Draft Genome Sequence of Ralstonia syzygii subsp. celebesensis from Indonesia, the Causal Agent of Blood Disease of Banana.

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HomePhytopathology®Vol. 112, No. 7Draft Genome Sequence of Ralstonia syzygii subsp. celebesensis from Indonesia, the Causal Agent of Blood Disease of Banana PreviousNext Resource Announcement OPENOpen Access licenseDraft Genome Sequence of Ralstonia syzygii subsp. celebesensis from Indonesia, the Causal Agent of Blood Disease of BananaAdy B. Prakoso, Tri Joko, Alan Soffan, Juli P. Sari, Jane D. Ray, André Drenth, and Siti SubandiyahAdy B. Prakosohttps://orcid.org/0000-0002-7753-2748Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaSearch for more papers by this author, Tri JokoDepartment of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaSearch for more papers by this author, Alan SoffanDepartment of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaSearch for more papers by this author, Juli P. SariDinas Pangan, Tanaman Pangan dan Hortikultura Provinsi Riau, Pekanbaru, 28142, IndonesiaSearch for more papers by this author, Jane D. RayCentre for Horticultural Science, University of Queensland, Brisbane, 4001, AustraliaSearch for more papers by this author, André Drenthhttps://orcid.org/0000-0002-8510-1534Centre for Horticultural Science, University of Queensland, Brisbane, 4001, AustraliaSearch for more papers by this author, and Siti Subandiyah†Corresponding author: S. Subandiyah; E-mail Address: sitisubandiyah@ugm.ac.idhttps://orcid.org/0000-0002-7718-2362Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaResearch Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta, 55281, IndonesiaSearch for more papers by this author AffiliationsAuthors and Affiliations Ady B. Prakoso1 Tri Joko1 Alan Soffan1 Juli P. Sari2 Jane D. Ray3 André Drenth3 Siti Subandiyah1 4 † 1Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia 2Dinas Pangan, Tanaman Pangan dan Hortikultura Provinsi Riau, Pekanbaru, 28142, Indonesia 3Centre for Horticultural Science, University of Queensland, Brisbane, 4001, Australia 4Research Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Published Online:6 May 2022https://doi.org/10.1094/PHYTO-10-21-0443-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementRalstonia syzygii subsp. celebesensis, the causal agent of blood disease of banana (BDB), was first reported in the early 1900s in Selayar Island, South Sulawesi Province, Indonesia. Since the 1980s, R. syzygii subsp. celebesensis has been spreading to most islands in Indonesia, and it was recently reported in Malaysia. R. syzygii subsp. celebesensis causes wilt of the banana plant and rot of fruit. A draft genome assembly of R. syzygii subsp. celebesensis isolate UGMSS_Db01, isolated from BDB-symptomatic banana in Indonesia, was generated using the Oxford Nanopore Technologies GridION Platform. The genome assembly was 3,568,564 bp, with 92× genome coverage for the chromosome and 1,614,128 bp, with 102× genome coverage for the plasmid. This draft genome will enrich resources to study the banana–R. syzygii subsp. celebesensis interaction and enhance development of research to control BDB.Blood disease of banana (BDB), caused by Ralstonia syzygii subsp. celebesensis, has significant impact on banana (Musa spp.) production, and banana is an important food crop in the world. In Indonesia, there is a high diversity of cultivated banana for both fresh and cooking use (Blomme et al. 2017; Ekasari et al. 2012; Li et al. 2013; Ploetz et al. 2007). Symptoms include wilting of the leaves, brown-red vascular staining, and rotten fruit pulp (Blomme et al. 2017; Tjahjono and Eden-Green 1988). BDB was first reported in South Sulawesi in the 1900s and, since the 1980s, the disease has been spreading around the country; recently, it was reported in Malaysia (Ray et al. 2021; Teng et al. 2016; Rijks 1916; Safni et al. 2014). The disease is transmitted by banana-visiting insects, birds, and bats; contaminated tools; and in other mechanical ways (Buddenhagen 2009). The movement