DNA Base Sequence Homology in Rhizoctonia solani Kuhn IV. Genetic Relatedness within AG-4

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1 DNA Base Sequence Homology in Rhizoctonia solani Kuhn IV. Genetic Relatedness within AG-4 Shiro KUNINAGA* and Ryozo YOKOSAWA* Abstract DNA base sequence homology among AG-4 isolates of Rhizoctonia solani was determined by DNA-DNA reassociation kinetics. Six of 14 isolates examined shared high homology ranging from 88.5 to 92.6% [AG-4 homogeneous group I (HG-I)]. There was also a high homology among the remaining 8 isolates, ranging from 88.7 to 101.3% [AG-4 homogeneous group II (HG-II)]. Relatively low values (30.9 to 47.9%) of DNA homology were found when the above two groups were compared. The genetic divergence between the two groups was also clearly demonstrated by the electrophoretic patterns of proteins and cultural appearances. These results indicate that AG-4 involved two types of isolates that are genetically divergent. (Received December 12, 1983) Key Words: Rhizoctonia solani (AG-4), DNA homology, genetic relatedness. Introduction A previous study7) demonstrated that anastomosis group 2-1 (AG-2-1) of Rhizoctonia solani Kuhn includes isolates that are genetically homogeneous. This was also true for AG-3, AG-5, AG-7 and AG-BI8) and for the genetically divergent isolates within the respective groups AG-16) and AG-2-27). Several researchers examined genetic relationships among isolates within the AG of R. solani by biochemical analysis. Adams & Butler1), who studied serologic comparisons, reported genetical uniformity among isolates in AG-4. Matsuyama et al.13) reported that isolates of this AG gave two kinds of esterase zymogram (Zym-4A and Zym-4B). More recently, Reynolds et al.16) reported that AG-4 isolates do not show homologous protein patterns in electrophoresis. In this study we attempted to clarify the genetic relationship among isolates within AG-4 on the basis of DNA-DNA reassociation kinetics. A preliminary report was published9). Materials and Methods Isolates. Fourteen isolates of AG-4 were used. The origins of these isolates are

2 Ann. Phytopath. Soc. Japan 50 (3). July, Table 1. Isolates of Rhizoctonia solani used given in Table 1. DNA homology. Methods for fungal cultivation, preparation of DNAs, measurement of melting temperature (Tm) of DNAs, shearing of DNAs and determination of DNA fragment size as well as for calculation of DNA homology values were described in previous papers5,6). DNA-DNA reassociation kinetics were observed in 5 ~SSC containing 20% DMSO at a temperature about 25C below Tm. Protein extraction and electrophoresis. Six-day-old mycelial mats were homogenized in 0.05M Tris-HCl buffer (ph 7.5) at 4C. This mixture was centrifuged for 30 min. at 16,000 ~g. The protein concentration in the supernatant solution was determined by the method of Lowry et al.10) This sample was subjected to polyacrylamide gel disc electrophoresis. Three hundred Đg of protein was layered onto a polyacrylamide gel tube, 10 ~0.5cm in size and electrophored at a constant current of 3mA/tube for about 90min. After electrophoresis, gels were stained for 6hrs in a mixture of ethyl alcohol: acetic acid: water (9:2:9, v/v) containing 0.25% of Coomassie brilliant blue, then destained by rinsing with several changes of ethyl alcohol: acetic acid: water (25:8:65, v/v). Results Thermal denaturation of DNA of AG-4 isolates The Tm of DNA of AG-4 isolates was examined to determine the optimum reaction temperature for DNA-DNA reassociation studies. Thermal denaturation was observed in 5 ~SSC containing 20% DMSO. Fig. 1 shows the thermal denaturation profiles of DNAs of representative isolates. Degrees of hyperchromicity of DNAs of AG-4 isolates ranged from 34.2 to 40.1%. The Tm values and base compositions of the DNAs are presented in Table 2. DNA base compositions of isolates in AG-4 showed no significant differences.

3 Fig. 1. Thermal denaturation profiles of DNA of AG-4 isolates of Rhizoctonia solani and Escherichia coli in 5 ~SSC containing 20% DMSO. Bar designates Tm value. Table 2. Thermal denaturation and base compositions of DNA of AG-4 isolates of Rhizoctonia solani a) The temperature corresponding to the midpoint of absorbance rise (Marmur & Doty, 1962)11). b) E. coli DNA (GC mole %=50) was used as a standard. The base composition of R. solani DNA was calculated on an equation modified from Marmur & Doty (1968)11) : GC mole %=50+(Tm R. solani DNA-Tm E. coil DNA/0.41). c) Base composition determined by chemical analysis (Kuninaga & Yokosawa, 1980)5).

