ARTICLE

New report of additional enterobacterial species causing wiltin West Bengal, IndiaShamayeeta Sarkar and Sujata Chaudhuri

Abstract: Ralstonia solanacearum is known to be the most prominent causal agent of bacterial wilt worldwide. It has a wide host rangecomprising solanaceous and nonsolanaceous plants. Typical symptoms of the disease are leaf wilt, browning of vascular tissues, andcollapsing of the plant. With the objective of studying the diversity of pathogens causing bacterial wilt in West Bengal, we collectedsamples of diseased symptomatic crops and adjacent symptomatic and asymptomatic weeds from widespread locations in WestBengal. By means of a routine molecular identification test specific to “R. solanacearum species complex”, the majority of these strains(68 out of 71) were found to not be R. solanacearum. Presumptive identification of these isolates with conventional biochemicals,extensive testing of pathogenicity of a subset involving greenhouse trials fulfilling Koch’s postulate test, and scanning electronmicroscopic analysis for the presence of pathogen in diseased plants were done. 16S rDNA sequencing of a subset of these strains(GenBank accession Nos. JX880249–JX880251) and analysis of sequences with the nBLAST programme showed a high similarity(97%–99%) to sequences of the Enterobacteriaceae group available in GenBank. Molecular phylogeny further established the taxonomicposition of the strains. The 3 bacterial strain cultures have been submitted to MTCC, Institute of Microbial Technology, Chandigarh,India, and were identified as Klebsiella oxytoca, Enterobacter cowanii, and Klebsiella oxytoca, respectively. Although Enterobacter sp. haspreviously been reported to cause wilt in many plants, susceptibility of most of the dedicated hosts of R. solanacearum to wilt caused byEnterobacter and other bacteria from Enterobacteriaceae is being reported for the first time in this work.

Key words: pathogenicity, bacteria, molecular identification, diversity, molecular phylogeny.

Résumé : Ralstonia solanacearum est assurément l’un des principaux agents du flétrissement bactérien a l’échelle mondiale. Sa largegamme d’hôtes comprend des plantes de la famille des solanacées et d’autres plantes. Les symptômes caractéristiques de la maladiesont le flétrissement des feuilles, le brunissement des tissus vasculaires et l’effondrement de la plante. Dans l’optique d’étudier ladiversité des pathogènes causant le flétrissement bactérien dans l’ouest du Bengale, on a effectué des prélèvements chez des culturessymptomatiques malades et des mauvaises herbes adjacentes avec et sans symptômes un peu partout dans cette région. Un testcourant de dépistage moléculaire de bactéries du complexe de R. solanacearum a révélé que la majorité des souches (68 sur 71) n’étaientpas R. solanacearum. On a procédé a une identification présomptive de ces isolats par biochimie conventionnelle, a une analyse détailléede la pathogénicité d’un sous-groupe ayant réussi un test de vérification des postulats de Koch d’après des essais en serre, et a uneanalyse par MET recherchant le pathogène dans des plantes affectées. Un séquençage de l’ADNr 16S d’un sous-ensemble de ces souches(numéros d’entrée GenBank JX880249–JX880251) et l’analyse des séquences par le programme nBLAST ont fait ressortir un degré desimilitude élevé (97–99 %) avec les séquences du groupe des Enterobacteriaceae présentes dans GenBank. La phylogénie moléculaire acorroboré la position taxonomique des souches. Les trois cultures de souches bactériennes ont été envoyées au MTCC de l’Institut deTechnologie Microbienne a Chandigarh, Inde, lequel les a identifiées comme étant Klebsiella oxytoca, Enterobacter cowanii et Klebsiellaoxytoca, respectivement. Bien qu’on ait rapporté précédemment qu’Enterobacter sp. causait la flétrissure chez nombre de plantes, cetouvrage expose pour la première fois la susceptibilité de la plupart des hôtes liés a R. solanacearum a la flétrissure causée par Enterobacteret d’autres bactéries de la famille des Enterobacteriaceae. [Traduit par la Rédaction]

Mots-clés : pathogénicité, bactéries, identification moléculaire, diversité, phylogénie moléculaire.