and distribution of R. syzygii subsp. celebesensis-contaminated animals, and human mobility in transporting infected fruit and suckers for replanting have contributed to long-distance spread of BDB (Gäumann 1921; Safni et al. 2018). R. syzygii subsp. celebesensis is a nonfluorescent Gram-negative bacterium, rod shaped, not spore forming, slow growing, and does not have a capsule structure (Denny and Baek 1991; Ray et al. 2021). A control method for the disease is urgently needed to stop the spread and reduce the yield loss; however, information about the pathogen is still limited compared with that of the related pathogen, R. solanacearum, which has been widely studied. The first genome of blood disease bacterium was published in 2017, isolated from Perak, Malaysia as the expansion of the disease from Indonesia (Badrun et al. 2017; Ray et al. 2021; Teng et al. 2016).The molecular identity of R. syzygii subsp. celebesensis from Indonesia as the origin of the pathogen is still unclear. This genome sequence of R. syzygii subsp. celebesensis strain UGMSS_Db01 will enrich the information about the pathogen and contribute to further research on BDB and the establishment of disease management strategies.R. syzygii subsp. celebesensis UGMSS_Db01 (isolate identifier: JR3412) was selected for whole-genome sequencing. The strain was isolated from a Musa balbisiana ‘Kepok’ plant showing BDB symptoms, located in Yogyakarta, Indonesia (geocoordinates −7.835059, 110.388721) (Prakoso et al. 2020; Ray et al. 2021). The isolate was grown on casein peptone glucose (CPG) medium (casein hydrolysate at 1 g liter−1, peptone at 10 g liter−1, glucose at 5 g liter−1, and bacto agar at 15 g liter−1) supplemented with2,3,5 triphenyl tetrazolium chloride at 50 mg liter−1 and cycloheximide at 100 mg liter−1 to inhibit fungal contamination (Ray et al. 2021). The isolate was identified using a Gram test, Kovac’s Oxidation Test, and confirmed with PCR using specific primers for R. syzygii subsp. celebesensis (121F: 5′-AAGTTCATTGGTGCCGAATCA-3′ and 121R: 5′-CGTATTGGATGCCGTAATGGA-3′) (Alexander and Strete 2001; Dickey and Kelman 1988; Edy et al. 2009; Suslow et al. 1982). To confirm the identity of the strain as the BDB causal agent, Koch’s postulates were carried out on M. balbisiana ‘Kepok’ and completely described by Ray et al. (2021). The genomic DNA (gDNA) was isolated from a 3-day-old culture on CPG medium at 25 ± 2°C by Presto Mini gDNA Bacteria Kit (Geneaid Biotech Ltd., Taiwan) protocols. DNA purification and concentration were determined using both NanoDrop spectrophotometers and Qubit fluorometers, resulting in a gDNA concentration of >300 ng µl−1 with a DNA purity ratio of absorbance at 260/280 nm of 1.82, and 9 µg of total amount of gDNA. Libraries were prepared with Ligation Sequencing Kit SQK-LSK110 (Oxford Nanopore Technologies, United Kingdom) following the manufacturer’s protocol. The sample was sequenced using GridION (Oxford Nanopore Technologies) with the MinKNOW software 20.06.9 (Oxford Nanopore Technologies). Base calling was done with Guppy 4.0.11 using high-accuracy mode (Wick et al. 2019). The sequencing produced 241,056 reads with an N50 read length of 18,605 bp for a total of 1,913,216,976 bp. The FASTQ file kept the best 500,000,000 bp by filtering with Fitlong (https://github.com/rrwick/Filtlong) and the quality was visualized using NanoPlot (De Coster et al. 2018), resulting in 19,819 reads, N50 read length of 28,267 bp, and 500,012,234 total bases. Flye software 2.8.1 was used for de novo assembly (Kolmogorov et al. 2019) with 10 threads, 3,000,000 genome size, and 5,000 minimum overlap. To polish the assembled sequence Medaka software v1.4.4 (https://github.com/nanoporetech/medaka) was used. Polished genomes were assessed using benchmarking universal single-copy orthologs (BUSCO) to evaluate completeness (Simão et al. 2015). The length of the draft genome assembly of UGMSS_Db01 was estimated at 3,568,564 bp (chromosome) and 1,614,128 bp (plasmid), with completeness of 96.5% using BUSCO v4.1.4 in genome mode with the