Detection of Virus and Viroid Pathogens in Plants

Abstract

Plant viruses differ significantly from other microbial pathogens in their simple structure and minute size, method of replication and obligate parasitism. Despite their simple structure, they cause numerous economically important plant diseases that still remain as threat to crop production in several countries. Depending on the biological, morphological, immunological and genomic properties, several approaches have been made for the detection, identification, quantification and differentiation of the viruses. Histochemical methods employing light and electron microscopy have been shown to be useful for detecting and studying the nature of intracellular inclusions characteristic of virus infection. Certain groups of viruses can be putatively identified by the type and nature of inclusion bodies in infected plant cells. Electron microscopy using appropriate stains has been useful to study the ultrastructure of viruses. Immunological techniques have been shown to provide more rapid, sensitive and precise detection of viruses compared to biological methods that depend on inoculation to diagnostic/assay hosts that exhibit characteristic symptoms. Nucleic acid-based techniques represent significant advancement in pathogen diagnostics. They offer more reliable, reproducible, sensitive and specific results for the detection, identification and quantification of the viruses in different host plant species that are symptomatic or asymptomatic. Attempts to detect two or more viruses simultaneously present in infected plants have been successful, resulting in saving time, labor and costs of testing. In spite of all the advantages, many nucleic acid-based techniques have not yet been demonstrated to be applicable in different locations, cost-effective and suitable for large scale use under field conditions. Viroids represent a group of most primitive pathogenic entities constituting exclusively of nucleic acids that are capable of independent replication and inducing diseases when introduced into susceptible plant cells. Biological methods have been useful in detecting the viroids based on their reaction on diagnostic host plants. Nucleic acid-based techniques are the ones that can provide reliable and rapid results for their detection and identification. As it is very difficult to cure the plants already infected by viruses or viroids, the practical option available for the management of diseases caused by them is the removal of infected plants. Such an eventuality will result in devasting adverse effect on crop production. To avoid such a catastrophic possibility, adequate attention has to be bestowed for the early and reliable detection of viruses and viroids in infected seeds and vegetatively propagated materials and eliminate all infected ­materials to build virus/viroid-free nucleus stocks for distribution to growers.

Appendices

Appendix 1: Detection of Plant Viruses by Different Formats of Enzyme-Linked Immunosorbent Assay (ELISA) (Clark and Adams 1977)

2.1.1 Double-Antibody Sandwich (DAS)-ELISA

• (i) Precipitate globulins from the antiserum using 36% sodium sulfate; wash the precipitate with 18% sodium sulfate and store at −70°C; conjugate a portion of globulin with alkaline phosphatase using glutaraldehyde as the coupling agent.

• (ii) Dilute the globulins fraction (unlabeled) with 0.05 M carbonate buffer at pH 9.6 to yield a concentration of 10 μg protein/ml; add 200 μl of antibody solution to each well in polystyrene ELISA plates and incubate at 37°C for 3–5 h; empty the wells, wash thrice with 0.15 mM phosphate buffered saline solution pH 7.2 containing 0.05% Tween 20 (PBS-Tween) and dry.

• (iii) Add samples (purified antigen or extracts of infected tissues) in 200 μl quantities in PBS-Tween; incubate at 4°C overnight or for 18 h and wash the wells as before.

• (iv) Add aliquots of 200 μl of enzyme-labeled antibody conjugate to each well; incubate for 4 h at 37°C and wash the wells as before.

• (v) Add enzyme substrate p-nitrophenyl phosphate at a concentration of 1 mg/ml in diethanolamine buffer at pH 9.8 at room temperature; stop the reaction after 30 min by adding 3 M NaOH at 50μl/well.

• (vi) Determine the color intensity (OD) at 405 nm in an ELISA reader.

2.1.2 Direct Antigen Coating (DAC)-ELISA

• (i) Add samples at 200 μl to each well in the ELISA plate, incubate at 37°C for 1 h and wash the wells with PBS-Tween.

• (ii) Add antiserum at suitable dilution at 200 μl/well; incubate for 1 h at 37°C and wash the wells with PBS-Tween.

• (iii) Add enzyme-labeled antirabbit IgG at 200 μl to each well; incubate for 1 h at 37°C and wash the wells with PBS-Tween.

• (iv) Follow steps (v) and (vi) as in DAS-ELISA.

2.1.3 Protein A-Coating ELISA

• (i) Dissolve protein A (1–10 mg/ml) in carbonate buffer; dispense 200 μl/well in ELISA plate; incubate for1 h at 37°C and wash in PBS-Tween.

• (ii) Dispense antiserum (at suitable dilution) at 200 μl/well; incubate for 1 h at 37°C and wash with PBS-Tween.

• (iii) Dispense 200 μl of samples (purified antigen/extracts of tissues at suitable dilutions); incubate at 37°C for 1 h and wash the wells with PBS-Tween.

• (iv) Dispense 200 μl of antiserum and proceed as in step (ii).

• (v) Dispense enzyme-labeled antirabbit IgG or Fc at 200 μl/well; incubate for 1 h at 37°C and wash with PBS-Tween.

• (vi) Follow steps (v) and (vi) as in DAS-ELISA.

2.1.4 Indirect ELISA

• (i) Dispense goat or chicken antivirus globulins (1–10 μl/ml) at 200 μl/well; incubate at 37°C for 1 h and wash with PBS-Tween.

• (ii) Dispense 200 μl of suitably diluted samples in each well; incubate at 37°C for 1–3 h and wash with PBS-Tween.

• (iii) Dispense 200 μl of antivirus rabbit globulin/well; incubate at 37°C for 1–3 h and wash with PBS-Tween.

• (iv) Dispense 200 μl of antirabbit globulin conjugate/well; incubate for 1 h at 37°C.

• (v) Follow steps (v) and (vi) as in DAS-ELISA.

Appendix 2: Detection of Grapevine leaf roll-associated virus-3 Using a Single-Chain Fragment Variable Antibody in ELISA (Cogotzi et al. 2009)

• (i) Coat the microtiter plates with anti-GLRaV-3 PAb (IgGLR3, 0.5 mg/ml) (Agritest, Italy) or purified scFvLR3 or C_L-LR3 at different concentrations (starting from 0.33 mg/ml) and incubate for 2 h at 37°C.

• (ii) Wash the plates three times with PBST containing 0.05% Tween-20 and incubate overnight at 4°C after adding extracts from infected grapevine petioles or phloem tissues that are crushed in ten volumes of extraction buffer [PBS containing 0.05% Tween-20, 20% polyvinylpyrrolidone (PVP), pH 7.4] and centrifuge at 300 g for 3 min.

• (iii) Detect the captured antigens with an alkaline phosphatase (AP)-conjugated anti-GLRaV-3 IgG (IgGLR3AP, Agritest s.r.1), scFvLR3 (scFvLR3AP) or C_L-LR3 (C_L-LR3AP), diluted in conjugate buffer (PBS containing 0.05% Tween-20, 20% PVP, 2% bovine serum albumin, pH 7.4) and incubate for 3 h at 37°C and wash the plate as done before [step (ii) above].

• (iv) Incubate the plates with 1 mg/ml of p-nitrophenylphosphate in substrate buffer (0.1 M Tris–HCl, pH 9.5).

• (v) Record the absorption values at 405 nm after 1 h using ELISA reader.

Appendix 3: Detection of Iris yellow spot virus (IYSV) by ELISA Incorporating a Blocking Agent (Smith et al. 2006)

• (i) Extract the virus from plant tissues in phosphate buffered saline (1g/20 ml) consisting of 10 mM potassium phosphate, 150 mM sodium chloride, pH 7.2, Tween 20 (5 ml/l) and polyvinylpyrrolidone (20g/l) (PBST) using a leaf press (Pollahne, Hannover, Germany).

• (ii) Follow double antibody-sandwich ELISA protocol (Appendix 1).

• (iii) Incorporate a blocking step by adding skim milk powder (5% w/v) to the conjugate buffer.

• (iv) Perform the tests with and without the blocking step to determine the test reliability for both sets of tests.

Appendix 4: Detection of Cucumber mosaic virus (CMV) by Dot Immunobinding Assay (DIBA) (Zein and Miyatake 2009)

• (i) Collect young upper leaves from healthy and CMV-infected plants; roll them longitudinally to a form a tight scroll; cut with a sharp razor blade at the center and hold the cut surface immediately on the surface of a PVDF membrane (Bio-Rad) for 6–8 s.

