Banana bunchy top virus is reported for the first time in Laos. Infected plants showed typical disease symptoms and the two complete genome sequences reported place the isolates in the Southeast Asian subgroup

Chittarath, Khonesavanh

Crew, Kathleen S.

Geering, Andrew D. W.

Kong, Gary

Pongsapich, Preyapan

Rungsawang, Wasana

Thomas, John E.

English

Queensland DAF eResearch Archive

Australasian Plant Disease Notes           (2022) 17:36 https://doi.org/10.1007/s13314-022-00482-yof banana bunchy top disease (Fig. 1) were observed in Bolikhamxay, Luang Namtha and Vientiane Provinces, and Vientiane Prefecture in Laos (Fig. 2) during surveys con-ducted between 2015 and 2017 (Table 1). Occurrences of this disease were mostly in scattered plants in household gardens and smallholder plantings and never at high inci-dence in any particular region. In this disease note, we pres-ent the complete genome sequences of two virus isolates from Laos and provide positive identifications for several other isolates from a range of cultivars and locations.Leaf samples of banana plants displaying symptoms typi-cal of banana bunchy top disease were dried over silica gel for later laboratory analysis, and a subset (n = 39) imported into Australia under licence and archived in the Queensland Department of Agriculture and Fisheries Plant Virus Col-lection (Ecosciences Precinct, Dutton Park, Queensland). Diagnoses of imported samples were done by either immu-nocapture (IC) PCR using DNA-R specific primers BBT1 and BBT2 (Sharman et al. 2000) or PCR using DNA-R fwd and rev primers (Stainton et al. 2012). For the latter, total plant DNA was extracted using an ISOLATE II Plant DNA kit (Bioline) according to the manufacturer’s instructions. PCR amplicons from six isolates were gel-purified using an ISOLATE II PCR and Gel kit (Bioline) and used for direct sequencing or sequenced after cloning using an Invitrogen TOPO TA cloning kit (Thermo Fisher Scientific). Sanger sequencing was outsourced to the Australian Genome Research Facility. As a limited confirmatory test, two sam-ples were also checked by triple antibody sandwich ELISA (Geering and Thomas 1996). All samples with banana bunchy top disease symptoms tested were positive by PCR       Banana bunchy top virus (BBTV) is the most damag-ing viral pathogen of bananas (Kumar et al. 2015). It has a multi-component, circular, single-stranded DNA genome and is horizontally transmitted by its primary vector, the banana aphid (Pentalonia nigronervosa), as well as verti-cally transmitted between plant generations by vegetative propagation practices (Thomas 2019). BBTV likely origi-nated in Southeast Asia (Stainton et al. 2015) but a notable gap in knowledge of the distribution of the virus within this region is Laos (Thomas 2019). Plants with symptoms Khonesavanh Chittarath, Kathleen Crew and Andrew Geering contributed equally to this work.  John E. Thomasj.thomas2@uq.edu.au1 Plant Protection Center, Department of Agriculture, Ministry of Agriculture and Forestry, P.O. Box 811, Vientiane, Lao PDR2 Department of Agriculture and Fisheries, 41 Boggo Road, 4102 Dutton Park, Queensland, Australia3 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, 4072 St Lucia, Queensland, Australia4 Plant Protection Division, Ministry of Agriculture and Cooperatives, 50 Paholyotin Rd, Ladyao Chatuchak,  10900 Bangkok, Thailand5 Redclaw Ink Pty Ltd, 110 Finley Rd, 4562 Eumundi, Queensland, Australia6 Department of