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.     

Penetration of Hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans Without the Formation of Appressoria

In a study using scanning electron microscopy (SEM), the mode of hyperparasitism of Coniothyrium minitans on its host Sclerotinia sclerotiorum was investigated. The SEM micrographs confirm previous reports, from light microscopic studies, that hyphal tips of C. minitans invade the host hypha by direct penetration, without developing appressoria, and that indentation of the host cell wall at the point of penetration is often evident. There is no functional distinction between amain branch and a side branch hypha of the hyperparasite and tips of either type of hyphae are capable of invading host hyphae by direct penetration.     

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.     

Water potential affects Coniothyrium minitans growth, germination and parasitism of Sclerotinia sclerotiorum sclerotia

Water availability is an important environmental factor which has major effects on fungal activity. The effects of osmotic (KCl amended agar) and matric Polyethylene glycol ((PEG) 8000 amended agar) potentials over the range -0.1 to -5.0MPa on mycelial growth and conidial germination of eight isolates of the sclerotial parasite Coniothyrium minitans was assessed. The influence of soil water potential on the ability of three selected isolates (LU112, LU545, and T5R42i) to parasitise sclerotia of the plant pathogen Sclerotinia sclerotiorum was determined. For all eight C. minitans isolates, decreasing osmotic and matric potentials caused a reduction in mycelial growth and conidial germination. Isolates were more sensitive to decreasing matric potential than osmotic potential. Across the isolates, growth at an osmotic potential of -5.0MPa was 30-70% of the growth seen in the control, whereas less than 20% of the control growth was seen at the corresponding matric potential. Across all isolates no conidial germination was seen at matric potential of -5.0MPa. The C. minitans isolates varied in their sensitivity to decreasing water potentials. Mycelial growth and conidial germination of three isolates (LU112, Conio, and CH1) were more tolerant of low osmotic potential and matric potential with respect to mycelial growth. Isolates T5R42i and LU430 were least tolerant. In contrast, conidial germination of isolates Conio, LU545, and T5R42i were less sensitive to decreasing matric potential. Soil water potential was seen to affect infection and viability of sclerotia by the three C. minitans isolates. Isolate LU545 reduced sclerotial viability over a wider water potential range (-0.01 to -1.5MPa) compared with LU112 (-0.01 to -1.0MPa), with isolate T5R42i being intermediate. Indigenous soil fungi (Trichoderma spp. and Clonostachys rosea) were recovered from sclerotia but did not result in reduction in sclerotial viability. The relevance of these results in relation to biocontrol activity of C. minitans in soil is discussed.     

Effect of volume and concentration of conidial suspensions of Coniothyrium minitans on infection of Sclerotinia sclerotiorum sclerotia

White mould, caused by Sclerotinia sclerotiorum, is a serious disease affecting a wide range of agricultural and horticultural crops. Biological control is one option available to limit its damage. Field experiments to evaluate various concentrations and volumes of Coniothyrium minitans spore suspensions applied to S. sclerotiorum-infected bean crops were conducted in 1997 and 1998. Percentage sclerotia infected by C. minitans were scored. Three replicate experiments were performed in time in 1997 with 21 combinations of isolates, volumes and concentrations, including two controls. In 1998, 22 combinations of isolates, volumes and concentrations plus two controls were used, combined with the absence or presence of a maize buffer, with two replicates for each. Isolates as well as concentration and volume had no effect on infection by C. minitans, but there was a significant effect of total dose (volume×concentration) of inoculum applied over the full range from 100 L ha^-1 at 10⁴ conidia mL^-1 to 1000 L ha^-1 at 10⁷ conidia mL^-1. Percentage infected sclerotia increased linearly with log (dose) as well as from 1 to 4 weeks after application of C. minitans, and reached a level of about 100% at high doses under the humid conditions of 1998. Apothecia of S. sclerotiorum developing from sclerotia in collected soil samples from the 1997 experiment showed no significant effect of C. minitans inoculum dose, but there was a significant effect of the replicate experiments. The influence of weather conditions is highlighted, and the implications of the results for cost-effective biocontrol of S. sclerotiorum are discussed.     

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.     

