Genetic Similarity of Eggplant Shoot and Fruit Borer, Leucinodes orbonalis, Populations

ORIGINAL ARTICLE

Genetic Similarity of Eggplant Shoot and Fruit Borer,Leucinodes orbonalis, Populations

Murugan Marimuthu,1,2 Yasodha Perumal,1 Abida Puthenpeedikal Salim,1 and Gautam Sharma1

Shoot and fruit borer, Leucinodes orbonalis (Guenee) (Pyraustidae: Lepidoptera), has become a production con-straint in all eggplant (Solanum melongena Linn. [Solanaceae])–growing countries. In India, transgenic eggplantsexpressing Bacillus thuringiensis Cry toxins have been tested in fields by private- and public-sector agencies.Understanding population diversity is important in designing strategies for better pest management. In thepresent investigation, random-amplified polymorphic DNA markers were used to assess the genetic diversity ofL. orbonalis population collected from different field locations of Tamilnadu state in India. Of 17 random-amplifiedpolymorphic DNA primers screened, only 11 primers generated polymorphic bands (up to 14 bands). Accordingto their level of similarities, only two major clusters with no variation among population were deduced. Ourresults indicated that there is a steady genetic flow among the present population of L. orbonalis alleviating geneticvariation, which may be attributed to passive and active dispersal of the insect besides absence of host-inducedvariations among the population. As molecular variability of L. orbonalis population is an important considerationfor shoot and fruit damage of the eggplant, constant monitoring is essential to study the possible development ofCry protein resistance in L. orbonalis. Further, strict adhesion of refugia concept for transgenic eggplant cultivationmay be considered for delaying or alleviating Cry protein resistance development.

Introduction

The eggplant (synonyms: auburgine and brinjal),Solanum melongena Linn. (Solanaceae), is the most com-

monly planted vegetable crop in countries such as India,China, Japan, Egypt, Turkey, Italy, Indonesia, Iraq, Syria,Spain, and Philippines. As reported in other countries, highgenetic variability of eggplant has also been reported in India(Sidhu and Dhatt, 2007). Eggplant, native to India, is culti-vated in 31% area (5.1�105 ha) of the world, contributing atotal of 28% (82�105 tonnes) of world eggplant fruit pro-duction (FAO, 2003). In India, eggplant cultivation has beenconsidered a major source of income for resource-poorfarmers.

Leucinodes orbonalis (Guenee) (Pyraustidae: Lepidoptera),the causative agent of shoot and fruit damage in eggplants,has become a very serious production constraint in SouthEast Asia (Isahaque and Chaudhuri, 1984). Initial damages ofL. orbonalis are confined to petioles, midribs of large leaves,and auxiliary shoots (Banerjee and Basu, 1955), and thedamages restrict the plant growth (Frempong, 1979). Uponflowering of eggplants, the larva attack developing fruits,which are the primary food source for the pest comparedwith other plant parts (Atwal, 1976). The total yield damage

by this devastating pest has been reported from 40% to 80%depending upon the severity of pest incidence (Alam et al.,2003).

Insecticides have been proved to be effective against insectpests, but have failed to provide adequate control againsthidden pests such as L. orbonalis. Moreover, incessant use ofchemical pesticides causes health hazards, outbreaks of sec-ondary pests, environmental pollution, objectionable pesti-cide residues on plant products, adverse effect on nontargetorganisms, and resource degradation (Alam et al., 2003).Harmful pesticidal residues enter into food chain, and con-sumers suffer from chemical poisoning (Reddy and Srivas-tava, 2004). Overuse of pesticides also leads to the developmentof insecticide resistance among target and nontarget insects(Clark and Yamaguchi, 2002).

Transgenic overexpression of Bacillus thuringiensis Berlinerendotoxins (Bt Cry proteins) in several plant species has beenproved to be effective against lepidopteran insect pests, in-cluding L. orbonalis (Singh et al., 2005). Production and testingof Bt transgenic eggplants targeting L. orbonalis is gainingimportance in India. As genetic characterization of popula-tion of insect pests plays a pivotal role in determiningmanagement strategies (Guirao et al., 1997), it is thereforeessential to study the population structure of insect pests.

1Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University,Coimbatore, Tamilnadu, India.

2Department of Entomology, Kansas State University, Manhattan, Kansas.

DNA AND CELL BIOLOGYVolume 28, Number 00, 2009ª Mary Ann Liebert, Inc.Pp. 1–5DOI: 10.1089=dna.2009.0920

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Type: research-article

Present study describes the population structure of L. orbo-nalis from eggplants using random-amplified polymorphicDNA (RAPD)–PCR technique.

Materials and Methods

Insect population samples

The occurrence of shoot and fruit borer infestation wassurveyed during September–November 2007 in the eggplant-growing areas in Tamilnadu State of India. Eggplant fruitsand larvae of L. orbonalis were collected from different loca-tions of Tamilnadu, India (T1 c Table 1). The larvae were allowedto grow on the same fruits until pupation at the InsectBioassay Laboratory, Centre for Plant Molecular Biology,Tamil Nadu Agricultural University, Coimbatore, India. Thedeveloping live stages were maintained at 25� 28C and80–85% relative humidity, and pupae were maintained in theadult emergence cages (60�30�30 cm). Upon emergence,adults were sexed, and the newly emerged females wereseparated and immediately preserved individually in vialcontaining hexadecyl trimethyl ammonium bromide (C-TAB)buffer. The samples were maintained at �208C until DNAextraction. All reagents were purchased from BangaloreGenei (Bangalore, India).

DNA extraction

DNA was extracted from individual females of L. orbonalisusing a modified C-TAB method (Doyle and Doyle, 1987).Briefly, the adult female insects stored using C-TAB bufferwere ground individually in microfuge tubes containing100 mL of preheated C-TAB extraction buffer and incubatedat 658C for 30–45 min with occasional mixing. After incuba-tion, the tubes were cooled to room temperature and equalvolume of chloroform:isoamyl alcohol mixture (24:1) wasadded and mixed by inversion for 15 min. The suspensionwas centrifuged at 8000 g for 30 min at 48C. The clearaqueous phase was transferred to a new microfuge tube andequal volume of ice-cold isopropanol was added and mixedgently by inversion and kept at �208C overnight for DNAprecipitation. The DNA pellet was separated from aqueous

phase by brief spin and was air-dried. Then, the DNA wasdissolved in 100–150 mL of b AU1TE buffer. To eliminate the con-taminated RNA, RNase (10 mL=100mL) was added to ex-tracted DNA and incubated at 378C for 30 min. Equalvolume of chloroform:isoamyl alcohol mixture (100mL) wasadded and mixed thoroughly by repeated inversions. Themixture was centrifuged at 8000 g for 10 min at 48C, and theaqueous phase was transferred to another microcentrifuge towhich two volumes of absolute alcohol was added and in-cubated at �208C overnight. DNA was pelleted by brief spin,and supernatant was discarded. The pellet was washed with70% ethanol and centrifuged at 8000 g for 5 min at 48C. Thealcohol was discarded, and DNA pellet was air-dried com-pletely. Depending upon the size of the pellet, DNA wasdissolved in 25–50 mL of b AU1TE and stored at 48C. The concen-tration of DNA was measured using NanoDrop� ND-1000spectrophotometer ( b AU2Nanodrop Technologies), and the qual-ity was checked by 0.8% agarose gel electrophoresis beforebeing used as template in PCR.

RAPD assays

RAPD analyses were carried out according to Lima et al.(2000) with minor modifications. Briefly, amplification reac-tions were performed in a 20mL reaction volume containinga final concentration of 10 mM dNTPs, 25 mM MgCl2, di-methyl sulfoxide, Taq polymerase 3 U=mL, 10�Taq buffer,sterile water, primer 100 nM, and DNA 20–25 ng=mL. A totalof 17 decamer primers synthesized by Operon Technologies( b AU3CA) were screened for RAPD bands. Amplification wasperformed in a Thermocycler� ( b AU2BioRad) programmed for aninitial denaturation (958C for 2 min) followed by 30 cycles of1 min at 958C, 1 min at 408C, and 1 min at 728C, with finalextension at 728C for 7 min. The RAPD products were size-fractionated on a 1.5% agarose gel.

RAPD analysis

Based on log molecular weight of the comigrating 100 bpDNA marker (Bangalore Genei) and their migration dis-tances, scatter plots were established and trend lines with

Table 1. List of Sampling Location, Plant Variety, Ecosystem, and Coordinate and Population Code

for Assessing Diversity in Leucinodes orbonalis Populations from Eggplant

Plant variety=hybrid

Date ofsampling Location Ecosystem Coordinate

Populationcode

CO 2 15=09=2007 Thondamuthur, Coimbatore a 118000N–778000E COMDU 1 16=09=2007 Melur, Madurai b 098580N–788100E MDUMohinic 19=09=2007 Palacode, Dharmapuri a 128080N–788130E DMPMohinic 24=09=2007 Hosur, Krishnagiri a 128320N–788160E KRGMohinic 6=10=2007 Attur, Salem a 118390N–788120E SLMCO 2 15=10=2007 Karur b 108580N–788070E KRRAnnamalai 25=10=2007 Annamalai Nagar, Chidambaram b 118240N–798440E CDMPLR 1 6=10=2007 Vallanad, Thirunelveli b 088440N–778440E TNVPKM 1 2=11=2007 Oddanchatram, Palani a 108000N–778000E PLNCO 2 15=11=2007 Nagappattinam b 118430N–798490E NGPMDU 1 25=11=2007 Thanjavur b 108470N–798100E TNJCO 2 27=11=2007 Lalgudi, Thiruchirappalli b 108500N–788460E TPA

aPolycrop vegetable–based plain terrains.bPolycrop rice–based plain terrains.cF1 hybrid plants.

2 MARIMUTHU ET AL.


best fit were derived. Molecular weight of the fragmentcorresponding to their migration distances was calculatedbased on the mathematical expression of the trend lines. Theindividual DNA bands were scored as present or absent(1=0) in the amplification profile of each sample. Only clearbands with good resolution were scored. The scored markerdata matrix was analyzed using the standard procedure inNTsys Pc-2.0 package (Rohlf, 1998). The data matrix wasused to calculate Jaccard’s similarity coefficient (Sneath andSokal, 1973), which does not consider the joint absence of amarker as an indication of similarity. The similarity valueswere used for cluster analyses. Sequential agglomerativehierarchical nonoverlapping clustering was done using un-weighted pair-group method with arithmetic averages. Thepercentage of polymorphism was calculated as the propor-tion of the polymorphic markers to the total number ofmarkers. A dendrogram was constructed after cluster anal-ysis of the similarity coefficients by the unweighted pair-group method analysis (Sneath and Sokal, 1973) using NTsysPc-2.0.

Results

Of the 17 primers tested, only 11 primers produced de-tectable DNA banding profiles. These primer-amplifiedRAPD bands ranged from 2 to 14. Of which, the primersOPM 12 and OPM 14 generated the maximum number ofbands 5 and 14, respectively (T2 c Table 2). Except the 14 RAPDbands generated by OPM 14, all other bands are monomor-phic markers. The size of the amplicons ranged from 100 bpto more than 1000 bp (F1 c Fig. 1). The Jaccard similarity coeffi-cient showing relationship among the amplified RAPD pro-files is shown inT3 c Table 3.

The similarity coefficient of RAPD markers ranged from0.953 to 1.000. Of the pair-wise combination among 12samples of L. orbonalis, Palani location got the minimumsimilarity index (0.953), and the maximum index (1.000) wasseen in majority of the locations except Thirunelveli, Krish-nagiri, and Palani. The dendrogram (not shown) resulted intwo major clusters. When all the population (except Krish-nagiri and Thirunelveli) formed cluster I, the populationfrom Krishnagiri and Thirunelveli formed cluster II.

Discussion

Studies of genetic variability could help to determinewhether the entire L. orbonalis population is truly a singleinterbreeding population in which intense gene flow canoccur. Assessing the genetic variability could help to designthe present and future management strategies of the insectpest, particularly the key and regular pests of crop plants.Genetic variation, predominated by natural selection pro-cess, occurs through the interaction of genetic forces andchanging environmental factors through space and time, andprovides the basis for evolutionary change. Gene flow, ge-netic drift, and natural selection interact in balance to creategenetic differentiation between populations (Futuyma andPeterson, 1985). Although natural selection acts directly onphenotype, it is a major factor causing genetic differentiationat the protein and DNA level.

In the present study, the banding profiles generated byRAPD primers indicated that there is no distinct variationbetween the populations of L. orbonalis. Out of 11 primers,OPM 14 alone produced polymorphic markers. However,the unweighted pair-group method analysis dendrogramanalysis revealed that the overall population of L. orbonalis inTamilnadu has 100% similarity index. Our results are in ac-cordance with those of Karthikeyan et al. (2005), who earlierreported the genetic variability of L. orbonalis populationsthrough the pattern of carboxyl esterase isozyme and RAPDprofile and observed that carboxylesterase fraction amongpopulations resulted in the generation of 100% similarity.Karthikeyan et al. (2005) reported the scorable DNA frag-ments ranged from 1450 to 480 bp, whereas the present studydescribes the detectable RAPD fragments ranged from 1000to 100 bp.

L. orbonalis mainly damages eggplants (Atwal, 1976),though its occurrence was recorded on other solanaceousplants also, for example, Solanum tuberosum L., S. nigrum L.,Lycopersicon esculentum Mill (Alam et al., 2003), S. sisym-briifolium Lam ( b AU4Dhankar et al., 1977), S. indicum L. (Nayaret al., 1976), S. torvum L. and S. myriacanthum L. (Isahaqueand Chaudhuri, 1984), and S. khasianum Clarke (Sundara-moorthy, 1984). Beyond solanaceae, it was also reported toinfest Mamordica charantia L. (Nayar et al., 1976) and Pisumsativum L. (Atwal, 1976; Nayar et al., 1976). It was found even

Table 2. Random-Amplified Polymorphic DNA

Polymorphism of Leucinodes orbonalis Population

from Different Locations of Tamilnadu, India

Primername

Numbersof markersgenerated

Polymorphicmarkers

Monomorphicmarkers

Percentpoly-

morphism

OPM 12 5 – 5 0OPM 13 4 – 4 0OPM 14 14 14 0 100OPM 15 3 – 3 0OPE 04 4 – 4 0OPE 08 4 – 4 0OPE 15 4 – 4 0OPC 15 3 – 3 0OPA 08 3 – 3 0OPA 13 2 – 2 0OPA 01 3 – 3 0

FIG. 1. A representative random-amplified polymorphicDNA profile amplified by OPA 01 for the differentiation ofLeucinodes orbonalis from different locations of Tamilnadu,India. Lane M, 100 bp DNA ladder (Bangalore Genei); lanes1–12, individual female moths from Coimbatore, Madurai,Dharmapuri, Krishnagiri, Salem, Karur, Chidambaram, Thir-unelveli, Palani, Nagapattinam, Thanjavur, and Thiruchir-appalli.

RAPD ANALYSIS OF L. ORBONALIS POPULATIONS 3


in single eggplants raised in the backyards of houses aturban areas, indicating its dispersal and migratory ability. Itcan infest eggplants in four ways. First, newly planted egg-plant crops can be infested by L. orbonalis moths fromneighboring eggplant fields that have already been dam-aged. This is the most common source of infestation.AU5 c Second,eggplant seedlings used for transplantation can sometimescarry eggs or tiny larvae, wherein seedlings are subjected forlong-distance transports. This is especially true if one usesslightly older seedlings that are raised near an L. orbonalis–damaged older crop or heaps of dried eggplant stubble.Third, if the previous crop grown in the field was also egg-plant, the L. orbonalis pupae from the previous crop restingwithin the soil would become adults and infest the neweggplant crop. Fourth, if old, uprooted eggplants are storednearby, the pupae living underneath such plant debris candevelop into adults and infest the eggplant crop.

Infected eggplant fruits are generally transported to longdistances to find markets for human consumption, and thefruits that carry different life stages of L. orbonalis are leftunattended at vegetable markets. Consumers discard thedamaged fruits with insects, providing a chance for thesurvival of insects. Once become adults, the moths are able tosearch eggplants to colonize. L. orbonalis moths are swiftfliers. Thus, the active and passive migration of L. orbonalismight have helped the insect to maintain a steady state ofgene flow and thereby reducing the genetic variation.

