Ralstonia solanacearum virulence in eggplant seedlings by the leaf-clip inoculation
- 810 Downloads
Ralstonia solanacearum causes a lethal bacterial wilt disease in numerous plants including important vegetable crops such as eggplant and tomato. One of the difficulties in studying virulence of this bacterium in different host plants is the development of an easy and stable pathogenicity assay. Recently we described a leaf-clip inoculation method to study its pathogenicity at the cotyledon stage of tomato seedlings. Hereafter, we demonstrated the leaf-clip inoculation method to be equally efficient for studying R. solanacearum pathogenicity in the cotyledon stage of eggplant seedlings. Our study revealed eggplant seedlings to be highly susceptible to R. solanacearum as compared to tomato seedlings, illustrated by appearance of disease symptoms in significantly higher number of seedlings. We also tested the virulence of several global transcription regulator mutants of R. solanacearum including hrpB, hrpG and phcA in eggplant seedlings. The phcA mutant was found to be only moderately virulence deficient in eggplant seedlings but was significantly reduced in virulence in tomato. This is indicative of some host specific responses towards certain pathogenicity functions of R. solanacearum, which are markedly different in tomato and eggplant seedlings. Apart from being economical in requiring less labor, time and space, this simple gnotobiotic leaf-clip inoculation method is anticipated to be helpful in further exploring the interaction between R. solanacearum and eggplant seedlings at the cotyledon stage.
KeywordsBacterial wilt Virulence Pathogenicity assay Leaf-clip inoculation Eggplant
Days post inoculation
Ralstonia solanacearum causes a lethal bacterial wilt disease in 200 plant species of 53 botanical families including agronomically important crop plants such as tomato, potato, eggplant, olive, banana, peanut, ginger, etc. (Hayward 1991). R. solanacearum is a soil borne bacterium. Under natural conditions, this pathogen infects the host plants through root, colonizes in the xylem vessels and then spreads systemically till causing wilting in its hosts (Genin 2010). Tomato and Arabidopsis plants are mainly used as model hosts to describe its pathogenicity functions at the molecular level (Vasse et al. 1995; Yang and Ho 1998). The pathogen uses an elaborate sensory and regulatory network to regulate its virulence and pathogenicity functions (Schell 2000; Mole et al. 2007).
Though, the pathogen causes the wilt disease in different hosts, its aggressiveness is not identical (Genin 2010; Genin and Denny 2012). There are plants, referred to as distant hosts, where R. solanacearum colonizes but fails to cause any disease symptom (Guidot et al. 2014). Its host range is expanding further with recent findings (Coutinho et al. 2000; Ozaki and Watabe 2009; Jiang et al. 2016; Weibel et al. 2016). Its differential virulence behavior in varied hosts still remains poorly understood. Recent studies of experimental evolution in this bacterium have given crucial insight into the role of transcription regulators in its host adaptation and colonization (Marchetti et al. 2010; Guidot et al. 2014).
Bacterial wilt in eggplant is a common disease in tropical and subtropical regions (Shekhawat et al. 1978; Ramesh 2006; Antony et al. 2015; Sakthivel et al. 2016; Singh et al. 2017). Studies regarding R. solanacearum pathogenicity in eggplant have mainly focused on identifying resistant cultivars against bacterial wilt and understanding the resistance mechanism of eggplant against the pathogen (Artal et al. 2012; Gopalakrishnan et al. 2014; Pensec et al. 2015; Salgon et al. 2017). The well-known soil drenching and stem inoculation methods are used to study R. solanacearum pathogenicity in eggplant (Salgon et al. 2017; Tjou-Tam-Sin et al. 2017). In these approaches, four to five weeks old (3–5 leaves stage) eggplants are inoculated with the pathogen and the inoculated plants remain under surveillance for about 1 month till completion of the pathogenicity assay (Lebeau et al. 2013; Cho et al. 2018). These pathogenicity assays are quite labor intensive and time consuming. In addition, requirement of sufficient space for incubation of large number of inoculated plants in experiments such as screening for resistant cultivars and screening of R. solanacearum mutants poses limitation to these protocols. The other complexity with R. solanacearum infection of soil-grown host plants is the colonization of unwanted bacteria from the soil in plant root and xylem, which may interfere with R. solanacearum pathogenicity assays in the host plants.
