ITS1 region of the nuclear ribosomal DNA of the mycoparasitePythium periplocum, its taxonomy, and its comparison

with related species

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

Received 30 March 2000; accepted 6 May 2000

Abstract

Pythium periplocum Drechsler was isolated from some soil samples taken in the botanical garden of Tenerife, Canary Islands. This fungushas been found to be an aggressive mycoparasite of Botrytis cinerea. It is unique amongst the members of the genus Pythium because of thecharacter combination of inflated filamentous type of sporangia and ornamented oogonia. The taxonomic description of this fungus and itscomparison with related species, together with the polymerase chain reaction of the internal transcribed spacer of its nuclear ribosomalDNA, are discussed in this article. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Allrights reserved.

Keywords: Sporangium; Antheridium; Oogonium; Oospore; ITS1 region; Ribosomal DNA; Pythium periplocum

1. Introduction

More than 200 species have been described within thegenus Pythium which was described by Pringsheim in1858. These are widely distributed throughout the world.Many are well-known as plant parasites, however not allare necessarily phytopathogens. Most of these fungi canlive as saprophytes, while others as facultative saprophytesbecoming plant pathogens from time to time and causingextensive damage to economically important crops worldwide [1]. Some of these can also behave as parasites ofmosquito larvae [2] and one is known to be a mammalianparasite [3]. Unlike most of the eumycetes, the members ofthis genus remain diploid throughout their life cycles withmeiosis occurring in the gametangia before fertilization [4].The female gametangia (oogonia) are usually smooth-walled, but species having ornamented oogonia are notrare for the genus, the most commonly occurring specieswith such oogonia is Pythium echinulatum. The wall orna-mentations or spines are merely extensions of the oogonialwall. The spines may be short or long, conical or curved,

with sharp or blunt apices, or may be digitate, mammi-form, or papillar [5]. The oogonia of Pythium periplocumare covered with short, blunt spines.

Monographs on the taxonomic descriptions of the genusPythium have appeared from time to time and the mostwidely used are those given by Middleton [5], Waterhouse[6] and Plaats-Niterink [7]. These taxonomic references arebased on the comparison of morphological characteristicsof di¡erent members of the genus. P. periplocum belongsto the group of Pythium having ¢lamentous in£ated typeof sporangia and ornamented oogonia. Such charactercombination is not found in any other species of the ge-nus.

Increasingly the taxonomic description of fungi combinemorphological descriptions and molecular data [8]. Molec-ular taxonomy may be approached in various ways includ-ing DNA probes [9^12] ; restricted fragment length poly-morphism analysis (RFLP) of polymerase chain reaction(PCR)-ampli¢ed rDNA [13,14], RFLP of mitochondrialDNA [15], RFLP of total DNA [16], species-speci¢c prim-ers [17], karyotype analysis using ¢eld gel electrophoresis[18], and RAPD [19]. PCR-RFLP is a useful tool in fungaltaxonomy and is currently used to identify di¡erent speciesof Pythium [20^22]. Ampli¢cation of the ribosomal gene isused for the genetic identi¢cation of many organisms sincethey comprise both highly conserved sequences during

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 5 2 - 4

* Corresponding author. Tel : +33 (3) 80396341;Fax: +33 (3) 80396326; E-mail : bernard.paul@u-bourgogne.fr

FEMSLE 9479 14-7-00

FEMS Microbiology Letters 189 (2000) 61^65

www.fems-microbiology.org

evolution and highly variable sequences which resolve atvarious taxonomic scales. The ribosomal nuclear DNAconsists of transcribed and non-transcribed regions [20].The internal transcribed spacer (ITS1) is a non-conservedregion and has been ampli¢ed with the PCR method usinguniversal primers ITS1 and ITS2.

P. periplocum is not a very common species as it has notbeen frequently reported from many places around theworld. Since its original discovery by Drechsler in USA[23], it has rarely been isolated. One report from TheNetherlands [7] indicates its presence in Europe. This is

the second occasion when it has been isolated in Europefrom soil samples taken in the Canary islands. Recently ithas been shown to be an aggressive mycoparasite to Bo-trytis cinerea [24]. P. periplocum when grown together withthe former, enters into its mycelium, branches freely with-in, coagulates its cytoplasm, and ¢nally pierces out fromits hyphae. Such mycoparasitism may be exploited tobring about biological control of B. cinerea, the causalorganism of the gray mould disease of the grape-vine[24]. In the present paper, the morphological details ofP. periplocum together with the sequences of the ITS1

Fig. 1. a^c: Filamentous in£ated type of sporangia; d: spherical oogonia ornamented with blunt spines; e,f : terminal oogonia; g: catenulate oogonia;h^k: oogonia provided with antheridia; l^p,r: aplerotic oospores; q: plerotic oospores. All ¢gures: bar = 25 Wm.

