Identification of sugarcane grassy shoot-associatedphytoplasma and one of its putative vectors in India

Govind Pratap Rao & Madhupriya &

Ajay Kumar Tiwari & Shailendra Kumar &

Virendra Kumar Baranwal

Received: 29 May 2013 /Accepted: 7 November 2013# Springer Science+Business Media Dordrecht 2013

Abstract During a survey of sugarcane fields at theSugarcane Research Institute, Shahjahanpur, UttarPradesh, India, in August–September 2012, 6% to28% incidence of sugarcane grassy shoot disease wasobserved in different fields of sugarcane variety CoS7250. The association of phytoplasma with symptomat-ic sugarcane was confirmed by direct and nested PCRamplification of phytoplasma ribosomal gene. Four dif-ferent delphacid leafhopper species, viz. Cofanaunimaculata Signoret, Exitianus indicus (Distant),Sogatella kolophon Kirkaldy and Hishimonus phycitis(Dist.) were the prevalent feeding species of theAuchenorrhyncha fauna in the symptomatic sugarcanefields. Out of these four leafhopper species, onlyE. indicus tested positive for phytoplasma presence.Phylogenetic analysis suggested that the phytoplasmasfrom sugarcane and E. indicus in the present study weremembers of 16Sr XI. The confirmation of association ofsugarcane grassy shoot phytoplasma in E. indicus pop-ulation is important to understand the secondary spreadof this phytoplasma in sugarcane plants.

Keywords Exitianus indicus . Leafhopper . Moleculardetection . Sugarcane grassy shoot disease

Introduction

The sugarcane grassy shoot (SCGS) is a major phyto-plasma disease of sugarcane in Asian countries, viz.,Bangladesh, India,Malaysia, Nepal, Pakistan, Sri Lankaand Vietnam (Rao et al. 2012). The SCGS is character-ized by the production of a large number of thin, slender,adventitious tillers from the base of the affected plants.This profuse growth gives rise to a dense or crowdedbunch of tillers bearing pale yellow or chlorotic leaveswhich remain thin, narrow, reduced in size and have asoft texture. Each stalk that is produced from the affect-ed stool shows shortened internodes and the develop-ment of side shoots from the bottom to the top. Affectedplants do not produce millable canes. The disease isparticularly pronounced in the ratoon crop, where theclusters of slender tillers with reduced but erect leavesgive an appearance of a field full of perennial grass, andfrom which it has derived its popular name "grassyshoots". Vegetative propagation in sugarcane facilitatessugarcane grassy shoot phytoplasma transmission in thefield (Rao et al. 2012).

In India, SCGS in a sugarcane crop has been associ-ated with phytoplasma presence (Nasare et al. 2007;Rao et al. 2008; Viswanathan et al. 2011). In the stateof Uttar Pradesh, the disease appearance in sugarcanefields is a regular feature (Rao et al. 2008). A surveyconducted during three consecutive years (2008–11) in17 districts of Uttar Pradesh, revealed a gradual increasein the GSD incidence, reaching 56% in many commer-cial sugarcane varieties. The SCGS infection causescane yield reduction up to 60% (Rao et al. 2012). It alsoreduces cane height, cane weight and juice quality

PhytoparasiticaDOI 10.1007/s12600-013-0366-1

G. P. Rao (*) :Madhupriya : S. Kumar :V. K. BaranwalDivision of Plant Pathology, Indian Agricultural ResearchInstitute,New Delhi 110012, Indiae-mail: gprao_gor@rediffmail.com

A. K. TiwariCentral Lab, Sugarcane Research Institute,Shahjahanpur 242 001, Uttar Pradesh, India

(Tiwari et al. 2012). Planting of the infected setts andspread of phytoplasma by vectors may be responsiblefor an increase in disease incidence (Srivastava et al.2006; Viswanathan & Rao 2011).

PCR assays have successfully detected the SCGSphytoplasma in sugarcane and in its leafhopper vectorDeltocephalus vulgaris in India (Rao et al. 2003;Srivastava et al. 2006; Viswanathan et al. 2005). Asso-ciation of phytoplasma with SCGS disease was con-firmed by amplification of the 16S rRNA gene. Nucle-otide sequence analysis of 16S rRNA genes revealedthat SCGS phytoplasma affecting sugarcane crops inIndia is very closely related to 'Candidatus Phytoplasmaoryzae' (16Sr XI group) (Nasare et al. 2007; Rao et al.2008; Viswanathan et al. 2011).

Several vectors are associated with phytoplasma dis-eases in sugarcane. SCGS is transmitted by a leafhopperDeltocephalus vulgaris in India (Srivastava et al. 2006),while sugarcane white leaf (SCWL) phytoplasma wastransmitted by the leafhopper vectors, Matsumuratettixhiroglyphicus and Yamatotettix flavovittatus in Thailand(Hanboonsong et al. 2002, 2006). The other vectors ofphytoplasma diseases of sugarcane include thedelphacid Eumetopina flavipes for Ramu stunt diseasein Papua New Guinea (Suma & Jones 2000) anddelphacid planthopper Saccharosydne saccharivora forsugarcane leaf yellows in Cuba (Arocha et al. 2005),respectively.

As limited information is available on vectors ofSCGS disease in India, hence, an attempt was made onthe detection of phytoplasma in the four predominantleafhopper species in sugarcane fields.

Materials and methods

Sugarcane crop was surveyed at Sugarcane ResearchInstitute, Shahjahnapur in August- September 2012 forgrassy shoot disease incidence and leafhopper popula-tion. The leafhopper species were collected from thegrassy shoot infected as well as from healthy sugarcanefields using sweep-netting method during early morninghours. The collected leafhopper species were identifiedin the Department of Entomology, University of Agri-cultural Sciences, Bangalore and plant and insect sam-ples were stored in a deep freezer at –20°C for furtherstudy.

The nucleic acid from leafhopper and leaf midribs ofsymptomatic and asymptomatic sugarcane leaves was

extracted following the procedure described by Ahrens& Seemüller (1992). Amplification of phytoplasma ri-bosomal DNA (rDNA) was performed with the univer-sal phytoplasma primer pairs P1/P7 (Deng & Hiruki1991) and the nested PCR was done with primer pairR16F2n/R16R2 (Gundersen & Lee 1996). The DNAisolated from periwinkle infected with toria phyllodyphytoplasma (group 16SrIX, pigeon pea witches' broomphytoplasma; Azadvar et al., 2009) and maintained in agreenhouse was used as positive control. The DNAextracted from healthy sugarcane was used as negativecontrol.

Reactions were carried out in a Minicycler(Biometra, USA) and the cycling protocol used for thefirst round PCR was: initial denaturation at 94°C for 5min, followed by 35 cycles consisting of denaturation at94°C for 45 sec, annealing at 55°C for 1 min, andextension at 72°C for 2 min, with extension in the finalcycle for 10 min.

Two μl of product of the first round of PCR was usedin nes ted PCR using internal pr imer pai rsR16F2n/R16R2. Reaction mixture and condition ofnested PCRwere similar as above except for the anneal-ing at 56°C for 1 min. Twenty-five μl of each PCRproduct was subjected to electrophoresis in a 1.0%(w/v) agarose gel, stained with ethidium bromide andobserved under UV transilluminator.

The ~1.8 and ~1.2 kb amplified PCR products ofSCGS was purified using the WizardR SV Gel andPCR Clean-up System (Promega). Both the purifiedPCR products were sequenced directly in both direc-tions. The sequences obtained from PCR products ofsugarcane and the leafhopper were assembled usingDNA Baser online tool.

A database search of homologous sequences wasperformed by BLAST analysis at NCBI. 16S rRNAand partial 23S rDNA gene sequences were aligned withphytoplasma group/subgroup representatives availablein GenBank using Clustal W (Thompson et al. 1994).The sequence generated from the present study andreference phytoplasma strains sequence retrieved fromGenBank were used to construct phylogeny throughMEGA 4.0 version software (Tamura et al. 2007). The16SrXI ‘Candidatus Phytoplasma oryzae’ (GenBankAc. No.AB052873, Lee et al. 1998) was used as refer-ence isolate for 16Sr XI group.

A restriction fragment length polymorphism (RFLP)analysis of the nested PCR products was performed (Leeet al. 1998) (with endonucleases BamHI, TaqI and BfaI;

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Fermentas, Glen Burnie, MD, USA). Restriction frag-ments were separated by electrophoresis through 2.8%agarose gel, stained with ethidium bromide and visual-ized under UV illumination. The obtained band patternswere compared with DNA standard markers,Ø174/HaeIII marker (Fermentas) and with the referencephytoplasmas of different groups. The 1.2 kb of 16SrDNA sequences (R16F2n/R16R2 primer primed) de-rived from SCGS phytoplasma from sugarcane andleafhopper were submitted to iPhyClassifier online tool(http://www.plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) as described by Zhao et al.(2009).

Results and discussion

During a survey of sugarcane crops in the year 2012 atSugarcane Research Institute fields in Shahjahnapur,Uttar Pradesh, India, 6% to 28% incidence of sugarcanegrassy shoot disease was recorded in sugarcane varietyCoS 7250 at ten different experiment plots (data notshown). Of the ten different plant and ratoon plots ofvar. Cos 7250 surveyed, all the ratoon plots were foundinfected with SCGS disease (Fig. 1), with an incidenceof 6-28% (Fig. 1). A total of 82 leafhoppers were col-lected from the SCGS infected sugarcane fields. Fourmajor species of leafhoppers were identified, viz.,Cofana unimaculata Signoret, Exitianus indicus(Distant), Sogatella kolophonKirkaldy and Hishimonusphycitis (Dist.). The predominant species were H.

phycitis and E. indicus, which were nearly 70% of thetotal leafhopper population.

A product of ca. 1800 bp was amplified from all theSCGS-infected sugarcane var. CoS7250 in the firstround of PCR assay with primer pair P1/P7. However,no amplification was observed in any of the four leaf-hopper species in the first round of PCRwith primer pairP1/P7. Nested PCR with primer pair R16F2n/R16R2yielded bands of ~1200 bp in E. indicus and symptom-atic sugarcane leaf (Fig. 2). This result confirmed theassociation of phytoplasma with E. indicus. No ampli-fication was observed with the DNA extracted from theother three leafhopper species (C. unimaculata, S.kolophon, H. phycitis).

16S rDNA sequences of the SCGS isolate fromsymptomatic sugarcane and E. indicus (GenBank Acc.

Fig. 1 Grassy shoot disease symptoms on ratoon of sugarcane variety CoS 7250. a: Excessive tiller proliferations at the base of the clump;b: Chlorosis and necrosis of tillers in affected stalks

Fig. 2 Nested PCR results of phytoplasma DNA amplificationfrom Exitianus indicus and sugarcane. Lane 1: Exitianus indicusleafhopper collected from SCGS infected sugarcane plant; Lane 2:E. indicus leafhopper collected from healthy sugarcane plant; Lane3: healthy sugarcane leaf; Lane 4: SCGS infected leaf; 5: Positivecontrol of Toria phyllody phytoplasma from periwinkle (Azadvaret al., 2009); Lane 6: 1 kb DNA ladder (G Biosciences, USA)

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JX862179 and KC595881) were compared and found tobe identical. Comparison of 1.2 kp of R16F2n/R2primed sequence of 16S rDNA of SCGS from diseasedsugarcane showed 99–100% similarity with isolates ofSugarcane grassy shoot phytoplasma (DQ459438,DQ459439), Sugarcane white leaf phytoplasma(AB646271, AB052874 ), Coconut root wilt phytoplas-ma (FJ794816), Goose grass white leaf phytoplasma(AB741629) and Sorghum grassy shoot phytoplasma(AF509324), all belonging to the 16Sr XI group. Phy-logenetic analysis of the 16S rDNA sequence fromSCGS phytoplasma and E. indicus with those of sub-mitted sequences of 16SrXI phytoplasma isolates inGenBank also revealed its close relationship with sug-arcane grassy shoot, coconut root wilt, sugarcane whiteleaf, areca nut yellow leaf, all members of Rice yellowdwarf group (16Sr XI group) (Fig 3).

Analysis of R16F2n/R16R2 primed 16S rDNA se-quences of SCGS phytoplasmas using iPhyClassifier

online tool with 17 selected restriction enzymes (Zhaoet al. 2009) and actual RFLP banding patterns with TaqIand BfaI endonucleases (Figs. 4 and 5) confirmed thatSCGS phytoplasma isolate collected from sugarcaneplants and leafhopper vector in India (GenBank Acc.No. JX862179) is identical to the reference phytoplasmaof 16SrXI subgroup B (sugarcane white leaf phytoplas-ma, rice yellow dwarf group, 16SrXI; GenBank Acc.No. X76432; Lee et al. 1997) and identified it as amember of 16SrXI-B subgroup phytoplasma. On theother hand, both the sugarcane and E. indicus 16SrDNA sequences shared 98% similarity with that ofthe ‘Ca. P. oryzae’ reference strain (AB052873,D12581), which indicates that SCGS phytoplasma inIndia is a ‘Ca. P. oryzae’-related strain.

Earlier SCGS isolates in India were grouped in riceyellow dwarf (XI) group on the basis of 16SrDNAsequence analysis and group based detection (Nasareet al. 2007; Rao et al. 2008; Viswanathan et al. 2011).

EF614269 Sugarcane grassy shoot

JX862179 Sugarcane grassy shoot isolate SO-SJP

GQ850122 Coconut root wilt

FJ794816 Coconut root wilt

DQ459439 Sugarcane grassy shoot

KC595881 Exitianus indicus (leafhopper)

HF586636 Sugarcane grassy shoot SC-Phy004

AB052874 Sugarcane white leaf

JX273772 Coconut root wilt

AM261831 Sugarcane grassy shoot VD7

HF586654 Sugarcane grassy shoot SC-Phy475

JN967911 Indian arecanut yellow leaf

FM208258 Sugarcane white leaf

AB646271 Sugarcane white leaf

AY736374 Napier grass stunt

AB052873 ‘Ca.P. oryzae’

D12581 ‘Ca.P. oryzae’

AF509324 Sorghum grassy shoot

AB741629 Goosegrass white leaf

M23932 A.laidlawii

99

59

76

62

78

56

0.01

Fig. 3 Phylogenetic tree showing the relationships among theSCGS phytoplasma, Exitianus indicus phytoplasma and referencephytoplasma strains. Accession numbers are specified in the tree.

‘Ca. P.’ stands for ‘Candidatus Phytoplasma sp.’ A. laidlawii wasused as an out group. Mega 4.0 software was used to construct thetree

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There are three subgroups in group XI (rice yellowdwarf phytoplasma (D12581) in rice from Japan :XI-A; sugarcane white leaf phytoplasma in sugarcane(X76432) from Thailand: XI-B; Leafhopper-borneBVK phytoplasma (X76429) from Germany: XI-C)(Lee et al. 1998) . The present study could assign SCGSisolate from Uttar Pradesh, India, in 16Sr XI-B sub-group. Prior to this study only D. vulgaris was reportedto transmit SCGS phytoplasma in India (Srivastava et al.2006). However, the detection of SCGS phytoplasma in

another leafhopper, E. indicus under natural conditions,suggests the occurrence of another potential vector ofSCGS disease in India. As the incidence of SCGSdisease is increasing in many commercial sugarcanevarieties in Uttar Pradesh (Tiwari et al. 2012;Viswanathan & Rao 2011), the detection and identifica-tion of a vector for secondary spread of SCGS phyto-plasma is important for epidemiological studies anddevelopment of suitable and timely managementpractices.

Fig. 4 Comparison of virtual RFLP patterns derived from in silicodigestions of 1.25 kb 16S rDNA sequences of (left) SCGS phyto-plasma (JX862179) and (right) sugarcane white leaf (X76432)

digested using 17 different restriction endonucleases indicatingthat the SCGS belonged to 16Sr XI-B phytoplasma subgroup

Fig. 5 Actual RFLP analysisof 1.2 kb of 16Sr DNAPCRproduct of sugarcane grassyshoot phytoplasma (ampli-fied using primer pairR16R2n/R16F2n) digestedwith BamHI, TaqI and BfaIrestriction enzyme, M: Øx174/HaeIII marker

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Acknowledgments The authors are thankful to the Departmentof Science and Technology, New Delhi, India, for providingfinancial assistance during the course of the study. The authorswish to express sincere thanks to the Head, Division of PlantPathology, and the Director, Indian Agricultural Research Insti-tute, for partial funding and the facilities for analysis of sequencedata. The authors also wish to thank Dr. C.A. Viraktamath, Prin-cipal Investigator, Network Project on Insect Biosystematics, De-partment of Entomology, GKVK, Bangalore, India, for identifyingthe taxonomy of leafhopper species. The help rendered by Direc-tor, UP Council of Sugarcane Research Shahjahanpur for provid-ing facilities to survey the experimental sugarcane fields work atShahjahanpur is sincerely acknowledged.

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