Identification and characterization of the EXPA7, EXPA18 and EXT10 genes in Turbinicarpus lophophoroides (Werderm.) Buxb. & Backeb; and their expression analysis in the root under abiotic stress.

Expansin and extensin are proteins involved in resistance to various abiotic stresses by processes of cell wall modification and in the formation and elongation of the hairy root. They are located in several organs of the plant included root epidermis. Turbinicarpus lophophoroides is a cactus model to studies these genes in adventitious and transformed roots. In this study, we identified and characterized the expansin7, expansin18 and extensin10 genes in T. lophophoroides. Bioinformatic analysis indicated that the expansin sequences contained the motifs: HTFYG, HFD, YRR, VPC and YW; and certain conserved cysteine (C) residues. Regarding extensin10, the sequence contains the conserved SPPPP (SP4), YYS and YV motifs. The expression analysis in adventitious and transformed roots under osmotic stress (300 mM mannitol), heat (37 °C) and cold (4 °C); shows a higher expression of TlExpA18 in both roots, a decrease in TlExpA7 in transformed roots and a null expression in TlExt10 in both roots. In addition, a morphological comparison of the maturation/differentiation zone, meristem and cap between adventitious and transformed roots by SEM was performed, finding differences in the quantity and length of the hairy roots and the shape of the root cap. Overall, the study concluded that TlExpA18 and TlExpA7 belong to expansin family and TlExt10 belong to extensin family. The expression characteristics of TlExpA18, TlExpA7 and TlExt10 will facilitate the investigation of its function in stress response and other physiological processes in T. lophophoroides.

Predictive factors of COVID-19 in patients with negative RT-qPCR.

OBJECTIVE: To evaluate the factors associated with false negatives in RT-qPCR in patients with mild-moderate symptoms of COVID-19. MATERIALS AND METHODS: This was a cross-sectional study that used a random sample of non-hospitalized patients from the primary care management division of the Healthcare Area of Leon (58 RT-qPCR-positive cases and 52 RT-qPCR-negative cases). Information regarding symptoms was collected and all patients were simultaneously tested using two rapid diagnostic tests - RDTs (Combined - cRDT and Differentiated - dRDT). The association between symptoms and SARS-CoV-2 infection was evaluated by non-conditional logistic regression, with estimation of Odds Ratio. RESULTS: A total of 110 subjects were studied, 52% of whom were women (mean age: 48.2±11.0 years). There were 42.3% of negative RT-qPCRs that were positive in some RDTs. Fever over 38°C (present in 35.5% of cases) and anosmia (present in 41.8%) were the symptoms most associated with SARS-CoV-2 infection, a relationship that remained statistically significant in patients with negative RT-qPCR and some positive RDT (aOR=6.64; 95%CI=1.33-33.13 and aOR=19.38; 95% CI=3.69-101.89, respectively). CONCLUSIONS: RT-qPCR is the technique of choice in the diagnosis of SARS-CoV-2 infection, but it is not exempt from false negatives. Our results show that patients who present mild or moderate symptoms with negative RT-qPCR, but with fever and/or anosmia, should be considered as suspicious cases and should be evaluated with other diagnostic methods.

Prevalence of SARS-CoV-2 infection in general practitioners and nurses in primary care and nursing homes in the Healthcare Area of León and associated factors.

OBJECTIVE: To evaluate the prevalence of and factors associated with SARS-CoV-2 infection in general practitioners and nurses from primary care centers and nursing homes in the Healthcare Area of León (Spain). MATERIALS AND METHODS: Cross-sectional study in a convenience sample of professionals from 30 health centers and 30 nursing homes from the primary care management division of the Healthcare Area of Leon. The work center, type of profession, COVID-19 infection, level of exposure, compliance with preventive measures, isolation (if required) and diagnostic tests carried out were collected. The determination of infection was made by differentiated rapid diagnostic test (dRDT), using a finger-stick whole-blood sample. The association of variables with infection was assessed by multivariable non-conditional logistic regression. The true prevalence of SARS-CoV-2 infection was calculated according to two scenarios for RDT (Sensitivity=0.6 and Specificity=0.985; Sensitivity=0.8 and Specificity=1). RESULTS: The true prevalence of SARS-CoV-2 infection was between 4.9% and 11.0%. The observed prevalence was 5.9% and was higher in nursing homes than in primary care centers (9.5% vs. 5.5%). No statistically significant differences were observed by sex, type of professional, level of exposure or compliance with preventive measures. CONCLUSIONS: The prevalence of SARS-CoV-2 infection in this group is low. A high number of professionals remain susceptible to SARS-CoV-2 infection and therefore protective measures should be taken, especially for professionals working in nursing homes.

[Effect of intensive nutritional intervention in patients refractory to weight loss]. / Efecto de una intervención nutricional intensiva en pacientes refractarios a la pérdida de peso.

