Banana Streak Research Articles

First Report of Banana bract mosaic virus in 'Cavendish' Banana in Ecuador.

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of bract mosaic disease. The disorder has been considered a serious constraint to banana and plantain production in India and the Philippines, where the virus was first identified (3). To date, the presence of BBrMV has been reported only in a few banana-growing countries in Asia (3). In the Americas, BBrMV has been detected by ELISA tests in Colombia only (1). The efficient spread of BBrMV through aphids and vegetative material increases the quarantine risk and requires strict measures to prevent entrance of the virus to new areas. In Ecuador-the world's number one banana exporter-the banana industry represents the main agricultural income source. Thus, early detection of banana pathogens is a priority. In June of 2012, mosaic symptoms in bracts and bunch distortion of 'Cavendish' banana were observed in a commercial field in the province of Guayas, Ecuador. Leaves from 35 symptomatic plants were tested for Cucumber mosaic virus (CMV), Banana streak virus (BSV), and BBrMV using double antibody sandwich ELISA kits from Adgen (Scotland, UK). Twenty-one plants tested positive for BBrMV but not for CMV or BSV. In order to confirm the ELISA results, fresh or lyophilized leaf extracts were used for immunocapture reverse transcription (IC-RT)-PCR. In addition, total RNA was extracted from the ELISA-positive samples and subjected to RT-PCR. The RT reactions were done using both random and oligo dT primers. Several sets of primers, flanking conserved regions of the virus coat protein (CP), have been used for PCR-detection of BBrMV (2,3,4). The Ecuadorian BBrMV isolate was successfully detected by three primer sets with reported amplification products of 324, 280, and 260 nucleotides long, respectively (3,4). Amplification products of the expected size were purified and sequenced. All the nucleotide sequences obtained from 20 PCR-positive symptomatic plants were 100% identical between each other. However, 99% identity was observed when PCR products from the Ecuadorian isolate were compared with the corresponding fragment of a BBrMV isolate from the Philippines (NCBI Accession No. DQ851496.1). PCR products of the Ecuadorian isolate, amplified by the different CP primers described above, were assembled into a 408-bp fragment and deposited in the NCBI GenBank (KC247746). Further testing confirmed the presence of BBrMV in symptomatic plants from four different provinces. To our knowledge, this is the first report of BBrMV in Ecuador and the first BBrMV partial nucleotide sequence reported from the Americas. It is worth mentioning that primer set Bract 1/Bract 2, which amplifies a 604-bp product (2), was not effective in detecting the Ecuadorian isolate. It is hypothesized that nucleotide variation at the reverse primer site is the cause of the lack of amplification with this primer set, since the forward primer is part of the sequenced product and no variation was found. Sequencing of the entire CP region is underway to conduct phylogenetic analysis and determine genetic relationships across several other BBrMV isolates.

Bacilliform DNA‐containing plant viruses in the tropics: commonalities within a genetically diverse group

Plant viruses, possessing a bacilliform shape and containing double-stranded DNA, are emerging as important pathogens in a number of agricultural and horticultural crops in the tropics. They have been reported from a large number of countries in African and Asian continents, as well as from islands from the Pacific region. The viruses, belonging to two genera, Badnavirus and Tungrovirus, within the family Caulimoviridae, have genomes displaying a common plan, yet are highly variable, sometimes even between isolates of the same virus. In this article, we summarize the current knowledge with a view to revealing the common features embedded within the genetic diversity of this group of viruses. Virus; order Unassigned; family Caulimoviridae; genera Badnavirus and Tungrovirus; species Banana streak viruses, Bougainvillea spectabilis chlorotic vein banding virus, Cacao swollen shoot virus, Citrus yellow mosaic badnavirus, Dioscorea bacilliform viruses, Rice tungro bacilliform virus, Sugarcane bacilliform viruses and Taro bacilliform virus. Bacilliform in shape; length, 60-900 nm; width, 35-50 nm; circular double-stranded DNA of approximately 7.5 kbp with one or more single-stranded discontinuities. Each virus generally limited to its own host, including banana, bougainvillea, black pepper, cacao, citrus species, Dioscorea alata, rice, sugarcane and taro. Foliar streaking in banana and sugarcane, swelling of shoots in cacao, yellow mosaic in leaves and stems in citrus, brown spot in the tubers in yam and yellow-orange discoloration and stunting in rice. http://www.dpvweb.net.

