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     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

The use of yeasts for biological control of the plant pathogen Sclerotinia sclerotiorum

Yeasts are promising biological control agents(BCAs) for a number of plant diseases. Studieswere carried out to evaluate various adjuvantsand nutrients for their ability to supportgrowth of a yeast BCA (Cryptococcusalbidus). Hydroxyethylcellulose (HEC) andinvert emulsions were found to stimulate growthof C. albidus in vitro. Severalcommercial spray adjuvants were compatible withC. albidus although they did not markedlystimulate growth. Other adjuvants were lethalto the yeast. In controlled environmentand field trials, the yeasts C. albidusand Pichia anomala provided low levels ofcontrol of white mould, a disease of bean (Phaseolus vulgaris) caused by the fungus Sclerotinia sclerotiorum. However, they weregenerally inferior in performance when comparedto either the biocontrol fungus Epicoccumnigrum or to the fungicide iprodione.     

Diagnosis and management of Sclerotinia stem rot (white mould) of lentils in Greece

The paper describes the field-level symptoms, the identification and management of Sclerotinia stem rot of lentil caused by the soilborne plant pathogen Sclerotinia sclerotiorum in Greece. Regarding symptoms at a field level, initially plants before flowering turn yellow with roots and the base of the plants become brown; then rotten plants exhibit a dry stem and die. On the diseased tissue, at the base of the stem, the typical white mycelium and the resting bodies (sclerotia) were observed. According to our pathogenicity studies in vitro, on the infected plant tissues the fungus first develop a characteristic fluffy white mycelium which will give rise to large black sclerotia, the most obvious evidence of plants infected with S. sclerotiorum. Finally, concerning evaluation of fungicides, isolates of S. sclerotiorum were sensitive to thiophanate-methyl and to triazole fungicides. Thiophanate-methyl and triazole fungicides proved to be most effective in controlling the disease emerged from mycelium or sclerotia.     

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.     

Mapping quantitative trait loci conferring partial physiological resistance to white mold in the common bean RIL population Xana?×?Cornell 49242

White mold, caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary, is a devastating disease in common bean (Phaseolus vulgaris L.). Resistance to this pathogen can be due to physiological or avoidance mechanisms. We sought to characterize the partial physiological resistance exhibited by Xana dry bean in the greenhouse straw test using quantitative trait locus (QTL) analysis. A population of 104 F7 recombinant inbred lines (RILs) derived from an inter-gene pool cross between Xana and the susceptible black bean Cornell 49242 was evaluated against five local isolates of Sclerotinia. The effect of morphological traits (plant height, first internode length, and first internode width) on response to white mold was examined. The level of resistance exhibited by Xana to five isolates of S. sclerotiorum was similar to that of the well-known resistant lines PC50, A195, and G122. Eighteen QTL, involving the linkage groups (LG) 1, 3, 6, 7, 8, and 11, were found to be significant in at least one evaluation and in the mean of the two evaluations. The number of significant QTL identified per trait ranged from one to five. Four major regions on LG 1, 6, and 7 were associated with partial resistance to white mold, confirming the results obtained in other populations. A relative specificity in the number and the position of the identified QTL was found depending on the isolate used. QTL involved in the control of morphological traits and in the response to white mold were co-located at the same relative position on LG 1, 6, and 7. The role of these genomic regions in physiological resistance or avoidance mechanisms to white mold is discussed.     

The Role of White Mold-Infected White Bean (Phaseolus vulgaris L.) Seeds in the Dissemination of Sclerotinia sclerotiorum (Lib.) de Bary

Sclerotinia sclerotiorum survived in infected seeds of white beans as dormant mycelium in testa and cotyledons. The rate of survival averaged 85 to 89% and did not change appreciably over a 3-year period. When the infected bean seeds were sown in soil or sand, 88 to 100% failed to germinate. The seeds that failed to germinate, depending on the severity of seed infection, were rotted by S. sclerotiorum. In place of each seed, 3 to 6 sclerotia were formed. A low percentage of these sclerotia germinated carpogenically with or without preconditioning, (2.5 and 11.5% respectively). Myceliogenic germination of sclerotia with and without preconditioning was 35.5% and 70.5% on water agar and 81.0% and 93.0% on glucose agar, respectively. Both, preconditioning and nonpreconditioned sclerotia which were scattered on soil surface could germinate myceliogenically and infect bean leaves by contact. It is therefore, concluded that dormant mycelia in the infected seeds play an important role notonly in dissemination of the fungus but also in epidemiology of the disease.     

