Indigenous and introduced species of the Bemisia tabaci complex in sweet potato crops from Argentina

Sweet potato (Ipomoea batatas (L.) Lam) is one of the most important crops worldwide. Recently, the appearance of severe viral symptoms has been observed in sweet potato crops in the pampas region of Argentina and both begomovirus and crinivirus, exclusively transmitted by whiteflies, have been identified. The aim of this study was to identify B. tabaci species from sweet potato crops in Colonia Caroya by analysing mitochondrial cytochrome c oxidase subunit I (mtCOI) sequences. Two previously described haplotypes were identified: New World2 (indigenous species) and MEAM1 (introduced species). The results indicate the presence of both species, which are potential vectors of begomovirus and crinivirus in Argentina.

Middle East-Asia Minor 1 (hereafter MEAM1 and commonly referred to as the B biotype in the literature) is known worldwide as one of the most invasive and destructive species in the complex (Perring, 2001).In Argentina, MEAM1 was first detected on horticultural crops, weeds and cotton samples (Viscarret et al., 2003).Furthermore, recent studies have demonstrated the presence of an indigenous species belonging to the B. tabaci cryptic species complex in Argentina, a new species affecting soybean and bean crops named New World2 (Alemandri et al., 2012).
Sweet potato (Ipomoea batatas (L.) Lam) is one of the most important crops worldwide, with great potential not only for human consumption but also for animal feeding and industrial use (Bovell-Benjamin, 2007).Argentina produced 340,105 metric tons in 2009 (USDA Economics and System, 2011), mainly in the pampas region (Buenos Aires, Córdoba and Santa Fe provinces).Recently, severe viral symptoms have been observed in this area.Previous studies suggest that more than one etiologic agent is involved in this new viral disease, including both begomovirus and crinivirus exclusively transmitted by whiteflies (Di Feo et al., 2000;Rodríguez Pardina et al., 2012).Although there are records of presence of whiteflies, the species of B. tabaci present in this area have not been identified.The aim of this work was to identify species of B. tabaci present in sweet potato crops from Colonia Caroya in Argentina by analysing mitochondrial cytochrome c oxidase subunit I (mtCOI) sequences.
Adult whiteflies (Bemisia tabaci) were collected from five sweet potato fields in Colonia Caroya, Colón department, Córdoba province (31º 02´ 02´´ S, 64º 04´ 46´´ W), in the pampas region, an important sweet potato growing area in Argentina.Live adult whiteflies were collected and placed in tubes containing 100% ethanol and maintained at 4°C until DNA extraction.
Whitefly individuals were sexed, and DNA was extracted from females, following the protocol of De Barro & Driver (1997) and Truol et al. (2003).A total of 10 females were analysed.Females were individually homogenized in 60 ml filtered lysis buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.3% Triton X-100, 60 mg/ml proteinase K) using 1.5 ml microcentrifuge tubes.The homogenate was incubated at 65 ºC for 15 min and at 98 ºC for 6 min.Then, it was centrifuged at 9600 rpm for 1 min and maintained at -20 ºC.The primers C1-J-2195 and L2-N-3014 were used for amplification (Simon et al., 1994;Frohlich et al., 1999).PCR reactions were carried out in a final volume of 25 μl containing 1 U Taq DNA polymerase (Invitrogen), 2.5 mM MgCl 2 , 0.20 mM dNTP, 0.6 μM of each primer and 2 μl of DNA.The reactions were performed with a thermal cycler programmed for one cycle of 2 min at 94 °C, followed by 30 cycles (1 min at 94 °C, 1 min at 52 °C and 1 min at 72 °C) and a final 5-min extension at 72 °C.Amplification products were analysed by 1.5% agarose gel electrophoresis.The amplified products of about 800 base pairs were purified using illustra MicroSpin S-200 HR Columns (GE Healthcare, UK) and sequenced in both directions with the amplification primers using an ABI 3130XL (Applied Biosystems) automated sequencer.
A total of 438 sequences were used to identify unique haplotypes of B. tabaci mtCOI sequences, which included 10 from our own whitefly collection and 428 unique haplotypes retrieved from GenBank.The latter comprised the sequences used in Firdaus et al. (2013) (which in turn included nine outgroups), newly proposed groups (Chowda-Reddy et al., 2012;Parrella et al., 2012;Esterhuizen et al., 2013) and the New World2 sequences recently detected in Brazil (Marubayashi et al., 2012).All the mtCOI sequences were 657 bases in length and were aligned using the CLUSTAL W Multiple alignment procedure (Thompson et al., 1994) implemented in the BIOEDIT software, version 7.0.4(Hall, 1999).A total of 428 unique B. tabaci mtCOI sequence haplotypes were identified from the analysis of 438 sequences using DnaSP v5 (Librado & Rozas, 2009).
Of the 10 mtCOI sequences generated in this study, two haplotypes were identified (Hap-1 and Hap-2) that belong to two species of the B. tabaci complex in sweet potato crops in Argentina.
Hap-1 was identical to a New World2 haplotype (JF901844) previously described in bean and soybean crops in Argentina (Alemandri et al., 2012).Hap-2 was identical to a MEAM1 haplotype (GU086344) previously described in USA, Dominican Republic, Trinidad and Tobago, Brazil, Spain, Italy and South Africa (Firdaus et al., 2013).Because the sequences generated in this study were not new haplotypes, phylogenetic analyses or pairwise comparison of genetic distances were not performed.Haplotypes previously described and identical to the sequences presented here were correctly identified using the new criteria proposed for B. tabaci (Alemandri et al., 2012;Firdaus et al., 2013).These two whitefly species were found to co-occur in three fields (Table 1).
The analysis of mtCOI supports the existence of two species of the B. tabaci complex in sweet potato fields in Argentina: an indigenous species belonging to the New World2 putative species, and an introduced species belonging to the MEAM1 putative species.This is the first report of MEAM1 in sweet potato in Argentina, and is in agreement with a previous study reporting the presence of MEAM1 in sweet potato in Cuba using information on the mtCOI gene (Muniz et al., 2011).Furthermore, several species, Asia II 1 (Liu et al., 2010), Asia II 6 (Ueda et al., 2009), Asia II 9, Asia II 10 (Hu et al., 2011), Indian Ocean (Delatte et al., 2005), Uganda (Maruthi et al., 2004), Mediterranean (De la Rua et al., 2006) and New World (Muniz et al., 2011) were found in I. batatas in other countries.To the best of our knowledge, this is the first report of New World2 in sweet potato from Argentina.
On the other hand, the results presented here suggest that New World2 and MEAM1 cooccurred in sweet potato under field conditions (Table 1).Similar events have been noted in Brazil, where specimens of the MEAM1 and New World2 species were found colonizing a single plant of Glycine maxima, or the same plot on Euphorbia heterophylla (Marubayashi et al., 2012).Previous studies indicate that the development of a hybrid whitefly between two species such as MEAM1 and Mediterranean is unlikely (Sun et al., 2011;McKenzie et al., 2012).There are no records of the development of a hybrid between New World2 and MEAM1.
The identification of the two species of the B. tabaci complex in sweet potato crops suggests that they would be the insect vectors responsible for sweet potato begomovirus and crinivirus transmission in Argentina.The present results highlight the need to further explore the diversity of the B. tabaci species present in sweet potato crops in the pampas region, as well as to conduct virus transmission experiments.

Table 1 .
Populations of Bemisia tabaci sampled in sweet potato crops in Argentina and identified by sequencing of the mtCOI gene.