Overstory #131 - Microsymbionts
Microsymbionts encompass soil-living organisms that form symbiosis with plant roots. There are three types of organisms that are important for cultivated plants: mycorrhizas, rhizobia, and frankiae. Mycorrhiza (meaning 'fungus-root') is formed by virtually all forest trees. Many trees grow poorly, especially under infertile soil conditions, without a mycorrhizal symbiont. A large group of important forest and agroforestry trees of the legume family (Leguminosae) depends on the bacterial symbionts, rhizobia (largest genus Rhizobium), which cause the formation of nitrogen-fixing root nodules. Some trees like Alnus and Casuarina species form nitrogen-fixing symbiosis with the bacteria Frankia. The bacterial associations rhizobia and Frankia are exclusively linked to nitrogen fixation while mycorrhiza play multiple roles in nutrient uptake (mainly phosphorus) and in protecting roots from infection and stress. Many leguminous and actinorhizal (associated with Frankia) trees depend on an association with both mycorrhiza and rhizobia or Frankia and must be inoculated with both.
Microsymbionts are often present in the soil at the planting site if the site has borne trees of the same or a closely related species within a fairly recent past. In these cases seedlings will normally be infected and form symbiosis with the organism soon after outplanting. Where forest soil is used as sowing or potting medium, seedlings may easily be inoculated via the soil, and some types of microsymbiont may be naturally dispersed to the nursery plants from other host plants or from a closely located forest. However, in modern nursery and planting practices, microsymbionts are often absent, and must consequently be applied by active inoculation, e.g.:
- Where species are grown on a site for the first time, and the species need specific types of symbiont not likely to be naturally present.
- Where seedlings are raised on sterile medium such as vermiculite or fumigated soil.
- Where planting is undertaken on denuded and eroded land, poor in nutrients and depleted of natural soil microsymbionts. Generally the survival of symbionts is short when their host species has disappeared.
Failure to establish appropriate symbiosis may cause complete crop failure, or production may be very low, especially on poor soil. On the other hand, productivity may increase significantly by using selected inoculant species or strains instead of naturally occurring ones. For example, in Pseudotsuga menziesii, wood production in trees inoculated with a superior strain was more than 100% above the naturally inoculated control after an 8-year study period (Le Tacon et al. 1992). In Paraserianthes (former Albizia) falcataria, the best Rhizobium strain gave 48% better height growth than the poorest strain (Umali-Garcia et al. 1988). Smaller, yet significant differences have been found between different strains of Frankia on inoculation of Casuarina (Rosbrook and Bowen 1987) and Alnus species (Prat 1989).
Because microsymbionts are associated with established trees and often species specific, they are often conveniently collected at the same time as the seed. Since application ('inoculation') is normally undertaken in connection with propagation (whether vegetatively or by seeds), microsymbiont management forms a natural extension of seed handling, and often runs parallel with seed handling. Many forest seed centres, seed banks and other seed and propagule suppliers, who collect, store and distribute seeds and propagules also supply inoculants. Effective management of microsymbionts implies the technical skill of and facilities for identification, collection, extraction, propagation, storage, distribution and inoculation. Detailed descriptions and guidelines have been elaborated for many temperate species, for mycorrhizae especially on pines, for rhizobia especially for agricultural crops. Many of these methods can be generalised to other species and conditions.
Terminology and classification
Microsymbionts are either bacteria or fungi that form a close association with a host plant. The association is denoted a symbiosis, which strictly means 'living together', but often implicitly means 'to mutual benefit'. Microsymbionts infect the feeder root of the host. However, unlike pathogenic infection there are no disease symptoms, and in contrast to a parasitic infection there is a two-way benefit, a nutrient exchange: the plant provides the infecting organism with photosynthesates (e.g. sugar); the microsymbiont in turn provides nitrogen or phosphorus depending on infection type. In the two types of bacterial symbiosis the infection is concentrated in special parts of the root, where the host plant forms root nodules, which are bacterial colonies surrounded by host tissue. The symbioses exist both in herbal and woody plants, and many plant species have both bacterial and fungal symbiosis.
Fungal symbionts are the mycorrhizas, which form the most wide-spread symbiosis between plants and microorganisms. There are two types of bacterial symbionts: rhizobia, named after the most important genus, Rhizobium, which forms symbiosis with host species of the family Leguminosae, and frankiae with the one genus, Frankia, which lives in association with a number of tree species from different families. Frankiae are actinomycetic bacteria which infect roots of their host plants; therefore the hosts are collectively called actinorhizal plants.
Mycorrhizal symbiosis functionally forms an extension of the plant root system. A fine net of fungal hyphae in close contact with the plant roots extend their threads into a large volume of soil where they explore and extract nutrients from the soil beyond the reach of the plant roots. The nutrients are translocated through the fungal hyphae, hence bringing them to the plant roots, where they can be assimilated and used by the plant. The fungus, in return, is provided with simple sugars and possibly other compounds from the plants' photosynthesis. Some mycorrhizal fungi produce plant hormones, which stimulate root development, e.g. Pisolithus tinctorius on poplars (Navratil and Rachon 1981).
Mycorrhiza is known to protect the roots of the host plant against pathogens and certain toxins, and mycorrhizal plants generally have a higher resistance to drought, soil acidity, and high soil temperatures (Redhead 1982). The fungal sheath surrounding the feeder roots of ectomycorrhiza often has a higher resistance to toxins (acids, etc.) than the plant root and can consequently form a physical barrier to the soil. Further, soil will adhere to the mycorrhizal net thereby decreasing 'shock' when the seedlings are exposed to field conditions; that is especially important for bare-root seedlings, where mycorrhiza may also reduce the risk of desiccation of the roots during transportation. Mycorrhizal symbionts are grouped into two main types according to the symbiotic structure of the root system: ectomycorrhiza and vesicular-arbuscular mycorrhiza (VAM).
Rhizobia are a group of soil-living bacteria, which are able to live in symbiosis with and nodulate members of the plant family Leguminosae. Leguminosae is subdivided into three subfamilies, Caesalpinoideae, Mimosoideae and Papilionoideae1. More than 30% of species of the Caesalpinoideae and more than 90% of the species in the other subfamilies form nodules (Brewbaker et al. 1982, Dart 1988). Within the subfamilies some genera are characterised by high frequency of nodulated species and others by low. There are also species within an otherwise highly nodulating genus which fail to nodulate. Most acacias, for example, nodulate but there are exceptions (Dommergues 1982). The species-specific capability of nodulation and N-fixation is, however, subject to uncertainty since many species capable of nodulation do not form nodules in some areas, either because of absence of the proper symbiont or because environmental conditions are unfavourable to the symbiosis. There are also differences between provenances in their susceptibility to nodulation by rhizobia (Dart 1988).
Some rhizobium - legume associations are very specific and the legume will form nodules only when infected with a specific rhizobium. Others will form nodules with a range of rhizobia. That means in practice that for the first group, inoculants must be collected from the same host species, for the second group a broad range of host species can be used as inoculant sources. Therefore, for practical purposes, legumes have been assembled into cross-inoculation groups. A cross inoculation group consists of species that will form nodules when inoculated with rhizobia obtained from nodules from any member of the group. A cross inoculation group may, in the extreme, consist of one species only. Cross inoculation groups are well established among agricultural crops but only superficially established among tree crops.
Obviously, host-specific rhizobia must be applied as inoculant when the host species is grown on a site for the first time. For other species the requirement depends on the possible available rhizobium in the soil, that is, whether other compatible legume hosts have grown on the site within a fairly recent past. Some Australian Acacia spp. grown in Africa nodulate freely with the indigenous rhizobia.
Frankia are bacteria which infect roots of their host plants; the hosts are collectively called actinorhizal plants. Frankia are filamentous, branching, aerobic, gram-positive bacteria. They differentiate into three different cell types viz. (1) vegetative cells which develop into mycelia almost like mycorrhizal fungi, (2) sporangia forming numerous spores, and (3) vesicles which are the site of nitrogen fixation (Lechevalier and Lechevalier 1990).
Frankia may live free in the soil as saprophytes. They are dispersed in the soil via the vegetative hyphae. The long-distance dispersal probably takes place via spores or vegetative cells in moving soil or by wind dispersal of spores; spores are relatively resistant to desiccation (Torrey 1982).
