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Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin are entomopathogenic fungi commonly used in microbial control of arthropods. In this study, we evaluated the insecticidal potential of six isolates of B. bassiana (BIITAC10.3.3, BIITAC6.2.2, and BIITAC8.1.5) and M. anisopliae (MIITAC11.3.4, MIITAC6.2.2, and MIITAC6.4.2) from Cameroon, against the banana aphid Pentalonia nigronervosa Coquerel, the vector of the banana bunchy top virus (BBTV). Pathogenicity tests were initially conducted using B. bassiana and M. anisopliae isolates at a concentration of 3.2 × 106 conidia/ml on P. nigronervosa adults sourced from four agroecologies in Cameroon. Four isolates (BIITAC6.2.2, BIITAC10.3.3, BIITAC8.1.5, and MIITAC6.2.2) were highly pathogenic, causing greater than 75% aphid mortality in all populations. A significant decrease in aphid fecundity was observed with BIITAC6.2.2, MIITAC6.2.2, and BIITAC10.3.3. These three isolates were in a test of a series of four fungal concentrations (3.2 × 101, 3.2 × 102, 3.2 × 104, and 3.2 × 106 conidia/ml). produced LC50 of 1.31 × 101 and 3.12 × 10-2 for BIITAC10.3.3 and MIITAC6.2.2, respectively. MIITAC6.2.2 had the lowest LC90 (1.55 × 103). Our results strongly support the continued development of biopesticides based on one or more of the three fungal entomopathogens for the control of banana aphids as a component of an Integrated Pest Management (IPM) strategy for the reduction of the prevalence and transmission of BBTV under field conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Enoh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bananas hold significant economic importance as an agricultural commodity, serving as a primary livelihood source, a favorite fruit, and a staple crop in various regions across the world. However, Banana bunchy top disease (BBTD), which is caused by banana bunchy top virus (BBTV), poses a considerable threat to banana cultivation. To understand the resistance mechanism and the interplay of host suitability factors in the presence of BBTV, we conducted RNA-seq-based comparative transcriptomics analysis on mock-inoculated and BBTV-inoculated samples from resistant (wild Musa balbisiana) and susceptible (Musa acuminata 'Lakatan') genotypes. We observed common patterns of expression for 62 differentially expressed genes (DEGs) in both genotypes, which represent the typical defense response of bananas to BBTV. Furthermore, we identified 99 DEGs exclusive to the 'Lakatan' banana cultivar, offering insights into the host factors and susceptibility mechanisms that facilitate successful BBTV infection. In parallel, we identified 151 DEGs unique to the wild M. balbisiana, shedding light on the multifaceted mechanisms of BBTV resistance, involving processes such as secondary metabolite biosynthesis, cell wall modification, and pathogen perception. Notably, our validation efforts via RT-qPCR confirmed the up-regulation of the glucuronoxylan 4-O-methyltransferase gene (14.28 fold-change increase), implicated in xylan modification and degradation. Furthermore, our experiments highlighted the potential recruitment of host's substrate adaptor ADO (30.31 fold-change increase) by BBTV, which may play a role in enhancing banana susceptibility to the viral pathogen. The DEGs identified in this work can be used as basis in designing associated gene markers for the precise integration of resistance genes in marker-assisted breeding programs. Furthermore, the findings can be applied to develop genome-edited banana cultivars targeting the resistance and susceptibility genes, thus developing novel cultivars that are resilient to important diseases., (© 2023. The Author(s).)

Banana bunchy top disease (BBTD) is caused by banana bunchy top virus (BBTV), the most important virus affecting banana. Currently, no cultivar or accession of banana has complete resistance to BBTD. A total of 36 wild Musa spp. accessions, including 34 Musa balbisiana and 2 M. acuminata subsp. errans ("Agutay"), were screened for resistance against BBTV. In greenhouse tests using viruliferous banana aphids (Pentalonia nigronervosa ), all M. balbisiana accessions remained symptomless, and BBTV was not detected in any of these plants by PCR at 3 and 6 months postinoculation. In contrast, 100% disease incidence was recorded in M. acuminata subsp. errans and in cv. Lakatan susceptible control plants. The PCR-negative M. balbisiana plants were then transferred to a field with high BBTV inoculum pressure where they remained symptomless and PCR-negative for up to 5 years, while all cv. Lakatan developed BBTD. Wild M. balbisiana accessions showed a high level of resistance and possibly immunity to BBTV and are expected to provide a resource for conventional and marker-assisted breeding.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Banana bunchy top disease is the most devastating viral disease of bananas worldwide and is caused by banana bunchy top virus (BBTV). The disease is spread by the banana aphid Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and through infected propagation material. In 2016, the virus was detected for the first time in an isolated area in the South Coast region of KwaZulu-Natal Province (KZN), South Africa. The aim of this study was to conduct surveys across all banana-producing regions in South Africa, viz. KwaZulu-Natal, Mpumalanga, and Limpopo provinces. Over 1700 plant and aphid samples were collected from commercial farms and rural households in the three provinces, and more-intense sampling was done in the affected KZN region. A BBTV-specific PCR targeting DNA-R (encoding the master replication initiation protein, M-Rep) was used to detect virus-infected samples, and amplicons of the expected size were sequenced. Comparative phylogenetic analysis showed that the South African BBTV isolates clustered within the Pacific Indian Oceans genomic group, which includes isolates from India and other regions in Africa, with a bootstrap value of 94%. To date, the virus has been identified only in the South Coast region of KwaZulu-Natal Province. Intense management strategies, including scouting, removal of infected plants, and control of aphids, have been implemented in areas where positive samples were identified to minimize the spread of the virus., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Banana bunchy top virus (BBTV) is a six-component ssDNA virus (genus Babuvirus, family Nanoviridae) transmitted by aphids, infecting monocots (mainly species in the family Musaceae) and likely originating from South-East Asia where it is frequently associated with self-replicating alphasatellites. Illumina sequencing analysis of banana aphids and leaf samples from Africa revealed an alphasatellite that should be classified in a new genus, phylogenetically related to alphasatellites of nanoviruses infecting dicots. Alphasatellite DNA was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads, altering relative abundance (formula) of viral genome components and interfering with virus transmission by aphids. BBTV and alphasatellite clones infected dicot Nicotiana benthamiana, followed by recovery and symptomless persistence of alphasatellite, and BBTV replication protein (Rep), but not alphasatellite Rep, induced leaf chlorosis. Transcriptome sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands of the entire viral genome, monodirectional Pol II transcription units of viral mRNAs and pervasive transcription of each component and alphasatellite in both directions, likely generating double-stranded precursors of viral siRNAs. Consistent with the latter hypothesis, viral DNA formulas with and without alphasatellite resembled viral siRNA formulas but not mRNA formulas. Alphasatellite decreased transcription efficiency of DNA-N encoding a putative aphid transmission factor and increased relative siRNA production rates from Rep- and movement protein-encoding components. Alphasatellite itself spawned the most abundant siRNAs and had the lowest mRNA transcription rate. Collectively, following African invasion, BBTV got associated with an alphasatellite likely originating from a dicot plant and interfering with BBTV replication and transmission. Molecular analysis of virus-infected banana plants revealed new features of viral DNA transcription and siRNA biogenesis, both affected by alphasatellite. Costs and benefits of alphasatellite association with helper viruses are discussed., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no conflict of interest.

