Intraspecific DNA polymorphisms of Pythium irregulare

Mycol. Res. 104 (11) : 1333–1341 (November 2000). Printed in the United Kingdom.

1333

Intraspecific DNA polymorphisms of Pythium irregulare

Chieko MATSUMOTO, Koji KAGEYAMA, Haruhisa SUGA and Mitsuro HYAKUMACHI Laboratory of Plant Pathology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan. E-mail : chiekom!mbd.sphere.ne.jp Received 29 April 1999 ; accepted 15 February 2000.

Forty-seven isolates of Pythium irregulare from different hosts and geographic origins were compared from molecular, morphological and physiological viewpoints. They were divided into four groups (I–IV) based on ITS-RFLP analysis and RAPD analysis. Groups I and II included 32 and eight isolates, respectively, collected from diverse hosts and geographic origins, and groups III and IV comprised seven isolates derived from sugar beet and sugar beet field soil. Group I had smaller oogonia and oospores than did the other three groups. In groups I and II, a significantly higher percentage of the oogonia produced multiple projections compared to groups III and IV which occasionally produced one projection. The growth rate of the four groups was similar at 5–30 mC. At 33 m, many isolates of group I grew rapidly but most of the isolates of other groups grew slowly, and at 35 m, the former grew but the latter did not. In phylogenetic analysis based on sequences of the ITS region, four groups of P. irregulare were included in one cluster with P. sylvaticum. Groups I–II and III–IV clustered more tightly in the same branch, respectively. The genetic divergence between I–II and III–IV was higher than between each group (I–II and III–IV) and P. sylvaticum, indicating that groups I–II and III–IV may represent two different species.

INTRODUCTION Pythium is a large genus of the Oomycota including more than 80 species (van der Plaats-Niterink 1981). Pythium species have wide host ranges and mainly cause seed rot, dampingoff, and root rot of seedlings. P. irregulare is one of the most important pathogenic species and is distributed worldwide. In Japan, root rot of tulip (Ichitani et al. 1988), carnation (Kimishima, Kobayashi & Nishio 1991), and welsh onion (Sako et al. 1997), and the damping-off of onion (Tanaka, Kishikawa & Nonaka 1984) have been reported. In other countries, the fungus causes severe damage to the roots of wheat and ryegrass (Dewan & Sivasithamparam 1988), and olive trees (Hernandez, Davila & Casas 1997, Hernandez et al. 1998). Furthermore, the fungus is a well-known causal agent of complex root disease in corn (Mao, Carroll & Whittington 1988), bean (Pieczarka & Abawi 1978), sugarcane, and pineapple (Klemmer & Nakano 1962), along with other species of Pythium or other genera such as Phytophthora (Hendrix & Campbell 1973). P. irregulare is distinguished on the basis of oogonium morphology, which has an irregular number (0–5) of projections, and spherical sporangia (van der Plaats-Niterink 1981). P. spinosum, P. sylvaticum, P. paroecandrum, and P. ultimum are closely related to P. irregulare, as they have similar spherical sporangia and oogonia. Although P. spinosum has an ornamented oogonium, it is distinguished by the finger-like

ornamentation that forms regularly, and by the plerotic oospore. P. sylvaticum, P. paroecandrum, and P. ultimum have smooth oogonia. P. sylvaticum is a heterothallic species that is identified by paired mating with ex-type strains, and it is differentiated from P. irregulare, which is homothallic. P. paroecandum is one of the most difficult species to identify, and is differentiated from P. irregulare on the basis of the more regular and slightly larger, mostly intercalary oogonia that are arranged in chains. Moreover, the germinability of the sporangia with zoospores separates it from P. irregulare. P. ultimum is characterized by a thick oospore wall and a sac-like, sometimes hypogynous but mostly monoclinous, antheridium originating from immediately below the oogonium. P. irregulare has mostly stalked antheridia originating at some distance from the oogonia. However, it is well-known in Pythium species that morphological characteristics are influenced by cultural conditions such as growth media, temperature regimes, and age of the culture. P. irregulare also shows considerable intraspecific variability in morphological characteristics, including the proportion of ornamented oogonia, the number of oogonial projections, and the ratio of aplerotic to plerotic oospores (Biesbrock & Hendrix 1967, van der Plaats-Niterink 1981). Barr, Warwick & Desaulniers (1997) reported that 125 isolates of P. irregulare formed two groups based on isozyme analysis, but the two groups did not differ in morphology or growth rate. They emphasized the need for a non-

