Construction of infectious clones of banana bunchy top virus (bbtv) in vietnam

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - UNDERGRADUATE THESIS TITLE: CONSTRUCTION OF INFECTIOUS CLONES OF BANANA BUNCHY TOP VIRUS (BBTV) IN VIETNAM Student name : LE THI THUY LINH Class : K63CNSHE Student code : 637415 Department : BIOTECHNOLOGY Supervisor : Assoc.Prof HA VIET CUONG Dr NGUYEN THI THUY HANH HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - UNDERGRADUATE THESIS TITLE: CONSTRUCTION OF INFECTIOUS CLONES OF BANANA BUNCHY TOP VIRUS (BBTV) IN VIETNAM Student name : LE THI THUY LINH Class : K63CNSHE Student code : 637415 Department : BIOTECHNOLOGY Supervisor : Assoc.Prof HA VIET CUONG Dr NGUYEN THI THUY HANH HANOI – 2022 DECLARATION I hereby declare that the graduate thesis work is mine All research results have been results during the implementation of the topic The results, the data are completely true, never appeared in any scientific report I also guarantee that the references and useful information for the topic are clearly cited and all help is appreciated Hanoi, June 27th, 2022 Student Le Thi Thuy Linh i ACKNOWLEDGEMENTS During the process of implementing my graduation project, I have received a lot of attention and help from individuals and groups First of all, I would like to express my respect and deep gratitude to Dr Nguyen Thi Thuy Hanh and Assoc Prof Ha Viet Cuong for giving me the opportunity to carry out this work, and their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis Secondly, I would like to thank the teachers in the Faculty of Biotechnology have helped and taught me during my training at university Especially the teachers of the Biology department who gave me advice during carrying out Finally, I would like to sincerely thank my family members and friends who always trust, support and encourage me to complete this report Sincerely thank! Hanoi, June 27th, 2022 Student Le Thi Thuy Linh ii CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS xi PART I INTRODUCTION 1.1 Preface 1.2 Objective and Requirements 1.2.1 Objective 1.2.2 Requirements PART II LITERATURE REVIEW 2.1 Banana (Musa SPP.) 2.1.1 Origin and distribution 2.1.2 Morphology 2.1.3 Banana economy 2.1.4 The importance of banana fruit in Vietnam 2.2 Banana bunchy top disease 11 2.2.1 Host plants and disease symptoms 11 2.2.2 Disease history and current epidemiological status 12 2.2.3 Disease transmission and spread 13 2.2.4 Disease detection and control 13 2.3 Banana bunchy top virus 14 2.3.1 Scientific classification 14 2.3.2 Genome organization and Replication cycle 15 2.3.3 Origin, evolution and diversity of BBTV isolates 17 2.4 Agroinoculation method 18 2.4.1 Principle and applications of agroinoculation in plant virology studies 18 iii 2.4.2 Agroinoculation using infectious clones of double strand DNA viruses and Nanovirus 20 2.5 Gibson Assembly technology: Principle and applications 22 2.5.1 Principle 22 2.5.2 Application and advantages of Gibson Assembly® 24 PART III MATERIAL AND METHODS 29 3.1 Location and time for research 29 3.1.1 Research location 29 3.1.2 Time for research 29 3.1.3 Subjects 29 3.2 Material 29 3.2.1 BBTV isolate 29 3.2.2 Reagents and commercial kit 29 3.2.3 Vectors 36 3.2.4 Key equipments 37 3.2.5 Chemicals, buffers and media 37 3.2.6 Markers and Primers 37 3.2.7 E coli strain 37 3.3 Methods 38 3.3.1 BBTV DNA extraction 38 3.3.2 PCR, Cloning and sequencing of BBTV DNA-R in pTZ57 vector 38 3.3.3 Sequence analysis of BBTV DNA-R 39 3.3.4 Design of BBTV primers for Gibson Assembly 39 3.3.5 Amplification 12 fragments (1mer, 0.5mer) by Phusion Polymerase Kit (NEB) 40 3.3.6 Rolling circle amplification 40 3.3.7 Amplification of 1-5 mer fragments using RCA product 41 3.3.8 Amplification of 1-5 mer fragments by fused 1-mer and 0.5-mer fragments 42 3.3.9 Optimization of overlap extension reaction 42 3.3.10 Phusion 1.5mer fragments in pairs to form fragments