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Plant Protection Service, Department of Mycology, P.O. Box 9102, 6700 HC Wageningen, the Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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Isozyme analysis and cytochrome oxidase sequences were used to examine whether differentiation of P. fragariae var. fragariae and P. fragariae var. rubi at the variety level is justified. In isozyme studies six strains of both P. fragariae varieties were analyzed with malate dehydrogenase (MDH), glucose phosphate isomerase (GPI), aconitase (ACO), isocitrate dehydrogenase (IDH) and phosphogluconate dehydrogenase (PGD), comprising altogether seven putative loci. Five unique alleles (Mdh-1A, Mdh-2 B, Gpi A, AcoB and Idh-1B) were found in strains of P. fragariae var. fragariae, whereas five unique alleles (Mdh-1B, Mdh-2 A, GpiB, Aco A and Idh-1A) were present in strains of P. fragariae var. rubi. It was inferred from these data that there is no gene flow between the two P. fragariae varieties. Cytochrome oxidase I (Cox I ) sequences showed consistent differences at 15 positions between strains of Fragaria and Rubus respectively. Based on isozyme data, cytochrome oxidase I sequences, and previously published differences in restyriction enzyme patterns of mitochondrial DNA, sequences of nuclear and mitochondrial genes, AFLP patterns and pathogenicity, it was concluded that both specific pathogenic varieties of P. fragariae are reproductively isolated and constitute a distinct species. Consequently strains isolated from Rubus idaeus are assigned to Phytophthora rubi comb. nov.
Key words: Cytochrome oxidase I, isozymes, Phytophthora rubi comb. nov
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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). Being homothallic, this species produces oospores autonomously that can rest in the soil up to 15 y without losing the ability to germinate and to infect strawberry plants. P. fragariae is assigned a quarantine status in EU countries on which a "nil tolerance" is placed. Later morphologically similar isolates were obtained from Rubus idaeus (raspberry) and described as P. fragariae var. rubi (Wilcox et al 1993). These isolates were highly pathogenic to raspberry, but caused only small amounts of necrosis in strawberry roots. The typical symptoms of red core root rot could not be produced in strawberry by isolates obtained from raspberry in pathogenicity experiments. Several molecular studies were done to define the delimitation of both varieties. Total protein patterns obtained with SDS-PAGE of the raspberry isolates were almost identical with those of P. fragariae (Duncan et al 1991). Restriction enzyme analysis of mitochondrial DNA (Förster et al 1992, StammLer et al 1993) with seven restriction enzymes showed no difference between strains from strawberry and strains from raspberry, whereas seven other restriction enzymes revealed fragment length differences between the two populations. It was inferred from these data that mtDNA of the two populations have a similarity value of 55%, indicating that they probably originate from a common ancestor. It was concluded that the strains from strawberry and the strains from raspberry represent two different homogeneous groups which however are closely related. Förster et al (1992) suggested that, considering the intraspecific diversity of P. capsici, P. citricola, P. citrophthora and P. megakarya, the strains from strawberry and the strains from raspberry might represent the genetic diversity of one single morphological species. Wilcox et al (1993) therefore decided to assign the raspberry isolates to P. fragariae. However it was also their opinion that there were sufficient differences between the two populations to justify separation at the variety level. It was noted by Wilcox et al (1993) that "should reproductive isolation be demonstrated between the raspberry and the strawberry forms of P. fragariae in future studies, it may be necessary to recognize them as separate biological species". Based on the high sequence homology of the ITS regions of the ribosomal DNA gene repeat of the two P. fragariae varieties, Cooke and Duncan (1997) concluded that taxonomical separation at the variety level was justified. The purpose of this paper was to test whether P. fragariae var. fragariae and P. fragariae var. rubi indeed are reproductively isolated by genetic barriers to gene flow by subjecting strains of both varieties to isozyme analysis. In addition cytochrome oxidase I sequences of some strains were determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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Enzyme extraction.—. Frozen mycelium was thawed at 4 C for 3 h before enzyme extraction. Routinely about 0.5 g of mycelium was ground in a chilled mortar with sand and 70 µL of extraction medium. The extraction medium consisted of 0.1 M Tris-HCl (pH 7.0), 1 mM dithiothreitol, 50 mM ethylene diamine tetra-acetic acid (EDTA, disodium salt, M.W.: 372.2), 10% polyvinyl pyrrolidone (PVP, M.W.: 25.000) w/v, 50 µg/mL soybean trypsin inhibitor, 0.1 mM phenyl methyl sulfonyl fluoride (PMSF) and 5% glycerol (v/ v). All mycelia were ground for 3 min. The homogenate was transferred to Eppendorf tubes and centrifuged 10 min at 14 000 rpm (4 C). The supernatant (40–80 µL) was collected and stored at –80 C before use.
