Phytopythium: molecular phylogeny and systematics.
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Abstract
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Phytopythium: molecular phylogeny and systematics
Abstract
The genus Phytopythium (Peronosporales) has been described, but a complete circumscription has not yet been presented. In the present paper we provide molecular-based evidence that members of Pythium clade K as described by Lévesque & de Cock (2004) belong to Phytopythium. Maximum likelihood and Bayesian phylogenetic analysis of the nuclear ribosomal DNA (LSU and SSU) and mitochondrial DNA cytochrome oxidase subunit 1 (COI) as well as statistical analyses of pairwise distances strongly support the status of Phytopythium as a separate phylogenetic entity. Phytopythium is morphologically intermediate between the genera Phytophthora and Pythium. It is unique in having papillate, internally proliferating sporangia and cylindrical or lobate antheridia. The formal transfer of clade K species to Phytopythium and a comparison with morphologically similar species of the genera Pythium and Phytophthora is presented. A new species is described, Phytopythium mirpurense.
INTRODUCTION
The genus Pythium as defined by Pringsheim in 1858 was divided by Lévesque & de Cock (2004) into 11 clades based on molecular systematic analyses. These clades are generally well supported by morphological features. In particular, Pythium species belonging to clade K were observed to be phylogenetically distinct from the rest of the Pythium spp. and showed combined features of both Pythium and Phytophthora. The unique phylogenetic placement of species belonging to clade K has been recognised since the beginning of sequence-based phylogenetics. Briard et al. (1995) and Cooke et al. (2000) showed that Pythium vexans was clearly different from other Pythium spp. and Phytophthora using the ribosomal large subunit (LSU) and internal transcribed spacer (ITS), respectively. In a multigene study, Villa et al. (2006) showed that Pythium species belonging to clade K were closely related to Phytophthora. The uniqueness of this clade was also supported by Bedard et al. (2006) by analysis of the organisation of the 5S gene family. In species in clade K, the 5S rRNA genes were predominantly linked to the rDNA repeat mostly in tandem arrays in the same orientation as the rRNA genes.
Phytopythium is a new genus in the family Pythiaceae, order Peronosporales that was described with Phytopythium sindhum as the type species by Bala et al. (2010b). They showed that Phytopythium sindhum is a member of clade K. Uzuhashi et al. (2010) divided Pythium into five new genera and assigned the name Ovatisporangium to the members of clade K, this name, however, is a later synonym of Phytopythium. Phytopythium therefore has priority. The objective of the present study is to establish which species belong to clade K and to make new taxonomic combinations for these species. To achieve this goal, phylogenies based on nuclear LSU rRNA (28S), SSU rRNA (18S) and mitochondrial DNA cytochrome oxidase1 (COI) as well as statistical analyses of the pairwise distances from these datasets were prepared with an extensive coverage of the oomycetes containing almost all Pythium and Phytophthora species available in culture. The ITS gene region was also used to ascertain the position of all possible species in clade K but not for phylogeny since it is too variable to align sequences between Pythium and Phytophthora. Diagnostic morphological features of the group are also presented and discussed.
MATERIALS AND METHODS
Morphological studies
The strains used for the phylogenetic study were morphologically examined to verify their identity and to find the characteristic features of the group. The methods used for cultivation of the strains for study of morphology and zoospore development are the same as described by de Cock & Lévesque (2004).
DNA extraction, amplification and sequencing
Almost 300 strains of Pythium, Phytopythium, Phytophthora, Halophytophthora and Albugo were used in this study (Table 1). DNA was extracted using the protocols as described in Bala et al. (2010a). PCR amplifications for the rDNA LSU and ITS1-5.8S-ITS2 regions and mitochondrial DNA COI were done using the protocols and primer sequences as provided in Robideau et al. (2011). The SSU region was amplified using forward primer NS1 (5’-TAGTCATATGCTTGTCTC-3’) (White et al. 1990) and reverse primer OomLo5.8S47B (3’-CGCATTACGTATCGCAGTTCGCAG-5’) (Mazzola et al. 2002), with an initial denaturation at 95 °C for 3 min, 35 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 45 s, elongation at 72 °C for 2 min and final elongation at 72 °C for 8 min. Sequencing primers used for the SSU region were NS1, NS2 (5’-GGCTGCTGGCACCAGACTTGC3’), NS3 (5’-GCAAGTCTGGTGCCAGCAGCC), NS4 (5’-CTTCCGTCAATTCCTTTAAG3’), NS5 (5’-AACTTAAAGGAATTGACGGAAG3’) and NS8 (5’-TCCGCAGGTTCACCTACGGA3’) (White et al. 1990) as well as Oom_Lo5.8S47 (5’-ATTACGTATCGCAGTTCGCAG3’) (Man in ’t Veld et al. 2002) for full bidirectional coverage. Sequencing reactions were prepared using the Big Dye Terminator (BDT) v. 2 protocols (Applied Biosystems, Foster City, CA). Sequencing of the PCR product was performed in an Applied Biosystems Prism Genetic Analyzer model 3130XL.
Table 1
Species and isolates included in the study, showing GenBank accession numbers for each gene.
| GenBank Accessions | ||||||||
|---|---|---|---|---|---|---|---|---|
| Species | Strain Number | Clade | SSU_ITS_28S | SSU_ITS | SSU | COI | LSU | ITS |
| Albugo candida | AC2V | – | – | – | HQ708184 | HQ665049 | – | |
| AC7A | – | – | HQ643110 | HQ708183 | HQ665050 | – | ||
| ACCS | – | – | KF853245 | – | – | – | ||
| Halophytophthora avicenniae | CBS188.85 | Halophytophthora | – | – | – | HQ708219 | HQ665146 | – |
| Halophytophthora operculata | CBS241.83 | Phytopythium | – | – | GU994173 | KF853238 | KJ128038 | KJ128038 |
| Halophytophthora polymorphica | CBS680.84 | Halophytophthora | – | – | – | – | HQ665288 | – |
| Phytophthora alni | P10564 | Clade 7 | – | – | JN635200 | – | – | – |
| Phytophthora alticola | P16053 | Clade 4 | – | – | JN635264 | – | – | – |
| Phytophthora andina | P13660 | Clade 1 | – | – | JN635253 | – | – | – |
| Phytophthora arecae | CBS305.62 | Clade 4 | – | – | – | HQ708218 | HQ665200 | – |
| Phytophthora austrocedrae | P16040 | Clade 8 | – | – | JN635271 | – | – | – |
| Phytophthora batemanensis | CBS679.84 | Halophytophthora | – | – | – | HQ708220 | HQ665286 | – |
| Phytophthora bisheria | P10117 | Clade 2 | – | – | – | – | EU080746 | – |
| P11311 | Clade 2 | – | – | JN635246 | HQ261249 | – | – | |
| Phytophthora boehmeriae | CBS291.29 | Clade 10 | – | – | – | HQ708221 | HQ665190 | – |
| P1257 | Clade 10 | – | – | JN635228 | – | – | – | |
| P6950 | Clade 10 | – | – | – | – | EU080166 | – | |
| Phytophthora botryosa | P1044 | Clade 2 | – | – | JN635168 | – | – | – |
| Phytophthora brassicae | CBS178.87 | Clade 8 | – | – | – | HQ708225 | HQ665144 | – |
| P10155 | Clade 8 | – | – | JN635172 | – | – | – | |
| P3273 | Clade 8 | – | – | JN635066 | – | – | – | |
| Phytophthora cactorum | CBS108.09 | Clade 1 | – | – | – | KJ128035 | KJ128036 | – |
| P0714 | Clade 1 | – | – | JN635210 | – | – | – | |
| P10365 | Clade 1 | – | – | JN635194 | – | – | – | |
| Phytophthora capsici | CBS554.88 | Clade 2 | – | – | – | HQ708250 | HQ665266 | – |