using Phusion® High-Fidelity DNA Polymerase with HF buffer 44 iv 3.3.11 Gibson assembly of 1.5mer fragments by Gibson Assembly® Master Mix (NEB), Gibson Assembly Master Mix Homemade, NEBuilder® HiFi DNA Assembly Master Mix 44 PART IV RESULTS AND DISCUSSION 47 4.1 Sequence analysis of the DNA-R component of the BBTV VN2018 isolate 47 4.1.1 PCR to amplify complete DNA-R of VN2018 isolate 47 4.1.2 Sequencing of DNA-R of VN2018 isolate 48 4.1.3 BLAST search 50 4.1.4 Phylogenetic analysis based on DNA-R 50 4.2 In silico construction of infectious clone of BBTV on pcambia2300 vector using Gibson Assembly Technology 52 4.2.1 Selection of templates of BBTV genomic components for in silico construction of infectious clone 52 4.2.2 In silico construction of BBTV infectious clone using Gibson Assembly Technology 54 4.3 Amplification mer and 0.5mer BBTV components 58 4.3.1 PCR to check the specificity of all primer pairs 58 4.3.2 PCR to amplify all 12 fragments 1mer and 0.5mer BBTV components 58 4.3.3 Agarose electrophoresis to estimate the concentration of the purified 12 fragments using ImageJ program 60 4.4 Amplification of 1.5 mer fragments using fused 1-mer and 0.5-mer fragments 65 4.4.1 PCR to amplify 1.5mer fragments using fused 1mer and 0.5mer without primers 65 4.4.2 Agarose electrophoresis to estimate the concentration of the purified 1.5mer fragments using ImageJ program 66 4.5 Amplification of 1.5 mer fragments using RCA product as template 69 4.5.1 Rolling circle amplification 69 4.5.2 PCR to amplify 1.5mer fragments using RCA products as template 70 4.6 Optimization of overlap extension reaction 71 4.7 Phusion 1.5mer fragments in pairs to form large fragments 73 4.8 Gibson Assembly all 1.5mer fragments BBTV 74 v PART V CONCLUSION AND SUGGESTION 78 5.1 Conclusion 78 5.2 Proposal for further work 79 REFERENCES 80 vi LIST OF TABLES Table 2.1: The proteins encoded by the integral genome components of BBTV 16 Table 2.2: Begomoviruses whose infectivities were demonstrated by agroinoculation 21 Table 3.1: Reagents required for 5X ISO Buffer and 1.33x ISO Assembly Mix 34 Table 3.2: Final Concentration of all reagents in Gibson Assembe Master Mix Homemade 35 Table 4.1: Five GenBank BBTV isolates closest to the VN2018 isolate identified by Blast search using the complete sequence of DNA-R of the VN2018 isolate as query 50 Table 4.2: Selection of templates of BBTV genomic components for in silico construction of infectious clone 53 Table 4.3: Primers and assembled fragment of BBTV to construct infectious clone of BBTV in pCAMBIA2300 vector 56 Table 4.4: DNA quantify (ng) correspond to typical band and pixel values on ImageJ 62 Table 4.5: Final concentraion (pmol/µl) of all fragments mer and 0.5mer used for phusion 64 Table 4.6: DNA quantify (ng) correspond to typical band and pixel values on ImageJ 67 Table 4.7: Final concentraion (pmol/µl) of fragments 1.5mer used for phusion 68 Table 4.8: Final concentraion (pmol/µl) of fragments 1.5mer used for phusion 69 Table 4.9: Gibson assembly products in figure 4.22, from left to right (lane 1-12) 76 vii LIST OF FIGURES Figure 2.1: World production of bananas in 2020, by region (in 1,000 metric tons) Figure 2.2: Some particular symptoms of banana caused by BBTV 11 Figure 2.3: Babuvirus Classification 14 Figure 2.4: Genome organization of BBTV 16 Figure 2.5: Summary of the methodology of constructing an infectious clone for Begomovirus 19 Figure 2.6: Gibson assembly overview 23 Figure 2.7: The Gibson Assembly® method is faster and more efficient than traditional cloning 25 Figure 2.8: Overview of Gibson Assembly® SDM of a circular template Following mutagenesis, DNA fragments of various lengths are uniformly assembled using complementary overlaps between fragments 26 Figure 3.1: Rolling circle amplification principle Random hexamers bind to the circular template and are extended by phi29 DNA polymerase Strand-displacing activity of phi29 