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Double-stranded RNA transmission through basidiospores of Heterobasidion annosum
Mycol. Res. 108 (2): 149–153 (February 2004). f The British Mycological Society
149
DOI: 10.1017/S0953756203008839 Printed in the United Kingdom.
Double-stranded RNA transmission through basidiospores of Heterobasidion annosum
Katarina IHRMARK, Elna STENSTRO¨M and Jan STENLID Swedish University of Agricultural Sciences, Department of Forest Mycology and Pathology, P.O. Box 7026, SE-750 07 Uppsala, Sweden. E-mail : [email protected] Received 7 May 2003; accepted 28 October 2003.
A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14 % of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10–84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P<0.05) ; germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant ; germination was 3 and 10 % for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.
INTRODUCTION Fungi can be infected by dsRNA mycoviruses, some of which cause negative effects for the host, while others are neutral (Ghabrial 1998). dsRNA mycoviruses in fungi do not leave the cytoplasm and are transmitted intracellularly through spores and hyphal contacts (Ghabrial 1998). Spread through anastomosis is partly restricted by vegetative incompatibility. The efficiency of transmission through spores varies with spore type and taxonomic grouping of the fungi. In ascomycetes, asexual spores can carry dsRNA infection while sexual ascospores normally are free of dsRNA elements (Buck 1986, 1998). In basidiomycetes, transmission into sexual basidiospores occurs readily in Ustilago maydis (Day & Dodds 1979), Rhizoctonia solani (Castanho & Butler 1978, Castanho, Butler & Shepherd 1978), Agaricus bisporus (Romaine, Ulhrich & Schlagnhaufer 1993) and the yeast Phaffia rhodozyma (Pfeiffer et al. 1996), although the frequencies varies considerably (33–100%). However, in Agrocybe aegerita evidence for the transmission of dsRNA to basidiospores is weak or non-existent (Barroso & Labarere 2000), which is contrary to transmission patterns found in other basidiomycetes. An increased frequency of germination and viability of basidiospores infected with
dsRNA has been found in A. bisporus (Romaine et al. 1993). Transmission to conidia has only been studied in Heterobasidion annosum (Ihrmark et al. 2002), where it occurs at a much lower frequency than in most ascomycetes. H. annosum is a tree pathogen that causes root and butt rot in conifers in boreal forests. Spores land on newly exposed wood such as fresh stump surfaces, colonise the stump root system and spread via root contacts into neighbouring healthy trees (Korhonen & Stenlid 1998). H. annosum is a complex of several intersterility (IS) groups. In Northern Europe two IS groups are found, the S and P groups, named after their main host species (spruce and pine, respectively). Karjalainen & Fabritius (1993) have shown that the variation within the ITS (internal transcribed sequence) region can be used to distinguish between the main IS groups by PCR-RFLP. H. annosum has been found to harbour doublestranded RNA (dsRNA) elements with genetic information resembling partitiviruses (Ihrmark 2001, Ihrmark et al. 2001), a common group of mycoviruses. Most of the dsRNA elements in H. annosum seem to be two-segmented with one segment coding for an RNA dependent RNA polymerase (RDRP) with homology to RDRPs from Partitiviridae. dsRNA has mainly been
dsRNA in basidiospores
150
Table 1. Origin of tissue isolates from fruit bodies tested for presence of dsRNA, number of fruit bodies collected and the number of fruit bodies that contained dsRNA.
Origin
Number of fruit bodies
Number of fruit bodies with dsRNA
Fiby, Uppland, Sweden Fredrikslund, Uppland, Sweden Hammarheden, Uppland, Sweden Kvill, Sma˚land, Sweden Lunsen, Uppland, Sweden Omberg, O¨stergo¨tland, Sweden Ra¨mso¨n, Uppland, Sweden Svensho¨ngen, Bohusla¨n, Sweden Ycke, O¨stergo¨tland, Sweden Vilnius forest district, Lithuania
1 3 1 1 22 7 1 1 1 9
0 0 0 0 8 0 0 0 0 1
Total
47
9
found in isolates from decayed wood and only rarely in isolates from incipient decayed wood or in isolates that have recently passed through conidiogenesis (Ihrmark et al. 2001). Furthermore, in an earlier study we found that dsRNA in H. annosum is transmitted efficiently through anastomosis, also between incompatible isolates, and that transmission through conidia is less efficient (Ihrmark et al. 2002). This has led to the speculation that dsRNA is mainly spread horizontally, via anastomosis, in H. annosum and that spores play only a minor role in transmission of dsRNA in this species. The aim of this study was to further investigate presence and transmission of dsRNA in H. annosum. In particular we wanted to study the potential for transmission through basidiospores, which is of considerable interest since H. annosum is mainly established in new areas or on fresh substrates by basidiospores (Korhonen & Stenlid 1998).
