Studies on the asparagus-bean mosaic virus

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1 Studies on the asparagus-bean mosaic virus Toshihiko HINO* I. Introduction The mosaic disease of asparagus-bean (Vigna sesquipedalis) and cowpea (V. sinensis) is widely distributed in every section of Japan where Vigna culture is done, and is prevalent at the end of growing season. As for the mosaic disease of these species in Japan, the first report concerning the symptoms was published in ). The studies on the seed transmission of the causal virus gave different results; its high transmissibility was reported on one hand9) and negaitve result was obtained on the other hand16). It was also reported that Cucumber Mosaic Virus was never isolated from mosaic-diseased Vigna10). The author already presented a brief account of the studies on the virus causing mosaic of asparagus-bean and cowpea in his preliminary report8). The present report deals with a full detail of the results obtained from that time on. The author wishes to express his appreciation to Prof. Dr. H. Yoshii and Asst. Prof. Dr. S. Koba of Kyushu University, for their advice during the course of the work, and for their criticism of the manuscript. II. Symptoms of the mosaic of asparagus-bean The diseased leaves usually show a mosaic symptom, dark and light green in colour, keeping dark green area along the veins in contrast with interveinal light green area. The dark green area swells on the upper surface, the leaves being apt to roll downwards. In summer, small reddish-brown ringspots are formed on the cotyledons inoculated with the virus in five days after inoculation. The symptom then systemically spreads. On V. sesquipedalis, (variety Tsurunashijuhachi-sasage), under glass-house condition, the systemic symptom appears in a week in summer, although in a month in winter. (Fig. 1). These symptoms are almost consistent with those described by Matsumoto13), and also with those described by McLean15) and Snyder25). Intracellular inclusion bodies are not found in the tissues of diseased plants. III. Materials and Methods The isolate VQ-O was obtained from a mosaic-diseased Vigna sesquipedalis, which were collected at Kasuya-cho, Kasuya-gun, Fukuoka Prefecture, in September The isolate 112 was obtained from a seed borne diseased plant grown from seed purchased on the market. The isolate 117 was obtained from a seedling grown from seed produced on an asparagus-bean plant, which had been infected by mechanical inoculation with the isolate VQ-O. These isolates were ascertained not to be mixed with Cucumber Mosaic Virus, by means of inoculation to Nicotiana glutinosa. The virus isolates were kept on V. sesquipedalis by successive inoculations. As for test plants, Vigna sesquipedalis (variety Tsurunashi-juhachi-sasage) and sometimes V. sinensis (variety Blackeye) were used throughout the experiments. Among six varieties of Vigna spp., Tsurunashi-juhachi-sasage was found to be the best for the purpose because of rapid appearance of the symptom and good growth in winter under glass-house condition. In host-range tests, plants were inoculated by the convetional rubbing method, and symptoms were observed and recorded after 30 days. From all the plants thus inoculated, back-inoculations were made to healthy asparagus-bean or cowpea. In the physical property tests, supernatant of cetrifugate (6,000 r. p. m. for 5min.) of sap obtained from fully-grown diseased plants was used as the original source of inoculation. This preparation was immediately used for the test of determine the dilution-end point, the same preparation was diluted with distilled water. As for the aphid-transmission tests, aphids were fed on diseased plants for two days, and were then * Laboratory of Plant Pathology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

2 T. HINO: Studies on the asparagus-bean mosaic virus 179 removed and fed on a healthy plant for two days in groups of five individuals per plant. Tests for acquisition and inoculation feeding thresholds were done using single individual of Myzus persicae for each plant. The duration of acquisition and inoculation feeding thresholds was denoted by the duration of actual penetration of the aphid's stylet into the plant tissue. IV. Experimental results (a) Host range 38 species of leguminous plants and 8 species of non-leguminous plants were used for the experiment. The names of the plants tested, the symptoms induced in 30 days after the inoculation, and the results of back-inoculation tests, are tabularly shown in Table 1. The symptoms on susceptible plants were observed as follows:- Table. 1. Host range of the virus of asparagus-bean mosaic

3 Remarks: O means no symptoms and that the virus was not found by the back-inoculation. Aeschynomene indica did not show any symptom even after two months, although the virus was found, by the back-inoculation test, to be present in a latent state. Astragalus sinicus showed vein-clearing in fifteen days in winter. Interveinal area of the diseased leaflet was, to some extent, darker than the healthy leaflet, and swelled up, while the area along veins was yellowish green. After three months, the symptom vanished, becoming indistiguishable from healthy leaflet. (Fig. 5) Canavalia ensiformis showed a mosaic symptom, and slight leaf deformation. Large irregular dark-reddish spots were often observed on inoculated leaves, in twenty days after the inoculation. Canavalia gladiata showed no symptom, although the virus was detected in back-inoculation tests. Cassia tora formed small black primary local lesions on primary leaves, in ten days after the

