Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

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.     

Metal stress selects for bacterial ARDRA-types with a reduced catabolic versatility

In a previous study, 76 out of 899 bacteria, isolated from uncontaminated and adjacent metal-contaminated experimental plots, were found to fall within 12 clusters on the basis of a fourfold ‘amplified ribosomal DNA restriction analysis’ (ARDRA). Eight of these 12 ARDRA-clusters were re-investigated in the present study by sequencing about 500 bp of the 16S rDNA of the pertaining 51 isolates. Sequencing confirmed that, with the stringent fourfold ARDRA applied, all isolates of a given ARDRA-cluster did indeed have identical 16S rDNA. Seven ARDRA-clusters had 16S rDNA similarities of 98% or more with reference species suggesting identification was at least at the genus level. With one ARDRA-cluster, having a similarity of only 93% with Sphingobacterium heparium, identification was less certain. An unequivocal assignment of the eight ARDRA-clusters to distinct species was not possible. However, similarities >99.3% of five ARDRA-clusters with five reference species from two genera suggested identification was eventually even at the species level.Isolates of seven ARDRA-clusters from increasingly metal-contaminated soil samples were decreasingly versatile with respect to aromatic acids they could utilise as sole sources of carbon and energy. Only in one ARDRA-cluster exhibiting 99.8% similarity with Arthrobacter nicotinovorans was this effect less pronounced. The data strongly suggest that the decrease in catabolic versatility observed with metal-affected bacterial communities also applies to the genus level, if not to the species level.     

高产水稻土细菌多样性的培养法与非培养法比较研究

利用细菌的通用引物扩增江西余江县高产水稻土红壤细菌总DNA和平板培养细菌混合总DNA的16S rDNA基因片段,在此基础上分别建立两种16S rDNA文库(文库a和文库b)。从两个文库中各随机挑选100个克隆,扩增出阳性克隆中的插入片段后选用HhaⅠ和RsaⅠ两种四碱基酶进行ARDRA(amplified rDNA restriction analysis)分析。统计比较分析发现,文库a的Shannon-Wienner指数、Simpson指数、丰富度、均一度分别为4.432、0.987、18.885和0.973,均高于文库b中相应的多样性参数(分别为2.271、0.758、5.736和0.501),即平板培养方法所展现的细菌群落结构多样性低于土壤中原始的多样性。结果表明,传统培养方法存在着很大的局限性,必须结合新的分子生物学技术手段才能更全面完善地认识土壤微生物群落结构多样性,以期充分利用其中丰富的微生物资源。     

Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

Consensus nematode 18S ribosomal DNA primers were designed by aligning available 18S sequences and identifying a variable region flanked by highly conserved regions. These primers were then used to amplify nematode 18S rDNA from whole soil community DNA extracted from a range of European grassland types. Cloning of the PCR amplicons (778 bp) followed by restriction digest analysis (RFLP) resulted in the recovery of 34 unique nematode sequences from the four grasslands studied. Comparison of these data with the limited number of 18S rDNA nematode sequences currently held in on-line databases revealed that all of the sequences could be assigned to known nematode taxa albeit tentatively in some cases. Two of the sequences recovered from the site in the Netherlands (wet, hay-grassland) were recovered in a clade that included a sequence of the genus Trichodorus whilst other sequences from this site showed similarity with 18S rDNA sequences of the genus Prismatolaimus (five sequences), Xiphinema (one sequence) and Enoplus (one sequence). Of the remaining sequences, two showed some affinity with Mylonchulus (UK, upland peat), four with Steinernema (UK) and one sequence with Mesorhabditis (Hungary, east European Steppe). Three sequences from the Netherlands and one from Hungary were recovered in a clade that included a sequence of the genus Pratylenchoides whilst three further sequences from the Netherlands and two from Hungary were recovered in a clade encompassing the genus Globodera. Of the remaining nine sequences, two (NL6, NL62) formed a distinct lineage within the Adenophorea with 90% bootstrap recovery in a paraphyletic clade that included sequences of Prismatolaimus and Trichodorus. Seven sequences (three from the Netherlands, three from the UK and one from Greece) were left unassigned though the tree topology suggested some relationship (58% bootstrap recovery) with the genus Cephalobus. To assess whether primers used to amplify 18S rDNA might be used to fingerprint genetic diversity in nematode communities in soil, the environmental sequence data were used to design a second set of primers carrying a GC-clamp. These primers amplified a 469 bp fragment internal to the region flanked by the primer set used to derive the nematode trees and were used to amplify 18S rDNA for subsequent analysis using denaturing gradient gel electrophoresis (DGGE). DGGE analysis of six major European grassland types revealed considerable genetic diversity between sites. However, the relationships seen with the DGGE data were inconsistent with previous studies where the same soils had been characterized with respect to functional and morphological diversity. To confirm that this second set of primers was amplifying nematode sequences, selected bands on the DGGE gels were extracted, PCR amplified and sequenced. The final alignment was 337 bases. These analyses revealed the presence of sequence signatures from the genera Paratrichodorus, Plectus, Steinernema, Globodera, Cephalobus and Pratylenchoides.     

