A reliable qPCR technique for detecting viable Xanthomonas arboricola pv. pruni cells.

Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot of stone fruits and almond (Prunus spp). Detection of Xap is typically carried out using quantitative real-time PCR (qPCR) combined with culture-based isolation. However, qPCR does not differentiate between viable and dead cells, potentially leading to an overestimation of the infective population in a sample. Such overestimation could result in unnecessary phytosanitary measures. The present study aims to develop a specific protocol ideally targeting to detection of only live Xap bacterial cells. To address this challenge, the viable quantitative PCR (v-qPCR) method was evaluated using three nucleic acid-binding dyes: propidium monoazide (PMA), a combination of PMA and ethidium monoazide (EMA), and PMAxx™, an improved version of PMA. PMAxx™ proved to be the most suitable dye for the detection and quantification of living bacterial cells. This methodology was also evaluated in infected plant material over time and can be considered a rapid and reliable alternative to PCR methods for detecting only those putative infective Xap that may pose a risk for Prunus crops. KEY POINTS: • Protocol to detect biofilm and planktonic viable X. arboricola pv. pruni cells. • Host validated protocol. • Benefits, reduction of chemicals in disease control.

New Approaches to Plant Pathogen Detection and Disease Diagnosis.

Detecting plant pathogens and diagnosing diseases are critical components of successful pest management. These key areas have undergone significant advancements driven by breakthroughs in molecular biology and remote sensing technologies within the realm of precision agriculture. Notably, nucleic acid amplification techniques, with recent emphasis on sequencing procedures, particularly next-generation sequencing, have enabled improved DNA or RNA amplification detection protocols that now enable previously unthinkable strategies aimed at dissecting plant microbiota, including the disease-causing components. Simultaneously, the domain of remote sensing has seen the emergence of cutting-edge imaging sensor technologies and the integration of powerful computational tools, such as machine learning. These innovations enable spectral analysis of foliar symptoms and specific pathogen-induced alterations, making imaging spectroscopy and thermal imaging fundamental tools for large-scale disease surveillance and monitoring. These technologies contribute significantly to understanding the temporal and spatial dynamics of plant diseases.

Graft Survival in Liver Transplantation: An Artificial Neuronal Network Assisted Analysis of the Importance of Comorbidities.

OBJECTIVES: Liver transplant represents a widespread therapeutic option for patients with end-stage liver failure. Up to now, most of the scores describing the probability of liver graft survival have shown poor predictive performance. With this in mind, the present study seeks to analyze the predictive value of recipient comorbidities on liver graft survival within the first year. MATERIALS AND METHODS: The study included prospectively collected data from patients who received a liver transplant at our center from 2010 to 2021. A predictive model was then developed through an Artificial Neural Network that included the parameters associated with graft loss as identified by the Spanish Liver Transplant Registry report and comorbidities with prevalence >2% present in our study cohort. RESULTS: Most patients in our study were men (75.5%); mean age was 54.8 ± 9.6 years. The main cause of transplant was cirrhosis (86.7%), and 67.4% of patients had some associated comorbidities. Graft loss due to retransplant or death with dysfunction occurred in 14% of cases. Of all the variables analyzed, we found 3 comorbidities associated with graft loss (as shown by informative value and normalized informative value, respectively): antiplatelet and/or anticoagulants treatments (0.124 and 78.4%), previous immunosuppression (0.110 and 69.6%), and portal thrombosis (0.105 and 66.3%). Remarkably, our model showed a C statistic of 0.745 (95% CI, 0.692-0.798; asymptotic P < .001), which was higher than others found in previous studies. CONCLUSIONS: Our model identified key parameters that may influence graft loss, including specific recipient comorbidities. The use of artificial intelligence methods could reveal connections that may be overlooked by conventional statistics.

Plant Pathogenic Microorganisms: State-of-the-Art Research in Spain.

Pathogenic microorganisms, including fungi, oomycetes, bacteria, viruses, and viroids, constitute a serious threat to agriculture worldwide [...].

Taxonomic Refinement of Xanthomonas arboricola.

