Virus Research 197 (2015) 8–12
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Changes in the mycovirus (LeV) titer and viral effect on the vegetative growth of the edible mushroom Lentinula edodes Jung-Mi Kim a , Ha-Yeon Song a , Hyo-Jin Choi a , Suk-Hyun Yun b , Kum-Kang So b , Han-Kyu Ko c , Dae-Hyuk Kim b,∗ a
Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, South Korea Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, South Korea c Forest Mushroom Research Center, Yeoju, Gyeonggi 469-803, South Korea b
a r t i c l e
i n f o
Article history: Received 15 September 2014 Received in revised form 9 November 2014 Accepted 12 November 2014 Available online 20 November 2014 Keywords: Lentinula edodes dsRNA Isogenic strain Mycovirus
a b s t r a c t This study attempted to cure the edible mushroom Lentinula edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygendepleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. These results indicate that LeV infection has a deleterious effect on mycelial growth. © 2014 Elsevier B.V. All rights reserved.
The presence of viruses in fungi is now accepted as not “exceptional” but “general” (Dawe & Nuss, 2001; Ghabrial, 1998; Pearson et al., 2009) since the first definitive description of mycovirus more than 50 years ago (Hollings, 1962). Viral infection occurs in all major taxa belonging to the kingdom Fungi. However, it is difficult to determine the biological consequences of mycoviral infection for following reasons. First, although there is compelling evidence that an increasing number of viruses induce symptomatic infections, mycoviral infections, in general, have been considered not to cause measurable phenotypic changes in the fungal hosts. If anything, such changes are cryptic or latent. Second, in some cases, although mycoviral infection causes considerable morphological and physiological changes, including debilitation- and virulencerelated phenotypes (Castro et al., 2003; Dawe & Nuss, 2001), the varied genetic backgrounds of fungal strains may respond
∗ Correspondence to: Department of Molecular Biology, Chonbuk National University, Dukjindong 664-14, Jeonju, Chonbuk 561-756, South Korea. Tel.: +82 63 270 3440; fax: +82 63 270 4312. E-mail address: [email protected] (D.-H. Kim). http://dx.doi.org/10.1016/j.virusres.2014.11.016 0168-1702/© 2014 Elsevier B.V. All rights reserved.
differently to the same mycovirus, which makes it desirable to have identical genetic backgrounds (with the exception of the presence or absence of the virus) for the study of fungus–mycovirus interactions. Establishment of such virus-free and virus-infected isogenic lines to explore direct mycovirus–fungal host interactions has been hampered due to several reasons. Firstly, although several attempts have been described, successfully curing an infected fungal host of a mycovirus infection requires a great deal of meticulous endeavors (Aoki et al., 2009; Carroll & Wickner, 1995; Herrero & Zabalgogeazcoa, 2011; Romo et al., 2006; Souza et al., 2000). Secondly, although viral infection to virus-free strain via purified virion or RNA transfection is the most direct and simple way of horizontal virus transmission, the case is very limited. In addition, hyphal anastomosis, the most commonly known and probably the only naturally occurring mechanism of horizontal virus transmission, can accompany many cytoplasmic inheritances during the virus transmission. This makes it difficult to determine whether the changes in phenotypes are due to the mycovirus, other cytological inheritance, or both (Nuss, 2005). The edible mushroom Lentinula edodes (Berk.) Pegler, commonly known as the shiitake, is the second most popular edible mushroom
J.-M. Kim et al. / Virus Research 197 (2015) 8–12
in terms of worldwide production and economic value (Hadeler, 1995) and is one of two most important commercial mushrooms in Korea (Ko et al., 2012). Several reports on the discovery of mycovirus in L. edodes exist (Kim et al., 2013; Magae, 2012; Rytter et al., 1991; Won et al., 2013). However, because these mycoviruses are commonly found in healthy and asymptomatic fruiting bodies, it is unclear whether they are the direct cause of abnormalities (Rytter et al., 1991). In addition, it has not been determined if a relationship exists between the presence or absence of a particular mycovirus and the vegetative characteristics of mycelia. In our initial survey of the abnormal browning of the medium surface and fruiting body of a popular commercial strain of L. edodes FMRI0339, we confirmed the presence of the mycovirus LeV (LeVFMRI0339) (Kim et al., 2013) and demonstrated its meiotic stability. In the current study, we attempted to cure L. edodes FMRI0339 of LeV-FMRI0339 in order to establish virus-free and virus-infected isogenic lines. By pair-wise comparison of the isogenic lines, we sought to better understand the relationship between LeV infection and vegetative characteristics of L. edodes. Based on the electrophoretic band pattern and Northern blot analysis (Park et al., 2008), the LeV concentration in L. edodes FMRI0339 varied significantly depending on whether the fungi were grown on a solid plate (Supplementary Fig. S1A–D; lane 1) or in submerged liquid culture (Supplementary Fig. S1A–D; lane 2). When the LeV-infected L. edodes FMRI0339 strain was grown in the potato dextrose broth (PDB) at 25 ◦ C with continuous agitation (200 rpm) in darkness, the concentration of LeV from the same amount of mycelia was significantly reduced. More than a 20-fold decrease in the virus titer, as measured by ImageJ software, was observed from the mycelia submerged in a liquid culture (Supplementary Fig. S1A–D; lanes 1 and 2). When we tested different media, such as V8 juice, similar results were observed (data not shown). Together, these results indicate that the decreased viral titer is a result