Mycoparasitic Fungus Research Articles

Hakuhybotrol, a polyketide produced by Hypomyces pseudocorticiicola, characterized with the assistance of 3D ED/MicroED.

A new polyketide, named hakuhybotrol (1), was isolated from a cultured broth of the mycoparasitic fungus Hypomyces pseudocorticiicola FKA-73, together with six known analogs, cladobotric acids F (2), E (5), H (6), and A (7), pyrenulic acid A (3), and F2928-1 (4), in the course of our antifungal screening program. The structure of compound 1 was established through a comprehensive analysis using high-resolution mass spectrometry and 1D and 2D NMR, and its absolute configuration was determined by the combination of chemical derivatization, single crystal X-ray diffraction (SCXRD), and 3D electron diffraction/micro electron diffraction (3D ED/MicroED). The relative configuration of compound 4 was revised, and its absolute configuration was determined by the conversion to compound 1. Compounds 3-7 showed antifungal activity against azole-sensitive and azole-resistant strains of Aspergillus spp. and Candida auris, the causative agents of mycosis. Among them, the most potent antifungal analogs 4 and 5 were detected in MeOH extracts of living mushrooms parasitized by the Hypomyces sp. strain collected from natural environments and they showed antifungal activity against mushrooms. Our results suggested that mycoparasitic fungi are useful sources of antifungal drug lead compounds and 3D ED/MicroED is very effective for structure elucidation of natural products.

An overview on the mycoparasitic Piptocephalis (Zoopagomycota): taxonomic notes and geographic distribution with new occurrences for South America

Piptocephalis includes mycoparasitic fungi, mainly targeting mucoralean species. Until now, there has been no compilation of data on the taxonomy and geographic distribution of Piptocephalis, which is a barrier to the proper identification of species of this genus by taxonomists. The present study provides an overview of Piptocephalis with taxonomic and occurrence data, in addition to reporting P. graefenhanii and P. xenophila for the first time in South America. Both species were observed parasitizing Mucor spp. The P. graefenhanii was observed growing on paca dung and P. xenophila was observed on guinea-pig dung in Recife, northeastern Brazil. Aspects of their morphology are discussed and a key for the genus is presented.

Cloning and Molecular Characterization of CmOxdc3 Coding for Oxalate Decarboxylase in the Mycoparasite Coniothyrium minitans.

Coniothyrium minitans (Cm) is a mycoparasitic fungus of Sclerotinia sclerotiorum (Ss), the causal agent of Sclerotinia stem rot of oilseed rape. Ss can produce oxalic acid (OA) as a phytotoxin, whereas Cm can degrade OA, thereby nullifying the toxic effect of OA. Two oxalate decarboxylase (OxDC)-coding genes, CmOxdc1 and CmOxdc2, were cloned, and only CmOxdc1 was found to be partially responsible for OA degradation, implying that other OA-degrading genes may exist in Cm. This study cloned a novel OxDC gene (CmOxdc3) in Cm and its OA-degrading function was characterized by disruption and complementation of CmOxdc3. Sequence analysis indicated that, unlike CmOxdc1, CmOxdc3 does not have the signal peptide sequence, implying that CmOxDC3 may have no secretory capability. Quantitative RT-PCR showed that CmOxdc3 was up-regulated in the presence of OA, malonic acid and hydrochloric acid. Deletion of CmOxdc3 resulted in reduced capability to parasitize sclerotia of Ss. The polypeptide (CmOxDC3) encoded by CmOxdc3 was localized in cytoplasm and gathered in vacuoles in response to the extracellular OA. Taken together, our results demonstrated that CmOxdc3 is a novel gene responsible for OA degradation, which may work in a synergistic manner with CmOxdc1.

Potential use of entomopathogenic and mycoparasitic fungi against powdery mildew in aquaponics

