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Citation: Tingfu Zhang, Na Li, Yongze Yuan, Qianwen Cao, Yanfen Chen, Binglan Tan, Guoqi Li, Deli Liu. Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum .VIROLOGICA SINICA, 2019, 34(6) : 688-700.

Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum

  • Corresponding author: Deli Liu,;
  • Electronic supplementary material The online version of this article ( contains supplementary material, which is available to authorized users.
  • Received Date: 16 February 2019
    Accepted Date: 14 May 2019
    Published Date: 02 August 2019
    Available online: 01 November 2019
  • Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum (P. italicum). In this study, we isolated and characterized a new double-stranded RNA (dsRNA) virus, designated Penicillium italicum chrysovirus 1 (PiCV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that PiCV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1ΔpksP defective in the polyketide synthase gene (pksP), which is involved in pigment biosynthesis, and these mutants formed albino (white) colonies. Then we applied hyphal anastomosis method to horizontally transmit PiCV1 from the white virus-donors (i.e., HSPi-YN1 mutants) to wild-type recipients (i.e., P. italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable PiCV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic dsRNA profile analysis. This blue-white colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized PiCV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen PiCV1-infected isogenic recipients.

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    Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum

      Corresponding author: Deli Liu,;
    • 1. Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China
    • 2. Yunnan Higher Education Institutions, College of Life Science and Technology, Honghe University, Mengzi 661199, China

    Abstract: Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum (P. italicum). In this study, we isolated and characterized a new double-stranded RNA (dsRNA) virus, designated Penicillium italicum chrysovirus 1 (PiCV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that PiCV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1ΔpksP defective in the polyketide synthase gene (pksP), which is involved in pigment biosynthesis, and these mutants formed albino (white) colonies. Then we applied hyphal anastomosis method to horizontally transmit PiCV1 from the white virus-donors (i.e., HSPi-YN1 mutants) to wild-type recipients (i.e., P. italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable PiCV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic dsRNA profile analysis. This blue-white colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized PiCV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen PiCV1-infected isogenic recipients.

    • Penicillium is a major pathogen causing huge losses during post-harvest handling of citrus fruits. P. digitatum and P. italicum are representative Penicillium pathogens because they have the strongest pathogenicity in citrus fruits (Prusky et al. 2004; Boubaker et al. 2009). Artificial synthetic fungicides, including demethylase inhibitors, have been used to control these phytopathogens (Korsten 2006; Liu et al. 2015). However, directed evolution of resistant strains and the failure to control green and blue molds have been attributed to the excessive use of fungicides (Wang et al. 2014). Therefore, it is necessary to develop alternative strategies, such as hypovirulent viruses, to control these post-harvest diseases.

      Viruses have been discovered in all major groups of fungi since the first definitive report of a virus infecting the cultivated button mushroom Agaricus bisporus 56 years ago (Hollings 1962). In 1967, the first evidence of mycoviruses infecting P. stoloniferum was reported (Ellis and Kleinschmidt 1967). Subsequently, mycoviruses were identified in P. funiculosum, P. chrysogenum, P. cyaneofulvum, P. brevi-compactum, P. citrinum, and P. variable (Banks et al. 1969; Wood et al. 1971. Borré et al. 1971;Border et al. 1972). Nevertheless, few studies have reported the identification and classification of viruses at the molecular level. Kim et al. (2003) reported that the complete genome sequence of P. stoloniferum virus (PsV-S) contained S1 and S2 double-stranded RNA (dsRNA). Phylogenetic analysis revealed that PsV-S was a definite partitivirus in the family Partitiviridae (Kim et al. 2003). With regard to Chrysovirus, Jiang and Ghabrial first published the whole-genome sequence of P. chrysogenum virus (PcV) and suggested that PcV may be subordinate to the genus Chrysovirus rather than the family Partitiviridae (Jiang and Ghabrial 2004). With the rapid development of sequencing technology, more than 10 Penicillium viruses infecting P. aurantiogriseum (Nerva et al. 2016), P. janczewskii (Nerva et al. 2016), and P. digitatum (Niu et al. 2016, 2018; Yang et al. 2018) have been successively identified, some of which are still unclassified or have not been approved by the International Committee on Taxonomy of Viruses (ICTV). We recently isolated viruses from P. crustosum and P. italicum (Niu et al. 2018). To date, mycoviruses have been found in more than 12 species of Penicillium. These results have expanded our understanding of taxonomic groups of Penicillium mycoviruses.

      Mycoviruses have been reported to include dsRNA, single-stranded RNA, and single-stranded DNA genomic types (Yu et al. 2010). Chrysoviridae family members belong to the dsRNA virus. Phylogenetic analysis of them and related, unclassified viruses showed that all these viruses can be divided into two clusters; cluster Ⅰ contains viruses with three or four genome segments, whereas cluster Ⅱ contains viruses with four or five genome segments (Ghabrial et al. 2018). Nine classified species of Chrysoviridae can infect ascomycetous or basidiomycetous fungi, harboring a typical quadripartite genome (11.5–12.8 kb) that is individually encapsidated with virions (isometric, nonenveloped, and approximately 40 nm in diameter) (Ghabrial et al. 2018). The capsid of PcV, a representative species of Chrysoviridae, comprises 60 subunits of a 109 kDa polypeptide arranged on a T = 1 icosahedral lattice (Castón et al. 2003). Unclassified, chrysovirus-related viruses with three-segmented dsRNA genomes infect plants but do not induce obvious damage (Li et al. 2013). However, some chrysovirus-related viruses with five-segmented dsRNA genomes cause deleterious effects in their fungal hosts (Urayama et al. 2014).