Xanthomonas arboricola comprises a number of economically important fruit tree pathogens classified within different pathovars. Dozens of nonpathogenic and taxonomically unvalidated strains are also designated as X. arboricola, leading to a complicated taxonomic status in the species. In this study, we have evaluated the whole-genome resources of all available Xanthomonas spp. strains designated as X. arboricola in the public databases to refine the members of the species based on DNA similarity indexes and core genome-based phylogeny. Our results show that, of the nine validly described pathovars within X. arboricola, pathotype strains of seven pathovars are taxonomically genuine, belonging to the core clade of the species regardless of their pathogenicity on the host of isolation (thus the validity of pathovar status). However, strains of X. arboricola pv. guizotiae and X. arboricola pv. populi do not belong to X. arboricola because of the low DNA similarities between the type strain of the species and the pathotype strains of these two pathovars. Thus, we propose to elevate the two pathovars to the rank of a species as X. guizotiae sp. nov. with the type strain CFBP 7408T and X. populina sp. nov. with the type strain CFBP 3123T. In addition, other mislabeled strains of X. arboricola were scattered within Xanthomonas spp. that belong to previously described species or represent novel species that await formal description.

Encephalitis as a neurological manifestation of COVID-19.

INTRODUCTION: In the context of the global COVID-19 pandemic, the different clinical manifestations of this infection pose a challenge for healthcare professionals. Respiratory involvement, the main symptom of SARS-CoV-2 infection, means that other manifestations, such as neurological, take a back seat, with the consequent delay in diagnosis and treatment. MATERIAL AND METHODS: All COVID-19 patients admitted with neurological symptoms or diagnosed with encephalitis since March 2020 in a tertiary hospital in Zaragoza, Spain. RESULTS: Two patients with COVID-19 infection confirmed by nasopharyngeal PCR and whose clinical picture consisted of neurological alterations compatible with encephalitis. Cerebrospinal fluid (CSF) microbiology was negative for bacteria and viruses, including SARS-CoV-2 but, given the clinical suspicion of encephalitis due to the latter, antiviral treatment with immunoglobulins and plasmapheresis was started early. Despite this, the evolution was not satisfactory. CONCLUSIONS: COVID-19 encephalitis is a recently described clinical entity, whose pathophysiology is still unknown and no treatment with clinical evidence is available to date.

INTRODUCCIÓN: En el contexto de la pandemia mundial por COVID-19, las distintas manifestaciones clínicas de esta infección suponen un reto para los profesionales sanitarios. La afectación respiratoria, síntoma principal de la infección por SARS-CoV-2, hace que otras manifestaciones, como las neurológicas, pasen a un segundo plano, con el consecuente retraso en el diagnóstico y tratamiento. MATERIAL Y MÉTODOS: Todo paciente COVID-19 que ha ingresado con sintomatología neurológica o diagnosticado de encefalitis desde Marzo de 2020 en un hospital de tercer nivel en Zaragoza, España. RESULTADOS: Dos pacientes con infección COVID-19 confirmada por PCR nasofaríngea y cuyo cuadro clínico consistía en alteraciones neurológicas compatibles con encefalitis. La microbiología del líquido cefalorraquídeo (LCR) fue negativa para bacterias y virus, incluido el SARS-CoV-2 pero, ante la sospecha clínica de encefalitis por este último, se instauró tratamiento antiviral, con inmunoglobulinas y plasmaféresis de forma precoz. A pesar de ello la evolución no fue satisfactoria. CONCLUSIONES: La encefalitis por COVID-19 es una entidad clínica descrita recientemente, cuya fisiopatología aún se desconoce y no se dispone, hasta la fecha, de un tratamiento con evidencia clínica.

[Encephalitis as a neurological manifestation of COVID-19]. / Encefalitis como manifestación neurológica del COVID-19.

INTRODUCTION: In the context of the global COVID-19 pandemic, the different clinical manifestations of this infection pose a challenge for healthcare professionals. Respiratory involvement, the main symptom of SARS-CoV-2 infection, means that other manifestations, such as neurological, take a back seat, with the consequent delay in diagnosis and treatment. MATERIAL AND METHODS: All COVID-19 patients admitted with neurological symptoms or diagnosed with encephalitis since March 2020 in a tertiary hospital in Zaragoza, Spain. RESULTS: Two patients with COVID-19 infection confirmed by nasopharyngeal PCR and whose clinical picture consisted of neurological alterations compatible with encephalitis. Cerebrospinal fluid (CSF) microbiology was negative for bacteria and viruses, including SARS-CoV-2 but, given the clinical suspicion of encephalitis due to the latter, antiviral treatment with immunoglobulins and plasmapheresis was started early. Despite this, the evolution was not satisfactory. CONCLUSIONS: COVID-19 encephalitis is a recently described clinical entity, whose pathophysiology is still unknown and no treatment with clinical evidence is available to date.

