RESEARCH ARTICLE

Genetic Diversity of Sugarcane Grassy Shoot(SCGS)-Phytoplasmas Causing Grassy Shoot Disease in India

R. Viswanathan • C. Chinnaraja •

R. Karuppaiah • V. Ganesh Kumar •

J. Jenshi Rooba • P. Malathi

Received: 3 January 2011 / Accepted: 15 July 2011 / Published online: 11 August 2011

� Society for Sugar Research & Promotion 2011

Abstract Sugarcane grassy shoot (SCGS) caused by

SCGS-phytoplasmas is a serious disease of sugarcane.

Since the diseased plants exhibit various phenotypic

symptoms under field conditions, diagnosis of the disease

becomes difficult. A detailed study was conducted on

validating phytoplasma specific primers for the diagnosis

of the disease and to identify the extent of variations in

rDNA genome of SCGS-phytoplasmas. A total of 37 sug-

arcane samples with different phenotypic symptoms of

SCGS were collected from various states of India. Nested-

PCR amplification using two sets of forward (P1/P7) and

reverse primers (P4/P7) of 16S rRNA gene, 16S–23S

rRNA spacer region (SR) and 23S rRNA gene sequences

amplified SCGS-phytoplasmas in all the suspected sam-

ples. Nucleotide sequence analysis clearly revealed that 18

isolates of SCGS-phytoplasmas from different locations/

varieties showed no variation in the targeted gene

sequences. Although there were significant variations in

phenotypic expression of SCGS phytoplasmas on sugar-

cane, we could not establish any genotypic variation among

the pathogenic isolates in rDNA region. All our sequences

matched exactly with other reported phytoplasmal isolates

of SCGS-Pun (DQ459439), SCGS (DQ380341), SCGS-

CM1 (AM269740) and SCGS-SV1 (AM269742) from

India. Among the other reported SCGS phytoplasmas it

shared sequence similarity ranging from 99.7 to 94.3%.

Our SCGS phytoplasmas showed 99.6% similarity with

other phytoplasmas infecting sugarcane such as sugarcane

white leaf (SCWL) and 97.8% with SCYL. The closely

related phytoplasmas such as BraWL and SGS

phytoplasmas showed 99.6% similarities with our SCGS-

phytoplasmas. However, distantly related phytoplasmas

FBP and CBY1 and StLL showed sequence similarities

between 84.3 and 82.4%. Restriction of the amplicons with

a set of restriction enzymes did not show any polymor-

phism among them. 16S rRNA, 16S–23S rRNA SR and

23S rRNA sequencing and restriction fragment length

polymorphism (RFLP) results proved that the phytoplasma

isolates infecting sugarcane in India with phenotypic

variations from different regions have similar ITS region

and further studies are needed to locate other genes in

phytoplasma genome which determine variations in phe-

notypic expression in the SCGS disease.

Keywords Grassy shoot disease � SCGS-phytoplasma �Nested-PCR � RFLP � Ribosomal DNA �Phylogenetic analysis

Introduction

The sugarcane grassy shoot (SCGS) and sugarcane white

leaf (SCWL) are the two major phytoplasma diseases of

sugarcane and SCGS has been reported from Bangladesh,

India, Malaysia, Nepal, Pakistan and Sri Lanka. In India,

SCGS is a major disease and its importance comes next to

the fungal diseases such as red rot, wilt, and smut and

causes cent percent losses in susceptible varieties in Uttar

Pradesh, Maharashtra, Haryana and Tamil Nadu and hence

is of great economic concern to both the farmers and sugar

industry (Viswanathan 2000). Vegetative propagation in

sugarcane facilitates transmission of phytoplasmas to the

field and recently they are reportedly transmitted by

R. Viswanathan (&) � C. Chinnaraja � R. Karuppaiah �V. Ganesh Kumar � J. Jenshi Rooba � P. Malathi

Plant Pathology Section, Division of Crop Protection,

Sugarcane Breeding Institute, Indian Council of Agricultural

Research, Coimbatore 641007, India

e-mail: rasaviswanathan@yahoo.co.in

123

Sugar Tech (July-Sept 2011) 13(3):220–228

DOI 10.1007/s12355-011-0084-2

phloem feeding leafhopper vectors (Srivastava et al. 2006).