Three Infectious Viral Species Lying in Wait in the Banana Genome

Plant pararetroviruses integrate serendipitously into their host genomes. The banana genome harbors integrated copies of banana streak virus (BSV) named endogenous BSV (eBSV) that are able to release infectious pararetrovirus. In this investigation, we characterized integrants of three BSV species-Goldfinger (eBSGFV), Imove (eBSImV), and Obino l'Ewai (eBSOLV)-in the seedy Musa balbisiana Pisang klutuk wulung (PKW) by studying their molecular structure, genomic organization, genomic landscape, and infectious capacity. All eBSVs exhibit extensive viral genome duplications and rearrangements. eBSV segregation analysis on an F1 population of PKW combined with fluorescent in situ hybridization analysis showed that eBSImV, eBSOLV, and eBSGFV are each present at a single locus. eBSOLV and eBSGFV contain two distinct alleles, whereas eBSImV has two structurally identical alleles. Genotyping of both eBSV and viral particles expressed in the progeny demonstrated that only one allele for each species is infectious. The infectious allele of eBSImV could not be identified since the two alleles are identical. Finally, we demonstrate that eBSGFV and eBSOLV are located on chromosome 1 and eBSImV is located on chromosome 2 of the reference Musa genome published recently. The structure and evolution of eBSVs suggest sequential integration into the plant genome, and haplotype divergence analysis confirms that the three loci display differential evolution. Based on our data, we propose a model for BSV integration and eBSV evolution in the Musa balbisiana genome. The mutual benefits of this unique host-pathogen association are also discussed.

English

Musa (banana and plantain) provides a major source of carbohydrates for about 400 million people of whom 20 million are from East Africa. Yet, banana is threatened by number constraints, banana streak virus inclusive. Banana streak virus (BSV) was monitored in Rakai and Ntungamo, Uganda for up to 72 months after planting (MAP) and 29MAP respectively. BSV incidence increase over time was fitted into exponential model and spatial spread analysed by 2DCLASS and 2DCORR. BSV infection was initiated in Rakai 29 months after planting (MAP), but only 6 MAP in Ntungamo. BSV incidence then increased at a rate of 0.10 plants respectively / infected plant / month at a rate 0.23 plants / infected plant / month in Rakai and Ntungamo respectively. In both sites, spatial analysis showed that there were significant aggregated BSV spatial patterns. New infections were more likely to occur within a 10 rows/coloumns from an old infection. Significant edge effects were also detected in Ntungamo, indicating that there was significant spread from the immediate surroundings (infected established field suggesting need for separation of new fields from old infected fields to delay onset of BSV. Roguing should be frequent enough to offset rate of BSV incidence increase. The study shows that BSV is a slow spreading disease; however, there is sufficient time in this perennial cropping system for it to increase to epidemic levels. It is however, possible to check the advance of the BSV epidemic through phytosanitary measures.  Key words: Banana streak virus (BSV), spatial and temporal spread, phytosanitation.

First Report of Banana mild mosaic virus Infecting Plantain in Ivory Coast.

