Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

Chest radiography predictor of COVID-19 adverse outcomes. A lesson learnt from the first wave.

AIM: To assess the role of a severity score based on chest radiography (CXR) in predicting the risk of adverse outcomes in coronavirus disease 2019 (COVID-19). MATERIALS AND METHODS: Of the patients who presented to L. Sacco Hospital (Milan, Italy) between 21 February and 31 March 2020, patients with a laboratory confirmation of COVID-19 who also underwent a CXR were included in the study. To quantify the extent of lung involvement, each CXR image was given a score (Milan score), ranging from 0 to 24, depending on the presence of reticular pattern and/or ground-glass opacities and/or extensive consolidations in each of the 12 areas in which the lungs were divided. The score was calculated by an expert radiologist, blinded to laboratory tests. The ability of the Milan score to predict hospital admission and mortality, after adjusting for some variables (age; gender; comorbidities; time between symptoms onset and admission), using univariate and multivariate statistical analysis was investigated retrospectively. RESULTS: Among the 554 patients, 115 of which (21%) had a negative CXR, the in-hospital mortality was 16% (90/554). At univariate analysis, age, gender, and comorbidities were significant predictors of mortality and hospital admission. At multivariate analysis, adjusting for age and gender, the Milan score was an independent predictor of mortality and hospitalisation. In particular, patients with a Milan score ≥ 9 had a mortality risk five-times higher than those with a lower score. Other independent predictors of mortality were gender and age. CONCLUSIONS: The CXR Milan score was an independent predictive factor of both in-hospital mortality and hospital admission.

An endophytic Kocuria palustris strain harboring multiple arsenate reductase genes.

Aiming at revealing the arsenic (As) resistance of the endophytic Kocuria strains isolated from roots and stems of Sphaeralcea angustifolia grown at mine tailing, four strains belonging to different clades of Kocuria based upon the phylogeny of 16S rRNA genes were screened for minimum inhibitory concentration (MIC). Only the strain NE1RL3 was defined as an As-resistant bacterium with MICs of 14.4/0.0125 mM and 300/20.0 mM for As3+ and As5+, respectively, in LB/mineral media. This strain was identified as K. palustris based upon analyses of cellular chemical compositions (cellular fatty acids, isoprenoides, quinones, and sugars), patterns of carbon source, average nucleotide identity of genome and digital DNA-DNA relatedness. Six genes coding to enzymes or proteins for arsenate reduction and arsenite-bumping were detected in the genome, demonstrating that this strain is resistant to As possibly by reducing As5+ to As3+, and then bumping As3+ out of the cell. However, this estimation was not confirmed since no arsenate reduction was detected in a subsequent assay. This study reported for the first time the presence of phylogenetically distinct arsenate reductase genes in a Kocuria strain and evidenced the possible horizontal transfer of these genes among the endophytic bacteria.

Changes in the Microbial Community of Pinus arizonica Saplings After Being Colonized by the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae).

The death of trees is an ecological process that promotes regeneration, organic matter recycling, and the structure of communities. However, diverse biotic and abiotic factors can disturb this process. Dendroctonus bark beetles (Curculionidae: Scolytinae) are natural inhabitants of pine forests, some of which produce periodic outbreaks, killing thousands of trees in the process. These insects spend almost their entire life cycle under tree bark, where they reproduce and feed on phloem. Tunneling and feeding of the beetles result in the death of the tree and an alteration of the resident microbiota as well as the introduction of microbes that the beetles vector. To understand how microbial communities in subcortical tissues of pines change after they are colonized by the bark beetle Dendroctonus rhizophagus, we compare both the bacterial and fungal community structures in two colonization stages of Pinus arizonica (Arizona pine) employing Illumina MiSeq. Our findings showed significant differences in diversity and the dominance of bacterial community in the two colonization stages with Shannon (P = 0.004) and Simpson (P = 0.0006) indices, respectively, but not in species richness with Chao1 (P = 0.19). In contrast, fungal communities in both stages showed significant differences in species richness with Chao1 (P = 0.0003) and a diversity with Shannon index (P = 0.038), but not in the dominance with the Simpson index (P = 0.12). The ß-diversity also showed significant changes in the structure of bacterial and fungal communities along the colonization stages, maintaining the dominant members in both cases. Our results suggest that microbial communities present in the Arizona pine at the tree early colonization stage by bark beetle change predictably over time.

