Distribution and biodiversity of soybean rhizobia in the soils of Shennongjia forest reserve,China

Soil samples were collected at an altitude of 500, 1,060, 1,500, 1,950, 2,400 and 3,100 m, respectively, from Shennongjia, a forest reserve in Hubei province (central China). Their corresponding pHs were 5.50, 4.91, 5.64, 5.28, 5.49 and 4.60. By using a plant trap method, a total of 25 soybean rhizobia were isolated from the soil above an altitude of 1,500 m and all identified to be Sinorhizobium fredii. Their genetic biodiversity was characterized by 16S–23S rDNA internally transcribed spacer (ITS) region polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and random amplification DNA (RAPD) analysis. All the tested strains produced a 2.1 kb 16S–23S rDNA ITS fragment. After digestion with three restriction endonucleases (HaeIII, MspI and CfoI), respectively, great variations in 16S–23S rDNA ITS PCR-RFLP patterns were observed. The tested strains could be differentiated into 11 ITS genotypes. The genotypes of rhizobia were not related to geographical location. Twelve primers were applied to RAPD analysis and a dendrogram was obtained, showing that all the strains (including reference strain S. fredii USDA205) were divided into two diverging groups. Moreover, each group could be further divided into two subgroups. Both RAPD and 16S–23S rDNA ITS PCR-RFLP analysis indicated that a high degree of genetic diversity existed among S. fredii strains isolated from Shennongjia virgin soils. Since Shennongjia is an unexploited forest region in central China and the gene centre of soybean is located in China, the symbiotic genes harboured by these strains may be of great importance and the rich diversity of these strains might contribute to the adaptation of soybean to an alpine environment.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

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.     

植物促生菌接种对中国芥蓝根际固有微生物群落结构的影响

Plant growth-promoting rhizobacteria (PGPR) have been widely recognized as an important agent,especially as a biofertilizer,in agricultural systems.The objectives of this study were to select efective PGPR for Chinese kale (Brassica oleracea var.alboglabra) cultivation and to investigate the efect of their inoculation on indigenous microbial community structure.The Bacillus sp.SUT1 and Pseudomonas sp.SUT19 were selected for determining the efficiency in promoting Chinese kale growth in both pot and field experiments.In the field experiment,PGPR amended with compost gave the highest yields among all treatments.The Chinese kale growth promotion may be directly afected by PGPR inoculation.The changes of microbial community structure in the rhizosphere of Chinese kale following PGPR inoculation were examined by denaturing gradient gel electrophoresis (DGGE) and principal coordinate analysis.The DGGE fingerprints of 16S rDNA amplified from total community DNA in the rhizosphere confirmed that our isolates were established in the rhizosphere throughout this study.The microbial community structures were slightly diferent among all the treatments,and the major changes depended on stages of plant growth.DNA sequencing of excised DGGE bands showed that the dominant species in microbial community structure in the rhizosphere were not mainly interfered by PGPR,but strongly influenced by plant development.The microbial diversity as revealed by diversity indices was not diferent between the PGPR-inoculated and uninoculated treatments.In addition,the rhizosphere soil had more influence on eubacterial diversity,whereas it did not afect archaebacterial and fungal diversities.     

Phylogenetic positions of Mn<Superscript>2+</Superscript>-oxidizing bacteria and fungi isolated from Mn nodules in rice field subsoils

We isolated manganous ion (Mn²⁺) oxidizing bacteria and fungi from Mn nodules collected from two Japanese rice fields. The phylogenetic position of the Mn-oxidizing bacteria and fungi was determined based on their 16S rDNA and 18S rDNA sequences, respectively. Among 39 bacterial and 25 fungal isolates, Burkholderia and Acremonium strains were the most common and dominant Mn²⁺-oxidizing bacteria and fungi, respectively. Majority of the Mn-oxidizing bacteria and fungi isolated from the Mn nodules belonged to the genera that had been isolated earlier from various environments. Manganese oxide depositions on Mn²⁺-containing agar media by these microorganisms proceeded after their colony developments, indicating that the energy produced from Mn²⁺ oxidation is poorly used for microbial growth.     

