Rhizosphere Engineering of Biocontrol Agents Enriches Soil Microbial Diversity and Effectively Controls Root-Knot Nematodes.

The root-knot nematode (RKN) causes significant yield loss in tomatoes. Understanding the interaction of biocontrol agents (BCAs)-nematicides-soil microbiomes and RKNs is essential for enhancing the efficacy of biocontrol agents and nematicides to curb RKN damage to crops. The present study aimed to evaluate the in vitro effectiveness of BACa and nematicide against RKN and to apply the amplicon sequencing to assess the interaction of Bacillus velezensis (VB7) and Trichoderma koningiopsis (TK) against RKNs. Metagenomic analysis revealed the relative abundance of three phyla such as Proteobacteria (42.16%), Firmicutes (19.57%), and Actinobacteria (17.69%) in tomato rhizospheres. Those tomato rhizospheres treated with the combined application of B. velezensis VB7 + T. koningiopsis TK and RKN had a greater frequency of diversity and richness than the control. RKN-infested tomato rhizosphere drenched with bacterial and fungal antagonists had the maximum diversity index of bacterial communities. A strong correlation with a maximum number of interconnection edges in the phyla Proteobacteria, Firmicutes, and Actinobacteria was evident in soils treated with both B. velezensis VB7 and T. koningiopsis TK challenged against RKN in infected soil. The present study determined a much greater diversity of bacterial taxa observed in tomato rhizosphere soils treated with B. velezensis VB7 and T. koningiopsis TK than in untreated soil. It is suggested that the increased diversity and abundance of bacterial communities might be responsible for increased nematicidal properties in tomato plants. Hence, the combined applications of B. velezensis VB7 and T. koningiopsis TK can enhance the nematicidal action to curb RKN infecting tomatoes.

Synergistic Effect of Two Peptaibols from Biocontrol Fungus Trichoderma longibrachiatum Strain 40418 on CO-Induced Plant Resistance.

Trichoderma longibrachiatum is a filamentous fungus used as a biological control agent against different plant diseases. The multifunctional secondary metabolites synthesized by Trichoderma, called peptaibols, have emerged as key elicitors in plant innate immunity. This study obtained a high-quality genome sequence for the T. longibrachiatum strain 40418 and identified two peptaibol biosynthetic gene clusters using knockout techniques. The two gene cluster products were confirmed as trilongin AIV a (11-residue) and trilongin BI (20-residue) using liquid chromatography coupled with tandem mass spectrometry. Further investigations revealed that these peptaibols induce plant resistance to Pseudomonas syringae pv tomato (Pst) DC3000 infection while triggering plant immunity and cell death. Notably, the two peptaibols exhibit synergistic effects in plant-microbe signaling interactions, with trilongin BI having a predominant role. Moreover, the induction of tomato resistance against Meloidogyne incognita showed similarly promising results.

Cobalt stress enhanced dendrobine-type total alkaloids biosynthesis of Trichoderma longibrachiatum UN32 through reactive oxygen species formation.

Trichoderma longibrachiatum UN32 is a well-documented mutant strain known to produce dendrobine-type total alkaloids (DTTAs). It was serendipitously observed that the addition of Co2+ to the medium resulted in a notable enhancement in DTTAs production in the T. longibrachiatum UN32 strain, accompanied by an upregulating effect on the expression of antioxidase-related genes. Hence, the objective of the present work was to ascertain whether ROS (intracellular levels of hydrogen peroxide) induced by Co2+ treatment has a beneficial or detrimental impact on DTTAs biosynthesis. A comparison of the intracellular levels of hydrogen peroxide (H2O2) and DTTAs treated with CoCl2 and CH3COOH revealed that CoCl2 was the optimal inducer for investigating the relationship between ROS formation and DTTAs production. This was due to the observation that ROS formation was reduced by approximately 4% and DTTAs production was increased by 12.55% in comparison to the CH3COOH treatment. The physiological results revealed that the introduction of Co2+ resulted in the oxidative damage and activation of the expression of intracellular superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Furthermore, it was confirmed that ROS induced by Co2+ was beneficial to DTTAs production by adding exogenous ROS scavengers. The inclusion of all ROS scavengers, including vitamin C, tocopherol, melatonin, mannitol, and sesamol, resulted in a reduction in ROS accumulation and a concomitant decrease in DTTAs production. Specifically, the addition of melatonin at a concentration of 0.4 mg/L demonstrated significant effects, resulting in a 32.53% (P < 0.01) decrease in ROS accumulation and a 45.22% (P < 0.01) reduction in DTTAs production. Subsequently, the timelines of accumulation of intracellular H2O2 and DTTAs content indicated that ROS are also crucial for normal fermentation without CoCl2 addition. Specifically, the proper H2O2 dose for DTTAs accumulation is between 8.82 and 18.86 µmol/g. The present study offers the initial experimental evidence indicating that CoCl2 enhance DTTAs production during the culture of T. longibrachiatum UN32 via leading an increase in intracellular ROS, which is conductive to DTTAs production and can be inhibited by the ROS scavengers. Our results provide insights into the mechanistic study of DTTAs biosynthesis.

Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides.

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Expression of Trichoderma spp. endochitinase gene improves red rot disease resistance in transgenic sugarcane.

Sugarcane (Saccharum spp.)is an economically useful crop grown globally for sugar, ethanol and biofuel production. The crop is vulnerable to fungus Colletotrichum falcatum known to cause red rot disease. The pathogen hydrolyses stalk parenchyma cells where sucrose is accumulated resulting in upto 75% losses in sugar recovery. In this study, transgenic sugarcane having resistance against red rot was developed by introducing Trichoderma spp. endochitinase following Agrobacterium mediated transformation. The transgene introduction and expression in genetically modified plants were verified through qRT-PCR revealing upto 6-fold enhancement in endochitinase expression than non-transgenic plants. Hyperspectral Imaging of transgenic plants displayed altered leaf reflectance spectra and vegetative indices that were positively correlated with ransgene expression. The bioassay with virulent pathotypes of C. falcatumCF08 and CF13 known for epiphytotic occurrence resulted in identification of resistant plant Chit 3-13.The plants with higher reflectance also displayed improved disease resistance, implying their early classification into resistant/susceptible. The losses in sucrose content were minimized (up to 4-fold) in inoculated resistant plant Chit 3-13 as compared to susceptible non-transgenic plant, and a fewer pathogen hyphae were detected in vascular cells of the former through optical microscopy. The electron micrographs confirmed sucrose-filled stalk parenchyma cells in Chit 3-13; in contrast, cells of non-transgenic inoculated plant were depleted of sucrose. The active sites involved in cleaving 1-4 ß-glycoside bonds of N-acetyl-d-glucosaminein the pathogen hyphal walls were detected through endochitinase protein structural modelling. The transgenic sugarcane is an important source for in trogressingred rot resistance in plant breeding programs.

Antifungal mechanism of nanosilver biosynthesized with Trichoderma longibrachiatum and its potential to control muskmelon Fusarium wilt.

Fusarium oxysporum (Schl.) f.sp. melonis, which causes muskmelon wilt disease, is a destructive filamentous fungal pathogen, attracting more attention to the search for effective fungicides against this pathogen. In particular, Silver nanoparticles (AgNPs) have strong antimicrobial properties and they are not easy to develop drug resistance, which provides new ideas for the prevention and control of muskmelon Fusarium wilt (MFW). This paper studied the effects of AgNPs on the growth and development of muskmelon, the control efficacy on Fusarium wilt of muskmelon and the antifungal mechanism of AgNPs to F. oxysporum. The results showed that AgNPs could inhibit the growth of F. oxysporum on the PDA and in the PDB medium at 100-200 mg/L and the low concentration of 25 mg/L AgNPs could promote the seed germination and growth of muskmelon seedlings and reduce the incidence of muskmelon Fusarium wilt. Further studies on the antifungal mechanism showed that AgNPs could impair the development, damage cell structure, and interrupt cellular metabolism pathways of this fungus. TEM observation revealed that AgNPs treatment led to damage to the cell wall and membrane and accumulation of vacuoles and vessels, causing the leakage of intracellular contents. AgNPs treatment significantly hampered the growth of mycelia in the PDB medium, even causing a decrease in biomass. Biochemical properties showed that AgNPs treatment stimulated the generation of reactive oxygen species (ROS) in 6 h, subsequently producing malondialdehyde (MDA) and increasing protective enzyme activity. After 6 h, the protective enzyme activity decreased. These results indicated that AgNPs destroy the cell structure and affect the metabolisms, eventually leading to the death of fungus.

