Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.     

Influence of Organic Amendments on the Accumulation of 137Cs and 90Sr from Contaminated Soil by Three Grass Species

Bahia grass (Paspalum notatum), johnson grass (Sorghum halpense) and switchgrass (Panicum virginatum) werecompared for their ability to accumulate ¹³⁷Csand ⁹⁰Sr from three different contaminated soilsin the presence and absence of either sphagnum peator poultry litter amendments. Above-ground plantbiomass did not differ between plants that were notexposed to these radionuclides and those that wereexposed to soil containing ¹³⁷Cs or ⁹⁰Sr.After three harvests, bahia, johnson and switchgrassplants accumulated from 17.2 to 67.3% of the¹³⁷Cs and from 25.1 to 61.7% of the ⁹⁰Sradded to the soil. Poultry litter and peat mossamendments increased aboveground plant biomass,activity of ¹³⁷Cs or ⁹⁰Sr in plant tissue, %accumulation of ¹³⁷Cs or ⁹⁰Sr from soil andthe plant bioconcentration ratio at each harvestcompared to the control (no amendment) treatment. Thegreatest increases in plant biomass, and radionuclideaccumulation were observed with poultry litter foreach of the three grass species. Johnson grass hadgreater aboveground plant biomass, activity of¹³⁷Cs and ⁹⁰Sr in plant tissue, %accumulation of ¹³⁷Cs or ⁹⁰Sr from soil andbioconcentration ratio in each soil amendment, at eachharvest compared to bahia and switchgrass. Thegreatest accumulation of ¹³⁷Cs and ⁹⁰Sr wasmeasured in johnson grass grown in soil that wasamended with poultry litter. These results suggestthat plant species selection and agronomic practicesmay need to be considered to maximize phytoremediationof radionuclide contaminated soils.     

Potential Remediation of 137Cs and90 Sr Contaminated Soil by Accumulation in Alamo Switchgrass

Cesium-137 (¹³⁷Cs) and Strontium-90 (⁹⁰Sr) are radionuclides characteristic of nuclear fallout from nuclear weapons testing and nuclear reactor accidents. Alamo switchgrass (Panicum virginatum L.) is a perennial C⁴ species native to central North America that produces exceptionally high biomass yields in short periods of time. In three separate experiments, Alamo switchgrass plants were tested for their ability to accumulate ¹³⁷ Cs and⁹⁰ Sr from a contaminated growth medium. Plants in experiment 1 were grown in 33 × 20 × 7 cm plastic pans containing 2.5 kg sand. Plants in experiments 2 and 3 were grown in 30 × 3 cm diameter test tubes containing 0.3 kg growth medium. After 3 months of plant growth, either 102 Bq ¹³⁷Cs or 73 Bq⁹⁰ Sr g^?1 soil were added to the growth medium. Plants in all three experiments were grown within a greenhouse that was maintained at 22 ± 2 °C with a photosynthetic active radiation of 400–700 µmol m^?2 s^?1 and a 14–16 h photoperiod. Above-ground plant biomass did not differ between plants that were not exposed to these radionuclides (controls) and those that were exposed to growth medium containing ¹³⁷Cs or⁹⁰ Sr over the course of the experiment. Plants accumulated 44 and 36% of the total amount of ⁹⁰Sr and¹³⁷ Cs added to growth medium after the first 5 harvests. After the first two harvests, the concentration of ¹³⁷Cs and⁹⁰ Sr in plant tissue and the amount of ¹³⁷Cs or⁹⁰ Sr removed from growth medium declined with each successive harvest. Duration of exposure correlated curvilinearly with accumulation of both ⁹⁰Sr and ¹³⁷Cs by plants (r² = 0.95 and 0.78, respectively). As concentration of both ¹³⁷Cs and ⁹⁰Sr in growth medium increased, plant accumulation of both radionuclides increased and correlated curvilinearly in seedlings (r² = 0.83 and 0.89 respectively).     