4 T. HINO: Studies on the asparagus-bean mosaic virus 181 inoculation. These lesions were, however, not always found under a certain seasonal condition. The virus did not systemically move in this host. (Fig. 6) Phaseolus angularis showed light mosaic, with pale yellow and light green mottle, in a month in winter. In summer, a severe mottle with leaf deformation, which closely resembled the symptom observed in the field, appeared in fifteen days. Phaseolus lunatus formed irregular dark-reddish primary local lesions on the inoculated leaves, and then showed a systemic symptom of light and green mosaic. The diseased leaves were apt to roll downwards. (Fig. 3) Phaseolus vulgaris showed no sign of infection in the autumn of 1956 and 1957, but in the spring of 1958, the variety "Master Piece" showed faint irregular chlorotic primary local leasions on the inoculated leaves, from which back-inoculation was successful. Some other varieties showed either no symptom or only faint chlorotic spots, the virus having been detected only from the inoculated leaves. Systemic infection was not noticed in 30 days after the inoculation on these varieties. So far as this experiment concerns, this virus seems not to be infectious to the variety "Hirasaya-shakugosun-ingen". (Fig. 2) Vicia atropurpurea rarely caused symptomless systemic infection, with the isolate 112. Vicia faba showed reddish-brown primary local lesions on the inoculated leaves, in ten days after the inoculation, but was not systemically infected. (Fig. 4) Vigna sesquipedalis and V. sinensis showed a systemic symptom, as already mentioned in the previous article. (b) Physical properties Throughout the experiment, tests using the isolate VQ-O were repeated three times, but tests using other isolates were not repeated. The results obtained are shown in Table 2. Table 2. Physical properties of the virus of asparagus-bean mosaic Remarks; The denominator shows the number of plants inoculated, and the numerator shows that of plants affected.

5 five degrees. The results showed that the end point of the virus activity lay between 55 and 112 were shown to be inactivated between 1/1,000 and 1/2,500; the isolate 117 between 1/1,000 between 12 and 24 hours, while the isolate 112 and the isolate 117 were inactivated between 0 and 24 hours. It seems likely from these results that the longevity of the virus lies between 12 and 24 hours. (c) Transmission The virus was easily transmitted by mechanical inoculation. Aphid-transmission experiments were done in a net-house. The tests with green peach aphid (Myzus persicae) and bean aphid (Aphis medicaginis) were successful in transmitting all the isolates, while the tests with cucumber aphid (Aphis gossypii) were unsuccessful in transmitting the isolate VQ-O. The aphid last mentioned was not used for tests with other isolates. Table 3. Acquisition and inoculation feeding thresholds of Myzus persicae using the isolate VQ-O of the asparagus-bean mosaic virus Remarks: The denominator shows the number of plants tested, and the numerator shows that of plants affected. Table 4. Seed transmission of the virus of asparagus-bean mosaic The results of tests to determine acquistion and inoculation feeding thresholds, using the isolate VQ-O with the green peach aphid, are shown in Table 3. Both the acquisition and the inoculation feeding thresholds were found to be between 10 and 15 seconds. As to the seed transmission of the isolate VQ-O, 2314 seedlings from the seeds produced on diseased Tsurunashi-juhachi-sasage (Vigna sesquipedalis), which had been infected by mechanical inoculation, were observed until bifoliate stage. Only 6 plants were found to be infected. In the cases of Kurodane-juhachi-sasage, Juroku-sasage (V. sesquipedalis), Blackeye (V. sinensis), and Dainagon (Phaseolus angularis), evidence of transmission through seeds was not obtained. The results are shown in Table 4. Although the isolates 112 and 117 were originally obtained from seed-borne diseased plants, virus transmission through the seeds of Tsurunashi-juhachi-sasage was not observed among 359

T. HINO: Studies on the asparagus-bean mosaic virus 183 and 31 seedlings respectively. The isolates 112 and 117 seem to be the same with the isolate VQ-O, in their behavior toward seed transmission.

