites
Actinoplanes missouriensis and Humicola fuscoatra after

14 days of incubatiOn on water agar ......................... 83

 

 

 

Parasitism of PhytOphthora megasperma var. sojae oospores
by six actinomycetes species after 14 days of incubation
on water agar 0.0.000000000000000000000000...OOOOOOOOOOOOOOOO 85

Invasion of living vs. killed oospores of Phytophthora
megasperma var. so ae by Actinoplanes missouriensis or
Humicola fuscoatra a ter 14 days of incubation on water

agar ......OOOOOOOOOOOOOOO......00............OOOOOOOOOOOOOOO 87

 

 

 

Susceptibility of PhytOphthora megasperma var. sojae
(Pms) oospores of three ages of Actin0planes missouriensis
or Humicola fuscoatra after 7 days of incubation on water

agar ......OOOOOOOOOOOOOOOOO00.0.0000.........OOOOOOO0.00.0... 88

 

Parasitism of oospores of Phytophthora megasperma var.
so ae by Actinoplanes missouriensis or Humicola fuscoatra
of different ages after 7 days of incubation on water

agarCOOOOOOOOOOOOO0.0.............OOCOOOOOOO......OOOOOCOOOO. 89

 

Effect of pH on parasitism of Phytophthora megasperma
var. sojae oospores by ActinOplanes missouriensis
after 7 days of incubation in autoclaved soil ............... 91

 

Biological control of Phytophthora root rot of soybean
in the laboratory using hyperparasites in naturally
infested $01.] OI...0.00.00.00.00.COO...OOOOOOOOOOOOOOOOOOOOOO101

 

iv

Table
11

12

13

14

15

Page

The effect of Eythium acanthicum or killed Phytophthora
megasperma var. sojae (Pmsl oospores on PhytOphthora
root rot in soils naturally infested with Amé ............... 103

 

 

Effect of potting medium on Phytophthora root rot of

soybean in a greenhouse experiment using oospores

produced in V-B juice broth at a concentration of

approximately 114 oospores/g potting medium ................. 106

 

Comparison of virulence of Phytophthora megasperma var.

sogae 005pores produced on millet seed or in clarified
- JUice brOth 00.0.0.0....-0.0.00.0.........OOOOOOOOOOO0.0.107

Lack of-biological control of Phytophthora megasperma

var. sojae (Pms) in the greenhouse by Actinoplanes

missouriensis applied as a mycelial/spore suspension

under different conditions .................................. 110

 

Biological control of Phythophthora megasperma var.

sgjae (Pms) in the greenhouse by Actinoplanes

missouriensis and Humicola fuscoatra applied as seed

treatments .................................................. 112

 

Figure

5-8

9-11

LIST OF FIGURES

Staining of oospores of three Oomycetes during 2-72 h
incubation in a 0.1% suspension of tetrazolium bromide
(MW) at 35C ......OOOOOOOOOO00.00.000.000.......OOOOOOOOOO

The effect of pH on parasitism of Phytophthora megasperma
var. sojae oospores by Humicola fuscoatra after 7 days of
incubation in autoclaved soil. Standard errors ranged from
1-3%. Soil pH was adjusted as described in Table 9 ........

 

Hyperparasitism of Phytophthora megasperma var. sojae
oospores by Actinoplanes missouriensis and Humicola
fuscoatra in autoclaved soil adjusted to different matric
potentials, after 7 days of incubation. The values
represent averages (:_standard errors of 2-6%) of six
experiments with A. missouriensis and averages (:_standard
errors of 2-5%) of five experiments with A. fuscoatra ......

The effect of temperature on parasitism of PhytOphthora
megasperma var. sojae oospores by Actinoplanes
missouriensis or Humicola fuscoatra after 7 days of
incubation in autoclaved soil. Standard errors ranged

from 0-4% in the A. missouriensis experiments and 1-4%

in the A. fuscoatra experiments ............................

 

 

Invasion of oospores of Phytophthora megasperma var.
sojae by Actinoplanes missouriensis ........................

Figure 5. Overview showing hyphae of the hyperparasite on
the oogonial wall, in the periplasmic space
between the oogonial and oospore wall, the

oospore wall, and the oospore cyt0plasm. X 2,592.

Figure 6. Penetration of the oogonial wall. X 17,280.

Figure 7. Concentration of A. missouriensis hyphae in the
periplasmic space. X 4,320.

Figure 8. Penetration of the oospore wall. X 17,280.

 

Invasion of oospores of PhytOphthora megasperma var.
SOJae by Humico1a fuscoatra 00......OOOOOOOIOOOOOOOOOOOOOOOO

 

Figure 9. Overview showing hyphae of the parasite
penetrating the oogonial wall, within the
periplasmic Space, and in the oospore wall.

X 8,250.
Figure 10. Penetration of the oogonial wall. X 20,000.
Figure 11. Penetration of the oospore wall. X 15,000.

V!

Page

78

92

93

95

97

99

INTRODUCTION

Soybean root rot caused by Phytophthora megasperma Drechs. var.
ggjgg Hildb., is the most important disease of soybeans in Michigan. Due
to the ability of the pathogen to fonn races that will infect once
resistant varieties, breeding programs may offer only a partial solution
of control. Chemical control has not been practiced because it is
expenseive, ineffective, and potentially hazardous. Biological control
may offer a third alternative to the perplexing battle of controlling
this pathogen.

Parasites of A. megasperma var. £913; oospores were discovered in
studies of oospore germination in soil (187). In further studies, Humble
and Lockwood (118) demonstrated the presence of oospore hyperparasites in
each of 15 field soils tested. However, there was no relationship
between the frequency of hyperparasitism and disease potential as
determined by a seedling assay of soils from the various fields. When

hyperparasites were applied to soil naturally infested with A. megasperma

 

var. sojae, biocontrol was inconsistent, but Hyphochytrium catenoides
caused significant disease reduction in two out of three experiments
(117).

Evaluation of biocontrol potential of A. megasperma var. sojae by

 

hyperparasites was the main objective of this study. This assessment was
made in several ways. The efficacy of the oospore hyperparasites

identified by Sneh et al. (187) was determined in laboratory experiments.

Host range studies using the parasites and plant pathogenic Oomycetes

other than A, megasperma var. sojae were undertaken to determine the

 

extent of hyperparasitic ability. Optimal environmental conditions for
hyperparasitic activity were studied. Greenhouse and field tests to

control Phytophthora root rot were conducted in artificially and

 

naturally infested soils.

LITERATURE REVIEW

Parasitism of one microorganism by another has been documented as
early as the late nineteenth century by workers such as deBary in Germany
(67), Bainier in France (17) and Thaxter in America (194). Numerous
reports of mycoparasitism have since been made, and several excellent
reviews have collated most of this material (24, 25, 26, 28, 40, 71,
169). The parasitism by one microbe on another parasite is known as
hyperparasitism, while the parasitism of one fungus by another is termed
mycoparasitism (5). Lacking within the current usage of hyper- and myco—
parasitism is a term describing parasitism of a saprophytic microorganism
by another microorganism. Myc0parasitism and hyperparasitism have been
used almost interchangeably to describe mycoparasitic interactions. In
this review, the term hyperparasites will be frequently used since
parasites of plant pathogens will be emphasized. Myc0parasitism is not
entirely appropriate here because not all parasites of plant pathogens
are fungi.

Hyperparasitism can be subdivided into two groups based on the mode
of parasitism. If the parasite kills the host cells prior to utilization

of host nutrients, the relationship is termed necrotrgphic (24, 40). If

 

the parasite obtains nutrients from the living host cell, the relation-
ship is biotrophic. Further interpretation of these categories is

dependent upon the researcher. Boosalis (40) subdivided the biotrophic

group into those parasites whic are more destructive and kill the host,

and those parasites which are balanced and can obtain nutrients from the
living host causing little or no harm. Barnett (24), on the other hand,
used the term biotrophic parasite interchangeably with balanced parasite,
and necrotrOphic interchangeably with destructive parasite. Barnett and
Binder (28) subdivided biotrOphic parasitism to include three types of
parasitism: 1) internal, parasites developing within the host cell,

2) contact, parasitism occurring without invasion of host cells, and

3) haustorial, parasites forming haustoria within host cells. Because
there are examples of internal, contact, and haustorial parasites that
will kill the host cell [e.g., numerous internal oospore parasites (188),
hyphal interference (120), and haustorial parasites such as Rhizidium

richmondense (221) and Rhizidiomycopsis stomatosa (190) in which the

 

rhizoidal system acts as the absorptive apparatus], it is deemed
necessary to retain Boosalis' interpretation of biotrOpic parasitism to
include both destructive and balanced parasites. Examples of internal,
contact, and haustorial parasites may then be categorized for both
destructive and balanced parasites. One might argue that classification
of hyperparasitic relations is unwarranted until a nutritive exchange
between host and parasite is proven experimentally. Terminology may
need to be refined as information becomes available on host-parasite
physiology.

Studies of hyperparasitism have had two major emphases: i) to
understand basic anatomical and physiological processes of the parasitic
relationship, and ii) to utilize the parasitic relationship to reduce
natural populations of the host organism. Biological control has long
been observed in nature by plant pathologists and has been held responsi-

ble for keeping powdery mildew and rust diseases in check during certain

years, as will be discussed later. It is probable that biological
control via hyperparasitic phenomena is already responsible for limiting
populations of plant pathogens. Recently, there are increased efforts
to introduce natural parasites of economically threatening plant
pathogens into agronomic cr0pping systems. Accordingly, this literature
review will emphasize the biological control potential of different

hyperparasites.

Parasitism of microorganisms causing rots of fruits and vegetables

Members of the Mucorales are important economically because of their
role in blossom blights and storage decay. These plant pathogens are
susceptible to parasites that are members of the Mucorales. The
literature dealing with hyperparasitic Mucorales is extensive, and much
of it has been reviewed by Benjamin (33).

Choanephora cucurbitarum, the causal agent of blossom end rot of
vegetables in the southern and eastern United States, is susceptible to

attack by Piptocephalis unispora (125) and A, virginiana (34). Other

 

hosts are Rhizopus nigricans which causes soft rot of vegetables,
particularly sweet potatoes and fruits, and A3325 spp. which can cause
decay of fruits. Most Piptocephalis spp. are limited in host range to
the Mucorales except, however, A, xenophila is known to attack some

species of Penicillium, Aspergjllus niger, A. repens, Cephalosporium sp.

 

and Venturia inaegualis (72).

On agar culture media, germ tubes of A. virginiana were attracted to
‘C. cucurbitarum hyphae; upon contact, an appressorium developed on the
host hypha (35). Penetration was accomplished by an infection peg and

was thought to be largely due to mechanical forces (156). An haustorium

then developed within the host hypha. A compatible interaction was

established between A. virginiana and C. cucurbitarum only when the host

 

 

hyphae were young (1-day-old) (155). The host cell wall was one layer in
thickness after 24 h, but was composed of two layers by the time the
hyphae were 7 days of age and resistant to attack. In the incompatible
reaction, the hyperparasite dissolved the outer wall layer of the host.
The inner layer became thickened and a papilla was developed by the host
which limited the develOpment of the hyperparasite (53). Haustoria of A.
virginiana were excluded from host cyt0plasm by a sheath in the early
stages of penetration. Host origin of the sheath was demonstrated with
autoradiography (157), and by the use of an enzyme inhibitor (158).

In the compatible interaction, no papilla formation was detected and

sheath formation was delayed (153). Direct contact of A. virginiana was

 

made with the protoplast of C. cucurbitarum (155). Cisternae of the

 

endoplasmic reticulum surrounded the young parasitic haustorium, the
disappearance of which appeared to coincide with sheath develOpment.
Lipid-containing Sphaerosome-like bodies were found in the host and

parasite (156).

A comparison was made between axenic cultures of A. virginiana and

 

cultures of the parasite with its host (151). Piptocephalis virginiana

 

developed to a very limited extent in sterile culture, and the few

spores that were produced would not germinate in the absence of a host
(143). Comparisons between spores produced with and without the host
revealed the absence of lipid bodies in the axenically-cultured spores.
Analyses using thin-layer and gas-liquid chromatography demonstrated the
presence of the fatty acid, v—linolenic acid, in spores produced in mixed

culture but not in those produced in sterile culture (151). Further

investigation by Manocha and Deven (154) revealed that environmental
conditions including media composition, temperature and light conditions,
and age of the culture affected the amount of y-linolenic acid detected
in C, cucurbitarum which in turn determined the degree and rate of
parasitism by_E. virginiana (152). Gamma linolenic acid is a major
constituent of the phospholipid, diphosphatidylglycerol, which was also
lacking in axenically cultured spores. Evidence was presented to suggest
that diphosphatidylglycerol was closely associated with the mitochondrial
system of yeasts (123); therefore, its absence may explain the poor

growth of A. virginiana in the absence of a hoSt (152).

 

Other workers also have shown that the medium composition (34, 38)
and incubation temperature (34) will affect hyperparasitism. Jeffries

and Young (124) investigated parasitism of the saprOphyte Cokeromyces

 

recurvatus by A. unispora. The same events occur in this relationship as

just described for g. cucurbitarum and A. virginiana, although the role

 

 

of v-linolenic acid has not yet been established.

Piptocephalis virginiana is a biotrophic, balanced, haustorial
parasite causing little if any damage to its hosts (35). Therefore, it
does not appear to have biological control potential.

Dispira cornuta, a plant parasite itself (9), will infect Q,
cucurbitarum, A2225 spp., and Rhizogus nigricans (10). Unlike
Pitocephalis, Q. cornuta may be cultured axenically on an egg medium and
still retain the ability to parasitize the Mucorales (19). Dispira
cornuta has demonstrated a growth requirement for high levels of
thiamine, biotin, and nitrogen (22). The parasitic ability of Q. cornuta
was altered when a host was grown on a culture medium with different

carbon and nitrogen sources (10, 140). When near a suitable host,

germinating spores of g. cornuta exhibited positive tr0pism (10).
Penetration followed by haustorial develOpment within the host occurred
only if host hyphae were young. Dispira cornuta is a biotrophic,
balanced, haustorial parasite, and probably has no value as a biological
control agent.

Syncephalis spp. have been noted as parasites of Choanephora, Mucor,

 

and Rhizopus. Syncephalis is a biotrophic, haustorial parasite as are
Piptocephalis and Dispira. Some Syncephalis spp. may suppress host
sporulation. Bainier (17) described the sporangial develOpment of S.
curvata on a hypha of A. nigricans. Syncephalis sphaerica was found to
parasitize A. mggggg_among other fungi, but did not appear to affect its
host appreciably (20). 0f the Mucorales tested, Rhizopus spp. were the
most susceptible to infection RY.§° californica (119).

No evidence of tropism of germinating spores of §. californica has
been observed (119). A single or multiple lobed appressorium, several
times the diameter of vegetative hyphae, encircled the host hypha after
chance contact, and an infection hyphae penetrated the hyphal wall.
Localized swellings of the host near the appressorium_were observed after
infection. The size of the swellings was inversely proportional to their
number. Once through the wall, the hyphae branched sparsely. Eventually
the host cytoplasm disappeared to reveal the parasitic hyphae tightly
packed in the swollen portions of the host. Some of these hyphae emerged
from the host, and penetrated other portions of the host thallus.
Although the host was not killed, infection by s. californica reduced
sporulation of A. ggygge in agar culture. Ellis (85) was able to grow

Syncephalis spp. on liver agar. In vitro, pH Optima ranged from 7 to 8,

 

and temperature Optima from 18-22 C.

Biological control of Rhizogus decay may be possible using S,

californica, but further work is needed to determine if the parasite is

 

destructive enough to prevent mycelial spread of the pathogen. Cold
storage is the major treatment presently used to prevent losses by
Rhizogus.

Chaetocladium and Parasitella cause swellings or galls in their

hosts much as Syncephalis does. Both parasites will infect Choanephora,

 

A3593, and Rhizogus (43). The similarity of the galls to the
progametangia of the Zygomycetes caused Burgeff (44) to suggest that an
imperfect sex reaction between host and parasite had initiated the
development of the parasitic relationship. He presented evidence that
the (+) strain of Parasitella would parasitize only a (-) strain Of the
host and vice versa. Satina and Blakeslee (180) tested a large number Of
host strains and concluded that the mating sex type was not entirely
responsible for attraction between host and parasite, although there did
seem to be a parasitic preference for Opposite sex strains. The
hyperparasite Chaetocladium is itself susceptible to attack by Dispira

simplex (41) and Piptocephalis (209).

 

Other hyperparasites of the Mucorales include Ampelomyces quisqualis

on A. mucedo (146) and Fusarium lini on A. racemosus (8). Trichoderma

 

spp. have been reported to penetrate hyphae of Rhizopus oryzae (80, 81).
The chytrid, Rhizophydium mycetophagum, was described as a
hyperparasite of the conidiophores and hyphae of an unidentified species
of Choanephora (130). It produced numerous epibiotic Sporangia on the
host cell. The zoospores were released, then encysted on the host
hyphae, penetrated, and formed a delicate but highly branched rhizoidal

system within the host cell. The host protOplasm degenerated by the time

10

the parasitic sporangia released their zoospores.
Another organism causing cucumber fruit decay, Rhizoctonia solani,
and its hyperparasite, Laetisaria arvalis, will be discussed later with

Rhizoctonia diseases.

Parasitism of organisms infecting aerial plant parts
Peronosporaceae. While the Oomycetes are generally known for

causing root diseases, one family, the Peronosporaceae, contains a

 

highly specialized group of obligate parasites causing downy mildews.
Phlyctochytrium punctatum has been observed infecting the oospores of
Peronospora tabacina, the cause of blue mold of tobacco (136). Sporangia
formed epibiotically on the oospores. An apOphysis developed within the
OOSpore as the result of swelling of the base of the rhizoidal axis. The

rhizoidal system was lobed or irregular. Oospores of Sclerospora sorghii

 

(the causal agent of downy mildew of corn and sorghum) were attacked by
an unidentified Phylyctochytrium sp. (133). Parasitism occurred as
described for A, punctatum, although apOphyses in the oospores were not
consistently formed. Healthy and heat-killed oospores were attacked
equally. The hyperparasite was cultured on agar and in liquid media, and
survived storage on agar at 6 C for 28 months. Optimum temperature range
for growth was 23-27 C. This hyperparasite was considered to have a high
potential as a biological control agent.

Another chytrid, RhiZOphidium pollinis, parasitized oospores of s.
graminicola, which causes downy mildew of grasses and millet (159).
Hyperparasite sporangia were sessile on the oospore, and a delicate
rhizoidal system formed within. Zoospores encysted on contact with an

OOSpore, germinated and penetrated the oospore, and eventually functioned

11

as sporangia. Resting spores were produced inside Old sporangia.
Sclerospora graminicola is also susceptible to attack by the imperfect
fungus, Fusarium semitectum (175), which invaded the oospores within the
infected millet head and rendered them, for the most part, ungerminable.
Fusarium semitectum was suggested as a potential biological control
agent.

Ascomycetes and fungi imperfecti. Claviceps purpurea causes a
disease of cereals known as ergot. This fungus replaces some of the

cereal grain with its own sclerotia. An isolate of Fusarium roseum

 

'Sambucinum' was found to be an effective hyperparasite of g, purpurea in
both laboratory and field tests (162). In addition to reducing the
number of sclerotia formed by g, purpurea, E. gggggm degraded ergotamine
into inert compounds which did not cause deleterious effects on rats or
rabbits. This A, gggggm isolate did not cause disease on rye, and
preliminary tests did not show that E. [ggggm was itself producing toxic
substances. The biological control potential of E. gggegm is readily
apparent due to its ability to infect g. purpurea, and to inactivate the
toxin produced by the plant pathogen.

Two plant pathogens that infect corn ears, Fusarium moniliforme and

 

Diplodia 533g, were found to be hosts of the contact biotrOphic
hyperparasite Acladium tenellum (141). Acladium tenellum made contact
with its host by the formation of specialized contact cells. Nutritional
studies have demonstrated the requirement for thiamine and biotin in
axenic culture. The destructiveness of A. tenellum was not alluded to,
but if like other biotrOphic contact parasites, it probably does not
cause enough damage to be of potential value as a biological control

agent.

12

Helminthosporium spp. cause leaf spots and blights of small grains
and corn. Sphaeronemella helvellae, a perithicial ascomycete, was found
to infect conidia and mycelia of_A. carbonum, A. ‘mpygip, and A. turcicum
(208). Infected conidia of A. carbonum were distorted, while mycelia
were vacuolated and had a slower growth rate. Biological control
experiments were not performed.

Trichoderma lignorum was isolated from soil collected from field
plots of barley that had remained healthy despite heavy inoculation with
pathogenic A, sativum (36). In biological control experiments, plants
were less diseased in pots containing both A. sativum and I. lignorum
then in pots containing only A. sativum. Parasitism was the mechanism
implicated in disease control, but this was not established.

Alternaria diseases are common on many plants, usually occurring
when plants are physiologically stressed. Gonatobotrys simplex is a
biotrophic parasite of A. ppppip (217). In culture work on the
host-parasite interaction, hyphae of p. simplex demonstrated trOpic
growth towards host spores. The parasite produced short, bulboUs
branches which contacted the host hyphae. Ultrastructural study of the
host-parasite interface showed membrane-lined plasmodesmata connecting
the fungi (106). The host wall was thickened. The contact cell of Q.
simplex was delimited from the main hypha by either an open or occluded
septal pore. The parasite caused only a slight reduction in growth of
its host (217). Gonatobotrys simplex would not grow in the absence of a
host unless a water extract of the host or an extract of certain other
fungi was added to the medium. A growth factor was partially purified
from fungus extracts and named mycotrOphein. This substance(s) has

apparently never been characterized.

13

Nectria inventa and its Verticillium state have been cited as

 

destructive parasites of Alternaria brassicae (198, 199, 201). In
cultural studies of their interactions, usually the hyperparasite hyphae
grew towards the host then grew along side or coiled around the host
hyphae forming appressorium-like structures. Degeneration of contacted
cells occurred with or without penetration; conidia were penetrated more
Often than hyphae. Penetration often occurred at septa of hyphae and
mature conidia resulting in separation Of cells. Because of the
formation of large holes in the cell wall and the lack Of indentation,
invasion was thought to be enzymatic in nature. The host plasma membrane
was invaginated beneath the point of contact in both hyphal and conidial
cells. The area between the invaginated membrane and the host cell wall
was electron transparent and contained electron-dense tubule structures
that in later stages appeared as irregular deposits. Septal plugs were
formed by the host hyphae delimiting the infected from the healthy area.
When conidial cells were penetrated, an enlargement of the parasitic
hyphae occurred at each septum. The hyphae became constricted with
passage into the adjoining cell. Complete vacuolation of infected host

cells eventually occurred. No biological control experiments have been

 

performed to study Nectria as a hyperparasite of Alternaria in nature,
but such attempts would appear promising since both hyphae and conidia
are susceptible to hyperparasitic attack.

