More than two hundred and fifty-five monoclonal antibody (MoAb)-secreting hybridomas against 3 plant luteoviruses, 2 plant reoviruses and a potyvirus were produced.The hybridomas for potato leafroll virus, beet western yellows virus, tobacco necrotic dwarf virus, rice dwarf virus, rice ragged stunt virus and potato virus Y-ordinary strain, were screened by four different procedures of enzyme-linked immunosorbent assay (ELISA); procedure 1, antigen adsorption indirect ELISA (AAI-ELISA) in which purified virus in phosphate buffered saline (PBS) at pH 7.4 was adsorbed onto the microplate wells, procedure 2, AAI-ELISA in which purified virus in sodium carbonate-bicarbonate buffer at pH 9.6 was adsorbed onto the microplate wells, procedure 3, indirect double antibody sandwich ELISA (IDAS-ELISA) in which polyclonal antibody was used as trapping antibody and purified virus preparations diluted in PBS-T (containing Tween-20) as antigens were used, procedure 4, IDAS-ELISA in which polyclonal antibody was used for trapping antibody and crude saps of virus infected plants extracted in PBS-T as antigens were used. Based on the MoAb reactivities against homologous viruses in four different ELISA procedures, MoAb-secreting hybridomas were divided into ten groups. Using purified MoAbs from ascitic fluids, direct double antibody sandwich ELISA (DAS-ELISA) was examined for the detection of virus antigens in infected plants. All the MoAbs reacted in DAS-ELISA belonged to group 1 in which the MoAbs were reactive in each four of the screening procedures 1-4, or group 2 in which MoAbs were reactive in three of screening procedures 1, 3 and 4. These results indicate that MoAbs being reactive in DAS-ELISA can be readily selected in hybridomas in group 1 or 2.
ABSTRAKPenelitian dilakukan untuk membedakan virulensi isolat Rice tungro virus (RTV) dari beberapa daerah endemis penyakit tungro di Indonesia berdasarkan reaksinya pada tanaman inang diferensial. Delapan isolat RTV dikumpulkan dari beberapa daerah endemis penyakit tungro di Indonesia, yaitu Bali (Denpasar), Kalimantan Selatan (Tanah Laut), Sulawesi Selatan (Lanrang), Jawa Barat (Subang dan Bogor), Jawa Tengah (Solo), Jawa Timur (Jember), dan Nusa Tenggara Barat (Mataram). Semua isolat dipelihara pada kultivar padi TN1 di rumah kaca melalui penularan serangga vektor Nephotettix virescens, selanjutnya masing-masing isolat ditularkan pada kultivar padi diferensial FK135 dan TN1. Empat minggu setelah inokulasi pada kultivar padi diferensial diamati gejala yang muncul dan infektifitas masing-masing isolat RTV. Tingkat virulensi isolat RTV dari beberapa daerah endemis penyakit tungro di Indonesia dapat dikelompokkan menjadi 3 tingkat. Virulensi paling tinggi ialah isolat RTV dari Jawa Barat; tingkat virulensi moderat ialah isolat RTV dari Jawa Timur, Kalimantan Selatan, Sulawesi Selatan dan Nusa Tenggara Barat; tingkat virulensi rendah ditunjukkan oleh isolat RTV dari Bali dan Jawa Tengah.Kata kunci: kultivar diferensial, Nephotettix virescens, padi, penyakit tungro ABSTRACTThe research was conducted to distinguish the virulence of Rice tungro virus (RTV) isolates from several tungro disease endemic areas in Indonesia based on their response in differential cultivars of rice. Eight isolates of RTV were collected from several tungro endemic regions in Indonesia i.e. Bali (Denpasar), South Kalimantan (Tanah Laut), South Sulawesi (Lanrang), West Java (Subang and Bogor), Central Java (Solo), East Java (Jember), and West Nusa Tenggara (Mataram). The isolates were maintained on TN1 rice cultivar in greenhouse by successive transfers via Nephotettix virescens. Each isolate was subsequently passed on to FK135 and TN1 rice cultivars. Four week after inoculation, plants responses were observed to compare their symptom and infectivity on the differential cultivars. The results showed that the level of virulence of RTV isolate from tungro disease endemic areas in Indonesia can be classified into 3 levels. The highest virulence were RTV isolates from West Jawa, the moderate level of virulence were RTV isolates from East Java, South Kalimantan, South Sulawesi, and West Nusa Tenggara, the low level of virulence were RTV isolates from Bali and Central Java.
Rice ragged stunt virus (RRSV) causes serious disease in South and Southeast Asian Countries and occurs also in China, Japan and Taiwan1,3). RRSV is transmitted in a persistent manner by the brown planthopper Nilaparvata lugens (Stal). The virus particles consist of polyhedral core ones approx. 50nm in diameter to which are attached flat spikes about 20nm wide and 10nm high2). RRSV has 10 segments of double-stranded RNA7) and several proteins2). In plant reovirus group, non-vector transmissible isolates have been obtained for leafhopper-borne wound tumor virus (WTV)9) and rice dwarf virus (RDV)5) after maintaining the virus for many years in plants without leafhopper vector transmission. This paper reports loss of vector transmissibility of RRSV after maintaining it for 3 years in rice plants without planthopper vector transmission. Symptoms, particle structure, RNAs and proteins of the non-vector-transmissible isolate are also described. In March 1981, seedlings of rice plants (Oriza sativa L. cv. Taitung Native 1) were inoculated with RRSV using viruliferous N. lugens. One infected seedling was selected and grown in a greenhouse. Three months after infection, tillers were divided from the selected plant and transplanted separately in pots. Every three months thereafter, tillers were divided from the vegetatively propagated plants and transplanted separately. After the first winter season was over, one plant was selected from a set of vegetatively propagated infected plants and used for vegetative propagation in the following year. The same cycle of selection and vegetative propagation was repeated for seven years. In March 1982, seedlings were similarly infected and the same cycle of selection and vegetative propagation was repeated on one selected plant for six years. On plants infected with RRSV in 1983with RRSV in , 1984with RRSV in , 1985with RRSV in , 1986with RRSV in or 1987, the same cycle of selection and propagation was repeated for five, four, three, two or one years, respectively. In June 1988, seven sets of RRSV-infected plants maintained for one to seven years by vegetative propagation were used for virus transmission. A plant newly infected in 1988 served as the control.The second or third-instar nymphs of the brown planthopper were fed for 2 days on each of three plants selected from each set of vegetatively propagated plants and then they were reared on healthy rice seedlings. Two weeks after the initiation of the acquisition feeding, rice seedlings were inoculated by one planthopper per seedling for 2 days. Inoculated seedlings were grown in a greenhouse. RRSV symptoms on inoculated seedlings were observed one month later. As shown in Table 1, vector transmissibility of RRSV from vegetatively propagated RRSV-infected plants decreased with the advance of years after inoculation for the first two years and it was lost in the third year and thereafter. Percentage of transmission of RRSV by the colony of N. lugens used in this study was 45% in the first year, 37% in the third year and was 17...
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