Biodiversity Data Journal 9: e65227 CO) doi: 10.3897/BDJ.9.e65227 open access Taxonomic Paper Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular phylogeny and pathogenicity Jia-Jia Chen*, Hui Feng§, Jian Yu*, Wenwu Ye§, Xiaobo Zheng§ + College of Landscape Architecture, Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang 212400, China § Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China Corresponding author: Jia-Jia Chen (jiaj.chen19@yahoo.com) Academic editor: Marco Thines Received: 28 Feb 2021 | Accepted: 18 Apr 2021 | Published: 22 Apr 2021 Citation: Chen J-J, Feng H, Yu J, Ye W, Zheng X (2021) Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular phylogeny and pathogenicity. Biodiversity Data Journal 9: e€65227. https://doi.org/10.3897/BDJ.9.e65227 Abstract Background Soybean (Glycine max) is a major source of edible oil and protein. A novel species of the genus Pythium, Pythium huanghuaiense, isolated from soybean seedlings in China, is described and illustrated on the basis of morphological characters and molecular evidence. New information Pythium huanghuaiense sp. nov. is closely related to species of the genus Pythium in clade F, as evidenced by the presence of hyphal swellings and its relatively rapid morphological growth. However, it differs by having relatively small sporangia and plerotic or nearly plerotic and thin-walled oospores. A pathogenicity test confirmed the newly-identified species as a pathogen of soybean. © Chen J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Chen J et al Keywords Cox1, ITS, oomycete, Pythium clade F Introduction Species of the genus Pythium Pringsheim are diverse, occupying a variety of habitats (van der Plaats-Niterink 1981). The genus was established by Pringsheim (1858), based on Pythium monospermum Pringsh. and is characterised by globose, oval, ellipsoidal, elongated, filamentous or toruloid sporangia and the development of zoospores in a vesicle formed at the tip of a discharge tube derived from a sporangium (van der Plaats-Niterink 1981). There are more than 160 species of Pythium (Long et al. 2012, Long et al. 2014, Uzuhashi et al. 2015, Ueta and Tojo 2016, Chen et al. 2020), which includes many important plant pathogens that frequently cause seed, seedling and root rot in economically-important crops, such as soybean (Glycine max), wheat (Triticum spp.) and corn (Zea mays) (Wang et al. 2003, Wrather and Koenning 2006). Some Pythium spp. are important pathogens of animals, while others are beneficial as biological control agents that protect against pathogenic fungi (van der Pladats-Niterink 1981, Ali-Shtayeh and Saleh 1999). To date, 74 species of Pythium have been reported in China (Ho 2013, Long et al. 2014, Chen et al. 2020). Huang-Huai Valley is one of the main areas of soybean farming in China, covering an enormous area in Shandong, Anhui, Jiangsu and Henan Provinces between the Yellow River and the Haihe River. During the studies on the diversity of Pythium in the Huang- Huai Valley, a novel species of clade F was identified, based on morphological characters and molecular phylogenetic analyses of internal transcribed spacer (ITS) and cytochrome c oxidase subunit | (Cox1) sequence data. The novel species is described and illustrated in this work. Moreover, comparisons of the novel species with morphologically and phylogenetically related species are also provided. Materials and methods Isolates During April and August 2016, 60 plants of soybean cultivar 'Hefeng 47' exhibiting seedling blight, damping off and root rot were collected from three fields in the Huang-Huai region of China. 