Molecular Characterization of a Chrysovirus Isolated From the Citrus Pathogen Penicillium crustosum and Related Fungicide Resistance Analysis

Penicillium sp. are damaging to a range of foods and fruits including citrus. To date, double-stranded (ds)RNA viruses have been reported in most Penicillium species but not in citrus pathogen P. crustosum. Here we report a novel dsRNA virus, designated as Penicillium crustosum chrysovirus 1 (PcCV1) and isolated from P. crustosum strain HS-CQ15. PcCV1 genome comprises four dsRNA segments, referred to as dsRNA1, dsRNA2, dsRNA3, and dsRNA4, which are 3600, 3177, 3078, and 2808 bp in length, respectively. Sequence analysis revealed the presence of four open reading frames (ORFs) in the PcCV1 genome. ORF1 in dsRNA1 encodes a putative RNA-dependent RNA polymerase (RdRp) and ORF2 in dsRNA2 encodes a putative coat protein (CP). The two remaining ORFs, ORF3 in dsRNA3 and ORF4 in dsRNA4, encode proteins of unknown function. Phylogenetic analysis based on RdRp sequences showed that PcCV1 clusters with other members of the genus Chrysovirus, family Chrysoviridae. Transmission electron microscope (TEM) analysis revealed that the PcCV1 visions are approximately 40 nm in diameter. Regarding biological effects of PcCV1, HS-CQ15 harboring the chrysovirus exhibited no obvious difference in colony morphology under fungicide-free conditions but decreased resistance to demethylation inhibitor (DMI)-fungicide prochloraz, as compared to PcCV1-cured strain. Here we provide the first evidence of a virus present in citrus pathogenic fungus P. crustosum and the chrysovirus-induced change in fungicide-resistance of its host fungus.


METHOD
The host of PcCV1, Penicillium HS-CQ15, was isolated from Penicillium-decayed citrus surface (Chongqing, China) and molecularly characterized as P. crustosum species, according to internal transcribed spacer (ITS) analysis described before (Gardes and Bruns, 1993;Pandey et al., 2018). HS-CQ15 conidial suspension stored in glycerol at −70 • C was initially cultured on potato dextrose agar (PDA) medium at 28 • C and 180 rpm for about 7 days, and the resulting mycelium fragments were transferred into potato dextrose broth (PDB) medium for additional 96 h cultivation at the same conditions. The fungal mycelia collected from the PDB cultures was exploited to extract viral dsRNAs, using phenol-chloroform-ethanol method (Sun and Suzuki, 2008;Sotaro et al., 2009). The obtained dsRNA mixture was purified by DNase I and S1 nuclease digestions at RNase-free conditions, separated by 1% (w/v) agarose gel electrophoresis, and finally recovered from individual EB-stained band using Gel Extraction kit (TaKaRa, Dalian, China). The amount of ∼5 µg dsRNA recovered was applied to construct cDNA libraries for Illumina high-throughput sequencing, according to protocols described before (Rwahnih et al., 2011;Niu et al., 2018). Afterwards, reads and contigs assembly, based on reference genome of PcV (Jiang and Ghabrial, 2004), provided sequences with partial length for PcCV1 dsRNAs, i.e., 2437 bp for dsRNA1, 3096 bp for dsRNA2, 2909 for dsRNA3, and 2726 bp for dsRNA4. Thirteen pairs of specific primers (Table S1) were designed to full-fill gaps by RT-PCR, generating ORF-included sequences, and then, 4 pairs of adaptor primers (Table S1) were designed to PCR-amplify 5 ′ -and 3 ′ -UTRs, finally generating full-length PcCV1 genomic dsRNAs. According to the full-length sequences, specific RNA probes were designed as shown in Figure 1C and used for digoxigenin-labeled northern blots, as previously described (Streit et al., 2009;Niu et al., 2018).
Open reading frames (ORFs) in putative viral sequences were identified using the NCBI ORF finder (http://www. ncbi.nlm.nih.gov/gorf/gorf.html) and further confirmed by simulated translation in DNAMAN software package version 6.0 (Lynnon Corporation, Quebec, Canada). The analysis of protein sequence similarity was initially performed using the BLASTp program at NCBI website (http://www.ncbi.nlm.nih. gov/). Multiple sequence alignments were further processed with CLUSTAL_X program (Thompson et al., 1997), and phylogenetic trees were constructed using the neighbor-joining method in MEGA version 6.0 (Tamura et al., 2013), and further confirmed by maximum likelihood approach in the same software.
Viral particles (VPs) were purified and characterized as described previously (Niu et al., 2016(Niu et al., , 2018. Approximately   Table S2. (E) Nucleotide sequence alignments of 5 ′ -and 3 ′ -UTRs of PcCV1 genomic dsRNAs. The identical nucleotides among dsRNA1 to 4 are especially color-shaded. The "box 1" is highlighted in black box, and the "CAA" repeats are highlighted with underlines. (F) Phylogenetic analysis of the RdRp sequences for PdGV1. The names (full and abbreviated) of selected chrysoviriuses as well as GenBank accession numbers of their RdRps are listed in Table S2. 100 g (wet weight) mycelia of HS-CQ15 were harvested from PDB cultures, mixed with 400 mL of 50 mM sodium phosphate buffer (pH 7.4), and ground into homogenates for VPs isolation using sucrose density gradient ultracentrifugation. The obtained VPs were suspended with sodium phosphate buffer (pH7.4) for the use of transmission electron microscopy (TEM) and SDS-PAGE analysis.
To investigate biological effects of PcCV1 on its host fungus, we prepared PcCV1-cured HS-CQ15 progenies by ribavirin protocol as described before (Niu et al., 2016(Niu et al., , 2018. In detail, the conidia of HS-CQ15 (PcCV1-infected) were incubated in PDB media containing 100 mmol·L −1 ribavirin for 12 h to generate virus-free progenies (PcCV1-cured). Five PcCV1cured strains were selected and independently subjected to the following experiments each containing three biological replicates. The vegetative growth and ability to resist DMI-fungicide for HS-CQ15 and its PcCV1-cured progenies were assessed on fungicide-free PDA media and 5.0 mg L −1 (final concentration) prochloraz-containing PDA media, respectively, according to the methods of Niu et al. (2018), and EC 50 values against prochloraz for the virus-infected and virus-cured strains were also measured. The average of colony diameters in each independent experiment was used for EC 50 calculation by SPSS software (version 10.0).

RESULTS AND DISCUSSION
Mycovirus PcCV1 was isolated from P. crustosum HS-CQ15 and its genomic segments (viral dsRNAs) were extracted and purified by using phenol-chloroform-ethanol method. Four dsRNA bands as PcCV1 genomic segments were designated to dsRNA1, dsRNA2, dsRNA3, and dsRNA4, according to their increasing electrophoresis mobility ( Figure 1A). The tetrasegmented genome of PcCV1 has been confirmed by northern blotting (Figure 1B).
The genome organization of PcCV1 is shown in Figure 1C. The sequence of dsRNA1 is 3,600 bp full-length containing a single open reading frame (referred to as ORF1) that encodes a 1,113-amino-acid (aa) protein with putative molecular weight ∼128 kDa. Blastn analysis revealed a high nucleotide sequence similarity (∼82%) between dsRNA1 and RdRp-encoding gene of a classical chrysovirus Penicillium chrysogenum virus (PcV), the earliest case of Penicillium-hosted chrysovirus (Jiang and Ghabrial, 2004). Blastp analysis also showed a high similarity (∼98%) in aa sequences between ORF1 and PcV RdRp. dsRNA2 with 3,177 bp size contained an ORF (referred to as ORF2) encoding a 981-aa protein (∼108.6 kDa) with ∼96% similarity to capsid protein (CP) of PcV. dsRNA3 and dsRNA4 with 3,078 bp and 2,808 bp fulllength, respectively, both contained a single ORF, encoding 912-aa and 847-aa protein with estimated molecular weight ∼101 and ∼95 kDa, respectively. These two relatively smaller proteins exhibited highest similarity to functionunknown proteins encoded by PcV dsRNA3 and dsRNA4, as previously documented (Jiang and Ghabrial, 2004), and also showed considerably high similarity to specific function-unknown proteins reported in other chrysoviruses (Jamal et al., 2010;Cao et al., 2011;Herrero, 2017).
Multiple alignment of the PcCV1 RdRp with other International Committee on Taxonomy of Viruses (ICTV)registered chrysoviruses belonging to the genus Chrysovirus (Jiang and Ghabrial, 2004;Urayama et al., 2010), listed in Table S2, showed eight conserved motifs (Figure 1D), as early verified to be typical structural traits of RdRps for dsRNA viruses in lower eukaryotes (Bruenn, 1993). Between the two closely related chrysoviruses (PcV and PcCV1), the amino acid sequence identities of their dsRNA-encoding proteins (P1, P2, P3, or P4) were summarized in Table S3. Specially, a conserved phytoreovirus S7 domain was observed in the upstream of both P1 (RdRp) and P3 sequences ( Figure S1). This agreed to the S7 domain reported in other chrysoviruses (Liu et al., 2012). In addition, we found highly conserved sequences at 5 ′ -UTR for the four PcCV1 genomic dsRNAs. As shown in Figure 1E, about 60 nt located at or close to 5 ′ -termini exhibited conserved among the present 4 dsRNAs, as named "box 1" in other reported chrysoviruses (Ghabrial, 2010). The second conserved region at PcCV1 5 ′ -UTR, locating downstream from the "box 1, " is characterized by a cluster of CAA-repeats (Figure 1E), as reported in almost all chrysoviruses (Jiang and Ghabrial, 2004;Urayama et al., 2010;Zhang et al., 2017;Okada et al., 2018). Such CAA-repeats, frequently identified at 5 ′ -UTRs of chrysoviruses, have been functionally associated with translational enhancer elements for tomaviruses (Gallie and Walbot, 1992), thus presumably contributing to control of genome replication and virion assembly of chrysoviruses that facilitated their persistent infections to fungal hosts (Ghabrial et al., 2018). Figure 1E indicated ∼13 nt sequence conserved in 3 ′ -UTRs of PcCV1 genomic dsRNAs. The locations of special conserved sequence at 3 ′ -UTRs have been identified in other chrysovirus genomes (Jiang and Ghabrial, 2004;Jamal et al., 2010;Zhong et al., 2016;Herrero, 2017), nevertheless exhibiting sequence diversities among these different chrysoviruses.
