Fusarium wilt of banana (FWB) is the main threatening factor for banana production worldwide. To explore bacterial biocontrol resources for FWB, the antagonistic effective strains were isolated from banana-producing areas in Yunnan Province, China. Two isolates (YN0904 and YN1419) displaying strong antagonism against Tropical Race 4 (TR4) were identified from a total of 813 strains of endophytic bacteria. TR4 inhibition rates of YN0904 and YN1419 were 79.6% and 81.3%, respectively. By looking at morphological, molecular, physiological and biochemical characteristics, YN0904 was identified as Bacillus amyloliquefaciens, while YN1419 was identified as as B. subtillis. The control effects of YN0904 and YN1419 on TR4 in greenhouse experiments were 82.6% and 85.6%, respectively. Furthermore, YN0904 obviously promoted the growth of banana plantlets. In addition, biocontrol marker genes related to the biosynthesis of antibiotics synthesized and auxin key synthetase genes could be detected in YN0904. Surprisingly, the marker gene sboA could be exclusively detected in YN1419, while other marker genes were all absent. Molecular characterization results could provide a theoretical basis for expounding the biocontrol mechanisms of these two strains. We concluded that natively antagonistic strains derived from local banana plantations could provide new biological control resources for FWB.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), especially Tropical Race 4 (TR4), seriously threatens banana production worldwide. There is no single effective control measure, although certain Bacillus strains secrete antibiotics as promising disease-biocontrol agents. This study identified five Bacillus strains displaying strong antibiotic activity against TR4, using a systemic assessment for presence/absence of genetic markers at genome level, and expression profiles at transcriptome level. A conventional PCR with 13 specific primer pairs detected biocontrol-related genes. An accurate, quantitative real-time PCR protocol with novel designed specific primers was developed to characterise strain-specific gene expression, that optimises strain-culturing and RNA-isolation methodologies. Six genes responsible for synthesising non-ribosomal peptide synthetase biocontrol metabolites were detected in all five strains. Three genes were involved in synthesising three Polyketide synthetase metabolites in all five strains, but the macrolactin synthase gene mln was only detected in WBN06 and YN1282-2. All five Bacillus strains have the genes dhb and bioA, essential for synthesising bacillibactin and biotin. However, the gene sboA, involved in subtilisin synthesis, is absent in all five strains. These genes’ expression patterns were significantly different among these strains, suggesting different mechanisms involved in TR4 biocontrol. Results will help elucidate functional genes’ biocontrol mechanisms.

Magnaporthe oryzae avirulence (AVR) genes are predicted to be involved in pathogen invasion and their virulence functions are restricted by the presence of the cognate resistance (R) genes. In this study, the distribution and variation of the avirulence (AVR‐Pii) gene of M. oryzae in Yunnan province, China were analysed to understand haplotype diversity of AVR‐Pii under field conditions. The presence of AVR‐Pii in 454 field isolates of M. oryzae collected in Yunnan province was examined using gene‐specific PCR markers. The results showed that 82 M. oryzae isolates carried AVR‐Pii. Among them, 39 (35.5%), 5 (15.2%), 4 (14.3%), 2 (13.3%), 25 (12.8%) and 7 (9.7%) of the M. oryzae isolates carried AVR‐Pii from central, southeastern, southwestern, northwestern, western and northeastern Yunnan province, respectively. Of these isolates, 55 were sequenced, while the remaining 27 isolates were not suitable for PCR‐based sequencing and were not used for further analysis. Moreover, three AVR‐Pii haplotypes were identified among the 55 isolates, in which H1 was identical with a previous sequence in GenBank (accession no. ) and H2 and H3 were novel variants. All DNA sequence variations were found to occur in the protein‐coding region resulting in amino acid substitutions. One virulent haplotype of AVR‐Pii to Pii was identified among 55 field isolates, suggesting that the AVR‐Pii gene has lost avirulence function through base substitution. These findings suggest that AVR‐Pii is under positive selection and AVR‐Pii mutations are responsible for overcoming race‐specific resistance in nature.

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Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.