Banana bunchy top virus (BBTV) is the most destructive etiological agent limiting banana cultivation areas globally. This study attempted BBTV elimination by traditional shoot-tip culture (control) and alternative shoot-tip + electrotherapy (treated) techniques. Shoot-tip culture from Musa acuminata cv. 'Grand Naine' infected sources were exposed to 100 mA electric current for different time intervals (20-60 min). Virus indexing (via PCR) and genetic fidelity (by ISSR assay) from the cultures were tested, alongside the physio-biochemical parameters. Exposure of electric current for less than 50 min was ineffective for BBTV elimination. Still, a rise in the duration from 50 min or more led to eradicating the virus from some explants. Elimination of BBTV was complete from 100 % of explants exposed to 100 mA for 60 min, as confirmed by lack of BBTV detection even at six months after acclimatization. In the control treatment, the maximum efficiency of BBTV elimination was 28 % after eight subcultures. On the other hand, improved survival % was observed in the treated culture. Moreover, homogenous ISSR patterns were there between the treated and the mother plant and similar physio-biochemical activities were seen in electro-exposed cultures and healthy ones. Thus, the study reports complete BBTV-elimination from banana with international compliances, for the first time, via electrotherapy while maintaining genomic template and biochemical stability., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The Banana Bunchy Top Disease (BBTD), caused by the Banana Bunchy Top Virus (BBTV) is the most important and devastating in many tropical countries. BBTD epidemiology has been little studied, mixed landscape smallholder systems. The relative risks associated with this disease vary between geographical areas and landscapes. This work analyzed the management and vegetation conditions in smallholder gardens to assess the factors linked to landscape-level BBTV transmission and management. Mapping was done in this study area which is in a BBTD-endemic region, involving farmers actively managing the disease, but with household-level decision making. A spatial scanning statistic was used to detect and identify spatial groups at the 5% significance threshold, and a Poisson regression model was used to explore propagation vectors and the effect of surrounding vegetation and crop diversity. Spatial groups with high relative risk were identified in three communities, Dangbo, Houéyogbé, and Adjarra. Significant associations emerged between the BBTD prevalence and some crop diversity, seed systems, and BBTD management linked factors. The identified factors form important candidate management options for the detailed assessment of landscape-scale BBTD management in smallholder communities., Competing Interests: The authors have declared that no competing interests exist.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus ) or eight (genus Nanovirus ) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus ), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus ). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae , which is available at ictv.global/report/nanoviridae.

The Banana Bunchy Top Virus (BBTV) is one of the most economically important vector-borne banana diseases throughout the Asia-Pacific Basin and presents a significant challenge to the agricultural sector. Current models of BBTV are largely deterministic, limited by an incomplete understanding of interactions in complex natural systems, and the appropriate identification of parameters. A stochastic network-based Susceptible-Infected-Susceptible model has been created which simulates the spread of BBTV across the subsections of a banana plantation, parameterising nodal recovery, neighbouring and distant infectivity across summer and winter. Findings from posterior results achieved through Markov Chain Monte Carlo approach to approximate Bayesian computation suggest seasonality in all parameters, which are influenced by correlated changes in inspection accuracy, temperatures and aphid activity. This paper demonstrates how the model may be used for monitoring and forecasting of various disease management strategies to support policy-level decision making., Competing Interests: The authors have declared that no competing interests exist.

Background: The genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear., Results: To understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, -M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR., Conclusions: Our data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.

An improved gold nanoparticle based Dot immunobinding assay (DIBA) was developed for the detection of Banana bunchy top virus (BBTV), that is more efficient, sensitive, rapid and simpler than conventional DIBA and ELISA. Instead of enzyme conjugates, gold nanoparticles were used as reporters owing to their unique optical properties. Antibody was raised against expressed recombinant coat protein of BBTV. The gold nanoparticles were conjugated to primary / detection antibody raised following immunization with recombinant coat protein, making it highly specific for the virus. Gold nanoparticle conjugated primary antibody (GCPab) based DIBA developed in this study has a detection efficiency comparable to ELISA. The results of using this assay format for detection of BBTV in banana plants from four geographical regions of India are also presented in this report. The test could detect the virus at sap dilution up-to 10 -2 . Using this improved DIBA, any lab with basic amenities can perform indexing on large numbers of samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

Viruses in the genus Babuvirus have multi-component ssDNA genomes and often associate with alphasatellite molecules containing two common motifs, a common-region stem-loop (CR-SL) involved in initiation of rolling-circle replication and a common-region major (CR-M) motif involved in secondary-strand synthesis. We compared known babuvirus genome components and alphasatellite CR-SL and CR-M sequences, defining five divergent CR-SL sequence classes. We identified iterated sequence elements in babuvirus genome components that have particularly conserved sequences and spatial arrangements between known babuviruses.

