High Frequency of Finding Double-stranded RNA in Naturally Occurring Isolates of Rhizoctonia solani

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1 J. gen. Virol. (1984), 65, Printed in Great Britain 1601 Key words: dsrna/fungal viruses/hypovirulence/rhizoctonia solani High Frequency of Finding Double-stranded RNA in Naturally Occurring Isolates of Rhizoctonia solani By D. H. ZANZINGER, B. P. BANDY AND S. M. TAVANTZIS* Department of Botany and Plant Pathology, University of Maine, Orono, Maine 04469, U.S.A. (Accepted 31 May 1984) SUMMARY Forty-nine out of 50 field isolates of the basidiomycete Rhizoctonia solani were found to contain dsrna. The presence of dsrna appeared to have no consistent association with the degree of pathogenicity (virulence) of the isolates. These findings contradict previous reports correlating the presence of dsrna with hypovirulence in R. solani. The pathogenic fitness or virulence of certain fungi has been shown to be diminished by factors such as mutant nuclear genes, hybrid nuclei or extrachromosomal genetic factors in the nuclei, or by cytoplasmic factors such as viruses, virus-like particles (VLPs), plasmids or organelles carrying genetic material (Elliston, 1982). The presence of dsrna has been associated with transmissible cytoplasmic hypovirulence (TCH) in fungi (Castanho et al., 1978 ; Day et al., 1977; Lapierre et al., 1970), With the exception of mushroom bacilliform virus (Tavantzis et al., 1980, 1983), all mycoviruses possess dsrna genomes (Bozarth, 1979). Naturally occurring hypovirulent strains of Endothia parasitica, the causal agent of chestnut blight, have been found to reduce the impact of virulent strains of the pathogen in Europe (Grente & Berthelay-Saurat, 1979). Double-stranded RNA and TCH are transmitted simultaneously during hyphal anastomosis between a virulent and a hypovirulent, dsrna-containing strain of E. parasitica (Anagnostakis & Day, 1979). However, evidence that dsrna may be the cause of hypovirulence in E. parasitica remains circumstantial. Cell-free transmission of dsrna into virulent strains has not been accomplished (Van Alfen, 1982). Pleomorphic, club-shaped particles have been associated with dsrna found in E. parasitica (Dodds, 1980). Rhizoctonia solani, an important pathogen of numerous chlorophyllous plant species, was found to have TCH similar to that of E. parasitica (Castanho & Butler, 1978 a, b; Castanho et al., 1978). The presence of dsrna in R. solani was associated with hypovirulence, expressed as a loss of dark pigmentation in the mycelium, a slower rate of growth, and the production of fewer sclerotia (isolate 189a). Healthy cultures lacking dsrna could be recovered at a low frequency by hyphal-tip isolation (isolate 189HT5). The hypovirulence condition as well as the dsrna were transmissible from 189a to 189HT5 but not to other isolates of the same anastomosis group (AG 1) (Castanho et al., 1978). The above findings were the motivation for us to study further the association of dsrna with hypovirulence in /L solani. We report here that dsrna is common in a given population of field isolates of R. solani, and that the presence of dsrna is not always associated with hypovirulence. Preliminary data of this study have been presented previously (Zanzinger et al., 1983). The procedure of Henis et al. (1978), and the selective medium of Ko & Hora (1970) were used for the isolation of R. solani from soil samples obtained from Maine potato fields. Nuclear staining with aniline blue (0"5~o, w/w, in lactophenol) (Herr, 1979) was used to differentiate multinucleate R. solani from binucleate Rhizoctonia-like fungi. Anastomosis grouping of the R. solani field isolates was conducted according to Parmeter et al. (1969) using known tester isolates. Fifty out of 60 isolates were multinucleate with mycelial characteristics of R. solani, and were distributed among AGs as follows: five in AG 1, eight in AG 2, 11 in AG 3, 13 in AG 4 and nine in AG 5. Four isolates failed to anastomose with any tester (Bandy et al., 1984). Doublestranded RNA was isolated from fungal mycelium essentially as described by Morris & Dodds /84/ $ SGM

