Manual on Arbuscular Mycorrhizal Fungus

[JM]

Published by the College of Tropical Agriculture and Human Resources (CTAHR) and issued in furtherance of Cooperative Extension work, Acts of May 8 and June
30, 1914, in cooperation with the U.S. Department of Agriculture. Andrew G. Hashimoto, Director/Dean, Cooperative Extension Service/CTAHR, University
of Hawaii at Manoa, Honolulu, Hawaii 96822. An Equal Opportunity / Affirmative Action Institution providing programs and services to the people of Hawaii without
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CTAHR publications can be found on the Web site <http://www.ctahr.hawaii.edu> or ordered by calling 808-956-7046 or sending e-mail to ctahrpub@hawaii.edu.
Soil and Crop Management
July 2003
SCM-5​

What are arbuscular​

mycorrhizal fungi?​

​

Mycorrhizal associations are formed by fungi. These associations should
not be confused with rhizobial associations,which are symbiotic associations
with bacteria that result in nitrogen-fixing nodules. Two major groups of fungi form
mycorrhizal associations: ectomycorrhizal fungi and endomycorrhizal fungi.
Ectomycorrhizas associate​

mainly with temperate-zone trees such​

as pine, poplar, and willow. These​

fungi form sheaths around their host’s​

root surfaces. Most ectomycorrhizas can be grown in

pure culture. ​

Endomycorrhizas form associations with​

most plants (approximately 80 percent of all plant species).​

These fungi cannot be grown in pure culture but​

must be grown in association with plant roots. They form​

branched structures called arbuscules within the host’s

root cells, and thus they are known as ​

arbuscular mycorrhizal​

fungi​

. The arbuscules are sites of nutrient exchange​

between the fungus and the host. This manual​

focuses on arbuscular mycorrhizal fungi.​

The associations that arbuscular mycorrhizal fungi​

form with plants are called symbiotic associations because

they are usually beneficial to both organisms. In

exchange for carbohydrates produced by the host through
photosynthesis, the fungi help the plant take up water
and immobile soil nutrients such as phosphorus (P), copper,
and zinc. The fungus extends from the plant root
and expands the volume of soil that the root system can
explore by itself.​

​

​

Manual on Arbuscular Mycorrhizal Fungus​

Production and Inoculation Techniques​

S.C. Miyasaka​

1, M. Habte1, J.B. Friday2, and E.V. Johnson1​

Departments of ​

1Tropical Plant and Soil Sciences and 2Natural Resources and Environmental Management​

To have beneficial associations​

between the fungus and plant roots, a​

low but sufficient ​

level of P in the soil​

or rooting medium is needed. If the​

soil P level is extremely low, the fungus​

can be parasitic (harmful to the​

plant) rather than beneficial, because

it will compete with the plant for

available P. When soil P is high (above
“sufficient”), the plant can obtain
enough P without the fungus, and the
association will not be formed.
For a more detailed explanation of
arbuscular mycorrhizal fungi, obtain
the CTAHR publication ​

Arbuscular​

Mycorrhizas: Producing and Applying Arbuscular Mycorrhizal​

Inoculum ​

by M. Habte and N.W. Osorio. This book​

may be purchased from CTAHR; obtain an order form at​

http://www.ctahr.hawaii.edu/oc/forsale/AMFflier.pdf​

.​

Why should plants be inoculated with​

arbuscular mycorrhizal fungus?​

In many tropical soils, P availability is limited due to Pfixation.​

Plants inoculated with arbuscular mycorrhizal

fungi in the nursery are better able to obtain P when

they are later planted into low-P soils. In addition, the
association helps the plant obtain water, which is critical
to plant survival and growth under dry conditions.
The amount of P fertilizer that needs to be applied
to a plant or crop is reduced when effective arbuscular
mycorrhizal associations have been formed. In addition
to the benefit of lowering fertilizer costs, reducing P
applications can help maintain environmental water
quality. Erosion of soil from fields with high P levels
Mycelia of an arbuscular mycorrhizal fungus
emerge from a root; the spherical bodies
are vesicles (fungal storage organs).
SCM-5 Arbuscular Mycorrhizal Fungus Production and Inoculation Techniques CTAHR — July 2003
2
often results in P enrichment of water bodies, which
causes excessive growth (“blooms”) of algae.
When should plants be inoculated with
arbuscular mycorrhizal fungus?
First, the particular plant species of interest must be able
to form effective associations with arbuscular mycorrhizal
fungi and be dependent on these associations for
nutrient exchange. A few plant families have species that
do not form mycorrhizal associations (e.g., Brassicaceae,
the mustard family, and Chenopodiaceae, the goosefoot
family). Table 1 lists selected food and tree crops that
are moderately, highly, or very highly dependent on associations
with arbuscular mycorrhizal fungi.
Second, an association between an arbuscular mycorrhizal
fungus and plant roots will be beneficial to the
plant when it is grown under low P or dryland (i.e., lowrainfall,
non-irrigated) conditions. If you are expecting
to plant seedlings in a soil that is known to be low in P
(e.g., volcanic ash soils along the Hamakua Coast of
Hawaii, or highly weathered red clay oxisols on Oahu
and Kauai) or in areas with low rainfall and no availability
of irrigation, then you should consider inoculating
seedlings in the nursery with an effective arbuscular
mycorrhizal fungus. For further information about soil
P in Hawaii, read ​

