Multiplex Detection of Pleurotus ostreatus Mycoviruses

Mycoviruses are viruses that specifically infect and replicate in fungi. Several mycoviruses have been previously reported in Pleurotus ostreatus, including the oyster mushroom spherical virus (OMSV), oyster mushroom isometric virus (OMIV), Pleurotus ostreatus spherical virus (POSV), and Pleurotus ostreatus virus 1 (PoV1). This study was designed to develop a multiplex RT-PCR for simultaneous detection and differentiation of the four P. ostreatus mycoviruses. Four pairs of primers were designed from conserved regions based on the reported sequences and the multiplex RT-PCR products were 672 bp for OMSV, 540 bp for OMIV, 310 bp for POSV, and 200 bp for PoV1. The optimal annealing temperature of the multiplex RT-PCR was 62 °C and the detection limits of the plasmids were 100 fg for OMSV and OMIV and 1 pg for POSV and PoV1. This technique was successfully applied for the detection of OMSV, OMIV, and POSV from different P. ostreatus strains and the plasmid containing the PoV1 sequence. This methodology can serve as a powerful diagnostic tool for the survey of the incidence and epidemiology of the four P. ostreatus mycoviruses, further contributing to the prevention and treatment of mycoviral diseases in P. ostreatus.

Mycoviruses have no known natural vectors and are usually horizontally transmitted via hyphal anastomosis and vertically transmitted via sporulation in nature [33]. Studies have shown that Lentinula edodes spherical virus (LeSV) was vertically transmitted by basidiospores in L. edodes [34]. A recent transmission study on A. bisporus revealed that the mushroom virus X (MVX) can be horizontally transferred via mycelia from an infected strain to five other uninfected strains [35]. Our previous studies have shown that the OMSV can be horizontally transmitted from the OMSV-infected strain to a virus-cured strain [31]. Due to these transmission patterns, it can be a challenge to prevent mycovirus diseases, especially in edible mushrooms. As reported, a few attempts to eliminate mycovirus infections have been described for several edible mushroom species. For example, the methods of growth on a limited nutrient medium containing cAMP and rifamycin, single hyphal tip cultures combined with high-temperature treatment, protoplast regeneration, or mycelial fragmentation were used for curing the OMSV, OMIV, POSV, and PoV-ASI2792 from the virus infected P. ostreatus strains [12,28,31,36]. Moreover, ribavirin treatment and mycelial fragmentation were also used for curing Lentinula edodes mycovirus HKB (LeV-HKB) and L. edodes partitivirus 1 (LePV1) from Lentinula edodes [37]. At present, the most effective control strategy is to detect and eliminate the virus from the virus-infected strain.
PCR is a commonly used technique for the detection and diagnosis of viruses due to its high sensitivity and specificity. Previously, the routine reverse transcription PCR (RT-PCR) technique has been used for the detection of viruses including OMSV, OMIV, PoV1, and POSV. Moreover, serological methods have also been used for the detection of OMSV and OMIV [15,38]. A surface plasmon resonance biosensor chip and a triple antibody sandwich-ELISA system have been successfully developed for the detection of OMSV and OMIV, respectively [15,38]. However, these methods could not distinguish the four P. ostreatus mycoviruses in one reaction. Multiplex RT-PCR has become a powerful tool for the simultaneous detection of several different viruses at a time [39,40]. In the present study, we developed a multiplex RT-PCR assay for the simultaneous detection of the four known P. ostreatus viruses. This is a simple and effective method to identify single or mixed viral infections in various P. ostreatus cultivars. This study represents the first time that the multiplex RT-PCR technique has been used to detect P. ostreatus mycoviruses.

RNA Extraction
The P. ostreatus strains presented in Table 2 were cultured in solid potato dextrose agar medium. After culturing at 25 • C for one week, 0.1 g mycelia of each strain were collected for RNA extraction by using the RNA Easy Fast Plant Tissue Kit (Tiangen, Beijing, China). The RNA was eluted in 50 µL RNase-free water and stored at −80 • C for future use.

