Banana bunchy top virus and the bunchy top disease

Banana bunchy top disease is one of the most serious diseases of bananas (Musa spp.) worldwide. The disease is caused by Banana bunchy top virus (BBTV), the type species of the Babuvirus genus; (family Nanoviridae). The genome of BBTV is comprised of at least six circular single-stranded DNA (ssDNA) components, each approximately 1 Kb in size. Symptoms of BBTV infection include plant stunting, foliar yellowing and most characteristically, dark green streaks on the pseudostem, petioles and leaves (Harding et al. 1993; Vetten et al., 2005). From 2004 to 2006, a series of field surveys were conducted in the major banana-growing areas in Iran: Mazandaran province (Sari, Babol, Behshahr, and Ghaemshahr) in the north and Hormozgan province (Jask and Varz-Abad) in the south. A total of 164 banana plant samples with bunchy top, stunting and leaf samples with green streak symptoms were collected. Samples were tested for the presence of BBTV with a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using positive control and polyclonal antibodies against BBTV, according to the supplier's instructions (Bio-Rad). Of the samples tested, 27 were positive for BBTV using DAS-ELISA. To confirm BBTV identification, polymerase chain reaction (PCR) was carried out using samples that previously tested positive by ELISA. Forward [F3: 5′-GGAAGAAGCCTCTCATCTGCTTCAGAGAR C-3′; nts 287 to 258] and reverse [FPCR4: 5′-TTCCCAGGCGCACACCTTGAGAAACGA AAG-3′; nts 284 to 313] primers were used to amplify complete BBTV DNA-1 (Furuya et al., 2005). PCR analysis showed fragments of approximately 1 Kb in 19 samples. Furthermore, using primer pairs designed to differentiate BBTV strains (Su et al., 2003), Iranian BBTV isolates were divided into two distinct strains. This is the first report of the occurrence of BBTV in Iran.

Banana Bunchy top virus (BBTV) is a multipartite circular single strand DNA virus that belongs to genus Babuvirus and family Nanoviridae. It causes significant crop losses worldwide and also in Pakistan. BBTV is present in Pakistan since 1988 however, till now only few (about twenty only) sequence of genomic components have been reported from the country. To have insights into current genetic diversity in Pakistan fifty-seven genomic components including five complete genomes (comprises of DNA-R, -U3, -S, -M, -C and -N components) were sequenced in this study. The genetic diversity analysis of populations from Pakistan showed that DNA-R is highly conserved followed by DNA-N, whereas DNA-U3 is highly diverse with the most diverse Common Region Stem-loop (CR-SL) in BBTV genome, a functional region, which previously been reported to have undergone recombination in Pakistani population. A Maximum Likelihood (ML) phylogenetic analysis of entire genomes of isolates by using sequence of all the components concatenated together with the reported genomes around the world revealed deeper insights about the origin of the disease in Pakistan. A comparison of the genetic diversity of Pakistani and entire BBTV populations around the world indicates that there exists a correlation between genetic diversity and recombination. Population genetics analysis indicated that the degree of selection pressure differs depending on the area and genomic component. A detailed analysis of recombination across various components and functional regions suggested that recombination is closely associated with the functional parts of BBTV genome showing high genetic diversity. Both genetic diversity and recombination analyses suggest that the CR-SL is a recombination hotspot in all BBTV genomes and among the six components DNA-U3 is the only recombined component that has extensively undergone inter and intragenomic recombination. Diversity analysis of recombinant regions results on average one and half fold increase and, in some cases up to four-fold increase due to recombination. These results suggest that recombination is significantly contributing to the genetic diversity of BBTV populations around the world.

