Characterization of the Mycovirome from the Plant-Pathogenic Fungus Cercospora beticola

Abstract

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (-)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future.

Keywords: Cercospora beticola; biocontrol; diversity; high-throughput sequencing; mycovirome.

Figure 1

The distribution and diversity of virus in C. beticola transcriptomes. The top graph describes the number of reads in each library and the full names of each library has been shown on the top of the bar (dodger blue). The bottom graph describes a summary of the classification of virus species found in this study. The viral families of mycoviruses were indicated by bars with different colors: Metaviridae, (−)ssRNA virus, Birnaviridae, Partitiviridae, Narnaviridae, Botourmiaviridae, Hypoviridae.

Figure 2

PCR confirmation of mycovirus contigs in C. beticola strains. (A) RT-PCR confirmation of mycovirus contigs in C. beticola strains. The viral primers were designed according to the contig sequences, and primers used are listed in Table S2. (B) DNA PCR amplification of each putative mycovirus contigs in C. beticola. Each line represented the specific strain of C. beticola contained the putative mycovirus. Line 1–13: CFNKY-BZ2-14, CFNKY-BZ2-13, WLCB0811-18, CFNKY-BZ2-8, CF1016SY-62, CFNKY-BZ2-13, XJYL-38, CF0901BZ-25, CF0901BZ-25, CF0901-6, CF0901-35, WLCB0807-13 and HEB-39. k141-52449 (related to partitiviruses) was amplified in strain HEB-39 which indicated the sequence had endogenized in genome of C. beticola. PCR amplification of TIS region with primers ITS1 and ITS4 were used as positive control for DNA quality.

Figure 3

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp of Hypovirus representative members. The insect pattern () represents viruses which can infect insects. Virus identified in this work is indicated by black arrows.

Figure 4

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp of Narnavirus representative members and other narnaviruses. Only bootstrap values above 50% are indicated. Virus identified in this work is indicated by black arrows.

Figure 5

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp of Botourmiaviridae representative members. The insect pattern () represents viruses which can infect insects, and the leaf pattern () represents viruses which can infect plants. Only bootstrap values above 50% are indicated. Viruses identified in this work are indicated by black arrows.

Figure 6

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp of Bunyavirales and Monoegavirales representative members. The insect pattern () represents viruses which can infect insects. Only bootstrap values above 50% are indicated. Viruses identified in this work are indicated by black arrows.