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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Trichoderma species isolated from grapevine with tolerance towards common copper fungicides used in viticulture for plant protection

Abstract BACKGROUND Copper‐containing fungicides are applied broadly in organic viticulture against downy mildew caused by Plasmopara viticola . Although long‐term application of copper‐based fungicides is associated with ecotoxic effects on the environment, their use in viticulture is required until sustainable alternatives are available. Trichoderma spp. might be a promising approach to fungicide reduction while promoting plant growth and development and displaying biocontrol activity. This study aims to examine the tolerance and compatibility of Trichoderma spp. to copper fungicides. This work contributes to the development of a spray application consisting of a copper‐tolerant Trichoderma sp. combined with a downscaled copper fungicide rate against P. viticola . RESULTS Trichoderma spp. isolated from grapevine wood in vineyards were identified and used for tolerance screening in various concentrations of copper fungicides. Copper hydroxide was identified as being highly compatible with Trichoderma . Two Trichoderma candidates, T. koningiopsis and T. harzianum , showed high copper tolerance in mycelial growth and germination tests, and were adapted to 2.85 g Cu L −1 of the selected fungicide. Microscopic investigations showed the attachment of copper compounds to fungal cell walls and copper uptake within the cytoplasm. In the case of high tolerance, large‐scale copper uptake was prevented. CONCLUSION Our findings identified two highly copper‐tolerant Trichoderma isolates with natural adaptation to the vineyard ecosystem, which could be further tested as biostimulants and biocontrol agents, combined with a reduced fungicide rate for sustainable plant protection. © 2022 Society of Chemical Industry.

Küpper, Verena↗
DOE OSTI2024

Potential biological control agents of Geosmithia morbida restrict fungal pathogen growth via mycoparasitism and antibiosis

Abstract Thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut) spp. (Fagales: Juglandaceae) is caused by the fungal pathogen Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat (Hypocreales: Bionectriaceae) and bark beetle pest/vector, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae). To further the development of biological management strategies for thousand cankers disease, we assessed the ability of 14 endophytic Trichoderma (Hypocreales: Hypocreaceae) isolates and the commercially available isolate T. afroharzianum strain KRL-AG2 to inhibit the in vitro growth of three different G. morbida isolates via mycoparasitism and antibiosis. To identify factors that may affect field success of candidate biological control agents, we quantified the growth responses of Trichoderma spp. and the commercially available entomopathogenic fungus, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) strain GHA, to the plant secondary metabolite and antimicrobial compound, juglone in vitro. A total of 12 Trichoderma isolates (from six different Trichoderma species) demonstrated antagonistic activity towards G. morbida in dual-plate assays. Juglone consistently reduced the growth of B. bassiana strain GHA and 14 of the 15 screened Trichoderma isolates in vitro. Additionally, one metabolite-producing Trichoderma isolate, TN4-47, completely inhibited the growth of all three G. morbida isolates across all tested metabolite concentrations and had comparatively greater tolerance to juglone compared to other Trichoderma isolates. Future lines of research should focus on characterizing the active antagonistic compound present in the metabolite filtrates, determine the mode of action of the active component(s), and elucidate how abiotic and biotic factors may influence the growth, persistence, and antagonistic activity of candidate biological control agents in planta .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Argonaute and Dicer are essential for communication between Trichoderma atroviride and fungal hosts during mycoparasitism

