Arbuscular Mycorrhiza Inoculum to Support Sustainable Cropping Systems

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    2004 Plant Management Network.Accepted fo r publication 14 January 2003. Published 1 March 2004.

    Arbuscular Mycorrhiza Inoculum to SupportSustainable Cropping Systems

    Yolande Dalp,Agriculture and Agri-Food Canada, ResearchBranch, 960 Carling Avenue Ottawa, Ontario, K1A 0C6; and MarciaMonreal,Agriculture and Agri-Food Canada, Research Branch, P.O.Box 1000A, Brandon, Manitoba, R7A 5Y3

    Corresponding author: Yo lande Dalp. [email protected]

    Dalp, Y., and Monreal, M. 2004. Arbuscular mycorrhiza inoculum to supportsustainable cropping s ystems . Online. C rop Management do i:10.1094/CM-2004-0301-09-RV.

    AbstractArbuscular mycorrhizae (AM) are symbiotic associations, formed between plantsand soil fungi that play an essential role in plant growth, plant protection, andsoil quality. The AM fungi expand their filaments in soil and plant roots. Thisfilamentous network promote bi-directional nutrient movement where soilnutrients and water move to the plant and plant photosynthates flow to thefungal network. AM fungi are ubiquitous in the soil and can form symbiosis withmost terrestrial plants including major crops, cereals, vegetables, andhorticultural plants. In agriculture, several factors, such as host cropdependency to mycorrhizal colonization, tillage system, fertilizer application,and mycorrhizal fungi inoculums potential can affect plant response and plantbenefits from mycorrhizae. Due to their obligate symbiotic status, AM fungineed to associate with plant for growth and proliferation. Consequently, thecultivation of AM fungal strains and the maintenance of reference collections

    require methodologies and infrastructures quite different from those used withother microbial collections and inoculum production. Interest in AM fungipropagation for agriculture is increasing due to their role in the promotion ofplant health, in soil nutrition improvement, and soil aggregate stability. Thecomprehensive life cycle of AM fungi and methods currently used for thepropagation of inoculum and the maintenance of in vivo and in vitro sourcecollections are described. Methods and regulations of large-scale production ofcommercial inoculum that provide users with products of high quality andefficiency are discussed.

    IntroductionArbusc ular my cor rhizae (AM) are sy mbiotic assoc iations formed

    between p lants and soil fungi that benefit both partners. The phy tobio nt

    cor respond to appro ximately 80% of plant species and the fungi are

    classified in the phy lum Glomeromy co ta, including nine genera; Glomus,

    Paraglo mus,Sclero cystis,Acaulospo ra,Entrophospo ra, Gigaspora,

    Scutellospo ra,Diversispora, Geosiphon, andArch aeospo ra(41). A M fungi

    (AMF) are ubiquitous in the soil with around 17 0 desc ribed species (46).

    The symbiosis is called arbuscular because the fungi form specialized

    tree-like struc tures (arbuscules = tree-like) inside root cells. Other

    structures pro duced by fungi are intra- and extraradical spores (which are

    germinating structures useful for long-term preserv ation of species,

    propagation, and species identification purposes), intraradical hyphae,

    extraradical hyphae, intracellular fungal storage structures called vesic les

    (which are lipid co ntaining bodies) and, for some genera, auxiliary cells

    branc hing from extraradic al hy phae. I ntraradical A M fungal my celium

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    form a network around and inside cortical ce lls of plant roots, ex traradical

    AM my celium c an spread thro ughout the soil surrounding the r oo t sy stem

    and increase the ability to explore soil areas, acce ssing water and

    nutrients for plant roo ts. Benefits to plants are improv ed water and

    nutrient uptake, enhanced P transport, and dro ught and disease

    resistance. Benefits to fungi are the supply of photosy nthates to the fungal

    network located in the cortical ce lls of the plant and the surrounding soil.

    All water, nutrients, and photosy nthates ex changes occur v ia the fungal

    filament network that bridged plant rhizosphere and plant roo ts.

