|Türk. entomol. bult., 2010, 1 (1): 000-000
Molecular diagnostic of two closely related mealybug species (Hemiptera: Pseudococcidae)
Istvan TÓBIÁS1 Ferenc KOZÁR1 M. Bora KAYDAN2
Planococcus citri (Risso, 1813) and Planococcus ficus (Signoret, 1875) (Hemiptera: Pseudococcidae) are important polyphagous pests species. Their distinguishing is very difficult by using morphological characters especially in male or larval stage. The aim of this study was to find a simple and fast PCR-based method to separate these two mealybug species. Thanks to the use of a short DNA extraction method and species-specific primer pairs P. citri and P. ficus can be distinguished at any developmental stages within three hours.
Key words: DNA extraction, PCR detection, Planococcus citri and Planococcus ficus
Molecular studies can be used for different purposes when studying mealybugs (Hemiptera: Coccoidea: Pseudococcidae): DNA barcoding, phylogeny, population genetics (Demontis et al., 2007; Gullan et al., 2010; Malausa et al., 2010; Hardy et al., 2008; Rung et al., 2008). On the other hand these methods can be used for fast mealybug identification. This is particularly important for identification of immature mealybug stages or adult males by quarantine services for example because morphological identification is restricted to adult females.
Planococcus citri (with 183 host plant species) and Planococcus ficus ( with 25 host plant species) are important pests infesting a wide range of crops and ornamental plants (Ben-Dov et al. 2010). These two species are often sympatric and display similar biological characters. Distinction between females of P. ficus and P. citri is mainly based on slight morphological differences, such as the length of certain setae, the number of tubular ducts, the presence of translucent pores on the hind legs, and the distribution of multilocular pores (Cox, 1989). Hence, identification of these two species by classical methods needs high level of expertise. This is why many molecular studies have recently been conducted to distinguish between closely related species (Demontis et al., 2007; Gullan et al. 2010; Malausa et al 2011; Rung et al, 2008). Very recently Tóbiás et al. (2010) studied on this subject and could get DNA extraction from dry males in the pheromone traps and could show differences (about 9 % differences) between sequences by using rDNA ITS2 region.
Material and Method
P. citri individuals were collected from an ornamental plant Aeonium arboreum (Crassulaceae) in Plant Protection Institute Hungarian Academy of Sciences (Budapest, Hungary). All development stages (egg, first second and third instar nymphs, adult females and males) were collected. P. ficus was obtained from mass culture on potato (Yüzüncü Yıl University, Van, Turkey). For this species only adult female, adult male and eggs were collected. All individuals were stored in 96 % ethanol at -20 oC.
Total genomic DNA was extracted from each individual using the REDExtract-N-AmplTM Tissue PCR Kit (Sigma) following manufacturer instructions with a slight modification: the individual was punched with a sterile needle and covered by 15-50µl of extraction solution (15 µl for eggs and 1st instar larvae, 20-30 µl for 2nd and 3rd instar larvae, 50 µl for adults) and incubated at room temperature for 10 minutes, and 3 minutes at 95 oC. Then, the same volume (15-50 µl) of neutralization solution was added to the sample and mixed through vortexing. One micro liter from this mixture was used as DNA matrix in the PCR. Amplification was performed in a 25 µL volume containing the PCR buffer (10 mM Tris-HCl, pH 9.5, 2.5 mM MgCl2, 50 mM KCl, 0.1 % Triton X100), 100 ng each of dATP, dCTP, dGTP and dTTP, 0.1 nM of each sense and antisense primers and 5 U Taq polymerase (Invitrogen). The forward primer was CAS5p8sFc (5’-GCGAACATCGACAAGTCGAACGCACAT-3’) (Kim and Lee 2008) and the reverse primers were designed from the ITS2 generated by Tóbiás et al. 2010 (Figure 1). The primers Pcitrirev1 (5’-GATCCTCCGTATATCTTCTC-3’) and Pficusrev1 (5’-GTGATGGTGGTAACGATAGG-3’) were used for P. citri and P. ficus, respectively. Twenty-five or thirty-five PCR cycles were performed (Eppendorf Mastercycler gradient) with the following parameters: initial denaturation at 95 oC for 4 min; denaturation at 94 oC for 30 s, hybridization at 54oC for 20 s and elongation at 70oC for 35 s; final extension at 70 oC for 7 min. PCR products were observed in a BioDoc-ItTM system (UV Transilluminator, UVP, USA) after electrophoresis in a 1 % agarose gel.
Results and Discussion
PCR using the two species-specific primers successfully specifically amplified the DNA of the species they targeted (Figure. 2). The sizes of PCR products were 459 bp for P. citri and 508 bp for P. ficus. Using a PCR of 25 cycles generated a small quantity of PCR product from the DNA extract of a first instar larva (Figure 2). Hence, we increased the cycle number from 25 to 35 in order to get more products (Figure 3). By this way, the quantity of the PCR product increased for small development stages.
This work also shows that the DNA extraction procedure used here was suitable for PCR detection. The DNA extraction by REDExtract-N-AmplTM Tissue PCR Kit (Sigma) is very simple and short (around 15 minutes) when compared to most commonly used extraction methods (Qiagen kit, or Phenol-chloroform) which take several hours. It is worthwhile to mention that Extract-N-AmpTM Plant PCR Kit and REDExtract-N-AmplTM Seed PCR Kit were also suitable for producing adequate quality and quantity DNA for PCR.
In conclusion, the quick DNA extraction method and species-specific primers used in this work are promising tools for distinguishing the two closely related species of P. citri and P. ficus.
İki yakın akraba unlubit (Hemiptera: Pseudococcidae) türünün moleküler tanısı
Planococcus citri (Risso, 1813) ve Planococcus ficus (Signoret, 1875) (Hemiptera: Pseudococcidae) polifag ve önemli zararlı türlerdir. Bu türlerin, özelliklede ergin öncesi ve erkek biyolojik dönemlerinin morfolojik açıdan ayrımı oldukça zordur. Bu çalışmanın amacı bu iki unlubit türünü birbirinden ayırabilmek için hızlı ve basit PCR tabanlı bir metod geliştirmektir. Çalışma sonucunda çok kısa DNA ekstraksiyon metodu kullanılarak, P. citri ve P. ficus için türe özgü belirteçler dizaynı sayesinde her iki türün tüm biyolojik dönemleri birkaç saat içerisinde ayırt edilebilmiştir.
The authors are grateful for skilful technical assistance to Zsuzsanna Benedicty Konczné, Kinga Fetykó. (Plant Protection Institute, Hungarian Academy of Sciences, H-1525, P.O.Box 102, Budapest, Hungary) and Didem Holat (Yüzüncü Yıl Üniversity, Agriculture Faculty, Plant Protection Department, 65080 Kampüs, VAN, Turkey). The authors thank to Thibaut Malausa (Chargé de Recherches INRA UMR 1301 IBSV 400 route des Chappes. BP 167 06903 Sophia Antipolis cedex. FRANCE) for reviewing and comments on the first draft of the manuscript. This research was partially supported by Hungarian Scientific Research Fund (OTKA 75889).
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