Cellulose-degrading potentials and phylogenetic classification of carboxymethyl-cellulose decomposing bacteria isolated from soil

In a previous study, culturable carboxymethyl-cellulose (CMC) decomposing soil bacteria isolated from different sampling positions across an agricultural encatchment have been classified into 31 pattern groups by digestion of amplified 16S rDNA using a single restriction enzyme (Ulrich and Wirth: Microb. Ecol. 37, 238-247, 1999). In order to reveal relationships between phylogenetic diversity and phenotypic functions, a further differentiation of two selected site-specific pattern groups (I and H) was performed, resulting in a sub-classification of four and three ARDRA groups, respectively. Based on sequencing a representative isolate of each ARDRA group, the isolates were assigned to the genus Streptomyces. The ARDRA groups were dispersed across various clades of the genus with a direct affiliation to species known for cellulolytic activity in one group, only. The isolates differed in potentials to degrade colloidal, native or highly crystalline cellulose derivatives. Out of 39 isolates, 11 were capable of degrading all substrates, 17 were restricted to degrade CMC only, and 11 were active decomposers of exclusively both CMC and colloidal cellulose. In most cases, the genetic classification of the isolates corresponded with groupings based on cellulose degrading capabilities. Thus, isolates of four ARDRA groups were restricted to the degradation of CMC, while two further isolates which efficiently degraded all cellulose derivatives formed two separate ARDRA groups. The major ARDRA group, however; displayed a high variability of degradation capabilities. The study of additional phenotypic features revealed a broad potential to decompose a set of various carbon substrates, which matched the phylogenetic classification in several cases.     

Diversity and Activity of Cellulose-Decomposing Bacteria,Isolated from a Sandy and a Loamy Soil after Long-Term Manure Application

The community of culturable cellulolytic bacteria was analyzed in two long-term experimental field sites on Albic Luvisol (silty sand) and Haplic Phaeozem (loam), with and without farmyard manure treatment. Against the backdrop of significant differences in soil properties, the bacterial community structure differed clearly between sites and was affected by manure application as analyzed by T-RFLP of 16S rDNA. The population densities of cellulolytic bacteria were significantly increased by manure application in Phaeozem. Cellulose decomposing potentials of 537 isolates were tested on soluble, colloidal, and crystalline cellulose. The results showed some evidence of a greater proportion of isolates with high decomposition activity in Luvisol, but no impact from manure application could be observed in both soils. Restriction analysis and sequencing of 16S rDNA of isolates revealed a rather simple community composition that was dominated by Streptomyces (67%). The composition of the RFLP groups was affected by manure application, which was most evident in Luvisol, whereas an effect of the soil type could not be found. Although abundant RFLP groups were assigned to phylogenetically different bacterial classes (Actinobacteria, Betaproteobacteria, and Gammaproteobacteria), cellulolytic activity could not consistently be differentiated. All in all, cellulolytic capabilities of the isolates were highly variable and did not map to phylogenetic affiliation.     

Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis

AIMS: To demonstrate the occurrence of cellulolytic bacteria in the termite Zootermopsis angusticollis. METHODS AND RESULTS: Applying aerobic cultivation conditions we isolated 119 cellulolytic strains from the gut of Z. angusticollis, which were assigned to 23 groups of aerobic, facultatively anaerobic or microaerophilic cellulolytic bacteria. 16S rDNA restriction fragment pattern and partial 16S rDNA sequence analysis, as well as numerical taxonomy, were used for the assignment of the isolates. The Gram-positive bacteria of the actinomycetes branch could be assigned to the order Actinomycetales including the genera Cellulomonas/Oerskovia, Microbacterium and Kocuria. The Gram-positive bacteria from the order Bacillales belonged to the genera Bacillus, Brevibacillus and Paenibacillus. Isolates related to the genera Afipia, Agrobacterium/Rhizobium, Brucella/Ochrobactrum, Pseudomonas and Sphingomonas/Zymomonas from the alpha-proteobacteria and Spirosoma-like from the "Flexibacteriaceae" represented the Gram-negative bacteria. CONCLUSIONS: A cell titre of up to 10(7) cellulolytic bacteria per ml, determined for some isolates, indicated that they may play a role in cellulose digestion in the termite gut in addition to the cellulolytic flagellates and termite's own cellulases. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of bacteria on cellulose degradation in the termite gut has always been a matter of debate. In the present survey we investigated the aerobic and facultatively anaerobic cellulolytic bacteria in the termite gut.     