Agriculture, Plant Quarantine Research Group, Plant Protection Research and Development Office, Bangkok, ThailandAbstractBanana bunchy top virus is reported for the first time in Laos. Infected plants showed typical disease symptoms and the two complete genome sequences reported place the isolates in the Southeast Asian subgroup.Keywords Nanovirus · Genome sequence · Lao PDR · Musa spp. · Viral diseaseReceived: 19 August 2022 / Accepted: 4 October 2022© The Author(s) under exclusive licence to Australasian Plant Pathology Society Inc. 2022Presence and distribution of banana bunchy top virus in LaosKhonesavanh Chittarath1 · Kathleen S. Crew2,3 · Andrew D.W. Geering3 · Wasana Rungsawang4 · Gary Kong5 · Preyapan Pongsapich6 · John E. Thomas31 3Australasian Plant Disease Notesand/or IC-PCR and the two samples tested by ELISA were also positive (Table 1).The two virus isolates selected for complete genome sequencing were DAF accession Q6624, collected from near the village of Ban Nakhorng (N18.303606, E102.565024), and DAF accession Q6634, collected from near the village of Patsum (N18.427711, E103.71643), both on 29 June 2016. Total plant DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN), and circular DNA molecules selec-tively enriched by rolling circle amplification using an Illus-tra TempliPhi 100 Amplification Kit (GE Biosciences), as per the manufacturer’s instructions. The TempliPhi products were then submitted to the Beijing Genomics Institute (BGI; Shenzen, China) for sequencing on an Illumina Hiseq 4000 platform, with a library construction size of 300–500 bp and pair-ended reads of 150 bp. Sequence trimming was done by BGI and all other analyses done in house using CLC Genomics Workbench 10.1.1.Following merging of the pair-ended reads, de novo genome assemblies were generated and six contigs obtained, each c. 1 kb in length and representing a large majority of the sequence reads (26,072–219,980 and 10,811–144,941 reads per DNA component for Q6624 and Q6634, respec-tively). BLASTN searches of GenBank suggested that each contig represented one component of the BBTV genome, with closest matches to isolates from Thailand. No evidence for the presence of satellite components or other viruses was obtained. Final genome assemblies were done by mapping reads to a previously sequenced genome of BBTV from Nong Khai Province in Thailand (GenBank Accessions KY427060.1- KY427065.1), which was among the high-est matching hits (97–99% nucleotide identity) in the pre-vious BLASTN analyses. Isolates Q6624 and Q6634 were 99.2 to 99.9% identical across the six genome components (Table 2).Other BBTV isolates from Laos were partially character-ised by sequencing of PCR amplicons and pairwise nucleo-tide identities of 97.3–100% were calculated among partial and complete DNA-R component sequences. The Laos isolates fell within the Southeast Asian subgroup of BBTV and were most closely related to isolates from neighbouring countries (Fig. 3).In conclusion, we report the presence of BBTV in Laos for the first time and confirm its presence in Vientiane Pre-fecture and the provinces of Bolikhamxay, Luang Namtha and Vientiane (Fig. 3), in both commercial Cavendish and local cultivars. There are no coordinated efforts to control BBTV in Laos, yet the incidence of BBTV has not reached a level that poses an existential threat to the local banana industry as it did when the virus was introduced into Aus-tralia in the early 1900s (Thomas 2019). It is possible that the local cultivars of banana have a degree of resistance to BBTV that prevents major epidemics from occurring.Fig. 1 Typical symptoms caused by banana bunchy top virus in field samples from Laos. Isolates Q6400 (A) and Q6653 (B) from Bolikxa-may Province, Q6644 (C, D) from Luang Namtha Province. Typical whole plant symptoms of stunting, narrow, erect bunched leaves with chlorotic margins in panels A, B, D; Characteristic dark green streaks on the petiole and minor leaf veins in panel C 1 3   36  Page 2 of 6Australasian Plant Disease NotesFig. 2 Distribution of confirmed banana bunchy top virus isolates from Laos described in this work indicated by red dots Table 1 Confirmed BBTV isolates from Laos. “+” signifies, as appropriate, positive PCR with BBT1/BBT2 or DNA-R fwd/rev primers obtained for each isolateIsolate Local cultivar namesGPS latitude GPS longitude Province BBTV BBT1,2 PCR bBBTV DNA-R PCRbGenBank codeQ6400c Hom 17.81853 102.69254 Vientianea nt +Q6401 Hom 17.81955 102.69254 Vientianea + ntQ6405c unknown 17.88909 102.61162 Vientianea + +Q6429 Knao 18.07235 102.53390 Vientianea nt +Q6617 Jeang 18.39402 103.64869 Bolikhamxay + ntQ6618 Klai 18.59825 102.33205 Vientiane nt +Q6619 Jeang 18.30361 102.56503 Vientiane + +Q6620 Klai 18.10906 104.27908 Bolikhamxay nt +Q6621 Hom 18.39446 103.64793 Bolikhamxay + ntQ6622 Jeang 18.39908 103.66482 Bolikhamxay nt +Q6623 Hom 18.60573 102.32871 Vientiane nt +Q6624 Jeang 18.30361 102.56503 Vientiane + + OP293737, OP297849-53Q6625 Nam 18.40741 103.70114 Bolikhamxay + ntQ6626 Cavendish 21.18041 101.15705 Luang Namtha + + OP485239Q6627 Klai 18.28380 102.71323 Vientiane + nt1 3Page 3 of 6    36 Australasian Plant Disease NotesTable 2 Nearest identities read numbers for the six genome components of banana bunchy top virus isolates Q6624 and Q6634Virus isolateDNA component GenBank codeReads1 Size (nt)Nearest BLAST match BBTV isolate2Nearest match % Identity Q6624 vs. Q6634GenBank code % IdentityQ6624 R OP293737 26,072 1104 Nakhon Ratchasima-TH16, DNA-RMF039879.1 99.6 99.8S OP297850 47,474 1060 Chaiyaphum-TH16 DNA-S MF039874.1 98.1 99.7M OP297852 195,794 1047 NongKhai-TH16, DNA-M KY427061.1 99.3 99.7C OP297851 76,170 1015 NongKhai-TH16, DNA-C KY427060.1 99.5 99.9N OP297853 247,470 1077 Chaiyaphum-TH16, DNA-N MF039872.1 99.9 99.9U3 OP297849 219,980 1053 NongKhai-TH16 DNA-U3 KY427065.1 99.0 99.2Total BBTV reads 812,960Total sample reads 2,365,796Q6634 R OP297854 27,151 1104 Nakhon Ratchasima-TH16, DNA-RMF039879.1 99.6S OP297858 10,811 1059 Chaiyaphum-TH16, DNA-S MF039874.1 97.9M OP297856 73,755 1047 NongKhai-TH16, DNA-M KY427061.1 99.6C OP297855 108,366 1015 NongKhai-TH16, DNA-C KY427060.1 99.6N OP297857 139,895 1077 Chaiyaphum-TH16, DNA-N MF039872.1 99.8U3 OP297859 144,941 1053 NongKhai-TH16, DNA-U3 KY427065.1 98.8Total BBTV reads 504,919Total sample reads 1,124,8381 Number of DNA component-specific reads2 All isolates are from ThailandTable 1 ContinuedQ6628 Hom 18.36763 103.19582 Bolikhamxay + + OP538684Q6629 Klai 18.91992 102.42963 Vientiane + + OP538685Q6630 Jeang 18.55261 102.50401 Vientiane + + OP538686Q6631 Hom 18.42771 103.71643 Bolikhamxay + ntQ6632 Klai 18.91874 102.45049 Vientiane nt +Q6633 Kung 18.55255 102.50410 Vientiane + +Q6634 Figle 18.42771 103.71643 Bolikhamxay + nt OP297854-59Q6635 Musi 18.56477 102.49913 Vientiane + + OP538687Q6636 Hom 18.28490 102.69923 Vientiane + ntQ6637 Klai 18.56387 102.49869 Vientiane nt +Q6638 Jeang 18.42829 103.71414 