'Hefeng 47' is commonly grown in the Huang-Huai Valley. The fields were located in Jining of Shandong Province, Suzhou of Anhui Province and Nanjing of Jiangsu Province, which are representative geographic locations in the Huang-Huai region. Soybean plants were sampled from fields at approximately 10 m intervals along a 150 m transect laid out in a “VW” pattern. Soybean plants were washed three times with sterile water and six sections of 0.5—-1 cm length were cut from the roots of each plants using a sterile scalpel. One section was taken Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular ... 3 from the root tip, one from the interface between the hypocotyl and soil and the others at either the middle of the root or a symptomatic area along the length of the root. The sections were blotted dry and embedded in selective V8 juice agar (V8A) containing rifampicin (50 mg/l), ampicillin (50 mg/l) and pentachloronitrobenzene (50 mg/l) and incubated for 2-3 days in the dark at 25°C. When mycelial growth was observed, cultures were purified by transferring a small piece of medium with mycelium at the edge of a colony to fresh medium or by transferring a single hyphal tip on to water agar three times. Morphology and growth rate The cultures studied were deposited in the Herbaria of the Institute of Microbiology, Beijing Forestry University (BJFC), Beijing, China; the College of Plant Protection, Nanjing Agricultural University (NJAU), Nanjing, China; and the College of Landscape Architecture, Jiangsu Vocational College of Agriculture and Forestry (JAFLA), Zhenjiang, China. Purified isolates were examined after incubation for 2-3 days at 25°C on V8A in the dark. Colony patterns of the representative isolate of the novel species were examined after incubation for 3 days at 25°C on corn meal agar (CMA), potato carrot agar (PCA) and V8A media (Miller 1955, van der Plaats-Niterink 1981). Isolates were transferred to sterilised distilled water to induce sporulation. Fifty measurements were taken for each morphological feature, such as sporangia, oogonia and oospores. Cardinal temperatures were examined on PCA as described by van der Plaats-Niterink (1981) and growth rates were measured after 24 h of incubation. Each isolate was incubated on PCA media at 5—40°C with intervals of 5°C. When no growth was observed, the intervals were reduced from 5°C to 2°C or 1°C and the culture was returned to room temperature to ensure that the strain could start growing again. The experiment was repeated twice using a single plate per repetition. Molecular phylogeny DNA extraction, amplification, sequencing and sequence alignment A cetyl trimethylammonium bromide (CTAB) rapid plant genome extraction kit (FH Plant DNA kit, Demeter Biotechnologies Co. Ltd, Beijing, China) was used to extract total genomic DNA from purified isolates and the polymerase chain reaction (PCR) was performed according to the manufacturer’s instructions (Cui et al. 2019). PCR amplification was Carried out in 30-ul volumes consisting of 1 ul of DNA template, 1 ul of each 10 uM forward and reverse primer, 15 ul of 2 x Taq PCR Master Mix and 12 ul of deionised water. The ITS region (approximately 900 bp) was amplified using the universal primers ITS5 (GGAAGTAAAAGTCGTAACAAGG) and ITS4 (TCCTCCGCTTATTGATATGC) (White et al. 1990). The Cox1 gene ((approximately 700 bp) was amplified using the universal primers OomCoxI-Levlo (CYTCHGGRTGWCCRAAAAACCAAA) and OomCoxl-Levup (TCAWCWMGATGGCTTTTTTCAAC) (Robideau et al. 2011) . PCR conditions for ITS were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of 94°C for 40 s, 54°C for 45 s and 72°C for 1 min and a final extension of 72°C for 10 min. PCR conditions for Cox1 were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 1 min and a final extension of 72°C for 4 Chen J et al 10 min (Rahman et al. 2014). PCR products were purified and