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International Plant Protection Convention diagnosis protocol for
Phytophthora ramorum
General comments
1. Include specific information on which methods are best (with references) and why the methods
included in the protocol were selected. Include the data on specificity, sensitivity and reliability.
2. Format the molecular methods in sentences (similar to the Thrips palmi approach)
3. Remove appendix – put in the text
4. Photographs – provide examples of symptoms on trees, twigs and leaves. For other examples,
refer to the SOD web site
5. It would be useful to have indications of symptoms of pathogens which could be confused with
SOD (general statement)
6. Remove first paragraph – not in the format of ISPM 27. Include any points in the text if required
7. Describe the positive and negative controls to be used in all assays
8. Is it OK to have a positive in real time PCR as the only method? Presumably for surveillance
purposes this is sufficient. A broken line in the flow diagram
9. It would be useful to have information on methods for samples with symptoms and symptomless
detection.
10. The DP should provide the minimum requirements for a positive diagnosis and then the text can
indicate the methods that can be used in critical cases such as first findings in a country. Delete
references to “confirmation” is it is not essential. If the DP requires an isolation after a very
sensitive and reliable method like real time PCR, then you may have cases that are negative
(because isolation is more likely to have false negatives). Put in the data on reliability etc.
11. A1 and A2 mating types have not been included. They should be referred to for information.
Secretariat note
Formatting will be required once the draft is in a more advanced form.
Check references are formatted in accordance with instructions to authors
This standard describes diagnostic protocols for the detection and identification of the plant
pathogen Phytophthora ramorum. It is not intended as a review document on P. ramorum or the
diseases it causes, neither is it a comprehensive review on methods available for diagnosis of P.
ramorum. Instead this document is intended as a tool to help diagnosticians unfamiliar with P.
ramorum to diagnoses it using well-established methods that can be used by most laboratories
worldwide. This document also gives details on where further information on P. ramorum can be
found including background literature, host records and where known positive cultures and
reference DNA can be sourced as well as contact details from whom further information on P.
ramorum can be obtained. All samples positive for P. ramorum and the material they have been in
contact with should be treated according to your local arrangements for dealing with quarantine
material.
Pest information
Phytophthora ramorum (Werres et al., 2001) causes high mortality of oak trees in California, where
the disease is known as ‘sudden oak death’ (SOD). The pathogen has also been found causing
‘ramorum dieback’ and ‘ramorum leaf blight’ on a range of other plants (www.suddenoakdeath.org;
www.defra.gov.uk/planth/pramorum.htm). It is unknown from where P. ramorum originated or
when it first entered Europe or North America. It is present in some countries in Europe and North
America. Werres & Marwitz (1997) report that they saw P. ramorum like symptoms in Germany as
early as 1994 and Rizzo et al., (2002) report that (use latin name first) Tanoaks in California were
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being killed from at least 1995. Indicate that the organism has been found in nurseries and
intercepted by some countries
It would be useful to indicate the host range – to make it clear the wide host range and also perhaps
to the main hosts in international trade
Refer to data sheets and databases (EPPO/CABI) for further information.
Taxonomic information
Name: Phytophthora ramorum Werres, De Cock & Man in’t Veld
Synonym: None
Taxonomic position: Straminipila, Oomycetes, Peronosporales, Pythiaceae
Detection
Symptoms
Three distinct disease syndromes caused by P. ramorum have been described (Hansen et al. 2002):
“Sudden oak death”, “ramorum shoot dieback” and “ramorum leaf blight”. The symptoms within
each “syndrome” can vary widely depending on host, the most commonly observed host symptoms
are described and illustrated below.
Sudden oak death
Typically this is expressed as a lethal cankers around the lower trunks of infected trees, which may
also ooze dark red to black sap (‘bleeding cankers’ or tarry spots) from their bark (Fig. 1).
Removing the outer bark under and around areas of ooze often reveals dead and discoloured inner
bark with a black ‘zone line’ present around edges of necrosis. The foliage of infected trees may die
prematurely with leaves remaining on the branches after death.