Genus-wide analysis of Trichoderma antagonism toward Pythium and Globisporangium plant pathogens and the contribution of cellulases to the antagonism.

Parasitism is an important lifestyle in the Trichoderma genus but has not been studied in a genus-wide way toward Pythium and Globisporangium hosts. Our approach screened a genus-wide set of 30 Trichoderma species in dual culture assays with two soil-borne Pythium and three Globisporangium plant-parasitic species and used exo-proteomic analyses, with the aim to correlate Trichoderma antagonism with potential strategies for attacking Pythium and Globisporangium. The Trichoderma spp. showed a wide range of antagonism from strong to weak, but the same Trichoderma strain showed similar levels toward all the Pythium and Globisporangium species. The Trichoderma enzymes from strong (Trichoderma asperellum, Trichoderma atroviride, and Trichoderma virens), moderate (Trichoderma cf. guizhouense and Trichoderma reesei), and weak (Trichoderma parepimyces) antagonists were induced by the autoclaved mycelia of one of the screened Pythium species, Pythium myriotylum. The variable proportions of putative cellulases, proteases, and redox enzymes suggested diverse as well as shared strategies amongst the antagonists. There was a partial positive correlation between antagonism from microscopy and the cellulase activity induced by autoclaved P. myriotylum mycelia in different Trichoderma species. The deletion of the cellulase transcriptional activator XYR1 in T. reesei led to lower antagonism toward Pythium and Globisporangium. The antagonism of Pythium and Globisporangium appears to be a generic property of Trichoderma as most of the Trichoderma species were at least moderately antagonistic. While a role for cellulases in the antagonism was uncovered, cellulases did not appear to make a major contribution to T. reesei antagonism, and other factors are also likely contributing.IMPORTANCETrichoderma is an important genus widely distributed in nature with broad ecological impacts and applications in the biocontrol of plant diseases. The Pythium and Globisporangium genera of fungus-like water molds include many important soil-borne plant pathogens that cause various diseases. Most of the Trichoderma species showed at least a moderate ability to compete with or antagonize the Pythium and Globisporangium hosts, and microscopy showed examples of parasitism (a slow type of killing) and predation (a fast type of killing). Hydrolytic enzymes such as cellulases and proteases produced by Trichoderma likely contribute to the antagonism. A mutant deficient in cellulase activity had reduced antagonism. Interestingly, Pythium and Globisporangium species contain cellulose in their cell walls (unlike true fungi such as Trichoderma), and the cellulolytic ability of Trichoderma appears beneficial for antagonism of water molds.

How carbohydrate-binding module affects the catalytic properties of endoglucanase.

The important role of Carbohydrate-binding module (CBM) in the cellulases catalytic activity has been widely studied. CBM3 showed highest affinity for cellulose substrate with 84.69 % adsorption rate among CBM1, CBM2, CBM3, and CBM4 in this study. How CBM affect the catalytic properties of GH5 endoglucanase III from Trichoderma viride (TvEG3) was systematically explored from two perspectives: the deletion of its own CBM(TvEG3dc) and the replacement of high substrate affinity CBM3 (TvEG3dcCBM3). Compared with TvEG3, TvEG3dc lost its binding ability on Avicel and filter paper, but its catalytic activity did not change significantly. The binding ability and catalytic activity of TvEG3dcCBM3 to Avicel increased 348.3 % and 372.51 % than that of TvEG3, respectively. The binding ability and catalytic activity of TvEG3dcCBM3 to filter paper decreased 51.7 % and 33.33 % than that of TvEG3, respectively. Further structural analysis of TvEG3, TvEG3dc, and TvEG3dcCBM3 revealed no changes in the positions and secondary structures of the key amino acids. These results demonstrated that its own CBM1 of TvEG3 did not affect its catalytic activity center, so it had no effect on its catalytic activity. But CBM3 changed the adsorption affinity for different substrates, which resulted in a change in the catalytic activity of the substrate.

The effects of EDTA and Trichoderma species on growth and Cu uptake of maize (Zea mays) plants grown in a Cu-contaminated soil.