IntroductionVascular wilts are widespread and occur as a result of the infec-

tion and activity of the wilt pathogen within the xylem of the hostplant. Wilt disease is caused by bacteria or fungi. Bacterial wiltsaffect mostly herbaceous plants, such as vegetable crops and or-namental and tropical plants. Visual symptoms of the disease in-clude wilting, browning, and death of leaves and succulent shootsfollowed by death of the entire plant (Buddenhagen and Kelman1964). In India and other regions of the world, bacterial wilt is oneof the devastating diseases. The various causal organisms of bac-terial wilt reported are Ralstonia, Erwinia, Xanthomonas, Pantoea,Clavibacter (Corynebacterium), Curtobacterium, and some species of

Enterobacter. Ralstonia solanacearum has one of the largest knownhost ranges for any pathogenic bacterium (Hayward 1991). In India,crop losses of 50% of potato (Mukherjee and Dasgupta 1989), up to75% of potato in some areas of Karnataka (Gadewar et al. 1991), and70% to 100% of tomato from the North Bengal districts of WestBengal (Kanjilal et al. 2000) have been reported to be caused byR. solanacearum.

In an attempt to screen the diversity of the wilt-causing pathogensin West Bengal, through molecular and biochemical identification,to study their pathogenicity on major hosts, and to determine thephylogenetic position of the strains among wilt-causing bacterialcommunity based on 16S rDNA sequence, we collected samples

Received 12 January 2015. Revision received 27 April 2015. Accepted 27 April 2015.

S. Sarkar. Post Graduate Department of Botany, Ramananda College, Bishnupur, Bankura 722122, West Bengal, India.S. Chaudhuri. Department of Botany, University of Kalyani, Kalyani 741235, West Bengal, India.Corresponding author: Shamayeeta Sarkar (e-mail: shamayeeta.s@gmail.com).

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Can. J. Microbiol. 61: 1–10 (2015) dx.doi.org/10.1139/cjm-2015-0017 Published at www.nrcresearchpress.com/cjm on 1 May 2015.

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Table 1. Origin, symptoms, and colony morphology on TTC (2,3,5-triphenyltetrazolium chloride) medium of bacterial isolates associated withwilt symptoms collected from West Bengal (WB), India.

SampleNo. Strain Host plant Host family Geographical location Symptom Colony morphology

1 Jat1 Jatropha curcas Euphorbiaceae Dumdum, Kolkata,North 24 Parganas, WB

Symptomatic White fluidal colony with pinkishred centre

2 Clero1 Clerodendrum sp. Verbenaceae Raniganj2, Malda, WB Symptomatic Fluidal white colony with brownishred centre

3 Sotu1 Solanum tuberosum Solanaceae Dwarika, Bankura, WB Symptomatic White fluidal colony with pink centre4 SomeMur Solanum melongena Solanaceae Murshidabad, WB Symptomatic Fluidal colonies with dark red centre5 Some3 Solanum melongena Solanaceae Raghunathpur, Purulia, WB Symptomatic Fluidal colonies with dark red centre6 LiesM Lycopersicon esculentum Solanaceae Raniganj2, Malda, WB Symptomatic Pink colony7 CapM Capsicum frutescens Solanaceae Raniganj2, Malda, WB Symptomatic White fluidal colony with pink centre8 Lies8 Lycopersicon esculentum Solanaceae South Dinajpur, WB Symptomatic Fluidal white colony with pink centre9 Euod1 Eupatorium odoratum Compositae Bishnupur, Bankura, WB Asymptomatic White rough colony with pink centre10 Gna1 Gnaphalium sp. Compositae Bhatar, Burdwan, WB Symptomatic Yellowish white rough colony

Fig. 1. Colony morphology of different bacterial strains on TTC (2,3,5-triphenyltetrazolium chloride) medium.

Table 2. Biochemical characteristics of the bacterial isolates.

SampleNo. Strain

Gramtest

Methylred Maltose Lactose Cellobiose Mannitol Sorbitol Dulcitol

Nitratereduction Fermentation

1 Jat1 – – + + + + + + + +2 Clero1 – – + + + + + + + +3 Sotu1 – – + + + + + + + +4 SomeMur – – + + + + + + + +5 Some3 – – + + + + + + + +6 LiesM – – + + + + + + + +7 CapM – – + + + + + + + +8 Lies8 – – + + + + + + + –9 Euod1 – – + + + + + + + +10 Gna1 – – + + + + + + + +

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from symptomatic and asymptomatic hosts in the years 2009–2010 and 2010–2011.