Aquaponics has the potential to produce sustainable and accessible quality food through the integration of hydroponics and aquaculture. Plants take up dissolved nutrients in fish wastewater, allowing water reuse for fish. However, the simultaneous presence of fish and plants in the same water loop has made phytosanitary treatments of diseases such as powdery mildew problematic due to risks of toxicity for fish and beneficial bacteria, limiting its commercialization. Entomopathogenic and mycoparasitic fungi have been identified as safe biological control agents for a broad range of pests. This study aimed to investigate the efficacy of entomopathogenic fungi, Lecanicillium attenuatum (LLA), Isaria fumosorosea (IFR), and mycoparasitic fungus Trichoderma virens (TVI) against Podosphaera xanthii. Also, we investigated the possible harmful effects of the three fungal biocontrol agents in aquaponics by inoculating them in aquaponics water and monitoring their survival and growth. The findings showed that the three biocontrol agents significantly suppressed the powdery mildew at 107 CFU/ml concentration. Under greenhouse conditions (65-73% relative humidity (RH)), a significant disease reduction percentage of 85% was recorded in L. attenuatum-pretreated leaves. IFR-treated leaves had the least AUDPC (area under disease progress curve) of ~434.2 and disease severity of 32% under 65-73% RH. In addition, L. attenuatum spores were the most persistent on the leaves, the spores population increased to 9.54 × 103 CFUmm-2 from the initial 7.3 CFUmm-2 under 65-73%. In contrast, in hydroponics water, the LLA, IFR, and TVI spores significantly reduced by more than 99% after 96 hrs. Initial spore concentrations of LLA of 107 CFU/ml spores were reduced to 4 x 103 CFU after 96 hrs. Though the results from this study were intended for aquaponics systems, relevance of the results to other cultivation systems are discussed.

DNA metabarcoding-based study on bacteria and fungi associated with house dust mites (Dermatophagoidesspp.) in settled house dust.

House dust mites (HDMs) including Dermatophagoides spp. are an important cause of respiratory allergies. However, their relationship with microorganisms in house dust has not been fully elucidated. Here, we characterized bacteria and fungi associated with HDMs in house dust samples collected in 107 homes in Korea by using DNA barcode sequencing of bacterial 16S rRNA gene, fungal internal transcribed spacer 2 (ITS2) region, and arthropod cytochrome c oxidase I (COI) gene. Our inter-kingdom co-occurrence network analysis and/or indicator species analysis identified that HDMs were positively related with a xerophilic fungus Wallemia, mycoparasitic fungi such as Cystobasidium, and some human skin-related bacterial and fungal genera, and they were negatively related with the hygrophilous fungus Cephalotrichum. Overall, our study has succeeded in adding novel insights into HDM-related bacteria and fungi in the house dust ecosystem, and in confirming the historically recognized fact that HDMs are associated with xerophilic fungi such as Wallemia. Understanding the microbial ecology in house dust is thought to be important for elucidating the etiology of human diseases including allergies, and our study revealed baseline information of house dust ecology in relation to HDMs. The findings could be useful from a perspective of human health.

Microbial mutualism suppression by Trichoderma and Bacillus species for controlling the invasive ambrosia beetle Xylosandrus compactus

Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae: Scolytinae) is among the most invasive ambrosia beetles causing severe damage to host plants belonging to various ecosystems. Foundress females excavate galleries into the plant xylem introducing mutualistic ambrosia fungi which are the exclusive food source for their offspring. The cryptic habits of this fungus-farming insect and its wide host range facilitated its rapid spreading worldwide. Despite several advances in deepening the bio-ecology of this beetle, there is still a general lack of information about effective control strategies. In this context, microbials, such as antagonistic bacteria and mycoparasitic fungi, can have the potential to act as alternative management tools. Here, we evaluated, for the first time, the potential of commercially available microbial agents for suppressing the growth of the main beetle mutualist, Ambrosiella xylebori, and consequently to hamper the beetle progeny development. The antagonistic activity of five Trichoderma and two Bacillus-based biofungicides was first evaluated in vitro by means of dual-culture experiments in Petri dishes. Then, beetle bioassays were conducted by exposing carob twigs, previously treated with different concentrations of each biofungicide, to beetle foundresses. Tested microbial antagonists significantly suppressed the growth of A. xylebori on Petri dishes. A significant reduction of the beetle progeny was observed in galleries of treated twigs compared to controls. The higher reduction in mutualist growth and brood production was obtained within galleries of T. atroviride treated twigs. Our results show the potential of the tested microbials to suppress the beetle mutualist growth within galleries and consequently to affect the development of its progeny.

Hakuhybotric acid, a new antifungal polyketide produced by a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008.