FEMSLE 9479 14-7-00

B. Paul, I. Masih / FEMS Microbiology Letters 189 (2000) 61^6562

region of its ribosomal nuclear DNA and its comparisonwith those of related species are given.

2. Materials and methods

2.1. Fungal material

Soil samples were collected in small plastic bags by thesenior author during a trip to the Canary islands. The soilsampling was done at random, and some samples camefrom the botanical garden of Tenerife. P. periplocum wasisolated from these by using the usual baiting techniquesdescribed elsewhere [5,25]. The fungus (CI056) is beingmaintained in the senior author's collection of pythiaceousfungi, at `Institut Jules Guyot', in Dijon, France. The col-onies isolated on hemp-seeds were puri¢ed by repeatedsub-culturing on fresh hemp-seeds in sterile distilled water.These were also maintained on potato dextrose agar(PDA), and potato carrot agar (PCA) media. For a com-parative study, other species such as Pythium oligandrum(F-960.2), Pythium acanthophoron (F-375), Pythium perii-lum (NC-65) were also taken from the same collection.

2.2. Culture conditions, DNA isolation, and PCR

The fungi were grown in potato dextrose broth (samecomposition as PDA except agar) in 30 ml sterile bottlescontaining 10 ml of the medium. These were incubated at25³C on a rotary shaker. After 5 days of rotation, themycelium was washed and kept at 320³C for 24 h, andDNA was isolated by methods described elsewhere [21].The ITS1 regions of the nuclear ribosomal DNA of fungiwere ampli¢ed by PCR using the universal primers, ITS1which is at the 3P end (TCCGTAGGTGAACCTGCGG)and ITS2 which is at the 5P end (GCTGCGTTCTTCATC-GATGC) as described earlier [22].

2.3. DNA sequences

The sequencing of the DNA was performed by `GenomeExpress', Paris. The sequences have been submitted to theGenBank under accession numbers AF203782 (P. periplo-cum), AF203783 (P. oligandrum), AF195769 (P. acantho-phoron) and AF203785 (P. periilum). The ITS1 sequence ofP. acanthicum was obtained from the GenBank(AF016502).

3. Results

3.1. Morphological description: P. periplocum (Figs. 1^4).

Mycelia are frequently branched at times bearing smallbranched appendages. Sporangia are of the ¢lamentousin£ated type, irregular in shape, terminal or intercalary,

formed of irregularly swollen elements which when spher-ical are up to 30 Wm in diameter (Figs. 1a^c and 2a).Zoospores are readily formed at room temperature(20³C) in vesicles which are in turn borne on long slenderdischarge tubes of various lengths. Encysted zoosporesmeasure between 8 and 10 Wm in diameter. Oogonia areterminal (Figs. 1e,f and 2c) or intercalary (Fig. 1f) rarelycatenulate (Figs. 1g and 2g), 16^28 Wm diameter (average25 Wm), oogonial walls are provided with small, bluntspines measuring 2^5 Wm in length and 1^3 Wm diameterat the base (Fig. 3). Antheridia 1^5 per oogonium (Figs.1h^k and 2d^f) closely applied to the latter and at timesencircling the oogonia. Antheridial cells in£ated, vanishing

Fig. 2. a: ¢lamentous in£ated type of sporangia; b,c: ornamented oogo-nia; d^f: oogonia with diclinous antheridia; g: catenulate oogonia; h:plerotic oospores; i : double oospores in an oogonium; j: aplerotic oo-spores. a, bar = 50 Wm; b^j, bar = 25 Wm

FEMSLE 9479 14-7-00

B. Paul, I. Masih / FEMS Microbiology Letters 189 (2000) 61^65 63

soon after fertilization, up to 30 Wm in length and 8 Wm indiameter. Oospores aplerotic (¢g. 1l^p,r and 2i,j) and pler-otic (Figs. 1q and 2g^h) measuring 15^26 Wm (average 21Wm), wall moderately thick (up to 3 Wm), sometimes twooospores (Figs. 1r and 2i) per oogonium were also ob-served.

The fungus grows well on solid media, on PCA itsgrowth is more than 30 mm day31 at 25³C, while onPDA the fungus produces a dense aerial mycelium givinga cottony aspect. On hemp seeds in water the fungusgrows luxuriant provided there is no bacterial contamina-tion. The mycelium of the fungus is very fragile and failsto grow on the slightest contamination.

The size of the PCR-ampli¢ed ITS1 region of the nu-clear ribosomal DNA of P. periplocum (AF203782) is 186bases: CCACACCTAAAAACTTTCCACGTGAACCG-TTATAACTATGTTCTGTGCTTCGTCGCAAGACTT-GAGGCTGAACGAAGGTGAGTCTGCGTCTATTTT-GGATGCGGATTTGCTGATGTTATTTTAAACACC-TATTACTTAATACTGAACTATACTCCGAATACGA-AAGTTTTTGGTTTTAACAATTAA

This was compared to the ITS1 regions of related my-coparasitic fungi having ornamented oogonia like P. oli-gandrum (186 bases), P. acanthophoron (192 bases),P. acanthicum (189 bases). The ITS1 sequence of P. peri-plocum was also compared with that of P. periilum (174bases), a species which is neither a mycoparasite, nor onewhich has the ornamented type of oogonia, the only com-mon character being the ¢lamentous in£ated type of spor-angia. The sequence alignment of these species is given inFig. 4. According to the ALIGN (version 2.0u) calculationthe ITS1 sequence of P. periplocum has a 99.5% identitywith P. oligandrum, 93.7% identity with P. acanthicum,89.2% identity with P. acanthophoron and 77.4% identitywith P. periilum.

4. Discussion

P. periplocum isolated from soil samples taken in theCanary islands resembles in most of its details that de-scribed by Drechsler [23]. This isolate however has someminor di¡erences worth mentioning: presence of mostlyplerotic oospores instead of aplerotic ones and at timesthe presence of two oospores per oogonium. The ITS1sequence of its nuclear ribosomal DNA is comprised of186 bases. The comparative study of the ITS1 regions ofthe mycoparasites, P. periplocum, P. oligandrum, P. acan-thicum, and P. acanthophoron as well as with a non-my-coparasite having smooth-walled oogonia, P. periilumshows a high degree of resemblance between the mycopar-asites (99.5, 93.7 and 89.2% respectively) while very littleresemblance with P. periilum (77.4%) a non-mycoparasite.

Fig. 4. CLUSTAL W (1.8) Multiple alignment of ITS1 sequences of P. periplocum, P. oligandrum, P. acanthicum, P. acanthophoron, and P. periilum.

Fig. 3. Scanning electron micrograph of an ornamented oogonium ofP. periplocum. Bar = 10 Wm.

FEMSLE 9479 14-7-00

B. Paul, I. Masih / FEMS Microbiology Letters 189 (2000) 61^6564

Moreover, all the mycoparasites are morphologically sim-ilar in the sense that they all possess ornamented type ofoogonia, while the non-mycoparasite, P. periilum hassmooth-walled oogonia. Hence the analysis of the ITS1region of the nuclear ribosomal DNA does not contradictthe morphological analysis and can be considered as asupplementary tool in the identi¢cation and comparisonbetween species of the same genera.

References

[1] Martin, F. (1994) Pythium. In: Pathogenesis and Host Speci¢city inPlant Diseases: Histopathological, Biochemical, Genetic and Molec-ular Bases, Vol.2, Eukaryotes (Komoto, K., Singh, U.S. and Singh,R.P., Eds.), pp. 17^36. Pergamon Press, Oxford.

[2] Saunders, G.A., Washburn, J.O., Egerter, D.E. and Anderson, J.A.(1988) Pathogenecity of fungi isolated from ¢eld collected larvae ofthe western treehole mosquito, Aedes sierrensis (Diptera: Culicidae).J. Invertebr. Pathol. 52, 360^363.

[3] De Cock, A.W.A.M., Mendoza, L., Padhye, A.A., Ajello, L. andKaufman, L. (1987) Pythium insidiosum sp. nov., the etiologic agentof pythiosis. J. Clin. Microbiol. 25, 344^349.

