| Plant parasitic nematode is one of the main pathogens causing plant diseases.Its wide host range,serious damage each year to agricultural production caused economic losses of 150 billion.At present,the control of plant-parasitic nematodes is still dominated by chemical control,but its toxicity is high and it pollutes the environment,which poses a potential threat to human and animal health.With the ban of highly toxic pesticides in our country,it is urgent to develop green and safe bio-control bacteria to control plant parasitic nematodes.In this study,different types of soil in Hunan Province were collected,and bacteria in the soil were isolated and screened.A strain D45 with significant inhibitory activity against both D.destructor))and M.graminicola was obtained.The morphological,physiological and biochemical and molecular biological techniques were used to identify them,and their fermentation conditions were optimized and preliminary analysis of the nematicidal substances produced by the fermentation was conducted.The main results are as follows:1)Using soil dilution method,186 strains of bacteria were isolated from different types of soil samples collected from areas where fertility in the soils of Zhangjiajie and Daweishan in Hunan Province were relatively abundant and where there were no microbial contamination.The primary screening of potato nematodes as the target nematode was carried out.The bacteria were screened by direct contact killing and 8 strains with higher nematicidal activity were screened.The rescreening was to target the second-instar larvae of the root-knot nematode larvae as the target nematode.The fermentation broth of D45 and D54 strains had the best insecticidal activity.After 24 h and 48 h incubation,the supernatant diluted five times of strain D45 culture resulted in a mortality of 69.8% and 72.1% to D.destructor,60.9% and 70.4% to M.graminicola,respectively.2)The results of pot experiment showed that the control effect of strain D45 fermentation liquor on rice root knot nematode was 79.1% and 64.8% respectively after dilution 2 times and 5 times.The control effect of strain D54 fermentation liquor on rice root knot nematode is relatively D45,and the control effect of 2 times and 5 times diluent is 66% and 45.7%% respectively.The field test results showed that the control effect of D45 fermentation broth diluted 2 times and 5 times to the rice quasi gramineous knot nematode was 56.3% and 53%,respectively,and the control effect of D54 fermentation broth diluted 2 times and 5 times of the strain was 52% and 44.3% respectively.3)Through the morphological observation,physiological and biochemical observation,16 S rDNA sequence phylogenetic analysis and other biological characteristics of the active strain D45 to determine the taxonomic status of the strain.It is preliminary thought that the active strain D45 is Bacillus megaterium and D54 is Bacillus aryabhattai.4)The fermentation conditions of the active strain D45 were optimized,and the formulation of the nitrogen and carbon sources was determined through the optimization of the culture medium: maltose 30 g/L,peptone 4 g/L.Then single factor fermentation conditions were selected to determine the optimum fermentation conditions for strain D45: temperature 30 °C,speed 180r/min,pH 7.55)In the virulence test of metabolites,it was found that strain D45 can effectively kill Caenorhabditis elegans and inhibit its locomotor activity.The fermentation broth of the strain in the sand column test can stimulate the diffusion movement of potato stem worms.The volatiles in the fermentation broth of strain D45 had a certain killing activity on potato stem worms. |
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