Two Novel Endornaviruses Co-infecting a Phytophthora Pathogen of Asparagus officinalis Modulate the Developmental Stages and Fungicide Sensitivities of the Host Oomycete

Abstract

Two novel endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) were found in isolates of a Phytophthora pathogen of asparagus collected in Japan. A molecular phylogenetic analysis indicated that PEV2 and PEV3 belong to the genus Alphaendornavirus. The PEV2 and PEV3 genomes consist of 14,345 and 13,810 bp, and they contain single open reading frames of 4,640 and 4,603 codons, respectively. Their polyproteins contain the conserved domains of an RNA helicase, a UDP-glycosyltransferase, and an RNA-dependent RNA polymerase, which are conserved in other alphaendornaviruses. PEV2 is closely related to Brown algae endornavirus 2, whereas PEV3 is closely related to Phytophthora endornavirus 1 (PEV1), which infects a Phytophthora sp. specific to Douglas fir. PEV2 and PEV3 were detected at high titers in two original Phytophthora sp. isolates, and we found a sub-isolate with low titers of the viruses during subculture. We used the high- and low-titer isolates to evaluate the effects of the viruses on the growth, development, and fungicide sensitivities of the Phytophthora sp. host. The high-titer isolates produced smaller mycelial colonies and much higher numbers of zoosporangia than the low-titer isolate. These results suggest that PEV2 and PEV3 inhibited hyphal growth and stimulated zoosporangium formation. The high-titer isolates were more sensitive than the low-titer isolate to the fungicides benthiavalicarb-isopropyl, famoxadone, and chlorothalonil. In contrast, the high-titer isolates displayed lower sensitivity to the fungicide metalaxyl (an inhibitor of RNA polymerase I) when compared with the low-titer isolate. These results indicate that persistent infection with PEV2 and PEV3 may potentially affect the fungicide sensitivities of the host oomycete.

Keywords: asparagus phytophthora rot fungus; attenuation; endornavirus; fungicide sensitivity; zoosporangium formation; zoospore transmission.

FIGURE 1

Detection of the dsRNA genomes of PEV2 and PEV3 in Phytophthora sp. isolates. (A) Agarose gel electrophoresis of dsRNAs purified from each of the isolates. Lane designation: M, DNA marker (250 ng of λ DNA digested with EcoT14I); 1, CH98ASP060; 2, CH98ASP060-a; 3, CH98ASP051; 4, CH98ASP059; 5, CH98ASP059-L; 6, CH98ASP066; 7, Ku-1; 8, Ak-6-1; and 9, Fk-3. The dsRNAs derived from each of the isolates (0.1 g dry weight) were electrophoresed on a 0.8% agarose gel for 18 h at 20 V and stained with ethidium bromide (0.5 μg/ml). Arrows indicate the positions of the 14.3 kb dsRNAs. (B) Hyphal morphologies of four isolates. The original isolate CH98ASP060 and its derivative, CH98ASP060-a, the original isolate CH98ASP059 and its derivative CH98ASP059-L were grown on PDA plates at 25°C for 14 days. (C) Results of one-step duplex RT-PCR amplification using PEV2 and PEV3-specific primers (Supplementary Table 2). Lane designation: M, DNA size markers; 1, CH98ASP060; 2, CH98ASP060-a; 3, CH98ASP051; 4, CH98ASP059; 5, CH98ASP059-L; 6, CH98ASP066; 7, Ku-1; 8, Ak-6-1; and 9, Fk-3.

FIGURE 2

Properties of the Phytophthora endornavirus (PEV) genomes. The total nucleotide length of each dsRNA genomes is shown below the virus name. The predicted amino acid numbers are shown. The boxes represent the large ORFs, whereas lines depict UTRs. Hel-1, viral helicase 1; UGT, UDP-glycosyltransferase; RdRp, viral RNA dependent RNA polymerase. (A) Genome organization of PEV2. The viral genome is 14,345 nt in length. (B) Genome organization of PEV3. The viral genome is 13,810 nt in length. (C) Genome organization of PEV1. The viral genome is 13,883 nt in length.

