Mycoparasitic Fungus Research Articles

The biology and chemistry of a mutualism between a soil bacterium and a mycorrhizal fungus

Arbuscular mycorrhizal (AM) fungi (e.g., Rhizophagus species) recruit specific bacterial species in their hyphosphere. However, the chemical interplay and the mutual benefit of this intricate partnership have not been investigated yet, especially as it involves bacteria known as strong producers of antifungal compounds such as Bacillus velezensis. Here, we show that the soil-dwelling B.velezensis migrates along the hyphal network of the AM fungus R.irregularis, forming biofilms and inducing cytoplasmic flow in the AM fungus that contributes to host plant root colonization by the bacterium. During hyphosphere colonization, R.irregularis modulates the biosynthesis of specialized metabolites in B.velezensis to ensure stable coexistence and as a mechanism to ward off mycoparasitic fungi and bacteria. These mutual benefits are extended into a tripartite context via the provision of enhanced protection to the host plant through the induction of systemic resistance.

Molecular identification of Hypomyces chrysospermus mycoparasitic fungus isolated from mushrooms in a local market of Iraq.

In this study, mycoparasitic fungus was identified by using the rDNA internal transcribed spacer barcode marker, i.e. ITS rDNA gene. Amplicons were sequenced and identified by NCBI - BLAST (Basic local alignment search tool). The BLAST results revealed that NOOR strain matched 100% with accession numbers [MH854754.1]. The phylogenetic tree shows close relationship between NOOR strain and H. chrysospermus [MH854754-1], H. chrysospermus [MZ389105-1], H. chrysospermus [MK605327- 1], H. chrysospermus [MT595227-1], H. [EU816370-1], H. chrysospermus [MG685885-1]. The Iraqi isolate was recorded as NOOR strain in the National Centre for Biotechnology Information for the first time in Iraq.

Are1-mediated nitrogen metabolism is associated with iron regulation in the mycoparasite Trichoderma atroviride

Trichoderma atroviride is a mycoparasitic fungus with antagonistic activity against fungal pathogens and is used as a pathogen control agent alternative to synthetic fungicides. Sensing nutrient availability in the environment and adjusting metabolism for optimal growth, development and reproduction is essential for adaptability and is relevant to its mycoparasitic activity. During mycoparasitism, secondary metabolites are produced to weaken the fungal prey and support the attack. Are1-like proteins act as major GATA-type transcription factors in the activation of genes subject to nitrogen catabolite repression. Since the quality and quantity of nitrogen has been proven particularly relevant in remodeling the biosynthesis of secondary metabolites in fungi, we decided to functionally characterize Are1, the ortholog of Aspergillus nidulans AreA, in T. atroviride. We show that the growth of the T. atroviride ∆are1 mutant is impaired in comparison to the wild type on several nitrogen sources. Deletion of are1 enhanced sensitivity to oxidative and cell-wall stressors and altered the mycoparasitic activity. We were able to identify for the first time a link between Are1 and iron homeostasis via a regulatory mechanism that does not appear to be strictly linked to the nitrogen source, but rather to an independent role of the transcription factor.

Accumulation of pathogens in soil microbiome can explain long-term fluctuations of legumes in a grassland community.

All plant populations fluctuate in time. Apart from the dynamics imposed by external forces such as climate, these fluctuations can be driven by endogenous processes taking place within the community. In this study, we aimed to identify potential role of soil-borne microbial communities in driving endogenous fluctuations of plant populations. We combined a unique, 35-yr long abundance data of 11 common plant species from a species-rich mountain meadow with development of their soil microbiome (pathogenic fungi, arbuscular mycorrhizal fungi and oomycetes) observed during 4 yr of experimental cultivation in monocultures. Plant species which abundance fluctuated highly in the field (particularly legumes) accumulated plant pathogens in their soil mycobiome. We also identified increasing proportion of mycoparasitic fungi under highly fluctuating legume species, which may indicate an adaptation of these species to mitigate the detrimental effects of pathogens. Our study documented that long-term fluctuations in the abundance of plant species in grassland communities can be explained by the accumulation of plant pathogens in plant-soil microbiome. By contrast, we found little evidence of the role of mutualists in plant population fluctuations. These findings offer new insights for understanding mechanisms driving both long-term vegetation dynamics and patterns of species coexistence and richness.

