Mycoparasitism by Coniothyrium minitans on Sclerotinia sclerotiorum and its Effect on Sclerotial Germination

Scanning electron microscopy showed that hyphae of Coniothyrium minitans produced appressorium-like swellings when they came in contact with Sclerotinia sclerotiorum in dual culture on PDA. The parasitized hyphae gradually skrank and collapsed, and hyphae of the mycoparasite were found inside the host hyphae. The mycoparasite hyphae grew inter- and intracellularly within the sclerotia of S. sclerotiorum. In the later stages of parasitism, hyphae of the mycoparasite proliferated extensively within the sclerotia and formed pycnidia near the sclerotial surface. At this stage, the sclerotia became flattened, soft and disintegrated. Sclerotia parasitized by C. minitans failed to germinate either myceliogenically or carpogenically.     

Control of the mushroom pests Lycoriella auripila (Diptera: Sciaridae) and Megaselia halterata (Diptera: Phoridae) by Steinernema feltiae (Nematoda: Steinernematidae) in field experiments

The nematode Steinernema feltiae (Nematoda: Steinemematidae) was tested for its ability to control two main mushroom pests i.e. the sciarid Lycoriella auripila (Diptera: Sciaridae) and the phorid Megaselia halterata (Diptera: Phoridae) in growing-rooms filled with spawned compost. A clear difference between female and male sciarid control was observed. A nematode application 1 day after casing preceded by an application 1 day before casing on the compost caused an almost complete control (97%) of the F1-generation of female sciarids. The F2-generation of females was similarly controlled (95%) by an application 7 days after casing. A dosage of 1 × 10⁶nematodes m^-2was found to be equally effective as higher dosages. Diflubenzuron remained active throughout entire the cropping period with high sciarid mortality rates varying from 72% to 99%. Phorid control was variable and seemed to depend on the presence of sciarids. In one occasion the control rate of F2-generation phorid larvae was 75% and was possibly caused by the presence of new infective juvenile nematodes recycled in F2-generation sciarid larvae. Diflubenzuron did not significantly reduce phorid numbers.     

Potential for biocontrol of sclerotinia rot of carrot with foliar sprays of Contans® WG (Coniothyrium minitans)

A glasshouse and field trial were conducted to evaluate foliar sprays of Contans® WG (Coniothyrium minitans) conidial suspensions for control of sclerotinia rot of carrot and infection of Sclerotinia sclerotiorum sclerotia by C. minitans. In the glasshouse trial, foliar sprays (1×10⁴–10⁸ conidia mL^?1) decreased the viability of sclerotia recovered from diseased plants and increased infection by C. minitans. In the field trial, three successive foliar sprays applied at 14-day intervals failed to reduce foliage disease severity, but significantly reduced viability of sclerotia recovered from diseased plants/crop debris and increased infection by C. minitans. No significant differences in sclerotial viability or infection were observed between two conidial concentrations (2.4 and 4.8×10⁶ conidia mL^?1). Foliar sprays of Contans® WG have potential for reducing viability of sclerotia produced on diseased foliage.     

Use of Coniothyrium minitans and other microorganisms for reducing Sclerotinia sclerotiorum

Biological control agents (BCAs) Bacillus subtilis QST 713, Coniothyrium minitans CON/M/91-08, Streptomyces lydicus WYEC 108, and Trichoderma harzianum T-22 were evaluated for their efficacy in the reduction of survival of sclerotia and production of apothecia of Sclerotinia sclerotiorum under controlled environments. A growth chamber assay was conducted where 25 sclerotia were buried in pots containing potting soil, and BCAs were drenched into the soil at various concentrations, and five soybean seeds were planted in each pot. The presence and number of S. sclerotiorum apothecia were recorded daily. Sclerotinia sclerotiorum sclerotia were retrieved six weeks after seeding and viability was assessed on water agar plates. All BCAs were effective in reducing S. sclerotiorum inoculum at various efficacies. In general, efficacy was positively correlated with the rate of application. At the rate of application when the efficacy did not change significantly by increasing the rate, the BCAs had various reductions of apothecia and sclerotia. B. subtilis reduced apothecia and sclerotia by 91.2 and 29.6%, respectively; C. minitans reduced apothecia and sclerotia by 81.2 and 50%, respectively; Streptomyces lydicus reduced apothecia and sclerotia by 100 and 29.6%, respectively; Trichoderma harzianum reduced apothecia and sclerotia by 80.5 and 31.7%, respectively. In addition, the commercial strain of C. minitans CON/M/91-08, and a wild Michigan strain of C. minitans W09 were compared for their growth and sclerotial reduction. W09 had faster growth rate than the commercial strain, indicating potential diversities of biological control strains to be studied.     