Intraspecific polymorphisms of Pythium irregulare morphological method in the accurate identification of P. irregulare and suggested that isozyme analysis is a useful method in this regard. Recently, the use of molecular methods has become popular in taxonomic studies of microorganisms, as these methods provide a great deal of accurate information regarding genetic background. In particular, the analysis of nucleotide sequences of ribosomal DNA (rDNA) genes permits the clarification of phylogenetic relationships over a wide range of taxonomic levels (Cantrell & Hanlin 1997, Ko, Hong & Jung 1997, Okada, Takematsu & Takamura 1997). Coding regions consisting of large subunit rDNA (LrDNA), small subunit rDNA (SrDNA), and 5n8S rDNA evolve relatively slowly and are useful for studying distantly related organisms at kingdom and\or genus levels. Non-coding regions consisting of internal transcribed spacers (ITS) and intergenic spacers (IGS) evolve more rapidly and can be useful for comparisons at species and\or population levels. Furthermore, universal primer sets (White et al. 1990) which amplify each region of rDNA have allowed the easy and rapid analysis of the regions based on restriction banding patterns (Berbee et al. 1995, Peterson 1995, Harrington & Potter 1997). Chen (1992) and Chen, Hoy & Schneider (1992) suggested that P. irregulare is very closely related to some other Pythium species based on RFLP analysis of ITS and SrDNA. RAPD analysis using about 10 mer random primers is also used to clarify the relationships of species and isolates. Phytophthora species were separated on the basis of RAPD patterns (Cooke et al. 1996). In Penicillium nodositatum, intraspecific variation that was not detected in an ITS-RFLP analysis was observed in the RAPD patterns (Sequerra et al. 1997). The purpose of this paper is to examine polymorphism between P. irregulare isolates derived from different hosts and geographic origins, using DNA characteristics such as ITSRFLP and RAPD, morphological characteristics, and hyphal growth rate. ITS sequence data was also used to compare P. irregulare to closely related species, so as to clarify the phylogenetic position of this fungus. MATERIALS AND METHODS Isolates Forty-seven isolates of Pythium irregulare and three isolates of each of P. paroecandrum, P. spinosum, P. sylvaticum and P. ultimum were used in this study (Table 1). The isolates of P. irregulare were collected from different hosts and geographic origin. All isolates were maintained on cornmeal agar (CMA) at 5 mC.

1334 DNA was extracted according to the procedure of Lee & Taylor (1990). RFLP analysis of rDNA-ITS region Primers ITS1 (5h-GTAGTCATATGCTTGTCTC-3h) and ITS4 (5h-CTTCCGTCAATTCCTTTAAG-3h) described by White et al. (1990) were used to amplify the nuclear rDNA region of internal transcribed spacers (ITS) including the 5.8S gene. Total volume of 50 µl reaction mixture contained 1 µ of each primer, 1n25 units of Taq DNA polymerase (Takara Shuzo Co. Ltd, Shiga, Japan), 0n2 m dNTP mixture, 1iPCR buffer (10 m tris HCl, pH 8n3, 50 m KCl and 1.5 m MgCl ), and # 200 ng of DNA template. Reactions were carried out with a DNA Thermal Cycler (Perkin–Elmer Cetus Instruments, Norwalk, Connecticut). The temperature cycling parameters were programmed for one cycle of 3 min at 94 m, followed by 30 cycles of 1 min at 94 m, 1 min at 55 m, 2 min at 72 m, and one cycle of 10 min at 72 m. PCR products were electrophoresed in 1n2 % Agarose LO3 (Takara Shuzo) gel in TAE buffer (40 m tris HCl, pH 7n5, 19 m glacial acetic acid, and 2 m EDTA) and then stained with ethidium bromide. The amplified DNA was used for restriction enzyme analysis. Digestions with four restriction enzymes, EcoRI, TaqI, HaeIII, and HinfI (Toyobo Co Ltd, Osaka), were carried out according to the manufacturer’s specifications. The restriction fragments were electrophoresed in 3n5 % NuSieve (3 : 1) agarose gel (FMC BioProducts, Rockland, MN, USA) in TAE buffer followed by staining with ethidium bromide and visualizing under ultraviolet light. Random amplified polymorphic DNA (RAPD) analysis Seven arbitrary primers (Table 2) were used for RAPD analysis. A total volume of 25 µl reaction mixture contained 1 µ of each primer, 0n625 units of Taq DNA polymerase (Takara Shuzo), 0n2 m dNTP mixture, 1iPCR buffer (10 m tris HCl, pH 8n3, 50 m KCl, 1n5 m MgCl ), and 50 ng of # DNA template. Reactions were carried out with a DNA Thermal Cycler (Perkin–Elmer). The temperature cycling parameters were programmed for one cycle of 3 min at 94 m, followed by 40 cycles of 1 min at 94 m, 1 min at 35 m, 3 min at 72 m, and one cycle of 10 min at 72 m. The RAPD products were electrophoresed in 1n2 % Agarose LO3 (Takara Shuzo) gel in TAE buffer (40 m tris\HCl, pH 7n5, 19 m glacial acetic acid, 2 m EDTA) and stained with ethidium bromide. Amplification of random fragments was confirmed with Image Master VDS (Pharmacia Biotech, San Francisco). A dendrogram was constructed from the cluster analysis generated by similarity coefficients using the unweighted pair group method with arithmetic averages (UPGMA) (Sneath & Sokal 1973). Morphological studies

DNA extraction Three agar plugs were removed from the growing margin of 2day-old cultures on CMA with a 1 cm cork borer and transferred to a flask containing 50 ml of 20 % V8-juice broth containing 2n5 g l−" CaCO . After 4–7 d incubation at 25 m, $ mycelial mats were collected on filter paper, washed with sterile distilled water, and frozen at k80 m. The total genomic

The morphology of oogonia, oospores and antheridia of P. irregulare were observed after 2 weeks growth on CMA (25 m). For each isolate, 60 random oogonia were examined microscopically and the diameter of oogonia and oospores, the ratio of oogonia with projections, the number of projections per oogonia, and the number of antheridia were recorded.

