|Techniques in Mycorrhizal Research|
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.
Ectomycorrhizae (EM) are a type of fungus that develop association with the roots of forest tree species, forming unique structures called Mantle, Hartig net.
Presence of Mantle and Hartig net (most important) are the most distinguishing features that confirm mycorrhization.
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
Wash bottles containing water
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.
4. Collect the sievings in jars.
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)
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
Potassium hydroxide solution (5-10%)
0.01 % acid fuschin : 0.01 g acid fuschin in 100ml actoglycerol
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.
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.
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.
Plastic plate with grids for measuring root length
1. A randomly selected aliquot of stained root segments (1 cm in length) suspended in lactoglycerol are spread in a petri dish.
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
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
Methods and Principles of Mycorrhizal Research
Manual for the identification of VA mycorrhizal fungi
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
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.
Image analyser system
Calibrate the Image analyser system with the help of stage micrometer at a given x of compound microscope.
Collecting mycorrhizal fungi should be undertaken in a wall-planned and ordered fashion to achieve a high standard of retrievable information.
Equipments and Reagents:
Divide the sample field into four blocks
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.