Comparative Small RNA and Degradome Sequencing Provides Insights into Antagonistic Interactions in the Biocontrol Fungus Clonostachys rosea.

Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact, and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small RNAs (sRNAs) are vital components of posttranscriptional gene regulation, although their role in gene expression regulation during mycoparasitisms remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tag sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant-pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were downregulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing desuppression (upregulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated potential gene targets for 282 differentially expressed sRNAs. Analysis of sRNA potential gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. The regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum, highlighting common and mycohost-specific gene regulation of C. rosea. By combining these results with transcriptome data collected during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism. IMPORTANCE Small RNAs (sRNAs) are emerging as key players in pathogenic and mutualistic fungus-plant interactions; however, their role in fungus-fungus interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and the plant-pathogenic mycohosts Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs, highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel microRNAs (miRNAs) and their potential gene targets at the endogenous level and at a cross-species level in B. cinerea and F. graminearum, indicating a role of cross-species RNA interference (RNAi) in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.

Geosmithia-Ophiostoma: a New Fungus-Fungus Association.

In Europe as in North America, elms are devastated by Dutch elm disease (DED), caused by the alien ascomycete Ophiostoma novo-ulmi. Pathogen dispersal and transmission are ensured by local species of bark beetles, which established a novel association with the fungus. Elm bark beetles also transport the Geosmithia fungi genus that is found in scolytids' galleries colonized by O. novo-ulmi. Widespread horizontal gene transfer between O. novo-ulmi and Geosmithia was recently observed. In order to define the relation between these two fungi in the DED pathosystem, O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. The impact of Geosmithia on DED severity was tested in vivo by co-inoculating Geosmithia and O. novo-ulmi in elms. A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on O. novo-ulmi. These results prove the existence of a new component in the complex of organisms involved in DED, which might be capable of reducing the disease impact.

Genome sequence and transcriptome profiles of pathogenic fungus Paecilomyces penicillatus reveal its interactions with edible fungus Morchella importuna.

Paecilomyces penicillatus is one of the pathogens of morels, which greatly affects the yield and quality of Morchella spp.. In the present study, we de novo assembled the genome sequence of the fungus P. penicillatus SAAS_ppe1. We analyzed the transcriptional profile of P. penicillatus SAAS_ppe1 infection of Morchella importuna at different stages (3 days and 6 days after infection) and the response of M. importuna using the transcriptome. The assembled genome sequence of P. penicillatus SAAS_ppe1 was 39.78 Mb in length (11 scaffolds; scaffold N50, 6.50 Mb), in which 99.7% of the expected genes were detected. A total of 7.48% and 19.83% clean transcriptional reads from the infected sites were mapped to the P. penicillatus genome at the early and late stages of infection, respectively. There were 3,943 genes differently expressed in P. penicillatus at different stages of infection, of which 24 genes had increased expression with the infection and infection stage, including diphthamide biosynthesis, aldehyde reductase, and NAD (P)H-hydrate epimerase (P < 0.05). Several genes had variable expression trends at different stages of infection, indicating P. penicillatus had diverse regulation patterns to infect M. importuna. GO function, involving cellular components, and KEGG pathways, involving glycerolipid metabolism, and plant-pathogen interaction were significantly enriched during infection by P. penicillatus. The expression of ten genes in M. importuna increased during the infection and infection stage, and these may regulate the response of M. importuna to P. penicillatus infection. This is the first comprehensive study on P. penicillatus infection mechanism and M. importuna response mechanism, which will lay a foundation for understanding the fungus-fungus interactions, gene functions, and variety breeding of pathogenic and edible fungi.

A natural association of a yeast with Aspergillus terreus and its impact on the host fungal biology.

Several fungi have been shown to harbor microorganisms that regulate the key components of fungal metabolism. We explored the symbiotic association of an endophyte, Aspergillus terreus, which led to the isolation of a yeast, Meyerozyma caribbica, as its symbiont. An axenic fungal culture, free of the symbiont, was developed to study the effect of this association on the endophytic fungus. The symbiotic yeast partner was found to play an important role in the adaptation of A. terreus to thermal as well as osmotic stress. Under these stress conditions, the symbiont enhanced the production of lovastatin and the growth of the host fungus. The symbiotic yeast was found to induce the expression of the global regulator gene, the key genes involved in the lovastatin biosynthetic pathway as well as those involved in general growth and development, under stress conditions, in the fungal partner. Analysis by PCR and fluorescent in situ hybridization microscopy indicated that the yeast may be present inside the hyphae of the fungus. However, a direct method like transmission electron microscopy may help to better understand the dynamics of this association, including the distribution of the yeast cells in/on the fungal hyphae and spores.

Experimental Analysis of Interactions Among Saprotrophic Fungi from A Phosphorous-Poor Desert Oasis in the Chihuahuan Desert.

Fungal ecological interactions play a key role in structuring community assemblages. These associations may involve both antagonistic and synergistic relationships, which are commonly influenced by abiotic factors such as nutrient conditions. However, information for extreme, oligotrophic systems remain poor. Herein, interactions among key members of the aquatic transient fungal community (Aspergillus niger, Cladosporium sp., and Coprinellus micaceus) of a low-nutrient freshwater system in the Cuatro Ciénegas Basin, Mexico were studied. Pairwise interaction bioassays were explored in vitro under different nutrient conditions, including carbohydrates-rich, carbohydrates and amino peptides-rich, and low nutrients. Our results indicated that antagonistic patterns prevail among the studied taxa. However, nutrient-dependent changes were observed in Cladosporium sp. shifting to synergy under carbohydrates-rich conditions, suggesting changes in the fungal community composition as a result of nutrient enrichment. Remarkably, our findings contrast with previous work demonstrating mainly synergistic interactions between our tested fungal isolates and co-occurring autochthonous bacteria (Aeromonas spp. and Vibrio sp.) under low-nutrient conditions. This observation may indicate that bacteria and fungi exhibit distinct community-level responses, driven by nutrient conditions. This contributes to the knowledge of fungal community dynamics and interspecific interactions in an oligotrophic ecosystem, highlighting the relevance of nutrient-based shifts and antagonistic interactions in ecosystem dynamics.