    AM Interaction with Soil and CropsThe increased capac ity o f plant roots for water and nutrients uptake

    from the soil when colonized by AMF is the main mechanisms propo sed to

    explain the effect of AM in plant performance. This behavior is

    particularly ev ident with soil nutrients that are more immobile such as

    phosphorus (P), zinc (Zn), and coppe r (Cu) . Improv ed phosphorus

    nutrition when co lonized with AMF has been demo nstrated for hundreds

    of cultivated plants. By ex tending past the P-depletion zone formed

    around the ro ot sy stems, 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 suc h, a reduction in P applications isreco mmended in order to stimulate and maintain symbiosis efficiency .

    Most agricultural cro ps such as flax, c orn, sorghum, wheat, barley ,

    potatoes, and sunflower can benefit from myc orrhizal association. Some

    other crop plants do not form AM symbiosis; those belong to the

    Cruciferae, Brassicaceae, Chenopodiaceae , and Caryophy llaceae families

    (3). Canola (Brassicaceae family), an important cro p in wester n Canada

    does not form A M. Efficiencies and limitations o f registered my cor rhizal

    inoculum, in terms of the cultivated cro ps, are clearly po sted on sale

    products together with reco mmendation for use.

    Interaction of AM and Agricultural PracticesAgricultural pr actic es such as fertilizer applic ations, c ro p ro tat ion,

    tillage, and liming affect field AM potential and ro ot c olonization leve ls.

    For ex ample, high lev els of P fertilization have been found to slow do wn or

    inhibit mycor rhizal efficiency in soybean fields (12). Cropping of a soil

    with cano la, a non-ho st plant spec ies, delay ed my cor rhiza dev elopment of

    maize and of flax (1 5; Monreal et al., unpublished data). Higher so il

    infectiv ity was observ ed under reduced or no tillage practices (31 ) and

    liming increased mycor rhizal colo nization of barley ro ots and soil

    infectiv ity (17 ). Plant spec ies differ in their fertilization requirements, and

    consequently their dependency o n AMF vary considerably from one crop

    to another (33). For ex ample, under field conditions, beans, corn, and leek

    have a much higher mycor rhizal dependency than potato and wheat. Thisrange of plant response to A MF has to be taken into acc ount when

    managing a cro pping system o r a cro p rotation. Data on the potential of

    cro p plants to benefit from myc orrhizal symbiosis are available at the

    mycor rhizal produc ers lev el. Table 1 , taken from Plenchette et al. (33) and

    personal investigations, gives ex amples of Relative Field Mycorrhizal

    Dependency (RFMD for some plants. Equation 1 gives the formula for

    calc ulating RFMD.

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

    Plant name RFMD* (%)

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    Cabbage (Brassicaceae)* 0

    Carrot 99.2

    Chicory (witloof) 82.4

    Faba bean 93.5

    Garden beet (C henopodiaceae)* 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.

    RFMD =

    DM of mycorrhizal plant - DM of non-mycorrhizal plant

    100 [1]DM of mycorrhizal plant

    Impact on Plant Protection and Microbial InteractionsAM fungi are recognized as high potential agents in plant protec tion

    and pest management (34,43,48). In several cases direct bioc ontrol

    potential has been demonstrated, especially for plant diseases caused by

    Phytophtora, Rhizoctonia, andFusariumpathogens (1,49,5 2). Sev eral

    studies have co nfirmed synergism between A MF and biocontro l agents

    such asBurkho lde ria cepacia Palleroni & Holmes (37 ),Pseudomonas

    fluo rescensMigula (11 ), Trichoderma harzianumRifai (7 ), and

    Ve rticillium chlamydosporiumKamysc hko ex Barron & Onions (35).

