Susceptibility of Citrus Varieties, Species, Citrus Relatives, and Non-Rutaceous Plants to Slash-cut Mechanical Inoculation with Citrus Tristeza Virus (CTV)
Susceptibility of Citrus Varieties, Species, Citrus Relatives, and Non-Rutaceous Plants to Slash-cut Mechanical Inoculation with Citrus Tristeza Virus (CTV)
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柑橘品种、物种、柑橘近缘种和非芸香植物对柑橘 Tristeza 病毒 (CTV) 切线机械接种的敏感性
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发表时间：
1984
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影响因子：
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通讯作者：
S. Garnsey
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文献类型：
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作者：
G. W. Müller;S. Garnsey
采用浓缩、部分纯化的柑桔tristeza virus （CTV）制剂对47个柑桔品种、栽培品种、杂交种和近缘种进行了CTV的茎条接种试验。用症状和/或酶联免疫吸附试验（ELISA）检测了31个柑橘品种中26个的侵染情况。ELISA程序对于检测无症状感染是有价值的。ctv诱导的静脉清除在一些正常潜伏宿主（如Rangpur lime）中观察到，直到全身感染开始。其中，香橼、朗普莱姆、菖蒲、墨西哥莱姆、甜橙和枸杞感染率较高。葡萄柚和古巴柚子难以感染。16个机械接种的柑桔近缘种中有6个被感染：Corr., chevalieri Swing。，凤尾花（学名：Afraegle paniculata）心血管病。，香茅。& M.凯尔。，南柑桔（蔓生）摇摆。和Pamburus mission (Wt.) Swing。经ELISA检测，55株不同木本和草本非芸香植物均未检测到CTV感染。Tristeza从柑橘机械地重新传播到Etrog citron。自20世纪40年代以来，巴西坎皮纳斯农业研究所（Instituto agronmic0）对大量柑橘品种和一些柑橘亲缘品种进行了柑橘tristeza病毒（CTV）易感性测试（3,5,10,11）。通过嫁接、蚜虫载体和几种菟丝子进行了接种（1,3）。CTV的嫁接传播仅限于与柑橘嫁接相容的寄主（13），但CTV已通过蚜虫传播给几种柑橘近亲，如chevalieri Aeglopsis。人们也多次尝试将CTV的寄主范围扩展到芸香科以外（6,12），但这只在西番莲（Passiflora gracilis Jacq）中取得了成功。由蚜虫接种（1,14）。最近开发的将CTV机械传播到柑橘的程序(7)和对ELISA测试的调整以快速检测CTV感染(2)提供*美国农业部使用公司或产品名称并不意味着批准或推荐该产品而排除其他可能合适的产品。进一步试验柑橘、柑橘亲缘植物和非芸香植物作为CTV寄主的可能性。我们希望能够发现草本寄主，以便更好地检测和/或增加CTV的植株。本文报道了用机械接种法测定柑橘、柑橘近缘种和非芸香植物对CTV感染的易感性的结果。材料和方法植物材料和生长条件。试验是在佛罗里达州奥兰多的一个风冷的、部分遮阳的温室中进行的。冬季和春季的气温为22至27摄氏度，夏季为22至35摄氏度。植物生长在灭菌的盆栽混合物中，根据需要施肥和喷洒，以保持健康，旺盛的生长。植株由无性系、无病毒源的种子或根茎扦插培养而成。选择了多种非芸香植物进行试验。一些是木质的，一些是其他病毒、类病毒和原核病原体的宿主（1516），一些是柑橘果园中发现的杂草，还有一个被描述为T. citricida的非壳宿主（17）。每次实验均选取机械接种CTV易感的柚子植株(7)，验证接种物的感染性。接种准备。用CTV分离物T-3（幼苗黄（SY）分离物）或CTV分离物T-4（非SY分离物）感染温室生长的Etrog citron或sweet lime植株的幼茎皮去皮，并以“Parafilm@.”包裹成5克捆。在Hooker@显微镜上切片，用剃须刀片按1:3 （w/v）的比例在提取缓冲液（0.05 M TrisHC1缓冲液[Tris（羟甲基）氨基甲烷盐酸]，pH为7.8，含100 mg/l蔗糖和2 pl/ml 2-巯基乙醇）中切碎。用手压机将提取液挤过粗棉布，用少量缓冲液重新提取。在Sorvall@ SS-34转子中以12,000 RPM离心15分钟澄清提取物，并在2.5 x 7.5或2.5 x 8.75 cm离心管中分层，离心管中含有新鲜制备的蔗糖步骤梯度（5ml 250mg /ml和5ml 600mg /ml蔗糖在Tris缓冲液中）(8)。试管在Beckmans SW25.1转子中以每分钟21,500转的速度或在SW28转子中以每分钟19,000转的速度离心过夜。通过刺穿梯度管的底部，在预先标记的试管中收集半ml的分数。采用酶联免疫吸附试验（ELISA）测定梯度馏分CTV含量（头法），将2-3个含CTV含量最低的馏分混合，用0.05 M Tris稀释1:1，接种5次。从25克树皮组织中回收约6至9毫升接种物。接种。大多数接种是在1980年1月至4月和9月至10月期间进行的，但也有一些是在1981年、1982年和1983年春季进行的。接种采用茎割法(7)，茎上3个部位各割30次。接种时，大部分茎的直径为0.4 ~ 0.8 cm。通常每种类型至少接种10株。每次接种试验使用新鲜的、部分纯化的制剂，每批用柚子植株验证感染性。最初，柑橘和柑橘近亲的切口是用Stericrepea （Beacon & Janis Ltd., London, w.c.i., England）包裹的。然而，后来发现这是不必要的。一些多肉非壳类植物用塑料袋覆盖，另一些接种切口用“Parafilm”包裹，植物不被覆盖。一些草本植物的叶中肋也被割开。接种后，将草本植物置于阴凉处48小时后，取下塑料袋。柑橘类和大多数木本植物在接种2周后立即被剪除以迫使新的生长，草本植物被剪除。在检测CTV感染之前，快速生长的植物被修剪了几次。每个试验种保留3 ~ 5株未接种的对照植株。感染试验。定期检查所有柑橘品种、种和近缘种的静脉清除情况，检查非柑橘类植物的静脉清除情况和其他异常情况。当对照植株出现清脉症状后，采集接种柑橘Tristexa及相关病害及柑橘近缘种的多肉茎皮组织。从肉质非乳质寄主上采集整个幼茎。将切碎的组织（0.5 g）放入直径2.5 cm的玻璃管中，加入5ml PBST-2 PVP缓冲液（含0.05% tween20和2%聚乙烯吡咯烷酮的磷酸盐缓冲盐水，m.w 40000）（2,4），用sdttissumiser @ （Tekmar Co., Cincinnati, Ohio 45222）均质约15秒。提取液经玻璃棉过滤，双抗体夹心法进行ELISA检测(4)。制备了CTV抗血清，使T-4分离株的整个病毒不固定(9)。IgG被纯化并结合到碱性磷酸酶上，如前所述（2,4）。我们使用聚苯乙烯微！带有圆底孔的ELISA@板（Dynatech Labs, Inc., Alexandria, Virginia 22314）。健康提取液、ctv感染提取液和缓冲对照液分别放在每个培养皿中作为参考。对底片进行目测评分，并通过照相记录结果。在某些情况下，稀释后的样品也用分光光度法测定了405nm的吸光度，以验证评估。样本不被认为是ctv阳性，除非用药剂量是2倍
The susceptibility of 47 citrus species, cultivars, hybrids and relatives to citrus tristeza virus (CTV) infection was tested by stem-slash inoculation of greenhouse-grown plants with concentrated, partially purified preparations of CTV. Infection in 26 of 31 citrus selections inoculated was detected by symptoms and/or by ELISA (enzyme-linked immunosorbent assay). The ELISA procedure was valuable for detecting symptomless infections. CTV-induced vein clearing was observed in several normally latent hosts such as Rangpur lime a t the onset of systemic infection. High rates of infection were observed in Etrog citron, Rangpur lime, calamondin, Mexican lime, sweet orange and Citrus hystrix. Grapefruit and Cuban Shaddock were difficult to infect. Six of 16 citrus relatives mechanically inoculated were infected: Aegle marmelos (L.) Corr., Aeglopsis chevalieri Swing., Afraegle paniculata (Schum.) Engl., Citropsis gilletiana Swing. & M. Kell., Microcitrus australis (Planch.) Swing., and Pamburus missionis (Wt.) Swing. No CTV infections were detected by ELISA in the 55 different woody and herbaceous nonrutaceous plants inoculated. Tristeza was retransmitted mechanically from A. marmelos to Etrog citron. Since the 1940's a large number of citrus types and a few citrus relatives have been tested for susceptibility to the citrus tristeza virus (CTV), a t the Instituto Agron6mic0, Campinas, Brazil (3, 5, 10, 11). Inoculations have been made by grafting, by aphid vectors, and with several species of dodder (1, 3). Graft transmission of CTV has been limited to hosts graft-compatible with citrus (13), but CTV has been transmitted to several citrus relatives, such as Aeglopsis chevalieri by aphids. Repeated attempts also have been made to extend the host range of CTV outside the Rutaceae ( 6 , 12), but this has been successful only with Passiflora gracilis Jacq. inoculated by aphids (1, 14). The recently developed procedure to mechanically transmit CTV to citrus (7) and the adaptation of the ELISA test to quickly detect CTV infections (2) offered *Use of a company or product name by the U.S. Department of Agriculture does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. further possibilities to test citrus, citrus relatives and nonrutaceous plants as hosts of CTV. We hoped that herbaceous hosts could be discovered which would be better assay and/or increase plants for CTV. In this paper, we report the results of inoculating and indexing numerous citrus, citrus relatives, and nonrutaceous plants to determine their susceptibility to CTV infection by mechanical inoculation. MATERIALS AND METHODS Plant materials and growing conditions. Tests were conducted in an air-cooled, partly shaded glasshouse in Orlando, Florida. Temperatures ranged from 22 to 27 C in winter and spring and 22 to 35 C in summer. Plants were grown in a sterilized potting mix, fertilized and sprayed as needed to maintain healthy, vigorous growth. Plants were grown from seeds or from rooted cuttings of clonal, virus-free sources. A variety of nonrutaceous plants were chosen for testing. Ninth IOCV Conference Some were woody, some were hosts for other viruses, viroids, and prokaryotic pathogens (1516), some were weeds found in citrus orchards, and one has been described as a nonrutaceous host of T. citricida (17). Etrog citron plants which are susceptible to CTV infection by mechanical inoculation (7) were included in every experiment to verify infectivity of the inoculum. Inoculation preparation. Young stem bark from greenhouse-grown Etrog citron or sweet lime plants infected with CTV isolate T-3 (a seedling yellows (SY) isolate) or CTV isolate T-4 (a non-SY isolate), was peeled and wrapped in 5 g bundles with "Parafilm@." The bundles were sectioned on a Hooker@ microtome and finely diced with a razor blade a t a 1 :3 (w/v) ratio in extraction buffer (0.05 M TrisHC1 buffer [Tris (hydroxymethyl) aminomethane hydrochloride], pH 7.8 which contained 100 mg/l sucrose and 2 pl/ml of 2-mercaptoethanol). The extract was squeezed through cheesecloth by hand press and reextracted with a small volume of buffer. All extraction steps were done a t 0 to 4 C. Extracts were clarified by centrifugation for 15 minutes a t 12,000 RPM in a Sorvall@ SS-34 rotor and layered in 2.5 x 7.5 or 2.5 x 8.75 cm centrifuge tubes containing a freshly prepared sucrose step gradient (5 ml of 250 mg/ml and 5 ml of 600 mg/ml sucrose in Tris buffer) (8). Tubes were centrifuged overnight a t 21,500 RPM in a Beckmans SW25.1 rotor or a t 19,000 RPM in a SW28 rotor. Half ml fractions were collected in premarked test tubes by puncturing the bottom of the gradient tube. Gradient fractions were tested for CTV content by ELISA (bead procedure) and the leading (lowest) 2-3 CTV-containing fractions were combined and diluted I :l with 0.05 M Tris to use a s inoculum. Approximately 6 to 9 ml of inoculum was recovered from 25 g of bark tissue. Inoculation. Most inoculations were done from January through April and during September and October 1980, but some were also done in the spring of 1981,1982 and 1983. Inoculations were done by the stem-slash method (7) with 30 cuts a t each of 3 sites on the plant stem. Most stems were 0.4 to 0.8 cm in diam a t the point of inoculation. Usually a minimum of 10 plants of each type were inoculated. Fresh, partially purified preparations were used for each inoculation test and Etrog citron plants were used to verify infectivity of each batch. Initially, cuts on citrus and citrus relatives were wrapped with Stericrepea (Beacon & Janis Ltd., London, W.C.I., England). Later, however, this was found unnecessary. Some succulent nonrutaceous plants were covered with plastic bags and on others the inoculation cuts were wrapped with "Parafilm" and the plants were not covered. Leaf midribs were also slashed on some herbaceous plants. After inoculation, the herbaceous plants were put in the shade for 48 hours before the plastic bags were removed. Citrus and most woody plants were cut back immediately to force new growth and the herbaceous plants were cut back 2 weeks after inoculation. Rapidly growing plants were cut back several times before testing for CTV infection. Three to 5 uninoculated control plants were kept for each species tested. Assay for infection. All citrus cultivars, species and relatives were examined periodically for vein clearing and the noncitrus plants were examined for vein clearing plus any other abnormalities. After the Etrog citron control plants showed vein-clearing symptoms, succulent stem bark tissue was collected from inoculated citrus Tristexa and Related Diseases and citrus relatives. Whole young stem was collected from succulent nonrutaceous hosts. Chopped tissue (0.5 g) was placed in a 2.5 cm-diameter glass tube with 5 ml of PBST-2 PVP buffer (phosphatebuffered saline with 0.05% Tween@ 20 and 2% polyvinylpyrrolidone,, m.w. 40,000) (2, 4) and homogenized about 15 seconds with a SDTTissumizer@ (Tekmar Co., Cincinnati, Ohio 45222). The extracts were filtered through glass wool and the ELISA tests were conducted by the double antibody sandwich method (4). The CTV antiserum used was prepared to unfixed, whole virus of the T-4 isolate (9). IgG was purified and conjugates to alkaline phosphatase prepared as described previously (2, 4). We used polystyrene Micro !ELISA@ plates (Dynatech Labs, Inc., Alexandria, Virginia 22314) with round-bottom wells. Healthy extracts, CTV-infected extracts, and buffer controls were included in each plate for reference. Plates were scored visually and results recorded by photography. In some cases, the absorbance a t 405 nm was also determined spectrophotometrically on diluted samples to verify assessment. Samples were not considered CTV-positive unless the O.D.,,, was 2X that of the