Ramorum shoot dieback
On Rhododendron aerial dieback is often seen with diseased twigs having brown to black lesions
that usually begin at the tip and move towards the base (Fig. *). The cambial tissue of diseased
twig is also often discoloured. Shoots and stems may also similarly be affected with cankers near
ground level resulting in rapid wilting of shoots, causing the leaves, which remain attached, to hang
down (Fig. *). On Viburnum infection usually occurs at the stem base causing plants to wilt and
collapse very quickly (Fig. ). Brown necrosis can often be seen spreading into the stem and
twig/shoot blight and leaf spots may also occur (Fig. *). On Pieris infection tends to cause petiole
blackening leading to stem cankers and vice versa leading to aerial dieback.
Ramorum leaf blight
On Rhododendron, Camellia, Kalmia and Pieris brown spots occur on leaves, usually at their tip.
Disease develops across infected leaves into their petioles often following the midrib eventually
leading to premature leaf fall.
Sampling procedures
Different techniques as described below are recommended depending on the material being tested.
When possible samples should be kept cool and sent to the diagnostic laboratory in strong plastic
bags or containers for next day isolation as prolonged transit times or raised temperatures can
reduce the likelihood of isolation. Placing a small amount of damp tissue with plant material can
reduce sample desiccation and increase the chance of isolation. Storage at 4C may also prolong
sample life.
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Plant material
When sampling bleeding cankers from trees the outer bark around the canker should be removed to
reveal the inner bark and the margin of necrosis. Excise pieces of phloem and xylem from across the
margin and placed this in a sealed container for subsequent isolation. For shoots and twigs a sample
approximately 15 cm long spanning the leading edge of an infection should be taken, while for
leaves place 4-6 leaves showing a range of symptoms. Do not specify the number of leaves –
provide information as examples.
Soil
About 500 g of soil and or plant debris should be taken from test sites.
Water
Water samples should be of at least 1L in volume. Samples should be skimmed from collection
surfaces [what does this mean? Take the sample from the upper layer of water] and include any
sediment or floating debris present. Various workers have used “baits” placed in water systems to
detect P. ramorum as described below.
Preliminary detection method; serological methods
If required serological methods may be used to pre-screen samples for the presence of Phytophthora
spp. but false negatives and positives may occur (Kox et al., 2007). Different formats are available
including lateral flow devices available from Forsite diagnostics, York, UK which are suitable for
field use and larger format assays available from Neogen, Lexington, USA which are more suitable
for laboratory use.
Isolation
Plant samples
Samples should be surface sterilised to eliminate diagnosis of cross-contamination between samples
and to confirm internal infection. For plant material wash off large amounts of any soil with water
then excise at least three 1 cm2 pieces from the leading edge of infection from each sample and dip
these in aqueous solution of thin bleach (1 % active sodium hypochlorite) for 2-5 min. Finally rinse
off the bleach with sterile water.
Water samples
In the laboratory water samples should be baited with health rhododendron leaf according to
Themann et al., 2002. In summary place the test water in a container (plastic box) with a large
surface area. Onto the water surface place at least three 1 cm2 pieces of leaf material from healthy
rhododendron leaf which have not been treated with fungicide; preferably Rhododendron hybrid cv
Cunningham’s white which is known to be highly susceptible to P. ramorum. Seal the box and
incubate on the laboratory bench (20-25 C). Within 3 to 7 days symptoms of P. ramorum infection
will develop and these should be cut out for pathogen isolation or direct testing, for example by
PCR. Baiting such as using pear fruit () or rhododendron in muslin bags containing polystyrene to
aid flotation (Beales, 2007) can also be used in field situations to check water sources including
streams and irrigation ponds.
Soil
The Rhododendron leaf test (see above) should be used with about a hand full of test soil being is
placed in a large plastic box and covered with about 500 mL of Petri’s mineral solution (Appendix
*). Again after 3-7 days incubation at what temp?, the presence of P. ramorum is checked as
described for the water samples.
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Media and incubation
Sometimes, presence of P. ramorum can be seen directly on the collected material (i.e. presence of
typical sporangia). If not, material can be incubated for 3-5 days in a closed box with a small piece
of damp tissue on the bottom to promote sporulation.
For isolation, P5ARP [H] media is recommended (Appendix 1) as this is semi-selective for
Phytophthora spp. and on it characteristic features of P. ramorum are readily observed. Test
material should be treated as described above then at least four small pieces (about 2 mm2) of tissue
from each sample should be aseptically transferred onto the P5ARP [H] media; incubate plates on
the laboratory bench (20-25 C) for up to 10 days by which time P. ramorum present should grow
out. For extended culturing isolates should be transferred to carrot piece agar (CPA, Appendix).