Metal contamination in soil poses a significant environmental concern worldwide, necessitating effective remediation strategies such as phytoremediation. The present study investigated the effects of EDTA dosage (1.5 and 3 mmol kg-1) and two Trichoderma species (T. harzianum and T. aureoviride) on copper (Cu) content and growth of maize plants grown in a Cu-contaminated soil, as well as Cu fractionation in the soil. In the absence of EDTA, only inoculation with T. harzianum led to a significant increase in shoot biomass. Combining fungal inoculum with EDTA only yielded a significant increase in shoot biomass when using T. aureoviride at a low EDTA rate, highlighting the interplay between fungal species and EDTA rates on plant growth. Results also indicated that EDTA application increased Cu bioavailability, enhancing Cu dissolution and root (not shoot) Cu concentrations. Conversely, inoculation with both Trichoderma species reduced Cu mobility and bioavailability in soil, thereby decreasing the shoot Cu concentrations of plants. When combined with EDTA, only application of T. harzianum resulted in an enhanced shoot Cu concentration, whereas combined application of T. aureoviride and EDTA did not make a significant change compared to the corresponding control (no fungal inoculation, no EDTA), possibly due to a lower compatibility of the T. aureoviride isolate with EDTA. Our results demonstrated that EDTA application, in both non-inoculated and inoculated treatments, increased Cu availability by facilitating its redistribution and transformation from less plant-available fractions (residual, Fe/Mn oxide-bound, and carbonate-bound) to the more readily plant-available forms (water-soluble and exchangeable fractions). In conclusion, although individual Trichoderma application proved beneficial for phytostabilization by reducing Cu content and mitigating Cu toxicity in plants, the combined application of EDTA and a compatible Trichoderma isolate (here, the T. harzianum isolate) holds promise for enhancing the phytoextraction capacity of plants. Although using maize has the advantage of being a food crop, to optimize phytoextraction, plant species with superior metal tolerance and phytoextraction capabilities should be selected, exceeding those of maize.

Trichoderma based formulations control the wilt disease of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris, better when inoculated as consortia: findings from pot experiments under field conditions.

Background: Commercial/chemical pesticides are available to control Fusarium wilt of chickpea, but these antifungals have numerous environmental and human health hazards. Amongst various organic alternatives, use of antagonistic fungi like Trichoderma, is the most promising option. Although, Trichoderma spp. are known to control Fusarium wilt in chickpea but there are no reports that indicate the biocontrol efficacy of indigenous Trichoderma spp. against the local pathogen, in relation to environmental conditions. Methods: In the present study, biological control activity of Trichoderma species formulations viz., Trichoderma asperellum, Trichoderma harzianum (strain 1), and Trichoderma harzianum (strain 2), either singly or in the form of consortia, was investigated against Fusarium oxysporum f. sp. ciceris, the cause of Fusarium wilt in chickpea, in multiyear pot trials under open field conditions. The antagonistic effect of Trichoderma spp. was first evaluated in in vitro dual culture experiments. Then the effects of Trichoderma as well as F. oxysporum, were investigated on the morphological parameters, disease incidence (DI), and disease severity (DS) of chickpea plants grown in pots. Results: In dual culture experiments, all the Trichoderma species effectively reduced the mycelial growth of F. oxysporum. T. asperellum, T. harzianum (strain 1), and T. harzianum(strain 2) declined the mycelial growth of F. oxysporumby 37.6%, 40%, and 42%. In open field pot trials, the infestation of F. oxysporum in chickpea plants significantly reduced the morphological growth of chickpea. However, the application of T. asperellum, T. harzianum (strain 1), and T. harzianum (strain 2), either singly or in the form of consortia, significantly overcome the deleterious effects of the pathogen, thereby resulted in lower DI (22.2% and 11.1%) and DS (86% and 92%), and ultimately improved the shoot length, shoot fresh weight and shoot dry weight by 69% and 72%, 67% and 73%, 68% and 75%, during the years 1 and 2, respectively, in comparison with infested control. The present study concludes the usefulness and efficacy of Trichoderma species in controlling wilt disease of chickpea plants under variable weather conditions.

Cytotoxic Peptaibols from Trichoderma guizhouense, a Fungus Isolated from an Urban Soil Sample.

