Limitations of In Vitro Strain Screening Methods for the Selection of Sclerotinia spp. as Potential Mycoherbicides against the Perennial Weed Ranunculus acris

Nineteen isolates of Sclerotinia sclerotiorum and three isolates of S. minor were inoculated on to excised tissues and intact plants of Ranunculus acris (giant buttercup), to evaluate their pathogenicity. All isolates proved pathogenic, with S. sclerotiorum being more pathogenic than S. minor on both excised tissues and intact plants. Seven of the S. sclerotiorum isolates were more pathogenic than the others on excised tissues, but no significant differences in pathogenicity were found between any of the isolates when they were inoculated on to intact plants. The results of this study indicate that the excised tissue method cannot be used to predict whole plant mortality, nor, therefore, the mycoherbicide potential of strains of S. sclerotiorum for this perennial weed.     

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: Sclerotium Survival

The plurivorous ascomycete, Sclerotinia sclerotiorum , is being evaluated as a mycoherbicide for the biocontrol of Cirsium arvense in pastures. Increased inoculum levels potentially add to the risk of disease in susceptible crops sown at or near the biocontrol site. This paper summarises: (1) two series of experiments that quantify the survival of soilborne sclerotia in sheep-grazed pastures in the Canterbury province of New Zealand; (2) a survey of the natural sclerotium population densities in four contrasting agricultural activity strata; and (3) a model for predicting the time period for which susceptible crops should be withheld from a site following the cessation of biocontrol. In the first series of two experiments, mycoherbicide-induced populations of sclerotia were sampled annually for four years. Half-lives were estimated to be 395 and 218 days. In the second series of eight experiments, sclerotia were buried in mesh bags in four different soils for three to four years, and exhibited half-lives of 206-506 days. In the survey, the four strata were permanent pasture, arable crop/pasture rotation, continuous cropping and market garden. The estimated sclerotium density in the market garden stratum was 9m -2 ; few sclerotia were found in the other three strata. Using the withholding period model, the average time for a biocontrol-induced sclerotium population to decay exponentially to a natural 'market garden' density of 9 sclerotia m -2 was estimated to be four years, assuming an average initial density of 125 sclerotia m -2 and an average half-life of 351 days. A conservative upper limit for the withholding period was estimated to be ten years.     

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.     

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.     

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.     

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.     

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.     

菌核病防治研究进展

菌核病是一种寄主种类广泛的重大植物病害,可侵染450多种重要作物和草类,在我国每年给油菜、大豆以及多种蔬菜带来10～30亿元的损失.介绍了菌核病的症状、危害以及致病机理等,概述了主要的防治措施,并报道了国内外在关于菌核病生物防治、转基因育种、分子机理等方面的研究进展.     

Enzymatic oxalate decarboxylation in isolates of Sclerotinia sclerotiorum

Abstract Sclerotinia sclerotiorum isolates B24 (virulent) and SS41 (hypovirulent) possess oxalate decarboxylase. Production was regulated by composition and pH of culture medium and required the presence of oxalate or its precursor, succinic acid, as inducers. Mycelia of both isolates contain equivalent amounts of enzyme.     

组学技术在核盘菌研究中的应用

核盘菌Sclerotinia sclerotiorum是一种典型的死体营养型植物病原真菌,全球分布且寄主范围广泛,严重危害多种植物,对农业生产造成严重损失。核盘菌研究主要集中在真菌生物学及病理学等方面。近年来,随着高通量分析技术的不断改进,多种组学技术为系统生物学研究提供了平台。文中主要综述利用多种组学研究方法在植物病原真菌核盘菌研究中的应用及研究进展,探讨开展植物病原物及病害发展的系统性研究思路,以期为核盘菌的分子生物学及致病机理等研究提供参考,同时也为其他植物病原物及病害系统研究提供理论依据。     

Characterization of microsatellites in the fungal plant pathogen,Sclerotinia sclerotiorum

Twenty‐five primers produced unambiguous amplification products of 23 microsatellite‐containing loci and two microsatellite‐like polymorphic loci, with 2–10 alleles at each locus in the plant pathogenic fungus, Sclerotinia sclerotiorum. Haplotypes are polymorphic among individuals sharing the same DNA fingerprint and DNA sequence haplotype, facilitating epidemiological monitoring worldwide. Fourteen of these primers also successfully amplified the closely related S. trifoliorum and S. minor.     

