13 May2002

Overstory #102 - Mycorrhizas: Producing and Applying Arbuscular Mycorrhizal Inoculum

Written by M. Habte and N.W. Osorio.

Introduction

To one degree or another, most plants in their natural habitats function under the influence of a special group of soil fungi known as arbuscular mycorrhizal fungi ("AM fungi" or AMF). The existence of these fungi has been recognized for more than a century, although they did not receive the attention they deserve until approximately 40 years ago. Worldwide, interest in AM fungi has now reached a point wherein any discussion of agricultural biotechnology that does not include their role in plant productivity can hardly be considered complete.

Many individuals and organizations concerned with managing native plant species, restoring natural ecosystems, and producing agronomic, horticultural, and forest plants with minimal chemical inputs are interested in applying AMF technology. A major challenge to the large-scale utilization of AMF is the unavailability of large quantity of high quality inoculum to introduce the fungi into plant growing media. The problem of producing inoculum is largely due to that AM fungi are "obligate symbionts," which means they require the presence of actively growing plants during their reproduction. Therefore AMF cannot be cultured on laboratory media in the same manner as other beneficial soil microorganisms such as Rhizobium bacteria. Fortunately, specialized techniques for AMF inoculum production have been in development at the University of Hawaii and elsewhere.

Arbuscular mycorrhizal associations

The term "mycorrhiza" was coined by A. B. Frank, a researcher in Germany, more than 100 years ago. It means "fungus-root," and stands for the mutualistic association existing between a group of soil fungi and higher plants. There are many types of mycorrhizal associations (15) of which the endomycorrhizal association of the vesicular arbuscular (VA) type are the most widespread geographically as well as within the plant kingdom. VA mycorrhizal fungi invade cortical cells inter- and intra-cellularly and form clusters of finely divided hyphae known as arbuscules in the cortex. They also form membrane-bound organelles of varying shapes known as vesicles inside and outside the cortical cells.

Arbuscules are believed to be sites of exchange of materials between the host and the plant. Vesicles generally serve as storage structures, and when they are old, they could serve as reproductive structures. Because vesicles are absent in two of the seven genera containing these fungi, the term that is currently preferred by many researchers to represent the association is arbuscular mycorrhizal (AM) fungi rather than vesicular-arbuscular (VA) mycorrhizal fungi. Arbuscular mycorrhizal fungi occur on a wide spectrum of temperate and tropical plant species and are absent in less than 30 plant families (26, 42).

AMF functions

Roles in plant nutrition

AM fungi absorb N, P, K, Ca, S, Fe, Mn, Cu, and Zn from the soil and then translocate these nutrients to the plants with whose roots they are associated (11, 16, 33, 43). Their most consistent and important nutritional effect is to improve uptake of immobile nutrients such as P, Cu, and Zn (29, 35). AM fungi have their greatest effect when a host plant not associated with them is deficient in P. They are also very useful to plant species that inherently lack morphological or physiological mechanisms for efficient P uptake (26, 30). Consequently, enhancement of growth of plants associated with AMF is explained in most instances by improved P nutrition (5).

Another advantage to associated plants is improved maintenance of a balanced supply of nutrients. This occurs because plants grown in association with AMF can grow with only a fraction of the P required for growth by plants lacking a mycorrhizal association. Moreover, when P is applied at high concentrations, as is commonly done when growing plants in soil where AMF are absent, it can cause nutritional disorders because of its antagonistic interactions with other nutrients, or because it inhibits mycorrhizal formation (27). Studies with the forage tree Leucaena leucocephala, which is highly dependent on mycorrhizal association, have shown that the AMF symbiosis can decrease the plant's external P requirement, reducing it to as much as 40 times less than the plant would require for good growth in the absence of AMF (MH, unpublished).

The ability of AMF to reduce plants' external P requirement has an important environmental benefit. High levels of P in soils can result in pollution of bodies of water when eroded soil rich in P is deposited in them. P enrichment of water bodies causes eutrophication (7, 38) due to excessive development of algae, cyanobacteria, and aquatic plants, and this condition impairs the usefulness of these waters. When plants rely on AMF association rather than heavy P fertilization, risks to water quality are reduced. Arbuscular mycorrhizal fungi, therefore, are an important component of nutrient management programs that aim to reduce environmental pollution.

Mechanisms of enhanced P uptake

In soils not adequately supplied with P, plant demand for this nutrient exceeds the rate at which it diffuses into the root zone, resulting in zones of P depletion surrounding roots. It is believed that AMF help overcome this problem by extending their external hyphae from root surfaces to areas of soil beyond the P depletion zone, thereby exploring a greater volume of the soil than is accessible to the unaided root (17, 21). The external hyphae of some AMF may spread 10--12 cm from the root surface. Assuming a radial distribution of hyphae around roots, it has been estimated that the volume of soil explored by the mycorrhizal root exceeds that explored by the unaided root by as much as 100 times (39).

AM fungal hyphae are 2.5--5 times smaller in diameter than plant roots and therefore have a greater surface area per unit volume. This surface area makes the fungi much more efficient than roots in the uptake of P (5). Moreover, the smaller diameter of AMF hyphae allows them to explore micropores in the soil that are not accessible to roots. And, studies carried out in solution culture have shown that AMF hyphae have a higher affinity for P than do roots (20).

AM fungi may have biochemical and physiological capabilities for increasing the supply of available P or other immobile nutrients. These mechanisms may involve acidification of the rhizosphere (2), increases in root phosphatase activity (10) and excretion of chelating agents.

Roles not directly related to nutrition

A growing body of research suggests that AMF could contribute to plant health and productivity independently of their role in enhancing nutrient uptake. For example, the fungi have been found to be involved in the suppression of plant diseases, (19, 33, 44) including nematode infection (6,13). AMF stimulate hormone production in plants, aid in improving soil structure (4, 46, 47), enhance leaf chlorophyll levels, (45) and improve plant tolerance to water stress, salinity, soil acidity, and heavy metal toxicity (3). Some of these functions may be the indirect effects of improved P nutrition (34, 39).

Factors influencing the AMF inoculation effect

The degree to which mycorrhizal fungi enhance the nutrition and health of associated plants depends on many biotic and abiotic soil factors, as well as other environmental factors that influence the host, the fungi, and their association. The most important factors include abundance of AMF infective propagules, soil P status, variation in the degree to which target plant species rely on the mycorrhizal condition at the prevailing soil-solution P concentration, and soil treatment, including the type of previous crop or native vegetation.

Abundance of AMF Propagules

Effectiveness of mycorrhizal fungi may not be rapidly expressed if the number of infective propagules contained in an inoculum is low. Many instances of poor inoculum performance may in fact be a result of a low level of infective propagules. All other things being equal, if high-quality inoculum is introduced into a soil containing a very low density of indigenous AMF fungi, the probability of obtaining a positive response to inoculation is high (12). However, if the soil contains high levels of infective propagules to begin with, it is unlikely that plants will respond to additional inoculation.

Soil P Status

There are critical ranges of soil-solution P concentration at which the host-fungus association is truly mutualistic, i.e., where the benefit each partner derives from the association outweighs the costs (9).

Mycorrhizal inoculation will have its maximum effect on plant growth at soil P concentrations near-optimal for mycorrhizal activity or at soil P concentrations that are barely accessible to the unaided root. Consequently, AMF play crucial roles in certain conditions:

- native ecosystems (e.g., forests) where applications of large quantities of fertilizer P to extensive land areas is not usually done or is not practical
- agricultural systems on soils with strong P-fixing capacity, or where P-fertilizer is unavailable or prohibitively expensive
- situations where it is essential to reduce soil fertilizer applications because of environmental concerns such as nutrient pollution of surface waters
- situations in which rock phosphate is readily available and used instead of more soluble P sources.

