First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.
First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.
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DOI：
10.1094/pdis-06-13-0588-pdn
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发表时间：
2014-03
期刊：
影响因子：
4.5
通讯作者：
M. Melzer;J. Shimabukuro;M. Long;Scot Nelson;Anne M. Alvarez;W. Borth;John S. Hu
中科院分区：
文献类型：
--
作者：
M. Melzer;J. Shimabukuro;M. Long;Scot Nelson;Anne M. Alvarez;W. Borth;John S. Hu
2013年2月，在夏威夷州檀香山的社区花园中观察到一种观赏蜡花（Hoya calycina Schlecter），其叶片显示出围绕番茄斑萎病毒感染典型的凹陷坏死病变的同心褪绿和坏死环。根据生产商的说明书，使用TSWV ImmunoStrips（AgDia，埃尔克哈特，印第安纳州）测定，症状叶组织对番茄斑萎病毒（TSWV）（夏威夷常见的番茄斑萎病毒）检测为阴性。从有症状的叶片中分离双链RNA，并使用随机引物进行逆转录（2）。然后将cDNA用作通用番茄斑萎病毒PCR测定中的模板，使用引物gL 3637和gL 4435 c，其扩增编码番茄斑萎病毒的RNA依赖性RNA聚合酶的L片段的序列（1）。使用pGEM-T Easy（Promega，麦迪逊，WI）扩增和克隆~800-bp产物。通过在夏威夷大学的基因组学、蛋白质组学和生物信息学高级研究实验室进行的染料终止子测序，选择三个克隆并发现它们是相同的。在引物序列修剪后，发现773-bp序列（GenBank登录号KF 030938）分别与辣椒褪绿病毒（CaCV;布尼亚病毒科，番茄斑萎病毒属的暂定种）菌株Ch-Har（GU 199334）、TwTom 1（HM 021140）和AIT（DQ 256124）具有97、88和87%的同一性。为了确认CaCV的存在，还将cDNA用作通用番茄斑萎病毒PCR测定中的模板，其中引物3 'T12和TsMCR 2扩增番茄斑萎病毒的S区段的区域（3）。如上所述克隆和测序来自该测定的扩增产物，发现其与GenBank中存在的CaCV核苷酸序列具有93 - 98%的同一性。尝试使用西瓜银斑驳病毒和花生芽坏死病毒三抗体夹心ELISA（AgDia）检测蜡花中的CaCV菌株是不成功的。社区花园中没有其他植物具有典型的番茄斑萎病毒样症状;然而，来自番茄（Solanum lycopersicum L.;两个样品）、辣椒（Capsicum spp.; 4个样品）、茄子（Solanum melongena L.;一个样品）和具有病毒样症状的西番莲（西番莲西姆斯;一个样品），并使用NucleoSpinRNAII试剂盒（Macherey-Nagel，Bethlehem，PA）分离RNA。使用引物gL 3637和gL 4435进行RT-PCR试验，在这些样品中均未检出番茄斑萎病毒。感染CaCV的蜡花植物立即被社区花园成员移走并销毁，防止进行任何额外的血清学或生物学测定。CaCV由几种蓟马传播，包括存在于夏威夷的蓟马帕尔米。蜡花不是原产于夏威夷，目前还不清楚CaCV是否在这种植物中进入夏威夷，或者它是否被含病毒的蓟马感染。调查CaCV在已知的主机是必不可少的，以确定CaCV在夏威夷的地理分布，因为这种病毒构成了相当大的威胁，番茄，辣椒，蝴蝶兰生产在夏威夷和美国。参考文献：（1）F.- H. Chu等人，Phytopathology 91：361，2001. (2)M. J. Melzer等人，Virus Genes 40：111，2010. (3)M. Okuda和K.花田J. Virol. Methods 96：149，2001.
In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States. References: (1) F.-H. Chu et al. Phytopathology 91:361, 2001. (2) M. J. Melzer et al. Virus Genes 40:111, 2010. (3) M. Okuda and K. Hanada. J. Virol. Methods 96:149, 2001.