P.J. Dart
Introduction
Non-symbiotic Nitrogen Fixation
Symbiotic Nitrogen Fixation
Mycorrhizae
Conclusion
References
Many forest trees are legumes which nodulate with the bacterium Rhizobium (fast growing) or Bradyrhizobium (slow growing) and fix gaseous nitrogen thereby utilising some of the 84,000 tonnes of nitrogen gas in the air above each hectare of land. There are more than 18,000 species of legumes of which about 7,200 species are woody. Only about 18% of these woody species have been examined for nodulation and of these 92-94% of the mimosoids and papilionoids nodulated, but only about 34% of the caesalpinoids (Allen and Allen 1981, Brewbaker et al. 1982, Dobereiner 1984). It is not easy to determine if tree legumes nodulate as nodules are both difficult to find in forest soils and difficult to assign to a particular tree. Hence, observations on young plants are helpful to assess the nodulation status of the plant.
Besides legumes, the humid tropical forest genus Parasponia (family Ulmaceae, order Urticales) also nodulates and fixes much nitrogen with a specific group of Bradyrhizobium bacteria (Trinick and Hadobas 1988). However, the closely related Trema spp., Celtis spp., Ulmus parviflora and Gironniera subequalis do not appear to nodulate.
Nodulated plants also occur in six other families; about 160 species are involved. The nodule forming endophyte is an actinomycete bacterium known as Frankia which can now be grown in pure culture and used as an inoculum. The best known of these species in tropical situations are Casuarina equisetifolia, C. junghuhniana, C. cunninghamiana, Alnus nepalense and Eleagnus philippensis.
Biological nitrogen fixation also occurs through non-symbiotic bacteria growing on roots and in degrading litter; blue-green algae or cyanobacteria on soil and plant surfaces; and associations of cyanobacteria with fungi and lichens, or with higher plants such as liverworts, mosses, cycads and the angiosperm Gunnera. Most of the nitrogen in forest ecosystems is derived from biological nitrogen fixation. These systems are very efficient in recycling nitrogen leached to lower depths in the soil through uptake by deep roots, and through leaf fall, concentrating this nitrogen in the litter and upper soil horizons.
Disturbing this natural cycle which conserves scarce nutrients so effectively can lead to rapid loss of soil fertility. Maintenance of the litter layer as a soil mulch to reduce erosion as well as to conserve nutrients is a very important aspect of maintaining soil fertility around trees and shrubs.
Inoculant strain selection
There are varying degrees of specificity in the requirement of tree species for effective rhizobium strains. There are even differences among provenances in the effectiveness of particular strains. Soils contain several types of rhizobium, and in some soils, populations of appropriate strains may be absent or too small for nodulation to occur. It is in these situations where a response to nodulation inoculation with rhizobium might be expected. Strains for use as inoculants therefore need to be selected for a particular legume accession (Table 3.4.1). This process usually starts with the assembly of a collection of strains by isolation from nodules, usually obtained from the legume under consideration. This is followed by an assessment of their ability to fix nitrogen in a strain trial in pots using a rooting medium which does not contain rhizobia.
Tree legumes are nodulated by two kinds of rhizobia. One type now known as Bradyrhizobium is slow growing on laboratory media, but is the prevalent kind of rhizobium in most tropical soils, nodulating many tropical plants effectively. All three legume families are nodulated by Bradyrhizobium. Another group is the fast growing Rhizobium type which produces more gum and larger, more distinctive colonies than Bradyrhizobium strains. Colonies of such fast-growing strains appear on agar about three days after inoculation compared with seven or more days for the slow growers. Fast growing Rhizobium strains have been isolated from Mimosa caesalpiniaefolia, Mimosa invisa, Mimosa pudica, Leucaena leucocephala, Acacia farnesiana and species of Sesbania, and form a distinctive preferred host group (Campelo and Dobereiner 1969, Trinick 1980). Both fast and slow growing rhizobia nodulate several Acacia species, but some are specific for either fast or slow growers (Dreyfus and Dommergues 1981, Roughley 1987). The non-legume Parasponia seems to be nodulated by a distinctive group of Bradyrhizobium strains (Trinick and Hadobas 1988).