Though eggplant is an important vegetable crop, and bacterial wilt is a serious disease of it, there has not been any report describing the virulence functions of R. solanacearum in this host plant. There may be several reasons why eggplant has not been used as a model host for this pathogen. As eggplant is closely related to tomato, it might have been thought that the pathogen behavior towards eggplant will be similar to that of tomato. The other possible reason may be that the pathogenicity assays used till date are more consistent and reproducible in tomato than in eggplant. In this present study we have demonstrated that the leaf-clip inoculation method is an easy and stable method for evaluating the virulence of R. solancearum in eggplant seedlings, which was ever successfully used to test R. solanacearum virulence in tomato seedlings (Kumar et al. 2017). This is an easy and stable method of inoculation in which the juvenile seedlings are maintained in 1.5 or 2.0 mL microfuge tubes. The seedlings are then inoculated with the pathogen by clipping only a part of the cotyledon leaves with a pair of scissors dipped in bacterial suspension. This method of inoculation takes care of the limitations encountered with the inoculation of the soil-grown plants. In this study we have demonstrated that R. solanacearum is more aggressive in eggplant seedlings than in tomato seedlings by the leaf-clip inoculation. Eggplant being a different host, as well as from economical viewpoint, understanding eggplant and R. solanacearum interaction is of significant importance. In addition, pathogenicity study in different hosts would enable researchers to explore novel host specific pathogenicity functions as well as host specific responses towards pathogenicity functions.
R. solanacearum F1C1 causes wilt disease at the cotyledon stage in eggplant seedlings inoculated by the leaf-clip method
The leaf-clip inoculation method is efficient to study the pathogenicity functions of R. solanacearum in eggplant seedlings
Eggplant seedlings are more susceptible to R. solanacearum F1C1 infection than tomato seedlings
To further ascertain that the eggplant seedlings were more susceptible than tomato seedlings to R. solanacearum infection, we inoculated the seedlings with lower concentrations of the pathogen (104 and 105 CFU/mL), respectively. In both the concentrations, F1C1 caused higher death in eggplant seedlings than that of tomato seedlings. The number of dead eggplant seedlings inoculated with a bacterial concentration of 104 CFU/mL was more than that of tomato seedlings inoculated with 105 CFU/mL of the pathogen (Additional file 1: Figure S7).
In this work we have demonstrated that the leaf-clip inoculation is a stable and consistent method to study R. solanacearum pathogenicity in the cotyledon stage of eggplant seedlings. With this inoculation method, several global transcription regulator mutants involved in its pathogenicity viz. hrpG, hrpB and phcA could be differentiated from each other as well as from the wild type strain in regard to their pathogenicity functions in eggplant seedlings. As of now, this is the first study of these R. solanacearum pathogenicity functions in eggplant.
It is pertinent to note that R. solanacearum pathogenicity in host plants is not so simple. Sometimes the bacterium colonizes its hosts without causing wilting symptom (Van der Linden et al. 2013; Guidot et al. 2014; Zuluaga et al. 2015). Therefore, a stable and consistent pathogenicity assay effective in any particular host is an important requirement for host-pathogen interaction study. The limitation may be a reason for the use of only a few model host plants for this pathogen out of a large number of hosts. Though the leaf-clip method is already established in tomato seedlings (Kumar et al. 2017), its applicability in eggplant seedlings is important for studying R. solanacearum pathogenicity functions in this plant. Even though tomato and eggplant are phyllogenetically close, there is significant difference in the germination processes of their seeds: germination of eggplant seeds takes more time than that of tomato seeds (Methods). We found eggplant seedlings to be more susceptible to R. solanacearum infection in comparison to tomato seedlings in terms of the duration of disease progression, the number of seedlings killed after inoculation with the pathogen as well as the with-host growth rate of the pathogen. The leaf-clip inoculation method described here is easy, simple and consistent. In future, it may allow a large scale screening of eggplant specific virulence deficient mutants of R. solanacearum. It may also be helpful for researchers interested in screening large number of disease resistant eggplant cultivars. The cotyledon leaves are short lived in seedlings unlike true leaves. Therefore, this mode of inoculation has the limitation to be recruited only in the cotyledon stage and can’t be done in grown-up stage of eggplant seedlings. Inoculation by clipping the true leaves will be interesting for a future study.
