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In this article we will discuss about:- 1. Isolation of VAM Spores from Soil 2. Measurement of Root Infection 3. Most Probable Number (MPN) 4. Identification of VAM Spores 5. Pure Inoculum Production 6. Mass Production 7. Pot Cultures 8. Aeroponic Cultures.
Endotrophic mycorrhiza or vesicular arbuscular mycorrhizae are known so, since they produce vesicles and arbuscules in the roots of plants. However, some genera like Gigaspora produce only arbuscules in the roots of plants, and are known as arbuscular mycorrhiza. Almost 90% of the earth’s vegetation has VAM fungi associated with the root system.
They increase absorption of water and minerals, provide growth hormones like auxins, cytokinins and gibbereilins. In-crease tolerance during adverse conditions to plants, accelerate nitrogen fixation and are useful in ex vitro establishment of tissue culture plantlets. With all these benefits, use of VAM in agriculture is limited since they cannot be grown in vitro without plant parts.
Isolation of VAM Spores from Soil:
1. Wet Sieving and Decanting:
Principle:
When soil suspension is passed through sieves having smaller mesh size than VAM spores, they will be retained in the sieve and can be collected.
Requirements:
a. Rhizosphere soil sample.
b. Beaker, glass rod, water, slides, etc.
c. Set of sieves (710-45 µm mesh size).
d. Binocular and microscope.
e. Lactophenol.
Procedure:
1. Place approximately 250 g of rhizosphere soil in a 500 ml Erlenmeyer beaker and add 400 ml of water.
2. Stir well in order to disperse the soil particles and allow heavier particles to settle down.
3. Decant the suspension through the set of sieves, 710-45 µm placed one below the other with 710 µm one on top and 45 µm and one at bottom.
4. Collect the residue of sieves 75 µm and 45 µm after removing them and washing them on a filter paper.
5. Examine the filter paper under a binocular and remove spores gently with the help of a flat needle to a drop of water on a slide and observe.
6. Blot water, place a drop of lactophenol on the slide, place a coverslip gently and observe under a microscope.
7. Count the number of spores and calculate for one gram of soil.
8. Permanent slides can be made by mounting spores in Hoyer s medium which preserves all characters of the spores in original form. Staining is not essential since most of the spores are brightly coloured.
Examine the slides of spores and note morphological characters like presence or absence of subtending hypha, spore wall, ornamentation, etc.
2. Sucrose Centrifugation:
Principle:
Spore suspension when placed in 2(M) sucrose solution and centrifuged, they easily float in the supernatant and these are filtered and collected.
Requirements:
1. Suspension of VAM spores from sieves.
2. Centrifuge.
3. 2(M) sucrose solution.
4. Slides and coverslips.
5. Distilled water.
Procedure:
1. Place the suspension from 75 and 45 µm sieves in a 50 ml centrifuge tube and make up the volume to 35 ml with distilled water.
2. Centrifuge at 2000 rpm for 10 minutes and filter supernatant.
3. Suspend the pellet in the centrifuge tube in 2(M) sucrose solution and make up the volume to 35 ml.
4. Stir well and centrifuge at 2000 rpm for 10 minutes.
5. Filter the supernatant and collect the spores.
6. Mount the spores and examine them.
3. Mass Collection VAM Spores:
Principle:
Larger spores like Gigaspora also can be collected by taking the fractions from 425 and 250 µm sieves besides 75 and 45 µm.
Requirements:
a. Rhizosphere soil sample.
b. Beakers glass rod, etc.
c. Set of sieves 710-45 µm.
d. 30% (W/V) aqueous sucrose gradients.
e. Aspirator.
f. Centrifuge.
g. Distilled water.
h. Sucrose (2M).
Procedure:
1. Subject the soil sample to wet sieving and decanting with cold water.
2. Collect spore fraction from 425 and 250 µm sieve with debris for Gigaspora margarita and for other genera from sieves 75 and 45 µm.
3. Decant and remove debris or vacuum aspirate the surface of the suspension in a 4 litre beaker which has the debris and spores from 250 µm sieve.