Cloning and Sequencing
Each of the mycoviruses infecting P. ostreatus was validated by RT-PCR and sequencing. The amplified target fragments of each mycovirus with specific primers were purified and ligated into pMD19-T vector and then transformed into Escherichia coli DH5α competent cells. At least three positive recombinant clones were sequenced by Sangon Biotech (Shanghai, China) Co., Ltd. Heiping Huimei LD-0328 Yantai ----Note: "+" means infected, and "-" means non-infected.

Establishment of the Multiplex RT-PCR Assay
The primers for OMSV, OMIV, POSV, and PoV1 produced amplicons of 672, 540, 310, and 200 bp, respectively. In order to investigate the effect of the annealing temperature on uniplex and multiplex RT-PCR, a gradient PCR was then performed over a range of 60.0-70.0 • C. Eight annealing temperatures (60.0, 61.0, 62.3, 64.0, 66.3, 68.1, 69.3, and 70.0 • C) were tested for amplification optimization. Gel electrophoresis using ethidium bromidestained agarose gels were used to separate and visualize the amplified DNA products corresponding to the targeted mycoviruses ( Figure 1). When the annealing temperature was 62.3 • C, the amplified bands were bright and clear. However, when the annealing temperatures increased to 64.0 • C, the multiple bands amplified by the multiplex RT-PCR became faint (Figure 1). According to the specificity and efficiency of amplification, the optimal annealing temperature for multiplex RT-PCR was determined to be 62 • C. The PCR program was optimized as follows: initial denaturation at 95 • C for 5 min; 30 cycles of amplification (95 • C for 30 s, annealing at 62 • C for 30 s; elongation at 72 • C for 60 s); and a final extension at 72 • C for 10 min. the annealing temperatures increased to 64.0 °C, the multiple bands amplified by the multiplex RT-PCR became faint (Figure 1). According to the specificity and efficiency of amplification, the optimal annealing temperature for multiplex RT-PCR was determined to be 62 °C. The PCR program was optimized as follows: initial denaturation at 95 °C for 5 min; 30 cycles of amplification (95 °C for 30 s, annealing at 62 °C for 30 s; elongation at 72 °C for 60 s); and a final extension at 72 °C for 10 min.

Sensitivity of Multiplex RT-PCR
To evaluate the sensitivity of uniplex and multiplex PCR, 10-fold serial dilutions of the purified plasmids pTOMIV, pTPOSV, pTPoV1, and pTOMSV were used as DNA templates in the uniplex and multiplex PCR amplification. The results demonstrated that the detection limits of the DNA quantity for uniplex PCR were 100 fg for OMSV, POSV, PoV1, and 10 fg for OMIV ( Figure 2). Using an equal mixture of the four plasmids as templates for multiplex PCR, the detection limits were 100 fg for OMSV and OMIV and 1 pg for POSV and PoV1 (Figure 2).

Sensitivity of Multiplex RT-PCR
To evaluate the sensitivity of uniplex and multiplex PCR, 10-fold serial dilutions of the purified plasmids pTOMIV, pTPOSV, pTPoV1, and pTOMSV were used as DNA templates in the uniplex and multiplex PCR amplification. The results demonstrated that the detection limits of the DNA quantity for uniplex PCR were 100 fg for OMSV, POSV, PoV1, and 10 fg for OMIV ( Figure 2). Using an equal mixture of the four plasmids as templates for multiplex PCR, the detection limits were 100 fg for OMSV and OMIV and 1 pg for POSV and PoV1 (Figure 2).

Specificity of Multiplex RT-PCR
To validate the specificity of the multiplex RT-PCR assay, a PCR was conducted by using the mixture of four pairs of primers in one tube, including single or mixed plasmids as templates. It was determined that in the presence of single plasmid, mixtures of primer pairs only amplified the virus corresponding single infection (Figure 3, lines 2-5). Mixed plasmids using dual, triple or quadruple combinations of different templates were also amplified simultaneously using multiplex RT-PCR (Figure 3, lines 6-16). Of all the cases we tested, amplicons were produced by their corresponding mycovirus templates.

Detection of the Four Mycoviruses in Different P. ostreatus Strains
To evaluate specificity for the P. ostreatus mycoviruses detection, the established multiplex RT-PCR assay was used to determine the incidence of mycoviruses. The healthy P. ostreatus sample (virus-cured strain 8129 [31]) acted as a negative control with no observed virus amplification, as expected. The incidence of both single or mixed infections in different P. ostreatus strains produced clear and specific target bands on an agarose gel ( Figure 4). Among the 36 P. ostreatus strains presented in Table 2, twelve strains were singly infected with OMSV, three strains were singly infected with POSV, and six strains were dually infected with OMSV and OMIV ( Figure 4). Although, neither triple nor quadruple viral infection was detected, mixed infections of OMSV, OMIV, and POSV using combinations of the P. ostreatus strains PG-ZP20 and TD300 were successfully amplified (Figure 4, line M1). None of the collected 36 strains were infected with PoV1. The above results were confirmed by the four uniplex RT-PCR assays (data not shown). These results demonstrated that the multiplex RT-PCR technique specifically detected the four different mycoviruses in P. ostreatus.

Specificity of Multiplex RT-PCR
To validate the specificity of the multiplex RT-PCR assay, a PCR was conducted by using the mixture of four pairs of primers in one tube, including single or mixed plasmids as templates. It was determined that in the presence of single plasmid, mixtures of primer pairs only amplified the virus corresponding single infection (Figure 3, lines 2-5). Mixed plasmids using dual, triple or quadruple combinations of different templates were also amplified simultaneously using multiplex RT-PCR (Figure 3, lines 6-16). Of all the cases we tested, amplicons were produced by their corresponding mycovirus templates.

Specificity of Multiplex RT-PCR
To validate the specificity of the multiplex RT-PCR assay, a PCR was conducted by using the mixture of four pairs of primers in one tube, including single or mixed plasmids as templates. It was determined that in the presence of single plasmid, mixtures of primer pairs only amplified the virus corresponding single infection (Figure 3, lines 2-5). Mixed plasmids using dual, triple or quadruple combinations of different templates were also amplified simultaneously using multiplex RT-PCR (Figure 3, lines [6][7][8][9][10][11][12][13][14][15][16]. Of all the cases we tested, amplicons were produced by their corresponding mycovirus templates.  triple nor quadruple viral infection was detected, mixed infections of OMSV, OMIV, and POSV using combinations of the P. ostreatus strains PG-ZP20 and TD300 were successfully amplified (Figure 4, line M1). None of the collected 36 strains were infected with PoV1. The above results were confirmed by the four uniplex RT-PCR assays (data not shown). These results demonstrated that the multiplex RT-PCR technique specifically detected the four different mycoviruses in P. ostreatus.  Table 2. Lane M1 represents the mixed strains of PG-ZP20 and TD300. Lane M2, combined strains of PG-ZP20 and TD300 with the plasmid pTPoV1. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000.

Discussion
Unlike other pathogens such as fungi and bacteria, chemical methods are difficult to employ to control mycoviruses. Therefore, the establishment of a rapid and sensitive detection method is necessary to control the mycovirus disease during the latent stage, or before the ejection of virulent spores. The multiplex RT-PCR assay has the advantage of easy operation, sensitivity, speed, and cost-effectiveness. Thus, this technology has been widely used for the simultaneous identification of several viruses, viral strains, isolates, or genotypes in plant viruses. Previously, this method has been successfully used for simultaneous detection of the two L. edodes mycoviruses in China [41]. In the present study, a multiplex RT-PCR assay for simultaneous detection of OMSV, OMIV, POSV, and  Table 2. Lane M1 represents the mixed strains of PG-ZP20 and TD300. Lane M2, combined strains of PG-ZP20 and TD300 with the plasmid pTPoV1. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000.

