Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

<p>Abstract</p> <p>Background</p> <p>Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is <it>Xanthomonas citri </it>subsp. <it>citri</it>, while <it>Xanthomonas fuscans </it>subsp. <it>aurantifolii</it>, strain B causes type B CBC and <it>Xanthomonas fuscans </it>subsp. <it>aurantifolii </it>strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment.</p> <p>Results</p> <p>A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure <it>Xcc </it>DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes.</p> <p>Conclusions</p> <p>The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide.</p

Ectopic expression of GmNHX3 and GmNHX1, encoding two Glycine max Na+/H+ vacuolar antiporters, improves water deficit tolerance in Arabidopsis thaliana

The importance of Na+/H+ antiporters in salt tolerance in plants has been demonstrated in many studies, but much less is known about their protective role during drought stress. To study their possible contribution to water deficit tolerance, two closely related soybean Na+/H+ antiporters belonging to the intracellular NHX exchanger protein family, GmNHX3 and GmNHX1, were evaluated in transgenic Arabidopsis thaliana. A. thaliana plants ectopically expressing GmNHX3 or GmNHX1 displayed a more drought-tolerant phenotype compared to wild-type plants, which was accompanied by an increase in relative water content and chlorophyll content during stress conditions. Both GmHNX1 and GmHNX3 transgenic lines accumulated higher amounts of Na+ and K+ cations, showed increased antioxidant enzyme activities and less membrane damage due to lipid peroxidation under water deficit, as compared to non-transformed plants. Furthermore, plants expressing GmNHX3 showed an increased sensitivity to abscisic acid as deduced from stomatal closure and seed germination inhibition studies. Finally, a significant up-regulation of abiotic stress-related genes was observed in both transgenic lines compared to wild-type plants in response to abscisic acid and mannitol treatments. These results demonstrate that GmNHX3 and GmNHX1 antiporters confer protection during drought stress in A. thaliana and hence are potential genetic targets to improve drought tolerance in soybean and other crops.Fil: Pardo, Esteban Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Pérez Borroto, Lucía Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Fleitas, L.. Universidad de la República; UruguayFil: Gallino, J. P.. Universidad de la República; UruguayFil: Machi, S.. Universidad de la República; UruguayFil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Welin, Björn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentin

Controlled synthesis of the DSF cell–cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell–cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence

Diversidad genética del complejo de virosis asociadas a la enfermedad del mosaico de la caña de azúcar en Tucumán, Argentina Genetic diversity of sugarcane mosaic virus complex in Tucuman, Argentina

El mosaico de la caña de azúcar es una de las enfermedades sistémicas de mayor importancia de este cultivo. El virus del mosaico de la caña de azúcar (SCMV) y el virus del mosaico del sorgo (SrMV) son los principales agentes causales de esta enfermedad. Mediante RT-PCR-RFLP (siglas del "reverse transcriptase-polymerase chain reaction"-"restriction fragment length polymorphism") y secuenciación del gen de la proteína de la cápside, se analizaron muestras de caña de azúcar con síntomas de mosaico para evaluar la presencia y estimar la diversidad genética de ambos virus en el área cañera de Tucumán. Noventa y tres por ciento de las muestras resultaron positivas para SCMV al utilizar los cebadores específicos SCMVF4/R3 y 33% de ellas exhibió el perfil RFLP de la raza E. El resto de las muestras presentó nueve perfiles diferentes que no se correspondieron con los de las razas descritas. El análisis de la secuencia indicó que 20% de las muestras de perfil RFLP desconocido presentó una alta identidad con la raza D, mientras que las muestras restantes resultaron divergentes entre sí. Mediante microscopía electrónica de transmisión, se confirmó la presencia de los típicos viriones flexuosos de los potyvirus. Asimismo, se detectó la presencia de SrMV en 90% de las muestras con los cebadores específicos y la coexistencia de ambos virus en 85% de las mismas. Mediante RFLP, se identificaron las razas M e I del virus del sorgo en 68% y 14% de las muestras, respectivamente, y en 18% de los casos se detectó coexistencia entre las razas M y H de esta virosis. Solo una de las muestras no amplificó para ninguno de los dos virus, lo que sugeriría la existencia de otro agente responsable de una sintomatología similar.Sugarcane mosaic is one of the most important systemic diseases of sugarcane. Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) are the causal agents of the disease. Sugarcane leaves with mosaic symptoms were analysed by RT-PCR-RFLP (reverse transcriptase-polymerase chain reaction- restriction fragment length polymorphism) and the nucleotide sequences of the coat protein (CP) genes amplified to assess the presence and genetic diversity of both viruses in the sugarcane producing area in Tucumán. Using the primers SCMVR3/F4, 93% of samples were SCMV positive and 33% of them had the E strain RFLP profile, while the rest produced nine different profiles that did not match those of any known strains. Sequence analyses showed that 20% of the samples with the unknown profiles were highly identical to the SCMV D strain, while the rest differed significantly from each other. The presence of the flexuous virions typical of potyviruses was confirmed by transmission electron microscopy. Also, using the specific primers, the presence of SrMV was detected in 90% of the samples, and co-existence of both viruses was found in 85% of the samples. RFLP analysis determined the presence of SrMV strains M and I in 68% and 14% of the samples, respectively, while in approximately 18% of the cases, both M and H strains were present. No RT-PCR product was produced by either SCMV or SrMV primer pairs in one symptomatic sample, suggesting the presence of another pathogen producing similar symptomatology

Diagnóstico molecular de enfermedades sistémicas de la caña de azúcar en la Argentina: ajuste metodológico y aplicaciones Molecular diagnosis of sistemic sugarcane diseases in Argentina: methodology adjustment and applications

Las enfermedades sistémicas representan uno de los principales factores que afectan la producción de caña de azúcar. El conocimiento del estado fitosanitario del cultivo y la identificación correcta de los fitopatógenos son claves para reducir las pérdidas por enfermedades. En este sentido, es fundamental contar con técnicas de diagnóstico sensibles, rápidas y fáciles de ejecutar, para realizar un diagnóstico preciso y precoz. A partir del año 2005, en la Estación Exper imental Agroindustrial Obispo Colombres se incorporó el diagnóstico molecular basado en la reacción en cadena de la polimerasa, para la detección específica de cuatro enfermedades sistémicas de la caña de azúcar: raquitismo de la caña soca (Leifsonia xyli sp. xyli), escaldadura de la hoja (Xanthomonas albilineans), mosaico de la caña de azúcar (Sugarcane mosaic virus, ScMV) y síndrome de la hoja amarilla (Sugarcane yellow leaf virus, ScYLV). En este trabajo, se presenta la optimización metodológica del diagnóstico molecular y se compara su eficiencia con la de la técnica inmunoquímica ELISA. El método molecular mostró mayor sensibilidad para las enfermedades evaluadas, tanto bacterianas como virales. El establecimiento del diagnóstico molecular constituye un avance tecnológico de gran importancia para la industria azucarera regional, ya que no solo ayudará a disminuir la incidencia de dichas enfermedades, sino que también evitará el ingreso de otras nuevas al introducir germoplasma de caña de azúcar desde otras regiones.Systemic diseases represent one of the main factors affecting sugarcane production. The knowledge of crop sanitary conditions and the correct identification of phytopathogens are key factors to reduce losses caused by them. To diagnose diseases as early as possible is crucial, so techniques that are sensitive, fast, accurate and easy to use are essential. Since 2005, molecular diagnosis based on polymerase chain reaction has been applied at Estación Experimental Agroindustrial Obispo Colombres, for detecting four systemic sugarcane diseases: ratoon stunting (Leifsonia xyli sp xyli), leaf scald (Xanthomonas albilineans), Sugarcane mosaic virus (ScMV) and yellow leaf syndrome (Sugar cane yellow leaf virus, ScYLV). This paper presents the methodological optimization of molecular diagnosis and compares its efficiency with that of ELISA immunochemistry technique. The molecular approach showed greater sensitivity for the detection of both the bacterial and viral diseases evaluated. The implementation of molecular analysis constitutes a technological advance for regional sugar industry, which will not only help to reduce disease incidence, but also avoid the entrance of new pathogens whenever sugarcane germplasm is introduced from other regions

Elicitor-Based Biostimulant PSP1 Protects Soybean Against Late Season Diseases in Field Trials