Predicting Soil Phosphorus Requirements​

by N.V. Hue, H. Ikawa, and X. Huang in ​

Plant​

Nutrient Management in Hawaii’s Soils: Approaches for​

Tropical and Subtropical Agriculture​

. The article is available​

on the Web at ​

http://www.ctahr.hawaii.edu/freepubs​

under the category Soil and Crop Management.​

Third, the presence of existing populations of mycorrhizal​

fungi in the field soil will determine whether​

you need to inoculate seedlings in the nursery. If the

soil contains a large population of arbuscular mycorrhizal

fungi that will form effective associations with
the plants you are growing, then you do not need to inoculate
seedlings in the nursery. If the soil has low populations
of indigenous mycorrhizal fungi, you should inoculate
your seedlings in the nursery.
Low soil P can occur because the soil layers rich in
P have been eroded. Low populations of indigenous
mycorrhizal fungi can occur when the soil has previously
supported either a predominance of plants that are
non-mycorrhizal (e.g., cabbage, broccoli) or plants that
are not dependent on mycorrhizal associations (e.g.,
kikuyugrass).
How can you tell if your plant is dependent on mycorrhizal
associations or if your site contains effective
arbuscular mycorrhizal fungi? One way is to inoculate
half of a batch of seedlings in the nursery with mycorrhizal
fungi and leave the other half uninoculated. Then,
observe the growth of the seedlings after planting in the
field. If there are visible differences, with inoculated
seedlings showing greater growth, then in the future you
should inoculate all the seedlings in the nursery. Alternatively,
you could take soil samples from at least three
locations within the field, mix them, place the mixture
in three 6-inch pots, plant corn seeds (or seeds of another
known host species, such as sorghum), and after
six weeks have a laboratory determine whether mycorrhizal
associations have developed. To determine the
presence of mycorrhizal associations, wash a sample of
fine roots from the nursery-grown plants and send it to
CTAHR’s Agricultural Diagnostic Service Center.
(Contact ADSC for information on service fees; call
808-956-5434 or e-mail ​

adsc@ctahr.hawaii.edu. Put the​

sample in a plastic bag with a damp paper towel to maintain​

moisture, and keep it as cool as possible. On Oahu,​

samples can be delivered to ADSC on the UH-Manoa​

campus at 1910 East-West Road, Sherman Laboratory,

Room 134. On Kauai, Maui, Molokai, or Hawaii, deliver

samples to the nearest office of the CTAHR Cooperative
Extension Service.)​

​

​

Table 1. Dependency of selected trees and food crops on arbuscular mycorrhizal associations (Miyasaka and Habte 2001).​

Moderately dependent Highly dependent Very highly dependent​

Acacia koa, koa Allium cepa, onion Leucaena leucocephala, koa haole

Acacia mangium Carica papaya, papaya Manihot esculenta, cassava

Colocasia esculenta, kalo, taro Senna siamea, pheasantwood Sophora chrysophylla, m​

ämane​

Gliricidia sepium, madre de cacao Coffea arabica, coffee​

Sesbania grandiflora, katurai Falcataria moluccana, albizia​

3​

SCM-5 Arbuscular Mycorrhizal Fungus Production and Inoculation Techniques CTAHR ​