Electrophoresis and enzyme staining.—. Isozymes were separated by electrophoresis with the automated PhastSystem of Amersham Pharmacia Biotech (Roosendaal, the Netherlands). Crude extracts, obtained as described before, were loaded on native polyacrylamide gels and subsequently electrophoresis was performed at 4 C for about 40 min. Bromphenol blue marker dye was added to the samples to monitor the progress of electrophoresis. The gels were made with 0.11 M Tris-acetate buffer (pH 6.4). The running buffer, contained in 2% agarose gel, consisted of a 0.25 M Tris and 0.88 M L-alanine buffer (pH 8.8). At completion of electrophoresis, gels were immersed in freshly prepared staining solutions in the dark at 37 C. Five enzymatic stains gave clearly interpretable bands, notably those for aconitase (ACO, EC 4.2.1.3 [EC] ), glucose-phosphate isomerase (GPI, EC 5.3.1.9 [EC] ), isocitrate dehydrogenase (IDH, EC 1.1.1.42 [EC] ), malate dehydrogenase (MDH, EC 1.1.1.37 [EC] ) and 6-phosphogluconate dehydrogenase (PGD, EC 1.1.1.44 [EC] ). The reaction ingredients for each enzyme were as follows (between brackets the geltype used in the assay):
ACO contained 25 mL of 0,2 M Tris-HCl pH 8.0, 50 mg of cis-aconitic acid (Sigma A 3412), 100 mg of MgCl2, 6 units of isocitrate dehydrogenase (Sigma I 2002), 12.5 mg of ß-nicotinamide adenine dinucleotide phosphate (NADP), 7.5 mg of nitro blue tetrazolium (NBT) (Sigma N 6876) and 1 mg of phenazine metho sulfate (PMS) (Sigma P 9625). (10–15% gradient gel)
GPI contained 25 mL of 0.1 M Tris-HCl pH 8.0, 20 mg of fructose-6-phosphate, 50 mg of MgCl2, 10 units of G6PDH (Sigma G 5760), 12.5 mg of ß-nicotinamide adenine dinucleotide (NAD), 7.5 mg of NBT and 1 mg of PMS. (12.5% homogeneous gel)
IDH contained 25 mL of 0.1 M Tris-HCl pH 8.0, 12.5 mg of isocitrate (trisodium salt), 100 mg of MgCl2, 10 mg of NADP, 7.5 mg of NBT and 1 mg of PMS. (8–25% gradient gel)
MDH: 25 mL 0.2 M Tris-HCl pH 8.0, 440 mg L-malic acid (disodium salt), 12.5 mg NAD, 7.5 mg NBT, 1 mg PMS. (8–25% gradient gel)
PGD contained 25 mL of 0.1 M Tris-HCl pH 8.0, 10 mg of 6-phosphogluconic acid (trisodium salt) 50 mg of MgCl2, 10 mg of NADP, 7.5 mg of NBT and 1 mg of PMS. (10–15% gradient gel)
Isozyme activity was recorded based on the relative mobility of the banding patterns. Each band was considered to be an allele belonging to a specific locus, and alleles were numbered alphabetically according to their relative mobilities. Because the genus Phytophthora is diploid, two identical letters were assigned to one band. When two zones of activity were present on the gel, the slowest zone was assigned the first locus and the fastest zone the second locus. It is known from isozyme analysis of several Phytophthora species that malate dehydrogenase and isocitrate dehydrogenase generate two loci (Oudemans and Coffey 1991b).
Sequence analysis of cytochrome oxidase I (Cox I).—.
