Antarctica's hidden mycoviral treasures in fungi isolated from mosses: A first genomic approach.

This study investigates the presence of mycoviruses in Antarctic fungi and elucidates their evolutionary relationships. To achieve this, we aligned mycoviral gene sequences with genomes of previously sequenced Antarctic endophytic fungi, made available by our research group and accessible via Joint Genome Institute. Our findings reveal that the most prevalent genetic regions in all endophytic fungi are homologous to Partitiviruses, Baculoviridae, and Phycodnaviridae. These regions display evidence of positive selection pressure, suggesting genetic diversity and the accumulation of nonsynonymous mutations. This phenomenon implies a crucial role for these regions in the adaptation and survival of these fungi in the challenging Antarctic ecosystems. The presence of mycoviruses in Antarctic endophytic fungi may indicate shared survival strategies between the virus and its host, shedding light on their evolutionary dynamics. This study underscores the significance of exploring mycoviruses within endophytic fungi and their contributions to genetic diversity. Future research avenues could delve into the functional implications of these conserved mycoviral genetic regions in Antarctic endophytic fungi, providing a comprehensive understanding of this intriguing association and genomic retention of viral region in fungi.

Isolation, molecular characterization and virome analysis of culturable wood fungal endophytes in esca symptomatic and asymptomatic grapevine plants.

Europe is the world largest grape producer, but in recent years, the report of diseases due to infection by grapevine trunk pathogens (GTPs) is becoming one of the main constraints for viticulture. Among grapevine trunk diseases (GTDs), esca syndrome is one of the most complex, characterized by simultaneous infection of several fungi, which leads to important reduction in yield and quality. Previous characterization of fungal isolates associated with esca syndrome leads to the recognition of at least three important players: Phaeocremonium minimum, Phaemoniella chlamydospora and Fomitiporia mediterranea. Here we isolated and characterized molecularly fungal endophytes directly from field wood tissues of plants showing or not esca symptoms. In addition, to better characterize such collection, a deep RNA sequencing (100 M reads in paired-end) to screen for mycovirus presence was performed. Thirty-nine viral genomes were detected, 38 of which were putative new viral species; some of these viruses infected GTPs, including P. minimum and F. mediterranea. In this work, we reported for the first time a curated collection of grapevine fungal endophytes identifying the associated mycoviruses some of which could be employed in future biotechnological exploitation as biological control agents for sustainable plant protection.

Production of fumonisins by endophytic strains of Tolypocladium cylindrosporum and its relation to fungal virus infection.

Fumonisins were first discovered in Fusarium verticillioides, a fungus associated to disease and asymptomatic infections in maize. Afterwards, other fungal taxa have been found to produce fumonisins. The entomopathogenic ascomycete Tolypocladium cylindrosporum has been isolated from soil and also as an endophyte from leaves of grasses. The objectives of this work were to determine the in vitro production of fumonisin B (FB) mycotoxins and the immunosuppressive compound cyclosporine A (CyA) in several strains of T. cylindrosporum, and to examine the effect of fungal virus infection and temperature in FB production. FB1 was detected in 30% of the strains, ranging from 0.16 to 5.52 µg cm-2 in solid media, and FB2 was detected in 78% of the strains, ranging from 0.764 to 40.92 µg cm-2. CyA was not detected in any strain. The mean FB2 concentration of the endophytic strain Tc37W was three times greater (p < 0.05) than that of any other strain. Up to 34% more of FB2 was detected in strains infected by the virus TcV3 than in the corresponding virus-free versions. The effect of temperature on FB2 content was interactively significantly dependent on fungal strain and growth medium; in the YES medium, the FB2 of virus-infected strains Tc37-1V and Tc37W increased by 67 and 16%, respectively, at 26 °C as compared to 20 °C. The FB concentration in some fungal strains was similar to that in fungi associated to food and feed intoxications.

Mycoviruses of an endophytic fungus can replicate in plant cells: evolutionary implications.

So far there is no record of a specific virus able to infect both fungal and plant hosts in nature. However, experimental evidence shows that some plant virus RdRPs are able to perform replication in trans of genomic or DI RNAs in the yeast Saccharomyces cerevisiae. Furthermore, tobacco mosaic virus was recently shown to replicate in a filamentous ascomycetous fungus. Thus, at least experimentally, some plant viruses can infect some fungi. Endophytic fungi have been reported from many plants and several of these fungi have been shown to contain viruses. Here we tested if mycoviruses derived from a marine plant endophyte can replicate in plant cells. For this purpose, we used partially purified viral particles from isolate MUT4330 of Penicillium aurantiogriseum var. viridicatum which harbors six virus species, some having dsRNA and some positive-strand ssRNA genomes. These were transfected into three distinct plant protoplast cell systems. Time-course analysis of absolute RNA accumulation provided for the first time evidence that viruses of two species belonging to the Partitiviridae and Totiviridae families, can replicate in plant cells without evidence of host adaptation, i.e, changes in their nucleotide sequence.

Investigation of mycoviruses in endophytic and phytopathogenic strains of Colletotrichum from different hosts.

Fungi belonging to the Colletotrichum genus can be categorized as endophytic or phytopathogenic. These fungi can be infected by viruses, termed mycoviruses, which are know to promote hypovirulence in infected fungi. However, there are few studies that have described mycoviral infections of endophytes. The production of secondary metabolites by endophytes with antimicrobial potential in inhibiting numerous pathogens has gained increasing attention. The aim of the current study was to investigate the presence of mycoviruses in endophytic and phytopathogenic fungi of the Colletotrichum genus, as well as to analyze the antimicrobial activity of crude extracts obtained from these samples. To detect the presence of mycoviruses in the samples, dsRNA was extracted, treated with enzymes, and analyzed following electrophoresis in agarose gel. Furthermore, isometric mycoviral particles were observed by transmission electron microscopy. Serial microdilution methodology was used to test crude extracts of Colletotrichum spp for antibacterial activity against Escherichia coli and Staphylococcus aureus, and antifungal activity against Fusarium solani. The results of the molecular and microscopic analyses indicated that a phytopathogenic strain presented infection by mycovirus. The antibacterial activity analysis revealed that the minimum inhibitory concentrations and minimum bactericidal concentrations were low for the fungal extracts of the two endophytes, indicating that these extracts were effective antibacterial agents. However, their antifungal activity against F. solani was not statistically different compared to that of the negative control.

Multiplexed interactions: viruses of endophytic fungi.

Mycoviruses have been detected from all four classes of fungal endophytes. The virus species richness is probably extremely high in endophytes. The incidence and diversity of mycoviruses may be affected by transmission modes, virus-fungus-plant interactions, and endophyte population structures. Endophyte viruses are unlikely to be strong antagonists to their fungal hosts and can clearly play mutualistic roles in the multiplex symbioses with endophytes and plants under some environmental conditions. A better understanding of fungal endophyte viruses will help prospects of future applications for sustainable agriculture.

[Plasmid pCQ4 and its phage phiCQ4 of endophytic Streptomyces sp. from Artemisia annua L].

OBJECTIVE: Large plasmid pCQ4 was detected in Streptomyces sp. W75 from Artemisia annua L. We clonined, sequenced, analyzed and characterized pCQ4. METHODS: Southern hybridization was used to determine restriction map of pCQ4. To clone the full-length of pCQ4, conjugation and recombinational cloning in a BAC vector were used. RESULTS: The complete nucleotide sequence of pCQ4 consisted of 84833-bp, encoding 129 ORFs which 40 ORFs resembled these of bacterial phages. W75 culture could infect W75 cured of pCQ4 and formed plaques on plate. Phage particle (phiCQ4) was observed by transmission electron microscopy. LinearphiCQ4 DNA was detected on pulsed-field gel electrophoresis. Comparison to Streptomyces plasmid-phage pZL12, genes encoding major phage structural proteins resembled that of pCQ4. CONCLUSION: Streptomyces plasmid pCQ4 could be transformed into lytic phagephiCQ4, and the phage segment on pCQ4 might be a mobile unit.

Are communities of microbial symbionts more diverse than communities of macrobial hosts?

In this study, fungal viruses (mycoviruses) of plant-associated fungi were used to test the general assertion that communities of parasitic and mutualistic symbionts may be more species-diverse than communities of their hosts. Mycoviruses are poorly studied in general, but can affect the fitness and ecology of the fungi and plants with which they associate. To date, mycovirus incidence and diversity in natural communities remain largely unaddressed. Here, we compared the incidence and diversity of fungi associated with tallgrass prairie plants to the diversity and incidence of mycoviruses within those fungi. Specifically, we sampled viruses from fungi associated with a parasitic plant (Cuscuta cuspidata) and its most frequent host plant (Ambrosia psilostachya) in a tallgrass prairie habitat in Oklahoma. For each plant sample we cultured fungal endophytes from surface-sterilized above-ground tissues. From the cultured fungi we extracted DNA to identify fungi, and extracted double-stranded RNA (dsRNA) to detect mycoviruses. Mycoviruses were further characterized using reverse transcription-PCR and sequence analyses. We found at least 25 fungal taxa associated with the two plants, and 10 % of these fungi contained readily detectable viruses. Several mycovirus types were shared among fungal taxa, indicating that mycoviruses may be less specialized than originally thought. Although the virus community was not as diverse as the fungal endophyte community (16 taxa), species accumulation rates of mycoviruses (inferred from rescaled rarefaction curves) may be higher than those of their associated fungal hosts. Thus, mycoviruses represent a further layer of undocumented biodiversity in ecological communities.