The loss of organic material from the roots provides the energy for the development of active microbial populations in the rhizosphere around the root. Generally, saproptrophs or biotrophs such as mycorrhizal fungi grow in the rhizosphere in response to this carbon loss, but plant pathogens may also develop and infect a susceptible host, resulting in disease. This review examines the microbial interactions that can take place in the rhizosphere and that are involved in biological disease control. The interactions of bacteria used as biocontrol agents of bacterial and fungal plant pathogens, and fungi used as biocontrol agents of protozoan, bacterial and fungal plant pathogens are considered. Whenever possible, modes of action involved in each type of interaction are assessed with particular emphasis on antibiosis, competition, parasitism, and induced resistance. The significance of plant growth promotion and rhizosphere competence in biocontrol is also considered. Multiple microbial interactions involving bacteria and fungi in the rhizosphere are shown to provide enhanced biocontrol in many cases in comparison with biocontrol agents used singly. The extreme complexity of interactions that can occur in the rhizosphere is highlighted and some potential areas for future research in this area are discussed briefly.

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Microbial interactions and biocontrol in the rhizosphere

Fungal species belonging to the genus Trichoderma are worldwide in occurrence and easily isolated from soil, decaying wood, and other forms of plant organic matter. They are, for the most part, classified as imperfect fungi, in that they have no known sexual stage. Rapid growth rate in culture and the production of numerous spores (conidia) that are varying shades of green characterize fungi in this genus. The reverse side of colonies is often uncolored, buff, yellow, amber, or yellow-green, and many species produce prodigious quantities of thick-walled spores (chlamydospores) in submerged mycelium (8). The potential of Trichoderma species as biocontrol agents of plant diseases was first recognized in the early 1930s (31), and in subsequent years, control of many diseases has been added to the list (1,3,5,7,9,11,19, 23,29,34,37,40). This has culminated in the commercial production of several Trichoderma species for the protection and growth enhancement of a number of crops in the United States (24), and in the production of Trichoderma species and mixtures of species in India, Israel, New Zealand, and Sweden (D. R. Fravel, personal communication). One of the most interesting aspects of the science of biological control is the study of the mechanisms employed by biocontrol agents to effect disease control. Past research indicates that the mechanisms are many and varied, even within the genus Trichoderma. In order to make the most effective use of biocontrol agents for the control of plant diseases, we must understand how the agents work and what their limitations are. We can then develop effective means of culturing, storing, applying, and utilizing biocontrol agents so that we harness their best effort for disease control. The selected research papers cited in this article were chosen because they illustrate what has been learned about mechanisms involved in biocontrol with Trichoderma species.

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Mechanisms Employed by Trichoderma Species in the Biological Control of Plant Diseases: The History and Evolution of Current Concepts.

The rhizosphere is a hot spot of microbial interactions as exudates released by plant roots are a main food source for microorganisms and a driving force of their population density and activities. The rhizosphere harbors many organisms that have a neutral effect on the plant, but also attracts organisms that exert deleterious or beneficial effects on the plant. Microorganisms that adversely affect plant growth and health are the pathogenic fungi, oomycetes, bacteria and nematodes. Most of the soilborne pathogens are adapted to grow and survive in the bulk soil, but the rhizosphere is the playground and infection court where the pathogen establishes a parasitic relationship with the plant. The rhizosphere is also a battlefield where the complex rhizosphere community, both microflora and microfauna, interact with pathogens and influence the outcome of pathogen infection. A wide range of microorganisms are beneficial to the plant and include nitrogen-fixing bacteria, endo- and ectomycorrhizal fungi, and plant growth-promoting bacteria and fungi. This review focuses on the population dynamics and activity of soilborne pathogens and beneficial microorganisms. Specific attention is given to mechanisms involved in the tripartite interactions between beneficial microorganisms, pathogens and the plant. We also discuss how agricultural practices affect pathogen and antagonist populations and how these practices can be adopted to promote plant growth and health.

/pdf/the-rhizosphere-a-playground-and-battlefield-for-soilborne-1yufhjdsuc.pdf

The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms

Fungi in the genus Trichoderma have been known since at least the 1920s for their ability to act as biocontrol agents against plant pathogens. Until recently, the principal mechanisms for control have been assumed to be those primarily acting upon the pathogens and included mycoparasitism, antibiosis, and competition for resources and space. Recent advances demonstrate that the effects of Trichoderma on plants, including induced systemic or localized resistance, are also very important. These fungi colonize the root epidermis and outer cortical layers and release bioactive molecules that cause walling off of the Trichoderma thallus. At the same time, the transcriptome and the proteome of plants are substantially altered. As a consequence, in addition to induction of pathways for resistance in plants, increased plant growth and nutrient uptake occur. However, at least in maize, the increased growth response is genotype specific, and some maize inbreds respond negatively to some strains. Trichoderma spp. are beginning to be used in reasonably large quantities in plant agriculture, both for disease control and yield increases. The studies of mycoparasitism also have demonstrated that these fungi produce a rich mixture of antifungal enzymes, including chitinases and beta-1,3 glucanases. These enzymes are synergistic with each other, with other antifungal enzymes, and with other materials. The genes encoding the enzymes appear useful for producing transgenic plants resistant to diseases and the enzymes themselves are beneficial for biological control and other processes.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-96-0190

Overview of Mechanisms and Uses of Trichoderma spp.

