Colorimetric LAMP Assay for Detection of Xanthomonas phaseoli pv. manihotis in Cassava Through Genomics: A New Approach to an Old Problem.

Bacterial blight caused by Xanthomonas phaseoli pv. manihotis (Xpm) is considered the main bacterial disease that affects cassava, causing significant losses when not properly managed. In the present study, a fast, sensitive, and easy-to-apply method to detect Xpm via colorimetric loop-mediated isothermal amplification (LAMP) was developed. To ensure the use of a unique-to-the-target pathovar core region for primer design, 74 complete genomic sequences of Xpm together with different bacterial species and pathovars were used for comparative genomics. A total of 42 unique genes were used to design 27 LAMP primer sets, from which nine primers were synthesized, and only one (Xpm_Lp1 primer set) showed sufficient efficiency in preliminary tests. The sensitivity, assessed by a serial dilution of the type strain (IBSBF 278) DNA, yielded high sensitivity, detecting up to 100 fg. The LAMP primers showed high specificity, did not cross-react with other bacterial species or other pathovars tested, and amplified only the Xpm isolates. Tests confirmed the high efficiency of the protocol using infected or inoculated macerated cassava leaves without the need for additional sample treatment. The LAMP test developed in this study was able to detect Xpm in a fast, simple, and sensitive way, and it can be used to monitor the disease under laboratory and field conditions.

Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

RXam2, a NLR from cassava (Manihot esculenta) contributes partially to the quantitative resistance to Xanthomonas phaseoli pv. manihotis.

KEY MESSAGE: The overexpression of RXam2, a cassava NLR (nucleotide-binding leucine-rich repeat) gene, by stable transformation and gene expression induction mediated by dTALEs, reduce cassava bacterial blight symptoms. Cassava (Manihot esculenta) is a tropical root crop affected by different pathogens including Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of cassava bacterial blight (CBB). Previous studies have reported resistance to CBB as a quantitative and polygenic character. This study sought to validate the functional role of a NLR (nucleotide-binding leucine-rich repeat) associated with a QTL to Xpm strain CIO151 called RXam2. Transgenic cassava plants overexpressing RXam2 were generated and analyzed. Plants overexpressing RXam2 showed a reduction in bacterial growth to Xpm strains CIO151, 232 and 226. In addition, designer TALEs (dTALEs) were developed to specifically bind to the RXam2 promoter region. The Xpm strain transformed with dTALEs allowed the induction of the RXam2 gene expression after inoculation in cassava plants and was associated with a diminution in CBB symptoms. These findings suggest that RXam2 contributes to the understanding of the molecular basis of quantitative disease resistance.

Complete Genome of Xanthomonas arboricola pv. corylina Strain A7 Isolated from Southern Chile.

The complete genome sequence of Xanthomonas arboricola pv. corylina A7 was obtained by a hybrid approach combining PacBio and Illumina HiSeq sequence data. A single circular chromosome of 5.1 mb with 65.47% G + C content was obtained, including 4,344 coding sequences identified as well as some genes involved in copper resistance. The information obtained corresponds to the first report of a high-quality whole genome of X. arboricola pv. corylina, isolated from infected hazelnut trees in southern Chile.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Molecular, Phenotypical, and Host-Range Characterization of Robbsia andropogonis Strains Isolated from Bougainvillea spp. in Mexico.

Characteristic leaf spot and blight symptoms caused by Robbsia andropogonis on bougainvillea plants were found in three locations in different provinces of Mexico from 2019 to 2020. Eleven bacterial isolates with morphology similar to R. andropogonis were obtained from the diseased bougainvillea leaves. The isolates were confirmed as R. andropogonis by phenotypic tests and 16S rRNA, rpoD, and gyrB gene sequencing. In addition to bougainvillea, the strains were pathogenic to 10 agriculturally significant crops, including maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), coffee (Coffea arabiga), carnation (Dianthus caryophilus), Mexican lime (Citrus × aurantifolia), common bean (Phaseolus vulgaris), broadbeans (Vicia faba), and pea (Pisum sativum), but not runner bean (Phaseolus coccineus). The haplotypes network reveals the genetic variability among Mexican strains and its phylogeographic relationship with Japan, the U.S.A., and China. The presence of this pathogen represents a challenge for plant protection strategies in Mexico.