Banana bunchy top virus (BBTV) (the genus Babuvirus, the family Nanoviridae) is a single-stranded circular DNA virus with a genome composed of six components designated as DNA-R, -U3, -S, -M, -C, and -N. This study analyzed the nucleotide identities of the DNA-R of 23 isolates from banana-producing provinces of China, including Guangdong, Hainan, Guangxi, and Yunnan. Results showed that the nucleotide identity of DNA-R was 72.3-100%. Phylogenetic analysis indicated that these BBTV isolates were clustered in different subgroups within the Asian group (AG). Sequence analysis of the five other components (DNA -U3, -S, -M, -C, and -N) of the five isolates from China confirmed the results established for DNA-R of these BBTV isolates. This study suggested that the variation of DNA-R from Chinese BBTV isolates was considerably higher than the variation of other AG isolates, but their genetic diversity was low.

Banana bunchy top virus (BBTV), belonging to the genus Babuvirus, is the most devastating and widespread banana virus. Banana and plantain are major crops in terms of household income and food security in Democratic Republic of Congo (DRC). Despite the large area under banana and plantain cultivation in the country, before this study, the genetic characterization of BBTV isolates had only been undertaken for two provinces. In the study presented here, genetic variation in BBTV was assessed from 52 BBTV isolates collected in five out of 11 provinces in DRC (Bandundu, Bas-Congo, Katanga, Kinshasa and Kasaï Oriental) and in two provinces using sequences previously described in databases. Full genome sequencing of DNA-R components was performed, revealing low genetic variation (98-100 % nucleotide identity) among the BBTV isolates detected. The phylogenetic analyses showed that all the DRC isolates were clustered in the South Pacific clade of BBTV. Based on the coding region for the replication initiator protein, haplotype diversity was estimated to be 0.944 ± 0.013, with 30 haplotypes from 68 isolates in DRC. Such diversity shows a haplotype distribution mainly at the sub-regional level in DRC. In addition, the sequence determination from the whole genome of selected isolates confirmed low genetic variation among isolates from seven DRC provinces (97-100 % nucleotide identity). This study strengthened the hypothesis of a single BBTV introduction some time ago, followed by the spread of the virus in the country.

The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests.

Banana bunchy top virus (BBTV) is a multi-component single-stranded DNA virus. From 267 potentially infected Musa plants, 24 apparently 'defective' BBTV components have been identified. Interestingly, 23/24 of these defective molecules were apparently derived from DNA-R. All of the identified defective molecules had retained at least part of the CR-SL and CR-M but had insertions and/or deletions that in most cases resulted in open reading frame disruptions. Our detection of three monophyletic but diverse (and therefore likely circulating) defective DNA-R lineages suggests that, in many cases, defective DNA-R molecules might remain associated with BBTV genomes for prolonged periods.

Plant viruses cause significant production and economic losses in the agricultural industry worldwide. Rapid and early identification of contagious plant viruses is an essential prerequisite for the effective control of further spreading of infection. In this work, we describe a miniaturized paper-based gene sensor for the rapid and sensitive identification of a contagious plant virus. Our approach makes use of hybridization-mediated target capture based on a miniaturized lateral flow platform and gold nanoparticle colorimetric probes. The captured colorimetric probes on the test line and control line of the gene sensor produce characteristic red bands, enabling visual detection of the amplified products within minutes without the need for sophisticated instruments or the multiple incubation and washing steps performed in most other assays. Quantitative analysis is realized by recording the optical intensity of the test line. The sensor was used successfully for the identification of banana bunchy top virus (BBTV). The detection limit was 0.13 aM of gene segment, which is 10 times higher than that of electrophoresis and provides confirmation of the amplified products. We believe that this simple, rapid, and sensitive bioactive platform has great promise for warning against plant diseases in agricultural production.

