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.

Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids.

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a 'calibrator'. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

Banana Tree Infected with Banana Bunchy Top Virus Attracts Pentalonia nigronervosa Aphids Through Increased Volatile Organic Compounds Emission.

Banana plants are affected by various viral diseases, among which the most devastating is the "bunchy top", caused by the Banana bunchy top virus (BBTV) and transmitted by the aphid Pentalonia nigronervosa Coquerel. The effect of BBTV on attraction mechanisms of dessert and plantain banana plants on the vector remains far from elucidated. For that, attractiveness tests were carried out using a two columns olfactometer for apterous aphids, and a flight cage experiment for alate aphids. Volatile Organic Compounds (VOCs) emitted by either healthy or BBTV-infected banana plants were identified using a dynamic extraction system and gas-chromatography mass-spectrometry (GC-MS) analysis. Behavioral results revealed a stronger attraction of aphids towards infected banana plants (independently from the variety), and towards the plantain variety (independently from the infection status). GC-MS results revealed that infected banana plants produced VOCs of the same mixture as healthy banana plants but in much higher quantities. In addition, VOCs produced by dessert and plantain banana plants were different in nature, and plantains produced higher quantities than dessert banana trees. This work opens interesting opportunities for biological control of P. nigronervosa, for example by luring away the aphid from banana plants through manipulation of olfactory cues.

Infectivity of an Infectious Clone of Banana Streak CA Virus in A-Genome Bananas (Musa acuminata ssp.).

We have characterized the complete genome sequence of an Australian isolate of banana streak CA virus (BSCAV). A greater-than-full-length, cloned copy of the virus genome was assembled and agroinoculated into five tissue-cultured plants of nine different Musa acuminata banana accessions. BSCAV was highly infectious in all nine accessions. All five inoculated plants from eight accessions developed symptoms by 28 weeks post-inoculation, while all five plants of M. acuminata AA subsp. zebrina remained symptomless. Symptoms were mild in six accessions but were severe in Khae Phrae (M. acuminata subsp. siamea) and the East African Highland banana accession Igisahira Gisanzwe. This is the first full-length BSCAV genome sequence reported from Australia and the first report of the infectivity of an infectious clone of banana streak virus.

Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin.

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.

Identification of putative viroplasms within banana cells infected by banana streak MY virus.

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.

A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants.

Banana bract mosaic virus (BBrMV) is a serious pathogen threatening the cultivation of banana and plantain worldwide. This study reports the development of a practical, rapid, sensitive, specific and user-friendly lateral flow immunoassay (LFIA) test for the on-site detection of BBrMV. The BBrMV coat protein (CP) was expressed in Escherichia coli and purified and used to immunize rabbits to produce a polyclonal antiserum (anti-BBrMVCP). The test was based on a double-antibody sandwich format. Protein-A affinity column-purified anti-BBrMVCP Immunoglobulins (IgG) (16 µg/mL), conjugated to ∼30 nm gold nanoparticles, was applied onto the conjugate pad. The anti-BBrMVCP IgG and goat anti-rabbit IgG were printed on the surface of a nitrocellulose filter membrane as the test line and control line, respectively. A positive result could be confirmed visually by the presence of a pink band that developed on the LFIA strip within 5-10 min. The detection limit of the test was 10 ng of the expressed recombinant BBrMV CP (rBBrMVCP), and a 1:20 dilution of the BBrMV-infected crude extract. This LFIA test was validated using 114 banana leaf samples randomly collected from the field and the results indicated a very high diagnostic sensitivity (99.04 %) and specificity (100 %) for the test. A Cohen's kappa coefficient of 0.861 obtained also indicated a very good agreement between the LFIA developed in this study and ELISA. This assay could be adopted by farmers, tissue culture industries and quarantine departments for surveys and surveillance. This is the first report on the development of a LFIA-based test for BBrMV detection.

Estimating a novel stochastic model for within-field disease dynamics of banana bunchy top virus via approximate Bayesian computation.

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.

What are the Main Sensor Methods for Quantifying Pesticides in Agricultural Activities? A Review.

In recent years, there has been an increase in pesticide use to improve crop production due to the growth of agricultural activities. Consequently, various pesticides have been present in the environment for an extended period of time. This review presents a general description of recent advances in the development of methods for the quantification of pesticides used in agricultural activities. Current advances focus on improving sensitivity and selectivity through the use of nanomaterials in both sensor assemblies and new biosensors. In this study, we summarize the electrochemical, optical, nano-colorimetric, piezoelectric, chemo-luminescent and fluorescent techniques related to the determination of agricultural pesticides. A brief description of each method and its applications, detection limit, purpose-which is to efficiently determine pesticides-cost and precision are considered. The main crops that are assessed in this study are bananas, although other fruits and vegetables contaminated with pesticides are also mentioned. While many studies have assessed biosensors for the determination of pesticides, the research in this area needs to be expanded to allow for a balance between agricultural activities and environmental protection.