of growth conditions (e.g., solid vs. liquid), not media components. However, it was possible to isolate LeV from the limited amount (≥0.1 g of powder per preparation) of ground powder of lyophilized mycelia grown in liquid culture. Thus, no loss of virus from the preparation using mycelia grown in the submerged liquid culture was observed. Interestingly, when the mycelia grown in liquid culture were transferred to the top of a plate containing the same medium, the freshly growing-mycelia on plate recovered a relatively high concentration of LeV (Supplementary Fig. S1A–D; lanes 3, 4, and 5). In addition, when the fungi were cultured by placing them gently on the surface of the liquid media without agitation, and were then used to prepare the virus from the floated mycelia, a high concentration of virus was maintained (Supplementary Fig. S1A–D; lanes 6, 7, and 8). These results indicate that growth conditions (e.g., submerged or not submerged) affect the viral replication significantly. Since the submerged culture condition substantially decreased viral titer, we further examined viral titer under oxygenlimited conditions. For oxygen-depleted culture conditions, the Anaeropack-Anaero system (Mitsubishi Gas Chemical, Tokyo, Japan) was used to create a hypoxic environment in an air tight jar, as previously described (Van Horn et al., 1997). Cultures containing actively growing mycelia were placed in an air tight jar with the Anaeropack-Anaero system and incubated in darkness at 25 ◦ C until use. Since the mycelial growth in an air tight jar with the Anaeropack-Anaero system was considerably decreased, we cultured for 20 days instead of 13 days in the air tight jar. No significant difference in the virus titer was observed between samples cultured for 13 days and 20 days on PDA. When the LeV-infected L. edodes FMRI0339 strain was inoculated on potato dextrose agar (PDA) and cultured for 20 days in an air tight jar using the Anaeropack-Anaero system, the concentration of LeV was significantly reduced (Supplementary Fig. S2A–D; lane 3). Densitometry indicated a two-fold
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Table 1 Growth rate and mycelial mass of virus-infected and virus-cured lines. Mean ± SD are shown with the range of measurement in parentheses.
Virus-infected L. edodes FMRI0339 Virus-cured L. edodes FMRI0339-vf-1 Virus-cured L. edodes FMRI0339-vf-14
Growth rate (cm)
Mycelial mass (mg of dried weight)
6.95 ± 0.83a * (5.10–8.20) 7.42 ± 0.83b (6.40–8.40)
11.73 ± 8.5a (2.2–44.6) 14.73 ± 11.4a,b (0.6–47.8)
7.29 ± 0.75b (5.70–8.40)
17.67 ± 8.1b (1.9–36.3)
* Mean numbers followed by same letters in each column are not significantly different at the 5% level by t-test.
decrease in the virus titer of the mycelia cultured under hypoxic conditions (Supplementary Fig. S2A–D; lane 1 vs. lane 3). Similarly, when the LeV-infected L. edodes FMRI0339 strain was cultured for 3 days in ambient air prior to transfer to hypoxic conditions, a two-fold decrease in the concentration of LeV was observed (Supplementary Fig. S2A–D; lane 1 vs. lane 4). These results indicated that the oxygen-depleted culture conditions affected LeV replication in fungal mycelia. However, considering the more than a 20-fold decrease observed in the submerged liquid culture compared to the two-fold decrease observed in the oxygen-limited condition, depletion of oxygen was not the only factor affecting viral titer in submerged liquid culture conditions. Further studies should examine what conditions other than hypoxia limit viral replication in fungal host. Viral titer reflects the host–parasite interaction. Besides differences in the genetic susceptibility of host, viral titer depends substantially on host cell physiology (Schoffelen et al., 2013) and culture conditions (Hillman et al., 1990; Jung et al., 2004). In nature, not only the genotypes of the host-parasite species but also environmental factors are important to select the outperforming interactions, which were hypothesized by the geographic mosaic theory of coevolution (Gomulkiewicz et al., 2000; Thompson, 1999). Accordingly, environmental factors were important to determine the outcome of the fungus–mycovirus interaction (Bryner and Rigling, 2011; Hyder et al., 2013). Thus, our study showing the variation in viral titer depending on culture conditions contributes to increase our understanding of the mechanism of variation in natural selection among ecosystems. Substantial changes in LeV titer depending culture condition may explain why it was not easy to determine the outcomes of L. edodes–LeV interaction. Therefore, it is desirable to establish virusfree and virus-infected isogenic L. edodes lines to determine the effect of LeV infection on L. edodes. Several methods have been applied to cure L. edodes FMRI0339 of LeV infection, such as hyphal tip transfer, cycloheximide treatment, incubation at low (20 ◦ C) or high (30 ◦ C) temperature, and mycelial fragmentation (Kim et al., 2013). Among these, the mycelial fragmentation method resulted in cured fungal lines. Based on the electrophoretic band pattern and Northern blot analysis of preparations of dsRNA by conventional CC41 cellulose chromatography, a high ratio (>40%) of colonies from mycelial fragments consisting of one to five cells lacked the LeV virus. This was further confirmed by RT-PCR using total nucleic acid and gene-specific primers corresponding to a part of RNA dependent RNA polymerase (RdRp) of the LeV in L. edodes FMRI0339 (GenBank Accession no. AB646992). The primers used were LeVRDRP (forward) 5 -TTA TGG TCT GGA TGG CGT-3 and LeV-RDRP (reverse) 5 -TGT CAC TCC AAA ACC TCC-3 . PCR was conducted as described previously (So et al., 2012). This simple mycelial fragmentation method of curing is extremely efficient and dependable as evidenced by repeated tests resulting in similar curing efficacy.
