| Entomopathogenic nematodes(EPNs)have strong ability to kill most insect pests because they carry pathogenic intestinal symbiotic bacteria.In vitro experiments,the symbiotic bacteria showed a strong ability to inhibit plant pathogenic fungi,so the exploitation of such microbial resources is an important issue in the field of biological control.In the present study,high-quality and efficient methods were employed and modified to isolate symbiotic bacteria from 10 species of Steinernema and 3 species of Heterorhabditis.Among them,Xenorhabdus bovienii NN6,showing broad-spectrum antifungal effect,was selected to for further study on the antifungal components and antifungal mechanism involved in,which laid a foundation for the follow-up research and utilization of such biocontrol resources.The major results obtained as following.The grinding method,hemolymph coating method and modified hemolymph coating method were used to separate the symbiotic bacterial strains from EPNs.By comparing the separation results,it was found that the grinding method had low separation efficiency.fewer colonies and was easy to be contaminated.Hemolymph coating method had low separation efficiency.fewer colonies and was difficult to observe due to blackening reaction.The modified hemolymph coating method had high separation efficiency,large number of colonies and easy observation.Seven strains of five species of the genus Xenorhabdus and 3 strains of 2 species of Photorhabdus were isolated by the modified hemolymph coating method.Plate mycelium inhibition method was conducted to test the antifungal spectrum of symbiotic bacteria.Different strains had different antagonistic effects on different fungi however.the inhibition efficiency of these symbiotic bacterial strains of EPNs against Magnaporthe grisea GuyⅡ Phytophthora capsici NPC1 were extremely high,and their inhibition efficiency to Botrytis cinerea NBC1 were relatively low.By parallel comparison of the antifungal rates of the 9 symbiotic strains tested,X.bovienii NN6 showed the highest comprehensive inhibition efficiency,among which the inhibition efficiency to Magnaporthe grisea GuyⅡ and Fusarium graminearum B4-1 reached 93.6%and 93.1%,respectively.This owns to it the broad exploitation potency.The inhibitory mechanism of X.bovienii NN6 against F.graminearum was investigated.Firstly,the dose effect of NN6 inhibited B4-1 was determined by the multiple dilution test of NN6 fermentation liquid,and then the cell suspension and sterile filtrate were obtained after the high-speed centrifugation and filtration of the fermentation liquid.The results showed that the inhibition rate of NN6 cells against B4-1 mycelium was 93.4%,and the inhibition rate against the conidia germination was 54.22%,while the sterile filtrate had no inhibition on the growth of mycelium and the inhibition rate of spore germination was 93.45%.Further study found that NN6 can inhibit the growth of B4-1 mycelium,and secrete intracellular substance when contacting with fungi,which eventually destroys the integrity of mycelium membrane structure and forms antifungal effect.In this study,we found that the modified hemolymph coating method was the most high-quality and efficient method to isolate EPNs symbiotic bacteria,and the strain X.bovienii NN6 was of broad-spectrum,whose cells had the ability to inhibit the mycelial growth and conidial germination of F.graminearum,while the fermentation filtrate could only inhibit conidial germination.NN6 cells secreted intracellular substances to destroy the integrity of mycelium membrane when contacting with fungi,thus inhibiting mycelium growth.The specific components of intracellular secretions are worth further study and discussion. |
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