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Persoonia 34, 2015: 25 – 39
www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158515X685382
RESEARCH ARTICLE
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 phylogeneti-
cally distinct from the rest of the Pythium spp. and showed com-
bined 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 phylo-
genetics. 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 multi-
gene 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 morphologi-
cally examined to verify their identity and to find the character-
istic 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’-CGCATTACG-
TATCGCAGTTCGCAG-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. Sequenc-
ing primers used for the SSU region were NS1, NS2 (5’-GGCT-
GCTGGCACCAGACTTGC3’), NS3 (5’-GCAAGTCTGGTGC-
CAGCAGCC), NS4 (5’-CTTCCGTCAATTCCTTTAAG3’), NS5
Phytopythium: molecular phylogeny and systematics
A.W.A.M. de Cock1, A.M. Lodhi2, T.L. Rintoul 3, K. Bala 3, G.P. Robideau3,
Z. Gloria Abad4, M.D. Coffey5, S. Shahzad6, C.A. Lévesque3
Key words
COI
LSU
Oomycetes
Oomycota
Peronosporales
Phytopythium
Pythiales
SSU
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.
Article info Received: 28 January 2014; Accepted: 27 September 2014; Published: 30 October 2014.
1 CBS-KNAW Fungal Biodiversity Centre, P. O. Box 85167, 3508 AD Utrecht,
The Netherlands.
2 Department of Plant Pathology, Sindh Agriculture University, Tandojam,
Pakistan.
3 Agriculture and Agri-Food Canada, 960 Carling Ave, Ottawa, ON K1A 0C6,
Canada; corresponding author e-mail: Andre.Levesque@agr.gc.ca.
4 USDA-APHIS-PPQ-Center of Plant Health Science and Technology, Bldg
580, BARC-e, Powder Mill Road, Beltsville, MD 20705, USA.
5 Department of Plant Pathology and Microbiology, University of California,
Riverside, CA 92521, USA.
6 Department of Agriculture, University of Karachi, Karachi, Pakistan.
26 Persoonia – Volume 34, 2015
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 –
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
27
A.W.A.M. de Cock et al.: The genus Phytopythium
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 P10443 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
ATCC66501/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
Table 1 (cont.)
GenBank Accessions
Species Strain Number Clade SSU_ITS_28S SSU_ITS SSU COI LSU ITS
28 Persoonia – Volume 34, 2015
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 –
DAOM230386 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 –
Table 1 (cont.)
GenBank Accessions
Species Strain Number Clade SSU_ITS_28S SSU_ITS SSU COI LSU ITS
29
A.W.A.M. de Cock et al.: The genus Phytopythium
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 –
Table 1 (cont.)
GenBank Accessions
Species Strain Number Clade SSU_ITS_28S SSU_ITS SSU COI LSU ITS
(5’-AACTTAAAGGAATTGACGGAAG3’) and NS8 (5’-TCCGCA-
GGTTCACCTACGGA3’) (White et al. 1990) as well as Oom_Lo-
5.8S47 (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 proto-
cols (Applied Biosystems, Foster City, CA). Sequencing of the
PCR product was performed in an Applied Biosystems Prism
Genetic Analyzer model 3130XL.
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 align-
ments of each gene region were generated using MAFFT
(Katoh et al. 2005). The genera included in the phylogenetic
analyses were Albugo, Halophytophthora, Phytophthora, Phyto-
pythium 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 Py-
thium the invalid species Pythium sterile and Pythium mega-
carpum as well as two strains of the novel species Phytopy-
thium 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 Re-
versible (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 infer-
ences (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 phylo-
30 Persoonia – Volume 34, 2015
genetic 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 con-
verged 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 phylo genetic trees. Consensus trees were generated
using the 50 % majority rule tree criteria and rooted to Albugo
(LSU, COI and SSU) or Pythium (ITS).
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 Devel-
opment Core Team, 2011). All pairwise distances involving a
Phytopythium species against Pythium or Phytophthora were
extracted, i.e. all pairwise distances involving any two Phyto-
pythium 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)
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
31
A.W.A.M. de Cock et al.: The genus Phytopythium
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
Fig. 1 Sporangia of Phytopythium species. a. P. sindhum, four stages of sporangium development showing a young, globose sporangium, a mature, papil-
late 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. outgrow-
ing papilla; j. empty sporangium with intermediate sized discharge tube (arrow indicating tip). — Scale bars = 20 µm.
32 Persoonia – Volume 34, 2015
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 Phy-
tophthora 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, g, i, j), which does not occur in Phy-
tophthora. 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 spo-
rangium, 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 Phy-
tophthora: zoospores developed (partly) inside a sporangium
and partly in a vesicle.