Comparative Analysis of the Gut Bacterial Community of Four Anastrepha Fruit Flies (Diptera: Tephritidae) Based on Pyrosequencing.

Fruit flies are the most economically important group of phytophagous flies worldwide. Whereas the ecological role of bacteria associated with tephritid fruit fly species of the genera Bactrocera and Ceratitis has been demonstrated, the diversity of the bacterial community in Anastrepha has been poorly characterized. This study represents the first comprehensive analysis of the bacterial community in the gut of larvae and adults of Anastrepha ludens, A. obliqua, A. serpentina, and A. striata using 454 pyrosequencing. A total of four phyla, seven classes, 11 families, and 27 bacterial genera were identified. Proteobacteria was the most represented phylum, followed by Firmicutes, Actinobacteria, and Deinococcus-Thermus. The genera Citrobacter, Enterobacter, Escherichia, Klebsiella, and Raoultella were dominant in all samples analyzed. In general, the bacterial community diversity in adult flies was higher in species with a broader diet breadth than species with a restricted number of hosts, whereas it was also higher in adults versus larvae. Differences in bacterial communities in adults might be determined by the number of fruit species infested. Lastly, the predictive functional profile analysis suggested that community members may participate in metabolic pathways related to membrane transport and metabolism of carbohydrates, amino acids, cofactors, and lipids. These results provide the basis for the study of unexplored functional roles of bacteria in this insect group.

Gut Bacterial Communities of Dendroctonus valens and D. mexicanus (Curculionidae: Scolytinae): A Metagenomic Analysis across Different Geographical Locations in Mexico.

Dendroctonus bark beetles are a worldwide significant pest of conifers. This genus comprises 20 species found in North and Central America, and Eurasia. Several studies have documented the microbiota associated with these bark beetles, but little is known regarding how the gut bacterial communities change across host range distribution. We use pyrosequencing to characterize the gut bacterial communities associated with six populations of Dendroctonus valens and D. mexicanus each across Mexico, determine the core bacteriome of both insects and infer the metabolic pathways of these communities with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to evaluate whether these routes are conserved across geographical locations. Our results show that the ß-diversity with UniFrac unweighted varies among locations of both bark beetles mainly due to absence/presence of some rare taxa. No association is found between the pairwise phylogenetic distance of bacterial communities and geographic distance. A strict intraspecific core bacteriome is determined for each bark beetle species, but these cores are different in composition and abundance. However, both bark beetles share the interspecific core bacteriome recorded previously for the Dendroctonus genus consisting of Enterobacter, Pantoea, Providencia, Pseudomonas, Rahnella, and Serratia. The predictions of metabolic pathways are the same in the different localities, suggesting that they are conserved through the geographical locations.

Massilia violacea sp. nov., isolated from riverbank soil.

A bacterial strain designated CAVIOT was isolated during the course of a study of culturable bacteria in a riverbank soil sample from Tlaxcala, Mexico. The strain was subjected to a polyphasic taxonomic characterization. Strain CAVIOT was aerobic, Gram-stain-negative, non-spore-forming and rod-shaped. Colonies grown on R2A agar at 28 °C were pale violet, mucoid, rounded, smooth and glossy. The strain was motile and catalase- and oxidase-positive, and maximum growth temperature was 35 °C. Strain CAVIOT was classified within the genus Massilia as its 16S rRNA gene sequence was closely related to those of Massilia umbonata LP01T (97.5 % similarity), Massilia dura 16T (97.2 %) and Massilia plicata 76T (97.1 %). The predominant respiratory quinone was Q8. The major fatty acids were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c). The predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and an unknown phospholipid. The DNA G+C content was 65.0 mol% (Tm). DNA-DNA hybridization results showed values below 25 % with respect to the type strains of the closest related species. Therefore, strain CAVIOT can be differentiated from previously described species of the genus Massilia and represents a novel species, for which the name Massilia violacea sp. nov. is proposed. The type strain is CAVIOT ( = CECT 8897T = LMG 28941T).

Helicobacter pylori Infection in Children. Antimicrobial Resistance and Treatment Response.

BACKGROUND: The aim of this study was to determine the appropriateness of the recent recommendations for managing Helicobacter pylori infection in children in a university hospital in Southern Europe. Antimicrobial resistance and response to eradication therapy were also determined. MATERIALS AND METHODS: The presence of H. pylori was studied in 143 children: by gastric biopsy culture (GBC), (13)C-urea breath test (UBT) and stool antigen immunochromatography test (SAIT) in 56 children; by GBC and UBT in 20, by GBC and SAIT in 18, and by GBC alone in 49. Antimicrobial susceptibility was determined by E-test. Infection was defined as a positive culture or positivity in both UBT and SAIT. Disease progression was studied in 118 patients. First evaluation of symptoms was carried out at 3-6 months after diagnosis and/or after treatment of the infection. RESULTS: H. pylori was detected in 74 from the 143 children analyzed (100% GBC positive, 98.1% UBT positive, and 58.1% SAIT positive). The main symptom was chronic abdominal pain (n = 121). Macroscopic antral nodularity was observed in 29.7% of infected patients and in 5.8% of uninfected patients, respectively. Resistance to clarithromycin and metronidazole was found in 34.7 and 16.7%, respectively. Eradication when susceptible antimicrobials were used occurred in 78.7% (48/61) versus 37.5% (3/8) when the treatment included a drug with resistance (p = .024). In patients with recurrent abdominal pain, symptoms resolved in 92.9% (39/42) patients with HP eradication versus 42.9% (6/14) without HP eradication (p < .001). CONCLUSION: Treated patients often failed to meet the criteria established in the guidelines for H. pylori diagnostic screening and treatment because most of them had only recurrent abdominal pain, but remission of their symptoms was associated with H. pylori eradication.

Comparative metabarcoding and biodiversity of gut-associated fungal assemblages of Dendroctonus species (Curculionidae: Scolytinae).

The genus Dendroctonus is a Holarctic taxon composed of 21 nominal species; some of these species are well known in the world as disturbance agents of forest ecosystems. Under the bark of the host tree, these insects are involved in complex and dynamic associations with phoretic ectosymbiotic and endosymbiotic communities. Unlike filamentous fungi and bacteria, the ecological role of yeasts in the bark beetle holobiont is poorly understood, though yeasts were the first group to be recorded as microbial symbionts of these beetles. Our aim was characterize and compare the gut fungal assemblages associated to 14 species of Dendroctonus using the internal transcribed spacer 2 (ITS2) region. A total of 615,542 sequences were recovered yielding 248 fungal amplicon sequence variants (ASVs). The fungal diversity was represented by 4 phyla, 16 classes, 34 orders, 54 families, and 71 genera with different relative abundances among Dendroctonus species. The α-diversity consisted of 32 genera of yeasts and 39 genera of filamentous fungi. An analysis of ß-diversity indicated differences in the composition of the gut fungal assemblages among bark beetle species, with differences in species and phylogenetic diversity. A common core mycobiome was recognized at the genus level, integrated mainly by Candida present in all bark beetles, Nakazawaea, Cladosporium, Ogataea, and Yamadazyma. The bipartite networks confirmed that these fungal genera showed a strong association between beetle species and dominant fungi, which are key to maintaining the structure and stability of the fungal community. The functional variation in the trophic structure was identified among libraries and species, with pathotroph-saprotroph-symbiotroph represented at the highest frequency, followed by saprotroph-symbiotroph, and saprotroph only. The overall network suggested that yeast and fungal ASVs in the gut of these beetles showed positive and negative associations among them. This study outlines a mycobiome associated with Dendroctonus nutrition and provides a starting point for future in vitro and omics approaches addressing potential ecological functions and interactions among fungal assemblages and beetle hosts.

A polyphasic taxonomy analysis reveals the presence of an ecotype of Rahnella contaminans associated with the gut of Dendroctonus-bark beetles.

