Advances in the morphological and molecular identification of Pythiogeton species

Z. Gloria AbadUSDA-APHIS-PPQ-PSPI, Beltsville, MD, USA

Motoaki TojoOsaka Prefecture University, Sakai, Osaka, Japan

Cytoplasm evacuation of sporangium of Pythiogeton

Background and objectives1. 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: StraminipilaPhylum: HeterokontaClass: PeronosporomycetesOrder: PythialesFamily: PythiaceaeGenus: Pythium

PhytophthoraHalophytophthoraPythiogeton

Patterns of sporangium development in Pythiaceae

Cysts

Zoospores

Vesicle

Sporangium

Flagellum synthesis

&Cytoplasmic

cleavage

Flagellum synthesis

&Cytoplasmic

cleavage

Flagellum synthesis

&Cytoplasmic

cleavage

Pythium Phytophthora，Halophytophthora

Pythiogeton

Clear Non or UnclearNon or Unclear

0:30

1:30 2:00

0:00

3:00

4:00 5:00

1:00

18:00

Zoospore development inPythiogeton sp. 1

Bars = 20μm

0:00 0:10 0:20

0:30 0:40 0:50

0:60 1:20 2:00

Zoospore development inPythiogeton sp. 3

Bars = 20μm

Sporagium of Pythiogeton sp. 1produced in water culture

Sporagium of Pythiogeton sp. 1 produced in radish root hairs. No obvious symptom was observed

1

2 3 4

5 6

10

9

8

7

11 12

1-5. Sporangia

6. Appressoria

7, 8. Cytoplasm evacuation and zoospore development through vesicles (arrowhead)

9. Zoospore with flagella

10. Encysted and germinated zoospores

11. Sporangia formed in cypress roots after artificial inoculation (arrow heads)

12. Sporangia formed around cypress roots after artificial inoculation

Bars: 1-10 = 20 µm

Bars: 11, 12 = 50 µm

Morphology of Pythiogeton sp. 1

22

23

24

26

25

27

28

30

31 32 33

29

22-25. Sporangia

26. Appressoria

27, 28. Development of zoospores out and inside of sporangia

29. Empty sporangium with a vesicle (arrowhead)

30. Encysted zoospores

31-33 Sporangia formed in and around English Ivyroots after artificial inoculation

Bars: 22-30 = 20 µm

Bars: 31-33 = 50 µm

Morphology of Pythiogeton sp. 2.

34

35

36 37

38

39

40

41

42

43 44

34-39. Sporangia

40. Appressoria

41. Empty sporangium with vesicle (arrowhead)

42. Encysted and germinated zoospores

31-33 Sporangia formed in and around root of Alyssum roots after artificial inoculation

Bars: 34-42 = 20 µm

Bars: 43, 44 = 50 µm

Morphology of Pythiogeton sp. 3

0.1

P.arrhenomanes.39

P.volutum.64P.vanterpoolii.61

P.periilum.54P.graminicola.29

P.inflatum.63

P.aphanidermatum.71P.deliense.42

P.porphyrae.06P.destruens.74

P.insidiosum.64P.grandisporangium.91Pythiogeton.English.Ivy.03.287

Pythiogeton.Cypress.02.1000Ph.cambivoraPh.cinnamomi

Ph.ilicisPh.cactorum

Ph.capsiciHalophytophthora.batemanensis

P.viniferum.94P.violae.63

P.rhizosaccharum.78P.ramificatum.17P.middletonii.49

P.rostratum.56P.longisporangium.93P.bifurcatum.35

P.orthogonon.53P.nunn.51

P.campanulatum.04P.acanthophoron.52Pythium.sp.HS.66

P.ultimum.var.sporangiiferum.6P.splendens.42

P.glomeratum.39P.heterothallicum.62

P.undulatum.30Achlya.bisexualis

Pythiogeton sp. 2

Pythiogeton sp. 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.

Summary1. 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 rDNAshows that all Pythiogeton are a solid species and the closest are members that belong to Pythium (P. grandisporangium) than Phytophthora.