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- Materials and methods
Pathogenicity tests were carried out on the bark of Alnus glutinosa with 19 isolates of the standard (near-tetraploid) hybrid alder phytophthora, nine isolates representing its known heteroploid variants and 11 isolates of P. cambivora, a probable parent species of the hybrid. Over a 4-year period, 12 experiments were conducted on living alder logs incubated at 20°C. Most isolates of the standard hybrid and those of the ‘Dutch variant’ were highly aggressive to alder bark. Isolates of the ‘Swedish’, ‘UK’ and ‘German variants’, and of P. cambivora, were only weakly pathogenic. Also, isolates of P. fragariae, P. cinnamomi, P. sp. ‘O-group’, P. cryptogea, P. megasperma, P. gonapodyides and P. citricola were either weakly or nonpathogenic. Rates of lesion development were greatest on logs cut during July–October, slower on logs cut between November and March and zero on logs cut during April, indicating a strong seasonal effect. Other evidence indicated that lesion development was subject to critical thresholds of host resistance. The standard hybrid was nonpathogenic to the bark of four other hardwood and two conifer species, indicating that it is relatively host specific. In contrast, P. cambivora was an aggressive pathogen on live bark of Quercus and Castanea. The significance of these results is discussed.
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- Materials and methods
During 1993–95, an unusual Phytophthora, morphologically similar to P. cambivora, was isolated from dead and dying alder (Alnus) in Britain. On the basis of its self-fertility and developmental instability, including a high level of zygotic abortion, it was postulated that this Phytophthora might be a species hybrid involving P. cambivora (Brasier et al., 1995). A study of chromosome numbers, nuclear behaviour and the sequence of the internal transcribed spacer (ITS) region of the rDNA was carried out on similar Phytophthora isolates from Britain and continental Europe. This confirmed that the common, ‘standard’ form of the alder phytophthora is a near-tetraploid species hybrid, and that it is probably of recent origin, with P. cambivora and a Phytophthora closely related to P. fragariae as parents (Brasier et al., 1999). P. cambivora is a common pathogen of Castanea, Fagus and other hardwoods in Europe (Peace, 1962), whilst P. fragariae is a major pathogen of strawberry and raspberry (Wilcox et al., 1993).
The standard alder phytophthora type is widely distributed across Britain, France, Germany and Austria. In addition, a range of unstable variant types, showing unique combinations of morphological and behavioural characters, chromosome numbers intermediate between diploid and tetraploid, different ITS profiles and variable amplified fragment length polymorphism (AFLP) of genomic DNA, were identified amongst isolates from the UK, The Netherlands, Germany and Sweden. These natural variants may be segregants from the standard alder phytophthora resulting from chromosome loss and homogenization of the ITS arrays. Even standard isolates are probably still evolving, since their ITS arrays show evidence of continuing recombination (Brasier et al., 1999).
These newly recognized phytophthoras can cause a rapid necrosis of the inner bark of the collar and stems and roots of alder trees (Brasier et al., 1995), the lesions sometimes reaching 2 m above soil level. Since April 1995, representative standard and variant alder phytophthora isolates and a wide range of P. cambivora isolates have been tested, at different times, for their comparative ability to cause lesions on living alder logs. Also tested have been isolates of P. fragariae var. rubi;P. cinnamomi, a common tree pathogen in Europe related to P. cambivora and P. fragariae (Cooke & Duncan, 1997); P. gonapodyides, Phytophthora sp. ‘O-group’ (Brasier et al., 1993a), P. citricola and P. megasperma. These last four Phytophthora morphospecies are frequently found in ponds, rivers or wet soil on riverbanks in Europe, and therefore often inhabit the same riparian ecosystems as the alder phytophthoras. More limited pathogenicity tests were also carried out on logs of other tree species. In all, 12 pathogenicity experiments were carried out over 4 years, spread over different times of the year. The results of these experiments are reported here.