C. Matsumoto and others

1335

Table 1. Pythium species\isolates used in the present study. Species

DNA groupa

Isolate

Host\habitat

Origin

Source

P. irregulare

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II II II II II II II III III III III IV IV IV

10-A 2W-1 2W-2 6K4SSDP A-1 A-2 BH4 BH6 BH30 BH40 BH45 BS12-1 250n28 287n31 265n38 461n48 469n50 749n96 751n96 EP-2 KT16-2 KZ23-4 305572 305894 425327 MI16-2 NG15-3 P-2 PM12-17 T-1 T-4 UOP359 74-22 42912 263n30 492n86 493n86 733n94 PM3-3 PoinB K4-2 K6-2 Py 26 Py 63 Py 61 Py 64 Py 66 157n64 203n79 651n79 OD231 Py 75 TN254 OM121 Py 77 305869 MK2-2-1V OF231 Py 79

Barnyard grass Welsh onion Welsh onion Zoysia grass coarse soil Welsh onion Welsh onion Soil Soil Soil Soil Soil Soil Bean Pea Sugar beet

Gunma, Japan Gifu, Japan Gifu, Japan Gifu, Japan Tottori, Japan Tottori, Japan Australia Australia Australia Australia Australia Aichi, Japan Netherlands Germany USA Australia

K. Kageyama S. Amano S. Amano K. Kageyama I. Sako I. Sako CSIRO CSIRO CSIRO CSIRO CSIRO K. Kageyama CBS CBS CBS CBS CBS CBS CBS K. Kageyama K. Kageyama K. Kageyama MAFF MAFF MAFF K. Kageyama K. Kageyama K. Kageyama K. Kageyama I. Sako I. Sako T. Ichitani K. Kageyama ATCC CBS CBS CBS CBS K. Kageyama S. Amano C. Matsumoto C. Matsumato K. Kageyama K. Kageyama K. Kageyama K. Kageyama K. Kageyama CBS CBS CBS K. Kageyama K. Kageyama K. Kageyama K. Kageyama K. Kageyama MAFF K. Kageyama K. Kageyama K. Kageyama

P. paroecandrum

P. spinosum

P. sylvaticum

P. ultimum

* Based on the composite rDNA genotype.

Lepidum sativum Soil Lepidum sativum Carrot Soil Soil Soil Soil Soil Soil Soil Cucumber Bean Welsh onion Welsh onion Tulip Bean

UK UK Gifu, Japan Nara, Japan Gifu, Japan Kanagawa, Japan Kagawa, Japan Hokkaido, Japan Gifu, Japan Gifu, Japan Gifu, Japan Hokkaido, Japan Tottori, Japan Tottori, Japan Kyoto, Japan Hokkaido, Japan

Tobacco Malus sylvestris Sweet cherry Wheat Bean Poinsettia Sugar beet field soil Sugar beet field soil Sugar beet Sugar beet Sugar beet Sugar beet Sugar beet Soil Water Soil Carrot Sugar beet Carrot Carrot Sugar beet Soil Bentgrass Carrot Sugar beet

USA Poland Poland Canada Hokkaido, Japan Gifu, Japan Hokkaido, Japan Hokkaido, Japan Hokkaido, Japan Hokkaido, Japan Hokkaido, Japan Hokkaido, Japan Hokkaido, Japan Australia Netherlands Netherlands Gifu, Japan Hokkaido, Japan Gifu, Japan Gifu, Japan Hokkaido, Japan Fukuoka, Japan Gifu, Japan Gifu, Japan Hokkaido, Japan

Intraspecific polymorphisms of Pythium irregulare

1336

Table 2. Sequence of oligonucleotide primers used in the present study.

a b c

Primer name

Sequence 5h–3h

A01a A07a A10a A11a OPR08b OPR13b R28c

TGC ACT ACA ACA TGC CTC GCA CCA ACT GGC CGA GGG GAT GGA TTT GGG CCC GTT GCC T GGA CGA CAA T ATG GAT CCG C

M

I

II

III

IV

bp 800 350

EcoRI

Wako Pure Chemical Industry Ltd, Osaka. Operon Technologies Inc., Alameda. Duncan et al. (1993). 800

Growth rates

350

Growth rates at different temperatures were examined for isolates of Pythium irregulare. Three agar plugs (6 mm in diam) taken from the edge of colonies growing on CMA were transferred to the edge of a 9 cm Petri dish containing 10 ml of potato-carrot agar (PCA). The cultures were incubated for 24 h at the test temperature (5–40 m), and the edge of the colony was marked and re-examined 24 h later.

