Multifunction And Diversity Analysis Of Bacillus Spp. In Vegetable Field Soils

In this study 120 soil samples were collected from tomato,cucumber,leek, Chinese cabbage,garlic,green onion and other vegetable fields in Liaocheng, Shouguang,Linyi and Zhangqiu areas.A number of 947 Bacillus isolates were obtained from these soil samples.Among the 947 isolates,350 isolates were found to fix nitrogen,solubilize insoluble-potassium and/or insoluble-phosphate by agar plate method.Among which 104 isolates were of nitrogen fixation, insoluble-potassium solubilization and insoluble-phosphate solubilization simultaneously.By sole carbon source method,14 Bacillus isolates were isolated to be chlorpyrifos degraders,no omethoate degrader were isolated,but these 14 isolates did not fix nitrogen,solubilize phosphate and potassium.The above 104 isolates were verified by replicate screening test and 24 were found to be better and stable in the three functions.These 24 isolates and two reference bacteria strain P418 and YKT41 which were known to solubilize K,P and fix nitrogen,were quantified in their ability of nitrogen fixation, insoluble-potassium solubilization and insoluble-phosphate solubilization by the total nitrogen colorimetry method,the sodium tetraphenylboron turbidity and the phospho-vanadic-molybdic colorimetry method,respectively.Compared with the strain P418 and YKT41,isolate SJ2-7,SJ3-7,SH5-6,SH6-1,LJ4-2 and CS6-4 showed the strongest ability in the three function combination.It was also found that isolate CS6-4 showed phytase activity;isolate SH5-4 and SH6-3 were able to inhibit the growth of Fusarium moniliforme on agar plate,isolate SJ7-3,SJ2-3 and SJ1-7 were able to inhibit the growth of Botrytis cinerea on agar plate.Based on physiological and biochemical characteristics,combined with 16S rDNA sequencing results,isolate SJ2-7,SJ3-7,SH5-6,SH6-1,LJ4-2 and CS6-4 were identified as Bacillus megaterium.These results showed that Bacillus spp.in vegetables field soils were functionally diversified,and Bacillus megaterium was shown as the best species in diversed functions.It was considered that these isolates were potential in development of microbial fertilizers after further research.Using primers specific to Bacillus spp.,DNA fragments were amplified from soil total DNA samples by nested PCR,and then denaturing gradient gel electrophoresis(DGGE) was adopted to analyse the DNA fragment profiles.It was found that Bacillus spp.molecularly diversified in these soil samples.PCR-DGGE electrophoresis was performed on the six isolates which showed the best functions and on the DNAs from their corresponding soil samples,results showed that the profiles from single isolate DNA matched the bands from the corresponding soil DNA,and thus supported the isolation and screening results.