• (ii) Homogenize the sample tissues with extraction buffer; use the extract for diluting the purified preparations (1:100) and gently transfer 2 μl of the mixture onto a PVDF membrane (0.45 μm pore size, Bio-Rad).

• (iii) Air-dry the membranes; block for 30 min with TBS buffer consisting of 10 mM Tris–HCl, pH 7.4, 0.15 M NaCl) containing defatted milk powder (50 g/l) (TBS-milk buffer) or in PBS buffer consisting of 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl, 3 mM NaN₃, pH 7.4) containing 20 g/l Triton X-100 and defatted milk powder (PBS-milk buffer); wash the membranes three times in distilled water, if PBS-milk buffer is used.

• (iv) Incubate the membrane for 60 min in 1 μg/ml MAbs diluted with TBS-milk buffer or PBS-milk buffer; wash times of 5 min each with TBST or PBST (TBS or PBS + Tween-20, 3g/l) followed by two washes with distilled water.

• (v) Incubate the membranes for 60 min in 1/5,000 dilution in TBS-milk or PBS-milk buffer with appropriate goat anti-mouse alkaline phosphatase (AP)-conjugated antibody (Amersham Pharmacia Biotech).

• (vi) Wash the membranes as before and equilibrate in substrate buffer (0.1 M Tris–HCl, pH 9.5) for 5 min and the chromogenic substrate 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium chloride (BCIP/NBT).

• (vii) Observe the development of purple color indicating positive reaction.

Appendix 5: Detection of Tomato spotted wilt virus (TSWV) in Different Tissues of Infected Plants by Tissue Blot Immunoassay (TBIA) (Whitefield et al. 2003)

• (i) Roll the healthy and infected leaves of herbaceous plants into a tight bundle; cut with a razor blade; gently blot the cut surface onto 0.45 μm Nitro ME nitrocellulose (NC) membranes (Micron Separations, Inc., USA) for 3–5 s.

• (ii) Incubate the rootlets sampled from dried test plant tubers in deionized water of 16–20 h at room temperature (RT) in 24-well microtiter plates (Costar Corp., USA); cut a cross section of each rootlet with the razor blade and press the cut surface onto the NC membrane.

• (iii) Air-dry the blotted membranes at RT; place them in glass hybridization tubes; allow reaction with reagents as detailed below: perform all incubations and washing steps at RT in a hybridization oven (Lab-Line, USA) at 16 rpm and expose the membranes sequentially to reagents and pour the reagents in and out of the tube without removing the membrane.

• (iv) Block membranes with 10 ml of 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) consisting of 0.14 M NaCl, 1.0 mM potassium phosphate, 8.0 mM sodium phosphate and 2.5 mM KCl, pH 7.5 for 1 h.

• (v) Treat the membranes with the appropriate dilution of anti-NSs MAb from stock solution (1 mg/ml) based on preliminary experiments, as working dilutions may vary from 1:100 to 1: 200; dilute the antibody (Abs) in 4 ml of Ab dilution buffer consisting of PBS, 0.1% Empigen BB (Calbiochem, USA) and 0.1% BSA and incubate membranes with Ab for 2 h.

• (vi) Wash the membranes three times in PBS with 0.5% Tween (PBST) for 5 min for each wash and incubate in 4 ml of a 1:2,000 dilution of rabbit anitmouse alkaline phosphatase (AP) conjugate (Bio-Rad) for 1 h.

• (vii) Wash the membranes again in PBST three times for 5 min/wash; add AP substrate (Sigma Fast BCIP/NBT tablets) and incubate the membranes for 5–15 min.

• (viii) Rinse the membranes in water and examine after air-drying at RT.

• (ix) Look for the development of purple precipitate on the membrane, indicating the positive reaction.

• (x) Under field conditions, perform the entire procedure in disposable 50 ml polypropylene tubes (Sarstedt, USA); incubate the tubes in pocket or backpack and agitate periodically.

• (xi) Actively growing plant samples do not require presoaking step followed for testing dried tubers and perform all other steps without any change as mentioned above.

Appendix 6: Detection of Citrus psorosis virus (CPsV) by Direct Tissue Blot Immunoassay (DTBIA) (Martin et al. 2002)

• (i) Select young tender leaves or shoots; cut transversely; roll the leaf blade tightly to form a cylinder and gently press the freshly cut surface onto nitrocellulose membrane of 0.45 μm pore size (Bio-Rad, Spain) or nylon membrane (Amersham, Spain).

• (ii) Air-dry the prints; block with TBS buffer (10 mM Tris–HCl, pH 7.4, 0.5 M NaCl) containing 50 g/l defatted milk powder (TBS-milk powder) or in PBS buffer (8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl,0.14M NaCl,3 mM NaN₃, pH 7.4) containing 20 g/l Triton X-100 and 50 g/l defatted milk powder (PBS-milk buffer) and wash the membranes in the latter case with distilled water before incubation with antibodies.

• (iii) For indirect detection, incubate the membranes in 1/10,000–1/50,000 dilution of ascites fluid containing MAb 13C5 or 2A3 or a 1:1 (v/v) mixture of the two MAbs in TBS-milk buffer for 90 min.

• (iv) Wash three times with TBST or PBST [TBS or PBS plus Tween 20 (3g/l)] and two washes (first and last) with distilled water and incubate in 1/20,000 dilution of TBS-milk or PBS-milk buffer with appropriate alkaline phosphatase (AP) conjugated antibody.

• (v) Wash the membranes as done earlier and equilibrate in substrate buffer (0.1 M Tris–HCl, pH 9.5) for 5 min and add the substrate.

• (vi) For direct detection, block the membrane as described earlier; incubate in a 1/10,000 dilution of the A322 antibodies conjugated with AP in TBS-milk buffer for 3 h and wash.

• (vii) Equilibrated in substrate buffer as indicated earlier; add the substrate and incubate at room temperature; use the chromogenic substrate 6-bromo-4-chloro-3-indolylphosphate (BCIP) or nitroblue tetrazolium (NBT) or chemiluminescent substrates [SPD or CPD- star (Roche Diagnostics)] as per manufacturer’s instructions.

Appendix 7: Detection of Citrus psorosis virus (CPsV) by Western Blotting Technique (Loconsole et al. 2006)

• (i) Grind the infected leaf samples (100 mg) from citrus or Chenopodium quinoa in 2.5 volumes of extraction buffer consisting of 0.5 M Tris–HCl, pH 8.8, 2% sodium dodecyl sulfate (SDS), 40% sucrose, 4% 2 mercaptoethanol; boil the extracts for 3 min and centrifuge at 4,000 rpm for 4 min.

• (ii) Analyze the aliquots of 5 μl of the supernatant by electrophoresis in 10% SDS slab gels together with recombinant virus coat protein (CP) (5 μg/μl) and 1 μg PC of purified virus preparation from C. quinoa.

• (iii) Electroblot the gels on polyvinyl difluoride membranes (PVDF, Immobilon-P, Millipore, USA); block with 1% bovine serum albumin (BSA), 5% non-fat dry milk, 0.05% Tween-20 in PBS buffer 1× for 2 h at room temperature and incubate the membranes with crude As.Ps.Rc1 serum diluted 1:2,000, 1:1,000 or 1:500 for 1 h.

• (iv) Wash three times of 10 min each in 1 × TBS, 0.1% Tween-20, incubate the membranes for 30 min with a 1:12,000 dilution in blocking solution of a goat antirabbit IgG-AP conjugate (Sigma, USA).

• (v) Wash three times of 15 min each; add 1 ml of CDP-star substrate (1:100) (Roche Applied Science, Switzerland) and detect the protein bands by exposure to x-ray films for 30 min.

Appendix 8: Detection of an Ampelovirus by Western Blot Analysis (Valverde et al. 1990; Maliogka et al. 2009)

2.8.1 Extraction of ds-RNA from Virus-Infected Plants

• (i) Grind 80 g cortical scrappings of grapevine in liquid nitrogen; transfer to a flask along with two volumes of 2 × STE (0.1 M NaCl, 0.05 M Tris-base, 0.001 M EDTA) also containing 2% ß-mercaptoethanol, 1% SDS and 0.7% Na₂SO₃ and shake well for 30 min.

