© 2004 Plant Management Network.

Accepted for publication 14 January 2003. Published 1 March 2004.

Arbuscular Mycorrhiza Inoculum to Support

Sustainable Cropping Systems
Yolande Dalpé, Agriculture and Agri-Food Canada, Research Branch,
960 Carling Avenue Ottawa, Ontario, K1A 0C6; and Marcia Monreal,
Agriculture and Agri-Food Canada, Research Branch, P.O. Box 1000A,
Brandon, Manitoba, R7A 5Y3

Corresponding author: Yolande Dalpé. [email protected]

Dalpé, Y., and Monreal, M. 2004. Arbuscular mycorrhiza inoculum to support sustainable
cropping systems. Online. Crop Management doi:10.1094/CM-2004-0301-09-RV.

Abstract
Arbuscular mycorrhizae (AM) are symbiotic associations, formed between plants
and soil fungi that play an essential role in plant growth, plant protection, and soil
quality. The AM fungi expand their filaments in soil and plant roots. This
filamentous network promote bi-directional nutrient movement where soil
nutrients and water move to the plant and plant photosynthates flow to the
fungal network. AM fungi are ubiquitous in the soil and can form symbiosis with
most terrestrial plants including major crops, cereals, vegetables, and
horticultural plants. In agriculture, several factors, such as host crop dependency
to mycorrhizal colonization, tillage system, fertilizer application, and mycorrhizal
fungi inoculum’s potential can affect plant response and plant benefits from
mycorrhizae. Due to their obligate symbiotic status, AM fungi need to associate
with plant for growth and proliferation. Consequently, the cultivation of AM fungal
strains and the maintenance of reference collections require methodologies and
infrastructures quite different from those used with other microbial collections and
inoculum production. Interest in AM fungi propagation for agriculture is increasing
due to their role in the promotion of plant health, in soil nutrition improvement,
and soil aggregate stability. The comprehensive life cycle of AM fungi and
methods currently used for the propagation of inoculum and the maintenance of
in vivo and in vitro source collections are described. Methods and regulations of
large-scale production of commercial inoculum that provide users with products of
high quality and efficiency are discussed.

Introduction
Arbuscular mycorrhizae (AM) are symbiotic associations formed between
plants and soil fungi that benefit both partners. The phytobiont correspond to
approximately 80% of plant species and the fungi are classified in the phylum
Glomeromycota, including nine genera; Glomus, Paraglomus, Sclerocystis,
Acaulospora, Entrophospora, Gigaspora, Scutellospora, Diversispora,
Geosiphon, and Archaeospora (41). AM fungi (AMF) are ubiquitous in the soil
with around 170 described species (46). The symbiosis is called “arbuscular”
because the fungi form specialized tree-like structures (arbuscules = tree-like)
inside root cells. Other structures produced by fungi are intra- and extraradical
spores (which are germinating structures useful for long-term preservation of
species, propagation, and species identification purposes), intraradical hyphae,
extraradical hyphae, intracellular fungal storage structures called vesicles (which
are lipid containing bodies) and, for some genera, auxiliary cells branching from
extraradical hyphae. Intraradical AM fungal mycelium form a network around
and inside cortical cells of plant roots, extraradical AM mycelium can spread
throughout the soil surrounding the root system and increase the ability to
explore soil areas, accessing water and nutrients for plant roots. Benefits to
plants are improved water and nutrient uptake, enhanced P transport, and
drought and disease resistance. Benefits to fungi are the supply of
photosynthates to the fungal network located in the cortical cells of the plant
and the surrounding soil. All water, nutrients, and photosynthates exchanges
occur via the fungal filament network that bridged plant rhizosphere and plant
roots.

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 AM Interaction with Soil and Crops
The increased capacity of plant roots for water and nutrients uptake from the
soil when colonized by AMF is the main mechanisms proposed to explain the
effect of AM in plant performance. This behavior is particularly evident with soil
nutrients that are more immobile such as phosphorus (P), zinc (Zn), and copper
(Cu) . Improved phosphorus nutrition when colonized with AMF has been
demonstrated for hundreds of cultivated plants. By extending past the P-
depletion zone formed around the root systems, the fungal soil network is able
to maintain P transport to plant for longer periods (19,21,27). Under high P soil
conditions, AMF are almost of no use to the plants and the symbiosis is
temporarily inhibited. As such, a reduction in P applications is recommended in
order to stimulate and maintain symbiosis efficiency.
Most agricultural crops such as flax, corn, sorghum, wheat, barley, potatoes,
and sunflower can benefit from mycorrhizal association. Some other crop plants
do not form AM symbiosis; those belong to the Cruciferae, Brassicaceae,
Chenopodiaceae, and Caryophyllaceae families (3). Canola (Brassicaceae
family), an important crop in western Canada does not form AM. Efficiencies
and limitations of registered mycorrhizal inoculum, in terms of the cultivated
crops, are clearly posted on sale products together with recommendation for
use.

