Fungal-fungal Interactions Research Articles

Visualizing liquid distribution across hyphal networks with cellular resolution.

Filamentous fungi and fungal-like organisms contribute to a wide range of important ecosystem functions. Evidence has shown the movement of liquid across mycelial networks in unsaturated environments, such as soil. However, tools to investigate liquid movement along hyphae at the level of the single cell are still lacking. Microfluidic devices permit the study of fungal and fungal-like organisms with cellular resolution as they can confine hyphae to a single optical plane, which is compatible with microscopy imaging over longer timescales and allows for precise control of the microchannel environment. The aim of this study was to develop a method that enables the visualization and quantification of liquid movement on hyphae of fungal and fungal-like microorganisms. For this, the fungal-fungal interaction microfluidic device was modified to allow for the maintenance of unsaturated microchannel conditions. Fluorescein-containing growth medium solidified with agar was used to track liquid transported by hyphae via fluorescence microscopy. Our key findings highlight the suitability of this novel methodology for the visualization of liquid movement by hyphae over varying time scales and the ability to quantify the movement of liquid along hyphae. Furthermore, we showed that at the cellular level, extracellular movement of liquid along hyphae can be bidirectional and highly dynamic, uncovering a possible link between liquid movement and hyphal growth characteristics. We envisage that this method can be applied to facilitate future research probing the parameters contributing to hyphal liquid movement and is an essential step for studying the phenomenon of fungal highways.

Stimulating Novel and Bioactive Metabolite Production by Cocultivation of Two Fungi─Aspergillus oryzae and Epicoccum dendrobii.

Fungi produce various bioactive secondary metabolites (SMs) as protective and weaponized tools to enhance survival in shared ecological niches. By mimicking a competitive ecosystem, cocultivation has been proven to be particularly successful in stimulating SM discovery. Here, we reported the identification of four novel metabolites, epiclactones A and B, epioxochromane and aoergostane, from the coculture of two biotechnologically important strains, Aspergillus oryzae and Epicoccum dendrobii. Transcriptome and metabolome analyses revealed widespread silent gene activation during fungal-fungal interaction. The majority of differentially expressed gene clusters were summarized for both strains. Based on these highly activated biosynthetic pathways, we suggested that a bidirectional chemical defense occurred under cocultivation. E. dendrobii enhanced the production of the spore inhibitor, fumigermin. Moreover, A. oryzae highly accumulated the antifungal agent kojic acid with a yield of up to 1.10 g/L. This study provides an excellent example for the discovery of hidden natural products by cocultivation.

Community Assembly Processes of Deadwood Mycobiome in a Tropical Forest Revealed by Long-Read Third-Generation Sequencing

Despite the importance of wood-inhabiting fungi on nutrient cycling and ecosystem functions, their ecology, especially related to their community assembly, is still highly unexplored. In this study, we analyzed the wood-inhabiting fungal richness, community composition, and phylogenetics using PacBio sequencing. Opposite to what has been expected that deterministic processes especially environmental filtering through wood-physicochemical properties controls the community assembly of wood-inhabiting fungal communities, here we showed that both deterministic and stochastic processes can highly contribute to the community assembly processes of wood-inhabiting fungi in this tropical forest. We demonstrated that the dynamics of stochastic and deterministic processes varied with wood decomposition stages. The initial stage was mainly governed by a deterministic process (homogenous selection), whereas the early and later decomposition stages were governed by the stochastic processes (ecological drift). Deterministic processes were highly contributed by wood physicochemical properties (especially macronutrients and hemicellulose) rather than soil physicochemical factors. We elucidated that fine-scale fungal-fungal interactions, especially the network topology, modularity, and keystone taxa of wood-inhabiting fungal communities, strongly differed in an initial and decomposing deadwood. This current study contributes to a better understanding of the ecological processes of wood-inhabiting fungi in tropical regions where the knowledge of wood-inhabiting fungi is highly limited.

