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Advances in the
morphological and
molecular
identification of
Pythiogeton species

Z. Gloria Abad
USDA-APHIS-PPQ-PSPI,
Beltsville, MD, USA

Motoaki Tojo
Osaka Prefecture University,
Sakai, Osaka, Japan
                               Cytoplasm evacuation of
                               sporangium of Pythiogeton
Background and objectives
1. Pythiogeton in the Kingdom Straminipila with
   it's only nine species is a not widely studied
   genus.
2. In the USA, P. ramosum (Minden 1916) and P.
   autosytum (Drechsler 1932) have been
   reported.
3. Three putative new Pythiogeton species were
   isolated from ornamental plants (cypress,
   English-ivy and alyssum) which showed root rot
   symptoms during the summer 2003 in NC, USA.
4. These Pythiogeton species were characterized
   on morphology and the ITS rDNA sequences.
Kingdom: Straminipila
Phylum: Heterokonta
Class: Peronosporomycetes
Order: Pythiales
Family: Pythiaceae
Genus: Pythium
        Phytophthora
        Halophytophthora
        Pythiogeton
Patterns of sporangium development in Pythiaceae

   Cysts


   Zoospores

                  Flagellum                                       Flagellum
                  synthesis                                       synthesis
                      &                                               &
   Vesicle       Cytoplasmic                                     Cytoplasmic
                  cleavage                                        cleavage


                               Clear      Flagellum    Non or
                                          synthesis    Unclear
                                              &
                                         Cytoplasmic
                                          cleavage
   Sporangium




                 Pythium               Phytophthora,    Pythiogeton
                                       Halophytophthora
0:00   0:30          1:00




1:30   2:00          3:00




                            Zoospore
                            development in
                            Pythiogeton sp. 1
4:00   5:00         18:00
              Bars = 20μm
0:00   0:10         0:20




0:30    0:40         0:50




                             Zoospore
                             development in
                             Pythiogeton sp. 3
0:60    1:20        2:00


               Bars = 20μm
Sporagium of
Pythiogeton sp. 1
produced in water
culture

                    Sporagium of Pythiogeton sp. 1 produced in radish
                    root hairs. No obvious symptom was observed
Morphology of Pythiogeton sp. 1
1                       7         1-5. Sporangia
                                  6. Appressoria
                                  7, 8. Cytoplasm evacuation
                                  and zoospore development
2       3      4        8         through vesicles (arrowhead)
                                  9. Zoospore with flagella
                                  10. Encysted and germinated
5       6               9         zoospores
                                  11. Sporangia formed in
                                  cypress roots after artificial
                        10        inoculation (arrow heads)
                                  12. Sporangia formed around
                                  cypress roots after artificial
11                 12
                                  inoculation
                                  Bars: 1-10 = 20 µm
                                  Bars: 11, 12 = 50 µm
Morphology of Pythiogeton sp. 2.
22      25             28          22-25. Sporangia
                                   26. Appressoria
                       29
        26                         27, 28. Development of
                                   zoospores out and inside
                                   of sporangia
                       30
                                   29. Empty sporangium
23                                 with a vesicle (arrowhead)

        27                         30. Encysted zoospores
                                   31-33 Sporangia formed in
                                   and around English Ivy
24
                                   roots after artificial
                                   inoculation
31               32         33
                                   Bars: 22-30 = 20 µm
                                   Bars: 31-33 = 50 µm
Morphology of Pythiogeton sp. 3
34            38             41        34-39. Sporangia
                                       40. Appressoria
              39                       41. Empty sporangium
35                                     with vesicle (arrowhead)
                                       42. Encysted and
                                       germinated zoospores
36     37       40                42
                                       31-33 Sporangia formed
                                       in and around root of
                                       Alyssum roots after
                                       artificial inoculation
                                       Bars: 34-42 = 20 µm
43                     44
                                       Bars: 43, 44 = 50 µm
P.arrhenomanes.39
                 P.volutum.64
                 P.vanterpoolii.61
              P.periilum.54
                  P.graminicola.29
            P.inflatum.63
            P.aphanidermatum.71
            P.deliense.42
                P.porphyrae.06
                  P.destruens.74
                  P.insidiosum.64
                                     P.grandisporangium.91
                                       Pythiogeton.English.Ivy.03.287                    Pythiogeton sp. 1
                                          Pythiogeton.Cypress.02.1000
                                                   Ph.cambivora
                                                   Ph.cinnamomi
                                                  Ph.ilicis
                                              Ph.cactorum
                                          Ph.capsici
                                         Halophytophthora.batemanensis                                       -
                  P.viniferum.94
                  P.violae.63                                                                 .
                            P.rhizosaccharum.78
                       P.ramificatum.17
                        P.middletonii.49                                                Pythiogeton sp. 2
                                           P.rostratum.56
                     P.longisporangium.93
                     P.bifurcatum.35
                    P.orthogonon.53
                    P.nunn.51
                     P.campanulatum.04
                  P.acanthophoron.52
                   Pythium.sp.HS.66
                     P.ultimum.var.sporangiiferum.6
                  P.splendens.42
                 P.glomeratum.39
                P.heterothallicum.62
            P.undulatum.30
                                                                    Achlya.bisexualis
      0.1



Phylogenetic tree based in internal transcribed spacer ribosomal DNA
region [ITS rDNA (ITS1, 5.8S, ITS2)] sequences constructed using
Neighbour-joining analysis. Achlya bisexualis was used as outgroup.
Summary
1. Three putative new species, Pythiogeton sp. 1,
   2 and 3 were isolated from ornamental plant
   roots in North Carolina, USA.
2. The all species infected to roots of their host
   plants and radish, but had no visible symptom
   in an inoculation test performed in Petri dish.
3. The phylogenetic tree based in ITS rDNA
   shows that all Pythiogeton are a solid species
   and the closest are members that belong to
   Pythium (P. grandisporangium) than
   Phytophthora.

