Characteristics and Production

Arbuscular
Technology
Arbuscular Mycorrhizae
Mycorrhizae Fungi
Fungi

Sushil K. Sharma
Principal Scientist (Ag. Microbiology)
2nd July, 2025
CONTENTS :
•Introduction
•Characteristics
•Types of mycorrhizae
•AM Spore germination
•Isolation and mass production
•Application rates
•Conclusion
•References
 Introduction
 Mycorrhizae are the symbiotic associations between plant root and
fungi, with bidirectional nutrient exchange between the partners.

 Vitadini (1842) was the first to recognize the possible beneficial role of
fungal mycelia that mantle the root of higher plants.

 This association is named as mycorrhiza (pl. mycorrhizae), i.e., the

fungal root, by Albert Bernard Frank (1885).

 The autotrophic host plant acts as the carbon source for the fungus,
while the fungus supplies mineral nutrients to the plant. About 90% of
all land plants are associated with mycorrhiza. The mycorrhizal
association is not available in Cruciferae, Chenopodiaceae, Resedaceae
and Hydrophytes.
 Mycorrhizal symbiosis is the most prevalent and
important mutualistic association in the plant
kingdom.

 Frank, a German Botanist (1885), was the first

person to use the term “Mycorrhiza”.

 A mycorrhiza (from Ancient

Greek μύκης (myco) 'fungus' and ῥίζα (rhíza) 'root';
pl. mycorrhizae, mycorrhiza, or mycorrhizas) is
a symbiotic association between a fungus and
a plant.

 Both paleobiological and molecular evidence

indicate that AM is an ancient symbiosis that
 Characteristics of Mycorrhizae
(i) Absence of any phytopathological symptoms in the partners during
the active phase of mutualism.

(ii) Presence of complex interfaces between cells of the partners with a

predominant type of perisymbiotic membrane, surrounding intracellular
symbionts.

(iii) Presence of various types of phagocyte-like structures during the

establishment of symbionts and during harvesting phase to control the
symbiotic population by the host.
 Types of Mycorrhiza
Peterson andFarquhar (1994) classifiedthe mycorrhizae into seven (7) distinct
types.
These are :
(1) Ectomycorrhizae
(2) Vesicular-arbuscular mycorrhizae
(3) Ectendomycorrhizae (Arbutoid)
(4) Ericoid mycorrhizae
(5) Centianoid mycorrhizae
(6) Orchidoid mycorrhizae
(7) Monotropoid mycorrhizae
Ectomycorrhizae
Ectomycorrhiza is commonly called “sheathing mycorrhiza”. They occur in 3% of all
seed plants in forests of temperate regions, especially on pine, beech, spruce, birch
etc.
Vesicular-arbuscular mycorrhizae (VAM)
• It is a type of endomycorrhizal association, where both vesicles and arbuscules are
developed together. VAM is by far the commonest of all mycorrhizae and has been
reported in more than 90% of land plants.
• They are found in bryophytes, pteridophytes, gymnosperm (except Pinaceae) and
most of angiosperms, commonly in Leguminosae (Fabaceae), Rosaceae,
Gramineae (Poaceae) and Palmae (Arecaceae). VAM has even been reported in the
Lower Devonian plant, Rhynia.
• VAM is produced by aseptate mycelial fungi that belong to Endogonaceae under
Mucorales of Zygomycotina and those members produce zygospores. The
important genera involved in VAM are Glomus, Gyrospora, Acaulospora, etc. Most
of the members are not cultivable.
Arbuscular mycorrhizae (AM)
Structures
AMF Spore Germination & Infection
 Spores: These are the "fruiting bodies" of the fungi and
are formed both inside the roots and externally in the soil.