An apoptosis-inducing factor controls programmed cell death and laccase expression during fungal interactions

Apoptotic-like programmed cell death (PCD) is one of the main strategies for fungi to resist environmental stresses and maintain homeostasis. The apoptosis-inducing factor (AIF) has been shown in different fungi to trigger PCD through upregulating reactive oxygen species (ROS). This study identified a mitochondrial localized AIF homolog, CcAIF1, from Coprinopsis cinerea monokaryon Okayama 7. Heterologous overexpression of CcAIF1 in Saccharomyces cerevisiae caused apoptotic-like PCD of the yeast cells. Ccaif1 was increased in transcription when C. cinerea interacted with Gongronella sp. w5, accompanied by typical apoptotic-like PCD in C. cinerea, including phosphatidylserine externalization and DNA fragmentation. Decreased mycelial ROS levels were observed in Ccaif1 silenced C. cinerea transformants during cocultivation, as well as reduction of the apoptotic levels, mycelial growth, and asexual sporulation. By comparison, Ccaif1 overexpression led to the opposite phenotypes. Moreover, the transcription and expression levels of laccase Lcc9 decreased by Ccaif1 silencing but increased firmly in Ccaif1 overexpression C. cinerea transformants in coculture. Thus, in conjunction with our previous report that intracellular ROS act as signal molecules to stimulate defense responses, we conclude that CcAIF1 is a regulator of ROS to promote apoptotic-like PCD and laccase expression in fungal-fungal interactions. In an axenic culture of C. cinerea, CcAIF1 overexpression and H2O2 stimulation together increased laccase secretion with multiplied production yield. The expression of two other normally silent isozymes, Lcc8 and Lcc13, was unexpectedly triggered along with Lcc9.Key points• Mitochondrial CcAIF1 induces PCD during fungal-fungal interactions• CcAIF1 is a regulator of ROS to trigger the expression of Lcc9 for defense• CcAIF1 overexpression and H2O2 stimulation dramatically increase laccase production

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Fungal-fungal cocultivation leads to widespread secondary metabolite alteration requiring the partial loss-of-function VeA1 protein.

Microbial communication has attracted notable attention as an indicator of microbial interactions that lead to marked alterations of secondary metabolites (SMs) in varied environments. However, the mechanisms responsible for SM regulation are not fully understood, especially in fungal-fungal interactions. Here, cocultivation of an endophytic fungus Epicoccum dendrobii with the model fungus Aspergillus nidulans and several other filamentous fungi triggered widespread alteration of SMs. Multiple silent biosynthetic gene clusters in A. nidulans were activated by transcriptome and metabolome analysis. Unprecedentedly, gene deletion and replacement proved that a partial loss-of-function VeA1 protein, but not VeA, was associated with the widespread SM changes in both A. nidulans and A. fumigatus during cocultivation. VeA1 regulation required the transcription factor SclB and the velvet complex members LaeA and VelB for producing aspernidines as representative formation of SMs in A. nidulans. This study provides new insights into the mechanism that trigger metabolic changes during fungal-fungal interactions.

Deletion of all three MAP kinase genes results in severe defects in stress responses and pathogenesis in Fusarium graminearum

Mitogen-activated protein kinase (MAPK) cascades are activated by external stimuli and convert signals to cellular changes. Individual MAPKs have been characterized in a number of plant pathogenic fungi for their roles in pathogenesis and responses to biotic or abiotic stresses. However, mutants deleted of all the MAPK genes have not been reported in filamentous fungi. To determine the MAPK-less effects in a fungal pathogen, in this study we generated and characterized mutants deleted of all three MAPK genes in the wheat scab fungus Fusarium graminearum. The Gpmk1 mgv1 Fghog1 triple mutants had severe growth defects and was non-pathogenic. It was defective in infection cushion formation and DON production. Conidiation was reduced in the triple mutant, which often produced elongated conidia with more septa than the wild-type conidia. The triple mutant was blocked in sexual reproduction due to the loss of female fertility. Lack of any MAPKs resulted in an increased sensitivity to various abiotic stress including cell wall, osmotic, oxidative stresses, and phytoalexins, which are likely related to the defects of the triple mutant in environmental adaptation and plant infection. The triple mutant also had increased sensitivity to the biocontrol bacterium Bacillus velezensis and fungus Clonostachys rosea. In co-incubation assays with B. velezensis, the Gpmk1 mgv1 Fghog1 mutant had more severe growth limitation than the wild type and was defective in conidium germination and germ tube growth. In confrontation assays, the triple mutant was defective in defending against mycoparasitic activities of C. rosea and the latter could grow over the mutant but not wild-type F. graminearum. RNA-seq and metabolomics analyses showed that the MAPK triple mutant was altered in the expression of many ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes and the accumulation of metabolites related to arachidonic acid, linoleic acid, and alpha-linolenic acid metabolisms. Overall, as the first study on mutants deleted of all three MAPKs in fungal pathogens, our results showed that although MAPKs are not essential for growth and asexual reproduction, the Gpmk1 mgv1 Fghog1 triple mutant was blocked in plant infection and sexual reproductions. It also had severe defects in responses to various abiotic stresses and bacterial- or fungal-fungal interactions.