Effect of Carbon Source on Production of Lytic Enzymes by the Sclerotial Parasites Trichoderma atroviride and Coniothyrium minitans

Three isolates of Trichoderma atroviride and two isolates of Coniothyrium minitans known to efficiently degrade sclerotia of Sclerotinia sclerotiorum were cultured on minimal medium with sucrose, carboxymethyl cellulose (CMC), xylan, laminarin, colloidal chitin or powdered sclerotia as carbon source. The activity of endochitinase, endo‐β‐1,3‐glucanase, endoxylanase and endocellulase in culture filtrates was determined after 7 and 15 days of culture using dye‐labelled substrates. The strongest inducers of chitinase were colloidal chitin and sclerotia powder. Chitinase activity appeared to be faster induced in the isolates of T. atroviride than in the isolates of C. minitans, but the maximum level of activity present in culture filtrates of the two species was similar. With CMC and xylan as carbon source, concurrent production of the corresponding enzymes was observed for the Trichoderma isolates. The isolates of C. minitans produced cellulase on xylan but not on CMC, whereas xylanase was produced on both carbon sources. Laminarin induced the formation of glucanases in the three isolates of T. atroviride but not the isolates of C. minitans. However, in the sclerotia‐containing cultures of C. minitans glucanase activity was even higher than in the respective cultures of the Trichoderma isolates. During the 31‐day study period, the pattern of enzyme production in shake cultures containing sclerotia powder was very similar for the isolates of T. atroviride and C. minitans. Glucanase activity generally reached a peak 24 days after inoculation of the flasks, whereas the activity of chitinase, cellulase and xylanase remained fairly constant throughout the experiment.     

Foliar Application of Fungal Biocontrol Agents for the Control of White Mold of Dry Bean Caused by Sclerotinia sclerotiorum

Field experiments were conducted during 1992–1994 to evaluate the effectiveness of five indigenous fungi for control of white mold (Sclerotinia sclerotiorum) of dry bean (Phaseolus vulgaris). The five fungi consisted of one antagonist, Epicoccum purpurascens, and four mycoparasites, Coniothyrium minitans, Talaromyces flavus, Trichothecium roseum, and Trichoderma virens. Spore suspensions of each fungus were sprayed onto bean plants two or three times during the early bloom to midbloom period. Incidence of white mold of dry bean was significantly reduced by all biocontrol agents. C. minitans and E. purpurascens, the most effective agents, reduced the proportion of plants infected by an average of 56 and 43%, respectively (P < 0.001). C. minitans was the only biocontrol agent recovered consistently from sclerotia and diseased seed present in harvested samples. It was recovered at similar frequencies in samples from all treatments. Of the sclerotia of S. sclerotiorum collected from harvested seed, 59% were infected by C. minitans in 1993 and 20% were infected by C. minitans in 1994. In three additional trials in 1994, comparing C. minitans with the fungicide benomyl, the fungus was not effective in any of the experiments, whereas benomyl reduced disease incidence relative to the control in one trial. The study suggests that, among the five indigenous fungi, C. minitans is the most promising agent for control of white mold of dry bean under Canadian prairie conditions.     

