Abstract

1. NON TRANSMISSIBILITY OF RADISH MOSAIC COMOVIRUS (RAMV) EUROPEAN STRAIN THROUGH SEED OF WILD TURNIP RAPE (Brassica campestris L.) Dj. Mamula (1), E. Djermic (2), Z. Stafa (3), B. Cvjetkovic (2), M. Pospisil (3), Z. Liber (1) and T. Milicevic (2) (1) Faculty of Science, Department of Botany, (2,3) Faculty of Agriculture, (2) Department of Plant Pathology, (3) Department of Field Crop Production, (1,2,3) University of Zagreb, Zagreb, Croatia Abstract Inocula prepared from the seed and respective seedlings of wild turnip rape infected with RaMV European strain/isolate HZ, tested on turnip as assay plant, showed the non-transmissibility of the virus by the seed of this species Aim The European strain of radish mosaic virus (RaMV) is harmful to Brassica (turnip) and to some other species. From biological and practical points of view this prompted the authors to investigate whether virus transmission by the seed of infected plants is present in this virus strain, the more so in that it was not found in another, different (neotype), strain of the virus infecting especially Raphanus spp. Fig. 7. Similar as in figures 5 and 6; left are siliquas of diseased plant Fig. 6. Similar as in figures 5 and 7 Material and Methods Source of seed.Seed was taken in late October/November from wild (weed) plants of wild turnip rape (Brassica campestris L.; synonyms: B. rapa L. subsp. campestris L. /Clapham/; etc) infected spontaneously - cca 15-30 % of the plants - with RaMV European strain/Croatian HZ isolate (unrecorded as /natural/ host of RaMV so far), in three distant fields (Zagreb region, central Croatia) (Figs. 5-7). The infection was demonstrated serologically (Fig.8). Assay. Biological method, using 'Kranjska' turnip (necrotic local lesions, also systemic infection) was applied. Samples for inoculation. About 800 fertile seeds of more or less normal shape but smaller than in healthy plants were chosen from nearly 4,000 seeds and tested. Seed, not washed (2 samples, each of 100 seeds) or washed (sample of 100 seeds, and two others, germinated for 2 days, one containing 100 and the other 250 seeds) with tap water, was ground in a mortar with buffer added and inoculated onto 10 carborundum-dusted leaves (3 or 4 plants) in a glasshouse. Seedling samples, contained 1- and 3-month seedlings (one sample) and 14-day seedlings, altogether 200 seedlings ground in buffer. The buffer used was 1/15 M, pH 7.0 Sörensen’s phosphate (3 ml per sample). Inoculations were performed during October-December (first 4 samples) and March. Fig. 1. Particles of radish mosaic virus (RaMV) in purified preparation. The barrepresents 50 nm. (From: Goldbach et al. Family Comoviridae. In: F.A. Murphy et al. (Eds) 1995: Virus Taxonomy. Springer-Verlag/Wien, p. 341. Fig. 2. Symptoms (veinal necrosis and malformations in younger leaves) produced by RaMV in field turnip plant (arrows) leading to rosetted appearance of plants and plant death (early stage of infection); beetle-produced damages are indicated (dashed arrow) Introduction Transmission through seed has been reported in about 25% of the investigated viruses from different plant virus groups, including both types, i.e. the presence of the virus in the embryo and in or on the seed coat (testa). Biologically, this mode of transmission is important for virus survival and for its dissemination (e.g.intercontinental transport of commercial seed). Regarding plant production, it enables an early (in terms of vegetation development) establishment of plant (crop) primary infection and then massive secondary infection by means of vectors6. In Comoviridae, a virus family known for many economically important (harmful) viruses, to which RaMV belongs, seed transmission of viruses is fairly frequent. These isometric viruses of 28-30 nm diameter (Fig. 1) with a positive-sense single stranded bipartite RNA genome, known for capsid organization with 12 pentamers and 20 trimers and one to four capsid protein species, include three genera with different vectors: Comovirus genus (Comoviruses), 14 viruses (also RaMV), Coleoptera(beetle) vectors Nepovirus genus (Nepoviruses), 27-39 viruses, nematode vectors, seed transmission in 27 viruses Fabavirus genus (Fabaviruses), 2 viruses, aphid vectors, not seed – transmissible (BBWV) or unknown1,2,4 European (including Moroccan isolate) and neotype (type Californian, Japanese and possibly Russian Far East isolates) RaMV strains differ clearly serologically and in natural hosts: Brassica, Eruca, Sinapis spp., etc (the first one), Raphanus spp (neotype strain). The first one is deleterious or harmful to the turnip B. rapa L. var. rapa (Fig. 2, 3), and also to E. sativa L., Chinese cabbage, hybrid Perko PVH, perennial kale, etc3.(Fig. 4). The neotype strain is deleterious to Raphanussativus L. varieties: radish, daikon, Chinese radish1,5. 3 2 4 Fig. 3. RaMV field infection in turnip plants. Leaf malformation and stunting are present (older infection) As 1 5 6 Fig. 8. Serological DRID (in 1% bacto agar gel) reaction of RaMV from wild turnip rapeplants (1-3) and HZ isolate from turnip (4, 6); healthy plant sap in 5. Uncentrifuged sap in 2, 4– 6. Antiserum to RaMV European strain, isolate HZ. Results and Discussion Symptoms did not occur in the test plants in any case. Thus, the experimental procedure used, which involved about 800 seeds, did not reveal the transmission of RaMV in wild turnip rape seed. No virus transmission was found in the seed of radish plants infected with the neotype, i.e. Californian3 and Japanese6 isolates of RaMV. The experiments are continued. Fig. 4. Symptoms of RaMV infection on rutabaga (Brassica napus var. napobrassica, f. rutabaga); leaf necrosis and malformation can be seen References (1) Brunt, A. A., K. Crabtree, M. J. Dalwitz, A. J. Gibbs, L. Watson, 1997: Viruses of Plants. Descriptions and Lists from the VIDE Database. CAB INTERNATIONAL. (2) Büchen-Osmond, C. (Ed), 2003. 00.018. Comoviridae. In: ICTVdB – The Universal Virus Database, version 3. ICTVdB Management, Columbia University, New York, USA. (2) Campbell, R. N. 1988. Radish mosaic comovirus. In: A. A. Brunt et al. 1997: Viruses of Plants. Descriptions and Lists from the VIDE Database. CAB INTERNATIONAL. (3) Campbell, R. N., Colt, W. M., 1967. Transmission of radish mosaic virus. Phytopathology 57, 502-504. (4) Goldbach, R., Martelli, G. P., Milne R.G.: Family Comoviridae. In: F.A. Murphy et al. (Eds) 1995: Virus Taxonomy. Springer-Verlag/Wien, p. 341-347. (5) Mamula, Dj., Stefanac, Z. (submitted for press). Radish mosaic. In: Rimmer/Shattuck, Compendium of Brassica Crop Diseases. APS Press, St. Louis. (6) Tochihara, H., 1968. Radish enation mosaic virus. Ann. Phytopathol. Soc. (Japan) 34, 129-136. (7) Walkey, D. G. A., 1991. Applied Plant Virology. Second edition.Chapmann and Hall, London, etc. Fig. 5.Wild turnip rape showing moderate ochre chlorosis, mosaic, necrosis and malformation leaf symptoms (arrow) of RaMV infection. Seed for examination was taken from these or similar plants Contact Information: Dj. Mamula, University of Zagreb, Faculty of Science, Department of Botany, Marulićev trg 20/II, 10000 Zagreb, Croatia, Phone: +385-1-4843851, FAX: +385-1-4844001, Email: dmamula@ croatica.botanic.hr