Banana and plantations (Musa sp.) are the most fourth most important global food crop after rice, wheat and maize (Piertersen and Thomas, 2001). Banana plantations are subjected to various natural climates but diseases; in particular, viral diseases constitute a major setback to this crop worldwide. Among viral infections, Banana Bunchy Top Virus (BBTV) is the most serious virus affecting banana in Egypt as well as the world (Dale, 1987; Othman et al., 1996; Harish et al., 2008) and is responsible for the massive reduction in banana crop yield. The symptoms of BBTD are yellowing, dark green streaks on midrib, petioles, extending down into pseudostem, dark green dots and dashes along the minor leaf veins, stunted growth with bunchy top appearance (Magee, 1940; Vetten et al., 2005). BBTV belong to genus Babuvirus and family Nanoviridae with isometric particles of 18-20 nm in diameter. It is naturally transmitted, in persistence manner, by banana aphid Pentalonia nigronervosa (Hu et al., 1996). BBTV is identified as a phloem-limited virus used to occur in low concentration in banana-infected plants (Harding et al., 1991 and 2000). The virus has a single coat protein with molecular weight (Mr.) of about 20-21kDa (Dietzgen and Thomas, 1991; Harding et al., 1991; Sadik et al., 1999).The viron consists of at least six components of circular ssDNA each component of the BBTV genome contains one large (monocistronic) transcriptional active open reading frame (ORF) except BBTV DNA 1 contains two ORFs one master replication protein (Rep) ORF (Hafner et al., 1997) and a small ORF internal to major ORF (Beetham et al., 1997). Additionally, two conserved region: stem-loop common region (CR-SL) and major common region (CR-M), potential TATA box at 3 ' of the stem-loop and polyadenylation signal (Burns et al., 1995; Beetham et al., 1997 and 1999).The nucleotide sequence of the component six of Australian isolate BBTV has 1089 nt. (Burns et al., 1995). The encoded protein of BBTV DNA-6 is a nuclear shuttle protein and is preferentially targeted to the nucleus when expressed alone however in the presence of the movement protein it targeted to the cell periphery (Wanitchakorn et al., 2000a).The aim of this study is to amplify, clone and sequence the full length of BBTV DNA-6 and compare it with other published sequences of the corresponding sequence.

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

Banana bunchy top disease is caused by a single-stranded circular DNA virus, banana bunchy top virus (BBTV), which is a member of the genus Babuvirus (family Nanoviridae). We have cloned and sequenced five components (DNA-R, DNA-S, DNA-N, DNA-M and DNA-C) of a BBTV isolate originating from Pakistan. In addition, the DNA-R and several other components of five further isolates, originating from geographically distinct sites across the banana-growing area of Sindh province, Pakistan, were cloned and sequenced. Analysis of the sequences indicates that BBTV present in Pakistan belongs to the "South Pacific" group of isolates and that the genetic diversity of the virus in the country is very low. The virus shows the highest levels of sequence identity to BBTV isolates originating from Egypt, India and Australia. The significance of these results with respect to the possible origin of the virus in Pakistan and the prospects for obtaining genetically engineered resistance to the virus are discussed.

Banana is a horticultural crop that has economic value and is widely cultivated in tropical countries. Banana Bunchy Top Virus (BBTV) is the cause of dwarf disease in banana plants (Musa. sp). The vector for the spread of this banana dwarf disease is the banana aphid (Pentalonia nigronervosa). BBTV has been reported in various regions of Indonesia, having been reported in Bengkulu in 2020. However, data on the resistance of BBTV infections in Indonesia are still very limited. This study aimed to evaluate the resistance of three types of bananas (Barangan, Kepok, and Ambon Hijau) to BBTV infection. In this study, two treatments were carried out, namely inoculated and without BBTV virus inoculation with three types of bananas. BBTV detection was carried out using the PCR method. BBTV transmission using aphids succeeded in infecting the test plants with a disease incidence of 12.5% ​​to 75.0% at 30 DAI. The most common symptom seen is, slightly yellowed leaf edges. the severity of BBTV on the Barangan banana plant was 20.83%, kepok was 4.17% and Ambon Hijau was 25.00%. Samples on the leaves of the test banana plant that showed symptoms of BBTV were taken for detection using Polymerase Chain Reaction (PCR) with a pair of related NSP primers in the BBTV genome with a size of ± 500 bp. The results of the detection of BBTV virus showed that the types of bananas that were resistant to BBTV were Kepok bananas, Barangan bananas and Ambon Hijau types which showed moderate resistance to BBTV

