Co-Infection of Three Novel Deltaflexiviruses in an Isolate of the Edible Fungus Pleurotus ostreatus Reduces Fruiting Body Yield by Inhibiting Cellulase Activity

Abstract

Pleurotus ostreatus, a globally cultivated oyster mushroom, is susceptible to viral infections that threaten yield and quality. This study reports the identification and characterization of three novel viruses from a symptomatic P. ostreatus strain K3: Pleurotus ostreatus deltaflexivirus 2, 3, and 4 (PoDFV2, PoDFV3, PoDFV4). Complete genome sequencing revealed that they are single-stranded, positive-sense RNA viruses with lengths of 7809 nt, 7771 nt, and 7786 nt, encoding 5, 2, and 4 open reading frames (ORFs), respectively. The largest open reading frame (ORF1) encodes a putative replication-associated polyprotein (RP) containing three conserved domains—viral RNA methyltransferase (Mtr), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp). Based on genomic sequence analysis, multiple sequence alignments, and phylogenetic analysis, PoDFV2–4 were identified as novel viruses of the genus Deltaflexivirus within the family Deltaflexiviridae. PoDFV2–4 had no significant effects on mycelial growth rate, plate mycelial biomass, or laccase activity. However, they significantly inhibited mycelial cellulase activity and resulted in malformed fruiting bodies, as well as a substantial reduction in yield.

1. Introduction

Mycoviruses (fungal viruses) infect and replicate in all major fungal taxa, including yeasts, pathogenic fungi, and edible fungi [1]. According to the 2025 virus taxonomy released by the International Committee on Taxonomy of Viruses (ICTV) (https://talk.ictvonline.org/, accessed on 15 January 2025), mycoviruses comprise 47 families and 93 genera. Mycoviruses are divided into 17 families with double-stranded (ds) RNA genomes, 15 families with positive single-stranded (+ss) RNA genomes, 5 families with negative single-stranded (-ss) RNA genomes, one family with ssDNA genomes, and 2 families with RT-ssRNA genomes. The order Tymovirales consists of 5 approved families: Alphaflexiviridae, Betaflexiviridae, Deltaflexiviridae, Gammaflexiviridae, and Tymoviridae [2]. The family Deltaflexiviridae contains a single genus, Deltaflexivirus, whose members are primarily found in fungi [3]. Viruses in this genus generally carry a monopartite genome of 6–8 kb in length, which comprises four to five open reading frames (ORFs). The gene encoding the RNA replicase is the only one that is conserved among all deltaflexiviruses.

Most mycoviruses cause asymptomatic infections in their fungal hosts; however, mycovirus infections can cause significant economic losses in mushroom production [4,5,6,7]. Pleurotus ostreatus cultivation has become a key project in China for rural economic development, poverty alleviation, and rural revitalization, with an annual production of approximately 6 million tons [8]. Characterized by its high protein and low fat content, it offers numerous health benefits [9]. Furthermore, this fungus holds considerable economic value due to its robust enzyme system, which enables efficient degradation of cellulose and lignin. This capability allows P. ostreatus to convert agricultural waste into viable cultivation substrates. However, the cultivation of P. ostreatus faces increasing challenges, including reduced mycelial growth, delayed fruiting body development, poor fruiting, and severe fruiting body abnormalities associated with viral infections, which significantly hinder high-efficiency production [10]. It has been reported that 6 viruses have been identified in P. ostreatus, including 4 double-stranded RNA (dsRNA) viruses: Oyster Mushroom Isometric Virus (OMIV) [11], Pleurotus ostreatus virus 1 (PoV1) [12], Pleurotus ostreatus spherical virus (POSV) [13], and Pleurotus ostreatus ASI2792 mycovirus (PoV-ASI2792); and 2 +ssRNA viruses: Oyster mushroom spherical virus (OMSV) [14] and Pleurotus ostreatus deltaflexivirus 1 (PoDFV1), the latter belonging to the Deltaflexiviridae family.