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Fig. 1. Analysis of growth rate (A) and dry mass (B). Data are the means ± standard deviations from four replications. Mean separation by Duncan’s multiple range test at p — 0.05. The same letters above bars indicate no significant difference between strains. Media and strains are indicated at the top and bottom of the each panel, respectively. Strains represented by P0, F1, and F14 indicate LeV-infected L. edodes FMRI0339, and two cured lines of L. edodes FMRI0339-vf-1 and L. edodes FMRI0339-vf-14, respectively.
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Two cured lines, L. edodes FMRI0339-vf-1 and L. edodes FMRI0339vf-14, were selected for further analysis. Once cured, there was no reappearance of LeV in any of cured colonies which were successively transferred to new media in 10-day intervals for six months. Furthermore, over the course of two years of cultivation of these cured lines, there has been no sign of viral resurrection via dsRNA analysis. Growth rate and mycelial dry weight of virus-infected L. edodes FMRI0339 were compared with two virus-cured lines (L. edodes FMRI0339-vf-1 and -vf-14) using 11 different media (Supplementary Table S1). To compare growth rate and mycelial mass, the colony diameters at 10-day-old culture, representing the cultivation time to cover more than 85% of plate, and the corresponding mycelial dried weight were measured as previously described (Lee et al., 2007). In order to determine the viral effects on both growth rate and mycelial mass, statistical analysis were conducted by ANOVA using SPSS version 15 (SPSS, Chicago, IL, USA), with significant differences between strains determined by the magnitude of the F value at p— — 0.05. When a significant F value was obtained, the significance of the effects of the strains was determined using Duncan’s multiple range test at p — — 0.05. Statistical analysis indicated that, when combined all tested media and cured lines, significant differences in growth rate and dry weight between virus-infected L. edodes FMRI0339 and its combined cured lines were observed at the level of p < 0.001 and p — — 0.013, respectively, confirming that virus infection made significant effects on both growth rate and mycelial mass. The growth rate of the cured-lines was significantly greater than that of L. edodes FMRI0339 and there was no difference between the two cured lines. This indicates that the presence of LeV affected the growth rate of the fungi (Table 1). However, differences in mycelial mass between virus-infected L. edodes FMRI0339 and each of the cured-line were not always statistically supported when combined all tested media (Table 1). The average dry weight of L. edodes FMRI0339-vf-14 was significantly larger than L. edodes FMRI0339; however, the difference between L. edodes FMRI0339-vf-1 and L. edodes FMRI0339 was not significantly supported although the average dry weight of L. edodes FMRI0339-vf-1 was larger than that of the L. edodes FMRI0339 (Table 1). Thus, we further examined the differences between virus-infected L. edodes FMRI0339 and each of the cured-lines depending on each media. Regarding the growth rate (Fig. 1A), the virus-cured lines showed colony diameters greater than those of the virus-infected control on all but two media (Hamada and V8 juice). Of the 11 media tested, six (MCM, ME, MYPA, PDA, YM, and YMPG) showed that all cured lines had a significant increase in colony diameters compared to that of the L. edodes FMRI0339 control. Three media (Czapek-dox, sawdust, and YMG) showed that, although the colony diameters of all cured-lines were greater than that of the L. edodes FMRI0339 control, not all differences were statistically supported. The colony diameter of the L. edodes FMRI0339 on the Hamada media was greater than the cured-lines but the differences were not statistically supported. In the V8 juice media, the L. edodes FMRI0339 control exhibited a significant increase in colony diameter compared to the cured L. edodes FMRI0339-vf-14 line. However, unlike other strains showing a stationary growth rate after the 10-day culture, the cured L. edodes FMRI0339-vf-14 line showed a linear growth rate until it reached the end of the plate. This suggests that there are no significant differences in growth rate because the diameters were close to equal after the 10-day culture. Therefore, growth rate analysis indicates that the overall growth rate of the cured lines was greater than that of the L. edodes FMRI0339 and this result was statistically significant. Regarding the corresponding mycelial mass (Fig. 1B), eight media (Czapek-dox, Hamada, MCM, MYPA, PDA, Sawdust, YM, and YMG) revealed that the average mass of the L. edodes FMRI0339
