Mycol Progress DOI 10.1007/s11557-011-0778-6

ORIGINAL ARTICLE

Two new species from China based on the morphology and DNA sequence data

Yan-Yan Long & Ji-Guang Wei & Xiang Sun & Yue-Qiu He & Ji-Tong Luo & Liang-Dong Guo

Received: 31 March 2011 /Revised: 27 June 2011 /Accepted: 11 July 2011 # German Mycological Society and Springer 2011

Abstract In an investigation of Pythium species in lawn Keywords Pythium breve . Pythium baisense . grassland of south China, two new species, Pythium Morphology. ITS . COXI breve and P. baisense, were identified based on morpho- logical characteristics and DNA sequence data. These two new species differ morphologically from the other Introduction Pythium species by the oogonia encompassed by many antheridia and with 1–3 antheridia on the wavy and The genus Pythium Pringsheim, established by Pringsheim curved stalks. Furthermore, P. baisense with complexly in 1858, belongs to , Pythiales, , lobed antheridial stalks differs from P. breve with Oomycota, and Straminipila (Hulvey et al. 2010). antheridial stalks entwining the hyphae nearby the Pythium species are widely distributed throughout the oogonia with several turns. Results of phylogenetic world, and most species are saprobic in soil, water and analyses showed that these two new species were clearly remains of dead plants and animals. Some species are separated from morphologically similar Pythium species important pathogens of plants and animals, and some can based on the internal transcribed spacer region (ITS1, be beneficial as biological control agents protecting 5.8S, ITS2) and cytochrome c oxidase subunit I (COXI) against pathogenic fungi (van der Plaats-Niterink 1981; gene sequences using maximum parsimony and Bayesian Ali-Shtayeh and Saleh 1999). methods. These two new species are described and Van der Plaats-Niterink (1981) revised the genus illustrated in detail. Pythium according to morphological characteristics, occur- rence and pathogenicity, and accepted 87 species in this genus. More than 60 new Pythium species have been described since van der Plaats-Niterink’s monograph (Paul Y.-Y. Long : J.-G. Wei College of Agriculture, Guangxi University, 1986, 1991, 1999, 2000, 2001, 2002, 2003a, b, 2004; Nanning 530005, People’s Republic of China Mathew et al. 2003;Pauletal. 1998, 2005; Nechwatal and Oßald 2003; Allain-Boulé et al. 2004; Nechwatal et al. : * X. Sun L.-D. Guo ( ) 2005; Garzón et al. 2007; Belbahri et al. 2008; De Cock et State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, al. 2008; Karaca et al. 2008, 2009; Moralejo et al. 2008; Beijing 100101, People’s Republic of China Senda et al. 2009; Uzuhashi et al. 2009; Bala et al. 2010a; e-mail: [email protected] Long et al. 2010). The of Pythium was mainly based on the Y.-Q. He Key Laboratory of Agricultural Biodiversity and Pests Control, morphological characteristics, such as the size and shape of Ministry of Education, Yunnan Agricultural University, oogonia, antheridia and sporangia. However, some impor- Kunming 650201, People’s Republic of China tant morphological structures are highly variable, overlap considerably and are absent in many species. These J.-T. Luo Forest Pest and Disease Control Station of Guangxi, disadvantages, therefore, contributed to the historical Nanning 530022, People’s Republic of China lack of consensus on the most important morphological Mycol Progress characteristics for species identification (Lévesque and de distilled water for sporulation. Fifty measurements were Cock 2004). taken for each morphological characteristics, such as Molecular techniques have been employed in the species sporangia, oogonia and oospores. identification and systematics of Pythium. Internal tran- The cardinal temperatures were examined on potato scribed spacer regions (ITS1, 5.8S, ITS2) of rDNA agar (PCA) according to the method of van der sequences have been successfully used in the identification Plaats-Niterink (1981), and growth rates were measured at of Pythium species (e.g., Paul et al. 1998; Nechwatal and 24 h incubation. Each isolate was incubated at 5–40°C with Oßald 2003; Allain-Boulé et al. 2004; De Cock et al. 2008; intervals of 5°C on PCA media. When no growth was Karaca et al. 2009), while there were identical ITS observed, the intervals were reduced from 5 to 2 or 1°C and sequences in some distinctly morphological species the culture was returned to room temperature to check (Lévesque and de Cock 2004;Longetal.2010)and whether or not growth resumed. intra-specific heterogeneity of ITS sequences in some species (Kageyama et al. 2007; Belbahri et al. 2008; DNA extraction, PCR amplification and sequencing Mcleod et al. 2009; Senda et al. 2009). Bala et al. (2010a) pointed out that ITS region and cytochrome c oxidase Isolates of Pythium species were cultured on sterilized subunit I (COXI) gene could provide good species cellophane sticked on potato dextrose agar (PDA) at 25°C recognition and were used as DNA barcodes for Pythium for 3–5 days. Fresh cultures were harvested by sterilized species identification. Therefore, Pythium species can be spatulas. Genomic DNA was extracted from fresh cultures accurately identified by the combination of morphological following the protocol of Guo et al. (2000). The ITS characteristics and DNA sequence data (e.g., Nechwatal et regions were amplified with universal primer pairs ITS5 al. 2005; Garzón et al. 2007; Moralejo et al. 2008; and ITS4 (White et al. 1990). COXI gene was amplified Uzuhashi et al. 2009). Recently, Bala et al. (2010b) with primer pairs FM52R (5′-TTAGAATGGAATTAGCA established a new genus Phytopythium Abad, de Cock, CAAC-3′, reversed the complementation of FM52) and Bala, Robideau & Lévesque comprising the Pythium FM55 (5′-GGCATACCAGCTAAACCTAA-3′, Martin species from clade K in Lévesque and de Cock (2004), 2000).Amplificationwasperformedina50-μL reaction and Hulvey et al. (2010) transferred this new genus from volume which contained PCR buffer (20 mM KCl, 10 mM