Materials and methods

Bacterial collection and preliminary characterizationOn the basis of information gathered on the occurrence of wilt

disease from the Agricultural Development Offices of the differ-ent districts of West Bengal, samples were collected from one ormore regions from each district over a period of 2 years (2009–

2010, 2010–2011) during the winter period. Since R. solanacearumwas the highest reported wilt causal organism, available data onsusceptible host range and identification of this pathogen wasused as an initial benchmark during the sample collection andlater stages. Crops were collected by observing the wilt symptoms.Surrounding weeds were also taken. The water streaming test wasdone for bacterial oozing to confirm the bacterial wilt. Surfacesterilization of samples (stems) of collected plants was done bydipping in 1% NaOCl for 10 min, followed by several washings with

Fig. 2. Biochemical and other tests for the bacterial strains. (a) Bacterial streaming. (b) Positive KOH test (Gram negative). (c) Sugar utilizationtest (Hayward 1964, 1976). Tubes: 1, control (no inoculation); 2, maltose; 3, lactose; 4, cellobiose; 5, mannitol; 6, sorbitol; 7, dulcitol. In the leftpanel, tubes 2, 3, and 4 show a positive reaction; in the right panel, none of the tubes show a positive reaction. (d) Methyl red test. (e) Nitratereduction test.

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sterilized distilled water. The stems were cut into small pieces andplaced in Petri plates containing Kelman’s triphenyl tetrazoliumchloride (TTC) agar medium and incubated for about 48 h in thedark at 28 °C. Colonies were then selected and assessed in freshTZC plates for purity. The appearance of colonies on TTC mediumwas recorded. This was followed by bacterial streaking of the iso-lated single colonies on casamino acid – peptone – glucose (CPG)slants, since growth of bacteria on selective medium, such as TZC,is known to lower their pathogenicity. The 3% KOH test was doneas a presumptive indication of Gram reaction. Biochemical testslike anaerobic growth test, carbohydrate utilization test, and ni-trate reduction test were also done.

DNA isolation and PCR analysis with R. solanacearum-specificprimers OLI1 and Y2 (Seal et al. 1993)

DNA was isolated using the protocol of Chen and Kuo (1993). A1.5 mL volume of saturated culture was harvested and lysed with20 �L of lysis buffer, 40 mmol/L Tris–acetate, pH 7.8, 20 mmol/L

sodium acetate, 1 mmol/L EDTA, 1% SDS (sodium dodecyl sulfate)by vigorous pipetting; 66 �L of 5 mol/L NaCl solution was thenadded and mixed well. The viscous mixture was then centrifugedat 12 000 r/min for 10 min at 4 °C. To the clear supernatant, anequal volume of chloroform was added and shaken. After centri-fuging at 12 000 r/min for 3 min, the upper layer was carefullycollected into a fresh vial, and DNA was precipitated with 100%

Fig. 3. PCR analysis of bacterial samples using primers OLI1/Y2.(a) Lanes: 1, 100 bp DNA ladder; 2, GMI1000 as positive control; 3, Jat1;4, Clero1; 5, Sotu1; 6, SomeMur; 7, Some3; 8, LiesM; 9, CapM; 10, Euod1;11, Gna1; 12, Lies2; 13, Sovi1; 14, Lies1; 15, LiesDD; 16, Arahy1; 17, GMI1000as positive control; 18, 100 bp DNA ladder. (b) Lanes: 1, 100 bp DNAladder; 2, GMI1000 as positive control; 3, Lies4; 4, Lies6; 5, Lies8.

Table 3. PCR results using primers OLI1 and Y2 specific for Ralstoniasolanacearum.

SampleNo. Strain

288 bpamplicon Inference

1 Jat1 – Not R. solanacearum species complex2 Clero1 – Not R. solanacearum species complex3 Sotu1 – Not R. solanacearum species complex4 SomeMur – Not R. solanacearum species complex5 Some3 – Not R. solanacearum species complex6 LiesM – Not R. solanacearum species complex7 CapM – Not R. solanacearum species complex8 Lies8 + R. solanacearum species complex9 Euod1 – Not R. solanacearum species complex10 Gna1 – Not R. solanacearum species complex

Table 4. Deposit identification at MTCC, Chandigarh, India.

Test Clero1 Gna1 Un4

Morphological testsColony morphology

Configuration Circular Circular CircularMargin Entire Entire EntireElevation Raised Raised RaisedSurface Smooth Smooth SmoothPigment Pale yellow Off-white Off-whiteOpacity Translucent Translucent Translucent

Gram reaction – – –Cell shape Rods Rods RodsArrangement Scattered Scattered ScatteredSpore(s) – – –Motility + – –

Physiological and biochemical testsGrowth on MacConkey + + +Indole – + +Methyl red + – –Voges–Proskauer + + +Citrate utilization + + +H2S production – – –Gas production from glucose + + +Casein hydrolysis – – +Gelatin hydrolysis – – –Starch hydrolysis + – –Nitrate reduction + + +Catalase + + +Oxidase – – –Urea hydrolysis – + +Esculin hydrolysis + + –Arginine dihydrolase – – –Tween 20 hydrolysis – – –Tween 40 hydrolysis – – –Tween 60 hydrolysis – – –Tween 80 hydrolysis – – –Acid production from:

