Detection of Viral and Viroid Plant Pathogens in the Environment

Abstract

Plant viruses and viroids with obligate parasitism as the mode of existence, have to be in association with living cells of plant hosts always. However, some highly stable viruses have been detected in a free state in soil, water or air. Other viruses depend on the vectors such as insects, mites, nematodes or fungi present in the environment for their spread from infected plants to healthy plants which may be available in the same field or far away. The role of alternative/additional host plant species functioning as sources of virus infection has been demonstrated by applying various diagnostic techniques. The presence of viruses has been detected by employing biological, immunological and nucleic acid-based techniques. The effectiveness and reliability of detection varies significantly. However, molecular techniques have been shown to be more sensitive, specific and fast in providing the results, although they are unable to indicate the level of pathogenic potential of the viruses detected in the environmental samples. Viroids lacking natural vectors and stability outside living cells have been detected in several additional/alternative plant species which may function as reservoir of viroid infection.

Appendices

Appendix 1: Detection of Tobacco ringspot virus (TRSV)in the Nematode Vector by Immunofluorescence Technique (Wang and Gergerich 1998)

• (i) Select individually active nematodes (about 200) after giving adequate acquisition periods of feeding on virus-infected plants; transfer them to 1.5-ml centrifuge tube containing tap water; centrifuge at 14,000 × g for 4 min and remove the water from the tube.

• (ii) Incubate for fixation in 1 ml of 2% formaldehyde for 1 h at 4°C; remove the fixative solution after centrifugation; place the nematodes on a clean glass slide in a small amount of 2% formaldehyde and cut the nematodes into pieces, using a razor blade.

• (iii) Suspend the nematode fragments in two or five drops of blocking buffer consisting of 0.14 M NaCl, 0.01 M phosphate buffer, 3% bovine serum albumin (BSA) and 0.2% Triton X-100, pH 7.2, for 15 min at 4°C; gently pipette out the solution into a clean microcentrifuge tube; centrifuge for pelleting the nematode fragments and remove the supernatant.

• (iv) Dilute the purified primary antibody specific to the target virus to 1:50 in blocking buffer; transfer 200 μl to the microcentrifuge tube containing nematode fragments; incubate in an orbital shaker at 28°C for 18 h and wash the fragments four times for 10 min each with blocking buffer at room temperature.

• (v) Incubate the fragments in 200 μl of a 1: 50 dilution of fluoroscein isothiocyanate (FITC), conjugated goat anti-rabbit immunoglobulin G (Sigma Chemical Co., USA) in blocking buffer in an orbital shaker for 20 h at 28°C and wash the fragments four times for 10 min each in blocking buffer and dry in a vacuum dryer for 15 min.

• (vi) Mount the dried pellet on a glass slide in 10 μl of 50% glycerol in phosphate-buffered saline and gently tease the nematodes for their dispersal in the buffer and examine under an epifulorescent microscope.

Appendix 2: Detection of Plant Viruses in Vector Nematodes by Nested PCR Assay (Martin et al. 2009)

3.2.1 Extraction of Viral RNA from Nematodes

• (i) Hand-pick nematodes into 100 μl sterile water in 1.5-ml microfuge tubes; add 100 μl collagenase solution containing 10 mg/ml in 50 mM Tris, pH 7.4, 1 mM CaCl₂ and incubate the tubes for 1 h at 37°C.

• (ii) Add 50 mg glass beads (acid-washed 425–600 μm, Sigma); then add 200 μl 2 × extraction buffer consisting of 400 mM Tris, pH 8.5, 600 mM LiCl, 200 mM EDTA, 3% lithium dodecyl sulfate, 2% deoxycholic acid and 2% tergitol and add 2% ß-mercaptoethanol just before use.

• (iii) Vortex the closed tubes at full speed for 1 min; add 400 μl of 6 M potassium acetate, pH 6.5, vortex again and chill on ice for 30 min.

• (iv) Spin the tubes in a microfuge at the maximum speed for 5 min; transfer the supernatant to a new tube; add 1 μl glycogen; mix well; add an equal volume of isopropanol; mix well by inverting the tubes four or five times and chill for 30 min at −20°C.

