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Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China
f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 5 0 7 e5 1 3
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Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China Liyou QIU*, Yanpeng LI, Yingmiao LIU, Yuqian GAO, Yuancheng QI, Jinwen SHEN College of Life Sciences, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, Henan, China
article info
abstract
Article history:
Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms
Received 3 September 2009
of degeneration. A spherical virus POSV and four dsRNA segments were extracted from
Received in revised form
mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5 kb
17 March 2010
dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa.
Accepted 6 April 2010
Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The
Available online 10 April 2010
1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences
Corresponding Editor:
of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases
Daniel C. Eastwood
(RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid
Keywords:
sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and
Naked virus
capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the
Particle virus
amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs
Pleurotus ostreatus
A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus
RNA mycovirus
TD300, the symptoms of degeneration completely disappeared. The results reveal that
Strain degeneration
P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Oyster mushroom (Pleurotus ostreatus) is the most popular mushroom in China and Korea. Its annual production is about 4 000 000 tons and accounts for about 40 % of the total mushroom fruiting body in China. It is a very healthy food ingredient since it contains enriched proteins, various biologically active polysaccharides, and other nutrients (Khatun et al. 2007; Gern et al. 2008; Jayakumar et al. 2006; Li et al. 2007). Furthermore it gets attention for its beneficial impacts of bioremediation on the environment and waste bioconversion (Rodrı´guez Pe´rez et al. 2008; Locci et al. 2008; Salmones et al. 2005).
However, P. ostreatus cultivation has been facing increasing challenges due to viral infections and strain degenerations. The symptoms of viral infection and strain degenerations were similar, including reduced mycelial growth, delayed fruiting body development, poor fruiting and severe fruiting body abnormalities (Yu et al. 2004; Go et al. 1992; van der Lende et al. 1995; Ro et al. 2006). P. ostreatus virus diseases were discovered to be related with several mycoviruses, such as double-stranded RNA (dsRNA) viruses (van der Lende et al. 1995), oyster mushroom isometric virus OMIV-I and OMIV-II (Yu et al. 2004; Ro et al. 2006), and oyster mushroom spherical virus (OMSV) (Yu et al. 2003). In
* Corresponding author. Tel.: þ86 371 6355 5175; fax: þ86 371 6355 5790. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.04.001
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contrast, infection with PoV1 did not show any distinct morphological or growth phenotypes (Ro et al. 2006). On the other hand, the cause of strain degeneration of P. ostreatus is still unknown. Besides P. ostreatus, virus diseases and strain degenerations were also found on many other mushrooms. La France isometric virus (LIV) (Hollings 1962), mushroom bacilliform virus (MBV) (Tavantzis et al. 1980), and mushroom virus X (MVX) (Gaze et al. 2000) were found to be the causative agents of virus diseases of Agaricus bisporus, as well as virus-like particles (VLPs) of Lentinus edodes (Ushiyama & Nakai 1975), and PeSV of Pleurotus eryngii (Ro et al. 2006). Strain degenerations of A. bisporus and Flammulina velutipes (Enokitake) have become a serious problem to industrial cultivation, and the chromosomal abnormalities responded to strain degenerations of A. bisporus (Horgen et al. 1996; Magae et al. 2005). P. ostreatus TD300 is a main cultivating strain in China, but in recent years it displayed serious degeneration. In this study, particle and dsRNA mycovirus in mycelium of P. ostreatus TD300 were isolated, their genome nucleic acid and capsid protein (CP) were characterized. One particle and two naked RNA mycoviruses were identified in P. ostreatus TD300.
Materials and methods Strain and media Strain of Pleurotus ostreatus TD300 was a commercial sample collected by Mushroom Cultivation Base of Henan Agricultural University. The media used were: PDA (potato dextrose agar) and PD (potato dextrose); WSP (filtrate from 100 g wheat bran boiled for 30 min, sugar 10 g, peptone 5 g, MgSO4$7H2O 0.5 g, KH2PO4 0.1 g, and distilled water added to 1 l); RCM (glucose 20 g, yeast extract 2.0 g, peptone 2.0 g, K2HPO4 1.0 g, KH2PO4 0.46 g, MgSO4$7H2O 0.5 g, mannitol 10.93 g, agar 15 g, and distilled water added to 1 l) (Kim et al. 1997). CCM (cottonseed hull 100 g, lime 1 g, gypsum 1 g, tap water 110 mL).
Purification and transmission electronic microscopy of mycovirus Purification of mycoviruses was carried out as described previously (Preisig et al. 1998). Mycelia were grown in WSP medium at 25 C on a rotary shaker at 150 rpm for 14 d. Mycelia were harvested and 100 g (wet weight) was powdered with liquid nitrogen and was mixed with two volumes of STE buffer (0.2 M NaCl, 0.1 M Tris, 0.002 M EDTA, pH 7.4), and then centrifuged at 80 000 g for 30 min at 4 C (Beckman, USA). The supernatant was mixed with polyethylene glycol 6000 (PEG 6000) to the final concentrations of 4 %, placed for 2 h at 4 C. After centrifugation at 100 000 g for 30 min at 4 C, the pellet was resuspended in 3 mL 1.5 STE buffer to obtain partially purified mycoviruses. And 3 mL those mycoviruses were layered onto 10 mL CsCl solution (1 g/2.5 mL) and ultracentrifuged at 400 000 g for 2 h at 4 C. The pellet was dissolved in 3 mL of 1.5 STE buffer, layered onto 10e40 % (w/v) CsCl density gradient solution, and centrifuged at 60 000 g for 3 h at 4 C. The collected virus band was
L. Qiu et al.
mixed with 5 volumes of ultrapure water, and then centrifuged at 60 000 g for 2 h at 4 C. The purified virus particles were examined with an H-8100 transmission electronic microscope (Hitachi, Japan) after been stained with 2 % phosphotungstic acid for 2 min.
Determinations of molecular weight of viral protein and genome size Molecular weight of the viral coat protein was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 5 mL loading buffer (100 mg SDS, 0.1 mL b-mercaptoethanol, 1 mL glycerol, 2 mg bromophenol blue, 0.5 mL 0.2 mol/L, pH 7.2 phosphate buffer solution, using double distilled water adjusted to 10 mL) was added to viral particles solution, and boiled for 5 min. The samples were analyzed using SDS-PAGE with 5 % concentration gel and 12 % separation gel. Coomassie brilliant blue G-250 was used for staining. Viral nucleic acids were extracted from the purified virus with equal volume phenol/chloroform (1:1) and then centrifuged at 15 000 g for 10 min. This step was repeated twice. Equal volume chloroform was added to the supernatant and centrifuged at 15 000 g for 10 min. The supernatant was mixed with 2 volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2), placed at 20 C for 1 h. After centrifuging at 15 000 g for 10 min, the pellet was washed with 70 % ethanol and centrifuged again. The viral nucleic acid (500 ng) was treated with RNase A (10 mg/mL) in 0.5 or 0.05 M NaCl or with DNase I (10 units/mL) in 10 mM MgCl2 at 37 C for 30 min. The nucleic acid samples were analyzed using 1 % agarose gel electrophoresis.