FIGURE 3

A phylogenic tree based on the putative RdRp regions of PEV2, PEV3, and related endornaviruses. The evolutionary history was inferred by using the Maximum Likelihood method based on the Le_Gascuel_2008 model (Le and Gascuel, 2008). A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 1.2113)]. The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 4.0463% sites). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Support for nodes was assessed by a reliability percentage after 1,000 bootstrap iterations. Branches with less than 50% bootstrap values were collapsed with TreeGraph 2 ver. 2.14.0-771 beta (Stöver and Müller, 2010). The GenBank accession numbers of the analyzed genes are provided in Supplementary Table 5. Grapevine leafroll-associated virus 1 is an ampelovirus in the family Closteroviridae and was used as an outgroup. Undefined virus names are shown in italics.

FIGURE 4

Detection of the PEV2, PEV3 dsRNAs in monozoospore isolates. (A–C) Agarose gel electrophoresis of the dsRNA genomes of PEV2 and PEV3 in CH98ASP060 (A), CH98ASP060-a (B), and CH98ASP059 (C). The dsRNAs derived from 0.1 g dry weight of each isolate were electrophoresed in 0.8% agarose gels for 16 h at 20 V and stained with ethidium bromide (0.5 μg/ml). Arrows indicate the positions of the PEV2 and PEV3 dsRNAs. (D–F) RT-PCR detection of the PEV2 and PEV3 dsRNA genomes in CH98ASP060 (D), CH98APS060-a (E), and CH98ASP059 (F). RT-PCR was performed using specific primers to amplify the PEV2 and PEV3 dsRNA genomes. The amplified DNA fragments were subjected to electrophoresis in 1% agarose gels for 0.5 h at 100 V. Lane M, 100 bp DNA ladder. Lanes 1–10, individual monozoospore isolates designated numbers 1–10.

FIGURE 5

The presence of PEV2 and PEV3 stimulates sporangium production in Phytophthora sp. Images show zoosporangium formation by the high-titer isolates CH98ASP060 (A), CH98ASP060-a (B), and CH98ASP059 (C) and the low-titer isolate CH98ASP059-L (D). Inserts show hyphal growth on PDA after incubation at 25°C for 7 days. Bars show 50 μm. The # in the dsRNA label under the isolate name is not visible by EtBr staining (0.5 mg/ml) but is detected by RT-PCR. PEV2; 14.3 kb, PEV3; 13.8 kb. This is used in the same mean for the subsequent Figures 6–9.

FIGURE 6

Hyphal growth in on media containing benthiavalicarb-isopropyl (0.03 μg ml^–1). CH98ASP060, CH98ASP060-a, CH98ASP059, and CH98ASP059-L were grown in the presence (right) or absence (left) of the fungicide. The minimum inhibitory concentration (MIC) for the high-titer isolates (CH98ASP060, CH98ASP060-a, and CH98ASP059) was used.

FIGURE 7

Hyphal growth on media containing famoxadone (1.5 μg ml^–1). CH98ASP060, CH98ASP060-a, CH98ASP059, and CH98ASP059-L were grown in the presence (right) or absence (left) of the fungicide. The MIC for the high-titer isolates was used. PG (1 mM) was included in all media.

FIGURE 8

Hyphal growth on media containing metalaxyl (10 μg ml^–1). CH98ASP060, CH98ASP060-a, CH98ASP059, and CH98ASP059-L were grown in the presence (right) or absence (left) of the fungicide. The MIC for the low-titer isolate (CH98ASP059-L) was used.

FIGURE 9

Hyphal growth on media containing chlorothalonil (400 μg ml^–1). CH98ASP060, CH98ASP060-a, CH98ASP059, and CH98ASP059-L were grown in the presence (right) or absence (left) of the fungicide. All four isolates had MIC values >400 μg ml^–1.