Hanstrdia sp. has been discovered in nature as a hyperparasite of

two foliar pathogens, Cercosporidium personatum which causes late leaf

 

spot on peanuts and Fulvia fulvum (Cladosporium fulvum) which causes leaf
mold of tomatoes (192, 193). Spores and hyphae of the hosts were

penetrated by the parasite. Cercosporidium personatum was colonized by

14

Hansfordia in greenhouse tests. When the fungicides benomyl, maneb, or
chlorothalonil were used in the greenhouse, Hansfordia Sp. was not found
on fplyip-infected plants.

Several other examples of hyperparasites of fungi causing leaf Spots

are A. tenellum, a hyperparasite of Diplocarpon rosae (141), the causal

 

agent of black spot of rose, and Fusarium semitectum, itself a secondary

 

plant parasite, parasitizing three species of Cercospora (176). Fusarium

 

semitectum coiled around host conidiophores and conidia sending out
infection pegs at contact points. HypertrOphy of host cells was
frequently observed.

Powdery mildews. Hyperparasitism of the powdery mildews by
Ampelomyces quisqualis (Cicinnobolus cesatii) is perhaps one of the most
frequently observed hyperparasitic relations in nature. DeBary in 1870
(87) gave a detailed account of the destructive internal parasitic
action. Ampelomyces guisqualis invaded the host hyphae directly and
quickly ramified through the hyphal cells passing through septa without
constriction. Hyperparasite pycnidia commonly formed in conidiOphores or
ascocarps, replacing ascospores with pycnidiospores. Only mature conidia
or ascocarps were not invaded by the parasite. The particular host and
stage of development influenced the size and shape of the parasite
pycnidium. Eventually the host protOplasm was degraded, and the parasite

hyphae became shorter and thicker. Ampelomyces quisqualis then lived

 

saprOphytically on the dead tissue of the host plant.

Ampelomyces quisqualis has been reported on mildews of the genera
Erysiphe, Leveillula, Microsphaera, Oidium, Podosphaera, Phyllactinia,
and Sphaerotheca (7, 191). Biological control of all these genera except

Microsphaera, which was not tested in the greenhouse, was Obtained when

15

repeated inoculations were made with the hyperparasite at 10-day

intervals (191). It has been suggested that A. quisqualis has held in

 

check powdery mildew of clover in Indiana (223) and of several herbaceous

plants in Iowa (7) during certain years. One reason A. quisqualis is not

 

used commercially for powdery mildew control may be because sulfur, an
inexpensive chemical control, is readily available. In addition, powdery
mildews will infect and cause disease at moisture conditions much lower

than A. quisqualis may tolerate.

 

Biotrophic, contact hyperparasitism has been observed between

Tilletiopsis sp. and the powdery mildew fungus, Sphaerotheca fuliginea

 

(110). The hyperparasitic hyphae entwined the hyphae, conidiophores, and
conidia of the host. Powdery mildew control was obtained in the

greenhouse by Spraying a spore suspension of Tilletiopsis onto detached

 

cucumber leaves infected with g. fuliginea. Development of §: fUligenea

was prevented when Tilletiopsis Spores were applied up to 8 days prior to

 

inoculation with S. fuligenea.

Apgpg. The observations of naturally occurring biological control
of the powdery mildews can only be matched in number by accounts of
hyperparasitic associations with the rusts. The hyperparasite Darluca
.filpp_has been isolated from a large number of rust species belonging to

the genera Puccinia, Uromyges (49, 131) and Peridoermium (1), and has

 

 

been reported to attack the pycnial (=spermatial), aecial, uredial, and
telial stages of the rust life cycle (1). Infection of the uredial stage
has been reported most frequently. .2. jilpm entered the host cell by
direct penetration (52). A study on the ultrastructural relationship of

Puccinia graminis and p. filum demonstrated the invasion of uredospores

 

but not the sub-basal cells or mycelium Of the host. After penetration

16

of the spore walls, electron transparent channels were observed next to
the walls of the hyperparasite. A haustorium was not formed. Complete
destruction of the spores followed penetration. Darluca filum replaces
the rust sporocarp with its own pycnidium. and can be recognized easily
in nature by the abnormal black appearance of a normally orange-red rust
lesions (166). The hyperparasite can be cultured on common laboratory
media (32). Growth rates Of Q. fllflfl conidial or mycelial isolates vary
with different carbon and nitrogen sources (32, 163, 174). Due to the
relatively common occurrence of conidia in nature, it was suggested that
the conidial stage be used for nutritional and physiological studies.
Darluca jjlpp_was thought to keep the spread of corn rust in check in
Iowa (66).

Tuberculina maxima, like Q. filum, is a fungus that has long been

 

reported to be closely associated with the rusts on woody plants. Field
surveys of several pine rusts have indicated the presence of I. maxima,
but the hyperparasite's significance in natural rust control has been

controversial (134, 160). Cronartium spp. and Uredinopsis mirabilis are

 

hosts for I. maxima (116). The hyperparasite has been thought to
directly attack the rust as a hyperparasite, but Wicker and Woo (220)
could find no penetration of rust cells, nor could they find any evidence

of direct attack upon Cronartium ribicola. However, the destruction of

 

rust-infected white pine cells by l. ppéipp was apparent in histological
sections. Evidence was presented to indicate that I. ppéjpp was
parasitic on the rust-parasitized plant cells and not directly on the
rust. The implied role of the rust was to weaken the middle lamella
which would allow secondary invasion of plant cells by _T_. m_ax_i_m_p. _T_.

maxima invaded healthy pine tissue in vitro but not in vivo (219), but

17

rust-free pine tissues grown in vitro have poorly deveIOped middle
lamellae (220). Wicker and Woo (220) claimed that Q, filpm may act in
a similar manner to I. mpéipp, but since that time, an ultrastructural
study showed direct penetration of rust cells by A, jjlpm (52). An
attempt to inoculate naturally-occurring rust cankers with I. ppéjpp
met with little success (116).

Scytalidium uredinicola has been cited as a destructive

hyperparasite of the rusts Cronartium guercum f. sp. fusiforme (139)

 

and Endocronartium harknessii (220). The mode of action of S.
uredinicola is similar to that 0f.Ir.flééflflé because direct contact

with the rust is not required for the rust to be harmed. Rust hyphae
and Spores were not penetrated, but rather disintegrated by the action
of §. uredinicola much like those of 1. mpéipp. It would be interesting
to know whether the two hyperparasites operate by the same mode of
action. Scytalidium uredinicola hyphae penetrated the rust-infected
galls and were able to destroy rust hyphae within the gall, even below
the invaded area, suggesting the involvement of a diffusible compound.

Surveys in some areas of Alberta indicated that S. uredinicola was

 

associated with greater than 80% of the rust galls (200). Scytalidium

 

uredinicola may decrease aeciospore production by Q. guercum f. Sp.
fusiforme by as much as 72% (138). The fact that the modes of action

for I. maxima and §, uredinicola may involve the inadvertent destruction

 

of the rusts does not diminish their value as biological control
agents.

Monocillium nordinii was found as a parasite of Cronartium

 

 

coleosporioides and Endocronartium harknessii on pine (197). Monocillium

 

 

 

nordinii formed appressorium-like structures on the host spores, but

18

penetration did not occur until after the host cells were devoid of

most contents. Another hyperparasite, Fusarium trichothecioides (E.

 

bactridioides), was found occurring on galls produced by Cronartium
spp. (222). White pine blister rust cankers inoculated with E.
trichothecioides were dead one year later. The hyperparasite was found

sporulating on Q. harknessii and C. filamentosum cankers several months

 

after inoculation.

Rust diseases can be very devastating. Control is usually through
resistant varieties, or removal of alternate hosts, although several
systemic fungicides have been very effective. Biological control would
be very useful if found effective.

Pathogens of woody plant parts. Hyperparasites of canker-producing

organisms have been noted. Gonatorrhodiella highlei, a biotrophic,

 

contact parasite has been found in close association with Nectria

coccinea, causal agent of beech bark disease, and A. cinnabarina, a

 

fungus reSponsible for dieback and canker of Shade trees (11, 38). In
agar culture, the germ tube upon contact with the host formed an
appressorium which gave rise to hyphal hooks which wrapped around the
host hyphae. A branched hyphal system developed from the appressorium and
hook cells, giving rise to new appressoria and hook cells (37, 38). In
several instances, Blyth (38) observed reversed parasitism, Nectria
causing vacuolation of p. highlei hyphae. Gain and Barnett (90) isolated
the hyperparasite by incubating cultures in the dark, and allowing the
hyphae of the hyperparasite to grow from the colony edge of the host then
transferring to an agar medium. To obtain an axenic culture of p.
highlei, it was necessary to add mycotrOphein, and a water soluble

compound isolated from beech bark (90). Some nitrogen sources at

19

concentrations greater than 42 mg/l caused inhibition of the
hyperparasite due to the liberation of NH3 by the host. Concentrations
of thiamine had to be greater than the Optimum for host development in
order for parasitism to occur. Other plant parasitic hosts of the
hyperparasite include A. galligena (11), Graphium sp., and Verticillium
2192-22112 <90) -

Calcarisporium parasiticum was isolated from a culture of Dothiorel-
.lp guercina (Ppysalospora glandicola) which causes twig blight of oak

(23). Several other Physalospora spp., Botryosphaeria ribis, Guignardia

 

 

bidwelli, Diplodia pinea, Coniothyrium sp., and Dothiorella Sp. were

 

also hosts of p. parasiticum (29). Calcarisporium parasiticum is a

 

 

biotrophic, contact mycoparasite which forms buffer or contact cells at
the host-parasite interface. Unlike the positive trOpic growth exhibited
by some of the other contact hyperparasites, in this case the host hyphae

were attracted to the spores of C. parasiticum. The host either produced

 

lateral branches, or occasionally the main hypha would turn and grow to
the parasite. After contact, a septum was formed in the germ tube of p.
parasiticum (105). The septal pore was eventually plugged, leaving
mitochondria, endoplasmic reticula, and vesicles contained in the contact
cell. A pore was dissolved in the host wall and cytoplasmic exchange
occurred between the two organisms. No movement of organelles was
observed through the septum of the contact cell. Very little effect on
the host was Observed, although dry weights Of the host cultures were
lower when parasitized (29).

Gonatobotryum fuscum, another biotrOphic, contact, balanced

hyperparasite, was found to parasitize Ceratocystis Spp. and related

 

Deuteromycetes (186). Ceratocystis iS important as the causal agent of

 

20

several tree wilts and one species, p, fimbriata, causes sweet potato
black rot. Graphium is one of the imperfect forms of Ceratocystis and

was used frequently as a host for p. fuscum (27, 186). Gonatobotryum

 

prgppp_developed contact cells on host hyphae (186). Mycotrophein was
necessary for axenic culture of the hyperparasite. A medium containing
glucose, yeast extract, thiamine, and biotin plus mycotrophein favored
good growth (48). Darkness enhanced development of the parasite on its
hosts (186), the inhibitory effects of light being related to the
destruction of vitamin 85 (27). Vitamin 85 production by the
hyperparasite apparently stimulates the host to produce biotin needed for
growth of p. M.

The destruction of Ceratocystis fagacearum and Q. fimbriata in agar

 

culture by Gliocladium roseum has been studied (30, 185). Coiling around
host cells was common and penetration, if it occurred, was after the
death of host cells. Biological control of oak wilt using this hyper-
parasite was attempted by Shigo (30). Sixty-two infected red, black and
scarlet oak trees were inoculated with p, Eppgpm. The hyperparasite was
reisolated from 38 of the trees 2 months later. When samples were taken
further from the inoculation point, there was no indication that p.

roseum had spread within the trees. Ceratocystis fagacearum was

 

recovered from all of the hyperparasite-inoculated trees, demonstrating

the failure of g. roseum to inhibit Q. fagacearum 1p vivo.

 

Necrotrophic hyperparasitism of C. fimbriata and Q. fagacearum by 15
wood-rotting basidiomycetes was reported to occur in culture (98).
Contact by the parasites resulted in the death of spores or mycelium of
the hosts. A role for hyperparasitism in microbial succession in stumps

or logs was speculated. The necrotrOphic hyperparasitism of p. fimbriata

21

by Hirschioporus pargamenus, the causal agent of white rot of sapwood of
several tree species was noted (195). Contact was not essential for this

antagonistic interaction.

Parasitism of organisms causing root diseases

Chytrids. Black wart of potato, caused by Synchytrium endobioticum,
is worldwide in distribution, but most severe in Europe. Kohler, in 1924,
reported the hyperparasite, Phlyctochytrium synchytrii, as a parasite of

the resting sporangia of.§. endobioticum (189). .3. synchytrii produced

 

sporangia and resting spores epibiotically. The endobiotic portion was
described as irregularly expanded or subspherical haustoria, flattened
against the host wall. More research in this area is necessary before the
prospects of A. synchytrii as a biological control agent can be assessed.
Oomycetesgparasitized by slime molds. Two endoparasitic slime molds,

Sorodiscus cokeri and Woronina pythii, have been Shown to infect Pythium

 

spp. Both are obligate parasites on species of Pythium (94, 95).
Sorodiscus cokeri parasitized A, irregglare (94) which is sometimes

associated with pea, lupine, or cucumber roots. Woronina pythii infected

 

the plant pathogens 3. aphanidermatum, A. debaryanum, A. irregulare, and

 

 

 

.3. ultimum, causing galls delimited by septa in the host cells (95). Both
hyperparasites have similar life cycles. Zoospores encysted on host
hyphae and then produced Short penetration tubes through which the
contents of the spores emptied into the host cell. Plasmodia eventually
appeared, one or more per gall. After the plasmodia increased in Size,
zoospores were delimited and bulbous exit tubes released the zoospores to

the outside. Woronina pythii is distinguished from S. cokeri by the

 

formation of galls in basipetal succession and by the types of cystosorus.

22

Not enough is known about these slime molds to speculate on their use in
biological control.

Oomycetes and their chytrid parasites. The aquatic nature of both
the chytrids and the Oomycetes has undoubtedly led to the coexistence of
the parasitic chytrids with the lower fungi. Many chytrids have been
described as parasites of oomycetous hyphae. Reports have included
parasites of saprOphytic as well as plant parasitic oomycetes. It is not
known if any of the mycoparasitic chytrids are pathogenic to the plant
pathogenic oomycetes. The chytrids parasitic on Oomycete hyphae are
listed here (189).

App; Parasite

Aphanomyces spp. Chytriomyces parasiticus
Olpidiopsis aphanomycis
gyluxurians
Phlyctidium mycetophagum

Pythium spp. Lagenidium pythii
OTpidiopsiS brevispinosa
.9: curvispinosa

. O. gracile
_O_'. pythii
Pythiella vernalis
Rhizophydium pythii
Rozella cuculus
'3. irregularis
A, laevis
Rozellopsis inflata
Solutoparies pythii

 

 

 

 

 

 

 

Phytophthora Spp. Rozella barretti (also sporangia)
RozellOpSis waterhouseii (also sporangia)

Olpidigpsis aphanomycis was observed as a hyphal parasite of
Aphanomyces cladogamus (218), a relatively unfamiliar plant pathogen

cited as causing tomato rootlet injury. Olpidiopsis aphanomycis, an

 

endobiotic hyperparasite, produced zoosporangia and resting spores in

swellings of the host hyphae.

23

Pythium gracile, the cause of ginger rot, was a host for Rozella

 

laevis (129). The hyperparasite caused marked hypertrOphy of the host
hyphae, and either filled or partially filled the swollen portion with

its Sporangium or resting spore. Pythium intermedium causes a

 

damping-off of fern and was susceptible to attack by four chytrids:

Olpidiopsis gracile, p, pythii, Rozella cuculus, and Rozellopsis inflata

 

 

 

(47, 129, 189). Olpidiopsis gracile and Q, pyppii infected the host
hyphae, and A. cuculus and A. inflata infected the sporangia. All were
endobiotic parasites inducing hypertrophy. Hyphae of Pyphium ygéppg, a
weak parasite which will sometimes cause seed decay and seedling disease

in corn may become infected by p. pythii and Rozella irregularis, causing

 

local swellings. Rozella irregularis may induce septation and abnormal

 

branching (47).

Rozella barretti was reported as a parasite of Phytophthora cactorum

 

(129), the causal agent of collar rot of apples. The hyperparasite
caused local hypertrOphy of the hyphae and, in addition, infected the

sporangia. Rozella barretti sporangia were not detectable in the host

 

sporangia until zoospores formed. Rozellopsis waterhouseii invaded the

 

sporangia of the root rotters E. cryptogea and A. megasperma, and
occasionally caused hypertrophy (129).

Rhizophidium apophysatus and A. carpgphilum were found to be

 

epibiotic parasites of oogonia and oospores of nonpathogenic Oomycetes
(130, 189). Sparrow (189) commented that species of Olpidiopsis and
Rozella occur endobiotically in the hyphae of water molds, while

Rhizidiomyces and Rhizophidium occur epibiotically on oogonia and

 

oospores.

Sneh et al. (188) isolated of wide range of hyperparasites Of

24

oospores of Phytophthora megasperma var. soj e (#3, megasperma f. Sp.

 

glycinea) from soil. These included three different chytrids:
Canteriomyces stigeoclonii, Hyphochytrium catenoides, and
Rhizidiomycopsis japonicus. Both 9. stigeoclonii and A. catenoides were
found to be endobiotic hyperparasites. The thalli of Q. stigeoclonii
released zoospores through a discharge tube in the presence of free

water. The thalli of A. catenoides develOped into sporangia, and

 

released zoospores through a discharge tube that terminated in a vesicle.
.3: japonicus, an epibiotic parasite, was observed previously parasitizing
oogonia of Aplanes Sp. (189). Sporangia of the hyperparasite developed
on the oospore while a rhizoidal system develOped within. These three

hyperparasitic chytrids were able to infect oospores of Aphanomyces

 

euteiches, Pythium sp., and Phytophthora cactorum, in addition to E.
megasperma var. gpjpp, In a survey of soil samples from 33 soybean
fields, A, catenoides was identified most frequently, followed by Q.
stigeoclonii and A. japonicus. when soils were flooded (118).

Soil moisture influenced the kinds of oospore hyperparasites
obtained (188). Chytrids were found only in flooded soils, while
hyphomycetes dominated when the soils were drier. To determine if a
relationship existed between hyperparasitism and disease potential, soil
was collected from a site in an apple orchard where trees were infected
with A. cactorum, and also from a site with healthy trees. Soil samples

were baited with oospores of A. euteiches, Pythium sp., 3. cactorum, and

 

‘3. megasperma var. sojae. After 1 or 3 weeks, significantly more
oospores were parasitized by chytrids in the soil collected from the
healthy site. In a separate study by Humble and Lockwood (118), chytrid

parasitism of oospores incubated in different soils could not be

25

correlated with A. megasperma var. sojae root rot potentials.

 

The oospore hyperparasite, A. catenoides, has received more
attention than any other chytrid as a potential biological control agent.

It was identified as a parasite of the oospores of Aphanomyces, Pythium,

 

and Phytophthora species (15, 188). The infection process began when
zoospores of A. catenoides encysted on the host cell. An infection peg
penetrated the oogonial and oospore walls

and entered the oospore cytOplasm. Sporangia of A. catenoides eventually

 

developed within the oospore (15, 188). Greatest zoospore production and
motility occurred in a pH range of 6.0 to 7.0, and at temperatures fron

20-30 C (15). Growth rate of A. catenoides in agar culture increased

 

with increasing temperatures, 16 C being the minimum and 28 C the maximum
(118). Invasion of A. myriotylum oospores was optimum between 20 and 30
C after Six days (15). Parasitism of A. megasperma var. gpjpg oospores
by A, catenoides in flooded Steamed soil increased with temperature to a
maximum of 85% at 28 C after 10 days (Humble & Lockwood, in press). When

tested in distilled water, oospores of A. aphanidermatum. A, ultimum, and

 

.fi- euteiches, and A. myriotylum were infected (Ayers & Lumsden, 1977).

 

Infection percentages of A. aphanidermatum and A, ultimum oospores were

 

21-26%, and for A, euteiches and_£. mygiotylum 65%, after 2 weeks.

 

OOSpore infection in soil by A. catenoides was slower, but after 60 days,

 

77% of the oospores Of E. myriotylum were infected compared to 13% for A.
aphanidermatum and 11% for A. ultimum. An isolate of A. megasperma var.
ppjpg race 7 was more resistant than isolates of races 1 and 3 to attack
by A, catenoides (118). Hyphochytrium catenoides was detected in five of
15 tested agricultural soils. This chytrid is being evaluated as a

biological control agent for Phytophthora megasperma var. sojae (Hsu &

 

26

Lockwood, unpublished results).

Oomycetous hyperparasites of other Oomycetes. Members of the Oomy-
cetes may, on occasion, act as mchparasites. Aphanomyces exoparasiticus
parasitized many Phycomycetes tested, including members of the
Mucoraceae, Peronosporaceae and Saprolegniaceae, but did not infect
selected members of the Ascomycetes and Basidiomycetes (60). The
parasite coiled around the host hyphae and developed superficial
rhizoidal attachments. Cross-walls were developed by the host where
infection occurred. Another Oomycete, Pythiella vernalis, developed
galls containing 1-4 Sporangia in the hyphae of Pythium gracile and 3,
di ctyosporum (61) .

Pythium spp. may be hyperparasites Of other Pythium spp. Hyphae of

Pythium debaryanum, A. irregulare, A. myriotylum, and A. ultimum were

 

susceptible to attack by A. acanthicum, A. oligandrum, A. periplocum

 

(75). Invasion of A. myriotylum hyphae was initiated by appressoria.
The parasite either penetrated and formed haustorial hyphae which were
delimited by host septa, or the host responded by increasing the
thickness of the outer and inner walls. In the latter case, the hyphae
either remained healthy or the cytOplasm degenerated.

Biological control of damping-off of sugar beet was achieved by one
of the aforementioned Pythium antagonists, A. oligandrum (210). Beet
seed emergence and plant weights were increased in pot trials by
treatment of beet seed with a suspension of 4 x 106 oospores of A.
oligandrum/ml. The biocontrol treatment was superior to a chemical
seed-dressing with Hermal (70% thiram).