Phytoremediation of soil contaminated with low concentrations of radionuclides

Primary sludge, secondary sludge, and wood ash from a pulp and paper mill were combined with sand to create a synthetic topsoil (C:N ratio of 18:1) to restore an abandoned gravel pit. Synthetic topsoil was applied to field microcosms at rates equivalent to 0, 2170, 4341, or 6511 kg N/ha; each was seeded with grass. Fifteen chemical constituents in leachate were measured during two field seasons. Cadmium, Ni and Zn were mobilized rapidly by soil disturbance. Chloride and SO₄-S eluted rapidly from the sludge along with Na. Nitrate leached with Ca late in each field season when sludge N-mineralization and nitrification exceeded plant uptake and microbial immobilization. Ammonium elution was negligible. Dissolved organic carbon (DOC) was mobilized by decomposition of organic matter in the sludge, as were Mg and K. Copper eluted with DOC, probably as an organic ligand. Lead and ortho-P were below our detection limits. We concluded that a synthetic topsoil with a 30:1 C:N ratio applied at a rate of 2100-4300 kg N/ha should provide adequate plant nutrition while minimizing water quality hazards.     

Ecological Risk Assessment of Alfalfa Medicago Varia L.) Genetically Engineered to Express a Human Metallothionein (hMT) Gene

The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO₄ by addition of CuSO₄ solutions to soil and (2) to identify potential unintended effects of the genetic engineering on root and shoot biomass, shoot nutrient content, arbuscular mycorrhizal infection and on the metabolic functions of microbial communities in the rhizosphere. In the absence of exogenous CuSO₄ additions to soil shoot biomass and the macronutrient (C, P, K, Ca, Mg and N) content of plants expressing hMT were not significantly different from the parental control. In the 0.5 mM and 1.0 mM CuSO₄ treatments transgenic plants expressing the commonly used transgenic β-glucuronidase (GUS) marker had significantly higher Fe content than the parental genotype. Significant differences were observed in the carbon substrate utilization patterns of rhizosphere microbial communities among the transgenic plants; no significant differences were observed in the percent mycorrhizal infection of parental and transgenic plants. Shoot biomass increased significantly in all genotypes treated with 0.5 mM CuSO₄ and decreased in all genotypes at CuSO₄ concentrations of 1.5 mM and 2.0 mM. Root dry weights decreased significantly in all genotypes at concentrations of 1.0 mM, 1.5 mM and 2.0 mM CuSO₄. The largest decreases in root dry weight were observed in hMT genotypes grown in soil treated with 1.5 and 2.0 mM CuSO₄. In plants treated with 1.5 mM CuSO₄, shoots of transgenic plants expressing the hMT gene accumulated nominally, but not statistically significantly higher levels of Cu in shoot tissue. Our results were surprising with regard to lack of sufficient efficacy of the current hMT constructs for significant accumulation of Cu from soil treated with CuSO₄. However, our results suggest the utility of applying adverse levels of CuSO₄ or other environmental stressors to identify potential unintended effects of genetic engineering that may not be apparent under typically more optimal plant growth test conditions.     

Response of Six Potato Cultivars to Amount of Applied Water and Verticillium dahliae

ABSTRACT Six potato cultivars were grown with or without the addition of Verticillium dahliae inoculum and were watered at 50, 75, or 100% estimated consumptive use. The applied water x cultivar interaction was significant (P = 0.009 and P = 0.001 for 1996 and 1997, respectively) for the relative area under the senescence progress curve (RAUSPC). With a decrease in water, there was an increase in RAUSPC. A significant interaction of inoculum density x cultivar also was found, based on RAUSPC (P = 0.0194 and P = 0.0033 for 1996 and 1997, respectively). In V. dahliae-infested plots, 'Katahdin' and 'Ranger Russet' were resistant to Verticillium wilt. Population size of V. dahliae in stem apices was significantly lower in 'Katahdin' in both 1996 and 1997 (P = 0.0001) and in 'Ranger Russet' in 1997 (P = 0.0001) than in the other cultivars. 'Russet Burbank' and 'Shepody' had large apical stem populations of V. dahliae and higher RAUSPC values associated with both V. dahliae inoculum and decreased amount of applied water. Marketable tuber yield was unaffected by V. dahliae in both years. Cultivar resistance to Verticillium wilt was related to cultivar tolerance to moisture deficit stress. Results suggest that moisture deficit stress response has the potential to be a useful tool in protocols for screening potato for Verticillium resistance.