Transboundary Introduction of Potato-Infecting Ralstonia

J Biosci (2021)46:80 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-021-00195-x (0123456789().,-volV)(0123456789().,-volV) Brief communication

Transboundary introduction of potato-infecting Ralstonia solanacearum in the Andaman Islands revealed by multilocus sequence typing: A potential threat to island agriculture

1,5 2,3 3,6 KSAKTHIVEL ,ABALAMURUGAN ,MASHAJYOTHI , 4 4 4 1,7 SOOBEDAR YADAV ,VBASKARAN ,KABIRAMI ,RKGAUTAM and 3 AKUMAR * 1Division of Field Crop Improvement and Protection, ICAR- Central Island Agricultural Research Institute, Port Blair 744 105, India 2Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu 641 003, India 3Division of Plant Pathology, ICAR - Indian Agricultural Research Institute, New Delhi 110 012, India 4Division of Horticulture and Forestry, ICAR- Central Island Agricultural Research Institute, Port Blair 744 105, India 5Present Address: Crop Protection Section, ICAR-Indian Institute of Oilseed Research, Hyderabad, Telangana 500 030, India 6Present Address: Plant Protection Lab, ICAR-Central Agroforestry Research Institute, Uttar Pradesh, Jhansi 284 003, India 7Present Address: Division of Germplasm Evaluation, ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012, India

*Corresponding author (Email, [email protected])

MS received 15 December 2020; accepted 30 June 2021

Wilting of potato plants with an incidence of 20–30 % was observed for the first time in the agricultural farms of Andaman Islands, India. The infected plants showed wilting syndrome that included downward drooping of leaves, yellowing, and collapse of the entire plants. Characteristic milky-white exudate from the infected stem indicated bacterial etiology of the disease. Upon streaking onto 2, 3, 5 triphenyl-tetrazolium chloride amended nutrient medium, the bacterial exudate yielded characteristic creamy-white, fluidal, irregular colonies with the pink center. Upon inoculation, the randomly picked bacterial colonies, AN_PRSGr and AN_PRSCh, representing the two locations, incited wilt symptoms on one-month-old potato plants. The host range studies revealed that the isolates were pathogenic on tomato and eggplant but non-pathogenic to chili and Solanum torvum (wild eggplant). The 16S rRNA gene sequencing and the Ralstonia-specific PCR test confirmed the identity of AN_PRSGr and AN_PRSCh as Ralstonia solanacearum. Intra-species level classification revealed their identity as strains of race 1, biovar 3, and phylotype-I. Multilocus sequence typing (MLST)-based in-depth sequence alignment for phylogenetic analysis revealed the isolates AN_PRSGr and AN_PRSCh clustered with two mainland race 1/biovar 3/phylotype-I isolates of Kerala, India. However, the allelic profile-based goe- BURST-analysis placed them as singletons in the global collection of Ralstonia solanacearum, conforming intra-racial/ intra-phylotype diversity within race 1/biovar3/phylotype-I strains. The molecular characterization

Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/ s12038-021-00195-x. http://www.ias.ac.in/jbiosci 80 Page 2 of 10 K Sakthivel et al by MLST revealed that the isolates shared several alleles with isolates reported from mainland India suggesting the transboundary movement of the pathogen, its introduction, and subsequent spread in the island.

Keywords. Potato bacterial wilt; R. solanacearum; host range; Phylotype-I; MLST; Andaman islands