An unidentified Pythium sp. was observed parasitizing oospores of A.

megasperma var. sojae in studies performed by Sneh et al. (188). This

27

organism was subsequently identified as‘A. monospermum (118). In steamed

 

soil, A. monospermum parasitized more than 70% of A. megasperma var.

 

 

sojae oospores. A, monospermum was an effective parasite over the range
of 16-28 C, but maximum parasitism occurred at 24 C. In agar culture,

linear growth of A. monospermum increased as temperature increased up to

 

28 C. Oospores of races 1 and 3 of A. megasperma var. sojae were more

 

susceptible to attack by A. monospermum than those of race 7. The basis

 

for the difference in susceptibility was not determined.

Leptolegnia sp. is another oomycete that was found infecting
oospores of A. megasperma var. gpjpp (188). Hyphae of this hyperparasite
emerged from the infected oospore to form Sporangia and zoospores.

Pythium acanthicum has a microbial host range that is too numerous
to list (102, 109). The hyperparasite hyphae were attracted to the host.
When A. aphanidermatum was parasitized, the hyperparasite hyphae coiled
around the host causing cytoplasmic changes, but no penetration occurred.
The hyphae at the contact zones appeared dead.

Phycomyces blakesleeanus, a saprOphyte, reacted to A. acanthicum

 

contact by producing very large papillae with a fibrillo-granular matrix,
which coalesced in response to multiple attacks (109). The host plasma
membrane separated the papillae from the cytoplasm. The papillae
increased in size until the hyperparasite penetrated the papillae and the
plasma membrane, at which time the rate of develOpment of the papillae
decreased, By the time penetration of cytOplasm occurred, the host cells
appeared dead. In coenocytic hosts, septa-like structures Slowed
hyperparasitic advancement. In Basidiomycete hosts, the cytOplasm

appeared dead a few minutes after papilla formation. Sclerotinia

 

sclerotiorum and Sclerotium rolfsii caused A, acanthicum hyphae to lyse

 

 

28

before contact was made. Deacon (64) also observed lysis of A.

oligandrum by some basidiomycetes.

 

Preliminary evidence indicates that hyperparasitic Pythium Spp.
merit further investigation as biological control agents. Caution will
be needed to find genetically stable isolates that will not pose the

threat of eventual develOpment of phytopathogenicity.

Fungi Imperfecti as hyperparasites of Oomycetes
Other hyperparasites of Pythium oospores include several Deuteromy-
cetes. Drechsler reported six species of Dactylella as parasites of

Pythium oospores: .p. anisomeres, p. helminthodes, Q. passalgpaga, p.

 

stenocrepis, Q, stenomeces, and Q, spermatqphaga (73, 76, 77, 78).
Parasitism by all species was accomplished in essentially the same
manner. Appressoria of the hyperparasite developed when contact was made
with oogonia or oospores. Penetration pegs then penetrated the oogonial
wall. Occasionally, a second appressorium formed on the oospore wall
preceded penetration of this structure. Eventually, after the oospore
cytoplasm was absorbed, the assimilatory hyphae became visible.

Dactylella helminthodes infected A. debaryanum oospores (76). Q,

 

 

stenocrepis parasitized A, butleri. Dactylella anisomeres was found to

parasitize A. butleri and_E. debaryanum (77). Pythium debaryanum, A.

 

 

irregulare, and A, ultimum were hosts for Q. stenomeces (78) and D,

passalOpaga (76). Dactylella spermatOphaga was destructive to A.

 

acanthicum, A. arrhenomanes, A, butleri, A. debaryanum, A. graminicola,

 

A, myriotylum, A. oligandrum, A. paroecandrum, A. periplocum, A. ultimum,

 

 

 

 

‘A. vexans, and many other saprOphytic Pythium spp. (73). In addition, Q.

spermatophaga parasitized oospores of Aphanomyces euteiches, PhytOphthora

29

cactorum and A. megasperma, and chlamydospores of A. cinnamomi.

 

Drechsler suggested that the role of Q. spermatophaga would be minimal in

 

controlling the pythiaceous fungi in nature because of the slow growth
rate of this hyperparasite (73). However, in the same paper he also
suggested that by manipulation, Q, spermatophaga had great potential for
attainment of "soil sanitation“. Drechsler also described two species Of

Trichothecium as oospore parasites (75, 76). I, arrhenopum invaded A.

 

ggaminicola oospores (74), and I. polyctonum was destructive to oospores
of the saprophyte A. spinosum (76). In addition, A. butleri oospores
were parasitized by Trinacrium subtile (73). Parasitism occurred
essentially as described for Dactylella. Hoch and Abawi (107) isolated

Fusarium merismoides from oospores of A. ultimum. Thirty to 74% of the

 

oospores of A. ultimum were parasitized by this hyperparasite over an 8
week period in sterile or natural soils.

Sneh et al. (188) identified Alternaria alternata, Cephalosporium
Sp., Diheterospora chlamydosporia, Fusarium oxysporum, and Humicola

fuscoatra as parasites of E. megasperma var. sojae oospores. These

 

Hyphomycetes were found to be the dominant hyperparasites of oospores of

.3. cactorum (188) and A, megasperma var. sojae (118, 188) when soil

 

moistures were held below saturation.
Biological control of A. ultimum was obtained when cotton seed was

treated in the furrow with Gliocladium virens (113). Seedling emergence

 

and survival increased significantly with the treatment. In addition to
acting as a hyperparasite, one of two antibiotics isolated fran culture
filtrates of p. yiggpp was inhibitory to E. ultimum. Mutants deficient
for antibiotic production did not control damping-off, while those with

enhanced antibiotic production were superior to the parent strain. The

30

The mode of parasitism was not elucidated. Gliocladium has been

 

associated with a complex of fungi causing seedling blights; therefore,‘
it would be advisable to test a wide range of higher plants as potential
hosts prior to field application.

Trichoderma spp. may be promising as biological control agents for

Pythium diseases. A wide range of species and isolates of Trichoderma

 

were assessed for their coiling response to potential host fungi (70).
In each species the majority of isolates would coil around hyphae of A.
ultimum inducing vacuolation and coagulation of cytoplasm. Occasionally,
hyphae of'f, ultimum were penetrated by isolates of I. harzianum and I.
yigipg. Two isolates of I. ‘yipipg also on occasion penetrated hyphae of
Phytophthora cactorum and A. erythroseptica. Growth of Epmgg annosus,
Fusarium oxysporum, and Rhizoctonia solani halted after contact with the
hyperparasite, regardless of whether antibiotics were being produced.
Reisolation of host fungi was usually not possible after contact with the
hyperparasites.

A non-antibiotic producting isolate of I. hamatum in a methyl
cellulose Slurry was effective in controlling root rot of peas planted in

a soil naturally-infested with E. aphanidermatum, A. oligandrum, and A,

 

ultimum (100). A similar finding was reported by Chet and Baker (55).
Better protection was obtained when pea seed was treated with a

concentration of Trichoderma conidia greater than 105/ml than at lower

 

concentrations, and when soil temperatures were between 17 and 34 C.
Trichoderma hamatum seed treatment amended with powdered, unbleached

chitin decreased disease incidence (101). Other amendments of peat or

Rhizoctonia solani cell walls increased the pOpulation Of I. hamatum, but

did not reduce Pythium seed rot. Rhizobium inoculum added to the I.

31

hamatum pea seed treatment did not interfere with control. Trichoderma
hamatum controlled pre-emergence damping off as well as chemical seed
treatments, but did not protect pea as well against post-emergence
damping off (100). In a replanting with untreated pea seed into the same
pots, disease control by I, hamatum was comparable to that by chemicals.
In a second replanting with untreated pea seed, plants in soils
originally treated with I, hamatum or fenaminosulf (Dexon) showed less
disease than captan-treated or untreated soils. Chaetomium globosum has
also been applied to seed as a potential biocontrol agent both alone and
in combination with I, hamatum (100). Although some control was
achieved, 9. globosum was less effective than I. hamatum, and it did not
augment control by I, hamatum.

Another Trichoderma sp., 1, harzianum, did not control pea seed rot

as a seed treatment (100), or pineapple root rot caused by Phytophthora

 

cinnamoni when applied to soil as a granule preparation (132).

Hyperparasitism of some of the lower fungi by Rhizoctonia.

 

Rhizoctonia solani and other Rhizoctonia spp. pathogenic to strawberry

 

 

roots were also hyperparasites of the Mucorales, Peronosporales and

 

Ambylosporium botrytis, a Deuteromycete (45). Included as hosts were

Mucor mucedo, Pythium butleri, A. debaryanum, A. irregular, E, vexans,

 

 

Phytophthora cinnamomi and (+) and (-) strains of Rhizopus nigricans (45,

 

 

46). On the basis of pathogenicity to E. debaryanum and A, nigricans,
Butler (45) recognized four physiological races of 3°.291201-

Penetration of the host occurred with or without coiling (45). When
parasitism occurred without coiling, a few infection hyphae penetrated
the host cell and develOped a hyphal system within the host cell.

Penetration with coiling took place at frequent intervals with many

32

infection pegs. An extensive mycelium ramified throughout the infected
area. Although the parasitic hyphae replaced the host protOplasm in
infected cells, noninfected portions survived. When A. gplppi parasi-
tized Syncephalastrum sp., young hyphae were the most susceptible (45).

Resistance of the host was expressed by walling off infection hyphae
or by inducing lysis of the A. gplppi hyphae (45). Barrel- shaped cells
and sclerotia were produced in high numbers in parasitized cultures
(46). There was no relationship between myCOparaSitic abilities and
plant pathogenicity (45).

Rhizoctonia solani will probably never intentionally be used as a
biological control agent because it is a plant parasite. The study of
the host-hyperparasite relationship of the lower fungi with‘A..§plppi is
interesting because this ability may be of survival value for Rhizoctonia
Spp.

Ascomycetes and Fungi Imperfecti. Gliocladium gpggpm, already

discussed as a parasite of Ceratocystis, was also a hyperparasite of

 

hyphae and sclerotia of Botrytis allii, an organism commonly associated

with neck rot of onion (211). Gliocladium roseum exhibited tropic growth

 

towards B. 31111 hyphae, appressoria were formed on host hyphae, and
haustoria-like structures appeared within the B. 31111 hyphae. Further
development of the absorptive hyphae took place intracellularly. 0f 40
naturally occurring 8. 31111 sclerotia plated on agar, 8 were infected
with p. gpggpm. In a controlled environment of 21 C and 100% relative
humidity, the rate of.B.‘pllii_infection of both wounded and healthy
onion bulbs decreased when p. gpggpm was placed in close contact with_§.
.pllii inoculum.

A wide range of economically important diseases are caused by

33

Fusarium spp. including wilts, and root and Stem rots. Gliocladium
catenulatum was tested as a hyperparasite of f. eguiseti, E. oxysporum,
.5. poae, and E. sporotrichioides (114). Macroconidia and hyphae of all

species were susceptible to p. catenulatum, but chlamydospores did not

 

show signs of infection. Host cells were killed on contact. ,p.

catenulatum formed pseudoappressoria which produced hyphae that did not

 

penetrate host cells. Sclerotinia sclerotiorum was also susceptible to

 

this hyperparasite.
Stephanoma phaeospora was found associated with a Fusarium sp. in
nature (173). Other hosts were Ceratocystis fimbriata, responsible for

sweet potato black rot, Septoria apii, causal agent of late blight of

 

celery, Thielaviopsis basicola, cause of black root rot, and the smuts,

Ustilago avena and p. maydis. Stephanoma phaeospora, a contact,

 

biotrophic hyperparasite, caused no visible damage to its hosts. ‘Aggpig
culture of g. phaeospora has been possible with the addition of a growth
factor from specific fungi, a condition much like that previously
mentioned for the hyperparasite Gonatobotrys simple . The contact cells
of S. phaeospora are of two shapes - hooked, or elongated and flattened.
No attraction of the parasite hyphae towards the host was observed.
Trichoderma spp. have been reported to be antagonistic to Fusarium.

Chi (57) noted hyphal coiling of Trichoderma and its hosts around each

 

other, and cytoplasmic changes in the hyphae of E. oxysporum, E. roseum,

and E. solani infected by two Species of Trichoderma. Pots of sterilized

 

soil infested with E. culmorum and I, lignorum contained fewer diseased
barley plants than pots with E. culmorum alone (36).

Pythium acanthicum, A. oligandrum and other mycoparasites were

 

easily isolated by placing small pieces of soil organic matter on agar

34

plates precolonized by Phialophora radicicola var. radicicola (65, 66).

 

Pythium acanthicum and E. oligandrum were recognized by spiny oogonia
produced on these plates. The interaction of A. oligandrum and
susceptible host fungi was evaluated 1p yipgp using amount of suppression
of filter paper cellulolysis as a measurement of parasitism, A.
radicicola var. radicicola was classified as highly susceptible, Fusarium
lgygypp f. Sp. cerealis, Gaeumannomyces graminis var. tritici, and A,
radicicola var. graminicola were moderately susceptible, A, graminis var.
graminis was resistant, and several basidiomycetes were antagonistic to
‘A. oligandrum (64). Susceptibility of host hyphae was greatest when
young, declined as hyphae matured, and then increased as hyphae aged.

The narrow, hyaline hyphae of Phialophora and Gaeumannomyces were more
susceptible to lysis by the parasite than the broad, darkly pigmented
hyphae. Deacon (64) suggested that melanization might be related to
resistance. Hyphal coiling by the parasite was observed only on
resistant hyphae. .E- acanthicum, like A. oligandrum, parasitized p.
graminis var. tritici, A, radicicola var. graminicola, and A, radicola

var. radicola. All A. acanthicum isolates but one, a plant pathogen,

 

caused a reduction in filter paper cellulolysis by host fungi (66). It
was suggested that Pythium isolates that are highly phytOpathogenic may
be only Slightly mycoparasitic and vice versa. This relationship was not

shown for mycoparasitic Rhizoctonia isolates (45).

 

Another hyperparasite of p. graminis was identified as the
Ascomycete, Didymella exitialis (187). Disease was reduced by 50% in
the greenhouse under sterile conditions when wheat seedlings were
co-inoculated with both host and hyperparasite. The hyphae of p.

graminis were penetrated and destroyed by p. exitialis in the rhizophere.

35

Combinations of free amino acids from culture filtrates of.g. exitialis
reduced pathogenicity of A. graminis by 40%, while single amino acids
caused a 20-30% reduction.

An isolate of Macrophomina phaseolina, causal agent of damping-off
and charcoal rot of slash pine, was found to be parasitized by an
unidentified Basidiomycete (69). The hyperparasite either directly
penetrated the host cell and colonized it, or first formed an
appressorimn from which a penetration peg developed. In the laboratory,
aseptically grown slash pine seedlings inoculated with agar disks of A.
phaseolina plus the hyperparasite Showed no disease symptoms, whereas A.
phaseolina alone caused disease. In a field experiment, equal amounts of
inoculum of the pathogen or parasite produced on vermiculite supplemented
with potato-dextrose broth, were spread over nursery plots. The

treatments included A. phaseolina, A, phaseolina + hyperparasite, and the

 

hyperparasite. Pine seeds germinated equally well in all treatments, but
seedling survival was increased 27% with the addition of the hyperpara-
site. Hyperparasitism by this organism also occurred on Cylindrocladium
floridanum, A. ultimum, A, cinnamomi, and A, pplppi.

Sclerotia. Fungi of the genus Sclerotinia cause root and stem

 

diseases, and are destructive to many crops. Mycelium infects suscepti-
ble tissue, and as it spreads, sclerotia are formed in cavities or on the
surface of infected plant parts. Sclerotia (and/or mycelium on plant
tissue) overwinter to produce apothecia in the spring. Many studies have
been done on the survival of sclerotia in soil (58). Sclerotia of §.

sclerotiorum were buried in soil or left on the soil surface and

 

retrieved at different times of the year to study the sclerotial

microflora (172). Organisms associated with the sclerotia varied with

36

the time of year. Buried sclerotia were colonized less than those at the
soil surface. Of those organisms isolated, Cunninghamella echinulata,

Fusarium ggysporum, Rhizopus orrhizus, and Trichoderma lignorum caused a

 

20-35% decrease in sclerotia germination in vivo. The parasitic nature
of these organisms was not established, but antibiosis was not detected

in culture. Penicillium citrinum, A. funiculosum, A. pallidum, and A.

 

steckii were aggressive sclerotia parasites. Aspergillus niger and A,

 

ustus were also parasitic, but less so than the Penicillium spp. Both
Aspergillus- and Penicillium-colonized sclerotia did not produce
apothecia in the field.

Species of Chaetomium, Codinaea, Fusarium, Gliocladium, Paecilo-

 

 

pypg_, Talaromyces, and Trichoderma isolated from surface-sterilized
sclerotia were associated with a decline in populations of sclerotia of
“S. pippg in four agricultural soils (89). In preliminary tests,
Chaetomium fusiforme and A, elegans decreased disease of lettuce caused
by §, EEDQE.I" the greenhouse.

Gliocladium virens parasitized sclerotia and mycelium of §. 351352-
‘pipppp (202). Appressorium—like structures were formed on the host. By
the use of transmission electron microscopy, the presence of A. .Aiggpg
inside the sclerotial cells was demonstrated. The hyperparasite
prevented the formation of sclerotia in culture, and those sclerotia that
were parasitized did not germinate. ‘g..11£pp§ also parasitized
Rhizoctonia solani. -

Trichoderma viride was isolated from sclerotia of §. sclerotiorum

 

(127). Four single spore isolations were made, of which one decayed the
majority of sclerotia on moist sand or in buried soil, while the other

three decayed only a few sclerotia on sand and none on soil. In a

37

culture medium, however, all four isolates parasitized S. sclerotiorum

 

hyphae and sclerotia. These results indicate the importance of isolate
selection.

Coniothyrium minitans, another hyperparasite of Sclerotinia spp.,

 

 

directly penetrated the host cell wall of A. sclerotiorum and caused

 

cytoplasmic disintegration of hyphae and sclerotia (115). In hyphal
infections, cells adjoining the infected area appeared vacuolated and

unhealthy. In response to infection, new hyphae of §. sclerotiorum were

 

produced within the infected areas but showed only limited growth.
Coniothyrium minitans produced pycnidia on infected sclerotia (204).
Sclerotia of s. cepivorum, A. pippp, S. trifoliorum and some isolates of
Botrytis cinerea were also susceptible to Q. minitans (204). Apothecia
were also parasitized. The first signs of infection occurred as brown
Spots which expanded and turned black. The apothecium remained
functional for some time before being destroyed (196). The optimum
temperature for sclerotial infection was 20 C, but parasitism occurred as
low as 10 C. Isolates of A. sclerotiorum demonstrated great variability
in susceptibility (204). Sclerotia were more susceptible to infection by
.Q. minitans when produced in agar culture than on autoclaved carrots
(204), or on brussel sprouts than on sterile oat seeds (50).

In an attempt to reduce numbers of sclerotia of A. sclerotiorum,
pycnidiospore preparations Of_g. minitans were placed in direct contact
with sclerotia in sterile sand or were applied to sterile sand containing
sclerotia. Apothecial formation was inhibited by both treatments (50).
In other work, a cornmeal-sand culture of p. minitans was mixed into a
loamy sand or a heavy clay field soil in which sclerotia of A.

trifoliorum were buried (196). Within 11 weeks, 85-99% of the sclerotia

38

were killed. Coniothyrium minitans was not pathogenic to several species

 

of plants (204). .Q. minitans appears to be very promising as a
biological control agent.

Sporidesmium sclerotivorum was recently described as a sclerotial
parasite of g. sclerotiorum (205). Glucose or mannitol as a carbon
source, casamino acids or glutamine as a nitrogen source, thiamine, and
an initial pH of 5.0-5.5 provided a suitable nutritional environment for

axenic culture of the hyperparasite (31). Sporidesmium sclerotivorum

 

infected §. cepivorum (12),_§. minor (13) and Sclerotium sclerotivorum,

in addition to S. sclerotiorum (12). The hyperparasite grew through

 

moist sand from one §. sclerotivorum sclerotium substrate to another
(12). Conidia as far as 9 mm from a sclerotium of.§.lpippg in moist
natural soil were stimulated to germinate (13). In 10 weeks or less, 95%
of the sclerotia were destroyed by the hyperparasite in sand or soil
(Steamed or natural) infested with sclerotia at a rate of 1% (w/w) (12).
Maximum infection occurred at 20-25 C, pH 5.5-7.5, and a soil moisture of
-8 bars matric potential or higher (12). Concentrations as low as 2 X
103 A, sclerotivorum Spores per 100 g of soil caused infection and

decay of host sclerotia.

Naturally occurring §. sclerotivorum caused a decline of §. minor

 

populations in a field soil (3). Populations of sclerotia were 90% in
June and 30% in September, of that in March. The hyperparasite was
observed on 13-22% of the sclerotia recovered for each assay from May to
September. In a field test where the hyperparasite was introduced at a
rate of 100 Spores/g soil, a similar decline was observed. Selected
naturally infested onion fields in New Jersey demonstrated an association

between naturally occurring A. sclerotivorum and decreased populations of

 

39

§, cepivorum. In five potato fields in Oregon, infection of introduced

sclerotia of S. sclerotivorum by A. sclerotiovorum was low in four fields

 

which had a moderate to high incidence of stem rot, while it was low in
the fifth field where little stem rot had occurred. Commercial
production of the hyperparasite may be the next step for use of §.
sclerotivorum as a biological control agent.

Sclerotium spp. infects a wide range of crOps as seedlings or mature
plants, and can also cause severe losses during the transport of fruits
and vegetables. It is a problem primarily in warm climates, and in the

United States diseases caused by Sclerotium are prominent in the South.

 

Control of Sclerotium is difficult, and an effective biological control
agent would help in reducing losses caused by this pathogen. Two genera
of hyperparasites that have been studied in terms of biological control

are Coniothyrium and Trichoderma.

 

Coniothyrium minitans forms its own pycnidia within the infected
sclerotia of A. cepivorum (92). Control of white rot of onion by p.
minitans was studied in the greenhouse (4). The hyperparasite was
prepared as a pycnidial dust and used in soil treatments at rates of 50
or 100 times that of §. cepivorum sclerotia, or as a seed dressing at
one-third the rate of sclerotial inoculum. .p. minitans gave disease
control equal to that of the fungicide Calomel, and the biological
control was recommended for further evaluation.

Trichoderma Spp. have been effective both in field and greenhouse in

reducing diseases caused by Sclerotium spp. Biological control of §.

 

rolfsii on beans was achieved in the greenhouse using a wheat bran
culture of I. hazianum (56, 82). I, harzianum decreased disease

approximately 40-60% over a temperature range of 22-34 C (82). Control

40

in the greenhouse was enhanced when I. harzianum was applied in
combination with PCNB (56), or when soil was pretreated with solar heat
at 47 or 50 C (83). A wheat bran preparation of I. harzianum also
reduced bean disease caused by s. rolfsii about 15% and increased green
pod yield by 20%, 48 days after planting in a field test (82).

Trichoderma hamatum also has been evaluated for control of §,
rolfsii on beans (55). Although no antagonism was observed between 1.
hamatum and §. rolfsii in agar culture, disease incidence in the
greenhouse was decreased by about 40% after 14 days when I, hamatum was
added to the soil at 106 propagules per g. A second planting into the
treated soil 14 days after the first provided 15% greater disease control
than was achieved in the first planting, after 14 days.

A.I: harzianum isolate capable of parasitizing §. rolfsii,

Sclerotinia trifoliorum and Botrytis cinerea, but not Rhizoctonia solani,

 

 

was tested as a biological control agent in greenhouse and field tests
(216). Trichoderma harzianum grown in a rye seed-soil mixture gave 100%
control of §. rolfsii on tomato or peanuts and 91% control on lupine,
when equal weights of.§. rolfsii on the same material was used in a

greenhouse experiment (216). Trichoderma harzianum gave 70% or more

 

disease control in field tests in soil naturally infested with §. rolfsii
when applied when plants were 4 cm high, again 9 days later, and finally
7 days later. In another field experiment, plants from I, harzianum
plots were at least 88% disease-free, almost twice that of untreated
plots, when the hyperparasite was applied at the time tomatoes were cut
back to approximately 2 cm. Elad et al. (82) also achieved a 20%
reduction in A. rolfsii disease of tomatoes in the field using a

different I, harzianum isolate.

41

A hyperparasitic isolate of I. harzianum gave significant control of
.A. rolfsii in naturally-infested peanut fields using wheat bran inoculum
(56) or molassses-impregnated diatomaceous earth granules (18) on which
the fungus was cultured. I, harzianum applied as granules increased
peanut yield equal to that of PCNB treatment.

Two Species of Trichoderma were selected for field trials on the
basis of their ip 11259 abilities to degrade the sclerotia of an A.

rolfsii isolate responsible for Sclerotium blight of turf (171).

 

Trichoderma grown on molasses-impregnated diatomaceous earth was sprayed
at rates of 110 kg/ha and 140 kg/ha onto three diseased golf greens.
Populations of Trichoderma increased over time, but biological control
was inconsistent.

Other organisms also have been reported to degrade sclerotia.

Teratosperma oligocladum parasitized sclerotia of Sclerotinia minor, A.

 

sclerotiorum, A. trifoliorum, Sclerotium cepivorum and Botrytis cinerea

 

(14). In soil infested with I, oligocladium, sclerotia of Sclerotinia

 

Spp. were infected within 2 weeks, and sclerotia of §. minor were
destroyed by 10 weeks. Acrostalagmus roseus, Gliocladium roseum,
Gliocladium Sp., Fusarium Sp., 1, viride, and Verticillium sp. were able

to infect the sclerotia of Botrytis cinerea, Claviceps purpurea,

 

Sclerotinia borealis, §, sclerotiorum, and §, trifoliorum ifl vitro (150).

Resistance of the Sclerotinia spp. to the parasites was related to the

 

rate of regeneration of the sclerotial cortex.

Rhizoctonia. Rhizoctonia solani is world-wide in distribution and

 

causes seedling rot and root rot on a wide range of crOp plants. Many
hyperparasites have been observed on A. solani and attempts have been

made to utilize them in biological control of this pathogen.

42

Gliocladium roseum caused the hyphae of A. pplppi to collapse in
dual culture, presumably without penetration (170). Another isolate of
the hyperparasite did not control A. gplppi in field trials with
cucumbers (144). Another Species of Gliocladium, p. ‘yiggpg, parasitized
A, pplppi hyphae (203), in much the same manner as this hyperparasite
infected Sclerotinia sclerotiorum (203) (see previous section). Hyphae
of §-.!i£Efl§ were found within sclerotial cells of A. pplppi in thin
sections (203). In culture, sclerotial formation was deterred by p.
‘Aipgpg. Disease of white beans caused by A. 221221 was decreased
proportionately to the amount of hyperparasite inoculum added to
artificially infested soil in greenhouse tests.

Penicillium vermiculatum was investigated as a potential biological
control agent of-A.'§plppi by Boosalis (39). .A. vermiculatum parasitized
53.321211 hyphae by the develOpment of penetration pegs at contact sites.
The hyphae of the hyperparasite either ramified throughout the host or
was limited within an area. Of 30 isolates of A. pplppl tested for their
susceptibility to A. vermiculatum, all were susceptible, though in
different degrees. The amount of dextrose in the agar medium influenced
the severity of infection in mixed cultures. An insignificant amount of
parasitism occurred when 10 g dextrose was used, while 20 9 resulted in
severe infection.

Biological control experiments were performed by simultaneous
artificial infestation of sterile or non-sterile soil with A. gplppi and
A, vermiculatum. After 10 days of incubation, pea seeds were planted.
Control Of A. gplppi was obtained in sterilized soil system but not in

unsterile soil. Use of_A. vermiculatum as a biological control agent

 

does not appear promising.

43

Papulospora stoveri was isolated from A, pplppi—infected sugar beet
seedlings (212). .A. stoveri parasitized A. pplppl hyphae ipuyipgp by
rapidly coiling around the host hyphae causing the host cytoplasm to
retract and eventually disintegrate at contact points. Hyphae of the
hyperparasite were often observed growing within the host hyphae. The
same mode of parasitism was seen when the host-parasite interaction was
studied in soil using buried Slides. In biological control experiments,
corn meal-sand cultures of the host and hyperparasite were mixed with
soil. After 4, 24, and 55 days of incubation, sugar beet seed pieces
were planted. Control efficiency increased with incubation time, and was
dependent on the strain of A. gplppi.

Laetisaria arvalis [Corticium Sp. (42)] was investigated as a
control for A, éplppi (144, 164, 165). Plantings of_L. arvalis-treated
seed into nonsterilized soil infested with A. pplppi resulted in
increased stands of dry beans, soybeans, and sugar beets in greenhouse
and field tests (164, 165). A second planting gave continued protection,
although results varied depending on the A. gplppi isolate and soil type
(164). Population density of A, pplppi in soils with L. arvalis was
decreased by 28%, while that in soil treated with PCNB was decreased by
35%. The addition of sugar beet pulp to_L. arvalis inoculum gave better
protection of sugar beets than either treatment alone (165). The
mechanism of control of A, pplppi was not determined, but other A.
arvalis isolates were hyperparasites of A. gplppi (165).

Field tests using A. arvalis as a control for Rhizoctonia fruit rot
of cucumber gave variable results (144). Control appeared to be
dependent on the method of infestation. When A. arvalis was disked into

the soil, treatments were as effective as the fungicide captafol, but

44

about 20% less effective than the fungicide chlorothalonil 6F. If the
hyperparasite inoculum was plowed into the soil, control was approximate-
ly 20% less than in the case of disking in the inoculum. There may be
better biological control agents than A. arvalis for A. EQléfli-

Recently, much information has been published on the use of
Trichoderma Spp. for the biological control of plant diseases. There

have been more experiments done with Trichoderma than with any other

 

hyperparasite. There are several reasons for this. First, Trichoderma

 

is commonly found in nature. Second, unlike many of the hyperparasites
discussed, it is easy to grow axenically. Third and most important, it
is an effective hyperparasite. Weindling, in the 1930's, was the first
to describe the parasitic nature of Trichoderma and to test this
hyperparasite as a biological control agent for control of A. pplppi.
Antibiotic production by Trichoderma strains will not be discussed here.
There are hyperparasitic Trichoderma strains that control plant pathogens
that do not produce antibiotics effective against the test organism

(54, 55).

The mode of parasitism appears to be the same for different species
of Trichoderma (147, 213). Trichoderma responded to the host by coiling
around A, pplppi hyphae. The host protOplasm degenerated and hyphal
cells separated at the septa. .1. lignorum hyphae were observed to
flourish at points where host cell contents were released into the
environment (213). Penetration of A. gplppi by I, harzianum was not
Observed (147). However, Weindling (213) Observed internal parasitism by
‘1. lignorum of a strain of. . pplppi parasitic in potatoes. Differences
in host susceptibility to parasitism by I. lignorum have been found among

isolates of A. solani, Phytophthora parasitica, Pythium spp., RhiZOpus

45

Spp., and Sclerotium rolfsii. The extent of the host range of
Trichoderma has been dependent on the isolates of both hyperparasite
and host (55, 82, 99).

Soil conditions play a large role in determining the effectiveness
of biological control by Trichoderma, the fungus being most favored by
moist soils and high acidity (147). Hyperparasitism of A. gplppi by I.
harzianum was Significantly greater at -0.3 or -0.1 bars matric potential
than at -O.5 bars (97). Trichoderma harzianum was more effective in
suppressing disease caused by A, pplppi_and over a longer time period at
high than low soil moisture levels (147).

Biological control experiments with sour orange seedlings indicated
that acidification of soil with aluminum sulfate, in conjunction with the
natural presence of Trichoderma spp., provided better control of damping-

off caused by A. solani than either aluminum sulfate or Trichoderma alone

 

(215). As pH decreased, growth of I, harzianum increased, and disease
incidence decreased (54, 147). It has been suggested that pH affects
antibiotic production by Trichoderma strains which in turn affects their
parasitic ability (214). The influence of lower pH on parasitic ability

may, however, be due to growth stimulation of Trichoderma and not to

 

increased antibiotic production. Biological control of A. gplppi by an
isolate of I. harzianum that did not produce antibiotics in vitro was
increased by acidic conditions (54, 147).

Trichoderma harzianum has been used in greenhouse and/or field
tests to control A, pplppi diseases of alfalfa (54), bean (56, 83, 99),
carnation (84), cotton (62, 82), cucumber (147), eggplant (56, 99),
potatoes (83), radishes (54, 55, 103, 147), sugarbeets (54), and tomatoes

(99). Trichoderma hamatum has been used to control A. solani as a

46

pathogen of radishes (55, 100, 101).

In most of the biological control experiments cited, Trichoderma was

 

cultured on sterilized wheat bran or wheat bran-sawdpst mixtures. An
increase in concentration of Trichoderma inoculum resulted in decreased
disease. For example, Elad et al. (84) investigated rate and placement
of I, harzianum for control of Rhizoctonia root rot of carnation.
Significant disease reduction was achieved when I, harzianum was
broadcast in the field at 100 g/m2 or added to the rooting medium at‘
13.5 g/mz. The rooting medium treatment gave the best disease control,
and may be appealing to conmercial carnation growers because of the ease
with which this method may be used.

Rhizoctonia solani on beans (99) and on eggplant (56, 99) grown in
the greenhouse was controlled by I, harzianum inoculum. Disease
incidence of beans after a second planting in artificially infested soil
was greatly decreased when soil was infested with 1.7, 2.3, and 2.9 x
1010 I, harzianum Spores/kg soil prior to planting (99). Bean disease in
naturally infested soil was significantly reduced by 21% using I,
harzianum at a rate of 3 g preparation/kg soil (56), or in other work by
41% at 6 g/kg, but not at 3 g/kg (99). Disease on beans caused by A.
'gplppi was very low (only about 13% after 48 days) in a field test, but
disease onset was delayed and disease incidence was reduced by about 50%
in the I, harzianum treatment. Three different concentrations of I.
harzianum delayed development of A. gplppi on eggplant and significantly
reduced disease by approximately 15% after 3 days (99).

In a field experiment, Rhizoctonia damping-off of cotton was reduced

 

— approximately 50% by the use of the hyperparasite, I. harzianum (82).

PCNB at 2 and 10 g/g soil, when combined with I. harzianum, provided an

47

additive control effect, but the cotton herbicides, prometryne,
fluometuran, and trifluralin interfered with control by Trichoderma (62).

Trichoderma harzianum was investigated in field trials as a
biological control for potato diseases caused by A. gplppi and in addition
by Sclerotium rolfsii or Verticillium dahliae (83). In 1977, I, harzianum
was used alone at 400 kg inoculum/ha, or was applied in conjunction with
solar heating. Trichoderma harzianum caused a significant decrease in
disease symptoms in potato Shoots at 86 days, but no significant symptom
decrease in shoots after 54 days, or in potato tubers after 133 days.
Solar heating in combination with I. harzianum gave a Significant decrease
in disease, but the decrease cannot be attributed to I, harzianum Since
solar heating alone gave equal control. Trichoderma harzianum did not
decrease Verticillium wilt at 86 days, but solar heating alone or with I.
harzianum caused a decrease in this disease. There were no differences in
potato yields between any of the treatments.

In 1978, I. harzianum was used alone, or following solar heating or
methyl bromide treatment. There was no disease reduction on potato
tubers or beans planted in the greenhouse in soil from the field, using
I, harzianum alone. Methyl bromide or solar heating followed by I.
harzianum did not cause a decrease in A. gplppi in tubers or in beans.
However, the combination of methyl bromide with I. harzianum resulted in
a significant increase in bean yields . There were no differences in
incidence of §. rolfsii or A. dahliae diseases on potatoes that could be
related to control by I, harzianum. Yields of potatoes were not different
among the treatments. Results from these field experiments do not
indicate that I. harzianum is an adequate control agent for A. EQliflia.§-

rolfsii, or A. dahliae on potatoes. Possibly, A, solani isolates from

48

potatoes are more resistant to [. harzianum than other isolates.

Soils taken from the potato plots in a field experiment at 0, 90,
and 135 days after potato planting, were planted with beans three
consecutive times at 28 day intervals (83). Disease in soil taken from
I, harzianum, solar heating, or solar heating plus I. harzianum
treatments, was approximately 10-60% less than that in the control,
except in the sample collected at 135 days, in which disease incidence
was the same in the I. harzianum treatment and the control in all three
consecutive plantings.

Hyperparasitic Trichoderma Spp. may induce soil suppressiveness.
Suppressive soils can be defined as soils that resist the introduction
a plant pathogen; or in which establishment of a plant pathogen is
successful but no disease occurs; or in which the pathogen is
established, disease occurs, but decreases in intensity with continuous
crOpping (19). This characteristic may be either expressed as soil
conduciveness or soil suppressiveness (103). Soil conduciveness is
expressed by the equation CI = 1 --§, where CI = conducive index, A =
number of healthy plants in the noninoculated control, and X = number of
healthy plants in the inoculated control. The formula used to express
the suppressiveness index (SI) is SI = 1 --§ where X = number of diseased
plants in the inoculated treatment and A = number of healthy plants in
the uninoculated treatment. The SI value will range from 0 to 1. The
lower the SI, the greater number of diseased plants. Henis et al. (103)
found that suppressiveness of a soil to A. gplppi could be increased by
successive monoculture of radish plants. In studying control of radish
damping-off caused by A. pplppi, 8.2 X 109 I, harzianum conidia/kg

soil, pentachloronitrobenzene (PCNB) at 4 g/kg soil, OF.I- harzianum plus

49

PCNB at those concentrations were added to a loamy sand soil and
incubated for 1 week. Soils were seeded with radish five consecutive
times at 1 week intervals. All of the treatments resulted in a decrease
in disease with time, but the combination of I, harzianum and PCNB gave
the most effective control of radish damping-off. POpulationS of A.
gplppi followed the same trends as disease incidence.

In further work, soils suppressive to A. pplppi were developed by
successive replantings of radish and cucumber (147). Mycelial mats of A,
gplppi were buried in untreated soil, soil made suppressive by successive

weekly replanting of radishes, or soil planted with radishes in the same

 

manner but without A. solani. Trichoderma Spp. were retrieved from
almost all of the mycelial mats incubated in the suppressive soil, but
only from a small percentage from those incubated in the other two treat-

ments. Aspergillus Sp., 1, aureoviride, I, koningii, and two isolates of

 

I, harzianum were compared for their ability to induce suppressiveness in
a conducive soil. The Fort Collins isolate of I. harzianum was most
effective in inducing suppressiveness. Suppressiveness has also been
developed in low pH soils consecutively replanted to alfalfa or sugarbeet
(54). Natural populations of I. harzianum increased as suppressiveness
increased.

Trichoderma hamatum also has been under investigation as a
biological control agent (55, 100, 101). In agar culture, the
hyperparasite coiled around the hyphae of A. gplppi, and eventually
caused the lysis and separation of the host cells (55).

A pH preference for slightly acid soils was also indicated for I.
hamatum (55), as for I, harzianum (147). In a clay loam soil that was

artificially infested with I. hamatum and A. solani, damping-off of

50

radishes in the first three plantings was Significantly lower in soil
acidified to pH 6.0 than in soil at pH 8.1 (55). By the fourth planting,
disease was very low in both treatments. Possibly, the increase in
Trichoderma populations induced by consecutive plantings overcame the pH
effect.

Soil temperature influenced the efficacy of I. hamatum as a
biological control agent (101). Radish seeds treated with I, hamatum
were planted into A. gplppi-infested soil and damping-off was assessed
at temperatures from 20 C to 35 C. Disease was controlled at all
temperatures tested except 35 C, which also is not conducive to growth
of I, hamatum in culture. Concentration of I, hamatum inoculum affected.
the degree of biological control obtained (55, 101).

Trichoderma hamatum as a seed treatment was superior to two
fungicides, Captan and PCNB, for control of A. gplppi damping-off of
radishes (100). PCNB gave as much control as I, hamatum 3 days after
planting, but was not as effective by 5 or 7 days. In a replanting with
untreated seeds, the I, hamatum treatment still was effective in reducing
disease. By 7 days, almost all of the plants were diseased in the
chemical treatments.

Trichoderma hamatum was compared with Chaetomium globosum for

 

 

controlling damping-off of radish (101). Radish seeds treated with I.
hamatum gave the least disease. The combination 0f.I- hamatum and Q.
globosum was not Significantly different from I, hamatum alone, and A,
globosum by itself provided no protection. Results were similar when the
same soil was replanted with untreated seeds. Soil seeded with [.
hamatum-treated seed contained the fewest A. pplppi propagules, and the

most propagules of Trichoderma Spp. Soil sown with seeds treated with I,

 

51

hamatum and Q. globosum contained about half as many Trichoderma Spp.

 

propagules and almost three times the number of A. .gplppi prOpagules as
soil sown with seeds treated with only I, hamatum.

The percentage of diseased radish seedlings decreased when seed were
treated with I, hamatum plus chitin, A, pplppi cell walls, or nonpatho-

genic A, solani (101). Trichoderma hamatum alone did not significantly

 

reduce disease in this experiment. Cellulose or peat did not augment the
effect of I, hamatum, yet the population density of Trichoderma was very
high in these treatments. The authors suggest that_I. hamatum may have
utilized organic compounds in peat, and that peat should also decrease
the pH of the soil environment. Trichoderma hamatum produces cellulase,
therefore, the ability to utilize cellulose as a carbon source was
reflected in the increased in I. hamatum population (100). It may be
possible that cellulose or peat enhanced the saprOphytic but not the
hyperparasitic qualities of I. hamatum during this experiment. In other
work, I, hamatum added to A. gplppi-infested soil decreased incidence of
stem rot of beans (55).

Studies have indicated that I, hamatum was as effective as_l.
harzianum as a biological control agent against Rhizoctonia diseases
(55, 100, 101).

Basidiomycetes. There are very few examples of hyperparasites of

the root-infecting basidiomycetes. Armillaria mellea was parasitized by

 

Trichoderma via coiling followed by lysis in agar culture at pH'S of 3.4
-5.1, but not at pH 7.0 (16). When studying the interaction of

Penigphora gigantea and Heterobasidium annosum (Fomes annosus), no

 

evidence was found that the inhibition caused by A. gigantea was due to

antibiotics (121, 122). However, A, annosum hyphae in direct contact

52

with A. gigantea became granulated and vacuolated. This interaction was
termed hyphal interference, and may be likened to destructive contact
hyperparasitism. Peniophora gigantea is used in EurOpe as a biological

stump treatment for control of A. annosum.

Parasitism of beneficial root-infecting fungi, the endomycorrhizae

When spores were first observed within the chlamydospores of
Endogonaceae spp., researchers weren't sure if these Spores were part Of
the chlamydospores or were those of a hyperparasite (91, 161). Godfrey
(93) isolated a slow-growing, non-sporulating fungus with septate
mycelium from infected Spores of a mycorrhizal fungus, then identified as
Endogone. Sparrow (189) described the chytrid, Rhizidiomycopsis

stomatosa as an epibiotic parasite of Gigaspora margarita, the rhizoidal

 

system Of which arose from an apOphysis inside the parasitized
chlamydospore. This parasite was first isolated by Schenck & Nicolson
(181), who found that the chytrid was prevalent in chlamydospores after
very heavy rains. The hyperparasitic host range included other Gigaspora

spp., Glomus spp., and Acaulospora laevis.

 

Another chytrid, Phlyctochytrium sp., has been observed as a

parasite of Glomus macrocarpus var. geosporus (179), A, epigaeces, A,

 

fasiculatus, and Gigaspora margarita (63). Spores of A, epigaeus were
more susceptible to parasitism by Phlyctochytrium Sp. after soaking in
H202, NaOCl, or KOH, but less susceptible if soaked in HCl (63). It
was suggested that melanin content of Spore walls may be related to
susceptibility of chlamydospores to hyperparasites.

Two members of the Fungi Imperfecti, Anguillospora pseudolongissisma

and Humicola fuscoatra, were identified as parasites of Glomus spp.

53

chlamydospores (63). .A. pseudolongissima infected 69.3% of the g.
epigaeus spores or 94.6% of the A. fasciculatus Spores in autoclaved soil
while A. fuscoatra infected 70.9% of A. epigaeus spores, or 91.2% of A.

fasciculatus spores in autoclaved soil. Parasitism was less in water

 

agar. 'A, pseudolongissima-infected spores contained sausage-shape
hyphae while A. fuscoatra-infected spores contained slender hyphae
and/or aleurospores. .A. fuscoatra has also been noted as a parasite
Phytophthora megasperma var. gpjpg_oospores (188).