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was the smallest among tested strains. However, although some cured-lines showed a significant increase of mycelial mass, not all cured-lines were statistically supported in all media. In ME and V8 juice media, the L. edodes FMRI0339 control had a larger mass than either one or two cured-lines, but these differences were not significant. The YMPG medium showed that the L. edodes FMRI0339 control had a significantly larger mass than the cured L. edodes FMRI0339-vf-1 line but it was significantly smaller than the other cured-line. While the mycelial mass varied among test strains depending on culture media, the overall dry weight of the cured lines was no less than that of the L. edodes FMRI0339. As a consequence, it was possible to obtain the virus-free L. edodes FMRI0339 showing the significantly increased growth rate and mycelial mass. Thus, studies to understand the effects of the LeV-FMRI0339 on fruiting body initiation and production are warranted in the future. In this study we showed that L. edodes FMRI0339 mycelia could be cured of the mycovirus LeV-FMRI0339 using mycelial fragmentation followed by single colony isolation. The titer of LeV was affected by the culture condition and greater titer was obtained from solid plate-cultured mycelia as opposed to liquid culture. Although some variations existed depending upon media, curing the fungus of LeV resulted in better growth rate and improved mycelial mass. Therefore, LeV appeared to cause decreased mycelial growth in L. edodes. We are currently exploring effects of LeV on fruiting body formation and mushroom yield. Acknowledgments This work was supported by the Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and in part by NRF grants from MSIP (No. 2008-0061897and NRF-2013R1A1A2012433). This research was also supported in part by the Korea Research Council of Fundamental Science & Technology (Joint Degree and Research Center for Biorefinery) and in part by “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ00999801)” from the Rural Development Administration, Republic of Korea. We thank the Institute of Molecular Biology and Genetics at Chonbuk National University for kindly providing the facilities for this research. K.K. So was supported by the BK21 PLUS program in the Department of Bioactive Material Sciences. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.016. References Aoki, T., Moriyama, H., Kodama, M., Arie, T., Teraoka, T., Fukuhara, T., 2009. A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata. Virus. Res. 140, 179–187. Bryner, S.F., Rigling, D., 2011. Temperature-dependent genotype-by-genotype interaction between a pathogenic fungus and its hyperparasitica virus. Am. Nat. 177, 65–74. Carroll, K., Wickner, R.B., 1995. Translation and M1 double-stranded RNA propaga— tion: MAK18 RPL41B and cycloheximide curing. J. Bacteriol. 177, 2887–2891. Castro, M., Kramer, K., Valdivia, L., Ortiz, S., Castillo, A., 2003. A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea. Fed. Eur. Microbiol. Soc. Microbiol. Lett. 228, 87–91. Dawe, A.L., Nuss, D.L., 2001. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu. Rev. Genet. 35, 1–29. Ghabrial, S.A., 1998. Origin, adaptation and evolutionary pathways of fungal viruses. Virus Genes 16, 119–131. Gomulkiewicz, R., Thompson, J.N., Holt, R.D., Nuismer, S.L., Hochberg, M.E., 2000. Hot spots, cold spots, and the geographic mosaic theory of coevolution. Am. Nat. 156, 156–174. Hadeler, H., 1995. Medicinal Mushrooms You Can Grow. The Cariaga Publishing House, Sechelt, BC, Canada.
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Herrero, N., Zabalgogeazcoa, I., 2011. Mycoviruses infecting the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum. Virus Res. 160, 409–413. Hillman, B.I., Shapira, R., Nuss, D.L., 1990. Hypovirulence-associated suppression of host functions in Cryphonectria parasitica can be partially relieved by high light intensity. Phytopathology 80, 950–956. Hollings, M., 1962. Viruses associated with a die-back disease of cultivated mushroom. Nature 196, 962–965. Hyder, R., Pennanen, T., Hamberg, L., Vainio, E.J., Piri, T., Hantula, J., 2013. Two viruses of Heterobasidion confer beneficial, cryptic or detrimental effects to their hosts in different situations. Fungal Ecol. 6, 387–396. Jung, S.I., Behie, L.A., Lee, P.W., Farrell, P.J., 2004. Optimization of reovirus production from mouse L-929 cells in suspension culture. Biotechnol. Bioeng. 85, 750–760. Kim, J.M., Yun, S.H., Park, S.M., Ko, H.G., Kim, D.H., 2013. Occurrence of dsRNA mycovirus (LeV-FMRI0339) in the edible mushroom Lentinula edodes and meiotic stability of LeV-FMRI0339 among monokaryotic progeny. Plant Pathol. J. 4, 460–464. Ko, H.K., Choi, S.K., Kim, S.C., Kim, J.H., Lee, W.H., Roh, J.H., Lee, B.S., Kim, I.Y., Kim, W.S., 2012. Cultural techniques of Lentinula edodes. Report of Forest Mushroom Research Center, Korea. Lee, J.K., Kim, B.T., Kim, J.A., Chung, H.J., Park, S.M., Yang, M.S., Hwang, K.J., Kim, D.H., 2007. Cultural characteristics and extraction of the fungal pigment phleichrome from a phytopathogenic fungus Cladosporium phlei. Bioetchnol. Bioprocess Eng. 12, 508–515. Magae, Y., 2012. Molecular characterization of a novel mycovirus in the cultivated mushroom Lentinula edodes. Virol. J. 9, 60. Nuss, D.L., 2005. Hypovirulence: mycoviruses at the fungal–plant interface. Nat. Rev. Microbiol. 3, 632–642.
Park, S.M., Kim, J.M., Chung, H.J., Lim, J.Y., Kwon, B.R., Lim, J.G., Kim, J.A., Kim, M.J., Cha, B.J., Lee, S.H., Kim, K.H., Lee, Y.S., Yang, M.S., Kim, D.H., 2008. Occurrence of diverse dsRNA in a Korean population of the chestnut blight fungus Cryphonectria parasitica. Mycol. Res. 12, 1220–1226. Pearson, M.N., Beever, R.E., Boine, B., Arthur, K., 2009. Mycoviruses of filamentous fungi and their relevance to plant pathology. Mol. Plant Pathol. 10, 115–128. Romo, M., Leuchtmann, A., García, B., Zabalgogeazcoa, I., 2006. A totivirus infecting the mutualistic fungal endophyte Epichloë festucae. Virus Res. 124, 38–43. Rytter, J.L., Royse, D.J., Romaine, C.P., 1991. Incidence and diversity of doublestranded RNA in Lentinula edodes. Mycologia 83, 506–510. Schoffelen, A.F., Wensing, A.M., Tempelman, H.A., Geelen, S.P., Hoepelman, A.I., Barth, R.E., 2013. Sustained virological response on second-line antiretroviral therapy following virological failure in HIV-infected patients in rural South Africa. PLos One 8, e58526. So, K.K., Kim, J.M., Nguyen, N.L., Park, J.A., Kim, B.T., Park, S.M., Hwang, K.J., Kim, D.H., 2012. Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei. J. Microbiol. Methods 91, 412–419. Souza, I.E., Azevedo, M.L., Succi, R.C., Machado, D.M., Diaz, R.S., 2000. RNA viral load test for early diagnosis of vertical transmission of HIV-1 infection. J. Acquir. Immune Defic. Syndr. 23, 358–360. Thompson, J.N., 1999. Specific hypotheses on the geographic mosaic of coevolution. Am. Nat. 153, 1–14. Van Horn, K.G., Warren, K., Baccaglini, E.J., 1997. Evaluation of the AnaeroPack system for growth of anaerobic bacteria. J. Clin. Microbiol. 35, 2170–2173. Won, H.K., Park, S.J., Kim, D.K., Shin, M.J., Kim, N., Lee, S.H., Kwon, Y.C., Ko, H.K., Ro, H.S., Lee, H.S., 2013. Isolation and characterization of a mycovirus in Lentinula edodes. J. Microbiol. 51, 118–122.
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Changes in the mycovirus (LeV) titer and viral effect on the vegetative growth of the edible mushroom Lentinula edodes Jung-Mi Kim a , Ha-Yeon Song a , Hyo-Jin Choi a , Suk-Hyun Yun b , Kum-Kang So b , Han-Kyu Ko c , Dae-Hyuk Kim b,∗ a
Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, South Korea Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, South Korea c Forest Mushroom Research Center, Yeoju, Gyeonggi 469-803, South Korea b
a r t i c l e
i n f o
Article history: Received 15 September 2014 Received in revised form 9 November 2014 Accepted 12 November 2014 Available online 20 November 2014 Keywords: Lentinula edodes dsRNA Isogenic strain Mycovirus
a b s t r a c t This study attempted to cure the edible mushroom Lentinula edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygendepleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. These results indicate that LeV infection has a deleterious effect on mycelial growth. © 2014 Elsevier B.V. All rights reserved.
The presence of viruses in fungi is now accepted as not “exceptional” but “general” (Dawe & Nuss, 2001; Ghabrial, 1998; Pearson et al., 2009) since the first definitive description of mycovirus more than 50 years ago (Hollings, 1962). Viral infection occurs in all major taxa belonging to the kingdom Fungi. However, it is difficult to determine the biological consequences of mycoviral infection for following reasons. First, although there is compelling evidence that an increasing number of viruses induce symptomatic infections, mycoviral infections, in general, have been considered not to cause measurable phenotypic changes in the fungal hosts. If anything, such changes are cryptic or latent. Second, in some cases, although mycoviral infection causes considerable morphological and physiological changes, including debilitation- and virulencerelated phenotypes (Castro et al., 2003; Dawe & Nuss, 2001), the varied genetic backgrounds of fungal strains may respond
∗ Correspondence to: Department of Molecular Biology, Chonbuk National University, Dukjindong 664-14, Jeonju, Chonbuk 561-756, South Korea. Tel.: +82 63 270 3440; fax: +82 63 270 4312. E-mail address: [email protected] (D.-H. Kim). http://dx.doi.org/10.1016/j.virusres.2014.11.016 0168-1702/© 2014 Elsevier B.V. All rights reserved.