Effects of intercropping and Rhizobium inoculation on yield and rhizosphere bacterial community of faba bean (Vicia faba L.)

The effects of intercropping with maize and Rhizobium inoculation on the yield of faba bean and rhizosphere bacterial diversity were analyzed by terminal restriction fragment length polymorphism, amplified 16S rDNA restriction analysis (ARDRA), and 16S rDNA sequencing. The results showed that intercropping but not Rhizobium inoculation significantly increased the faba bean yield. Probably the relatively high level of native rhizobia in soil annulled the effect of rhizobia inoculation. ARDRA results showed that intercropping did not affect bacterial diversity whereas Rhizobium inoculation decreased bacterial diversity. The canonical correspondence analysis showed that the composition of bacterial community was changed apparently by intercropping, and there was a positive correlation (P = 0.724) between faba bean yields and intercropping and an apparent correlation (P = 0.648) between intercropping and total N. The available content of K and P had a lower effect on the bacterial community composition than did the total N content, Rhizobium inoculation, and microbial biomass C. Rhizobium inoculation negatively correlated with microbial biomass C (P = −0.827). These results revealed a complex interaction among the intercropped crops, inoculation with rhizobia, and indigenous bacteria and implied that the increase of faba bean production in intercropping might be related to the modification of rhizosphere bacterial community.     

Characterization of cultivable heterotrophic bacterial communities in Cr-polluted and unpolluted soils using Biolog and ARDRA approaches

The bacterial communities of two soils with different chromium levels were characterized by Biolog carbon substrate utilization patterns and amplified 16S ribosomal DNA restriction analysis (ARDRA). For each bacterial community sample, cell suspensions containing 10,000 or 100 colony-forming units (CFU) were inoculated in each well of Biolog-GN microplates. The number of carbon compounds utilized by the bacterial community consisting of 100 CFU from unpolluted soil was significantly lower than that detected for the bacterial community consisting of 10,000 CFU. The size of inoculum did not substantially influence the percentage of carbon sources utilized by the Cr-polluted soil bacterial community. ARDRA approach was applied to about 100 bacterial isolates for each soil sample. A similar number of clusters for Gram-negative bacteria were found in both soils, but there were differences in percentages of isolates belonging to each group and specific genomic groups were found in each soil. Pseudomonas was the dominant taxon in both soils. Comparing the ARDRA clusters obtained from Gram-positive isolates it was evident that the culturable bacterial communities of Cr-polluted and unpolluted soils were dominated by the genus Arthrobacter and the genus Bacillus, respectively.     

Microbial community structure and function in a soil contaminated by heavy metals: effects of plant growth and different amendments

We studied the effects of in situ remediation of a heavy metal (HM) contaminated soil on some soil chemical properties, microbial function and microbial structural diversity after 18 months. The experiment was carried out at semifield scale in containers filled with HM contaminated soil from the Aznalcóllar mine accident (Southern Spain, 1998). The remediation measures consisted of the application of different amendments and/or establishment of a plant cover (Agrostis stolonifera L.). Seven treatments were established: four organic treatments (municipal waste compost (MWC), biosolid compost (BC), leonardite (LEO) and litter (LIT)), one inorganic treatment (sugar beet lime (SL)) and two controls (control with plant cover (CTRP) and control without plant cover (CTR)). Several soil chemical (pH, soluble HM, total organic C (TOC), water-soluble C (WSC) and available-P) and biochemical properties (microbial biomass C (MBC), MBC/TOC ratio and enzyme activities) were determined. Microbial community structure was studied by means of ARDRA (amplified ribosomal DNA restriction analysis). The SL, MWC and BC treatments were the most efficient to raise soil pH and decrease soluble HM concentrations. Total organic C was increased in the organic treatments by 2 to 4-fold, whereas water-soluble C was statistically similar in the CTRP, SL and the organic treatments, probably due to the presence of a root system in all these treatments. Available-P was also increased in the BC, SL and MWC treatments due to the higher P content of the amendments applied in these treatments. Soil microbial function was generally enhanced in the amended and CTRP treatments. The MWC, BC and SL treatments were particularly efficient to increase microbial biomass C, the MBC/TOC ratio and the dehydrogenase and aryl-sulphatase enzyme activities. These results could be attributed to the amelioration of some of the soil chemical properties: increase in soil pH and water-soluble C and decrease of HM soluble concentrations. ARDRA analyses showed changes in structural diversity in both the bacterial and fungal community under the different treatments. Fingerprinting patterns of the 16S rDNA obtained with Hinf-I and of the 18S rDNA with Hpa-II revealed higher similarity percentages among samples from the same treatment compared with samples from the other treatments. In addition, a higher similarity was found between samples from all treatments under the Agrostis influence. The use of certain amendments and/or a plant cover is important for in situ remediation of HM contaminated soils, since these practices can affect soil chemical properties, as well as the microbial community function and structure.     

