Molecular ecological methods to quantify biocontrol of Sclerotinia sclerotiorum by Trichoderma harzianum, and inhibitory effects of fungivorous nematodes on the biocontrol agent.

摘要

Fungi of the genus Trichoderma have received considerable attention as biocontrol agents against a wide range of plant pathogenic fungi. Sclerotinia sclerotiorum, a destructive plant pathogen with a wide host range, forms sclerotia that serve as over-wintering survival structures and the primary source of pathogen inoculum. Several Trichoderma spp. have shown the ability to mycoparasitize sclerotia and/or hyphae of S. sclerotiorum. Conventional methods to determine colonization of sclerotia by biocontrol fungi only indicate incidence of colonization, but cannot indicate the extent of colonization.{09}Real-time quantitative PCR assays with specific primer/Tagman-probe sets for the genus Trichoderma, T. harzianum ThzID1-M3, and S. sclerotiorum were developed. The assays sensitively and precisely quantified biocontrol activity of total Trichoderma spp. and the introduced fungal agent T. harzianum ThzID1-M3 against sclerotia, and measured DNA of S. sclerotiorum in sclerotia. The introduced fungus T. harzianum ThzID1-M3 significantly increased the extent and incidence of colonization of sclerotia by total Trichoderma spp. in the soil.{09}The extent of colonization of sclerotia by ThzID1-M3 was significantly correlated with hyphal density of the fungus in soil. The added ThzID1-M3 significantly stimulated population growth of fungus-feeding nematodes that fed on hyphae of the fungus in the soil. Predation by fungivorous nematodes decreased hyphal proliferation and establishment of ThzID1-M3, and reduced the extent of colonization of sclerotia. Fungivorous nematodes had little effect on ThzID1-M3 once it was inside sclerotia; thus, sclerotia provide spatial refuges for the biocontrol fungus from the feeding activity of nematodes. A mathematical model was developed to investigate the dynamics of interactions between fungivorous nematodes, the introduced biocontrol fungus, and S. sclerotiorum in soil. The model quantitatively predicted nematode population dynamics, biomass of ThzID1-M3, and extent of sclerotial colonization. Model predictions closely matched experimentally observed patterns. In conclusion, fungivorous nematodes may be a significant constraint on hyphal proliferation and biocontrol activity of fungal agents in natural soils.