Characterisation of endobacterial communities in ectomycorrhizas by DNA- and RNA-based molecular methods

The diversity of endobacteria associated with ectomycorrhizas of Suillus variegatus and Tomentellopsis submollis, in two Corsican pine (Pinus nigra) stands was analysed by cultivation-dependent and cultivation-independent molecular methods. Denaturing gradient gel electrophoresis (DGGE) analysis revealed the cultivable endobacterial communities associated with S. variegatus were similar within the same stand. The most abundant cultivable bacterial species belonged to the genera Pseudomonas and Burkholderia. Cultivation-independent molecular analysis indicated that the structure of the endobacterial communities in ectomycorrhizas was consistent across all samples regardless of ECM fungal species or the pine stand from which the samples were collected. However, comparison between rDNA- and rRNA-derived DGGE gels showed that metabolically active endobacterial species were not always detected in rDNA-based profiles. Clone libraries constructed from rRNA molecules indicated that Pseudomonas and Burkholderia spp. were metabolically active bacteria. As some of the most abundant cultivable bacteria, including Bacillus/Paenibacillus spp., were not detected in cultivation-independent DGGE profiles, a combination of cultivation-dependent and -independent approaches provided a more complete assessment of the diversity of endobacteria associated with ectomycorrhizas.     

Comparison of Bacterial Communities Associated with Microcrustaceans from the Floodwater of a Paddy Field Microcosm: Estimation Based on DGGE Pattern and Sequence Analyses

Bacterial communities associated with five kinds of microcrustaceans ( Tanycypris sp., Moina sp., Mesocyclops sp., Cypretta sp. and Heterocypris sp.) from the floodwater of a paddy field microcosm were examined by the application of denaturing gradient gel electrophoresis (DGGE) to PCR-amplified 16S rDNA products with universal bacterial primers and by sequencing of characteristic DGGE bands. The number of DGGE bands of the associated bacteria was small, indicating the association of specific bacterial members with the microcrustaceans studied, among which Tanycypris sp. showed the smallest number of bands. Principal component analysis (PCA) demonstrated that the community structure of the associated bacteria could be divided into three groups: Podocopida ( Tanycypris sp., Cypretta sp. and Heterocypris sp.), Moina sp. and Mesocyclops sp., and further analysis separated Tanycypris sp. and Heterocypris sp. into different clusters. The duration of the incubation period affected the bacteria associated with Tanycypris sp., Moina sp. and Cypretta sp. only. Nearly all of the associated bacteria belonged to Gram-negative bacteria, especially the Cytophaga-Flavobacterium-Bacteroides (CFB) group. Closest relatives of the DGGE bands common to three Podocopida and Mesocyclops sp. belonged to an invertebrate endosymbiont.     

Molecular Monitoring of SRB Community Structure and Dynamics in Batch Experiments to Examine the Applicability of in situ Precipitation of Heavy Metals for Groundwater Remediation (15 pp)