DNA polymerase causes nascent strand to be displaced, exposing new recognition sites for the hexamers in a process known as branching 31 Figure 3.2: Map of pCambia2300 Plant Expression Vector (ab275758) (Abcam) 36 Figure 3.3: Amplification of 1-5 mer fragments using RCA product as template 41 Figure 3.4: Amplification of 1-5 mer fragments using fused 1-mer and 0.5-mer fragments as templates 42 Figure 4.1: Left: BBTV VN2018 isolate Right: amplification of the complete DNA-R component (arrowed) of the BBTV VN2018 isolate using back-to-back primers M is GeneRuler kb DNA ladder (Thermo Scientific) 47 Figure 4.2: Complete sequence and key molecular features of DNA-R of the VN2018 isolate Note: Grey: common region-stem loop, Pink: Stem loop, Green: common region-major, Blue: TATA box, Yellow: open reading frame 49 viii Figure 4.16: Check 2µl RCA product on agarose 1%, M is GeneRuler kb DNA ladder (Thermo Scientific) Note: The obtained product are RCA product (arrowed) 4.5.2 PCR to amplify 1.5mer fragments using RCA products as template After that, PCR to amplify 1.5mer fragments using RCA products as template and run PCR products on gel agarose 1% 0.5X TAE buffer in Labnet Gel Xl Ultra V Electrophoresis System, 20 minutes at 100V The results showed in figure 4.18 Figure 4.17: Amplification of 1.5 mer fragments (R, U3, S, M, C, N), using RCA product as template, M is GeneRuler kb DNA ladder (Thermo Scientific) Note: The obtained products are 0.5-mer fragments (arrowed) 70 All amplifications using RCA templates always produced the 0.5-mer bands (the noted arrow) This was caused by primers anneal to the internal homologous sequence and produced unwanted 0.5-mer products representing in figure 4.18 Figure 4.68: Primer 1mer-forward and 0.5mer-reverse anneal to internal homologous sequence That the reason why even when amplify 1.5mer fragments on its 1.5mer template, we still got 0.5mer bands on gel electrophoresis We did not get any band 1.5mer, this can be explained due to primers anneal to the internal homologous sequence and block the elongation step of other primers However, the reason lead to this phenomenon need to be more research to explain 4.6 Optimization of overlap extension reaction After doing phusion reaction 1mer and 0.5mer to form 1.5mer fragments BBTV, it proved that fused products reduce half, therefore, it is important to optimize of overlap extension reaction to get better phusion yield 71 We conducted experiments: doing overlap extension reaction 1.5mer C and 1.5mer N with different kinds of kit listed in below table Titan One Tube PCR Kit Long Template PCR Phusion® High-Fidelity DNA Polymerase 5X Phusion HF Buffer Phusion® High-Fidelity DNA Polymerase 5XPhusion GC Buffer Figure 4.19: Phusion 1.5mer C and 1.5mer N using different kinds of buffer, M is GeneRuler kb DNA ladder (Thermo Scientific) Note: The arrow (lane 3) is fused products of 1.5mer C and N fragments The arrow (lane 4) is 1.5mer C and N fragments Based on the smallest quantity of 1.5mer DNA appear on gel electrophoresis (lane 3), it proves that there is a small amount of 1.5mer fusing with each other in reaction using phusion HF buffer We can see a fuzzy band approximately 3.2kilobase pairs as expected band In addition, lane 1,2,4 appear band 1.5mer lighter than band 1.5mer in lane 3, meaning that unfused 1.5mer remaining with larger amount in these lane than lane In conclusion, PCR showed that phusion HF buffer have the best yield in comparison with other kinds 72 Use Phusion HF Buffer to fuse fragments 1.5mer in pairs to form fragments 4.7 Phusion 1.5mer fragments in pairs to form large fragments Based on the result of optimization of overlap extension reaction, we conducted phusion reaction 1.5mer fragments in pairs to form large fragments using Phusion® High-Fidelity DNA Polymerase 5X Phusion HF Buffer After phusion 1.5 mer fragments in pairs, run electrophoresis on gel agarose 1%, 0.5X TAE in