Effects of colonisation by different strains of Coniothyrium minitans on the viability of sclerotia of Sclerotinia sclerotiorum

White mold is a major disease in commercial soybean production. An effective measure to reduce the negative effects of Sclerotinia sclerotiorum is the use of bio-fungicides. Strains of Coniothyrium minitans were isolated and efficacy tests against S. sclerotiorum was studied. The efficacy of pycnidiospores sprays of strain N09 (GenBank Accession No HQ908274) from Iowa, USA and strain CON/M/91-08 of Contans® WG were compared in a series of experiments. Sclerotia viability was significantly (P < 0.05) lower in both sclerotia-infested-sterilized-soils (SISS) and sclerotia-infested-unsterilized-soils (SIUS) sprayed with N09 compared with CON/M/91-08 and control at 3°C for 75d and 90d sampling. Similarly, sclerotia viability was significantly (P < 0.05) lower at 23°C for 45, 60 and 75d sampling in SISS and 45, 75 and 90 d sampling in SIUS compared with CON/M/91-08 and control. In contrast, viability of N09 colonies were significantly (P < 0.05) higher than that of CON/M/91-08 both at 3°C and 23°C in SISS across sampling periods. While in SIUS, N09 colonies were significantly higher at 3°C for 15, 30, 45, 75 and 90 d sampling, and at 23°C for 30, 60 and 75 d sampling. Also, (1) N09 had a faster growth rate and produced 1.5 times more pycnidiospores than CON/M/91-08; (2) mycoparasitism by N09 was faster than CON/M/91-08; and (3) co-inoculation of sclerotia and the strains, N09 showed lower sclerotia reproduction than CON/M/91-08. Our data suggest that the new strain N09 has a greater efficiency than CON/M/91-08 in killing sclerotia.     

Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens

Coniothyrium minitans is a potential biological control agent of the plant pathogenic fungus Sclerotinia sclerotiorum. In this research, T-DNA insertional transformation of strain ZS-1 of C. minitans mediated by Agrobacterium tumefaciens was obtained, with optimization of spore maturity for transformation. After confirmation by PCR, transformants were subjected to Southern blot analysis, and results showed that more than 82.7% of transformants had single T-DNA insertions, and 12.1% of transformants had two copies T-DNA insertions. The genomic DNA segments of transformants flanking the T-DNA could be amplified from both borders with TAIL-PCR. Four types of mutants were screened and identified from the T-DNA insertional library, which comprised sporulation deficient mutants, pathogenicity deficient mutants, pigment change mutants and antibiotic deficient mutant, and some of the mutants were described; the number and frequency of each type of mutant from the library were calculated, and the frequency of each type is 3.27 x 10(-3), 1.0 x 10(-4), 1.4 x 10(-4), 2.5 x 10(-4), respectively. The successful creation of the T-DNA insertional transformation library may help us to unravel the interaction between a parasite and its host at a molecular level, to clarify the differentiation and development of this fungus, and to analyze and clone functional genes from the biocontrol microorganism in tripartite associations.     

盾壳霉在油菜菌核病菌生物防治中的应用

油菜核病菌(Sclerotiniasclerotiorum)是一种世界性病原菌,其分布广、危害大、难根治。盾壳霉(Coniothyriumminitans)是该病原菌的破坏性寄生真菌,可以有效、专一地降低病原菌菌核的形成与萌发,在该病原菌的生物防治方面具有较大的应用潜力。从油菜核盘菌的致病过程与特点、盾壳霉的生长特性、盾壳霉和油菜核盘菌间相互作用的规律及途径等几个方面阐述了盾壳霉对油菜核盘菌的生防特性,讨论了盾壳霉在生产实践中的应用潜力及存在问题,并提出了一些解决问题的可能途径及需要进一步研究的内容与方向 。     

植病生防菌盾壳霉的分子生物学研究进展

盾壳霉是一种重要的核盘菌寄生茵.近年来,该菌在分子水平的研究取得了一定的进展.本文概述了盾壳霉在产孢调控、与核盘菌互作、遗传转化以及动态检测和遗传多样性等方面的研究现状,并对研究中存在的问题进行了讨论.希望在此基础上能够促进该茵分子生物学研究的不断深入,更好地开发利用该菌的基因资源.     

Production, Survival and Evaluation of Liquid Culture-produced Inocula of Coniothyrium minitans Against Sclerotinia sclerotiorum

Growth of Coniothyrium minitans on potato dextrose broth was compared with that on an inexpensive molasses-yeast liquid medium at 18-22°C in static culture. Biomass and conidial production were, in general, similar, although the rate of biomass production was quicker and conidial production was slightly greater per unit volume of medium in the molasses-yeast medium. Air-dried biomass from molasses-yeast liquid culture containing mycelia, pycnidia and conidia of C. minitans was mixed (12%, w/w) with kaolin to give a kaolin-biomass dust. The ability of C. minitans to survive and subsequently infect and reduce the viability of sclerotia of Sclerotinia sclerotiorum from this kaolin-biomass dust was found to be little affected by storage for 48 weeks between 4 and 15°C but was decreased by higher storage temperatures. The kaolin-biomass dust preparation did not differ from a standard maizemeal-perlite inoculum of C. minitans in its ability to infect sclerotia of S. sclerotiorum or reduce their viability or carpogenic germination in glasshouse and field pot bioassays. Further, when either inoculum was applied once to glasshouse soil naturally infested with S. sclerotiorum prior to planting three successive crops of lettuce, the pattern of disease control, reduction of sclerotial numbers/ plot, infection of sclerotia, reduction of sclerotial viability and survival in soil were similar for both inocula. The potential for the commercial development of liquid-culture-produced inocula of C. minitans is discussed.     

