electron microscopy and molecular characterization of phytoplasmas associated with little leaf...

Department of Physiology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki-305, Japan1

Electron Microscopy and Molecular Characterization of Phytoplasmas Associatedwith Little Leaf Disease of Brinjal (Solanum melongena L.) and Periwinkle(Catharanthus roseus) in Bangladesh

A. B. M. SIDDIQUEIDDIQUE1*, G. K. AGRAWALGRAWAL

2*, N. ALAMLAM3 and M. KRISHNARISHNA REDDYEDDY

4

Authors' addresses: 1,3Department of Botany, Jahangirnagar University, Savar, Bangladesh; 2Department of MolecularGenetics, National Institute of Agrobiological Resources, Tsukuba, Ibaraki-395, Japan; 4Division of Plant Pathology, IndianInstitute of Horticultural Research, Hessaraghatta Lake PO, Bangalore, 560 089, India (correspondence to: A. B. M. Siddique,E-mail: [email protected])

With 4 ®gures

Received February 21, 2000; accepted October 12, 2000

Keywords: Bangladesh, brinjal, electron microscopy, periwinkle, phytoplasma

AbstractIn Bangladesh little leaf disease was observed in brinjal(Solanum melongena L.) and in periwinkle (Catharan-thus roseus). Phloem-inhabiting phytoplasmas2 wereconsistently detected in both species of diseased plantsusing transmission electron microscopy (TEM) andpolymerase chain reaction (PCR) techniques. Theshape, size and within-tissue distribution of phytoplas-mas appears to be similar in both hosts. Furthermore,the molecular characterization and identi®cations ofobserved phytoplasmas were carried out based onrestriction fragment length polymorphism (RFLP)patterns of PCR-ampli®ed products (1200 bp) usingphytoplasma-speci®c universal primers and sequencinganalysis of both 16S ribosomal DNA (rDNA) andintergenic spacer region (ISR) of 16S-23S rDNAphytoplasma genes. The patterns of RFLP analysiswith seven restriction enzymes exhibited a similarpattern for both phytoplasma strains. The sequencehomology between these two strains showed 100%similarity based on 16S rDNA and 16S-23S ISR.Therefore, in Bangladesh the causal agents of brinjallittle leaf (BLL-Bd) and periwinkle little leaf (PLL-Bd)are probably the same or closely related phytoplasmastrains. These strains, are very close or identical to thestrain of brinjal little leaf phytoplasma in India (BLL-In), belonging to the clover proliferation group (Leeet al., Int. J. Syst. Bacteriol. 48, 1153±1169, 1998;Seemuller et al., J. Plant Pathol. 80, 3±26, 1998).

ZusammenfassungElektronenmikroskopische und molekulare Charakterisierung

von Phytoplasmen, die mit der KleinblaÈ ttrigkeit von Aubergine

(Solanum melongena L.) und Catharanthus roseus in Bangla-

desch assoziiert sind

In Bangladesch wurde KleinblaÈ ttrigkeit bei Auberginen(Solanum melongena L.) und Catharanthus roseus beo-bachtet. Bei erkrankten P¯anzen beider Arten wurdenmit transmissionselektronenmikroskopischen Unter-suchungen (TEM) und PCR-Techniken das Phloembesiedelnde Phytoplasmen festgestellt. Die Form undGroÈ ûe der Phytoplasmen sowie ihre Verteilung imGewebe waren bei beiden Wirtsp¯anzen gleich. Diemolekulare Charakterisierung und Identi®zierung derbeobachteten Phytoplasmen erfolgte auf Grundlage vonRFLP-Mustern PCR-ampli®zierter Produkte (1200 bp)unter Verwendung phytoplasmaspezi®scher universellerPrimer und Sequenzanalysen der 16S-ribosomalen DNA(rDNA) sowie der intergenischen Spacerregion (ISR)der phytoplasmatischen 16S-23S-rDNA-Gene. DieMuster der mit 7 Restriktionsenzymen durchgefuÈ hrtenRFLP-Analyse waren bei beiden PhytoplasmastaÈ mmengleich. Die Sequenzen beider StaÈ mme waren zu 100%homolog, basierend auf 16S-rDNA und 16S-23S-ISR.In Bangladesch handelt es sich bei den Erregern derKleinblaÈ ttrigkeit von Aubergine (BLL-Bd) und C. roseus(PLL-Bd) demnach um nur einen Phytoplasmastammoder um nahe miteinander verwandte StaÈ mme. DieseStaÈ mme sind mit dem indischen Erreger derKleinblaÈ ttrigkeit der Aubergine (BLL-In) sehr naheverwandt oder identisch; letzterer gehoÈ rt der Clover-Proliferation-Gruppe an (Lee et al., Int. J. Syst. Bacte-riol. 48, 1153±1169, 1998; Seemuller et al., J. PlantPathol. 80, 3±26, 1998).

