Literature Review

General Mycorrhizal Function and Diversity

Mycorrhiza (myco = fungi, rhiza = root) describes a relationship that is most appropriately defined as “a structure in which symbiotic union between a fungus and the absorbing organs of a plant confers increase of fitness on one or both partners” (Read 1999). Arbuscular mycorrhizal fungi (AMF) have been shown in the fossil record since the establishment of plants on land during the Ordovician Period (Simon, Bousquet et al. 1993; Remy, Taylor et al. 1994). Today, it is estimated that mycorrhizae are associated with 70 to 95% of all plant species are mycorrhizal (Hardie and Leyton 1981; Allen 1991; Smith and Read 1997; Smith, Smith et al. 2003); however further research is still required. This long history has provided ample time for the co-evolution of plants and their endophytic symbiotic fungi, resulting in dependency, benefits, and instances of host-specificity. The foundation of the relationship is based on bi-directional nutrient transfer that is beneficial and in some cases, necessary for survival (Smith and Smith 1990). AMF are completely dependent upon host plants to provide carbohydrates they require, but ultimate control of colonization belongs to the plant (Thompson, Robson et al. 1986; Anderson and Liberta 1989; van der Heijden and Sanders 2002). It is this dependency that has so far staved the ability of scientists to grow, study, and manufacture these organisms in pure culture. Moreover, mycorrhizal symbioses are impacted by light intensity and edaphic conditions (Mosse 1972; Mosse 1972; Menge, Davis et al. 1978; Nemec 1978; Menge and Timmer 1982). In exchange for metabolites, AMF offer their hosts a multitude of “physiological and morphological” benefits (Allen, Sexton et al. 1981) including: increased nutrient uptake, improved water relations, increased rates of photosynthesis and transpiration, modified carbon and nutrient allocation, defense from plant pathogens, reduced transplant stress, resistance to metal toxicity, and broad ecological effects such as mediation of competition and succession in disturbed areas.

Edaphic conditions

Soil is a complex amalgamation of organic and mineral matter, water, and air; the exact amounts and concentrations of which affect drainage, water-holding capacity, quantity and size of pores, and root development (Mukerji, Manoharachary et al. 2002). Soils differ from site to site in color, depth, horizon arrangement, and microbial population (Waksman and Starkley 1931). AMF are sensitive to edaphic factors (Bethlenfalvay, Brown et al. 1982; Mukerji, Bhattacharjee et al. 1982; Stahl, Christensen et al. 1990) and vary in their tolerance of these conditions (Bethlenfalvay, Brown et al. 1982; Kothari, Marschner et al. 1991). Plant responses and degree of colonization are ultimately influenced by the same environmental conditions of the soil, primarily: temperature, moisture, pH, disturbance, and nutrient content.

Soil temperature plays a major role in spore germination and root colonization (Mosse 1973; Tommerup 1984; Anderson, Ebbers et al. 1986; Koske 1987). Some studies have shown maximum VAM formation occurs between 18.5 and 24.0˚C and although increased soil temperature stimulated root colonization, it was inhibited above 29.5˚ C (Parke, Linderman et al. 1983). Others have demonstrated maximum arbuscule development and root colonization to occur between 30 and 40 C (Furlan and Fortin 1973; Schreiner and Koide 1993). Spores of the Genus Glomus germinate between 20-25 C and Gigaspora around 30C (Daniels and Trappe 1980). Low temperatures correlate with low spore germination.

Soil type affects mycorrhizal associations by controlling the moisture content and drainage of the environment through factors such as clay content. Poorly draining soils result in anaerobic conditions of water-logged areas theoretically inhibiting the growth and beneficial associations of mycorrhizal fungi (Miller 2000; Purakayastha and Chhonkar 2001).

Soil acidity seems to highly influence mycorrhizae (Yost, Uehara et al. 1982; Porter, Robson et al. 1987; Porter, Robson et al. 1987; Vierheilig and Ocampo 1991; Wang, Stribley et al. 1993). Soil pH over a range of 4.8 to 8.0 and 5.6 to 6.9 has been shown to significantly influence the germination of Glomus epigaeum and Glomus fasciculatum, respectively (Read, Koucheki et al. 1976). pH may also significantly affect survival and growth of hyphae in soil, the penetration and colonization of roots, and propagule formation (Porter, Robson et al. 1987; Porter, Robson et al. 1987).