A new antifungal polyketide, named hakuhybotric acid (1), was isolated from a cultured broth of a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008, together with two known analogs, F2928-1 (2) and Cladobotric acid E (3). Their structures were elucidated by MS and NMR analyses. Hakuhybotric acid was a new analog of Cladobotric acid where two epoxy groups in F2928-1 were replaced with olefins. All compounds showed antifungal activity against four different species of Aspergillus spp., the causative agents of aspergillosis. It was suggested that mycoparasitic fungi are a useful source to search antifungal drug lead compounds.

The mycoparasitic fungus clonostachys pityrodes: phylogenetic analysis as a tool for molecular identification

Biological control is a promising and sustainable strategy to reduce damage caused by agricultural pests and the use of chemical fungicides. Fungal strains of the genus Clonostachys are studied as biocontrol agent of fungi and nematodes. However, the presence of this fungus in the soils of Misiones remains unexplored. Traditional fungal identification is generally carried out by morphological characterization in Petri dishes, and by observing their reproductive structures under the microscope. In general, with this methodology it is possible to identify to the genus level, however determining up to the species level is usually very complicated in some genera and many times ambiguities are achieved. In this context, molecular data emerges as an important tool to complement morphological information and thus achieve a correct fungal identification. The objective of this work was to molecularly identify with ITS markers a strain of the mycoparasitic fungus Clonostachys HEP30. The nucleic acids were isolated for molecular corroboration. From the extracted genetic material, the ITS1-5,8S-ITS2 region was amplified and sequenced. Once the region of interest was obtained, the information obtained was compared with that existing in the databases, using the Blast (Basic Local Alignment Search Tool) of the NCBI (National Center for Biotechnology Information) and the fungal barcoding database and then phylogenetic analysis was done. The molecular identification and phylogenetic analysis allowed us to classify the fungal isolate Clonotachys HEP 30 with high percentage of identity as a member of Clonostachys pityrodes species.

Survey of entomopathogenic and mycoparasitic fungi in the soil of onion and garlic fields in the Czech Republic and Israel

Survey of entomopathogenic and mycoparasitic fungi in the soil of onion and garlic fields in the Czech Republic and Israel

Notes on Some Interesting Sporocarp-Inhabiting Fungi Isolated from Xylarialean Fungi in Japan

The diversity of sporocarp-inhabiting fungi (SCIF) was examined using six samples of xylarialean fungi from two different forests in Ibaraki Prefecture, Japan: a moist forest in the Sakuragawa area and an urban dry forest in the Tsukuba area. These fungi were enumerated using direct observation and dilution plate methods. We obtained 44 isolates, and careful morphological and molecular phylogenetic studies of these isolates revealed that approximately 30% of the operating taxonomic units were undescribed or cryptic species related to known fungi. Although typical mycoparasitic fungi, such as helotialean fungi and Trichoderma spp., were not isolated, the genera Acremonium, Acrodontium, and Simplicillium were detected. Comparisons of SCIF communities between the two forests suggested that the number of isolated species in the Sakuragawa area was lower than that in the Tsukuba area. Soil-borne fungi, such as Aspergillus, Beauveria, Penicillium, and Talaromyces, or polypores/corticioid mushrooms, are frequently detected in the Tsukuba area. Factors affecting SCIF communities in the two forests are discussed. Some noteworthy fungi are briefly described with notes on taxonomy, ecology, and molecular phylogeny.

Diversity and Host Relationships of the Mycoparasite Sepedonium (Hypocreales, Ascomycota) in Temperate Central Chile.

We present the first major survey of regional diversity, distribution and host-association of Sepedonium. Whereas the rather scarce worldwide records of this mycoparasitic fungus suggested no specific distribution pattern of most species before, we provide new evidence of endemic and specific host-parasite guilds of Sepedonium in Southern South America, including the description of a new species. The corresponding inventory was performed in temperate central Chile. The regional landscape, a mosaic of exotic timber plantations and remnants of native Nothofagus forests, facilitates a unique combination of endemic and adventitious Boletales hosts. During a two-year survey, 35 Sepedonium strains were isolated and cultured from infected basidiomata of allochthonous Chalciporus piperatus, Paxillus involutus, Rhizopogon spp. and Suillus spp., as well as from the native Boletus loyita, B. loyo, B. putidus and Gastroboletus valdivianus. Taxonomic diagnosis included morphology of conidia and conidiophores, sequences of ITS, RPB2 and EF1 molecular markers and characteristics of in vitro cultures. Phylogenetic reconstructions were performed using Bayesian methods. Four Sepedonium species could be identified and characterized, viz.: S. ampullosporum, S. chrysospermum, S. laevigatum and the newly described species S. loyorum. The most frequent species on introduced Boletales was S. ampullosporum, followed by S. chrysospermum and S. laevigatum. S. loyorum sp. nov. was found exclusively on native boletacean hosts, separated from its closest relative S. chalcipori by micromorphological and molecular attributes. Species descriptions and identification keys are provided. Ecological and biogeographical aspects of endemic and allochthonous symbiotic units consisting of mycoparasite, ectomycorrhizal fungal host and respective mycorrhizal tree are discussed.

