高产杀粉蝶菌素的链霉菌鉴定及其拮抗水稻白叶枯菌活性研究

A piericidin-producing Streptomyces strain:identification and antagonistic activity on Xanthomonas oryzae

【目的】为保证农业生产可持续性发展,研发和使用环境友好的生物农药受到全社会的高度重视。微生物代谢产物农药是我国目前应用最广的生物农药,也是未来发展绿色农药的一个重要方向。【方法】利用包含水稻白叶枯菌(Xanthomonas oryzae pv.oryzae,Xoo)PXO99A的NA培养基琼脂平板,从水稻根际土壤中筛选能抑制Xoo生长的链霉菌。通过高效液相色谱和质谱分析活性代谢产物的化学结构;采用剪叶法接种Xoo到水稻叶片后,再喷施杀粉蝶菌素溶液(0.1 g/L),2周后测定叶枯症状;采用响应面分析法优化高产杀粉蝶菌素的发酵培养基;采用PacBio SMRT测序平台+Illumina HiSeq X Ten平台开展全基因组测序。平均核苷酸一致性(average nucleotide identity,ANI)用于比较HSW2009与其他链霉菌在全基因组水平的亲缘关系。【结果】分离到一株对Xoo生长有强抑制活性的链霉菌HSW2009,其活性代谢产物为杀粉蝶菌素A1(piericidin A1,简称PIE);喷施PIE可以减轻Xoo在水稻叶片内的侵染;优化HSW2009高产PIE的发酵条件,得到MFM培养基;HSW2009在MFM培养中生长快、PIE发酵效价最高可以达到0.72 g/L;菌落表型分析、PIE生物合成基因簇分析、全基因组测序和ANI分析表明HSW2009与目前有效描述的PIE产生菌不同。【结论】HSW2009是一株高产杀粉蝶菌素A1的链霉菌属候选新种,命名为绍武链霉菌,可作为杀粉蝶菌素产业化研究的出发菌株。

[Objective]The research and development and application of eco-friendly biopesticides for the sustainable agricultural development has received widespread concern.Microbial metabolite pesticide(MMP)is one of the most popular biopesticides in China and also one of the most important green pesticides to be developed.[Methods]The nutrient agar(NA)containing Xanthomonas oryzae pv.oryzae(Xoo)strain PXO99A was used to screen the anti-Xoo Streptomyces strain HSW2009.High performance liquid chromatography(HPLC)and mass chromatography(MS)were employed to characterize the chemical structure of the anti-Xoo metabolite produced by HSW2009.Xoo was inoculated into adult rice plants(Oryza sativa L.subsp.japonica)by leaf-clipping,followed by spraying with the piericidin solution(0.1 g/L).The length of spot was measured 2 weeks after inoculation.Response surface methodology was employed to optimize the fermentation medium.PacBio SMRT together with Illumina HiSeq X Ten was applied for genome sequencing.Average nucleotide identity(ANI)was compared to analyze the relationship between HSW2009 and other Streptomyces strains.[Results]A Streptomyces strain HSW2009 was detected to have strong inhibitory effect on Xoo growth and it produced the active metabolite piericidin A1(PIE).PIE application mitigated Xoo infection on rice leaves.An optimized MFM medium was developed for PIE production.In MFM,HSW2009 grew well,with the highest fermentation titer of PIE reaching 0.72 g/L.HSW2009 was different from the known PIE-producing Streptomyces strains in colony phenotype,PIE biosynthetic gene cluster,whole genome sequence,and ANI.[Conclusion]HSW2009 is a representative of a candidate novel species of Streptomyces producing high level of PIE and is named as Streptomyces shaowuensis sp.nov.It has potential to be developed as an industrial strain for piericidin production.