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L. Karanja1, A. Wangai2, G. Harper3, J. Stanley3 and R.S. Pathak4 1 KARI Njoro, P.O. Njoro, Kenya, 2KARI-NARL Biotechnology Program, P.O. Box 14733, Nairobi 3 Dept. of Disease and Stress Biology, John Innes Centre, Norwich Research, Park, Colney, Norwich NR4 7UH, UK. 3 Egerton University Department of Plant and Soil Science, P.O. Box 536 Njoro, Kenya Abstract Banana Streak Virus (BSV) is one of the recent constraints of banana production in Kenya. Results of preliminary surveys carried out previously in various parts of the country had indicated the presence of BSV in all the banana cultivars through symptoms and Enzyme Linked Immunosorbent Assay (ELISA) Tests. The ability to quickly and reliably detect BSV is important for the management of the virus and for availing virus-free material to the farmers. Hence, detection of the variability of BSV was necessary for the development of a suitable diagnostic protocol for routine screening of tissue cultured material. To develop the protocol, 215 samples were randomly collected from 40 farms at 8 locations across Kenya. Banana streak virus was found present in 200 of the field samples using ELISA technique. The possible variation of the virus was assessed on 80 infected banana samples using immune capturepolymerase chain reaction (IC-PCR) technique and sequence analysis. Both degenerate and specific primer sets were used on extracted viral DNA, and used a procedure previously described by Harper et al., (1998). Some of the samples did not give a product with the degenerate primers but did with specific primer sets. Banana streak virus was confirmed in a total of 29 samples. Virus found in the samples was compared to Nigerian isolate Obino l’ Ewai (BSOEV) and on the basis of the observed size of amplicons, some Kenyan isolates were different from BSOEV. Nucleotide sequence analysis showed that even the same size PCR products had differing sequences. Variation of the isolates was confirmed through sequencing and use of different primer sets for specific isolates. A total of 6 isolates were detected. Results confirmed presence of BSV in Kenya and its genetic diversity. Introduction Bananas and plantains (Musa) are the fourth most important food crop worldwide based on gross value of production (Anon, 2000) and are subsistence and cash crop for many smallholder farmers, particularly in West, Central and East Africa. In Kenya, the annual production is approximately 210,000 metric tonnes for banana and 830,000 tons for plantain (FAO, 2004). One limiting factor to banana production is banana streak disease caused by Banana Streak Virus (BSV). Banana streak disease was first reported in the Cote d’Ivoire in 1966 (Yot-Dauthy and Bové, 1966; Lassoudière, 1974) and is found in all countries where bananas are grown (Lockhart and Jones, 2000). Banana Streak Virus-infected Musa plants frequently express broken or continuous chlorotic or necrotic streaks on the leaves, stunting of diseased plants and occasionally heart-rot of the pseudostem and plant death. However, the disease symptom varies and is thought to depend upon a variety of factors such as virus isolate, host genotype, level of management and environmental conditions (Lockhart, 1986; Lockhart and Jones, 2000; Dahal et al., 1998a). Little is known about the effects of host genotype on symptom expression but expression of symptoms can be intermittent possibly due to environmental conditions such as temperature (Dahal et al., 1998a). This intermittent symptom expression was correlated with virus titre in the leaves with symptomatic leaves containing higher levels of virus (Dahal et al., 1998a). Yield loss due to BSV infection range from 6% to 90% (Lassoudière, 1974; Dahal et al., 2000; Daniells et al., 2001). This range probably reflects difference in cultivars and cultural conditions. Banana Streak Virus is a member of the family Caulimoviridae, genus Badnavirus having a circular, non-covalently closed double-stranded DNA genome of 7.5 kbp encapsidated in bacilliform particles approximately 120 X 30 nm (van Regenmortel et al., 2000). The virus is serologically and genomically very variable with many different isolates. Lockhart and Olszewski, (1993) recognised at least 3 distinct serotypes and four distinct isolates based on PCR amplification with degenerate primers followed by a DNA hybridisation analysis. The most widely used techniques to detect the viruses are ELISA, immunosorbent electron microscopy (ISEM) and PCR (Ndowora and Lockhart, 1997, 2000; Dahal et al., 1998b; Thottappilly et al., 1998; Harper et al., 1999a; Geering et al., 2000). The problem with using PCR-based diagnostics is that BSV sequences are integrated into the host genome of certain banana genotypes (LaFleur et al., 1996; Harper et al., 1999b; Ndowora et al., 1999), hence it is necessary to ensure that the sample is not contaminated with chromosomal DNA. (Geering et al., 1999, 2001). Banana streak virus is transmitted by several species of mealybug (Lockhart and Jones, 2000; Kubiriba et al., 2001b). Mealybug transmission is slow but a more significant transmission is likely to be through vegetative propagation material (Lockhart and Jones, 2000).