Table 3.4.1. Total dry matter yield of five lines of Sesbania sesban inoculated with two strains of Rhizobium or fertilised with inorganic nitrogen (M. Masafu, unpublished data).
|
S. sesban line |
Rhizobium strain |
Nitrogen treatment | |||
|
|
PMA-295/2 |
CB-3023 |
+N* |
-N |
Mean |
|
9266 |
8.6 |
8.8 |
19.9 |
7.4 |
11.2 |
|
10895 |
9.7 |
6.4 |
19.2 |
6.6 |
10.2 |
|
15022 |
7.0 |
8.4 |
18.6 |
6.6 |
10.2 |
|
15036 |
9.2 |
7.4 |
18.9 |
6.7 |
10.6 |
|
30071 |
14.0 |
6.8 |
18.5 |
6.5 |
11.5 |
|
Mean |
9.7 |
7.6 |
19.0 |
6.6 |
|
LSD (P < 0.06) Rhizobium and nitrogen means = 1.0 g; line means = 1.1 g; interaction 2.1 g.
* +N = 42.5 kg N/ha
Nodules that are nitrogen-fixing contain haemoglobin very similar to the pigment in muscles. This haemoglobin is concerned with maintaining an adequate oxygen supply to the bacteria at low oxygen tension to maintain the nitrogen fixing process which is sensitive to oxygen. The colour of a sliced nodule is thus a useful indicator of its nitrogen fixing activity. Brown, green or white nodules are probably non-fixing while red or pink nodules are usually formed by strains effective in nitrogen fixation. The more red the colour of a legume nodule, usually the more active it is.
Sometimes soils contain strains which nodulate but which are not very effective in fixing nitrogen. In these situations, inoculating plants with a superior strain may supplant the indigenous soil population in forming the nodules. This is not easy because only a small number of bacteria can be added in the inoculum relative to the large soil population of rhizobia. To overcome competition from the indigenous population, the inoculum strain needs to have an intrinsic competitive ability to form nodules and to be able to colonise root systems readily, so that the strain is present in the zone of the root susceptible to nodulation.
Legumes are often grouped into species which reciprocally nodulate with a 'homologous' group of rhizobium strains isolated from those species (the cross inoculation group). 'Heterologous' strains isolated from outside this group usually do not initiate infections within this group.
Rhizobium infection and nodule development
Root infection and nodule development involves colonisation of the rhizosphere by rhizobia, their attachment to the root at the site of infection, and uptake of plant flavonoids by rhizobium that modify gene expression to initiate production of specific factors enabling infection and nodulation to occur. Rhizobia induce specific changes in plant cell wall development at this stage that enable the bacteria to cross the cell wall barrier.
In the first pattern of infection, rhizobia invade the small emerging root hairs by causing them to curl during growth and enclose a rhizobium microcolony. This then induces production of an infection thread within the root hair cell. The infection thread grows into the root cortical cells carrying the rhizobia.
The second method of infection occurs through cortical cells or the epidermal cells, which do not form root hairs, usually at the break in the cortex integrity caused by the emergence of a lateral root. The rhizobia invade the root growing in the intercellular spaces, a microcolony causes the cortical cell wall to thin and grow around the bacteria, depositing cell wall materials on the internal wall surface, and the bacteria appear to grow through this barrier. Once released, the rhizobia induce host cell division and divide along with the chromosomes in the process. All the invaded cells are then produced by continued host cell division. Nodules produced in this way usually have a round appearance. Parasponia nodules seem to be formed in this way.
Most tree nodules appear to develop from infection threads, and are also characterised by a terminal meristem which continues to form cells for nodule growth. Behind the meristem the expanding cells are invaded by infection threads which release the rhizobia. The rhizobia then divide and multiply, at the same time remaining enclosed within a plant derived membrane. This membrane may contain one or several bacteroids which may be rod shaped but for some associations they may be very enlarged and pleomorphic. Only a small proportion of the nodule cortical cells in some tree nodules is invaded. Little is known about the effect this has on nodule performance. Because of continued growth, perennial nodules can be very large (more than 3 cm long), often with several meristems developed from the original one, giving the nodule a coralloid appearance.