800 350

TaqI

HinfI

Sequencing of rDNA-ITS region Primers ITS1 and ITS4 were used in PCR reactions as previously described. Following electrophoresis, targeted bands were cut out of agarose gels and the DNA containing the bands were extracted using TE-saturated phenol and phenol\chloroform\isoamyl alcohol (25 : 24 : 1, v\v\v). The Big Dye4 Terminater Cycle Sequencing Ready Reaction Kit (Perkin–Elmer) was used for the sequence reaction according to the instructions of the manufacturer. Reactions were carried out with a Gene Amp PCR system 2400 (Perkin–Elmer). Electrophoresis of the sequencing products was carried out with an ABI 377 DNA sequencer (Perkin–Elmer) or an ABI 310 DNA sequencer (Perkin–Elmer). Phylogenetic analysis The sequence data of Phytophthora megasperma, used an outgroup, were drawn from the EMBL databank. All sequences were first aligned using the multiple sequence alignment program CLUSTAL W ver. 1.60 (Thompson, Higgins & Gibson 1994), and the alignment was optimized. Alignment gaps were treated as missing data and ambiguous positions were excluded from the analysis. Nucleotide variation of the sequences were analyzed by the maximum-parsimony method of the Phylogenetic Analysis Using Parsimony (PAUP) program 3.1.1 (Swofford 1993). The bootstrap analysis was implemented using 100 replicates of heuristic searches to determine the confidence levels of the inferred phylogenies (Felsenstein 1988). RESULTS RFLP of rDNA-ITS regions The amplified ITS regions were about 1020-bp long for P. irregulare, although there were slight differences between some groups of isolates. Digestion with EcoRI resulted in

800

HaeIII

350

Fig. 1. Restriction banding patterns of polymerase chain reactionamplified internal transcribed spacer of rDNA from selected Pythium isolates after digestion with EcoRI, TaqI, Hinfl and HaeIII. Key : M l size marker from a 50 bp DNA ladder (Gibco–BRL) ; Pythium isolates were : I l CBS 287.31 and EP-2 ; II l ATCC 42912 and PM3-3 ; III l K6-2 and Py26 ; IV l Py61 and Py64.

similar banding patterns for all isolates (Fig. 1). Digestion with TaqI, HaeIII, and HinfI did reveal variation, however two and three banding patterns were observed in digestion with TaqI and HinfI, respectively (Fig. 1). Seven of 47 isolates had one restriction site for HaeIII, but others had no restriction sites. Based on the combinations of banding patterns of these four enzymes, the 47 isolates were divided into four groups (I–IV) (Table 1). Thirty-two of the 47 isolates were included in group I, and eight isolates were in group II. The isolates of these groups come from diverse hosts and geographic origins. Four and three isolates were included in groups III and IV, respectively, and all were derived from sugar beet or field soil of sugar beet in Hokkaido in Japan. RAPD analysis Each of the seven primers produced a variety of banding

C. Matsumoto and others M

1337 I

II

III

IV

bp 2072

600 A01

2072

600

A07

2072

A11 600

2072

600

OPR08

Fig. 2. Random amplified polymorphic DNA analysis of the selected isolates of Pythium irregulare using the primers A01, A07, A11 and OPR08. Key : M l size marker from a 100 bp DNA ladder (Gibco BRL) ; Pythium isolates were : I l 2W-1, 6K4-SSDP, A-1, CBS 265.38, CBS 751.96, EP-2, KT16-2, MAFF 305572, Ml16-2, PM12-17 and UOP 359 ; II l 74-22, ATCC 42912, CBS 263.30, PM3-3 and PoinB ; III l K6-2, Py26 and Py63 ; IV l Py61 and Py64.

patterns for 21 isolates as representative of the four groups identified by RFLP analysis. In particular, there were four clear groups of similar banding patterns with the A01, A07, A11, and OPR08 primers, each group corresponding to the RFLP groups I–IV defined above (Fig. 2). Two to three groups of

banding patterns were obtained using the A10, OPR13, and R28 primers. The variable banding patterns for all seven primers are summarized by the cluster analysis shown in Fig. 3. The isolates were first separated into two clusters based on a similarity value of 0n16, and these two clusters were then

Intraspecific polymorphisms of Pythium irregulare 0

1338 0.5

0.5 2W-2 A-1 6K4SSDP MAFF 305704 PM12-7 KT16-2 MI16-2 UOP359 CBS 265.38 CBS 751.96 EP-2 ATCC 42912 74-22 CBS 263.30 PM3-3 PoinB K6-2 Py26 Py63 Py61 Py64

Cluster I

Cluster II

Cluster III

Cluster IV

Fig. 3. Dendrogram showing relationships among 21 isolates of Pythium irregulare based on RAPD patterns. The dendrogram was constructed from the similarity coefficient by using UPGMA. Table 3. Morphological characteristics of Pythium irregulare. Oogonia diameter (µm)

a b c d e

Oospore diameter (µm)

Projection rating (%)b

Number of projectionsc

Number of antheridiad

DNA groupa

Range

Mean

Range

Mean

Mean

Range

Mean

Range

Mean

I II III IV

14n1–24n7 14n9–26n3 16n0–22n7 16n3–25n4

17n62 ae 19n43 b 19n06 b 20n25 b

13n3–23n2 14n6–24n4 15n0–20n7 14n7–23n2

16n39 a 18n16 b 17n34 b 18n13 b

48n69 b 45n47 b 11n04 a 14n20 a

1–8 1–9 1 1–2

1n72 b 1n74 b 1n00 a 1n25 ab

1–4 1–4 1–2 1–4

1n22 a 1n33 a 1n38 a 1n25 a

Based on the composite rDNA genotype. The percentage of oogonia with projection for each isolate. Number of projections per oogonium that have some projection. Number of antheridia associated with oogonium. Means carrying same letters in a column are not significantly different (P l 0n05) according to Duncan’s multiple range test.