The parasitism of beneficial mycorrhizae may be a negative side
aspect to the use of hyperparasites as biological control agents. An
attempt to control hyperparasites of A. egigaeus in sterile sand with
fungicides interfered with germination of mycorrhizal spores (63). It is
suggested that mycorrhizae be included in host range tests of potential

hyperparasitic biological control agents.

Potential Biological Control Agents

Not one of the promising hyperparsitic agents discussed is used in
commercial agricultural production to date. This is not to say that
biological control will not be effective on a commercial scale, or is not
taking place in certain microenvironments. The advancement of biological
control in plant pathology has only recently attracted the renewed
interest of researchers.

A wide range of organisms have been cited as hyperparasites. The
biotrophic, contact or haustorial hyperparasites do not appear to cause
enough damage to their hosts to be of value as biological control agents.
Other hyperparasites are themselves plant pathogens, which may preclude

their use. The destructive hyperparasites, however, can actively

54

suppress and in some cases destroy their hosts. This is the group that
plant pathologists will need to exploit as hyperparasitic biological
control agents.

Once the efficacy of a potential biological control agent is
established, other considerations become important. The variability of
isolates of parasites and the host must be considered. For example,
some Strains of I. harzianum infect only A, gplppi, whereas others will
infect other plant pathogens as well (55, 82, 99). Strains of A. pplppi
may differ in susceptibility to Trichoderma (215). Strain selection
could be important to the success of biological control.

A few researchers are investigating the inclusion of biological
control agents in integrated pest management systems. Already, the
additive effect of I, harzianum combined with PCNB in control of g.
rolfsii (56) or A. gplppi (83, 103), and of I. harzianum following methyl
bromide for A. gplppi control (83) has been demonstrated.

Biological control efficacy may be adversely affected by the use of

pesticides. The detrimental effects of cotton herbicides on the effec-

 

tiveness of Trichoderma in controlling A. solani was revealed by Curl et

al. (62). Fungicides prevented the establishment of Hansfordia as a

 

biological control of leaf spot in the greenhouse (193). Presently, I.
harzianum mutants are being screened for tolerance to fungicides (167).
Information obtained on the interaction of biological and chemical
controls may aid in establishing a successful disease control program.
Although there is not yet a hyperparasitic biological control agent

in commercial use, it may not be long before Trichoderma will be the

 

first.

MATERIALS AND METHODS

Host fungi

 

The Oomycetes selected for study were Aphanomyces cochlioides

 

Drechs. obtained from C. L. Schneider, Michigan State University, East
Lansing; Aphanomyces euteiches Drechs. isolate L30 from W. F. Pfender,

University Of Wisconsin, Madison, Wisconsin; Phytophthora cactorum Leb.

 

and Cohn (Schroet.) from J. E. Mitchell, University of Wisconsin,

Madison, Wisconsin; Phytophthora megasperma (Drechs.) var. sojae Hildb.

 

[eA, megasperma (Drechs.) f. sp. glycinea (Kuan and Erwin) (137)] races
1, 3, and 4 from A. F. Schmitthenner, Ohio Agricultural Research and
Development Center, Wooster, Ohio; A, megasperma var. gpjpg race 7 from
F. A. Laviolette, Purdue University, Lafayette, Indiana; Pythium
aphanidermatum (Edson) Fitzpatrick from R. D. Lumsden, USDA, Soilborne

Diseases Laboratory, Beltsville, Maryland. Pythium ultimum Trow was

 

isolated from diseased soybeans (182), and was maintained in our
laboratory. The following were obtained from the University of

California, Riverside, California: Phytophthora capsici Leonian, A1

 

isolate P504 and A2 isolate P505; Phytophthora citrophthora (Smith and

 

Smith) Leonian, isolate P405; Phytophthora megasperma Drechs. [3A.

 

megasperma (Drechs.) f. Sp. medicaginis (Kuan and Erwin) (137)] isolate

 

 

P1057; Phytgphthora parasitica A1 isolate P991 and A2 isolate P731.

Aphanomyces spp. and Pythium spp. were maintained on water agar. A,

 

megaspmera var. sojae (Pms) and A, cactorum were normally maintained on

 

55

56

Schmitthenner's Phytophthora selective medium (83). The other
Phytophthora spp. were maintained on modified V-8 juice agar (178). To
Obtain oospores, two agar disks from a culture plate of the colonized
maintenance medium were transferred to Opposite sides of petri dishes
containing the appropriate medium. .A: cochlioides and A, euteiches were

grown for 40-45 days in oatmeal broth (184). All PhytOphthora spp. were

 

grown for 30-34 days in clarified V-8 juice supplemented with 30 mg/l of
cholesterol (15). .A. aphanidermatum and A. ultimum were grown in
clarified V-8 juice for three weeks. All cultures were incubated in the
dark at 23:2 C.

Cultures of A. cochlioides, A, euteiches, A. cactorum, and A,

 

megasperma var. sojae were frozen at -12 C to -20 C for 1 hour to kill
mycelium and to aid in oospore separation. Mycelial mats of A.
aphanidermatum were dried on filter paper in sterile petri dishes prior

to oospore extraction. Cultures of A. cochlioides, A, euteiches, A.

 

aphanidermatum, and A. ultimum were ground in an Omni-mixer (Ivan
Sorvall, Norwalk, Connecticut) at a rheostat setting Of 70 using 10,

5-second pulses. Those of all PhytOphthora spp. were similarly ground

 

using a rheostat setting of 40 for 10 min. The oospores were passed
through an 80 "m nylon mesh screen which retained mycelial fragments.
The fragments were reground using the same procedure, to release more
oospores. The oospore suspension was centrifuged for 15 seconds at 3100
.g. The supernatant was removed by suction, leaving the oospores in the
pellet. The oospores were resuspended in sterile deionized distilled
water (SDDW) and centrifuged again. The oospores were stored in SDDW at
4!2 C until use.

Chlamydospores of the mycorrhizal fungus Glomus etunicatus Becker

 

57

Gerdeman, as identified by B. A. Daniels, Kansas State University,

Manhattan, were isolated from soil by N. Bolgiano and C. E. Nelsen,
Michigan State University, East Lansing, using methods described by
Caveness and Jensen (53). Chlamydospores were collected on an 8 um
membrane filter (Millipore Filter Corporation, Bedford, Massachusetts) in
a Millipore Swinnex-13 holder. The spores were surface-sterilized in the
filter holder with 5.25% NaOCl for three minutes, and then rinsed three
to five times with SDDW. The membrane filter was placed in SDDW in a
sterile petri dish to release the spores. Chlamydospores were used

immediately.

Maintenance of,potential hyperparasites

The parasites previously isolated from Ag; oospores (118, 188) that
were used in laboratory, greenhouse and/or field experiments, included
Diheterospgra chlamydosporia (Komishko) Barron and Onions, Humicola

fuscoatra Traaen., and Hyphochytrium catenoides Karling which were

 

maintained on potato-dextrose agar (PDA), and Pythium monospermum

 

Pringsh. which was maintained on water agar. F4 was maintained on PDA.
F4 was a fungus isolated from an infected oospore by the author. It had
septate hyphae, but failed to sporulate on various culture media. Other
test organisms that had no past history as hyperparasites were several

members of the Actinoplanaceae: Amorphosporangium auranticolor Couch

 

 

isolate 255, Ampullariella regularis Couch isolate 31, and Spirillospora

 

albida Couch isolate 163; a member of the Micromonosporaceae, Micromono-
spora sp. isolate 44, all provided by J. N. Couch, University of North
Carolina, Chapel Hill, North Carolina. Another oospore hyperparasite

(88) used was Actinoplanes missouriensis Couch isolate 431. Two other

 

58

Species of Actinoplanes tested for their ability to parasitize Am;

oospores were A. philippinensis Couch isolate 455, and A, utahensis Couch

 

isolate 37. The cultures of the Actinoplanes Spp. were Obtained from

 

M. M. Hoehn, Lilly Research Laboratories, Indianapolis, Indiana. All
actinomycete isolates had been lypholized and in storage since 1957 or
1958. They were easily recovered by transfer to chitin agar (145). All
isolates were maintained on Czapek's sucrose-nitrate agar (59) made with
soil water (J. N. Couch, personal communication). Soil water was
prepared by heating, but not boiling, 100 g of field soil in 1000 ml of
deionized distilled water for 1.5 h. The soil water was filtered through

Whatman #1 filter paper.

Production of potential hyperparasites for laboratory use

.A. catenoides was grown in potato-dextrose broth for 30 days. The

 

culture was ground in an Omni-mixer at a rheostat setting of 40 for 10
min, then centrifuged for 15 seconds at 3100 g. The supernatant broth
was removed by suction, and the sporangia were resuspended in SDDW. .A.
monospermum was grown in clarified V-8 juice broth for 4 weeks. The

mycelial mats of A. monospermum were harvested and ground in an

 

Omni-mixer at a rheostat setting of 70 using 10, 5-Second pulses. .2-
chlamydosporia and A. fuscoatra were cultured on PDA for 30-32 days.
Suspensions were prepared by scraping hyphae and spores from agar plates,
and grinding with SDDW in an Omni-mixer at a rheostat setting of 40 for
10 minutes. All the actinomycetes were grown on Czapek's sucrose nitrate
agar for 30-32 days. Sporangia and mycelia were harvested by scraping
the agar plates and grinding the scrapings in an Omni-mixer at a rheostat

setting of 30 for 10 min. All test organisms were stored at 4:2 C as

-59
aqueous suspensions until use.

Evaluation of hyperparasitism 1p vitro

 

The membrane filter-water agar technique used by Jimenez-A (126) to
assess oospore germination, was adopted here for host-parasite Studies.
Approximately 103 spores of the host fungi were applied using mild
suction to 0.4 pm polycarbonate membrane filters (Nuclepore, Pleasanton,
California), 1 X 1 cm Square. Membranes bearing the oospores were
floated on SDDW until used. Two percent water agar was poured into
5-cm-diameter plastic petri dishes. Just before the agar solidified,
membrane filters were placed, spores down, on the agar surface. After
the agar had solidified, the outline of the membrane was traced with a
marking pen on the bottom of each plate to allow for easy location of the
spores when viewing microscopically. The membranes were removed from the
agar, which was inoculated with a suspension of the test hyperparasite,
at a concentration of 104 propagules per plate. The propagule types

were sporangia for A. catenoides, oospores for A, monospermum,

 

 

chlamydospores for A. chlamydosporia, and aleuriospores for A. fuscoatra.
' Sporangial counts were used to determine actinomycete concentrations for
Actinoplanes species, while concentrations of the other actinomycetes
were determined by plating on Czapek's sucrose-nitrate agar and counting
colony forming units.

In accordance with moisture conditions that were thought to favor
each hyperparasite (118, 188), the agar was either flooded with 2 ml of
SDDW or a thin water film was obtained by using 0.5 ml of SDDW. Plates
were incubated for two weeks at 2521 C in the dark. All experiments were

arranged in a completely randomized design. There were 2-4 replications

60

per test organism per experiment, and each experiment was repeated 1-6
times. At the end of the incubation period plates were viewed
immediately, or were held at 4:2 C for no longer than a week and then
viewed. On each plate, 100 spores were evaluated for infection by the
test hyperparasite. Oospores were considered infected when mycelia or
spores of the hyperparasite had replaced the oospore cytoplasm, or if the
cytoplasm was granulated and disorganized. A vital stain was sometimes

used to aid in assessment of hyperparasitism.

Evaluation of host spore viability using tetrazolium bromide

Tetrazolium bromide [3-(4,5-dimethyl-thiazol-2-yl)-2A-tetrazolium-
bromide] (MTT) was used by Zimmerman and Young (224) to assess oospore
viability from crossed pairings of Phytophthora palmivora and A. capsic'.
Ribeiro (177) used the stain for detection of viable oospores of

Phytophthora infestans, and also recommended MTT for other Phytophthora

 

species. MTT blocks the electron transport chain, and results in a
rose-colored formazan product (6). It was tested as a stain for oospores

of A. cochlioides, A, euteiches, A, cactorum, Pms, A. aphanidermatum, and

 

 

A, ultimum. A 1:1 mixture of a 0.1% aqueous solution of MTT (Aldrich
Chemical Company, Inc., Milwaukee, Wisconsin) and an oospore suspension
(usually 0.5 ml of each) was prepared in a sterile test tube. The
oospores were incubated at 23:2 C or 35:1 C for 2, 24, 47, and 72 hr to
determine the optimum time period and temperature for staining. Two
replications were prepared per treatment. Each replication was sampled
three times, with 100 oospores counted in each sample. Rose-colored

oospores were scored as healthy.

61

Environmental factors affecting hyperparasitism of E. megasperma var.
sogae oospores by A. missouriensis and A, fuscoatra

The soil used was Capac loam from a family of fine-loamy, mixed,
mesic Aeric Ochraqualfs (206, 207). The moist bulk density ranged
from 1.43-1.73 g/cm3 and organic matter, 1-3%. The soil pH was 5.4
as measured in water (1:1, v/v). The soil was collected from a
Phytophthora-infested field in Shiawassee County in 1980. It was
necessary to sterilize the soil prior to use because of interference
by natural hyperparasites. Nine cm-diameter glass petri dishes were
used as experimental units.

MES, MOPS, and HEPPS buffers (0.05M) in SDDW were used to adjust
soil pH (96). The buffers were adjusted with NaOH to a pH dependent on
the buffering capacity of the soil such that when added to soil the
desired soil pH would be obtained. A range of pH from 4.7 to 8.0 was
obtained at approximately 0.5 unit intervals. MES was used to obtain pH
4.7-4.8, 5.4-5.5 and 6.0. MOPS was used to obtain pH 6.5-6.6 and
6.7-7.2. HEPPS was used to Obtain pH 7.3-7.7 and 7.9-8.0. Twenty grams
of air-dried soil were weighed into each of 21 petri dishes which were
autoclaved for 1 h.[ Thirty ml of the buffer solutions were added to each
dish. Equilibration for at least one week was required for pH to
stabilize.

Approximately 103 Am§_oospores were applied with suction to 1 X 1
cm squares of 25 um pore size monofilament nylon fabric (Tetko, Elmsford,

New York) (149). A suspension of A, missouriensis or A. fuscoatra was

 

applied directly on top of the oospores at a concentration of 104
propagules per square. Mesh squares with only oospOres served as

controls. For each pH treatment, six mesh squares were placed on the

62

soil in each petri dish. After one week of incubation in the dark at
25:1 C, the membranes were retrieved and 100 oospores on each membrane
were assessed for parasitism. In some experiments, oospores were stained
with MTT.

Soil that had not been air-dried to avoid error caused by hysteresis
(104) was used in experiments to study the effect of soil moisture on
hyperparasitism. The soil was autoclaved, and 700 g was placed on a
ceramic pressure plate (Soil Moisture Equipment Company, Santa Barbara,
California) and adjusted to -0.3, -1, -5, -10, and -15 bars matric
potential. The soil was then removed from the plate and quadruple-bagged
in plastic until used. Soil weights showed no significant loss of water
over time. Two soil plates were prepared for each moisture treatment per
parasite. For the 0 bar treatment, 40 g of soil were weighed into each
petri dish and flooded with 40 ml deionized distilled water. For
treatments less than 0 bars, 20 g of the adjusted soil was placed in a
petri dish, on top of which were placed a 9 cm diameter circle of 0.05 mm
mesh nylon fabric. Twenty grams of soil was placed on top of the mesh.

Three mesh squares with oospores and sporangia of A. missouriensis, or

 

Spores of A. fuscoatra, or with only oospores were placed under the 9
cm-diameter mesh fabric for water potentials less than 0 bars or were
immersed under the water for 0 bars.

To study the effect of temperature on parasitism of oospores by A.
missouriensis, soil was flooded and adjusted to a pH range of 6.6-6.8
with MOPS buffer, as described previously. For A. fuscoatra, the soil
was adjusted to -0.3 bars, but the pH was left at 5.4. The petri dishes
were placed in incubators at 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1 C. One

dish was prepared per temperature per hyperparasite or control. Six mesh

63

squares bearing oospores plus parasite were placed in each dish.
After one week of incubation, parasitism was assessed by counting
100 oospores per square. Control oospores were stained with MTT for

viability determination.

Electron microscope studies of oospore penetration

Transmission electron microscopy was used to investigate the para-
sitism of Ap§_oospores by A, missouriensis and A, fuscoatra.

Membrane filter-water agar plates were prepared with Ap§_oospores

and suspensions of A, missouriensis or A. fuscoatra as described in the

 

section Evaluation of Hyperparasitism 1p.yitgg. After 4 days Of incuba-
tion in the dark at 25 C, a square of agar including the interaction zone
was removed and prepared for transmission electron microscopy by K. K.
Baker (Director, Electron Optics Laboratory, Michigan State University,
East Lansing) using methods of Hooper et al. (111). Specimens were
examined with a Phillips 300 transmission electron microscope operated at

60 kV.

Biological control experiments 1A_vitro

 

A variety of soils was collected from soybean fields with plants
infected with App. Each of these samples was tested in the laboratory
for the presence of Ap§_using two-day-old Hark sOybean seedlings as baits
(89). The Hark variety is susceptible to all races of Ap§_known to be
present in Michigan (148). Twenty grams of coarsely-sieved (5 mm) soil
was placed in a glass petri dish and flooded with 30 ml distilled water
for five days to stimulate oospore germination. Five seedlings approxi-
mately 36 h old, germinated in vermiculite, were placed in each plate, 5

seedlings/plate. Ten soils were evaluated and five replications were

64

used per sample. Disease was rated after 3-4 days on a scale from 0 to 3
with 0 = no disease, 1 = mild disease identified by slight browning of
(the roots, 2 = moderate disease identified by root browning and the ab-
sence of secondary roots, and 3 = severe disease identified by stunting,
browning and absence of secondary roots (118). Positive identification
of Phytophthora infection was obtained by observing the sporangia of Am;
on the root surface with the aid of a dissecting microscOpe. The natural
soils causing severe disease were used in later experiments.

The same petri dish bioassay was used to assess biological control

by A, fuscoatra, A. catenoides, and A. monospermum. Aqueous suspensions

 

 

of each of the hyperparasites were added to 20 g of naturally infested
Apg soil in petri dishes at a concentration of 104 propagules/dish.
The volume was brought up to 30 ml of liquid. Plates were incubated 7
days before seedlings were added. Disease was assessed as described

above after 3-4 days.

Production of inoculum for greenhouse and field experiments

Am§_inoculum was produced in clarified V-8 juice broth (15), on
millet seed (126), or in soybean seedlings (118). Inoculum was usually
grown for approximately 30 days, and frozen for 1-4 h before use to kill
the mycelium. In an attempt to identify suitable media for production of
hyperparasite inoculum, different substrates were inoculated with A.

fuscoatra, A, catenoides, or A. monospermum. These included seeds of

 

 

barley, millet, soybean, wheat, and rye; ground preparations of wheat
bran, alfalfa hay, linseed meal, beet pulp, sawdust, and crab shell;
ground stems and leaves of wheat, corn, and barley; and onion leaves.

Onion leaves had been found to be a suitable substrate for A. catenoides

65

(221). Fifty ml of each substrate was placed in 125 ml Erlenmeyer
flasks. Fifteen ml distilled water was added to flasks inOculated with
A, fuscoatra and 30 ml to flasks inoculated with A. catenoides or A.
monospermum. The flasks were autoclaved for 1 h each on successive days.
After 3-4 weeks, the cultures were assessed for growth of the respective
parasite.

Difficulties in growing A, catenoides stimulated an attempt to grow

 

this chytrid on an Oomycete that had already colonized a substrate.

Aythium aphanidermatum, a known host of E. catenoides (15) was selected.

 

Barley seed, prepared as above except with 40 ml of distilled water, was

inoculated with A. aphanidermatum. After 8 weeks when the barley was

 

completely colonized, the cultures were inoculated with A. catenoides.

 

Growth of A. catenoides was also tested on the following liquid

 

media: Chytrid Medium [(=Emerson's YpSS (86)] prepared without agar;
corn meal extract prepared by steaming 20 9 corn meal in 400 ml of
distilled water for 30 min, straining, and autoclaving; diluted V-8 juice
broth made with 50 ml of V-8 juice and 950 ml distilled water; potato-
dextrose broth; and Am§_extract prepared by grinding six 3-week-old
mycelial mats of Ap§_in 200 ml distilled water in an Omni-mixer at a
rheostat setting of 40 for 10 min, autoclaving, centrifuging and using
the supernatant liquid. Fifty ml of each medium was dispensed into 250
ml Erlenmeyer flasks. Each medium was inoculated with a colonized agar

disk of A. catenoides.

 

A coarse grade of perlite (Krum Horticultural Perlite, Silbrico
Corporation, Chicago, Illinois) was used as a substratum (J. A. Lewis,
personal communication) for A. missouriensis and A, fuscoatra. A given

amount of perlite in flasks or autoclavable plastic bags was saturated

66

with either Czapek's broth node with soil water for A, missouriensis or

 

potato-dextrose broth for A. fuscoatra. The flooded perlite was
autoclaved, the excess solution decanted, and the media were inoculated

with sporangial or spore suspensions, respectively.

Carriers for biological control agents

Carriers used for A. missouriensis, A, fuscoatra, A, catenoides, A,

 

monospermum, or F4, an unidentified parasite, included soybean seed (as a
seed treatment), vermiculite (Grace Horticultural Products, Cambridge,
Massachusetts), Turface (=Montmorillonite clay, International Minerals
and Chemical Corporation, Des Plaines, Illinois), attapulgus clay 18/35
AA RVM (The Upjohn Company, Kalamazoo, Michigan), and Readi-earth mix
(Grace Horticultural Products, Cambridge, Massachusetts). Spores and
mycelial suspensions prepared as described under the section on "Produc-
tion of Potential Hyperparasites for Laboratory Use" were applied to each
carrier by mixing in a plastic bag. The treated material was spread in a
thin layer and allowed to air-dry. Normally when seed were treated with
a given hyperparasite, no sticking agents were used. Soybean seeds
absorbed water very rapidly, and the seeds were dried as quickly as
possible by rolling the treated seeds on filter paper. Colonized barley
seed and perlite also were used as carriers for A, fuscoatra. A given
volume (800-1000 ml) of barley was autoclaved for 1 h in an autoclavable
bag, fitted with a 0.05 cm diameter glass tube plugged with cotton for
air exchange. The barley was inoculated with a spore suspension, and the
bags were occasionally shaken to provide for inoculum dispersal. The
method for growing inoculum on impregnated perlite was described under

Production of Inoculum for Greenhouse and Field Experiments.

67

To determine concentrations of propagules on the seeds, 10 seeds
were soaked in 10 ml water for 15-30 min. Seed coats were removed from
the seed and added to the liquid which was brought up to 50 ml, then
ground in an Omni-mixer for 10 min at a rheostat setting of 30. The
suspension was poured through an 80 m sieve and enumerated by the
microsyringe method (135), or by counting colonies of A. missouriensis on
Czapek's agar and A. fuscoatra on potato-carrot agar (1:1, potato-
dextrose agar:carrot agar). Concentrations of organisms on vermiculite
or Readi-earth mix were determined by microsyringe counts, whereas those

on clays or perlite were determined by counting colony forming units.

Greenhouse experiments

The soybean cultivar Hark was used in all experiments. Conover loam
from a family of fine-loamy, mixed, mesic Udollic Ochraqualfs (206),
collected from a severely diseased soybean field in 1978, was used
initially in experiments. Soils were diluted with coarse sand (1:1 and
1:3, v/v). Other potting media were coarse sand, Michigan State
University greenhouse mix (1:1:1, sand:peat:soil, v/v), and Readi-earth
potting mix. Pots were either 8 cm-diameter styrofoam cups, 20 x 14 x 5
cm diameter styrofoam containers, 20 x 10 x 5.5 cm plastic containers, or
11 cm diameter clay pots.

[Amp OOSpores were added to the potting media with continuous mixing
to ensure uniform distribution of the plant pathogen. Clay or barley
seed bearing the hyperparasites was mixed into the potting media
following infestation with Ap§_inoculum. .5- missouriensis, A, fuscoatra,

A, catenoides, and A, monospermum were also tested without carriers. The

 

containers were incubated on greenhouse benches for 3-7 days, and kept

68

flooded or just below saturation (preferable for A. fuscoatra treatments)
with tap water. The excess water in the flooded treatments was allowed
to evaporate for approximately 24 h before planting to improve emergence
of the soybeans. Treated or untreated seeds were directly seeded into
the infested potting media, or when carriers other than seed were used,
untreated seeds were germinated in vermiculite for about 36 h before
transplanting. Controls in biological control experiments included soil
with or without Apg, Ap§_inoculum was always frozen for 1-4 h at -12 C
to -20 C to kill the mycelium prior to use. The effects of culture
medium, oospore concentration, and watering regimes on disease production
by Am§_oospores were investigated independently of biological control

tests.

Field experiments: Biological control with hyperparasites

During the summer of 1978, soybean fields were observed for
Phytophthora root rot in Ingham, Genesee, and Shiawassee Counties,
Michigan. Two severely diseased fields were observed. One field was
located on the east Side of Reed Road, almost midway between Cronk and
Juddville Roads in Section 32 of Shiawassee County. This property
belongs to Mr. John Majzel. The other field was located on the east
side of Byron Road, between Lansing and Newburg Roads, north of the
railroad tracks, near the intersection of Lansing and Byron Roads in
Section 14 of Shiawassee County. This area was being farmed by the Leo
Shuman family. Soil samples were collected from both farms, and disease
potential was assessed using the petri dish assay. S. 0. Cohen, Senior
Research Assistant, Michigan State University, determined that only race

1 of App was present in each field using the petri dish assay (148) and

69

differential cultivars (142). Arrangements were made to place a field
plot in each of these areas in 1979.

1212, In Mr. Majzel's field, a plot 6.4 m X 18.3 m (21' X 60') was
laid out in a split-plot design consisting of four whole plots, each
including four hyperparasites. The whole plots were replicated three
times, and two carrier systems comprised subplots. The rows were 3 m
(10') in length with 0.4 m (15") row spacing. Approximately 16 seeds were
planted per meter using a belt seeder. In Mr. Shuman's field the plot was
4.6 m X 54.8 m (15' X 180'). The rows were 3 m (10') in length with 0.6 m
(24") row spacing. Seeds were sown at a rate of 25 seeds per meter using
a belt seeder. The experiment was designed as a split-plot with five
whole plots, each including five hyperparasites. The whole plots were
replicated three times, and two carrier systems comprised subplots.

A, missouriensis, A, fuscoatra, A. catenoides, A, monospermum, and an

 

unidentified oospore parasite (F4) were tested as biological control
agents. F4 was used only at the Shuman Farm. The other hyperparasites
were cultured as described in Production of Potential Hyperparasites for
Laboratory Use. Cultures of all organisms were ground for about 10 min
with sterile distilled water using an Omni-mixer. The suspensions were
then washed with repeated centrifugation and reground for 1 min. All
parasites were used to coat soybean seeds, cv. Hark, by applying 45 ml of
a hyperparasite suspension plus 0.45 g of Cellulose Gum (Hercules Powder
Company, Wilmington, Delaware) to 450 seeds. Excess liquid was removed
rapidly from the seeds by drying them on filter paper. Controls included
seed treated with cellulose gum and untreated seed.

Vermiculite was used as a carrier for all five hyperparasites.

Czapek's broth was added to the vermiculite for A. missouriensis and P08

 

70

for A. catenoides and A, monospernum, but not for A, fuscoatra and F4.

 

Aqueous suspensions of the hyperparasites were mixed with vermiculite in
a plastic bag. The ratio of liquid to vermiculite was 1:2 (v/v) in all
treatments. The treated vermiculite was air-dried at 23:2 C. For each
row, 50 ml vermiculite inoculum of A. missouriensis, AA fuscoatra, and F4

or 40 ml for A. catenoides or A. monospermum was Spread on top of 75

 

untreated Hark seed on the belt of the seeder, and incorporated in the
.row with the seed. Untreated vermiculite was included as a control.

Inoculum also consisted of A. fuscoatra and F4 grown on barley seed.
After the seed was thoroughly colonized (1.5 months) the seed was dried
at 23:2 C. Sixty ml of the F4-colonized barley seed or 35 ml of A.
fuscoatra-colonized barley was mixed with 75 seed and applied to each row
with the belt seeder.

Soybean emergence was counted and post-emergence disease was also
evaluated in each plot.

AAAQ, Four separate biological control experiments were done. In
one experiment, field soils was naturally infested with AA§_and in
another, Amp-colonized millet seed was used to artificially infest the
soil. The third and fourth experiments were designed to test the
efficacy of two potential biological control agents when applied in the
fall or spring prior to planting.

An experimental plot, 4.9 m X 53.3 m (16' X 175'), was put in
Mr. Shuman's field, naturally infested with App, and adjacent to last
year's plot. A similar experiment was set up at the Botany and Plant
Pathology Farm, Michigan State University, in a 9.1 m X 24 m (30' X 80')
area, in which the soil was artificially infested with Amp. In both

experiments a completely randomized design was utilized. The rows were

71

6.1 m (20') in length with a 0.9 m (3') spacing between rows. Seeding was

done with a belt seeder at a rate of 33 seeds/m.

A, missouriensis, A, fuscoatra, and A, catenoides were applied as

 

seed treaments using methods previously described. The average concentra-

tion of inoculum per seed was 2.5 x 105 sporangia for A, missouriensis, 1

 

x 105 spores for A. fuscoatra, and 2.5 x 104 sporangia for A, catenoides.

 

Other carriers used were Turface for A, missouriensis, colonized barley

 

seed for A. fuscoatra, and vermiculite for A. catenoides. Mycelium and

 

sporangia of A, missouriensis grown in nutrient broth were used to treat
the Turface. Two hundred ml of Turface inoculum was applied per row. One
hundred ninety-five ml of barley seed colonized by A, fuscoatra (30 days
old) was applied per row. A control of barley seed colonized by
Trichoderma harzianum Rifai was applied at the rate of 195 ml/row.
Inoculum of A. catenoides (prepared by Su-chan Hsu, Michigan State
University, East Lansing, Michigan) was grown in suspension cultures of
0.2 to 0.5% PDA which were later added to vermiculite. Approximately 1 l
of the inoculum was applied per row. Untreated controls were included.
Inoculum of App, for the plot at the Botany and Plant Pathology Farm, was
produced on millet seed and was applied to each row with a belt seeder at
seeding. The final concentration of oospores (mycelium killed by
freezing) was 105/row or 30/g of treated area 5 cm wide X 7 cm deep X
3 m long (2“ X 3" X 120").

In the other two field experiments, additional time between appli-
cation of the hyperparasites and seeding was allowed. Inoculum of A.
missouriensis and A. fuscoatra was incorporated into two plots in the

Phytophthora-infested field of Mr. Shuman. Untreated rows served as

 

controls. One plot was treated November 19, 1979 and the other May 28,

72

1980, prior to planting in June 1980. The size of each plot was 9 m X

9 m (30' X 30'). Rows were 2.4 m (8') in length with a row Spacing 0.8 m
(36"). Both plots were arranged in a randomized complete block design
with 9 replications.

An aqueous suspension of A. missouriensis was obtained by grinding
scrapings from 85 agar plates for 5 min in 60 ml sterile distilled water
in an Omni-mixer at a rheostat setting of 40. A, fuscoatra inoculum was
prepared from 60 PDA plates in a similar manner. For each treatment, 60
ml of the hyperparasite suspension was Sprayed onto 500 ml Readi-earth
mix after it was finely ground dry in a Waring Blendor. Sterile
distilled water was sprayed on the mix to serve as the control. All
preparations were air-dried at 2312 C. Five hundred ml of inoculum was
spread on the soil surface by hand in a band about 0.15 m (6") in width,
then was rota-tilled into the rows of both plots to a depth of 0.15 m
(6"). Based on microscopic counts made in the laboratory before
application to the soil, final concentrations were 84 Sporangia/g soil
for A, missouriensis and 2.6 X 103 spores/g soil for A. fuscoatra.

Weed control was provided by periodic rototilling, but prior to planting,
Round-up herbicide (Monsanto Agricultural Products Company, St. Louis,
Missouri) was used at a rate of 0.9 kg/ha (2 lbs/A) in the fall-treated
plot. Both plots were seeded at the same time with a belt seeder at a
rate of 33 untreated seeds cv. Hark/m.

Soybean emergence was counted and post emergence disease was
evaluated in each plot. .

The relative concentration of AA§_waS roughly estimated using the
soybean seedling assay (89), and using soybean stem segments as a

substitute for leaf disks as baits (51). Usually, three soil subsamples

73

were assayed per row. For the seedling assay, 10 seedlings cv. Hark were
tested with 20 9 soil samples flooded with 30 ml distilled water.

Soybean stem segments, 3-4 mm in length, were cut from 2-week-old Hark
soybean plants between the cotyledons and primary leaves. Ten segments
were placed on 20 9 soil in each plate and incubated for 18-22 h. The
segments were then transferred to a selective medium (183). After three
days, the agar plates were flooded with DW, rinsed after 3-4 h, and then
reflooded. Plates were viewed under a dissecting microscope at 75X.

Each stem segment was rated for presence or absence of App sporangia.
AAAA, In an effort to establish a higher concentration of REE.

oospores in soil, microplots (21), employing clay tiles 0.2 m X 0.3 m (8"
X 12") were used. The tiles were buried vertically 0.9 m (3') apart in
the field at the Botany and Plant Pathology Farm, with about 2 cm
extending above the soil surface. Treatments were arranged in a
completely randomized design with 5 replicates per treatment.

The hyperparasites A, missouriensis and A, fuscoatra were applied as
seed treatments at concentrations of 4.5 X 103 and 3 X 104 sporangia or
spores/seed, respectively. Inoculum of hyperparasites on carriers and of
App inoculum were thoroughly mixed into the top 10 cm of field Soil in
each tile by hand using a trowel. A, missouriensis on attapulgus clay
was added at a final concentration of 4 X 104 CFU/g soil. Thirty-day-old
A, fuscoatra-colonized perlite was added to the soil to give a final

concentration of 2 X 105 CFU/g. Inoculum of A. catenoides was produced

 

by Su-chan Hsu in suspension cultures in 0.2-0.5% PDA for approximately

40 days. Five hundred ml of cultures of A. catenoides were mixed into

 

the soil in each tile.

A, megasperma var. sojae was grown in clarified V-B juice broth with

74

30 mg/l of cholesterol for 2-4 weeks. The mycelial mats were harvested,
frozen for two hours at -20 C, and ground in a Waring Blendor for ten
minutes. The final concentration Of oospores was 53/g soil. The control
tiles were untreated. Twenty-five soybean seeds (cv. Hark) were planted
per tile. The relative concentration of App was roughly estimated using
the soybean seedling assay (89).

During the growing season of 1981, soybean emergence was counted and

post emergence disease was also evaluated.

Statistical Analyses
Where apprOpriate, tests for statistical Significance were performed
using analysis of variance. Differences between means were detected with

Duncan's Multiple Range Test, or in some cases by Student's t-test.

RESULTS

Tetrazolium bromide as an oospore stain
Staining efficacy varied with the Oomycete (Table 1). Low

percentages of Aphanomyces cochlioides, A, euteiches, and Aythium ultimum

 

oospores stained 2-4 days, while 65% or more of the oospores of

Phytophthora cactorum, A. megasperma var. sojae (Pms) race 1, and Pythium

 

aphanidermatum stained after 1-2 days. The color reaction of oospores
was categorized using the Horticultural Colour Chart (112). Color
intensity varied slightly with different oospore lots of each Oomycete,
and was always within the limits of the specific colors listed in

Table 1. The difference in color may have been due to contamination of

MTT with other tetrazolium salts. A, aphanidermatum oospores varied in

 

the shade of Spirea Red manifested within a given batch.
In a time course study done at 23:2 C, none of the test organisms
showed significant staining up to the 72 h sampling time. At 35:1 C, not

more than 9-10% of the oospores of A, euteiches and A. cochlioides, or

 

30% of the oospores of A. ultimum were stained after 72 h. In one exper-
iment, sample tubes of these three organisms were left to incubate at
35:1 C for 20 days. After this time period, still only very few oospores

of A. euteiches were stained, but 99% of the oospores of A. cochlioides

 

stained Rose Red. Many of the A. ultimum oospores were stained black.
An average of 87% and 79% of the OOSpores of AA§_and A,

aphanidermatum, respectively, were stained after 8 h, and more than 85%

75

76

Table 1. Stain reactions of several Oomycetous fungi with tetrazolium

bromide (MTT)

 

 

 

 

 

 

 

Fungus Stained oospores, %a Colorb Time
Aphanomyces cochlioides 4 Rose Red 3 days
A, euteiches 4 Spirea Red 3 days
Phytophthora cactorum 89 China Rose 1-2 days
.A. megasperma var.

sojae 85 China Rose 1-2 days
Pythium aphanidermatum 85 Spirea Red 2 days
.A. ultimum 30 Spirea Red 2 days
Rose Red 3 days

Magenta Rose 4 days

 

aAverage % of oospores stained from subsamples of two replicate tubes.

bColor reaction of the oospores were identified using Horticultural

Colour Chart.

77

of both species were stained after 24 h (Figure 1). The remainder were
either not stained or were stained black. The oospores of App were
stained more intensively nearer the oospore wall than in the center,
whereas those of E. aphanidermatum were evenly stained. At 24 h, the
China Red oospores of AA§_changed to Spirea Red and the cytoplasm became
evenly stained in most of oospores viewed. After 24 h, the population of
A, aphanidermatum oospores began to display different intensities of
Spirea Red, and uneven color within a given oospore, even though total
numbers of stained oospores increased from that at 8 h. Eighty-nine
percent of the A, cactorum oospores were stained China Rose by 24 h. The
cytoplasm near the oospore wall was more intensely stained in several A,
cactorum oospores, but most oospores were evenly stained. After 24 h,
the numbers of black oospores of App and A, cactorum increased causing a
decrease in numbers of rose colored oospores. There was little change in
the number of unstained oospores. Staining of A. aphanidermatum oospores
remained essentially the same. In preliminary experiments, MTT staining
of Phytophthora oospores after 48 h at 35 C averaged 71, 88, and 77% for
‘A. capsici, A. citrophthora and A, parasitica, respectively. Sixty two %
of the oospores of the hyperparasite, Pythium monospermum, stained Spirea
Red in a 0.1% solution of MTT at 23:2 C.

To test whether MTT was acting as a vital stain, dead oospores
killed by autoclaving were compared with living oospores. Three hundred
autoclaved or untreated oospores were stained in each of Six tubes.
Eighty-one % of untreated oospores were stained, whereas only 5% of
autoclaved oospore were stained. Oospores not stained rose color were
unstained and were not black. Similar results were obtained in another

experiment. Staining of viable oospores with MTT also has been

% OOSPORES STAINED

Figure 1.

100

80

60

4O

20

 

    

P. aphanidermatum:

R cactorum

 

 

 

Staining of oospores Of three Oomycetes during 2-72 h

incubation in a 0.1% suspension of tetrazolium bromide

(MTT) at 35 C.

79

correlated with oospore germinability (Sutherland and Cohen, unpublished
results).

It was discovered in host range experiments that MTT also Stained
the mycelia and Sporangia of Actinoplanes missouriensis within infected
oospores stained purple, after approximately 15 min. at 23:2 C. Since
healthy oospores never stain purple this permitted selective staining of
the parasite and facilitated microscopic evaluation of parasitism,
especially in the presence of soil particles. The actinomycetes,
Actinoplanes philippinensis, Actinoplanes utahensis, Ampullaria

regularis, Amorphosporangium auranticolor, and Spirillospora albida,

 

within infected oospores, also were stained purple in MTT at 23:2 C after

about 15 min.

Host range experiments
All possible combinations of the following potential hosts and
parasites were tested: Aphanomyces cochlioides, A, euteiches,

Phytophthora citrophthora, A, megasperma var. sojae race 1, Pythium

 

aphanidermatum, and A, ultimum as potential hosts, and Actinoplanes

 

missouriensis, Diheterospora chlamydosporia, Humicola fuscoatra,

Hyphochytrium catenoides, and Aythium monospermum as potential oospore

 

parasites. All host species were parasitized by all parasites except A.

ultimum by A, catenoides (Table 2). Invasion of A, cochlioides and A.

 

euteiches by A. catenoides and A, monospermum after two weeks was less

 

than 50%, while parasitism by the other organisms was greater than 66%.

 

.E- citrophthora and AA§_appeared to be equally susceptible to A,

missouriensis, A. chlamydosporia, and A. fuscoatra, parasitism being

 

greater than 90% in all cases. Susceptibility of App oospores to A.

8O

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mgu *o :owum>gmmno uwaoumocupa An um:_scmumu we: sm_u_mmcma .mum_a cog umucsoo mew: mmconmoo oofi

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.ammmmxm Enguxcuocmxr mfimmdamm mcoamocmum;_a mace—mocwuu<

 

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81

catenoides and A. monospermum was about 20% less than the susceptibility

of A. citrophthora to the same parasites. A, aphanidermatum and A,

 

ultimum were not parasitized as frequently as the other hosts (45% or
less), except in the interactions between A. aphanidermatum and A.
fuscoatra or A, ultimum and A, monospermum where oospore infection
averaged 79% and 77%, respectively.

Glomus etunicatus was also tested as a host for A. missouriensis and

 

A, fuscoatra to determine the susceptibility of a mycorrhizal fungus to
oospore hyperparasites. The deep pigmentation of chlamydospores made
assessment of parasitism by A, fuscoatra difficult. Empty chlamydospores
were rated as infected by A. fuscoatra. After 34 days of incubation, an
average of 47% of the chlamydospores were parasitized by A. fuscoatra in
one experiment and 38% in a second experiment. After 2 weeks 39% of the
chlamydospores were infected in a third experiment. Chlamydospores
incubated with A. missouriensis remained healthy for at least 4 weeks.

Phytophthora cactorum, A. capsici, A. megasperma, and A, parasitica

 

 

also were tested as hosts for A. missouriensis and A, fuscoatra

(Table 3). Oospore infection of each species by both parasites was
approximately the same, average percentages ranging from 89-97% for
parasitism of A. parasitica to 95-99% for A, capsic . Races 1, 3, 4, and
7 of A. megasperma var. gpjpg were also tested to determine their

susceptibility to A, missouriensis and A. fuscoatra. Parasitism of each

 

BEE race averaged 95% or higher (Table 4). Similar results were obtained
in a second experiment.

Two other Actinoplanes Species, A, philippinensis and A, utahensis,

 

and Amorphosporangium auranticolor, Ampullariella regularis, and

Spirillospora albida, all members of the Actinoplanaceae, and

 

82

Table 3. Parasitism of oospores of four Phytophthora species by

 

Actinoplanes missouriensis or Humicola fuscoatra after 14 days

 

of incubation on water agar.

 

 

Oospores parasitized, %a

 

 

 

 

Host A, missouriensis A, fuscoatra
Phytophthora cactorum 97 : 1 98 : 1
‘A. capsici 95 : 1 99 : O
A, megasperma 93 : 2 96 : 3
.A. parasitica 97 : 1 89 : 2

 

 

aMean % of oospores parasitized : standard error on 3-4 replicate plates;

100 oospores were counted per plate. Parasitism was determined as

described in Table 2.

83

Table 4. Comparative susceptibilities Of oospores of four races of
Phytophthora megasperma var. sojae to the hyperparasites
Actinoplanes missouriensis and Humicola fuscoatra after 14 days

of incubation on water agar.

 

 

 

 

 

Hyperparasite Oospore race Oospores parasitized, %a
Actinoplanes missouriensis 1 99 : 1
3 99 : 1
4 95 : 3
7 96 : 2
Humicola fuscoatra 1 96 : 4
3 95 : 2
4 98 : 1
7 95 : 1

 

aMean % of parasitized oospores : standard error on three replicate
plates; 100 oospores were counted on each plate. Parasitism was

determined as described in Table 2.

84

Micromonospora sp. of the Micromonosporaceae, were not known to be
oospore parasites. These organisms were tested as potential hyperpara-
sites by incubating each actinomycete with BEE oospores for two weeks.
Colony forming units per petri dish used in testing hyperparasitism were
5 x 105 for A- auranticolor, 2 x 105 for A. regularis, 7 x 104 for
Micromonospora Sp., and 9 X 105 for A, plpipp. Concentrations of 104

sporangia per petri dish were used for A. philippinensis and A,

 

utahensis. All Of the test organisms were successful oospore parasites
with percentages Of parasitism ranging from 94-99% (Table 5).

AA§_oospores were inoculated with four different Single spore
isolates obtained from a culture of AA fuscoatra to test the hyperpara-
sitic stability of this organism. There were no gross morphological
differences among the isolates. No significant differences were detected
in parasitism of Am; oospores by the isolates in either of two
experiments.

Pigmentation ranging from white to gray to black was observed in
culture of A, fuscoatra. Since the occurrence of cultures bearing albino
spores was rather high (roughly 1 in 10), the parasitic capability of
albino and dark spores was compared. Hyperparasitism of App oospores was
compared using aleuriospores derived from albino and black (wild type)
cultures of A. fuscoatra. Parasitism by albino Spores was 80% and 97% in

two experiments whereas that by pigmented spores was 78% and 93%.

Invasion of living vs. killed oospores
To determine whether killed App oospores could be used to stimulate
an increase in the populations of indigenous oospore parasites, the

extent to which autoclaved oospores were invaded was tested. Oospores of

85

Table 5. Parasitism of Phytophthora megasperma var. sojae OOSpores by six

actinomycetes species after 14 days of incubation on water agar.

 

 

 

 

 

 

 

Hyperparasite % Oospores infecteda
Actinoplanes philippinensis 95 : 2
A. utahensis 99 : 1
Amorphosporangium auranticolor 95 : 2
Ampullariella regularis 99 : 1
Micromonospora Sp. 95 : 3
Spirillospora albida 94 : 1

 

 

aAverage % of parasitized oospores : standard error, on three replicate
plates; 100 oospores were counted per plate. Parasitism was determined by

microscopic observation of the hyperparasite within the oospore.

86

AA§_were autoclaved for 15 min in a 1 ml volume of water. Identical
batches of oospores were not autoclaved. Using the membrane filter-water
agar technique, plates were inoculated with suspensions of A.
missouriensis or A. fuscoatra. Invasion of killed oospores by A.
fuscoatra was nearly equal to that of healthy oospores, but A.
missouriensis invaded only about one-third the number of killed oospores
(Table 6). Vital staining of oospores initially, as indicated by
staining in MTT, was 88% for the untreated oospores, but after killing
was 6%. The experiment was repeated with Similar results. The results
suggested that killed oospores of App might be useful to stimulate
natural parasites, especially A. fuscoatra.
Effect of Oomycete culture age on susceptibility of oospores to
hyperparasites

,App oospores were harvested from cultures 16, 22, or 30 days of age.
There was no significant difference in hyperparasitism of the oospores by
A, missouriensis or A, fuscoatra (Table 7). As an indication of
viability prior to the addition of hyperparasites, oospore staining with
0.1% MTT was 89% or greater for each age. A second experiment gave

similar results.

Effect of hyperparasite culture age on hyperparasitism
To determine if culture age of the hyperparasites would have any

bearing on biological control potential, A, missouriensis sporangia were

 

collected from cultures 13, 29, or 45 days of age, and A. fuscoatra
aleuriospores were collected from cultures 12, 28, or 78 days Of age and
used as inoculum. Oospore parasitism by both organisms was greater than
95% at all ages tested (Table 8). Results were Similar in a second

experiment.

87

Table 6. Invasion of living vs. killed oospores of Phytophthora
megasperma var. sojae by Actinoplanes missouriensis or Humicola

fuscoatra after 14 days of incubation on water agar.

 

 

Oospores invaded, %a

 

 

 

Actinoplanes Humicola MTT staining of

Treatment missouriensis fuscoatra control oosporesb
Untreated oospores 94 : 2 97 : 1 88 : 3
Autoclaved oospores 31** : 6 99 : 1 6 : 2

 

aMean % of oospores invaded : standard error on three replicate plates;
100 oospores were counted per plate. The value with double asterisks
(**) is significantly lower (A?0.01) than that for untreated oospores.
Parasitism was determined as described in Table 2.

bMean % of oospores stained as an estimate of oospore viability; 100

oospores were counted in each of three replicate plates.

88

Table 7. Susceptibility of Phytpphthora megasperma var. sojae (Pms)
oospores of three ages to parasitism by Actinoplanes

missouriensis or Humicola fuscoatra after 7 days of incubation

 

on water agar.

 

 

 

 

 

 

REE Oospores parasitized,%a
culture age, Actinoplanes Humicola MTT staining of
days missouriensis fuscoatra of oosporesb
16 91 t 4 94 t 2 89 i 3
22 86 i 4 93 t 4 90 i 2
30 95 : 2 90 : 4 94 : 2

 

aAverage % of oospores parasitized : standard error on three replicate

plates; 100 oospores were counted per plate. Parasitism was determined

as described in Table 2.

bMean % of oospores stained as an estimate of viability on three replicate

plates; 100 oospores were counted per plate.

89

Table 8. Parasitism of oospores of Phytophthora megasperma var. sojae by

Actinoplanes missouriensis and Humicola fuscoatra of different

 

ages after 7 days of incubation on water agar.

 

 

 

 

 

Hyperparasite Culture age, days Oospores parasitized, %a
Actinoplanes missouriensis 13 96 : 1

29 98 : 1

45 99 : 1
Humicola fuscoatra 12 98 : 1

28 » 97 : 2

78 97 : 1

 

aAverage % of oospores parasitized : standard error on three replicate
plates; 100 oospores were counted per plate. Parasitism was determined

as described in Table 2.

90

The influence of soil pH, moisture, and temperature on hyperparasitism of
A, megasperma var. sojae oospores by A. missouriensis and A, fuscoatra

 

 

Environmental parameters were tested in order to assess the range of

conditions that A. missouriensis and A. fuscoatra would tolerate as

 

hyperparasites. As soil pH increased from 4.7 to 8.0, parasitism of App
oospores by A, missouriensis, in Experiment 1, increased from 4 to 100%
(Table 9). At the lower pH'S, 4.7 to 6.0, parasitism was not greater
than 16%. Rasing the pH to 6.6, greatly increased oospore infection to
91%. Infection remained high (98-100%) at pH'S 7.0 to 8.0. In a second

experiment, oospore parasitism by A, missouriensis increased with

 

increasing pH up to 98% oospore infection at pH 8.0. However, in this
experiment parasitism greatly increased at pH 5.5 to 83% and steadily
increased thereafter. Parasitism of AA§_oospores by A, fuscoatra
increased from 5 to 50% as soil pH increased from 4.7 to 7.9 (Figure 2).
Parasitism was 6% or less at pH'S 4.7 to 6.0 then increased to 27% at pH
6.6, and 50 to 53% at pH'S 7.2 to 7.9. Results were similar in a second

experiment. Parasitism by A. missouriensis or A. fuscoatra in soils

 

adjusted with MES, MOPS, or HEPPS buffers and NaOH to a common pH (6.7
to 7.0) did not differ. It is not known why oospore parasitism 9Y.B-
missouriensis differed between experiments at the low pH'S, 4.7 to 6.0.

Parasitism of Pms oospores by A, missouriensis and A. fuscoatra was

 

studied at matric potentials from 0 to -15 bars (Figure 3). Mean values
for A, missouriensis in six experiments ranged from 59-85%, and for A.
fuscoatra from 67 to 91% in five experiments. Oospore infection by A.
missouriensis was 85% at 0 bars and gradually decreased with decreasing
matric potential to 59% at -15 bars. Parasitism of AA§_by A. fuscoatra
was 67% at 0 bars, increased to 91% at -0.3 bars, then decreased to 67%

at -15.0 bars.

91

Table 9. Effect of pH on parasitism of Phytophthora megasperma var.
sojae oospores by Actinoplanes missouriensis after 7 days of

incubation in autoclaved soil.

 

 

Oospores parasitized, %b

 

pHa Experiment 1 Experiment 2
4.7 4 : 1 36 : 1
5.5 11 : 1 83 : 1
6.0 16 : 1 89 : 1
6.6 91 : 1 93 : 1
7.0 98 : 1 94 : 1
7.7 98 : 1 96 : O
8.0 100 : 0 98 : 1

 

aSoil pH was adjusted using 0.05 M concentrations of the following
buffers: MES (pH 4.7, 5.5, and 6.0), MOPS (pH 6.6 and 7.0), and HEPPS
(pH 7.7 and 8.0).

bAverage % per plate Of soil for six replicate membranes : standard error;
100 oospores were counted per membrane. Parasitism was determined by

microscopic observation of the hyperparasite within the oospore.

92

 

 

 

 

1 00 r r I I
80 - .
a"
a.
lull
560- -
t
0‘!
< .-.-0“.
a: [I '
E I
U, 40 - I, -
“‘ I
a: I
O I
n5
0’ .’
8 i
20 ' I, 'l
/.
I
I
I
I
0 ---o -— o
1 l l l
5.0 6.0 20 8.0
Figure 2. The effect of pH on parasitism of Phytophthora megasperma var.

 

sojae oospores by Humicola fuscoatra after 7 days of
incubation in autoclaved soil. Standard errors ranged from

1-3%. Soil pH was adjusted as described in Table 9.

 

100

CO
C)
I

OOSPORES PARASITIZED, %

 

Ch
(3
I

A
CD
I

20-

A.MISSOURIENSIS °\o ..

 

l_

 

Figure 3.

o -o:3 -1.0 -so -1oo -15.o

SOIL MOI STU RE, BARS

Hyperparasitism of Phytophthora megasperma var. sojae

oospores by Actinoplanes missouriensis and Humicola fuscoatra

 

in autoclaved soil adjusted to different matric potentials,
after 7 days of incubation.. The values represent averages
(: standard errors of 2-6%) of Six experiments with A.
missouriensis and averages (: standard errors of 2-5%) of

five experiments with A. fuscoatra.

94

AS soil temperature increased from 5 to 30 C, parasitism of Pms

oospores by A; missouriensis and A. fuscoatra increased from a minimum of

 

4% at 5 C to a maximum of 99% for A. missouriensis or 92% for A. fuscoatra
at 30 C (Figure 4). Very little hyperparasitism (4 to 6%) occurred at 5

and 10 C by either oospore parasite. At 15 C, oospore infection increased

 

to 63% for A, missouriensis but remained low (9%) for A. fuscoatra. From
20 to 30 C, both hyperparasities parasitized greater numbers of oospores
as the temperature increased. The same trends were shown in two
additional experiments for each hyperparasite. The effect of temperature

on hyperparasitism of Pms oospores by A- missouriensis also was tested on

 

water agar over a two-week period. After 7 days results were similar to
those in autoclaved soil, but by 14 days hyperparasitism at 10 and 15 C
had approached that at 20 to 25 C. The hyperparasite did not appear to be
active at 5 C. Therefore, parasitism in autoclaved soil at 10 and 15 C

may also have increased with a longer inoculation time.

Electron microscgpic studies of the host-parasite interaction

 

A, missouriensis hyphae were found throughout the infected BEE

 

oospore four days after incubation of oospores with the hyperparasite

(Figure 5). ‘A, missouriensis hyphae were observed to directly penetrate

 

the oogonial wall (Figure 6) and the oospore wall (Figure 8), with little
change in the diameter of the penetration hyphae. High concentrations of
hyperparasite hyphae commonly occurred in the periplasmic space between
the oogonial and oospore walls (Figure 7). ‘A. fuscoatra also penetrated
directly through the oogonia wall, but the penetration hyphae appeared
narrower than other hyphae (Figure 10). A hypha grew through the

periplasmic space (Figure 9), penetrated the oospore wall then immediately

95

 

100

$8
9 S 3

OOSPORES PARASITIZED, %

to
O
T

 

 

 
  
 

.0
I
l
o I
I ..
I
I
I
I .
I
I
[I
[RHJUSCOATRA
I
l
l
I
I
I "
I
I
I

 

n L l 1 L

 

Figure 4.

5 10‘ 15 ‘ 20 25 3O
TEMPERATURE, °C

The effect of temperature on parasitism of PhytOphthora
megasperma var. ppjpg oospores by Actinoplanes missouriensis
or Humicola fuscoatra after 7 days of incubation in
autoclaved soil. Standard errors ranged from 0-4% in the

.A, missouriensis experiments and 1-4% in the A, fuscoatra

experiments.

96

Figures 5-8. Invasion of oospores of Phytophthora megasperma var. soj e

 

by Actinoplanes missouriensis.

Figure 5. Overview showing hyphae of the hyperparasite on the
oogonial wall, in the periplasmic Space between the
oogonial and oospore wall, the oospore wall, and the
oospore cytoplasm. X 2,592.

Figure 6. Penetration of the oogonial wall. X 17,280.

Figure 7. Concentration of A. missouriensis hyphae in the
periplasmic space. X 4,320.

Figure 8. Penetration of the oospore wall. X 17,280.
KEY TO LETTERING 0N FIGURES 5-8.

HP = hyperparasite, CC = oospore cytOplasm, OgW = oogonial wall, OSW =

oospore wall, PS = periplasmic space.

97

 

98

Figures 9-11. Invasion of oospores of Phytophthora megasperma var. soj e

by Humicola fuscoatra.

Figure 9. Overview showing hyphae of the parasite penetrating the
oogonial wall, within the periplasmic space, and in the
oospore wall. X 8,250.

Figure 10. Penetration of the oogonial wall. 'X 20,000.

Figure 11. Penetration of the oospore wall. X 15,000.

KEY TO LETTERING ON FIGURES 9-11.

HP = hyperparasite, 0C = oospore cytoplasm, OgW = oogonial wall, OSW =

oospore wall, PS = periplasmic Space.

 

99

 

100

branched (Figure 11). Parasitism by both hyperparasites was similar in
that no specialized cells, e.g., appressoria or haustoria, were formed
and penetration occurred directly. Indentations of the host walls were
not observed at penetration sites of either hyperparasites. There was no
c‘l earing of the host wall adjacent to the parasite suggesting that the
hyperparasite may penetrate mechanically without enzymatic degradation.
The 3115 oospores appeared to lack a physical defense system as no change

in oospore morphology or internal structure was detected in response to

hyperparasite penetration.

fiiological control experiments using the soybean seedling assay

Soil naturally infested with A111_s_ was used to evaluate the biological
control potential of four hyperparasites in the laboratory. A.
flscoatra, A. catenoides, A. monospermum, A. catenoides in combination
with A. monospermum, or an unidentified hyperparasite, F4, were tested at
concentrations of 104 propagules/petri dish (20 9 soil). Partial
control of Phytophthora root rot was Obtained in two of three experiments
by all hyperparasites tested except A. fuscoatra which reduced disease in
one experiment (Table 10). A lower disease index was Obtained with the
combination of A. catenoides and A. monospermum in the first, but not in
the second or third experiments. Disease incidence and severity were

decreased 38-66% in Experiment 1, and 38-56% in Experiment 2.

fittempts to enhance indigenous hyperparasites through soil treatment with
oospores

An alternative approach to the addition of hyperparasites in
biological control is to enhance indigenous hyperparasitic organisms. To
test whether such an approach might be feasible, oospores of Pythium

_gcanthicum, itself a hyperparastie, and autoclaved oospores of Pms were

101

Table 10. Biological control of Phytophthora root rot of soybean in the

laboratory using hyperparasites in naturally infested soil.

 

 

Disease indexa

 

 

 

Treatment Experiment 1b Experiment 2 Experiment 3
Control 2.9 A 2.7 A 2.9 A
'A. fuscoatra - 1.5 B 2.9 A
Pythium monospermum 1.8 B 1.6 B 2.7 A
Ayphochytrium catenoides 1.5 B 1.2 B 2.9 A
.A. monospermum 1.0 B 1.7 B 2.9 A

 

+ A, catenoides

F4 1.3 B 1.8 B 2.5 A

 

aMean values for three replicate plates each containing five seedlings.
Seedlings were rated on a scale from O = no disease to 3 = severe root
rot. Numbers followed by the same letter are not significantly different
(£50.05, Experiment 1; A;0.10, Experiment 2).

PA. fuscoatra was tested in unsaturated soil and was compared with an

unsaturated control. The disease index was 1.7 for both treatments.

102

added to soils naturally infested with Agg.

Oospores of A. acanthicum (2 X 105) were added to petri dishes
containing 10 g of soil naturally infested witthmg. The soil was
flooded to bring the final volume of water to 20 ml, and petri dishes
were incubated 10 days. Four replications of treated and untreated soil
were prepared. The disease indices after 5 days were identical in the

control and A, acanthicum-amended soils.

 

Autoclaved Am; cultures were added to two soils naturally infested
‘with Am§_at the rate of 3 X 103 oospores/g soil in five replicate petri
dishes per soil. All soils including control soils without added
autoclaved Am; were flooded and incubated 10 days. No differences in

disease were detected in the soils (Table 11).

Inoculum substrates for hyperparasites

Humicola fuscoatra grew successfully on barley, millet, soybean,

 

wheat, or rye seed; wheat bran, linseed meal, beet pulp; ground wheat,
corn, or barley stems and leaves; or onion leaves. Barley seed was
selected as the food base for A. fuscoatra in greenhouse and field tests.

The same substrates were not colonized by A. catenoides or A. monospermum

 

under the conditions used.

Because of the difficulty in growing A, catenoides, attempts were

 

made to culture this hyperparasite on oospores of Pythium aphanidermatum,

a previously identified host (15). .E- aphanidermatum which had been

 

cultured on sterilized barley seed was inoculated with A. catenoides.

 

However, A, catenoides failed to colonize the culture. A, catenoides was

 

 

grown successfully in several liquid media, including Emerson's Chytrid

medium, corn meal extract, diluted V-B juice broth, P08, and Amg extract.

103

Table 11. The effect of Pythium acanthicum or killed Phytophthora

megasperma var. sojae (Pms) oospores on Phytophthora root rot

 

in soils naturally infested with Pms.

 

 

Disease index for indicated soila

 

 

Brookston-
Conover loam Parkhill Conover
Treatment association loam loam
Control 2.8 2.4 2.5
Eythium acanthicum 2.8 - -
Autoclaved Am; - 2.3 2.4

 

aAverage numbers of five replicate plates each containing 10 seedlings.

Plants were rated on a scale from 0 = no disease to 3 = severe root rot.

104

Although colony counts were not made, it was obvious that the greatest
number of new A. catenoides colonies formed in the shortest period of
time in the Emi extract. Fewer new colonies formed in the Chytrid

medium, but they were much larger. Corn meal extract and diluted V-8

broth supported approximately equal growth of A. catenoides, but were

 

inferior to A95 extract and Chytrid medium. No new A. catenoides

 

colonies formed in PDB, but there appeared to be more growth than in the
other media.

Perlite saturated with Czapek's broth made with soil water or with
PDB were tested as substrates for A. missouriensis and A. fuscoatra,
respectively. “A. missouriensis did not colonize the perlite very
rapidly. A. fuscoatra grew well on perlite with PDB, and this medium was

used in further work.

Greenhouse experiments

Effect of potting medium on disease. Several different potting
media were used to compare disease using culturally grown oospores at a
concentration of 114 oospores/g soil. Two hundred ml of Michigan State
University greenhouse mix (1:1:1, sand:peat:soil), Readi-earth potting
mix, Dshtemo-Boyer loamy sand, or riverbed coarse sand were measured into
pots. The potting medium in each of five pots was infested with Amg,
while five other pots of the potting media were not infested. The
potting media were kept flooded for 4 days after which time five,
2-day-old seedlings were tranSplanted to each pot. An additional
treatment was included to test the effect of direct seeding on disease
incidence in riverbed coarse sand. Emergence was recorded as an

indication of disease. Plants grown in non-infested potting media were

105

healthy (Table 12). Pre-emergence disease was significantly higher
(A;0.05) and only 1-2 seedlings/5 survived when greenhouse mix,
Readi-earth mix, riverbed coarse sand, or Oshtemo-Boyer loamy sand were
used as the potting media. Emergence was less when seed were directly
planted in riverbed coarse sand.

In a similar experiment, six pots of sterilized Capac loam (a soil
naturally infested with Amg) each were infested with 100 oospores/g soil.
The oospores had been produced in V-8 juice broth. Details were as
described above. N0 disease was obtained in the artificially infested
soil.

Effect of culture medium on infectivity of A. megasperma var. sojae

 

oospores. Am§_oospores produced on millet seed or in clarified V-B juice
broth were harvested when cultures were 35 days old. Eight pots of
Readi-earth mix were infested (50 oospores/g) with each oospore type.
Eight pots of mix were left untreated. All pots were flooded for five
days. On the seventh day, two-day-old soybean seedlings were
transplanted into all pots. The experiment was repeated using an oospore
concentration of 100/9 soil, and four replications per treatment.
Oospores produced on V-8 juice broth caused the same amount of disease as
those produced on millet seed in both experiments (A§0.01, Experiment 1;
.Aé0.05, Experiment 2) (Table 13).

Effect of inoculum density on disease using two different potting

 

Aggig. Two hundred ml of riverbed coarse sand or Readi-earth potting
mix were infested with Am; oospores produced in V-8 juice broth at
concentrations of 1, 10, 30 or SO/g soil. Each treatment was replicated
seven times. Potting media were kept flooded for five days to stimulate

oospore germination. Two days later, four 2-day-old seedlings were

106

Table 12. Effect of potting medium on Phytophthora root rot of soybean in

 

a greenhouse experiment using oospores produced in V-B juice
broth at a concentration of approximately 114 oospores/g

potting medium.

 

 

Mean emergence/five

Potting medium seedlings/pota

 

Transplanted seedlingsb

Controlsc 5 A
Greenhouse mix 2 B
Readi-earth mix 2 B
Riverbed coarse sand 1 B
Oshtemo-Boyer loamy sand 3 B

Direct seeding
Control 4 A

Riverbed coarse sand
Directly seeded 4 A

 

aMean values for five replicate pots, each with five seedlings. Numbers
followed by the same letter are not significantly different (A;0.05).

bTransplanted seedlings were two days old with the radicle approximately

1 cm in length.

CControls included five pots of each potting medium without Phytophthora.

 

107

Table 13. Comparison of virulence of Phytophthora megasperma var. soj e

oospores produced on millet seed or in clarified V-8 juice

 

 

 

 

broth.
Mean emergence/five seedlings/pota
Inoculum source Experiment 1 Experiment 2
Control 5 A 5 A
Millet seed 3 B 4 AB
V-8 juice broth 3 B 3 B

 

aSeedlings two days old with the radicle approximately 1 cm in length were
transplanted. Means for eight replicate pots in Experiment 1 and four
replicate pots in Experiment 2, each with five seedlings. Values
followed by the same letter were not significantly different (A§0.01,

Experiment 1; A;0.05, Experiment 2).

108

tranSplanted into the pots. Plant stand was significantly reduced 25-50%
(A;0.05) at oospore concentrations of 10, 30 and 50/g soil in Readi-earth
potting mix, but not in riverbed coarse sand.