Molecular techniques can be used as a detection method (as well as an identification method), so
they should be included here.
Identification
P. ramorum may be identified at species level by either its growth characteristics in culture and
morphology or by appropriate molecular methods. A flow diagram indicating equivalence or
combinations of methods is given in Figure **.
Growth characteristics in culture and morphology
The growth characteristics on agar and morphological features are described in Werres et al. (2001).
The most essential features on a selective and a non-selective medium are given in Table 1.
Generally, the unique morphological features described make P. ramorum a relatively easy
organism to identify in culture. Possible confusion in morphology/cultural characteristics is most
likely to occur with P. palmivora. The key characteristics are illustrated in Figs. *.
Molecular biological methods
Molecular methods have been developed to identify P. ramorum from culture plates as well as
direct in planta using either conventional or real-time PCR, many of these methods are compared by
Kox et al., 2007 and Martin et al., 200*. In general, the conventional PCR primers are quite
specific for P. ramorum with a small number of cross-reactions and the real-time assays show no
cross-reaction. Each assay relies on amplification of P. ramorum DNA from sample material,
conventionally this is extracted from test material using commercially kits or following published
methods. Test samples should be surface sterilised to reduce the chance of sample cross
contamination following one of the methods described above.
Experimental controls
The ability of a method to extract DNA from sample material should always be confirmed by the
use of control exactions, the use of internal amplification controls or the parallel amplification of
extracts with universal primers such as described by White et al., 1990. With all molecular methods
appropriate controls should be used. Ideally these include extraction controls including known
positive and negation material. For PCR a negative control spiked with molecular grade water
(MGW) should be included in every experiment to test for contamination as well as a positive
control containing P. ramorum DNA to monitor the performance of the PCR.
Pre extraction preparation
Samples should be broken-up to help with DNA extraction, several methods can be used depending
on the material being tested these include place samples in thick walled plastic bags with a small
amount of MGW or DNA extraction buffer if this is supplied with the commercial DNA extraction
kit to be used. Rolling the bag with a wallpaper seam roller or similar device to crush the material.
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First freezing the material in liquid nitrogen can also help the crushing process. Alternatively,
samples may be ground in mechanical mills, for example cut samples 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) and either 1mL MGW or extraction buffer as outlined above.
The tube is closed with a screw-fitting lid containing an o-ring and the tube is oscillated in a MiniBeadbeater (Biospec products) at full power for at least 20 s.
DNA extraction
Different commercial kits have been used to extract P. ramorum DNA from plant tissue and
cultures including NucleoSpin plant extraction kit (Macherey-Nagel, Düren, Germany) others are
also available. More traditional method such as described in Hughes et al. (2000) may also be used.
Further purification using polyvinylpolypyrrolidone (PVPP) columns (Cullin et al., 199*) following
DNA extraction may also be beneficial. 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 MWG is applied to the PVPP column which is centrifuged for 5 min at 4000
rpm 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 rpm. Approximately 20-100 ng/L
DNA should be yielded from each sample and this should be stored at 4C for immediate use or at 20C if testing is not to be performed on the same day.
Identification by conventional PCR
Two similar primer sets are in common usage, these have similar activity (Hayden et al., 2004 &
Lane et al., 2003). Here only the assay by Hayden et al., is described.
Primers:
Phyto 1: 5’CAT GGC GAG CGC TTG A3’
Phyto 4: 5’GAA GCC GCC AAC ACA AG3’.
Reaction mix (25L): 5 L DNA sample, 2.5 L 10x reaction buffer inc. 15 mM MgCl2 (Qiagen);
0.5 L 10 mM dNTPs, 0.1 L 100 M of each primer, 0.1 L HotStarTaq (5
U/µl; Qiagen), and 16.7 µL molecular grade water (MWG)
Cycling 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
completed with one cycle of 72°C for 10 min.
After amplification, 10 µL from the cycled reactions is mixed with 2 µL of loading dye (25µg
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.
Visualisation of conventional PCR products and interpretation of results
Following electrophoresis, stain the gel for 30 min with ethidium bromide [0.5µg/mL] then wash
off excess stain and view the gel on a UV transilluminator. No bands should be produced for the
negative controls while the positive control and positive samples should produce a single band of c.
