16S rRNA-encoding Gene Sequences Research Articles

Identification, characteristics and rice growth promotion of a highly efficient cellulolytic bacterial strain, Cellulomonas iranensis ZJW-6, isolated from paddy soil in central China.

The microbial degradation of lignocellulose is the best way to treat straw, which has a broad application prospect. It is consistent with the idea of agricultural sustainable development and has an important impact on the utilization of biomass resources. To explore and utilize the microbial resources of lignocellulose degradation, 27 lignocellulose degrading strains were screened from 13 regions in China. ZJW-6 was selected because of its 49.6% lignocellulose weight loss rate. According to the theoretical analysis of the experimental results, the following straw degradation conditions were obtained by ZJW-6: nitrogen source input of 8.45 g/L, a pH of 8.57, and a temperature of 31.63°C, the maximum weight loss rate of rice straw could reach 54.8%. It was concluded that ZJW-6 belonged to Cellulomonas iranensis according to 16S rRNA-encoding gene sequence comparison and identification. ZJW-6 is a Gram-positive bacterium that grows slowly and has a small yellowish green colony. To explain the degradation mechanism of lignocellulose, the experiment of enzymatic properties of the strain was prepared and carried out. It was discovered that ZJW-6 has an excellent ability to degrade cellulose, hemicellulose, and lignin, with cellulose and hemicellulose loss rates reaching almost 50% in 4 days and lignin loss rates reaching nearly 30%. Furthermore, ZJW-6 demonstrated lignocellulose degradation under aerobic and anaerobic conditions, indicating the strain's broad application potential. ZJW-6 was found to be more effective than ordinary humic acid in improving rice soil (available phosphorus, available nitrogen, organic matter) and promoting rice growth in a rice pot experiment (increasing root-shoot ratio, root activity, chlorophyll content and net photosynthetic rate). ZJW-6 plays an important role in promoting the development and utilization of straw resources. It has important significance for the advancement of green agriculture.

First report of a ‘Candidatus Phytoplasma asteris’ strain affecting lingonberry (Vaccinium vitis‐idaea) and causing lingonberry stunt phytoplasma disease

