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1.
Soil nematodes are both taxonomically and functionally diverse, respond quickly to soil perturbation and have much potential as indicators of soil health. However, because of the perceived difficulty of identifying nematodes to species level morphologically, they are frequently neglected in soil ecological studies. Recently, extraction of soil DNA, amplification of 18S rDNA genes using nematode consensus primers and subsequent separation by denaturing gradient gel electrophoresis (DGGE) has been used to estimate nematode diversity in soil. Here, we investigate an alternative approach whereby nematodes are first extracted from the soil prior the 18S rDNA gene amplification using universal primers. We used this system to estimate nematode species richness in 10 soil samples—five from Scotland and five from the Netherlands. There was no direct correlation between species richness as estimated morphologically and by the PCR-DGGE method. However, inspection of the data suggested that the samples fell into two discrete groups, which was confirmed by canonical and stepwise discriminant function analysis; the values for the Shannon and equitability indices being important discriminators. Further analysis revealed a significant relationship between morphological species richness and DGGE estimates for species that represented greater than 1% of the sample biomass.  相似文献   

2.
DGGE法与常规培养法对稻田蓝细菌多态性分析结果比较   总被引：1，自引：0，他引：1  
研究运用蓝细菌和硅藻16SrDNA特异引物,将晚季水稻生长后期稻田土壤中提取的总DNA进行PCR扩增后,以DGGE技术对PCR产物进行分析结果表明,14条DGGE带经克隆测序,经NCBI基因库比对得晚季水稻生长后期存在10种蓝细菌,包括4种Leptolyngbya、1种Chamaesiphon、1种Nostoc、1种Oscillatoria、2种Syne-chococcus和1种Chroococcidiopsis。同层不同位置土壤中蓝细菌种群亦有所不同,但每个取样点都有一些特有的蓝细菌种类。用常规方法对同一稻田土壤样品进行分离培养,根据蓝细菌鉴定图谱观察到类似Lyngbya、Oscillatori-a、Chroococcidiopsis及Nostoc的蓝细菌,但显微镜下无法准确分类。比较结果表明采用DGGE法比常规培养法能更准确进行蓝细菌多态性鉴定。  相似文献   

3.
The use of molecular approaches based on 16S rDNA-PCR in microbial ecology has revealed a tremendous prokaryotic diversity in environmental samples. However, there is little or no systematic evaluation of the impacts of hypervariable (V) regions of rrs genes choice on microbial community analysis in soil samples, especially the detailed information about the dominant groups preferentially amplified by different primer pairs. In the present study, eight primer pairs were detected to compare the different V regions for fingerprinting microbial communities in a paddy soil irrigated with petroleum-wastewater, using denaturing gradient gel electrophoresis (DGGE) and amplified ribosomal DNA restriction analysis (ARDRA) techniques. Results reveal the obvious PCR bias produced by different V regions. Both ARDRA analysis of 16S rDNA clone library and DGGE suggest that V1-V3 region amplified with primer pair 8f-519r produced the most informative fingerprinting profiles. Additionally, V3-V5 region amplified with 341f-907r was another preferable choice for microbial diversity in petroleum-contaminated soil. The V4-V5 region and single V region (V1, V3, and V8) were not recommended for the future study of microbial diversity in soil samples. Phylogenetic analysis of 123 sequences from libraries constructed by amplicons generated from six different V regions suggests that different dominant groups were amplified with distinct primer sets. In detail, V1-V3 library (amplified with 8f-519r) and V3-V5 library were dominated by Actinobacteria (20.4%) (particularly in genus Arthrobacter), V1-V3 library (amplified with 63f-518r) was dominated by γ-Proteobacteria (25.0%) and α-Proteobacteria (22.0%) (particularly in genus Brevundimonas), V3 library was dominated by β-Proteobacteria (22.3%) (particularly in genus Gallionella) and α-Proteobacteria (20.0%), V6-V8 library was dominated by Chlamydiae (20.4%) and β-Proteobacteria (20.4%), V8 library was dominated by γ-Proteobacteria (27.2%) (particularly in genus Acinetobacter) and β-Proteobacteria (14.0%). The present work strongly recommends that primer pairs should be chosen cautiously in community diversity analysis based on PCR amplification of 16S rDNA, and involving at least two different 16S rDNA universal primer pairs would perform better.  相似文献   

