Detection of Fungal Pathogens in Plants

Abstract

Among the microbial plant pathogens, fungus-like and fungal pathogens have well developed thallus consisting of hyphae, asexual and sexual reproductive structures. The morphological characteristics of these structures and various kinds of spores produced by them have been the basis of identification up to genus/species level and classification of these pathogens into family, order and class. However, the formae speciales, strains, varieties or biotypes within a morphologic species have to be identified using other characteristics such as pathogenicity, biochemical and immunological properties or nucleotide sequences of the genomic DNA. Isozyme analysis, vegetative compatability group (VCG) analysis and electrophoretic mobility of cell wall proteins have been shown to be useful for the detection of strains of some fungal pathogens. The usefulness of immunoassays for early detection and precise identification has been significantly enhanced following the development of enzyme-linked immunosorbent assay (ELISA) and monoclonal antibodies which exhibit greater sensitivity and specificity compared with Appendix 1 based methods which are laborious and time-consuming. Nucleic acid-based diagnostic techniques depending on the variations in the nucleotide sequences of the pathogen DNA have become the preferred ones, because of their greater speed, specificity, sensitivity, reliability, and reproducibility of the results obtained, following the development of polymerase chain reaction (PCR). Several variants of PCR and commercial kits for on-site adoption under field conditions, away from the laboratory, are now available, providing the results in a short time. The possibility of detecting two or more pathogens simultaneously has become bright after the development of DNA array technology. A wide range of diagnostic techniques can be applied for detection, identification and quantification of fungal pathogens present in the infected plants, propagative plant materials and postharvest produce. Speed, specificity, sensitivity and cost-effectiveness are the primary factors that may determine the suitability and choice of the diagnostic tests.

Appendices

Appendix 1: General and Selective Media for Isolation of Fungal Pathogens

A. General media
Czapek Dox agar	Solution A
Sodium nitrate	40 g
Potassium chloride	10 g
Magnesium sulphate (hydrous)	10 g
Ferrous sulfate (hydrous)	0.2 g
Distilled water	1 liter
	Solution B
Dipotassium hydrogen phosphate	20 g
Distilled water	1 l
(store the solutions A and B separately in a refrigerator)
Prepare the mixture of A and B
Stock solution A	50 ml
Stock solution B	50 ml
Distilled water	900 ml
Sucrose (analar)	30 g
Oxiod agar No.3	20 g
Just before autoclaving add for 1 l
Zinc sulfate (1.0 g/100 ml water)	1.0 ml
Cupric sulfate (0.5 g/100 ml water)	1.0 ml
Malt extract agar
White bread malt extract	20 g
Oxoid agar No.3	20 g
Tap water	1 l
Oat agar
Oat meal ground	30 g
Oxoid agar No.3	20 g
Tap water	1 l
Potato carrot agar
Grated potato	20 g
Grated carrot	20 g
Oxoid agar No.3	20 g
Tap water	1 l
Potato dextrose agar
Potatoes	200 g
Oxoid agar No.3	20 g
Dextrose	15 g
Tap water	1 l
V8 agar
V8 vegetable juice	200 ml
Oxoid agar No.3	20 g
Distilled water	800 ml
B. Selective media
CW medium for Alternaria brassicola (Wu and Chen 1999)
Galactose	30 g
Calcium nitrate	3 g
Dipotassium hydrogen phosphate	1 g
Magnesium sulfate	1 g
Benomyl	100 ppm
Chloramphenicol	100 ppm
Agar	20 g
Distilled water	1 l
Media for Botrytis cinerea (Edwards and Seddon 2001)
(i) Botrytis selective medium
Glucose	2 g
NaNO3	0.1 g
K2HPO4	0.1 g
MgSO4.7H2O	0.2 g
KCl	0.1 g
Chloramphenicol	0.02 g
Maneb 80 (80% manganese ethyl bisdithiocarbamate)	0.02 g
Rose Bengal	0.05 g
Tannic acid	5.0 g
Oxoid agar No.3	20 g
Water	1 l
Adjust the pH to 4.5 using 1M NaOH prior to addition of agar
(ii) Botrytis spore trap medium (BSTM)
Rubigan (12% fenarimol) is used intead of Rose Bengal usedin BSM above
(iii) Diluted supplemented Malt extract agar (dsMEA)
Malt extract broth (Oxoid)	4.0 g
Chloramphenicol	0.2 g
Rose Bengal	0.05 g
Oxoid agar No.3	15 g
Water	1 l
Semi-selective medium for Colletotrichum gloeosporioides (Ekefan et al. 2000)
Basal medium – PDA amended with
Pencycuron	50 mg/l
Tolclofos-methyl	10 mg/l
Streptomycin sulfate	100 mg/l
Chloramphenicol	100 mg/l
Chlortetracycline	100 mg/l
Adjust the pH to 5.0
Komada’s Selective medium for Fusarium spp. (Arie et al. 1995)
K2HPO4	1 g
KCl	0.5 g
MgSO4.7H2O	0.5 g
Fe-EDTA	10 mg
L-asparagine monohydrate	2.0 mg
D-(+) galactose	20 g
Pentachloronitrobenzene (PCNB)	0.75 g (a.i.)
Sodium chlorate	0.5 g
Sodium tetraborate decahydrate	1 g
Chloramphenicol	0.25 g
Agar	15 g
Distilled water	1 l
Fusarium selective (FS) medium for Fusarium circinatum (Schweigkofler et al. 2004)
Bacto peptone	15 g
Agar	20 g
KH2PO4	1 g
MgSO4.7H2O	0.5 g
Streptomycin sulfate	0.3 g
Ampicillin	0.1 g
Pentachloronitrobenzene	0.2 g
PDA	39 g
Water	1 l
R-PDA medium for Gaeumannomyces graminis var. tritici (Duffy and Weller 1994)
Peeled boiled potatoes	40 g
Dextrose	4 g
Agar	18 g
Deionized water	1 l
Add after autoclaving and adjusting the pH to 6.0–6.5
Rifampicin	100 μg/ml
Tolclofos-methyl	10 μg/ml

Appendix 2: Assessment of Vegetative Compatibility Relationships Among Verticillium dahliae Strains (Joaquim and Rowe 1990; Daayf et al. 1995)

A. Generation of nit mutants

• (i) Prepare monoconidial subcultures of isolates of the test pathogen in small culture vials containing sterilized soil, perlite and peat moss (1:1:1 v/v/v) and store at 5°C.

• (ii) Cut agar discs (5 mm diameter) using a cork borer from the edge of wild-type colonies of the pathogen growing on potato dextrose agar (PDA); transfer to chlorate minimal medium (CMM) amended with 30 g/l of potassium chlorate kept in petridishes (9 cm diameter) and incubate for 10 days at 25°C.

• (iii) Cut a small segment (1 cm diameter) from the margin of the chlorate-resistant colony; transfer into a tube containing sterile water (8 ml); agitate well to disperse the conidia from the medium and adjust the conidial concentration to 1 × 10⁴/ml using a hemacytometer.

• (iv) Dispense aliquots of 50 μl of conidial suspension into petridishes containing CMM; incubate for about 40 h and transfer individual germinated conidia onto minimal medium (MM) placed in the petridishes to select distinct chlorate-resistant monoconidial strains.

• (v) Observe the colony morphology; thin and expansive mycelium on MM medium indicates the inability to metabolize nitrate; these colonies are considered as nit mutatnts.

B. Characterization of nit mutants

• (i) Transfer a segment of the mycelium of each chlorate-resistant mutant onto basal medium (MM without nitrogen source) supplemented with one of the following nitrogen sources: (a) sodium nitrate (0.2 g/l), (b) sodium nitrite (0.4 g/l), (c) hypoxanthine (0.5 g/l), or (d) ammonium tartarate (0.8 g/l) buffered with calcium carbonate and examine the growth response of each mutant in each nitrogen source.

• (ii) Assign nit mutants the phenotype identity as follows:

  • (a) Nit1 mutants – unable to utilize nitrate, but capable of using nitrite, ammonium, hypoxanthine and uric acid.

  • (b) Nit mutants – unable to use nitrate and hypoxanthine, but can use the other three nitrogen sources.

C. Assignment of strains to vegetative compatibility groups (VCGs)

• (i) Prepare complementary tester strains (Nit1 and NitM) for the strains to be assigned to VCG.

• (ii) Pair the tester strains with nit mutants by placing a Nit1 or NitM mutant derived from one strain at the center of each plate containing MM; place Nit1 and NitM (derived from a tester strain representing a specific VCG) each 1.0–1.5 cm apart on either side.

• (iii) Perform pairing twice for each mutant.

• (iv) Observe the prototrophic growth developing at the mycelial interface between the nit mutant positioned at the center; score the density of growth after 18–24 days of inoculation.

Appendix 3: Media for Generation and Selection of Vegetative Compatibility Groups (VCGs) of Fusarium oxysporum (Correll et al. 1987)

• A. Basal medium: Sucrose – 30 g; KH₂PO₄ – 1 g; MgSO₄.7H₂O – 0.5 g; KCl – 0.5 g; FeSO₄.7H₂O – 10 mg; agar – 20 g; trace element solution 0.2 ml; water 1,000 ml

  (Trace element solution: ZnSO₄.7H₂O – 5.0 g; Fe(NH₄)₂(SO₄)₂.6H₂O – 1.0 g; CuSO₄.5H₂O – 0.25 g; MnSO₄.H₂O – 50 mg; H₃BO₄ – 50 mg; NaMoO4.2H₂O – 50 mg; water – 95 ml)

• B. Complete medium: Basal medium – 1 l; NaNO – 2.0 g; N-Z amine (Sheffield) – 2.5 g; yeast extract (Difco) – 1 g; vitamin solution – 10 ml (Vitamin solution: Thiamine HCl – 100 mg; riboflavin – 30 mg; pyridoxine HCl – 75 mg; D-pantothenate-Ca – 200 mg; p-aminobenzoic acid – 5 mg; nicotinamide – 75 mg; cholineCl – 200 mg; folic acid – 5 mg; D-biotin – 5 mg; myoinositol – 4 mg; ethanol (50%) – 100 ml)

• C. Minimal medium: Basal medium 1 l; NaNO₃ – 2 g

• D. Minimal agar medium with chlorate (MMC): Minimal medium 1 l; L-asparagine 1.6 g; NaNO₃ − 2 g; KClO₃ – 15 g

• E. Potato dextrose agar medium with chlorate (PDC): Potato dextrose broth (Difco) – 24 g; agar – 20 g; KClO₃ – 15 g; water – 1,000 ml

Appendix 4: Generation of Antibodies Against Fungi (Banks et al. 1992)

A.Preparation of antigen

• (i) Prepare spore suspensions using 0.01% Tween 80; wash thrice by centrifugation; inoculate 1 ml of spore suspension (10⁶ spores/ml) into 100 ml of liquid medium supplemented with NaCl (100 g/l) and incubate at 25°C for 7 days in the dark by placing the flask with contents on a rotary shaker.