Extraction and electrophoresis of dsRNA dsRNA was isolated from mycelium using CF-11 enrichment technique modified of Morris & Dodds (1979). Pleurotus ostreatus mycelium (3 g per tube) was homogenized on ice in a plastic pestle with 3 mL GPS buffer (0.2 M glycine, 0.1 M Na2PO4, 0.6 M NaCl, pH 9.5), 3 mL saturated phenol, 1.5 mL chloroform/isoamyl alcohol (25/1), 0.3 mL 10 % SDS, and 0.03 mL b-mercaptoethanol. The homogenate was centrifuged at 12 000 g for 20 min at 4 C, and the aqueous layers of supernatant liquid were adjusted to 15 % ethanol. CF-11 celluose (Sigma) was added and mixed, and then loaded into a 10 mL syringe barrel (plugged with siliconised glass wool) and washed with 150 mL STE buffer (0.1 M NaCl, 0.05 M TriseHCl, 1 mM EDTA, pH 7.0) containing 15 % ethanol (STE/15). The dsRNA was eluted with 10 mL of STE, added with 2 volumes ethanol and 0.1 volumes 2 M sodium acetate to the eluant, precipitated for over night at 20 C, and centrifuged at 12 000 g for 20 min at 4 C. 1 % agarose gel electrophoresis was used to analyze dsRNA. The viral dsRNAs were confirmed by using DNase I and S1 Nuclease (Cheng et al. 2003). The viral nucleic acid (500 ng) was treated with 1 mL DNase I (60e80 units/mL) and in DNase I buffer (TaKaRa, Dalian, China) at 37 C for 30 min, or 10 U S1 Nuclease (TaKaRa) in S1 Nuclease buffer at 23 C for 15 min. The nucleic acid samples were analyzed by 1 % agarose gel electrophoresis.
Particle and naked RNA mycoviruses in industrially cultivated mushroom
Fig 1 e Electron microcopy of virus particle POSV from Pleurotus ostreatus TD300, it had a diameter of 23 nm.
cDNA synthesis, cloning and sequencing of dsRNAs Randomly primed cDNAs from every dsRNA element of Pleurotus ostreatus TD300 were synthesized using Invitrogen SUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat. 18248-013; Gibco/BRL). dsRNA was firstly denatured at 100 C for 8 min. cDNAs were cloned into plasmid pMD18-T (TaKaRa) and sequenced. Sequence information was analyzed with DNAstar software. Homologies of both nucleic acid and predicted amino acid sequences were determined using DNAMAN software. Encoded amino acid sequences were analyzed using ExPASy.
The preparation of virus free strain Virus free strains of Pleurotus ostreatus TD300 were prepared by protoplast regeneration. Mycelia of P. ostreatus TD300 was
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Fig 3 e Agarose gel analysis of dsRNA of particle virus POSV from mycelium of P. ostreatus TD300.
cultured as starting stain in 500 mL flasks containing 100 mL PD broth at 25 C for 5 d. The mycelia were harvested and washed twice with distilled water and 0.6 M mannitol. The mycelia were dried on absorbent paper and then suspended in filter-sterilized Lywallzyme solution (20 mg/mL in 0.6 M mannitol; Guangdong Institute of Microbiology, China) at 30 C on a rotary shaker (100 rpm). The lytic enzyme treated mycelia were sampled half hourly until a large quantity of protoplast formed. The remaining hyphal fragments were removed by filtration through a 0.5 cm column of absorbent cotton placed in the bottom of a syringe. Protoplasts were collected from the filtrate by centrifugation at 4500 rpm for 20 min. Pellets were washed twice with 0.6 M mannitol. After suspending the final pellet in 0.6 M mannitol, the concentration of the protoplast was determined by using a hemocytometer. For regeneration, 0.1 mL of protoplast suspension (about 105/mL) was plated on RCM medium. The plates were incubated at 25 C for 7 d and stellate aristae colonies grew out. Dikaryotic mycelium colonies were identified by the presence of clamp connections and picked to PDA plates. The plates were incubated at 25 C for 7 d. The virus free strains were screened by using CF-11 enrichment technique.
Mycelia growth rate Agar plugs of 1 cm diameter from the inoculation plates of the virus free strain and starting strain of Pleurotus ostreatus TD300 were transferred onto five freshly prepared PDA plates. The plates were incubated at 25 C and colony diameters were measured after 3 d.
Biomass and laccase activity
Fig 2 e SDS-PAGE analysis of coat proteins (CP) of particle virus POSV from mycelium of P. ostreatus TD300.
Agar plugs of 1 cm diameter from the inoculation plates of the virus free strain and starting strain of Pleurotus ostreatus TD300 were transferred into five 500 mL flasks containing 100 mL PA broth, and then incubated at 25 C on a rotary shaker
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parts and were placed separately in the two end and middle sections of plastic bags containing 3000 g CCM. The virus free and starting strain of P. ostreatus TD300 was cultivated in 30 bags at 25 C. Soon after the hyphae had fully spread, the bags were kept in a cold room at 18 C, 95 % RH and under incandescent lighting of 1000 Lux to induce primordia formation. Basidiomata were harvested and weighed at 7/8th ripe. The yield of mushroom production was recorded by the weight (g) of freshly harvested basidiomata per bag. Biological efficiency was defined as the percentage of the fresh weight of harvested mushrooms over the dry weight of inoculation substrates.
Statistical analysis Student’s t test was used to analyze the data for differences in growth rate, biomass, enzyme activity, or biological efficiency between the virus free strain and starting strain of Pleurotus ostreatus TD300.
Results Characterization of mycovirus particles
Fig 4 e Agarose gel analysis of extraction dsRNA (lane 1) from mycelium of P. ostreatus TD300.
Viral particles purified from Pleurotus ostreatus TD300 mycelium were observed and a spherical virus with 23-nm diameter was found and named POSV (Fig 1). Proteins and nucleic acids of the viral particle were analyzed by SDS-PAGE and agarose gel electrophoresis. The molecular weights of the coat proteins were 39 kDa and 30 kDa, respectively (Fig 2). An encapsidated 2.5 kb segment was confirmed as a dsRNA fraction by using DNase I and S1 Nuclease degradation (Fig 3).
