Fluorescent marker of biocontrol actinomycetes Ahn75 and its colonization in rice

Abstract:

[Background] At present, marker gene gfp has become an important tool to study the interaction between target microorganism and host. Using gfp to mark the biocontrol strain, we can track the survival and colonization of the biocontrol strain effectively. [Objective] The colonization of Ahn75 on rice was studied by making fluorescence labeling in the biocontrol actinomycete Ahn75, to lay a foundation for the resistant mechanism study of Ahn75 against rice blast. [Methods] Plasmid pIJ8655 with green fluorescent marker gene (gfp) was introduced into E. coli ET12567 by electroporation, then the gfp gene was incorporated into the genome of Ahn75 through conjugal transfer. The inhibitory activity of Ahn75-GFP against rice blast pathogens was tested by the dural culture method. Ahn75-GFP colonized in rice by spraying spores was observed by fluorescence microscopy, and re-isolated from the rice and counted, to explore the distribution of Ahn75-GFP in rice tissues. [Results] The PCR amplification of gfp and the fluorescence observation of Ahn75-GFP showed that, the green fluorescent marker gene was successfully integrated into Ahn75. The inhibitory activity of Ahn75-GFP against rice blast pathogens by the dural culture method was not significantly different from the original strain. Ahn75-GFP colonized in the root, stem and leaf, could be observed by a fluorescent microscope, and the re-isolation results of endophytic strains in rice indicated that the strains had the strongest colonization ability in stems. [Conclusion] A green fluorescence-labeled biocontrol strain Ahn75-GFP was successfully obtained, and showed good colonization ability in rice, which was of great significance for studying the control of rice blast by Ahn75 strain.