Molecular and phenotypic characterization of Agrobacterium species from vineyards allows identification of typical Agrobacterium vitis and atypical biovar 1 strains.

AIM: To molecularly and phenotypically characterize a selection of Agrobacterium-like isolates from grapevine canes, crowns, soil and tumours in plants grown under cold conditions. METHODS AND RESULTS: Most of the strains were biovar 3 (Agrobacterium vitis), and the remaining were atypical biovar 1 (Agrobacterium tumefaciens). All of them were tumourigenic on grapevine plants but differences in other hosts were observed. Chromosomal and plasmid-borne traits were analysed by gene amplification with four primer sets. Detection of the pectin enzyme hydrolase gene clearly distinguished A. vitis from the atypical A. tumefaciens. Regarding the virulence sensor gene, limited host range tumour-inducing plasmids were found in the atypical isolates. About opine utilization, most A. vitis and some A. tumefaciens contained octopine/cucumopine plasmids, but the nopaline-type was only detected in one A. tumefaciens. CONCLUSIONS: The A. vitis strains were molecularly and phenotypically more homogeneous than those of A. tumefaciens, the latter displaying some typical A. vitis characteristics, suggesting an adaptation to life in grapevine. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work will help to improve detection procedures of the pathogen, and demonstrate the pathogen diversity in cold vineyards, laying the groundwork for epidemiological studies and development of control strategies of the crown and cane gall disease.

[Validation, adaptation and translation of the MacCAT-T into Spanish: a tool to assess the ability to make health decisions]. / Validación, adaptación y traducción al castellano del MacCAT-T: herramienta para evaluar la capacidad en la toma de decisiones sanitarias.

INTRODUCTION: Capacity assessment is an essential element of the informed consent process and is the duty of the physician. The MacCAT-T instrument explores four skills needed to consent a treatment. There is no Spanish version, and the main objective of this work is to validate, adapt and translate the MacCAT-T into Spanish. MATERIAL AND METHODS: The MacCAT-T was translated into Spanish and then back-translated into English. It was validated as regards its appearance and content (by 15 experts), construct (inter-rater reliability and internal consistency) and criteria (the validity of an instrument by comparing it to some external criterion, in this case the Mini Examen Cognoscitivo de Lobo). Ninety medical and surgical outpatients over 18 years were included with no deficits of expression and/or severe disorders of consciousness that did not allow them to be interviewed. RESULTS: They have been optimal considering different types of validity. The average application time was between 9 and 13minutes. DISCUSSION: Data are consistent with those obtained in other applications of MacCAT-T in the English language and facilitate the provision of a Spanish tool for assessing capacity.

Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods.

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.

Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material.

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 microl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction.

A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol-chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri) with a sensitivity of 10(2)-10(3) cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies.

A simple and efficient PCR method for the detection of Agrobacterium tumefaciens in plant tumours.

A simple PCR protocol was developed for identifying Agrobacterium as the causal agent of the tumours produced by this bacterium in plant material. The sensitivity of this method was compared with that of bacterial isolation using common and selective media with a previous enrichment step. More than 200 samples from tumours of naturally infected and inoculated plants from several hosts including almond, peach x almond hybrids, apricot, rose, tobacco, tomato, raspberry, grapevine and chrysanthemum, were analysed by both methods. PCR was the most efficient method for detecting the bacterial aetiology of the plant tumours. Agrobacterium tumefaciens was better detected in crown and root tumours than in aerial tumours with all the methods assayed in inoculated plants. A comparison between the efficiency of the diagnosis by analysing pieces from the external and internal part of the tumour showed no differences between them.