• Spores of diameters
ranging from 50 to 400
μm

• Depending upon the

season and conditions,
spores can make up a
significant amount of
mycorrhizal biomass.
AMF structures in legume roots observed under a binocular microscope. A)
Spores, B) Intraradical hyphae, C) Vesicles, D) Arbuscules, E) MTT for viable
spores.
 Arbuscule Formation by Arbuscular Mycorrhizal Fungi (AMF) in
Root Cortex (Light Microscopy)

An arbuscule of Glomus Arbuscule of Gigaspora margarita Developing arbuscule of

versiforme in a root cortex cell with an elongated trunk hypha (T) Glomus mosseae in a root
with branch hyphae densely and tufts of fine branch hyphae cell with fine branch hyphae
packed in the cortex cell of the (arrows) (arrows). The trunk (T) of
host this arbuscule branched
from an intercellular hyphae.
 FUNCTIONS
Mycorrhiza refers to an association or symbiosis between plants and fungi that
colonize the cortical tissue of roots during periods of active plant growth.

The association is characterized by the movement of plant-produced carbon to

the fungus and fungal-acquired nutrients to the plant.

Out of these, arbuscular mycorrhizae (AM) are commonly used in moisture-

deficient soils, and nutrients taken up by the extramatrical hyphae can lead to
improved plant growth and reproduction.

As a result, mycorrhizal plants are often more competitive and better able to
tolerate environmental stresses than are non-mycorrhizal plants.
 FUNCTIONS
• The AM fungi are important to their hosts as they enhance the ability of plants to
absorb phosphorus from soil, which is relatively inaccessible to the plants.
• However, the AM association may also increase the phytoavailability of
micronutrients, e.g., copper and zinc.
• In a study, absorption of trace elements, such as boron and molybdenum, was found
to be enhanced by VA mycorrhizae.
• In addition, it has been suggested that some AM associations can mobilize
organically bound nitrogen (Arginine), which the plants are unable to absorb.
• Symbiotic association of AM fungi with the plants seems to influence the
composition of bacterial communities in the mycorrhizosphere due to changes in
root exudation patterns induced by AM colonization.
Mycorrhizal
hyphae (1)
interconnect
roots with soil
particles (3),
provide direct
connections of
root systems of
different plant
individuals (2),
and interact with
several soil
microbes (4).
FACTS……….
Though AM fungi have enormous potential and practical utility in agriculture,
horticulture, and forestry, the main constraint is the lack of ability to grow in
artificial media.

The common genera of AM fungi include Glomus, Gigaspora, Acaulospora,

Sclerocystis, and Scutellospora.
AM fungi exist in soil as chlamydospores.

AM cultures will be isolated from the soil by collecting the chlamydospores
present in the soil.

The spores are collected from soil using the wet sieving and decantation
technique described by Gardemann and Nicolson (1963).
• The commercial utilization of mycorrhizal fungi has become difficult because of
the obligate symbiotic nature and difficulty in culturing on laboratory media.

• Production of AM inoculum has evolved from the original use of infested field
soils to the current practice of using pot culture inoculum derived from the
surface disinfected spores of single AM fungus on a host plant grown in
sterilized culture medium.

• Several researches in different parts of the world resulted in different methods

of production of AM fungal inoculum as soil based culture as well as carrier
based inoculum.

• Root organ culture and nutrient film technique provide scope for the production
of soil less culture.
Culturing of AMF
• The obligate symbiotic nature of the fungus makes axenic cultivation
an important challenge for both scientific and practical point of view.
Inability to culture AM in the laboratory is the major limiting factor in
their application in agriculture. Though AMF has very broad specificity
towards plants including various agricultural horticultural and forestry
plant species, but the ability to produce AM in bulk quantities is a major
bottleneck. AM biofertilizer is currently recommended only for
transplanted and nursery raised crops because of the difficulty in
inoculum production as well as the bulk requirement of the inoculum.
• Various methods were developed for mass production of AM fungi
world wide.
i) Isolation
A ) Sieving method :

Soil sample + sterile water

Hot water

Filter and sieve

( 719μm → 250μm → 50μm → 45μm )

Spores separated from soil particles

Mix with carrier material

Use when required as biofertilizer

wet sieving and decantation technique described by Gardemann and Nicolson

(1963).
IV. Observation Under Stereo Zoom Microscope
Materials Required:


Stereo zoom microscope (40–100x)


Petri dishes


Fine forceps


Needle probe
Relatively small white
spores of a Glomus
species.