Plantain (Musa sp., genomic group AAB) is an important crop for millions of the world's poorest people. In Ivory Coast, it is the second most consumed food and an important source of income for farmers. Between 2010 and 2011, a survey for viruses infecting plantain (AAB) was conducted in 10 major plantain-growing regions located in eastern (Abengourou), middle-western (Bouaflé, Daloa, Issia, Oumé, Sinfra, Zuenoula), central (Yamoussoukro), and southern (Aboisso, Gagnoa) Ivory Coast. Leaf samples showing yellow streaks or mild chlorotic streaks were collected and dried on CaCl2 for storage. A representative sample from each location was selected and tested for the presence of Cucumber mosaic virus (CMV, genus Cucumovirus), Banana streak virus (BSV, genus Badnavirus), Banana mild mosaic virus (BanMMV, family Flexiviridae), and Banana bract mosaic virus (BBrMV, genus Potyvirus). Immunocapture (IC)-PCR was used for the detection of BSV while reverse transcription (RT)-PCR was used for the detection of CMV, BanMMV, and BBrMV. The following primers sets were used: BSV cl1 and BSV cl2 (1), CMV 3' and CMV 5' (3), BanMMV BanCP1 and BanCP2 (4), BBrMV Bract N2 and Bract NR (2). BanMMV was detected as mixed infections with BSV in the 10 tested samples, one of which also contained CMV. To confirm the identity of the amplification products from the BanMMV primers, one cDNA fragment was directly sequenced in the forward direction (Macrogen Inc., Seoul, South Korea). BLAST search in GenBank revealed that the partial coat protein (CP) sequence of the Ivorian isolate shared 80 to 88% nucleotides and 81 to 92% deduced amino acid similarities with BanMMV isolates. In contrast, partial CP sequence of the Ivorian isolate had less than 40% deduced amino acid sequence identity with other Flexiviridae CP sequence. The partial CP sequence of the Ivorian BanMMV isolate was deposited in GenBank under Accession No. JX014304. To further confirm the identification, all the samples were tested by plate trapped antigen (PTA)-ELISA with rabbit polyclonal antiserum specific to BanMMV (obtained from B. E. Lockhart, University of Minnesota, U.S.A.) and anti-rabbit IgG (Sigma-Aldrich, Belgium/A3687). The 10 samples reacted positive for BanMMV by ELISA. CMV and BSV have been reported in Ivory Coast, but to our knowledge, this is the first report of BanMMV in the country. The detection of BanMMV in association with BSV or CMV in mixed infection in 10 locations which are important plantain growing areas is a first step in the evaluation of the impact of virus diseases on plantain production in this country.

English

  Banana (Musa spp.) is grown for subsistence and income generation by 80% of small scale farmers all year round in Kenya hence it is an important food security crop. However viral diseases such as banana streak disease (BSD), caused by Banana streak virus, hamper the production of the crop. BSV has been reported to be present in all the commercial banana cultivars in Kenya. Tissue culture materials of 15 cultivars were evaluated for BSV expression so as to determine the tolerant cultivars under field and greenhouse conditions and stages of disease expression. A number of factors including plant age, cultivar, season and growth site were investigated in the greenhouse and field to assess their effect on BSD symptom expression. Statistical analysis system (SAS) for ANOVA was used for analysis of data for the experiments. Results indicated that BSD symptom expression is significantly influenced by all the factors under investigation. Significant correlation was also observed between symptom severity and plant height, girth, and number of leaves. Findings of this study will be invaluable to stakeholders and researchers in banana industry by laying a foundation for development and adoption of viable BSD management strategies.   Key words: Banana streak virus, Banana streak disease, symptoms expression, tissue culture, ELISA.

Genetic diversity of Sugarcane bacilliform virus isolates infecting Saccharum spp. in India

Sugarcane bacilliform virus (SCBV), which causes leaf freckle in sugarcane, is a member of the genus Badnavirus. Studies were conducted to characterize SCBV in Saccharum officinarum germplasm and cultivated varieties in India by sequencing the complete genomes of five isolates. Genome lengths ranged from 7,553 to 7,884 nucleotides. Duplications in ORF3 and insertions in the RNase H-domain in some of the isolates were found to contribute to the large size of their genomes. The Indian SCBV isolates share identities of 69-85 % for the complete genomic sequence, indicating wide genetic diversity among them, and share 70-82 % identity with Sugarcane bacilliform Ireng Maleng virus (SCBIMV) and Sugarcane bacilliform Morocco virus (SCBMV), as well as 43-46 % identity with Banana streak virus (BSV) and BSV-related SCBV species from Guadeloupe, indicating that the Indian SCBV isolates are distinct from SCBV isolates reported to date. Irrespective of the region compared, SCBV isolates from India, Australia, and Morocco clustered together. BSV and BSV-related SCBV isolates from Guadeloupe formed another cluster. A phylogenetic analysis based on the partial RT/RNase H-sequence separated SCBV and BSV-related SCBV sequences into 11 SCBV groups viz. SCBV-A to -K. Among the 11 groups, the SCBV sequences separated under H, I, J, and K are newly identified in this study, representing three new species and are tentatively named as SCBBBV, SCBBOV, and SCBBRV. Thus, the PASC and phylogenetic analyses evidenced that the symptoms associated with badnaviruses in sugarcane in India are caused by at least three new species, SCBBBV, SCBBOV, and SCBBRV, besides SCBIMV and SCBMV represented by SCBV-BT and SCBV-Iscam, respectively.

