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

Patil, Virupaksh U.; Girimalla, Vanishree; Sagar, Vinay; Chauhan, Rajinder Singh; Chakrabarti, Swarup Kumar

doi:10.1016/j.bjm.2016.10.016

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Genome Announcements • Braz. J. Microbiol. 48 (2) • Apr-Jun 2017 • https://doi.org/10.1016/j.bjm.2016.10.016 copy

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

Authorship SCIMAGO INSTITUTIONS RANKINGS

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.

Thumbnail

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".

References

¹
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.
²
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.
³
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.
⁴
Genin S, Denny TP. Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol 2012;50:67-89.
⁵
Hayward AC. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 1991;29:65-87.
⁶
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.
⁷
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.
⁸
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/s10658-013-0299-z
⁹
Ailloud F, Lowe T, Cellier G, Roche D, Allen C, Prior P. Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity. BMC Genomics 2015;16:270.
¹⁰
Raman R, Gaitonde S, Achari G, et al. Genome sequencing of Ralstonia solanacearum Biovar 3, Phylotype I, strains Rs-09-161 and Rs-10-244, isolated from eggplant and Chili in India. Genome Announc 2014;2(3):e00323-e414.
¹¹
Salanoubat M, Genin S, Artiguenave F, et al. Genome sequencing of the plant pathogen Ralstonia solanacearum. Nature 2002;415:497-502.
¹²
Li Z, Wu S, Bai X, et al. Genome sequence of the tobacco bacterial wilt pathogen Ralstonia solanacearum. J Bacteriol 2011;193(21):6088-6089.
¹³
Cao Y, Tian B, Liu Y, et al. Genome sequencing of Ralstonia solanacearum FQY_4, isolated from a bacterial wilt nursery used for breeding crop resistance. Genome Announc 2013;1(3):e00125-e213.
¹⁴
Remenant B, Babujee L, Lajus A, Médigue C, Prior P, Allen C. Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum. J Bacteriol 2012;194(10):2742-2743.
¹⁵
Remenant B, Goutaland BC, Guidot A, et al. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence. BMC Genomics 2010;11:379.
¹⁶
Xu J, Zheng HJ, Liu L, et al. Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82. J Bacteriol 2011;193(16):4261-4262.
¹⁷
Remenant B, de Cambiaire J-C, Cellier G, et al. Ralstonia syzygii, the blood disease bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles. PLoS ONE 2011;6(9):e24356.

Publication Dates

Publication in this collection
Apr-Jun 2017

History

Received
6 May 2016
Accepted
7 Oct 2016

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivative License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited and the work is not changed in any way.

Strain	Origin	Isolated from	Phylotype^a a Sequevar numbers are in parenthesis.	Sequence status	Genome size (Kb)^b b CHR - chromosome, MLP - Megaplsmid, ND - not available.			GC%^c c G + C content in percent.	CDS^d d CDS - number of coding sequences.	rRNA^e e Number of genes coding for ribosomal RNAs.	tRNA^f f Number of genes coding for transfer RNAs.	T3E^g g Number of predicted type III effectors.	Reference
					CHR	MPL	Total
Grenada9-1	Grenada	Banana	IIA(6)	Draft	NA	NA	5479	66.60	5365	3^h h ARAGON tRNA detection.	56^h h ARAGON tRNA detection.	NA	⁹9 Ailloud F, Lowe T, Cellier G, Roche D, Allen C, Prior P. Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity. BMC Genomics. 2015;16:270.
IBSBF1503	Peru	Cucumber	II(4)NPB	Draft	NA	NA	5514	66.70	5452	3^h h ARAGON tRNA detection.	54^h h ARAGON tRNA detection.	NA
CBF1416	Costa Rica	Plantain	IIB(3)	Draft	NA	NA	5744	66.60	5722	3^h h ARAGON tRNA detection.	59^h h ARAGON tRNA detection.	NA
Rs-09-161	India	Eggplant	I/R1b3	Complete	3741	1985	5726	66.82	5213	3^h h ARAGON tRNA detection.	66^h h ARAGON tRNA detection.	71	¹⁰10 Raman R, Gaitonde S, Achari G, et al. Genome sequencing of Ralstonia solanacearum Biovar 3, Phylotype I, strains Rs-09-161 and Rs-10-244, isolated from eggplant and Chili in India. Genome Announc. 2014;2(3):e00323-e414.
Rs-10-244	India	Chilli	I/R1b3	Complete	3716	2025	5741	66.98	5202	3^h h ARAGON tRNA detection.	63^h h ARAGON tRNA detection.	76
RS-2 ⁱ i Isolated from the infected stem.	Indore (MP), India	Potato	IIB(1)	Draft	3481	1608	4768	57.36	4590	2	53	63	This study
RS-25 ^j j Isolated from infected tubers.	Shimla (HP), India	Potato	I(45)	Draft	3065	1950	5232	60.11	4732	3	57	91
RS-48 ^j j Isolated from infected tubers.	Shimla (HP), India	Potato	I(30)	Draft	3065	1727	5300	60.10	4817	3	57	89
RS-75 ⁱ i Isolated from the infected stem.	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	¹¹11 Salanoubat M, Genin S, Artiguenave F, et al. Genome sequencing of the plant pathogen Ralstonia solanacearum. Nature. 2002;415:497-502.
Y45	China	Tomato	IB	Draft	3726	1986	5712	NA	5496	5	53	ND	¹²12 Li Z, Wu S, Bai X, et al. Genome sequence of the tobacco bacterial wilt pathogen Ralstonia solanacearum. J Bacteriol. 2011;193(21):6088-6089.
FYQ_4	China	Tomato	I	Complete	3715	2089	5805	66.82	5153	9	62	ND	¹³13 Cao Y, Tian B, Liu Y, et al. Genome sequencing of Ralstonia solanacearum FQY_4, isolated from a bacterial wilt nursery used for breeding crop resistance. Genome Announc. 2013;1(3):e00125-e213.
K60	USA	Tomato	IIA(7)	Draft	3717	1773	5490	66.70	5213	3^h h ARAGON tRNA detection.	51^h h ARAGON tRNA detection.	ND	¹⁴14 Remenant B, Babujee L, Lajus A, Médigue C, Prior P, Allen C. Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum. J Bacteriol. 2012;194(10):2742-2743.
CFBP2957	Fr. West Indies	Tomato	IIA(36)	Complete	3539	2144	5683	69.90	5310	1	56	72	¹⁵15 Remenant B, Goutaland BC, Guidot A, et al. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence. BMC Genomics. 2010;11:379.
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	¹⁶16 Xu J, Zheng HJ, Liu L, et al. Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82. J Bacteriol. 2011;193(16):4261-4262.
BDB R229	Indonesia	Banana	IV	Draft	3574	1585	5159	66.50	4629	1	45	57	¹⁷17 Remenant B, de Cambiaire J-C, Cellier G, et al. Ralstonia syzygii, the blood disease bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles. PLoS ONE. 2011;6(9):e24356.
R. syzygii R24	Indonesia	Clove	IV	Draft	3681	1743	5424	65.90	4867	2	50	48

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Acompanhe os números deste periódico no seu leitor de RSS

[1] ¹
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.

[2] ²
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.

[3] ³
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.

[4] ⁴
Genin S, Denny TP. Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol 2012;50:67-89.

[5] ⁵
Hayward AC. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 1991;29:65-87.

[6] ⁶
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.

[7] ⁷
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.

[8] ⁸
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/s10658-013-0299-z

[9] ⁹
Ailloud F, Lowe T, Cellier G, Roche D, Allen C, Prior P. Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity. BMC Genomics 2015;16:270.

[10] ¹⁰
Raman R, Gaitonde S, Achari G, et al. Genome sequencing of Ralstonia solanacearum Biovar 3, Phylotype I, strains Rs-09-161 and Rs-10-244, isolated from eggplant and Chili in India. Genome Announc 2014;2(3):e00323-e414.

[11] ¹¹
Salanoubat M, Genin S, Artiguenave F, et al. Genome sequencing of the plant pathogen Ralstonia solanacearum. Nature 2002;415:497-502.

[12] ¹²
Li Z, Wu S, Bai X, et al. Genome sequence of the tobacco bacterial wilt pathogen Ralstonia solanacearum. J Bacteriol 2011;193(21):6088-6089.

[13] ¹³
Cao Y, Tian B, Liu Y, et al. Genome sequencing of Ralstonia solanacearum FQY_4, isolated from a bacterial wilt nursery used for breeding crop resistance. Genome Announc 2013;1(3):e00125-e213.

[14] ¹⁴
Remenant B, Babujee L, Lajus A, Médigue C, Prior P, Allen C. Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum. J Bacteriol 2012;194(10):2742-2743.

[15] ¹⁵
Remenant B, Goutaland BC, Guidot A, et al. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence. BMC Genomics 2010;11:379.

[16] ¹⁶
Xu J, Zheng HJ, Liu L, et al. Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82. J Bacteriol 2011;193(16):4261-4262.

[17] ¹⁷
Remenant B, de Cambiaire J-C, Cellier G, et al. Ralstonia syzygii, the blood disease bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles. PLoS ONE 2011;6(9):e24356.