MATERIALS AND METHODS Isolates We screened 59 isolates of Heterobasidion annosum from the culture collection of the Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, for dsRNA. The isolates originated from Siaro¨, Sweden (20 isolates), Norway (30) and Russia (9), and were all originally isolated as mycelium from decayed wood. Fruit bodies of H. annosum were collected from different parts of Sweden and Lithuania (Table 1) during Sept.–Oct. 2000. Mycelia were isolated from 47 fruit bodies by placing small pieces from the interior of fresh fruit bodies on Hagem agar (Stenlid 1985) with 50 mg lx1 ampicillin, 50 mg lx1 streptomycin and 5 mg lx1 benomyl. After a few days incubation in the dark at 21 xC the newly outgrown mycelia were transferred to new Hagem agar plates without antibiotics.
Table 2. The transmission frequency of dsRNA into basidiospores from fruit bodies containing dsRNA and germination frequency of basidiospores in water and malt extract. All fruit bodies were from Lunsen, Uppland, Sweden.
Fruit body
dsRNAa
2 3 5 6 11 12 13 15 17 18 19 20 23 Mean valued 8 10 14 16 21 22 25 26 Mean valued
x x x x x x x x x x x x x + + + + + + + +
Germination Germination Transmission in water in malt frequency (%)b (%)c extract (%)c
n.a. n.a. 40 (6/15) 67 (8/12) 10 (2/21) 11 (2/19) n.a. 84 (21/25)
0 11 0 8 2 17 4 7 36 17 26 0 6 10 a n.a. 1 11 n.a. 0 1 3 4 3a
n.a. n.a. n.a. 78 n.a. 94 27 n.a. 59 96 98 94 n.a. 78 b n.a. 30 55 n.a. 14 58 n.a. 15 34 c
+, fruit bodies containing dsRNA; x, dsRNA-free fruit bodies. n.a. : not analysed; in brackets: number of infected basidiospores/number of single basidiospore isolates analysed. c Germination after 24 h among 100 randomly chosen basidiospores, percentage; n.a.: not analysed. d Comparisons of germination frequencies of basidiospores from fruit bodies with and without dsRNA. The means followed by the same letter are not significantly different (P<0.05). a
b
Basidiospores Spores were collected from 33 of the fruit bodies by placing a piece of plastic close to the hymenium for one hour prior to collecting the fruit body. Spores were kept at 4 x during 7–10 d, whereupon the spores were suspended in water and incubated at 21 x for 20–24 h and the proportion of germinated spores was counted under a light microscope. Between 100 and 500 spores were counted from each fruit body. Spores were collected anew twice from Lunsen, Uppland, Sweden. In April 2001, spores were collected from fruit bodies adjacent to five of the fruit bodies that were found to contain dsRNA. The spores were immediately suspended in water and spread onto Hagem agar, incubated 36 h and thereafter wellseparated germinated spores were transferred to new Hagem agar plates. From each fruit body, 12–25 single basidiospore isolates were checked for the presence of dsRNA. In August 2001, spores were again collected from fruit bodies adjacent to twelve of the analysed fruit bodies and both fruit bodies containing dsRNA and dsRNA free isolates were included (Table 2). The spores were immediately suspended in 1.25 % malt
K. Ihrmark, E. Stenstro¨m and J. Stenlid extract medium, incubated at 21 x for 21 h and the proportion of germinated spores was counted as above. Differences in germination frequencies and transmission frequencies were statistically analysed by chisquare tests and one-way analysis of variance. Extraction of dsRNA All isolates were subcultured in 30 ml liquid malt syrup (2 %) in Petri dishes for two to four weeks in the dark at 21 x prior to extraction of nucleic acids. Freeze-dried mycelium, 200–1000 mg of each isolate, was ground to a fine powder with a small quantity of aluminium oxide powder in a mortar and pestle. Nucleic acids were extracted in 6 ml CTAB-buffer (3 % cetyltrimethylammoniumbromide, 2 mM EDTA, 150 mM Tris-HCl, 2.6 M NaCl, pH 8) and 5 ml phenol :chloroform : isoamyl alcohol (49.5: 49.5 :1, pH 8), followed by chloroform extraction and precipitation with isopropanol. The nucleic acids were dissolved in one millilitre STE (0.1 M NaCl, 50 mM Tris-HCl, 1 mM EDTA, pH 8) and dsRNA was separated from other nucleic acids by cellulose CF-11 chromatography as described by Sonnenberg, van Kempen & van Griensven (1995). The dsRNA pellets were dissolved in 30 ml water and treated with single-strand specific S1 nuclease, 330 units mlx1 (Roche, Basel) and DNase I, 330 units mlx1 (Roche), to ensure that only bands consisting of dsRNA would appear on gels. The digestions were conducted in 0.1 M sodium acetate buffer with 50 mM MgSO4 and 5 mM ZnCl2, pH 4.6, at 37 x for 2 h. The dsRNA was precipitated with isopropanol and the pellets dissolved in water. The entire samples were analysed on 1% agarose gels (D-1 ; Hispanlab, Madrid) for 90 min at 4.3 V cmx1 in 0.5rTBE (Sambrook, Fritsch & Maniatis 1989). The gels were stained in ethidium bromide and photographed under UV light. 1 Kb DNA Ladder (Gibco/Invitrogen, Carlsbad, CA) was used on each gel as a size standard. Intersterility group affiliation To certify IS affiliation of all isolates found to contain dsRNA, a PCR-RFLP analysis of the ITS region was performed as described by Ihrmark et al. (2001).