Sequence analysis of Cox I was performed with COXF4N and COXR4N primers according to the protocol of Kroon et al (2004). One strain of P. fragariae var. fragariae (P965-DQ674735
[GenBank]
) and two strains of P. fragariae var. rubi (R49-DQ674736
[GenBank]
and P1282-DQ674737
[GenBank]
) were sequenced and their sequences were aligned with sequences in GenBank. Sequences were edited with Seqman 4.05 (DNASTAR, Madison, Wisconsin). MegAlign (DNASTAR) was used to perform the final alignment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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). All enzymes generated clear bands except at Mdh-1, which showed a smeary appearance in three strains (TABLE I). Two isozyme loci, notably IDH-2 and PGD, were monomorphic for all isolates in this study. Five other loci, notably ACO, GPI (FIG. 1), IDH-1, MDH-1 and MDH-2 (FIG. 2), were polymorphic and resolved the population into two groups: one comprised the P. fragariae isolates from Fragaria and the second those from Rubus idaeus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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). If however, in spite of almost similar morphology and identical ITS sequences, P. fragariae strains from strawberry and those from raspberry do not share a common gene pool and hence apparently are reproductively isolated by genetic barriers to gene flow, separating the two populations at the species level is justified (Wilcox et al 1993). It is clear from this study that P. fragariae strains from strawberry as well as those from raspberry each contain several unique isozyme alleles, indicating that they indeed are isolated reproductively. In Southern hybridization the probes pPS2 and pPS42 generated different hybridization patterns in both P. fragariae varieties, whereas probe pPS39 hybridized exclusively with total DNA from strains isolated from Rubus (including R49, which also is analysed in the present study) (StammLer et al 1993). Molecular analysis based on AFLP patterns of both varieties of P. fragariae (including P965 and P823, which also are analysed in the present study) also indicated that both varieties represent different genetic entities (Brasier et al 1999). Moreover both taxa contained different Cox I sequences as well (FIG. 3
). In addition sequence analysis of four nuclear genes (ASF-like, GPA1, Ras-Ypt and TRP1) and two mitochondrial genes (Nadh and Cox II ) showed that in all cases both taxa contained different sequences (Martin and Tooley 2003, Ioos et al 2006). Hence it is concluded that it is justified to raise the rank of P. fragariae var. rubi to the species level for which the name P. rubi is proposed.
The question whether both populations are reproductively isolated by pre- or post mating barriers awaits an answer. Attempts to infect each other’s host hitherto have been unsuccesful, and this lack of success in itself indicates a strong premating reproductive isolating mechanism. Restriction enzyme analysis of mitochondrial DNA revealed that both populations have homogeneous but different DNA profiles with a similarity level of ~55% (Förster et al 1992) indicating that it is unlikely that reticulation has taken place recently. The sequences of Cox II differed also between strains from Fragaria and Rubus (Martin and Tooley 2003) and restriction enzyme analysis of a region spanning the Cox I and Cox II genes could distinguish isolates from Fragaria and Rubus as well (Martin and Tooley 2004), thus supporting the results of isozyme analysis. Sequence analysis of Cox I showed variation in strains ex Fragaria as well as in strains ex Rubus idaeus, indicating that both taxa are not clonal.
A third variety is reported by Wang and Lu (1978), P. fragariae var. oryzobladis Wang & Lu. According to Ho and Jong (1998) the type strain no longer is available for examination. In contrast to strains isolated from Fragaria and Rubus, this variety is characterized by rapid growth and the abundance of chlamydospores. The assignment to P. fragariae by Wang and Lu (1978), according to Ho and Jong (1998), is not justified at all by morphological data. Therefore P. fragariae var. oryzobladis has no affinity with P. fragariae at all and hence varieties of P. fragariae automatically cease to exist.
A combination of isozyme analysis and mitochondrial DNA analysis has been used before to separate taxa with high morphological similarity. Based on host range, nutritional, biochemical and morphological criteria, it was proposed by Galindo and Hohl (1985) to name strains from Mirabilis jalapa as a variety of P. infestans. The sequences of the ITS regions of the ribosomal DNA gene repeat were identical between the two varieties, but small yet distinct differences were found in mtDNA restriction profiles. Möller et al (1993) therefore proposed assigning the isolates obtained from Mirabilis jalapa to P. infestans forma specialis mirabilis. Goodwin (1999) however concluded that, based on isozyme analysis and mitochondrial haplotypes, isolates obtained from Mirabilis jalapa and P. infestans sensu stricto were reproductively isolated by genetic barriers to gene flow, and hence he proposed to assign isolates obtained from Mirabilis jalapa to be P. mirabilis. Isozyme analysis and mitochondrial DNA analysis also were used to demonstrate that P. porri-like strains isolated from Brassica constitute a distinct species: P. brassicae (Man in ’t Veld et al 2002). Isozymes, separated by electrophoresis under native conditions, are well defined by their mobility and identity. Isozyme analysis is one of the oldest techniques used to characterize Phytophthora species genetically. Due to rapid developments of DNA-related techniques in the past decade, isozyme analysis is not widely used anymore. However its potential in studying the population structure (Oudemans and Coffey 1991a, b), delineating species (Goodwin et al 1999, Man in ’t Veld et al 2002) and, by using dimeric enzymes, detecting crossings and hybrids (Man in ’t Veld et al 1998, Brasier et al 2004) will remain valuable in the future.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYLITERATURE CITED |
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Basionym Phytophthora fragariae var. rubi Wilcox & Duncan, Mycological Research 97(7):830. 1993.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: w.a.man.in.t.veld{at}minlnv.nl
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LITERATURE CITED |
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———, Kirk SA, Delcan J, Cooke DEL, Jung T, Man in ’Veld WA. 2004. Phytophthora alni sp. nov. and its variants: designation of emerging heteroploid hybrid pathogens spreadng on Alnus trees. Mycol Res 108:1172–1184.[CrossRef][Medline]
Cooke DEL, Duncan JM. 1997. Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycol Res 101(6): 667–677.[CrossRef]
Duncan JM, Kennedy DM, Scott PH. 1991. Relationships between non-papillate, soilborne species of Phytophthora: root rot of raspberry. In: Lucas JA, Shatlock RC, Straw DS, Cooke LR, eds. Phytophthora. Symposium of the British Mycological Society, the British Society for Plant Pathology and the Society for Irish Plant Pathologists held at Trinity College, Dublin, Sep 1989. Cambridge, UK: Cambridge University Press. p 129–147.
