Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 15, 2012
Publication Date: February 21, 2013
Citation: Maruthachalam, K., Klosterman, S.J., Anchieta, A.G., Mou, B., Subbarao, K.V. 2013. Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments. Phytopathology. 103:268-280. Interpretive Summary: Verticillium wilt is a plant disease caused by fungi in the genus Verticillium. The losses caused by the disease caused by Verticillium dahliae, the primary causal agent of Verticillium wilt, are of economic importance in the US and worldwide. The fungus can also be transmitted in plant seeds. It is well established that spinach seeds, obtained from Europe and the Pacific Northwest of the US, are commonly infected with V. dahliae. There is also concern that the seedborne fungus may be transmitted to other crops grown in rotation with spinach. In this study, a strain of V. dahliae was prepared that fluoresces due to the expression of green fluorescent protein and the localization of this fungal strain was examined by microscopy in spinach plants and the seeds from infected plants to gain insight on the colonization process and the efficacy of seed treatments. The results indicate that the fungus does not colonize the most centrally located perisperm tissue in the core of the spinach seeds obtained from the infected plants. However, the thick pericarp immediately external to the seed coat was heavily colonized with the fungus, as was the seed coat itself. One type of fungicide seed treatment was applied to eliminate the fungus, and the effect on the fungus within the seed was analyzed. The results indicated that some fungus remains viable in the inner seed parts. This information contributes to our understanding of how V. dahliae colonizes the spinach host, and will be particularly useful for developing effective seed treatments to reduce the levels of seedborne V. dahliae.
Technical Abstract: Verticillium wilt is caused by the soilborne fungus V. dahliae on spinach (Spinacia oleracea L.) but the disease is a serious problem only in seed production fields. Spinach crops are harvested well before symptom expression, and thus, Verticillium wilt is not a significant threat in fresh and processed spinach production. The pathogen is seedborne and transmission of V. dahliae through seed is a major concern because of the dispersal potential of the pathogen to areas where fresh and processing spinach crops are grown in rotation with susceptible crops. Reduction in seedborne inoculum minimizes pathogen spread, and therefore knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Spinach seedlings were inoculated with conidial suspension of a green fluorescent protein (GFP)-tagged strain of V. dahliae and colonization events were followed through seed production by confocal laser scanning microscopy. Between 24 to 96 hr post-inoculation (PI), conidia germinated and formed hyphal colonies on root tips and in root elongation zones. Hyphae colonized root cortical tissues both intra and intercellularly by two wk and eventually colonized taproot xylem with abundant mycelia and conidia that led to vascular discoloration coincident with foliar symptom expression by 8 wk PI. At 10 weeks PI, xylem of the upper stem, inflorescence and various spinach seed parts, including the pericarp, seed coat, cotyledons and radicle had been colonized by the pathogen but not the perisperm (the diploid maternal tissue). Maximum concentration of the fungus was in the seed coat, the outermost layer with the vasculature. Efficacy of a seed treatment was assessed on V. dahliae-infested commercial seed lots in a separate experiment. Seed treatments significantly reduced detectable levels of the pathogen, but did not eliminate it from the seed. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. This information will be particularly useful for administering effective seed treatments that in turn reduce the seedborne inoculum transmission to crop production fields.