• (ii) Transfer the mycelium by filtering into a sintered glass filter; wash with sterile water and then with sterile phosphate-buffered saline (PBS) containing 2.9 g Na₂HPO₄.12H₂O, 0.2 g KH₂PO₄, 8.0 g NaCl and 0.2 g KCl and 1,000 ml distilled water; freeze overnight at −20°C; thaw and transfer to centrifuge tubes and dry in vacuum dryer.

• (iii) Collect the mycelium and add 50 ml of liquid nitrogen; mince the mycelium in a blender for 1 min and grind in a mortar with a pestle to a fine powder.

• (iv) Suspend the mycelial powder in PBS (200 mg in 100 ml); centrifuge at 4,500 rpm (3,000 g) for 10 min at 4°C and divide the supernatant containing soluble nitrogen into 0.5 ml aliquots and store at −20°C.

• (v) Estimate the total protein content of the antigen preparation.

B. Production of polyclonal antiserum

• (i) Mix soluble antigen preparation with equal volumes of Freund’s complete adjuvant (Difco) to produce a final protein concentration of the mixture at 1 mg/ml.

• (ii) Inject rabbits intramuscularly with 1 ml of the mixture at predetermined intervals.

• (iii) Bleed the animal at 4 weeks after the first injection and subsequently at 14, 16 and 18 weeks.

• (iv) Separate the serum after completion of clotting of blood cells followed by centrifugation.

C. Production of monoclonal antiserum

• (i) Mix soluble antigen preparation with an equal amount of Freund’s ­complete adjuvant to yield a final protein concentration of 1 mg/ml.

• (ii) Inject a BALB/c mouse, after anaesthetization with 0.1 ml of the immunogen intraperitoneally and subsequently at 2, 4, 6 and 8 weeks after the first injection with PBS and remove the spleen after sacrificing the animal by cervical dislocation.

• (iii) Carry out fusion of splenocytes with selected myeloma cell line (P3-NS-1-Ag4) at a ratio of 1 × 10⁸: 5 × 10⁷ by gentle addition of 2 ml of 30% polyethylene glycol (PEG) (w/v) over 60 s.

• (iv) Add 10 ml of warm serum-free RPMI 1640 medium (Gibco) over next 60 s with gentle stirring; add another 20 ml of RPMI and centrifuge for 3 min at 400 g at room temperature.

• (v) Suspend the pellet of cells in 50 ml of growth medium (RPMI 1640) with 20% Myclone fetal calf serum (FCS) (v/v); dispense cell suspension into five 96-well microplates at 100 μl/well.

• (vi) Add 110 μl of hypoxanthine aminopterin–thymidine (HAT) medium diluted to 1:50 in growth medium to each well in the fusion plates.

• (vii) Add growth medium + HAT on 2, 4, 7 and 10 days by removing 100 μl of the medium and replacing with 100 μl of fresh medium.

• (viii) Screen the hybridoma cells for efficiency of antibody production by indirect ELISA procedure.

• (ix) Clone healthy growing hybridoma twice by limiting dilution in a non-selective medium; preserve by freezing slowly in 7.5% dimethyl sulfoxide (DMSO) and store in liquid nitrogen.

Appendix 5: Detection of Botrytis cinerea by Enzyme-linked Immunosorbent Assay (ELISA) Test (Bossi and Dewey 1992)

A. Preparation of antigen

• (i) Prepare surface washings of the pathogen (B. cinerea) grown on PDA for 17–20 days at 21°C, using 5 ml/petridish of phosphate-buffered saline (PBS) containing 8.0 g NaCl, 0.2 g KCl, 1.15 g Na₂HPO₄, 0.25 g KH₂PO₄, and water 1,000 ml at pH 7.2 and remove the wash suspension by suction.

• (ii) Centrifuge the wash fluid for 3 min at 13,000 g to remove the fungal debris and dilute the supernatant with PBS to have tenfold dilutions.

• (iii) Remove the high MW carbohydrates and glycoproteins by passing the cell-free wash fluid through a Centricon 30-kDa filter (Amicon No. 4208) to prevent induction of nonspecific antibodies; freeze-dry the ­filtrate and redissolve the contents in 1 ml of distilled water and use it as the antigen.

B. Enzyme-linked immunosorbent assay (ELISA)

• (i) Coat the wells (in triplicate) in the 96-well microtiter plates with PBS surface washing fluid (50 μl/well) overnight and wash the wells four times allowing two min for each washing followed by a brief washing with distilled water.

• (ii) Air-dry the plates in a laminar flow hood and seal them in a polythene bag and store at 4°C.

• (iii) Incubate the plates successfully with hybridoma supernatants for 1 h, then with a 1/200 dilution of a commercial goat antimouse polyvalent (IgG + IgM) peroxidase conjugate and finally with PBS with 0.05% Tween-20 (PBST) for 1 h more.

• (iv) Add the substrate solution containing tetramethyl benzidine (100 μg/ml) for 30 min.

• (v) Maintain the controls incubated tissue culture medium containing 5% fetal bovine serum (FBS) in place of hybridoma supernatant.

• (vi) Stop the reaction by adding 3 M H₂SO₄ (50 μl/well); determine the intensity of color developed in each well using ELISA reader at 450 nm.

• (vii) Absorbance levels more than three times greater than those of controls indicate positive reaction and presence of antigen protein.

Appendix 6: Quantitative Detection of Mycosphaerella fijiensis by Triple Antibody Sandwich (TAS)-ELISA (Otero et al. 2007)

A. Preparation of antigens

• (i) Cultivate the fungal pathogen in appropriate medium under optimal growth conditions required; transfer the mycelial disks from the nutrient medium to 250 ml tissue culture flasks containing 50 ml of sterile potato dextrose broth (Difco) and incubate for 3–7 days at room temperature.

• (ii) Transfer the mycelial suspensions aseptically to 250 ml flasks; incubate with shaking (80 rpm) at 26°C and harvest the mycelia using cellulose filters in a Buchner funnel under vacuum.

• (iii) Dry the harvested mycelia (10 g) of 18 days old; mince in liquid nitrogen and resuspend the homogenate in protein extraction buffer containing 50 mM Tris HCl, pH 8.0, 1 mM phenylmethane sulphonyl fluoride (PMSF), 2 mM ethylenediaminetetraacetic acid (EDTA) and 2 mM 1-4-dithio-DL-threitol under agitation at room temperature.

• (iv) Precipitate the protein at 80% ammonium sulfate followed by destalting (Sephadex G-25) into phosphate-buffered saline (PBS) and determine the protein contents of the samples by the Coomassie method (Bradford 1976).

• (v) Prepare the secreted protein antigen from the metabolized pathogen culture suspension media by concentration, precipitation at 80% ammonium sulfate and dialysis against PBS and determine protein content of the sample as done earlier (step iv).

• (vi) Prepare the antigens from the leaves by washing with distilled water; cut them into fragments; powder the fragments (10 g) using liquid nitrogen followed by mixing (1:3 w/v) with alkaline extraction buffer containing Tris (50 mM), EDTA (10 mM), ascorbic acid (0.2%), sodium chloride (150 mM), 2-mercaptoethanol (20 mM), PMSF (0.57 mM), Tritox X-100 (1.5%, pH 7.5); keep the mixture at 4°C for 1 h; centrifuge at 2,000 × g for 10 min; precipitate the protein at 80% ammonium sulfate; dialyze against PBS and estimate the protein content as done earlier (step iv).

B. Preparation of polyclonal antiserum

• (i) Emulsify mycelial antigen preparation (100 μg) in 2 ml of complete Freund’s adjuvant (Sigma) and inject into female New Zealand adult rabbits subcutaneously and inject the same dose of antigen emulsified in incomplete Freund’s adjuvant (Sigma) at 2-week interval until the titer rises to 1:32 by Ouchterlony double immunodiffusion method.

• (ii) Bleed the animal; precipitate at 50% ammonium sulfate, desalt and fractionate on a matrix of diethyl aminoethyl Sepharose (Amersham-Bioscience) to have suitable IgG fraction.

• (iii) Determine the protein content following bicinchroninic acid method (Smith et al. 1985).

C. Preparation of monoclonal antiserum

• (i) Immunize 6–8 weeks old female BALB/c mice by intraperitoneal route with 50 μg of mycelial antigen in 1:1 (v/v) emulsion of complete Freund’s adjuvant (Sigma) and PBS and administer subsequent doses of mycelial antigen (50 μg) emulsified with incomplete Freund’s adjuvant (1:1 v/v) and PBS by subcutaneous route at 2 and 4 weeks after initial immunization.

• (ii) Obtain serum before and at 2 weeks after each immunization and analyze for antibodies reacting to pathogen mycelial antigen and assess the antibody response using an indirect ELISA.

• (iii) Give booster injection by intravenous route to the mouse with the highest antibody titer, with 25 μg of antigen in PBS; sacrifice the mouse by CO₂ asphyxiation and remove the spleen.