(200 rpm) for 8 d. The cultured fluids were centrifuged at 3000 rpm for 10 min. Pellets were dried at 60 C until their weight kept constant. The supernatant liquids underwent laccase assay. Lactase activity was determined by oxidation of 2, 20 -azmobis-(3-ethyl benzthiazoline-6-sulphonate) (ABTS). The reaction mixture contained 0.5 mM ABTS, 0.1 M sodium acetate buffer (pH 4.5) and the supernatant liquid. Oxidation of ABTS was followed by absorbance increase by 420 nm (3420 ¼ 3.6 104 M1 cm1). Enzyme activity was expressed in units (U ¼ mmol/min) (Bourbonnais & Paice 1990).
dsRNA profiles of mycelium Four dsRNA bands were extracted from Pleurotus ostreatus TD300 mycelium using CF-11 enrichment technique, and confirmed by DNase I and S1 Nuclease degradation. Their sizes were 8.2 kb (doublet), 2.5 kb, 2.0 kb, and 1.1 kb, respectively (Fig 4). The 8.2 kb, 2.0 kb, and 1.1 kb dsRNAs were not detected in RNA sample obtained from the virus particles, suggesting that these three dsRNAs are naked viral genomes. The 1.1 kb dsRNA band may not always be present.
Basidiomata formation Fungal inocula were prepared first. Agar plugs of 1 cm diameter from the inoculation plates of the virus free strain and starting strain of Pleurotus ostreatus TD300 were transferred into plastic bags containing 500 g CCM, and then incubated at 25 C until the hyphae spread all over bags. The inocula were broken into suitable sizes. Inocula (300 g) were divided into three equal
dsRNAs sequence homology analysis Two cDNA sets of 264 bp and 3479 bp were obtained from the 8.2 kb dsRNA segment. Their nucleic acid and amino acid
Table 1 e Summary of sequence analysis of dsRNA elements from Pleurotus ostreatus TD300. dsRNA (kb)
bp sequenced (GenBank accession no.)
8.2
264 (GQ505290) 3479 (GQ505290) 1558 (GQ505291) 1046 (GQ505292)
2.5 2.0
Homologies
Identities of cDNA sequence (%)
Identities of the amino acid sequence of cDNA (%)
RDRP in OMSV RDRP in OMSV
93 95
89 91
RDRP in dsRNA-1 of PoV1 CP in dsRNA-2 of PoV1
83 73
90 74
OMSV: oyster mushroom spherical virus; PoV1: P. ostreatus virus 1.
Conserved motifs
RDRP conserved motifs A to D and helicase motif GXGKS(T) RDRP conserved motifs A to C
Particle and naked RNA mycoviruses in industrially cultivated mushroom
A
B
511
C
D
Fig 5 e Alignments of the four conserved motifs A to D within RDRP sequences in mushroom virus genomes. The sequence accession number in GeneBank is AY182001 (oyster mushroom spherical virus, OMSV), AY533038 (P. ostreatus virus 1, PoV1), AY308801 (oyster mushroom isometric virus II, OMIV-II), X94361 (Agaricus bisporus virus 1, ABV1), NC_001633 (mushroom bacilliform virus, MBV), AB429554 (Lentinula edodes mycovirus HKB), GQ505290 (P. ostreatus dsRNA virus, 8.2 kb segment), and GQ505291 (P. ostreatus dsRNA virus, 2.5 kb segment), respectively. Amino acids that are conserved are highlighted. The length separating the motifs is indicated. Italic sequence name means that it was from ssRNA.
sequences were around 90 % identical to those of RDRP in oyster mushroom spherical virus (OMSV), an ssRNA virus (Yu et al. 2003) (Table 1). In particular, the amino acid sequence of the 3479 bp set had four conserved motifs A to D of RNAdependent polymerases in viruses (Poch et al. 1989), and the region containing the motifs was nearly identical to that of OMSV (Fig 5). Furthermore, the conserved helicase motif GXGKS(T) (Kadare & Haenni 1997) was also present in the 3479 bp set. A 1558 bp cDNA set was obtained from the 2.5 kb dsRNA segment. It was homologous to the RDRP gene in PoV1 dsRNA-1 (Lim et al. 2005) (Table 1), and contained three conserved motifs A to C, which were very similar to those in RDRP of PoV1 dsRNA-1 (Fig 5). From the 2.0 kb dsRNA segment we sequenced a 1046 bp cDNA set. Its nucleic and amino acid sequences were homologous to those of the capsidic protein of PoV1 dsRNA-2 (Lim et al. 2005) (Table 1).
Characterization and comparison between the virus free strain and starting strain of Pleurotus ostreatus TD300 A lot of protoplast regenerated strains were gained. Some, such as PR09, did not show the 8.2 kb dsRNA segment.
Fig 6 e dsRNA detection of virus eliminated or free strain of P. ostreatus TD300 by protoplast regeneration. M. DNA molecular marker; 1. Virus eliminated strain PR09; 2. Virus eliminated strain PR22; 3. Virus free strain PR15.
Some, such as PR22, had neither the 8.2 kb nor 2.1 kb segments. And some, such as PR15, had none of the 8.2 kb, 2.5 kb and 2.1 kb segments (Fig 6). Similar result was described elsewhere (van der Lende et al. 1995). Compared to the starting strain, the hyphae of the virus free strain PR15 grown on PDA plate were whiter and denser, and the hyphae growth rate was 18.18 % higher (Table 2); Similarly, the hyphae biomass and laccase activity of PR15 grown on PD broth for 8 d were 145.70 % and 134.36 % higher than those of the starting strain. In both cultivated bags, the period from incubation to primordia appearance of PR15 was 5 d shorter than that of starting strain, the basidiomata looked more even in appearance and size, and had thicker pileus, and the biological efficiency was 20.70 % higher (Table 2).
