Isolation Of Antimicrobial Compounds From Bacillusamyloliquefaciens NJN-6 And Their Effects On Soil Microbial Community

Fusarium wilt disease and bacterial wilt are the vital soil-born disease in agricultural production, and limited the sustainable development of agriculture. For the bioconrol approach,a Bacillus myloliquefaciens NJN-6 (NJN-6 in abbreviation in the whole thesis)was isolated and indentified from a health banana rhizosphere n a soil-born disease area. In previous studies, this strain showed a talented inhibition of pathogen in vitro and significant biocontrol and growth promotion effects both in pot and field conditions. This study was performed to finger out the antagonist mechanism, especially the active compounds produced by NJN-6 strain. Firstly, we isolated and identified several water soluble antimicrobial compounds and a large group of volatile organic compounds from the fermentation of NJN-6. Then, we evaluated the ability of colonization and active compounds production in the rhizosphere of banana plant. At last, the effects of active compounds from NJN-6 strain on soil microbial community and some functional genes were investigated by high throughput sequencing technology and quantitive PCR. The main results obtained were summarized as follows:1. Antifungal compound iturin A was firstly extracted by n-butanol from fermentation of NJN-6, and then identified by high pressure liquid chromatography (HPLC) through comparing retention time of target compounds with standard samples. The molecular weight of three iturin A was also measured by HPLC/ESI-MS as 1043, 1057 and 1071 Da,which were consistence with the standard samples, respectively. Another five compounds were extracted by XAD-16 resin and then separated using HPLC equipped with automatical collector. Two of them showing antifungal effect were identified as bacillomycin D with molecular weight of 1030 Da (C14) and 1044 Da (C15); other three of them showing antibacterial effect were identified as macrolactin A, 7-O-malonyl macrolactin A and 7-O-succinyl macrolactin A with the molecular weight of 402, 487 and 502 Da, respectively.All the identified active compounds showing excellent antifungal or antibacterial effect indicated NJN-6 strain a talented biocontrol agent.2. This study addresses the use of an aqueous two-phase system (ATPS) using ethanol/ammonium sulfate to extract iturin A from Bacillus amyloliquefaciens NJN-6 fermentation broth and the quantification of iturin A by HPLC. Baseline separation of iturin A homologues was performed using an RP-C18 column with a mixture of water and acetonitrile. The results showed that the correlation coefficient between integral area and concentration was 0.9961 within the range of 20-140 mg/1. The RSD of the retention time and the peak area were 1.29% and 1.45%, respectively. The effects of some operating parameters in ATPS, e.g., pH, temperature and centrifugation time, were also studied. This method can be successfully used for the rapid quantification of iturin A.3. The strain NJN-6 has an ability to inhibit the growth and the spore germination of Fusarium oxysporum without directly contaction, indicating that volatile organic compounds (VOCs) from NJA-6 were in function. When spores in soil were suffocated in chemosynthesis cultures with VOCs produced by NJN-6, the concentration of spores was almost 100-fold lower than that of non-suffocated cultures. Fungi-static volatiles were extracted using solid-phase micro-extraction (SPME) and identified by gas chromatography-mass spectrometry (GC-MS). All of the 36 detected VOCs, including 12 benzenes, 7 alkyls, 3 alcohols, 7 ketones, 2 aldehydes, 3 naphthyls, 1 ester and 1 ether, were assayed (200 ?l or 0.2 g) for antifungal activity using divided Petri plates for three days.Eleven compounds completely inhibited the growth of Fusarium oxysporum, 7 VOCs showed >80% antifungal activity and 17 VOCs showed inhibitory activity, ranging from 6.12% to 78.8%. The antifungal activity of these compounds suggested an important role of VOCs produced by NJN-6 in suppressing Fusarium oxysporum growth. In addition, VOCs produced by NJN-6 may be useful in the long-distance inhibition of Fusarium oxysporum.These findings provide novel information regarding the biocontrol mechanism of NJN-6 and indicate NJN-6 as a promising candidate for use as a biocontrol agent.4. NJN-6 is an important plant growth-promoting rhizobacteria (PGPR) which can