First Report of Banana bunchy top virus in Banana and Plantain (Musa spp.) in Nigeria

Plantain and banana (Musa spp.) are among the most important staple crops for food and income generation for the rural and urban populations in the humid forest agroecological zone of West Africa. Until recently, Cucumber mosaic virus (genus Cucumovirus) and Banana streak virus (genus Badnavirus) were the only viruses reported to occur in Musa spp. in West Africa. In 2011, an outbreak of banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV; genus Babuvirus, family Nanoviridae) was reported in Ouémé Département (6°30'N and 2°36'E) in the Republic of Benin (2). BBTV is one of the most economically important pathogens of Musa spp. It is well established in Central Africa and also in Angola, Malawi, and Zambia in Southern Africa (2). Plants infected at early growth stages are severely dwarfed and do not bear fruit. BBTV is transmitted by the banana aphid Pentalonia nigronervosa, which is widespread in Africa (1). The regions in the Republic of Benin affected by BBTV border Ogun State (7°00'N and 3°35'E) of Nigeria. Epidemiological investigations were conducted during May 2012 at 31 locations in Ogun State to determine the potential risk of BBTV spreading into Nigeria. Plants with typical symptoms of BBTD (stunting, narrow and shortened leaves, chlorotic streaks on petioles and pseudostem) were observed in four locations: Ilashe, Odan-Itoro, Ido-Ologun, and Igbogila. Total DNA was extracted from 90 leaf samples randomly collected from symptomatic and asymptomatic banana and plantain plants in these areas. Samples were tested for BBTV by polymerase chain reaction (PCR) using primer pairs, mREP-F and mREP-R, which amplifies a 241-bp of BBTV DNA-mRep segment (1), and Scp-F and Scp-R specific for approximately 1075-bp BBTV DNA-S that encodes coat protein gene (1). The amplicons of expected size were obtained from 17 of 90 samples analyzed (18.8%). BBTV in the symptomatic plants was further confirmed by nucleic acid spot hybridization (NASH) assay using DIG-labeled 1,075-bp probe corresponding to coat protein gene and chromogenic detection as per the previously described protocol (3). The DIG-probe specifically reacted with nucleic acid from the symptomatic plants, but not with negative controls, providing conclusive evidence for the BBTV. The PCR products of DNA-mRep segment amplified from three banana plants infected with BBTV collected in Ilashe (Ipokia Local Government Area) were purified and sequenced in both directions. The sequences of these isolates were 100% identical with each other (GenBank Accession Nos. JX290301, JX290302, and JX290303). A BLASTn search revealed 100% nucleotide sequence identity with a BBTV isolate from Benin (JQ437548) and 99 to 100% identity with DNA-mRep sequences of several other BBTV isolates from Africa, Australia, India, and the South Pacific. Further analysis of the 241-bp mRep gene sequences with Neighbor-Joining phylogenetic analysis grouped the BBTV isolate with the South Pacific isolates. To our knowledge, this is the first report of BBTV in Nigeria. This underscores need for surveys to assess the extent of BBTV spread in Nigeria and strict implementation of phytosanitary measures, including restrictions on the movement of planting material from disease-affected regions, to prevent further spread of this important disease.

English

  Banana is the fourth most important food crop worldwide. However, its production has been threatened by banana streak disease, caused by banana streak virus (BSV). Despite this situation, little is known about the inter-relationships between symptom severity and cultivar/virus genotypes. Symptom severity assessment for sixty five symptomatic samples was carefully done. The rolling circle amplification technique was used to identify the virus species (isolate) infecting each sample. The Mysore virus isolates (BSMysV) and the banana cultivars containing the balbisiana (B) component were associated with the most severe banana streak disease symptoms.   Key words: Banana streak disease, symptom severity, banana streak virus (BSV) isolate, rolling circle amplification.

