Notes on some Phytopythium and Pythium species occurring in oak forests in southern Poland

Phytopythium and Pythium species are known to be soil-born oomycete pathogens of forest trees in Europe. Little is known, however, about the presence of these micro-organisms in Polish oak forests. During the period 2007–2009 a comprehensive study of Phytophthora species in soils of oak forests in southern Poland was conducted using baiting technique. In this study, baits were also colonized by oomycete resembling Pythium species. Based on morphological characteristics and the ITS sequences comparisons, 10 species of Phytopythium and Pythium were isolated from the soil-root samples, including three putative new species belonging to the genus of Phytopythium. The most commonly encountered Pythium species was Pythium anandrum. The present study demonstrates for the first time that Phytopythium citrinum and Pythium diclinum can also act as soil-borne organisms in oak forests. In addition, these species were reported for the first time in Poland.


Introduction
The genus Pythium Pringsh.including "fungus-like organisms" or "pseudo-fungi" is placed in the kingdom Chromista [1] or kingdom Straminipila [2].The species of Pythium are cosmopolitan, widely distributed throughout the world, and occupy several diverse ecological niches.Some species of Pythium are known as pathogens of various plants, including forest and fruit trees; they lead to rot of fruit, rot of roots, and stems, and pre-or postemergence damping-off.For example Pythium undulatum H.E. Petersen, causes root rot of Abies procera Rehder and Pseudotsuga menziesii (Mirb.)Franco in northern Germany [3].
The genus Pythium is characterized by filamentous or globose sporangia with zoospores develop in a vesicle, oospores formed in smooth or ornamented oogonia with paragynous or hypogynous antheridia [4].Many reports have shown that Pythium is composed of few morphological groups, whereas recent molecular analyses have shown that the genus Pythium is a polyphyletic group that includes several monophyletic groups [5,6].In a recent study, Uzuhashi et al. [7] restricted the genus Pythium to those species with filamentous sporangia and created four new genera to accommodate species with non filamentous sporangia: Ovatisporangium, Elongisporangium, Globisporangium and Pilasporangium.In the same year Bala [8] proposed a new genus Phytopythium for those species with globose to ovoid, often papillate and internally proliferating sporangia.Recently, de Cock et al. [9] provided molecular-based evidence that members of Pythium clade K as described by Lévesque and de Cock [5] belong to the Phytopythium genus.While recognizing the genus status of remaining species of Pythium clades (A-J) is still unclear we prefer to use to the definition of Pythium sensu Lévesque and de Cook [5].
Little is known about the occurrence of Phytopythium and Pythium species in Europe, particularly in forest soils.There were only four reports about isolation of Pythium spp.from soils under oak forests [10][11][12][13].These studies reported occurrence of P. undulatum, P. anandrum Drechsler, P. aphanidermatum (Edson) Fitzp., P. irregulare Buisman, P. middletonii Sparrow, P. rostratum Butler and P. intermedium de Bary in oak stands in Austria, Germany, Turkey and Sweden.Of all Pythium species recorded in these studies, P. anandrum and P. undulatum are the most commonly reported and are known as fine root pathogens which may affect the health of oak trees in Europe [9][10][11][12][13] [14].However, information about the oak-associated Pythium and Phytopythium species is still very limited.
During the period 2007-2009 a comprehensive study of Phytophthora species in soil samples collected from oak forests in southern Poland was conducted using baiting (oak leaves) and selective agar medium technique [15].In this study, baits were also colonized by other oomycetes, particularly by Pythium species.For this reason, our objective was to identify the Pythium isolates obtained during Phytophthora detecting in oak forests in southern Poland.

Study sites
The survey was conducted during May-June and September-October 2007-2009 in 29 pedunculate oak (Quercus robur L.) stands in the southern part of Poland.Sampling sites were selected from areas characterized by different tree health status and site conditions.The characteristics of the study sites are given in Tab. 1.