Soil sustains human life by nourishing crops, storing food sources, and housing microbes, which may affect the nutrition and biosynthesis of secondary metabolites, some of which are used as drugs. To identify lead compounds for a new class of drugs, we collected soil-derived fungal strains from various environments, including urban areas. As various human pathogens are assumed to influence the biosynthetic pathways of metabolites in soil fungi, leading to the production of novel scaffolds, we focused our work on densely populated urban areas and tourist attractions. A soil-derived fungal extract library was screened against MDA-MB-231 cells to derive their cytotoxic activity. Notably, 10 µg/mL of the extract of Trichoderma guizhouense (DS9-1) was found to exhibit an inhibitory effect of 71%. Fractionation, isolation, and structure elucidation efforts led to the identification of nine new peptaibols, trichoguizaibols A-I (1-9), comprising 14 amino acid residues (14-AA peptaibols), and three new peptaibols, trichoguizaibols J-L (10-12), comprising 18 amino acid residues (18-AA peptaibols). The chemical structures of 1-12 were determined based on their 1D and 2D NMR spectra, HRESIMS, electronic circular dichroism data, and results of the advanced Marfey's method. The 18-AA peptaibols were found to exhibit cytotoxicity against MDA-MB-231, SK-Hep1, SKOV3, DU145, and HCT116 cells greater than that of the 14-AA peptaibols. Among these compounds, 10-12 exhibited potent sub-micromolar IC50 values. These results are expected to shed light on a new direction for developing novel scaffolds as anticancer agents.

Trichoderma produces methyl jasmonate-rich metabolites in the presence of Fusarium, showing biostimulant activity and wilt resistance in tomatoes.

Bioactive secondary metabolites from fungi, including Trichoderma, are an excellent source of plant biostimulants. Although production of novel biostimulants from known microbes is critical, challenging them may produce novel bioactive compounds. With this hypothesis, the study used live Fusarium chlamydosporum (FOL7) culture as the inducer during T. harzianum (IF63) growth in broth. Plate assays and gas chromatography-mass spectrometry (GC-MS) analysis were used to characterise the metabolites. Microscopy, pot experiments and, biochemical estimations of the defence-related enzymes in tomato plants established the biostimulant activity of the induced Trichoderma metabolites. Fungal crude metabolites (FCM) obtained from IF63+FOL7 extracts (TF.ex) showed increased antimicrobial activity. TF.ex at 50 µg mL-1, inhibited the FOL7 growth by 68.33% compared to the Trichoderma alone extract. Scanning electron microscopy (SEM) revealed morphological disruption of FOL7 mycelia by TF.ex. GC-MS analysis of the extracts revealed the presence of approximately 64 compounds, of which at least 13 were detected explicitly in TF.ex. Methyl (3-oxo-2-pentylcyclopentyl) acetate (Methyl dihydrojasmonate), a lipid functionally related to jasmonic acid, was the major metabolite (∼21%) present in TF.ex. Tomato seed dressing with TF.ex promoted plant growth and induced systemic resistance against FOL7 compared to alone Trichoderma and Fusarium extracts. The TF.ex treatment increased the superoxide dismutase (33%) and catalase activity by 2.5-fold in tomato plants. The study concludes that fungal secondary metabolites may be modulated by providing appropriate challenges to produce effective metabolite-based biostimulants for agricultural applications.

Neg1 overexpression in Trichoderma harzianum T4 enhanced its ability to produce spores and antagonistic activity against phytopathogenic fungi.

Trichoderma harzianum T4 is a soil fungus that plays an important role in the biological control of plant diseases. The aim of this study was to functionally characterize the ß-1,6-glucanase gene Neg1 in T. harzianum T4 and to investigate the effect of its overexpression on biocontrol traits, especially antagonism against pathogenic fungi. We found that overexpression of Neg1 did not affect growth of T. harzianum but enhanced sporulation of T. harzianum T4 cultures. Generally, spores are closely related to the defense ability of defense fungi and can assist their proliferation and improve their colonization ability. Secondly, overexpression of Neg1 also increased the secretion level of various hydrolytic enzymes and enhanced the antagonistic ability against phytopathogenic fungi of Fusarium spp. The results suggest that Neg1 is a key gene for improving the biocontrol effect of T. harzianum T4, which contributes to a better understanding of the mechanism of action of T. harzianum T4 as a fungal biocontrol agent.

Three Ecological Models to Evaluate the Effectiveness of Trichoderma spp. for Suppressing Aflatoxigenic Aspergillus flavus and Aspergillus parasiticus.

Chemical pesticides help reduce crop loss during production and storage. However, the carbon footprints and ecological costs associated with this strategy are unsustainable. Here, we used three in vitro models to characterize how different Trichoderma species interact with two aflatoxin producers, Aspergillus flavus and Aspergillus parasiticus, to help develop a climate-resilient biological control strategy against aflatoxigenic Aspergillus species. The growth rate of Trichoderma species is a critical factor in suppressing aflatoxigenic strains via physical interactions. The dual plate assay suggests that Trichoderma mainly suppresses A. flavus via antibiosis, whereas the suppression of A. parasiticus occurs through mycoparasitism. Volatile organic compounds (VOCs) produced by Trichoderma inhibited the growth of A. parasiticus (34.6 ± 3.3%) and A. flavus (20.9 ± 1.6%). The VOCs released by T. asperellum BTU and T. harzianum OSK-34 were most effective in suppressing A. flavus growth. Metabolites secreted by T. asperellum OSK-38, T. asperellum BTU, T. virens OSK-13, and T. virens OSK-36 reduced the growth of both aflatoxigenic species. Overall, T. asperellum BTU was the most effective at suppressing the growth and aflatoxin B1 production of both species across all models. This work will guide efforts to screen for effective biological control agents to mitigate aflatoxin accumulation.

Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates.

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.

Whole genome sequencing and analysis of the weed pathogen Trichoderma polysporum HZ-31.

In order to resolve the key genes for weed control by Trichoderma polysporum at the genomic level, we extracted the genomic DNA and sequenced the whole genome of T. polysporum strain HZ-31 on the Illumina Hiseq platform. The raw data was cleaned up using Trimmomatic and checked for quality using FastQC. The sequencing data was assembled using SPAdes, and GeneMark was used to perform gene prediction on the assembly results. The results showed that the genome size of T. polysporum HZ-31 was 39,325,746 bp, with 48% GC content, and the number of genes encoded was 11,998. A total of 148 tRNAs and 45 rRNAs were predicted. A total of 782 genes were annotated in the Carbohydrase Database, 757 genes were annotated to the Pathogen-Host Interaction Database, and 67 gene clusters were identified. In addition, 1023 genes were predicted to be signal peptide proteins. The annotation and functional analysis of the whole genome sequence of T. polymorpha HZ-31 provide a basis for the in-depth study of the molecular mechanism of its herbicidal action and more effective utilization for weed control.

Metabolic picture of microbial interaction: chemical crosstalk during co-cultivation between three dominant genera of bacteria and fungi in medicinal plants rhizosphere.

INTRODUCTION: Microbial communities affect several aspects of the earth's ecosystem through their metabolic interaction. The dynamics of this interaction emerge from complex multilevel networks of crosstalk. Elucidation of this interaction could help us to maintain the balance for a sustainable future. OBJECTIVES: To investigate the chemical language among highly abundant microbial genera in the rhizospheres of medicinal plants based on the metabolomic analysis at the interaction level. METHODS: Coculturing experiments involving three microbial species: Aspergillus (A), Trichoderma (T), and Bacillus (B), representing fungi (A, T) and bacteria (B), respectively. These experiments encompassed various interaction levels, including dual cultures (AB, AT, TB) and triple cultures (ATB). Metabolic profiling by LC-QTOFMS revealed the effect of interaction level on the productivity and diversity of microbial specialized metabolites. RESULTS: The ATB interaction had the richest profile, while the bacterial profile in the monoculture condition had the lowest. Two native compounds of the Aspergillus genus, aspergillic acid and the dipeptide asperopiperazine B, exhibited decreased levels in the presence of the AT interaction and were undetectable in the presence of bacteria during the interaction. Trichodermarin N and Trichodermatide D isolated from Trichoderma species exclusively detected during coexistence with bacteria (TB and ATB). These findings indicate that the presence of Bacillus activates cryptic biosynthetic gene clusters in Trichoderma. The antibacterial activity of mixed culture extracts was stronger than that of the monoculture extracts. The TB extract exhibited strong antifungal activity compared to the monoculture extract and other mixed culture treatments. CONCLUSION: The elucidation of medicinal plant microbiome interaction chemistry and its effect on the environment will also be of great interest in the context of medicinal plant health Additionally, it sheds light on the content of bioactive constituents, and facilitating the discovery of novel antimicrobials.

Effectiveness of local isolates of Trichoderma spp. in imparting drought tolerance in rice, Oryza sativa.

