New rapid DNA extraction method with Chelex from Venturia inaequalis spores

Journal of Microbiological Methods 115 (2015) 139–143

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New rapid DNA extraction method with Chelex from Venturia inaequalis spores Ceren Turan, Irene Maja Nanni, Agostino Brunelli, Marina Collina ⁎ Department of Agricultural Sciences, University of Bologna, Viale G. Fanin 46, 40127 Bologna, Italy

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Article history: Received 27 April 2015 Received in revised form 10 June 2015 Accepted 11 June 2015 Available online 14 June 2015 Keywords: Chelex Venturia inaequalis DNA extraction CTAB

a b s t r a c t The objective of this study was to develop a rapid method to isolate DNA from Venturia inaequalis spores for use in diagnostic DNA mutation analysis. Chelex-100 resin was evaluated and compared with a well established DNA exctraction method, utilizing CTAB in order to have a robust comparison. In this research we demonstrated that Chelex-100 efficiently makes extraction of the DNA from V. inaequalis spores available for direct use in molecular analyses. Also, the quantity and quality of extracted DNA were shown to be adequate for PCR analysis. Comparatively, the quality of DNA samples isolated using Chelex method was better than those extracted using CTAB. In conclusion, the Chelex method is recommended for PCR experiments considering its simplicity and costeffectiveness. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, advances in molecular methods have provided fast and accurate detection and identification of phytopathogenic fungi. Isolation of good quality DNA from fungi is the initial and essential step in molecular research. The good quality and quantity of recovered DNA affect the success of PCR-based method applications. Numerous reports have described procedures for the extraction and purification of fungal DNA from plants (Rogers and Bendich, 1985) and from fungi (Cenis, 1992; Chi et al., 2009; Feng et al., 2010; González-Mendoza et al., 2010; Min et al., 1995; Zhang et al., 2010). Many fungal DNA isolation methods disrupt and lyse the conidia and hyphae through mechanical (freezing in liquid nitrogen, grinding with pestle, enzyme digestion, glass bead milling, microwaving) and non-mechanical (alkaline chemicals, detergents, xanthogenates) methods in order to increase good DNA quality (Karakousis et al., 2006). Genomic DNA is usually extracted with the CTAB (cetyl trimethyl ammonium bromide) (Doyle and Doyle, 1987) method and its modifications, and then purified by toxic organic substances such as phenol/chloroform and isopropanol or ethanol precipitation (Ashktorab and Cohen, 1992). The CTAB method has been widely used for extraction of DNA from leaves, seeds, processed food samples and fungal mycelium (Capote et al., 2012). Commercial genomic DNA extraction kits have also been developed but they are generally more expensive (Ahmed et al., 2009; Feng et al., 2010). Molecular methods have also been successfully used to detect fungicide resistant genotypes of several plant pathogens, such as Venturia inaequalis (Cooke), the causal agent of apple scab disease (Koenraadt ⁎ Corresponding author. E-mail address: [email protected] (M. Collina).

http://dx.doi.org/10.1016/j.mimet.2015.06.005 0167-7012/© 2015 Elsevier B.V. All rights reserved.

and Jones, 1992; Schnabel and Jones, 2001; Zheng and Köller, 1997). The evolution of resistance to strobilurins, which represent one of the most important fungicide classes used in V. inaequalis management, must be monitored to develop the most adequate strategies for apple scab disease control. Such monitoring is often based on microbiological assays, such as conidial germination tests and molecular assays for the detection of amino acid substitution that occurs in cytochrome b (cytb) gene at codon 143 (G143A). Since G143A substitution conferring resistance to strobilurins is known (Steinfeld et al., 2002), molecular monitoring methods can be considered for monitoring V. inaequalis resistance. For many years, the CTAB extraction method has been used for V. inaequalis DNA isolation from frozen mycelia as well as from mixed and unique lesion samples ground in liquid nitrogen using a mortar and pestle (Fiaccadori et al., 2011; Lê Van et al., 2013; Manafu et al., 2014; Michalecka et al., 2011; Vejl et al., 2003). In addition to CTAB, SDS (basic sodium dodecyl sulfate) and its modified methods (Xiaoping et al., 2004) and some commercial extraction kits (Fontaine et al., 2008; Villiani and Cox, 2014) have been successfully used for extraction of V. inaequalis DNA from mycelium of bulk population and monoconidial isolates for molecular characterization (Khajuria et al, 2012), genetic diversity (Tenzer and Gessler, 1999) and fungicide resistance studies (Fiaccadori et al., 2011; Michalecka et al., 2011; Steinfeld et al., 2001). Although these methods generally produce consistent results for DNA quantity and quality, most of them take a long time, are expensive and involve the use of toxic and hazardous chemical solvents. For this reason, we developed a rapid, inexpensive and reliable procedure utilizing Chelex-100 for the DNA extraction in order to have the same starting material for in vitro sensitivity tests and molecular assays, and to process large amounts of samples per day. This method was

