Studies On The Production Of Nematocidal Substances By Endophytic Bacteria In Plants And Their Colonization And Control Effects

Plant parasite nematode is one of the important plant pathogens,it has a serious impact on agriculture and forestry production.In order to acquire the plant endophytic bacteria of having high antagomistic effect on plant parasitic nematodes,in this study,81 strains endophytic bacteria zymotic fluid which were handled with 121? for 20min were determined nematicidal activit in 12 hours,these strains were preserved in 4?.The nematicidal endophytic bacteria culture medium was optimized;Extracting with insecticidal protein efficient nematicidal activity of endophytic bacterial strain BCM2 and purification in order to do determination;Colonization dynamics of 3 strains endophytic bacteria BCM2,CCM7 and SZ5 were detected in tomato plant for 45d and the control effect of root knot nematode of these strains.The results were as follows:1 The pine wood nematode was used as a target,using the mearsure of direct contact killing to detect the nematicidal activit of 81 strains plant endophytic bacteria which saved in the laboratory,the bacteria zymotic fluid was determined nematicidal activity in 12 hours,there are 9 strains bacteria 12h nematicidal activity achieve at 80%above;By using single factor method to determine the best carbon source and the optimum nitrogen source for 5 strains bacterias in order to produce much more nematicidal activity.The results showed that the best carbon source bacteria is beef extract and the best nitrogen source is yeast extract and peptone of 5 strains for producing nematicidal substance.The optimum nitrogen source and the best carbon source of orthogonal experiment,the results of bacteria that endogenous 12h nematicidal activity stablely results in more than 85%.2 By using ammonium sulfate extract nematicidal protein,with the 80%saturation concentration of ammonium sulfate extract nematicidal crude protein,there are2L BCM2 fermentation liquid extract 0.99g is to extract nematicidal crude protein;Crude protein was analyzed in physical and chemical properties,the results indicate that no heat and alkali resistant of insecticidal protein;The purified protein by Sephadex G-75 and SDS-PAGE,and sequence analysis of the protein amino acid with MALDI-TDF-TOF mass spectrometer,The results showed that SDS-PAGE is one band,by mass spectrometry in combination with phosphoric acid has 100%similarity protein,a total of 376 amino acid residues,relative molecular mass(Mr)of 38.5kD,its isoelectric point(pI)value was 7.77.3 Using the methods of putting the root in bacterial suspension for 3-5min to inoculate,three strains(BCM2?CCM7 and SZ5)could colonize in the tomato and all their colonization dynamics were in a 'first increased and then decreased,then increase to decrease again' trends.Inoculated Id,the colonization of 3 strains endophytic bacteria is more than 103cfu/g,strain BCM2 and CCM7 reached the maximum colonization amount in the 7d,were 4.48 x 106cfu/g and 2.19×106cfu/g,while the SZ5 is in the 9d to achieve maximum colonization which is 7.44×106cfu/g.Colonize after 45d,3 strains still have higher colonization,which all maintained at more than 103cfu/g;3 strains endophytic bacteria to control root knot nematodes include the prevention experiment and treatment experimental,the prevention experiment is divided into pre inoculated endophytic bacteria and simultaneous inoculation of endophytic bacteria and transplanting to soil containing nematodes in experiment,the results show that the prevention of experimental results better than the treatment experiment,early inoculation experiments than simultaneous inoculation and transplanting the experiment,in which 3 strains of endophytic bacteria BCM2,SZ5 and CCM7 pre inoculation experiment of root knot inhibition rates were 96.69%,90.26%and 76.89%.Also found 3 strains of bacteria have certain promoting effect on tomato seedlings.