Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees
Article first published online: 12 DEC 2003
Volume 52, Issue 6, pages 694–702, December 2003
How to Cite
Balcì, Y. and Halmschlager, E. (2003), Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees. Plant Pathology, 52: 694–702. doi: 10.1111/j.1365-3059.2003.00919.x
- Issue published online: 12 DEC 2003
- Article first published online: 12 DEC 2003
- Accepted 3 June 2003
- oak decline;
- pathogenicity test;
- Phytophthora quercina;
- Phytophthora cinnamomi;
- Phytophthora citricola;
From 1999 to 2001, a survey on the occurrence of Phytophthora spp. in the rhizosphere soil of healthy and declining oak trees was conducted in 51 oak stands in Turkey. Seven Phytophthora spp. were recovered from six out of the nine oak species sampled: P. cinnamomi, P. citricola, P. cryptogea, P. gonapodyides, P. quercina, Phytophthora sp. 1 and Phytophthora sp. 2. The most frequently isolated species, P. quercina, was very common on slopes susceptible to drought. It occurred in four different climatic zones and on six Quercus spp., suggesting that it is native to oaks. The second most common species, P. citricola, was separated into three subgroups: type C was recovered only in Anatolia, whereas A and B occurred only in the European part of Turkey. Phytophthora cinnamomi was recovered at one site only, and may not be involved in oak decline in Turkey. The other four species were recovered sporadically. On affected sites there was a significant association between deteriorating crown status and the presence of Phytophthora spp., particularly P. quercina. The occurrence of Phytophthora species was significantly influenced by soil pH. Stem inoculation tests on oak seedlings revealed that Q. petraea was the most susceptible species.
The genus Phytophthora, which comprises over 60 species, causes major damage in both agricultural and forest plants (Erwin & Ribeiro, 1996). The emergence of new taxa in the genus Phytophthora– such as P. ramorum, which causes tremendous mortality on several tree species in California (Rizzo et al., 2002), or a hybrid Phytophthora which is killing alder trees (Alnus spp.) along river banks in Europe (Brasier et al., 1995; Brasier & Kirk, 2001; Streito et al., 2002) – are current examples demonstrating their impact on forest and river ecosystems. There is some evidence that Phytophthora spp. are associated with oak decline in Europe. Mortality of cork and holm oak (Quercus suber and Q. ilex, respectively) in Spain and Portugal was associated with the widespread pathogen P. cinnamomi (Brasier et al., 1993a; Gallego et al., 1999; Sánchez et al., 2002). A diverse Phytophthora population, including hitherto unknown species, thrives in the rhizosphere soil of European oak forests (Jung et al., 1996; Jung et al., 2000; Jung et al., 2002; Thomas et al., 2002; Vettraino et al., 2002; Delatour, 2003; Balcì & Halmschlager, 2003a; Balcì & Halmschlager, 2003b). Among these species the frequently recovered P. quercina, which causes fine-root destruction of Q. robur and Q. petraea, has been suggested to be involved in oak decline syndrome in Europe (Jung et al., 1999; Jung et al., 2000). In France and Italy, however, no association was found between the overall presence of Phytophthora spp. and the health status of oak trees (Robin et al., 1998a; Hansen & Delatour, 1999; Vettraino et al., 2002), but there was a significant association of P. quercina with declining oak trees in Italy (Vettraino et al., 2002).
Oak trees cover a very wide range of woodlands in Turkey. From the 18 oak species native to Turkey, nine were included in this study, comprising the most common species managed for timber production (Q. petraea and Q. cerris), but also less common (Q. robur, Q. frainetto, Q. hartwissiana, Q. virgiliana, Q. ithaburensis ssp. ithaburensis) and endemic oak species (Q. vulcanica, Q. macranthera ssp. syspirensis). Oak decline was most apparent in the European part of Turkey during the past decade in Thrace (H. Topaloğlu, Demirköy Forest Management Direction, Karacadağ Office, Turkey, personal communication), and decline symptoms were similar to those described in previous reports, including yellowing of leaves, thinning of the crown, dieback of small and large branches, and development of epicormic shoots on the stem (Siwecki & Liese, 1991; Luisi et al., 1993; Dreyer & Aussenac, 1996). To date, no research has been conducted in Turkey on the possible involvement of Phytophthora spp. in oak decline. The present study aimed to investigate the assemblage of Phytophthora spp. in native oak ecosystems in Turkey and to examine the relationship between dieback symptoms of oak trees and the presence of Phytophthora spp. Furthermore, stem inoculation tests were conducted to assess the pathogenicity of the recovered Phytophthora spp.
