Draft Annex to ISPM 27: Phytophthora ramorum 2004-013 (3.3)
DRAFT ANNEX TO ISPM27: Phytophthora ramorum (2004-013)
Status boxThis is not an official part of the standard and it will be modified by the IPPC Secretariat after adoption
Date of this document / 2015-05-26
Document category / Draft new annex to ISPM27 (Diagnostic protocols for regulated pests)
Current document stage / To TPDPJune 2015
Origin / Work programme topic: Fungi and fungus-like organisms, CPM-1 (2006)
Original subject: Phytophthora ramorum
Major stages / 2004-11: SC added original subject: Phytophthora ramorum (2004-013)
2015-03 Expert Consultation
Discipline leads history / Hans de Gruyter (NL, Discipline Lead)
Robert TAYLOR (NZ, Referee)
Consultation on technical level / The first draft of this diagnostic protocol was prepared by:
× K.J.D. Hughes, (Fera, York UK)
× M. E. Palm, (APHIS PPQ Molecular Diagnostic Lab, Beltsville USA)
× S. C. Brière, (Canadian Food Inspection Agency, Ottawa, CAN)
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, (Fera, York UK); S. Werres and S. Wagner, (Federal Biological Research Centre for Agriculture and Forestry, Braunschweig DE). A. Schlenzig, (Science and Advise for Scottish Agriculture, Edinburgh (UK) reviewed the protocol.
This current draft is being updated by Patricia Giltrap and Jennifer Tomlinson (Fera, York, UK), S. C. Briere and Gloria Abad (USDA-APHIS-PPQ-Center of Plant Health Science & Technology, Beltsville, USA). Lynn Laurenson (Fera, York, UK) has assisted with reviewing of molecular comments
Main discussion points during development of the diagnostic protocol
[to be updated throughout DP development] / (Note: Especially after experts have been consulted at early stages of development, the cover note should indicate substantial comments that were not incorporated in the draft. Include as bullet points)
Notes / This is a draft document.
Content [to be added later]
Adoption
[1] This diagnostic protocol was adopted by the Commission on Phytosanitary Measures in 20--.
[2] The annex is a prescriptive part of ISPM 27.
1. Pest Information
[3] Phytophthora ramorum Werres, de Cock & Man in ’t Veld (Werres et al., 2001) is an Oomycete pathogen considered to have been introduced into western North America and western Europe in the late twentieth century by the ornamental plant trade (Goss et al., 2011; Grunwald et al., 2012; Mascheretti et al., 2008; Prospero et al., 2007; Van Poucke et al., 2012). Phytophthora ramorum attacks a wide range of trees and shrubs, causing leaf blights, stem cankers, bleeding stem lesions, dieback in nurseries and in the field. In North America the pathogen was found early in the 1990s causing mortality of oak trees and tanoaks mainly in California and Oregon (Rizzo et al 2002), where the disease, known as ‘sudden oak death’ (SOD), has reached epidemic proportions. The pathogen has been also observed since 1993 causing twig blight of rhododendron in nurseries and mature bushes in gardens in Germany and The Netherlands and since 1998 on diseased Viburnum sp. (Werres et al., 2001, Werres & Marwitz (1997)). Rizzo et al. (2002) reported that Lithocarpus densiflorus (tanoaks) in California were being killed since at least 1995. More recent findings including lists of the known host for P. ramorum can be found on the following (Cabi 2014, COMTF 2014, Fera 2014a, Fera 2014b, USDA 2014). Disease symptoms and host plants are listed and regularly updated on several websites (COMTF 2014, Fera 2014a).
[4] In the USA the pathogen was originally considered a woodland disease but since 2003 nursery plants in several US states have been affected. The disease has also been found in Canada. In Europe, the pathogen has been recorded in over 20 European countries, predominantly on ornamental plants in nurseries and a few managed gardens. In 2009, however, P. ramorum was unexpectedly found infecting and killing large numbers of Larix kaempferi (Japanese larch) trees in south-west England. Heavy dieback and mortality of plantation Larix kaempferi trees in western Britain and Northern Ireland have resulted in the felling of millions of trees (Brasier & Webber 2010; Webber et al., 2010). This emphasising that although many hosts are known, the main threat of P. ramorum is to tree species and other ecologically important plants such as heathland species.
[5] The origin of P. ramorum is unknown although it was speculated this may be in the Yunnan province of China or Taiwan (Brasier et al. 2004a).