Bolikhamxay nt +Q6639 Hom 18.19197 104.22105 Bolikhamxay + ntQ6644 Cavendish 21.11860 101.09366 Luang Namtha + + OP538688Q6650 Hom 18.47652 103.68278 Bolikhamxay + +Q6651 Cavendish 18.47652 103.68278 Bolikhamxay + ntQ6652 Hom 18.47791 103.67943 Bolikhamxay + ntQ6653 Hom 18.47763 103.67988 Bolikhamxay + ntQ6654 Klai 18.19101 104.24592 Bolikhamxay + ntQ6655 Klai 18.19100 104.24587 Bolikhamxay + +Q6656 Hom 18.19104 104.24551 Bolikhamxay + ntQ6658 Hom 18.42494 103.61960 Bolikhamxay + ntQ6659 Klai 18.57740 102.35112 Vientiane + ntQ6660 Klai 18.57728 102.35129 Vientiane + +Q6661 Klai 18.57723 102.35122 Vientiane + ntaVientiane Prefectureb nt – not testedc Also positive for BBTV by ELISA1 3   36  Page 4 of 6Australasian Plant Disease NotesReferencesGeering ADW, Thomas JE (1996) A comparison of four serological tests for the detection of banana bunchy top virus in banana. Aust J Agric Res 47:403–412Kumar PL, Selvarajan R, Iskra-Caruana M-L, Chabannes M, Hanna R (2015) Biology, Etiology, and Control of Virus Diseases of Banana and Plantain. In: Gad L, Nikolaos IK (eds) Advances in Virus Research, Volume 91. Academic Press, p 229–269. https://doi.org/10.1016/bs.aivir.2014.10.006Acknowledgements We gratefully acknowledge support from ACIAR Project HORT/2010/069 (Enabling improved plant biosecurity prac-tices in Cambodia, Lao PDR and Thailand) and the Plant Biosecu-rity Cooperative Research Centre through the Australian Cooperative Research Centres’ Program.DeclarationsConflict of interest The authors declare that they have no conflict of interest.Fig. 3 Maximum likelihood phylogenetic tree showing the relation-ships between banana bunchy top virus isolates from Laos (bold) and representatives of the two virus subgroups, based on the DNA-R nucleotide coding sequence. The related nanovirus abaca bunchy top virus (ABTV) serves as an outgroup. Bootstrap values >70% are shown at the nodes of the branches. The scale bar represents the num-ber of substitutions per site  1 3Page 5 of 6    36 Australasian Plant Disease NotesWalters M, Collings D, Mabvakure B, Lemey P, Harkins G, Thomas J, Varsani A (2015) The global distribution of Banana bunchy top virus reveals little evidence for frequent recent, human-mediated long distance dispersal events. Virus Evol 1(1):vev009. https://doi.org/10.1093/ve/vev009Thomas J (2019) Banana bunchy top. In: Jones D (ed) Handbook of Diseases of Banana, Abacá and Enset. CAB International, Wall-ingford, Oxfordshire, UK, pp 362–376Publisher’s Note Springer Nature remains neutral with regard to juris-dictional claims in published maps and institutional affiliations. Sharman M, Thomas JE, Dietzgen RG (2000) Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. J Virol Methods 89:75–88Stainton D, Kraberger S, Walters M, Wiltshire EJ, Rosario K, Halafihi Mi, Lolohea S, Katoa I, Faitua TaH, Aholelei W, Taufa L, Thomas JE, Collings DA, Martin DP, Varsani A (2012) Evidence of inter-component recombination, intra-component recombination and reassortment in banana bunchy top virus. J Gen Virol 93(Pt 5):1103–1119. https://doi.org/10.1099/vir.0.040337-0Stainton D, Martin D, Muhire B, Lolohea S, Halafihi Mi, Lepoint P, Blomme G, Crew K, Sharman M, Kraberger S, Dayaram A, 1 3   36  Page 6 of 6

Presence and distribution of banana bunchy top virus in Laos

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Presence and distribution of banana bunchy top virus in Laos

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