Trehalose + + +Xylose + + +L-Arabinose + + +Lactose + + +Maltose + + +D-Sorbitol + + +

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ethanol. The pellet was washed twice with 70% ethanol, dried, anddissolved in HPLC water. The primers used were OLI1 (5=-GGG-GGTAGCTTGCTACCTGCC-3=) and Y2 (5=-CCCACTGCTGCCTCCCGT-AGGAGT-3=). PCR amplifications were performed using a thermalcycler (Mastercycler Personal, Eppendorf, Germany). The reactionmixture of 50 �L contained 1× PCR buffer (with 1.5 mmol/L MgCl2),0.2 mmol/L (each) dNTP, 1 U of Taq DNA polymerase, 1 �mol/L(each) primers OLI1 and Y2, and 10–20 ng of template DNA. Thereaction was initially heated to 96 °C for 120 s, then cycled35 times through phases of denaturation (94 °C for 20 s), anneal-ing (68 °C for 20 s), and extension (72 °C for 30 s), followed by afinal period of 600 s at 72 °C. From 50 �L of the reaction mixture,15 �L was examined by electrophoresis using 1.5% agarose gel.

16S rDNA sequencing for molecular identification of thenon-R. solanacearum group

A subset of 3 strains from the pathogens that were negative tothe R. solanacearum-specific PCR analysis were chosen for 16S rDNAsequencing. The selection of these 3 pathogens was done based ontheir pathogenicity on brinjal and tomato. The universal primers27f (5=-AGAGTTTGATCMTGGCTCAG-3=) and 1525r (5=-AAGGAGGTG-ATCCAGCC-3=) were used to amplify the 16S rDNA. PCR amplifica-tion was done using 50 �L of reaction mixture containing 1× PCRbuffer (with 1.5 mmol/L MgCl2), 0.2 mmol/L (each) dNTPs, 1 U of TaqDNA polymerase, 1 �mol/L (each) primers 27f and 1525r. The reac-tion cycle was as follows: initial denaturation for 300 s at 95 °C,30 cycles with denaturation (95 °C for 60 s), annealing (54 °C for60 s), and extension (72 °C for 60 s). This was followed by a finalextension at 72 °C for 600 s.

The �1.5 kb PCR products of a subset of the bacterial isolateswere sent to Genomics Services Xcelris Labs Ltd., Ahmedabad, India,where they were sequenced using the Sanger sequencing method.Analyses of sequences for bacterial identification were performed

using the basic sequence alignment nBLAST program run againstthe nucleotide database GenBank (http://www.ncbi.nlm.nih.gov/blast). The sequences were submitted to the GenBank for obtain-ing accession numbers.

Table 5. BLAST analysis data of sequenced samples.

SampleNo. Strain

Host plant (sourceof the pathogen)

Genome regionsequenced

GenBankacc. No.

Sequencesize (bases) Sequence identity with:

Nucleotidesequence identity %

1 Jat1 Jatropha curcas 16S ribosomal RNA gene,partial sequence

JX880249 727 Klebsiella pneumoniae 99Enterobacter cancerogenus 99Enterobacter sp. 99

2 Clero1 Clerodendrum sp. 16S ribosomal RNA gene,partial sequence

JX880250 1472 Escherichia hermannii 99Enterobacter sp. 99Enterobacter cowanii 99Enterobacter cloacae 99

3 Gna1 Gnaphalium sp. 16S ribosomal RNA gene,partial sequence

JX880251 1000 Klebsiella oxytoca 97Klebsiella sp. 97

Fig. 4. The phylogenetic tree compiled from 16S rRNA gene sequence data inferred using the neighbour-joining method from the Clustal Wprogramme of MEGA software version 5. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionarydistances used to infer the tree. Tamura and Nei parameter of substitution model has been applied. All positions containing gaps and missingdata has been eliminated. Bootstrap analyses (1000 replicates) for node values from 50% are indicated.

Table 6. Percentage of disease incidence on tomato andbrinjal plants due to pathogens over a period of 28 days andtheir significance values deduced by DMRT (� = 0.05).

