无色杆菌Am77绿色荧光蛋白标记菌株的构建及其菌丝际定殖和迁移特性研究

Construction of a Green Fluorescent Protein-labeled Strain of Achromobacter Am77 and Its Colonization and Migration Characteristics in Hyphosphere

黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum,Foc)是黄瓜枯萎病的病原菌,该病害可导致黄瓜严重减产甚至绝收。无色杆菌(Achromobacter sp.)Am77由黄瓜枯萎病菌菌丝诱集获得,推测其在Foc菌丝际具有强定殖能力。为明确菌株Am77在Foc菌丝际的定殖及迁移能力,采用CaCl_(2)法将pRK415-EGFP质粒转化进入菌株Am77感受态细胞,从20株转化子中筛选出一株荧光表达强烈的GFP标记菌株,命名为GFP-77;对GFP-77菌株生长曲线的测定结果表明GFP-77与原始菌株Am77生长特性一致;连续传代遗传稳定性检验结果表明,连续传代10次的GFP-77菌株发光率均在99%以上;为明确GFP-77菌株是否与Foc存在特异互作,选取了Foc、与其同属的茄病镰刀菌(Fs)以及生防真菌哈茨木霉(Tr),测定了GFP-77菌株在3种真菌菌丝际的定殖和迁移能力,结果表明,GFP-77菌株在3种真菌菌丝际的定殖和迁移能力不同,在Foc菌丝际定殖能力最强,随菌丝迁移距离最远,培养3 d的平均迁移距离达21.5 mm。研究表明菌株Am77与Foc关系密切,研究结果为探究无色杆菌Am77与枯萎病菌Foc的互作提供了依据。

Fusarium oxysporum f.sp.cucumerinum(Foc)is the causal pathogen of cucumber Fusarium wilt disease,which causes great yield loss or destruction in cucumber production.Achromobacter sp.Am77 was isolated from Fusarium oxysporum f.sp.cucumerinum(Foc)hyphosphere by a baiting technique,and was suggested to be a bacterium which could strongly colonize in Foc hyphosphere.To clarify the colonization and migration capacity of strain Am77 in Foc hyphosphere,GFP labeling of strain Am77 was performed by CaCl_(2) method through transforming of pRK415-EGFP plasmid into the competent cells of strain Am77,and a strong fluorescence expression strain GFP-77 was screened from 20 transformants.The growth characteristics of GFP-77 strain were investigated,and the results showed that GFP-77 had the same growth characteristics to original strain Am77.The genetic stability of GFP-77 strain was tested,and the luminescence rate of GFP-77 strain was above 99%after sub-culturing for 10 generations.The colonization and migration of GFP-77 in the hyphosphere of Foc,Fusarium solani(Fs)and Trichoderma harzianum(Tr)were examined to determine whether GFP-77 strain had specific interaction with Foc.GFP-77 strain performed different colonization and migration characteristics in hyphosphere of the three tested fungi.It had the strongest colonization ability in Foc hyphosphere,and migrated farthest with Foc hyphae growth at an average migration distance of 21.5 mm after 3 d incubation.The results showed that strain Am77 was closely related to Foc,which provided a basis for exploring the interaction between Achromobacter Am77 and Foc.