Sporocarp of Glomus
invermaium typical of the
dead spores often found
in field-collected soil.
Genus Spore Shape Wall Pigmentation Image
Size Characteristics
(µm)
Glomus 80–15 Globose, 1–3 layers, Hyaline to
0 subglobos smooth or yellow-
e slightly brown
ornamented
Acaulospora 100–2 Globose to Multiple wall Yellow to
50 ovoid layers; brown
laminated
walls; no
subtending
Genus Spore Shape Wall Pigmentation Image
Size (µm) Characteristics

Gigaspora 200–450 Globose, Thick-walled Yellow to

ellipsoid spores with orange-
bulbous brown
sporogenous
cell
Scutellospora 150–400 Globose, Multiple Brown to dark
subglobos layered wall brown
e with
germination
shield; often
Genus Spore Shape Wall Pigmentatio Image
Size Characteristics n
(µm)
Claroideoglomu 80–16 Globose Thin to Hyaline to
s 0 moderately light yellow
thick wall;
smooth
surface;
straight hypha
Diversispora 120–2 Variable Multilayered Pale to dark
50 (globose wall; often brown
to finely
B ) Floatation method :

Soil sample + sterile water

Separate the soil particles using

membrane filter

Centrifuge
( Density gradient centrifuge = at
3000rpm
for 30 min )

Spores separated from soil particles

Mix with carrier material

Use when required as biofertilizer

In-Vitro Culture/Axenic Culture
Techniques
i)Solution culture
• Involves growing infected roots in aqueous medium enriched
with mineral nutrients required for the growth of the roots
under controlled biotic and abiotic conditions.

ii)Aeroponic culture
• Involves applying a fine mist of nutrient solutions to colonized
roots for AM fungal inoculum production.
iii)Root organ culture
• Use of a modified agar
medium (MS rooting
medium)/ liquid
medium for creation of
increased amount of
roots from callus tissue
and these roots are
infected by AM spores
or by surface sterilized
root bits obtained from
mycorrhizal plant.
Taxonomic Rank Classification
Kingdom Fungi
Phylum Glomeromycota
Subphylum Glomeromycotina
Class Glomeromycetes
Order Glomerales, Diversisporales, Paraglomerales,
Archaeosporales
Families Glomeraceae, Gigasporaceae, Acaulosporaceae,
Diversisporaceae, Claroideoglomeraceae,
Paraglomeraceae, Archaeosporaceae
Genera Glomus, Funneliformis, Claroideoglomus,
Acaulospora, Gigaspora, Scutellospora, Diversispora,
Paraglomus, Archaeospora
Common
Hosts
Mass production of Mycorrhizal biofertilizer
 MASS PRODUCTION

1. Tank for mass multiplication 2. Sprinkling of water in tank 3. Making of furrows to sow maize
of AM with vermiculite seeds
 MASS PRODUCTION

5. View of the maize sown 6. Vermiculite contained raised AM

4. Sowing the seeds in furrows
AM pit infected maize plants
Multiplication under soil less substrate
 Applied 20g urea, 20g super phosphate and 10g muriate of potash for
each trench at the time of sowing seeds. Further 10g urea is applied twice
on 30 and 45 days after sowing for each trench.
 Quality test on VAM colonization in root samples is carried out on 30th
and 45th day.
 Stock plants are grown for 60days (8 weeks). The inoculum is obtained by
cutting all the roots of stock plants. The inoculum produced consists of a
mixture of vermiculite, spores, pieces of hyphae and infected root pieces.
• As a carrier based inoculum, pot culture is widely adopted method for production.

• The AM inoculum was prepared by using sterilized soil and wide array of host
crops were used as host.

