Genome sequencing of four strains of Phylotype I, II and IV of Ralstonia solanacearum that cause potato bacterial wilt in India

Virupaksh U. Patil Vanishree Girimalla Vinay Sagar Rajinder Singh Chauhan Swarup Kumar Chakrabarti About the authors

Abstract

Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.

Keywords:
Potato wilt; Ralstonia; Species Complex; Phylotype; Bipartite; Genome

Genome announcement

Ralstonia solanacearum11 Smith EF. A bacterial disease of tomato, pepper, eggplant and Irish potato (Bacillus solanacearum nov. sp.) US Dep. Div Veg Phys Pathol Bull. 1896;12:1-28.,22 Yabuuchi E, Kosako Y, Yano I, Hotta H, Nishiuchi Y. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: praposal of Ralstonia solanacearum (Smith 1896) comb. Nov. and Ralstonia eutropha (Davis 1969). Microb Immunol. 1995;39:897-904. formally known as Pseudomonas solanacearum and Burkholderia solanacearum is a gram-negative, chemo-organotroph phytopathogenic β-proteobacterium with an unusual broad host range.33 Fegan M, Prior P. How complex is the "Ralstonia solanacearum species complex. In: Allen C, Prior P, Hayward AC, eds. Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. St. Paul, MN: American Phytopathological Society; 2005:449–462. The pathogen not only affects solanaceous but many plants of other dicot and monocot families. The extensive genetic diversity of strains responsible for various wilt diseases has in recent years led to the concept of an R. solanacearum "species complex" (RsSC).44 Genin S, Denny TP. Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol. 2012;50:67-89. As R. solanacearum strains have been isolated from virgin forest-soils of all five continents, the origin of the species complex is believed to predate the geographical separation of continents.55 Hayward AC. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol. 1991;29:65-87. The pathogen is hierarchically classified into four phylotypes according to newly proposed phylotype sub-classification system based on 16S-23S ITS region, egl and hrpB genes and on comparative genomic hybridization (CGH) which reflect their origin as Asia (Phylotype I), America (II), Africa (III) or Indonesia (IV).66 Wicker E, Grassart L, Beaudu RC, et al. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential. Appl Environ Microbiol. 2007;73:6790-6801. These phylotypes are further classified into sequevars, containing isolates with similar virulence patterns or common geographic origin.33 Fegan M, Prior P. How complex is the "Ralstonia solanacearum species complex. In: Allen C, Prior P, Hayward AC, eds. Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. St. Paul, MN: American Phytopathological Society; 2005:449–462. Despite their considerable diversity, R. solanacearum strains are unified by their common etiology resulting in disease.77 Patil VU, Gopal J, Singh BP. Improvement for bacterial wilt resistance in potato by conventional and biotechnological approaches. Agric Res. 2012;1(4):299-316. Three of the four phylotypes of R. solanacearum are known to cause bacterial wilt of potato in India.88 Sagar V, Arjunan J, Mian S, et al. Potato bacterial wilt in India caused by strains of Phylotype I, II and IV of Ralstonia solanacearum. Eur J Plant Pathol. 2013, http://dx.doi.org/10.1007/s10658-013-0299-z.
http://dx.doi.org/10.1007/s10658-013-029...
In the present study four strains, RS2 (Phylotype II), RS25 and RS48 (Phylotype I) and RS75 (Phylotype IV) isolated from brown-rot infected potato tubers obtained from different parts of the country were taken for complete genome sequencing and to analyze their relationship complexity.

We sequenced the genomes of all four strains using shotgun approach and Roche-454, GSflx-Titanium platform yielding appx. 2.88 million reads (>500 bp) of which nearly 99.5% reads were of high quality. The genome coverage ranged from 18X (RS48) to 76X (RS75). The high quality reads were aligned using GS De Novo Assembler (version 2.5.3) and gene prediction using the prokaryotic GeneMark.hmm (Version 2.2a) and AUGUSTUS (http://bioinf.uni-greifswald.de/augustus/submission/) revealed a total of 4590, 4732, 4817 and 4867 protein coding regions (CDSs) respectively for RS2, RS25, RS48 and RS75 spread over megaplasmid as well as chromosomal genomes. High quality reads were mapped on to publically available reference genomes, GMI1000 (RS25 & RS48), Po82 (RS2) and PSI07 (RS75) (www.ncbi.nlm.nih.gov/genome/) using gsMapper with optimized mapping parameters and obtained total genome coverage and per cent GC content for all four strains. The total protein coding regions, rRNA and tRNA coding, regulatory and pathogenicity genes including the Type III secretary genes were obtained from the consensus using the .gff (from public database) file with the help of in-house perl scripts (Table 1). The presence of repetitive elements was analyzed using MISA (http://pgrc.ipk-gatersleben.de/misa/) and was observed that nearly 90% of the elements were of di or tri and 7.3% hexa mer repeats. Chromosomes carried higher portion (60–70%) of the repeat elements than megaplsmids in all the four strains. The availability of the reference genomes of more and more strains of RsSC would greatly aid in epidemiological/quarantine studies and in gaining understanding on their origin, evolution, intra and inter-relationship within the complex and their interactions with plants.

Table 1
General features and distribution of CDS, tRNA, rRNA, regulatory genes and pathogenic genes between Chromosome and Megaplsmid of R. solanacearum strains including Rs2, Rs25, Rs48 and Rs75.

Nucleotide sequence accession numbers. This Whole Genome Shotgun project has been deposited at NCBI/GenBank under Bio-Project PRJNA221562 with Accession Nos. SRX360515, SRX365373, SRX365374 and SRX365375.

Acknowledgements

This work was supported by Indian Council of Agriculture Research (ICAR), New Delhi, Government of India under the network programme "PhytoFuRa – Phytophthora, Fusarium and Ralstonia Diseases of Horticulture and Field Crops".

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Publication Dates

  • Publication in this collection
    Apr-Jun 2017

History

  • Received
    6 May 2016
  • Accepted
    7 Oct 2016
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
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