Detection of Bacterial and Phytoplasmal Pathogens

Abstract

Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.

Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acid-based techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on DNA sequences of the target phytoplasma(s) to be detected. Precise, rapid and reliable results obtained from molecular methods can be used for detection, quantification and classification of phytoplasmas infecting whole plants, as well as those present in propagative plant materials which form the primary sources of infection for the crops to be planted in the subsequent seasons.

Appendices

Appendix 1: Media for Isolation of Bacterial Plant Pathogens

A. General Media
Brinkerhoff’s medium (Brinkerhoff 1960)
Dextrose	20 g
K2HPO4	50 g
Calcium carbonate	10 g
Agar	15 g
Water	1 l
Dye’s medium (Dye 1962)
Glucose	10 g
K2HPO4	2 g
Ammonium phosphate	1 g
MgSO4	0.2 g
NaCl or KCl	0.2 g
Water	1 l
King’s B agar medium (Matsuyama et al. 1998)
Peptone	20 g
K2HPO4	1.5 g
MgSO4.7H2O	1.5 g
Agar	15 g
Glycerol solution (1%)	1,000 ml
PH	7.2
Medium 523 (Kado 1971)
Sucrose	10 g
Casein acid hydrolysate	8 g
Yeast extract	4 g
KH2PO4	50 g
Magnesium sulfate	0.3 g
Agar	15 g
Water	1 l
Nutrient broth
Bactopeptone	5 g
Beef extract	3 g
Water	1 l
Potato–peptone–glucose–agar (PPGA) medium
Potato extract	500 ml
Bactopeptone	5 g
Glucose	5 g
NaCl	3 g
Na2HPO4	3 g
K2HPO4	0.5 g
Agar	18 g
Water	500 ml
Tetrazolium medium (TTC) (Kelman 1954)
Dextrose	10 g
Peptone	10 g
Cis amino acids	1 g
Agar	18 g
Water	1 l
Wakimoto’s medium (Wakimoto 1960)
Potato	200 g
Surcrose	15 g
Peptone	5 g
Na2HPO4.12 H2O	2 g
Calcium nitrate	0.5 g
Water	1 l
B. Semi-selective/selective media
Cefazolin trehalose agar (CTA) medium (Fessehaie et al. 1999)
K2HPO4	3 g
Na2HPO4	1 g
MgSO4.7H2O	0.3 g
NH4Cl	1 g
D (+)-trehalose	9 g
D (+)-glucose	1 g
Yeast extract	1 g
Cefazolin	0.025 g
Lincomycin	0.0012 g
Phosphomycin	0.0025 g
Cycloheximide	0.25 g
Agar	14 g
Distilled water	1 l
MMG semiselective medium (Toussaint et al. 2001)
Maltose	10 g
Tryptone	5 g
K2HPO4	2.75 g
Trace elements	0.02–1 mg
Methyl green (1% aqueous solution)	2 ml
Amoxicillin	32 mg
Cephalothin	32 mg
Cycloheximide	50 mg
Agar	15 g
Distilled water	1 l
MSCFF medium (Maringoni et al. 2006)
Peptone	5 g
Meat extract	3 g
Sucrose	5 g
Agar	15 g
Skim milk powder	5 g
Congo red	0.05 g
Chlorothalonil	0.01 g
Thiophanate methyl	0.01 g
Nalidixic acid	0.01 g
Nitrofurantoin	0.01 g
Oxacillin	0.001 g
Sodium azide	0.001 g
Distilled water	1 l
PCCG medium (Hara et al. 1995; Ito et al. 1998)
Potato semisynthetic agar (PSA) medium
Potato decoction from	300 g
Peptone	5 g
Sucrose	15 g
Ca(NO3)2	0.5 g
Na2HPO4.12H2O	2 g
Agar	18 g
PCCG medium
PSA	1,000 ml
Gella gum	18 g
Crystal violet	5 mg
Polymyxin B	4 × 105 units
Chloramphenicol	7.5 mg
Cycloheximide	50 mg
Tetrazolium chloride	2.5 mg
T-5 Semi-selective agar medium (Gaitaitis et al. 1997)
NaCl	5 g
Ammonium phosphate	1 g
K2HPO4	1 g
MgSO4.7H2O	0.2 g
d-tartaric acid	3 g
Phenol red	0.01 g
Agar	20 g
Water	1 liter
After autoclaving add
Bacitracin	10 mg
Vaniomycin	6 mg
Cycloheximide	75 mg
Novobiocin	45 mg
Penicillin G	5 mg
Adjust pH to 7.4
XTS selective medium (Schaad and Forster 1985)
Nutrient agar (Difco)	23 g
Glucose	5 g
Add the following antibiotics after autoclaving:
Cycloheximide (stock solution of 100 mg/ml in 75% ethanol)	200 mg
Gentamycin (stock solution of 10mg/ml in 75% ethanol)	8 mg
Cephalexin (stock solution of 10 mg/l in 75% ethanol)	10 mg

Appendix 2: Detection of Acidovorax avenae subsp. citrulli (Aac) by Pulsed-Field Gel Electrophoresis (PFGE) (Walcott et al. 2000; Burdman et al. 2005)

• 1. Grow the target bacteria (Aac) on nutrient agar (NA) at 28°C for 48 h; suspend the cells in a buffer containing 100 mM EDTA and 100 mM NaCl, pH 8 and adjust the bacterial cells to an OD₆₀₀ of 0.3 (approximately 5 × 10⁸ cfu/ml at 600 nm using a colorimeter).

• 2. Centrifuge the suspension at 10,780 × g for 2 min; resuspend the pellet in 300 μl of the buffer used earlier (step 1) and add 60 μl of 10 mg/ml lysozyme solution.

• 3. Prepare agarose plugs as follows: mix 500 μl of molten low-melting point ­agarose (10 mM Tris, pH 8, 10 mM MgCl₂, 10 mM EDTA, pH 8 and 2% (w/v) Sea Plaque GTG agarose (FMC BioProducts, Rockland, USA) with cell suspensions and pipette the mixture into disposable plastic plug-molds (Bio-Rad Laboratories, USA).