• (ii) Centrifuge at 8,000 g for 15 min; transfer 10 ml of the upper aqueous phase to a 50-ml centrifuge tube (if 10-ml tube is not available, adjust the volume by adding 1 × STE); add 2.1 ml 95% ethanol to each tube and mix well.

• (iii) For chromatography, add the sample to cellulose (CF-11, Sigma-Aldrich); agitate for 1 h at room temperature and centrifuge at 16,000 g for 5 min.

• (iv) Discard the upper phase; dilute the pellet in 200 ml STE 16 (STE 10 × + 16% ethanol) and repeat centrifugation three times with the last one performed at 23,000 g for 7 min.

• (v) Dissolve the pellet in STE 16 and transfer to a column and repeat chromatography cycle before the final ds-RNA precipitation.

• (vi) Treat with RNase, DNAse I and proteinase K; analyze in 1.3% low-melting-point agarose (Applichem, Germany) for 3.5 h at 100 v and visualize using SYBR gold (Molecular probes, Eugene, USA).

2.8.2 Production of Virus-Specific Antiserum with Recombinant Coat Protein (CP)

• (i) Amplify the full-length CP gene using the primer set M3CpF/M3CpR which includes the SphI and PstI restriction sites respectively at the 5′ end; ligate the amplified DNA fragment directionally in the pQE31 protein expression vector (Qiagen, Germany) bearing 6 His residues at the N-terminus and introduce the plasmid by transformation into Escherichia coli strain M15 (Qiagen, Germany).

• (ii) Add 1 mM isopropylthiogalactopyranoside (IPTG) to the bacterial culture for induction of protein expression and incubate for 4 h.

• (iii) Purify the expressed CP protein under denaturing conditions using affinity chromatography with Ni-NTA resin (Qiagen) as per the manufacturer’s recommendations.

• (iv) Immunize a New Zealand white rabbit with three intramuscular injections of 150, 272 and 300 mg of purified recombinant protein respectively in an equal volume of incomplete Freund’s adjuvant at 2-week intervals.

• (v) Collect the blood at 10 days after the last injection and purify the antibodies using protein A affinity chromatography.

• (vi) Perform Western blotting analysis using the antiserum at a dilution of 1/2,000 and extracts from mature leaf petioles or cortical scrappings prepared by grinding in five volumes of loading buffer 2 × 100 mM Tris–HCl, pH 6.8, 2% ß-mercaptoethanol, 4% SDS, 0.2% bromophenol blue and 20% glycerol.

Appendix 9: Detection of Phalaenopsis Orchid Viruses by Immunoblot Technique (Jan and Yeh 1995; Zheng et al. 2008a,b)

• (i) Grind the leaves from mock-inoculated and virus-infected plants with three volumes (v/w) of dissociation buffer containing 100 mM Tris–HCl, pH 7.2, 2% ß-mercaptoethanol, 10% sucrose, 0.005% bromophenol blue and 10 mM EDTA.

• (ii) Boil the crude antigen extract and separate electrophoretically on a 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel.

• (iii) Transfer to a 0.45 μm nitrocellulose membrane (Bio-Rad, USA) and pre-incubate with TSW buffer containing 10 mM Tris–HCl, pH 7.4, 0.9% NaCl, 0.25% gelatin, 0.1% Triton X-100 and 0.2% SDS.

• (iv) Incubate with primary antiserum against target virus (CP or NP) at required dilution (1:4,000) in TSW buffer for 1 h and then with alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (Jackson Immuno Research Labs, USA) for 1 h (1:5,000 dilution in TSW buffer).

• (v) Develop color by incubating with chromogenic substrates-nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolylphosphate paratoluidine salt in 100 mM NaCl, 5 mM MgCl₂ and 100 mM Tris–HCl, pH 9.5, at room temperature for 10–30 min.

• (vi) Stop the reaction by rinsing the membrane in distilled water.

• (vii) Use extracts from healthy leaves and normal serum as controls.

Appendix 10: Detection of Passion fruit woodines virus (PWV) by Light Microscopic Staining Technique (Jan and Yeh 1995)

• (i) Prepare fresh epidermal strips from healthy and virus-infected leaves of golden passionfruit and treat them with 5% Triton X-100 in distilled water for 5 min.

• (ii) Float the epidermal strips on a solution of 10% pectinase and 4% cellulose in 20 mM phosphate-buffered saline (PBS, pH 7.2) for 30–60 min.

• (iii) Incubate the strips first with antisera against cylindrical inclusion protein (CIP) or the 51K protein (amorphous inclusion) at 1:400 dilution for 30 min at 37°C and incubate with protein A-gold complex (Sigma, P-1039 diluted at 1:6) for 30 min at 37°C.

• (iv) Rinse the strips extensively with PBS after each treatment; mount the slides in 10% glycerine and view the strips under a light microscope with a blue filter.

Appendix 11: Detection of Potato virus Y (PVY) by Immunohistochemistry Method (Ryang et al. 2004)

• (i) Immerse the sampled plant tissues immediately in fixative consisting of 50% ethanol, 5% acetic acid and 3.7% formalin.

• (ii) Dehydrate and infiltrate the tissues in a graded series of ethanol solutions – 50%, 70%, 90% and 100% each for 30 min and embed them in paraffin (Paraplast-plus, Sigma).

• (iii) Cut sections of 12 μm thick of stem tissue using a rotary microtome and place them on glass slides.

• (iv) Dewax the sections in xylene and wash in 100% ethanol.

• (v) Dehydrate in a graded series of ethanol (70%, 50% and 30%) and distilled water for 10 min each.

• (vi) Incubate the sections in PBST and 1% BSA for 1 h and then incubate with PVY-CP-specific antibody diluted to 1:200 in PBST/BSA for 2 h at 37°C.

• (vii) Incubate the sections with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) diluted to 1:200 in PBST/BSA for 2 h at 37°C.

• (viii) Wash the sections and stain with BCIP/NBT liquid substrate system.

• (ix) Wash the stained sections in distilled water and observe under the microscope.

Appendix 12: Detection of Grapevine leaf roll-associated virus 3 (GLRaV-3) by Electron Microscopic Decoration Technique (Cogotzi et al. 2009)

• (i) Crush the tissues infected by target virus in 0.1 M phosphate buffer, pH 7.2 containing 2% nicotine.

• (ii) Sensitize the carbon grids by floating on drops of rabbit IgG capable of recognizing GLRaV-1, GLRaV-3 and GLRaV-7 or purified C_L-LR3 diluted in 0.1 M phosphate buffer, pH 7.2 for 1 h at 37°C.

• (iii) Wash the grids with buffer; float the grids on drops of plant tissue extracts at 4°C for 48 h to allow trapping of target virus particles.

• (iv) For virus decoration, rinse pre-coated grids exposed to the antibodies described above or C_L-LR3 diluted in PBS.

• (v) Allow reaction of C_L-LR3 with antibodies conjugated with alkaline phosphatase (C_LAP) diluted to1:50 and wash thrice with PBS.

• (vi) Negatively stain the trapped and decorated virus particles with 2% uranyl acetate and examine under transmission electron microscope (TEM).

Appendix 13: Detection of Grapevine fan leaf virus (GFLV) by RT-PCR Assay (Bashir and Hajizadeh 2007)

2.13.1 Extraction of Total RNA

• (i) Incubate 200 mg leaf tissue sample in 1 ml cold extraction buffer consisting of 91 mM K₂HPO₄, 30 mM KH₂PO₄, 292 mM sucrose, 0.22 mM bovine serum albumin (BSA) fraction II, 0.8 mM PVP 25, 30 mM ascorbic acid, pH 7.6 in a mortar for 20 min; grind well; add 1 ml more of extraction buffer and grind to get a homogenous extract.

• (ii) Centrifuge at 1,050 g for 4 min; transfer the supernatant to a fresh 1.5 ml tube; centrifuge at 16,800 g for 10 min; discard the supernatant and dissolve the pellet in 50 mM Tris, pH 8.0 containing 10 mM EDTA, 0.1% ß-mercaptoethanol and 1.25% SDS and incubate for 10 min at 60°C.

• (iii) Add 80 μl 5 M potassium acetate; place the tube on ice for 30 min and centrifuge for 15 min at 16,800 g.

