Appendix 2 Media for Phytophthora ramorum
Vegetable juice agar (V8): vegetable juice 250 mL; CaCO3 5 g; agar 15 g; distilled water 1000 mL. Add CaCO3 to the vegetable juice and stir firmly during 15 min. Centrifuge the mixture for 20 min at 5000 r.p.m., and pour off the supernatant. Make up the resultant to 1 L with distilled water, and autoclave at 120°C for 20 min.
P5ARP[H](Jeffers & Martin, 1986): cornmeal agar 17 g; distilled water 1000 mL. Autoclave, then cool to 50°C in a water bath. Then prepare pimaricin 5 mg; ampicillin (Na salt) 250 mg; rifampicin (dissolved in 1 mL 95% Ethanol) 10 mg; PCNB 100 mg; hymexazol 22.5 mg and dissolve all in 10 mL sterile distilled water. Add to cooled media, poor, store at 4°C in the dark, use within 5 days.
P5ARP If hymexazol is unavailable, then PARP is still very useful.
PARB [H] (Robin et al., 1998): cornmeal agar 17 g; distilled water 1000 mL. Autoclave, then cool to 50°C in a water bath. Then prepare pimaricin 10 mg; ampicillin 250 mg; rifampicin 10 mg; benomyl 15 mg; hymexazol 50 mg.
Carrot Piece Agar (Werres et al., 2001): agar 22 g, carrot pieces 50 g, distilled water 1000 mL.
Carrot Juice Agar 5% (Kröber, 1985): agar 5–22 g; carrot juice (without honey) 50 mL; distilled water 950 mL.
CSL Dark Carrot Agar (DCA): carrots 200 g; agar Oxoid No. 3 15 g; distilled water 1000 mL.
Slice the fresh carrots and comminute in a blender with 500 mL of distilled water for 1 min at high speed. Filter through four layers of cheesecloth and squeeze out the juice from the residue. Make up the resultant filtrate to 1 L and add the agar. Heat to dissolve the agar, pour into bottles and autoclave at 121°C for 15 min.
Cherry decoction Agar (CHA): agar 60 g; distilled water 3600 mL; cherry juice 400 mL. Filter the cherry juice, and adjust the pH to 4.4 with KOH. Dissolve the agar thoroughly first, then add cherry juice. Autoclave at 102°C for 5 min.
Snyder and Nash Agar (SNA): KH2PO4 1 g; KNO3 1 g; MgSO4·7H2O 0.5 g; KCl 0.5 g; glucose 0.2 g; saccharose 0.2 g; agar 15 g; distilled water 1000 mL. Autoclave for 15 min at 120°C.
Appendix 3 Identification on basis of isozymes
Culturing: isolates are grown in 50 mL of Tryptone Soy Broth (TSB, Oxoid) medium in 250 mL Erlenmeyer flasks on a rotary shaker at 40 r.p.m. in the dark at 23°C. Flasks are inoculated with three V8-agar plugs (diameter 5 mm) bearing mycelium, from the margin of actively growing colonies (three days old). After 7 days, the mycelium is collected by pouring the content of the flask through a sieve (pore size approximately 1 mm). The fungal tissue is dried by pressing it between filter paper. The mycelium is stored overnight at −70°C before extracting enzymes.
Extraction: frozen mycelium is thawed at 4°C for 3 h before enzyme extraction. Then approximately 0.5 g of mycelium is ground for three minutes in a chilled mortar with sand and 70 µL of extraction medium (0.1 m Tris-HCl (pH 7.0), 1 mm dithiothreitol, 50 mm Ethylene Diamine Tetra-acetic Acid (EDTA), 10% Poly Vinyl Pyrrolidone (PVP; M.W. 25000) w/v, 50 µg/mL soybean trypsin inhibitor, 0.1 mm Phenyl Methyl Sulfonyl Fluoride (PMSF) and 5% glycerol (v/v)). The homogenate is transferred to microcentrifuge tubes and centrifuged for 10 minutes at 14 000 r.p.m. (4°C). The supernatant (40–80 µL) is collected, and stored at −70°C before use.