[6] Although many hosts are known for P. ramorum, findings are most commonly observed on Camellia, Magnolia, Pieris, Quercus spp (i.e. Q. acuta, Q. agrifolia, Q. cerris Q. chrysolepis, Q. ilex, Q.rubra (‘red oak’ species in particular), Rhododendron and Viburnum.
[7] Phytophthora ramorum has a complex life-cycle and is adapted to cool temperatures with 20 ºC being optimal. Although P. ramorum is soil-borne, deciduous, asexually produced sporangia are formed on the surface of infected leaves or twigs and depending upon environmental conditions, are locally splash-dispersed or spread over long distances by wind and wind-driven rain (Davidson et al., 2005). Sporangia which land on suitable hosts germinate to produce hyphae or in the presence of water will release motile zoospores which encyst on the host surface, germinate and penetrate the host tissue forming a colony from which more sporangia are produced. These sporangia repeat the cycle and if this occurs enough times, in the right environmental conditions, can lead to an epidemic. Different asexual spores, chlamydospores, are produced in abundance within infected plant tissue and allow P. ramorum to survive adverse conditions in infected stems and leaves on the plant, in plant debris on the soil surface or in the soil (Grünwald et. al., 2012).
[8] Phytophthora ramorum may produce sexual oospores but this requires both mating types to be together (heterothallic). No evidence exists that natural crossing of these mating types has occurred in nature although this has been achieved in the laboratory (Brasier & Kirk, 2004b). Currently, mating type A1 is predominantly found in Europe while type A2 is the predominant type observed in North America (Werres & Kaminski, 2005). There are four clonal lineages known to date, designated as: NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (mating type A2; North America; nurseries), and EU1 (mating type predominantly A1, rarely A2; Europe and North America; nurseries and gardens) (Grünwald et al., 2009). A new lineage EU2 has been discovered recently in Northern Ireland and western Scotland associated to four different host plants, including Larix kaempferi (Van Poucke et al., 2012).
2. Taxonomic Information
[9] Name: Phytophthora ramorum Werres, de Cock & Man in ’t Veld, 2001.
[10] Synonyms: None
[11] Taxonomic position: Chromista, Oomycota, Oomycetes, Pythiales, Pythiaceae
[12] Common name: Sudden oak death (SOD), ’ramorum leaf blight’ ‘ramorum shoot dieback’ and ‘sudden larch death’.
[13] Reference: Mycobank MB474485
3. Detection
[14] Laboratory studies have shown that the time between foliage infection and visible disease expression is typically between 3 and 14 days depending on host and temperature. However, this may be longer in the field and on different plant parts (DEFRA, 2007). There is still the possibility though that symptomless material could carry infection surface contamination. If this is a concern then suspect host material should be grown under quarantine conditions for at least two weeks to see if any symptoms for P. ramorum develop. Leaves selected at random can also be checked for latent infection by baiting or molecular methods as described below. The use of fungicides can make it more difficult to detect infested plant material by culture and PCR. They may suppress symptom development as well as viability of the pathogen, that may lead to false negative test results.
[15] This standard describes well established methods for the detection and identification of P. ramorum. It is not a comprehensive review of all methods available for diagnosis of P. ramorum. Where available, comments on the specificity, sensitivity and reliability of each method is given. 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.
[16] In this diagnostic protocol, methods (including reference to brand names) are described as published, as these defined the original level of sensitivity, specificity and/or reproducibility achieved. The use of names of reagents chemicals or equipment in these diagnostic protocols implies no approval of them to the exclusion of others that may also be suitable. Laboratory procedures presented in the protocols may be adjusted to the standards of individual laboratories, provided that they are adequately validated.
3.1 Symptoms
[17] Several disease syndromes caused by P. ramorum have been described. The symptoms within each “syndrome” can vary widely depending on the host. The most commonly observed host symptoms are described below and illustrated (Figs 2-6), additional disease symptoms can be found on several websites (COMTF 2014, EPPO, 2014, Fera 2014c, USDA 2009).