Susceptibility of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> strains different in oxalate production to infection by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

Three strains of Sclerotinia sclerotiorum, namely Ep-1PB (PB), Ep-1PK (PK) and Ep-1PNA5 (A5), were compared for the production of oxalic acid (OA) on potato dextrose agar (PDA) and Maxwell agar medium (MAM) and for mycelial susceptibility to infection by the mycoparasite Coniothyrium minitans on PDA. Results showed that strain PB produced negligible oxalate, whereas strain PK was detected to produce oxalate, but much less than that produced by strain A5. The three investigated strains differed slightly in mycelial growth rates and mycelial biomass on PDA. However, colonies of strains PB and PK formed on PDA were more susceptible to invasion by C. minitans than colonies of strain A5. Meanwhile, amendment of synthetic oxalate in PDA at 0.25–2.00 mg g⁻¹ medium suppressed aggressiveness of C. minitans in invasion of colonies of S. sclerotiorum strain PB developed on this medium. These results suggest that infection of hyphae of S. sclerotiorum is negatively affected by the presence of oxalate. The importance of oxalate degradation by C. minitans in its mycoparasitism on hyphae of S. sclerotiorum provides a clue for improvement of the biocontrol efficacy of C. minitans in the future.     

Field management of Sclerotinia stem rot of soybean using biological control agents

Biological control agents (BCAs) were evaluated for their efficacy on reducing the number of sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary in the soil and on Sclerotinia stem rot in soybean production systems in Michigan. BCAs included Coniothyrium minitans CON/M/91–08 (Product name: Contans®WG), Streptomyces lydicus WYEC 108 (Actinovate®AG), Trichoderma harzianum T-22 (PlantShield®HC), and Bacillus subtilis QST 713 (Serenade®MAX). At two field locations, soil artificially infested with S. sclerotiorum sclerotia, was treated by incorporating the above BCAs in the topsoil before planting and boscalid was applied as a foliar fungicide at growth stage R1 as a positive control. C. minitans was the most effective BCA and reduced the disease severity index (DSI) by 68.5% and the number of sclerotia of S. sclerotiorum in the soil by 95.3%. S. lydicus and T. harzianum reduced DSI by 43.1% and 38.5% and sclerotia in soil by 90.6% and 70.8%, respectively. B. subtilis only had a marginal effect on S. sclerotiorum. Populations of Bacillus, Streptomyces, Trichoderma spp., and C. minitans collected from soil samples and at 3, 28, 71, and 169 days after BCA application indicated that the population of Streptomyces, Trichoderma spp., and C. minitans did not change significantly throughout the season, which may be the reason for their effectiveness.     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.Revisions requested 27 July 2004; Revisions received 7 September     

Biocontrol of Sclerotinia sclerotiorum infection of cabbage by Coniothyrium minitans and Trichoderma spp.

Nine fungal isolates [Clonostachys rosea (1), Coniothyrium minitans (1), Trichoderma crassum (1), T. hamatum (4), T. rossicum (1) and T. virens (1)] were tested in two bioassays for their ability to degrade sclerotia and reduce apothecial production and carpogenic infection of cabbage seedlings. C. minitans LU112 reduced apothecial production in both bioassays, with T. virens LU556 significantly reducing apothecial production in the sclerotial parasitism assay. Both isolates also reduced sclerotial viability in this assay to 5% for C. minitans and 22% for T. virens. C. minitans LU112 and T. virens LU556 reduced the infection of cabbage seedlings in the pot bioassay 126 days after sowing but not after 147 days, partly due to ascospore cross-infection between treatments. C. minitans LU112, T. virens LU556 and T. hamatum LU593 as maizemeal-perlite (MP) soil incorporation and transplant potting mix incorporation were evaluated for their ability to control Sclerotinia sclerotiorum disease of cabbage in field experiments. S. sclerotiorum infection of cabbage was reduced by 46–52% and 31–57% by both C. minitans LU112 and T. hamatum LU593 as MP soil incorporations, respectively, in the two field experiments. T. virens LU556 MP soil incorporation and C. minitans LU112 and T. hamatum LU593 transplant potting mix incorporations reduced S. sclerotiorum disease in the first experiment but not in the second experiment. A commercial C. minitans LU112 formulation, C. Mins LU112 WG, also significantly reduced S. sclerotiorum disease by 59%. Soil incorporation of C. minitans and T. hamatum was shown to have potential to control S. sclerotiorum disease in cabbage.     

