Culture-dependent and culture-independent microbial investigation of pine litters and soil in subtropical Australia

Background, aim, and scope  Forest plantations, widely grown for wood production, involve the selective promotion of single-tree species or replacement of natural species by exotic tree species. Slash pine (Pinus elliottii) has been chosen for reforestation of infertile sandy soils in southeast Queensland, Australia. These exotic pine plantations minimize soil and water losses and are important scientific study sites. The soil environment of these plantations, though devoid of sufficient nutrients, organic carbon and other factors, harbors innumerable bacteria that may play a crucial role in maintaining soil quality and ecosystem functions. These soil microorganisms also have the potential for use as sensitive biological indicators to reflect environmental changes. It is therefore essential to understand the interrelationships among bacterial communities and their environment by assessing their structural and functional diversity and their responses to disturbances. The main aim of our investigation was to determine the diversity of bacterial communities in forest litters and soil during the forest leaf litter decomposition using culture-dependent and culture-independent techniques. Materials and methods  A 25-cm (diameter) × 40-cm core sample was collected and fractionated into three subsamples designated E1 (L leaf litter layer), E2 (F leaf litter layer), and E5 (0–10 cm soil layer). Both culture-dependent and culture-independent methods were applied in this study. In the culture-independent study, a strategy of whole-community DNA extraction, polymerase chain reaction (PCR) amplification followed by cloning and 16S rDNA sequence analysis was used; for culture-dependent study, the strategy included sample plating and bacteria isolating, DNA extraction, PCR amplification, and 16S rDNA sequence analysis. The diversity similarities between two bacterial communities and two methods are quantified using Jensen–Shannon divergence. Results  From culture-dependent study, 336 colonies in total were isolated and grouped from the three subsamples, and the 16S rRNA sequence analysis from a representative isolate from each morphogroup (21 isolates) indicated that they belonged to the phyla Actinobacteria, Firmicutes, and Proteobacteria. Culture-independent assessment based on 16S rRNA gene library comprising 194 clones revealed that members of the phylum Actinobacteria were absent in the culture-independent studies. Clones in libraries from E1 consisted exclusively of members of the Firmicutes. The majority of clones from E2 were related to Firmicutes (79%) and Proteobacteria (21%). Clones derived from E5 were mostly affiliated with Acidobacterium (42%), followed by unclassified bacteria (27%), Verrucomicrobiales (12%), Proteobacteria (11%), and Planctomycetes (8%). Discussion  This study showed that bacterial culturabilities in different fractions of leaf litters were similar, and both of them were higher than the bacterial culturability in the soil. Unculturable bacterial diversity in the soil, however, was much higher than the leaf litter bacterial diversity. The bacterial diversity on the top layer of leaf litters was slightly less than that on the bottom layer of leaf litters. This might indicate that forest soils are a more complex environment than leaf litters are and also that they might inhabit more unculturable microorganisms in the forest soils, which would need to be further investigated. The leaf litter layer samples also demonstrate the significant difference between the bacterial community diversity discovered by these two methods in this study. The information provided by assessing the different fractions of leaf litters and forest soil has improved our understanding of the bacterial community distributions within the forest soil and the above-leaf litters in an exotic pine plantation of subtropical Australia. Conclusions  This study represents the first attempt to examine the bacterial community in the different fractions of forest leaf litters and soil in subtropical Australia. The data from this study show that the 16S rDNA clone libraries provided more comprehensive phylogenetic diversity in the soil and leaf litter samples than the culture collections provided, and both the culture-dependent and culture-independent studies revealed that the bacterial diversity present in the leaf litters was very different to that present in the soil. The comparative analysis of bacterial communities in different fractions of leaf litters and soil samples has also provided important baseline information about the bacterial diversity and composition in the exotic pine forest plantations. Recommendations and perspectives  The experimental data provided important information on the bacterial diversity in forest leaf litter and soil samples, though additional surveys and comparisons at different locations would be needed to further characterize. In addition, combined methods that can provide different parts of information on bacterial diversity are encouraged to be used in bacterial community study. The established libraries of diverse 16S rRNA gene fragments from slash pine leaf litters and forest soil can be used to construct specific DNA primers and probes to target bacterial groups of interest. It may then be possible to study the ecology of these bacterial communities and the role of specific bacterial groups that contribute to the many interesting properties of these environments.     

紫色水稻土水稳性团聚体土壤微生物基因组DNA提取方法探讨

为了找到提取土壤微生物总DNA的最佳方法,通过OD值检验、凝胶电泳、PCR和DGGE分析,比较了Reddy法、基于DNAout kit试剂盒改进的实验方法、以及Kuske修订法、Edgcomb改进法、SDS高盐提取法、Eichner调整法等常用的不同土壤微生物基因组的DNA提取方法在亚热带地区长期免耕紫色水稻土水稳性团聚体0.25～2.0 mm粒径上的提取效果.结果表明,6种方法都可以从团聚体中提取到长度大于23.1 kb的DNA片段,但不同方法提取的DNA的产量存在明显差异,土壤总DNA均不需纯化就可以用于PCR扩增,使用细菌16S rDNA基因V3区的通用引物可扩增得到相应的片段.研究表明,改进的DNAout kit试剂盒法是长期免耕紫色水稻土水稳性团聚体中微生物基因组DNA的最佳提取方法.     

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响,为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取,该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA,并对这两种方法的提取DNA的效果进行了比较。结果表明,试剂盒法不能用于提取土壤中的微生物DNA;可以从猪粪中提取到DNA,PCR扩增能得到目的产物,但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低,纯化后纯度增加,但DNA有所损失,用于PCR扩增时结果不理想;处理猪粪样品,提取的DNA浓度较低但纯度较高,PCR扩增结果比较理想。由此可见,化学裂解法用来提取猪粪样品中的微生物DNA是可行的,但需寻求更好的土壤样品微生物DNA的提取方法。     

不同保存方法和提取方法对扁桃基因组DNA质量的影响

以扁桃（Amygdalus conmmuis L.）幼叶为试材,通过7种保存方法并采用改良2×CTAB法、3×CTAB法和SDS法对其DNA进行提取,用琼脂糖凝胶电泳检测其DNA完整性,分光光度计测其产量和纯度,以探讨不同保存方法和不同提取方法对扁桃DNA提取质量的影响。结果表明,扁桃新鲜幼叶、压干幼叶后在-75℃保存、45℃烘干幼叶后在-75℃保存以及硅胶干燥幼叶后在-75℃保存三个月均可以用三种提取方法可获得完整的DNA,纯度和产量均高,其保存和提取方法简便易行且获得高质量DNA。扁桃鲜叶直接放在-20℃保存比-75℃保存更易获得DNA,而压干后的扁桃叶片-20℃保存三个月并采用SDS法提取,获得高产量、高纯度的DNA,不影响提取效果。SDS法和改良3×CTAB法均可获得扁桃DNA,但SDS法优于3×CTAB法,其操作简单、耗时少;改良2×CTAB法提取扁桃成功率较低,不稳定,产量比其它两种方法低。     

Purification and isotopic signatures (<Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C, <Emphasis Type="Italic">δ</Emphasis><Superscript>15</Superscript>N, Δ<Superscript>14</Superscript>C) of soil extracellular DNA

The aim of this work was to obtain pure extracellular DNA molecules so as to estimate their longevity in soil by an isotope-based approach. Extracellular DNA molecules were extracted from all horizons of a forest soil and purified by the procedure of Davis (Purification and precipitation of genomic DNA with phenol–chloroform and ethanol. In: Davis LG, Dibner MD, Battey JF (eds) Basic methods in molecular biology. Appleton & Lange, Norwalk, 16–22, 1986) without (DNA1) or with (DNA2) a successive treatment with binding resins followed by elution. The two differently purified DNA samples were compared for their A₂₆₀/A₂₈₀ ratio, polymerase chain reaction (PCR) amplification and natural abundance of stable (¹³C and ¹⁵N) and radioactive (¹⁴C) isotopes. The purity index and the PCR amplification did not differentiate the efficiency of the two purification procedures. The isotopic signature of DNA was more sensitive and was strongly affected by the purification procedures. The isotopic measurements showed that the major contaminant of extracellular DNA1 was the soil organic matter (SOM), even if it is not possible to exclude that the similar δ ¹³C, δ ¹⁵N and Δ¹⁴C values of DNA and SOM could be due to the use of SOM-deriving C and N atoms for the microbial synthesis of DNA. For extracellular DNA2, extremely low values of Δ¹⁴C were obtained, and this was ascribed to the presence of fossil fuel-derived substances used during the purification, although in amounts not revealed by gas chromatography-mass spectrometry analysis. The fact that it is not possible to obtain contaminant-free DNA molecules and the potential use of soil native organic compounds during the microbial synthesis of DNA make it not achievable to estimate the age of soil extracellular DNA by radiocarbon dating.     

