Isolation And Identification Of Endophytic Nitrogen-fixing Bacteria From Rice And Investigation Of The Antifungal Mechanisms Of Strain BV6

Endophytes are microorganisms that board in plant tissues at part or whole stages of their life cycles but do not cause obvious damage to host plants.Endophytic nitrogen-fixing bacteria are endophytic microorganisms that beneficial to human production,they can colonize in host plants and fix nitrogen in combination with host plants to provide nitrogen source for their hosts.In addition,some endophytic nitrogen-fixing bacteria have excellent biological control ability,which can effectively inhibit the growth of pathogenic microorganisms,thereby promoting crop growth and increasing yield.Compared with Rhizosphere microorganisms,endophytic bacteria have natural advantages,occupy the comfortable micro-environment niche of host plant tissue which is conducive to nutrient acquisition,as well as survival and reproduction of endophytic bacteria.At present,the known endophytic nitrogen-fixing bacteria are bacteria,and no fungi have been reported.In this study,the endophytic bacteria were isolated and purified by DN nitrogen-free medium and YMA low-nitrogen medium with surface-disinfected rice roots.The isolated and purified endophytic bacteria were identified by morphological observation,16S rRNA gene and conservative nifH gene of nitrogen-fixing bacteria sequencing.Finally,23 endophytic bacteria of 10 genera were isolated from rice root tissue.Among them,five strains with nifH gene were Burkholderia sp.BV6、Novosphingobium sp.CC1、Acidovorax sp.CC2、Pleomorphomonas sp.CC3 and Rhizobium sp.CC4.Nitrogenase activity of five endophytic nitrogen-fixing bacteria was determined by acetylene reduction method using gas chromatography.The results showed that their nitrogenase activities were 331.76 μmol·mL-1·h-1,54.78 μmol·mL-1·h-1,5.48 μmol·mL-1·h-1,3.36μmol·mL-1·h-1 and 5.24 μmol·mL-1·h-1,respectively.Metabolites such as dissolve phosphorus,siderophores and indole acetic acid secreted by five nitrogen-fixing bacteri had been tested.The results showed that strain BV6 had the ability to dissolve phosphorus and secrete siderophores;CC1 is able to secrete siderophores and indole acetic acid;strain CC2 had no ability to produce these three substances;strain CC3 has the ability to secrete siderophores、indole acetic acid and phosphorus solubilization;CC4 only produced indole acetic acid.The growth-promoting effect of five nitrogen-fixing bacteria on rice seedlings showed that the plant height of rice seedlings inoculated with strain CC4 was significantly higher than that of the control group at 17 days,while the rice plants inoculated with the other four nitrogen-fixing bacteria had no significant change compared with the control group.In this study,the biological control ability of five strains of nitrogen-fixing bacteria was preliminarily explored by plate confrontation method and double-plate method.Only BV6 showed antifungal effect.Its fermentation broth and VOCs have strong inhibitory effects on plant pathogenic fungi such as Botrytis cinerea,Magnaporthe oryzae,Penicillium digitatum and Fusarium oxysporum(FOC).In view of excellent bacteriostatic ability of strain BV6,the antifungal substances in fermentation broth and VOCs were searched.In order to isolate and identify the antifungal substances in fermentation broth,the basic properties of antifungal substances were studied.When fermentation broth was extracted by ethyl acetate,the antifungal substance was still retained in the fermentation broth,and no inhibitory activity was detected in the ethyl acetate,which indicated that the substance was a hydrophilic substance.The molecular weight of the antifungal substances in fermentation broth was estimated between 0.5 KD and 1 KD by isolation test using dialysis bags.Thermal stability experiments showed that the antifungal activity of the antifungal substance was not loss when it was treated at 70℃ for 10h and 100℃for 1h,which proved that the substance had high temperature endurance and thermal stability.When treated at pH 3,7,9 for 2 h,its antifungal activity did not lose.Strain BV6 has a broad antifungal spectrum,not only inhibits the four pathogenic fungi studied,but also inhibits Phytophthora infestans and Rhizoctonia solani.The secretion of antifungal substances in BV6 fermentation broth had no significant difference with the presence or absence of pathogenic fungi,which indicated that the production of antifungal substances was non-inductive.In conclusion,the antifungal substance is a small molecule heat-resistant substance.The VOCs of strain BV6 were identified by headspace solid phase microextraction(SPME)coupling with gas chromatography-mass spectrometry(GC-MS).A total of 17 compounds were identified and 2-nonanone,oleic acid were selected to perform antifungal experiment.2-nonanone showed the best antifungal effect.The minimum completely inhibitory concentration of 2-nonanone on B.cinerea,M.oryzae,P.digitatum and FOC was 4.0,2.0,6.0 and 4.0 μL/plate,respectively.In liquid culture the minimum completely inhibition concentration of 2-nonanone on B.cinerea spores was 300 ppm.In addition,the inhibition mechanism of 2-nonanone on B.cinerea was studied preliminarily.When treated with 2-nonanone,the mycelium reactive oxygen species(ROS)of B.cinerea began to produce at 2 h and burst out at 5 h.In the cell membrane integrity test,after 2-nonanone treatment,a small amount of damage was detected in the cell membrane at 1h,and the cell membrane was severely damaged at 3 h.These results are consistent with the phenomena observed in previous experiments that the growth of B.cinerea was inhibited by 2-nonanone treatment.In conclusion,strain BV6 is an endogenous nitrogen-fixing bacteria with excellent antagonism to pathogenic fungi.