Evidence of recombination in the Banana bunchy top virus genome

Among the various factors responsible for low yield in banana (Musa spp.), bunchy top disease caused by Banana bunchy top virus (BBTV) is the most important and devastating disease in most of the tropical countries including India. Several approaches were advocated to control or combat the bunchy top virus in banana transmitted by banana aphid. However, no tactics are able to completely protect these plants against virus infections. The selection of BBTV-free banana plants is the prerequisite for the production of disease free plants and hence early detection of this disease in these plantlets is considered necessary. So, PCR-based detection of BBTV in the current study will be useful in eliminating the infected plants in the early stage. An extensive survey was conducted in Tamil Nadu, India to collect Banana bunchy top virus (BBTV) isolates from hill banana cv. Virupakshi (AAB) which was found to be severely affected by bunchy top virus. Cloning and sequencing of the coat protein (CP) and nuclear shuttle protein (NSP) gene sequences of BBTV were carried out to elucidate the phylogenetic relationship of the hill banana virus isolates with other existing BBTV isolates. Two species-specific primers viz. CP and NSP were used for the amplification of CP and NSP genes of BBTV, respectively. Polymerase chain reaction (PCR) amplified 510 bp CP and 250 bp NSP gene sequences of BBTV. Complete and partial sequences of CP and NSP genes of BBTV were obtained. The analysis revealed that CP gene shared 90% nucleotide sequence identity with other Indian isolates and one Burundi isolate and 89% identity with an Egyptian isolate of BBTV. The comparison of the NSP with the other isolates revealed a 98% identity with three Indian isolates, 97% with an Australian isolate and 95% with a Pakistan isolate of BBTV, whereas only 88% identity was shared with China and Taiwan isolates. The present study suggests that the hill banana isolate falls within the South Pacific group of BBTV isolates.

Bananas (Musaceae, Zingiberales) are considered to be one of the most important fruit crops and an important staple food in many developing tropical and subtropical regions (Kumar et al., 2011). In Southern Africa, banana production is under threat from a number of viral diseases, including Banana bunchy top virus (BBTV) (family Nanoviridae, genus Babuvirus). BBTV is the cause of Banana bunchy top disease, the most devastating virus disease of banana. In Sub-Saharan Africa the disease is endemic to 13 countries, including Malawi and Zambia in the Southern African sub-region. Previously, a detection survey for BBTV was done in South Africa in 1996 and this study confirmed the absence of BBTV in the Kiepersol region of Mpumalanga Province (Pietersen et al., 1996). No other surveys were conducted since then in South Africa. The disease is spread by the banana aphid Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), that is present in South Africa, and through infected propagation material. Pentalonia nigronervosa feeds on the phloem of host plants, and potentially picks up the BBTV particles from the phloem and spreads them in plantations as they feed. In June 2015, five 'Williams' plants, showing symptoms resembling BBTV, including streaks on the pseudostem and upright bunchy appearance, were submitted for identification to the Virology Diagnostic Lab at Agricultural Research Council-Plant Protection Research Institute (ARC-PPRI). The plants originated from a farm close to Hibberdene in the KwaZulu Natal South Coast production region (GPS coordinates: S 300 30, 636', E 300 30,653'). A BBTV-specific PCR was done using the protocol published by Thomson and Dietzgen (1995), using primer pairs BBT-1 and BBT-2, amplifying a fragment of the putative replicase gene. The five plants tested positive for BBTV, and the PCR products (~349 bp) of two of the accessions, 15/1408 and 15/1410, were directly sequenced using Sanger sequencing and 321 bp of both sequences were deposited in GenBank (GenBank Accession Nos: KU196167, KU196168). A non-infected banana plant was included in the test as a healthy control. Phylogenetic analyses of the partial replicase genomic region grouped accessions 15/1408 and 15/1410 with the South Pacific group that included accessions from India, Pakistan and other regions in Africa with a sequence identity of 99% (Kumar et al., 2011). A second confirmation test for the presence of BBTV in these plants was conducted using a BBTV-specific Enzyme-linked immunosorbent assay (ELISA) supplied by Agdia (Catalog number: SRA24700) using the manufacturer's instructions. The five plants tested positive for BBTV in the ELISA test, confirming the previous positive results. Forty-four additional samples were included in the ELISA test. These samples were collected randomly from 14 blocks on the same farm (GPS coordinates: S 300 30, 636', E 300 30,653') and comprised of symptomatic and non-symptomatic plants. BBTV was detected with ELISA in 34 of these plants, ten plants tested negative for BBTV. In one of these blocks, C17, all the sampled plants (4), tested negative. In three other blocks (A9, C8 and C15), positive and negative plants were recorded. In the remaining 10 blocks, all collected plants tested positive. Intense management strategies, including removal of infected plants and spraying for aphids, were implemented on the farm to prohibit the spread of the virus to neighbouring plantations. The ELISA test confirmed the earlier detection of BBTV with PCR and sequencing. This is the first report of BBTV on Musa spp. from South Africa. Strict implementation of phytosanitary measures will be necessary to prevent the spread of this virus to other regions in the country. The need to do extensive surveys for the presence of BBTV in other banana production regions in South Africa is highlighted by this finding.