• (iv) Follow the polyethylene glycol-based procedure for fusion of spleen cells with murine plasmacytoma cells Sp. 2/0-Ag 14 (ATCC No. CRL-1581) and identify positive clones by evaluating the supernatant using indirect ELISA method, using hybridoma culture medium.

• (v) Clone the hybridoma-producing antibody reactive to both mycelial and secreted antigens of the fungal pathogen using limiting dilution to recover homogeneous hybridoma cell line.

• (vi) Inoculate the selected hybridoma (2H6H8) into BALB/c mice by intraperitoneal route; collect the ascetic fluid and purify the antibody using affinity chromatography in a Protein G Sepharose Fast Flow (Amersham-Biosciences) column as per the manufacturer’s recommendations.

• (vii) Dialyze the antibody against PBS and determine the protein concentration as done earlier (Step B. iii); sterilize the antibody solution by filtration through a 0.22 μm nitrocellulose membrane.

D. Double antibody sandwich (DAS)-ELISA test

• (i) Coat the microtiter plates with 100 μl of a solution containing 10 μg/ml of the target pathogen in carbonate–bicarbonate buffer pH 9.6 and incubated at 4°C for 16 h.

• (ii) Wash the plates thrice with PBS-T after completion of the incubation period; block the wells using 3% BSA in PBS for 1 h to prevent nonspecific binding; dispense PAb solution (50–200 μg/ml) diluted in PBS-T solution to the antigen coated wells and incubate at 37°C for 1 h.

• (iii) Wash the wells as done before; transfer a 1:10,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit antibody (Sigma) to the wells and incubate for 1 h.

• (iv) Add p-nitrophyenyl phosphate disodium hydrate in diethanolamine buffer, pH 9.8 and determine absorbance at 405 nm, after stopping the reaction by adding concentrated sulfuric acid.

E. Triple antibody system (TAS)-ELISA test

• (i) Coat the microplates with 100 μl of a solution containing 10 μg/ml of MAb recognizing the mycelial antigen in carbonate-bicarbonate buffer, pH 9.6; incubate for 16 h at 4°C; wash the plates thrice with PBS-T after completion of incubation period.

• (ii) Block the wells as done before (step D iii); dilute the antigen (1.25–40 μg/ml) in the blocking solution and transfer to the wells and incubate for 2 h at 37°C.

• (iii) Dispense 20 μg/ml of the second antibody PAb (anti-pathogen) and follow other steps as in DAS-ELISA test.

Appendix 7: Detection of Resting Spores of Plasmodiophora brassicae in Plant Tissues by ELISA (Orihara and Yamamoto 1998)

A. Preparation of immunogen

• (i) Homogenize club root-infected roots and hypocotyls (850 g) in distilled water for 5 min using a blender; filter the homogenate through eight layers of gauze; centrifuge the filtrate at 3,000 rpm for 20 min; resuspend the pellet in distilled water and repeat centrifugation cycle five times.

• (ii) Prepare a sucrose density column (with 20% and 40% sucrose solutions) in a transparent centrifuge tube; overlayer the final suspension containing resting spores and plant cell debris on sucrose gradient column and centrifuge at 3,000 rpm for 20 min.

• (iii) Collect the layer containing resting spores; wash with distilled water five times and store at −20°C.

B. Preparation of antiserum

• (i) Inject the rabbit intramuscularly with 0.5 ml of immunogen preparation (6 × 10⁷) purified resting spores/ml of 0.85% NaCl solution); inject again intramuscularly with a mixture of 1 ml of immunogen and 1 ml of Freund’s complete adjuvant after an interval of 2 weeks; administer additional dose of immunogen (0.5 ml containing 5.4 × 10⁷ resting spores/ml) intravenously.

• (ii) Collect the blood serum after a rest period of 2 weeks; purify the antibodies by ammonium sulfate preparation and DEAE-cellulose column chromatography.

C. Indirect ELISA

• (i) Collect the club root-infected roots and hypocotyls and similar healthy tissues and store at −20°C; homogenize the samples separately in distilled water for 5 min; filter as done earlier (step A i above) and adjust the spore concentration to 1 × 10⁶ spores/ml and dilute healthy samples to the same volume.

• (ii) Suspend the sample extracts in coating buffer (carbonate) and dilute to required level and transfer 200 μl of each sample to two wells of microtiter plates and incubate overnight at 4°C.

• (iii) Wash the wells thrice with PBS-containing polyvinyl pyrrolidone (2%) and BSA (2%) and incubate for 1 h at room temperature.

• (iv) Dispense to each well PBS containing 2 μg/ml of anti-resting spore IgG; incubate at 37°C for 4 h and wash the wells as done earlier.

• (v) Add PBS containing goat anti-rabbit IgG-alkaline phosphate conjugate at a dilution of 1/2,000; incubate for 4 h at 37°C and wash the wells as done earlier.

• (vi) Add 1 ml of diethanolamine (10%), pH 9.8 containing p-nitrophenyl phosphate (enzyme substrate) to each well; incubate for 5 min at 37°C in the dark and record the absorbance values at 405 nm using an ELISA reader.

D. Dot immunobinding assay (DIBA)

• (i) Spot the samples of 2 μl onto a 40 cm² nitrocellulose membrane sheet (Trans-Blot, BIO-RAD, USA); air dry and block nonspecific binding sites by immersing the membrane in a buffer solution consisting of 20 mM Tris-HCl, 500 mM NaCl, and 0.05% Tween-20, pH 7.5 (TTBS), 2% polyvinyl pyrrolidone (PVP) and 2% BSA.

• (ii) Treat the membrane with 0.1–0.2 μg/ml of anti-resting spore IgG in TTBS containing 2% PVP and 0.2% BSA (TTBSPB) for 1 h at room temperature.

• (iii) Treat the membrane with alkaline phosphatase conjugated goat anti-rabbit IgG in TTBSPB for 1 h and then with buffer consisting of 0.1 M Tris-HCl, 0.1 M NaCl and MgCl₂, pH 9.5 containing 0.33 mg/ml of nitroblue tetrazolium substrate and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt prediluted with N,N-dimethylformamide.

Appendix 8: Detection of Fusarium spp. by Direct Tissue Blot Immunoassay (DTBIA) (Arie et al. 1995; Arie et al. 1998)

• (i) Prepare cross sections of stems of infected plants (3 mm thick); place them on nitrocellulose membrane (0.45 μm, pore size) (Trans-Blot Transfer Medium, BIO-RAD USA) saturated with Tris-buffered saline (TBS), pH 7.0 for 10–30 min for direct tissue blotting.

• (ii) Immerse the membrane in a blocking solution containing fetal calf serum (FCS) (10% v/v) and BSA 1.0% w/v) in TBS (FB-TBS) for 1 h at room temperature.

• (iii) Incubate with specific MAb (AP 19–2) diluted in FB-TBS for 1 h at room temperature and wash the membrane thrice in TBS containing Tween-20 (0.1%) (TBST).

• (iv) Incubate the membrane with a mixture of biotinylated anti-mouse IgM-goat IGg, diluted to 1:500 in FB-TBS and horseradish peroxidase-avidin D ­conjugates (diluted 500 times) for 1 h at room temperature.

• (v) Wash the membrane with TBST for 5 min and repeat washing twice; immerse the membrane in substrate solution containing 4-chloro-1-naphthol and 0.02% hydrogen peroxide (v/v).

• (vi) Observe for the development of blue color indicating positive reaction.

Appendix 9: Detection of Fusarium spp. in Tomato by Immunofluorescence Assay (Arie et al. 1995)

• (i) Cut transverse sections (3 cm diameter, 0.3 mm thick) from fresh stem, crown or root of test plants using a sharp razor blade.

• (ii) Immerse the sections in blocking solution containing 1% gelatin and 10% BSA in 0.1 M phosphate buffered saline (PBS, pH 7.0) for 2 h at room temperature.

• (iii) Soak the sections in the MAb (AP19-2) solution diluted in PBS containing 0.1% Tween-20 (PBST) for 2 h at room temperature and then wash the sections thrice in PBST.

• (iv) Incubate the sections with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgM diluted 500 times with PBST for 2 h at room temperature.

• (v) Observe the sections under a reflecting fluorescence microscope (BHS-RF-A, Olympus, Japan) by ß-excitation.

Appendix 10: Detection of Polymyxa graminis by Fluorescent Antibody Technique (FAT) (Delfosse et al. 2000)

• (i) Prepare root fragments (∼5 mm long) from healthy and infected plants; fix them in 3% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.2 prepared under vacuum); wash in phosphate buffer; dehydrate in graded series of ethanol and embed in epoxy resin (Ladd Research Industries, USA).

• (ii) Cut transverse sections (∼8 μm thick) of embedded fragments; transfer to glass multispot slides and heat briefly on a hot plate at 50°C for the sections to adhere to the glass.

• (iii) Conjugate GAR-IgG (Sigma R-3128, Sigma Chemical Co., USA) with fluorescein 5-isothiocyanate (FITC) (Sigma F-7250) by the procedure detailed below.

• (iv) Dissolve 15 mg FITC in 1 ml of dimethyl sulfoxide (Sigma D-2650) and mix with 14 ml of 0.1 M sodium carbonate buffer, pH 9.6; dissolve one mg of GAR-IgG in 1 ml of carbonate buffer; dialyze against 15 ml of FITC solution overnight in cold room and remove excess FITC by dialysis against PBS.

• (v) Cross-absorb crude antiserum against the pathogen (P. graminis) with an equal volume of healthy sorghum root extract (dried roots at 0.4% w/v) prepared in conjugate buffer; remove the immuoprecipitate by centrifugation and repeat the cross-absorption process three times.

• (vi) Extract IgG for the pathogen from the supernatant with neutral ammonium sulfate and use at a concentration (100 μg/ml).

• (vii) Stain the thin sections in glass multispot slides using 20 μl of reagent/window in each step; soak sections in PBS-Tween containing 10% low fat milk (blocking buffer) for 1 h at 37°C and wash the sections under a gentle stream of distilled water.