4.
A potentially diagnostic 18S rDNA (ribosomal DNA) gene was amplified reliably from red-listed ethnomedicinal species of Myristica and its wild and related genera. Individuals from nine species of Myristicaceae were utilized for the study. The sequences ranged from 1,767 to 1,794 nucleotide (nt) in length. The GC content (%) varied from 52.77 to 51.04. The frequencies (%) of nt were A (23.31), T (23.82), C (24.48) and G (28.39). The alignment of all sequences produced 195/1,516 variable sites and 1,257/1,516 conserved sites. Total numbers of single nucleotide polymorphism (SNP) sites found in the alignment were 146/1,516. Knema andamanica (Warb.) W.J. de Wilde was the most distinct that included 18 variable regions and 15 InDel with 27 SNP sites, specific to this species. The identified regions from nine species of Myristica and its wild and closely related genera were deposited in the GenBank Database (Accession numbers JN228257-JN228265). Comparison of morphological identifications and phylogenetic analysis indicated that the specimens were correctly assigned on the basis of a short stretch of 18S rDNA (~1,600 bp) making this a potentially useful marker for the rapid molecular assignment of an unknown related species also. Significant sequence homology ranging from 72 to 99 % was observed on comparison with 18S rDNA genes of other plants in the public domain. A comparison of intraspecific data information of nine 18S with that of 73 matK and 86 rbcL sequences from GenBank revealed that polymorphism, divergence and conservation is higher in 18S locus for Myristicaceae. Hence these markers may be utilized for phylogenetic analysis, evaluation of species richness during ecological surveys or for environmental assessments. These molecular markers are especially important due to the fact that the species studied are mostly vulnerable and red-listed with limited availability in endangered ecological niches.  相似文献   

5.
The structure of the β-proteobacterial autotrophic ammonia-oxidizing bacterial (AOB) communities in a microcosm of submerged paddy soil was determined by denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments amplified using AOB-selective primers. Shift in the community composition was observed 4 weeks after submergence. The communities from the surface layers (0–1, 2–3 mm) of the soil microcosm were different from those of the subsurface layers (6–9, > 15 mm) and DGGE bands specific to each layer were detected. The majority of the retrieved sequences were Nitrosospira-like, whereas no Nitrosomonas-like sequences were obtained. The 16S rDNA primer set also amplified sequences that were not related to the known Nitrosospira-Nitrosomonas group, although they showed a close relationship with other groups of β-proteobacteria. The results suggest that Nitrosospira-like populations are dominant AOB populations in the submerged paddy soil, and that the oxic layer of submerged paddy soil harbours the specific AOB.  相似文献   

6.
Nucleic acid-based techniques allow the exploration of microbial communities in the environments such as the rhizosphere. Azospirillumbrasilense, a plant growth promoting rhizobacterium (PGPR), causes morphological changes in the plant root system. These changes in root physiology may indirectly affect the microbial diversity of the rhizosphere. In this study, the changes in the rhizobacterial structure following A. brasilense inoculation of maize (Zea mays) plants was examined by PCR-denaturating gradient gel electrophoresis (DGGE) and automated ribosomal intergenic spacer analysis (ARISA), using two universal primers sets for the 16S rRNA gene, and an intergenic 16S-23S rDNA primer set, respectively. Similar results were obtained when using either ARISA or DGGE performed with these different primer sets, and analyzed by different statistical methods: no prominent effect of A. brasilense inoculation was observed on the bacterial communities of plant roots grown in two different soils and in different growth systems. In contrast, plant age caused significant shifts in the bacterial populations.  相似文献   

