Arbuscular Mycorrhizal Fungi

Ectomycorrhizal Fungi

Recovery and quantitative estimation of arbuscular mycorrhizal (AM) spores from soil

Quantitative estimation of intraradical colonization by AM fungi

Image Analysis and Analysis of Data

Sample collection for AM fungal analysis

Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizal fungi (AMF) is an important component of biodiversity particularly in tropical & sub-tropical ecosystems. About 80% of the total plant species are associated with AMF and are thus, potential factors determining diversity in the ecosystems. They can modify the structure and functioning of a plant community. Many reports document the occurrence of AM in diverse crops of intensively cultivated arable soils. These fungi can increase plant growth under low-fertility conditions and of particular interest for well-fertilized agricultural soils, can improve tolerance towards different kinds of stress such as drought or resistance towards root pathogens. These fungi can enhance mineral nutrient acquisition in host plants. Phosphorus, nitrogen, zinc, and copper acquisition are most commonly reported being enhanced by AMF in plants, but the acquisition of other mineral nutrients required for plant growth may be enhanced. AMF are not only a major component of soil fertility they also play a crucial role in the regulation of soil biological activity because of their abundance throughout the uppermost soil layer. AMF represent upto 20% of the dry biomass of the mycorrhizae (Bethlenfalvay et al., 1982), can account for 25% of the biomass of the soil microflora and microfauna combined and can extend as mycelium more than 9 cm beyond the roots . AMF have direct access to plant-fixed carbon, and constitute a major input of carbon and energy in soil. They distribute this carbon throughout the soil of the rooting zone for use by soil animals and microbes. In addition to their contribution to soil carbon, AMF play an important role in soil aggregation and stability, the key-stone of agricultural sustainability.

AMF do not induce distinctive alterations of root morphology. Therefore, the detection of their presence in the root system, the extent of colonization of the roots as well as development and anatomical features of colonization are all dependent on the identification of typical internal and external structures formed by the fungus. The AMF form a two-phase mycelial system: an internal mycelium within the cortex of the mycorrhizal roots and an external mycelium in the soil.

The external or extraradical mycelium is dimorphic and consists of i) permanent, coarse, thick-walled, generally aseptate hyphae which comprise a major portion of the mycelial phase & ii) numerous, fine, thin-walled and highly branched, lateral hyphae which become septate at maturity and are ephemeral in nature. The thick-walled external hyphae penetrate and cause internal colonization of the roots. At the entry point, the penetrating hypha forms appressoria in the host plants. An appressorium is a lens shaped multinucleate structure, 20-40 mm long, which develops between adjacent epidermal cells. The penetrating hyphae spread inter and intra-cellularly in the root cortex. In the cortical cells they form arbuscules which are branched haustoria-like structures. They are formed early in the association by repeated dichotomous branching of fungal hyphae. When fully developed, arbuscules frequently occupy a large proportion of the host cell lumen. Throughout its entire life span which is only 4-15 days , the arbuscule is surrounded by an intact host plasmalemma and increased amount of host cytoplasm containing elevated number of host organelles . The arbuscules are considered to be the primary structures involved in the bidirectional transfer of nutrients between the fungal symbiont and host plant. The vesicle development occurs later as terminal or intercalary swellings of the inter or intra-cellular hyphae.

They are thick-walled structures and their shape ranges from spherical to oval or they may become lobed. They store oil and polyphosphate granules, which are utilized by the plant under phosphorus deficiency.

Ectomycorrhizal Fungi

Ectomycorrhizae (EM) are a type of fungus that develop association with the roots of forest tree species, forming unique structures called Mantle, Hartig net.

Fruiting structure of a mushroom

WH Freeman (http://www.whfreeman.com/); http://www.fao.org/docrep/%20007/y5489e/y5489e06.htm

Vegetative mycelium of EM growing on agar substrate Source: http://www.geocities.com

Presence of Mantle and Hartig net (most important) are the most distinguishing features that confirm mycorrhization.

A section of root showing Mantle and Hartignet

Recovery and quantitative estimation of arbuscular mycorrhizal (AM) spores from soil

Propagules of AM fungi can consist of chlamydospores or azygospores, vesicles and mycelium or infected root pieces. Used together, as they occur in soil, these propagules may be termed mixed inoculum as compared with spores that have been separated from soil and represent a ‘purer’ inoculum. Before a propagule recovery technique can be selected, the desired form of propagule must be determined.

Various techniques are used to recover AM propagules from soil. The most basic of these is wet sieving and decanting (Gerdemann and Nicolson, 1963) to remove the clay and sand fractions of the soil while retaining spores and other similar-sized soil and organic matter particles on sieves of various sizes.

This technique is relatively fast but further purification of the spores is usually necessary particularly if spore numbers in a soil are low. Other methods involving sucrose density layers or gradients can also be used. This method has also been used successfully with a wide range of soil types.

