牛蒡上发生的根结线虫种类鉴定

2007～2009年,对我国牛蒡主产区的牛蒡及其根际土壤中的植物寄生线虫进行调查研究,发现根结线虫危害最为严重。根据根结线虫的形态学特征和分子生物学检测方法对其进行鉴定,结果表明,我国牛蒡主产区根结线虫种类为北方根结线虫(Meloidogyne hapla)。     

不同保存方法对光皮桦总DNA提取效果的影响

光皮桦是一种顽拗类植物,其鲜叶组织中富含酚类、糖类及其他次生代谢物。这些物质很容易使鲜叶发生褐变,从而严重影响植物基因组DNA的提取质量。本文以新鲜嫩叶为对照,比较了-20℃冷藏、硅胶干燥、改进的饱和NaCl-CTAB溶液保存和核分离缓冲液固定4种鲜叶保存方法对提取光皮桦基因组总DNA效果的影响,寻找最佳的光皮桦嫩叶保存方法。并以提取的核酸得率、纯度、片段分布情况、是否含有PCR反应抑制剂等指标来评价其保存效果。结果表明:-20℃冷藏的样品未能提到DNA;新鲜样品、改进的饱和NaCl-CTAB溶液保存和核分离缓冲液保存的样品均提到纯度较好的DNA,大小在48Kb左右,得率为400!g/g鲜叶左右,并获得条带清晰、多态性好的PCR扩增图谱;用硅胶干燥保存的样品也能提到DNA,但得率低,为51.2!g/g鲜叶,且未获得条带完整、清晰的PCR扩增图谱。     

Indica-japonica differentiation in Chinese rice landraces

Summary Ninety Chinese rice landraces were examined with special reference to the indica-japonica differentiation in terms of traditional criteria, isozyme analysis and PCR analysis of the chloroplast DNA (cpDNA). Cultivars were separated into indica and japonica defined by a discriminant function (Z) based on key characters, as well as by isozyme genotypes. Most indica landraces had chloroplast DNAs with a deletion at the Pst-12 fragment, while most japonica landraces had cpDNAs without the deletion. Two traditionally recognized varietal groups in China, keng and hsien, corresponded largely to the respective japonica and indica revealed in our study. The results obtained in this study showed good agreement for classification of indica and japonica types by the three methods: discriminant analysis by Z value, isozyme analysis, and PCR analysis for cpDNA.     

Microbial diversity in soil: ecological theories, the contribution of molecular techniques and the impact of transgenic plants and transgenic microorganisms

This review mainly discusses three related topics: the application of ecological theories to soil, the measurement of microbial diversity by molecular techniques and the impact of transgenic plants and microorganisms on genetic diversity of soil. These topics were debated at the Meeting on Soil Emergency held in Erice (Trapani, Italy) in 2001 for the celebration of the 50th anniversary of the Italian Society of Soil Science. Ecological theories have been developed by studying aboveground ecosystems but have neglected the belowground systems, despite the importance of the latter to the global nutrient cycling and to the presence of life on the Earth. Microbial diversity within the soil is crucial to many functions but it has been difficult in the past to determine the major components. Traditional methods of analysis are useful but with the use of molecular methods it is now possible to detect both culturable and unculturable microbial species. Despite these advances, the link between microbial diversity and soil functions is still a major challenge. Generally studies on genetically modified bacteria have not addressed directly the issue of microbial diversity, being mainly focused on their persistence in the environment, colonization ability in the rhizosphere, and survival. Concerns have been raised that transgenic plants might affect microbial communities in addition to environmental factors related to agricultural practice, season, field site and year. Transgenic plant DNA originating from senescent or degraded plant material or pollen has been shown to persist in soil. Horizontal transfer of transgenic plant DNA to bacteria has been shown by the restoration of deleted antibiotic resistance genes under laboratory in filter transformations, in sterile soil or in planta. However, the transformation frequencies under field conditions are supposed to be very low. It is important to underline that the public debate about antibiotic resistant genes in transgenic plants should not divert the attention from the real causes of bacterial resistance to antibiotics, such as the continued abuse and overuse of antibiotics prescribed by physicians and in animal husbandry.     