• (viii) Soak the slides in pathogen IgG for 3 h at 37°C or overnight at 5°C prepared in blocking buffer and wash the sections in distilled water.

• (ix) Add FITC-labeled GAR-IgG at a dilution of 1:20 prepared in blocking buffer; incubate for 3 h at 37°C and wash the sections in distilled water.

• (x) Mount the sections in 90% glycerol in 0.1 M PBS; examine under the microscope with a provision for epifluorescence and photograph the desired tissues of healthy and infected roots for inference, using a Kodak 400 ASA color reversal film at a magnification of × 80 or × 100.

Appendix 11: Detection of Botrytis cinerea by Protein A-Gold Labeling Technique (Svircev et al. 1986)

A. Immunogen preparation

• (i) Separate the mat of mycelia and spores; treat with 0.5% formalin; centrifuge the suspension of fungal cells at 1,700 g for 10 min; resuspend the pellet in distilled water; repeat washing and centrifugation cycle three times and resuspend the mycelial mass in 2 ml of Freund’ complete adjuvant to have a concentration of 10⁶ cells/ml.

• (ii) Adminster the fungal preparation intramuscularly followed by the booster dose (second injection ) after an interval of 2 weeks.

• (iii) Collect the blood, separate the antiserum after centrifugation and store at −20°C.

B. Preparation of proteina-gold label

• (i) Prepare the colloidal gold particles (15 mm diameter) by adding 4 ml of aqueous sodium citrate (1%) to 100 ml of a boiling solution of chloroauric acid (0.01); cool the mixture for 5 min until a wine red color develops and store at 4°C in the dark.

• (ii) Prepare the protein A-gold complex by adjusting the pH of colloidal gold suspension (10 ml) to pH 6.9 using potassium carbonate and add 0.3 ng protein A (Sigma, USA) in 0.2 ml of distilled water.

• (iii) Centrifuge at 48,000 g at 4°C to remove the excess unbound protein A.

• (iv) Resuspend the dark red protein A-gold pellet in 10 ml of 0.01 M PBS, pH 7.4 and store at 4°C.

Protein A-gold label may be stable for 6–8 weeks.

C. Protein A-gold labeling

• (i) Float thin sections of plant tissue to be tested onto a saturated sodium periodate solution to remove osmium tetroxide used as a fixative for 2–3 min.

• (ii) Wash the sections with distilled water three times and treat with 1% ovalbumin for 5 min to block non-specific binding sites.

• (iii) Float the sections on drops of specific antiserum placed on coated electron microscope grids and wash the sections thoroughly by passing the grids through a series of water drops.

• (iv) Treat the sections with protein A-gold solution for 30 min; wash with drops of water as done earlier and stain with 3% uranyl acetate for 20 min.

• (v) Examine the grids under the electron microscope.

Appendix 12: Detection of Fungal Pathogens with DNA Probes (Tisserat et al. 1991)

• (i) Transfer 200–400 mg of infected plant tissues in a 1.5 ml microfuge tube; freeze by adding liquid nitrogen and grind with smooth lipped steel rod.

• (ii) Suspend the macerate in 600 μl 2 × CTAB buffer (2 × CTAB = 1.4 M NaCl, 2% hexadecyl triethyl ammonium bromide, 1% 2-mercaptoethanol, 10 mM Tris-HCl, pH 8.0 and extract with chloroform.

• (iii) Precipitate by adding 0.8 volume of isopropanol and resuspend the pelleted DNA in 40 μl TE buffer (TE = 10 mM Tris, pH 7.6; 1 mM EDTA).

• (iv) Denature DNA at 95°C for 4 min; transfer 20 μl to a nylon membrane in a slot-blot apparatus and bake at 80°C for 2 h.

• (v) Hybridize with as for Southern hybridizations.

Appendix 13: Identification of Fungal Pathogens by Repetitive DNA Polymorphism (Panabieres et al. 1989)

A. Fungal DNA preparation

• (i) Cultivate the target fungus in an appropriate medium; harvest the cultures by filtration on filter paper under vacuum; rinse the mycelia in 250 ml of distilled water and store by freezing.

• (ii) Grind the frozen mycelium (250 mg) in liquid nitrogen; suspend the powdered mycelium in 0.5 ml of NIB buffer containing 100 mM NaCl, 30 mM Tris-HCl, pH 8.0; 10 mM ß-mercaptoethanol; 0.5% NP-40 9v/v) and centrifuge for 1 min at 12,000 g.

• (iii) Resuspend the pellet in NIB buffer; repeat the procedure in (ii) above; resuspend the pellet in 0.8 ml of homogenization buffer consisting of 0.1 M NaCl, 0.2 M sucrose and 10 mM EDTA; add 0.2 ml of lysis buffer containing 0.25 M EDTA; 0.5 M Tris, pH 9.2 and 2.5% sodium dodecylsulfate and incubate at 55°C for 30 min.

• (iv) Extract twice with one volume of phenol-chloroform isoamyalcohol (50:48:2) and then with one volume of ether.

• (v) Add one volume of ethanol; centrifuge for one min in a microcentrifuge at room temperature and collect the DNA as pellet.

• (vi) Wash the pellet with 70% ethanol; centrifuge again; resuspend in 50 μl of TE (10 mM Tris, pH 8.0; 1 mM EDTA) and store at −20°C.

B. Digestion of DNA and electrophoresis analysis

• (i) Digest 5 μg of total DNA overnight with 20 units of restriction enzyme as per the manufacturer’s instruction.

• (ii) Separate DNA fragments on 1% agarose gels at 5 V/cm in 90 mM Tris borate buffer, pH 8.3.

• (iii) Stain the gels with ethidium bromide; view under UV light.

Appendix 14: Rapid Extraction of DNA from Fusarium oxysporum (Plyler et al. 1999)

• (i) Remove 1-cm² block of fungal growth from the colony edge of single-spore cultures; place the mycelium in a 1.5-ml Eppendorf tube containing 150 μl Tris-EDTA (TE) buffer and grind the mycelium using sterile wooden sticks.

• (ii) Place the tubes in liquid nitrogen for 3–4 min; thaw the tubes in a water bath at 22°C for 5 min; return the tube to liquid nitrogen followed by thawing at 22°C and repeat the cycle three or four times.

• (iii) Place the tubes at 65°C in a water bath for 15 min; centrifuge at 11,500 g for 10 min; transfer the supernatant to new Eppendorf tube; add an equal volume of chloroform-octanol (24:1) to the supernatant and mix the contents by ­vigorous shaking.

• (iv) Centrifuge at 12,000 g for 10 min and dilute the contents by tenfold for use in PCR assays. If necessary adopt the following additional steps for further purification.

• (v) Mix 24 μl of the supernatant with 16 μl of isopropanol in a separate tube to precipitate DNA; centrifuge for 5 min and drain isopropanol from the tubes.

• (vi) Add 16 μl of 70% ethanol to wash the pellet; centrifuge for 5 min; dry the pellet under a laminar flow hood; resupend the pellet in 2 μl TE buffer and use it for PCR amplification.

Appendix 15: Extraction of Genomic DNA from Claviceps sp. by Magnetic Separation (Scott Jr et al. 2002)

• (i) Cultivate the fungus (Claviceps africana) in YM broth (Difco) at 22°C in darkness and lyophilize the mycelium.

• (ii) Grind 5–10 mg of lyophilized mycelium to a fine powder in liquid nitrogen; resuspend the ground material in 250 μl of DNA X –Tract^TM solution 1 (D2 BioTechnologies Inc., USA); mix the suspension with an equal volume of DNA X-Tract^TM solution 2 (high salt buffer) in a 1.5 ml microfuge tube and add 500 μl of chloroform/isoamyl alcohol (24:1).

• (iii) Vortex the mixture vigorously; centrifuge at 10,000 g for 5 min; transfer the aqueous phase to a new tube; mix the suspension with 250 μl of DNA X Tract^TM precipitation solution and 250 μl of DNA X-Tract^TM solution 3 and incubate for 30 min on ice.

• (iv) Precipitate the DNA by centrifuging at 10,000 g for 15 min in a microcentrifuge and save the pellet.

• (v) Resuspend the maganetic particles (Dynabeads, DNA Direct System Dynal Inc., USA) by gentle swirling to get a homogenous dispersion of magnetic microparticles in solution and equilibrate to room temperature as per manufacturer’s instructions.

• (vi) Transfer 200 μl of magnetic particle solution to a sterile 1.5 ml microcentrifuge tube and place the tube in a magnetic stand (Dynal MPC) to allow the magnetic particles to complex to the sides of the tube and transfer the supernatant, after 2 min, to the tube containing the pellet of DNA (step iv above).

• (vii) Resuspend the pellet by flicking and breaking up with a pipette tip; transfer back the contents to the tube containing the magnetic microparticles (step vi above) and incubate the magnetic microparticles-DNA mixture for 10 min at room temperature.

• (viii) Place the tube again in the magnetic stand to allow the DNA-magnetic microparticle complex to aggregate to the sides of the tube and carefully pipette out the supernatant solution.

• (ix) Resuspend the complex in 200 μl of washing buffer; place the tube in the magnetic stand; allow it stand till the supernatant becomes clear and repeat washing once again.

• (x) Resuspend the complex in 30 μl of resuspension buffer and use the suspension either directly or after dilution (1:10) in PCR reactions.

• (xi) Alternatively, elute the DNA by incubation at 65°C for 5 min and place the tube in the magnetic stand to allow the magnetic microparticles to complex to the sides of the tube.

• (xii) Transfer the supernatant containing the DNA to new tube for use in PCR.

Appendix 16: Extraction of DNA from Fungal Cultures (Griffin et al. 2002)

• (i) Grow the test fungal pathogen in suitable medium; transfer 1 ml of culture suspension to a sterile cryogenic storage tube containing 200 μl of sterile glycerol and store at −70°C.

• (ii) Streak out the fungus onto plates containing R2A agar (Fisher Scientific, USA) and incubate for 2 days at room temperature.