Labnet Gel Xl Ultra V Electrophoresis System, 20 minutes at 100V The results showed in figure 4.20 Figure 4.20: Phusion 1.5mer fragments (R, U3, S, M, C, N), in pairs to form large fragments, M is GeneRuler kb DNA ladder (Thermo Scientific) PCR results on gel agarose showed fuzzy expected band in lane 2+3 (3.2kb) (fragments R+U3) and lane 8+9 (3.5kb) (fragments C+N) However, lane 5+6 did not show any expected band even fuzzy band, only smear To check whether these lanes have right band, we cut gel at expected band and purified products Next, PCR with specific primer to check products after gel purification 73 Figure 4.71: PCR product of fused 1.5mer fragments in pairs Lane 1,2,3 correspond to fragments R+U3, S+M, C+N, respectively, M is GeneRuler kb DNA ladder (Thermo Scientific) PCR results on gel agarose in figure 4.21 showed that lane and lane appear some unexpected band but these band produced due to primers anneal to the internal homologous sequence, meaning that it must have key template (fused product) for amplification these unwanted band It proved that fused fragments R+U3 (lane 1) and C+N (lane 3) successfully, except fragments S+M, it did not show any band, gave the same result with phusion reaction these fragment before Therefore, to increase phusion yield 1.5mer in pairs, we changed procedure for construction that using Gibson Assembly to fuse 1.5mer fragments 4.8 Gibson Assembly all 1.5mer fragments BBTV To get better phusion yield 1.5mer fragments, especially fragment S+M, we changed procedure for construction that is Gibson Assembly 1.5mer fragments of BBTV with kinds of Gibson Master Mix Buffer: NEBuilder, NEB, Homemade 74 First, Gibson Assembly was done with phusion 1.5mer fragments in pairs ( R+U3, S+M, C+N), incubate in 15 minutes in thermocycler, followed by combining all tubes with each other to continue phusion reaction in 15 minutes Gibson Assembly products after that was check by PCR reaction with primers and run electrophoresis on gel agarose 1%, 0.5X TAE in 20 minutes The results showed in below figure Figure 4.82: Gibson Assembly all 1.5mer fragments (R, U3, S, M, C, N), BBTV M is GeneRuler kb DNA ladder (Thermo Scientific) Lane 1-12 showed in table below 75 Table 4.9: Gibson assembly products in figure 4.22, from left to right (lane 1-12) Kit NEBuilder® HiFi DNA Lane Fused fragments R+U3 S+M C+N R+U3 S+M C+N R+U3 S+M C+N 10 R+U3+S+M+C+N 11 R+U3+S+M+C+N 12 R+U3+S+M+C+N Assembly Master Mix Gibson Assembly® Master Mix (NEB) Gibson Assembly Master Mix Homemade NEBuilder® HiFi DNA Assembly Master Mix Gibson Assembly® Master Mix (NEB) Gibson Assembly Master Mix Homemade It showed that Gibson Assembly work better when phusion small number of fragments (2 fragments 1,5mer lane 1->9) in comparison with large number of fragments (lane 10,11,12) We can also see that all kinds of Gibson Assembly 76 (NEBuilder, NEB, Homemade) gave nearly same results After analysis gel electrophoresis picture, it proved that we got expected products (lane 1,2,3,4,5,6) based on analysis unexpected bands which produced due to primers anneals the internal homologous sequence of key template (expected band) This concluded that Gibson Assembly happened but with very low yield, it can be caused due to the low level of endonuclease activity of T5 endonuclease and 3´→5´ exonuclease activity of phusion polymerase active too strong leading to chew 3‟ ends before annealing 77 PART V CONCLUSION AND SUGGESTION 5.1 Conclusion DNA-1 BBTV isolate VN2018 was PCR, cloned in pTZ57, sequenced and blast search proved that the VN2018 is a BBTV isolate typical for BBTV population of the Northern Vietnam within the Asian population The phylogenetic and Blast search identified that the Q529-4 from China is the GeneBank isolate that is closest to VN2018 Construction of infectious clone of BBTV on pcambia2300 vector using Gibson Assembly Technology, specifically, DNA-R (VN2018), DNA-S the sequence