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Assessment of river environment
    using Pythium species


             Koji Kageyama

             River Basin Research ...
Research background



  Recently, it is important to assess the effect of human activity
  on natural ecosystem.

  Previ...
Objectives                               Human activity
                                         strongly affect
         ...
Why Pythium as
target genus?

  The genus consists of more than 120
species and inhabits in freshwater and
seawater as wel...
Sampling sites


                  Nagara River




                   Kiso River




                 Chikugo River
Nagara River
A: Owashi       D: Mino




B: Kamagahora   E: Gifu




C: Yamato       F: Kaizu
Soil Sampling




                                                   4

                                   3              ...
Identification
 Morphological characterization
                    Asexual structure                           Sexual stru...
Pythium species from riverbed soils in three rivers


13 species including 3 new        Chikugo River
  species and 4 grou...
Population density of Pythium species in three rivers

                                                                   ...
16
                                                                                                       Kiso River




 ...
Is Pythium irregulare
available as indicator of river
environmental assessment ?

To examine the relationships between the...
Nagara river
                      35
Farmland area (km2)


                      30                                  r2= ...
To verify if P. irregulare will be available for
      assessment of the impact of agriculture




•   The growth rate of ...
Mycelial growth rate of Pythium irregulare

                                   30            Isolate from farmland soil

 ...
Pathogenicity of Pythium irregulare from riverbed soil

                      Cucumber          Tomato         Chinese cab...
Population structure in
                                     farmland soil of the river basin


                          ...
Conclusion
                                  Soil pH                       ×

➢ Population density of P.        Soil textu...
Future perspective




More ecological studies are necessary to clarify
impacts of human activities on environment
includi...
Our Research Group



Kuniaki Hanai, River Basin Research Center, Gifu
University

Tomohiro Tanahashi, River Basin Researc...
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O21 Kageyama

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2008 Phytophthora Workshop in Italy

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O21 Kageyama

  1. 1. Assessment of river environment using Pythium species Koji Kageyama River Basin Research Center, Gifu University
  2. 2. Research background Recently, it is important to assess the effect of human activity on natural ecosystem. Previous researches have tried environmental assessment in view of changes on plant flora and animal fauna. There are few reports to be focused on soil microorganisms Soil microorganisms play important roles on preservation of natural ecosystem and have a potential to quickly response to environmental changes.
  3. 3. Objectives Human activity strongly affect river environment. Impact To determine potential assessment of human activity on river environment using soil microorganism Pythium
  4. 4. Why Pythium as target genus? The genus consists of more than 120 species and inhabits in freshwater and seawater as well as soil. The genus have a wide range of functional species, such as saprophytes, animal and plant pathogens, mycoparasites. The selective medium for isolation from environmental samples is available. Identification of the species is practicable based on the morphological characteristics and molecular techniques.
  5. 5. Sampling sites Nagara River Kiso River Chikugo River
  6. 6. Nagara River A: Owashi D: Mino B: Kamagahora E: Gifu C: Yamato F: Kaizu
  7. 7. Soil Sampling 4 3 5 2 1 ・ Select five spots in 100m area along the river
  8. 8. Identification Morphological characterization Asexual structure Sexual structure Molecular identification Phylogenic analysis PCR-RFLP helicoides SOS helicoides GF48 helicoides RoPh3 helicoides RoTo helicoides H5 helidoides CBS167 68 helicoides CBS286 31 helicoides CBS343 72 chamaehyphon CBS259 30 ostracodes CBS768 73 vexans 2D111 oedochilum CBS292 37 ultimum DQ071398
  9. 9. Pythium species from riverbed soils in three rivers 13 species including 3 new Chikugo River species and 4 groups P. nagaii Pythium spp. P. spinosum Nagara River P. vexans P. dissotocum Kiso River P. acanthicum P. parvum P. irregulare P. inflatum Group P Pythium spp. P. rostratifingens P. perplexum Group T P. torulosum P. rostratum Pythium spp. Group F P. vanterpoolii Group HS
  10. 10. Population density of Pythium species in three rivers a Population density (cfu/g dry soil) Sampling site aca dis inf irr nag par per rof ros spi tor van vex HS P F T spp. Total Nagara A Up 0.0 3.3 0.0 0.0 0.0 0.0 0.0 8.6 0.0 0.0 2.7 0.0 44.7 156.2 0.0 0.0 0.0 2.6 218.0 2001.11 B 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.0 3.1 0.0 0.0 42.9 0.0 15.7 0.6 20.7 84.3 C 0.0 1.6 0.6 0.1 0.0 0.0 0.0 0.6 0.0 0.0 0.6 0.1 0.1 10.5 0.1 0.4 1.5 0.1 16.1 D 0.0 0.8 0.0 0.2 0.0 0.0 0.0 3.6 2.7 0.0 0.1 0.0 0.8 3.7 0.0 0.0 0.0 0.1 11.9 E 0.0 22.6 0.0 70.2 0.0 3.2 0.1 3.2 0.0 0.0 0.0 0.0 19.0 200.0 0.0 0.0 4.8 0.0 323.1 F Down 0.0 0.0 0.0 0.6 0.0 0.0 0.0 29.2 0.0 8.9 0.0 0.0 0.5 3.4 0.0 0.0 0.0 0.0 42.5 Nagara A Up 0.0 18.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 90.7 2404.4 0.0 0.0 0.0 0.0 2513.5 2002.05 B 0.0 5.9 0.0 0.0 0.0 0.0 0.0 0.0 5.9 0.0 5.9 0.0 0.0 650.4 0.0 10.3 0.0 3.0 681.5 C 0.0 5.4 0.0 0.0 0.0 0.0 0.0 2.4 0.0 2.4 0.0 0.0 1.3 30.9 0.0 0.0 0.9 0.1 43.2 D 0.3 0.7 0.0 0.0 0.0 0.0 0.0 4.2 0.6 0.3 0.2 0.0 0.1 1.7 0.0 0.3 0.0 0.1 8.4 E 0.0 3.4 0.0 1.5 0.0 0.0 0.0 59.6 0.0 0.0 0.0 0.6 0.0 40.3 0.0 0.0 0.0 0.0 105.7 F Down 0.0 37.6 3.1 110.5 0.0 0.0 12.3 0.0 0.0 0.0 15.0 81.6 0.0 753.8 0.0 0.0 56.4 8.9 1079.2 Kiso G Up 0.0 57.8 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 16.7 0.0 0.0 1416.7 0.0 33.3 0.0 0.3 1526.5 2003.6 H 0.0 29.5 0.0 2.9 0.0 0.0 0.0 2.8 0.0 0.0 2.6 0.0 0.0 15.0 0.0 1.9 0.0 0.3 55.0 I Down 0.0 0.0 0.0 14.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 874.1 0.0 32.7 0.0 0.0 921.3 Chikugo J Up 0.0 3.5 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 1.1 0.0 0.0 3.2 0.0 4.6 0.0 0.9 13.4 2003.6 K 0.0 0.3 0.0 0.4 0.0 0.0 0.0 0.1 0.0 0.5 0.1 0.0 0.1 2.7 0.0 0.4 0.0 0.9 5.7 L 0.0 2.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.1 0.8 0.0 0.0 3.7 0.0 2.8 0.0 1.7 12.1 M Down 0.0 0.8 0.0 1.3 0.3 0.0 0.0 0.0 0.0 0.0 1.2 0.0 0.0 14.3 0.0 2.0 0.0 0.0 19.8 aaca, P. acanticum; dis, P. dissotocum; het, P. heterothallicum; inf, P. inflatum; irr, P. irregulare; nag, P. nagaii; par, P. parvum; per, P. perplexum; rof, P. rostratifingens; ros, P. rostratum; spi, P. spinosum; tor, P. torurosum; van, P. vanterpoolii; vex, P. vexans; HS, Pythium group HS; G, Pythium group G; P, Pythium group P; F, Pythium group F; T, Pythium group T; spp, Pythium spp.
  11. 11. 16 Kiso River Density (cfu/gsoil) 12 8 120 Nagara River 4 100 Density (cfu/gsoil) 2001.11 0 80 2002.5 G H I 60 Sampling site 40 2 Chikugo River 20 Density (cfu/gsoil) 0 A B C D E F Sampling site 1 0 J K L M Sampling site Population density of Pythium irregulare strain in riverbed soil
  12. 12. Is Pythium irregulare available as indicator of river environmental assessment ? To examine the relationships between the distribution of P. irregulare and environmental factors 1. Soil pH 2. Soil texture 3. Carbon/nitrogen ratio in soil 4. Farmland area
  13. 13. Nagara river 35 Farmland area (km2) 30 r2= 0.5227* 25 20 15 Soil pH 10 Soil texture 5 C/N ratio 0 X 0 10 20 30 40 50 60 Population density of P. irregulare (cfu/g soil) P. irregulare The distribution of P. irregulare will correlate with agricultural practice
  14. 14. To verify if P. irregulare will be available for assessment of the impact of agriculture • The growth rate of P. irregulare from riverbed soil • Pathogenicity of P. irregulare from riverbed soil to crop plants • The influence of Pythium flora in farmland soil on the flora in riverbed soil.
  15. 15. Mycelial growth rate of Pythium irregulare 30 Isolate from farmland soil Mycelial growth (mm/24h) Isolate from riverbed soil 25 20 15 10 5 0 5 10 15 20 25 30 35 Temperature (℃) P. irregulare in riverbed soil will have similar growth habit of that in farmland soil
  16. 16. Pathogenicity of Pythium irregulare from riverbed soil Cucumber Tomato Chinese cabbage Isolate EMa DIb EM DI EM DI Nagara isolate 0 0 17 100 90 0 Kiso isolate 73 59 5 100 75 0 Non-inoculated 67 0 93 0 90 0 a: Percentage of emergency b: Disease incidence P. irregulare in riverbed soil is pathogenic to crops
  17. 17. Population structure in farmland soil of the river basin Ⓔ P. irregulare Ⓕ P. irregulare P. irregulare The population structure of Pythium irregulare in farmland soil will affect that in riverbed soil.
  18. 18. Conclusion Soil pH × ➢ Population density of P. Soil texture × irregulare is correlated with Soil Carbon/Nitrogen ratio × Farmland area ○ ➢ P. irregulare from riverbed soil shows similar growth response to temperature to the response from farmland soil. ➢ P. irregulare from riverbed soil also has pathogenicity to crops. ➢ Population structure of Pythium species in farmland soil influences that in riverbed soil. P. irregulare will be available as an indicator for assessment of the environmental impact of agriculture.
  19. 19. Future perspective More ecological studies are necessary to clarify impacts of human activities on environment including agriculture. Molecular quantitative and qualitative detection methods are strongly required to make ecological studies easier and common.
  20. 20. Our Research Group Kuniaki Hanai, River Basin Research Center, Gifu University Tomohiro Tanahashi, River Basin Research Center, Gifu University Masako Senda, River Basin Research Center, Gifu University Haruhisa Suga, Life Science Research Center, Gifu University

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