Characterization of Sclerotinia and mycoparasite Coniothyrium minitans interaction by microscale co-culture

Aims:  To characterize the interaction of Sclerotinia sclerotiorum and S. minor with strains of the mycoparasite and commercial biocontrol agent Coniothyrium minitans using novel perfusion chamber gasket co-culture.
Methods and Results:  Sclerotinia were cultured in perfusion chamber gaskets and then flooded with Coniothyrium conidia. After germination, Coniothyrium failed to show any form of directed growth, making contact with Sclerotinia hyphae in a random manner. In turn, some Coniothyrium hyphae coiled round Sclerotinia counterparts and although no intracellular growth was observed, Coniothyrium proliferated, while the hyphae of Sclerotinia became vacuolated and lost the cytoplasm. When co-cultures of Sclerotinia with Coniothyrium were flooded with FITC-lectins, small difference in fluorescence between the fungi was found with FITC-Con A suggesting that cell walls of both the species exposed mannose. In contrast, Coniothyrium fluoresced poorly in comparison with Sclerotinia when FITC-wheat germ agglutinin was used, indicating a marked paucity of N -acetylglucosamine exposure by cell walls of Coniothyrium, hence reduced exposure to chitinolytic enzyme action.
Conclusions, Significance and Impact of the Study:  The approach employed supported direct sequential microscopic observation of Coniothyrium and Sclerotinia as well as the utilization of representative fluorescent moieties to characterize relative carbohydrate cell wall exposure.     

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy‐1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy‐1, the Cmoxdc1‐disrupted mutants had reduced expression levels of two mycoparasitism‐related genes chitinase (Cmch1) and β‐1,3‐glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1‐disrupted mutants in OA‐amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1‐mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.     

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.     

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.     

Structural Aspects of the Parasitism of Sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary by Coniothyrium minitans Campb.

Sclerotia of Sclerotinia sclerotiorum inoculated with pycnidiospores of Coniothyrium minitans were studied by means of light microscopy and transmission and scanning electron microscopy. The hyperparasite penetrated the walls of the rind cells by means of physical pressure and destroyed the cell contents. Penetration of medullary hyphae was by enzymic lysis and physical pressure; there was evidence to suggest that the hyperparasite may coil around the host cells before inserting infection hyphae.     

Overexpression of the chitinase gene CmCH1 from Coniothyrium minitans renders enhanced resistance to Sclerotinia sclerotiorum in soybean

Transgenic Research - Pathogenic fungi represent one of the major biotic stresses for soybean production across the world. Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a...     

Effect of Sclerotial Damage of Sclerotinia sclerotiorum on the Mycoparasitic Activity of Trichoderma hamatum

Damaged sclerotia of Sclerotinia sclerotiorum buried in soil infested with Trichoderma hamatum isolate TMCS - 3 were degraded rapidly when the medulla of sclerotia was com pletely exposed by the feeding activity of larvae of the fungus gnat Bradysia coprophila. These heavily damaged sclerotia also enhanced , in vitro, the growth of TMCS - 3 . Growth of TMCS - 3 in liquid culture was studied using different carbon sources as substrates , including sclerotia of S. sclerotiorum. Significantly more biomass of TMCS - 3 was recovered using sclerotia as a substrate compared to other carbon sources tested . Exudates from sclerotia whose melanized rinds had been completely removed by feeding larvae accelerated the germination of conidia of TMCS - 3 . Concentrations of amino acids , carbohydrates and proteins in the sclerotial exudates were not increased as damage to sclerotia was increased . Exudation of electrolytes was higher in undamaged than damaged sclerotia . Glucanase activity of TMCS - 3 was slightly increased when the fungus was exposed to damaged sclerotia . However , chitinase activity was not increased by damaging the sclerotia . Larval damage altered the sclerotia not only physically but also chemically , thereby enhancing the activity of the fungus T. hamatum.     

Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum

Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S. sclerotiorum. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development.