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developed for the first time for DNA extraction from spore suspensions of V. inaequalis monoconidial isolates to be used in CAPS–PCR assays to detect the G143A substitution and to quantify the percentage of mutated allele by Real Time PCR. Chelex-100 is a chelating resin with a high affinity for multivalent metal ions. It is frequently used to release DNA from low numbers of cells by a boiling treatment, at the same time protecting the DNA from the boiling effects with resin beads (Walsh et al., 1991). Chelex has been used previously for DNA amplification from forensic specimens (Walsh et al., 1991), blood of mice (Wingberg, 1991), dried blood spots for virology testing (Kain and Lanar, 1991) and cultures or clinical samples for microbiology testing (de Lamballerie et al., 1992). This resin has also been successfully used to recover DNA from phytopathogenic fungi such as Erysiphe necator (Brewer and Milgroom, 2010; Frankel et al., 2012), Alternaria alternata (Nemsa et al., 2012) and arbuscular mycorrhizal fungal spores (Wyss and Bonfante, 1993). The same samples were also extracted through the well-established CTAB method to compare the efficiency of Chelex 100 in extracting DNA as determined by the presence of high molecular weight DNA.

2. Material and methods 2.1. Sampling Conidia were harvested from about 20 apple scab lesions per sample by rinsing with sterile water; the suspension was diluted to 20–30 conidia/microscope field (100 ×). Suspension aliquots of 0.2 mL were streaked on Petri dishes of water agar (2% agar grade A; Becton, Dickinson and Company, Sparks, MD, USA) amended with 400 mg L−1 of streptomycin sulfate (Sigma-Aldrich, Steinheim, Germany). After 24 h incubation at 20 °C, single germinated spores were selected under stereomicroscope, drawn and placed on PDA (3.9%; Becton, Dickinson and Company) amended with 300 mg L−1 of streptomycin sulfate and chlortetracycline (Sigma-Aldrich, Steinheim, Germany), chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA). After 60 days, the colonies were ready for conidial production. Cellophane-covered surfaces of PDA were used for high yields of conidia of V. inaequalis (Parker et al., 1995). After incubation for 2 to 4 weeks, the cellophane was removed from the agar surface and placed in a 150 mL beaker containing 50 mL water. The beaker was stirred rapidly for approximately 5 min to remove the conidia from the cellophane. The conidial suspension was poured through filter into another beaker in order to remove residual mycelium. The number of conidia was determined with a Thoma cell counting chamber and the conidial concentration was adjusted to 1–3 × 105 spores mL for amplification. After conidia had been harvested, they were used immediately for DNA extraction. Fifty-eight monoconidial isolates were used to assess the efficiency of DNA extraction methods.

2.2. DNA extraction 2.2.1. Chelex method 200 μL of conidial suspension from each of the fifty-eight samples was extracted by adding 150 μL 5% Chelex-100 resin. Chelex-100 was purchased from Sigma-Aldrich, St Louis, MO, USA. Samples were vortexed for 2 min and spun for 30 s at 12,000 rpm. The mixture was incubated at 90 °C for 30 min, then vortexed for 2 min and spun for 30 s at 12,000 rpm. Samples were incubated at 90 °C for 15 min and spun for 15 s at 12,000 rpm. Amount purity and integrity of DNA samples were assessed on the basis of absorbance ratio of 1.80–1.90 at 260/280 using an Infinite 200 NanoQuant spectrophotometer (Tecan® Group Ltd., Grödig, Austria). Aliquots of the supernatant were transferred into clean tubes and purified through a Sepharose (Sigma-Aldrich, St Louis, MO, USA); samples were stored at −20 °C and amplified through PCR.

