Some fungi in the Chytridiomycota can assimilate both inorganic and organic sources of nitrogen Alana L.DIGBY, FrankH.GLEASON*, PeterA.MCGEE School of Biological Sciences A12,University of Sydney, NSW2006, Australia Speaker: 洪萱慈 Advisor: 藍清龍 老師

Introduction Chytrids(Chytridiomycota) 壺菌 Blastocladiomycota 芽枝菌 GrowthNitrate(NO3−) Nitrogen source pH Zoosporic fungi PYG agar(peptone, yeast extract,glucose)

Introduction 650 × Allomyces arbuscula Butler (Blastocladiales)

Introduction

Introduction

Chytridiomycota( 壺菌門 ) The name is from the Greek chytridion, meaning "little pot", describing the structure containing unreleased spores. Zoospore of Chytridiales.

Chytridiomycota( 壺菌門 ) The most primitive Heterotrophic Many chytrids are aquatic

Chytridiomycota( 壺菌門 ) 蛙壺菌 (Batrachochytrium dendrobatidis)

Introduction Nitrogen is requirement for growth in all fungi. Fungi that utilize nitrate can also utilize ammonium salts, but not all fungi that utilize ammonium can utilize nitrate. Fungi that utilize nitrate can also utilize ammonium salts, but not all fungi that utilize ammonium can utilize nitrate. Nitrate and ammonium salts may be more readily available in soil. Nitrate and ammonium salts may be more readily available in soil.

Introduction To understand zoosporic fungi ecology we need to know that they how to utilize nitrogen in soil. we predict that fungi will have a greater response to organic sources of nitrogen. We examined the growth of eleven genera of zoosporic fungi on a range of nitrogen sources.

Meterials and methods selection of isolates Meterials: Nineteen isolates of zoosporic fungi. Nineteen isolates of zoosporic fungi. From vegetated and cultivated soils. From vegetated and cultivated soils. PYG agar

Meterials and methods Growth on solid medium with nitrate as the sole nitrogen source CSM: Chytrid Synthetic Medium Petri dishes.

Meterials and methods Growth on solid medium with nitrate as the sole nitrogen source The medium contained : Mg(NO 3 ) 2 Number of newly produced thalli. produced thalli. Diameter of the colonies. Subcultured 7 day at 20 ℃

Meterials and methods Growth on solid medium with nitrate as the sole nitrogen source Subculture (X5) 7 d at 20 ℃

Meterials and methods different sources of nitrogen in liquid 1.The basal CSM : without nitrogen. 2.Addition nitrogen or glucose. or glucose. 3. Addition different nitrogen source nitrogenglucose

Meterials and methods different sources of nitrogen in liquid 25 ml PYG agar / 8ml sterile deionised (DI) water Ratek orbital shaker set at speed 2 centrifuge 2 weeks 20 ℃ Four replicates

Meterials and methods different sources of nitrogen in liquid

Meterials and methods Meterials and methods Growth response to amino acids at different pH values Liquid CSM with either aspartate or arginine Using NaOH and HCl to pH6.5 pH7.5 pH8.5 Four replicate After growth for two weeks at 20 ℃

Meterials and methods use amino acids as a carbon source To test whether the amino acids could be the sole sources of energy. glucose was removed from the CSM. glucose was removed from the CSM. Use carbon-free basal medium. alaninehistidinearginine Aspartate or phenylalanine

Results nitrogen source Results nitrogen source Neither of the two isolates in the Blastocladiomycota grew. Three isolates of Chytridiomycota didn’t growth.

When grown on different nitrogen sources,isolates have diversity preferences. Urea was the best source of nitrogen for most fungi. The un-neutralised aspartate was the least favoured. Results Growth response to different sources of nitrogen in liquid media

Fig 1–The growth of tenisolates of zoosporic fungi on eight nitrogen sources.Nitrate(Ni), ammonium(Am), urea(Ur), aspartic acid(As), alanine(Ala), phenylalanine(Phe), histidine(His) and arginine(Arg). Bars with same letter are not significantly different at P>0.05.

Results Growth response to different sources of nitrogen in liquid media

Fig 1–The growth of tenisolates of zoosporic fungi on eight nitrogen sources. Nitrate(Ni), ammonium(Am), urea(Ur), aspartic acid(As), alanine(Ala), phenylalanine(Phe), histidine(His) and arginine(Arg). Bars with same letter are not significantly different at P>0.05.

In pH 6.5: Asp > Arg Exception of AUS6 and AUS11, all growth on the aspartate medium at pH6.5 was above 10 mg. Results Growth response to amino acids at different pH values

Fig 2–The effect of pH on growth on media with aspartate(As) and arginine (Ar) as nitrogen sources.Barswithsamelett er are not significantly different at P>0.05.No significant differences were found for A6 (P[0.162)

Results Growth response to amino acids at different pH values

Fig 2–The effect of pH on growth on media with aspartate(As) and arginine (Ar) as nitrogen sources.Barswithsameletter are not significantly different at P>0.05.No significant differences were found for A6 (P[0.162)

Results use amino acids as a carbon source AUS 6AUS 12AUS 14 Dec CC 4-10 Z AUS 6 AUS 6 、 AUS 12 、 AUS 14 、 Dec CC 4-10 Z can growth in CSM ， Use Aspartic acid/Alanine as source of carbon and energy. Aspartic acidAlanine None of the other amino acids supported growth of the isolates tested.

Discussion nitrogen source Nitrate appears to be a good source of nitrogen for some zoosporic fungi in the Chytridiomycota. Allo CW 16 and Poly Ad 2.0 : need organic sulphur. AUS6,AUS11, AUS13,AUS14 : urea and/or several amino acids urea and/or several amino acids grew well grew well nitrates nitrates not well not well

Discussion The growth of the zoosporic fungi were varied. In Rhizophydiales: AUS 6 grew on all five amino acids. AUS 12 grew on none.

Discussion In Rhizophlyctis rosea assimilation of nitrate: AUS 13 grew very poorly. BV80H grew significantly better on nitrate.

Discussion The nitrogen assimilated unrelated to the sites from which the fungi were isolated. Spizellomyces isolates came from alkaline soils, but grew better at pH6.5 than at pH7.5.

Discussion Growth on amino acids as sole sources of both carbon and nitrogen was poor. metabolic machinery: single amino acids carbon and nitrogen. Significant relation between nutrition and phylogeny in zoosporic fungi. Significant relation between nutrition and phylogeny in zoosporic fungi.

Discussion Higher order classification: Most Chytridiomycota utilized nitrate nitrate, ammonium salts, amino acids as a sulphur source. Some Blastocladiomycota and Neocallimastgomycota can use ammonium salts but can’t use nitrate.

Discussion ecology Fungi must compete with plants and other microbes for nitrate in the soil. Zoosporic fungi may have a competitive advantage over those microbes. Wide range of available in organic and organic sources of nitrogen. Wide range of commonly available in organic and organic sources of nitrogen.

Discussion We expect that Differences in the amount of growth due to differences in cultural conditions,such as pH Some zoosporic fungi may be widely distributed in poor soils.