— July 2003​

How is arbuscular mycorrhizal​

fungus inoculum produced?​

1. ​

Surface-sterilize seeds of a host plant, such as corn​

or sorghum, using a 10% solution of household bleach:​

1 volume of bleach (containing 5% sodium hypochlorite)​

plus 9 volumes of water. Soak the seeds in the solution​

for approximately 5–10 minutes.​

​

​

2. ​

Germinate the seeds by placing them between moistened​

paper towels on a plate placed in a loosely sealed​

plastic bag. Corn seeds will germinate within two to three​

days.​

​

​

3. ​

It is essential to select a medium with low phosphorus
.​

An appropriate nursery medium for producing​

arbuscular mycorrhizal inoculum contains a low level of​

P. Do not use a nursery medium that contains P fertilizer.​

If there is much P available in the planting medium, the

fungi will not form associations with the seedlings. Our

recommended medium is crushed basaltic rock with a
diameter less than ​

1⁄8 inch (available from some concrete​

or aggregate suppliers). This medium was selected because​

it contains very low levels of available plant nutrients​

(including P). Alternative media are quartz sand (not​

coral sand), available from some agricultural suppliers,

or a mixture of 1 volume of peat moss with 3 volumes of

either vermiculite, perlite, or volcanic cinder.​

​

​

4. ​

It is critical that the medium be sterilized to ensure​

good mycorrhizal infection​

. If it is not sterilized, there​

is a chance that microorganisms present in the medium​

will inhibit the formation of mycorrhizal associations.​

In an autoclave or pressure cooker, heat the medium for​

60 minutes. In a steam-sterilizer, heat it at 200​

°F for 60​

minutes. Let the medium cool and use it immediately.​

5. ​

Place the medium in 6–10-inch diameter pots. To prevent​

escape of medium, seal the large bottom holes with​

tape, but poke small holes to allow drainage of water.​

​

​

6. ​

Obtain a starter-culture of arbuscular mycorrhizal​

inoculum from a commercial source or researchers working​

on mycorrhizal fungi. A starter culture of ​

Glomus​

aggregatum ​

may be obtained from Dr. Mitiku Habte,​

CTAHR, but this is subject to availability. Thoroughly​

mix the culture into the medium at 1 volume of inoculum​

to 20 volumes of medium. Or, put ​

1⁄2 ounce (5–10​

grams) of inoculum into each planting hole when planting​

the germinated seeds.​

​

​

7. ​

Plant 2–6 germinated seeds in each pot. The object​

is to maximize root development in the pot so that the​

medium is completely filled with a mass of roots.​

​

​

8. ​

Place the pots in a greenhouse or in a well lighted​

area under a rain shelter to minimize contamination from​

microorganisms carried by rain or wind.​

​

​

9. ​

Water the plants once a day until water begins to drip​

from the bottom of the pot.​

10. ​

Apply 1 teaspoon per pot of a low-P, slow-release​

fertilizer, such as 19-5-12 Apex​

® Foliage Fertilizer or​

another low-P (e.g., 17-6-10), slow-release fertilizer. Or,​

make your own low-P fertilizer using the following​

recipe (adapted from a manual by J.N. Gemma and R.E.​

Koske, available on the Web at ​

http://www.hawaii.edu/​

scb/scinativ_mycor.html​

). Mix into 2 gallons of water​

1​

1⁄3 teaspoons (6 g) of Peters Professional® 15-0-15 fertilizer,​

1​

⁄5 teaspoon (0.9 g) of epsom salts (MgSO4), and​

1.5 milliliters of a concentrated phosphate fertilizer solution​

called Quick Start​

TM (4-12-4) made by Miracle-​

Gro​

®. (These materials are available from some garden​

shops and fertilizer companies). You can measure milliliters​

using a simple measuring spoon for liquid medicines​

obtainable at most drug stores.) Apply 1 cup of​

fertilizer solution once a week to each pot.​

​

​

11. ​

Associations between the host plant roots and the​

arbuscular mycorrhizal fungi should form in approximately​

six weeks, depending on temperature and sunlight.​

To confirm that mycorrhizal associations have​

formed, excavate several roots containing fine branches

after six weeks and send to CTAHR’s Agricultural Diagnostic

Service Center for determination of mycorrhizal
associations (see instructions for sending on page 2).​

​

​

12. ​

To produce a mycorrhizal inoculum containing​

spores, continue growing the host plant for 16 weeks.​

Withhold water beginning at 14 weeks after planting.​

At 16 weeks, remove the plant tops and discard them.​

Dump the medium and roots from the pots onto a clean

tray. Cut up the dried roots with scissors, mix the fragments

with the medium, and store this as a “crude inoculum”
in a refrigerator or a cool, dry location. It can
be stored for up to one year.​

​

​

SCM-5 Arbuscular Mycorrhizal Fungus Production and Inoculation Techniques CTAHR ​

— July 2003​

4​

How are plants inoculated in the nursery?​

1. ​

Surface-sterilize the seeds. For example, for koa seeds,​

soak them in a 10% solution of household bleach (1 volume​

of bleach plus 9 volumes water) for 5–10 minutes.​

For other plant species, you may need to try more dilute​

concentrations of bleach solution or a shorter soaking duration

to ensure surface-sterilization without harming the

seeds. Test a few seeds before treating large batches to be
sure that the treatment will not inhibit their germination.
For seeds that have seed-coat dormancy, additional
treatment may be needed to ensure good germination.
For example, koa seeds can be immersed in hot water
removed from the stove after it reaches boiling, and
soaked overnight.​

​

​

2. ​

Germinate the seeds between moistened paper towels​

on a plate enclosed in a loosely sealed plastic bag.​

Alternatively, if you have developed an effective surface​

sterilization technique, you could plant ungerminated​

seeds directly into the potting media.​

​

​

3. ​

Selection of a medium with low P is critical to successful​

development of mycorrhizal fungus associations​

with plant roots​

. We have successfully used a mixture​

of 1 volume of volcanic ash soil (a highly P-fixing​

soil collected along the Hamakua Coast) with 1 volume​

of Sunshine​

® Mix #2 from Sun GroTM, or a mixture of 1​

volume of volcanic ash soil with 1 volume of Sunshine​

Mix #2 and 1 volume of crushed basaltic rock. Or, use 1​

volume of peat moss (for example, Sunshine Mix #2)​

mixed with 3 volumes of either vermiculite, perlite, or

volcanic cinder.​

​

​

4. ​

Sterile medium is critical to success in developing​

mycorrhizal fungus associations with plant​

roots​

. To prevent pathogen problems or competition​

from other microorganisms, sterilize the nursery​

medium in an autoclave or pressure cooker for​

60 minutes. Or, use a steam-sterilizer and raise the​

temperature of the medium to 200​

°F for 60 minutes.​

In the case of soil, let it cool and aerate for 2–​

7 days to allow release of possible toxins generated​

by heating.​

​

​

5. ​

Inoculate the nursery medium with arbuscular​

mycorrhizal inoculum at a rate of 1 volume of inoculum​

to 20 volumes of medium.​

​

​

6. ​

Plant surface-sterilized seeds in pots containing​

the sterilized medium. Or, put ​

1⁄2 ounce (5–10​

grams) of inoculum into each planting hole before sowing​

the seeds.​

​

​

7. ​

Water daily until water begins to flow out of the bottom​

of the pot.​

8. ​

Apply a low-P, slow-release fertilizer such as 19-5-​

12 Apex​

® Foliage Fertilizer or another low-P (such as​

17-6-10), slow-release formulation. Alternatively, make​

your own low-P fertilizer as described earlier (Step 10​

under ​

How is arbuscular mycorrhizal fungus inoculum​

produced?​

). The amount of fertilizer applied depends​

on your plant species and pot size. You will need to determine​

the proper amount through careful observation​

of plant growth. For example, for koa seedlings grown​

in Super Cell tubes (10 cubic inches, #SC-10, Steuwe

and Sons, Inc.), we applied 10 milliliters per tube per

week.​

​

​

9. ​

After six weeks, confirm presence of arbuscular mycorrhizal​

fungus associations by submitting root samples​

to ADSC as described on page 2.​

​

​

References​

Miyasaka, S.C., and M. Habte. 2001. Plant mechanisms and mycorrhizal​

symbioses to increase phosphorus uptake efficiency.

Commun. Soil Sci. Plant Anal. 32: 1101–1147.

Habte, M., and N.W. Osorio. 2001. Arbuscular mycorrhizas: producing
and applying arbuscular mycorrhizal inoculum. CTAHR,
Univ. of Hawaii, Honolulu. 47 pp.
Silva, J.A., and R.S. Uchida (eds.). 2000. Plant nutrient management
in Hawaii’s soils: approaches for tropical and subtropical
agriculture. CTAHR, Univ. of Hawaii, Honolulu. 158 pp.
Gemma, J.N., and R.E. Koske. <www.hawaii.edu/scb/
scinativ_mycor.html>​

Corn in a 10-inch pot ready to be harvested for inoculum.
The koa at right grew better in a low-P​

soil when inoculated with fungus

​

​

.​

from: http://www.ctahr.hawaii.edu/oc/freepubs/pdf/SCM-5.pdf​