Melanin production and laccase mediated oxidative stress alleviation during fungal-fungal interaction among basidiomycete fungi

Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. In the present study, we performed two sets of fungal-fungal interaction—Trametes coccinea (F3) with Leiotrametes lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) to understand the changes in the metabolite profile during the interaction process and how this process impacts the hyphal/mycelial morphology of the participating fungi. The metabolites produced during interaction of T. coccinea (F3) with L. lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) was analysed through liquid chromatography coupled to mass spectroscopy (LC-MS). Most of the metabolites secreted or produced during interaction are associated with defensive response. Further, visualization with scanning electron microscopy revealed that interaction between the tested fungi led to the changes in the hyphal morphology. The bipartite fungal interaction resulted in the production of a dark brown colour pigment—melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal–fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further, increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal–fungal interaction. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production.

Conventional and conservation tillage practices affect soil microbial co-occurrence patterns and are associated with crop yields

A comprehensive understanding of the effects of tillage practices on soil microbial communities is fundamental to a better understanding of their roles in maintaining or improving the stability of agroecosystems. However, little is known regarding soil microbial co-occurrence patterns and their function in ecosystems shaped by long-term tillage practices. The goal of the present study was to investigate the impact of no-tillage (NT), reduced tillage (RT) and moldboard plow tillage (MP) practices on soil microbial interactions, central species and functional taxa as well as to investigate their associations with crop yields. The results showed that the average node degree was lower in the RT network than in the NT and MP networks of bulk and rhizosphere soils, indicating inefficient mutual effects among taxa and a more stable network structure induced by RT. Moldboard plow tillage resulted in the most fungal-fungal interactions (17.6%), while RT yielded the most bacterial-fungal interactions (32.3%) in the rhizosphere soils. The potential plant pathogens Fusarium were identified as hub node with most connections, and pathogen Sphingomonas and Clonostachys were found as central speices with those simultaneously termed as hub node and generalist in the MP network of rhizosphere soils. Intriguingly, these potential plant pathogens had negative correlations with crop yields in both bulk and rhizosphere soils, as determined by structural equation modeling (SEM). In contrast, RT might help to alleviate the ecological risks of the potential plant pathogens and increased the availability of soil nutrients mediated by central species, such as Nitrospira. These findings suggested RT as an optimal practice that could stabilize microbial network structure, alleviated the potential transmission of pathogens and finally enhance crop yields, providing a new perspective for tillage application in agroecosystems.

LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea.

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Δlysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Δlysm2 and Δlysm1Δlysm2 strains displayed reduced ability to colonize wheat roots, while only Δlysm1Δlysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol.

Tropical Palm Endophytes Exhibit Low Competitive Structuring When Assessed Using Co-occurrence and Antipathogen Activity Analysis