Equipment and Reagent

Stalking sieves with nylon or stainless steel mesh and a large range of pore sizes for isolating spores from the soil sample

40-50 micron (0.04 mm) for small sized spores
100 micron (0.10 mm) for medium sized spores
250 micron (0.25 mm) for very large spores and sporocarps

Wash bottles containing water
Jars for collecting the sieving
Stereo zoom (stereomicroscope)
Petri dishes ( 11 cm ) for observing the sieving under stereomicroscope
Micropipettes for spore picking
Sodium hexametaphosphate
Centrifuge

Separating spores from soil

1. Air-dry and weigh the samples. This will allow spore number to be expressed relative to the soil weight. Remove the coarse materials like straw, debris and rocks should be removed with a 2-mm sieve.
2. The size of the soil samples that can be expressed will depend on spore numbers and soil texture. Generally less than 100 g of soil is best, but larger samples of up to 1 kg can be used, if care is taken to ensure that fine sieves do not become clogged. A dispersant, such as Calgon, can be used with clay soils, but this may kill spores. Vigorous washing with water is necessary to free spores from aggregates of clay or organic materials.
3. Mix the Soil in a substantial volume of water and decant through a series of sieves arranged in descending order of mesh size. Roots and coarse debris are collected on a coarse (750-µm) sieve, while spores are captured on one or more finer sieves.

Figure 1 Sieve set for AMF spore extraction	Figure 2 Sieved material with coarse debris collected at the top sieve ---------

4. Collect the sievings in jars.
5. Transfer the sievings into centrifuge tubes and centrifuge for 5 minutes at 1750 rpm in a horizontal rotor.
6. Decant the supernatant liquid carefully and resuspend pellet in 60% sucrose solution. Again centrifuge for 2 –5 minutes
7. Pour the supernatant (with spores) onto a 300BSS sieve size and rinse with water to remove the sugar.
8. Transfer the sieving onto the grided petri dishes/plate and observe it under stereomicroscope. Count the number of spores in plate/dish and express it as spores/g of the soil sample.

Preservation of spores

After extracting the spores if you do not want to observe immediately the spores can be stored for few days in Ringer’s solution.

Composition of Ringer’s solution (100 ml)

Reference:

Gerdemann JW & Nicolson TH (1963) “Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting”. Transactions of British Mycological Society 46: 235-244.

Practical Methods in Mycorrhizal Research Ed: M. Beundrett, L. Melville, L. Peterson Mycologue

Methods and Principles of Mycorrhizal Research Ed: N. C. Schenck, The American Phytopathological Society

Manual for the identification of VA mycorrhizal fungi Ed: N. C. Schenck and Perez, The American Phytopathological Society

Quantitative estimation of intraradical colonization by AM fungi

Equipments

Beakers
Test tubes
Weighing balance
Forceps
Needles
Plastic capsules/Test tubes
Water bath
Coarse sieve to prevent root loss during washing/changing solutions
Plastic vials with tight-sealing lids for storage of stained samples in 50% glycerol.

Reagents

Potassium hydroxide solution (5-10%)
Alkaline H₂O₂ - 25% Ammonia solution : 3 ml
- 10% H₂O₂ : 30 ml
1% HCl
50% glycerol-water (v/v) solution for destaining and storage of stained roots.
Lactoglycerol
- Lactic acid : 876 ml
- Glycerine : 64 ml
- Distilled water : 60 ml

Staining solutions

0.01 % acid fuschin : 0.01 g acid fuschin in 100ml actoglycerol
0.05% trypan blue : 0.05 g trypan blue in 100ml actoglycerol
0.03% Chlorozol black E ( CBE) in lactoglycerol ( 1:1:1 lactic acid, glycerol and water ). Dissolve CBE in water before adding equal volumes of lactic acid and glycerol.

Clearing and staining root specimens (Modified procedure of Phillips and Hayman, 1970)

Clearing and staining procedures requires root samples that should be washed free of soil. It is important that KOH and staining solution volumes are sufficient for the amount of roots being processed and that roots are not tightly clumped together for uniform contact with solutions. To ensure uniform staining, the roots should be chopped in to smaller (1-2 cm) segments.

1) Wash root specimens under running tap water thoroughly. Place them in beaker containing 5-10% KOH solution for about 15-30 minutes. The concentration of KOH and time of incubation of roots depend upon the age and tenderness of the roots.

Plastic cassettes to hold the root samples

2) Pour off the KOH solution and rinse the roots well in a beaker using at least three complete changes of tapwater or until no brown colour appears in the rinse water.
3) Cover the roots with alkaline H₂O₂ at room temperature for 10 minutes or until roots are bleached.
4) Rinse the roots thoroughly using at least three complete changes of tap water to remove the H₂O₂.
5) Cover the roots with 1% HCl and soak for 3-4 min. And then pour off the solution. DO NOT rinse after this step because the specimens must be acidified for proper staining.
6) Incubate the roots with staining solution (0.01% acid fuchsine in lactoglycerol or 0.05% trypan blue in lacto phenol) and keep them overnight for staining.
7) Place the root specimens in glass petriplate /multiwell plate for destaining. The destaining solution (50% glycerol) is the standard used in step 6, but of course, without the stain.