OBJECTIVE: To assess the effect of an intensive nutritional intervention on the body weight and waist circumference in adults refractory to weight loss, by applying a personalised low-calorie Mediterranean diet. MATERIAL AND METHODS: A prospective study was conducted for 6 months on 100 participants with an age range between 55 and 75 years, a BMI ≥ 27 and < 40 kg/my, and fulfilled 3 or more criteria of metabolic syndrome. The intervention consisted of prescribing a low-calorie Mediterranean diet, exercise recommendations, and motivational talks. Anthropometric variables were recorded 11a month during the entire intervention. An analysis was made of the results using the Chi-squared and Student-t tests. The Odds Ratio of the variables associated with weight loss and their 95% confidence intervals was calculated using a non-conditional logistic regression. RESULTS: After the "rescue" intervention, the patients lost an average of 2.9% of the body weight and 2.1% of waist circumference, the target of the loss ≥ 3% of the weight and 26% of the sample the target of reduction ≥ 3% of waist circumference being achieved 38% of the sample. No statistically significant differences were observed in weight and waist circumference loss in any of the variables analysed. CONCLUSIONS: Intensive intervention, based on a low-calorie Mediterranean diet, with recommendations of physical activity and motivational talks, achieved a moderate weight loss in patients refractory to treatments for obesity.

Chronic intake of moderate fat-enriched diet induces fatty liver and low-grade inflammation without obesity in rabbits.

Non-Alcoholic Fatty Liver Disease (NAFLD) is the cause of chronic liver disease. Even though NAFLD is strongly associated with obesity and metabolic syndrome, there is a proportion of patients who develop this condition in the absence of obesity and the underlying mechanisms are poorly understood. We investigated early events in the pathogenesis of non-obese NAFLD, analyzing the impact of the chronic intake of a moderate fat-enriched diet on hepatic lipid accumulation and their relationship with inflammation. Rabbits fed with a moderate Fatty-Acid- Enriched Diet 3% palmitic acid (FAED), were evaluated for body weight, biochemical parameters, and liver function. Liver samples were analyzed by histology and RT-qPCR to measure lipid accumulation, the expression of inflammation-related genes IL-1ß, IL-6, IL-10, IL-13, IL-18, COX-2, TNF-α, and TLR-4. Chronic consumption by 6-months of FAED did not generate metabolic changes, but it induced fatty liver. We also observed the development of low-grade inflammation characterized by the up regulation of TNF-α, IL-13 and IL-18. The consumption by 12-months of FAED caused the overexpression of IL-6, IL-10, IL-13, COX-2, and TLR-4. We show that hepatic steatosis is an early consequence of fat-enriched diets, and that it is accompanied by an immune response that exerts protective effects that prevent the development of metabolic disorders, such as overweight/obesity and metabolic syndrome. However, the excessive intake of fatty acids renders these mechanisms less efficient for delaying the start of metabolic alterations. Rabbits fed with FAED can be used as a model of NAFLD in non-obese and obese groups, especially at early stages of the disease.

Evaluation of the impact of live pig trade network, vaccination coverage and socio-economic factors in the classical swine fever eradication program in Peru.

Classical swine fever (CSF) is a viral infectious disease of swine with significant economic impact in the affected countries due to the limitation of trade, culling of infected animals and production losses. In Latin America, CSF is endemic in several countries including Ecuador, Bolivia, Brazil and Peru. Since 2010, the National Veterinary Services of Peru have been working to better control and eradicate the disease with an intensive vaccination program. The aim of this study was to evaluate the effectiveness of the vaccination program and determine which factors are still contributing to the persistence of the disease in certain regions of Peru. We integrated the data from the vaccination campaign, the live pig movement network and other socioeconomic indicators into a multilevel logistic regression model to evaluate their association with CSF occurrence at district level. The results revealed that high vaccination coverage significantly reduces the risk of CSF occurrence (OR = 0.07), supporting the effectiveness of the vaccination program. Districts belonging to large and medium pig trade network communities (as identified with walktrap algorithm) had higher probability to CSF occurrence (OR = 2.83 and OR = 5.83, respectively). The human development index (HDI) and the presence of a slaughterhouse in the district was also significantly associated with an increased likelihood of CSF occurrence (OR = 1.52 and OR = 3.25, respectively). Districts receiving a high proportion of the movements from districts that were infected in the previous year were also at higher risk of CSF occurrence (OR = 3.30). These results should be useful to guide the prioritization of vaccination strategies and may help to design other intervention strategies (e.g., target education, movement restrictions, etc.) in high-risk areas to more rapidly advance in the eradication of CSF in Peru.

Ambient Computing to Support the Association of Contextual Cues with Medication Taking / Cómputo Ambiental para Facilitar la Asociación de Pistas Contextuales con la Toma de Medicamento

The most common reason for non-adherence to medication among older adults is forgetfulness. Contextual cues, such as daily routines, serve as implicit situational information that increases the retrieval process of the intended action. The main contribution is an overview of the process and the technical details of Ambient Computing displays we developed to help seniors use contextual cues to remember actions associated with medication intake (i.e. remember to take medications or remember having taken them earlier). Through a qualitative study, we obtained evidence about the potential of our technological approach to make seniors more responsible and independent for taking medications.

Las razones más comunes para que adultos mayores no se apeguen a la medicación es el olvido. Las rutinas de vida diaria sirven como pistas contextuales que mejoran el proceso cognitivo relacionado con recordar realizar una acción planeada. Nuestro propósito es presentar el proceso de desarrollo, así como detalles técnicos, de Sistemas de Cómputo Ambiental que proveen pistas contextuales al adulto mayor para ayudarle a recordar acciones de su medicación (e.g., recordar medicarse o recordar que se medicaron). Mediante un estudio cualitativo, obtuvimos evidencia del potencial de nuestra tecnología para que el adulto mayor sea más responsable e independiente para medicarse.