| P6522 | Clade 2 | – | – | JN635061 | – | – | – | |
| Phytophthora captiosa | P10719 | Clade 9 | – | – | JN635227 | – | – | – |
| P10720 | Clade 9 | – | – | JN635229 | – | – | – | |
| Phytophthora cinnamomi | CBS144.22 | Clade 7 | – | – | – | HQ708257 | HQ665126 | – |
| Phytophthora cinnamomi var. parvispora | CBS411.96 | Clade 7 | – | – | – | HQ708268 | HQ665231 | – |
| Phytophthora cinnamomi var. robiniae | P16351 | Clade 7 | – | – | JN635269 | – | – | – |
| Phytophthora citricola | CBS221.88 | Clade 2 | – | – | – | HQ708269 | HQ665161 | – |
| Phytophthora citrophthora | CBS950.87 | Clade 2 | – | – | – | HQ708272 | HQ665305 | – |
| P1212 | Clade 2 | – | – | JN635223 | – | – | – | |
| Phytophthora clandestina | P3942 | Clade 1 | – | – | JN635111 | – | – | – |
| Phytophthora colocasiae | P6102 | Clade 2 | – | – | JN635058 | – | – | – |
| Phytophthora cryptogea | P16165 | Clade 8 | – | – | JN635259 | – | – | – |
| CBS468.81 | Clade 8 | – | – | – | HQ708276 | HQ665238 | – | |
| Phytophthora drechsleri | P10331 | Clade 8 | – | – | – | – | EU079511 | – |
| P1087 | Clade 8 | – | – | – | HQ261299 | – | – | |
| P1087 | Clade 8 | – | – | JN635260 | – | – | – | |
| Phytophthora erythroseptica | CBS129.23 | Clade 8 | – | – | – | HQ708286 | HQ665121 | – |
| P1693 | Clade 8 | – | – | JN635249 | – | – | – | |
| Phytophthora europaea | P10324 | Clade 7 | – | – | JN635189 | – | – | – |
| Phytophthora fallax | P10722 | Clade 9 | – | – | JN635219 | – | – | – |
| Phytophthora foliorum | P10969 | Clade 8 | – | – | – | HQ261307 | EU079704 | – |
| Phytophthora fragariae | CBS209.46 | Clade 7 | – | – | – | HQ708294 | HQ665150 | – |
| P1435 | Clade 7 | – | – | JN635233 | – | – | – | |
| Phytophthora frigida | P16051 | Clade 2 | – | – | JN635162 | – | – | – |
| Phytophthora gonapodyides | CBS363.79 | Clade 6 | – | – | – | – | HQ665216 | – |
| CBS554.67 | Clade 6 | – | – | – | HQ708297 | HQ665265 | – | |
| P10337 | Clade 6 | – | – | JN635201 | – | – | – | |
| P3700 | Clade 6 | – | – | JN635141 | – | – | – | |
| Phytophthora hedraiandra | CBS118732 | Clade 1 | – | – | – | HQ708300 | – | – |
| PDA331 | Clade 1 | – | – | – | – | EU080880 | – | |
| Phytophthora heveae | CBS296.29 | Clade 5 | – | – | – | HQ708301 | HQ665194 | – |
| P10167 | Clade 5 | – | – | JN635090 | – | – | – | |
| Phytophthora hibernalis | P3822 | Clade 8 | – | – | JN635091 | – | – | – |
| Phytophthora himalayensis | CBS357.59 | Clade 8 | – | – | – | – | HQ665215 | – |
| Phytophthora humicola | CBS200.81 | Clade 6 | – | – | – | – | HQ665148 | – |
| P3826 | Clade 6 | – | – | JN635108 | – | – | – | |
| Phytophthora idaei | P6767 | Clade 1 | – | – | JN635116 | – | – | – |
| Phytophthora ilicis | P3939 | Clade 3 | – | – | JN635092 | – | – | – |
| Phytophthora infestans | CBS366.51 | Clade 1 | – | – | – | HQ708309 | HQ665217 | HQ643247 |
| Phytophthora insolita | P6703 | Clade 9 | – | – | JN635140 | – | – | – |
| Phytophthora inundata | CBS215.85 | Clade 6 | – | – | – | HQ708311 | HQ665154 | – |
| P8478 | Clade 6 | – | – | JN635083 | – | EU079946 | – | |
| Phytophthora ipomoeae | P10225 | Clade 1 | – | – | JN635181 | – | – | – |
| Phytophthora iranica | CBS374.72 | Clade 1 | – | – | – | HQ708314 | HQ665219 | – |
| Phytophthora katsurae | CBS587.85 | Clade 5 | – | – | – | HQ708315 | HQ665278 | – |
| P10187 | Clade 5 | – | – | JN635173 | – | – | – | |
| Phytophthora kelmania | P10613 | Clade 8 | – | – | JN635103 | – | – | – |
| Phytophthora kernoviae | P10958 | Clade 10 | – | – | – | HQ261349 | EU080057 | – |
| P10958 | Clade 10 | – | – | JN635237 | – | – | – | |
| Phytophthora lateralis | CBS168.42 | Clade 8 | – | – | – | – | KJ128037 | – |
| Lev1213 | Clade 8 | – | – | – | HQ708320 | – | – | |
| Phytophthora macrochlamydospora | P1026 | Clade 9 | – | – | JN635190 | – | – | – |
| Phytophthora meadii | CBS219.88 | Clade 2 | – | – | – | HQ708324 | HQ665159 | – |
| Phytophthora medicaginis | P7029 | Clade 8 | – | – | JN635096 | – | – | – |
| Phytophthora megakarya | P1672 | Clade 4 | – | – | – | HQ261357 | – | – |
| P1672 | Clade 4 | – | – | JN635250 | – | – | – | |
| P8516 | Clade 4 | – | – | – | – | EU079974 | – | |
| Phytophthora megasperma | CBS402.72 | Clade 6 | – | – | – | HQ708329 | HQ665228 | – |
| Phytophthora megasperma | P10340 | Clade 6 | – | – | JN635176 | – | – | – |
| Phytophthora melonis | CBS582.69 | Clade 7 | – | – | – | HQ708336 | HQ665274 | – |
| P3609 | Clade 7 | – | – | JN635049 | – | – | – | |
| Phytophthora mengei | P10139 | Clade 2 | – | – | JN635038 | – | – | – |
| Phytophthora mirabilis | CBS678.85 | Clade 1 | – | – | – | HQ708339 | HQ665285 | – |
| P10231 | Clade 1 | – | – | JN635179 | – | – | – | |
| Phytophthora multivesiculata | CBS545.96 | Clade 2 | – | – | – | HQ708340 | HQ665257 | – |
| Phytophthora multivora | P1233 | Clade 2 | – | – | JN635155 | – | – | – |
| Phytophthora nemorosa | P10288 | Clade 3 | – | – | JN635183 | – | – | – |
| Phytophthora nicotianae | CBS303.29 | Clade 1 | – | – | – | HQ708352 | – | – |
| P10297 | Clade 1 | – | – | JN635184 | – | – | – | |
| P7146 | Clade 1 | – | – | – | – | EU079560 | – | |
| Phytophthora palmivora | CBS298.29 | Clade 4 | – | – | – | HQ708357 | HQ665195 | – |
| P0113 | Clade 4 | – | – | JN635188 | – | – | – | |
| P0255 | Clade 4 | – | – | JN635186 | HQ261382 | EU080343 | – | |
| Phytophthora parsiana | P21281 | Clade 9 | – | – | JN635161 | – | – | – |
| P21282 | Clade 9 | – | – | JN635160 | HQ261384 | – | – | |
| Phytophthora phaseoli | CBS556.88 | Clade 1 | – | – | – | HQ708359 | HQ665267 | – |
| P10145 | Clade 1 | – | – | JN635167 | – | – | – | |
| Phytophthora pinifolia | P16100 | Clade 6 | – | – | – | HQ261390 | – | – |
| P16100 | Clade 6 | – | – | JN635272 | – | – | – | |
| Phytophthora polonica | P15004 | Clade 9 | – | – | – | HQ261394 | EU080268 | – |
| P15005 | Clade 9 | – | – | JN635240 | – | – | – | |
| Phytophthora porri | CBS567.86 | Clade 8 | – | – | – | HQ708368 | HQ665271 | – |
| P10728 | Clade 8 | – | – | JN635236 | – | – | – | |
| Phytophthora primulae | P10220 | Clade 8 | – | – | JN635180 | – | – | – |
| P10333 | Clade 8 | – | – | JN635187 | HQ261397 | EU080403 | – | |
| Phytophthora pseudosyringae | P1 0443 | Clade 3 | – | – | – | – | EU080026 | – |
| P16355 | Clade 3 | – | – | JN635257 | HQ261399 | – | – | |
| Phytophthora pseudotsugae | CBS444.84 | Clade 1 | – | – | – | HQ708381 | HQ665234 | – |
| P10218 | Clade 1 | – | – | JN635207 | – | – | – | |
| Phytophthora quercetorum | P15555 | Clade 4 | – | – | – | HQ261404 | – | – |
| PD01105 | Clade 4 | – | – | – | – | EU080905 | – | |
| Phytophthora quercina | P10334 | Clade 4 | – | – | JN635198 | – | – | – |
| Phytophthora quininea | CBS407.48 | Clade 9 | – | – | – | HQ708386 | HQ665230 | – |
| P3247 | Clade 9 | – | – | JN635110 | – | – | – | |
| Phytophthora ramorum | CBS101553 | Clade 8 | – | – | – | HQ708387 | HQ665053 | – |
| P10301 | Clade 8 | – | – | JN635185 | – | – | – | |
| Phytophthora richardiae | P3876 | Clade 8 | – | – | JN635045 | – | – | – |
| Phytophthora rosacearum | P8048 | Clade 6 | – | – | JN635062 | – | – | – |
| P8049 | Clade 6 | – | – | JN635057 | – | – | – | |
| Phytophthora rubi | CBS967.95 | Clade 7 | – | – | – | – | HQ665306 | – |
| Phytophthora sansomea | P3163 | Clade 8 | – | – | JN635047 | – | – | – |
| Phytophthora sinensis | CBS557.88 | Clade 7 | – | – | – | – | HQ665269 | – |
| Phytophthora siskiyouensis | P15122 | Clade 2 | – | – | – | HQ261421 | HQ665311 | – |
| P15123 | Clade 2 | – | – | – | – | HQ665312 | – | |
| Phytophthora sojae | CBS382.61 | Clade 7 | – | – | – | – | HQ665224 | – |
| Phytophthora sp aacrimae | P15880 | Clade 6 | – | – | JN635255 | – | – | – |
| Phytophthora sp asparagi | P10707 | Clade 6 | – | – | JN635226 | – | – | – |
| Phytophthora sp canalensis | P10456 | Clade 6 | – | – | JN635174 | – | – | – |
| Phytophthora sp cuyabensis | P8213 | Clade 9 | – | – | JN635084 | – | – | – |
| Phytophthora sp lagoriana | P8220 | Clade 9 | – | – | JN635085 | – | – | – |
| Phytophthora sp napoensis | P8225 | Clade 9 | – | – | JN635082 | – | – | – |
| Phytophthora sp niederhauserii | P10617 | Clade 7 | – | – | JN635212 | – | EU080247 | – |
| Phytophthora sp novaeguinee | P3389 | Clade 5 | – | – | JN635067 | – | – | – |
| Phytophthora sp ohioensis | P16050 | Clade 4 | – | – | JN635265 | – | – | – |
| Phytophthora sp personii | P11555 | Clade 6 | – | – | JN635134 | – | – | – |
| Phytophthora sp sulawesiensis | P6306 | Clade 6 | – | – | JN635095 | – | – | – |
| Phytophthora syringae | CBS132.23 | Clade 8 | – | – | – | HQ708404 | HQ665123 | – |
| P10330 | Clade 8 | – | – | JN635193 | – | – | – | |
| Phytophthora tabaci | CBS305.29 | Clade 1 | – | – | – | HQ708411 | HQ665198 | – |
| Phytophthora tentaculata | CBS552.96 | Clade 1 | – | – | – | HQ708413 | HQ665264 | – |
| P10363 | Clade 1 | – | – | JN635192 | – | – | – | |
| Phytophthora thermophilum | P1896 | Clade 9 | – | – | JN635117 | – | – | – |
| Phytophthora trifolii | P1462 | Clade 8 | – | – | JN635065 | – | – | – |
| Phytophthora tropicalis | CBS434.91 | Clade 2 | – | – | – | HQ708417 | HQ665233 | – |
| Phytophthora tropicalistype | P10329 | Clade 2 | – | – | JN635099 | – | – | – |
| Phytophthora uliginosa | P10328 | Clade 7 | – | – | JN635175 | – | – | – |
| P10413 | Clade 7 | – | – | JN635202 | – | – | – | |
| Phytopythium boreale | CBS551.88 | Phytopythium | AY598662 | – | – | HQ708419 | – | – |
| Phytopythium carbonicum | CBS112544 | Phytopythium | HQ643373 | – | – | HQ708420 | – | – |
| Phytopythium chamaehyphon | CBS259.30 | Phytopythium | AY598666 | – | – | HQ708421 | – | – |
| Phytopythium citrinum | CBS119171 | Phytopythium | HQ643375 | – | – | HQ708422 | – | – |
| Phytopythium delawarense | OH382/CBS123040 | Phytopythium | KF853241 | – | – | KF853240 | – | EU339312 |
| Phytopythium helicoides | CBS286.31 | Phytopythium | AY598665 | – | – | HQ708430 | – | – |
| Phytopythium kandeliae | CBS113.91 | Phytopythium | – | – | – | HQ708206 | HQ665079 | HQ643133 |
| AT CC66501 /P11614 | Phytopythium | – | – | GU994166 | – | – | – | |
| Phytopythium litorale | CBS118360 | Phytopythium | HQ643386 | – | – | HQ708433 | – | – |
| CBS122662 | Phytopythium | – | – | – | – | HQ665114 | HQ643385 | |
| Phytopythium mercuriale | A89 (GENBANK) | Phytopythium | – | – | – | – | – | JN630486 |
| CBS122443 | Phytopythium | KF853243 | – | – | KF853239 | KF853236 | – | |
| Phytopythium mirpurense | CBS124523 | Phytopythium | KJ831613 | – | – | KJ831612 | – | – |
| CBS124524 | Phytopythium | – | – | – | – | KJ831614 | KJ831614 | |
| Phytopythium montanum | CBS111349 | Phytopythium | HQ643389 | – | – | HQ708436 | – | – |
| Phytopythium oedochilum | CBS292.37 | Phytopythium | AY598664 | – | – | HQ708439 | – | – |
| Phytopythium ostracodes | CBS768.73 | Phytopythium | AY598663 | – | – | HQ708442 | – | – |
| Phytopythium sindhum | CBS124518 | Phytopythium | HQ643396 | – | – | HQ708443 | – | – |
| Phytopythium vexans | CBS119.80 | Phytopythium | HQ643400 | – | – | HQ708447 | – | – |
| Pythium abappressorium | CBS110198 | Clade F | HQ643408 | – | – | HQ708455 | – | – |
| Pythium acanthicum | CBS377.34 | Clade D | AY598617 | – | – | HQ708456 | – | – |
| Pythium acanthophoron | CBS337.29 | Clade J | AY598711 | – | – | HQ708460 | – | – |
| Pythium acrogynum | CBS549.88 | Clade E | – | – | – | – | HQ665258 | – |
| Pythium adhaerens | CBS520.74 | Clade B | AY598619 | – | – | HQ708462 | – | – |
| Pythium amasculinum | CBS552.88 | Clade D | AY598671 | – | – | HQ708481 | – | – |
| Pythium anandrum | CBS285.31 | Clade H | AY598650 | – | – | HQ708482 | – | – |
| Pythium angustatum | CBS522.74 | Clade B | AY598623 | – | – | HQ708484 | – | – |
| Pythium aphanidermatum | CBS118.80 | Clade A | AY598622 | – | – | HQ708485 | – | – |
| Pythium apiculatum | CBS120945 | Clade E | HQ643443 | – | – | HQ708490 | – | – |
| Pythium apleroticum | CBS772.81 | Clade B | AY598631 | – | – | HQ708491 | – | – |
| Pythium aquatile | CBS215.80 | Clade B | AY598632 | – | – | – | HQ665153 | – |
| Pythium aristosporum | CBS263.38 | Clade B | AY598627 | – | – | HQ708494 | HQ665179 | – |
| Pythium arrhenomanes | CBS324.62 | Clade B | – | – | – | HQ708499 | HQ665208 | – |
| Pythium attrantheridium | DAOM230383 | Clade F | – | – | – | HQ708524 | HQ665308 | – |
| DAO M 230386 | Clade F | HQ643476 | – | – | – | – | – | |
| Pythium buismaniae | CBS288.31 | Clade J | AY598659 | – | – | – | HQ665188 | – |
| Pythium camurandrum | CBS124096 | Clade E | – | – | – | HQ708527 | – | – |
| Pythium canariense | CBS112353 | Clade G | – | – | – | HQ708528 | HQ665069 | – |
| Pythium capillosum | CBS222.94 | Clade B | AY598635 | – | – | HQ708529 | HQ665164 | – |
| Pythium carolinianum | CBS122659 | Clade E | – | – | – | HQ708530 | HQ665111 | – |
| Pythium catenulatum | CBS842.68 | Clade B | AY598675 | – | – | HQ708540 | HQ665302 | – |
| Pythium chondricola | CBS203.85 | Clade B | – | – | – | HQ708544 | HQ665149 | – |
| Pythium coloratum | CBS154.64 | Clade B | AY598633 | – | – | HQ708547 | HQ665128 | – |
| Pythium conidiophorum | CBS223.88 | Clade B | AY598629 | – | – | HQ708555 | HQ665166 | – |
| Pythium contiguanum | CBS221.94 | Clade B | – | – | – | HQ708560 | HQ665162 | – |
| Pythium cryptoirregulare | CBS118731 | Clade F | HQ643515 | – | – | HQ708561 | HQ665083 | – |
| Pythium cylindrosporum | CBS218.94 | Clade F | AY598643 | – | – | HQ708562 | HQ665157 | – |
| Pythium cystogenes | CBS675.85 | Clade J | HQ643518 | – | – | HQ708564 | HQ665284 | – |
| Pythium debaryanum | CBS752.96 | Clade F | AY598704 | – | – | HQ708565 | HQ665294 | – |
| Pythium deliense | CBS314.33 | Clade A | AY598674 | – | – | HQ708568 | HQ665204 | – |
| Pythium diclinum | CBS664.79 | Clade B | – | – | – | HQ708570 | HQ665282 | – |
| Pythium dimorphum | CBS406.72 | Clade H | AY598651 | – | – | HQ708571 | HQ665229 | – |
| Pythium dissimile | CBS155.64 | Clade B | AY598681 | – | – | HQ708572 | HQ665130 | – |
| Pythium dissotocum | CBS166.68 | Clade B | AY598634 | – | – | HQ708574 | HQ665139 | – |
| Pythium echinulatum | CBS281.64 | Clade E | AY598639 | – | – | HQ708577 | HQ665183 | – |
| Pythium emineosum | BR479 | Clade F | – | – | – | GQ244423 | – | – |
| Pythium erinaceus | CBS505.80 | Clade E | – | – | – | HQ708578 | HQ665243 | – |
| Pythium flevoense | CBS234.72 | Clade B | AY598691 | – | – | HQ708580 | HQ665170 | – |
| CBS278.81 | Clade B | – | – | – | – | HQ665182 | – | |
| Pythium folliculosum | CBS220.94 | Clade B | – | – | – | HQ708584 | HQ665160 | – |
| Pythium glomeratum | CBS120914 | Clade I | HQ643543 | – | – | – | HQ665091 | – |
| Pythium graminicola | CBS327.62 | Clade B | AY598625 | – | – | HQ708589 | HQ665211 | – |
| Pythium grandisporangium | CBS286.79 | Clade C | AY598692 | – | – | HQ708590 | HQ665187 | – |
| Pythium helicandrum | CBS393.54 | Clade H | AY598653 | – | – | HQ708592 | HQ665225 | – |
| Pythium heterothallicum | CBS450.67 | Clade I | AY598654 | – | – | HQ708597 | HQ665235 | – |
| Pythium hydnosporum | CBS253.60 | Clade D | AY598672 | – | – | HQ708608 | HQ665175 | – |
| Pythium hypogynum | CBS234.94 | Clade E | AY598693 | – | – | HQ708609 | HQ665171 | – |
| Pythium inflatum | CBS168.68 | Clade B | AY598626 | – | – | HQ708610 | HQ665140 | – |
| Pythium insidiosum | ATCC 58643 | Clade C | AF289981 | – | – | – | – | – |
| CBS574.85 | Clade C | – | – | – | HQ708614 | HQ665273 | – | |
| Pythium intermedium | CBS266.38 | Clade F | AY598647 | – | – | HQ708616 | HQ665180 | – |
| Pythium irregulare | CBS250.28 | Clade F | AY598702 | – | – | HQ708640 | HQ665172 | – |
| Pythium iwayamai | CBS156.64 | Clade G | AY598648 | – | – | HQ708713 | HQ665131 | – |
| Pythium kashmirense | ADC0819 | Clade B | – | HQ643671 | – | – | – | – |
| CBS122908 | Clade B | – | – | – | HQ708715 | HQ665118 | – | |
| Pythium kunmingense | CBS550.88 | Clade F | AY598647 | – | – | – | HQ665259 | – |
| Pythium longisporangium | CBS122646 | Clade E | – | – | – | HQ708724 | HQ665099 | – |
| Pythium lucens | CBS113342 | Clade F | HQ643681 | – | – | HQ708725 | HQ665077 | – |
| Pythium lutarium | CBS222.88 | Clade B | – | – | – | HQ643682 | HQ665163 | – |
| Pythium lycopersici | CBS122909 | Clade D | – | – | – | HQ708727 | HQ665119 | – |
| Pythium macrosporum | CBS574.80 | Clade F | AY598646 | – | – | HQ708728 | HQ665272 | – |
| Pythium marsipium | CBS773.81 | Clade E | – | – | – | HQ708734 | HQ665297 | – |
| Pythium mastophorum | CBS375.72 | Clade J | AY598661 | – | – | HQ708735 | HQ665220 | – |
| Pythium megacarpum | CBS112351 | Phytopythium | – | – | – | – | – | HQ643388 |
| Pythium middletonii | CBS528.74 | Clade E | – | – | – | HQ708738 | HQ665249 | – |
| Pythium minus | CBS122657 | Clade E | – | – | – | HQ708739 | HQ665109 | – |
| CBS226.88 | Clade E | AY598698 | – | – | HQ643696 | – | – | |
| Pythium monospermum | CBS158.73 | Clade A | HQ643697 | – | – | HQ708741 | HQ665137 | – |
| Pythium multisporum | CBS470.50 | Clade E | AY598641 | – | – | HQ708744 | HQ665239 | – |
| Pythium myriotylum | CBS254.70 | Clade B | AY598678 | – | – | HQ708745 | HQ665176 | – |
| Pythium nagaii | CBS779.96 | Clade G | AY598705 | – | – | HQ708749 | HQ665299 | – |
| Pythium nodosum | CBS102274 | Clade J | – | – | – | HQ708753 | HQ665055 | – |
| Pythium nunn | CBS808.96 | Clade J | AY598709 | – | – | HQ708755 | HQ665300 | – |
| Pythium okanoganense | CBS315.81 | Clade G | AY598649 | – | – | – | HQ665205 | – |
| Pythium oligandrum | CBS382.34 | Clade D | AY598618 | – | – | HQ708759 | HQ665223 | – |
| Pythium oopapillum | BR632 | Clade B | – | – | – | FJ655178 | – | – |
| Pythium ornacarpum | CBS112350 | Clade E | HQ643721 | – | – | HQ708762 | HQ665066 | – |
| Pythium ornamentatum | CBS122665 | Clade D | – | – | – | HQ708763 | HQ665117 | – |
| Pythium orthogonon | CBS376.72 | Clade J | – | – | – | HQ708764 | HQ665221 | – |
| Pythium pachycaule | CBS227.88 | Clade B | – | – | – | HQ708765 | HQ665169 | – |
| Pythium paddicum | CBS698.83 | Clade G | AY598707 | – | – | HQ708769 | HQ665290 | – |
| Pythium paroecandrum | CBS157.64 | Clade F | AY598644 | – | – | – | HQ665133 | – |
| Pythium parvum | CBS225.88 | Clade E | AY598697 | – | – | HQ708779 | HQ665167 | – |
| Pythium pectinolyticum | CBS122643 | Clade B | HQ643739 | – | – | HQ708780 | HQ665096 | – |
| Pythium periilum | CBS169.68 | Clade B | – | – | – | HQ708781 | HQ665141 | – |
| Pythium periplocum | CBS289.31 | Clade D | AY598670 | – | – | HQ708784 | HQ665189 | – |
| Pythium perplexum | CBS674.85 | Clade J | AY598658 | – | – | HQ708785 | HQ665283 | – |
| Pythium pleroticum | CBS776.81 | Clade E | AY598642 | – | – | HQ708789 | HQ665298 | – |
| Pythium plurisporium | CBS100530 | Clade B | AY598684 | – | – | HQ708790 | HQ665052 | – |
| Pythium polymastum | CBS811.70 | Clade J | AY598660 | – | – | HQ708793 | HQ665301 | – |
| Pythium porphyrae | CBS369.79 | Clade A | AY598673 | – | – | HQ708794 | HQ665218 | – |
| Pythium prolatum | CBS845.68 | Clade H | AY598652 | – | – | HQ708795 | HQ665303 | – |
| Pythium pyrilobum | CBS158.64 | Clade B | AY598636 | – | – | HQ708796 | HQ665136 | – |
| Pythium radiosum | CBS217.94 | Clade E | – | – | – | – | HQ665156 | – |
| Pythium rhizooryzae | CBS119169 | Clade B | HQ643757 | – | – | HQ708798 | HQ665087 | – |
| Pythium rhizosaccharum | CBS112356 | Clade E | – | – | – | HQ708801 | HQ665072 | – |
| Pythium rostratifingens | CBS115464 | Clade E | HQ643761 | – | – | HQ708802 | HQ665080 | – |
| Pythium rostratum | CBS533.74 | Clade E | AY598696 | – | – | HQ708808 | HQ665252 | – |
| Pythium salpingophorum | CBS471.50 | Clade B | AY598630 | – | – | HQ708809 | HQ665240 | – |
| Pythium scleroteichum | CBS294.37 | Clade B | AY598680 | – | – | HQ708812 | HQ665192 | – |
| Pythium segnitium | CBS112354 | Clade E | HQ643772 | – | – | HQ708813 | HQ665070 | – |
| Pythium senticosum | CBS122490 | Clade H | HQ643773 | – | – | HQ708814 | HQ665093 | – |
| Pythium sp balticum | CBS122649 | Clade F | – | – | – | HQ708525 | – | – |
| Pythium sp | CBS113341 | Clade F | KF853244 | – | – | – | – | – |
| Pythium sp CAL-2011a | CBS122647 | Clade D | – | – | – | HQ708815 | – | – |
| Pythium sp CAL-2011e | CBS122648 | Clade E | – | – | – | HQ708770 | HQ665101 | – |
| Pythium sp CAL-2011f | CBS101876 | Clade J | HQ643778 | – | – | HQ708819 | – | – |
| Pythium spiculum | CBS122645 | Clade F | KF853242 | – | – | – | HQ665098 | – |
| Pythium spinosum | CBS275.67 | Clade F | AY598701 | – | – | HQ708834 | HQ665181 | – |
| Pythium splendens | CBS462.48 | Clade I | AY598655 | – | – | HQ708836 | HQ665237 | – |
| Pythium sterilum | B09 | Phytopythium | – | – | – | – | – | EU240096 |
| Pythium sukuiense | CBS110030 | Clade B | – | – | – | HQ708877 | HQ665059 | – |
| Pythium sylvaticum | CBS453.67 | Clade F | AY598645 | – | – | HQ708886 | HQ665236 | – |
| Pythium takayamanum | CBS122491 | Clade E | HQ643854 | – | – | HQ708895 | HQ665094 | – |
| Pythium terrestris | CBS112352 | Clade F | – | – | – | HQ708898 | HQ665068 | – |
| Pythium torulosum | CBS316.33 | Clade B | AY598624 | – | – | HQ708900 | HQ665206 | – |
| Pythium tracheiphilum | CBS323.65 | Clade B | – | – | – | HQ708903 | HQ665207 | – |
| Pythium ultimum var. sporangiiferum | CBS219.65 | Clade I | AKYB02045405 | – | – | HQ708920 | HQ665158 | – |
| Pythium ultimum var. ultimum | CBS398.51 | Clade I | AY598657 | – | – | HQ708906 | HQ665227 | – |
| Pythium uncinulatum | CBS518.77 | Clade J | AY598712 | – | – | HQ708985 | HQ665244 | – |
| Pythium undulatum | CBS157.69 | Clade H | AY598708 | – | – | HQ708987 | HQ665134 | – |
| Pythium vanterpoolii | CBS295.37 | Clade B | AY598685 | – | – | HQ708993 | HQ665193 | – |
| Pythium viniferum | CBS119168 | Clade F | HQ643956 | – | – | HQ708997 | HQ665086 | – |
| Pythium violae | CBS132.37 | Clade G | AY598717 | – | – | – | – | – |
| CBS159.64 | Clade G | AY598706 | – | – | HQ708999 | HQ665138 | – | |
| Pythium volutum | CBS699.83 | Clade B | AY598686 | – | – | HQ709012 | HQ665291 | – |
| Pythium zingiberis | CBS216.82 | Clade B | – | – | HQ709014 | HQ665155 | – | |
Phylogenetic analyses
Sequences were edited manually using the DNAStar Lasergene 9 Suite (Bioinformatics Pioneer DNAStar, Inc., WI) or Geneious v. 6.1.6 (Biomatters http://www.geneious.com/). Multiple alignments of each gene region were generated using MAFFT (Katoh et al. 2005). The genera included in the phylogenetic analyses were Albugo, Halophytophthora, Phytophthora, Phytopythium and Pythium. Isolates of Albugo candida from the order Albuginales were included as an outgroup.