Microwave irradiation is a useful tool for improving isolation of actinomycetes from soil

Actinomycetes are an important source of novel, biologically active compounds. New methods need to be developed for isolating previously unknown actinomycetes from soil. The objective of this experiment was to study microwave irradiation of soil as a means for isolating previously unknown actinomycetes. Soil samples were collected at ten elevations between 800 m and 3670 m on Taibai Mountain, Shaanxi Province, China. Moistened soil samples were irradiated at 120 W heating power (2450 MHz) for 3 min using a household microwave oven. Irradiation increased total actinomycete, streptomycete, and antagonistic actinomycete counts on three types of culture media. Irradiation also increased the number of culturable actinomycete isolates. Some actinomycete isolates were culturable only after the soil was irradiated, whereas other isolates could not be cultured after irradiation. Irradiation of soil from elevations >3000 m increased actinomycete counts significantly but had little effect on the number of culturable actinomycete isolates. In contrast, irradiation of samples from elevations <3000 m had relatively little effect on actinomycete counts, but significantly increased the number of culturable actinomycete isolates. We used 16S rDNA sequence analysis to identity 14 actinomycete isolates that were only culturable after irradiation. Microwave irradiation of soil was helpful for isolating Streptomyces spp., Nocardia spp., Streptosporangium spp., and Lentzea spp. Slightly more than 90% of the identified actinomycete species were biologically active. In conclusion, microwave irradiation is a useful tool for isolating biologically active actinomycetes from soil.     

Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil.

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

Assessment of bacterial community structure in a long-term copper-polluted ex-vineyard soil

The influence of long-term copper contamination on the diversity of bacterial communities was investigated in an ex-vineyard soil. Two sites of the same area but exhibiting different 3-fold exchangeable copper (Ex-Cu) concentrations were analysed. Culturable bacterial community structure was assessed using a variety of approaches: determination of culturable bacteria number, analyses of 132 isolates, and denaturing gradient gel lectrophoresis (DGGE) patterns of bacterial biomass grown on agar plates and of soil DNA. There was no significant difference in the number of total heterotrophs at the two sites, whereas the percentage of fast-growing bacteria growing in 1 day, was lower at the site with the higher Ex-Cu content. A high percentage of Cu-tolerant bacteria was found in both sites (63-70%) and it was relatively independent of the Cu content. Shifts in species composition of the culturable bacterial community were detected by analysing isolates from the two soils, Gram-positive bacteria prevailed in the less-polluted soil while Gram-negative bacteria in the more-polluted soil. Each sample site had a community with a different metal resistance pattern. Our study seems to indicate that in this soil ecosystem, copper influenced the culturable bacterial communities, affecting the structural diversity and altering some of the metal resistance of the microorganisms. The Sorensen similarity index calculated on DGGE profiles of 16S rDNA of total and culturable bacterial communities indicated a different species composition at the two sites, although both sites had the same biodiversity degree and different dominance.     

Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 10⁷ to 2.5 × 10⁸ cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site

In this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences.     

Impact of cultivation on characterisation of species composition of soil bacterial communities

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.     

繁茂膜海绵中可培养稀有放线菌的多样性

摘要：【目的】本文旨在尝试改进分离培养方法从大连海域繁茂膜海绵中筛选稀有放线菌,并对其多样性进行研究。【方法】根据繁茂膜海绵元素组成配制微量元素溶液,加入到放线菌分离培养基中,同时将部分培养基稀释成寡营养培养基,结合富集培养法,对繁茂膜海绵中放线菌进行分离培养。采用16S rDNA的限制性片断长度多态性(Restriction Fragment Length Polymorphism, RFLP)分析和序列分析,揭示其多样性。【结果】共获得可培养放线菌59株,通过形态、颜色观察,将其归为27个类群。RFLP分析表现为15种不同的图谱类型。16S rDNA序列分析表明：它们分别属于放线菌的10个属,其中布劳氏菌属（Prauseria）和糖单胞菌属（Saccharomonospora）是首次报道从海绵中分离培养。【结论】改进的分离培养基适合于繁茂膜海绵中稀有放线菌的分离培养,进一步揭示了该海绵中丰富的稀有放线菌,同样的方法有可能应用于其他海绵放线菌的分离培养。     

