Pythium glomeratum, a new species isolated from agricultural soiltaken in north-eastern France, its ITS region and its comparison

with related species

Bernard Paul �

Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite¤ de Bourgogne, BP 27877, 21078 Dijon, France

Received 21 May 2003; received in revised form 5 June 2003; accepted 5 June 2003

First published online 28 June 2003

Abstract

Pythium glomeratum sp. nov. is described here. It was isolated from soil samples taken in the northern France in 1992, was wronglyidentified as Pythium heterothallicum and was kept aside. Recently, the ITS region of the rDNA of this oomycete was amplified andsequenced. The differences between the sequence and a more detailed study of the morphological characters of these two species, revealedthat both are related but different species. P. glomeratum is characterized by the presence of branched antheridia that wrap around theoogonia, aplerotic to almost plerotic oospores, and the lack of zoosporangia and zoospores. Taxonomical description of this new species,its comparison with related oomycetes, polymerase chain reaction (PCR) amplification of the internal transcribed region (spacers ITS1,ITS2, and the gene 5.8S) of its ribosomal nuclear DNA and the nucleotide sequence of this region are given here.4 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Antheridia; Oogonia; Oospore; ITS region; Ribosomal DNA; Pythium

1. Introduction

During the course of investigation of soil borne oomy-cetes in France, a number of Pythiaceous oomycetes wereisolated between 1990 and 1994. These were maintained inthe author’s personal collection. Some of these were di⁄-cult to identify because of the lack of reproductive struc-tures. The advent of molecular techniques has now made itpossible to identify these isolates.A number of soil samples were collected from cultivated

¢elds in the region of Rue near the Atlantic coast ofnorthern France in the year 1992. Out of these, manymembers of the genus Pythium were detected. Four iso-lates having close resemblance and having the same tem-perature^growth relationship were kept aside as thesefailed to sporulate and to produce sex organs. This lot

has now been taken out and grown on grass blades and onsolid media supplemented with sterols. The four isolatesbelong to the same species and produce antheridia andoogonia on grass blades in water cultures after prolongedincubation. The type specimen is F-304 and is closely re-lated to Pythium heterothallicum. However, it has its owncharacteristics of having very large oogonia that arewrapped around with four to six antheridia. It is a non-sporulating type of oomycete and is described as a newspecies here. Morphological details, ITS sequences of itsrDNA, and a comparison of its morphological and molec-ular characteristics with related species, are also given.

2. Materials and methods

Soil samples were collected in sterilized capped bottlesand brought to the laboratory. Oomyceteous organismswere isolated from these by using the usual baiting tech-niques described elsewhere [1^3]. The baits used wereboiled hemp-seed halves introduced to a watery solutionof the soil. Temperature^growth relations were observedon solid media like potato carrot agar (PCA) and potatodextrose agar (PDA). Benomyl was used to suppress the

0378-1097 / 03 / $22.00 4 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.doi :10.1016/S0378-1097(03)00478-6

* Tel./Fax: +33 3 80396326.E-mail address: bernard.paul@u-bourgogne.fr (B. Paul).

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growth of Fusarium-like fungi [4]. To induce sexual repro-duction, 2 mg L-sitosterol was dissolved in 10 ml ethanoland was either poured into 1 l solid media (usually PCA)before sterilization, or 10 Wl of this solution was asep-tically pricked at various points on the PCA [5]. Inwater, natural baits other than the hemp seeds werealso used. Small pieces of autoclaved grass leaves were£oated in water, which were then inoculated with the oo-mycete. Identi¢cation was done with the help of keys pro-vided by Middleton [6], Waterhouse [7] and Plaats-Niter-ink [1].

2.1. DNA isolation and polymerase chain reaction (PCR)

The oomycetes were grown in PDB (potato dextrose

broth), which is prepared in the same manner as PDAwithout the addition of agar^agar. The culture conditions,DNA isolation and the PCR of the internal transcribedspacers (ITS1 and ITS4) of the ribosomal nuclear DNAwere done using the procedures described earlier [8]. Uni-versal primers ITS1 (TCC GTA GGT GAA CCT GCGG) and ITS4 (TCC TCC GCT TAT TGA TAT GC) weresynthesized and the DNA sequence realized by GenomeExpress (Paris). ITS1 is at the 3P end of the 18S rDNAgene and ITS4 is at the 5P end of the 28S rDNA gene. TheITS1 sequence of the isolate F-304 was compared with theITS1 sequences of related species. The sequence of the ITSand the £anking regions of the nuclear ribosomal DNA ofPythium glomeratum (F-304) has been deposited to theGenBank as accession number AY263339.

Fig. 1. Morphological and reproductive structures of P. glomeratum A: Hyphal bodies. B,C: Catenulate oogonia. D^K: Oogonia provided with dicli-nous branched antheridia. L^O: Aplerotic to almost plerotic (O) oospores. Bar = 30 Wm.

