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Arbuscular mycorrhiza fungi

Lab Report, Rhizosphere Ecology (weberv)

Background

Arbuscular mycorrhiza (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments.

AM use C from plants and improve their growth through:

  • Improve of nutrients supply such as P.
  • Increase of the resistance to disease by physical protection and reduce of C compounds to pest and pathogens.
  • Increase of the level of phytohormones in plants such as cytokinin and gibberellin-like substances.
  • Improve of the drought resistance of the plants.

Design of experiment

lab-report-rhizosphere-ecology-iii_p1.jpg

Aim

  • To assess the P uptake by Glomus intraradices and Gigaspora margarita.
  • To study the effects of these fungus on plant growth.
  • To understand the competition between these two species for roots colonization.

Hypothesis

  1. Glomus i. improves growth compared to Gigapora m. because Gigaspora has high carbon requirements.
  2. Both AMF supply P to plants and Glomus supply more.
  3. Glomus is a better competitor than Gigaspora, meaning that amount of Glomus biomass in roots will be higher in a plant colonized first by Gigaspora and then by Glomus.

Preparation for root dry weight and DNA-extraction
The soil around the plant roots was collected. Then the roots were cleaned in ice water and dried afterwards. The roots were weighted (about 300 mg) and then cut in 1cm pieces and mixed. Then the roots were frozen in fluid nitrogen. Additionally some roots were put into an Eppendorf vial for DNA-extraction. The rest of the roots were measured and put into an aluminum pot for drying to measure the humidity of the roots. The shoot was weighted. The soil was used separated for roots staining, DNA extraction and drying and P extraction.

Results
Root weight is highest without colonization. So without colonization it is important for the plant to produce roots in order to find nutrients. For this the root surface is most important. The shoot dry weight is highest for Glomus and smaller for Gigaspora. This result matches with hypothesis 1.
Additionally Gigaspora has a negative effect on the plant because the shoot dry weight (growth) with Gigaspora is smaller than with no inoculation (control). With Glomus the shoot dry weight is increased.

Extraction of Mycelium and length density assessment in substrate
5g of soil substrate were weighted and sieved. As a result 99% of mycelium stays on second sieve. The rest of the soil was weighted (ca. 20g) for dry weight measurement. To the suspension from the 40 µm sieve was water added and then mixed in a blender. The suspension was filled up to 500ml with water and then stirred for 1 min. After the stirring at different time points 5ml sample were taken. These samples ware filtered through a grid. Then the filter was stained with 2ml Typan Blue solution and washed with water under vacuum. After 2 min the vacuum was removed and then the grid was placed on a microscope slide.
The number of mycelium was computed according to the lines on the grid. If the mycelium crossed a line, it was counted.

lab-report-rhizosphere-ecology-iii_p2.jpg

Results

  • NM-NM: Some mycelium is found even if plant was not inoculated with AMF
  • Primary inoculum Glomus leads to the greatest development of AMF mycelium. Whereas the primary inoculum of NM and Gigaspora show a similar development of mycelium which is about half of amount compared to Glomus. This result matches with the third hypothesis.
  • Gigaspora:NM does only slightly increase the development of mycelium in comparison to NM:NM, but Gigaspora:Gigaspora yields about the double amount of mycelium
  • Glomus:Gigaspora yield the highest amount of mycelium
  • > Glomus is very efficient as a primary inoculum. Colonizes roots rapidly, stays there and spreads fast further
  • > Once Glomus has colonized the roots it says there and spreads further
  • > Gigaspora is efficient as a secondary inoculum. Colonizes roots more slowly, colonization does not continue if removed from plant (needs spores for initiation)
  • > Glomus:Gigaspora yields much higher development of mycelium than other treatments.
  • > Occasionally, the substrate of this treatment was diluted too much (but corrected for calculations) – any effect (if any, opposite would be expected)?

Root DNA extraction
Roots where homogenized and then the internal standard was added. Afterwards the Buffer API and RNAse were added. The Buffer protects the DNA whereas the RNAse destroys the RNA which is more frequent in organisms than DNA. The suspension was transferred into tube and then incubated at 65°C. At this temperature the proteins denaturize and the histone depart from the DNA. Then the Buffer AP2 was added which denaturizes the sugar proteins. With this step the membrane precipitates. After incubation the lysate was centrifuged several times. 400 µl of lysate was than recovered without disturbing the cell-debris pellet. Then 600 µl Buffer AW was added, although AP3 E should have been added. Because of the ethanol the DNA binds to the membrane. The mixture was transferred into a DNeasy Mini spin column and again centrifuged. Afterwards Buffer AW was added twice. The mixture was transferred in a new column and Buffer AE was added to the membrane. After incubation the flow was transferred onto the membrane and centrifuged again to improve DNA yield. Then the DNA was stored at -20°C.