Oxalic acid production and its role in pathogenesis of Sclerotinia sclerotiorum

Abstract Sunflower plants were inoculated with a virulent isolate of Sclerotinia sclerotiorum and with the same isolate nutritionally conditioned to produce small amounts of oxalic acid. The preconditioned isolate behaved as hypovirulent. Tomato plants were inoculated with four S. sclerotiorum isolates of increasing virulence. A close correlation among disease severity, accumulation of oxalic acid, decrease in pH and inhibition of polyphenoloxidase in both infected host tissues was demonstrated. Oxalic acid production as an important factor of virulence in S. sclerotiorum is emphasized and its effect on the phenolic metabolism of the host via inhibition of polyphenoloxidase is suggested.     

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.     

稀土元素对油菜菌核病菌生长及其生化性状影响的研究

研究了氧化镧 (La2 O3 )对油菜菌核病菌 (Sclerotiniasclerotiorum ,Ss)生长及其生化性状的影响 .结果表明 ,La2 O3 浓度在 3 0～ 45 0mg·L-1时 ,固体培养和液体培养中对Ss生长均呈明显抑制作用 ,且生长抑制率 (Y)随La2 O3 浓度增加而相应增加 .液体培养中油菜菌核病菌产生的果胶酶活性随溶液中La2 O3 浓度增加而相应增加     

Detection of Sclerotinia sclerotiorum in Planta by a Real-time PCR Assay

Stem rot caused by Sclerotinia sclerotiorum is a very serious disease on oilseed rape worldwide. In this study, a pair of polymerase chain reaction (PCR) primers was designed based on the nucleotide sequence of a DNA region amplified by a microsatellite primer M13. The primer pair amplified a 252-bp fragment from all S. sclerotiorum isolates collected from oilseed rapes at different locations in different years, but not from any other fungus tested. Using this pair of primers, a real-time PCR assay was developed to rapidly detect early infection of S. sclerotiorum on petals of oilseed rape. The real-time PCR assay developed in this study could help growers make a timely decision on fungicide application.     

酿酒酵母INVSC1对油菜菌核病菌抑制作用的研究

以油菜菌核病菌为供试病原菌,在摇瓶发酵备件下,研究了酿酒酵母菌INVSC1在各种培养条件下对油菜菌核病菌的抑菌效果,确定了酿酒酵母的最佳培养条件：最佳培养温度为28℃,培养时间为20h,接种量为5％。酿酒酵母能抑制油菜菌核病可能与它发酵产乙醇有关。酿酒酵母抑制油菜菌核病的发现使生物防治又多了一类新的可供选择的生防菌。     

油菜菌核病菌对速克灵的抗药性风险研究

在室内条件下通过菌丝生长速率法测定了分离自安徽省10个县市的油菜菌核病菌（Sclerotinia scleroti-orum）对速克灵的敏感性。结果表明,速克灵对各供试菌株的EC50值分布范围为0.0899-0.4966μg/mL,平均为0.2541μg/mL,且供试菌株在含速克灵质量浓度为10 000μg/mL的PDA平板上菌丝生长几乎完全被抑制。表明各供试菌株对速克灵十分敏感,但其敏感程度地区间存在较大差异。通过室内药剂直接诱变法,获得了抗速克灵突变株。抗性突变株抗性测定结果表明,某些地区的抗性菌株抗性消失,有些地区的抗性菌株抗性继续保持。结果显示安徽省油菜菌核病菌对速克灵具有潜在的抗药性风险。     

两株致病力不同的核盘菌形态变化研究

比较了强弱致病型不同的核盘菌Ep-1PNA5和Ep-1PN的菌落、菌丝、细胞的形态变化。结果发现强致病型的Ep-1PNA5在平板上的菌落呈圆形均匀扩展,气生菌丝少,形成的菌核多,细胞的原生质均匀浓密,内部结构完整。弱致病型的Ep-1PN菌落呈扇形扩展,不均匀,气生菌丝发达,形成的菌核少,菌丝顶端分枝异常,有原生质外渗,且原生质不均匀,内部结构不完整。Ep-1PN致病力减弱和生长抑制可能与其菌丝结构和细胞形态变异有关。