Molecular Evidence for Persistence of Anaplasma phagocytophilum in the Absence of Clinical Abnormalities in Horses after Recovery from Acute Experimental Infection

Background: Anaplasma phagocytophilum infects several mammalian species, and can persist in sheep, dogs, and calves. However, whether this organism persists in horses or induces long-term clinical abnormalities is not known.
Objectives: To evaluate whether A. phagocytophilum can persist in horses and to document clinical findings for 3 months after complete recovery from acute disease.
Animals: Five clinically normal adult horses that had recovered spontaneously from experimentally induced acute disease caused by a Swedish equine isolate of A. phagocytophilum .
Methods: Horses were monitored for up to 129 days post inoculation (PI) by daily clinical examination and at least alternate day blood sampling for evidence of A. phagocytophilum on polymerase chain reaction (PCR) and blood smears. All horses were euthanized and underwent postmortem examination.
Results: All horses were periodically PCR positive after recovery from acute infection. Before day 66 PI 2 horses were persistently PCR negative whereas 3 horses were intermittently PCR positive. Subsequently, 4 of 5 horses were intermittently PCR positive, particularly after stress mimicking interventions. One animal was positive immediately before postmortem examination. Clinical abnormalities related to persistence of anaplasma were not observed. No specific changes were found at postmortem examination, and all sampled tissues from all horses were negative on PCR for A. phagocytophilum .
Conclusions and Clinical Importance: Infection with A. phagocytophilum can persist in the horse for at least 129 days. However, the continued presence of the organism is not associated with detectable clinical or pathological abnormalities.     

Genetic diversity in Sorghum bicolor(L.) Moench accessions from different ecogeographical regions in Malawi assessed with RAPDs

Genetic variation within and among several Sorghum populations from different agroecological zones in Malawi were investigated using random amplified polymorphic markers (RAPDs). DNA samples from individual plants were analyzed using 35 oligonucleotides of random sequence. Twenty five of these primers allowed amplifications of random polymorphic (RAPD) loci. Overall, 52% of the scored loci were polymorphic. Every accession was genetically distinct. The analysis of molecular variance revealed that the within-region (among accessions) variations accounted for 96.43% of the total molecular variance. Observed variations in allelic frequency was not related to agroecological differences. The degree of band sharing was used to evaluate genetic distance between accessions and to construct a phylogenetic tree. Further analysis revealed that the sorghum accessions analyzed were genetically close despite considerable phenotypic diversity within and among them. It is suggested that all the sorghum landraces currently available in Malawi should be conserved both ex situ and in situ to maintain the current level of genetic diversity.     

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.     

Root-nodule bacteria from indigenous legumes in the north-west of Western Australia and their interaction with exotic legumes

Bacteria were isolated from root-nodules collected from indigenous legumes at 38 separate locations in the Gascoyne and Pilbara regions of Western Australia. Authentication of cultures resulted in 31 being ascribed status as root-nodule bacteria based upon their nodulation of at least one of eight indigenous legume species. The authenticated isolates originated from eight legume genera from 19 sites. Isolates were characterised on the basis of their growth and physiology; 20 isolates were fast-growing and 11 were slow-growing (visible growth within 3 and 7 d, respectively). Fast-growers were isolated from Acacia, Isotropis, Lotus and Swainsona, whilst slow-growers were from Muelleranthus, Rhynchosia and Tephrosia. Indigofera produced one fast-growing isolate and seven slow-growing isolates. Three indigenous legumes (Swainsona formosa, Swainsona maccullochiana and Swainsona pterostylis) nodulated with fast-growing isolates and four species (Acacia saligna, Indigofera brevidens, Kennedia coccinea and Kennedia prorepens) nodulated with both fast- and slow-growing isolates. Swainsona kingii did not form nodules with any isolates. Fast-growing isolates were predominantly acid-sensitive, alkaline- and salt-tolerant. All slow-growing isolates grew well at pH 9.0 whilst more than half grew at pH 5.0, but all were salt-sensitive. All isolates were able to grow at 37 °C. The fast-growing isolates utilised disaccharides, whereas the slow-growing isolates did not. Symbiotic interactions of the isolates were assessed on three annual, one biennial and nine perennial exotic legume species that have agricultural use, or potential use, in southern Australia. Argyrolobium uniflorum, Chamaecytisus proliferus, Macroptilium atropurpureum, Ononis natrix, Phaseolus vulgaris and Sutherlandia microphylla nodulated with one or more of the authenticated isolates. Hedysarum coronarium, Medicago sativa, Ornithopus sativus, Ornithopus compressus, Trifolium burchellianum, Trifolium polymorphum and Trifolium uniflorum did not form nodules. Investigation of the 31 authenticated isolates by polymerase chain reaction with three primers resulted in the RPO1 primer distinguishing 20 separate banding patterns, while ERIC and PucFor primers distinguished 26 separate banding patterns. Sequencing the 16S rRNA gene for four fast- and two slow-growing isolates produced the following phylogenetic associations; WSM1701 and WSM1715 (isolated from Lotus cruentus and S. pterostylis, respectively) displayed 99% homology with Sinorhizobium meliloti, WSM1707 and WSM1721 (isolated from Sinorhizobium leeana and Indigofera sp., respectively) displayed 99% homology with Sinorhizobium terangae, WSM1704 (isolated from Tephrosia gardneri) shared 99% sequence homology with Bradyrhizobium elkanii, and WSM1743 (isolated from Indigofera sp.) displayed 99% homology with Bradyrhizobium japonicum.     

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