6 T. HINO: Studies on the asparagus-bean mosaic virus 183 and 31 seedlings respectively. The isolates 112 and 117 seem to be the same with the isolate VQ-O, in their behavior toward seed transmission. (d) Viruses obtained from mosaic-diseased asparagus-bean plants in the field Viruses of the mosaic-diseased asparagus-bean plants collected in the suburbs of Fukuoka were tested for their possible identity with the virus here described. The collected samples were inoculated to Vigna sesquipedalis and Nicotiana glutinosa, and symptoms produced were examined. 14 samples of V. sesquipedalis and 4 samples of V. sinensis were tested. Out of these samples, one from V. sesquipedalis was found to be infective both to V. sesquipedalis and N. glutinosa, while all the others were found to be infective only to V. sesquipedalis, but not to N. glutinosa. The former sample was proved to contain both the virus in question and the Cucumber Mosaic Virus. The Cucumber Mosaic Virus isolated from this V. sesquipedalis, through N. glutinosa, caused feeble leaf distortion and faint mottling, when back-inoculated to V. sesquipedalis. These symptoms, however, were quite different from those caused by the virus described in this report. From these results, it is considered that actual damages caused by the mosaic disease of asparagus-bean in the field are mainly due to the virus above described. V. Discussion and conclusion Virus diseases of leguminous plants in Japan are reported to be Adzuki-bean mosaic13)19)21), Broad-bean mosaic7), Red-clover mosaic7), Pea mosaic7), Broad-bean necrotic mosaic6), Soybean mosaic11), Alfalfa mosaic3), Asparagus-bean and Cowpea mosaic9), French-bean mosaic12), Milk-vetch dwarf14), and Lupine virus disease17). As for the four diseases last mentioned, their properties have not yet been reported. The other viruses, except the Adzuki-bean mosaic, seem to be different from the virus described in the present report in respect to their host ranges. As for Adzuki-bean mosaic disease, Matsumoto reported, in 1922, 29 susceptible varieties of adzuki-bean. He also described certain anatomical change in the diseased leaf tissue13). Otani reported the properties of the causal virus, in detail. According to him, the causal virus was not mechanically transmissible, and Myzus persicae and Aphis medicaginis acted as vector insects. Adzuki-bean, lima-bean, and sword-bean were reported to be susceptible19). Tasugi and Fukuda reporrted that the causal virus of Adzuki-bean mosaic was mechanically transmissible, and also by means of aphids, Myzus persicae, Aphis medicaginis, and A. gossypii. This virus was not infectious to asparagus-bean, pea, and Falcata comosa21). The virus dealt with in the present report and the virus described by Otani19) closely resemble with each other, in respect to the species of vectors, acquisition and inoculation feeding thresholds, and also host range, although they differ from each other in respect to mechanical transmission. The Otani's virus is highly transmissible through seeds16), while the virus in question is not so readily transmitted through seeds of asparagus-bean, or adzuki-bean. Cowpea Mosaic Virus reported by McLean is pathogenic to cowpea and lima-bean, and Anderson's Blackeye Cowpea Mosaic Virus causes systemic infection to cowpea, lima-bean, pea, broad-bean, sword-bean, Crotalaria spectabilis, and Desmonium spp.; forms local lesions on French-bean, Cassia tora, and Dolichos lablab; also causes symptomless local infection on soybean Mosaic Virus is pathogenic to asparagus-bean, and rarely to French-bean and is inactivated at although not by aphids5). Catjang Mosaic Virus reported by Capoor and Varma is inactivated at The virus described in the present report differs from that of Snyder20) in respect to its seed transmission ratio, and from that of McLean15) in respect to its thermal inactivation point. Murayama reported that seeds from mosaic-diseased Vigna plants only produced healthy seedlings16), although Kawamura observed 3 infected plants out of 37 seedlings in his survey of seed transmission of Vigna mosaic disease9). In the author's experiments, only 0.24 per cent of seed transmission was observed. Snyder concluded that his virus and that of McLean were the same, and the difference observed might be due to the strains used20). According to Anderson's