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O12 Motoaki

  • 1. Advances in the morphological and molecular identification of Pythiogeton species Z. Gloria Abad USDA-APHIS-PPQ-PSPI, Beltsville, MD, USA Motoaki Tojo Osaka Prefecture University, Sakai, Osaka, Japan Cytoplasm evacuation of sporangium of Pythiogeton
  • 2. Background and objectives 1. Pythiogeton in the Kingdom Straminipila with it's only nine species is a not widely studied genus. 2. In the USA, P. ramosum (Minden 1916) and P. autosytum (Drechsler 1932) have been reported. 3. Three putative new Pythiogeton species were isolated from ornamental plants (cypress, English-ivy and alyssum) which showed root rot symptoms during the summer 2003 in NC, USA. 4. These Pythiogeton species were characterized on morphology and the ITS rDNA sequences.
  • 3. Kingdom: Straminipila Phylum: Heterokonta Class: Peronosporomycetes Order: Pythiales Family: Pythiaceae Genus: Pythium Phytophthora Halophytophthora Pythiogeton
  • 4. Patterns of sporangium development in Pythiaceae Cysts Zoospores Flagellum Flagellum synthesis synthesis & & Vesicle Cytoplasmic Cytoplasmic cleavage cleavage Clear Flagellum Non or synthesis Unclear & Cytoplasmic cleavage Sporangium Pythium Phytophthora, Pythiogeton Halophytophthora
  • 5. 0:00 0:30 1:00 1:30 2:00 3:00 Zoospore development in Pythiogeton sp. 1 4:00 5:00 18:00 Bars = 20μm
  • 6. 0:00 0:10 0:20 0:30 0:40 0:50 Zoospore development in Pythiogeton sp. 3 0:60 1:20 2:00 Bars = 20μm
  • 7. Sporagium of Pythiogeton sp. 1 produced in water culture Sporagium of Pythiogeton sp. 1 produced in radish root hairs. No obvious symptom was observed
  • 8. Morphology of Pythiogeton sp. 1 1 7 1-5. Sporangia 6. Appressoria 7, 8. Cytoplasm evacuation and zoospore development 2 3 4 8 through vesicles (arrowhead) 9. Zoospore with flagella 10. Encysted and germinated 5 6 9 zoospores 11. Sporangia formed in cypress roots after artificial 10 inoculation (arrow heads) 12. Sporangia formed around cypress roots after artificial 11 12 inoculation Bars: 1-10 = 20 µm Bars: 11, 12 = 50 µm
  • 9. Morphology of Pythiogeton sp. 2. 22 25 28 22-25. Sporangia 26. Appressoria 29 26 27, 28. Development of zoospores out and inside of sporangia 30 29. Empty sporangium 23 with a vesicle (arrowhead) 27 30. Encysted zoospores 31-33 Sporangia formed in and around English Ivy 24 roots after artificial inoculation 31 32 33 Bars: 22-30 = 20 µm Bars: 31-33 = 50 µm
  • 10. Morphology of Pythiogeton sp. 3 34 38 41 34-39. Sporangia 40. Appressoria 39 41. Empty sporangium 35 with vesicle (arrowhead) 42. Encysted and germinated zoospores 36 37 40 42 31-33 Sporangia formed in and around root of Alyssum roots after artificial inoculation Bars: 34-42 = 20 µm 43 44 Bars: 43, 44 = 50 µm
  • 11. P.arrhenomanes.39 P.volutum.64 P.vanterpoolii.61 P.periilum.54 P.graminicola.29 P.inflatum.63 P.aphanidermatum.71 P.deliense.42 P.porphyrae.06 P.destruens.74 P.insidiosum.64 P.grandisporangium.91 Pythiogeton.English.Ivy.03.287 Pythiogeton sp. 1 Pythiogeton.Cypress.02.1000 Ph.cambivora Ph.cinnamomi Ph.ilicis Ph.cactorum Ph.capsici Halophytophthora.batemanensis - P.viniferum.94 P.violae.63 . P.rhizosaccharum.78 P.ramificatum.17 P.middletonii.49 Pythiogeton sp. 2 P.rostratum.56 P.longisporangium.93 P.bifurcatum.35 P.orthogonon.53 P.nunn.51 P.campanulatum.04 P.acanthophoron.52 Pythium.sp.HS.66 P.ultimum.var.sporangiiferum.6 P.splendens.42 P.glomeratum.39 P.heterothallicum.62 P.undulatum.30 Achlya.bisexualis 0.1 Phylogenetic tree based in internal transcribed spacer ribosomal DNA region [ITS rDNA (ITS1, 5.8S, ITS2)] sequences constructed using Neighbour-joining analysis. Achlya bisexualis was used as outgroup.
  • 12. Summary 1. Three putative new species, Pythiogeton sp. 1, 2 and 3 were isolated from ornamental plant roots in North Carolina, USA. 2. The all species infected to roots of their host plants and radish, but had no visible symptom in an inoculation test performed in Petri dish. 3. The phylogenetic tree based in ITS rDNA shows that all Pythiogeton are a solid species and the closest are members that belong to Pythium (P. grandisporangium) than Phytophthora.