Purification of groundnut bud necrosis virus and antiserum production
1997
Sopone Wongkaew | Jutharat Chuapong (Khon Kaen Univ. (Thailand). Faculty of Agriculture. Dept. of Plant Pathology)
Groundnut bud necrosis virus (GBNV) sample from Laemsingh, Chantaburi (GBNV-LS1) was isolated using lesion isolation technique. The virus was propagated in Necotianum tabacum CV. White Burley or Tainan 9 peanut. A purification procedure as described by the International Crop Research Institute for Semi-Arid Tropic (ICRISAT) was employed with some modification and found suitable for the GBNV-LS1 isolate. The procedure was less time consuming compared to that of the ICRISAT. The highest virus concentration was located in the 2.8-3.6 cm band (from the tube bottom) after the last density gradient centrifugation instead of 2.5-3.1 cm as specified by the ICRISAT protocol. The virus yield of 20-25 A260 was obtained from 100 g of infected peanut leaves. Because only trace amount of virus was obtain when tobacco was used as a virus source it was considered a poor propagative host of GBNV. An antiserum was prepared from 2 intramuscular and 1 booster intravienous injections in a rabbit using the purified preparations. A titer or 1:12800 was obtained by DAC-ELISA when 5 percent GBNV infected peanut sap was used as a test antigen for the antiserum. The end point was above 1:400 if the healthy sap was used but this positive background disappeared when 1:3200 dilution was used or the antiserum was crossed absorbed with healthy sap prior to use.
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