Effect of length of time of floodingon disease. Oospore germination
by Am§_requires free water. To determine the optimum time of flooding
soil, 20 pots each containing 200 ml of Readi-earth mix were infested with
oospores (lDO/g) produced in V-B juice broth, then were flooded. Twenty
additional pots of flooded soil without Amg were left to serve as
controls. Pots of mix were planted 1, 2, 3 and 4 days after flooding.
Five pots containing either infested or non-infested soil were directly
seeded with soybeans, five seeds per pot; after seeding, soils were
watered as needed. No disease occurred in any treatment. After the
experiment was concluded, 10 9 mix was taken from one pot each of one of
the Amsfinfested treatments. Disease potential, assessed using soybean
seedlings (89), showed severe root rot of seedlings. It is not known why
disease was not obtained in the greenhouse.

Biological control in the greenhouse by A. missouriensis applied as

 

an aqueous suspension. Mycelial/spore suspensions of A. missouriensis
were tested for biological control under different conditions. In one
experiment, ground Am§fcolonized millet seed was used as inoculum.

Fifty ml of the colonized seed was ground in 50 ml ow in an Omni-mixer

at a rheostat setting of 30 for 1 min. The Amsfseed suspension was
incorporated into Readi-earth mix by thorough mixing in a plastic bag.
This infestation procedure was repeated for 11 bags. The average oospore
concentration was 7 X 103 oospores/g soil. Millet seed controls were
prepared in the same manner except that Amgecolonized millet seed was

autoclaved for 20 min. at 121 C, before grinding. .A. missouriensis was

 

109

added to 6 pots of mix infested with Am§_or mix without Am§_by adding the
A. missouriensis while mixing the potting medium. The final concentra-
tion of A. missouriensis was approximately 103 sporangia/g soil. Soils
were flooded for 4 days. On the fifth day, seedlings germinated in
vermiculite for 36 h were transplanted to the soils at S/pot.

In a second experiment, Am; oospore inoculum was produced in V-8
juice broth. Washed and steamed riverbed coarse sand was infested with
Am§_oospores at a concentration of 450/9. An aqueous suspension of A.
missouriensis was applied at a concentration of 38 sporangia/g. After 4
days of flooding, water was withheld and five seedlings were transplanted
to each pot.

Both kinds of Am; inocula caused severe seedling rot (Table 14). .A.
missouriensis applied as a mycelial/spore suspension did not control
pre-emergence seedling rot in either experiment (A;0.Dl). In experiment
1 where Aggycolonized millet seed was used, emergence in the millet seed
control and A. missouriensis treatments was less than that in the
control, but this was due to toxicity from the millet seed. Soybean
emergence in the A. missouriensis control was not different from
emergence in the untreated control.

Biological control witth. missouriensis and A, fuscoatra as seed

treatments. In three experiments, A. missouriensis and A. fuscoatra

 

were evaluated as seed treatments for soybeans grown in Michigan State
University greenhouse mix. After Am§.inoculum was mixed into the soil
and prior to planting, soils were flooded 4-5 days. Watering was then
withheld until needed. Five replications were prepared per treatment.
.Amg oospores from cultures grown 11 and 14 days in V-8 juice broth,

were used to infest soil in two experiments at a concentration of

110

Table 14. Lack of biological control of Phytophthora megasperma var.

sojae (Pms) in the greenhouse by Actinoplanes missouriensis

 

applied as a mycelial/spore suspension under different

 

 

 

 

 

conditions.
Mean emergence/five
seedlings/pota

Inoculum source Experiment 1b Experiment 2c
Control (no treatment) 5 A 5 A
Millet seed controld 4 A -

‘33; on millet seed 1 B -
Am§_from V-8 juice broth - 1 B
Actinoplanes missouriensis 4 A 5 A

.A. missouriensis + BEE. 1 B 2 B

 

 

aSeedlings two days old with the radicle approximately 1 cm in length
were transplanted. Means of six replicate pots in Experiment 1 or five
replicate pots in Experiment 2, each with five seedlings. Values
followed by the same letter were not significantly different (£F0-01)-
PAm§_was grown on millet seed for 30 days then ground with water. Fifty
ml of Amg suspension was used to infest Readi-earth potting mix at a
concentration of 7 X 103 oospores/g mix. ‘A. missouriensis was
incorporated at a concentration of 103 sporangia/g mix.

cAm_s__oospores grown in V-B juice broth for 30 days were used to infest
steamed riverbed coarse sand at a concentration of 450 oospores/g sand.
A. missouriensis was applied as a drench at a concentration of 38
Sporangia/g sand.

dAutoclaved Amgfcolonized millet seed.

111

approximately 3 X 103/g. Populations of A. missouriensis or A. fuscoatra
on the seed averaged 105 CFU or 5 X 104 CFU, respectively. In a third
experiment, Am; oospores produced in soybean tissue (117) were used at a
concentration of 200 oospores/g soil. The cultures were 6-8 months old,
and unlike other Am§_cultures used were frozen for 3 days instead of 1-4
h prior to use. Concentrations on the seed were approximately 3 X 104

CFU's of A, missouriensis or A. fuscoatra.

 

In all three experiments plant stand was increased significantly
(A30.05) with the hyperparasites as seed treatments (Table 15). The
reason for the poor emergence of controls in Experiments 1 and 2 is
unexplained, but may have been due to poor seed quality or seedling decay
pathogens present in the mix. Twice the number of plants emerged when A.

missouriensis-treated seed were planted in soil artificially infested

 

with Am; in Experiment 1. In Experiment 2 when seeds were treated with
A. fuscoatra, almost one-third more plants emerged. In Experiment 3,

A? missouriensis increased emergence by 5 times, while A. fuscoatra

 

increased plant stand 3 times, as compared to stand in soil artificially
infested with Amé,

Additional biological control experiments. Many experiments that
were done to test hyperparasites failed because Am§_inoculum did not
’cause disease. Briefly, the agents, method of delivery, and numbers of
experiments done were as follows:

A, missouriensis: aqueous suspension, 5
attapulgus clay, I
4

seed,

it fuscoatra: aqueous suspension, 5
attapulgus clay, 2
barley seed, 1
seed, 2

112

Table 15. Biological control of Phytophthora megasperma var. sojae (Pms)

in the greenhouse by Actinoplanes missouriensis and Humicola

fuscoatra applied as seed treatments.

 

 

Emergence/12 seeds planted/pota

 

 

 

Treatment Experiment 1 Experiment 2 Experiment 3
Control 6 A 10 A 4 A
Am§_ 3 B , 7 B 1 B
Actinoplanes

missouriensis

+ BEE 6 A - 5 A
Humicola

fuscoatra

+ BEE - 10 A 3 A

 

aMeans of five replicate pots, each with 12 seeds. Values followed by the
same letter are not significantly different (A;D.05, Duncan's multiple
range test). AA§_inoculum for Experiments 1 and 2 was produced in V-8

juice broth; that for Experiment 3 was produced in soybean tissue.

113

A, catenoides: aqueous suspension, 2
.A. monospermum: aqueous suspension, 1
Failure was independent of Pms inoculum age, culture medium, or concen-

tration; potting medium; or time period of flooding prior to planting.

Effect of viable Pms mycelium on disease incidence and biological control

 

In three different experiments in which Pms inoculum was not frozen
to kill the mycelium, significant disease was obtained. ‘A, missouriensis

as a seed treatment was tested once, and A, missouriensis and A,

 

fuscoatra on clay were tested twice in attempts to obtain biological
control of both mycelium and oospores. No disease control was obtained,

probably because the mycelium is not susceptible to parasitism.

Field experiments

In the summer of 1979, biological control was tested in fields
naturally infested with Am; on John Majzel's farm. Phytophthora root
rot incidence was very low (0-3 infected plants/50 seeds planted)
despite severe disease being present the preceding year. No significant
differences were found among the treatments. Noodchuck damage to soybean
plants was considered too extensive for the data collected at the Shuman
farm to be meaningful. _

. Biological control was evaluated in 1980 in an artifically infested
plot at the Michigan State University Botany and Plant Pathology Farm,
and in a naturally infested plot at the Leo Shuman Farm. Disease
incidence was low in both areas, only 0-3 infected plants/200 seeds
planted at the Botany Farm and 4-12 infected plants/200 seeds planted at
the Shuman Farm. The addition of hyperparasites caused no statistically
detectable beneficial or detrimental effects. Using the soybean seedling

assay, AA§_was detected in the soil samples collected from the plot at

114

the Shuman Farm. There were no differences in the disease indices among
soil samples from rows treated with A, missouriensis-treated Turface

(2.0), A, fuscoatra-colonized barley seed (1.6), and A, catenoides-

 

treated vermiculite (2.2) and controls (1.9). Therefore, no disease
control was indicated using these hyperparasites. The soybean stem
segnent assay proved to be an unreliable indicator of Am_s_ occurrence.
Disease incidence was very low in naturally infested field plots in
which fall or spring hyperparasite application prior to seeding was
evaluated in 1980. No significant differences in numbers of infected
plants were found among the control or A, missouriensis- and A.
fuscoatra-infested soils at either time of hyperparasite application.
Disease incidence in the Fall plot ranged 3-4 plants/80 seeds planted and
in the Spring plot ranged from 4-6 plants/80 seeds planted. Soil samples
were collected from the rows of the spring plot, and baited with soybean
seedlings or stems to estimate the occurrence oqumg. Results showed no
differences in the presence of Am§_among treatments (range = 1.5-1.7),
and therefore indicate no biological control had taken place with
hyperparasite application in the spring prior to planting. The stem
baiting method gave results too inconsistent to be interpreted.
Biological control was assessed in 1981 in microplots at the
Michigan State University Botany and Plant Pathology Farm. Microplots
were artificially infested with Ami. 0f 25 seeds planted per microplot,
0-3 plants emerged even in uninfested controls. Stems and epicotyls of
some of the seedlings that emerged appeared red and shriveled, and
subsequently died. Soil samples collected from each microplot were
indirectly rated for disease potential using the soybean seedling assay.

All seedlings in the plates were rapidly destroyed, but microscopic

115

examination revealed Fusarium macroconidia, and two oospore types,
neither similar to Pms OOSpores. Plants in a soybean plot adjoining the
hyperparasite trial were diseased due to Fusarium. It was therefore

concluded that soybean losses in the microplots were not due to Pms.

DISCUSSION

The most significant new findings of this research are: i) the
extension of the host range of Am§_oospore parasites to include other
Oomycetes, ii) the discovery of several new oospore hyperparasites,

iii) the apparent direct penetration and rapid colonization of oospores
by Actinoplanes missouriensis and Humicola fuscoatra as seen with
electron microscopy, iv) the definition of the range of soil temperature,
water potential, and pH that influence oospore parasitism by A,
missouriensis and A. fuscoatra, and v) the demonstration of partial
biological control of Phytophthora root rot of soybean in the laboratory
and greenhouse.

The wide host ranges of the hyperparasites suggest that in nature
many Oomycetes are susceptible to hyperparasitic attack. The plant
pathogenic Oomycetes are, in general, difficult to control because host
resistance and chemical methods are not always available. The diversity
of the hyperparasites and the wide host range of many of them should lend
encouragement to their further study as potential biological control
agents.

Ayers and Lumsden (15) found in tests using agar that Hyphochytrium

 

catenoides infected 26% of Pythium aphanidermatum oospores, 21% of A.

 

ultimum oospores, and 64% of A, euteiches oospores. My work showed that
A, catenoides parasitized 20:7% of the A. aphanidermatum oospore

population, but only 17:6% of A. euteiches oospores and no A. ultimum

116

117

oospores. Humble and Lockwood (118) reported that approximately 64% of
Am§_oospores were infected by A. catenoides in steamed soil after 14 days.
.A. catenoides parasitized 54:4% of the oospores of Am§_in experiments on
water agar after 14 days in this study. The same isolates of host and
parasite as used by Humble and Lockwood (118) were also used in my work.
Differences between my results and those of Ayers and Lumsden (15) may
have been due to somewhat different methods employed, or to the use of
different isolates of hyperparasites and hosts.

Humble and Lockwood (118) determined that nearly 90% of Am§_oospores

were infected in steamed soil by Humicola fuscoatra after three weeks. In

 

my work, using different test media, but the same isolates of host and
parasite as Humble and Lockwood (118), parasitism of Am§_oospores by A,
fuscoatra was greater than 90% on water agar after 14 days, and in
autoclaved soil after only 7 days. Persistent microorganisms in the
steamed soil may have delayed parasitic action DY.fl- fuscoatra in the
studies of Humble and Lockwood (118). Sterile conditions and a higher
hyperparasite concentration may account for the higher rate of parasitism
of Ems oospores by A, fuscoatra in my work. Daniels and Menge (63)
detected more infection of Algmg§ spores by Anguillospora pseudolongissima
and A, fuscoatra in autoclaved soil than on water agar. In the present
work, agar was used because of its convenience. It is not known if
parasitism was underestimated using water agar, but in any case, host
range results are useful for comparative purposes.

Several new hyperparasites, all members of the actinomycetes, were

discovered. Two Actinoplanes species, A, philippinensis and A? utahensis,

 

 

and Amorphosporangium auranticolor, Ampullariella regularis, and

Spirillospora albida, members of the Actinoplanacae, and Micromonospora

 

118

Sp. of the MicromonOSporaceae, were found for the first time to infect

Phytophthora oospores. Hyperparasitism by Actinoplanes missouriensis was

 

 

commonly observed in flooded natural soil (188). The role of these
organisms, other than A. missouriensis, in nature has not been
established, but it may be possible that they play a role in the
population dynamics of Oomycetes in soil. The aquatic nature of the
Actinoplanes spp., Spirillospora albida, Hyphochytrium catenoides, and
Pythium monospermum may have led to the co-evolution of these organisms
with the plant pathogenic Phycomycetes. The production of zoospores by
these hyperparasites may be expected to increase the chances of contact
with oospores in soil.

Tetrazolium bromide (MTT) stain aided the assessment of parasitism,
and was frequently used when Amg was the test host. High percentages of
oospores of Am§_and the other Phytophthora spp. were stained pink or red
in a 0.1% MTT solution at 3521 C after 24 h. In contrast, only low
numbers of oospores of Aphanomyces spp. and Eythium ultimum stained with
MTT, presumably due to the lack of stain penetration. This was suggested
by the observation that oospores of these species reacted to MTT when in
the initial stages of invasion by a hyperparasite. A higher temperature,
higher MTT concentration, a longer incubation time, or a combination of
these conditions may allow for increased staining of A? euteiches, A?
cochlioides, or A. ultimum. The majority of A. cochlioides oospores
were stained rose-colored after an extended period of time, 20 days, in
0.1% MTT. '

In addition to the value of MTT as an oospore stain, hyphae,‘
sporangia, and spores of the hyperparasites stained blue-black or black

and were thus highlighted by the stain. Sporangia of the Oomycetes and

119

A, catenoides stained pink after approximately 15 min at 2312 C and
turned blue-black within 1 h or even sooner at 3511 C. Mycelia and
sporangia of the hyperparasitic actinomycetes turned a deep purple after
15 min incubation in MTT. The differential staining response of host and
parasite greatly facilitated the determination of oospore parasitism,
and MTT was used frequently in parasitic assessment of oospores by A?
missouriensis and A. fuscoatra.

Little is known about the process of infection by oospore hyperpara-
sites. All descriptions to date have been based upon observations made

with the light microsc0pe. Parasitism of oospores of Pythium myriotylum

 

by A. catenoides was described briefly by Ayers and Lumsden (15).
Zoospores of the hyperparasite encysted on the host wall. An infection
peg entered the cytoplasm. Sporangia developed within the host oospore
and released zoospores via short exit tubes into the environment. In the

case of the Imperfect Fungi, Dactylella anisomeres, A, helminthodes, A,

 

 

stenocrepis, A, stenomeces, Tricothecium arrhenOpum, and I, polyctonum

 

(74, 76, 77, 78), appressoria of various shapes were usually formed,

except occasionally with Q. helminthodes, Q, stenomeces or I, polyctonum,

 

where no differentiation occurred (76). An infection peg penetrated the
oogonial wall. Sometimes, a second enlargement or appressorium occurred
prior to penetration of the oospore wall by A, helminthodes, Q,
stenocrepis, A, stenomeces, and I. polyctonum. Assimilative branches
of the parasite became visible as the oospore cytoplasm was absorbed.

. In the present work, no evidence was found to indicate that

appressoria were formed by A, missouriensis or A, fuscoatra by light or

 

electron microscopy. Indentations of the host walls were not observed at

penetration sites, possibly due to the inherent rigidity of the walls.

120

The hyphae of A. missouriensis directly penetrated the oogonial and
oospore walls, and entered the cytoplasm without any noticeable change in
the diameter of the hyphae. However, penetrating hyphae of A. fuscoatra
appeared narrower than the vegetative hyphae (Figure 10). There was no
indication of enzymatic degradation of the oogonial or oospore wall
adjacent to penetrating hyphae of either hyperparasite, but without bio-
chemical tests, the role of enzymes in oospore parasitism cannot be ruled
out. The host did not respond to invasion by papillae formation, which
may possibly be explained by the dormancy of the oospores. Once the
hyperparasites infected the oospore cytoplasm, it rapidly disappeared,
and was replaced with the thallus of the hyperparasite. The mechanism(s)

of host-parasite recognition by A. missouriensis and A, fuscoatra is

 

still completely unknown. It is interesting to note, however, that
infection by these two organisms, a prokaryote and a eukaryote, appears
to be similar.

Experiments to determine the effect of environmental parameters on
parasitism by A, missouriensis and A, fuscoatra were done in sterile
soil. The exclusion of indigenous parasites by autoclaving the soil was
necessary to clarify the hyperparasitic relationship. Conditions highly

conducive to parasitism of Am§_oospores by A. missouriensis after 7 days

 

were 0 bars matric potential, soil temperatures of 15-30 C, and soil pH's
5.5 or 6.6-8.0. Optimum conditions for parasitism by A. fuscoatra were
-0.3 bars matric potential, soil temperature of 20-30 C, and soil pH's of
6.6-8.0. A parasitic preference for flooded soil by A, missouriensis and
unsaturated soil for A. fuscoatra had been alluded to by other workers
(118, 188). Soil pH in my experiments could not be maintained with

Good's buffers at -D.3 bars matric potential, the optimum soil moisture

121

for oospore parasitism by A. fuscoatra. To resolve this difficulty, pH
was tested at 0 bars where it could be stabilized. Parasitism of Am§_
oospores by A. fuscoatra was less than would be expected had soils been
at -O.3 bars.

It is difficult to eliminate variables that could interfere with
assessment of pH effects on hyperparasitism. The addition to soil of
buffers may bring about changes due to microbial utilization, degrada-
tion, or specific toxicity. The collection of various soils with a wide
pH span, such as 5-8, would likely introduce variability due to other
soil properties (e.g., structure, texture). Good's buffers were selected
for use because of their relative stability. The buffers MES, MOPS, and
HEPPS, when adjusted to a common pH, did not appear to interfere with
hyperparasitism of Am; oospores by A. missouriensis or A, fuscoatra.
Thus, it appears that the buffers used were not differentially inhibitory
to the hyperparasites. The results, however, should be verified using
different buffer systems.

It was hoped that results from the experiments on environmental
parameters might be used to define the limits of biological control with
Am; hyperparasites. Since there was such difficulty in obtaining
Phytophthora root rot, this could not be accomplished.

There was never any difficulty obtaining disease using the soybean
seedling-petri dish assay in early biological control work. On the
contrary it was thought disease might be too severe to detect biological
control. Therefore, greenhouse experiments were initiated using
naturally infested Am; soil. Although a reduction of plant height and
root length was demonstrated in one experiment, there was not enough

disease to evaluate biological control. The problem was never solved

122

throughout the course of this work in spite of much effort directed
toward this goal.

Biological control of Am§_in the greenhouse was demonstrated using
.fi- missouriensis- and A, fuscoatra-treated seed. These results suggest
that under some conditions biological control of Am§_may be feasible.
Difficulties obtaining disease in the greenhouse may reflect the
inconsistent nature of disease in the field. Most often soybean plants
in the field are infected in a linear pattern. Plants adjacent to each
other, in groups of three to five, frequently show cankers. Mr. Shuman's
soybean field in 1978, however, was evenly infected with Am§_and it was
difficult to find a healthy plant in approximately 50 acres. The soil in
this area was assayed in June of 1979, and at that time the disease index
averaged 2.7 on a scale from O to 3. In the summer of 1980, soil samples
from selected areas caused root rot in the seedling assay indexed at 1.7
to 1.9. If the assay is reliable, indigenous Am; populations would
appear to have declined from 1979 to 1980. Credibility of the assay
would have been strengthened had there been more disease in the field
plots.

Very little disease was obtained during the three years of field
experiments using natural or artificially grown Am§_inoculum. It is
possible that the environment was not conducive to severe infection of
soybeans, or to symptom expression. It may also be possible that Am§_
populations were reduced by indigenous parasites, and that biological
control was taking place without the intervention of man. These
alternative hypotheses need to be evaluated in future work.

The difficulty in obtaining Phytophthora root rot in the greenhouse

and field experiments does not preclude biological control as a viable

123

means to control disease and diseases caused by related pathogens.
Several findings of this research lend support to the feasibility of

using hyperparasites to reduce disease caused by Phytophthora. These

 

entail the wide range of oospore hyperparasites now identified (15, 74,
76, 77, 78, 118, 188), including several actinomycetes found for the
first time to be oospore hyperparasites. The broad host range of these :
parasites also offers many options for their applied usage. Many of
these hyperparasites grow well on common laboratory media and, therefore,
the mass production of inoculum in fermentation vats designed for
commercial production of microorganisms may be feasible. Large scale
fermentation may be especially adaptable to the culture of the
hyperparasitic actinomycetes due to their bacteria-like metabolism. .
Culture age of the hyperparasites A. missouriensis and A, fuscoatra did
not affect their hyperparasitic abilities, which allows flexibility in
the duration of inoculum production. The parasitic ability of A.
fuscoatra may be a genetically stable trait, since each of four single
spore isolates, and an albino isolate, were highly virulent. The

population densities of A, missouriensis and A, fuscoatra on treated seed

 

and other carriers remained stable for 2-12 months after drying. This
stability suggests that the shelf-life of biocontrol preparations of
these organisms may be adequate. Finally, the reduction in Phytophthora
root rot obtained by hyperparasites introduced into naturally infested
soil in the laboratory, and by hyperparasite-treated seeds in the
greenhouse suggests that this approach to control Phytophthora diseases

merits continued research effort.

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