1. Introduction cropping season, wilt incidence ranged from 20 to 30% was recorded in potato cultivar, Kufri-Badsha –an Bacterial wilt or brown rot disease in potato (Solanum Indian mainland variety in the research farms and tuberosum L.) caused by Ralstonia solanacearum is an South Andaman Island. The results of symptomato- economically significant bacterial disease worldwide. logical investigations and preliminary ooze tests indi- The disease occurs in warm-temperate regions with cated bacterial association in the potato wilt. The high soil moisture where the epidemiological factors present work describes in-depth phenotypic, genetic, favor the bacterium to survive, colonize, and thereby and pathological characters of potato bacterial wilt facilitate the wilt infection (Kumar et al. inciting strains in the Andaman Islands. 2013, 2014a, 2017; Charkowski et al. 2020). Ralstonia solanacearum is touted as a quarantine pathogen owing to its lethal effect on its host plants (Elphinstone 2005) 2. Materials and methods and causes severe crop losses in almost 450 different plant species (Peeters et al. 2013). In India, the bac- 2.1 Collection of bacterial wilt affected potato, terial wilt pathogen causes 50% crop loss in potatoes and isolation of the pathogen and other solanaceous vegetables (Mukherjee and Dasgupta 1989). The wilted potato plants collected from Garacharma Traditionally R. solanacearum was classified into (11.6021° N, 92.7046° E) and Ferrargunj (11.7144° N, five races (Buddenhagen et al. 1962) and five biovars 92.6544° E) were washed in water, blot dried, and (Hayward 1964) based on its host range and carbon allowed to ooze cells (bacterial exudation) in sterile source utilization. The global collection of Ralstonia water. Thus obtained bacterial ooze was streaked onto solanacearum can be classified into four phylotypes casamino acid peptone glucose agar (CPG; g L-1, reflective of their geographical origin (Prior and Fegan Casamino acid 1, Peptone 10, Glucose 10, Agar 15, pH 2005). Nucleotide polymorphism in the endoglucanase 7.2) amended with 2, 3, 5 triphenyl-tetrazolium chlo- (egl) gene sequence is used to classify R. solanacearum ride (1.0 %) (Kelman 1954; Kumar et al. 2004). The into sequevars (sequence variants) (Fegan and Prior streaked plates were incubated at 28°C for 72 h until 2005). Bacterial wilt of potato is incited by one of the the appearance of bacterial colonies. two races of R. solanacearum: (i) race 1/ biovar 3 or race 1/biovar 4 (r1/b3 or r1/b4) in the tropics (warm and lower elevations) and (ii) race 3 biovar 2 (r3/b2) in 2.2 Pathogenicity test, race, and biovar temperate (cool and higher elevation) regions world- identification wide (Martin and French 1985). In India, brown rot of potato or bacterial wilt was first documented by Cappel The pathogenicity test was conducted by adopting the in 1892 for the first time in Pune (Cappel 1892), and soil inoculation method as described by Kumar (2006). presently it is widely present in many potato-growing The freshly grown bacterial suspension (20 ml) con- states such as Himachal Pradesh, West Bengal, Madhya taining 19108 CFU/ml (absorbance 600 nm = 0.5–1.0 Pradesh, Uttar Pradesh, and Meghalaya in an endemic OD) was soil inoculated around the root zone of form (Shekhawat et al. 1992; Sagar et al. 2014; Kumar 30-day-old potato plants (cv. Kolar). Similarly, a host et al. 2014a). range study was performed on major solanaceous crops In recent years, potato cultivation is being promoted like tomato cv. Pusa Ruby (Solanum lycopersicum L.), among the farmers in Andaman and Nicobar Islands eggplant cv. CO-2 (Solanum melongena L.), chili cv. mainly to meet the local demand, and for this purpose, CO-1 (Capsicum annuum L.) and Turkey berry (Sola- the potato seed tubers suitable for tropical climates num torvum). All the experiments were conducted with have been introduced from mainland