Currently, fungicide application in soybean production accounts for an important amount of global pesticide use, and it is therefore most desirable to find new healthier and more environmental friendly alternatives for the phytosanitary management in this crop. In this study, we present convincing evidence for effective induction of disease protection by the agricultural biostimulant PSP1, a formulation based on the plant-defense eliciting activity of the fungal protease AsES (Acremonium strictum elicitor subtilisin), in multiple field trials in Argentina.PSP1 was shown to combine well with commercial spray adjuvants, an insecticide, a herbicide and fungicides used in Argentinian soybean production without losing any defense-inducing activity, indicating an easy and efficient adaptability to conventional soybean production and disease management in the region. Results from multiple soybean field trials conducted with different elite genotypes at several locations during two consecutive growing seasons, showed that PSP1 is able to induce an enhanced pathogen defense which effectively reduced late season disease (LSD) development in field-grown soybean. This defense response seems to be broad-range as disease development was clearly reduced for at least three different fungi causing LSDs in soybean (Septoria glycines, Cercospora kikuchii and Cercospora sojina). It was noteworthy that application of PSP1 in soybean alone gave a similar protection against fungal diseases as compared to the commercial fungicides included in the field trials and that PSP1 applied together with a fungicide at reproductive stages enhanced disease protection and significantly increased grain yields.PSP1 is the first example of an elicitor-based strategy in order to efficiently control multiple fungal diseases under field conditions in the soybean crop. These results show the feasibility of using induced resistance products as complements or even full-good replacements to currently used chemical pesticides, fulfilling a role as important components of a more sustainable crop disease management system

Evaluación de la variación somaclonal en vitroplantas de caña de azúcar mediante marcadores moleculares Evaluation of somaclonal variation in in vitro produced sugarcane plants through molecular markers

El cultivo in vitro de tejidos vegetales puede producir variación somaclonal, fenómeno que consiste en modificaciones genéticas en las células y tejidos cultivados. Esto puede limitar la aplicación de dicha técnica para la micropopagación masiva, especialmente si la variación provoca un cambio fenotípico de importancia agronómica. En este trabajo se optimizó una metodología basada en la comparación de perfiles de marcadores moleculares AFLP (del inglés "Amplified Fragment Length Polymorphism"), para la detección de la variación somaclonal en vitroplantas de caña de azúcar. Para la optimización de la técnica de AFLP en caña de azúcar, se utilizaron plantas de seis genotipos propagados convencionalmente y dos tipos de muestras: hojas tiernas y meristemas. La variación somaclonal fue evaluada en líneas de vitroplantas de los mismos genotipos al final del cultivo in vitro, luego de seis meses de micropropagación. Con las 19 combinaciones de cebadores utilizadas, se diferenciaron los perfiles moleculares de los seis genotipos. En los plantines micropropagados se detectaron perfiles diferenciales en las variedades LCP85-384 y TUCCP77-42 con 3 de las 19 combinaciones de cebadores utilizadas. Este resultado muestra la validez de la técnica para detectar variantes somaclonales, y deja en evidencia la diferencia de susceptibilidad de los genotipos al cultivo in vitro. Esto permitió ajustar la metodología de micropropagación para cada genotipo multiplicado y asegurar la pureza genética de cada vitroplanta.<br>In vitro culture of plant tissue can produce somaclonal variation, which consists of genetic modifications in cultured cells and tissues. This may constrain the use of this technique in massive micropopagation, especially if such change causes an agronomically relevant phenotypical modification. In this work, a methodology based on the comparison of AFLP (Amplified Fragment Length Polymorphism) molecular marker profiles was developed for detecting somaclonal variation in in vitro propagated sugarcane plants. To optimize AFLP technique application to sugarcane plants, six conventionally propagated genotypes and two types of samples (tender leaves and meristems) were used. Somaclonal variation was determined in micropropagated lines of these genotypes after six months of micropropagation. Molecular profile differentiation of the selected genotypes was achieved with 19 primer combinations. Differential profiles were detected in LCP85-384 and TUCCP77-42 micropropagated lines with 3 of the 19 primer combinations. This result demonstrated that the technique can be used to detect somaclonal variants and that there are different susceptibility levels to in vitro culture among genotypes. Therefore, micropropagation methodology was adjusted to each multiplied genotype so as to ensure genetic purity of in vitro propagated plants

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm forma tion and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.Fil: Conforte, Valeria P. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: Malamud, Florencia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina.Fil: Yaryura, Pablo M. Universidad de Villa María. Centro de Investigaciones y Transferencia de Villa María (CIT - CONICET); Argentina.Fil: Toum Terrones, Laila. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina.Fil: Toum Terrones, Laila. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA - CONICET); Argentina.Fil: Torres, Pablo S. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: De Pino, Verónica. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: Chazarreta, Cristian N. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: Gudesblat, Gustavo E. Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA – CONICET -Estación Experimental Agroindustrial Obispo Colombres (EEAOC); Argentina.Fil: Castagnaro, Atilio P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina.Fil: Castagnaro, Atilio P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA - CONICET); Argentina.Fil: Marano, María Rosa. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Marano, María Rosa. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Vojnov, Adrián A. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina