Paul T. Rygiewicz

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

BackgroundThe Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.ResultsNumerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers.ConclusionThese plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.

Influence of adverse soil conditions on the formation and function of Arbuscular mycorrhizas

Abstract The majority of plants have mycorrhizal fungi associated with them. Mycorrhizal fungi are ecologically significant because they form relationships in and on the roots of a host plant in a symbiotic association. The host plant provides the fungus with soluble carbon sources, and the fungus provides the host plant with an increased capacity to absorb water and nutrients from the soil. Adverse conditions are a pervasive feature in both natural and agronomic soils. The soil environment is constantly changing with regard to moisture, temperature and nutrient availability. In addition, soil properties are often manipulated to improve crop yields. In many cases, soils may be contaminated through disposal of chemicals that are toxic to plants and microorganisms. The formation and function of mycorrhizal relationships are affected by edaphic conditions such as soil composition, moisture, temperature, pH, cation exchange capacity, and also by anthropogenic stressors including soil compaction, metals and pesticides. Arbuscular mycorrhizal fungi are of interest for their reported roles in alleviation of diverse soil-associated plant stressors, including those induced by metals and polychlorinated aliphatic and phenolic pollutants. Much mycorrhizal research has investigated the impact of extremes in water, temperature, pH and inorganic nutrient availability on mycorrhizal formation and nutrient acquisition. Evaluation of the efficacy of plant–mycorrhizal associations to remediate soils contaminated with toxic materials deserves increased attention. Before the full potential benefits of arbuscular mycorrhizal fungi to reclaim contaminated soils can be realized, research advances are needed to improve our understanding of the physiology of mycorrhizae subjected to adverse physical and chemical conditions. This paper will review literature and discuss the implications of soil contamination on formation and function of arbuscular mycorrhizal associations.

Time-dependent responses of soil CO2 efflux components to elevated atmospheric [CO2] and temperature in experimental forest mesocosms

We previously used dual stable isotope techniques to partition soil CO2 efflux into three source components (rhizosphere respiration, litter decomposition, and soil organic matter (SOM) oxidation) using experimental chambers planted with Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] seedlings. The components responded differently to elevated CO2 (ambient + 200 μmol mol−1) and elevated temperature (ambient + 4 °C) treatments during the first year. Rhizosphere respiration increased most under elevated CO2, and SOM oxidation increased most under elevated temperature. However, many studies show that plants and soil processes can respond to altered climates in a transient way. Herein, we extend our analysis to 2 years to evaluate the stability of the responses of the source components. Total soil CO2 efflux increased significantly under elevated CO2 and elevated temperature in both years (1994 and 1995), but the enhancement was much less in 1995. Rhizosphere respiration increased less under elevated temperature in 1995 compared with 1994. Litter decomposition also tended to increase comparatively less in 1995 under elevated CO2, but was unresponsive to elevated temperature between years. In contrast, SOM oxidation was similar under elevated CO2 in the 2 years. Less SOM oxidation occurred under elevated temperature in 1995 compared with 1994. Our results indicate that temporal variations can occur in CO2 production by the sources. The variations likely involve responses to antecedent physical disruption of the soil and physiological processes.

Lifetime and temporal occurrence of ectomycorrhizae on ponderosa pine (Pinus ponderosa Laws.) seedlings grown under varied atmospheric CO2 and nitrogen levels

Climate change (elevated atmospheric CO2, and altered air temperatures, precipitation amounts and seasonal patterns) may affect ecosystem processes by altering carbon allocation in plants, and carbon flux from plants to soil. Mycorrhizal fungi, as carbon sinks, are among the first soil biota to receive carbon from plants, and thereby influence carbon release from plants to soil. One step in this carbon release is via fine root and mycorrhizal turnover. It is necessary to know the lifetime and temporal occurrence of roots and mycorrhizae to determine the capacity of the soil ecosystem to sequester carbon assimilated aboveground. In this study, ponderosa pine (Pinus ponderosa Laws) seedlings were grown under three levels of atmospheric CO2 (ambient, 525 and 700 μmol CO2 mol-1) and three levels of annual nitrogen additions (0,100 and 200 kg N ha-1) in open-top chambers. At a two-month frequency during 18 months, we observed ectomycorrhizal root tips observed using minirhizotron tubes and camera. The numbers of new mycorrhizal root tips, the numbers of tips that disappeared between two consecutive recording events, and the standing crop of tips at each event were determined. There were more mycorrhizal tips of all three types seen during the summer compared with other times of the year. When only the standing crop of mycorrhizal tips was considered, effects of the CO2 and N addition treatments on carbon allocation to mycorrhizal tips was weakly evident. However, when the three types of tips were considered collectively, tips numbers flux of carbon through mycorrhizae was greatest in the: (1) high CO2 treatment compared with the other CO2 treatments, and (2) intermediate N addition treatment compared with the other N addition treatments. A survival analysis on the entire 18 month cohort of tips was done to calculate the median lifetime of the mycorrhizal root tips. Average median lifetime of the mycorrhizal tips was 139 days and was not affected by nitrogen and CO2 treatments.

Stress Interactions and mycorrhizal plant response: Understanding carbon allocation priorities

In this paper, a framework is presented for studying responses of mycorrhiza to external stresses, including possible feedback effects which are likely to occur. The authors review recent literature linking carbon allocation and host/fungal response under natural and anthropogenic stress, and present a conceptual model to discuss how carbon may be involved in singular and multiple stress interactions of mycorrhizal seedlings. Due to an integral integral role in metabollic processes, characterizing carbon allocation in controlled laboratory environments could be useful for understanding host/fungal responses to a variety of natural and anthropogenic stresses. Carbon allocation at the whole-plant level reflects an integrated response which links photosynthesis to growth and maintenance processes. A root-mycocosm system is described which permits spatial separation of a portion of extramatrical hyphae growing in association with seedling roots. Using this system, it is shown that root/hyphal respiratory release of pulse-labeled 14C follows a sigmoidal pattern, with typical lag, exponential and saturation phases. Total respiratory release of 14C per mg root and the fraction respired of total 14C allocated to the root is greater in ponderosa pine inoculated with Hebeloma crustuliniforme than in noninoculated controls. Results illustrate the nature of information than can be obtained using this system. Current projects using the mycocosms include characterizing the dynamics of carbon allocation under ozone stress, and following the fate of organic pollutants. The authors believe that the system could be used to differentiate fungal- and host-mediated responses to a large number of other stresses and to study a variety of physiological processes in mycorrhizal plants.

Ecological and water quality consequences of nutrient addition for salmon restoration in the Pacific Northwest

Salmon runs have declined over the past two centuries in the Pacific Northwest region of North America. Reduced inputs of salmon-derived organic matter and nutrients (SDN) may limit freshwater production and thus establish a negative feedback loop affecting future generations of fish. Restoration efforts use the rationale of declining SDN to justify artificial nutrient additions, with the goal of reversing salmon decline. The forms of nutrient addition include introducing salmon carcasses, carcass analogs (processed fish cakes), or inorganic fertilizers. While evidence suggests that fish and wildlife may benefit from increases in food availability as a result of carcass additions, stream ecosystems vary in their ability to use nutrients to benefit salmon. Moreover, the practice may introduce excess nutrients, disease, and toxic substances to streams that may already exceed proposed water quality standards. Restoration efforts involving nutrient addition must balance the potential benefits of increased foo...

90sr uptake by 'pinus ponderosa' and 'pinus radiata' seedlings inoculated with ectomycorrhizal fungi

Strontium-90 ((90)Sr) is a radionuclide characteristic of fallout from nuclear reactor accidents and nuclear weapons testing. Prior studies have shown that Pinus ponderosa and P. radiata seedlings can remove appreciable quantities of (90)Sr from soil and store it in plant tissue. In this study, we inoculated P. ponderosa and P. radiata seedlings with one of five isolates of ectomycorrhizal fungi. Inoculated and noninoculated (control) seedlings were compared for their ability to remove (90)Sr from an organic growth medium. Seedlings were grown in a growth chamber in glass tubes containing 165 cm(3) of sphagnum peat moss and perlite (1 : 1 (v/v)) and, except in the controls, the fungal inoculum. After 3 months, 5978 Bq of (90)Sr in 1 ml of sterile, distilled, deionized water was added. Seedlings were grown for an additional month and then harvested. P. ponderosa seedlings with ectomycorrhizae accumulated 3.0-6.0% of the (90)Sr; bioconcentration ratios (Bq (90)Sr cm(-3) plant tissue/Bq (90)Sr cm(-3) growth medium) ranged from 98-162. Ectomycorrhizal P. radiata seedlings accumulated 6.0-6.9% of the (90)Sr; bioconcentration ratios ranged from 88-133. Nonmycorrhizal P. ponderosa and P. radiata seedlings accumulated only 0.6 and 0.7% of the (90)Sr and had bioconcentration ratios of 28 and 27, respectively. Ectomycorrhizal P. ponderosa and P. radiata seedlings are able to remove 3-5 times more (90)Sr from contaminated soil than seedlings without ectomycorrhizae.

Isotopic estimates of new carbon inputs into litter and soils in a four-year climate change experiment with Douglas-fir

Because soil is a major reservoir of terrestrial carbon and a potential sink for atmospheric CO2, determining plant inputs to soil carbon is critical for understanding ecosystem carbon dynamics. We present a modified method to quantify the effects of global climate change on plant inputs of carbon to soil based on 13C:12C ratio (δ13C) analyses that accounts for isotopic fractionation between inputs and newly created soil carbon. In a four-year study, the effects of elevated CO2 and temperature were determined for reconstructed Douglas-fir (Pseudotsuga mensiezii (Mirb.) Franco) ecosystems in which native soil of low nitrogen content was used. The δ13C patterns in litter and mineral soil horizons were measured and compared to δ13C patterns in live needles, fine roots, and coarse roots. From regression analyses, we calculated the isotopic enrichment in 13C of newly incorporated soil carbon relative to needle and root carbon at 4‰ and 2‰, respectively. These enrichments must be considered when using shifts in soil δ13C to calculate inputs of plant carbon into the soil, and are probably a major factor in the progressive enrichment in 13C with increasing depth in soil profiles. Relative to the total carbon in each layer, the proportion of new carbon from recent photosynthate in each soil layer was 13–15% in the A horizon, 7–9% in litter layers, and 4% in the B2 and C horizons. New carbon in the A horizon was estimated at 370xa0g Cxa0m−2. Carbon concentrations and new carbon in A horizons were correlated (r2=0.78, n=12), but with a slope of 0.356, indicating that about 36% of net carbon accumulation in the A horizon was from inputs via roots, root exudates or mycorrhizal fungi and 64% of carbon was derived from surface litter decomposition. Under the nitrogen-limited growth conditions used in this study, neither elevated CO2 nor temperature affected soil carbon sequestration patterns.

Timber harvesting and long-term productivity: weathering processes and soil disturbance

Abstract Both timber harvesting and amelioration practices can cause chemical and physical changes to the soil. These changes can affect factors which alter soil mineral stability and weathering rates, potentially changing inputs to the nutrient cycle. This paper discusses possible effects of harvesting and ameliorative practices on soil mineral stability and weathering. It also presents data from a case study of harvesting impacts in New Zealand. A soil disturbance study established in 1981 was examined in 1990 for potential effects of soil disturbance on weathering and soil nutrient availability. Post-harvesting site treatments included O horizon preserved, O horizon removed, and O and A horizons removed followed by compaction. Results showed that removal of the O horizon greatly reduced the available nutrient pool. Changes in concentrations of solution Si also indicated that mineral equilibrium had been affected in the surface soil horizons of the two disturbance treatments. Fine-root and mycorrhizae biomass was reduced with both disturbance treatments. A comparison of soil nutrient inputs and outputs suggests that weathering inputs must provide most of the available nutrients with the disturbance treatments.

Screening and selecting arbuscular mycorrhizal fungi for inoculating micropropagated apple rootstocks in acid soils

Santa Catarina state is the largest producer of apples in Brazil. Soils in this region have low pH and high levels of aluminum and manganese, requiring high inputs of fertilizers and amendments increasing costs of apple production. Inoculation of arbuscular mycorrhizal fungi can improve the establishment of micropropagated apple plants in such adverse soil conditions. Soil samples were collected from apple orchards in the Caçador, Fraiburgo and São Joaquim regions to develop a corn bioassay to identify mycorrhizal communities with high infectivity. Eleven fungal species were identified from one Caçador soil with the highest infectivity. Glomus etunicatum SCT110, Scutellospora pellucida SCT111, Acaulospora scrobiculata SCT112 and Scutellospora heterogama SCT113 were brought into single-species culture and used in a plant growth and nutrient uptake experiment using micropropagated apple (Malus prunifolia), cultivated at three soil pH. Colonization by fungal isolates significantly affected plant height, shoot and root dry weights, and root:shoot ratio. Soil pH also significantly affected all growth parameters except shoot dry weight. Mycorrhizal inoculation also significantly altered tissue concentrations of P, Zn, Cu, Ca, S, Na, N, K, Fe and Al. Association with mycorrhizal fungi increased P concentration and also decreased Al concentrations in the shoots. Overall, G. etunicatum and S. pellucida were the most effective isolates to promote plant growth and nutrient uptake. Inoculation of apple rootstock with selected fungal isolates during the acclimatization stage represents a useful strategy for producing micropropagated apples that can withstand acidic soil conditions.