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 –d, ar-
rows). Occasionally club-shaped antheridia with apical attach-
ment occur. In P. vexans, the antheridia are often very broadly
attached to the oogonium and lobed (Fig. 2e, f ).
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 mem-
bers 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 map-
ped onto the trees are shown (Fig. 3A – 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
Fig. 2 Oogonia and antheridia of Phytopythium species. a. P. sindhum, slightly elongated antheridium; b –c. P. oedochilum, long cylindrical antheridia; d. P. mir-
purense, 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.
33
A.W.A.M. de Cock et al.: The genus Phytopythium
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 pro-
ba bilities (< 50 % / 0.65). This suggests that given the SSU
data set, the major clades are unresolved in relation to the
outgroup.
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 sup-
ported by both ML bootstrap replicates and Bayesian proba-
bility 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).
Statistical analyses of pairwise distances
Markers, genera and clades as well as interactions between
them all had a significant effect on pairwise distances of Phytopy-
thium 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 Phyto-
pythium 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.
0.2
Albugo candida AC7A
Phytopythium chamaehyphon CBS259.30
Albugo candida AC2V
Phytopythium vexans CBS119.80
Phytopythium citrinum CBS119171
Phytopythium ostracodes CBS768.73
Phytopythium kandeliae CBS113.91
Phytopythium boreale CBS551.88
Phytopythium montanum CBS111349
Phytopythium delawarense CBS123040
Phytopythium helicoides CBS286.31
Phytopythium mirpurense CBS124523
Phytopythium oedochilum CBS292.37
Pythium Clade H
Phytophthora Clades 1-10
and Halophytophthora
Phytopythium carbonicum CBS112544
Phytopythium mercuriale CBS122443
Phytopythium litorale CBS118360
Pythium Clades E-G, I, J
Phytopythium sindhum DAOM238986
Halophytophthora operculata CBS241.83
Pythium Clades A-D
-/0.67
95/1
68/0.99
-/o
66/0.68
-/o
100/1
-/0.67
-/o
93/
0.99
85/
0.99
-/0.67
-/0.56
100/1
-/o
79/
0.98
90/0.96
95/1
-/0.89
99/1
-/0.99
100/1
96/1
0.04
Phytopythium citrinum CBS119171
Pythium Clades A-D
Phytopythium boreale CBS551.88
Phytopythium mirpurense
CBS124523
Phytopythium vexans CBS11980
Halophytophthora
operculata CBS241.83
Phytopythium montanum
CBS111349
Phytopythium oedochilum CBS29237
Albugo candida AC7A
Phytopythium delawarense
CBS123040
Phytopythium kandeliae P11614
Phytopythium litorale CBS118360
Albugo candida ACCS
Phytopythium carbonicum
CBS112544
Phytopythium sindhum CBS124518
Phytopythium mercuriale LEV3286A
Pythium Clades E-J
Phytopythium helicoides CBS286.31
Phytophthora
Clades 1-10
Phytopythium chamaehyphon
CBS259.30
-/o
99/1
-/o
-/o
-/o
-/o
-/o
-/o
100/1
-/o
-/
0.65
72/0.67
94/1
-/o
-/0.87
-/0.60
-/0.71
-/0.77
99/1
-/o
A) LSU C)SSUB)COI
0.2
-/o
54/0.90
100/1
-/o
-/o
92/o
-/0.78
-/o
70/0.98
-/o
-/o
-/o
-/o
-/o
61/0.67
100/1
67/0.92
79/0.99
-/0.97
92/0.99
-/0.51
53/o
Albugo candida AC2V
Phytophthora Clades 1-10
and Halophytophthora
Phytopythium kandeliae CBS113.91
Phytopythium delawarense CBS123040
Phytopythium mirpurense CBS124523
Phytopythium litorale CBS118360
Pythium Clades A-D
Phytopythium citrinum CBS119171
Phytopythium chamaehyphon CBS259.30
Phytopythium ostracodes CBS768.73
Phytopythium helicoides CBS286.31
Pythium Clades E-J
Phytopythium carbonicum CBS112544
Pythium insidiosum CBS574.85
Phytopythium vexans CBS455.62
Phytopythium mercuriale CBS122443
Phytopythium montanum CBS111349
Halophytopthora operculata CBS241.83
Albugo candida AC7A
Phytopythium sindhum CBS124518
Phytopythium boreale CBS551.88
Phytopythium oedochilum CBS292.37
Fig. 3 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.
