Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods

Several biochemical and molecular methods are used to investigate the microbial diversity and changes in microbial community structure in rhizospheres and bulk soils resulting from changes in management. We have compared the effects of plants on the microbial community, using several methods, in three different types of soils. Pots containing soil from three contrasting sites were planted with Lolium perenne (rye grass). Physiological (Biolog), biochemical (PLFA) and molecular (DGGE and TRFLP) fingerprinting methods were employed to study the change in soil microbial communities caused by the growth of rye grass. Different methods of DNA extraction and nested PCR on TRFLP profiles were examined to investigate whether they gave different views of community structure. Molecular methods were used for both fungal and bacterial diversity. Principal component analysis of Biolog data suggested a significant effect of the plants on the microbial community structure. We found significant effects of both soil type and plants on microbial communities in PLFA data. Data from TRFLP of soil bacterial communities showed large effects of soil type and smaller but significant effects of plants. Effects of plant growth on soil fungal communities were measured by TRFLP and DGGE. Multiple Procrustes analysis suggested that both methods gave similar results, with only soil types having a significant effect on fungal communities. However, TRFLP was more discriminatory as it generated more ribotype fragments for each sample than the number of bands detected by DGGE. Neither methods of DNA extraction nor the nested PCR had any effect on the evaluation of soil microbial community structure. In conclusion, the different methods of microbial fingerprinting gave qualitatively similar results when samples were processed consistently and compatible statistical methods used. However, the molecular methods were more discriminatory than the physiological and biochemical approaches. We believe results obtained from this experiment will have a major impact on soil microbial ecology in general and rhizosphere–microbial interaction studies in particular, as we showed that the different fingerprinting methods for microbial communities gave qualitatively similar results.     

西北地区耕地土壤真菌DNA提取方法比较

由于西北土壤理化性质的复杂性和真菌特殊性,所以从土壤中提取真菌基因组DNA就相对细菌更困难。在2种常用的土壤微生物基因组DNA提取方法与在传统提取方法的基础上,结合了一种专门适用于真菌的提取方法进行了比较,并且利用真菌28SrDNA通用引物U1/U2进行扩增。三种提取方法比较结果表明:SDS法提取的DNA纯度最低,传统CTAB-SDS的DNA产量最低,实验室的提取方法既可以提高DNA产量又可以保证DNA的片段完整性,并且本实验室的提取方法扩增效果最好,可广泛应用于西北地区土壤真菌的分子生物学研究。     

Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996     

Surveying soil faunal communities using a direct molecular approach

Soil faunal communities are often phylogenetically diverse and the accurate assessment of the taxonomic structure of these communities is both time-consuming and requires a high level of taxonomic expertise. Here we describe a DNA sequence-based methodology for characterizing soil micro- and mesofaunal communities that is similar to the molecular approaches commonly used to survey soil microbial diversity. The technique involves the direct extraction of faunal DNA from soil, PCR amplification of the extracted DNA with metazoan-specific primers, followed by the construction of clone libraries and direct sequencing of individual PCR products. We used this technique to characterize micro- and mesofaunal community composition from six individual soils representing two land-use types. The technique captured the more abundant faunal groups in the soils (nematodes, Collembola, Acari, tardigrades, enchytraeids) and provided sufficient taxonomic resolution to describe the overall structure of the communities. We compared the results obtained using this molecular approach to results obtained using a traditional, microscopy-based approach and found that the results were broadly similar. However, since biases are inherent in both methods it remains unclear which method provides a more accurate assessment of soil faunal community composition. Although this molecular approach has some distinct disadvantages over the more widely-used direct extraction methods, one advantage is that the taxonomic identification it can provide will be more accurate and consistent across research groups, facilitating effective comparisons of mesofaunal surveys.     

Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis

The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grinding-freezing-thawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel electrophoresis. This indicates the importance of the DNA extraction protocol on data for evaluating soil bacterial diversity. Consistently, the bead-beating procedure gave rise to the highest number of DNA bands, indicating the highest number of bacterial species. Supplementing the bead-beating procedure with additional cell-rupture steps generally did not change the bacterial community profile. The same consistency was not observed when evaluating the efficiency of the different methods on soil DNA yield. This parameter depended on soil type. The DNA size was of highest molecular weight with the sonication and grinding-freezing-thawing procedures (approx. 20 kb). In contrast, the inclusion of bead beating resulted in more sheared DNA (approx. 6-20 kb), and the longer the bead-beating time, the higher the fraction of low-molecular weight DNA. Clearly, the choice of DNA extraction protocol depends on soil type. We found, however, that for the analysis of indigenous soil bacterial communities the bead-beating procedure was appropriate because it is fast, reproducible, and gives very pure DNA of relatively high molecular weight. And very importantly, with this protocol the highest soil bacterial diversity was obtained. We believe that the choice of DNA extraction protocol will influence not only the determined phylogenetic diversity of indigenous microbial communities, but also the obtained functional diversity. This means that the detected presence of a functional gene—and thus the indication of enzyme activity—may depend on the nature of the applied DNA extraction procedure.     

