Spatial parameters associated with the risk of banana bunchy top disease in smallholder systems.

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.

Miniaturized paper-based gene sensor for rapid and sensitive identification of contagious plant virus.

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.

Asymmetric patterns of reassortment and concerted evolution in Cardamom bushy dwarf virus.

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.

Identification and characterization of a distinct banana bunchy top virus isolate of Pacific-Indian Oceans group from North-East India.

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.

Nine novel DNA components associated with the foorkey disease of large cardamom: evidence of a distinct babuvirus species in Nanoviridae.

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.

High-throughput detection of banana bunchy top virus in banana plants and aphids using real-time TaqMan(®) PCR.

Banana bunchy top virus (BBTV) is the causal agent of banana bunchy top disease. Current diagnostic methods for BBTV are laborious and prone to generate false-negative results. A simple, reliable, and high-throughput method for detecting BBTV in plants and aphids has been developed, which involves tissues disruption from banana plants and viruliferous aphids followed by real-time TaqMan(®) PCR. Extraction of BBTV single-stranded DNA using this method is simple and less prone to contamination than using the CTAB (hexadecyltrimethylammonium bromide) method. The high throughput TaqMan(®) PCR system was highly sensitive, detecting as few as 2.76 copies of BBTV genomic DNA or 1.0 ng-1.0mg of infected banana leaves. The entire assay could be completed within 2h. Regression analysis showed that the quantitative results of TaqMan(®) PCR and copies of the virus have good correlation for plasmids (R(2)=0.966) and for infected leaves (R(2)=0.979). The method developed in this study can quantify BBTV in aphids and plants, even before the appearance of symptoms of banana bunchy top disease.

Predicting the benefits of banana bunchy top virus exclusion from commercial plantations in Australia.

Benefit cost analysis is a tried and tested analytical framework that can clearly communicate likely net changes in producer welfare from investment decisions to diverse stakeholder audiences. However, in a plant biosecurity context, it is often difficult to predict policy benefits over time due to complex biophysical interactions between invasive species, their hosts, and the environment. In this paper, we demonstrate how a break-even style benefit cost analysis remains highly relevant to biosecurity decision-makers using the example of banana bunchy top virus, a plant pathogen targeted for eradication from banana growing regions of Australia. We develop an analytical approach using a stratified diffusion spread model to simulate the likely benefits of exclusion of this virus from commercial banana plantations over time relative to a nil management scenario in which no surveillance or containment activities take place. Using Monte Carlo simulation to generate a range of possible future incursion scenarios, we predict the exclusion benefits of the disease will avoid Aus$15.9-27.0 million in annual losses for the banana industry. For these exclusion benefits to be reduced to zero would require a bunchy top re-establishment event in commercial banana plantations three years in every four. Sensitivity analysis indicates that exclusion benefits can be greatly enhanced through improvements in disease surveillance and incursion response.

Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification.

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.

Cloning and sequence analysis of two banana bunchy top virus genomes in Hainan.

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 in sub-Saharan Africa: investigations on virus distribution and diversity.

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.

Molecular characterization of an Indian isolate of Banana bunchy top virus based on six genomic DNA 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.

Molecular characterisation of banana bunchy top virus (BBTV) from Pakistan.

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.

Abacá bunchy top virus, a new member of the genus Babuvirus (family Nanoviridae).

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.