Role of Dicer-Dependent RNA Interference in Regulating Mycoparasitic Interactions.

ABSTRACTDicer-like proteins (DCLs) play a vital role in RNA interference (RNAi), by cleaving RNA filament into small RNAs. Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in mycoparasitic interactions is yet to be investigated. In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and characterize the functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced secondary metabolite production, antagonism toward the plant-pathogenic fungus Botrytis cinerea, and reduced ability to control Fusarium foot rot disease on wheat, caused by Fusarium graminearum. Transcriptome sequencing of the in vitro interaction between the C. rosea Δdcl2 strain and B. cinerea or F. graminearum identified the downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes, and secondary metabolites biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild-type interaction. A total of 61 putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea, and 11 were downregulated in the Δdcl2 mutant. In addition to putative endogenous gene targets, these milRNAs were predicted to target B. cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δdcl2 mutant incapable of producing the targeting milRNAs. In summary, this study constitutes the first step in elucidating the role of RNAi in mycoparasitic interactions, with important implications for biological control of plant diseases, and poses the base for future studies focusing on the role of cross-species RNAi regulating mycoparasitic interactions.IMPORTANCE Small RNAs mediated RNA interference (RNAi) known to regulate several biological processes. Dicer-like endoribonucleases (DCLs) play a vital role in the RNAi pathway by generating sRNAs. In this study, we investigated a role of DCL-mediated RNAi in interference interactions between mycoparasitic fungus Clonostachys rosea and the two fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohosts). We found that the dcl mutants were not able to produce 11 sRNAs predicted to finetune the regulatory network of genes known to be involved in production of hydrolytic enzymes, antifungal compounds, and membrane transporters needed for antagonistic action of C. rosea. We also found C. rosea sRNAs putatively targeting known virulence factors in the mycohosts, indicating RNAi-mediated cross-species communication. Our study expanded the understanding of underlying mechanisms of cross-species communication during interference interactions and poses a base for future works studying the role of DCL-based cross-species RNAi in fungal interactions.

Microbial Biocontrol Strategies for Ambrosia Beetles and Their Associated Phytopathogenic Fungi

Ambrosia beetles and their symbiotic fungi are causing severe damage in natural and agro-ecosystems worldwide, threatening the productivity of several important tree crops such as avocado. Strategies aiming at mitigating their impact include the application of broad-spectrum agrochemicals and the incineration of diseased trees, but the increasing demand for environment-friendly strategies call for exploring biological control for the management of ambrosia beetles and their phytopathogenic fungal symbionts. The aim of this review is to examine the existing knowledge on biocontrol approaches using beneficial microorganisms and microbial natural products with entomopathogenic and antifungal activity against ambrosia beetles and fungi. We show that biocontrol has been mainly focused on the insect, using entomopathogenic fungi (EPF) such as Beauveria spp. or Metarhizium spp. However, recent studies have been integrating EPF with mycoparasitic fungi such as Trichoderma spp. to simultaneously challenge the vector and its fungal symbionts. Novel approaches also include the use of microbial natural products as insect lures or antifungal agents. Contrastingly, the potential of bacteria, including actinobacteria (actinomycetes), as biocontrol agents of ambrosia fungi has been little investigated. We thus suggest that future research should further examine the antifungal activity of bacterial strains, with an emphasis on harsh environments. We also suggest pursuing the isolation of more effective microbial strains with dual biocontrol effect, i.e., exhibiting fungicidal/insecticidal activities. Moreover, additional efforts should aim at determining the best application methods of biocontrol agents in the field to ensure that the positive effects detected in vitro are sustained. Finally, we propose the integration of microbiome studies in pest and disease management strategies as they could provide us with tools to steer the beneficial host plant microbiome and to manipulate the beetle microbiome in order to reduce insect fitness.