Materials and methods
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- Materials and methods
The origins of the Phytophthora isolates studied are given in Table 1. For experimental work, isolates were maintained on carrot agar (CA) (Brasier, 1969) at 20°C and subcultured at 3-week intervals. For long-term storage, cultures were grown for 4 days on oatmeal agar slopes in universal bottles and submerged in sterile paraffin oil. Synthetic mucor agar (SMA) + rifamycin selective medium comprised SMA agar (Elliott et al., 1966) containing a 4% solution of methyl benzimidazol-2-ylcarbamate (MBC) (4 g MBC heated in 47·2 mL H2O + 2·8 mL HCl, then made up to 100 mL with H2O), 0·1 g L−1 pimaricin powder and 3·0 mL L−1 of a 1% w/v aqueous solution of rifamycin.
Table 1. Key Phytophthora isolates studied and experiments in which they were used
|Phytophthora species|| Isolate||Location, country|| Sampled/isolated by||Sampled from||Sampling or receipt date|| I|| II|| III|| IV|| V|| VI|| VII|| VIII|| IX|| X|| XI|| XII|
|Standard form||P668||Worcestershire, UK||J. Rose||ablb||1994||•||•|| || ||•|| || || || || || || |
| ||P669||Worcestershire, UK||J. Rose||asc||1994|| || ||•|| ||•|| || || || || || |
| ||P670||Gwent, UK||J. Rose||abl||1994|| || ||•||•||•|| || || || || || |
| ||P677||West Sussex, UK||J. Rose||abl||1994||•|| || || || || || || || || || |
| ||P765||West Sussex, UK||J. Rose||abl||1994|| || || || ||•|| || || || || || |
| ||P766||Oxfordshire, UK||J. Rose||abl||1994|| || ||•|| ||•|| || || || || || |
| ||P768||Essex, UK||J. Rose||abl||1994||•|| || || || || || || || || || |
| ||P772||South Yorkshire, UK||G. MacAskill||abl||1994||•||•||•||•||•||•||•||•|| || ||•||•|
| ||P773||Dyfed, UK||J. Rose||abl||1994|| ||•|| || ||•|| || || || || || |
| ||P785||Norfolk, UK||J. Rose||abl||1995|| || ||•||•||•|| || || || || || |
| ||P806||Berkshire, UK||D. Kennedy||abl||1995|| || || || || ||•|| || || || || |
| ||P807||Hampshire, UK||M. Lipscombe||abl||1995|| || ||•||•||•||•||•||•|| || || |
| ||P817||North Germany||G. Hartmann||abl||1994|| || ||•|| ||•|| || || || || || |
| ||P818||North Germany||G. Hartmann||abl||1995|| || ||•||•|| ||•||•||•|| ||•||•|
| ||P834||Léon, France||C. Delatour||abl||1996|| || || || || || ||•||•|| || ||•||•|
| ||P844||Upper Austria||T. Cech||abl||1996|| || || || || || || || ||•|| || |
| ||P891||North Yorkshire, UK||G. MacAskill||abl||1997|| || || || || || || || || ||•|| || |
| ||P938||Hampshire, UK||J. Delcan||abl||1997|| || || || || || || || || ||•||•||•|
| ||P976||Grampian, UK||J.N. Gibbs||abl||1998|| || || || || || || || || || ||•|
|Sector variant||P818vd||n/a||n/a||Cultural variant of P818||1997|| || || || || || || || || ||•|| || |
|Dutch variant||P770||Wageningen, Netherlands||H. Van Kesteren||abl||1994||•||•||•||•||•||•||•||•|| || || ||•|
| ||P972||De Wieden, Netherlands||C. Van Dyck||abl||1998|| || || || || || || || || || ||•||•|
|UK variant||P841||North Yorkshire, UK||S.C. Gregory||abl||1996|| || || || || || || || || ||•|| ||•|
|Swedish variant||P875,||Gothenburg, Sweden||C. Olsson||abl||1997|| || || || || || || || ||•||•|| || |
| ||P876|| || || || || || || || || || || || || || || |
| ||P887||Save River, Sweden||C. Olsson||abl||1997|| || || || || || || || || || || ||•|