Discussion
Unlike other pathogens such as fungi and bacteria, chemical methods are difficult to employ to control mycoviruses. Therefore, the establishment of a rapid and sensitive detection method is necessary to control the mycovirus disease during the latent stage, or before the ejection of virulent spores. The multiplex RT-PCR assay has the advantage of easy operation, sensitivity, speed, and cost-effectiveness. Thus, this technology has been widely used for the simultaneous identification of several viruses, viral strains, isolates, or genotypes in plant viruses. Previously, this method has been successfully used for simultaneous detection of the two L. edodes mycoviruses in China [41]. In the present study, a multiplex RT-PCR assay for simultaneous detection of OMSV, OMIV, POSV, and PoV1 in P. ostreatus from one reaction was developed based on the conserved regions of RdRp (for OMIV, POSV, and PoV1) and the CP (for OMSV) genes. The method presented here demonstrated high sensitivity and specificity for the detection of the four mycoviruses. This methodology can serve as a valuable tool for the diagnosis of the four mycoviruses at the early stages of P. ostreatus production. This method can therefore contribute to the prevention and treatment of mycovirus diseases. To the best of our knowledge, this study is the first to report multiplex RT-PCR detection of P. ostreatus mycoviruses. Previous reports have demonstrated that some mycoviruses such as Cryphonectria hypovirus 1 (CHV1), Cryphonectria naterciae fusagravirus 1 (CnFGV1), and Cryphonectria nitschkei chrysovirus 1 (CnCV1) can cross the barrier of incompatibility and infect different related species of fungi [42][43][44]. To date, it is not clear whether the four known P. ostreatus mycoviruses (OMSV, OMIV, POSV, and PoV1) could infect other closely-related host species. Thus, the multiplex RT-PCR assay can be further used for a comprehensive survey on new host species of the four mycoviruses.
In nature, multiple viral infections are common in plants and this phenomenon also can occur in fungi. An increasing number of fungal species are infected with different mycoviruses. In the plant pathogen Sclerotium rolfsii, 21 virus-like sequences have been identified in a hypovirulent strain [45]. In the L. edodes mushroom, distinct viral sequences have been previously identified [46]. Co-infection of Lentinula edodes mycovirus HKB (LeV-HKB) with Lentinula edodes partitivirus 1 (LePV1) is prevalent in Chinese L. edodes germplasm resources [26,47]. In southern Korea, dual infections of OMSV and OMIV were detected in the P. ostreatus cultivars Suhan and Chunchu [15,28]. In the current study, the incidences of the four mycoviruses in different P. ostreatus strains were investigated using the multiplex RT-PCR assay. Single infection with OMSV or POSV and dual infection with OMSV and OMIV were detected. Previous research has shown that the incidence rate of OMSV in Beijing suburbs was 32.8% [48]. In the detection of the 36 collected P. ostreatus strains, the incidence rate of OMSV was higher (47.2%, 17/36) than OMIV (6/36, 16.7%), POSV (3/36, 8.3%), or PoV1. In order to obtain an accurate prevalence pattern of the four mycoviruses, a larger number of Chinese P. ostreatus germplasm resources should be further collected for multiplex RT-PCR detection. In this study, OMIV was found to occur in the form of co-infection with OMSV. Therefore, the possibility that two mycoviruses have synergistic interactions cannot be ruled out. However, whether or not the two mycoviruses have direct interactions requires further study.
Our newly developed multiplex RT-PCR assay offers a simple, quick, and sensitive technique for the diagnosis of the four different mycoviruses infecting P. ostreatus. This method can be used as a powerful diagnostic tool for a large-scale survey of the occurrence, distribution, and epidemiology of known P. ostreatus mycoviruses. This methodology can contribute to the future prevention and control of mycoviral diseases.

Conclusions
In this study, we developed a multiplex RT-PCR assay for simultaneous detection and differentiation of the four P. ostreatus mycoviruses. This methodology can serve as a powerful diagnostic tool for the survey of the incidence and epidemiology of the four P. ostreatus mycoviruses, further contributing to the prevention and treatment of mycoviral diseases in P. ostreatus.

Data Availability Statement:
The data presented in this study are available within the article.