Banana bunchy top virus (BBTV) DNA-3 to 6 have each previously been shown to contain one large open reading frame in the virion sense, whereas no large ORF had been identified in BBTV DNA-2. RNAs transcribed from the BBTV genome were mapped using northern hybridisation and 3' RACE. One mRNA was transcribed from each of BBTV DNA-2 to 6 and four of these mRNAs mapped to the ORFs previously identified in BBTV DNA-3 to 6. The mRNA of BBTV DNA-2 was transcribed from a virion sense ORF probably using a TATA box sequence different to that in BBTV DNA-1, and DNA-3 to 6. This ORF encoded a 10 kDa protein of unknown function. The 3' untranslated region of the five mRNAs varied from 25 nucleotides (BBTV DNA-6) to 167 nucleotides (BBTV DNA-4) and each contained putative polyadenylation signals with associated GT rich sequence together with a possible termination signal (C/T/A)TGTAA conserved in all five mRNAs.

Banana bunchy top virus (BBTV) is a single-stranded circular DNA virus of the genus Babuvirus, belonging to family Nanoviridae. The six genomic DNA components of Indian (Lucknow) isolate of BBTV were amplified by polymerase chain reaction (PCR) with specific primers using total DNA extracted from banana tissues showing typical symptoms of banana bunchy top disease (BBTD). The resulting ~1.1 Kb amplicons were cloned and sequenced. Analysis of sequence data revealed the presence of six full-length components of BBTV: DNA-R (1111 bp), DNA-U3 (1060 bp), DNA-S (1075 bp), DNA-M (1048 bp), DNA-C (1018 bp), and DNA-N (1096 bp). Comparisons of sequence data of the six DNA components of the BBTV Lucknow isolate revealed highest identities with sequences of other BBTV isolates from the South Pacific group: [DNA-R (98%), DNA-U3 (93%), DNA-S (100%), DNA-M (98%), DNA-C (97%), and DNA-N (99%)]. A phylogenetic analysis revealed a close relationship of the Lucknow isolate with BBTV isolates of South Pacific group rather than those of the Asian group. Based on these analyses the virus has been classified as BBTV Lucknow, a new member of South Pacific group.

Banana bunchy top disease is caused by banana bunchy top virus (BBTV). BBTV is transmitted locally by aphids (Pentalonia spp.), but the long-distance spread is through the movement of infected planting materials. This study investigated potential alternative hosts of BBTV in ornamental Musa and related species in the Zingiberales in the Philippines. Artificial inoculation of BBTV, molecular detection and transmission assay were used to evaluate 15 plant test species. The potential for seed transmission of BBTV through Canna indica seeds was also investigated. Seed samples were validated and quantified for BBTV presence using molecular tools, and then grown for transmission assay. Typical symptoms of BBTV in bananas, including dark green streak on the midrib and petiole and rosetting were observed on inoculated Musa coccinea (banana blossom), M. velutina (velutina), M laterita. (bronze banana) and Canna indica (Bandera Espanola). PCR assays confirmed BBTV infection in these symptomatic test plants, as well as in Curcuma longa (turmeric) which exhibited large chlorotic blotches on the leaf. BBTV was detected from both seeds and germinated seedlings of artificially inoculated and field-collected C. indica samples. This study identified M. laterita as a new host of BBTV. The susceptibility to BBTV of M. coccinea, M. velutina, C. indica, and C. longa was also confirmed. The study also provided the first evidence of seed transmission of BBTV. C indica is an ornamental plant popularly used for landscaping in the Philippines and seeds were shown to be an efficient mode of transmission of the virus with rates up to 34%. The discovery of natural infection in ornamental plants and seeds poses a risk to the banana industry and responsible propagation and appropriate quarantine protocols must be implemented.