Interaction of AM and Agricultural Practices

Agricultural practices such as fertilizer applications, crop rotation, tillage,
and liming affect field AM potential and root colonization levels. For example,
high levels of P fertilization have been found to slow down or inhibit mycorrhizal
efficiency in soybean fields (12). Cropping of a soil with canola, a non-host plant
species, delayed mycorrhiza development of maize and of flax (15; Monreal et
al., unpublished data). Higher soil infectivity was observed under reduced or no
tillage practices (31) and liming increased mycorrhizal colonization of barley
roots and soil infectivity (17). Plant species differ in their fertilization
requirements, and consequently their dependency on AMF vary considerably
from one crop to another (33). For example, under field conditions, beans, corn,
and leek have a much higher mycorrhizal dependency than potato and wheat.
This range of plant response to AMF has to be taken into account when
managing a cropping system or a crop rotation. Data on the potential of crop
plants to benefit from mycorrhizal symbiosis are available at the mycorrhizal
producers level. Table 1, taken from Plenchette et al. (33) and personal
investigations, gives examples of Relative Field Mycorrhizal Dependency (RFMD
for some plants. Equation 1 gives the formula for calculating RFMD.

Table 1. Relative Field Mycorrhizal Dependency (RFMD) for selected plants.

Plant name RFMD* (%)
Cabbage (Brassicaceae)* 0
Carrot 99.2
Chicory (witloof) 82.4
Faba bean 93.5
Garden beet (Chenopodiaceae)* 0
Garden pea 96.7
Kentucky blue grass 72.4
Kidney bean 94.7
Leek 95.7
Pepper 66.1
Potato 41.9
Tomato (according cultivars) 59.2 - 78.0
Sweet corn 72.7
Wheat (according cultivars) 44.5 - 56.8
* Non-mycorrhizal plant.

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 DM of mycorrhizal plant - DM of non-mycorrhizal plant
RFMD = × 100 [1]
DM of mycorrhizal plant

Impact on Plant Protection and Microbial Interactions

AM fungi are recognized as high potential agents in plant protection and pest
management (34,43,48). In several cases direct biocontrol potential has been
demonstrated, especially for plant diseases caused by Phytophtora, Rhizoctonia,
and Fusarium pathogens (1,49,52). Several studies have confirmed synergism
between AMF and biocontrol agents such as Burkholderia cepacia Palleroni &
Holmes (37), Pseudomonas fluorescens Migula (11), Trichoderma harzianum
Rifai (7), and Verticillium chlamydosporium Kamyschko ex Barron & Onions
(35). These interactions suggest that AM might affect plant and soil microbial
activity by stimulating the production of root exudates, phytoalexins, and
phenolic compounds (30,32). A small increase of activity of plant defence genes,
especially for the production of chitinases, glucanases, flavonoid biosysthesis,
and phytoalexins, has been observed during mycorrhizal growth; however these
mycorrhizal defence induction mechanisms remain transitory (18).

AMF Impact on C Sequestration and Soil-aggregate Stability

Over the years, a body of research has accumulated showing the effects of
AMF biomass accumulated in the roots of colonized plants and in the
surrounding soil. The AMF soil hyphae spread into the rhizosphere where they
develop a network of microscopic filaments that make up to 80% of the total
hyphae content in soil (24). For example, in 4- to 5-week-old inoculated faba
beans plants (Vicia fava L.), mycorrhizal fungi biomass varied from 0.5 to 5%
associated with low (16%) and high (62%) root colonization levels, respectively
(25). Also, AMF spore biomass, measured in nine soil-field samples grown with
cassava for six months, was estimated between 89 to 93 lb/acre (44).
Soil-aggregates stability is an important soil physical property that can be
affected by AMF. Recently, a glycoprotein produced by AMF that promotes soil
aggregation, “glomalin,” has been discovered. Furthermore, higher than normal
carbon dioxide concentrations help to promote soil aggregation by increasing
the production of glomalin (38). These findings could have important future
implications in the use of mycorrhizal fungi to promote the production of soil
stable aggregates, improve water infiltration, and soil C sequestration in
agricultural systems.

Propagation Cycle of Arbuscular Mycorrhizal Fungi

The major biological characteristic of AMF is their obligate biotrophic
nature. This means that each of their life cycle steps requires the association
with a living plant. As with most of the filamentous fungi, AMF propagation can
occur either by spores differentiation and germination (Figs. 1a, 1b) or by
mycelium extension through soil and roots (Figs. 1c, 1d). Spores are
differentiated by budding intercalary or apically on hyphae. AMF species
identification is based on spore characters, spore wall architecture, and the
morphology of subtending hyphae. Some molecular tools to differentiate among
AMF species and strains have been developed. However these new technologies
remain to be tested for a variety of AM fungal strains and species (26,29,42,53).
Sexual reproduction has not yet been observed for these symbiotic fungi;
therefore they are considered asexual.
Fungal filaments grow through soil particles and come in contact with young
plant roots, the fungus threads its way through root surface, and then grow
between and inside cortical cells (Figs. 1e, 1f, 1g). The wide dispersal of the
fungal network through its filaments gives the plant-root mycorrhizae access to
a much larger volume of soil than the root system itself (Fig. 1d). The
establishment of mycorrhizal networks in roots and soil constitute a soil-root
fungal continuum, which is required for beneficial symbiotic exchanges between
fungi and plant.

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 Fig. 1. Propagation cycle of AMF. a. Spores of (i) Gigaspora, (ii) Glomus, (iii)
Entrophospora, and (iv) Acaulospora; b. germinating spore; c. hyphal network
and spores; d. hypha and spores around root; e. hyphal penetration inside root;
f. intracellular arbuscules; g. intraradical vesicles; h. colonized plant.

Inoculum Propagation
The main obstacle in the production of efficient and reliable AM fungal
inoculum lies in their symbiotic behaviour, the fungi obligatory requiring a host
plant for growth. Traditionally, mycorrhizal fungi are propagated through pot-
culture. Starting fungal inoculum, usually made of spores and colonized root
segments, are incorporated to a growing substrate for seedling production (5).
The fungi spread in the substrate and colonize root seedlings. Both colonized
substrates and roots can then serve as mycorrhizal inoculum. Soilless similar
culture systems such as aeroponic cultures enable the production of cleaner
spores and facilitate uniform nutrition of colonized plants (20). The successful
propagation of some AM fungal strains on root-organ culture allowed the
cultivation of monoxenic strains that can be used either directly as inoculum or
as starting inoculum for large-scale production (13).
(i) Pot-culture propagation. Unlike saprophytic fungi, the large-scale
production of AMF inoculum, due to their obligate symbiotic status, requires
control and optimization of both host growth and fungal development. The
microscopic sizes of AMF, together with the complex identification processes
also contribute to the pitfalls of inoculum propagation. The inoculum
propagation process entails the following stages.
Isolation of AMF pure culture strain. Pure culture strains can be obtained
originally from a single spore that germinate and colonize roots of a host plant.
AM fungal strains can also be generated from colonized root segments isolated
directly from field plants. Monospecific cultures will then be obtained through
subsequent pot-culture generation, using isolated spores or fine root segments
as starting inoculum. A technical problem usually encountered with AMF is that
spores can easily fall into dormancy and germination rates decrease dramatically
(16). A cold-temperature treatment can be used to break dormancy (23,39).
Research culture collection can provide users with reliable fungal cultures
appropriate to start AM fungus propagation, accompanied with detailed
information on species origin, spore morphology, and sometimes strain
molecular biology and biochemistry.
Choice of a host plant. The most important criteria required for the host
plant is its high mycorrhizal potential (i.e., its capacity to be colonized by the
AMF strain and to promote its growth and sporulation), a tolerance to growth
under growth chamber and greenhouse conditions, and an extensive root system
made of solid but non-lignified roots. Leek (Allium porrum L.), Sudan grass
(Sorghum bicolor (L.) Moench), corn (Zea mays L.), and bahia grass (Paspalum
notatum Flugge) are the most frequently used plant host for inoculum
propagation (50).

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 Optimum growing conditions. Pasteurized, steamed, or irradiated growing
substrates are required in order to avoid culture contamination which could
affect the quality of the inoculum. A well-aerated substrate is recommended,
such as coarse texture sandy soil (14) mixed with vermiculite or perlite or
Turface (8). Inadequate mineral nutrient composition may affect fungal
development. Optimum P levels vary with the host plant and cultivated fungal
strains and an excess of available phosphorus can inhibit AMF propagation.
Potassium, nitrogen, magnesium, and a selection of micro-element ratios may
also affect inoculum development, especially when inert growing substrates are
used and plant fertilization is performed artificially (9,45). Other edaphic factors
such as pH, soil temperature (36), light intensity, relative humidity, and
environment aeration must also be controlled to optimize AMF propagation.
(ii) In vitro propagation on root-organ culture (Fig. 2). Root-organ
cultures consist of excised roots that proliferate under axenic conditions on a
synthetic nutrient media (Fig. 2d) supplemented with vitamins, minerals, and
carbohydrates. Continuous cultures of vigorous root-organ cultures have been
obtained through transformation of roots by the soil bacterium Agrobacterium
rhizogenes Conn. (51). Since 1988 (4), several dozen species and strains have
been successfully propagated in vitro with various synthetic growth media and
growth conditions (13), and tested with compartmentalized solid and liquid
vessels. The mono-specific strains available can be used directly as starting
material for large-scale inoculum production, a sole Petri dish culture being
enough to generate several thousand of spores and meters of hyphae within 4
months.

Fig. 2. In vitro propagation. a. Isolated spores; b. germinating colonized root

segment; c. carrot root in culture; d. AMF root-organ culture; e. closer view of an
AMF root-organ culture.

In vitro bulk production of AMF inoculum is promising, offering clean,

viable, contamination-free fungi (Glomeromycota in vitro collection, or GINCO)
(Fig. 2e). The cost of in vitro inoculum may appear prohibitive compared to the
cost of a greenhouse-propagated one, but its use as starting inoculum is a
warranty of purity.

Research Collections (In Vivo and In Vitro)

Three major research collections (Table 2) manage inoculum maintenance
and distribution. Their respective activities, services, and availability in AMF
strains are posted on their respective websites.

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 Table 2. Major research collections of AMF inoculum.
Name and
internet address Propagation mode
Banque Européenne des Glomeromycota (BEG) Pot-culture
www.kent.ac.uk/bio/beg
Glomeromycota In Vitro Collection (GINCO) Root-organ culture
res2.agr.gc.ca/ecorc/ginco-can/index_e.htm
www.mbla.ucl.ac.be/ginco-bel
International Culture Collection of Pot-culture
Vesicular-Arbuscular Mycorrhizal Fungi (INVAM)
invam.caf.wvu.edu

Their common purpose is mainly to provide research and industry scientists

with pure and reliable material for starting inoculum production for both
fundamental researches and applied technologies.
Several other laboratory and industry collections are distributed throughout
the world to support either fundamental and applied researches or commercial
activities.

Long-term Preservation of AMF Inoculum

Large-scale production of mycorrhizal inoculum requires inventory of
product and the ability to provide clients with products of high and consistent
quality. Although the detailed procedure for inoculum preservation is
proprietary, methodologies for its preservation remain simple and inexpensive.
Fungal viability and mycorrhizal efficiency can be maintained for several
months at room temperature (68 to 77°F) especially when semi-dry inocula are
kept in their plastic containers or packaging. The major inconvenience of such a
storage period is the occurrence of spore dormancy. Long-term storage (up to 1
to 2 years) may be conducted at 41°F cold temperature storage (Dalpé et al.,
unpublished results). This method is efficient for both in vivo and in vitro
propagated strains. As spore germination and mycelium potential may be
stimulated by cold treatment, strain vigour can usually be recovered after long-
term storage at cold temperature. Liquid inoculums should react similarly to the
traditional dry ones.
More sophisticated and expensive preservation techniques are performed by
research culture collections. These include the maintenance of inoculum on
living plant-host grown on sterile growth substrate with regular check for mono-
specificity of the cultivated strains, storage in liquid nitrogen tanks (10), and
freeze-drying under vacuum. The last two techniques are the usual techniques
used in repository culture collections (DAOM/CCFC; ATCC).

Commercial Inoculum Production

Small scale AMF inoculum production began in the 1980s followed by large
scale production in the 1990s. At present, several companies have officially
registered and commercialized AMF inoculum.
(i) Methodologies. The first generation of commercial inoculum appeared
in the early 1980s. Since then, basic methodologies used for in vivo inoculum
propagation have evolved gradually. Pot-cultivation remains the preferred
propagation technique, as it provides a convenient and relatively economic
method to produce mycorrhizal inoculum on a large scale (40). Generally,
mycorrhizal fungi propagules, such as colonized roots, spores, and hyphae, are
mixed with a growing substrate, and the pots are seeded and incubated under
controlled conditions (Fig. 3). The in vitro propagation on root-organ culture
may not change drastically the traditional procedures but will certainly facilitate
the quality control of strain purity and improve the supply of massive amounts
of spores as starting inoculum.

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 Fig. 3. In vivo propagation. a. Seeding mycorrhizal substrates; b. mycorrhizal
seedling production; c. growth chamber inoculum propagation; d. root growth
and colonization; e. colonized seedlings; f. field inoculum propagation.

(ii) Obtaining mother inoculum for large-scale production. Both

segments of colonized roots (0.08 to 0.16 inches long) containing hypha and/or
vesicles and fungal spores may be used as starting fungal propagules for the
production of mother inoculum. Research collections can provide such material
from either in vivo or in vitro propagated fungi. Since root-organ culture
technology has become available, fungal propagules may be extracted from in
vitro cultures grown at large scales on solid or semi-liquid growing media.
(iii) Establishment of cultures. The establishment of mycorrhizal
cultures may proceed in different ways (Fig. 3):

· Mother inoculum added directly at seeding in large trays or pots;

· Mother inoculum incorporated at seedling transplantation of 4-to-6-
week old plantlets;
· Mother inoculum added at transplantation of micro-propagated
plantlets;
· Colonized seedlings produced in greenhouses and transplanted to the
field.

Composition of Commercial Inoculum

The inoculum sold on the market are provided as granular substrates made
from mixed materials such as peat, compost, vermiculite, perlite, sand, and/or
expanded clay in which segments of colonized roots, spores, and filamentous
networks are distributed. Most of the time these roots, spores, and hyphal
networks are not detectable because of their microscopic sizes. In terms of
fungal content, the tendency is to introduce a mix of several AMF in commercial
inoculum. The most frequently used AMF species for commercial inoculum is
typically Glomus intraradices Schenck & Smith. This species is well adapted to
both in vivo and in vitro propagation, can colonize a large variety of host plants,
survive to long-term storage, and is geographically distributed all over the world.
These characteristics make the G. intraradices species an excellent candidate for
commercial inoculum. Several other AMF belonging mainly to Glomus species,
but also to Gigaspora, Scutellospora, and Acaulospora genera, are gradually
used for commercial inoculum production. These AMF are sometimes in a
mixture with growth-promoting bacteria and with ectomycorrhizal fungi,
making a potentially better inoculum for plant protection and production.

Innovations and Future Developments

One innovative technique is the ready- and easy-to-use inoculum in which
fungal propagules are extracted from growing media, concentrated, and mixed
with carriers such as peat, sand, vermiculite, or expanded clay. Products are
available in powdered form containing a specified number of active fungal
propagules per volume of inoculum. Liquid inoculum dedicated to horticultural
use and isolated spores are also available. Aeroponic inoculum production at

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 large scale has been investigated by Souza et al. (47) but has not reached
commercialization. Bioreactor assays with liquid AMF root-organ culture
propagation (22) may eventually become suitable for commercialization for
research needs. However, as the fungi are produced in association with
Agrobacterium-transformed roots, it is unlikely that its used can be allowed for
field inoculation.
Knowing the performance variability between AM fungal strains, the
improvement of commercial inoculum quality will almost certainly come from
the selection of higher performance mycorrhizal fungal strains better adapted to
the plant host or crop to be colonized and to specific environmental growing
conditions (2,28).

Constraints and Regulations

(i) Cost of inoculum versus fertilizers. Again, the obligate biotrophic
nature of AMF which, unlike other fungi, implies the establishment of a plant
propagation system, either under greenhouse conditions or in vitro laboratory
propagation. These techniques result in high inoculum production costs, which
still remains a serious problem since they are not competitive with production
costs of phosphorus fertilizer. Even if farmers understand the significance of
sustainable agricultural systems, the reduction of phosphorus inputs by using
AM fungal inocula alone cannot be justified except, perhaps, in the case of high
value crops. This could be the case of organic crop farmers, which can sell their
products at premium price.
(ii) Sanitary control. Another serious problem in commercializing
inoculum comes from the need to control the biological composition of the
product, especially from invading phytopathogenic microorganisms. At present,
the inoculum produced using the pot-culture variants, either in greenhouses,
growth chambers, or fields, is never completely free from external
microorganisms. This is a problem even though the producers attempt to control
pathogens with various agrochemicals. Farmers are usually aware of the risk of
pathogens, so they avoid using inoculum containing host root residues. In most
commercial inoculum, colonized root segments are chopped into 0.08-to-0.12-
inch-long pieces so segments that remain are difficult to detect. When colonized
roots are directly incorporated to carriers, their surface sterilization with a light
solution of disinfecting product can be done without affecting the effectiveness
of an inoculum. When roots and rhizosphere material are used for inoculum
preparation, handling with clean apparatus is advised.
(iii) Efficiency of inoculum. In the field application of any microbial
inoculum, it is essential to verify that the inoculated microorganisms possess the
characteristics and the potential described by the inoculum manufacturers. With
AM inoculum, such evaluations can be done using several approaches such as
morphological identification of AM spores to confirm the fungus identity, and by
estimating the mycorrhizal root colonization level of test plants (6). Tentative
molecular techniques have been developed for the detection of AMF inoculum
strains and discrimination from indigenous AMF strains naturally occurring in
soils. These techniques are not yet totally reliable due to the large genetic
heterogeneity in AMF and, as such, these techniques are not routinely used for
the detection of AMF. Similar situations are observed with the discrimination
among strains when using internal transcribed spacer (ITS) sequences of
ribosomal DNA genes (rDNA). Reliable molecular techniques to trace the
inoculated strains using rep-PCR and specific primers developments are under
study (42). Such a technological breakthrough would greatly facilitate both
fundamental and applied research on mycorrhizae as well as improve quality
control of commercial inoculum.
(iv) Official registration for commercial products. The
commercialization of mycorrhizal inoculum is subjected to regional or national
registration at agriculture departments and usually falls under the country’s
Fertilizer Act. In Canada, mycorrhizal inoculum are considered to be
supplements: products, “other than fertilizers, manufactured, sold or
represented for use in the improvement of the physical condition of the soil or to
aid plant growth or crop yields.” In the USA, registration of an AM inoculum
may fall either in the fertilizer or the pesticide sectors, depending on the
vocation of the proposed mycorrhizal product.

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 Application for registration is required for such products and extensive
information is attached to the registration request: (a) a list of ingredients and
possible contaminants in the proposed inoculum; (b) the minimum
concentration of each ingredient including the active mycorrhizal fungi and the
purpose of each of them; (c) official material safety data sheets; (d) the product
label, showing the name and address of producer, the number of viable fungal
propagules or the symbiotic efficiency expressed as percentage of colonization
expected by the inoculum, recommended plant host, soil conditions for
effectiveness, recommended application rate, storage conditions, and expiration
date; (e) manufacturing process; and (f) the testing protocol. Such quality
control is important to exclude poor quality microbial inocula from the market.
Attached with previous information, statistically significant efficacy data
from field tests done under different soil and climatic conditions are usually
required in order to support the claims being made regarding the performance
of the proposed inoculum. A detailed taxonomic description may also be
requested together with strain history, geographic distribution, and existing
literature on the mycorrhizal potential of the fungal strain. In most countries,
mycorrhizal fungi are no longer considered detrimental for human and animal
health. As such, no environmental infectivity or toxicity tests are required. The
creation of an International Association of Mycorrhizal Inoculum Producers was
discussed at the last International Conference on Mycorrhizae held August 10-
15, 2003 in Montreal, in order to establish rules and regulations which would
stimulate industrial production of high quality inoculum. Organic crop farmers
since they have already started using AMF inoculum in larger-scale production,
are potentially a new clientele base. However, at this point, they are having
difficulties with inoculum application and with a clear measurement of
beneficial effect in their crops (personal communication). For large-scale
application of inoculum, future research focusing on achieving good contact
between seed an inoculum is needed. Regardless of the method of inoculum
application, new users should establish a portion of their crop without inoculum
in order to assess the benefits obtained in the crop established with inoculum.

Acknowledgments
The authors wish to thank Dr. Mary Leggett (Philom Bios Inc., Saskatoon,
Saskatchewan, Canada) who organized the Inoculum Forum Conference; Mr.
Clifford Hamilton for his technical assistance on image preparation; and S.
Séguin, J. Cayouette, and S. Redhead for comments on the manuscript.

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