680 bp. As DNA concentration can affect PCR amplification it maybe that highly concentrated
DNA preparations need first diluting ten-fold before adding to PCR reaction mixes before
amplification occurs. If amplification is expected but still does not occur then the DNA should be
tested with the universal ITS primers ITS1 and ITS4, the use of which are described below for
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sequencing (‘Identification by sequencing part of the ITS-region’). If amplification is still not
produced, fresh DNA should be extracted and retested.
Identification by real-time PCR
As for conventional PCR several real-tme PCR assays are in usage for diagnosis of P. ramorum
(Hayden et al., 2004, Martin et al. 2007). Here only the assay by Hughes et al. 2007 is described.
Explain why this assay was chosen – include information on specificity sensitivity and reliability so
that the reader can understand the usefulness and limitations of the test.
Primers:
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’
Probe:
Pram 1527-134-T: 5’ TTC GGG TCT GAG CTA GTA G 3’. The
TaqMan-probe is labelled at the 5’ end with the fluorescent reporter dye 6carboxyfluorescein (FAM) and at the 3’ end with the quencher dye, 6carboxy-tetramethyl-rhodamine (TAMRA).
Reaction mix (25L): 1.0 L DNA sample; 12.5 L of 2 X Taqman Universal master mix
(Applied Biosystems); 1.5 L 5 M of each primer; 0.5 L 5 M probe; 8.0
L MGW.
Cycling conditions:
Cycle reactions in a suitable instrument for detection of reporter
fluorescence, for example an ABI Prism 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.
Interpretation of results
Data from real-time PCR runs should be analyzed as per manufacturers instructions. Samples with
cycle threshold (Ct) values less than 36 are considered as positive for P. ramorum, typically values
are between 25-36. Ct values between 36-40 are rare and usually result from aerosol contamination
these should be retested and the presence of P. ramorum confirmed by another means if the result is
still in doubt. Samples with Ct values of 40 are considered negative.
Identification by real-time PCR including internal control
To aid confidence in test results an internal control assay can be used. The assay described here
uses internal control primers and probe based on a plant sequences described Weller et al. (2000).
This assay will only therefore work as a control assay when DNA is extracted from plant material.
Primers:
COX-F 5’ CGT CGC ATT CCA GAT TAT CCA 3’,
COX-RW 5’ CAA CTA CGG ATA TAT AAG RRC CRR AAC TG 3’
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.
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.
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Reaction mix (25L): 1.0 L DNA sample; 12.5 L of 2 X Taqman Universal master mix
(Applied Biosystems); 1.5 L 5 M of each primers Pram 114-FC & Pram
1527-190-R; 0.5 L 5 M probe Pram 1527-134-T; 1.0 L 5 M of each
primer COX-F & COX-RW; 0.5 L 5 M probe COX-P and 5.5 L MWG.
Cycling conditions:
Cycle reactions as described for the real-time assay above.
Assessment of real-time PCR results:
Samples containing amplifiable P. ramorum DNA produce FAM fluorescence as recorded by Ct
FAM values of < 36. These samples may also produce VIC fluorescence as recorded by Ct values of
< 36 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. If no FAM fluorescence (< Ct 36) is recorded and
VIC florescence is between Ct 36-40 then the sample consideration should also be ma Please
complete this sentence!!
If COX PCR RFLP is a valid method for identification of isolates (or direct testing of plant
material), then it should be included here with the circumstances that it can be used for, otherwise,
delete reference to it in the Figure.
Identification by ITS sequencing
The identity of P. ramorum isolates can be confirmed by sequencing. Several gene regions from
different genes have been used including ITS 1&2 (Hughes et al. 2007), COX (Martin et al., 2007)
and the Ypt1 gene of the Ras gene (Schena et al.,2006). Only DNA from pure isolates can be tested
using this method, otherwise sequences from multiple organisms may be amplified in the same
reaction. The method for sequencing the ITS 1 &2 regions of the nrRNA gene is described below.
Primers:
ITS 1: 5’ TCC GTA GGT GAA CCT GCG G 3’
ITS 4: 5’ TCC TCC GCT TAT TGA TAT GC 3’.
Reaction mix (100L): 1.0 L DNA sample; 10.0 L 10 X reaction buffer containing 15 mM
MgCl2 (Applied Biosystems); 8.0 L 10 mM dNTPs; 10 L5 M of each
primer; 0.5 L AmpliTaq (Applied Biosystems) (5 U/L), and 60.5 µL
MGW.
Cycling conditions:
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 then one cycle for 10 min at 72°C.
