Common but Nonpersistent Acquisitions of Plant Viruses by Plant-Associated Fungi

Abstract

Investigating a virus's host range and cross-infection is important for better understanding the epidemiology and emergence of viruses. Previously, our research group discovered a natural infection of a plant RNA virus, cumber mosaic virus (genus Cucumovirus, family Bromoviridae), in a plant pathogenic basidiomycetous fungus, Rhizoctonia solani, isolated from a potato plant grown in the field. Here, we further extended the study to investigate whether similar cross-infection of plant viruses occurs widely in plant-associated fungi in natural conditions. Various vegetable plants such as spinach, leaf mustard, radish, celery, and other vegetables that showed typical virus-like diseases were collected from the fields in Shandong Province, China. High-throughput sequencing revealed that at least 11 known RNA viruses belonging to different genera, including Potyvirus, Fabavirus, Polerovirus, Waikavirus, and Cucumovirus, along with novel virus candidates belonging to other virus genera, infected or associated with the collected vegetable plants, and most of the leaf samples contained multiple plant viruses. A large number of filamentous fungal strains were isolated from the vegetable leaf samples and subjected to screening for the presence of plant viruses. RT-PCR and Sanger sequencing of the PCR products revealed that among the 169 fungal strains tested, around 50% were carrying plant viruses, and many of the strains harbored multiple plant viruses. The plant viruses detected in the fungal isolates were diverse (10 virus species) and not limited to particular virus genera. However, after prolonged maintenance of the fungal culture in the laboratory, many of the fungal strains have lost the virus. Sequencing of the fungal DNA indicated that most of the fungal strains harboring plant viruses were related to plant pathogenic and/or endophytic fungi belonging to the genera Alternaria, Lecanicillium, and Sarocladium. These observations suggest that the nonpersistent acquisition of plant viruses by fungi may commonly occur in nature. Our findings highlight a possible role for fungi in the life cycle, spread, and evolution of plant viruses.

Keywords: cross-infection; fungi; plant viruses; virus acquisition; virus transmission.

Figure 1

Schematic diagram illustrating the experimental procedures for the screening of fungal strains with plant virus infections.

Figure 2

Representative vegetable plants showing typical virus-like disease symptoms collected as the sample material.

Figure 3

Schematic genome structures of four novel RNA viruses (named Qingdao RNA virus 1–4, QRV1–4) detected in the leaf samples. The name and length of sequence contigs from which the virus genomes were built are presented. A putative missing region (or segment) of ORV1 and possible RNA segments of QRV3 and ORV4 are also presented. The bold line and dashed line represent the genome sequence and unknown sequences of the 5′ and 3′ terminal regions. The colored boxes represent predicted open reading frames (ORFs). The conserved domains in the predicted viral proteins are shown with a blue bar along with the domain name and its E-value according to the NCBI conserved domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 26 August 2022).

Figure 4

Phylogenetic relationships of QRV1, QRV2, QRV3, and QRV4 with their related viruses. The virus names are followed by their accession numbers. Poleroviruses, narnaviruses, and peribunyaviruses were used as outgroups in the tree for QRV1, QRV2, and QRV3, respectively. The trees were refined using FigTree ver. 1.3.1 and the scale bar represents amino acid distances. The numbers at the nodes indicate aLRT values determined using an SH-like calculation (>0.9 are displayed). Some phylogroups were collapsed into a triangle. The names and accession numbers of these viruses are shown below the trees.

Figure 5

Detection of plant viruses in the fungal strains. (A) Nested RT-PCR detection of BrYV in the fungal strains isolated from the napa cabbage sample. (B) Detection of CMV in the fungal strains by RT-PCR and Northern blotting. * indicates undefined bands.

Figure 6

Phenotypic growth of representative fungal strains infected with plant viruses. The colonies were grown on PDA medium for 7 days and then photographed.