Discussion A spherical virus POSV, which was 23 nm in diameter and encapsidated a 2.5 kb dsRNA genome, was found in mycelium of Pleurotus ostreatus TD300. Similarly, three other dsRNA spherical viruses were extracted in the mycelium of P. ostreatus var. florida and P. ostreatus strain ASI2596 (van der Lende et al. 1995; Lim et al. 2005). And three dsRNA isometric viruses were found in P. ostreatus (Yu et al. 2004) and P. ostreatus cultivar “Suhan” (Ro et al. 2006). However, only one ssRNA spherical virus OMSV was found in P. ostreatus (Yu et al. 2003). So we proposed that the particle viruses infected in P. ostreatus were mostly dsRNA viruses. A 2.5 kb dsRNA segment encapsidated in POSV and a 2.0 kb dsRNA segment isolated from the mycelium of P. ostreatus TD300 had high identities to the genome of Partiviridae virus PoV1, respectively. However, POSV only encapsidated 2.5 kb dsRNA and was not a Partiviridae virus. It needed further research to determine whether the 2.5 kb and 2.0 kb dsRNA segments belong to a Partiviridae. Naked dsRNA segments around 8.0 kb had been found in P. ostreatus strain Shin-Nong (Kim et al. 2008), P. ostreatus var. florida (van der Lende et al. 1995), but their sequences were not determined. We also extracted an 8.2 kb dsRNA segment from mycelium of P. ostreatus TD300, and obtained two cDNA sequences, 264 bp and 3479 bp, from the dsRNA segment. Their nucleic and amino acid sequences were highly similar to that of RDRP of ssRNA particular virus OMSV
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Table 2 e Biological character comparison between the virus free strain PR15 and starting strain of P. ostreatus TD300a. Strain PR15 Starting strain
Growth rateb (mm/d)
Biomassc (g Dry Wt/l)
Laccase activityc (U/mL)
Days taken from incubation to primordia appearanced
Biological efficiencyd (%)
6.5** 5.5
45.7** 18.6
193.75** 82.67
38 43
94.5** 78.3
a Paired numbers within a column with ** are significantly different at the 0.01 level from corresponding values of starting strain. b Hyphae grown on PDA plates. The values of growth rate are arithmetic means of the five replicate measurements. c Hyphae grown on PD broth. The values of biomass or laccase activity are arithmetic means of the five replicate measurements. d Hyphae grown on cottonseed hull cultivation medium (CCM). The values of days taken from incubation to primordia appearance or biological efficiency are arithmetic means of the 30 replicate measurements.
(Table 1 and Fig 5). The length separating the conserved motifs A to B was smaller than 45 amino acids in most of the positive strand ssRNA virus, but greater than 50 amino acids in dsRNA viruses (Fig 5) (Poch et al. 1989). Thus, the 8.2 kb dsRNA segment may be the replicative form of an ssRNA virus genome, and most closely related to Botrytis virus F (BVF) whom OMSV is very similar to (Yu et al. 2003). Only one gene is common to all RNA viruses. It encodes an RDRP or an RNA-dependent DNA polymerase (Bruenn 1993). Although there is low sequence similarity over the entire length of RDRPs among dsRNA viruses (Wiener & Joklik 1989), four common motifs A to D are conserved with the same linear arrangement in RDRPs encoded by RNA viruses (Poch et al. 1989). The four conserved motifs present in the 8.2 kb, 2.5 kb, and other mushroom virtual genomes were compared in Fig 5. It demonstrated that the conserved motifs were homologous between OMSV and the 8.2 kb dsRNA, and between PoV1 and the 2.5 kb dsRNA, in spite of being isolated from different cultures in South Korea and China. The result suggested that the viruses of P. ostreatus had same origin. Furthermore, the conserved motifs in RDRP of dsRNA viruses displayed higher degree of identity within the dsRNA viruses than between the dsRNA and ssRNA viruses of P. ostreatus. Similarly the conserved motifs in dsRNA viruses ABV1 had a low identity with that of ssRNA virus MBV of Agaricus bisporus, so were the mycoviruses infecting P. ostreatus, A. bisporus, and Lentinula edodes (Fig 5). Chromosomal abnormalities were discovered to associate with strain degeneration in the cultivated mushroom, A. bisporus (Horgen et al. 1996). Although there was no clear evidence for a relationship between the presence of viruses and mushroom strain degeneration, we found that after eliminating the viruses of P. ostreatus TD300, the symptoms of degeneration disappeared. The hypha of the virus free strain from protoplast regenerant grew denser and faster than the starting strain. Also, the biomass, laccase enzyme activity, and mushroom biological efficiency of the virus free strain were all higher (Table 2). Magae et al. (1985) selected six strains from the dikaryotic protoplast regenerants of P. ostreatus at random, and all strains were able to form normal fruiting bodies, and the yield was 18 % greater than the parent. They suggested that a protoplast regeneration process may yield useful variations. We propose that virus was probably eliminated in the protoplast regeneration process. Our results revealed that mycoviruses infection is a probable cause of mushroom strain degeneration.
Acknowledgements This research was supported by the Science and Technology Development Program of Henan Provincial Department of Science and Technology, and the Doctorial Innovation Fund of Henan Agricultural University.
Supplementary material Supplementary material for this article is available, in the online version, at doi:10.1016/j.funbio.2010.04.001.
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Ro HS, Lee NJ, Lee CW, Lee HS, 2006. Isolation of a novel mycovirus OMIV in Pleurotus ostreatus and its detection using a triple antibody sandwich-ELISA. Journal of Virological Methods 138: 24e29. Rodrı´guez Pe´rez S, Garcı´a Oduardo N, Bermu´dez Savo´n RC, Ferna´ndez Boiza´n M, Augur C, 2008. Decolourisation of mushroom farm wastewater by Pleurotus ostreatus. Biodegradation 19: 519e526. Salmones D, Mata G, Waliszewski KN, 2005. Comparative culturing of Pleurotus spp. on coffee pulp and wheat straw: biomass production and substrate biodegradation. Bioresource Technology 96: 537e544. Tavantzis SM, Romaine CP, Smith SH, 1980. Purification and partial characterization of a bacilliform virus from Agaricus bisporus: a single stranded RNA mycovirus. Virology 105: 94e102. Ushiyama R, Nakai Y, 1975. Viruses associated with Hymenomycetes. II. Presence of polyhedral virus-like particles in shiitake mushroom, Lentinus edodes (Berk.) Sing. Reports of the Tottori Mycological Institute (Japan) 12: 53e60. van der Lende TR, Harmsen MC, Go SJ, Wessel JGH, 1995. Doublestranded RNA mycoviruses in mycelium of Pleurotus ostreatus. FEMS Microbiology Letters 125: 51e56. Wiener JR, Joklik WK, 1989. The sequences of the reovirus serotype 1, 2 and 3 L1 genome segments and analysis of the mode of divergence of the reovirus serotypes. Virology 169: 194e203. Yu HJ, Lee JS, Lee NJ, Ha SJ, Kim SY, Gang EJ, Bae DW, Chang MU, Lee HS, 2004. Identification of three isometric viruses from Pleurotus ostreatus (Running Title: ssRNA and dsRNA Viruses of Oyster Mushroom, Plerortus ostreatus). Journal of Huazhong Agricultural University 23: 150e156. Yu HJ, Lim D, Lee HS, 2003. Characterization of a novel singlestranded RNA mycovirus in Pleurotus ostreatus. Virology 314: 9e15.