种植模式和坡位对茶园土壤细菌群落结构及功能类群的影响

茶树（Camellia sinensis L.）种植是亚热带丘陵山区主要的土地利用类型,茶园种植模式是影响土壤细菌群落结构的主要人为因素。为揭示种植模式和坡位对土壤细菌群落结构和功能的影响,选取两种不同种植模式（常规和有机种植模式）和3个坡位（上、中、下坡位）表层土壤（0-20cm）为对象,采用野外调查、Illumina Miseq高通量测序和PICRUSt2功能预测相结合的研究方法,研究不同种植模式和坡位下土壤细菌群落结构和功能特征,阐明土壤理化性质对土壤细菌群落结构的影响,预测土壤细菌功能特征。研究结果表明：（1）与常规种植模式相比,有机种植模式茶园土壤细菌Alpha多样性有所降低,其中中坡位常规茶园土壤细菌Sobs和Simpson指数显著高于有机茶园（P<0.05）;从坡面尺度看,两种种植模式下土壤细菌Alpha多样性指标均以中坡位最高,其中常规茶园中坡位土壤细菌Ace和Simpson指数均显著高于下坡位（P<0.05）。（2）各样地茶园土壤细菌共获得29个门82个纲190个目316个科517个属929个种,主要细菌优势门为绿弯菌门（Chloroflexi）、放线菌门（Actinobacterita）、变形菌门（Proteobacteria）、酸杆菌门（Acidobacteria）。土壤细菌群落优势属以AD3、热酸菌属（Acidothermus）、norank_f__norank_o__Elsterales和norank_f__Xanthobacteraceae为主。（3）主坐标分析（PCoA）显示,不同种植模式的茶园土壤细菌群落结构存在明显差异,常规种植模式下不同坡位之间的土壤细菌群落结构有显著差异（P<0.05）,有机种植下不同坡位之间的土壤细菌群落结构无显著差异。置换多元方差分析（PERMANOVA）结果表明不同种植模式的土壤细菌群落结构差异显著（P<0.05）,而不同坡位土壤细菌群落结构无显著差异（P>0.05）,说明种植模式对茶园土壤细菌群落结构的影响更大。组间群落差异分析（LEfSe）表明,57个差异物种对种植模式非常敏感,不同种植模式富集了不同的细菌类群。（4） PICRUSt2功能预测共获得6个一级功能层和46个二级功能层,表现出功能上的丰富性,土壤细菌群落在代谢、遗传信息处理和环境信息方面功能活跃。有机种植模式提高了土壤细菌碳水化合物代谢、氨基酸代谢、膜运输、信号转导、脂质代谢及外源生物降解与代谢功能。（5）相关分析和冗余分析结果表明,土壤碱解氮、速效磷、全磷、全钾和pH是影响土壤细菌群落丰度和多样性的主要影响因子。总体而言,有机种植模式改变了茶园土壤细菌群落结构和代谢功能,增加土壤有益细菌的数量,有利于保持茶园土壤可持续的生态环境。     

Diversity and Antimicrobial Activity of Actinomycetes Isolated from Lut Desert: The Extremely Arid Climatic Zones of Iran

Lut desert is situated in one of the extremely arid climatic zones of Iran and is one of the hottest deserts in our plant with the extreme fluctuation of temperature over a day. The main objective of this study is to characterize the diversity of the culturable actinomycetes and preliminary evaluation of their extracts as antimicrobial components on drug resistant pathogens. Twenty-four soil samples were collected, successively diluted and inoculated into the different culture media to support the growth of most culturable bacteria including actinomycetes. Phenotypic and molecular methods were used for accurate identification of recovered isolates particularly actinomycetes at the genus and species levels. The isolates were also evaluated for their inhibitory activities against drug resistant Acinetobacter baumannii, Enterococcus faecium, Klebsiella pneumoniae and Staphylococcus aureus. A total of 56 isolates recovered from the samples. Based on phenotypic tests, 41 isolates were identified as actinomycetes, amongst them 8 isolates were active against drug resistant pathogens. Our study revealed Lut desert, as one of the hottest deserts in the world, is the habitat to diverse taxa of bacteria particularly actinomycetes which have potential novel antimicrobial components.