Nanoviruses are single-stranded DNA (ssDNA) plant viruses which have multipartite genomes consisting of discrete, individually encapsidated components. This multipartite strategy may lead to high rates of reassortment, whereby entire genome components are exchanged among different strains. However, few studies have explored the extent to which reassortment shapes the genetic diversity of nanovirus populations. Here we present an extensive analysis of reassortment among 163 Cardamom bushy dwarf virus (CBDV; Nanoviridae family, Babuvirus genus) isolates collected in Northeast India. We also examined evidence of recombination, which is known to play a role in the evolutionary dynamics of nanovirus populations. By sequencing six discrete genome components for each isolate, we demonstrate that over 40% of the isolates display evidence of at least one reassortment event during their evolutionary histories. Nevertheless, a bias in the frequencies at which different genome components reassort was observed, with the DNA-M and DNA-N components being the most predisposed to reassortment. This may reflect variation in the ability of different genome components to function efficiently in a foreign genomic background. Comparisons of the common regions of different genome components revealed signatures of concerted evolution mediated by frequent inter-component homologous recombination. This process, which has previously been reported in nanoviruses and other multipartite ssDNA viruses, may allow proteins which initiate replication to maintain control over distinct genome components. Notably, DNA-N, one of the genome components most prone to reassortment, also exhibited the most frequent inter-component homologous recombination. This supports the idea that inter-component homologous recombination may promote the efficient replication of novel components which are introduced into a genome via reassortment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Banana bunch top virus (BBTV) is considered to be a serious threat to banana production. A new isolate of the virus (BBTV-Umiam) was identified and characterized from local banana mats growing in mid-hills of Meghalaya in North-East India. The complete nucleotide sequence analysis revealed the presence of six full-length ssDNA components (DNA R, DNA U3, DNA S, DNA M, DNA C and DNA N) sharing major common region (CR-M) and a stem-loop common region (CR-SL). BBTV-Umiam showed a unique deletion of 20 nucleotides in the intergenic region of DNA R, the absence of predicted open reading frame (ORF) in DNA U3 and probability for a small ORF in DNA U3 expecting functional evidence at transcriptional level. Phylogenetic analysis based on 88 complete nucleotide sequence of BBTV DNA R available in GenBank generated two broad clusters of Pacific-Indian Oceans (PIO) and South-East Asian (SEA) groups including BBTV-Umiam within PIO cluster. However, BBTV-Umiam was identified as the most distinct member of the PIO group with 100% bootstrap support. This was further supported by the phylogenetic grouping of each genomic component of BBTV-Umiam at the distant end of PIO group during clustering of 21 complete BBTV sequences. BBTV-Umiam shared relatively less nucleotide identity with PIO group for each genomic component (85.0-95.4%) and corresponding ORF (93.8-97.5%) than that of earlier PIO isolates (91.5-99.6% and 96.0-99.3%, respectively). Recombination analysis revealed two intra-component and five inter-component recombination events in BBTV-Umiam, but none of them was unique. Moreover, the isolate was identified as major parental sequence for intra-component recombination event spanning the replication-associated protein encoding region in Tongan BBTV DNA R. The current study indicated differential evolution of BBTV in North-East India (Meghalaya). The natural occurrence of hybrids of Musa balbisiana and M. acuminata in this geographically isolated region could be the contributing factor in accumulating genetic distinctiveness in BBTV-Umiam which need further characterization., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aphids are known to live in symbiosis with specific bacteria called endosymbionts that have positive or negative impacts on their hosts. In this study, six banana aphid (Pentalonia nigronervosa Coquerel) strains from various geographical origins (Gabon, Madagascar, and Burundi) were screened to determine their symbiotic content, using complementary genomic (16S rDNA sequencing and specific polymerase chain reaction) and proteomic (two-dimensional difference gel electrophoresis coupled with protein identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) approaches. Despite the geographical heterogeneity, the combined methods allowed us to identify the same two symbionts in the six aphids strains tested: Buchnera aphidicola and Wolbachia. Although B. aphidicola is found in almost all aphid species, the systematic presence of Wolbachia in banana aphids is particularly interesting, as this bacterium usually has a low prevalence in aphid species. Phylogenetic analyses showed that the Wolbachia sp. strain found in P. nigronervosa was very similar to the strain present in aphids of the genus Cinara, known to have developed a strong and long-term symbiotic association with Wolbachia. The high level of asexual reproduction in P. nigronervosa could be linked to the presence of Wolbachia, but its prevalence also suggests that this symbiotic bacterium could play a more essential role in its aphid host.

Foorkey disease is a serious constraint to the production of large cardamom (Amomum subulatum, family Zingiberaceae). The disease is characterized by profuse proliferation of excessive stunted shoots, which makes the clump totally unproductive. The disease has been known in India since 1936 but the complete genome of the virus had not yet been characterized. In a preliminary study, an associated virus tentatively named as Cardamom bushy dwarf virus (CBDV) was identified based on the partial sequence of a single DNA component (DNA-R). In the present study, a high incidence (37.2-39.3%) of foorkey was recorded in certain plantations in the Darjeeling hills located at lower altitudes (300-1380 m) and CBDV was detected in several field samples by PCR. Nine novel DNA components were isolated and characterized from foorkey affected plants. CBDV contained six major DNA components (DNA-R, -S, -M, -C, -N and -U3) similar to the integral genome components known for the members of the genus Babuvirus in the family Nanoviridae. Additional components, satellite Rep (DNA-sRep1) and unknown components (DNA-Uf1 and -Uf2) were also identified. The size of the genome components ranged from 1028 to 1127. The sequence identity and phylogeny based on the individual components as well as overall genome (59.8-62% identity) distinguished CBDV from the two existing babuvirus species, Banana bunchy top virus and Abaca bunchy top virus. CBDV is the first distinct babuvirus species that affects plant species outside family Musaceae. This study shows further diversity in the genus Babuvirus., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

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.

A sensitive loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Banana bunchy top virus (BBTV) infection. The reaction was performed in a single tube at 63°C for 90 min, with an improved closed-tube detection system by adding the SYBR Green I dye to the inside of the tube lid prior to amplification. The detection limit of the LAMP assay was approximately 1 pg/μl plasmid DNA when mixed with extracted DNA from healthy banana plant, and no cross-reaction with other banana-infected pathogens was observed. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this LAMP assay was about 100-fold more sensitive than PCR. The results demonstrated that this LAMP method should be useful for both banana disease monitoring and mass propagation of virus-free banana plantlets., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The genome of Banana bunchy top virus (BBTV) consists of six segments of single-stranded DNA of approximately 1 kb in length. We identified and sequenced the complete genomes of two BBTV isolates, one with and one without satellite DNA, from Haikou, Hainan, China. The Haikou-2 isolate contains six genomic segments and an additional satellite DNA while the Haikou-4 isolate contains only six genomic segments. Typical of other babuviruses, each genomic segment encodes a single open reading frame and contains the highly conserved stem-loop and major common regions. Phylogenetic analysis of the two Haikou isolates together with existing sequence records in GenBank confirmed the grouping of BBTV into two large groups and further refined the geographical distribution of each group. To accommodate the changes in the BBTV geographical distribution, the two groups are proposed as the Southeast Asian group and the Pacific-Indian Oceans group. Both the Haikou-2 and Haikou-4 isolates belong to the newly proposed Southeast Asian group.

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component, ssDNA virus, which causes widespread banana crop losses throughout tropical Africa and Australasia. We determined the full genome sequences of 12 BBTV isolates from the Kingdom of Tonga and analysed these together with previously determined BBTV sequences to show that reassortment and both inter- and intra-component recombination have all been relatively frequent occurrences during BBTV evolution. We found that whereas DNA-U3 components display evidence of complex inter- and intra-component recombination, all of the South Pacific DNA-R components have a common intra-component recombinant origin spanning the replication-associated protein gene. Altogether, the DNA-U3 and DNA-M components display a greater degree of inter-component recombination than the DNA-R, -S, -C and -M components. The breakpoint distribution of the inter-component recombination events reveals a primary recombination hotspot around the 5' side of the common region major and, in accordance with recombination hotspots detectable in related ssDNA viruses, a secondary recombination hotspot near the origin of virion-strand replication.