7 classification of Vigna and Crotalaria Viruses1), the viruses of Anderson1,2), Snyder20), McLean15), Oliveira18), and Yu24) ought to be included in the Group 2 along with the author's virus, which should be called Asparagus-bean Mosaic Virus. VI. Resume The causal virus of mosaic disease of asparagus-bean and cowpea in Japan was investigated and the following results were obtained: 1) This virus was easily transmissible by pressed juice and also by aphids, Myzus persicae and Aphis medicaginis. Transmission tests with A. gossypii were unsuccessful. Both acquisition and inoculation feeding thresholds of Myzus persicae were between 10 and 15 seconds. 2) Seed transmission rate of this virus was 0.24 per cent in the case of Vigna sesquipedalis, when 2914 seedlings were used for the test. 3) Among 38 species of Leguminosae and 8 species of non-leguminous plants tested, the susceptible plants were shown to be as follows: Astragalus sinicus, Canavalia ensiformis, Phaseolus angularis, P. lunatus, Vigna sesquipedalis, and V. sinensis. All these species produced systemic mottling. Cassia tora formed only black local lesions. Phaseolus vulgaris formed faint chlorotic local lesions, or caused symptomless local infection, but some varieties were immune from the disease. Aeschynomene indica and Canavalia gladiata were symptomless carriers. Vicia faba formed reddish-brown local lesions, but was not systemically infected. found to be between 12 and 24 hours. Dilution-end point was between 1: 1,000 and 1: 2,500. 5) Strains isolated from seed-borne diseased asparagus-bean plants found to be almost the same in respect to host range, physical properties, and transmission. References (Received Sept. 3, 1960) 1) Anderson, C. W. (1954). Plant Dis. Reptr. 38: ) Anderson, C. W. (1955). Plant Dis. Reptr. 39: ) Asuyama, H., Komuro, Y, and Syoda, K. (1955). Jubilee Publ. Comm. 60th Birthdays Prof. Y. Tochinai & T. Fukushi: ) Capoor, S. P. and Varma, P. M. (1956). Indian Jour. Agric. Sci. 26: ) Dale, W. T. (1949). Ann. Appl. Biol. 36: ) Fukano, H. and Yokoyama, S. (1952). Kyushu Agric. Res. 10: ) Fukushi, T. (1937). Jour. Sapporo Soc. Agric. Forest. 29: ) Hino, T. and Yoshii, H. (1956). Proc. Asoc. Plant Prot. Kyushu 2: ) Kawamura, E. (1941). Ann. Phytopath. Soc. Japan 11: ) Komuro, Y. and Asuyama, H. (1952). Ann. Phytopath. Soc. Japan 16: ) Koshimizu, S. and Iizuka, N. (1955). Ann. Rept. Soc. Plant Prot. North Japan 6: ) Kuribayashi, K. (1926). Jour, Plant Prot. (Japan) 13: ) Matsumoto, T. (1922). Jour. Plant Prot. (Japan) 9: ) Matsuura, Y. (1953). Ann. Phytopath. Soc. Japan 17: ) McLean, D. M. (1941). Phytopath. 31: ) Murayama, D. (1940). Ann. Phytopath. Soc. Japan 10: ) Nishida, T. (1951). Kyushu Agric. Res. 8: ) Oliveira, M. A. (1947). Bol. Tech. Agron. Sul. (Pelotas) 1: ) Otani, Y. (1942). Ann. Phytopath. Soc. Japan 12: ) Snyder, W. C. (1942). Phytopath. 32: ) Tasugi, H, and Fukuda, K. (1956). Ann. Rept. Soc. Plant Prot. North Japan 7: ) Warid, W. A. and Plakidas, A. G. (1950). Phytopath. 40: ) Warid, W. A. and Plakidas, A. G. (1952). Plant Dis. Reptr. 36: ) Yu, T. F. (1946). Ann. Appl. Biol. 33: Explanation of plate Fig. 1. Mosaic symptom on Vigna sesquipedalis var. Tsurunashi-juhachi-sasage. (VQ-O) Fig. 2. Chlorotic local lesions on the inoculated leaf of Phaseolus vulgaris var. Master Piece. (VQ-O)

8 T. HINO: Studies on the asparagus-bean Fig. 3. Mosaic symptom on Phaseolus lunatus var. Fig. 4: Red (VQ-O) Fig. 5. Vein Fig. 6. Black local lesions on clearing on the inoculated Astragalus mosaic virus Fordhook Lima. leaf of (VQ-O) Vicia faba var. Boshu-wase-soramame. sinicus. (VQ -O) Left: healthy. Right: leaf of Cassia tora. (117) local lesions on the inoculated diseased.

MILD MOSAIC AND FAINT MOTTLE RINGSPOT, TWO FLORIDA

MILD MOSAIC AND FAINT MOTTLE RINGSPOT, TWO FLORIDA 444 FLORIDA STATE HORTICULTURAL SOCIETY, 1964 are rarely found colonizing on papayas has little bearing on the problem, as was demonstrated by Dickson et al. (3) for cantaloupe mossaic. Winged adults which