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Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent., Plan Nacional de I+D+I of the former Ministerio de Ciencia e Innovación, Spain. Grant Number: AGL2010-21848-C02-01

The purpose of this study was to assess the stability of mRNA and rRNA for evaluation of viability for Xanthomonas citri subsp. citri (Xcc). Total RNA from Xcc suspensions subjected to different stress treatments (high temperature or chemical treatment with sodium orthophenylphenate at different concentrations) was extracted at different time periods post-treatment (0, 3, 24 and 48h) and analysed by quantitative real-time reverse transcription PCR (Q-RT-PCR). Primers were designed from selected fragments of rRNA and mRNA from genes involved in bacterial fitness, virulence or general metabolic mechanisms (gumD, rpfB, avrBs2 and gyrB). After stress treatment, only a 445-bp fragment from the gumD mRNA was detected in live Xcc cells specifically, whereas other RNA fragments, as well as DNA targets, were detected in both viable and nonviable cells. Statistical analyses demonstrated that the amount of some transcripts from genes involved in xanthan synthesis, pathogenicity factor regulation and DNA processing was significantly reduced after lethal treatments. The amplification of the 445-bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc; the product was detected specifically from viable bacteria on leaf and citrus fruit surfaces and in citrus canker lesions. Instability of long RNA fragments can be used as a practical tool for the study of survival of citrus canker bacteria or for diagnostic purposes when the presence of viable bacteria needs to be confirmed. © 2011 The Authors. Plant Pathology © 2011 BSPP.

Aims To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. Methods and Results Citrus fruits with canker-like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real-time PCR with SYBR Green or a TaqMan probe, were compared. Canker-like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real-time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39-52 lesions depending on the protocol employed. Conclusions An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real-time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. Significance and Impact of the Study This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present. © 2007 The Authors.