[4] Martin, F. (1995) Meiotic instability of Pythium sylvaticum as dem-onstrated by inheritance of the nuclear markers and karyotype anal-ysis. Genetics 139, 1233^1246.

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

[6] Waterhouse, G.M. (1967) Key to Pythium Pringsheim. Mycol. Pap.109, 1^15.

[7] Van der Plaats-Niterink, A.J. (1981) Monograph of the genusPythium. Studies in Mycology, Vol. 21, pp. 1^242. Centraal-bureauvoor Schimmelcultures, Baarn.

[8] Levesque, C.A., Harlton, C.E. and de Cock, A.W.A.M. (1998) Iden-ti¢cation of some oomycetes by reverse dot blot hybridisation. Phy-topathology 88, 213^222.

[9] Martin, F.N. (1991) Selection of DNA probes useful for isolate iden-ti¢cation of two Pythium spp. Phytopathology 81, 742^746.

[10] Levesque, C.A., Vrain, T.C. and De Boer, S.H. (1994) Developmentof species-speci¢c probe for Pythium ultimum using ampli¢ed riboso-mal DNA. Phytopatholgy 84, 474^478.

[11] Matthew, J., Hawke, B.G. and Pankhurst, C.E. (1995) A DNA probefor the identi¢cation of Pythium irregulare in soil. Mycol. Res. 99,579^584.

[12] Klassen, G.R., Balcerzak, M. and de Cock, A.W.A.M. (1996) 5SRibosomal RNA gene spacers as species-speci¢c probes for eightspecies of Pythium. Phytopathology 86, 581^587.

[13] Chen, W. and Hoy, J.W. (1993) Molecular and morphological com-parison of Pythium arrhenomanes and P. graminicola. Mycol. Res. 97,1371^1378.

[14] Ra¢n, C., Brygoo, Y. and Tirilly, Y. (1995) Restriction analysis ofampli¢ed ribosomal DNA of Pythium spp. isolated from soilless cul-ture systems. Mycol. Res. 99, 277^281.

[15] Martin, F.N. and Kistler, H.C. (1990) Species-speci¢c banding pat-terns of restriction endonuclease-digested mitochondrial DNA fromthe genus Pythium. Exp. Mycol. 14, 33^46.

[16] Descalzo, R.C., Punja, Z.K., Levesque, A.C. and Rahe, J.E. (1996)Identi¢cation and role of Pythium species as glyphosate synergists onbean (Phaseolus vulgaris) grown in di¡erent soils. Mycol. Res. 100,971^978.

[17] Wang, P.H., White, J.G. (1996) Development of a species-speci¢cprimer for Pythium violae. In: BCPC Symposium Proceedings 65:Diagnostics in Crop Production, pp. 205^210.

[18] Martin, F.N. (1995) Electrophoretic karyotype polymorphisms in thegenus Pythium. Mycologia 87, 333^353.

[19] Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. andTingey, S.V. (1990) DNA polymorphism ampli¢ed by arbitrary prim-ers are useful as genetic markers. Nucleic Acids Res. 18, 6531^6535.

[20] Chen, W. (1992) Restriction fragment length polymorphisms in enzy-matically ampli¢ed ribosomal DNA of 3 heterothallic Pythium spe-cies. Phytopathology 82, 1467^1472.

[21] Chen, W., Hoy, J.W. and Schneider, R.W. (1992) Species-speci¢cpolymorphisms in transcribed ribosomal DNA of ¢ve Pythium spe-cies. Exp. Mycol. 16, 22^34.

[22] Paul, B., Galland, D. and Masih, I. (1999) Pythium prolatum isolatedfrom soil in the Burgundy region: A new record for Europe. FEMSMicrobiol. Lett. 173, 69^75.

[23] Drechsler, C. (1930) Some new species of Pythium. J. Wash. Acad.Sci. 20, 398^418.

[24] Paul, B. (1999) Pythium periplocum, an aggressive mycoparasite ofBotrytis cinerea causing the grey mould disease of grape-vine. FEMSMicrobiol. Lett. 181, 277^280.

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

FEMSLE 9479 14-7-00

B. Paul, I. Masih / FEMS Microbiology Letters 189 (2000) 61^65 65