Preserved human remains from Militello Rosmarino (Sicily, 18th19th centuries AD): assessing the microbial status of a late modern Italian mummy assemblage.

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

AimThe aim of this study was to understand the effects of revegetation on the diversity of bacteria and fungi in soil by sowing a single species and exploring the underlying mechanism.LocationBeijing, China.TaxonPlants and Microbes.MethodsIn a short‐term ecological restoration experiment, one natural recovery treatment and three seed sowing treatments were chosen to assess their effects on the alteration of fungal and bacterial diversity. Plant species richness, abundance, and height were investigated. The diversity of fungi and bacteria was analyzed by high‐throughput sequencing technologies. Linear mixed‐effects model analysis was used to examine the effects of different restoration methods on biodiversity and ecosystem functions. Pearson's correlation analysis, analysis of covariance, and structural equation modeling (SEM) were used to examine the relationship between biodiversity and environmental factors.ResultsSpecies richness and the Shannon–Wiener Index (H′) of plants in the sown treatments were lower than in the natural recovery treatment, especially with sowing of Medicago sativa L. Similarly, the sum of the observed species and H′ of fungi and bacteria significantly decreased in the sown treatments. Moreover, plant density, community coverage, and soil moisture increased markedly, while soil bulk density decreased in the sown treatments. Importantly, SEM showed that sown treatments reduced the diversity of plants through increasing plant density, while it decreased the diversity of fungi and bacteria through decreasing the plant diversity and increasing soil moisture.Main conclusionsOur findings confirm that ecological restoration by sowing could improve soil conditions, but may be unfavorable to the amelioration of soil microbial diversity in the short‐term. Restoration practitioners should consider long‐term studies on the dynamics of biodiversity in the above‐ and belowground after revegetation by native species to achieve goals related to biodiversity conservation.

Endophytic bacteria colonize the internal tissues of plants without causing infection or negative effects on their hosts. This study investigates the occurrence and diversity of culturable endophytic bacteria in the fruits of Coffea canephora at three developmental stages. Isolation and quantification were performed in R2A culture medium, and the diversity was established using molecular methods and analysis of fatty acid methyl esters (FAME). α- and γ-Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were identified in the investigated community. Kocuria turfanensis and Pantoea vaganswere identified as endophytes for the first time. Of the 18 species that were found, the following seven had not been previously described as endophytic in coffee fruits: Bacillus thuringiensis, Bacillus licheniformis, Agrobacterium tumefaciens, Escherichia coli,Enterobacter hormaechei, Chryseobacterium sp., and Ochrobactrum sp. The diversity of endophytic bacteria varied during the three developmental stages that were investigated, and the diversity was greatest in fruits during the green stage, during which Bacillus subtilis predominated. The number of Gram-positive bacteria was larger than the number of Gram-negative bacteria during the two earliest developmental stages, whereas their numbers were similar during the ripe stage. The diversity was lowest during the ripe stage, and Klebsiella oxytoca was the predominant species at this stage, probably due to the higher caffeine and sugar contents in the fruits. Key words: Coffee, bacterial community, sequencing, 16S rDNA, FAME

BackgroundThere has been considerable confusion concerning the number and classification of Sarcocystis spp. in chickens. Scarce nucleotide data of Sarcocystis spp. from chickens are provided in GenBank. The study aimed to investigate the morphological and molecular characteristics of Sarcocystis spp. found in chickens in China.MethodsTissues from 33 chickens were collected in 2019. Sarcocysts were observed using light (LM) and transmission electron microscopy (TEM). Individual sarcocysts from different chickens were selected for DNA extraction, and five loci, 18S rDNA, 28S rDNA, ITS1 region, the mitochondrial cox1 gene and the apicoplastic rpoB gene, were amplified from each sarcocyst, sequenced and analyzed.ResultsOnly S. wenzeli was found in 14 of 33 (42.4%) chickens. Under LM, the sarcocysts were microscopic and exhibited palisade-like villar protrusions measuring 1.5–2.8 μm. Ultrastructurally, the sarcocyst wall contained numerous stubby hill-like villar protrusions. The protrusions included scattered microtubules, which extended from the tips of the protrusions into the ground substance. The five loci were successfully sequenced and the sequences deposited in GenBank. At 18S rDNA, ITS1 and cox1, the most similar sequences in GenBank were those of Sarcocystis sp. obtained from the brains of chickens, i.e. 99.9–100%, 98.1–98.5% and 99.3% identity, respectively. The five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) showed different levels of interspecific sequence similarity with other closely related species of Sarcocystis (e.g. 99.8%, 99.0–99.2%, 89.3–89.7%, 98.5%, and 97.5%, respectively, with S. anasi). Phylogenetic analysis based on four of the loci (18S rDNA, cox1, rpoB and ITS1) revealed that S. wenzeli formed an independent clade with Sarcocystis spp. that utilize geese or ducks as intermediate hosts and canines as the known or presumed definitive host.ConclusionsTo our knowledge, the sequences of 28S rDNA and rpoB reported here constitute the first records of genetic markers of Sarcocystis spp. in chickens. Based on molecular analysis, S. wenzeli might be responsible for the neurological disease in chickens, and ITS1 and rpoB are more suitable for discriminating it from closely related Sarcocystis spp. Phylogenetic analysis revealed that S. wenzeli presents a close relationship with Sarcocystis spp. in geese or ducks.

We have analysed the diversity of culturable sulphate-reducing bacteria (SRB) in Zostera noltii colonized sediments from Bassin d'Arcachon (France). Four organic substrates have been tested as well as the combination of H2 and CO2 to select for lithotrophic SRB. All energy sources were supplied in parallel cultures that were amended with yeast extract plus NH4+ and prepared without a source of combined nitrogen, the latter to select for diazotrophic SRB. The 10 different enrichment media were inoculated from serial dilution of rhizosphere samples. The highest dilution cultures yielding positive growth (i.e. 10-7) were studied by molecular techniques (16S rDNA clone libraries, RISA and ARDRA). Lactate as a single organic substrate in combination with a source of combined nitrogen resulted in selection of members of the Desulfovibrionaceae. Surprisingly, when lactate was added without a source of combined nitrogen, Desulfobacteriaceae were selected. A strong influence of the presence or absence of combined nitrogen was also observed for the substrates sucrose and fructose. Whereas the liquid culture growing on sucrose and NH4+ systematically yielded 16S rDNA clones related to an environmental unidentified green sulphur bacterium (OPS185), on plates we were able to isolate a SRB related to Desulfovibrio dechloracetivorans, which likely represents a non-described species. Under diazotrophic conditions, sucrose selected for SRB clones related to the cluster formed by Desulfovibrio zosterae, Desulfovibrio salexigens and Desulfovibrio bastinii. The corresponding isolate obtained on plates showed only low sequence similarity with this closest neighbour (93.8%), and we suggest that it also represents a non-described species. Surprisingly, a 16S rDNA sequence corresponding to an archaeon, i.e. a non-extremophile Crenoarchaeota, was retrieved from several of the SRB enrichment cultures even after subsequent transfers.

Little is known of the effects of ionizing radiation exposure on soil biota. We exposed soil microcosms to weekly bursts of 60Co gamma radiation over six weeks, at three levels of exposure (0.1 kGy/hr/wk [low], 1 kGy/hr/wk [medium] and 3 kGy/hr/wk [high]). Soil DNA was extracted, and shotgun metagenomes were sequenced and characterised using MG-RAST. We hypothesized that with increasing radiation exposure there would be a decrease in both taxonomic and functional diversity. While bacterial diversity decreased, diversity of fungi and algae unexpectedly increased, perhaps because of release from competition. Despite the decrease in diversity of bacteria and of biota overall, functional gene diversity of algae, bacteria, fungi and total biota increased. Cycles of radiation exposure may increase the range of gene functional strategies viable in soil, a novel ecological example of the effects of stressors or disturbance events promoting some aspects of diversity. Moreover, repeated density-independent population crashes followed by population expansion may allow lottery effects, promoting coexistence. Radiation exposure produced large overall changes in community composition. Our study suggests several potential novel radiation-tolerant groups: in addition to Deinococcus-Thermus, which reached up to 20% relative abundance in the metagenome, the phyla Chloroflexi (bacteria), Chytridiomycota (fungi) and Nanoarcheota (archaea) may be considered as radiation-tolerant.