Control of nodule number and nodule senescence
The development of a root nodule involves a complex and continuing exchange of signals between host plant and bacterium. It is a finely tuned interaction and the process may abort at any stage. Nodulation is under the genetic control of both partners but little is known of the way in which one symbiont modifies the expression of genes in the other partner.
The existing nodules on a root seem to influence the formation of other nodules. For soybean this appears to be through a shoot factor which translocates to the root to control further nodulation. For some tree species, the level of this control varies from plant to plant and between species. Thus nodule number on Acacia mangium may vary from about 200 per 8 month old plant to several thousand nodules on older plants. Nodulation on plants with many nodules seems to be more robust in the face of stresses than on plants with fewer nodules.
For some species there is a cyclical growth of nodules in the rainy season followed by senescence and decay in the dry season, the senescence starting at the base of the nodule in the oldest formed cells which turn green or brown due to chemical changes in the haemoglobin (Homchan et al. 1989). The degeneration continues to the meristem, and the nodule then dries out leaving an empty shell which sloughs off the root, releasing rhizobia back into the soil. Nodules will only form again when new roots develop. Little is known about the factors resulting in nodule senescence or the perenniality of other nodules. The pattern of nodulation is influenced to a certain extent by the rhizobium strain; some less effective strains form nodules which senesce earlier, but the process seems to be host plant determined.
Many tree legume nodules are characterised by a thick protective husk layer containing a peripheral sheet of suberised and thickened cells. The husk cells also contain much tannin. Both characteristics are presumably protective adaptations against desiccation and pest attack. Both NH4+ and NO3- at high concentrations in soil solution inhibit nodulation of many legume species including some tree species such as Leucaena and Albizia. However, small amounts of combined nitrogen can be synergistic with nodulation in providing for the nitrogen requirements of plants. Very little is known of the tolerance of tree species to soil nitrogen levels, although nodulation of L. leucocephala is stimulated by small amounts of nitrogen fertiliser and inhibited by large amounts (see Figure 2.1.2). Adding small amounts of nitrogen fertiliser (30-50 kg N/ha) may boost seedling growth and produce a more extensive nodulation. In an experiment with 20 Acacia species grown in an acidic soil (pH 4.5 in water), nitrogen fertiliser applied at 100 kg N/ha produced more rapid growth than plants which were unfertilised, relying on nodulation for their nitrogen supply. This was partly a reflection of the long time taken to form nodules on tree seedlings so that the N supply from nodules was only available over about half the 5 month growth period (N. Ashwath, P.J. Dart and D.G. Edwards, unpublished data).
Response to inoculation
In recent experiments in northeast Thailand, inoculation of L. leucocephala at six sites with the highly effective Rhizobium strain TAL 1145 resulted in most of the nodules being formed by the inoculant strain over the first 72 weeks (Homchan et al. 1989). Virtually no nodules were formed in the uninoculated plots in the first 20 weeks. At 20 weeks, inoculated plants were between 66 and 280% larger, depending on the site. The strain TAL 1145 proved to be very effective in nitrogen fixation and well adapted to the soils in six of the eight sites tested, moving to uninoculated plots and forming nodules. At 72 weeks, there was no apparent effect of inoculation on plant dry matter production and height (Homchan et al. 1989). However, the increased vigour in the establishment phase would justify the effort and small cost of inoculating plants. By 64 weeks at some sites, some indigenous populations of rhizobia had developed sufficiently to nodulate the Leucaena as well as the inoculant strain.
In a similar trial in peninsular Malaysia on four acid soils, the inoculated plants were also much better nodulated in the early stages of growth up to about 200 days after planting when plants became too large for further meaningful sampling. The response to inoculation resulted in a substantial increase in plant growth and nitrogen uptake at two of the four sites over the first 300 days (Chee et al. 1989).
