Preliminary Studies on the Effects of Oyster Mushroom Spherical Virus China Strain on the Mycelial Growth and Fruiting Body Yield of the Edible Mushroom Pleurotus ostreatus

Abstract

Oyster mushroom spherical virus (OMSV) is a positive-sense single-stranded RNA mycovirus which is associated with a devastating oyster mushroom die-back disease. However, little is known about its diversity, and the effects of OMSV infection on its fungal host are not well understood. In this study, we determined the nearly complete nucleotide sequence of OMSV isolated from cultivated oyster mushrooms in China. Sequence analysis suggested that the virus represents a new strain of OMSV (referred to here as OMSV-Ch). A GenBank BLAST search of the genomic sequences demonstrated that the OMSV-Ch had the highest identity (74.9%) with the OMSV from Korea (OMSV-Kr). At the amino acid-sequence level, these two strains shared 84.1% identity in putative replication protein (RP) and 94.1% identity in coat protein (CP). Phylogenetic analysis based on RP showed that OMSV-Ch clustered with OMSV-Kr, closely related to Tymoviridae. Phylogenetic analysis based on both the RP and CP showed that OMSV had a distant clade relationship with tymoviruses, marafiviruses, and maculaviruses. We obtained the OMSV-Ch-free Pleurotus ostreatus strain via single hyphal tip cultures combined with high-temperature treatment. Preliminary studies indicate that OMSV-Ch can significantly inhibit mycelial growth, cause malformations of the fruiting bodies, and reduce the yield of P. ostreatus. Co-cultivation resulted in horizontal transmission of the OMSV-Ch to a virus-cured strain. The findings of our study contribute to the prevention and control of mycoviral diseases in the future.

Keywords: Pleurotus ostreatus; genome sequence; oyster mushroom spherical virus; phylogenetic analysis; virus curing.

Figure 1

RT-PCR detection of OMSV in malformed fruiting bodies of P. ostreatus. (a) The collected fruiting body samples from P. ostreatus cultivar 8129 display symptoms of funnel-shaped or morning glory–shaped caps. (b) RT-PCR detection for the presence or absence of OMSV, OMIV, POSV, or PoV1 from fruiting body samples described in (a). Lanes 1–4, four fruiting body samples of P. ostreatus that were positive for OMSV by RT-PCR. N, negative control, the healthy P. ostreatus sample; M, GL DNA Marker2000. (c) Schematic representation of the OMSV-Ch genome structure. Boxes represent the ORFs. Numbers represent nucleotide positions.

Figure 2

Phylogenetic tree based on the amino acid sequences of the replication protein of OMSV-Ch and 17 representatives of the order Tymovirales. The OMSV-Ch sequence from the present study is marked with a red dot. Potato leafroll virus (PLRV) was used as an outgroup.

Figure 3

Phylogenetic analysis of the OMSV-Ch replication protein (a) and coat protein (b) with corresponding proteins of tymoviruses, marafiviruses, and maculaviruses. The OMSV-Ch sequence from the present study is marked with a red dot. Potato virus X (PVX) was used as an outgroup.

Figure 4

Curing of OMSV-Ch of P. ostreatus on PDA plates. (a) Colony morphology of the OMSV-Ch-infected strain compared with the OMSV-Ch-cured strain after 7 days of incubation at 25 °C. (b) Semi-quantitative RT-PCR detection of OMSV-Ch accumulation from each generation. The primers OMSV-4959F/OMSV-5605R (upper panel) and OMSV-CPF/OMSV-CPR (middle panel) were used for RT-PCR detection of OMSV-Ch. Actin served as an internal control (lower panel). N, negative control, the healthy P. ostreatus sample; P, positive control, the OMSV-Ch-infected P. ostreatus sample; M, GL DNA Marker2000. (c) Effects of OMSV-Ch curing on mycelial growth. Values are significantly different at p ≤ 0.05, by Student’s t test. (d) Microscopic observation of the colony margin in the OMSV-Ch-infected strain compared with the OMSV-Ch-cured strain. The fungal hyphae were observed at 400× magnification (lower panel). Bars = 20 μm.

Figure 5

The cultivation test to examine the effects of the OMSV-Ch infection on P. ostreatus fruiting bodies. (a) The morphological characteristics of fruiting bodies of the OMSV-Ch-cured (upper panel) and OMSV-Ch-infected (lower panel) strains. (b) Semi-quantitative RT-PCR analysis for OMSV-Ch in fruiting bodies of OMSV-Ch-infected and OMSV-Ch-cured strains. Different bags of fruiting bodies were collected from the OMSV-Ch-infected (lanes 1–4) and OMSV-Ch-cured strains (lanes 5–8). The primers OMSV-4959F/OMSV-5605R (upper panel) and OMSV-CPF/ OMSV-CPR (middle panel) were used for RT-PCR detection of OMSV-Ch. Actin served as an internal control (lower panel). N, negative control, the healthy P. ostreatus sample; P, positive control, the OMSV-Ch-infected P. ostreatus sample; M, GL DNA Marker2000.

Figure 6

Pairwise co-cultivation of OMSV-Ch-infected (+) and OMSV-Ch-cured (−) strains of P. ostreatus. (a) After incubation for 5 days at 25 °C, one inoculum from the donor side (I) and two inocula from the recipient (II and III) were sub-cultivated for 5 days. (b) RT-PCR detection for the presence of OMSV. Negative controls of pairing tests were OMSV-Ch-cured strains. M, GL DNA Marker2000. (c) Colony morphology of the OMSV-Ch-cured strain compared to the newly infected OMSV-Ch strain after 5 days of incubation at 25 °C. (d) Growth rate of OMSV-Ch-cured and newly infected OMSV-Ch strains of P. ostreatus. ** p < 0.01 highlight significant growth differences by independent sample t-test.