In order to include the maximum molecular data for clade K Pythium the invalid species Pythium sterile and Pythium megacarpum as well as two strains of the novel species Phytopythium mirpurense are considered in a cladogram generated based on ITS sequence data. Pythium ultimum from clade I and Pythium dimorphum from clade H are outgroups in these analyses and representatives of Phytophthora, P. infestans, P. ramorum and P. sojae are included. The aligned data matrix from 23 strains contained 1 096 characters from the ITS1, ITS2 and the 5.8S gene.
The aligned data matrices were assessed to find the best-fit model of nucleotide substitution using jMODELTEST (Posada 2008). In each case this was identified as General Time Reversible (GTR+I+G). Redundant sequences were identified and those with 100 % identity to other included taxa were removed from the analyses. These duplicates are catalogued in Table 2. The aligned data matrices contained 1 374 bp of D1–D3 regions of LSU with 176 strains, 1 724 bp of SSU rRNA with 159 strains and 680 bp of COI with 174 strains. The sequence alignments were subjected to maximum likelihood analysis using the GTR+I+G substitution model and the Best option for tree topology search with PhyML v. 3.0 (Guindon & Gascuel 2003) to obtain ML trees which were rooted to Albugo (LSU, COI and SSU) or Pythium (ITS). Nonparametric ML bootstraps were calculated with 1 000 bootstrap replicates. Bayesian inferences (BI) were generated using MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003) with Markov Chain Monte Carlo (MCMC) methodology to calculate posterior probabilities of the phylogenetic trees. The program was run for 20 M generations for the LSU, 40 M generations for the COI, 50 M generations for the SSU and 10 M for the ITS datasets with the GTR+I+G model of evolution for each gene. The first 25 % of the iterations were discarded as burn-in and every 1 000th iteration was sampled from the remainder. The trees were considered to be fully converged when the average standard deviation of split frequencies reached a level less than 0.01. FigTree v. 1.3.1 (http://tree.bio. ed.ac.uk/software/figtree/) was used to view and edit ML and Bayesian phylogenetic trees. Consensus trees were generated using the 50 % majority rule tree criteria and rooted to Albugo (LSU, COI and SSU) or Pythium (ITS).
Table 2
Species and isolates not included in the study for strains that were 100 % identical for certain genes and therefore not included in the phylogenetic analyses.
| Sequence included in phylogeny | Identical sequences not included in phylogenies | ||||||
|---|---|---|---|---|---|---|---|
| Species | Strain | Clade | GenBank | Species | Strain | Clade | GenBank |
| SSU | |||||||
| Phytophthora alticola | P16053 | Clade 4 | JN635264 | Phytophthora frigida | P16051 | Clade 2 | JN635162 |
| Phytophthora asparagi | P10707 | Clade 6 | JN635226 | Phytophthora rosacearum | P8048 | Clade 6 | JN635062 |
| Phytophthora cactorum | P0714 | Clade 1 | JN635210 | Phytophthora cactorum | P10365 | Clade 1 | JN635194 |
| Phytophthora captiosa | P10719 | Clade 9 | JN635227 | Phytophthora captiosa | P10720 | Clade 9 | JN635229 |
| Phytophthora cryptogea | P16165 | Clade 8 | JN635259 | Phytophthora pseudosyringae | P16355 | Clade 3 | JN635257 |
| Phytophthora erythroseptica | P1693 | Clade 8 | JN635249 | Phytophthora gonapodyides | P3700 | Clade 6 | JN635141 |
| Phytophthora richardiae | P3876 | Clade 8 | JN635045 | ||||
| Phytophthora sansomea | P3163 | Clade 8 | JN635047 | ||||
| Phytophthora trifolii | P1462 | Clade 8 | JN635065 | ||||
| Phytophthora europaea | P10324 | Clade 7 | JN635189 | Phytophthora uliginosa | P10328 | Clade 7 | JN635175 |
| Phytophthora uliginosa | P10413 | Clade 7 | JN635202 | ||||
| Phytophthora lagoriana | P8220 | Clade 9 | JN635085 | Phytophthora lagoriana | P8223 | Clade 9 | JN635086 |
| Phytophthora parsiana | P21282 | Clade 9 | JN635160 | ||||
| Phytophthora palmivora | P0113 | Clade 4 | JN635188 | Phytophthora palmivora | P0255 | Clade 4 | JN635186 |
| Phytophthora primulae | P10220 | Clade 8 | JN635180 | Phytophthora primulae | P10333 | Clade 8 | JN635187 |
| Pythium flevoense | CBS23472 | Clade B | AY598691 | Pythium pectinolyticum | CBS122643 | Clade B | HQ643739 |
| Pythium minus | CBS22688 | Clade E | AY598698 | Pythium pleroticum | CBS776.81 | Clade E | AY598642 |
| Pythium parvum | CBS225.88 | Clade E | AY598697 | ||||
| Pythium porphyrae | CBS36979 | Clade A | AY598673 | Pythium adhaerens | CBS520.74 | Clade B | AY598619 |
| Pythium salinum | CBS113341 | Clade F | KF853244 | Pythium attrantheridium | DAOM230386 | Clade F | HQ643476 |
| Pythium spinosum | CBS27567 | Clade F | AY598701 | Pythium violae | CBS132.37 | Clade G | AY598717 |
| Pythium lucens | CBS113342 | Clade F | HQ643681 | ||||
| Pythium kunmingense | CBS55088 | Clade F | AY598647 | ||||
| Pythium uncinulatum | CBS51877 | Clade J | AY598712 | Pythium buismaniae | CBS288.31 | Clade J | AY598659 |
| LSU | |||||||
| Phytophthora arecae | CBS30562 | Clade 4 | HQ665200 | Phytophthora palmivora | CBS29829 | Clade 4 | HQ665195 |
| Phytophthora boehmeriae | CBS29129 | Clade 10 | HQ665190 | Phytophthora boehmeriae | P6950 | Clade 10 | EU080166 |
| Phytophthora brassicae | CBS17887 | Clade 8 | HQ665144 | Phytophthora brassicae | CBS178.87 | Clade 8 | HQ665144 |
| Phytophthora erythroseptica | CBS12923 | Clade 8 | HQ665121 | Phytophthora himalayensis | CBS35759 | Clade 8 | HQ665215 |
| Phytophthora fragariae | CBS20946 | Clade 7 | HQ665150 | Phytophthora rubi | CBS96795 | Clade 7 | HQ665306 |
| Phytophthora gonapodyides | CBS55467 | Clade 6 | HQ665265 | Phytophthora gonapodyides | CBS36379 | Clade 6 | HQ665216 |
| Phytophthora inundata | P8478 | Clade 6 | EU079946 | Phytophthora humicola | CBS20081 | Clade 6 | HQ665148 |
| Phytophthora inundata | CBS21585 | Clade 6 | HQ665154 | ||||
| Phytophthora melonis | CBS58269 | Clade 7 | HQ665274 | Phytophthora sinensis | CBS55788 | Clade 7 | HQ665269 |
| Phytophthora sp “niederhauserii" | P10617 | Clade 7 | EU080247 | Phytophthora sojae | CBS38261 | Clade 7 | HQ665224 |
| Phytophthora siskiyouensis | P15123 | Clade 2 | HQ665312 | Phytophthora siskiyouensis | P15122 | Clade 2 | HQ665311 |
| Pythium amasculinum | CBS55288 | Clade D | HQ665263 | Pythium lycopersicum | CBS122909 | Clade D | HQ665119 |
| Pythium oligandrum | CBS38234 | Clade D | HQ665223 | ||||
| Pythium apleroticum | CBS77281 | Clade B | HQ665296 | Pythium aquatile | CBS21580 | Clade B | HQ665153 |
| Pythium buismaniae | CBS28831 | Clade J | HQ665188 | Pythium polymastum | CBS81170 | Clade J | HQ665301 |
| Pythium capillosum | CBS22294 | Clade B | HQ665164 | Pythium flevoense | CBS27881 | Clade B | HQ665182 |
| Pythium flevoense | CBS23472 | Clade B | HQ665170 | ||||
| Pythium catenulatum | CBS84268 | Clade B | HQ665302 | Pythium rhizo-oryzae | CBS119169 | Clade B | HQ665087 |
| Pythium viniferum | CBS119168 | Clade F | HQ665086 | Pythium debaryanum | CBS75296 | Clade F | HQ665294 |
| COI | |||||||
| Phytophthora arecae | CBS30562 | Clade 4 | HQ708218 | Phytophthora palmivora | CBS29829 | Clade 4 | HQ643307 |
| Pythium amasculinum | CBS55288 | Clade D | HQ708481 | Pythium lycopersicum | CBS122909 | Clade D | HQ643683 |
| Pythium ornamentatum | CBS122665 | Clade D | HQ708763 | ||||
| Pythium conidiophorum | CBS22388 | Clade B | HQ708555 | Pythium salpingophorum | CBS47150 | Clade B | HQ643768 |
| Pythium debaryanum | CBS75296 | Clade F | HQ708565 | Pythium viniferum | CBS119168 | Clade F | HQ643956 |
| Pythium diclinum | CBS66479 | Clade B | HQ708570 | Pythium lutarium | CBS22288 | Clade B | HQ643682 |
| Pythium erinaceus | CBS50580 | Clade E | HQ708578 | Pythium ornacarpum | CBS112350 | Clade E | HQ643721 |
| Pythium folliculosum | CBS22094 | Clade B | HQ708584 | Pythium torulosum | CBS31633 | Clade B | HQ643859 |
| Pythium minus | CBS122657 | Clade E | HQ708739 | Pythium pleroticum | CBS77681 | Clade E | HQ643748 |
| Pythium myriotylum | CBS25470 | Clade B | HQ708745 | Pythium zingiberis | CBS21682 | Clade B | HQ643973 |
Statistical analyses of pairwise distances
The alignments of COI, LSU and SSU used for phylogeny were also used to generate pairwise distance as was done for DNA barcode analyses (Robideau et al. 2011, Schoch et al. 2012). Statistical analyses and plots were performed with R (R Development Core Team, 2011). All pairwise distances involving a Phytopythium species against Pythium or Phytophthora were extracted, i.e. all pairwise distances involving any two Phytopythium species were excluded. An arcsine transformation of the distances was done to improve the variance homogeneity. ANOVA using ’lm’ was done with markers (COI/LSU/SSU), genera (Phytophthora/Pythium) or clades (clade 1–10 and A–J) as variables. Plots were generated with ’ggplot’ for R. The 0.05 confidence interval for 60 multiple comparisons was adjusted using the Bonferoni method. The average pairwise distance by marker was normalised to remove the bias from the difference in number of species between Pythium and Phytophthora.
Isolation and identification of Phytopythium mirpurense
Stagnant water was collected and immediately brought to the laboratory for the isolation of oomycetous fungi by the baiting technique of Harvey (1925). Grass blades, dicot leaves, hemp seeds, sesame seeds, lemon leaf and young cucumber stems were used as baits. Plates were incubated at room temperature, between 22–25 °C. Hyphae were observed on the baits after 5–8 days of incubation. The baits were rinsed in sterilised water to remove excess contaminants and transferred to fresh plates half-filled with sterile water. New fresh baits were then added and monitored daily for colonisation by oomycetes. After 2 d of incubation, the baits colonised by oomycetous fungi were transferred onto corn-meal agar (CMA) medium for purification by hyphal tip transfer. To obtain a pure culture a small disc of the CMA culture was placed into the centre of water agar plates. After 15–24 h growing apical hyphae were cut with the aid of a microscope in the laminar flow hood and transferred onto the surface of a fresh plate containing culture media.
For the assessment of cardinal temperatures, the isolates from this study were sub-cultured in two replicates on CMA in 90 mm Petri plates, and incubated at 10, 15, 20, 25, 30, 35 and 40 °C for 5 d. Radial growth was measured daily along two lines intersecting the centre of the inoculum. Isolates were also grown on potato dextrose agar (PDA), potato carrot agar (PCA), CMA and corn meal dextrose agar (CMDA) in 90 mm Petri plates (recipes according to Crous et al. 2009), and colony characteristics were assessed after incubation for 5 d at 25 °C.
Water cultures for zoospore and sporangial production were prepared by adding an inoculum disc and a grass blade to sterile water in a Petri plate and incubating at 25 °C. Biometric values i.e aplerotic index, ooplast index and wall index were determined for 20 oogonia with the method described by Shahzad et al. (1992).
RESULTS AND DISCUSSION
Morphological comparison of Phytopythium with Phytophthora and Pythium
Most species in the genus Phytopythium produce papillate, internally proliferating sporangia (Fig. 1). The shape of the sporangia is more or less similar to the shape of papillate Phytophthora sporangia: (sub-)globose to ovoid and papillate (Fig. 1). However, in Phytophthora the papillate sporangium type never shows internal proliferation. The combination of internal proliferation and papillation (Fig. 1) is unique to sporangia of Phytopythium and some Pythium species (see below). Also, the papillae in Phytopythium are different from the papillae in Phytophthora sporangia. In Phytopythium the sporangia are initially non-papillate, and the papillae develop at maturity and do not consist of a hyaline ’apical thickening’ as in Phytophthora (Blackwell 1949). They may grow out to form a shorter or larger discharge tube (Fig. 1d, ,f,f, ,g,g, ,i,i, ,j),j), which does not occur in Phytophthora. In some species the papilla is not the place where the plasma flows out, rather one or more discharge tubes are formed more basally of the sporangium. In some species the papilla grows out and develops branches (Fig. 1e). Another difference with Phytophthora is the zoospore discharge which is pythium-like in Phytopythium: the plasma flows out of the sporangium through a discharge tube to form a plasma-filled vesicle at the tip. Zoospores are developed outside the sporangium, within the vesicle membrane and are released after rupture of the membrane (Fig. 1a). According to Marano et al. (2014), Phytopythium kandeliae has zoospore release mostly like Pythium and occasionally in between Pythium and Phytophthora: zoospores developed (partly) inside a sporangium and partly in a vesicle.
Sporangia of Phytopythium species. a. P. sindhum, four stages of sporangium development showing a young, globose sporangium, a mature, papillate sporangium, internal proliferation and pythium-like zoospore development; b. P. vexans, subglobose, non-papillate sporangia; c–g. P. citrinum: c. normal sporangia; d. outgrowing papillae; e. outgrowing and branching papilla; f. empty sporangium with internal proliferation and short discharge tube; g. empty sporangium with internal proliferation and long discharge tube (arrow indicating tip); h–j. P. helicandrum: h. sessile, globose, papillate sporangium; i. outgrowing papilla; j. empty sporangium with intermediate sized discharge tube (arrow indicating tip). — Scale bars = 20 μm.
Another unique characteristic of Phytopythium is the shape of the antheridium (Fig. 2). In most species the antheridia are elongate, cylindrical, often with constrictions. The fertilisation tube is mostly not apical but in ’navel position’ (Fig. 2a, ,b,b, ,c,c, ,d,d, arrows). Occasionally club-shaped antheridia with apical attachment occur. In P. vexans, the antheridia are often very broadly attached to the oogonium and lobed (Fig. 2e, ,ff).
Oogonia and antheridia of Phytopythium species. a. P. sindhum, slightly elongated antheridium; b–c. P. oedochilum, long cylindrical antheridia; d. P. mirpurense, elongate antheridium with slight constriction; e–f. P. vexans: e. elongate antheridium with distinct constrictions; f. antheridium with two lobes. Arrows indicate the fertilisation tube in navel position (a–d). — Scale bars = 10 μm.
Papillate sporangia with internal proliferation also occur in a small number of Pythium species: three members of clade E (P. marsipium, P. middletonii, P. multisporum), one member of clade G (P. nagaii) and clade C (P. grandisporangium) and all members of clade H (P. anandrum, P. dimorphum, P. helicandrum, P. prolatum, P. undulatum). However, none of these species except three has elongate, cylindrical or lobate antheridia. Only P. helicandrum has elongate antheridia, however, this species has ornamented oogonia and much bigger sporangia than any of the species in Phytopythium. Pythium marsipium has bell-shaped antheridia as they occur in Phytopythium vexans, however, its sporangia are utriform instead of ovoid. Pythium grandisporangium has lobate antheridia but this is a marine species with extremely large sporangia with a tapering neck rather than a distinct papilla.
Phylogenetic position of Phytopythium
Maximum likelihood analyses of nuclear (LSU and SSU) and mitochondrial DNA (COI) with Bayesian probability values mapped onto the trees are shown (Fig. 3A, ,B,B, ,C).C). These cladograms place all the strains belonging to the genus Phytopythium as a monophyletic group with bootstrap support (85–100 %) and high probabilities (0.99–1.00). Phylogenetic trees of the LSU and COI regions support this group as intermediary between Phytophthora and Pythium. There is phylogenetic support with two of the genes to group Phytopythium with Phytophthora (95 % / 1.00 for LSU and 79 % / 0.99 for COI). The SSU tree has Pythium clades A–D as grouping closer to Phytophthora and Halophytophthora, with very low bootstrap support and probabilities (< 50 % / 0.65). This suggests that given the SSU dataset, the major clades are unresolved in relation to the outgroup.
Maximum likelihood phylogenetic trees of: a. LSU ribosomal RNA region; b. mitochondrial COI; c. SSU ribosomal RNA region. Maximum likelihood bootstrap support values larger than 50 % are indicated numerically, those under 50 % are marked with (–). Posterior probability values larger than 0.50 are labelled numerically, those under 0.50 are marked (–) on each branch, those clades which were not present in the Bayesian trees are marked as (o), the scale bars represent the average number of substitutions per site.
Our results from phylogenetic analysis of nuclear (LSU and SSU) and mitochondrial (COI) genes with all available species of Pythium and Phytophthora support that Phytopythium is a distinct genus. Its placement as intermediate between Pythium and Phytophthora is supported by two of these datasets. In the three gene trees, this new genus clade was strongly supported by both ML bootstrap replicates and Bayesian probability values, which unambiguously confirmed the status of Phytopythium as a novel monophyletic genus. The maximum likelihood and Bayesian analyses did not clearly delineate the relationships between the different groups in the part of the oomycete evolutionary tree we focused on. Inclusion of some of the more basal groups such as the Salisapiliaceae (Hulvey et al. 2010) and additional markers in future analyses would likely lead to greater resolution of these relationships.
The ITS tree (Fig. 4) shows that the two strains of species P. mirpurense are both well embedded within Phytopythium with strong support (91 % / 0.96) and demonstrated the close relationships between P. litorale and Pythium sterile (100 / 1) as well as Phytopythium boreale and Pythium megacarpum (99 / 1).
Maximum likelihood tree of internal transcribed spacer (ITS) region of Phytopythium spp., Pythium spp. and Phytophthora spp. Maximum likelihood bootstrap support values larger than 50 % are indicated numerically, those under 50 % are marked with (–). Posterior probability values larger than 0.50 are labelled numerically, on each branch those clades which were not present in the Bayesian trees are marked as (o), the scale bars represent the average number of substitutions per site.