Materials and methods
Study sites and sample trees
The survey was conducted during September–November 1999, August–September 2000 and April–May 2001 in 51 oak stands in various parts of Turkey (Fig. 1). With the exception of sites 21 and 33, which were examined each year, all other sites were surveyed only once during the 3-year period. The stands were located in four climatic zones: the Mediterranean and sub-Mediterranean winter rain areas, the warm humid zone, and the mountain climate zone. Sites 12, 13, 19, 23, 24, 25, 28, 36 and 46 were located in nature-protection areas. Except for site 15, which was characterized by periodic flooding and a high groundwater level, almost all sites were located on slopes. Sampling sites were chosen from areas where oaks showed decline symptoms, or where dieback was recorded by local forest officers and authorities. Ideally, half the sample trees were healthy and the other half showed symptoms of dieback (crown transparency >15%). However, some stands were totally healthy, whereas in other areas all trees had dieback symptoms. In each stand, two to 16 trees (mean for all sites, 5·7 trees) were selected for further study. Sample trees were generally selected from mature trees ( 60 years old) because it is difficult to assess crown status on young trees. Trees were selected to be representative at each site and had a scattered distribution within each stand. In total, 291 trees were examined.
The crown status of the sample trees was assessed as follows: class 1, no symptoms of decline, crown transparency less then 10–15%; class 2, slight damage, dieback of some branch tips and small gaps in lateral branch system of crown, crown transparency 15–35%; class 3, moderate damage, apparent transparency in all parts of crown, dieback of twigs and branches and large gaps in lateral branch system, yellowing and wilting of leaves, epicormic shoots often present, crown transparency 35–55%; and class 4, severe damage, considerable transparency of crown and mainly large gaps, many dead twigs and branches, leaves mainly restricted to shoot tips, yellowing of leaves and many epicormic shoots, crown transparency 55–75% (Neumann & Pollanschütz, 1988; Anonymous, 1998).
Field sampling and isolation of Phytophthora spp.
Four to five soil samples with fine roots (soil-root-monoliths c. 30 × 30 × 30 cm) were taken 50–150 cm away from the trunk at four equidistant positions around the base of each tree. After the organic layer was removed from each soil sample, they were mixed, and a subsample was placed in a plastic bag and transported to the laboratory. A 300 mL soil sample from each tree was flooded with deionized water in a plastic box (15 × 11 × 6 cm) and baited with 2–4-day-old leaflets from seedlings of Q. petraea and Q. hartwissiana. After removing the litter with filter paper, 10–15 leaflets were placed on the water surface. Baited soils were incubated at 17–20°C in diffuse daylight. After 3–5 days, brownish leaflets were mounted on glass slides in tap water and examined for Phytophthora sporangia. If they were present, leaflets were washed in tap water, dried on filter paper, cut into 1–3 mm wide pieces and placed on PARPNH selective agar [V8 juice agar media amended with 10 µg mL−1 pimaricin, 200 µg mL−1 ampicillin, 10 µg mL−1 rifampicin, 25 µg mL−1 pentachloronitrobenzene (PCNB), 50 µg mL−1 nystatin and 50 µg mL−1 hymexazol]. Plates were incubated in the dark at 20–22°C and examined within 2–5 days. Phytophthora colonies were purified by transferring outgrowing mycelia onto V8 juice agar [(100 µL mL−1 multivitamin juice, 3 µg mL−1 CaCO3 and 20 µg mL−1 agar); Jung et al., 1996]. If no Phytophthora was isolated from the soil sample, it was completely air-dried then rebaited with oak leaflets (Jeffers & Aldwinckle, 1987).