Indian states three replications for each host where uninoculated (Soobedar Yadav and Baskaran 2018). During the 2018 plants served as mock. All the inoculated plants were Potato-infecting R. solanacearum in Andaman Islands Page 3 of 10 80 incubated and maintained under a glasshouse set for dehydrogenase oxidoreductase; adk – adenylate kinase; temperature ranging from 26 to 32°C with alternate ppsA – phosphoenolpyruvate synthase, and gyrB – light and dark photoperiods. DNA gyrase, subunit B) and three-virulence genes in Race identification by the tobacco leaf infiltration megaplasmid (fliC – encoding flagellin protein; hrpB - method was performed according to Buddenhagen regulatory transcription regulator and egl – endoglu- et al.(1962). The bacterial suspension prepared (19108 canase precursor) were nucleotide-sequenced. The CFU/ml, absorbance 600 nm = 0.5–1.0 OD) from both PCR amplification of the MLST loci was performed as the isolates was injected using a hypodermic needle suggested by Kumar et al (2014b) which is as follows; onto fully expanded leaves of Nicotiana tabacum. the reaction mixture (50 ll) contained 100 ng template- Plants injected with sterile water served as mock. DNA, 1X PCR buffer, 1.5 mM MgCl2,50lM each Biovar identification was performed based on the uti- dNTP, 6.0 % DMSO, 10 pmol primers and 1 Unit Taq- lization of disaccharides and sugar alcohols using the polymerase. PCR was preceded with 9.0 min denatu- protocol suggested by Hayward (1964). ration step at 96° C, followed by 30 cycles at 95°C/1.0 min, the appropriate annealing temperature for 1.0 min and extension temperature of 72°C/2.0 min, with a final 2.3 Molecular identification of Ralstonia extension step at 72°C/10 min. Sequences were solanacearum assembled using DNA baser v4 software package and the curated partial sequences of all seven genes were The species identity of the bacterial isolates was con- submitted to GenBank (https://www.ncbi.nlm.nih.gov/) firmed by a comparison of 16S rRNA gene sequences and the accession numbers were assigned. Sequences generated using universal primers 27F and 1492R were also analyzed in PAMDB software (http:// (Taghavi et al. 1996; Poussier et al. 2000). PCR genome.ppws.vt.edu/cgi-bin/MLST/home.pl) and allele amplicon of about 1465 bp were obtained using 50 lL numbers assigned by comparing allele sequences doc- reaction mixtures, which consisted of 100 ng of tem- umented in the database. New allele number was pro- plate DNA, 1X PCR buffer, MgCl2 1.5 mM, each posed for new base variations in the MLST loci and dNTP 50 lM, 5.0 pmol of each primer, and 1 U of Taq submitted to PAMDB. Further, the concatenated DNA polymerase. PCR was performed in a thermo- nucleotide sequences representing the seven loci (810 cycler (Eppendorf, Germany) with an initial denatura- bp (hrpB) ? 774 bp (gapA) ? 873 bp (gyrB) ? 774 bp tion of 96°C/2.0 min, followed by 35 cycles of 94°C/ (ppsA) ? 420 bp (adk) ? 318 bp (fliC) ? 686 bp (egl) 30s, 60°C/1.0 min, and 72°C/1.0 min. PCR was com- = 4655) of the two isolates was used for phylogenetic pleted with 10 min/72°C. The amplicons were analysis in Mega X using Maximum Likelihood sequenced bidirectionally and the nucleotide sequence method along with 88 entries of R. solanacearum obtained was assembled using DNA-Baser v4 software strains worldwide. Additionally, for population genetic and subjected to BLAST analysis in GenBank (https:// analysis, goeBURST program (http://www.phyloviz. www.ncbi.nlm.nih.gov/). Species identity was further net/goeburst/), an algorithm exclusively developed for confirmed using R. solanacearum specific primers analyzing microbial MLST data was used to analyze (Opina et al. 1997). Further, the isolates were subjected the allele data and visualize the population genetic to phylotyping analysis using multiplex-PCR assay relationship (Francisco et al. 2009). (Fegan and Prior 2005).

2.4 Genotyping by multilocus sequence typing 3. Results and discussion (MLST) Typical creamy white, thread-like bacterial ooze was Having identified the pathogen as Ralstonia solana- obtained in sterile water from wilt infected potato stem cearum, the MLST-based genetic analysis (Castillo and which confirmed their bacterial etiology. The colony Greenberg 2007) was adopted to decipher the genetic morphology of both the R. solanacearum isolates col- relationship of AN_PRSGr and AN_PRSCh with a lected from two different locations of Andaman global R. solanacearum collection including previously Islands, India [Garacharma and Ferrargunj] showed reported ones from the Andaman Islands. Seven-ge- characteristic creamy-white, fluidal, irregular-colony nomic loci comprising of four-housekeeping genes in with a pinkish center (figure 1A, B, C) and further the chromosome (gapA – glyceraldehyde 3-phosphate preserved under the names AN_PRSGr, and 80 Page 4 of 10 K Sakthivel et al

Figure 1. Pathogenicity of Ralstonia solanacearum isolates AN_PRSGr and AN_PRSCh on potato (cv. Kolar). (A) White milky bacterial ooze obtained from the infected stem used for the isolation on 2, 3, 5 triphenyl-tetrazolium chloride amended CPG media. (B & C) Typical culture morphology of R. solanacearum isolates AN_PRSGr and AN_PRSCh. (D & E) Typical bacterial wilt symptoms on potato upon soil inoculation of the isolate AN_PRSGr and AN_PRSCh characterized by downward drooping of leaves lead to plant death. (F) Plant inoculated with sterile water remained healthy in mock.

Table 1. Pathogenicity test and host range studies of Ralstonia solanacearum

Pathogenicity and Host range assay*

Potato (Kolar) Tomato (Pusa Ruby) Brinjal (CO-2) Chilli (CO-1) S. torvum (Wild sp.)

R. solanacearum isolates DW PDI (%) DW PDI (%) DW PDI (%) DW PDI (%) DW PDI (%) AN_PRSGr 13 50.00 5 100.00 7 73.33 NP – NP – AN_PRSCh 10 66.66 4 100.00 5 86.66 NP – NP –

*Mean of three replications; DW, days to wilt; PDI, percent disease incidence; NP, non-pathogenic.

AN_PRSCh in glycerol stock (30 % v/v) at -80°C, on chilies and S. torvum even upon prolonged incu- and characterized in further studies. bation up to 60 days, and also by pinprick-aided The results of pathogenicity tests revealed that both inoculation (Kumar 2006); this indicated the non- the isolates, AN_PRSGr and AN_PRSCh, were pathogenic nature of the bacterial isolates on these pathogenic on potatoes and caused typical bacterial solanaceous plants (figure 2G, H, J, K; table 1). Our wilt symptoms within 10–13 days post-inoculation results are in agreement with earlier reports of low (dpi) (figure 1D–F; table 1). The host range assays infectivity of certain R. solanacearum isolates on chili conducted on other solanaceous crops indicated that (Sakthivel et al. 2016a, b; Balamurugan et al. 2018). both the isolates induced wilt symptoms in tomato and The bacterial pathogen was re-isolated from the wilted eggplant seedlings within 4 to 7 dpi (figure 2A, B, D, plants and found them sharing similar phenotypic E; table 1). Interestingly, the isolates did not cause wilt characters with those used for the inoculation. The Potato-infecting R. solanacearum in Andaman Islands Page 5 of 10 80