of DNA-S (Acc number AF1498945) of the V14 isolate was selected as template, with the sequences of DNA-U3, DNA-M, DNA-C and DNA-N (Acc numbers KM607807, KM607239, KM607099 and KM607387, respectively) of the isolate Q529-4 was selected as corresponding templates for in silico construction All 12 fragments 1mer and 0.5mer were specifically amplified which were fused together without primers, creating 1.5mer fragments by Phusion™ HighFidelity DNA Polymerase (NEB) with GC buffer After optimization of overlap PCR, Phusion™ High-Fidelity DNA Polymerase (NEB) with HF buffer + DMSO 3% showed better phusion yield compared to GC buffer, Long Expand Template PCR and Titan One Tube PCR Phusion short fragments (1mer+0.5mer) was easier than longer fragments (1.5mer+1.5mer) and phusion without primers need to be prepare large amount of DNA fragments because after phusion even with 100% yield, the amount of fused products reduce by half Gibson Assembly efficiency was very low, the reason can be phusion DNA polymerase have 3´→5´ exonuclease activity, it chews 3‟ ends before they anneal with each other In addition, T5 exonuclease activity possess a low level of 78 endonucleolytic activity specific for single-stranded DNA, this activity could cleave the single-stranded 3‟ overhangs revealed by the exonuclease activity, resulting in undesired 3‟ single-stranded DNA (ssDNA) ends which lead to assemble improperly or not at all 5.2 Proposal for further work Due to the limited time in the research process, the report has some limitations Because of this, we would like to give some recommendations: Produce large amount of mer, 0.5 mer and 1.5mer products for phusion Added a fourth protein to the Gibson Assembly reaction, the singlestranded DNA-binding protein (SSB) from a thermophilic organism (ET SSB from New England Biolabs) SSB is known to bind ssDNA, protecting it from cleavage by endonucleases Furthermore, SSB has been found to reduce secondary structure of ssDNA, improve PCR specificity, and improve the processivity of DNA polymerases Thus, we hypothesized that ET SSB would protect the ssDNA ends from cleavage and might additionally improve the efficiency and specificity of these ends annealing, resulting in a more accurate and efficient Gibson Assembly reaction 79 REFERENCES Adegbola R O., Ayodeji O., Awosusi O O., Atiri G I & Kumar P L (2013) First Report of Banana bunchy top virus in Banana and Plantain (Musa spp.) in Nigeria Plant Disease 97(2): 290-290 Amin I., Qazi J., Mansoor S., Ilyas M & Briddon R W (2008) Molecular characterisation of Banana bunchy top virus (BBTV) from Pakistan Virus Genes 36(1): 191-198 Anhalt M & Almeida R (2008) Effect of temperature, vector life stage, and plant access period on transmission of Banana bunchy top virus to banana Phytopathology 98(6): 743-748 Aubry F., Nougairède A., Gould E A & de Lamballerie X (2015) Flavivirus reverse genetic systems, construction techniques and applications: A historical perspective Antiviral Research 114: 67-85 Banerjee A., Roy S., Behere G T., Roy S S., Dutta S K & Ngachan S (2014a) Identification and characterization of a distinct banana bunchy top virus isolate of Pacific-Indian Oceans group from North-East India Virus research 183: 41-49 Banerjee A., Roy S., Behere G T., Roy S S., Dutta S K & Ngachan S V (2014b) Identification and characterization of a distinct banana bunchy top virus isolate of PacificIndian Oceans group from North-East India Virus Res 183: 41-9 Beetham P R., Hafner G J., Harding R M & Dale J L (1997) Two mRNAs are transcribed from banana bunchy top virus DNA-1 Journal of General Virology 78(1): 229-236 Blomme G., Ploetz R., Jones D., De Langhe E., Price N., Gold C., Geering A., Viljoen A., Karamura D & Pillay M (2013) 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 Annals of applied Biology 162(1): 426 Borth W., Perez E., Cheah K., Chen Y., Xie W S., Gaskill D., Khalil S., Sether D., Melzer