Förster H, Coffey MD. 1992. Molecular characterization of Phytophthora isolates with nonpapillate sporangia causing root rot of raspberry using mtDNA restriction fragment length polymorphisms. Mycol Res 96:571–577.
Galindo AJ, Hohl HR. 1985. Phytophthora mirabilis, a new species of Phytophthora. Sydowia, Annal Mycol II 38:87–96.
Goodwin SB, Legard DE, Smart CD, Levy M, Fry WE. 1999. Gene flow analysis of molecular markers confirms that Phytophthora mirabilis and P. infestans are separate species. Mycologia 91(5):796–810.[CrossRef]
Hickman CJ. 1940. The red core root disease of the strawberry caused by Phytophthora fragariae. J Pomol Hortic Sci 18:89–119.
Ho HH, Jong SC. 1988. Phytophthora fragariae. Mycotaxon 31(2):305–322.
Ioos R, Andrieux A, Marcais B, Frey P. 2006. Genetic characterization of the natural hybrid species Phytophthora alni as inferred from nuclear and mitichondrial DNA analysis. Fungal Genet Biol 43:511–529.[CrossRef][Medline]
Kroon LPNM, Bakker FT, van den Bosch GBM, Bonants PJM, Flier WG. 2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences. Fungal Genet Biol 41:766–782.[CrossRef][Medline]
Man in ’t Veld WA, Veenbaas-Rijks WJ, Ilieva E, de Cock AWAM, Bonants PJM, Pieters R. 1998. Natural hybrids of Phytophthora nicotianae and Phytophthora cactorum demonstrated by isozyme analysis and random amplified polymorphic DNA. Phytopathology 88:922–929.[Medline]
———, de Cock AWAM, Ilieva E, Lévesque CA. 2002. Gene flow analysis of Phytophthora porri reveals a new species: Phytophthora brassicae sp. nov. Eur J Plant Pathol 108: 51–62.[CrossRef]
Martin FN, Tooley PW. 2003. Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes. Mycologia 95:269–284.
———, ———. 2004. Identification of Phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction-amplified region of mitochonrial DNA. Phytopathology 94:983–991.[CrossRef][Medline]
Möller EM, de Cock AWAM, Prell HH. (1993). Mitochondrial and nuclear DNA restriction enzyme analysis of the closely related Phytophthora species P. infestans and P. mirabilis, and P. phaseoli. J Phytopathol 139:309–32.[CrossRef]
Oudemans P, Coffey MD. 1991a. Isozyme comparison within and among worldwide sources of three morphologically distinct species of Phytophthora. Mycol Res 95(1):19–30.
———, ———. 1991b. A revised systematics of twelve papillate Phytophthora species based on isozyme analysis. Mycol Res 95(9):1025–1046.
StammLer G, Seemüller E, Duncan JM. 1993. Analysis of RFLPs in nuclear and mitochondrial DNA and the taxonomy of Phytophthora fragariae. Mycol Res 97:151–156.
Wang J, Jia-yun L. 1978. Phytophthora leaf blight of rice seedlings—a new disease of rice. Acta Microbiol Sinica 18:95–101.
Wilcox WF, Scott PH, Hamm PB, Kennedy DM, Duncan JM, Brasier CM, Hansen EM. 1993. Identity of a Phy-Phytophthora species attacking raspberry in Europe and North America. Mycol Res 97(7):817–831.
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