• (v) Centrifuge the tubes for 10 min at maximum speed; dry the pellet after decanting the supernatant; wash the pellet with 500 μl 70% EtOH and dry the final pellet under vacuum for 5 min.

• (vi) Dissolve the pellet in 20 μl Molecular Biology Grade (MBG) water (Invitrogen); chill on ice for immediate use or store at −20°C.

3.2.2 Nested PCR Assay

• (i) For DNA preparation, transfer 2 μl RNA preparation to a 200-μl tube for 20 μl RT reaction as follows: combine 10.2 μl MBG water, 4 μl 5 × first strand buffer, 1 μl 0.1 MDTT, 1 μl 10 mM each dNTP mix, 0.7 μl RNase OUT (Invitrogen), 0.4 μl of 10 μM primer and 0.7 μl Superscript III (Invitrogen) and incubate at room temperature for 1 min and then at 50°C for 60 min.

• (ii) Use 1 μl of RT as a template in a 50 μl PCR using the first primer pair specific for target virus and Taq polymerase (Invitrogen) as per the manufacturer’s instructions (or alter concentration, if required).

• (iii) Provide the following conditions for amplification: 2 min at 94°C, ­followed by 40 cycles each at 94°C for 40 s, at 56°C for 40 s, at 72°C for 1 min and finally at 72°C for 4 min.

• (iv) For nested PCR, use 1 μl of the first PCR amplicon as template in a 50 μl reaction with the same parameters and conditions as in the first round PCR except for replacement with nested primer pair.

• (v) Analyze 15 μl of nested PCR amplicon by agarose gel electrophoresis and ­staining with ethidium bromide.

Appendix 3: Detection of Raspberry bushy dwarf virus (RBDV) in the Nematode Vector Longidorus juvenilis by Nested PCR Assay (Pleško et al. 2009)

• (i) Extract the nematodes from soil samples; store them at 4°C; extract the total RNA from the nematodes using RNeasy Plant Mini Kit (Qiagen).

• (ii) Use RBDV-specific primers U1, L3 and L4.

• (iii) For cDNA synthesis, add 10 μl of extracted total RNA to 15 μl of reaction mix containing 50 pmol of primer L4, 5 μl × 5M-MLV RT buffer (Promega), reverse transcriptase (Promega), 200 U RNasin (Promega) and incubate for 1 h at 50°C.

• (iv) Add 10 μl of RT reaction to 40 μl of reaction mix consisting of 75 mM Tris–HCl, pH 8.8, 20 mM ammonium sulfate, 0.01% Tween-20, 2 mM MgCl₂, 0.2 μM dNTPs, 50 μmol of each of the primers U1 and L4 and 2.5 U Taq DNA polymerase (Fermentas) for the first round PCR.

• (v) Provide the following conditions for PCR: initial denaturation for 5 min at 94°C, followed by 40 cycles of 1 min at 95°C, 1.5 min at 55°C and 1.5 min at 72°C and final elongation for 10 min at 72°C.

• (vi) Use 1 μl of the amplicon for second amplification using the primers U1 and L3 in place of the primer L4 and provide all conditions as mentioned above.

• (vii) Analyze the amplified products on 1% agarose gels, after staining with ethidium bromide.

Appendix 4: Detection of Polymyxa DNA in the Soil by Real-Time PCR Assay (Ward et al. 2004)

3.4.1 Extraction of DNA from Soils

• (i) Collect soils with Polymyxa betae from sugar beet fields and soils with P. graminis from cereal fields; mix 5 g soil samples with 10 ml of EDTA-based lysis buffer (containing 50 mM Tris–HCl, pH 7.2, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol) to form a slurry; transfer 1-ml aliquots to 2-ml tubes (five replicates) containing five 3-mm tungsten carbide balls (Qiagen) and centrifuge at 13,000 g for 2 min.

• (ii) Transfer the clarified soil extract to a fresh 2-ml Eppendorf tube; extract the DNA using a Wizard ManeSil DNA Purification System for food kit (Promega) and a Kingfisher Magnetic Particle Processor as per the manufacturer’s recommendations; determine DNA purity using a spectrophotometer at 260 nm (DNA), 280 nm (protein) and 320 nm (background turbidity) and calculate absorbance ratio = (A₂₆₀ − A₃₂₀)/(A₂₈₀ − A₃₂₀) (the ratio of pure DNA is 1.8).

