Production and turnover of ectomycorrhizal extramatrical mycelial biomass and necromass under elevated CO2 and nitrogen fertilization.

Extramatrical mycelia (EMM) of ectomycorrhizal fungi are important in carbon (C) and nitrogen (N) cycling in forests, but poor knowledge about EMM biomass and necromass turnovers makes the quantification of their role problematic. We studied the impacts of elevated CO2 and N fertilization on EMM production and turnover in a Pinus taeda forest. EMM C was determined by the analysis of ergosterol (biomass), chitin (total bio- and necromass) and total organic C (TOC) of sand-filled mycelium in-growth bags. The production and turnover of EMM bio- and necromass and total C were estimated by modelling. N fertilization reduced the standing EMM biomass C to 57% and its production to 51% of the control (from 238 to 122 kg C ha(-1)  yr(-1) ), whereas elevated CO2 had no detectable effects. Biomass turnover was high (˜13 yr(-1) ) and unchanged by the treatments. Necromass turnover was slow and was reduced from 1.5 yr(-1) in the control to 0.65 yr(-1) in the N-fertilized treatment. However, TOC data did not support an N effect on necromass turnover. An estimated EMM production ranging from 2.5 to 6% of net primary production stresses the importance of its inclusion in C models. A slow EMM necromass turnover indicates an importance in building up forest humus.

Effects of tropospheric ozone on loblolly pine seedlings inoculated with root infecting ophiostomatoid fungi.

Seedlings from four loblolly pine (Pinus taeda L.) families were exposed in open-top chambers to charcoal-filtered air (CF), non-filtered air (NF) or air amended with ozone to 2 times ambient (2×). Two of the families used were selected for their tolerance to fungi associated with Southern Pine Decline while two were selected for their susceptibility. Seedlings were treated with five inoculation treatments: no wound (NW), wound only (W), wound + media (WM), Grosmannia huntii (GH) and Leptographium terebrantis (LT). After 118 days of exposure (AOT40 = 31 ppm-hr(-1) for 2× ozone) seedling volume, dry matter, chlorophyll content, water potential and lesions were measured and analyzed using ANOVA procedures. Our results indicate that seedlings selected for their susceptibility to root infecting ophiostomatoid fungi were also more sensitive to ozone. Overall lesion length was greater on seedlings exposed to elevated ozone concentrations but was not specific to either root infecting ophiostomatoid fungi.

On-site enzymes produced from Trichoderma reesei RUT-C30 and Aspergillus saccharolyticus for hydrolysis of wet exploded corn stover and loblolly pine.

Cellulase production by two filamentous fungi Trichoderma reesei RUT-C30 and novel fungal strain, Aspergillus saccharolyticus on pretreated corn stover was investigated. Cellulase production was followed by the hydrolysis of two feedstocks, wet-exploded corn stover (WECS) and wet-exploded loblolly pine (WELP) by on-site produced enzyme cocktails containing cellulase from T. reesei RUT-C30 and ß-glucosidase from A. saccharolyticus. The sugar yields using the on-site enzyme cocktails were compared with commercial enzymes preparations, Celluclast 1.5L and Novozym 188 at two substrate concentrations, 5% and 10% (w/w) and enzyme loading at 5 and 15 FPU/g glucan for WECS and WELP. The highest sugar yields were obtained at 5% (w/w) substrate concentration and 15 FPU/g glucan for both feedstocks. Glucose yields of 81% and 88% were obtained from on-site and commercial enzymes, respectively using WECS as feed stock. The sugar yields were 55% and 58% for WELP samples hydrolyzed with on-site and commercial enzymes, respectively.

Reaction kinetics of hydrothermal carbonization of loblolly pine.