Assessment of results:
Samples (10 L) should be run on a gel as described above with DNA from Phytophthora spp.
producing single amplicons of c. 900 bp in size. The remaining 90 µL from positive test reactions
should be purified using a suitable PCR purification kit such as QIAquick PCR purification kit
(Qiagen, Crawley, GB, Cat. ref. 28106) following the manufacturers instructions. The purified
amplicon should then be two-way sequenced with forward primer ITS1 and reverse primer ITS4.
Finally, consensus sequences should be compared for test samples with those on GenBank
(www.ncbi.nlm.nih.gov ).
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Positive identification
For a positive identification the fungus should be identified unambiguously using one or preferably
more than one of the methods described above i.e.
1) Morphological examination: determination of the presence of semi-papillate, caducous
sporangia with short or no stalk, and large chlamydospores (up to 80 μm), hyaline to pale
brown;
2) Conventional PCR yielding an amplicon of 680 bp, and the positive but not the negative
controls should have produced an amplicon of this size.
3) Real-time PCR with Ct-values less than 36 and positive and negative controls reacting as
expected
4) ITS-PCR sequencing.
A flow-diagram describing the appropriate tests necessary for a positive diagnosis is given in Fig.
**.
Section on reporting findings keeping positive samples ?
Reference cultures
Type strain of P. ramorum: BBA 9/95 (A1) = CBS 101553. Available from BBA-Braunschweig,
DE (Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection
in Horticulture, Messeweg 11/12, D- 38104, Braunschweig, DE)), or from CBS, Utrecht, the
Netherlands.
Contact points for further information
Further information can be obtained from :
Kelvin Hughes, Mycology, Central Science Laboratory, YO41 1LZ York, England, GB (fax: 44
1904 462147, el: 44 1904 462000, e-mail: [email protected])
Mary Palm, PPQ Molecular Diagnostic Lab, B-580, BARC-East
Powder Mill Rd., Beltsville MD 20705, USA (fax 301-504-6124, tel. 301-504-7154, email
[email protected]
Stéphan Brière, Phytopathology Laboratory, Canadian Food Inspection Agency, 3851 Fallowfield
Road | 3851, Fallowfield, Ottawa, Canada ON K2H-8P9 (fax (613) 228-6676, tel. (613) 228-6698
ext.5911 email [email protected].
Acknowledgements
This protocol was written by K.J.D. Hughes, Central Science Laboratory, York (GB), Mary Palm,
APHIS PPQ Molecular Diagnostic Lab, Beltsville USA and Stéphan Brière, Canadian Food
Inspection Agency, Ottawa, Canada. It was adapted from the EPPO diagnostic protocol on P.
ramorum which originally drafted by G.C.M. van Leeuwen, Plant Protection Service, Wageningen
(NL); C.R. Lane and K.J.D. Hughes, Central Science Laboratory, York (GB); S. Werres and S.
Wagner, Federal Biological Research Centre for Agriculture and Forestry, Braunschweig (DE).
References
Beales (2007) PH0182 Water baiting ***
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Hansen E, Sutton W, Parke J & Linderman R (2002) Phytophthora ramorum and Oregon forest
trees- One pathogen, three diseases. Abstract in proceedings of “Sudden Oak Death, a Science
Symposium”, December 2002, Monterey, CA (US) pp. 78.
Hayden KJ, Rizzo D, Tse J & Garbelotto M (2004) Detection and quantification of Phytophthora
ramorum from California forests using a real-time polymerase chain reaction assay.
Phytopathology 94, 1075-1083.
Hughes KJD, Inman AJ & Cooke DEL (2000) Comparative testing of nested PCR-based methods
with bait-plant tests for detecting Phytophthora fragariae var. fragariae in infected strawberry
roots from fruit crops in the UK. Bulletin OEPP/EPPO Bulletin 30, 533-538.
Hughes KJD, Griffin RL, Tomlinson JA, Boonham N, Inman AJ & Lane CR (2005) Development
of a One-Step Real-Time PCR Assay for Diagnosis of Phytophthora ramorum. Phytopathology
2007.
Kox L.F.F., van Brouwershaven I.R., Van de Vossenberg B.T.L.H., van den Beld H. E., Bonants
P.J.M. and de Gruyter J. (2007). Diagnostic values and unity of immunological, morphological
and molecular methods for in planta detection of Phytophthora ramorum. Phytopathology
97:1119-1129.
