Mechanisms of phyllosphere biological control of Leptosphaeria maculans, the blackleg pathogen of canola, using antagonistic bacteria.

摘要

Blackleg, caused by Leptosphaeria maculans (Desm.) Ces & De Not (anamorph Phoma lingam (Tode:Fr./Desm.)), is an economically important disease of canola (Brassica napus L.) in Canada and worldwide. In W. Canada, a shift in the pathogen population to more virulent races (PG3, PGT and PG4) has increased the risk of a blackleg epidemic, which is a huge threat for the canola industry. Biological control using non-pathogenic, antagonistic bacteria has been successfully used in other cropping systems for the control of plant diseases. This study identified that bacteria originating from the stubble and leaf tissues (endophytes) had the highest antifungal activity, against L. maculans, in plate and plant assays, respectively. Bacteria with the highest disease suppression in cotyledon assays were also effective at the 3-4 leaf stage. In field assays, bacteria applied at the cotyledon leaf stage suppressed the disease. PCR screening for antibiotic biosynthetic genes identified Pseudomonas chlororaphis (DF 190, DF202, DF210) and P. aurantiaca (DF200) isolates harboring biosynthetic genes of phenazine and pyrrolnitrin. Similarly, PCR-screening also detected the presence of biosynthetic genes of: iturin A and bacillomycin D in Bacillus cereus strain DFE4, B. amyloliquefaciens strains DFE16 and BS6; surfactin in strains DFE4 and DFE16; zwittermicin A and the self-resistance protein (ZmaR) in B. cereus isolates DFE4, DFE8 and DFE13. The GC-MS analysis of the methanol-broth extract of strain DF 190 revealed the presence of phenazine, 2-hydroxyphenazine and 2-acetamidophenol. MALDI-TOF-MS analysis of the cell surface extracts of strains DFE4 and DFE16 detected the production of antifungal lipopeptide antibiotics iturin A, bacillomycin D and surfactin, confirming the PCR detection of the biosynthetic genes of these antibiotics. The production of 2-acetamidophenol by a P. chlororaphis isolate, and lipopeptide antibiotics iturin A, bacillomycin D and surfactin by a B. cereus isolate has not been reported before. Antifungal antibiotics-producing bacteria were also tested for elicitation of ISR in blackleg control. Bacterial cells and broth extracts, when inoculated away from the pathogen, failed to elicit ISR towards the suppression of blackleg lesions on cotyledons. However, the bacteria and broth extracts, when inoculated locally with the pathogen, significantly suppressed the pycnidiospores and lesion development. The absence of localized host antifungal defense enzyme activity further indicated direct antifungal activity of the bacteria and metabolites present in their broth extracts. A gacS mutant of strain PA23 lost its secondary metabolite production and biocontrol activity, which was restored with the complementation of the gacS gene. Interestingly, a phenazine non-producing mutant of strain PA23, producing same levels of pyrrolnitrin as the wild type, exhibited same or better levels of blackleg disease suppression. This suggests the potential role of pyrrolnitrin in antibiosis mediated by strain PA23, along with extracellular lytic enzyme activity for the biocontrol of L. maculans in canola. Overall, this study has demonstrated the potential of bacterial biocontrol agents to mediate phyllopshere control of L. maculans, which could be incorporated in an integrated disease management system.