This study revealed non-pathogenic behavior of the hrpB mutant both in eggplant and tomato seedlings. This is in concordance with our earlier findings in tomato seedlings using the same leaf-clip inoculation procedure (Kumar et al. 2017). The type III protein secretion system, whose expression is positively regulated by the HrpB, is fundamental to R. solanacearum pathogenicity, and is therefore important for the pathogenicity in eggplant seedlings too. The hrpG mutant was observed to cause disease in a few seedlings of tomato as well as in eggplant. This result is different from the earlier study in tomato seedlings (Kumar et al. 2017), where the hrpG mutant was non-pathogenic like hrpB. In R. solanacearum GMI1000, hrpG positively regulates the expression of hrpB, as well as several other important virulence functions (Valls et al. 2006). So, it can be predicted that hrpG mutant would be less pathogenic than hrpB mutant. This was indeed found to be true in a recent study that described differential impact of hrpB and hrpG mutants on root growth of Arabidopsis thaliana (Lu et al. 2018). In this regard the disease caused by hrpG mutant in a few seedlings of tomato and eggplant in this study seemed intriguing. It need mention that GMI1000 possesses a homologue of hrpG, known as prhG which regulates the expression of hrpB under special circumstances as well as in the absence of hrpG (Plener et al. 2010). The genome sequence of F1C1 strain also revealed presence of a prhG homologue (unpublished result). It may be possible that prhG homologue contributes differently in the expression of genes in the F1C1 strain even when hrpG is defective. In addition, the hrpG mutant used in this study was observed to elicit a delayed hypersensitive response in tobacco leaves (Additional file 1: Figure S9). Therefore perplexing virulence phenotype of hrpG mutant may be attributed to different aspects such as the strain background, inoculation mode, and type of mutation or any other unknown factors. In future, the transcriptomics of the hrpG mutant will be of significant interest.
Further, difference between eggplant and tomato seedlings with regard to R. solanacearum infection was prominent in the case of phcA mutant. The phcA mutant exhibited virulence deficiency in tomato seedlings observed in this study is in concordance with earlier results (Kumar et al. 2017). But, for the eggplant seedlings, the phcA mutant was observed to be only moderately virulence deficient. PhcA is a known global transcription regulator in R. solanacearum and has been described as the largest regulon of the pathogen, which is involved in the regulation of unusually a large number of genes (~ 30% genes in the genome) including important pathogenicity determinants such as exopolysaccharides, extracellular enzymes, motility and type III secretion system (Perrier et al. 2018). In planta gene expression study in tomato has revealed PhcA as an important regulator for the strategic switch between attachment/spread and growth/virulence in this pathogen (Khokhani et al. 2017). Therefore, its differential virulence behavior in the two hosts indicates that factors associated with PhcA may contribute differently towards the pathogen adaptation inside different hosts. Differential expression of R. solanacearum virulence functions in laboratory and in plant environments is already known (Jacobs et al. 2012; Khokhani et al. 2017; Lowe-Power et al. 2018; Mori et al. 2018; Perrier et al. 2018). The disparity in virulence due to phcA amidst tomato and eggplant seedlings further demonstrates relevance of leaf-clip inoculation procedure for pathogenicity study in eggplant seedlings. In future, in planta gene expression studies in this pathogen with regard to eggplant and tomato seedlings may draw out mechanism of differential virulence of the phcA mutant between the two hosts.
Unlike the leaf-clip inoculation method, disease occurrence in the eggplant seedlings by a recently described root inoculation method (Singh et al. 2018) was observed to be inconsistent and time consuming (Unpublished data). But in those seedlings the leaf-clip inoculation method was efficient to study R. solanacearum pathogenicity (Unpublished data). We believe that optimization of the root inoculation method will be required in future for efficient pathogenicity study. As root inoculation is a natural mode of infection, therefore inoculation by this mode might necessitate a greater physiological tuning between the host and the pathogen for infection to occur (Singh et al. 2018). It is already reported that root entry mechanism of the pathogen is complex (Tran et al. 2016; Lu et al. 2018). However, in the case of the leaf-clip inoculation, the pathogen is directly deposited at the cut end of the leaf and disease symptom appeared in the inoculated leaves soon after pathogenic colonization and growth. Although, it is known that the natural mode of entry of this pathogen is through root regions of its host, looking at the severity of symptoms developed in the cotyledon stage of seedlings by leaf-clip inoculation, entry of R. solanacearum into its host through damaged epiphytic regions such as leaves or by other means in natural environments, can’t be eliminated. We anticipate this study would open new windows of investigations towards issues related to host specific pathogenic functions and responses in the immediate future.