3.4.2 TaqMan Real-Time PCR

• (i) Design primers to correspond to unique areas of sequence using Primer Express software (Applied Biosystems, USA); use primer pair and probe Pgraminis 690F/758R/713T specific to P. graminis and Pbetae689F/760R/718T specific to P. betae.

• (ii) Set up TaqMan reactions in 96-well reaction plates using a PCR core-reagent kit (Applied Systems) as per the manufacturer’s recommendations; for each reaction add 1 μl DNA to 24 μl mastermix in the appropriate well, giving a final reaction volume of 25 μl.

• (iii) Provide the following conditions: 50°C for 2 min, 95°C for 10 min and 40 cycles of 60°C for 1 min, 95°C for 15 s within the 7,700 Sequence Detection System (Applied Biosystems) using real-time data collection.

Appendix 5: Detection of Impatiens necrotic spot virus (INSV) by DAS-ELISA in Thrips Vectors (Sakurai et al. 2004)

• (i) Coat each well of microtiter plate with 100 μl coating buffer (containing 0.05 M sodium carbonate, pH 9.6) with 1.5 μg/ml IgG of PAb (raised against N protein of INSV-J) and incubate for 3 h at 37°C.

• (ii) Block nonspecific binding sites with 100 μl 1% BSA in PBS consisting of 0.14 M NaCl, 1 mM KH₂PO₄, 8 mM Na₂HPO₄, 2.5 mM KCl, pH 7.4; load the wells with 100 μl thrips extracts homogenized with a micropestle in a sample buffer containing 2% polyvinyl pyrrolidone( MW 40,000), 0.2% BSA in PBS including 0.05% Tween-20 (PBST) and incubate overnight at 4°C.

• (iii) Maintain controls with comparable thrips without access to infected source plants.

• (iv) Add 100 μl IgG alkaline phosphatase conjugate (1.5 μg/ml) in a sample buffer to each well; incubate for 3 h at 37°C and rinse the wells with PBST three times between each step above.

• (v) Add substrate 100 μl of 1 μg/ml p-nitrophenyl phosphate in 0.01 M ­diethanolamine buffer, pH 9.8 to each well and record absorbance values at 405 nm after 1 h reaction using an ELISA plate reader.

• (vi) Absorbance values greater than the mean plus four times the SD of controls are considered as positive reaction.

Appendix 6: Detection of Citrus tristeza virus RNA Targets by Tissue Squash Real-Time RT-PCR Assay in Aphids (Bertolini et al. 2008)

3.6.1 Preparation of Single Aphid Squash Samples

• (i) Squash single aphid species (Aphis gossypii) on 3 MM paper or nylon membranes with the rounded end of an Eppendorf tube and insert the pieces of paper or membrane harboring/squashed sample into Eppendorf tubes.

• (ii) Add 100 μl of 0.5% Triton X-100 or 100 μl buffer containing 0.1 M glycine, 0.05 NaCl and 1 mM EDTA and incubate at 95°C for 10 min.

• (iii) Vortex the contents in the tube; place the tubes on ice and use 5 μl of this extract as template for real-time RT-PCR.

3.6.2 Real-Time RT-PCR Assay

• (i) Select nucleotide sequence flanked by universal primers Pin1 and Pin2 to design the primers and probes and use Primer Express software (Applied Biosystems) to obtain the optimal oligonucleotide probe sequences.

• (ii) Perform TaqMan assays for real-time RT-PCR in ABI Prism 7,000 Sequence Detection System software (Applied Biosystems) using the reaction cocktail containing IX TaqMan Universal PCR Master Mix (Applied Biosystems) consisting of 1 μM 3′ UTR, 150 nM TaqMan probe 18IT and 5 μl of purified RNA or extracted RNA targets from immobilized samples.

• (iii) Provide the following conditions: one-step at 48°C for 30 min and 95°C for 1 min followed by 45 cycles of amplification at 95°C for 15 s and 60°C for 1 min.

• (iv) Perform data acquisition and analysis using ABI Prism 7,000 software and repeat the assays six times for determining the sensitivity and reliability of the assay.