Several important trees and shrubs are nodulated by specific strains of Rhizobium Sesbania sesban lines differed in their response to two strains of Rhizobium with a marked strain x line interaction (M. Masafu, unpublished data) (Table 3.4.1). Thus it may be necessary to select specific strains to effectively nodulate lines or cultivars within a species. Acacia species appear to be nodulated by a diverse range of rhizobia which may be fast growing Rhizobium or slow growing Bradyrhizobium species. Only a restricted range of rhizobia will nodulate some species (e.g. A. holosericea, A. cincinnata, A. polystachya). Other species such as A. auriculiformis are more promiscuous in their nodulation habits, nodulating with a range of Rhizobium and Bradyrhizobium strains present in many tropical soils. Strains that infect and nodulate a particular Acacia species often vary a great deal in their effectiveness in fixing nitrogen.
Forty-eight strains of rhizobia were isolated from Acacia species from different countries and were tested for their effectiveness in nodulating and fixing nitrogen with A. auriculiformis and A. mangium in sand culture. Strain PMA 311/1 from northern Australia was outstandingly effective on both, and is now a recommended inoculant strain (Dart et al. 1991).
Acacia mangium is much more specific in its Rhizobium affinities than A. auriculiformis
Habish and Khairi (1970) found that of ten Acacia spp. occurring in Sudan, some were nodulated by Bradyrhizobium and others by Rhizobium spp. Some species nodulated freely with rhizobia isolated from other species, while others only nodulated with a very restricted range of isolates. Dreyfus and Dommergues (1981) extended these observations showing that some Africian species nodulated effectively only with slow growing Bradyrhizobium strains (e.g. Faidherbia albida), whereas others nodulated only with fast growing Rhizobium strains (e.g. A. nilotica, A. raddiana and A. Senegal). Acacia seyal was effectively nodulated by both types of rhizobia. Similar patterns of specificity were obtained for introduced Acacia spp. of Australian origin.
Roughley (1987) showed that a great deal of host species x strain specificity existed among Australian acacias, with some species being very specific, nodulating with a few strains isolated from those species, while others nodulated freely with strains isolated from a wide range of Acacia spp. nodules.
The large degree of interaction between soils, strains and host species indicates that selection of rhizobium strains will be best done on a species by species basis and then tested for provenance interactions.
Moisture, acidity and salinity effects on nodulation
Rhizobia move through the soil with a wetting front after rain. Soil moisture levels affect the ability of rhizobia to move along the root system and to colonise young roots with emerging, infectable root hairs. Thus nodulation is limited in dry soils.
Nodulation of A. auriculiformis and A. ampliceps was much affected by the soil moisture tension in a pot experiment. Pots were watered to weight twice daily to maintain levels of 0.008 MPa moisture deficit (near to field capacity), 0.08 MPa or 0.8 MPa. Virtually no nodules were formed at 0.8 MPa and nodule number at 0.08 MPa was about half that in the moist soil at 0.008 MPa (N. Aswathappa, P.J. Dart, D.G. Edwards and P.K. Kanna, unpublished data).
Trees such as Acacia spp. are often grown in adverse soil conditions and methods of selecting rhizobium and host provenances tolerant of acidity and salinity are currently being investigated.
In a field trial in Malaysia, L. leucocephala strain TAL 1145, selected for acid tolerance, was less competitive in forming nodules than strain CB81. The latter strain was isolated from an alkaline soil and was less acid tolerant in growth in vitro. However, a Malaysian isolate was the most persistent and competitive strain. This suggests that it may be beneficial to select strains for adaptation to particular soils as well as for ability to fix nitrogen. The ability of inoculant strains to form nodules in competition with indigenous populations is an important criterion for strain selection.
There are differences between rhizobium strains nodulating Acacia species in their tolerance of salinity. Strains isolated from nodules grown in saline soil were more tolerant on agar and in broth of higher salt levels than other strains. Some strains could grow in the presence of 2% salt.
Inoculation of Acacia ampliceps, a salt tolerant species, with a salt tolerant strain, resulted in a large increase in nitrogen fixation in the presence of 200 mg salt compared with a non-tolerant strain (N. Zou, P.J. Dart, N. Marcar and C.J.. Asher, unpublished data).
Rhizobium populations are usually small or absent in saline soils, and salinity tolerant strains survive better than intolerant strains when added to such soils.
Rhizobium inoculation techniques
At present, limited seed availability restricts most reforestation and some agroforestry activity to nursery reared plants. This makes it easy to inoculate the plants by dipping the seed before sowing in a slurry of peat carrier plus rhizobium inoculum plus a glue such as 1.5% (w/v) carboxymethylcellulose. Since rhizobia are killed by UV light, the seed should be dried in the shade after inoculation, and planted as soon as possible. Another method is to suspend the peat carrier plus rhizobium in water and sprinkle this mixture over the seedlings.
Since rhizobia are killed by most fungicides and many insecticides it is good practice not to inoculate treated seed using the seed coating method.
At present there is no commercial production of inoculants for forest tree species. Commercial inoculants are available for Leucaena.
Nitrogen fixation
The amount of nitrogen fixed by trees can be estimated in several ways (Peoples et al. 1991, Danso et al. 1992, Ladha et al. 1993). The simplest method is a relative measure based on plant dry matter accumulation. The nitrogen balance method uses the Kjeldahl digestion process to measure N contents of soil and plant material at the beginning and end of the growth period. Alternatively nitrogen fixation is estimated as the difference in N uptake by a nodulated and a non-nodulated control plant. This method is suitable for plants in pots or small plants in the field, but obviously has limited use for larger trees. Variability in soil N content spatially is difficult to handle, requiring many samples to provide an accurate estimate of the overall N level.
The isotope 15N which occurs at an abundance of about 0.366% in air can be used to measure nitrogen fixation in several ways by isotope dilution. These include measurement of 15N uptake by plants grown in an atmosphere containing 15N; comparison of the uptake by fixing and non-fixing plants of 15N containing fertiliser added to soil; and the natural abundance method.
In the natural abundance method, a very sensitive mass spectrometer is required to measure the small differences in 15N and 14N content of fixing and non-fixing plants when no external N is supplied. The method relies on the different ratio of 15N in nitrate and ammonium in the soil and in N2 in the atmosphere. Nitrogen fixing plants will take up more atmospheric N and hence will end up with a different 15N:14N ratio than the non-fixing plants.
Ladha et al. (1993) used this technique to measure nitrogen fixation by Gliricidia sepium in an alley cropping or hedgerow system in the Philippines. About 50% of the nitrogen in the plants was derived from nitrogen fixation except in the dry season when this fell to 30%. Acacia auriculiformis and Paraserianthes falcataria fixed about 55% of their nitrogen uptake. In northern Australia, Desmodium rensonii fixed about 70%, and Gliricidia sepium 26-75%. In Indonesia, Sesbania sesban fixed about 80% (Peoples et al. 1991).
In a glasshouse experiment, nitrogen fixation by L. leucocephala amounted to about 65% of N uptake whereas Faidherbia albida derived about 20% from the atmosphere. Provenances within these species varied a great deal in the proportion and amount of nitrogen fixation (Sanginga et al. 1990). Extrapolation from nitrogen balance experiments and cutting trials in the field suggest that Leucaena can fix up to 300 kg N/ha/year, a very large amount indeed.
Mycorrhizae are beneficial, symbiotic fungal associations with plant roots which benefit plant growth by increasing the effective absorbing zone of the root through the hyphae which explore the soil away from the root surface.
Mycorrhiza benefit growth through uptake of 'immobile' nutrients such as P, Zn, Cu and NH4 usually present in soil in low concentrations in soluble form. The nutrients travel inside the hyphae via cytoplasmic streaming to the root cells where uptake by the plant occurs. In return, the plant supplies carbohydrates and amino acids for the fungal growth. There are two major forms of mycorrhizal development in higher plants - ectomycorrhizal and vesicular arbuscular mycorrhizae (VAM).
Vesicular arbuscular mycorrhizal hyphae found in the top 5 cm of the soil around subterranean clover roots were up to 30 m in length for every centimetre of root length (Abbott and Robson 1985). Ectomycorrhizae can form between 20 and 39% of the weight of tree roots.
Ectomycorrhizae do not penetrate the root cells but form a 'Hartig net' of fungal mycelium round the root, often distorting root growth by increasing lateral number and stunting and thickening the roots. These roots proliferate at the soil surface beneath the litter layer and play an important role in recycling nutrients from the litter back into the trees. Ectomycorrhizae develop on woody plants and are particularly important in establishment of pines commercially.
