不同因子對桃蚜媒介之矮南瓜黃化嵌紋病毒協助成分之影響

矮南瓜黃化嵌紋病毒危害世界各地之葫蘆科作物且造成經濟上嚴重的損失。蚜蟲媒介矮南瓜黃化嵌紋病毒需有由病毒轉譯之協助成分(helper component; HC)的存在才能媒介成功。本文探討不同酸鹼值之緩衝液或溶液種類、離子濃度、還原劑或鉗合劑添加物、濃縮方式和在不同溫度下之保存時間對協助成分活性的影響。分別利用不同酸鹼值之醋酸鹽緩衝液、檸檬酸鹽緩衝液、磷酸緩衝液、磷酸氫二鉀溶液、Tris-HCl緩衝液, Tris-H_2SO_4,緩衝液、硼酸緩衝液和碳酸鈉緩衝液等研磨病葉，在100,000 g離心90分鐘後，所得含協助成分之上澄液添加等體積濃度爲0.2 mg/ml之純化病毒，再經由桃蚜以人工膜餵食方式測試其傳毒能力，結果顯示其活性在pH值爲7、8和9時之緩衝液內較佳，其中以在pH 9之磷酸氫二鉀溶液時的活性爲最高，可達77%之傳播率。比較協助成分在磷酸氫二鉀溶液不同離子濃度中之活性，結果顯示其活性在離子濃度爲0.3 M時較佳。經添加0.1%之亞硫酸鈉或二羌基乙二醇(2-mercaptoethanol)還原劑以及0.01 M之EDTA, EGTA和DIECA等鉗合劑至0.3 M之K-_2HPO_4溶液中，對協助成分之活性並無顯著提高。將含協助成分之上澄液置於室溫(24-28 C)、4 C和-20 C下，結果發現置於室溫下處理之協助成分的活性，從對照組(0小時)之77%，經24小時後急降爲13%，48小時後只餘3%，而置於4 C者經48小時後仍有17%之活性存在，然而經96小時後除了置於-20 C之試液仍有30%之活性存在外，其它處理皆已失去活性。利用飽和硫酸銨溶液和聚乙醇6,000 (PEG MW 6,000)溶液分級沉降含協助成分之試液，以及冷凍乾燥法和聚乙二醇20,000 (PEG MW 20,000)粉末濃縮處理含協助成分之試液，得知協助成分之活性可以在30%-50%之硫酸銨溶液或4%-8%之PEG 6,000溶液中沉降，而經冷凍乾燥及PEG 20,000粉末包埋濃縮處理之協助成分其活性分別爲60%和63%，其中以含8% PEG 600沉降之協助成分活性77%爲較佳。

關鍵字

矮南瓜黃化嵌紋病毒；桃蚜；協助成分；媒界傳播

並列摘要

Zucchini yellow mosaic virus (ZYMV) is a member of potyviruses and causes severe losses of cucurbit crops throughout the world. Transmission of potyviruses in nature is almost accomplished by aphid. However, aphid transmission of ZYMV depends on the presence of a virus-encoded protein termed as helper component (HC). This study was to determine the effects of values of various buffers or K_2HPO_4 solution, K_2HPO_4 concentrations, additives of antioxidants and chelating agents concentration methods, and storage temperature on the activity of ZYMV HC in mediating the transmission of purified ZYMV by Myzus persicae. A series of 0.1 M extraction buffers or K_2HPO_4. solution at pH values ranging from 4 to 10 were used to homogenize the ZYMV infected squash leaf tissues. The activity of ZYMV HC in the supernatant after a 100,000 g, 90 min, centrifugation was examined by feeding the aphids on the tested solution through a parafilm membrane. The tested solution contained 10% sucrose and an equal volume of undiluted supernatant and purified ZYMV (0.2 mg/ml). The results showed that the activity of ZYMV HC was higher in neutral or alkaline extraction buffers or KHPO4 solution than in acid buffers. Among them, K2HPO4 solution, pH 9, resulted in the est aphid transmission rate, 77%. The activity of ZYMV HC in K_2HPO_4 solution was higher in the concentration of 0.3 M than in other concentrations tested. Addition of 0.1% two antioxidants, sodium sulphite and 2-mercaptoethanol, and 0.01 M of three chelators, EDTA, EGTA and DIECA to 0.3 M K HPO4 did not increase the activity of ZYMV HC. The activity of ZYMV HC in the supernatant rapidly decreased from 77% (transmission rate) to 13% and 3% after kept at room temperature for 24 hr and 48 hr, respectively those at 4 C for 48 hr were 17%; no activity of ZYMV HC was detected om temperature or 4 C for 96 hr. However, ZYMV HC activity remained 30% after kept at ransmission rate after storage at -20 C for 96 hr. Most of ZYMV HC activity in the supernatant v saturated ammonium sul fate or by 4%-8% PEG 6,000. The rates of reciptated by 30%-50 transmission of the HC in the supernatant after lyophilization and embedding w h PEG 20,000 were respectively. Precipitation with 8% PEG 6,000 was the best concentration m whic 60% and 63%, rate of aphid transmission (7%) resulted in the highest ni yellow mosaic virus, Myzus persicae, helper componen ransmission (77%).

並列關鍵字

zucchini yellow mosaivirus ； Myzus persicae ； helper component ； tramission

國際替代計量

不同因子對桃蚜媒介之矮南瓜黃化嵌紋病毒協助成分之影響

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