      RNA mycoviruses can be transmitted vertically via sporulation and horizontally via hyphal anastomosis between mycelial compatible strains of the same fungus (Pearson et al. 2009). Transfection of virions into recipient protoplasts or protoplast fusion between donors and recipients has greatly improved the efficiency of virus infection between host fungi (Hillman et al. 2004; Sasaki et al. 2006; Lee et al. 2014; Niu et al. 2016). In addition, dual culture based on mycelia anastomosis of virus donors and recipients has become the main technique for studying mycovirus infections between fungi. Although mycoviruses are thought to exhibit host specificity, the ability of fungal viruses to transmit among fungal individuals (even via interspecies transmission) is determined by virus characteristics and differences in host mycelial compatibility (Ghabrial and Suzuki 2009). Mycovirus transmission via hyphal anastomosis has been reported in the same fungal species, including Botrytis cinerea (Wu et al. 2010), Fusarium graminearum (Lee et al. 2014), Ustilaginoidea virens (Zhang et al. 2014), and Rosellinia necatrix (Sasaki et al. 2016), and via interspecies transmission in Aspergillus (Coenen et al. 1997), Sclerotinia (Melzer et al. 2002), Cryphonectria (Liu et al. 2003), Heterobasidion (Vainio et al. 2010), and P. aurantiogriseum (Nerva et al. 2017) under laboratory experiment conditions. Chrysoviruses could be transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis (Ghabrial et al. 2018). However, exploration of host/ chrysovirus interactions has been hampered in Penicillium spp. because of difficulties in curing fungi of viral infections and a lack of simple methods for artificial inoculation of mycoviruses (Kim et al. 2013). Based on hyphal anastomosis or protoplast fusion, established methods for screening virus-infected isogenic recipient strains are mainly dependent on antibiotic (hygromycin B or geneticin G418) resistance selection after coculture (Lee et al. 2011; Kim et al. 2013). Therefore, optimization or construction of a simple and convenient screening method/system for horizontal transmission of viruses is essential for studies of the interactions between Penicillium spp. and chrysoviruses.

      Accordingly, in this study, we isolated and sequenced a chrysovirus, designated PiCV1, from P. italicum (HSPi-YN1). Molecular characterization and phylogenetic analysis of PiCV1 were performed to evaluate the taxonomic status. Based on the PiCV1 (virus)-HSPi-YN1 (host) system, we then established a blue–white colony screening method using a PiCV1-infected HSPi-YN1 mutant defective in the pksP gene. Overall, our findings provided important insights into the detection, isolation, and characterization of P. italicum isolates infected by PiCV1.

    • In total, 148 strains of Penicillium were isolated from the epidermis of mildew citrus collected from markets or fruits packing houses in Hubei, Jiangxi, and Yunnan provinces and Chongqing municipality, China from November 2015 to March 2016. These strains included 28 P. italicum strains, such as the virus-infected strain HSPi-YN1 and the virus-free strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1. The three virus-free strains were selected as recipients in dual culture for the current study. Conidia and mycelia from all strains were cultured on potato dextrose agar (PDA) medium and in potato dextrose broth on a rotary shaker (180 rpm) at 28 ℃. Total genomic DNA was extracted from ground mycelia powder in liquid nitrogen using a Biospin Fungus Genomic DNA Extraction Kit (Bio Flux) following the manufacturer's protocol. The virusharboring strains were primarily screened through genomic composition detection and confirmation using nuclease digestion analysis by agarose gel electrophoresis and staining with ethidium bromide. For species determination of fungal isolates, internal transcribed spacer (ITS) sequences of ribosomal DNA were amplified by PCR using universal primer pairs (Supplementary Table S1). PCR products were sequenced using an ABI3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

    • Mycelia were frozen with liquid nitrogen and ground to a fine powder in a mortar. dsRNA was then extracted with phenol–chloroform and isolated by CF-11 cellulose (Sigma, St. Louis, MO, USA) chromatography, according to a previously described method (Morris and Dodds 1979). The nucleic acid sample was treated with RNasefree DNase I and S1 nuclease (Thermo Fisher Scientific, Waltham, MA, USA) at 37 ℃ for 30 min to digest genomic DNA and single-stranded RNAs (rRNAs and mRNAs) and then separated on 1% agarose gels stained with ethidium bromide. The dsRNA gel bands were excised and purified with a gel extraction kit (Axygen, Union City, CA, USA), dissolved in appropriate volume of RNase-free double-distilled water, and stored at - 70 ℃. This dsRNA sample was later used for high-throughput sequencing and as a template for synthesizing cDNA.

    • Purified dsRNA (8 μg) was used for cDNA library construction with a TruSeq RNASample Preparation Kit (Illumina) following the manufacturer's protocol. Paired-end sequencing with a 150-bp read length was conducted on an Illumina HiSeq 2500 machine. cDNA library construction, sequence assembly, and evaluation of deep sequencing were carried out by GENEWIZ Solid Science. Sequenced reads matching the host P. italicum databases (isolate B3) were removed, and the remaining reads were de novo assembled using Velvet (Zerbino and Birney 2008) (version 1.2.10) with default parameters. All contigs were subjected to BLASTN and BLASTX alignment against the NCBI database to search for viral sequences.

