Recent characterization of cowpea aphid-borne mosaic virus ( CABMV ) in Bahia State , Brazil , suggests potential regional isolation

Woodiness disease is the most important disorder of passion fruit worldwide. The causal agent in Brazil is the Cowpea aphid-borne mosaic virus (CABMV), and despite the economic relevance of passion fruit for agriculture there have been recently very few studies about this virus in Brazil and worldwide. This work reveals the phylogenetic relationships of 10 newly identified CABMV isolates from Bahia State, the region where CABMV was first identified (in that time reported as PWV) in South America before its outbreak. The coat protein of 10 CABMV isolates (CABMV-Lns1 CABMV-Lns10) from Livramento de Nossa Senhora Country, Bahia State, were sequenced and presented very close identity between themselves (nucleotide: 97 to 99%, amino acid: 95 to 100%). They are phylogenetically closely related to Brazilian CABMV, however forming an isolated cluster within the Brazilian clade. According to previous evidences, our data demonstrate that CABMV-Lns are more closely related to isolates from Southern rather than from Northern Africa. Other two isolates from Bahia State clustered separately from CABMVLns, but together with isolates from other Brazilian regions thus suggesting that CABMV-Lns are a strain likely restricted to Bahia. The characterization of new populations of CABMV enables greater resolution of the evolution of viruses causing woodiness disease in passion fruit vines. Our data shed light on an as yet unexplored population of CABMV in Brazil and contributes to the understanding of its evolutionary history.


INTRODUCTION
The passion fruit is one of the most important crops in Brazil.It belongs to Passiflora genus and Passifloraceae family.Viral diseases are the most severe in terms of passion fruit yield loss (Sokhandan et al., 1997;Gibbs et al., 2008b).This is mainly due to the lack of both quick and accurate diagnosis as well as effective methods for disease control (Andrade and Pio-Ribeiro, 2001).In Brazil and Africa woodiness disease is caused by Cowpea aphid-borne mosaic virus (CABMV) (McKern et al., 1994;Nascimento et al., 2006;Maciel et al., 2009).This is in contrast to Australia where the passion fruit woodiness virus (PWV) is the most common causal agent (Sokhandan et al., 1997) and also to Asia where the same disease is caused by the East asia passiflora virus (EAPV) (Iwai et al., 2006).Its genus Potyvirus, family Potyviridae, contains more agriculturally important plant viruses than any other (Adams et al., 2005).Although in the past 15 years molecular studies about Brazilian CABMV have been carried out (Novaes and Rezende, 2003;Nascimento et al., 2004;Barros et al., 2011;Cerqueira-Silva et al., 2012;Nicolini et al., 2012), surprisingly none of them has phylogenetically investigated the virus from the region of largest passion fruit production in Brazil, the Livramento de Nossa Senhora County.Exploring the molecular variation of the coat protein (CP) in CABMV helps to better understand variability among strains, isolates and related species, to elucidate the evolutionary history of CABMV as well as assists to shed light on disease outbreaks in the course of passion fruit dispersion (Iwai et al., 2006;Gibbs et al., 2008a;Gibbs et al., 2008b).Bahia State is the place where passion fruit woodiness disease was firstly identified in Brazil during the 70's (Chagas et al., 1981) before its dissemination to most of the countries' fields.This led us to hypothesize whether Bahia State might host phylogenetically different isolates than the rest of Brazil that could be due to genomic mutation, recombination with other viral species, selective pressure or other evolutionary events.In this study we report a phylogenetic characterization of a population of CABMV from the State of Bahia, in comparison to other viruses causing fruit woodiness disease in passion fruit vines around the world.

Virus and plant resources
Ten CABMV isolates were collected from different passion fruit crop fields in the farming region of Livramento de Nossa Senhora Country, Bahia State, Brazil (13° 38′ 34″ S, 41° 50′ 27″ W).Ground extract from symptomatic leaves was used for inoculation on healthy passion fruit plants for maintenance, following the method described by Novaes and Rezende (2003).The 10 CABMV isolates here reported were named: CABMV-Lns1 to CABMV-Lns10.Several sequences from passion fruit woodiness disease-related viruses were acquired from public databases (Supplementary Table 1 and Figure 1).

RNA extraction, PCR, cloning and sequencing
RNA extraction was performed following the Arabidopsis Functional Genomics Consortium Trizol protocol (Arabidopsis Functional Genomics Consortium -AFGC, 2013).cDNA synthesis and PCR (Promega), DNA purification (PCR Purification Kit -NORGEN, Wizard SV Gel and PCR Clean-Up System -Promega) and cloning (pGEM-T Easy Vector -Promega, Max Efficiency DH5α Competent Cell -Invitrogen) were performed as essentially as described in the manufacturer's protocol.Newly designed primers for both cDNA synthesis and PCR were:

Sequencing and phylogenetic analysis
Sequencing of at least three independent clones from each isolate was performed twice by Macrogen (South Korea).The sequences were manipulated using DNA Baser Sequence Assembler v2 (DNA Baser Sequence Assembler v2.x, 2010).Phylogenetic tree was constructed using MEGA5 (Tamura et al., 2011).For nucleotidebased tree, Neighbor-Joining (NJ) and BioNJ pairwise distances matrix were estimated by maximum composite likelihood (MLC), estimated under bootstrap values higher than 70% (1,000 replications).