sequenced by Genscript (Nanjing, China) using the same primers. Sequences, generated in this study, were aligned with additional sequences downloaded from GenBank (Table 1) using ClustalX (Thompson et al. 1997) and manually adjusted in BioEdit (Hall 1999). The sequence alignment has been deposited in TreeBase (http://purl.org/phylo/treebase; submission ID $24209). Table 1. A list of species, cultures and GenBank accession numbers of sequences used in this study. Species name Sample no. Locality GenBank accession no. ITS Cox1 Pythium abappressorium CBS 110198 USA HQ643408 HQ708455 P. acanthophoron CBS 337.29 USA HQ643413 HQ708460 P. alternatum CBS 139279 Japan AB998876 AB998877 P anandrum CBS 285.31 - HQ643435 HQ708482 P. attrantheridium DAOM 230383 Canada HQ643477 HQ708524 P. baisense HMAS 242232 China FR775440 FR774198 P. barbulae CBS 139569 Japan LC028389 LC028392 P. brachiatum UZ00736 Japan KJ995581 KJ995593 P. canariense CBS 112353 Spain HQ643482 HQ708528 P. cryptoirregulare CBS 118731 USA HQ643515 HQ708561 P. cylindrosporum CBS 218.94 Germany HQ643516 HQ708562 P. debaryanum CBS 752.96 UK HQ643519 HQ708565 P. emineosum BR 479 UK GQ244427 GQ244423 P. grandisporangium CBS 286.79 USA HQ643546 HQ708590 P. huanghuaiense Chen 94 China MF984118 MF984155 P. huanghuaiense Chen 95 China MF984119 MF984156 P. huanghuaiense Chen 96 China MF984120 MF984157 P. huanghuaiense Chen 99 China MF984121 MF984158 P. huanghuaiense Chen 100 China MF984122 MF984159 P inflatum CBS 168.68 USA HQ643566 HQ708610 P. insidiosum CBS 574.85 Costa Rica HQ643570 HQ708614 P. intermedium CBS 266.38 Netherlands HQ643572 HQ708616 P irregulare CBS 250.28 Netherlands HQ643596 HQ708640 P junctum UZ00732 Japan KJ995576 KJ995595 P. kunmingense CBS 550.88 China HQ643672 HQ708716 P. lucens CBS 113342 UK HQ643681 HQ708725 Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular ... Species name Sample no. Locality GenBank accession no. ITS Cox1 P. macrosporum CBS 574.80 Netherlands HQ643684 HQ708728 P. mamillatum CBS 251.28 Netherlands HQ643687 HQ708731 P. marsipium CBS 773.81 Netherlands HQ643690 HQ708734 P minus CBS 226.88 United Kingdom HQ643696 HQ708740 P monospermum CBS 158.73 United Kingdom HQ643697 HQ708741 P. nodosum CBS 102274 France HQ643709 HQ708753 P nunn CBS 808.96 USA HQ643711 HQ708755 P. oligandrum CBS 382.34 United Kingdom HQ643715 HQ708759 P. paroecandrum CBS 157.64 Australia HQ643731 HQ708772 P. periplocum CBS 289.31 USA HQ643743 HQ708784 P plurisporium CBS 100530 USA HQ643749 HQ708790 P. prolatum CBS 845.68 USA HQ643754 HQ708795 P recalcitrans CBS 122440 Spain DQ357833 EF426549 P. sp. "balticum" CBS 122649 Sweden HQ643478 HQ708525 P. spiculum CBS 122645 France HQ643790 HQ708831 P. spinosum CBS 122663 India HQ643791 HQ708832 P. splendens CBS 462.48 USA HQ643795 HQ708836 P. sukuiense CBS 110030 Taiwan HQ643836 HQ708877 P. sylvaticum CBS 453.67 USA HQ643845 HQ708886 P terrestris CBS 112352 France HQ643857 HQ708898 P. ultimum var. ultimum CBS 398.51 Netherlands HQ643865 HQ708906 P. viniferum CBS 119168 France HQ643956 HQ708997 P. wuhanense HMAS 243736 China HE862398 HE862402 Saprolegnia parasitica CBS 113187 Russia HQ644005 HQ709046 S. parasitica CBS 540.67 United Kingdom HQ644000 HQ709041 New sequences are shown in bold. Phylogenetic analyses Phylogenetic analysis was conducted as descibed by Cui et al. (2019). Maximum Likelihood (ML) and Bayesian Inference (Bl) methods were also used to generate phylogenetic trees from the combined ITS and Cox1 dataset. Two isolates of Saprolegnia parasitica Coker were used as outgroups (Villa et al. 2006). Substitution models suitable for ITS partition and Cox’ partition of the dataset were determined using the Akaike Information Criterion implemented in MrMODELTEST2.3 (Nylander 2004). The General Time Reversible + proportion Invariant + Gamma (GTR+I+G) substitution model was selected for each partition. RAXML v.7.2.6 (Stamatakis 2006) was used for ML analysis. All parameters in the ML analysis used the default setting and statistical support values were 6 Chen J et al obtained using non-parametric bootstrapping with 1000 replicates. A Bayesian tree was inferred using MrBayes3.1.2 (Ronquist and Huelsenbeck 2003), with a general time reversible model of DNA substitution and an invgamma distribution rate variation across sites. Eight Markov chains were run from the random starting tree for 2 million generations of the combined ITS and Cox1 dataset and sampled every 100 generations. Chain convergence was determined using Tracer v.1.5 (http://tree.bio.ed.ac.uk/software/tracer/) to confirm sufficiently large ESS values (> 200). The burn-in parameter was set to discard the first 25% of trees. A majority rule consensus tree of all remaining trees was generated for each analysis. Branches receiving bootstrap values for ML and Bayesian posterior probabilities (BPP) greater than or equal to 75% (ML) and 0.95 (BPP) were considered significantly supported. Phylogenetic trees were visualised using FigTree v.1.4.2 (http:// tree.bio.ed.ac.uk/software/figtree/). Pathogenicity Pathogenicity was confirmed using the hypocotyl slit inoculation method (Dorrance et al. 2008). Three-day-old V8A plugs (1.5 cm diam.) of isolate Chen 94 were used to infect the soybean cultivar 'Hefeng 47'. Five soybean seedings inoculated with uncolonised agar plugs served as controls. The inoculated soybean seedings (five plants for the isolate) were incubated at 25°C with a 12-h photoperiod in a greenhouse for 4-5 days. Experiments were conducted in triplicate. Taxon treatment Pythium huanghuaiense Jia J. Chen & X.B. Zheng 2021, sp. n. ° MycoBank 822954 Materials Holotype: a. scientificName: Pythium huanghuaiense; class: Oomycetes; order: Pythiales; family: Pythiaceae; genus: Pythium; country: China; stateProvince: Jiangsu; locality: Nanjing, Jiangning District, Pengfu Village; year: 2016; month: 4; day: 29; habitat: on seedlings of Glycine max; recordedBy: Jiajia Chen; identifiedBy: Jiajia Chen; type: chen 94 (BUFC-C 1993, metabolically inactive culture); language: en Paratypes: a. scientificName: Pythium huanghuaiense; class: Oomycetes; order: Pythiales; family: Pythiaceae; genus: Pythium; country: China; stateProvince: Jiangsu; locality: Nanjing, Jiangning District, Pengfu Village; year: 2016; month: 4; day: 1; habitat: on seedlings of Glycine max; recordedBy: Jiajia Chen; identifiedBy: Jiajia Chen & Xiaobo Zheng; type: Chen 95 (NJAU-JN18, JAFLA 95; metabolically inactive culture) & Chen 96 (NJAU-JN19, JAFLA 96; metabolically inactive culture); |anguage: en b. scientificName: Pythium huanghuaiense; class: Oomycetes; order: Pythiales; family: Pythiaceae; genus: Pythium; country: China; stateProvince: Anhui; locality: Suzhou; year: 2016; month: 7; habitat: on seedlings of Glycine max; recordedBy: Jiajia Chen; Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular ... 7 identifiedBy: Jiajia Chen & Xiaobo Zheng; type: Chen 99 (NJAU-JN30, JAFLA 99; metabolically inactive culture); language: en Cc. scientificName: Pythium huanghuaiense; class: Oomycetes; order: Pythiales; family: Pythiaceae; genus: Pythium; country: China; stateProvince: Shandong; locality: Jining; year: 2016; month: 8; habitat: on seedlings of Glycine max; recordedBy: Jiajia Chen; identifiedBy: Jiajia Chen & Xiaobo Zheng; type: Chen 100 (NJAU-JN65, JAFLA 100; metabolically inactive culture); language: en Other material: a. scientificName: Pythium huanghuaiense; class: Oomycetes; order: Pythiales; family: Pythiaceae; genus: Pythium; country: China; stateProvince: Jiangsu; locality: Nanjing, Jiangning District, Pengfu Village; year: 2016; month: 4; day: 29; habitat: on seedlings of Glycine max; recordedBy: Jiajia Chen; identifiedBy: Jiajia Chen & Xiaobo Zheng; type: JAFLA 94, NJAU-JN11 (isotypes, metabolically inactive culture); language: en Description Pathogenic on soybean. Colonies submerged, with a cottony pattern on CMA, a rosette pattern on PCA and a cottony pattern on 10% V8A (Fig. 1). Average growth rates of 32.8 mm/day at 25°C on PCA (Fig. 2). Cardinal temperatures: minimum 4°C, optimum 25°C, maximum 37°C. Main hyphae hyaline, aseptate, up to 5.0 um wide. Hyphal swellings globose, sub-globose, obturbinate to pyriform, mostly terminal or sometimes intercalary, 15-22.5 x 13.5-20 (mean 19 x 17.5) um. Sporangia and zoospores not observed. Homothallic; oogonia globose, smooth or with a projection, terminal or intercalary, 12.5-18 ym (mean 15.5 ym) in diameter. Antheridia mostly monoclinous, sometimes hypogynous, one to two per oogonium; antheridial stalks unbranched, arising at various distances from oogonia; antheridial cells sub-globose, club-shaped or fist-shaped, making broad or narrow apical contact with oogonia. Oospores plerotic or nearly plerotic, globose, 11.5-17 um (mean 14.5 ym) in diameter, hyaline. Oospore wall 0.5—1.5 um (mean 1.1 pm) thick (Fig. 3). Figure 1. EESl Colony patterns of Pythium huanghuaiense (Chen 94) on A. CMA; B. PCA; and C. V8A. Etymology With reference to the distribution of the species in the Huang-Huai area of China. Chen J et al Notes Pythium huanghuaiense can be distinguished morphologically from its closest relatives, including P mamillatum Meurs, P. paroecandrum Drechsler, P spiculum B. Paul and P wuhanense Y.Y. Long, J.G. Wei & L.D. Guo, by its narrower hyphae and relatively higher maximum growth rate. Additional differences between the novel species and other related species are listed in Table 2. Table 2. Morphological description of Pythium huanghuaiense and the most closely related species. Pythium P mamillatum P P. spiculum P. wuhanense huanghuaiense paroecandrum (Chen 94) Width of Up to 5 Up to 6.5 Up to 9 Up to 6 Up to 7.5 hyphae (um) Sporangia/ Globose, sub- Globose, Globose or Globose, ovoid, Globose, hyphal globose, broadly ovoid __ ellipsoidal, cylindrical and sometimes swellings obturbinate to or ellipsoidal, —_intercalary or at times cylindrical to pyriform, intercalary or __ terminal peanut-shaped, elongated, mostly terminal lateral mostly mainly intercalary, or sometimes intercalary to often catenulate intercalary catenulate, with oogonia, rarely terminal occasionally terminal or lateral Oogonia (um) 12.5-18 (av. 15-18 (av. 16), 17-24 (av. 19), 13-22 (av. 10-20 (av. 17.7), 15.5), intercalary or __intercalary, often 15.6), mostly intercalary, terminal or terminal in chains and mostly often catenulate intercalary rarely terminal intercalary orin with sporangia and chains antheridia, sometimes terminal or lateral Oogonium Absent Present Absent Present Absent ornamentation Antheridia Mostly Mostly Mostly Monoclinous Monoclinous, monoclinous, monoclinous, §monoclinous, hypogynous or sometimes infrequently sometimes diclinous hypogynous diclinous diclinous Oospores (um) Plerotic or nearly Plerotic , Aplerotic , Plerotic or Aplerotic , plerotic, 12-15 (av.14) 15-21 (av.17) — aplerotic , 7.5-17.5 (av. 14.5) 11.5-17 (av. 8-18 14.5) Oospore wall 0.5-1.5 0.8-1.4 1-1.5 0.5—1 0.5-1 thickness (ym) Double Absent Absent Absent Present Present oospores Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular ... Pythium P mamillatum P P. spiculum P. wuhanense huanghuaiense paroecandrum (Chen 94) Cardinal Min 4°C, Min 5°C, Min 5°C, Unknown Min 4°C, optimum temperature optimum 25°C optimum 25°C — optimum 25°C 28-30°C and max and max 37°C and max 30—.