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

Abstract

Nonpathogenic Fusarium oxysporum strain SA70, a biological control agent against tomato fusarium wilt, was genetically transformed with both hygromycin B resistance (Hph) and p-glucuronidase (GusA) genes. Transformant 70T01 was selected for further testing based on its stable expression of (3-glucuronidase (GUS) activity, resistance to hygromycin B (HmB), and retention of wild-type characteristics, including growth rate, root colonization ability, 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 mycelial dry weight. Assayable GUS activity was highest in young mycelia and was detectable 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 protoplasts, CFPs). Fungal biomass in 70T01 -infected tomato plant roots, as determined by the GUS-CFP method, was compared with that determined by the conventional method of plating macerated, infected plant material. The results 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 determining fungal biomass in natural ecological settings.