Banana bunchy top virus (BBTV) was first reported from sub-Saharan Africa (SSA) from Democratic Republic of Congo (DRC) in the 1950s, has become invasive and spread into 11 countries in the region. To determine the potential threat of BBTV to the production of bananas and plantains (Musa spp.) in the sub-region, field surveys were conducted for the presence of banana bunchy top disease (BBTD) in the DRC, Angola, Cameroon, Gabon and Malawi. Using the DNA-S and DNA-R segments of the virus genome, the genetic diversity of BBTV isolates was also determined from these countries relative to virus isolates across the banana-growing regions around the world. The results established that BBTD is widely prevalent in all parts of DRC, Malawi, Angola and Gabon, in south and western part of Cameroon. Analysis of the nucleotide sequences of DNA-S and DNA-R indicate that BBTV isolates from these countries are genetically identical forming a unique clade within the 'South Pacific' phylogroup that includes isolates from Australia, Egypt, South Asia and South Pacific. These results imply that farmers' traditional practice of transferring vegetative propagules within and between countries, together with virus spread by the widely prevalent banana aphid vector, Pentalonia nigronervosa, could have contributed to the geographic expansion of BBTV in SSA. The results provided a baseline to explore sanitary measures and other 'clean' plant programs for sustainable management of BBTV and its vector in regions where the disease has already been established and prevent the spread of the virus to as yet unaffected regions in SSA., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Banana bunchy top virus (BBTV), family Nanaviridae, genus Babuvirus, is a single stranded DNA virus (ssDNA) that causes banana bunchy top disease (BBTD) in banana plants. It is the most common and most destructive of all viruses in these plants and is widespread throughout the Asia-Pacific region. In this study we isolated, cloned and sequenced a BBTV sample from Hainan Island, China. The results from sequencing and bioinformatics analysis indicate this isolate represents a satellite DNA component with 12 DNA sequences motifs. We also predicted the physical and chemical properties, structure, signal peptide, phosphorylation, secondary structure, tertiary structure and functional domains of its encoding protein, and compare them with the corresponding quantities in the replication initiation protein of BBTV DNA1.

Viruses serve as good model for evolutionary studies, owing to their short generation times and small genomes. Banana bunchy top virus (BBTV) is a significant subject being multicomponent circular single stranded DNA virus. BBTV belongs to family Nanoviridae and contains DNA-R, -U3, -S, -M, -C, and -N as integral genomic components. Evolutionary studies have shown genetic re-assortment of components among its isolates and revealed a concerted type evolution in non-coding regions of its genome. The DNA U3 having been shown as the most diverse component in our previous studies, was subjected to sequencing from some Pakistani isolates for the first time. Sequence analysis revealed intergenomic recombination in DNA-U3 among the isolates of two sub-groups and a very rare intragenomic recombination in Pakistani BBTV population. This indicates that like other evolutionary processes including intergenomic recombination, intragenomic recombination among the genomic components of the same isolate may also have a significant contribution in the evolution of BBTV genome. Intragenomic recombination therefore appears to be a unique way to generate genetic diversity in the multicomponent ssDNA viruses., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Five genes encoded by Banana bunchy top virus (BBTV) originating from Pakistan were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. Expression of the master replication-associated protein (mRep) and movement protein (MP) resulted in necrotic cell death of inoculated tissues, as well as leaf curling and necrosis along the veins in newly emerging leaves. The systemic necrosis induced by the expression of MP was discolored (dark) in comparison to that induced by mRep. Expression of the cell-cycle link protein (Clink), the coat protein (CP), and the nuclear shuttle protein from the PVX vector induced somewhat milder symptoms, consisting of mild leaf curling and mosaic, although expression of the CP caused a necrotic response in inoculated leaf. The accumulation of viral RNA was enhanced by MP, Clink, and CP. Of the five BBTV-encoded gene products two, the MP and Clink, stabilized GFP-specific mRNA and reduced GFP-specific small interfering RNA in N. benthamiana line 16c when expressed under the control of the 35S promoter and co-inoculated with a construct for the expression of GFP hairpin RNA construct. These results identified MP and Clink as suppressors of RNA silencing. Taken together the ability of MP to induce severe symptoms in plants and suppress RNA silencing implicates this product as a major pathogenicity determinant of BBTV.

Banana bunchy top virus (BBTV) can be transmitted by aphids and consists of at least six integral components (DNA-R, -U3, -S, -M, -C, and -N). Several additional replication-competent components (additional Reps) are associated with some BBTV isolates. A collected BBTV strain (TW3) that causes mild symptoms was selected to study the processes in BBTV evolution. Southern blot hybridization, polymerase chain reaction (PCR), and real-time PCR did not detect DNA-N in TW3. Real-time PCR quantification of BBTV components revealed that, except for the copy number of TW3 DNA-U3, each detected integral component of BBTV TW3 was at least two orders lower than that of the severe strains. No infection was observed in plants inoculated with aphids, which were first given acquisition access to the TW3-infected banana leaves. Recombination analysis revealed recombination between the integral component TW3 DNA-U3 and the additional Rep DNA-Y. All BBTV integral components contain a replication initiation region (stem-loop common region) that share high sequence identity. Sequence alignment revealed that TW3 DNA-R, -S, -M, and -C all have a stem-loop common region containing a characteristic 9-nucleotide deletion found only in all reported DNA-N. Our data suggest that the additional Rep DNAs can serve as sources of additional genetic diversity for integral BBTV components.