• 4. Solidify the agar plugs at 4°C for 20 min; cut the agar plugs into 5 × 5 × 1 mm pieces; rinse four times with 1 ml of sterile 1 × TAE (30-min rinses); replace 1 × TAE (40 mM Tris, 40 mM acetic acid and 1 mM EDTA) with 200 μl of 1 × restriction enzyme (SpeI, New England Biolabs, USA), buffer B (Promega Corp, USA) and incubate the plugs at 25°C for 15 min.

• 5. Replace restriction buffer with 200 μl of fresh restriction buffer containing 1 μl of SpeI and incubate at 37°C overnight.

• 6. Replace the restriction buffer with 500 μl of lysing solution without proteinase K; incubate at 55°C for 2 h; replace the lysing solution with fresh lysing solution ­without proteinase K and incubate the agarose plugs at 25°C for an additional 2 h.

• 7. Load the agarose plugs into wells of a 1% (w/v) pulsed-field certified agarose (BioRad Laboratories) gel (15 × 15 cm 1% Seakem Gold agarose); seal the molten low-melting-point agarose and use concatemeric lambda DNA (New England Biolabs) as a molecular marker.

• 8. Incubate the loaded gel in 0.5 × Tris-borate-EDTA (TBE) buffer (45 mM Tris; 45 mM boric acid and 1 mM EDTA, pH 8) for 30 min prior to electrophoresis and perform electrophoresis on a contour-clamped homogeneous electric field gel electrophoresis unit (CHEF-DRIII, Bio-Rad Laboratories) for 22 h at 6V/cm in 0.5 × TBE buffer at 14°C with initial and final switch times of 5 and 45 s respectively.

• 9. Stain the gels with a solution of 0.5 μg of ethidium bromide per ml for 30 min and capture the images under ultraviolet (at 295 nm) transillumination using a Kodak DC-40 digital camera.

• 10. Perform two independent PFGEs for each isolate for comparison of results for similarity/variation.

Appendix 3: Detection of Bacterial Pathogens by Slide Agglutination Test (Lyons and Taylor 1990)

2.3.1 Preparation of Somatic Antigen

• 1. Harvest the growth of target bacteria from 20 petriplates into 100 ml distilled water; centrifuge at 12,000 × g for 30 min; resuspend the bacterial cells in 50 ml cold acetone added with constant stirring to a total volume of 200 ml; centrifuge at 250 × g for 5 min; resuspend the bacterial cells in 10 ml cold acetone; lyophilize and store at −20°C.

• 2. Transfer approximately 2 g lyophilized bacterial cells to 100 ml cold distilled water; stir well; add slowly 100 ml 1 N trichloroacetic acid; stir the mixture for 18 h in the cold and centrifuge at 12,000 × g for 30 min at 4°C.

• 3. Collect the supernatant; neutralize with saturated sodium hydroxide; add four volumes of cold 95% ethanol (−20°C) and mix thoroughly.

• 4. Centrifuge at 200 × g for 10 min; remove the white floccular precipitate after discarding the supernatant; dissolve the precipitate in 5 ml distilled water; add 20 ml cold 95% ethanol; centrifuge at 200 × g for 5 min; resuspend the precipitate in 5 ml cold 95% ethanol; lyophilize and store dried powder at −20°C.

2.3.2 Production of Antiserum

• 1. Immunize New Zealand white rabbits with 0.5 ml of thick suspension of bacterial cells (10⁸ –10⁹ /ml) in saline or 10 mg of dried somatic antigen in 0.5 ml sterile distilled water emulsified with 0.5 ml Freund’s incomplete adjuvant intramuscularly, after bleeding for obtaining normal control serum; ­provide a second injection after 7 days and a third one, if necessary; collect the antiserum after bleeding; mix the antiserum with equal part of glycerol and store at 2–4°C.

2.3.3 Production of Staphylococcus aureus Reagent

• 1. Grow the authentic Cowan 1 strain of S. aureus on nutrient agar at 37°C for 24–48 h; prepare a cloudy suspension of bacterial cells (10³ –10⁷ ) in glycerol broth containing 1.6 g nutrient broth (Difco), 30 ml glycerol and 170 ml distilled water; transfer 1–2 ml aliquots to sterile storage containers; add an equal volume of sterile 3 mm hollow glass beads (Creative Beadcraft Ltd, Amersham, UK) and place the containers directly at −80°C without pre-cooling.

• 2. Thaw one container; transfer the contents to 10 ml nutrient broth; distribute the culture uniformly by shaking well to 40 nutrient agar plates and incubate at 37°C for 72 h.

• 3. Harvest the bacteria from the plates by flooding with phosphate-buffered saline (PBS) containing 0.02% sodium azide and gently scrapping surface of plates with a glass streaker.

• 4. Centrifuge for 30 min at 300 × g; resuspend the deposit, after discarding the supernatant, in 20 ml 1.5% formaldehyde solution; mix well for 90 min; heat at 80°C for 30 min and cool rapidly to room temperature.

• 5. Wash twice by centrifugation; resuspend in PBS containing 0.05% sodium azide and mix with PBS containing 0.05% sodium azide at the rate of one volume of packed cells to nine volumes of buffer.

• 6. Sonicate the suspenstion gently for 30 s to eliminate clumps and store at 2–4°C until required for use.

2.3.4 Conjugation of Working Reagent

• 1. Use 170 μl antiserum (1:1 mixture with glycerol), 4 ml PBSA, 830 μl S. aureus reagent and 100 μl filtered saturated alcoholic basic fuchsin 100 μl and store this preparation at 2–4°C.

