Isolation And Metabolomics Of Endophytic Bacteria Antagonistic To Rice Seedling Blight

Rice seedling blight is one of the most important diseases in dry rice-nursery field,which seriously threatens the yield and quality of rice.The environmenta l safety problems caused by chemical control methods are worrying.In addition,the problem of pathogen resistance to chemical fungicides is becoming more and more prominent.Therefore,the production of new environmentally friendly agents is urgently needed to solve this problem.In this study,endophytic bacteria that can effectively control rice blight were isolated and screened from rice.The fermentation medium and fermentation conditions of endophytic bacteria were optimized by single factor and orthogonal experiment.The control effect of rice seedling blight was verified,and the potential antimicrobial metabolites were identified by metabonomics.The results are as follows:（1）A total of 93 strains were isolated from the roots,stems and leaves of healthy rice seedlings in the vicinity of the plot where rice seedling blight occurred,and 15 strains of endophytic bacteria with strong antagonistic activity against Fusarium oxysporum were screened by filter paper method.The inhibitory rate of endophytic mdj-15 was 55.95±1.72%,which was significantly higher than that of other strains,and it was taken as the dominant strain for subsequent studies.mdj-15 was identified as Bacillus subtilis by morphological observation,physiological and biochemical analysis and 16S RNA gene sequence analysis.（2）The effect of each component in the medium on the antibacterial activity of fermentation broth was A（soluble starch）>C（Na Cl）>E（KCl）>D（KH₂PO₄）>B（yeast cream）.The optimal fermentation medium for endophytic bacteria mdj-15 to produce antibacterial substances was as follows:soluble starch 20g/L,yeast extract 2g/L,Na Cl 10g/L,KH ₂PO₄ 1g/L and KCl 1g/L,Mg SO₄0.5 g/L,Mn SO₄ 5.0 mg/L,Cu SO₄ 0.16 mg/L,Fe SO₄ 0.15 mg/L.The effects of fermentation conditions on the antibacterial activity of fermentation broth were B（time）>A（inoculation amount）>D（ph）>C（temperature）>E（revolution）,the optimal fermentation conditions of endophytic mdj-15 were as follows:inoculation amount was 5%,fermentation time was 20h,temperature was 30℃,p H was 6,and revolution was 180rpm.The inhibition rate of the optimized fermentation broth against Fusarium oxysporum was67.77%,which was 20.59%higher than that before the optimization.（3）Endophytic mdj-15 fermentation broth had significant control effect on rice blight.At15℃,20℃and 25℃,the control effect was 69.23±2.09%,74.57±0.43%,73.57±6.11%,respectively,which was significantly higher than that of hymexazol,thiophanate-methyl.At 30℃,the control effect of endophytic strain mdj-15 was not significantly different from that of the chemical agents.15℃（4）Under the conditions of,20℃,25℃and 30℃,endophytic bacteria mdj-15 fermentation broth could induce the increase of SOD activity in rice leaves.Except for 15℃and 20℃,the SOD activity was not significantly different from that of hymexazol treatment,but was significantly higher than that of hymexazol treatment,thiophanate-methyl treatment and water treatment under other conditions.Under different temperature conditions,with the decrease of temperature,the overall SOD activity increased at first and then decreased.Under 20℃,the SOD activity of endophytic bacteria mdj-15 reached the maximum value,which was increased by 75%compared with that of water treatment.Under the conditions of 15℃,20℃,25℃and 30℃,the POD activity of rice leaves treated with endophytic mdj-15 fermentation broth was compared with that treated with hymexazol,thiophanate-methyl and water,except at 20℃,there was no significant difference between hymexazol treatment,but the others were significantly increased.With the decrease of temperature,the POD activity increased first and then decreased,and the highest POD activity was found at 20℃.At 15℃,20℃,25℃and 30℃,POD activity of endophytic mdj-15 was increased respectively by 51%,51%,54%and 25%compared with water treatment.Under the conditions of 15℃,20℃,25℃and 30℃,the CAT activity of rice leaves treated with endophytic bacteria mdj-15 fermentation broth was not significantly different from that of hymexazol treatment,but significantly higher than that of thiophanate-methyl and water.Similar to the changing trend of SOD and POD,with the decrease of temperature,CAT enzyme activity in all treatments increased at first and then decreased.Under the conditions of 15℃,20℃,25℃and30℃,POD activity of endophytic mdj-15 was increased respectively by 71%,70%,62%and 49%compared with water treatment.（5）The germination force of seeds treated with endophytic mdj-15 fermentation broth was10.53%and 12.00%higher than that treated with water and liquid medium,respectively,and showed significant difference.The germination rate of seeds treated with endophytic mdj-15fermentation broth was 4.83%and 3.58%higher than that treated with water and liquid medium,respectively.The results showed that mdj-15 fermentation broth could promote the germination of rice seeds plant height,root length,root number,fresh weight,and dry weight of rice seedlings treated with mdj-15 fermentation broth were 18.44±0.14cm,6.61±0.04cm,13.43±0.17,28.36±0.15g,and 5.11±0.02g,respectively.Compared with the treatment with water and endophytic culture medium,mdj-15 had significant promoting effects.（6）Metabolomics was used to detect all metabolites in the fermentation broth of endophytic strain mdj-15.The metabolites in the fermentation broth of strain mdj-15 and control strain mdj-34were significantly different,and the samples of the two groups were well differentiated with obvious clustering.In the two groups of mdj-15 and mdj-34 samples,166 metabolites with significant differences were screened in the positive ion mode,and 57 metabolites with sig nificant differences were screened in the negative ion mode.Among them,the metabolites with the most significant difference mainly included amino acids,organic acids,ketones,alcohols,nitrogen compounds and so on.Through KEGG Pathway enrichment analysis,further studies on differential metabolites were conducted.Determine the difference between the main biochemical pathways including secondary bile acid biosynthesis,glycerophospholipid metabolism,biosynthesis of unsaturated fatty acids,arginine and proline metabolism,fatty acid biosynthesis,benzoate degradation,taurine and hypotaurine metabolism,sphingolipid metabolism,xylene degradation,glyoxylate and dicarboxylate metabolism,sulfur metabolism,carbon metabolism,bacterial chemotaxis,purin e metabolism,glycine,serine and threonine metabolism,beta-Alanine metabolism,biosynthesis of antibiotics,degradation of aromatic compounds,and Vancomycin resistance.Among these pathways,the secondary bile acid biosynthesis and glycerophospholipid metabolism were the most significant enrichment pathways.