JEB Journal of Environmental Biology Website : www.jeb.co.in ISSN: 0254-8704 (Print) ISSN: 2394-0379 (Online) E-mail : [email protected] CODEN: JEBIDP Molecular phylogenetic analysis of mango , mangiferae from Punjab

Geetika Banta, Vikas Jindal*, Bharathi Mohindru, Sachin Sharma, Jaimeet Kaur and V.K. Gupta Molecular Biology Laboratory, Department of Entomology, Punjab Agricultural University, Ludhiana - 141 004, India Corresponding Author Email: [email protected]

Abstract (Hemiptera: Pseudococcidae) are major pests of a wide range of crops and ornamental plants worldwide. Their high degree of morphological similarity makes them difficult to identify and limits their study and management. In the present study, four Indian populations of mango Publication Info mealybug (mango, litchi, guava from Gurdaspur and mango from Jalandhar) were analyzed. The mtCOI region was amplified, cloned, the nucleotide sequences were determined and analysed. All Paper received: the four species were found to be D. mangiferae. The population from Litchi and Mango from 12 May 2014 Gurdaspur showed 100% homologus sequence. The population of Guava-Gurdaspur and Mango- Jalandhar showed a single mutation of 'C' instead of 'T' at 18th and 196 th position, respectively. Revised received: Indian populations were compared with populations from Pakistan (21) and Japan (1). The 13 April 2015 phylogenetic tree resulted in two main clusters. Cluster1 represent all the 4 populations of Punjab, India, 20 of Pakistan (Punjab, Sind, Lahore, Multan, Faisalabad and Karak districts) with Accepted: homologous sequences. The two population collected from Faisalabad district of Pakistan and Japan made a separate cluster 2 because the gene sequence used in analysis was from the COI-3p 22 April 2015 region. However, all the other sequence of D. mangiferae samples under study showed a low nucleotide divergence. The homologus mtCO1 sequence of Indian and Pakistan population concluded that the genetic diversity in mealybug population was quite less over a large geographical area. Keywords Diversity, Drosicha mangiferae, , Phylogenetic tree

Introduction Coccoidae is a serious, dilapidating, polyphagus, dimorphic and notorious pest of mango in India and neighboring Mango (Mangifera indica L.), known as the king of countries. Its nymphs and female bugs suck plant sap from fruits, is an important commercial crop grown in tropical and roots, tender leaves, petioles and fruits leading to fruit drop. sub-tropical countries (Abdullah and Shamsulaman, 2008). These bugs also exude sticky honey dew over the mango tree Mango crop is subjected to a number of diseases and insect leaves, on which sooty mold fungus develops which pests at all stages of its development i.e. from nursery to ultimately reduces the photosynthetic efficiency of tree consumption of fruits. The crop is attacked by about 492 (Pruthi and Batra, 1960 ). species of , 17 species of mites and 26 species of nematodes world wide of which 188 species have been reported All plant parts viz., trunk, branch, twig, leaf, petiole, from India (Tandon and Verghese, 1985; Srivastava, 1998). flower and fruit are attacked by different pests among which Among these, mealybugs (: Pseudococcoidae) are mealybugs are the important pest of mango. Twenty species important group of phytophagous insects that cause significant of mango mealybug have been reported of which D. Onlinemangiferae (G.), D. Copy stebbingi (G.) and Rastrococcus damage worldwide (Miller et al., 2002). iceryoides (G.) are considered to be key destructive species Mango mealybug (Drosicha mangiferae), also of mangoes in subcontinent of South East Asia (Karar et al., known as giant mealy bug belonging to superfamily 2012). The mango mealybugs (R. iceryoides and R. invadens)

© Triveni Enterprises, Lucknow (India) Journal of Environmental Biology, Vol. 37, 49-55, January 2016 50 G. Banta et al. have been reported on a number of economically important microcentrifuge tube. The suspension was incubated at 65°C plants causing economic damage in fruit crops, ornamental for 45 min and then thoroughly mixed with equal volume of plants and forest trees (Moore, 2004; Sundararaj and Devaraj, Sevag solution (chloroform: isoamyl alcohol:: 24:1) by 2010). vortexing. The suspension was centrifuged at 12,000 rpm for 5 min at room temperature. The upper aqueous layer was Identification of scale insects to the species level is carefully transferred to fresh microcentrifuge tube and the often challenging due to their small size. DNA barcoding is a extracted DNA was precipitated by adding equal volume of taxonomic system structured on sequence information from a ice-cold isopropanol in the presence of 1/10th volume of 2.5 short stretch of core DNA sequence (Santos and Faria, 2011). mM sodium acetate solution. The precipitated DNA was A region of approximately 658-bp of the mitochondrial gene collected in pellet by centrifugation. DNA pellet was washed cytochrome c oxidase I (COI) was proposed as the barcode with 70% ethanol, air dried at room temperature and source to identify and delimit all species (CBOL, dissolved in 100 ml TE (Tris EDTA, 100 mM) buffer Consortium for the Barcode of Life, available at containing DNAse free pancreatic RNAse (10 mg ml−1 ) and http://www.barcoding.si.edu/DNABarCoding.htm). It stored at −20 °C until used. The quality of isolated DNA was involves sequencing of this particular portion of DNA, determined using horizontal agarose (0.75% agarose followed by comparison with other sequences previously containing ethidium bromide 1 mg ml-1 ) gel electrophoresis in deposited in the database. Species are identified by matching 1× Tris–acetate–EDTA buffer at 75 V for 1 hr. DNA bands the obtained sequence with sequences of known identity were visualized and recorded using a UV Gel Documentation already in the database (Hebert et al., 2003). system (Ultra Cam). DNA barcoding, nucleotide sequencing of PCR amplification : Mitochondrial cytochrome oxidase I mitochondrial cytochrome oxidase I (mtCOI) gene, has been (mtCOI) gene region from total DNA of mealybug nymph also established as standard for species level identification of was PCR amplified with specific primers set (F- mealybugs. However, only few reports on molecular attcaaccaatcataaagatattgg and R taaacttctggatgtccaaaaaatca) identification, genetic relationships and species composition (Hajibabaei et al., 2006). Each PCR reaction mixture in mealybugs on various host plants across different consisted of insect DNA- 10 ng, primers (10 μM)- 1.0 μl each, geographical areas is available. Among mango mealybug, 10× Taq reaction buffer- 2.0 μl, Taq polymerase 2 U, 5 mM the ITS and CO1 gene of different mealybug species from dNTPs mix- 1.0 μl and distilled water to make- 20 μl. PCR Pakistan has been studied by Ashfaq et al. (2011). Keeping in amplification was accomplished in a programmable DNA view the severity of the problem and importance of molecular thermolcycler (Mastercycler Gradient, Eppendorf) using PCR techniques in identification of insects at species level as program: 95°C- 5 min (95°C- 1 min, 52°C- 1min, 72°C- 2 mentioned above, the present study was conducted to min)×30 cycles, 72°C-10 min, and stored at 4°C. PCR product genetically characterize the composition of mango mealybug was analyzed by horizontal agarose gel electrophoresis by co- species and asses the molecular phylogenetic relationship of running a molecular weight standard (100 bp DNA ladder mango mealybug, D. mangiferae infesting mango in Punjab plus, Fermentas, Life Sciences) along with the samples. and other parts of the world. Cloning and sequencing of mtCO1 gene : Amplified PCR Materials and Methods product was purified from agarose gel block using 'QIAquick Gel Extraction Kit' (Qiagen) as per manufacturer's protocol. Collection of mealybug population : The nymphal The purified DNA fragment was cloned in a sequencing populations of mango mealybug were collected from three different hosts viz., mango, litchi and guava from two vector pTZ57R/T using 'InsT/A Clone PCR product cloning locations of Punjab i.e. Gurdaspur (adjoining Pakistan kit' (Fermentas Life Sciences) and transformed into border) and Jalandhar (Table 1). The nymphs were preserved Escherichia coli DH5α host cells. Inserted DNA (amplified in absolute alcohol in glass vials (15mm dia and 50mm PCR product) in the respective recombinant clones was height) till further used for isolation of genomic DNA. custom sequenced for both strands, using sequencing services of M/S Xcelris (Ahmedabad, India). Final sequence Extraction of total DNA : Single individual nymph from of all the individual mtCOI gene fragments from mealybug each population was thoroughly washed with sterile double populations were edited using DNA software Chromaslite distilled water followed by 90% ethanol. The genomic DNA 201 and CLC Sequence Viewer 6.5.4 (CLC bio A/S) and was extracted from single nymph using standardOnline cetyl submitted to BOLD databaseCopy and GenBank (Table 1). trimethyl ammonium bromide (CTAB) method (Cubero et al., 1999). Two nymphs from each population were used as Molecular analysis of mtCOI sequences for genetic two replications. The nymph was ground with a micropestle variation and phylogeny: All the mtCO1 sequences in the presence of 0.5 ml of 2% CTAB buffer in 1.5 ml representing four different populations were aligned using CLC

Journal of Environmental Biology, January 2016 Molecular phylogenetic analysis of mango mealybug 51 Sequence Viewer 6.5.4 (CLC bio A/S) and the sequence Multiple alignments of mtCOI sequences of four divergence was observed. The comparative analysis of different mealybug populations from Gurdaspur and Jalandhar mtCOI sequences from different mealybug samples was done showed that D. mangiferae of Punjab were almost similar using mealybug isolates from Pakistan (21) and Japan (1) that are available in GenBank Database. The sequences were aligned and processed to homologous 521bp region to develop comparative genetic analysis of D. mangiferae. The sequences were analysed, aligned and phylogenetic tree was created using maximum likelihood statistical method with the MEGA6 program (Tamura et al., 2013).

Results and Discussion

Mitochondrial cytochrome oxidase1 (mtCO1) is preferred ~700bp universal barcode for animal kingdom. Using total genomic DNA from individuals of mealybug populations as template, mtCOI specific primers resulted in amplification of single amplicon of ~700 bp (Fig 1). Blast hits of our sequences in NCBI GenBank database showed 99 to 100% homology with sequences of Drosicha mangiferae, and thus all the four samples from Punjab were identified as D. mangiferae. The L1234 sequences were submitted to Barcode of Life Database Fig. 1: PCR amplification of mtCOI gene from mealybug, Drosicha (www.barcodeoflife.org) and were assigned respective mangiferae samples. Lane1: Lichi-Gurdaspur, lane 2: Mango-Gurdaspur, BOLD IDs (Table 1). lane 3: Mango-Jalandhar, lane 4: Guava-Gurdaspur, L: 100bp DNA Ladder

Table 1.: Source of mealybug, Drosicha mangiferae used for analysis of genetic diversity Location Genbank Acc No GPS coordinates Barcode Index Number Host Gurdaspur, Punjab, India KP759556 32° 1' N / 75° 23' E HEMP 007-13 Mango Gurdaspur, Punjab, India KP759559 32° 1' N / 75° 23' E HEMP 006-13 Litchi Gurdaspur, Punjab, India KP759558 32° 1' N / 75° 23' E HEMP 005-13 Guava Jalandhar, Punjab, India KP759557 31° 19' N / 75° 34' E HEMP 008-13 Mango Faisalabad, Pakistan AB523736.1 31° 25' N / 73° 4' E - Mango Sind, Pakistan HM891565 25° 53' N / 68° 31' E MAIMB214-09 Mango Shiekhupura, Pakistan HM388808 31° 42' N / 73° 59' E MAIMB210-09 Mango Faisalabad, Pakistan HM891564 31° 25' N / 73° 4' E MAIMB213-09 Mango Faisalabad, Pakistan HM891563 31° 25' N / 73° 4' E MAIMB211-09 Alstonia sp. Higashi-ku, Fukuoka, Japan AB439511 33° 37' N / 130° 25' E - - Karak, Pakistan HM388807 33° 6' N / 71° 6' E MAIMB209-09 Ziziphus jujube Lahore, Pakistan HM388809 31° 33' N / 74° 21' E MAIMB215-09 Eugenia jambolana Lahore, Pakistan JF792878.1 31° 33' N / 74° 21' E MAIMB449-09 Mango Nankana Sahab, Pakistan JF792881.1 31° 27' N / 73° 42' E MAIMB446-09 Mango Punjab, Multan, Pakistan JF792876.1 30° 11' N / 71° 28' E MAIMB452-09 Mango Punjab, Multan, Pakistan JF792874.1 30° 11' N / 71° 28' E MAIMB454-09 Mango Sindh, Pakistan JF792873.1 25° 53' N / 68° 31' E MAIMB455-09 Mango Karak, Pakistan JF792872.1 35° 53' N / 72°45' E MAIMB456-09 Mango Karak, Pakistan JF792871.1 35° 53' N / 72°45' E MAIMB457-09 Mango Karak, Pakistan JF792870.1 35° 53' N / 72°45' E MAIMB458-09 Mango Punjab, Multan, Pakistan JF792875.1 30° 11' N / 71° 28' E MAIMB453-09 Mango Karak, Pakistan JF792869.1Online 35° 53' N / 72°45' E MAIMB459-09 Copy Mango Punjab, Lahore, Pakistan JF792877.1 31° 33' N / 74° 21' E MAIMB450-09 Mango Faisalabad, Gatwala, Pakistan JF792880.1 31° 28' N / 73° 12' E MAIMB447-09 Mango Faisalabad, Jhal, Pakistan JF792879.1 31° 24' N / 73° 5' E MAIMB448-09 Mango Nawan Lahore, Pakistan JF792882.1 31° 19' N / 72° 43' E MAIMB445-09 Mango

Journal of Environmental Biology, January 2016 52 G. Banta et al.

Fig. 2: Parts of multiple alignment of mtCOI gene sequences from different mealybug, Drosicha mangiferae populations. Consensus sequence represents the most common sequence based upon all individual nucleotide positions. Highlighted are the differences in gene sequences.

(Fig. 2). The population from Litchi and Mango from ranging 0 to 0.6 % representing 12 and 7 haplotypes, Gurdaspur showed 100 % homologus sequence of mtCOI respectively. Beltra et al. (2012) analysed 33 mealybug gene. The population from Guava-Gurdaspur and Mango- populations from Spain and reported intraspecific variations Jalandhar showed a single mutation of 'C' instead of 'T' at 18th in the population of five mealybug species. and 196th position of mtCOI sequence, respectively from mealybug population collected from Litchi. The mtCOI Sequence data of COI barcode region for D. nucleotide sequence variability amongst different D. mangiferae specimens collected from different regions and mangiferae populations was quite less (<0.1%) which host plants were aligned and phylogenetic relation was indicated that all the populations of mealybugs in the present determined using maximum likelihood statistical method. study were almost genetically similar and belonged to same Based upon multiple alignment mtCO1 sequences from four species. Hebert et al. (2003) reported COI appeared to population of Punjab, India and 21 of Pakistan and one of possess enough sequence divergence to regularly allow Japan, the developed phylogenetic tree formed two major differentiation between closely related species. The only clusters (Table 1 and Fig. 3). The cluster1 consist of all the 4 report on genetic variations in D. mangiferae from different population of Punjab, India (this study) and twenty of geographical locations and host plants available is from Pakistan. These populations were from Punjab, Sind, Lahore, Pakistan (Ashfaq et al., 2011). Only minor genetic Multan, Faislabad and Karak districts of Pakistan. The differences in both ITS1 and COI barcode region were districts of Punjab in Pakistan were close to Indian territories reported among D. mangiferae that corroborate our findings. i.e. J&K, Punjab and Rajasthan, which in turn showed In another mealybug genus Ferrisia spp. average genetic similarity with biological attributes between Pakistan and distance in COI between four divided clades was 4 %, which India. Cluster 2 consist of population from Faisalabad district showed greater genetic diversity than recognizedOnline by the of Pakistan and Japan (AB523736.1Copy and AB439511 ) which current morphology-based (Gullan et al., 2010). showed divergence from other populations in cluster1 (Table Rung et al. (2008) reported genetic distances among 1 and Fig. 3). These two population formed a separate cluster individuals of mealybug i.e., Planococcus citri and P. minor because the gene sequence was from COI-3p region.

Journal of Environmental Biology, January 2016 Molecular phylogenetic analysis of mango mealybug 53

Fig. 3: Maximum likelihood tree of Drosicha mangiferae specimens collected from Punjab with 21 other D. mangiferae available in NCBI GenBank database based on mtCOI sequences. Bootstrap values (500 replicates) for each node are shown next to each branch. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 26 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 509 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

However, all the sequences of D. mangiferae samples under mitochondrial genes from mango mealybug populations study showed a low nucleotide divergence within Punjab and were sequenced and compared with sequence data of other Pakistan populations. This indicated that mealybug mealybug from different geographical regions to assess population in Indian and Pakistan were almost genetically phylogenetic relationships. Ashfaq et al. (2011) also similar. analyzed 20 mealybug populations collected from different regions of Pakistan using mtCO1, 18S and ITS1 and DNA based characterization and species composition compared with other mealybug and scale species available in of mealybug species can provide valuable informationOnline to GenBank database. TheirCopy analysis did not included any carry out further research or to study mealybug association Indian mealybug population. The Pakistan population and interaction with its natural enemies using molecular tools showed maximum homology to species of family and to make full use of the information for a successful . As the Indian population are also biological control program. In the present study, genetically similar to population of Pakistan, thus the Indian

Journal of Environmental Biology, January 2016 54 G. Banta et al. mealybugs also belongs to family Monophlebidae. providing facilities to conduct the experiments. We would Morphological studies have suggested that D. mangiferae also like to Dr Muhammad Ashfaq, Biodiversity Institute of and D. stebbingi exists in Pakistan and India, but the present Ontario, University of Guelph, Canada for helping in study showed that all the four samples from Punjab were D. analysis of data. The financial assistance for this work was mangiferae. funded by Scientific and Engineering Research Board (SERB), New Delhi. Using molecular techniques, very limited work has been published in Drosicha spp. or Rastrococcus spp. References however. these techniques are being used widely to identify and solve the cryptic taxa of mealybugs belonging to family Abd-Rabou, S., H. Shalaby, J.F. Germain, N. Ris, P. Kreiter and T. Pseudococcidae (Park et al., 2010; Malausa et al., 2011; Malausa: Identification of mealybug pest species (Hemiptera: Pseudococcidae) in Egypt and France, using a DNA barcoding Ashfaq et al., 2010; Hardy et al., 2008) and Diaspididae approach. Bull. Ent. Res., 102, 515-523 (2012). (Morse and Normark, 2006). Using mtCO1, ITS1, 28S gene Abdullah, F. and K. Shamsulaman: Insect pests of Mangifera indica sequence analysis, the complexes and identification of plantation in Chuping, Perlis, Malaysia. J. Entomol., 5, 239-251 different mealybugs genera/species viz., Planococcus, (2008). Phenacoccus, Pseudococcus, Dysmicoccus, Ferrisia, Ashfaq, M., J. Ara, A.R. Noor, P.D.N. Hebert and S. Mansoor: Molecular Maconellicoccus, Saccharicoccus, Rhizoecus, phylogenetic analysis of a (Drosicha mangiferae; Delottococcus, Hypogeococcus from USA, Australia, Hemiptera: Monophlebidae) infesting mango orchards. Pak. Euro. Ecuador, Indonesia, Israel, Mexico, Taiwan, South Africa, J. Entomol., 108, 553–559 (2011). Spain, Egypt, France, Brazil were studied (Rung et al., 2008; Ashfaq, M., A.R. Noor and S. Mansoor: DNA-based characterization of an invasive mealybug (Hemiptera: Pseudococcidae) species Gullan et al., 2010; Malausa et al., 2011; Abd-Rabou et al., damaging cotton in Pakistan. Appl. Entomol. Zool. 45, 395-404 2012; da Silva et al., 2014). Rung et al. (2008) indicated the (2010). existence of third clade from Hawaiian Islands, which Beltra, A., A. Soto and T. Malausa : Molecular and morphological differed from Planococcus citri and P. minor clades. da-Silva characterisation of Pseudococcidae surveyed on crops and et al. (2014) identified 17 different species of mealybug ornamental plants in Spain. Bull. Ent. Res., 102, 165–172 (2012) infesting vineyards collected from different locations in Cubero, O.F., A. Crespo, J. Fatehi and P.D. Bridge: DNA extraction and Brazil. Beltra et al. (2012) characterized 33 mealybug PCR amplification method suitable for fresh, herbarium and populations infesting crops and ornamental plants in eastern lichenized fungi. Pl. Syst. Evol., 216, 243-49 (1999). Spain, using a combination mtCOI gene sequence and da Silva, V.C.P., A. Bertin, A. Blin, J.F. Germain, D. Bernardi, G. Rignol, M. Botton and T. Malausa: Molecular and morphological morphological techniques. This led to identification of ten identification of mealybug species (Hemiptera: Pseudococcidae) in species and provided sequence data for three previously Brazilian vineyards. PloS one, 9, e103267 (2014). unsequenced species of the family Pseudococcidae. These Gullan, P.J., M. Kaydan and N.B. Hardy: Molecular phylogeny and studies have lead to successful development of some species recognition in the mealybug genus Ferrisia Fullaway molecular markers to identify mealybug species. Hence, (Hemiptera: Pseudococcidae). System. Entomol., 35, 329-339 there is a need to generate more gene sequence information (2010). on Indian mango mealybug so that the species diversity of Hajibabaei, M., D.H. Janzen, J.M. Burns, W. Hallwachs and P.D.N. mango mealybug could be mapped. Hebert: DNA barcodes distinguish species of tropical Lepidoptera. Proc. Natl. Acad. Sci. USA, 103, 968–71 (2006). Till date, there are no reports from India regarding Hardy, N.B., P.J. Gullan and C.J. Hodgson: A subfamily-level molecular identification or genetic/phylogenetic classification of mealybugs (Hemiptera: Pseudococcidae) based on relationships and species composition in mango mealybugs integrated molecular and morphological data. Syst. Entomol., 33, from different geographical areas on different host plants. 51-71 (2008). Hebert, P.D.N., A. Cywinska, L. Ball Shelley and R. deWaard Jeremy: This is the first report to the best of our knowledge on genetic Biological identifications through DNA barcodes. Proc Biol Sci., diversity of mealybug in India and its comparison with other 270, 313–321 (2003) population from different countries using molecular Karar, H., M. J. Arif, A. Ali, A. Hameed, G. Abbas and Q. Abbas: techniques. The resultant information and gene sequences Assessment of yield losses and impact of morphological markers of submitted to GenBank and BOLD database will serve as various mango (Mangiferae indica) genotypes on mango preliminary data for future research plan. It may also aid pest Mealybug (Drosicha mangiferae Green)(Homoptera: management programs by allowing implementation of ). Pak. J. Zool., 44, 1643-1651 (2012). species specific management strategy on mango mealybug. Malausa, T., A. Fenis, S. Warot, J.F. Germain, N. Ris, E. Prado, M. OnlineBotton, F. Vanlerberghe-Masutti, Copy R. Sforza, C. Cruaud, A. Acknowledgments Couloux and P. Kreiter: DNA markers to disentangle complexes of cryptic taxa in mealybugs (Hemiptera: Pseudococcidae). J. Appl. We would like to thank to the Head, Department of Entomol., 135, 142-155 (2011). Entomology, Punjab Agricultural University, Ludhiana for Miller, D.R., G.L. Miller and G.W. Watson: Invasive species of

Journal of Environmental Biology, January 2016 Molecular phylogenetic analysis of mango mealybug 55

mealybugs (Hemiptera: Pseudococcidae) and their threat to U.S. Planococcus (Homoptera: Pseudococcidae). Annals Ent. Soc. Am., agriculture. Proc. Entomol. Soc. Washington, 104, 825–836 101, 525-532 (2008). (2002). Santos, L.M. and L.R.R. Faria: The taxonomy's new clothes: a little more Moore, D.: Biological control of Rastrococcus invadens. Biocontr. News about the DNA-based taxonomy. Zootaxa., 3025, 66-68. (2011) Inform., 25, 17N–27N (2004). Srivastava, R.P.: Pests of Mango. In: Mango Cultivation. International Morse, G.E. and B.B. Normark: A molecular phylogenetic study of Book Distribution company, Lucknow, pp.175-299 (1998). armoured scale insects (Hemiptera: Diaspididae). Syst. Entomol., Sundararaj, R. and R. Devaraj: Record of mango mealybug, Rastrococcus 31, 338-349 (2006). iceryoides (Green) (Pseudococcidae: Hemiptera) on Pongamia Park, D. S., Y.J. Leem, K.W. Hahn, S.J. Suh, K.J. Hong and H.W. Oh: pinnata (L.) Pierre in Karnataka. Indian Forest. 136: 269–270 Molecular identification of mealybugs (Hemiptera: (2010). Pseudococcidae) found on Korean pears. J. Econ. Entomol., Tamura K., G. Stecher, D. Peterson, A. Filipski and S. Kumar: MEGA6: 103(1), 25-33 (2010). Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Pruthi , H.S. and H.N. Batra: Some important fruit pests of north-west Evol., 30, 2725-2729 (2013). India. ICAR Bull., 80, 1-113 (1960). Tandon, P.L. and A. Verghese: World list of Insect, Mite and other pests Rung, A., S.J. Scheffer, G. Evans and D. Miller: Molecular identification of Mango. Technical Document No.5, Indian Institute of of two closely related species of mealybugs of the genus Horticultural Research, Banglore, India (1985).

Online Copy

Journal of Environmental Biology, January 2016