Frankia form symbiosis with plant species from a number of distinct genera and families, many of which have no close taxonomic relation. So far, around 200 actinorhizal plants are known, distributed over 8 families and 25 genera. The most important forest trees with symbiotic relationship with Frankia belong to the plant family Casuarinaceae, a family that comprises almost exclusively actinorhizal plants. Apart from Casuarina, the family includes two other actinorhizal genera viz. Allocasuarina and Gymnostoma. Betulaceae contains only one actinorhizal genus, Alnus. The genus Rubus contains only one known actinorhizal species viz. Rubus ellipticus (Gauthier et al. 1984). Frankia also form symbiosis with species of the genera Aelaeagnus and Hippophae.
Some actinorhizal plants can be inoculated with a range of Frankia strains while others are very specific. For example the genus Allocasuarina can be inoculated with strains obtained from that genus only, while Gymnostoma are the least specific one and can be inoculated with inoculants even from species outside Casuarinaceae; Casuarina spp. are intermediate between those two in terms of specificity (Torrey 1990, Gauthier et al. 1984).
Collection and handling
Mycorrhizae, rhizobia and frankiae are soil-living organisms and spend their entire or the greater part of their life cycle under the soil surface. They are adapted to moist, dark and relatively cool conditions with small temperature fluctuations. These conditions should be maintained during handling. Some microsymbionts form dispersal units, e.g. spores which are relatively resistant to above-soil conditions. They can survive desiccation, higher temperatures and light and have relatively long viability. Generally however, the viability of most microsymbionts is short in comparison to seeds, but there is a great variation between species within the three types. Proper handling and storage conditions can greatly improve the viability of microsymbionts.
Where inoculant material, whether soil, nodules or spores, is collected from the field, a site with mature, healthy and vigorous trees should be selected. Mature trees are likely to support the largest amount of symbionts, healthy and vigorous trees may also be an indication of good inoculation, and the risk of collecting material infected with pathogens, which could be a nuisance later on, is smaller.
Collection should be made from or under trees of the same species or species with compatible microsymbionts, for rhizobia and Frankia within the same cross inoculation group (Baker 1987). Collection should be made from trees growing on typical growth sites; these are likely to contain symbionts adapted to the prevailing soil type. Exceptionally good or poor sites should be avoided unless the trees to be inoculated are supposed to be grown on similar sites (Benoit and Berry 1990).
The best time of collection differs for different types of inoculant material. Soil usually contains a reasonable amount of inoculant and can be collected at almost any time of the year. Sporocarps of ectomycorrhizal fungi are only available for a short period of the year. The moist season normally supports the greater number of sporocarps, but both duration and season of sporocarp formation vary with species. Inoculant collected from or together with host roots should generally be collected during the most active growth season, which normally is the rainy season. This is also practical as the soil is easier to dig up and there is less risk of damage to both the host tree and the inoculant.
Rhizobium nodules should preferably be collected from young roots. The nodules of older roots are likely to be senescent and contain few infective bacteria. Seedlings or young trees are the best source of nodules. Cutting and examining the interior of a few nodules with a hand lens gives an indication of the condition: fresh and active N2-fixing rhizobium nodules are typically pink, red or brown, Frankia whitish or yellowish; senescent nodules are typically greyish green (Benoit and Berry 1990).
Inoculant types and inoculation techniques
Inoculant types vary from simple forms in which microsymbiont-infected soil is applied to the nursery soil, to sophisticated production of pure culture inoculants, incorporated into carriers and applied to seeds as pellets or beads. Which species and method is used is a result of balanced consideration of various factors:
- Some commercial pure culture inoculants contain microsymbiont species which promote productivity under particular environmental conditions, but may be less productive than local species under other conditions.
- Different methods of inoculant production and inoculation apply to different species and situations. Some tree species may only form symbiosis with specific bacteria or fungal species. Sometimes compatibility between the two organisms varies with the environment.
- Pure culture production is usually both technically complicated and expensive. In many cases, inoculants purchased from specialised manufacturers and dealers may be more economical than starting independent production or using unselected material.