burkholderiales_odb10 dataset. Species verification by orthoANI genome distance to R. syzygii strain LLRS-1 (Lu et al. 2021) as reference was carried out using OAT v0.93.1 (Lee et al. 2016).UGMSS_Db01 has 98.44% orthoANI similarity to the chromosome and 96.76% to the plasmid. The annotation was added using the NCBI Prokaryotic Genome Annotation Pipeline with 4,719 total genes, 4,651 total coding sequences, and 54 transfer RNAs. Compared with that reported previously in Malaysia, blood disease bacterium A2-HR MARDI, this genome of R. syzygii subsp. celebesensis UGMSS_Db01 is longer in size, with a larger total number of genes and a shorter chromosome but longer plasmid. The genome comparative parameters to blood disease bacterium A2-HR MARDI are shown in Table 1.Table 1. Genome comparison of Ralstonia syzygii subsp. celebesensis UGMSS_Db01 and blood disease bacterium A2-HR MARDIResultParametersUGMSS_Db01A2-HR MARDIaGenome length5,185,1885,089,660Genome coverageChromosome92×98.4×Plasmid102×UnavailableGC content (%)66.566.4Chromosome length3,568,5643,603,619Plasmid length1,614,1281,486,041Total genes4,7194,650Total coding sequences4,6514,581Transfer RNAs5455aBlood disease bacterium A2-HR MARDI (Badrun et al. 2017).Table 1. Genome comparison of Ralstonia syzygii subsp. celebesensis UGMSS_Db01 and blood disease bacterium A2-HR MARDIView as image HTML The genome sequence of R. syzygii subsp. celebesensis isolate UGMSS_Db01 will provide an essential resource for researchers around the world working on blood disease of banana, molecular science, and pathogenesis-related gene exploration and will also contribute to the development of control methods against the disease which has been currently spreading in Southeast Asia. Sequence data of R. syzygii subsp. celebesensis isolate UGMSS_Db01 are available at the NCBI database under BioProject ID PRJNA580360, BioSample ID SAMN17266611, assembly GCA_016743075.1.The author(s) declare no conflict of interest.Literature CitedAlexander, S. K., and Strete, D. 2001. Microbiology: A Photographic Atlas for the Laboratory. Benjamin Cummings, San Francisco, CA, U.S.A. Google ScholarBadrun, R., Abu Bakar, N., Laboh, R., Redzuan, R., and Bala Jaganath, I. 2017. Draft genome sequence of blood disease bacterium A2 HR-MARDI, a pathogen causing banana bacterial wilt. Genome Announce. 5:e00408-e00417. https://doi.org/10.1128/genomeA.00408-17 Crossref, Medline, Google ScholarBlomme, G., Dita, M., Jacobsen, K. S., Vicente, L. P., Molina, A., Ocimati, W., Poussier, S., and Prior, P. 2017. Bacterial diseases of bananas and enset: Current state of knowledge and integrated approaches toward sustainable management. Front. 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Genome Biol. 20:129. https://doi.org/10.1186/s13059-019-1727-y Crossref, Medline, ISI, Google ScholarFunding: This research is funded by a PMDSU-DIKTI scholarship under the contract number of 2201/UN1.DITLIT/DIT-LIT/PT/2020 for bacterial isolation, identification, and pathogenicity test, and by PTUPT Simlitabmas number 2881/UN1.DITLIT/DIT-LIT/PT/2020 for WGS and bioinformatic analysis.The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 112, No. 7 July 2022SubscribeISSN:0031-949Xe-ISSN:1943-7684 DownloadCaptionA sideview showing short, orange aecia (aecial cups) produced on the backside of Mahonia fortunei (alternate host) seedling after inoculation with Puccinia striiformis f. sp. tritici (the causal pathogen of wheat stripe rust) basidiospores that were derived from germinated teliospores (Cheng et al.). Photo credit: Jie Zhao Metrics Downloaded 576 times Article History Issue Date: 2 Jul 2022Published: 6 May 2022Accepted: 19 Jan 2022 Pages: 1584-1586 Information© 2022 The American Phytopathological SocietyFundingPMDSU-DIKTIGrant/Award Number: 2201/UN1.DITLIT/DIT-LIT/PT/2020PTUPT SimlitabmasGrant/Award Number: 2881/UN1.DITLIT/DIT-LIT/PT/2020Keywordsbacterial pathogensbioinformaticsblood diseasedata sciencegenomegenomicsMusa balbisianaRalstonia syzygii subsp. celebesensisThe author(s) declare no conflict of interest.PDF download