| ||P888||Alingsas, Sweden||C Olsson||abl||1997|| || || || || || || || || || || ||•|
|German variant||P889,||Freising, Germany||T. Jung||abl||1997|| || || || || || || || || ||•|| ||•|
| ||P890|| || || || || || || || || || || || || || || |
|P. cambivora||P28||Cheshire, UK||R.G. Strouts||Chamaecyparis root/soil ||1975|| ||•|| || || || || || || || || || |
| ||P199||Unknown, UK||M.G. Griffin||Fagus sp.||1969|| ||•||•||•||•||•||•||•||•|| || |
| ||P203||Surrey, UK||R. Reeves||Castanea sativa||1969||•|| ||•|| || || || || || || || |
| ||P219||Hampshire, UK||R.G. Strouts||Acer soil||1971||•|| || || || || || || || || || |
| ||P238||Unknown, UK||R. Reeves||Fagus sp.||1971||•|| || || || || || || || || || |
| ||P239||Surrey, UK||R.G. Strouts||Fagus sp.||1972||•||•||•|| || || || || || || || |
| ||P281||Hampshire, UK||C. Gulliver||Fagus soil||1975|| || ||•|| || || || || || || || |
| ||P315||Bristol, UK||C. Gulliver||Platanus soil||1976|| || ||•||•||•||•||•||•|| || || |
| ||P819||Perthshire, UK||D. Cooke||Rubus sp.||1985|| || ||•||•||•||•||•||•||•||•||•||•|
| ||P820||Somerset, UK||D. Cooke||Rubus sp.||1995|| || ||•||•|| ||•||•||•|| || || |
| ||P821||Unknown, Italy||T. Turchetti||Castanea sativa||1980|| || || || ||•||•||•||•|| || ||•||•|
|P. cinnamomi||P382||Surrey, UK||R.G. Strouts||Nothofagus procera soil||1980|| || || ||•|| ||•||•||•||•|| || || |
| ||P402||Unknown, Canary Islands||L. Gallo||Persea sp.||1976|| ||•|| || || || || || || || || |
| ||P404||Unknown, Malaysia||L.B. Siew||Syzygium sp.||1990||•|| || || || || || || || || || |
| ||P592||Yorkshire, UK||R.G. Strouts||Crataegus roots||1991|| || || ||•|| || || || || || || |
| ||P596||Badajoz, Spain||C.M. Brasier||Quercus ilex roots/soil ||1992||•||•|| ||•|| ||•||•||•|| || || |
| ||P612||Western Cape, South Africa||S. von Broembsen||Leucospermum comosum||1994||•|| || || || || || || || || || |
| ||P724||Lagos, Portugal||E. Sanchez||Quercus suber soil||1995|| || || ||•|| || ||•||•|| || || || |
|P. citricola||P812||Oregon, USA||C.M. Brasier and E.M. Hansen||Alnus roots||1995|| || || ||•|| ||•||•||•|| || || || |
| ||P813||Oregon, USA||C.M. Brasier and E.M. Hansen||River water||1995|| || || ||•|| || || || || || || |
|P. cryptogea||P187||Surrey, UK||M.G. Griffin||Hebe sp. roots/stems ||1972||•|| || || || || || || || || || || |
| ||P241||Kent, UK||R.G. Strouts||Prunus sp. stems||1972||•||•||•|| || || || || || || || |
| ||P242||Kent, UK||R.G. Strouts||Viburnum sp. roots||1972||•|| || || || || || || || || || |
|P. fragariae var. rubi||P823||Scotland, UK||D. Cooke||Rubus sp.||1971|| || || ||•||•||•|| || || || || || |
|P. gonapodyides||P236||Gloucestershire, UK||C.M. Brasier and R.G. Strouts||Prunus sp. roots||1971||•|| || || || || || || || || || || |
| ||P245||Kent, UK||C.M. Brasier and R.G. Strouts||Salix sp. roots||1972||•||•|| || || || || || || || || |
| ||P501||Surrey, UK||T. Reffold||Ilex sp.||1995||•||•|| || || || || || || || || |
| ||P786||Norfolk, UK||J. Rose||abl||1995|| || || || || || || || || || || ||•|
| ||P878||Unknown, Denmark||K. Thingaard||Alder debris in pond||1995|| || || || || || || || || || || ||•|
|P. megasperma||P426||Unknown, UK||C. Brasier||Populus sp.||1984|| || ||•|| || || || || || || || || |
| ||P922||Hampshire, UK||J. Delcan||as||1997|| || || || || || || || || || || ||•|