M., Wang M., Manshardt R., Gonsalves D & Hu J S (2011) Transgenic banana plants resistant to banana bunchy top virus infection International Society for Horticultural Science (ISHS), Leuven, Belgium 449-457 Bressan A & Watanabe S (2011) Immunofluorescence localisation of Banana bunchy top virus (family Nanoviridae) within the aphid vector, Pentalonia nigronervosa, suggests a virus tropism distinct from aphid-transmitted luteoviruses Virus research 155(2): 520-525 Burns T M., Harding R M & Dale J L (1995) The genome organization of banana bunchy top virus: analysis of six ssDNA components Journal of General Virology 76(6): 1471-1482 Chabi M C & Dassou A G (2018) Banana and plantain production systems in Benin: ethnobotanical investigation, varietal diversity, pests, and implications for better production 14(1): 78 Chen Y & Hu X (2013) High-throughput detection of banana bunchy top virus in banana plants and aphids using real-time TaqMan(®) PCR J Virol Methods 193(1): 177-83 Chiaki Y., Nasir N., Herwina H., Jumjunidang, Sonoda A., Fukumoto T., Nakamura M & Iwai H (2015) Genetic structure and diversity of the Banana bunchy top virus population on Sumatra Island, Indonesia European Journal of Plant Pathology 143(1): 113-122 Dale J L (1987) Banana bunchy top: an economically important tropical plant virus disease Advances in virus research 33: 301-325 80 De Langhe E., Vrydaghs L., De Maret P., Perrier X & Denham T (2009) Why bananas matter: an introduction to the history of banana domestication Ethnobotany Research and Applications 7: 165-177 Elayabalan S., Kalaiponmani K., Subramaniam S., Selvarajan R., Panchanathan R., Muthuvelayoutham R., Kumar K K & Balasubramanian P (2013) Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease World Journal of Microbiology and Biotechnology 29(4): 589-596 Espino R., Johns A., Juanillo C & Magnaye L (1993) Evaluation of Philippine banana cultivars for resistance to bunchy-top and fusarium wilt FAO (2019) Banana Market Review Preliminary Results for 2019 Food and Agriculture Organization of the United Nations Ferreira S (1991) The status of moko and bunchy top diseases in Hawaii Research extension series-College of Tropical Agriculture and Human Resources, University of Hawaii, Cooperative Extension Service (USA) Frison E A & Taher M (1991) FAO/IBPGR technical guidelines for the safe movement of citrus germplasm Bioversity International pages pages Furuya N., Bahet N., Kawano S & Natsuki K (2003) Detection of viruses and genetic analysis of Banana bunchy top virus associated with Abaca Jpn J Phytopathol 69: 326-327 Geering A & Thomas J (1996) A comparison of four serological tests for the detection of banana bunchy top virus in banana Australian Journal of Agricultural Research 47(3): 403-412 Geering A & Thomas J (1997) Search for alternative hosts of banana bunchy top virus in Australia Australasian Plant Pathology 26(4): 250-254 Grigoras, I T., T K., L., , Grande-Pérez A & Vetten H J., & Gronenborn, B , (2009) Reconstitution of authentic nanovirus from multiple cloned DNAs Journal of virology 83(20), 10778-10787 Hafner G J., Harding R M & Dale J L (1995) Movement and transmission of banana bunchy top virus DNA component one in bananas Journal of general virology 76(9): 2279-2285 Hailu M., Seyoum Workneh T & Belew D (2013) Review on postharvest technology of banana fruit African Journal of Biotechnology 12: 635-647 Hallam D (1995) The world banana economy In: Bananas and Plantains Gowen, S (ed.) Springer Netherlands Dordrecht: 509-533 pages Harding R M., Burns T M & Dale J L (1991) Virus-like particles associated with banana bunchy top disease contain small single-stranded DNA J Gen Virol 72 ( Pt 2): 225-30 Harding R M., Burns T M., Hafner G., Dietzgen R G & Dale J L (1993) Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene Journal of General Virology 74(3): 323-328 Horser C L., Karan M., Harding R M & Dale J L (2001) Additional Rep-encoding DNAs associated with banana bunchy top virus Archives of Virology 146(1): 71-86 Hu J.