Genome-Scale Metabolic Model of Xanthomonas phaseoli pv. manihotis: An Approach to Elucidate Pathogenicity at the Metabolic Level.

Xanthomonas phaseoli pv. manihotis (Xpm) is the causal agent of cassava bacterial blight, the most important bacterial disease in this crop. There is a paucity of knowledge about the metabolism of Xanthomonas and its relevance in the pathogenic process, with the exception of the elucidation of the xanthan biosynthesis route. Here we report the reconstruction of the genome-scale model of Xpm metabolism and the insights it provides into plant-pathogen interactions. The model, iXpm1556, displayed 1,556 reactions, 1,527 compounds, and 890 genes. Metabolic maps of central amino acid and carbohydrate metabolism, as well as xanthan biosynthesis of Xpm, were reconstructed using Escher (https://escher.github.io/) to guide the curation process and for further analyses. The model was constrained using the RNA-seq data of a mutant of Xpm for quorum sensing (QS), and these data were used to construct context-specific models (CSMs) of the metabolism of the two strains (wild type and QS mutant). The CSMs and flux balance analysis were used to get insights into pathogenicity, xanthan biosynthesis, and QS mechanisms. Between the CSMs, 653 reactions were shared; unique reactions belong to purine, pyrimidine, and amino acid metabolism. Alternative objective functions were used to demonstrate a trade-off between xanthan biosynthesis and growth and the re-allocation of resources in the process of biosynthesis. Important features altered by QS included carbohydrate metabolism, NAD(P)+ balance, and fatty acid elongation. In this work, we modeled the xanthan biosynthesis and the QS process and their impact on the metabolism of the bacterium. This model will be useful for researchers studying host-pathogen interactions and will provide insights into the mechanisms of infection used by this and other Xanthomonas species.

Sources, Spectrum, Genetics, and Inheritance of Phaseolus vulgaris Resistance Against Xanthomonas citri pv. fuscans.

Common bean (Phaseolus vulgaris) is one of the most consumed agricultural products in the world. Its production is affected by common bacterial blight (CBB) caused by Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli. In this work, we investigated the spectrum, genetics, and inheritance of common bean resistance to X. citri pv. fuscans. Inoculation of nine selected cultivars with an X. citri pv. fuscans strain showed that BRS Radiante and IAPAR 16 were resistant. These two cultivars were also resistant to six X. phaseoli pv. phaseoli strains of different geographic origins, demonstrating their broad-spectrum resistances. BRS Radiante sustained smaller X. citri pv. fuscans populations than two susceptible cultivars. Stomatal densities of IAPAR 16 and BRS Radiante were significantly higher than or not different from susceptible cultivars. BRS Radiante showed the lowest general combining ability values and the combination BRS Radiante × Carioca MG the lowest specific combining ability (SCA) values, revealing the capacity of BRS Radiante to increase resistance to X. citri pv. fuscans. Positive and negative parental SCA values indicated dominant and recessive genes involved in X. citri pv. fuscans resistance. Resistance of the BRS Radiante × Carioca MG cross segregated in a 9:7 ratio in the F2 population, indicating that it is governed by two complementary dominant genes. Maximum likelihood analysis showed that the resistance of BRS Radiante to X. citri pv. fuscans is conferred by a gene of major effect with contribution of additional polygenes. This study contributes with important knowledge on the resistance against CBB in Brazilian common bean cultivars as well as with molecular tools for confirmation of common bean hybrids.

Imidazolium salts as alternative compounds to control diseases caused by plant pathogenic bacteria.