The pH of soil samples was measured with a glass electrode in a 0·01 m CaCl2 suspension and in deionized H2O.
Identification of Phytophthora species
Identification of Phytophthora spp. was based on their cultural and morphological characteristics (e.g. Stamps et al., 1990; Erwin & Ribeiro, 1996). The heterothallic species were paired with A1 and A2 tester strains of P. cinnamomi, P. cambivora and P. cryptogea from CBS (Utrecht, the Netherlands). Representative strains were compared with authenticated isolates, kindly provided by T. Jung (Bavarian State Institute of Forestry, LWF, Germany) and CBS. A subset of isolates of different morphotypes of P. citricola, unknown isolates, and strains of P. quercina was selected for DNA sequence analysis in order to confirm their identity. Sequencing of the internal transcribed spacer (ITS) region of the rDNA operon and comparisons with known sequences of various Phytophthora spp. in GenBank were done by D. Cooke (SCRI, Dundee, UK). Isozyme analyses of different P. citricola strains were carried out by W.A. Man in ′t Veld (PPS, Wageningen, the Netherlands). Reference strains of all Phytophthora spp. were deposited at CBS and are also being maintained in the Phytophthora collection of the Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF) of BOKU – University of Natural Resources and Applied Life Sciences, Vienna.
Pythium species were identified according to Kröber (1985).
Stem inoculation tests were carried out on 2-year-old seedlings of Q. petraea and 3-year-old seedlings of Q. cerris, the most common and widespread oak species in Turkey. Seedlings were inoculated with seven fungal isolates with 10 replicates (Table 4). The seedlings were grown in a glasshouse in natural soil from a Phytophthora-free oak forest and were regularly watered. Inoculation was carried out in June 2002, before the plants started a second shoot-growth cycle. On each seedling, a 4 mm diameter bark plug was removed with a cork borer approximately 5 cm above the collar. The inoculum was applied to the exposed surface and the bark plug was replaced afterwards. The wound was covered with wet, autoclaved cotton wool and sealed with Parafilm. Inoculum consisted of plugs of V8 juice agar bearing mycelium from the margin of a 7-day-old culture of the test isolate. In addition, 10 control plants of each oak species were treated similarly with sterile V8 juice agar plugs. Nine weeks after inoculation, seedlings were harvested and visible lesion length was measured on the cambial surface after removing the bark. Reisolation of inoculated Phytophthora spp. onto PARPNH selective medium was attempted from the lesion margin.
|Phytophthora spp.||Isolate number||Q. petraea||Q. cerris|
|P. cinnamomi||Tr/25 Bel.||86·4 (32·6)||9·8 (3·76)|
|P. cambivora||Tr/47 Kep.||9·7 (1·49)||9·7 (1·49)|
|P. citricola A||Tr/1 Kar.||11·3 (1·15)||7·4 (1·17)|
|P. citricola B||Tr/12 Bel.||12·2 (3·15)||11·6 (1·5)|
|P. citricola C||Tr/38 Vez.||8·6 (0·84)||7·6 (1·35)|
|Phytophthora sp. 1||Tr/22 Yen.||8·4 (0·84)||7·8 (0·92)|
|P. quercina||Tr/41 Tok.||6·8 (0·63)||–|
|Control||6·7 (0·44)||6·5 (0·85)|
The relationship between the crown status of sample trees and the presence/absence of Phytophthora spp. in the rhizosphere soil was analysed using logistic regression analyses. The relative risk that oaks develop severe above-ground symptoms was tested with contingency tables by comparing all the healthy (crown damage class 1) and declining (crown damage classes 2–4) trees. Furthermore, the soil pH data of all Phytophthora-affected stands and all Phytophthora-free stands were combined, and the two groups compared using a t-test. Significant differences in lesion length of inoculated seedlings compared to control plants, as well as differences in susceptibility of tested oak species to Phytophthora infection, were tested with the nonparametric Mann–Whitney test. All statistical analyses were made using the programme package spss for Windows 10·0.
