Microbial Diversity In Tobacco Rhizosphere Soil And Antagonistic Microorganisms Against Phytophthora Nicotianae

Tobacco black shank caused by Phytophthora nicotianae is one of the main diseases of tobacco.The long-term continuous cropping of tobacco caused the enrichment of Phytophthora,the reduction of the number of beneficial microorganisms,and the imbalance of soil microbial community structure and composition,resulting in the frequent occurrence of tobacco black shank.Therefore,analyzing the structural characteristics and diversity of microbial community in tobacco rhizosphere soil,enriching and screening beneficial microorganisms,obtaining antagonistic strains and analyzing the action mechanism are of positive significance for the use of biocontrol bacteria to control tobacco black shank.In this study,the bacterial community structure and diversity in rhizosphere soil of healthy and diseased tobacco plants were compared,the beneficial microbial flora inhibiting Phytophthora nicotianae was enriched,and the main antagonistic strains were screened.The antibacterial active substances of biocontrol strains were analyzed by genome,transcriptome and targeted metabolic analysis,which provided a theoretical basis for the use of antagonistic strains to control tobacco black shank.The main results are as follows:1.Based on Miseq high-throughput sequencing of 16 S r RNA,the soil microbial structure and diversity in the rhizosphere of healthy and diseased tobacco plants were compared and analyzed.It was found that the occurrence of black shank disease significantly changed the soil bacterial community structure,and Shannon diversity and Pielou evenness decreased.Compared with healthy group(CK),actinomycetes(Streptomyces and arthrobacteria)were the OTU with the greatest decrease in relative abundance,and Proteus and Acidobacteria were the OTU with increased relative abundance.The results of molecular ecological network analysis showed that the occurrence of black shank destroyed the interaction between bacteria.Functional prediction analysis showed that the relative abundance of antibiotic biosynthesis related genes(such as ansamycin and streptomycin)in rhizosphere microorganisms in the pathogenesis group decreased significantly.2.Enrich soil microorganisms in tobacco roots and rhizosphere,and screen antagonistic bacteria against Phytophthora nicotianae by plate confrontation culture,so as to study the effects of antagonistic strains on the structure and diversity of soil microbial community in tobacco rhizosphere.The results showed that 39 antagonistic bacterial groups were isolated from rhizosphere and rhizosphere soil,and the inhibitory effect of endophytic antagonistic group(RFM)on Phytophthora nicotianae was stronger than that of soil antagonistic group(SFM);The application of RFM and SFM changed the diversity,composition,structure and interaction of soil bacterial community;The results of molecular ecological network analysis showed that the application of RFM enhanced the interaction between soil microorganisms,and the relative abundance of OTU of Acinetobacter,Enterobacter and Bacillus was positively correlated with the control effect of tobacco black shank.3.The antagonistic strains were isolated from the enriched antagonistic flora,classified and identified,and the effect was evaluated by pot experiment.The results showed that three strains 4-9,B44R-1 and D7 with strong antibacterial activity against Phytophthora nicotianae were isolated.The three antagonistic strains were identified as Streptomyces acidiscabies,Acinetobacter calcoaceticus and Brevibacillus brevis by morphological observation and 16 S r RNA gene sequence analysis,In the greenhouse pot experiment,the control effects on tobacco black shank were 60.59%,55.47% and 59.42%,respectively;Inoculation of antagonistic strains had an obvious effect on the root growth and development of tobacco plants.Thirty days after treatment,the root development was significantly better than that of the control.The fresh weight,dry weight,plant height,stem circumference,leaf number and maximum leaf area of tobacco plants were improved.4.The whole genome of antagonistic strains 4-9,B44R-1 and D7 was sequenced.The biosynthetic gene clusters of secondary metabolites were excavated by Antismash.Combined with transcriptome sequencing and targeted metabolic analysis of strain fermentation broth,the antibacterial active substances of biocontrol strains were analyzed.Biosynthetic gene clusters of secondary metabolites were excavated from the three strains by antismash analysis.The results of transcriptome sequencing showed that the biosynthetic gene clusters of pactamycin,tyrocidine,interferopeptin and a type I lanthopeptide(region17)of strain 4-9 were highly expressed in 24-96 h;The expression abundances of fengycin(region 4)and RIPP like lanthipeptide(region 5)in the genome of strain B44R-1 were relatively high at each time point;The expression abundance of edeine,tyrocidine and gramacidin biosynthetic gene clusters in the genome of strain D7 was high.The fermentation broth was analyzed by LC-MS/Ms.no known antibacterial substances were found in the fermentation broth of strains 4-9 and B44R-1.The non ribosomal peptide antibiotic edeine A/B was excavated from the fermentation broth of D7.The compound has broad-spectrum antibacterial activity.