Lane CR, Barnes AV, Beales PA, Griffin RL, Hughes KJD, Inman AJ, Townend, VC, Brasier CM
&Webber JF (2003b) First report of Phytophthora ramorum in the UK. ICPP Paper 19.35, 8th
International Congress of Plant Pathology, Christchurch, New Zealand, 2-7th February 2003.
ISBN 086476 151-1.
Rizzo DM, Garbelotto M, Davidson, JM, Slaughter, GW & Koike ST (2002) Phytophthora
ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in
California. Plant Disease 86, 205-214.
Themann K, Werres S, Diener H-A & Lüttmann, R (2002) Comparison of different methods to
detect Phytophthora spp. in recycling water from nurseries. Journal for Plant Pathology, 84, 4150.
Weller SA, Elphinstone JG, Smith NC, Boonham N & Stead DE (2000). Detection of Ralstonia
solanacearum strains with a quantitative, multiplex, real-time flurogenic PCR (TaqMan) assay.
Applied and Environmental Microbiology 66, 2853-2858.
Werres S & Marwitz R (1997). Unbekannte Phytophthora. Deutscher Gartenbau 21, 1166-1168.
Werres S, Marwitz R, Man in ‘t Veld WA, de Cock AWAM, Bonants PJM, De Weerdt M, Themann
K, Ilieva E & Baayen RP (2001) Phytophthora ramorum sp. nov., a new pathogen on
Rhododendron and Viburnum. Mycological Research 105, 1155-1165.
White TJ, Bruns T, Lee S. & Taylor J (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In : PCR Protocols, a guide to methods and
applications. Eds. Innis M.A., Gelfand D.H., Sninsky J.J. & White T.J. Academic Press, London
(GB). pp 315-322.
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Appendix I
Appendix II
Media for Phytophthora ramorum
P5ARP[H] (Jeffers & Martin, 1986): cornmeal agar 17 g; distilled water 1000 mL. Autoclave, then
cool to 50oC 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.
Carrot Piece Agar (Werres et al., 2001): agar 22 g, carrot pieces 50 g, distilled water 1000 mL.
Table 1 Growth characteristics on a selective and non-selective medium
Character
Colony
P5ARP(H) *
relatively slow growing,
approximately 2 mm per day
Mycelium
Carrot piece agar *
weak rosette-like pattern,
pronounced concentric rings,
growth rate approximately 3 mm
per day
aerial mycelium sparse, no hyphal
swellings
weakly coralloid, growing
within the agar with little
superficial growth, no hyphal
swellings
Sporangia
produced abundantly on the agar surface, semi-papillate, caducous
with short or no stalk. Size: 20-32 x 40-80 µm, average 24 x 52 µm;
average length/width ratio 2.16
ellipsoid, frequently in small
ellipsoid, spindle-shaped or
clusters and relatively narrow,
elongated-ovoid, single or in
initial sporangium commonly
clusters
producing secondary, smaller
sporangia
Chlamydospores more common in older colonies
after 3 days incubation in the
(7-10 days), very large (up to 80
dark, in the older parts but very
μm diameter), hyaline to pale
often also in the young parts of
brown to brown
the colony, thin-walled, hyaline
to pale brown up to 88 µm
Sexual
can be observed on carrot piece agar after pairing with the opposite
structures
mating type, e.g. that of P. cryptogea (Werres and Zielke, 2003).
P. ramorum x P. ramorum pairing is also possible in vitro (not with
all isolates) (Brasier, pers. comm.) and in Rhododendron twigs
(Werres and Zielke, 2003)
* On P5ARP(H) characters can be observed after 4-6 days incubation on the bench at 20C, 12h
light/12h dark. On carrot piece agar this is after 3-5 days incubation at 20°C in darkness.
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2007-TPDP-21 REV1
Modified by the TPDP and with IPPC secretariat comments
Agenda: 7
Suggested web Figures ( Which could be used).
Web Fig. 1 Oak bleeding canker
Web Fig. 2 Canker on Quercus rubra
Web Fig. 3 Canker on Quercus falcata
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 non-selective medium (CPA)
Web Fig. 21 Sporangia (bar = 20 µm) on a non-selective medium (CPA)
Web Fig. 22 Chlamydospore (bar = 20 µm) on a non-selective medium (CPA)
Fig. 23 Flow diagram for diagnosis of Phytophthora ramorum on plants and plant products
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