Electrophoresis and enzyme staining: electrophoresis is performed at 4°C, for example by using an automated PhastSystem®. Crude extracts, obtained as above are analyzed for any one of the following enzymes:
- 1malic enzyme (MDHP, EC 22.214.171.124) on native, 12.5% homogeneous polyacrylamide gels, or
- 2malate dehydrogenase (MDH, EC 126.96.36.199) on native, 8–25% gradient polyacrylamide gels.
Gels are prepared with 0.11 m Tris-acetate buffer (pH 6.4). The running buffer, contained in 2% agarose gel, consists of a 0.25 m Tris and 0.88 m l-alanine buffer (pH 8.8). Glycine is added to the samples to produce a final concentration of 0.22 m. MDH activity is assayed by diluting 5–10 times to avoid smearing. Following electrophoresis, gels are immersed in freshly prepared staining solutions in the dark at 37°C for 5 and 20 min for MDH and MDHP, respectively. The reaction ingredients of the enzymatic stains are as follows:
MDH: 25 mL 0.2 m Tris-HCl pH 8.0, 440 mg l-malic acid (di-sodium salt), 12.5 mg Nicotineamide-Adenine-Dinucleotide (NAD), 7.5 mg Nitro Blue Tetrazolium (NBT) (Sigma N 6876), 1 mg Phenazine Metho Sulfate (PMS) (Sigma P 9625).
MDHP: 25 mL 0.2 m Tris-HCl pH 8.0, 440 mg l-malic acid (di-sodium salt), 12.5 mg Nicotinamide Adenine Dinucleotide Phosphate (NADP), 7.5 mg NBT, 1 mg PMS.
Analysis: the bands appearing on the gel are identified by using a reference strain of P. ramorum.
Appendix 4 Identification at species level by conventional PCR
Primers: a primer pair (Phyto 1 and Phyto 4) has been developed by M. Garbelotto (Hayden et al., 2004) for the detection of P. ramorum by conventional PCR. The primer sequences are:
Sample handling: plant material (leaves, twigs, stems, stem base) is disinfected superficially with a tissue using 1% sodium hypochlorite. Small pieces of material (0.5–1 cm2 each) are cut from the edge of symptomatic tissue with a sharp knife. In general 6–10 pieces are collected and washed in a laminar flow cabinet according to the following procedure: tap water (10 s), alcohol 50% (10 s), tap water (10 s), 1% sodium hypochlorite (10 s), and finally tap water (10 s). The material is then dried on sterile filter paper for 20–30 s. The pieces are cut into very small parts (each approximately 1 mm2) with a sterile scalpel, and placed in a clean 1.5 mL screw cap tube. When not immediately processed, the tubes are stored with material in a freezer (−20°C). Cultures can also be used as starting material for extraction. A piece of approximately 1 cm2 from the agar (taking as little agar as possible) is cut, and put it into a 1.5 mL screw cap tube.
DNA extraction: 500 µL extraction buffer (0.02 m PBS, 0.05% Tween T25, 2% polyvinylpyrolidone, 0.2% bovine serum albumin) and beads are added to the tube which is beated for 80 s at speed 5 (Hybaid Ribolyzer). The tube is centrifuged for 5 s in a microcentrifuge at maximum speed (16 100 g) and 75 µL of the supernatant is transferred to a new 1.5 mL tube.
DNA isolation: DNA for PCR is isolated using the Dneasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions (Qiagen, 2000), and is eluted with 100 µL AE buffer. It is further purified using polyvinylpolypyrrolidone (PVPP) columns. These columns are prepared by filling a Micro Bio-Spin chromatography column (Biorad, cat no: 732–6204) with 0.5 cm PVPP, and placed in an empty tube. Then 250 µL RNAse- and DNAse-free water are applied to the PVPP column which is centrifuged for 5 min at 4000 r.p.m. in a microcentrifuge. This last step is repeated. The columns are put on a clean tube, and DNA is applied to the column which is centrifuged for 5 min at 4000 r.p.m.
Amplification and analysis: the reaction mixture (25 µL) should contain: 5 µL DNA suspension; 2.5 µL of 10 × concentrated reaction buffer containing 15 mm MgCl2 (Qiagen); 0.5 µL 10 mm dNTPs; 0.1 µL 100 µm of each primer Phyto 1 and Phyto 4; 0.1 µL HotStarTaqTaq DNA polymerase (5 U/µL; Qiagen), and 16.7 µL DNAse- and RNAse-free water to give a final volume 25 µL.
