RESEARCH LETTER
Pythium stipitatum sp. nov. isolated from soil and plant debris taken in France, Tunisia,Turkey, and India ¨ Gursel Karaca1, Rinita Jonathan2 & Bernard Paul3 1
Plant Protection Department, Faculty of Agriculture, Suleyman ¨ Demirel University, Isparta, Turkey; 2Department of Biotechnology, Hislop College, Civil 3 Lines, Nagpur, India; and Laboratoire de Mycology et de Phytopathologie, Institut Jules Guyot, Universite´ de Bourgogne, Dijon, France
Correspondence: Bernard Paul, Laboratoire de Mycology et de Phytopathologie, Institut Jules Guyot, Universite´ de Bourgogne, 21000 Dijon, France. Tel.: 133 3 8039 6326; fax: 133 3 8039 6265; e-mail:
[email protected] Received 16 February 2009; accepted 6 March 2009. First published online 20 April 2009. DOI:10.1111/j.1574-6968.2009.01579.x Editor: Richard Staples Keywords Pythium stipitatum; oogonia; antheridia; oospores; ITS region; rRNA.
Abstract Pythium stipitatum is a slow-growing oomycete and has been isolated from soil samples and plant materials from France, Tunisia, Turkey and India. Its morphological characteristics are reminiscent of those of Pythium ramificatum, discovered in Algeria by the corresponding author. Unfortunately, the Algerian isolate was not deposited in any culture collection and ultimately got lost. Those were the days when molecular description of fungi was not a fashion; hence, no molecular characteristics of the Algerian isolates were deposited to the GenBank. Moreover, its coralloid antheridial branches made it an easy prey to be considered as synonymous to Pythium minus. Because there are no living strains of P. ramificatum, and no sequence at the GenBank, it is being treated as ‘nomen invalidum’ here. However, we have now isolated the same type of oomycete from four different countries and we have sufficient evidence, both molecular and morphological, to describe it as a new species, quite different from P. minus. In this article, we are giving the morphological and molecular evidence to separate it as a distinct species, P. stipitatum, belonging to the ‘Clade E’ of the genus Pythium. Taxonomic description of this oomycete, its comparison with related species, and the sequence of the internal transcribed spacer region of its rRNA gene, are discussed here.
Introduction The genus Pythium consists of 4 160 recognized species distributed worldwide (DeCock et al., 2008), 4 35 of which have been described by the corresponding author. These are mostly saprophytes, living on dead organic matter found in the soil; however, some species of the genus are phytopathogenic, mammalian pathogens, sea weed pathogens, mycoparasites, and plant disease-resistant inducing agents (Hendrix & Campbell, 1973; De Cock et al., 1987; Lodha & Webster, 1990; Laing & Deacon, 1991; Jones & Deacon, 1995; Paul, 1999a, b; Karaca et al., 2008; Paul et al., 2008; Bala et al., 2009). The taxonomy of the genus Pythium is widely considered to be difficult because a range of morphological criteria have been used in the past to classify members of this genus (van der Plaats-Niterink, 1981); however, the high variability among different structures and considerable overlap of some of these features among different species complicates accurate species identification. In recent years, molecular 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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analysis is increasingly being used in fungal taxonomy and disease diagnostics. The genes coding for rRNA that have been used as molecular markers for inferring the evolutionary history of organisms at different taxonomic levels have come to the rescue of taxonomists and this has become a useful tool to supplement the morphological differences between species (DeCock & Le´ vesque, 2004). The internal transcribed spacer (ITS) region of the rRNA operon is often studied as a potential source of sequence variation among closely related organisms, because it has a lower degree of conservation when compared with those of the rRNAcoding genes. The analysis of the sequences of ITS region of the rRNA has now made it possible to separate the oomycetes from true fungi. Although, morphologically, they are ‘fungus-like,’ they no longer enjoy the place of ‘true fungi’ but are now supposed to be closer to ‘algae’ than to the fungal world, and in recent classifications the oomycetes are classified as belonging to the stramenopile lineage in the eukaryotic domain, together with brown algae (Paul et al., 2006; Belbahri et al., 2008). FEMS Microbiol Lett 295 (2009) 164–169
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During the course of this study, Pythium stipitatum sp. nov. was isolated six times in France (Type material F-1516), thrice in Turkey (Type material TUR 14-3) and Tunisia (Type TUN 30.2), and twice from India (F-1515.1). All these oomycetous organisms are morphologically similar and closely resemble the almost forgotten oomycete, Pythium ramificatum, which was described from water samples obtained from a water dam in Sig in north-western Algeria (Paul, 1986). It is a nonsporulating oomycete that produces a much branched, coralloid type of antheridial filaments that approach the oogonia without making any knot-like structure around the female gametangia. This feature and the lack of molecular characteristics prompted some specialists of the genus Pythium to incorporate this taxon within Pythium minus (Dick, 1990). Because we no longer have the living cultures of P. ramificatum, we consider it as nomen invalidum. However, we have now isolated 14 strains of an oomycete that is very similar to the Algerian isolate. These were obtained from soil samples taken in the Burgundian region of France, vineyard soil in Tunisia, sugar beet (Beta vulgaris) from Isparta in Turkey and from grass roots (Cyanodon dactylon) in Nagpur, India. The morphological characteristics of all the 14 isolates fit well with those described in 1986. The type strain F-1516 is now being deposited at the CBS culture collection in Utrecht, Holland, and its ITS sequences have already been submitted to the GenBank. Morphological details and the molecular characteristics based on the rRNA sequence of the P. stipitatum are being described here and compared with related species.