• The sterilization process is a cumbersome one and scientists started using inert
materials for production of AM fungi.

• The researchers tried use of perlite, montmorillonite clay, vermiculite as

substrate for the replacement of soil sterilization, which resulted in the best
method of inoculum production.
 MASS PRODUCTION

A trench (1m x 1m x 0.3m) is formed and lined with black polythene sheet to be
used as a plant growth tub.


Mixed 50 kg of vermiculite and 5 kg of sterilized soil and packed in the trench up
to a height of 20 cm


Spread 1 kg of AM inoculum (mother culture) 2-5 cm below the surface of
vermiculite


Maize seeds surface sterilized with 5% sodium hypochlorite for 2 minutes are
sown


Applied 2 g urea, 2 g super phosphate and 1 g muriate of potash for each trench
at the time of sowing seeds. Further 10 g of urea is applied twice on 30 and 45
 MASS PRODUCTION


Quality test on AM colonization in root samples is carried out on 30th and 45th
day


Stock plants are grown for 60 days (8 weeks). The inoculum is obtained by
cutting all the roots of stock plants. The inoculum produced consists of a mixture
of vermiculite, spores, pieces of hyphae and infected root pieces.

Thus within 60 days 55 kg of AM inoculum could be produced from 1 sq meter area.

This inoculum will be sufficient to treat 550 m2 nursery area having 11,000
seedlings.
Mycorrhizal products are available commercially……
Application of AM fungi
 Nursery application: 100 g bulk inoculum is sufficient for one metre square. The
inoculum should be applied at 2-3 cm below the soil at the time of sowing. The seeds/
cutting should be sown/planted above the VAM inoculum to cause infection.

 For polythene bag raised crops: 5 to 10 g bulk inoculum is sufficient for each packet.
Mix 10 kg of inoculum with 1000 kg of sand potting mixture and pack the potting
mixture in polythene bag before sowing.

 For out –planting: Twenty grams of VAM inoculum is required per seedling. Apply
inoculum at the time of planting.
For existing trees: Two hundred gram of VAM inoculum is required for inoculating one
tree. Apply inoculum near the root surface at the time of fertilizer application.
Mycorrhizal Application Rates
• Row Crop Application : side dress seed furrows or transplants @ 6 to 9 kg/ha.
Incorporate at 2-6 inches (5 to 15cm) below soil surface.

• Seed Drilling: incorporate into the soil at a depth below the seed.

• Broadcast and Till: Evenly distribute across seedbed after seeding. Cover the
exposed seed and inoculum by harrowing , chain dragging or applying an
organic topdressing .

• Nursery Medium: Evenly blend @ 3 kg/ m3.

• Do not leave inoculum exposed to sunlight.

 Benefits
• Increase P & N Nutrition and other elements: N, K, Ca, Mg, Zn, Cu, S, B, Mo,
Fe, Mn, Cl
• Increase diversity of plant.
• Significant role in nutrient recycling.
• More tolerant to adverse soil chemical constraints which limit crop
production.
• Increase plant resistance to diseases and drought. Stimulate the growth of
beneficial microorganisms. Improve soil structure.
• Stable soil aggregate – hyphal polysaccharides bind and aggregate soil
particles.
• Increases absorption of phosphate by crops. uptake of zinc also increases.
• Increases uptake of water from soil. Increases uptake of sulphur from the
soil
 Benefits
• Increases absorption of phosphate by crops. uptake of zinc also increases.
• Increases uptake of water from soil. Increases uptake of sulphur from the
soil
• Increases the concentration of cytokinins and chloroplast in plants.
• Produce more vigorous and healthy plants.
• Increase plant establishment and survival at seedling or transplanting.
• Enhance flowering and fruiting.
• Increase yields and crop quality.
• Optimize fertilizers use, especially Phosphorus.
• Contribute to maintain soil quality and nutrient cycling.
• Contribute to control soil erosion.
THANK YOU