4. For the remaining portion from 75 and 45 µm sieve separate spores by using discontinuous 30% (W/V) aqueous sucrose gradients.
5. Take a one litre beaker add 600 ml of water over 200 ml sucrose.
6. Gently place the sieved material in layers on this large gradient.
7. Spores and debris get collected at the interface due to gravity.
8. Remove this layer by vacuum aspiration and rinse with cold water.
9. Centrifuge at 1600 rpm for 2 to 5 minutes on 2nd gradient (15 ml water and 20 ml sucrose).
10. Aspirate spore fraction from gradient interface and store in cold water.
4. Modification in Our Laboratory:
A. Wax Coated Plates:
Principle:
Spores floating on the surface will move and stick to paraffin from .where it can easily be collected.
Requirements:
1. Debris from sieves.
2. Petri dishes coated on the sides (inside) with paraffin.
Procedure:
1. Suspend debris and spores in Petri plates coated with paraffin wax.
2. Due to gravity debris gets settled and spores float on the surface and gets stuck to the paraffin coating.
3. Decant water and collect spores in cold water.
B. Funnel Assembly:
Principle:
As water is let out, little by little, the air bubble that comes up push the spores through waves to the periphery.
Requirements:
1. Washing from sieves.
2. Funnel fitted to a suction pump with closed stopcock.
Procedure:
1. Place water in the funnel fined to a suction pump and with the stopcock closed.
2. Suspend washings with water from sieve in funnel. Most of the spores move to the periphery and stick to the sides of the funnel.
3. Open the cock gently and allow a bit of water to go.
4. Spores present in the central region also now move to the periphery.
5. Repeat this process to reduce the quantity of water in the funnel and ultimately drain off the whole water by opening the cock.
6. Rings of spores get stuck to the sides of the funnel. Collect them in cold water.
Assessment of Mycorrhizal infection in roots.
Principle:
Using KOH the tissues of roots are macerated and fungal element which remains intact is stained and observed.
Requirements:
a. Feeder roots of plants with VAM infection.
b. 10% KOH.
c. 1(N) HCl.
d. 0.5% trypan blue/cotton blue in lactophenol.
e. H2O2
f. Slides, cover slips.
g. Needles and forceps.
h. Distilled water.
i. Petri dish.
Procedure:
1. Wash infected roots thoroughly in water.
2. Add 10% KOH and place them covered in a Petri dish in an oven at 90°c for 30 minutes to 1 hour.
3. Wash the root segments gently two to three times with distilled water.
4. Add H2O2 and leave aside for one to two hours.
5. Acidify by immersing in dilute in HCl for a few minutes.
6. Stain the roots with cotton blue in lactophenol and remove excess of stain with lactophenol. Mount on slides in lactophenol or acetic acid: glycerol (l:lv/v)
7. Place a cover slip seal and observe.
For Pigmented Roots:
Principle:
Pigmented roots are decolourised by treating it with stronger solution of KOH and heating it for a longer time.
Requirements:
1. 10-20% KOH.
2. VAM infected feeder roots.
3. 1(n) (HCI).
4. 0.5% cotton blue in lactophenol.
5. H2O2.
6. Slides and coverslips.
7. Needles, forceps.
8. Distilled water.
9. Petri plates.
Procedure:
Same as above but depending on the colour of roots 10 or 20% KOH is used and this is kept for a longer period of at least one hour.
Measurement of Root Infection:
Requirements:
1. Stained segments of infected root.
2. Calibrated microscope.
3. Slides and cover slips.
4. Needles.
Procedure:
1. Select at random, stained segments of roots and mount them on microscopic slides in groups of ten.
2. Examine under the microscope and assess length of cortical colonization of VAM in µm for each segment.
3. Take the average of ten segments and express as percentage.
Mycorrhizal colonisation
= Number of root bits colonized / Total number of root bits analysed × 100
Gridline Intersect Method
Modified Method
Requirements
1. Petri dish 10 cm (4″) with a grid of lines marked at the bottom of the dish forming 1.2 cm (0.5″) squares.
2. Infected roots (softened with KOH) stained with acid fuchsin.
3. Lactophenol.
4. Microscope.
Procedure:
1. Place 1 cm pieces of roots, stained with acid fuchsin lactic acid solution, in Petri dishes that have gridlines marked at the bottom forming 1.2 cm squares.
