Isolation And Identification Of Bauhinia Blakeana Leaf Spot Pathogens And Its Biological Control

In recent years,Bauhinia blakeana leaf spot disease has occurred in a large area of Chengdu City,Sichuan Province,causing leaf perforation,death and defoliation,reducing the ornamental properties of Bauhinia blakeana and affecting its ecological and medicinal values.In this paper,we identified the pathogenic fungus of Bauhinia blakeana leaf spot,isolated and screened the best antagonistic bacteria from the healthy leaves of Bauhinia blakeana,and optimized the fermentation conditions of the antagonistic bacteria by orthogonal test,and tested the biological control effect of the antagonistic bacteria on Bauhinia blakeana leaf spot by potting test.The antifungal proteins of the antagonistic bacteria were extracted,stability tests and mass spectrometry were conducted to predict the structure and function of the antifungal proteins,and the following main findings were obtained:1.The pathogenic fungus YTJ-1 was isolated and purified from diseased Bauhinia blakeana leaves by conventional tissue isolation and Koch’s rule.based on morphology and molecular biology identification of rDNA-ITS,ACT,GAPDH,TUB2 gene sequences in tandem to construct a multigene phylogenetic tree,the pathogenic fungus YTJ-1 was identified as Colletotrichum endophytic,whose rDNA-ITS,ACT,GAPDH,and TUB2 gene sequences were registered on GenBank as OK560626,OK562583,OK562584,and OK562585,respectively.2.Nine bacterial isolates were isolated from healthy Bauhinia blakeana leaves by dilution isolation method,and the antagonistic bacteria SF9 with good antagonistic effect against the pathogenic fungus YTJ-1 and stable antagonistic effect were screened based on the plate standoff growth method and growth rate method.Their average inhibition rate was 69.07%in the plate standoff growth method and 65.41%in the growth rate method.The mycelial growth of the pathogenic fungus near the antagonist was significantly inhibited,with regression or no growth of mycelium,sparse mycelium and marginal mycelium turning green.The antagonistic strain SF9 was identified as Bacillus atrophaeus by combining morphology and molecular biology based on the 16S rRNA gene sequence to construct a phylogenetic tree,and its 16S rRNA gene sequence GenBank accession number was OQ451561.3.Single-factor tests and orthogonal tests based on the results of the single-factor tests were performed in fixed 250 mL conical flasks to optimize the growth conditions of Bacillus atrophaeus SF9.The results of the one-way test showed that the optimal inoculum level for strain SF9 was 2%,where OD600 was 1.520;the optimal loading volume was 100 mL/250mL,where OD₆₀₀ was 1.517;the optimal pH was 6.8,where OD₆₀₀ was 1.922;the optimal temperature was 30℃,where OD600 was 1.761;the optimal shaking speed was 200 r/min,where OD₆₀₀was 1.720./The orthogonal test showed that the optimal combination of growth conditions for strain SF9 was 220 r/min,pH 6.4 and 30℃,the average OD₆₀₀ was 2.220,and the average inhibition rate was 76.60%under these conditions.4.In a pot experiment,the control effect was determined by inoculating healthy Bauhinia blakeana leaves with the pathogenic fungus YTJ-1 and then spraying sterile water and optimized Bacillus atrophaeus SF9 fermentation solution at different concentrations.The results showed that Bauhinia blakeana plants sprayed with sterile water had 96.67%disease incidence,while Bauhinia blakeana plants sprayed with optimized Bacillus atrophaeus SF9fermentation solution had effective control of the disease.Bauhinia blakeana leaves treated with the original solution showed an 86.67%reduction in incidence and 82.5%reduction in disease index.However,the control effect gradually decreased with the increase of the dilution of the sterile fermentation solution,where the optimal dilution was between 50 and 500 times of the solution,when the control effect could reach more than 70%.5.The antifungal proteins was extracted from Bacillus atrophaeus SF9 by ammonium sulfate salting at different saturation levels,and the antibacterial activity was measured.The results showed that the antifungal proteins with 60%ammonium sulfate saturation level had the best antibacterial effect on pathogenic fungus,and the inhibition rate was about 66.93%.The stability test showed that the antifungal proteins still had a reasonable inhibition rate at 40～80℃and pH 6.0～8.0,and the UV irradiation did not affect the inhibition rate,i.e.,the antifungal proteins was stable.The proteins in the samples were detected using LC-MS mass spectrometry,and the raw data obtained by mass spectrometry were analyzed to match the proteins database to get the proteins identification results for quality assessment and functional annotation.Based on the proteins identification results,the amino acid sequences of proteins with high coverage（%）were downloaded,including DNA-binding protein,Thioredoxin,Septation protein SpoVG,Phosphocarrier protein,Chaperonin GroEL Ferredoxin,ABC transporter domain-containing protein,and CSD domain-containing protein,and the secondary and tertiary structures of each protein were inferred from the amino acid sequences.