4 DNA homology among AG-4 isolates Ann. Phytopath. Soc. Japan 50 (3). July, DNA homology was first examined by comparing DNAs of 6 isolates selected based on their cultural characteristics. Table 3 shows the cot1/2 values and DNA homology values among these isolates. High DNA homology (88.5 to 91.7%) was found among 3 isolates (AH-1, Chr-3 and 78-23R-3). This group was classified as AG-4 homogeneous group I (HG-I). We also observed high DNA homology (91.1 to 99.5%) among the remaining 3 isolates RR5-2, Rh-165 and HI521-21, and this group was classified as AG-4 HG-II. Comparatively low values of DNA homology (33.3 to 47.9%) were observed when the above two groups were compared. Fig. 2 and Fig. 3 show representative profiles of DNA-DNA reassociation kinetics. The homology of DNAs of eight other isolates with the two groups was examined. These results are presented in Table 4. R97, R101 and GM-3 showed high homology values (90.4 to 96.2%) with isolates from AG-4 HG-I, but low homology (31.6 to 41.2 %) with isolates from AG-4 HG-II. UHBC, 77-20R-1, Rh-131, Rh-154 and Rh-317 showed high homology values (88.7 to 101.3%) with isolates from AG-4 HG-II, but low Table 3. Reassociation kinetics and DNA homology among isolates of Rhizoctonia solani AG-4 a) All reactions were carried out in 5 ~SSC containing 20% DMSO at 62C. b) The range represents results from three determinations. c) Homology values were calculated from the equation by Seidler & Mandel (1971)17), and are average of three determinations.

5 Fig. 2. Reassociation kinetics of the mixture of DNA from isolates of Rhizoctonia solani AG-4. All reactions were carried out in 5 ~SSC containing 20% DMSO at 62C. homology (30.9 to 42.1%) with isolates from AG-4 HG-I. Therefore, R97, R101 and GM-3 were classified as AG-4 HG-I. UHBC, 77-20R-1, Rh-131, Rh-154 and Rh-317 were classified as AG-4 HG-II. Protein similarity among AG-4 isolates Electrophoretic patterns of soluble protein among AG-4 isolates were compared. We found similar electrophor etic profiles among AG-4 HG-I isolates and among AG-4 HG-II isolates, respectively. Distinct differences were found between these two groups. As shown in Fig. 4, deeply stained protein bands were observed at Ef 0.8 to 0.95 (cathode side) in AG-4 HG-I and at Ef 0.5 to 0.75 in AG-4 HG-II. Fig. 3. Reassociation kinetics of the mixture of DNA from isolates of Rhizoctonia solani AG-4. All reactions were carried out in 5 ~SSC containing 20% DMSO at 62C

6 Ann. Phytopath. Soc. Japan 50 (3). July, Table 4. DNA homology among isolates of Rhizoctonia solani AG-4 a) Homology values are average of two determinations. All reactions of DNA-DNA reassociation kinetics were carried out in 5 ~SSC containing 20% DMSO at 62C. Fig. 4. Electrophoretic profiles of mycelial proteins from AG-4 isolates of Rhizoctonia solani. Cultural appearance of AG-4 isolates As shown in Fig. 5, distinct differences were consistently observed between AG-4 HG-I and AG-4 HG-II with respect to colony colour on potato dextrose agar. AG-4 HG-I isolates (R101, Chr-3 and AH-1) were dark brown while AG-4 HG-II isolates (RR 5-2, UHBC and HI521-21) were grey to whitish brown. Discussion Adams & Butler1) reported that AG-4 in R. solani is a genetically uniform group. Also Anderson2) could not detect subgroups in this anastomosis group. The results of this study, however, indicate that AG-4 involves two groups with a distinct partial non-homology in DNA base sequence. The two groups detected were named AG-4 HG-I