2. Scan vertical and horizontal gridlines for the presence or absence of infection and record at each point where the roots intersect a line.
3. Calculate percentage of infectivity using Newman’s (1966) formula relating the total length of roots spread out in a given area to the number of times they intersect a number of straight lines placed randomly within this area.
Newman’s formula R = r × n / 2H
R = Total length of root.
K = The area in which roots are distributed.
n = Number of intersections between roots and straight lines and
H = The total number of straight lines.
% Infectivity = No. of infected roots /Total number of roots × 100
Most Probable Number (MPN):
Principle:
The inoculum potential of VAM spores is found out by this method where dilutions of the inoculum are made in sterile soil and host plants are grown.
Requirements:
a. Plastic pots 8 X 10 cm.
b. Sterile garden soil after two successive steaming at 90°C for 3-4 hours.
c. Test soil (with VAM spores).
d. A tube 1-2 cm in diameter.
e. Pregerminated jowar seeds.
f. Phosphorus deficient nutrient solution.
g. Lids to seal the pots.
Procedure:
1. Using sterilised garden soil dilute test soil (with VAM) to 2 to 10 fold. Sieve the mixture in order to mix them thoroughly and also to have an even distribution of VAM spores.
Dilutions of this are made as follows:
i. 5g sterile garden soil + 5 g diluent = 2-3 dilution
ii. 5g2-1 dilution +5g sterile garden soil = 2-2 dilution.
iii. 5g 2-2 dilution +5 g sterile garden soil = 2-3 dilution.
iv. 5 g 2-3 dilution +5 g sterile garden soil = 2-4 dilution.
v. 5 g2-4 dilution +5 g sterile garden soil . = 2-5 dilution.
vi. 5 g 2-4 dilution +5 g sterile garden soil = 2-6 dilution.
vii. 5g2-6 dilution +5 g sterile garden soil = 2-7 dilution.
2. Fill pots in replicates and controls with steamed garden soil and seal pots with lids.
3. Remove a 1-2 cm core of soil from the centre of the pot by vertically pushing tubes through a hole made in the centre of the lid.
4. Return approximately 3 g of the removed soil to the hole and 5 g of the diluted soil on top of it.
5. Return the remaining garden soil that was removed from the centre of the pot to the top of the diluted soil.
6. In controls only steamed soil is to be added in the holes.
7. Plant two pregerminated seeds into the centre of the hole and water pots to field capacity for 8 weeks.
The spores of Glomus (VAM) were added in such a fashion that the number decreases from 10-2 to 10-7 with increasing dilution. Hence there will be decrease in the biomass with decreasing spore number.
Identification of VAM Spores :
i. ELISA Method (EISA)
Requirements:
a. Freund’s complete adjuvant.
b. P04 buffer.
c. Carbonate coating buffer.
d. 0.05% Tween 20 in phosphate buffered saline (PBST).
e. Sterile saline.
f. lg G-Horse radish peroxidase-labellcd immunoconjugate.
g. Orthophenylenediamine.
h. H2S04.4N
i. Cultures of VAM in sterile sand grown host plant maintained in glass house with 16 hour light per day.
j. Incubator.
k. Polyvinyl microlitre trays.
l. Rabbits.
m. Needle and syringe.
n. Centrifuge.
Procedure:
1. Place roots of infected plant on a 250 µm sieve and wash with a strong jet of water (to remove external hyphae) into the 250 µm sieve.
2. Place sieving in a Petri plate and remove soil particles by observing under a sterioscan binocular.
3. Take hyphal mass, lyophilize, grind and store in desiccator at room temperature.
This will be approximately 20 mg (dry weight) of hyphae from a single heavily infected host.
4. Antigen when required for immunisation is prepared by resuspending 5 mg of powder in 1ml sterile saline and emulsifying it with equal volume of Freund’s complete adjuvant.
5. Inject antigen into white rabbits.
6. A booster dose of 2 mg is given after 3 weeks in sterile saline intravenously.
7. Take 5 ml of blood after seven days by cardiac puncture, allow the blood to clot and draw off the serum.
8. 5 ml of pre-immune serum serves as control.