Hydrothermal carbonization (HTC) is a pretreatment process to convert diverse feedstocks to homogeneous energy-dense solid fuels. Understanding of reaction kinetics is necessary for reactor design and optimization. In this study, the reaction kinetics and effects of particle size on HTC were investigated. Experiments were conducted in a novel two-chamber reactor maintaining isothermal conditions for 15s to 30 min reaction times. Loblolly pine was treated at 200, 230, and 260°C. During the first few minutes of reaction, the solid-product mass yield decreases rapidly while the calorific value increases rapidly. A simple reaction mechanism is proposed and validated, in which both hemicellulose and cellulose degrade in parallel first-order reactions. Activation energy of hemicellulose and cellulose degradation were determined to be 30 and 73 kJ/mol, respectively. For short HTC times, both reaction and diffusion effects were observed.

Dry weight partitioning and hydraulic traits in young Pinus taeda trees fertilized with nitrogen and phosphorus in a subtropical area.

Plants of Pinus taeda L. from each of four families were fertilized with nitrogen (N), phosphorus (P) or N + P at planting. The H family had the highest growth in dry mass while the L family had the lowest growth. Measurements of plant hydraulic architecture traits were performed during the first year after planting. Stomatal conductance (gs), water potential at predawn (Ψpredawn) and at midday (Ψmidday), branch hydraulic conductivity (ks and kl) and shoot hydraulic conductance (K) were measured. One year after planting, dry weight partitioning of all aboveground organs was performed. Phosphorus fertilization increased growth in all four families, while N fertilization had a negative effect on growth. L family plants were more negatively affected than H family plants. This negative effect was not due to limitations in N or P uptake because plants from all the families and treatments had the same N and P concentration in the needles. Phosphorus fertilization changed some hydraulic parameters, but those changes did not affect growth. However, the negative effect of N can be explained by changes in hydraulic traits. L family plants had a high leaf dry weight per branch, which was increased by N fertilization. This change occurred together with a decrease in shoot conductance. Therefore, the reduction in gs was not enough to avoid the drop in Ψmidday. Consequently, stomatal closure and the deficient water status of the needles resulted in a reduction in growth. In H family plants, the increase in the number of needles per branch due to N fertilization was counteracted by a reduction in gs and also by a reduction in tracheid lumen size and length. Because of these two changes, Ψmidday did not drop and water availability in the needles was adequate for sustained growth. In conclusion, fertilization affects the hydraulic architecture of plants, and different families develop different strategies. Some of the hydraulic changes can explain the negative effect of N fertilization on growth.

Biomarker genes highlight intraspecific and interspecific variations in the responses of Pinus taeda L. and Pinus radiata D. Don to Sirex noctilio F. acid gland secretions.

Sirex noctilio F., a Eurasian horntail woodwasp recently introduced into North America, oviposits in pines and other conifers and in the process spreads a phytopathogenic fungus that serves as a food source for its larvae. During oviposition the woodwasp also deposits mucus produced in its acid (venom) gland that alters pine defense responses and facilitates infection by the fungus. A 26,496-feature loblolly pine cDNA microarray was used to survey gene expression of pine tissue responding to S. noctilio venom. Six genes were selected for further assessment by quantitative real-time polymerase chain reaction (qRT-PCR), including one that encoded an apparent PR-4 protein and another that encoded a thaumatin-like protein. Expression of both was strongly induced in response to venom, while expression of an apparent actin gene (ACT1) was stable in response to the venom. The pattern of gene response was similar in Pinus taeda L. and Pinus radiata D. Don, but the magnitude of response in P. radiata was significantly stronger for each of the induced genes. The magnitude of the biomarker gene response to venom also varied according to genotype within these two species. The qRT-PCR assay was used to demonstrate that the primary bioactive component in S. noctilio venom is a polypeptide.

The effect of induced heat waves on Pinus taeda and Quercus rubra seedlings in ambient and elevated CO2 atmospheres.