Sampling and isolation methods
In each stand 6 mature trees (tree age >50 years), which were considered to be representative for the health status of the stand, were chosen.The crown status of these trees was assessed according to Balcì and Halmschlager [11].Three soil samples were taken with fine roots (soil-root monoliths without the organic part ca. 25 × 25 × 25 cm), 100-150 cm from the trunk and spaced in three directions around the stem base of each tree.Soil from three monoliths was mixed and a sub-sample of this mixture was put in a plastic bag and transported to the laboratory.
Isolation was performed using the oak leaf baiting method described by Jung et al. [10,16].Each sample was mixed thoroughly, then two 200 mL subsamples were flooded with 400 mL of distilled water in a plastic boxes (18 × 11 × 7 cm), and baited by floating 2-to 6-day-old Q. robur leaves (10-15 leaves per each box) at room temperature.After 3-6 days, discolored leaves were taken to Phytophthora spp.isolation.For this purpose, leaves were washed under tap water, dried on filter paper, cut into small pieces (2 × 2 mm) and placed on PARPNH medium (V8 juice agar amended with 10 mg/L pimaricin, 200 mg/L ampicillin, 10 mg/L rifampicin, 25 mg/L PCNB, 50 mg/L nystatin and 50 mg/L hymexazol).Emerging cultures were purified by transferring small pieces of mycelium from individual colonies to fresh V8 juice agar (100 mL/L Vega's juice (Tymbark®, Poland), 900 mL/L distilled water, 15 g/L agar, 3 g/L CaCO 3 ).
Tab. 1 Characteristics of the study sites (in bold the sites where Phytopythium/Pythium spp.were found).  The pH was measured with a glass electrode in a 0.01 m CaCl2 suspension and in deionized H2O. 5 H -healthy (class 1 according to Balcì and Hamschlager [10]); SD -slightly damaged (class 2); D -declining (class 3 and 4).

Identification of Pythium and phylogenetic analysis
All Pythium cultures were grouped according to morphological characters using a Nikon Eclipse 50i microscope (Nicon® Corporation, Tokyo, Japan) and an Invenio 5S digital camera (DeltaPix®, Maalov, Denmark) with Coolview 1.6.0software (Pre-coptic®, Warsaw, Poland).Pythium structures and colony characteristics were compared with the descriptions of species given in the literature [17].The morphology of colonies was described from 7-day-old colonies growing on carrot (CA), cornmeal agar (CMA), potato-dextrose agar (PDA), malt agar (MEA) and V8 agar medium.Sporangia were obtained by flooding agar discs taken from growing margins of 7-dayold colonies with unsterile soil extract (ratio 1:10).From each morphological group, isolates were selected for DNA sequencing and were deposited in the Fungal Culture Collection of the Department of Forest Pathology, Mycology and Tree Physiology, Hugo Kołłątaj University of Agriculture, Cracow, Poland.Genomic DNA from 18 strains (Tab.2) was isolated using ArchivePure DNA Yeast/Gram-positive Bacteria Kit (5 PRIME, Inc. Gaithersburg, MD) with modified time of incubation with lytic enzyme solution (2 h, 37°C) and cell lysis solution (4 h, 64°C).Nuclear ITS rDNA region internal transcribed spacers (ITS1 and ITS2) and 5.8S subunits were amplified with primer set ITS5/ITS4 [18].The reaction mixtures and conventional PCR protocols were the same as in Hubka and Kolarik [19].Custom purification of PCR amplicons and sequencing was conducted at Macrogen Inc. (Seoul, South Korea) using the same primers.
Tab. 2 Cultures used in this study and GenBank accession numbers for sequences.Sequences were compared with the data from GenBank using a BLAST search.All sequences were aligned online with MAFFT v6 [20], using the E-INS-i option with a gap-opening penalty of 1.53 and an offset value of 0.00.
Datasets were analyzed using maximum likelihood (ML) and Bayesian inference (BI).For the ML and Bayesian analyses, the best-fit substitution models for each data set were established using the corrected Akaike information criterion (AICc) in jModelTest 0.1.1 [21].The selected model for the ITS was GTR+I+G.
All sequences generated in this study were deposited in the NCBI GenBank (Tab.2) and are presented in the phylogenetic tree (Fig. 1, Fig. 2).Pythium clades were designated according to Lévesque and de Cock [5].

Results
Morphological investigation showed that ten groups producing sporangia and sexual structures in culture were collected.The ITS data confirmed that these groups represented taxa belonging to the phylogenetically related genera, Phytopythium and Pythium.Groups with affinity to the genus Phytopythium represented one known species (Phytopythium citrinum) and five unknown species named here as Phytopythium cf.citrinum A, Phytopythium cf.citrinum B, Phytopythium sp. 1, Phytopythium sp. 2 and Phytopythium sp. 3.In turn, the genus Pythium was represented by four species including Pythium anandrum, P. diclinum Tokun., P. intermedium and P. undulatum (Fig. 1, Fig. 2).Polygenetic analyses of the ITS sequences of Pythium isolates placed them in three clades (B, F, H) within Pythium (in Lévesque, de Cock [5]).Phytopythium cf.citrinum A and Phytopythium cf.citrinum B in phylogenetic analyses were most closely related to isolate Pythium sp.GD33b, while the three unknown species were most closely related to different species in the genus Phytopythium (Fig. 2, Tab.2).Their identity and morphological features are still under investigation and descriptions of these taxa will be provided in a later publication.
Among the six identified soil textures, Phytopythium spp.and Pythium spp.were most frequently isolated from soil developed from sandy loam.These organisms occurred on sites with a mean soil pH range (CaCl 2 ) ranging from 3.29 to 5.39, and were most often isolated from soil samples taken from declining trees (14 out of 36) and from pure Q. robur stands (4 out of 29; Tab. 1).

Discussion
This is the first extensive report demonstrating the presence of Phytopythium and Pythium species in oak forests in Poland.Based on morphological characteristics and ITS rDNA sequence analysis, 10 species of Phytopythium spp.and Pythium spp.were isolated from the soil-root samples, including three putative new species.The most commonly encountered Pythium species was Pythium anandrum.The present study demonstrates for the first time that Phytopythium citrinum and Pythium diclinum can also act as soil-borne oomycete species in oak forests.In addition, these species were reported for the first time in Poland.
This study has shown that the assemblage of Phytopythium/Pythium spp.occurring in the rhizosphere soil of oak forests in Poland was quite diverse.The occurrence of  Pythium species in oak stands is in accordance with results from previous studies [10,11,13,14].Of all the Pythium species recorded in association with the rhizosphere soil of oak stands in Europe, P. anandrum and P. undulatum are the most commonly reported.Consistent with Jung et al. [10], Balcì and Halmshlager [11] and Jönsson et al. [13], we isolated P. anandrum most frequently.However, in contrast to these studies but similar to Cordier et al. [14], we additionally relatively often isolated species resembling Phytopythium citrinum.The level of Pythium diversity found in our survey was certainly underestimated because the typically selective medium for Pythium has not been used.Therefore further studies will be needed to fully characterize the oak-associated Pythium species.
Among the species of the genus Pythium, only P. undulatum is known to be pathogenic to oak [16].Weber et al. [3] showed also the aggressiveness of P. undulatum on roots of A. procera and P. menziesii.In addition, Shafizadeh and Kavanagh [25] have also shown the pathogenicity of P. undulatum on Picea sitchensis (Bong.)Carr., Picea abies (L.) Karst.and Pinus contorta Dougl.ex Loud.In the present study, this root pathogen has been recorded only on one site suggesting that rather does not play important role in the destruction of root systems of oaks.However, the majority of Phytopythium spp.and Pythium spp.isolates have been obtained from declining stands indicating a possible association between the presence of these organisms and health status of trees.Similar relationships have been revealed for soil-borne Phytophthora spp. in several European countries [10][11][12]26,27].
It was shown that Phytopythium and Pythium species are widespread on a range of different soil textures with a mean soil pH (CaCl 2 ) between 3.29 and 5.39.Our results resembled those of Jung et al. [10] and Balcì and Halmshlager [11] who mentioned that Pythium species in Germany and Austria occurred in similar site conditions like Phytophthora species in oak forests.

Fig. 1
Fig.1Phylogram obtained from the analyses of ITS sequence data, revealing the identity of Pythium spp.isolated from soil in oak stands in the southern part of Poland.Sequences obtained during this study are presented in bold type.The phylogram was obtained from maximum likelihood (ML) analyses.The bootstrap values (>75%) for ML and posterior probabilities (>75%) that were obtained from Bayesian (BI) analyses are presented at nodes as follows: ML/BI.* Bootstrap values <75%.

Fig. 2
Fig.2Phylogram obtained from the analyses of ITS sequence data, revealing the identity of Phytopythium spp.isolated from soil in oak stands in the southern part of Poland.Sequences obtained during this study are presented in bold type.The phylogram was obtained from maximum likelihood (ML) analyses.The bootstrap values (>75%) for ML and posterior probabilities (>75%) that were obtained from Bayesian (BI) analyses are presented at nodes as follows: ML/BI.* Bootstrap values <75%.

No. Site Longitude Latitude Forest Type 1 Tree age Soil texture 2 Soil moisture 3 Soil pH 4 CaCl2 Crown status 5
1 Q -pure Quercus robur stand; P -mixture with Pinus sylvestris; D -mixture with other deciduous species.2Soilstextureaccording to USDA (United States Department of Agriculture).3M-moist; MM -moderately moist; D -dry; MD -moderately dry; PFWT -periodically fluctuating water table.
3ab.3Isolation frequencies of Phytopythium spp.and Pythium spp.from soil samples in oak stands in Poland and soil pH ranges.