Diversity of Paenibacillus polymyxa populations in the rhizosphereof wheat (Triticum durum) in Algerian soils

The diversity of Paenibacillus polymyxa populations associated with the rhizosphere of durum wheat was investigated in Algerian soils sampled in regions where wheat had been cultivated for 5 and 26 years (Hamiz, H5 and Z26), 70 years (Algiers, D70), and more than 2 000 years (Tiaret, T2000 and K2000). A total of 111 strains were isolated by immunotrapping and identified as P. polymyxa using an API50CHB kit and restriction analysis of the amplified 16S rDNA gene. The phenotypic characteristics of the P. polymyxa populations were compared and the strains found not to cluster according to their origin. The longer the history of wheat cultivation, the lower the phenotypic diversity and the higher the frequency of nitrogen-fixing strains. Population genetic diversity, evaluated by ERIC-PCR, showed the same trends as phenotypic diversity. The distribution of ERIC genotypes among the different populations studied were compared using Pearson’s Chi-squared test. The strains isolated in D70, Z26, and H5 derived from soil populations sharing the same genetic structure, but those isolated in T2000 and K2000 each stemmed from a population with a specific genetic structure. These data suggest that the genetic structure of P. polymyxa populations has been affected by long-term wheat cultivation.     

Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobiumleguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

Genetic diversity and phylogeny of indigenous rhizobia from cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.]

16S rRNA RFLP, 16S rRNA sequencing, 16S-23S rRNA Intergenetic Spacer (IGS) RFLP and G-C rich random amplified polymorphic DNA (RAPD) assays were conducted to genetically characterise indigenous cowpea [Vigna unguiculata (L.) Walp.] rhizobia from different geographic regions of China. Isolated cowpea rhizobia comprised six 16S rRNA genospecies. Genotype I was composed of 14 isolated strains and the reference strains of B. japonicum and B. liaoningense. This group was divided into two sub-groups respectively related to B. japonicum and B. liaoningense by 16S rRNA sequencing, IGS restriction fragment length polymorphism and RAPD assays. Genotype II composed of 27 isolates from a variety of geographic regions. Four different assays confirmed this group was genetically distinct from B. japonicum and B. liaoningense and probably represent an uncharacterised species. Strains isolated from Hongan, Central China and B. elkanii were grouped to genotype III. Strain DdE4 was solely clustered into genotype IV and related to Rhizobium leguminosarum. Genotypes V and VI consisted of six fast-growing isolates and clustered with reference strain of Sinorhizobium fredii. Comparing with the miscellaneous slow-growing isolates, fast-growing isolates mainly isolated from cowpea cultivar Egang I exhibited strict microbe–host specificity except SjzZ4. Nucleotide sequences reported were deposited in the GenBank with the accession numbers DQ786795–DQ786804.     

Diversity of Paenibacillus durus strains isolated from soil and different plant rhizospheres evaluated by ARDRA and gyrB-RFLP analysis

Strains belonging to Paenibacillus durus isolated from the rhizosphere of various grasses and from bulk soil were previously divided into five phenotypic groups (A1–A5) based on the fermentation pattern of six carbohydrates (A1: sorbitol (+), A2: dulcitol and tagatose (+), A3: starch and glycogen (+), A4: starch, glycogen and d-arabitol (+) and A5: negative for these carbohydrates). This study aimed to assess whether plant types select for specific P. durus phenotypic groups. For that purpose, polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA (ARDRA) and DNA gyrase subunit B (gyrB-RFLP) were used to produce genetic fingerprints. ARDRA and gyrB-RFLP data were clustered together to generate a dendrogram and two main clusters were observed. Cluster I showed a predominance of strains isolated from wheat, maize and sugarcane rhizospheres. Strains isolated from maize were distributed among the five patterns of carbohydrate metabolism, while strains isolated from sugarcane showed to be predominantly able to metabolize starch and glycogen. Neither sorbitol- nor arabitol-metabolizing strains were found in cluster II, which consisted of strains isolated from soil and from all plant species used. Our results suggest that the plants influenced the diversity of P. durus in their rhizospheres.     

