| Gastrodia elata Bl.is a saprophytic perennial herb in the Orchidaceae,which has the effect of treating headache,dizziness,epilepsy,infantile convulsion and tetany.It is one of the most valuable Chinese herbal medicines in China.However,it is often endangered by diseases during its growth and cultivation,which seriously affects its yield and quality.With the new requirements of sustainable agricultural development for the prevention and control of traditional diseases,microbial pesticides have been widely concerned by researchers,with high efficiency,low toxicity,environmental protection,and wide sources.Pathogenic microorganisms are an important cause of G.elata disease.To find out the main types of G.elata pathogenic microorganisms and the strength of their infection are the primary problems to be solved in the prevention and control of diseases.Finding out antagonistic endophytes of these pathogenic microorganisms is an important means to solve the G.elata disease.Therefore,in this study,the diseased G.elata tubers were used as experimental materials to isolate and identify the pathogens,then the epidemiology of the main pathogenic microorganisms were compared,so as to determine the main types of G.elata pathogenic microorganisms and the strains with a wide range of epidemics.Secondly,the isolation and identification of endophytes were carried out with healthy G.elata tubers as experimental materials,then the main species of G.elata endophytes were determined and the highly targeted biological control bacteria were obtained.Thirdly,the interactions between endophytes and pathogens were determined,and the antimicrobial effect of endophytes and the infectious effect of pathogens were determined.Finally,the bacteriostatic effects of endophytes were verified again by a back infestation test.The results we concluded are as follows:1.Isolation and identification of G.elata pathogens and its epidemiological study:The diseased G.elata,A.membranaceus and M.officinalis were collected,and the tubers and leaves were separately sterilized to remove the attached bacterias,cultured by tissue imprinting,and purified by hyphae top purification method and line culture method.Five strains of pathogens were isolated from the different appropriate mediums,then they were identified by colony morphology,microscopic identification,staining and molecular biology.The five pathogens were Penicillium oxalicum of G.elata JY.TM-2,Pseudomonas of G.elata JY.TM-1,Candida vartiovaarae of G.elata JY.TM-3,Penicillium oxalicum of A.membranaceus CC.HQ-1,Penicillium oxalicum of M.officinalis CC.CMX-1.Among them,the genetic distances of strains JY.TM-2?CC.HQ-1 and CC.CMX-1 were the closest,and the genetic distances between strains JY.TM-1and JY.TM-2?CC.HQ-1?CC.CMX-1 were the furthest.Penicillium oxalicum of G.elata was the pathogenic microorganism with wide prevalence.2.Isolation and identification of G.elata endophytes: The attached bacterias on the surface of healthy G.elata tissues were removed by conventional surface sterilization method,cultured by tissue imprinting,and purified by hyphae top purification method and line culture method.Three strains of endophytes were isolated from the appropriate medium,then they were identified by colony morphology,microscopic identification,staining and molecular biology.The three endophytes were Herbaspirillum of G.elata JY.TM-4,Serratia plymuthica of G.elata JY.TM-5,Acremonium of G.elata JY.TM-6.The genetic distance between strain JY.TM-4 and strain JY.TM-5 was close.3.Study on the interaction between G.elata endophytes and pathogens: Inhibition zones diameters were determined by filter paper to explore the interaction between endophytes and pathogens.The results showed that,The effects of endophytes on pathogens were:compared with the PDB group,Herbaspirillum of G.elata,Serratia plymuthica of G.elata and Acremonium of G.elata had significant antibacterial activity against Penicillium oxalicum of G.elata,A.membranaceus and M.officinalis(P<0.05).Among them,the bacteriostatic effect of Herbaspirillum of G.elata was the best(P<0.05),and the inhibition rates were 87.64 %,85.98%,75.73%;Serratia plymuthica of G.elata and Acremonium of G.elata had significant antibacterial activity against Candida vartiovaarae of G.elata,and Serratia plymuthica of G.elata had the best antibacterial effect(P<0.05),the inhibition rate was 86.94%;Acremonium of G.elata had significant antibacterial activity against Pseudomonas of G.elata(P<0.05),and the inhibition rate was 45.77%.The effects of pathogens on endophytes were:compared with the PDB group,the degree of infection that Pseudomonas of G.elata on the three endophytes was,Serratia plymuthica of G.elata,Herbaspirillum of G.elata > Acremonium of G.elata;the degree of infection that Candida vartiovaarae of G.elata on the three endophytes was,Herbaspirillum of G.elata>Acremonium of G.elata>Serratia plymuthica of G.elata;the degrees of infection that Penicillium oxalicum of G.elata,A.membranaceus and M.officinalis on the three endophytes were Acremonium of G.elata>Serratia plymuthica of G.elata>Herbaspirillum of G.elata.4.Back infestation test of interaction between G.elata endophytes and pathogens: Healthy G.elata was collected,then the endophytes of G.elata and pathogens of three plants were respectively inoculated on the surface-sterilized G.elata tubers under sterile conditions to observe the incidence.The results showed that,the results of endophytes inoculated on pathogens were: compared with the PDB group,Herbaspirillum of G.elata had significant antibacterial activity against Penicillium oxalicum of G.elata,A.membranaceus and M.officinalis(P<0.05),and the inhibition rates were 83.33%,50.00%,66.67%;Serratia plymuthica of G.elata had antibacterial activity against Candida vartiovaarae of G.elata,Penicillium oxalicum of G.elata,A.membranaceus and M.officinalis,among which the best antibacterial effect against Penicillium oxalicum A.membranaceus and M.officinalis(P<0.05);Acremonium of G.elata had antibacterial activity against Candida vartiovaarae of G.elata,Pseudomonas of G.elata,Penicillium oxalicum of G.elata,A.membranaceus and M.officinalis,and the inhibition rates were 16.67%,66.67%,33.33%,50.00%,33.33%,the best antibacterial effect against Pseudomonas of G.elata(P<0.05).The results of pathogens inoculated on endophytes were: the degree of infection of 5 kinds of pathogens on Herbaspirillum of G.elata was,Pseudomonas of G.elata>Candida vartiovaarae of G.elata>Penicillium oxalicum of A.membranaceus>Penicillium oxalicum of M.officinalis>Penicillium oxalicum of G.elata;the degree of infection on Serratia plymuthica of G.elata was,Penicillium oxalicum of G.elata> Pseudomonas of G.elata > Candida vartiovaarae of G.elata > Penicillium oxalicum of A.membranaceus,Penicillium oxalicum of M.officinalis;the degree of infection on Acremonium of G.elata was,Pseudomonas of G.elata>Penicillium oxalicum of G.elata,Penicillium oxalicum of M.officinalis>Penicillium oxalicum of A.membranaceus>Candida vartiovaarae of G.elata.In conclusion,3 strains of pathogens and 3 strains of endophytes were isolated and identified from G.elata.Among them,Penicillium oxalicum was the most widely prevalent pathogen,and the inhibition of Herbaspirillum was the most widely used,and the interaction rule between the isolated endophytes and pathogens was clarified. |
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