New PCR-Based Assay for the Identification of Pectobacterium carotovorum Causing Potato Soft Rot.

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management because different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is Pectobacterium carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of Pectobacterium versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen Pectobacterium parmentieri. This should be useful for the routine diagnosis of potato soft rot.

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain.

Liberibacter is a bacterial group causing different diseases and disorders in plants. Among liberibacters, Candidatus Liberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future works.

Dual disorders in individuals under treatment for both alcohol and cocaine: Madrid study on the prevalence of dual disorders / Patología dual en individuos en tratamiento por uso de cocaína y alcohol: estudio Madrid sobre prevalencia de patología dual

Abstract: Introduction: Descriptive data about co-occurrence of alcohol and cocaine consumption is scarce, despite its important prevalence. Dual disordes shows high prevalence in clinical samples, and patients report worse evolution and need more health services. Objective: To compare psychopathology in patients in treatment with lifetime alcohol and cocaine (Alc + Coc) substance use disorder (SUD) with subjects with alcohol but not cocaine (Alc) lifetime SUD and cocaine but not alcohol (Coc) SUD. Method: The sample consisted of 837 outpatients from Madrid, Spain, under treatment in substance misuse or mental health units. Two analyses were made: we compared subjects in the Alc + Coc (n = 366) to the Alc group (n = 162), and then to the Coc group (n = 122). Socio-demographic variables were addressed by interview. The Mini International Neuropsychiatric Interview (MINI) was used to evaluate Axis I disorders and the Personality Disorder Questionnaire (PDQ) to evaluate Personality Disorders (PD). Results: Compared to Alc group, patients in the Alc + Coc group were younger, had different socio-demographic characteristics, had more proportion of cannabis and opioid SUD, had less proportion of major depressive disorder, obsessive and depressive PD, more proportion of antisocial PD and lower suicide risk. Compared to the Coc group, they had more cannabis SUD and lower opioid SUD, showed higher prevalence of bipolar disorder, general anxiety disorder, paranoid, histrionic and dependent PD. Discussion and conclusion: We present a cross-sectional study describing comorbidity of dual disordes on treatment-seeking concurrent alcohol and cocaine problematic users. This concurrence showed different dual disordes prevalence profile than single users in some specific mental disorders.

Resumen: Introducción: La patología dual presenta una alta prevalencia en muestras clínicas. Estos pacientes sufren mayor comorbilidad y requieren más servicios. Existen escasos datos de comorbilidad por alcohol y cocaína. Objetivo: Valorar datos sociodemográficos, prevalencias de trastorno por uso de sustancias (TUS), trastorno mental y trastornos de personalidad en pacientes con trastorno por uso de alcohol y cocaína frente a pacientes con uno de los dos TUS. Método: La muestra incluyó 837 pacientes procedentes de Centros de Salud Mental y Centros de Atención a Drogodependientes. Se realizaron dos análisis: se comparó el grupo de alcohol y cocaína (Alc + Coc, n = 366) con el grupo de alcohol (Alc, n = 162), y posteriormente con el grupo de cocaína (Coc, n = 122). Se recogieron variables sociodemográficas y sobre TUS mediante entrevista, diagnósticos de trastorno mental del eje I mediante la entrevista estructurada Mini International Neuropsychiatric Inerview (MINI) y los trastornos de personalidad mediante el Personality Disorder Questionnaire (PDQ). Resultados: Los pacientes del grupo Alc + Coc frente al grupo Alc presentaban diferencias sociodemográficas significativas, mayor prevalencia de TUS asociados, riesgo más alto de suicidio y mayor proporción de trastorno antisocial de personalidad. Además, tenían menor prevalencia de trastornos depresivos. Frente al grupo Coc presentaron mayor prevalencia de trastorno por consumo de cannabis, pero menor de opioides y mayor prevalencia de trastorno bipolar, trastorno de ansiedad generalizada y trastornos de personalidad. Discusión y conclusión: Los pacientes comórbidos para alcohol y cocaína presentaron un distinto perfil de prevalencia de algunos trastornos mentales específicos.

Fontibacillus solani sp. nov. isolated from potato (Solanum tuberosum L.) root.