|P. sp. ′O'-group||P210||Buckinghamshire, UK||C.M. Brasier and R.G. Strouts||Aesculus hippocastanum||1970|| || || || || || || || || || || ||•|
| ||P246b||Kent, UK||C.M. Brasier and R.G. Strouts||Salix sp. roots||1970|| || || || || || || || || || || ||•|
| ||P845||Nancy, France||J.C. Streito||Alnus sp.||1996|| || || || || || || || || || || ||•|
| ||P877||Unknown, Denmark||K. Thingaard||Alnus debris in pond||1995|| || || || || || || || || || || ||•|
For inoculation tests, 1·2 m long × 20–30 cm diameter billets were cut from stems of living Alnus glutinosa trees 24–48 h before the experiment and the cut ends sealed with Isoflex (bitumen) (Ronseal Ltd, Sheffield, UK). A modification of the elm inoculation protocol of Webber & Hedger (1986) was used. A 5 mm diameter hole was punched through the bark to the wood surface with a cork borer. A 5 mm agar plug from the margin of an actively growing colony on CA was then inserted and the bark plug replaced. Moist cottonwool was placed over the wound, and covered with a 5 × 5 cm piece of aluminium foil secured by adhesive PVC tape. Inoculation points were staggered as shown in Fig. 1. There were eight replicates per isolate (10 in the first experiment), inoculated so as to ensure that the different Phytophthora species were evenly distributed over the logs. Control inoculations (one per log) were as above, but of plain CA. Inoculated logs were stood upright, covered individually in loose polythene sleeves (sealed at both ends) and incubated at 18–22°C (setting 20°C) in an air-conditioned laboratory for 26–55 days (see Table 4 for details of duration).
Table 4. Seasonal influence on lesion sizes produced by standard alder phytophthora isolatesa
| Experiment|| Date of inoculation||Length of experiment (days)||No. of isolates tested||Mean longitudinal lesion extension rate (mm day−1) and SE||Mean lesion area (cm2) and SE||Largest mean lesion area of any isolate (cm2)|
|XI||14 July 1998||42||5||5·8 ± 0·2||354 ± 32||415·6|
|VII||8 October 1996||42||4||5·5 ± 0·3||408 ± 55||517·5|
|XII||21 October 1998||26||3||5·0 ± 0·1||99 ± 6||108·0|
|VIII||22 October 1996||42||4||4·3 ± 0·3||240 ± 56||353·4|
|IV||12 February 1996||42||3||3·2 ± 0·7||100 ± 35||139·6|
|II||4 October 1995||55||3||3·1 ± 1·2||225 ± 107||385·7|
|IX||19 December 1996||35||1||2·0 ||26 ||26·0|
|X||2 February 1998||35||3||1·9 ± 0·1||25 ± 2||27·3|
|III||13 December 1995||43||8||1·6 ± 0·4||26 ± 8||71·0|
|V||4 March 1996||45||10||0·8 ± 0·2||12 ± 4||36·7|
|I||17 April 1995||42||4||Nil/trace||Nil/trace||(1·0) nil/trace|
|VI||15 April 1996||43||4||Nil/trace||Nil/trace||(1·0) nil/trace|
The logs were destructively sampled by removing the periderm with a drawknife to expose the phloem. Any lesion or stained necrotic area around an inoculation point was quickly outlined with a marker pen prior to the oxidative staining of the surrounding live bark tissue. Lesion outlines were then traced onto tracing paper. Maximum length and average breadth measurements were made from photocopies of the traced lesions. Areas were calculated by cutting and weighing the tracings. The mean lesion length, width and area for each isolate, together with the SE, were then calculated. Bark shavings removed during assessment were bagged and autoclaved. The logs were air dried and burnt.