2.3.5 Slide Agglutination Test

• 1. Mix the conjugated working agent and the target organism either from pure ­culture or infected plant tissue extracts for several seconds on a glass microscope slide (Multitest slides) using a sterile wood toothpick; use 7 μl conjugated ­working reagent + sufficient bacteria to produce a thin suspension or5 μl plant extract + 5 μl conjugated reagent for mixing on the slide.

• 2. Maintain positive and negative controls with homologous bacteria and test reagent alone respectively.

• 3. Observe for the formation of granular agglutination indicating the positive reaction.

Appendix 4: Detection of Xanthomonas campestris pv. vesicatoria by Enzyme-Linked Immunosorbent Assay (ELISA) Formats (Tsuchiya and d’Ursel 2004)

2.4.1 Preparation of Bacterial Cells (Antigen)

• 1. Grow the target bacterial species on suitable (YPA) medium ­containing 7 g yeast extract, 16 g agar, 1 l distilled water, pH 7.2 by incubating at 28°C for 2 days; collect the bacteria with 10 ml of 0.01 M phosphate-buffered saline (PBS), pH 7.2 and wash the cells with PBS three times by centrifugations at 10,000 × g for 10 min.

• 2. Weigh the wet cell precipitates; adjust the stock concentration of 10 mg/ml and prepare the standard cell suspension spectrophotometrically to about 10⁸ cfu/ml.

2.4.2 Preparation of Plant Samples

• 1. Collect the leaves with symptoms at random in the fields; cut them separately; treat them as described below, maintaining healthy controls.

• 2. Collect leaves from inoculated plants; wash them by successively soaking them in water, 30 s in 70% EtOH and then 5 min in 0.5% NaClo and rinse thoroughly with sterile distilled water.

• 3. Excise two disks for each sample with a 10 mm diameter cork borer; homogenize in 1 ml of 0.1 M phosphater buffer, pH 7.0 with a tissue grinder; transfer to 1 ml microcentrifuge tubes; centrifuge for 1 s at 5,000 rpm; store the supernatant at 4°C and adjust the fractions for coating with 1 M sodium carbonate buffer, pH 9.6 just before use.

2.4.3 ABC-Enzyme-Linked Immunosorbent Assay

• 1. For noncompetitive immunoassay, coat 96-well plates first with samples (200 μl/well) suspended in 0.1 M carbonate buffer at 37°C for 4 h and wash three times with PBS buffer and also in subsequent steps as described below:

• 2. Block unspecific reactive sites with a solution of 1% skim milk powder for 2 h at room temperature (RT); add monoclonal antibodies (MAbs) (100 μl/well); incubate at RT and wash.

• 3. Characterize the MAbs by following one of the two methods described below:

  For avidin/biotin peroxidase-conjugate (ABC) method, use an ABC Kit (Vector Laboratories, USA) with 2,2′-azino-di-3-ethyl-benzothiazoline-b-sulfonic acid (ABTS) (Boehringer, Germany) as substrate for peroxidase (A_max + 405 nm)

  For the second method, incubate with goat anti-mouse immunoglobulin M (IgM) peroxidase conjugate (Chemi-ConInternational, USA) for 30 min; add a solution of 0.04% O-phenylenediamine dihydrochloride (OPD) (A_max = 492 nm) and 0.12% hydrogen peroxidase in 0.5 M phosphate–citrate buffer pH 5, as substrate.

• 4. Competitive immunoassay allows reaction between MAbs and the samples diluted in PBS for 4 h in microcentrifuge tubes at RT; dispense in duplicates to the antigen precoated 96-will plate; determine the amount of MAbs retained on the wells as per the second method described above (step 3).

Appendix 5: Detection of Erwinia chrysanthemi Using Fimbria-Specific Antibody in ELISA Technique (Singh et al. 2000)

2.5.1 Production of Antiserum

• 1. Immunize New Zealand white rabbit for producing polyclonal antibodies (PAbs) with glutaraldehyde-fixed cells of target bacterial species intramuscularly; provide five injections at 2-week intervals; collect the blood by cardiac puncture and separate the antiserum.

• 2. Immunize the mice for producing monoclonal antibodies (MAbs) by intraperitoneal injection of glutaraldehyde-fixed bacterial cells.

• 3. Fuse the splenocytes from immunized mice with FOX-NY cells using 50% polyethylene glycol (PG) and dimethyl sulphoxide as fusogens.

• 4. Use Dulbeccos modified Eagles Medium supplemented with hypoxanthine, aminopterin and thymidine to select suitable hybridomas for screening for specificity of reaction.

2.5.2 Screening Hybridomas for Specific MAbs

• 1. Wash 24-h old pathogen cells from pure cultures; suspend them in 0.01 M ­phosphate buffered saline (PBS), pH 7.2 and adjust the cell population to an OD of 1.0 at 620 nm.

• 2. Transfer 100 μl bacterial cells diluted to 1:1 in 0.1 M carbonate buffer, pH 9.6 to each of the microplate well and incubate overnight at 4°C.

• 3. Block the monspecific sites with 5% skim milk powder for 20 min; wash and add hybridoma fluid containing MAbs.

• 4. Carry out all incubations (one h at 37°C) and washing steps as in standard ELISA protocol.

• 5. Detect the reactive MAbs using goat ant-mouse antibodies conjugated with ­alkaline phosphatase and p-nitrophenol phosphate at 0.5 mg/ml in 1 M diethanolamine buffer, pH 9.8 and incubate for 45–60 min.

• 6. Determine the color intensity at 405 nm using an ELISA reader.

2.5.3 Detection of Target Pathogen in Plant Tissues

• 1. Prepare the 1% plant extracts using the sample buffer consisting of 2 g KH₂PO_4. 11.5 g Na₂HPO₄, 0.14 g disodium EDTA, 0.02 g thimerosal and 0.2 g lysozyme in 100 ml water.

• 2. Coat the microtiter wells with PAbs (1:5,000 dilution of raw serum in 0.1 M carbonate buffer, pH 9.6).

• 3. Transfer the plant extracts to each well to capture the antigen by PAbs present in the wells.