First Report of Fusarium temperatum Causing Seedling Blight and Stalk Rot on Maize in Spain.

In Europe, several diseases of maize (Zea mays L.) including seedling blight and stalk rot are caused by different Fusarium species, mainly Fusarium graminearum, F. verticillioides, F. subglutinans, and F. proliferatum (3). In recent years, these Fusarium spp. have received significant attention not only because of their impact on yield and grain quality, but also for their association with mycotoxin contamination of maize kernels (1,4). From October 2011 to October 2012, surveys were conducted in a maize plantation located in Galicia (northwest Spain). In each sampling, 100 kernels and 10 maize stalks were collected from plants exhibiting symptoms of ear and stalk rot. Dried kernels and small stalk pieces (1 to 2 cm near the nodes) were placed onto potato dextrose agar medium and incubated in the dark for 7 days. Fungal colonies displaying morphological characteristics of Fusarium spp. (2) were subcultured as single conidia onto SNA (Spezieller Nahrstoffarmer agar) (2) and identified by morphological characteristics, as well as by DNA sequence analysis. A large number of Fusarium species (F. verticillioides, F. subglutinans, F. graminearum, and F. avenaceum) (1,2) were identified. These Fusarium species often cause ear and stalk rot on maize. In addition, a new species, F. temperatum, recently described in Belgium (3), was also identified. F. temperatum is within the Gibberella fujikuroi species complex and is morphologically and phylogenetically closely related to F. subglutinans (2,3). Similar to previous studies (3), our isolates were characterized based on the presence of white cottony mycelium, becoming pinkish white. Conidiophores were erect, branched, and terminating in 1 to 3 phialides. Microconidia were abundant, hyaline, 0 to 2 septa; ellipsoidal to oval, produced singly or in false heads, and on monophialides, intercalary phialides, and polyphialides. Microconidia were not produced in chains. No chlamydospores were observed (3). Macroconidia in carnation leaf agar medium (2) were hyaline, 3 to 6 septate, mostly 4, falcate, with a distinct foot-like basal cell (2,3). DNA was amplified with primers ITS1/ITS4 and EF1/EF2 (3). Partial sequences of gene EF-1α showed 100% homology with F. temperatum (3) (GenBank Accession Nos. HM067687 and HM067688). DNA sequences of EF-1α gene and ITS region obtained were deposited in GenBank (KC179824, KC179825, KC179826, and KC179827). Pathogenicity of one representative isolate was confirmed using a soil inoculation method adapted from Scauflaire et al., 2012 (4). F. temperatum isolate was cultured on sterile wheat grains. Colonized wheat grains (10 g) were mixed with sterilized sand in 10 cm diameter pots. Ten kernels per pot were surface disinfected in 2% sodium hypochlorite for 10 min, rinsed with sterilized water, drained (4), placed on the soil surface, and covered with a 2 cm layer of sterilized sand. Five pots were inoculated and five uninoculated controls were included. Pots were maintained at 22 to 24°C and 80% humidity for 30 days. Seedling malformations, chlorosis, shoot reduction, and stalk rot were observed on maize growing in inoculated soil and not from controls. F. temperatum was reisolated from the inoculated seedlings but not from the controls. References: (1) B. J. Bush et al. Phytopathology 94:88, 2003. (2) J. F. Leslie et al. The Fusarium Laboratory Manual, page 388. Blackwell Publishing, 2006. (3) J. Scauflaire et al. Mycologia 103:586, 2011. (4) J. Scauflaire et al. Eur. J. Plant Pathol. 133:911, 2012.

How Important is Working with an Ordered Electrode to Improve the Charge Collection Efficiency in Nanostructured Solar Cells?

The collection efficiency of carriers in solar cells based on nanostructured electrodes is determined for different degrees or morphological one-dimensional order. The transport process is modeled by random walk numerical simulation in a mesoporous electrode that resembles the morphology of nanostructured TiO2 electrodes typically used in dye-sensitized solar cells and related systems. By applying an energy relaxation procedure in the presence of an external potential, a preferential direction is induced in the system. It is found that the partially ordered electrode can almost double the collection efficiency with respect to the disordered electrode. However, this improvement depends strongly on the probability of recombination. For too rapid or too slow recombination, working with partially ordered electrodes will not be beneficial. The computational method utilized here makes it possible to relate the charge collection efficiency with morphology. The collection efficiency is found to reach very rapidly a saturation value, meaning that, in the region of interest, a slight degree of ordering might be sufficient to induce a large improvement in collection efficiency.

First Report of Phytophthora alni subsp. uniformis on Black Alder in Spain.

Phytophthora alni is the causal organism responsible for devastating losses occurring on riparian alders stands in Europe. This emergent hybrid pathogen has multiple variants that have been placed in three subspecies (1). P. alni subsp. uniformis and P. alni subsp. multiformis are reported to be less aggressive than P. alni subsp. alni, though all are considered pathogenic. In Spain, P. alni subsp. alni was detected for the first time in 2009 in Galicia (northwestern Spain) causing root and collar rot on riparian alder populations (3,4), but other subspecies had not been identified. In April 2011, a survey along the Deza River in Galicia was carried out to clarify the Phytophthora sp. associated with the alder decline. Thirty riparian Alnus glutinosa stands, from both sides of the river, were surveyed. Samples of bark and roots of 18 alder stands that showed symptoms of Phytophthora rot and soil from all 30 stands were collected. Roots and tissue from fresh, active, inner bark lesions from 54 trees were transferred to selective medium V8-PARPH agar and incubated for 7 days at 22°C in the dark. P. alni subsp. alni (1) was isolated from roots, bark, or soil in five alder stands. Another Phytophthora sp. was isolated from the bark of one symptomatic tree located in Silleda (Pontevedra), transferred to carrot agar (CA), and incubated in the dark. On CA, the isolate produced irregular and appressed colonies with an optimum growth temperature of 22 to 23°C. The isolate was homothallic with smooth-walled oogonia with a diameter ranging from 36 to 50 µm and two-celled, amphigynous antheridia (1). In soil extract, noncaducous, nonpapillate, ellipsoid-to-ovoid sporangia were produced. Average sporangium were 43.4 × 30.1 µm with a length/breadth ratio of 1.43. Internal proliferation occurred. Amplification of DNA was accomplished by sequence characterized amplified region (SCAR)-PCR primers (2). The amplicon sizes obtained were identical to P. alni subsp. uniformis. Internal transcribed spacer (ITS) (DC6-ITS6/ITS4) and nadh1 (NADHF1/NADHR1) mitochondrial gene regions were also amplified and deposited in GenBank (Nos. JN880411 and JN880410). Comparison of the sequences showed 100% homology with P. alni subsp. uniformis (GenBank Nos. GU259293 and DQ202489). Pathogenicity was tested on 10 3-year-old black alder plants grown in pots. A shallow wound was made with a scalpel at the root collar level of each plant. A 5-mm-diameter mycelia plug, taken from the margin of a 7-day-old culture grown on CA, was inserted in every wound and sealed with Parafilm. Five black alder control plants received only sterile CA agar plugs. Plants were kept at 24°C and 80% humidity. After 3 months, wilting of shoots, dead leaves, and dark stained necroses of the bark tissue varying in length from 0.8 to 5 cm were observed on inoculated plants. Control plants remained healthy. P. alni subsp. uniformis was recovered from inoculated plants, but not from controls. To our knowledge, this is the first time that P. alni subsp. uniformis has been reported in Spain. The presence of a new subspecies in a new region can result in hybridization between individuals of different species or subspecies. This process may allow the rapid evolution and adaptation of these species to new hosts or environmental conditions. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (3) C. Pintos et al. Plant Dis. 94:273, 2010. (4) A. Solla et al. Plant Pathol.59:78, 2010.

First Report of Dieback on Hybrid Rhododendrons Caused by Neofusicoccum luteum and N. parvum in Spain.

During the conducting of Phytophthora ramorum surveys at Galician public parks (northwestern Spain) in 2010, established Rhododendron spp. plants were observed to be exhibiting leaf spots and necrosis, shoot blight, and cankers and dieback of shoots and branches. Branches and leaves of affected rhododendrons contained pseudothecia with bitunicate asci and hyaline pseudoparaphyses, and pycnidia were observed within the same stromatic masses. Symptomatic samples were disinfested in 0.5% sodium hypochlorite for 3 min. Tissues were cut from the margin of lesions, placed onto malt extract agar amended with streptomycin (25 µg ml-1), and incubated at 25°C in the dark. Cultures displaying morphological characteristics associated with Botryosphaeriaceae species were subcultured on 2% water agar with sterilized Pinus pinaster needles as a substrate and incubated at 25°C under near-UV light to encourage pycnidial production (1). Single conidial cultures gave rise to two distinct colonies on potato dextrose agar (PDA) at 25°C. In type 1, isolates produced a sparse, aerial mycelium and a characteristic yellow pigment that was more intense after 3 days, thereafter becoming violaceous and gradually turning dark gray. Growth occurred in the range of 4 to 38°C with an optimum at 29°C. Conidia were hyaline, fusiform, aseptate, thin walled, and averaged 21.1 (14.3 to 25.0) × 5.7 (4.3 to 6.8) µm with a length/width (L/W) ratio of 3.7 ± 0.4 (n = 100). On the basis of these characteristics, isolates were identified as Neofusicoccum luteum (1,3). Colonies of type 2 produced a dense, white-to-yellowish mycelium that rapidly became gray followed by marked diurnal zonation. Mycelial growth occurred in the range of 6 to 38°C with an optimum at 29 to 30°C. Conidia were hyaline, elliptical or fusiform, aseptate, thin walled, and averaging 18.3 (14.1 to 20.7) × 5.8 (4.6 to 7.0) µm with a L/W ratio of 3.2 ± 0.4 (n = 100). These isolates were identified as N. parvum (1,2). Identity was confirmed by DNA sequences analysis of internal transcribed spacer (ITS) regions. Comparison of the sequences of type 1 and 2 showed 100% homology with N. luteum and N. parvum (GenBank Accession Nos. EU673311 and GU251146, respectively). Representative sequences were deposited at GenBank (Accession Nos. HQ197352 and HQ197351). Pathogenicity of each isolate of N. luteum and N. parvum was confirmed by inoculating four 3-year-old Rhododendron spp. seedlings grown in pots. Shallow cuts were made in three branches of each plant. A colonized 6-mm agar plug, removed from the margin of an actively growing colony, was inserted beneath the flap and sealed with Parafilm. Four control seedlings received only sterile PDA agar plugs. Plants were maintained at 26°C and 70% humidity for 21 days. Inoculated plants began showing symptoms after 3 days. Necrosis progressed quickly and bidirectionally from the wound, resulting in death of leaves and wilting of shoots. N. luteum and N. parvum were reisolated from all inoculated plants but not from the controls. To our knowledge, this is the first report of N. luteum and N. parvum on Rhododendron spp. in Spain. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) S. R. Pennycook et al. Mycotaxon 24:445, 1985. (3) A .J. L. Phillips et al. Sydowia 54:59, 2002.

