Study On The Pathogen Identification Of Stem Rot In Dendrobium Officinale And The Plant Growth Promotion And Biocontrol Effect Of Bacillus Velezensis D-12

Dendrobium officinale Kimura et Migo is a precious herb.However,as the rapid development of its cultivation,the disease is becoming a sreious problem.During our investigation in Zhejiang province,the disease with stem rot symptom was observed on commercial cultivation areas,which seriously affected the growth of this plant.In this study,the pathogen of stem rot disease of D.officinale was detcted and identified;the endophytes bacteria from D.officinale were isolated and it’s antagonistic activity and plant growth promoting mechanism of biocontrol bacterial strain were studied.The main results are mentioned as follows:1.The four isolates were obtained from symptamatic tissues.Results of Koch’s postulates confirmed that strain A-612 is the pathogen,which was identified as Athelia rolfsii based on rDNA-ITS gene and morphological charactristics.The temperature has important effect on its growth rate.2.Fourteen strains of endophytic bacteria isolated from healthy leaves of D.officinale.Antimicrobial activity of bacteria against phytopathogen was screened using dual culture assay.The strain D-12 was found to has strong antagonistic ability against A-612,with inhibition zones up to 4.235 mm,and it could inhibit the growth of all tested fungi,which showed its widely antifungal spectrum.The strain D-12 was identified as Bacillus velezensis by physiological,biochemical properties,16 S rDNA and gyrB gene sequence analysis.The D-12 strain sequence was deposited in the NCBI（Gen Bank Accession No.MW673741）.3.The results of mycelium growth rate and enclosed chamber test showed that sterile fermented liquid of D-12 could effectively inhibit the mycelium growth of A.rolfsii A612,A.tenuissima,B.dothidea and F.oxysporum.After adding 10%fermentation broth on medium,the inhibition rate for the four pathogens were61.22%,68.72%,48.88%and 41.41%respectively.In addition,the mycelium vitality of A.rolfsii A-612 decreased after be cultured with D-12.Microscopic observation demonstrated that A.rolfsii mycelium was swollen and distorted by confrontation with D-12,while the mycelium of F.oxysporum was wrinkled and the sporulation structure was deformed.In addition,with increasing the time of confrontation culture,cytoplasmic cell wall separation,cell wall thickness and organelle degradation were observed in A.rolfsii mycelium.After 72 h post confrontation culture,the intact organelle was not exist basically,and also after 84 h,most of the space in mycelium was occupied by the cell wall,and the organelle destruction was sever.4.Biochemical and physiological tests indicated that D-12 is able to produce cellulase,β-1,3-glucanase,siderophore and protease,but was negative for chitinase production.Furthermore,five pairs of primers were designed to amplify lipopeptides genes（itu A、fen A、fen B、sfp、bmy A）corresponding on the genome of B.velezensis D-12.The presence of those above mentioned genes were confirmed,which showed D-12 might produce abundant lipopeptide metabolites including Surfactins,Iturins,Fengycin and Bacillomyicin.5.The biocontrol effect of D-12 was evaluated on the pot experiment,and showed that plants treated with D-12 had significantly lower disease incidence and disease index values than control.6.During the growth-promoting experiment,we found out that strain D-12 had a good influence on the growth of rice and rape seeds.Compared with the control group,the length of germ and radicle of rice increased 6.95%and 43.28%respectively in the 8th day after the treatment of strain D-12 culture with OD₆₀₀=1.0;and the length of hypocotyl and radicle of rape increased by 18.51%and 138.01%respectively.By using MRM（multiple reaction monitoring）,the fermentation broth of D-12 can be detected the hormones including GA₄,cis-OPDA,SA,t Z,i PR,i P and IAA.When the concentration of fermentation broth increased from OD₆₀₀=0.5 to OD₆₀₀=1.0,the content of GA₄、i P、IAA decreased,while the content of i PR increased.