Disease Control and Plant Growth Promotion of \u3cem\u3e Miscanthus \u3c/em\u3e × \u3cem\u3e giganteus \u3c/em\u3e with \u3cem\u3e Trichoderma \u3c/em\u3e Bio-Inoculants

The second-generation bioenergy crop Miscanthus (Miscanthus × giganteus) is being assessed in New Zealand for its potential to provide shelter on irrigated dairy farms. Miscanthus is a perennial sterile hybrid vegetatively propagated rhizomatous C4 grass and the young rhizomes and roots are prone to infection by soil-borne fungal pathogens (Glynn et al., 2015) which can cause deleterious effects on plant establishment and growth. In Europe, several species of Fusarium such as F. avenaceum, F. culmorum, F. moniliforme and F. oxysporum have been implicated as causal agents of root and rhizome rot (Thinggaard, 1997; Covarelli et al., 2012) leading to poor field establishment of in-vitro propagated Miscanthus plants. When tested for their ability to cause disease of Miscanthus, Rhizoctonia solani (Kuhn) was reported as the most aggressive species among nineteen fungal pathogens of cereal crops (Glynn et al. 2015). In New Zealand, R. solani reduces seedling emergence and plant establishment of several herbage species and the problem may be alleviated through biocontrol using Trichoderma fungi (Kandula et al., 2015). In a glass-house study, the effect of four T. atroviride isolates on growth of tissue culture propagated Miscanthus plants in a soil naturally infested with R. solani was investigated

The NADPH Oxidases Nox1 and Nox2 Differentially Regulate Volatile Organic Compounds, Fungistatic Activity, Plant Growth Promotion and Nutrient Assimilation in Trichoderma atroviride

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity

The NADPH oxidases Nox1 and Nox2 differentially regulate volatile organic compounds, fungitastic activity, plant growth promotion and nutrient assimilation in Trichoderma atroviride

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity

Development and validation of a species-specific marker for Trichoderma atroviride

Biocontrol agents can use a range of strategies to control pathogens, such as plant growth promotion, induced resistance, mycoparasitism, antibiosis and competition. The mechanisms of biocontrol used by any particular agent vary depending on the conditions they experience. In recent years, it has become more apparent how complex these mechanisms are in respect to a combination of abiotic and biotic factors such as soil type, pH, temperature, host plant and pathogen. In order to study biocontrol activity by Trichoderma atroviride in a natural environment, it is important to distinguish it from other Trichoderma species present and to track and quantify the strains in the environment. Here we present the development and validation of a species-specific molecular marker for T. atroviride. The Trichoderma endochitinase-42 encoding gene chit42 is a single-copy gene and was selected as it has only low sequence homology to chitinases in other fungi. We looked for sequence variations in chit42 that distinguished T. atroviride from other Trichoderma species. Genomic sequences of chit42 from 66 Trichoderma isolates, representing 9 species, were compared to identify T. atroviride-specific polymorphisms. Based on these sequence polymorphisms, primers were designed that only amplify T. atroviride. A species-specific PCR amplification of chit42 using these primers was optimised to find the most stringent PCR conditions. Using these primers in real-time PCR, standard curves were generated to quantify the amount of T. atroviride chit42 DNA in natural non-sterilised soil inoculated with spores or mycelial tissue. This species-specific marker will allow us to study T. atroviride’s mode of action in more detail in pot and field trials and natural ecosystems