Sample storage and slide preparation

If clearing and staining is not possible immediately then fresh roots can be kept moist and stored at 5 °C (for several days), or may be preserved in 50% ethanol for months together in tightly sealed vials.

Staining quality is subsequently improved by destaining roots in 50% glycerol for several months prior to observation to allow excess stain to leach from roots. Semi-permanent slides of stained roots can be made with PVLG mountant. For temporary slide the stained roots can be observed in plain lactoglycerol.

Assessment of Root length colonized by AM fungi

Mycorrhizal colonization is assessed using Biermann and Linderman (1981) method (frequency distribution method) in which the colonization is assessed as proportion of root length colonized by mycorrhizal fungi using a compound microscope.

Equipments

Plastic plate with grids for measuring root length
Beakers
Fine forceps
Microscopic glass slides, cover slips and lactoglycerol
Compound microscope
Stereomicroscope
Stage and ocular micrometer
Petri dish (1 cm grid)

1. A randomly selected aliquot of stained root segments (1 cm in length) suspended in lactoglycerol are spread in a petri dish.
2. Calibrate the ocular micrometer with the stage micrometer by placing it on the eyepiece of compound microscope.
3. Mount the root pieces on the glass slides (5-10 pieces) and calibrate the ocular micrometer with the stage micrometer at the particular x of compound microscope and observe the root pieces.
4. The proportion of the length of each root segment consisting vesicles, arbuscules or hyphae are estimated to the nearest 10%.
5. Data are recorded frequency distributions from samples containing 25, 50, 100 root segments. The percentage of the root length with mycorrhizal fungi in the sample is calculated from the frequency distribution.

Calculation of the percentage of the root length with mycorrhizal colonization in a sample of 25 root segments (1 cm) from a frequency distribution of the percentage of segment lengths with mycorrhizal colonization

Reference:

Biermann B & Linderman RG (1981) “Quantifying vesicular-arbuscular mycorrhizae: Proposed method towards standardization”. New Phytologist 87: 63-67.

Phillips SJM & Hayman DS (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of British Mycological Society 55: 158-160.

Practical Methods in Mycorrhizal Research
Ed: M. Beundrett, L. Melville, L. Peterson Mycologue

Methods and Principles of Mycorrhizal Research
Ed: N. C. Schenck, The American Phytopathological Society

Manual for the identification of VA mycorrhizal fungi
Ed: N. C. Schenck and Perez, The American Phytopathological Society

Image Analysis and Analysis of Data

Image analysis is the science of making geometric and densitometric measurements on images from any source. Image analyser system comprises of a PC having the facility of capturing video images and software for performing various analysis. The system provides several classes of measurements ranging from semi-manual planimetry to semi-automatic particle sizing, including:

Planimetry of length, distance and area
Phase percent,area fraction and volume fraction
Calibrated densitometry
Particle shape and size analysis

The system is well equipped, having many image processing functions used for image filtering and enhancement, for acquiring digital images of the specimen AMF spores. The measurement of AMF spores (spore diameter, wall thickness etc.) is very important for the correct identification of these fungi. Image analyser system is used for this work. This system has many advantages over the conventionally used method of micrometer for measurements. The system provides rapid, accurate and statistically significant data and has an added benefit of acquiring digital images.

Equipments

Image analyser system
CCOD video camera
Compound microscope
Diagnostic Slide

Procedure

Calibrate the Image analyser system with the help of stage micrometer at a given x of compound microscope.
Prepare the diagnostic slides of the specimen spores and analyse for taxonomic characteristics under a compound microscope linked to image analyser. The main morphological variables used for the identification of AM fungi are:
a) sporocarp occurrence, shape, colour & size;
b) peridium occurrence, & characteristics;
c) spore colour, size & shape;
d) spore walls number, colour, thickness & ornamentation;
e) hyphal attachment, shape & type of occlusions.
In order to record the data, use the worksheet (INVAM) given in the manual.
For identifying the AM fungal species, consult the manual of Schenck & Perez (1990).

Observation sheet

  Sample collection for AM fungal analysis

Collecting mycorrhizal fungi should be undertaken in a wall-planned and ordered fashion to achieve a high standard of retrievable information.

Equipments and Reagents:

Sampler
Spade
Zipped Polythene bags
Scissors
Blotting Sheets

Sample collection

Divide the sample field into four blocks
Take the samples from the rhizosphere of host plants at a depth of 0-30 cm in each block.
Number of samples from each block should be decided on the basis of the site topography. The number can vary from 3-15.
Air dry the sample to a point where there is no free moisture and then fill the sample in plastic bags, sealed and stored at 40C in cold room until further processed.
These samples then can be screened for mycorrhizal parameters (spore count, species richness, percent root colonization, infectivity potential of AMF, etc.) or can be used for initiating the trap cultures for their further multiplication.

Data Recording

Data on fungi and their environments, particularly associated plants and soil, need to be recorded at the time of collection as it is often impossible to retrieve such data at a later date.