Near real-time quantification of microbial volatile organic compounds from mycoparasitic fungi: Potential for advanced monitoring and pest control

Microbial volatile organic compounds (MVOCs) are thought to play a key role in the interactions between mycoparasitic fungi, such as the biocontrol agent Trichoderma atroviride (T. atroviride), and their environment. However, the analysis of MVOC emissions from fungal samples is challenging because of low analyte concentrations, typically in the ppbV-range, and the complex chemical nature of biological samples. In a recent study using proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) to determine MVOC emissions from T. atroviride, many product ions were unspecific, as they could arise from a large number of possible analytes. The aim of the present study was to determine whether fast gas chromatography (fast-GC) coupled to PTR-ToF-MS could be used to overcome this issue and constitute a suitable on-line, near real-time method to identify and quantify fungal MVOC emissions in the ppbV-to-ppmV regime. Using gas standards of eleven MVOCs known to be emitted by T. atroviride such as 6-amyl-α-pyrone (6-PP), 2-pentylfuran, 1-octen-3-ol, 2-heptanone, 3-octanone, 2-methyl-1-propanol, 2-pentanone, 3-methyl-1-butanol, 3-methylbutanal, acetone and ethanol, we developed a fast-GC method with a total runtime of 180 s which significantly enhances the analytical specificity of PTR-ToF-MS compared to conventional PTR-ToF-MS without fast-GC separation. Limits of detection were on the order of 0.1–4 ppbV. The increased analytical specificity demonstrated notable benefits, especially for MVOCs having partially overlapping distributions of product ions when analyzed directly using PTR-ToF-MS.In order to demonstrate the applicability of the analytical method, we analysed T. atroviride samples in four biological replicates twice daily over a duration of five days. Using the fast-GC method, nine out of the eleven MVOC species considered in this study in the headspace of T. atroviride could be identified and quantified and their time evolution over the five-day incubation period determined. The measured volume mixing ratios (VMRs) ranged from single-digit ppbV (2-pentylfuran) up to few ppmV (6-PP and ethanol), with the other compounds in the 10-to-100-ppbV range (1-octen-3-ol, 2-heptanone, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-methylbutanal and acetone).Our results suggest that fast-GC-PTR-ToF-MS is a method well-suited for the analysis of gas-phase samples of biological origin, including but not limited to (mycoparasitic) fungi, in a wide range of VMRs from sub-ppbV to few-ppmV.

Demonstrating the Applicability of Proton Transfer Reaction Mass Spectrometry to Quantify Volatiles Emitted by the Mycoparasitic Fungus Trichoderma atroviride in Real Time: Monitoring of Trichoderma-Based Biopesticides.

The present study aims to explore the potential application of proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for real-time monitoring of microbial volatile organic compounds (MVOCs). This investigation can be broadly divided into two parts. First, a selection of 14 MVOCs was made based on previous research that characterized the MVOC emissions of Trichoderma atroviride, which is a filamentous fungus widely used as a biocontrol agent. The analysis of gas-phase standards using PTR-ToF-MS allowed for the categorization of these 14 MVOCs into two groups: the first group primarily undergoes nondissociative proton transfer, resulting in the formation of protonated parent ions, while the second group mainly undergoes dissociative proton transfer, leading to the formation of fragment ions. In the second part of this investigation, the emission of MVOCs from samples of T. atroviride was continuously monitored over a period of five days using PTR-ToF-MS. This also included the first quantitative online analysis of 6-amyl-α-pyrone (6-PP), a key MVOC emitted by T. atroviride. The 6-PP emissions of T. atroviride cultures were characterized by a gradual increase over the first two days of cultivation, reaching a plateau-like maximum with volume mixing ratios exceeding 600 ppbv on days three and four. This was followed by a marked decrease, where the 6-PP volume mixing ratios plummeted to below 50 ppbv on day five. This observed sudden decrease in 6-PP emissions coincided with the start of sporulation of the T. atroviride cultures as well as increasing intensities of product ions associated with 1-octen-3-ol and 3-octanone, whereas both these MVOCs were previously associated with sporulation in T. atroviride. The study also presents the observations and discussion of further MVOC emissions from the T. atroviride samples and concludes with a critical assessment of the possible applications and limitations of PTR-ToF-MS for the online monitoring of MVOCs from biological samples in real time.