journal homepage: www.elsevier.com/locate/funbio
Particle and naked RNA mycoviruses in industrially cultivated mushroom Pleurotus ostreatus in China Liyou QIU*, Yanpeng LI, Yingmiao LIU, Yuqian GAO, Yuancheng QI, Jinwen SHEN College of Life Sciences, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, Henan, China
article info
abstract
Article history:
Many cultivated mushroom strains, such as Pleurotus ostreatus TD300, displayed symptoms
Received 3 September 2009
of degeneration. A spherical virus POSV and four dsRNA segments were extracted from
Received in revised form
mycelium of P. ostreatus TD300. POSV had a diameter of 23 nm and encapsidated a 2.5 kb
17 March 2010
dsRNA segment with coat proteins whose molecular weights were 39 kDa and 30 kDa.
Accepted 6 April 2010
Four dsRNA segments were 8.2 kb, 2.5 kb, 2.0 kb, and 1.1 kb in size, respectively. The
Available online 10 April 2010
1.1 kb dsRNA segment often escaped detection. The cDNA and the amino acid sequences
Corresponding Editor:
of the 8.2 kb dsRNA were homologous to those of RNA-dependent RNA polymerases
Daniel C. Eastwood
(RDRP) of ssRNA oyster mushroom spherical virus (OMSV), and contained conserved motifs A to D which were almost identical to those in RDRP of OMSV. The cDNA and amino acid
Keywords:
sequences of the 2.5 kb and 2.0 kb dsRNA segments were homologous to that of RDRP and
Naked virus
capsid protein of dsRNA virus P. ostreatus virus 1 (PoV1), respectively. In particular, the
Particle virus
amino acid sequence of 2.5 kb dsRNA segment had high identity with the conserved motifs
Pleurotus ostreatus
A to C in RDRP of PoV1, a Partiviridae virus. After eliminating the viruses in P. ostreatus
RNA mycovirus
TD300, the symptoms of degeneration completely disappeared. The results reveal that
Strain degeneration
P. ostreatus TD300 was at least infected by a particle virus POSV, and two naked viruses, one was a dsRNA virus with a 2.0 kb dsRNA segment, the other was an ssRNA virus whose replicating form of genome was an 8.2 kb dsRNA segment. Mycoviruses infection is a causative agent of mushroom strain degeneration. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Oyster mushroom (Pleurotus ostreatus) is the most popular mushroom in China and Korea. Its annual production is about 4 000 000 tons and accounts for about 40 % of the total mushroom fruiting body in China. It is a very healthy food ingredient since it contains enriched proteins, various biologically active polysaccharides, and other nutrients (Khatun et al. 2007; Gern et al. 2008; Jayakumar et al. 2006; Li et al. 2007). Furthermore it gets attention for its beneficial impacts of bioremediation on the environment and waste bioconversion (Rodrı´guez Pe´rez et al. 2008; Locci et al. 2008; Salmones et al. 2005).
However, P. ostreatus cultivation has been facing increasing challenges due to viral infections and strain degenerations. The symptoms of viral infection and strain degenerations were similar, including reduced mycelial growth, delayed fruiting body development, poor fruiting and severe fruiting body abnormalities (Yu et al. 2004; Go et al. 1992; van der Lende et al. 1995; Ro et al. 2006). P. ostreatus virus diseases were discovered to be related with several mycoviruses, such as double-stranded RNA (dsRNA) viruses (van der Lende et al. 1995), oyster mushroom isometric virus OMIV-I and OMIV-II (Yu et al. 2004; Ro et al. 2006), and oyster mushroom spherical virus (OMSV) (Yu et al. 2003). In
* Corresponding author. Tel.: þ86 371 6355 5175; fax: þ86 371 6355 5790. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.04.001
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contrast, infection with PoV1 did not show any distinct morphological or growth phenotypes (Ro et al. 2006). On the other hand, the cause of strain degeneration of P. ostreatus is still unknown. Besides P. ostreatus, virus diseases and strain degenerations were also found on many other mushrooms. La France isometric virus (LIV) (Hollings 1962), mushroom bacilliform virus (MBV) (Tavantzis et al. 1980), and mushroom virus X (MVX) (Gaze et al. 2000) were found to be the causative agents of virus diseases of Agaricus bisporus, as well as virus-like particles (VLPs) of Lentinus edodes (Ushiyama & Nakai 1975), and PeSV of Pleurotus eryngii (Ro et al. 2006). Strain degenerations of A. bisporus and Flammulina velutipes (Enokitake) have become a serious problem to industrial cultivation, and the chromosomal abnormalities responded to strain degenerations of A. bisporus (Horgen et al. 1996; Magae et al. 2005). P. ostreatus TD300 is a main cultivating strain in China, but in recent years it displayed serious degeneration. In this study, particle and dsRNA mycovirus in mycelium of P. ostreatus TD300 were isolated, their genome nucleic acid and capsid protein (CP) were characterized. One particle and two naked RNA mycoviruses were identified in P. ostreatus TD300.
Materials and methods Strain and media Strain of Pleurotus ostreatus TD300 was a commercial sample collected by Mushroom Cultivation Base of Henan Agricultural University. The media used were: PDA (potato dextrose agar) and PD (potato dextrose); WSP (filtrate from 100 g wheat bran boiled for 30 min, sugar 10 g, peptone 5 g, MgSO4$7H2O 0.5 g, KH2PO4 0.1 g, and distilled water added to 1 l); RCM (glucose 20 g, yeast extract 2.0 g, peptone 2.0 g, K2HPO4 1.0 g, KH2PO4 0.46 g, MgSO4$7H2O 0.5 g, mannitol 10.93 g, agar 15 g, and distilled water added to 1 l) (Kim et al. 1997). CCM (cottonseed hull 100 g, lime 1 g, gypsum 1 g, tap water 110 mL).
