The Study On The Mechanism Of Bacillus Licheniformis BL06 Secretion Proteins In Inhibiting The Growth And Pathogenicity Of Magnaporthe Oryzae

Rice is the most important staple food for more than half people worldwide.However,the causal agent Magnaporthe oryzae caused worldwide mutil-cycle disease of rice blast,which bring about severe yield losses.As the disadvantages of the resistant rice varieties and chemical pesticides application in rice production become more and more obvious,the status of microbial pesticides are gradually prominent.Bacillus licheniformis BL06 was isolated from cucumber rhizosphere in Huai’an city,Jiangsu Province.We recently evaluated BL06 as a promising biocontrol agent,the results showed that BL06 did have a good biocontrol effect that was up to 63.05%in a greenhouse inoculation assay with Magnaporthe oryzae strain guy 11.In order to understand the biological control mechanism of Bacillus licheniformis BL06 against rice blast,we conducted a series of experiments in vitro to explore the effect of BL06 on the mycelia growth and conidia development of guy11.We found that swelling of mycelium was caused by BL06 at a concentration of 1×107 CFU/mL,and many ballooning of mycelia were observed.Then conidium germination was significantly inhibited by BL06 bacteria solution at the concentration of 1×107 CFU/mL.Conidium germination rate significantly reduced in a concentration-dependent manner by BL06 bacteria solution.Interestingly,compared with Bacillus cereus HS24 as the control,there was no significant difference in the inhibition of appressorium formation between BL06 and HS24 at the same concentration of bacterial solution,but BL06 was not better than HS24 in the inhibition of conidium germination.By comparing effective components of BL06 and HS24,it was found that the secretion proteins in the supernatant of BL06 bacteria solution had strong inhibitory effect on conidia growth than that of HS24.We named the secretion proteins in the supernatant of BL06 bacteria solution as BL06-SP.BL06-SP had a significant antagonistic effect on the growth of mycelia of M oryzae.The resulting phenomenon is the same as the effect of BL06 bacterial solution on mycelia,observing some of mycelia swell into a round shape effected by BL06-SP at 5 mg/mL,and it significantly suppressed conidium germination and appressorium formation.The inhibitory effect of BL06-SP on conidium germination and appressorium formation were dependent on its concentration,and BL06-SP at 50 μg/mL supported similar levels of germination to the control of sterile water,however,it affected the functional development of appressorium and further weakened the pathogenicity of M.oryzae,which confirmed by the incipient collapse assay and infection assay.The most important discovery is that BL06-SP against tip of conidium has emerged many swollen structure.It has been reported that swollen structure is due to blocked chitin synthesis of the cell wall.The transcriptional level of CHS1,CHS2,CHS4,CHS6 and CHS7,which are key genes in chitin synthesis,were significantly down-regulated in BL06-SP-treated mycelia and conidia.These results suggest that the inhibition by BL06-SP may be through interfering with cell wall integrity in M.oryzae.We then measured the chitin content in mycelia and conidia of M.oryzae.We found that chitin content of mycelia and conidia treated with BL06-SP at 5 mg/mL decreased significantly,compared with control water-treated.CFW-28 fluorescence staining in conidia were treated with sterile water and BL06-SP at 5000,50 μg/ml.In comparison with water-treated,relative fluorescence intensity of chitin treated with BL06-SP at 5 mg/mL had approximately 3-fold reduction in conidia.However,BL06 at 50 μg/mL supported similar levels of fluorescence intensity to the control.The above results indicated that B.licheniformis BL06-SP interfered with cell wall integrity by inhibiting chitin synthesis of M.oryzae,thus playing an important role in biocontrol.In this study,we found that the inhibitory effect of the biocontrol bacteria BL06 on the growth and development of M.oryzae is related to the synthesis of chitin.The results will promote the analysis of the biocontrol mechanism of B.licheniformis,indicating that BL06-SP provides potential new leads in the control of this devastating pathogen by targeting specific chitin syntheses.It will lay a theoretical foundation for the industrial application of BL06 secretion proteins in preventing and controlling rice blast.