Freitas D M S, Nardin I, Shimoyama N, Souza-Dias J A C, Rezende J A M
Departamento de Fitopatologia e Nematologia, ESALQ/USP, 13418-900 Piracicaba, SP, Brazil.
Grupo MH Agro Horiguchi, 73850-000 Cristalina, Goiás, Brazil.
Plant Dis. 2012 Apr;96(4):593. doi: 10.1094/PDIS-12-11-1068-PDN.
Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for 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 ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system. References: (1) J. C. Barbosa et al. Plant Dis. 92:1709, 2008. (2) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (3) F. R. Fernandes et al. Trop. Plant Pathol. 35:43, 2010. (4) G. C. Wisler et al. Plant Dis. 82:270, 1998.
2011年6月,在巴西戈亚斯州克里斯塔利纳县的一片商业马铃薯作物中,发现了表现出卷叶和脉间失绿症状的马铃薯植株(Solanum tuberosum cv. Ágata),尤其是老叶上。该作物受到烟粉虱Bemisia tabaci生物型B的严重侵染。对有症状植株的四个马铃薯块茎进行了以下病毒检测:番茄褪绿病毒(ToCV)、马铃薯卷叶病毒(PLRV)、番茄严重皱缩病毒(ToSRV)和马铃薯Y病毒(PVY)。从每个块茎中分别提取总RNA,并使用HS-11/HS-12引物对进行逆转录(RT)-PCR,该引物对从报道的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。随后,通过巢式PCR用特异性引物ToC-5/ToC-6对RT-PCR产物进行ToCV检测(2)。从分别从三个块茎中提取的总RNA中扩增出的463 bp扩增子进行了纯化并直接测序。448 bp的三个共有序列(GenBank登录号JQ288896、JQ288897和JQ288898)之间的比较分别显示,与巴西ToCV番茄分离株的报道序列(GenBank登录号EU868927)(1)的同一性为98%、100%和100%。对于ToSRV检测,从两个块茎中提取总DNA,并用特异性引物通过PCR扩增出一段约820 bp的片段(3)。通过双抗体夹心ELISA(SASA,爱丁堡,苏格兰)分别对两个和三个块茎中的PLRV和PVY进行了检测。将无毒的烟粉虱生物型B分别转移到感染ToCV的马铃薯和番茄叶片上,获取期为24小时。将30只带毒粉虱转移到四株幼嫩的、已发芽的马铃薯植株cv. Ágata上(每种病毒分离株接种两株),接种期为24小时。接种37天后,一株接种ToCV马铃薯分离株和番茄分离株的植株分别在老叶上出现了卷叶和脉间失绿症状,与PLRV诱导的症状相似。Wisler等人(4)在美国报道了用ToCV对马铃薯植株进行实验性感染,诱导出了类似于PLRV感染的卷叶症状。ToCV的马铃薯分离株也通过烟粉虱传播到了两株接种的番茄植株中的一株上。如上所述,通过巢式RT-PCR检测了所有接种植株中ToCV的存在。据我们所知,这是世界上关于在田间马铃薯植株中检测到ToCV的首次报道。鉴于ToCV在世界上许多国家都有发生,它由一种世界性昆虫传播,并且它诱导的症状与PLRV相似,这一发现对依赖田间的种薯繁殖、病毒检测员和认证系统发出了警报。参考文献:(1)J. C. Barbosa等人,植物病害92:1709,2008。(2)C. I. Dovas等人,植物病害86:1345,2002。(3)F. R. Fernandes等人,热带植物病理学35:43,2010。(4)G. C. Wisler等人,植物病害82:270,1998。