    These interactions suggest that A M might affect plant and soil micro bialactiv ity by stimulating the production of root exudates, phy toalex ins, and

    phenolic co mpounds (30,32). A small increase of activity o f plant defence

    genes, especially for the pro duction of chitinases, glucanases, flavo noid

    bio sy sthesis, and phy toalexins, has been observed during my cor rhizal

    growth; howev er these mycor rhizal defence induction mechanisms

    remain transitory (18).

    AMF Impact on C Sequestration and Soil-aggregate StabilityOver the years, a body o f research has acc umulated showing the effects

    of AMF biomass accumulated in the roo ts of colonized plants and in the

    surrounding soil. The A MF soil hyphae spread into the rhizosphere where

    they dev elop a network of micro scopic filaments that make up to 80% ofthe total hyphae co ntent in soil (24). For ex ample, in 4- to 5-week-old

    inoculated faba beans plants (Vic ia fava L.), myc orrhizal fungi biomass

    varied from 0.5 to 5% assoc iated 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 mo nths,

    was e stimated between 89 to 93 lb/acre (44).

    Soil-aggregates stability is an important soil physical property that can

    be affec ted by AMF. Recently , a gly copro tein produc ed by AMF that

    promotes soil aggregation, glomalin, has been discov ered. Furthermore,

    higher than normal carbon diox ide conce ntrations help to promote soil

    aggregation by increasing the produc tion of glomalin (38). These findings

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    could have important future implications in the use o f myco rrhizal fungi

    to promote the pro duction of soil stable aggregates, improv e water

    infiltration, and soil C sequestration in agricultural systems.

    Propagation Cycle of Arbuscular Mycorrhizal FungiThe 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. A s with most o f the filamentous fungi, AMF

    propagation can occur either by spores differentiation and germination

    (Figs. 1a, 1b) or by my celium ex tension through soil and roots (Figs. 1c ,1d). Spores are differentiated by budding intercalary or apically o n

    hyphae. AMF species identification is based on spore characters, spore

    wall architec ture, and the morphology of subtending hy phae. Some

    molecular to ols to differentiate among AMF species and strains have been

    dev eloped. However these new technologies remain to be tested for a

    variety of A M fungal str ains and spec ies (26,29,42,53). Sex ual

    reproduction has not yet been observ ed for these symbiotic fungi;

    therefore they are considered asexual.

    Fungal filaments grow through soil particles and come in c ontact with

    young plant ro ots, the fungus threads its way thr ough roo t surfac e, and

    then grow between and inside co rtical cells (Figs. 1e , 1f, 1 g). The wide

    dispersal of the fungal network through its filaments gives the plant-rootmyc orrhizae access to a much larger volume of soil than the root sy stem

    itself (Fig. 1d). The establishment of my co rrhizal networks in roots and so il

    constitute a so il-root fungal continuum, which is required for beneficial

    symbiotic ex changes between fungi and plant.

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

    Inoculum PropagationThe main obstacle in the produc tion of efficient and reliable A M fungal

    inoculum lies in their symbiotic behaviour, the fungi obligatory requiring

    a host plant for growth. Traditionally, mycor rhizal fungi are propagated

    through pot-culture. Starting fungal inoculum, usually made of spores and

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    colo nized root segments, are incorpor ated to a growing substrate for

    seedling production (5 ). The fungi spread in the substrate and colonize

    root seedlings. Both co lonized substrates and roots can then serv e as

    mycor rhizal inoculum. Soilless similar culture sy stems such as aeroponic

    cultures enable the produc tion of cleaner spores and facilitate uniform

    nutrition of co lonized plants (20). The successful propagation of some A M

    fungal strains on roo t-organ culture allowed the c ultivation of monox enic

    strains that can be used either directly as inoculum or as starting

    inoculum for large-scale production (13).

    (i) Pot-cu lture propagation.Unlike sapro phytic fungi, the large-scale produc tion of AMF inoculum, due to their obligate symbiotic status,

    requires c ontrol and o ptimization of both host gro wth and fungal

    dev elopment. The micro scopic sizes of AMF, together with the complex

    identification proc esses also c ontribute to the pitfalls of inoculum

    propagation. The inoculum propagation pro cess e ntails the following

    stages.

