Plants are the cornerstone of the global ecosystem. Being primary producers they convert energy from the sun into the creation of carbon compounds that serve as the fuel for life. However this cornerstone has an unappreciated yet vital set of foundations, mycorrhizal fungi that aid in the collection of nutrients needed for assimilation in return for some of the plants organic compounds.
In this paper I shall describe the pathway science has taken as it tries to understand more about the complex interactions between plants and fungi, from the initial questions raised by Grime (1987) regarding the necessity of mycorrhizal fungi, to the landmark studies by Simard (1997) and Heijden (1998) and finally onto the effects man is having on mycorrhizal biodiversity and its implications for the future Lilleskov (2001). Mycorrhizal fungi are fungi that form associations with plant roots, either externally, ectomycorrhizae, or internally endomycorrhizae.
Ectomycorrhizae ensheaf the plant root (creating the mantle) and send out projections called mycelia which scavenge the soil at a level inaccessible to plant roots (>3i?? m). This opens up a new world of resources for the symbiont to exploit, which would usually be limiting factors such as Nitrogen (N) and Phosphorous (P). They are abundant in nearly all land plant families (~80%); up to 90% of most trees “feeding” roots have such an association (Read 1997).
Ectomycorrhizae assist plants in a variety of ways, through solubilising N and P from litter as well as P from rock, translocation of N and P over long distances, store P and polyphosphate and N as amino acids and release nutrient to the plant across a specialised interface, the hartig net. Endomycorrhizal fungi form intracellular associations with plant root cells and have very small mycelia. They are found together with ~90% of land plant families. This mutualistic symbiosis is undertaken by Arbuscular Mycorrhiza Fungi (AMF), order Glomales, a Zygomycete.
They assist in plant uptake and mobilisation of N and P. A major aim in ecology is to understand the determinants of plant community structure, the factors affecting the diversity of plant species, their spatial distribution and relative abundance. In 1987 the major factors were thought to be; Plant soil interactions, Plant climate interactions, Interactions among plants, Interactions of plants with herbivores and Pathogens. The interactions of plants with microbes had been overlooked.
The reasons for this lack of research were probably due to the small number of people in the field at the time, the complexity of the analysis of the experiment, and the trouble identifying and comparing fungal morphotypes without precise molecular tools. Grime (1987) conducted a study to identify the factors affecting floral diversity in a model system. Three variables were investigated in 40 turf microcosms (5 replicates per combination), soil heterogeneity, grazing by a herbivore and the addition of AMF.
Results showed that the presence of AMF increased diversity (using the Shannon index). The relative growth of the canopy species Festuca ovina did not appear to alter with the addition of AMF but the subordinate species underwent an increase in biomass. For example Centauium erythrea seedlings had an increased rate of growth in 25% of the population. Grime hypothesised that an export of assimilate from the canopy species to the subordinate species through a mycelia network was likely, a notion that went directly against the Darwinian concept of individual fitness being paramount.
Over the next ten years, research continued into the distribution of nutrients through the mycelial network in the laboratory using isotopic pulse chase analysis. It was noted that cultured AMF species were able to associate with a wide range of plant species and many species could be present on a single plant. This provided evidence for a Wood Wide Web (Nature 1997), where groups of plants are associated through their fungal symbionts and provide a pathway for the transmission of nutrients between them.
These multi-plant associations are referred to as guilds. However there was no direct evidence that these cultured AMF species had any bearing on the plant fungal interactions in the field, whether they do associate with more than one species and if so is there a transfer of assimilates between plants? I. e. the presence of the Wood Wide Web. Simard (1997) executed a landmark study to validate the common held hypothesis of the existence of the Wood Wide Web and extend earlier field results.
Simard set out to ascertain whether the transfer of Carbon (C) occurred in the field, if it was bidirectional between plants, if a net gain occurred in one plant, and if the transfer affected the plants performance in the field. So far lab experiments had suggested movement of nutrients occurred in a source sink relationship and the magnitude of this transfer cold be influenced through the shading of recipient plants or fertilisation with P of donor plants.
The experiment used reciprocal labelling of carbon isotopes (13CO2 and 14CO2) in a pulse chase analysis between two tree species Betula papyrifera and Pseudotsuga menziesii to study below ground transfer. These species share 7 ectomycorrhizal morphotypes covering 90% of their root tips. Thuja plicata was used as a control subject that had no mycorrhizal associations to monitor the amount of Carbon that was transferred through the non mycelial pathways. The three 1 year old species were planted in seedling groups 0. m apart in a randomised block design of 24 experimental units (3×2 factorial set of treatments with 4 replicates). The species were all found together naturally in the same forest. B. papyrifera and P. menziesii were already ectomycorrhizal at time of planting. It was assumed that the plants would have colonies of the compatible ectomycorrhiza present. The seedlings were tested twice during the experiment over two growing seasons. 4-6 weeks before labelling P. menziesii was subjected to different light treatment, full shade, half shade and full ambient light.