The amplification is performed in thin-walled 200 µL PCR tubes in a Peltier type thermal cycler with a heated lid using the following conditions: 15 min at 95°C; then 35 cycles of 15 s at 94°C, 60 s at 62°C, 45 s at 72°C. One cycle for 10 min at 72°C should be conducted after the 35 cycles, and finally 1 s at 20°C. After amplification, 10 µL of the reaction mixture is loaded onto a 1.0% agarose gel in 0.5 × TBE buffer, separated by electrophoresis, stained with ethidium bromide, and viewed and photographed under UV light. A negative control (DNAse- and RNAse-free water) should be included in every experiment to test for contamination as well as a positive control (DNA from a reference strain of the pathogen) to monitor the performance of the PCR. The positive control should yield an amplicon of 687 bp, but not the negative control. Strains yielding an amplicon of this size are positive for P. ramorum. Samples not yielding such an amplicon can be considered negative for P. ramorum. If either the negative or positive control fails the electrophoresis and/or the PCR should be repeated. To monitor for false negatives resulting from inhibition of the PCR reaction, duplicate reactions are spiked with P. ramorum DNA. If the spiked reaction gives a negative result, the PCR should be repeated with diluted DNA extract.
For Garbelotto's primers see above.
Sampling and sample preparation: if possible at least five twigs or leaves with disease symptoms are taken per plant. About five tissue pieces per twig or leaf (each 0.5 cm2, per sample about 100–200 mg total weight) are cut out from the discoloured tissue using a sterile knife.
DNA extraction: 500 µL CTAB-buffer (2%) and 50 mg of sterile quartz sand are added to the sample, which is then homogenised with a pestle and mortar for 2 min. This preparation is transferred into a 2 mL tube and vortex for 20 s. It is then freezed at −20°C defrosted at 75°C. The operation is repeated two times for two min, and a third time for 30 min 350 µL of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) is added and the preparation is vortexed for 1 min at maximum speed. It is then centrifuged for 15 min in a microcentrifuge at maximum speed (16 100 g), and the supernatant is removed and pipetted into a new sterile 1.5 mL tube.
DNA isolation: the DNA is purified with GeneClean Turbo Kit (Nucleic Acid Purification Kit: ObioGene).
Amplification and analysis: the reaction mixture (25 µL) should contain: 6.25 µL DNA suspension; 2.5 µL of 10 × concentrated reaction buffer containing 7.5 mm MgCl2 (Invitrogen); 2.5 µL 2 mm dNTPs; 0.25 µL 50 µm of each primer Phyto 1 and Phyto 4; 0.25 µL Taq DNA polymerase (Invitrogen, recombinant cloned), and 12.25 µL DNAse- and RNAse-free water.
Thermocycler conditions should be as follows: 1.25 min at 94°C; then 34 cycles of 35 s at 93°C, 55 s at 62°C, 50 s at 72°C adding 5 s at each cycle. One cycle for 10 min at 72°C should be conducted after the 34 cycles. Separate the PCR product after amplification. Load 10 µL of the reaction mixture onto a 1.0% agarose gel in 1 × TAE buffer, separate by electrophoresis, stain with ethidium bromide, and view and photograph under UV light.
A negative and a positive control should be included in every PCR. For the negative control 6.25 µL DNAse- and RNAse-free water and 18.75 µL PCR mix are taken. For the positive control DNA from the type strain is used. The DNA of the type strain should yield an amplicon of 687 bp. Samples yielding an amplicon of this size should be identified as P. ramorum, assuming that the controls have reacted properly.
The following protocol is for the conventional PCR identification of P. ramorum from cultures and plant material.
Primers: a primer pair (Pram F1 and Pram R1) has been developed by Hughes (Lane et al., 2003b) for conventional PCR. The primer sequences are:
Sample handling: a 0.5 cm × 1 cm sample from a test culture is cut aseptically, or several small pieces of tissue from the leading infection edge of suspect plant material are removed and placed in a thick-walled plastic bag.