differently to the same mycovirus, which makes it desirable to have identical genetic backgrounds (with the exception of the presence or absence of the virus) for the study of fungus–mycovirus interactions. Establishment of such virus-free and virus-infected isogenic lines to explore direct mycovirus–fungal host interactions has been hampered due to several reasons. Firstly, although several attempts have been described, successfully curing an infected fungal host of a mycovirus infection requires a great deal of meticulous endeavors (Aoki et al., 2009; Carroll & Wickner, 1995; Herrero & Zabalgogeazcoa, 2011; Romo et al., 2006; Souza et al., 2000). Secondly, although viral infection to virus-free strain via purified virion or RNA transfection is the most direct and simple way of horizontal virus transmission, the case is very limited. In addition, hyphal anastomosis, the most commonly known and probably the only naturally occurring mechanism of horizontal virus transmission, can accompany many cytoplasmic inheritances during the virus transmission. This makes it difficult to determine whether the changes in phenotypes are due to the mycovirus, other cytological inheritance, or both (Nuss, 2005). The edible mushroom Lentinula edodes (Berk.) Pegler, commonly known as the shiitake, is the second most popular edible mushroom
J.-M. Kim et al. / Virus Research 197 (2015) 8–12
in terms of worldwide production and economic value (Hadeler, 1995) and is one of two most important commercial mushrooms in Korea (Ko et al., 2012). Several reports on the discovery of mycovirus in L. edodes exist (Kim et al., 2013; Magae, 2012; Rytter et al., 1991; Won et al., 2013). However, because these mycoviruses are commonly found in healthy and asymptomatic fruiting bodies, it is unclear whether they are the direct cause of abnormalities (Rytter et al., 1991). In addition, it has not been determined if a relationship exists between the presence or absence of a particular mycovirus and the vegetative characteristics of mycelia. In our initial survey of the abnormal browning of the medium surface and fruiting body of a popular commercial strain of L. edodes FMRI0339, we confirmed the presence of the mycovirus LeV (LeVFMRI0339) (Kim et al., 2013) and demonstrated its meiotic stability. In the current study, we attempted to cure L. edodes FMRI0339 of LeV-FMRI0339 in order to establish virus-free and virus-infected isogenic lines. By pair-wise comparison of the isogenic lines, we sought to better understand the relationship between LeV infection and vegetative characteristics of L. edodes. Based on the electrophoretic band pattern and Northern blot analysis (Park et al., 2008), the LeV concentration in L. edodes FMRI0339 varied significantly depending on whether the fungi were grown on a solid plate (Supplementary Fig. S1A–D; lane 1) or in submerged liquid culture (Supplementary Fig. S1A–D; lane 2). When the LeV-infected L. edodes FMRI0339 strain was grown in the potato dextrose broth (PDB) at 25 ◦ C with continuous agitation (200 rpm) in darkness, the concentration of LeV from the same amount of mycelia was significantly reduced. More than a 20-fold decrease in the virus titer, as measured by ImageJ software, was observed from the mycelia submerged in a liquid culture (Supplementary Fig. S1A–D; lanes 1 and 2). When we tested different media, such as V8 juice, similar results were observed (data not shown). Together, these results indicate that the decreased viral titer is a result of growth conditions (e.g., solid vs. liquid), not media components. However, it was possible to isolate LeV from the limited amount (≥0.1 g of powder per preparation) of ground powder of lyophilized mycelia grown in liquid culture. Thus, no loss of virus from the preparation using mycelia grown in the submerged liquid culture was observed. Interestingly, when the mycelia grown in liquid culture were transferred to the top of a plate containing the same medium, the freshly growing-mycelia on plate recovered a relatively high concentration of LeV (Supplementary Fig. S1A–D; lanes 3, 4, and 5). In addition, when the fungi were cultured by placing them gently on the surface of the liquid media without agitation, and were then used to prepare the virus from the floated mycelia, a high concentration of virus was maintained (Supplementary Fig. S1A–D; lanes 6, 7, and 8). These results indicate that growth conditions (e.g., submerged or not submerged) affect the viral replication significantly. Since the submerged culture condition substantially decreased viral titer, we further examined viral titer under oxygenlimited conditions. For oxygen-depleted culture conditions, the Anaeropack-Anaero system (Mitsubishi Gas Chemical, Tokyo, Japan) was used to create a hypoxic environment in an air tight jar, as previously described (Van Horn et al., 1997). Cultures containing actively growing mycelia were placed in an air tight jar with the Anaeropack-Anaero system and incubated in darkness at 25 ◦ C until use. Since the mycelial growth in an air tight jar with the Anaeropack-Anaero system was considerably decreased, we cultured for 20 days instead of 13 days in the air tight jar. No significant difference in the virus titer was observed between samples cultured for 13 days and 20 days on PDA. When the LeV-infected L. edodes FMRI0339 strain was inoculated on potato dextrose agar (PDA) and cultured for 20 days in an air tight jar using the Anaeropack-Anaero system, the concentration of LeV was significantly reduced (Supplementary Fig. S2A–D; lane 3). Densitometry indicated a two-fold
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Table 1 Growth rate and mycelial mass of virus-infected and virus-cured lines. Mean ± SD are shown with the range of measurement in parentheses.
