新疆棉花黄萎病株内生真菌荧光定量检测及时空动态分析

Quantitative detection and spatiotemporal dynamic analysis of endophytic fungi in cotton infected by verticillium wilt using Real-time PCR

摘要:

【背景】棉花黄萎病严重制约新疆棉花持续高产和稳产，内生菌在棉花黄萎病生物防治中潜力巨大。棉花黄萎病发生与内生菌有密切关系，但棉花黄萎病株内生真菌含量的研究鲜见报道。【目的】了解棉花黄萎病株内生真菌数量的时空动态变化及其与黄萎病病原数量的关系。【方法】用Taqman探针实时荧光定量PCR方法对棉花黄萎病株内生真菌数量进行周年动态测定，分析棉花内生真菌数量与黄萎病原菌数量的关系。【结果】不同生育时期棉花植株根部内生真菌数量表现出不同变化趋势。库尔勒棉花吐絮期根部最大值达1.46×109 copies/g FRW，阿拉尔棉花根部内生真菌数量表现为蕾期缓慢上升，花铃期达到最大值，为8.30×107 copies/g FRW。棉花根部内生真菌数量以南疆棉区库尔勒的数量最高，吐絮期平均达1.46×109 copies/g FRW；其次为阿拉尔，花期平均达8.30×107 copies/g FRW；精河最少，苗期平均为1.85×104 copies/g FRW。棉花根部内生真菌数量的空间变化趋势是南疆、东疆、北疆依次递减：南疆库尔勒和阿拉尔内生真菌数量较高，库尔勒吐絮期达到最大值1.46×109 copies/g FRW，其次为阿拉尔8.30×107 copies/g FRW，精河最低1.85×104 copies/g FRW。精河棉花内生真菌数量与黄萎病病原菌数量显著正相关，其皮尔逊相关系数高达0.639。石河子和哈密棉花内生真菌与黄萎病病原菌呈负相关，其相关系数分别为?0.180和?0.275。其他内生真菌与黄萎病病原菌之间存在正相关，但相关性不显著。【结论】棉花黄萎病株根部内生真菌含量较高，内生真菌数量均随采样棉花生育时期和采样地点不同而呈现波动性变化，内生真菌数量最大值出现在库尔勒花铃期。

Abstract:

[Background] Verticillium wilt has seriously restricted the sustainable yield of cotton in Xinjiang, China. Endophyte in cotton (Gossypium hirsutum L.) have great potential for biological control of cotton verticillium Wilt. There are the close relation to cotton verticillium wilt and endophytic microorganisms, but no study has investigated the content of fungal endophyte in different branches and leaves as well as roots of Verticillium Wilt affected cotton. [Objective] To study the spatiotemporal dynamic of endophytic fungi number and the relationship between the number of endophytic fungi and the pathogen of Verticillium Wilt in cotton infected by Verticillium Wilt. [Methods] Primers and probes specific to Internal Transcribed Spacer (ITS) of fungi were designed. A TaqMan probe-based, Real-time fluorescence quantitative PCR was established. Taqman probe real-time fluorescence quantitative PCR method was used to determine the total amount of endophytic fungi in cotton Verticillium Wilt, and to analyze the relationship between the number of endophytic fungi and the pathogen of Verticillium dahlia. [Results] The number of total endophytic fungi in cotton roots infected by Verticillium Wilt showed different trends in different growth period of cotton in Korla, Xinjiang. The maximum number of total endophytic fungi in cotton roots at boll opening stage infected by Verticillium Wilt was 1.46×109 copies/g in Korla cotton root fresh fungi (FRW). The number of total endophytic fungi showed increased slowly in bud stage, and reached the maximum value, 8.30×107 copies/g FRW of cotton roots in boll opening stage. The number of endophytic fungi was the highest in South Xinjiang Korla, and was average up to 1.46×109 copies/g FRW in boll opening period; followed by an average of 8.30×107 copies/g FRW in flower stage; at least by an average of 1.85×104 copies/g FRW in seedling stage, Jinghe. The spatial variation trend of number of endophytic fungi is decreasing in the Southern, Eastern, Northern Xinjiang; the number of endophytic fungi was higher in Korla and Alar, the number of endophytic fungi was the highest in South Xinjiang Korla, and was average up to 1.46×109 copies/g FRW in boll opening period; followed by an average of 8.30×107 copies/g FRW in flower stage; at least by an average of 1.85×104 copies/g FRW in seedling stage, Jinghe. The correlation between number of endophytic fungi and of Verticillium dahliae was significantly positive in cotton infected by Verticillium Wilt in Jinhe, Xinjiang. The correlation index PCC value is as high as 0.639. The correlation between number of endophytic fungi and of V. dahliae was negative correlation in cotton infected by Verticillium Wilt in Shihezi and Hami, Xinjiang. The correlation index PCC value was ?0.180 and ?0.275, respectively. The correlation between number of the other endophytic fungi and of V. dahliae was positive, but the correlation is not significant. [Conclusion] There were numerous endophytic fungi in cotton infected by Verticillium Wilt in Xinjiang. The higher concentration of endophytic fungi was in roots. The number of total endophytic fungi in cotton roots infected by Verticillium Wilt showed different trends in different growth period and sampling sites of cotton in Xinjiang. The maximum value number of total endophytic fungi in cotton roots infected by Verticillium Wilt appeared during the blooming period in Korla, Xinjiang.