0261-2194(95)00017-8 C,oP Protection Vol. 14. No. 3, pp. 179-186. 1995

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Biological control of Rhizoctonl’a solani by binucleate Rhizoctonia spp. and hypovirulent R. solani agents Leonard J. Herr

Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and

Deveiopment Center Wooster, OH 44691, USA

Despite research on biological control dating back to the early 193Os, reliable, economical biocontrols of diseases caused by Rhizoctonia solani are not generally available commercially. New approaches for detection and use of novel agents and development of broadly applicable biological control management systems are needed, especially for field crops. During the past 10 years, new sources of agents from within the diverse groups of binucleate Rhizoctonia spp. and hypoviwlent R. solani isolates have been demonstrated to be effective in biocontrol of a range of host - R. soluni disease combinations. Although these agents include isolates from several different binucleate Rhizocfonia anastomosis groups (AG) and hypovirulent R. solani AG, neither mycoparasitism nor antibiosis is involved in biocontrol of R. solani by any of these isolates. Postulated mechanisms of biocontrol include induction of systemic host resistance, and/or competition for recognition and invasion sites or nutrients. Tested collections of binucleate Rhizoctonia spp. and hypovirulent R. solani differ markedly in effectiveness as biocontrol agents for diseases caused by R. soluni. Reportedly, plant surface-colonizing isolates (i.e. on or superficially within outer tissues of roots, crowns, hypocotyls, stems or petioles) are effective biocontrol agents, whereas, non-colonizers are ineffective.

Keywords: plant surface colonization; mechanisms of biocontrol; induced systemic host resistance; competition for invasion sites; competition for nutrients; protected hosts

Rhizoctonia diseases in field, vegetable, ornamental, nursery and greenhouse crops cause large economic losses to growers (Baker, 1970), which result in incresed costs to consumers. Strains of Rhizocroniu solani Kiihn, and certain other Rhizoctonia spp. are capable of attacking a wide range of hosts, causing seed decays, damping-off of seedlings, stem cankers, root rots, fruit decays and foliage diseases (Raker, 1970; Ogoshi, 1985). Moreover, R. sofani occurs world-wide in most cultivated soils and is indigenous in many uncultivated areas (Ogoshi, 1987).

At present, Rhizoctonia diseases on some hosts are treatable with fungicides and a very few diseases can be controlled by use of resistant varieties. Many of these diseases, however, are ameliorated by cultural and cropping practices only (Leach and Garber, 1970; Cook and Baker, 1983). For small acreages of high value crops, such as greenhouse and nursery crops, pathogen eradication by heat treatment or chemical fumigation is useful, but re-infestation often presents renewed problems prior to harvest (Baker, 1957). Fungicides pose some risk to health, water quality and other aspects of the environment. Additionally some fungicides vary in effectiveness among, and within, anastomosis groups (AG) of R. soluni (Kataria, Verma and Gisi, 1991), suggesting that their extensive use could result in development of resistance as occurs within other plant pathogen populations (Cook and Baker, 1983). In theory, biological control could

reduce dependence on fungicides, by replacing or reducing fungicide applications on crops. Additionally, there is a necessity to develop biological controls for diseases for which there are no controls or only partial controls (Cook, 1993). However, most existing bio- logical controls fail to give consistent results or adequate levels of control of target diseases, except in specialized crop production {Cook and Baker, 1983). Special use exceptions for Rhizoctonia diseases include a Glio- cfudium virens biofungicide (Lumsden, Locke and Walter, 1991) marketed as Glio GuardTM by 0. M. Scott in the USA for control of damping-off diseases of greenhouse vegetables and ornamental plants. Myco- stop, a formulation of Srreptomyces sp. marketed by Kemira Oy in Finland is reported to protect against damping-off and other diseases. New approaches for detection and use of novel agents are called for in addition to continued studies of the extensively investigated traditional agents (e.g. Trichoderma , BUCillus) Streptomycetes, Penicillium, and Pseudomonas).

Several binucleate Rhizoctonia spp. (BNR) and hypovirulent R. solani isolates show promise as new agents. Hypovirulence is used herein sensu Marcus et al (1986). Isolate within pathogen populations in general (Marcus et al., 1896) and of R. solani AGs in particular, exhibit a continuum between high virulence to avirulence (Herr and Roberts, 1980; Herr, 1982, 1992; Ichielevich-Auster et al. 1985a). Often it is

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difficult to differentiate low virulence from avirulence among isolates. Such naturally occurring, low virulent to avirulent, healthy isolates of R. solani were collect- ively designated as hypovirulent isolates by Marcus et al. (1986). A number of plant diseases caused by pathogenic isolates of various R. solani AGs were controlled by these agents, in field as well as in greenhouse tests and at rates of application within practical and economic limits (Cardoso and Echandi 1987a; Escande and Echandi, 1991a; Herr, 1988c, 1989b; Harris, Schisler and Ryder, 1993; Harris et al., 1994). These Rhizoctonia spp. agents are related to pathogenic R. solani types and therefore are probably similar ecologically and physiologically in many respects, except that they are either: (a) phytoplane (plant surface) non-parasitic non-pathogens; (b) mild, symptomless parasites (Daniels, 1963); or (c) mildly virulent pathogens (Cook and Baker, 1983). More explicitly, phytoplane non-parasitic non-pathogens denote fungal agent-plant surface colonizers demonstrably growing on plant surfaces without penetration of the outer tissues of plant organs. A mild symptomless state of parasitism (mild symptomless parasitism) of R. solani isolates on many weed hosts was described by Daniels (1963) as consisting of ‘Many . . . hyphae closely applied to root surfaces . . . (with) penetration of the root surface here and there without (formation of) appressoria or other (penetration) structures and without . . . visible symptoms on the host.’ A third category of agent-host relationship is mild pathogenicity characterized by invasion of plants by hyphae, which is accompanied by limited depression of plant growth (Daniels, 1963). Subsequently, Cook and Baker (1983) referred to the invasive agents of biocontrol as mildly virulent pathogens. Disease severity is limited to lesions of little consequence to plant integrity relative to the protection conferred on the infected plants (biocontrol) against highly virulent pathogens. Agents possessing any of these capabilities may compete with virulent R. solani isolates for rhizosphere nutrients and/or colonize plant surfaces and compete with virulent isolates for host recognition and invasion sites, or may be capable of inducing systemic host resistance. One or more of these charac- teristics may serve as mechanisms of biocontrol.

The purpose of this review is to develop some broad concepts of biological control of R. solani by BNR and hypovirulent R. solani agents based on data accumu- lated over the past 10 years. Despite the pathogenic heterogeneity of R. solani, the wide range of hosts and diseases, and the diversity of BNR and hypovirulent R. solani agents of biocontrol, some commonalities, relevant terminology and potential research approaches will be explored and expounded upon.

Agents and crops protected

Binucleate Rhizoctonia agents have been demonstrated to control R. solani diseases in a variety of hosts and cropping systems over the past 10 years. The Cerato- basidium anastomosis grouping (CAG) system of Burpee et al. (1980b), consisting of seven groups (CAG-1 through CAG-7), was used to designate the BNR agents in early biological control studies in the

USA., More recently, however, the more extensive binucleate Rhizoctonia anastomosis classification system (AG-A through AG-S) developed by Ogoshi et al. (1979) and Ogoshi (1985) has largely superseded the CAG system. Ogoshi (1985) ascertained equivalents for the specific CAG, under his system and has listed them (Ogoshi, 1985; Sneh, Burpee and Ogoshi, 1991). BNR agents will be reported here as given by their authors. In later sections equivalents of CAGs in Ogoshi’s system (Sneh et al., 1991) will be given as needed.

Among the first reports of the use of BNR as biocontrol agents against R. solani are those of Bell, Sumner and Mullinix (1984) and of Burpee and Goulty (1984). Bell et al. (1984) reported that heat-treated soil artificially infested with R. solani AG-4 plus a CAG-2 BNR agent and planted to snapbean 2 or 5 weeks later had a significantly (P = 0.01) higher percentage of normal bean plants than did soil infested only with AG-4. Furthermore, higher numbers of normal corn brace and crown roots were present in soil infested with R. solani AG-2-2 plus a CAG4 agent and planted to corn 2 or 5 weeks later, than in soil with AG-2-2 alone. Burpee and Goulty (1984) found a close associa- tion between non-pathogenic BNR and R. solani on turfgrasses and investigated the possible role of BNR in disease suppression. In short-term (8 day) field experi- ments, creeping bentgrass had significantly (P = 0.05) less disease when treated with BNR inoculum (un- specified CAG) 24 h before inoculation with R. solani AG-4 than when inoculated with R. solani alone. BNR isolates differed significantly in their ability to suppress disease.

Beginning in 1985, Cardoso and Echandi published a series of articles on protection of snapbean seedlings from infection by R. solani AG-4 with BNR (Cardoso and Echandi, 1985, 1987a, 1987b). Of 11 BNR isolates investigated for control of bean root rot in greenhouse and field tests, four gave significant disease control in greenhouse tests and were effective in one or more field tests (Cardoso and Echandi, 1987a). Isolate BN 160 gave significant (P = 0.05) disease control in all field tests. BN 160 was characterized as a CAGJ in a subsequent study (Cardoso and Echandi, 1987b).

Biological control of Rhizoctonia canker on potato, caused by R. solani AG-3, with BNR was studied by Escande and Echandi (1991a, 1991b). In greenhouse screening tests, eight of 15 BNR isolates reduced the incidence and severity of stem canker in artificially- infested pasteurized soil by an average of 78 and 85%, respectively. Four isolates, selected by greenhouse tests, also reduced (P = 0.05) canker severity in one or more field experiments. BNR isolates 232 CG and J-3S4-3 protected potato against Rhizoctonia stem canker throughout the season, in a field naturally infested with R. solani AG-3. Stolon canker was reduced by 232 CG and JF-3S4-3 by 23 and 45%, respectively, at harvest. Protection was similar to PCNB and superior to Tops 2.5D (thiophanate methyl) fungicides (P = 0.05). -A non-significant interaction of potato cultivar (six cultivar) times biocontrol agents on disease control indicates protection against stem canker is not cultivar dependent.

Cubeta and Echandi (1991) found in greenhouse tests using pasteurized soil artificially infested with R.

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solani AG-4, that treatment of the soil with eight of 19 BNR isolates significantly (P = 0.05) reduced pre- emergence damping-off caused by R. solani compared with the R. solani-infested control. In naturally-infested soils, both R. solani and Pythium spp. caused damping- off of cucumber and, therefore, the BNR agents, which were effective against R. soluni only did not adequately control Pythium damping-off. Integrated approaches to control of both R. solani and Pythium spp. were investigated in field soils. Several strains of Enterobacter cloacae combined with AG-G isolate 232-CG, the latter also used by Escande and Echandi (1991b), failed to control damping-off in field soils. Planting of Pseudo- monas fluorescens-treated seeds of cucumber in BNR 232 CG-infested soil also did not protect the seedlings against damping-off in the field. Both of these bacterial agents were previously reported to control Pythium damping-off diseases (Howell and Stipanovic, 1980; Nelson, 1988). Amendment of naturally-infested soils in the field with agent 232 CG and metalxyl gave the most consistent and effective protection against both Rhizoctonia and Pythium damping-off.

Herr (1986) reported that in initial greenhouse assays of BNR isolates, GM 460 (= BN-l), applied to 6-9 week-old plants, consistently gave control of Rhizoctonia crown and root rot of sugar beet, caused by R. soluni AG-2-2 IV. Of 10 BNR isolates obtained from Ohio sugar beet production areas, seven effect- ively controlled crown and root rot in greenhouse tests (Herr, 1986, 1988~). All 10 of these Ohio BNR isolates belong in AG-B(o) (Herr, 1991) of Ogoshi’s BNR anastomosis system (Ogoshi et al., 1979; Ogoshi, 1985). Ogoshi’s AG-B(o) tester isolate (Og 177) from Japan was as effective in greenhouse tests as GM 460, the selected standard Ohio reference AG-BcO) agent (Herr, 1988~). Two BNR isolates, (applied either post- thinning or pre-layby) were effective in a 1986 field test (Herr, 1987, 1988~) and increased yields ca. 22% in comparison to the no agent-R. solani infested control. In a 1988 field test, two effective BNR isolates (GM 460 and W 157), applied pre-layby gave a >50% increase (~1.5 fold) in sugar beet yield over that of the no agent-R. soluni infested control treatment on a silty clay loam soil; whereas on a sandy loam soil the best BNR biotic agent (W 157) gave a 383% increase (ca. 4.8 fold) in yield over the no agent-R. solani infested control (Herr, 1989b).

In Nebraska tests, the Ohio GM 460 isolate signific- antly (P = 0.05) controlled brown patch disease of tall fescue caused by R. solani AG-l-1A in a growth chamber and field experiments (Yuen and Craig, 1992; Yuen, Craig and Giesler, 1994). GM 460 applied to tall fescue in the field persisted for 1 month and reduced brown patch from 35% blighted turf in the control to less than 20%. Other BNR isolates from turfgrass in Nebraska were not as effective as the Ohio GM 460 isolate from sugar beet field soil. This use of a BNR biotic agent from Ohio, effective in biocontrol of Rhizoctonia (AG-2-2 IV) crown and root rot of sugar beet in Ohio to control Rhizoctonia (AG-l-1A) brown patch on tall fescue turf under Nebraska conditions indicates that isolate GM 460 is a robust agent effective against different R. solani AGs on different hosts under presumably quite different sets of environmental conditions.

Nine non-pathogenic BNR isolates obtained from greenhouse potting media and nursery plants in South Australia (Schisler, Neate and Masuhara, 1993; Masuhara, Neate and Schisler, 1993) were screened as putative agents for biocontrol of Rhizoctonia damping- off caused by R. solani AG-4 or AG-8 on seedlings of Capsicum annuum, Celosia argentea and Petunia hybrida in an artificially-infested pasteurized potting medium in growth chamber assays (Harris et al. 1994). All nine BNR isolates reduced (P = 0.05) damping-off of Capsicum caused by AG-4. In glasshouse experi- ments two isolates, BNR 1 and BNR 2, controlled damping-off (AG-4) in Cupsicum (P d 0.01) as well as the fungicide PCNB and better than Bacillus subtilis A 13, an Australian commercial bacterial seed treatment for Rhizoctonia damping-off. In another test, both isolates reduced damping-off in Celosia and Viola seedlings and one isolate reduced damping-off in Petunia. These two BNR isolates are of particular interest because they also control Pythium damping-off (Harris, Schisler and Ryder, 1993) and thus have great potential usefulness for control of both Rhizoctonia and Pythium damping-off of nursery crops. The AG(s) to which BNR 1 and BNR 2 belong were not reported (Harris et al., 1993, 1994).

A hypovirulent R. solani AG-4, isolate 521, protected cotton and radish seedlings (P = 0.01) against damping- off caused by a virulent AG-1 and three AG-4 isolates, when the 521 agent was applied 8 days before the virulent isolates and 5 days before seedling (Ichielevich-Auster et al., 1985b). Isolate 521 was the most effective of the eight hypovirulent R. soluni tested in these greenhouse tests in nonsterile potting soil mix. Wheat seedlings were also protected against damping- off indicating that a diverse group of crops (radish, cotton and wheat) were protected against R. soluni damping-off by the 521 agent.

Characterization and pathogenicity of agents

With exceptions, many of the effective BNR isolates used for biocontrol of R. solani and fewer of the less effective or ineffective BNR have been characterized by AG and intraspecific groupings (ISG). Bell et al. (1984) used a CAG-2 to control AG-4 on bean and a CAG-4 to control AG-2-2 on corn. In the AG system of Ogoshi (1985) for BNR, these CAG designations (system of Burpee et al., 1980b) are relegated to AG-A and AG-F, respectively. While these BNR (Bell et al., 1984) were undoubtedly non-pathogenic on the protected hosts (bean, corn), each has been reported to be pathogenic on other hosts. Ogoshi (1985) attributed the following diseases to isolates within AG-A: straw- berry root rot, damping-off of sugar beet seedlings, tortoise shell-like symptoms of potato tubers, and browning of peanut pods. Although Ogoshi (1985) and Sneh et al. (1991) list no pathogens in AG-F, Burpee et al. (1980a) reported CAG-4 (AG-F) caused pre- and post-emergence damping-off of bean, pea and tomato seedlings. An isolate belonging to CAG-5 was highly effective in controlling bean root rot (Cardoso and Echandi, 1987b). CAG-5, which is AG-R in the system of Ogoshi (Sneh et al., 1991) was reported by Burpee et al. (1980a) as a damping-off pathogen of

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bean, pea and tomato seedlings. Two BNR agents effective in control of Rhizoctonia canker of potato were characterized as AG-G by Escande and Echandi (1991a). According to Ogoshi (1985), AG-G isolates constitute one of the three binucleate AG within R. fruguriue Husain and McKeen, which is pathogenic on strawberry. Moreover, isolates of AG-Gare pathogenic on peanut, causing browning of peanut pods (Ogoshi, 1985). Although these BNR groups (AG) that include biocontrol agents can include pathogenic isolates, with few exceptions little information is available on the incidence and severity of the diseases caused by virulent isolates on many of the reported plant hosts. Furthermore, it does not follow that all isolates within given AG are necessarily virulent on all or any of the reported hosts. Support for this opinion is given by use of a hypovirulent R. soluni AG-4 isolate as an effective biocontrol agent for suppression of damping-off in cotton, radish and wheat seedlings caused by virulent isolates of R. soluni AG-4 and R. zeae (Ichielevich- Auster et al., 1985b).

In this context, Ichielevich-Auster et al. (1985a) assessed the virulence and host specificity of 129 isolates of Rhizoctoniu spp., within AG-1,2,3,4,5 and 6 of R. soluni, two groups of R. zeue, and AG-A, F and K of binucleate Rhizoctuniu spp. on six to 11 different host plants. Most tests were run in plates of agar or sterile filter paper on seedling hosts, which may or may not relate to older plants or to field tests. However, radish, carrot, lettuce and cotton plants remained symptomless throughout the growing season in field plots infested with the hypovirulent AG-4 isolate 521. R. soluni (presumably isolate 521) was reisolated from roots and hypocotyls of treated plants. Approximately 30% of all collected Rhizoctoniu spp. isolates were non-pathogenic on all hosts tested. Thus, hypovirulence is not a rarity in natural Rhizoctoniu populations. Ichielevich-Auster et al. (1985a) concluded that, in Israel, natural populations of R. soluni consist of both virulent and hypovirulent strains and that the hypo- virulence observed was non-transmissible cytoplasmic- ally among hypovirulent strains.

The 10 -Ohio BNR isolates tested for biocontrol of Rhizoctonia crown and root rot of sugar beet (Herr, 1988~) all belonged to a single subgroup, AG-B(o), of AG-B (Herr, 1991). On the basis of variation in cultural characteristics and efficacy as biocontrol agents, this group of 10 biotic agents was quite heterogeneous. Herr (1989a) studied the host range of the Ohio BNR isolates in greenhouse pathogenicity tests and found them to be non-pathogenic on both seedlings and older plants of cabbage, cucumber, corn, soybean, sugar beet, tobacco, tomato and wheat. However, other isolates of AG-B(o) were reported to be pathogenic, causing sheath blight-like lesions of rice (Ogoshi et al., 1979; Ogoshi, 1985). Although rice was not tested as a host by Herr (1989a), the Ohio AG-B(o) isolates were non-pathogenic on corn and wheat (Herr, 1989a) and isolate GM 460 was reported to be non-pathogenic on tall fescue (Yuen et al., 1994), bluegrass and bentgrass (Yuen, personal communication).

Three AG-K BNR isolates, which significantly decreased the severity of R. soluni AG-2-2 damping-off and stem canker on soybean in North Dakota, were nonpathogenic on soybean (Khan, Nelson and Helms,

1992). No hosts or diseases were reported for this AG by Ogoshi (1985) or Sneh et al. (1991), However, isolates within AG-K were reported to be pathogenic on seedlings of radish, tomato, carrot, onion, lettuce, cucumber and cantaloupe (Ichielevich-Auster et al., 1985a). Schisler et al. (1993) mention the ‘potential’ pathogenicity of isolates in AG-I and AG-K, but their results indicate only low virulence for AG-K isolates tested on brussels sprouts, bell ptpper and wheat. Successful BNR biocontrol agents will have to be phytoplane non-parasitic non-pathogens, mild symptomless parasites or, possibly, mild pathogens on other potential hosts in crop rotations as well as effective in biocontrol.

Survival of BNR

Crop to crop survival of BNR biotic agents under field conditions also relates to this general question of effects on ‘non-target’ crops in the rotation. Apropos is a statement, modified from Cook (1993) that ‘(because of) . . . the ephemeral nature of microorganisms introduced into the environment, . . . it is difficult to make a scientifically based (a priori) case for significant risk of these (biotic) agents to non-target organisms in the environment’. Survival and effects of agents on nontarget crops (organisms) have to be determined experime@ally. Cubeta, Echandi and Gumpertz (1991) investigated the survival of two isolates of AG-A in soil and in precolonized bean stems. Survival was affected by depth of burial of inoculum and by isolate. Populations of both isolates initially increased after 1 month, but did not persist more than 9 months in soil and 11 months in bean stems. Survival of isolates of CAG-2 (AG-A), CAG-3 (AG-E), CAG4 (AG-F) and CAG5 (AG-R) in pots of pasteurized soil buried in fallow field plots was investigated by Bell and Sumner (1987). Isolates of AG-E, AG-F and AG-R were re- isolated after 283 days. AG-A was not recovered after 211 days. Subsequently, Sumner and Bell (1994) investigated survival of the same BNR in soil, initially fumigated, in microplots cropped to corn, snapbean and peanut. Population densities of AG-A and AG-F were variable after 5 weeks and 3 months, respectively, and not different among treatments. AG-R populations were detectable for 19 months. Biological control effects of AG-F were evident in the first year, but not in the third year of cropping. Whereas, biocontrol effects of AG-R on corn yields, but not on yields of peanut and snapbean were evident in the third year of cropping. Reliance on surviving field populations of BNR agents for disease control appears to be uncertain and risky. As noted by Sumner and Bell (1994) and Cubeta et al. 1991), extended survival studies must be designed to prevent re-introduction of the isolates under test from outside sources during the course of the experiment to avoid misleading survival results.

Colonization of plant surfaces

There is compelling evidence of the ability of BNR and hypovirulent R. soluni biotic agents to colonize plant surfaces. Burpee and Goulty (1984) isolated BNR from

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symptomless leaves 8-10 days after inoculation. Isolate BN 160, effective in biocontrol, was easily re-isolated from uninfected, unsterilized bean stems (Cardoso and Echandi, 1987a). The BNR grew in the presence of root exudates and colonized the bean plant rhizosphere, rhizoplane and hypocotyl surface of healthy, un- damaged plant tissues (Cardoso and Echandi, 1987b). Herr (1988b) devised a tissue disk method for assaying plant surface colonization of individual large sugar beet plants and beet plant populations to ascertain and evaluate the effects of various factors on establishment of BNR on sugar beet plants in greenhouse and field. It was reported that BNR effective in biocontrol colonized 60-96% of beet crowns, whereas ineffective BNR isolates did not (0%) or did so poorly (27%). These results indicate that biocontrol effectiveness is directly related to plant colonization effectiveness. A new term, phytoplane competence, to describe plant surface colonizing ability, derived from Ahmad and Baker’s (1988) ‘rhizosphere competence’ used in relation to the effectiveness of Trichoderma harzianum Rifai isolates in biocontrol of Pythium damping-off, is suggested for BNR agents. Yuen et al. (1994) followed colonization of tall fescue grass by GM 460 and other agents by sectioning leaf blades and plating them on selective media. Suppression of brown patch by GM 460 was related to decreased occurrence of the virulent R. solani isolate on agent-treated and subsequently pathogen inoculated leaf blades. Eayre and Echandi (1988) examined BNR-colonized bean roots and hypo- cotyls by use of light and scanning EM microscopy and reported that protection from R. sofani was associated with small hypersensitive lesions. BNR penetrated intercellularly though two to three cell layers of the epidermis and cortex (mild symptomless parasitism). Colonization of the plant surface by BNR was sparse and only on hypocotyls at the soil surface. Contrasting results were reported by Sneh, Ichielevich-Auster and Shomer (1989) who examined 3-day-old cotton seed- lings colonized by the hypovirulent R. solani AG-4 isolate 521. Hyphae of isolate 521 were attached to the surface of the epidermis, but no appressoria or infection cushions were observed. Although isolate 521 densely colonized the seedling outer surface it did not penetrate the cortical tissues (phytoplane non-parasitic non- pathogen). Harris et al. (1994) noted BNR 1 and BNR 2 agents formed dense hyphal mats on the underground portions of Capsicum seedlings.

Mechanisms of biocontrol

The mechanisms of biocontrol by which these various BNR and hypovirulent R. solani isolates suppress diseases caused by R. solani, and some other pathogenic Rhizoctonia spp., have been investigated and speculated on since the early studies of Burpee and Goulty (1984). Direct antagonism of pathogens does not appear to be involved in biocontrol by these biotic agents. Burpee and Goulty (1984) noted that the BNR they used were not mycoparasitic on R. solani. Similar findings regard- ing absence of mycoparasitism were reported by Ichielevich-Auster et al. (1985b), Cardoso and Echandi (1987a), Herr (1988a) and Harris et al. (1993, 1994), although in each study the particular biocontrol agents

used differed. Further, no evidence of antibiosis or inhibition, the other manifestation beside hyper- parasitism of direct antagonism to pathogens, was found by any of these investigators (Burpee and Goulty, 1984; Cardoso and Echandi, 1987a, 1987b; Herr, 1988a; Ichielevich-Auster et al., 1985b; Harris et al., 1993, 1994.

As a consequence of the well-documented obser- vations of plant surface colonization by BNR and hypovirulent R. solani agents effective in suppressing various diseases caused by R. solani, two hypotheses of the mechanism of biocontrol, competition and induced host resistance, have been advanced. Burpee and Goulty (1984) hypothesized that either of these two mechanisms could be responsible for disease control. Repeated additions of external nutrients (medium A of Weinhold, Bowman and Ordman, 1969) to host hypo- cotyls treated with hypovirulent R. solani, did not reduce the protective effect against pathogenic R. sofani (Ichielevich-Auster, etal., 1985b). Consequently, they concluded that the disease suppression probably was not induced by competition for nutrients. Cardoso and Echandi (1987b) reported that R. solani developed fewer infection cushions on BNR-treated bean seed- lings than on the nontreated controls. Additionally, they reported root exudates (ethanolic extracts) from BNR-treated seedlings inhibited germination of sclerotia and hyphal growth. More importantly, they reported that surface-sterilization of roots and hypocotyls killed BNR, but the seedlings were still protected against R. soluni infection. These latter observations were regarded as evidence for the induced host resistance mechanism of biocontrol. Sneh, Ichielevich-Auster and Plaut (1989) made a detailed investigation of the mechanisms of biocontrol induced by a hypovirulent isolate of R. solani AG-4 on cotton seedlings with the following results: (a) the amount of exudates from protected seedlings was not reduced, indicating competition for root exudates (nutrition) was not a mechanism of biocontrol; (b) the hypovirulent R. solani did not induce production of inhibitory substances of host origin (i.e. did not induce host resistance; and (c) removal of hyphae of the hypovirulent isolate from the seedling surface nullified the protective effect. The apparent contradiction of the latter finding compared to Cardoso and Enchandi’s (1987b) report that surface- sterilized seedlings retained the protective effect was explained by the suggestion that nullification of protec- tion requires the removal of the biotic agent hyphae (living or dead) from recognition and infection sites, which must be unoccupied for host penetration by the virulent isolate. Thus, the mechanism of protection advocated was that colonization of roots and hypo- cotyls by the hypovirulent isolate provides a protective, ‘masking’ barrier at recognition and infection sites which prevented host penetration by the pathogen (Sneh et al., 1989). In considering the results of these investigations of the mechanism of biocontrol, one should not forget that different isolates have been used in each case, some of which may have the ability to cause hypersensitive cell reactions and induce host resistance (i.e. mild symptomless parasites or mild pathogens) while other isolates may lack this ability (phytoplane non-parasitic non-pathogens). Thus, in some cases only competition for recognition and

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infection sites may be involved, whereas in other cases both competition and induced host resistance, or only induced host resistance may be involved.

Enhancement of biocontrol

Despite the ambiguous results from investigations into the mechanism(s) of biocontrol by these fungi, the evidence that colonization of plant surfaces by BNR and hypovirulent R. sofuni is prerequisite for biocontrol of diseases caused by R. soluni is clear. Consequently, selection of those isolates most competent in colonizing plant surfaces (phytoplane competence) represents one approach to improvement of levels of biocontrol and greater biocontrol reliability and reproducibility. Herr (1988b) reported effective BNR isolates colonized sugar beet to a greater extent than did BNR isolates ineffective in biocontrol. Others (Burpee and Goulty, 1984; Cardoso and Echandi, 1986, 1987a; Ichielevich- Auster et al., 1985; Sneh et al., 1989; Harris et al., 1993, 1994) have reported on colonization by effective biotic agents, but comparisons with colonization by ineffective agents, of the same AG apparently were not made. The approach used by Ahmad and Baker (1987, 1988) with Trichoderma biocontrol agents, in which mutant isolates were found to be more rhizosphere competent than wild type strains has not been attempted with BNR and hypovirulent R. soluni agents. Rhizosphere competence of Trichoderma isolates correlated with celluloytic activity (Ahmad and Baker, 1987). Comparison of cellulolytic activities between effective and ineffective BNR and hypovirulent R. solani have not been reported, but Sneh et al. (1989) reported the hypo- virulent R. solani isolate 521 had lower cellulolytic activity than the virulent R. solani isolate. Further, the report of Marcus et al. (1986) of the occurrence of the enzyme endopectinlyase in virulent and absence of hypovirulent R. soluni isolates should be examined for any relevance to biocontrol by BNR agents.

Another approach to enhancement of biocontrol activity and reliability is based on investigation of factors which favorably affect colonization of plant surfaces. Frequently, non-pathogenic BNR and R. soluni are isolated from plants either in association with virulent R. so&i or from healthy plants. Daniels (1963) who reported on the saprophytic and parasitic activities of R. solani, mentioned that R. solani could frequently be isolated from healthy, normal plants and suggested this was a survival mechanism. Some Rhizoctonia spp_ form mycorrhizal associations with orchids (Sneh et al., 1989, 1991; Warcup, 1981) and perhaps the association of other Rhizoctonia spp. with healthy plants other than orchids represents a transition from mycorrhizal to rhizoplane types. Evidence of growth enhancement of host plants induced by BNR and hypovirulent R. solani agents in the absence of virulent R. solani pathogens (Sneh, et al., 1986; Harris et al., 1993, 1994) manifests certain parallels with mycorrhizal growth promotion effects. The nature of this growth enhancement is presently based only on various speculations. Not all BNR are effective in growth promotion (Schisler, Neate and Masukara,

In the case of mycorrhizae, Groth and Martinson (1983) reported that soil incorporation of metalaxyl

increased vesicular-arbuscular mycorrhizal (VAM) infection of maize and soybean roots. Possibly revelant is the treatment of soil with metalaxyl prior to planting. Cardoso and Echandi (1987a) reported reduced incidence of Pythium root rot and increased effective- ness of the biocontrol agents, probably due to a reduction in microbial competition in the rhizosphere. BNR selective fungicides (Kataria and Gisi, 1989) merit testing for selective enhancement of applied BNR agent colonization of plant surfaces. Other substances may be effective in enhancing colonization and/or biocontrol, such as the polysaccharides, polyhydroxy alcohols and organic acids reported by Nelson, Harman and Nash (1988) for enhancement of Trichoderma- induced biocontrol of Pythium seed rot of peas.

Cultural practices may favorably effect biocontrol. In greenhouse assays, additional ‘crown covering’ soil applied to sugar beet in pots 30 days after treatment with isolate GM 460 at seeding enhanced crown rot disease control compared with similar at planting treatments not given applications of additional crown covering soil (Herr, 1988~). Row-hilling cultivation (i.e., thowing additional soil onto beet crowns) follow- ing BNR agent applications appeared to improve BNR colonization of beet plant surfaces and crown rot disease control in the field. Similar soil deposition in and on crowns in the absence of applied agents increased the severity of Rhizoctonia root rot of sugar beet (Schneider et al., 1982). Alteration of the crown environment, especially occurrence of higher moisture levels probably is involved (Herr, unpublished).

Combinations of two biocontrol agents with different attributes may be beneficial for disease control. Yuen et al. (1994) found a combination of BNR agent GM 460 with Gliocladium virens, isolate TRBG, was as or more effective (P = 0.05) than the individual isolates in controlling brown patch of tall fescue. Isolate TRBG exhibited antibiosis and mycoparasitism against R. soluni in vitro. However, Yuen et al. (1994) found that not all combinations of biocontrol agents were bene- ficial. Some combinations of agents were incompatible, as were all combinations of the bacterial antagonists of Pythium damping-off, E. cloacae and P. jkorescens, with the two BNR isolates Cubeta and Echandi (1991) used for contiol of Rhizoctonia and Pythium damping- off of cucumber in field soil.

The substantial progress made in the discovery and development of BNR and hypovirulent R. soluni biotic agents for biocontrol of diseases caused by R. solani is most encouraging. Further elucidation of the mechan- isms of biocontrol and the ecology of the agents should aid in the selection and development of superior biocontrol agents from within these fungi.

Acknowledgements

I wish to thank the late Dr Ralph Baker, Department of Plant Pathology and Weed Science, Colorado State University for suggesting this review. Thanks to all authors cited for the information upon which this review is based. All research support was provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript number 67-94.

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Received 29 April 1994 Revised 1 August 1994 Accepted 3 August 1994

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