水稻内生放线菌OsiRt-1的分离鉴定及对稻瘟病的防治作用

Identification of an endophytic actinomyce OsiRt-1 isolated from rice and its effect against rice blast disease

摘要:

【目的】从水稻中分离、筛选并鉴定出对稻瘟病真菌具有拮抗性能的内生放线菌,对高抗菌株进行稻瘟病生防效果评定,初步探讨其作用机制。【方法】采用4 种分离培养基对水稻内生放线菌进行分离,用平板对峙法筛选出对稻瘟病菌拮抗性能最好的菌株,结合其菌落形态、生理生化及16S rRNA 基因序列分析进行鉴定;用环境扫描电镜(SEM)观察拮抗菌对稻瘟病菌菌丝形态影响,用菌丝生长速率法对拮抗菌发酵滤液进行抗菌活性评价;大田条件下,喷洒拮抗菌孢子液于水稻,检测其稻瘟病控制效果;同时,对拮抗菌进行产酶和次级代谢产物分析,检测其聚酮合酶基因(PKSⅡ型)和非核糖体多肽合成酶基因(NRPS)。【结果】从1 800 个水稻样品中分离出117 株内生放线菌,从中筛选出拮抗性能最好的菌株OsiRt-1,鉴定为米修链霉菌Streptomyces misionensis。该菌株使稻瘟病菌菌丝出现畸形,其无菌滤液对病菌菌丝生长的抑制率为28.06%;大田条件下,OsiRt-1 对水稻苗瘟、穗瘟均有较好防效,其中对苗瘟防效为7.76%,穗瘟防效高达25.65%,损失率降低了17.46%。与之对应,OsiRt-1 处理过的水稻地上部分,OsiRt-1 所占内生放线菌的比例明显高于未处理水稻。该菌株具有可能降解真菌细胞壁的纤维素酶、蛋白酶活性,同时可产生植物生长促进剂铁载体、IAA、ACC 脱氨酶。菌株OsiRt-1 呈现PKSⅡ型和NRPS 阳性。【结论】菌株OsiRt-1 是一株具生防潜力的内生放线菌,在农业上具有实际研发价值。

Abstract:

[Objective] To isolate, screen and identify rice endophytic actinomyces with antagonistic activity against rice blast pathogen, to evaluate the disease control effect of the selected antagonist and to study its potential antagonistic mechanism. [Methods] We used 4 isolation media to isolate endophytic actinomycetes from surface sterilized rice. Using dual culture assay, we screened the strain with antagonistic activity and identified it by morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. We observed the effect of the antagonist on the hyphae of pathogen under scanning electron microscopy and tested the in vitro antagonistic activity of fermentation broth by growth rate method. In field condition, we evaluated the disease control efficiency after spraying the antagonist spore solution on rice. Meanwhile, we analyzed enzymes and secondary metabolites production of the antagonist, and amplified its polyketide synthases (PKSⅡ) and nonribosomal peptide synthetases (NRPS) genes. [Results] From 1 800 rice samples, a total of 117 endophytic actinomycetes were isolated, among which, OsiRt-1 exhibited remarkable antagonistic activity. OsiRt-1 was identified as Streptomyces misionensis. The pathogen Magnaporthe oryzae treated by OsiRt-1 revealed significant morphological alterations in hyphe and its mycelial growth was inhibited by culture filtrates of OsiRt-1 with a percent of 28.06%. In field condition, OsiRt-1 spore solution could reduce seedling blast severity by 7.76%, panicle blast disease severity by 25.65% and lose rate by 17.46%. The separation rate of OsiRt-1 from above-ground portion of rice treated with OsiRt-1 is higher than the not treated ones. Besides, OsiRt-1 is positive for PKSⅡ and NRPS genes and can produce cellulase, protease, siderophore, IAA and ACC deaminase. [Conclusion] Endophytic Streptomyces misionensis OsiRt-1 has the application potential in agriculture as a biocontrol agent against rice blast disease.