The association of PcCV1 with family Chrysoviridae was verified by phylogenetic tree analysis (Figure 1F), based on aa sequences of RdRps between PcCV1 and ICTV-annotated dsRNA chrysoviruses. PcCV1 (with the closest association with PcV) was also closely clustered with other previously reported members of the genus Chrysovirus. Under TEM scanning, the purified virions of PcCV1 were isometric in ∼40 nm diameter ( Figure S2A) that was consistent with other reported chrysoviruses virions in size (Urayama et al., 2010;Ghabrial et al., 2018), also supporting the evolutionary position of PcCV1. Here, the purified virions were confirmed to be extracted from PcCV1 by gel analysis of genomic dsRNA segments ( Figure S2B) and SDS-PAGE analysis of CP ( Figure S2C).
To evaluate biological effects of PcCV1, we compared vegetative growth between PcCV1-infected HS-CQ15 and its virus-cured progenies, as well as fungicide-resistance based on PDA experiments. The colony diameters of PcCV1-infected HS-CQ15 strains were similar to those of PcCV1-cured progenies under fungicide-free conditions (Figure 2A), but obviously smaller under prochloraz conditions ( Figure 2B). These results indicated that PcCV1 had little effect on the vegetative growth but deduced prochloraz resistance of its host fungus. Considering almost no effect of PcCV1 on host growth, as indicated by statistics analysis of colony diameters (Figure 2C), the present chrysovirus is not assumed as a hypovirus to decrease HS-CQ15 virulence, and this assumption needs support by citrus-based pathogenicity assessments. Here, we emphasized the PcCV1induced decrease in prochloraz resistance for its host fungus. As shown in Figure 2D, prochloraz EC 50 value of PcCV1-infected HS-CQ15 was 2.53 ± 0.41 mg L −1 , significantly lower than those of PcCV1-cured progenies (4.57 ± 0.29 mg L −1 ). The similar effects were at first time reported in PdPmV1/PdNLV1coinfected P. digitatum strains (Niu et al., 2018). Now we provided another evidence of mycovirus-induced fungicideconditioned hypovirulence that would enhance drug efficacy to control citrus pathogen P. crustosum.
In conclusion, according to molecular features of genomic dsRNAs, homolog and phylogenetic analysis, and characteristics of their 5 ′ -and 3 ′ -UTRs, this report identified the first mycovirus (PcCV1) found in citrus pathogen P. crustosum as a variant of PcV in the Chrysovirus genus (Chrysoviridae family) and revealed that PcCV1-infection decreased prochloraz-resistance of its host fungus (HS-CQ15).

AUTHOR CONTRIBUTIONS
YY and DL conceived this study, acquired project funding, revised to complete final version of manuscript, and supervised all research activities. SW, ZY, and TZ designed experiments and conducted experimental procedures. QC and GL contributed to data curation and bioinformatics analysis. NL contributed to P. crustosum isolation and characterization.

FUNDING
This work was financially supported by the National Natural Science Foundation of China (No.31371893) and Natural Science Fund of Hubei Province (No. 2018CFB676).