| Black spot disease can cause the leaves of Eucommia ulmoides to turn wither and yellow,and the leaves fall early,causing the branches to die.In recent years,the black spot disease of E.ulmoides has occured in a large area in the E.ulmoides plantation area of Dayi County,Chengdu City,Sichuan Province,which has seriously affected the growth and quality of E.ulmoides,restricted the economic value and ecological benefits of E.ulmoides,and caused losses.At present,there are few studies and reports on the black spot of E.ulmoides.In this paper,through the isolation,identification and biological characteristics of pathogens in E.ulmoides leaves,on the basis of the separation and screening of antagonistic bacterial strain,the fermentation parameters of antagonistic bacterial strain were optimized by response surface method,and the biocontrol effect of antagonistic bacterial strain on black spot of E.ulmoides was explored by pot experiments.The main research results obtained are as follows:1.The pathogen DZHBB-1 was isolated and purified from the leaves of black spot of E.ulmoides by conventional tissue separation and single spore purification.The pathogenicity of DZHBB-1 was verified by Koch’s postulates,and identificated by morphological characteristics and molecular method.It was identified as Pestalotiopsis trachicarpicola,and its r DNA-ITS,β-tubulin and tef1 gene sequences Gen Bank accession numbers were MG827240,MG833724,and MG833725,respectively.2.The biological characteristics of the colonies and conidia of DZHBB-1 were determined by plate assays and germination on glass slides,respectively.The results show the mycelial growth was the best in the PDA medium,p H 7,the temperature is 20-25 °C,and glucose and beef extract are the best for carbon and nitrogen,respectively.The spore suspension treated with 1% glucose solution and 1% sucrose solution was propitious to improve the germination rate of conidia.It was propitious to the growth of hyphae under full light condition and propitious to the germination of conidia under full dark condition.The lethal temperature of hyphae and conidia were 52 °C and 72 °C,respectively.3.Sixty-two strains of Bacillus were isolated from the healthy leaves of E.ulmoides by dilution isolation method.Fourteen strains of antagonistic bacteria were screened by PDA sputum experiments method,which had better antagonistic effect on Pestalotiopsis trachicarpicola DZHBB-1.The growth rate method was used to re-screen out the antagonistic bacteria strain J1,which had the best antagonistic activity and was stable.The average width of the inhibitory zone of the antagonistic bacteria at the initial screening of the pathogens was 1.28 cm;the average inhibition rate of the rescreening was 66.67%.Combining morphological,physiological and biochemical properties,and molecular methods,the antagonistic bacteria was identified as Bacillus amyloliquefaciens,whose 16 S r RNA,gyr A,and gyr B gene sequences Gen Bank accession numbers were MK737747,MK789290,and MK789291,respectively.4.The fermentation parameters of Bacillus amyloliquefaciens J1 were optimized according to the single factor test and response surface design.The results showed that the optimal fermentation conditions of strain J1 were p H=6.2,rotating speed of shaking table of 179 r/min,culture temperature of 32.6 °C,inoculum volume of 4%,and liquid loading volume of 100 m L/250 m L.5.The results of pot experiments showed that the Eucommia ulmoides plant without spraying Bacillus amyloliquefaciens J1 fermentation broth had a very high incidence of96.67%,and the disease was effectively controlled after spraying the fermentation broth of B.amyloliquefaciens J1.Among them,the control effect of response surface optimized fermentation broth of B.amyloliquefaciens J1 on the black spot of E.ulmoides was better than that of the non-optimized suspension,and the control effect of the two stock solutions on the black spot of E.ulmoides was 90.99% and 83.78% respectively.After diluted with sterile water,the control effect of B.amyloliquefaciens J1 fermentation broth will be reduced,but the control effect of B.amyloliquefaciens J1 suspension with 50-200 times and B.amyloliquefaciens J1 fermentation broth with 50-500 times on the black spot of E.ulmoides will be more than 70%. |
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