Purification and transmission electronic microscopy of mycovirus Purification of mycoviruses was carried out as described previously (Preisig et al. 1998). Mycelia were grown in WSP medium at 25 C on a rotary shaker at 150 rpm for 14 d. Mycelia were harvested and 100 g (wet weight) was powdered with liquid nitrogen and was mixed with two volumes of STE buffer (0.2 M NaCl, 0.1 M Tris, 0.002 M EDTA, pH 7.4), and then centrifuged at 80 000 g for 30 min at 4 C (Beckman, USA). The supernatant was mixed with polyethylene glycol 6000 (PEG 6000) to the final concentrations of 4 %, placed for 2 h at 4 C. After centrifugation at 100 000 g for 30 min at 4 C, the pellet was resuspended in 3 mL 1.5 STE buffer to obtain partially purified mycoviruses. And 3 mL those mycoviruses were layered onto 10 mL CsCl solution (1 g/2.5 mL) and ultracentrifuged at 400 000 g for 2 h at 4 C. The pellet was dissolved in 3 mL of 1.5 STE buffer, layered onto 10e40 % (w/v) CsCl density gradient solution, and centrifuged at 60 000 g for 3 h at 4 C. The collected virus band was
L. Qiu et al.
mixed with 5 volumes of ultrapure water, and then centrifuged at 60 000 g for 2 h at 4 C. The purified virus particles were examined with an H-8100 transmission electronic microscope (Hitachi, Japan) after been stained with 2 % phosphotungstic acid for 2 min.
Determinations of molecular weight of viral protein and genome size Molecular weight of the viral coat protein was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 5 mL loading buffer (100 mg SDS, 0.1 mL b-mercaptoethanol, 1 mL glycerol, 2 mg bromophenol blue, 0.5 mL 0.2 mol/L, pH 7.2 phosphate buffer solution, using double distilled water adjusted to 10 mL) was added to viral particles solution, and boiled for 5 min. The samples were analyzed using SDS-PAGE with 5 % concentration gel and 12 % separation gel. Coomassie brilliant blue G-250 was used for staining. Viral nucleic acids were extracted from the purified virus with equal volume phenol/chloroform (1:1) and then centrifuged at 15 000 g for 10 min. This step was repeated twice. Equal volume chloroform was added to the supernatant and centrifuged at 15 000 g for 10 min. The supernatant was mixed with 2 volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2), placed at 20 C for 1 h. After centrifuging at 15 000 g for 10 min, the pellet was washed with 70 % ethanol and centrifuged again. The viral nucleic acid (500 ng) was treated with RNase A (10 mg/mL) in 0.5 or 0.05 M NaCl or with DNase I (10 units/mL) in 10 mM MgCl2 at 37 C for 30 min. The nucleic acid samples were analyzed using 1 % agarose gel electrophoresis.
Extraction and electrophoresis of dsRNA dsRNA was isolated from mycelium using CF-11 enrichment technique modified of Morris & Dodds (1979). Pleurotus ostreatus mycelium (3 g per tube) was homogenized on ice in a plastic pestle with 3 mL GPS buffer (0.2 M glycine, 0.1 M Na2PO4, 0.6 M NaCl, pH 9.5), 3 mL saturated phenol, 1.5 mL chloroform/isoamyl alcohol (25/1), 0.3 mL 10 % SDS, and 0.03 mL b-mercaptoethanol. The homogenate was centrifuged at 12 000 g for 20 min at 4 C, and the aqueous layers of supernatant liquid were adjusted to 15 % ethanol. CF-11 celluose (Sigma) was added and mixed, and then loaded into a 10 mL syringe barrel (plugged with siliconised glass wool) and washed with 150 mL STE buffer (0.1 M NaCl, 0.05 M TriseHCl, 1 mM EDTA, pH 7.0) containing 15 % ethanol (STE/15). The dsRNA was eluted with 10 mL of STE, added with 2 volumes ethanol and 0.1 volumes 2 M sodium acetate to the eluant, precipitated for over night at 20 C, and centrifuged at 12 000 g for 20 min at 4 C. 1 % agarose gel electrophoresis was used to analyze dsRNA. The viral dsRNAs were confirmed by using DNase I and S1 Nuclease (Cheng et al. 2003). The viral nucleic acid (500 ng) was treated with 1 mL DNase I (60e80 units/mL) and in DNase I buffer (TaKaRa, Dalian, China) at 37 C for 30 min, or 10 U S1 Nuclease (TaKaRa) in S1 Nuclease buffer at 23 C for 15 min. The nucleic acid samples were analyzed by 1 % agarose gel electrophoresis.
Particle and naked RNA mycoviruses in industrially cultivated mushroom
Fig 1 e Electron microcopy of virus particle POSV from Pleurotus ostreatus TD300, it had a diameter of 23 nm.
cDNA synthesis, cloning and sequencing of dsRNAs Randomly primed cDNAs from every dsRNA element of Pleurotus ostreatus TD300 were synthesized using Invitrogen SUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat. 18248-013; Gibco/BRL). dsRNA was firstly denatured at 100 C for 8 min. cDNAs were cloned into plasmid pMD18-T (TaKaRa) and sequenced. Sequence information was analyzed with DNAstar software. Homologies of both nucleic acid and predicted amino acid sequences were determined using DNAMAN software. Encoded amino acid sequences were analyzed using ExPASy.
The preparation of virus free strain Virus free strains of Pleurotus ostreatus TD300 were prepared by protoplast regeneration. Mycelia of P. ostreatus TD300 was
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Fig 3 e Agarose gel analysis of dsRNA of particle virus POSV from mycelium of P. ostreatus TD300.
cultured as starting stain in 500 mL flasks containing 100 mL PD broth at 25 C for 5 d. The mycelia were harvested and washed twice with distilled water and 0.6 M mannitol. The mycelia were dried on absorbent paper and then suspended in filter-sterilized Lywallzyme solution (20 mg/mL in 0.6 M mannitol; Guangdong Institute of Microbiology, China) at 30 C on a rotary shaker (100 rpm). The lytic enzyme treated mycelia were sampled half hourly until a large quantity of protoplast formed. The remaining hyphal fragments were removed by filtration through a 0.5 cm column of absorbent cotton placed in the bottom of a syringe. Protoplasts were collected from the filtrate by centrifugation at 4500 rpm for 20 min. Pellets were washed twice with 0.6 M mannitol. After suspending the final pellet in 0.6 M mannitol, the concentration of the protoplast was determined by using a hemocytometer. For regeneration, 0.1 mL of protoplast suspension (about 105/mL) was plated on RCM medium. The plates were incubated at 25 C for 7 d and stellate aristae colonies grew out. Dikaryotic mycelium colonies were identified by the presence of clamp connections and picked to PDA plates. The plates were incubated at 25 C for 7 d. The virus free strains were screened by using CF-11 enrichment technique.
Mycelia growth rate Agar plugs of 1 cm diameter from the inoculation plates of the virus free strain and starting strain of Pleurotus ostreatus TD300 were transferred onto five freshly prepared PDA plates. The plates were incubated at 25 C and colony diameters were measured after 3 d.
