Banana Bract Mosaic Virus Research Articles

Immunodiagnostic detection of banana bract mosaic virus (BBrMV) infecting abaca through the recombinant coat protein and its sequence diversity analysis in the Philippines

Abaca is an indigenous fiber crop in the Philippines and is an important source of raw fiber worldwide. Banana bract mosaic virus (BBrMV) causes the abaca bract mosaic disease which results in decreased fiber yield and quality. Out of the 261 abaca samples tested, 175 tested positive via ELISA using raw antiserum against the recombinant BBrMV coat protein (CP). To further validate the antisera produced, a total of 48 samples were screened for BBrMV CP fragment via RT-PCR. Out of the 26 positives for RT-PCR, 22 were cloned and sequenced to validate the RT-PCR amplified fragments. Nucleotide analysis showed a high percentage identity (97–98%) towards BBrMV CP Pacific and Indian isolates while phylogenetic analysis revealed clustering of BBrMV CP Philippine abaca isolates with the Pacific group. The results validate the application of polyclonal antibodies against the recombinant coat protein of BBrMV (rCP) for its serological detection in infected abaca.

Characterization of Two Novel Viruses Within a Complex Virome from Flowering Ginger in Hawaii.

Flowering ginger (Alpinia purpurata) is economically and culturally important in Hawaii. In the past decade, a slow decline syndrome has impacted the production of this crop in the state. RNA sequencing analyses and virus indexing surveys were done on samples collected from four of the Hawaiian Islands. Viral sequences corresponding to six viruses were recovered from transcriptomic data from samples with virus-like symptoms. Canna yellow mottle virus (CaYMV, genus Badnavirus) and two novel viruses, Alpinia vein clearing virus (ApVCV, genus Ampelovirus) and Alpinia vein streaking virus (ApVSV, genus Betanucleorhabdovirus), were found at a moderate incidence in diseased plants. Conversely, three other viruses, including the two potyviruses, banana bract mosaic virus and bean common mosaic virus, and a badnavirus, banana streak GF virus, were also found but at a low incidence. Virus detection in potential insect vectors and transmission assays identified the mealybug Planococcus citri as a vector of CaYMV and ApVCV, whereas the aphid Pentalonia caladii was identified as a vector of the novel ApVSV. Both P. citri and P. caladii are common pests of flowering ginger in Hawaii. Transmission of ApVSV was achieved using P. caladii colonies either established in the laboratory or naturally feeding on infected plants, although no transmission was obtained using viruliferous aphids originally reared on taro (Colocasia esculenta). Our study provides insights into the potential association between viral infections and the observed decline symptoms of flowering ginger in Hawaii. However, more definitive studies are needed to link single or mixed viral infections with decline symptoms.

Piriformospora indica suppresses the symptoms produced by Banana bract mosaic virus by inhibiting its replication and manipulating chlorophyll and carotenoid biosynthesis and degradation in banana.

The online version contains supplementary material available at 10.1007/s13205-024-03983-y.

Beneficial Fungal Root Endophyte Piriformospora indica Diminishes Yield Loss Without Compromising Quality of Banana Fruits Due to Banana bract mosaic virus Infection through Better Soil Nutrient Mobilization