• (iv) Transfer the supernatant to a fresh tube; add 0.1 volume of 3 M sodium acetate and 1 volume of ice-cold isopropanol; keep the tube for 1 h at −20°C and centrifuge at 16,800 g for 30 min.

• (v) Wash the pellet with 200 μl 80% ethanol; dry and suspend in 30 μl sterile distilled water.

2.13.2 Synthesis of First Strand cDNA

• (i) Incubate 2.5 μl aliquot containing 50 pmol oligo d(T)₁₆, 1.0 μl RNA and 1.1 μl RNAase-free water at 70°C for 5 min and chill immediately on ice.

• (ii) Add 7.5 μl of reverse transcription mix containing 1 × reverse transcription buffer (Fermentas, Lithuania), 10 mM of each dNTP, 10 U RNAase inhibitor and 100 U M-MuLV reverse transcriptase (Fermentas) and incubate at room temperature for 15–20 min, followed by incubation at 42°C for 60 min.

• (iii) Stop the reaction by heating at 70°C for 10 min.

2.13.3 Polymerase Chain Reaction (PCR)

• (i) Use appropriate pairs of primers (S2515/A3300 and CP433V/912C).

• (ii) Dispense 12.5 μl PCR mix containing 2 mM magnesium chloride, 0.5 pmol each of the primer, 1 × Taq DNA polymerase buffer, 0.3 U Taq DNA polymerase (Fermentas) and 2.5 μl of cDNA.

• (iii) Provide the following thermocycling conditions:

For S2515/A3300 primer set: one cycle at 94°C for 1 min, followed by 35 cycles of 94°C for 30 s, 40 or 50°C for 30 s, 72°C for 60 s and finally one cycle of 72°C for 10 min.

For CP433V/912C primer set: same as for S2515/A3300 primer set, except performing annealing at 40°C or 48°C for 45 s and extension at 72°C for 45 s.

Appendix 14: Detection of Strawberry Viruses by Multiplex RT-PCR Assay (Chang et al. 2007)

2.14.1 Total Nucleic Acid Extraction

• (i) Grind 0.05 g leaves in a mortar with liquid nitrogen; transfer the powdered tissue to a microfuge tube containing 500 μl CTAB buffer consisting of 2% CTAB, 100 mM Tris, pH 8.0, 20 mM EDTA, 1.4 M NaCl; add 2% ß-mercaptoethanol just before use and incubate for 20 min at 65°C.

• (ii) Extract the suspensions with chloroform/isoamyl alcohol (24:1) twice and separate the phases by spinning.

• (iii) Add 1/10 volume NaAc, pH 5.2 and 2.5 volume ethanol to water phase; mix well; remove the pellet; wash with 70% ethanol and dry on a clean bench.

• (iv) Resuspend in 50 μl of DEPC (diethylpyrocarbonate) water.

2.14.2 Extraction of Total RNA

• (i) Follow the steps A(i) and A(ii) above.

• (ii) Add ¼ volume 10 M LiCl to water phase; precipitate RNA at −20°C for 60 min; redissolve the precipitate in TE buffer and extract with phenol/chloroform/isoamylalcohol and chloroform/isoamylalcohol successively.

• (iii) Precipitate RNA; wash twice; dry on a clean bench and resuspend in 30 μl DEPC water.

2.14.3 Reverse Transcription (RT) Reaction

• (i) Incubate 30–50 ng (1 μl) of total RNA or 90–130 ng (1 μl) of total nucleic acid extract in 11.5 μl of sterile distilled water plus 1 μl of dNTPs (each 2.5 mM), 0.5 μl random primer (9-mer) (50 μM) and 0.5 μl oligo d(T)₁₈ (50 μM) (TaKaRa, Japan) at 65°C for 5 min.

• (ii) Add 4 μl buffer 5 × (TaKaRa, Japan), 0.5μl RNasin and 1 μl AMV (5 U/μl) TaKaRa, Japan) and incubate at 37°C for 2.5 h.

• (iii) Inactivate the enzyme by incubating at 70°C for 15 min.

2.14.4 Polymerase Chain Reaction (PCR)

• (i) Use primer pairs Y1/Y2 for Strawberry mild yellow edge virus (SMYEV), D1/D3 for Strawberry mottle virus (SMoV) and I2/SM2 for Strawberry vein banding virus (SVBV).

• (ii) Use single PCR reaction mixture containing 1 μl of first strand cDNA, 1 × PCR buffer, 0.2 mM dNTPs, 0.2 μM each primer, 1 U Taq polymerase in a total volume of 20 μl.

• (iii) Provide the following conditions for amplification:

For Y1/Y2 or D1/D3 primer pair: initial denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s and final elongation at 72°C for 5 min.

For I2/SM2 primer pair set: initial denaturation step at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 55°C for 60 s, and 72°C for 2 min and final elongation at 72°C for 5 min.

2.14.5 Duplelx PCR Assay

• (i) For SMYEV and SMoV use reaction mixture containing 1μl of first strand cDNA, 2 × PCR buffer, 0.2 mM dNTPs, 0.27 μM D1/D3 primers, 1 U Taq polymerase in a total volume of 20 μl.

• (ii) Provide the following cycling conditions: denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 57°C for 40 s and 66°C for 2 min and final elongation at 72°C for 5 min.

2.14.6 Multiplex PCR Assay

• (i) For SMYEV, SMoV and SVBV, use reaction mixture containing 1μl of first strand cDNA, 2 × PCR buffer, 0.2 mM dNTPs, 1 U Taq polymerase and use primer concentration 0.17 μM D1/D3, 0.25 μM I2/SM2 and 0.15 μM Y1/Y2 in a total volume of 20 μl.

• (ii) Provide the following cycling conditions: initial denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55°C for 40 s and 68°C for 2.5 min and final elongation step at 72°C for 5 min.

• (iii) For duplex and multiplex assays, resolve the PCR products by electrophoresis in 1.5% agarose gels in 1 × TBE buffer and after staining with ethidium bromide visualize under UV light.

Appendix 15: Detection of Pepino mosaic virus (PepMV) by Multiplex One-Step RT-PCR Assay (Alfaro-Fernández et al. 2009)

2.15.1 Extraction of Total Nucleic Acids

• (i) Place 0.5–0.6 g of leaf tissue in a sample bag (Agadia, USA) suitable for use on a Homex grinder (Bioreba, USA) and homogenize with 5 ml of lysis buffer consisting of 4 M guanidine isothiocyanate, 0.2 M sodium acetate, pH 5.0, 25 mM EDTA, 2.5% PVP-40 (w/v) and 1% 2-mercaptoethanol (v/v) (added just before use).

• (ii) Transfer the homogenate (1 ml) to a graduated microfuge tube using a disposable plastic transfer pipette; mix with 100 μl 20% sarkosyl (w/v) and incubate at 70°C for 10 min with agitation (1,200 rpm) using an Eppendorf thermometer.

• (iii) Transfer approximately 600–650 μl homogenate to QIA Shredder (Qiagen) column using a new disposable plastic pipette and centrifuge at 14,000 × g for 2 min in an Eppendorf microcentrifuge.

• (iv) Transfer 25 μl of column flowthrough to a new 1.5 ml microfuge tube; mix with 225 μl 95% ethanol; load onto an RNeasy (Qiagen) column and centrifuge at 8,000 × g for 45 s.

• (v) Discard the flowthrough; apply 700 μl RW1 buffer (Qiagen) to the column and wash thoroughly by centrifugation at 8,000 × g for 15 s.

• (vi) Transfer the column to a clean 2 ml collection tube; apply 500 μl RPE buffer (Qiagen) to the column and wash through by centrifugation at 8,000 × g for 15 s.

• (vii) Discard the flowthrough; apply additional 0.5 ml RPE buffer to the column and centrifuge at 14,000 × g for 5 min to remove all traces of ethanol.

• (viii) Transfer the column to a new 1.5 ml microfuge tube and elute RNA by applyi ng 100 ml RNA-free water and centrifuge at 8,000 × g for 1 min.

• (ix) Label the collection tubes appropriately and store the RNA samples at −80°C till use for RT-PCR assay.

2.15.2 Primers Employed

• (i) Select specific region of the RNA polymerase gene for identification of PepMV isolates; three specific primers – PepMV-DEP, PepMV-D1 and PepMV-D2 – and a common antisense primer (PepMV-R) for differentiation of EU/PE, CH1/US1 and CH2/US2 groups of PepMV respectively.