Biomass and laccase activity
Fig 2 e SDS-PAGE analysis of coat proteins (CP) of particle virus POSV from mycelium of P. ostreatus TD300.
Agar plugs of 1 cm diameter from the inoculation plates of the virus free strain and starting strain of Pleurotus ostreatus TD300 were transferred into five 500 mL flasks containing 100 mL PA broth, and then incubated at 25 C on a rotary shaker
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parts and were placed separately in the two end and middle sections of plastic bags containing 3000 g CCM. The virus free and starting strain of P. ostreatus TD300 was cultivated in 30 bags at 25 C. Soon after the hyphae had fully spread, the bags were kept in a cold room at 18 C, 95 % RH and under incandescent lighting of 1000 Lux to induce primordia formation. Basidiomata were harvested and weighed at 7/8th ripe. The yield of mushroom production was recorded by the weight (g) of freshly harvested basidiomata per bag. Biological efficiency was defined as the percentage of the fresh weight of harvested mushrooms over the dry weight of inoculation substrates.
Statistical analysis Student’s t test was used to analyze the data for differences in growth rate, biomass, enzyme activity, or biological efficiency between the virus free strain and starting strain of Pleurotus ostreatus TD300.
Results Characterization of mycovirus particles
Fig 4 e Agarose gel analysis of extraction dsRNA (lane 1) from mycelium of P. ostreatus TD300.
Viral particles purified from Pleurotus ostreatus TD300 mycelium were observed and a spherical virus with 23-nm diameter was found and named POSV (Fig 1). Proteins and nucleic acids of the viral particle were analyzed by SDS-PAGE and agarose gel electrophoresis. The molecular weights of the coat proteins were 39 kDa and 30 kDa, respectively (Fig 2). An encapsidated 2.5 kb segment was confirmed as a dsRNA fraction by using DNase I and S1 Nuclease degradation (Fig 3).
(200 rpm) for 8 d. The cultured fluids were centrifuged at 3000 rpm for 10 min. Pellets were dried at 60 C until their weight kept constant. The supernatant liquids underwent laccase assay. Lactase activity was determined by oxidation of 2, 20 -azmobis-(3-ethyl benzthiazoline-6-sulphonate) (ABTS). The reaction mixture contained 0.5 mM ABTS, 0.1 M sodium acetate buffer (pH 4.5) and the supernatant liquid. Oxidation of ABTS was followed by absorbance increase by 420 nm (3420 ¼ 3.6 104 M1 cm1). Enzyme activity was expressed in units (U ¼ mmol/min) (Bourbonnais & Paice 1990).
dsRNA profiles of mycelium Four dsRNA bands were extracted from Pleurotus ostreatus TD300 mycelium using CF-11 enrichment technique, and confirmed by DNase I and S1 Nuclease degradation. Their sizes were 8.2 kb (doublet), 2.5 kb, 2.0 kb, and 1.1 kb, respectively (Fig 4). The 8.2 kb, 2.0 kb, and 1.1 kb dsRNAs were not detected in RNA sample obtained from the virus particles, suggesting that these three dsRNAs are naked viral genomes. The 1.1 kb dsRNA band may not always be present.
Basidiomata formation Fungal inocula were prepared first. Agar plugs of 1 cm diameter from the inoculation plates of the virus free strain and starting strain of Pleurotus ostreatus TD300 were transferred into plastic bags containing 500 g CCM, and then incubated at 25 C until the hyphae spread all over bags. The inocula were broken into suitable sizes. Inocula (300 g) were divided into three equal
dsRNAs sequence homology analysis Two cDNA sets of 264 bp and 3479 bp were obtained from the 8.2 kb dsRNA segment. Their nucleic acid and amino acid
Table 1 e Summary of sequence analysis of dsRNA elements from Pleurotus ostreatus TD300. dsRNA (kb)
bp sequenced (GenBank accession no.)
8.2
264 (GQ505290) 3479 (GQ505290) 1558 (GQ505291) 1046 (GQ505292)
2.5 2.0
Homologies
Identities of cDNA sequence (%)
Identities of the amino acid sequence of cDNA (%)
RDRP in OMSV RDRP in OMSV
93 95
89 91
RDRP in dsRNA-1 of PoV1 CP in dsRNA-2 of PoV1
83 73
90 74
OMSV: oyster mushroom spherical virus; PoV1: P. ostreatus virus 1.
Conserved motifs
RDRP conserved motifs A to D and helicase motif GXGKS(T) RDRP conserved motifs A to C
Particle and naked RNA mycoviruses in industrially cultivated mushroom
A
B
511
C
D
Fig 5 e Alignments of the four conserved motifs A to D within RDRP sequences in mushroom virus genomes. The sequence accession number in GeneBank is AY182001 (oyster mushroom spherical virus, OMSV), AY533038 (P. ostreatus virus 1, PoV1), AY308801 (oyster mushroom isometric virus II, OMIV-II), X94361 (Agaricus bisporus virus 1, ABV1), NC_001633 (mushroom bacilliform virus, MBV), AB429554 (Lentinula edodes mycovirus HKB), GQ505290 (P. ostreatus dsRNA virus, 8.2 kb segment), and GQ505291 (P. ostreatus dsRNA virus, 2.5 kb segment), respectively. Amino acids that are conserved are highlighted. The length separating the motifs is indicated. Italic sequence name means that it was from ssRNA.
sequences were around 90 % identical to those of RDRP in oyster mushroom spherical virus (OMSV), an ssRNA virus (Yu et al. 2003) (Table 1). In particular, the amino acid sequence of the 3479 bp set had four conserved motifs A to D of RNAdependent polymerases in viruses (Poch et al. 1989), and the region containing the motifs was nearly identical to that of OMSV (Fig 5). Furthermore, the conserved helicase motif GXGKS(T) (Kadare & Haenni 1997) was also present in the 3479 bp set. A 1558 bp cDNA set was obtained from the 2.5 kb dsRNA segment. It was homologous to the RDRP gene in PoV1 dsRNA-1 (Lim et al. 2005) (Table 1), and contained three conserved motifs A to C, which were very similar to those in RDRP of PoV1 dsRNA-1 (Fig 5). From the 2.0 kb dsRNA segment we sequenced a 1046 bp cDNA set. Its nucleic and amino acid sequences were homologous to those of the capsidic protein of PoV1 dsRNA-2 (Lim et al. 2005) (Table 1).