-M., Fu H.-C., Lin C.-H., Su H.-J & Yeh H.-H (2007) Reassortment and Concerted Evolution in Banana Bunchy Top Virus Genomes J Virol 81(4): 1746-1761 HU J S., Wang M., Sether D., Xie W & Leonhardt K W (1996) Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa) Annals of applied Biology 128(1): 55-64 81 Jones D R (2002) Risk of spread of banana diseases in international trade and germplasm exchange In Proceedings XV ACORBAT meeting 2002, Colombia: Cartagena de Indias Kagy V., Thomas J., Sharman M & Mademba-Sy F (2001) First record of banana bunchy top disease in New Caledonia Australasian Plant Pathology 30(1): 71-71 Kannan M., Zainal Z., Ismail I., Baharum S N & Bunawan H (2020) Application of Reverse Genetics in Functional Genomics of Potyvirus Viruses 12(8) Karamura D., Karamura E & Blomme G (2011) General Plant Morphology of Musa In.: 1-20 pages Karan M., Harding R M & Dale J L (1994) Evidence for two groups of banana bunchy top virus isolates Journal of General Virology 75(12): 3541-3546 Kenyon L., Brown M & Khonje P (1997) First report of banana bunchy top virus in Malawi Plant Disease 81(9): 1096-1096 Kesavamoorthy R (1980) Radical changes in ecosystem in the Pulney hills Proceedings of the 13th national seminar on banana production technology Coimbatore: TNAU 23-28 Khalid S., Soomro M & Stover R (1993) First report of banana bunchy top virus in Pakistan Lakatan (2016) Banana flesh extract as an antibacterial liniment Retrieved from https://graduateway.com/lakatan-banana-flesh-extract-as-an-antibacterial-liniment/ Larkin M A., Blackshields G., Brown N P., Chenna R., McGettigan P A., McWilliam H., Valentin F., Wallace I M., Wilm A., Lopez R., Thompson J D., Gibson T J & Higgins D G (2007) Clustal W and Clustal X version 2.0 Bioinformatics 23(21): 2947-8 Magee C (1948) Transmission of bunchy top to banana varieties Journal of the Australian Institute of Agricultural Science 14: 18-24 Mansoor S., Qazi J., Amin I., Khatri A., Khan I A., Raza S., Zafar Y & Briddon R W (2005) A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana Molecular Biotechnology 30(2): 167-169 Morton J (1987a) Banana In: Fruits of warm climates p 29–46 Morton J 1987b Fruits of warm climates Banana Miami Florida NelsonSC (2004) Banana bunchy top: detailed signs and symptom Cooperative Extension Service, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa pp 22 Peng J., Fan Z & Huang J (2012) Rapid Detection of Banana Streak Virus by Loop-mediated Isothermal Amplification Assay in South China Journal of Phytopathology 160(5): 248250 Perrier X., Langhe E D., Donohue M., Lentfer C., Vrydaghs L., Bakry F., Carreel F., Hippolyte I., Horry J.-P., Jenny C., Lebot V., Risterucci A.-M., Tomekpe K., Doutrelepont H., Ball T., Manwaring J., Maret P d & Denham T (2011) Multidisciplinary perspectives on banana (Musa spp.) domestication Proceedings of the National Academy of Sciences 108(28): 11311-11318 Ploetz R & Evans E (2015) The Future of Global Banana Production In.: 311-352 pages Qazi J (2016) Banana bunchy top virus and the bunchy top disease Journal of General Plant Pathology 82(1): 2-11 Ram R & Summanwar A S (1984) Colocasia esculenta (L) Schott a reservoir of bunchy top disease of banana Current Science 53(3): 145-146 82 Robson J D., Wright M G & Almeida R P (2014) Biology of Pentalonia nigronervosa (Hemiptera, Aphididae) on banana using different rearing methods Environmental Entomology 36(1): 46-52 Saad M F M., Sau A R., Akbar M A., Baharum S N., Ramzi A B., Talip N & Bunawan H (2021) Construction of Infectious Clones of Begomoviruses: Strategies, Techniques and Applications Biology 10(7): 604 Selvarajan R & Balasubramanian V (2014a) Chapter - Host–virus interactions in bananainfecting viruses In: Plant Virus–Host Interaction Gaur, R K., Hohn, T & Sharma, P (eds.) Academic Press Boston: 57-78 pages