Staffordshire University
An investigation into the underlying microbial factors governing plant recruitment and competition in a grassland creation scheme on a capped landfill site.

Aims of the investigation

Objectives of the project

Context of the investigation

Theoretical basis

Methods of investigation

References

Bibliography

AIMS OF THE INVESTIGATION
The aim of this investigation is to gain an understanding of the underlying factors governing plant recruitment and competition through the study of vesicular-arbuscular mycorhiza (AM) and its relationship with other soil characteristics (including organic content, earthworm activity, bulk density) in order to provide an insight into how grassland creation and restoration techniques can be improved to reflect a more natural community structure.

OBJECTIVES OF THE PROJECT
The objectives of the project are as follows:
i To assess the study sites soil in terms of its existing AM, earthworm population, water holding capacity (WHC) and organic matter content by spring 2004.
ii To create an area of grassland using known quantities of a number of grassland species of local genotype by winter 2004.
iii To manipulate the variables being investigated on plots within the created grassland, including inoculating plots with AM and introducing three levels of green compost by autumn 2004.
iv To monitor earthworm populations and WHC within the grassland plots.
v To monitor germination, growth and infection of AM of plants within the plots.
vi To establish a glasshouse experiment in pots to investigate findings from the field experiment under controlled conditions by autumn 2005.
vii To monitor germination, growth and infection of AM in plants in pots within the glasshouse.
viii To monitor the outcome of competition between plants within the grassland plots.

CONTEXT OF THE INVESTIGATION
Semi-natural grasslands of high botanical value constitute only 3% of permanent grassland in the British lowlands. 97% of unimproved grasslands in England and Wales were lost between 1930 and 1984, and losses are continuing at a high rate (Dryden 1997). This can largely be attributed to the intensification of agricultural practices and loss to developments. The current socio-economic climate means that there are greater opportunities and requirements for grassland re-creation and restoration schemes. This is not only due to an increase in the availability of agri-environment schemes but also to an increase in pressure through planning policy, legislation and case law for effective mitigation for developments and through achieving targets set as part of the Biodiversity Action Plans for grassland habitats (for example Lowland Meadows are identified as priority habitats). Other opportunities for habitat creation exist through a legal obligation within the planning permission to ensure that nature conservation is fully considered are landfill and opencast mining. This can include both creation on adjacent land and on the site itself once mining or landfill operations are completed.

As a result of the development of this niche, a number of research projects on grassland creation and restoration were initiated during the late 1980’s and 1990’s. These were largely concerned with the effect of nutrient enrichment on semi-natural grasslands, methods of reducing the nutrient status of soils and techniques of grassland creation. An increasing number of investigations into the influence of micro-organisms within the soil, such as AM, on the success of creation projects are being undertaken as the importance of these factors is being realised. Relatively little is, however, known about these microbial elements and is referred to as the ‘black box’.

The floristics of unimproved grasslands are rarely uniform in structure with species being locally abundant or dominant whilst others are constant throughout the sward but in low densities. Although the objective of the majority of grassland (re)creation schemes is to establish communities similar to those that historically occupied the site or a particular geographical area, little research appears to have been undertaken into the factors dictating the patch dynamics. Research has shown that parasitic plants such as yellow rattle (Rhianthus minor) and eye-brights (Euphrasia spp.) have an impact on the productivity of grasslands which is accompanied by characteristic changes in sward composition with herbaceous species occurring at the expense of grasses (Crofts et al. 1999). It is also known that microtopography (which in turn affects factors such as pH and nutrient status on a micro-scale), aspect and localized edaphic variations within grasslands have an effect on the patch dynamics, little is known about the role microbiology of the soil. The microbial component is known to alter competition between plants by enhancing the competitive abilities through symbiotic relationships, whilst soil fauna such as earthworms improve the structure of the soil.

Many habitat creation schemes are proving to be unsuccessful with planted trees and sown grass and herbaceous plants failing to thrive. Merryweather (2001) identified the depleted soil biology of substrates commonly used for creation schemes, such as arable, post-industrial, landfill or mined land, to be a major factor in the failure of these schemes.

An investigation into the function that AM plays in the patch dynamics of grasslands, and in turn the interaction between AM and other soil properties such as organic content, bulk density and earthworm populations, would provide a useful insight into how AM could be used as a tool within grassland restoration and (re)creation schemes to produce a sward with patch dynamics that most closely resemble a semi-natural unimproved sward.