RESULTS dsRNA in isolates Of the 59 isolates from the culture collection that were screened for dsRNA, eight isolates contained dsRNA. Five of the 20 isolates from Siaro¨, Sweden, and three of the 30 isolates from Norway contained dsRNA. None of the isolates from Russia contained dsRNA. All of the dsRNA positive isolates belonged to the S intersterility group of Heterobasidion annosum. Of the 47 fruit body isolates, nine contained dsRNA (Table 1). Eight isolates came from the same area,
151 Lunsen, Uppland, Sweden, and belonged to the S group. The ninth isolate that contain dsRNA came from the Vilnius forest district, Lithuania, and belonged to the P group. Each isolate that contained dsRNA had one or two dsRNA fragments and all fragments were in the range 1.8 to 2.5 kbp. In the isolates yielding two dsRNA fragments, the smaller fragment was present at a lower concentration than the larger fragment, as could be visually seen on the agarose gels. The isolates yielding only one visible dsRNA fragment, all had a very faint dsRNA band and a second fragment might have been below the detection level. dsRNA in basidiospores All five fruit bodies that were used for testing dsRNA transmission frequency to basidiospores contained two dsRNA fragments and both dsRNA fragments were also present in the infected single basidiospore isolates from these fruit bodies. On average, of those basidiospores that germinated, 40 % contained dsRNA, from infected fruit bodies. Presence of dsRNA in germinated basidiospores varied considerably between the five fruit bodies tested. dsRNA was detected in 10, 11, 40, 67 and 84 % of the basidiospore isolates, respectively, from the five fruit bodies. There was no correlation between the proportion of germinated spores from the fruit bodies containing dsRNA and the proportion of germinated spores containing dsRNA (Table 2), i.e. both high and low frequency of transmission of dsRNA to basidiospores could be found from infected fruit bodies with a low basidiospore germination frequency (fruit bodies 26 and 21, respectively) as well as from infected fruit bodies with a high germination frequency (fruit bodies 14 and 22, respectively). Germination of basidiospores Eight of the nine fruit bodies containing dsRNA came from the same area, Lunsen, Uppland, Sweden, and we therefore limited the study of spore germination to fruit bodies from this area, to exclude differences in spore germination due to differences in geographical origin. Of the 22 fruit bodies from Lunsen, 21 produced spores at the time of collection (Table 2). Spore germination in malt extract was higher than in water and varied between 14 and 98 % (Table 2). There was a statistically significant difference in average germination frequency for basidiospores in malt extract from fruit bodies containing dsRNA, 34 %, and for dsRNA free fruit bodies, 78 % (P<0.05). Spore germination in water was low, compared with malt extract, and varied considerably between isolates, from 0 to 36 %, with an average of 6.4 % germinated spores (Table 2). Spores from fruit bodies with dsRNA germinated at a slightly lower frequency than those from dsRNA free fruit bodies, 3 and 10%, respectively, although the difference was not statistically significant.