First Report of Cankers and Dieback Caused by Neofusicoccum mediterraneum and Diplodia corticola on Grapevine in Spain.

In November 2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla (south Spain) revealed trunk cankers. Several pathogens were isolated, including Cylindrocarpon liriodendri (2), Phaeoacremonium aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum, and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi isolated on potato dextrose agar (PDA) were two types that did not fit the above mentioned species. Isolates of type 1 produced an abundant, gray mycelium with a diurnal zonation that gradually became dark olivaceous. Mycelium growth occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline, fusiform, aseptate, thin walled, but gradually became obscured and septate with age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) µm with a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1 were identified as N. mediterraneum (3). Single-spore cultures of type 2 developed a whitish, dense, aerial mycelium and remained white up to 10 days on PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to 37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate, thick walled, oblong to cylindrical, sometimes becoming light brown and one or two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-) 14.3 (-18.6) µm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates of type 2 were identified as Diplodia corticola (1). Nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region and the -tubulin genes were used to confirm the identifications through BLAST searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to 100% homology with N. mediterraneum (HM443604 (4) and GU251836) and D. corticola (AY268421 (1) and EU673117), respectively. Representative sequences of N. mediterraneum (JF949757 and JF949756) and D. corticola (JF949758 and JF949759) were deposited in GenBank. The pathogenicity of one representative isolate of each of N. mediterraneum and D. corticola was confirmed by inoculating 10 detached grapevine canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the margin of an actively growing colony, was inserted in every wound and sealed with Parafilm. Ten grapevine canes controls received only sterile PDA agar plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all inoculated canes developed cankers and pycnidia around the inoculation site. Vascular necroses that developed on the inoculated canes were an average of 28.6 mm for N. mediterraneum and 27.7 mm for D. corticola. One-way analysis of variance and Tukey's test confirmed significant differences in the extent of vascular necroses. The average necroses length in the inoculated canes was significantly greater (P < 0.05) than the average length of discoloration induced by the simulated inoculation process in the control. Both pathogens were reisolated from all inoculated plants but not from controls. To our knowledge, this is the first report of N. mediterraneum and D. corticola as pathogens on grapevine in Spain. References: (1) A. Alves et al. Mycologia 96:603, 2004. (2) A. Aroca and D. Gramaje et al. Eur. J. Plant. Pathol. 126:165, 2010. (3) P. W. Crous et al. Fungal Planet. No. 19, 2007. (4) F. P. Trouillas et al. Plant. Dis. 94:1267, 2010.

The membrane-bound quinohemoprotein alcohol dehydrogenase from Gluconacetobacter diazotrophicus PAL5 carries a [2Fe-2S] cluster.

Gluconacetobacter diazotrophicus stands out among the acetic acid bacteria as it fixes dinitrogen and is a true endophyte. It has a set of constitutive enzymes to oxidize ethanol and acetaldehyde which is upregulated during N(2)-dependent growth. The membrane-bound alcohol dehydrogenase (ADH) is a heterodimer (subunit I approximately 72 kDa, subunit II approximately 44 kDa) and constitutes an important component of this organism. ADH of Ga. diazotrophicus is a typical quinohemoprotein with one pyrroloquinoline quinone (PQQ) and four c-type cytochromes. For the first time, a [2Fe-2S] cluster has been identified by EPR spectroscopy in this type of enzyme. This finding is supported by quantitative chemical analysis, revealing 5.90 +/- 0.15 Fe and 2.06 +/- 0.10 acid-labile sulfurs per ADH heterodimer. The X-band EPR spectrum of ADH (as isolated in the presence of dioxygen, 20 K) showed three broad resonances at g 2.007, 1.941, and 1.920 (g(av) 1.956), as well as an intense narrow line centered at g = 2.0034. The latter signal, which was still detected at 100 K, was attributed to the PQQ semiquinone radical (PQQ(sq)). The broad resonances observed at lower temperature were assigned to the [2Fe-2S] cluster in the one-electron reduced state. The oxidation-reduction potentials E(m) (pH 6.0 vs SHE) of the four c-type cytochromes were estimated to E(m1) = -64 (+/-2) mV, E(m2) = -8 (+/-2) mV, E(m3) = +185 (+/-15) mV, and E(m4) = +210 (+/-10) mV (spectroelectrochemistry), E(mFeS) = -250 (+/-5) mV for the [2Fe-2S] cluster, and E(mPQQ) = -210 (+/-5) mV for the PQQ/PQQH(2) couple (EPR spectroscopy). We propose a model for the membrane-bound ADH of Ga. diazotrophicus showing hypothetical intra- and intermolecular electron pathways. Subunit I binds the PQQ cofactor, the [2Fe-2S] cluster, and one c-type cytochrome. Subunit II harbors three c-type cytochromes, thus providing an efficient electron transfer route to quinones located in the cytoplasmic membrane.