Graft Risk Index After Liver Transplant: Internal and External Validation of a New Spanish Indicator.

OBJECTIVES: Scarcity of liver grafts has led to the use of marginal donors, consequently increasing the number of complications posttransplant. To prevent this situation, several indicators have been developed. However, important differences remain among countries. Here, we compared an early-risk liver transplant indicator based on the Spanish Liver Transplant Registry, called the Graft Risk Index, versus the US donor risk index and the Eurotransplant donor risk index. MATERIALS AND METHODS: The new indicator was based on prospectively collected data from 600 adult liver transplants performed in our center. We considered 2 events to compare the indexes: graft survival and rejection-free graft survival, with Cox proportional regression for analyses. Power to predict graft survival was evaluated by calculating the receiver operating characteristic area under the curve. RESULTS: We found no differences between the US and Eurotransplant donor risk indexes in prediction of patients with and without early graft failure. With regard to early survival, only the Graft Risk Index allowed better survival discrimination, in which survival progressively decreased with values ≥ 3 (with probability of graft survival at 1 month of 68%; 95% confidence interval, 46.2-82.5). This increase in risk was significant compared with the standard group (hazard ratio of 10.15; 95% confidence interval, C 3.91- 26.32; P < .001). We calculated powers of prediction of 0.52 (95% confidence interval, 0.43-0.62), 0.54 (95% confidence interval, 0.45-0.65), and 0.69 (95% confidence interval, 0.61-0.77) for donor risk index, Eurotransplant donor risk index, and early Graft Risk Index, respectively. CONCLUSIONS: Neither the US donor risk index nor the Eurotransplant donor risk index was valid for our Spanish liver donation and transplant program. Therefore, an indicator to predict posttransplant graft survival that is adapted to our environment is necessary. This national Graft Risk Index can be a useful tool to optimize donor-recipient matching.

Graft survival after liver transplantation: an approach to a new Spanish risk index.

INTRODUCTION: several indicators are available to assess liver graft survival, including the American DRI and the European ET-DRI. However, there are significant differences between transplant programs of different countries, and the previously mentioned indicators might be not valid in our setting. OBJECTIVES: the aim of the study was to describe a new national liver graft risk indicator based on the results obtained from the Registro Español de Trasplante Hepático (RETH) and to validate the DRI and ET-DRI indicators. METHODS: the RETH includes a Cox analysis of factors associated with graft survival; the graft risk index (GRI) indicator was defined based on these results. The variables considered are dependent upon the donation conditions (age, cause of death, blood compatibility and cold ischemia time) and the transplant recipient (age, underlying disease, hepatitis C virus, transplant number, UNOS status and surgical technique). A logistic regression curve was obtained and graft survival curves were calculated by stratification. Precision was assessed using the ROC analysis. RESULTS: a GRI of 1 represents a probability of graft loss of 23.25%; each point increase in the GRI score multiplies this probability by 1.33. The best discrimination of GRI was obtained by stratification. The DRI ROC area was 0.54 (95% CI, 0.50-0.59) and the ET-DRI ROC area was 0.56 (95% CI, 0.51-0.61), compared to 0.70 (95% CI, 0.65-0.73) (p < 0.0001) for the GRI. CONCLUSIONS: both the DRI and ET-DRI do not seem to be useful in our setting. Hence a national indicator is more desirable. The GRI requires a national study in order to further streamline and assess this indicator.

Xanthomonas arboricola pv. pruni, causal agent of bacterial spot of stone fruits and almond: its genomic and phenotypic characteristics in the X. arboricola species context.

