Attenuated Isolates of Cucumber Mosaic Virus Produced by Satellite RNA and Cross Protection between Attenuated Isolates and Virulent Ones

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1 Attenuated Isolates of Cucumber Mosaic Virus Produced by Satellite RNA and Cross Protection between Attenuated Isolates and Virulent Ones Kouji YOSHIDA*, Tadanori GOTO* and Norio IIZUKA* Key Words: attenuated cucumber mosaic virus, satellite RNA, cross protection, tomato necrosis. Cucumber mosaic virus (CMV) has four major RNA segments, which are named RNA 1, 2, 3 and 4 in order of decreasing molecular weight. The largest three RNAs are essential for the manifestation of the infectivity of the virus, and RNA 4 is a subgenomic fragment of RNA 3. Recently, the existence of another low molecular weight satellite RNA, i.e. the fifth RNA, associated with the replication of CMV has been reported1-8). Kaper and Waterworth4) showed that CMV-S containing the satellite RNA (CARNA 5) induced lethal necrotic symptoms on tomato, and Takanami7) also reported striking changes in the symptoms on CMV-infected plants by the addition of the satellite RNA. Mossop and Francki6) showed that Sat-RNA attenuated symptoms of CMV on Rutgers tomato plants. In 1982, necrotic disease of tomato plants (Fig. 1) was observed in Ohno and Nanae, southern Hokkaido, and an isolate of CMV, designated as CMV-P(n), was found to be the causal agent of the disease9). A small RNA was detected among the RNAs of CMV- P(n)10), and it had a satellite nature similar to that of CARNA 54) or satellite RNA7) previously reported. This satellite RNA which was isolated from CMV-P(n), and designated as (n)rna 5, caused necrosis on tomato plants only when it was simultaneouly inoculated with satellite-free CMV. Another satellite RNA, designated (fl)rna 5, was obtained from CMV-PF(fl) which induced fern-leaf symptoms on tomato plants10) (Fig. 2). In this case, however, the satellite RNA caused an attenuation of the symptoms on tomato plants induced by CMV when it was simultaneously inoculated with satellite-free CMV. The present paper dealt with the cross protection between the attenuated isolates containing (fl)rna 5 and the virulent ones in order to determine whether the attenuated isolates can be used for controlling CMV on tomato plants. 1) Gonsalves, D., Provvidenti, R. and Edwards, M.C. (1982). Phytopathology 72: ) Gould, A.R., Palukaitis, P., Symons, R.H. and Mossop, D.W. (1978). Virology 84: ) Kaper, J.M., Tousignant, M.E. and Lot, H. (1976). Biochem. Biophys. Res. Commun. 72: ) Kaper, J.M. and Waterworth, H.E. (1977). Science 196: ) Kaper, J.M., Tousignant, M.E. and Thompson, S.M. (1981). Virology 114: ) Mossop, D.W. and Francki, R.I.B (1979). Ibid. 95: ) Takanami, Y. (1981). Ibid. 109: ) Waterworth, H.E., Kaper, J.M. and Tousignant, M.E. (1978). Science 204: ) Goto, T., Yoshida, K. and Iizuka, N. (1984). Ann. Phytopath. Soc. Japan 50: ) Yoshida, K., Goto, T. and Iizuka, N. (1984). Ibid. 50:

2 Ann. Phytopath. Soc. Japan 51 (2). April, Table 1. Symptoms induced on tomato plants inoculated with CMV isolates with or without satellite RNA designated as (fl)rna 5 a) YM; severe yellow mosaic, M; severe mosaic, st; stunting, nl; slightly narrow leaflets, m; mild mosaic, -; no symptoms, (); symptoms occasionally present. Table 2. Cross protection tests between the attenuated isolates produced by adding (fl)rna 5 and virulent isolates of CMV a) M; severe mosaic, m; mild mosaic, SN; stem necrosis, D; died, st; stunting, nl; slightly narrow leaflets, -; no symptoms, (); symptoms occasionally present. b) -; no virus inoculation. c) Number of plants which showed symptoms 30 days after inoculation with challenge virus, out of 6 tested.