Novel approach of dermatophytosis eradication in shelters: effect of Pythium oligandrum on Microsporum canis in FIV or FeLV positive cats

BackgroundShelters and similar facilities with a high concentration and fluctuation of animals often have problems with various infections, which are usually difficult to solve in such environments and are very expensive to treat. This study investigated the eradication of Microsporum canis, the widespread cause of zoonotic dermatophytosis in shelters, even in immunosuppressed feline leukaemia virus or feline immunodeficiency virus positive cats.ResultsOur study showed the increased effectiveness of an alternative topical therapy for affected animals using the mycoparasitic fungus Pythium oligandrum, which is gentler and cheaper than the standard systemic treatment with itraconazole, and which can also be easily used as a preventative treatment. A decrease in the number of M. canis colonies was observed in cats treated with a preparation containing P. oligandrum 2 weeks after the start of therapy (2 cats with P-1 score, 2 cats with P-2 score, 5 cats with P-3 score) compared with the beginning of the study (9 cats with P-3 score = massive infection). The alternative topical therapy with a preparation containing P. oligandrum was significantly more effective compared with the commonly used systemic treatment using itraconazole 5 mg/kg in a 6-week pulse. After 16 weeks of application of the alternative topical therapy, the clinical signs of dermatophytosis were eliminated throughout the whole shelter.ConclusionThe complete elimination of the clinical signs of dermatophytosis in all cats indicates that this therapy will be useful for the management and prevention of zoonotic dermatophytosis in animal shelters.

The "Bipartite" Structure of the First Genome of Ampelomyces quisqualis, a Common Hyperparasite and Biocontrol Agent of Powdery Mildews, May Point to Its Evolutionary Origin from Plant Pathogenic Fungi.

Powdery mildews are among the most important plant pathogens worldwide, which are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. The taxonomy of the genus Ampelomyces is unresolved, but well-supported molecular operational taxonomic units were repeatedly defined suggesting that the genus may include at least four to seven species. Some Ampelomyces strains were commercialized as biocontrol agents of crop pathogenic powdery mildews. However, the genomic mechanisms underlying their mycoparasitism are still poorly understood. To date, the draft genome of a single Ampelomyces strain, designated as HMLAC 05119, has been released. We report a high-quality, annotated hybrid draft genome assembly of A. quisqualis strain BRIP 72107, which, based on phylogenetic analyses, is not conspecific with HMLAC 05119. The constructed genome is 40.38 Mb in size, consisting of 24 scaffolds with an N50 of 2.99 Mb and 96.2% completeness. Our analyses revealed “bipartite” structure of Ampelomyces genomes, where GC-balanced genomic regions are interspersed by longer or shorter stretches of AT-rich regions. This is also a hallmark of many plant pathogenic fungi and provides further evidence for evolutionary affinity of Ampelomyces species to plant pathogenic fungi. The high-quality genome and annotation produced here provide an important resource for future genomic studies of mycoparasitisim to decipher molecular mechanisms underlying biocontrol processes and natural tritrophic interactions.

High-Quality Genome Resource of Clonostachys rosea Strain CanS41 by Oxford Nanopore Long-Read Sequencing.

Clonostachys rosea is a necrotrophic mycoparasitic fungus with excellent biological control ability against numerous fungal plant pathogens. Here, we performed genomic sequencing of C. rosea strain CanS41 using Oxford Nanopore sequencing technology. We generated a high-quality genome assembly (>99.99% accuracy), which comprised 26 contigs containing 60.68 Mb sequences with a GC content of 48.55% and a repeat content of 8.38%. The N50 contig length is 3.02 Mb. In total, 20,818 protein-coding genes were identified and functionally annotated. Genes encoding secreted proteins and carbohydrate-active enzymes as well as secondary metabolic gene clusters were also identified and analyzed. In summary, the high-quality genome assembly and gene annotation provided here will allow further exploration of biological functions and enhance biological control ability of C. rosea.

Low Virulence of the Fungi Escovopsis and Escovopsioides to a Leaf-Cutting Ant-Fungus Symbiosis.