ABSTRACT Trichoderma species are known for their mycoparasitic activity against phytopathogenic fungi that cause significant economic losses in agriculture. During mycoparasitism, Trichoderma spp. recognize molecules produced by the host fungus and release secondary metabolites and hydrolytic enzymes to kill and degrade the host’s cell wall. Here, we explored the participation of the Trichoderma atroviride RNAi machinery in the interaction with six phytopathogenic fungi of economic importance. We determined that both Argonaute-3 and Dicer-2 play an essential role during mycoparasitism. Using an RNA-Seq approach, we identified that perception, detox, and cell wall degradation depend on the T. atroviride -RNAi when interacting with Alternaria alternata, Rhizoctonia solani AG2 , and R. solani AG5. Furthermore, we constructed a gene co-expression network that provides evidence of two gene modules regulated by RNAi, which play crucial roles in essential processes during mycoparasitism. In addition, based on small RNA-seq, we conclude that siRNAs regulate amino acid and carbon metabolism and communication during the Trichoderma -host interaction. Interestingly, our data suggest that siRNAs might regulate allorecognition ( het ) and transport genes in a cross-species manner. Thus, these results reveal a fine-tuned regulation in T. atroviride dependent on siRNAs that is essential during the biocontrol of phytopathogenic fungi, showing a greater complexity of this process than previously established. IMPORTANCE There is an increasing need for plant disease control without chemical pesticides to avoid environmental pollution and resistance, and the health risks associated with the application of pesticides are increasing. Employing Trichoderma species in agriculture to control fungal diseases is an alternative plant protection strategy that overcomes these issues without utilizing chemical fungicides. Therefore, understanding the biocontrol mechanisms used by Trichoderma species to antagonize other fungi is critical. Although there has been extensive research about the mechanisms involved in the mycoparasitic capability of Trichoderma species, there are still unsolved questions related to how Trichoderma regulates recognition, attack, and defense mechanisms during interaction with a fungal host. In this work, we report that the Argonaute and Dicer components of the RNAi machinery and the small RNAs they process are essential for gene regulation during mycoparasitism by Trichoderma atroviride .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T . atroviride exudates. In contrast, P . simiae and P . putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δ tmk 3) gene of T . atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T . atroviride , bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δ tmk3 mutant. Unexpectedly, exudates from wild-type T . atroviride and the Δ tmk 3 mutant rescued purine auxotrophic mutants of H . seropedicae , K . michiganensis and P . simiae . Metabolomic analysis on exudates from wild-type T . atroviride and the Δ tmk 3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma -metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Trichoderma harzianum transcriptome in response to the nematode Pratylenchus brachyurus

The root-lesion nematode Pratylenchus brachyurus causes extensive damage in several crops of economic importance. Fungi of Trichoderma genus have been highlighted as biopesticide agents in the control of several plant diseases. Although it is already widely used in agriculture, there are few studies, especially at the molecular level, that evaluate T. harzianum in the control of P. brachyurus . The aim of the present study was to investigate how interaction with the nematode P. brachyurus influences gene expression of T. harzianum , by using RNA-Seq analysis. Of the 13,932 predicted genes in the T. harzianum genome, 2,922 (21%) were differentially expressed in the presence of P. brachyurus , in relation to the absence of the nematode. Among the differentially expressed genes, we found genes encoding Carbohydrate Active EnZymes (CAZy), MEROPS peptidases, and proteins related to secondary metabolite synthesis. 118 pathways were identified as related to the biosynthesis of secondary metabolites. Additionally, the analysis identified 136 metabolic pathways related to these differentially expressed genes, among which we highlight: aminobenzoate degradation, xenobiotic metabolism by cytochrome P450, and sesquiterpenoid and triterpenoid biosynthesis. Our results contribute to a better understanding of the response of T. harzianum to the nematode P. bracyurus , the potential for biocontrol by this fungus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Rhizosphere Microbiome Diversity Potentially Supports Robust Nature of Field Pennycress ( Thlaspi arvense L.) in Dryland Cropping Systems of Eastern Washington

ABSTRACT Field pennycress ( Thlaspi arvense L.) is an annual in the Brassicaceae family and is currently being developed as an oilseed intermediate crop suitable for renewable biodiesel and jet fuel. It displays many desirable characteristics for this role including cold tolerance, a rapid life cycle, and a seed fatty acid profile conducive to bioenergy generation. These traits make field pennycress favorable for winter oilseed cultivation in the inland Pacific Northwest (iPNW). Simultaneously, intermediate crops are an increasingly recognized component of both agronomic sustainability and soil health management. Intermediate crops enhance soil microbial diversity, which benefits both soil and plant health. To understand the impact of field pennycress on soil microbial diversity, two natural accessions and seven experimental accessions were grown at three sites in Eastern Washington. Aboveground biomass and rhizosphere soil were then collected. Soil genomic DNA was extracted from rhizosphere samples and used to generate an amplicon library for bacterial (16S) and fungal (ITS) rRNA sequences. The resulting libraries were analyzed in QIIME2, which revealed that not only did the fad2 deficient line from the Spring32‐10 background have significantly increased aboveground biomass production compared to other pennycress genotypes, but also displayed significantly higher β‐diversity in the rhizosphere community specifically at the site experiencing the driest conditions. ANCOM analysis showed that multiple sequences similar to beneficial plant and soil health enhancing organisms such as Trichoderma spirale , Pseudomonas spp., and Methylobacterium goesingense were found to be enriched in the microbiome of the fad2 Spring32‐10 background also at that site. To add additional context to rhizosphere community data, root exudates from two pennycress genotypes were captured in magenta boxes and analyzed using HPLC. Future work will expand our understanding of the mechanisms by which field pennycress creates diversity in the rhizosphere, thus expanding our ability to cultivate this crop in the iPNW.

54 ENVIRONMENTAL SCIENCES↗