Detection of Badnavirus in pineapple in northeastern Brazil Detecção de Badnavirus em abacaxizeiro no Nordeste do Brasil

Brazil is one of the main global producers of pineapple (Ananas comosus L. Merril), with emphasis in the Northeastern region of the country. Pineapple is exposed to several phytosanitary problems, including viruses. Pineapple badnaviruses are caused by two distinct species: Pineapple bacilliform CO virus (PBCoV) and Pineapple bacilliform ER virus (PBErV). The present study aimed to detect possible species of the genus Badnavirus in pineapple samples in Northeastern Brazil, via PCR and sequencing of the RT/RNaseH region. Leaf samples of pineapples were collected in the states of Alagoas, Maranhão, Paraiba and Pernambuco, and subsequently subjected to total DNA extraction and amplification via PCR. Badnavirus positive samples were selected for sequencing. Analysis of pairwise comparisons revealed that all sequences obtained in this work showed an identity greater than 80% with the sequence of the species PBCoV (EU377664), from Australia, fully corroborating with phylogenetic analyzes. These results suggest the widespread of PBCoV in Northeastern Brazil and record the first report of Badnavirus in pineapple culture in Brazil.


INTRODUCTION
Pineapple (Ananas comosus L. Merril) is a perennial, herbaceous monocotyledon, belonging to the family Bromeliaceae, with approximately 50 genera and 200 species.
From the economic point of view the genus Ananas is the most important in the family (CUNHA & CABRAL, 1999).
Brazil is one of the biggest pineapple producers in the world, with a production ranging from 2.2 to 2.7 million tons in the last decade (MATOS, 2018). In 2018, the Northeastern Brazilian region produced 590 thousand tons of pineapple, with the state of Paraiba standing as the biggest national producer (IBGE, 2018). Despite the high volume of pineapple produced in Brazil and in the world, this crop is exposed by many phytosanitary problems causing significant economic losses (MATOS et al., 2009), and viral diseases are included amongst these.
The genus Badnavirus in the most diverse within the family, with 59 recognized species by the International Committee on Taxonomy of Viruses (ICTV, https://talk.ictvonline.org/taxonomy/). The transmission occurs mainly by mealybugs (some by aphids) in a semi-persistent manner (BATH et al. 2016;GEERING & HULL, 2012) and is among the most important plant viruses groups with a DNA genome.
In 1995 the first occurrence of Pineapple bacilliform virus was reported in pineapple hybrids in Australia (WAKMAN et al., 1995) (GAMBLEY et al., 2008;SETHER et al., 2012). An association between the symptoms and the infection by Badnavirus in pineapple was not yet demonstrated (GAMBLEY et al., 2008).
In this context, the objective of the present study was to detect species from the genus Badnavirus in pineapple samples from the Northeastern Brazil via PCR and sequencing of the RT/RNaseH region.

Analysis and comparison of sequences
The sequences obtained were assembled using the CodonCode Aligner v. 4.1.1 (www.codoncode.com) and, initially, analyzed using the algorithm BLASTn (ALTSCHUL et al., 1990) and GenBank non-redundant nucleotide database

RESULTS AND DISCUSSION
A total of 287 samples were analyzed, with 24 of them being positive for badnavirus and amplification of fragments with the expected size (around 580 bb). Form these positive samples, 20 were selected for sequencing, from each sample collecting area, with a total of 100 samples, however, quality sequences were obtained only for 83 or them. Preliminary analysis using the algorithm BLASTn and comparing paired sequences with SDT revealed that sequences obtained in the present work have nucleotide identity above 80% with the isolate from the species Pineapple bacilliform CO virus -PBCoV (EU377664), from Australia (GAMBLEY et al., 2008b). This result shows that all sequences characterized in the present study, correspond to new isolates of PBCoV ( Figure 1).

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Phylogeny based in nucleotide sequences from domain RT/RNAseH of the badnaviruses ORF3, revealed that isolates in the present study were clustered with the sequence of PBCoV_EU377664 (Figure 2). This analysis showed that all 83 isolates formed a monophyletic branch (Group I) including the species PBCoV, which have a common ancestral with the species PBErV ( Figure 2). Subgroup I contains isolates collected from all sampled municipalities, while subgroup II includes the majority of isolates coming from the municipalities of Arapiraca and Coruripe, in the State of Alagoas. Group II includes all other species belonging to the genus Badnavirus ( Figure   2).

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The geographic proximity between the municipality of Arapiraca and the village of Pindorama (Coruripe), separated by only 66 km, is the probable reason for the gathering of isolates, once such circumstance fosters the exchange of seedlings between farmers in the region. Paired analysis integrally supports the results obtained by phylogeny, that is to say, groups I in the phylogenetic tree includes only isolates obtained in the present work with the sequence PBCoV, from Australia, which share more than 80% of nucleotide sequence identity in the paired analysis, a decisive criteria to determine species form the genus Badnavirus, according to ICTV (GEERING & HULL, 2012). Thomson et al. (1996) (DALLOT et al., 2001;DAHAL et al., 2000). Some stress conditions such as tissue culture, hybridization and recombinant events which occur in endogenous sequences, may lead to the reconstitution of the viral genome in the activated form, thus, resulting in episomal infections (CÔTE et al., 2010;DALLOT et al., 2001;NDOWORA et al., 1999 neobrevipes was already observed in many fruit crops planted in Brazil (COSTA, 2002).
However, the transmission rate of Badnavirus by mealybugs is generally considered low, with higher dissemination risk caused by the use of infected propagative material (LOCKHART & OLSZEWSKI, 1993). Therefore, it is believed that pineapple vegetative propagation is one of the main factors contributing to the prevalence of PBCoV in the Brazilian Northeastern region.

CONCLUSIONS
This is the first record of Badnavirus infection in pineapple in Brazil. The presence of PBCoV in the states of Alagoas, Maranhão, Paraiba and Pernambuco, suggests a wide distribution and prevalence of this species in the Brazilian Northeastern region, due to the geographic proximity of the cultivation areas evaluated and the absence of phytosanitary barriers, allowing the common practice of exchanging infected seedlings between farmers in cropping areas within these Brazilian states.