Isolation And Identification Of Two Strains Of Endophytic Bacteria From Passiflora Edulis And Their Effect On Growth And Inhibition

Plant endophytes can promote plant growth,inhibit plant pathogens,and expel insect pests,and will not cause a series of polution problems to environment such as soil and water as traditional chemical fertilizers do.Therefore,they are made into environmentally friendly microbial inoculants and are widely used.In this study,86 strains of endophytic bacteria were isolated from different tissues of Passiflora edulis plants.All the isolated endophytic bacteria were tested on plate with various pathogenic fungi of Passiflora edulis to observe their pathogenic fungi-inhibiting effect.Simultaneously,the growth-promoting characteristics of these isolated endophytic bacteria such as dephosphorization,nitrogen fixation,and IAA production were also determined under the condition of lab environment.Then the selected endophytic bacteria were subjected to a pot test,and finally the strains with both two good characteristics were screened out.These two target strains were applied in the field.The screened target strains were identified through morphological characteristics observation,physiological and biochemical index determination and molecular detection,and their growth curves and endogeneity were measured.This research has laid a theoretical foundation for future application in agricultural production.The main research contents and results are as follows:1.86 endogenous bacteria were isolated from the roots,stems,leaves,and fruits of Passiflora edulis plants.The number of endogenous bacteria in each tissue was fruit> root> stem> leaf.All endophytic bacteria include 14 different bacterial genera,of which Bacillus(Bacillus)accounts for 33%,which is the dominant genus.2.The 86 isolated endophytic bacteria were tested for flat-panel confrontation assay with the five pathogenic fungi(Fusarium oxysporum,Fusarium proliferatum,Fusarium solani,Colletotrichum gloeosporioides,Alternaria tenuissima).Screened 4 strains of endophytic bacteria(N22,N28,3YJ1,H5-3)with broad-spectrum resistance to five pathogenic bacteria of Passiflora edulis.Among them,the strain N22 has the most obvious bacteriostatic effect on 5 pathogenic fungi,the bacteriostatic range is 56.78%～72.17%,and the bacteriostatic range of strains H5-3and 3YJ1 against 5 pathogenic fungi is 58.43%～67.65% and 54.90%～68.60%,the bacteriostatic range of strain N28 is 57.08%～66.98%.The effects of 4 strains of antibacterial bacteria on the mycelium morphology of 5 passionfruit pathogenic fungi were observed under an optical microscope and it was found that hyphae of Fusarium proliferatum showed the phenomenon of shrink,deform,split ends,expand locally,etc.but no obvious effect on the mycelium of the other 4 pathogenic fungi.After fermention for 12 h,24 h and 48 h respectively,the fermentation broth of the 4 strains of antibacterial bacteria were filtered to get rid of bacterium,which was applied to test the inhibiting effect on the five pathogenic fungi.The results showed that the sterilization filtrate had a better inhibitory effect on Alternaria tenuissima but not on other pathogenic fungi.Total proteins of the 4 antagonistic bacteria were also crudely extracted and applied to pathogenic fungi-inhibiting assay,among which N22 and N28 had better inhibitory effects on Fusarium solani.3.All the isolated endophytic bacteria were applied to check phosphate-dissolving and IAA-producing characteristics,and 9 strains with growth-promoting characteristics were screened out.Among them,6strains(C3,C5,C6,C9,N22,3YJ1)have phosphate-dissolving properties,and 3 strains(C10,C14,N28)have the ability to produce IAA.The endophytic bacteria screened out were used for pot experiment,and the plant height,root length,fresh weight and dry weight of the plant were measured after treatment.It was found that plants treated with C3,C5,C6 and N22 were much taller than the control group.Strains C3 and N28 significantly increased the root length of Passiflora edulis plants.Strains C3,C5,C6,N22,and N28 significantly increased the fresh weight of the plants.Strains C3,C5,and C6 increased dry weight of the plants.In general,the strains C3,C5,C6,N22,and N28 have a promoting effect on the Passiflora edulis plants as a whole.4.Strains N22 and N28,which have broad-spectrum resistance to Passiflora edulis pathogens and have obvious growth promoting effects on the plants,were used as target strains for field trials.Field test results showed that the plant height,root weight and fresh weight of Passiflora edulis seedlings treated with strain N22 fermentation broth were 1.98,1.69 and 1.69 times,respectively,higher than those of the control group treated with liquid LB medium.Similarly,the plant height,root weight and fresh weight of Passiflora edulis seedlings treated with strain N28 fermentation broth were 1.92,1.89 and 1.94,respectively,higher than the control group treated with liquid LB medium.The results indicate that these two beneficial endophytic bacteria have a good growth-promoting effect on Passiflora edulis plants.5.Strains N22 and N28 that have broad-spectrum resistance to Passiflora edulis pathogens and have obvious growth promoting effects on the plant are used as target strains.According to the analysis of morphological characteristics,physiological and biochemical tests and16 Sr RNA sequencing,the two beneficial bacteria were classified into Bacillus,and their growth curves and endophytes were further measured.In summary,this experiment obtained endophytic bacterial strains N22 and N28 with characteristics of both promoting growth of plants and inhibiting growth of pathogenic fungi from Passiflora edulis,which will lay a theoretical foundation for future application in fields.