石油污染土壤鞘氨醇单胞菌遗传多样性16S rDNA-PCR-DGGE分析

鞘氨醇单胞菌是降解多环芳烃(PAHs)的重要功能微生物。然而,采用属特异引物直接从PAHs污染土壤中检测鞘氨醇单胞菌进而研究鞘氨醇单胞菌在污染土壤中的多样性、种群结构及其与PAHs污染的关系鲜有报道。本研究自行设计鞘氨醇单胞菌属特异性引物,通过16S rDNA-PCR-DGGE技术对沈抚灌区石油污染土壤鞘氨醇单胞菌遗传多样性进行了研究。结果表明,自行设计的鞘氨醇单胞菌属引物732f-982r能够特异、灵敏的从环境样品中检出鞘氨醇单胞菌。当沈抚灌区原位污染土壤的PAHs浓度小于5 mg kg-1时,鞘氨醇单胞菌多样性随着PAHs浓度的增加而增加;而初始PAHs浓度高的实验室模拟实验土壤,其鞘氨醇单胞菌多样性较低。鞘氨醇单胞菌种群结构受污染土壤总石油烃(TPH)含量的影响,TPH含量接近的土壤,鞘氨醇单胞菌的群落结构较为相似。     

Changes in chemical and biological soil properties as induced by anthropogenic disturbance: A case study of an agricultural soil under recurrent flooding by wastewaters

Monitoring the environmental impact of anthropogenic disturbance on soil ecosystem is of great importance for optimizing strategies for soil use, conservation and remediation. The aim of this study was to assess whether and to what extent a long-term, human-induced disturbance could have affected main chemical and biological properties in an agricultural soil. The study site was a hazel (Corylus avellana L.) orchard located in the area surrounding the volcanic apparatus of Somma-Vesuvius (Southern Italy). For the last two decades, the site has been repeatedly subjected to floodings by wastewaters containing not only alluvial sediments but also potentially hazardous compounds from illegally disposed wastes. Soil disturbance was assessed by a multitechnique approach, which combined chemical, biochemical and physiological (Biolog^®) methods together with community fingerprinting by denaturing gradient gel electrophoresis (DGGE) and amplified ribosomal DNA restriction analysis (ARDRA). A hazel site never subjected to flooding provided the control soil. Soil sampling was repeated three times over a 1-year period. The effect of flooding by wastewaters, sampling time and their interaction were statistically evaluated. Under wastewater flooding, soil pH and most organic matter-related pools, i.e. total organic C, total N, and active soil C-resources such as basal (SBR) and substrate-induced respiration (SIR) and microbial biomass C (MBC) were all increased; whereas sampling time mostly affected two active-N pools, namely K₂SO₄-extractable N (Extr-N) and potentially mineralizable N that varied unconcurrently in tested soils. Also the electrical conductivity varied across samplings. Parameters related to microbial maintenance energy (ATP and qCO₂) were higher in the flooded soil, even though they were not statistically affected by wastewater flooding or by sampling time. The Biolog^® method evidenced that under recurrent flooding, soil microbial populations became functionally more uniform when compared to the control soil. Molecular fingerprinting of PCR-amplified 16S rDNA targets revealed that, along with seasonal shifts, a marked change in the genetic structure of total bacterial community occurred in the flooded soil. Furthermore, compositional shifts in the actinomycete community were less marked and mostly influenced by seasonal effects. Yet, a decreased genetic diversity in the ammonia-oxidizing bacteria community was evidenced in the flooded soil by ARDRA. Thus both the genetic and the functional structure of native soil bacterial populations were changed under repeated flooding by wastewaters. Repeated sampling over a 1-year period allowed us to reveal soil disturbance effects beyond seasonal variations.     