DNA extraction: the bagged sample is placed in liquid nitrogen. Once the sample is frozen, the bagged sample is put on the laboratory bench and ground by rolling the bag with a wallpaper seam roller or similar device. Alternatively, samples may be ground up by cutting them into small pieces and placing these in a 2 mL centrifuge tube containing approximately 150 mg siliconised 0.5 mm glass beads (Biospec products, Bartlesville, USA). The tube is closed with a screw-fitting lid containing an o-ring and the tube is oscillated in a Mini-Beadbeater (Biospec products) at full power for at least 20 s.
DNA isolation: DNA is extracted from ground-up samples using a suitable kit such as the NucleoSpin plant extraction kit (Macherey-Nagel, Düren, DE, Cat. ref. 740 570.250) or a more traditional method such as described in Hughes et al. (2000). Extracted (neat) DNA is stored at 4°C for immediate use or at −20°C if testing is not to be performed on the same day.
Amplification and analysis: extracted DNA is defrosted if necessary and a 10-fold dilution of each extract is prepared in sterile molecular grade water. Then in an area dedicated for PCR work and using dedicated pipettes with filtered tips, enough reaction mix for testing at least two replicates of the neat and 10-fold dilution for each extract is prepared. For each PCR run positive control reactions of master mix plus P. ramorum DNA and negative control reactions of reaction mix loaded with water rather than DNA are included.
The reaction mixture (25 µL) should contain: 1.0 µL DNA suspension; 2.5 µL 10 X reaction buffer containing 15 mm MgCl2 (Applied Biosystems); 2.0 µL 10 mm dNTPs; 2.5 µL 5 µm of each primer Pram F1 and Pram R1; 0.125 µL AmpliTaq (Applied Biosystems) (5 U/µL), and 14.375 µL sterile molecular grade water to give a final volume of 25 µL.
Amplification is performed in thin-walled PCR tubes in a PCR thermocycler programmed as follows: 2 min at 94°C; then 30 cycles of 30 s at 94°C, 30 s at 57°C, 30 s at 72°C. One cycle for 10 min at 72°C should be conducted after the 30 cycles. After amplification, 10 µL from the cycled reactions is mixed with 2 µL of loading dye (25 µg bromophenol blue and 25 µg xylene cyanol FF in 10 mL 50% glycerol) and amplification products are resolved by electrophoresis on a 1.5% agarose gel made with 1X TBE buffer at pH 8.0 (9.0 mm Tris, 8.9 mm boric acid and 2.5 mm EDTA). At least one replicate of a 100 base pair (bp) marker is added to each gel for amplicon size determination. Following electrophoresis, stain the gel for 30 min with ethidium bromide [0.5 µg/mL] then wash off excess stain and view the gel on a UV transilluminator.
Assessment of PCR: reactions containing amplifiable DNA from P. ramorum produce a single c. 700 bp amplicon while no bands should be produced for the negative controls. Following extensive testing, some isolates of other Phytophthora species simultaneously amplify two bands, one between 100 and 500 bp and the second at c. 700 bp. Samples should only be considered positive for P. ramorum if a single 700 bp band is amplified. As DNA concentration can affect PCR amplification it may be that only one concentration of positive test samples is amplified, this is normal and the reason why two concentrations of test DNA are tested. If neither concentration is amplified, the DNA should be tested with the universal ITS primers ITS1 and ITS4 (White et al., 1990), and their cycling conditions are described below (‘Identification by sequencing part of the ITS-region’). Amplification with these primers shows that the test DNA is of an amplifiable quality and that a true negative for P. ramorum has occurred. However, if amplification is still not produced, fresh DNA should then be extracted and retested.
Appendix 5 Identification at species level by Real-time PCR
The following two equivalent and validated methods may be used for TaqMan®-PCR identification of P. ramorum from cultures and plant material.
Primers/TaqMan®-probe: the primer sequences are:
Pram 114-FC: 5′-TCA TGG CGA GCG CTG GA-3′,
Pram 1527-190-R: 5′-AGT ATA TTC AGT ATT TAG GAA TGG GTT TAA AAA GT-3′,
and the TaqMan®-probe is:
The TaqMan®-probe is labelled at the 5′ end with the fluorescent reporter dye 6-carboxyfluorescein (FAM) and at the 3′ end with the quencher dye, 6-carboxy-tetramethyl-rhodamine (TAMRA).
Sample handling/DNA extraction/DNA isolation: DNA from samples is prepared at approximately 20–100 ng/µL as described above for the conventional PCR-method C.
Amplification and analysis: in optical quality reaction tubes/plates (Applied Biosystems) at least two replicate reactions for each test sample and control samples of known P. ramorum DNA (positive control) and water (negative control) are prepared.
The reaction mixture (25 µL) should contain: 12.5 µL of 2 X Taqman Universal master mix (Applied Biosystems); 1.5 µL 5 µm primer Pram 114-FC; 1.5 µL 5 µm primer Pram 1527-190-R; 0.5 µL 5 µm probe Pram 1527-134-T; 1.0 µL c. 20–100 ng DNA test suspension; 8.0 µL sterile molecular grade water.
Test reactions are cycled in a suitable instrument for detection of reporter fluorescence, for example an ABI Prism 7700 or 7900 Sequence Detection System (Applied Biosystems) using the following conditions: 10 min at 94°C; then 40 cycles of 15 s at 94°C and 60 s at 60°C.
Assessment of PCR: data from the TaqMan® run are analyzed as per manufacturer's instructions. Samples with cycle threshold (Ct) values less than 36 are considered as positive for P. ramorum, typically Ct values are between 25 and 35. A Ct value of 36 indicates a negative result.
Internal control primers should be used when plant material is tested directly; this is not necessary when using pure cultures. The internal primers and probe are based on sequences by Weller et al. (2000) and amplify plant DNA present in the test extracts. Their use confirms that amplifiable DNA is present in test extracts from plants which are PCR- negative for P. ramorum.
Internal control primers: the primer sequences are:
N.B. Primer COX-RW contains degenerative nucleotides indicated by the IUPAC code R, indicating that both adenine and guanine are inserted at these positions in equal amounts.
Internal control TaqMan®-probe:
COX-P 5′-AGG GCA TTC CAT CCA GCG TAA GCA-3′
The TaqMan®-probe is labelled at the 5′ end with the fluorescent reporter dye VIC (Applied Biosystems) and at the 3′ end with the quencher dye TAMRA.
Amplification and analysis: test reactions and positive/negative controls are prepared using the master mix as described below, and each sample is cycled as described above for testing cultures by TaqMan® PCR.
The reaction mixture (25 µL) should contain: 12.5 µL 2 X Taqman Universal master mix (Applied Biosystems); 1.5 µL 5 µm primer Pram 114-FC; 1.5 µL 5 µm primer Pram 1527-190-R; 0.5 µL 5 µm probe Pram 1527-134-T; 1.0 µL 5 µm primer COX-F; 1.0 µL 5 µm primer COX-RW; 0.5 µL 5 µm probe COX-P; 1.0 µL c. 20–100 ng DNA test suspension; 5.5 µL sterile molecular grade water.
Assessment of PCR: samples containing amplifiable P. ramorum DNA produce FAM fluorescence as recorded by Ct FAM values of < 40. These samples may also produce VIC fluorescence as recorded by Ct values of < 40 as should all other samples NOT containing P. ramorum DNA. VIC fluorescence indicates that the COX primer/probe set has amplified viable DNA present in the test sample. If neither FAM or COX fluorescence is recorded this indicates that the sample contains no amplifiable DNA and that sample should be re-extracted and tested again.
Primers/TaqMan®-probe: the primer sequences are:
Pram-5: 5′-TTA GCT TCG GCT GAA CAA TG-3′,
Pram-6: 5′-CAG CTA CGG TTC ACC AGT CA-3′,
and the TaqMan®-probe is:
The hybridization oligonucleotide is labeled with 6-FAM at the 5′ end (reporter) and TAMRA at the 3′ end (quencher).
Sample handling/DNA extraction/DNA isolation: DNA from samples are prepared as described in section ‘Identification at species level by conventional PCR’, Method (A).
Amplification and analysis: the reaction mixture (15 µL) should contain: 1 X Taqman Universal PCR Master Mix (Applied Biosystems part no. 4324018); 0.2 µm of each primer; 0.2 µm of probe; 5.0 µL template DNA.