Figure 2. Host range studies of Ralstonia solanacearum isolates AN_PRSGr and AN_PRSCh on other solanaceous crops. Typical bacterial wilt symptoms including drooping of leaves downwards to extensive wilting of whole plants induced by the isolates AN_PRSGr and AN_PRSCh on tomato cv. Pusa Ruby (A, B); and eggplant cv. CO-2 (D, E). Both the isolates showed non-pathogenic nature on chili cv. CO-1 (G, H) and wild species of S. torvum (J, K). Plants inoculated with sterile water alone on tomato (C), brinjal (F), chili (I), and S. torvum (L) remained healthy. 80 Page 6 of 10 K Sakthivel et al uninoculated plants remained healthy and unaffected were assigned (table 2). The sequences were also (figure 2C, F, I, L; table 1). compared with the allele database in PAMDB, and the Tobacco leaf infiltration assay showed that the iso- allele numbers were assigned for each locus. Allele lates could induce brown necrotic reaction within 48 numbers not matching in PAMDB for the new hours post inoculation (hpi) on all inoculated sites that sequence were assigned a new allele number, and later turned yellow at 72 hpi followed by extensive submitted (table 2). wilting of whole plants in a week that confirmed their Further, phylogenetic analysis of concatenated identity as race 1 of R. solanacearum (supplementary sequences from 88 entries of R. solanacearum isolates figure 1). No such reaction was observed in the mock. from worldwide was used and compared (supplemen- Biovar test conducted revealed the identity as biovar-3 tary tables 2 and 3). The phylogenetic tree constructed as the isolates utilized/oxidized all carbon sources used by the Maximum Likelihood method using concate- in the assay (supplementary figure 2; supplementary nated nucleotide sequences representing the seven loci table 1). (4655 bp) in Mega X displayed that the two isolates For 16S rRNA gene sequence-based identification, AN_PRSGr and AN_PRSCh, clustered with the phy- the 1400 bp sequence generated was used for BLAST lotype-I clade of R. solanacearum (figure 3). analysis which revealed 99% similarity with other R. Interestingly, it was also found that both the isolates solanacearum isolates, and the sequences were were genetically close with two Sequence Types (STs), deposited in the NCBI database with accession num- TRs-Cal (ST 918) and GRs-Pvl (ST 913), reported bers MT277400 (AN_PRSGr) and MT277401 from the mainland Indian state of Kerala (figure 3). The (AN_PRSCh) (NCBI Resource Coordinators 2016). goeBURST analysis revealed that the potato infecting The species identification was performed using R. two Sequence Types shared four alleles with Ralstonia solanacearum specific primers that yielded R. solana- isolates from mainland India. The population snapshot cearum-specific amplicon of 281 bp (supplementary displayed the isolates, AN_PRSGr (ST1343) and figure 3). Further, phylotyping by multiplex-PCR AN_PRSCh (ST1344), in proximity with tomato yielded 144 bp amplicon and confirmed the isolates (ST1806) and eggplant (ST1341) infecting R. solana- belong to phylotype-I known for its ‘Asian origin’ cearum also reported from Andaman Island (figure 4). (supplementary figure 4). Transmission of R. solanacearum through succulent In the MLST approach, sequences of seven MLST vegetatively propagated planting materials such as loci were assembled using DNA Baser v4, and the rhizomes, tubers, and suckers is already established and curated partial sequences of all the genes were sub- reported (Kumar et al. 2004; Kumar and Abraham, mitted to NCBI GenBank, and the accession numbers 2008). The prevalence of genetically identical isolates

Table 2. Molecular characterization of Ralstonia solanacearum causing potato bacterial wilt disease in Andaman Islands, India

NCBI GenBank accession numbers Allele numbers* Details AN_PRSGr AN_PRSCh a. 16S rRNA gene sequence MT277400 MT277401 AN_PRSGr AN_PRSCh b. Multilocus sequence typing (MLST) analysis isolate isolate I. Four chromosomal housekeeping genes (i) ppsA (phosphoenolpyruvate synthase) MT277408 MT277409 24 24 (ii) gyrB (DNA gyrase) MT277404 MT277405 27 27 (iii) adk (adenylatekinase) MT277406 MT277407 7 7 (iv) gapA (glyceraldehyde 3-phosphate dehydrogenase MT277396 MT277397 24 32* oxido reductase) II. Three megaplasmid borne virulence-related genes (v) hrpB (regulatory transcription regulator) MT277410 MT277411 10 33* (vi) ﬂiC (encoding ﬂagellin protein) MT277398 MT277399 24* 25* (vii) egl (endoglucanase precursor) MT277402 MT277403 35* 26

*New alleles identiﬁed in this study based on BLAST search in Plant Associated and Environmental Microbes Database (http:// genome.ppws.vt.edu/cgi-bin/MLST/home.pl) Potato-infecting R. solanacearum in Andaman Islands Page 7 of 10 80 b Figure 3. Molecular phylogenetic analysis of Ralstonia solanacearum based on concatenated sequence length of 4655-bp representing 7 loci by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). The tree with the highest log likelihood (-17979.77) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likeli- hood (MCL) approach and then selecting the topology with a superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 90 nucleotide sequences. There were a total of 4655 positions in the ﬁnal dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018).

To curtail the transboundary movement of r3/b2, strict quarantine laws are enacted in several countries (El Sayed et al. 2019). In India, the prevalence of two races such as r1/b3 and r3/b2 that infect potatoes has been reported earlier (Sagar et al. 2014; Kumar et al. 2014a). Herein, r1/b3 strain was found to incite bacterial wilt in potatoes in the remote island clusters in Andaman; this indicated the potential of locally adapted r1/b3 Asian strain of R. solanacearum to cause pre- harvest losses in potato. Adaptation of r1/b3 strain on other solanaceous and non-solanaceous hosts is cur- rently known, as isolates classiﬁed under race 1 are broad-host-range plant pathogens. The island is also known for the extensive cultivation of tomatoes, eggplant, and chili where bacterial wilt is reported earlier (Sakthivel et al. 2016a, b). The incidence of bacterial wilt of solanaceous crops in the Andaman Islands is attributed to the inadvertent introduction of the pathogen from the mainland of India by early settlers who introduced and practiced agriculture in the islands upon their arrival during the mid-20th century (Sakthivel in well-separated geographical locations is attributed to et al. 2016a). The early settlers traced their origin in the horizontal transmission of plant pathogens, per- West Bengal, Tamil Nadu, and Kerala – the mainland haps, aided by planting materials (Kumar et al. Indian states who continue to contribute to the agri- 2004, 2014b, 2020; Balamurugan et al. 2018). cultural economic activities of the island. Intensiﬁca- Worldwide, r3/b2 strains of R. solanacearum, pre- tion of agricultural practices in the land-scarce sumably, originated in the Andes regions of South Andaman Islands has prompted overlapping cropping America, are reported to incite bacterial wilt on pota- sequences, especially in the cultivation of solanaceous toes. Furthermore, the potato-infecting wilt pathogen is vegetables. Continuous cropping coupled with conge- presumed to have spread throughout the world, possi- nial environmental and climatic conditions throughout bly, by elite-tubers imported for potato (genetic) the year might have supported the r1/b3 strain to incite improvement programs in various countries. wilt in potatoes on the island. 80 Page 8 of 10 K Sakthivel et al

Figure 4. Population snapshot of Ralstonia solanacearum prevalent in Andaman Island generated by goeBURST 1.2.1 using allelic differences in MLST genes. ST1343 and ST1344 isolates caused bacterial wilt in potatoes in the Andaman Islands.

