pythium ornacarpum: a new species with ornamented oogonia isolated from soil in france

Pythium ornacarpum : a new species with ornamented oogoniaisolated from soil in France

Bernard Paul *Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Universite de Bourgogne, P.O. Box 138, 21004 Dijon, France

Received 13 September 1999; received in revised form 25 September 1999; accepted 27 September 1999

Abstract

Pythium ornacarpum sp. nov. was isolated from a soil sample taken from Genlis in the Burgundy region of France. Thisspecies is unique because of its ornamented oogonia which are completely surrounded by antheridial filaments. The fungus isclosely related to Pythium echinulatum Matthews. Morphological and reproductive aspects of this species as well as a study byPCR of the sequence of the internal transcribed spacer (ITS1) of the nuclear ribosomal gene and its comparison with relatedspecies are described here. The nucleotide sequence of the ITS1 region flaking the 5.8S rRNA of this species and other relatedspecies are also given here. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Allrights reserved.

Keywords: Pythium ornacarpum ; Sporangium; Antheridium; Oogonium; Oospore; Ornamentation; Internal transcribed spacer;

Polymerase chain reaction

1. Introduction

The genus Pythium, belonging to the family Py-thiaceae, order Peronosporales, and class Oomycetes,was created by Pringsheim in 1858 and is widelydistributed throughout the world. More than 200species of this genus have been described out ofwhich 130 have been recognised recently [1]. Fungibelonging to this genus are often known as phyto-pathogens, but not all are necessarily plant parasites.Most of these fungi can live as saprophytes, whileothers are facultative saprophytes becoming plant

pathogens from time to time and causing extensivedamage to economically important crops worldwide[2]. The pathogenic members of the genus Pythiuminfect mainly juvenile or succulent tissues, thus re-stricting their parasitic behaviour to seedlings or tothe feeder roots or root tips of older plants, and towatery fruits or stem tissues [3]. Some of them canalso behave as parasites of mosquito larvae [4] andone is known to be a mammalian pathogen [5].Several species of this genus have also been studiedfor their production of human dietary supplements[6,7].

The members of the genus Pythium are true fungihaving coenocytic branched mycelium, but phyloge-netic comparisons place them close to algae andhigher plants [8,9]. Unlike most of the eumycetes,the members of this genus remain diploid through-

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 0 4 - 2

* Tel: +33 3 80396341; Fax: +33 3 80396326;E-mail: [email protected]

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out their life cycles with meiosis occurring in thegametangia before fertilisation [2,10].

Taxonomic descriptions of these fungi have ap-peared from time to time but the most widely usedones are those given by Middleton [11], Waterhouse[12] and Van der Plaats-Niterink [13]. These taxo-nomic references are based on the comparison ofmorphological characteristics of di¡erent membersof the genus. In recent years attempts have beenmade towards a molecular taxonomy by the compar-ison of mitochondrial DNA (mtDNA) and the nu-clear-encoded genes encoding the production of ri-bosomal RNA (rDNA) [2]. The morphologicaldescription of di¡erent species of Pythium can becomplemented by the polymerase chain reactioncoupled to restricted fragment length polymorphismanalysis (PCR-RFLP). This method has become auseful tool in fungal taxonomy and is currentlyused to identify di¡erent species of Pythium [14^16]. Ampli¢cation of the ribosomal gene is used forthe genetic identi¢cation of many organisms becausethey comprise both highly conserved sequences dur-ing evolution and highly variable sequences amongspecies and even within species. The ribosomal nu-clear DNA consists of transcribed and non-tran-scribed regions [14]. The ITS1 (internal transcribedspacer) is a non-conserved region and has been am-pli¢ed with the PCR method using primers ITS1 andITS2.

Quite a number of species of Pythium are knownto possess ornamented oogonia. The commonest ofthem is P. echinulatum. These species are di¡erentfrom the non-ornamented ones morphologically asthey have ornamentations or spines on their oogoni-al wall. This trait is mostly of taxonomic value, theirbehaviour being identical to those with smooth-walled oogonia. The ornamented species are also dis-tributed throughout the world. Some of them havebeen reported from France [17] and a new species, P.radiosum, has also been described from this country[18].

A new species, isolated from soil samples taken inGenlis in the Burgundy region of France, is de-scribed here. The fungus is a saprophyte and closelyresembles P. echinulatum. It is unique as its anther-idial branches surrounds the spiny oogonia making aknot around the latter. This trait is found in thosespecies that have smooth-walled oogonia, but is

unique in the group of Pythium having ornamentedoogonia. In the present paper, the morphological de-tails of the new species are given together with thesequences of the ITS1 region of its nuclear ribosomalDNA and it is compared with ITS1 sequences ofrelated species.