BACKGROUND: Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of stone fruits and almond, an important disease that may reduce the yield and vigour of the trees, as well as the marketability of affected fruits. Xap lies within the Xanthomonas genus, which has been intensively studied because of its strain specialization and host range complexity. Here, we summarize the recent advances in our understanding of the complexities of Xap, including studies of the molecular features that result after comparative phenotypic and genomic analyses, in order to obtain a clearer overview of the bacterial behaviour and infection mechanism in the context of the X. arboricola species. TAXONOMIC STATUS: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species X. arboricola; Pathovar pruni. HOST RANGE AND SYMPTOMS: Xap infects most Prunus species, including apricot, peach, nectarine, plum and almond, and occasionally cherry. Symptoms are found on leaves, fruits, twigs and branches or trunks. In severe infections, defoliation and fruit dropping may occur. DISTRIBUTION: Bacterial spot of stone fruits and almond is worldwide in distribution, with Xap being isolated in Africa, North and South America, Asia, Europe and Oceania. It is a common disease in geographical areas in which stone fruits and almonds are grown. Xap is listed as a quarantine organism in several areas of the world. GENOME: The genomes of six isolates from Xap have been publicly released. The genome consists of a single chromosome of around 5 000 000 bp with 65 mol% GC content and an extrachromosomal plasmid element of around 41 000 bp with 62 mol% GC content. Genomic comparative studies in X. arboricola have allowed the identification of putative virulence components associated with the infection process of bacterial spot of stone fruits and almond. DISEASE CONTROL: Management of bacterial spot of stone fruits and almond is based on an integrated approach that comprises essential measures to avoid Xap introduction in a production zone, as well as the use of tolerant or resistant plant material and chemical treatments, mainly based on copper compounds. Management programmes also include the use of appropriate cultivation practices when the disease is already established. Finally, for the effective control of the disease, appropriate detection and characterization methods are needed for use in symptomatic or asymptomatic samples as a first approach for pathogen exclusion. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTPR; http://www.cost.eu/COST_Actions/ca/CA16107; http://www.xanthomonas.org.

Elevation of three subspecies of Lonsdalea quercina to species level: Lonsdalea britannica sp. nov., Lonsdalea iberica sp. nov. and Lonsdalea populi sp. nov.

Four subspecies of Lonsdalea quercina (L. quercina subsp. quercina, L. quercina subsp. britannica, L. quercina subsp. iberica and L. quercina subsp. populi) were studied by genome sequence-derived average nucleotide identity (ANI), phylogenetic analysis based on 16S rRNA gene sequences, multilocus sequence analysis (MLSA) and phenotypic characteristics. In phylogenetic trees, based on 16S rRNA gene sequences, and in MLSA data, the four subspecies were divided into four subclusters in the Lonsdalea clade with high boot strap support. The ANI values between the four subspecies were 88.71-93.38 %, respectively, lower than the proposed species boundary ANI cut-off (95-96 %) that is considered the most important criterion to reclassify these subspecies at the species level. It is proposed that three subspecies be elevated to the species level as Lonsdalea britannica sp. nov. (type strain R-43280T=LMG 26267T=NCPPB 4481T=CFCC 10822T), Lonsdalea iberica sp. nov. (type strain R-44166T=LMG 26264T=NCPPB 4490T=CFCC 10824T) and Lonsdalea populi sp. nov. (type strain NY060T=DSM 25466T=NCAIM B 02483T=LMG 27349T=CFCC 13125T).

Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors.

Xanthomonas arboricola is a plant-associated bacterial species that causes diseases on several plant hosts. One of the most virulent pathovars within this species is X. arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruit trees and almond. Recently, a non-virulent Xap-look-a-like strain isolated from Prunus was characterized and its genome compared to pathogenic strains of Xap, revealing differences in the profile of virulence factors, such as the genes related to the type III secretion system (T3SS) and type III effectors (T3Es). The existence of this atypical strain arouses several questions associated with the abundance, the pathogenicity, and the evolutionary context of X. arboricola on Prunus hosts. After an initial characterization of a collection of Xanthomonas strains isolated from Prunus bacterial spot outbreaks in Spain during the past decade, six Xap-look-a-like strains, that did not clustered with the pathogenic strains of Xap according to a multi locus sequence analysis, were identified. Pathogenicity of these strains was analyzed and the genome sequences of two Xap-look-a-like strains, CITA 14 and CITA 124, non-virulent to Prunus spp., were obtained and compared to those available genomes of X. arboricola associated with this host plant. Differences were found among the genomes of the virulent and the Prunus non-virulent strains in several characters related to the pathogenesis process. Additionally, a pan-genomic analysis that included the available genomes of X. arboricola, revealed that the atypical strains associated with Prunus were related to a group of non-virulent or low virulent strains isolated from a wide host range. The repertoire of the genes related to T3SS and T3Es varied among the strains of this cluster and those strains related to the most virulent pathovars of the species, corylina, juglandis, and pruni. This variability provides information about the potential evolutionary process associated to the acquisition of pathogenicity and host specificity in X. arboricola. Finally, based in the genomic differences observed between the virulent and the non-virulent strains isolated from Prunus, a sensitive and specific real-time PCR protocol was designed to detect and identify Xap strains. This method avoids miss-identifications due to atypical strains of X. arboricola that can cohabit Prunus.