Genomic, Morphological and Biological Traits of the Viruses Infecting Major Fruit Trees.

Banana trees, citrus fruit trees, pome fruit trees, grapevines, mango trees, and stone fruit trees are major fruit trees cultured worldwide and correspond to nearly 90% of the global production of woody fruit trees. In light of the above, the present manuscript summarizes the viruses that infect the major fruit trees, including their taxonomy and morphology, and highlights selected viruses that significantly affect fruit production, including their genomic and biological features. The results showed that a total of 163 viruses, belonging to 45 genera classified into 23 families have been reported to infect the major woody fruit trees. It is clear that there is higher accumulation of viruses in grapevine (80/163) compared to the other fruit trees (each corresponding to less than 35/163), while only one virus species has been reported infecting mango. Most of the viruses (over 70%) infecting woody fruit trees are positive-sense single-stranded RNA (+ssRNA), and the remainder belong to the -ssRNA, ssRNA-RT, dsRNA, ssDNA and dsDNA-RT groups (each corresponding to less than 8%). Most of the viruses are icosahedral or isometric (79/163), and their diameter ranges from 16 to 80 nm with the majority being 25-30 nm. Cross-infection has occurred in a high frequency among pome and stone fruit trees, whereas no or little cross-infection has occurred among banana, citrus and grapevine. The viruses infecting woody fruit trees are mostly transmitted by vegetative propagation, grafting, and root grafting in orchards and are usually vectored by mealybug, soft scale, aphids, mites or thrips. These viruses cause adverse effects in their fruit tree hosts, inducing a wide range of symptoms and significant damage, such as reduced yield, quality, vigor and longevity.

"Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay".

Cucumber mosaic virus (CMV) is a widespread plant virus infecting important vegetables, plantation and flower crops. Currently, CMV is detected by enzyme-linked immunosorbent assays (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. ELISA requires polyclonal antibodies and is time-consuming. PCR requires skilled manpower and complex procedures of RNA isolation as well as a thermal cycler. To overcome these difficulties, a portable rapid, simple and visual fluorescence-based reverse transcription-recombinase polymerase amplification (portable RT-exo-RPA) assay for the detection of CMV was developed. A specific primer pair of 30-33 bp targeting a conserved region of the coat protein (CP) gene of CMV and a probe to function in the RT-exo-RPA assays were designed and synthesized. A total of 62 symptomatic as well as 58 asymptomatic banana plant samples, collected from banana orchards located in Jalgaon, Maharashtra, India, were evaluated for CMV infections using crude leaf extracts as templates by a reverse transcription-recombinase polymerase amplification (RT-RPA) assay as well as a real-time RT-exo-RPA assay and the results were compared with those of a reverse transcription-polymerase chain reaction (RT-PCR) assay using purified total plant RNAs as templates. CMV was as efficiently detected using the crude leaf extract template in the RT-RPA and real-time RT-exo-RPA assays as using the purified RNA template in the RT-PCR assay. To dispense with the use of real-time PCR, a portable RT-exo-RPA assay was developed and the alternative methods for the visualization of CMV detection using either a fluorometer or direct viewing with a UV transilluminator were evaluated. To our knowledge, this is the first report of the rapid and reliable diagnosis of CMV infections by a real-time RT-exo-RPA assay using a crude leaf extract as template.

Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence.

Banana bunchy top virus (BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO (RbcL) and elongation factor 2 (EF2), involved in protein synthesis. Therefore, the peptide released from B4 (also a precursor) may act as a non-canonical modifier to influence host-pathogen interactions involving BBTV and plants.

CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding.

Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.

Alphasatellitidae: a new family with two subfamilies for the classification of geminivirus- and nanovirus-associated alphasatellites.

Nanoviruses and geminiviruses are circular, single stranded DNA viruses that infect many plant species around the world. Nanoviruses and certain geminiviruses that belong to the Begomovirus and Mastrevirus genera are associated with additional circular, single stranded DNA molecules (~ 1-1.4 kb) that encode a replication-associated protein (Rep). These Rep-encoding satellite molecules are commonly referred to as alphasatellites and here we communicate the establishment of the family Alphasatellitidae to which these have been assigned. Within the Alphasatellitidae family two subfamilies, Geminialphasatellitinae and Nanoalphasatellitinae, have been established to respectively accommodate the geminivirus- and nanovirus-associated alphasatellites. Whereas the pairwise nucleotide sequence identity distribution of all the known geminialphasatellites (n = 628) displayed a troughs at ~ 70% and 88% pairwise identity, that of the known nanoalphasatellites (n = 54) had a troughs at ~ 67% and ~ 80% pairwise identity. We use these pairwise identity values as thresholds together with phylogenetic analyses to establish four genera and 43 species of geminialphasatellites and seven genera and 19 species of nanoalphasatellites. Furthermore, a divergent alphasatellite associated with coconut foliar decay disease is assigned to a species but not a subfamily as it likely represents a new alphasatellite subfamily that could be established once other closely related molecules are discovered.