dsRNA in basidiospores DISCUSSION Vertical transmission of dsRNA to basidiospores has been investigated in only a few species. With this report we present transmission frequency of dsRNA to basidiospores on an average of 40%, in Heterobasidion annosum. This number is lower than for other species that have been investigated, where transmission frequencies have been close to 100 % (Castanho & Butler 1978, Day & Dodds 1979, Pfeiffer et al. 1996). The exception is Agaricus bisporus, where transmission frequencies of 33–100 % (average 65–75 %) have been found (Romaine et al. 1993) and Agrocybe aegerita where transmission could not be detected (Barroso & Labarere 2000). The transmission frequencies from A. bisporus are still higher than that found in H. annosum, where the transmission frequencies in two isolates were as low as 10–11%. The reason for this difference is unclear, but the concentration of dsRNA in mycelia and fruit bodies might be a possible explanation. If there is a low concentration of dsRNA in the fruit bodies, there may be less chance for dsRNA to be transmitted into the spores. Another possible explanation for differences in transmission frequencies of dsRNA into basidiospores between species and individuals could be that some fungi might to some extent actively exclude dsRNA from their spores. Basidiospores from fruit bodies containing dsRNA had a significantly lower germination frequency in high nutrient media compared with basidiospores from dsRNA-free fruit bodies, although the presence of dsRNA in the spores did not inhibit germination totally. This is the first phenotypic effect of dsRNA we have seen in H. annosum, but the variation in basidiospore germination frequency among fruit bodies was large even within groups, with or without dsRNA, and this difference could be due to genetic variations between the isolates. Further studies of germination frequency of basidiospores from isogenic strains, with or without dsRNA, could discern whether genetic variation between isolates or presence of dsRNA causes the difference in germination frequency. This study indicates that dsRNA is more common in H. annosum than what was reported by Ihrmark et al. (2001), where 4 % of the isolates contained dsRNA. Many of the isolates in that study were isolated through conidia, but dsRNA was almost exclusively found in isolates that had been isolated as mycelium. Here we found dsRNA in 14 % of tissue isolates from fruit bodies and in 19 % of isolates from decayed wood. None of these isolates had passed through a conidial stage during isolation or subculturing, thus eliminating the risk of the dsRNA having been lost through poor transmission into conidiospores. In the previous study dsRNA was only rarely found in isolates from incipient decayed wood, which led to the hypothesis that basidiospores rarely contain dsRNA, since the incipient decay is often started by basidiospore establishment on fresh stump surfaces. However, the data presented
152 here excludes the hypothesis that basidiospores do not contain dsRNA. Rather, the lack of dsRNA in isolates from incipient decayed wood in the previous study is probably due to the fact that those isolates were all isolated through conidia (Ihrmark et al. 2001). As in earlier studies the dsRNA elements found in this study seem to belong to the virus family Partitiviridae, since most, if not all, of them are two-segmented and sequence data from several of the dsRNAs show strong homologies with partitiviruses (Ihrmark 2001). H. annosum is spread mainly by basidiospores, long and short distances, and vegetatively by mycelium to neighbouring trees (Korhonen & Stenlid 1998). Both means provide an opportunity for dsRNA to disseminate, since dsRNA is transmitted into basidiospores and through anastomoses directly between mycelia (Ihrmark et al. 2002). The effect on spore germination detected in our study slightly reduces the fitness of basidiospores, which limits the possibilities for dsRNA to spread throughout the whole H. annosum population. The role of conidia for spread of H. annosum is unclear (Korhonen & Stenlid 1998). Since the vertical transmission frequency of dsRNA into conidia is lower (Ihrmark et al. 2002) than into basidiospores, conidia are probably of little importance for transmission of dsRNA. ACKNOWLEDGEMENTS This work was financially supported by the Foundation for Strategic Environmental Research (MISTRA). Thanks are due to Hans-Olov Pettersson and Maria Jonsson for assistance with the experiments and Ma˚rten Gustafsson, Kari Korhonen, Halvor Solheim, Vaidotas Lygis and Rimvydas Vasiliauskas for providing isolates.
REFERENCES Barroso, G. & Labarere, J. (2000) Distribution and transmission of Agrocybe aegerita dsRNAs. In Science and Cultivation of Edible Fungi (L. J. L. D. van Griensven, ed.) 1: 271–280. Balkema, Rotterdam. Buck, K. W. (1986) Fungal virology – an overview. In Fungal Virology (K. W. Buck, ed.) : 1–84. CRC Press, Boca Raton. Buck, K. W. (1998) Molecular variability of viruses of fungi. In Molecular Variability of Fungal Pathogens (P. Bridge, Y. Couteaudier & J. Clarkson, eds): 53–72. CAB International, Wallingford. Castanho, B. & Butler, E. E. (1978) Rhizoctonia decline: a degenerative disease of Rhizoctonia solani. Phytopathology 68: 1505–1510. Castanho, B., Butler, E. E. & Shepherd, R. J. (1978) The association of double-stranded RNA with Rhizoctonia decline. Phytopathology 68: 1515–1519. Day, P. R. & Dodds, J. A. (1979) Viruses of plant pathogenic fungi. In Viruses and Plasmids in Fungi (P. A. Lemke, ed.): 201–238. Marcel Dekker, New York. Ghabrial, S. A. (1998) Origin, adaptation and evolutionary pathways of fungal viruses. Virus Genes 16 : 119–131. Ihrmark, K. (2001) Double-stranded RNA elements in the root rot fungus Heterobasidion annosum. PhD thesis. Swedish University of Agricultural Sciences, Uppsala. Ihrmark, K., Johannesson, H., Stenstro¨m, E. & Stenlid, J. (2002) Transmission of double-stranded RNA in Heterobasidion annosum. Fungal Genetics and Biology 36: 147–154.