—s_ and max 35°C 35°C 35°C Daily growth 32 25 20-25 25 50 rates on PCA at 25°C (mm) Reference This study van der Plaadts- vander Pladts- Paul et al. Long et al. (2014) Niterink (1981) Niterink (1981) (2006) 35 —— Chen 94 A »eem>> Chen95 S ~§ 25 —— Chen 96 z —— Chen 100 rau Be “ 3 € 10 oe) 5 0 4 5 10° 25" 20 =25. 30 35° 37 Temperature (°C) Figure 2. EES Mycelial growth rate of isolates of Pythium huanghuaiense Chen 94, 95, 96, 99 and 100 on PCA at different temperatures. Analysis Isolates Five cultures of Pythium (Chen 94-96, Chen 99 and Chen100), representing an unknown species of Pythium, were obtained from soybean plant samples collected from three fields in three cities during April and August 2016. Molecular phylogeny Five ITS and Cox1 sequences were newly generated for this study and their accession numbers are available in GenBank (Table 1). BLAST analyses of the ITS and Cox 10 Chen J et al sequences of the five isolates, described here as Pythium huanghuaiense, showed the best phylogenetic matches with species of clade F in Pythium (Levesque and Cock 2004). o =x Asexual and sexual reproductive bodies of Pythium huanghuaiense (Chen 94). A. Obturbinate hyphal swelling; B. globose hyphal swelling; C. sub-globose hyphal swelling; D. pyriform hyphal swelling; E, F. intercalary hyphal swellings; G, H. oogonia with a projections; I. nearly plerotic oospore; J. elongated antheridial cell wavy in contour; K. intercalary oogonium; L. Nearly plerotic oospore and two antheridia. Bars: A-E 10 ym; G—J 5 um. Figure 3. EESl ML and BI analyses yielded similar tree topologies and only the ML tree is shown (Fig. 4). The five isolates of the novel species, P huanghuaiense, formed a well-supported lineage (100% ML and 1 BPP), indicating that they are phylogenetically distinct from other species of clade F in Pythium (Fig. 4). Pathogenicity Pythium huanghuaiense (Chen 94) significantly stunted and reduced the growth of soybean seedlings compared with uninoculated controls (Fig. 5). To fulfil Koch’s postulates, pieces of diseased tissues obtained from inoculated plants were placed on V8A to re- isolate the causal agent. Pythium huanghuaiense could be recovered from the diseased soybean seedlings and was identified, based on morphological characteristics and comparisons of ITS and Cox1 sequences. According to Feng et al. (2020), pathogenicity tests, using dish cultures of FP) huanghuaiense isolates and pots containing P huanghuaiense cultures on soybean cultivar 'Zhonghuang 13', respectively, showed that P huanghuaiense significantly reduced the germination rates of soybean and was highly Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular ... 11 pathogenic on this plant. These results confirm that P huanghuaiense is a soybean pathogen with a high degree of pathogenicity. um CBS 122663 HMAS 243736 ito q P_ terrestris CBS 112352 egy © vin CBS 119168 P CBS 158,73 um CBS 286,79 m CBS $74.85 P peri AS 289.31 P. oligandrum CBS 382,34 Saprolegnia parasitica CBS (13187 Saprolegnia parasitica CBS $40,67 Figure 4. EES] Phylogeny of Pythium huanghuaiense and related species generated by Maximum Likelihood, based on ITS+ Cox1 sequences. Branches are labelled with parsimony bootstrap proportions (before slanting line) higher than 50% and Bayesian posterior probabilities (after slanting line) more than 0.95. The branch of the new species is highlighted in pink. Uninoculation Chen 94 Figure 5. EES] Pathogenicity of Pythium huanghuaiense (Chen 94) on the soybean cultivar Hefeng 47. A. Control; B. disease symptoms caused by P huanghuaiense. 