A bacterial strain designated A4STR04(T) was isolated from the inner root tissue of potatoes in Spain. Phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate into the genus Fontibacillus, being most closely related to Fontibacillus panacisegetis KCTC 13564(T) with 99% identity. The isolate was observed to form Gram-positive, motile and sporulating rods. The catalase test was found to be negative and oxidase positive. Nitrate was found to be reduced to nitrite. ß-Galactosidase and caseinase were observed to be produced but the production of gelatinase, urease, arginine dehydrolase, ornithine and lysine decarboxylase was negative. Aesculin hydrolysis was found to be positive and acetoin production was negative. Growth was found to be supported by many carbohydrates and organic acids as carbon source. MK-7 was the only menaquinone detected and the major fatty acid (61.5%) was identified as anteiso-C(15:0), as occurs in the other species of genus Fontibacillus. The strain A4STR04(T) was found to display a complex lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a glycolipid, two phospholipids, a lipid and two aminophospholipids. Mesodiaminopimelic acid was detected in the peptidoglycan. The G+C content was determined to be 50.5 mol% (Tm). Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain A4STR04(T) (=LMG 28458 (T) = CECT 8693(T)) should be classified as representing a novel species of genus Fontibacillus, for which the name Fontibacillus solani sp. nov. is proposed.

[Space-time suicide clustering in the community of Antequera (Spain)]. / Clusters de casos de suicidio espacio-temporal en la comunidad de Antequera (España).

INTRODUCTION: Approximately 3,500 people commit suicide every year in Spain. The main aim of this study is to explore if a spatial and temporal clustering of suicide exists in the region of Antequera (Málaga, España). METHODS: Sample and procedure: All suicides from January 1, 2004 to December 31, 2008 were identified using data from the Forensic Pathology Department of the Institute of Legal Medicine, Málaga (España). Geolocalisation. Google Earth was used to calculate the coordinates for each suicide decedent's address. Statistical analysis. A spatiotemporal permutation scan statistic and the Ripley's K function were used to explore spatiotemporal clustering. Pearson's chi-squared was used to determine whether there were differences between suicides inside and outside the spatiotemporal clusters. RESULTS: A total of 120 individuals committed suicide within the region of Antequera, of which 96 (80%) were included in our analyses. Statistically significant evidence for 7 spatiotemporal suicide clusters emerged within critical limits for the 0-2.5 km distance and for the first and second semanas (P<.05 in both cases) after suicide. There was not a single subject diagnosed with a current psychotic disorder, among suicides within clusters, whereas outside clusters, 20% had this diagnosis (X2=4.13; df=1; P<.05). CONCLUSIONS: There are spatiotemporal suicide clusters in the area surrounding Antequera. Patients diagnosed with current psychotic disorder are less likely to be influenced by the factors explaining suicide clustering.

[Risk and protective factors in adolescents' drug use, and differences by age and sex]. / Factores de riesgo y de protección en el consumo de drogas en adolescentes y diferencias según edad y sexo.

Adolescents' drug use has huge social and personal implications, so it is essential to identify risk and protective factors. In this research, the CTCYS was used with 2440 adolescents to detect risk and protective factors for drug use in the community, family, school and peers/individual; differences in risk and protective factors by age and sex; and relationships between risk and protective factors and substance use. Protective factors are high. Risk factors are high in the community, the school and the individual. Older adolescents have more risks and less protection than the youngest; and there are sex differences, because males have less protection and more risks. The risk factors more closely related to drug use are availability of drugs in the community, family attitudes favourable to drug use, family history of antisocial behaviour, early start and use of drugs by friends, perceived risk and attitudes favourable to drug use. In the protective factors, the role played by social skills for alcohol use is important.

Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes.

Some strains of the former genus Agrobacterium have high biotechnological interest and are currently misclassified. Consequently, in this study, the taxonomic status of the non-pathogenic strain Agrobacterium radiobacter K84, used in biological control, and the tumourigenic strain Agrobacterium tumefaciens AKE10, able to regenerate tobacco transgenic plants, was revised. The phylogenetic analysis of the chromosomal genes rrs, atpD and recA showed that they should be reclassified into Rhizobium rhizogenes. The analysis of virulence genes located in the Ti plasmid (pTi) outside T-DNA showed a common phylogenetic origin among strains AKE10, R. rhizogenes 163C and A. tumefaciens (currently R. radiobacter) C58. However, the genes located inside the T-DNA, mainly the 6b gene, of strain AKE10 were phylogenetically close to those of strain 163C but divergent from those of strain C58. Furthermore, the T-DNA of tumourigenic strains from R. rhizogenes conferred on them the ability to regenerate tumour tissue resembling fasciation in tobacco plants. These results showed the existence of a highly mosaic genetic organization in tumourigenic strains of the genus Rhizobium and provided evidence of the involvement of T-DNA from tumourigenic strains of R. rhizogenes in fasciation of Nicotiana leaves. The data further suggested that pathogenic strains of Rhizobium could be good models to analyse bacterial evolution.

An Indigenous Virulent Strain of Erwinia amylovora Lacking the Ubiquitous Plasmid pEA29.

