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© 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. dalpey@agr.gc.ca

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.

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.

* Non-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.

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).

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.

Table 2. Major research collections of AMF inoculum.

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.

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 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.

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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