*These authors contributed equally to this paper.

J. Phytopathology 149, 237±244 (2001)Ó 2001 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-1785

U.S. Copyright Clearance Centre Code Statement: 0931±1785/2001/4905±0237 $ 15.00/0 www.blackwell.de/synergy

IntroductionThe term phytoplasma refers to a group of mycoplasma-like organisms that are associated with plant diseases ofseveral hundred species including fruits, vegetables,cereals, trees and legumes (McCoy et al., 1989; Daviset al., 1997; Seemuller et al., 1998). The most commonsymptoms of this disease are little leaf, stunting growth,yellows, bushy structure, axillary bud proliferation,phyllody, big bud and so on. Numerous studies havebeen published on phytoplasma disease from NorthAmerica, South America, Europe, Australia, NewZealand, Africa and some parts of Asia. This providesvaluable information on distribution and genetic diver-sity of the pathogens in relation to their geographicallocation. Irrespective of host speci®city, the di�erentstrains of phytoplasmas are known to be associated withdi�erent plant species. A number of examples are knownwhere multiple distinct phytoplasmas are associatedwith a particular disease in di�erent hosts (Davis et al.,1993; Alma et al., 1996). Likewise, a particular speciesof phytoplasma can also causes distinct diseases(Liefting et al., 1998). Sometimes a single host plantgets infected doubly or multiple with di�erent strains ofphytoplasmas (Bianco et al., 1993; Prince et al., 1993;Lee et al., 1994).

Phytoplasmas have yet to be cultured in vitro, andlittle is known about their pathogenicity or transmis-sion. The inability to culture phytoplasmas in vitro, andtheir variable titre levels and uneven distribution canmake detection di�cult. The in vitro ampli®cation ofphytoplasma DNA by the polymerase chain reaction(PCR) has proved to be a sensitive technique fordetection of phytoplasma infections in plants. A recentapproach to phytoplasma detection and classi®cationhas been the analysis of highly conserved genes codingfor the 16S rRNA, and the spacer region between 16Sand 23S rRNA (Lee et al., 1993, 1998). By using primerpairs based on the 16S rRNA gene, selective ampli®-cation of phytoplasma DNA from mixtures with hostDNA has been achieved (Deng and Hiruki, 1991; Leeet al., 1993). More recently, nested PCR employingcombinations of rDNA primer pairs have enhanceddetection of phytoplasmas (Lee et al., 1995). Restrictionsite analysis or sequencing of rDNA products hasprovided an important means of identifying and char-acterizing phytoplasmas for taxonomic purposes(Seemuller et al., 1994; Gundersen et al., 1994; Kirk-patrick et al., 1994; Lee et al., 1998).

Diseases possibly due to phytoplasma in Bangladeshhave not been investigated in detail in terms of theiraetiology and molecular characterization of the patho-gens. To our knowledge, based on the symptomatologya few plant species have been recorded as phytoplasma-hosts without further con®rmation of the causal agent.The non-culturable nature of phytoplasma unlike otherplant pathogens and the limited access to moderntechnology such as PCR might be the key barriers tothe study of this disease in Bangladesh and also in otherdeveloping countries.

Recently plant samples of brinjal (Solanum melongenaL.) a vegetable plant, and periwinkle (Catharanthusroseus) an ornamental plant, that displayed little leafsymptoms resembling those of phytoplasma diseaseswere collected from Bangladesh. These phytoplasmainfections have not been identi®ed and never examinedfor their taxonomic position. The present investigationswere undertaken to characterize, classify and comparethe genetic relatedness of the associated phytoplasmaswith those of other reported phytoplasmas using elec-tron microscopy and molecular methods of restrictionfragment length polymorphism (RFLP) analysis andgene sequencing of both 16S rRNA and 16S-23S ISR.This is the ®rst study of naturally infected phytoplasmadiseases of brinjal and periwinkle in Bangladesh.

Materials and MethodsPlant material

Both symptomatic and asymptomatic plant samples ofbrinjal and periwinkle were collected from Bangladeshin mid-February of 1999. The diseased brinjal werefound in a farmer's ®eld in Gajipure, about 80 km northof Dhaka city and the diseased periwinkle was observedin Jahangirnagar University campus, 30 km north-westof Dhaka city. For microscopy the collected leafmaterials were preserved in 4.0% formaldehyde andfor the PCR studies the collected materials were store at)20°C in a small polybag until further processing.

Transmission electron microscopy

Before processing the tissues (leaf mid rib) for trans-mission electron microscopy (TEM), as described bySiddique et al. (1998), the collected leaf materials werewashed with 50 mMM phosphate bu�er (pH 7.0) forseveral times to remove formaldehyde. Small pieces oftissues cut from leaf mid rib, ®xed over-night in a®xative, washed with bu�er, post-®xed in 1% osmium,dehydrated in ethanol series, in®ltrated and embeddedwith Spurr's resin (Spurr, 1969). Thin sections includingvascular region were cut from the tissue block, post-stained with uranyl acetate and lead citrate beforeobserving under TEM at 75 kb volt. (Hitachi H-7100,Hitachi Ltd., Tokyo, Japan).

DNA extraction

DNA was extracted from the leaves of symptomatic andasymptomatic healthy plants using the Qiagen DNAisolation kit (Qiagen, Tokyo,4 Japan). Before extraction,the material (about 1 g of each) was washed with sterilewater several times and then powdered in liquid nitrogen.The quality and quantity of DNA was checked on 1%(w/v) agarose gel in 1 ´ TAE5 bu�er. DNA bands werevisualized on an UV transilluminator after ethidiumbromide staining.

PCR ampli®cation

Extracted DNA was diluted in sterile milliQ water toobtain a ®nal concentration of 10 ng/ll. The speci®c anduniversal primers used in this study for phytoplasmalDNA ampli®cation are mentioned below. Both non-

238 SIDDIQUEIDDIQUE et al.

nested and nested PCR were conducted. In non-nestedPCR, genomic DNA was used as a template withuniversal primer pairs P4/P7 and P1/P6. In nested PCR,non-nested diluted (1 : 20 in sterile milliQ) PCR productof P1/P6 (about 1500 bp) was used as a template DNAwith speci®c primer pairs R16F2n/R16R2. For bothnon-nested and nested PCR the ampli®cations wereperformed in 50 ll ®nal reaction mixture each contain-ing 50 ng DNA template, 0.4 lMM of each primer, 200 lMM

of dNTP, 1.0 U of Ex Taq polimerase (TaKaRa Co.,Osaka,6 Japan) and 5% DMSO6;7 (Dimethyl Sulfoxide).After 5 min of initial annealing at 97°C the reactionmixtures were subjected to 30 cycles at followingparameters: 45 s of denaturation at 95°C, 45 s ofannealing at 65°C (53°C for R16F2n/R16R2 and P4/P7), 3 min of extension at 72°C. After completion of 30cycles, ampli®cation was accomplished with a ®nalincubation step at 72°C for 5 min. Reaction mixturecontaining template DNA from healthy plants, knownphytoplasma-infected hydrangea plant or sterile deion-ized water substitute for template DNA, served ascontrols in each experiment. The PCR products (5 ll)were electrophoresed on a 1% agarose (1.5% for P4/P7)followed by ethidium bromide staining, visualization ofthe DNA bands under UV transilluminator and photo-graphy. The oligonucleotide sequences of the primersused in this work were P1: 5¢-AAG AGT TTG ATCCTG GCT CAG GAT-3¢ (Deng and Hiruki, 1991), P4:5¢-GAA GTC TGC AAC TCG ACT TC-3¢ (Kirkpa-trick et al., 1994), P6: 5¢-CGG TAG GGA TAC CTTGTT ACG ACT TA-3¢ (Deng and Hiruki, 1991), P7:5¢-CGT CCT TCA TCG GCT CTT-3¢ (Smart et al.,1996), R16F2n: 5¢-GAA ACG ACT GCT AAG ACTGG-3¢ (Lee et al., 1993), R16R2: 5¢-TGA CGG GCGGTG TGT ACA AAC CCC G-3¢ (Lee et al., 1993),SAGUN-1: 5¢-GGG CGA GCG TTA TCC GGA ATTATT G-3¢ (This study).

RFLP analysis of PCR products

R16F2n/R16R2 primed 6.0 ll nested PCR product(about 1200 bp) was digested separately with AluI,EcoRI, HincII, HindIII, HinfI, KpnI, and Sau3AIrestriction enzymes according to the manufacturers'instructions. The restriction enzymes were purchasedfrom either Life-Technology (Rockville, MD,8 USA),TaKaRa Co., or Toyobo Co. (Tokyo,9 Japan). Thedigested products were separated by electrophoresisthrough 3%10 Nusieve 3 : 1 agarose gel (FMC, Rockland,ME,11 USA). The resulting rDNA fragment pro®les in gelwere visualized and photographed as before.

Sequence analysis

Ampli®ed phytoplasma rDNA products (about 1200and 500 bp) with primer pairs (R16F2n/R16R2 andP4/P7) were puri®ed using PCR puri®cation kit (QiagenCo., Hilden, Germany12 ). The puri®ed product (10±12 ng)was used for PCR cycle sequencing using Big DyeTerminator ready reaction Mixture kit (PerkinElmer,Boston, MA, USA)13 and analysed with 310 GeneticAnalyser capillary machine. In addition to universal

primers, a primer (called SAGUN-1) speci®c to brinjaland periwinkle was also used for sequencing.

Phylogenetic analysis

Sequences (both 16S rDNA and ISR) were aligned withsimilar reference sequences of other phytoplasma avail-able in the GenBank database (Table 1) using CLUSTALLUSTAL

X (Thompson et al., 1994). The regions of sequenceambiguity and positions that were not available for allof the sequences compared were omitted before under-taking the phylogenetic analysis. Phylogenetic treeswere constructed from the aligned sequences withoriginal data set and 100 bootstrap data sets generatedby the CLUSTALLUSTAL X. The trees were generated14 for rDNAsequence and 16S-23S spacer region using Acheloplasmalaiwiddi, Acholeplasma palma and Clostridum innocuumas the outgroup sequences to allow the trees to berooted. The ®nal trees were displayed using the TREE-TREE-

VIEWVIEW (Page, 1996) and NJNJ PLOTPLOT programs (Perriere andGouy, 1996).

Nucleotide sequence accession number

The nucleotide sequences of both the 16S rDNA andISR obtained from phytoplasma strains in Bangladesh,brinjal little leaf (BLL-Bd) and periwinkle little leaf(PLL-Bd), were submitted to the GenBank databaseunder the accession numbers AF228052 and AF228053,respectively.

ResultsSymptomatology and electron microscopy

The observed little leaf disease symptoms in both brinjaland periwinkle plants were similar. The a�ected plantsexhibited small yellowish leaves that roll upward inbrinjal, stunted growth with shortened internodes andprofuse branching that sometimes gave bushy structureto the plants. Infected plants of both species did notproduce ¯owers (Fig. 1A,B). Cell-wall-less,15 irregular,round-shaped structures resembling phytoplasmas wereobserved exclusively in phloem sieve elements of dis-eased plants of both species. Thin sections from healthyplants did not show such structures. In both hosts(brinjal and periwinkle) the shape, size and within-tissuedistribution of these organisms was similar. Within thesame vascular bundle some sieve cells were fully packedwith the structures (Fig. 2A,C) whereas others werealmost empty (Fig. 2B). Similarly, the cell content ofputative phytoplasmas was also variable from one toanother within the same infected sieve element (Fig. 2A,D). In most cases the16 DNA strands were at the centreand electron-dense materials (cytoplasm) were at theperiphery of the phytoplasma cells.

PCR and RFLP analysis

The universal primer pair P1/P6 ampli®ed the targetDNA of expected size 1500 bp along with other minorbands in the PCR mixture containing DNA template ofinfected brinjal, periwinkle and known phytoplasma-infected hydrangea (as a reference sample). The same pri-mer pairs failed to amplify any DNA fragments in PCR

Characterization of Phytoplasmas of Little Leaf Disease of Brinjal and Periwinkle in Bangladesh 239

Table 1List of phytoplasma strains, original sources and GenBank accession number of their respective 16S rRNA and 16S-23S rRNA spacer regionsequences used to make phylogenetic tree in this paper

Accession number

Acronyms Strain Original source 16S rRNA 16S-23S

ASHY Ash yellows ashY Ash: USA X68339 U54986Ash Y1 Ash yellows Fraxinus: USA AF092209 AF 092209BGWL Bermuda grass white leaf Bermuda grass: Italy Y16388 AF 025423BLL-Bd Brinjal little leaf Eggplant: Bangladesh AF228052 AF228052BLL-In Brinjal little leaf Eggplant: India X83431 X83431CiPh Cirsium arvense phyllody Cirsium arvense: Germany X83438 X83438CLY Coconut lethal yellows Palm: Florida U18747 AF024639CP Clover proliferation Clover: Canada L33761 L33761CYE Clover yellow edge Clover: Canada F173558 AF 173558ESFY European stone fruit yellow PPER Peach: Germany X68374 U54988EY Elm yellows Elm: USA L33763 U54991FBP Faba bean phyllody Faba bean: Sudan X83432 X83432FD Flavescence doree Grapevine: France X76560 X76560IAWB Italian alfalfa witches-broom Alfalfa: Italy Y16390 Y16390PD Pear decline Pear: Italy Y16392 U54989PLL-Bd Periwinkle little leaf Periwinkle: Bangladesh AF228053 AF228053PpDB Papaya dieback disease Papaya: Australia Y10095 Y08176PPWB Pigeon pea witches broom Pigeon pea: USA L33735 AF025427SAY Western Aster Yellows Celery: USA M86340 M86340SCWL Sugarcane white leaf Sugarcane: Thailand X76432 AF037595StLL Stylosanthes little leaf Stylosanthes scabra: Australia Y17055 Y17055STOL Stolbur STOL Pepper: Serbia X76427 AF035361SUNHP Slunhemp witches broom Sunhemp; Thailand X76433 AF037596SWB Spartium witches broom Buckthorn: Germany X932869 X92869TLD Tanzanian lethal decline Palm: Tanzania, Africa X80117 Y13913ViLL Vigna little leaf Vigna lanceolata: Australia Y15866 Y15866WX Western X diseases Peach: California L04682 U54992A. laidlawii Acholeoplasma laidlawii M23932 D13260A. palmae Acholeplasma palmae L33734 L33734C. innocuum Clostridium innocuum M23732 M23732

Fig. 1 Symptoms of little leaf disease on brinjal and periwinkle host. (A) Diseased brinjal plant. (B) Diseased (on right) and healthy (on left)periwinkle plants


mixture containing target DNA of asymptomatic healthyplant (Fig. 3A). The ampli®ed product of P1/P6 primerswas re-ampli®ed with speci®c primer pairs (R16F2n/R16R2) in nested PCR and the resultant ampli®edproducts (about 1200 bp) were the same in brinjal andperiwinkle (Fig. 3B). Similarly non-nested PCR primedby P4/P7 on genomic DNA resulted the same ampli®ca-tion, 500 bp of ISR, of diseased brinjal and periwinkle butdi�erent for reference sample (Fig. 3C). The ampli®ednested PCR products of 16S rDNA from diseased brinjaland periwinkle showed similar RFLP patterns (Fig. 4)following separate digestion with AluI, EcoRI, HincII,HindIII, HinfI, KpnI and Sau3AI.

Nucleotide sequence comparison

Nearly complete sequencing of the 16S rDNA gene andISR was performed in phytoplasma strains of BLL-Bdand PLL-Bd and the percentage of sequence homologywere compared with that of other closely related strains.The BLL-Bd and PLL-Bd had 100% sequence homo-

logy at 16S rDNA and ISR region. Both strains were99% similar at 16S rDNA and 100% similar at the ISRregion with brinjal little leaf strain (BLL-In) from India,whereas, the clover proliferation strain had similarity ofless than 98% with BLL-Bd and PLL-Bd at 16S rDNA.Both strains had homology of 94% with AshY strain atthe ISR region.

Phylogenetic analysis

Phylogenetic trees were constructed on the basis of both16S rRNA and 16S-23S ISR of BLL-Bd and PLL-Bdalong with other phytoplasma strains listed in Table 1.The bootstrap analysis generated a tree (Fig. 5) which isin good agreement with the trees constructed previously(Kenyon et al., 1998; Lee et al., 1998; Seemuller et al.,1998). Both trees formed a separate branching withBLL-Bd, PLL-Bd and BLL-In as a subgroup, due tohigh similarity among them, from the clover prolifer-ation group. Furthermore, phylogenetic analysis of ISRof BLL-Bd and PLL-Bd along with several other

Fig. 2 Electron micrographs of leaf mid rib from diseased plants. (A) and (B) Infected sieve cells of brinjal. Arrows indicate phytoplasmas thatare variable in number from one cell to another. (C) and (D) Infected sieve cells of periwinkle. Note the densely packed phytoplasmas in C (shownby arrows). (D) Square marked portion of C at higher magni®cation. Sieve cell (s), Companion cell (c), Cell wall (cw). Bar� 1.0 lm in (A), 2.0 lm(B), 1.5 lm in (C) and 0.25 lm in (D)


phytoplasmas revealed that the strains BLL-Bd, PLL-Bd and BLL-In are identical forming one cluster. Thephylogenetic trees for the 16S rRNA gene and 16S-23SISR look identical in this study. Therefore, the tree forthe 16S rRNA gene is presented in Fig. 5.17

DiscussionSince the ®rst report of little leaf of brinjal from India(Verma et al., 1969), this syndrome has also beenobserved in other plants in several parts of the Indiansubcontinent (Mitra, 1988). In Bangladesh, little leafsyndrome has been noticed and identi®ed as a phyto-plasma disease based on symptoms only. The presentstudies have revealed that little leaf of brinjal andperiwinkle are due to phytoplasma infections and theobserved symptoms are very similar to those describedpreviously (Verma et al., 1969; Mitra, 1988). Theconsistent presence of phytoplasmas in sieve elementsof diseased brinjal and periwinkle and their absencefrom healthy or asymtomatic plants strongly supportsthe phytoplasma aetiology, although it is not directproof of the disease. The pleomorphic bodies seen in thephloem sieve cells by TEM are similar to those reportedfor other phytoplasma diseases (Pisi et al., 1990; Credi,1994).The primer combinations of P1/P6 for the ®rst round

of PCR followed by R16F2n and R16R2 gave the mostconsistent results of ampli®cation of all the samplestested. A 1200 bp product could be ampli®ed frominfected plants of both species. The RFLP analysis ofthese products showed that little leaf-associated phyto-plasmas BLL-Bd and PLL-Bd are indistinguishable.These phytoplasmas together with BLL-In from Indiafall under the clover proliferation (CP) group accordingto the classi®cation proposed by Lee et al. (1998) andSeemuller et al. (1998). This is the ®rst report of a phy-toplasma member of this group in Bangladesh.The gene sequence analysis of phytoplasma associated

with little leaf symptoms of brinjal (BLL-Bd) andperiwinkle (PLL-Bd) in Bangladesh have made itpossible to further distinguish and phylogeneticallyclassify them. Sequence data obtained for the 16SrRNA and ISR con®rmed the RFLP grouping. On thebasis of our sequencing data, the strains BLL-Bd andPLL-Bd can be considered identical or closely related tothe strain BLL-In from India (Schneider et al., 1995).Earlier, Schneider et al. (1995) reported 97% similaritybetween BLL-In and ash yellows strain (AshY) phyto-plasma sequences and they consider them to represent asubgroup of elm yellows strain cluster (Seemuller et al.,199418 ). Similarly, periwinkle little leaf phytoplasma isthought to be interrelated with AshY, although they canbe di�erentiated from one another (Lee and Davis,1988; Davis et al., 1990). In the present study the BLL-Bd and PLL-Bd also indicate similar sequence homol-ogy (96.2%) with AshY; however, we consider themunder the CP group because of the 99% homology withthe strains belonging to this group compared with thestrains belonging to AshY. In addition to RFLPanalysis, sequence homology of BLL-Bd and PLL-Bd

Fig. 3 Non-nested and nested polymerase chain reaction (PCR)ampli®cation of phytoplasma 16S rDNA and intergenic spacer region(ISR) of 16S-23S rDNA. (A) non-nested PCR primed by P1/P6 ongenomic DNA of healthy and diseased plants. Note the major bands1500 bp (virtually the complete 16S rDNA) indicated by arrowhead,was ampli®ed along with other minor bands. (B) nested PCR primedby R16F2n/R16R2 universal primer pairs using PCR product shownin (A) as template. (C) non-nested PCR primed by P4/P7 on genomicDNA of healthy and diseased plants. Note ampli®cation of 500 bp(arrow) of 16S-23S intergenic spacer region. Lane 1, healthyperiwinkle; 2, diseased periwinkle; 3, healthy brinjal; 4, diseasedbrinjal; 5, healthy hydrangea; 6, diseased hydrangea; M� k/styIDNA ladder, MI� 100 bp DNA ladder. Hydrangea was taken asreference sample


at 16S rRNA and ISR indicate that these two strains ofphytoplasma are indeed the same and infecting bothbrinjal and periwinkle with similar symptoms. From thephylogenetic analysis it is clear that the BLL-Bd, PLL-Bd and BLL-In together make a separate branch as asubgroup from CP indicating these strains may be thesame species.

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electron microscopy and molecular characterization of phytoplasmas associated with little leaf...

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