MOLECULAR DETECTION AND VARIABILITY OF BANANA STREAK VIRUS ISOLATES IN KENYA

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 dIvoire in 1966 (Yot-Dauthy and Bov, 1966; Lassoudire, 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% (Lassoudire, 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).

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., 2002). The objectives of this study were to characterise BSV isolates from diverse locations in Kenya and to develop a protocol for routine diagnostics. Materials and Methods Two hundred and fifteen leaf samples with BSV-like symptoms were collected from 8 locations of Kenya including Mt Elgon, Bungoma, Kakamega, Kisii, Njoro, Embu, Nyeri and JKUAT (Table 1 shows only representative samples). Serological tests were performed using a broad spectrum polyclonal BSV antiserum (Ndowora and Lockhart, 1997, 2000). Double-antibody sandwich (DAS) ELISA followed the procedure of Ndowora and Lockhart (1997) with plates being read on Elisa Reader, E-Max molecular devices. Immunosorbent electron microscopy (ISEM) was used as described by Ahlawat et al. (1996). Grids were viewed in a JEOL JEM 1200EX electron microscope at 15 000 x magnification and particle counts were made on 10 fields.
Table 1Analysis of Kenyan banana leaf samples for the presence of BSV Locality Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Mt. Elgon Bungoma Bungoma Bungoma Bungoma Bungoma Kakamega Kisii Kisii Kisii R.C Kisii Kisii Kisii Kisii Kisii Kisii Kisii Kisii R.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, A/B= Apple Banana, CC= Chinese Cavendish, GB=Golden beauty; 2 ve=Negative/ No particles/no bands; + = very weak detection / 1 particle/field; ++ = moderate detection / 2 particles/field; +++ = strong detection / 3particles

Mini-preps of virus were prepared from fresh-frozen and freeze-dried material using the procedure by Harper et al. (2002). The IC-PCR method described by Harper et al. (2002) was used and the PCR program followed the manufacturers recommendations (Invitrogen). The primers used to screen the BSV isolates were as indicated in Table 2. PCR products obtained from 8 samples Likhaho, Lisulya1, Lisulya2, Khabusi, Sialamule, Nshule, Gold-Finger, and Kampala were purified using QIAquick PCR purification kit protocol (Invitrogen). The products were cloned into the vector pCRII-TOPO (Invitrogen) and used to transform competent E. coli. Clones containing inserts of the expected size were identified by restriction analysis. Selected clone inserts were analysed using the Big Dye 3.1 sequencing protocol (Applied Biosystems) and automated sequencing (Lark Technologies UK). Sequences were compared to other BSV and Badnavirus. Sequence alignments and construction of bootstrapped, neighbour-joining trees were carried out using Clustal X (Thompson et al., 1997). Results and Discussion The 215 collected samples had streaks that were considered fine, long and narrow to short and broad similar to those described by Harper et al. (2002) (Table 1 shows only representative samples). Fifty percent of the sampled Musa genotypes belonged to East African Highland AAA (EAHAAA) genotype. Symptoms observed ranged from mild to severe with chlorotic and necrotic streaking on the leaves. The mostly affected areas were in western part of Kenya mainly Mount Elgon, Bungoma, and Kisii. Although the intensity of BSV was influenced by genotypes, there was no relationship between them. Disease severity was more pronounced in western than central and eastern regions (Table 1). In virus extraction the sap-extraction method yielded both high and low virus yield scores of +++ and +, respectively (Table 1). This method was used to extract virus in all the 80 samples. There was no direct relationship between the severity of the symptoms and virus yield. Enzyme Linked Immunosorbent Assay were conducted in all the 215 samples where 197 confirmed positive (Data not shown). Immunosorbent electron microscopy results were limiting because only eight samples were done, however these were representative samples. Characteristic virions of approximately 120 nm x 30 nm (Figure 1) were detected in seven samples (Likhaho 1, Likhako 2, Lisulya 1, Lisulya 2, Khabusi, Bururu, and Nshule). These tests confirmed that samples with typical BSV symptoms contained bacilliform particles.

Fig. 1: ISEM of a virus preparation from Kenyan banana leaf cv. Likhaho from Mt. Elgon The arrow indicates a bacilliform particle of approximate size 120 nm x 30nm.

Although amplification of a specific viral DNA fragment was successful using both standard PCR and IC-PCR techniques, better amplification was achieved using IC-PCR, as shown for Khabusi (Figure 2 A and B). It is possible that compounds which interfere with PCR amplification are removed during the immune-capture process. The immune capture-PCR amplified a total of twenty nine BSV samples (Figure 3 shows a few of the amplified samples). 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.

Fig. 2: Comparison of standard PCR and IC-PCR for detection of BSV. Samples of cv. Khabusi of Mt. Elgon

0.1

0.1
Fig. 3: Phylogenetic analysis of partial badnavirus sequences including Kenyan BSV isolates. The Kenyan isolates are P1_5REV; P3_2, P3_5, P3_7, P3_8 and P3_9; 4_1F,., 4_3R, 4_4R, 5_3R,; 5B_1F, 5B_2R 5B_3R, 9_4F, 9_5F, 9_9R, 9_10R; GF13_1, GF13_2, GF13_3 GF13_4, GF13_6, GF13_7, GF13_8; Amplified sequences were aligned with other database badnavirus sequences, and neighbour-joining trees calculated (and bootstrapped) using ClustalX. The phylogenetic tree was rooted with RTBV, the caulimovirus most closely related to the badnaviruses, as outgroup.

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. Sample 5B_1F of cv. Khabusi was similar to Banana Streak Goldfinger Virus (BSGfV) isolate, sample P3_5 of cv. Lisulya was similar to Banana streak Imove virus (BSImV) isolate, and samples GF13_1 to GF13_8 of Gold-Finger cultivar were similar to Banana streak Obino lEwai virus (BSOEV)species (Fig 4) 1). All the three species have previously been identified as integrated and activatable sequences associated with the Musa B genome (Harper, 1999). Uganda A virus (BSUgAV), was previously detected as an episomal virus in Uganda and was restricted to the Mt. Elgon region. It seems likely that this isolate was recently introduced into Kenya, possibly from Uganda. 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). All were of comparable size and compared well to the results obtained with sequencing.
Table 2Molecular 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'- ATCTGAAGGTGTGTTGATCAATGC-3'' BSV-RD GenBankORFIII 522 (AF215816) polyprotein RD - 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' 5CTNTAYGARTGGYTNGTNATGCCNTTY GG3 5TCC 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 - 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 +

Ngombe -

Libururu + -

++ -

+ nd +

++ +

++ -

++ -

+ +

+ +

BSV-mys

++ ++ -

+ + -

+ +

+ -

Conclusion and Recommendation

The presence of BSV in Kenyan samples has been confirmed using ISEM, IC-PCR and sequencing techniques. A protocol was developed to detect the virus from field and tissue culture materials. The transmission of BSV is poorly understood, particularly under field conditions and over large areas. Strategies to minimise re-infection of clean planting material and so maximise banana production in Kenya should be developed. A practical control measure would be to avoid propagating identified infected mother plants and use of BSV screened tissue culture material. Acknowledgement We are grateful to Director KARI, to Rockefeller Foundation for funding this study, Professor Lockhart for providing us with the antisera and John Innes Centre for allowing us to use the labouratory facilities. References
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