2.2.2. CTAB (cetyl-trimethyl ammonium bromide) method The genomic fungal DNA was extracted directly from the same conidial suspensions by a modified CTAB-based method (Murray and Thompson, 1980). 600 μL of CTAB buffer (2% CTAB (w/v) (Sigma-Aldrich, Steinheim, Germany), 1.4 mol/L NaCl, 20 mmol/L EDTA pH 8 100 mmol/L Tris–HCl pH 8) was added to 200 μL conidial suspension, and vortexed for 25 s. The suspension was then heated at 65 °C for 45 min. Subsequently, Table 1 Spectrophotometric measurements of amount (concentration) and purity of DNAa extracted from the same aliquots of conidial suspension using Chelex and CTAB methods. Chelex extraction

CTAB extraction

Sample ID

DNA ng/μL

Ratio A260/A280

DNA ng/μL

Ratio A260/A280

62-1 62-4 62-8 62-10 62-15 62-26 62-30 62-33 62-35 62-36 62-38 62-39 1202-27 1202-29 1202-42 1202-46 1202-47 1202-56 1202-64 1202-72 1202-75 1202-77 1202-83 1202-100 202-7 202-31 202-35 202-62 202-66 202-76 202-77 202-89 156-1 156-2 156-3 156-4 156-5 156-6 156-7 156-8 156-9 156-12 156-17 102-5 102-9 102-10 102-13 102-44 102-55 102-68 102-1 102-3 503-6 503-10 503-13 503-18 503-19 503-23 X ± S.D

20.8 25.8 28.5 27.6 30.2 26.4 31.1 28.5 28.7 25.9 26.9 26.3 22.0 29.0 19.6 16.8 33.5 25.4 29.8 30.5 28.7 27.9 29.0 32.3 29.5 28.6 31.1 30.6 32.5 26.5 25.5 27.6 29.6 28.9 29.3 29.5 28.9 28.5 30.6 32.1 22.7 29.1 27.6 25.9 26.3 28.4 27.5 27.9 27.1 26.9 29.7 24.5 27.1 26.6 25.6 25.5 24.7 29.4 28.15 ± 3.06

1.99 1.91 1.95 1.97 1.93 1.97 1.96 1.92 1.93 1.91 1.92 1.90 1.93 1.96 1.89 1.93 1.99 1.94 1.93 1.98 1.97 1.96 1.88 1.99 1.91 1.99 1.97 1.96 1.93 1.96 1.87 1.99 1.97 1.95 1.95 1.98 1.96 1.92 1.96 1.94 1.96 1.97 1.96 1.86 1.93 1.92 1.99 1.99 1.87 1.86 1.93 1.95 1.95 1.96 1.97 1.96 1.96 1.97 1.94 ± 0.03

15.5 9.5 16.8 14.5 17.6 20.5 25.6 15.5 14.7 12.3 11.9 18.8 19.2 24.9 13.5 14.8 24.5 19.6 12.6 19.8 15.9 20.3 20.6 15.9 15.9 16.0 16.5 16.9 17.0 19.5 18.3 18.9 17.6 19.3 19.6 14.6 18.9 18.6 18.7 17.9 17.8 18.6 18.3 20.8 21.3 16.8 17.5 17.3 18.1 18.4 18.6 18.9 17.1 16.5 16.7 19.3 20.1 15.5 17.85 ± 2.93

1.77 1.79 1.75 1.69 1.85 1.70 1.70 1.68 1.59 1.77 1.74 1.80 1.73 1.80 1.77 1.72 1.65 1.55 1.46 1.63 1.55 1.78 1.82 1.81 1.59 1.66 1.78 1.79 1.75 1.65 1.63 1.69 1.64 1.80 1.81 1.74 1.54 1.63 1.51 1.81 1.79 1.78 1.78 1.76 1.59 1.62 1.79 1.80 1.81 1.80 1.77 1.77 1.79 1.78 1.79 1.69 1.58 1.80 1.71 ± 0.09

P ≤ 0.001. a In all samples, DNA was extracted from 200 μL conidial suspension (1–5 × 105 spores mL−1).

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600 μL of chloroform was added to the solution and centrifuged for 15 min at 12,000 rpm. at 4 °C. DNA precipitation was carried out by adding 600 μL of isopropanol to the supernatant and centrifuging for 20 min at 14,000 rpm. The pellet was then dried under vacuum, washed with 200 μl of cold 70% ethanol and centrifuged for 5 min at 12,000 rpm. Finally, the pellet was air dried and re-suspended in 50 μl of sterile MilliQ water (AppliChem, Darmstadt, Germany). The extracted DNA was transferred into clean tubes and purified through a Sepharose (Sigma-Aldrich, St Louis, MO, USA) mini-column. DNA concentration and A260/A280 ratio were assessed using the Infinite 200 NanoQuant spectrophotometer. 2.3. DNA analysis