produce secondary metabolites antagonistic to several soil-borne pathogens. In this study, to determine the beneficial mechanism of the strain, the colonization of NJN-6 strain on banana roots was evaluated using scanning electron microscope (SEM). The plant growth promoting hormones, Indole-3-Acetic Acid (IAA) and Gibberellin A3 (GA3) along with antifungal lipopeptides iturin A were detected when the NJN-6 strain was incubated in root exudates of banana. In addition, we also found transcription level of four genes (ituA,bamD, epsD and yqxM) in NJN-6 living in the hydroponic system were upgrade especially in the rhizosphere of banana seedling roots. Therefore, the NJN-6 could colonize the roots of banana after the application and produce active compounds which were beneficial for the growth of banana plants.5. Macrolactin has been proven to be the efficient antibacterial agent against many bacterial pathogens. However, no studies about the effect of macrolactin on soil bacterial community as well as the aboriginal antibiotic producing genes have been reported so far.In this study, the macrolactin mixture was isolated from the NJN-6, and applied to soil once a week (four weeks in total). The 16S rRNA Illumina MiSeq sequencing and qPCR showed that continuous application of macrolactin reduced the copy number of 16S rRNA gene and a-diversity of bacterial community, changed the relative abundance of microbes at both phyla and genus levels. Three significantly increased genera (Burkholderia, Dyella, and Rhodanobacter) from Beta-Proteobacteria and Gamma-Proteobacteria may be the macrolactin degrader in soil. Also, the native macrolactin producing gene - polyketide synthase (PKS) gene was reduced in copy number after the application of macrolactin. This suggested that the broad-spectrum microbiological source bactericide could reduce the non-target microbes and decrease the diversity of the soil bacterial community similar to the performance of agricultural chemicals.6. Lipopeptides have been proven to be efficient antifungal agents against many fungal pathogens extensively. No studies, however, have been reported so far, referring to their effect on soil fungal community as well as the aboriginal antibiotic producing genes. In this study, the lipopeptides mixture from the NJN-6 was applied to soil to investigate the intriguing effects. Results showed that lipopeptides reduced the copy number of 18S rRNA gene and a-diversity of fungal community, but significantly increased the relative abundance of Basidiomycota as well as its further taxons Cryptococcus and Trichosporon after detection by ITS rRNA Illumina MiSeq sequencing platform. Meanwhile, the Fusarium spp.,especially Fusarium oxysporum was significantly inhibited in proportion.Also, the native lipopeptides producing gene - Nonribosomal peptides synthase (NRPS)gene was also reduced in diversity and in abundance. This confirmed that microbiological source fungicide could change soil fungal community aimlessly. Moreover, these fungicides application may also break out the balance of native antibiotic producing genes community.7. As a talent antibiotics producer, NJN-6 could also release volatile organic compounds (VOCs) suppressive pathogen Fusarium oxysporum, while their effects on soil microbial community and some functional genes are still unknown. Here, VOCs produced by NJN-6 were used to influence the soil microbial community in divided plates (NJN-6 was cultured in one side, and soil was placed on the other side of divided plate). Results showed that the number of bacteria was promoted by the present of VOCs, while the number of fungi was inhibited. MiSeq sequencing data showed that VOCs significantly decreased the a-diversity of bacterial community and fungal community. VOCs from NJN-6 also showed inhibition effects on antibiotics producing genes- PKS and NRPS. We also evaluated the effect of VOCs on N-cycling related genes, and found the inhibition on abundance of AOA, nifH and nirS gene, while promotion on abundance of AOB gene.Conclusion: Bacillus amyloliquefaciens NJN-6 isolated from this lab can produce a large amount of antagonistic compounds to soil-born diseases and thus is a good biocontrol agent. Application of the antagonistic compounds to soils can significantly affect the soil microbiome. The paper confirms that soil microbiome can be manipulated or shaped by application of biocontrol agents or their bioorganic fertilizers.