农田土壤铅有效态分析法(AB-DTPA)的优化与验证

中国土壤类型丰富多样，不同区域土壤重金属赋存形态各异，难以实现对不同区域不同类型土壤重金属有效性的比较与评价。为筛选建立适宜区域农田土壤重金属的有效性评价的方法，该研究选择滇东地区5种性质差异较大的典型农地土壤（黄棕壤、黄壤、红壤、石灰土和紫色土）运用正交试验法将AB-DTPA法的提取条件（固液比、提取剂pH值和振荡时间）进行正交组合，建立土壤-作物Pb迁移模型与总量法、CaCl₂-DTPA法和AB-DTPA法进行拟合优度对比，并以白菜和菠菜为指示作物开展盆栽试验，探讨不同分析方法与作物Pb累积能力的相关性，进而综合评价不同方法的适宜性。结果表明，1）固液比（g/mL）1:3、提取剂pH值7.6以及振荡时间120 min对Pb有效态的提取量有较大的影响；2）与其他方法相比，优化AB-DTPA法（固液比1:3、pH值7.6和振荡时间120 min）能更好地预测作物对Pb的吸收能力；3）盆栽试验表明，优化AB-DTPA法提取的土壤 Pb有效态含量与白菜和菠菜吸收Pb含量具有更好的相关性（R²=0.898, R²=0.752），Pb的加标回收率范围为99.5%～113.0%，准确度高。4）将土壤pH值加入预测模型表明，土壤pH值对土壤Pb与在作物可食部分中相关性影响很小，表明优化AB-DTPA法很稳定。因此，优化AB-DTPA法适用于滇东地区农田土壤Pb有效态的提取，该法具有可行性、广普性和准确性。研究结果为区域农田土壤修复技术效果及污染风险评价提供基础依据。     

茶园土壤微生物总DNA不同提取方法的比较

传统的微生物分离培养方法,在反映茶园土壤微生物基因信息上有很大的局限性,因此,目前逐步被分子生态学的方法替代,而获得高质量、大片段、无偏好的土壤微生物总DNA则是茶园土壤微生物分子生态学研究的基础。本文采用SDS高盐法、变性剂加SDS高盐法、脱腐SDS高盐法、CTAB法和Krsek改进法5种土壤微生物DNA提取方法分别从茶园土壤微生物中提取总DNA,并对5种方法提取的DNA的片段大小、质量和产量进行了综合评价。结果表明,Krsek改进法提取到的DNA片段最大（〉23kb）、纯度最高（OD2UOD280〉1．70;OD2UOD230〉1．35）、产量较高（〉34．50μg／gdrysoil）且不需纯化就可以用于PCR扩增和RFLP分析。因此,Krsek改进法是一种高效、可靠且适合于茶园土壤微生物分子生态学研究的DNA提取方法。     

Optimizing the indirect extraction of prokaryotic DNA from soils

The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme-SDS-proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.     

Importance of DNA quality in comparative soil microbial community structure analyses

This study assessed how different in-situ lysis soil DNA extraction methods influence the DNA yield, quality and hence the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA). Of the methods tested in three soils, a modified hexadecyltrimethylammonium bromide-dithiotreitol (CTAB-DTT)-based method produced ?3 times more DNA of higher quality than the other methods (260/230 nm ratios=1.64–1.82 and 260/280 nm ratios=1.82–1.89 and extracts were less inhibitory of PCR). DNA extracted by this method also yielded more reproducible ARISA ribotypes (89?119 for bacteria and 48?88 for fungi; P<0.05) than DNA extracted by other methods, and consequently produced more reliable estimates of bacterial and fungal diversity in all three test soils. The significant correlations observed between the numbers of reproducible ribotypes and DNA extract 260/230 nm ratios (r=0.88 and 0.72 for bacteria and fungi, respectively; P<0.001) reaffirmed the strong influence of DNA quality on the reliability of microbial diversity indices determined based on PCR-based DNA fingerprinting technique. Results of discriminant function analysis (DFA) and multivariate analysis of variances (MANOVA) performed on ARISA profiles (number and relative abundance of ribotype) revealed that the variability associated with DNA extraction methods did not exceed the biological variability among soil types; this supports the conclusion that high-quality DNA underpins DNA fingerprinting techniques.     

Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

 We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods, i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples. The profiles generated by all four methods were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community. However, the patterns of clustering also indicated that different populations of bacteria could be detected in the same soil using different soil DNA extraction methods. The application of two dilution levels of DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods. The indirect methods gave clustered profiles at both dilution levels. It is likely that these methods extracted DNA from a major, easily desorbed, bacterial fraction, consisting of low-density populations. PCR-DGGE was found to be a suitable technique with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination of microbial community diversity at the molecular level. Received: 22 June 1999     

Analysis of total inositol phosphates in municipal solid waste compost-treated soils by two extraction methods

 Humic substances are the most dynamic component of agricultural soils. In this study, humic substances were extracted from soils based on standard acid/base solubility and by gel filtration using Sephadex G25. Organic P is a component of humic substances, and inositol phosphates are considered to have high prominence in organic P. The objectives of the study were to determine the effects of municipal solid waste (MSW) compost on the amount of inositol phosphates (IP) present in soils, and compare methods of extracting humic substances from soils. Total IP (IP_T) in soil extracts was determined following separation using a AG-1× 8 anion exchange resin, acid digestion and ICAP analysis for P. The percentage of IP (%IP) of the total P in the soil was also determined by colorimetric analysis. The soils were part of an experiment to study the effects of three rates of MSW compost and fertilizers on three crops grown in a Pugwash sandy loam (Humo-Ferric Podzol). The data were compared using ANOVA atP≤0.05; extraction methods, fractions [humic/fulvic, high/low molecular (MW)], and five treatments (three different rates of compost, fertilizer, and untreated soil) were compared. The gel filtration method extracted significantly more IP_T and %IP than the solubility method. In the solubility method, humic and fulvic acids contained the same amounts of IP_T and %IP, while the high MW fraction always contained more IP_T and %IP than the low MW fraction in the gel filtration method. Fulvic acids and the low MW fraction contained similar amounts of IP_T and %IP. There were differences in %IP between compost-treated soils and the non-compost-treated soils, although there were no differences in IP_T due to rate of compost addition. Received: 20 October 1999     

AB‐DTPA cation extraction in Spanish soil samples

Abstract

Since only one extraction is required to determine a large number of nutrients, many laboratories employ universal extractants to determine the available nutrients in a soil sample. This paper compares the universal ammonium bicarbonate‐DTPA (AB‐DTPA) method developed by Soltanpour and Schwab (1977) with the traditional methods, ammonium acetate (NH₄OAc) test for exchangeable cations and the Lindsay and Norwell (1969) test for the micronutrients. Results from the analysis of 28 soils by these methods were compared. Most soils were selected from those used by the Spanish Working Group for the Standardization of Analytical Methods. In most cases, statistical correlations between methods presented good agreement for each element, but depending on the soil pH range, some elements needed two correlations. Also, when results for wet and dry soils were compared, variability was lower when the AB‐DTPA extraction method was used. We concluded that, besides being faster, the AB‐DTPA method is valid for Spanish soils, even for calcium (Ca) extraction in calcareous soils, where the ammonium acetate method fails due to excessive Ca solubilization.     

Comparison of Various Chemical Extraction Methods used for Determination of the Available Iron Amounts of Calcareous Soils

This study was conducted to compare the most appropriate method for the evaluation of available iron (Fe) status of calcareous soils by using nine different chemical extraction methods. Leaf and soil samples were collected from nine peach (Prunus persica L.) orchards, each of which included green, slightly chlorotic, and severely chlorotic peach trees. According to the chlorosis degrees of the leaves, total and active Fe contents and some soil properties were determined. Relationships between these parameters and Fe amounts obtained from the methods were correlated. Among the methods tested, method 3 (M3) [0.05 N hydrochloric acid (HCl) + 0.025 N sulfuric acid (H₂SO₄)] and method 8 (M8) 0.05 M ethylenediaminetetraacetic acid (EDTA) (pH 7.0) were the most suitable methods to indicate the available Fe status of the soils.     