Phytophthora spp. were recorded from 38 out of 51 sites, and from soil samples around 117 out of 291 examined trees (Table 1). They were recovered from 22% of samples at the first attempt, and from 78% following air-drying and rebaiting. Seven Phytophthora species were recovered. These were P. quercina, P. citricola, P. cinnamomi, P. cryptogea, P. gonapodyides, Phytophthora sp. 1 and sp. 2 (Table 1). Additional Phytophthora sporangia were observed on leaflets used for baiting, but could not be recovered in pure culture and thus were not identified (P. unknown). Phytophthora cambivora was isolated from stem necroses and soil samples from beech trees (Fagus orientalis) in a mixed stand with oak (site 47), but was not recovered from oak.
|Phytophthora spp.||pH range (CaCl2)||Quercus spp.||No. positive trees||Frequency of isolation (%) (291 trees)||Percentage of positive stands||Site number|
|Q. cerris||11||1, 3, 4, 5, 7, 11,|
|Q. frainetto||2||14, 15, 17, 18,|
|Q. hartwissiana||3||20, 21, 24, 28,|
|P. quercina||3·4–7·1||Q. petraea||70||31·2||56·8||29–33, 35, 39,|
|Q. robur||1||40–42, 44–47,|
|P. citricola A||4·9–5·8||Q. cerris||4||1, 51|
|P. citricola B||4·0||Q. frainetto||2||2·7||7·8||12|
|P. citricola C||5·7||Q. petraea||2||38|
|P. cinnamomi||3·4||Q. petraea||5||1·7||2||25|
|P. cryptogea||5·7||Q. cerris||4||1·4||2||38|
|P. gonapodyides||6·5||Q. robur||1||0·3||2||16|
|Phytophthora sp. 1||3·4–5·0||Q. petraea||9||3·1||3·9||11, 22|
|Phytophthora sp. 2||4·0||Q. frainetto||1||0·3||2||12|
|P. ‘unknown’a||4·1–5·6||Q. hartwissiana||3||5·8||11·8||2, 8, 15, 18, 23,|
The highest overall isolation frequency was recorded in 1999 in late autumn, followed by spring 2001, and isolation frequency was lowest in mid-summer 2000. All soil samples collected in the first and second years were baited again the following year, after storage for 10 and 8 months, respectively. In this repeated baiting, only Phytophthora sp. 1 and P. quercina were recovered; no additional Phytophthora isolations were made.
At sites 21 and 33, which were examined each year, there was considerable variation between years. Isolation frequency was highest in 1999, whereas in 2000 no Phytophthora was recovered, and in 2001 only one out of seven soil samples at site 33 yielded Phytophthora.
Phytophthora species were isolated most frequently from soils of declining oak trees. On Phytophthora-affected sites at least one species was isolated from 40% of the healthy trees (class 1), 59% of class 2 and 67% of combined classes 3 and 4 (Table 2). On affected sites there was a significant association between crown class and the occurrence of Phytophthora (logistic regression: odds ratio = 0·594, P = 0·005; Table 2), with increasing recovery of Phytophthora spp. with deteriorating crown status. Phytophthora quercina showed a similar trend (odds ratio = 0·505, P = 0·001; Table 2). Oaks with P. quercina in their rhizosphere were 1·9 times more likely to exhibit decline symptoms than oaks without P. quercina (relative risk = 1·894; 95% CI: 1·333–2·691). The relative risk decreased to 1·6 when all recovered Phytophthora species were considered (relative risk = 1·620, 95% CI: 1·184–2·216).
|Status of Phytophthora spp. for the 38 affected sites|
|Logistic regression: Odds ratio = 0·594; 95% CI = 0·411–859, P = 0·005|
|Status of P. quercina for the 29 affected sites|
|Logistic regression: Odds ratio = 0·505; 95% CI = 0·328–776, P = 0·001|
In general, dieback symptoms occurred on single, scattered trees or in small patches. There was neither an association of decline with valleys nor high incidence of decline throughout large areas in Turkey. Stands that were examined for 2 or 3 years showed no changes in crown class. No symptoms of decline were observed on understorey plants or oak seedlings in the stands surveyed.
Among all the isolated Phytophthora species, P. quercina occurred most frequently and was recovered from 29 sites (56·8%) and 91 out of 291 sample trees (31·2%). It also showed the widest geographical distribution, occurring in four different climatic regions and on six oak species (Fig. 1, Table 1).
Phytophthora citricola, the second most common species, was isolated from four sites (7·8%) and eight trees (2·7%). This species comprised three subgroups (A–C) based on colony morphology, different optimum growth temperatures and other morphological features, as well as sequences of internal transcribed spacer (ITS) regions of ribosomal DNA (Y. Balcì & D. Cooke, unpublished data) and isozyme patterns (Y. Balcì and W. A. Man in ′t Veld, unpublished data). The three P. citricola types also showed different geographical distributions: type C occurred only in Anatolia, whereas the other two types (A and B) occurred only in the European part of Turkey (Fig. 1; Table 1).
Phytophthora cinnamomi was isolated only in the Belgrade forest (site 25), from the rhizosphere soil of Q. petraea, but not from that of the admixed tree species F. orientalis and Carpinus betulus. Phytophthora cryptogea was recovered from the same site as P. citricola type C (site 38) in a coppice stand (Q. cerris) with loamy soil. Phytophthora gonapodyides was isolated once from a soil sample from Q. robur on a wet site (site 16) in a mixed stand of oak and beech. However, nonpapillate sporangia and nested proliferation resembling P. gonapodyides were also observed on baits from another wet site (site 15), but could not be obtained in pure culture. Phytophthora sp. 1, which may be an undescribed species, was obtained from the rhizosphere soil of Q. petraea on one site in the European and one in the Asian part of Turkey (Fig. 1; Table 1). Phytophthora sp. 2, an unidentified P. citricola-like taxon, was recovered once (Table 1). On six sites (sites 2, 8, 15, 18, 23 and 44), papillate or/and nonpapillate sporangia of Phytophthora were observed on the leaflets, but did not grow in culture and were designated P. ‘unknown’.
Pairing tests revealed that P. cinnamomi and P. cambivora belonged to the mating type A2. Phytophthora cryptogea and P. gonapodyides did not produce oogonia.
Among the isolated Phytophthora spp., P. quercina occurred on sites showing the widest range of mean soil pH (CaCl2), extending from 3·4 to 7·1 (Table 1). All other recovered species showed different ranges, which lay within the pH range of P. quercina. The mean pH value of Phytophthora-affected sites was significantly higher (P = 0·01) than that of Phytophthora-free sites (Table 3).
|Phytophthora status||pH (CaCl2)|
Besides Phytophthora, sporangia of a variety of Pythium spp. were frequently observed on the oak leaflets used as bait, and although a Phytophthora-selective media was used, the following taxa were recovered from 36 sites: Pythium undulatum, P. anandrum, P. aphanidermatum, P. intermedium, P. middletonii, P. rostratum and members of Pythium Group P. At sites with Pythium spp. present, isolation of Phytophthora spp. often proved to be very difficult or even impossible, even though sporangia of the latter were observed on the leaflet baits. Cultures of Pythium were often dominant in the isolation plates from these sites and often overgrew Phytophthora spp.
Stem inoculation test
With the exception of P. quercina, the mean length of necrotic lesions was significantly higher in all tested isolates than in the control (Table 4). Overall, Q. petraea proved to be more susceptible than Q. cerris (P = 0·008). On Q. petraea, the largest lesions were caused by P. cinnamomi, with a mean lesion length of 86·4 mm. All other Phytophthora spp. caused smaller lesions, from 6·8 mm (P. quercina) to 12·2 mm (P. citricola type B).