      The complete 5'- and 3'-terminal sequences were further determined using rapid amplification of cDNA ends (RACE)-PCR. First, the 3'-terminus of each strand of dsRNA was ligated to the closed adaptor primer PC3-T7 loop (Supplementary Table S1) by T4 RNA ligase (Thermo Fisher Scientific) at 16 ℃ for 18 h. Next, the oligonucleotide-ligated dsRNA was reverse-transcribed using primer PC2 (Supplementary Table S1), which was complementary to the oligonucleotide used for RNA ligation, and sequence-specific primers corresponding to the 5'- and 3'-end sequences of the dsRNA in the presence of M-MLV reverse transcriptase, as described previously (Liu et al. 2009), with minor modifications. Additionally, fragments of dsRNAs between the 5'- and 3'-ends were subjected to reverse transcription (RT)-PCR using sequencespecific primer pairs to obtain cDNA that overlapped with RACE sequences of the 5'- and 3'-ends. All amplified cDNA products were cloned into the pMD18-T vector (TaKaRa, Shiga, Japan), sequenced using an ABI3100 DNA sequencer, and subsequently assembled by DNAMAN (version 7.0) to obtain and confirm the full-length sequences of all dsRNAs. At least three independent clones were analyzed for sequence determination at all nucleotide positions.

      For Northern blot hybridization analysis, dsRNAs isolated from mycelia of virus hosts were separated on 1% agarose gels for 3 h at 80 V. The gels were then soaked for 20 min in 0.1 mol/L NaOH and neutralized twice in 0.1 mol/L Tris–HCl (pH 8.0) for 20 min, and the RNAs were transferred by capillary action to a Hybond-N+ nylon membrane in 20 × SSC buffer overnight. The RNA was fixed to the membrane by ultraviolet (UV) crosslinking following exposure to UV irradiation for 60 s at 120 mJ/c2. Prehybridization and hybridization were performed under stringent conditions in hybridization solution using a BCIPNBT DIG Detection Kit (TIANDZ, Beijing, China) according to the manufacturer's instructions. The RNA blots were probed with digoxigenin-labeled probes (500–700 bp) prepared by oligolabeling of cloned cDNA to the four dsRNAs (Jamal et al. 2010).

    • Sequence similarity was assessed using the BLAST tool on the NCBI website. Analysis of conserved sequences at the 5'- and 3'-untranslated regions (UTRs) in dsRNAs was performed with DNAMAN (version 7.0) by manually adjustment. Coding open reading frames (ORFs) in dsRNA sequences were detected with ORF Finder (, deduced, and translated by DNAMAN (version 7.0) with default parameters. Multiple sequence alignments of the amino acid sequences were generated using CLUSTAL_X (Thompson et al. 1997). Phylogenetic tree construction was based on the neighbor-joining method, as described previously with boot-trapping analysis of 1000 replicates by MEGA (version 5.0) (Tamura et al. 2011).

    • Approximately 80 g of mycelia mixed with 320 mL of 0.05 mol/L sodium phosphate buffer (pH 7.4) was levigated for 3 min using a precooled grinder. Isolation of virus-like particles (VLPs) was performed as previously described using differential centrifugation and ultracentrifugation in sucrose density gradients (Niu et al. 2016). Briefly, the crude extracts were obtained by differential centrifugation, and the supernatants were subjected to ultracentrifugation in sucrose density gradients (200–600 mg/mL with intervals of 100 mg/mL). The UV absorbance at 260 nm for all 12 fractions was measured using a spectrophotometer, and fractions with a peak absorption corresponding to the sucrose gradient were supplemented with 0.05 mol/L sodium phosphate buffer (pH 7.4) and subjected to ultracentrifugation once again to remove the sucrose. The VLPs were suspended in 100 μL of 0.01 mol/L sodium phosphate buffer (pH 7.4) and then used for transmission electron microscopy observations and SDS-PAGE analysis.

    • The method of blue–white colony screening was established based on hyphal anastomosis for virus transmission between P. italicum individuals. Briefly, the virus-free recipient was streak-inoculated at an interval of 0.5 cm with virus donor harboring deletion of the pksP gene, resulting in the albino colony phenotype. In order to obtain the pksP-knockout P. italicum strain, we introduced approximately 1.0 μg of the pksP L-hyg-R fragment (i.e., approximately 1.2-kb upstream and downstream sequences of the pksP ORF were selected as the L- and R-arms, respectively, establishing a knockout site to fuse with the hyg cassette) into wild-type donor (HSPi-YN1) protoplasts (100 μL, 1 × 108 protoplasts/mL) by polyethylene glycol (PEG)-mediated transformation (Zhao et al. 2016). Regenerative transformants with the albino phenotype were verified using negative and positive-selection by PCR amplification. Primer pairs used to construct and verify the HSPi-YN1ΔpksP mutant are listed in Supplementary Table S1.

