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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.48 no.2 São Paulo April./June 2017

http://dx.doi.org/10.1016/j.bjm.2016.10.016 

Genome Announcements

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

Virupaksh U. Patila  * 

Vanishree Girimallaa 

Vinay Sagarb 

Rajinder Singh Chauhanc 

Swarup Kumar Chakrabartia  b 

aCentral Potato Research Institute, Division of Crop Improvement, Bemloe, Shimla, Himachal Pradesh, India

bCentral Potato Research Institute, Division of Plant Protection, Bemloe, Shimla, Himachal Pradesh, India

cJaypee University of Information and Technology, Waknaghat, Solan, Himachal Pradesh, India


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 solanacearum1,2 formally known as Pseudomonas solanacearum and Burkholderia solanacearum is a gram-negative, chemo-organotroph phytopathogenic β-proteobacterium with an unusual broad host range.3 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).4 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.5 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).6 These phylotypes are further classified into sequevars, containing isolates with similar virulence patterns or common geographic origin.3 Despite their considerable diversity, R. solanacearum strains are unified by their common etiology resulting in disease.7 Three of the four phylotypes of R. solanacearum are known to cause bacterial wilt of potato in India.8 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. 

Strain Origin Isolated from Phylotypea Sequence status Genome size (Kb)b GC%c CDSd rRNAe tRNAf T3Eg Reference
CHR MPL Total
Grenada9-1 Grenada Banana IIA(6) Draft NA NA 5479 66.60 5365 3h 56h NA 9
IBSBF1503 Peru Cucumber II(4)NPB Draft NA NA 5514 66.70 5452 3h 54h NA
CBF1416 Costa Rica Plantain IIB(3) Draft NA NA 5744 66.60 5722 3h 59h NA
Rs-09-161 India Eggplant I/R1b3 Complete 3741 1985 5726 66.82 5213 3h 66h 71 10
Rs-10-244 India Chilli I/R1b3 Complete 3716 2025 5741 66.98 5202 3h 63h 76
RS-2i Indore (MP), India Potato IIB(1) Draft 3481 1608 4768 57.36 4590 2 53 63 This study
RS-25j Shimla (HP), India Potato I(45) Draft 3065 1950 5232 60.11 4732 3 57 91
RS-48j Shimla (HP), India Potato I(30) Draft 3065 1727 5300 60.10 4817 3 57 89
RS-75i Shillong (Meghalaya), India Potato IV(8) Draft 2903 1720 5045 60.06 4867 2 54 73
GMI1000 Fr. Guyana Tomato I (18) Complete 3716 2094 5811 67.00 5120 4 57 74 11
Y45 China Tomato IB Draft 3726 1986 5712 NA 5496 5 53 ND 12
FYQ_4 China Tomato I Complete 3715 2089 5805 66.82 5153 9 62 ND 13
K60 USA Tomato IIA(7) Draft 3717 1773 5490 66.70 5213 3h 51h ND 14
CFBP2957 Fr. West Indies Tomato IIA(36) Complete 3539 2144 5683 69.90 5310 1 56 72 15
Molk2 Phillippines Banana IIB(3) Draft NA NA 5961 66.70 5061 1 34 75
CMR15 Cameroon Tomato III(29) Complete 3594 1963 5593 69.90 5149 3 59 67
Psi07 Indonesia Tomato IV(10) Complete 3508 2085 5606 66.30 5247 1 49 74
Po82 Mexico Potato IIB(4) Complete 3481 1949 5430 66.65 5019 3 54 75 16
BDB R229 Indonesia Banana IV Draft 3574 1585 5159 66.50 4629 1 45 57 17
R. syzygii R24 Indonesia Clove IV Draft 3681 1743 5424 65.90 4867 2 50 48

aSequevar numbers are in parenthesis.

bCHR - chromosome, MLP - Megaplsmid, ND - not available.

cG + C content in percent.

dCDS - number of coding sequences.

eNumber of genes coding for ribosomal RNAs.

fNumber of genes coding for transfer RNAs.

gNumber of predicted type III effectors.

hARAGON tRNA detection.

iIsolated from the infected stem.

jIsolated from infected tubers.

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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Received: May 6, 2016; Accepted: October 7, 2016

*Corresponding author at: Division of Crop Improvement, Central Potato Research Institute, Shimla 171 001, Himachal Pradesh, India. E-mails: veerubt@gmail.com virupakshagouda.patil@icar.gov.in (V.U. Patil).

Conflicts of interest

The authors declare no conflicts of interest.

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