First Report of Phytophthora Rot on Alders Caused by Phytophthora alni subsp. alni in Spain.

Phytophthora alni, a soil- and waterborne pathogen, causes aggressive root and collar rot on riparian alder populations (1,2,4). The disease has been described from several European countries with a destructive impact in Great Britain (1,2). All European alder species and the red alder (Alnus rubra) are highly susceptible. P. alni has multiple variants that have been placed in three subspecies: P. alni subsp. alni, P. alni subsp. uniformis, and P. alni subsp. multiformis (1). In July 2009, a survey of symptoms of Phytophthora rot from A. glutinosa at 20 riparian stands along the Avia River in Galicia (northwest Spain) was conducted. Affected trees showed symptoms of Phytophthora rot including abnormally small, sparse, and yellowish foliage, dieback in the canopy, necroses of the inner bark and cambium, and bleeding cankers on the trunks (2,4). Phytophthora spp. were baited from saturated rhizosphere soil and watercourses using oak leaflets (4). Roots and tissue from fresh active inner bark lesions were transferred to selective medium V8-PARPH agar (4) and incubated for 7 days at 22°C in the dark. A Phytophthora sp. was isolated, transferred to carrot agar (CA), and incubated in the dark. Colonies were appressed, often irregular in outline, and with limited aerial mycelium (1). Growth on CA occurred from 4 to 31°C with optimum growth at 23 to 25°C. Chlamydospores were not observed. Ellipsoid, nonpapillate, noncaducous sporangia had a length/breadth average ratio of 1.4. Nesting and extended internal proliferation occurred. Oogonia, antheridia, and oospores were abundantly produced in a single culture. Oogonia with tapered stalks were spherical (mature oogonia 38 to 50 µm in diameter) and some had ornamented walls or bullate protuberances (1,2). Antheridia were large, amphigynous, and predominantly two-celled (23 to 37 × 16 to 23 µm). Oospores were plerotic. Distorted comma-shaped or smaller oogonia and aborted oospores were observed (1). Amplification of DNA was accomplished by using sequence-characterized amplification region-PCR primers (3). The amplicon sizes obtained were identical to P. alni subsp. alni (3). Internal transcribed spacer (ITS)-DNA and nadh1 mitochondrial gene were also amplified. DNA sequences of ITS and mt-DNA regions were deposited in GenBank (Nos. GU108602 and GU108603). Comparison of the sequences showed 100% homology with P. alni subsp. alni (GenBank Nos. FJ746679 and DQ202490). P. alni subsp. alni was recovered from trees at 3 of 20 riparian alder stands with symptoms. Pathogenicity of one representative isolate was confirmed by inoculating 10 3-year-old A. glutinosa seedlings grown in pots. One shallow cut was made into the bark at the collar level. A colonized agar plug, from the margin of an actively growing colony of P. alni subsp. alni, was inserted beneath the flap that was sealed with Parafilm. Five controls seedlings received only sterile CA agar plugs. Plants were incubated at 24°C and 95% humidity for 30 days. On inoculated plants, necroses progressed bidirectionally from the wound, and dead leaves and wilting of shoots were observed. P. alni subsp. alni was recovered from inoculated seedlings, but not from controls. To our knowledge, this is the first report of Phytophthora rot on alder caused by P. alni subsp. alni in Spain. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) J. Gibbs et al. For. Comm. Bull. 126, 2003 (3) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (4) T. Jung et al. Plant Pathol. 53:197, 2004.

Random walk numerical simulation for hopping transport at finite carrier concentrations: diffusion coefficient and transport energy concept.

The random walk numerical simulation (RWNS) method is used to compute diffusion coefficients for hopping transport in a fully disordered medium at finite carrier concentrations. We use Miller-Abrahams jumping rates and an exponential distribution of energies to compute the hopping times in the random walk simulation. The computed diffusion coefficient shows an exponential dependence with respect to Fermi-level and Arrhenius behavior with respect to temperature. This result indicates that there is a well-defined transport level implicit to the system dynamics. To establish the origin of this transport level we construct histograms to monitor the energies of the most visited sites. In addition, we construct "corrected" histograms where backward moves are removed. Since these moves do not contribute to transport, these histograms provide a better estimation of the effective transport level energy. The analysis of this concept in connection with the Fermi-level dependence of the diffusion coefficient and the regime of interest for the functioning of dye-sensitised solar cells is thoroughly discussed.

First Report of Cylindrocladium buxicola on Buxus sempervirens in Spain.

