V. Synergisms That May Affect the Effectiveness of Microbial Agents

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death of leaves and shoots or even entire plants whereas infection by the rust

alone seldom caused death. Rust-free plants, even when severely wounded, were

not infected by F. roseum. Thus it appears that a facultative parasite, incapable of

infecting a plant alone, contributed to increased disease severity by invading

lesions produced by another pathogen. This phenomenon has been extended to

biological control of weeds.

Alternaria macrospora Zimm. has been investigated as a potential mycoherbicide to control spurred anoda (Anoda crisrata (L.) Schlecht.) in the United

States (Crawley et al., 1985; Walker, 1981; Walker and Sciumbato, 1979). The

susceptibility of spurred anoda to A . rnacrospora is highly correlated with plant

age. Approximately 100% of seedlings inoculated at the cotyledonary stage were

killed by infection, but less than 10% were killed at the three- to four-leaf stage

(Walker and Sciumbato, 1979). However, 100% of plants inoculated at the fourto five-leaf stage were killed by the interaction of A . rnacrospora and Fusarium

lateritium Nees ex Fr. Fusarium lateritium is a weak pathogen of spurred anoda

and causes less than 20% mortality when inoculated to seedlings alone, although

F. laterilium usually killed wound-inoculated spurred anoda seedlings (Crawley

et al., 1985). The highest mortality occurred when Afternaria was inoculated 5

days before Fusarium. When Fusarium was inoculated 5 days before Alternaria,

only 1 1 % of the seedlings were killed. One suggested explanation for the Afternarial Fusarium interaction was that F . lateritium penetrated and infected

through the lesions caused by Alternaria. These results indicate that sequential

applications of both fungi were more effective than either fungus used alone for

control of spurred anoda.

Mortality of groundsel infected by P . lagenophorae has been attributed to

invasion of rust lesions by Botntis cinerea Pers. (Hallett et al., 1990a,b). Inoculation of healthy groundsel with B . cinerea caused only 10% mortality and only

40% mortality of abiotically wounded plants: however, all plants previously

infected by P . fagenophorae died after inoculation with Botrytis. Death of plants

was attributed to growth of Botryris into stems. The time necessary to kill plants

was dependent upon several factors, including the inoculum concentration of

Botrytis and initial pustule numbers of the rust (Hallett, 1990a.b).



B. SYNERGISM

OF PATHOGENS

WITH INSECTS

Insects and plant diseases may have played a role in the control of prickly pear

in Australia and other countries (Wilson, 1969). However, the relative importance of pathogens in biological control of prickly pear is not clear since most of

the intensive research focused on the role of insects and Cactoblastis cacrorum

Berg rather than on potential fungal and bacterial pathogens (Wilson, 1969).

Similarly, introduction of the insect Proceidorchures utilis Stone into Australia



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127



from Hawaii in 1952 may have coincidentally introduced a pathogen, Cercospora eupatorii, that resulted in the build-up of a leaf spot disease of crofton

weed, Eupatorium adenophorum Spreng. The combined effect of this insect and

pathogen (with native insects) may have contributed to control of crofton weed

(Dodd, 1961).

Cercospora rodmanii is a pathogen of waterhyacinth, Eichornia crassipes,

with biological control potential as first described by Conway (1976a). This

fungus causes a leaf spot disease, is highly virulent to water hyacinth and is

capable of inflicting severe damage on waterhyacinth (Conway, 1976b; Charudattan, 1986). Waterhyacinths are infested with a variety of fungi and bacteria

and at least two insects, the weevil Neochefina eichorniae Warner, and a mite,

Orthogaliimna terebrantis Wallwork (Charudattan et al. , 1978). It was generally

concluded that arthropod-infested plants were more diseased than plants without

insects (Charudattan el al., 1985). Charudattan (1986) concluded that insects and

Cercospora in combination could control up to 99% of the waterhyacinth plants

in treated areas within 7 months after treatment and that their interaction was

synergistic in nature.

More recently, Yang and TeBeest (1992b, 1994) showed that green treefrogs

(Hyla cinerea Schneider) and grasshoppers, Conocephalis spp. and Melanoplus

diTerentialis (Thos.), may be important vectors in the dispersal of inoculum and

disease caused by C . gloeosporioides f.sp. aeschynornene on northern jointvetch.

Their reports, however, did not attempt to quantify the role these two vector

groups played in the control of northern jointvetch by this commercial biological

pesticide. It was noted, however, that most of the treefrogs carried the pathogen

on their bodies, while only a small but significant fraction of the grasshoppers

captured in rice fields carried the pathogen on their mouthparts or feet.

A strain of the fungus Colletotrichum gloeosporioides has been associated

with mortality of St. John’s-wort, Hypericum perjoratum L., and is under investigation as a potential biological control agent for that weed (Hildebrand and

Jensen, 1991) in Canada. An insect, Chrysolina hyperici Forst., has been intoduced and is established in Nova Scotia; it is often observed feeding on St. John’swort but has not given adequate control. Although the fungus effectively spread

to control plots in field tests, Hildebrand and Jensen (1991) did not address

whether this dispersal resulted from insect or environmental factors or if the

disease and insect acted together to help control this weed.

It is also possible that even while insects and pathogens may occur on the same

host plant, an interaction is not established. For example, Linders (1994) studied

the interaction between fungal pathogens and an insect herbivore on Planfago

lanceoluta L. After a thorough study, it was shown that a weevil, Trichosirocalus

froglodytes Fabr., had only marginal if any interaction with a pathogen, Diaporthe adunctr (Rob.) Niessl., that affected the ability of the pathogen to infect

and control this weed.



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C. SYNERGISM

OF PATHOGENS

WITH CHEMICALS

A promising consideration concerning possible chemical interactions with

plant pathogens is the utilization of various chemicals, including herbicides, to

increase the effectiveness of biological agents by weakening host resistance to

infection by a pathogen (Charudattan, 1993).

Colletorrichurn coccodes (Walk). Hughes is a pathogen of two nightshade

species, Solanurn ptycantlium Dun. and Solanum sarrachoides Sendt., and velvetleaf, Aburilon rheophrasti Medik. (Anderson and Walker, 1985; Eberlein et

al., 1991; Wymore et al., 1987, 1988). Although the fungus infected velvetleaf

over a wide range of dew period durations and temperatures, disease was most

rapid and destructive at 24°C following a 24-hr dew period. Velvetleaf plants at

all growth stages were susceptible and were reduced in vigor following inoculations, but only plants at the cotyledonary stage were killed (Wymore et a [ . ,

1988). Under less than optimal conditions, the fungus was unable to kill seedlings in the field. Tank mix applications of C . coccodes and thidiazuron ( N phenyl-N'- 1,2,3-thidiazol-5-yl-urea)

acted synergistically to increase velvetleaf

mortality when compared with the fungus alone (Wymore et a f . , 1987; Hodgson

el al., 1988).

Gohbara and Yamaguchi (1993) showed that the combined use of the herbicide

pyrazosulfuron-ethyl and the fungus D . monoceras showed significant synergisim

in controlling barnyardgrass in rice in Japan. Application of pyrazosulfuron-ethyl

at 180 mg/a with 109 conidia/a caused 100% mortality of seedlings, exceeding

control of seedlings by pyrasulfuron alone or with similar concentrations of

conidia alone. Similarly, Scheepens ( 1987) showed that atrazine was synergistic

with the pathogen Cochliobolus lunutus for control of Echinochlou crus-galli (L.)

Beauv. in maize (Zea mays L.) in The Netherlands. In controlled experiments in a

greenhouse or growth chambers, barnyardgrass seedlings could be controlled with

the fungus after treatment with a sublethal dose of atrazine.

Alternuria cassiae Jurair & Khan was intensively studied as a potential bioherbicide for sicklepod, Cassiae obtusifolia L, in the United States. Sharon et al.

(1992) showed that suppression of a phytoalexin biosynthesis with a sublethal

dose of glyphosate increased the susceptibility of C . obtusifolia to infection by A .

cassiue. Also, five times less inaculum was needed to control sicklepod when

applied with glyphosate than without the herbicide, without affecting its specificity. This technique offers an intriguing approach to enhance the pathogenic

effects of many pathogens and, therefore, also to increase their potential effectiveness as microbial pesticides.

In yet another unique approach to increase the effectiveness of biological

pesticides by aiding the establishment of infections in the abscence of conducive

environmental conditions, Boyette et al. (1993) and Egley er a f . (1993) used

invert emulsions to reduce the free moisture requirements for germination of



BIOLOGICAL CONTROL OF WEEDS



129



conidia of Colletotrichum truncatum (Schw.) Andrus & W. 0. Moore to control

hemp sesbania, Sesbania exaltata L. When sesbania seedlings were sprayed with

aqueous suspensions of conidia and were not given a postinoculation dew treatment, only a few seedlings were killed by infection. However, 100% of the

seedlings sprayed with invert emulsions of condia that did not receive a dew

treatment were killed within 7 days after inoculation. These results were as

expected because the moisture requirements for conidial germination were satisfied by the invert emulsion.

Similarly, Amsellem et al. (1990, 1991) showed that the inoculum thresholds

needed to control C . obtusifolia and Datura stramonium L. with A . cassiae and

Alternariu crassa (Sacc.) Rands, respectively, were reduced to one conidium per

droplet when an invert emulsion was used as an adjuvant for the inoculum.

However, invert emulsions also have the potential for crop damage, and the

significance of potential crop damage by mycoherbicides in invert emulsions is

not clear (Amsellem, 1991).

A concern with the use of C . gloeosporioides f.sp. aeschynomene, and potentially for other mycoherbicides on other crops as well, is integration with fungicides used to control rice and soybean diseases. Proper timing of fungicide

treatments with C . gloeosporioides f.sp. aeschynomene enabled the effective use

of both pesticide groups (Klerk et al., 1985). Colletotrichum gloeosporioides

f.sp. aeschynomene has been integrated into the rice management system in

Arkansas, which utilizes chemical pesticides to control weeds, insects, and diseases (Kenney, 1986; Klerk et al. 1985).



VI. THE ENVIRONMENTAL IMPACT OF

MICROBIAL HERBICIDES

A. EFFECTS

ON NONTARGET

BENEFICIAL

PLANTSPECIES

The ability of plant pathogens of weeds to infect cultivated and noncultivated

plant species is a serious and important part of every effort to develop a commercial product for biological control of weeds. Almost without exception, these

pathogens can and do infect cultivated and horticulturally important plant species

in controlled experiments. And of course, numerous pathogens of crop plants

also infect weeds. Because of necessity and importance, various schemes to

identify the susceptible plant species and the host range of each plant pathogen

under consideration for weed control have been developed (Wapshere, 1974).

Colletotrichum gloeosporioides f. sp. aeschynomene has been used in a registered product, COLLEGO, since 1982 to control a single weed species, A .

virginica, in rice, Oryza saliva L., and soybeans, Glycine max Merr., in the



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United States. This fungal pathogen infects several species within the genus

Aeschynomene. However, it also infects species within eight other genera, including Cicer, Indigofera, Lathyrus, Lens, Lotus, Lupinus, Vicia, and Pisum. It

is important to note, however, that the fungus is highly virulent only to A .

virginica and Lathyrus arboreus L., although certain cultivars of Pisurn sativurn

L. were also severely infected (TeBeest, 1988; Weidemann et a l . , 1988).

Similarly, the host range of P . palmivora, the fungus formulated as DeVine,

includes species other than M. odoruta, the intended target (Ridings et a l . ,

1976). The host range of P . palmivora includes onions, cantaloupe, watermelon,

okra, tomato, endive, cucumber, english pea, and carrot, and even certain citrus

root stocks based on greenhouse tests of preemergence, postemergence, and

foliage inoculations.

On the other hand, the host specificity of C. gloeosporioides f.sp. malvae

appears to be more limited than either of the other two commerically available

bioherbicides. Colletotrichum gloeosporioides f. sp. malvae appears to be specific to plants within a single family, the Malvaceae, except for two species,

safflower (Carthamus tinctorius) and white mustard (Brassica hirta) (Mortenson,

1988). However, infections of either of these two species did not affect the

plants. The fungus infected weedy and ornamental species within the Malvaceae

with disease being most severe on certain Malva species and Abutilon theophrasti Medic, which is also a weed.

These three examples are sufficient to illustrate two obvious facts. First, weed

pathogens can and do infect nontarget cultivated and noncultivated species.

Second, infections of nontarget species are occasionally severe. Host range

testing protocols, however, have been gradually developed and adopted to identify potential nontarget cultivated and noncultivated host species. Similarly, nearly

all host-range tests have been conducted in greenhouses or growth chambers

under carefully controlled conditions. It is not certain if host ranges tests conducted and determined in this manner have any direct relationship to similar tests

conducted in the field.



B. EFFECTS

ON NONTARGET

SPECIES

AND FOOD

WEBCOMPONENTS

Colletotrichum gloeosporioides f. sp. aeschynomene has been used in a variety

of test systems to evaluate the infectivity of microbial pest control agents on

nontarget species (Fournie et a l . , 1988; Genthner and Foss, 1991, 1994). Fournie

et al. (1988) showed that this plant parasitic fungal species did not cause adverse

effects to a series of test species including fish (Cyprinodon variegatus), a crustacean (Paleomonetes pugio), or a bivalve mollusc (Crassostrea virginica). The

test system employed in this work could be used effectively to evaluate other