2 1602 Short communication (a) (b) (c) t'.- Fig. 1. Ethidium bromide-stained bands of dsrna mol. wt. standards (S) from Helminthosporium maydis (Hm 9, 5.7 x 106), Penicilliumchrysogenum(PeV, ), P. stoloniferum(psv, 1-0 x l06 and 0.46 x 106) and dsrna components of the following R. solani isolates: (a) 23 and 52 (AG 1), 7 and 25 (AG 4), 1 and 53 (AG 5); (b) 28, 57 and 58 (AG 2), 14 and 43 (AG 3), 26 (AG 4); (c) 27 (AG 3) and 3, 4, 32, 33, 35 and 39 (AG 4). The numbers on the left are the mol. wt. (x 10-6) of the dsrna standards. (1979). Mycelia of a known hypovirulent (ATCC 38771), dsrna-containing, and a known virulent (ATCC 22508), dsrna-free strain of E. parasitica, grown and extracted in the same manner as R. solani, were used as controls for the d s R N A isolation and characterization procedures. Aspergillusfoetidus (ATCC 10254), and reovirus serotype 3 (strain Dearing) were also used as sources of d s R N A controls. Prior to electrophoresis, samples were treated with pancreatic DNase I (free of RNase; Worthington) and pancreatic RNase A at high ionic strength (0.3 M-NaC1). Electrophoresis was in 2. 4 ~ polyacrylamide gels containing 0"5~o agarose (Tavantzis et al., 1980). Molecular weights of d s R N A species were determined with an accuracy of +5~ as described by Bozarth & Harley (1976) using mol. wt. standards obtained from reovirus, A. foetidus (ATCC 10254), or a set of d s R N A markers donated by R. F. Bozarth. Some gels were treated with nucleases after electrophoresis (Morris & Dodds, 1979). Pathogenicity and/or virulence of the R. solani isolates was determined in greenhouse tests. Inocula were 10-day-old fungal cultures grown on sterile wheat or corn seed, and were mixed into the top half layer of autoclaved sterile soil mixture (Metromix) in plastic pots. Seed of the appropriate host of each A G (Anderson, 1982) was planted 1 to 2 cm deep, and damping-off was determined 2 to 4 weeks after planting. Controls, treated with uninoculated wheat or corn seed, or with known virulent isolates (of the same AG) were included in each experiment. The degree of pathogenicity of the different R. solani isolates was determined by the percentage of dampingoff. Forty-nine of the 50 isolates of R. solani were found to contain detectable dsrna. Several isolates contained six, seven or eight d s R N A segments. A representative analysis, which includes members from all AGs, is shown in Fig. 1. Repeated extractions and electrophoretic analysis of d s R N A from a small number of isolates resulted in identical banding patterns. Nuclease treatment, alkali treatment (0.4 M-KOH at 20 C for 18 h) and thermal melting all showed the nucleic acid segments to be dsrna. The amount of purified d s R N A per 20 g of fresh

3 Short communication 1603 Table 1. Frequency and mol. wt. of the most commonly occurring dsrna segments in the different anastomosis groups of R. solani Anastomosis Mol. wt. group ( x 10-6) Frequency* AG /5 AG / / /8 AG / / / /11 AG / / /13 AG /9 * Number of isolates possessing the designated dsrna segment over the dsrna content. number of isolates analysed for Table 2. Comparison of dsrna content, growth, and virulence of eight AG 4 members of R. solani Growth R. solani at 24 C Virulence* Mol. wt. of dsrna isolate (mm) (% damping-off) segments (x 10 6) Rs >5.7,4.3,1.5,1.3,1.1,0.5 Rs ,3.1,1.4,1.3,1-1,1-0 Rs ,1.6,1.4,1.3,1.1,0.6 Rs ,1.4,1.2 Rs ,1.5,1-4,1.2,1.1,1.0 Rs ,3.1,1.2, < 0.5 Rs ,3.1,1.6,1.4,1.2,1.1, <0.5 Rs ,1.6,l.4 * Phaseolus vulgaris is a compatible host of AG 4 (Anderson, 1982). Thirty surface-sterilized seeds of the cultivar 'Topcrop' were planted in sterilized soil mix, which had been inoculated with each isolate. Virulence was estimated as percent damping-off relative to the number of emerged, uninoculated plants (26 out of 30). Percent damping-off caused by known pathogenic AG 4 strains ranged from 12 to 67. The relative virulence of the above isolates remained the same when it was expressed on the basis of size and severity of stem lesions. A highly virulent and a less virulent AG 4 isolate (Rs 4 and Rs 33, respectively) caused discrete areas of superficial discoloration in the hypocotyl tissue of potato, which is an incompatible host of AG 4 strains (Reynolds et al., 1983). mycelial tissue varied from 0 to 125 Bg depending on the isolate, and was not related to the degree of virulence. The dsrna segments occurring in the various R. solani isolates were classified into three mol. wt. categories, which ranged from less than 0.46 x 106 to greater than 5.7 x 106. Ten percent were less than (small), 49~ were between and (medium) and 41 ~ were greater than 2.8 x 106 (large). The frequency of the major dsrna components and their mol. wt. are presented in Table 1. Double-stranded RNA segment(s) with an approximate mol. wt. of were the most frequent and were found in members of AG 2, AG 3 and AG 4. Table 2 shows a representative sample of isolates, members of AG 4, which all produced greyish-white colonies and similar numbers of sclerotia. Growth and relative degree of pathogenicity (virulence) of the above isolates were fairly consistent. The isolates were similar in their growth at 24 C, but the percent damping-off (i.e. virulence) caused by these isolates varied from 0 to almost 50. However, more than one segment of dsrna was detected in each isolate. Thus, the hypothesis that the presence of dsrna is associated with hypovirulence in R. solani (Castanho et al., 1978) appears to be incorrect. The only isolate (Rs 29) that contained no

