Transformation of a non-defoliating strain of Verticillium dahliae with the green fluorescent protein, and its colonisation on Upland cotton

Verticillium wilt caused by the soilborne fungus Verticillium dahliae, is one of the most challenging and economically significant diseases of cotton in Australia and worldwide. Host resistance is regarded as the most effective control strategy, however the biological complexity of the pathogen and controversy regarding the mechanisms of resistance hinder plant breeding efforts. Previous studies have utilised GFP-tagged isolates of V. dahliae to investigate the host – pathogen interaction on cotton, providing insights into the host resistance response and little understood areas of the disease cycle. Here, we establish GFP-tagged isolate Vd71-3 as a tool for evaluating infection and colonization on cotton cultivars tolerant and susceptible to Verticillium wilt. Isolate Vd71-3 was obtained by transforming a GFP vector construct into highly virulent non-defoliating strain Vd71171 isolated from a diseased Upland cotton plant in NSW, Australia. Prior to study on cotton, pathogenicity of Vd71-3 was deemed consistent with that of the parent wildtype, indicating that GFP expression does not dramatically alter virulence. Confocal laser scanning microscopy observations confirmed existing descriptions of early infection on cotton, including germination of conidia by 24 hours post-inoculation, formation of an infection peg, intercellular colonisation of the root tips but not lateral root junctions, preferential colonisation of the xylem vessels, and acropetal movement of conidia in vessels. Extensive fungal occlusion of the vessels was also observed, not previously captured elsewhere on cotton. V. dahliae was recovered from six of the eight weed species that were inoculated with the transformed VCG 1A and 2A strains. The VCG 2A transformant was recovered more frequently from weeds than the VCG 1A transformant, suggesting that V. dahliae VCG 2A may have higher infectivity towards weed hosts in Australian cotton fields. V. dahliae was not recovered from seeds from cotton plants that were subject to direct stem inoculation, although vascular tissue adjacent to the site of inoculation was colonised. Further investigation is needed to understand whether V. dahliae VCG 1A and 2A strains are capable of infecting Australian cotton seed using alternative inoculation techniques.