    Isolation of AMF pure culture strain. Pure culture strains can be

    obtained originally from a single spore that germinate and co lonize roots

    of a host plant. AM fungal strains can also be generated from co lonized

    root segments isolated directly from field plants. Monospec ific cultures

    will then be obtained through subsequent pot-culture generatio n, using

    isolated spores or fine roo t segments as starting inoculum. A technicalproblem usually enco untered with AMF is that spores c an easily fall into

    dormancy and germination rates decrease dramatically (1 6). A cold-

    temperature treatment can be used to break dormancy (23,39). Research

    culture collection can prov ide users with reliable fungal cultures

    appropriate to start AM fungus propagation, acco mpanied with detailed

    information on spec ies 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 myco rrhizal potential (i.e., its capacity to be

    colo nized by the AMF strain and to promote its growth and sporulation), a

    tolerance to gro wth under growth chamber and greenhouse conditions,

    and an extensive roo t system made o f solid but non-lignified roots. Leek(Allium po rrumL.), Sudan grass (Sorghum bicolor(L.) Moench), cor n

    (Zea mays L.), and bahia grass (Paspalum notatum Flugge) are the most

    frequently used plant host for inoculum propagation (50).

    Optimum growing c onditions. Pasteurized, steamed, or irradiated

    growing substrates are required in order to avoid culture co ntamination

    whic h could affect the quality of the inoc ulum. A well-aerate d substrate is

    recommended, such as co arse texture sandy soil (14) mixed with

    vermic ulite or perlite or Turface (8). Inadequate mineral nutrient

    compo sition may affect fungal developme nt. Optimum P levels vary with

    the host plant and cultivated fungal strains and an exc ess of available

    phosphorus c an inhibit A MF propagation. Potassium, nitrogen,

    magnesium, and a selection of micro-element ratios may also affectinoculum dev elopment, espec ially 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 o ptimize

    AMF propagation.

    (ii) In vitro propagation on root-organ c ulture(Fig. 2). Root-

    organ cultures co nsist of exc ised roots that proliferate under axe nic

    conditions on a synthetic nutrient media (Fig. 2d) supplemented with

    v itamins, minerals, and carb ohydrates. Continuo us c ultures o f vigoro us

    root-organ cultures hav e been obtained through transformation of roots

    by the soil bacteriumAgrobacterium rhizogenesConn. (51). Since 1 988

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    (4), sev eral dozen species and strains have been successfully propagated

    in vitro with various sy nthetic growth media and growth co nditions (13),

    and tested with compartmentalized solid and liquid vessels. The mono-

    specific strains available can be used direc tly as starting material for large-

    scale inoculum production, a sole Petri dish culture being enough to

    generate sev eral thousand of spores and meters of hyphae within 4

    months.

    Fig. 2. In vitro propagation. a.Isolated spores; b.germinating colonized rootsegment; c.carrot root in culture; d.AMF root-organ culture; e.closer view ofan AMF root-organ culture.

    In v itro bulk production of AMF inoculum is promising, offering clean,

    v iable, contaminat ion-free fungi (Glomeromy cota in v itro c ollection, o r

    GINCO) (Fig. 2e). The co st of in vitro inoculum may appear prohibitive

    compared to the co st 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 collec tions (Table 2) manage inoculum

    maintenance and distribution. Their respectiv e activ ities, serv ices, and

    availability in AMF strains are posted on their respec tive websites.

    Table 2. Major research collections of AMF inoculum.

    Name andinternet address Propagation mode

    Banque Europenne des Glomeromycota (BEG)www.kent.ac.uk/bio/beg

    Pot-culture

    Glomeromycota In Vitro Collection (GINCO)res2.agr.gc.ca/ecorc/ginco-can/index_e.htmwww.mbla.ucl.ac.be/ginco-bel

    Root-organ culture

    International Culture Collection ofVesicular-Arbuscular Mycorrhizal Fungi (INVAM)invam.caf.wvu.edu

    Pot-culture

    Their commo n purpose is mainly to pro vide research and industry

    scientists with pure and r eliable material for starting inoculum pro duction

    for both fundamental researches and applied technologies.