2. Materials and methods

2.1. Fungal isolates

The new fungus was isolated from a soil sampletaken in Genlis in the Burgundy region of France bythe usual baiting techniques using boiled hemp seedhalves [13,19^21]. A bit of the fungal mycelium wasinoculated upon solid media like potato carrot agar(PCA) and potato dextrose agar (PDA). All the cul-tures were incubated at 25³C. The fungus was iden-ti¢ed with the help of keys provided by Middleton[11], Van der Plaats-Niterink [13], and Dick [1]. Thenew species was compared with the related fungi P.echinulatum (spiny oogonia) and P. nodosum(smooth-walled oogonia) which were taken fromthe author's personal collection.

2.2. Culture conditions and genomic DNA isolation

The fungi were grown in a liquid medium (10 g ofpeptone, 20 g of glucose, 8 g of yeast extract, 5 g ofNaCl per liter of distilled water) in 30-ml £asks con-taining 10 ml of medium. After a 5-day incubationon a rotary shaker at 25³C, the mycelium waswashed on TE bu¡er (10 mM Tris-HCl pH 8,1 mM EDTA). The mycelium was kept at 320³Cfor 24 h and DNA was isolated from this using themethod described earlier [22].

2.3. PCR

Universal primers ITS1 and ITS2 were synthesisedby the Genome Express Society (Paris). ITS1 (TCCGTA GGT GAA CCT GCG G) is at the 3P end ofthe 17S rDNA gene and ITS2 (GCTGCGTTC TTATC GAT GC) is at the 5P end of the 5.8S rDNAgene. The ITS1 region of P. ornacarpum was ampli-¢ed by PCR as described earlier [17]. For compar-ison the ITS1 regions of P. echinulatum and P. no-

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Fig. 1. P. ornacarpum. a1ÿ8 : Sporangia or hyphal bodies. a9;10 : Sporangia bearing spines. b1ÿ3 : Terminal oogonia with acute ornamenta-tions. b4ÿ7 : Oogonia having hypogynous antheridia. b8 : Oogonia with hypogynous and monoclinous antheridia. b9;10 : Intercalary oogo-nia. c1ÿ6 : Plerotic or almost plerotic oospores. d1;2 : Oogonia with monoclinous antheridia. d3 : Diclinous antheridia surrounding oogonia.Bar = 20 Wm.

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B. Paul / FEMS Microbiology Letters 180 (1999) 337^344 339

dosum were also ampli¢ed. All the sequencing of theampli¢ed ITS1 regions was performed by GenomeExpress, Paris.

3. Results

3.1. Morphological description

Pythium ornacarpum sp. nov. (Figs. 1^5): Myce-lium bene rami¢catum, sine loculis, hypha principalis3^8 Wm diam. Sporangia globosa, subglobosa velovata, intercalaria, interdum terminalia, 13^26 Wmdiam. Oogonia intercalaria vel terminalia, globosa,13^30 Wm diam., ornata spiculis 3^12 Wm longis.Antheridia hypogynata, monoclinata vel diclinata,

interdum circa singulum oogonium formans nodumcircum. Oosporae pleuroticae, globulus, 11^28 Wmdiam. Incrementum radiale quotidianum 8.5 mm ad25³C in agaro Solani tuberosi et Dauci carotae(PCA). Secretum ex terra in Genlis (Bourgogne)France. Holotypus in herbario Universitatis Bour-gogne conservatus (F-93).

Mycelium is well branched, hyaline, main hyphaemeasuring 3^8 Wm diam. and at times bearing con-ical spines measuring 3^7 Wm in length (Fig. 2a). OnPCA the colonies are submerged showing a chrysan-themum pattern. The average daily growth on thismedium at 25³C is 8.5 mm. The fungus grows lux-uriantly on boiled hemp seed halves in water givingthe characteristic whitish colonies of mycelial aquaticfungi.

Fig. 2. a: Hypha bearing spines. b: Sporangia with papilla. c: Terminal oogonia with pointed spines. d: Oogonia with ornamentations.e: Intercalary oogonia. f^i : antheridia making a complex knot around oogonia. Bars (a^g) 10 Wm, (h, i) 20 Wm.

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Sporangia or hyphal bodies are globose to oval,mostly intercalary, at times terminal, measuring 13^26 Wm (av. 20.17) diam. (Fig. 1a1ÿ8). Occasionallythese structures bear conical spines measuring 3^4Wm (Figs. 1a9;10 and 2b). Sporangial vesicles andzoospores were not observed.

Oogonia are both intercalary (Figs. 1b8ÿ10 and 2e)and terminal (Fig. 1b1ÿ7) and are covered with acuteconical ornamentations (Figs. 1b1ÿ10 and 2c^e). Theoogonia (excluding spines) are 13^30 Wm (av. 22.7)diam. and are provided with spines which measure3^12 Wm.