Background, Aims and Scope   Sulfate-reducing bacteria (SRB) are known for their capacity to reduce and precipitate heavy metals (HM) as metal sulfides, offering the opportunity to create an in situ reactive zone for the treatment of heavy metal-contaminated groundwater, a process called in situ metal precipitation (ISMP). The applicability of the ISMP technology first has to be investigated at a laboratory scale before going into an on site application. The evaluation and optimization of the ISMP process is facilitated when physical/chemical analysis techniques are combined with molecular tools that specifically monitor the abundance, diversity and dynamics of the indigenous sulfate reducing microbial community. In this study, batch experiments were conducted in order to investigate the feasibility of ISMP as a groundwater remediation strategy for an industrial site contaminated with elevated levels of Zn, Cd, Co and Ni. Methods   The potential of different types of carbon source/ electron donor (lactate, acetate, methanol, ethanol, Hydrogen Release Compound?, molasses) to stimulate the sulfate reduction and metal precipitation activity of the naturally present (or indigenous) SRB community was explored. In addition, the effect of amending vitamin B12 and yeast extract was evaluated. The ISMP process was monitored by combining analytical analyzes of process parameters (SO42-concentration, heavy metal concentrations, pH, Eh) with molecular tools such as SRB subgroup and genus specific PCR, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis of clone sequences, based on either the 16S rRNA or the dsr (dissimilatory sulfite reductase) gene. Results and Discussion   The efficiency of different carbon-sources to stimulate the ISMP process followed the order HRC 〉 molasses 〉 methanol 〉 lactate 〉 ethanol 〉 acetate. Within 10 weeks, the highest sulfate and metal removal efficiencies ranged from 85% to 99%. Addition of yeast extract boosted the ISMP process, whereas vitamin B12 negligibly affected SRB activity. Analysis of the sulfate reducing population by SRB subgroup and genus specific PCR demonstrated that members of the genus Desulfosporosinus dominated in all batch tests, while 16S rDNA DGGE profiles additionally revealed the presence in the microbial communities of non-sulfate reducing bacteria within the family Clostridium and the -proteobacteria. The dsrB-based DGGE profiles allowed us to assess the diversity and dynamics of the sulfate reducing community and added to a better understanding of the effects of different batch conditions on the ISMP process. Remarkably, all dsrB sequences affiliated with the dsrB gene sequence cluster found in Desulfotomaculum, which received their xenologous dsrB gene from the -proteobacteria. Conclusions   The batch experiments, which aimed at stimulating the activities of the indigenous SRB communities, demonstrated that these communities were present and that their activities could be used to obtain efficient in situ precipitation of the contaminating heavy metals. This opens the possibility to test this concept in the future as an on site demonstration as part of the groundwater strategy for the heavy metal contaminated site. Although batch setups are suitable for preliminary feasibility studies for ISMP, they do not reflect the in situ situation where sulfate and heavy metal and metalloid polluted groundwater are supplied continuously. A sulfate reducing strain JG32A was isolated from whose 16S rRNA gene affiliated with the genus Desulfosporosinus, while its dsrB gene sequence clustered with Desulfotomaculum dsrB gene sequences, which received their xenologous dsr genes from -proteobacteria. Therefore we hypothesize that the batch experiments enrich members of the Desulfosporosinus genus that possess a non-orthologous dsrB gene. Recommendation and Perspective   The next step towards an on site pilot test for ISMP will be the setup of a series of column experiments, with process conditions that are selected based on the above mentioned results. This will allow to define optimal ISMP process conditions and to test its long-term efficacy and sustainability before going into an on site bioremediation application. By applying the described molecular tools together with physical-chemical analyzes, it can be investigated whether the same SRB community is enriched and which type of C-source is most effective in promoting and sustaining its growth and sulfate-reduction activity.     

添加乳酸菌和葡萄糖对苜蓿青贮品质的影响

初花期收获的苜蓿经过0h、8h和32h的晾晒(干物质含量分别为27.15%、38.45%、50.87%),添加不同含量的乳酸菌 葡萄糖(0、105cfu/g 20g/kg、106cfu/g 15g/kg、107cfu/g 10g/kg)进行青贮,其品质测定结果表明:无添加剂直接青贮时,苜蓿低水分(干物质含量为50.87%)青贮的效果最好,其青贮综合评定为82分;添加乳酸菌 葡萄糖青贮时,3种不同干物质水平中以凋萎苜蓿(干物质含量为38.45%)青贮后青贮料的青贮品质和综合评定最好;苜蓿较低干物质含量(27.15%)条件下,适中的乳酸菌和葡萄糖添加量(106cfu/g 15g/kg)可以得到最好的青贮效果和最高的综合评分;而在干物质含量为38.45%和50.87%条件下,乳酸菌和葡萄糖添加量为107cfu/g 10g/kg时,可以得到较好的青贮效果和最高的综合评分。     

基于显微图像处理的稻瘟病菌孢子自动检测与计数方法

稻瘟病菌孢子的检测通常在显微镜下由人工目测完成,该方法费时、费力、自动化程度低。因此,该研究提出了一种基于显微图像处理技术的稻瘟病菌孢子自动检测和计数方法。首先,采用显微图像系统获取稻瘟病菌孢子图像;然后提出一种分块背景提取法对其进行光照校正;根据显微图像中孢子的边缘特征,利用Canny算子进行边缘检测,其中Canny边缘检测过程中的阈值应用模糊C均值算法在梯度图上自动确定;接着对边缘检测后的二值图像进行数学形态学闭开运算处理。根据孢子和主要杂质的形态特征,利用椭圆度、复杂度和最小外接矩形宽度等形态特征参数对目标物进行分类,提取只含孢子的二值图像。最后,提出了基于距离变换和高斯滤波的改进分水岭算法对粘连孢子进行分离。测试结果表明:在100幅测试的显微图像样本中,孢子检测的平均准确率为98.5%,满足稻瘟病菌孢子自动检测和计数要求。     

Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique

Few studies of the inoculation of cereal crops with N₂-fixing bacteria have included more than one or two plant genotypes. In a recent study performed in Argentina using 12 different maize genotypes, it was found in 2 consecutive field experiments that several of them responded consistently, either negatively or positively, to inoculation with a mixture of strains of Azospirillum spp. The present study in post was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes. Two of these genotypes were grown in ¹⁵N-labelled soil with the aim of quantifying any contributions of biological N₂ fixation. Two genotypes (Morgan 318 and Dekalb 4D-70) produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp. strains or fertilized with the equivalent of 100 kg N ha^-1. The other genotypes (Dekalb 2F-11 and CMS 22) showed little response to inoculation or N fertilization. The Morgan 318 and Dekalb 4D-70 genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR- mutants generally caused plant responses similar to those of the parent strains. Despite problems with the stabilization of the ¹⁵N enrichment in the soil, the ¹⁵N isotope dilution results indicated that there were very significant biological nitrogen fixation (BNF) contributions to the Dekalb 4D-70 and CMS 22 maize genotypes.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday     

抗生素/抗性细菌/抗性基因在土壤-植物系统中迁移转化及阻控消减的研究进展

抗生素在土壤环境中的迁移转化过程及生态毒理效应已成为各国土壤学、植物学、环境科学和食品科学等领域学者及政府关注的热点。阐明抗生素及其复合抗性污染物在土壤-植物系统中的环境行为及生态毒理学机理,对管控生态环境风险及保障食品安全具有重要的意义。本文综述了近年来国内外关于土壤-植物系统中抗生素的迁移转化规律,抗生素/抗性细菌/抗性基因的生态风险以及复合抗性污染物阻控消减技术的研究进展。本综述可以为土壤-植物系统中抗生素抗性污染风险的管控和消减提供科学支撑。     

Bacteria associated with Glomus clarum spores influence mycorrhizal activity

The effects of bacteria associated with arbuscular mycorrhizal fungal (AMF) spores on spore germination, growth in vitro and on the pea-AMF symbiosis were evaluated. Bacterial colonies were recovered from untreated Glomus clarum NT4 spores and NT4 spores decontaminated with 5% chloramine-T for 30, 45 and 60 min on five different media. Both G+ and G− bacteria were recovered from untreated NT4 spores, whereas only G+ bacteria were isolated from decontaminated spores. An in vitro assessment of the effect of spore-associated bacteria on clean, decontaminated NT4 spores revealed that (i) most of the bacteria isolated from untreated spores generally did not significantly alter spore function, (ii) some bacteria isolated from clean, decontaminated spores inhibited or stimulated NT4 spore germination, (iii) stimulation of spore germination occurred only when bacteria were in contact with spores, and (iv) inhibition of spore germination was the result of volatile bacterial metabolites. A stimulatory bacterial isolate, Bacillus pabuli LA3, significantly (P<0.05) enhanced the shoot growth, AMF-colonization, shoot N content and P use efficiency of NT4-inoculated 6 week-old pea plants over that of plants co-inoculated with an inhibitory bacterial isolate, Bacillus chitinosporus LA6a and NT4.     

Gram-negative bacterial flora on the root surface of wheat (Triticum aestivum) grown under different soil conditions

We identified 161 Gram-negative bacterial strains isolated from the root surface of wheat grown under different soil conditions. The strains were divided into seven groups based on major morphological and physiological properties. Taxonomic allocation of the groups was verified by guanine+cytosine contents of DNA. Except for one group, which may be assumed to include bacteria belonging to the genera Flavobacterium and Cytophaga, the various groups were taxonomically united. The distribution of the groups changed with soil improvement. Pseudomonads predominated in unimproved soil, but Flavobacterium and Cytophaga spp. were predominant in the most improved soil. As all the strains were non-fermentative by Hugh and Leifson's test, API 20NE identification was applied. However, many strains were misidentified by this system, especially in the Flavobacterium and Cytophaga spp. group. For ecological studies, the strains were classified to species level by the API 20 NE system and by the results of a combination of guanine+cytosine (mol%) and isoprenoid quinone data. The pattern of distribution of the bacteria on the root surface of wheat varied at species level within one genus depending on soil conditions.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday