Functional Gene Analysis Of Bacillus Amyloliquefaciens WK1 And Its Colonization Possibility In Carya Cathayensis Tree

Carya cathayensis is a famous local nut with high economic benefits to planters in Zhejiang and Anhui Province.However,long-term intensive management with high rate of fertilizer and herbicide application has caused a severe problems,such as soil acidification,nutrient imbalance and serious pathogen,especial the canker disease caused by Botryosphaeria dothidea in Carya cathayensis forest land.A strain of Bacillus amylolyticus WK1(CGMCC 11640)isolated by our early research was of ability to inhibit against B.dothidea and other broad-spectrum fungi pathogen.However,manually painting WK1 on the stem of Carya cathayensis is huge work as to wide range of practical management.Therefore,it is equivalent to that the plant obtains autoimmune ability if strains WK1 can colonize inner plant.The objective of this study was to analyze the gene clusters related to antibiotic synthesis and strain colonization based on the whole genome information by sequencing.The green fluorescent protein(GFP)technology was used to to label WK1 in order to track its path and dynamics of colonization inner Carya cathayensis as well as rhizosphere soil.A pot trail with three levels of pH by planting young seedlings of Carya cathayensis and field experiment in Carya cathayensis stands by applying GFP inoculation in root soil,stem and leaf were conducted to obtain appropriate soil pH and inoculation methods for WK1 colonization.The main results are as follows:1.Genomic analysis revealed that WK1 has synthesis gene clusters of lipopeptide and polyketide antibiotics,as well as a series of colonized-related gene clusters.Strain WK1 has similar functional gene clusters as model strain FZB42.2.The labeled WK1(GFP-WK1)has the similar growth curve and inhibition ability against B.dothidea with WK1.The plasmid PNW33 had genetic stability in GFP-WK1 strain so that labeled strain met the requirement of tracking study in natural environment..3.The pot trail discovered that GFP-WK1 successful habited in soil and inner seedling root,stem and leaf of hickory.The highest number of GFP-WK1 was detected in treatment with soil pH 6.8.At 60 days,the number of GFP-WK1 individuals corresponding to fresh weight of tissue unit was 105,105,104 and 105 cfu·g-1 respectively.4.The field experiment in hickory stands showed that the colonization amount of GFP-WK1 in leaves decreased with increasing time after inoculation by leaves spraying and root watering,and it tended to be stable after 30 days,while the dynamics was opposite in the trunk instill treatment.The highest amount of GFP-WK1 was observed in leaves of trunk instill treatment and reached a stable number of 105 cfu·g-1 after 30 days.Conclusion:WK1 has the complete gene cluster of antibiotic synthesis and plant colonization,which can be stably colonized in plants.The best method of bacterial solution application is trunk suspension.