They are formed by higher fungi, mainly Basidiomycetes and Ascomycetes, and produce above ground fruiting bodies such as truffles. There are about 2,000 species of fungi associated with Douglas fir tree roots many of which are potential ectomycorrhizal fungi. There is some host species fungal species specificity in the effectiveness of the association. Nurseries propagating trees often adopt practices which ensure development of the ectomycorrhizae by using a fungal spore inoculum or mixing forest soil in the root medium in which the tree seedlings are grown.
VAM are much more widespread and can form associations with most herbaceous and many woody species. They are called vesicular arbuscular because they invade the plant root and form characteristic, large, thick-walled vesicles in the roots and invade the cortical cells to form arbuscules or tree-like growths which are the surface for interchange of metabolites between fungus and host plant. VAM form spores, often in specialised sporophyte structures in the soil, and these germinate to form hyphae attracted chemotactically to the root surface, which they then penetrate. The hyphae colonise the root, form arbuscules and develop more hyphae which penetrate the soil. Some groups of plants, such as the Cruciferae, are poorly infected. VAM infections start 2-3 weeks after germination of seedlings.
There are more than 100 species of VAM now recognised in soil. Because the fungi cannot be cultured in vitro, species are classified on the basis of morphological characters such as spore size, wall thickness and form, spore contents, form of the hyphal connection to the spore, vegetative hyphal thickness and form. VAM survive in soils as spores and perhaps hyphae. Strains within species can be distinguished by differences in their effects on plant growth. Soils usually contain up to six spores per gram of soil. Spores vary in size but are usually in the range 50-150 m m in diameter.
Mycorrhizal strains and host species interact to determine the amount of infection and the level of colonisation. Strains giving good growth responses with one plant species are generally effective with others, but strains differ in their tolerance of acid soil related stresses. At CIAT in Colombia, scientists have selected inoculant strains which are effective on both their pasture legume and grass selections, are adapted to low pH soils and give plant growth responses even with increasing levels of rock phosphate addition to the soil.
Because most soils contain some mycorrhizal propagules already, competition between indigenous strains and introduced strains is a key factor in the response to inoculation. Very little is known of the competitive ability of strains because they are so hard to identify since they cannot be grown in culture. At the moment, competitive strains are selected empirically on the basis of plant growth response to inoculation in non-sterile soil. If the plant responds the strain is considered more competitive than the native strains in forming mycorrhizae.
Work at the University of Queensland has demonstrated the importance of VAM to the phosphorus nutrition of Leucaena leucocephala. In sterilised soil, growth of Leucaena was poor without the benefit of the VAM symbiosis, but could be restored to normal levels by very high applications of phosphorus fertiliser (Figure 3.4.1). Examination of phosphorus concentrations in whole tops of Leucaena seedlings growing in unsterilised soil showed that seedlings experienced a period of P deficit prior to effective VAM infection which may limit early growth. This was avoided by application of very high rates of phosphorus (Figure 3.4.2). Brandon (1993) argued that the slow seedling growth of leucaena observed in many soils could be partially attributed to this period of P starvation during early establishment.
Fig. 3.4.1. Plant height of Leucaena grown at four phosphorus rates in (a) unsterilised and (b) sterilised soil from Mt Cotton in southeast Queensland (Brandon 1993).
(a) Unsterilised
(b) Sterilised
Brandon (1993) also showed that soils of southeast Queensland vary in their mycorrhizal activity as estimated by a bioassay which measured infection levels on the roots of leucaena (Figure 3.4.3). He concluded that soils low in mycorrhizal activity were likely to have been continuously cultivated, regularly waterlogged or to have come from areas supporting native eucalypt forest.
Fig. 3.4.2 Phosphorus concentration in whole tops of leucaena grown at four phosphorus rates in unsterilised soil from Mt Cotton in southeast Queensland (Brandon 1993).
Fig. 3.4.3. Mycorrhizal infection level of leucaena after 7 weeks' growth in eight soils from southeast Queensland dilated in a sterilised sandy loam soil.
Other work (Ruaysoongnern 1989) has shown that nodulation and therefore N nutrition of leucaena may also be dependent on the VAM symbiosis. No nodulation of rhizobium-inoculated leucaena occurred in sterilised soil, and even at high phosphorus levels, nodulation was lower in sterilised than in unsterilised soil (Table 3.4.2).
