| Polygonatum cyrtonema Hua,included in the Chinese Pharmacopoeia(2015 edition),is a perennial herhaceous plant,which belongs to Liliaceae Polygonatum,and is a Chinese herbal medicine with diet and medicine value.It has the functions of strengthening the spleen,moistening the lungs.At present,artificial cultivation of P.cyrtonema Hua has became the mainstay of the market supply,but it is often affected by various diseases during the cultivation,such as leaf spot disease and anthracnose disease.In the planting base in ZheJiang province,a serious disease was found with the symptom as that:the lesion was formed at first at the tip of the leaf,and then expanded continuously,finally,the whole plant leaves died.In order to distinguish this symptom from that of other disease,the disease was named as leaf blight disease.This disease seriously affects the growth and yield of P.cyrtonema Hua.In this study,the pathogen of leaf blight disease was isolated and identificated.While,the endophytes of P.cyrtonema Hua were isolated and the biocontrol bacterial strain was screened out.The antagonistic mechanism and growth promoting mechanism of biocontrol bacterial strain were studied.The main results are as follows:1.The pathogen was isolated and identified from the infected plants of P.cyrtonema Hua.Five fungi were isolated and the pathogenicity was detected.It was found that strain PC-4 conformed to Koch's law and was identified as Fusarium oxysporum by the analysis of morphology,the rDNA-ITS and TEF-1? gene sequence.2.Eleven strains of endophytes were isolated from the roots,stems and leaves of P.cyrtonema Hua.The endophytes belonged to 3 genera,among which Bacillus was the dominant genus.The strain ZJU-3 was found to be antagonistic to F.oxysporum PC-4 and was identified as Bacillus velezensis by the morphological,physiological,biochemical properties and the analysis of the 16S rDNA and gyrB gene sequence.3.The pot experiment was carried out to determine the biocontrol effect of B.velezensis ZJU-3.The results showed that the disease index of OD600=0.5 fermentation liquid of strain ZJU-3 was 39.5%,while the disease index of the control group was 70.8%and the biocontrol effect was 44.2%.4.In the tissue culture test of P.cyrtonema Hua and P.kingianum,it was found that the strain ZJU-3 could promote the growth of plant height and fresh weight,but had no effect on the chlorophyll content in leaves.In pot experiment,the strain ZJU-3 had a significant effect on the growth of P.cyrtonema Hua,and the root length and root number were significantly higher than that of the control group.The analysis by LC-MS showed the strain ZJU-3 could produce plant hormone IAA,kinetin,zeatin and GA3,and their concentrations were 8.29 ng/mL,21.34 ng/mL,0.02 ng/mL,0.07 ng/mL.5.The rifampicin-resistant mutant Rif-B.velezensis ZJU-3,which maintains antibacterial activity,was obtained by concentration gradient method,and its resistance to rifampicin reached 150 ?g/mL.The leaf inoculation and tissue separation showed that the number of Rif-B.velezensis ZJU-3 strain in the leaves was 2x 105 per gram.6.The detection results of mycelial growth rate showed that sterile fermented liquid of B.velezensis ZJU-3 inhibited the mycelium growth of F.oxysporum PC-4.As the contentration of sterile fermented liquid in the medium increased,the antagonistic activity increased.When 5%sterile fermented liquid was added,the colony diameter was 3.78 cm,although the control was 4.87 cm,and the inhibition rate was 22.3%.The stability analysis showed that the antagonistic efficacy of B.velezensis ZJU-3 sterile fermented liquid was not affected by temperature,pH,proteinase K and metal salt ions.7.The antimicrobial activity assay showed that the fermented liquid had strong inhibitory effects on Colletotrichum gloeosporioides,C.truncatum,F.graminearum,F.solani,Rhizoctonia solani,F.oxyporum f.sp.niveum,and the antimicrobial activity are 71.8%,79%,55%,58.4%,75.1%and 66.4%,and the hyphae of some pathogens became thicker,twisted,blackened,wrinkled on the surface,and the amount of sporulation was significantly reduced,which indicating that the strain has broad-spectrum resistance.8.B.velezensis ZJU-3 strains can produced protease,?-gluconase,cellulase and siderophore,but not chitinase.Three pairs of primers designed based on the known lipopeptides genes were used to amplify the corresponding genes,srf,ituA and fenA from the genome of B.velezensis ZJU-3.The results showed that B.velezensis ZJU-3 contained srfA,itu A and fenA.The production of lipopeptide surfactin,fengycin and iturin were comfirmed by the detection and anylysis of MALDI-TOF-MS.The crude extract of lipopeptide,obtained by the hydrochloric acid precipitation and methanol extraction,could inhibit the mycelial growth of F.oxysporum with the inhibition rate of 51.6%. |
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