      We used three virus-free P. italicum strains, i.e., HSPiCQ54, HSPi-HB4, and HSPi-HN1, as original recipients in dual cultivation with the albino donor (HSPi-YN1ΔpksP). Virus donors and recipients were incubated at 28 ℃ for one week on dishes containing 20 mL PDA. When the spores and hyphae were grown together, spores were randomly selected at the edge of the border to separate single spore isolates on fresh PDA plates by spore gradient dilution with dilution ratios of 103–108. Next, 10 blue isolates derived from single spores were visually selected for every recipient and cultured on PDA plates for DNA and dsRNA extraction and subsequent virus detection by gel electrophoresis analysis and RT-PCR. In addition, the wild-type virus donor HSPi-YN1 was evaluated using the same methods as for the recipient strains (CQ54hyg, HB4hyg, and HN1hyg) harboring the hygromycin B resistance gene. Recipients overexpressing the hygromycin B-resistant gene were obtained by Agrobacterium-mediated genetic transformation (Wu et al. 2016). Single spore isolates separated from cocultures were selected on PDA containing 50 μg/mL hygromycin B by spore gradient dilution with dilution ratios of 103–108. Virus detection was performed using the blue–white colony screening method.

    • The conidial suspension (1 × 107 spores/mL) of each isolate strain (10 μL) was inoculated into a hole in the epidermis of citrus fruit and cultured several days at 25 ℃. Lesions in the citrus fruits were measured at 7 days after inoculation. In addition, 100 μL of the conidial suspension was coated onto PDA media plates and precultured overnight at 28 ℃. Mycelia plugs (~ 8 mm in diameter) were shaped from the plate using a punch and transplanted on the center of fresh PDA or treated with different concentrations of prochloraz (i.e., 0–0.01 mg/mL for HSPi-HN1, 0–0.1 μg/mL for HSPi-CQ54, and 0–10 μg/mL for HSPiHB4) for assays of vegetative growth and prochloraz EC50, respectively. The plaques were measured at 7 days after inoculation, and the colony areas were measured once every day for 7 consecutive days at 28 ℃ or according to the experimental need. The diameters of different colonies were measured. Each assay was performed with three replicates and independent samples t test was applied in the SPSS Statistics 17.0 context to assess the significance of differences between the means.

    • In total, 148 strains of Penicillium were isolated from the epidermis of post-harvest mildew citrus fruit in Hubei, Jiangxi, and Yunnan provinces and Chongqing municipality, China (Fig. 1A). There were 39 isolates harboring extra genome segments in profile analyses of the fungal total genome, suggesting that these fungi harbored viruses (Fig. 1B). Phylogenetic analysis of ITSs showed that one, four, and 34 isolates were clustered to branches with previously identified P. italicum, P. crustosum, and P. digitatum based on sequences of accession numbers KU561924.1, KU714642, KX507075.1, MF188258.1, KJ834506.1, and AY373910.1 (Fig. 1C). The extra genome fragments of P. italicum strain HSPi-YN1 were confirmed to be the dsRNA genome of a virus infecting the host fungus HSPi-YN1 by nuclease digestion analysis (Fig. 2A).

      Figure 1.  Screening and identification of Penicillium strains harboring virus. A Citrus fruits were decayed by infection with Penicillium pathogens during the post-harvest stage. (a) The decayed citrus in the fruit heap; (b) citrus fruits infected by P. italicum; (c) citrus fruits infected by P. digitatum and P. italicum; (d) citrus fruits infected by P. digitatum. B Total genome extracts was detected using 1% agarose gel electrophoresis. Lanes 2, 4, 5, 6, 9, 16, 17 correspond to isolates WH7, YN1, WH2, WH3, CQ8, CQ15, and CQ16, respectively, which had extra genome elements; other lanes corresponded to virus-free strains. Lane M: DS1500 DNA marker. C Thirty-nine wild-type strains of Penicillium harboring virus were clustered using ITS sequences by the neighbor-joining method. Black triangles, blue squares, and red dots represent P. digitatum, P. crustosum, and P. italicum, respectively.

      Figure 2.  Genome organization and UTR sequence characterization of PiCV1. A Electrophoretic profile of dsRNA preparations on 1% agarose gels. The dsRNA extracted from HSPi-YN1 (lanes 1–2) and after treatment of purified digestion products with S1 nuclease (lanes 3–4); dsRNA digestion with S1 nuclease and DNase I (lane 5). Lane M: 1-kb ladder DNA marker. B Northern blot analysis of HSPi-YN1 dsRNA was electrophoresed under denaturing conditions, blotted onto nylon membranes, and probed by DIG-labeled DNA fragments. Lanes S1–S4 corresponded to genomic dsRNA segments 1–4, respectively. The positions of probes are shown in panel C. C Schematic representing the genome organization of PiCV1 dsRNA segments. D Comparison of the 5'-and 3'-UTRs and display of (CAA)n repeats upstream of the initiation codon (AUG) in four dsRNA segments of PiCV1. Multiple sequence alignments were obtained in the nucleotide sequences of the 5'- and 3'-UTRs. The red background signifies identical bases at the positions, and orange (pink) indicate that three of four (two of four) bases were identical at the positions.

    • dsRNA extracts from the fungal mycelia of P. italicum strain HSPi-YN1 were treated with DNase I and S1 nuclease, and four clear bands of approximately 3.0–4.0 kb were observed after 1% agarose gel electrophoresis (Fig. 2A). The purified dsRNA sample was used to construct a cDNA library for cluster preparation and subsequent sequencing using an Illumina next-generation sequencing platform. Sequences from the fungal host were removed, and the remaining reads were de novo assembled to four segments, which were similar to those of PcV, with sequence identity of approximately 83%–84% by BLAST. For convenience, the virus from HSPi-YN1 strains was named PiCV1, and these four dsRNAs were numbered dsRNA1–4 based on their molecular masses. The complete 5'- and 3'-terminal sequences of each segment were further determined using RACE-PCR, and all full-length sequences were verified by PCR amplification and cloning. The sequences of these dsRNAs were deposited in GenBank (accession numbers: MK214380, MK214381, MK214382, and MK214383). Four dsRNA bands for PiCV1 from HSPi-YN1 were also detected and confirmed using specific probes (500–700 bp) labeled with digoxigenin by Northern blot analysis (Fig. 2B). The genomic organization of the four dsRNAs is shown in Fig. 2C.

      Sequence analysis of cDNAs derived from the four dsRNAs indicated that the full-length sequences were 3570, 3189, 2975, and 2857 bp in size and that each dsRNA contained a single large ORF (ORF1–4; Fig. 2C). A comparison of nucleotide sequences between PiCV1 and PcV revealed relatively high identity (83%, 84%, 83%, and 84% for dsRNA1, dsRNA2, dsRNA3, and dsRNA4, respectively). ORF1 from dsRNA1 nt positions 146–3497 encoded a putative protein (1117 amino acids) with a molecular mass of 128.3 kDa, which showed extremely high sequence identity (99%) to the RNA-dependent RNA polymerase (RdRp) encoded by PcV (Fig. 2C). ORF2 from dsRNA2 nt positions 161–3107 encoded a putative protein (982 amino acids) with a molecular mass of 108.4 kDa, which showed high sequence identity (97%) to the capsid protein (CP) encoded by PcV (Fig. 2C). ORF3 (from dsRNA3 nt positions 162–2898) and ORF4 (from dsRNA4 nt positions 154–2695) encoded functionally unknown proteins that had calculated molecular masses of 100.7 (912 amino acids) and 94.6 kDa (847 amino acids), respectively, and high identity (both reached 97%) to the corresponding proteins encoded by PcV (Fig. 2C). Analysis of the UTRs of four dsRNAs showed that their 5'- and 3'-termini were highly conserved, and analogous (CAA)n repeats (tobamovirus-like translational enhancer elements) were found upstream of the AUG initiator codon in PiCV1 (Fig. 2D). Similar results were reported for approved chrysoviruses, such as PcV and Hv145SV (Jiang and Ghabrial 2004; Ghabrial and Suzuki 2009). Multiple sequence alignment revealed that the 5'- terminus included a 10-nt A-rich conserved sequence and a CCUGAGGA conserved sequence region, whereas the 3'- terminus included a 9-nt U-rich conserved sequence and a 12-nt UA-rich conserved sequence in UTRs of the four segments (Fig. 2D).

    • A multiple amino acid sequence alignment and conserved domain database search of PiCV1 RdRp revealed the presence of eight conserved motifs (Fig. 3A). The genomic and amino acid identities between PiCV1 and PcV were more than 83% and 97%, respectively; however, the hosts were different. Phylogenetic analysis based on multiple alignments of the deduced amino acid sequences of the RdRp in PiCV1 and other selected viruses in the families Chrysoviridae, Totiviridae, and Partitiviridae (Supplementary Table S2) demonstrated that PiCV1 was clustered closely to Chrysovirus member PcV, with a support coefficient of 100 in the same sub-branch (Fig. 3B). According to this analysis, we propose that PiCV1 may be a new strain of P. chrysogenum virus from P. italicum.

      Figure 3.  Comparison of RdRp conserved motifs among different members in the Chrysoviridae family and phylogenetic analysis of dsRNA mycovirus RdRps from three related families. A Comparison of RdRp conserved motifs encoded by PiCV1 dsRNA1 and other selected mycoviruses. The Roman numerals Ⅰ–Ⅷ refer to the eight conserved motifs characteristic of RdRps in the selected dsRNA viruses. Alignment was performed, and the accession numbers used to analyze conserved motifs are listed in Supplementary Table S2. B Phylogenetic analysis of the PiCV1 RdRp sequence. A phylogenetic tree was constructed using the neighbor-joining method. Member information for the three families and six genera is listed in Supplementary Table S2 in the phylogenetic tree.

    • PiCV1 particles were purified by differential centrifugation and sucrose density gradient centrifugation. The absorbance profile at 260 nm showed a single peak for the fractions corresponding to 300 mg/mL sucrose (Supplementary Figure S1). Observation of these gradient fractions using transmission electron microscopy uncovered isometric virus-like particles with a diameter of approximately 40 nm (Fig. 4A), consistent with the morphology and size of virions from chrysoviruses (Jiang and Ghabrial 2004; Ghabrial et al. 2018). Profile analysis showed that dsRNAs extracted from the purified particles were indistinguishable from dsRNAs isolated from the mycelia of the same HSPi-YN1 culture used for particle preparations (Fig. 4B). SDS-PAGE analysis of the purified virions showed a major band at ~ 116 kDa representing the CP, which was close to the putative molecular weight (108.4 kDa) of the major CP encoded by PiCV1 genome segment dsRNA2 (Fig. 4C).

      Figure 4.  Compositions of virus particles separated from P. italicum strain HSPi-YN1. A Electron micrograph of virus particles. The purified virus particles were negatively stained with 2% phosphomolybdic tungstic acid and observed by transmission electron microscopy. Scale bar: 100 nm. B dsRNA detection in HSPi-YN1 virus particles. Lane 1: dsRNA isolated from the same hyphae of HSPi-YN1 after virion extraction and purification; lane 2: dsRNA isolated from virus particles. Lane M: 1-kb ladder DNA marker. C SDS-PAGE was used to detect protein components of purified particles. The purified virus particles were mixed with loading buffer, denatured, and electrophoresed on 10% polyacrylamide gels (lane 1). Proteins were stained with Coomassie brilliant blue R-250. Lane M: protein molecular weight marker.

    • In order to generate a morphological selection marker phenotype, the cassette pksP L-hyg-R was amplified using overlapped PCR and introduced into protoplasts of the wild-type virus donor (HSPi-YN1) by PEG-mediated transformation. The pksP disruption mutant (HSPi-YN1ΔpksP) was generated through homologous recombination and exhibited albino colonies that could be visually distinguished from the wild-type colonies (Fig. 5A, 5C). A segment of approximately 1.1 kb in the knockout sequence was amplified in the wild-type strain or mutant harboring ectopic expression of hyg but not in mutants with pksP gene knockout by negative PCR. The hyg cassette (~ 2.1 kb) was amplified from a mutant of pksP gene disruption or ectopic expression of hyg but not the wildtype strain by positive PCR (primers at two terminuses of the hyg cassette; Fig. 5B). These results demonstrated that the pskP gene was successfully knocked out in the wildtype strain HSPi-YN1. Subsequently, viral genome profile analysis and RT-PCR verification revealed that the albino mutant YN1ΔpksP still harbored PiCV1 and became a modified virus donor (Fig. 5D), which was easily distinguished from the wild-type recipient strain based on colony color. Thus, we established a blue–white colony screening system by dual culture of the albino virus donor strain with wild-type recipient strains (blue colony) for the first time.

      Figure 5.  pksP gene disruption in the wild-type strain HSPi-YN1. A Schematic showing that the target gene pksP was replaced with the hyg cassette though homologous recombination (HR). The black and red arrows represent negative and positive PCR primers, respectively. B Mutants with pksP gene disruption were confirmed by negative and positive PCR. Lane M: 1-kb DNA marker. H and KO represent lanes loaded with amplification products using positive and negative PCR primers, respectively. C Wild-type HSPi-YN1 and HSPi-YN1ΔpksP morphological phenotypes on PDA. D Viral genomic profile analysis and RT-PCR verification confirmed that the modified virus donor (albino mutant) harbored PiCV1. (a) dsRNA genome of PiCV1 isolated from the wild-type strain and albino mutant, as detected by electrophoresis analysis. (b) PiCV1 found in the wild-type strain and the modified virus donor by RT-PCR. Lanes 1–4: PiCV1 dsRNA segments; lane M: DNA marker DS5000.

      Based on colony color differences combined with genomic profile analyses, we observed PiCV1 horizontal transmission among P. italicum individuals with different genetic backgrounds, generating isogenic and virus-infected fungal isolates derived from wild-type recipients. After coculture of the albino mutant (HSPi-YN1ΔpksP) with wild-type recipients (HSPi-CQ54, HSPi-HB4, and HSPi-HN1), isogenic and virus-infected recipient candidates from these cocultures were selected based on colony color (blue) by spore gradient dilution on PDA (Fig. 6A). All candidate single-spore isolates (30 isolates in three groups of paired cultures) were screened and identified by total genome and viral dsRNA profile analyses. The results indicated that PiCV1 could be horizontally transmitted between different individuals of P. italicum via hyphal anastomosis (Fig. 6B). The proportion of virus-infected isolates was 10%–50% (1–5 of 10 randomly picked singlespore isolates) in every plate coated with an appropriate dilution ratio (105–106) of the spore suspension after dual culture (Fig. 6B). These virus-infected P. italicum isolates were further confirmed by RT-PCR with specific probe primers targeting the four dsRNA segments of the PiCV1 genome (Fig. 6C).

      Figure 6.  Isogenic isolates infected with PiCV1 among wild-type recipients with different genetic backgrounds were screened based on the blue–white colony screening method. A Experimental procedure for the blue–white colony screening method based on the albino phenotype of the donor via hyphal anastomosis. (a–c) Wild-type recipients CQ54, HN1, and HB4 were cocultured with the albino virus donor YN1ΔpksP, and corresponding single-spore isolates were generated by gradient dilution of the mixture spores whose blue isolates were selected as candidates for PiCV1 infection. The area in the red square is the colony border for the coculture. B PiCV1- infected isogenic isolates were screened and identified from candidates (blue colony) by total genome and viral genomic dsRNA profile analyses. (a) The total genome profile analysis of all blue colony candidates derived from CQ54, HN1, and HB4. ''V?'' represents the status of PiCV1 infection, and lane numbers 1–10 represent corresponding blue colony isolate serial numbers. Lane M: DS5000 DNA marker. Red arrow: dsRNA from isolates infected with PiCV1. (b) Electrophoresis profile of dsRNA extracts from virus-free recipient strains and corresponding isogenic virus-infected isolates. Lane M: 1-kb ladder DNA marker. C Virus-infected isolates were further verified by RT-PCR. Lanes 1–4: serial numbers of PiCV1 dsRNA segments; lane M: DNA marker DS5000. For brevity, the strain numbers HSPi-CQ54, HSPi-HB4, and HSPi-HN1 are abbreviated as CQ54, HB4, and HN1, respectively, in C.

