| Streptomyces venezuelae Snea253 has nematocidal activity, while its metabolites may be water soluble compounds of oligosaccharides with amino that are difficult to separate and purification in our previous research. So, in order to lay theoretical foundation for bio-nematicidal development and industrialization, the key metabolic genes of nematicidal activity production from Snea253 were researched by using nutrition and gene metabolic regulation in here. At the same time, soil nematode and microbes community in microecological habitas by Snea253 treatment was explored to determine its environment safety. The results were as follows:1. The nutrition metabolic regulation of Snea253 on carbon and nitrogen sources:the best carbon and nitrogen medium were obtained by Plackeet-Burman and Box-Benhnken experimental design methods:soluble starch 4%, glucose 3%, peanut meal 3.5%, (NH4)SO4 0.3%, NaCl 0.04%, K2HPO4 0.02%, MgSO4·7H2O 0.02%, FeSO4 0.00125% in midia. Nematicidal activity of Snea253 optimization broth was 89.4%, which was higher 26.3% than that of the original strain. The optimized fermentation conditions were confirmed by Central composite experimental design method:seed age (72 h), inoculums (5%), initial pH 6.6, fermentation time (5.25 d), rotation speed (175 r/min), loaded liquid (20%). Nematicidal activity of Snea253 optimization broth was 91.2%, which was higher 29.7% than that of the original strain.2. The gene metabolic regulation research of Snea253:Transcriptome gene analysis between high and low virulence strain of Snea253 was sequenced by De Novo transcriptomic analysis of RNA-Seq. About 3 234 differentially expressed Unigenes were detected, including 2 668 up-regulated Unigenes and 566 down-regulated Unigenes in high virulent strains. GO analysis of differential expression genes (DEGs) showed that 2 160 DEGs were annotated to 45 GO terms. Pathway enrichment analysis revealed that there were 2 796 DEGs enriched to 161 pathways, mainly involved in Metabolic pathway, Biosynthesis of secondary metabolites, Microbial metabolism in diverse environments, Ribosome, Starch and sucrose metabolism and so on.Through the large data analysis, about 8 genes related with carbon and nitrogen metabolism were screened, which included 5 up-regulated genes Unigene556_All, Unigene609_All, Unigene1016_All, Unigene989_All and Unigene1006_All and 3 down-regulated gene Unigene1272_All, Unigene1090_All, CL20. Contig2_All. The results of gene expression by Q-PCR in high virulence strain were consistent with the results of RNA-Seq. Compared with the low virulence strain, Unigene556_All was high up-regulated 22.86 times, and Unigene1006_All also up-regulated 3.48 times gene expression. Meanwhile Unigene1272_All, Unigene1090_All and CL20.Contig2_Allwas down-regulated 2.32,1.40 and 1.38 times in the high virulence strain. Streptomyces expressed vector pIB139 were successfully used to construct overexpression strains Snea253-Unigene556 and Snea253-CL20.Contig2 by PEG-mediated method. The gene expression were higher respectively 1.68 times and 2.16 times than that of wild strain.3. Diversity change of soil nematodes community on Snea253 treatment microecological habitat:Through identifying the soil nematodes with experiment treatments in the greenhouse of northeast China, we found that Snea253 treatment could inhibit total soil nematodes. Plant-parasites (PP) and Bacterivores (BF) nematodes than the untreatment control (CK). But the inhibition degree was weaker than Avermectin treatment. Compared to Avermectin treatment, Omnivore-predators (OP) nematode increased in Snea253 treatment significantly. Plant-parasites (PP) and Meloidogyne incognita in Snea253 treatment were slightly more than in Avermectin treatment, however, it is effective to keep PP and Meloidogyne at low level.Shannon-Weiner diversity index (H’) in Snea253 treatment was higher, while Simpson’s dominance index (λ) was lower than that in Avermectin treatment. Compared to Avermectin treatment, Soil nematodes community diversity was rich, dominant species was not prominent, and the species were well-distributed in Snea253 treatment. The nematode faunal analysis and Principal component analysis (PCA) found that the datum of Snea253 treatment mainly distributed in the quadrant B.It showed that soil nutrient status was better and soil nematodes community was less disturbed and had better mature and stable food web in Snea253 treatment microecological habitat.4. Dynamic change of soil microbial community on Snea253 treatment microecological habitat:Using high throughput sequencing technology to detect soil microbe community dynamic changes, we found that soil bacterial had 12 dominant phylum,9 dp,omamt family and 11 dominant genera, meanwhile soil fungal community had 6 dominant phylum,9 dominant family and 12 dominant genera. Compared to Avermectin treatment, Snea253 treatment had significantly increased the relative abundance of the top dominant phylum Proteobacteria, the top dominant family Xanthomonadaceae, the top dominant genus Rhodanobacter, and also had significantly increased the relative abundance of the top dominant phylum Ascomycota, the top dominant family Pyronemataceae, the top dominant genus Pseudaleuria and Pyronemataceae-unknown, and no significantly increase the relative abundance of the second top dominant family Sordariales-unknown, dominant genus Sordariales-unknown in soil fungi. There were no significant difference between Snea253 treatment and CK.Biolog-Eco data analysis indicated that both Snea253 treatment and CK had the similar average well colour development (AWCD), Shannon index (H’) and McIntosh index (U), which were higher than that of Avermectin treatment. It revealed that soil microbial community treated by Snea253 was beneficial to using carbon source and soil microbial diversity and evenness, better than Avermectin treatment. Combined Biolog-Eco analysis and high-throughput sequencing results, Snea253 as a biological nematicide was safe on soil microbial and neamtodes community, also the research provided lots of test data for biological nematicides industrialization like Snea253. |
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