After shading the plants were pulse labelled with the isotopes and a 9 day chase followed. Seedlings were then harvested and subjected to analysis for isotopes using combustion for 13C and liquid scintillation for 14C. Results of the first readings indicated bidirectional transfer between the two species with no net gain and transfer to T. plicata was >1% of overall transfer (4%). The second year provided more interesting results an average 6% total (3-10%) isotope fixed transfer occurred from B. papyrifera to P. menziesii. T. plicata did not receive significant amounts of C.
However this is not substantiated (Read 1997) due to the fact that a shortage of replicate seedling groups (which themselves have inherent random effects on the data) only one labelling scheme was applied in the second year 14C B. papyrifera with 13C P. menziesii. The greater effect of shading on magnitude of bidirectional transfer and the occurrence of net transfer in the second rather than the first year coincided with the greater root extension and potential for interplant hyphal linkages, as well as increased vigour of seedlings in the second year (Simard 1997).
Another critique of the experiment was that the sampling times differed by 3 weeks which may have had an effect if the climate had changed significantly of if the species composition of the ecosystem had altered. These findings indicate that the greater transfer was due to the increased sink strength of P. menziesii suggesting that Carbon travels between species down Carbon gradients. The magnitude of transfer is indicative of a tightly linked plant-fungus-soil system and that transfer is possibly governed by resource gradients other than light.
This mechanism could explain how species rich communities are able to maintain productivity and stability in conditions where nutrients or resources are limiting. Read (1997) called for less emphasis on competition as being the primary factor in determining plant community structure and more on the distribution of resources within the community. If mycorrhizal colonisation results in an equalisation of resource availability it would be expected to reduce the dominance of aggressive species and promote co-existence and greater biodiversity.
Problems with the correct identification of fungal species has led to confusion and mismatching of data in previous experiments (Helgason 1998) has been alleviated thanks to molecular PCR identification techniques and has yielded some interesting results about plant mycorrhizal diversity. In arable sites 92% of obtained fungal sequences represented Glomus mossae or closely related taxa whereas sequences obtained from were much more diverse.
However in both wood and field identical sequences from different plant species were found, suggesting the wide range of associations noted in culture may also be realised in nature. Therefore the low diversity of fungi in arable fields could not be due to monoculture per se but could reflect other aspects of the agronomic process such as ploughing, fertilising or the addition of fungicide. The evidence was now in place for the existence of a common mycelial network of ectomycorrhizal fungi creating guild in plant ecosystems.
Although the transfer of nutrients down resource gradients had been confirmed and that AMF presence increases biodiversity (through subordinate donation of assimilates Grime 1987) and productivity, the effects that fungal (particularly AMF) communities had on plant community structure had been paid little attention. Heijdens’ paper in Ecology provided evidence for the hypothesis that AMF community structure could potentially influence plant community structure.
Based on Grimes’ work Heijden hypothesised that although AMF species have a wide range of possible symbiont species if a differential response according to the species occurred the AMF community could directly affect the plant community. A pot experiment with three calcareous grassland species, Hieracium pibsella, Bromus erectus and Festuca ovina inoculated with a combination of 4 AMF species all obtained from a similar habitat, produce three pieces of evidence that AMF communities can determine plant community structure.
Mycorrhizal dependency of plant species The three plant species differed in their dependency based on biomass to control ratio at the end of the experiment. Ratios of 0. 23, 0. 39 and 0. 98 for Bromus Festuca and Hieracium respectively, suggesting that Hieracium may be obligately dependent on AMF for their growth. This provides evidence of how plant species differ in their response to AMF thus varying in the amount of benefit received.
However a pot experiment like this and many of the others that preceded it may not have much ecological relevance as this kind of presence or absence assay would not occur naturally in the field. This is because almost all plant communities have succeeded beyond a primary community and contain a community of AMF species, making this question irrelevant. So ecological interpretation of how species may co-exist based solely on their mycorrhizal dependencies should be avoided or should include a measurement of their response to different AMF species in which they naturally occur.
Effects of different AMF species Differential responses of plant species to AMF in the experiments is interesting as two of the three species, Bromus and Festuca, are frequently co-dominants of calcareous grasslands. This indicates that the co-existence among dominants may also potentially be affected depending on which AMF are colonising their roots. Variation in responses among plant species Hieracium displays a large variation in response to different AMF species, while Bromus exhibits a low amount of variance to different AMF.
The ability of Hieracium to co-exist with other plant species could therefore be highly dependant on which AMF species it forms a symbiosis with. These three pieces of evidence are very important as they support the hypothesis that AMF community structure can directly influence the structure of the plant community by altering the competitiveness of certain species they form more favourable associations with, hence effecting both species composition and community productivity and stability.
However these results did not close the book on the effects of mycorrhizal fungi on plant communities. Read (1998) noted that even with the evidence for the effects that AMF have on structure there is great uncertainty about how these fungal partners bring about these changes on a proximal level. Also that this situation is much more complex as additive effects of single AMF in the laboratory become multiplicative effects in the field and are hard to predict unless complete knowledge of the situation is obtained.
Heijdens next paper to appear in Nature (1998) provided more evidence towards the AMF community being a major determinant of the plant community. Two independent but complementary experiments were performed, one using microcosms the other macrocosms. The first experiment used microcosms simulating European calcareous grassland. 48 plots consisting of 8 blocked replicates were planted with 70 seedlings from 11 different native species at fixed distances in a random order and subjected to combinations of 4 native AMF species.
The blocks were randomised every two weeks and the soil water content was kept constant between microcosms through weighing. This was a good experimental design, much more robust than previous greenhouse experiments mentioned as a large number of replicates and plants have been used to reduce random error and increase estimates towards a population mean rather than a sample mean. Also the use of stringent randomising and water control reduces error due to natural conditions within the greenhouse. The results from the first experiment were very informative.
It appeared that 8 out of the 11 species to be added to the microcosms were completely dependant on the presence of AMF to be successful at all. This supplies credence to Heijdens first experiment in his Ecology paper. Also the biomass produced from different species varied greatly depending on which AMF species was present in the single AMF inoculation. This affirms Heijdens second previous statement and it can be inferred that at low AMF diversities the structure of the plant community is highly dependent on the AMF species present.
Altering AMF taxa in multiple AMF species microcosms did not really alter the biomass of the dominant species Bromus erectus but significantly increased relative proportions of subordinate species also supplying more evidence to ideas proposed by Grime (1987) and his own work. Overall conclusions from this experiment was that in it’s superior design and execution it supported previous work and affirmed that overall presence of mycorrhizal fungi is required to maintain a basic level of biodiversity and stability.
As with all laboratory experiments they do supply a lot of reliable information but whether that information is answering the correct questions is difficult to ascertain, the best place to ask questions concerning plant communities is in their natural habitat. Field studies are harder to control and execute allow the researcher to ask more context specific questions and hopefully get more reliable answers. The second experiment was performed on a macrocosm level simulating North American old-field ecosystems. 70 macrocosms were used each inoculated with 1kg of soil containing 1,2,4,8 or 14 randomly selected AMF species from a list of 23.
The soil was then sown with a seed rain of 100 seeds from 15 of the most abundant native species. After one growing season the plants were harvested and dry biomass calculated. This experimental design is good, although it is important with field studies to remember the constraints imposed by the nature of the experiment itself. Randomising blocks every two weeks and control of growing conditions is not possible but the unseen factors and interactions that cannot be created in the lab are possible in the field and so the results may yield more informative data.
The results showed that increasing AMF species richness resulted in an increase in plant biodiversity, (measured by Simpson’s diversity index) and an increase in productivity above and below ground. Also increased AMF species richness led to a significant increase in length of mycorrhizal hyphae in the soil, to a decreased soil P concentration and an increased P content in plant material. Resulting in a more efficient exploitation of soil P and resources available to the system. The effects of increasing AMF diversity have been shown here to clearly have an effect on plant community structure and ecosystem productivity.
The present situation is described by Sen (2000) in their paper, stating that there is an increasing (and long overdue) appreciation for the central role played by mycorrhizal symbiosis in plant communities, a large progression from the thinking about plant communities in the 80’s where they were not usually considered. Regarding current methodology, the analysis of intact mycorrhizal systems has greatly improved in the laboratory by the use of two dimensional microcosms developed by Read and co-workers. They provide an ideal solution for determining mycorrhizal driven cycling in soils.
Studies in the field still apply and provide the field with large quantities of data and applications for this data have become very important when considering conserving biodiversity. In almost all of the papers cited here the conclusions have called for mycorrhizal communities to play a more important role when considering appropriate methods for conserving species. One worrying paper produced by Lilleskov (2001) states that ectomycorrhizal communities are losing diversity due to the increased use of fertilisers in the soil.
Although plants welcome the additional Nitrogen, it upsets the balance of the symbiosis and hence the balance of the ecosystem. Possible proximal reasons for this could be the increased carbon cost of assimilation of the Nitrogen for the fungi or the fact that since Nitrogen is no longer the limiting factor functional shifts can be seen towards species that specialise in other resource limiting factors. This clearly demonstrates the importance of plant-fungal interactions and if one of the members of this symbiosis is perturbed then the effects will echo thought the global ecosystem.