Splash dispersal of Coniothyrium minitans in the glasshouse

Coniothyrium minitans, a mycoparasite with biocontrol activity against Sclerotinia sclerotiorum, was found to disperse during glasshouse trials where overhead irrigation was used. Consequently, the role of water splash in dispersal of C. minitans was investigated using soil-incorporated inoculum and a range of irrigation regimes found to occur in the glasshouse. The resulting inoculum deposition over horizontal distances up to 2 m was measured. Using drops < 6 mm diameter at 680 mm h^-1, C. minitans was splash-dispersed at least 2.0 m, whereas with drops > 6 mm diameter at 30 mm h^-1 it was dispersed to only 1.75 m. Irrigation with droplets < 1mm diameter at 49 mm h^-1 failed to disperse inoculum beyond 0.5 m. The dispersal gradient produced by drops < 6 mm diameter at 680 mm h^-1 was best described mathematically by the power function, whereas irrigation with drops > 6 mm diameter at 30 mm h^- resulted in a gradient described well by power or exponential functions. The latter regime produced a significantly steeper gradient than irrigation with drops < 6 mm diameter at 680 mm h^-1. C. minitans was isolated using an Andersen air sampler at concentrations of 2839 cfu m^-3 or 22 cfu m^-3 during irrigation with drops < 6 mm diameter at 680 mm h^-1 or > 6 mm diameter at 30 mm h^-1, respectively. After irrigation, deposition of C. minitans-canying aerosol particles declined exponentially and distance from source had no effect on the amount of inoculum isolated. Conidia of C. minitans, splash-dispersed by irrigation with drops < 6 mm diameter at 680 mm h^-1 were able to infect sclerotia of S. sclerotiorum such that almost all sclerotia at 0.5 m from the inoculum source, and c. 50% of those at 2.0 m, became infected with the mycoparasite.     

Importance of Pollen and Senescent Petals in the Suppression of Alfalfa Blossom Blight (Sclerotinia sclerotiorum) by Coniothyrium minitans

The effect of pollen and senescent petals on the suppression of alfalfa (Medicago sativa L.) blossom blight (Sclerotinia sclerotiorum) by the mycoparasite Coniothyrium minitans was investigated. When incubated at 20°C for 39 h, germination of conidia of C. minitans and ascospores of S. sclerotiorum was 99.9 and 98.6%, respectively, in the presence of alfalfa pollen (9×10⁴ pollen grains mL^?1), whereas spore germination of both organisms was &lt;0.5% in the absence of pollen (in water). In the presence of a commercial pollen product, Swiss? pollen granules (mainly bee pollen), germination was 99.6% for C. minitans and 98.3% for S. sclerotiorum when the pollen concentration was 1.0% (w/v). When the pollen concentration was reduced to 0.1% (w/v), germination was reduced to 13.0% for C. minitans and 10.8% for S. sclerotiorum. Tests on detached alfalfa florets showed that the colonization of alfalfa florets by S. sclerotiorum was significantly suppressed by C. minitans in the presence of pollen (1.0% Swiss? pollen granules), especially when C. minitans was inoculated 1-day before S. sclerotiorum. In vivo inoculation tests revealed that the efficacy of C. minitans in the protection of alfalfa pods from the infection by S. sclerotiorum was affected by the time at which C. minitans was applied. When C. minitans was applied on young blossoms of alfalfa at the anthesis stage, pod infection was 96.6% for the treatment of C. minitans+S. sclerotiorum and 99.6% for the treatment of S. sclerotiorum alone. However, when C. minitans was applied on senescent petals of alfalfa at the pod development stage, pod infection was 8.0% for the treatment of C. minitans+S. sclerotiorum compared to 90.8% for the treatment of S. sclerotiorum alone. These results suggest that timing of the application of C. minitans is critical for the mycoparasite to compete with S. sclerotiorum for the source of nutrients from pollen and senescent petals, and for its control of alfalfa blossom blight caused by S. sclerotiorum.     

Biological control of white rot of onion

Summary Interactions of 123 isolates of fungi, 17 of bacteria and 22 of actinomycetes, respectively, withSclerotium cepivorum were studied in agar culture. They were grouped into 4 different types of reactions. Amongst themTrichoderma viride, Fusarium graminearum, Coniothyrium minitans andGliocladium roseum inhibited the growth ofS. cepivorum and later grew over its colony.T. viride showed a characteristic coiling around the hyphae ofS. cepivorum. T. viride andF. graminearum prevented the development of sclerotia.C. minitans was found to parasitize the sclerotia ofS. cepivorum and produced its pycnidia within them.Aleurisma carnis, Cladosporium elatum, Penicillium expansum, P. nigricans, P. notatum, P. piscarium, P. puberulum, P. rolfsii, P. urticae, P. variabile, Tilachlidium humicola andHelminthosporium sp. inhibited the growth ofS. cepivorum at a distance. Eleven isolates of bacteria and 3 ofStreptomyces sp. showed pronounced antagonistic properties againstS. cepivorum.Experiments were carried out to study the effects on white rot development in soil of organisms selected from agar plate tests. None of the antagonistic micro organisms had any deleterious effects on onion growth. Of the organisms testedP. nigricans gave the best results in controlling white rot infection.This work was carried out at the Department of Botany, The University, Birmingham, England. I wish to thank Prof.C. J. Hickman for his invaluable advice and encouragement.     

Comparison ofRhizoctonia solani isolates from web blight and basal canker of cowpea and from soil

Summary Isolates ofRhizoctonia solani from web blight and stem basal canker of cowpea and those obtained from soil had similar linear growth rates on potato dextrose agar (PDA) at various temperatures but differed in other features. The web blight isolates differed from the basal canker and the soil isolates in cultural appearance on PDA and on potato marmite agar (PMA). The web blight isolates readily formed discrete sclerotia on PDA, PMA and soil but the other isolates did not. In greenhouse tests, the former were generally the most virulent in inciting foliage and stem basal necrosis and damping-off of seven crop species. Of the plants tested, the legumes were the most susceptible toR. solani.     

Abundance of Collembola (Insecta) inhabiting the hyphal mat of an ectomycorrhizal fungus,Sarcodon scabrosus,in a Pinus densiflora forest

The total number of collembolans collected from the hyphal mat of the ectomycorrhizal fungus Sarcodon scabrosus was less than half of that collected from the adjacent non-mat soil. The same was true of all families of collembolans except one, although not all differences were significant. The exception was the Hypogastruridae, with more individuals in the hyphal mat on the sampling day in the fruiting season; these were also the most abundant collembolans on the fruiting bodies. These results indicate that most collembolans avoid the hyphal mat of S. scabrosus in a Japanese red pine forest.     

Phoretic relationship between the myceliophagous mite Microdispus lambi (Acari: Microdispidae) and mushroom flies in Spanish crops

We studied the role played by the phorid Megaselia halterata (Wood) and the sciarid Lycoriella auripila (Winnertz) in the phoretic dispersion of the myceliophagous mite Microdispus lambi (Acari: Pygmephoridae). Twenty‐four crops were monitored during 18 months in commercial mushroom farms in Castilla‐La Mancha (Spain). Adults of both species were collected weekly and the mites they carried were counted and identified. Both phorids (19.6%) and sciarids (4.4%) carried the mite M. lambi. The calculated load of each was 3.4 M. lambi mites per phorid and 1.9 per sciarid. The same percentage of male and female phorid was used as vector, but the load was slightly higher for females (1.86 mites per female compared with1.48 mites per male). A mean of 7.2% of the phorids examined in winter were vectors of M. lambi, while in spring and autumn of the first year the average was more than 22%. The mean load did not vary significantly between seasons. Inside the mushroom farms, less than 10% of a small initial population of phorids carried mites (less than two mites per phorid). As the cycle progressed, more than 35% of a larger population of emerging flies did so (average 3.5 mites per phorid vector). At the end of the growth cycle, the flies may fly off to colonise nearby farms, favouring the propagation of M. lambi from infested to uninfested crops. Megaselia halterata is the principal vector of M. lambi in the mushroom farms of Castilla‐La Mancha due to their high numbers, the high percentage carrying mites and the number of M. lambi they carry.     

Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum

Fungal isolates, with known activity against Sclerotinia spp. in laboratory assays, were tested for their ability to control Sclerotinia minor in four field experiments (1998–2000). In the first experiment, eight fungal isolates (Trichoderma hamatum LU595, LU593, LU592, Trichoderma virens LU555 and LU556, Coniothyrium minitans LU112, Clonostachys rosea LU115 and Trichoderma rossicum LU596) were evaluated by incorporating spore suspensions into transplant potting mix and planting lettuce seedlings into a S. minor infested field site. At harvest, Trichoderma hamatum LU595, LU593, T. virens LU555 and C. minitans LU112 reduced disease by 30–50% compared with the untreated control under very high disease pressure (100%). In further field experiments C. minitans LU112 and T. hamatum LU593, applied as maizemeal–perlite soil amendments or incorporated into the potting mix, reduced S. minor disease over a range of disease pressures (29–91%). Disease control was equivalent or greater than that achieved with the standard carbendazim fungicide treatment. Both isolates were shown to effectively colonize the lettuce rhizosphere and surrounding soil and this colonization may have protected the roots from infection by S. minor. Multiple applications of C. minitans LU112 or T. hamatum LU593 formulations gave no added disease control compared with a single application at planting. Commercial formulations of both C. minitans LU112 and T. hamatum LU593 applied as transplant treatments, solid substrate soil amendments or as a spore drench gave consistent disease control and are currently being developed further.     

Biological control of sciarid and phorid pests of mushroom with predatory mites from the genus Hypoaspis(Acari: Hypoaspidae) and the entomopathogenic nematode Steinernema feltiae

In small-scale experiments, the predatory mites, Hypoaspis aculeifer (Canestrini) and H. miles Berlese, applied at 700 mites m(-2), and the entomopathogenic nematode, Steinernema feltiae (Filipjev) applied at 3 x 10(-6) nematodes m(-2) controlled sciarids and phorids in mushroom compost and casing substrates. For both mite species, earliest application to the growing substrate following sciarid infestation reduced sciarid emergence. In contrast, later application of each biological control agent provided more effective control of phorid emergence. The behaviour of adult mites suggested that H. aculeifer were more positively geotactic than H. miles although both species could penetrate compost and casing substrates to a depth of 2-12 cm. A majority of S. feltiae nematodes resided at a depth of 2-4 cm in both substrate types. Independent application of H. aculeifer provided more comprehensive control of sciarids and phorids than the other biological agents studied, owing to its better dispersal within compost and casing, and ability to attack larvae of differing ages.     

Physiological characterization of the dandelion bioherbicide,Sclerotinia minor IMI 344141

The fungus Sclerotinia minor (IMI 344141) is being developed as a biological control for dandelion and other broadleaf weeds in turfgrass environments. Being a microbial pest control agent (MPCA), the S. minor strain must be characterized to show relatedness to like organisms and to distinguish the MPCA from related microorganisms. Phenotypic variation among 30 isolates of S. minor, collected from different regions and hosts, was studied on potato dextrose agar (PDA) and oatmeal agar (OMA). Isolates varied significantly in sclerotia shape (length/width ratio) and number, but did not vary in colony morphology or growth rates. There was high diversity (0.6) among the mycelial compatibility groups (MCG) as seven multi-member and 11 single member groups were recognized. Isolates were categorized into highly virulent, virulent, moderately virulent, and hypo virulent based on 48 h post mycelial growth on detached dandelion leaves. When assessed on dandelion plants in the greenhouse, isolate IMI 344141 ranked the highest in biocontrol efficacy, reduction of above- and below-ground biomass, and reduction in dandelion survival. Oxalic acid production was not correlated with isolate aggressiveness or growth rate and did not vary among isolates of the same MCG. IMI 344141 can be phenotypically distinguished from the other tested S. minor isolates by performing vegetative compatibility testing and counting sclerotia produced on standard 9-cm diameter PDA plates. IMI 344141 produces <100 sclerotia/plate.