K. Ihrmark, E. Stenstro¨m and J. Stenlid Ihrmark, K., Zheng, J., Stenstro¨m, E. & Stenlid, J. (2001) Presence of double-stranded RNA in Heterobasidion annosum. Forest Pathology 31 : 387–394. Karjalainen, R. & Fabritius, A. L. (1993) Identification of intersterility groups of Heterobasidion annosum by restriction analysis of PCR products. Journal of Phytopathology 139: 193–200. Korhonen, K. & Stenlid, J. (1998) Biology of Heterobasidion annosum. In Heterobasidion annosum: biology, ecology, impact and control (S. Woodward, J. Stenlid, R. Karjalainen & A. Hu¨ttermann, eds): 43–70. CAB International, Wallingford. Pfeiffer, I., Kucsera, J., Varga, J., Parducz, A. & Ferenczy, L. (1996) Variability and inheritance of double-stranded RNA viruses in Phaffia rhodozyma. Current Genetics 30: 294–297. Romaine, C. P., Ulhrich, P. & Schlagnhaufer, B. (1993) Transmission of La France isometric virus during basidiosporogenesis in Agaricus bisporus. Mycologia 85: 175–179.
153 Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning : a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, New York. Sonnenberg, A. S. M., van Kempen, I. P. J. & van Griensven, L. J. L. D. (1995) Detection of Agaricus bisporus viral dsRNAs in pure cultures, spawn and spawn-run compost by RT-PCR. In Science and Cultivation of Edible Fungi (T. J. Elliott, ed.) 2: 587–594. Balkema, Rotterdam. Stenlid, J. (1985) Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility, and isoenzyme patterns. Canadian Journal of Botany 63: 2268–2273.
Corresponding Editor: D. J. Bond
149
DOI: 10.1017/S0953756203008839 Printed in the United Kingdom.
Double-stranded RNA transmission through basidiospores of Heterobasidion annosum
Katarina IHRMARK, Elna STENSTRO¨M and Jan STENLID Swedish University of Agricultural Sciences, Department of Forest Mycology and Pathology, P.O. Box 7026, SE-750 07 Uppsala, Sweden. E-mail : [email protected] Received 7 May 2003; accepted 28 October 2003.
A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14 % of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10–84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P<0.05) ; germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant ; germination was 3 and 10 % for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.
INTRODUCTION Fungi can be infected by dsRNA mycoviruses, some of which cause negative effects for the host, while others are neutral (Ghabrial 1998). dsRNA mycoviruses in fungi do not leave the cytoplasm and are transmitted intracellularly through spores and hyphal contacts (Ghabrial 1998). Spread through anastomosis is partly restricted by vegetative incompatibility. The efficiency of transmission through spores varies with spore type and taxonomic grouping of the fungi. In ascomycetes, asexual spores can carry dsRNA infection while sexual ascospores normally are free of dsRNA elements (Buck 1986, 1998). In basidiomycetes, transmission into sexual basidiospores occurs readily in Ustilago maydis (Day & Dodds 1979), Rhizoctonia solani (Castanho & Butler 1978, Castanho, Butler & Shepherd 1978), Agaricus bisporus (Romaine, Ulhrich & Schlagnhaufer 1993) and the yeast Phaffia rhodozyma (Pfeiffer et al. 1996), although the frequencies varies considerably (33–100%). However, in Agrocybe aegerita evidence for the transmission of dsRNA to basidiospores is weak or non-existent (Barroso & Labarere 2000), which is contrary to transmission patterns found in other basidiomycetes. An increased frequency of germination and viability of basidiospores infected with
dsRNA has been found in A. bisporus (Romaine et al. 1993). Transmission to conidia has only been studied in Heterobasidion annosum (Ihrmark et al. 2002), where it occurs at a much lower frequency than in most ascomycetes. H. annosum is a tree pathogen that causes root and butt rot in conifers in boreal forests. Spores land on newly exposed wood such as fresh stump surfaces, colonise the stump root system and spread via root contacts into neighbouring healthy trees (Korhonen & Stenlid 1998). H. annosum is a complex of several intersterility (IS) groups. In Northern Europe two IS groups are found, the S and P groups, named after their main host species (spruce and pine, respectively). Karjalainen & Fabritius (1993) have shown that the variation within the ITS (internal transcribed sequence) region can be used to distinguish between the main IS groups by PCR-RFLP. H. annosum has been found to harbour doublestranded RNA (dsRNA) elements with genetic information resembling partitiviruses (Ihrmark 2001, Ihrmark et al. 2001), a common group of mycoviruses. Most of the dsRNA elements in H. annosum seem to be two-segmented with one segment coding for an RNA dependent RNA polymerase (RDRP) with homology to RDRPs from Partitiviridae. dsRNA has mainly been
dsRNA in basidiospores
150
Table 1. Origin of tissue isolates from fruit bodies tested for presence of dsRNA, number of fruit bodies collected and the number of fruit bodies that contained dsRNA.
