Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens

Abstract

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.

Importance: Plant-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.

FIG 1

Genome organizations and phylogenetic relationships of putative negative-stranded RNA virus genomes detected from Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. (A) Comparison of the organizations of putative monopartite negative-stranded RNA viruses Sclerotinia sclerotiorum negative-stranded RNA virus 2 (SsNSRV2), Sclerotinia sclerotiorum negative-stranded RNA virus 3 (SsNSRV3), and Sclerotinia sclerotiorum negative-stranded RNA virus 4 (SsNSRV4) to Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV1). Open reading frames (ORFs) are shown as boxes and staggered to indicate the reading frame. (B) Maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of SlaMyfV1 and other confirmed and proposed members of the order Mononegavirales. Predicted RdRp amino acid sequences were aligned with MUSCLE, and trees were inferred using MEGA6. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Bootstrap percentages greater than 50% are shown.

FIG 2

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of assembled sequences from Macrophomina phaseolina and Sclerotinia sclerotiorum with similarities to members of the Fusariviridae or Hypoviridae. (A) Comparisons of the organizations of Cryphonectria hypovirus 1 (CHV1), Cryphonectria hypovirus 3 (CHV3), Macrophomina phaseolina hypovirus 1 (MpHV1), Macrophomina phaseolina single-stranded RNA virus 1 (MpSRV1), Rosellinia necatrix fusarivirus 1 (RnFV1), and Sclerotinia sclerotiorum hypovirus 2-L (SsHV2L). Open reading frames (ORFs) are shown as boxes and staggered to indicate the reading frame. (B) For the maximum likelihood tree, predicted RdRp amino acid sequences were aligned, and phylogenetic trees were constructed as described in the legend to Fig. 1. The Plum pox virus RdRp amino acid sequence was used as an outgroup to root the tree.

FIG 3

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of contigs assembled from Rhizoctonia solani and Sclerotinia sclerotiorum and confirmed and proposed members of the Endornaviridae. (A) Comparisons of the organizations of Rhizoctonia solani endornavirus 2 (RsEV2), Sclerotinia sclerotiorum endornavirus 1 Lactuca (SsEV1L), and Sclerotinia sclerotiorum endornavirus 2 (SsEV2-IL). (B) Predicted RdRp amino acid sequences were aligned and phylogenetic trees were constructed as described in the legend to Fig. 1. The Barley yellow mosaic virus RdRp amino acid sequence was used as an outgroup to root the tree. Asterisks indicate incomplete sequences.

FIG 4

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of contigs assembled from Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum with confirmed and proposed members of the family Narnaviridae and genus Ourmiavirus. (A) Comparisons of the organizations of Epirus cherry virus (EpCV), Phytophthora infestans RNA virus 4 (PiRV4), and Sclerotinia sclerotiorum mitovirus 20 (SsMV20). (B) Predicted RdRp amino acid sequences were aligned and phylogenetic trees were constructed as described in the legend to Fig. 1.

FIG 5

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of an assembled sequence from Sclerotinia sclerotiorum with members of the Tombusviridae. (A) Comparisons of the organizations of carnation mottle virus (CarMV), Diaporthe ambiqua RNA virus 1 (DaRV1), Magnaporthe oryzae RNA virus (MoRV), and Sclerotinia sclerotiorum umbra-like virus 1 (SsULV1). Open reading frames (ORFs) are shown as boxes and staggered to indicate the reading frame. (B) For the maximum likelihood tree, predicted RdRp amino acid sequences were aligned and phylogenetic trees were constructed as described in the legend to Fig. 1.

FIG 6

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of sequences assembled from two isolates of Macrophomina phaseolina with members of the Virgaviridae. (A) Comparisons of the genome organizations of Obuda pepper virus (ObPV), Macrophomina phaseolina tobamo-like virus (MpTLV1), and Macrophomina phaseolina tobamo-like virus a (MpTLV1a). Open reading frames (ORFs) are shown as boxes and staggered to indicate the reading frame. (B) For the maximum likelihood tree, predicted RdRp amino acid sequences were aligned and phylogenetic trees were constructed as described in the legend to Fig. 1. Regions encoding the coat protein (CP), helicase (Hel), methyltransferase (Met), movement protein (MP), and RdRp are indicated in the corresponding ORFs.

FIG 7

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) of sequences assembled from Rhizoctonia solani with members of the order Tymovirales. (A) Comparisons of the organizations of grapevine fleck virus (GFkV), mushroom bacilliform virus (MBV), Rhizoctonia solani barnavirus 1 (RsBV1), and Rhizoctonia solani positive-stranded RNA virus 1 (RsPSV1). Open reading frames (ORFs) are shown as boxes and staggered to indicate the reading frame. (B) For the maximum likelihood tree, predicted RdRp amino acid sequences were aligned, and phylogenetic trees were constructed as described in the legend to Fig. 1. Regions encoding the coat protein (CP), helicase (Hel), methyltransferase (Met), movement protein (MP), and RdRp are indicated in the corresponding ORFs.

FIG 8

Genome organizations and maximum likelihood tree depicting the relationships of the predicted amino acid sequences of RNA-dependent RNA polymerases (RdRps) from contigs assembled from Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum and mycoviruses with double-stranded RNA genomes. (A) Comparisons of the genome organizations of Colletotrichum truncatum partitivirus 1 (CtParV1), Diaporthe longicolla totivirus 1 (DpTotV1), Fusarium graminearum double-stranded RNA mycovirus 3 (FgV3), Macrophomina phaseolina double-stranded RNA virus 2 (MpDSRV2), Macrophomina phaseolina chrysovirus 1 (MpChrV1), Rhizoctonia solani partitivirus 1 (RsParV1), Saccharomyces cerevisiae virus L-A (ScV-LA), Sclerotinia sclerotiorum double-stranded RNA virus 3 (SsDSRV3), Sclerotinia sclerotiorum partitivirus S (SsPVS), and Verticillium dahliae partitivirus 1 (VdPV1). Open reading frames are shown as boxes. (B) Predicted RdRp amino acid sequences were aligned and phylogenetic trees were constructed as described in the legend to Fig. 1. Regions encoding the coat protein (CP) and RdRp are indicated in the corresponding ORFs.