On farm production of Mycorrhizae

Yanglem Herojit Singh, K Mamocha Singh, Kennedy Ninthoujam, Ng Taibangnganbi Chanu *

Mycorrhiza are symbiotic relationships between fungi and roots of higher plats and such fungus-roots are called mycorrhizae. Perhaps more than 80% plant species are having mycorrhizal association. The exceptions are many members of the Cruciferae family (e.g., broccoli, mustard), and the Chenopodiaceae family (e.g., lambs quarters, spinach, beets), which do not form mycorrhizal associations.

The level of dependency on mycorrhizae varies greatly among varieties of some crops, including wheat and corn. Mycorrhizae increases the root’s ability to absorb the nutrients (phosphorus (P), zinc (Zn), manganese (Mn), copper (Cu)) and water from the soil by increasing the surface absorbing area of roots from 100 to 1,000 times.

All the cultivated plants remove large quantity of nutrients from soil, particularly NPK. Among these major nutrients, the efficiency of applied P through chemical fertilizers is very low i.e., 15 to 20%. This is due to fixation of applied phosphorous in the soil into unavailable form. Therefore, there is need to enhance the phosphorous availability by using phosphorous solubilising microorganisms (PSM).

Among PSM, different species of mycorrhizal fungi have been reported to be effective in increasing growth and uptake of phosphorous by different plant species. Mycorrhiza filaments extend from the roots into the soil, reaching several times beyond the root hairs. Nutrients are taken up by the hyphae and carried to the plant.

Types of Mycorrhizae

On the basis of morphological and anatomical features, mycorrhizae are divided into three types:
a) Endomycorrhizae
b) Ectomycorrhizae
c) Ectendomycorrhizae

Endomycorrhizae

It is a mycorrhizal association in which the fungal hyphae are present on root surface as individual threads that may penetrate directly into root hairs, other epidermal cells and into cortical cells.

Endomycorrhizae further classified into five types:
i. VAM fungi (vesicular arbuscular mycorrhizae)
ii. Orchidoid mycorrhizae
iii. Monotropoid mycorrhizae
iv. Ericoid Mycorrhizae
v. Arbutoid mycorrhizae

VAM fungi (vesicular Arbuscule mycorrhizae)

o Fungi formed VAM association with plants may belong to Zygomycetes.
o All VAM fungi are obligate biotrophic, as they are completely dependent on plants for their survival.
o Some of the fungi forming Endomycorrhizae are Endogone, Glomus, Sclerocystis, Gigaspora etc.

Ectomycorrhizae

o Ectomycorrhizae (ECM) are association, where fungi form a mantle around roots.
o There is no hyphal penetration of cells.
o Fungal hypha is generally separate.
o A distinct Hartig’s net is present between the cells

Ectendomycorrhizae

The fungi belong to Basidiomycotina, which covers both gymnosperms and Angiosperms plants. Ectendomycorrhizae show many of the same characteristics of Endomycorrhizae but also show extensive intercellular penetration.

Mass Production

Obligate symbionts AM fungi could be mass produced only in the presence of living roots. There are several techniques reported for mass production of AM inoculums.

Aim of “On Farm Production Technique”

o To ensure high quality AM inoculums in large quantities at farmers field
o Very little cost technology.

Method of Mycorrhiza production

a) Pit method
A nursery area of about 75 m2 (5 m x 15 m) is selected and split into 3 beds of equal sizes (0.8 m x 15 m).

Key points in procedure

o Development of mycorrhizal starter culture prepared by either isolating resident A fungi or by procuring commercial starter culture.
o Multiplying the culture on trap plant roots at farmer’s fields.

Steps involved
Selection of site

o The site should be water logging free, levelled.
o Low in available phosphorous.
o The site is prepared by ploughing and hoeing to make raised beds (80 cm wide, 20 cm high and 15 m length) with a spacing of 0.6-0.8 m between rows.
o The soil in these beds needs to be amended with well decomposed compost/vermicompost @ 4 kg m-1 or fine powdered FYM @ 8 kg m-1.

AM fungal inoculation

o AM fungal inoculum @ 8,000 infective propagules m-2 is placed 2-3 cm deep in furrows just below the seed at the time of seed sowing.
o Direct seeded crops like methi, maize, sorghum, marigold, barseem etc., can be used as trap plants.
o Transplanted crops like tomato, brinjal, onion etc., can also be used as trap plants in between routine multiplication cycles.

