Research On The Control Of Endophytic Bacillus On Meloidogyne Incognita

Tomato(Solanum lycopersicum)is used in this study to research the biological control and mechanism of endophytic Bacillus spp.against Meloidogyne incognita.Three endophytic bacteria strains were isolated from plant tissues and showed high nematicidal activity against M.incognita 2nd stage juveniles(J2)in vitro.Through the observation of colony morphology,microscopic structure characteristics,16S rDNA and gyrB gene amplification and phylogenetic analysis of three isolates,identified three endophytic bacterium strains are Bacillus cereus BCM2,B.cereus SZ5 and B.altitudinis CCM7,respectively.Construct mutant strains with streptomycin tag,detecting colonization dynamic of 3 isolates in tomato root with root-stabbing methods,through organization grinding and dilution coated on plate.The results indicated that the population of 3 isolates were stably over 103 cfu/g in tomato root within 50 days,BCM2 was found to have the strongest colonization ability,and the maximum density of cells was 3.53×106 cfu per gram fresh weight at 9 dpi on the tomato root.The pot and disease nursery experiment were used to detect the potential of endophytic bacteria in biocontrol of M.incognita.Pre-inoculation with BCM2 was shown to have the highest IR with respect to galls and egg masses on tomato roots by up to 81.2%and 75.6%,there is a positive correlation between colonization ability and biological control effect.The pre-inoculated treatment with B.cereus BCM2 enhanced shoot length and fresh weight significantly.The result of disease nursery show that endophytic bacteria BCM2 reduce the gall severity caused by M.incognita,the biological control effect is the same as abamectin.B.cereus BCM2 which was found to have strongest colonization ability was chosen to study the mechanism of endophytic bacteria against nematode.Firstly,the isolate BCM2 tagged with green fluorescent protein(GFP)to construct.B.cereus BCM2-str’-pBCgfp-1,tissue distribution of endophytic bacteria were monitored with CLSM and SEM technology.The result indicated that the green fluorescent cells of GFP-tagged colonizers around the tissue of tomato roots containing the epidermis,cortex and vascular cylinder,they preferentially colonized around the lysed plant cells and root hair in the root epidermis gathered in large numbers in lateral roots and junctions,then a single or clusters distributed in the root phloem and vascular bundles.In addition,more GFP-tagged cell clusters were detected between the giant cells in the root of diseased tomato caused by M.incognita.There were no endophytic bacteria in tomato stem and leaves,suggesting that B.cereus BCM2 specially colonized in tomato roots.Endophytic bacteria also gathered on the surface of galls which was caused by nematode.Therefore,endophytic bacteria B.cereus BCM2 can permanently gathered in plant roots and effectively occupy the niche to defense root-knot nematode.Secondly,extraction of B.cereus BCM2 extracellular protein components,and detected hydrolysis ability of crude protein to nematode cuticle and intestinal by using scanning electron microscope(SEM)and transmission electron microscopy(TEM).Separation and purification of nematicidal protein by using ion exchange technology and sephadex gel filtration method,MALDI-TOF/TOF identified that three separate proteins were chitosanase,alkaline serine protease and neutral protease,respectively.Thirdly,plate and two-pots experiment were used to detected repellent action of root exudate which was extracted from BCM2 colonized tomato.Hexane was used to extract root secretion,and then the eight different components were identified with Gas Chromatograph-Mass Spectrometer-computer(GC-MS).Five different components are 2,5-dimethyl-Noname,2,4-dimethylethyl-Phenol,3,3-dimethyl-Octane,Tridecane and Heneicosane.Fourthly,in order to study the role of endophytic B.cereus BCM2 in host immune response against root-knot nematodes infection,we explored from three aspects to study the endophytic bacterium BCM2 effect on plant immunity and function mechanism.1)the level of organization structure:detect the influence of endophytic bacterium BCM2 to callose deposition;2)the level of physiology and biochemistry:detect the influence of endophytic bacterium BCM2 to ROS concentration and defense related enzyme ativity of POD,PAL and PPO;3)the level of molecular mechanism:study the influence of endophytic bacterium BCM2 to salicylic acid(SA),Jasmonic acid(JA)and ethylene(ET)signal pathway.The results indicated that endophytic bacteria inoculation do not cause callose deposition to resistant nematode.POD and PAL activity increased in M.incognita infection early and PPO activity increased in infection later period with pre-inoculated with endophytic BCM2,indicating an intense physiology and biochemistry immune reaction.SA,JA and ET levels change showed that root knot nematodes infection could prompt JA has always been maintained at a higher level,and in the later stages of the nematode infection,SA and ET levels increased significantly.BCM2 colonization changed the expression of hormone signals,to participate in the host’s immune response.At last,to determine effection and reaction of the endophytic bacteria mediated tomato against M.incognita,and screen the defense-related genes which were triggered with BCM2.Split-root method was used to detect B.cereus BCM2 induced systemic resistance.Inoculated BCM2 water did not reduce the galls and egg masses compare with sterile water treatment,the highest reduction was obtained when inoculated by M.incognita J2 with pre-inoculated B.cereus BCM2 in inducer side.Significant less galls and egg masses were detected at 50 dpi.RNA-Seq was introduced to investigate the plant defense-related genes that are specifically activated by endophytic B.cereus BCM2.Four sequenced libraries samples from tomato roots treated respectively with sterile water,BCM2,M.incognita and M.incognita with pre-inoculated BCM2 were developed,obtaining four high quality total clean bases ranging from 6.64 to 6.75 Gb.Pair-wise comparisons were conducted in each library,in which differentially expressed genes and candidate defense target genes were identified.Our data indicated that endophytic B.cereus BCM2 alone did not induce the systemic resistance and kept harmonious horst-microbe relationship,while endophytic bacteria activated many differentially expression of defense and stress-related genes under nematode attack.Thirty four candidate genes were acquired as targets for endophytic B.cereus BCM2 triggered intense plant immunity or defense-related factors in resistance against M.incognita on tomato.