Origin
Number of fruit bodies
Number of fruit bodies with dsRNA
Fiby, Uppland, Sweden Fredrikslund, Uppland, Sweden Hammarheden, Uppland, Sweden Kvill, Sma˚land, Sweden Lunsen, Uppland, Sweden Omberg, O¨stergo¨tland, Sweden Ra¨mso¨n, Uppland, Sweden Svensho¨ngen, Bohusla¨n, Sweden Ycke, O¨stergo¨tland, Sweden Vilnius forest district, Lithuania
1 3 1 1 22 7 1 1 1 9
0 0 0 0 8 0 0 0 0 1
Total
47
9
found in isolates from decayed wood and only rarely in isolates from incipient decayed wood or in isolates that have recently passed through conidiogenesis (Ihrmark et al. 2001). Furthermore, in an earlier study we found that dsRNA in H. annosum is transmitted efficiently through anastomosis, also between incompatible isolates, and that transmission through conidia is less efficient (Ihrmark et al. 2002). This has led to the speculation that dsRNA is mainly spread horizontally, via anastomosis, in H. annosum and that spores play only a minor role in transmission of dsRNA in this species. The aim of this study was to further investigate presence and transmission of dsRNA in H. annosum. In particular we wanted to study the potential for transmission through basidiospores, which is of considerable interest since H. annosum is mainly established in new areas or on fresh substrates by basidiospores (Korhonen & Stenlid 1998).
MATERIALS AND METHODS Isolates We screened 59 isolates of Heterobasidion annosum from the culture collection of the Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, for dsRNA. The isolates originated from Siaro¨, Sweden (20 isolates), Norway (30) and Russia (9), and were all originally isolated as mycelium from decayed wood. Fruit bodies of H. annosum were collected from different parts of Sweden and Lithuania (Table 1) during Sept.–Oct. 2000. Mycelia were isolated from 47 fruit bodies by placing small pieces from the interior of fresh fruit bodies on Hagem agar (Stenlid 1985) with 50 mg lx1 ampicillin, 50 mg lx1 streptomycin and 5 mg lx1 benomyl. After a few days incubation in the dark at 21 xC the newly outgrown mycelia were transferred to new Hagem agar plates without antibiotics.
Table 2. The transmission frequency of dsRNA into basidiospores from fruit bodies containing dsRNA and germination frequency of basidiospores in water and malt extract. All fruit bodies were from Lunsen, Uppland, Sweden.
Fruit body
dsRNAa
2 3 5 6 11 12 13 15 17 18 19 20 23 Mean valued 8 10 14 16 21 22 25 26 Mean valued
x x x x x x x x x x x x x + + + + + + + +
Germination Germination Transmission in water in malt frequency (%)b (%)c extract (%)c
n.a. n.a. 40 (6/15) 67 (8/12) 10 (2/21) 11 (2/19) n.a. 84 (21/25)
0 11 0 8 2 17 4 7 36 17 26 0 6 10 a n.a. 1 11 n.a. 0 1 3 4 3a
n.a. n.a. n.a. 78 n.a. 94 27 n.a. 59 96 98 94 n.a. 78 b n.a. 30 55 n.a. 14 58 n.a. 15 34 c
+, fruit bodies containing dsRNA; x, dsRNA-free fruit bodies. n.a. : not analysed; in brackets: number of infected basidiospores/number of single basidiospore isolates analysed. c Germination after 24 h among 100 randomly chosen basidiospores, percentage; n.a.: not analysed. d Comparisons of germination frequencies of basidiospores from fruit bodies with and without dsRNA. The means followed by the same letter are not significantly different (P<0.05). a
b
Basidiospores Spores were collected from 33 of the fruit bodies by placing a piece of plastic close to the hymenium for one hour prior to collecting the fruit body. Spores were kept at 4 x during 7–10 d, whereupon the spores were suspended in water and incubated at 21 x for 20–24 h and the proportion of germinated spores was counted under a light microscope. Between 100 and 500 spores were counted from each fruit body. Spores were collected anew twice from Lunsen, Uppland, Sweden. In April 2001, spores were collected from fruit bodies adjacent to five of the fruit bodies that were found to contain dsRNA. The spores were immediately suspended in water and spread onto Hagem agar, incubated 36 h and thereafter wellseparated germinated spores were transferred to new Hagem agar plates. From each fruit body, 12–25 single basidiospore isolates were checked for the presence of dsRNA. In August 2001, spores were again collected from fruit bodies adjacent to twelve of the analysed fruit bodies and both fruit bodies containing dsRNA and dsRNA free isolates were included (Table 2). The spores were immediately suspended in 1.25 % malt
