Molecular and Taxonomic Diversity of Cotton ...

VEGETOS

Vol. 27 (2) : 225-228 (2014)

DOI: 10.5958/2229-4473.2014.00036.6

Molecular and Taxonomic Diversity of Cotton Leafhopper, Amrasca biguttula biguttula (Ishida) in Major Cotton Growing Districts of Karnataka D Sagar*, R A Balikai and Ramesh S Bhat1 Received: 21.6.13 / Revised: 19.5.14/ Accepted: 12.7.14 / Published online: 30 September, 2014 This article is published in open access at www.vegetosindia.org

Abstract Cotton leafhopper, Amrasca biguttula biguttula (Ishida) is an alarming pest causing both quantitative and qualitative losses on Bt cotton. Taxonomic identification results revealed that composite sample of adult cotton leafhopper population collected from Dharwad, Belgaum, Haveri, Mysore, Raichur, Yadgir districts was Amrasca biguttula biguttula (Ishida). Fragment of mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced from the cotton leafhoppers collected from six districts. COI gene sequences of A. biguttula of Belgaum, Haveri and Mysore districts showed 99 per cent similarity with NCBI published sequence of A. biguttula COI gene (HQ113231.1), while those from Raichur and Yadgir districts showed 98 per cent similarity. Population from Dharwad district showed 96 per cent similarity. Sequence analysis showed high similarity among the leafhopper population of major cotton growing districts of Karnataka. Key words: Amrasca biguttula biguttula, COI gene, dendrogram, sequence similarity, taxonomy Introduction Among the sucking pest complex of Bt cotton, the cotton leafhopper, Amrasca biguttula biguttula (Ishida) (Homoptera: Cicadellidae) is an alarming pest causing both quantitative and qualitative losses. After the introduction of Bt cotton, there has been a check to the bollworm complex. But, the sucking pest population especially leafhoppers increased gradually reaching economic injury level in many parts of India (Mohan and Nandini 2011). Earlier this pest was considered as a serious pest during vegetative phase of crop. At present, it is a serious pest during reproductive phase too, prevailing up to 120 days after sowing and has become one of the limiting factors in economic productivity of

the crop (Balakrishnan et al. 2007). The taxonomic tools for identification of insect pest are often laborious, time consuming and requires considerable skill; whereas biochemical markers, such as esterase isozymes are influenced by the environment, food plants (hosts) and stage of insect. In contrast, molecular markers are relatively recent, reasonably accurate and can be complimented with the other methods. Molecular markers are more powerful tools to assess relationships at the population level (Folmer et al., 1994). Genetic differentiation and gene flow between the species can be analyzed by using various molecular markers likely RAPD, microsatellite, mitochondrial and ribosomal markers (Kim et al. 2000). At present, there is scanty information available on molecular variation among the local population of cotton leafhopper. Hence, the present investigation was carried out using cytochrome oxidase I (COI) region of mitochondrial DNA to understand the genetic difference in leafhopper populations of major cotton growing districts of Karnataka. Materials and Methods Adult cotton leafhoppers were collected from farmers’ field in major cotton growing districts of Karnataka viz., Dharwad, Belgaum, Haveri, Mysore, Raichur and Yadgir and the cultures were designated as CLH-I, CLH-II, CLH-III, CLHIV, CLH-V and CLH-VI, respectively. These insects were stored in 70 per cent alcohol at -20°C for further analysis. Total genomic DNA was extracted from adults using the CTAB protocol described by Marzachi et al. (1998). A 710 bp fragment of the mitochondrial cytochrome oxidase I gene was amplified with the primers LCO-1490 (5´-GGTCAACAAATCATAAAGATATTGG-3´) and H C O - 2 1 9 8 ( 5 ´ TAAACTTCAGGGTGACCAAAAAATCA-3´) described as conserved primers for invertebrate

Department of Agricultural Entomology, University of Agricultural Sciences, Dharwad-580 005, Karnataka, India 1 Department of Biotechnology, University of Agricultural Sciences, Dharwad-580 005, Karnataka, India *Corresponding author: e-mail: [email protected]

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Molecular and Taxonomic Diversity of Cotton Leafhopper Fig 1. PCR amplification of COI region (710 bp) of cotton leafhopper (Amrasca biguttula biguttula) collected from six major cotton growing districts of Karnataka

DNA (Folmer et al. 1994). The polymerase chain reaction (PCR) reaction volume (20 µl) contained 13.7 µl water, 2.0 µl dNTP’s (2 mM), 2.0 µl Taq buffer (10X), primers (10 pmoles per µl) of 0.5 µl each, 1.0 µl DNA template and Taq polymerase (5 U/µl) of 0.3 µl. Amplification of COI gene was carried out in a thermal cycler with the following conditions, 95°C for five minutes as initial denaturation followed by 32 cycles each of denaturation at 94°C for one minute, annealing at 57°C for one minute, extension at 72°C for two minutes. Final extension was kept at 72°C for 20 minutes. The PCR amplified product was electrophoresed on one per cent agarose gel along with 100 bp DNA as ladder and gel image was visualized and documented in the gel documentation system (UV Doc Image Master® VDS). The intense band of 710 bp PCR products obtained using primers were excised from agarose gel and were subjected for QIAGEN MinElute® PCR

purification kit to elute the PCR product from agarose gel. The purification of PCR product was done as described in the user’s manual. The purified product was sequenced using both forward (LCO-1490) and reverse (HCO2198) primers at Bangalore Genei (India) Pvt. Ltd., Bangalore using the ABI 3130 XL sequencer. The sequences obtained using forward and reverse primers were assembled using Vector NTI software. The sequences were subjected to BLAST analysis at http://www.ncbi.nlm.nih.gov. Sequence similarity was studied by dendrogram, which was constructed using unweighted pairgroup arithmetic average (UPGMA) method available in MEGA 4.0.2 software for the nucleotide sequences of COI region of all six district’s populations. Published sequence of A. biguttula biguttula COI gene (HQ113231.1) was downloaded from NCBI (National Center for Biotechnology Information) and used in the sequence similarity analysis. Helicoverpa armigera (KC340924.1) nucleotide sequence was used as out group in the construction of dendrogram. To study the taxonomic diversity, the population of adult cotton leafhopper collected from farmers’ field at Dharwad, Belgaum, Haveri, Mysore, Raichur and Yadgir districts were stored in 70 per cent alcohol and sent for identification to Dr. C. A. Viraktamath, Professor (Emeritus), Insect Systematics Unit, Department of Entomology, UAS, GKVK, Bangalore, Karnataka. Results and Discussion The total genomic DNA isolated from cotton leafhopper collected from different districts of Karnataka was confirmed by running on 0.8 per cent agarose gel electrophoresis. The concentration of whole genomic DNA was 250-

Fig 2. Dendrogram based on cytochrome oxidase I (COI) gene sequence of cotton leafhoppers (Amrasca biguttula biguttula) collected from major cotton growing districts of Karnataka

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D Sagar et al. Table 1. Comparison and identity of fragment of the cytochrome oxidase I (COI) gene of Amrasca biguttula biguttula of major cotton growing districts of Karnataka with that of referred gene bank available in NCBI Sample ID

District

CLH-I

Dharwad

CLH-II

Belgaum

CLH-III

Haveri

CLH-IV

Mysore

CLH-V

Raichur

CLH-VI

Yadgir

Identified as

Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial

Gene Bank Accession Number HQ113231.1

Reference

% Similarity

Kranthi et al., 2010

96

HQ113231.1


99

HQ113231.1


99

HQ113231.1


99

HQ113231.1


98

HQ113231.1


98

Table 2. Taxonomic identification results of cotton leafhopper in major cotton growing districts of Karnataka

Districts / Locations Dharwad Belgaum Haveri Mysore Raichur Yadgir

Species identified as

Order : Family

Amrasca biguttula biguttula (Ishida) A. biguttula biguttula A. biguttula biguttula A. biguttula biguttula A. biguttula biguttula A. biguttula biguttula

300 ng/µl. The polymerase chain reaction with LCO-1490 and HCO-2198 primers amplified expected 710 bp products in all the six samples (Fig 1). The amplicon was excised and eluted successfully in all the samples. The single pass sequence of these products obtained with forward and reverse primers were used to get the longest sequence by assembling them using Vector NTI software. These nucleotide sequences were BLAST searched at NCBI. The sequences from the isolates of all the six districts showed high similarity with published sequence of A. biguttula COI gene HQ113231.1). This was a confirmation at molecular level that the isolates belonged to cotton leafhopper, A. biguttula biguttula supporting the taxonomic identification. The sequence similarity of the COI gene from the six isolates with those acces-

Hemiptera: Cicadellidae

Number of insects identified 27 40 18 35 48 47

sions at NCBI ranged from 96 to 99 per cent. Fragment of COI gene sequences of A. biguttula of Belgaum, Haveri and Mysore showed 99 per cent similarity with NCBI published sequence of A. biguttula COI gene (HQ113231.1), while Raichur and Yadgir A. biguttula showed 98 per cent similarity and Dharwad A. biguttula showed 96 per cent similarity (Table 1). Similar studies were made by Asokan et al. (2012) to study the genetic variation of actin gene in H. armigera. Sequence similarity was analysed between the COI gene sequences of six district’s leafhopper population. Dendrogram results of six district’s A. biguttula population for COI region revealed that, there are two major clusters, cluster A comprising CLH-I (Dharwad), CLH-III (Haveri), CLH-V (Raichur) and CLH-VI (Yadgir) 227

Molecular and Taxonomic Diversity of Cotton Leafhopper

district population, whereas cluster B comprises CLH-II (Belgaum) and CLH-IV (Mysore) (Fig 2). In cluster A, CLH-I and CLH-III are closely related; these two are related to CLH-VI population and CLH-V population showed intimate relation with CLH-VI population. In cluster B, CLH-II and CLHIV population are related and CLH-IV population is more closely related to published sequence, HQ113231.1. As expected, branching of H. armigera was altogether different having wide divergence with separate branch in dendrogram. Similar studies were made by Kranthi et al. (2011) for mitochondrial COI region of the key pest Empoasca devastans (Distant) and the results revealed that north Indian leafhopper population on cotton although morphologically and taxonomically similar, they were genetically distinct from leafhopper of south and central India. Anonymous (2012) studied the genetic diversity using COI region of leafhopper (E. devastans) collected across the country. Results depicted that leafhopper population of north Indian region is different from south Indian population, while central India showed mixed population. The central Indian leafhopper population formed the polyphyletic groups while the north and south Indian population formed the monophyletic groups. From the dendrogram, it could be concluded that there is a narrow molecular diversity among the leafhopper population of major cotton growing districts of Karnataka. The higher similarity among leafhopper population might be due to less environmental variation with not much variation in the cropping pattern. Taxonomic identification revealed that composite collection of adult cotton leafhopper population of Dharwad, Belgaum, Haveri, Mysore, Raichur, Yadgir districts was only A. biguttula biguttula (Table 2). Similar studies were made by Hanumantappa and Pramod (2010) in sunflower to document five species of leafhopper and A. biguttula biguttula was the predominant species. Acknowledgements Senior author thankfully acknowledges Department of Science and Technology, Government of India for financial assistance under INSPIRE fellowship and to Dr. C. A. Viraktamath, Professor (Emeritus), Insect Systematics Unit, Department of Entomology, UAS, GKVK, Bangalore, for his help in identification of cotton leafhopper.

Station, Coimbatore, Tamil Nadu 641 003, India. Asokan R, Nagesha SN, Krishnakumar NK and Manamohan M (2012) Genetic variation of actin gene of Helicoverpa armigera Hubner (Lepidoptera: Noctuidae). J Ent Res 36(2): 123127. Balakrishnan N, Murugesan N, Vanniarajan C, Ramalingam A and Suriachandraselvan M (2007) Screening of cotton genotypes for resistance to leafhopper, Amrasca biguttula biguttula (Ishida) in Tamil Nadu. J Cotton Res Dev 21(1): 120-121. Folmer O, Black M, Hoeh W, Lutz R and Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Marine Biol Biotechnol 3: 294-299. Hanumantappa M and Pramod K (2010) Incidence and diversity of leafhopper on sunflower. Karnataka J Agric Sci 23(1): 149-150. Kim I, Bae JS, Choi KH, Jin BR, Lee KR and Sohn HD (2000) Haplotype diversity and gene flow of diamond back moth, Plutella xylostella (L.) (Yponomeutidae: Lepidoptera) in Korea. Korean J Appl Ent 39: 43-52. Kranthi S, Kranthi KR, Rishi Kumar, Dharajothi, Udikeri SS, Rao GMVP, Zanwar PR, Nagrare VN, Naik CB, Singh V, Ramamurthy VV and Monga D (2011) Emerging and key insect pests on Bt cotton - their identification, taxonomy, genetic diversity and management. Paper presented in: World Cotton Research Conference-5, Mumbai, India, 7-11 November, 2011, pp. 281-286. Kranthi S, Mandle MG, Pareek R, Sangode V, Zanwar P, Udikeri SS, Padwad S and Kranthi KR (2010) Amrasca biguttula cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial (Gene Bank accession number: HQ113231.1). http://www. ncbi.nlm.nih.gov/ nuccore/ 308737210. Marzachi C, Veratti F and Bosco D (1998) Direct PCR detection of phytoplasmas in experimentally infected insects. Ann Appl Biol 133: 45-54. Mohan S and Nandini S (2011) A promising entry for cotton leafhopper. Pestology 35(6): 1113.

References Anonymous (2012) Project Report (2007-12), Technology Mission on Cotton, Published by Director and Member Secretary, CICR, Regional 228