Analysis of coat protein sequence
Complete CP coding sequence containing 828 nucleotides and 275 amino acids were identified in all ten CABMV-Lns isolates, in agreement with the sequenced CABMV genomes (Mlotshwa et al., 2002;Barros et al., 2011).Very little variation was found amongst the nucleotide sequence of CABMV-Lns isolates (identity from 97 to 99%) (Table 1).Comparison of CABMV-Lns and other CABMV isolates showed sequence identity varying from 78 to 94%, where the lowest identity was found with CABMV-Mor, CABMV-Iba and CABMV-Monguno isolates (78%) and the highest with CABMV-Brs isolate (94%).The identity of amino acid sequences amongst CABMV-Lns isolates ranged from 95 to 100% identity (Table 1).When compared to other CABMV isolates, it ranged from 80 to 96%, when the least identity was found with CABMV-Bnt isolate (80%) and the highest with CABMV-TC1 isolate (96%).The CP conserved motifs of potyviruses were entirely conserved in nearly all CABMV-Lns isolates, with exception of CABMV-Lns6 which has a substitution of aspartic acid by an alanine residue at the DAG motif (Supplementary Figure 2).

CABMV-Lns9
CABMV  1).Interestingly they clustered separately from CABMV-Jgr and CABMV-Itb isolates also identified in Bahia State, which are more closely related to isolates from the States of Pernambuco and Paraíba, located around 1,200 km and 1,350 km away from Livramento de Nossa Senhora, respectively.PWV and EAPV (Iwai et al., 2006), which are well known different species from CABMV, formed a distinct clade also separately from CABMV-Lns (Figure 1).

DISCUSSION
The high identity of nucleotide and amino acid sequences amongst the ten CABMV-Lns isolates is strong evidence that they belong to the same strain of CABMV.This information is of great relevance for purposes of disease control, once most of the attempts to overcome woodiness disease in passion fruit such as premunization with mild strains of CABMV/PWV or pathogen-derived resistance have been hindered by variant strains of the same virus (Novaes and Rezende, 2003;Alfenas et al., 2005, Trevisan et al., 2006;Cerqueira-Silva et al., 2014).On the other hand, CABMV-Lns clustered separately from the isolates CABMV-Jgr and CABMV-Itb, which were also collected in Bahia State.As CABMV-Jgr and CABMV-Itb clustered together with isolates from rather far regions, the data suggest that CABMV-Lns are likely a strain restricted to Bahia State.This finding proposes either a single introduction of this strain to the area or a constraint in dissemination of this strain to other places by plant material, since there is no evidence that the virus is seed-transmitted in Passion fruit.According to the demarcation criteria for the Potyvirus genus (King et al., 2011), our data support the hypothesis that CABMV-Lns isolates are a different strain from CABMV-Prp, CABMV-Cr, CABMV-Vni, CABMV-Z, CABMV-Iba, CABMV-Monguno, CABMV-Mor, CABMV-MG-Avr, CABMV-PE-Bnt (80 to 89% amino acid identity).Additionally, our data confirm the higher sequence identity and closer phylogenetic relationship of CABMV-Lns with Southern African CABMV rather than isolates from the North of Africa, which was also previously suggested (Nascimento et al., 2006).The slave trade between Africa and South America is the likely event for introduction of CABMV in Brazil (Gibbs et al., 2008b), by which infected biological material could have been transported from the South rather than the North of Africa.However, we are aware that the limited number of CABMV sequences currently available, associated to the little information about African CABMV infecting Passion fruit plants, significantly narrow down the possibilities of drawing more precise conclusion on this regard.With reference to the important CP conserved motifs of potyviruses, the DAG motif is related to transmissibility of potyviruses by insects (Abdullah et al., 2009).Isolate CABMV-Lns6 shows a substitution of aspartic acid by alanine it to the DAG motif (Supplementary Figure 1).Mutations in this region significantly reduced transmissibility by insects in previous studies (Abdullah et al., 2009;López-Moya et al., 1999).However, unfortunately transmissibility experiments to investigate the consequences of the mutated DAG motif on the transmissibility capacity of CABMV-Lns6 were not suitable before the submission of this work.

Conclusion
Our data precisely demonstrate that CABMV-Lns belong also to CABMV species.These isolates can be classified as a strain of CABMV thus far occurring only in Bahia State and phylogenetically more distant from other isolates occurring in the same region.In addition and accordingly to previous reports, they are more closely related to Southern rather than Northern African CABMV.The exploitation of a larger number of viral samples from other as yet unexplored places in the promising State of Bahia will help to shed light on the events contributing to the observed regional isolation of CABMV-Lns.The whole genome sequencing of CABMV-Lns is ongoing and will allow us to comprehend their evolutionary history with regard to other related viral species around the world.Sokhandan et al. (1997) Abbreviations: CABMV, Cowpea aphid-borne mosaic virus; PWV, passion fruit woodiness virus; EAPV, East asia passiflora virus.

Figure 1 .
Figure 1.Molecular phylogenetic tree of viral species that cause woodiness disease in Passion fruit plants.The nucleotide coding sequence for the Coat Protein was used to generate the tree.The relationships were inferred using the Maximum Likelihood method based on the General Time Reversible model (highest log likelihood -10235.3876 is shown).A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.8776)).The branch length is measured by number of substitutions per site.■ CABMV-Lns, • CABMV from Bahia State, CABMV from Brasília, ○ Other CABMV, □ EAPV and ∆ PWV.

Table 1 .
Percentage identity of nucleotide (above the diagonal) and amino acid (below the diagonal) sequences of the coding CP gene from ten CABMV-Lns isolates.