Strain Day 7 Day 14 Day 21 Day 28

TomatoJat1 19b 40.1ab 66.16cd 75dClero1 23.6a 33.33c 77a 87bSotu1 15.6c 43.3a 62.3de 70eSomeMur 11.3d 25e 49.66g 58gSome3 17.7bc 31.67cd 67cd 70eLiesM 18.67b 35c 64.6cd 75.67dCapM 8.3e 22.3e 58.3ef 64fLies8 15c 35.66bc 68.33bc 97aEuod1 10de 27de 56.7f 68.3defGna1 15.67c 41.6a 72.7ab 81.33c

BrinjalJat1 16.23c 28.9c 51.31d 64.4dClero1 23.3b 52.38a 57.14c 71.43cSotu1 10.78d 22.3e 44.9e 50eSomeMur 11.14d 26.22d 29.5f 35.5fSome3 16.37c 26.95cd 61.21b 80.5bLiesM 11d 20.8e 49.21d 65.2dCapM 15.01c 20.43e 44.9e 50eLies8 25.2a 45.66b 63.13b 90.1aEuod1 11.5d 12f 18.79g 38.5fGna1 22.74b 45.27b 68.23a 79.94b

Note: Means with the same letters in the same column are notsignificantly different.

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Phylogenetic analysisA phylogenetic distance tree was compiled from 16S rDNA se-

quence data using MEGA5 Software (Tamura et al. 2011).The sequences were aligned using the program MUSCLE, and

phylogenetic analysis was done through Clustal W where theneighbour-joining distance method was used with the Tamuraand Nei model of substitution. The significance of branching wasevaluated by bootstrap analysis with 1000 replicates. Bootstrapnode values >50% are indicated in the tree.

Pathogenicity and hypersensitivity testsA greenhouse test for pathogenicity was done on 5 known

host plants: potato (Solanum tuberosum ‘Kufri chandramukhi’),tomato (Lycopersicon esculentum ‘Patharkuchi’), brinjal (Solanummelongena var. esculentum), chilli (Capsicum frutescens ‘Dhani’),and tobacco (Nicotiana tabacum ‘White burley’). Three replicasets were used for each pathogen. The pots were arranged in arandomized block design pattern in the greenhouse. A controlset of uninoculated plants was also maintained. The inoculatedplants were observed regularly and disease severity was assessedas follows:

% Disease incidence� (Number of leaves showing wilt symptoms

÷ Total number of leaves) × 100

The percentage of disease incidence was calculated for each setover a period of 28 days at 7-day intervals. From the convertedarcsine values of disease incidence, area under disease progresscurve (AUDPC) values were calculated for each treatment. Statis-tical significance of the percentage of disease incidence betweenthe most effective treatments on same host was calculated atP = 0.05. Furthermore, Koch’s postulate was confirmed by re-isolation of the bacterium from symptomatic tissues.

A hypersensitivity test on tobacco leaves was done with each bac-terial isolate. The bacterial suspension was prepared and adjusted toa concentration of about 1 × 108 colony-forming units (cfu)/mL. Oneside of completely expanded tobacco leaves was injected with 1.0 mLof bacterial suspension. The leaves were observed daily for 4 daysafter inoculation for hypersensitivity reaction.

SEM (scanning electron microscope) study of bacteria in vivoThe infected plants were uprooted, surface sterilized, cut into

thin sections using a blade, and treated with fixatives. A healthy

Fig. 5. Disease symptoms under greenhouse conditions. The plants are 40 days old. Inoculation was done at the 3-leaf stage. (a) Controland diseased tomato (Lycopersicon esculentum ‘Patharkuchi’) plants: uninoculated (left plant) and inoculated with strain Clero1 (rightplant). (b) Symptoms in close view: wilting and chlorosis (left plant), a wilted branch (middle plant), wilting of the apical part of theplant (right plant).

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plant was used as a control. After treatment with glutaraldehyde–cacodylate buffer, the sections were dehydrated by passing throughan increasing gradation of different ethanol concentrations. Theywere air-dried, and gold coating of the air-dried sections was doneusing an IB2 Ion Coater. SEM photographs were taken with a S530Hitachi Scanning Electron Microscope, Japan.

Results

Collection and culture of bacterial isolatesThe collection was done on the basis of known visible symp-

toms. Most of the strains were isolated from symptomatic plants,while a few were collected from asymptomatic weeds likeAmaranthus spinosus growing in the vicinity of infected symptom-atic crop plants. Some of the weed hosts like Clerodendrum sp. andJatropha curcas showed notable symptoms (Table 1).

While all the isolates formed creamy white colonies in CPGmedium, variable colony morphologies were observed when grownon TTC medium at 28 °C for 48 h. Three distinct types of colonieswere observed, viz., white fluidal colonies with pink centre, darkred colonies, fluidal colonies with dark red centre (Table 1, Fig. 1).All the isolates were tolerant to 0.5% TTC and could reduce it towater-soluble formazan.

