Detection and molecular characterization of a new bipartite begomovirus infecting asystasia (Asystasia gangetica) in Benin Republic in West Africa

Australasian Plant Disease Notes (2023) 18:16 https://doi.org/10.1007/s13314-023-00501-6 Detection and molecular characterization of a new bipartite begomovirus infecting asystasia (Asystasia gangetica) in Benin Republic in West Africa Bahoya JA Lauryn1 · Ekwa Y Monono1 · Jean CM Piau1 · Boniface D Kashina2 · Vincent N Fondong3 · Walter N Leke1,3 Received: 5 March 2023 / Accepted: 27 April 2023 © The Author(s) under exclusive licence to Australasian Plant Pathology Society Inc. 2023 Abstract Asystasia gangetica (A. gangetica) leaves exhibiting yellow mosaic symptoms were tested for the presence of begomovirus infection. The putative full-length of a bipartite begomovirus components A and B molecules, respectively were amplified, sequenced, and analyzed. This represents a distinct begomovirus species infecting Asystasia in Benin Republic in West Africa. Keywords Acanthaceae: Asystasia · Geminiviridae: begomovirus · West Africa Asystasia virus Asystasia gangetica (L.) T. Anderson, belongs to the family Acanthaceae which is an annual or perennial invasive semi-woody dicot herb (weed) wildly distributed across West and Central Africa and is used for food and medicine (Irvine 1938).Yellow mosaic disease symptoms are commonly observed on Asystasia gangetica throughout West and Central Africa (Fig. 1); these plants are also observed to be heavily infested by whiteflies Bemisia tabaci (Genn.), which are vectors of viruses in the genus Begomovirus (family Geminiviridae). This suggests that these yellow mosaic symptoms may be caused by begomoviruses. Thus, in 2014, we collected leaf samples randomly from three symptomatic and three asymptomatic A. gangetica plants from Cotonou (6o2213.05N and 2o2328.45E) in Benin Republic in West Africa. The leaves were press-dried and stored until DNA extraction. Total DNA was isolated as described by Shepherd et al. (2008). The viral circular, ssDNA was enriched in each of the samples by rolling cycle amplification (RCA) (GE Healthcare Bio-Science, Piscataway, NJ). The RCA products * Walter N Leke walter.leke@fulbrightmail.org 1 Present Address: Institute of Agricultural Research for Development (IRAD), Ekona, PMB 25, Buea, Southwest Region, Cameroon 2 Department of Crop Protection, Ahmadu Bello University, Zaria, Nigeria 3 Department of Biological Sciences, Delaware State University, 1200 N. Dupont Highway, Dover, DE 19901-2277, USA were pooled for both the symptomatic plants (BNAsy58) and asymptomatic plants (BN_C), respectively. The two samples were subjected to Illumina sequencing (MisEquation 2 × 150-bp configuration). The sequences were assembled using SeqMan NGen software (DNASTAR, Madison, WI). The putative full-length begomovirus sequences for BNAsy58 were 2,728-bp for DNA-A and 2, 661-bp for DNA-B. As expected, no viral sequences were detected in BN_C from asymptomatic plants. The BLASTn (Altschul et al. 1990) and pairwise SDT (Muhire et al. 2013) analyses of the full-length begomovirus sequences revealed the closest identity of BNAsy58 putative DNA-A (KT444609) (89.4%) and DNA-B (KT444610) (93.8%) to the West African Asystasia virus1 (WAAV1) DNA-A and DNA-B, respectively (Wyant et al. 2015). Sequence analysis showed that BNAsy58 sequences exhibit typical bipartite begomovirus genome organization. Thus, DNA-A contained four open reading frames (ORFs): AV1 and AV2 in the virion sense strand, and AC1, AC2, AC3, and AC4 in the complimentary sense strand. Correspondingly, DNA-B contained two ORFs: BV1 in the virion sense strand and BC1 in the complimentary sense strand. No begomovirus-associated satellites (alphasatellite or betasatellite) genome and other virus-specific contigs were present in the Illumina reads. Based on the Illumina-derived viral sequences, the endonucleases SacI (DNA-A) and ApaI (DNA-B) were used to digest the RCA products of the three symptomatic and three asymptomatic samples. This digestion yielded ~2.8-bp fragments for all three samples from the symptomatic plants, in contrast no 13 Vol.:(0123456789) 16 Page 2 of 3 Australasian Plant Dis. Notes (2023) 18:16 Fig. 1 Asystasia gangetica with yellow mosaic symptoms, typical of begomovirus infection such fragment was