K. Ihrmark, E. Stenstro¨m and J. Stenlid extract medium, incubated at 21 x for 21 h and the proportion of germinated spores was counted as above. Differences in germination frequencies and transmission frequencies were statistically analysed by chisquare tests and one-way analysis of variance. Extraction of dsRNA All isolates were subcultured in 30 ml liquid malt syrup (2 %) in Petri dishes for two to four weeks in the dark at 21 x prior to extraction of nucleic acids. Freeze-dried mycelium, 200–1000 mg of each isolate, was ground to a fine powder with a small quantity of aluminium oxide powder in a mortar and pestle. Nucleic acids were extracted in 6 ml CTAB-buffer (3 % cetyltrimethylammoniumbromide, 2 mM EDTA, 150 mM Tris-HCl, 2.6 M NaCl, pH 8) and 5 ml phenol :chloroform : isoamyl alcohol (49.5: 49.5 :1, pH 8), followed by chloroform extraction and precipitation with isopropanol. The nucleic acids were dissolved in one millilitre STE (0.1 M NaCl, 50 mM Tris-HCl, 1 mM EDTA, pH 8) and dsRNA was separated from other nucleic acids by cellulose CF-11 chromatography as described by Sonnenberg, van Kempen & van Griensven (1995). The dsRNA pellets were dissolved in 30 ml water and treated with single-strand specific S1 nuclease, 330 units mlx1 (Roche, Basel) and DNase I, 330 units mlx1 (Roche), to ensure that only bands consisting of dsRNA would appear on gels. The digestions were conducted in 0.1 M sodium acetate buffer with 50 mM MgSO4 and 5 mM ZnCl2, pH 4.6, at 37 x for 2 h. The dsRNA was precipitated with isopropanol and the pellets dissolved in water. The entire samples were analysed on 1% agarose gels (D-1 ; Hispanlab, Madrid) for 90 min at 4.3 V cmx1 in 0.5rTBE (Sambrook, Fritsch & Maniatis 1989). The gels were stained in ethidium bromide and photographed under UV light. 1 Kb DNA Ladder (Gibco/Invitrogen, Carlsbad, CA) was used on each gel as a size standard. Intersterility group affiliation To certify IS affiliation of all isolates found to contain dsRNA, a PCR-RFLP analysis of the ITS region was performed as described by Ihrmark et al. (2001).
RESULTS dsRNA in isolates Of the 59 isolates from the culture collection that were screened for dsRNA, eight isolates contained dsRNA. Five of the 20 isolates from Siaro¨, Sweden, and three of the 30 isolates from Norway contained dsRNA. None of the isolates from Russia contained dsRNA. All of the dsRNA positive isolates belonged to the S intersterility group of Heterobasidion annosum. Of the 47 fruit body isolates, nine contained dsRNA (Table 1). Eight isolates came from the same area,
151 Lunsen, Uppland, Sweden, and belonged to the S group. The ninth isolate that contain dsRNA came from the Vilnius forest district, Lithuania, and belonged to the P group. Each isolate that contained dsRNA had one or two dsRNA fragments and all fragments were in the range 1.8 to 2.5 kbp. In the isolates yielding two dsRNA fragments, the smaller fragment was present at a lower concentration than the larger fragment, as could be visually seen on the agarose gels. The isolates yielding only one visible dsRNA fragment, all had a very faint dsRNA band and a second fragment might have been below the detection level. dsRNA in basidiospores All five fruit bodies that were used for testing dsRNA transmission frequency to basidiospores contained two dsRNA fragments and both dsRNA fragments were also present in the infected single basidiospore isolates from these fruit bodies. On average, of those basidiospores that germinated, 40 % contained dsRNA, from infected fruit bodies. Presence of dsRNA in germinated basidiospores varied considerably between the five fruit bodies tested. dsRNA was detected in 10, 11, 40, 67 and 84 % of the basidiospore isolates, respectively, from the five fruit bodies. There was no correlation between the proportion of germinated spores from the fruit bodies containing dsRNA and the proportion of germinated spores containing dsRNA (Table 2), i.e. both high and low frequency of transmission of dsRNA to basidiospores could be found from infected fruit bodies with a low basidiospore germination frequency (fruit bodies 26 and 21, respectively) as well as from infected fruit bodies with a high germination frequency (fruit bodies 14 and 22, respectively). Germination of basidiospores Eight of the nine fruit bodies containing dsRNA came from the same area, Lunsen, Uppland, Sweden, and we therefore limited the study of spore germination to fruit bodies from this area, to exclude differences in spore germination due to differences in geographical origin. Of the 22 fruit bodies from Lunsen, 21 produced spores at the time of collection (Table 2). Spore germination in malt extract was higher than in water and varied between 14 and 98 % (Table 2). There was a statistically significant difference in average germination frequency for basidiospores in malt extract from fruit bodies containing dsRNA, 34 %, and for dsRNA free fruit bodies, 78 % (P<0.05). Spore germination in water was low, compared with malt extract, and varied considerably between isolates, from 0 to 36 %, with an average of 6.4 % germinated spores (Table 2). Spores from fruit bodies with dsRNA germinated at a slightly lower frequency than those from dsRNA free fruit bodies, 3 and 10%, respectively, although the difference was not statistically significant.