Rice is a crop that requires high amount of water, and the drought is a major constraint in paddy cultivation. Water stress condition frequently prevails due to shortage of rain which results in significantly reduced plant growth and yield of rice. In the present study capability of Trichoderma spp. in imparting drought tolerance to rice, Oryza sativa was explored. Eleven local strains of Trichoderma spp. were applied to rice cv. Swarna Sub-1 through soil application (2 g/kg soil) and seed treatment (20 g/kg seed) under 0, 25, 50 and 75% less watering of the recommended amount. The soil application of T. harzianum AMUTHZ84 significantly promoted the shoot and root length (23.6 and 21.3%) followed by seed treatment (19.7 and 18.2%) under recommended level of irrigation condition (100% irrigation). Next in effectiveness was T. viride AMUTVR73 (21.5 and 18.1%) over untreated control. However, under 75% water availability, soil application with T. harzianum AMUTHZ82 was found superior over other isolates in enhancing shoot and root length (17.7 and 16.4%). The same isolate was also recorded to be superior under 50% (12.4 and 10.1%) and 25% water availability (9.3 and 8.1%) in enhancing the plant growth and biomass of rice cv. Swarna Sub-1. The isolate also significantly enhanced the leaf pigments, and photosynthesis in the rice plants grown under 25-75% water stress condition. In general, soil application of Trichoderma isolates was found more effective than seed treatment, and the T. harzianum AMUTHZ82 provided 8-17% enhancement in the plant growth, biomass, leaf pigments and photosynthesis of rice cv. Swarna Sub-1 grown under 25-75% water stress condition.

Cytotoxic Peptaibols from Trichoderma strigosum.

Five new lipopeptaibols (1-5) and eight new 19-residue peptaibols (8-15) along with two known lipopeptaibols, lipovelutibols C (6) and D (7) were isolated from Trichoderma strigosum. The planar structures of the newly discovered peptaibols (1-5, 8-15) were elucidated using 1D and 2D NMR, and UPLC-MS/MS data. The absolute configurations for new peptaibols (1-5, 8-15) were elucidated using the advanced Marfey's method and GITC (2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization. Through analysis of CD spectra, these peptabols were found to have right-handed helical conformations. While most of the new compounds were significantly more active than the positive control, 9, 10, 12, and 15 containing Ser and Leu at positions 10 and 11, respectively, were the most cytotoxic against MDA-MB-231, SNU449, SKOV3, DU145, and HCT116 cancer cell lines, and the 19-residue peptaibols were generally more potent than lipopeptaibols.

Combination of irradiated chitosan and microbial agent to reduce downy mildew on grapevine cv. Thompson seedless.

Globally sustainable disease management ensuring high quality in grapes is in demand as it holds significant importance as a versatile fruit for consumption, winemaking, and production of various products such as grape juice, raisin, and grape-seed oil. The present paper reports a combination of nano-biotechnology as a promising strategy for enhancing plant health and fruit productivity in grapes combining Irradiated chitosan nanoparticles and bio-control agents. The Irradiated Chitosan with Bacillus subtilis and Trichoderma viridae and pesticides were evaluated for disease management. Percent disease index, percent disease control, and percent yield enhancement in Cymoxanil 8% + Mamcozeb 64% WP @ 0.2% treatment were as 17. 24%, 67.97% and 33.91% in 150 ppm Irradiated chitosan+B. subtilis were 19.83, 63.16, 30.41 and in Trichoderma 150 ppm Irradiated chitosan were 24.58, 54.33, and 27.40, respectively as compared to untreated crop with disease severity 53.84% PDI. Thus, irradiated chitosan and Bacillus subtilis elucidated a synergistic combination for residue-free efficient phytosanitary measures, which harnessed the strength of chitosan and bio-control agents for sustainable grape productivity. These findings will also pave the way for a deeper understanding of the synergistic interaction between Irradiated nanochitosan and bio-control agents for an eco-friendly and economically viable disease management strategy. The minimum temperature and morning relative humidity (RH I) had positive significance, with correlation coefficients of 0.484 and 0.485, respectively. The evening relative humidity (RH II) had a positive highly significant positive correlation coefficient of 0.664. Chitosan merits as a multiple stress tolerance enhancing agent that will further help in mitigating climate change adaptations in grapevines reducing reliance on chemical agro-inputs.