Statistical analyses of pairwise distances
Markers, genera and clades as well as interactions between them all had a significant effect on pairwise distances of Phytopythium against Pythium and Phytophthora species (p < 10−15). The average pairwise distance of all Phytophthora species against all Phytopythium species using COI was 13.7 % whereas it was 14.5 % for all Pythium species against all Phytopythium, showing that Phytopythium is significantly closer to Phytophthora than Pythium (p < 10−16). For LSU, these differences were 10.4 % and 10.9 %, respectively, and were also significant (p < 10−16). For SSU, the trend was reversed, still significant, with the average pairwise distance between Pythium and Phytopythium being 2.5 % whereas the average between Phytophthora and Phytopythium was 2.7 %. The clade effect was significant, including a significant interaction with markers; therefore, the results are presented by clades and markers in Fig. 5. Each clade is compared against Phytopythium to show clades that have a significant difference from the average pairwise distance. The significant trend of Phytopythium being closer to Phytophthora clades than Pythium clades can be seen with COI and LSU whereas it is more difficult to visualise the reverse trend in SSU. With all markers, Pythium clades H and I were significantly closer to Phytopythium than were the other Pythium clades but for SSU there were three additional clades (B, F and G) that were significantly closer to Phytopythium than were the other clades.
Analysis of all pairwise distances containing only one representative of each Phytopythium sp. The dotted lines represent the average of all these pairwise distances for each marker, adjusted to remove the bias for the difference in species number between Pythium and Phytophthora. The bars represent 95 % confidence intervals corrected by the Bonferoni method for 60 comparisons. The analysis was done with arcsine transformation, therefore, the averages as well as the upper and lower boundaries of the intervals were transformed back to actual pairwise distances for the plot. Intervals that are not touching the average dotted line are significantly below or above the average, i.e. closer to or more distant to Phytopythium, respectively.
Strains used in circumscription of the genus
There are two invalid species that were investigated for the sake of examining the complete range of Pythium species from clade K, namely Pythium megacarpum and P. sterile. Pythium megacarpum is an invalid species because no type was indicated at the time of publication. Lévesque & de Cock (2004) placed it as potentially synonymous with Phytopythium boreale and in the barcode analyses of Robideau et al. (2011) these two species were only distinguishable through COI sequence data analysis, not by ITS. Pythium sterile is an invalid taxon based on the nomination of two herbarium specimens as the type of this species; this contravenes Art. 40.3 of the Melbourne convention (McNeill et al. 2012). Pythium sterile possesses identical ITS sequences to Phytopythium litorale. Other sequences from this organism could not be compared since no strain of Pythium sterile is available. Both species do not produce sexual stages. A more extensive study of these pairs of species, namely, Phytopythium boreale / Pythium megacarpum and Phytopythium litorale / Pythium sterile including more isolates and more DNA regions should reveal whether P. sterile and P. megacarpum should be validated as legitimate species.
There were some clade K species which were not included in the phylogenetic analyses presented here. In the studies by Lévesque & de Cock (2004) and Robideau et al. (2011) the species Pythium indigoferae appeared in clade K, which is now the genus Phytopythium. In stark contrast to the other species in clade K, Pythium indigoferae produces filamentous sporangia according to its original description (Butler 1907). The strain of Pythium indigoferae in the study of Lévesque & de Cock (2004) was the strain CBS 261.30 which was used by van der Plaats-Niterink (1981) in her publication ’Monograph of the genus Pythium’, as the ex-type strain was no longer available. However, CBS 261.30 is also no longer viable. Under observation by van der Plaats-Niterink and more recently while it was still culturable, this strain did not sporulate. The identity of this strain can therefore not be confirmed. Other strains with DNA sequences very close to CBS 261.30 have been identified (unpubl. data) which produced, however, subglobose, proliferating, papillate sporangia. These findings agree with Spies et al. (2011) who suggested that this strain be re-identified as Pythium vexans. CBS 261.30 and related strains are clearly part of a Phytopythium vexans complex that needs to be resolved through further phylogenetic study. This P. vexans complex also contains the invalid taxon Pythium cucurbitacearum, which was not included in our analyses. This taxon is invalid as it is missing a Latin diagnosis and based on Art. 36 of the Melbourne convention (McNeill et al. 2012). The representative strain of P. cucurbitacearum CBS 748.96 is no longer viable. The ITS sequence of this strain was reported by Spies et al. (2011), to be related yet distinct from a novel strain isolated from Acacia which was very different among the isolates in the monophyletic Phytopythium vexans complex studied. Most likely strain CBS 748.96 represents a distinct species from the P. vexans complex, which as of yet is not validly described. Once this complex is resolved it is likely that it will represent a number of new species for the genus Phytopythium.
Two other Pythium species not included in the phylogenetic analyses are P. palingenes and P. polytylum. Because no living strains of these species are available, they could not be included in the DNA studies. Morphological data for P. palingenes and P. polytylum show the typical characters of Phytopythium: ovoid, papillate, internally proliferating sporangia and cylindrical antheridia. Therefore we consider P. palingenes and P. polytylum as members of Phytopythium.
A new species of Phytopythium was isolated from water samples collected in District MirpurKhas of Sindh province, Pakistan. It is described and illustrated here as P. mirpurense (see section New Species). Genetically, Phytopythium mirpurense is shown to nestle within the genus Phytopythium, in all of the phylogenetic trees presented. The most obvious morphological characters of this new species are the proliferating, subglobose sporangia, terminal and intercalary oogonia, antheridia with lengthwise application to oogonia over their entire length, aplerotic to nearly plerotic oospores, and high optimum temperature for growth. These characters are shared with many other members of Phytopythium. The main differentiation of this species is shown through the molecular analyses of DNA sequences and the phylogenetic trees (Fig. 3, ,44).
Halophytophthora s.l. is a heterogenous, polyphyletic genus (Hulvey et al. 2010) with species of marine origin. Two species of this genus clustered within the clade of Phytopythium: H. operculata (originally described as Phytophthora operculata) and H. kandeliae. Further, only species of Halophytophthora s.str. (Hulvey et al. 2010) show some morphological similarity to Phytopythium. However, their sporangia are in average two or more times the size of sporangia in the Phytopythium species (length av. 64–117 μm, resp. 20–40 μm). They develop zoospores inside the sporangium and not in a vesicle like Pythium, though the formation of a vesicle may be part of the release process. Moreover, no internal proliferation was observed in these species. Halophytophthora kandeliae was previously transferred to Phytopythium (Marano et al. 2014, Thines 2014). The strains of Halophytophthora kandeliae used in barcode analyses of ITS and COI regions were CBS 111.91 and CBS 113.91 and they were both found to be associated with the Phytopythium clade (Robideau et al. 2011). However, neither of these strains is the type strain of this species. Marano et al. (2014) have published the ITS sequence of the type strain of H. kandeliae from ATCC and this sequence was identical to that of CBS 111.91 and 113.91. We have then included data from strain CBS 113.91 in our analyses here and are certain that it well represents the systematic placement of Phytopythium kandeliae. There are some difficulties with Halophytophthora operculata’s lack of fit in this clade by morphological measures and we have decided not to rename it at this time. This marine species has zoospore development fully within the sporangium; no vesicle occurs. Zoospore discharge is unique, via an operculum at the apex of the sporangium and no internal proliferation was observed. The size of the sporangia is significantly much larger than those of the Phytopythium species (up to 175 um). The strain CBS 241.83, which is the ex-type strain of H. operculata, did not sporulate during our investigations, so the identity of the strain could not be confirmed. However the current molecular data available about this strain, the sequence data presented here and the organisation of the 5S gene family as reported by Bedard et al. (2006), does indicate that it belongs in a monophyletic circumscription of Phytopythium. More investigation of this species is clearly required in order to confirm its identity.
New combinations were deposited in MycoBank (see below in section Taxonomic and Nomenclatural Changes; Crous et al. 2004).
CONCLUSIONS
The genus Phytopythium was first proposed to the community in 2008 (see www.phytophthoradb.org/pdf/O8LevesquePM.pdf) and it was formally published in June 2010 (Bala et al. 2010b), with Phytopythium sindhum as the type species. In 2010, Uzuhashi et al. (2010) proposed another name Ovatisporangium for clade K using a partial sampling of Pythium and Phytophthora species and published their findings in September of 2010. Comparison of their circumscription of the genus Ovatisporangium to our molecular analyses clearly shows that the type of Phytopythium, P. sindhum is a member of the group described as Ovatisporangium (Fig. 1, ,2).2). Ovatisporangium is thus recognised as a synonym of Phytopythium.
We demonstrated with three different phylogenetic markers that all species belonging to Pythium clade K represent a monophyletic genus that includes the type species of the previously described genus Phytopythium. The taxonomic circumscription of other Pythium clades remains unresolved. The species with filamentous and globose sporangia are well separated as reported before in many studies, however, both LSU and COI suggest that clades A–J could be divided into subgroups but provide no support for any particular arrangement. The inclusion of species from other genera closely related to Pythium such as Pythiogeton, Lagenidium or Myzocytiopsis can change these conclusions but clade support remains very low (Schroeder et al. 2013, Hyde et al. 2014). Therefore, we recommend avoiding any further changes in the generic status of Pythium Pringsheim species belonging to clade A–J until better phylogenetic markers are found and multigene phylogenies are available with the closely related genera.
TAXONOMIC AND NOMENCLATURAL CHANGES
Phytopythium Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, Persoonia 24: 137. 2010
Type species. Phytopythium sindhum, Lodhi, Shahzad & Lévesque, Persoonia 24: 137. 2010.
Etymology. Named after combined features of the genera Phytophthora and Pythium.