Vaccinium vitis-idaea is a red berry-producing woody shrub, native to North America, Europe, and Scandinavia, known commonly as lingonberry (Hendrickson, 1997). During the spring/summer seasons of 2019 and 2020, producers from Quebec, Canada, have noticed symptoms of yellowing leaves and stunted growth, resembling symptoms caused by phytoplasma. To confirm whether phytoplasmas were indeed associated with these reported symptoms, fifteen lingonberry plants from different producers from the region of Chaudière-Appalaches were analysed (Table 1). Of these, nine had symptoms of stunting, little leaves and yellowing, while the rest showed no visible symptoms when collected (Figure 1). For each plant, total DNA was extracted from leaves and stem tissue using a CTAB-based method. The DNA was used as a template for a cpn60UT PCR with primers H279p/H280p (Dumonceaux et al., 2014), and the positive samples were subjected to direct PCR with phytoplasma 16S primers R16F2n/R16R2 as previously described (Perez-Lopez et al., 2019). Amplicons of the expected size (∼600 bp for cpn60UT) were obtained for 9/15 samples, while those for R16F2n/R2 sequences were obtained for 8/9 samples positives using the cpn60UT assay (Table 1). The cpn60UT and R16F2n/R2 amplicons obtained from two symptomatic samples (named LbSP14 and LbSP16 for lingonberry stunt phytoplasma) were purified and sequenced directly with the amplification primers (Centre Hospitalier de l'Université Laval de Quebec, Canada). The R16F2n/R2 sequence from LbSP14 was cloned into the pGEMT-Easy Vector (Promega, USA), and two clones were sequenced. Sequences were deposited in GenBank with Accession Nos OL739234 and OL739235 for LbSP14 and LbSP16 cpn60UT, respectively, and OL773369 and OL773370 for LbSP14 and LbSP16 R16F2n/R2 sequences. Accession Nos. OL773367 and OL773368 were used to register the sequences obtained from clone 1 (LbSP14c1) and clone 2 (LbSP14c2), respectively. A group/subgroup classification was performed on the LbSP cpn60UT sequences using CpnClassiPhyR (Muirhead et al., 2019) and R16F2n/R2 sequences with iPhyClassifier (Zhao et al., 2009). Phylogenetic relationships were determined using cpn60UT and R16F2n/R2 sequences of the LbSP phytoplasma and those from GenBank with the MEGA program version X (Kumar et al., 2018) and the neighbour-joining method with 1000 bootstraps. The CpnClassiPhyR classification suggested that the LbSP phytoplasma strain is a ‘Candidatus Phytoplasma asteris’-related strain and a member of the cpn60UT I-I(E/AI)AI subgroup (Figure 2a), with a similarity coefficient of 1.00 compared with blueberry stunt phytoplasma (BbSP; MH279496). The phylogenetic tree showed that LbSP strains branches with cpn60UT I, closely related to BbSP isolates from Quebec, Nova Scotia and New Brunswick (Figure 3). The 16S classification linked to the cpn60UT identified is 16SrI- (E/AI)AI (Perez-Lopez et al., 2019). We also found that LbSP displays 16S rRNA-encoding gene sequence heterogeneity with two distinct rrn operons, rrnE (OL773367) and rrnAI (OL773368), as previously reported for BbSP (Figure 2b and Figure 4). To our knowledge, this is the first report of lingonberry as a possible phytoplasma host. Other Vaccinium species, for example blueberry (Vaccinium corymbosum), are known to be affected by phytoplasmas in the same 16SrI- (E/AI)AI subgroup in Quebec (Perez-Lopez et al., 2019), and by 16SrI-E phytoplasmas in Canada and the USA (Lee et al., 2004; Arocha-Rosete et al. 2019). The fact that blueberries and lingonberries can be affected by phytoplasma strains in the same phytoplasma subgroup may suggest a common insect vector with variable feeding habits. Our findings are the basis for the understanding of the colonisation of a new plant host in the family Ericaceae by phytoplasmas. We would like to thank the MAPAQ and Les Airelles des Frères, specially to F. Philippe Roy for the support and collaboration, and to Prof. Jean Collin who through the class PLG-3305 supported the completion of the research.

Isolating and characterizing bacteria in the intestine of wild sandfish, Holothuria scabra as probiotics candidate

The research aims to isolate bacteria as potential probiotics for rearing of sandfish, H. scabra. The procedures were isolating bacteria from nature sandfish’s intestines, characterizing, identifying, enzymatic hydrolysis activity and pathogenic testing, and in vivo testing of candidate probiotics. Identification of probiotic bacteria was based on 16S rRNA encoding gene sequence. Similarity identification was conducted by using BLAST on NCBI. The enzymatic activity test was carried out through Extra Cellular Product (ECP) of isolated bacteria. The in vivo test was done in twelve 1.2 m3 tanks. Initial mean body weight of juvenile was 6.0 ± 4.3 g and total length 4.3 ± 0.6 cm. The results, there were three isolated bacteria as candidates of probiotics with code M-4, Q-1, and E-2 which had high ability in hydrolyzing gelatin, casein, amylase, lecithin, and lipase enzymes. The M-4 was identified as Gamma proteobacterium with a 99% similarity, Q-1 and E-2 were identified as Bacillus subtilis and Bacillus sp with 98% and 97% similarity. The in vivo trial of probiotic candidate in feed for juvenile gave higher survival rate 95% compare to control 91%. The growth performance was (7.3 ± 2.1 g; 4.5 ± 0.7 cm) in probiotic which higher compare to control (6.0 ± 1.8g; 4.3 ± 0.8 cm).

Genetic diversity and plant growth-promoting activity of the dominant bacteria from canola plants in Western Iran

BackgroundPlant growth-promoting bacteria (PGPB) can be used as a safe and sustainable agricultural measure to increase plant yield. In this study, cultivable rhizospheric, endophytic, and epiphytic bacteria associated with canola were isolated on nutrient agar medium. The SDS-PAGE method was used for grouping the bacterial strains. Bacterial strains with more than 80% similarity in their protein patterns were placed in the same species and selected as a representative for partial 16S rRNA encoding gene sequence analysis.ResultsIt was found that the bacteria used in this study belonged to Pseudomonas, Frigoribacterium, Sphingomonas, Sphingobacterium, Microbacterium, Bacillus, and Rhodococcus genera. Three PGP bioactivities including siderophore production, nitrogen fixation ability, and phosphate solubilization were evaluated for 18 Pseudomonas representative strains. All tested strains were able to produce siderophore, although only 5 strains could fix nitrogen and none was able to solubilize phosphate. Greenhouse experiments showed that 4 bacterial strains (i.e., 8312, 642, 1313W, and 9421) significantly affected canola seed germination and seedling growth. Bacterial strain 8312, which was identified as Pseudomonas sp., showed the highest effect on the growth of the canola plant. These results indicated the potential of strain 8312 to be used as a biofertilizer in canola cultivation.ConclusionsThe results of this research indicated that canola-related bacteria were diverse during the flowering stage. One isolate had a significant effect on all canola plant growth factors in the greenhouse. Hence, it is recommended to investigate this strain further in field infestation experiments.

Semidry acid hydrolysis of cellulose sustained by autoclaving for production of reducing sugars for bacterial biohydrogen generation from various cellulose feedstock.

Cellulosic biowastes are one of the cheapest and most abundant renewable organic materials on earth that can be, subsequent to hydrolysis, utilized as an organic carbon source for several fermentation biotechnologies. This study was devoted to explore a semidry acid hydrolysis of cellulose for decreasing the cost and ionic strength of the hydrolysate. For semidry acid hydrolysis, cellulose was just wetted with HCl (0 to 7 M) and subjected to autoclaving. The optimum molar concentration of HCl and period of autoclaving for semidry acid hydrolysis of cellulose were 6 M and 50 min respectively. Subsequent to the semidry acid hydrolysis with a minimum volume of 6 M HCl sustained by autoclaving, the hydrolysate was diluted with distilled water and neutralized with NaOH (0.5 M). The reducing sugars produced from the semidry acid hydrolysis of cellulose was further used for dark fermentation biohydrogen production by Escherichia coli as a representative of most hydrogen producing eubacteria which cannot utilize non-hydrolyzed cellulose. An isolated E. coli TFYM was used where this bacterium was morphologically and biochemically characterized and further identified by phylogenetic 16S rRNA encoding gene sequence analysis. The reducing sugars produced by semidry acid hydrolysis could be efficiently utilized by E. coli producing 0.4 mol H2 mol−1 hexose with a maximum rate of hydrogen gas production of 23.3 ml H2 h−1 L−1 and an estimated hydrogen yield of 20.5 (L H2 kg−1 dry biomass). The cheap cellulosic biowastes of wheat bran, sawdust and sugarcane bagasse could be hydrolyzed by semidry acid hydrolysis where the estimated hydrogen yield per kg of its dry biomass were 36, 18 and 32 (L H2 kg−1 dry biomass) respectively indicating a good feasibility of hydrogen production from reducing sugars prepared by semidry acid hydrolysis of these cellulosic biowastes. Semidry acid hydrolysis could also be effectively used for hydrolyzing non-cellulosic polysaccharides of dry cyanobacterial biomass. The described semidry acid hydrolysis of cellulosic biowastes in this study might be applicable not only for bacterial biohydrogen production but also for various hydrolyzed cellulose-based fermentation biotechnologies.

Molecular characterization of group 16SrI and 16SrIII phytoplasmas associated with loofah witches’ broom disease in Sinaloa, Mexico

Loofah [Luffa acutangula (L.) Roxb.] is grown in some regions of Asia, Africa and the Americas. This vegetable is well known for its high nutritional and medicinal value. Loofah plants showing typical symptoms of phytoplasma infection, such as witches’ broom, fruit deformation, flower proliferation and phyllody were observed in a commercial field in Sinaloa, Mexico, in January 2015. Nineteen symptomatic loofah plants and seven asymptomatic weeds (Helianthus annuus, Sarcostemma sp., Chenopodium sp., Amaranthus palmeri, Rumex sp., Ambrosia sp., and Raphanus sp.) growing nearby were collected and analyzed by phytoplasma-specific nested PCR. Phytoplasmas were detected in 79% of symptomatic loofah plants and in two of the seven weeds. Conventional and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rRNA-encoding gene sequences indicated the presence of strains of the 16SrIII and 16SrI phytoplasma groups in loofah, which occurred as both single and mixed infections. The phylogenetic analysis confirmed four new phytoplasma strains associated with loofah diseases, two ‘Candidatus Phytoplasma asteris’-related strains (16SrI-AG and 16SrI-AH), and two ‘Candidatus Phytoplasma pruni’-related strains (16SrIII-AA, and 16SrIII-AB). Furthermore, phytoplasmas belonging to groups 16SrI and 16SrIII also were identified in the weeds H. annuus and A. palmeri, respectively; 16SrIII-AC represents a new phytoplasma subgroup described in A. palmeri in Sinaloa. To our knowledge, this is the first report of loofah disease associated with phytoplasmas belonging to the 16SrI and 16SrIII groups in Mexico.

Occurrence of ‘Candidatus Phytoplasma omanense’-related strains and other phytoplasmas in Sophora alopecuroides plants showing dwarfing and yellowing

Sophora alopecuroides dwarfing and yellowing symptoms were observed at the edge of several Damaneh and Fereydun Shahr (Isfahan province, Iran) fields. Polymerase chain reaction using primers P1/P7 and nested PCR using the same primers followed by R16mF2/R16mR2 and R16F2n/R16R2 primer pairs amplified the 16S ribosomal RNA-encoding gene in all the symptomatic plants tested, but not in the asymptomatic ones. RFLP analysis of R16F2n/R16R2 amplicons with AluI, HaeIII, HhaI, HpaI, HpaII, RsaI, MseI and TaqI restriction enzymes showed profiles referable to 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX phytoplasma ribosomal groups indicating also the presence of mixed infection in several samples. The identity percentage and the phylogeny obtained after direct sequencing of selected 16S rRNA-encoding gene sequences (R16F2n/R2 amplicons) from symptomatic S. alopecuroides confirmed the presence of phytoplasma strains related to ‘Candidatus Phytoplasma omanense’ (group 16SrXXIX), ‘Ca. P. australasia’ (group 16SrII), and ‘Ca. P. trifolii’ (16SrVI). Moreover, in a few samples/amplicons, 16SrXII-A, 16SrIX-H, 16SrVI-F and 16SrXXIX-A phytoplasma subgroups were identified by real and virtual RFLP analyses. The other phytoplasmas detected were classified in subgroups 16SrII-D and 16SrVI-A. This is the first report of the presence of 16SrII and 16SrXXIX phytoplasma groups and of mixed infection of phytoplasma strains belonging to groups 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX in S. alopecuroides worldwide.

First Report of Association of a ‘Candidatus Phytoplasma aurantifolia’ 16SrII-D Related Phytoplasma with Geranium (Pelargonium hortorum) Little Leaf Disease in Iran