Soil prokaryotic and fungal biome structures associated with crop disease status across the Japan Archipelago.

Archaea, bacteria, and fungi in the soil are increasingly recognized as determinants of agricultural productivity and sustainability. A crucial step for exploring soil microbiomes with important ecosystem functions is to perform statistical analyses on the potential relationship between microbiome structure and functions based on comparisons of hundreds or thousands of environmental samples collected across broad geographic ranges. In this study, we integrated agricultural field metadata with microbial community analyses by targeting 2,903 bulk soil samples collected along a latitudinal gradient from cool-temperate to subtropical regions in Japan (26.1-42.8 °N). The data involving 632 archaeal, 26,868 bacterial, and 4,889 fungal operational taxonomic units detected across the fields of 19 crop plant species allowed us to conduct statistical analyses (permutational analyses of variance, generalized linear mixed models, randomization analyses, and network analyses) on the relationship among edaphic factors, microbiome compositions, and crop disease prevalence. We then examined whether the diverse microbes form species sets varying in potential ecological impacts on crop plants. A network analysis suggested that the observed prokaryotes and fungi were classified into several species sets (network modules), which differed substantially in association with crop disease prevalence. Within the network of microbe-to-microbe coexistence, ecologically diverse microbes, such as an ammonium-oxidizing archaeon, an antibiotics-producing bacterium, and a potentially mycoparasitic fungus, were inferred to play key roles in shifts between crop-disease-promotive and crop-disease-suppressive states of soil microbiomes. The bird's-eye view of soil microbiome structure will provide a basis for designing and managing agroecosystems with high disease-suppressive functions.IMPORTANCEUnderstanding how microbiome structure and functions are organized in soil ecosystems is one of the major challenges in both basic ecology and applied microbiology. Given the ongoing worldwide degradation of agroecosystems, building frameworks for exploring structural diversity and functional profiles of soil microbiomes is an essential task. Our study provides an overview of cropland microbiome states in light of potential crop-disease-suppressive functions. The large data set allowed us to explore highly functional species sets that may be stably managed in agroecosystems. Furthermore, an analysis of network architecture highlighted species that are potentially used to cause shifts from disease-prevalent states of agroecosystems to disease-suppressive states. By extending the approach of comparative analyses toward broader geographic ranges and diverse agricultural practices, agroecosystem with maximized biological functions will be further explored.

Potential application of a fungal co-culture crude extract for the conservation of post-harvest fruits.

Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.

The histone deacetylase Hda1 affects oxidative and osmotic stress response as well as mycoparasitic activity and secondary metabolite biosynthesis in Trichoderma atroviride.

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride, regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens.

First Description of Simplicillium lanosoniveum, a Potential Antagonist of the Coffee Leaf Rust from Cuba

(1) The fungal genus Simplicillium (Cordycipitaceae: Hypocreales) has an extensive distribution and a broad spectrum of hosts and substrates. The species Simplicillium lanosoniveum is a mycoparasite with potential for biological control of coffee leaf rust, Hemileia vastatrix. Morphologically, Simplicillium closely resembles mycoparasitic and entomopathogenic Lecanicillium fungi, often resulting in misidentification. A fungal isolate was obtained from leaf-rust-infested coffee plants from Cienfuegos Province, Cuba. (2) Combined analyses of morphology and molecular markers (ITS, LSU, EF-1alpha) were used for fungal identification. (3) In the NJ, ML, and BI phylogenies which were reconstructed, the isolate LBSim-01 was located in the Simplicillium lanosoniveum clade. This species-level identification was supported by morphological features. (4) The isolate LBSim-01 was assigned to the species Simplicillium lanosoniveum. This is the first description of a Simplicillium fungus associated with coffee leaf rust in Cuba. The presented results hold implications for the biological control of this economically relevant plant disease.

New treasures in Cordycipitaceae: Fungicolous fungi associated with Pseudocercospora fijiensis and P. musae in Brazil, including Matutinistella gen. nov.

Cordycipitaceae is a large family belonging to the order Hypocreales, including cosmopolitan species found on different substrates. This family includes species with different lifestyles such as entomopathogenic, endophytic, and mycoparasitic fungi. Some mycoparasitic species in this family attack phytopathogenic fungi. We identified species of Cordycipitaceae found during a survey of fungicolous fungi associated with Pseudocercospora fijiensis and P. musae in Brazil. Based on morphological characters combined with phylogenetic analysis of ITS, LSU, SSU, RPB1, RPB2, and TEF1-α, we propose a new genus in Cordycipitaceae, namely Matutinistella, with its type species M. brasiliensis, and a new species of the genus Simplicillium, namely S. pseudocercosporicola. Furthermore, we report P. fijiensis as a new host of the mycoparasitic fungus Simplicillium lanosoniveum. In this study we newly report fungicolous fungi of Cordycipitaceae associated with the causal agents of Black Sigatoka and Yellow Sigatoka on banana crops. We provide relevant information for future work involving control measures for these diseases that cause major losses in banana crops.

Solid state culture conditions for improved conidial production of the mycoparasitic fungus Escovopsis weberi

ABSTRACT We evaluated culture conditions (temperature, substrate moisture content, agitation and light conditions) for optimising conidia production of Escovopsis weberi Ew1 using solid state culture. In addition, several substrates such as agricultural waste and food industry by-products were evaluated with the aim of reducing spore production costs in a strategic way towards circular bioeconomy. Finally, we selected substrate combinations in order to achieve a suitable system for the production of conidia.

Identification and evaluation of suitable reference genes for RT-qPCR analyses in Trichoderma atroviride under varying light conditions

BackgroundTrichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms.ResultsUnder cyclic light–dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1.ConclusionThe data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.

Surveying potentially antagonistic fungi to myrtle rust (Austropuccinia psidii) in Brazil: fungicolous Cladosporium spp.

The myrtle rust (MR), caused by Austropuccinia psidii, is a worldwide threat to the cultivated and wild Myrtaceae. Originally from the neotropics, it has spread to North America, Africa, and Asia and has reached geographically isolated areas in the Pacific and Australasia. It is attacking native species in those new ranges and is still spreading and causing great concern for the damage caused to endemic Myrtaceae, and to the environment. Classical biological control is regarded as the most sustainable management option for mitigating such biological invasions. However, there are no examples of introductions of host-specific co-evolved natural enemies of plant pathogens, from their native range, as a management strategy for plant pathogens. In order to explore this neglected approach, a survey of potential fungal natural enemies of A. psidii was initiated recently in the state of Minas Gerais (Brazil). Several purported mycoparasites have been collected from A. Psidii pustules formed on myrtaceous hosts. This included some isolates of dematiaceous fungi recognized as having a Cladosporium-like morphology. Here we present the results of the investigation aimed at elucidating their identity through a polyphasic taxonomic approach. Besides morphological and cultural features, molecular analyses using sequences of translation elongation factor 1-α (EF1) and actin (ACT) were performed. The combination of data generated is presented herein and placed all Cladosporium-like isolates in six species of Cladosporium, namely, Cladosporium angulosum, C. anthropophilum, C. bambusicola, C. benschii, C. guizhouense, and C. macadamiae. None of these have ever been recorded in association with A. psidii. Now, with the identification of these isolates at hand, an evaluation of biocontrol potential of these fungi will be initiated. In contrast with the ready finding of fungicolous (possibly mycoparasitic) fungi on MR in this study, no evidence of those was recorded from Australasia until now.

Enhancing Soil Organic Matter Transformation through Sustainable Farming Practices: Evaluating Labile Soil Organic Matter Fraction Dynamics and Identifying Potential Early Indicators