Elgon Mt. A/B= Apple Banana. 2002). Elgon Mt. Elgon Mt. Bungoma. ++ = moderate detection / 2 particles/field. Immunosorbent electron microscopy (ISEM) was used as described by Ahlawat et al. Elgon Mt. Materials and Methods Two hundred and fifteen leaf samples with BSV-like symptoms were collected from 8 locations of Kenya including Mt Elgon. 2 –ve=Negative/ No particles/no bands. Table 1–Analysis of Kenyan banana leaf samples for the presence of BSV Locality Mt.C Kisii Kisii Kisii Kisii Kisii Kisii Kisii Kisii R. Embu. Elgon Bungoma Bungoma Bungoma Bungoma Bungoma Kakamega Kisii Kisii Kisii R.Recent studies in Kenya have shown that BSV is prevalent in all the banana growing regions and all the popular cultivars grown by farmers are susceptible (Wangai et al. GB=Golden beauty.C Njoro Njoro Njoro Njoro Njoro Njoro Njoro Njoro Embu JKUAT JKUAT JKUAT JKUAT 1b Cultivar Name Likhaho Likhaho Likhaho Lisulya Lisulya Lisulya Khabusi Khabusi Bururu Libururu Libururu Murure Chilume Valary Nasirembe D/C 1 Genotype EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA AAA EAHAAA AAA EAHAAA AAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA EAHAAA AB AB AAA AAA EAHAAA AAAB Symptom Expression Severe Severe streaks Severe Severe Moderate streaks Severe Severe streaks Moderate Severe Severe streaks Severe streaks Severe Moderate Moderate Moderate Moderate Moderate Moderate Severe Moderate Severe Severe streaks Severe Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Severe streaks Mild streaks Mild streak Moder streaks Mild streaks Mild streaks Mild streaks Moderate Severe Moderate Moderate Moderate ELISA ++ nd nd ++ + nd + + nd nd + + + + + + + + + + nd + + + + + + + nd + nd nd nd nd nd nd nd + + + + + Direct virus PCR +++ ++ + nd + + +++ nd + -ve nd nd nd nd nd nd nd + + nd nd nd nd nd nd nd + nd + + nd nd -ve nd nd IC-sap nd + + + + + + + ++ + + -ve + ++ +++ + + -ve -ve ++ ++ + -ve -ve -ve -ve -ve ++ -ve + ++ + + + + + + + -ve -ve -ve -ve -ve Suriat Grand Naine Mysore Sialamule Nshule Nshule Ngombe Ngombe Kisukari A/B Bogoya Kampala 1 U/G Mbuki Gold Finger Namukhila Valary U/Green Grandnaine Lacatan Lacatan Lacatan Paz 1 GB CC 1 1 AAA AB AAA AAA AAA Valary Williams AbbreviationD/C=Dwarf Cavendish. Grids were viewed in a JEOL JEM 1200EX electron microscope at 15 000 x magnification and particle counts were made on 10 fields. CC= Chinese Cavendish. Kisii. Kakamega. +++ = strong detection / 3particles 2 . Nyeri and JKUAT (Table 1 shows only representative samples). Elgon Mt. The objectives of this study were to characterise BSV isolates from diverse locations in Kenya and to develop a protocol for routine diagnostics.. Elgon Mt. + = very weak detection / 1 particle/field. Njoro. E-Max molecular devices. Elgon Mt. Serological tests were performed using a broad spectrum polyclonal BSV antiserum (Ndowora and Lockhart. 1997. Elgon Mt. Double-antibody sandwich (DAS) ELISA followed the procedure of Ndowora and Lockhart (1997) with plates being read on Elisa Reader. Elgon Mt. Elgon Mt. 2000). (1996). Elgon Mt. Elgon Mt.