In this study, we report the identification of three novel mycoviruses from P. ostreatus strain K3, designated as “Pleurotus ostreatus deltaflexivirus 2” (PoDFV2), “Pleurotus ostreatus deltaflexivirus 3” (PoDFV3), and “Pleurotus ostreatus deltaflexivirus 4” (PoDFV4). We further investigated the effects of these mycoviruses on the biological properties of the host strain K3 and elucidated the taxonomic classification of PoDFV2–4.

2. Materials and Methods

2.1. Fungal Isolates and Culture Conditions

The P. ostreatus strain K3, used in this study, was acquired from the Shouguang Institute of Edible Fungi (Shandong, China). For long-term preservation, mycelial plugs were stored in a sterile 25% (v/v) glycerol solution at −80 °C. To activate the culture, a mycelial plug was inoculated onto potato dextrose agar (PDA) and incubated at 25 °C in the dark for 7 days. Subsequently, mycelial plugs from the PDA culture were transferred to potato dextrose broth (PDB) and incubated in a shaking incubator at 28 °C and 180 rpm for 7 days to obtain fresh mycelia for subsequent experiments.

2.2. Detection and Purification of Viral Double-Stranded RNA (dsRNA)

To screen for the presence of mycoviruses, double-stranded RNA (dsRNA) was extracted from frozen mycelial samples using the CF-11 cellulose chromatography method, as previously described with modifications [15,16]. Briefly, the mycelia were ground into a fine powder in liquid nitrogen. The powder was homogenized in 1 × STE buffer (pH 8.0, containing 100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA) supplemented with 2% SDS and Tris-saturated phenol. The homogenate was then subjected to adsorption onto CF-11 cellulose, followed by a series of washing and elution steps. The purified dsRNA was treated with DNase I to remove any contaminating DNA and analyzed by electrophoresis on a 1% agarose gel to confirm the presence of dsRNA molecules.

2.3. Molecular Cloning and Sequencing

Viral genomes were identified through high-throughput sequencing of the total RNA extracted from the mycelia. The cDNA library was constructed and sequenced on an Illumina NovaSeq 6000 platform (performed by Shanghai Bohao Biotechnology Company, Shanghai, China). The raw reads were assembled de novo, and the resulting contigs were subjected to BLASTx (version 2.15.0) analysis against the NCBI non-redundant (NR) protein database. This analysis identified three contigs with significant similarity to known mycoviruses, designated as PoDFV2, PoDFV3, and PoDFV4. The presence of these viruses in the original strain was validated by RT-PCR using specific primers. To determine the complete genome sequences, the 5′- and 3′-terminal regions were amplified using rapid amplification of cDNA ends (RACE) kits (PC3–T7 Loop RACE and the Invitrogen 5′ RACE System, respectively). All PCR products were cloned into a plasmid vector and Sanger sequenced. The full-length genomes were assembled by integrating the terminal sequences with the internal contigs.

2.4. Sequence and Phylogenetic Analysis

Nucleotide sequences were assembled and analyzed using DNAMAN version 7. Open reading frames (ORFs) were predicted using the NCBI ORF Finder tool (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 25 January 2025). Reference sequences of related mycoviruses were retrieved from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 25 January 2025). Homology searches were performed using BLAST on the NCBI website (https://blast.ncbi.nlm.nih.gov/, accessed on 25 January 2025). Deduced amino acid sequences were aligned using DNAMAN version 7. Phylogenetic analyses were performed using MEGA version 11 [17]. Neighbor-joining trees were constructed with 1000 bootstrap replicates. Maximum likelihood phylogenetic trees were generated based on the best-fit substitution model for each dataset, with branch support evaluated using 1000 bootstrap replicates.