    Several other laboratory and industry collections are distributed

    http://invam.caf.wvu.edu/http://www.mbla.ucl.ac.be/ginco-belhttp://res2.agr.gc.ca/ecorc/ginco-can/index_e.htmhttp://www.kent.ac.uk/bio/beg
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    throughout the world to support either fundamental and applied

    researches or commercial activities.

    Long-term Preservation of AMF InoculumLarge-scale production of mycorrhizal inoculum requires inventory of

    product and the ability to prov ide clients with products o f high and

    consistent quality. Although the detailed procedure for inoculum

    preserv ation is proprietary , methodologies for its preserv ation remain

    simple and inexpensive. Fungal v iability and mycor rhizal efficiency can be

    maintained for several months at room temperature (68 to 7 7 F)especially when semi-dry inocula are kept in their plastic c ontainers or

    packaging. The major inconv enience o f such a storage period is the

    occ urrence of spore dormancy . Long-term storage (up to 1 to 2 y ears)

    may be co nducted at 41 F cold temperature storage (Dalp et al.,

    unpublished results). This method is efficient for both in v ivo and in vitro

    propagated strains. As spore germination and myce lium potential may be

    stimulated by c old treatment, strain vigour can usually be reco vered after

    long-term storage at cold temperature. Liquid inoculums should react

    similarly to the traditional dry o nes.

    More sophisticated and expensive preservation techniques are

    performed by research culture co llections. These include the maintenance

    of inoculum on liv ing plant-host grown o n sterile growth substrate withregular check for mono -specificity of the cultivated strains, storage in

    liquid nitrogen tanks (10), and freeze-drying under v acuum. The last two

    techniques are the usual techniques used in repository culture co llections

    (DAOM/CCFC; ATCC).

    Commercial Inoculum ProductionSmall scale AMF inoculum pro duction began in the 1 980s followed by

    large scale production in the 1990s. At present, sever al companies have

    officially registered and c ommerc ialized AMF inoculum.

    (i) Methodologies.The first generation of co mmercial inoculum

    appeared in the early 1 980s. Since then, basic methodo logies used for in

    v iv o inoc ulum propagation hav e evolv ed gradually . Pot-cultiv ation

    remains the preferred propagation technique, as it prov ides a convenient

    and relatively economic method to produc e my corrhizal inoculum on a

    large scale (40). Generally , myco rrhizal fungi propagules, such as

    colo nized roots, spores, and hyphae, are mixed with a growing substrate,

    and the pots are seeded and incubated under c ontrolled co nditions (Fig.

    3). The in vitro pro pagation on root-organ culture may not change

    drastically the traditional procedures but will certainly facilitate the

    quality co ntrol of strain purity and improv e the supply of massiv e

    amounts of spores as starting inoculum.

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

    (ii) Obtaining m other inocu lum for large-scale production .

    Both segments of colonized roots (0.0 8 to 0.1 6 inches long) containing

    hypha and/or v esicles and fungal spores may be used as starting fungalpropagules for the production of mother inoculum. Research collections

    can prov ide such material from either in viv o or in v itro propagated fungi.

    Since roo t-organ culture technology has become av ailable, fungal

    propagules may be extracted from in v itro cultures grown at large scales

    on so lid or semi-liquid growing media.

    (iii) Establishmen t of c ultures.The establishment of myc orrhizal

    cultures may proceed in different ways (Fig. 3):

    Mother inoculum added directly at seeding in large trays or po ts;

    Mother inoculum incorporated at seedling transplantation of 4-

    to-6-week old plantlets;

    Mother inoculum added at transplantation of micro-propagatedplantlets;

    Colonized seedlings produced in greenhouses and transplanted to

    the field.

    Composition of Commercial InoculumThe inoculum sold o n the market are provided as granular substrates

    made from mixed materials such as peat, compost, v ermiculite, perlite,

    sand, and/or expanded clay in which segments of colo nized roots, spores,

    and filamentous networks are distributed. Most of the time these roots,

    spores, and hyphal networks are not detectable bec ause of their

    microscopic sizes. In terms of fungal content, the tendency is to introduc e

    a mix of several AMF in co mmercial inoculum. The most frequently used

    AMF spe cies for commerc ial inoc ulum is typic ally Glomus intraradices

    Schenck & Smith. This species is well adapted to both in v ivo and in vitro

    propagation, can colonize a large variety o f host plants, survive to long-

    term storage, and is geographically distributed all over the wor ld. These

    characteristics make the G. intraradicesspecies an exc ellent candidate for

    commercial inoculum. Sev eral other AMF belonging mainly to Glomus

    species, but also to Gigaspora,Scutellospo ra, andAcaulospo ragenera,

    are gradually used for co mmercial inoculum produc tion. These AMF are

    sometimes in a mixture with growth-promoting bacteria and with

    ecto myco rrhizal fungi, making a potentially better inoculum for plant

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    protection and production.

    Innovations and Future DevelopmentsOne innovativ e technique is the ready - and easy-to-use inoculum in

    whic h fungal pro pagules are extracted fro m growing media, c oncentrated,

    and mixed with carriers such as peat, sand, vermiculite, or ex panded clay .

    Products are av ailable in powdered form containing a specified number of

    active fungal propagules per v olume of inoculum. Liquid inoculum

    dedicated to hor ticultural use and isolated spores are also av ailable.

    Aeroponic inoculum produc tion at large scale has b een inv estigated bySouza et al. (47 ) but has not reached co mmercialization. Bioreac tor assays

    with liquid AMF ro ot-organ culture pro pagation (22) may ev entually

    become suitable for commerc ialization for research needs. Howev er , as

    the fungi are produced in association withAgrobacterium-transformed

    roots, it is unlikely that its used c an be allowed for field inoculation.

    Knowing the performance variability b etween A M fungal strains, the

    improvement of commercial inoculum quality will almost certainly co me

    from the selection of higher performance my cor rhizal fungal strains better

    adapted to the plant host or cro p to be c olonized and to specific

    env ironmental growing conditions (2,28).

    Constraints and Regulations(i) Cost of inoc ulu m v ersus fertilizers.A gain, the obligate

    bio trophic nature of AMF which, unlike other fungi, implie s the

    establishment of a plant propagation system, e ither under greenhouse

    conditions or in vitro laboratory propagation. These techniques result in

    high inoculum pro duction costs, which still remains a serious problem

    since they are not compe titive with produc tion 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 ex cept, perhaps, in the case of high

    value c rops. This c ould be the case of organic crop farmers, whic h can sell

    their products at premium price.

    (ii) Sanitary con trol.Another serious problem in commercializing

    inoculum comes from the need to co ntrol the biological composition of

    the product, espec ially from invading phytopathogenic micro organisms.

    At present, the inoculum produced using the pot-culture v ariants, eithe r

    in greenhouses, growth chambers, or fields, is never co mpletely free from

    external microorganisms. This is a problem even though the producers

    attempt to co ntrol pathogens with various agrochemicals. Farmers are

    usually aware of the risk of pathogens, so they avo id using inoculum

    containing host root residues. In most co mmercial inoculum, colonized

    root segments are chopped into 0.08-to-0.1 2-inch-long pieces so

    segments that remain are difficult to detect. When colo nized roots are

    directly incorporated to carriers, their surface sterilization with a lightsolution of disinfecting product can be done without affecting the

    effectiv eness of an inoculum. When roots and r hizosphere material are

    used for inoculum preparation, handling with clean apparatus is advised.

    (iii) Efficiency of inocu lum .In the field application of any

    microbial inoculum, it is essential to v erify that the inoculated

    microor ganisms possess the characteristics and the potential described by

    the inoculum manufacturers. With AM inoculum, such ev aluations can be

    done using several approaches such as morphological identification of AM

    spores to c onfirm the fungus identity, and by estimating the myc orrhizal

    root c olonization level of test plants (6). Tentative molec ular techniques

    have been dev eloped for the detection of AMF inoculum strains and

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    discrimination from indigenous A MF strains naturally o cc urring in soils.

    These techniques are not y et totally re liable due to the large genetic

    heterogeneity in AMF and, as such, these techniques are not ro utinely

    used for the detection o f AMF. Similar situations are observ ed with the

    discrimination among strains when using internal transcribed spac er (ITS)

    sequences of ribosomal DNA genes (rDNA). Reliable molec ular techniques

    to trac e the inoculated strains using rep-PCR and specific primers

    dev elopments are under study (42). Such a technological breakthrough

    would greatly facilitate bo th fundamental and applied researc h on

    mycor rhizae as well as improv e quality contro l of commercial inoculum.(iv) Official registration for com m ercial produc ts.The

    commercialization of mycorrhizal inoculum is subjected to regional or

    national registration at agriculture departments and usually falls under

    the country s Fertilizer A ct. In Canada, myco rrhizal inoculum are

    considered to be supplements: products, other than fertilizers,

    manufactured, sold o r represented for use in the improvement of the

    physical condition of the soil or to aid plant growth or crop y ields. In the

    USA, registration o f an AM inoculum may fall either in the fertilizer or the

    pesticide sectors, depending on the vocation of the proposed my cor rhizal

    product.

    Applicatio n for registration is required for such produc ts and

    extensive information is attached to the registration request: (a) a list ofingredients and possible contaminants in the proposed inoculum; (b) the

    minimum co ncentration of each ingredient including the active

    mycor rhizal fungi and the purpose of each o f 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 co lonization ex pected by the

    inoculum, reco mmended plant host, soil conditions for effectiveness,

    reco mmended application rate, storage co nditions, and expiration date;

    (e) manufacturing proc ess; and (f) the testing proto co l. Such quality

    contro l is important to exc lude poor quality micro bial inocula from the

    market.

    Attached with prev ious information, statistically significant efficacydata from field tests done under different so il and climatic co nditions are

    usually required in order to support the claims being made regarding the

    performance of the proposed inoculum. A detailed taxo nomic description

    may also be requested to gether with strain history , geographic

    distribution, and ex isting literature on the my co rrhizal potential of the

    fungal strain. In most c ountries, myco rrhizal fungi are no longer

    considered detrimental for human and animal health. As such, no

    env ironmental infectivity o r tox icity tests are required. The creation of an

    International Association of My cor rhizal Inoculum Producers was

    discussed at the last International Conference o n Myco rrhizae held August

    10 -15 , 200 3 in Montreal, in order to establish rules and regulations which

    would stimulate industrial pro ductio n of high quality inoculum. Organiccro p farmers since they hav e already started using AMF inoculum in

    larger-scale production, are po tentially a new clientele base. Howev er, at

    this point, they are hav ing difficulties with inoculum application and with

    a clear measureme nt of beneficial effect in their cro ps (personal

    communication). For large-scale application of inoculum, future research

    focusing on achieving good co ntact between seed an inoculum is needed.

    Regardless of the method of inoculum application, new user s should

    establish a portion of their crop without inoculum in order to assess the

    benefits obtained in the c rop established with ino culum.

    Acknowledgments

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    The authors wish to thank Dr. Mary Leggett (Philom Bios Inc.,

    Saskatoon, Saskatchewan, Canada) who organized the Inoc ulum Forum

    Conference; Mr. Clifford Hamilton for his technical assistance on image

    preparation; and S. Sguin, J. Cayouette , and S. Redhead for c omments on

    the manuscript.

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