7.
The 18S–28S and 5S rDNA sites are useful chromosome landmarks and provide valuable evidence about genome organization and evolution. This investigation was the first attempt to study the dynamics, distribution and directionality of rDNA gains and losses, as well as to understand the contribution of site number variation in the speciation of the genus Citrullus. In this study, we employed fluorescent in situ hybridization (FISH), using the18S–28S and 5S rDNA gene loci, to evaluate the differences between the (1) cultivated type watermelon C. lanatus var. lanatus (sweet watermelon), (2) the “bitter” desert watermelon C. colocynthis (colocynth) that is indigenous to the deserts of northern Africa, the Middle East and Asia, (3) the C. lanatus var. citroides (citron) “Tsamma” or “cow watermelon” that is known as and is indigenous to southern Africa, (4) and C. rehmii that thrive in the Namibian Desert. The FISH analyses showed that the sweet watermelon and colocynth have similar rDNA configuration. The sweet watermelon and colocynth genomes contain two 18S–28S rDNA gene loci, each located on a different chromosome, and one 5S rDNA locus which is co-localized with one of the 18S–28S rDNA gene loci. On the other hand, the C. rehmii has one 18S–28S rDNA locus and one 5S rDNA locus positioned on different chromosomes, while the citron has one18S–28S rDNA and two 5S rDNA loci, each located on a different chromosome. A FISH analysis of F1 (citron × sweet watermelon) chromosome spreads revealed uniparental homeologous rDNA gene copies pertaining to the sweet watermelon versus the citron chromosomes, with the sweet watermelon chromosome containing the 18S–28S and 5S rDNA locus versus the citron homologue chromosome that has the 5S rDNA locus, but not the 18S–28S rDNA locus. Genomic in situ hybridization (GISH) analysis, using the entire citron genome as a probe to be differentially hybridized on sweet watermelon chromosome spreads, revealed that the citron genomic probes mainly hybridize to subtelomeric and pericentromeric regions of the sweet watermelon chromosomes, suggesting extensive divergence between the citron and sweet watermelon genomes. The FISH and GISH cytogenetic analysis here indicate major differences in genome organization between the cultivated watermelon type sweet watermelon and its counterpart citron that thrive in southern Africa and considered a useful germplasm source for enhancing disease and pest resistance in watermelon cultivars.  相似文献   

8.
The narG gene is frequently used as a molecular marker for bacterial nitrate-reducing community analysis. In this study, a new set of primers targeting the narG gene was designed and applied to semi-nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) assay. The potential of the new primers was verified on DNA directly extracted from soils from five different experimental sites distributed in Central and Southern Italy. Specificity of the primers was determined by excision, amplification, and sequencing of bands resolved by DGGE. A phylogenetic analysis showed the correlation between the sequences retrieved from the soils studied and the narG sequences from β and γ-Proteobacteria. These primers expanded the existing molecular tools for ecological study on the size and diversity of nitrate-reducing bacterial community in soil.  相似文献   

9.
Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N2-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.  相似文献   

10.
The autotrophic ammonia-oxidising bacterial (AOB) community composition was studied in acid coniferous forest soil profiles at a site in southwestern Sweden 6 years after liming. Liming caused a significant increase in pH in the organic horizons, while the mineral soil was unaffected. The AOB communities were studied by single-strand conformation polymorphism (SSCP) in parallel with denaturing gradient gel electrophoresis (DGGE) analysis of partial 16S rRNA genes amplified by PCR using primers reported to be specific for β-Proteobacteria AOB, followed by nucleotide sequencing. High genetic diversity of Nitrosospira-like sequences was found in the limed soil profiles, whereas no AOB-like sequences were detected in the control soil at any depth, according to both the SSCP and DGGE analyses. This clearly showed that liming induced growth of a diverse flora of AOB at this forest site. Both Nitrosospira cluster 2 and cluster 4 sequences were present in the limed soil profiles, regardless of soil pH, but we found a higher number of sequences affiliated with cluster 4. The high lime dose seemed to affect the AOB community more than the low dose, and its effects reached deeper into the soil profile. Seven different Nitrosospira-like sequences were found 10 cm under the litter layer in the soil limed with the high dose, but only two in the soil amended with the low lime dose.  相似文献   

11.
We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.  相似文献   

12.
Using peptides as energy sources, H2 as electron donor, thiosulfate as electron acceptors, we isolated, from four ricefield soils originating from France and the Philippines, 52 strains of anaerobes, among which 18 reduced thiosulfate but not sulfate. These 18 strains were strict proteolytic asaccharolytic anaerobes producing H2S when grown on thiosulfate + H2. They exhibited the same restriction fragment length polymorphism (RFLP) profile (11 restriction enzymes tested). Partial sequencing of the 16S rDNA showed that they belonged to the genus Clostridium and were phylogenetically related to C. subterminale. DNA–DNA hybridization of a representative strain with the closest C. subterminale strain (DSM 6970T) yielded a value of 68.9%. Previous counts of thiosulfate reducers unable to reduce sulfate (TSRnSR) in ricefield soils, their identification as Clostridium strains, and the known ubiquity of this genus in such soils indicate that TSRnSR of the genus Clostridium may play a significant role in S cycling in some wetland soils.  相似文献   