Xanthomonas arboricola es una especie de bacterias asociada a plantas. Dentro de la misma se han descrito nueve grupos infraespecíficos o patovares causantes de enfermedad en diversas especies vegetales. Asimismo, se han identificado tanto cepas patógenas como no patógenas que no se incluyen en ninguno de estos grupos. Entre los grupos definidos como fitopatógenos se encuentra X. arboricola pv. pruni, agente causal de la mancha bacteriana de los frutales de hueso y el almendro. Este patógeno de aparición reciente en España, está considerado como organismo de cuarentena en la Unión Europea. En este trabajo se ha caracterizado, tanto molecular como fenotípicamente, una serie de cepas de X. arboricola aisladas de Prunus spp. Un análisis de secuencias multilocus determinó que junto a las cepas de X. arboricola pv. pruni, existían, cohabitando en los mismos huéspedes, algunas cepas de la misma especie que no pertenecían a ninguno de los grupos que se integran en X. arboricola. Además, según la caracterización fenotípica basada en aspectos relacionados con la infección bacteriana, estas cepas no presentaban la capacidad para producir enfermedad en Prunus spp. La caracterización inicial de las cepas de Xanthomonas aisladas de Prunus spp. fue aprovechada para seleccionar una cepa patógena de almendro y otra de ciruelo, CITA 33 e IVIA 2626.1 respectivamente, y tres cepas no patógenas, CITA 14, aislada de P. mahaleb, así como CITA 44 y CITA 124, aisladas de melocotonero. El estudio de genómica comparativa de estas cepas permitió identificar factores que podían estar relacionados con el desarrollo de la enfermedad de la mancha bacteriana de los frutales del género Prunus. El análisis filogenético basado en el “core genome” de la especie, así como un análisis de similitud basado en la presencia de los genes del pan genoma entre las diversas cepas de X. arboricola, permitió corroborar que las cepas no patógenas formaban un grupo filogenético diferente al del patovar pruni, junto con otras cepas de baja virulencia o no patógenas aisladas de diferentes plantas huésped. Además, la comparación de los diferentes genomas permitió encontrar diferencias en los genes relacionados con mecanismos implicados en los estados iniciales de la infección, tales como transportadores TonB-dependientes (TBDTs), sensores del sistema regulador de dos componentes (STCRs) y proteínas aceptoras de grupos metilo (MCPs). Asimismo, se observó en el patovar pruni la presencia de adhesinas no fimbrilares, como FhaB2, que no estaban presentes en las cepas no patógenas. De la misma manera, se encontró variación en cuanto al contenido de genes asociados al pilus tipo IV entre ambos grupos bacterianos. Respecto a la estructura del flagelo, tanto las cepas patógenas como las no patógenas presentaron el mismo perfil en aquellos genes relacionados con su estructura y biogénesis, sin embargo, se observó un cambio en la secuencia de aminoácidos de la proteína flagelina de X. arboricola pv. pruni, la cual podría estar relacionada con la respuesta de la bacteria frente a los mecanismos de defensa de la planta. De igual manera, se encontraron diferencias entre las cepas patógenas y no patógenas en caracteres asociados con etapas tardías de la infección. Una de ellas fue la presencia de diferentes perfiles de genes codificantes de enzimas degradadoras de la pared celular. Otra diferencia encontrada entre ambos grupos de organismos fue la referente al sistema de secreción tipo III y sus efectores. Las cepas patógenas, identificadas como pertenecientes al patovar pruni, presentaron todos los genes asociados a los componentes estructurales y de regulación de este sistema de secreción, así como un total de 21 efectores tipo III. Sin embargo, dos de las cepas no patógenas CITA 44 y CITA 124, no presentaron sistema de secreción tipo III, ni ninguno de los efectores tipo III descritos en Xanthomonas. La cepa no patógena CITA 14 presentó los componentes génicos para un sistema de secreción tipo III funcional pero se identificó solamente un repertorio de seis efectores. Se seleccionó además el gen xopE3, que solamente está presente en el patovar pruni y se desarrolló un protocolo de PCR tiempo real que permitió aumentar la especificidad del protocolo de diagnóstico para la mancha bacteriana de los frutales de hueso y el almendro, al permitir diferenciar de manera específica y sensible las cepas causantes de la enfermedad de aquellas cepas no patógenas que cohabitan los frutales del género Prunus. Este estudio ha permitido conocer una serie de componentes genéticos que podrían estar asociados con el desarrollo de la enfermedad de la mancha bacteriana de los frutales del género Prunus. De la misma manera ha permitido profundizar en el conocimiento sobre la diversidad biológica de X. arboricola y ha generado datos que pueden marcar las pautas de cómo ha podido ser la evolución de la patogenicidad y especificidad de huésped en esta especie de bacterias. Los datos generados han permitido además el desarrollo de un nuevo método de diagnóstico de esta enfermedad más preciso basado en PCR en tiempo real. ABSTRACT Xanthomonas arboricola is a species of plant-associated bacteria, conformed by at least nine infraspecific groups or pathovars. The species encompasses groups of bacteria with diverse plant host range, as well as pathogenic and non-pathogenic strains that does not belong to any of these nine pathovars. Within the plant pathogenic groups, X. arboricola pv. pruni, which is the causal agent of bacterial spot disease of stone fruits and almond, has been recently determined as present in Spain and it is regulated as a quarantine pathogen in the European Union. In this work, a group of Spanish strains of X. arboricola, isolated from Prunus spp. has been characterized using molecular and phenotypic approaches. An initial molecular characterization, conducted by using a multilocus sequence type analysis, determined that there were two groups of strains that cohabited in these hosts. The first one was composed by strains of X. arboricola pv. pruni and the second one by atypical strains that did not belong to any of the nine well-stablished pathovars of X. arboricola. In addition, according to the phenotypic analysis, none of the atypical strains were able to cause disease on Prunus spp. Based on these results, two pathogenic strains of X. arboricola pv. pruni, CITA 33 and IVIA 2626.1, isolated from peach and plum respectively, and three non-pathogenic strains of X. arboricola, CITA 14, isolated from P. mahaleb and CITA 44 and CITA 124, both isolated from peach, were selected for whole genome sequencing. The genome comparative analysis of these strains permitted to find those genomic characters that could be associated with the development of bacterial spot disease of stone fruits and almond. A phylogenetic analysis based in the core genome of X. arboricola as well as a similarity analysis based on the pan-genome of this species, corroborated that the atypical non-pathogenic strains formed cluster which was different to the one composed by the strains of the pathovar pruni. These non-pathogenic strains clustered in a group with other non-pathogenic or low-pathogenic strains isolated from a wide range of plant hosts. Additionally, according to the gene content comparison, variants in the genes related to the different stages of the disease process were found. For instance, both groups showed a different repertoire of genes associated with the sensing of the environmental stimuli, such as those related to the Ton-B dependent transporters (TBDTs), the sensors of the two-component regulatory system (STCRs) and the methyl accepting chemotaxis proteins (MCPs). Beside this, in X. arboricola pv. pruni, some non-fimbrial adhesins, like FhaB2, as well as some components related to the type IV pilus that were absent in the non-pathogenic strains, were found. According to the gene content associated with the flagella structure and regulation, no differences among gene profile of both groups of organisms were elucidated, however, in the pathovar pruni, a variant in the flagellin protein sequence. This variant could be associated with the ability of this bacterial group to avoid the initial steps of the plant defence process. In the same way, variations in the gene content of some key characters related to late stages of the disease process were found. One of the differences was the dissimilar profile of cell wall degrading enzymes between pathogenic and non-pathogenic strains. Moreover, the most important difference observed between these two groups was found in the gene content associated with the type III secretion system and its related effectors. Plant pathogenic strains of X. arboricola pv. pruni harboured all the genetic components related to the structure and regulation of the type III secretion system as well as in a repertoire of 21 type III effectors. Nevertheless, non-pathogenic strains CITA 44 and CITA 124 did not harbour this secretion system or none of the type III effectors described in Xanthomonas. In the case of the non-pathogenic strain CITA 14, this contained all the genes related to a functional type three secretion system but a reduced profile of effectors composed by only six genes was determined. In addition, the gene xopE3, which is present only in X. arboricola pv pruni, was selected and used to design a real-timer PCR protocol which permitted to improve the specificity of the available protocol to identify the bacteria associated to the spot disease of stone fruit and almond. This protocol allowed to perform a specific and sensitive differentiation of the disease-associated strains from those non-pathogenic strains that cohabited on Prunus spp. This study provides a global overview of the genetic components that could be associated with the development of the bacterial spot disease of Prunus. At the same time, it reveals new insights in some other elements of the biology of this bacterial group such as its biological diversity and the potential process associated with the evolution of pathogenicity and host specificity. All the data generated in this investigation has also permitted to develop a new and more accurate real time PCR diagnostic tool for this disease.