ABSTRACT An atypical strain of Erwinia amylovora was isolated near an outbreak of fire blight at a nursery in Spain in 1996. It was obtained from a Crataegus plant showing typical symptoms and was identified as E. amy-lovora by biochemical tests and enrichment-enzyme-linked immuno-sorbent assay, but not by polymerase chain reaction using primers based on the pEA29 sequence. Nevertheless, with primers from chromosomal regions, the isolate gave the expected amplification band. This strain carries one plasmid of approximately 70 kb, with no homology with the 29-kb plasmid common to all pathogenic strains, or with a large plasmid present in some E. amylovora strains. Growth of the strain in minimal medium without thiamine was slower compared with cultures in the same medium with thiamine, a characteristic typical of strains cured of the 29-kb plasmid. Nevertheless, aggressiveness assays on pear, apple, and Pyracantha plants and in immature pear fruit showed that this strain exhibited a virulence level similar to other strains containing pEA29. To the best of our knowledge, this is the first report of the isolation from naturally infected plant material of a pathogenic strain of E. amylovora without pEA29, but with a plasmid of approximately 70 kb not previously described.

Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures.

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14 degrees C or higher, but its isolation was usually unsuccessful at temperatures below 9 degrees C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35 degrees C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35 degrees C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.

The coexistence of symbiosis and pathogenicity-determining genes in Rhizobium rhizogenes strains enables them to induce nodules and tumors or hairy roots in plants.

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.

Bradyrhizobium betae sp. nov., isolated from roots of Beta vulgaris affected by tumour-like deformations.

Some varieties of sugar beet, Beta vulgaris, cultivated in northern Spain have large deformations that resemble the tumours produced by Agrobacterium species. In an attempt to isolate the agent responsible for these deformations, several endophytic slow-growing bacterial strains were isolated, the macroscopic morphology of which resembled that of Bradyrhizobium species. These strains were not able to produce tumours in Nicotiana tabacum plants and, based on phylogenetic analysis of their 16S rRNA, they are closely related to the genus Bradyrhizobium. Phenotypic and molecular characteristics of these strains revealed that they represent a species different from all Bradyrhizobium species previously described. Sequence analysis of the 16S-23S rDNA intergenic spacer region indicated that these novel strains form a homogeneous group, related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense. DNA-DNA hybridization confirmed that these strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium betae sp. nov. is proposed. The type strain is PL7HG1T (=LMG 21987T=CECT 5829T).

A preliminary report on multiple family discussion groups for patients with chronic medical illness and its repercussions in the management of the hemodialysis process.

The hemodialysis (HD) process involves an important degree of stress, not only for the patient but also for the family. The available data suggest that the quality of the family's performance predicts the degree of commitment that the patient acquires with the dialysis center. The establishment of a program of multiple family discussion groups allows exploration of the effect of the treatment on the patient in their context, not only regarding the illness, but also regarding the quality of life related to health, satisfaction and functional state. After the startup in the Hospital Virxe da Xunqueira of the multiple family discussion group (MFDG) for patients with chronic medical illness, the objective of this work was to evaluate the repercussions of the MFDG over the therapeutic fulfillment, the quality of life and the expectations of the patients in the chronic HD program, through the assessment of these indicators before and after their participation in this group. The MFDG was performed for a total of eight people, the families of four patients in the chronic HD program. Six weekly 1.5 hour sessions were performed and structured according to the following general contents: chronic illness impact component (2 sessions), family development component (3 sessions) and family illness integration component (1 session). Although no objectives were made for changes in relation to the therapeutic fulfillment, the average auto-effectiveness, locus of control, success and family general expectations went up slightly after the participation in the MFDG. The average specific self-effectiveness and family expectations in the presence of the illness reflected a modest increase, while the specific expectations of control locus and success in the presence of the illness decreased slightly. The scores obtained regarding the general state of health reflected a small decrease, while the evaluation of the quality of life of patients and family members showed a slight increase. We can conclude in the first place highlighting the viability of the MFDG, since no impediments were found either in recruitment of the families of the participants, or in preventing their excellent participation in the beginning and through the course of the group. Although no objectives were made for changes in relation to the therapeutic fulfillment, the high indices of satisfaction which the group obtained indicate that the discussion group is useful for the patients to find more support from their families, to change their view of the illness, to learn from other families new ways to resolve the difficulties and to increase their perception of capacity in the presence of the illness. The obtained data are preliminary and derived from only four families, but are encouraging as far as the improvement in the quality of life and the adjustment of the participants to the illness. Studies with the inclusion of more families are still pending in order to be able to arrive at conclusions based on a greater empiric basis. The records of MFDG for the families of HD patients are scarce. With this work it is attempted to reveal that these types of groups can be applied with these patients and their families, and they seem to prove beneficial for all those involved: patients, family and health professionals.

Enrichment double-antibody sandwich indirect enzyme-linked immunosorbent assay that uses a specific monoclonal antibody for sensitive detection of Ralstonia solanacearum in asymptomatic potato tubers.

Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29 degrees C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.