Isolates of Banana bunchy top virus (BBTV), which causes bunchy top disease in bananas, were collected in field surveys on seven islands in Okinawa Prefecture, Japan. From 44 banana samples, one isolate from each island was selected, and the DNA-1 and DNA-3 components were sequenced. Analysis of the major common region of DNA-1 showed that BBTV in Okinawa belongs to the Asian group of BBTV. DNA-1 and DNA-3 analysis revealed that Okinawan BBTV had a closer relationship with isolates from Taiwan and the Philippines than with some isolates from China and Vietnam. All the Okinawan BBTV isolates had high homology in the nucleotide sequences of DNA-1 and DNA-3 (%) because of a single, recent BBTV invasion of this area.

Banana bunchy top disease (BBTD) can be caused by the infection of two different viruses, Banana bunchy top virus (BBTV) or Abaca bunchy top virus (ABTV). Both viruses can be transmitted persistently by aphid Pentalonia nigronervosa Coq. The research was conducted to detect and to differentiate the virus bypolymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) techniques. Infected plants were collected from Yogyakarta (Sleman, Yogyakarta city, Bantul, Gunung Kidul, and KulonProgo). Nucleon Phytopure DNA Extraction Kit method was used to extract the total DNA of infected plants. Universal primers of Common DNA region (S-CRF and S-CRR) and specific primers DNA-R (C1-CRF and CI-CRR) were used for PCR amplification. PCR products were analyzed by RFLP technique using the restriction enzyme of DraI. The results reconfirm previous reports that bunchy top disease of banana in Yogyakarta is caused by BBTV. The ABTV was not detected in this present study. Based on the RFLP analysis it was concluded that BBTV collected in this study could be divided into three groups. Group 1 consisted of BBTV isolate from Sleman and Yogyakarta city with two fragments DNA of 400 and 388 bp. Group 2 consisted of isolate BBTV from Kulon Progo and Gunung Kidul with three fragments DNA of 400, 388, and 323 bp. Group 3 consisted of isolate from Bantul with two fragments DNA of 723 and 376 bp. Further study on the complete characteristics of these groups is still needed.

Plant viruses of the families Luteoviridae and Geminiviridae rely on hemipteran vectors for the infection of their hosts. Several lines of evidence have revealed that these viruses are transmitted by competent vectors in a circulative manner, involving entry into the vector's body and the crossing of epithelial tissues forming the alimentary tract and the salivary glands. Similar to luteovirids and geminiviruses, a third family of plant viruses, the family Nanoviridae, have also been reported to be transmitted by aphids in a circulative manner. However, there is limited direct evidence of a possible path of translocation through the aphid vectors. Here, we used time-course experiments and transmission assays coupled with real-time PCR and immunofluorescence assays on dissected tissues to examine the translocation, compartmentalization and retention of banana bunchy top virus (BBTV) into the aphid vector Pentalonia nigronervosa. Our results indicate that BBTV translocates rapidly through the aphid vector; it is internalized into the anterior midgut in which it accumulates and is retained at concentrations higher than either the haemolymph or the principal salivary glands. Despite the large increase in viral concentration, we have failed to detect BBTV transcripts with RT-PCR. When tissues were not permeabilized, BBTV localized as distinct puncta in the proximity of the basal surface of the cells forming the anterior midgut and principal salivary glands, suggesting an on-going process of virion escape and internalization, respectively. Interestingly, we document that those organs can have direct contact within the aphid body, suggesting a possible haemolymph-independent translocation path.