Aims: Banana bract mosaic virus (BBrMV) is a significant viral disease that adversely impacts the yield and quality of banana (Musa acuminata) fruits. Endophytes have been recognized as a sustainable tool to enhance plant nutrient availability and uptake. Piriformospora indica is a beneficial fungal root endophyte that can improve nutrient utilization and confer tolerance to diverse abiotic and biotic stresses in banana plants. The effect of P. indica colonization on nutrient assimilation and fruit quality of BBrMV-infected banana plants still needs to be better understood, particularly in the humid tropical environments of southern laterites of Kerala. P. indica promotes both fruit size and quality, leading to an overall improvement of fruit attributes viz., fruit length, width, colour, TSS, acidity, moisture, and ash content etc., An experiment comprising four treatments with five replications revealed that P. indica colonization enhanced potassium concentration at the rhizosphere (603 Kg ha-1), whereas decreased in the diseased plants (318 Kg ha-1). The nutrient concentration in the rhizosphere recorded a substantial relation with disease severity. Compared to P. indica colonized plants, the uptake of N, P, K, Ca, Mg, S and B by BBrMV infected plants exhibited 36.5, 37.6, 57.5, 44.1, 23.3, 43.7 and 48.9 per cent reduction respectively. Hence, pre-colonization of P. indica improved the nutrient mobilization (in soil), uptake and accumulation (in fruit) in the BBrMV infected banana plants compared to the virus alone infected plant. Fruits from P. indica pre-colonized plants challenged with the virus exhibited an enhanced K (515.5 ± 6.25 mg 100g -1) and Fe (0.42 ± 0.04 mg 100 g-1) content along with high total soluble sugars (27.25oBrix), titrable acidity (0.48%) and ash content (1.14%) compared to healthy and virus infected plants. Hence, this can instigate the appropriate sustainable virus disease management strategies in banana plants, emphasizing its improved fruit quality through nutrient assimilation.

One-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) for closed-tube colorimetric detection of banana bract mosaic virus in Banana (Musa spp.).

The banana bract mosaic virus (BBrMV) is a major virus affecting bananas and plantains. Banana being propagated vegetatively, there arises a high risk of virus transmission through planting materials. Available molecular detection technique like the Reverse Transcriptase Polymerase Chain Reaction needs post-amplification sample handling, predisposing to sample cross contamination. A one-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) assay coupled with colorimetric detection was optimised for easy and quick detection of BBrMV in banana. The viral coat protein gene was amplified under isothermal conditions at 65 ºC. The RT-LAMP assay was optimised with respect to concentrations of MgSO4, dNTP, Bst polymerase enzyme and HNB dye. The total RNA purified from symptomatic samples was directly amplified under isothermal conditions by including 100 U M-MLV reverse transcriptase and 20 U RNasin® plus RNase inhibitor in the reaction. With the addition of 120µM of Hydroxy Naphthol Blue (HNB) dye in the RT-LAMP reaction, the BBrMV-positive samples had a colour change from violet to sky blue after the reaction. The RT-LAMP assay detected BBrMV in 0.1pg of total RNA isolated from symptomatic plants. Molecular characterisation of RT-LAMP products was done using restriction profiling and sequence analysis. The RT-LAMP assay was validated using field-collected banana leaf samples. The assay successfully detected the virus from symptomatic samples while the healthy samples showed no amplification. Samples sourced from banana plants with symptoms of banana bunchy top virus, banana streak virus and cucumber mosaic virus tested negative in the RT-LAMP assay, thus ensuring the specificity of the assay.

Occurrence of Banana bract mosaic virus on Musa ornata Roxb based hybrids in India.

The online version contains supplementary material available at 10.1007/s13337-022-00788-6.

Banana bract mosaic virus (banana bract mosaic disease)

Banana bract mosaic virus (banana bract mosaic disease)

Computationally predicted virus responsive miRNAs in banana (Musa AAB)

The bioinformatics tool NovoMIR was used to predict miRNAs in Musa acuminata genome. NovoMIR predicted 85 pre-miRNAs from the 11 chromosomes of Musa acuminata DH Pahang and BLAST analysis of the predicted pre-miRNAs against annotated miRNAs of miRBase identified 52 mature miRNAs belonging to 38 different families. psRNATarget server identified 124 protein-coding sequences as potential targets for 40 mature miRNAs. Based on the role of the target genes in biological processes related to biotic stress, five miRNAs were selected for analyzing their response to Banana bract mosaic virus (BBrMV) infection. Three-month-old in vitro raised banana plants of var. Nendran (Musa AAB) infected with BBrMV showed the presence of all the five selected miRNAs in both healthy and BBrMV infected plants. Expression analysis using RT-qPCR showed changes in the expression of miR-3900-5p, miR-2172-5p, miR-6928-5p and miR-971-5p and their targets during BBrMV infection.