Cylindrocladium buxicola Henricot, included in the EPPO alert list until November 2008, causes a dangerous foliar disease on Buxus spp. that has been recorded in several European countries and New Zealand (3,4). Buxus sempervirens L. (common boxwood) is one of the oldest ornamental garden plants in Europe. In September 2008, we received 10 2- to 3-year-old potted plants of B. sempervirens cv. Suffruticosa from a nursery in Galicia (northwest Spain) where ≈60% of the plants were affected and had finally defoliated. Diseased plants showed dark brown-to-black spots on the leaves and black streaks on the stems (3,4). To induce sporulation, diseased leaves and stem pieces were incubated in damp chambers at 22°C. A Cylindrocladium sp. was obtained. Four single conidial isolates were plated onto carnation leaf agar under near-UV light at 25°C for 7 days (2,3). Only conidiophores of the isolates growing on the surface of the carnation leaves were examined microscopically (1,3). Macroconidiophores were comprised of a stipe, a stipe extension, a terminal vesicle, and a penicillate arrangement of fertile branches (2). The stipe extension was septate, hyaline, and 90 to 165 × 2 to 4.5 µm (from the highest primary branch to the vesicle tip) (1) terminating in an ellipsoidal vesicle (6 to 11 µm in diameter) with a papillate apex. The widest part of the vesicle was above the middle. Primary branches were mainly aseptate or one septate (12 to 35 × 3 to 6 µm), secondary branches were aseptate (11 to 21 × 3 to 6 µm), and tertiary branches were rare. Each terminal branch produced two to five phialides (9 to 20 × 2.5 to 5 µm) that were reniform and aseptate. Conidia were cylindrical, straight, and one septate (56 to 75 × 4 to 6 µm). Chlamydospores were dark brown and aggregated to form microsclerotia. Cardinal temperatures of Cylindrocladium isolates on 2% malt extract agar ranged from 7 to 28°C (optimum 25°C). The 5' end of the ß-tubulin gene was amplified using primers T1 and Bt2b (3), and PCR products were sequenced directly and deposited in GenBank (Accession No. FJ696535). Comparison of the sequence with others available in GenBank showed 100% homology with those previously identified as C. buxicola (Accession Nos. AY078123 and AY078118). Pathogenicity of one representative isolate was confirmed by inoculating stems and leaves of four 3- to 4-year-old plants of B. sempervirens cv. Suffruticosa. Leaves were inoculated by spraying a spore suspension of the fungus (1 × 106 conidia per ml). For the stems, agar pieces of 1-week-old cultures grown on malt extract agar were placed and sealed with Parafilm. As a control, four plants were inoculated with agar malt plugs and sterile distilled water. Plants were incubated at 22°C and 95% humidity. Symptoms identical to ones previously described appeared 4 days after inoculation. C. buxicola was reisolated from inoculated plants but not from the controls. On the basis of morphological and physiological characteristics, pathogenicity, and the DNA sequencing of the ß-tubulin gene, the isolates obtained from B. sempervirens were identified as C. buxicola (3). To our knowledge, this is the first report of C. buxicola on B. sempervirens in Spain. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) P. W. Crous and M. J. Wingfield. Mycotaxon 51:341, 1994. (3) B. Henricot and A. Culham. Mycologia 94:980, 2002. (4) B. Henricot et al. Plant Pathol. 49:805, 2000.

First Report of Phytophthora pseudosyringae on Chestnut Nursery Stock in Spain.

Phytophthora pseudosyringae causes stem necrosis and collar rot of deciduous tree species (Quercus spp., Fagus silvatica, and Alnus glutinosa) in several European countries (1,2). In November 2006, we received diseased Castanea sativa seedlings from a nursery in Galicia (northwest Spain). These plants had tongue-shaped necroses of the inner bark and cambium. Reddish, sunken lesions occurred on the surface of the bark, either in the stem base or higher on the stem. Tissue from the leading edge of the lesions was transferred to a selective V8 agar medium (4) and incubated for 7 days at 20°C in the dark. A Phytophthora sp. was isolated, transferred to cornmeal agar (CMA) and V8 agar, and incubated in the dark. Colonies were appressed with stellate to rosaceous growth patterns on CMA and stellate, limited aerial mycelium on V8 agar. Growth on V8 occurred from 2 to 25°C with an optimum at 20°C and a radial growth rate of 4.5 mm per day at 20°C. Chains of inflated spherical to deltoid hyphal swellings with radiating hyphae were abundantly produced in water (2). Chlamydospores were not observed on agar media. The deciduous, sympodial, semipapillate, rarely bipapillate sporangia with pedicels had a length/breadth average ratio of 1.55. Oogonia, antheridia, and oospores were produced within a single culture. Oogonia were spherical and smooth walled, antheridia were predominantly paraginous, but some were amphyginous, and oospores were plerotic that turned golden yellow with age (2). Internal transcribed spacer (ITS)-rDNA and mitochondrial DNA (mtDNA) regions were amplified by nested-PCR and sequenced with DNA extracted from mycelium. The amplicon sizes obtained were similar to those reported for P. pseudosyringae (2,3). DNA sequences showed 99 to 100% homology with those previously identified as P. pseudosyringae and deposited in GenBank. Pathogenicity of the isolate was confirmed by inoculating 10 C. sativa seedlings, as well as three detached leaves from each of another 10 young plants growing in containers. For the seedlings, one shallow cut was made into the bark on the main stem. A colonized agar plug was inserted beneath the flap that was sealed with Parafilm. Unwounded and wounded detached leaves of C. sativa were dipped into a zoospore aqueous suspension (1 × 105 zoospores ml-1) for 10 s., seedlings and leaves were incubated at 20°C and 95% humidity for 60 and 7 days, respectively. After 7 days, foliar lesions that developed exceeded 25 mm, and the pathogen was consistently reisolated. Leaves inoculated with sterile water did not develop symptoms. On inoculated seedlings, the external surface of the bark was reddish and sunken. Stem lesions progressed bidirectionally from the wound. P. pseudosyringae was recovered from inoculated seedlings but not from controls. On the basis of its unique combination of morphological and physiological characters, pathogenicity, and ITS and mtDNA sequences, the Phytophthora isolated from chestnut was identified as P. pseudosyringae. To our knowledge, this is the first report of P. pseudosyringae on C. sativa in Spain. References: (1) EPPO Reporting Service. Online publication. No. 10 2005/162, 2005. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) F. N. Martin et al. Phytopathology 94:621, 2004. (4) C. Pintos Varela et al. Plant. Dis. 87:1396, 2003.

First Report of Phytophthora ramorum on Camellia japonica in Spain.

Phytophthora ramorum causes shoot and foliar blight on Rhododendron spp., Viburnum spp. (4), Pieris spp. (2), Kalmia latifolia, and Camellia spp. in several European countries (1-4). In December 2002, we received diseased C. japonica growing in containers from several nurseries in Galicia (northwestern Spain). These young camellia plants had leaves with brown-to-black, water-soaked lesions with diffuse borders that expanded into larger blotches resulting in dead leaves and necrotic lesions on the petioles. Eventually the entire plant wilted and died. Tissue from the leading edge of the lesions was transferred to a selective medium (V8 agar supplemented with pimaricin (10 µg/ml), rifampicin (25 µg/ml), hymexazol (5 µg/ml), and benomyl (5 µg/ml)) and incubated for 3 to 4 days at 20°C in the dark. A Phytophthora sp. was isolated, transferred to carrot piece agar (CPA) (4), and incubated in alternating light. Isolates exhibited coralloid mycelium with concentric rings and a radial growth of 2.5 to 3 mm per day at 20°C. The hyaline-to-yellowish chlamydospores were terminal and intercalary, occasionally lateral, and 24 to 74 µm in diameter. The caducous, sympodial, semipapillate sporangia had a length/breadth ratio of 1.8 to 2.1 and a short pedicel (<5 µm) or no pedicel. Oogonia, antheridia, and oospores were produced by pairing the isolates with P. cryptogea A2 tester BBA 63651 (3,4) provided by S. Werres. Oogonia were subspherical and smooth-walled, antheridia were amphyginous, and oospores were plerotic. The internal transcribed spacer-rDNA polymerase chain reaction (PCR) product obtained by using DNA extracted from mycelium and nested PCR with P. ramorum-specific primers was the size reported for P. ramorum (1). Pathogenicity of the isolates was confirmed by inoculating detached leaves of C. japonica. Five isolates were tested on leaves from 15 young plants growing in containers. Three leaves of each plant were detached and inoculated with each isolate. Leaves were dipped for 5 min into a suspension of sporangia and mycelial fragments and maintained at 20°C and 95% humidity. After 15 days, lesions that developed from the petiole base exceeded 25 mm, and the pathogen was consistently reisolated from the lesions. Leaves inoculated with water from sterile CPA plates did not develop symptoms. A C. japonica isolate has been deposited in the Spanish Type Culture Collection (CECT 20519). To our knowledge, this is the first report of P. ramorum on C. japonica in Spain, though the pathogen has been isolated from Rhododendron spp. and Viburnum tinus growing in several nurseries in Galicia. References: (1) J. M. Davidson et al. On-line publication doi:10.1094. Plant Health Progress, PHP, 2003-0707-01-DG, Plant Management Network. (2) A. J. Inman et al. First Report of Ramorum Dieback (Phytophthora ramorum) on Pieris in England. On-line publication. New Dis. Rep. Vol. 7, British Society for Plant Pathology, 2003. (3) E. Moralejo et al. Plant Dis. 86, 9:1052, 2002. (4) S. Werres et al. Mycol.Res.105:1155, 2001.

Caracterización de una población bruxista y evaluación de la terapéutica mediante análisis cluster / Characterization of a bruxist population and therapeutical evaluation by means of a cluster analysis

El efecto de la férula oclusal y el TENS ha sido estudiado en una muestra de 24 pacientes bruxistas (apretadores y rechinadores) que fueron diagnosticados por medio de un cuestionario y anamnesis. Se estudiaron diferentes variables tanto cualitativas como cuantitativas, atendiendo a aspectos psicológicos, de oclusión tanto estática como dinámica, actualidad electromiográfica e índices electromiográficos en músculos posturales y elevadores y presencia de desarreglos internos de la articulación temporomandibular. El método estadístico utilizado fue el análisis cluster. Los resultados no mostraron claramente una discriminación entre grupos tanto entre apretadores y rechinadores como entre los dos tipos de tratamiento...

DMFT, dmft and treatment requirements of schoolchildren in Asturias, Spain.

An epidemiological survey was carried out in 1992 to study the dental health status of schoolchildren aged 6, 9 and 12 in Asturias, Spain. It focused on the caries prevalence, dmtf, DMFT, restoration indices and dental treatment needs of this population. A representative sample of 1839 subjects, randomly selected and proportionally assigned by age group (6, 9 and 12) with the classroom as the sample unit, was examined. Analysis of the data showed that in 6-year-old children the caries prevalence in primary teeth was 45.8%. The mean caries indices were 2.10 dmft and 0.25 DMFT. At 9 years old the prevalence of caries in primary teeth was 62.8% and in the permanent teeth 49.1%. The mean level of caries was 2.38 dmft and 1.50 DMFT. At 12 years old the caries prevalence in permanent teeth was 71% and in first molars 64.2%. The mean caries experienced was 3.30 DMFT. In all groups the D-component constituted the major part of the caries index. The results for girls were higher than for boys in almost all age groups. Surface fillings were the treatment most required in all age groups.