A historical overview of the appearance and spread of Musa pests and pathogens on the African continent: highlighting the importance of clean Musa planting materials and quarantine measures

AbstractThe genus Musa is not native to Africa. It evolved in tropical Asia, from southwest India eastward to the island of New Guinea. There is a growing circumstantial evidence which suggests that the East African Highland banana and the tropical lowland plantain were cultivated on the African continent since before 1 AD. It is also probable that ABB cooking and AB and AAB dessert cultivars were brought to the continent from India by Arabian traders from 600 AD, and that these were disseminated throughout East Africa. During the colonial era, the main centres of distribution for banana cultivars were botanical gardens, such as Zomba in Malawi, Entebbe in Uganda and Amani in Tanzania. It appears that the very early introductions of Highland banana and plantain arrived in Africa as a relatively clean material without the conspicuous pests and diseases that affect them in Asia. In contrast, several devastating problems now impact the crop in Africa, including nematodes, the borer weevil and diseases, most notably banana bunchy top, banana streak, Sigatoka leaf spots, Xanthomonas wilt and Fusarium wilt. We (a) provide chronological overviews of the first reports/observations of different Musa pests and pathogens/diseases in Africa, (b) highlight specific examples of when a pest or pathogen/disease was introduced via planting materials and (c) give recent examples of how the pests and pathogens spread to new regions via planting materials. In total, these production constraints threaten banana and plantain production throughout the continent and impact those who can ill afford lost production, the small‐holder producer. Our intent in this review is to highlight the significance of these problems and the great importance that infested planting materials have played in their development.

Simultaneous Detection of Four Banana Viruses by Multiplex PCR

AbstractBanana bunchy top virus (BBTV), Cucumber mosaic virus (CMV), Banana bract mosaic virus (BBrMV) and Banana streak virus (BSV) are four viruses known to infect banana corp. On the basis of the establishment of the optimal PCR and reverse transcription (RT)‐PCR for the detection of a single virus, a quadruplex RT‐PCR method that employed virus‐specific primers was developed for the simultaneous detection and differentiation of all the four viruses in banana. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by simplex and quadruplex RT‐PCR. The assay was then validated using banana samples infected with one or more viruses collected from fields and tissue‐culture banana industries in Hainan Island.

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

AbstractBanana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.

Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.

Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based Methods in Detection of <i>Banana streak virus</i> in <i>Musa</i> Germplasm

Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.

Molecular characterization of Banana streak virus isolate from Musa Acuminata in China

Banana streak virus (BSV), a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantations has previously been investigated, but there are relatively few reports of the genetic characteristic of episomal (non-integrated) BSV genomes isolated from China. Here, the complete genome, a total of 7722bp (GenBank accession number DQ092436), of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAcYNV) in Yunnan, China was determined. The genome organises in the typical manner of badnaviruses. The intergenic region of genomic DNA contains a large stem-loop, which may contribute to the ribosome shift into the following open reading frames (ORFs). The coding region of BSAcYNV consists of three overlapping ORFs, ORF1 with a non-AUG start codon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein. Besides the complete genome, a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants. Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vietnam (BSAcVNV). The corresponding coding regions shared identities of 88% and -95% at nucleotide and amino acid levels, respectively. Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.

High molecular variability of sugarcane bacilliform viruses in Guadeloupe implying the existence of at least three new species

Thirty-five unique partial sugarcane bacilliform virus (SCBV) sequences extending over 529bp were identified in sugarcane samples from Guadeloupe diagnosed by Immunocapture-PCR (IC-PCR) using specific badnavirus primers. Phylogenetic analysis of these sequences along with the two known genome sequences of Sugarcane bacilliform Mor virus (SCBMV) and Sugarcane bacilliform IM virus (SCBIMV) revealed high molecular variability in the SCBV genome. Seven phylogenetic groups, named A to G, were characterized: virus isolates from groups A-B, C and D are proposed to be members of three additional SCBV species.The two (7446 and 7444bp) and one (7317bp) complete sequences of SCBV isolates from groups A and D, respectively, likely represented the genome of two new species. Phylogenetic analysis of the complete genome and RT/RNase H sequences confirmed the polyphyletic structure of SCBV isolates and the absence of a clear separation between SCBV and Banana streak virus (BSV) isolates within badnavirus group 1. These results showed that reconsideration of taxonomy and classification of SCBV and BSV are necessary.

