Isolation,Screening And Identification Of Endophytes From Medicago Sativa L. For Biocontrol Agents

Alfalfa is one of most important forage for dairy cows,which is known as “the king of forage”.Endophytes are microorganisms(usually bacteria,fungi or actinomycetes)that are colonized in plant tissues.As potential candidates for anti-microbial,anti-insect and anti-cancer and more properties,they store a large number of new bioactive secondary metabolites,such as alkaloid,phenolic acids,quinone,steroid,saponin,tannin and terpenoids,and they are also an important source of microbial pesticides.In this study,the endophytes isolated from alfalfa of Hebei,Inner Mongolia and Ningxia were used as experimental materials to evaluate the biocontrol effect,the results are as the followings:1.Isolation of endophytes from AlfalfaIn 2015-2016,108 plants of alfalfa samples meeting to collection standard from Hebei,Inner Mongolia and Ningxia,were collected,from which 409 strains endophytes were isolated.Among the endophytes,306 strains,101 strains and 2 strains were bacteria,fungi and actinomycetes respectively.The endophytic bacteria were the dominant endophytes.Moreover,125 strains were isolated from leaves of alfalfa,101 strains were got from stems of alfalfa,and 183 strains isolated from roots of alfalfa.The data showed that endophytes from root were the most.2.Evaluation on Antagonism of endophytes isolated from alfalfaIn 2015,362 strains of endophytes were isolated.Their antagonism to four pathogens Rhizoctonia solani,Fusarium oxysporum f.sp.cucumerinum,Fusarium oxysporum f.sp.medicaginis and Phytophthora capsici were tested.35 strains of endophytes were antagonistic to at least one pathogen and 12 strains of which antagonistic to the four pathogens.They were 3 endophytic bacterial strains,8endophytic fungal strains and 1 endophytic actinomycete strain.Endophytic bacterium NX36L-1possessed most strong antagonism to the four pathogens,which was obtained from alfalfa collected in Ningxia.Meanwhile,the average inhibition rates of PDA NM2S-4 to four pathogens were the highest with 81.2% among the endophytic fungi,which was obtained from alfalfa from Inner Mongolia.3.Evaluation on control effect of endophytes to cucumber wilt in vivoThe control effect of 362 strains of endophytes isolated in 2015 were evaluated by cucumber seedling inoculated Fusarium oxysporum f.sp.cucumerinum.37 strains’ biocontrol effect on leaves was more than 60%,among them NA NX51R-5,NA HB1S-3 and NA NM1S-1’ biocontrol effect was best(84%).21 strains biocontrol effect on roots is more than 60%,among them NA NX90R-8 was best(84%).The strains showed good effect on both in vitro and in vivo screening mentioned above were selected to do evaluation on biocontrol effect against Cucumber Fusarium Wilt assay in vivo again.3strains showing better biocontrol effect were selected,which were NA NX51R-5,NA NX90R-8and NA NM1S-1,respectively.4.Control effect of the selected strains to alfalfa root rot.The three strains of endophytic bacteria with excellent biocontrol activity were screened by alfalfa seedling in culture test,experiments in the test tube seedlings and pot culture experiment,showed that the alfalfa root rot caused by Fusarium oxysporum F.sp.medicaginis has obvious effect of protection.The relative control effect of potted plants was 57.1%.5.Identification of endophytesThe use of modern molecular biology techniques,26 strains of endophytic bacteria,which had antagonism,identified belonging to 6 species by 16 s rDNA,moreover Bacillus spp.And Pseudomonas spp.are dominant strains.Through the molecular identification,the 8 endophytic fungi belong to 3species by ITS(internal transcribed spacer),in which the dominant species is Trichoderma harzianum.The three strains of endophytic bacteria possessing high biocontrol effect were identified by 16 S rDNA,NA NM1S-1 and NA NX51R-5 were Bacillus spp.,and NA NX90R-8 was Pseudomonas spp.