Bungoma. Although amplification of a specific viral DNA fragment was successful using both standard PCR and IC-PCR techniques. Fig. Our results confirms that episomal virus can be trapped by the antibodies and the viral DNA detected by IC-PCR with high sensitivity and specificity. long and narrow to short and broad similar to those described by Harper et al. neighbour-joining trees were carried out using Clustal X (Thompson et al. respectively (Table 1). Lisulya 2. coli. Sequence alignments and construction of bootstrapped. Disease severity was more pronounced in western than central and eastern regions (Table 1). The IC-PCR method described by Harper et al. as shown for Khabusi (Figure 2 A and B). however these were representative samples. There was no direct relationship between the severity of the symptoms and virus yield. (2002) was used and the PCR program followed the manufacturer’s recommendations (Invitrogen). Enzyme Linked Immunosorbent Assay were conducted in all the 215 samples where 197 confirmed positive (Data not shown). Lisulya1. (2002) (Table 1 shows only representative samples). better amplification was achieved using IC-PCR. These tests confirmed that samples with typical BSV symptoms contained bacilliform particles. and Kisii. Lisulya 1. Selected clone inserts were analysed using the Big Dye 3. The immune capture-PCR amplified a total of twenty nine BSV samples (Figure 3 shows a few of the amplified samples). The mostly affected areas were in western part of Kenya mainly Mount Elgon. Results and Discussion The 215 collected samples had streaks that were considered fine. Khabusi. Although the intensity of BSV was influenced by genotypes. Bururu. Nshule. and Kampala were purified using QIAquick PCR purification kit protocol (Invitrogen). 1997). It is possible that compounds which interfere with PCR amplification are removed during the immune-capture process. In virus extraction the sap-extraction method yielded both high and low virus yield scores of +++ and +.Mini-preps of virus were prepared from fresh-frozen and freeze-dried material using the procedure by Harper et al. Clones containing inserts of the expected size were identified by restriction analysis.. (2002). there was no relationship between them. PCR products obtained from 8 samples Likhaho. Khabusi. Lisulya2. Sialamule. and Nshule). Likhaho from Mt. Elgon The arrow indicates a bacilliform particle of approximate size 120 nm x 30nm. Symptoms observed ranged from mild to severe with chlorotic and necrotic streaking on the leaves. Gold-Finger. Sequences were compared to other BSV and Badnavirus.1 sequencing protocol (Applied Biosystems) and automated sequencing (Lark Technologies UK). 1: ISEM of a virus preparation from Kenyan banana leaf cv. The products were cloned into the vector pCRII-TOPO (Invitrogen) and used to transform competent E. Characteristic virions of approximately 120 nm x 30 nm (Figure 1) were detected in seven samples (Likhaho 1. Immunosorbent electron microscopy results were limiting because only eight samples were done. This method was used to extract virus in all the 80 samples. Likhako 2. Fifty percent of the sampled Musa genotypes belonged to East African Highland AAA (EAHAAA) genotype. 3 . The primers used to screen the BSV isolates were as indicated in Table 2.