Grouping of Bradyrhizobium USDA strains by sequence analysis of 168 rDNA and 168-238 rDNA internal transcribed spacer region

In order to select appropriate Bradyrhizobium USDA reference strains for primary grouping of indigenous soybean bradyrhizobia, we systematically constructed phylogenetic trees of 20 USDA strains based on DNA sequence analysis and PCR-restriction fragment length polymorphism (RFLP) targeted to 16S rDNA and the internal transcribed spacer (ITS) region between 16S and 23S rDNAs. The phylogenetic trees of 16S rDNA showed 3 major groups, cluster USDA 110 (USDA 62, 110, 122, 125, and 129), cluster USDA 6 (USDA 4, 6^T, 38, 115, 123, 127, 135, and 3622^T) and cluster B. elkanii (USDA 31, 46, 61, 76^T, 94, and 130), as well as the phylogenetically independent strain USDA 124. The topology of the ITS trees was almost similar to that of 16S rDNA, although the positions of two extra-slow-growing strains, USDA 135 and USDA 3622^T were variable among the ITS sequences, PCR-RFLP of the ITS region and 16S rDNA. Only two strains, USDA 110 and USDA 122, harbored hup genes and they fell into the USDA 110 cluster. These results suggest that PCR-RFLP analysis of 16S rDNA and the 16S-23S rDNA ITS region may be useful for the grouping of bradyrhizobia and for the first screening of hup-positive strains. Based on the above results, we propose a minimum set of USDA strains reflecting Bradyrhizobium diversity that includes B. japonicum USDA 6^T, B. japonicum USDA 110, B. japonicum USDA 124, and B. elkanii USDA 76^T. In addition, an extra-slow-growing strain with the serotype USDA 135 might be necessary for genomic diversity analysis of bradyrhizobia, because their phylogenetic positions were variable.     

Use of random amplified DNA markers to analyse genetic variability and relationships of Coffea species

Summary The use of random amplified DNA fragments as genetic markers in Coffea was investigated. Arbitrary oligonucleotides were used as primers to amplify genomic DNA of different coffee accessions representing major Coffea species by polymerase chain reaction. Intraspecific variation was easily detected in C. canephora and C. liberica whereas the primers assayed failed to reveal polymorphism between C. arabica accessions. Extensive interspecific variation was observed. Genetic relationships between Coffea species are deduced from the degrees of similarity in amplified product profiles. Random amplified DNA markers appeared to be of high value for characterization, analysis and utilization of coffee genetic resources.     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.     

RAPD and AFLP assessment of genetic variation in a landrace of pepper (Capsicum annuum L.), grown in North-West Italy

In several regions of Italy as well as other parts of southern Europe, the heterogeneity of the land, the climate and the soil favour the survival in cultivation of a large number of landraces specifically adapted to local conditions. Knowledge on the level and distribution of their genetic variation can help to develop appropriate strategies, in order to suistainably manage in situ these germplasm resources at risk of genetic erosion. C. annuum is an herbaceous diploid species and is considered to be self-pollinating, although different rates of out-crossing have been recorded. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess genetic diversity within and between five populations of a landrace of Capsicum annuum L., grown in a limited area in north-west Italy and locally known as Cuneo pepper. Partitioning the genetic variation with Shannon's diversity index revealed that 41.6% occurred between and 58.4% within populations. Analogous results were obtained when the analysis was based only on RAPD or AFLP markers. However, AFLP was more reliable, since a lower range of variation was observed among primer combinations in detecting the two components of genetic variation. Notwithstanding the rather high level of within genetic variation detected, the five populations were clearly differentiated and differed in the frequency of alleles exclusive and/or present at very low frequencies. Our results show the need for accurate estimation of allele frequencies, in order to identify populations to which priority should be given for dynamic conservation of landraces.     