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3. Results

3.1. P. glomeratum PAUL sp. nov. (Figs. 1^3)

Mycelium bene rami¢catum, sine loculis, hyphae princi-pales 2.5^5 Wm diametro. Tumores hyphales globosi, subglo-bosi vel prolati ; intercalaires vel catenaires 5^21 Wm diam.et ad 50 Wm elongatae. Oogonia intercalaria raro terminalia,globosa vel subglobosa, 16^43 Wm diam. Antheridia diclina-ta, rami¢cata, complectentia aut circumdantia oogonium,interdum constricta. Oosporae apleroticea, globosae 15^32Wm diam., paries 1.5^2.5 Wm crassus. Incrementum radialequotiadianum 13 mm 25‡C in agaro Solani tuberosi et Daucicarotae (PCA). Secretum ex terra in Rue, France. Holoty-

pus in herbario Universitatis Burgundiae conservatus (F-304) distractum.Etymology: The oomycete is being named as P. glom-

eratum because of the presence of antheridia that wraparound the oogonia forming a woollen ball-like structure(glomus= ball).Mycelium is well branched measuring up to 5 Wm in

diameter. Colonies on solid media are submerged, givinga chrysanthemum pattern on PCA. Average radial growthof the oomycete at 25‡C on this medium is 13 mm per day.The oomycete does not produce sporangia and zoospores,even after prolonged incubation at di¡erent temperaturesand being £ooded with sterile distilled water, tap water,and pond water.Hyphal bodies (Fig. 1A: A1^A13, Fig. 2a,b) are pro-

duced plentifully in water cultures as well as on solid me-dia. These are globose, subglobose, ovoid, to cylindrical.Sometimes these structures are hardly thicker than thevegetative mycelium, measuring 5^21 Wm in diameter(average 14.4 Wm). When elongated these can attain alength of up to 50 Wm (Fig. 1, A12, Fig. 2b).

P. glomeratum does not easily form reproductive struc-tures on hemp-seed halves in water. However on grassblades in water and also on solid media supplementedwith L-sitosterol, the oomycete produces antheridia andoogonia plentifully.Oogonia are smooth-walled (Fig. 1B^O), usually spher-

ical, but at times ovoid, rarely elongated (Fig. 1C,D);mostly intercalary at times catenulate (Fig. 1B,C,H),sometimes terminal ; measuring 16^43 Wm in diameter(average 26.9 Wm). These are ¢lled with densely granulatedcytoplasm.Antheridia are mostly diclinous, providing one to six

antheridial branches per oogonium (Fig. 1D^K), curvedelongate, often wavy in contour, applied lengthwise tothe oogonium over their entire length. Antheridial cellsare in£ated which are persistent and remain attached tothe oogonia even after fertilization. Sometimes a singleantheridial branch may bear two to four antheridial cellsapplied laterally to the oogonium. The clustering of an-theridia around the oogonia gives a woollen ball-like ap-pearance and makes it extremely di⁄cult to observe thenumber and the type of contact of the antheridia (Fig.2c^f).Oospores are aplerotic to almost plerotic. In all cases

Table 1Sequence of the ITS and £anking regions of the rDNA of P. glomera-tum

1 gacattacca cacctaaaaa actttccacg tgaactgtca

aacctgttct gtgcttgtgc

61 tgggtctgcg ttttcggacg cggacacggn ggaggctgaa

cgaaggttgg ttttcttctt

121 gtgagagacc agctgatata tttttcaaac ccctttttac

aaaatgactg atcaatactg

181 ggagaacgaa agttcttgct ttaaactaga taacaacttt

cagcagtgga tgtctaggct

241 cgcacatcga tgaagaacgc tgcgaactgc gatacgtaat

gcgaattgca gaattcagtg

301 agtcatcgaa attttgaacg catattgcac tttcgggtta

gacctggaag tatgtctgta

361 tcagtgtccg tacatcaaac ttgcctctct tttttctgtg

tagtcaggat tggagacgtg

421 cagatgtgaa gtgtctcgcg cacttgcgtc ttcggacgac

aagctgtcga gtccttttaa

481 agcgacacga tctttctatt gtttctgtga agcgtattgc

tcgaacgcgg tgattttcgg

541 atcgctcgca gtcgtcggcg acttcggtga gaacataaag

gaggaaacct caattcgcgg

601 tatgttcggc ttcggctcga caatgttgct tattgtgtgt

ggaatctgtt ttcgccttga

661 ggtgtactga tggttgtgtg cttgaactgg gagttggtgt

gtagtagagc ggtgcagcat

721 gcatggttac gccttttata tagagagatg tctatttggg

aaaatggtac tttttggcaa

781 gcatcttgcc gactgtatct caattggacc

1^7= 18S gene, partial sequence; 8^213= ITS1, complete sequence; 214^372= 5.8 gene, complete sequence; 373^803= ITS2, complete sequence;804^810= 28S gene, partial sequence.

Table 2Comparison of some characters of P. glomeartum and P. heterothallicum

P. heterothallicum P. glomeratum

Average daily growth on PCA 20 mm 13 mmPattern on PCA radiate chrysanthemalSporangia and zoospores readily formed absentSex organs in single cultures not formed formedOogonial position terminal and intercalary intercalary and catenulateOogonial diameter 20^32 Wm 16^43 WmOospore diameter 18^28 Wm 15^32 Wm

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Fig. 2. Morphological and reproductive structures of P. glomeratum. a: Spherical intercalary hyphal body. b: Elongated hyphal body. c^f: Oogonia sur-rounded by antheridia. g: Aplerotic oospores. h: Almost plerotic oospore. a,b,d^h: bar = 30 Wm; c: bar = 72 Wm.

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there is a clear space between the oospore and the oogo-nial wall (Fig. 1L^O, Fig. 2g,h). These are usually spher-ical but at times ovoid to slightly elongated, one per oo-gonium, measuring 15^32 Wm in diameter (average 22.4Wm). The oospore wall is comparatively thinner rangingbetween 1.5 and 2.5 Wm in thickness.ITS1 and ITS2 sequences and their £anking regions of

P. glomeratum (F-304) are comprised of 810 bases. Thesequence of this region of the rDNA of P. glomeratum isgiven in Table 1.The comparison of the ITS1 sequences of P. glomeratum

and related species is given in Fig. 3 in the form of CLUS-TAL multiple alignment.

4. Discussion

P. glomeratum was mistakenly identi¢ed as P. hetero-

thallicum by the author for quite a long time. Its anther-idial branches surrounding the oogonia and its unwilling-ness to produce sexual structures on hemp-seed halves ledthe author to this conclusion. During the course of aninvestigation on the ITS region, the isolates identi¢ed asP. heterothallicum were taken out from the personal col-lection of the author, and the PCR ampli¢cation of thisITS regions was performed. The sequence obtained wasdi¡erent from the sequence of P. heterothallicum on theGenBank (AF330186). A BLAST search with ITS se-quence of P. glomeratum (GenBank accession numberAY263339) gives close resemblance with P. heterothallicum(AF452162), Pythium intermedium (AJ233447), Pythiumorthogonon (AJ233452), Pythium middletonii (AJ233449),Pythium sylvaticum (AF452140), and Pythium paroecan-drum (AJ233453).ALIGN sequence alignments with the entire ITS se-

quence of P. glomeratum and P. heterothallicum gives

Fig. 3. CLUSTAL W (1.81) multiple sequence alignment of ITS1 region of the rDNA of P. glomeratum (AY263339) with P. heterothallicum(AF452162), P. orthogonon (AJ233452), P. middletonii (AJ233449), P. intermedium (AJ233447), P. sylvaticum (AF452140) and P. paroecandrum(AJ233453).

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97.5% similarity. This is the closest match while all othersare distant relatives with a score of 74% with P. interme-dium, 72% with P. orthogonon and P. middletonii, 70.7%with P. paroecandrum and only 70% with P. sylvaticum. Ascore of 97.5% identity with P. heterothallicum brings thisspecies very close to P. glomeratum. However the smalldi¡erences in the ITS sequences of both the oomycetes issigni¢cant for the genus Pythium, in which the di¡erentspecies may vary by only 1 or 2 bp [9]. The results of thesequence alignment support the morphological observa-tions that the oomycete is very closely related to P. hetero-thallicum. However, a close look at the morphologicalfeatures as well as reproductive structures reveals thatwhile P. glomeratum is related to P. heterothallicum, it isan entirely di¡erent species of oomycete. A comparison ofthe two species is given in Table 2.The di¡erences in the morphological characteristics,

supplemented by the di¡erences in the ITS region of therDNA of P. glomeratum justify the creation of this newtaxon.

References

[1] Plaats-Niterink, A.J. (1981) Monograph of the genus Pythium. Stud.Mycol. Baarn 21, 1^242.

[2] Paul, B. (1986) An aquatic species Pythium toruloides sp. nov. fromAlgeria. Trans. Br. Mycol. Soc. 86, 330^334.

[3] Paul, B. (1987) A new species of Pythium with ornamented oogoniafrom Algeria. Mycologia 79, 797^802.

[4] Paul, B. (1991) Pythium folliculosum : A new species from the bank oflake Zurich. Mycol. Helv. 4, 203^208.

[5] Paul, B. (1990) A new aquatic species of Pythium from Algeria with¢lamentous sporangia and thick walled oospores. Condollea 45, 575^579.

[6] Middleton, J.T. (1943) The taxonomy, host range, and geographicaldistribution of the genus Pythium. Mem. Torrey Bot. Club 20, 1^171.

[7] Waterhouse, G.M. (1968) The genus Pythium Pringsheim. Mycolog-cal papers 110, CMI.

[8] Paul, B. (2000) ITS1 region of the rRNA of Pythium megacarpum sp.nov., its taxonomy, and its comparison with related species. FEMSMicrobiol. Lett. 183, 105^110.

[9] Paulitz, T.Z., Adams, K. and Mazzola, M. (2003) Pythium abappres-sorium ^ a new species from eastern Washington. Mycologia 95, 80^86.

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