9. For ELISA suspend 10 mg of powdered antigen in sterile distilled water, centrifuge at 15000 rpm for 20 minutes, discard surface lipids and lyophilize supernatant, suspend the powder in carbonate coating buffer (pH 9.6) at a concentration of 5 µg-ml-1.
10. Disperse 50 µl aliquots of the antigen suspension into polyvinyl microlitre trays. Allow it to dry at 37°C for 12 hours.
11. Wash tray three times with phosphate buffered saline containing 0.05% Tween 20 (PBST).
12. Make serial dilutions of serum in phosphate buffered saline containing 0.05% Tween 20 (PB$T) (50µl) and add in quadruplicates to the wells.
13. Seal the plates with tape, incubate for one hour at 37°C.
14. Repeat washing with PBST.
15. Add 50 µl of goat anti-rabbits Ig G-horse raddish peroxidase labelled immuno conjugate diluted to 1:1000 in PBST in each well and incubate at 37°C for 30 minutes.
16. Give a final wash to the trays and add 50 µl of orthophenylene diamine substrate.
17. Using NH2SO4 stop the reaction after 15 minutes.
18. Read wells on a spectrophotometer at 490 nm using normal rabbit serum as control.
19. From the absorbance of the test serum, the absorbance of control serum is to be deducted.
This gives specific reading.
20. Select a positive serum as reference to give an absorbance of 1.0. Compare other antisera to this reference.
Pure Inoculum Production of VAM:
Principle:
Since VAM cannot be grown in vitro, they are multiplied on the root system of host plants.
Requirements:
1. Seeds of host plant-Jowar.
2. Plastic pots 7 X 10 X 10 cm.
3. Sand (sterile).
4. Inoculum obtained by wet sieving and decanting.
5. Inorganic nutrient solution—Hoglands.
6. Starter inoculum.
7. Growth chamber.
Procedure:
1. Wash Jowar seeds under running tap water to remove pesticides.
2. Soak them over night and germinate.
3. Add a 3 cm layer of sand (sterile) to the clean plastic pots.
4. Then place 1 cm depth of substrate with VAM inoculum (starter).
5. Place maximum 5-6 seeds on top of the inoculum per pot.
6. Cover them with sand.
7. Irrigate pots with Hogland’s solution (composition given under N2 fixation) at the rate of 20 ml per pot.
8. Keep the pots at 28°-30°C (room temperature) with a photo period of 14 hours light and 10 hours dark with a light intensity of 100 lux supplied with fluorescent tubes for 3 weeks. Now prepare second phase with expanded clay.
Mass Production of VAM:
Principle:
The pure inoculum produced must be transferred to expanded clay along with roots to be supplied as VAM inoculum.
Requirements:
1. Plants grown for pure inoculum.
2. Ethanol 70%.
3. Na hypochlorite-2%.
4. Sterile expanded clay.
5. 5 litre pots (clay ones).
6. Growth room with photoperiod of 14 hours light and 10 hours dark.
7. P free medium (liquid) / Hogland’s solution.
Procedure:
There are several steps involved in the production of VAM on a large scale.
a. Separation of host plants:
Plants grown with pure inoculum in pots in the above experiment should be uprooted carefully.
b. Sterilisation of host plants:
Wash thoroughly the uprooted host plants and surface sterilise the root system of these uprooted plants to prevent the transfer of pathogenic organisms to the expanded clay. For this, dip the root system in 70% ethanol for 2 minutes or 2% Na hypochlorite solution. (This is optional for plants grown under controlled conditions in growth chamber or the ones that are upto three weeks old).
c. Preparation of expanded clay:
Either pasteurize or expose the clay to steam for 4 hours and cool to 20°C.
Place this in 5 litre or larger pots and plant two surface sterilised plants (Jowar) having maximum VAM colonisation in each pot carefully without damaging the root system and water the plants.
d. Parameters:
Five parameters are to be followed:
i. Optional illumination of host plant.
ii. Optional temperature range for fungi in the root.
iii. No water logging or drought.
iv. Plant nutrition should be optional for symbiosis.
v. Plants should be treated for pathogen.