Here in this work, we are reporting for the first time about susceptibility of the cotyledon stage (~ 14 days old) eggplant seedlings towards bacterial wilt pathogen R. solanacearum under gnotobiotic condition. The pathogenicity test conducted via leaf-clip inoculation procedure has indicated it to be an efficient method to study R. solanacearum virulence functions in eggplant seedlings too, as was shown for tomato seedlings earlier (Kumar et al. 2017). Our findings further demonstrate higher susceptibility of eggplant seedlings towards R. solanacearum (F1C1) virulence than tomato seedlings, when the pathogen was inoculated by the same leaf-clip method. We believe that the efficacy of the R. solanacearum leaf-clip inoculation mode in tomato and eggplant seedlings is expected to provide fertile ground for its potential utility in the pathogenicity tests of other hosts in near future. The important virulence regulator, phcA (so far the known largest regulon of R. solanacearum) that controls plethora of pathogenicity functions downstream seems to have distinct roles in tomato and eggplant seedlings. We anticipate, present study will stir more critical investigations on R. solanacearum virulence functions in association with eggplant which would immensely assist in understanding R. solanacearum host specific virulence behavior, in coming days.
Bacterial strains and growth conditions
Bacterial strains used in this study
Ralstonia solanacearum strains
Wild type virulent R. solanacearum strain (Phylotype I), isolated from wilted chili plant collected from a nearby field of Tezpur University, Tezpur, India.
rif-1zxx::Tn5gusA11; Gus + ve, Rifr, Spcr, Vir+, derived after Tn5gusA11 insertion in an unknown locus in the genome
hrpB::Ω; Spcr, HrpB deficient, Vir−, hypersensitive response deficient (HR−), derived from F1C1
Singh et al. 2018
phcA::Ω; Spcr, PhcA deficient, exopolysaccharide deficient (EPS−), hypermotile, derived from F1C1
Singh et al. 2018
Genr, mCherry tagged F1C1
Singh et al. 2018
hrpB::Ω; Spcr, Genr, HrpB deficient, Vir−, HR−, derived from TRS1016
phcA::Ω; Spcr, Genr, PhcA deficient, EPS−, hypermotile, derived from TRS1016
hrpG::pCZ367; Ampr, Genr, HrpG deficient, Vir−, HR−, derived from F1C1
Other bacterial strains
Wild type E. coli
Isolated from tomato seedling
Germination of eggplant seedlings
Eggplant seeds of respective varieties (viz. Devgiri, Devkiran, Param Hybrid) recruited in this study were surface sterilized with 70% ethanol by submerging for 2 min, followed by washing twice with sterile distilled water, then kept on sterile wet tissue paper and incubated for germination inside a growth chamber (Orbitek, Scigenics, India) maintained at 28 °C, 75% relative humidity (RH) with a 12 h photoperiod. The tissue paper bed was kept wet by adding sterile water every day. Germination of eggplant seeds took more than 2 weeks (14–15 days) to reach two leaves cotyledon stage. In the case of tomato, it took only 1 week (6–7 days) (Durga; Ruby variety) to reach two leaves cotyledon stage (Kumar et al. 2017). In this study we referred to the germinated seedlings with only cotyledon leaves (without true leaves) as cotyledon stage seedlings.
Preparation of bacterial inoculum
For inoculum preparation, freshly grown R. solanacearum (F1C1) colonies were transfered to 10 mL BG broth and incubated in a shaking incubator (Orbitek, Scigenics, India) at 28 °C, 150 rpm for 24 h. Cultures were resuspended in sterile distilled water to obtain a bacterial concentration of 109 CFU/mL after centrifugation at 4000 rpm for 10 min at 28 °C (5804R; Eppendorf, Germany). Inoculum of P. putida and E. coli were prepared in a similar way except the growth temperature for E. coli was 37 °C.
Pathogenicity assay by leaf-clip method
Pathogenicity assay in eggplant seedlings was done by the leaf-clip method as described previously for tomato seedlings (Kumar et al. 2017). The leaf-clip inoculation method used to study R. solanacearum pathogenicity in tomato seedlings in our earlier work and here in the eggplant seedlings, was inspired from the work of Kauffman et al. (1973), who studied pathogenicity of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight in rice, in leaves of grown-up host plant. Briefly, 14–15 days old cotyledon stage eggplant seedlings were gently transferred from the germination tray to 1.5 mL microfuge tubes containing 1.0 mL of sterile distilled water (Fig. 1). Then a pair of sterile scissors were dipped in the bacterial suspension (~ 109 CFU/mL or other concentrations required) and ~one-third of both the cotyledon leaves from the tips were clipped off in each eggplant seedling, and 6-7 days old tomato seedlings at cotyledon stage were recruited for inoculation in the same way.