34 Persoonia – Volume 34, 2015
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 mega-
carpum 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 nomi-
nation 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
0.3
Phytophthora infestans CBS366.51
Phytopythium sindhum DAOM238986
Pythium sterile B09
Phytopythium mercuriale A89
Phytopythium montanum CBS111349
Phytopythium boreale CBS551.88
Phytopythium mirpurum CBS124523
Halophytophthora operculata CBS241.83
Phytopythium mirpurum CBS124524
Phytopythium ostracodes CBS768.73
Pythium megacarpum CBS112351
Phytopythium carbonicum CBS112544
Phytopythium chamaehyphon CBS259.30
Phytophthora ramorum CBS101553
Phytopythium oedochilum CBS292.37
Phytopythium citrinum CBS119171
Phytopythium vexans CBS119.80
Pythium ultimum CBS398.51
Phytopythium helicoides CBS286.31
Phytopythium litorale CBS118360
Phytopythium delawarense CBS123040
Phytopythium kandeliae CBS113.91
Pythium dimorphum CBS406.72
Phytophthora sojae CBS382.61
99/1
99/1
100/1
51/0.95
99/1
-/o
100/1
63/0.87
100/1
-/o
63/0.94
96/1
100/1
98/0.99
68/0.99
98/1
100/1
100/1
-/o
59/0.98
-/o
-/0.78
Fig. 4 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.
35
A.W.A.M. de Cock et al.: The genus Phytopythium
van der Plaats-Niterink (1981) in her publication ‘Monograph of
the genus Pythium’, as the ex-type strain was no longer avail-
able. 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, prolife-
rating, 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 miss-
ing 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 monophyl-
etic 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 an-
theridia. Therefore we consider P. palingenes and P. polytylum
as members of Phytopythium.
A new species of Phytopythium was isolated from water sam-
ples collected in District MirpurKhas of Sindh province, Paki-
stan. 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, subglo-
bose sporangia, terminal and intercalary oogonia, antheridia
with lengthwise application to oogonia over their entire length,
aplerotic to nearly plerotic oospores, and high optimum tem-
perature 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, 4).
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. oper-
culata (originally described as Phytophthora operculata) and H.
kandeliae. Further, only species of Halophytophthora s.str. (Hul-
vey et al. 2010) show some morphological similarity to Phyto-
pythium. 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 spe-
cies. 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
COI
LSU
SSU
0.13
0.14
0.15
0.100
0.105
0.110
0.115
0.120
0.021
0.024
0.027
0.030
Clade A
Clade B
Clade C
Clade D
Clade E
Clade F
Clade G
Clade H
Clade I
Clade J
clade 01
clade 02
clade 03
clade 04
clade 05
clade 06
clade 07
clade 08
clade 09
clade 10
Phytopythium against
pairwise distance
Fig. 5 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 dif-
ference 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.
36 Persoonia – Volume 34, 2015
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 mono-
phyletic 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 Ovatispo-
rangium for clade K using a partial sampling of Pythium and
Phytophthora species and published their findings in Septem-
ber 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). 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 mono-
phyletic genus that includes the type species of the previously
described genus Phytopythium. The taxonomic circumscrip-
tion 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 mark-
ers 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, Per-
soonia 24: 137. 2010.
Etymology. Named after combined features of the genera Phytophthora
and Pythium.
Common morphological characteristics of the species of Phyto-
pythium are globose to ovoid shape of sporangia, often with a
more or less distinct papilla or non-papillate and often prolife-
rating 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., Myco-
science 51: 360. 2010 (MB517560).
Representative strain — China, soil under Brassica caulo-
rapa, 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., Myco-
science 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., Myco-
science 51: 360. 2010 (MB517563).
Representative strain — FranCe, Marsaunay la cote, vin-
yeard 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 & Dor-
rance, Mycologia 104: 789. 2012 (MB563353).
Representative strain — USA, Ohio, Delaware county, Gly-
cine max, CBS 123040 (ex-type strain).
37
A.W.A.M. de Cock et al.: The genus Phytopythium
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., Myco-
science 51: 360. 2010 (MB517566).
Representative strain — Germany, Lake Konstanz, rhizo-
sphere 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., Myco-
science 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., Myco-
science 51: 360. 2010 (MB517571).
Representative strain — Spain, clay soil, CBS 768.73 (strain
used by van der Plaats-Niterink (1981), ex-type strain not avail-
able).
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., Myco-
science 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).
= Pythium piperinum 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
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, prolifera-
ting, 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 oogo-
nium 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, 3). Aplerotic index 66.7 %,
ooplast index 23 % and wall index 47 %.
Colony characteristics — Phytopythium mirpurense pro-
duces 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').
38 Persoonia – Volume 34, 2015
Acknowledgements We thank Nicole Désaulniers for assistance in main-
taining Phytopythium cultures, Rafik Assabgui and Julie Chapados from Agri-
culture and Agri-Food Canada, Ottawa for sequencing these strains. Strains
were received from Anne Dorrance from The Ohio State University, Food, Agri-
cultural, 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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