Biochemical characteristics of the bacteriaBiochemical studies of all the isolates showed a wide range of

variation, especially in the utilization of sugars and sugar alco-hols. All the bacterial strains were Gram negative and facultativeanaerobes. Out of all the bacterial isolates studied, more than 50%of all the isolates showed a similar pattern, i.e., were methyl rednegative, oxidized all sugars (maltose, lactose, and cellobiose),utilized all sugar alcohols (mannitol, sorbitol, dulcitol), and re-duced nitrates (Table 2, Fig. 2).

Molecular identification of pathogensUsing the R. solanacearum-specific primers OLI1 and Y2, PCR ampli-

fication yielded the expected amplicons from the R. solanacearumGMI1000 control and 3 of the collected isolates (Lies4, Lies6, andLies8) (Fig. 3b). These 3 strains were confirmed as R. solanacearum.However, the majority (68) of the isolates did not yield the desiredamplicon, indicating that they were not R. solanacearum (Fig. 3a).The PCR results obtained for a selected subset are shown inTable 3. The experiment was repeated several times to negateerrors during PCR.

Partial 16S rDNA sequencing of 3 virulent isolates (Jat1, Clero1,and Gna1) was done from the non-R. solanacearum subset. These3 strains were chosen for molecular identification on the basisof their performance in the pathogenicity test in the green-house. The sequences were submitted to GenBank (accessionNos. JX880249, JX880250, and JX880251). The sequences of the16S rDNA fragment were analyzed by NCBI BLAST and found toshow 97%–99% similarity to the Enterobacteriaceae group. The bac-terial cultures were sent to MTCC, Chandigarh, where they wereidentified on the basis of phenotypic characterization followingmorphological and biochemical features (Table 4). The samplesJat1 (MTCC Un4) and Gna1 (MTCC gna1) were identified as Klebsiellaoxytoca, and Clero1 (MTCC Clero1) was identified to be Enterobactercowanii. This was nearly in congruence to the BLAST results(Table 5).

Phylogenetic cluster analysis of the 3 sequenced strains wasdone using relevant phytopathogenic strains (Hauben et al. 1998;Wang et al. 2010) to see whether the samples cluster in the waythat would be expected from the present view about the taxon-omy of the isolates (Fig. 4). Since previous reports of K. oxytoca as aphytopathogen were not available, some environmental strains ofthe genus were obtained from GenBank (Liao et al. 2014). In thephylogenetic tree compiled using the neighbor-joining methodand Tamura and Nei correction, the bootstrap nodal valuesgreater than 50% are shown. It was observed that our strains clus-

tered with the other enterobacterial strains used in the study.Pseudomonas syringae pv. mori and R. solanacearum appeared as dis-tant outgroups.

Strain Clero1 (JX880250) showed 99% identity with Enterobactercowanii, indicating that the strain belonged to this species. Whilestrain Gna1 (JX880251) clustered with K. oxytoca with 97% identityand 99% query cover, strain Jat1 (JX880249) showed 97% identitywith 50% query cover, thus rendering a different cluster. HenceGna1 and Jat1 are pathogenic strains of Klebsiella but might besome other species, as neither gave an identity greater than 97%with K. oxytoca (Stephan et al. 2007).

Pathogenicity testsAmong all the bacterial strains used, R. solanacearum Lies8 was

found to be more virulent, with the highest disease incidence overa period of 28 days. This strain had previously been isolated fromsymptomatic Lycopersicon esculentum isolated from South Dinajpurdistrict of West Bengal. Most of the non-R. solanacearum strainsshowed high pathogenicity when inoculated in tomato (Lycopersi-con esculentum ‘Patharkuchi’). Almost all plants died after 30 daysof inoculation. Disease incidence was moderate to high in brinjaland potato. It was mostly low in chilli. However, bacterial isolatescollected from wilted chilli plants in fields were moderate tohighly pathogenic on chilli when observed in greenhouse experi-ments. The results of pathogenicity tests in tomato and brinjal bya selected pathogen subset are shown in Table 6.

Wilting was accompanied by discoloration of leaves and defolia-tion (Fig. 5). Defoliation was maximum in potato (Solanum tuberosum‘Kufri chandramukhi’) and brinjal (Solanum melongena var. esculentum).In chilli, the symptoms varied among the strains. While somestrains caused wilting in the apical part of the plant, strains likeClero1 did not show such localization. Defoliation was either ac-companied by wilt or occurred before wilting of leaves.