2.15.3 RT-PCR Assay

• (i) Perform RT-PCR reaction using the SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase kit (Invitrogen Life Technologies, Spain); use a mixture of all primers at a final concentration of 0.25 pmol/μl and the primers of internal control Rbc1 gene at 0.05 pmol/μl corresponding to the partial sequence of ribulose 1,5-biphosphate carboxylase chloroplast gene.

• (ii) Provide the following conditions for amplification: initial incubation at 50°C for 30 min, followed by 94°C for 2 min and 40 cycles of 94°C for 15 s, 50°C for 30 s and 68°C for 1 min and a final incubation at 68°C for 10 min.

• (iii) Resolve the amplified PCR products by electrophoresis, using 1.2% agarose/TAE gels stained with ethidium bromide.

• (iv) To confirm the viral origin of the amplified fragment, purify them with High Pure PCR Product Purification Kit (Roche Diagnostics, Germany) and directly sequence the amplicons.

• (v) Digest 10 μl of PCR amplicon directly with SacI enzyme (MBI Fermentas, Lithuania) in a total volume of 20 μl as per the manufacturer’s recommendations.

• (vi) Analyze the digestion products in a 5% TAE polyacrylamide gel followed by staining with ethidium bromide.

Appendix 16: Detection of Orchid Viruses by Multiplex RT-PCR Assay (Lee and Chang 2006)

2.16.1 Simplex RT-PCR Assay

• (i) Extract total RNA and viral RNA from plant tissues using Plant Total RNA Extraction Miniprep System (Viogene, USA) as per the manufacturer’s instructions.

• (ii) Use primer pair CymMV CP-F1/CymMV CP-R1, ORSV CP-F1/ORSV CP-R1 and mt-F2/mt-R1 for the amplification of Cymbidium mosaic virus Odontoglossum ringspot virus and the internal control respectively.

• (iii) For RT reaction with a total volume of 12 μl, mix 2 μl extracted plant total RNA (200 ng) with/without viral RNA, with 0.5 μl of 5 μM reverse primer, CymMV CP-R1, ORSV CP-R1 or mt-R1; add 5μl double distilled (dd) water; heat the solution for 10 min at 70°C and cool immediately on ice for 5 min.

• (iv) Add RT mixture containing 3.25 μl dd water, 2.5 μl 5 × first strand buffer (Promega, USA), 1.2 μl dNTPs (10 mM), 0.25 μl rRNasin (40 U/μl) (Promega) and 0.25 μl AMV reverse transcriptase (10 U/μl, Promega) and incubate at 42°C for 60 min.

• (v) For PCR use 20 μl reaction volume containing 2 μl RT product, 2 μl 10 × DyNAzyme^TM II DNA polymerase buffer (Finnzymes Inc., Finland), 2 μl forward and reverse primers 5 μM CymMV CP-F1/CymMV CP-R1, ORSV CP-F1/ORSV CP-R1 or mt-F2/mt-R1, 2 μl dNTPs (2 mM), 0.4 μl DyNAzyme^TM II DNA polymerase (2 U/μl, Finnzymes Inc.) and 11.6 μl dd water.

• (vi) Perform amplification providing the following conditions: initial denaturation at 96°C for 5 min, followed by 30 cycles of 96°C for 30 s, 50°C for 30 s, 72°C for 30 s, and a final extension step at 72°C for 7 min.

• (vii) Analyze the PCR products on agarose gels by electrophoresis, as described in subdivision C below.

2.16.2 Multiplex RT-PCR Assay

• (i) Perform RT reaction in the same manner as described above except that the three reverse primers- CymMV CP-R1, ORSV CP-R1 and mt-R1 are to be added simultaneously.

• (ii) Carry out PCR with 20 μl volume comprising of 11.6 μl dd water, 2 μl of 10 × DyNAzyme^TM II polymerase buffer, 2 μl multiplex primer set (including 2.5 μM mt-F2/mt-R1, 1.25 μM CymMV CP-F1/CymMV CP-R1, 1.25 μM ORSV CP-F1/ORSV CP-R1), 2 μl dNTPs (2 mM); mix 0.4 μl DyNAzymeTM DNA polymerase and assay in the same PCR conditions.

• (iii) Analyze the PCR products on agarose gels after electrophoresis as in subdivision C below.

2.16.3 Agarose Gel Electrophoresis

• (i) Resolve aliquots of 10 μl of RT-PCR products in 2% agarose gel in TAE buffer consisting of 40 mM Trisacetate and 1 mM EDTA.

• (ii) Stain the gel with ethidium bromide (05 μg/ml) and illuminate with UV light.

• (iii) Determine the fragment sizes with Kodak Digital Science^TM ID image analysis software (Eastman Kodak Co., USA) by comparison with DNA molecular weight markers (Invitrogen, USA).

Appendix 17: Detection of Orchid Viruses by Multiplex RT-PCR Assay Using Simple-Direct-Tube (SDT) Method for RNA Extraction (Suehiro et al. 2005; Yamane et al. 2008)

2.17.1 Extraction of Total RNA by SDT Method

• (i) Grind 0.1 g infected leaves in 0.3 ml of phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST); transfer 50 μl crude sap to a polypropylene PCR tube (0.5 ml) using a truncated tip (cut 5 mm of the top portion) and incubate for 15 min at room temperature.

• (ii) Remove the crude sap using a truncated tip; wash twice with 50 μl PBST; add 30 μl diethylpyrocarbonate water to the tube and incubate at 95°C for 1 min and cool on ice.

• (iii) Use the RNA solution for RT-PCR assay.

2.17.2 Multiplex RT-PCR Assay

• (i) Use the primer pair CymMV-5034F/CymMV-5189R for CymMV and primer pair ORS CP-F/ORS CP-R for ORSV.

• (ii) Use the reaction mixture containing a total 5 μl consisting of 2.375 μl RNA solution, 0.25 μl of 5 μM reverse primer, 1 μl 5 mM MgCl₂, 0.5 μl 10 × RT buffer, 0.5 μl dNTP mixture, 0.125 μl RNase inhibitor and 0.25 μl AMV reverse transcriptase; incubate for 30 min at 50°C, 5 min at 99°C and 5 min at 5°C.

• (iii) Perform PCR amplification with 5 μl 5 × PCR buffer, 0.25 μl of 5 μM forward primer, 0.125 μl TaKaRa Taq HS and 14.5 μl sterile distilled water added to the cDNA solution.

• (iv) Heat the tubes at 94°C for 2 min; provide 35 cycles of amplification-denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s.

• (v) Mix aliquots of 8 μl amplicon with 2 μl 5 × loading buffer; electrophorese in 4% agarose gel in Tris-acetate-EDTA (TAE) buffer at 100 V for 40 min; include a DNA ladder as molecular weight markers.

• (vi) Stain the gels with ethidium bromide and photograph on UV transilluminator.

Appendix 18: Detection of Pepper Tobamoviruses by Immunocapture (IC)-RT-PCR Assay (Kim et al. 2006)

2.18.1 Double Antibody Sandwich (DAS)-ELISA

• (i) Coat the microtiter plates (Polystyrene plates, Costar) with 100 μl/well of IgG diluted to 1 ng/ml in coating buffer, pH 9.6 at 37°C for 3 h and wash the wells with PBS-T three times for 3 min each.

• (ii) Add 150 μl plant extract to each well; incubate overnight at 4°C and wash for three times as done earlier with PBS-T.

• (iii) Add 100 μl IgG-enzyme conjugate; incubate for 3 h at 37°C and wash as done before.

• (iv) Add 100 μl of substrate (1 μg/ml of p-nitrophenylphosphate in 10% diethanolamine, pH 9.8) to each well.

• (v) Record absorbance values at 405 nm using microplate reader.

2.18.2 Immunocapture RT-PCR Assay

• (i) Coat microtubes (200 μl) with 50 μl antibodies to both viruses [Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV)] at 1 μg/ml for each tube; incubate at 37°C for 2 h and wash the tubes three times with PBS-T.

• (ii) Add 50 μl leaf extract or purified virus preparation to each tube; incubate for 2–3 h at 37°C or overnight at 4°C and remove the solution by pipetting; add 80 μl of PBS-T/tube; centrifuge and remove the wash solution by pipetting.