Apart from soil mixtures which may contain all types of organisms, both type of inoculant and application methods vary with type of symbiont. Mycorrhizal inoculants can be applied as spores or mycelium. Mycelium inoculates usually give faster infection but are more sensitive to desiccation and other environmental factors. They have short viability and are relatively bulky as compared to spores. Some ectomycorrhizal fungi can be grown in pure culture on a nutrient medium to obtain a mycelial culture. The spores are often initially germinated on agar prior to cultivation. Some ectomycorrhizal fungi can be multiplied by applying spores directly to the nutrient medium (Marx 1980).
VAM fungi cannot be grown in pure culture on nutrient media and are therefore multiplied by infecting roots of an intermediate host e.g. sorghum or sweet potato with the spores of VAM. Both rhizobia and Frankia can be grown in pure culture but the method is too slow and too expensive for most Frankia. Many plants need dual inoculation with mycorrhiza and either rhizobia or Frankia. Generally the two types of organism are not antagonistic to each other and can sometimes be mixed. However, in many cases it is difficult to control the application rate if the two inoculants are mixed, and they are therefore usually handled separately throughout.
Inoculation rate, i.e., the amount of inoculant used per seedling, varies with application method, and the concentration of infective bacteria, spores or mycelium in the inoculant. Increasing amount of applied inoculum generally speeds up the colonisation process and symbiont formation. Plants are usually inoculated in the nursery rather than during planting in the field. Nursery inoculation has the following rationale:
- Inoculated seedlings are generally much more competitive and able to withstand the inevitable stress they will be exposed to immediately after outplanting especially if the plants are planted under harsh environmental conditions.
- Early inoculation usually reduces requirement for fertilizer and pesticides in the nursery. In addition to reducing the cost and possible harmful effects of these applicants, mycorrhizal seedlings are known to be generally more resistant to pests, diseases and adverse environments.
- Nursery inoculation opens the potential for selective inoculation with superior microsymbiont strains or types, specifically adapted to the species and the planting site and is hence potentially more effective (Trappe 1977, Marx et al 1982).
Field inoculation has its main advantage in that the seedlings are exposed to the future environment when inoculated and may consequently preferably form symbiotic association with the species that are better adapted to that particular field condition. It is known for mycorrhiza that even if seedlings are inoculated in the nursery, fungal associations often change when the plants are transplanted into the field, provided that a microsymbiont is present at the planting site (Marx et al. 1982). Where seedlings are inoculated with several species or strains, one or few usually become dominant under the prevailing field conditions.
Forest soil or litter collected under appropriate tree species often contains a balanced population of adapted microsymbionts. As freshly collected forest soil is often used as planting medium because of its physical properties, seedlings are naturally exposed to inoculation in this way. It may also be used deliberately as inoculation material, e.g. by applying a small amount of inoculated soil or litter (leaves, needles and root fragments) to the planting medium. This method is often used for mycorrhizal inoculation, whereas for rhizobia and Frankia the amount of inoculant provided this way is often too small. Soil collected from nursery beds previously supporting seedlings with good mycorrhiza is appropriate. About 10 - 15% by volume of soil is mixed into the top approximately 10 cm of the nursery bed or mixed in the same ratio into the potting soil (Molina and Trappe 1984). If soil is scarce, a handful of soil can be placed at root level during pot filling.
Problems of using soil as inoculant material are the bulk to be transported and that soil may contain infective pathogens. Fumigation and other soil sterilisation normally kill microsymbionts.
Infected roots and nodules
Problems with bulk and potential pathogens may be reduced by collecting infected mycorrhizal roots and bacterial nodules only. Roots are chopped and nodules crushed before application. However, for mycorrhiza relatively large quantities are needed if the material is used as inoculant directly. Cruz (1983) estimated that at least one kg of ectomycorrhizal roots should be used per cubic meter of nursery soil to assure proper inoculation. VAM is often applied as chopped roots of intermediate hosts after being multiplied in pot culture. Crushed nodules are rarely used as inoculant for rhizobia because the number of bacteria that can be applied in this way is too small. The nodules are more often used as a source for cultured inoculants. Because the root nodules of Frankia are relatively large, and because culture of Frankia is slow, crushed nodules are often used directly as inoculant for this type. Both fresh and stored Frankia nodules can be used, but dry nodules should be rehydrated before crushing.
