链霉菌ZH-356的植病生防作用

Biocontrol effects of Streptomyces sp. ZH-356 on plant diseases

摘要:

目的 对具有拮抗植物病原真菌作用的链霉菌ZH-356进行定殖能力和生防作用评价，并结合组学分析初步揭示其植病生防机制。方法 采用绿色荧光蛋白(green fluorescent protein, GFP)荧光标记法，检测链霉菌ZH-356在植物体内的定殖情况；同时，通过测定不同剂型菌剂(种子包衣剂、可湿性粉剂、胶水菌剂和骨胶菌剂)对植物真菌病害的生防效果来评价链霉菌ZH-356的植病生防作用及潜力；利用三代测序技术分析链霉菌ZH-356的全基因组信息，并对其基因功能进行注释；结合转录组学对比分析链霉菌ZH-356在拮抗植物病原真菌过程中表达发生显著改变的基因，预测其拮抗活性物质的合成基因。结果 通过GFP荧光标记的植物定殖分析，发现链霉菌ZH-356能在番茄和小麦的根茎中稳定定殖；同时，基于ZH-356制备的不同剂型菌剂对番茄早疫病和苹果树腐烂病均具有良好防效作用。其中，链霉菌ZH-356制成的种子包衣剂处理后不影响番茄种子的发芽率，且能有效保护番茄苗免受番茄早疫病菌的侵染；基于ZH-356制备的可湿性粉剂对番茄早疫病具有良好的预防与治疗效果，且预防作用优于治疗作用；由链霉菌ZH-356制成的液体菌剂，无论是否刮除发病处的树皮，对苹果树腐烂病均具有一定的防治效果，刮除病斑上的树皮后防治效果更好，且这种防治效果优于甲基硫菌灵农药。通过对链霉菌ZH-356的全基因组测序分析发现，其基因组含有一条线状染色体，大小为9 435 898 bp，G+C含量平均为70.82%，共预测到8 432个编码基因、69个tRNA基因和18个rRNA基因。物种注释结果显示，链霉菌ZH-356不属于任何已完成基因组测序的链霉菌种类。经全基因组分析预测，链霉菌ZH-356中含有32个次级代谢产物合成基因簇。通过转录组学分析发现，在拮抗植物病原真菌过程中，NRPS/T1PKS基因簇ZH_356_GM000343-ZH_356_GM000422的表达显著上调，推测它可能是链霉菌ZH-356中介导其抗植物病原真菌活性物质生物合成基因簇，而ZH_356_GM000409可能是该活性物质的核心生物合成基因。结论 链霉菌ZH-356能在植物体内稳定定殖，基于该菌株制备的生防菌剂对植物真菌病害具有良好防治效果，其拮抗植物病原真菌的活性物质可能由ZH_356_GM000343-ZH_356_GM000422基因簇负责合成。本研究为菌株ZH-356的产业化应用和拮抗植物病原真菌的机制研究奠定了基础。

关键词:链霉菌;定殖;菌剂;基因组测序;转录组测序;次级代谢产物

Abstract:

Objective To evaluate the colonization ability and biocontrol effects of Streptomyces sp. ZH-356 with antagonistic effects on plant pathogenic fungi and reveal the biocontrol mechanism of Streptomyces sp. ZH-356 by omics analysis.Methods The colonization of Streptomyces sp. ZH-356 in plants was detected by the GFP fluorescent labeling method. The biocontrol effects and potential of Streptomyces sp. ZH-356 on plant fungal diseases were evaluated based on the biocontrol effects of the inoculant in different dosage forms (seed coating agent, wettable powder, gum inoculant, and bone glue inoculant). The whole genome information of Streptomyces sp. ZH-356 was analyzed by third-generation sequencing, and its gene functions were annotated. The comparative transcriptome analysis was performed to screen the differentially expressed genes during the antagonizing process of Streptomyces sp. ZH-356 against plant pathogenic fungi, and thus the genes involved in the synthesis of antagonistic substances were predicted.Results Streptomyces sp. ZH-356 stably colonized the roots and stems of tomato and wheat plants. Different dosage forms of inoculants prepared based on Streptomyces sp. ZH-356 demonstrated strong control effects on tomato early blight and apple valsa canker. Among them, the seed coating agent prepared with Streptomyces sp. ZH-356 did not affect the germination rate of tomato seeds after treatment while protecting tomato seedlings from the infection of Alternaria solani. The wettable powder prepared with Streptomyces sp. ZH-356 showed both prevention and treatment effects on tomato early blight, with the prevention effect stronger than the treatment effect. The liquid inoculants prepared from Streptomyces sp. ZH-356 had control effects on apple Valsa canker, regardless of whether the diseased bark was scraped or not, while the control effect was better when the diseased bark was scraped and better than that of thiophanate-methyl. The whole genome sequencing results showed that Streptomyces sp. ZH-356 contained only one linear chromosome with a size of 9 435 898 bp and the average G+C content of 70.82%. A total of 8 432 coding genes, 69 tRNA genes, and 18 rRNA genes were predicted. Species annotation results showed that Streptomyces sp. ZH-356 did not belong to any Streptomyces species whose genome has been sequenced. Genome-wide analysis showed that there were 32 biosynthetic gene clusters (BGCs) for secondary metabolites in ZH-356. Transcriptomic analysis showed that the expression of the NRPS/T1PKS gene cluster ZH_356_GM000343-ZH_356_GM000422 was significantly up-regulated in the process of antagonizing plant pathogenic fungi, suggesting that it may be a BGC mediating the biosynthesis of active substances against plant pathogenic fungi in Streptomyces sp. ZH-356. Moreover, ZH_356_GM000409 may be the core biosynthetic gene of the active substances.Conclusion Streptomyces sp. ZH-356 can colonize plants, and the biocontrol agents prepared based on this strain demonstrate good control effects on plant fungal diseases. The active substances for the antagonistic effects may be synthesized by the gene cluster ZH_356_GM000343-ZH_356_GM000422. The above work lays a foundation for the industrial application of strain ZH-356 and the research on the mechanism of antagonizing plant pathogenic fungi.

Key words:Streptomyces;colonization;inoculant;genome sequencing;transcriptome sequencing;secondary metabolites