Isolation and identification of locally isolated bacterial strains effective against whitefly Bemisia tabaci

Potent bacterial strains effective against the whitefly, Bemisia tabaci, nymphs (second instar), were isolated from tomato cultivated fields at Fayoum governorate, Giza, Egypt. Of 72 isolates, 12 with the most morphologically distinct-looking bacterial colonies were selected and named A1, A2, A3, A6, A7, A9, A12, A13, A107, B37, B45 and B100. All isolates were preliminarily identified as members of the genus Bacillus based on morphological, physiological and biochemical characteristics. When tested for their pathogenicity against Bemisia tabaci, the 12 isolates revealed varying efficiencies with isolates A1 and A9 being superior, exhibiting maximum mortality of 92.2 and 90.8% on day 10, respectively. Isolate A7 recorded the lowest percentage at 18.3%. Further genetic characterization of the 12 isolates was performed using inter simple sequence repeat (ISSR), randomly amplified polymorphic DNA (RAPD) and 16S rDNA gene sequencing analysis. RAPD and ISSR results confirmed each other. The combined ISSR and RAPD phylogenetic tree showed two major clusters. With 16S rRNA gene analysis, isolate A1 and A12 sequences recorded 100% identity with Bacillus thuringiensis, while isolates A7 and B100 showed 95.7% and 95.6% identity with Bacillus cereus and Bacillus sphaericus, respectively.     

新疆灰霉病菌多态性及其致病力分化分析

灰霉病菌(Botrytis cinerea)是引起植物病害的重要病原。本研究将来自新疆3个主要生态区域的12个灰霉病菌菌株, 经人工接种于7个寄主的离体叶片上测定病原菌的致病性, 并采用RAPD技术分析了12个菌株的遗传多态性。研究结果表明, 12个灰霉菌株的致病力存在明显差异, 可分为强、中、弱3种致病型, 其中来自和田的黄瓜灰霉菌和伊犁特克斯的番茄灰霉菌致病力最强; 阿克苏番茄灰霉菌的致病力最弱。根据在不同寄主上的致病反应, 12个菌株被划分为4个菌群。RAPD分析结果表明, 在0.65阈值下, 12个灰霉菌株可分为4个菌群, 说明供试灰霉菌株间有高度的遗传分化。灰霉病菌致病力强弱与菌株寄主、地理位置均无直接相关性, 但灰霉菌株的遗传距离与菌株来源存在明显相关性。     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Molecular phylogeny of Chinese vegetable mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) based on the internal transcribed spacers (ITS) of nuclear ribosomal DNA

Sequence variation of nuclear internal transcribed spacer regions of ribosomal DNA (ITS1, 5.8S rRNA and ITS2) from Chinese vegetable mustards (AB-genome) and its putative parents Brassica rapa (the A-genome) and Brassica nigra (the B-genome) were used to investigate the molecular phylogeny and the probable evolutional pattern of this amphidiploid species that uniquely formed in China. Totally, 16 accessions of Chinese vegetable mustard those covering nearly all the diverse variations were included in this study, and together with three accessions of B. rapa and one accession of B. nigra. The results disclosed two strongly supported clades, one containing four accessions of vegetable mustard which have closer relationship with B-genome species “B.nigra” lineage and the other containing 12 accessions of B. juncea and three A-genome accessions. This classification was in disagreement with the evidence from chloroplast DNA, mitochondrial DNA, nuclear DNA restriction fragment length polymorphism (RFLP), which suggested that B. juncea was closely related to the A-genome type. For the incongruence, we speculated that the B. juncea crops derived from Chinese have evolved through different recombined events of the diploid morphutypes and evolved unidirectional concerted evolution. The traditional phenotypic classification of B. juncea was not wholly supported by ITS results, and hence the phylogenetic relationships among these subspecies need to be reconsidered on molecular level.     

