Publisher Summary There has been a large upsurge in interest in biological disease control recently, reflecting increasing environmental concern over pesticide use. This chapter discusses the ecological considerations, which comprises of suppressive soils, organic amendments and cornposts, monoculture decline, physical and chemical practices, and role of fauna in natural biological disease control. There has been a gradual increase in the number of commercial biocontrol agents on the market for the control of soil–borne plant pathogens over the last few years and only one product, Dagger–G, a Pseudomonaspuorescens marketed by Ecogen, Inc, Lanhorne, PA, USA, for the control of Rhizoctonia and Pythium damping–off of cotton, has been withdrawn from sale because of poor shelf–life of the material. The chapter discusses that chemicals still dominate the disease control market and some consideration should be given to research areas likely to improve further the commercial uptake of biological disease control agents. Consequently, optimizing realistic screening procedures is a first step, which can be refined to target specific types of antagonists. Once specific antagonists have been identified, they can be developed further by consideration of inoculum production, formulation, storage and application, ideally linking the expertise of scientific researchers with industrial partners. Molecular biology has helped identify the modes of action of many biocontrol agents. A considerable effort is currently underway to improve biocontrol efficacy of microorganisms through the use of molecular biology.

Developments in the Biological Control of Soil-borne Plant Pathogens

Improving the efficacy of biocontrol agents against soilborne pathogens

This review addresses current environmental applications of naturally occur­ ring, nonrecombinant microorganisms and potential future genetic modifica­ tions of such organisms, as well as releases of recombinant microorganisms that have occurred to date. Awareness of the current uses of nonrecombinant microorganisms provides insight into the diversity of habitats in which recombinant microorganisms may be released in the future, while an exami­ nation of pot,ential and realized genetic modifications provides insight into the variety of applications for which recombinant microorganisms may be used.

Release of Recombinant Microorganisms

Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), beta-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.

Cotransformation of Trichoderma harzianum with β-Glucuronidase and Green Fluorescent Protein Genes Provides a Useful Tool for Monitoring Fungal Growth and Activity in Natural Soils

(1987). Microbial Inoculants as Crop-Yield Enhancers. Critical Reviews in Biotechnology: Vol. 6, No. 1, pp. 61-85.

M. N. Shatla

Papers

Induction of new isolates of Trichoderma harzianum tolerant to fungicides and their experimental use for control of white rot of onion.