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, ß and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.

Metabarcoding of mycetangia from the Dendroctonus frontalis species complex (Curculionidae: Scolytinae) reveals diverse and functionally redundant fungal assemblages.

Dendroctonus-bark beetles are associated with microbes that can detoxify terpenes, degrade complex molecules, supplement and recycle nutrients, fix nitrogen, produce semiochemicals, and regulate ecological interactions between microbes. Females of some Dendroctonus species harbor microbes in specialized organs called mycetangia; yet little is known about the microbial diversity contained in these structures. Here, we use metabarcoding to characterize mycetangial fungi from beetle species in the Dendroctonus frontalis complex, and analyze variation in biodiversity of microbial assemblages between beetle species. Overall fungal diversity was represented by 4 phyla, 13 classes, 25 orders, 39 families, and 48 genera, including 33 filamentous fungi, and 15 yeasts. The most abundant genera were Entomocorticium, Candida, Ophiostoma-Sporothrix, Ogataea, Nakazawaea, Yamadazyma, Ceratocystiopsis, Grosmannia-Leptographium, Absidia, and Cyberlindnera. Analysis of α-diversity indicated that fungal assemblages of D. vitei showed the highest richness and diversity, whereas those associated with D. brevicomis and D. barberi had the lowest richness and diversity, respectively. Analysis of ß-diversity showed clear differentiation in the assemblages associated with D. adjunctus, D. barberi, and D. brevicomis, but not between closely related species, including D. frontalis and D. mesoamericanus and D. mexicanus and D. vitei. A core mycobiome was not statistically identified; however, the genus Ceratocystiopsis was shared among seven beetle species. Interpretation of a tanglegram suggests evolutionary congruence between fungal assemblages and species of the D. frontalis complex. The presence of different amplicon sequence variants (ASVs) of the same genus in assemblages from species of the D. frontalis complex outlines the complexity of molecular networks, with the most complex assemblages identified from D. vitei, D. mesoamericanus, D. adjunctus, and D. frontalis. Analysis of functional variation of fungal assemblages indicated multiple trophic groupings, symbiotroph/saprotroph guilds represented with the highest frequency (∼31% of identified genera). These findings improve our knowledge about the diversity of mycetangial communities in species of the D. frontalis complex and suggest that minimal apparently specific assemblages are maintained and regulated within mycetangia.

Rahnella sp., a Dominant Symbiont of the Core Gut Bacteriome of Dendroctonus Species, Has Metabolic Capacity to Degrade Xylan by Bifunctional Xylanase-Ferulic Acid Esterase.

Rahnella sp. ChDrAdgB13 is a dominant member of the gut bacterial core of species of the genus Dendroctonus, which is one of the most destructive pine forest bark beetles. The objectives of this study were identified in Rahnella sp. ChDrAdgB13 genome the glycosyl hydrolase families involved in carbohydrate metabolism and specifically, the genes that participate in xylan hydrolysis, to determine the functionality of a putative endo-1,4-ß-D-xylanase, which results to be bifunctional xylanase-ferulic acid esterase called R13 Fae and characterize it biochemically. The carbohydrate-active enzyme prediction revealed 25 glycoside hydrolases, 20 glycosyl transferases, carbohydrate esterases, two auxiliary activities, one polysaccharide lyase, and one carbohydrate-binding module (CBM). The R13 Fae predicted showed high identity to the putative esterases and glycosyl hydrolases from Rahnella species and some members of the Yersiniaceae family. The r13 fae gene encodes 393 amino acids (43.5 kDa), containing a signal peptide, esterase catalytic domain, and CBM48. The R13 Fae modeling showed a higher binding affinity to ferulic acid, α-naphthyl acetate, and arabinoxylan, and a low affinity to starch. The R13 Fae recombinant protein showed activity on α-naphthyl acetate and xylan, but not on starch. This enzyme showed mesophilic characteristics, displaying its optimal activity at pH 6.0 and 25°C. The enzyme was stable at pH from 4.5 to 9.0, retaining nearly 66-71% of its original activity. The half-life of the enzyme was 23 days at 25°C. The enzyme was stable in the presence of metallic ions, except for Hg2+. The products of R13 Fae mediated hydrolysis of beechwood xylan were xylobiose and xylose, manifesting an exo-activity. The results suggest that Rahnella sp. ChDrAdgB13 hydrolyze xylan and its products could be assimilated by its host and other gut microbes as a nutritional source, demonstrating their functional role in the bacterial-insect interaction contributing to their fitness, development, and survival.

Phylogenetic and multivariate analyses to determine the effects of different tillage and residue management practices on soil bacterial communities.

Bacterial communities are important not only in the cycling of organic compounds but also in maintaining ecosystems. Specific bacterial groups can be affected as a result of changes in environmental conditions caused by human activities, such as agricultural practices. The aim of this study was to analyze the effects of different forms of tillage and residue management on soil bacterial communities by using phylogenetic and multivariate analyses. Treatments involving zero tillage (ZT) and conventional tillage (CT) with their respective combinations of residue management, i.e., removed residue (-R) and kept residue (+R), and maize/wheat rotation, were selected from a long-term field trial started in 1991. Analysis of bacterial diversity showed that soils under zero tillage and crop residue retention (ZT/+R) had the highest levels of diversity and richness. Multivariate analysis showed that beneficial bacterial groups such as fluorescent Pseudomonas spp. and Burkholderiales were favored by residue retention (ZT/+R and CT/+R) and negatively affected by residue removal (ZT/-R). Zero-tillage treatments (ZT/+R and ZT/-R) had a positive effect on the Rhizobiales group, with its main representatives related to Methylosinus spp. known as methane-oxidizing bacteria. It can be concluded that practices that include reduced tillage and crop residue retention can be adopted as safer agricultural practices to preserve and improve the diversity of soil bacterial communities.

Seasonal changes in the bacterial community structure of three eutrophicated urban lakes in Mexico city, with emphasis on Microcystis spp.

Artificial urban lakes commonly have physicochemical conditions that contribute to rapid anthropogenic eutrophication and development of cyanobacterial blooms. Microcystis is the dominat genus in most freshwater bodies and is one of the main producter of microcystins. Using 454-pyrosequencing we characterized the bacterial community, with special emphasis on Microcystis, in three recreational urban lakes from Mexico City in both wet and dry seasons. We also evaluated some physicochemical parameters that might influence the presence of Microcystis blooms, and we associated the relative abundance of heterotrophic and autotrophic bacterial communities with their possible metabolic capacities. A total of 14 phyla, 18 classes, 39 orders, 53 families and 48 bacterial genera were identified in both seasons in the three urban lakes. Cyanobacteria had the highest relative abundance followed by Proteobacteria and Actinobacteria. Microcystis was the dominant taxon followed by Arthrospira, Planktothrix and Synechococcus. We also found heterotrophic bacteria associated with the blooms, such as Rhodobacter, Pseudomonas, Sphingomonas and, Porphyrobacter. The highest richness, diversity and dominance were registered in the bacterial community of the Virgilio Uribe Olympic Rowing-Canoeing Track in both seasons, and the lowest values were found in the Chapultepec Lake. The canonical correspondence analysis showed that dissolved oxygen and NO3-N concentrations might explain the presence of Microcystis blooms. The metabolic prediction indicated that these communities are involved in photosynthesis, oxidative phosphorylation, methane metabolism, carbon fixation, and nitrogen and sulfur metabolism. The lakes studied had a high prevalence of Microcystis, but average values of microcystins did not exceed the maximum permissible level established by the United States Environmental Protection Agency for recreational and cultural activities. The presence of cyanobacteria and microcystins at low to moderate concentrations in the three lakes could result in ecosystem disruption and increase animal and human health risks.

Primary hyperparathyroidism: can ultrasonography be the only preoperative diagnostic procedure?