Between April 1995 and November 1998, a total of 19 standard alder phytophthora isolates from different countries and nine isolates representing the UK, Dutch, German and Swedish variant types were tested, plus isolate P818v, a morphologically unique sector from a colony of standard isolate P818. In addition, 11 isolates of P. cambivora were tested, together with 21 isolates of other species. The experiments in which each isolate was used are indicated in Table 1. Some alder isolates (e.g. standard type P772 and Dutch variant P770) were used repeatedly to maintain a level of continuity between experiments. However, a balance also had to be struck between repeating the same isolates, the need to test freshly collected alder phytophthora isolates or isolates of other Phytophthora species, and the practical limits on the size of the experiments.
The different experiments and isolates resulted in very different extents of lesion development. To allow a direct visual comparison to be made of the results between experiments (see Figs 2 and 3), the mean lesion areas of all the isolates and the controls within each experiment are shown as a percentage of the largest mean lesion area caused by any isolate. In consequence, SE cannot be shown in Figs 2 and 3. Statistical analyses (either analysis of variance (anova) or general linear models) of lesion sizes, however, were performed on the raw data sets, with lesion areas transformed to square roots. Because of considerable replicate variation within isolates, skewed distributions of the data sets and the unequal group sizes, suitable error and link functions were applied as appropriate.
Figure 2. Lesion sizes caused by Phytophthora isolates on Alnus glutinosa. Area in cm2 is the largest mean lesion area of any isolate in that experiment. st, isolates of standard, near-tetraploid alder phytophthora; *, isolate P772; nl, d, uk, sw, isolates of the Dutch, German, UK and Swedish variants; d2, cultural variant of standard German isolate P818; cam, P. cambivora; cin, P. cinnamomi; cry, P. cryptogea; fra, P. fragariae var. rubi; cit, P. citricola; o, Phytophthora sp. ‘O’-group; pg, P. gonapodyides; m, P. megasperma; C, control.
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In Experiment II, re-isolation of all the inoculated fungi onto SMA + rifamycin selective medium was attempted from the lesion margins.
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- Materials and methods
The heteroploid alder phytophthoras are believed to be hybrids between P. cambivora and a taxon closely related to P. fragariae (Brasier et al., 1999). P. cambivora and P. fragariae are related through ecologically distinct taxa, the former being a pathogen of various hardwood trees, the latter a specialized pathogen of strawberry and raspberry. In the present study, isolates of the standard alder phytophthora from several European countries were shown to be potentially highly aggressive pathogens of alder bark. However, they were nonpathogenic to the bark of six other tree genera, including tree species such as Castanea sativa and Chamaecyparis lawsoniana that are otherwise susceptible to a range of phytophthoras including P. cambivora. The standard alder phytophthora therefore appears to be relatively specific to alder. In contrast, 11 isolates of P. cambivora caused very limited lesion development on alder, whereas selected P. cambivora isolates caused substantial lesions on other tree genera, such as Castanea and Quercus, under the same conditions. An isolate of P. fragariae var. rubi caused almost no lesion development on alder, Quercus or Castanea.
The genetically and phenotypically unique Swedish, Dutch, German and UK natural variants are suggested to be either genetic breakdown products or backcross products of the standard type (Brasier et al., 1999). Only one of these, the Dutch variant, was also a strong pathogen of alder bark. The others were comparatively weak pathogens. If the variant types are breakdown products of the standard type, three of them appear to have lost the high level of aggressiveness to alder bark in the process. All the natural variants, however, were originally isolated either from alder bark or from a root lesion on alder. They must therefore have some ability to attack alder in the field. Nonetheless, the present results suggest that the disease is likely to be more aggressive in regions where the standard type is predominant, such as the UK, France and Austria.
