Cheng Y H, Deng T C, Chen C C, Liao J Y, Chang C A, Chiang C H
Plant Pathology Division, Agricultural Research Institute, Wufeng, Taichung, Taiwan.
Graduate Institute of Biochemical Science and Technology, Chaoyang University of Technology, Wufeng, Taichung, Taiwan.
Plant Dis. 2011 May;95(5):617. doi: 10.1094/PDIS-10-10-0721.
Bell pepper (Capsicum annuum L.) plants exhibiting systemic mild mosaic, vein yellowing, and leaf malformation were collected from Puli City in 2006. Double-antibody sandwich (DAS)-ELISA was used to test these samples for Chilli veinal mottle virus (ChiVMV) infection using polyclonal antibodies. In addition, Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana plants were mechanically inoculated with sap extracted from collected samples. Ten days postinoculation, chlorotic local lesions were observed on inoculated leaves of C. quinoa and C. amaranticolor plants, whereas, systemic mosaic and foliar distortion symptoms were developed on upper leaves of N. benthamiana plants. The DAS-ELISA test showed that field-collected pepper samples and inoculated leaves of C. quinoa and C. amaranticolor were infected with ChiVMV, while N. benthamiana with mosaic symptoms did not react with ChiVMV antibodies. To confirm ChiVMV, field-collected samples as well as mechanically inoculated plants were tested by reverse transcription (RT)-PCR using the potyvirus degenerate primers Hrp5/Pot1 (2). Amplified RT-PCR products were cloned and sequenced. Sequence analysis of amplified fragments (1.4 kb) revealed that field-collected pepper samples were infected with ChiVMV and Pepper mottle virus (PepMoV). The DNA fragment amplified from C. quinoa and C. amaranticolor showed high (99.2%) sequence identities with the CP gene of ChiVMV (3) (GenBank Accession No. AM909717). However, amplicons obtained from N. benthamiana plants (GenBank Accession No. HQ329082) that showed mosaic symptoms showed 83.6% to 98.7% nucleotide identities with PepMoV (GenBank Accession Nos. AB126033, AF227728, AF440801, AF501591, EU586133, and M96425). Next, a pure isolate of PepMoV was established on N. benthamiana by mechanical inoculation of diluted plant sap obtained from a PepMoV-infected N. benthamiana plant. Bell pepper plants inoculated with the Taiwan isolate of PepMoV developed mosaic and leaf distortion symptoms. Antiserum against the PepMoV Taiwan isolate was subsequently prepared by immunizing rabbits with purified virus particles. Using the prepared antiserum and specific primers (1) to detect PepMoV, ChiVMV, and Pepper veinal mottle virus (PVMV), three viruses could be readily detected and differentiated from diseased bell peppers in the field. In a survey done in 2007, 18 of 33 pepper samples from southern Taiwan were found with mixed infections of PepMoV and ChiVMV, seven samples were infected with PepMoV and PVMV, five samples were infected with PVMV, and another three samples were infected with ChiVMV. To our knowledge, this is the first report of the occurrence of PepMoV in bell peppers in Taiwan. References: (1) Y. H. Cheng et al. Plant Dis. 93:107, 2009. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.
2006年，从埔里市采集到表现出系统性轻度花叶、叶脉黄化和叶片畸形的甜椒（Capsicum annuum L.）植株。使用双抗体夹心（DAS）-ELISA方法，利用多克隆抗体检测这些样品是否感染辣椒脉斑驳病毒（ChiVMV）。此外，将藜麦（Chenopodium quinoa）、苋色藜（C. amaranticolor）和本氏烟草（Nicotiana benthamiana）植株用采集样品中提取的汁液进行机械接种。接种后10天，在藜麦和苋色藜植株的接种叶片上观察到褪绿局部病斑，而本氏烟草植株上部叶片出现系统性花叶和叶片扭曲症状。DAS-ELISA检测表明，田间采集的辣椒样品以及藜麦和苋色藜的接种叶片感染了ChiVMV，而出现花叶症状的本氏烟草与ChiVMV抗体无反应。为确认ChiVMV，使用马铃薯Y病毒属简并引物Hrp5/Pot1（2）通过逆转录（RT）-PCR对田间采集的样品以及机械接种的植株进行检测。对扩增的RT-PCR产物进行克隆和测序。对扩增片段（1.4 kb）的序列分析表明，田间采集的辣椒样品感染了ChiVMV和辣椒斑驳病毒（PepMoV）。从藜麦和苋色藜扩增的DNA片段与ChiVMV的CP基因（3）（GenBank登录号AM909717）显示出高度（99.2%）的序列同一性。然而，从表现出花叶症状的本氏烟草植株（GenBank登录号HQ329082）获得的扩增子与PepMoV（GenBank登录号AB126033、AF227728、AF440801、AF501591、EU586133和M96425）的核苷酸同一性为83.6%至98.7%。接下来，通过用从感染PepMoV的本氏烟草植株获得的稀释植株汁液进行机械接种，在本氏烟草上建立了PepMoV的纯分离物。接种PepMoV台湾分离物的甜椒植株出现花叶和叶片扭曲症状。随后，用纯化的病毒粒子免疫兔子制备了针对PepMoV台湾分离物的抗血清。使用制备的抗血清和特异性引物（1）检测PepMoV、ChiVMV和辣椒脉斑驳病毒（PVMV），可以很容易地从田间患病甜椒中检测和区分这三种病毒。在2007年进行的一项调查中，台湾南部33个辣椒样品中有18个被发现同时感染了PepMoV和ChiVMV，7个样品感染了PepMoV和PVMV，5个样品感染了PVMV，另外3个样品感染了ChiVMV。据我们所知，这是台湾甜椒中出现PepMoV的首次报道。参考文献：（1）Y. H. Cheng等人，《植物病害》93:107，2009年。（2）S. S. Pappu等人，《植物病害》82:1121，1998年。（3）W. S. Tsai等人，《植物病理学》58:408，2008年。
