With Killing Nematode Active Plant Endophytic Bacteria And Rhizosphere Actinomycetes Screening And Control Efficiency

Plant-parasite nematode is one of the most important pathogens in plant.In this paper,6 economic plants and 17 soil samples has been selected to isolate its endophytic bacteria and rhizosphere actinomycetes, screened the antinematode activity strains and studied the strain with better activities. The results were as follows:Endophytic bacteria were isolated and purified from the 6 species of plant by the technique of tissue isolation, and were screened by testing their ability of colonizing in sterile cucumber seedlings and their metabolite against Bursaphelenchus xylophilus in vitro. Among the 112 isolates there were in total 64 strains have broad-spectrum of colonization in sterile cucumber seedlings experiment, and among them there were 13 strains of endophytic bacteria secreting antinematode metabolite. The supernatant of the fermentation broth of these endophytic bacteria gave 100% mortality of nematodes after treated as the follows:1ml each was mixed with.0.2ml of the sunspension of nematodes (2000 nematodes/ml) then incubated at 25℃for 24 h, some of which could led to leakage or dissolution of nematodes. Among them, four strains, BCM2,SZ5,CCM7 and DP1, showed stronger activity than others. The supernatants diluted three times also gave not less than 95% mortality after 24 h treatment, and those from DPI and SZ5 even gave 100% mortality.Rhizosphere actinomycetes were also isolated and purified from the 17 soil samples by the technique of spreading dilute. Those isolates were also screened by testing their metabolite against B.xylophilus in vitro.In total 326 strains were isolated and there were 21 strains secreting antinematode metabolite, the supernatant of the fermentation broth of these 21 strains gave more than 80% mortality of nematodes after treated with the same methods in screening the endophytic bacteria,some of which also could led to leakage or dissolution of nematodes. In the repeat screen assay to the 16 strains with higher activity, we found that the activity was increased by degrees with the extending of the treating time, and five strains, NK17,NK19,NK27,NK29 and V21, showed stronger activity than others in the 6h treatment assay and 12h treatment assay.In addition, four endophytic bacteria with high activity were identified by physiology-biochemistry analysis and 16SrDNA sequence analysis.DPl and CCM7 were identified to be Bacillus subtilis, while SZ5 and BCM2 to be Bacillus cereus.The physico-chemical property and the stability of the antinematode substances were evaluated by various. The antinematode substance secreted by rhizosphere actinomycetes was a kind of micromolecule alkaline substance which was evaporable. But antinematode substance of those endophytic bacteria were found stable when treated with protease or heating or stored at 4℃after 100 days, while instable when treated with acid or alkali, we conjectured that it was a kind of peptide with low molecular weight.Those strains inhibiting B._xylophilus were selected to culture in liquid medium and conditions were optimized by mono-factor test for the best carbon source and nitrogen source, and orthogonal test was done to optimize the fermentation condition. In the reasults we found that the best carbon source for the 4 strains secreting antinematode substance was peptone, and the best nitrogen source was beef extract. The fermentation conditions of the four strains were optimized and the antinematode activity grew up four times after optimization.Tomato seedlings and potting plants were utilized to study these active bacterium and actinomycete strains' biological control function and their combination function. The results indicated that the strains NK29,NK17 were most effective in reducing the number of galls and egg masses caused by the root-knot nematode by 38.17% and 37.63%, respectively. Compared to the 7 strains of endophytic bacteria, all of the strains except DPI could prevent the growth of the root-knot nematode population by some degree, and BCM2,DP24 were the best two strains, with the control efficiency were 57.35% and 50%, respectively. The control efficiency of CCM7 and DLJ1 were also good but was lower than BCM2 and DP24. BCM2 and CCM7 were selected to cure the tomato plants which were affected by root-knot nematodes, but the result was not ideal. While in the exprement that exert their fermentation broth to the healthy tomato's root which grow in the soil contained nematodes, we found that the two endophytic bacteria could prevent nematodes affecting the root of the tomatos by some degree.The combinated biological control efficiency of the bacteria and actinomycete were not much better than exerting single fermentation broth. Compared to the CK of exerting water, the combination of BCM2 and NK17, CCM7 and NK29 could prevent nematodes affecting the root of the tomatos by some degree while not influenced the growth of the plant, their biological control efficiency were 57.81% and 37.89% respectively, the growth of the plants in these groups were at the same level with exerting Avermectin group. The effects of the fermentation broth of the endophytic bacteria and rhizosphere actinomycetes on B._xylophilus in vitro, the antinematode substance and their biological control function were studied in this paper. The results showed that there were endophytic bacteria secreting antinematode metabolite in economic crops. The metabolite of some strains showed strong and stable antinematode activity. Our results indicate the real potential of biocontrol by endophytic bacteria.