Study On The Species Diversity Of Urea-producing Bacteria And Their Application In Biocontrol Of Root-knot Nematode

Root-knot nematode disease is one of the important diseases that seriously harm crops.A variety of chemical nematocides have been banned due to their high toxicity and residues,and biological control has attracted much attention.Urea-producing bacteria arethenovel resource for biological control of nematodes for their abilities to release nematicidal ammonia under the catalysis of urease.However,scarce information is available on the species diversity,biocontrol potential and urea biosynthesis of urea-producing bacteria.In this paper,the following studies were carried out on the resourceexploitation of urea-producing bacteria and their application in the control of root knot nematode disease:(1)Screen of urea-producing bacteria from vermicompost and analysis on their species diversity;(2)Identification of new species of urea-producing bacteria;(3)Evaluationof the biocontrol potential of urea-producing bacteria against nematode disease;(4)Illustration of the molecular mechanism of bacteria producing urea.The main results obtianed were concluded as the follow.1.A total of 246 bacterial strains were isolated from vermicompost by culturable method.Of which,53 strains of urea-producing bacteria were screened out based on the principle that ammonia was released when urea was catalyzed by urease.Of the53 striains,38 were confirmed to have the function of producing urea when quantitatively determine the urea content in the thier fermentation broth using oxime method.There were 7 and 6 strains could produce urea with concertration?100?g/mL when cultured using LB and NB media,respectively.According to phylogenetic analysis,these 38 urea-producing bacteria were included in four groups:Firmicutes,Actinobacteria,Proteobacteria,and Bacteroidetes.The dominant genus is Bacillus,which including14 strains from 10 species.2.Based on polytaxonomicmethod,the two strains isolated from vermicompost,1.1416~T and 1.3611~T,were identified as new species,and designated as Luteimonas lumbrici and Sphingobacterium lumbrici,respectively.3.The nematocidal activities of 38 urea-producing bacteria was evaluated by plate assay.Using Panagrellus redivivusas target,30 strains showed thier nematicidal fatality rates between 17%and 100%.Among them,Bacillus included the most active strains(14 strains of 10 species)with fatality rate of 71-100%,and 11 strains showed fatality rate?90%.Using Meloidogyne incognita as target,27 strains showed thier fatality rates between 5.34%and 85.53%,in which incuding 6 strains with fatality rate?70%.The results of pot experiment showed that among the five urea-producing bacteria tested,B.zhangzhouensis 1.4517 showed the best control effect against root knot nematode disease on tomato,and the proportion of root with nodules in natural soil and sterilized soil were 27.91%(46.5%by CK)and 5.13%(11.97%by CK),respectively.4.The mechanism of ureabiosynthesis in B.zhangzhouensis 1.4517 was investigated.In this strain,arginidase gene(RocF)was present and expressed normally.Arginine of this strain was generated by glutamate under the action of multiple synthases and kinases,which followed an economical pathway and had an arginase metabolic pathway.When cultured B.zhangzhouensis 1.4517 using LB and NB madiea,results of transcriptome analysis suggested that the differentially expressed genes related to urea formation were found in the synthesis of arginine and arginase metabolic pathways.The arginine of B.zhangzhouensis 1.4517 was metabolized to urea by the arginase pathway,and the accumulation of arginine in the strain was benefical for increasing the urea yield.