Illumination:
In temperate countries mass production should be done in green houses with an intensity of 5000 Lux for 16 hours per day with sodium vapour high pressure lamps; 10,000 Lux or more by mercury vapour lamps for 14 hours. In tropical countries 1000 Lux supplied by fluorescent mercury vapour tubes for 14 hours per day is sufficient.
Temperature:
Pots should be shaded by placing them in wide well-aerated trays.
Irrigation:
Since expanded clay has larger particles on top and smaller ones below, more moisture will be present in the lower layers. To avoid this, use clay pots instead of plastic ones and cover them with thin plastic sheets with holes to allow exchange of gases.
Plant nutrition:
Modified Hogland and Arnon (1938)
Composition already given.
Treatment against pathogens Water, plants once a month. Treat with fungicide (Previeur 0.15%) to prevent pathogens like Pythium.
e. Growth period:
It takes about 3—4 months for mass production of inoculum in expanded clay. Stop irrigation, after checking if growth and colonisation is found good. A drought stress caused by stopping irrigation will induce formation of VAM spores.
f. Hence expose plants to drought for a week and then cut the plants.
g. After three weeks spread the substrate in a layer and air dry. Test for contamination.
h. Store the inoculum under cool dry conditions. They retain infectivity for several years.
Pot Cultures:
In pot cultures pure inoculum can be multiplied by inoculating the roots with one species of VAM.
Requirements:
1. Starter culture.
2. Potting mixture by Perrus etal. (1988) who introduced the concept of substrate receptiveness.
3. Allium porrum in pots with pre-inoculated propagules of Glomus having varying concentrations.
Procedure:
1. Prepare potting mixture of clay loam, calcareous soils and calcinised clay.
2. Medium should be disinfected to destroy indigenous VAM spores as well as pathogens by fumigation, irradiation, pasteurization or microwave treatment.
3. Plant Allium porrum in pots with various potting mixtures, having VAM (Glomus) propagules in varying concentrations.
4. Use P deficient nutrient solution to water pots.
5. Measure colonisation after 14—24 days. Relative growth rate will be more in clay loam, than calcareous soils and calcinised clay. Calcinised clay plus vermiculite mixtures will be more conductive than peat based mixtures. Peat based mixes supported colonisation level of VAM upto 25-44% of root length where P was minimum according to Caron and Patent (1988).
6. Recover soil with roots and spores, air dry and pack in polythene bags.
Aeroponic Cultures:
Nutrient solution is supplied as a spray to the root system that has no soil or any other material.
Requirements:
1. Culture chamber, waterproof box.
2. Plant-Ipomoea batatas.
3. Pure inoculum-precolonised plants with surface sterilised VAM spores grown in vermiculite for a short period.
4. Nutrient solution.
5. Pump or spray.
Procedure:
1. Make a hole on top of the chamber and introduce the plant with root system hanging into the chamber.
2. Adjust variable like concentration of nutrient solution, light, temperature and water quantity as in pot culture. P concentration 0.3 mM and pH 5.0-7.5.
3. Apply nutrient solution from a reservoir present at the bottom, to the root system as a mist or fog by pump/spray method.
Nutrient Film Technique:
Precolonised seedlings are placed on an inclined tray over which a layer of nutrient solution flows.
Soilless culture:
Sand culture.
Requirements:
1. Drip irrigation system.
2. Plastic pots (15 cm).
3. Hogland’s solution modified with 20 mM P04 and 0.5 mM MES buffer.
4. Host plants-need not be precolonised (Sorghum).
5. Inoculum (Glomus sp.).
Procedure:
1. Fill 15 cm pots with sand and add inoculum (Glomus sp.) to the sand.
2. Plant the host plants (Sorghum) in it.
3. Add 60 ml Hogland’s solution (each per pot).
4. Examine after 20—25 days, and cut the crown.
5. Air dry sand with spores and roots and use as inoculum.
Other methods:
1. Rock wool as substrate.
2. Polymeric hydrogel crystals soaked in water and mixed with vermiculite.