Little is known about the difference between bacterial and fungal genetic and functional diversity in karst regions of south China. In this study, the genetic and functional diversity of bacteria and fungi in nine types of soil microenvironments in the karst region in Maolan National Nature Reserve in Guizhou were investigated by PCR-DGGE (Denaturing Gradient Gel Electrophoresis) and BIOLOG EcoPlates. Maolan National Nature Reserve is a UNESCO Biosphere Reserve and plays an important role in protecting the karst forest ecosystem and rare and endangered wild animals and plants in central Asia. The results showed that the diversity of both bacteria and fungi was high and the main factors influencing the diversity of bacteria and fungi were different. The bacterial community structure from different microhabitats under the same vegetation type had higher similarity than similar microhabitats in different vegetation types, which could indicate that the bacterial community structure was mainly controlled by vegetation. For fungi, similar microhabitat species under different vegetation types had higher similarities than different microhabitats species under the same vegetation type, which could indicate that the fungal community structure is mainly controlled by microhabitats. In addition, the metabolic patterns of similar microhabitats in different vegetation were different, while the metabolic patterns of different microhabitats in the same vegetation were not obviously different. In conclusion, the effect of vegetation types on soil microbial functional diversity was greater than that of microhabitats, and this difference was reflected by the different degrees of influence on soil microbial genetic diversity and community structure.

Introduction One of the most destructive diseases of pome fruit species which causes irreparable damage to gardening products worldwide is fire blight disease caused by Erwinia amylovora. In Iran, it was first observed in pear trees in Alborz province and then spread across fruit orchards in the country. One of the main problems in fire blight management is evaluating causal agent genotypic characteristics. Thus, the present research was conducted to characterize genotypic features of E. amylovora as the cause of fire blight disease in pome fruit species in Hamadan province, Iran. Material and methods In this study, the samples with symptoms of canker on shoots and blight blossoms were observed and collected from the orchards in Hamadan province located in the west of Iran. After isolation, the isolates were purified for further studies. Phenotypic, biochemical, and pathogenicity tests were performed due to standard bacterial criteria. Phenotypic and biochemical tests of strains were examined by Ntsys-pc 2.02 software. A total of 15 representative isolates were selected and analyzed due to the size of amplified DNA using primers Ea71 and genetic features of the rep-PCR test using primers ERIC and BOX. For more accuracy and higher reliability of the specific primers results, the 16S rDNA_ gene of two representative isolates was amplified and subjected to sequencing. Results Based on the biochemical, pathogenicity and molecular tests, a total of 34 isolates were identified as E. amylovora. Due to the numerical analysis, the data obtained by phenotypic and biochemical tests were similar at 89% level. Therefore, the isolates obtained from a specific host or region was grouped very close to each other. In the PCR tests survey, the isolates amplified 187 base pair expected fragments. The results of determining sequences indicated that both isolates were similar to E. amylovora showing 97% identity to the type of strains in the NCBI database. Due to data from the rep-PCR analysis, the isolates were divided into three groups at the similarity level of 77%. Discussion The results of genetic diversity using rep-PCR showed that there is no significant difference among E. amylovora bacterial isolates from different regions of Hamadan Province. Moreover, they showed high similarity to each other and placed close to each other. Our results confirm other studies regarding phenotypic characteristics of E. amylovora. Our results confirm that the isolates are homogenous in Hamadan province of Iran. To summary, these findings can be applied to breeding programs to better management of the bacterial disease.

Bauxite residue (red mud), generated during the extraction of alumina from bauxite ore is characterized by high pH, high concentrations of soluble ions with low or virtually no organic matter. These extreme conditions along with numerous nutrient deficiencies, limit the microbial growth and vegetation establishment. In the present study, diversity of both cultivable and non-cultivable bacteria present in the red mud was investigated by 16S rDNA sequence analyses. The cultivable bacteria were identified as Agromyces indicus, Bacillus litoralis, B. anthracis, Chungangia koreensis, Kokuria flava, K. polaris, Microbacterium hominis, Planococcus plakortidis, Pseudomonas alcaliphila and Salinococcus roseus based on their 16S rDNA sequence analysis. These isolates were alkali tolerant, positive for one or more of the enzyme activities tested, able to produce organic acids and oxidize wide range of carbon substrates. For non-cultivable diversity of bacteria, DNA was extracted from the bauxite residue samples and 16S rDNA clone library was constructed. The 16S rDNA clones of this study showed affiliation to three major phyla predominant being betaproteobacteria (41.1%) followed by gammaproteobacteria (37.5%) and bacteroidetes (21.4%). We are reporting for the first time about the bacterial diversity of this unique and extreme environment.