Characterization and comparison between the virus free strain and starting strain of Pleurotus ostreatus TD300 A lot of protoplast regenerated strains were gained. Some, such as PR09, did not show the 8.2 kb dsRNA segment.
Fig 6 e dsRNA detection of virus eliminated or free strain of P. ostreatus TD300 by protoplast regeneration. M. DNA molecular marker; 1. Virus eliminated strain PR09; 2. Virus eliminated strain PR22; 3. Virus free strain PR15.
Some, such as PR22, had neither the 8.2 kb nor 2.1 kb segments. And some, such as PR15, had none of the 8.2 kb, 2.5 kb and 2.1 kb segments (Fig 6). Similar result was described elsewhere (van der Lende et al. 1995). Compared to the starting strain, the hyphae of the virus free strain PR15 grown on PDA plate were whiter and denser, and the hyphae growth rate was 18.18 % higher (Table 2); Similarly, the hyphae biomass and laccase activity of PR15 grown on PD broth for 8 d were 145.70 % and 134.36 % higher than those of the starting strain. In both cultivated bags, the period from incubation to primordia appearance of PR15 was 5 d shorter than that of starting strain, the basidiomata looked more even in appearance and size, and had thicker pileus, and the biological efficiency was 20.70 % higher (Table 2).
Discussion A spherical virus POSV, which was 23 nm in diameter and encapsidated a 2.5 kb dsRNA genome, was found in mycelium of Pleurotus ostreatus TD300. Similarly, three other dsRNA spherical viruses were extracted in the mycelium of P. ostreatus var. florida and P. ostreatus strain ASI2596 (van der Lende et al. 1995; Lim et al. 2005). And three dsRNA isometric viruses were found in P. ostreatus (Yu et al. 2004) and P. ostreatus cultivar “Suhan” (Ro et al. 2006). However, only one ssRNA spherical virus OMSV was found in P. ostreatus (Yu et al. 2003). So we proposed that the particle viruses infected in P. ostreatus were mostly dsRNA viruses. A 2.5 kb dsRNA segment encapsidated in POSV and a 2.0 kb dsRNA segment isolated from the mycelium of P. ostreatus TD300 had high identities to the genome of Partiviridae virus PoV1, respectively. However, POSV only encapsidated 2.5 kb dsRNA and was not a Partiviridae virus. It needed further research to determine whether the 2.5 kb and 2.0 kb dsRNA segments belong to a Partiviridae. Naked dsRNA segments around 8.0 kb had been found in P. ostreatus strain Shin-Nong (Kim et al. 2008), P. ostreatus var. florida (van der Lende et al. 1995), but their sequences were not determined. We also extracted an 8.2 kb dsRNA segment from mycelium of P. ostreatus TD300, and obtained two cDNA sequences, 264 bp and 3479 bp, from the dsRNA segment. Their nucleic and amino acid sequences were highly similar to that of RDRP of ssRNA particular virus OMSV
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Table 2 e Biological character comparison between the virus free strain PR15 and starting strain of P. ostreatus TD300a. Strain PR15 Starting strain
Growth rateb (mm/d)
Biomassc (g Dry Wt/l)
Laccase activityc (U/mL)
Days taken from incubation to primordia appearanced
Biological efficiencyd (%)
6.5** 5.5
45.7** 18.6
193.75** 82.67
38 43
94.5** 78.3
a Paired numbers within a column with ** are significantly different at the 0.01 level from corresponding values of starting strain. b Hyphae grown on PDA plates. The values of growth rate are arithmetic means of the five replicate measurements. c Hyphae grown on PD broth. The values of biomass or laccase activity are arithmetic means of the five replicate measurements. d Hyphae grown on cottonseed hull cultivation medium (CCM). The values of days taken from incubation to primordia appearance or biological efficiency are arithmetic means of the 30 replicate measurements.
(Table 1 and Fig 5). The length separating the conserved motifs A to B was smaller than 45 amino acids in most of the positive strand ssRNA virus, but greater than 50 amino acids in dsRNA viruses (Fig 5) (Poch et al. 1989). Thus, the 8.2 kb dsRNA segment may be the replicative form of an ssRNA virus genome, and most closely related to Botrytis virus F (BVF) whom OMSV is very similar to (Yu et al. 2003). Only one gene is common to all RNA viruses. It encodes an RDRP or an RNA-dependent DNA polymerase (Bruenn 1993). Although there is low sequence similarity over the entire length of RDRPs among dsRNA viruses (Wiener & Joklik 1989), four common motifs A to D are conserved with the same linear arrangement in RDRPs encoded by RNA viruses (Poch et al. 1989). The four conserved motifs present in the 8.2 kb, 2.5 kb, and other mushroom virtual genomes were compared in Fig 5. It demonstrated that the conserved motifs were homologous between OMSV and the 8.2 kb dsRNA, and between PoV1 and the 2.5 kb dsRNA, in spite of being isolated from different cultures in South Korea and China. The result suggested that the viruses of P. ostreatus had same origin. Furthermore, the conserved motifs in RDRP of dsRNA viruses displayed higher degree of identity within the dsRNA viruses than between the dsRNA and ssRNA viruses of P. ostreatus. Similarly the conserved motifs in dsRNA viruses ABV1 had a low identity with that of ssRNA virus MBV of Agaricus bisporus, so were the mycoviruses infecting P. ostreatus, A. bisporus, and Lentinula edodes (Fig 5). Chromosomal abnormalities were discovered to associate with strain degeneration in the cultivated mushroom, A. bisporus (Horgen et al. 1996). Although there was no clear evidence for a relationship between the presence of viruses and mushroom strain degeneration, we found that after eliminating the viruses of P. ostreatus TD300, the symptoms of degeneration disappeared. The hypha of the virus free strain from protoplast regenerant grew denser and faster than the starting strain. Also, the biomass, laccase enzyme activity, and mushroom biological efficiency of the virus free strain were all higher (Table 2). Magae et al. (1985) selected six strains from the dikaryotic protoplast regenerants of P. ostreatus at random, and all strains were able to form normal fruiting bodies, and the yield was 18 % greater than the parent. They suggested that a protoplast regeneration process may yield useful variations. We propose that virus was probably eliminated in the protoplast regeneration process. Our results revealed that mycoviruses infection is a probable cause of mushroom strain degeneration.
Acknowledgements This research was supported by the Science and Technology Development Program of Henan Provincial Department of Science and Technology, and the Doctorial Innovation Fund of Henan Agricultural University.
Supplementary material Supplementary material for this article is available, in the online version, at doi:10.1016/j.funbio.2010.04.001.
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Particle and naked RNA mycoviruses in industrially cultivated mushroom
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