In all the pathogenicity experiments, 40 seedlings were inoculated in a set and each experiment was performed three times independently with two replicates. Seedlings inoculated with sterile distilled water were kept as control in all experiments. Inoculated seedlings along with control were transferred to a growth chamber (Orbitek, Scigenics, India) maintained at 28 °C, 75% RH under 12 h photoperiod and observed for disease progression next day onwards till 10 dpi. Statistical analysis of virulence data were done by Kaplan-Meier survival curve (Kaplan and Meier 1958) and log-rank test.
Eggplant seedlings of three different cultivars namely Devkiran (Bangalore), Param Hybrid (Hyderabad) and Devgiri (Kolkata) were tested for susceptibility to R. solanacearum F1C1 by the leaf-clip inoculation..
Leaf-clip inoculation of non-pathogenic bacteria such as P. putida and E. coli in eggplant seedlings was done also as described above.
Inoculation of eggplant and tomato seedlings within a single microfuge tube
One of the difficulties in R. solanacearum pathogenicity test is to make close comparison between infected susceptible host plants. A close comparison of R. solanacearum F1C1 pathogenicity between eggplant and tomato seedlings was made by keeping the two seedlings in a single microfuge tube. The seedlings were then inoculated with F1C1 and the mutants including hrpB, hrpG and phcA, respectively at concentrations ~ 109 CFU/mL by the leaf-clip method. The disease progression was recorded till 10 dpi.
Inoculation of eggplant seedlings with different concentration of R. solanacearum
To determine the effect of different titers of the pathogen on disease progression, the eggplant seedlings were inoculated with different titers of R. solanacearum F1C1 (~ 109, 107, 105, 104 and 103 CFU/mL). A set of 40 seedlings were used for each dilution inoculation and the experiment was repeated three times independently with two replicates. Inoculated seedlings were analyzed for disease progression till 10 dpi.
Creation of mCherry tagged hrpB and phcA mutant strains of R. solanacearum F1C1 and colonization study in eggplant and tomato seedlings
The transformation protocol used in R. solanacearum F1C1 was the same as described previously (Singh et al. 2018). To create mCherry marked hrpB and phcA mutant of F1C1, genomic DNA of TRS1012 and TRS1013 was used to naturally transform mCherry-marked F1C1, respectively (TRS1016). Both types of transformants were selected on BG agar medium supplemented with gentamycin and spectinomycin. The hrpB mutant was found to be deficient in eliciting hypersensitive response when infiltrated inside the leaves of the Nicotiana tabaccum (Additional file 1: Figure S8).
Inoculum of mCherry labelled hrpB mutant (TRS1017) and phcA mutant (TRS1018) of F1C1 were used for leaf inoculation of eggplant seedlings. After 4 dpi, the infected seedlings were surface sterilized as described previously (Kumar et al. 2017) and were observed for red fluorescence under the fluorescence microscope (EVOS FL, Life technologies) at 4× magnification.
Creation of hrpG insertion mutant of R. solanacearum F1C1
We created hrpG mutant by using the insertional vector pCZ367 (Cunnac et al. 2004) which also results in the lacZ reporter gene fusion. Taking reference sequence of GMI1000, primers were designed for partial amplification of hrpG homologue in F1C1 strain. Forward primer oFhrpG (5′-GCCAAGCTTGCGTACCGAGGCATTCAGTC-3′) incorporated with HindIII restriction site and reverse primer oRhrpG (5′-GCCTCTAGATCTTGCGCAGCTTGTAGATGT-3′) incorporated with XbaI restriction site at their 5′ ends, respectively were used to amplify approximately 500 bp amplicon of hrpG homologue in F1C1. Amplicon was cloned into promoter less, insertional vector pCZ367 and the recombinant hrpG::pCZ367 construct was then naturally transformed into F1C1 following the protocol described earlier (Singh et al. 2018). Its integration into the genome of F1C1 was confirmed by performing PCR with forward primer (5′-GCCAAGCTTTCCAATCCATCCAGCTTCGC-3′) designed upstream of the hrpG cloned fragment and olacR1 (5′-AAGGGGGATGTGCTGCAAGG-3′) designed downstream of the lacZ gene. One of the successful transformants TRS1027 was recruited in the further experiments and was deficient in eliciting hypersensitive response (Additional file 1: Figure S8).
