Editor: Bernard Paul
Phytophthora polonica, a new species isolated from declining Alnus glutinosa stands in Poland
Article first published online: 6 JUL 2006
FEMS Microbiology Letters
Volume 261, Issue 2, pages 165–174, August 2006
How to Cite
Belbahri, L., Moralejo, E., Calmin, G., Oszako, T., García, J. A., Descals, E. and Lefort, F. (2006), Phytophthora polonica, a new species isolated from declining Alnus glutinosa stands in Poland. FEMS Microbiology Letters, 261: 165–174. doi: 10.1111/j.1574-6968.2006.00349.x
- Issue published online: 6 JUL 2006
- Article first published online: 6 JUL 2006
- Received 6 March 2006; revised 28 April 2006; accepted 14 May 2006.First published online 6 july 2006.
- alder decline;
- Phytophthora polonica;
- elongation factor 1α;
- mitochondrial DNA;
- phylogenetic analysis
In a survey of Phytophthora associated with alder decline in Poland, several isolates of a homothallic Phytophthora sp., which could not be assigned to other taxa including Phytophthora alni subspecies, were consistently recovered from rhizosphere soil samples. Their morphology and pathogenicity, as well as sequence data for three nuclear regions (internal transcribed spacer rDNA, elongation factor-1α and β-tubulin) and a coding mitochondrial DNA region (nadh1), were examined. The new Phytophthora species is characterized by the moderate to slow growth rate of its colony in carrot agar at 20°C, high optimal (c. 30°C) and maximum (c. 38°C) growth temperatures, formation of catenulate, often lateral, hyphal swellings, large chlamydospores in agar media and in soil extract, persistent, ovoid to ellipsoid nonpapillate sporangia and large oogonia with paragynous and sometimes amphigynous antheridia. Phytophthora polonica was slightly pathogenic to alder twigs and not pathogenic to trunks of several tree species. In a phylogenetic analysis using either Bayesian inference or maximum likelihood methods, P. polonica falls in clade 8 ‘sensu Kroon et al. (2004)’ of Phytophthora.
Species within the genus Phytophthora are well-known plant pathogens causing important diseases in agriculture, arboriculture and natural ecosystems (Erwin & Ribeiro, 1996). Although traditionally studied by mycologists, Phytophthora spp. are unrelated to the true fungi (Eumycota), and nowadays are classified in a distant phylogenetic position within the diploid, algae-like Oomycetes in the Straminipila lineage of the Eukaryota domain (Cavalier-Smith, 1986; Hawksworth et al., 1995). The genus represents over 70 species, most of the discoveries in the past 10 years including notable disease agents of ornamentals (e.g. Cacciola et al., 1996; Ilieva et al., 1998; Werres et al., 2001; De Cock & Lévesque, 2004) and forest trees (e.g. Jung et al., 1999, 2003; Werres et al., 2001; Brasier et al., 2003, 2004, 2005; Hansen et al., 2003). Interest in surveying for Phytophthora in natural ecosystems has increased after awareness of the implication of several Phytophthora spp. in some extensive cases of forest decline and tree mortality in Europe (Brasier et al., 1993, 2004; Jung et al., 2000), sudden oak death in America (Rizzo et al., 2002) and ‘Jarrah dieback’ in Australia (Weste & Marks, 1987).
Recent molecular analysis (Crawford et al., 1996; Cooke et al., 2000; Martin & Tooley, 2003) has largely improved our understanding of the phylogenetic relationships between Phytophthora species. These studies have been, however, hampered by the use of sequence information on single DNA regions and by the limited subset of Phytophthora species covered. Cooke et al. (2000), on the basis of neighbour-joining analysis of internal transcribed spacer (ITS) data, proposed that Phytophthora is paraphyletic, with the main cluster consisting of eight clades and two additional closely related ones comprising Phytophthora macrochlamydospora, Phytophthora richardiae (‘clade 9’) and Phytophthora insolita (‘clade 10’). Such a tree topology has also been noticed by Kroon et al. (2004) in a separate analysis of mitochondrial DNA. However, in the same study, the nuclear DNA and the combined nuclear and mitochondrial data set analysis showed that P. insolita and P. richardiae were situated within the main Phytophthora clade. The use of a significantly larger data set has resulted in a robust phylogeny, largely supporting those of Cooke et al. (2000) and Martin & Tooley (2003).