Draft Genome Sequence of Two Strains of Xanthomonas arboricola Isolated from Prunus persica Which Are Dissimilar to Strains That Cause Bacterial Spot Disease on Prunus spp.

The draft genome sequences of two strains of Xanthomonas arboricola, isolated from asymptomatic peach trees in Spain, are reported here. These strains are avirulent and do not belong to the same phylogroup as X. arboricola pv. pruni, a causal agent of bacterial spot disease of stone fruits and almonds.

Multilocus Variable Number of Tandem Repeat Analysis Reveals Multiple Introductions in Spain of Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot Disease of Stone Fruits and Almond.

Xanthomonas arboricola pv. pruni is the causal agent of the bacterial spot disease of stone fruits, almond and some ornamental Prunus species. In Spain it was first detected in 2002 and since then, several outbreaks have occurred in different regions affecting mainly Japanese plum, peach and almond, both in commercial orchards and nurseries. As the origin of the introduction(s) was unknown, we have assessed the genetic diversity of 239 X. arboricola pv. pruni strains collected from 11 Spanish provinces from 2002 to 2013 and 25 reference strains from international collections. We have developed an optimized multilocus variable number of tandem repeat analysis (MLVA) scheme targeting 18 microsatellites and five minisatellites. A high discriminatory power was achieved since almost 50% of the Spanish strains were distinguishable, confirming the usefulness of this genotyping technique at small spatio-temporal scales. Spanish strains grouped in 18 genetic clusters (conservatively delineated so that each cluster contained haplotype networks linked by up to quadruple-locus variations). Furthermore, pairwise comparisons among populations from different provinces showed a strong genetic differentiation. Our results suggest multiple introductions of this pathogen in Spain and redistribution through contaminated nursery propagative plant material.

Comparative Genomic and Phenotypic Characterization of Pathogenic and Non-Pathogenic Strains of Xanthomonas arboricola Reveals Insights into the Infection Process of Bacterial Spot Disease of Stone Fruits.

Xanthomonas arboricola pv. pruni is the causal agent of bacterial spot disease of stone fruits, a quarantinable pathogen in several areas worldwide, including the European Union. In order to develop efficient control methods for this disease, it is necessary to improve the understanding of the key determinants associated with host restriction, colonization and the development of pathogenesis. After an initial characterization, by multilocus sequence analysis, of 15 strains of X. arboricola isolated from Prunus, one strain did not group into the pathovar pruni or into other pathovars of this species and therefore it was identified and defined as a X. arboricola pv. pruni look-a-like. This non-pathogenic strain and two typical strains of X. arboricola pv. pruni were selected for a whole genome and phenotype comparative analysis in features associated with the pathogenesis process in Xanthomonas. Comparative analysis among these bacterial strains isolated from Prunus spp. and the inclusion of 15 publicly available genome sequences from other pathogenic and non-pathogenic strains of X. arboricola revealed variations in the phenotype associated with variations in the profiles of TonB-dependent transporters, sensors of the two-component regulatory system, methyl accepting chemotaxis proteins, components of the flagella and the type IV pilus, as well as in the repertoire of cell-wall degrading enzymes and the components of the type III secretion system and related effectors. These variations provide a global overview of those mechanisms that could be associated with the development of bacterial spot disease. Additionally, it pointed out some features that might influence the host specificity and the variable virulence observed in X. arboricola.