13.
Saccaromonospora viridis is a thermophilic actinomycetes organism which is found in mushroom compost, as well as being a causal agent of mushroom worker's lung (MWL) and other hypersensitivity pneumonitis conditions, including farmer's lung. Phenotypically, it is difficult to distinguish the seven species described for this genus based solely on chemtaxonomic characterization, therefore it was the aim of this study to examine partial 16S rDNA PCR amplification and direct sequencing, as an improved molecular means of identification of Saccharomonospora viridis, associated with MWL. The approach adopted in this study was to identify hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the seven individual species within this genus and to employ highly conserved flanking primers to allow initial PCR amplification, prior to direct DNA sequencing of the 16S rDNA amplicon, in a partial 16S rDNA-sequence typing technique. Four universal 16S rDNA primer combinations [P11P/P13P, PSL/PSR, XB1(SV)/PSR and XB1(SV)/P13P] were compared for their ability to identify an unknown thermophilic Saccharomonospora organism from MWL. All PCR primer combinations coupled with direct sequencing allowed for the successful identification of the MWL isolate as S. viridis, demonstrating that universal 16S rDNA PCR primer pairs examined, including the P11P/P13P primer pair, flank regions within the 16S rRNA gene, of sufficient hypervariability to be able to reliably differentiate S. viridis from the other species within this genus. This approach may therefore be useful in the identification of Saccharomonospora spp. associated with composting, as well as with allergic alveolitis or pneumonitis associated occupational exposure in agricultural and horticultural environments, including mushroom production.  相似文献   

14.
Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.  相似文献   

15.
The present study aimed to investigate microbial communities in seven Indian composts and their potential for biocontrol of Fusarium oxysporum f. sp. lycopersici. In addition, identification of bioactive substances in disease suppressive composts was also attempted. Composts were chosen based on disease suppressiveness and subjected to molecular microbial analyses. Total genomic DNA from the composts was extracted and amplified with polymerase chain reaction using primers targeting the 18S rRNA and 16S rRNA genes of fungi and bacteria, respectively. Denaturing gradient gel electrophoresis (DGGE) fingerprinting and DNA sequencing were used to identify the fungal and bacterial targets. Phylogenetic analysis of the fungal 18S rRNA ITS gene sequences showed that phylum Ascomycota was dominant in all composts, while in the bacterial 16S rRNA gene sequences, the phylum Proteobacteria was dominant. Some fungi in disease suppressive composts grouped phylogenetically close to F. oxysporum. Bacterial sequences with close similarity (>95% identity) with Actinobacterium showed a strong presence only in disease suppressive composts. Disease suppressive composts formed a separate group in the cluster analysis of 18S rRNA ITS and 16S rRNA gene sequences. Gas chromatography-time of flight-mass spectrometry was performed with compost extracts to determine if bioactive substances were present in disease suppressive composts. The analysis of compost organic matter showed a negative association of disease suppressiveness with phloroglucinol, sitosterol, and monoenoic fatty acid, while cholesterol and certain organic acids were positively associated with suppressiveness.  相似文献   

16.
Total and active soil fungal communities in a native eucalypt forest and first rotation Pinus elliotti plantation were investigated by direct extraction of DNA and RNA from soil. Terminal restriction fragment length polymorphism (T-RFLP) analysis of internal transcribed spacer (ITS) and 18S rRNA profiles indicated that total and active fungal communities differed significantly in both forest types. This was supported by DGGE profile analysis on an individual plot basis for both forest types and when groups in the canonical analysis were redefined to allow comparison between forest types. Analyses of both ITS and 18S T-RFLP profiles indicated that conversion from native eucalypt forest to P. elliottii plantation may significantly alter total and active soil fungal communities. ITS DGGE (DNA) and 18S (RNA) profiles also suggested differences in fungal communities in the two forest types. No significant separation of the fungal communities in the two forest types was observed, however, when ITS DGGE (RNA) profiles were compared. Overall, the data suggest that conversion from native eucalypt forest to P. elliottii plantation at the Beerburrum State Forest in subtropical Australia has significantly altered soil fungal communities.  相似文献   