The most interesting thing of L. orbonalis is its host, theeggplant; although highly diversified (Sidhu and Dhatt,2007), the commercially grown plants have no much widervariation in terms of their susceptibility to L. orbonalis(AU4 c Dhankar, 1988; Talekar, 2005). There is no selection pressurefrom the host plants on the key pest to undergo geneticchanges. Host-associated genetic differentiation had beendocumented in moth families such as the Noctuidae (Pash-ley, 1986; Subramanian and Mohankumar, 2006), Tortricidae(Emelianov et al., 1995), and Prodoxidae (Groman and Pell-myr, 2000). In the case of L. orbonalis, the specialized colo-nization on eggplants might have reduced such influencesfrom other host plants on differentiation of individualsamong the population.

The present finding suggests that there is no wider dif-ference in the case of existing population of L. orbonalis. In

insects, because of their huge diversity and generally fastrates of reproduction, it may be assumed that they wouldprovide many examples of intraspecific adaptations leadingto speciation. In an agricultural context, such radiations areimportant as pests and beneficial insects adapt to new hostswith potentially significant economic implications. Under-standing population structure of L. orbonalis may providecritical base-line information for developing sustainablemanagement strategies that include future application oftransgenic crops. The use of refuge crop for Bt-transgeniccrops is under debate, as in Bt-transgenic cotton targeting thepolyphagous insect cotton bollworm Helicoverpa armigeraHubner, at least in countries like India, which has polycropecosystems. However, our results clearly suggest the im-portance of maintaining refuge crops for Bt-transgenic egg-plants, if introduced, owing to the genetic similarity ofL. orbonalis. Besides, the results also suggest that in case ofemergence of L. orbonalis resistance in Bt eggplants, sufficientcounteraction will be taken to defer their spread to othergeographic locations.

Acknowledgment

The USAID supported ‘‘IPM CRSP Regional IPM Researchand Education for South Asia’’ scheme; Virginia Tech b AU2is ac-knowledged for the financial support. The Department ofPlant Molecular Biology and Biotechnology, Centre for PlantMolecular Biology, Tamil Nadu Agricultural University,Coimbatore, India, is acknowledged for providing the facil-ities to carryout the research.

Disclosure Statement

No competing financial interests exist.

References

Alam, S.N., Rashid, M.A., Rouf, F.M.A., Jhala, R.C., Patel, J.R.,Satpathy, S., Shivalingaswamy, T.M., Rai, S., Wahundeniya, I.,Cork, A., Ammaranan, C., and Talekar, N.S. (2003). Develop-ment of an Integrated Pest Management Strategy for Eggplant Fruitand Shoot Borer in South Asia (Shanhua, Taiwan: AVRDC-theWorld Vegetable Center), Technical Bulletin No. 28. AVRDCPublication No. 03–548.56pp.

Table 3. Jaccard’s Similarity Coefficient Showing Relationship Among Leucinodes orbonalis

Populations from Locations of Tamilnadu, India

L. orbonalispopulation CO MDU DMP KRG SLM KRR CDM TNV PLN NGP TNJ TPA

CO 1MDU 1 1DMP 1 1 1KRG 0.976 0.976 0.976 1SLM 1 1 1 0.976 1KRR 1 1 1 1 0.976 1CDM 1 1 1 0.976 1 1 1TNV 1 1 1 0.976 1 1 1 1PLN 0.976 0.976 0.976 0.953 0.976 0.976 0.976 0.953 1NGP 1 1 1 0.976 1 1 1 0.976 0.976 1TNJ 1 1 1 0.976 1 1 1 0.976 0.976 1 1TPA 1 1 1 0.976 1 1 1 0.976 0.976 1 1 1

4 MARIMUTHU ET AL.


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Address correspondence to:Murugan Marimuthu b AU7

Department of EntomologyKansas State University

123 Waters HallManhattan, KS 66506-4004

E-mail: [email protected]

Received for publication May 17, 2009; received in revisedform July 2, 2009; accepted July 6, 2009.

RAPD ANALYSIS OF L. ORBONALIS POPULATIONS 5



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