SCGS continues to be a serious threat to sugarcane culti-

vation in India. Planting of disease infected canes results in

significant decline in yield in plant crop however, more

severe losses are expected in the subsequent ratoons. The

infected plants show excess tillering with varying levels of

chlorosis in the leaves. However in some cases the tillers

show only green tillers (Viswanathan and Padmanaban

2008). Varying intensities of chlorosis in the leaves creates

confusion in disease diagnosis and also they resemble

symptoms of many deficiency diseases (Fig. 1). In addi-

tion, the disease causes bud sprouting, alteration in leaf

texture, and chlorosis of leaves in the whorl region with

bunching of leaves in the top. Hence there is a need for

developing diagnostic techniques to detect SCGS-phy-

toplasmas. In this regard earlier we reported use of ELISA,

immunofluorescent technique and PCR techniques to

diagnose SCGS phytoplasmas (Viswanathan 1997, 2000;

Viswanathan et al. 2005). Major limitations of ELISA were

with the purification and the specificity of phytoplasmas

and lack of sensitivity (Jones et al. 1997). At present PCR

assays using oligonucleotide primers based on sequences

derived from the phytoplasmal 16S rRNA gene provide one

of the most sensitive, reliable, rapid and accepted method

for detecting phytoplasma in infected plants. The sensi-

tivity of detection can be increased by the recent molecular

diagnostic technique of nested PCR, in which two universal

primer pairs are used.

The disease expresses in various forms under field

conditions and this creates confusion in disease diagnosis

and probably indicates variability in SCGS phytoplasmas.

Restriction fragment length polymorphism (RFLP) using

Fig. 1 Variation in symptoms

of GSD infected canes. a Plant

showing completely young

chlorotic leaves, b partial young

chlorotic leaves in the whirl,

c completely green bushy tillers,

d partial green/chlorotic tillers

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different restriction endonucleases and sequence analysis

of rDNA are used to elucidate the genetic relationship

among the phytoplasmas and between other related phy-

toplasmal groups. As the disease manifests in different

phenotypes it is yet to be known whether the disease is

caused by different phytoplasma genotypes, varietal inter-

actions or environment factors. Hence a detailed study was

conducted to diagnose the disease by PCR technique and to

assess molecular variability of SCGS-phytoplasmas by

sequencing rDNA and PCR–RFLP.

Materials and Methods

Sources of Phytoplasma

Leaf sample from 37 SCGS-suspected sugarcane varieties

were collected from the field of Sugarcane Breeding

Institute, Coimbatore and field samples from other states

viz. Haryana, Punjab, Orissa, Karnataka, Assam, Andhra

Pradesh and Uttar Pradesh during 2007–2008 cropping

season (Table 1).

DNA Extraction and PCR

Total DNA from healthy and infected sugarcane plants

were extracted from leaves using CTAB (2% CTAB, 2 M

NaCl, 20 mM EDTA, 100 mM Tris–HCl at pH 8.0 and 1%

PVP) buffer (Dellaporta et al. 1983). Quantity and quality

of DNA were estimated spectrophotometrically. The fol-

lowing primer combinations P1/P7 and P4/P7 (Table 2)

were used for the first and second PCR amplification in the

nested-PCR.

First round PCR (25 ll) reaction mixture contained

50 ng of DNA, 0.4 lM of each primer, 200 lM of each

dNTPs, 1 U of Taq polymerase with 19 PCR buffer and

this reaction was carried out for 35 cycles (denaturation at

94�C for 30 s; annealing at 58�C for 90 s; extension at

72�C for 90 s; and final extension at 72�C for 15 min) in a

thermocycler (Mastercycler Gradient, Eppendorf, Ger-

many). Nested PCR with P4/P7 primers was performed in

25 cycles (denaturation at 94�C for 30 s; annealing at 57�C

for 30 s; extension at 72�C for 30 s; and final extension at

72�C for 10 min) with 1 ll PCR product from the first

cycle. PCR products were electrophoresed in 1.5% agarose

gel containing ethidium bromide in 1X TAE buffer and

visualized and documented in gel documentation system

(Alphaimager, Alpha Innotech, USA).

RFLP Analysis

PCR products of *1,800 bp from 18 out of 37 isolates

were digested separately with three different restriction

endonucleases Rsa I, Alu I and Hpa II (Fermentas, USA).

30 ll of restriction reaction performed at 37�C for 90 min

comprised 25 ll of template, 1 ll of milli Q water, 3 ll of

19 Tango restriction buffer (MBI Fermentas, USA) and

1 ll of 5 U of restriction enzyme. RFLP products were

electrophoresed in 1.5% agarose gel containing ethidium

bromide, visualized and documented as before.

Cloning and Sequencing

530 bp from nested-PCR products were purified using

GenElute Gel Extraction Kit (Sigma, USA) and ligated to

pTZ57R/T vector. The recombinant plasmids were cloned

into Escherichia coli strain DH5a. Plasmid DNA was

extracted with Plasmid mini preparation kit (Sigma, USA).

Restriction digestion of plasmid DNA was performed with

Hind III (10 U/ll) and Xba I (10 U/ll) restriction enzymes

to check for the presence of insert. Sequencing of plasmid

DNA was carried out by ABI PRISM 377 DNA sequencer

using the primer pair M13F (-20) and M13R puc (-26)

with BigDye Terminator Cycle Sequencing Kit v3.0/v3.1

(Perkin Elmer). Nucleotide sequencing was performed at

the DNA sequencing facility of 1st base, Selangor Darul

Ehsan, Malaysia. Sequence alignments were performed in

ClustalW using the software BioEdit ver. 7.0.9.0. For

phylogenetic analysis, other related phytoplasmal sequen-

ces retrieved from GenBank (Table 3) were compared with

five of our phytoplasmal sequences and the phylogram was

constructed by neighbor-joining method using ClustalX

(1.8) software with 1,000 bootstrap replications.

Results and Discussion

Using universal primer pair P1/P7 in the first round

PCR, *1,800 bp product was amplified from all the 37

SCGS suspected samples and no such amplification was

found in healthy control. Although equal amount of tem-

plate was used in all the samples, we found mild amplifi-

cation of the target sequences in 5 samples of 4 varieties

viz CoC 671, Co 86032, CoVc 03165 (young chlorotic),

CoVc 03165 (young nonchlorotic) and CoH 07265. Four

different leaf samples of SCGS symptomatic leaves from

severely infected cv CoC 671 starting from top to bottom

showed that the amplification was mild in the top young

leaf and the intensity of the PCR product increased grad-

ually towards the bottom indicating that the phytoplasma

population in the younger leaf is low compared to matured

leaves where are green in the bottom. In Co 86032 and

CoH 07265 a mild amplification was found in PCR which

may be due to the initial stages of infection in the sampled

plants. Among the four leaf samples collected from the

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Table 2 Primers used in nested PCR assays to amplify SCGS-phytoplasmas

Primer Primer sequence (50–30) Coding region Product size Reference

P1 Forward (AAG AGT TTG ATC CTG GCT CAG GAT T) 16S rDNA P1/P7—1800 bp Deng and Hiruki (1991)

P4 Forward (GAA GTC TGC AAC TCG ACT TC) 16S rDNA P4/P7—530 bp Schneider et al. (1995)

P7 Reverse (CGT CCT TCA TCG GCT CTT) 23S rDNA Schneider et al. (1995)

Table 1 List of sugarcane isolates taken for the diagnosis of SCGS phytoplasma

S. no. Isolates (accession no) Source variety Place Symptom variation (visual observation)

1 CBJ87-(GU138385) CoJ 87 Karnal, Haryana GS with partial to complete chlorosis

2 CBSi776 CoSi 776 Sirugamani, TN Severe GS with complete chlorosis

3 CBM7704 CoM 7704 Padegaon, MS GS with mild chlorosis

4 94A109-(GU138386) 94A 109 Anakapalle, AP GS with nonchlorosis

5 CBJ84291-(GU138387) CoJ 84291 Karnal, Haryana GS with nonchlorosis to mild chlorosis

6 CBLk8002 CoLk 8002 Karnal, Haryana Partial chlorosis

7 CB86249 Co 86249 Coimbatore, TN GS, Young leaves with complete chlorosis

and matured green leaves

8 CB89003-(GU138388) Co 89003 Coimbatore, TN Severe GS, complete chlorosis

9 CB89003 Co 89003 Coimbatore, TN Severe GS, partial chlorosis

10 CB0238 Co 0238 Coimbatore, TN Leaves normal, partial chlorosis

11 CBSel922-(GU138389) Sel 922 Coimbatore, TN Young leaf: complete chlorosis

12 CBSP801842-(GU138390) SP-801842 Coimbatore, TN Matured leaf: green

13 CBPant97222 CoPant 97222 Uttarankhand Matured leaf: green

14 CBBln04174-(GU138391) CoBln 04174 Buralikshan, Assam Matured leaf: green

15 CBISH43 ISH 43 Coimbatore, TN Partial chlorosis

16 CBC671-(GU138392) CoC 671 Cuddalore, TN Young leaf: complete chlorosis

17 CBC671 CoC 671 Cuddalore, TN Matured leaf: green

18 CBC671 CoC 671 Cuddalore, TN First leaf from whorl: complete chlorosis

19 CBC671 CoC 671 Cuddalore, TN Second leaf from whorl: complete chlorosis

20 LG02100-(GU138393) LG 02100 Lucknow, UP Matured green leaves

21 CBC671 CoC 671 Cuddalore, TN Partial chlorosis

22 CBC99061 CoC 99061 Cuddalore, TN Young leaf: completely chlorosis

23 CB86032-(GU138394) Co 86032 Padegaon, MS Matured leaf: green

24 CB86032-(GU138395) Co 86032 Chinnamanur, TN Young leaf: completely chlorosis

25 CB94010-(GU138396) Co 94010 Markandeyankottai, TN Young leaf: completely chlorosis

26 CB92012 Co 92012 Markandeyankottai, TN Matured leaf: green

27 CB6907-(GU138397) Co 6907 Dhenkanal, Orissa Completely chlorosis

28 CB8014-(GU138398) Co 8014 Padegaon, MS Partial chlorosis

29 CB7508-(GU138399) Co 7508 Panipoila, Orissa Partial chlorosis

30 CBOr03152 CoOr 03152 Panipoila, Orissa Completely chlorosis

31 CBVc03165-(GU138400) CoVc 03165 Mandya, Karnataka Completely chlorosis

32 CBVc03165 CoVc 03165 Koppa, Karnataka GS with chlorosis

33 CBVc03165-(GU138401) CoVc 03165 Koppa, Karnataka GS with nonchlorosis

34 CBVc03165 CoVc 03165 Koppa, Karnataka GS with nonchlorosis

35 CB94008 Co 94008 Coimbatore, TN GS with complete chlorosis

36 CB62175-(GU138402) Co 62175 Coimbatore, TN GS with nonchlorosis

37 CBH07265 CoH 07265 Karnal, Hariyana GS with nonchlorosis

GS grassy shoot, TN Tamil Nadu, MS Maharashtra, AP Andhra Pradesh, UP Uttar Pradesh

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cv CoVc 03165, two were matured leaves and showed

chlorotic and nonchlorotic symptoms and another two were

young leaves with similar type of symptoms. Phytoplasma

infections were found confirmed by PCR in both matured

and young leaves. Among the young leaves, pathogen load

was found to be high in symptomatic chlorotic leaf when

compared to nonchlorotic, which revealed phytoplasmas

concentration was high in chlorotic leaves (Fig. 2).

Table 3 List of other phytoplasmas related to SCGS phytoplasma

Phytoplasma (strain) Geographical

origin

Group GenBank

accession no.

Similarity %

observed

Other SCGS phytoplasmas

Sugarcane grassy shoot (SCGS-Pun) India 16SrXI DQ459439 100

Sugarcane grassy shoot (SCGS) India 16SrXI DQ380341 100

Sugarcane grassy shoot (SCGS-CM1) India 16SrXI AM269740 100

Sugarcane grassy shoot (SCGS-SV1) India 16SrXI AM269742 100

Sugarcane grassy shoot (SCGS) India 16SrXI DQ380336 99.7

Sugarcane grassy shoot (SCGS) India 16SrXI AB243298 98.0

Sugarcane grassy shoot (SCGS) India 16SrXI AM085763 97.6

Sugarcane grassy shoot (SCGS) India 16SrXI AM085764 94.3

Other sugarcane phytoplasmas

Sugarcane white leaf (SCWL) Thailand 16SrXI X76432 99.6

Sugarcane yellows phytoplasma (SCYP) Mauritius 16SrIII AJ539178 97.8

Related groups of phytoplasma

Sorghum grassy shoot (SGS-1) Australia – AF509324 99.6

Brachiaria white leaf (BraWL-KK) Thailand – AB052872 99.6

Cynodon white leaf (CWL) Australia – AF509321 99.1

Rice yellow dwarf (RYD) Japan 16SrXI D12581 97.5

Mollicutes Germany – X76429 96.6

Sweet potato witches broom (SPWB) USA 16SrII L33770 94.4

Ethiopian gliricidia little leaf (EGLL) USA – AF361018 93.9

Bermuda grass white leaf (BGWL-C1) Italy 16SrXIV AJ550984 93.6

Ca.P.Pini Spain 16SrXXI AJ632155 85.7

Potato witches broom (PWB) USA 16SrV1 AY500818 84.7

Stylosanthus little leaf (StLL) Australia 16SrII AJ289192 84.3

China berry yellows (CBY) Bolivia 16SrXIII AF495882 84.3

Faba bean phylody (FBP) Bolivia 16SrII X83432 82.4

Fig. 2 Amplification of 1.80 kb DNA from phytoplasma infected

leaves of 38 isolates. Lanes 1 CoJ 87, 2 CoSi 776, 3 CoM 7704, 4 94A

109, 5 CoJ 84291, 6 CoLk 8002, 7 Co 86249, 8 Co 89003, 9 Co

89003, 10 Co 0238, 11 Sel 922, 12 SP 80-1842, 13 CoPant 97222, 14CoBln 04174, 15 ISH 4, 16 CoC 671, 17 CoC 671, 18 CoC 671, 19

CoC671, 20 LG 02100, 21 CoC 671, 22 CoC 99061, 23 Co 86032, 24Co 86032, 25 Co 94010, 26 Co 92012, 27 Co 6907, 28 Co 8014, 29Co 7508, 30 CoOr 03152, 31 CoVc 03165, 32 CoVc 03165, 33 CoVc

03165, 34 CoVc 03165, 35 Co 94008, 36 Co 62175, 37 CoH 07265,

38 CoC 671, M 100 bp Marker

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The phenotypic expressions of SCGS disease can be

noticed at all the growth stages in an infected plant (Edison

et al. 1977; Hughes and Abbott 1964; Rishi and Chen

1989). The appearance of a chlorotic leaf among the top

leaves in a healthy shoot is the earliest symptom in the

plant crop, then the number of white leaves appearing in

the infected plant increases gradually (Viswanathan 2000).

Results of this PCR assay clearly established that SCGS-

phytoplasmas causes different phenotypes such as infected

plant with either partial or completely chlorotic leaves,

grassy shoot with green tillers or chlorotic leaves and

leaves from infected plant showing varying disease

symptoms or ages. We have specifically amplified the PCR

product from all these suspected plant samples.

In the nested PCR reaction, DNA from all the 18

selected samples including 3 low titre PCR products viz

CoC 671, CoVc 03165 (young chlorotic) and CoVc 03165

(young non chlorotic) resulted in amplification

of *530 bp product for the primers P4/P7 which again

confirmed SCGS-phytoplasmas infection (Fig. 3). Nested-

PCR is used, only when samples with suspected phytopl-

asma symptoms were consistently negative in first round

PCR tests (Tran-Nguyen et al. 2000). The nested PCR

studies very clearly established that using this technique

SCGS-phytoplasma infection can be very precisely detec-

ted either in symptomatic or non symptomatic plants. Since

greater SCGS-symptom variation is exhibited in sugarcane,

this technique will be more reliable in detecting SCGS-

phytoplasmas. The results indicate that amplification of

SCGS-phytoplasma by PCR is specific from sugarcane

irrespective of the nature of symptoms. Also there is no

clear variation in maturity status of the suspected leaves in

phytoplasma amplification. Probably higher intensity of the

amplified product could be correlated with phytoplasma

titre. However this has to be validated further by quanti-

tative PCR assays.

The phylogenetic relationships of SCGS phytoplasma

strains to each other and to the most related phytoplasmas

were examined, by sequencing both 16S rRNA gene and

16S/23S rRNA spacer region (SR, Rao et al. 2008). The

phylogenetic relationships of SCGS phytoplasma isolates

among themselves and related phytoplasmas based on 16S

rRNA gene sequences showed 100% identity/no variation

among 16S rRNA gene sequences of 18 isolates. Although

there were significant variations in phenotypic expression

of SCGS phytoplasmas on sugarcane, we could not estab-

lish any genotypic variations. When compared with other

reported SCGS phytoplasmas in India, 16S rRNA gene

sequences of our 18 isolates exactly matched with SCGS-

Pun (DQ459439) and SCGS (DQ380341). Similarly IGS

regions of SCGS (AM269740) and SCGS (AM269742)

were also 100% similar with our 18 gene sequences. The

other isolates reported from India viz., DQ 380336, AB

243298, AM 085763 and AM 085764 showed sequence

relation of 99.7, 98.0, 97.6 and 94.3% respectively. Other

than SCGS-phytoplasmas, other phytoplasmas such as

SCWL-phytoplasmas causing white leaf disease and sug-

arcane yellows phytoplasmas (SCYP) causing leaf yellows

are also infecting the sugarcane. Comparison of SCGS-

phytoplasmas with these two phytoplasmas revealed 99.6%

similarity with SCWL (X76432) followed by 97.8% simi-

larity with SCYP (AM085764).

Comparison of our 16S rRNA gene sequences of 18

phytoplasma isolates with other related groups of phytopl-

asmas revealed close relation to SGS-1 (AF509324) and

BraWL-KK (AB052872) which shared 99.6% similarity

followed by CWL (AF509321) with 99.1% similarity. Our

phytoplasmas had moderate similarities of 97.5% with RYD

(D12581), 96.6% with Mollicutes (X76429), 94.4% with

SPWB (L33770), 93.9% with EGLL (AF36118) and 93.6%

with BGWL-C1 (AJ55984). The distantly related phytopl-

asmas were Ca.P.Pini (AJ632155) having 85.7% similarity

followed by PWB (AY500818) with 84.7%, StLL

(AJ289192) and CBY (AF495882) with 84.3% and FBP

(X83432) with 82.4% similarity. Phylogenetic tree was

constructed using neighbor-joining method with 16S rRNA

gene sequences from strains of SCGS phytoplasma and

related phytoplasmas (Fig. 4). Cluster 1 in phylogram

showed 100–99% similar isolates, where the least similar

phytoplasma CWL (AF509321), shared 99.1% similarity.

Cluster 2 had 99–94% similar isolates, where SCWL

(X76432) is the closely related phytoplasma with similarity

of 99.6% and SCGS (AM 085764) had the least similarity of

94.3%. Distantly related phytoplasmas of 94–82% similar

isolates were grouped in cluster 3, where SPWB (L33770)

shared 94.4% and the distantly related phytoplasma StLL

(AJ289192), shared 82.4% similarity.

Fig. 3 Nested PCR amplification of 530 bp product with P4/P7

primers. Lanes 1 CoJ 87, 2 94A 109, 3 CoJ 84291, 4 Co 89003, 5 Sel

922, 6 SP-801842, 7 CoBln 04174, 8 LG 02100, 9 CoC 671, 10 Co

86032, 11 Co 86032, 12 Co 94010, 13 Co 6907, 14 Co 8014, 15 Co

7508, 16 CoVc 03165, 17 CoVc 03165, 18 Co 62175, 19 CoC 671,

M 100 bp Marker

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Fig. 4 Phylogram was

constructed with 1,000

bootstrap replications by

comparing 16S rRNA gene

sequences of 5 SCGS-

phytoplasmas from this study

(GU138385, GU138386,

GU138388, GU138393 and

GU138397) with other related

phytoplasmas from GenBank

using neighbour-joining method

in ClustalX (1.8) software. The

bar represents the phylogenetic

distance of 10%. The details of

accession numbers are given in

Table 3

Fig. 5 a Rsa I, b Alu I and

c Hpa II restriction profiles of

18 SCGS phytoplasmal DNA

amplified by PCR using

universal primer pair P1/P7.

Lanes refer Fig. 3

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When RFLP was performed with the 3 different restric-

tion enzymes Rsa I, Alu I and Hpa II, 18 SCGS phytoplasma

isolates collected from various states/varieties have shown

the similar restriction patterns (Fig. 5). The Rsa I restriction

enzyme had given 3 different fragments such as 420, 520

and 830 bp. Similarly seven different fragments of nearly

980, 770, 720, 600, 390, 290 and 210 bp were given by Alu I

restriction enzyme. The restriction enzyme Hpa II gave five

products of *1400, 1000, 980, 500 and 330 bp. Similar

restriction profile of these 3 different restriction enzymes

concluded that SCGS samples showing different phenotypic

symptoms have similar gene sequences of 16S rDNA gen-

ome. Earlier Wongkaew et al. (1997) reported such Hpa II

showed similar banding pattern in SCGS phytoplasmas, but

when compared with SCWL, BGWL and Crowfoot white

leaf phytoplasma different banding profiles noticed, in case

of RsaI no polymorphism was observed for the other

reported phytoplasmas. Lee et al. (1997) reported no poly-

morphism was observed with Alu I restriction profiles of

SCGS-In1, RYD-In1, SCWL, BGWL and ABGWL phy-

toplasmas. To detect the polymorphism in 16S rDNA of

SCGS phytoplasmas, the PCR products were subjected to

RFLP analysis using five restriction enzymes (Hpa II, Sau

3AI, Taq I, Tas I, and Tau I) and no significant polymor-

phism was detected (Nasare et al. 2007). It is possible to

reliably characterize, differentiate, and classify the phy-

toplasmas mainly based on RFLP and sequence analysis of

ribosomal DNA (rDNA) by which various groups and sub-

groups could be distinguished (Lee et al. 1993; Schneider

et al. 1993; Gundersen et al. 1994; Seemuller et al. 1994).

Sequence analysis and RFLP restriction of the SCGS-phy-

toplasmas have revealed that some other region of the

phytoplasma genome may cause phenotypic variations in

SCGS infected canes. SCGS disease occurs in various forms

throughout sugarcane growing regions in India and at times

it seriously cause losses to cane production. Hence further

studies are needed to identify the respective phytoplasma

genome determining variations in phenotypic expression of

the disease and such information is totally lacking. Further,

characterization of pathogenicity genes of SCGS-phytopl-

asma also needs to be done to understand the disease epi-

demiology and presence of strains varying in virulence.

Acknowledgments Authors are grateful to Dr. N. Vijayan Nair,

Director of the Institute for providing the facilities to carry out this

research work and for his sustained encouragement.

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