Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata

Abstract

Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus, and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea, a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

Keywords: Alternaria alternata; apple; defective RNA; hypovirulence; hypovirus; leaf blotch disease; mycovirus.

Figure 1

Isolation of Alternaria alternata strains carrying mycovirus-like double-stranded RNA (dsRNA) elements. (A) Phenotypic growth of A. alternata strains (Yangling isolates) on PDA medium. Colonies were grown on PDA for 5 days and photographed. (B) DsRNA profiles of the fungal strains shown in (A). The entire gel image was provided in the Supplementary Figure S1B. The yellow arrow head shows the position of the band of a putative hypovirus. DsRNA samples were run on agarose gel and stained with EtBr. (C) Virus-like dsRNA element isolated from the YL-3C strain. After DNase treatment, the dsRNA sample was run on PAGE and stained. M, DNA size marker (DNA ladder VI, http://www.real-times.com.cn).

Figure 2

Genomic properties of Alternaria alternata hypovirus 1 (AaHV1). (A) Genome organization of AaHV1, Wuhan insect virus 14 (WhIV14) and Cryphonectria hypovirus 1 (CHV1). Dashed lines above HE579692 and HE579693 indicate the corresponding regions of similar virus-like sequences found in A. alternata pyrosequencing (Feldman et al., 2012). Small red or gray squares (strong and weak probabilities, respectively) indicate the position of predicted transmembrane domains (TM). (B) Amino acid sequence alignment of the region corresponding to RdRp domain. The position of nine core RdRp motifs and conserved residues (Koonin et al., 1991) were highlighted. (C) Amino acid sequence alignment of the region corresponding to cysteine protease. Conserved three cysteine protease core residues (cysteine, histidine, and glycine) (Koonin et al., 1991) are highlighted.

Figure 3

Phylogenetic relationships of AaHIV1 with other hypoviruses. (A) An ML phylogenetic tree based on the multiple amino acid sequence alignment of the replicase protein or its candidate protein sequences. Virus names and GenBank/Refseq accession numbers of replicase proteins are shown. The members of species for genus Hypovirus were shown with red circles. (B) An NJ tree based on cysteine protease or its candidate protein sequences. Numbers at the nodes indicate aLRT or bootstrap values (values less than 0.8 are not displayed).

Figure 4

Identification of the defective RNA (D-RNA) of AaHV1. (A) DsRNA profiles of YL-3C with D-RNA (YL-3C-D). DsRNA samples were run on PAGE and stained with EtBr. (B) RNA blot analysis of YL-3C-D strain carrying AaHV1 D-RNA. The genomic position of the probes used for detection is shown in C (N, M and C, blue lines). (C) The sequence of AaHV1 D-RNA. The additional 30 aa owing to the frame shift in D-RNA is shown in red letters.

Figure 5

Effects of AaHV1 infection on fungal growth and virulence. (A) Phenotypic growth of AaHV1-free and -infected YL-3C strains (YL-3C/dsF, YL-3C, and YL-3C-D) on PDA and stress-inducing mediums. Colonies were grown on PDA for 5 days and photographed. Data are means ± SD (n = 3). Different letters indicate a significant difference at p < 0.01 (one-way analysis of variance (ANOVA) using MATLAB anova1 program). (B) The colonies of AaHV1-free and -infected YL-3C strains on apple leaves. Colonies were grown on leaves for 5 days and photographed. Data are means ± SD (n = 3). Different letter indicates a significant difference at p < 0.01 (one-way ANOVA).

Figure 6

AaHV1 accumulation and pathogenicity in another A. altenaria strain YL-1P. (A) Relative AaHV1 dsRNA accumulation levels in the YL-1P and YL-3C strains. Total RNA samples were run on agarose gel and stained with EtBr. RT-PCR detection of AaHV1 using genome- and D-RNA-specific primer sets (Supplementary Table S1). (B) Phenotypic growth of AaHV1-free and -infected YL-1P strains on PDA mediums. Colonies were grown on PDA for 5 days and photographed. (C) The colonies of AaHV1-free and -infected YL-1P strains on apple leaves. Colonies were grown on leaves for 5 days and photographed. Data are means ± SD (n = 3).

Figure 7

AaHV1 infectivity and pathogenicity in Botryosphaeria dothidea. (A) AaHV1 RNA in B. dothidea YL5 strain (YL5-2-2 isolate) detected by dsRNA isolation and RNA blotting. (B) Phenotypic growth and virulence of AaHV1-free and -infected B. dothidea strains on PDA medium and apples. Colonies were grown on PDA (for 4 days) and leaves (for 4 days) and photographed. (C) The lesion area on apples described in B. Data are means ± SD (n = 3). Asterisk indicates p < 0.01 (Student’s t-test).