• (iii) Wash with PBS-T and finally with deoinized water.

• (iv) Perform RT-PCR procedure using ‘AccessQuick RT-PCR Kit (Promega); add reaction mix to microtubes after washing them; reaction mix contains: 12.5 μl mastermixture, 0.5 μl AMV reverse transcriptase; 1 μl (10 pmole) of each primer and nuclease-free distilled water up to a final volume of 25 μl.

• (v) Use appropriate primers (PM317-F/PM317-R for PMMoV; CPTMG-F/CPTMG-R for TMGMV) and centrifuge briefly.

• (vi) Carry out the RT reaction with one cycle at 42°C for 45 min and 35 cycles of PCR amplification using the step program: 95°C for 45 s; 50°C for 50 s; 72°C for 60 s, followed by final extension at 72°C for 10 min.

Appendix 19: Detection of Florida hibiscus virus by Immunocapture (IC)-RT-PCR Assay (Kamenova and Adkins 2004)

• (i) Coat sterile 0.6 ml polypropylene microcentrifuge tubes with 100 μl of virus specific IgG (1 mg/ml diluted in 0.05 sodium carbonate buffer, pH 9.6); incubate for 3 h at 37°C and wash three times with PBST in 200 μl/tube.

• (ii) Homogenize healthy and virus-infected plant tissues in ELISA sample buffer and centrifuge at 10,000 × g for 10 min at 4°C.

• (iii) Prepare suitable dilutions of supernatants and virion preparations; dispense 100 μl aliquots to the coated tubes; incubate overnight at 4°C and wash three times with PBST as done before.

• (iv) Design suitable specific primer pairs; synthesize first strand cDNA by Moloney murine leukemia virus reverse transcriptase using 200 U/μl (Promega, USA) at 47°C for 45 min in the buffer provided by the manufacturer.

• (v) Provide the following conditions: 30 cylces of PCR amplification with Taq polymerase (50 U/μl; Promega) at 94°C for 1 min, 59°C for 1 min and 72°C for 1 and 30 s in the manufacturer’s buffer.

• (vi) Analyze the amplified products by electrophoresis on native 2% agarose gels and detect the bands by ethidium bromide staining.

Appendix 20: Detection of Potato virus Y (PVY) by PCR-ELISA Test (Hataya et al. 1994; Varveri 2000)

• (i) Precipitate the PCR product using ethanol; dissolve in TNE buffer (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, pH 8.0) and denature by heating.

• (ii) Dilute the denatured product by 200-fold in 10 × SSC (1 × SSC: 15 mM NaCl, 0.015 M sodium citrate, pH 7.0) – 10 mM EDTA, pH 7.0 directly into the microplate wells; incubate for 2 h at 37°C and wash three times with PBS + Tween-20.

• (iii) Fill each well with 100 μl hybridization solution consisting of 5 × SSC, 10 mM EDTA, pH 7.0, ).1% Tween-20; 50% formamide containing heat-denatured DIG-labeled probe at a dilution of 1:500; incubate at 42°C for 12 h and wash three times.

• (iv) Fill the wells with 100 μl alkaline-phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim); incubate for 1 h at 37°C and wash three times.

• (v) Add p-nitrophenyl phosphate substrate at 1 mg/ml diethanolamine and incubate for 2 h.

• (vi) Record the absorbance values at 405 nm with a colorimeter (Dynatech MR 5000).

Appendix 21: Detection of Plum pox virus (PPV) by Spot Real-Time PCR Assay (Capote et al. 2009)

2.21.1 Preparation of Crude Extracts

• (i) Collect five to eight spurts or dards with dormant or swelling buds/tree in the winter and five to eight spurs or shoots with fully expanded leaves/tree in the following spring; grind the winter samples in plastic bags containing a heavy net (Plant Print Diagnostics, Valencia) using a hammer in the presence of 1:20 (w/v) sodium diethyldithiocarbamate and process the spring samples also in the same buffer in plastic bags containing a soft net (Bioreba), using the Homex-6 machine (Bioreba).

• (ii) Prepare serial dilutions of the extract from PPV-infected peach seedling with the extract of healthy seedling.

2.21.2 Spot Real-Time PCR Assay

• (i) Load 5 μl of crude extract onto 0.45 μm positively charged nylon membrane (Roche) or on Whatman 3 MM paper filter.

• (ii) Provide the reaction cocktail for SYBR Green chemistry: 1 × Power SYBR Green Master Mix (Applied Biosystems), 6.25 units of MultiScribe reverse transcriptase (Applied Biosystems), 10 U of RNase inhibitor, 0.3 μM primer P1, 0.05 μM primer P2.

• (iii) Provide RT-PCR variables as follows: 48°C for 30 min, 95°C for 10 min, 45 cycles of amplification at 95°C for 15 s and 60°C for 1 min.

• (iv) Prepare a melting point curve for temperatures between 60°C and 95°C.

• (v) Perform analysis using an ABI Prism 7000 and StepOnePlus software packages (Applied Biosystems).

Appendix 22: Detection of Strawberry vein banding virus (SVBV) by Real-Time NASBA Technique (Vašková et al. 2004)

2.22.1 Extraction of Nucleic Acids

• (i) Place leaves in extraction bags containing sample extraction buffer (1:10 ratio, w/v) consisting of 0.14 M NaCl, 2 mM KCl, 2 mM KH₂PO₄ 8 mM Na₂HPO₄.2H₂O, pH 7.4), 0.05% Tween-20 (v/v), 2% polyvinylpyrrolidone 40, 0.2% ovalbumin (w/v), 0.5% BSA (w/v) and 0.05% sodium azide and homogenize with the Bioreba HOMEX 6 homogenizer

• (ii) Transfer 100 μl of the homogenized solution for extraction of nucleic acids by the RNeasy Plant Mini Kit or DNeasy Plant Mini Kit (Qiagen, Germany)

• (iii) Elute the RNA in 100 μl RNase-free water and store at −60°C.

• (iv) Elute DNA in 200 μl low salt DNeasy dilution buffer and store at −20°C.

2.22.2 Real-Time Nucleic Acid Sequence-Based Amplification (NASBA)

• (i) Use appropriate primer pair and probe for amplification of template present in the purified preparations or plant tissue extracts containing nucleic acids.

• (ii) Add 3 μl sample to 12 μl NASBA premixture (final concentration in 20 μl reaction mixture: 40 mM Tris–HCl, pH 8.5, 0.5 mM DTT, 12 mM KCl, 15% DMSO (v/v), 1 mM dNTPs, 2 mM each of ATP, UTP and CTP, 1.5 mMGTP and 0.5 mM ITP, 0.2 μM of each primer).

• (iii) Add 9 ng molecular beacons for each reaction; carry out denaturation of the mixture at 65°C for 5 min and adjust the temperature to 41°C for 5 min.

• (iv) Add pre-mixed enzymes, 375 mM sorbitol, 21 μg BSA, 0.08 U RNase H, 32 U T7 RNA polymerase and 6.4 U AMV-RT (BioMerieux BV, the Netherlands) and incubate at 41°C for 90 min.

• (v) Perform real-time measurement using the iCycler iQ^TM (Real-time PCR Detection System, Bio-Rad, USA) and excite the fluorophore at 575 nm.

• (vi) Determine fluorescence emission at 620 nm at 2 min interval.

• (vii) Separate 3 μl NASBA amplification product using a nondenaturing gel electrophoresis; and blot on Z-probe nylon membrane.

• (viii) Hybridize to SVBV-specific biotinylated probe (Bio1) and visualize using enhanced chemi-luminescent (ECL) detection (Amersham Pharmacia Biotech).

• (ix) Expose the membrane to x-ray film.

Appendix 23: Detection and Differentiation of Citrus tristeza virus (CTV) Isolates by Single-Strand Conformation Polymorphism Analysis (Sambade et al. 2002)

2.23.1 cDNA Synthesis

• (i) Use ds-RNA enriched preparations for cDNA synthesis by RT-PCR as the template and specific primers for genes p18, p13 p20 and p23 to be designed using the sequences conserved in CTV isolates T36, VT and T385.

• (ii) Perform RT-PCR in an A20 air thermal cycler (Idaho Technologies, USA) using a reaction mixture (25 μl) containing 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 3 mM MgCl₂, 4 mM each of dATP, dCTP, dGTP, and dTTP, 500 μg/ml BSA, 1 μM primers, 4 U RNase OUT, 20 U SuperScript II reverse transcriptase (RT) and 1 U Taq DNA polymerase (Life Technologies, USA).