separated into two clusters with similarity values of 0n48 and 0n38, respectively. The isolates included in each cluster corresponded to the four groups in the RFLP analysis (Table 1). Isolates within each group had similarity values of 0n71–1n0 and were grouped more tightly. Morphology Each isolate of P. irregulare formed globose, subspherical, limonform oogonia that were intercalary or terminal. Oospores were mostly aplerotic and only rarely plerotic. The ratio of oogonia to sporangia varied within isolates. In isolates forming oogonia abundantly in CMA, the sporangia were relatively sparse. On the other hand, in isolates forming sporangia abundantly in CMA, the oogonia were sparse or rarely seen. This tendency was same as observations in grass blade culture (Waterhouse 1967). In particular, the isolates of groups III and IV produced few oogonia, which were large. The average of size of oogonia and oospores in group I was smaller than in groups II, III, and IV (Table 3). Considerable variation was observed among the groups in the percentage of oogonia with projections. In groups I and II,

the percentage was commonly high at 10–73 % for individual isolates (mean 47 %, Table 3), while in all the isolates of groups III and IV it was much lower at 9–16 % (mean 12 %, Table 3). All isolates of groups I and II formed some oogonia with plural projections, but in groups III and IV, the number of projections was single except for one isolate in group IV. Antheridia were mostly monoclinous, originating at some distance from the oogonia, although they were occasionally hypogynous or diclinous. Differences among the groups were not observed, and most of the isolates had one to two antheridia per oogonium, although some varied from three to four per oogonia. Growth rates Hyphal growth rates of the three representative isolates in each group are shown in Fig. 4. The four groups grew at similar rates at 5–30 m, and the optimum temperature was 28 m or 30 m. However, a clear difference was observed among the groups in the growth rate at 35 m. Therefore, further experiments were performed to compare growth rates at 33 and 35 m using all isolates that grew normally at 25 m (22–28 mm

C. Matsumoto and others

1339 Table 4. Growth rates (mm 24 h−") of Pythium irregulare isolates at 25m, 33m and 35 mC.

Growth rate (mm 24 h–1)

30

Temperature (mC) 20

Group I

10

Group II Group III Group IV

0

5

10

15

20 25 28 30 Temperature (°C)

35

40

Fig. 4. Growth rates of Pythium irregulare. Data was obtained as mean values for selected isolates of each group. Pythium isolates were : Group I l 6K4SSDP, EP-2 and MAFF 305894 ; Group II l ATCC 42912, CBS 263.30 and CBS 492.86 ; Group III l K6-2, Py26 and Py63 ; Group IV l Py61.

24 h−", on PCA). At 33 m, 18 of 25 isolates in group I grew rapidly (20n3–24n3 mm 24 h−"), while all isolates in group II grew comparatively slowly (4n0–18n5 mm 24 h−", Table 4). The above 18 isolates in group I grew at 35 m (3n0–12n0 mm 24 h−") ; in contrast, most of the isolates in groups II–IV, except for PoinB, did not grow at this temperature. None of the isolates grew at 40 m. Phylogenetic analysis based on the sequences of the rDNA-ITS regions Sequences of the ITS regions were resolved for twelve representative isolates of P. irregulare (four isolates from group 1, three each from groups II and III, and two from group IV) and three isolates each of P. paroecandrum, P. spinosum, P. sylvaticum, and P. ultimum. The length of the ITS regions including the 5n8S rDNA was 937 bp in group I of P. irregulare, and two sites of substitution were recognized among the four isolates. In group II, three isolates had ITS regions of different lengths, 938, 945, and 948 bp, and there were 29 sites of substitution or insertion\deletions. In group III, all three isolates had 916 bp sequences, and one site of substitution was recognized. Two isolates of group IV had the same ITS sequences with lengths of 926 bp. P. spinosum, P. sylvaticum, and P. ultimum had ITS regions 916, 907, 825 bp long, respectively, and there were one to three sites of substitution among the three isolates of each species. The three isolates of P. paroecandrum had ITS sequences completely distinct from one another. The data from 24 taxa provided 865 aligned sites. In these sites, 158 and 430 sites in ITS1 and ITS2 were used for phylogenetic analysis. The parsimony analysis of these characters produced one minimum length tree of 458 steps, with a consistency index (CI) and retention index (RI) of 0n857 and 0n879, respectively (Fig. 5). In the phylogenetic tree, four groups of P. irregulare and P. sylvaticum were included in Cluster A-1. In this cluster, groups I–II and groups III–IV were grouped more tightly, and the genetic divergence between groups I and II, and III and IV was very low (Table 5). The genetic divergence between groups

a

Isolate

DNA groupa

25

33

35

10-A 2W-1 2W-2 6K4SSDP A-1 A-2 BH4 BH6 BH40 BH45 BS12-1 CBS 469n50 CBS 749n96 EP-2 KT16-2 KZ23-4 MAFF 305572 MAFF 305894 MAFF 425327 MI16-2 NG15-3 P-2 T-1 T-4 UOP 359

I I I I I I I I I I I I I I I I I I I I I I I I I

24n5 27n5 26n8 26n5 28n0 25n5 22n3 22n5 23n8 23n5 28n0 25n5 25n5 26n8 22n3 25n0 25n3 26n0 23n3 28n0 25n8 25n5 26n5 27n0 23n8

23n8 23n3 22n5 23n5 22n5 24n3 6n8 6n0 1n5 2n3 24n3 7n0 2n3 22n5 23n5 22n5 22n0 23n0 17n5 23n5 23n5 23n5 22n8 23n0 20n3

8n5 11n0 11n5 10n5 12n0 9n5 0n0 0n0 0n0 0n0 8n5 0n0 0n0 10n1 9n5 4n3 9n5 9n3 0n0 4n3 11n3 7n8 10n5 10n5 3n0

74-22 ATCC 42912 CBS 263n30 CBS 492n86 CBS 493n86 CBS 733n94 PoinB

II II II II II II II

23n3 27n5 25n0 24n5 27n8 27n8 28n0

4n0 8n0 6n0 18n0 17n0 11n8 18n5

0n0 0n0 0n0 0n0 0n0 0n0 1n5

K4-2 K6-2 Py26 Py63

III III III III

26n0 23n5 24n5 25n0

8n5 9n0 0n0 4n0

0n0 0n0 0n0 0n0

Py61

IV

26n3

0n0

0n0

Based on the composite rDNA genotype.

I–II and III–IV, groups I–II and P. sylvaticum, and groups III–IV and P. sylvaticum were 6n01, 3n89, and 3n33 %, respectively. P. spinosum (Cluster A–2) and P. ultimum (Cluster B) were placed distant from P. irregulare. In the three isolates of P. paroecandrum, CBS 157n64 was closely related to groups I–II of P. irregulare, CBS 651n79 was included in the cluster of P. sylvaticum, and CBS 203.79 similar to Phytophthora, did not belong to any cluster. DISCUSSION In this study, P. irregulare was divided into four groups, I–IV, based on the phylogenetic analysis of ITS sequences and RAPD analysis, and groups I and II, III and IV were close related, respectively. In the four groups of P. irregulare, groups I and II included isolates having various hosts and geographic origins. Chen, Hoy & Schneider (1992) and Wang & White (1997) analyzed RFLP of ITS regions for several isolates and showed that the restriction patterns of these isolates were the

Intraspecific polymorphisms of Pythium irregulare

97

93

1340

MAFF 305572

I

EP-2

I

10-A

I

UOP359

I

83 61 60

ATCC 42912

II

74-22

II

PoinB

II

CBS 157.64 95 Cluster A-1

86

67

K6-2

III

Py26

III

Py63

III

99

Cluster A

55

100 66

99 Cluster A-2 100

Cluster B 100

par

Py61

IV

Py64

IV syl syl

CBS 651.79

par

MAFF 305869

syl

OD231

spi

TN254

spi

MK2-2-1V

ult

OF231

ult

Py79

ult

CBS 203.79

Within groups I and II of P. irregulare Within groups III and IV of P. irregulare Between groups I–II and III–IV of P. irregulare

Between groups III–IV of P. irregulare and P. sylvaticum

Py77

spi

Divergence

Between groups I–II of P. irregulare and P. sylvaticum

OM121

Py75

Table 5. Percentage of percent genetic divergence in ITS1jITS2 from pairwise comparisons among four groups of Pythium irregulare and P. sylvaticum.

par

Phytophthora

Fig. 5. Consensus tree of Pythium species based on the combined ITS1 and ITS2 sequences. The consensus tree was generated from maximum parsimony analysis using the heuristic search algorithm of PAUP 3.1.1. Numbers of the branches indicate the bootstrap values resulting from 100 bootstrap replications. I, II, III, and IV, represent groups of P. irregulare based on the composite rDNA genotype. The letters of par, sly, spi, and ult represent the species, P. paroecandrum, P. sylvaticum, P. spinosum, and P. ultimum, respectively.

same as those of group I, which is the largest group in this study. Barr et al. (1997) separated 125 isolates of P. irregulare into two groups, A and B, based on isozyme analysis. Group A consisted of 118 isolates, including five isolates (CBS 250.28, CBS 287.31 CBS 265.38, CBS 461.48, CBS 469.50) and two isolates (CBS 263.30, CBS 493.86), that belong to our groups I and II, respectively. These results suggest that group A may correspond to groups I and II and that these groups may be widespread throughout the world. On the other hand, groups III and IV consisted of seven isolates, all derived from sugar beet or sugar beet field soil in different areas of Hokkaido and all obtained at different sampling times. Two other isolates from Hokkaido belonged to groups I and II, but these were isolated from bean. An isolate collected from sugar beet was included in group I, but this isolate did not originate from Hokkaido. Therefore, groups III and IV might be specific to a combination of host and geographic origin. In the isozyme analysis conducted by Barr et al. (1997), seven isolates of group B collected worldwide had only a few oogonia with projections that grew slowly or could not grow