12 Chen J et al Discussion Pythium huanghuaiense is characterised by globose, sub-globose, ellipsoid, obturbinate to pyriform hyphal swellings; smooth and relatively small oogonia (12.5-18 wm); mostly monoclinous, sometimes hypogynous antheridia; sub-globose, club-shaped or fist-shaped antheridial cells; and plerotic or nearly plerotic and thin-walled oospores (0.5—1.5 ym). According to Levesque and Cock (2004), Pythium can be split into 11 clades (A-K), of which clade F is composed of species with either globose, non-proliferating sporangia or globose hyphal swellings (only P irregulare Buisman develops both) and a fast growth rate (often more than 25 mm/day; Levesque and Cock 2004). Phylogenetic analysis, based on ITS and Cox1 sequences, indicated that P huanghuaiense belongs to clade F of Pythium with full statistical support. Pythium huanghuaiense shares several morphological characteristics with other species of clade F, such as smooth oogonia and a fast growth rate. However, P huanghuaiense can be readily distinguished from other species by having narrower hyphae and a relatively higher maximum growth rate. Pythium huanghuaiense is similar to P wuhanense in its quick growth. The two species are phylogenetically closely related, belonging to clade F of Pythium (Fig. 4), but the former has narrower hyphae and plerotic or nearly plerotic oospores (Long et al. 2014; Table 2). Both P mamillatum and P. spiculum have similar sized oogonia and they share some similarity with P huanghuaiense; however, these two species can be readily distinguished from P. huanghuaiense by the ornamentation on their oogonia (van der Plaats-Niterink 1981, Paul et al. 2006;Table 2). In addition, these three species clustered in different lineages in the phylogenetic analysis. P huanghuaiense differs from P paroecandrum by its quicker growth rate, narrower hyphae and plerotic or nearly plerotic oospores (van der Plaats-Niterink 1981). Soybean is a major source of edible oil and protein and plays an important role in the human diet. Many species of Pythium are reported to be pathogens of soybean and some studies have documented the diversity of members of this genus, as well as their pathogenicity on soybean (such as Zhang and Yang 2000, Zitnick-Anderson and Nelson 2015, Coffua et al. 2016, Radmer et al. 2017). However, the diversity and importance of Pythium spp. as pathogens in China, particularly in soybean, are largely unknown. In a recent study on Pythium and Phytopythium spp. in a soybean—wheat rotation system in the Huang-Huai region, P huanghuaiense (as an undescribed candidatus species) was highly pathogenic on soybean and wheat (Feng et al. 2020). As part of an ongoing study on the diversity of Pythium spp. associated with soybean in China, the novel species, P huanghuaiense, was identified and described in this study on the basis of morphological characteristics and ITS and Cox1 sequence data. Additional pathogenicity tests and studies on the economic impact of P huanghuaiense on soybean and other crop plants will be conducted in the future. Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular ... 13 Acknowledgements The research was supported by the National Natural Science Foundation of China (32070022) and the Science Fund of the Jiangsu Vocational College of Agriculture and Forestry (2020kj003). Author contributions Conceived and designed the experiments: JJC and XBZ. Performed the experiments: JJC, HF and JY. Analyzed the data: JJC, HF, JY and WWY. Contributed reagents/materials/analysis tools: JJC, HF, JY and WWY. Wrote the paper: JJC, HF, JY and XBZ. References ° Ali-Shtayeh MS, Saleh AS (1999) Isolation of Pythium acanthicum, P. oligandrum, and P. periplocum from soil and evaluation of their mycoparasitic activity and biocontrol efficacy against selected phytopathogenic Pythium species. 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