Variation in the dependence of plants on AM fungi

Mycorrhizal dependency is a measure of the degree to which a plant species relies on the mycorrhizal condition for nutrient uptake and growth as the concentration of P in the soil solution is increased. It is well established that plant species and cultivars within a given species vary in their response to AMF colonization (36, 37, 18, 25). Most of the variation may have to do with the ability of plant species to take up P at very low soil-P concentrations in the absence of mycorrhizal fungi (1, 11, 31).

Soil disturbance

The activities of AM fungi can be severely curtailed by soil disturbance in both native and agricultural ecosystems. In native ecosystems, soil disturbances caused by land cleaving and mining operations can be so severe that mere inoculation of the affected areas with AMF may not be able to restore the symbiotic function of the fungi (14, 40). The impacts of disturbances that have been studied in agricultural ecosystems are generally less drastic (32). On the other hand, the activities of AMF are known to be adversely impacted even by disturbance such as mechanical planting operations in otherwise undisturbed soils (28). Numerous investigations have been undertaken over the past 15 years with the intent of understanding the mechanisms by which soil disturbance hampers AMF development and function. Soil disturbance due to tillage can adversely influence the abundance and diversity of AMF, but data on the subject is very scant at present. Nevertheless, there is evidence to indicate that the diversity of AMF communities tends to decline upon the conversion of native ecosystems into agricultural ecosystems and with the intensification of agricultural inputs (23). Pot studies involving the use of split compartments separated from each other by sealed nylon meshes have clearly demonstrated that tillage suppresses the effectiveness of AMF by destroying the hyphal network that develops in soil in association with the previous mycorrhizal crop (8, 22, 24).

In no-till and reduced-tillage systems, maintenance of the integrity of this hyphal network contributes to more rapid AMF infectivity and more efficient nutrient uptake than is possible in more severely disturbed soils. In soils severely disturbed by tillage, the native AMF populations are not likely to initiate AMF formation on the target crop rapidly, and the process can be enhanced by inoculating the soil with high-quality AMF inoculum.

Sources of amf inoculum

Soil as inoculum

Soil from the root zone of a plant hosting AMF can be used as inoculum. Such soil inoculum is composed of soil, dried root fragments, and AMF spores, sporocarps. and fragments of hyphae. Soil may not be a reliable inoculum unless one has some idea of the abundance, diversity, and activity of the indigenous AMF.

An additional concern with the use of soil as inoculum is the possible transfer of weed seeds and pathogens with the soil. Figuring out how much soil to add as inoculum to a growth medium or a field is another challenge, because the abundance and viability of AMF propagules in the soil is often uncertain. Soils are thus AMF inoculum sources of last resort, and their use should be avoided if other types of inoculum are available.

Crude inoculum

Crude inoculum is obtained after a known AMF and a suitable host are grown together in a medium optimized for AMF development and spore formation. Such inoculum is the most common type available for large-scale crop inoculation. It consists of spores, fragments of infected roots, pieces of AMF hyphae, and the medium in which the inoculum was produced.

Root inoculum

Infected roots of a known AMF host separated from a medium in which crude inoculum was produced can also serve as a source of inoculum.

Inoculum storage

Both root and crude inocula must be dried to a moisture content of less than 5% before they are stored. We recommend that inoculum be stored in closed plastic containers in a dehumidified room at 22°C. We have been able to store high-quality crude inoculum at 22°C for up to two years with minimal loss in viability. It is possible to extend the shelf life of root inoculum through cold storage (41). However, this can add substantially to the cost of inoculation.

Inoculum application

Methods of applying AMF inoculum include mixing inoculum with soil, placing inoculum as a layer at various soil depths, applying it as a core below the seed, banding it in much the same way as fertilizers are applied in bands, dipping roots of seedlings in a viscous suspension containing AMF propagules, and placing AMF propagules adjacent to roots at the time of transplanting.

Mixing inoculum thoroughly with the soil is the most straightforward method of applying inoculum in the field as well as in the greenhouse, but it is effective only when large amounts of inoculum are applied. This approach is better with crude inoculum than it is with root inoculum, because root fragments do not readily disperse in soil. Inoculum can be placed at various depths (up to 5 cm) from the surface of the soil as a layer or applied in bands near the seed row (generally 5 cm below and 5 cm to the side of it).

Any type of inoculum can be placed close to seedling roots at the time of transplanting. For example, spores can be applied directly onto roots either at the time of transplanting or to roots of an established plant after making a hole adjacent to the roots. Crude inoculum and root inoculum can also be applied to established plants by placing inoculum in holes bored into the soil where roots are likely to be contacted. Before planting, seedling roots can be inoculated by dipping them in a viscous medium (1% methyl cellulose or 10--20% gum arabic) containing AMF propagules, usually spores.

Seed application of AMF inoculum is rare, but has been tried with citrus in Florida with variable results and with Leucaena leucocephala at the University of Hawaii (MH, unpublished data).

Amount of inoculum to apply

The amount of inoculum to apply directly to soil is dependent on the quality of the inoculum. If a crude inoculum contains four to eight infective propagules per gram, application of 50 g/kg soil usually produces rapid initiation of AMF colonization of target plants with a minimal lag period. Root inocula are generally more effective in stimulating plant growth in quantities substantially lower than are normal for crude inocula. Our investigations (MH, unpublished data) showed that if root inoculum contains 4000 cm of infected root per gram, application of 0.5 - 1 g/kg of medium produced good results.

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Original source

This article was adapted from:

Habte, M. and N.W. Osorio. 2001. Arbuscular Mycorrhizas: Producing and Applying Arbuscular Mycorrhizal Inoculum. College of Tropical Agriculture & Human Resources (CTAHR), University of Hawaii at Manoa.

This publication answers common questions about AM fungi and provides information that will enable interested individuals to produce and then evaluate AMF inocula with minimal external assistance.

For information on purchasing the book, contact: Publications and Information Office CTAHR-UHM 3050 Maile Way Gilmore Hall 119 Honolulu, HI 96822 Tel: 808-956-7036, Fax: 808-956-5966 Email: ctahrpub@hawaii.edu. Web site: ctahr.hawaii.edu/ctahr2001/PIO/ForSalePubs.html

The authors wish to announce an upcoming workshop in producing and evaluating arbuscular mycorrhizal inoculum at the University of Hawaii, Manoa in the summer of 2002. For further information contact Dr. Mitiku Habte, Department of Tropical Plant and Soil Sciences, 1910 East West Road, University of Hawaii, Honolulu, HI 96822; Tel: 808-956-6498; Fax: 808-956-6539; Email: mitiku@hawaii.edu.

About the authors

Dr. Mitiku Habte is Professor of Soil Science at the Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa. His interests include the interactions of beneficial soil microorganisms (rhizobia, cyanobacteria, P solubilizing microorganisms, arbuscular mycorrhizal fungi) with economically and ecologically important plant species. The emphasis of his work is on arbuscular mycorrhizal fungi (AMF), with the goal of contributing to the understanding of the ecological interrelationships involved in order to manage populations of the fungi for enhanced plant productivity, and maintenance of soil and environmental quality. Address: Dr. Mitiku D. Habte, Professor of Soil Science, University of Hawaii at Manoa, Department of Tropical Plant and Soil Sciences, 3190 Maile Way, St. John 102, Honolulu, HI 96822, USA; Tel: 808-956-6498; Fax: 808-956-6539; Email: mitiku@hawaii.edu.

N.W. Osorio is a graduate student at the Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa. The focus of his dissertation research is the interaction of arbuscular mycorrhizal fungi with phosphate solubilizing microorganisms.