observed in samples collected from the asymptomatic plants. The fragments were cloned into a similarly digested pGEM-5Zf (+) plasmid vector (Promega) and sequenced bidirectionally by primer walking. The Sangerand Illumina derived asystasia begomovirus sequences were 100% identical for both DNA-A and DNA-B components. Recombination Detection Program (RDP5) (Martin et al. 2020) analysis of the begomovirus sequences subjected to phylogenetic analysis did not provide significant support for recombination for the newly discovered West African Asystasia begomovirus species, based on a significant p-value of <10-3 for a minimum of three RDP methods. Fig. 2 Phylogenetic relationships between the DNA-A and B components of West Africa Asystasia virus3 (WAAV3) identified in the Benin Republic in West Africa and related begomoviruses (abbreviations according to Brown et al. 2015). Trees constructed using the neighbour-joining algorithm in MEGA 11. The begomovirus sequences identified in this study are shown in bold. Horizontal lines are proportional to the number of nucleotide substitutions per site. Bootstrap values of ≥70% are shown at the major nodes. GenBank Accession Nos. and isolate names for each virus are indicated. The trees are rooted on yet another new bipartite begomovirus identified infecting cotton in Benin Republic (Leke et al. 2016b) 13 Australasian Plant Dis. Notes (2023) 18:16 According to the recommendations of the International Committee on Taxonomy of Viruses (Brown et al. 2015) and the phylogenetic relatedness of the Benin asystasia begomovirus with the previously described asystasia begomoviruses (Fig. 2), the results indicate that the begomovirus associated with asystasia yellow mosaic disease in Benin Republic (Fig. 1), is a new bipartite begomovirus and has been named by ICTV as West African Asystasia virus3 (WAAV3). West African Asystasia virus1 and West African Asystasia virus2 have been reported to infect asystasia and cassava in West and Central Africa (Wyant et al. 2015; Leke et al. 2016a; Bahoya et al. in press). Surprisingly, West African Asystasia virus2, reported to co-infect asystasia with West African Asystasia virus1 (Wyant et al. 2015) was not detected in this present study. The identification of WAAV3 which represents a new begomovirus species suggests asystasia is a major host to begomoviral complexes in West and Central Africa, which are predominantly bipartite in nature. Elsewhere in Madagascar in southern Africa, another begomovirus was identified infecting asystasia, with bipartite genome organization, named Asystasia mosaic Madagascar virus (AMMGV) (De Bruyn et al. 2015), just as WAAV1 and WAAV3 in this study. This study is the first to identify a begomovirus infecting asystasia in the Benin Republic in West Africa, representing a different begomovirus species. This study, together with previous studies strongly suggests that, apart from WAAV2, asystasia is predominantly infected by bipartite begomoviruses. Funding This research was partly funded by National Science Foundation award number IOS-1212576. Walter Leke was supported by the Fulbright Foundation. Declarations Conflict of interest The authors declare that they have no conflict of interest. Page 3 of 3 16 Brown JK, Zerbini FM, Navas-Castillo J, Moriones E, Ramos-Sobrinho R, Silva JCF, Briddon RW, Hernandez-Zepeda C, Idris AM, Malathi VG, Martin DP, Rivera-Bustamante R, Ueda S, Varsani A (2015) Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Arch Virol 160:1593–1619. https://doi. org/10.1007/s00705-015-2398-y De Bruyn A, Harimalala M, Hoareau M, Ranomenjanahary S, Raynaud B, Lefeure P, Lett JM (2015) Asystasia mosaic Madagascar virus: a novel bipartite begmovirus infecting the weed Asystasia gangetica in Madagascar. Arch Virol 160:1589–1591. https://doi.org/10. 1007/s00705-015-2413-3 Leke WN, Mignouna DB, Brown JK, Fondong VN (2016) First report of Soybean chlorotic blotch virus and West African Asystasia virus 1 infecting cassava and a wild cassava relative in Cameroon and Togo. New Dis Rep 33:24. https://doi.org/10.5197/j.2044-0588. 2016.033.024 Leke WN, Khatabi B, Mignouna DB, Brown JK, Fondong VN (2016) Complete genome sequence of a new bipartite begomovirus infecting cotton in the Republic of Benin in West Africa. 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