Virus-infected L. edodes FMRI0339 Virus-cured L. edodes FMRI0339-vf-1 Virus-cured L. edodes FMRI0339-vf-14
Growth rate (cm)
Mycelial mass (mg of dried weight)
6.95 ± 0.83a * (5.10–8.20) 7.42 ± 0.83b (6.40–8.40)
11.73 ± 8.5a (2.2–44.6) 14.73 ± 11.4a,b (0.6–47.8)
7.29 ± 0.75b (5.70–8.40)
17.67 ± 8.1b (1.9–36.3)
* Mean numbers followed by same letters in each column are not significantly different at the 5% level by t-test.
decrease in the virus titer of the mycelia cultured under hypoxic conditions (Supplementary Fig. S2A–D; lane 1 vs. lane 3). Similarly, when the LeV-infected L. edodes FMRI0339 strain was cultured for 3 days in ambient air prior to transfer to hypoxic conditions, a two-fold decrease in the concentration of LeV was observed (Supplementary Fig. S2A–D; lane 1 vs. lane 4). These results indicated that the oxygen-depleted culture conditions affected LeV replication in fungal mycelia. However, considering the more than a 20-fold decrease observed in the submerged liquid culture compared to the two-fold decrease observed in the oxygen-limited condition, depletion of oxygen was not the only factor affecting viral titer in submerged liquid culture conditions. Further studies should examine what conditions other than hypoxia limit viral replication in fungal host. Viral titer reflects the host–parasite interaction. Besides differences in the genetic susceptibility of host, viral titer depends substantially on host cell physiology (Schoffelen et al., 2013) and culture conditions (Hillman et al., 1990; Jung et al., 2004). In nature, not only the genotypes of the host-parasite species but also environmental factors are important to select the outperforming interactions, which were hypothesized by the geographic mosaic theory of coevolution (Gomulkiewicz et al., 2000; Thompson, 1999). Accordingly, environmental factors were important to determine the outcome of the fungus–mycovirus interaction (Bryner and Rigling, 2011; Hyder et al., 2013). Thus, our study showing the variation in viral titer depending on culture conditions contributes to increase our understanding of the mechanism of variation in natural selection among ecosystems. Substantial changes in LeV titer depending culture condition may explain why it was not easy to determine the outcomes of L. edodes–LeV interaction. Therefore, it is desirable to establish virusfree and virus-infected isogenic L. edodes lines to determine the effect of LeV infection on L. edodes. Several methods have been applied to cure L. edodes FMRI0339 of LeV infection, such as hyphal tip transfer, cycloheximide treatment, incubation at low (20 ◦ C) or high (30 ◦ C) temperature, and mycelial fragmentation (Kim et al., 2013). Among these, the mycelial fragmentation method resulted in cured fungal lines. Based on the electrophoretic band pattern and Northern blot analysis of preparations of dsRNA by conventional CC41 cellulose chromatography, a high ratio (>40%) of colonies from mycelial fragments consisting of one to five cells lacked the LeV virus. This was further confirmed by RT-PCR using total nucleic acid and gene-specific primers corresponding to a part of RNA dependent RNA polymerase (RdRp) of the LeV in L. edodes FMRI0339 (GenBank Accession no. AB646992). The primers used were LeVRDRP (forward) 5 -TTA TGG TCT GGA TGG CGT-3 and LeV-RDRP (reverse) 5 -TGT CAC TCC AAA ACC TCC-3 . PCR was conducted as described previously (So et al., 2012). This simple mycelial fragmentation method of curing is extremely efficient and dependable as evidenced by repeated tests resulting in similar curing efficacy.
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Fig. 1. Analysis of growth rate (A) and dry mass (B). Data are the means ± standard deviations from four replications. Mean separation by Duncan’s multiple range test at p — 0.05. The same letters above bars indicate no significant difference between strains. Media and strains are indicated at the top and bottom of the each panel, respectively. Strains represented by P0, F1, and F14 indicate LeV-infected L. edodes FMRI0339, and two cured lines of L. edodes FMRI0339-vf-1 and L. edodes FMRI0339-vf-14, respectively.
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Two cured lines, L. edodes FMRI0339-vf-1 and L. edodes FMRI0339vf-14, were selected for further analysis. Once cured, there was no reappearance of LeV in any of cured colonies which were successively transferred to new media in 10-day intervals for six months. Furthermore, over the course of two years of cultivation of these cured lines, there has been no sign of viral resurrection via dsRNA analysis. Growth rate and mycelial dry weight of virus-infected L. edodes FMRI0339 were compared with two virus-cured lines (L. edodes FMRI0339-vf-1 and -vf-14) using 11 different media (Supplementary Table S1). To compare growth rate and mycelial mass, the colony diameters at 10-day-old culture, representing the cultivation time to cover more than 85% of plate, and the corresponding mycelial dried weight were measured as previously described (Lee et al., 2007). In order to determine the viral effects on both growth rate and mycelial mass, statistical analysis were conducted by ANOVA using SPSS version 15 (SPSS, Chicago, IL, USA), with significant differences between strains determined by the magnitude of the F value at p— — 0.05. When a significant F value was obtained, the significance of the effects of the strains was determined using Duncan’s multiple range test at p — — 0.05. Statistical analysis indicated that, when combined all tested media and cured lines, significant differences in growth rate and dry weight between virus-infected L. edodes FMRI0339 and its combined cured lines were observed at the level of p < 0.001 and p — — 0.013, respectively, confirming that virus infection made significant effects on both growth rate and mycelial mass. The growth rate of the cured-lines was significantly greater than that of L. edodes FMRI0339 and there was no difference between the