Biodiversity Data Journal :
Taxonomic Paper
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Corresponding author: Jia-Jia Chen (jiajiachen@jsafc.edu.cn)
Academic editor: Marco Thines
Received: 28 Feb 2021 | Accepted: 18 Apr 2021 | Published: 22 Apr 2021
© 2021 Jia-Jia Chen, Hui Feng, Jian Yu, Wenwu Ye, Xiaobo Zheng
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.
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: e65227. https://doi.org/10.3897/BDJ.9.e65227
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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.
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.
Cox1, ITS, oomycete, Pythium clade F
Species of the genus Pythium Pringsheim are diverse, occupying a variety of habitats (
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 I (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.
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 “W” 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 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.
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 (
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 (
Sequences, generated in this study, were aligned with additional sequences downloaded from GenBank (Table
A list of species, cultures and GenBank accession numbers of sequences used in this study.
Species name |
Sample no. |
Locality |
GenBank accession no. |
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ITS |
Cox1 |
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Pythium abappressorium |
CBS 110198 |
USA |
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P. acanthophoron |
CBS 337.29 |
USA |
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P. alternatum |
CBS 139279 |
Japan |
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P. anandrum |
CBS 285.31 |
– |
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P. attrantheridium |
DAOM 230383 |
Canada |
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P. baisense |
HMAS 242232 |
China |
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P. barbulae |
CBS 139569 |
Japan |
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P. brachiatum |
UZ00736 |
Japan |
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P. canariense |
CBS 112353 |
Spain |
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P. cryptoirregulare |
CBS 118731 |
USA |
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P. cylindrosporum |
CBS 218.94 |
Germany |
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P. debaryanum |
CBS 752.96 |
UK |
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P. emineosum |
BR 479 |
UK |
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P. grandisporangium |
CBS 286.79 |
USA |
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P. huanghuaiense |
Chen 94 |
China |
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P. huanghuaiense |
Chen 95 |
China |
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P. huanghuaiense |
Chen 96 |
China |
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P. huanghuaiense |
Chen 99 |
China |
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P. huanghuaiense |
Chen 100 |
China |
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P. inflatum |
CBS 168.68 |
USA |
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P. insidiosum |
CBS 574.85 |
Costa Rica |
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P. intermedium |
CBS 266.38 |
Netherlands |
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P. irregulare |
CBS 250.28 |
Netherlands |
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P. junctum |
UZ00732 |
Japan |
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P. kunmingense |
CBS 550.88 |
China |
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P. lucens |
CBS 113342 |
UK |
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P. macrosporum |
CBS 574.80 |
Netherlands |
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P. mamillatum |
CBS 251.28 |
Netherlands |
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P. marsipium |
CBS 773.81 |
Netherlands |
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P. minus |
CBS 226.88 |
United Kingdom |
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P. monospermum |
CBS 158.73 |
United Kingdom |
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P. nodosum |
CBS 102274 |
France |
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P. nunn |
CBS 808.96 |
USA |
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P. oligandrum |
CBS 382.34 |
United Kingdom |
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P. paroecandrum |
CBS 157.64 |
Australia |
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P. periplocum |
CBS 289.31 |
USA |
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P. plurisporium |
CBS 100530 |
USA |
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P. prolatum |
CBS 845.68 |
USA |
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P. recalcitrans |
CBS 122440 |
Spain |
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P. sp. "balticum" |
CBS 122649 |
Sweden |
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P. spiculum |
CBS 122645 |
France |
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P. spinosum |
CBS 122663 |
India |
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P. splendens |
CBS 462.48 |
USA |
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P. sukuiense |
CBS 110030 |
Taiwan |
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P. sylvaticum |
CBS 453.67 |
USA |
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P. terrestris |
CBS 112352 |
France |
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P. ultimum var. ultimum |
CBS 398.51 |
Netherlands |
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P. viniferum |
CBS 119168 |
France |
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P. wuhanense |
HMAS 243736 |
China |
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Saprolegnia parasitica |
CBS 113187 |
Russia |
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S. parasitica |
CBS 540.67 |
United Kingdom |
New sequences are shown in bold.
Phylogenetic analyses
Phylogenetic analysis was conducted as descibed by
Pathogenicity was confirmed using the hypocotyl slit inoculation method (
Pathogenic on soybean. Colonies submerged, with a cottony pattern on CMA, a rosette pattern on PCA and a cottony pattern on 10% V8A (Fig.
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 μm; G–J 5 μm.
With reference to the distribution of the species in the Huang-Huai area of China.
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
Morphological description of Pythium huanghuaiense and the most closely related species.
Pythium huanghuaiense (Chen 94) |
P. mamillatum |
P. paroecandrum |
P. spiculum |
P. wuhanense |
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Width of hyphae (μm) |
Up to 5 |
Up to 6.5 |
Up to 9 |
Up to 6 |
Up to 7.5 |
Sporangia/hyphal swellings |
Globose, sub-globose, obturbinate to pyriform, mostly terminal or sometimes intercalary |
Globose, broadly ovoid or ellipsoidal, intercalary or lateral |
Globose or ellipsoidal, intercalary or terminal |
Globose, ovoid, cylindrical and at times peanut-shaped, mostly intercalary to catenulate, rarely terminal |
Globose, sometimes cylindrical to elongated, mainly intercalary, often catenulate with oogonia, occasionally terminal or lateral |
Oogonia (μm) |
12.5–18 (av. 15.5), terminal or intercalary |
15–18 (av. 16), intercalary or terminal |
17–24 (av. 19), intercalary, often in chains and rarely terminal |
13–22 (av. 15.6), mostly intercalary or in chains |
10–20 (av. 17.7), mostly intercalary, often catenulate with sporangia and antheridia, sometimes terminal or lateral |
Oogonium ornamentation |
Absent |
Present |
Absent |
Present |
Absent |
Antheridia |
Mostly monoclinous, sometimes hypogynous |
Mostly monoclinous, infrequently diclinous |
Mostly monoclinous, sometimes diclinous |
Monoclinous |
Monoclinous, hypogynous or diclinous |
Oospores (μm) |
Plerotic or nearly plerotic, 11.5–17 (av. 14.5) |
Plerotic, 12–15 (av. 14) |
Aplerotic, 15–21 (av. 17) |
Plerotic or aplerotic, 8–18 |
Aplerotic, 7.5–17.5 (av. 14.5) |
Oospore wall thickness (μm) |
0.5–1.5 |
0.8–1.4 |
1–1.5 |
0.5–1 |
0.5–1 |
Double oospores |
Absent |
Absent |
Absent |
Present |
Present |
Cardinal temperature |
Min 4°C, optimum 25°C and max 37°C |
Min 5°C, optimum 25°C and max 30–35°C |
Min 5°C, optimum 25°C and max 35°C |
Unknown |
Min 4°C, optimum 28–30°C and max 35°C |
Daily growth rates on PCA at 25°C (mm) |
32 |
25 |
20–25 |
25 |
50 |
Reference |
This study |
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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.
Five ITS and Cox1 sequences were newly generated for this study and their accession numbers are available in GenBank (Table
ML and BI analyses yielded similar tree topologies and only the ML tree is shown (Fig.
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.
Pythium huanghuaiense (Chen 94) significantly stunted and reduced the growth of soybean seedlings compared with uninoculated controls (Fig.
Pythium huanghuaiense is characterised by globose, sub-globose, ellipsoid, obturbinate to pyriform hyphal swellings; smooth and relatively small oogonia (12.5–18 μm); 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 µm).
According to
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.
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
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).
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.