In June 2011, extensive bleaching and numerous small whitish spots on leaves were observed in an onion (Allium cepa) seed crop as well as chlorotic spots and streaks in the neighboring garlic (A. sativum) bulb crop in the Aleksandrovo locality (Central Banat District, Serbia). Affected plants occurred throughout the field and disease incidence was estimated at 60% in the onion and 40% in the garlic crop. A high population of Thrips tabaci that was found in both crops, and local necrotic spots on Petunia × hybrida mechanically inoculated with infected onion or garlic sap by a chilled 0.01 M phosphate buffer, pH 7.0, containing 0.1% sodium sulfite (1), suggested the presence of a Tospovirus. For these reasons, sampled symptomatic onion and garlic plants were tested for the presence of Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) using commercial double-antibody sandwich-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy onion and garlic tissue were included in each ELISA. Of the 18 onion and 10 garlic plants tested, 16 and 7 samples, respectively, were positive for TSWV, and all were negative for IYSV. The identity of TSWV was further confirmed by conventional reverse transcription (RT)-PCR analysis. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using TSWV-specific forward (5′-GGTTAAGCTCACTAAGAAARCA-3′) and reverse primers (5′-TTTAACYCCRAACATTTCATAGA-3′), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs obtained from plants infected with a Serbian isolate of TSWV (GenBank Accession No. GQ373173) and healthy onion garlic plants were used as positive and negative controls, respectively. An amplicon of the expected size was produced from the 16 onion and 7 garlic ELISA-positive plants, but not from healthy controls. The amplified products derived from the two selected isolates, 114-11 from onion and 115-11 from garlic, were sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen); the sequences obtained were allocated GenBank Accession Nos. JQ619234 and JQ619235, respectively. Sequence analysis of the partial N gene, conducted with MEGA5 software (4), revealed 99.9% nucleotide identity (100% amino acid identity) between the two Serbian Allium isolates. Serbian onion and garlic isolates showed the highest nucleotide identities of 100% and 99.9% with Serbian summer squash isolate (JF303081) and tobacco isolate from Montenegro (GU369729), respectively. Well-established in many European countries, TSWV has been reported as an important constraint to the production of tomato, pepper, tobacco, and ornamentals (2), but the information on TSWV naturally infecting Allium spp. is limited. The presence of TSWV on onion and garlic in Serbia revealed that its known host range has expanded in Europe. To our knowledge, other than Marchoux's unpublished data (3), there are no other reports of garlic as a natural host of TSWV. The TSWV presence on Allium spp. represents a serious threat for these crops in Serbia, considering that it is prevalent in other crops in the area and its vectors are widespread. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) H. R. Pappu et al. Virus Res. 141:219, 2009. (3) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.
Efficient RT-PCR tool for tomato spotted wilt virus detection in its vectors Thrips tabaci and Frankliniella occidentalis