Towards bioremediation of toxic unresolved complex mixtures of hydrocarbons: identification of bacteria capable of rapid degradation of alkyltetralins

Background, aim and scope  Unresolved complex mixtures (UCMs) of aromatic hydrocarbons are widespread, but often overlooked, environmental contaminants. Since UCMs are generally rather resistant to bacterial degradation, bioremediation of UCM-contaminated sites by bacteria is a challenging goal. Branched chain alkyltetralins are amongst the individual classes of components of aromatic UCMs which have been identified in hydrocarbon-contaminated sediments and a number of synthetic alkyltetralins have proved toxic in laboratory studies. Thus, alkyltetralins should perhaps be amongst the targets for UCM bioremediation strategies. The slow degradation of several alkyltetralins by a microbial consortium has been reported previously; however, the bacteria involved remain unidentified and no single strain capable of alkyltetralin biodegradation has been isolated. The present project therefore aimed to enrich and identify bacterial consortia and single strains of bacteria from a naturally hydrocarbon-contaminated site (Whitley Bay, UK), which were capable of the degradation of two synthetic alkyltetralins (6-cyclohexyltetralin (CHT) and 1-(3’-methylbutyl)-7-cyclohexyltetralin (MBCHT)). Materials and methods  Bacteria were enriched from sediment collected from Whitley Bay, UK by culturing with CHT and MBCHT for a period of 4 months. Biodegradation experiments were then established and degradation of model compounds monitored by gas chromatography–mass spectrometry. Internal standards allowed the generation of quantitative data. 16S rRNA gene clone libraries were constructed from individual enrichments to allow assessment of microbial community structure. Selective media containing MBCHT were used to isolate single bacterial strains. These strains were then tested in liquid culture for their ability to degrade MBCHT. Results  The consortia obtained through enrichment culture were able to degrade 87% of CHT and 76% of MBCHT after only 46 days compared with abiotic controls. The 16S ribosomal RNA gene clone libraries of these bacteria were dominated by sequences of Rhodococcus spp. Using selective media, a strain of Rhodococcus was then isolated that was also able to biodegrade 63% of MBCHT in only 21 days. Discussion  The present report describes the isolation of a single bacterial strain able to degrade the resistant MBCHT. Although significant losses of MBCHT were observed, putative metabolites were not detectable. Rhodococcus sp. have been reported previously to be able to biodegrade a range of hydrocarbon compounds. Recommendations and perspectives  Due to their environmental persistence and toxicity, aromatic UCMs require bioremediation. The culturing and identification of such bacteria capable of rapid degradation of alkyltetralins may be an important step toward the development of bioremediation strategies for sites contaminated with toxic UCMs.     

The 5S rRNA gene diversity in the genus <Emphasis Type="Italic">Secale</Emphasis> and determination of its closest haplomes

We analyzed 127 rDNA sequences (5S DNA units) obtained from 23 seed accession samples from more or less 10 taxa in wild and cultivated rye, genus Secale L. The sequences fell into two known groups, here assigned to two unit classes, viz. long R1 and short R1 (designations to reflect on R haplome of rye). The different taxa could not be fully differentiated based on the 5S DNA units. We searched for 5S DNA sequences from known unit classes most closely similar to the long R1 and the short R1. One set with the long R1 unit class contained sequences of the long P1 unit class from Agropyron (P haplome) and from Kengyilia (StYP haplome), long J1 from Thinopyrum (J haplome), whereas the set with the short R1 included the long S1 from Pseudoroegneria (St haplome) and Kengyilia (StYP haplome), the short J1 from Thinopyrum (J haplome) and the short V1 from Dasypyrum (V haplome). Each of the two sets was analyzed separately by maximum likelihood (ML) phylogenetic analysis from which we were able to infer that the 5S DNA units of Secale differentiated in a non-clock fashion and followed the HKY substitution model in the gene tree with the long R1 unit class and the HKY + G in the gene tree with the short R1 unit class. A complementary Bayesian analysis yielded identical tree topologies to the ML ones for each of the two sequence sets. In the tree with the long R1 units the long P1 and long J1 unit classes were closest to the long R1 unit class, whereas in the tree with the short R1 units the long S1 and short J1 unit classes were closest to the short R1 unit class, indicating possibly a close relationship between the St, J and R haplomes.