Soil disturbance leads to the loss of species and loss of genotypes within a species resulting from the destruction of the expansive mycelial networks of mycorrhizae developed over time (van der Heijden and Sanders 2002). Disturbance leads to propagule attrition (McGee 1989; Pattinson and McGee 1997). Re-establishment of this network is determined by the remaining fungal propagules (Torrisi, Pattinson et al. 1999). Successful restoration of disturbed habitats may therefore require the re–introduction of mycorrhizal propagules to facilitate biodiversity, but knowledge and experimentation in this field is limited (Mukerji, Manoharachary et al. 2002; van der Heijden and Sanders 2002).

The growth of arbuscular mycorrhizal fungi is dramatically reduced by increased salinity; however, plant growth is still improved over non-mycorrhizal plants under the same conditions (Gupta and Krishnamurthy 1996). Mycorrhizal plants average greater biomass, carbohydrate, protein content (Charest, Dalpe et al. 1993), water content (Vauclin, Vieira et al. 1982),and P composition (Yost, Uehara et al. 1982) than non-mycorrhizal plants.

Nutrient quality and quantity of the soil broadly determines which category of mycorrhizae dominates the ecosystem (Kühn and Stasvaski 1990; Read 1991; Read 1991). It influences the responsiveness of the individual plants or species to mycorrhizal colonization (Murdoch, Jackobs et al. 1967). AMF derived from low P soil are much more sensitive to P supply than those from high P soils (Abbott and Robson 1978; Cooper 1978; Jasper, Robson et al. 1979).

Nutrient Uptake

The rate-limiting step for nutrient acquisition is the rate of diffusion through soil. Plants without fine root hair systems are frequently more infected and therefore it is believed that hyphae replace the functions of these systems (Powell and Bagyaraj 1984) . Their hyphae can provide access to remote areas, micropores, hornblende, and feldspar particles where roots are incapable of entering (Hatano, Iwanaga et al. 1988; Hildebrand 1994). Mycorrhizae are therefore able to increase plant fitness by improving nutrient uptake through mycelial networks by extending beyond nutrient depletion zones of the rhizosphere (Sanders and Tinker 1971), decreasing soil diffusion distances (Bauer, Kellogg et al. 2003), increasing the absorbent surface area (Allen, Sexton et al. 1981; Graham, Linderman et al. 1982), and producing of enzymes (Jones, Durall et al. 1991; Timonen, Finlay et al. 1996). Mycorrhizae provide particular access Cu (Gilmore 1971; La Rue, McClellan et al. 1975; Lambert, Baker et al. 1979; Gildon and Tinker 1983), Zn (Bowen, Skinner et al. 1974; Timmer and Leyden 1978; Lambert, Baker et al. 1979), but most notably, P (Asimi, Gianinazzipearson et al. 1980; Koide 1991).

Phosphate Uptake

Phosphate is present in the soil in three forms: soluble inorganic P, insoluble inorganic P, and organic P compounds. In soils of low P availability, whether due to low concentrations or tightly bound to soil particles, depletion zones quickly develop around the root (Sanders and Tinker 1971; Graham, Linderman et al. 1982). Mycorrhizae increase P availability through hyphae that extend beyond the depletion zones and providing more efficient pathways for nutrient uptake. The mycobionts take over direct pathways for P (Smith, Jakobsen et al. 2000), providing hyphal flow (uptake per unit length of hyphae per unit time) calculated to be approximately 6 times the rate of a single uninfected root (Powell and Bagyaraj 1984). Furthermore, conversion of inorganic to organic P form in the leaves (Allen, Sexton et al. 1981; Allen, Smith et al. 1981) also creates a sink which results in steep P gradients (Bieleski 1973) that also increase P flow and uptake. However, although theories have been developed, the exact means by which mycorrhizae increase nutrient availability seem to be unknown.

Phosphatase Activity

One of the most explored possibilities for increased P availability is the production of internal and external phosphatases by mycorrhizal hyphae (Joner and Johansen 2000; Joner, Ravnskov et al. 2000; Joner, van Aarle et al. 2000).Their production is important to the solubilization of sugars (Abbott and Robson 1991) and initial enzymatic studies reveal that they display kinetic uptake parameters with lower K_m, higher C_m, and V_max values up to 7 times higher than plant roots. According to Michaelis-Menton dynamics and repeated experimentation, colonized plants have a higher affinity for phosphorus and are able to absorb it at much lower concentrations than non-mycorrhizal plants (Van Tichelen and Colpaert 2000). However, they enhance P uptake through soil exploration despite their inability to solubilize unavailable forms of P (Barea, Azcon et al. 1975; Warnock, Fitter et al. 1982).

Organic Acid Production

Another possible reason for increased nutrient uptake stems from the oxalic, formic, lactic, citric, and malic acids produced by mycorrhizae to increase cation concentration of soil, resulting in mineral weathering in and beyond the rhizosphere (Jones, Durall et al. 1998; Arocena and Glowa 2000). Mineral weathering results in the degradation of organic compounds (Bending and Read 1995). Although still not fully understood, these features have made mycorrhizae an interesting potential alternative to fertilization or at the very least, a possibility for increasing its effectiveness and reducing the amounts required through large-scale field inoculation.

Improved Water Relations

AMF have been shown to improve water relations by increasing hydraulic conductivity (Russo and Bresler 1981; Safir and Nelson 1981) and reducing root resistance (Safir, Boyer et al. 1972) and may be associated with higher leaf water potentials (Hardie and Leyton 1981), higher transpiration rates (Hardie and Leyton 1981), and lower stomatal resistances (Levy and Krikun 1980; Allen, Smith et al. 1981; Allen 1982; Nelson and Safir 1982; Nelson and Safir 1982). This lower resistance can confer improved drought tolerance (Aldon 1975; Cress, Throneberry et al. 1979; Nelson and Safir 1982; Nelson and Safir 1982). Infected plants frequently appear to be less susceptible to wilting and transplant shock than uninfected plants (Barrows and Roncadori 1977; Menge, Davis et al. 1978; Sieverding 1979; Janos 1980; Levy and Krikun 1980; Hardie and Leyton 1981; Sieverding 1981; Cooper 1983). Accumulation of proline, as a consequence of water deficit and salinity stress, has been shown experimentally to be lower in mycorrhizal plants (Levy and Krikun 1980) and supports the belief that mycorrhizal associations improve water relations and decrease plant stress.

Stimulation of Plant Growth, Photosynthesis, and Transpiration

Mycorrhizal infection increases photosynthetic rates and boosts transpiration and uptake of CO₂ (Jones and Hutchinson 1988; Jones and Hutchinson 1988; Rygiewicz and Andersen 1994; Colpaert, VanLaere et al. 1996). Increased photosynthetic rates make up for the increased energy expenditure of infected plants (Powell and Bagyaraj 1984).

Mycorrhizal colonization stimulates plant growth through the production of auxins or expression of auxin related genes of the host plant (Martin, Lapeyrie et al. 1997; Karabaghli-Degron, Sotta et al. 1998). The extent to which mycorrhizal colonization increases growth rate will depend on (1) maximum growth rate achieved by a non-mycorrhizal plant under low nutrient condition, (2) extent to which colonization increases nutrient supply, (3) changes in nutrient efficiency, and (4) differences in root structures, root: shoot ratio (van der Heijden and Sanders 2002). Some AM fungi are more effective than others in stimulating plant growth (Hayman 1983; Menge 1983). This increase in plant growth has been directly correlated with P uptake and development of external hyphae (Sanders, Tinker et al. 1977; Graham, Linderman et al. 1982). However, whether the increased growth is a direct result of this nutrient uptake is still in question (Quoreshi and Timmer 1998).

Plant Pathogen Tolerance

Mycorrhizae also appear to confer an improved tolerance against plant pathogens (Newsham, Fitter et al. 1994). However, as plants with artificially improved nutrient status have also been shown to have higher disease resistance, it is unlikely that this benefit is much more than a result of the plant’s increased ability to secure nutrients (Safir 1968; Baltruschat and Schonbeck 1972; Schonbeck and Schninzer 1972; Daft 1973; Baltruschat and Schmitthenner 1975; Baltruschat and Schonbeck 1975; Schonbeck and Dehne 1977).

Carbon Flux and allocation

A single fungal mycelium may be associated with several plants (Molina, Massicotte et al. 1992). AMF may facilitate carbon and nutrient fluxes amongst them resulting or increasing interplant competition, ecosystem productivity (van der Heijden and Sanders 2002), and increased plant biodiversity (van der Heijden, Klironomos et al. 1998; Hartnett and Wilson 1999). Although the net C-flux is from plant to fungus, carbon moves from fungus to plant as well. The system relies on the conversion of glucose and fructose to glycogen, trehalose, and manitol (Smith, Muscatine et al. 1969; Jordy, Azemar-Lorentz et al. 1998). This can ultimately lead to increased photosynthesis (Dunham, Ray et al. 2003) through the demands of the symbiosis.

Arbuscular mycorrhizal fungi have played a large role in plant community structure, development, and diversity for more than 400 million years (Remy, Taylor et al. 1994). They influence recruitment into the community by means of carbon flux and nutrient transfer via their mycelial networks (van der Heijden and Sanders 2002) affecting seedling establishment near mature plants by reducing competitive dominance, allowing them to colonize rapidly, or link to other plant resources through an established AM network (Eissenstat and Newman 1990; Bidartondo, Kretzer et al. 2000). Disturbance effectively decreases biodiversity and community structure by destroying this network and interrupting the flow from sufficient to deficient plants (Grime, Mackey et al. 1987; Gange, Brown et al. 1993; Moora and Zobel 1996). However, mycorrhizae assist in the recovery of species following disturbance (Perry, Margolis et al. 1989; Horton, Bruns et al. 1999). In addition to the translocation of nutrient and carbon sources, AM fungi form storage vesicles late in development resulting in a C drain from plants (Bago, Pfeffer et al. 2000; van der Heijden and Sanders 2002). Therefore, lower root:shoot ratios can result from mycorrhizal associations as ongoing C drain can reduce plant dry matter (Smith 1980; Son and Smith 1988). Therefore, mycorrhizae benefit the plant community structure through a network of hyphae that allow the translocation of nutrients and carbon. By allowing more nutrient stores to be bound in the organic matter of living plants, they also help to avert nutrient loss through groundwater flow. This may help to explain their presence in aquatic areas once believed unsuitable for these organisms (Miller 2000).

Mycorrhiza are in Wetlands in Spite of Anaerobic Conditions

The exact role of mycorrhizal associations in wetland plants is not well understood. Although it was once believed the fungi could not survive in the low oxygen environments of saturated and inundated soils, several studies have shown there to be considerable colonization even under wet conditions (Sondergaard and Laegaard 1977; Anderson, Liberta et al. 1984; Farmer 1985; Newman and Reddell 1987; Rickerl, Sancho et al. 1994; Turner, Amon et al. 2000). Particular interest lies in their participation in the calcareous wetlands and fens. Fens are groundwater fed, peatlands rich in calcium and magnesium (Cornwell, Bedford et al. 2001). Their inherent alkaline and calcareous conditions cause plants in these habitats to be phosphorus limited due to its precipitation from the water table (Boyer and Wheeler 1989). Therefore, mycorrhizae may play a crucial role in wet, alkaline habitats by securing needed nutrients as they do in terrestrial systems (Allen 1991; Brundrett 1991; Smith and Read 1997; Van Hoewyk, Wigand et al. 2001).

Wetlands may not limit mycorrhizal associations through anaerobic conditions as once hypothesized. AMF may obtain the oxygen they need through extensive aerenchyma tissue of wetland plants. Research has shown that aerenchyma cells are more abundant in monocots (Crawford 1989). However, monocots lack the fibrous root systems of dicots that facilitate heavy colonization by mycorrhizae. This may provide another explanation why mycorrhizae do are not found throughout wetland systems to the same degree as upland plant communities, but can also explain individual colonization of roots most heavily in oxygenated regions near the stem.

Approximately 80% of all known plant species depend upon some extent of mycorrhizal colonization for competition and acquisition of nutrients from their habitats (Harley 1989; Smith and Read 1997). Although wetland mitigation and restoration has become mandatory under the Clean Water Act, the 95% of these restorations are unsuccessful and demand closer examination of the underlying reasons. One such suggestion, is that the land and soil disturbance and long term drainage of targeted areas remove the mycelial networks of mycorrhizal fungi and effectively inhibit the re-establishment of native plant species (Allen, Smith et al. 1981; Turner, Amon et al. 2000; Bauer, Kellogg et al. 2003). Therefore, testing the effectiveness of inoculation on transplants in various local wetland sites would be beneficial in determining their possible application in restoration of these ecosystems.

Methods

Soil

To simulate the natural soil conditions in local fen sites in the greenhouse, soil will be obtained from a supply of highly organic, hydric soil left over from the construction of fens at the ODNR site on New-Germany Trebein Rd.

Disinfestation

In order to assess the effectiveness of mycorrhizal inoculum, the native AM propagules in soil will be removed to create a baseline from which we can extrapolate data. The soil will be passed through a 2mm sieve to remove large plant debris and stones before being treated. Although steam treatment, γ radiation, solarization, and fungicide application have traditionally been used as a means soil sterilization, they are also eliminate the entire native soil flora, fungal components, and/or effectively change the physical and chemical properties of the soil, particularly organics (Bowen and Cawse 1964; Stribley, Read et al. 1975). Therefore, the soil will be treated with a 30 minute exposure at a core soil temperature of 65°C. This treatment will be repeated after 48 hours to allow AM propagules can germinate and be eliminated during the consecutive treatment. This soil will then be transported and stored in a sealed plastic tub until all is treated and can be homogenized for experimentation.

Re-introduction

As the soil micro flora, such as bacteria and protozoa, may exert important and distinctive effects upon the plant that would otherwise be falsely attributed to mycorrhizal colonization, re-establishment of these organisms must be addressed prior to experimentation (Kitt, Hetrick et al. 1988; Hetrick, Wilson et al. 1989). This will be accomplished by washing field samples, passing them through a 38μm sieve to remove any mycorrhizal spores, filtering the sievate through Whatman filter paper to remove hyphal propagules, and evenly distributing this final solution over the soil.

Plants

Selection

The following have been selected for experimentation based upon their natural tendency to form mycorrhizas as well as previous seed collection and germination studies in order to test the effects of greenhouse inoculation on local wetland plant species. Monocots and dicots will be represented. The plants proposed are Mimulus ringens (Monkey Flower), Typha latifolia L. (Common Cattail), Penthorum sedoides (Ditch Stonecrop), Pycanthemum pycanthemoides (Virginia Mountain Mint), Asclepias incaranata (Swamp Milkweed), Eupatorium perfoliatum L. (Boneset), Verbena Hastata (Blue Vervain), and possibly others.

Seeds of the selected species will be germinated in soil to obtain 300 or more seedlings of each. For each species, 6 flats of 28 potting quads will be planted with healthy seedlings. (Total 168 plants each.) One-half of these seedlings (3 flats) will be inoculated with mycorrhizal inoculum one week after transplantation. Greenhouse space requirements should not exceed 120 square feet.

Transplantation

Inoculated and control plants will be transplanted to sites of various land use histories to test the effectiveness of greenhouse inoculation on the success of wetland restoration and creation. Three control and inoculated plants of each species will be randomly chosen and sacrificed to determine percent colonization as another twenty-five others will be transplanted to pots in the greenhouse. Fifteen control and inoculated plants of each species will also be transplanted to wetland habitats. These sites are as follows: restored wetland habitat (Fairborn Marsh, Valle Greene, Fawn Ridge, Experimental Fens), natural wetland habitat (Sibenthaler Fen, Fairborn Marsh Fens), and a created wetland habitat (Hagenbuch preserve, Southdown,) which all vary in their levels of soil disturbance, edaphic characteristics, and hydric top soils.

Transplantation Success

The effectiveness of greenhouse inoculation will be based upon the comparison of the success of transplantation between control and treated plants. Transplantation success will be evaluated by the survival of the plant and shoot tissue composition of carbon, nitrogen, and phosphorus upon the dormant season on 5 randomly chosen control and inoculated plants of each species. Five greenhouse plants will be randomly chosen to periodically determine their tissue composition of C, N, and P. The size and number of leaves, production of propagules (seeds, suckers, anything leading to multiplication), and colonization by mycorrhiza will also be documented. Nutrient composition, size and number of leaves, and propagule production can all be associated with success; however, actual interpretation of results must take into consideration the fact that there is a cost for symbiosis.

Mycorrhizae

Production of Inoculum

In order to compare the effectiveness of commercial and natural sources of inoculum for the restoration of wetlands, AM mycorrhizal inoculum will be created and maintained from natural and commercial sources. A commercial mixture of known species was purchased in the form of MycoGrow^TM Soluble (www.fungiperfecti.com). This inoculum consists of the powdered spore mass of twelve endomycorrhizal fungi, including: Glomus mosseae, Glomus intraradices, Glomus clarum, Glomus monosporus, Glomus deserticola, Glomus brasilianum, and Gigaspora margarita. The commercial inoculum provides a “high-quality” inoculum of identified species at a low price.

A natural mixture of unknown mycorrhizal species will be obtained from experimental wetland areas in the form of soil and colonized root material.

A lab prepared mycorrhizal inoculum will be produced in order to avoid the direct use of field soils that may contain plant pathogens. Initial sources obtained from the field and commercial venders will be used to inoculate pot cultures of soybean and corn plants in sterilized sand media. Pot cultures will be maintained with a modified Hoagland’s nutrient solution lacking phosphate in order to stimulate mycorrhizal colonization. At harvest, mycorrhizal colonization or the lack thereof will be assessed from inoculated and control plants. Plants will then be harvested by pruning to the soil level, sieving the sandy mass for AM propagules, and chopping the infected roots into uniform segments. This inoculum will then be briefly air dried, placed in plastic bags, and stored at 4°C until use (Ferguson and Menge 1981; Ferguson and Woodhead 1982).

Inoculation

Plants will be inoculated with AM fungi shortly after transplantation of germlings in order to determine if greenhouse inoculation procedures will improve the successfulness of wetland restorations. Three flats of healthy plants of each species will be inoculated with soil mixtures, containing spores and infected root materials, created from pot culture conditions by placing a specified amount of inoculum in close contact with the roots at transplantation during the selection of healthy seedlings for experimentation (Menge and Timmer 1982).

Mycorrhizal Staining

As hyphae penetrate the root and spread through the absorptive organs of plants via the cortical tissues, it is not possible to detect and/or identify their structures without clearing the roots of tannins. However, I will subjectively seek out those clear roots near the growing tip that are the most active areas of live arbuscule interaction. These roots will be cut into 1cm segments and stained in order for optical examination. Trypan blue will be used to permit microscopic examination and quantification of infection (Brundrett, Melville et al. 1994; Smith, Jakobsen et al. 2000).

Percent Colonization

The relationship between mycorrhizal colonization and transplantation success will be exposed through the quantification of host root infection. Mycorrhizal colonization will be assessed from 5 randomly chosen treated and control plants immediately prior to transplantation, before the end of the first growing season, and on all remaining plants at the end of the experiment. Assessment of colonization will be conducted on 1cm root segments with the grid-line intersect method. Special attention will be paid to the differences in percent colonization between species and habitats, the percent colonization of control plants, and the apparent differences in mycorrhizal species noted among control and inoculated species (particularly field specimens) as well as differences between plant species.

The effectiveness of the inoculum will be tested by calculating the percentage of roots colonized at regular intervals for each plant species throughout the experiment. I will be using the grid-line intersect method (Giovannetti and Mosse 1980). Although this method has a tendency to over or underestimate the true colonization (McGonigle, Miller et al. 1990), it provides rapid and reproducible results that can be verified through a simple redistribution of the root fragments and examination thereof. Statistical analyses will therefore require less samples and experimental error will be thoroughly tested. Although, many methods exist for achieving similar results, the grid-line intersect method has been used in nearly two-thirds of mycorrhizal research. This method also helps to eliminate the possibility of experimenter bias throughout the process (McGonigle, Miller et al. 1990).

Identification

If time permits, the identification of the AM fungal species involved throughout experimentation would provide a means of reproducibility. Identification of mycorrhizal species used as inoculum throughout the experiment will be assessed by spore identification. This will involve the growing of pot cultures created from a mixture of soil and infected root material obtained from the experimental sites previously mentioned. Pot cultures raised in the greenhouse will provide a level of control that will allow numerous healthy spores to be obtained and identified through standard methods.

Quantification of Nutrients

Digestion of Plant Material and Soils

The digestion of plant material will be performed in order to prepare the specimens for elemental analysis, specifically, the concentration of total phosphorus, plant available phosphorus, and total Kjeldahl nitrogen. Plant material or soil samples will be added to sulphuric acid and oxidized with hydrogen peroxide in Hach Digesdahl digestion apparatuses. Actual digestion procedures will be modified from Hach, but the methods are still in development.

Detection of Phosphorus

The colorimetric detection of phosphorus will be performed on the Genesys 10 Spectrophotometer. The analysis of plant available phosphorus will be adapted from that of Watanabe and Olsen (Watanabe and Olsen 1965) using reagent packets calibrated for phosphorus detection in 5mL samples. Actual detection methods are still in development.

Detection of Nitrogen

The colorimetric detection of nitrogen will also be performed on the Genesys 10 Spectrophotometer using a method adapted from the Hach field procedure and considerations drawn from standard methods (Kjeldahl 1883). Reagent packets calibrated for nitrogen detection in 5mL samples will be used for the colorimetric assay. Actual detection methods are still in development.

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Hetrick, B. A. D., G. W. T. Wils

Proposal

Goal

Hypotheses and Objectives