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.     

Colonisation of sclerotia of Sclerotinia sclerotiorum by a fungivorous nematode

Aphelenchoides saprophilus nematodes fed on sclerotia, mycelium, and alginate-formulated pellets of Sclerotinia sclerotiorum, mycelium of Trichoderma harzianum, and mixed fungal cultures. As many as 500 nematodes were found inside individual sclerotia. Results suggest potential impacts of fungivory on S. sclerotiorum and its ecological interactions with plant hosts and biocontrol fungi.     

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.     

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.     

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.     

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.     

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.     

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.     

Trichoderma asperelloides antagonism to nine Sclerotinia sclerotiorum strains and biological control of white mold disease in soybean plants

Sclerotinia sclerotiorum is an important plant pathogen with worldwide distribution that causes severe economic losses of various agricultural crops such as soybean. This fungus is normally controlled with synthetic chemical fungicides that pose risks to the environment, and can be harmful to human health, and they can also induce resistance in pests. The aim of this study was to investigate the potential of Trichoderma asperelloides as a biocontrol agent towards white mold disease on soybeans crops. The antagonism of two strains of T. asperelloides (T25 and T42) isolated from soil samples was determined in-vitro by dual-culture confrontation testing on nine S. sclerotiorum strains obtained from sclerotia collected on diseased soybean plants. The mycelial growth and inhibition of carpogenic and ascospore germination by T. asperelloides extracts, as well as the efficacy of these on white mold control in soybeans were evaluated. Both strains of T. asperelloides exhibited high potential of antagonism. Methanolic and ethyl acetate extracts of the two T. asperelloides strains showed excellent growth inhibition (60–100%) on all of the pathogens tested. The ethyl acetate extracts of both T. asperelloides strains exhibited the highest efficacy against carpogenic germination, decreasing by 20–30% the number of ascospores per apothecium. Strains of T. asperelloides tested were more efficient in controlling white mold than two commercial products made from Trichoderma harzianum. The new strains of T. asperelloides have potential for successful biological control of white mold disease of soybean crops in the field.     

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.     

Colony Age of Trichoderma azevedoi Alters the Profile of Volatile Organic Compounds and Ability to Suppress Sclerotinia sclerotiorum in Bean Plants

Common bean (Phaseolus vulgaris L.) is one of the most important crops in human food production. The occurrence of diseases, such as white mold, caused by Sclerotinia sclerotiorum can limit the production of this legume. The use of Trichoderma has become an important strategy in the suppression of this disease. The aim of the present study was to evaluate the effect of volatile organic compounds (VOCs) emitted by Trichoderma azevedoi CEN1241 in five different growth periods on the severity of white mold in common bean. The in vitro assays were carried out in double-plate and split-plate, and the in vivo assays, through the exposure of the mycelia of S. sclerotiorum to the VOCs of T. azevedoi CEN1241 and subsequent inoculation in bean plants. Chemical analysis by gas chromatography coupled to mass spectrometry detected 37 VOCs produced by T. azevedoi CEN1241, covering six major chemical classes. The profile of VOCs produced by T. azevedoi CEN1241 varied according to colony age and was shown to be related to the ability of the biocontrol agent to suppress S. sclerotiorum. T. azevedoi CEN1241 VOCs reduced the size of S. sclerotiorum lesions on bean fragments in vitro and reduced disease severity in a greenhouse. This study demonstrated in a more applied way that the mechanism of antibiosis through the production of volatile compounds exerted by Trichoderma can complement other mechanisms, such as parasitism and competition, thus contributing to a better efficiency in the control of white mold in bean plants.     

Integrated control of white rot disease on beans caused by Sclerotinia sclerotiorum using Contans® and reduced fungicides application

This study was conducted to determine the compatibility of Contans® (Coniothyrium minitans) with fungicides against Sclerotinia sclerotiorum. Results showed that both Contans® and Topsin® significantly reduced the disease incidence caused by S. sclerotiorum by 90% and 95% survival plants, respectively when they were individually applied and compared to control. While, soil application of Contans® and Sumisclex mixture was the most effective in suppressing the white rot disease incidence that produced 100% survival plants, application of C. minitans combined with the reduced doses of fungicides would be advantageous in saving labor cost, thus increasing production efficiency of bean.