Multiplication

o The multiplication of AM fungi generally coincides with the host cycle comprising of kharif, rabi and summer cropping seasons.
o Regular hoeing, weeding, watering, and protection from animals should be done during the multiplication cycles.
o There should not be any water logging in the nursery beds especially during rainy season.

Harvesting of inoculum

o At the end of each cycle, the shoot of the trap plants are removed from the soil surface without disturbing the roots, the roots need to be retained in the beds but the soil should be hoed or loosened during second cycle of multiplication and so on.
o After 5 multiplication cycles the plants are uprooted gently to get maximum root biomass.
o The roots with adhering soil are then chopped finely and thoroughly mixed.
o This mixture is tested for percent root length mycorrhizal colonization and analysed for number of infectious propagules (IP) per g inoculum using standard protocols.

b) Pot method
Methodology

o Take required quantity of soil from own field to fill up the pots. Sterilize soil to minimize the presence of other fungus or pathogens in soil by heating for 2-4 h using a big metal pan or by drying under intense heat of the sun for 2-3 days.
o After cling, place the sterilized soil in thoroughly clean dry pots. For large scale production polythene bags, trough lined with plastic sheet may be used.
o Place a pinch of root mother culture in the pot and then cover with a thin layer of soil.
o Sow 3-5 seeds in each pot at such a depth that seedling roots should come in contact with culture.
o Grow the plants for three months under normal conditions.
o Stop watering the plants after 3 months and cut the upper part of plants or stalks when they are completely dried. Allow the soil in the pot to dry further.
o Remove the roots along with adhering soil from the pots and dry in shade.
o Cut the roots finely and save some root inoculants for future use. Mix the fine cut roots with the soil from the pot to produce VAM soil culture.
o Store the root and soil inoculants in sealed plastic bags in cool dry place.

Precautions for Production of Mycorrhiza

o Compulsory use quality Mycorrhiza mother culture for production (100 infective propagules per g)
o Create physical contact between the mycorrhizal culture and the plant root
o Care should be taken during plants grow

Benefited Crops

Mycorrhiza or VAM suitable for all cereals, legumes, fruits and horticultural crops

Benefits of Mycorrhiza

o Produce more vigorous and healthy plants. Increase plant establishment and survival at seedling or transplanting.
o Enhance flowering and fruiting.
o Increase yields and crop quality.
o Improve drought tolerance, allowing watering reduction
o Optimize fertilizers use, especially phosphorous
o Increase tolerance to soil salinity.
o Reduce disease occurrence.
o Contribute to maintain soil quality ad nutrient cycling
o Contribute to control soil erosion.

Application of VAM fungi

Nursery application

o 100 g bulk inoculum is sufficient for one m2
o The inoculum should be applied a 2-3 cm below the soil at the time of sowing.
o The seeds/cuttings should be sown/planted above the VAM inoculum to cause infection.
For polythene bag raised crops
o 5 to 10 bulk inoculums is sufficient for each packet.
o Mix 1 kg of inoculum with 100 kg of potting mixture and pack the potting mixture in polythene bag before sowing.

For out-planting
o 20 g of VAM inoculum is required per seedling
o Apply inoculum at the time of planting.

For existing trees
o 200 g of VAM inoculum is required for inoculating one tree. Apply inoculum near the root surface at the time of fertilizer application

Application Dose: 3-4 kg acre-1

Economics of AMF Production

o In this on-farm production technique, the left over rhizospheric soil (after harvesting of roots) contains fairly good number of infectious propagules (more than 10 IP g-1 soil) and can be used to continue subsequent production cycles, to produce 10 lakhs propagules of AM fungi during every cropping season.
o The average cost of production is Rs. 400.00 during each production cycle for producing sufficient inoculum for application in one ha.

Assessment of Root Colonization

o Wash the surface feeder roots and cut the roots up to 1 cm.
o Heat the roots at 700C for 10-15 min (depend upon the age and tenderness of the roots) with 10% potassium hydroxide (KOH) solution.
o Pour off the KOH solution and rinse the roots thoroughly with water.
o Acidify the roots by immersing in 5% HCl for 5 min.
o Pour out the HCI and add staining solution (0.05% lactophenol tryphan blue) and keep them overnight for staining or boil for 10-15 min at 700C.
o Destain the roots with destaining solution (50% glycerol).
o Mount the root pieces on the glass slides and observe under microscope for mycorrhizal colonization.

Micorrhizal colonization is expressed using the following formula: Percentage of Colonization = Number of root segments with Mycorrhiza x 100.