• (iii) Transfer the fungal tissue (∼2.5 mg) from each isolate/species in a sterile 1.5 ml microcentrifuge tube and add to each tube 400 μl of AP1 buffer (DNeasy Plant Mini Kit, Qiagen) and 4 μl RNase (supplied with the kit).

• (iv) Apply freeze/thaw cycle to lyse fungal cells using crushed ice/ethanol and a boiling water bath; repeat the cycle seven times and boil for 30 min in a water bath, after the last cycle of freeze/thaw cycle.

• (v) Use a sterile 1 ml micropipette tip to grind any visible tissue in the tubes briefly (5 s) between the tip and conical bottom of the microcentrifuge tube.

• (vi) Follow DNeasy Plant Mini Kit ‘Protocol for Appendix 1 of DNA from plant tissue procedure starting with step 4 (add 130 μl of buffer AP2…).

• (vii) Elute the DNA in 50 μl buffer AE and use 5 μl of diluted DNA for PCR amplification.

B. Bead-beating extraction of fungal DNA

• (i) Streak the isolates from storage (−70°C) onto plates containing R2A agar and incubate for 2 days at room temperature.

• (ii) Transfer the fungal tissue (2.5 mg) of each isolate/species to sterile 2 ml cryogenic/microcentirfuge tubes fitted with an O-ring; add to each sample 400 μl AP1 buffer (DNeasy Plant Mini Kit) and 4 μl of RNase A (from the kit); transfer sterile glass bead (∼100 μl, 0.1 mm diameter) (BioSpec Products Inc., USA) and load the tubes in a Mini-BeadBeater-8 (BioSpec Products).

• (iii) Allow the beater to work for 2 min at maximum speed and repeat the bead-beating/cooling cycle twice.

• (iv) Centrifuge the samples for 10 min at 14,000 rpm in microcentrifuge.

• (v) Transfer the supernatant fluid from each tube separately to sterile 1.5 ml microcentrifuge tubes.

• (vi) Perform DNeasy Plant Mini Kit-Protocol for Appendix 1 of DNA from plant tissues procedure starting from step 4 (add 130 μl of buffer AP2).

• (vii) Elute the DNA in 50 μl buffer AE and use 5 μl of eluted DNA in PCR assay.

Appendix 17: Extraction of Genomic DNA from Phytophthora spp. (Lamour and Finley 2006)

A. Growing and disruption of pathogen mycelium

• (i) Use appropriate medium kept in petridishes for multiplication of the target pathogen and after the required incubation period gently scrap the mycelium from the top surface of the medium.

• (ii) Dispense 1 ml of PARP-V6 broth amended with 25 ppm pimaricin, 100 ppm ampicillin, 25 ppm rifampicin and 25 ppm pentachloronitrobenzene (PCNB) into each of the 24-deepwell (DW) Uniplate microtitter plates (Whatman Inc., USA) containing 10-ml wells; transfer wefts of mycelium scrapped from culture plates; cover the plates with ryan breathable tape and incubate the plates for 6 days at room temperature.

• (iii) Dispense the glass balls using a Millipore dry dispensing plate; transfer the pathogen colonies developing into a 96-well 2-ml DW plate containing three 3 mm glass balls/well; cover the plates with Aeraseal ryon breathable tape (PGC Scientifics, USA) and freeze the contents at −80°C for at lease 1 h.

• (iv) Lyophilize the samples for a period of 48 h; use the Labconco stoppering tray drying systems (STDS) (Labconco Corp., USA) with incubation chamber at 0°C for 24 h, followed by 24 h with incubation at 23°C.

• (v) Remove the samples from the chamber and apply a capmat immediately to deepwell plates with a capmat applicator (CMA) (Fisher Scientific).

• (vi) Disrupt the samples with MM 300 for a total of 2 min on the highest setting of 30 rpm; rotate the 96-well deepwell plate 180°, after bashing 1 min and bash again for an additional minute.

B. Extraction of DNA (Adatation of Qiagen DNeasy 96 Plant Kit)

• (i) Centrifuge the plates containing pulverized dried mycelium at 4,600 g for 5 min and remove the capmat carefully.

• (ii) Transfer a total of 400 μl of lysis cocktail containing 100 mM Tris, pH 8.0, 50 mM EDTA, 500 mM NaCl, 1.33% SDS with 0.8% Fighter F antifoaming agent (Loveland Industries, Colorado, USA) and 0.2 mg/ml RNase A to each well using the Apricot and apply a new capmat.

• (iii) Agitate vigorously by inverting the plate five to ten times and incubate them in a 65°C chamber for 20 min.

• (iv) Centrifuge the plates at 4,600 g for 2 min; gently remove the capmat; add 150 μl of 5 M potassium acetate using the Apricot and apply a new capmat.

• (v) Agitate the inverted plates vigorously five to ten times; incubate at −20°C for 30 min to overnight and centrifuge the plates at 4,600 g for 30 min.

• (vi) Transfer 400 μl of the supernatant to a new 2 ml DW plate containing 600 μl of a 0.66 M guanidine hydrochloride and 6.33% ethanol solution using the Apricot (Handle hazardous guanidine chloride carefully and use mask for eye protection) and apply a new capmat.

• (vii) Agitate the plates as done earlier to mix the solution; transfer 1 ml of the mixture to a Nunc spin column plate (Nalge Nunc Inc., NY, USA) sitting on a 2 ml DW plate and centrifuge at 4,600 g for 5 min.

• (viii) Discard the flow through; wash the membrane by adding 500 μl wash solution consisting of 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl and 67% ethanol and centrifuge at 4,600 g for 5 min.

• (ix) Wash the membrane again by adding 500 μl of 95% ethanol; centrifuge at 4,600 g for 5 min and incubate the spin column plate at 65°C for 5 min to dry the membrane.

• (x) Add 200 μl of 10 mM Tris, pH 8.0, to each well using the Apricot and incubate plates at room temperature for 30–60 min.

• (xi) Elute the DNA into a clean 1 ml DW plate by centrifuging at 4,600 g for 2 min and assess the quality of DNA by separation on a 1% agar gel.

Appendix 18: Rapid PCR-Based Method for the Detection of Fungal Pathogen (Harmon et al. 2003)

A. Extraction of DNA of fungal pathogen

• (i) Grow the fungal pathogen (Magnaporthe oryzae) in appropriate medium (V 8 juice agar, Campbell Soup Co. USA) and maintain the culture conditions that favor optimal growth.

• (ii) Grind the mycelium in liquid nitrogen; suspend in 0.4 ml of phenol and 0.8 ml of fungal genomic DNA extraction buffer (100 mM LiCl, 10 mM EDTA, 10 mM Tris, pH 8.0, 0.5% SDS and 0.1% ß-mercaptoethanol) in tubes and incubate for 5 min at 60°C.

• (iii) Agitate the tubes gently; allow them to cool; add 0.4 ml of chloroform/isoamyl alcohol (24:1 v/v) and centrifuge at 16,000 g for 10 min.

• (iv) Extract 0.7 ml of the upper phase with an equal volume of chloroform/isoamyl alcohol (24:1, v/v); separate the upper phase and precipitate DNA with 1.0 ml of cold ethanol containing 150 mM sodium acetate.

• (v) Allow the pellet to dry for 5 min and dissolve in 0.5 ml of TE buffer ( 1 mM EDTA,10 mM Tris, pH 8.0).

• (vi) Treat with RNase (50 μg); extract with phenol/chloroform and precipitate with ethanol; dissolve the pellet in 0.1 ml of TE buffer, pH 8.0; determine the DNA contents spectrophotometrically and adjust the final concentration to 50 ng/μl.

B. Extraction of DNA from plant tissues

• (i) Place the pieces of infected leaf blades in 1.5 ml Eppendorf tubes; add sufficient extract solution (100 μl) (from the Extract-N-Amp Kit, Sigma Chemical Co. USA) to cover the leaf tissues and incubate for 10 min at 95°C.

• (ii) Add equal volume of dilution solution (from the kit); homogenize the sample in the tube using a polypropylene pestle; place in ice and dilute 5 μl aliquot tenfold in sterile distilled water.

C. PCR amplification and detection of diagnostic amplicon

• (i) Use primers pfh2a and pfh2b capable of amplifying the 687-bp region of the Pot2 transposon.

• (ii) Perform PCR in a 50 μl rection mixture with DNA Taq polymerase (Promega, Madison, USA) and purify genomic DNA from pathogen isolates.

• (iii) Perform PCR for plant samples in a 20 μl reaction mixture from the kit in a DNA thermal cycler (Perkin Elmer Cetus, CT, USA).

• (iv) PCR program consists of initial denaturation of 2 min at 94°C; 30 cycles of 45 s denaturation at 94°C; 45 s of annealing at 55°C; 45 s of extension at 72°C and final extension at 72°C for 10 min.

• (v) Resolve the amplicon after electrophoresis in a 1% agarose gel; stain for 10 min in an ethidium bromide solution (10 μg/ml) and visualize the bands with UV light.

• (vi) Use photoimaging system (Stratagene, CA, USA) for getting gel images.

Appendix 19: Detection of Powdery Mildew Pathogens by PCR-mediated Method (Chen et al. 2008)

A. Extraction of DNA from obligate fungal pathogens

• (i) Scrape fungal mycelium from diseased leaf tissues; transfer into 2-ml microfuge tube; freeze the mycelium in liquid nitrogen and grind it into a powder using a plastic pestle.

• (ii) Add 700 μl of lysis buffer containing 50 mM Tris-HCl, pH 7.2, 50 mM EDTA, pH 7.2, 3% SDS, 1% mercaptoethanol; vortex the contents and heat in a water bath at 65°C for 1 h.

• (iii) Extract DNA solution; mix well with 700 μl of phenol/chloroform; centrifuge at 12,000 × g for 10 min; separate the upper phase; mix with 500 μl of chloroform and centrifuge at 12, 000 × g for 4 min.

• (iv) Transfer the aqueous phase into a new 1.5 ml Eppendorf tube; add 50 μl of 3 M sodium acetate and 500 μl of isopropanol and centrifuge at 12, 000 × g for 20 min.

