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 <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.     

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.     

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.     

Some Nutritional Factors Affecting Production of Biomass and Antifungal Metabolites of Coniothyrium minitans

The ability of the mycoparasite Coniothyrium minitans to utilize a range of C and N sources and vitamins for growth, pycnidial formation and antifungal metabolite production was examined using a defined liquid medium. Coniothyrium minitans was able to use all the C sources tested, with the exception of D -xylose, and all the N sources tested, although growth was generally better on organic N sources rather than NO 3 -N. Increasing C:N ratios from 9:1-202:1 with N constant (2.0 g L -l L -alanine) resulted in steadily increasing yields, whereas increasing C:N ratios with C constant (40.0 g L -l D -glucose) gradually decreased yield. Addition of thiamine to the glucose-alanine basal medium resulted in the greatest increase in growth but biomass was still less than that achieved using an undefined molasses-yeast medium. Pycnidial production was generally low or failed to occur in the basal medium + C + N sources in the absence of vitamins, but addition of thiamine consistently led to abundant pycnidial formation. Molasses-yeast static culture provided greater biomass and conidial yields than molasses-yeast shaken culture. Incorporation of C. minitans culture medium into potato dextrose broth (10% v/v) resulted in consistent reduction in growth of Sclerotinia sclerotiorum irrespective of C, N or vitamin content of the basal medium or whether molasses-yeast medium was used. This is the first report of consistent production of antifungal metabolites by C. minitans .     

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.     

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.     

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.     

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.     

Production of macrosphelide A by the mycoparasite Coniothyrium minitans

Coniothyrium minitans, a mycoparasite of sclerotia of Sclerotinia sclerotiorum and Sclerotium cepivorum, produced four closely related metabolites inhibitory to fungal growth. The major metabolite, identified as macrosphelide A, had IG(50) values (the concentration of metabolite to inhibit growth by 50%) of 46.6 and 2.9 microgram ml(-1) against S. sclerotiorum and S. cepivorum, respectively. This is the first report of both antifungal activity due to macrosphelide A as well as isolation of macrosphelide A from C. minitans.     

Demographic and Biomass Production Consequences of Inundative Treatment of Cirsium arvense with Sclerotinia sclerotiorum

The adventitious shoots in three populations of Cirsium arvense in sheep-grazed pastures were treated in October (spring) 1991 with a mycelium/wheat formulation of Sclerotinia sclerotiorum and the fates of mapped shoots were followed over the growing season. In untreated plots, deaths through natural causes were compensated for by births (emergence of new shoots above the soil) throughout the growing season, but, on plots treated with S. sclerotiorum, deaths from the induced disease exceeded births for 35 days following treatment, causing the shoot population to decline markedly. Disease-induced deaths occurred only among shoots present at the time of treatment; there was no evidence of transfer of the pathogen to shoots emerging after the treatment was applied. A life-table analysis showed that only 8% of the adventitious shoots emerging during the growing season survived to seeding on treated plots, compared with 28% on the untreated plots; most mortalities occurred in shoots at the vegetative stage of development. The dry mass of propagative roots in autumn was reduced to 35% of that on the untreated plots by the pathogen and the density of shoots emerging the following spring was reduced to a similar extent. The results of this study indicate that S. sclerotiorum has potential as a mycoherbicide for C. arvense in sheep-grazed pasture in New Zealand.     

Risk Analysis of Sclerotinia sclerotiorum for Biological Control of Cirsium arvense in Pasture: Ascospore Dispersal

Natural levels of Sclerotinia sclerotiorum ascsospores in the Canterbury region were determined over 3 years by trapping depositing ascospores in dishes containing a selective agar. Mean levels in 'horticulture', 'biocontrol-pasture', 'mixed cropping' and 'pasture' strata were 115, 56, 10 and 3 ascospores m -2 day -1 , respectively. Ascospore deposition downwind of small experimental biocontrol sites was measured on 2 days in 1994 and 9 days in 1997 in late spring. Exponential depletion models scaled up to represent a 1 ha biocontrol site, revealed that dispersing ascospores declined to natural levels at downwind distances of 2.5-7.9 m. These results imply that biological weed control in pasture using S. sclerotiorum creates no greater risk of crop disease than does horticulture, and that under the conditions of our experiments, an isolation distance of 8 m would have sufficed. However, such a safety zone may be inadequate under certain meteorological conditions not encountered in the experiments when ascospores may disperse in larger numbers over longer distances. To complete the information required to build a mechanistic model of spore dispersal (beyond the scope of this paper) which would cope with a variety of meteorological conditions, two studies were conducted on the dynamics of apothecium formation and ascospore release. In a two-year study, apothecium formation was confined to the spring (September-November), and population size peaked in mid October. In a 5-day study, ascospore release occurred during the daytime, reaching a maximum late morning on frost-free days and a lower maximum mid afternoon on days with morning frost.     