Leaf-inhabiting fungal and bacterial endophytes are at their most diverse in tropical rainforest plant hosts, with some influencing host plant fitness as either symbionts or pathogens. Endophyte activity and community composition is thought to depend on competition amongst co-occurring species for resources. Here, we attempt to reveal the strength of competitive interactions between endophytes by combining e-DNA metabarcoding to characterize the community with culturing assays to ascertain their potential activity. The endophyte community associated with the understory palm Astrocaryum sciophilum was determined by extracting eDNA from 120 leaflets of eight plants located in a lowland rainforest of French Guiana. This revealed 516 fungal and 606 bacterial OTUs. Co-occurrence analysis of the most abundant OTUs revealed that direct fungal-fungal interactions were overall more negative than bacterial-bacterial interactions. This was confirmed by C-score calculations for the whole endophyte community, which revealed significantly greater levels of species segregation for fungi but not bacteria when compared with simulated random communities. Following the culturing of 131 fungal, and 66 bacterial Astrocaryum endophytes collected from the same leaves, Minimal Inhibitory Concentrations of ethyl acetate culture extracts were evaluated against the fungus Trichophyton rubrum and the bacterium Staphylococcus aureus. Overall, a minority of extracts demonstrated antipathogen activity, with greater antifungal activity expression for both fungi and bacteria when compared with antibacterial activity. In order to explore whether this activity translated into competitive structuring of the observed community, a BLAST was conducted to match metabarcoding sequences with Sanger derived culture sequences. This demonstrated that cultures displaying antipathogen activity were significantly more likely to co-occur with fungi if they were bacteria and bacteria if they were fungi. Nevertheless, overall correlation values averaged around zero. These results indicate that for Astrocaryum palms, fungal endophytes are likely to play a greater role in determining colonization success of subsequent endophytes whether these be fungal or bacterial, but that overall endophyte communities do not display strong patterns of community structuring through competition. Endophyte communities are thus likely to reach relative stasis in mature leaves, where the diverse community of fungi and bacteria and plant traits prevent the subsequent establishment of pathogens.

Comparative evolutionary histories of fungal proteases reveal gene gains in the mycoparasitic and nematode-parasitic fungus Clonostachys rosea

BackgroundThe ascomycete fungus Clonostachys rosea (order Hypocreales) can control several important plant diseases caused by plant pathogenic fungi and nematodes. Subtilisin-like serine proteases are considered to play an important role in pathogenesis in entomopathogenic, mycoparasitic, and nematophagous fungi used for biological control. In this study, we analysed the evolutionary histories of protease gene families, and investigated sequence divergence and regulation of serine protease genes in C. rosea.ResultsProteases of selected hypocrealean fungal species were classified into families based on the MEROPS peptidase database. The highest number of protease genes (590) was found in Fusarium solani, followed by C. rosea with 576 genes. Analysis of gene family evolution identified non-random changes in gene copy numbers in the five serine protease gene families S1A, S8A, S9X, S12 and S33. Four families, S1A, S8A, S9X, and S33, displayed gene gains in C. rosea. A gene-tree / species-tree reconciliation analysis of the S8A family revealed that the gene copy number increase in C. rosea was primarily associated with the S08.054 (proteinase K) subgroup. In addition, regulatory and predicted structural differences, including twelve sites evolving under positive selection, among eighteen C. rosea S8A serine protease paralog genes were also observed. The C. rosea S8A serine protease gene prs6 was induced during interaction with the plant pathogenic species F. graminearum.ConclusionsNon-random increases in S8A, S9X and S33 serine protease gene numbers in the mycoparasitic species C. rosea, Trichoderma atroviride and T. virens suggests an involvement in fungal-fungal interactions. Regulatory and predicted structural differences between C. rosea S8A paralogs indicate that functional diversification is driving the observed increase in gene copy numbers. The induction of prs6 expression in C. rosea during confrontation with F. graminearum suggests an involvement of the corresponding protease in fungal-fungal interactions. The results pinpoint the importance of serine proteases for ecological niche adaptation in C. rosea, including a potential role in the mycoparasitic attack on fungal prey.