Biological control involves the use of beneficial organisms, their genes, and/or products, such as metabolites, that reduce the negative effects of plant pathogens and promote positive responses by the plant. Disease suppression, as mediated by biocontrol agents, is the consequence of the interactions between the plant, pathogens, and the microbial community. Antagonists belonging to the genus Trichoderma are among the most commonly isolated soil fungi. Due to their ability to protect plants and contain pathogen populations under different soil conditions, these fungi have been widely studied and commercially marketed as biopesticides, biofertilizers and soil amendments. Trichoderma spp. also produce numerous biologically active compounds, including cell wall degrading enzymes, and secondary metabolites. Studies of the three-way relationship established with Trichoderma, the plant and the pathogen are aimed at unravelling the mechanisms involved in partner recognition and the cross-talk used to maintain the beneficial association between the fungal antagonist and the plant. Several strategies have been used to identify the molecular factors involved in this complex tripartite interaction including genomics, proteomics and, more recently, metabolomics, in order to enhance our understanding. This review presents recent advances and findings regarding the biocontrol-resulting events that take place during the Trichoderma –plant–pathogen interaction. We focus our attention on the biological aspects of this topic, highlighting the novel findings concerning the role of Trichoderma in disease suppression. A better understanding of these factors is expected to enhance not only the rapid identification of effective strains and their applications but also indicate the potentials for improvement of natural strains of Trichoderma .

Trichoderma–plant–pathogen interactions

Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi except T. harzianum strain P1. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungi. For most of the fungi tested, the ED50 values for the endochitinase and the chitobiosidase were 35-135 micrograms ml-1 and 62-180 micrograms ml-1, respectively. Complete inhibition occurred at 200-300 micrograms ml-1. Combining the two enzymes resulted in a synergistic increase of antifungal activity. The ED50 values for a 1:1 mixture of endochitinase and chitobiosidase were as low as 10 micrograms ml-1 for Botrytis cinerea, 34 micrograms ml-1 for Ustilago avenae, 13 micrograms ml-1 for Uncinula necator, and 30 micrograms ml-1 for Fusarium solani. T. harzianum strain P1 was resistant to its own chitinolytic enzymes up to 800 micrograms ml-1, with an ED50 value 1,000 micrograms ml-1. The chitinolytic enzymes from T. harzianum appeared to be biologically more active than enzymes from other sources and more effective against a wider range of fungi. The involvement of these chitinolytic enzymes in biocontrol is also discussed

Chitinolytic enzymes produced by trichoderma harzianum; antifungal activity of purified endochitinase and chitobiosidase

The culture filtrate from the biocontrol agent Gliocladium virens strain 41 grown in chitin-containing medium was strongly inhibitory to mycelial growth of different plant-pathogenic fungi. The antibiotic gliotoxin was isolated from the culture liquid. The culture filtrate also contained different types of chitinolytic enzyme activities, including endochitinase, chitin 1,4β-chitobiosidase, and glucan N-acetyl-β-D-glucosaminidase, as well as glucan 1,3-β-glucosidase activity. An endochitinase was purified to homogeneity []

Endochitinase from Gliocladium virens: isolation, characterization, and synergistic antifungal activity in combination with gliotoxin.

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Production of antifungal compounds by Chaetomium globosum and the role in suppression of Pythium damping-off of sugarbeet were evaluated. Two metabolites with antifungal activity against Pythium ultimum, 2-(buta-1,3-dienyl)-3-hydroxy-4-(penta-1,3-dienyl)-tetrahydrofuran (BHT) and the epidithiadiketopiperazine, chaetomin, were isolated from liquid cultures of C. globosum. BHT was produced in 1% malt extract medium by six of eight tested C. globosum strains, whereas chaetomin was produced in 1% corn steep medium by five of nine tested strains (...)

Role of antibiotics produced by Chaetomium globosum in biocontrol of Pythium ultimum, a causal agent of damping-off

Biocontrol strains from the genera Enterobacter and Pseudomonas and two chitinolytic enzymes from Trichoderma harzianum isolate P1 were combined and tested for antifungal activity in bioassays. Inhibitory effects on spore germination and germ tube elongation of Botrytis cinerea, Fusarium solani, and Uncinula necator were synergistically increased by mixing fungal enzymes and cells of Enterobacter cloacae but not of Pseudomonas spp. Culture filtrate of E. cloacae contained antifungal compounds and produced moderate levels of inhibition, either in plate assays or in bioassays conducted in potato-dextrose broth. However, the combination of bacterial culture filtrate with fungal chitinolytic enzymes generated only an additive response, indicating that the presence of bacterial cells was required for a synergistic effect. Chitinolytic enzyme activity in the presence of chitinous substrates enhanced the growth of E. cloacae and readily restored the ability of bacterial cells to bind to hyphae of the pathogens despite high concentrations of D-glucose or sucrose in the medium. The results of this study suggest that transgenic bacteria, capable of binding to fungal cell walls and expressing fungal genes encoding cell wall-degrading enzymes, may be powerful biocontrol agents

A. Di Pietro

Papers

Chitinolytic enzymes produced by trichoderma harzianum; antifungal activity of purified endochitinase and chitobiosidase

Endochitinase from Gliocladium virens: isolation, characterization, and synergistic antifungal activity in combination with gliotoxin.

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Role of antibiotics produced by Chaetomium globosum in biocontrol of Pythium ultimum, a causal agent of damping-off

Antifungal, synergistic interaction between chitinolytic enzymes from Trichoderma harzianum and Enterobacter cloacae