Since 1993, a new disease of alder trees (Alnus glutinosa) has been spreading across Europe (Brasier et al., 2004). The causal agent is an incipient species probably resulting from hybridization between Phytophthora cambivora and an unknown Phytophthora closely related to Phytophthora fragariae (Brasier et al., 2004). Its taxonomic status has been resolved by erecting the species Phytophthora alni, with several subspecies differing mainly in their karyotype, gametangial morphology and aggressiveness towards alder (Brasier et al., 2004).
In the framework of the present research on Phytophthora spp. associated with declining alder stands in Poland, an unidentified Phytophthora, superficially resembling P. insolita, was isolated from the soil samples of several investigated stands. This paper describes this species as Phytophthora polonica sp. nov. and provides details of its morphology, physiology and pathogenicity towards alder and Quercus spp. Molecular evidence is used to sustain its specific status. A full analysis of several nuclear and mitochondrial genetic markers elucidates its phylogenetic position.
Materials and methods
A wide survey and identification, by ITS sequencing, of numerous Phytophthora and Pythium isolates from different regions in Poland resulted in the identification of an undetermined Phytophthora (L. Belbahri, T. Oszako & F. Lefort, unpublished data). Fourteen isolates of this Phytophthora species were collected from soil samples associated with declining alder stands in the towns of Kolo, Adamowizna and Siestrzen by plating pieces of rhododendron leaves used as baits on PARP or PARPNH, selective media for Phytophthora (Erwin & Ribeiro, 1996). Occasionally, oak (Quercus robur) decline symptoms were also observed in the vicinity of the alder stands. Stock cultures were maintained in carrot agar (CA; Brasier, 1967). The isolates UASWS0197 and UASWS0198 have been deposited at the CBS (Centraalbureau voor Schimmelcultures). All others (UASWS0199, UASWS0205, UASWS0207, UASWS0209, UASWS0210, UASWS0211, UASWS0230, UASWS0231, UASWS0232, UASWS0233, UASWS0234 and UASWS0235) are maintained in the authors' culture collection at the University of Applied Sciences of Western Switzerland. Only the isolates from Kolo are described here because this undetermined Phytophthora sp. was particularly represented.
Morphology and physiology
Stock cultures were initiated from single zoospores. Isolates were grown at 20°C in the dark in 90 mm diameter Petri dishes on cornmeal agar (CMA), CA, malt extract agar (MEA) and potato dextrose agar (PDA), and colony morphologies were examined after 7 days. Colony radial growth rates in 90 mm Petri dishes on CA were measured at 5°C intervals between 5 and 40°C, with two replicates for each temperature and isolate combination. Two points on the colony margin, sited at right angles from the centre of a colony grown for 4 days at 20°C, were marked below the Petri dish with a grease pencil. Radial growth was measured after 48 h exposure at each temperature tested, and the daily radial-growth rate was then calculated.
The presence of sporangia, hyphal swellings and chlamydospores was checked in all media during 2 weeks. In a failed attempt to induce sporangial formation, three 12 mm diameter mycelial plugs taken from the edge of a 5-day-old colony grown on CA at 20°C were placed into a 60 mm diameter Petri dish previously flooded with 5 mL of a soil extract (Moralejo et al., 2005). The dishes were kept for 48–72 h at 20°C either under continuous white light or in darkness. Abundant hyphal swellings and chlamydospores were formed, but no sporangia. In a second attempt we assessed the requirement of nutrients (e.g. amino acids) in solution for the development of sporangia. A sparse sporangial crop was obtained by flooding mycelial plugs with 5 mL of a gelatine solution (10 g of commercial gelatine dissolved in 1 L of distilled water and autoclaved at 121°C for 15 min). The plates were incubated for 24 h at 20°C. Subsequently, the gelatine solution was decanted and replaced with tap water, and the dishes were incubated at 25°C for 48 h in darkness. Zoospore suspensions were obtained by chilling the plates at 7°C for 1 h, and then incubating at room temperature for 30 min. For the production of single zoospore isolates, 0.1 mL of the zoospore suspension was evenly spread on water agar in a 90 mm diameter Petri dish. The plates were checked for zoospore cyst germination under a dissecting microscope. After 24 h at 20°C in darkness, single germinated cysts were lifted with a sterile needle and plated individually on CA.
The development of oogonia, antheridia and oospores on CA at 20°C was examined during 10 days. The above structures as well as chlamydospores and hyphal swellings were lifted with a needle from cultures grown on CA or soil extract, mounted in distilled water and examined at × 400 magnification. About 20 samples of each fungal structure were chosen at random and measured using a calibrated eyepiece under an Olympus BX50F-3 compound microscope equipped with differential interference contrast (DIC) optics. An Olympus DP 12 digital camera adapted to the microscope was used for photography.
DNA extraction, PCR amplification, PCR products purification and sequencing
Mycelial DNA was purified from pure cultures grown in pea broth (Kroon et al., 2004) and checked for quality with a NanoDrop NT-100 UV spectrophotometer. DNA amplifications were performed for three nuclear and one mitochondrial loci. Ribosomal DNA ITS amplifications were carried out using the previously described universal primers ITS4 and ITS6 that target conserved regions in the 18S and 28S rDNA genes (White et al., 1990; Cooke et al., 2000). Amplifications for the translation elongation factor 1α gene (EF-1α), the β-tubulin (β-tub) gene and the NADH dehydrogenase subunit 1 gene (nadh1) were performed according to Kroon et al. (2004) using primers ELONGF1 and ELONGR1 for the EF-1α gene, TUBUF2 and TUBUR1 for the β-tub gene, and NADHF1 and NADHR1 for the nadh1 gene. PCR products were purified with a Minelute PCR Purification Kit (Qiagen, Switzerland) and quantity and quality were checked as reported above. Amplicons were sequenced directly in both sense and antisense directions. ITS amplicons from 14 isolates were sequenced twice and a consensus sequence was created from the duplicates. ITS sequences were registered in GenBank under accession numbers DQ396409 (UASWS0197), DQ396410 (UASWS0198), DQ396411 (UASWS0199), DQ396417 (UASWS0205), DQ396419 (UASWS0207), DQ396421 (UASWS0209), DQ396422 (UASWS0210), DQ396423 (UASWS0211), DQ500127 (UASWS0230), DQ500128 (UASWS0231), DQ500129 (UASWS0232), DQ500130 (UASWS0233), DQ500131 (UASWS0234) and DQ500132 (UASWS0235). The sequences for the genes β-tub, nadh1b and EF-1α were obtained for the isolates UASWS0197 and UASWS0198 and are registered under accession numbers DQ399843, DQ399844, DQ399845, DQ399846, DQ399847, DQ399848, DQ399849, DQ399850 and DQ399851, respectively.
Sequence data for three nuclear regions (ITS rDNA, EF-1α and β-tub) and a coding mtDNA region (nadh1) were compared with those of Phytophthora spp. listed in Kroon et al. (2004) and for P. polonica isolates UASWS0197 and UASWS0198. We conducted separate phylogenetic analyses for individual genes, for combined nDNA sequences and for combined mtDNA+nDNA sequences after a method already reported (Belbahri et al., 2005; Paul et al., 2006). Sequences were aligned manually using Seaview (Galtier et al., 1996). The maximum likelihood (ML) trees were obtained using the PhyML program (Guindon & Gascuel, 2003), with the HKY (Hasegawa et al., 1985) model allowing transitions and transversions to have potentially different rates and the general time reversible (GTR) model allowing all rates to be different (Lanave et al., 1984; Rodriguez et al., 1990). In order to correct the among-site rate variations, the proportion of invariable sites (I) and the parameter of g distribution (G), with eight rate categories, were estimated by the program and taken into account in all analyses.
Nonparametric ML bootstraps (BSs) (with 100 replicates) were calculated using PhyML. Bayesian inferences (BIs) were obtained with MrBayes v.3.0 (Huelsenbeck & Ronquist, 2001) using the same models of DNA evolution as for the ML analyses. The program was run for 2 000 000 generations, sampled every 100 generations, with four simultaneous chains. The trees, sampled before the chains reached stationarity, were discarded. Neighbour-joining plot and Treeview were used to view ML and Bayesian trees, respectively.
In a first trial, the pathogenicity of UASWS0197 and UASWS0198 was determined by wound-inoculating twigs of A. glutinosa c. 10 cm below the apex. A 4 mm long sliver of bark was lifted with a scalpel and a mycelial plug c. 4 mm2 from a 7-day-old colony was inserted beneath. For controls, a sterile CA plug was used. A moist cotton plug was placed on the wound and sealed with parafilm. Five twigs were used for each isolate, one of them as control. They were placed in sets of five in 250 mL Erlenmeyer flasks filled with 200 mL DW, closed with cotton plugs and incubated at 20°C for 10 days. The full length of the discoloured tissue was measured in millimetres. A small piece of discoloured tissue was plated on PARP medium for reisolation of the fungus. The identity of the emerged colonies was morphologically examined.
In the second pathogenicity test carried out in winter, we assessed the capacity of UASWS0198 for infecting trunks of other tree species. Taking advantage of material available at one of the labs in Spain (IMEDEA), the inner bark of logs of four tree species from NE Spain, i.e. Quercus canariensis, Quercus faginea, Quercus suber and Fraxinus angustifolia, was wound inoculated using the log inoculation method of Brasier & Kirk (2001). As positive controls, we used isolates of Phytophthora ramorum and one of Phytophthora cinnamomi, two well-known pathogens of oak. Eight freshly cut logs (1 m length × 20 cm diameter) of each species were inoculated with mycelial plugs. Plain agar plugs were inserted as controls. The logs were sealed with plastic sheet and incubated at 20°C. Lesion formation was examined by carefully shaving the outer bark 40 days after inoculating. The outline of the necrotic lesions was traced on a transparent paper with a pen. The image was scanned and the area was calculated using Olympus 12P software. Differences in aggressiveness among isolates were analysed with Fisher's least significant difference test of a one-way analysis of variance (anova) design using the GLM, with isolates as categorical predictors. Lesion area data were log transformed to meet the anova assumption of homogeneity of variance (Levene's test).
Phytophthora polonica Belbahri L, Moralejo E & Lefort F. sp. nov.
‘polonica’ refers to the country where it was isolated.
Coloniae in agaro Dauci carotae ‘carrot agar’ moderate crescentes, leviter rosaceae, adpressae. Hyphae principales 6–8 μm latae. Hyphae fumescentes in agaro et in extracto aquatico ex humo, hyphae inflatae irregulares, laterales, saepe catenulatae, interdum aggregatae. Chlamydosporae laterales vel terminales abundantes supra bracchiis curtis, 48.4 μm diam, subglobosae vel globosae, levies. Sporangia absentia in agaro et in extracto aquatico ex humo; in medio gelatinoso diluto, sed praesentes, non-papillosa, ovoidea vel ellipsoidea, 52–67 × 32–44 μm, porus plus quam 10 μm latus. Homothallica. Oogonia globosa, levia, 41.8 μm diam. Oosporae typice apleroticae, 38.1 μm latae, parietes moderate crassae. Antheridia solitaria, clavata vel doliiforma vel globosa, plerumque diclina et paragyna, aliquando amphygina, interdum cum productionis hyphalis.
Poland: Kolo, isol. ex solo et rhizosphaera Alni glutinosae, July 2004, T. Oszako, (cultura sicca in agaro Dauci carotae in herbario Universitatis Helvaticae Occidentalis conservatus) – holotypus; UASWS0198=CBS 119650 ex type-culture.
Phytophthora polonica was recovered from the soil of an alder stand in Poland during the summer of 2004.
Main hyphae up to 8 μm wide. Colony pattern: aerial mycelium on CA appressed to limited, slightly stellate to rosaceous; concentric growth rings somewhat noticeable on the underside of the Petri dish, colony on CMA submerged and somewhat radiate, on MEA aerial mycelium appressed, fairly felty, markedly rosaceous, on PDA felty and broadly lobed, rosaceous.
Readily and abundantly formed, usually large (up to c. 50 μm long), single or more frequently catenulate, intercalary or lateral, or aggregated, inflated, toruloid, irregularly shaped to globose (Figs 1a and b). Found either in agar or in soil water extract.
Abundant within 10 days on CA, CMA and in soil extract; spherical to subglobose or pyriform, average diameter 48.4 μm (ranging from 16 to 69 μm), moderately thin walled (1–2 μm), intercalary, lateral or terminal on short branches. In water, forming extensive networks of geniculate hyphae with lateral chlamydospores at the joints (Figs 1c and d).
Not observed on any culture media. None formed in water soil extract and only a few in gelatine solution. Borne on long nonbranching sporangiophores, mostly ovoid to ellipsoid, c. 52–67 × 32–44 μm, noncaducous, nonpapillate, proliferating internally, often nested or catenulate. Zoospores discharged through an exit pore 10–18 μm wide (Figs 1e and f).
Abundant in single-zoospore isolates on CA after 1 week. Mostly borne on stalked branches, spherical to subglobose, smooth- and thin-walled, 41.8±2.8 SD μm diameter (Figs 2a–d).
From aplerotic to nearly filling the oogonia, 38.1±2.5 μm diameter, moderately thick walled (average 2.9±0.8 μm) (Figs 2c and d). A high proportion of aborted oogonia were seen.
Mostly clavate to irregularly shaped, less frequently spherical to barrel shaped (Fig. 2a), 16.2±2.8 μm long × 13±2.1 μm wide; borne on long stalks, mostly attached near the oogonial base (Figs 2b–d), occasionally with hyphal extensions (Fig. 2d). Predominantly paragynous (Figs 2b–d) but sometimes amphigynous (Fig. 2a).
Colony growth rates
Moderately slow (Fig. 3) on CA and CMA at 20°C and slow on PDA and MEA. Optimum temperature c. 30°C, minimum c. 5°C and maximum c. 38°C. In cultures on CA at 20°C contaminated by bacteria, growth was stimulated on the side facing the bacterial colonies.
Sequence analysis and phylogenetic position of P. polonica
The rRNA gene ITS sequences of eight P. polonica isolates had 100% identity and only 90% identity with their closest match P. insolita over an 824 bp sequence run. On the basis of the ITS sequence, P. polonica falls within ‘clade 10’ of Cooke et al. (2000), together with P. insolita. Based on nuclear DNA analysis, as well as by combining mitochondrial and nuclear data sets, P. insolita, P. richardiae and P. polonica are located within the main Phytophthora clade (Figs 4 and 5). Phytophthora polonica shows a phylogenetic position close to or lying between Phytophthora quininea/P. richardiae and P. insolita within clade 8, as described by Kroon et al. (2004).
Phytophthora polonica isolates were slightly pathogenic to alder twigs, tissue discolouration progressing only a few millimetres beyond the inoculation wound after 10 days. There was no significant difference in pathogenicity between both isolates, and the data are thus here combined. The mean lesion length was 8.5 mm, ranging from 6 to 11 mm. No lesion was formed on controls. The oomycete was reisolated from all inoculated twigs when plated on PARP.
Phytophthora polonica was not pathogenic to the inner bark of F. angustifolia (mean lesion area 1.1±0.2 SD cm2), with necrotic areas not differing (P=0.69) from those of the negative controls, and not pathogenic to slightly pathogenic to the three Quercus species. Mostly, the lesions extended very little beyond the point of inoculation on Q. faginea (2.3±2.1 cm2), Q. suber (2.8±2.2 cm2) or Q. canariensis (2.8±1.8 cm2). The maximum lesion area in which the pathogen was recovered on Q. suber was 7.8 cm2. Lesion areas were always less than c. 10% of those formed by the positive controls, P. cinnamomi and P. ramorum (data not shown).
Phytophthora polonica sp. nov. exhibited a combination of unique morphological characters and distinctive nuclear and mitochondrial DNA sequences that easily enables distinction from other Phytophthora species. It belongs to group V of Waterhouse et al.'s (1983) morphological scheme of classification by being homothallic with paragynous antheridia, and bearing nonpapillate sporangia with internal proliferation. Other Phytophthoras within group V include P. fragariae var typ., P. fragariae var. rubi, P. humicola, P. insolita, P. medicaginis, P. megasperma, P. quininea, P. sojae and P. trifolii. Unlike P. polonica, both varieties of P. fragariae as well as species within the ‘megasperma complex’sensuHansen et al. (1986) can be readily distinguished by having lower cardinal growth temperatures; P. humicola has unusually high optimal temperatures like P. polonica but does not form chlamydospores (Ko & Ann, 1985). Of those species included in clade 8 sensuKroon et al. (2004), P. insolita (Ann & Ko, 1980) superficially resembles P. polonica in its cardinal temperatures and colony pattern and in the formation of hyphal swellings and small chlamydospores, but it is easily distinguished by its parthenogenetic oospores, i.e. without attached antheridia (Ho et al., 2002); P. quininea differs by producing larger oogonia (Crandall, 1947); P. richardiae is self-fertile and has a lower maximum growth temperature (Waterhouse, 1970); and P. macrochlamydospora does not form sexual structures (Irwin, 1991). It is noteworthy that all these species of clade 8 sensuKroon et al. (2004) form survival structures such as hyphal swellings, chlamydospores or oospores in pure culture. By having hyphal swellings and chlamydospores, P. cinnamomi and P. lateralis could be confused with P. polonica; however, the first has higher colony growth rates and the second lower cardinal temperatures. Although occupying the same niche, P. alni and its subspecies are distinguished by their almost exclusive amphigynous antheridia and often nonsmooth oogonial walls.
The ecology and pathogenic status of P. polonica still remain unclear. Many of its morphological characters are shared with other typical soil-inhabiting, root-infecting as well as riparian Phytophthora spp. (Brasier, 1983; Brasier et al., 2003). In addition, like most Phytophthoras within clade 6, it exhibits unusually high cardinal temperatures and an optimal growth temperature around 28–30°C (Brasier et al., 2003). This might indicate a physiological adaptation to some other ecological aspect such as litter breakdown, as suggested by Brasier et al. (2003). Our observation of colony growth stimulation by bacteria could be situated in this context and would require further studies. Although it has been consistently isolated from soils associated with alder decline, it is not clearly the causal agent: P. polonica and P. alni were isolated in several cases from the same soil sample. Despite being pathogenic to fruits in wound inoculations (data not shown), no reports on plant diseases attributable to P. polonica have appeared in Poland. On present evidence from pathogenicity tests on alder twigs and on a few Iberian trees, P. polonica is apparently a poor inner bark colonizer, although it is acknowledged that further pathogenicity tests on roots of riparian tree species, especially alder, and during different seasons, would be needed to solve this issue.
Phytophthora polonica clustered in a phylogenetic analysis within Cooke's clade 10 and was included in clade 8e according to the clade definition of Phytophthora by Kroon et al. (2004). It had a high ITS sequence similarity with P. insolita, only isolated from irrigated soils in China, Taiwan and California (Ho et al., 2002). Until now, the biodiversity in clade 8 had been considerably underestimated. Recently a new Phytophthora, Phytophthora kernoviae, alien to the UK, has emerged during recent surveys of P. ramorum in the latter country (Brasier et al., 2005). Based on the ITS sequence, it is related to Phytophthora boehmeriae, and clusters in Cooke's clade 10. The unusual taxon described here was only recently discovered during extensive Phytophthora surveys in Polish forest ecosystems. As such investigations of ‘natural ecosystems’ are being extended, it is expected that more taxa belonging to clade 8 will be found. We should determine if P. polonica exists in other parts of the world before speculating on its origin. Nevertheless, as Polish isolates were obtained from relatively undisturbed and protected forests, P. polonica could be indigenous to this area.
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