3 Table 3. Cross protection tests between the attenuated isolates CMV-P (No.2) containing (fl) RNA 5 and virulent isolates of CMV a) Interval between the primary inoculation and the challange inoculation. b) M; severe mosaic, SN; stem necrosis, -; no symptoms. c) -; no virus inoculation. d) Number of plants which showed symptoms 35 days after inoculation with challenge virus, out of 9 tested. Tomato plants were inoculated with crude extract from tobacco tissues infected with CMV and maintained in a greenhouse. The isolates of CMV used in this experiment were as follows: (1) CMV-Y, isolated from tobacco supplied by Dr. Kiriyama, (2) CMV- O (No.138), isolated from azuki bean11), (3) CMV-P (pepper), isolated from chilli pepper12), (4) CMV-P (n) and CMV-PF (fl), isolated from tomato10), (5) CMV-P (No.2) and CMV-P (No.7), derived from CMV-P(n) by single lesion isolation. These isolates were propagated in Nicotiana tabacum cv. Xanthi-nc and purified by the method of Tsuchizaki13). Satellite RNA was extracted from purified virus preparations by polyacrylamide slab gel electrophoresis according to Loening14) and recovered by phenol extraction excised band by staining with toluidine blue O15). Comparison of the symptoms induced on tomato plants (cv. Fukuju II) infected with satellite-free isolates of CMV and attenuated isolates containing (fl) RNA 5 is shown in Table 1. Tomato plants inoculated with satellite-free isolates of CMV developed severe symptoms, while the plants inoculated with three isolates of CMV-P containing (fl) RNA 5 showed only mild or no symptoms. Another experiment was conducted in which tomato plants were inoculated with each of three isolates of CMV-P (pepper), CMV-P (No.2) or CMV-P (No.7), with or without (fl)rna 5, as a primary virus, and subsequently inoculated with either isolate of CMV-O (No.138) or CMV-P (n) as a challenge virus 12 days later. All plants inoculated with the primary virus of any of the isolates failed to show mosaic symptoms except occasional mild mosaic symptoms, in spite of the inoculation with the challenge virus, CMV-O (No.138). The plants inoculated with CMV-P (pepper), CMV-P (No. 2) or CMV-P (No. 7) containing (fl)rna 5 also showed no necrotic symptoms, in spite of the inoculation with the challenge virus, CMV- 11) Yoshida, K. and Tsuchizaki, T. (1979). Ibid. 45: ) Goto, T., Tsuchizaki, T. and Iizuka, N. (1981). Ibid. 47: ) Tsuchizaki, T. (1973). Ibid. 39: ) Loening, U.E. (1969). Biochem. J. 113: ) Peden, K.W.C. and Symons, R.H. (1973). Virology 53: ) Tien, P. and Chang, X.H. (1984). Abstracts of Sixth International Congress of Virology (Sendai) 379.

4 Ann. Phytopath. Soc. Japan 51 (2). April, P (n) (Fig. 3). Three isolates of CMV-P (pepper), CMV-P (No.2) or CMV-P (No.7) which contained no (fl) RNA 5, however, were not protected from the infection with CMV-P (n) (Table 2). The plants inoculated with CMV-P (No.2) containing (fl)rna 5 showed no symptoms when they were inoculated with CMV-O (No.138) or CMV-P (n), at 12, 35 and 60 days after the primary inoculation (Table 3). These results suggest that the isolates of satellite-free CMV changed to attenuated ones by addition of satellite RNA, (fl) RNA 5, which induced only mild or no symptoms on tomato plants, and the infected plants were protected from the infection with virulent CMV. Very similar experiments were reported in the sixth International Congress of Virology held at Sendai in September 1984 from China by Thien and Chang16) in which they used the attenuated isolates with satellite RNA for control CMV on pepper plants. Although many problems, such as possible existence of variation of attenuated isolate or protection against other virulent isolates of CMV and the symptoms of other plants infected with attenuated isolate, still remain to be revealed, it appears that the satellite RNA, (fl) RNA 5, could be used practically as an attenuated virus to control CMV on tomato plants. We thank Dr. M. Ichinohe for help in preparation of the manuscript. Explanation of plate Plate I Fig. 1. Tomato necrosis disease, Symptoms on tomato cultivar Fukuju II. Fig. 2. Tomato (cv. Fukuju II) inoculated with CMV-PF (fl) showing fern-leaf symptoms. Fig. 3. Effect of an attenuated isolate, CMV-P (No.2) with (fl) RNA on tomato cultivar Fukuju II. Left: Tomato plants pre-inoculated with attenuated isolate and postinoculated with CMV-P (n). Right: Tomato plants inoculated with CMV-P (n).

5 Plate I