The growing global population and increasing demand for agricultural products have exerted significant pressure on agricultural systems. As a result, soil organic matter depletion and degradation have become prevalent issues, including in regions such as South Bohemia, Czech Republic, where conventional farming practices are predominant. Soil organic matter (SOM) plays a critical role in soil health, crop productivity, and the sustainability of agricultural systems, with changes occurring in both the total and labile fractions of the organic matter pools. However, changes in the total soil organic matter carbon pool (TOC) resulting from agricultural practices occur gradually and may become evident only after several years, posing challenges for timely management adjustments. Therefore, the identification of early indicators of SOM dynamics is crucial for implementing prompt corrective actions. The aim of this study was to evaluate the effects of sustainable management practices, such as cultivated crops (Pisum sativum and Lupinus albus), selected entomopathogenic and myco parasitic fungi (MEHA) (Trichoderma virens and Metarhizium brunneum), and lactic acid bacteria (LAB) on the labile fraction of the SOM pool (CLSOM) and identify potential early indicators. Our findings demonstrated that the type of crop and applied microorganisms (treatments) significantly affected the CLSOM in peas, and the crop growth stages affected the TOC in both pea and lupin. Growth stages also showed an impact on the CLSOM in lupin. Moreover, in both crops, the change in CLSOM correlated with changes in the SOM oxidation rate constant (k), carbon lability index (LI), carbon management index (CMI), and carbon enrichment ratio (ER). Conversely, changes in the TOC did not exhibit significant correlations, except for LI and CMI, which showed a positive correlation with the TOC in peas (p < 0.05). Furthermore, the separate application of MEHA and LAB on seeds or leaves resulted in increased SOM carbon pools compared with the combined application. The application of these beneficial microorganisms in pea and lupin crops showed potential in maintaining or increasing CLSOM, which can be assessed early through indicators such as k, LI, CMI, and ER. Contributing to the development of sustainable soil management strategies, future research should further investigate different crops and microorganisms—and the mechanisms underlying their observed relationships—and explore additional early indicators to refine and optimize sustainable agricultural practices.

First report of a rust fungus (Puccinia sp.) infecting lemongrass in Minnesota.

In July 2021 and July - Oct. 2022, in a community garden near Mankato, Minnesota, rust disease was observed on lemongrass (Cymbopogon citriatus). In 2022, all 20 plants in the garden plot were infected. Lemongrass is used in some Asian cuisines and for tea or medicine. It is not hardy in Minnesota but is grown in gardens and outdoors in small-scale production. Uredinia were cinnamon-brown on the abaxial surface of leaf blades. Pustules were small (0.2 - 0.5 x .01 - .05 mm) and numerous, causing large necrotic lesions and leaf dieback (Fig. 1A). Severity ranged from 5 - 50% leaf loss. Urediniospores were finely echinulate, slightly ovular (22-25 x 20-23 μm), thick-walled (2.5-4 μm), with 3-4 roughly equatorial, sometimes scattered germ pores (Fig 1B; 1C). Clavate paraphyses were abundant. Other spore types were not observed. The pycnidia of a mycoparasitic fungus were present within the uredinia. The specimen was submitted to the Arthur Fungarium at Purdue University (PUR N24011). Primers ITS1rustF10d (Barnes and Szabo, 2007) and ITSRu1 (Rioux et al., 2015) were used to generate amplicons for the rust fungus, and ITS4 and ITSF+ (White, 1990) for the mycoparasite. Amplicons were sequenced on an Oxford Nanopore MinIon with R9 flow cells following manufacturer instructions. Reads (PRJNA802078) were filtered for quality (> Q13) and length (> 200 bp), mapped to reference sequences, aligned, and separated based on similarity. Consensus sequences were generated for the amplicons of the rust fungus and of two other fungi. BLASTn searches of the ITS sequences, OM442990 and OM442991, identified an Alternaria sp. (99.8% match (597/598) with MT548677) and Sphaerellopsis filum (syn. Darluca filum; 98.3% (529/538 bp) match with EF600974), a common rust mycoparasite. A BLASTn search of the rust fungal ITS sequence (OM442989) yielded 98.9% (549/555) and 98.6% (633/642) match with MT955206 and MT955207, respectively, both Puccinia cesatii on Bothriochloa ischaemum. The third closest match is P. cymbopogonis on C. citriatus (97.1% (595/613) with KY764115). Urediniospore morphology is consistent with that of P. cesatii (Cummins, 1971). Available evidence suggests the fungus is P. cesatii or a closely related species. Puccinia cesatii has been reported infecting Cymbopogon spp. (Stevenson et al., 1926; Dhar and Rekha, 1984), but lemongrass is not generally considered a host-possibly due to confusion of P. cesatii with P. cymbopogonis, a closely related rust pathogen of lemongrass that is morphologically very similar to P. cesatii. P. cymbopogonis has not been reported in the U.S. Rust diseases of lemongrass have been reported in three states: Hawaii (Gardner, 1985), California (Koike and Molinar, 1999), and Florida (Ploetz et al., 2014). In each case, the rust was identified as Puccinia nakanishikii. Urediniospores of P. nakanishikii are larger (26-36 μm long) (Cummins, 1971) and the ITS2 has no significant sequence similarity. P. cesatii is widespread in Eurasia, the southwest U.S., and Mexico (Cummins, 1971). Cummins lists three genera closely related to Cymbopogon as telial hosts of P. cesatii: Bothriochloa, Capillipedium, and Dicanthium. He lists nine rust fungi that infect Cymbopogon but does not list P. cesatii. Of these nine species, only P. cymbopogonis is morphologically similar. Further research is needed to investigate the potential impact of rust fungi on lemongrass production and to elucidate phylogenetic relationships of rust fungi infecting lemongrass.

