Pythium huanghuaiense sp. nov. isolated from soybean: morphology, molecular phylogeny and pathogenicity

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.


Introduction
Species of the genus Pythium Pringsheim are diverse, occupying a variety of habitats (van der Plaäts-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 Plaäts-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 andKoenning 2006). Some Pythium spp. are important pathogens of animals, while others are beneficial as biological control agents that protect against pathogenic fungi (van der Plaäts-Niterink 1981, Ali-Shtayeh andSaleh 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 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.

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 "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.

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 Plaäts-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 Plaäts-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.

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-μl volumes consisting of 1 μl of DNA template, 1 μl of each 10 μM forward and reverse primer, 15 μl of 2 × Taq PCR Master Mix and 12 μl 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 OomCoxI-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 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 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 (BI) 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 Cox1 partition of the dataset were determined using the Akaike Information Criterion implemented in MrMODELTEST2.3 (Nylander 2004

Etymology
With reference to the distribution of the species in the Huang-Huai area of China.

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.

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 Cox1 sequences of the five isolates, described here as Pythium huanghuaiense, showed the best phylogenetic matches with species of clade F in Pythium (Lévesque and Cock 2004).
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 reisolate 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 P. 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 pathogenic on this plant. These results confirm that P. huanghuaiense is a soybean pathogen with a high degree of pathogenicity.
According to Lévesque 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; Lévesque 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 Plaäts-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 Plaäts-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.