PURPOSE: The purpose of our study was to assess the role of ultrasonography (US) before surgical treatment of primary hyperparathyroidism. MATERIALS AND METHODS: We retrospectively evaluated 77 patients (60 women, 17 men; mean age 59 years) with primary hyperparathyroidism who underwent parathyroid US prior to surgery. Sixty-five of 77 (84%) patients had undergone (99m)Tc- sestamibi (MIBI) scintigraphy. The results were correlated with the surgical and histopathological findings. RESULTS: Surgery revealed 85 abnormal parathyroid glands in 77 patients (70 adenomas, 15 hyperplasias). The locations of the parathyroid glands were typical cervical (n=77), thyrothymic ligament (n=3), carotid sheath (n=2), and mediastinum (n=3). In two patients, intrathyroid microadenoma was diagnosed by histopathology. Seventy-four enlarged glands in 64 patients were correctly identified at US. Per-patient sensitivity and positive predictive values, respectively, were 84% (64/76) and 99% (64/65) for US, 68% (44/65) and 100% (44/44) for scintigraphy and 91% (59/65) and 98% (59/60) for both techniques combined. We weighed 63 out of 85 glands, obtaining a value of 1,004+/-1,564 mg; 460 mg (mean+/-standard deviation; median). CONCLUSIONS: Preoperative detection and localisation of enlarged parathyroid glands can be based on US, an inexpensive and widely available method, limiting the use of scintigraphy to those cases with negative and/or doubtful findings on US.

An Overview of Genes From Cyberlindnera americana, a Symbiont Yeast Isolated From the Gut of the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae), Involved in the Detoxification Process Using Genome and Transcriptome Data.

Bark beetles from Dendroctonus genus promote ecological succession and nutrient cycling in coniferous forests. However, they can trigger outbreaks leading to important economic losses in the forest industry. Conifers have evolved resistance mechanisms that can be toxic to insects but at the same time, bark beetles are capable of overcoming tree barriers and colonize these habitats. In this sense, symbiont yeasts present in the gut of bark beetles have been suggested to play a role in the detoxification process of tree defensive chemicals. In the present study, genes related to this process were identified and their response to a terpene highly toxic to bark beetles and their symbionts was analyzed in the Cyberlindnera americana yeast. The genome and transcriptome of C. americana (ChDrAdgY46) isolated from the gut of Dendroctonus rhizophagus were presented. Genome analysis identified 5752 protein-coding genes and diverse gene families associated with the detoxification process. The most abundant belonged to the Aldo-Keto Reductase Superfamily, ATP-binding cassette Superfamily, and the Major Facilitator Superfamily transporters. The transcriptome analysis of non-α-pinene stimulated and α-pinene stimulated yeasts showed a significant expression of genes belonging to these families. The activities demonstrated by the genes identified as Aryl-alcohol dehydrogenase and ABC transporter under (+)-α-pinene suggest that they are responsible, that C. americana is a dominant symbiont that resists high amounts of monoterpenes inside the gut of bark beetles.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa trivialis in Florida.

Brown ring patch is a newly described disease of cool-season turfgrass first reported in Japan on creeping bentgrass (Agrostis palustris) (2) and later reported in California on annual bluegrass (Poa annua) (1). The disease is characterized by either patches or rings of discolored to blighted turfgrass that can range from a few centimeters to a meter in diameter. Affected turfgrass plants turn chlorotic and can be blighted from the crown to the leaf tips. Blight symptoms have been associated with fluffy white-to-cream aerial mycelium after extended incubation of the sample. Symptoms including patches of blighted turfgrass approximately 10 cm in diameter were observed on roughstalk bluegrass (Poa trivialis) that had been overseeded onto a dormant 'Tifdwarf' bermudagrass (Cynodon dactylon) putting green in Palatka, FL. A sample was submitted by the superintendent in June 2005 because symptoms were confused with dollar spot and a fungicide resistance issue was suspected. The sample produced abundant aerial mycelium after incubation. The pathogen was isolated on potato dextrose agar amended with rifampicin (100 ppm) and streptomycin (100 ppm) from Poa plants surface disinfested with 70% ethanol for 30 s. Colony and sclerotia morphology were consistent with Waitea circinata var. circinata as previously described (1,2). The teleomorph W. circinata var. circinata was not observed on plant material or culture plates. Amplified fragments of rDNA including internal transcribed spacers from the isolate were sequenced bidirectionally from four bacterial clones. The consensus sequences (GenBank Accession Nos. FJ029103, FJ029104, FJ029105, and FJ029106) matched with 99% homology (99% sequence overlap) isolate TRGC1.1 of W. circinata var. circinata described by Wong, NCBI Accession No. DQ900586 (1). Pots of 'Cypress' roughstalk bluegrass that were 1 week postemergence were inoculated with the pathogen using 10 infested wheat grains. Plants were incubated at 25°C in a sealed plastic bag with a moist paper towel in the bottom. Hyphae grew from the grains and colonized the grass. Individual plants began to turn chlorotic within 3 days and greater than 90% of the turf in pots was dead after 1 week. The fungus was reisolated from affected plants. Control pots were inoculated with uninfested wheat grains and showed no disease symptoms after 1 week. Inoculations were repeated twice more with the same results. To our knowledge, this is the first report of brown ring patch on P. trivialis in Florida. References: (1) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (2) T. Toda et al. Plant Dis. 89:536, 2005.