      Additionally, we performed hygromycin B screening for recipient strains with overexpression of the hyg marker (HSPi-CQ54hyg, HSPi-HN1hyg, and HSPi-HB4hyg) cocultured with the wild-type virus donor (HSPi-YN1). Based on hygromycin B resistance, genomic profile analysis, and RT-PCR, genetic backgrounds of donor and recipients were distinguished, and the horizontal transmission of PiCV1 between these strains of P. italicum was achieved via hyphal anastomosis. The efficiency was similar to that observed by blue–white colony screening (Supplementary Figure S2). Thus, pksP gene knockout did not affect the ability of PiCV1 to inhabit the virus host (P. italicum) or the efficiency of horizontal transmission between different P. italicum individuals via hyphal anastomosis. Furthermore, blue–white colony screening for generation of isogenic and virus-infected recipients was simpler and more economically feasible than antibiotic screening.

    • To investigate biological effects of PiCV1 infection, we compared vegetative growth and virulence between isogenic virus-free and virus-infected strains, i.e., HSPi-CQ54 versus HSPi-CQ54V, HSPi-HB4 versus HSPi-HB4V, and HSPi-HN1 versus HSPi-HN1V. The results showed no obvious difference in mycelia growth, colony morphologies and lesions on 7-day incubated citrus fruits for each comparison (Fig. 7A, 7C, 7D, 7F). There were also no difference in pigment accumulation between HSPi-CQ54 and its isogenic virus-infected strain HSPi-CQ54V (Fig. 7A). In addition, all the PiCV1-infected recipients including HSPi-CQ54V, HSPi-HN1V, and HSPi-HB4V strains exhibited little difference in fungicide prochloraz resistance as compared to the corresponding virus-free recipients, i.e., HSPi-CQ54, HSPi-HN1, and HSPi-HB4, respectively (Fig. 7B, 7E). These results in together indicated no significant hypovirulent and drug-conditioned hypovirulent effects of PiCV1 infection on the present P. italicum strains.

      Figure 7.  Comparison of biological phenotypes between PiCV1-infected and virus-free P. italicum strains. A Colony morphology of growth assays for tested strains grown on PDA for 7 days. The bottom row shows the pigment on the back of the PDA plate. B Sizes of colonies grown on PDA with different concentrations of prochloraz. C Lesion area for virus-infected strains and virus free-stains on the surface of citrus fruits. D Line charts showing the mean diameters at different time points for growth rate assays shown in (A). Strains were evaluated using three independent replicates. E Column charts showing the mean EC50s for all strains. All concentrations were measured with three plates for each strain (P > 0.05). F Column charts showing the mean diameters of lesions at 7 days after inoculation of P. itlicum on the citrus surface. All strains were inoculated into three oranges (P> 0.05).

    • Mycoviruses are widely distributed in different fungal groups (Ghabrial and Suzuki 2009). To the best of our knowledge, approximately 12 species of Penicillium fungi have been found to harbor viruses, and chrysoviruses have been detected in various Penicillium species, including P. cyaneofulvum, P. brevicompactum, P. chrysogenum, P. janczewskii P. crustosum, and P. italicum (Banks et al. 1969; Wood et al. 1971; Jiang and Ghabrial 2004; Nerva et al. 2016; Ghabrial et al. 2018; Niu et al. 2018). Interestingly, PiCV1 and Penicillium crustosum chrysovirus 1 (PcCV1), which were isolated from the citrus pathogens P. italicum and P. crustosum, respectively, shared approximately 83% identity in the quadripartite genome with the PcV. However, when the PiCV1 host P. italicum strain (HSPi-YN1 or HSPi-YN1ΔpksP) and virus-free P. crustosum strain (HSPc-CQ4) or PcCV1 wild-type host P. crustosum strain (HSPc-CQ15) and virus-free P. italicum strain (HSPi-HB4) were cocultured, no virus-infected isolates derived from HSPc-CQ4 or HSPi-HB4 were detected in single spore isolates of cocultures by hyphal anastomosis. Thus, taken together, our results indicated that PiCV1 could be horizontally transmitted between different individuals in intraspecies hosts but could not be transferred among different species of fungal hosts under natural culture conditions. These results suggested that P. italicum and P. crustosum each had their own unique chrysoviruses, regardless of the considerably high genome identity observed in this study. Chrysoviruses inhabiting different Penicillium species (i.e., P. italicum, P. chrysogenum, and P. crustosum) may be derived from the same original viral ancestor and adaptively evolved into new variants in different species of Penicillium hosts.