• 4. Follow the steps as per standard ELISA procedure.

Appendix 6: Detection of Erwinia chrysanthemi by Immunogold Labeling and Electron Microscopy Technique (Singh et al. 2000)

2.6.1 Immunogold Labeling

• 1. Collect the target bacterial cells from a 24-h old culture; suspend in PBS at a concentration of 1.0 OD at 620 nm; mix with an equal volume of hybridoma 6A6 culture fluid and incubate at 4°C overnight.

• 2. Wash the cells three times in PBS; suspend them in 200 μl of PBS and add 15 μl of gold beads conjugated with goat anti-mouse immunoglobulin G and immunoglobulin M (IgG + IgM, Bio/Can Scientific, Canada) and incubate the mixture for 1 h at room temperature with agitation.

• 3. Wash the cells with PBS three times and resuspend in 200 μl PBS.

2.6.2 Electron Microscopy

• 1. Place the bacterial cell suspension on copper-coated electron microscope grids at 25 μl/ grid and air dry.

• 2. Stain with 1% phosphotungstic acid and view under the electron microscope.

Appendix 7: Detection of Clavibacter michiganensis subsp. michiganensis by Flow Cytometry (Chitaara et al. 2006)

2.7.1 Labeling of Target Bacterial Cells

• 1. Grow the target pathogen on 1% glucose-nutrient agar medium (Oxoid) for 24 h at 25°C; harvest the bacterial cells; resuspend in 0.2 M sodium phosphate buffer (SP1), pH 7.4; adjust the OD at 620 nm to approximately to 0.35 to give 10⁶ –10⁷ cfu/ml concentration.

• 2. Prepare nonviable bacterial cells by exposing the bacterial cells to 80°C for 30 min.

• 3. Mix the viable (non-treated) and non-viable (heat-treated) cells in different ratios: 100/0, 80/20, 50/50, 20/80, 0/100 respectively.

• 4. Incubate the bacterial cells in SP1 for 1 h at 28°C in the presence of 10 μM cFDA (carboxy-fluorescein diacetate), 10 μM Calcein AM (Calcein acetoxy methyl ester) separately or in combination with 10 μM PI (propidium iodide) for double labeling; incubate the samples stained with PI alone for 20 min at room temperature.

• 5. Centrifuge the suspension at 11,000g; wash; resuspend in SP1, pH 7.4 and place them on ice till required.

2.7.2 Flow cytometric Analysis

• 1. Analyze individual cells using a FACS Calibur flow cytometer (Becton-Dickinson Benelux NV, Belgium) equipped with an air-cooled argon ion laser (excitation wave length 488 nm) operated at 15 mW.

• 2. Use a logarithmic amplification of the incoming signal and a sample analysis time of approximately 2 min.

• 3. Quantify based on the flow rate that is determined to be 4.8 μl/min.

• 4. Use non-treated (viable) cells as negative controls for Calcein AM and cFDA and heat-treated (non-viable) unstained cells for PI.

• 5. Sort out the bacterial cells labeled with Calcein AM, cFDA or PI based on the green and red fluorescence of cells at 530 and >670 nm respectively.

• 6. Note the high green fluorescence signals from Calcein AM- and cFDA-stained cells (viable) and high red fluorescence signal from PI-stained cells (non-viable).

Appendix 8: Detection of Erwinia amylovora by Polymerase Chain Reaction (PCR) (Stöger et al. 2006)

2.8.1 Extraction of DNA

• 1. Cut wood slices from naturally infected or inoculated plant samples; place 0.5–0.7 cm slices/leaf discs into 1.5-ml collection tube; add extraction solution of REDExtract-N-Amp^TM Plant PCR Kit and incubate at 95°C for 10 min; follow all steps as per the manufacturer’s recommendations.

• 2. Dilute the extract with 100 μl of dilution buffer; mix by vortexing and use 4 μl of the diluted extract for PCR.

• 3. Adopt the modified procedure for enhancement of sensitivity; place the wood slices (instead of leaf discs) into 1.5-ml collection tube; add 150 μl extraction solution supplemented with 0.1% Triton X-100 (v/v) and 0.05% Nonidet NP-40 and incubate at 95°C for 30 min.

• 4. Transfer 50 μl of the extract to a new tube; dilute it with 50 μl dilution buffer and store at 2–8°C till use.

• 5. Prepare a 1:30 dilution (v/v) of extract with a 1: 1 mixture of extraction: dilution solution and use 4 μl of diluted extract for PCR.

2.8.2 Polymerase Chain Reaction

• 1. Perform all steps as per manufacturer’s instructions by providing the following PCR conditions: 95°C for 5 min; 35 cycles each of 95°C for 15 s, 53°C for 30 s and 72°C for 45 s and final step at 72°C for 10 min.

• 2. Analyze amplified PCR products on 1.5% agarose gels stained with ethidium bromide and photographed.

Appendix 9: Detection of Xanthomonas campestris pv. vesicatoria (Xcv) by PCR Assay (Park et al. 2009)

2.9.1 PCR Amplification

• 1. Use the primers XCVF and XCVR designed using the sequences of rhs family gene of Xcv to amplify a product of 517-bp.

• 2. Perform the PCR amplification using a PTC-225^TM thermocycler (MJ Research, USA) in a final volume of 50 μl containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.01 % gelatin, 0.2 mM of each dNTP, 10 pM of each primer and 2 Units of Taq polymerase (Promega^TM, USA) and approximately 50 ng of genomic DNA of test microorganism.

• 3. Provide the following conditions: 25 cycles, each consisting of 60 s at 94°C, 30 s at 58°C and 60 s at 72°C with initial denaturation of 5 min at 94°C and final extension of 10 min at 72°C.

• 4. Resolve an aliquot of 8 μl of PCR product on a 1.0% agarose gel, stained with ethidium bromide and visualized on a UV transilluminator.

