钩状木霉 ACCC31649的GFP标记及其对辣椒定殖和促生作用

【目的】旨在建立农杆菌介导的绿色荧光蛋白 (GFP) 基因转化钩状木霉 (Trichoderma hamatum) ACCC31649的技术方法，筛选遗传稳定的GFP标记转化子，并研究该菌株在辣椒植株中定殖和对辣椒的促生作用，为进一步阐明木霉在辣椒根际定殖及其与辣椒病原菌在辣椒根际和植株中的定殖、互作和生防作用奠定基础。【方法】通过根癌农杆菌介导的遗传转化方法筛选遗传稳定的GFP标记转化子，通过灌根接种方法和组织切片的水玻片荧光观察研究了钩状木霉在辣椒植株中定殖过程和对辣椒的促生作用。【结果】获得了遗传稳定GFP标记的钩状木霉转化子。荧光显微观察表明，辣椒根、茎、叶组织中都检测到GFP标记菌株的定殖。标记菌株首先在根部定殖，然后通过根部维管束逐步定殖到茎和叶片组织中。野生型菌株和GFP标记菌株灌根接种4叶期辣椒幼苗，30天后，GFP标记菌株与水处理对照相比，辣椒的株高增长13.5%，根长增长16.2%，鲜重和干重分别增加了43.8%和45.3%，而且野生型菌株与GFP标记菌株对辣椒的促生作用没有显著差异。【结论】钩状木霉能够在辣椒植株根、茎和叶组织中定殖，并且对辣椒具有显著的促生作用。同时，GFP标记的钩状木霉将在进一步阐明该菌株在辣椒根际定殖及其对病原菌拮抗和互作研究中发挥重要作用。

GFP-labeled transformation of Trichoderma hamatum ACCC31649 and its promotion on colonization and growth of pepper plants

Objectives]The green fluorescent protein (GFP) gene was transferred into Trichoderma hamatum ACCC31649 by the Agrobacterium tumefaciens-mediated transformation. The growth-promoting effect and the colonizing patterns in pepper plants of this strain were investigated in this paper, which would provide a technical basis for the research of the rhizosphere colonization of T. hamatum in pepper plants and biocontrol mechanism of antagonistic fungus against pathogen.Methods]The Agrobacterium tumefaciens-mediated transformation was used to select the GFP-labeled strain T. hamatum-GF21 which inherited stably, and the inoculation of T. hamatum by pouring root treatment and the observation of water slides of pepper tissue sections by fluorescence microscopy were used for the growth- promoting effect of pepper plants and its colonizing patterns.Results]The stable GFP-labeled strain T. hamatum-GF21 was obtained. Fluorescence microscopy observation confirmed that the GFP-labeled strain T. hamatum-GF21 colonized in the roots, stems and leaves of the pepper. It colonized on theroots firstly and then moved to stems and leaves through the vascular bundle of the roots. After 30 days of pouring root treatment in the four-leaf pepper seedlings by the wide strain and the GFP-labeled strain T. hamatum-GF21 separately, the GFP-labeled strain T. hamatum-GF21 increased the pepper height by 13.5%, root length by 16.2%, fresh weight by 43.8% and dry weight by 45.3% respectively, compared with the water control. There is no significant difference between the wide strain and the GFP-labeled strain T. hamatum-GF21 in the effect on the plant growth promoting.Conclusions]The GFP-labeled strain T. hamatum can colonize in the roots, stems and leaves of the pepper, and show a significant growth-promoting effect on pepper plants. Therefore, the GFP-labeled strain T. hamatum-GF21 can be used on the research of the rhizosphere colonization in pepper plants and the biocontrol mechanism between pathogen and its antagonistic fungus.