Selvarajan R & Balasubramanian V (2014b) Host–virus interactions in banana-infecting viruses In: Plant Virus–Host Interaction Elsevier: 57-78 pages Shekhawat U K S., Ganapathi T R & Hadapad A B (2012) Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection Journal of General Virology 93(8): 1804-1813 Simmonds H (1931) Noxious weeds and their control in Fiji II biological control Fiji Agricultural Journal 4: 29 Stainton D., Kraberger S., Walters M., Wiltshire E J., Rosario K., Halafihi M., Lolohea S., Katoa I., Faitua T H., Aholelei W., Taufa L., Thomas J E., Collings D A., Martin D P & Varsani A (2012a) Evidence of inter-component recombination, intra-component recombination and reassortment in banana bunchy top virus J Gen Virol 93(Pt 5): 11031119 Stainton D., Kraberger S., Walters M., Wiltshire E J., Rosario K., Halafihi M i., Lolohea S., Katoa I., Faitua T a H., Aholelei W., Taufa L., Thomas J E., Collings D A., Martin D P & Varsani A (2012b) Evidence of inter-component recombination, intra-component recombination and reassortment in banana bunchy top virus Journal of General Virology 93(5): 1103-1119 Stover R H (1972) Banana, plantain and abaca diseases Commonwealth Mycological Institute pages pages Su H.-J., Tsao L.-Y., Wu M.-L & Hung T.-H (2003) Biological and Molecular Categorization of Strains of Banana bunchy top virus Journal of Phytopathology 151(5): 290-296 Sun S.-K (1961) Studies on the bunchy top disease of bananas Spec Publ Coll Agric Taiwan Univ 10: 82-109 Tamura K., Stecher G., Peterson D., Filipski A & Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0 Mol Biol Evol 30(12): 2725-9 Thomas J., Geering A., Dahal G., Lockhart B & Thottappilly G (2003) Banana and plantain In: Virus and virus-like diseases of major crops in developing countries Springer: 477-496 pages Thomas J & Iskra Caruana M.-L (2000) Bunchy top Thomas J E & Dietzgen R G (1991a) Purification, characterization and serological detection of virus-like particles associated with banana bunchy top disease in Australia Journal of general virology 72(2): 217-224 Thomas J E & Dietzgen R G (1991b) Purification, characterization and serological detection of virus-like particles associated with banana bunchy top disease in Australia J Gen Virol 72 ( Pt 2): 217-24 83 Timchenko, T K., L., , Aronson M., Vega-Arreguin J C & Ramirez B C., Vetten, H J., & Gronenborn, B (2006) Infectivity of nanovirus DNAs: induction of disease by cloned genome components of Faba bean necrotic yellows virus Journal of general virology 87(6), 1735-1743 Viet Nam Bananas production (2022) knoema.com Knoema Retrieved from https://knoema.com/atlas/Viet-Nam/topics/Agriculture/Crops-Production-Quantitytonnes/Bananas-production on 25 May VOV.VN Export prices of Vietnamese bananas to EU market sees upturn (n.d.) [Online] Retrieved from https://english.vov.vn/en/economy/export-prices-of-vietnamese-bananasto-eu-market-sees-upturn-840406.vov on May 11 Wanitchakorn R., Hafner G J., Harding R M & Dale J L (2000) Functional analysis of proteins encoded by banana bunchy top virus DNA-4 to -6 J Gen Virol 81(Pt 1): 299306 Wanitchakorn R., Harding R M & Dale J L (1997) Banana bunchy top virus DNA-3encodes the viral coat protein Archives of Virology 142(8): 1673-1680 Wardlaw C (1961) Mosaic, infectious chlorosis and other virus diseases: banana diseases, including plantains and abaca Long-mans, London 116-145 Watanabe S & Bressan A (2013) Tropism, compartmentalization and retention of banana bunchy top virus (Nanoviridae) in the aphid vector Pentalonia nigronervosa Journal of general virology 94(1): 209-219 Wu R Y & Su H J (1990) Purification and Characterization of Banana Bunchy Top Virus Journal of Phytopathology 128(2): 153-160 Yu N.-T., Zhang Y.-L., Feng T.-C., Wang J.-H., Kulye M., Yang W.-J., Lin Z.-S., Xiong Z & Liu Z.-X (2012) Cloning and sequence analysis of two banana bunchy top virus genomes in Hainan Virus Genes 44(3): 488-494 84