Using GUS expression in a nonpathogenic Fusarium oxysporum strain to measure fungal biomass

Nonpathogenic Fusarium oxysporum strain SA70, a biological control agent ag ainst tomato fusarium wilt, was genetically transformed with both hygromyci n B resistance (Nph) and beta-glucuronidase (GusA) genes. Transformant 70T0 1 was selected for further testing based on its stable expression of beta-g lucuronidase (GUS) activity, resistance to hygromycin B (HmB), and retentio n of wild-type characteristics, including growth rate, root colonization ab ility, and disease control efficacy. Single copies of each of the GusA and Hph genes were stably integrated into the 70T01 genomic DNA. Expression of GUS, as measured by fluorometry, was highly correlated with myeclial dry we ighs. Assayable GUS activity was highest in young mycelia and was detectabl e in as little as 1 ng of mycelia (dry weight). GUS activity was extracted from protoplasts generated from germinating spores and the activity in the extracts related. to the number of viable protoplasts (colony-forming proto plasts, CFPs). Fungal biomass in 70T01-infected. tomato plant; roots, as de termined by the GUS-CFP method, was compared with that determined by the co nventional method of plating macerated, infected plant material. The result s demonstrated that while similar changes in the relative levels of fungal biomass could be detected with both methods, GUS-CFP biomass estimates were typically 6- to 50-fold higher than those determined by the plating method . The GUS-CFP assay provides a rapid and sensitive technique For determinin g fungal biomass in natural ecological settings.