In general, tomato was observed to be the most susceptiblehost with maximum disease incidence. From the AUDPC values(Table 7), it was observed that among the bacterial strains used inthe pathogenicity study, Clero1, Gna1, and Jat1 had high AUDPCvalue in most of the hosts used in the greenhouse, viz., tomato,brinjal, and potato. Besides, these 3 strains were significantly dif-ferent in terms of percentage of disease incidence observed overthe maximum period of 28 days. Thus, they could be distinguished

Table 7. Arcsine values and area under disease progress curve(AUDPC) values obtained for the non-Ralstonia solanacearum bacterialsubset, in tomato and brinjal.

Strain Day 7 Day 14 Day 21 Day 28AUDPCvalue

TomatoJat1 0.19 0.41 0.72 0.85 11.59Clero1 0.24 0.34 0.88 1.06 13.05Sotu1 0.16 0.45 0.68 0.77 11.09SomeMur 0.11 0.25 0.52 0.62 7.97Some3 0.18 0.32 0.74 0.78 10.77LiesM 0.19 0.36 0.7 0.86 11.07CapM 0.08 0.23 0.62 0.69 8.67Euod1 0.1 0.27 0.6 0.75 9.1Gna1 0.16 0.43 0.82 0.92 12.49

BrinjalJat1 0.16 0.29 0.54 0.7 8.84Clero1 0.24 0.55 0.61 0.8 11.72Sotu1 0.11 0.22 0.47 0.52 7.04SomeMur 0.11 0.27 0.3 0.36 5.61Some3 0.16 0.27 0.66 0.94 10.37LiesM 0.11 0.21 0.51 0.71 7.94CapM 0.15 0.21 0.47 0.52 7.06Euod1 0.12 0.12 0.19 0.4 3.95Gna1 0.23 0.47 0.75 0.93 12.59

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as representatives of pathogenicity groups. These strains had beenoriginally isolated from symptomatic weeds, Clerodendrum sp.,Gnaphalium sp., and Jatropha curcas, from the districts Malda,Burdwan, and North 24 Parganas, respectively, of West Bengal.None of the strains caused wilting in tobacco (N. tabacum ‘Whiteburley’) even with repeated pathogen inoculation. Similarly,when a bacterial suspension (at a concentration of 1 × 108 cfu/mL)was injected into the tobacco leaf base, observation for 4 daysshowed zero to very low hypersensitivity response in the experi-ment.

SEM studyA distinctly visible difference was observed from the SEM im-

ages. While the healthy plants showed clear vessels with intactboundaries, those with severe symptoms had bacterial mass over-flowing from obstructed vessels. The boundary walls of the vesselsalso collapsed to varied degrees. Blockage of the vessels by bacte-ria was discernible (Fig. 6).

DiscussionThe incidence of bacterial wilt in a wide variety of crops and

weeds and its occurrence throughout the year in West Bengal hasbeen reported in recent years (Mondal et al. 2011, 2014). Theseworks further claim R. solanacearum to be the causal organism, onthe basis of the ooze test, isolation in modified selective medium,morphological and biochemical studies, and cross-inoculationstudy by stem injection and root inoculation method (Kelman1953, 1954; Hayward 1964; Palleroni 1984). However, as found inthis work, often morphological and physiological parameters alonecannot authenticate the causal organism to be R. solanacearum.

Several other cases have been reported all over the world,where molecular diagnosis has actually helped in identifyingpathogens that have either appeared newly or were present butmisdiagnosed.

One such recent addition is in mulberry, which was reported tobe infected by a pathogen from Enterobacter. Mulberry (Morus alba)is known to be a potential host of R. solanacearum phylotype

Fig. 6. Scanning electron micrographs of transverse sections of healthy and diseased tomato. (a) Xylem vessels in healthy tomato. (b) Diseasedtomato stems 30 days after inoculation with Clero1: panels A and B, bacterial mass overflowing from obstructed vessels; panels C and D,blockage of the vessels and wall collapse.

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(Mathew et al. 1993), which causes manifestation of wilt disease inthis plant. Studies done in China, on the basis of Biolog metabolicprofiles, fatty acid methyl ester analysis, and sequence analysis ofthe partial 16S rDNA and rpoB genes, reported a novel species ofpathogen and named it as Enterobacter mori. It was also observedthat another known species called Enterobacter asburiae also causedmulberry wilt disease (Wang et al. 2008). Similarly, in Zingiber-aceae family, Curcuma alismatifolia (pathumma), an ornamentalplant with a worldwide market, was reported to be susceptible tobacterial wilt caused by Enterobacter sp. (Promsai et al. 2012).