MV

1

141

2

156-1 156-9

3

4

5

6

7

202-7 102-5 102- 9 156-1 156-9

8

9

10

202-7 102-5 102- 9

1500 bp 1000 bp

500 bp 400 bp

2.3.1. Cleaved Amplified Polymorphic Sequence (CAPS) analysis The molecular analyses were performed after DNA extraction of monoconidial isolates. A 413 bp fragment of cytochrome b of V. inaequalis, including the region codifying the G143A site, was then amplified using the primers ANK 10 reverse (5′-CTG TTG TTA GGC TCT TCA ATG -3′) and ANK 283 forward (5′-CTG TAG TTG AAAGGC TAT TAG -3′). These primers were specific for V. inaequalis, as no amplification was observed with DNA of other fungi such as Venturia pyrina Aderh. and Alternaria species [Alternaria mali Roberts, A. alternata (Fr.) Keissler]. PCR amplifications were performed in an ICycler (Bio-Rad, Hercules, CA, USA) in 50 μL of mix reaction containing 5 μL of reaction buffer 10× (Takara, BIO INC, Japan), 0.2 mM of dNTPs, 0.25 μM of each primer, 1 μL of total DNA (20 ng/μL) and 1.25 units of rTaq DNA polymerase (Takara, BIO INC, Japan). The reaction started at 94 °C for 2 min and continued for 40 cycles at 94 °C for 45 s, 57 °C for 45 s and 72 °C for 45 s. The final extension was at 72 °C for 7 min. PCR products were then digested with TseI restriction enzyme (New England BioLabs, Ipswich, MA, USA) which is able to recognize its target site only when the mutation causing G143A substitution is present. After electrophoresis, the agarose gel (1.2%) was stained with ethidium bromide, and the sizes of DNA fragments were determined by comparison with the DNA ladder. This procedure works best on young fungal tissue with abundant conidia and the fungal colonies should be 2–3 weeks old and contamination free. 2.4. Statistical analysis Means and standard deviations were calculated using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). Differences

100 bp

Fig. 2. PCR amplification of the V. inaequalis DNA extracted by Chelex-100 and CTAB methods using the primer set ANK 10-ANK 283. Lanes 1 to 5 were extracted with Chelex, lanes 6 to 10 are the same DNA samples extracted with CTAB method. MW, 100 bp + 1.5 Kb DNA ladder (SibEnzyme Ltd., Russia).

between mean DNA yields were tested for significance using an unpaired student's t-test. P-values ≤ 0.001 were considered statistically significant. 3. Results 3.1. DNA yield and purity Results of Chelex and CTAB procedures are expressed as absolute quantity of DNA (ng/μL) in Table 1. DNA yields ranged from 16.8 to 33.5 ng per 200 μL of starting material and these yields were comparable with the CTAB method. The ratio from absorbency at A260/A280 ranged from 1.86–1.99. The results obtained using each method were compared and statistically significant differences were found between the two extraction methods. The average amount of DNA recovered using the Chelex method (28.15 ± 3.06) was significantly greater than that recovered using the CTAB method (17.85 ± 2.93). Differences between the DNA yields from the two extraction methods were highly significant

3000 bp

1000 bp 500 bp 400 bp

100 bp

Fig. 1. PCR results using the primer set ANK 10-ANK 283 for V. inaequalis monoconidial isolates using two DNA extraction methods. Lanes 1 to 6: DNA samples extracted with Chelex-100, lanes 7 to 12: The same DNA samples extracted with CTAB method. MW, 100 bp plus DNA ladder (Fermentas Life Sciences) and lane 13 is negative control (no DNA).

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156 -1

2 503-6

3

4

5

6

7

8

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503-10 503-13 503-18 503-19 503-23 102-5 102-9

10

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202-7

12

13

14

15

16

17

102-9 102-10 102-13 102-44 102-55 102-68 ( +)

18 102-1

1500 bp 1000 bp G143A specific fragment

500 bp 400 bp

100 bp

Fig. 3. TseI digestion of the 413 bp PCR fragment of the cytochrome b gene obtained by primers ANK 10-ANK 283 using DNA extracted by Chelex from QoI-sensitive Venturia inaequalis monoconidial isolates (lane; 1, 10), Qol-resistant monoconidial isolates (lane; 2 to 9 and lane 11 to 16 and lane 18). MW, 100 bp + 1.5 Kb DNA ladder (SibEnzyme Ltd., Russia). Lane 17 is the positive control (resistant isolate with G143A substitution).

