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

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.

Key words:Cotton, endophytic fungi, TaqMan probe, fluorescence quantitative PCR