Impacts of methamidophos on the biochemical,catabolic, and genetic characteristics of soil microbial communities

Methamidophos is an organophosphate pesticide with high toxicity and may significantly affect soil microbes. However, the magnitude of this effect is unclear. We examined the effect of low and high inputs of methamidophos on the structure of the soil microbial community, and the catabolic activity and the genetic diversity of the bacterial community using the polyphasic approaches of microbial biomass, phospholipid fatty acids (PLFAs), community-level catabolic profiles (CLCPs), and amplified ribosomal DNA restriction analysis (ARDRA) patterns. Our results indicated that high methamidophos inputs significantly reduced total microbial biomass carbon (C_mic) and fungal biomass, but increased Gram-negative bacteria with no significant effects on the Gram-positive bacteria. Interestingly, CLCPs patterns showed that high methamidophos inputs also significantly improved the catabolic activity of Gram-negative bacteria. The ARDRA pattern showed that the genetic diversity of the bacterial community decreased under chemical stress. Furthermore, changes in the microbial parameters examined were less significant under low inputs than high inputs of methamidophos, suggesting a dosage effect of methamidophos on the microbial community. Our results provide the first evidence that methamidophos differentially affected components of the soil microbial community through inhibiting fungal growth but enhancing the biomass and catabolic activity of Gram-negative bacteria.     

芽孢杆菌TS01的ARDRA评估和遗传分析

利用自主分离的芽孢杆菌菌株TS01和15种芽孢杆菌(地衣芽孢杆菌,枯草芽孢杆菌,短小芽孢杆菌,巨大芽孢杆菌,凝结芽孢杆菌,蜡状芽孢杆菌,迟缓芽孢杆菌,苏云金芽孢杆菌,嗜热脂肪芽孢杆菌,解淀粉芽孢杆菌,环状芽孢杆菌,球形芽孢杆菌,侧孢短芽孢杆菌,多粘类芽孢杆菌,泛酸枝芽孢杆菌)模式菌种进行ARDRA分析。采用16S rDNA通用引物16S-27和16S-1525进行PCR扩增,16S rDNA扩增片段经六种限制性酶（Alu I、Taq I、Mse I、Bst UI、Hha I和Tsp509 I）酶切电泳,获得了TS01菌株的特征性ARDRA指纹图谱。ARDRA图谱通过GelcomparⅡ软件进行聚类分析(UPGMA),结果表明菌株TS01和地衣芽孢杆菌处于同一分支,亲缘关系最近。ARDRA分析鉴定结果与实验室前期菌株TS01形态、生化鉴定和16S rDNA序列分析结果一致,TS01是一株地衣芽孢杆菌菌株,从而证明ARDRA技术在菌种水平上对芽孢杆菌TS01进行鉴别具有可靠性。     

Evaluation of genetic diversity in a Camelina sativa (L.) Crantz collection using microsatellite markers and biochemical traits

A genomic DNA library enriched with GA/TC repeats from Camelina sativa variety Calena has been analysed. After sequencing of about 200 randomly selected clones, approximately 60 % of them showed to contain simple or compound microsatellites with a high number of repeats. Among all microsatellite markers analysed 15 primer pairs amplified polymorphic fragments. Forty C. sativa accessions of different origin were genotyped with 15 microsatellite markers that generated 134 alleles with an average of 8.93 alleles per locus. The observed heterozygosity (Ho) among the accessions ranged from 0.0 to 0.15 with an average of 0.0370, whereas the average of expected heterozygosity (He) among accessions was 0.2769. The analysis of the average total heterozygosity (H_T = 0.651), the intrapopulation genetic diversity (H_S = 0.260), the interpopulation genetic diversity (D_ST = 0.391) and the coefficient of genetic differentiation among populations (G_ST = 0.574) demonstrated that 57.4 % of the genetic diversity is among the accessions, while 42.6 % resides within them. Phylogenetic tree of the 40 C. sativa accessions was constructed based on Nei’s genetic distance. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram shows, except for CAM108 and CAM170, a clear discrimination among C. sativa accessions grouping them in five subgroups. ANOVA analysis indicates significant differences in some biochemical and agronomic parameters among the C. sativa accessions grouped according to Nei’s genetic distance. The result of the Tukey HSD test demonstrated that the A4 subgroup showed a significant higher TWS and linoleic acid (LA) content, while the subgroup A1 showed a significant higher linolenic and lower LA content compared to the remaining groups.     

Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China

A total of 98 non-symbiotic endophytic bacterial strains isolated from soybean root nodules were classified into eight rDNA types in ARDRA analysis and 21 BOX types in BOX-PCR. The phylogenetic analysis of 16S rDNA identified these strains as Pantoea, Serratia, Acinetobacter, Bacillus, Agrobacterium, and Burkholderia. Limited genetic diversity was revealed among these bacteria since most of the strains (85.7%) were found in three very similar rDNA types corresponding to Pantoea agglomerans, and many strains shared the same BOX-PCR patterns. The inoculation of nodule endophytes had no significant effects on the growth and nodulation of soybean, but most of the strains produced indoleacetic acid (IAA), could solubilize mineral phosphate, and could fix nitrogen, implying that they are a valuable pool for discovering plant growth promoting bacteria. Our results demonstrated that the nodule endophytes were common in soybean and their diversity was affected by the plant's character and the soil conditions. The 99% similarities found in the nifH genes of Bradyrhizobium japonicum and of the endophytic Bacillus strains strongly indicated that horizontal transfer of symbiotic genes happened between the symbiotic bacteria and the endophytes.     

Diversity, abundance and vertical distribution of methane-oxidizing bacteria (methanotrophs) in the sediments of the Xianghai wetland, Songnen Plain, northeast China

Purpose

Methanotrophs in wetlands are of great importance because up to 90 % of the methane (CH₄) produced in such wetlands could be oxidized by methanotrophs before reaching the atmosphere. The Xianghai wetland of Songnen Plain represents an important ecosystem in northeast China. However, methanotrophic characteristics in this ecosystem have not been studied in detail. The aim of this study is to give an overview of methanotrophic diversity and vertical distribution in the sediments of this important wetland.

Materials and methods

Sediment cores were collected from three freshwater marshes, each dominated by a particular vegetation type: Carex alata, Phragmites australis and Typha orientalis. The diversity of methanotrophs was studied by phylogenetic analysis of both the 16S rRNA gene and the particulate methane monooxygenase (pmoA) gene. Methanotroph abundance was determined by quantitative PCR (qPCR) targeting the pmoA gene; group-specific pmoA gene quantification was also used to estimate the abundance of each methanotrophic group.

Results and discussion

16S rRNA and pmoA gene homological analysis revealed the presence of type Ia, Ib and II methanotrophs. Novel pmoA sequences distantly affiliated to cultured Methylococcus sp. were detected, implying the existence of novel methanotrophs in the wetland. Most obtained representatives of Methylobacter genus (both 16S rRNA and pmoA genes) were closely clustered in relation to sequences acquired from the Zoige wetland, Tibet and Siberia permafrost soils, therefore suggesting methanotrophs belonging to Methylobacter genus shared characteristics with methanotrophs in cold areas. The dominance of type I methanotrophs (especially the Methylobacter genus) was detected by both clone library analysis and group-specific qPCR assay. The relatively high methanotroph diversity and pmoA copy numbers measured in the T. orientalis marsh sediments indicated that vegetation type played an important role during CH₄ oxidation in the wetland.

Conclusions

We present the first data set on methanotroph diversity and vertical distribution in the sediments of the Xianghai wetland. DNA sequences information of Methylococcus-like methanotrophs in the wetland will facilitate the isolating of novel methanotrophs from the wetland. In a worldwide context, our study has enriched the database of genotypic diversity of methanotrophs, which will help in the understanding of the geographical distribution of methanotrophic communities.     

Genetic diversity and phylogeny of pink-pigmented facultative methylotrophic bacteria isolated from the phyllosphere of tropical crop plants

Genetic diversity of pink-pigmented facultative methylotrophic bacteria belonging to the genus, Methylobacterium, was assessed using 16S ribosomal RNA (rRNA) gene sequencing, amplified ribosomal DNA restriction analysis (ARDRA), and differential carbon-substrate utilization profile in the phyllosphere of cotton, maize, sunflower, soybean, and mentha plants. Methylobacterium populi, Methylobacterium thiocyanatum, Methylobacterium suomiense, M. aminovorans, and Methylobacterium fujisawaense were identified to colonize the phyllosphere of these crop plants. Among these, M. populi found to be the dominating species followed by M. aminovorans. The diversity indices like Shannon index of diversity, Pielou index of evenness, and Margalef index of richness calculated based on combined data of ARDRA, 16S rRNA gene sequence, and differential carbon-substrate utilization revealed that sunflower leaves showed richest methylobacterial diversity, followed by soybean, while cotton and mentha leaves recorded the lowest diversity. The outcome of this study indicates that diversity of Methylobacterium in phyllosphere depends upon the host plant species colonized.     

Season and management related changes in the diversity of nitrifying and denitrifying bacteria over winter and spring

This study assessed the effects that season and tillage practices have on the diversity of nitrous oxide producing bacteria (nitrifiers and denitrifiers) and to relate this to measured N₂O fluxes at our field site. Large-scale field plots (1.5 ha) were established in Elora, Ontario in 2000, and managed using conventional tillage (CT) or no-tillage (NT). Each field plot was instrumented with micrometeorological equipment to determine N₂O fluxes on a field scale. Soil samples were taken at four time points between the fall of 2005 and the spring of 2006. The diversity of the nitrifier and denitrifier communities was assessed by PCR–denaturing gradient gel electrophoresis (DGGE) using primer pairs targeting the amoA, nirS and nirK gene. Seasonal variation (a combination of soil temperature, available soil moisture, nutrient levels and other potential factors) had the largest influence on the diversity of nitrifier and denitrifier populations; while tillage practice also influenced the diversity of the microbial community at certain time periods. Tillage significantly affected all communities in March and affected denitrifiers on all other dates except for the nirS community in February. Further statistical analysis revealed that diversity of the nitrifying and denitrifying populations was the lowest in February, in frozen soils, and rapidly increased in March, corresponding with spring thaw N₂O emissions. Long-term soil nutrient, temperature and N₂O data taken at this site added additional information on the dynamics of the nitrogen cycle.     

Effect of Azospirillum brasilense inoculation on rhizobacterial communities analyzed by denaturing gradient gel electrophoresis and automated ribosomal intergenic spacer analysis

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.     

Diversity of heterotrophic aerobic cultivable microbial communities of soils treated with fumigants and dynamics of metabolic, microbial, and mineralization quotients

A combination of molecular and classical techniques was used to study the composition, structure, diversity, and dynamics of an aerobic heterotrophic cultivable bacterial community isolated from five different soil samples treated with the fumigant agent 1,3-dichloropropene (1,3-D) and further subjected to nitrogen–phosphorous–potassium (NPK) fertigation (F), amendment (C ₂ and C ₄), and NPK fertigation plus amendment (F + C) in two different periods (May and July). The restriction and sequence analysis of 16S rDNA from 189 isolates revealed a very high percentage (94%) of Gram-positive bacterial isolates, most of which (83%) belonging to the genus Bacillus. The degree of intraspecific genetic diversity was high, as shown by random amplified polymorphic DNA analysis. These data seem to be related with the increase in microbial biomass C (C _mic) content and the decrease in the total organic C (C _org) and metabolic quotient (qCO₂) values, especially in amended soils (C ₂, C ₄) where soil microflora mineralized the organic matter of the added fertilizers. In a short term, it is suggested that the presence of very high percentage of Gram-positive bacteria might be related to the ability of these bacteria to form spores so as to be resistant to fumigants rather than being the result of a selective pressure in the predominance of microbial species with a set of genes involved in biodegradation of 1,3-D. Stefano Mocali and Donatella Paffetti contributed equally to this work.     

Genetic diversity of wild banana (<Emphasis Type="Italic">Musa balbisiana</Emphasis> Colla) in China as revealed by AFLP markers

Wild banana Musa balbisiana Colla is one of the progenitors of cultivated bananas and plantains. It is native to Southeast Asia and the western Pacific. South China represents the northern limit of its distribution range. The genetic diversity of Musa balbisiana was assessed by the amplified fragment length polymorphism (AFLP) fingerprinting in 15 populations of China. Four primer pairs produced 199 discernible loci. High levels of genetic diversity were detected, with P = 78.5%, H _E = 0.241, and H _pop = 0.3684 at population level, and P = 100%, H _T = 0.3362 and H _sp = 0.5048 at species level. Significant genetic differentiation among populations was detected based on Hickory’s analysis (27.6%), Shannon’s diversity index (27.0%) and AMOVA (27.1%). The AFLP results are discussed and compared with data obtained by microsatellites method. The estimates of genetic diversity and differentiation between each pair of populations computed with microsatellites and AFLP markers were not significantly correlated. Conservation strategies for Musa balbisiana in China are proposed.     

Identification of an Organism Associated with Mushroom Worker's Lung

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.