Materials and methods Oomycete isolates Fourteen isolates of P. stipitatum (Type strain, F-1516) were isolated from soil samples obtained from France, Tunisia (vineyard soil samples), Turkey (B. vulgaris) and India (C. dactylon) using the usual baiting techniques, on hempseed halves, described elsewhere (van der Plaats-Niterink, 1981; Paul, 2004). Isolates were subsequently purified by repeated washing with sterile distilled water and subculturing on solid media such as potato carrot agar (PCA) and potato dextrose agar. All these isolates are maintained in the author’s personal collection of pythiaceous oomycetes at ‘Institut Jules Guyot’ in Dijon, France. The type culture is being deposited at the ‘CBS collection’ in Utrecht (Holland). Oomycete identification was performed using monographs of Middleton (1943), van der Plaats-Niterink (1981), Waterhouse (1967), and a senior author’s descriptions of new species (Paul, 2001, 2002a, b, 2003; Belbahri et al., 2006; Paul et al., 2006) and also through BLAST search, using the sequence of the ITS region of its rRNA. FEMS Microbiol Lett 295 (2009) 164–169
DNA extraction and PCR amplification DNA was isolated from mycelia using the DNA-Easy Plant Mini kit (Qiagen, Basel, Switzerland), according to the manufacturer’s specifications. Quality was checked by visualization under UV light following electrophoretic separation with a molecular mass standard (1-kb DNA ladder, Invitrogen, Switzerland) in 1% agarose (Biofinex, Switzerland) gel in 1 TBE, subjected to 100 V for 1 h and stained with ethidium bromide (0.5 mg mL 1). Concentrations were assayed in an S2100 Diode Array spectrophotometer (WPA Biowave, Cambridge, UK). ITS amplifications of Pythium samples were carried out using previously described universal primers ITS1 and ITS4, which target conserved regions in the 18S and 28S rRNA genes (White et al., 1990; Cooke et al., 2000). The reaction mixture contained 1 PCR buffer [75 mm Tris-HCl (pH 9.0), 50 mM KCl, and 20 mM (NH4)2SO4], 0.1 mM dNTPs, 0.25 mM of each primer, 1.5 mM MgCl2, 1 mL of Taq Polymerase (Biotools, Spain) and 1 mL of DNA in a total volume of 50 mL. Amplifications were carried out in a Master Gradient thermocycler (Techne, Progene, France) according to the following amplification program: an initial denaturation step of 95 1C for 2 min followed by 30 cycles including denaturation at 95 1C for 20 s, annealing for 25 s at 55 1C and extension for 50 s at 72 1C. Amplification was terminated by a final extension step of 10 min at 72 1C (Cooke et al., 2000). PCR products were separated in 1% agarose (Biofinex) gels in 1 TBE, subjected to 100 V for 1 h, stained with ethidium bromide (0.5 mg L 1) and visualized under UV light. Amplicons were purified using a Minelute PCR Purification Kit (Qiagen), according to the manufacturer’s specifications. Quantity and quality were checked as described above for DNA extraction. Amplicons were sequenced directly in both sense and antisense directions. All oomycete samples were sequenced twice, and a consensus sequence was created from the duplicates. DNA sequences have been deposited in GenBank under the accession number EU368671.