[18] Sudden oak death
[19] Despite this name, which is most common, the following symptoms can be observed on many different tree species and can take several years to completely kill mature trees. Typically, symptoms include lethal cankers around the lower trunks of infected trees from which dark red to black sap may ooze (‘bleeding cankers’ or tarry spots) (Fig. 2). Removing the outer bark under and around areas of ooze often reveals dead and discoloured inner bark with a black ‘zone line’ around edges of necrosis. The foliage of infected trees may die prematurely with leaves remaining on the branches after death. Trees that show these symptoms may suddenly completely die off. It should be noted that these symptoms are not restricted to an infection caused by P. ramorum, but may also be hastened by other plant pathogens (including other Phytophthora spp.), or associated with non-pathogenic disorders and/or insect pests.
[20] Ramorum shoot dieback
[21] On Rhododendron species diseased twigs often have brown to black lesions that usually begin at the tip and move towards the base (Fig. 3). Lesions mid stem can also be found. The cambial tissue of diseased twigs is often discoloured. Shoots and stems may have cankers near ground level resulting in rapid wilting of shoots, causing the leaves, which remain attached, to hang down (Fig. 4). Infection on Viburnum spp. usually occurs at the base of the stem causing plants to wilt and collapse very quickly (Fig. 5). Brown necrosis often can be seen spreading into stems and twigs and leaf spots may also be observed (Fig. 6). Infections on Pieris spp. tend to cause petiole-blackening leading to stem cankers and aerial dieback.
[22] Ramorum leaf blight
[23] On Rhododendron, Camellia, Kalmia and Pieris species black/brown lesions occur on leaves, usually at the tip but often at the petiole end. Disease develops across infected leaves often following the midrib eventually leading to premature leaf fall. On Magnolia, multiple small spots can also be observed eventually merging into larger necrotic areas.
[24] Symptoms on conifers and Sudden larch death
[25] On conifers the pathogen causes needle blight and dieback of young shoots of Pseudotsuga menziesii, Douglas fir, Sequoia sempervirens (coastal redwood) and Abies grandis (grand fir). Typical symptoms observed on Larch are needle infections, shoot dieback as well as branch and trunk cankers. Infected shoot tips wither and wilt and infected needles appear blackened. Early needle abscission of infected needles also occurs.
3.2 Sampling and sample preparation
[26] Different techniques as described below are recommended depending on the material being tested. Samples should be kept cool and sent to the diagnostic laboratory in strong closed plastic bags or containers or, double bagged for next day isolation as prolonged transit times or raised temperatures can reduce the likelihood of successful isolation and detection. Placing a small amount of damp tissue with the plant material will reduce sample desiccation and increase the chance of isolation. However others recommend that damp tissue not be placed into sealed self closing plastic bags where excessive moisture can hasten tissue degradation and saprophytic activity. Storage at 4 °C is highly recommended to prolong sample life but if storage is for longer than 7 days, this reduces ease of isolation.
[27] Plant material
[28] 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. Pieces of phloem and xylem can then be excised from across the leading edge and sent for testing. Symptomatic shoots and twig samples approximately 15 cm long, spanning the leading edge of an infection should be taken, while for leaves, several should be taken showing a range of typical symptoms.
[29] Water
[30] Water samples should be at least 1L in volume and from the surface of the area under testing. The water samples should be kept cold during storage and transport and tested within 48 hours of collection. Water baits are an alternative, very effective method of on-site test for water (DEFRA 2007, USDA-APHIS 2014b). The use of Rhododendron 'Catawbiense Grandiflorum', R. ‘Cunningham's White’ or R. ponticum cut leaves in muslin bags (bait bags) containing polystyrene to aid flotation has been used extensively in field situations to check water sources including streams and irrigation ponds (DEFRA, 2007). Bait bags should be placed preferably where the water is flowing, however slow, as opposed to where the water is still. Bait bags can be used at temperatures of 4° C and above (DEFRA, 2007).
[31] Soil and plant debris
[32] About 500g of soil and or plant debris should be taken from test sites. This should be placed in a sealed container or bag. Alternatively, cut rhododendron leaves in bait bags (without the polystyrene) can be buried in the soil/plant debris provided this is going to remain moist.
3.3. Preliminary detection method/ serological methods
[33] Serological methods may be used only to pre-screen samples for the presence of Phytophthora spp. but a low level of false negatives and false positives may occur (Kox et al., 2007). Different formats are available including lateral flow devices available from Forsite diagnostics, York, UK[1] and the Immunostrip® from Agdia[2] both suitable for field use primarily to screen out negative samples. Larger format ELISA assays are also available from Neogen[3], Lexington[4], or Agdia[5], Elkhart, USA which are more suitable for laboratory use.