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.     

Reaction of sunflower varieties to Sclerotinia sclerotiorum under various inoculation methods,time of field evaluation and phylogenic stages of host

Sunflower cultivation is affected seriously by Sclerotinia sclerotiorum (Lib.) de Bary in Iran, particularly north-western areas. Because of economic and environmental harms by chemical control, it is necessary to develop cultivars with adequate genetic resistance for reduction of yield losses. The purpose of this study was to find an effective method of inoculation with S. sclerotiorum under field evaluations. Three stem-inoculation techniques including: 1 – mycelium plug, 2 – oxalic acid solution (OAS) and 3 – infested wheat seeds with Sclerotinia mycelium were employed under field conditions. Four genotypes including Ghalami (local variety in market), Confeta, Allstar and Master were used in this study. The lesion length, lesion width and lesion as up and down leading on the stem from inoculation site were measured after 3, 7, 10 and 14 days of inoculation. The analysis of variance showed significant difference between all employed techniques and incubation days after inoculation. Mycelial plug (MP) inoculation technique produced significantly more developed lesions on the treated stems. In spite of this effect, Master variety demonstrated reasonable resistance reaction against the disease. The progress of disease in wounded treatments was also faster than the non-wounded ones. And, the shortest time to obtain significant differences between varieties was 10?days after inoculation. By comparison of results of lesion length at flowering and seed-filling stages, the more obvious effectiveness of the disease was observed at the second stage. Finally, there were negative correlations between mean temperature and mean lesion length in all three inoculation methods.     

Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

紫外光诱导的油菜菌核病菌抗扑海因菌株的特性研究

通过室内紫外光诱导,获得了油菜菌核病菌多株抗扑海因突变体。采用菌丝生长速率法.对诱变得到的抗扑海因菌株的抗药性进行了测定。结果显示,油菜菌核病菌抗扑海因菌株的EC_（50）值分布范围为0.1159-604.2200μg/mL,平均值为117.1363μg/mL,突变株UVIP＇SS-7、UVIP＇SS-8、UVIP＇SS-15、UVIP＇SS-16和UVIP＇SS-18的EC_（50）值均大于160μg/ML。油菜菌核病菌抗扑海因突变株在含扑海因质量浓度为1μg/mL时的菌核产生数量绝大多数都大于不含药PSA平板的菌核数量,而高浓度下菌核几乎不产生。     

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

AIMS: To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. METHODS AND RESULTS: The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. CONCLUSION: Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.     

The plant-associated Bacillus amyloliquefaciens strains MEP2 18 and ARP2 3 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease

Aims: This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP₂3 and MEP₂18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. Methods and Results: The antifungal compounds were isolated by acid precipitation of cell‐free supernatants, purified by RP‐HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP‐HPLC eluted fractions showed the presence of surfactin C₁₅, fengycins A (C₁₆–C₁₇) and B (C₁₆) isoforms in supernatants from strain ARP₂3 cultures, whereas the major lipopeptide produced by strain MEP₂18 was iturin A C₁₅. Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides‐containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide‐producing B. subtilis strain. Conclusions: Both strains, renamed as B. amyloliquefaciens ARP₂3 and MEP₂18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide‐producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. Significance and Impact of the Study: Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP₂3 and MEP₂18 to control plant diseases caused by S. sclerotiorum.