The Use of PCR to Detect a Pathogen in Compost

Testing compost for pathogens can be very difficult, time consuming and expensive. A method to purify DNA from compost for amplification by polymerase chain reaction (PCR) was sought. The published techniques used to purify DNA from soils or sediments did not work with compost samples in our laboratory. We have devised an extraction protocol specifically for compost samples. In a series of Escherichia coli-spiked compost samples, ranging from 10⁸ to 10³ organisms per gram of compost, the removal of substances that inhibit PCR amplification of DNA was accomplished by extracting the organisms from the compost and treating the extract with base resin. After cell lysis, the collected DNA was further purified on a 1 percent low melt agarose gel. A 260 base-pair region of the lac Z gene, unique to E. coli, was successfully amplified by PCR in the DNA extracted from the gel. The technique was sensitive to the level of 10⁴ E. coli cells per gram of compost.     

一种可用于PCR扩增的直接提取土壤细菌DNA的方法

本文以澳大利亚桉树林和松树林的土壤为例 ,采用Napp提取液和SDS直接溶解土壤细菌 ,并配合温浴 -玻璃珠震荡、苯酚 -氯仿萃取和异丙醇提取以及纯化DNA等步骤 ,直接从土壤样品中提取了土壤细菌DNA。所得DNA完全适用于酶解和PCR扩增的要求。该方法高效简单 ,费用低 ,在土壤微生物研究中具有重要的应用价值     

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 10⁴ CFU equivalents g⁻¹ lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10⁴ CFU equivalents g⁻¹. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.     

An empirical approach to target DNA quantification in environmental samples using real-time polymerase chain reactions

Recent advances in molecular techniques have allowed for the routine examination of nucleic acids in environmental samples. Although current methodologies are very sensitive, accurate target DNA quantification from environmental samples remains challenging. To facilitate high-throughput DNA quantification from environmental samples, we developed a novel DNA quantification method based on a non-linear curve-fitting approach to extract additional information from quantitative PCR amplification curves and used the fitted parameters to develop multiple regression standard equations for target DNA quantification. A 3-parameter sigmoidal function performed superior to a 4-parameter Weibull function for generating the multiple regression standard equations. In a verification experiment, target DNA was quantified in a series of ‘unknown’ samples in three soils using this approach and the results were compared to target DNA values determined using corrected and uncorrected Ct-based (threshold cycle) methods. For each method, the deviations from the expected target DNA content were determined. Results clearly showed that over all DNA concentrations, target DNA content determined by the non-linear curve-fitting method was more accurate and more precise than values predicted by all other methods. Analysis of variance conducted on the predicted DNA contents also revealed fewer statistical artifacts with the non-linear curve fitting method compared to the conventional Ct-based methods. The novel approach described here is accurate, inexpensive, and very amenable for automation and high-throughput applications.     

Novel preparation method of template DNAs from wine for PCR to differentiate grape (Vitis vinifera L.) cultivar

Template DNAs were extracted from wine and purified for use as samples for PCR to differentiate grape cultivars. It has been pointed out that the authentication of grape material by PCR using wine as a material is very difficult. The problems are (1) decomposition of DNAs during fermentation; (2) contamination of DNAs from microorganisms such as yeast; (3) interference of DNA extraction by polysaccharides and polypeptides in the beverages; and (4) coexistence of PCR inhibitors, such as polyphenols. For this study was developed a novel preparation method of template DNA from wine to differentiate grape cultivars using PCR by (1) lyophilizing and pulverizing the fermented beverage to concentrate the DNAs; (2) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction using heat-resistant amylase and proteinase K without DNA damage by endogenous DNase; and (3) separation of the template DNAs for PCR from PCR inhibitors, such as polyphenols, by purification using 70% EtOH extraction and isopropyl alcohol precipitation. To prevent the amplification of microorganisms' DNAs during PCR, suitable PCR primers closely related to the specific plant DNAs, such as chloroplast DNA and mitochondrial DNA, were selected. The sequences of the amplified DNAs by PCR were ascertained to be the same as those of grape materials.     

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     

土壤微生物多样性研究的新方法

传统的分离培养和鉴定土壤微生物方法所具有的困难性和局限性 ,是造成难以深入了解土壤微生物生态学特性和多样性组成方面的主要障碍。本文运用分子生物学技术 ,以澳大利亚两种主要森林类型的土壤微生物多样性研究为实例 ,介绍了从土壤中直接提取土壤微生物DNA的方法以及末端限制性酶切片段长度多态性 (T RFLP)分析的基本原理和方法。作者认为 ,用该方法提取的土壤真菌DNA的纯度高 ,完全适合PCR扩增和T RFLP分析的要求。T RFLP已成为国外深入研究土壤微生物多样性的理想方法之一     