Development of a gold-nano particle based novel dot immunobinding assay for rapid and sensitive detection of Banana bunchy top virus.

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.

Molecular characterization of two badnavirus genomes associated with Canna yellow mottle disease.

Members of the genus Badnavirus have a single non-covalently closed circular double-stranded DNA genome of 7.2-9.2kb. The genome encodes three open reading frames (ORFs) on the positive DNA strand. Canna yellow mottle virus (CaYMV) is a badnavirus that has been described as the etiological cause of yellow mottle disease in canna, although only a 565bp fragment of the genome has been previously reported from cannas. In this report, concentrated virions were recovered from infected canna plants and nucleic acids were extracted. Two full-length sequences represent two badnavirus genomes were recovered and were determined to be 6966bp and 7385bp in length. These DNAs represent a virus strain belonging to Canna yellow mottle virus and a novel species tentatively termed Canna yellow mottle associated virus. Phylogenetic analysis indicates that these two viruses are closely related to sugarcane bacilliform GD virus, pineapple bacilliform comosus virus, banana streak MY virus, and cycad leaf necrosis virus. We also showed naturally grown canna plants to be frequently co-infected by these two badnaviruses along with a potyvirus, Canna yellow streak virus.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

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.

Characterization of the banana streak virus capsid protein and mapping of the immunodominant continuous B-cell epitopes to the surface-exposed N terminus.

This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different capsid protein (CP) isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N or the C terminus. A three-dimensional model of the BSMYV-CP was constructed, which showed that the CP is a multi-domain structure, containing homologues of the retroviral capsid and nucleocapsid proteins, as well as a third, intrinsically disordered protein region at the N terminus, henceforth called the NID domain. Using the Pepscan approach, the immunodominant continuous epitopes were mapped to the NID domain for five different species of banana streak virus. Anti-peptide antibodies raised against these epitopes in BSMYV were successfully used for detection of native virions and denatured CPs in serological assays. Immunoelectron microscopy analysis of the virion surface using the anti-peptide antibodies confirmed that the NID domain is exposed on the surface of virions, and that the difference in mass of the two CP isoforms is due to variation in length of the NID domain.

Low genetic diversity of Banana bunchy top virus, with a sub-regional pattern of variation, in Democratic Republic of Congo.

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.

How endogenous plant pararetroviruses shed light on Musa evolution.

BACKGROUND AND AIMS: Banana genomes harbour numerous copies of viral sequences derived from banana streak viruses (BSVs) - dsDNA viruses belonging to the family Caulimoviridae.These viral integrants (eBSVs) are mostly defective, probably as a result of 'pseudogenization' driven by host genome evolution. However, some can give rise to infection by releasing a functional viral genome following abiotic stresses. These distinct infective eBSVs correspond to the three main widespread BSV species (BSOLV, BSGFV and BSIMV), fully described within the Musa balbisiana B genomes of the seedy diploid 'Pisang Klutuk Wulung' (PKW). METHODS: We characterize eBSV distribution among a Musa sampling including seedy BB diploids and interspecific hybrids with Musa acuminate exhibiting different levels of ploidy for the B genome (ABB, AAB, AB). We used representative samples of the two areas of sympatry between M. acuminate and M. balbisiana species representing the native area of the most widely cultivated AAB cultivars (in India and in East Asia, ranging from the Philippines to New Guinea). Seventy-seven accessions were characterized using eBSV-related PCR markers and Southern hybridization approaches. We coded both sets of results to create a common dissimilarity matrix with which to interpret eBSV distribution. KEY RESULTS: We propose a Musa phylogeny driven by the M. balbisiana genome based on a dendrogram resulting from a joint neighbour-joining analysis of the three BSV species, showing for the first time lineages between BB and ABB/AAB hybrids. eBSVs appear to be relevant phylogenetic markers that can illustrate theM. balbisiana phylogeography story. CONCLUSION: The theoretical implications of this study for further elucidation of the historical and geographical process of Musa domestication are numerous. Discovery of banana plants with B genome non-infective for eBSV opens the way to the introduction of new genitors in programmes of genetic banana improvement.