0n99a (0n00–1n80)b 1n20 (0n00–2n00) 6n01 (5n30–7n10) 3n89 (3n50–4n40) 3n33 (2n80–3n90)

Genetic divergence (%) l (number of substitution\total number of sequence)i100. a Average of percent genetic divergence. b Range of percent genetic divergence.

at 30–35 m. These characteristics are similar to those of our groups III and IV, suggesting that group B may correspond to groups III and IV. In view of these results, groups III and IV appear to have a wide distribution with a low frequency of isolation, although the isolates examined were obtained from sugar beet in Hokkaido. Further research with more isolates is needed to clarify this point. Biesbrock et al. (1967) examined morphology in P. irregulare and reported that the ratio of aplerotic to plerotic oospores and the number of oogonia with projections varied among isolates. In the present study, the size of oogonia and oospores in group I was smaller than in the other three groups, although the difference was very small. On the other hand, considerable variation in the ratio of oogonia with projections and in the number of projections were observed between the groups. Groups I and II showed a high percentage of oogonia with a number of projections. Isolates of groups III and IV, however, had little ability to form oogonia regardless of whether the culture was new or old. Three of the seven isolates in groups III and IV had few oogonia of abnormally large size when they were formed. P. sylvaticum is generally regarded to be heterothallic, but several isolates can produce a few sexual organisms in single culture (Kageyama, Ui & Uchino 1991). Therefore, the isolates of groups III and IV seemed to be P. sylvaticum. However, when these isolates were paired with female and male strains of P. sylvaticum, none reacted to either of the strains or developed sexual organs. The results demonstrate that these isolates are not P. sylvaticum. In terms of growth rate, 18 of 25 isolates of group I grew at 35 m, whereas 11 of 12 isolates of groups II–IV did not grow at this temperature regardless of culture age, suggesting that groups II–IV had little growth ability at high temperature (33–35 m). According to the description of van der PlaatsNiterink (1981), P. irregulare grows 25 mm 24 h−" on CMA at 25 m and the maximum temperature for growth is 35 m. In the present study, two neotype isolates, CBS 263.30 and CBS 469.50, used as type cultures by van der Plaats-Niterink (1981), grew 25n0–25n5 mm 24 h−" in PCA at 25m but did not grow at 35 m. Furthermore, according to the report of Barr et al. (1997), the neotype isolates CBS 250.28 and CBS 461.48

C. Matsumoto and others grew 8–11 mm 24 h−" in PCA at 25 m but did not grow at 35m. These results indicate that the growth rate at optimum temperature and maximum temperature might be unreliable as taxonomic criteria for the identification of P. irregulare. In DNA analysis, the isolates of P. irregulare were separated into four groups and clearly distinguished from other species. The genetic divergences were very low between groups I and II, and between III and IV, but not between groups I–II and III–IV, I–II and P. sylvaticum, and III–IV and P. sylvaticum. The phylogenetic distances between groups I–II and III–IV were higher than those between each of the groups (I–II and III–IV) and P. sylvaticum, suggesting that groups I–II and III–IV may represent two different species. There are differences in the ratio of oogonial projection and in the number of the projections between the I–II and III–IV groups, as mentioned above. However these groups cannot be differentiated on the basis of morphology because all isolates corresponded to the morphological taxonomic criterion that includes oogonium with an irregular number (0–5) of projections and spherical sporangia (Waterhouse 1967, van der Plaats-Niterink 1981). Further investigations based on pathological and biochemical approaches are needed to detect the variations in phenotype originating from genomic diversity. DNA analysis, however, is simply a method for examining intraspecific polymorphisms of P. irregulare and is useful for distinguishing this species from other morphologically similar species.

REFERENCES Barr, D. J. S., Warwick, S. I. & Desaulniers, N. L. (1997) Isozyme variation, morphology, and growth response to temperature in Pythium irregulare. Canadian Journal of Botany 75 : 2073–2081. Berbee, M. L., Yoshimura, A., Sugiyama, J. & Taylor, J. W. (1995). Is Penicillium monophyletic? An evaluation of phylogeny in the family Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence date. Mycologia 87 ; 210–222. Biesbrock, J. A. & Hendrix, F. F. jr (1967) A taxonomic study of Pythium irregulare and related species. Mycologia 59 : 943–952. Cantrell, S. A. & Hanlin, R. T. (1997) Phylogenetic relationships in the family Hyaloscyphaceae inferred from sequence of ITS regions, 5.8S ribosomal DNA and morphological characters. Mycologia 89 : 745–755. Chen, W. (1992) Restriction fragment length polymorphisms in enzymatically amplified ribosomal DNAs of three heterothallic Pythium species. Phytopathology 82 : 1467–1472. Chen, W., Hoy, J. W. & Schneider, R. W. (1992). Species-specific polymorphisms in transcribed ribosomal DNA of five Pythium species. Experimental Mycology 16 : 22–34. Cooke, D. E. L., Kennedy, D. M., Guy, D. C., Russell, J., Unkles, S. E. & Duncan, J. M. (1996) Relatedness of group I species of Phytophthora as assessed by randomly amplified polymorphic DNA (s) and sequences of ribosomal DNA. Mycological Research 100 : 297–303. Dewan, M. M. & Sivasithamparam, K. (1988) Pythium spp in roots of wheat and rye-grass in western Australia and their effect on root rot caused by Gaeumannomyces graminis var. tritici. Soil Biology and Biochemistry 20 : 801–808. Duncan, S., Barton, J. E. & O’Brien, P. A. (1993) Analysis of variation in isolates of Rhizoctonia solani by random amplified polymorphic DNA assay. Mycological Research 97 : 1075–1082. Felsenstein, J. (1988) Phylogenies from molecular sequences : inference and reliability. Annual Review of Genetics 22 ; 521–565. Harrington, F. A. & Potter, D. (1997) Phylogenetic relationships with Sarcoscypha bases upon nucleotide sequences of the internal transcribed spacer of nuclear ribosomal DNA. Mycologia 89 : 258–267.