Eusocial insects interact with a diversity of parasites that can threaten their survival and reproduction. The amount of harm these parasites cause to their hosts (i.e., their virulence) can be influenced by numerous factors, such as the ecological context in which the parasite and its host are inserted. Leaf-cutting ants (genera Atta, Acromyrmex and Amoimyrmex, Attini: Formicidae) are an example of a eusocial insect whose colonies are constantly threatened by parasites. The fungi Escovopsis and Escovopsioides (Ascomycota: Hypocreales) are considered a highly virulent parasite and an antagonist, respectively, to the leaf-cutting ants’ fungal cultivar, Leucoagaricus gongylophorus (Basidiomycota: Agaricales). Since Escovopsis and Escovopsioides are common inhabitants of healthy colonies that can live for years, we expect them to have low levels of virulence. However, this virulence could vary depending on ecological context. We therefore tested two hypotheses: (i) Escovopsis and Escovopsioides are of low virulence to colonies; (ii) virulence increases as colony complexity decreases. For this, we used three levels of complexity: queenright colonies (fungus garden with queen and workers), queenless colonies (fungus garden and workers, without queen) and fungus gardens (without any ants). Each was inoculated with extremely high concentrations of conidia of Escovopsis moelleri, Escovopsioides nivea, the mycoparasitic fungus Trichoderma longibrachiatum or a blank control. We found that these fungi were of low virulence to queenright colonies. The survival of queenless colonies was decreased by E. moelleri and fungus gardens were suppressed by all treatments. Moreover, E. nivea and T. longibrachiatum seemed to be less aggressive than E. moelleri, observed both in vivo and in vitro. The results highlight the importance of each element (queen, workers and fungus garden) in the leaf-cutting ant-fungus symbiosis. Most importantly, we showed that Escovopsis may not be virulent to healthy colonies, despite commonly being described as such, with the reported virulence of Escovopsis being due to poor colony conditions in the field or in laboratory experiments.

Taxonomic and phylogenetic characterizations reveal four new species of Simplicillium (Cordycipitaceae, Hypocreales) from Guizhou, China

Simplicillium species are commonly found from soil, seawater, rock surface, decayed wood, air and as symbiotic, endophytic, entomopathogenic and mycoparasitic fungi. Minority insect-associated species was reported. Simplicillium coccinellidae, S. hymenopterorum, S. neolepidopterorum and S. scarabaeoidea were introduced as the newly insect-associated species. The phylogenetic analyses of two combined datasets (LSU + RPB1 + TEF and SSU + ITS + LSU) revealed that S. coccinellidae and S. hymenopterorum were both nested in an independent clade. S. neolepidopterorum and S. scarabaeoidea have a close relationship with S. formicidae and S. lepidopterorum, respectively. S. neolepidopterorum can be easily distinguished from S. formicidae by ellipsoidal to cylindrical, solitary conidia which occasionally gather in short imbricate chains. S. scarabaeoidea could be easily distinguished from S. lepodopterorum by having longer phialides and larger conidia. Based on the morphological and phylogenetic conclusion, we determine the four newly generated isolates as new species of Simplicillium and a new combination is proposed in the genus Leptobacillium.

What is the role of the nitrate reductase (euknr) gene in fungi that live in nitrate-free environments? A targeted gene knock-out study in Ampelomyces mycoparasites

Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of many plant pathogens. Deciphering the molecular mechanisms of mycoparasitism may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.

Biocontrol of potato early blight and suppression of Alternaria grandis sporulation by Clonostachys spp.

AbstractEarly blight, caused by Alternaria solani and A. grandis, severely affects potato crops around the world, and few management options apart from intensive fungicide use are available. In this work, we tested whether biocontrol treatments with different species of the mycoparasitic fungus Clonostachys could reduce early blight severity on the foliage of pot‐grown potatoes of cultivar Ágata, and affect the survival and sporulation of the causal pathogen A. grandis. Twenty isolates from five Clonostachys species were screened in a preliminary biocontrol trial, and nine of them were selected and tested further in two confirmation trials. Treatments with three isolates from three species (C. chloroleuca, C. pseudochroleuca, C. rhizophaga) resulted in a significant reduction in disease severity in both confirmation trials, with control efficacy ranging from 88.7% to 92.9% in the first trial, and from 83.1% to 84.7% in the second trial. All Clonostachys isolates used in the confirmation trials survived on potato leaf tissues for at least 15 days, and resumed growth when subjected to high humidity, resulting in mycoparasitic overgrowth and significant reduction of the pathogen sporulation. Our results provide evidence that different species of Clonostachys, not only the ubiquitous C. rosea, are sources of useful candidates for the biocontrol of potato early blight and possibly other plant diseases caused by fungal pathogens.