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.     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

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.     

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.     

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.     

Population structure of Sclerotinia sclerotiorum in an Australian canola field at flowering and stem-infection stages of the disease cycle.

Populations of the ascomycete pathogen Sclerotinia sclerotiorum sampled from a canola field were analysed using microsatellite markers. Fifty isolates were collected from ascospore-infested canola petals and, later in the season, another 55 isolates were obtained from stem lesions; these isolates were used to compare inoculum and disease-causing populations. Fifty-five unique haplotypes were identified, with gene diversity ranging from 0.40 to 0.71. Genotypic diversity was higher in the inoculum population than it had been in the previous year, but analysis of molecular variance (AMOVA) showed that less than 10% of the variation was attributable to differences between the 2 years. Genotypic disequilibrium measures were consistent with the occurrence of both clonal reproduction and out-crossing. There was no significant population subdivision between the ascospore and stem-lesion populations, as measured with fixation indices (R(ST) = 0.015, p = 0.90) and AMOVA, suggesting that there are no genetically defined subgroups of isolates more likely to proceed from petal colonization to cause stem infection. This might be because S. sclerotiorum possesses wide-ranging pathogenicity mechanisms that account for the lack of host specificity observed to date.     

Evaluation of polyclonal-antibody-based immunoassays for detection of Sclerotinia sclerotiorum on canola petals, and prediction of stem rot

The potential for using polyclonal-antibody-based immunoassays for detection of Sclerotinia sclerotiorum on canola petals as part of a disease prediction model was investigated. A commercial ELISA kit designed for Sclerotinia homoeocarpa was evaluated for specificity to S. sclerotiorum in comparison to other Sclerotinia spp., and known phyllosphere fungi. This polyclonal-antibody-based kit cross-reacted with antigens from other Sclerotinia spp., and fungi, and absorbance values obtained from S. sclerotiorum-infested canola petals were poorly correlated with percentages of infested petals as determined by plating on semi-selective medium, except when petals were incubated at high humidity for 24 h at 20 degrees C-22 degrees C prior to ELISA evaluation. Additional polyclonal antibodies were prepared from mycelial and semi-purified cell wall antigens, and these antibodies were more specific to S. sclerotiorum than the ELISA kit. However, absorbance values obtained from S. sclerotiorum-infested canola petals were poorly correlated with percentages of infested petals as determined by plating on semi-selective medium. The results are discussed in relation to the use of polyclonal-antibody-based immunoassays for the prediction of epidemics or crop risk from sclerotinia stem rot of canola.     

1,8-Dihydroxynaphthalene monoglucoside,a new metabolite of Sclerotinia sclerotiorum,and the effect of tricyclazole on its production

Isolate SS7 of Sclerotinia sclerotiorum was previously shown to produce and excrete into agar medium copious amounts of the melanin precursor 1,8-dihydroxynaphthalene. Much reduced quantities of this product were produced in the presence of tricyclazole, an inhibitor of pentaketide melanin biosynthesis. In this study, we demonstrate that young cultures of isolate SS7 produce 1,8-dihydroxynaphthalene monoglucoside, a new natural product not previously reported from fungi. When cultured in the presence of tricyclazole, such young cultures also accumulated two new monoglucosides of 1,3,8-trihydroxynaphthalene, which, as well as 1,8-dihydroxynaphthalene monoglucoside, were also obtained from cultures of two other isolates of S. sclerotiorum. It is proposed that rapid glucosylation of 1,3,8-trihydroxynaphthalene in young tricyclazole-inhibited S. sclerotiorum cultures accounts for the failure to observe 2-hydroxyjuglone or other metabolites usually associated with blockage of the pentaketide pathway to melanin in fungi.     

Involvement of alternative oxidase in the regulation of growth, development, and resistance to oxidative stress of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a cosmopolitan, filamentous, fungal pathogen that can cause serious disease in many kinds of crops. Alternative oxidase is the terminal oxidase of the alternative mitochondrial respiratory pathway in fungi and higher plants. We report the presence of this alternative pathway respiration and demonstrate its expression in two isolates of S. sclerotiorum under unstressed, normal culture conditions. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, severely inhibited the mycelial growth of S. sclerotiorum both on potato dextrose agar plates and in liquid culture media. Inhibition of alternative oxidase could influence the growth pattern of S. sclerotiorum, as salicylhydroxamic acid treatment induced obvious aerial mycelia growing on potato dextrose agar plates. Under the treatment with salicylhydroxamic acid, S. sclerotiorum formed sclerotia much more slowly than the control. Treatment with hydrogen peroxide in millimolar concentrations greatly decreased the growth rate of mycelia and delayed the formation of sclerotia in both tested S. sclerotiorum isolates. As well, this treatment obviously increased their alternative pathway respiration and the levels of both mRNA and protein of the alternative oxidase. These results indicate that alternative oxidase is involved in the regulation of growth, development, and resistance to oxidative stress of S. sclerotiorum.     