1341 Hendrix, F. F., Jr. & Campbell, W. A. (1973) Pythium’s as plant pathogens. Annual Review of Phytopathology 11 : 77–98. Hernandez, M., Davila, A., Dealgaba, A., Lopez, M. & Casas, A. (1998) Occurrence and etiology of death of young olive trees in southern Spain. European Journal of Plant Pathology 104 : 347–357. Hernandez, M. E. S., Davila, A. R. & Casas, A. T. (1997) First report of Phytophthora megasperma and Pythium irregulare as olive tree root pathogens. Plant Disease 81 : 1216. Ichitani, T., Fukunishi, T., Kang, H. T. & Miyata, Y. (1988) Identification of root rot fungi of tulip. Annals of the Phytopathological Society of Japan 54 : 355. [In Japanese.] Kageyama, K., Ui, T. & Uchino, H. (1991) Pythium sylvaticum isolates from bean and sugarbeet, and their sexual behavior. Transactions of the Mycological Society of Japan 32 : 283–289. Kimishima, E., Kobayashi, Y. & Nishio, T. (1991) Root rot of carnation caused by Pythium irregulare and P. aphanidermatum. Annals of the Phytopathological Society of Japan 57 : 534–539. Klemmer, H. W. & Nakano, R. Y. (1962) Distribution and pathogenicity of Phytophthora and Pythium in pineapple soils of Hawaii. Plant Disease Reporter 48 : 848–852. Ko, K. S., Hong, S. G. & Jung, H. S. (1997) Phylogenetic analysis of Trichaptum based on nuclear 18S, 5.8S and ITS ribosomal DNA sequences. Mycologia 89 : 727–734. Lee, S. B. & Taylor, J. W. (1990) Isolation of DNA from fungal mycelia and single spores. In PCR Protocols : a guide to methods and applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds) : 282–287. Academic Press, San Diego. Mao, W., Carroll, R. B. & Whittington, D. P. (1998) Association of Phoma terrestris, Pythium irregulare, and Fusarium acuminatum in causing red root of corn. Plant Disease 82 : 337–342. Okada, G., Takematsu, A. & Takamura, Y. (1997) Phylogenetic relationships of the hyphomycete genera Chaetopsina and Kionochaeta based on 18S rDNA sequences. Myoscience 38 : 409–420. Peterson, S. W. (1995) Phylogenetic analysis of Aspergillus sections Cremei and Wentii, based on ribosomal DNA sequences. Mycological Research 99 : 1349–1355. Pieczarka, D. J. & Abawi, G. S. (1978) Effect of interaction between Fusarium, Pythium, and Rhizoctonia on severity of bean root rot. Phytopathology 68 : 403–408. Sako, I., Tojo, M., Kato, M. & Taguchi, Y. (1997) Root rot of welsh onion caused by Pythium irregulare. Annals of the Phytopathological Society of Japan 63 : 203. [In Japanese.] Sequerra, J., Marmeisse, R., Valla, G., Normand, P., Capellano, A. & Moiroud, A. (1997) Taxonomic position and intraspecific variability of the nodule forming Penicillium nodositatum inferred from RFLP analysis of the ribosomal intergenic spacer and amplified polymorphic DNA. Mycological Research 101 : 465–472. Sneath, P. H. A. & Sokal, R. (1973) Numerical Taxonomy. W. H. Freeman, San Francisco. Swofford, D. L. (1993) PAUP : Phylogenetic analysis using parsimony, Version 3.1.1. [Computer program] Illinois Natural History Survey, Champaign. Tanaka, K., Kishikawa, T. & Nonaka, F. (1984) Occurrence of damping-off of onion seedlings in Saga Prefecture. Proceedings of Association of Plant Protection Kyushu 30 : 177. [In Japanese.] Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) CLUSTAL W : improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22 : 4673–4680. Wang, P. H. & White, J. G. (1997). Molecular characterization of Pythium species based on RFLP analysis of the internal transcribed spacer region of ribosomal DNA. Physiological Molecular of Plant Pathology : 51 129–143. Waterhouse, G. M. (1967) Key to Pythium Pringsheim. Mycological Papers 109 : 1–15. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols : a guide to methods and applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds) : 315–322. Academic Press, San Diego. van der Plaats-Niterink, A. J. (1981) Monograph of the genus Pythium. Studies in Mycology 21 : 1–142. Corresponding Editor : R. F. Park