Real time-PCR
5 µl of DNA plus 45 µl PCR water was prepared for the samples 6-9 and 10 µl of DNA plus 40 µl PCR for the samples 1-5 (these samples had the double volume). Then the dilutions of the internal standards were prepared. For this 45 µl of water was added 5 µl of standard was added for the 108 dilution. From this dilution 5 µl was added to 45 µl of the 107 dilution and so on.
To the DNA 14 µl of the Taq-Polymerase was added. This polymerase is not active at room temperature. It needs to be headed up to 95°C in the PCR to be active. Afterwards three Mixes (for Gigaspora, Glomus and plasmid) were prepared. These mixes were pipette to the real time PCR capillaries for all plant-fungi treatments. To the mixes the master mix with the DNA was pipette. The capillaries were caped and transferred to a carousel. Then the carousel was centrifuged and real time PCR was started. Within this method the capillaries are heated first for denaturation (95°C) and then for amplification (72°C). Afterwards, the machine measures the amount of DNA by detecting the light which is reflecting back from the samples (the fluorescent mix binds to double stranded DNA). After 50 of these cycles the melting curves were computed. Within this step the temperature is slowly increasing which is increasing the distance between the DNA chains. The melting curve analysis is then based on the GC-content of the DNA. For Glomus the melting temperature is 83,8°C, for Gigaspora 84°C, for the internal standard 85,1°C and for the primer dimmers below 80°C (primer dimer are bindings of the primer, so that only small DNA  are amplified within the real time PCR).
The real time PCR was completed with cooling. With this the DNA chains are binding together again.
With the results the copy numbers as well as the cycling efficiency were calculated.

Results

  • highest amount of copies with Glomus/NM and Gigaspora/Gigaspora
  • with Glomus as primary inoculum is the amount of DNA highest which matches with the third hypothesis
  • with Gigaspora as secondary inoculum is the amount of DNA highest (same result like for mycelium extraction)
  • with both fungi present the amount of DNA is mixed with both fungi DNA  (Gig/Glomus, Glomus/Gig)

P extraction from plant biomass, P estimation by Malachit green method, 33P scintillation counting
200-400 mg plant samples were dried at 550°C for 6-15 hours. After this time the samples were left for cooling. Afterwards HNO3 was added, which dissolves heavy metals, and heated on a plate. The samples were cooled down and then deionized water was added. Then the dilution was transferred to funnels with filter papers. The crucibles were cleaned twice and then water was added up to 20 ml. At the end, the sample were mixed and kept at 4°C.
For the malachite green method water and extracted solution were pipetted in 50 times dilution. Then Malachit Green 1 was added. After 20 minutes malachite 2 was added. Then standards were prepared. After 60 minutes the P concentration was measured.

  • The lowest P concentration without any AMF. The introduction of Glomus intraradices in second period (after 3 weeks) increased P concentration in shoot and root of plants.
  • The highest P concentrations with Glomus intraradices inoculum in the first 3 weeks. When in the second period G. margarita was introduced or soil without AMF (NM), P concentrations for roots is higher.
  • P concentration with Gigaspora margarita comparable with non mycorrhizal during first 3 weeks period. Introduction of Glomus intraradices after 3 weeks increased P concentration in shoot and root.
  • > Glomus intraradices and Gigaspora margarita differ in their P uptake and transport efficiency. G. intraradices seems to be more active in this process.       G. margarita less efficient, values almost the same as in non mycorrhizal treatment.

For scintillation the acid extract was added to a vial and then scintillation cocktail was added.  The radioactivity was measured with a beta-counter. The radioactivity per plant and the transfer rates to the plant were computed.

Results

  • Glomus is very efficient in 33P-uptake whereas the contribution of Gigaspora for 33P uptake is  very low (Hypothesis 2)
  • 33P Glomus >>33P Gigaspora >33P NM
  • > Lower Root/Shoot ratio for NGo & GoGo than for GoN, GoGi, and GoGi due to reduced root growth
  • > Higher shoot[33P] in NGo than in GiGo à Competition limiting P-supply to  shoot?

Root staining
The roots were cleaned and immersed into 10% KOH, which opens the middle lamella. Then the roots were incubated in 80°C water bath. Afterwards the KOH was removed and HCL was added to neutralize the roots. Then the roots were stained and again incubated in 80°C water bath. Afterwards the roots were immersed with water and stored at 4°C.

Results

  • With no inoculum there is no root colonization
  • With Glomus root colonization increases (Hypothesis 3)
  • Root colonisation of Glomus highest after no inoculum
  • With glomus => gigaspora is the vesicles percentage highest
  • Interesting is, that the proportion of roots is highest when the roots are first not colonized with AM, followed with the colonization of Glomus
  • Hyphae are more abundant than arbuscules and vesicles => Results match with reality
December 24th, 2007
Topic: Crop Science, Plant nutrition Tags: None

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