Geranium, an important ornamental plant belong to the family Geraniaceae, is widely cultivated in urban landscapes and private gardens all over the world. During a survey in spring 2019, a few seed-propagated geranium plants exhibiting typical symptoms induced by phytoplasmas, including pale green and little leaves, phyllody, virescence, and witches’-broom symptoms, were observed in Jiroft, Kerman province of Iran. Total genomic DNA extracted from leaf midribs and petioles of the three symptomatic and two asymptomatic plants was used as a template in a nested polymerase chain reaction (PCR) assay using universal phytoplasma primer pairs P1/P7 followed by R16F2n/ R16R2 (Gundersen and Lee 1996) to amplify a part of the 16S rDNA phytoplasma genome as previously described by Azadvar et al. (2011). The generated 1.8- and 1.25-kb amplicons in the first and second round of the nested PCR assay, respectively, from each of the three symptomatic plant samples revealed phytoplasma infection of the plants. No amplicon was generated in PCR assays by using the DNA templates from symptomless plants. The purified PCR products were directly sequenced (Bioneer, South Korea) in both forward and reverse directions. The consensus sequences were analyzed by the iPhyClassifier tool (Zhao et al. 2009) and pDRAW32 software (AcaClone software, http://www.acaclone.com) for 16Sr group/subgroup phytoplasma identification. The 16S rDNA nucleotide sequences from the three diseased plants were identical (GenBank accession no. MT187639). iPhyClassifier analysis showed that the F2nR2 16S rRNA encoding gene sequence of the phytoplasma associated with geranium little leaf (GrLL) disease in Iran was identical (similarity coefficient 1.00) to those of the reference pattern of 16Sr group II subgroup D (GenBank accession no. Y10097) and had 98.7% similarity with the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession U15442). Virtual restriction fragment length polymorphism (in silico RFLP) analysis of the 16S rDNA F2nR2 sequence with the pDRAW32 program using AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, SspI, and TaqI endonucleases further confirmed that the GrLL phytoplasma belong to subgroup II-D. The 16SrII-D subgroup phytoplasma has been previously reported in association with Calendula officinalis (Esmailzadeh Hosseini et al. 2016), Helianthus annuus (Salehi et al. (2015), and Petunia hybrida (Faghihi et al. 2014) ornamental plants in Iran. We also found many petunia (P. hybrida) plants showing little leaf, yellowing, phyllody, and witches’-broom symptoms next to the GrLL-infected plants, which hosted the same phytoplasma (GenBank accession no. MT187637). A 16SrII group-related phytoplasma has previously been reported associated with Pelargonium capitatum weed plant exhibiting severe stunting and necrosis in Australia (Lee et al. 2010). To our knowledge, this is the first report of a ‘Ca. P. aurantifolia’ 16SrII-D subgroup-related phytoplasma associated with geranium (Pelargonium hortorum) little leaf disease in the world.

Forest Tree Associated Bacterial Diffusible and Volatile Organic Compounds against Various Phytopathogenic Fungi.

Plant growth-promoting rhizobacteria (PGPR) can potentially be used as an alternative strategy to control plant diseases. In this study, strain ST–TJ4 isolated from the rhizosphere soil of a healthy poplar was found to have a strong antifungal activity against 11 phytopathogenic fungi in agriculture and forestry. Strain ST–TJ4 was identified as Pseudomonas sp. based on 16S rRNA-encoding gene sequences. The bacterium can produce siderophores, cellulase, and protease, and has genes involved in the synthesis of phenazine, 1–phenazinecarboxylic acid, pyrrolnitrin, and hydrogen cyanide. Additionally, the volatile compounds released by strain ST–TJ4 can inhibit the mycelial growth of plant pathogenic fungi more than diffusible substances can. Based on volatile compound profiles of strain ST–TJ4 obtained from headspace collection and GC–MS/MS analysis, 1-undecene was identified. In summary, the results suggested that Pseudomonas sp. ST–TJ4 can be used as a biocontrol agent for various plant diseases caused by phytopathogenic fungi.

A novel 'Candidatus Phytoplasma asteris' subgroup 16SrI-(E/AI)AI associated with blueberry stunt disease in eastern Canada.

Phytoplasmas ('Candidatus Phytoplasma' species) are phytopathogenic bacteria vectored by insects and are associated with crop diseases that cause severe yield losses by affecting reproductive tissue development. Infection of northern highbush blueberry plants (Vaccinium corymbosum; Ericaceae) with phytoplasma leads to yield losses by altering plant development resulting in stunting and subsequent plant death. Samples collected from symptomatic blueberry plants in two important blueberry-producing areas in Canada, in the provinces of Québec and Nova Scotia, were analysed for the presence of DNA sequences associated with phytoplasma. Analysis of the 16S rRNA gene sequences demonstrated that the plants were infected with a strain of 'Candidatus Phytoplasma asteris', which was previously identified as blueberry stunt phytoplasma (BBS; 16SrI-E). Examination of further bacterial sequences revealed that two distinct 16S rRNA-encoding gene sequences were present in each sample in combination with a single chaperonin-60 (cpn60) sequence and a single rpoperon sequence, suggesting that this strain displays 16S rRNA-encoding gene sequence heterogeneity. Two distinct rrnoperons, rrnE and the newly described rrnAI, were identified in samples analysed from all geographic locations. We propose, based on the sequences obtained, delineating the new subgroup 16SrI-(E/AI)AI, following the nomenclature proposed for heterogeneous subgroups. To our knowledge, this is the first report of a heterogeneous phytoplasma strain affecting blueberry plants and associated with blueberry stunt disease.