Trichoderma koningiopsis (Hypocreaceae) has the smallest mitogenome of the genus Trichoderma.

Fungal mitogenomes exhibit remarkable variation in conformation, size, gene content, arrangement and expression, including their intergenic spacers and introns. The complete mitochondrial genome sequence of the mycoparasitic fungus Trichoderma koningiopsis was determined using the Illumina next-generation sequencing technology. We used data from our recent Illumina NGS-based project of T. koningiopsis genome sequencing to study its mitochondrial genome. The mitogenome was assembled, annotated, and compared with other fungal mitogenomes. T. koningiopsis strain POS7 mitogenome is a circular molecule of 27,560 bp long with a GC content of 27.80%. It harbors the whole complement of the 14 conserved mitochondrial protein-coding genes (PCG) such as atp6, atp8, atp9, cox1, cox2, cox3, cob, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6, also found in the same gene order to other Hypocreales. The mitogenome also contains 26 transfer RNA genes (tRNAs), 5 of them with more than one copy. Other genes also present in the assembled mitochondrial genome are a small rRNA subunit and a large rRNA subunit containing ribosomal protein S3 gene. Despite the small genome size, two introns were detected in the T. koningiopsis POS7 mitogenome, one of them in cox3 gene and the other in rnl gene, accounting 7.34% of this mitogenome with a total size of 2,024 bp. A phylogenetic analysis was done using the 14 PCGs genes of T. koningiopsis strain POS7 mitogenome to compare them with those from other fungi of the Subphyla Pezizomycotina and Saccharomycotina. T. koningiopsis strain POS7 was clustered together with other representatives of Trichoderma lineage, within the Hypocreales group, which is also supported by previous phylogenetic studies based on nuclear markers. The mitochondrial genome of T. koningiopsis POS7 will allow further investigations into the taxonomy, phylogenetics, conservation genetics, and evolutionary biology of this important genus as well as other closely related species.

An Outstandingly Rare Occurrence of Mycoviruses in Soil Strains of the Plant-Beneficial Fungi from the Genus Trichoderma and a Novel Polymycoviridae Isolate.