Inoculants
VAM fungi must be multiplied for use as inoculants by growing on plant roots in pots or in nursery beds on plants such as the grasses Cenchrus ciliaris (buffer grass), Paspalum notatum (Bahia grass), Panicum maximum (green panic), or maize. Inoculants are prepared by chopping up the roots and adding a mixture of roots, spores and hyphae to the seed or cutting to be inoculated using a glue as 'sticker'. In selecting inoculant strains for effectiveness, experiments are usually conducted by growing plants in sterilised rooting medium, with and without inoculation, with and without P addition. Effective strains are then tested in field experiments and have been shown to increase P, Zn and water uptake and dry matter yield of a range of crops.
Inoculation can enhance plant growth in the presence of relatively insoluble fertilisers such as rock phosphate and help produce a large residual effect of the fertiliser on plant growth in subsequent years after the fertiliser addition. VAM inoculation is practised commercially with citrus plantings into irrigated desert sands in California, where soils are sterilised by fumigation, and in rehabilitation of disturbed soils such as mine sites. Large responses have been obtained with Acacia auriculiformis planted in degraded hilly soils in the Philippines (Dart et al. 1991).
While inoculation of nursery plantings or transplanted materials is feasible, inoculants for broadacre crops have not yet been commercialised. Finding a suitable inoculant form is a major problem and the logistics of preparing inoculants for thousands of hectares is daunting! Some progress has been made in growing VAM in sterile conditions using plant cells in a tissue culture system to foster fungal growth. A few ectomycorrhizal strains can be grown in culture and this can serve as an inoculant. Methods to use mycorrhizae in tree production systems are being investigated in two research projects at the University of Queensland.
Mycorrhizal inoculation of nurseries should be considered as a routine practice to ensure good establishment of these beneficial associations.
Acacia species can form both ecto- and endo-mycorrhizal associations (Redder and Warren 1987). The ectomycorrhizal fungus Thelephora spp. forms a beneficial association in promoting growth of A. auriculiformis. Glomus etunicatum followed by G. macrocarpum and Gigaspora margarita were the most effective VAM isolates for A. auriculiformis and A. mangium. For A. mangium, the response to inoculation with G. etunicatum in the nursery persisted in the field for 2 years after planting at Pantabangan in the Philippines while all uninoculated trees died (R. de la Cruz, personal communication).
Table 3.4.2. Effect of VAM inoculation and phosphorus application on VAM infection of roots, concentration of P in youngest fully expanded leaves (YFEL) and nodulation of leucaena seedlings growing in a podzolic soil.
|
Parameter
|
Rate of phosphorus application (kg/ha) |
|||
|
150 |
450 |
|||
|
-VAM |
+VAM |
-VAM |
+VAM |
|
|
Dry wt. (g/pot) |
1.5 |
28.1 |
86.7 |
81.1 |
|
Nodules (mg/pot) |
0 |
297 |
1,334 |
2,029 |
|
% Root infection |
0 |
77 |
0 |
98 |
|
Y. P in YFEL |
0.07 |
0.31 |
0.17 |
0.23 |
Forage tree legumes form major symbiotic associations with two groups of microorganisms, namely, rhizobia and mycorrhizae. These associations play crucial roles in providing for the nitrogen and phosphorus nutrition of host plants. There is evidence that the two symbioses interact and that effective development of one symbiosis will enhance the development of the other. Much more needs to be known about the organisms, their ecology and their host x strain interactions to ensure that growth of tree legumes is maximised.
Abbott, L.K. and Robson, A.D (1985) Formation of external hyphae in soil by four species of vesicular-arbuscular mycorrhiza fungi. New Phytologist 99, 245-255.
Allen, O.N. and Allen, E.K. (1981) The Leguminosae. A Source Book of Characteristics, Uses and Nodulation. University of Wisconsin Press, Madison, Wisconsin, 812 pp.
Brandon, N.J. (1993) Establishment requirements of Leucaena leucocephala (Lam.) de Wit cv. Cunningham. PhD thesis, The University of Queensland.