Elgon 0. GF13_6. 4_1F. 4_3R. 9_10R. 5B_1F. Samples of cv. 4 . 5_3R. and neighbour-joining trees calculated (and bootstrapped) using ClustalX. GF13_8. The phylogenetic tree was rooted with RTBV. 5B_2R 5B_3R.1 Fig. 4_4R.... 2: Comparison of standard PCR and IC-PCR for detection of BSV. as outgroup. 9_5F.1 0. the caulimovirus most closely related to the badnaviruses. P3_7. 9_4F. GF13_7. P3_2. P3_5. 9_9R. The Kenyan isolates are P1_5REV.Fig. 3: Phylogenetic analysis of partial badnavirus sequences including Kenyan BSV isolates. GF13_2. GF13_3 GF13_4. Amplified sequences were aligned with other database badnavirus sequences. GF13_1. Khabusi of Mt. P3_8 and P3_9.
All were of comparable size and compared well to the results obtained with sequencing. Uganda A virus (BSUgAV). It seems likely that this isolate was recently introduced into Kenya. possibly from Uganda. Khabusi was similar to Banana Streak Goldfinger Virus (BSGfV) isolate. Sample 5B_1F of cv. In addition to the seven primer sets (Table 2) 6 of the Kenyan samples (Table 3) gave varying band size of between 400 – 700 corresponding to 6 different isolates (data not shown).R1 5'-GCTCACTCCGCATCTTATCAGTC-3' GF-F1 5'-ACGAACTATCACGACTTGTTCAAGC-3' BSV-GF GenBankORFIII 782 GF-R1 5'-TCGGTGGAATAGTCCTGAGTCTTC-3' (AF215814) polyprotein BSV467 3-F1 BSV531 7-R1 1A-F1 4’ -R1 L8235 BADNA T 5'-GGAATGAAAGAGCAGGCC-3' 5'-AGTCATTGGGTCAACCTCTGTC-3' 5’CTNTAYGARTGGYTNGTNATGCCNTTY GG3’ 5’TCC AYT TRC ANA YNS CNC CCC ANC C3’ 5’-TAAAAGCACAGCTCAGAACAAACC-3’ 5’-CTCCGTGATTTCTTCGTGGTC-3’ BSVONNE BADNA GenBank(AJ002234) (Harper et al 2002) GenBank(AF214005) ORFIII polyprotein Aspartic protease and RT 476 644 BSV-Mys 597 408 Primer RD . Lisulya was similar to Banana streak Imove virus (BSImV) isolate. Table 2–Molecular characterisation of the viruses isolated from 8 samples using amino acid and oligonucleotide sequence analysis and primer set screening Primer Sequence Virus Source Product Target isolate of product RD – F1 5'.F1 RD – R1 GF-F1 GF-R1 BSV4673F1 BSV5317R1 1A-F1 4’ -R1 BSVUg A – F1 BSVUg A –R1 L8238 BADNAT I-M – F1 I-M – R1 EK – OL– F1 EK – OL– R1 Isolate BSV-RD BSV-GF BSVONNE All Cultivars Khabusi + + + Chirume + - Lisulya +++ ++ + Likhaho - Nshul e + Ng’ombe - Libururu + - ++ - + nd + ++ + ++ - ++ - + + + + BSV-mys ++ ++ - + + - + + + + + - 5 .Molecular characterisation of the viruses isolated from 8 samples using amino acid and oligonucleotide sequence analysis and primer set screening has shown that they are closely related to 6 known virus isolates.ATCTGAAGGTGTGTTGATCAATGC-3'' BSV-RD GenBankORFIII 522 (AF215816) polyprotein RD . All the three species have previously been identified as integrated and activatable sequences associated with the Musa B genome (Harper. 1999). and samples GF13_1 to GF13_8 of Gold-Finger cultivar were similar to Banana streak Obino l’Ewai virus (BSOEV)species (Fig 4) 1). was previously detected as an episomal virus in Uganda and was restricted to the Mt. sample P3_5 of cv. Elgon region.
Plant Pathology. Cloning and sequence analysis of banana streak virus DNA . Harper G.E. Association of badnavirus with citrus disease in India.S. 17.E. and Jones D. France Lassoudière A. Archives of Virology Jones D. 24..E.W. 76..O.L. Musa fact sheet No. Vuylsteke D.L. relative concentration of viral antigen. (2002). (2004).L. Identification et purification de diverses souches du virus. Thottappilly G. particularly under field conditions and over large areas.J.E.K.. Relationship between natural occurrence of banana streak badnavirus and symptom expression. Karanja J. 247-257. FAO. IC-PCR and sequencing techniques. Bryde N. and Olszewski N. Ortiz R. (2002). Fruits. Hull R.. Annals of Applied Biology.. Thompson J.. Lockhart B.. Daniells J.. A practical control measure would be to avoid propagating identified infected mother plants and use of BSV screened tissue culture material. Harper G.. 29.D... 263-274. and yield characteristics of some micropropagated Musa spp. Lockhart B.. UK: CAB International.P. 449-465. Annals of Applied Biology.. Harper G. (2000). In: Ganry J (Ed)..R. Kimani S. and Hull R. Hughes J.E. (1993) Banana streak disease.D.. Phytopathology. Banana streak.. (2000). and Varma A. Analysis tools. Yot-Dauthy D. The effect of Banana streak virus on the growth and yield of dessert bananas in tropical Australia. Ed. Jeanmougin F. Preliminary results on surveys of banana streak virus (BSV) in Kenya. Harper G.E. Acknowledgement We are grateful to Director KARI.S. 2003-2004.L. Srivastrava M. Pant R. L. (2001). and Bové J. Lockhart B. Gibson T. Plant Disease 80... 349-357. (1986). (1997). Geering A. Chakraborty N. to Rockefeller Foundation for funding this study. Food and Agriculture Organisation of the United Nations. and Higgins D. Osuji J. Commodity market review. Wangai A. Hull1 R. The diversity of Banana streak virus isolates in Uganda. (1966). Mosaïque du bananier..G.W. (2005). (1974). Wallingford. 49. Ndung'u E. 590–592. Lockhart B.. Professor Lockhart for providing us with the antisera and John Innes Centre for allowing us to use the labouratory facilities. (1999) Integration of banana streak badnavirus into the Musa genome: molecular and cytogenetic evidence. Fruit.. Kilonzo F. 51-60.Conclusion and Recommendation The presence of BSV in Kenyan samples has been confirmed using ISEM.L. 68-79.. and R.. Nucleic Acids Res. La mosaïque dite “à tirets” du bananier Poyo en Côte d’Ivoire..International network for improvement of banana and plantain.L. Tenkouano A. 271-278. The transmission of BSV is poorly understood. pp. and Nguthi. Plewniak F. and Thomas J. Virology 255.. 139. Lockhart B..M. Lockhart B. References Ahlawat Y. (1998).W... In Diseases of banana abaca and enset. Hart D. 141. Purification and serology of a bacilliform virus associated with banana streak disease. 995-999. The ClustalX. Virus Genes.E.J. Rome. Egerton university/ KARI-NPBRC 2nd Annual Symposium 6 . A protocol was developed to detect the virus from field and tissue culture materials. R. Moult S. (1993) Serological and genomic heterogeneity of banana streak badnavirus: implications for virus detection in Musa germplasm. (1996).E. 1. Strategies to minimise re-infection of clean planting material and so maximise banana production in Kenya should be developed. Montpellier. d’A. Hart D. Geering A. Detection of banana streak virus in field samples of bananas from Uganda. D R Jones. Heslop-Harrison J. 4876–4882. Dahal G. Hull. Moult S. and Thomas J. 21..
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