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.     

A Comparison of Auxotrophic and Wild Strains of Sclerotinia sclerotiorum Used as a Mycoherbicide Against Californian Thistle ( Cirsium arvense )

Two auxotrophic mutant strains of Sclerotinia sclerotiorum were tested in the greenhouse for pathogenicity on Cirsium arvense (Californian thistle) with and without amino acid amendments. An arginine auxotrophic mutant, with an amendment of the amino acid, followed an identical disease progress curve to that of the wild strain of the pathogen from which it was derived. However, when deprived of the amino acid amendment it was still highly pathogenic. A leucine auxotrophic mutant demonstrated poor pathogenicity without a leucine amendment, but improved pathogenicity with the addition of the amino acid. However, both of these treatments were inferior to the two wild strains tested and the arginine auxotroph with and without amendments. A field experiment was conducted on C. arvense stems in permanent pasture to compare the pathogenicity of amended auxotrophic strains and wild strains of S. sclerotiorum applied as a granule in a wheat-based carrier. The two wild strains gave significant reductions in thistle cover within 3 months of treatment, and subsequent reductions in thistle stem height and density during the following season. There was no evidence that the auxotrophic strains reduced thistle cover in the season the treatments were applied, but they did reduce subsequent stem density in the following spring. To determine disease carry-over associated with the wild and auxotrophic strains of the pathogen, rape was planted into subplots over the next three consecutive seasons. Despite substantial populations of sclerotia being present in the soil, especially in the first season after treatment of the thistles, no disease of rape caused by S. sclerotiorum was detected over the three seasons in any of the plots. Sclerotium populations of S. sclerotiorum in the soil declined by over 50% between 20 and 32 months after treatment, but there was no decline over the subsequent 12 months. The trial demonstrated that the auxotrophic strains were less field fit compared with the wild strains and that the presence of inoculum and a susceptible host to S. sclerotiorum were not the only prerequisites for disease development. It was concluded that use of a trap crop following treatment is not a suitable method for determining the risk of using this pathogen as a mycoherbicide in pasture.     

Trichoderma harzianum T39 Preparation for Biocontrol of Plant Diseases-Control of Botrytis cinerea , Sclerotinia sclerotiorum and Cladosporium fulvum

Isolate T39 of Trichoderma harzianum (TRICHODEX) is a commercial biocontrol agent. It controls Botrytis cinerea (grey mould) in greenhouse crops and in vineyards, Sclerotinia sclerotiorum (white mould) in various greenhouse and field crops, Cladosporium fulvum (leaf mould) in tomato, and the powdery mildews Sphaerotheca fusca in cucurbits and Leveillula taurica in pepper. T. harzianum T39 was applied in vineyards and greenhouses as part of grey mould management programmes in alternation with chemical fungicides. In the present study, the effect of T39 on diseases of greenhouse crops was demonstrated. The biocontrol agent was applied in formulations containing two concentrations of the active ingredient, or in the presence of oil in cucumber and tomato greenhouses. Suppression of B. cinerea , C. fulvum and S. sclerotiorum was similar when T39 was applied at final active ingredient rates of 0.2 or 0.4 g l -1 , except for one sampling date in one experiment. The addition of JMS Stylet-Oil did not contribute to the control of the above mentioned diseases achieved by T39.     

向日葵菌核病拮抗菌XRK5的筛选与鉴定

从向日葵根际分离了640个细菌分离物,以向日葵菌核病菌(Sclerotinia sclerotiorum)为靶标菌,通过平板对峙法获得了18个具有拮抗性能的细菌,其中XRK5具有较强拮抗能力,且拮抗性能稳定,具有较好的生防应用潜力。经过形态观察、生理生化特征及16SrRNA序列分析,将XRK5鉴定为辣椒溶杆菌(Lysobacter capsici),XRK5的16SrRNA序列在GenBank中的注册号为FJ959348。     

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.     

Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans

Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance. Using REMI, up to 32 transformants mug DNA(-1) were obtained, while 37.8 transformants 5 x 10(5) germlings(-1) were obtained using ATMT. Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively. A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C. minitans transformants. Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified.