Received for publication: 29 October, 2013. Accepted for publication: 19 March, 2014. 1 National Phy tosanitary Laboratory Diagnosis, Tibaitatá Research Center, Instituto Colombiano Agropecuario (ICA). Mosquera (Colombia). jorge.angel@ica.gov.co; eeebraitr@unal.edu.co Agronomía Colombiana 32(1), 7-13, 2014 Comparison of DNA extraction methods for detection of citrus huanglongbing in Colombia Comparisión de métodos de extracción ADN para detección de huanglongbing en Colombia Jorge Evelio Ángel1, Erick Geovanni Hernández1, Néstor Andrés Herrera1, Linda Yhiset Gómez1, Ángela Patricia Castro1, Adriana Milena Sepúlveda1, and Everth Emilio Ebratt1 ABSTRACT RESUMEN Four DNA citrus plant tissue extraction protocols and three methods of DNA extraction from vector psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) were compared as part of the validation process and standardization for detection of huanglongbing (HLB). The comparison was done using several criterias such as integrity, purity and concentration. The best quality parameters presented in terms of extraction of DNA from plant midribs tissue of citrus, were cited by Murray and Thompson (1980) and Rodríguez et al. (2010), while for the DNA extraction from psyllid vectors of HLB, the best extraction method was suggested by Manjunath et al. (2008). En el proceso de validación y estandarización para la detec- ción de huanglongbing (HLB) se compararon protocolos de extracción de ADN, cuatro a partir de tejido vegetal de cítricos y tres a partir del psílido vector Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Para la comparación de los protocolos se utilizaron los criterios de integridad, pureza y concentración. Los protocolos citados por Murray and Thompson (1980) y Rodríguez et al. (2010) fueron los que presentaron los mejores parámetros de calidad en términos de extracción de ADN a partir de la nervadura central del tejido vegetal de cítricos. En la extracción de ADN a partir del psílido vector de HLB, el método que mejores resultados arrojó fue el reportado por Manjunath et al. (2008). Key words: Candidatus Liberibacter, Diaphorina citri, Psyllidae, vectors. Palabras clave: Candidatus Liberibacter, Diaphorina citri, Psyllidae, vectores. PLANT BREEDING, GENETIC RESOURCES AND MOLECULAR BIOLOGY Introduction The disease known as citrus huanglongbing (HLB) was first detected in Asian countries and Africa and more recently in American countries such as Argentina (SENASA, 2013), Costa Rica (SFE, 2011), Belize (Manjunath et al., 2010), Cuba (Martínez et al., 2009), Mexico (NAPPO, 2009), Dominican Republic (Matos et al., 2009), USA (Halberth, 2005; Manjunath et al., 2008) and Brazil (Teixeira et al., 2005), which implied important losses to the citrus indus- try (Bové, 2006). HLB is caused by alpha-proteobacteria non-cultivated genus Candidatus Liberibacter inhabiting the phloem of citrus plants (Da Graca, 1991; Tsai and Liu, 2000; Tsai et al., 2002) and is spread by vegetative propaga- tion and insect vectors, reason why an effective control is difficult (Hung et al., 2004; Manjunath et al., 2008). According to the etiological agent, its genome and the in- fluence of temperature on the expression of symptoms in the host plant, HLB has been divided into Asian, African and American variants (Halbert and Manjunath, 2004). The Asian and American variants are transmitted by the psyllid vector Diaphorina citri and are worldwide known as Candidatus Liberibacter asiaticus (Garnier et al., 2000) and Candidatus Liberibacter americanus (Teixeira et al., 2005), while the African variant Candidatus Liberibacter africanus is transmitted by the psyllid Trioza erytreae and Diaphorina citri (Bové, 2006; Lin et al., 2010). In 2007, the psyllid vector Diaphorina citri was first report- ed in Colombia by the Colombian Agricultural Institute (ICA) in citrus crops and seedlings in the departments of Valle del Cauca and Tolima; later the insect was found in the departments of Risaralda, Caldas, Quindio, Antioquia, Cordoba, Cesar, Bolivar, Atlantico, Norte de Santander, Santander, Casanare, Meta, Huila, Cauca, Nariño and Cun- dinamarca, infesting 95% of the citrus area in the Central Pacific, Orinoco and Atlantic regions of Colombia (Ebratt et al., 2011), due to Colombiań s proximity to countries where the presence of the insect vector and disease has been detected, puts the country at high risk for this serious disease of citrus. 8 Agron. Colomb. 