     

Phylogenetic analysis of different isolates of<Emphasis Type="Italic"> Sulfobacillus</Emphasis> spp. isolated from uranium-rich environments and recovery of genes using integron-specific primers

The isolation and phylogenetic characterization of acidophilic moderate thermophilic bacteria from different locations of uranium mines and a uranium processing mill in Pakistan is reported. The dominant culturable bacteria found were related to Sulfobacillus thermosulfidooxidans in all the samples analyzed. Different strains displayed different levels of identity (95–97%) to16S rDNA of known strains of this species, indicating group heterogeneity. Genomic DNA from five isolates was subjected to amplification using integron-specific primers HS286 and HS287. Recovery of different integron-linked genes from one of the isolates indicated the usefulness of this approach for gene mining in place of traditional gene recovery methodologies.Communicated by K. Horikoshi     

Molecular characterization of rhizospheric soil streptomycetes isolated from indigenous Turkish plants and their antimicrobial activity

As part of a research program whose aim is to determine the diversity of streptomycetes in order to discover new bioactive secondary metabolites, rhizosphere soils of three indigenous plants were analyzed. A total of 55 actinomycetes were isolated using three different medium from the samples. The rhizospheric soil of the plant Aethionema dumanii gave the highest number of actinomycetes, i.e., 42% versus 27% and 31% for the soils from Salvia aytachii and Achillea ketenoglui, respectively. The AIA is the most favorable medium for the isolation of the actinomycetes from different rhizospheric soils. 16S rDNA sequence analysis revealed that while some isolates belong to different cluster groups such as Streptomyces lydicus, S. rochei, S. microflavus, S. griseoflavus, S. albidoflavus and S. violaceusniger, the majority of the sequences did not considerable clustered with the member of different Streptomyces groups. The in vitro antimicrobial activities of the crude organic and aqueous extracts of isolates were screened using a disc diffusion assay against a panel of bacteria and C. albicans. A total of 22 isolates showed antimicrobial activity. The antibacterial action of the extracts is more pronounced on Gram-positive than on Gram-negative bacteria in most cases. About 18% of the actinomycetes showed also antifungal activity. Study of the influence of two different culture media on production of bioactive molecules showed that the higher antimicrobial activity was obtained in M2 when compared to TSB. The results from this study provide evidence that the streptomycetes in the rhizosphere soils could be promising sources for antimicrobial bioactive agents.     

Soil microbial counts and identification of culturable bacteria in an extreme by arid zone

Sixteen samples of two soil cores (about 550 and 180 cm in depth) were drilled at intervals in the lower reach of Heihe river basin (northwest of China) in order to illustrate soil microbial characteristics and diversity of culturable bacteria in an extreme by arid environment. Soil water content, organic matter, total nitrogen, pH, direct cell counts, and culturable microorganism counts were evaluated. The total cell concentration was 19-1120/microg (i.e. 0.19-11.2 x 10(8) per g) soil, the culturable bacteria count being 0.2-10.9 per microg (i.e. 2 x 10(5)-10.9 x 10(6) CFU/g) soil. The number of direct cell counts obtained by 4',6-diamidino-2-phenylindole-staining or the cound of culturable microbes after enrichment with different media were statistically significantly correlated with soil organic matters, total nitrogen content, soil water content and surface vegetation; this partly explained the larger number in the deeper first core than in the shallower one. As part of identification of 228 colonies isolated from the two cores, thirty-two were selected for 16S rDNA amplification, sequencing and molecular identification. These 32 isolates were affiliated to 5 major groups of bacteria: alpha-Proteobacteria, 5-Proteobacteria, gamma-Proteobacteria, the high-G+C G+-bacteria, the low-G+C G- -bacteria, and the Cytophaga-Flexibacter-Bacteroides group. Twenty-eight were rod- or short-rod shaped, which accounted for >87.5% of all species; only 4 of 32 species were cocci (<12.5%).     