In fungi, viral infections frequently remain cryptic causing little or no phenotypic changes. It can indicate either a long history of coevolution or a strong immune system of the host. Some fungi are outstandingly ubiquitous and can be recovered from a great diversity of habitats. However, the role of viral infection in the emergence of environmental opportunistic species is not known. The genus of filamentous and mycoparasitic fungi Trichoderma (Hypocreales, Ascomycota) consists of more than 400 species, which mainly occur on dead wood, other fungi, or as endo- and epiphytes. However, some species are environmental opportunists because they are cosmopolitan, can establish in a diversity of habitats, and can also become pests on mushroom farms and infect immunocompromised humans. In this study, we investigated the library of 163 Trichoderma strains isolated from grassland soils in Inner Mongolia, China, and found only four strains with signs of the mycoviral nucleic acids, including a strain of T. barbatum infected with a novel strain of the Polymycoviridae and named and characterized here as Trichoderma barbatum polymycovirus 1 (TbPMV1). Phylogenetic analysis suggested that TbPMV1 was evolutionarily distinct from the Polymycoviridae isolated either from Eurotialean fungi or from the order Magnaportales. Although the Polymycoviridae viruses were also known from Hypocrealean Beauveria bassiana, the phylogeny of TbPMV1 did not reflect the phylogeny of the host. Our analysis lays the groundwork for further in-depth characterization of TbPMV1 and the role of mycoviruses in the emergence of environmental opportunism in Trichoderma. IMPORTANCE Although viruses infect all organisms, our knowledge of some groups of eukaryotes remains limited. For instance, the diversity of viruses infecting fungi-mycoviruses-is largely unknown. However, the knowledge of viruses associated with industrially relevant and plant-beneficial fungi, such as Trichoderma spp. (Hypocreales, Ascomycota), may shed light on the stability of their phenotypes and the expression of beneficial traits. In this study, we screened the library of soilborne Trichoderma strains because these isolates may be developed into bioeffectors for plant protection and sustainable agriculture. Notably, the diversity of endophytic viruses in soil Trichoderma was outstandingly low. Only 2% of 163 strains contained traces of dsRNA viruses, including the new Trichoderma barbatum polymycovirus 1 (TbPMV1) characterized in this study. TbPMV1 is the first mycovirus found in Trichoderma. Our results indicate that the limited data prevent the in-depth study of the evolutionary relationship between soilborne fungi and is worth further investigation.

First Report of Clonostachys rosea as a Mycoparasite on Sclerotinia sclerotiorum Causing Head Rot of Cabbage in India.

Clonostachys rosea, an ascomycetous, omnipresent, cellulose-decaying soil fungus, has been reported to be a well-known mycoparasitic biological control agent. In this study, we isolated C. rosea, a mycoparasitic fungus for the first time in India from sclerotia of the notorious plant pathogen Sclerotinia sclerotiorum, causing head rot disease in cabbage. A total of five mycoparasitic fungi were isolated from the sclerotial bodies of S. sclerotiorum (TNAU-CR 01, 02, 03, 04 and 05). All the isolates were tested under morpho-molecular characterization. Among them, TNAU-CR 02 showed the greatest mycelial inhibition of 79.63% over the control. Similarly, the SEM imaging of effective C. rosea isolates indicated the presence of numerous conidia destroying the outer cortex layers of sclerotia. Metabolite fingerprinting of C. rosea TNAU-CR 02 identified 18 chemical compounds using GC-MS analysis. The crude antibiotics of C. rosea TNAU-CR 02 were verified for their antifungal activity against S. sclerotiorum and the results revealed 97.17% mycelial inhibition compared with the control. Similarly, foliar application of TNAU-CR 02 at 5 mL/litre on 30, 45 and 60 days after transplanting showed the lowest disease incidence of 15.1 PDI compared to the control. This discovery expands our understanding of the biology and the dissemination of C. rosea, providing a way for the exploitation of C. rosea against cabbage head rot pathogens.

Study on secondary metabolites of Trichoderma atroviride F742 and their role in antibiosis

Abstract Soil-borne pathogenic fungi responsible for serious damage in agriculture are widely distributed. Traditional approach to control these pathogens leads to the use of chemical fungicides, many of which have proven harmful side effects on the environment. Mycoparasitic fungi of the genus Trichoderma have been used for pathogens biocontrol as well as for their ability to promote plant growth. As increased mycoparasitic activity of mutant strain Trichoderma atroviride F742 has been observed, its use as a biocontrol agent might be considered. In this work, we focused on mycoparasitic activity of the strain T. atroviride F742 prepared by UV mutagenesis (from parental strain T. atroviride F534) and the physiological role of T. atroviride metabolites in antibiosis. T. atroviride F742 shows remarkable mycoparasitic activity attacking and colonizing phytopathogens (Alternaria alternata, Botrytis cinerea, Fusarium culmorum). Its isolated metabolites inhibit the growth of bacteria, yeasts, and fungi Candida albicans, A. alternata and F. culmorum. As different expression patterns in the secondary metabolites production of ABC transporters have been observed, we suggest their role in transport of secondary metabolites produced by T. atroviride F742.