Only the Dutch variant was tested on hosts other than alder. It was found, like the standard type, to be nonpathogenic on these hosts. The host ranges of the other variants remain to be tested. Of particular interest is the Swedish variant, which in several other respects shows the greatest similarity of all the variants to P. cambivora (Brasier et al., 1999). It might therefore have a more P. cambivora-like host range.
Isolate P818v was derived from a unique sector that arose in a colony of German standard isolate, P818. It differs from P818 in its densely aerial colony type, in producing many single-celled (as opposed to two-celled) antheridia, and in having the same ITS pattern as the UK, German and Dutch natural variants (D.L. Cooke and C.M. Brasier, unpublished results; Delcan & Brasier, 2001). P818v therefore shows phenotypic abnormalities comparable to the natural variants, and could have originated through the type of genetic rearrangement event in a standard isolate that has given rise to the variants in nature. In the present tests, P818v was found to have retained its high level of pathogenicity to alder.
Of the other Phytophthora species tested, none was a significant pathogen of alder bark. These included P. cinnamomi, an aggressive pathogen of certain tree species in warm, temperate ecosystems, and P. gonapodyides, Phytophthora sp. ‘O-group’, P. megasperma and P. citricola, four taxa commonly isolated from river water and wet soil in riparian ecosystems, including alder habitats, in Britain and Iberia (Brasier et al., 1993b; C.M. Brasier, J. Delcan and E.M. Sánchez-Hernandez, unpublished observations). They are therefore phytophthoras to which European Alnus spp. are likely to have been exposed prior to the appearance of the hybrid alder phytophthoras.
Evidence was obtained for marked seasonal variation in the susceptibility of alder logs to the standard alder phytophthora. If repeated in the field, development of lesions in the bark, postinfection, is likely to be most rapid between July and October, i.e. the period of leaf retention, restricted from November to March, after leaf fall, and strongly if not totally suppressed during April, the approximate time of leaf flushing. Sometimes, therefore, lesion development might cease during the winter, allowing an opportunity for a tree to recover. The physiological basis of these seasonal differences in host susceptibility is unknown. Low bark water content has been shown to restrict the development of Phytophthora pathogens (e.g. Tippett et al., 1987; Marcais et al., 1993). Hence, one factor involved (but unfortunately not measured in these tests) might be a lower bark water content during the autumn and winter months. Another factor is likely to be the bark's ability to mobilize stored photosynthates or antifungal metabolites for defence (cf. Cahill & McComb, 1992).
There was also evidence, within some experiments, for ‘higher’ and ‘lower’ levels of lesion development amongst the standard isolates and the Dutch variant. This was further indicated by the variable position of standard isolate P772 (used as a control) in relation to other standard isolates. These observations suggest that critical thresholds of host resistance were operating. In the field, such threshold effects could mean a difference between chronic, suppressed disease and acute, potentially lethal disease. Internal (host) and external influences, such as temperature or microbial competition, could be additional interactive variables in such a threshold phenomenon. P. cambivora, the UK, German and Swedish variants and the other Phytophthora species tested appeared unable to cross the threshold at any time.
In tests on other tree species, P. cambivora was highly and moderately aggressive to the bark of Castanea and Fagus, respectively. This is consistent with the well-known susceptibility of these taxa to P. cambivora under forest conditions (e.g. Day, 1938, 1939; Peace, 1962). P. cambivora was also shown here to be an aggressive pathogen of Quercus bark (cf. also Jung & Blaschke, 1996). Other recent research shows that P. cambivora can be isolated from small roots of Q. robur in the field, that it is a frequent associate of Q. robur on heavy soils in Germany and the UK and that it can cause significant fine root loss in inoculated oak seedlings (Jung et al., 1996, 1999; C.M. Brasier and J. Rose, unpublished results). These combined observations suggest that, under suitable conditions, P. cambivora is potentially a serious primary pathogen of Quercus.