Cycle test reactions should be performed in a suitable instrument, for instance ABI Prism 7700 or 7900 Sequence Detection System (Applied Biosystems) under the following conditions: 1 cycle at 95°C for 3 min; then 35 cycles at 95°C for 15 s, 60.5°C for 1 min. Store end products between 4 and −20°C.
A negative control (DNAse- and RNAse-free water) should be included in every experiment to test for contamination as well as a positive control (DNA from a reference strain of the pathogen) to monitor the performance of the PCR. If either the negative or positive control does not give the proper result, the PCR should be repeated. To monitor for false negatives resulting from inhibition of the PCR, duplicate reactions are spiked with P. ramorum DNA. If the spiked reaction gives a negative result, the PCR should be repeated with diluted DNA extract.
Assessment of PCR: samples containing amplifiable P. ramorum DNA produce FAM fluorescence as recorded by Ct FAM values of < 35.
Appendix 6 Identification at species level by sequencing part of the ITS region
The identity of P. ramorum isolates from new hosts or isolates that do not morphologically match published descriptions can be confirmed by sequencing. Only DNA from pure isolates can be tested using this method, otherwise sequences from multiple organisms may be amplified in the same reaction.
The primer sequences are
ITS 1 : 5′-TCC GTA GGT GAA CCT GCG G-3′ and
ITS 4 : 5′-TCC TCC GCT TAT TGA TAT GC-3′ (White et al., 1990).
Amplification and analysis: the reaction mixture should contain: 1.0 µL DNA suspension; 10.0 µL 10 X reaction buffer containing 15 mm MgCl2 (Applied Biosystems); 8.0 µL 10 mm dNTPs; 10 µL 5 µm of each primer ITS 1 and ITS 4; 0.5 µL AmpliTaq (Applied Biosystems) (5 U/µL), and 60.5 µL sterile molecular grade water to give a final volume of 100 µL.
Amplification is performed in thin-walled PCR tubes in a PCR thermocycler programmed as follows: 2 min at 94°C; then 30 cycles of 1 min at 94°C, 1 min at 53°C, 1.5 min at 72°C. One cycle for 10 min at 72°C must be conducted after the 30 cycles. Samples are resolved on a 1.5% agarose gel as previously described. Using this method samples containing Phytophthora DNA produce single amplicons of c. 900 bp in size.
Sequencing of amplicons: the remaining 90 µL from positive test reactions is purified using a suitable PCR purification kit such as QIAquick PCR purification kit (Qiagen, Crawley, GB, Cat. ref. 28106) following the manufacturer's instructions. Send samples for two-way sequencing with forward primer ITS1 and reverse primer ITS4. Finally, consensus sequences are compared for test samples with those on GenBank (http://www.ncbi.nlm.nih.gov).
Web Fig. 1 Oak bleeding canker.
Web Fig. 2 Canker on Quercus rubra.
Web Fig. 3 Canker on Quercus falcate.
Web Fig. 4 Oak – dead tree.
Web Fig. 5 Rhododendron shoot canker.
Web Fig. 6 Rhododendron – wilted leaves.
Web Fig. 7 Rhododendron – leaf blackening.
Web Fig. 8 Viburnum – stem base necrosis.
Web Fig. 9 Viburnum – leaf blight.
Web Fig. 10 Pieris – leaf blight.
Web Fig. 11 Syringa – bud/leaf blight.
Web Fig. 12 Kalmia – leaf blight.
Web Fig. 13 Camellia – leaf blight.
Web Fig. 14 Leucothoe – leaf blight.
Web Fig. 15 Taxus – needle dieback.
Web Fig. 16 Typical slow growing colony on a selective medium (P5ARP(H)).
Web Fig. 17 Typical coralloid mycelium on a selective medium (P5ARP(H)).
Web Fig. 18 Sporangia on a selective medium (P5ARP(H)).
Web Fig. 19 Chlamydospore on a selective medium (P5ARP(H)).
Web Fig. 20 Typical outgrowth of colony on a nonselective medium (CPA).
Web Fig. 21 Sporangia (bar = 20 µm) on a nonselective medium (CPA).
Web Fig. 22 Chlamydospore (bar = 20 µm) on a nonselective medium (CPA).
Web Fig. 23 Flow diagram for diagnosis of Phytophthora ramorum on plants and plant products.