VIRUS-INDEXING TECHNOLOGY FOR PRODUCTION OF QUALITY BANANA PLANTING MATERIAL: A BOON TO THE TISSUE-CULTURE INDUSTRY AND BANANA GROWERS IN INDIA

Banana and plantain (Musa spp.) are the most important fruit crop in India and play a major role in the livelihood of millions of resource-poor small farmers. Use of quality planting material is very important for increasing productivity. Though conventional suckers are still the primary planting material, use of tissue-culture plants has increased because of their advantages, like more uniform bunches with even maturity and increased yield. However, banana viral pathogens, which are economically important in India, can be inadvertently spread through tissue-culture plants. In order to control the spread of the viruses, virus-indexing techniques were developed at the National Research Centre for Banana, Tiruchirapalli for early detection in mother plants and tissue-culture plants used for mass propagation. Polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), non-radioactive probe-based nucleic acid spot hybridisation (NASH) and enzyme-linked immune-sorbent assay (ELISA) based techniques were developed and validated for routine testing. PCR and NASH tests are being done for detection of Banana bunchy top virus (BBTV) and Banana streak Mysore virus (BSMysV). RT-PCR and ELISA tests are being done for the detection of Banana bract mosaic virus (BBrMV) and Cucumber mosaic virus (CMV). Based on the work done at the centre, the Department of Biotechnology (DBT), Government of India, has accredited the Molecular Virology lab for testing for banana viruses in the country. In total, 15,850 tissue-culture and mother-plant samples were tested against viruses. The percentage of positive plants for BBTV, BSMysV, CMV and BBrMV were 3.83, 2.85, 17.3 and 0.95% respectively. Indexing was done mostly for the cultivars ‘Grande Naine’ (AAA, Cavendish subgroup), ‘Robusta’ (AAA, Cavendish subgroup) and ‘Hill Banana’ (syn. ‘Virupakshi’, AAB, Pome subgroup). Around 25 tissue-culture banana commercial laboratories are undertaking the services of virus testing. Our laboratory has also developed polyclonal antisera for CMV and BBrMV through recombinant DNA technology, which is currently being validated for ELISA-based testing.

Molecular characterisation of six badnavirus species associated with leaf streak disease of banana in East Africa

Banana leaf streak disease, caused by several species of Banana streak virus (BSV), is widespread in East Africa. We surveyed for this disease in Uganda and Kenya, and used rolling-circle amplification (RCA) to detect the presence of BSV in banana. Six distinct badnavirus sequences, three from Uganda and three from Kenya, were amplified for which only partial sequences were previously available. The complete genomes were sequenced and characterised. The size and organisation of all six sequences was characteristic of other badnaviruses, including conserved functional domains present in the putative polyprotein encoded by open reading frame (ORF) 3. Based on nucleotide sequence analysis within the reverse transcriptase/ribonuclease H-coding region of open reading frame 3, we propose that these sequences be recognised as six new species and be designated as Banana streak UA virus, Banana streak UI virus, Banana streak UL virus, Banana streak UM virus, Banana streak CA virus and Banana streak IM virus. Using PCR and species-specific primers to test for the presence of integrated sequences, we demonstrated that sequences with high similarity to BSIMV only were present in several banana cultivars which had tested negative for episomal BSV sequences.

Complete genome sequence of a novel badnavirus, banana streak IM virus

In 1999, banana streak disease outbreaks occurred at two locations in Australia in new banana hybrids that were being screened for fusarium wilt resistance. Two different badnaviruses, banana streak GF virus and a newly discovered virus called banana streak IM virus (BSIMV), were detected in these plants. The complete nucleotide sequence of the BSIMV genome was determined and comprised 7768 nt. Three open reading frames were detected, the first beginning with a non-conventional start codon (CUG). A 55-nt repetition in the putative pregenomic RNA promoter was also identified. Phylogenetic analysis suggests that BSIMV is most closely related to banana streak VN virus.

Development of a Novel Rolling-Circle Amplification Technique to Detect Banana streak virus that also Discriminates Between Integrated and Episomal Virus Sequences.

Banana plants are hosts to a large number of Banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity among BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence-nonspecific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterization of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV), and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV, and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.