Functional analysis of polyketide synthase genes in the biocontrol fungus Clonostachys rosea

Clonostachys rosea is a mycoparasitic fungus used for biological control of plant diseases. Its genome contains 31 genes putatively encoding for polyketide synthases (PKSs), 75% of which are arranged in biosynthetic gene clusters. Gene expression analysis during C. rosea interactions with the fungal plant pathogens Botrytis cinerea and Fusarium graminearum showed common and species-specific induction of PKS genes. Our data showed a culture media dependent correlation between PKS gene expression and degree of antagonism in C. rosea. The pks22 and pks29 genes were highly induced during fungal-fungal interactions but not during pigmentation, and gene deletion studies revealed that PKS29 was required for full antagonism against B. cinerea, and for biocontrol of fusarium foot rot on barley. Metabolite analysis revealed that Δpks29 strains has a 50% reduced production (P = 0.001) of an unknown polyketide with molecular formula C15H28O3, while Δpks22 strains lost the ability to produce four previously unknown polyketides named Clonorosein A-D. Clonorosein A and B were purified, their structures determined, and showed strong antifungal activity against B. cinerea and F. graminearum. These results show that PKS22 is required for production of antifungal polyketide Clonorosein A-D, and demonstrate the role of PKS29 in antagonism and biocontrol of fungal plant diseases.

Aspergillus piperis A/5 from plum-distilling waste compost produces a complex of antifungal metabolites active against the phytopathogen Pythium aphanidermatum

Adding compost to soil can result in plant disease suppression through the mechanisms of antagonistic action of compost microflora against plant pathogens. The aim of the study was to select effective antagonists of Pythium aphanidermatum from compost, to assess the effect of its extracellular metabolites on the plant pathogen, and to characterize antifungal metabolites. The fungal isolate selected by a confrontation test was identified as Aspergillus piperis A/5 on the basis of morphological features and the internal transcribed spacer (ITS) region, ?-tubulin and calmodulin partial sequences. Liquid chromatography-mass spectroscopy (LC-MS) analysis showed that gluconic and citric acid were the most abundant in the organic culture extract. However, the main antifungal activity was contained in the aqueous phase remaining after the organic solvent extraction. The presence of considerable amounts of proteins in both the crude culture extract as well as the aqueous phase remaining after solvent extraction was confirmed by SDS-PAGE. Isolated Aspergillus piperis A/5 exhibits strong antifungal activity against the phytopathogen Pythium aphanidermatum. It secretes a complex mixture of metabolites consisting of small molecules, including gluconic acid, citric acid and itaconic acid derivatives, but the most potent antifungal activity was associated with proteins resistant to heat and organic solvents. Our findings about the activity and characterization of antagonistic strain metabolites contribute to the understanding of the mechanism of interaction of antifungal metabolites as well as fungal-fungal interaction. The obtained results provide a basis for further application development in agriculture and food processing.

Filamentous fungi isolated from grape marc as antagonists of Botrytis cinerea

In this paper we report on the isolation and identification of three filamentous fungi from grape marc, and antifungal effect of their cell-free culture filtrates on the growth of Botrytis cinerea, causal agent of gray mold. Grape marc is a waste material that has been used as soil amendment in sustainable agriculture. Isolates originating from grape marc were identified on the basis of morphological features and internal transcribed spacer rDNA or ?-tubulin gene sequencing. The presence of three different species, Penicillium paneum, Penicillium chrysogenum and Aspergillus fumigatus has been detected expressing different effect on the growth of B. cinerea. The effect of crude culture filtrates of selected fungi on B. cinerea growth was tested. Heat sensitivity of the established inhibition effect was examined by autoclaving the crude culture filtrate prior to testing. Additional aim was to determine whether antifungal effect was influenced by previous exposure to B. cinerea in dual liquid cultures. Crude culture filtrate of A. fumigatus K16/2 showed the lowest suppression of B. cinerea growth. A maximal percentage inhibition achieved within the study was 38.2%, 39.8% and 23.8 for crude filtrates of P. paneum K7/1, P. chrysogenum K11/1 and A. fumigatus K16/2, respectively. Presence of B. cinerea in dual liquid culture induced significant increase in antifungal capacity of the culture filtrates in comparison to pure culture filtrates of the chosen isolates. The antifungal activity of all of the isolates? culture filtrates retained after heat treatment suggesting the presence of some thermostable antifungal metabolites. The results indicate the complexity and specificity of the interaction between filamentous fungi and B. cinerea. Grape marc is a good source for isolation od B. cinerea fungal antagonists and their antifungal metabolites. Specificity of fungal-fungal interactions suggests that further research on the antagonistic mechanisms and factors affecting them should be studied separately for each pair of antagonists.

Identifying glycoside hydrolase family 18 genes in the mycoparasitic fungal species Clonostachys rosea

Clonostachysrosea is a mycoparasitic fungal species that is an efficient biocontrol agent against many plant diseases. During mycoparasitic interactions, one of the most crucial steps is the hydrolysis of the prey's fungal cell wall, which mainly consists of glucans, glycoproteins and chitin. Chitinases are hydrolytic enzymes responsible for chitin degradation and it is suggested that they play an important role in fungal-fungal interactions. Fungal chitinases belong exclusively to the glycoside hydrolase (GH) family 18.These GH18 proteins are categorized into three distinct phylogenetic groups (A, B and C), subdivided into several subgroups. In this study, we identified 14 GH18 genes in the C. rosea genome, which is remarkably low compared with the high numbers found in mycoparasitic Trichoderma species. Phylogenetic analysis revealed that C. rosea contains eight genes in group A, two genes in group B, two genes in group C, one gene encoding a putative ENGase (endo-β-N-acetylglucosaminidase) and the ech37 gene, which is of bacterial origin. Gene expression analysis showed that only two genes had higher transcription levels during fungal-fungal interactions, while eight out of 14 GH18 genes were triggered by chitin. Furthermore, deletion of the C group chiC2 gene decreased the growth inhibitory activity of C. rosea culture filtrates against Botrytis cinerea and Rhizoctonia solani, although the biocontrol ability of C. rosea against B. cinerea was not affected. In addition, a potential role of the CHIC2 chitinase in the sporulation process was revealed. These results provide new information about the role of GH18 proteins in mycoparasitic interactions.

Influence of Hyphal Inoculum potential on the Competitive Success of Fungi Colonizing Wood.

The relative amounts of hyphal inoculum in forest soils may determine the capacity for fungi to compete with and replace early colonizers of wood in ground contact. Our aim in this study was to test the flexibility of priority effects (colonization timing) by varying the timing of inoculum introduction (i.e., precolonization) and amount of inoculum (i.e., inoculum potential). We controlled these variables in soil-block microcosms using fungi with known competitive outcomes in similar conditions, tracking isolate-specific fungal biomass, and residue physiochemistry over time. In the precolonization trial (experiment I), a brown rot fungus Gloeophyllum trabeum was given 1, 3, or 5weeks to precolonize wood blocks (oak, birch, pine, and spruce) prior the introduction of a white rot fungus, Irpex lacteus, a more aggressive colonizer in this set-up. In the inoculum potential trial (experiment II), the fungi were inoculated simultaneously, but with eightfold higher brown rot inoculum than that of experiment I. As expected, longer precolonization duration increased the chance for the less-competitive brown rot fungus to outcompete its white rot opponent. Higher brown rot fungal inoculum outside of the wood matrix also resulted in competitive success for the brown rot isolate in most cases. These temporal shifts in fungal dominance were detectable in a 'community snapshot' as isolate-specific quantitative PCR, but also as functionally-relevant consequences of wood rot type, including carbohydrate depolymerization and pH. These results from a controlled system reinforce fungal-fungal interaction and suggest that relative inoculum availability beyond the wood matrix (i.e., soils) might regulate the duration of priority effects and shift the functional trajectory of wood decomposition.

PacC and pH-dependent transcriptome of the mycotrophic fungus Trichoderma virens.