THEORETICAL BASIS
Mycorrhiza is a dynamic and integral functional feature in terrestrial ecosystems forming symbiotic associations with an estimated 90% of the world’s plants. Mycorrhiza mobilize soil nutrients making them, particularly phosphorus, more available to host plants whilst excess carbon from the plant is harnessed by the mycorrhiza (Smith & Read 1997). Mycorrhiza is also known to benefit host plants through functions such as increasing disease resistance and improving water relations (Harnett et al 2002; Sanchez-Diaz & Horunbia 1994 cited in O’Connor et al 2002). Mycorrhizal specificity (species of host to species of mycorrhiza) is extremely loose (Harley 1991 cited in Norris et al 1994) although as far back as 1900 it has been recognized that different host species responded differently to mycorrhizal infection (Allen 1991). The level of specificity of mycorrhiza can, however, have important consequences on plant ecology. As a result of the specificity of plant response to mycorrhiza, the diversity and composition of the mycorrhizal community has been shown to exert large effects on plant diversity and composition (Van der Heijden et al. 1998). Conversely, the composition of the plant community has similar effects on the diversity and composition of the mycorrhizal community (Bever et al. 1996 cited in Bever 2002).

The mycorrhizal component of natural soils is greatly reduced by both natural disturbance and human inflicted disturbance such as farming. Many crop plants are non-mycorrhizal (such as rape) resulting in no support for the mycorrhiza (Merryweather 2001). Similarly, during opencast mining, soil stripping and storage and replacement result in major disturbance of the native mycorrhizal populations (Scullion 1994). Similar storage and replacement of soil is seen in landfill practices resulting in a similar effect on the soil’s microbiology (Harris et al. 1989).

Other problems associated with the soil stripping, storage and replacement process were highlighted at an opencast mine at Bryngwyn. Reinstated soil was extremely impervious resulting in surface water logging after even light rainfall and the land becoming dry and cracked in periods of dry weather making it hostile for plants and animals alike. Earthworm populations were drastically reduced as a result of soil handling and storage of the soil, with Martin et al (1998) finding a mortality rate of >90%.

Up until recently research has concentrated on the relationship between mycorrhiza and plant diversity and productivity, implicating a positive correlation with these features (O’Connor et al 2002; Grime et al. 1987). More recent work has concentrated on the role of mycorrhiza in shaping plant community structure. There is increasing evidence that the effects of mycorrhiza on their host plant communities are context dependent, varying with host species, plant life history stage, resource availability and abiotic conditions (Harnett et al 2002). St. John et al (1983 cited in Norris et al 1994) found that the hyphae were more extensive in the presence of organic phosphorus than of inorganic substrates. Read et al (1985) described intensive mycorrhizal hyphal development associated with patches of decomposing organic matter (Allen 1991).

Experiments investigating the effects of AM are largely restricted to glasshouses, where soil variables can be manipulated through processes such as sterilization of the soil, whilst control over AM has been achieved using fungicide in field experiments (Carey et al. 1992, Fitter 1986, Fitter & Nichols 1998, Merryweather & Fitter 1986). Glasshouse experiments typically lack the realism necessary to draw conclusions about the functioning of mycorrhizas in natural communities, and field studies rarely reveal underlying mechanisms (Harnett et al. 2002). The effects of sterilization and use of fungicide on other microbial aspects of the soil, such as bacterial components, cause further limitations in experiments. This experiment will therefore embrace a complementary combination of field studies and glasshouse experiments. Furthermore, the proposed study site is ex-landfill that has been capped with lias clay, and therefore is likely to have a severely impoverished existing biological component to the soil, including AM, earthworm populations, and organic content and will therefore have a large potential for controlled manipulation of these features.

METHODS OF INVESTIGATION
The investigation will be conducted on a blue lias clay capped landfill site in Carymoor, Somerset which has collaborated with the University of Staffordshire over a number of years in establishing research. The investigation will essentially be divided into four phases:

PHASE I: Preliminary site investigation, development of methodologies and training programme
Preliminary site investigation and the development of methodologies will be conducted using experimental plots established by Elizabeth Poston in 2001. The aim of this project was to determine the most appropriate substrate for re-creating a hay meadow from seed. Plots were subjected to three different treatments: removal of topsoil (thus removing organic matter and the existing seed bank); removal of vegetation and a control plot (no treatment). A seed mix based on the MG5 National Vegetation Classification (NVC) grassland community (Rodwell 1991) was sown and growth monitored. The following baseline information will be established here: ·

• Existing AM component through assessing infection of vegetation within the plots by staining root samples;
• Existing earthworm populations through extraction using formaldehyde;
• Soil physical and chemical factors including WHC and organic content through loss on ignition (LOI).

Methodologies will be developed through conducting the preliminary tests and training will be undertaken including attending an AM workshop held by the University of York and the University of Staffordshire skills training and professional development module.

PHASE II: Field experiment
Phase II comprises the establishment and ongoing monitoring of the field experiment. A series of quadrats will be established within the study site in which known numbers of seeds of a number of grassland plant species will be sown. Species will be selected according to flora typical of the geographical area, of a local genotype and with root properties most compatible with staining techniques, such as spiny restharrow (Ononis spinosa), a locally scarce plant in Somerset that occurs naturally on the site at Carymoor. The quadrats will be subjected to a series of conditions including the addition of three known quantities of organic matter, varied ploughing depths (to manipulate WHC) and inoculation of AM (this will be obtained through extraction from more developed local soil through wet sieving). The following aspects of the plots will then be monitored over a period of four seasons:

• Germination success by counting the number of plants of known sown species;
• Infection of plants by AM by wet sieving the soil and staining root samples;
• Patch formation of plants by mapping locations/dominance of individuals and species within the quadrats;
• Soil physical and chemical factors including WHC and organic content through LOI.
• Earthworm populations through extraction using formaldehyde.

PHASE III: Glasshouse experiments
Glasshouse experimental pots will be established in University glasshouses following the completion of the field experiments first year. Following preliminary analysis and data manipulation of the results for the field experiments glasshouse pots will be established testing isolation of significant field variables. As in the field experiment known quantities of grassland species will be sown in pots containing sterilized soil. Pots will be subjected to a series of conditions according to the results of the field experiment. Manipulated variables will include organic matter, earthworms and inoculation of AM (known innoculum will be used in the glasshouse experiments). Testing of findings from the field in the glasshouse will be ongoing over a period of four seasons.

PHASE IV: Analysis
Parametric data statistical analysis will be conducted on results from this replicated block plot experimental design. Multi Variable Statistical Package (MVSP) will be used to ordinate plant data to analyse community dynamics. ANOVA and Principal Correspondence Analysis (PCA) will be conducted on the data set.

REFERENCES
Allen, M. F (1991), The ecology of mycorrhizae, Cambridge University Press.
Bever, J. D (2002), Host-specificity of AM fungal population growth rates can generate feedback on plant growth, Plant and Soil 244, 281 – 290.
Carey, P. D, Fitter, A. H, Watkinson, A. R (1992), A field study using the fungicide benomyl to investigate the effect of mycorrhizal fungi on plant fitness, Oecologica 90, 550-555.
Crofts, A, Jefferson. R. G (Ed) (1999), The lowland grassland management handbook, 2nd Edition, English Nature.
Dryden, R (1997), Habitat restoration project: Fact sheets and bibliographies, English Nature.
Fitter, A. H (1986), Effect of Benomyl on leaf phosphorus concentration in alpine grassland: a test of mycorrhizal benefit, New Phytologist 103, 767-776.
Fitter, A. H, Nichols, R (1988), The use of Benomyl to control infection by vesicular-arbuscular mycorrhizal mycelium, New Phytologist 110, 201-206.
Fitter, A. H, Merryweather, J (1996), Phosphorus nutrition of an obligately mycorrhizal plant treated with benomyl in the field, New Phytologist 132, 307-311.
Grime, J. P, Mackey, J. M, Hillier, S. H, Read, D. J (1987), Floristic diversity in a model system using experimental microcosms, Nature 328, 420 – 422.
Harnett, D. C, Wilson, G. W. T (2002) The role of mycorrhizas in plant community structure and dynamics: lessons from grasslands, Plant and Soil 244: 319 – 331.
Harris J. A, Birch, P (1989), Changes in the microbial community and physio-chemical characteristics of topsoils stockpiled during opencast mining, Soil use and management 5 161 – 168.
Martin, A. D, Humphries, R. N, Whittington, W. J (1988), Midland research project – invertebrate studies In: Land restoration investigation techniques, pp 47-56, British Coal Opencast Executive, Mansfield.
Merryweather, J (2001), Comment: Meet the Glomales -the ecology of mycorrhiza, British Wildlife Volume 13, 2: 86 – 93.
Norris, J. R, Read, D. J, Varma, A. K (1994), Techniques for mycorrhizal research, Academic press.
O’Connor, P. J, Smith, S. E, Smith, F. A (2002), Arbuscular mycorrhizas influence plant diversity and community structure in a semiarid herbland, New Phytologist 154, 209 – 218.
Poston, E (2001), Evaluating the creation of a species-rich hay meadow on a blue lias clay capped landfill site, Carymoor, Somerset, unpublished.
Scullion, J (1994), Restoring farmland after coal, The Bryngwyn project, British Coal Opencast.
Smith, S. E, Read, D. J (1997), Mycorrhizal symbiosis, 2nd Edition, Academic Press.
Van der Heijden, M. G. A, Kilironomos, J. N, Ursic, M, Moutoglis, P, Streitwolf-Engel, R, Boller, T, Wiemken, A, Sanders, I. R (1998), Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity, Nature 396, 69 – 72.

BIBLIOGRAPHY
Allen,M.F (1991), The ecology of mycorrhizae, Cambridge University Press.
Bever, J. D (2002), Host-specificity of AM fungal population growth rates can generate feedback on plant growth, Plant and Soil 244, 281 – 290.
Carey, P. D, Fitter, A. H, Watkinson, A. R (1992), A field study using the fungicide benomyl to investigate the effect of mycorrhizal fungi on plant fitness, Oecologica 90, 550-555.
Crofts, A, Jefferson. R. G (Ed) (1999), The lowland grassland management handbook, 2nd Edition, English Nature.
Dryden, R (1997), Habitat restoration project: Fact sheets and bibliographies, English Nature.
Fitter, A. H (1986), Effect of Benomyl on leaf phosphorus concentration in alpine grassland: a test of mycorrhizal benefit, New Phytologist 103, 767-776. Fitter, A. H, Nichols, R (1988), The use of Benomyl to control infection by vesicular-arbuscular mycorrhizal mycelium, New Phytologist 110, 201-206.
Fitter, A. H, Merryweather, J (1996), Phosphorus nutrition of an obligately mycorrhizal plant treated with benomyl in the field, New Phytologist 132, 307-311.
Gough, M, Marrs, R (1989), Trends in soil chemistry and floristics associated with establishment of a low-input meadow system on arable clay soil in Essex, England , Biological Conservation 52 (1990) 135-146.
Gough, M, Marrs, R (1989), A comparison of soil fertility between semi-natural and agricultural plant communities: Implication for the creation of species-rich grassland on abandoned agricultural land, Biological Conservation 51(1990) 83-96.
Grime, J. P, Mackey, J. M, Hillier, S. H, Read, D. J (1987), Floristic diversity in a model system using experimental microcosms, Nature 328, 420 – 422.
Harnett, D. C, Wilson, G. W. T (2002) The role of mycorrhizas in plant community structure and dynamics: lessons from grasslands, Plant and Soil 244: 319 – 331.
Harris J. A, Birch, P (1989), Changes in the microbial community and physio-chemical characteristics of topsoils stockpiled during opencast mining, Soil use and management 5 161 – 168.
Hutchings, M, Booth, K (1996), Studies on the feasibility of re-creating chalk grassland vegetation on ex-arable land, I. The potential roles of the seed bank and the seed rain, Journal of Applied Ecology, 33 (1996) 1171-1181.
Hutchings, M, Booth, K (1996), Studies on the feasibility of re-creating chalk grassland vegetation on ex-arable land, II. Germination and early survivorship of seedlings under different management regimes, Journal of Applied Ecology (1996) 33, 1182-1190.
Kilironomos, J. N, Hart, M. M (2002), Colonisation of roots by arbuscular mycorrhizal fungi using different sources of innoculum, Mycorrhiza 12, 181 – 184.
Marrs, R, Snow, C, Owen, K, Evans, C (1997), Heathland acid grassland creation on arable soils at Minsmere: Identification of potential problems and a test cropping to impoverish soils, Biological Conservation 85 (1998) 69-82.
Martin, A. D, Humphries, R. N, Whittington, W. J (1988), Midland research project – invertebrate studies In: Land restoration investigation techniques, pp 47-56, British Coal Opencast Executive, Mansfield.
McCrea, A, Trueman, I, Fullen (2000), A comparison of the effects of four arable crops on the fertility depletion of a sandy sit loam destined for grassland habitat creation, Biological Conservation 97 (2001) 181-187.
McCrea, A, Trueman, I, Fullen, M, Atkinson, M, Besenyei, L (2000), Relationships between soil characteristics and species richness in two botanically heterogenous created meadows in the urban English West Midlands, Biological Conservation 97 (2001) 171-180.
Merryweather, J (2001), Comment: Meet the Glomales -the ecology of mycorrhiza, British Wildlife Volume 13, 2: 86 – 93.
Norris, J. R, Read, D. J, Varma, A. K (1994), Techniques for mycorrhizal research, Academic press.
O’Connor, P. J, Smith, S. E, Smith, F. A (2002), Arbuscular mycorrhizas influence plant diversity and community structure in a semiarid herbland, New Phytologist 154, 209 – 218.
Poston, E (2001), Evaluating the creation of a species-rich hay meadow on a blue lias clay capped landfill site, Carymoor, Somerset, unpublished.
Scullion, J (1994), Restoring farmland after coal, The Bryngwyn project, British Coal Opencast.
Smith, S. E, Read, D. J (1997), Mycorrhizal symbiosis, 2nd Edition, Academic Press.
Van der Heijden, M. G. A, Kilironomos, J. N, Ursic, M, Moutoglis, P, Streitwolf-Engel, R, Boller, T, Wiemken, A, Sanders, I. R (1998), Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity, Nature 396, 69 – 72.

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Carymoor Environmental Trust March 2004