4 1604 Short communication detectable dsrna, was an AG 2 member, had a growth rate higher than average, a normal colony appearance, and virulence comparable to that of other dsrna-containing, AG 2 field isolates. The conclusion that the size of dsrna segments is 'isolate-specific' (Castanho et al., 1978), based on the dsrna content of three isolates, also proved to be premature. Our data showed that a number of dsrna segments of various mol. wt. are commonly found among isolates of the same or different AGs (Table 1). Furthermore, the frequency of dsrna being present in R. solani is much higher (49 out of 50 isolates) than that in the previous study (three out of 13 isolates) (Castanho et al., 1978). In this respect, our data agree with reports on the ubiquitous occurrence of dsrna or virus-like particles in Agaricus bisporus (Tavantzis & Smith, 1979; Ushiyama, 1979) or Gaeumannomyces graminis var. tritici (Rawlinson et al., 1973), as well as on the lack of a consistent association between the presence of dsrna and a reduction in fungal vigour or virulence. Elliston (1978) reported that at least one strain (EP 103) of E. parasitica, found in numerous tests to contain dsrna, had culture characteristics and virulence comparable to those of normal, dsrna-free strains. Frick & Lister (1978) found serotype variation in VLPs obtained not only from G. graminis isolates of different geographical origins, but also from isolates taken from the same field in the same year. They suggested that "the frequency and diversity of such serotype variation may reflect similar biotype variation in these viruses and could offer an explanation for inconsistencies in reports of the association of G. graminis viruses with fungal virulence." Results from work with A. bisporus (Tavantzis & Smith, 1979), E. parasitica (Elliston, 1978), G. gram&is (Lapierre et al., 1970; Buck et al., 1981), and R. solani (this report) lend support to the above hypothesis. Double-stranded RNA-containing isometric VLPs, 33 nm in diameter, have been purified from an isolate of R. solani (S. M. Tavantzis & B. P. Bandy, unpublished results). Based on the above evidence, as well as observations of the centrifugation behaviour of dsrna-containing particles in other isolates, including 189a (Castanho et al., 1978), we believe that all dsrna in R. solani is of viral nature. Data on the purification and characterization of VLPs from R. solani will be reported in a subsequent paper. We thank Drs D. K. Bell and E. E. Butler for providing us with the R. solani AG testers, Dr R. F. Bozarth for the dsrna mol. wt. standards, Dr J. E. Elliston for the E. parasitica cultures, and Dr W. K. Joklik for the reovirus. This work was supported by USDA Grant 82-CRSR REFERENCES ANAGNOSTAKIS, S. L. & DAY, P. R. (1979). Hypovirulence conversion in Eadothiaparasitiea. Phytopathology 69, ANDERSON, N. A. (1982). The genetics and pathology ofrhizoctonia solani. Annual Review of Phytopathology 20, BANDS', S. P., ZANZINGER, D. H. & TAVANTZIS, S. M. (1984). Anastomosis group 5 of Rhizoetonia solani Kuhn isolated from potato-cultivated soils in Maine. Phytopathology (in press). BOZARTH, R. F. (1979). The physicochemical properties of mycoviruses. In Viruses andplasmids in Fungi, pp Edited by P. A. Lemke. New York: Marcel Dekker. BOZARTH, g. F. & HARLEY, E. H. (1976). The electrophoretic mobility of double-stranded RNA in polyacrylamide gels as a function of molecular weight. Biochimiea et biophysica acta 432, BUCK, K. W., ALMOND, M. R., McFADDEN, J. J. P., ROMANOS, M. A. & RAWLINSON, C. J. (1981). Properties of thirteen viruses and virus variants obtained from eight isolates of the wheat take-all fungus, Gaeumannomyces gram&is var. tritici. Journal of General Virology 53, CASTANI-IO, B. & BUTLER, E. E. (1978a). Rhizoctonia decline: a degenerative disease of Rhizoetonia solani. Phytopathology 68, CASTAtqnO, B. & BUTLER, E. E. (I978b). Rhizoctonia decline: studies on hypovirulence and potential use in biological control. Phytopathology 68, CASTANItO, B., BUTLER, E. E. & SHEPHERD, R. J. (1978). The association of double-stranded RNA with Rhizoctonia decline. Phytopathology 68, DAY, P. R., DODDS, J. A., ELLISTON, J. E., JAYNES, R. A. & ANAGNOSTAKIS, S. L. (1977). Double-stranded RNA in Endothia parasitica. Phytopathology 6"/, DODOS, J. A. (1980). Association of type 1 viral-like dsrna with club-shaped particles in hypovirulent strains of Endothia parasitica. Virology 107, ELLISTON, J. E. (1978). Pathogenicity and sp0rulation of normal and diseased strains of Endothia parasitica. In Proceedings of the American Chestnut Symposium, pp Edited by W. L. MacDonald, F. C. Chech, J. Luchok & C. Smith. Morgantown: West Virginia University Press.

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