Protease-producing bacteria play a vital role in degrading organic nitrogen in marine environments. However, the diversity of the bacteria and extracellular proteases has seldom been addressed, especially in communities of coral reefs. In this study, 136 extracellular protease-producing bacterial strains were isolated from seven genera of scleractinian corals from Luhuitou fringing reef, and their protease types were characterized. The massive coral had more cultivable protease-producing bacteria than branching or foliose corals. The abundance of cultivable protease-producing bacteria reached 106 CFU g−1 of coral. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolates were assigned to 24 genera, from which 20 corresponded to the phyla Firmicutes and Proteobacteria. Bacillus and Fictibacillus were retrieved from all coral samples. Moreover, Vibrio and Pseudovibrio were most prevalent in massive or foliose coral Platygyra and Montipora. In contrast, 11 genera were each identified in only one isolate. Nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases; 45.83% of isolates also released cysteine or aspartic proteases. These proteases had different hydrolytic ability against different substrates. This study represents a novel insight on the diversity of cultivable protease-producing bacteria and their extracellular proteases in scleractinian corals.

The identification of Mycobacteria in the species level has great medical importance. Biochemical tests are laborious and time-consuming, so new techniques could be used to identify the species. This research aimed to the comparison of biochemical and sequencing 16S rDNA gene methods to identify nontuberculous Mycobacteria in patients suspected to tuberculosis in Golestan province which is the most prevalent region of tuberculosis in Iran. Among 3336 patients suspected to tuberculosis referred to hospitals and health care centres in Golestan province during 2010-2011, 319 (9.56%) culture positive cases were collected. Identification of species by using biochemical tests was done. On the samples recognized as nontuberculous Mycobacteria, after DNA extraction by boiling, 16S rDNA PCR was done and their sequencing were identified by NCBI BLAST. Of the 319 positive samples in Golestan Province, 300 cases were M.tuberculosis and 19 cases (5.01%) were identified as nontuberculous Mycobacteria by biochemical tests. 15 out of 19 nontuberculous Mycobacteria were identified by PCR and sequencing method as similar by biochemical methods (similarity rate: 78.9%). But after PCR, 1 case known as M.simiae by biochemical test was identified as M. lentiflavum and 3 other cases were identified as Nocardia. Biochemical methods corresponded to the 16S rDNA PCR and sequencing in 78.9% of cases. However, in identification of M. lentiflavum and Nocaria sp. the molecular method is better than biochemical methods.

Dinophilidae (Annelida) is most likely not a progenetic Eunicida: Evidence from 18S and 28S rDNA

The survival of the bacterial pathogen, Xanthomonas campestris pv. campestris (Xcc), was studied in plant debris-infested soil with different matric potentials (0, −10, −30, −50, −100, −200, and −900 kPa), and on the phylloplane of crops used for rotation with cabbage. Populations of cellulolytic and proteolytic microorganisms were studied in relation to soil matric potential and Xcc. The survival of Xcc was negatively correlated (r=−0.710; P=0.06) with soil matric potential and with the abundance of cellulolytic microorganisms (P=0.05). In saturated soil, Xcc survived for only 19–28 days, while at −900 kPa there was no significant change in the bacterial population within this period. Survival of Xcc on the phylloplane of cabbage (Brassica oleracea var. capitata) (host) was similar to that on mustard and lettuce (48 days), whereas the pathogen was detected for only 9 days on rice. It appears that high matric potentials reduced populations of Xcc in soil, whereas epiphytic survival of this pathogen depended on the plant species.

The effects of compost and liquid biofertiliser on the chemical and biological char-acteristics of soil cultivated with banana ( Musa spp. L.) cv. ‘Grand Naine’ were investigated. Four treatments (liquid bioferment (LBF), compost (COM), liquid bioferment + compost (LBF + COM) and unamended (CONTrOL)) were applied to banana plants in the fieldin a completely randomised design in the crop cycles of 2005-2006 and 2007-2008. The physico-chemical characteristics, enzymatic activity and microbial diversity were determined prior to the bioferment addition and 140 and 280 days after application (DAP). The microbial groups were deter-mined (i.e., bacteria, fungi, actinomycetes). Next, cellulolytic, proteolytic, starch hydrolysing, asymbiotic N 2 -fixing and phosphorous-solubilising microorganisms were determined. The application of LBF + COM increased organic matter 1.39 times, increased compost 1.33 times and increased LBF 1.16 times at 140 DAP. The greatest microbial populations were found in soil amended with LBF + COM at 140 DAP. The highest esterase and invertase enzyme activities were found in soil amended with LBF+COM at 140 DAP. The highest xylanase activity was found in soil amended with compost at 280 DAP. These results showed that the addition of compost and liquid bioferment improves soil fertility and stimulates microbial activ-ity and their capacity to metabolise complex organic molecules.Keywords: enzyme activities, cellulolytic microorganisms, proteolytic microorgan-isms, starch hydrolysing microorganisms, asymbiotic N

In the summer of 2011, in a nursery located in Viçosa City, Minas Gerais State, brownish, necrotic, irregular spots were observed on leaves of Mabea fistulifera Mart. (Euphorbiaceae), an indigenous forest species commonly found in Brazil. Around 6,300 seedlings were evaluated and as many as 60% of them showed disease symptoms, including severe defoliation and plant death. Leaves with coalescing lesions turned papery in texture and had a blighted appearance. Bacterial colonies were isolated from these symptomatic leaves on King B's medium and identified based on biochemical and molecular analysis, as a member of the Enterobacteriaceae family. Like other members of the Enterobacteriaceae family, the bacteria were facultative anaerobic, gram-negative, cream-colored on YDC medium, urease and oxidase negative, as well as catalase and asparagine positive. Bacterial DNA was extracted from pure culture grown overnight in liquid 523 medium at 28°C using the Wizard Genomic DNA Purification kit (Promega) and conserved sequences in 16S rDNA (3) and rpoB (1) were amplified by PCR. The sequence of the 1,300-bp 16S rDNA fragment and the 750-bp rpoB gene were analyzed by NCBI BLAST. Related sequences were aligned and analyzed by ClustalW in MEGA 5 software. Phylogenetic analysis by maximum likelihood, using PAUP version 4.0 and TBR algorithm with 1,000 bootstrap replications, grouped the isolate in a clade with Enterobacter cowanii and the result showed 99% and 98% identity to the 16s rDNA and rpoB, respectively. The isolate clustered closely with the type strain of E. cowanii in both phylogenetic trees constructed. Pathogenicity tests were carried out by inoculating leaves of healthy seedlings either by spraying or cutting with a scissor previously dipped into a 108 CFU/ml bacterial suspension. The experiment was in a completely randomized design, with six replications. A pot with one plant was considered one experimental unit. Control seedlings were sprayed or cut with a scissor treated with saline solution. Prior to and after inoculation, plants were kept in a humid chamber for 24 h at 26°C in the dark and at room temperature. Subsequently, plants were transferred to growth chamber at 26°C, under a 12-h photoperiod (40 μmol/s/m2). Consistent with the symptoms observed originally, 7 days after inoculation, all seedlings developed leaf spots. No characteristic symptoms could be observed in the negative control. Furthermore, Koch's postulates were confirmed by reisolation of the bacterium from symptomatic tissues. In summary, the phenotypic, biochemical, and molecular tests identified the pathogen as E. cowanii. Recently, E. cowanii was isolated from Eucalyptus trees with symptoms of bacterial blight, although its pathogenicity was not demonstrated (2). To the best of our knowledge, this is the first report of a member of the Enterobacteriaceae family causing disease in M. fistulifera. The result has a great importance to better understand the role of E. cowanii as a pathogen-causing disease on a forest species.