dsRNA in basidiospores DISCUSSION Vertical transmission of dsRNA to basidiospores has been investigated in only a few species. With this report we present transmission frequency of dsRNA to basidiospores on an average of 40%, in Heterobasidion annosum. This number is lower than for other species that have been investigated, where transmission frequencies have been close to 100 % (Castanho & Butler 1978, Day & Dodds 1979, Pfeiffer et al. 1996). The exception is Agaricus bisporus, where transmission frequencies of 33–100 % (average 65–75 %) have been found (Romaine et al. 1993) and Agrocybe aegerita where transmission could not be detected (Barroso & Labarere 2000). The transmission frequencies from A. bisporus are still higher than that found in H. annosum, where the transmission frequencies in two isolates were as low as 10–11%. The reason for this difference is unclear, but the concentration of dsRNA in mycelia and fruit bodies might be a possible explanation. If there is a low concentration of dsRNA in the fruit bodies, there may be less chance for dsRNA to be transmitted into the spores. Another possible explanation for differences in transmission frequencies of dsRNA into basidiospores between species and individuals could be that some fungi might to some extent actively exclude dsRNA from their spores. Basidiospores from fruit bodies containing dsRNA had a significantly lower germination frequency in high nutrient media compared with basidiospores from dsRNA-free fruit bodies, although the presence of dsRNA in the spores did not inhibit germination totally. This is the first phenotypic effect of dsRNA we have seen in H. annosum, but the variation in basidiospore germination frequency among fruit bodies was large even within groups, with or without dsRNA, and this difference could be due to genetic variations between the isolates. Further studies of germination frequency of basidiospores from isogenic strains, with or without dsRNA, could discern whether genetic variation between isolates or presence of dsRNA causes the difference in germination frequency. This study indicates that dsRNA is more common in H. annosum than what was reported by Ihrmark et al. (2001), where 4 % of the isolates contained dsRNA. Many of the isolates in that study were isolated through conidia, but dsRNA was almost exclusively found in isolates that had been isolated as mycelium. Here we found dsRNA in 14 % of tissue isolates from fruit bodies and in 19 % of isolates from decayed wood. None of these isolates had passed through a conidial stage during isolation or subculturing, thus eliminating the risk of the dsRNA having been lost through poor transmission into conidiospores. In the previous study dsRNA was only rarely found in isolates from incipient decayed wood, which led to the hypothesis that basidiospores rarely contain dsRNA, since the incipient decay is often started by basidiospore establishment on fresh stump surfaces. However, the data presented
152 here excludes the hypothesis that basidiospores do not contain dsRNA. Rather, the lack of dsRNA in isolates from incipient decayed wood in the previous study is probably due to the fact that those isolates were all isolated through conidia (Ihrmark et al. 2001). As in earlier studies the dsRNA elements found in this study seem to belong to the virus family Partitiviridae, since most, if not all, of them are two-segmented and sequence data from several of the dsRNAs show strong homologies with partitiviruses (Ihrmark 2001). H. annosum is spread mainly by basidiospores, long and short distances, and vegetatively by mycelium to neighbouring trees (Korhonen & Stenlid 1998). Both means provide an opportunity for dsRNA to disseminate, since dsRNA is transmitted into basidiospores and through anastomoses directly between mycelia (Ihrmark et al. 2002). The effect on spore germination detected in our study slightly reduces the fitness of basidiospores, which limits the possibilities for dsRNA to spread throughout the whole H. annosum population. The role of conidia for spread of H. annosum is unclear (Korhonen & Stenlid 1998). Since the vertical transmission frequency of dsRNA into conidia is lower (Ihrmark et al. 2002) than into basidiospores, conidia are probably of little importance for transmission of dsRNA. ACKNOWLEDGEMENTS This work was financially supported by the Foundation for Strategic Environmental Research (MISTRA). Thanks are due to Hans-Olov Pettersson and Maria Jonsson for assistance with the experiments and Ma˚rten Gustafsson, Kari Korhonen, Halvor Solheim, Vaidotas Lygis and Rimvydas Vasiliauskas for providing isolates.
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