Notes on some Phytopythium and Pythium species occurring in oak forests in southern Poland 1 of 10Published by Polish Botanical Society Acta Mycologica ORIGINAL RESEARCH PAPER Notes on some Phytopythium and Pythium species occurring in oak forests in southern Poland Robert Jankowiak1*, Hanna Stępniewska1, Piotr Bilański2 1 Department of Forest Pathology, Mycology and Tree Physiology, University of Agriculture in Krakow, Al. 29 Listopada 46, 31-425 Cracow, Poland 2 Department of Forest Protection, Entomology and Forest Climatology, University of Agriculture in Krakow, Al. 29 Listopada 46, 31-425 Cracow, Poland * Corresponding author. Email: rljankow@cyf-kr.edu.pl Abstract 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 compre- hensive 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 Phy- topythium 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. Keywords Quercus robur; oomycetes; soil; Phytopythium; Pythium This issue of Acta Mycologica is dedicated to Professor Maria Lisiewska and Professor Anna Bujakiewicz on the occasion of their 80th and 75th birthday, respectively. 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 zoo- spores 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 monophy- letic 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 accom- modate species with non filamentous sporangia: Ovatisporangium, Elongisporangium, Globisporangium and Pilasporangium. In the same year Bala [8] proposed a new genus DOI: 10.5586/am.1052 Publication history Received: 2014-11-28 Accepted: 2015-05-18 Published: 2015-08-05 Handling editor Maria Rudawska, Institute of Dendrology of the Polish Academy of Sciences, Poland Authors’ contributions RJ: writing the paper, identification of isolates and analyzing the data; HS: collecting of samples and isolates, identification of isolates; PB: analyzing the data Funding Research was carried out under the theme No. DS 3414/ ZFLM i FD funded by a grant to study awarded by the Polish Ministry of Science and Higher Education. Competing interests No competing interests have been declared. Copyright notice © The Author(s) 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits redistribution, commercial and non- commercial, provided that the article is properly cited. Citation Jankowiak R, Stępniewska H, Bilański P. Notes on some Phytopythium and Pythium species occurring in oak forests in southern Poland. Acta Mycol. 2015;50(1):1052. http://dx.doi. org/10.5586/am.1052 Digital signature This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website. mailto:rljankow%40cyf-kr.edu.pl?subject=Notes%20on%20some%20Phytopythium%20and%20Pythium%20species%20occurring%20in%20oak%20forests%20in%20southern%20Poland http://dx.doi.org/10.5586/am.1052 http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ http://dx.doi.org/10.5586/am.1052 http://dx.doi.org/10.5586/am.1052 2 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests Phytopythium for those species with globose to ovoid, often papillate and internally proliferating sporangia. Recently, de Cock et al. [9] provided molecular-based evi- dence 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 Eu- rope, particularly in forest soils. There were only four reports about isolation of Py- thium spp. from soils under oak forests [10–13]. 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 [9–13]. Recently, Pythium sterilum, P. spiculum B. Paul Belbahri & Lefort, and two Phytopythium species, Phytopythium citrinum-like (B. Paul) Abad, de Cock, Bala, Ro- bideau, Lodhi & Lévesque and Phytopythium mercuriale (Belbahri, B. Paul & Lefort) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque have been associated with soil in declining oak stands in Poland [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 bait- ing (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 de- tecting in oak forests in southern Poland. Material and methods 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. Sam- pling 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 rep- resentative 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 tem- perature. 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 transfer- ring 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 CaCO3). 3 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests Tab. 1 Characteristics of the study sites (in bold the sites where Phytopythium/Pythium spp. were found). Geographic coordinates Sites No. Site Longitude Latitude Forest Type1 Tree age Soil texture2 Soil moisture3 Soil pH4 CaCl2 Crown status5 1 Ispina 1 20.362721° 50.096364° Q 130 silty clay loam M 3.69 SD 2 Ispina 2 20.359564° 50.081254° D 165 silty clay loam M 3.75 SD 3 Wola Batorska 1 20.269651° 50.032460° P 120 sand M 2.83 SD 4 Wola Batorska 2 20.249682° 50.027453° Q 195 silty clay loam M 3.57 D 5 Stanisławice 1 20.335946° 49.991910° P 65 sand MM 3.55 SD 6 Stanisławice 2 20.324853° 49.932567° P 125 sand M 2.92 SD 7 Krzyszkowice 20.016746° 50.002342° D 150 silt loam MM 3.75 D 8 Ostrowy Tuszowskie 21.643837° 50.326383° Q 135 sand MM 3.61 SD 9 Przyborów 1 15°45'06" 51°47'27" Q 150 sandy loam PFWT 4.82 D 10 Przyborów 2 15°45'07" 51°46'58" Q 140 sandy loam PFWT 5.39 D 11 Babice 18°16'03" 50°08'01" Q 120 sandy loam MM 3.77 SD 12 Maleniska 22°19'59" 50°16'00" D 70 sand MM 3.32 H 13 Przedbórz 19°54'17" 51°05'41" P 60 sand MD 3.46 SD 14 Rączna 19°44'52" 50°00'26" D 70 sand MD 3.51 H 15 Radymno 23°04'00" 49°56'60" P 115 loamy sand MM 4.22 D 16 Pomorsko 15°28'47" 52°02'44" Q 150 sand PFWT 5.20 H 17 Piskorowice 22°34'40" 50°14'04" P 80 loamy sand MM 3.75 SD 18 Pociękarb 18°04'44" 50°18'58" Q 150 sandy loam MM 3.14 SD 19 Bierdzany 18°09'10" 50°48'19" D 80 loamy sand MM 3.33 H 20 Rzędzów 18°08'22" 50°44'18" D 50 sand MM 3.52 SD 21 Chełmiec 16°04'58" 51°02'13" Q 100 silt loam MM 3.6 H 22 Moszna 17°46'25" 50°25'31" D 90 loamy sand MM 3.32 SD 23 Leśniki 15°50'22" 52°07'39" P 105 sand MD 5.94 SD 24 Laski 15°48'32" 52°10'27" D 135 sandy loam MM 3.29 D 25 Kręcko 15°04'01" 52°10'27" D 125 sand MM 5.18 SD 26 Zwierzyniec 18°45'31" 50°55'30" Q 140 sandy loam MM 3.99 H 27 Głubczyce 17°38'39" 50°09'50" Q 135 silt loam M 3.68 H 28 Mikolin 17°40'48" 50°47'41" Q 120 silt loam M 3.89 SD 29 Czyżowice 18°22'39" 49°59'33" Q 90 silty clay loam MM 3.58 SD 1 Q – pure Quercus robur stand; P – mixture with Pinus sylvestris; D – mixture with other deciduous species. 2 Soils texture according to USDA (United States Department of Agriculture). 3 M – moist; MM – moderately moist; D – dry; MD – moderately dry; PFWT – periodically fluctuating water table. 4 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). 4 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests 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 1.6.0 software (Pre- coptic®, Warsaw, Poland). Pythium structures and colony characteristics were com- pared 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-day- old 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]. Cus- tom 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. Taxon Isolate number1 Site Accession No. Closest match in BLAST Accession of match Identity (%) Phytopythium citrinum 121HR09 Zwierzyniec KC602480 Phytopythium citrinum HQ 643379 100.0 143HR09 Zwierzyniec KC602481 Phytopythium citrinum HQ 643379 100.0 35HR09 Zwierzyniec KC602482 Phytopythium citrinum HQ 643379 100.0 48HR09 Zwierzyniec KC602483 Phytopythium citrinum HQ 643379 100.0 99HR09 Głubczyce KC602484 Phytopythium citrinum HQ 643379 100.0 Phytopythium cf. citri- num A 444HR08 Przyborów 2 KC602485 Pythium sp. GD33b EF152505 99.7 447HR08 Przyborów 2 KC602486 Pythium sp. GD33b EF152505 99.7 Phytopythium cf. citri- num B 428HR08 Przyborów 2 KC602487 Pythium sp. GD33b EF152505 100.0 520HR08 Przyborów 1 KC602488 Pythium sp. GD33b EF152505 100.0 Phytopythium sp. 1 68HR09 Zwierzyniec KC602492 Pythium sp. B57 JN863966 95.9 Phytopythium sp. 2 451HR08 Przyborów 2 KC602493 Pythium sp. PV So7 EU669081.1 94.0 Phytopythium sp. 3 474HR08 Przyborów 1 KC602494 Phytopythium sp. MAB-2011e AB690623.1 97.5 Pythium anandrum 304HR07 Ispina 2 KC602489 Pythium anandrum HQ643435 100.0 305HR07 Ispina 2 KC602490 Pythium anandrum HQ643435 100.0 Pythium diclinum 27HR09 Laski KC602491 Pythium diclinum JQ898459 100.0 Pythium intermedium 2HR09 Kręcko KC602495 Pythium intermedium AY083936 100.0 Pythium undulatum 719HR08 Radymno KC602496 Pythium undulatum EU240049 100.0 725HR08 Radymno KC602497 Pythium undulatum EU240049 100.0 1 KFL: Fungal Culture Collection, Department of Forest Pathology, Mycology and Tree Physiology, Hugo Kołłątaj University of Agriculture, Cracow, Poland. 5 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests 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. ML searches were conducted in PhyML 3.0 [22], via the Montpelier online server (http://www.atgc-montpellier.fr/phyml/) with 1000 bootstrap replicates. BI analyses based on a Markov chain Monte Carlo (MCMC) were performed with MrBayes v3.1.2 [23]. The MCMC chains were run for 10 million generations using the best fitting model. Trees were sampled every 100 generations, resulting in 100 000 trees from both runs. The burn-in value for each dataset was determined in Tracer v1.4.1 [24]. 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 desig- nated 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 rep- resented taxa belonging to the phylogenetically related genera, Phytopythium and Pythium. Groups with affinity to the genus Phytopythium represented one known spe- cies (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]). Phytopy- thium cf. citrinum A and Phytopythium cf. citrinum B in phylogenetic analyses were most closely related to isolate Pythium sp. GD33b, while the three unknown spe- cies 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. Ten different Phytopythium and Pythium species were isolated from rhizosphere soil in 8 of the 29 oak stands. Among them, Pythium anandrum was most frequently recorded (4.0%) and showed the widest geographical distribution (4 stands). Phyto- pythium citrinum, Phytopythium cf. citrinum B, Phytopythium sp. 1 and Phytopythium sp. 3 were found in two stands. On average, the isolation frequency of Phytopythium citrinum, Phytopythium cf. citrinum B, Phytopythium sp. 1 and Phytopythium sp. 3, in the oaks forests was 2.3%, 1.7%, 2.3% and 2.9%, respectively. Other species were sporadically isolated from rhizosphere soil (Tab. 3). Among the six identified soil textures, Phytopythium spp. and Pythium spp. were most frequently isolated from soil developed from sandy loam. These organisms oc- curred on sites with a mean soil pH range (CaCl2) 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 Py- thium 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 http://www.atgc-montpellier.fr/phyml/ 6 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests 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 Phytophthora ramorum HQ643339 Phytophthora lateralis HQ875390 Phytopythium carbonicum HQ643373 Phytopythium montanum HQ643391 Pythium violae HQ643968 Pythium splendens JN585826 Pythium ultimum JX191931 Pythium nagaii JN630511 Pythium okanoganense AB468817 Pythium paddicum HQ643728 Pythium attrantheridium JQ898456 Pythium intermedium KC602495 Pythium intermedium AY083936 Pythium sylvaticum AB499723 Pythium spinosum AB499701 Pythium cylindrosporum HQ643516 Pythium irregulare JQ898464 Pythium mamillatum HQ643687 Pythium spiculum HQ643790 Pythium debaryanum HQ643519 Pythium macrosporum AB507408 Pythium middletonii HQ643694 Pythium echinulatum HQ643531 Pythium radiosum HQ643756 Pythium mastophorum HQ643692 Pythium polymastum HQ643752 Pythium nunn HQ643711 Pythium perplexum HQ643744 Pythium undulatum EU240049 Pythium dimorphum AY598651 Pythium undulatum KC602496 Pythium undulatum KC602497 Pythium helicandrum AY598653 Pythium prolatum HQ643754 Pythium anandrum HQ643435 Pythium anandrum KC602489 Pythium anandrum KC602490 Pythium senticosum HQ643773 Pythium amasculinum HQ643434 Pythium hydnosporum HQ643564 Pythium aphanidermatum JX462954 Pythium deliense HQ643522 Pythium conidiophorum HQ643509 Pythium dissimile HQ643526 Pythium scleroteichum HQ643771 Pythium catenulatum HQ643494 Pythium torulosum HQ643859 Pythium aristosporum HQ643447 Pythium volutum HQ643971 Pythium graminicola HQ643545 Pythium inflatum HQ643566 Pythium monospermum HQ643697 Pythium pachycaule EF583439 Pythium flevoense HQ643538 Pythium aquatile HQ643445 Pythium oopapillum HQ643717 Pythium coloratum HQ643506 Pythium lutarium JQ898467 Pythium diclinum JQ898459 Pythium diclinum KC602491 Pythium dissotocum JN863965 Pythium marinum AY598689 100/100 100/100 88/99 100/100 100/100 77/98 100/ 100 100/100 98/100 88/98 */75 100/100 99/100 100/100 99/100 100/100 100/10096/100 95/100 95/100 96/100 */83 100/100 100/100 100/100 */96 */98 100/100 97/100 */96 */89 87/100 92/100 99/100 100/100 */100 100/100 0.2 F H B Fig. 1 Phylogram 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%. 7 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests 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 PythiumlutariumJQ898467 PythiumdiclinumJQ898459 PhytophthoraramorumHQ643339 PhytophthoralateralisHQ875390 Phytopythiummercuriale DQ916350 Phytopythiumkandeliae KJ399961 Phytopythiummirpurense KJ831614 Phytopythiumostracodes HQ643395 PhytopythiummegacarpumHQ643388 Phytopythiumboreale HQ643372 PhytopythiumoedochilumHQ643394 Phytopythiumsp. 3 KC602494 Phytopythiumsp. AB690623 Phytopythium sp. 2 KC602493 PhytopythiummontanumHQ643391 PhytopythiumcarbonicumHQ643373 Pythium sp. EU669081 Phytopythiumlitorale JQ898465 Pythiumsterilum EU240096 Phytopythiumcitrinum KC602483 Pythiumsp. DQ403787 Phytopythiumcitrinum KC602484 PhytopythiumcitrinumHQ643375 Phytopythiumcitrinum KC602480 Phytopythiumcitrinum KC602482 Phytopythiumcitrinum KC602481 Phytopythiumcf. citrinum A KC602486 Phytopythiumcf. citrinum A KC602485 Phytopythium cf. citrinum B KC602487 Phytopythium cf. citrinum B KC602488 Pythiumsp. EF152506 Pythiumsp. EF152505 Phytopythiumdelawarense EU339312 Phytopythiumhelicoides HQ643382 Phytopythiumchamaehyphon HQ643374 Phytopythium sp. 1 KC602492 Pythiumsp. JN863966 Phytopythiumvexans JX074739 100/100 100/ 100 100/100 */92 75/99 */87 83/91 78/84 82/100 100/100 96/100 */91 100/100 98/100 100/100 */81 */95 100/100 0.5 100/100 100/ 100 100/ 100 Fig. 2 Phylogram 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%. 8 of 10© The Author(s) 2015 Published by Polish Botanical Society Acta Mycol 50(1):1052 Jankowiak et al. / Pythium species occurring in oak forests Pythium has not been used. Therefore further studies will be needed to fully charac- terize the oak-associated Pythium species. Among the species of the genus Pythium, only P. undulatum is known to be patho- genic 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 im- portant role in the destruction of root systems of oaks. However, the majority of Phy- topythium 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 [10–12,26,27]. It was shown that Phytopythium and Pythium species are widespread on a range of different soil textures with a mean soil pH (CaCl2) 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. Tab. 3 Isolation frequencies of Phytopythium spp. and Pythium spp. from soil samples in oak stands in Poland and soil pH ranges. Taxon pH range (CaCl2) Number of positive soil samples Frequency of isolation (%) Percent of positive stands Site number Phytopythium citrinum 3.68–3.99 4 2.3 6.9 26, 27 Phytopythium cf. citrinum A 5.39 1 0.6 4.2 10 Phytopythium cf. citrinum B 4.82–5.39 3 1.7 6.9 9,10 Pythium anandrum 3.75–4.22 7 4.0 13.8 2, 15, 24, 26 Pythium diclinum 3.29 1 0.6 4.2 24 Pythium intermedium 5.18 1 0.6 4.2 25 Pythium undulatum 4.22 1 0.6 4.2 15 Phytopythium sp. 1 3.99–5.39 4 2.3 6.9 10, 26 Phytopythium sp. 2 4.82 2 1.1 4.2 9 Phytopythium sp. 3 4.82–5.39 5 2.9 6.9 9, 10 Total 23 13.2 27.6 2, 9, 10, 15, 24, 25, 26, 27 References 1. Kirk PM, Cannon PF, Minter DW, Staplers JA. Ainsworth & Bisby’s dictionary of the fungi. 10th ed. Wallingford: CAB International; 2008. 2. Webster J, Weber RWS. Introduction to fungi. 3rd ed. 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Vettraino AM, Barzanti GP, Bianco MC, Ragazzi A, Capretti P, Paoletti E. Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees. Forest Pathol. 2002;32:19–28. http://dx.doi.org/10.1046/j.1439-0329.2002.00264.x 27. Jönsson U, Jung T, Sonesson K, Rosengren U. Relationships between health of Quercus robur, occurrence of Phytophthora species and site conditions in southern Sweden. Plant Pathol. 2005;54:502–511. http://dx.doi.org/10.1111/j.1365-3059.2005.01228.x http://dx.doi.org/10.1046/j.1439-0329.2002.00264.x http://dx.doi.org/10.1111/j.1365-3059.2005.01228.x Abstract Introduction Material and methods Study sites Sampling and isolation methods Identification of Pythium and phylogenetic analysis Results Discussion References 2015-08-03T19:00:53+0100 Piotr Otręba