Evidence of viral genome linked protein of banana bract mosaic virus interaction with translational eukaryotic initiation factor 4E of plantain cv. Nendran based on yeast two hybrid system study.

Banana bract mosaic virus (BBrMV), belongs to the genus Potyvirus and it is an important viral pathogen of bananas and plantains. The eukaryotic translation initiation factor, eIF4E, and its isoform play key roles during the virus infection in plants, particularly Potyvirus. The present study was undertaken to determine the role of BBrMV-viral protein genome-linked (VPg) in virus infectivity by analyzing the interaction with the eukaryotic translation initiation factor eIF4E through yeast two-hybrid system. The results suggest that plantain cv. Nendran eIF4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus and may potentially contribute to BBrMV resistance.

Biochemical alterations in banana cultivars infected systemically by banana bract mosaic virus (BBrMV)

Virus infection in plants lead to the alteration of physiological, biochemical and metabolic processes such as total protein synthesis, activation or synthesis of defence peptides and proteins, rapid production of reactive oxygen species (ROS) as well as synthesis of phenols. Banana Bract Mosaic Disease (BBrMD) is one of the most destructive viral diseases of banana, however, information on physiological and biochemical changes during banana-BBrMV interaction is still unexplained. Therefore, the present investigation was conducted to find out the quantifiable changes in biochemical parameters such as total proteins, phenolic compounds, peroxidase (PO), polyphenol oxidase (PPO), catalase (CAT) ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) activities in leaves of commercial banana cultivar Poovan (AAB) and a plantain cv. Nendran (AAB). The amount of phenols, total protein, GPX and SOD activities were significantly higher in leaves of BBrMV infected plants of both the cultivars over the healthy plants, whereas PO, PPO, CAT and APX activities reduced in infected than healthy plants. Overall the results suggest that BBrMV infection induces significant changes in biochemical and enzyme levels leading to irreversible symptom development and significant yield loss.

Transgenic banana plants carrying ihpRNA cassette targeting viral replicase gene show resistance against Banana bract mosaic virus

ABSTRACT RNAi approach was attempted to impart resistance against Banana bract mosaic virus (BBrMV). An intron hairpin RNA (ihpRNA) vector targeting its replicase gene was constructed in pSTARLING vector. A fragment (421 bp) of the BBrMV replicase gene isolated from the infected sucker of banana cv. Nendran (Musa AAB) was used for making ihpRNA cassette. The ihpRNA cassette (NotI fragment) was mobilised to the binary vector pART27 in Agrobacterium tumefaciens strain LBA 4404 for transformation. Embryogenic calli derived from the immature male inflorescence of cv. Nendran maintained in MS medium supplemented with BA 0.1 mg L−1 and picloram 1 mg L−1 were transformed with LBA 4404. Transformed calli were subjected to embryo induction in semisolid MS medium supplemented with BA 2 mg L−1 and IAA 0.5 mg L−1 and germinated on half-strength MS medium with BA 2 mg L−1 and IAA 0.5 mg L−1, with a maximum regeneration of 25% under the selection pressure of 100 mg L−1 kanamycin. The regenerated shoots were confirmed for the presence of ihpRNA construct by PCR with primers specific for sense-intron-antisense fragment, npt II and cre intron. The transgenics challenged with BBrMV showed no infection. Untransformed plants showed infection and developed disease symptoms after 3 months.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV)

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl β-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Rapid, simple detection of banana bract mosaic virus in abaca using a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay

Banana bract mosaic virus (BBrMV) causes one of the major viral diseases of the fiber crop abaca at a great economic loss in the Philippines. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect BBrMV. A set of six primer pairs was designed based on the conserved coat protein gene sequences of BBrMV. The RT-LAMP can be completed in 30 min and was 100,000-fold more sensitive than RT-PCR. The RT-LAMP assay is suitable for field detection of BBrMV using crude RNA extracts and fluorescent DNA intercalating dyes that produce easily visualized results.

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.