4. Conclusion Acknowledgements

Taken together, it can be concluded that the intra- We are grateful to the Director, ICAR-Central Island regional spread of R. solanacearum r1/b3 is Agricultural Research Institute, Port Blair, and Direc- inevitable in the Andaman Islands owing to the tor, ICAR-Indian Agricultural Research Institute, New continuous cultivation of wilt susceptible solana- Delhi, for support and encouragement. We acknowl- ceous crops. The outcome of the present study edge the Consortium Research Project-Genomics further showed that the broad host range race-1 for funding the genomics work carried out in the strain of R. solanacearum is a potential threat to project. potato cultivation in the Andaman Islands even in the absence of the ‘potato-adapted’ South Ameri- can r3/b2 strain. The investigation highlights the References conducive nature of agricultural fields in the Andaman Islands for bacterial wilt incidence. Balamurugan A, Kumar A, Muthamilan M, et al. 2018 Hence, an integrated wilt management strategy Outbreak of tomato wilt caused by Ralstonia solana- with due emphasis on the production and distri- cearum in Tamil Nadu, India, and elucidation of its bution of certified pathogen-free tubers of geneti- genetic relationship using multilocus sequence typing cally improved potato cultivars must be promoted. (MLST). Eur. J. Plant Pathol. 151 831–839 The result of our study should also form a basis Buddenhagen IW, Sequeira L and Kelman A 1962 Desig- for formulating effective disease management nation of races of Pseudomonas solanacearum. Phy- strategies in the future. topathology 52 726 Potato-infecting R. solanacearum in Andaman Islands Page 9 of 10 80 Cappel EL 1892 A note on a potato disease prevalent in solanacearum race 3/ biovar 2/ phylotype IIB from Poona district elsewhere. Report of Department of Land potato. Indian Phytopath. 67 346–352 Records and Agriculture. Bombay Presidency Kumar A, Prameela TP, Suseelabhai R, Siljo A, Anandaraj Charkowski A, Sharma K, Monica L, Parker ML, Gary A, M and Vinatzer BA 2014b Host specificity and genetic Secor GA and Elphinstone J 2020 Bacterial diseases of diversity of race 4 strains of Ralstonia solanacearum. potato. The Potato Crop: Its Agricultural, Nutritional and Plant Pathol. 63 1138–1148 Social Contribution to Humankind (Springer) pp 351–388 Kumar A, Sarma YR and Anandaraj M 2004 Evaluation of Castillo JA and Greenberg JT 2007 Evolutionary dynamics genetic diversity of Ralstonia solanacearum causing of Ralstonia solanacearum. Appl. Environ. Microbiol. 73 bacterial wilt of ginger using REP-PCR and PCR-RFLP. 1225–1238 Curr. Sci. 87 1555–1561 El Sayed T, Samuel J, Nour E, Sorensen SJ and Smalla K Kumar A, Sharma J, Munjal V, et al. 2020 Polyphasic 2019 Biocontrol of bacterial wilt disease through complex phenotypic and genetic analysis reveals clonal nature of interaction between tomato plant, antagonists, the indige- Xanthomonas axonopodis pv. punicae causing pomegra- nous rhizosphere microbiota and Ralstonia solana- nate bacterial blight. Plant Pathol. 69 347–359 cearum. Front. Microbiol. 10 2835 Kumar S, Stecher G, Li M, Knyaz C and Tamura K 2018 Elphinstone JG 2005 The current bacterial wilt situation: A MEGA X: Molecular Evolutionary Genetics Analysis global view; in Bacterial Wilt Disease and the Ralstonia across computing platforms. Mol. Biol. Evol. 35 solanacearum Species Complex (Eds.) C. Allen, Ph. Prior, 1547–1549 & A. C. Hayward (St. Paul, Minnesota: APS Press) pp 9–28 Martin C and French ER 1985 Bacterial wilt of potato, Fegan M, Prior P 2005 How complex is the ‘‘Ralstonia Pseudomonas solanacearum. Technical bulletin 13. Lima: solanacearum species complex’’? in: Bacterial Wilt International Potato Centre Disease and the Ralstonia solanacearum Species Com- Mukherjee N and Dasgupta MK 1989 Udvider Rog [Plant plex (Eds) C. Allen, P. Prior, and A. C. Hayward Disease] Poschimbongo Rajyo Pustak Porsad (West (American Phytopathological Society. St. Paul, MN) Bengal State Book Board, Kolkata, India) pp 449–461 NCBI Resource Coordinators 2016 Database resources of Francisco AP, Bugalho M, Ramirez M and Carrico JA 2009 the National Center for Biotechnology Information. Global Optimal eBURST analysis of Multilocus typing Nucleic Acids Res. 44 D7–D19 data using a graphic matroid approach. BMC Bioinform. Opina N, Tavne F, Holloway G, et al. 1997 A novel method 10 152 for development of species and strain-specific DNA Hayward AC 1964 Characteristics of Pseudomonas solana- probes and PCR primers for identifying Burkholderia cearum. J. Appl. Bacteriol. 27 265–277 solanacearum (formerly Pseudomonas solanacearum). Kelman A 1954 The relationship of pathogenicity in Asia Pac. J. Mol. Biol. Biotech. 5 19–33 Pseudomonas solanacearum to colony appearance on a Peeters N, Guidot A, Vailleau F and Valls M 2013 tetrazolium medium. Phytopathology 44 693–695 Ralstonia solanacearum, a widespread bacterial plant Kumar A 2006 Methods for screening ginger (Zingiber pathogen in the post-genomic era. Mol. Plant Pathol. 14 officinale Rosc.) for bacterial wilt resistance. Indian 651–662 Phytopath. 59 281–286 Poussier S, Trigalet-Demery D, Vandewalle P, Goffinet B, Kumar A and Abraham S 2008 PCR based detection of Luisetti J and Trigalet A 2000 Genetic diversity of bacterial wilt pathogen Ralstonia solanacearum in ginger Ralstonia solanacearum as assessed by PCR-RFLP of the rhizomes and soil collected from bacterial wilt affected hrp gene region, AFLP and 16S rRNA sequence analysis field. J. Spices Aromatic Crops 1 109–113 and identification of an African subdivision. Microbiology Kumar A, Munjal V, Sheoran N, Prameela TP, Suseelabhai 146 1679–1692 R, Aggarwal R, Jain RK and Eapen SJ 2017 Draft Prior P and Fegan M 2005 Recent development in the genome sequence of highly virulent race 4/biovar 3 of phylogeny and classification of Ralstonia solanacearum. Ralstonia solanacearum CaRs_Mep causing bacterial wilt Acta Hortic. 695 17660 in Zingiberaceae plants in India. Genome Announc. 5 Sagar V, Jeevalatha A, Mian S, et al. 2014 Potato 01420–01516 bacterial wilt in India caused by strains of phylotype-I, Kumar A, Prameela TP and Suseelabhai R 2013 A unique II and IV of Ralstonia solanacearum. Eur. J. Plant DNA repair and recombination gene (recN) sequence for Pathol. 138 51–65 identification and intraspecific molecular typing of bac- Sakthivel K, Kumar A, Devendrakumar C, et al. 2016a terial wilt pathogen Ralstonia solanacearum and its Diversity of Ralstonia solanacearum strains on comparative analysis with ribosomal DNA sequences. J. the Andaman Islands in India. Plant Dis. 100 Biosci. 38 267–278 732–738 Kumar A, Prameela TP and Panja B 2014a Genetic Sakthivel K, Baskaran V, Abirami K, Manigundan K and characterization of an Indian isolates of Ralstonia Gautam RK 2016b Cross-Infectivity of Ralstonia 80 Page 10 of 10 K Sakthivel et al solanacearum from Marigold Grown in Andaman Taghavi M, Hayward C, Sly LI and Fegan M 1996 Analysis Islands. J. Hort. Sci. 11 179–181 of the phylogenetic relationships of strains of Burkholde- Shekhawat GS, Chakrabarti SK and Gadewar AV 1992 ria solanacearum, Pseudomonas syzygii and the blood Potato bacterial wilt in India. Tech. Bull. No. 38, Central disease bacterium of banana based on 16S rRNA gene Potato Research Institute (CPRI), Shimla, India sequences. Int. J. Syst. Bacteriol. 46 10–15 Soobedar Y and Baskaran V 2018 ICAR-CIARI (Central Tamura K and Nei M 1993 Estimation of the number of Island Agricultural Research Institute), Port Blair, nucleotide substitutions in the control region of mito- Annual report 2017-18 (https://ciari.icar.gov.in/ar. chondrial DNA in humans and chimpanzees. Mol. Biol. htm) Evol. 10 512–526

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