• (v) Was the DNA pellet with 500 μl of 70% ethanol and centrifuge at 12, 000 × g for 20 min.

• (vi) Air-dry the DNA pellet; dissolve in 0.5 ml of TE buffer containing 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 to have a concentration of ∼200–500 μg /ml.

B. Primers and PCR amplification

• (i) Use ITS universal primer pair PN23/PN34.

• (ii) Use PCR reaction mixture containing 0.15 mM dNTPs, 0.4 μM primers, 1 U Taq polymerase (BioBasic), 1 × PCR buffer with 1.5 mM MgCl₂ and 10 μg of template DNA.

• (iii) Add sterile distilled water to have a final volume of 25 μl.

• (iv) Perform PCR amplification using a thermal cycler under the following conditions: initial denaturation at 94°C for 5 min; 30 cycles consisting of denaturation at 94°C for 40 s; annealing at 62°C for 1 min; DNA synthesis at 72°C for 1.5 min; final extension at 72°C for 5 min.

• (v) Separate PCR product (5 μl) by gel electrophoresis on a horizontal 2% agarose gel and stain the bands with ethidium bromide (0.5 μg/ml).

• (vi) Visualize the bands under UV light and photograph.

Appendix 20: Detection of Rust Pathogen by PCR-Based Method (Wang et al. 2008)

A. Extraction of DNA from rust pathogen

• (i) Freeze pure samples of urediospores (from artificially inoculated wheat seedlings) in liquid nitrogen and store at −70°C till needed.

• (ii) Transfer 25 mg of urediospores to 2 ml tube; add 500 μl extraction buffer (50 mM Trsi-HCl, pH 8.0, 150 mM NaCl, 100 mM EDTA) and homogenize with a plastic pestle.

• (iii) Add 5 μl proteinase K (1 mg/ml); make up the volume to 1.0 ml with extraction buffer and incubate for 30 min at 65°C.

• (iv) Divide the mixture into two equal parts in two microfuge tubes; extract with phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0) and chloroform respectively; transfer the top aqueous phase to a clean tube; add an equal volume of cold isopropanol and incubate for 1 h at −20°C.

• (v) Centrifuge the contents at 12,000 rpm for 20 min at 4°C to precipitate the nucleic acid; rinse the pellet twice with cold 70% ethanol; dry and dissolve in 0.1 ml TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0).

• (vi) Add 1 μl of ribonuclease (10 mg/ml, final concentration 20 μg/ml) and incubate at 4°C overnight to digest the RNA completely.

• (vii) Reprecipitate the DNA; rinse it with cold 70% ethanol; dissolve in 50 μl of TE buffer and quantify the DNA spectrophotometrically.

B. PCR amplification

• (i) Use primers specific for the pathogen (Puccinia striiformis) Pst1 and Pst2.

• (ii) Perform amplification in aliquots of 25 μl containing 20 ng DNA template, 2.5 μl 10 × reaction buffer (750 mM Tris-HCl, 200 mM (NH₄)2SO₄, 0.1 Tween-20) 25 mM MgCl₂, 2.5 mM each of dATP, dCTP, dGTP, dTTP, 0.2 μM primer and 1 U Taq polymerase and make up the volume to 25 μl with sterile distilled water.

• (iii) Provide optimal conditions using a thermal cycler: initial denaturation at 94°C for 3 min; 34 cycles of amplification each consisting of denaturation at 94°C for 50 s, primer annealing at 50–60°C for 90 s and primer extension at 72°C for 2 min; final extension step at 72°C for 10 min.

• (iv) Resolve the amplicons in 1.5% agarose gels, electrophoresed at 10 V/cm for 60–90 min along with a molecular size marker set.

Appendix 21: Detection of Colletotrichum acutatum by Arbitrarily Primed (AP)-PCR (Yoshida et al. 2007)

• (i) Grow the fungal pathogen in potato dextrose agar (PDA) for 4–7 days at ∼20°C.

• (ii) Extract the genomic DNA from each isolate using the Wizard Genomic DNA Purification Kit (Promega) as per the manufacturer’s recommen­dations.

• (iii) Use a total volume of 25 μl of ∼100 ng of genomic DNA,Read-to-Go-RAPD Analysis Beads (GE Healthcare) and uu(CAG)₅ (5′-CAGCAGCAGCAGCAG-3′) or (GACAC)₃ (5′-GACACGACACGACAC-3′) primer.

• (iv) Carry out the reactions using the GeneampPCR System 9600 (Applied Biosystems) starting with a 2-min denaturation at 95°C, followed by 45 cycles consisting of 1 min at 95°C, 1 min at either 60°C (for (CAG)₅) or 48°C (for (GACAC)₃) and 2 min at 72°C.

• (v) Resolve the amplicons using 2% agarose gels in TBE buffer and view the bands under UV light after staining with ethidium bromide.

Appendix 22: Detection of Puccinia coronata by Real-Time PCR Assay (Jackson et al. 2006)

A. Pathogen DNA extraction and amplification

• (i) Place standard weights (10²–10⁵ μg) of uredinospores or sections of infected leaf tissues in 1.5 ml tubes (Qiagen); lyophilize the pathogen tissues and add ∼30 mg 0.1 mm diameter zirconia/silica beads (Biospec) and 100 mg of 0.5 mm diameter zirconia/silica beads to each tube.

• (ii) For leaf samples add additional 3.2 mm stainless steel beads (Biospec) and 2.3 mm stainless beads and grind the tissues on a vibration mill (Retsch MM 300 USA) for 30 min at 30 Hz (3 O oscillations/s).

• (iii) Centrifuge the tube-contents at ∼6,000 × g for 10 min; add 500 μl of CTAB extraction buffer and mix the contents well.

• (iv) Grind the samples for 15 min on the vibration mill; place in water bath at 65°C for 25 min.

• (v) Add chloroform-isoamyl alcohol (24:1); centrifuge at 6000 × g for 15 min and separate the supernatant.

• (vi) Precipitate DNA with isopropanol; wash the pellet with 70% ethanol and dissolve the pellet in 200 μl of TE buffer containing 10 μl/ml RNase.

• (vii) Determine the purity and quantity of the DNA spectrophotometrically at A₂₈₀ nm and A₂₆₀ nm and store the DNA preparations at 4°C.

B. Conventional and real-time PCR assays

• (i) Perform amplifications at 50°C for 60 s and 95°C for 10 min; then 40 cycles at 95°C for 15 s and 60°C for 60 s in a total volume of 50 μl containing 28.6 μl sterile double distilled (dd) water, 5.0 μl 10 × buffer, 3.0 μl 25 mM Mg²⁺, 0.4 μl 5 U/ml Ampli TaqDNA polymerase (Applied Biosystems), 1.0 μl 10 mM dNTPs, 1.0 μl of each of forward and reverse primer (300 nM) and 10 μl DNA template (20 ng/μl).

• (ii) Visualize the PCR amplicons on 2% agarose (SIGMA) gels stained with ethidium bromide after 2 h at 90 V (approximate distance of 5 cm from the wells) using a Fluorochem 8800 Image System (Alpha Innotech Corp. CA, USA).

• (iii) Perform real-time PCR amplifications using a 96-well optical reaction plate in an ABI Prism 7000 Sequence detection system; follow thermal cycling conditions as in conventional PCR amplification (step B (i) above).

• (iv) Use reaction volumes of 50 μl containing 13.4 μl sterile dd water, 25 μl TaqMan Universal master mix (Applied Biosystems), 0.3 μl of each forward and reverse primer (300 nM), 10 μl TaqMan probe (200 nM) and 10 μl DNA template.

Appendix 23: Detection of Colletotrichum spp. in Strawberry Plants by Real-Time PCR Assays (Garrido et al. 2009)

A. Extraction of DNA from strawberry plant tissues

• (i) Place the weighed samples (0.25–1.0 g) in extraction bags (Bioreba) with 8–10 volumes of CTAB lysis buffer containing 12% sodium phosphate buffer, pH 8.0, 2% CTAB, 1.5 M NaCl, supplemented with 2% antifoam B emulsion (Sigma Aldrich); homogenize the samples to a paste-like consistency using a Homex grinder (Bioreba); transfer the homogenate to clean 2-ml centrifuge tubes and centrifuge for 5 min at 10,000 g to pellet the cell debris.

• (ii) Dispense 600 μl of lysate (supernatant) to fresh 2-ml tubes containing 200 μl chloroform; mix by vortexing and centrifuge for 5 min at 13,000 g.

• (iii) Transfer 500 μl of aqueous layer to clean 2-ml tubes containing 500 μl isopropanol and 50 μl MagneSil^® Paramagnetic Particles (Promega); and incubate for 10 min at room temperature.

• (iv) Extract the DNA using a robotic magnetic particle processor (Kingfisher ML, ThermoScientific); load the Kingfiser 5-ml tube strips as detailed below:

  (a)tube 1: 1 ml sample containing the MagneSil^® beads; (b) tube 2: 1 ml GITC lysis buffer containing 5.25 M guanidiniumthiocyanate, 50 mM Tris HCl pH 6.4, 20 mM EDTA and 13 g/l Triton X-100; (c) tubes 3 and 4: 1 ml 70% ethanol; (d) tube 5: 200 μl sterile distilled water.

• (v) Use a total genomic DNA program (Kingfisher ML, ThermoScientific) to purify the DNA in each sample; transfer DNA collected in tube 5 to fresh 1.5-ml microcentrifuge tubes and store all DNA samples at −20°C, until needed for use in real-time PCR.

B. Real-time PCR formats

• (i) Set up all real-time PCR assays in 96- or 384-well reaction plates.

• (ii) Set up all SYBR^® green assays, use with Uni 58SSybr F1/Uni 58SSybr R2 primers with an Absolute^TM QPCR SYBR^® Green ROX (500 nM) Mix Kit (AB gene) as follows.