2.5. Impact of the Virus on the Host’s Biological Properties

2.5.1. Production of Virus-Free Isolates via Protoplast Regeneration and Molecular Validation

A systematic experimental approach was employed to assess the impact of viral infection on host biology. To generate virus-free (cured) and singly infected experimental materials, a virus elimination protocol based on protoplast regeneration was utilized, which exploits the uneven distribution of virus particles within host hyphal cells. Briefly, young hyphae of the target strain were digested enzymatically using lysozyme in a 0.6 mol/L mannitol solution to prepare protoplasts. The purified protoplast suspension was mixed with a PDA complex-sucrose hypertonic medium (supplemented with 100 µg/mL ampicillin) and pour-plated. Plates were incubated at 25 °C in the dark until single colonies appeared. Randomly selected regenerated colonies were subcultured. Total RNA was then extracted from the mycelia using the Trizol method and subjected to RT-PCR analysis with two pairs of virus-specific primers. A strain was confirmed as virus-free only if no target bands were amplified by either primer pair, thereby establishing a validated experimental system for subsequent studies.

2.5.2. In Vitro Assessment of Mycelial Growth Characteristics

Mycelial growth was evaluated based on colony morphology, radial growth rate, and biomass accumulation. To determine the growth rate, mycelial plugs (6 mm diameter) taken from the colony edge were inoculated at the center of fresh PDA plates. After incubation at 25 °C in the dark for 6 days, the colony diameter was measured, and the growth rate (mm/day) was calculated. Biomass was quantified using a cellophane overlay method: a mycelial plug was inoculated onto the center of a PDA plate overlaid with sterile cellophane. Following 7 days of incubation under identical conditions, the cellophane, now fully covered with mycelia, was carefully peeled off, residual medium was removed, and the fresh weight was recorded as a proxy for mycelial biomass. All plate assays were conducted with three biological replicates per sample, and the entire experiment was performed in three independent runs.

2.5.3. Evaluation of Reproductive Phenotypes Through Standardized Bag Cultivation

The effect of the virus on host reproductive growth was investigated using standardized bag-cultivation trials. The substrate was formulated with cottonseed hulls, wheat bran, and gypsum in a 78:20:2 (w/w) ratio, with moisture adjusted to 60–65%. Bags were filled with 500 g of substrate, sterilized at 121 °C for 2 h, and inoculated aseptically after cooling. Incubation proceeded at 25 °C and 60–70% relative humidity in darkness until full mycelial colonization. Subsequently, bags were transferred to a fruiting chamber. Primordia initiation and fruiting body development were induced by implementing a day–night temperature differential of 8–10 °C, increasing relative humidity to 85–95%, and providing scattered light. The first flush of fruiting bodies was documented morphologically and photographically. At harvest, the total fresh weight of fruiting bodies per bag was measured to evaluate viral effects on yield and morphology. The cultivation trial consisted of three biological replicates per treatment and was repeated three times independently.

2.5.4. Spectrophotometric Assay of Extracellular Lignocellulolytic Enzyme Activities

The activities of two key extracellular enzymes involved in lignocellulose degradation, laccase and cellulase, were assayed. Laccase activity was determined spectrophotometrically using ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] as the substrate. The reaction mixture contained 0.2 mL of 1.0 mmol/L ABTS, 2.7 mL of citrate-phosphate buffer (pH 5.0), and 0.2 mL of appropriately diluted enzyme extract. After incubation at 25 °C for 5 min, the increase in absorbance at 420 nm was recorded immediately. Enzyme activity (U) was calculated based on the molar extinction coefficient of ABTS (ε₄₂₀ = 36,000 L·mol⁻¹·cm⁻¹), with one unit defined as the amount of enzyme oxidizing 1 μmol of ABTS per minute. Cellulase activity was measured using a commercial assay kit (Cellulase Activity Assay Kit, Product No. BC2540, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) strictly according to the manufacturer’s protocol.

2.6. Statistical Analyses

All data were analyzed using GraphPad Prism version 9.5 and SPSS version 23.0 software. Subsequently, data (mean ± SE) from different experimental groups were analyzed using Student’s t-test and one- or two-way analysis of variance (ANOVA) followed by a least significant difference (LSD) test. A p-value of 0.01 or lower was considered statistically significant.