Electrophoretic studies of Australasian, North American and European isolates of Sclerotinia sclerotiorum and related species.

Forty-seven isolates of Sclerotinia species, collected from a variety of crops growing in Australia, New Zealand, North America and Europe, have been classified into three distinct groups on the electrophoretic patterns for soluble proteins, arylesterase, acid phosphatase, tetrazolium oxidase, glucose-6-phosphate dehydrogenase (NADP-linked) and reduced nicotinamide adenine dinucleotide phosphate dehydrogenase. There were only small intra-group differences. The electrophoretic patterns of an isolate of Whetzelinia (= Sclerotinia) tuberosa were characteristically different from those of the other isolates. These results support the findings from previous studies when ontogenetic, electrophoretic and mycelial-interaction criteria were used to group a smaller number of isolates from New South Wales, Australia. It is concluded that S. sclerotiorum, S. trifoliorum and S. minor are three distinct species.     

Pathogenicity of Sclerotinia sclerotiorum on Ranunculus acris in Dairy Pasture

Fifty-four isolates of Sclerotinia sclerotiorum from Ranunculus acris and other natural hosts were applied as mycelial infested kibbled wheat onto 6 month-old R. acris plants in two glasshouse screening experiments. Most isolates (90%) did not differ in their pathogenicity towards R. acris. One isolate, S. sclerotiorum G45, was selected based on its ability to cause severe disease and suppress regeneration of R. acris. A field experiment was conducted to determine the efficacy of S. sclerotiorum (G45) against R. acris in infested dairy pastures in the Takaka Valley, Golden Bay, New Zealand. Isolate G45 was formulated as a wettable powder and was applied as a slurry at 20 and 40 ml/plant in December 1995. After 10 weeks, regeneration from the crown of treated plants was apparent and a second application of S. sclerotiorum was made in February 1996. Best control of R. acris was obtained when the plants were inoculated in full flower in December. At the first time of treatment, the 40 ml application of S. sclerotiorum slurry reduced the total dry weight of R. acris by an average of 57%. The second application had no effect on total dry weight, possibly because moisture levels were not sufficient for S. sclerotiorum infection. This study confirmed S. sclerotiorum to be an aggressive pathogen of R. acris under both glasshouse and field conditions. As a result, this pathogen has potential as a mycoherbicide for R. acris. Further experiments are required to explore ways of enhancing the efficacy of S. sclerotiorum against R. acris by manipulation of the host, pathogen and environment.     

孜然种子提取物枯茗醛和枯茗酸抑菌作用研究

以番茄早疫病菌、棉花黄萎病菌、小麦纹枯病菌、烟草赤星病菌、小麦全蚀病菌、油菜菌核病菌、辣椒疫霉病菌、水稻纹枯病菌、小麦白粉病菌和番茄灰霉病菌等为供试菌种,采用离体与活体相结合的方法系统地测定了枯茗醛和枯茗酸的抑菌活性。离体抑菌活性测定结果表明,枯茗醛和枯茗酸对多种病原菌具有一定的抑制效果,其中对油菜菌核病菌菌丝生长的抑制效果高于其它供试病原菌,有效中浓(EC50)分别为2.1和7.3 mg/L;枯茗醛和枯茗酸对小麦白粉病的防治实验结果表明,供试浓度为1000 mg/L时,两种药剂的保护防效均高于50%;相同处理浓度下,枯茗酸对油菜菌核病的保护防效与速克灵处理相当,达到57.52%。     

核盘菌弱毒株Ep-1PN培养性状的有性遗传特性*

观察和测定了核盘菌（Sclerotiniasclerotiorum（Lib．）deBary）弱毒株Ep-1PN单孢分离物的生长、菌落扩展和菌落形态等培养性状。结果发现：来自7个子囊盘的1574个单孢分离物中有1560个分离物的培养性状与正常菌株的没有显著差异,与亲本相似的分离物只有6个,其它8个分离物介于弱毒株Ep-1PN与正常菌株之间。弱毒株Ep-1PN培养性状的有性遗传不遵循孟德尔核遗传规律。     

核盘菌弱毒株Ep-1PN培养性状的有性遗传特性

观察和测定了核盘菌(Sclerotinia sclerotiorum (Lib.) de Bary)弱毒株Ep-1PN单孢分离物的生长、菌落扩展和菌落形态等培养性状.结果发现来自7个子囊盘的1574个单孢分离物中有1560个分离物的培养性状与正常菌株的没有显著差异,与亲本相似的分离物只有6个,其它8个分离物介于弱毒株Ep-1PN与正常菌株之间.弱毒株Ep-IPN培养性状的有性遗传不遵循孟德尔核遗传规律.     

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。     

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742)

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

An optimized method for mycelial compatibility testing in Sclerotinia sclerotiorum

Classification of isolates into mycelial compatibility groups (MCGs) is used routinely in many laboratories as a quick marker for genotyping Sclerotinia sclerotiorum within populations. Scoring each new sample requires optimization of standardized conditions to support adequate growth of all paired isolates. Appropriate conditions for growth are especially important because diverse compatibility reactions are difficult to categorize and score (e.g., in samples from populations with high genetic diversity, such as those that receive immigration from genetically diverse sources or those that deviate from strict clonality). The current standard medium for MCG testing can be inhibitory to isolates from some samples, confounding scoring of compatibility. We identified two foci for optimization: (i) choice of medium, in this experiment, Patterson's medium amended with red food coloring (termed modified Patterson's medium, MPM, the current standard medium) versus potato dextrose agar (PDA) and (ii) amount of McCormick's red food coloring amended to the growth medium. The red food coloring often yields a red reaction line in incompatible interactions; alternative incompatible reactions are a line of thick or thin hyphae. Based on results to date, self-self pairings of S. sclerotiorum are compatible and are a reliable standard for scoring compatible self-nonself mycelial interactions. PDA amended with 75 microl/L of McCormick's red food coloring was identified as optimal for isolates inhibited by MPM from a highly diverse, recombining population sample. This precisely amended PDA was also suitable for isolates from highly clonal populations that were not inhibited by MPM or by higher concentrations of red food coloring. Under the optimized, standardized conditions all paired isolates grew together and produced interactions that could be scored in repeatedly identifiable categories, compatible or incompatible. Workers are advised to optimize conditions before screening a new population sample.     

Biology and mycovirus-assisted biological control of Sclerotinia sclerotiorum infecting vegetable and oilseed crops

Abstract

Plant disease caused by pathogenic fungal infection causes maximum crop damage. Among different fungal diseases, rot caused by Sclerotinia spp.; is a primary concern for vegetables and oilseed industry. Disease management using Chemical fungicides is a potential hazard and leads to the development of many fungicide-resistant strains. Hypovirulence associated mycoviruses is a possible environment-friendly solution, and current studies are aiming to exploit their potential as biocontrol agents. The use of the mycovirus mediated hypovirulent approach has emerged as a new technique to identify successful biocontrol agents. Most mycoviruses are known to have RNA genomes, double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA). A total of six dsRNA mycoviruses and a one ssDNA mycovirus have been reported from Sclerotinia sclerotiorum till date which includes the most recent entry as published by Hamid and his group in 2018. In contrast to dsRNA mycovirus, ssDNA mycovirus reported from Sclerotinia sclerotiorum has significant potential to be used as a biocontrol agent in the fields. Despite several reports on mycoviruses of Sclerotinia, not much could be done to explore its commercial importance. The present review describes the recent developments in the area of mycoviruses of Sclerotinia sclerotiorum and associated biocontrol potential.     

Phosphatase activity in susceptible and resistant cultivars of Brassica juncea inoculated with isolates of Macrophomina phaseolina and Sclerotinia sclerotiorum

Inoculations with isolates of Macrophomina phaseolina and Sclerotinia sclerotiorum resulted in a significant increase in the acid phosphatase activity of susceptible and resistant cultivars of Brassica juncea, which appeared to be related to the disease reaction of different host-pathogen combinations. After an increase on the 6th day of inoculation there was usually a fall in activity on the 12th and 18th days. Resistant cultivars showed very poor activity in comparison to their susceptible counterparts.