Ecological studies of transformed Trichoderma harzianum strain 1295-22 in the rhizosphere and on the phylloplane of creeping bentgrass

Abstract

A β-glucuronidase (GUS) reporter gene and a hygromycin B (hygB) phosphotransferase gene were integrated separately into the Trichoderma harzianum strain 1295-22 genome, using biolistic transformation. The mycelial growth and biocontrol ability of the transformed strains did not differ from that of the original strain. The transformed Gus+-kanamycin-resistant (Gus+KanR) strains were used to monitor growth and interactions with Rhizoctonia solani on creeping bentgrass plants. The hygB-resistant (hygBR) strains were used to selectively recover strain 1295-22 from the rhizosphere soil and phylloplane of creeping bentgrass after spray applications. The population levels of two hygBR strains and the original strain were very similar for all treatments. All three strains persisted for the duration of the experiment (28 days) in both the rhizosphere soil and on leaves, although population levels declined somewhat over the course of the experiment in unautoclaved soils. In this study, the results demonstrated that hygBR strains remained dominant over time when assayed on Trichoderma-selective medium containing hygB. The hygBR strains were not displaced by strains that colonized untreated plants. Microscopic observation showed that the Gus+KanR strains colonized the rhizoplane, seed coat, and phylloplane of creeping bentgrass. These results supported our earlier observation that strain 1295-22 was rhizosphere and phyllo-plane competent. Interactions between T. harzianum and R. solani were readily observed in situ and changed over time. Two types of reactions were found in these experiments. In the first type, sections of hyphae of R. solani near the hyphae of T. harzianum appeared damaged, and the pathogen appeared necrotic when viewed with a microscope. The second type, observed less frequently than the first type, was typical of myco-parasitism. The findings in this study provide new insight into the interactions between R. solani and T. harzianum, providing a basis for future research.