Efficacy of Pseudomonas chlororaphis subsp. aureofaciens SH2 and Pseudomonas fluorescens RH43 isolates against root-knot nematodes (Meloidogyne spp.) in kiwifruit

The Root-knot nematodes, Meloidogyne spp., are parasites of many crops and orchards, including kiwifruit trees. The Islamic Republic of Iran is among the leading kiwifruit producers in the world and M. incognita has been found as the dominant species responsible for severe loss of this crop. In order to evaluate the eff ectiveness of antagonistic bacteria on larval mortality, number of galls per plant and egg masses of nematode reduction, fifty local bacterial strains were isolated from root surrounding soils of kiwifruit plants in the northern production areas in Iran. Bacterial antagonists were characterized by morphological, physiological, biochemical and molecular methods. Two representative strains, showing the best nematicidal activity, were identif ed as Pseudomonas chlororaphis subsp. aureofaciens (isolate Sh2) and Pseudomonas fluorescens (isolate Rh43). They increased the percentage of larval mortality to 56:38% and 54:28% respectively in assays in vitro and showed excellent performance also in vivo with consistent reduction of number of galls (67:31% and 55:63%, respectively) and egg mass (86:46% and 84:29%, respectively) in plants. This study indicates that Pseudomonas chlororaphis subsp. aureofaciens isolate Sh2 and Pseudomonas fluorescens isolate Rh43 are good potential biocontrol agents for containing root-knot nematodes in kiwifruit trees.peer-reviewe

Caracterización molecular de la pérdida del poder patógeno en Agrobacterium tumefaciens

Agrobacterium tumefaciens es una bacteria fitopatógena causante de tumores en el cuello y raíces de muchas especies vegetales de interés económico. En los viveros e invernaderos es donde más daños produce, al aparecer episodios epidémicos que pueden impedir la comercialización de producciones enteras, porque las plantas afectadas por esta enfermedad no pueden ser comercializadas. Los estudios epidemiológicos y de dinámica de poblaciones del patógeno son poco abundantes y han incidido en aspectos del desarrollo y mantenimiento de la enfermedad producida por la bacteria. Aunque se ha señalado el predominio de cepas no patógenas frente a patógenas en ambientes en los que las cepas productoras de la enfermedad verían favorecido su mantenimiento, apenas se ha investigado la pérdida del poder patógeno en aislados de tumores. Por tanto, en esta memoria se ha pretendido ampliar el conocimiento sobre la aparición de cepas no patógenas a partir de cepas patógenas en tumores de diversas especies vegetales, huéspedes habituales y no habituales. El objetivo era comprobar si este fenómeno se presenta en la mayoría de cepas de A. tumefaciens, y si existen interacciones con plantas o de otro tipo que favorezcan su aparición. En primer lugar, se ha puesto a punto un método de identificación de cepas individuales de A. tumefaciens, por medio de un sistema de RAPDs, que emplea iniciadores inespecíficos, y que se ha comprobado que es eficaz para diferenciar entre cepas del género Agrobacterium sp. En segundo lugar, se han llevado a cabo varios experimentos de inoculación de diversos huéspedes vegetales y, de los tumores producidos, se han aislado colonias de las que se ha estudiado el fenotipo patógeno, para comprobar la frecuencia de aparición de este fenómeno y estudiar los cambios que a nivel molecular habían sufrido. También se han llevado a cabo experimentos in vitro, mediante siembras sucesivas en un medio general, para ver si la pérdida del fenotipo patógeno se produce de forma espontánea. El sistema de identificación mediante RAPDs ha permitido comprobar que, en el análisis de más de 5.000 colonias no patógenas procedentes de los aislamientos realizados en los tumores, solo siete de estas colonias se correspondía con la que previamente se había inoculado en alguno de los huéspedes. El resto eran cepas de Agrobacterium no patógenas de las que se desconoce su origen. Los resultados de este trabajo muestran por tanto, que el fenómeno de la pérdida del poder patógeno se produce, en las condiciones y cepas estudiadas, de forma muy escasa. La caracterización molecular de los mutantes ha mostrado que 6 de 7 presentan cambios en su plásmido Ti. Entre ellos, inserciones de elementos transponibles en genes de virulencia o mutaciones puntuales. El caso más llamativo es el de dos mutantes que presentan grandes cambios en su plásmido Ti, con un tamaño doble del de la cepa salvaje. El resto de mutantes presenta variaciones en el tamaño o número de plásmidos respecto a las cepas parentales. Los cambios ocurridos parecen implicar grandes reorganizaciones en el genoma de los mutantes, no sólo a nivel del plásmido Ti

Caracterización molecular de la pérdida del poder patógeno en Agrobacterium tumefaciens

Agrobacterium tumefaciens es una bacteria fitopatógena causante de tumores en el cuello y raíces de muchas especies vegetales de interés económico. En los viveros e invernaderos es donde más daños produce, al aparecer episodios epidémicos que pueden impedir la comercialización de producciones enteras, porque las plantas afectadas por esta enfermedad no pueden ser comercializadas. Los estudios epidemiológicos y de dinámica de poblaciones del patógeno son poco abundantes y han incidido en aspectos del desarrollo y mantenimiento de la enfermedad producida por la bacteria. Aunque se ha señalado el predominio de cepas no patógenas frente a patógenas en ambientes en los que las cepas productoras de la enfermedad verían favorecido su mantenimiento, apenas se ha investigado la pérdida del poder patógeno en aislados de tumores. Por tanto, en esta memoria se ha pretendido ampliar el conocimiento sobre la aparición de cepas no patógenas a partir de cepas patógenas en tumores de diversas especies vegetales, huéspedes habituales y no habituales. El objetivo era comprobar si este fenómeno se presenta en la mayoría de cepas de A. tumefaciens, y si existen interacciones con plantas o de otro tipo que favorezcan su aparición. En primer lugar, se ha puesto a punto un método de identificación de cepas individuales de A. tumefaciens, por medio de un sistema de RAPDs, que emplea iniciadores inespecíficos, y que se ha comprobado que es eficaz para diferenciar entre cepas del género Agrobacterium sp. En segundo lugar, se han llevado a cabo varios experimentos de inoculación de diversos huéspedes vegetales y, de los tumores producidos, se han aislado colonias de las que se ha estudiado el fenotipo patógeno, para comprobar la frecuencia de aparición de este fenómeno y estudiar los cambios que a nivel molecular habían sufrido. También se han llevado a cabo experimentos in vitro, mediante siembras sucesivas en un medio general, para ver si la pérdida del fenotipo patógeno se produce de forma espontánea. El sistema de identificación mediante RAPDs ha permitido comprobar que, en el análisis de más de 5.000 colonias no patógenas procedentes de los aislamientos realizados en los tumores, solo siete de estas colonias se correspondía con la que previamente se había inoculado en alguno de los huéspedes. El resto eran cepas de Agrobacterium no patógenas de las que se desconoce su origen. Los resultados de este trabajo muestran por tanto, que el fenómeno de la pérdida del poder patógeno se produce, en las condiciones y cepas estudiadas, de forma muy escasa. La caracterización molecular de los mutantes ha mostrado que 6 de 7 presentan cambios en su plásmido Ti. Entre ellos, inserciones de elementos transponibles en genes de virulencia o mutaciones puntuales. El caso más llamativo es el de dos mutantes que presentan grandes cambios en su plásmido Ti, con un tamaño doble del de la cepa salvaje. El resto de mutantes presenta variaciones en el tamaño o número de plásmidos respecto a las cepas parentales. Los cambios ocurridos parecen implicar grandes reorganizaciones en el genoma de los mutantes, no sólo a nivel del plásmido Ti

The Viable but Non-culturable State in Xanthomonas citri subsp. citri is a Reversible State Induced by Low Nutrient Availability and Copper Stress Conditions

Xcc (Xanthomonas citri subsp. citri) causes citrus bacterial canker, a leaf, stem and fruit spotting disease that affects most commercial citrus species and cultivars. Copper compounds, widely used for management of this pathogen, have been reported as inducers of a VBNC (viable but non-culturable state) in plant pathogenic bacteria. VBNC may be considered as a state preceding bacterial death or as a survival mechanism under adverse conditions. Several experiments were performed to characterize the reversibility and persistence of the VBNC state in Xcc. VBNC was induced in low nutrient medium or with amendment of copper at concentrations used for field disease control. The VBNC condition was demonstrated to persist up to 150 days after copper treatment and was reversed after the addition of culture media without copper or amendment with citrus leaf extract. Xcc viability was evaluated by recovery of colonies on culture media, confirmed by membrane integrity, respiratory activity and by real-time RT-PCR targeting a sequence from the gumD gene. Besides, the colonies recovered were pathogenic on citrus leaves. These results confirm that the VBNC state in Xcc is inducible and reversible and therefore may occur in the phyllosphere when Xcc is under copper stress or starvation

Innovative tools for detection of plant pathogenic viruses and bacteria

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative- PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is microarray technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms

Efficacy of Pseudomonas chlororaphis subsp. aureofaciens SH2 and Pseudomonas fluorescens RH43 isolates against root-knot nematodes (Meloidogyne spp.) in kiwifruit

The Root-knot nematodes, Meloidogyne spp., are parasites of many crops and orchards, including kiwifruit trees. The Islamic Republic of Iran is among the leading kiwifruit producers in the world and M. in- cognita has been found as the dominant species responsible for severe loss of this crop. In order to evaluate the effectiveness of antagonistic bacteria on larval mortality, number of galls per plant and egg masses of nematode reduction, fifty local bacterial strains were isolated from root surrounding soils of kiwifruit plants in the northern production areas in Iran. Bacterial antagonists were characterized by morphological, physiological, biochemical and molecular methods. Two representative strains, showing the best nematicidal activity, were identifed as Pseudomonas chlororaphis subsp. aureofaciens (isolate Sh2) and Pseudomonas fluorescens (isolate Rh43). They increased the percentage of larval mortality to 56.38% and 54.28% respectively in assays in vitro and showed excellent performance also in vivo with consistent reduction of number of galls (67.31% and 55.63%, respectively) and egg mass (86.46% and 84.29%, respectively) in plants. This study indicates that Pseudomonas chlororaphis subsp. aureofaciens isolate Sh2 and Pseudomonas fluorescens isolate Rh43 are good potential biocontrol agents for containing root-knot nematodes in kiwifruit trees

La instrucción en el proceso penal de menores como experiencia modelo para los procesos ordinarios

El Ministerio Fiscal en España ha sido una figura muy controvertida en los últimos años. Dicha controversia gira en torno al aumento de competencias que los Fiscales han experimentado y a una posible atribución de la función instructora que hasta ahora ha estado en manos del Juez Instructor de la caus

Comparative study of genetic diversity of Clavibacter michiganensis subsp michiganensis isolates from the Canary Islands by RAPD-PCR, BOX-PCR and AFLP

Molecular characterization of seedborne pathogens is an important issue when discerning their origin and tracking the spread of a disease. In the Canary Islands (Spain), Clavibacter michiganensis subsp. michiganensis (Cmm) was first detected in 2002, causing severe losses in many tomato‐growing areas. Fifty four strains of this bacterium isolated from 2002 to 2007 and 19 strains from different countries were characterized for genetic diversity. RAPD‐PCR, BOX‐PCR and AFLP provided differentiation among Cmm strains whereas no differences were observed with ERIC‐PCR, REP‐PCR and 16S‐23S ITS PCR‐RFLP. RAPD‐PCR and BOX‐PCR revealed high homogeneity among the Canary Island strains (>80 and >75% of similarity, respectively) which could not be grouped based on tomato cultivar, location or year of isolation. By contrast, strains of Cmm from other countries displayed high diversity, providing several clusters, most of which were composed of a single strain. Similarly, AFLP analysis of 29 selected strains of Cmm gave the same profile for the Canarian ones (>90% of similarity) whereas high polymorphism was obtained with strains from different countries. Moreover, two strains, one from the USA and another from Spain, were related to the Canarian strains, according to RAPD‐PCR (>60% of similarity), BOX‐PCR (>75%) and AFLP analysis (>90%), suggesting a common origin. The circumstances under which the Cmm outbreaks occurred in the Canary Islands and the high homogeneity observed among the Canarian strains would suggest that the bacterium was introduced into the region from only one origin

Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses?

Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future