蛋鸽早期性别DNA分子鉴定

蛋鸽早期性别的鉴定一直是制约蛋鸽业发展的瓶颈问题。在本研究中,我们采用PCR扩增和琼脂糖凝胶电泳检测的方法,对20日龄期的100只蛋鸽的性别连锁基因染色体解旋酶DNA结合基因CHD（chromo-helicase DNA binding）进行跨内含子扩增检测。蛋鸽早期DNA分子电泳检测结果与成年后的真实性别核对结果表明：所有雌性蛋鸽扩增结果含有两条带,其大小分别为280bp和250bp,雄性蛋鸽仅有一条280bp的条带;泊松统计分析发现,蛋鸽早期DNA分子判定结果达到统计学上显著水平,可以作为蛋鸽早期性别鉴定的一种方法。本实验实现了以DNA分子为基础的早期蛋鸽性别鉴定,将对蛋鸽业早期的饲养成本降低和早期雄性蛋鸽的淘汰起到准确的指导作用。     

干制羊肉基因组DNA不同提取方法的比较

为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL^-1,A₂₆₀/A₂₈₀值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。     

Can denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16s rDNA of soil bacterial populations be used in forensic investigations?

In criminal investigations, information on the origin of soils may be crucial for solving cases. The biological complexity of soil may potentially be used for sorting and differentiating between soil samples. Nucleic-acid based analyses of soil microbial populations are powerful tools, routinely used in studies of this habitat. Application of such approaches in forensics implies that a standardized DNA extraction method has to be applied to all samples. In this study, several DNA extraction protocols were compared. An improvement on the method proposed by Tsai and Olson (1991) was found to be most suited to extract DNA from various soil types, including from small samples. A blind test on soils from a crime, an alibi scene and unrelated locations was conducted to evaluate the potential of environmental PCR and denaturating gradient gel electrophoresis for use in forensic science. In most cases, soil patterns clustered according to soil type and location.     

Rapid PCR-based method for the direct analysis of fungal communities in complex environmental samples

Fungal community analysis using 18S rDNA primer pairs and denaturing gradient gel electrophoresis of PCR products (Vainio, E.J., Hantula, J., 2000. Mycological Research 104, 927–936) was applied to field studies of the forest ecosystem. We report a DNA extraction method producing high quality DNA allowing successful PCR amplification from problematic samples without use of nested polymerase chain reaction (PCR) procedures. The analysis was found to be applicable for samples from environments of varying fungal diversities and high organic matter content: wood samples from fallen branches of trees, laboratory mini-ecosystems and forest humus samples. When the method was tested using replicate forest soil samples, it was shown to be highly reproducible.     

短序十大功劳基因组总DNA提取方法研究

以短序大功劳嫩叶为材料,采用CTAB法、CTAB改良法1、CTAB改良法2、SDS法和试剂盒法五种方法提取短序十大功劳基因组总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的纯度和得率,用ISSR-PCR扩增的方法检测所得总DNA的质量。结果表明,五种方法均能从短序大功劳叶片中提取到基因组DNA,但不同方法提取得的基因组DNA的纯度、浓度和得率存在明显的差异。CTAB改良法2和试剂盒法提取的DNA纯度高,可直接用于下游分子生物学实验,CTAB法、CTAB改良法1和SDS法提取的总DNA质量较差,不利于下游的分子生物学实验;五种方法提取的总DNA的得率在10．836-451．709μg/g之间,呈CTAB法〉SDS法〉CTAB改良法1〉CTAB改良法2〉试剂盒法的现象。此实验获得的结果可以为短序十大功劳分子生物学研究提供基础。     

蜂蜜中转基因成分的PCR检测

我国是蜂蜜生产大国,棉花是主要蜜源植物之一。近些年来,我国种植的棉花大多为转基因抗虫棉,棉花蜜中是否含有转基因成分,引起国内外广大消费者的密切关注。本文以抗虫棉种植地采取的棉花蜜为原料,采用改良CTAB法,在DNA提取过程中,用PBS缓冲液除去其中大部分的可溶性多糖,并提高CTAB提取缓冲液中NaCl浓度以去除残余多糖,从蜂蜜中成功提取出片段完整、纯度较高的DNA,DNA得率为486ng/mL,OD260/OD280为2.01,能够满足后续PCR定性检测的需求。从以转基因棉花为蜜源的蜂蜜中检测出外源DNA片段,建立了蜂蜜中转基因成分的PCR检测技术。本研究对转基因食品标识制度的完善、转基因食品监管、减少贸易摩擦以及保证消费者权益等具有重要意义。     

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。     

Results of molecular analysis of an archaeological hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.) DNA sample from North West China

Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C. sativa sample showed that C. sativa races might have been introduced into China from the European–Siberian center of diversity.