We thank Dr. L. Sahoo (IIT Guwahati) for the kind gift of the tobacco plants to test the HR in this study. We are grateful to Drs. S. Genin (LIPM, France), M. Dickinson (University of Nottingham, UK), Prabhu B Patil (IMTECH, India), Gopaljee Jha (NIPGR) for their helpful comments on the manuscript. We are very much grateful to the two anonymous reviewers and to Dr. Hui Li, the Editor of this journal for their kind comments which helped us to improve the manuscript significantly. TP and KK are thankful to UGC, GoI for the BSR and the NET-JRF fellowships, respectively. RS/BRJ and NS are thankful to the DBT, GoI for the MSc fellowship and BET-JRF/SRF fellowships, respectively. PLS/AB are thankful to DBT UExcel grant for the SRF/RA fellowships. Research in SKR lab is funded by UExcel NER grant, DBT twinning, CEFIPRA, and Departmental project grants such as UGC-SAP (DSR II), DST-FIST.
TP performed and designed the experiments, analyzed the data, wrote the manuscript; KK performed the experiments, analyzed the data, wrote the manuscript; RS performed the experiments; PLS wrote the manuscript; NS wrote the manuscript; AB wrote the manuscript; BRJ performed the experiments; SKR designed the experiments, analyzed and interpreted the data and wrote the manuscript.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
- Artal RB, Gopalkrishnan C, Thippeswamy B. An efficient inoculation method to screen tomato, brinjal and chilli entries for bacterial wilt resistance. Pest Manag Hortic Ecosyst. 2012;18:70–3.Google Scholar
- Boucher CA, Barberis P, Demery DA. Transposon mutagenesis of Pseudomonas solanacearum: isolation of Tn5-induced avirulent mutants. J Gen Microbiol. 1985;131:2449–57.Google Scholar
- Cunnac S, Occhialini A, Barberis P, Boucher C, Genin S. Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system. Mol Microbiol. 2004;53:115–28.CrossRefGoogle Scholar
- Gopalakrishnan C, Singh TH, Artal RB. Evaluation of eggplant accessions for resistance to bacterial wilt caused by Ralstonia solanacearum (E.F. smith) Yabuuchi et al. J Hortic Sci. 2014;9:202–5.Google Scholar
- Kauffman HE, Reddy APK, Hsieh SPY, Merca SD. An improved technique for evaluation of resistance of rice varieties to Xanthomonas oryzae. Plant Dis Rep. 1973;57:537–41.Google Scholar
- Kumar R. Studying virulence functions of Ralstonia solanacearum, the causal agent of bacterial wilt in plants. PhD thesis. India: Tezpur University; 2014. http://hdl.handle.net/10603/48742.
- Ozaki K, Watabe H. Bacterial wilt of geranium and portulaca caused by Ralstonia solanacearum in Japan. Bul Minamikyushu Univ. 2009;39:67–71.Google Scholar
- Perrier A, Barlet X, Peyraud R, Rengel D, Guidot A, Genin S. Comparative transcriptomic studies identify specific expression patterns of virulence factors under the control of the master regulator PhcA in the Ralstonia solanacearum species complex. Microb Pathog. 2018;116:273–8.CrossRefGoogle Scholar
- Ramesh R. Field evaluation of biological control agents for the management of Ralstonia solanacearum in Brinjal. J Mycol Plant Pathol. 2006;36:327–8.Google Scholar
- Shekhawat GS, Singh R, Kishore V. Distribution of bacterial wilt and races and biotypes of the pathogen in India. J Indian Potato Assoc. 1978;5:155–65.Google Scholar
- Singh D, Chaudhary G, Yadav DK. Genetic diversity of Indian isolates of Ralstonia solanacearum causing bacterial wilt of eggplant (Solanum melongena). Indian J Agric Sci. 2017;87:1466–75.Google Scholar
- Weibel J, Tran TM, Bocsanczy AM, Daughtrey M, Norman DJ, et al. A Ralstonia solanacearum strain from Guatemala infects diverse flower crops, including new asymptomatic hosts vinca and sutera, and causes symptoms in geranium, mandevilla vine, and new host african daisy (Osteospermum ecklonis). Plant Health Prog. 2016;17:114–21.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.