two cured lines. This indicates that the presence of LeV affected the growth rate of the fungi (Table 1). However, differences in mycelial mass between virus-infected L. edodes FMRI0339 and each of the cured-line were not always statistically supported when combined all tested media (Table 1). The average dry weight of L. edodes FMRI0339-vf-14 was significantly larger than L. edodes FMRI0339; however, the difference between L. edodes FMRI0339-vf-1 and L. edodes FMRI0339 was not significantly supported although the average dry weight of L. edodes FMRI0339-vf-1 was larger than that of the L. edodes FMRI0339 (Table 1). Thus, we further examined the differences between virus-infected L. edodes FMRI0339 and each of the cured-lines depending on each media. Regarding the growth rate (Fig. 1A), the virus-cured lines showed colony diameters greater than those of the virus-infected control on all but two media (Hamada and V8 juice). Of the 11 media tested, six (MCM, ME, MYPA, PDA, YM, and YMPG) showed that all cured lines had a significant increase in colony diameters compared to that of the L. edodes FMRI0339 control. Three media (Czapek-dox, sawdust, and YMG) showed that, although the colony diameters of all cured-lines were greater than that of the L. edodes FMRI0339 control, not all differences were statistically supported. The colony diameter of the L. edodes FMRI0339 on the Hamada media was greater than the cured-lines but the differences were not statistically supported. In the V8 juice media, the L. edodes FMRI0339 control exhibited a significant increase in colony diameter compared to the cured L. edodes FMRI0339-vf-14 line. However, unlike other strains showing a stationary growth rate after the 10-day culture, the cured L. edodes FMRI0339-vf-14 line showed a linear growth rate until it reached the end of the plate. This suggests that there are no significant differences in growth rate because the diameters were close to equal after the 10-day culture. Therefore, growth rate analysis indicates that the overall growth rate of the cured lines was greater than that of the L. edodes FMRI0339 and this result was statistically significant. Regarding the corresponding mycelial mass (Fig. 1B), eight media (Czapek-dox, Hamada, MCM, MYPA, PDA, Sawdust, YM, and YMG) revealed that the average mass of the L. edodes FMRI0339
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was the smallest among tested strains. However, although some cured-lines showed a significant increase of mycelial mass, not all cured-lines were statistically supported in all media. In ME and V8 juice media, the L. edodes FMRI0339 control had a larger mass than either one or two cured-lines, but these differences were not significant. The YMPG medium showed that the L. edodes FMRI0339 control had a significantly larger mass than the cured L. edodes FMRI0339-vf-1 line but it was significantly smaller than the other cured-line. While the mycelial mass varied among test strains depending on culture media, the overall dry weight of the cured lines was no less than that of the L. edodes FMRI0339. As a consequence, it was possible to obtain the virus-free L. edodes FMRI0339 showing the significantly increased growth rate and mycelial mass. Thus, studies to understand the effects of the LeV-FMRI0339 on fruiting body initiation and production are warranted in the future. In this study we showed that L. edodes FMRI0339 mycelia could be cured of the mycovirus LeV-FMRI0339 using mycelial fragmentation followed by single colony isolation. The titer of LeV was affected by the culture condition and greater titer was obtained from solid plate-cultured mycelia as opposed to liquid culture. Although some variations existed depending upon media, curing the fungus of LeV resulted in better growth rate and improved mycelial mass. Therefore, LeV appeared to cause decreased mycelial growth in L. edodes. We are currently exploring effects of LeV on fruiting body formation and mushroom yield. Acknowledgments This work was supported by the Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and in part by NRF grants from MSIP (No. 2008-0061897and NRF-2013R1A1A2012433). This research was also supported in part by the Korea Research Council of Fundamental Science & Technology (Joint Degree and Research Center for Biorefinery) and in part by “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ00999801)” from the Rural Development Administration, Republic of Korea. We thank the Institute of Molecular Biology and Genetics at Chonbuk National University for kindly providing the facilities for this research. K.K. So was supported by the BK21 PLUS program in the Department of Bioactive Material Sciences. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.11.016. References Aoki, T., Moriyama, H., Kodama, M., Arie, T., Teraoka, T., Fukuhara, T., 2009. A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata. Virus. Res. 140, 179–187. Bryner, S.F., Rigling, D., 2011. Temperature-dependent genotype-by-genotype interaction between a pathogenic fungus and its hyperparasitica virus. Am. Nat. 177, 65–74. Carroll, K., Wickner, R.B., 1995. Translation and M1 double-stranded RNA propaga— tion: MAK18 RPL41B and cycloheximide curing. J. Bacteriol. 177, 2887–2891. Castro, M., Kramer, K., Valdivia, L., Ortiz, S., Castillo, A., 2003. A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea. Fed. Eur. Microbiol. Soc. Microbiol. Lett. 228, 87–91. Dawe, A.L., Nuss, D.L., 2001. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu. Rev. Genet. 35, 1–29. Ghabrial, S.A., 1998. Origin, adaptation and evolutionary pathways of fungal viruses. Virus Genes 16, 119–131. Gomulkiewicz, R., Thompson, J.N., Holt, R.D., Nuismer, S.L., Hochberg, M.E., 2000. Hot spots, cold spots, and the geographic mosaic theory of coevolution. Am. Nat. 156, 156–174. Hadeler, H., 1995. Medicinal Mushrooms You Can Grow. The Cariaga Publishing House, Sechelt, BC, Canada.
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