重金属复合污染农田土壤DNA的快速提取及其PCR-DGGE分析

国内首次运用FastPrep○R 核酸快速提取系统提取了重金属复合污染农田土壤的DNA ,并对其进行了聚合酶链反应—变性梯度凝胶电泳 (PCR DGGE)分析。结果表明 ,FastPrep○R核酸提取仪与相应的FastD NASPINKitforSoil试剂盒联用时 ,能有效地分离到纯度较高的重金属污染农田土壤的DNA。PCR DGGE电泳图谱表明 ,PCR产物经DGGE检测后得到的电泳条带清晰且分离效果好 ,可以明显反映出重金属复合污染导致了农田土壤微生物在基因上的损伤 ,影响到农田土壤生态系统的细菌丰富度 ,改变了土壤环境的优势菌群 ,从而使农田土壤微生物群落结构多样性发生变化。可见 ,FastPrep○R核酸提取系统同样适用于重金属污染农田土壤环境中微生物基因组DNA的快速分离和纯化 ,得到的DNA可直接用于PCR DGGE分析。     

Determination of carbon and nitrogen in microbial biomass of southern-Taiga soils by different methods

The results of methods for determining microbial biomass carbon vary in reproducibility among soils. The fumigation-extraction and substrate-induced respiration methods give similar results for Albic Luvisol and Gleyic Fluvisol, while the results of the rehydration method are reliably higher. In Histic Fluvisol, relatively similar results are obtained using the fumigation-extraction and rehydration methods, and the substrate-induced respiration method gives almost halved results. The seasonal dynamics of microbial biomass carbon also varies depending on the method used. The highest difference is typical for the warm period, when the concentrations found by the extraction and substrate-induced methods poorly agree between two out of three soils studied. The concentration of microbial biomass nitrogen is less sensitive to the analytical method: the differences between the results of the fumigation-extraction and rehydration methods are statistically insignificant in the all soils. To reveal stable relationships between the results of determining microbial carbon and the soil properties and analytical method, a large diversity of soils should be studied. This will allow for proposing of conversion factors for the recalculation of the obtained values to the concentrations of carbon and nitrogen in microbial biomass for different soils (or soil groups) and, hence, the more correct comparison of the results obtained by different methods.     

Calibration model of microbial biomass carbon and nitrogen concentrations in soils using ultraviolet absorbance and soil organic matter

There is a need for a rapid, simple and reliable method of determining soil microbial biomass (SMB) for all soils because traditional methods are laborious. Earlier studies have reported that SMB‐C and ‐N concentrations in grassland and arable soils can be estimated by measurement of UV absorbance in soil extracts. However, these previous studies focused on soils with small soil organic matter (SOM) contents, and there was no consideration of SOM content as a covariate to improve the estimation. In this study, using tropical and temperate forest soils with a wide range of total C (5–204 mg C g^?1 soil) and N (1–12 mg N g^?1 soil) contents and pH values (4.1–5.9), it was found that increase in UV absorbance of soil extracts at 280 nm (UV₂₈₀) after fumigation could account for 92–96% of the variance in estimates of the SMB‐C and ‐N concentrations measured by chloroform fumigation and extraction (P < 0.001). The data were combined with those of earlier workers to calibrate UV‐based regression models for all the soils, by taking into account their varying SOM content. The validation analysis of the calibration models indicated that the SMB‐C and ‐N concentrations in the 0–5 cm forest soils simulated by using the increase in UV₂₈₀ and SOM could account for 86–93% of the variance in concentrations determined by chloroform fumigation and extraction (P < 0.001). The slope values of linear regression equations between measured and simulated values were 0.94 ± 0.03 and 0.94 ± 0.04, respectively, for the SMB‐C and ‐N. However, simulation using the regression equations obtained by using only the data for forest profile soils gave less good agreement with measured values. Hence, the calibration models obtained by using the increase in UV₂₈₀ and SOM can give a rapid, simple and reliable method of determining SMB for all soils.     

A simplified risk assessment for losses of dissolved reactive phosphorus through drainage pipes from agricultural soils