Prediction and experimental confirmation of banana bract mosaic virus encoding miRNAs and their targets

BackgroundPotyviridae is the largest plant infecting family under the monophyletic group Riboviria, infects many of the food, fodder and ornamental crops. Due to the higher mutation and recombination rate, potyvirids are evolving rapidly, adapting to the environmental chaos and expanding their hosts. Virus control measures are need to be updated as the economic importance of potyvirids is massive. microRNAs (miRNAs) are well known for their functional importance in eukaryotes and many viruses. Regardless of its biogenesis, whether canonical or noncanonical, microRNA centric antivirus approaches attract the researchers to the hopeful future of next-generation broad-spectrum antiviral measures.MethodsIn this study, we predicted and screened banana bract mosaic virus (BBrMV) encoding miRNAs by computation approaches and their targets on banana transcriptome using plant small RNA target analysis server (psRNAtarget). The target gene functions were annotated by Blast2GO. The predicted BBrMV miRNAs were experimentally screened by stem-loop RT-PCR.ResultsThe results showed that, among the predicted BBrMV miRNAs, miRNA2 is conserved throughout BBrMV isolates and has multiple virus-specific target transcripts. In addition, primary experimental validation for the predicted miRNAs revealed that miRNA2 exists in the BBrMV infected banana leaf samples.ConclusionsThe existence of BBrMV miRNA2 is confirmed by stem-loop RT-PCR followed by cloning and sequencing. The presence of miRNA of Potyviridae is rarely addressed and would definitely spread the hope to understand the virus infectious cycle. Our report would also help to better understand and manipulate potyviral infections.

Multiplex reverse transcription-polymerase chain reaction for simultaneous detection of banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV) in abaca

The abaca (Musa textilis Nee) is an important fibre crop in the Philippines. Its fibre yield and quality is severely impacted by two potyviruses, namely, banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV). A multiplex RT-PCR assay was developed for the simultaneous detection of these two RNA viruses in abaca. Primer sets were designed to amplify fragments of BBrMV coat protein (743 bp), SCMV coat protein (595 bp) and Musa sp. elongation factor 1 (219 bp) as an internal control. The specificity of each primer pair was confirmed by cloning and sequencing of the RT-PCR products. The multiplex RT-PCR assay was optimized by varying primer concentration combinations, dNTP concentration, Mg2+ concentration, Taq polymerase concentration, annealing temperature and extension temperature. The developed multiplex RT-PCR protocol has the same level of sensitivity and specificity when compared with uniplex RT-PCR detection for each virus and internal control. The inclusion of internal control amplification eliminates the risk of obtaining false negative results. The use of the optimized multiplex RT-PCR in simultaneous detection of BBrMV and SCMV from field samples proves that it is reliable, robust and faster than uniplex RT-PCR detection of each virus. This method will be useful for large-scale surveys of these two RNA viruses in abaca and will be vital for epidemiological studies and disease prevention.

Evidence of seed transmission of Banana bract mosaic virus in Musa synthetic diploid H-201, a possible threat to banana breeding

Banana bract mosaic virus (BBrMV) is a serious constraint in Musa spp. and Musa textilis causing enormous economic losses to growers. The distribution of the virus in various parts of the seeds, flower and leaf tissues of seedlings from banana plants of a synthetic diploid, H-201, naturally infected in the field was determined by Plate-trapped antigen coating enzyme-linked immunosorbent assay (PTA-ELISA), reverse transcriptase (RT) polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR) analysis and sequencing. RT-PCR and qRT-PCR results revealed the presence of BBrMV in embryo, endosperm and seed coat of seeds from infected plants. Nucleotide sequencing of the RT-PCR amplicons confirmed the BBrMV infection in seed. Quantification of BBrMV in various parts of seed by qRT-PCR using SYBR Green chemistry revealed that the viral load was maximal in the endosperm followed by seed coat and embryo. In a seed growth test, thirteen seedlings, that were raised from embryos of infected seeds in vitro and twenty seedlings raised from infected seeds in pots, expressed typical symptoms of bract mosaic virus disease and were confirmed RT-PCR positive for the virus. This finding will have significant implications in banana breeding programmes.

Molecular characterization of banana bract mosaic virus from India reveals recombination and positive selection in the VPg gene

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is an important viral pathogen affecting banana and plantains in India. The present study was undertaken to determine the genetic variation and molecular evolution of BBrMV infecting different cultivars of banana and plantain in the Indian subcontinent, based on the viral genome-linked protein (VPg) gene. Sequence identity of this gene from 29 BBrMV isolates showed a range of nucleotide (nt) and amino acid (aa) identity of 75–100% and 95–100%, respectively. Phylogenetic analysis based on nt revealed that except for two isolates (TN1 and TN2), all the other Indian isolates clustered together. Different functional motifs of the VPg gene previously reported were found to be conserved. A single possible recombination event was detected using a recombination detection programme. A codon-based selection analysis revealed that most of the codons in the VPg gene were under purifying selection except for those at position 46, 47, 71, 107, 149, 153, 156, 175, 176, and 178, which were under positive selection. Gene flow between different Indian populations of BBrMV from banana was relatively low. This is the first report on genetic diversity and evolution of the VPg gene of BBrMV. Since VPg is identified as a virulence determinant in resistance mediated by the eukariotic intiation factor 4E (eIF4E) in several plant-potyvirus interactions, any mutation in the VPg interacting domain may abolish the interaction with eIF4E or its isoforms in vitro and prevent viral infection in planta. Therefore, understanding the population structure of BBrMV based on VPg would potentially provide insight into the diversification and infection cycle of this virus.

Complete genome sequencing of banana bract mosaic virus isolate infecting cardamom revealed its closeness to banana infecting isolate from India.

The complete genome of banana bract mosaic virus (BBrMV), a Potyvirus belonging to the family Potyviridae causing chlorotic streak disease of cardamom (Elettaria cardamomum) in India was determined for the first time from a naturally infected cardamom var. Njallani Green Gold through reverse transcription PCR using nine sets of primers designed to different overlapping regions of the genome. The complete genome has 9708 nucleotides excluding poly (A) tail and has the genome organization similar to that of BBrMV isolates infecting banana and flowering ginger (Alpinia purpurata). The virus has a single open reading frame of 9372 nucleotides that encodes for a polypeptide of 3124 amino acids which is later cleaved into ten matured proteins. The length and arrangements of different proteins in BBrMV-Cardamom was similar to other BBrMV isolates except for the P1 protein that showed a single amino acid deletion. Comparison with three available complete genome sequences revealed that, BBrMV-Cardamom isolate is more closer to BBrMV-Banana isolate from India (BBrMV-TRY) (96.7% identity) than to BBrMV-Banana isolate from Philippines and flowering ginger isolates from USA (94.5%). Analysis of polyprotein and their individual proteins also showed close identity of BBrMV-Cardamom and BBrMV-TRY. The phylogenetic analysis also suggested that BBrMV-Cardamom isolate is closely related to other BBrMV isolates.

Yield loss management through fertilizers in Banana bract mosaic virus affected French plantain cv. Nendran (AAB)

Plant virus infection significantly reduces the yield of many horticultural crops causing reduction in the production and productivity. Higher fertilizer application has often to overcome the yield reduction due to virus in many crops. A field experiment was conducted to find out the effect of increased doses of fertilizers on the banana bract mosaic virus (BBrMV) infected plants of French plantain cv. Nendran. The plant height, girth, number of leaves, number of fingers and the bunch weight had significantly increased with increased dose of fertilizer in the first and second crops with BBrMV infection. In the first crop, with the application of 125 and 150% of recommended dose of fertilizers (RDF), the BBrMV infected plants could produce bunches which were on par with that of healthy plants, receiving 100% RDF, but with prolonged days of flowering. In the second crop, though 125 and 150% RDF doses in the infected plants increased the bunch weight over 100% RDF by 14.60% and 18.33%, respectively, could not be able to compensate the yield loss due to virus infection. This may probably be due to the accumulation of virus over the period of two years. This is the first report on strategies for mitigating adverse effects of BBrMV in plantain.