2.9.2 Dot Blot Analysis

• 1. Spot aliquots of 100 ng of each sample of DNA isolated from test pathogen (Xcv) onto Hybond-N+ nylon membrane (Amersham Pharmacia Biotech UK).

• 2. Label the PCR product of Xcv as probe with ³² P dCTP using random primed method as per manufacturer’s recommendations(Ladderman^TM Labeling Kit, Takara, Japan).

• 3. Perform prehybridization and hybridization in hybridization buffer consisting of 0.75 M NaCl, 75 mM sodium citrate, 0.5% SDS, 0.1% BSA, 0.1% FiColl, 0.1% polyvinyl pyrrolidone and 50 μg/ml denatured salmon sperm DNA at 65°C for 18 h.

• 4. Wash the filter twice for 10 min each in 2 × SSC containing 0.1% SDS at room temperature and twice for 15 min each, in 0.1 × SSC containing 0.1% SDS at 65°C.

• 5. Carry out autoradiography at - 70°C with CUR1 X-ray film (AGFA, Belgium).

Appendix 10: Detection of Xanthomonas axonopodis pv. citri (Xac) by Immuno-Capture (IC) and Nested (N)-PCR Assay (Hartung et al. 1996)

2.10.1 Magnetic Immunocapture of Target Pathogen Cells

• 1. Quantify the selected MAb (A1 for Xac) spectrophotometrically at 280 nm and incubate with paramagnetic Dynabeads M-280 precoated with sheep anti-mouse IgG (Dynal) overnight at 4°C with shaking at 125 rpm.

• 2. Coat 40 μl (0.4 mg) of beads with 4 or 0.04 μg of MAb (10 or 0.1 μg of MAb/10⁶ beads) depending on the experiment; separate the bead-MAb complex from the solution by applying a magnet to the wall of the tubes; eliminated unbound MAb by four washes for 30 min/wash with two volumes of phosphate buffered saline (PBS) with 0.1 bovine serum albumin (BSA) and incubate the complex with 500 μl of serially diluted target bacterial suspensions (from 10 to 10⁸ cells/ml of Tris-HCl buffer, pH 7.2) for at least 1 h at 4°C with gentle shaking.

• 3. Separate the immunocaptured bacterial cells magnetically from the solution; wash three times with PBS buffer; suspend the bacteria in 10 μl sterile water; boil the beads for 5 min and centrifuge at 13,000 × g briefly.

2.10.2 PCR Reactions

• 1. Use primers based on the sequence of 572-nt of Xac insert in the plasmid pFL1 (Cruchem, USA).

• 2. Use reaction buffer with 3 mM MgCl₂, 0.5 μM each primer, 125 μM nucleotides and 1.25 units Taq polymerase per 25 μl of reaction.

• 3. Provide the following conditions for the first round of PCR: 35 cycles of 95°C for 30 s, 58°C for 60 s and 72°C for 60 s.

• 4. For nested (N)-PCR, use 1μl from the first reaction product as template with primers 94-3 bio (5′-biotin) and 94-4 lac for amplification of 315-bp product from DNA or cells of Xac.

• 5. Use 20 cycles at the same temperature profile mentioned above for the second round PCR.

• 6. Resolve the amplified products by electrophoresis through 2% NuSieve (FMC Bioproducts, USA) agarose (3:1) gels and staining with ethidium bromide.

Appendix 11Appendix 11: Detection of Xylella fastidiosa (Xf) in Xylem Exudates of Grapevine By IC-PCR Technique (Guo and Lu 2004)

2.11.1 Extraction of Xylem Exudates

• 1. Adopt the pressure chamber method as follows: collect 10–12 cm sections of infected and healthy shoots from vineyard between 09 and 10 AM; wrap them in moist paper towels and place them in plastic bags separately.

• 2. Extract the xylem fluids from the shoots or leaf petioles using a pressure chamber (PMS Instrument CO., USA) as per the manufacturer’s instructions; plate 1 μl xylem exudates onto PD3 medium and count the number of ­colonies after 3 weeks.

2.11.2 Preparation of Xf Suspension by Immunocapture Procedure

• 1. Add 100 μl of PBS-BSA (PBS plus 0.2% BSA) to 5–10 μl of xylem exudates; add 100 μl diluted whole rabbit serum (1/1,000 in PBS-BSA) and incubate the mixture for 45 min.

• 2. Centrifuge at 12,000 rpm for 5 min; reject the supernatant; add 5 μl of sheep anti-rabbt antibody-coated beads (Dynal, Oslo, Norway) to the pellet; incubate at 4°C for 45 min and wash the beads four or five times using PBS-BSA.

• 3. Recover the beads by centrifugation at 12,000 rpm and resuspend in 10 μl of sterile water.

• 4. Alternatively, centrifuge the xylem exudates at 12,000 rpm for 5 min; wash the pellet four or five times as described above and resuspend in 10 μl of sterile distilled water.

2.11.3 PCR Amplification

• 1. Use two oligonucleotide primer pairs XF766f/XF686r and RST31/RST33.

• 2. Perform PCR amplification using a volume of 25 μl containing 1 × reaction ­buffer, 1.5 mM MgCl₂, 160 μM each dNTP, 2 μM of each primer, 1 U of Taq DNA polymerase (Promega, Madison, USA) and 2 μl of bacterial suspension as the DNA template.

• 3. Provide the following conditions using a DNA termocycler (MJ Research, USA): initial denaturing at 92°C for 5 min, followed by 35 PCR amplification cycles; denaturing at 92°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min, final extension at 72°C for 8 min.

• 4. Resolve the PCR products on a 1.5% agarose gel; stain with ethidium bromide and photograph on a UV transilluminator (Polaroid Co., Cambridge, USA).

Appendix 12: Detection of Xanthomonas axonopodis pv. citri by Real Time PCR Assay (Mavrodieva et al. 2004)

2.12.1 Sample Preparation Using the IT 1-2-3 RAPID DNA Purification Kit

• 1. Collect the field samples from freshly infected lesions from leaves and fruits, in addition to uninfected samples; swip the leaves and fruits with moistened swabs provided with the kits; place the swabs inside tubes containing ceramic beads suspended in water provided with the kit and store the samples up to 1 week at 4°C prior to further processing.

• 2. Vortex the samples in 5 min in DNA extraction buffer provided in the kit; centrifuge and run the supernatant over a filtration spin column; wash the column once; elute the DNA in 100 μl of elution buffer and perform real-time PCR analysis.

2.12.2 Real-Time PCR Assay

• 1. Perform the assay on a mobile RAPID 7200 System (Idaho Technology, USA) using SYBR Green I (Molecular Probes, Eugene, UK) fluorescent dye detection system; run each sample in duplicates in glass capillary tubes (Roche Diaganostics, USA); include a negative (without target DNA) and positive (with target DNA) controls.

• 2. Add 2 μl of sample supernatant or purified DNA extracted from culture-grown cells or plant tissues to 18 μl of reaction mix containing 1 × PCR buffer with bovine serum albumin (BSA), 2mM MgCl₂, 0.5 μM each primer, 0.2 mM dNTPs, 2 μl of 1:3,000 SYBR Green I and 0.8 units of Taq polymerase (Roche Diganostics).

• 3. For hot-start PCR, incubate Taq polymerase with TaqStart Antibody (Clontech, CA) 1:28 (v/v) for 10 min at room temperature before adding to the reaction mixture.

• 4. Provide conditions as per manufacturer’s recommendations.

• 5. Detect total SYBR Green fluorescence after each amplification cycle which ­provides a measure of PCR product formation; use a melting curve analysis as a second criterion to determine the presence or absence of target, as it helps in detecting false positive PCR product formation; each PCR product shows a characteristic peak at its melting temperature (Tm) maxium.

Appendix 13: Detection of Phytoplasmal Pathogens by Histological Techniques (Galvis et al. 2007)

2.13.1 DAPI Fluorescence Test

• 1. Fix the petioles (5–8 mm long) healthy and diseased leaves in 2.5% glutaraldehyde dissolved in 0.1 M phosphate buffer, pH 7.0 and store at 4°C till required for staining.

• 2. Rinse the petiole tissues with water to remove the excess fixative; immerse in 4′6-diamidino-2 phenylindole [(DAPI), Sigma] at 0.4 g/ml; mount the tissues into carrots and prepare sections (15–20 μm thick).

• 3. Examine the sections under epifluorescence microscope (Zeiss Jenamed) with a set of one BP365/11 excitation filter and an LP395 suppressor filter.

2.13.2 Transmission Electron Microscopy (TEM)

• 1. Cut the petioles of healthy and diseased leaves into 1 × 2 mm pieces and prefix in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 24 h.

• 2. Prepare sections (500 nm) with a diamond knife using an MT 6000 Sorvall Ultramicrotome; post-fix and precontrast in acetate; dehydrate in an ethanol series of 25, 50, 75, 90 and 100% (20 min each) and continue with pure acetone (20 min, three times).

• 3. Embed in Spurr’s resin (60°C for 8 h), after a 60-min infiltration with acetone-Spurr (1:1) to facilitate entry of resin into the tissues.

• 4. Mount the utrathin sections on copper grids (1 mesh) and observe on a JEOL 100 CX transimission electron microscope; note the presence of pleomorphic bodies in the phloem cells.

Appendix 14: Detection of Spiroplasma Infecting Carrot by Nested PCR Assay (Green et al. 1999; Lee et al. 2006)

2.14.1 Extraction of Pathogen DNA

• 1. Prepare plant tissues (leaf petioles and midribs, scion wood or root tissues) for total DNA extraction from samples of 0.5–0.6 g; place them in a sample bag (Agadia) suitable for use with a Homex 5 homogenizer–extractor and homogenize with 5 ml of CTAB extraction buffer consisting of 100 mM Tris, pH 8.0, 1.4 M NaCl, 50 mM EDTA, pH 8.0, 2.5% (w/v), CTAB, 1% ­polyvinyl ­pyrrolidone (PVP-40) and 0.2% 2-mercaptoethanol (added just before homogenization).

• 2. Transfer 0.5 ml homogenate to a 1.5 ml microcentrifuge tube; mix with 22 μl RNase A (20 mg/ml) and incubate at 65°C with gentle shaking (800 rpm) using an Eppendorf Thermomixer for 25–35 min.

• 3. Add 162 μl AP2 buffer (Qiagen), mix by inversion; place on ice for 5 min; ­transfer the entire contents to QIA shredder (Qiagen) column sitting in a 2-ml collection tube and microcentrifuge at a maximum speed for 2 min in an Eppendorf mcirocentrifuge.

• 4. Transfer the column flow through (450 μl) without disturbing the cell debris ­pellet to a new 1.5 ml microcentrifuge tube and mix by inversion with 225 μl of AP 3 buffer (Qiagen) and 450 μl of 95% ethanol.

• 5. Transfer 650 μl of the mixture to a DNeasy (Qiagen) column; microcentrifuge at 8,000 × g for 1 min; discard the flowthrough and repeat the microcentrifugation with the remaining sample.

• 6. Transfer the column to a clean 2-ml collection tube; add 500 μl of AW (Qiagen) buffer to the column; wash throughly by microcentrifugation at 8000 × g for 1 min; discard the flowthrough and add additional 500 μl of AW buffer to the column.

• 7. Microcentrifuge at maximum speed for 2 min; transfer the column to a new 1.5 ml microcentrifuge tube; elute the DNA with 100 μl of pre-warmed (60°C) AE (Qiagen) buffer and microcentrifuge at 8,000 × g for 1 min.

• 8. Heat the DNA extracts at 95°C for 5 min; place them on ice for 5–10 min and store at −80°C until needed for PCR analysis.

2.14.2 Nested PCR and RFLP Analysis for Phytoplasmas

• 1. Perform the first amplification using primer pair P1/16S-Sr, followed by R16F2n/R162n in the second amplification and maintain a negative control devoid of DNA templates in the reaction mix in all PCR assays.

• 2. Use an automotive thermocycler for 38 cycles of amplification with AmpliTaq Gold polymerase (Applied Systems); carry out the reaction in 25 μl reaction mixtures containing 1 μl undiluted nucleic acid preparation (100–200 ng), 200 μM of each dNTP, and 0.4 μM of each primer.

• 3. Maintain the following conditions: denaturation at 94°C for 1 min (11 min for the first cycle to activate AmpliTaq Gold polymerase), annealing for 2 min at 55°C and primer extension for 3 min (7 min in the final cycle) at 72°C.

• 4. Use 1 μl of diluted (1:30) PCR product from the amplification as a template in the reaction mixture (50 μl) for nested PCR format.

• 5. Electrophorese the products (5 μl) on a 1% agarose gel; stain in ethidium bromide and visualize with UV-transilluminator.

• 6. Digest the PCR product (6 μl) individually with restriction enzymes AluI, HhaI MseI and Sau3AI (New England Biolabs) according to the manufacturer’s instructions; separate the restriction products by electrophoresis through a 5% polyacrylamide gel for 1 h at 150v; stain with ethidium bromide and visualize with UV transilluminator.

2.14.3 Nested PCR and RFLP Analysis for Spiroplasmas

• 1. Use the primer pair ScR1F1/ScR16R1 in the first amplification followed by ScR16F1A/ScR16R2 in the second amplification.

• 2. Adopt PCR conditions as described for nested PCR format except the annealing temperature of 50°C.

• 3. Digest PCR products (1.5 kb) obtained from second amplification with MseI, AluI, HhaI and Hae III.

• 4. Use appropriate reference strain(s) for comparison with test samples.

• 5. Follow the rest of the steps as described above.

Appendix 15: Detection of Phytoplasmas by Oligonucleotide Microarray-Based Assay (Nicolaisen and Bertaccini 2007)

2.15.1 PCR Amplification

• 1. Perform all PCR amplification using Taq DNA polymerase (Promega) according to the manufacturer’s recommendations, providing the following conditions: 94°C for 2.5 min followed by 30 cycles of 94°C for 15 s, 62°C for 30s and 72°C fir 15 min and finally 72°C for 10 min.

2.15.2 Labeling of Hybridization Probes

• 1. Label the PCR products (1 μl PCR) for hybridization using the BioPrime Array CGH Labeling Kit (Invitrogen) with Cy-3 dCTP (Amersham Biosciences) according to the manufacturer’s instructions (volume reduced to one fourth of the recommended quantity to reduce the costs).

• 2. Purify labeling reactions using MinElute Reaction Clean up Kit (Qiagen) according to the manufacturer’s recommendations.

2.15.3 Printing and Post-processing of Oligonucleotide Arrays

• 1. Prepare a 384-well plate with 10 μl of 20 μM capture oligonucleotide (CO) ­solutions in 30% DMSO, 1.5 × SSC and 0.005% SDS and spot the samples with a QArray Minispotter (Genetix) on NextronE slides (Schott UK Ltd) using one aQu printing pin K2801 (Genetix) at 45% humidity.

• 2. Spot each oligonucleotide in quadruplicate in each of the 16 fields and post-process as per the manufacturer’s recommendations.

2.15.4 Microarray Hybridization

• 1. Attach a FAST Frame (Schleicher & Schuell) array grid with 16 wells to printed slides to allow 16 individual hybridization reactions on each slide.

• 2. Heat the labeling reactions in 80 μl of 10% formamide, 5 × SSC, 0.1% SDS and 0.1 μg/μl sheared salmon sperm DNA to 95°C for 3 min before each hybridization mix is applied to individual wells; cover the slides and place them in a moist chamber with saturated NaCl solution and incubate at 48°C overnight.

• 3. After detaching the array grid, wash the slides in a 2 × SSC, 0.1% SDS at 42°C for 5 min, repeat washing twice in 1 × SSC, 0.1% SDS for 1 min at room ­temperature and dry the slides using compressed oil-free air.

2.15.5 Microarray Scanning

• 1. Scan the hybridized arrays using an ArrayWoRx^® Biochip Reader (Applied Precision) as per the manufacturer’s instructions with Cy3 settings (595 nm) and 1 s exposure; locate DNA spots on the array using ARRAYWoRx software and define the spots to be within a circle with a diameter of 200 μm.

• 2. Define a circumscribing square (300 μm) as a region of interest (ROI) and use it to determine local background of spots.

• 3. Process the results as mean pixel intensity within the defined spot area subtracted from local mean background as defined by the ROI.

Appendix 16: Detection of Clavibacter michiganensis subsp. michiganensis (Cmm) by Immunomagnetic Separation (IMS) – Plating Technique (de León et al. 2006)

2.16.1 Coating Magnetic Beads

• 1. Coat the immunomagnetic beads (IMBs) precoated with sheep anti-rabbit IgG (Dynabeads M-280, Dynal, Norway) with specific antisera for ­target pathogen (Cmm); wash the beads three times with phosphate buffer saline 0.1 M, pH 7.2 (PBS); collect the IMBs using magnetic particle concentrator (MPC-s, Dynal) and resuspend in PBS to have the desired concentration of IMBs.

• 2. Incubate the IMBs with anti-Cmm antiserum for 24 h at 4°C with gentle shaking as per the instruction of the manufacturer; wash three times with PBS containing 0.1% bovine serum albumin (PBS-BSA) and 0.05% Tween-20 to remove unattached antibodies and resuspend the beads in PBS-BSA to obtain a stock suspension (10³ IMBs/ml) and store at 4°C.

2.16.2 Standardization of IMS Technique

• 1. Prepare required dilutions of the pathogen (Cmm)-specific antiserum; add aliquots of anti-Cmm-coated IMBs to 1 ml of Cmm suspension (5.7 × 10³ cfu/ml) to obtain 10⁵ IMBs/ml and incubate for 1 h at room temperature with gentle shaking.

• 2. Wash the IMBs three times with 1 ml of PBS-BSA containing 0.05% Tween X-20 for 10 min; resuspend in 1 ml of PBS and spread 0.1 ml of suspension on petriplates containing appropriate nutrient medium (YPGA); incubate for 5 days at 25°C and enumerate the pathogen (Cmm) colonies.

2.16.3 Isolation of Target Pathogen from Naturally Infected Seeds by IMS

• 1. Soak 2 g of seeds in 20 ml of PBS + 0.1% Tween X-20; gently shake in a rotary shaker for 15 min; incubate for 18 h at 4°C; remove the supernatant and keep it separately.

• 2. Deposit the seeds in an extraction bag provided with a synthetic intermediate mesh (Bioreba); crush the seeds with a pestle and resuspend in the same supernatant.

• 3. Inoculate the extract (large debris is retained in the extraction bag) with the pathogen to obtain different dilutions; plate 100 μl of seed extract on appropriate medium (YPGA) before and after IMS and examine the plates for the development of the target pathogen.

• 4. Perform the assays in quadruplicates.

Appendix 17: Detection of Xanthomonas campestris pv. carotae by PCR Assay (Meng et al. 2004)

2.17.1 Preparation of Seed-wash

• 1. Incubate 10 g seed samples (about 10,000 seeds) in 100 ml 0.85% NaCl at 4°C for 16–18 h in a 250 ml flask; add two drops of Tween 20; place the flasks on a rotary shaker (250 rpm) for 2 min and filter through four layers of sterilized cheese cloth.

• 2. Centrifuge the filtrate at 1,000 × g for 10 min; resuspend the pellet in 10 ml 0.85% NaCl; add 0.5 or 1 ml of the suspension to a 1.5 ml Eppendorf tube and centrifuge at 10,000 × g for 10 min and use the pellet for DNA extraction.

2.17.2 DNA Extraction from Seed-Wash

• 1. Use the CTAB method for extraction by resuspending the pellet in 800 μl of 2% CTAB buffer and use 2 μl of this extract in 50 μl PCR.

• 2. Use the PCR primer pair 3SF/9BF designed on the sequences common to all strains of the pathogen species for amplification of ∼350-bp target fragment.

Appendix 18: Detection of Acidovorax avenae subsp. citrulli by Immuno-capture PCR Assay (Xiao et al. 2007)

2.18.1 Selection of Antibodies and Primers

• 1. Select suitable polyclonal antibodies (Ab and Ab1) specific to target pathogen raised in New Zealand white rabbits.

• 2. Design specific primers (WFB1 and WFB2) based on the sequences of 16S rRNA gene from the standard strain of the pathogen.

2.18.2 Detection by Standard PCR Assay

• 1. Use aliquots of 25 μl PCR mixture containing 200 μmol/l of dNTP, 0.25 μmol/l of each primer (WFB1 and WFB2), 1 μmol/l of Taq DNA polymerase, 2.5 μl 10 × PCR buffer with MgCl₂ and 2–4 μl of bacterial cell lysates as template.

• 2. Perform amplification in a thermal cycler providing the following conditions: initial denaturation at 95°C for 5 min followed by 30 cylcles of denaturation at 90°C for 30 s, primer annealing at 65°C for 30 s and elongation at 72°C for 30 s and incubation of tubes at 72°C for 5 min.

• 3. Resolve the PCR amplicons by electrophoresis on 1% agarose gels.

2.18.3 Detection by Immuno-capture (IC)-PCR Assay

• 1. Dilute the antiserum specific to the target pathogen to 1:2,000 with double sterilized distilled water; transfer 200 μl aliquots into each 0.25 ml Eppendorf tube and incubate for 3 h at 57°C.

• 2. Rinse thrice with washing buffer containing 0.01 mol/l phosphate-buffered saline (PBS) plus 0.2% Tween 20 (PBST); add 200 μl of 0.5% dried skimmed milk powder and incubate for 30 min at 37°C.

• 3. Add 200 μl bacterial cell lysates after removing the milk powder; incubate overnight at 4°C and rinse thrice with PBST followed by rinsing once with sterile distilled water.

• 4. All reagents except the template and all steps as in standard PCR protocol (Section B above) are to be adopted.

Appendix 19: Detection of Burkholderia glumae in Rice Seeds by Real-time PCR Assay (Sayler et al. 2006)

2.19.1 Preparation of Seed-wash

• 1. Wash 2 g seed samples ( approximately 20 seeds) in 2 ml distilled water ­containing 0.1% Tween-20; mix the aqueous suspensions on a rotary shaker at 150 rpm for 30 min and test 1 μl aliquots of wash solution in duplicates for each sample.

2.19.2 Real-time PCR Assay

• 1. Perform the assay using a Thermo-Fast 96-well reaction plate and Thermo-Fast Caps (AB gene Epsom, UK) in the Mx 3000 P real-time PCR machine (Stratagene Corporation, USA).

• 2. Transfer to each well a 25-μl reaction mixture containing 12.5 μl 2 × SYBR Green PCR Master Mix (Qiagen) and 12.5 μl primer (3 pmol each of forward and reverse primer and 1 μl of bacterial cells or seed wash/ plant extract).

• 3. Provide the following conditions for amplification: 95°C for 15 min; 40 cycles each of 95°C for 15 s, 60°C annealing for 30 s and 72°C for extension for 15 s, 95°C for 1 min and 55°C for 30 s.

• 4. Construct a standard curve to calculate the correlation between bacterial cell numbers and PCR Ct values by assaying a series of dilutions of bacterial culture; determine the bacterial cell number by turbidimetric assay using a spectrophotometer, followed by plate counting.