17.
The small subunit ribosomal gene (SSU rDNA) is used in both environmental and phylogenetic studies of nematodes. However, currently described SSU rDNA primers near to the 5′ end of the gene show mismatches with a number of different nematode sequences. Here a new SSU rDNA 5′ forward primer, Nem_SSU_F74, is designed from existing database sequences and its performance compared with a previously described forward primer, SSU_F04, by sequencing from nematode assemblage DNA, in combination with a previously described reverse primer. DNA was extracted from three nematode assemblages isolated from arable soil and, in total, six clone libraries were created: three amplified with established forward primer SSU_F04 and three with the novel primer Nem_SSU_F74. Ninety six clones were sequenced from each library. Nem_SSU_F74 libraries yielded a higher number of nematode sequences than SSU_F04 libraries, and a greater number of nematode taxa were found using the novel forward primer. The most abundant sequences were common to libraries created with either forward primer. Data from a morphological survey of the same samples revealed that biomass was more closely related to molecular analysis than simple counts of nematodes. For all but one nematode order (Aphelenchida), percentage assemblage composition was not significantly different between biomass and sequences obtained with the novel forward primer Nem_SSU_F74.  相似文献   

18.
Whole-cell fatty acids methyl ester (FAME) profile and 16S rDNA sequence analysis were employed to isolate and identify the bacterial groups that actively solubilized phosphates in vitro from rhizosphere soil of various crops of Korea. Out of several hundred colonies that grew on Pikovskaya's medium 13 best isolates were selected based on the solubilization of insoluble phosphates in liquid culture and further characterized and identified. They were clustered under the genera Enterobacter, Pantoea and Klebsiella and the sequences of three representative strains were deposited in the GenBank nucleotide sequence data library under the accession numbers AY335552, AY335553, AY335554.  相似文献   

19.
 Thirteen of the most abundant Gram-negative bacteria which are able to grow in N-depleted culture conditions were isolated from the rhizoplane and endorhizosphere of canola (Brassica napus) and identified by 16S rDNA sequence analysis. Eight of these bacteria induced a significant increase in root dry weight ranging from 11 to 52%. Phylogenetic positioning based on 16S rDNA sequences indicated that at least four genera are represented, Pseudomonas, Variovorax, Agrobacterium and Phyllobacterium. The most important direct plant growth-promoting-rhizobacteria effect was found with both isolates belonging to the Phyllobacterium. Received: 18 April 2000  相似文献   

20.
Nematode density and biodiversity in maize field soil treated with compost, chemical fertilizer and with no amendments were investigated in a multi-year field experiment at the Qu-Zhou experimental station, China Agricultural University. The soils were collected from the upper (0–20 cm) soil layer during the maize growing stages in 2004. The results demonstrated that significant differences for the total nematode density, bacterivores, fungivores, plant parasites and omnivores-predators density were found between treatments and between dates. The total nematode density and bacterivores density were greater in compost-treated soil than in chemical fertilizer-treated soil, and were greater in chemical fertilizer-treated soil than in control soil during all sampling periods. The total nematodes density ranged from 106 to 657 individuals per 100 g dry soil in the present study. Total 40 nematode genera were found in all treatments and sampling periods, and 12 genera were bacterivores, 4 genera were fungivores, 16 genera were plant parasites and 8 genera were omnivores-predators. Cephalobus, Rhabditis, Tylenchorhynchus, Pratylenchus, Helicotylenchus and Rotylenchus were dominant genera in present study. The plant parasites and bacterivores were dominant trophic groups. The ratio of bacterivores plus fungivores to plant parasites was higher in compost-treated soil compared to chemical fertilizer-treated soil except October. Maturity index and combined maturity index were lower in compost-treated soil compared to chemical fertilizer-treated and control soil except July. The plant parasite index was higher in compost-treated soil compared to chemical fertilizer-treated soil except July. The multi-year application of compost and chemical fertilizer had effected on soil nematode population density and community structure.  相似文献   

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