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Molecular Characterization of a Novel Begomovirus Infecting Malva (Gaya guerkeana K. Schum.).

The family Geminiviridae encompasses several species of phytoviruses that have a genome composed of circular single-stranded DNA (ssDNA) encapsidated in geminated quasi-icosahedral particles (Brown et al., 2012). The family consists of the genera Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus and Turncurtovirus, classified according to insect vector type, host range, genomic organization and phylogenetic relationship (Varsani et al., 2017; Zerbini et al., 2017). Begomoviruses are transmitted by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and are widely spread in tropical and subtropical regions, where they are considered a limiting factor for the production of economically important crops (Morales, 2010; Navas-Castillo et al., 2011). In Brazil, bean (Phaseolus spp.) and tomato (Solanum lycopersicon L.) crops are the most severely affected (Morales, 2010; Navas-Castillo et al., 2011).

Besides infecting cultivated plants, begomoviruses are found in a wide range of wild/non-cultivated hosts, especially those belonging to the families Malvaceae, Euphorbiaceae, and Fabaceae (Morales, 2010). Non-cultivated plants may act as alternative begomovirus hosts when cultivated species do not exist in the field or as a source of novel begomoviruses that arise via recombination in cases of mixed infections (Barreto et al., 2013; Rocha et al., 2013).

In this context, this work aimed at the molecular characterization of a novel begomovirus infecting the non-cultivated plant Gaya guerkeana.

A leaf sample of G. guerkeana presenting symptoms indicative of begomovirus infection (yellowing and mosaic) was collected in Caruaru, Pernambuco (PE) in 2010 (Figure 1), and the total DNA was extracted from plant tissue, according to the protocol described by Doyle & Doyle (1987). Viral genomic components were amplified via Rolling Circle Amplification (RCA) according to the method described by Inoue-Nagata et al., (2004). Aliquots of the amplification were individually submitted to cleavage through BamHI and HindIII endonucleases. The products of the cleavage reactions containing fragments of approximately 2600 nucleotides, corresponding to one copy of each genomic component, were used for binding to the pBluescript KS+ vector (Stratagene), previously linearized through the same enzyme and dephosphorylated. Potential recombinant plasmids were used to transform ultracompetent cells of Escherichia coli DH5[alpha] strain by the heat shock method. The obtained clones were commercially sequenced via primer walking.

Contigs of the complete viral genomic component were assembled using the DNAMAN software version 6 (Lynnon Biosoft Corporation). The obtained sequence was initially analyzed through the BLASTn algorithm and the non-redundant nucleotide database GenBank to determine the viral species with the highest percentage of sequence identity. Similar sequences from GenBank were used for species demarcation of the novel begomovirus isolate using the Sequence Demarcation Tool v. 1.2 (Muhire et al., 2013).

Multiple nucleotide sequence alignments for the DNA-A dataset of the G. guerkeana isolate and other begomoviruses from Brazil were prepared using the MUSCLE algorithm and manually adjusted in the MEGA6 package (Tamura et al., 2013). Bayesian Inference for DNA-A was performed on the CIPRES web portal (https://www.phylo.org/) using MrBayes v. 3.2.3 (Ronquist et al., 2012). The GTR+G+I model of evolution was used for the GaYMV DNA-A dataset. The analysis was run for 10 million generations using four chains and sampling at every 1,000 generations for a total of 10,000 trees. The first 2,500 trees were discarded as a burn-in phase. Posterior probabilities (Rannala & Yang, 1996) were determined from a consensus tree majority-rule generated with the 7,500 remaining trees. Trees were viewed and edited using the software FigTree v. 1.4 (ztreebio.ed.acuk/software/figtree) and Inkscape (https://inkscape.org/pt/).

A clone (BR-Car1-12A) was obtained from the G. guerkeana sample collected in Caruaru (PE). BLASTn analysis showed that the genomic component corresponded to DNA-A. Using pairwise comparisons and the [greater than or equal to]91% identity criteria for complete DNA-A sequences established by the Geminiviridae Study Group of International Committee on Taxonomy of Viruses (ICTV) for the demarcation of species within the Begomovirus genus (Brown et al., 2015), the clone obtained herein was classified as a putative new species of Begomovirus. BR-Car1-12A was more closely related to the Sida mottle Alagoas virus (SiMoAV, JX871386), displaying an 84.8% nucleotide identity, and the name Gaya yellow mosaic virus (GaYMV) is proposed.

GaYMV presented a genomic organization typical of New World begomoviruses, with five ORFs in DNA-A. The conserved nonanucleotide (5' TAATATT/AC 3') was detected in the common region, which is part of the stem-loop that contains the viral replication origin. We also identified repeated GGGGG/GGGGG sequences (iterons), which are essential to initiate viral replication.

In the Bayesian phylogenetic tree, GaYMV grouped with SiMoAV, Sida mosaic Alagoas virus (SiMAV), and Sida yellow blotch virus (SiYBV), all obtained from Sida spp. from the State of Alagoas (Tavares et al., 2012; Wyant et al., 2012) (Figure 2). These results agree with those found in pairwise comparison analysis, in which the BR-Car1-12A isolate has a higher identity percentage with the SiMoAV species.

Several studies have shown that, as it occurs with cultivated plants, the diversity of begomovirus species associated with weeds is high, especially those belonging to the families Malvaceae, Solanaceae and Fabaceae (Castillo-Urquiza et al., 2008; Silva et al., 2012; Tavares et al., 2012). Standing out among the malvae, members of the genera Sida and Abutilon show wide begomovirus genetic diversity, and some cultivated species are also infected by this virus group (Castillo-Urquiza, et al., 2008; Tavares et al., 2012; Wyant et al., 2012; Albuquerque et al., 2013).

In Brazil, Sida spp. have been frequently described as hosts for several begomovirus species, and may play an important epidemiological role as a source of begomovirus for cultivated species, especially tomato plants (Castillo-Urquiza et al., 2008; Tavares et al., 2012). This hypothesis is reinforced by the detection of Sida micranta mosaic virus, SiMoV and Tomato mild mosaic virus occurring in both tomato plants and Sida spp. in field conditions (Castillo-Urquiza et al., 2007; Cotrim et al., 2007; Rocha et al., 2013).

Although this is the first report of a begomovirus infecting the wild species G. guerkeana (Malvaceae), this result indicates that this uncultivated plant may constitute an important reservoir and source of novel begomoviruses. However, additional studies are needed to evaluate the epidemiological role of these hosts for begomovirus outbreaks in cultivated species.

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Mayra Machado de Medeiros Ferro (1); Arthur Alencar Raposo Tenorio (1); Sarah Jacqueline Cavalcanti da Silva (1); Janaine Rossane Araujo Silva (1); Roberto Ramos-Sobrinho (1*); Gaus Silvestre de Andrade Lima (1); Iraildes Pereira Assuncao (1*)

(1) Setor de Fitossanidade/Centro de Ciencias Agrarias, Universidade Federal de Alagoas, Rio Largo, AL, Brazil, 57100-000

(*) Corresponding authors: R. Ramos-Sobrinho, e-mail: cbrobertorsb@hotmail.com I.P. Assuncao, e-mail: iraildes.assuncao@ceca.ufal.br

Received: 12 May 2017

Accepted: 13 April 2018

DOI: 10.14295/CS.v9i3.2083
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Title Annotation:Article
Author:de Medeiros Ferro, Mayra Machado; Tenorio, Arthur Alencar Raposo; da Silva, Sarah Jacqueline Cavalca
Publication:Comunicata Scientiae
Article Type:Report
Geographic Code:3BRAZ
Date:Jul 1, 2018
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