Pythiaceae of Pythiales to Peronosporaceae of Peronospor- (NH4)2SO4, 2 mM MgCl2, 20 mM Tris-HCl, pH8.4), ales based on the analyses of ITS and partial 28S rDNA 200 μM of each deoxyribonucleotide triphosphate, 15 gene sequences. pmols of each primer, 100 ng template DNA, and 2.5 units In an ongoing study of Pythium species in China, two new of Taq DNA polymerase (Biocolor BioScience and Pythium species were identified based on morphological Technology, Shanghai, China). The thermal cycling pro- characteristics and ITS and COXI sequence data. gram was as follows: 3 min initial denaturation at 95°C, followed by 35 cycles of 40 s denaturation at 94°C, 50 s primer annealing at 54°C, 60 s extension at 72°C for ITS, Materials and methods 70 s extension at 72°C for COXI, and a final 10 min extension at 72°C. Isolation PCR products were purified using PCR Cleanup Filter Plates (MultiScreen® PCRμ96; Millipore, USA) according Soil samples were collected from lawn grassland in to the manufacturer’s protocol. Purified PCR products Baise, Guilin, Liuzhou and Nanning of Guangxi, China. were directly sequenced with primer pairs as mentioned The samples were taken to the laboratory for isolation above in an ABI 3730-XL DNA sequencer (Applied within 7 days. The particles of the soil samples were Biosystems, USA). put on Petri dishes with corn meal agar (CMA) and were incubated at 25°C for 2–3 days. When mycelial Phylogenetic analysis growth was observed, purification was carried out by cutting a small piece of media with mycelia at the edge The sequences were aligned with MAFFT using the G-INS- of a colony and then transplanted onto new medium i option (Katoh et al. 2005). All sites were treated as plates. unordered and unweighted, and gaps were treated as missing in the phylogenetic analyses. Maximum parsimony Morphology and growth rate analyses of the alignment ITS and COXI datasets were conducted with the heuristic search algorithm with tree- The purified isolates were grown on CMA for morphological bisection-reconnection branch swapping in PAUP 4.0b10 studies. Pythium isolates have been transferred to sterilized (Swofford 2002). For each search, 1,000 replicates of Mycol Progress random stepwise sequence addition were performed and all Herbario Mycologico Academiae Sinicae in Beijing trees were saved per replicate. The strength of the internal (HMAS242231) conservatur. branches of the trees was tested with bootstrap analyses Holotype China, Guangxi, Nanning, from the soil of using 1,000 replications with the same search settings. dawn, Y.Y. Long, 10.4.2009 HMAS242231. Living culture Bayesian analyses of the same alignment dataset were (CNN213) deposited in the Department of Plant Protection, conducted in MrBayes 3.1.2 (Huelsenbeck and Ronquist College of Agriculture, Guangxi University. 2001). The best-fit evolutionary model was determined The grows well on CMA, PCA and PDA. Its for each dataset by comparing different evolutionary mycelium in water hyaline, well branched, the main hyphae models via MrModeltest 2.3 (Nylander 2008). The up to 5 μm wide, and dendroid, often with short side-branches general time-reversible model of molecular evolution (Fig. 1a). Colonies on PCA submerged, without a special was used for both ITS and COXI datasets according to pattern. Average growth rates 3 mm day−1 at 15°C, the MrModeltest suggestion, in which a proportion of the 8mmday−1 at 20°C, 16 mm day−1 at 25°C, site was assumed to be invariable, while the rate for the 17.5 mm day−1 at 30°C, 7.5 mm day−1 at 35°C, no growth remaining sites was approximated from a gamma distri- at 7°C and 38°C, but when returned to room temperature bution with four categories (GTR + I + G), and a random both of them started to grow again. Cardinal temperatures: starting tree. The prior probability density is a flat minimum 7°C, optimum 30°C, maximum 38°C. Appressoria Dirichlet (all values are 1.0) for both Revmatpr and sausage-shaped, often catenulate, occasionally present Statefreqpr as default settings. Four simultaneous chains (Fig. 1b). Zoospores formed in sterile water at 20°C of Markov Chain Monte Carlo were run starting from (Fig. 1c). Sporangia globose or subglobose, sometimes random trees and sampling every 100 generations. The provided with papilla, occasionally oval to elongate or analyses were halted at 1,000,000 generations, when the gourd-shaped; mostly terminal, sometimes intercalary, calculation approached to stationarity. At the end of the 11.3–31.3 (average 21) μmindiameter(Fig.1d–i). The analysis, 10,000 trees were generated and 25% of them female gametangia (oogonia) globose, subglobose or sac- were excluded as the “burn in” when calculating the like, smooth-walled, at times with a papilla, mostly posterior probabilities. Bayesian posterior probabilities terminal, sometimes intercalary (Fig. 1j–k, o, s), 22.5– were obtained from the 50% majority rule consensus of 31.3 μm in diameter (average 28.2 μm). The female the trees kept. If more than 95% of the sampled trees gametangia filled with dense, coarsely granulated proto- contained a given clade, it was considered to be plasm. The male gametangia (antheridia) mostly diclinous, significantly supported by our data. occasionally monoclinous, sometimes crowding around the oogonia, making it very difficult to determine their origin, type of contact and numbers (Fig. 1k–l). Anther- Results idial stalks sometimes wavy and more or less curved, arising at various distances from the oogonium, at times Taxonomy entwining the hyphae nearby the oogonium with several turns (Fig. 1m, n). Antheridial cells club-shaped or Pythium breve Y.Y. Long, J.G. Wei & L.D. Guo sp. nov. elongated, sometimes wavy in contour or large, 1–8per (Fig. 1) oogonium, making apical or lateral contact with the oogonium (Fig. 1k, p–q, t). Oospores plerotic or nearly MycoBank. MB 561132 plerotic, at times aplerotic, globose, 17.5–28.8 μmin Etymology The oomycete is named as P. breve because of diameter (average 24.2 μm),generallysingle,rarely2in its short side-branches on hyphae. an oogonium, colorless (Fig. 1j–k, r). Latin description Hyphae praecipae usque ad 5 μm diam; Sporangia terminalia vel intercalaria, globosa vel Pythium baisense Y.Y. Long, J.G. Wei & L.D. Guo sp. nov. subglobosa, interdum papillis, 11.3–31.3 μm diam, non (Fig. 2) prolifis; Oogonia globosa, subglobosa, intercalaria, inter- dum terminalia, 22.5–31.3 μm; Antheridia diclinata, inter- MycoBank. MB 561133 dum monoclinata, uno vel duobus per oogonium, interdum Etymology The oomycete is named as P.baisense plures frequentatio inter singulum oogonium, clavatis vel because its holotype strain was isolated from lawn soil elongato, interdum crispans in contactus, stipite elongato collected from Baise city, Guangxi Province, P.R. China. curvo saepe sinuato praeditis; Oosporae singulae, pleuro- Latin description Hyphae praecipae usque ad 6.3 μm ticae, interdum apleuroticae, globosa, 17.5–28.8 μm diam. diam; Sporangia terminalia vel intercalaria, globosa vel Incrementum radiale quotiadianum 16 mm 25°C in agaro subglobosa, interdum papillis, 5–15 μmdiam,non Solani tuberosi et Dauci carotae (PCA). Holotypus in prolifis; Oogonia globosa, subglobosa, terminalia vel Mycol Progress

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Fig. 1 Asexual and sexual reproductive bodies of Pythium breve. a l many antheridia crowding around oogonia; m wavy and curved Hyphal dendroid structures; b sausage-shaped appressoria; c released antheridial stalk; n antheridial stalk entwining the hypha nearby the zoospores; d globose provided with papilla; e terminal oogonium with several turns; o diclinous antheridium; p elongate sporangium; f intercalary sporangium; g oval sporangium; h gourd- antheridial cell; q large antheridial cell; r 2 oospores in an oogonium; shaped sporangium; i elongate sporangium; j sac-like oogonium with s oogonium with a pappila; t antheridial cell wavy in contour. Scale aplerotic oospore; k intercalary oogonium with plerotic oospore; bar 10 μm emanio directus ex hyphae, interdum pleurogena, 13.8– Incrementum radiale quotiadianum 30 mm 25°C in agaro 28.8 μm; Antheridia diclinata vel monoclinata, interdum Solani tuberosi et Dauci carotae (PCA). Holotypus in Herbario emanio directus ex hyphae vel plures frequentatio inter Mycologico Academiae Sinicae in Beijing (HMAS242232) singulum oogonium, inflatae campanulatae, clavatis vel conservatur. incomposite, interdum elongate et crispans in contactus Holotype China, Guangxi, Baise, from the soil of dawn, antheridial coma interdum crispans quod pandus vel Y.Y. Long, 5. 10. 2009, HMAS242232. Living culture universa lobed; Oosporae singulae, interdum duobus, apleur- (QBS123) deposited in the Department of Plant Protection, oticae, interdum pleuroticae, globosa, 11.3–23.8 μmdiam. College of Agriculture, Guangxi University. Mycol Progress

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Fig. 2 Asexual and sexual reproductive bodies of Pythium baisense. a immediately from the hyphae; m wavy and curved antheridial stalk; Sausage-shaped appressoria; b released zoospores; c globose sporan- n intercalary oogonium with antherium on complexly lobed stalk; o gium; d lateral sporangium with a papilla; e intercalary sporangium; f antheridia originating immediately from the hyphae; q clustering of elongate sporangium; g globose oogonium; h–i, p sac-like oogonium several antheridial cells around the oogonium; r 2 oospores in an with aplerotic oospore; j, l antheridium contact with oogonium in the oogonium; s elongated antheridial cell wavy in contour; s 2 oogonia neighbourhood of the oogonial stalk; k oogonium originating clustering in a small group. Scale bar 10 μm

Except the type strain, isolate XGL324 collected from up to 6.3 μm diam. Colonies on PCA showed a vague the soil of lawn in Guilin, Guangxi, China, by Y.Y. Long, rosette pattern, average growth rates 9.5 mm day−1 at 15°C, 15.7.2009. Isolate CLZ223 collected from the soil of lawn 21 mm day−1 at 20°C, 30 mm day−1 at 25°C, 36 mm day−1 in Liuzhou, Guangxi, China, by Y.Y. Long, 3.5.2009. Living at 30°C, 20 mm day−1 at 35°C, no growth at 8°C and 39°C, culture all deposited in the Department of Plant Protection, but when returned to room temperature both of them started College of Agriculture, Guangxi University. to grow again. Cardinal temperatures: minimum 8°C, The oomycete grows well on CMA, PCA and PDA. Its optimum 30°C, maximum 39°C. Appressoria sausage- mycelium in water hyaline, well branched, the main hyphae shaped, sometimes catenulate, often present (Fig. 2a). Mycol Progress

Zoospores formed in sterile water at 25°C (Fig. 2b). contact with the oogonium in the neighbourhood of the Sporangia globose, subglobose to elongate, at times with oogonial stalk (Fig. 2g, j, l–q, s). Oospores mostly a papilla, terminal or intercalary, 5–15 μm in diameter aplerotic, sometimes nearly plerotic, globose, sometimes (average 12.3 μm) (Fig. 2c–f). The female gametangia subglobose, rarely peanut-shape or elongate, 11.3–23.8 μm (oogonia) globose or subglobose, sometimes sac-like, in diameter (average 18.5 μm), generally single, sometimes smooth-walled, originating directly from the hyphae or 2 in an oogonium, colorless (Fig. 2k–r). terminal on short side branches, occasionally lateral, 13.8– 28.8 μm in diameter (average 21.2 μm), sometimes 2–4 Phylogenetic analyses clustering in small groups (Fig. 2g–l, s–t). The female gametangia filled with dense, coarsely granulated proto- The ITS sequences of two new species, 32 reference taxa plasm. The male gametangia (antheridia) monoclinous and of Pythium (Pythiaceae) and Phytopythium boreale, P. diclinous, sometimes terminal on wavy and curved anther- helicoides and P. vexans (Peronosporaceae) used as the idial stalks or originating directly from the hyphae or outgroup were included in the phylogenetic analyses. In crowding around the oogonia, making it very difficult to the alignment of the 37 taxa, the data matrix comprised determine their origin, type of contact and numbers 1,279 characters, of which 432 (33.8%) characters were (Fig. 2g, m, o, q). Antheridium occasionally with complex- constant and 666 (52.1%) were parsimony informative. ly lobed stalk (Fig. 2n). Antheridial cells bell-shaped, club- The maximum parsimony (MP) analysis of the alignment shaped or irregular, occasionally elongated and wavy in dataset was subjected to a heuristic search for the most contour, 1–8 per oogonium, often making apical or lateral parsimonious trees and a strict consensus tree was

99/* AY598682 100/71 AY598625 100/97 Pythium scleroteichum AY598680 100/55 Pythium monospermum AY598621 Pythium porphyrae AY598673 99/58 55/* AY598622 98/70 Pythium acanthophoron AB355644 85/63 100/100 Pythium periplocum AY598670 100/100 AY598618 96/* AY598617 EF016855 58/* 100/100 Pythium helicandrum AY598653 Pythium dimorphum AY598651 100/100 var sporangiiferum AY598657 80/* AY598655 Pythiaceae 100/99 Pythium attrantheridium AY286014 71/* AY598701 91/* Pythium paddicum AY598707 73/* Pythium nagaii AY598705 100/100 Pythium takayamanum AB362163 99/* Pythium carolinianum AY987038 Pythium bifurcatum AY083935 100/100 Pythium buismaniae AY598659 100/100 Pythium polymastum AY598660 100/97 Pythium uncinulatum AY598712 98/97 Pythium mastophorum AY598661 100/98 Pythium megalacanthum AB512782 Pythium breve FR751317 100/97 Pythium nunn AY598709 94/69 100/100 Pythium orthogonon AY598710 99/* Pythium campanulatum AY256904 82/* Pythium baisense QBS123 100/100 Pythium perplexum AY598658 Pythium cystogenes AY707985 100/75 Phytopythium helicoides AY598665 100/100 Phytopythium boreale AY598662 Phytopythium vexans AY598713 Peronosporaceae 0.1

Fig. 3 Bayesian tree based on ITS (ITS1, 5.8S, ITS2) region support calculated from 1,000 replicates (right). *Indicates lack of sequences. The tree was rooted with Phytopythium helicoids, P. support or support less than 50% for a particular clade. Bar 0.1 boreale and P. vexans. The numbers at each branch point represented expected changes per site Bayesian posterior probabilities (left) and percentage bootstrap Mycol Progress obtained from the five equally most parsimonious trees and a strict consensus tree was obtained from four equally (CI=0.4612, RI=0.6611, RC=0.3049, HI=0.5388). The most parsimonious trees (CI=0.6113, RI=0.6113, RC= same alignment dataset was also performed using MrBayes 0.3736, HI=0.3887). The same alignment dataset was also program, and a similar topology as the strict consensus tree performed using MrBayes program, and a similar topology was obtained. Therefore, a Bayesian tree with posterior as the strict consensus tree was obtained. Therefore, a probabilities (BPP) and parsimony bootstrap values at Bayesian tree with posterior probabilities and parsimony branches is shown in Fig. 3. In the phylogenetic tree, P. bootstrap values at branches was shown in Fig. 4.Inthe breve formed a clade with P. buismaniae Plaats-Niterink, P. phylogenetic tree, P. breve formed a clade with P. buisma- mastophorum Drechsler, P. megalacanthum de Bary, P. niae, P. mastophorum and P. uncinulatum (BBP 93%, MP polymastum Drechsler and P. uncinulatum Plaats-Niterink 59%). P. baisense clustered together with P. perplexum with with strong support (BPP 100%, MP 98%). P. baisense relatively low support. formed a clade with P. cystogenes De Cock & Lévesque, P. perplexum H. Kouyeas & Theoh, P. campanulatum R. Mathew, K.K. Singh & B. Paul, P. orthogonon Ahrens and Discussion P. nunn Lifsh, Stangh & R.E.D. Baker with 100% support of BPP and MP.These two new species formed a single branch, The isolate of P. breve was obtained from the lawn soil respectively. collected in Nanning, Guangxi Province, China. This new Phylogenetic study was also performed based on COXI species is characterized by smooth oogonia crowded by sequences from two new species and ten reference Pythium many antheridia, the large antheridial cells to be sometimes species, with Phytopythium boreale, P. ostracodes and P. wavy in contour, and the antheridial stalks entwining the vexans as the outgroup. In the alignment of the 15 taxa, the hyphae nearby the oogonia occasionally with several turns. data matrix comprised 593 characters, of which 420 (70.8%) Although many Pythium species have some similarly characters were constant and 127 (21.4%) were parsimony morphological features with P. breve, the distinctly wavy informative. The maximum parsimony analysis was sub- antheridial stalks were reported only in P. bifurcatum B. jected to a heuristic search for the most parsimonious trees Paul, P. scleroteichum Drechsler, and P. takayamanum M.

Pythium buismaniae EU350527 100/98 100/89 Pythium uncinulatum EU350530

93/59 Pythium mastophorum EU350523

85/* Pythium breve FR774196

Pythium baisense FR774198 97/62 53/* Pythium perplexum EU350520 Pythiaceae Pythium nunn EU350522 68/67 100/98 Pythium orthogonon EU350521

Pythium echinulatum EU350526

75/84 Pythium acrogynum EU350528 EU350528

Pythium aphanidermatum AY129164 100/63 Pythium oopapillum FJ655181

Phytopythium boreale EF408872 91/84 100/100 Phytopythium ostracodes EF408874 Peronosporaceae

Phytopythium vexans EF426548 0.1

Fig. 4 Bayesian tree based on COXI sequences. The tree was rooted and percentage bootstrap support calculated from 1,000 replicates with Phytopythium boreale, P. ostracodes and P. vexans. The numbers (right). *Indicates lack of support or support less than 50% for a at each branch point represented Bayesian posterior probabilities (left) particular clade. Bar 0.1 expected changes per site Table 1 A comparison of Pythium breve with morphologically similar species

Character P. breve P. bifurcatum P. scleroteichum P. takayamanum P. palingenes

− − − − Growth 16 mm day 1 at 25°C 11 mm day 1 at 25°C 20 mm day 1 at 25°C 15.4 mm day 1 at 30°C Unknown rate on PCA Sporangia Globose or subglobose, 11.3–31.3 Globose to cylindrical, 25– Never observed Subglobose, 13–28 μm diam (av. Subglobose or ovoid, 24–42×18– μm diam (av. 21 μm), terminal, 50 μm diam (av. 35 μm), 19.4 μm), terminal or intercalary 33 μm diam (av. 33×29 μm), sometimes intercalary mostly intercalary papillate, terminal Oogonia Globose or subglobose, sometimes Globose, smooth, 17–36 μm Globose, smooth, (17) 21–29 Globose to subglobose or sac-like, Subglobose, smooth, (19) 28–40 (41) sac-like, smooth, 22.5–31.3 μm diam diam (av. 26.7 μm), terminal, (34) μm diam (av. ellipsoidal, smooth, 12–21 μm μm diam (av. 34 μm), terminal, (av. 28.2 μm), intercalary or terminal subterminal, rarely intercalary 25.3 μm), terminal or diam (av. 14.9 μm), terminal or sessile, intercalary or unilaterally intercalary intercalary Antheridia 1–8 per oogonium, diclinous, Often 2 per oogonium, 1–5 per oogonium, 1–2 per oogonium, monoclinous, (1) 2 (4) per oogonium, diclinous occasionally monoclinous hypogynous or monoclinous monoclinous, sometimes occasionally diclinous, sessile sessile or monoclinous diclinous Oospores Globose, 17.5–28.8 μm diam (av. Globose, 16–35 μm diam (av. Globose, (13) 16–25 (30) μm Globose, 11–19 μm diam (av. Subglobose, (18) 26–36 (37) μm 24.2 μm), plerotic or nearly plerotic, 24.9 μm), plerotic, sometimes diam (av. 20 μm,) 13.8 μm), aplerotic to nearly diam (av. 31.3 μm), aplerotic, 1 per sometimes aplerotic, 1 (2) per aplerotic, 1 (2) per oogonium aplerotic, 1 per oogonium plerotic, 1 per oogonium oogonium oogonium Reference This study Paul 2003a Van der Plaats-Niterink 1981 Senda et al. 2009 Van der Plaats-Niterink 1981

Table 2 A comparison of Pythium baisense with related species

Character P. baisense P. hypoandrum P. perplexum P. campanulatum

− − Growth 30 mm day 1 at 25°C Unknown 15 mm day 1 at 25°C 15 mm day−1 at 25°C rate on PCA Sporangia Globose, subglobose to elongate, 5–15 μm Globose or subglobose, 13–29 μm Globose, elongated, 13–41 μm Globose to oval, somewhat elongated, 11–25 μm diam (av. 12.3 μm) , terminal or intercalary diam (av. 20.4 μm), mostly terminal, diam (av. 17 μm), terminal or diam (av. 18 μm), terminal or intercalary sometimes intercalary intercalary, occasionally in series Oogonia Globose or subglobose, sometimes sac-like, Globose, smooth, 18–28 μm diam (av. Globose, smooth, 16–27 μm Globose, smooth, 13–27 μm diam (av. 20.6 μm), smooth, 13.8–28. 8 μm diam (av. 21.2 μm), 20.5 μm), mostly terminal, sometimes diam (av 20 μm), terminal on mostly terminal terminal or sessile, occasionally lateral intercalary lateral branches Antheridia 1–8 per oogonium, monoclinous and diclinous, 1 (4) per oogonium, monoclinous or 1–2 per oogonium, mostly 1–5 per oogonium, monoclinous or diclinous, sometimes sessile or crowding around the diclinous; Antheridial cells subelliptic or monoclinous, originating at a many wrap around the oogonia with a very oogonia; Antheridial cells bell-shaped, club- pseudofusiform, contact with the short distance from the complicated knot which disappears at last shaped or irregular, occasionally elongated and oogonium in the neighbourhood of the oogonium; Antheridial cell leaving 1–2 antheridial cell; Antheridial cells wavy in contour, often contact with the oogoni- oogonial stalk bell-shaped campanulate or at times elongated Progress Mycol um in the neighbourhood of the oogonial stalk Oospores Globose or subglobose, 11.3–23. 8 μm diam Globose, 11–25 μm diam (av. 16.7 μm), Globose, 12–20 μm diam Globose, 11–20 μm diam (av. 16.8 μm), aplerotic, (av. 18.5 μm), aplerotic or nearly plerotic aplerotic (av. 16 μm), aplerotic sometimes plerotic Reference This study Yu 1998 Galland Paul 2001 Mathew et al. 2003 Mycol Progress

Senda (van der Plaats-Niterink 1981; Paul 2003a; Senda et curved and waved antheridial stalks. However, P. baisense al. 2009). However, P. breve can be differentiated from P. with antheridial stalks occasionally complexly lobed is bifurcatum by the absence of hypogynous and monoclinous obviously distinguished from P. breve with antheridial antheridia, from P. takayamanum due to the absence of the stalks entwining the hyphae nearby the oogonia with ellipsoid oogonia with constricted area, and from P. several turns. The results of the phylogenetic analyses of scleroteichum by the lack of sporangia (Table 1). In ITS and COXI sequences indicated that P. baisense was addition, P. breve is morphologically similar to P. palin- clearly separated from P. breve (Figs. 3 and 4). Due to a genes Drechsler in the shape of wavy antheridial cells and lack of the molecular data of P. hypoandrum, we could not the oogonial stalks coiled by antheridial stalks in a few analyze the phylogenetic relationship between P. baisense turns, but P. breve differs from P. palingenes in the absence and P. hypoandrum. of sessile oogonia and proliferating or forming secondary In addition, P. baisense had closer relationships with P. sporangia on branches originating just below the septum of perplexum and P. campanulatum compared to the other the primary sporangia (van der Plaats-Niterink 1981). species in the phylogenetic analyses of ITS and COXI Furthermore, P. breve has smaller sporangia, oogonia and sequences (Figs. 3 and 4), but P. baisense had low DNA oospores compared to P. palingenes (Table 1). The results of sequence similarity with P. perplexum (84.4% ITS identity the phylogenetic analysis of ITS sequences indicated that P. with AY598658 and 93.7% COXI identity with EU350520) breve was clearly separated from these three morphologi- and with P. campanulatum (79.9% ITS identity with cally similar species. Due to the lack of the molecular data AY256904). Furthermore, in morphology, P. baisense with of P. palingenes, we could not analyze the phylogenetic monoclinous and diclinous antheridia and more (1–8) relationship between P. breve and P. palingenes. antheridia conspicuously differs from P. perplexum with P. breve had a closer relationship with P. megalacanthum mostly monoclinous antheridia and fewer (1–2) antheridia and P. mastophorum compared to the other species in the (Galland and Paul 2001) and from P. campanulatum with phylogenetic trees (Figs. 3 and 4), but P. breve had low fewer (1–5) antheridia which wrap around oogonia with a DNA sequence similarities with P. megalacanthum (64.9% very complicated knot (Mathew et al. 2003) (Table 2). ITS identity with AB512782) and with P. mastophorum In conclusion, the distinctively morphological and (65.4% ITS identity with AY598661 and 92.9% COXI molecular characteristics of P. breve and P. baisense, identity with EU350523). Furthermore, in morphology, P. compared to other Pythium species described, certify that breve, with smooth-walled oogonia and smaller sporangia, these two taxa are new to science. oogonia and oospores, conspicuously differs from P. megalacanthum and P. mastophorum with ornamented Acknowledgements This work was supported by the National Natural oogonia and bigger sporangia, oogonia and oospores (van Science Foundation of China (No. 30670012), the Guangxi Natural Science Foundation (No. 0640013) and the Plans for Construction of der Plaats-Niterink 1981). Scientific Topnotch and Innovation Team in Guangxi University. P. baisense (strains CLZ223, XGL324 and QBS123) were collected from the lawn soils in Guilin, Liuzhong and Baise of Guangxi in China, respectively. These three References isolates were isomorphic and had almost the same growth rates. 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