• (iii) Provide the following cycling conditions: reverse transcription for 30 min at 46°C, RT inactivation at 94°C for 2 min, 40 cycles of 94°C for 5 s, 55°C for 5 s, 72°C for 30 s and final extension at 72°C for 2 min.

• (iv) Analyze the cDNA by electrophoresis on 2% agarose gel.

2.23.2 Single-Strand Conformation Polymorphism (SSCP) Analysis

• (i) Mix 1 μl PCR amplicon with 9 μl of denaturing solution consisting of 95% formamide, 20 mM EDTA, pH 8.0, 0.05% bromophenol blue and 0.05% xylene-cyanol; heat at 90°C for 10 min and immediately chill the mixture on ice.

• (ii) Separate the DNA strands by electrophoresis in a nondenaturing polyacrylamide minigel (8% acrylamide) using TBE containing 89 mM Tris–borate, pH 8.2, 2 mM EDTA as electrophoresis buffer and a constant voltage of 300 V for 1.5 h or 200 V for 3 h at 4°C.

• (iii) Stain the gels with silver nitrate.

Appendix 24: Isolation of ds RNA of Plant Viruses (Zhang et al. 1998)

• (i) Freeze plant tissues (leaf, petiole or root) in liquid nitrogen; grind to a fine powder using a pestle and mortar and extract with a mixture of 14 ml 1 × STE (containing 0.05 M Tris pH 6.8), M NaCl and 1 mM EDTA, 2 ml 10% sodium dodecyl sulfate (SDS), 1 ml 2% bentonite and 18 ml 2 × STE-saturated phenol.

• (ii) Shake the extract well for 30 min; centrifuge for 15 min at 8,000 rpm and add ethanol to have 16.5% concentration.

• (iii) Pass through a cellulose column (Whatman CF-11) that binds ds-RNA; wash thoroughly with 1 × STE buffer at 16.5% ethanol and elute from the column with 1 × STE.

• (iv) Precipitate from the eluate with 1/10 volume of 3 M sodium acetate and 2. 5 volumes of ethanol and resuspend the precipitate in 50 μl water.

• (v) Electrophorese half of the above preparation through a 6% polyacrylamide gel and stain with ethidium bromide (1 μg/ml).

• (vi) Visualize the bands under UV light.

Appendix 25: Detection of Plant RNA Viruses by Macroarray Technique (Agindotan and Perry 2007)

2.25.1 Extraction of Total RNA from Plants

• (i) Extract total RNA from samples of 200 mg of leaf tissues using RNeasy Plant Mini Kit (Qiagen) as per the manufacturer’s instructions.

• (ii) Estimate the RNA concentration at 260 nm using a spectrophotometer.

2.25.2 Probes for Target cDNAs

• (i) Design probes (ca. 70 nts) based on the sequences of target viruses (CMV, PVY and PLRV).

• (ii) Evaluate virus specificity of each oligonucleotides using the Basic Local Alignment Search Tool (BLAST).

• (iii) Use commercially synthesized oligonucleotides (Integrated DNA Techno­logies, USA).

2.25.3 Printing of Oligonucleotide Probes on Membranes

• (i) Dilute the ribosomal and virus-specific DNA oligonucleotide probes to 10 and 20 μM respectively in 1 × spotting buffer containing 4 mM sodium carbonate buffer, pH 8.3, 3 × SSC (consisting of 0.15 M NaCl and 0.15 M sodium citrate), 50% dimethyl sulfoxide (DMSO), 0.01% N-laroylsarcosine and 1 mg bromophenol blue.

• (ii) Transfer the probes in 30 μl aliquots into the wells of a polypropylene 384-well microtiter plate (Nalge Nune International, NY) and a clean pin replicator (V & P Scientific Inc., USA) for 384-well plates as per manufacturer’s recommendations.

• (iii) Cut the Hybond-N Plus membrane (GE Healthcare Biosciences Corp, USA) to the size (12 × 10 mm pieces) of a multiprint device (V & P Scientific) and copier unit (V & P Scientific) aligned over the 384-well plate.

• (iv) Print the oligonucleotide probes on a membrane on the same spots twice (pin replicator delivers ca. 0.2 μl per print); allow 5 min for air-drying the membrane before the second printing and fix the printed oligonucleotides onto the membrane by UV-cross-linking for 2 min at 120 mJ/s.

2.25.4 Labeling and Hybridization

• (i) Use the labeling kit (AlkPhos Direct labeling kit, GE Healthcare Bio-Sciences Corp) as per the manufacturer’s recommendations.

• (ii) Wet the membrane by incubating in 0.5% sodium dodecylsulfate (SDS) (w/v) at 55°C for 1 h followed by a rinse in 0.1 M Tris–HCl, pH 8.4 for 5 min.

• (iii) Perform prehybridization and hybridization at 55°C using the protocol suggested by AlkPhos Direct kits in 50-ml polypropylene tubes.

• (iv) Use 10 ml of hybridization buffer for every 20–25 cm² with 200 ng labeled target DNA for prehybridization; use only 5 ml of the buffer for hybridization with 200 ng labeled target DNA and perform hybridization at 55°C overnight.

• (v) Wash the membranes twice in a primary wash buffer containing 2 M urea, 0.1% SDS, 50 mM sodium phosphate, 150 mM NaCl and 0.2% blocking agent supplied with the kit at 55°C for 15 min each, followed by washing in a secondary wash buffer containing 50 mM Tris–HCl, 100 mM NaCl, pH 10 for 10 min at room temperature on a shaker.

• (vi) Incubate the membranes with CDP-Star chemiluminescent (GE-Healthcare Bio-Sciences Corp) reagent for 5 min; drain and expose to chemiluminescence BioMax film (Kodak Co, NY) for 1 h to overnight.

Appendix 26: Detection of Peach latent mosaic viroid (PLMVd) by Hybridization Techniques (Hadidi et al. 1997)

2.26.1 Extraction of Total Nucleic Acids

• (i) Powder the leaf tissue sample (0.2 g) with liquid nitrogen; extract in TE buffer consisting of 10 mM Tris–HCl, 1 mM Na₂ EDTA, pH 8.0; treat with an equal volume of phenol/chloroform (1:1) for 15–20 min and centrifuge at 3,000 rpm for 5 min at 4°C.

• (ii) Precipitate the nucleic acids from the supernatant by adding 0.1 volume of 3 M sodium acetate, pH 5.3 and 2.5 volumes of absolute ethanol; keep the mixture at −70°C for at least 2 h and centrifuge at 12,000 rpm for 10 min at 4°C.

• (iii) Resuspend the pellet in TE buffer; concentrate the total nucleic acids with 20% polyethylene glycol (PEG) 6,000 and 2.5 M NaCl solution; add distilled water to make up the volume to 100 μl; incubate in ice water for 1 h and centrifuge at 12,000 rpm for 20 min in a microcentrifuge.

• (iv) Wash the pellet once in 70% ethanol; dry in vacuo and dissolve in 20 μl deionized water.

2.26.2 Dot Blot Hybridization

• (i) Mix 10 μl total nucleic acids of each sample with 10 μl 20 × SSC/formaldehyde solution (3:2, v/v) [1 × SSC = 0.015 M sodium citrate and 0.15 M NaCl, pH 7.0]; keep the mixture at 65°C for 30 min and chill the mixture on ice for 2 min.

• (ii) Blot 20 μl of each mixture on a wet 0.45 μM Nytran membrane (Schleicher and Schuell) that have been soaked in a 6 × SSC solution; place the membrane on a minifold apparatus under vacuum and wash with 200 μl 6 × SSC solution.

• (iii) Cross-link to the membrane by irradiation in a UV cross-linker.

2.26.3 Northern (RNA) Blot Hybridization

• (i) Mix 50 μl of total nucleic acids of each sample with 50 μl 20% PEG 6,000 and 100 μl water; keep on the ice for 1 h and centrifuge at 12,000 rpm for 10 min at 4°C in microcentrifuge.

• (ii) Wash the pellet with 70% ethanol; dry under vacuum; resuspend in 10 μl TE buffer.

• (iii) Load 10 μl of PEG-precipitated total nucleic acids on a 6% polyacrylamide gel and electrophoresce for 1.5 h.