Antheridia are hypogynous (Fig. 1b4ÿ7), monocli-nous (Fig. 1b8,d1;2) and diclinous (Fig. 1d3). At timesthe mono- and diclinous antheridia completely sur-round the oogonia producing a complicated knotaround the latter (Figs. 2f^i and 3) making it di¤cultto determine the number and type of antheridialcontacts with the oogonium. After fertilisation theproduced oospores are plerotic or almost, measuring11^28 Wm (av. 18.2) diam., with a relatively thin wall(Fig. 1c1ÿ6 : ).

3.2. Nucleotide sequence of ITS1 region

The nucleotide sequences of the ITS1 region £ank-

ing the 5.8S ribosomal gene of P. ornacarpum(273 bases), P. echinulatum (278 bases], and P. nodo-sum (261 bases) have been submitted to GenBankand are as follows:

P. ornacarpum (GenBank accession numberAF187840): TTAAAACTATCCACGTGAACCGT-TTGTACCAAGATTTGCGTCAAGATGTTTGT-GCATGTTTGTTGTATCACTGTGTATTCGTAC-GCGGTGTGTGGCAAGTATGTATGACGCTT-GGCTGATCGAAGGTCGTGTCGCACTTGATT-GTGTGTATCGGCTGACTTATTTTTCAAACC-CATTCTTTAGTACTGATTATACTGTGAAGA-CGAAAGTCTTTGCTTTTACTAGATAACAAC-TTTCAGCAGTGGATGTCTAGGCTCGCGCAT-CGATGAAGAA.

P. echinulatum (GenBank accession numberAF187841): CACCTTAAAACTATCCACGTGAA-CCGTTTGTACCAAGATTTGCGTCAAGATGT-TTGTGCATGTTTGTTGTATCACTGTGTATTC-GTACGCGGTGTGTGGCAAGTATGTATGACG-CTTGGCTGATCGAAGGTCGTGTCGCACTTG-ATTGTGTGTATCGGCTGACTTATTTTTCAAA-CCCATTCTTTAGTACTGATTATACTGTGAAG-ACGAAAGTCTTTGCTTTTACTAGATAACAA-CTTTCAGCAGTGGATGTCTAGGCTCGCGCA-TCGATGAAGAAC.

Fig. 3. P. ornacarpum. Complex antheridial knot around oogonia. Bar = 20 Wm.

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P. nodosum (GenBank accession numberAF187842): AACTTTCCACGTGAACCGTTTTG-TGCGTTTTGTGCTTGTTGTTTTTCGATCTGC-TCTCTGTGCCTTTCGGGTGTGGAGTGTGGG-GAAGAAAGCTCCAGGCTAAACGAAGGCTG-CGAGTTTCGTGCTTGCGGCCGATTTATTCT-TTCAAACCCATACATTAAACACTGAAGTAT-ACTGTGAGGACGAAAGTCCTTGCTTTGAAC-TAGATAGCAACTTTCAGCAGTGGATGTCTA-GGCTCGCGCATCGATGAAGAACGCAGCA.

The sequence alignments are shown in Figs. 4 and5.

4. Discussion

P. ornacarpum belongs to the group of Pythiumhaving spiny oogonia. Its morphological character-istics and temperature-growth relationship resemblethose of P. echinulatum. However, the lack of zoo-spores, the presence of sporangia and at times hy-phae with spines, and especially its antheridial traitsare quite di¡erent from any other species of Pythium

having spiny oogonia. The antheridial branches sur-rounding the spiny oogonia are the main distinguish-ing trait of this new species. When the ITS1 region ofits ribosomal DNA is compared with a species ofPythium having ornamented oogonia (P. echinula-tum) on the one hand, and with one havingsmooth-walled oogonia and complicated knot-likeantheridia (P. nodosum) on the other, it is clearthat P. ornacarpum is more closely related to theformer (98.2% identity) than to the latter (68.5%identity). Within the group of Pythium having spinyoogonia, P. ornacarpum di¡ers morphologically fromall described species. Its ITS1 sequences has only47.3% identity with P. acanthicum, 51.2% identitywith P. oligandrum and 48.9% identity with P. pro-latum (unpublished data). However, with specieshaving similar ornamentations (P. echinulatum) theresemblance is very high (98.2%). Such high resem-blances are not rare for the genus as in the case of P.echinulatum and P. erinaceus having 99.6% identity(author's personal observation, data unpublished).

Morphologically P. ornacarpum is di¡erent fromall these species; its antheridial traits, lack of zoo-

Fig. 4. Comparison of the ITS1 sequence of P. ornacarpum with P. echinulatum (ALIGN, Myres and Miller, CABIOS 1989).

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spores, and the sequence of its ITS1 region of theribosomal DNA justify the creation of the new tax-on.

Acknowledgements

The author wishes to thank Mr Isaac Masih forhis help in the preparation of the manuscript.

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pythium ornacarpum: a new species with ornamented oogonia isolated from soil in france

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