On Q. cerris, the largest lesions were caused by P. citricola type B (11·6 mm), whereas in the other two types (A and C), lesion length was shorter than for all other Phytophthora spp. tested, but still differed significantly from the control (Table 4).
Phytophthora cinnamomi caused long bleeding cankers on Q. petraea and one seedling died; lesions on Q. cerris were smaller. Phytophthora cambivora led to sunken necroses and eight out of the 10 Q. petraea seedlings were nearly girdled, whereas on Q. cerris only superficial bark necroses were formed. Phytophthora citricola type B was associated with dark bark necrosis on both oak species, whereas P. citricola type A caused similar necrosis only on Q. petraea, not on Q. cerris, where necrotic lesions were not apparent on the stem surface. Phytophthora sp. 1 and P. citricola type C caused necrosis mostly under the bark, and wounds were mostly closed after 9 weeks.
The results of this study revealed that a diverse population of Phytophthora spp. is abundant in oak forests in Turkey. Differences in the species assemblage between the present and previous studies (Jung et al., 1996; Hansen & Delatour, 1999; Jung et al., 2000; Thomas et al., 2002; Vettraino et al., 2002; Balcì & Halmschlager, 2003a; Balcì & Halmschlager, 2003b) refer only to the less frequently isolated species. As in Germany and Austria (Jung et al., 2000; Balcì & Halmschlager, 2003b), P. quercina was the most frequently recovered species in Turkey, and isolation frequency was markedly higher than in Italy (Vettraino et al., 2002). This species showed high plasticity concerning site factors and soil pH, as in the studies in Germany, Italy and Austria (Jung et al., 2000; Vettraino et al., 2002; Balcì & Halmschlager, 2003b). The recovery of P. quercina, as well as P. citricola and Phytophthora sp. 1, from dry upland sites suggests that these species are well adapted to nonhydromorphic site conditions, while the other species detected failed to survive on these sites. This behaviour was also reported in studies in France and Germany (Hansen & Delatour, 1999; Jung et al., 2000). Furthermore, the repeated isolation of P. quercina and Phytophthora sp. 1 after 8 or 10 months from soil samples from the first and second years provided evidence that both species are able to survive extended dry periods in the absence of a suitable host. The widespread occurrence of P. quercina in Turkey and other European countries, in very different soils and climates and on several oak species, including endemic taxa (Jung et al., 1996; Hansen & Delatour, 1999; Jung et al., 2000; Thomas et al., 2002; Vettraino et al., 2002; Balcì & Halmschlager, 2003a; Balcì & Halmschlager, 2003b) strongly suggests a native association of P. quercina with oak trees. In accordance with Bianco et al. (2003), stem inoculation tests revealed that P. quercina is not pathogenic to the stem and collar of Q. petraea seedlings. However, severe damage on fine roots caused by P. quercina was demonstrated by Jung et al. (1999, 2002) in soil inoculation tests.
Phytophthora citricola, the second most recovered species in Turkey, was found to be the most frequent Phytophthora spp. in Italian oak forests (Vettraino et al., 2002) and was also very common in oak forests in Germany (Jung et al., 1996; Jung et al., 2000) and north-eastern France (Hansen & Delatour, 1999). It occurs worldwide on a broad range of agricultural plants, as well as on trees (e.g. Erwin & Ribeiro, 1996). Cultures resembling P. citricola from Turkey could be divided genetically and morphologically into three types, which supports the general view that this widespread species forms a species or subspecies complex. These types also varied in their virulence in stem inoculation tests on young oak seedlings. Variability in aggressiveness of P. citricola isolates was also demonstrated on Eucalyptus marginata clones (Bunny, 1996) and Q. robur seedlings (Jung et al., 1996). However, the latter study did not separate the isolates by their cultural characteristics. Types A and B were encountered in the European part of Turkey and also in Austrian oak forests (Balcì & Halmschlager, 2003b), whereas type C was restricted to the Asian part of Turkey, suggesting the existence of ecological barriers. Brasier & Hansen (1992) consider that P. citricola may already have been geographically dispersed in ancient times, resulting in isolated biological species. Furthermore, Oudemans et al. (1994), investigating a worldwide collection of P. citricola, differentiated five distinct subgroups within the P. citricola complex, whereas Bunny (1996) separated P. citricola isolates from Australia into three electrophoretic subgroups, using isozyme analyses. Further investigations are therefore needed to clarify the taxonomy of the P. citricola complex.
Although the P. cinnamomi isolate obtained from the Belgrade forest (site 25) proved to be the most aggressive isolate to Q. petraea seedlings, no association between crown symptoms and the presence of this highly pathogenic species was found in the field. Similarly, Robin et al. (1998) and Vettraino et al. (2002) reported no association between the presence of P. cinnamomi and dieback of infected oak species in France and Italy, although its high pathogenicity on cork and holm oak was demonstrated in several studies (Robin & Desprez-Loustau, 1998; Robin et al., 1998; Gallego et al., 1999; Robin et al., 2001; Sánchez et al., 2002), and P. cinnamomi was found to be associated with Iberian oak mortality (Brasier et al., 1993a). The restricted occurrence of P. cinnamomi could be because it is possibly a recent introduction to Turkey, or because of unfavourable climatic conditions such as cold winter temperatures, which have a major effect on mycelium growth (Zentmyer, 1980; Brasier & Scott, 1994). Furthermore, fine roots of Q. petraea are probably less susceptible to P. cinnamomi infection than those of holm and cork oak, a point that has already been demonstrated for Q. cerris, Q. robur, Q. rubra and Q. palustris (Marçais et al., 1996; Maurel et al., 2001; Bianco et al., 2003).
Phytophthora gonapodyides was recovered only once, from soil in a site where water accumulates, in accordance with the findings of Hansen & Delatour (1999) who also isolated P. gonapodyides almost exclusively from aquatic habitats such as puddles, streams and submerged leaf litter. This suggests saprophytic behaviour and an ability to survive in water in the absence of a suitable host. Phytophthora gonapodyides was also encountered in similar habitats in other oak forests in Europe (Jung et al., 1996; Jung et al., 2000; Vettraino et al., 2002). It is considered a weak parasite, but proved to be moderately pathogenic to oak seedlings in stem and soil inoculation tests (Jung et al., 1996; Jung et al., 1999; Bianco et al., 2003). However, Delatour et al. (2000) did not find a significant relationship between root damage of oak seedlings and infection by P. gonapodyides. Brasier et al. (1993b) stated that in the field, P. gonapodyides may frequently attack the small or fine feeder roots of its woody hosts and may sometimes contribute to rapid decline if host stress ensues.
The rarely obtained P. cryptogea was recorded from Q. cerris trees in Italy (Vettraino et al., 2002), but was not recovered in other surveys in oak forests (Jung et al., 1996; Hansen & Delatour, 1999; Jung et al., 2000; Thomas et al., 2002). It is an important pathogen of ornamentals and its host range includes a large number of genera with worldwide distribution (Erwin & Ribeiro, 1996), but its impact on oak is still unknown. So far, it has not been reported from agricultural crops (Biçiçi & Çnar, 1990) and ornamentals in Turkey, thus it is possibly a recent introduction.
Hitherto unknown Phytophthora species (Phytophthora sp. 1 and sp. 2) were obtained in the present study, as in studies in Germany, France and Italy (Jung et al., 1996; Hansen & Delatour, 1999; Jung et al., 2000; Vettraino et al., 2002). Phytophthora sp. 1 proved to be pathogenic to both oak species in the stem inoculation test; however, its aggressiveness to fine roots needs further investigation. The low isolation frequencies of these rarely recovered species suggest that they require special microsite conditions. The fact that some Phytophthora spp. were observed on the leaves used as baits, but could not be recovered in pure culture (P. ‘unknown’), indicates that repeated isolations in different seasons would probably reveal additional new reports.
The extended baiting method provided evidence that soils initially assessed as Phytophthora-free could contain Phytophthora spp., thus single baiting could lead to some false-negative results. Germination and regrowth of viable units in soils, such as chlamydospores or oospores, being probably suppressed by microbial antagonists, needs to be induced for positive detection. On the other hand, it was shown that inoculum level can decrease to nearly undetectable levels in soils over time, which might be an effect of nonconducive soil conditions or antagonism.
The occurrence of Phytophthora spp. on sites with a soil pH (CaCl2) ≥ 3·4 and over a fairly wide pH range is consistent with the results of Jung et al. (2000), Thomas et al. (2002) and Vettraino et al. (2002). The statistically significant differences in mean soil pH between the Phytophthora-free and affected sites support the view that higher pH values favour Phytophthora spp. (Erwin & Ribeiro, 1996).
Logistic regression revealed that Phytophthora spp. may be involved in oak dieback at certain sites in Turkey. A significant association between the occurrence of Phytophthora spp., particularly the common species P. quercina, and deteriorating crown status has already been demonstrated in Germany and Italy (Jung et al., 2000; Vettraino et al., 2002). However, in the latter study a significant relationship did not exist if all recovered Phytophthora spp. were considered. This discrepancy could be the result of differences in sampling methodology, because most of the trees sampled in the Italian study were very young. The present results also contrast with the findings of Hansen & Delatour (1999), who found no correlation between the presence of Phytophthora spp. and tree health status. However, Hansen & Delatour stated that because of the limited number of trees examined and sites sampled, their study was inadequate for supporting firm conclusions concerning correlations between the occurrence of Phytophthora spp. and the decline status of oak trees.
The relative risk that oaks infected by P. quercina will exhibit above-ground symptoms of oak decline was similar in the present study and the German study (Jung et al., 2000). Furthermore, the odds ratios from the logistic regression of the present study were nearly the same as in a simultaneous study in Austria (Balcì & Halmschlager, 2003b), which indicates a similar association between deteriorating crown status and the presence of Phytophthora spp. in Turkey and Austria.
Decline symptoms were most severe in the present study at the two sites (41 and 42) which also exhibited the highest isolation frequencies of P. quercina (data not shown). Both sites were characterized by very shallow soils and the driest conditions among the sampled sites. Similarly, Moreira et al. (2000) pointed out that symptoms on P. cinnamomi-infected sites in Portugal were most severe in plantations of Q. suber and Q. rotundifolia on poor, shallow soils or poorly drained soils, which were also subjected to severe drought. Weste (1983) considered that shallow soils may contain a higher concentration of plant roots that become colonized more rapidly, resulting in the development of a high pathogen population.
Recent studies on oak seedlings revealed that the severity of Phytophthora root rot was higher and lesion size was greater on plants subjected to water stress such as drought or waterlogging (Marçais et al., 1993; Marçais et al., 1996; Moreira et al., 2000; Robin et al., 2001; Sánchez et al., 2002; Jung et al., 2003). The effect of drought is probably more severe on infected mature trees because of the substantial reduction in the root system, which results in reduced capacity to explore the soil (Maurel et al., 2001). Similarly Hansen & Delatour (1999) and Delatour (2003) pointed out that unusual combinations of environmental factors may be required for resident Phytophthora spp. to have a detrimental impact on oaks.
We are grateful to the forestry authorities in Turkey, the Forestry Faculty of University of Istanbul and the Austrian Exchange Service (ÖAD) for providing financial support to this project. The authors would also like to thank T. Kirisits (IFFF-BOKU) for his thorough review of the draft manuscript, H. Anglberger (IFFF-BOKU) and A. Ploner (BOKU) for their constructive advice on the statistical analyses, J. Pennerstorfer (IFFF-BOKU) for his help with the distribution map, T. Jung (LWF) for providing isolates of Phytophthora spp., and D. Cooke (SCRI) and W. A. Man in ′t Veld (PPS) for their help with the molecular genetic analyses. We further thank S. Oguz for assistance in laboratory work, and Y. Balcì and K. Asutay for their help with the field work.
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