7 Fig. 5. Cultural appearance of AG-4 isolates of Rhizoctonia solani grown on potato dextrose agar for 14 days at 28C. AG-4 HG-I isolates-top row (left to right): R101, Chr-3 and AH-1 AG-4 HG-II isolates-bottom row (left to right): RR5-2, UHBC and HI and AG-4 HG-II. Previous investigations6,7) reported that AG-1 and AG-2-2 comprise genetically divergent isolates of which DNA homology is 49.9 to 55.9% in AG-1 and 68.6 to 71.5% in AG-2-2, respectively. In this study, lower homology values (30.9 to 47.9%) were observed between AG-4 HG-I and AG-4 HG-II isolates. The evidence of remarkable divergence among isolates in AG-4 was also obtained from electrophoretic comparison of their mycelial soluble proteins. Although homologous protein pattern was observed within each of the two divergent groups (AG-4 HG-I and AG-4 HG-II), definite differences were observed between the two groups. Protein polymorphism among AG-4 isolates was also reported in studies by Reynolds et al.16) Our experiments may find support from the fact that AG-4 HG-I and AG-4 HG-II exhibit considerable base substitutions of DNAs. Previous studies6,7) demonstrated that the classification of groups in terms of DNA homology is consistent with the grouping based on the cultural characteristics reported by Matsuda & Watanabe19). The two divergent groups in AG-4 were also easily distinguished by their cultural appearances. In a previous publication9) we observed differences of mycelial growth rate and pathogenicity in AG-4 HG-I and AG-4 HG-II. These results may be understood on the basis that the phenotypic expressions of isolates in R. solani depend on nucleotide sequences of their DNAs. Further studies on phenotypic characteristics of isolates in AG-4 HG-I and AG-4 HG-II, however, may be

8 Ann. Phytopath. Soc. Japan 50 (3). July, needed to establish these points. In this study, AG-4 HG-I and AG-4 HG-II isolates were similar in DNA base compositions (GC contents). Other studies6,7) also detected different DNA homology values of isolates in AG-1 or AG-2-2. On the other hand, GC contents of isolates in each of these groups were not different. Baharaeen et al.3) reported similar results among strains of Cryptococus vishnicicii. These indicate that DNA base composition could not be used as a quantitative measure of divergence although it is useful as an initial and approximative indicator of relatedness. Little is known about the relationship between the phylogenic classification based on DNA homology and systematic taxonomy in fungi. The investigations suggest that AG-4 isolates of R. solani with less than about 35% homology values should be regarded as belonging to different AGs. This argument may be supported by an equivalent suggestion that gnegative homology values h (25 to 30%) indicate the presence of different species in yeast strains4,12,15,18). On the other hand, a previous study has shown that homology value slightly above 35% was found in comparing two different AGs (AG-2-1 and AG-2-2) which were subdivided from AG-2 because of frequency of anastomosis and other cultural characteristics14). Further studies on DNA homology among other AGs of R. solani will be required for establishing relationships among isolates of AG-4. The authors are grateful to Dr. A. Ogoshi, Hokkaido University, and Dr. R. Baker, Department of Botany and Plant Pathology, Colorado State University, for critical reading of this manuscript. The authors also thank Dr. T. Naiki, Gifu University, and Dr. S. Naito, Hokkaido National Agricultural Experiment Station, for kind supply of isolates of R. solani. Literature cited 1. Adams, G.C. Jr. and Butler, E.E. (1979). Phytopathology 69: Anderson, N.A (1982). Ann. Rev. Phytopathol. 20: Baharaeen, S., Bantle, J.A. and Vishniac, H.S. (1982). Can. J. Microbiol. 28: Fiol, J.B. and Poncet, S. (1980). Mycopathologia 70: Kuninaga, S. and Yokosawa, R. (1980). Ann. Phytopath. Soc. Japan 46: Kuninaga, S. and Yokosawa, R. (1982). Ibid. 48: Kuninaga, S. and Yokosawa, R. (1982). Ibid. 48: Kuninaga, S. and Yokosawa, R. (1983). Ibid. 49: Kuninaga, S. and Yokosawa, R. (1984). Ibid. 50: 100 (Abstr.). 10. Lowry, O.H., Rosebrough, H.J., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chem. 193: Marmur, J. and Doty, P. (1962). J. Mol. Biol. 5: Martini, A. and Phaff, H.J. (1973). Ann. Microbiol. 23: Matsuyama, N., Moromizato, Z., Ogoshi, A. and Wakimoto, S. (1978). Ann. Phytopath. Soc. Japan 44: Ogoshi, A. (1976). Bull. Natl. Inst. Agric. Sci. Series C. 30: 1-63 (In Japanese). 15. Price, C.W., Fuson, G.B. and Phaff, H.J. (1978). Microbiol. Rev. 42: Reynolds, M., Weinhold, A.R. and Morris, T.J. (1983). Phytopathology 73: Seidler, R. and Mandel, M. (1971). J. Bacteriol. 106: van der Walt, J.P. and Johannsen, E. (1979). Antonie van Leeuwenhoek 45: Watanabe, B. and Matsuda, A. (1966). Appointed Experiment (Plant Diseases and Insect Pests). No.7, Agriculture, Forestry and Fishery Research Council and Ibaraki Agric. Exp. Sta., pp. 131 (In Japanese).

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