BackgroundIn fungi, environmental pH is an important signal for development, and successful host colonization depends on homeostasis. Surprisingly, little is known regarding the role of pH in fungal-fungal interactions. Species of Trichoderma grow as soil saprobes but many are primarily mycotrophic, using other fungi as hosts. Therefore, Trichoderma spp. are studied for their potential in biocontrol of plant diseases. Particularly in alkaline soil, pH is a critical limiting factor for these biofungicides, whose optimal growth pH is 4–6. Gaining an understanding of pH adaptability is an important step in broadening the activity spectrum of these economically important fungi.ResultsWe studied the pH-responsive transcription factor PacC by gene knockout and by introduction of a constitutively active allele (pacCc). ΔpacC mutants exhibited reduced growth at alkaline pH, while pacCc strains grew poorly at acidic pH. In plate confrontation assays ΔpacC mutants showed decreased ability to compete with the plant pathogens Rhizoctonia solani and Sclerotium rolfsii. The pacCc strain exhibited an overgrowth of R. solani that was comparable to the wild type, but was unable to overgrow S. rolfsii. To identify genes whose expression is dependent on pH and pacC, we designed oligonucleotide microarrays from the transcript models of the T. virens genome, and compared the transcriptomes of wild type and mutant cultures exposed to high or low pH. Transcript levels from several functional classes were dependent on pacC, on pH, or on both. Furthermore, the expression of a set of pacC-dependent genes was increased in the constitutively-active pacCc strain, and was pH-independent in some, but not all cases.ConclusionsPacC is important for biocontrol-related antagonism of other fungi by T. virens. As much as 5% of the transcriptome is pH-dependent, and of these genes, some 25% depend on pacC. Secondary metabolite biosynthesis and ion transport are among the relevant gene classes. We suggest that ΔpacC mutants may have lost their full biocontrol potential due to their inability to adapt to alkaline pH, to perceive ambient pH, or both. The results raise the novel possibility of genetically manipulating Trichoderma in order to improve adaptability and biocontrol at alkaline pH.

Bacterial and fungal chitinase chiJ orthologs evolve under different selective constraints following horizontal gene transfer

BackgroundCertain bacteria from the genus Streptomyces are currently used as biological control agents against plant pathogenic fungi. Hydrolytic enzymes that degrade fungal cell wall components, such as chitinases, are suggested as one possible mechanism in biocontrol interactions. Adaptive evolution of chitinases are previously reported for plant chitinases involved in defence against fungal pathogens, and in fungal chitinases involved in fungal-fungal interactions. In this study we investigated the molecular evolution of chitinase chiJ in the bacterial genus Streptomyces. In addition, as chiJ orthologs are previously reported in certain fungal species as a result from horizontal gene transfer, we conducted a comparative study of differences in evolutionary patterns between bacterial and fungal taxa.FindingsChiJ contained three sites evolving under strong positive selection and four groups of co-evolving sites. Regions of high amino acid diversity were predicted to be surface-exposed and associated with coil regions that connect certain α-helices and β-strands in the family 18 chitinase TIM barrel structure, but not associated with the catalytic cleft. The comparative study with fungal ChiJ orthologs identified three regions that display signs of type 1 functional divergence, where unique adaptations in the bacterial and fungal taxa are driven by positive selection.ConclusionsThe identified surface-exposed regions of chitinase ChiJ where sequence diversification is driven by positive selection may putatively be related to functional divergence between bacterial and fungal orthologs. These results show that ChiJ orthologs have evolved under different selective constraints following the horizontal gene transfer event.

Sphaerodes mycoparasitica biotrophic mycoparasite of 3-acetyldeoxynivalenol- and 15-acetyldeoxynivalenol-producing toxigenic Fusarium graminearum chemotypes

Fusarium spp. are economically important crop pathogens and causal agents of Fusarium head blight (FHB) of cereals worldwide. Of the FHB pathogens, Fusarium graminearum 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) are the most aggressive mycotoxigenic chemotypes, threatening food and feed quality as well as animal and human health. The objective of the study was to evaluate host specificity and fungal-fungal interactions of Sphaerodes mycoparasitica- a recently described mycoparasite - with F. graminearum 3- and 15-ADON strains by employing in vitro, microscopic and PCR techniques. Results obtained in this study show that the germination of mycoparasite ascospore in the presence of F. graminearum 3- and 15-ADON filtrates was greatly improved compared with Fusarium proliferatum and Fusarium sporotrichioides filtrates, suggesting a compatible interaction. Using quantitative real-time PCR with Fusarium-specific (Fg16N) and trichothecene Tri5 (Tox5-1/2)-specific primer sets, S. mycoparasitica was found to reduce the amount of F. graminearum 3-ADON and 15-ADON DNAs under separate coinoculation assays. Sphaerodes mycoparasitica was not only able to germinate in the presence of F. graminearum filtrates, but also established biotrophic mycoparasitic relations with two F. graminearum chemotypes and suppressed Fusarium growth.