First report of Mesocriconema sphaerocephalum (Taylor, 1936) Loof, 1989 associated with wild grass in Botswana.

During a survey on the biodiversity of plant-parasitic nematodes of natural areas in Botswana, Bitylenchus ventrosignatus was discovered around the rhizosphere of wild grass. The nematodes were extracted using the tray method and then fixed according to the available protocols. The morphological characters fit well with the description of B. ventrosignatus. In addition, molecular analysis using 18 S and 28 S rDNA indicated 98% (KJ461617) and 95% (KJ461567) similarity with the Spanish population of B. ventrosignatus. The phylogenetic analysis of 18 S and 28 S rDNA placed the examined population with other populations of B. ventrosignatus in a group with a posterior probability support value of 100. According to published literature, this is the first report of B. ventrosignatus from Botswana.

During a survey on the biodiversity of plant-parasitic nematodes in Limpopo Province, South Africa, Bitylenchus ventrosignatus was discovered around the rhizosphere of a tomato in Dalmada. The nematodes were extracted using the tray method and then fixed according to available protocols. The morphological characters fit well with the description of B. ventrosignatus. In addition, molecular analysis using 18S and 28S rDNA indicated 99% similarity (MW255611; MW255613) with the Botswana population of B. ventrosignatus. The phylogenetic analysis of 18S and 28S rDNA placed the examined population with other populations of B. ventrosignatus in a group with a posterior probability support value of 1.00. According to published literature, this is the first morphological and molecular characters of 18S and 28S rDNA of B. ventrosignatus from South Africa.

Phylogeny of Tunicata inferred from molecular and morphological characters

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

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens

Kiwi is becoming one of the most important fruit in subtropical regions of South Africa with altitudes that confer sufficient chilling requirements. During a survey on biodiversity of plant-parasitic nematodes of kiwi in Magoebaskloof in Limpopo Province, several plant-parasitic nematodes were discovered, with Meloidogyne species occurring at the highest frequency. Nematodes were sampled from roots and the rhizosphere of one stunted Kiwi tree, extracted using the tray method and then fixed. The morphological characters fit well with those of M. hapla. The molecular approach using ITS and 28S rDNA, along with the related phylogenetic analysis, placed the examined population in a group with other populations of M. hapla. Kiwi is being reported as a new host for M. hapla in South Africa.

BackgroundLake Magadi and little Magadi are hypersaline, alkaline lakes situated in the southern part of Kenyan Rift Valley. Solutes are supplied mainly by a series of alkaline hot springs with temperatures as high as 86 °C. Previous culture-dependent and culture-independent studies have revealed diverse groups of microorganisms thriving under these conditions. Previous culture independent studies were based on the analysis of 16S rDNA but were done on less saline lakes. For the first time, this study combined illumina sequencing and analysis of amplicons of both total community rDNA and 16S rRNA cDNA to determine the diversity and community structure of bacteria and archaea within 3 hot springs of L. Magadi and little Magadi.MethodsWater, wet sediments and microbial mats were collected from springs in the main lake at a temperature of 45.1 °C and from Little Magadi “Nasikie eng’ida” (temperature of 81 °C and 83.6 °C). Total community DNA and RNA were extracted from samples using phenol-chloroform and Trizol RNA extraction protocols respectively. The 16S rRNA gene variable region (V4 – V7) of the extracted DNA and RNA were amplified and library construction performed following Illumina sequencing protocol. Sequences were analyzed done using QIIME while calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS) redundancy analysis (RDA) and diversity indices were carried out using the R programming language and the Vegan package.ResultsThree thousand four hundred twenty-six and one thousand nine hundred thirteen OTUs were recovered from 16S rDNA and 16S rRNA cDNA respectively. Uncultured diversity accounted for 89.35 % 16S rDNA and 87.61 % 16S rRNA cDNA reads. The most abundant phyla in both the 16S rDNA and 16S rRNA cDNA datasets included: Proteobacteria (8.33–50 %), Firmicutes 3.52–28.92 %, Bacteroidetes (3.45–26.44 %), Actinobacteria (0.98–28.57 %) and Euryarchaeota (3.55–34.48 %) in all samples. NMDS analyses of taxonomic composition clustered the taxa into three groups according to sample types (i.e. wet sediments, mats and water samples) with evident overlap of clusters between wet sediments and microbial mats from the three sample types in both DNA and cDNA datasets. The hot spring (45.1 °C) contained less diverse populations compared to those in Little Magadi (81–83 °C).ConclusionThere were significant differences in microbial community structure at 95 % level of confidence for both total diversity (P value, 0.009) based on 16S rDNA analysis and active microbial diversity (P value, 0.01) based on 16S rRNA cDNA analysis, within the three hot springs. Differences in microbial composition and structure were observed as a function of sample type and temperature, with wet sediments harboring the highest diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0748-x) contains supplementary material, which is available to authorized users.

Pear psyllids are among the most damaging pests in pear orchards, but little knowledge exists of psyllid species in cultivated pear orchards in China. In this study, DNA sequence analyses of the 16S rDNA and cytochrome oxidase I (COI) DNA regions were performed to identify pear psyllids from 28 regions of 20 provinces in China and to classify their genetic relationships to understand the origin of the species. The results showed that Cacopsylla chinensis Yang et Li (Hemiptera: Psyllidae) was found in most pear orchards in China, but Cacopsylla qianli (Hemiptera: Psyllidae) was found in only the cities of Guiyang (Guizhou province) and Xiangyang (Hubei province). The results for the 16S rDNA and COI regions were similar. Based on the nucleotide sequences and phylogenetic analyses of 16S rDNA and COI, C. chinensis could be divided into three groups: lineages I, II, and III. Based on 16S rDNA and COI, lineage II showed approximately 4% and 3% difference from lineage I, and lineage III showed approximately 12% and 9% difference from lineage I, respectively. C. chinensis lineage I was found in most provinces of China, while C. chinensis lineage II samples were mainly found in the Bohai rim region of China, and lineage III samples were found in Northeast China. The results of this study will provide information to pear producers regarding effective control measures to prevent further damage from pear psyllids.

RFLP analyses of 16S rDNA nested PCR products from 34 phytoplasma strains with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16Sr groups) and 32 sub-groups. The similarity coefficients of RFLP patterns between distinct groups were 90% or below. By including additional groups and sub-groups from which RFLP analyses were not performed but for which 16S rDNA sequence data were available to predict restriction sites, a total of 14 groups and 41 sub-groups were proposed. By combined RFLP analyses of 16S rRNA and ribosomal protein gene sequences, thus far, a total of 46 subgroups have been recognized. The phytoplasma 16Sr groups were consistent with the phylogenetic groups (subclades) defined by phylogenetic analysis of near-full-length 16S rRNA gene sequences, indicating that the RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple, reliable and rapid means for differentiation and classification of unknown phytoplasmas.

Most veterinary and milk hygiene laboratories identify streptococci and enterococci based on serological and biochemical tests. The analysis of 16S rDNA was suggested to be used for more exact identification; however, its use has not been considered so far in monitoring studies. The objective of the present study was to compare a conventional phenotypic method with restriction fragment length polymorphism analysis of 16S rDNA (16S rDNA RFLP) for identification of streptococci isolated from composite milk samples collected in connection with intramammary infection (IMI) in six Argentinean dairy farms. Composite milk samples (n = 1223) from cows belonging to six herds were collected for bacteriological analysis. Twelve reference strains and fifty streptococci or streptococcuslike isolates were identified to species level by the API 20 Strep system, conventional biochemical tests and 16S rDNA RFLP in a blind assay. The remaining streptococci or streptococcus-like isolates (n = 40) were identified to the species level both by 16S rDNA RFLP and conventional biochemical tests. As indicated by Kappa values, agreement between the 16S rDNA RFLP and the conventional scheme for identification of Streptococcus agalactiae, S. dysgalactiae, S. uberis, S. equinus and Enterococcus faecalis was 0.91, 0.73, 0.92, 0.81 and 0.85, respectively. Together with the less frequently isolated streptococcal species, the conventional scheme correctly identified 77 out of 90 isolates (85.5%). Thus, the use of 16S rDNA RFLP is considered valuable for monitoring studies due to its affordable cost for standard laboratories.

Postglacial Ace Lake (Vestfold Hills, Antarctica) was initially a freshwater lake, then an open marine system, and finally the present-day saline, stratified basin with anoxic, sulfidic, and methane-saturated bottom waters. Stratigraphic analysis of carotenoids and ancient 16S rDNA in sediment cores revealed that almost immediately after marine waters entered the palaeo freshwater lake as a result of post-glacial sea-level rise, Ace Lake became meromictic with the formation of sul.dic bottom waters and a chemocline colonized by obligate anoxygenic photolithotrophic green sulfur bacteria (Chlorobiaceae). Ancient 16S rDNA stratigraphy revealed that the fossil source of chlorobactene throughout the Holocene as well as in the present-day chemocline of Ace Lake was a species with 99.6% sequence similarity to the 16S rDNA sequence of Chlorobium phaeovibrioides DSMZ 269T. Comparison of the ratio between rDNA and chlorobactene of the latter species in the water column and in Holocene sediment layers revealed that the degradation of DNA was mostly influenced by the preservation conditions of the ancient water column. Within the sulfidic Holocene sediments, the remaining ancient DNA of green sulfur bacteria was more stable than intact carotenoids. We showed the development of anoxygenic photosynthesis with our previous stratigraphic analysis of 16S rDNA and lipid biomarkers indicative for prokaryotes involved in the cycling of methane in order to get a more complete picture of anoxygenic processes in Ace Lake during the Holocene.

The Bradyrhizobium species currently admitted are three (B. japonicum, B. elkanii and B. liaoningense), but this number is expected to increase with the analysis of more strains isolated from non-previously studied legumes. In the Canary Islands a large number of endemic shrub legumes of great agronomic interest grow. Tagasaste (Chamaecytisus proliferus) has specially been used for centuries as fodder on the Canaries and other parts of the world. In previous works with some rhizobia isolates from tagasaste and other endemic woody legumes, it was shown that these legumes were nodulated by a group of Bradyrhizobium strains phenotypically and genetically diverse (Leon-Barrios et al., 1991; Santamaria et al., 1997; Vinuesa et al., 1998). In this study, thirty-six strains of slow-growing rhizobia isolated from endemic woody legumes were characterized by analysis of PCR-amplified and restricted 16S rDNA, and by LMW RNA profiles. The isolates, incapable of nodulating soybean, were compared with reference strains of genus Bradyrhizobium. Both techniques showed similar results, clustering the isolates in two main groups. The PCR-amplified 16S rDNA was individually restricted with four endonucleases, and the combined RFLP patterns used for cluster analysis by UPGMA. This analysis showed a larger group (29 strains) containing only Canarian isolates (BTA-1 group), and two strains of Lupinus. One smaller group of nine strains (BGA-1 group) clustered with B. japonicum strains. None of the isolates grouped with the strains of B. elkanii. RNA profiles in the 5S rRNA zone showed a pattern identical to B. japonicum for both groups. However, tRNA profiles of both groups were different from that of B. japonicum, although BGA-1 group profiles are more similar to this species. According to previous results (Velazquez et al., 1998), the different species present different tRNA profiles. Thus, the results obtained from both techniques suggest that some Canarian isolates appear to be new species of genus Bradyrhizobium. This idea is also supported by partial sequencing of 16S rRNA gene (Vinuesa et al., 1998). However, new data will be needed to confirm it, especially total DNA hydridization. The finding of two strains of Bradyrhizobium sp. (Lupinus) clustering at a similar level of 100% with the BTA-1 group by ARDRA analysis was a surprise. It had been found (Vinuesa, 1998) that RFLP analysis of rDNA operon could separate a group of ten Canarian isolates among a vast collection of bradyrhizobia, isolates from different parts of the world, giving strong evidence that they could represent a local population of bradyrhizobia confined to the Canary Islands. However, the fact of two strains of Lupinus collected in geographically unrelated locations (Sevilla and Madrid, Spain-mainland), that can nodulate tagasaste and clustering in the same ARDRA group as the “Canarian group”, means that more investigation should be carried out to clear up this matter. Data reflecting total genome variation will be very interesting.

Alnus glutinosa (alder) is widespread in Europe and is an important component of biological diversity in natural forest ecosystems in the Baltic Region. In 2000, diseased trees of A. glutinosa exhibiting characteristically phytoplasmal disease symptoms of shoot proliferation and leaf yellowing were observed in Aukstaitija National Park, Lithuania. In other parts of Europe, alder is affected by a phytoplasmal disease known as alder yellows, which is characterized by symptoms that include yellowing and reduced leaf size, die-back of branches, and decline of trees (2,3). Proliferation of shoots has not been previously reported with this disease. An association between alder yellows and infection by a phytoplasma has been reported in A. glutinosa in Germany and Italy, and a phytoplasma has been found in A. glutinosa in France and Hungary (2,4). We examined symptomatic alder from Lithuania using nested polymerase chain reaction (PCR) (1), primed by P1/P7 and followed by R16F2n/R16R2 (F2n/R2), for amplification of phytoplasmal ribosomal (r) DNA. The results indicated the presence of a phytoplasma, designated ALY-L, in the diseased alder. We classified the ALY-L phytoplasma through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA. A 1.2-kbp fragment (F2n-R2 segment) of rDNA, amplified in PCR primed by F2n/R2, was analyzed using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, Sau3AI, BfaI, and ThaI. On the basis of collective RFLP patterns, phytoplasma ALY-L was classified as a member of group 16SrV (group V, elm yellows group), subgroup C. The amplified 16S rDNA was cloned in Escherichia coli and sequenced, and the sequence was deposited in the GenBank data library (Accession No. AY028789). Nucleotide sequence alignment revealed that 16S rDNA from phytoplasma ALY-L shared 100% sequence similarity with 16S rDNA (GenBank Accession No. Y16387) from a phytoplasma associated with alder yellows (ALY) disease in Italy. The results support the conclusion that a strain of ALY phytoplasma is present in Lithuania. Phytoplasmas belonging to groups 16SrI (aster yellows phytoplasma group) and III (X-disease phytoplasma group) have been found in herbaceous plant species in Lithuania. This report records the first finding of a group V phytoplasma, and the first finding of a phytoplasma in a tree species in the eastern Baltic Region. These findings contribute knowledge about the diversity of phytoplasmas in the Baltic Region and the distribution of ALY phytoplasma in Europe. Apparently, A. glutinosa may be infected by the phytoplasma but not develop obvious disease symptoms, as has been reported elsewhere (3). Thus, it is possible that ALY-L phytoplasma is widespread, but as yet undetected, throughout the geographic range of alder in the Baltic Region. This possibility is supported by the finding of the monophagous leafhopper vector (Oncopsis alni) of ALY phytoplasma throughout Europe (cited in Maixner and Reinert [3]). Further research is needed to assess the impact of phytoplasmal infections such as those by ALY-related phytoplasma strains on trends in biological diversity in the natural forest ecosystems of the Baltic Region and elsewhere in Europe.

The genus Burkholderia (β-Proteobacteria) currently comprises more than 60 species, including parasites, symbionts and free-living organisms. Several new species of Burkholderia have recently been described showing a great diversity of phenotypes. We examined the diversity of Burkholderia spp in environmental samples collected from Caatinga and Atlantic rainforest biomes of Bahia, Brazil. Legume nodules were collected from five locations, and 16S rDNA and recA genes of the isolated microorganisms were analyzed. Thirty-three contigs of 16S rRNA genes and four contigs of the recA gene related to the genus Burkholderia were obtained. The genetic dissimilarity of the strains ranged from 0 to 2.5% based on 16S rDNA analysis, indicating two main branches: one distinct branch of the dendrogram for the B. cepacia complex and another branch that rendered three major groups, partially reflecting host plants and locations. A dendrogram designed with sequences of this research and those designed with sequences of Burkholderia-type strains and the first hit BLAST had similar topologies. A dendrogram similar to that constructed by analysis of 16S rDNA was obtained using sequences of the fragment of the recA gene. The 16S rDNA sequences enabled sufficient identification of relevant similarities and groupings amongst isolates and the sequences that we obtained. Only 6 of the 33 isolates analyzed via 16S rDNA sequencing showed high similarity with the B. cepacia complex. Thus, over 3/4 of the isolates have potential for biotechnological applications.

We assessed whether 16S rDNA and gyrB gene sequences, alone or combined, were suitable for determining the phylogenetic relationship among Salmonella enterica strains isolated from Tehran, Iran. Patients over five years of age enrolled in an acute diarrheal surveillance project in Tehran province between May 2004 and October 2006 were selected as our study group. 16S ribosomal DNA (rDNA) and gyrB genes from 40 Salmonella isolates obtained from patients with acute diarrhea were sequenced and the data was used to generate phylogenetic trees that facilitated isolate comparison. Salmonella strains clustered into five to seven phylogenetic groups, dependent on analysis of 16S rDNA (1546 bp), gyrB (1256 bp) or a combination of the two genes. By 16S rDNA sequence analysis, only strains of Salmonella enterica serovar Typhi ( S. Typhi) clustered exclusively together. gyrB sequences permitted clustering of all the S. Typhi and S. Paratyphi A isolates, and clustering of S. Enteritidis into two separate but exclusive groups. Concatenation of the two data sets did not significantly improve the resolution of the strains compared to the gyrB gene. None of the analyses completely resolved S. enterica Paratyphi B and C into mutually exclusive groups. Sequencing of gyrB represents a potentially useful tool for determining the phylogenetic relationship of S. enterica strains in Tehran, Iran. Genetic analysis of the 16S rRNA gene alone or in combination with gyrB did not increase the resolution between serotypes of S. enterica. We speculate that inclusion of additional genetic markers would improve the sensitivity of the analysis.