Colonization on Cotton Plants with a GFP Labeled Strain of Bacillus axarquiensis

Verticillium dahliae was one of the most important diseases caused Verticillium wilt of cotton. In our previous study, Bacillus axarquiensis TUBP1 was screened and found to be an antagonistic strain against V. dahliae with 43% biocontrol effect in the cotton field. In order to uncover the functional mechanism of B. axarquiensis against Verticillium wilt in cotton, the colonization of B. axarquiensis labeled with a green fluorescent protein (GFP) was investigated in cotton plants and the rhizosphere soil. Firstly, a plasmid (pHT-315) containing gfp gene was successfully transformed into wild B. axarquiensis TUBP1 and labeled a green fluorescence by electroporation, which didn't change the bioactivity in vitro. In gnotobiotic conditions, cotton seeds were then inoculated with the gfp-labeled strain and grown in green house. Observation with a confocal laser scanning microscope and a scanning electron microscope showed that GFP-labeled B. axarquiensis TUBP1 infected cotton roots and widely distributed in epidermis, cortical parenchyma, intercellular spaces, the xylem vessels, and pith cells as well as root hair cells through cracks formed at the lateral root junctions, followed by a slow migration from roots to stems and leaves. Quantitative fluorescence and flow cytometry (FACS) approaches showed a gradual decrease in the number of TUBP1-315gfp with increasing inoculation time. However, TUBP1-315gfp levels were detectable till 45 days after planting. In contrast, no fluorescence signal was detected in the non-inoculated groups. Therefore, GFP-labeled B. axarquiensis TUBP1 exhibited colonization in different parts of cotton plants from the rhizosphere soil.