Results Pythium stipitatum G. Karaca, & B. Paul, sp. nov. (Figs 1–5) Etymology: The oomycete is named as Pythium stipitatum because of its branched antheridial stalks (latin ‘stipes’ = branches). Hypha hyalina 5–7 mm crassa. Sporangia non observata. Conidium (aut corpora mycelia) globosa intercalaria vel terminalia, subglobosa, vel cylindrosa, 11–24 mm diam; zoosporae non observata. Oogonia globosa, terminalia vel intercalaria, glabra, interdum papillosa 14–25 mm diam. Antheridia monoclinata unum aut plurima, profondus stipes, cellulae antheridiales clavatae. Oogonia continentia unam, duas raro trias oosporas, pleroticae et interdum apleroticae, 11–23 mm diam, paries 0.5–1 mm crassus. 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Fig. 1. (a, b) Intercalary hyphal bodies, (c) subterminal hyphal body, and (d) dumb-bell-shaped hyphal body. (a–c) Scale bar = 25 mm, (d) scale bar = 20 mm.
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Fig. 3. (a) An elongated antheridial cell having an apical contact with an oogonium, (b) monoclinous antheridia, (c) sessile antheridia and (d) monoclinous and diclinous antheridia. (a–c) Scale bar = 15 mm, (d) scale bar = 35 mm.
Fig. 4. (a) Plerotic oospore, (b) aplerotic oospore and (c, d) double oospores. (a, c) Scale bar = 20 mm, (b, d) scale bar = 18 mm. Fig. 2. (a, b) Papillated terminal oogonia and (c, d) corolloid antheridial branches surrounding oogonia. (a, b) Scale bar = 15 mm, (c, d) scale bar = 20 mm.
Incrementum radiale quotiadianum 15 mm 25 1C in agaro Solani tuberosi et Dauci carotae (PCA). Secretum ex terra in, France, Turkey, Tunisia et India. Holotypus in herbario Universitatis Bourgogne conservatus (F-1516).
Morphological description The oomycete grows well both on solid media as well as hemp-seed halves in water. Its mycelium in water is hyaline, well branched, with the main hyphae 5–7 mm wide. Colonies 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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on PCA are submerged, and shows a radiate pattern on this medium. Average radial growth of the oomycete at 25 1C on PCA is 15 mm day 1. It reproduces in water on hemp-seed halves and on PCA, giving plenty of asexual and sexual structures. However, zoospore formation was not observed in spite of repeated flooding of the cultures with distilled water, pond water, and soil extract water. The hyphal bodies or sporangia are globose, cylindrical to dumb-bell shaped (Fig. 1a–d), terminal and intercalary, measuring between 10 and 24 mm in diameter (average 15 mm). These germinate, directly producing one to many germ tubes to give a new mycelium. FEMS Microbiol Lett 295 (2009) 164–169
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Fig. 5. Phylogenetic tree with bootstrap values drawn with ITS sequences of Pythium stipitatum and related species.
The female gametangia (oogonia) are mostly terminal (Fig. 2a and b), occasionally intercalary, smooth walled, measuring from 14 to 25 mm in diameter (average 20 mm). These are usually smooth walled but at times provided with one to many papillae (Fig. 2b). The male gametangia (antheridia) are usually much branched (Figs 2c, d and 3d). The antheridia are mostly monoclinous, one to many arising from various distances from the oogonia. The antheridial branches emerge toward the oogonia and give one to many antheridial cells to the oogonia (Fig. 3a–c). The branched antheridia are reminiscent of the male gametangia found in P. minus. The antheridial cells are conspicuous, long and have an apical contact with the oogonia (Fig. 3a). Sometimes the antheridia are devoid of the branches and are monoclinous sessile (Fig. 3c). The oospores are both plerotic and aplerotic (Fig. 4a and b), usually one per oogonium (Fig. 4a), but often two (Fig. 4c and d), and rarely three oospores can be formed. These measure from 11 to 23 mm in diameter (average 16.5 mm); the double oospores can measure up to 45 30 mm (Fig. 4d). The oospore walls are moderately thin, measuring up to 1 mm in thickness.
Discussion The isolates of P. stipitatum were collected from different countries during 2006–2008. All the isolates are typical soil FEMS Microbiol Lett 295 (2009) 164–169
saprophytes. The morphological characters of this oomycete are reminiscent of P. ramificatum described previously by the corresponding author from Algeria (Paul, 1986). Unfortunately, we have to treat this oomycete as ‘nomen invalidum’ because there are no living cultures of this oomycete anywhere and also because nothing is known about its molecular characteristics. However, the old oomycete is literally being revived as P. stipitatum as we think that the isolates collected in France, Turkey, Tunisia, and India are identical to the Algerian isolate. Pythium stipitatum has a coralloid type of antheridia and hence can be easily mistaken with P. minus, as in the case of P. ramificatum (Dick, 1990). However, these two are different oomycetes. The only character they share in common is the formation of coralloid type of antheridial branches. Apart from this, the temperature–growth relationship, and the size of sporangia, oogonia, and oospores are all different. Papillate oogonia were frequently found in our new species, which were completely absent in the latter. The main differences between P. minus and the new species are outlined in Table 1. The ITS sequence of P. stipitatum consists of 874 bases while those of P. minus have 869 bases. The ALIGN comparison of the two sequences presents only 93% similarity between these two species (P. minus, GenBank AY598698). Hence, these two cannot be considered as the same species. Pythium stipitatum is closer to species such as 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Table 1. Comparison of the characteristics of Pythium stipitatum and Pythium minus Characteristics
P. stipitatum
P. minus
ITS size (bp) Growth on PCA at 25 1C (mm) Sporangia or hyphal bodies
874 15 Globose, cylindrical to dumb-bell–shaped, terminal and intercalary, measuring between 10 and 24 mm in diameter (average 15 mm) Branched at times, forming corolloid branches around the oogonia, mostly monoclinous, at times diclinous and at others sessile. one to many antheridial branches emerge toward the oogonia. Antheridial cells conspicuous, long and have an apical contact with the oogonia Oogonia are mostly terminal occasionally intercalary, smooth-walled, measuring from 14 to 25 mm in diameter (average 20 mm). These are usually smooth-walled but at times provided with one to many papillae The oospores are both plerotic and aplerotic, usually one per oogonium, rarely 2–3 oospores can also be found. These measure from 11 to 23 mm in diameter (average 16.5 mm)
869 11 Globose, intercalary, sometimes terminal, measuring 20–40 mm in diameter (average 22.6 mm)
Antheridia
Oogonia
Oospores
Pythium rhizosaccharum (96%, AY207378), two undescribed species F-162 and F-166 (98%, DQ218335 and EF583443), Pythium takayamanum (96%, AB362163), Pythium parvum (95%, AY598697), Pythium pleroticum (93%, AY598642), Pythium cystogenes (93%, AY707985), and Pythium carolinianum (89%, AY987038). A ‘CLUSTAL multiple alignment’ is shown in Supporting Information, Fig. S1, and a phylogentic tree generated with the ITS sequences of these species is shown in Fig. 5 (http://www.genebee.msu.su/services/ phtree_reduced.html). The molecular and morphological characteristics, different from all other species of the genus Pythium, justify the creation of this new taxon. The new oomycete fits in the ‘Clade E’ or ‘Pythium segnitium clade’ as defined by DeCock et al. (2008), although this clade may already be outdated as new members have been added ever since the creation of the clade.
References Bala K, David DR, Paul B & Elad Y (2009) Pythium elicitors in biological control of Botrytis cinerea multitrophic interactions in soil. IOBC/Wprs Bull 42: 11–14. Belbahri L, Calmin G, Sanchez-Hernandez E, Oszako T & Lefort F (2006) Pythium sterilum sp. nov. isolated from Poland, Spain and France: its morphology and molecular phylogenetic position. FEMS Microbiol Lett 255: 209–214. Belbahri L, McLeod A, Paul B et al. (2008) Intra-specific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae). FEMS Microbiol Lett 284: 17–27. Cooke DEL, Drenth JM, Duncan JM, Wagels G & Brasier CM (2000) Fungal genetics and biology. 30: 17–32.
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1–2 ( 5) per oogonium, monoclinous and usually stalked; arising at a distance from the oogonium base, often forming a corolloid development. Antheridial cells clavate
Smooth-walled, globose to subglobose, intercalary or terminal, 10–16 mm in diameter (average 13.3 mm)
Modal number of oospores, 1–6 per oogonium. The oospores measure 10–16 mm (average 13.3) and are plerotic
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region of its ribosomal RNA, and its comparison with related species. FEMS Microbiol Lett 254: 317–323. Paul B, Mathew R, Bala K, Paul A, Henry M, Lefort F & Belbahri L (2008) Morphology, taxonomy, and phylogenetic analysis of a new species of Pythium isolated from France. Fungal Divers 28: 55–63. van der Plaats-Niterink AJ (1981) Monograph of the genus Pythium. Stud Mycol Baarn 21: 1–242. Waterhouse GM (1967) Key to Pythium Pringsheim. Mycological Papers 109: 1–15. White TJ, Bruns T, Lee S & Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications (Innis MA, Gelfand DH, Sninsky JJ & White TJ, eds), pp. 315–322. Academic Press, San Diego.
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. CLUSTAL multiple alignments of the ITS 1 sequences of Pythium stipitatum and related species. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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