Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae. More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts. IMPORTANCEPlant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution. R ecent metatranscriptomic and metagenomic studies of animals, fungi, insects, plants, and environmental samples have shown that mycoviruses are ubiquitous in nature (1-10). Analyses of viral metagenomes (i.e., viromes) of environmental samples suggest that the field of virology has discovered less than 1% of the existing viral diversity, and the rate of discovery by metage...

Atmospheric change studies conducted in free air concentration enrichment (FACE) systems and open-topped chambers have increased understanding of how factors, such as rising CO 2 and O 3 levels, impact the development of plant disease epidemics. Using these systems, plant scientists have been able to study host ⁄ pathogen systems under real-world conditions where variations in multiple environmental parameters impact disease outcomes. Results from these studies are useful for evaluating earlier predictions on plant responses to climate-change parameters and the resulting impacts on plant disease epidemics. Some of these predictions have been verified, whilst others have yet to be tested. Significant interactions among climate-change parameters are highlighting the importance of conducting studies under real-world conditions. The development of molecular and gene expression tools is allowing the fine scale mechanisms responsible for the observed reactions to be determined, and should increase the ability to predict plant disease outcomes under future climatic conditions.

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Human driven changes in the Earth's atmospheric composition are likely to alter plant disease in the future. We evaluated the effects of elevated carbon dioxide (CO 2 ) and ozone (O 3 ) on three economically important soybean diseases (downy mildew, Septoria brown spot and sudden death syndrome-SDS) under natural field conditions at the soybean free air concentration enrichment (SoyFACE) facility. Disease incidence and/or severity were quantified from 2005 to 2007 using visual surveys and digital image analysis, and changes were related to microclimatic variability and to structural and chemical changes in soybean host plants. Changes in atmospheric composition altered disease expression, but responses of the three pathosystems varied considerably. Elevated CO 2 alone or in combination with O 3 significantly reduced downy mildew disease severity (measured as area under the disease progress curve-AUDPC) by 39-66% across the 3 years of the study. In contrast, elevated CO 2 alone or in combination with O 3 significantly increased brown spot severity in all 3 years, but the increase was small in magnitude. When brown spot severity was assessed in relation to differences in canopy height induced by the atmospheric treatments, disease severity increased under combined elevated CO 2 and O 3 treatment in only one of the 3 years. The atmospheric treatments had no effect on the incidence of SDS or brown spot throughout the study. Higher precipitation during the 2006 growing season was associated with increased AUDPC severity across all treatments by 2.7 and 1.4 times for downy mildew and brown spot, respectively, compared with drought conditions in 2005. In the 2 years with similar precipitation, the higher daily temperatures in the late spring of 2007 were associated with increased severity of downy mildew and brown spot. Elevated CO 2 and O 3 induced changes in the soybean canopy density and leaf age likely contributed to the disease expression modifications.

A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5=-and 3=-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5= terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related to Valsa ceratosperma hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strain Sclerotinia sclerotiorum hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession number KF898354. Sclerotinia sclerotiorum isolate 328 was coinfected with a strain of Sclerotinia sclerotiorum endornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in S. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized in vitro and transfected into a virus-free isolate of S. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L. IMPORTANCEA cosmopolitan fungus, Sclerotinia sclerotiorum infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in S. sclerotiorum were lacking. By establishing a cause-and-effect relationship between Sclerotinia sclerotiorum hypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and S. sclerotiorum, a widespread fungal pathogen of plants. Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan fungal plant pathogen that causes necrotic diseases (e.g., Sclerotinia stem rot) in more than 400 plant species, which result in yield losses in major crops each year (1-3). However, the diseases caused by S. sclerotiorum have not been adequately controlled by conventional technologies thus far (4, 5). A number of mycoviruses have been molecularly characterized and ...

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Virulence and double-stranded RNA (dsRNA) profiles of 44 isolates of Fusarium virguliforme were compared. When grouped according to dsRNA profiles, isolates with large dsRNAs were significantly (P≤0.05) less virulent than isolates without dsRNAs. High-throughput sequence analysis of total RNA prepared from cultures with large dsRNAs identified two novel RNA viruses with genome sequences of approximately 9.3 kbp, which were named Fusarium virguliforme dsRNA mycovirus 1 and Fusarium virguliforme dsRNA mycovirus 2. The new viruses were most closely related to a group of unclassified viruses that included viruses of F. graminearum and Phlebiopsis gigantea and are related to members of the family Totiviridae.