3. Result

3.1. Detection and Sequencing of dsRNA in P. ostreatus Strain K3

The P. ostreatus strain K3 was found to harbor two dsRNA species with lengths of approximately 7.5 kb and 10 kb (Figure 1). The band was resistant to digestion by DNase I and S1 nuclease, confirming it to be dsRNA. Analysis of the dsRNA extraction results indicated a potential mixed viral infection in the P. ostreatus strain K3. To identify the viral species present and assess their complexity, high-throughput sequencing was performed on qualified total RNA samples of strain K3. Based on the annotation information, only four virus-related contigs (Contig300, Contig1049, Contig1298, and Contig1926) were identified (Figure S1). BlastX searches showed that Contig300, Contig1049, and Contig1298 all exhibited the highest similarities (with amino acid sequence identities of 75.92%, 76.86%, and 83.59%, respectively) to the deltaflexivirus Pleurotus ostreatus deltaflexivirus 1 (PoDFV1). We have tentatively designated the viruses associated with these three contigs as Pleurotus ostreatus deltaflexivirus 2 (PoDFV2), Pleurotus ostreatus deltaflexivirus 3 (PoDFV3), and Pleurotus ostreatus deltaflexivirus 4 (PoDFV4), respectively. Additionally, Contig1926 is related to a member of the family Tymoviridae, the Oyster mushroom spherical virus Chinese strain (OMSV-Ch). To validate the accuracy of the high-throughput sequencing results, we designed specific primers for RT-PCR amplification targeting known sequences of three viruses (Figure S2). Subsequently, through cloning and sequencing, we obtained nearly complete genomes of PoDFV2 (7649 nt), PoDFV3 (7337 nt), and PoDFV4 (5400 nt). We employed the RACE technique to clone the unknown 5′ and 3′ terminal sequences of the virus in order to fill the gaps at the genome ends (Figure S3). Complete nucleotide sequences of PoDFV2, PoDFV3, and PoDFV4 were determined and deposited in GenBank under accession numbers PV467741.1, PV467742.1, and PV467743.1, respectively.

3.2. Molecular Characterization of PoDFV2–4

The genome organization of PoDFV2–4 is shown in Figure 2. Analysis of the nucleotide sequence of PoDFV2 revealed the presence of five ORFs (designated as ORF1–5) and 5′ and 3′ untranslated regions (UTRs) of 20 and 190 nt (excluding the polyA tail) (Figure 2 and

Table 1. Sequence identity (%) between PoDFV2–4/K3 and PoDFV1 based on multiple alignments of complete nt sequences, polyprotein sequences, and nt sequences of the 5′UTR and 3′UTR.

Virus	Genome Information
	Full Length (bp)	nt Identities (%)	ORF Numbers	ORF1 Length (nt)	Protein Molecular Weight (kDa)	aa Identities (%)	5′UTR (nt)	3′UTR (nt)
PoDFV2	7809	67.4	5	6000	221.4	76.64	20	190
PoDFV3	7771	66.8	2	5994	221.4	76.62	21	673
PoDFV4	7786	67.7	4	5958	219.1	76.82	66	287

). However, PoDFV3 was found to harbor only two putative ORFs (ORF1, 2), with 5′ and 3′ UTRs measuring 21 and 672 nucleotides, respectively. In the case of PoDFV4, four putative ORFs (ORF1–4) were identified, accompanied by 5′ and 3′ UTRs of 66 and 287 nucleotides in length, respectively. PoDFV2–4 ORF1 encodes a ~220 kDa replication-associated polyprotein (RP), respectively. The RP contains three conserved domains: viral RNA methyltransferase (Mtr), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp), which are present in all members of the family Deltaflexiviridae. BLASTp analysis revealed no significant homology for the amino acid sequences encoded by ORF2–5 of PoDFV2, ORF2 of PoDFV3, or ORF2–4 of PoDFV4. Consequently, these ORFs are predicted to encode small (15–20 kDa) hypothetical proteins with currently unknown biological functions.

