Study On The Main Biocontrol Mechanisms Of Bacillus Amyloliquefaciens PG12 Against Apple Ring Rot Disease

Bacillus spp. are safe microorganisms which can be found ubiquitously in the environment. They exhibit versatility in protecting plants from pathogen infection, and have excellent abilities to colonize and to sporulate. B. amyloliquefaciens are promising candidates for biological control of postharvest diseases. Apple ring rot disease, caused by Botryosphaeria dothidea, was one of the most important diseases during the postharvest period on apple fruit. In order to reveal the main machanisms of biocontrol activity of B. amyloliquefaciens PG12 against apple ring rot, chemistry and molecular methods were used to identify the main antimicrobial compounds and their phenotypic functions involved in its antifungal activity. This study layed the foundation for field application of bio-pesticides.1)B. amyloliquefaciens PG12 effectively controlled apple ring rot disease.B. amyloliquefaciens PG12 exhibited outstanding inhibitory effects in control of mycelia and spore infection of B. dothidea. When apples were sprayed with PG12 before harvest or only after harvest, the disease incidence was significantly lower than that for control in the postharvest period. Lumpy appearance and abnormal structure of the mycelia from the edge of inhibition zone were observed using scanning electron microscopy (SEM) in in vitro assays. According to vitro assay results, antibiosis was considered as one of the most important mechanisms in biocontrol of B. dothidea.2) Lipopeptides were responsible for the inhibitory activity of B. amyloliquefaciens PG12 against B. dothidea.To further investigate biocontrol mechanisms of B. amyloliquefaciens PG12 against B. dothidea, antimicrobial compounds were considered as an important breach. Results obtained from charateristics analysis of cell-free supernatant of PG12 showed thatthe active compounds have a high stability to ultra-vioket radiation, proteinase K, high temperature, strong acid and alkali conditions. The lipopeptide crude extracts from cell-free supernatant of PG12 had remarkable antifungal activity against B. dothidea, indicating that lipopeptides played a major role in the biocontrol ability of PG12. PCR detection revealed that PG12 harbored the gene clusters required for the biosynthesis of the three main families of lipopeptides, including iturin, fengycin and surfactin. The iturin-like (R/0.4) and fengycin-like (Rf 0.1-0.2) compounds showed inhibitory activity against B. dothidea using thin layer chromatography (TLC)-bioautography analysis. The iturin-like compounds were further fractionated by semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC). The fraction with a molecular weight of 1043.55 m/z was identified as iturin A by electrospray ionization quadrupole-time-of-flight mass spectrometry/mass spectrometry (ESI-Q-TOF MS/MS). According to some reports, a technological strategy for gene manipulationin B. amyloliquefaciens strains was summarized. B. amyloliquefaciens PG12 mutants lacking production of one, two, or three lipopeptides were used for study of antimicrobial activity. In in vitro assay, the srfAA, fenA and ituD mutants significantly decreased antifungal activity. Although these single mutants still retained antifungal activity against B. dothidea, a double mutant lacking iturin A and fengycin was heavily impaired in its ability to control mycelia and spore infection of B. dothidea, suggesting that iturin A and fengycin act in a synergistic manner.3) Lipopeptides promoted the multicellular behaviors of B. amyloliquefaciens PG12 in different degrees.Recently, besides antimicrobial activity of lipopeptides, some research has showed that lipopeptides also contribute to the multicellular behaviors of B. subtilis, such as biofilm formation, colony morphology, swimming and swarming motility. As relatively little was known about B. amyloliquefaciens, this study further revealed the connections between lipopeptides and their function in multicellular behaviors in PG12. The deficiency of three lipopeptides resulted in the change of pellicle and colony morphology. Iturin A, fengycin and surfactin potentially participated at different stages of biofilm formation. Surfactin played an important influence on the stage of both initial attachment and maturation, while iturin A and fengycin had more influence on the stage of initial attachment than that of maturation. B. amyloliquefaciens PG12 mutants lacking production of these three lipopeptides lost the ability of swarming motility. Compared with surfactin, iturin A and fengycin contributed differently to the swimming motility. In addtion, B. amyloliquefaciens PG12 mutants lacking production of these three lipopeptides performed very poorly in biocontrol of apple ring rot in the postharvest period.Taken together, the three lipopeptides contributed differently and comprehensively to the antifungal activity and multicellular behaviors of B. amyloliquefaciens PG12. Surfactin, iturin A and fengycin were important factors in excellent biocontrol activity of B. amyloliquefaciens PG12 against apple ring rot disease.