32(1) 2014 Conventionally, visual symptoms indicate the presence of HLB (Roistacher, 1991), but over time more reliable de- tection systems have been developed based on electronic microscopy, specific fluorescent marker substances to HLB (Schwarz, 1968) and enzyme linked immunosorbent assays with monoclonal antibodies (ELISA) (Gao et al., 1993). De- veloped detection methods using PCR and real time PCR, were based on the 16S ribosomal DNA region analysis and several other regions of the bacterium,s genome (Hocquellet et al., 1999; Lin et al., 2010; Morgan et al., 2012). In order to standardize and apply molecular methods for detection of HLB in citrus regions around the country and to establish a rapid and reliable method for diagnosis, four DNA extraction protocols were compared from citrus leaf tissue, and three DNA extraction methods from Diapho- rina citri psyllids. The basic parameters evaluated in each method were: concentration, purity, efficiency and integrity. Methods and materials Plant material Ten samples of lemon mandarin (Citrus aurantifolia) leaf tissue from trees between 10 and 15 years old were collected in the area of the Instituto Colombiano Agropecuario of Mosquera (ICA-Tibaitata), and were then transported in styrofoam coolers with the appropriate cooling conditions until their final storage location. The next day, the leaves were washed with sterilized water and dried with paper towels, to begin the DNA extraction process. Capture of Diaphorina citri psyllids Adult individuals were collected using entomological nets, while the nymphs of D. citri were directly taken from symptomatic trees in different phenological stages (vege- tative, flowering or harvest) such as lemons (Citrus limon), oranges (Citrus sinensis) and mandarins (Citrus reticulata) between 4 and 20 years old, located in the municipalities of Sasaima and La Mesa, in the department of Cundina- marca. Afterwards, they were placed in 1.5 mL Eppendorf tubes with alcohol (96% concentration) and taken to the National Laboratory of Phytosanitary Diagnosis LNDF- ICA-Tibaitata, where they were stored at room temperature until the DNA extraction was performed. DNA extraction from plant tissue of citrus Four DNA extraction methods previously reported were compared. Three of them were based on CTAB and 2-mer- captoethanol (Thompson et al., 1983; Murray and Thomp- son, 1980; Rodríguez et al., 2010) and one reported by Qiagen (a commercial kit). Ten samples were tested by each method. The whole DNA was extracted from the midrib of each leave of the collected samples, which were finely cut and pulverized using liquid nitrogen according to the four protocols. All were compared through four quality parame- ters: concentration, purity, efficiency and integrity, which depend on detection of the disease during the analytical procedures based on PCR (García-Cañas et al., 2004), and the pollutants coming from the extraction process that can inhibit these reactions (Hughes and Moody, 2007). Extraction of DNA from D. citri psyllids To perform the extraction of DNA from psyllids three methods were compared: Manjunath et al. (2008), Alja- nabi et al. (1998) and Teixeira et al. (2005). In all the cases, the sample was made up by six nymphs or six adults. The concentration, purity and integrity of the three quality indicators were evaluated. Determination of the integrity, concentration, and purity of DNA extracts Extracted DNA was run on an agarose gel 1.2% to de- termine its integrity (undegraded DNA, and without DNA presence scanning) according to a scale from one to five (one meaning completely degraded DNA and five non-degraded DNA). The concentration of all samples was determined by a spectrophotometric analysis using a NanoDrop ND-1000 (Thermo Fisher Scientific, Wilming- ton, DE), according to the manufacturer’s instructions and procedure. Its purity was assessed by a 260/280 nm ratio, with and approximate value of 1.8. This value indicates the DNA is free of contaminants. The effectiveness of the DNA extraction process was calculated by the following equation: A = (B) (C) (1) D where, A is the DNA performance (ng of ADN/mg of plant tissue); B, is the volumen of the DNA extract (µL of H20 or buffer in which the extracted DNA pellet was resuspended), in accordance to the used extraction method); C, is the DNA concentration in the volume of extract (ng µL-1); D, is the plant tissue weight used in each DNA extraction method (mg). Statistical analysis The statistical analysis was performed using the "t-Student" test, specifically the couple comparison products of SAS (Statistical Analysis System) software version 8.0 (SAS Institute, 2002), which integrated both plant and insect 9Ángel, Hernández, Herrera, Gómez, Castro, Sepúlveda, and Ebratt: Comparison of DNA extraction methods for detection of citrus huanglonbing in Colombia samples, previous verification of the assumptions of nor- mality and homoscedasticity. Discussion and results Extraction effectiveness and integrity of the DNA absorbance range 260/280 nm, from leaf tissue In each of the methods for extracting DNA from leaf tissue different factors were evaluated: concentration (ng µL-1), performance (ng mg-1 DNA from plant tissues), the purity by the absorbance ratio 260/280 nm, indicating the presen- ce or absence of protein contaminants, and the integrity of the relation to the non-degradation of the total DNA. Once the parameters were evaluated, it was observed that the DNA obtained from the Murray and Thompson (1980) protocol, showed the required parameters, and its concen- tration and purity determined via equipment Nanodrop 1000 (Figs. 1 and 2) was adequate. Thompson,s protocol (1983), was a good indicator which enabled establishing that although its concentration was optimized for the needs of amplification (396.8 ng DNA/mg plant tissue, on average), its purity (1.2) indicated the presence of polyphenols due probably to the absence of PVP for DNA extraction, and its integrity (2.4) was also deficient, as degradation was evidenced, which can decrease the efficiency of the PCR (Holden et al., 2003). The protocol reported by Rodríguez et al. (2010), starting from 500 mg of plant tissue, had optimal quality param- eters with a high purity of the DNA (1.77 to 2.06), and an appropriate integrity (4.5) defined by non degraded DNA, the DNA concentrations extracted were relatively high compared with the other extraction methods evaluated (Fig. 2). Figure 3 shows the integrity of DNA extractions obtained by this protocol, which was the best of the four methods analyzed. Finally, it was demonstrated that when assessing the commercial Qiagen method, yield was inferior to the other procedures tested in this study. Additionally, the absorbance rate indicated the presence of contaminants such as not hydrolyzed proteins and polyphenols, which could not be completely removed during the process. How- ever, the integrity of the method had an acceptable value compared to the other protocols (4.1). FIGURE 1. Distribution data with respect to the average A, concentration; B, absorbance; C, integrity; D, yield of four DNA extraction protocols from citrus leaf tissue. Positive asymmetric in concentration parameters, integrity and yield was observed, while for the absorbance distribution was symmetrical. Fr eq ue nc y Group 0 2.0 1.0 2.5 1.5 0.5 4.70 40.53 76.37 112.20 148.03 Fr eq ue nc y Group 0 2.0 1.0 2.5 1.5 0.5 2.40 3.10 3.80 4.50 5.20 Fr eq ue nc y Group 1.20 1.47 1.73 2.00 2.27 0 2.0 1.0 2.5 1.5 0.5 Fr eq ue nc y A Group 0 2.0 1.0 2.5 1.5 0.5 9.50 380.20 750.90 1,121.60 1,492.30 C B D 10 Agron. Colomb. 32(1) 2014 1Kb M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 FIGURE 3. Visualization of total DNA from ten samples of citrus plants in agarose gel 1.2%, extracted by the method of Rodríguez et al. (2010). Line 1 shows a molecular weight marker (1 kb). Lines M1 to M10, show analyzed samples of citrus plant tissues. FIGURE 2. Comparison of the concentration parameters A, concentration; B, purity; C, integrity; D, performance of the four DNA leaf tissue extraction protocols cited by Rodríguez et al. (2010) (1), Qiagen (2), Murray and Thompson (1980) (3) and Thompson (1983) (4). Means with different letters indicate significant differences according to t-Student test (P≤0.05) (n = 10). Error bars indicate standart deviation. 41.3 4.7 112.2 99.2 c b a aa c b a 4.5 4.1 3.1 2.4 a b a b 2 1.4 2 1.2 413.4 9.5 1121.6 396.8 b c a b Pe rf or m an ce (n g D N A /m g le af ti ss ue ) Protocols for DNA extraction from leaf tissue 0 100 60 120 80 40 20 1 2 3 4 In te gr ity Protocols for DNA extraction from leaf tissue 0 4.5 3.5 5.0 4.0 3.0 2.5 2.0 1.5 1.0 0.5 1 2 3 4 A bs or ba nc e (2 60 /2 80 ) 1 2 3 4 0 2.0 1.0 2.5 1.5 0.5 C on ce nt ra tio n of D N A ( ng µ L- 1 ) 0 1,000 600 1,200 800 400 200 1 2 3 4 DC BA Concentration and integrity of the DNA extractions, and the absorbance range 260/280 nm, from psyllids The visualization of the results for each of the evaluated parameters (Figs. 4 and 5) to compare DNA extraction methods from psyllid D. citri, reported by Manjunath et al. (2008), Aljanabi et al. (1998) and suggested by Teixeira et al. (2005), shows that the latter did not provide good quality parameters, such as the low concentration and poor inte- grity of the DNA. This is similar to observations reported by Aljanabi et al. (1998), where although the concentration of nucleic acids was high (230.6 ng μL-1), the quality of DNA was degraded (1.1) and its absorbance (0.9) indicated the presence of contaminants in the samples. Finally, the protocol reported by Manjunath et al. (2008) allowed establishing the samples proven with this method had the best integrity among all the other tested protocols (Fig. 6), and outstanding concentration of nucleic acids and a better absorbance ratio that indicated that the samples were free of contamination. Because the concentration of the bacteria,s variants that causes HLB can be very low in the insect vector and es- pecially in citrus leaf tissue, causing that the symptoms of this disease are not always easily observed, consequently it is very important to have a method that give the best indi- cators of purity, quantity and quality, so although certain protocols in this study showed good results in one or more of the parameters evaluated, should not be used as a starting point for the diagnosis of HLB, because contaminants such as polyphenols and no hydrolyzed proteins could lead to inhibition of the PCR reactions and lead to false negatives. 11Ángel, Hernández, Herrera, Gómez, Castro, Sepúlveda, and Ebratt: Comparison of DNA extraction methods for detection of citrus huanglonbing in Colombia According this, the statistical analysis conducted by the testing method "t-Student ", significant differences were found between the methodologies evaluated for both DNA extractions made from leaf tissue and those made from D. citri. The DNA extraction method for psyllids reported by Manjunath et al. (2008), was the best quality indicators obtained and presented significant difference (p≤0.0001) compared to the methods reported by Aljanabi et al. (1998) and Teixeira et al. (2005). Regarding the protocols used from plant tissue, the difference between the methods which presented the best quality parameters (Murray and Thompson, 1980; Rodríguez et al., 2010), with the protocols FIGURE 5. Comparison of the concentration parameters A, concentra- tion; B, purity; C, integrity; of DNA extraction protocols psyllids from D. citri cited by Manjunath et al. (2008) (1), Aljanabi et al. (1998) (2) and Teixeira et al. (2005) (3). Means with different letters indicate significant differences according to t-Student test (P≤0.05) (n = 10). Error bars indicate standart deviation. a b b 3.6 1.1 1.1 b c a 1.9 0.9 2.1 a b a 227.9 230.6 66.8 In te gr ity Protocols for DNA extraction from psyllids 0 3.5 4.0 3.0 2.5 2.0 1.5 1.0 0.5 C 1 2 3 A bs or ba nc e (2 60 /2 80 ) 0 2.0 1.0 2.5 1.5 0.5 B 1 2 3 C on ce nt ra tio n of D N A ( ng µ L- 1 ) 0 200 100 250 150 50 1 2 3 A 1Kb M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 FIGURE 6. Total DNA from D. citri psyllids in agarose gel 1.2%, extracted by the method of Manjunath et al. (2008). Line 1, shows a molecular weight marker 1 kb. Samples M1-M5, show DNA obtained from adult insect. M6-M10 samples, show DNA obtained from insect nymph. Fr eq ue nc y Group 0 2.0 1.0 2.5 1.5 0.5 1.10 1.93 2.77 3.60 4.43 Fr eq ue nc y 0.90 1.30 1.70 2.10 2.50 0 2.0 1.0 2.5 1.5 0.5 Fr eq ue nc y 0 2.0 1.0 2.5 1.5 0.5 66.80 121.40 176.00 230.60 285.20 C B A FIGURE 4. Distribution data with respect to the average A, concentra- tion; B, absorbance; C, integrity of three DNA extraction protocols from D. citri psyllids. Negative asymmetric in concentration and absorbance parameters was observed, while for the integrity its distribution was positive asymmetrical. of Qiagen and the reported by Thompson (1983), was highly significant (p≤0.0001). 12 Agron. Colomb. 32(1) 2014 Conclusions The protocols with best results as quality indicators to perform DNA extractions from leaf tissue of citrus plants were cited by Murray and Thompson (1980) and Rodríguez et al. (2010). Also, the method by which the best results were obtained for this procedure from psyllids, was the proposed by Manjunath et al. (2008). Acknowledgements The authors express their gratitude to Keremane Man- junath, researcher at the American Department of Agri- culture (USDA) and Chandrika Ramadugu, researcher with the Department of Botany and Plant Sciences at the University of Riverside (CA), for their technical support and for providing positive control DNA citrus plants infected with bacteria Ca. L. americanus, Ca. L. asiaticus and Ca. 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