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.

Emerging diseases are increasing in incidence; therefore, understanding how pathogens are introduced into new regions and cause epidemics is of importance for the development of strategies that may hinder their spread. We used molecular data to study how a vector-borne banana virus, Banana bunchy top virus (BBTV), spread in Hawaii after it was first detected in 1989. Our analyses suggest that BBTV was introduced once into Hawaii, on the island of Oahu. All other islands were infected with isolates originating from Oahu, suggesting that movement of contaminated plant material was the main driving factor responsible for interisland spread of BBTV. The rate of mutation inferred by the phylogenetic analysis (1.4 x 10(-4) bp/year) was similar to that obtained in an experimental evolution study under greenhouse conditions (3.9 x 10(-4) bp/year). We used these values to estimate the number of infections occurring under field conditions per year. Our results suggest that strict and enforced regulations limiting the movement of banana plant material among Hawaiian islands could have reduced interisland spread of this pathogen.

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.

Two isolates of a novel babuvirus causing "bunchy top" symptoms were characterised, one from abacá (Musa textilis) from the Philippines and one from banana (Musa sp.) from Sarawak (Malaysia). The name abacá bunchy top virus (ABTV) is proposed. Both isolates have a genome of six circular DNA components, each ca. 1.0-1.1 kb, analogous to those of isolates of Banana bunchy top virus (BBTV). However, unlike BBTV, both ABTV isolates lack an internal ORF in DNA-R, and the ORF in DNA-U3 found in some BBTV isolates is also absent. In all phylogenetic analyses of nanovirid isolates, ABTV and BBTV fall in the same clade, but on separate branches. However, ABTV and BBTV isolates shared only 79-81% amino acid sequence identity for the putative coat protein and 54-76% overall nucleotide sequence identity across all components. Stem-loop and major common regions were present in ABTV, but there was less than 60% identity with the major common region of BBTV. ABTV and BBTV were also shown to be serologically distinct, with only two out of ten BBTV-specific monoclonal antibodies reacting with ABTV. The two ABTV isolates may represent distinct strains of the species as they are less closely related to each other than are isolates of the two geographic subgroups (Asian and South Pacific) of BBTV.

The nanovirus Banana bunchy top virus (BBTV) has six standard components in its genome and occasionally contains components encoding additional Rep (replication initiation protein) genes. Phylogenetic network analysis of coding sequences of DNA 1 and 3 confirmed the two major groups of BBTV, a Pacific and an Asian group, but show evidence of web-like phylogenies for some genes. Phylogenetic analysis of 102 major common regions (CR-Ms) from all six components showed a possible concerted evolution within the Pacific group, which is likely due to recombination in this region. The CR-M of additional Rep genes is close to that of DNA 1 and 2. Comparison of tree topologies constructed with DNA 1 and DNA 3 coding sequences of 14 BBTV isolates showed distinct phylogenetic histories based on Kishino-Hasegawa and Shimodaira-Hasegawa tests. The results of principal component analysis of amino acid and codon usages indicate that DNA 1 and 3 have a codon bias different from that of all other genes of nanoviruses, including all currently known additional Rep genes of BBTV, which suggests a possible ancient genome reassortment event between distinctive nanoviruses.

Banana bunchy top virus (BBTV), a multi-component circular ssDNA virus, replicates via a dsDNA intermediate that also serves as a template for virion sense transcription. Seven virus-derived transcripts have been previously identified and analysed in BBTV-infected bananas by northern analysis and 3' rapid amplification of cDNA ends (3' RACE). In this study, we have used RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-RACE) to complete the mapping of the BBTV gene transcripts and have now fully mapped the transcribed regions of each BBTV component and effectively defined the upstream regulatory region.