Common morphological characteristics of the species of Phytopythium are globose to ovoid shape of sporangia, often with a more or less distinct papilla or non-papillate and often proliferating internally like those in Phytophthora with non-papillate sporangia. Zoospore discharge is like Pythium. Most species have large, smooth oogonia, thick-walled oospores, and 1–2 elongate or lobate antheridia, laterally applied to the oogonium. Cultures are mostly homothallic, occasionally sterile.
Notes — Phytopythium (Bala et al. 2010b) is emended to include species of Pythium in clade K from Lévesque & de Cock (2004) and described after that. It is morphologically and phylogenetically between Pythium and Phytophthora.
NEW COMBINATIONS
Phytopythium boreale (R.L. Duan) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563326
Basionym. Pythium boreale R.L. Duan, Acta Mycol. Sin. 4: 1. 1985 (as ‘borealis’) (MB105742).
≡ Ovatisporangium boreale (R.L. Duan) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517560).
Representative strain — CHINA, soil under Brassica caulorapa, CBS 551.88 (ex-type strain not available).
Phytopythium carbonicum (B. Paul) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563328
Basionym. Pythium carbonicum B. Paul, FEMS Microbiol. Lett. 219: 270. 2003 (MB489329).
≡ Ovatisporangium carbonicum (B. Paul) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517561).
Representative strain — FRANCE, soil on top of spoil heap, CBS 112544 (ex-type strain).
Phytopythium chamaehyphon (Sideris) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563329
Basionym. Pythium chamaehyphon Sideris, C.P, Mycologia 24: 33. 1932 (as ‘chamaihyphon’) (MB260414).
≡ Ovatisporangium chamaehyphon (Sideris) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517562).
Representative strain — USA, Hawaii, Carica papaya, CBS 259.30 (ex-type strain).
Phytopythium citrinum (B. Paul) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563330
Basionym. Pythium citrinum B. Paul, FEMS Microbiol. Lett. 234: 273. 2004 (MB368597).
≡ Ovatisporangium citrinum (B. Paul) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517563).
Representative strain — FRANCE, Marsaunay la cote, vinyeard soil, CBS 119171 (ex-type strain).
Phytopythium delawarense (Broders, P. E. Lipps, M L. Ellis & Dorrance) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB807542
Basionym. Pythium delawarense Broders, P.E. Lipps, M.L. Ellis & Dorrance, Mycologia 104: 789. 2012 (MB563353).
Representative strain — USA, Ohio, Delaware county, Glycine max, CBS 123040 (ex-type strain).
Phytopythium helicoides (Drechsler) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563332
Basionym. Pythium helicoides Drechsler, J. Wash. Acad. Sci. 20: 413. 1930 (MB266912).
≡ Ovatisporangium helicoides (Drechsler) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517559).
= Phytophthora fagopyri S. Takim. ex S. Ito & Tokun., Trans. Sapporo Nat. Hist. Soc. 14: 15. 1935 (MB472184).
Representative strain — USA, Phaseolus vulgaris, CBS 286.31 (authentic strain).
Phytopythium litorale (Nechw.) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563335
Basionym. Pythium litorale Nechw., FEMS Microbiol. Lett. 255: 99. 2006 (MB521454).
≡ Ovatisporangium litorale (Nechw.) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517566).
Representative strain — GERMANY, Lake Konstanz, rhizosphere soil (Phragmites australis), CBS 118360 (ex-type strain).
Phytopythium mercuriale (Belbahri, B. Paul & Lefort) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563337
Basionym. Pythium mercuriale Belbahri, B. Paul & Lefort, FEMS Microbiol. Lett. 284: 20. 2008 (MB511433).
≡ Ovatisporangium mercuriale (Belbahri, B. Paul & Lefort) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517568).
Representative strain — SOUTH AFRICA, Limpopo Province, ex rhizosphere Macadamiae integrifoliae, CBS 122443 (ex-type strain).
Phytopythium montanum (Nechw ) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563338
Basionym. Pythium montanum Nechw., Mycol. Progr. 2: 79. 2003 (MB373239).
≡ Ovatisporangium montanum (Nechw.) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517569).
Representative strain — GERMANY, Bavarian Alps, wet soil under Picea abies, CBS 111349 (ex-type strain).
Phytopythium oedochilum (Drechsler) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563339
Basionym. Pythium oedochilum Drechsler, J. Wash. Acad. Sci. 20: 414. 1931 (MB272763).
≡ Ovatisporangium oedochilum (Drechsler) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (as ‘oedichilum’) (MB517570).
Representative strain — USA, CBS 292.37 (authentic strain).
Phytopythium ostracodes (Drechsler) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563340
Basionym. Pythium ostracodes Drechsler, Phytopathology 33: 286. 1943 (MB290364).
≡ Ovatisporangium ostracodes (Drechsler) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517571).
Representative strain — SPAIN, clay soil, CBS 768.73 (strain used by van der Plaats-Niterink (1981), ex-type strain not available).
Phytopythium palingenes (Drechsler) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB807543
Basionym. Pythium palingenes Drechsler, J. Wash. Acad. Sci. 20: 416. 1930 (MB273284).
Representative strain — None available.
Phytopythium polytylum (Drechsler) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB807544
Basionym. Pythium polytylum Drechsler, J. Wash. Acad. Sci. 20: 415. 1930 (MB275012).
Representative strain — None available.
Phytopythium vexans (de Bary) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque, comb. nov. — MycoBank MB563322
Basionym. Pythium vexans de Bary, J. R. Agric. Soc. 12 (Ser. 2,1): 255. 1876 (MB174427).
≡ Ovatisporangium vexans (de Bary) Uzuhashi, Tojo & Kakish., Mycoscience 51: 360. 2010 (MB517573).
= Pythium complectens M. Braun, J. Agric. Res. 29: 415. 1924 (MB261556).
= Pythium allantocladon Sideris, Mycologia 24: 27. 1932 (MB256394).
= Pythium ascophallon Sideris, Mycologia 24: 29. 1932 (MB257476).
= Pythium polycladon Sideris, Mycologia 24: 32. 1932 (MB274913).
= Pythium euthyhyphon Sideris, Mycologia 24: 34. 1932 (MB536649).
= Pythiumpiperinum Dastur, Proc. Indian Acad. Sci., B 1, 11: 803. 1935 (MB274563).
Representative strain — IRAN, soil, CBS 119.80 (strain used by van der Plaats-Niterink (1981) ex-type strain not available).
NEW SPECIES
Phytopythium mirpurense Lodhi, De Cock, Lévesque & Shahzad, sp. nov. — MycoBank 809691; Fig. 6
Phytopythium mirpurense sp. nov. sporangia (a–g) and gametangia (h–o). a–b. Papillate sporangia; c. sporangium proliferation outside of empty sporangium; d. internally proliferating sporangium; e. early stage of vesicle formation; f. vesicle with zoospore development inside; g. empty sporangium with remnants of vesicle membrane still attached; h. oogonium on short lateral stalk; i. unilaterally intercalary oogonium; j–o. oogonia with aplerotic oospores and long, cylindrical antheria. Arrows indicate constrictions in antheridia. — Scale bar in all figures 10 μm, except panel d (20 μm).
Etymology. Name refers to the District MirpurKhas of Sindh province, Pakistan from where this species was frequently isolated.
Main hyphae up to 6 μm wide. Sporangia papillate, proliferating, subglobose, limoniform, obovoid or ovoid 20–25 μm diam. Discharge tube short 5–8 × 5–6 μm diam. Oogonia large smooth globose, terminal, intercalary, occasionally unilaterally intercalary, (27-)34–37(-40) (av. 34) μm diam. Antheridia 1–3 per oogonium, mostly monoclinous or distantly monoclinous, occasionally diclinous. Oogonia and antheridial stalk originate from same hyphae. Antheridia apply lengthwise to the oogonium producing lateral or occasionally apical fertilisation tubes. Oospores aplerotic or nearly plerotic (22-)29–32(-34) (av. 29.45) μm diam. Oospore wall thickness is 2.5–3 (av. 2.8) μm. Ooplast 13–16 μm diam (Fig. 2, Fig. 3). Aplerotic index 66.7 %, ooplast index 23 % and wall index 47 %.
Colony characteristics — Phytopythium mirpurense produces profuse white cottony growth on PDA and CMDA, on PCA submerged without any patterns, and on CMA with a rosette pattern. The optimum growth occurred at 30 °C. Daily growth at 25 °C on PDA 19 mm, PCA 20 mm, CMA 23.5 mm and CMAD 26 mm. The maximum growth temperature was 35 °C.
Material examined. PAKISTAN, Sindh, District MirpurKhas, MirWah, N25°23’ E69°02’, stagnant water, 12 Jan. 2006, A.M. Lodhi (holotype CBS 124523, maintained in inactive state. Culture ex-type also deposited as DAOM 238991 in CCFC).
Additional material examined. PAKISTAN, Sindh, from water pond at Sindhri, District MirpurKhas (DAOM 238992, CBS124524) (N25°37’ E69°12’).
Acknowledgments
We thank Nicole Désaulniers for assistance in maintaining Phytopythium cultures, Rafik Assabgui and Julie Chapados from Agriculture and Agri-Food Canada, Ottawa for sequencing these strains. Strains were received from Anne Dorrance from The Ohio State University, Food, Agricultural, and Environmental Sciences, Plant Pathology, Columbus, OH, USA. We thank Marjan Vermaas for composing the photo plates. This research was supported through funding to the Consortium for the Barcode of Life Network (CBOL) from Genome Canada (through the Ontario Genomics Institute), NSERC and other sponsors listed at http://www.BOLNET.ca.
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Genes & Proteins (Showing 55 of 55)
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