  12.5 μl SYBR^® Green Mix, 0.375 μl ROX, passive reference (diluted 1:50), 300 nM primers and 10 μl diluted DNA extract made up to 25 μl using molecular grade water; carry out SYBR^® green assays in duplicate with generic cycling conditions: 95°C for 10 min and 40 cylcles of 60°C for 1 min and 95°C for 15 s, followed by a dissociation step consisting of a single transfer from 60°C to 95°C at a ramp rate of 2% within an ABI Prism 7900 HT Sequence Detector Systen (PE Biosystems).

• (iii) Analyze the melting curves, after each run, to check for the presence of non-specific amplification products.

• (iv) Set up all TaqMan^® assays using PCR Core Reagent Kits (PE Biosystems) consisting of 1 × buffer A, 0.025-U μl⁻¹ AmpliTaq Gold, 0.2 mM dNTPs and 5.5 mM MgCl₂.

• (v) Use all sets of primers at 300 nM and probes at 100 nM; add 1 μl DNA extract, giving a final volume of 25 μl/reaction.

• (vi) Maintain negative controls containing nuclease-free water instead of DNA for each run.

• (vii) Carry out TaqManR PCR reaction in duplicate at 50°C for 2 min and 45 cycles of 95°C for 15 s and 60°C for 1 min.

• (viii) Assess the C_T values for each reaction using SEQUENCE DETECTION SOFTWARE v2.2.2 (PE Biosystems).

Appendix 24: Detection of Phytophthora cactorum by RAPD-PCR Technique (Causin et al. 2005)

A. Extraction of DNA of fungal pathogen

• (i) Crush the fungal mycelium (∼200 mg wet weight) in liquid nitrogen using pestle and mortar; transfer immediately the macerate into a microcentrifuge tube; add 1 ml of lysis buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, pH 8.0, 1.4 mM NaCl, 2% cetyltrimethylammonium bromide (CTAB), 1% polyvinylpyrrolidone (PVP), 1% mercaptoethanol) and incubate at 65°C for 60 min.

• (ii) Add 1 ml chloroform/isoamyl alcohol (24:1 v/v), shake the contents for 1 h in ice for completion of protein denaturation and centrifuge at 17,300 g for 10 min to separate the phases.

• (iii) Recover the aqueous phase carefully; precipitate the DNA by adding 2/3 volume of isopropanol and 1/10 volume 3 M sodium acetate, pH 5.2 and allow the sample to remain at −20°C for 20 min.

• (iv) Centrifuge at 17,300 g for 10 min; wash the pellet with 70% ethanol (v/v); repeat the cycle of pelleting and washing processes and dry the pellet at room temperature.

• (v) Resuspend the pellet in 100 μl of TE buffer consisting of 10 mM Tris-HCL and 1 mM EDTA) and store at −20°C till required.

B. Screening for RAPD markers

• (i) Use the DNA extracted from the test fungus for testing the 10-mer RAPD primers of the OPA series (OPA-1 – OPA-11) (Operon Technologies Inc., CA, USA).

• (ii) Perform the reactions in 25 μl volumes with 10–15 ng of template DNA, 100 μM of each dNTP, 2.5 μl of 10 × buffer (200 mM Tris-HCl, pH 9.0, 500 mM KCl, 1% Triton^® × 100), 2 mM Mg Cl₂, 0.2 μM of RAPD primer and 1 Unit of Taq DNA polymerase (Promega Corp. USA); overlay a drop of sterile mineral oil on the reaction mix and maintain negative controls without the template DNA to check for DNA contamination of the reagents.

• (iii) Provide the following conditions using the Thermo Cycler (Cycler TM, Bio-Rad, Italy): initial denaturation at 94°C for 2 min and 30 s; 45 cycles of amplification consisting of 94°C for 30 s, annealing at 38°C for 1 min, extension at 72°C for 2 min; final extension of 5 min at 72°C after cycling.

• (iv) Separate the amplicons using 1.5% TBE buffer (45 mM Tris-borate, 1 mM EDTA) in agarose gels for 2 h followed by staining with ethidium bromide and photographing under UV illuminator (302 nm).

• (v) Elute the RAPD band specific for the target pathogen directly from the agarose gel using the Agarose gel DNA Extraction Kit (Boehringer Mannheim Corp. USA).

• (vi) Ligate the purified DNA into a plasmid pGEM-T Vector System (Promega) as per the manufacturer’s instructions.

• (vii) Transform Escherichia coli strain JM 109 competent cells using the plasmids and identify recombinant column by the blue-white color selection after 12 h of growth at 37°C on LB agar medium (1.8% trypan-NaCl, 0.5% yeast extract, 1.6% agar agar) containing ampicillin, IPTG (isopropyl ß-D-1-thigalactopyranoside) and X-Gal (5-Bromo-4-chloro-3-indolyl ß-D-galactopyranoside) as per manufacturer’s recommen­dation.

• (viii) Purify the plasmids from LB/ampicilling liquid cultures of selected colonies using a High Pure Plasmid Isolation Kit (Boehringer Mannheim Corp.) following the manufacture’s instructions.

Appendix 25: Detection of Macrophomina phaseolina in Cowpea Seeds by DAS-ELISA Technique (Afouda et al. 2009)

A. Preparation of antigen

• (i) Grow the pathogen in polysulfon membrane filters (HT-200 Tuffryn, Gelman, Germany) supported by inert fibre for 4 days in petridishes containing 10 ml of potato dextrose broth (PDB); harvest the mycelium from the filter; homogenize in phosphate buffered saline (PBS, pH 7.4) and centrifuge at 30,000 × g for 10 min and at 130,000 × g for 30 min.

• (ii) Estimate the protein concentration by the method of Bradford (1976); adjust the protein concentration to 1 mg/ml and store aliquots of 1 ml in 2-ml Eppendorf centrifuge tubes at −20°C, until required.

• (iii) Dialyze the culture filtrate, after harvesting the mycelium against PBS overnight at 7°C; concentrate by ultracentrifugation; determine the protein concentration; adjust the protein concentration to 0.1 mg/ml and store at −20°C, until required.

B. Preparation of antiserum

• (i) Immunize the rabbits by injecting 1 ml of antigen emulsified with Fruend’s adjuvant (Difco); space the first three injections at 2-week intervals and fourth after 4 months, as booster injection.

• (ii) Bleed the rabbits at 1 week after each injection; store the antiserum ­supplemented with 0.05% sodium azide at 4°C and label the antisera generated against the mycelium and culture filtrate separately before storing.

• (iii) Purify the immunoglobulins (IgG) in the antiserum by precipitation with 50% ammonium sulfate, followed by suspension in half-strength PBS and passage through a DEAE-Fractogel columns (Merck, Germany).

• (iv) Collect 1 ml aliquots from the column; adjust their final OD to 1.45 at 280 nm corresponding ∼1 mg IgG/ml and store at −20°C.

• (v) For biotinulation of IgG, dialyze 1 ml of purified IgG overnight at 7°C against coupling buffer (containing 10 g NaCl, 10 g NaHCO₃ and 1,000 ml water, pH 7.5) with three changes of buffer solution; add 50 μl of biotinylation reagent (1 mg X-NHS-Biotin, Sigma, Germany) to the IgG; incubate for 30 min at room temperature; stop the reaction by adding 50 μl 1 M Tris-HCL, pH 7.4; dialyze the product overnight in saline (0.85% NaCl); add 50% glycerol and 1% bovine serum albumin (BSA) and store the mixture at −20°C.

C. Preparation of seed extract

• (i) Soak the seeds singly in the wells of microtiter plates containing 1 ml PBS-T (PBS with 0.05% Tween 20) and 2% polyvinyl pyrrolidone (PVP); incubate the plates for 24 h at 4°C ;crush the seed into the buffer solution and incubate for a further period of 24 h at 4°C.

• (ii) Centrifuge the plates and use the supernatant for detection of the target pathogen.

D. Double-Antibody Sandwich (DAS)-ELISA technique

• (i) Coat the wells of microplates with IgG-diluted to 1: 1,000 (v/v) in 0.05 M carbonate buffer, pH 9.6; incubate at 4°C overnight and wash the plates thrice with half-strength PBS-T and subsequently between each step mentioned below.

• (ii) Block unspecific reactive surfaces of each well with 200 μl of 0.2% BSA dissolved in coating buffer (0.05 M sodium carbonate buffer, pH 9.6); incubate the plates at room temperature for 2 h.

• (iii) Dilute the antigen preparation suitably with PBS-T with 2% PVP; add 200 μl antigen solution to each well; incubate at 4°C overnight.

• (iv) Dilute the biotinylated IgG (1:1,000, v/v) in PBS-T buffer with 0.2% BSA; incubate at 4°C overnight.

• (v) Add streptavidin-alkaline phosphatase (Sigma) conjugate diluted (1:1,000, v/v) in conjugate buffer (half-strength PBS-T with 0.2% BSA and incubate at 37°C for 30 min.

• (vi) Add 100 μl/well the substrate (1 mg.ml of p-nitrophenyl phosphate) in 10% diethanolamine, pH 9.8 and record the absorbance values at 405 nm using the ELISA reader after allowing the reaction for 1–2 h at 37°C; absorbance values that are more than twice that of healthy control are considered to be positive reactions.

Appendix 26: Detection of Fungal Pathogens in Soybean Seeds by PCR-RFLP Technique (Zhang et al. 1999)

A. Extraction of DNA from soybean seeds

• (i) Treat the soybean seeds with 95% ethanol for 30 s, 0.5% NaOCl for 1 min, 2.5% paraquat (Gramoxone, Zeneca Corp. USA) for 2 min and rinse the seeds three times in double-distilled (dd) water.

• (ii) Squeeze the disinfected seeds to release the seed coats that are individually placed into a 1.5 ml microfuge tube with 250 μl of extraction buffer containing 50 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0, 100 mM NaCl, 1.0% sodium dodecyl sulfate and 10 mM ß-mercaptoethanol.

• (iii) Break the seed coats using an ultrasonic processor (Biospec Products) and a tapered microtip (5 mm diameter) for 10 s; mix with 150 μl of potassium acetate (5 M, pH 5.2) and incubate on ice for 20 min.

• (iv) Centrifuge at 12,000 × g for 10 min; determine the DNA concentrations of the supernatant (extracted from the seed coat) by measuring OD at 260 nm in an UV spectrophotometer (Perkin-Elmer Applied Biosystems).

B.Extraction of DNA from the mycelium growing out of seeds on potato dextrose agar (PDA)

• (i) Plate 100 surface-disinfested seeds from each seed lot on PDA and incubate at 27°C for 24–36 h.

• (ii) Cut out 5 × 10 × 2 mm PDA plugs with mycelial growth originating from individual seeds; place into a microfuge tube (1.5 ml) containing 250 μl of extraction buffer and break the cells using the ultrasonic processor.

• (iii) Centrifuge and save the supernatants as DNA extracts.

C. PCR-RFLP Assay

• (i) Plate 100 seeds from each seed lots on PDA medium and extract the DNA from the pathogens as per steps B (i)–(iii).

• (ii) Perform PCRs with a DNA thermal cycler (Perkin-Elmer Applied Biosystems) using the reaction mixture containing 50 mM KCl, 2.5 mM MgCl2, 10 mM Tris-HCl, pH 8.3, 0.2 mM each of dTTP, dATP, dGTP and dCTP, 50 pmol of the primers, 2.5 units of Taq polymerase and 25 ng of genomic DNA in a final volume of 50 μl.

• (iii) Incubate the reactants at 96°C for 3 min followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min.

• (iv) Check the amplification efficiency by subjecting to agarose gel electrophoresis using 5 μl of PCR amplicons.

• (v) Digest the PCR amplicons (7 μl) with suitable restriction enzymes (5–10 units of Alu I, Mse I, Hha I, Rsa I and ScrF I) as per the manufacturer’s instructions using 1.5 μl of buffer (10×) and 6.5 μl of double distilled water at 37°C for 2–4 h.

• (vi) Size fractionate the enzyme-digested PCR amplicons on a mixed agarose gel of 1% ultra pure agarose (Amresco, USA) at 3.5 V/cm and stain with ethidium bromide.

• (vii) Visualize on a UV-transilluminator and photograph.

Appendix 27: Detection of Rhynchosporium secalis in Barley Seeds by Competitive PCR (Lee et al. 2002)

A. Extraction of DNA from seeds

• (i) Surface sterilize the seeds (100/sample) with ethanol for 30 s; wash them with several times with distilled water; dry at 22°C and grind to a fine powder using a mixer mill grinder.

• (ii) Extract total DNA from the seed powder (0.1 g) and adopt the cetyltrimethyl ammonium bromide (CTAB) procedure.

• (iii) Use the DNA equivalent of 0.1 mg dry seed weight in 1 μl for PCR.

B. PCR amplification

• (i) Use a reaction mixture (25 μl) containing 1 × PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂), 0.2 mM dNTPs, 250 nM of each forward and reverse primer and 0.6 units of TaqDNA polymerase.

• (ii) Use primer sets designed from ITS regions of target pathogen DNA.

• (iii) Perform PCR amplification with the following conditions: initial denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min with final extension at 72°C for 10 min.

• (iv) Use 8 μl of PCR amplicons for separating them on a 1.5 agarose gel in Tris-borate-EDTA buffer at 100 V for 1 h; stain the gel with ethidium bromide for 15 min and visualize the PCR products on a UV transilluminator.

C. Competitive PCR

• (i) Prepare a heterologous internal control using a competitive DNA Construction Kit (Takara Shuzo Co. Ltd., Japan) which has 5′-and 3′-termini identical to the fungal target primary sites (RS1 and RS3), but no internal sequence homology to the target sequence.

• (ii) Generate the competitor fragment (445-bp) as per the manufacturer’s recommendation.

• (iii) Use 0.1 g milled seed powder (from 100-seed sample) for DNA extraction and use the extracts in the presence of constant amount of the internal control template DNA.

• (iv) Calculate the mean PCR product ratios obtained from three replicates of each level of infection (disease intensity); plot the ratios against percentage of seed infection and generate a standard curve by reference to which the quantification of fungal DNA in field-infected barley seed can be made.

• (v) Calculate the mean levels of PCR product ratios of samples and subsamples of naturally infected seeds and convert to ng of fungal DNA/mg seed material using the standard calibration curve.

Appendix 28: Detection of Verticillium dahliae in Olive Seeds by Nested PCR (Karajeh 2006)

A. Extraction of pathogen DNA from infected seeds

• (i) Grind the seed samples (10 seeds/sample) in liquid nitrogen using a Phillips screwdriver; homogenize the seed powder in 1.5 ml preheated 65°C) extraction buffer consisting of 50 mM Tris-HCl, pH 8.0, 700 mM NaCl, 10 mM EDTA-Na₂; 2% CTAB, 0.5% 2-mercaptoethanol (v/v) and 1.0% polyvinyl pyrrolidone (PVP).

• (ii) Dispense the homogenate into two 1.5 ml microtubes; incubate at 65°C for 15 min with occasional mixing and extract with 0.6 ml chloroform-isoamyl alcohol (24:1) and centrifuge at 14,000 g for 5 min.

• (iii) Transfer the top aqueous phase to a new tube containing 2 μl RNase A (10 mg/ml water); incubate for 15 min at room temperature and precipitate protein using 0.5 volume of 3 M sodium acetate, pH 5.2.

• (iv) Add 0.3 ml isopropanol to each microtube; incubate at −20°C overnight and centrifuge at 14,000 g for 5 min.

• (v) Wash the DNA pellet with 1 ml 70% ethanol, air-dry and dissolve in 100 μl TE buffer consisting of 10 mM Tris, 1 mM EDTA, pH 8.0.

• (vi) Estimate DNA concentration using agarose gel electrophoresis and with a spectrophotometer at 260 nm.

B. DNA amplification using nested PCR assay

• (i) Carry out the first round amplification with the primer pair NESF 18S and NESR 28S from the highly conserved DNA sequences of 18S amd 28S genes that flank ITS region of the pathogen DNA and identify the product about 480-bp in size.

• (ii) Transfer one microliter of the product of first amplification; perform second amplification with pathogen-specific ITS primers FVD and RVD and identify the product 330-bp in size.

• (iii) Perform the PCR in a total volume of 25 μl with each reaction containing the following: 0.2 mM dNTPs (an equal molar mixture of dATP, dGTP. dCTP and dTTP), 0.5 U of Taq DNA polymerase, 0.25 mM of each forward and reverse primer, 1 × PCR buffer (10×: 500 mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl, pH 9.0 at 25°C) and 1 Triton X-100, 1 μl of the final DNA extract (about 50 ng).

• (iv) Maintain a negative control (water) for each round to detect contamination with template DNA and olive DNA extract.

• (v) Provide the following conditions using Eppendorf Master cycler: initial DNA denaturation for 2 min at 94°C, followed by 35 cycles each consisting of denaturation at 95°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 30 s and the final extension for 3 min at 72°C.

• (vi) Analyze the PCR products by agarose gel electrophoresis with 0.5 × TBE buffer consisting of 10 × buffer of 0.9 M Tris, 0.9 M boric acid and 20 mM EDTA; stain with ethidium bromide and visualize on a UV transilluminator.

Appendix 29: Detection of Pythium spp. in Carrot Tissue by PCR Assay (Klemsdal et al. 2008)

A. Extraction of DNA from the oomycete and carrot tissue

• (i) Cultivate the pathogen in potato dextrose agar (PDA) covered with cellophane at 20°C for 4–6 days; harvest the mycelium; grind it to a fine powder in liquid nitrogen and extract the DNA using DNeasy Plant Mini Kit (Qiagen Inc.) as per manufacturer’s recommendations.

• (ii) Collect carrots with and without symptoms of infection at harvest from the fields; wash them with water; take the peels carefully from the top to the tip in each carrot; freeze dry the peels overnight and grind them to a fine powder using pestle and mortar.

• (iii) Transfer carrot tissue powder (50 mg) to a microcentrifuge tube; extract the DNA using the GenElute Plant Genomic DNA Kit (Sigma-Aldrich) according to the manufacturer’s recommendations, except that elution of DNA from the binding column once with 100 μl TE buffer pH 7.5 and pre-warm the extract to 65°C.

• (iv) Purify the DNA further using Micro BioSpin Chromatography columns (BioRad Laboratories Ltd.) filled with insoluble polyvinyl polypyrrolidone (PVPP).

• (v) Load each column with 400 μl sterile water placed in a microcentrifuge tube and centrifuge for 5 min at 1,500 g.

• (vi) Transfer the column to a new centrifuge tube and load the DNA onto the PVPP surface.

• (vii) Collect the purified DNA as pellet after centrifuging at 1,500 g for 5 min.

B. Polymerase Chain Reaction Assay

• (i) Perform the assay in a total volume of 25 μl with final concentration of 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.2 mM dNTPs, 0.1 mg/ml bovine serum albumin (BSA) and 0.1 mM MgCl₂.

• (ii) Use 25 pmol of each primer and 0.6 U Ampli Taq polymerase (Applied Biosystems).

• (iii) Use 1 μl of the DNA extracted from carrot tissue as template; use universal primers ITS3 and ITS4 as positive control for DNA extracted from carrot tissues.

• (iv) Perform amplifications in a GeneAmp^® PCR System 9700 thermal cycler (Applied Systems) programmed for initial denaturation at 94°C for 5 min followed by 45 cycles of 20 s at 94°C, and 30 s annealing at 72°C.

• (v) Use the following annealing temperatures for Pythium sylvaticum: 56°C; P. ‘vipa’: 57°C; P. sulcatum and P. intermedium: 60°C and P. violae: 61°C.

• (vi) Separate the PCR amplicons by electrophoresis through 1.2% agarose gels; stain with ethidium bromide and photograph in UV light on a GelDoc 1000 (BioRad Laboratories Ltd. USA).