Genome Sequence of a Plant-Pathogenic Bacterium, "Candidatus Phytoplasma asteris" Strain TW1.

A draft genome sequence is presented for a strain of "Candidatus Phytoplasma asteris" affecting canola plants in Saskatoon, Canada. This phytopathogenic bacterium was determined to be a 16SrI strain and features 16S rRNA-encoding gene sequence heterogeneity.

Variable responses of human microbiomes to dietary supplementation with resistant starch

BackgroundThe fermentation of dietary fiber to various organic acids is a beneficial function provided by the microbiota in the human large intestine. In particular, butyric acid contributes to host health by facilitating maintenance of epithelial integrity, regulating inflammation, and influencing gene expression in colonocytes. We sought to increase the concentration of butyrate in 20 healthy young adults through dietary supplementation with resistant starch (unmodified potato starch—resistant starch (RS) type 2).MethodsFecal samples were collected from individuals to characterize butyrate concentration via liquid chromatography and composition of the microbiota via surveys of 16S rRNA-encoding gene sequences from the Illumina MiSeq platform. Random Forest and LEfSe analyses were used to associate responses in butyrate production to features of the microbiota.ResultsRS supplementation increased fecal butyrate concentrations in this cohort from 8 to 12 mmol/kg wet feces, but responses varied widely between individuals. Individuals could be categorized into three groups based upon butyrate concentrations before and during RS: enhanced, high, and low (n = 11, 3, and 6, respectively). Fecal butyrate increased by 67 % in the enhanced group (from 9 to 15 mmol/kg), while it remained ≥11 mmol/kg in the high group and ≤8 mmol/kg in the low group. Microbiota analyses revealed that the relative abundance of RS-degrading organisms—Bifidobacterium adolescentis or Ruminococcus bromii—increased from ~2 to 9 % in the enhanced and high groups, but remained at ~1.5 % in the low group. The lack of increase in RS-degrading bacteria in the low group may explain why there was no increase in fecal butyrate in response to RS. The microbiota of individuals in the high group were characterized by an elevated abundance of the butyrogenic microbe Eubacterium rectale (~6 % in high vs. 3 % in enhanced and low groups) throughout the study.ConclusionsWe document the heterogeneous responses in butyrate concentrations upon RS supplementation and identify characteristic of the microbiota that appear to underlie this variation. This study complements and extends other studies that call for personalized approaches to manage beneficial functions provided by gut microbiomes.

Dynamics of the fecal microbiome in patients with recurrent and nonrecurrent Clostridium difficile infection.

BackgroundRecurrent Clostridium difficile infection (CDI) remains problematic, with up to 30 % of individuals diagnosed with primary CDI experiencing at least one episode of recurrence. The success of microbial-based therapeutics, such as fecal microbiota transplantation, for the treatment of recurrent CDI underscores the importance of restoring the microbiota. However, few studies have looked at the microbial factors that contribute to the development of recurrent disease. Here we compare microbial changes over time in patients with or without recurrence to identify microbial signatures associated with the development of recurrence.MethodsWe used 16S rRNA-encoding gene sequence analysis to compare the fecal microbiota of 93 patients with recurrent and nonrecurrent CDI, sampled longitudinally. Cross-group and intra-individual differences in microbial community diversity and similarity were compared prior to the development of recurrent disease and over time.ResultsSamples from these patient groups exhibited variable community profiles, clustering into four distinct community groups. Cross-group comparison of the index sample collected from patients that did or did not develop recurrence revealed differences in diversity and community structure (analysis of molecular variance, p < 0.05). Intra-individual comparisons of the microbiota were more informative and samples from recurrent patients were less likely to recover in diversity (Chi-square test, p < 0.005), exhibiting less community similarity overall (Kruskal–Wallis test, p < 0.05). Interestingly, patients with severe disease harbored a significantly less diverse community, a trend that was observed across both nonrecurrent and recurrent patient groups (Wilcoxon test, p < 0.05).ConclusionsTo date, this study represents one of the largest studies focused on the relationship between predictive signals from the gut microbiota and the development of recurrent CDI. Our data demonstrate that specific microbiota-derived characteristics associate with disease severity and recurrence and that future studies could incorporate these characteristics into predictive models.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0298-8) contains supplementary material, which is available to authorized users.

Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

In the 2013–2014 growing season, field surveys were conducted in native corn fields located in high altitude agricultural communities in the ‘Sierra Norte de Puebla’ in Mexico. Symptoms typical of maize bushy stunt (MBS) disease were observed and DNA extracted from symptomatic native corn plants was used as template to confirm the presence of phytoplasmas. Amplification and sequencing of 16S rRNA-encoding sequences and chaperonin 60 universal target (cpn60 UT) sequences followed by in vitro restriction fragment length polymorphism and phylogenetic analyses revealed that the phytoplasma detected belongs to the subgroup 16SrI-B, ‘Candidatus Phytoplasma asteris’. Based on 16S rRNA-encoding gene sequence analysis and on a single nucleotide polymorphism within the cpn60 UT sequence, two MBS strains, MBS-Puebla and MBS-Veracruz, were identified. This is the first detection of MBS phytoplasma (MBSP) affecting native corn and the first molecular survey made in corn fields in Mexico to detect and characterize MBSP. We discuss these results in light of the potential evolutionary relationship between corn and MBSP.

O15.6 Impact of the lng-ius on cervical persistence ofchlamydia trachomatisand vaginal microbiota in a baboon model

Introduction Alterations in vaginal microbiota associated with intrauterine contraception may impact host susceptibility to sexually transmitted infection. We evaluated the effect of the levonorgestrel intrauterine system (LNG-IUS) on cervical persistence of Chlamydia trachomatis (CT) in a baboon model and whether CT persistence was correlated with vaginal microbial community structure. Methods 20 wild caught female olive baboons ( Papio abubis ) were randomly assigned to receive either LNG-IUS and CT inoculation (n = 8), LNG-IUS and sham inoculum (n = 2), CT inoculation alone (n = 8), or sham inoculation (n = 2). Animals were acclimated to the LNG-IUS for 24 weeks after which animals were cervically inoculated once weekly for 5 weeks. Vaginal swabs were collected weekly for microbiome analysis by 16S rRNA-encoding gene sequence analysis. Presence of CT in the cervical epithelium was confirmed with weekly nucleic acid amplification testing (NAAT) and culture. Results Use of the LNG-IUS was significantly associated with positive CT culture (p = 0.04) but not NAAT (p = 0.07). Median time to cervical clearance of CT as detected by NAAT was 12.5 days (range 5–16) for LNG-IUS animals in comparison to 7 days (range 3–10) for non-implanted animals (p = 0.14). Similarly, median time to cervical clearance of CT by culture was 12 days (range 5–15) for LNG-IUS animals and 5 days (range 1–10) for non-implanted animals (p = 0.05). We did not detect significant within group differences between vaginal microbial community structure at baseline and following LNG-IUS insertion, CT inoculation, or LNG-IUS and CT in combination. Conclusions Use of the LNG-IUS was associated with a trend towards cervical persistence of CT in a baboon model. However, this persistence is not explained by alterations in vaginal microbial communities. Disclosure of interest statement The authors have no disclosures to report.

Clostridium difficile Infection.

Clostridium difficile Infection.

Application of a neutral community model to assess structuring of the human lung microbiome.

ABSTRACT DNA from phylogenetically diverse microbes is routinely recovered from healthy human lungs and used to define the lung microbiome. The proportion of this DNA originating from microbes adapted to the lungs, as opposed to microbes dispersing to the lungs from other body sites and the atmosphere, is not known. We use a neutral model of community ecology to distinguish members of the lung microbiome whose presence is consistent with dispersal from other body sites and those that deviate from the model, suggesting a competitive advantage to these microbes in the lungs. We find that the composition of the healthy lung microbiome is consistent with predictions of the neutral model, reflecting the overriding role of dispersal of microbes from the oral cavity in shaping the microbial community in healthy lungs. In contrast, the microbiome of diseased lungs was readily distinguished as being under active selection. We also assessed the viability of microbes from lung samples by cultivation with a variety of media and incubation conditions. Bacteria recovered by cultivation from healthy lungs represented species that comprised 61% of the 16S rRNA-encoding gene sequences derived from bronchoalveolar lavage samples.Importance Neutral distribution of microbes is a distinguishing feature of the microbiome in healthy lungs, wherein constant dispersal of bacteria from the oral cavity overrides differential growth of bacteria. No bacterial species consistently deviated from the model predictions in healthy lungs, although representatives of many of the dispersed species were readily cultivated. In contrast, bacterial populations in diseased lungs were identified as being under active selection. Quantification of the relative importance of selection and neutral processes such as dispersal in shaping the healthy lung microbiome is a first step toward understanding its impacts on host health.