Brewbaker, J.L., van den Beldt, R. and MacDicken, K. (1982) Nitrogen-fixing tree resources: potentials and limitations. In: Graham, P.H. and Harris, S.C. (eds), Biological Nitrogen Fixation Technology for Tropical Agriculture. CIAT, Cali, Colombia, pp. 413-425.
Campelo, A.B. and Dobereiner, J. (1969) Estudo sobre inoculacao cruzada de algumas leguminosas florestais. Pesquisa Agropecuaria Brasiliera 4, 67-72. (In Portuguese.)
Chee, C.W., Sundram, J., Date, R.A. and Roughley, R.J. (1989) Nodulation of Leucaena leucocephala in acid soils of Peninsular Malaysia Tropical Grasslands 23, 171-178.
Danso, S.K.A., Bowen, G.D. and Sanginga N. (1992) Biological nitrogen fixation in trees in agro-ecosystems. Plant and Soil 141, 177-196.
Dart, P.J., Umali-Garcia M. and Almendras, A. (1991) Role of symbiotic associations in the nutrition of tropical Acacias. In: Turnbull, J.W. (ed.), Advances in Tropical Acacia Research. ACIAR Proceedings No. 35, Canberra, pp. 13-19.
Dobereiner, J. (1984) Nodulation and nitrogen fixation in legume trees. Pesquisa Agropecuaria Brasiliera 19, 83-90.
Dreyfus, B.C. and Dommergues, Y. (1981) Nodulation of Acacia species by fast and slow-growing strains of Rhizobium. Applied and Environmental Microbiology, 41, 97-99.
Habish, H.A. and Khairi, Sh.M. (1970) Nodulation of legumes in the Sudan II. Rhizobium strains and cross-inoculations of Acacia spp. Experimental Agriculture 6, 171-176.
Homchan, J., Date, R.A and Roughley, R.J. (1989) Responses to Inoculation with Root-Nodule Bacteria by Leucaena leucocephala in soils of N.E. Thailand. Tropical Grasslands 23, 92-97.
Ladha, J.K., Peoples, M.B., Garrity, D.P., Capuno, V.T. and Dart, P.J. (1993) Measurement of the N2 fixation of hedgerow vegetation in an alley-crop. Journal of the Soil Science Society of America. (In press.)
Peoples, M.B., Bergersen, F.J., Turner, G.L., Sampet, C., Rerkasem, B., Bhromsiri, A., Nurhayati, D.P., Faizah, A.W., Sudin, M.N., Norhatati, M. and Herridge, D.F (1991) Use of the natural enrichment of 15N in plant available soil N for the measurement of symbiotic N2-fixation. In: Stable Isotopes in Plant Nutrition, Soil Fertility and Environmental Studies. IAEA, Vienna pp. 117-129.
Reddell, P. and Warren, R. (1987) Inoculation of Acacias with mycorrhizal fungi: potential benefits. In: Turnbull, J.W. (ed.), Australian Acacias in Developing Countries. ACIAR Proceedings No. 16, pp. 50-53.
Roughley, R.J. (1987) Acacias and their root-nodule bacteria. In: Turnbull J.W. (ed.), Australian Acacias in Developing Countries. ACIAR Proceedings No. 16, pp. 45-49.
Ruaysoongnern, S. (1989) A study of seedling growth of Leucaena leucocephala (Lam.) de Wit cv. Cunningham with special reference to its nitrogen and phosphorus nutrition in acid soils. PhD thesis, The University of Queensland.
Sanginga, N., Bowen, G.D. and Danso, S.K.A. (1990) Assessment of genetic variability for N2 fixation between and within provenances of Leucaena leucocephala and Acacia albida estimated by 15N labelling techniques. Plant and Soil 127, 169-178.
Trinick, M.J. (1980) Relationships amongst the fast growing rhizobia of Lablab purpureus, Leucaena leucocephala, Mimosa spp, Acacia farnesiana and Sesbania grandiflora and their affinities with other rhizobial groups. Journal of Applied Bacteriology 49, 39-53.
Trinick, M.J. and Hadobas, P.A. (1988) Biology of the Parasponia - Bradyrhizobium symbiosis. Plant and Soil 110, 177-185.