      The Chrysoviridae family contains approved chrysoviruses and chrysovirus-related viruses. The genome of the former viruses is comprise of four monocistronic dsRNA segments, whereas chrysovirus-related viruses infecting fungi have either four or five segment (Ghabrial et al. 2018). The PiCV1 genome includes four linear dsRNAs encoding proteins having high identify to corresponding proteins in PcV, as a classic chrysovirus. dsRNA1 encodes RdRp, which harbors conserved regions (motif Ⅰ–Ⅷ), and dsRNA2 encodes a major CP (approximately 109 kDa in size), which assembles virions with a diameter of ~ 40 nm. p3 and p4 encoded by dsRNA3 and dsRNA4, respectively, are unknown functional proteins containing the phytoreo S7 domain (found in phytoreovirus P7 core proteins) and a putative cysteine protease motif found in the ovarian tumor gene-like superfamily. All four dsRNA segments of PiCV1 were highly conserved in both the 5'- and 3'-UTRs. The 5'-terminus contained a 10-nt A-rich conserved sequence and a CCUGAGGA conserved sequence region, whereas the 3'-terminus contained a 9-nt U-rich conserved sequence and a 12-nt UA-rich conserved sequence. These conserved elements play important roles in virus replication, transcription, and packaging (Wei et al. 2003; Jiang and Ghabrial 2004). The analogous (CAA)n repeats were similar to tobamovirus-like translational enhancer elements upstream of the AUG initiator codon of each dsRNA monocistronic in the PiCV1, similar to the typical chrysoviruses PcV and Hv145SV, consistent with previously reported chrysoviruses (Gallie and Walbot 1992; Jiang and Ghabrial 2004; Ghabrial and Suzuki 2009; Ghabrial et al. 2018). These enhancer elements may regulate genome replication and virion assembly for chrysoviruses in their Penicillium hosts, which would explain why chrysoviruses cause latent persistent infections in fungal hosts (Ghabrial et al. 2018). In this study, we found it difficult to obtain PiCV1-abolished isolates from the host HSPi-YN1 (P. italicum) through ribavirin-based protoplast regeneration. Phylogenetic analysis based on RdRp indicated that the Chrysoviridae family included two clade groups; cluster I consisted of PiCV1 and typical chrysovirus members (PcV, Hv145S, IjCV1, CnCV1, VdCV1, and ACCV), whereas cluster Ⅱ members were unclassified chrysovirus-related viruses, such as MoCV1, BdCV1, and PjCV1. These results were consistent with a recent report by the ICTV describing the Chrysoviridae family phylogenetic tree (Ghabrial et al. 2018). According to the PiCV1 selectivity of the host in Penicillum species, genomic characteristics, and phylogenetic analyses, we concluded that this was a new strain of P. chrysogenum virus.

      To date, the reported mycoviruses in most cases cause little or no obvious symptoms in their fungal hosts, and some phytopathogenic fungi infected by chrysoviruses can change their pathogenicity and pathogenic traits (Ghabrial and Suzuki 2009; Aihara et al. 2018; Okada et al. 2018). However, the PiCV1 infection caused no obvious change in the host P. italicum phenotypes including mycelia growth, pigment production, virulence and fungicide resistance (Fig. 7). Our results were consistent with the previous report that some typical chrysoviruses in Penicillium species (P. chrysogenum, P. cyaneo-fulvum, and P. brevicompactum) did have no obvious effects on their host fungi (Border et al. 1972). Infrequently, infection of specific mycovirus can enhance their host resistance to some abiotic stresses such as heat and salinity (Márquez et al. 2007; Nerva et al. 2017). Thus more biological effects of PiCV1 infection like those would be further studied.

      In the current study, we constructed and applied two methods, i.e., the blue–white colony screening method and the hygromycin B screening method to generate recipients infected with PiCV1 via hyphae anastomosis. The former was based on the visual morphological marker of albino donor colonies to distinguish genetic backgrounds between virus donors and recipients, whereas the latter was based on the hygromycin B resistance of the recipient. Although these methods showed the same efficiency for detection of horizontal transmission between different P. italicum individuals via hyphal anastomosis, antibiotic screening was not economical in terms of cost and required complex steps. In contrast, the white–blue colony screening method showed the following advantages. First, virus-infected recipient isolates were hyg-marker-free and had the same genetic background as the recipient strain before virus infection. Second, there was no need to consider the adverse effects of integration of the hyg gene into the fungal genome in the fungus/virus interaction system (Lee et al. 2014). In contrast, in hygromycin B screening, the virus must be retransmitted from the virus-infected recipient with overexpression of the hyg gene to the wild-type recipient (hyg sensitive) to exclude the influence of the hyg gene by back-introduction (Lee et al. 2014). Third, virus-infected isogenic candidate recipient isolates (blue) were visually selected based on the albino colony phenotype (white) of the virus donor in the coculture mixture, making this approach much simpler and more economical. Indeed, the process of hygromycin B screening was tedious and ineffective owing to the absence of a selective marker after coculture in back-introduction (Lee et al. 2014). Overall, our method could be tentatively applied to coculture screening of the horizontal transmission of mycoviruses to other filamentous fungi with pigmented colonies, such as Penicillium species and Aspergillus.

      To make a conclusion, the present study isolated and characterized a new chrysovirus PiCV1 from the citrus pathogen P. italicum (HSPi-YN1), constructed pksP-knockout HSPi-YN1 and verified its horizontal transmission among dually cultured P. italicum isolates, and established a blue–white colony method to select virusinfected P. italicum recipients (blue colonies) using HSPi-YN1ΔpksP (white colonies) as virus donors. Here we provide an effective and simplified approach to select virus-infected isogenic recipients based on the distinguishable colony-colors (i.e., blue virus-recipients versus white virus-donors), which would be applied to develop screening methods of virus-infected isogenic recipients for other filamentous fungi in future studies.

    • This work was supported by the National Natural Science Foundations of China (No. 31371893), the Natural Science Fund of Hubei Province (No. 2018CFB676) and the Project of Hubei Key Laboratory of Genetic Regulation and Integrative Biology (Grant No. GRIB20184).

    • DL and YY conceived this study, acquired project funding, revised to complete final version of manuscript, and supervised all research activities. TZ and NL designed experiments and completed the data analysis. QC, YC, BT and GL performed most of the experiments. TZ wrote the paper draft. All authors read and approved the final manuscript.

    • The authors declare that they have no competing interests.

    • This article does not contain any studies with human or animal subjects performed by any of the authors.

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