In the present study, a R. solanacearum-specific PCR test hasshown that wilt-causing bacterial strains in a wide range of so-lanaceaous and nonsolanaceaous plants isolated from West Ben-gal consisted predominantly of strains belonging to the non-“R. solanacearum species complex”. However, there was a lot of sim-ilarity between these pathogenic strains and the “R. solanacearumspecies complex”, especially the overlapping host range, growthconditions in laboratory, colony morphology on TZC medium(Kelman 1954), and disease symptoms. A molecular method ofidentification at the generic level was hence applied for this un-identified group of wilt-causing bacteria. 16S rRNA gene sequenc-ing is the most widely used (Stackebrandt and Goebel 1994)molecular method in bacterial identification. Partial 16S rDNAsequencing followed by BLAST result of the subset samples(GenBank accession Nos. JX880249, JX880250, and JX880251) usingthe GenBank database showed 97%–99% similarity to enterobacte-rial members Klebsiella and Enterobacter. The phylogenetic dendro-gram of these strains along with close taxons showed that theycluster together with enterobacterial strains. While Pantoeastewartii resides as a separate cluster, the different species ofEnterobacter and Klebsiella are scattered into different subclusterswithin the same cluster. In a separate work by Hauben et al. (1998),done using almost complete 16S rDNA sequences of phytopatho-genic strains of Enterobacteriaceae, 3 phytopathogenic species ofthe genus Enterobacter were found to be scattered among the gen-era Citrobacter and Klebsiella in the dendrogram.

A parallel characterization was done by MTCC, Chandigarh,where the cultures were submitted for preservation and who onthe basis of phenotypic characters identified strains Jat1 (MTCCUn4) and Gna1 (MTCC Gna1) as K. oxytoca and strain Clero1 (MTCCClero1) as Enterobacter cowanii. These results complement the in-ference obtained from molecular identification up to the genericlevel. While Enterobacter cowanii has been reported to be a phyto-pathogen causing bacterial blight in Eucalyptus (Brady et al. 2009),K. oxytoca has no report till now of pathogenicity in plants.

A subsequent determination of the pathogenic potential ofthe enterobacterial strains in greenhouse experiments hadbeen done by inoculating the pathogens in potted plants, viz.,tomato, potato, brinjal, chilli, and tobacco. Minimally less se-vere than R. solanacearum (Table 6), most of the strains were highlypathogenic on tomato, irrespective of the initial host from wherethey were isolated and showed moderate to high severity on brin-jal and potato. Chilli was least affected. The isolates obtained fromsymptomatic and asymptomatic weeds retained high virulencewhen inoculated in the solanaceous greenhouse plants. This showsthat there exists a trend of survival of the pathogens in weedsthroughout the year even after the crops are harvested withoutloss in virulence.

The biochemical test profile in this experiment has shown someheterogeneity among the non-R. solanacearum enterobacterialstrains. All these bacterial strains were Gram-negative rods andfacultative anaerobes. Most of the isolates were methyl red nega-tive (55 out of the 68 strains), could reduce nitrate (61 out of68 strains), and hydrolysed cysteine (61 out of the 68 strains). Ofthe isolates, 50% oxidized all sugars (maltose, lactose, and cellobi-ose) and utilized all sugar alcohols (mannitol, sorbitol, and dulci-tol).

True wilt bacteria, according to Buddenhagen and Kelman(1964), primarily invade the xylem tracheary elements and affectwater transport in the host. SEM studies of severely infected to-mato plants from greenhouse inoculation with strain Clero1 haveconfirmed that the wilting was caused by blockage of the xylemvessels by bacterial mass. There was also notable collapsing of thevessel wall that may be due to degradation by enzymes or toxineffect.

From the results of this article, it can be concluded thatR. solanacearum is not the principal wilt-causing pathogen in WestBengal but rather members from Enterobacteriaceae, some ofwhich are newly reported as phytopathogens cause similar symp-toms. Identification of these pathogens has been made possiblemainly using molecular diagnosis methods. However, the use of16S rRNA gene sequence alone for identification has been ques-tioned especially in the Enterobacteriaceae (Dahllöf et al. 2000) dueto various reasons, including multiple heterogeneous copies ofthe 16S rRNA gene within a genome (Crosby and Criddle 2003;Case et al. 2007). The use of alternative core housekeeping genes,such as the RNA polymerase subunit gene (rpoB) (Zhu et al. 2011;Yabuchhi et al. 1996), is hence suggested for further identification ofthese isolates.

AcknowledgementsThe authors thank Sanjoy Guha Roy (Department of Botany,

West Bengal State University, West Bengal) and Prasenjit Majumder(Dhirubhai Ambani Institute of Information and CommunicationTechnology, Gujarat) for their valuable suggestions. They also ac-knowledge the support of DST–FIST and DST–PURSE.

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