Determination of organochlorine compounds in neuston from the Mediterranean.

A reliable analytical procedure was developed to determine trace levels of organochlorine compounds in neuston samples. Freeze-dried samples were extracted by sonication (3 x 15 ml cyclohexane, 15 min) followed by gel permeation chromatography (GPC) and gas chromatography electron capture detection (GC-ECD) determination. Neuston samples may present great differences in their lipid content, which may become a drawback for the analytical determination of hydrophobic pollutants. In this way, GPC fractionation combined with Florisil cleanup was successfully used to avoid potential lipidic interferences in the GC-ECD determination. Organochlorine compounds were determined by GC-ECD and a standard addition method was performed to evaluate the recoveries of 41 PCB individual congeners (80 +/- 20%). The congeners with lower K(ow) (octanal water partition coeeficient) values showed lower recoveries (54 +/- 7%) than the more lipophylic ones (80 +/- 7%). The limits of detection ranged from 0.001 to 0.211 ng g(-1) of wet samples. The reproducibility of the developed analytical methodology for independent replicates (n = 3) at low pg g(-1) levels was quite satisfactory (RSD 18 +/- 10%).

Bacteriome from Pinus arizonica and P. durangensis: Diversity, Comparison of Assemblages, and Overlapping Degree with the Gut Bacterial Community of a Bark Beetle That Kills Pines.

Symbioses between plants and microorganims have been fundamental in the evolution of both groups. The endophytic bacteria associated with conifers have been poorly studied in terms of diversity, ecology, and function. Coniferous trees of the genera Larix, Pseudotsugae, Picea and mainly Pinus, are hosts of many insects, including bark beetles and especially the Dendroctonus species. These insects colonize and kill these trees during their life cycle. Several bacteria detected in the gut and cuticle of these insects have been identified as endophytes in conifers. In this study, we characterized and compared the endophytic bacterial diversity in roots, phloem and bark of non-attacked saplings of Pinus arizonica and P. durangensis using 16S rRNA gene pyrosequencing. In addition, we evaluated the degree of taxonomic relatedness, and the association of metabolic function profiles of communities of endophytic bacteria and previously reported gut bacterial communities of D. rhizophagus; a specialized bark beetle that colonizes and kills saplings of these pine species. Our results showed that both pine species share a similar endophytic community. A total of seven bacterial phyla, 14 classes, 26 orders, 43 families, and 51 genera were identified. Enterobacteriaceae was the most abundant family across all samples, followed by Acetobacteraceae and Acidobacteriaceae, which agree with previous studies performed in other pines and conifers. Endophytic communities and that of the insect gut were significantly different, however, the taxonomic relatedness of certain bacterial genera of pines and insect assemblages suggested that some bacteria from pine tissues might be the same as those in the insect gut. Lastly, the metabolic profile using PICRUSt showed there to be a positive association between communities of both pines and insect gut. This study represents the baseline into the knowledge of the endophytic bacterial communities of two of the major hosts affected by D. rhizophagus.