3.3. Sequence Alignment and Phylogenetic Classification of PoDFV2–4/K3

The amino acid sequence alignments were obtained using the putative Mtr, Hel, and RdRp domains of PoDFV2–4/K3 and selected viruses in the family Deltaflexiviridae (Figure 3). Specifically, putative Mtr domains were identified at the N-terminus of the replication-associated polyprotein in PoDFV2 (75 aa, nt 256–480), PoDFV3 (75 aa, nt 255–479), and PoDFV4 (72 aa, nt 247–463). The Hel domain was identified at the N-terminus of the replication-associated polyprotein, downstream of the Mtr domain, in PoDFV2 (81 aa, nt 1180–1425), PoDFV3 (80 aa, nt 1174–1413), and PoDFV4 (84 aa, nt 1162–1415). An RdRp domain was detected at the C-terminal end of the RP of PoDFV2–4/K3, downstream from the Mtr domain. Multiple sequence alignments revealed that the Mtr domain of PoDFV2–4 contains six conserved motifs (I–VI, Figure 3a). The Hel domain of PoDFV2–4 also comprises six conserved motifs (I–VI, Figure 3b). In comparison, the RdRp domain of PoDFV2–4 contains six conserved viral RdRp motifs (I–VI, Figure 3c). To better understand the taxonomic status of PoDFV2–4, phylogenetic trees were generated and visualized with MEGA (Version 7.0). A phylogenetic tree based on the RP amino acid sequences of the members in the family Deltaflexiviridae showed that PoDFV2–4 clustered with very high bootstrap confidence with SsDFV1, SsDFV2, SsDFV3, and PoDFV1, which are members of the genus Deltaflexivirus, family Deltaflexiviridae (Figure 4). According to the species demarcation criteria of the ICTV, the Deltaflexiviridae family, a nucleotide sequence identity of <72% or an amino acid sequence identity of <80% in the replication-associated polyprotein (RP) is considered indicative of a new species [18]. These values fall below the established threshold for demarcating species. Therefore, we identify PoDFV2, PoDFV3, and PoDFV4 as three new species in the genus Deltaflexivirus.

3.4. Biological Effects of PoDFV2–4/K3 on P. ostreatus

To assess the impact of PoDFV2–4/K3 infection on the biological properties of P. ostreatus strain K3, an isogenic virus-free strain (designated K3–VF) was required via protoplast regeneration (Figure S4a). Additionally, we obtained singly infected strains harboring only PoDFV2, PoDFV3, or PoDFV4, designated K3–V2, K3–V3, and K3–V4, respectively (Figure S4b). The mycelial growth rate of K3–VF was 12.07 mm/d, while the growth rates of the virus-infected strains K3–V2, K3–V3, and K3–V4 were 11.84 mm/d, 12.15 mm/d, and 11.54 mm/d, respectively. No significant differences in mycelial growth rates were observed among the different virus-infected strains or between these strains and either the wild-type K3 or K3-VF control, indicating that these three viruses do not affect the mycelial growth rate. (Figure 5b). Similarly, the mycelial biomass of K3-VF was 0.81 g, with no significant differences detected among the infected strains, suggesting that viral infection also does not influence mycelial biomass accumulation (Figure 5c). Observation and yield measurement of the first flush of fruiting bodies at the primordia stage revealed that virus-infected strains exhibited abnormal fruiting body morphology, primarily characterized by smaller pilei and stunted development, whereas the K3-VF strain displayed larger pilei and well-developed fruiting bodies (Figure 6a). The yield of the K3-VF strain was 62.39 g per bag, whereas the average yields for strains K3–V2, K3–V3, and K3–V4 were 42.56 g, 40.19 g, and 41.36 g per bag, respectively. This result demonstrates that PoDFV2–4 infection significantly reduces fruiting body yield (p < 0.01) (Figure 6b).

Fruiting bodies of the healthy-looking P. ostreatus strains show typical phenotypes of the oyster mushroom, such as a long and thick stipe and a middle slate-gray pileus with a low funnel shape [19]. To investigate whether the yield reduction and abnormal morphology of the fruiting body were associated with intracellular enzyme activity, we measured laccase and cellulase activity in virus-free and infected strains using the ABTS method. No significant differences in laccase activity were found between the K3-VF strain and the virus-infected strains or the wild-type K3 strain (Figure 6c). However, determination of cellulase activity revealed significant differences both between K3-VF and each of the virus-infected strains (K3–V2, K3–V3, and K3–V4) and between K3-VF and the wild-type K3 (p < 0.01) (Figure 6d). In contrast, no significant differences were observed among the infected strains themselves. These findings suggest that PoDFV2–4 may impair the strain’s nutrient absorption and utilization by inhibiting cellulase activity in the strain K3, leading to malformed fruiting bodies and a significant reduction in yield.

4. Discussion

This study characterizes three novel mycoviruses—PoDFV2, PoDFV3, and PoDFV4—from Pleurotus ostreatus, expanding the known diversity within the Deltaflexiviridae family. Beyond their molecular identification, we systematically investigated their biological impact, revealing a distinct and nuanced phenotype that advances our understanding of virus–fungus interactions in economically important basidiomycetes.

All three viruses displayed significant homology to PoDFV1 (Deltaflexiviridae), with full-length nucleotide and ORF1 amino acid sequence identities ranging from 66.8% to 67.7% and from 76.62% to 76.82%, respectively (Table 1). Genome structure analysis, multiple sequence alignments, and phylogenetic analysis showed that PoDFV2–4 were novel viruses of the genus Deltaflexivirus within the family Deltaflexiviridae. The genomic organizations of PoDFV2 and PoDFV4 resemble those of other known deltaflexiviruses, comprising 4 to 5 putative ORFs, including one large ORF (ORF1) and 3 to 4 small, extensively overlapped ORFs. In contrast, the genome of PoDFV3 comprises only 2 putative ORFs: ORF1 and an additional small ORF. ORF1 encodes 3 conserved domains—Mtr, Hel, and RdRp—each of which contains 6 conserved motifs that are characteristic of viruses belonging to the family Deltaflexiviridae.

While mycovirus infections are generally asymptomatic in their fungal hosts, notable exceptions exist. The symptoms of certain mycovirus infections in P. ostreatus include reduced mycelial growth, fruiting body abnormalities, and decreased fruiting body yield [20,21,22]. Although there has been considerable research on viruses in P. ostreatus, reports on the Deltaflexiviridae family are limited to PoDFV1. Only the genomic sequence of PoDFV1 has been reported, and its biological characteristics have not yet been fully characterized [23]. Virus-free and virus-infected strains harboring PoDFV2, PoDFV3, and PoDFV4, respectively, were generated via protoplast regeneration. A comparative analysis was then conducted to evaluate key phenotypic traits, including colony morphology, growth rate, biomass, fruiting body morphology, and yield. Lignocellulolytic enzymes produced by cultivated mushrooms play a crucial role in the degradation of complex plant biomass into soluble substances [24]. In this study, PoDFV2–4 had no significant effects on the colony morphology, growth rate, mycelial biomass, or laccase activity of strain K3. This result is consistent with the finding that SsDFV2 infection did not cause significant differences in the growth rate of the Ep-1PNA367hph strain [25]. Crucially, however, the viruses imposed severe abnormalities in fruiting body morphology and a significant reduction in yield. This dissociation between asymptomatic vegetative growth and dysfunctional reproduction is significant. Fruiting body development in basidiomycetes like P. ostreatus is a highly complex, energy-demanding process regulated by intricate genetic and environmental signaling pathways [26]. The virus-induced failure at this stage suggests a targeted interference with these developmental cascades, rather than a general metabolic burden. Furthermore, we observed a specific suppression of cellulase activity, while laccase activity remained unchanged. This selective inhibition of enzymatic activity parallels the effects reported for the double-stranded RNA virus PoV-ASI2792 on extracellular enzyme activity [27]. It implies that PoDFV2–4 may disrupt a specific node in the lignocellulose degradation pathway or its regulation. Given the critical role of carbon metabolism and nutrient reallocation from substrate mycelium to developing fruiting bodies, such targeted interference with cellulose degradation could directly compromise the energy supply necessary for proper morphogenesis. Here, PoDFV2–4 was found to suppress cellulase activity specifically, without imparting significant changes to laccase activity.