(P b 0.001). The confidence levels of the averages (at 95%) were 0.80 and 0.77 for the Chelex and CTAB methods, respectively. 3.2. PCR analysis The quantity and quality of extracted DNA by Chelex was shown to be adequate for PCR-based analysis. Successful amplifications obtained by using primers ANK 10-ANK 283 (Figs. 1, 2, 3) demonstrated that DNA extracted with this procedure is suitable for PCR assays. No differences in amplification patterns were obtained for the same isolates when DNA was extracted with the two extraction methods (Fig. 1). In most cases, DNA extracted by CTAB did not show amplifications (Fig. 2). Fig. 3 is an example of digested DNA with the restriction enzyme (TseI) that makes it possible to recognize the G143A substitutions. 4. Discussion The objective of this study was to design a protocol using Chelex-100 that is quick and efficient for V. inaequalis DNA isolation with the ability to obtain good quality from spores and to provide an alternative to the CTAB-based extraction method. Chelex-100 is a chelating resin that has a high affinity for polyvalent metal ions. It was proposed that DNA degrades while a sample is boiling in the presence of Chelex by chelating metal ions that may act as catalysts in DNA breakdown at high temperatures in low ionic strength solutions (Singer-Sam et al., 1989). It was aimed to use the same template in biological assays and molecular assays to evaluate the sensitivity of V. inaequalis isolates to strobilurins and detect the mutated allele. Therefore, we established an efficient method utilizing Chelex 100 for DNA extraction from V. inaequalis spores of monoconidial isolates tested in biological assay. It was sometimes difficult to obtain PCR amplifications at the first attempt with a DNA Chelex extract, therefore a second extraction beginning from the previously extracted DNA was needed in order to increase the yield; however, the extraction time was still less than using the CTAB method. Sufficient quantities of DNA were extracted from V. inaequalis spores by Chelex 100 and this DNA was suitable for both CAPS PCR and Real Time PCR (data not shown). However, in most cases DNA quantity using the CTAB method was too low to be used directly for PCR amplification. We also found that the efficiency of extraction diminishes with the age of the V. inaequalis cultures (data not shown). The extraction with fresh material was more successful.

Mycelial and/or scabbed leaf sample extraction using the CTAB based method has been performed by many researchers for molecular studies of V. inaequalis (Fiaccadori et al., 2011; Lê Van et al., 2013; Manafu et al., 2014; Michalecka et al., 2011; Vejl et al., 2003). However, this procedure is very time consuming compared to the Chelex protocol and it involves grinding in liquid nitrogen and the use of toxic chemicals (e.g., chloroform, phenol). The Chelex extraction method is feasible, quick to obtain DNA from isolates and to be used in molecular analyses, it does not require either liquid nitrogen or toxic chemicals, thus being safe for operators, and does not require the disposal of harmful wastes. It is well-established and has been used by molecular scientists for over a decade. Indeed, it is inexpensive, technically easy, requires three reagents and takes only 40 min to complete, while the CTAB protocol involves many reagents and nearly 2 day processing time. Therefore, Chelex-100 could be useful, especially in the routine processing of large amounts of samples. In addition, contamination of DNA can cause significant problems with PCR (Kwock and Higuchi, 1989) but this method omits many of the surface contacts (e.g., contact between DNA and pestle, mortar and other equipment) that occur with CTAB and other methods. The results show that the Chelex method is comparable to the preexisting CTAB method and could be substituted as a simple and sustainable approach especially when resources are limited. In conclusion, the Chelex method is recommended for molecular studies of V. inaequalis considering its simplicity and cost-effectiveness. Acknowledgments The authors wish to thank Dr Annette Freund (BASF, Agricultural Research Centre of Limburgerhof, Germany) for her help with the protocol for the molecular characterization of strobilurin-resistant isolates. References Ahmed, I., Islam, M., Arshad, W., Mirza, B., 2009. High quality plant DNA extraction for PCR: an easy approach. J. Appl. Genet. 50, 105–107. Ashktorab, H., Cohen, R.J., 1992. Facile isolation of genomic DNA from filamentous fungi. Biotechniques 13, 198–200. Brewer, M.T., Milgroom, M.G., 2010. Phylogeography and population structure of the grape powdery mildew fungus, Erysiphe necator, from diverse Vitis species. BMC Evol. Biol. 10, 268. Capote, N., Pastrana, A.M., Aguado, A., Sánchez-Torres, P., 2012. Molecular tools for detection of plant pathogenic fungi and fungicide resistance. In: Cumagun, C.J. (Ed.), Plant. Pathol., pp. 151–202

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