• (iv) Wash the gel with 1 × TAE ( 40 mM Tris–HCl, pH 8.0, 20 mM sodium acetate and 2 mM EDTA) for 10 min; electrotransfer to a TAE-equilibrated 0.2 μm Nytran membrane at 0.5 A for 14 h at 4°C.

• (v) Cross-link to the membrane by irradiation in UV cross-linker.

Appendix 27: Detection of Coconut cadang-cadang viroid (CCCVd) by Dot Blot Hybridization Technique (Vadamalai et al. 2009)

2.27.1 Extraction of Nucleic Acids Enriched with the Viroid

• (i) Blend the chopped leaf samples (10–20 g) in 120 ml chilled 100 mM NaSO₃; filter through cotton muslin; shake vigorously for 30 min at 4°C with polyvinyl pyrrolidone (20 g/l); mix vigorously with 50 ml chloroform for 5 min and centrifuge at 10,000 × g for 4 min.

• (ii) Add polyethylene glycol (PEG) (80 g/l) to the supernatant; dissolve by stirring at 4°C and collect the precipitate after centrifugation as done earlier.

• (iii) Extract the nucleic acids by resuspending the precipitate in 2 ml of SDS (10 g/l); add 2 ml aqueous phenol (900 g/l) containing 8-hydroxyquinoline (1 g/l) and shake vigorously.

• (iv) Collect the upper phase after centrifugation at 10,000 g for 10 min; reextract with 1 ml phenol and 1 ml chloroform for 5 min; add NaCl to 0.1 M; add cetyltrimethyl ammonium bromide (CTAB) (3.3 g/l) and incubate for 30 min on ice.

• (v) Centrifuge at 10,000 × g for 10 min; wash the pellet with 0.1 M sodium acetate in 75% ethanol and once with 100% ethanol and air-dry.

• (vi) Resuspend the pellet in 500 μl sterile double distilled water (SDDW) ; add an equal volume of 4 M LiCl; incubate the mixture at 4°C for 15–18 h and centrifuge at 12,000 × g for 15 min.

• (vii) Recover LiCl-soluble component; add 2.5 volumes of ethanol; allow the mixture to stand for 2–3 h; collect the precipitate by centrifugation at 12,000 g for 15 min; air-dry the pellet; resuspend the precipitate in 500 μl SDDW and store at −20°C.

2.27.2 Dot Blot Hybridization Assay

• (i) Cut leaf samples (1 g) into 50 mm lengths; crush in a plastic bag with 2 ml buffer (2 M NaCl, 100 mM sodium acetate, 10 mM EDTA, 50 mM Tris–HCl, pH 7.5 and 0.1% monothioglycerol) and extract 0.5 ml aliquots with 1% SDS and phenol-chloroform-isoamyl alcohol.

• (ii) Precipitate nucleic acids with isopropanol; wash the pellets with 95% ethanol; air-dry and redissolve in sterile water.

• (iii) Apply 1 μl samples (at different dilutions 1, 1/10 and 1/100) to nylon membrane (Zeta-Probe, Bio-Rad, USA) and cross-link by UV irradiation.

• (iv) Prehybridize the membranes at 65°C for 1 h in buffer containing 5 × SSC (750 mM NaCl, 75 mM sodium citrate, pH 7), 2.5% SDS, 0.3% PVP 40 and 100 μg/ml autoclaved herring sperm DNA.

• (v) Exchange the buffer for the same buffer containing 160 pmoles/ml of an oligonucleotide probe labeled with digoxingenin at the 5′ terminus; perform the hybridization at 65°C for 3 h; then at 40°C for 45 h.

• (vi) Wash the membrane sequentially in 3 × SSC, 2.5% SDS for 15 min at 38°C; 0.5 × SSC, 2.5% SDS for 15 min at 35°C; then 0.2 × SSC, 1% SDS for 15 min at 60°C.

• (vii) Block the membranes and detect the DIG-labeled probe with commercial monoclonal antibodies-phosphatase-conjugated antibody and substrate.

Appendix 28: Detection of Coconut cadang-cadang viroid (CCCVd) by Ribonuclease Protection Assay (Vadamalai et al. 2009)

• (i) Use a complementary sense RNA (cRNA) probe labeled with ³²P transcribed from a pGEM^®-T Easy vector (Promega Corp, USA) containing a monomeric insert of the 246-nt form of CCCVd.

• (ii) Precipitate the nucleic acids from leaf samples (5 g from oil palm or 1 g from coconut) with three volumes of ethanol in the presence of 0.1 M sodium acetate; air-dry the pellets; resuspend in 30 μl hybridization buffer containing 80% formamide, 40 mM PIPES (1,4-piperazine-diethanesulphonic acid), pH 6.5, 400 mM NaCl, 1 mM EDTA, pH 8.0, probe (2 × 10⁵ c.p.m); heat for 10 min at 95°C and incubate at 55°C for 12–18 h.

• (iii) Include a tube containing 100 μg cRNA from wheat germ (Sigma, Australia) as negative control.

• (iv) Add to each tube, 350 μl digestion buffer consisting of 300 mM NaCl, 10 mM Tris, pH 7.5 and 5 mM EDTA, pH 8.0 containing 10 μg/ml of RNAse A and 10 U/ml RNAse T1 (Roche) and incubate at 30°C for 1 h.

• (v) Stop the RNAse digestion by adding 10 μl of 20% SDS and 2.5 μl of proteinase K (10 mg/l) (Amresco, USA) to each tube and incubate at 37°C for 20 min.

• (vi) Add tRNA to a final concentration of 0.025 μg/μl and 400 μl of phenol-chloroform-isoamyl alcohol mix (1:1) followed by final extraction.

• (vii) Transfer 300 μl from the supernatant to a new tube; precipitate the nucleic acid with three volumes of ethanol at −70°C for 30 min and collect the pellet by centrifugation.

• (viii) Resuspend the pellets in 10 μl of denaturing loading buffer consisting of 0.25% bromophenol blue, 0.25% xylene cyanol, 10 mM sodium EDTA, pH 8.0 and 80% formamide; heat for 5 min at 95°C and chill on ice.

• (ix) Fractionate by 5% denaturing PAGE (8 M urea) at 40 mA for 1 h; fix the gel for 10 min in 0.5% acetic acid and 10% ethanol and wash once with double-distilled water.

• (x) Transfer to a wet sheet of 3-mm Whatman filter paper; dry for 2 h at 80°C in a gel dryer (Bio-Rad) and autoradiograph at −70°C for 1–72 h with an intensifying screen.

Appendix 29: Extraction of Citrus Viroids for Multiplex RT-PCR Assay (Wang et al. 2009)

2.29.1 Extraction of Total Nucleic Acids

• (i) Place the 5–10 mg samples (leaf, bark, fruit skin or root tissues) in 1.5 ml Eppendorf tubes immersed in liquid nitrogen ; grind the tissues with sterile plastic pestle (Bio-Rad); homogenize with 60 μl TES buffer [100 mM Tris–HCl, 2 mM EDTA and 2% SDS (w/v)] and 60 μl of a mixture composed of water saturated phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v); incubate in a water bath at 70°C for 5–10 min; centrifuge at 12,000 × g for 5–10 min.

• (ii) Make a hole at the bottom of a 0.5 ml Eppendorf tube using a hot 25 gauge needle; add a small quantity of glass beads (425–600 μM, Sigma) maintained in TNE buffer (10 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA) for covering the hole in the tube; fill this minicolumn with a slurry of Sephadex G50-80 ) (Amersham Biosciences) pre-equilibrated with the above TNE buffer; place inside a 2.2-ml centrifuge tube and centrifuge at 5,000 × g for 3 min to pack the matrix.

• (iii) Place this packed minicolumn into a sterile 1.5-ml Eppendorf tube; transfer 40 μl of the aqueous phase of the extract (from step (i) above) into the matrix, discarding the pellet and centrifuge the mini-column at 5,000 × g for 4 min to retrieve the eluate (about 20–80 μl).

• (iv) Use this total nucleic acid extract directly for RT-PCR or store at −20°C for periods up to 1 year.

• (v) Assess the purity of RNA extracted using a spectrophotometer and determine the OD₂₆₀/OD₂₈₀ ratio (ranging from 1.6 to 1.8).