The principle of 'nurse seedlings' is that microsymbionts from already inoculated seedlings will spread naturally to neighbouring seedlings in the nursery. Hence a precondition is that there is likelihood of movement in the soil which is the case with both ectomycorrhiza and VAM. The inoculated seedlings are planted in the nursery bed at intervals of one to two meters before the seeds are sown. Mycorrhiza will spread from the infected to the newly germinated seedlings. Alternatively, chopped roots of mycorrhizal seedlings are incorporated into the soil of the nursery bed (Castellano and Molina 1989, Cruz 1983, Marx 1980, Mikola 1970, and Molina and Trappe 1984).
The main advantage of nurse seedlings is that fresh inoculant material is always available and that the inoculant is adapted to the prevailing climate and nursery soil. However, the method also has certain drawbacks:
- The nurse seedlings may compete with the young established seedlings for nutrients and light.
- The nurse seedlings may interfere with the preparation and management of the seed bed.
- Inoculation may be slow and uneven.
- Fumigation or other soil sterilisation procedures cannot be undertaken after the nurse seedlings have been planted. Therefore there is a higher risk of soil pathogens and competition with naturally disseminated mycorrhizal fungi.
Baker, D.D. 1987. Relationships among pure cultured strains of Frankia based on host specificity. Physiologia Plantarum. 70: 2, 245-248.
Benoit, L.F. and Berry, A.M. 1990. Methods for production and use of actinorhizal plants in forestry, low maintenance landscapes, and revegetation. In: The Biology of Frankia and Actinorhizal Plants (Schwintzer, CR. and Tjepkema, J.D., eds.). 281-298. Academic Press.
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Castellano, MA. and Molina, R. 1989: Mycorrhiza. In: The container tree nursery manual, Vol. 5. Agric. Handbook 674. (Landis, T.D., Tinus, R.W., McDonald, SE. and Barnett, J.P., eds.). 101-167. US Department of Agriculture, Forest Service. Washington DC.
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Dommergues, Y.R. 1982. Ensuring effective symbiosis in nitrogen-fixing trees. In: Biological Nitrogen Fixation Technology for Tropical Agriculture. (Graham, P.H. and Harris, S.C., eds.). 395-411.
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Marx, D.H. 1980. Ectomycorrhizal fungus inoculations: a tool for improving forestation practices. In: Tropical Mycorrhiza Research. (Mikola, P., ed.). 13-71. New York: Oxford University Press.
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Torrey, J.G. 1990. Cross-inoculation groups within Frankia and host endosymbiont association: In: The Biology of Frankia and Actinorhizal Plants. (Schwintzer, C.R. and Tjepkema, J.D., eds.). Academic Press. 83-106.
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Umali-Garcia, M., Libuit, J.S. and Baggayan, R.L. 1988. Effects of Rhizobium inoculation on growth and nodulation of Albizia falcatarta (L.) Fosh. and Acacia mangium Willd. in the nursery. Plant and Soil 108, 71-78.
This article was adapted with the kind permission of the author and publisher from:
Schmidt, L. 2000. Guide to Handling of Tropical and Subtropical Forest Seed. Danida Forest Seed Centre. Humlebaek, Denmark.
This exceptional guide covers forest tree seed handling from scientific, practical and administrative perspectives. Much of this text is available online at: http://www.dfsc.dk/Guidechapters.htm.
For further information about the book and a wide range of other publications contact:
About the author
Lars Schmidt is Chief Technical Adviser of the Indonesia Forest Seed Project, a Danish-Indonesian support project to the Indonesian forest seed sector. Lars is a biologist specialising in tropical forest ecosystems and tropical forest seed. He has been adviser to international and bi-lateral forestry projects in Malawi, the Philippines and Indochina. In Indochina he was Technical Adviser for Vietnam, regional training adviser and regional coordinator on conservation of Forest Genetic Resources. He is presently on leave from Danida Forest Seed Centre, Denmark. His publications include mainly technical guidelines and articles. Address: Indonesia Forest Seed Project, Taman Hutan Raya Ir. H. Juanda No. 120, Dago Pagar, Bandung 40198, Jawa Barat, PO Box. 6919 Bandung 40135, Indonesia. Tel/fax: 62-22-2515895. E-mail: firstname.lastname@example.org.
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