Here, we investigated the effect of different heat-wave intensities applied at two atmospheric CO2 concentrations ([CO2]) on seedlings of two tree species, loblolly pine (Pinus taeda) and northern red oak (Quercus rubra). Seedlings were assigned to treatment combinations of two levels of [CO2] (380 or 700 µmol mol(-1)) and four levels of air temperature (ambient, ambient +3°C, or 7-d heat waves consisting of a biweekly +6°C heat wave, or a monthly +12°C heat wave). Treatments were maintained throughout the growing season, thus receiving equal heat sums. We measured gas exchange and fluorescence parameters before, during and after a mid-summer heat wave. The +12°C heat wave, significantly reduced net photosynthesis (Anet) in both species and [CO2] treatments but this effect was diminished in elevated [CO2]. The decrease in Anet was accompanied by a decrease in Fv'/Fm' in P. taeda and ΦPSII in Q. rubra. Our findings suggest that, if soil moisture is adequate, trees will experience negative effects in photosynthetic performance only with the occurrence of extreme heat waves. As elevated [CO2] diminished these negative effects, the future climate may not be as detrimental to plant communities as previously assumed.

Effects of predicted future and current atmospheric temperature and [CO2] and high and low soil moisture on gas exchange and growth of Pinus taeda seedlings at cool and warm sites in the species range.

Predicted future changes in air temperature and atmospheric CO(2) concentration ([CO(2)]), coupled with altered precipitation, are expected to substantially affect tree growth. Effects on growth may vary considerably across a species range, as temperatures vary from sub-optimal to supra-optimal for growth. We performed an experiment simultaneously at two locations in the current range of loblolly pine, a cool site and a warm site, to examine the effect of future climate conditions on growth of loblolly pine seedlings in contrasting regions of the species range. At both sites 1-year-old loblolly pine seedlings were grown in current (local ambient temperature and [CO(2)]) and predicted future atmospheric conditions (ambient +2 °C temperature and 700 µmol mol(-1) [CO(2)]). Additionally, high and low soil moisture treatments were applied within each atmospheric treatment at each site by altering the amount of water provided to the seedlings. Averaged across water treatments, photosynthesis (A(net)) was 31% greater at the cool site and 34% greater at the warm site in elevated temperature and [CO(2)] compared with ambient temperature. Biomass accumulation was also stimulated by 38% at the cool site and by 24% at the warm site in that treatment. These results suggest that a temperature increase of 2 °C coupled with an increase in [CO(2)] (predicted future climate) will create conditions favorable for growth of this species. Reduced soil moisture decreased growth in both current and predicted atmospheric conditions. Biomass accumulation and A(net) were reduced by ∼39 and 17%, respectively, in the low water treatment. These results suggest that any benefit of future atmospheric conditions may be negated if soil moisture is reduced by altered precipitation patterns.

Enhanced root exudation induces microbial feedbacks to N cycling in a pine forest under long-term CO2 fumigation.

The degree to which rising atmospheric CO(2) will be offset by carbon (C) sequestration in forests depends in part on the capacity of trees and soil microbes to make physiological adjustments that can alleviate resource limitation. Here, we show for the first time that mature trees exposed to CO(2) enrichment increase the release of soluble C from roots to soil, and that such increases are coupled to the accelerated turnover of nitrogen (N) pools in the rhizosphere. Over the course of 3 years, we measured in situ rates of root exudation from 420 intact loblolly pine (Pinus taeda L.) roots. Trees fumigated with elevated CO(2) (200 p.p.m.v. over background) increased exudation rates (µg C cm(-1) root h(-1) ) by 55% during the primary growing season, leading to a 50% annual increase in dissolved organic inputs to fumigated forest soils. These increases in root-derived C were positively correlated with microbial release of extracellular enzymes involved in breakdown of organic N (R(2) = 0.66; P = 0.006) in the rhizosphere, indicating that exudation stimulated microbial activity and accelerated the rate of soil organic matter (SOM) turnover. In support of this conclusion, trees exposed to both elevated CO(2) and N fertilization did not increase exudation rates and had reduced enzyme activities in the rhizosphere. Collectively, our results provide field-based empirical support suggesting that sustained growth responses of forests to elevated CO(2) in low fertility soils are maintained by enhanced rates of microbial activity and N cycling fuelled by inputs of root-derived C. To the extent that increases in exudation also stimulate SOM decomposition, such changes may prevent soil C accumulation in forest ecosystems.

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine.

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state (13)C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS (13)C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.

Elevated CO2 increases root exudation from loblolly pine (Pinus taeda) seedlings as an N-mediated response.

The degree to which forest ecosystems provide a long-term sink for increasing atmospheric CO(2) depends upon the capacity of trees to increase the availability of growth-limiting resources. It has been widely speculated that trees exposed to CO(2) enrichment may increase the release of root exudates to soil as a mechanism to stimulate microbes to enhance nutrient availability. As a first test to examine how the atmospheric CO(2) and nitrogen availability affect the rates of root exudation, we performed two experiments in which the exudates were collected from loblolly pine (Pinus taeda L.) seedlings that were grown in controlled growth chambers under low and high CO(2) and at low and high rates of N supply. Despite the differences in experimental design between the two studies, plants grown at high CO(2) were larger, and thus whole plant exudation rates were higher under elevated CO(2) (P = 0.019), but the magnitude of this response depended on the N level in both studies. Seedlings increased mass-specific exudation rates in response to elevated CO(2) in both experiments, but only at low N supply. Moreover, N supply had a greater impact on the exudation rates than did CO(2), with mass-specific exudation rates significantly greater (98% and 69% in Experiments 1 and 2, respectively) in the seedlings grown at low N supply relative to high N supply. These results provide preliminary evidence that loblolly pines alter exudation rates in response to both CO(2) concentration and N supply, and support the hypothesis that increased C allocation to root exudates may be a mechanism by which trees could delay progressive N limitation in forested ecosystems.

Identification and characterization of a matrix metalloproteinase (Pta1-MMP) expressed during Loblolly pine (Pinus taeda) seed development, germination completion, and early seedling establishment.

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. Pta1-MMP protein has the structural characteristics of other plant MMPs, the recombinant protein exhibits Zn(2+)-dependent protease activity, and is inhibited by EDTA and the active site-binding hydroxamate inhibitor GM6001. The Pta1-MMP gene is expressed in both embryo and megagametophyte, with transcript levels increasing in both during the period from proembryo to early cotyledonary stage, then declining during late embryogenesis and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed germination was stimulated by GA(3) and inhibited by ABA, and the timing of germination completion was mirrored by the presence of MMP-like protease activity in both water- and GA(3)-imbibed embryos. Pta1-MMP gene transcript levels increased in association with radicle protrusion for both GA(3)- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 11 days after imbibition, Pta1-MMP gene transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed germination inhibited radicle protrusion. Our results suggest that MMP activity may be involved in ECM modification, facilitating the cell division and expansion required during seed development, germination completion, and subsequent seedling establishment.

Resin flow responses to fertilization, wounding and fungal inoculation in loblolly pine (Pinus taeda) in North Carolina.

Resin flow is the primary means of natural defense against southern pine beetle (Dendroctonus frontalis Zimm.), the most important insect pest of Pinus spp. in the southern United States. As a result, factors affecting resin flow are of interest to researchers and forest managers. We examined the influence of fertilization, artificial wounding and fungal inoculation on resin flow in 6- and 12-year-old stands of loblolly pine (Pinus taeda L.) and determined the extent of that influence within and above the wounded stem area and through time. Fertilization increased constitutive resin flow, but only the younger trees sustained increased resin flow after wounding and inoculation treatments. An induced resin flow response occurred between 1 and 30 days after wounding and inoculation treatments. Wounding with inoculation resulted in greater resin flow than wounding alone, but increasing amounts of inoculum did not increase resin flow. Increased resin flow (relative to controls) lasted for at least 90 days after wounding and inoculation. This increase appeared to be limited to the area of treatment, at least in younger trees. The long-lasting effects of fungal inoculation on resin flow, as well as the response to fertilization, suggest that acquired resistance through induced resin flow aids in decreasing susceptibility of loblolly pine to southern pine beetle.

Isolation and characterization of a molecule stimulatory to growth of somatic embryos from early stage female gametophyte tissue of loblolly pine.

Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements. We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65 nmoles per stage 2-3 FG) and the amount of citric acid that stimulates colony growth (25-50 nmoles) when applied topically to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes can provide valuable information leading to further applications of these molecules to improve LP SE protocols.

Liquid medium and liquid overlays improve embryogenic tissue initiation in conifers.

Somatic embryogenesis (SE) is expected to play an important role in increasing productivity, sustainability, and uniformity of future US forests. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (Pinus taeda L.), the main commercial US forest species, is often recalcitrant for desirable genotypes. Liquid initiation medium with no or low gelling agent or placement of the explant on gelled medium followed later by a liquid medium overlay during the initiation process increased initiation for loblolly pine and Norway spruce (Picea abies). Loblolly pine liquid medium required reduction of NAA from 2 mg/l in gelled medium to 0.3 mg/l in liquid medium. Once the NAA concentration was adjusted, loblolly pine initiation occurred in liquid medium with fully immersed megagametophytes, explants supported at the liquid medium surface, or on gelled medium overlaid with liquid medium. Liquid overlays (0.25 ml) consisting of medium with NAA reduced to 0.3 mg/l, 9 mg/l ABA and no gelling agent applied to explants on 2 ml of gelled medium provided excellent initiation results. Greatest initiation percentages occurred when the liquid overlay was applied 14 days after placement of the megagametophyte on gelled medium. Initiation increases ranged from +8.5% with high-value cross-pollinated seed sources to +6.5 to +9.9% with open-pollinated and often recalcitrant seed sources. Liquid medium addition allows rapid replenishment of nutrients and adjustment or change of pH, hormones, or other parameters without disturbing the tissue.

Light quality treatments enhance somatic seedling production in three southern pine species.

Embryogenic cultures of loblolly pine (Pinus taeda L.), slash pine (Pinus elliottii Engelm.), longleaf pine (Pinus palustris Mill.) and slash pine x longleaf pine hybrids were initiated from immature seeds on an initiation medium containing 13.57 microM 2,4-dichlorophenoxyacetic acid and 2.22 microM benzylaminopurine. Embryogenic cultures proliferated and somatic embryos developed, matured and germinated following a modified protocol and media originally developed for radiata pine (Pinus radiata D. Don.) somatic seedling production. A discrete, light-sensitive pre-germination stage and a later germination (radicle emergence) stage were identified by the differential response of somatic embryos to light of different wavelengths. Different light quality treatments were applied during the pre-germination and germination steps, using cool white fluorescent bulbs or light-emitting diodes (LEDs), or both. In general, red wavelengths provided by LEDs during these steps resulted in higher frequencies of somatic embryo germination (up to 64%) and conversion (up to 50%), longer tap roots and more first-order lateral roots than the standard cool white fluorescent treatments or treatment with blue wavelengths from LEDs. In addition, exposure to red light allowed germination of somatic embryos of some clones that failed to produce germinants under fluorescent light. Germination and conversion were further enhanced by sequential application of cool white fluorescent light and red light, resulting in up to 100% germination and conversion in one experiment. Longleaf pine somatic embryos were especially responsive to the light quality treatments, resulting in the first report of somatic seedling production for this species.

Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation with abscisic acid and silver nitrate.

Loblolly pine ( Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7 micro M), silver nitrate (20 micro M), and guanosine 3',5'-cyclic monophosphate, 8-bromo-, sodium salt (10 micro M) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4-6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.

Brassinolide improves embryogenic tissue initiation in conifers and rice.

Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine ( Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce ( Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005-0.25 micro M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 micro M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice-15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 micro M cGMP, 0.1 micro M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.