Phytophthora cinnamomi is associated with root rot and mortality of Q. suber, Q. ilex and other Quercus spp. in Mediterranean Europe, and with stem canker of Q. rubra in France (e.g. Brasier et al., 1993b; Marcais et al., 1993). Australian and South African P. cinnamomi isolates showed considerable variation in aggressiveness when inoculated into Eucalyptus spp. (Dudzinski et al., 1993; Linde et al., 1999). In addition, Robin & Desprez-Loustau (1998) observed a wide range in aggressiveness of P. cinnamomi isolates on excised bark of Q. rubra. In the present tests, isolate P382 of P. cinnamomi, from Nothofagus soil in the UK, was a particularly aggressive bark pathogen on all hosts tested except Acer and Alnus. Two other P. cinnamomi isolates (P596 and P724), from Quercus roots and associated soil in Iberia, were considerably less aggressive than P382 on the susceptible hosts, including Q. robur. This again demonstrates marked variation in the aggressiveness of individual isolates of this species. P. cinnamomi was the only Phytophthora tested that was an aggressive bark pathogen of Taxus. This is consistent with the known susceptibility of Taxus to P. cinnamomi in the field (cf. Brasier, 2000).
P. citricola isolate P812 was obtained from soil around roots of red alder, Alnus rubra, in Oregon, USA during a preliminary (negative) search for the new alder phytophthoras in the Pacific North-west. Similar P. citricola isolates were obtained by baiting from the water of adjacent alder-lined rivers (C.M. Brasier and E.M. Hansen, unpublished results). Isolate P812 was only a weak pathogen of alder bark in the present test, but proved to be an extremely aggressive pathogen of Acer bark, producing broad, diamond-shaped lesions. Along with another P. citricola isolate, it was also a significant pathogen of bark of Castanea, Fagus (cf. also Jung & Blaschke, 1996) and Chamaecyparis. In the UK, P. citricola tends to be associated with wet soils, including soil around oak trees (Brasier, 2000; J. Rose and C.M. Brasier, unpublished results), and, as in Oregon, with rivers (J. Delcan and C.M. Brasier, unpublished results). It is also found in flooded alder carr sites in the Netherlands (C. van Dyck, personal communication). It may therefore be well adapted to attacking tree roots on wet soils or in flooded situations.
The present pathogenicity tests were conducted at approximately 20°C, close to the temperature growth optimum for the standard alder phytophthora of approximately 22°C (Brasier et al., 1995). P. cinnamomi is reported to be most active as a pathogen at 25–30°C (Shearer & Tippett, 1989), and is therefore unlikely to have been at its maximum aggressiveness in these tests. This might also apply to P. cambivora, which has an optimum temperature for growth of approximately 27–30°C (Brasier et al., 1995). It should also be emphasized that the log inoculation method used here tests an isolate's capacity for spread in the host, not for infection. The common mode of entry of phytophthoras into the host is via the fine root tips, which could involve a different set of pathogenicity and resistance attributes. The comparative ability of the standard and variant alder phytophthoras to colonize such roots therefore needs to be investigated.
If, as proposed, the standard alder phytophthora is a recently evolved hybrid involving P. cambivora and a P. fragariae-like species as parents (Brasier et al., 1999), it appears to have become pathogenic to alder in the process, whilst the wide host range of the P. cambivora parent has apparently been lost. That Phytophthora interspecific hybrids can acquire host specificities that are not expressed in the parent species has recently been demonstrated via laboratory crosses (Ersek et al., 1995). However, little is known about the genetic basis of host specificity in Phytophthora pathogens of trees, or about their natural variability in aggressiveness. These aspects are in need of study: firstly, so that the risk of damage by existing or newly evolving Phytophthora tree pathogens can be better assessed; secondly, to provide information of use in breeding trees for resistance. The log inoculation method used here provides a simple assay that could be used in assessing pathogenic variation amongst progeny of Phytophthora crosses.