Abstract

Soil tests with extractions are commonly used for risk assessments of phosphorus (P) leaching. Procedures for routine analysis of crop-available soil P by extraction with acid ammonium lactate (P-AL) have been used for nearly 50 years in Sweden, Norway and several East European countries. Aluminium and iron (Al-AL and Fe-AL) were determined in the same extract for 40 well known clayey, loamy or sandy soils from the Swedish long-term studies. Average outcome was 16.8 and 6.0% for the two elements related to extraction with chelating ammonium oxalate (Al-AO and Fe-AO) and concentrations had a correlation coefficient of 0.947 and 0.891, respectively, when the two extraction agents were compared. On average, P-AL determination using inductive coupled plasma (ICP) resulted in 19% higher soil P concentrations compared to analysis using a colorimetric method based on non-calcareous and calcareous soils from the southern counties in the Swedish soil survey, represented mainly by sandy loam soils. Degree of P saturation on a molar basis in the AL extract (DPS-AL) was determined for 22 Nordic observation fields with drained clayey, loamy and sandy soils. Results were used together with long-term flow-weighed concentration of dissolved reactive P (DRP) concentration in drainage water. These parameters were correlated (r=0.918, p=0.000) and could be fitted to a linear regression model (R²=84.3). In addition, two fields with unusually high DPS-AL values could clearly be identified as those with lowest P sorption index and highest DRP concentrations in drainage water. This demonstrates DPS-AL to have the potential as an environmental risk indicator for Swedish acid soils. A set of 230 non-calcareous soils in the southern counties of Sweden from the Swedish soil survey indicated that 3% of the soils had a high DPS-AL in the topsoil or subsoil, from which high DPS leaching probably occurs.     

Determination of bentazone, dichlorprop, and MCPA in different soils by sodium hydroxide extraction in combination with solid-phase preconcentration

A method for the extraction of bentazone, dichlorprop, and MCPA in three selected Norwegian soils of different textures is described. Initially three different extraction methods were tested on one soil type. All methods gave recoveries >80% for the pesticide mixture, but extraction with sodium hydroxide in combination with solid-phase preconcentration was used for further recovery tests with soils of different properties spiked at four herbicide concentration levels (0.001-10 microg/g of wet soil). The method was rapid and easy and required a minimum of organic solvents. The recoveries were in the range of 82-109, 80-123, and 45-91% for the soils containing 1.4 (Hole), 2.5 (Kroer), and 37.8% (Froland) organic carbon, respectively. Limits of quantification using GC-MS were 0.0003 microg/g of wet soil for bentazone and 0.0001 microg/g of wet soil for both dichlorprop and MCPA.     

A method for obtaining the relationship between the amount of DNA and the fine root weight from mixtures of fine roots and soil particles

Fine root biomass can be estimated from the quantity of DNA of a target plant extracted from fine root samples using regression analysis. However, the application of this method to fine root samples mixed with soil particles (mixed samples) is difficult due to the high DNA adsorption capacity of some clay minerals. Our aim in this study was to clarify the enhancement level of the DNA extraction efficiency of an improved method, and to obtain a regression line between the amount of DNA and the root biomass from a mixed sample with similar reliability as for fine roots alone (pure root sample). We examined the amount of DNA extracted from a mixture of Zea mays L. fine roots and highly adsorbent Kanuma soil using various concentrations of a skim milk solution, which acts as an adsorption competitor for the soil particles during the DNA extraction process. The amount of DNA of Zea mays extracted from the mixed sample using 0% skim milk was lower than from the pure root sample. However, the amount of DNA extracted from the mixed sample increased with increasing concentrations of skim milk, reaching the same level as for the pure root samples and resulting in a regression line that was similar to the pure root samples. Optimal DNA extraction levels were obtained with the addition of 20?µL of a 20% skim milk solution to 30?mg of a mixed sample. We also discuss the applicability of this method to other plant species and soil types.     

Pressurized Hot Water and DTPA‐Sorbitol as Viable Alternatives for Soil Boron Extraction. II. Correlation of Soil Extraction to Responses of Boron‐Fertilized Alfalfa

Abstract

Pressurized hot water and DTPA‐Sorbitol are two relatively new, proposed alternative soil boron (B) extraction methods for which no data on yield or plant nutrient uptake have been reported for validation. Both methods initially have shown significant correlation with the hot water extraction method in untreated soils as well as soils incubated with various levels of B. The objective of the research was to extract samples of B‐treated soils by using all three extraction methods and correlate the B values obtained to yield, B tissue concentration, and total B removal of alfalfa (Medicago sativa). Greenhouse and field experiments on alkaline and limed acid soils naturally low in hot water‐extractable B were conducted to test alfalfa response to B fertilizer. In the greenhouse, highly significant relationships exist between plant uptake and extractable B with all three methods at varying levels of applied B, but no alfalfa yield response was observed. All three methods result in accurate predictions of plant B tissue concentrations and total B removal. The field experiment exhibited a significant positive relationship between total alfalfa yield and extractable B using hot water and pressurized hot water extractions. Extractable B using DTPA‐Sorbitol was not related to total alfalfa yield in the field experiment. This work, coupled with the earlier incubation studies, supports the pressurized hot water extraction method as an improvement over hot water in diverse soil types. The lack of relationship in the acid soil supports DTPA‐Sorbitol as an improvement over hot water in alkaline soils.