Barbosa J C, Teixeira L D D, Rezende J A M
Departmento de Fitopatologia e Nematologia, ESALQ/USP, 13418-900 Piracicaba, SP, Brazil.
Plant Dis. 2010 Mar;94(3):374. doi: 10.1094/PDIS-94-3-0374C.
In June of 2009, sweet pepper (Capsicum annuum cvs. Elisa and Prador) plants exhibiting interveinal chlorosis, some necrosis, and mild upward leaf curling on the intermediate leaves were found in three protected crops in the municipality of São Miguel Arcanjo, São Paulo state, Brazil. Incidence of symptomatic plants varied from 70 to 100%. Abundant whitefly adults were seen in all crops. Initially, total DNA was separately extracted from seven symptomatic plants and submitted to a PCR reaction using the universal primer pairs PAL1v1978/PAR1c496 and PBL1v2040/PCRc1 for begomovirus (3). The results were negative. The same samples were also analyzed for infection with Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). Total RNA was extracted separately from leaves of each symptomatic plant and used for one-step reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for TICV and ToCV (1). The RT-PCR product was subsequently tested by nested-PCR for single detection of TICV and ToCV using primer pairs TIC-3/TIC-4 and ToC-5/ToC-6, respectively (1). Only one fragment of approximately 463 bp was amplified from the five plants with the primer pair specific for ToCV. No amplification was obtained with the primers specific for TICV. Four purified amplicons of 463 bp were directly sequenced in both directions. Sequence comparisons of the 419-bp consensus sequence, encompassing nucleotides 750 and 1,167 of the HSP-70 homolog gene, revealed 98% identity with the reported sequences of tomato infecting isolates of ToCV from Brazil (GenBank Accession No. EU868927) and the United States (GenBank Accession No. AY903448). Virus-free adults of Bemisia tabaci biotype B were confined on symptomatic pepper leaves for a 48-h acquisition access period. Twenty adults were transferred to one plant of sweet pepper cv. Magda for a 24-h inoculation access period. The sweet pepper plant exhibited the original symptoms on the leaves 67 days after inoculation under greenhouse conditions. Infection by ToCV was confirmed by RT-PCR. The susceptibility of sweet pepper plants to ToCV was previously reported in Spain (2), whereas in the United States, this species was experimentally found as nonhost for this virus (4). Further studies are needed to better understand the variable susceptibility of sweet pepper to ToCV. References: (1) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (2) G. Lozano et al. Plant Dis. 88:224, 2004. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) W. M. Wintermantel et al. Plant Dis. 90:814, 2006.
2009年6月，在巴西圣保罗州圣米格尔·阿坎茹市的三个保护地作物中，发现甜椒（辣椒品种伊丽莎和普拉多）植株出现脉间黄化、一些坏死以及中部叶片轻度向上卷曲的症状。有症状植株的发病率在70%至100%之间。在所有作物中都发现了大量的烟粉虱成虫。最初，从七株有症状的植株中分别提取总DNA，并使用针对双生病毒的通用引物对PAL1v1978/PAR1c496和PBL1v2040/PCRc1进行PCR反应（3）。结果为阴性。对相同样本也进行了番茄感染性褪绿病毒（TICV）和番茄褪绿病毒（ToCV）（纤毛病毒属，长线形病毒科）感染情况的分析。从每株有症状植株的叶片中分别提取总RNA，并使用HS - 11/HS - 12引物对进行一步逆转录（RT）-PCR，该引物对从已报道的TICV和ToCV热休克蛋白（HSP - 70）同源基因的高度保守区域扩增出一段587 bp的片段（1）。随后，通过巢式PCR分别使用引物对TIC - 3/TIC - 4和ToC - 5/ToC - 6对RT - PCR产物进行检测，以单独检测TICV和ToCV（1）。仅用针对ToCV的引物对从五株植株中扩增出了一条约463 bp的片段。用针对TICV的引物未获得扩增产物。对四个纯化的463 bp扩增子进行双向直接测序。对包含HSP - 70同源基因核苷酸750至1167的419 bp共有序列进行序列比较，发现与来自巴西（GenBank登录号EU868927）和美国（GenBank登录号AY903448）的已报道的感染番茄的ToCV分离株序列具有98%的同一性。将烟粉虱生物型B的无病毒成虫限制在有症状的甜椒叶片上48小时的获毒期。将20只成虫转移到一株甜椒品种玛格达植株上进行24小时的接种期。在温室条件下接种67天后，甜椒植株的叶片出现了最初的症状。通过RT - PCR证实了ToCV的感染。此前在西班牙报道过甜椒植株对ToCV的易感性（2），而在美国，通过实验发现该品种对这种病毒是非寄主（4）。需要进一步研究以更好地了解甜椒对ToCV易感性的差异。参考文献：（1）C. I. Dovas等人，《植物病害》86:1345，2002年。（2）G. Lozano等人，《植物病害》88:224，2004年。（3）M. R. Rojas等人，《植物病害》77:340，1993年。（4）W. M. Wintermantel等人，《植物病害》90:814，2006年。