宁夏黄土高原马铃薯连作栽培对土壤可培养细菌遗传多样性的影响

采用平板培养、BOXAIR-PCR和16S rDNA RFLP技术对宁夏黄土高原马铃薯连作栽培土壤可培养细菌遗传多样性进行研究。结果表明,4个连作年限2个生育期8份土样共分离到91株细菌菌株, BOXAIR-PCR分析发现,91株细菌菌株的遗传相似系数为0.531～0.939,相同连作年限不同生育期根际土细菌菌群分布不同,不同连作年限同一生育期根际土细菌菌群的分布也不同,随着连作年限增加,可培养细菌遗传多样性呈现下降趋势;结合16S rDNA 的序列分析,从91株菌株中筛选出的41个代表菌株可分为23个物种,分属于细菌域的12个属,其中,芽孢杆菌属（Bacillus）占同一连作年限菌株数的53.6%。连作导致土壤细菌菌群结构发生变化,出现各自特有的菌属。系统发育分析表明,23个细菌物种分布于6个系统发育群。     

Bacterial diversity associated with subalpine fir (Abies lasiocarpa) ectomycorrhizae following wildfire and salvage-logging in central British Columbia

To assess the effect of fire and salvage logging on the diversity of mycorrhizal-bacterial communities, bacteria associated with Cenococcum, Thelephora, Tomentella, Russulaceae, and E-strain ectomycorrhizae (ECM) of Abies lasiocarpa seedlings were characterized using two approaches. First, bacteria were isolated and characterized by Biolog, gas chromatography fatty acid methyl ester (GC-FAME), and amplified 16S rDNA restriction analysis (ARDRA). The bacterial communities retrieved from ECM from both sites were dominated by Proteobacteria (groups gamma and beta). Pseudomonas was the most common genus isolated, followed by Variovorax, Burkholderia, and Xanthomonas. Gram-positive isolates (mostly high-G+C Gram-positive bacteria) were more frequently retrieved on the burned-salvaged site, many commonly associated with the two ascomycete ECM, Cenococcum and E-strain. Pseudomonas species were retrieved more frequently from Thelephora. Although actinomycetes were isolated from all sites, almost no actinomycetes or other Gram-positive bacteria were isolated from either Thelephora or Tomentella. Second, amplified 16S rRNA gene sequences were amplified directly from root tips and then cloned into the plasmid vector pAMP1, followed by restriction analysis. This technique distinguished more genotypes than isolates retrieved by culturing methods, but generally, results were similar in that the largest proportion of the bacteria were putatively Gram-negative; putative Gram-positive bacteria were fewer and most were from the burned-salvaged site. Direct cloning resulted in many patterns that did not match any identified isolates, suggesting that a large proportion of clones were unique or not culturable by the methods used. Analysis for both protocols showed no significant difference in bacterial diversity between the burned-salvaged and unburned sites.     

Diversity of culturable <Emphasis Type="Italic">Streptomycetes</Emphasis> from wheat cropping system of fertile regions of Indo-Gangetic Plains,India

Streptomycetes is one of the most important genera, representing up to 70% population among all soil actinomycetes. We investigated the genotypic diversity of culturable Streptomycetes, freshly isolated from thirty-eight different soil samples (wheat cropping system) from Indo-Gangetic Plains of India. A total of 238 morphologically distinct actinomycetes colonies were isolated and clustered together by restriction fragment length polymorphism analysis of 16S rRNA gene. Identification of representative isolates from each cluster was carried out by micro/macroscopic analysis as well as biochemical characterization, and further confirmed by 16S rRNA sequencing (sequences were submitted in public database, NCBI).     

Nuclease-producing bacteria in soil cultivated with herbicide resistant transgenic white poplars

This study was carried out using soil cultivated, under greenhouse conditions, with transgenic white poplars expressing thebar gene for tolerance to the Basta® herbicide. The occurrence of extracellular nucleolytic activity was monitored in soil samples collected at four different times over a 26-month period. The fraction of nuclease producing bacteria (NPB) ranged from 62.5 to 100% of the total culturable bacterial population. The DNA-methyl green plate assay allowed to distinguish five groups of bacteria showing increasing levels of extracellular DNase activity. The NPB isolates were classified by 16S rDNA sequence analysis as members of theBacillus, Brevibacillus, Microbacterium, Pseudomonas andStenotrophomonas genera. For each genus, NPB isolates were cultured in liquid medium and the nucleolytic activity during different growth phases was monitored. Production of extracellular nucleases was observed only during the mid-exponential growth phase of theBrevibaccillus Microbacterium andStenotrophomonas isolates, while no activity was evidenced for isolates classified within theBacillus andPseudomonas genera.