The nasal cavity microbiota of healthy adults

BackgroundThe microbiota of the nares has been widely studied. However, relatively few studies have investigated the microbiota of the nasal cavity posterior to the nares. This distinct environment has the potential to contain a distinct microbiota and play an important role in health.ResultsWe obtained 35,142 high-quality bacterial 16S rRNA-encoding gene sequence reads from the nasal cavity and oral cavity (the dorsum of the tongue and the buccal mucosa) of 12 healthy adult humans and deposited these data in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) (Bioproject: PRJNA248297). In our initial analysis, we compared the bacterial communities of the nasal cavity and the oral cavity from ten of these subjects. The nasal cavity bacterial communities were dominated by Actinobacteria, Firmicutes, and Proteobacteria and were statistically distinct from those on the tongue and buccal mucosa. For example, the same Staphylococcaceae operational taxonomic unit (OTU) was present in all of the nasal cavity samples, comprising up to 55% of the community, but Staphylococcaceae was comparatively uncommon in the oral cavity.ConclusionsThere are clear differences between nasal cavity microbiota and oral cavity microbiota in healthy adults. This study expands our knowledge of the nasal cavity microbiota and the relationship between the microbiota of the nasal and oral cavities.

Biofilm community dynamics in bench-scale annular reactors simulating arrestment of chloraminated drinking water nitrification.

Annular reactors (ARs) were used to study biofilm community succession and provide ecological insight during nitrification arrestment through simultaneously increasing monochloramine (NH2Cl) and chlorine to nitrogen mass ratios, resulting in four operational periods (I-IV). Analysis of 16S rRNA-encoding gene sequence reads (454-pyrosequencing) examined viable and total biofilm communities and found total samples were representative of the underlying viable community. Bacterial community structure showed dynamic changes corresponding with AR operational parameters. Period I (complete nitrification and no NH2Cl residual) was dominated by Bradyrhizobium (total cumulative distribution: 38%), while environmental Legionella-like phylotypes peaked (19%) during Period II (complete nitrification and minimal NH2Cl residual). Nitrospira moscoviensis (nitrite-oxidizing bacteria) was detected in early periods (2%) but decreased to <0.02% in later periods, corresponding to nitrite accumulation. Methylobacterium (19%) and members of Nitrosomonadaceae (42%) dominated Period III (complete ammonia and partial nitrite oxidation and low NH2Cl residual). An increase in Afipia (haloacetic acid-degrading bacteria) relative abundance (<2% to 42%) occurred during Period IV (minimal nitrification and moderate to high NH2Cl residual). Microbial community and operational data provided no evidence of taxa-time relationship, but rapid community transitions indicated that the system had experienced ecological regime shifts to alternative stable states.

Diversity of endophytic bacteria in Malaysian plants as revealed by 16S rRNA encoding gene sequence based method of bacterial identification

Bacterial endophytes do have several potential applications in pharmacy, medicine and agricultural biotech industry. The main objective of this study was to understand types of bacterial endophytes associated with dicotyledonous (dicot) and monocotyledonous (monocot) plant species. Isolation of the endophytic bacteria was performed using surface-sterilized various tissue samples, and identification of the endophytic bacterial isolates (EBIs) was completed using 16S rRNA encoding gene sequence similarity based method. In total, 996 EBIs were isolated and identified from 1055 samples of 31 monocot and 65 dicot plant species from Peninsular Malaysia. The 996 EBIs represented 71 different types of bacterial species. Twelve (12) out of 71 species are reported as endophytes for the first time. We conclude that diverse types of bacterial endophytes are associated with dicot and monocot plants, and could be useful in pharmacy, medicine and agricultural biotechnology for various potential applications.