AIMS: To evaluate the inhibitory effect of five structurally different imidazolium salts on the in vitro growth of plant pathogenic bacteria that belong to divergent taxonomic genera as well as their ability to reduce the severity of common bacterial blight of common bean caused by Xanthomonas axonopodis pv. phaseoli and bacterial speck of tomato caused by Pseudomonas syringae pv. tomato. METHODS AND RESULTS: Growth inhibition of Xanthomonas, Pseudomonas, Erwinia, Pectobacterium and Dickeya strains by imidazolium salts was assessed in vitro by radial diffusion on agar medium and by ressazurin reduction in liquid medium. The reduction of common bacterial blight and bacterial speck symptoms and the area under de disease progress curves were determined by spraying two selected imidazolium salts on healthy plants 48 h prior to inoculation with virulent strains of the bacterial pathogens. All imidazolium salts inhibited the growth of all plant pathogenic bacteria when tested by radial diffusion on agar medium. The strength of inhibition differed among imidazolium salts when tested on the same bacterial strain and among bacterial strains when tested with the same imidazolium salt. In liquid medium, most imidazolium salts presented the same minimum inhibitory concentration (MIC) and minimum bactericidal concentration values (200 µmol l-1 ), the most notable exception of which was the MIC (at least 1000 µmol l-1 ) for the dicationic MImC10 MImBr2 . The imidazolium salts C16 MImBr and C16 MImCl caused significant reductions in the severity of common bacterial blight symptoms when compared with nontreated plants. CONCLUSION: Imidazolium salts inhibit the in vitro growth of plant pathogenic bacteria and reduce plant disease symptoms to levels comparable to an authorized commercial antibiotic product. SIGNIFICANCE AND IMPACT OF THE STUDY: New compounds exhibiting broad-spectrum antibacterial activity with potential use in agriculture were identified.

Rapid Detection of Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli in Common Bean by Loop-Mediated Isothermal Amplification.

A single loop-mediated isothermal amplification (LAMP) assay was developed for specific detection of both pathogens that cause bacterial blight in common bean, Xanthomonas phaseoli pv. phaseoli (Xpp) and Xanthomonas citri pv. fuscans (Xcf). The objective was to provide a simple, easy-to-use, specific, and sensitive method to investigate the presence of one or both pathogens in plant material and seeds for routine diagnosis. The detection limits for both pathogens were 10 CFU/ml for cell suspensions and 1 fg of DNA, whereas in conventional PCR, the primers detected up to 105 CFU/ml and 1 ng of DNA. Specificity was confirmed by testing DNA from bean leaves, other Xanthomonas species, common fungal and bacterial bean pathogens, and bacteria from the leaf microbiota. The method was tested with bean leaves inoculated with Xpp, and the pathogen could be detected from 4 h up to 15 days postinoculation, even before disease symptoms were visible. When the method was applied to bacterium detection (Xpp or Xcf) in seed lots from infected plants, the bacterium detection rate was 100% (24 of 24). The pathogens were detected in seeds incubated for just 1 h in saline solution (0.85%), reducing the time needed for bacterium detection. The LAMP assay could be useful as a tool in bean bacterial blight management. Rapid and sensitive detection of bacteria in bean seed lots would reduce the risks of planting highly contaminated seeds in environments favorable to blight multiplication.

Leveraging barrel medic genome sequence for the development and use of genomic resources for genetic analysis and breeding in legumes

BACKGROUND: A total of 62,591 cowpea expressed sequence tags (ESTs) were BLAST aligned to the whole-genome sequence of barrel medic (Medicago truncatula) to develop conserved intron scanning primers (CISPs). The efficacy of the primers was tested across 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea. Genetic diversity was assessed using the same primers on different cowpea genotypes. Singlenucleotide polymorphisms (SNPs) were detected, which were later converted to length polymorphism markers for easy genotyping. CISPs developed in this study were used in tagging resistance to bacterial leaf blight disease in cowpea. RESULTS: A total of 1262 CISPs were designed. The single-copy amplification success rates using these primers on 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea were approximately 60% in most of the legumes except soybean (47%) and peanut (37%). Genetic diversity analysis of 35 cowpea genotypes using 179 CISPs revealed 123 polymorphic markers with PIC values ranging from 0.05 to 0.59. Potential SNPs identified in cowpea, chickpea, and pigeon pea were converted to PCR primers of various sizes for easy genotyping. Using the markers developed in this study, a genetic linkage map was constructed with 11 linkage groups in cowpea. QTL mapping with 194 F3 progeny families derived from the cross C-152 × V-16 resulted in the identification of three QTLs for resistance to bacterial leaf blight disease. Conclusions: CISPs were proved to be efficient markers to identify various other marker classes like SNPs through comparative genomic studies in lesser studied crops and to aid in systematic sampling of the entire genome for well-distributed markers at low cost

The STK domain of the cassava bacterial blight resistance protein RXam1 interacts with an E3 Ubiquitin Ligase / El dominio stk de la proteína de resistencia a la bacteriosis vascular de yuca rxam1 interactúa con una e3 ubiquitin ligasa

RESUMEN La yuca (Manihot esculenta) representa el pilar de la seguridad alimentaria para cerca de mil millones de personas, principalmente en las zonas tropicales. Uno de los factores limitantes de la producción de yuca es la bacteriosis vascular causada por la bacteria Xanthomonasaxonopodis pv. manihotis (Xam). Recientemente se identificó el gen RXaml el cual confiere resistencia parcial de yuca a cepas de Xam. RXaml codifica una proteína con un dominio LRR (Leucine Rich Repeats) extracelular y un dominio STK (Serina Treonina Kinasa) citoplasmático; estas proteínas son conocidas como RLKs (Receptor Like Kinases). En este estudio se realizó el tamizaje de una librería de ADNc de yuca mediante doble híbrido de levadura para identificar las posibles proteínas que interactúan con el dominio STK de RXam1. El tamizaje de 3x108 clones permitió identificar y confirmar cinco clones de ellos los cuales corresponden al mismo gen, el cual codifica para una proteína que presenta un dominio central de dedos de zinc CHY, seguido por un dominio C-terminal "RING finger" y un "Zinc ribbon" el cual fue denominado CRFE3-1 (Cassava RING Finger E3 ligase). La interacción entre STK y CRFE3-1 fue altamente especifica ya que se demostró también por doble híbrido que STK no interactúa con una E3 ligasa de Arabidopsis, altamente similar a CRFE3-1, así como tampoco CRFE3-1 interactúa con el dominio STK de un RLK de lechuga similar a RXam1. La identificación de CRFE3-1 sugiere que mecanismos de degradación proteica son importantes para regular la actividad de RXam1.

ABSTRACT Cassava (Manihot esculenta) represents food security support for nearly one billion people, mainly in the tropics. One of the limiting factors of cassava's production is cassava bacterial blight, caused by the bacterium Xanthomonas axonopodis pv. manihotis (Xam). Recently, the RXam1 gene was identified, which confers partial resistance to some Xam strains. RXam1 encodes a protein with an extracellular LRR (Leucine Rich Repeats) domain and a cytoplasmic STK (Serine Threonine Kinase) domain; these proteins are known as RLK (Receptor-like Kinases). In this study, a cassava cDNA library was screened using a yeast Two-hybrid assay to identify possible proteins interacting with the STK domain of RXam1. Screening of 3x108 clones allowed identifying and confirming five of them, which correspond to the same gene, and code for a protein that has a core domain of zinc fingers CHY, followed by a C-terminal "RING finger" domain and a "Zinc ribbon". This gene was called CRFE3-1 (Cassava RING Finger E3 ligase). It was also demonstrated by yeast Two-hybrid that STK does not interact with an E3 ligase of Arabidopsis that is highly like CRFE3-1. CRFE3-1 did not show interaction with the STK domain of an RLK of lettuce related to RXam1, indicating a highly specific interaction between cassava RXam1 STK and CRFE3-1. The identification of CRFE3-1 suggests that protein degradation mechanisms are important to regulate the activity of RXam1.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain Xff49: a new isolate obtained from common beans in Southern Brazil.

The genus Xanthomonas comprises Gram-negative bacteria, many of which are phytopathogens. Xanthomonas fuscans subsp. fuscans is one of the most devastating pathogens affecting the bean plant, resulting in the common bacterial blight of bean (CBB). The disease is mainly foliar and affects a wide variety of bean species, thus acting as the yield-limiting factor for the bean crop. Here, we report the whole-genome sequencing of a new strain of X. fuscans subsp. fuscans, named Xff49, isolated from the infected and symptomatic beans from Capão do Leão, Southern Brazil. The genetic analysis demonstrated the presence of single-nucleotide variants (SNVs) in this strain, potentially affecting the mobilome, cell mobility, and inorganic ion metabolism. In addition, the analysis resulted in the identification of a new plasmid similar to the pAX22 derived from Achromobacter denitrificans, which was named plX, along with plA and plC, previously reported in other strains of X. fuscans subsp. fuscans. Xff49 represents the first Brazilian genome of X. fuscans subsp. fuscans and might provide useful information applicable to the studies of phylogenetics, evolution, and pathogenomics, thereby allowing a better understanding of the genomic features present in the Brazilian strains.

Green propolis ethanolic extract in bean plant protection against bacterial diseases / Extrato etanólico de própolis verde na proteção de feijoeiro contra bacterioses

The aim of this study was to evaluate the ethanolic extract of green propolis (EEP) in the protection of common bean plants against two main bacterial cultures, bacterial blight (Xanthomonas axonopodis pv. phaseoli) and wildfire (Pseudomonas syringae pv. tabaci). Experiments on antimicrobial activity were performed, inducing phytoalexins, defense-related enzymes, and disease severity, under concentrations of 0, 0.5, 1.0, 2.5, and 5.0%. The EEP presented antimicrobial activity on both phytobacteria, causing a decrease in their development. It has also promoted a linear accumulation of phaseolin in bean hypocotyls according to the EEP concentration used. There was a reduction in the lesion area, which was caused by bacterial blight on bean leaves treated with EEP, and local and systemic effect were observed. Polyphenoloxidase was activated with 5% EEP, reaching the maximum activation time 62.5 h after application. An increase was observed in the activity of phenylalanine ammonia-lyase in plants treated with EEP, with local and systemic effect. Results indicated the potential of EEP in the control of these diseases.(AU)

Este trabalho teve como objetivo avaliar o extrato etanólico de própolis verde (EEP) na proteção de plantas de feijoeiro contra as duas principais bacterioses da cultura, crestamento bacteriano (Xanthomonas axonopodis pv. phaseoli) e fogo selvagem (Pseudomonas syringae pv. tabaci). Foram realizados experimentos sobre atividade antimicrobiana indutora de fitoalexinas, de enzimas relacionadas à defesa, e na severidade da doença, usando as concentrações de 0, 0,5, 1,0, 2,5 e 5,0%. O EEP apresentou atividade antimicrobiana sobre ambas fitobactérias, causando uma redução em seu desenvolvimento. O EEP também promoveu um acúmulo linear de faseolina em hipocótilos de feijoeiro conforme a concentração usada. Houve uma redução na área lesionada pelo crestamento bacteriano em folhas de feijoeiro tratadas com EEP, com efeito local e sistêmico. A enzima polifenoloxidase foi ativada pelo EEP a 5%, com ponto máximo de ativação 62,5 horas após aplicação. Houve um aumento na atividade de fenilalanina amônia-liase em plantas tratadas com EEP, com efeito local e sistêmico. Os resultados indicam o potencial do EEP no controle dessas doenças.(AU)

Green propolis ethanolic extract in bean plant protection against bacterial diseases / Extrato etanólico de própolis verde na proteção de feijoeiro contra bacterioses

ABSTRACT: The aim of this study was to evaluate the ethanolic extract of green propolis (EEP) in the protection of common bean plants against two main bacterial cultures, bacterial blight (Xanthomonas axonopodis pv. phaseoli) and wildfire (Pseudomonas syringae pv. tabaci). Experiments on antimicrobial activity were performed, inducing phytoalexins, defense-related enzymes, and disease severity, under concentrations of 0, 0.5, 1.0, 2.5, and 5.0%. The EEP presented antimicrobial activity on both phytobacteria, causing a decrease in their development. It has also promoted a linear accumulation of phaseolin in bean hypocotyls according to the EEP concentration used. There was a reduction in the lesion area, which was caused by bacterial blight on bean leaves treated with EEP, and local and systemic effect were observed. Polyphenoloxidase was activated with 5% EEP, reaching the maximum activation time 62.5 h after application. An increase was observed in the activity of phenylalanine ammonia-lyase in plants treated with EEP, with local and systemic effect. Results indicated the potential of EEP in the control of these diseases.

RESUMO: Este trabalho teve como objetivo avaliar o extrato etanólico de própolis verde (EEP) na proteção de plantas de feijoeiro contra as duas principais bacterioses da cultura, crestamento bacteriano (Xanthomonas axonopodis pv. phaseoli) e fogo selvagem (Pseudomonas syringae pv. tabaci). Foram realizados experimentos sobre atividade antimicrobiana indutora de fitoalexinas, de enzimas relacionadas à defesa, e na severidade da doença, usando as concentrações de 0, 0,5, 1,0, 2,5 e 5,0%. O EEP apresentou atividade antimicrobiana sobre ambas fitobactérias, causando uma redução em seu desenvolvimento. O EEP também promoveu um acúmulo linear de faseolina em hipocótilos de feijoeiro conforme a concentração usada. Houve uma redução na área lesionada pelo crestamento bacteriano em folhas de feijoeiro tratadas com EEP, com efeito local e sistêmico. A enzima polifenoloxidase foi ativada pelo EEP a 5%, com ponto máximo de ativação 62,5 horas após aplicação. Houve um aumento na atividade de fenilalanina amônia-liase em plantas tratadas com EEP, com efeito local e sistêmico. Os resultados indicam o potencial do EEP no controle dessas doenças.

ESTUDIO DE LA EXPRESIÓN DE GENES QUE CODIFICAN PARA PUTATIVAS PROTEÍNAS PR EN YUCA (Manihot esculenta Crantz) / Studying the Expression of Genes Coding for Putative PR Proteins in Cassava (Manihot esculenta Crantz)

RESUMEN Posterior al reconocimiento de agentes patógenos las plantas activan una serie de cascadas de señalización que culminan con la activación de factores de transcripción. Esto genera una concomitante reprogramación de la expresión génica que incluye la activación de la transcripción de los genes PR (relacionados con patogenicidad). Las proteínas PR son conocidas por poseer actividad antimicrobiana y evitan la posterior colonización del patógeno. En este estudio se empleó una aproximación bioinformática para identificar el repertorio de posibles proteínas PR en el genoma de yuca. Adicionalmente, se evaluó la expresión de nueve genes PR a lo largo del tiempo en variedades de yuca resistentes y susceptibles en respuesta a la inoculación con la bacteria Xanthomonas axonopodis pv. manihotis (Xam) mediante RT-PCR. Se encontró que varios genes PR fueron inducidos producto de la herida que se realiza durante el proceso de inoculación. Con el fin de evaluar cuantitativamente la contribución real de la infección bacteriana en la expresión de estos genes, se llevó a cabo una RT-PCR en tiempo real (QRT, Quantitative Real-Time PCR). Se encontró que en la variedad resistente el gen que codifica para MePR1 (Manes06G026900.1) presentó una inducción en su expresión a diferentes tiempos post-inoculación, lo cual no se observó en la variedad susceptible. De esta manera, este gen se constituye en un excelente marcador para evaluar la respuesta molecular de resistencia en plantas de yuca.

ABSTRACT Once pathogens are perceived by plants a signal transduction pathway is activated leading to the induction of transcription factors, which in turn reprogram the host gene expression including the transcription of PR (Pathogenesis-Related) genes. The PR proteins are well known for their antimicrobial activity and for contributing to arrest the invasion of pathogens. In this work, a bioinformatics approach was used to identify the repertoire of possible PR proteins in the cassava genome. Additionally, the expression of nine PR genes was evaluated over a time course in resistant and susceptible cassava varieties in response to inoculation with the bacterium Xanthomonas axonopodis pv. manihotis (Xam) by semiquantitative RT-PCR. It was found that several PR genes were induced as a result of the wound that is made during the inoculation process. In order to evaluate quantitatively the real contribution of the bacterial infection in the expression of the genes, a Real Time RT-PCR (qRT, quantitative Real-Time PCR) was carried out. In the resistant variety the gene coding for MePR1 (Manes06G026900) was induced at different post-inoculation times, which was not observed in the susceptible variety. Therefore, this gene constitutes an excellent marker to evaluate the molecular resistance response in cassava plants.

The overexpression of RXam1, a cassava gene coding for an RLK, confers disease resistance to Xanthomonas axonopodis pv. manihotis.

MAIN CONCLUSION: The overexpression of RXam1 leads to a reduction in bacterial growth of XamCIO136, suggesting that RXam1 might be implicated in strain-specific resistance. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is a prevalent disease in all regions, where cassava is cultivated. CBB is a foliar and vascular disease usually controlled through host resistance. Previous studies have found QTLs explaining resistance to several Xam strains. Interestingly, one QTL called XM5 that explained 13% of resistance to XamCIO136 was associated with a similar fragment of the rice Xa21-resistance gene called PCR250. In this study, we aimed to further identify and characterize this fragment and its role in resistance to CBB. Screening and hybridization of a BAC library using the molecular marker PCR250 as a probe led to the identification of a receptor-like kinase similar to Xa21 and were called RXam1 (Resistance to Xam 1). Here, we report the functional characterization of susceptible cassava plants overexpressing RXam1. Our results indicated that the overexpression of RXam1 leads to a reduction in bacterial growth of XamCIO136. This suggests that RXAM1 might be implicated in strain-specific resistance to XamCIO136.

Molecular detection of Erwinia psidii in guava plants under greenhouse and field conditions / Detecção molecular de Erwinia psidii em goiabeira em condições de casa de vegetação e campo

Erwinia psidii causes bacterial blight of guava ( Psidium guajava ), an important disease of this crop in Brazil. The pathogen affects branches and twigs of guava trees, reducing yield significantly. Bacterial dissemination often occurs through contaminated but asymptomatic propagating plant material. The objectives of this research were to evaluate the use of BIO-PCR and conventional PCR to detect E. psidii in inoculated guava plants grown in a greenhouse and in symptomatic and asymptomatic trees from guava orchards. Erwinia psidii strain IBSBF 1576 was inoculated (107CFU mL-1) into young guava shoots and plant tissue was analysed at 0, 5, 10, and 15 days after inoculation. Symptoms were observed after 5 days and all inoculated shoots were PCR positive at all times, by both BIO-PCR and conventional PCR. Under natural infection conditions, 40 samples were tested by BIO-PCR from each of three guava orchards, 20 showing symptoms and 20 asymptomatic. PCR was positive for 58 out of 60 symptomatic samples (96.7%) and for 6.7% of asymptomatic samples, showing that the method can be used to detect the pathogen at early stages of infection. This PCR method may be used as a diagnostic tool to assess bacterial survival, dissemination and disease outbreaks.(AU)

Erwinia psidii é o agente causal da seca dos ponteiros da goiabeira ( Psidium guajava ), uma importante doença dessa cultura no Brasil. O patógeno afeta folhas, frutos, ramos e brotações, reduzindo significativamente a produtividade da cultura. A disseminação do patógeno ocorre por meio de material propagativo contaminado, porém assintomático. Os objetivos do trabalho foram avaliar o uso da BIO-PCR e da PCR convencional para detectar E. psidii em plantas inoculadas em casa de vegetação e em plantas sintomáticas e assintomáticas em pomares de goiabeira. A estirpe IBSBF 1576 de E. psidii foi inoculada (107UFC mL-1) em brotações novas de mudas de goiabeira e o tecido foi analisado nos tempos 0, 5, 10, e 15 dias após a inoculação. Sintomas foram observados após 5 dias e todas as plantas inoculadas foram positivas por PCR em todos os tempos avaliados, pelos dois métodos (BIO-PCR e PCR convencional). Sob condições de infecção natural em campo, três pomares foram avaliados por BIO-PCR. De cada pomar, foram coletadas 40 amostras, sendo 20 com e 20 sem sintomas. PCR foi positiva para 58 das 60 amostras sintomáticas (96,7%) e para 6,7% das amostras assintomáticas, demonstrando que o método pode ser usado para detectar o patógeno nos estágios iniciais da infecção. Este método poderá ser útil como uma ferramenta para a diagnose e para monitorar a sobrevivência e disseminação da bactéria e, consequentemente, novos focos da doença.(AU)

Molecular detection of Erwinia psidii in guava plants under greenhouse and field conditions / Detecção molecular de Erwinia psidii em goiabeira em condições de casa de vegetação e campo

ABSTRACT: Erwinia psidii causes bacterial blight of guava ( Psidium guajava ), an important disease of this crop in Brazil. The pathogen affects branches and twigs of guava trees, reducing yield significantly. Bacterial dissemination often occurs through contaminated but asymptomatic propagating plant material. The objectives of this research were to evaluate the use of BIO-PCR and conventional PCR to detect E. psidii in inoculated guava plants grown in a greenhouse and in symptomatic and asymptomatic trees from guava orchards. Erwinia psidii strain IBSBF 1576 was inoculated (107CFU mL-1) into young guava shoots and plant tissue was analysed at 0, 5, 10, and 15 days after inoculation. Symptoms were observed after 5 days and all inoculated shoots were PCR positive at all times, by both BIO-PCR and conventional PCR. Under natural infection conditions, 40 samples were tested by BIO-PCR from each of three guava orchards, 20 showing symptoms and 20 asymptomatic. PCR was positive for 58 out of 60 symptomatic samples (96.7%) and for 6.7% of asymptomatic samples, showing that the method can be used to detect the pathogen at early stages of infection. This PCR method may be used as a diagnostic tool to assess bacterial survival, dissemination and disease outbreaks.

RESUMO: Erwinia psidii é o agente causal da seca dos ponteiros da goiabeira ( Psidium guajava ), uma importante doença dessa cultura no Brasil. O patógeno afeta folhas, frutos, ramos e brotações, reduzindo significativamente a produtividade da cultura. A disseminação do patógeno ocorre por meio de material propagativo contaminado, porém assintomático. Os objetivos do trabalho foram avaliar o uso da BIO-PCR e da PCR convencional para detectar E. psidii em plantas inoculadas em casa de vegetação e em plantas sintomáticas e assintomáticas em pomares de goiabeira. A estirpe IBSBF 1576 de E. psidii foi inoculada (107UFC mL-1) em brotações novas de mudas de goiabeira e o tecido foi analisado nos tempos 0, 5, 10, e 15 dias após a inoculação. Sintomas foram observados após 5 dias e todas as plantas inoculadas foram positivas por PCR em todos os tempos avaliados, pelos dois métodos (BIO-PCR e PCR convencional). Sob condições de infecção natural em campo, três pomares foram avaliados por BIO-PCR. De cada pomar, foram coletadas 40 amostras, sendo 20 com e 20 sem sintomas. PCR foi positiva para 58 das 60 amostras sintomáticas (96,7%) e para 6,7% das amostras assintomáticas, demonstrando que o método pode ser usado para detectar o patógeno nos estágios iniciais da infecção. Este método poderá ser útil como uma ferramenta para a diagnose e para monitorar a sobrevivência e disseminação da bactéria e, consequentemente, novos focos da doença.

Xanthomonas fuscans subsp. fuscans causing wilt symptoms in bean plants (Phaseolus vulgaris) in Brazil

Common and fuscous blights of bean are diseases widely distributed in the world. The most commonly observed symptoms are spots on leaves, stems, pods and seeds. In December 2009, bean plants cv. Uirapuru showing symptoms of wilt similar to those induced by Curtobacterium flaccumfaciens pv. flaccumfaciens were observed in a commercial crop located in the county of Itararé, State of São Paulo, Brazil. The plants were at the last cycle stage with mature pods and these symptoms were noted in the majority of the growing area. Optical microscopic observations of discolored vascular tissue from diseased stems revealed the presence of bacterial masses oozed from infected tissue, indicating that the disease was caused by bacterial pathogen. Isolations on nutrient agar showed circular, convex, yellow colonies with smooth edges. The causal bacterium was Gramnegative and produced a dark brown pigment in culture medium. Biochemical, cultural and physiological tests confirmed its identity as Xanthomonas fuscans subsp. fuscans(syn. Xanthomonas campestris pv. phaseolivar. fuscans"). The pathogenicity of the isolates was confirmed by artificial inoculations. Systemic infection has been reported in the literature but these kinds of symptoms are not currently observed in Brazilian fields. Bacterial strains were deposited on the Phytobacteria Culture Collection of Instituto Biológico (IBSBF - www.biologico.sp.gov.br/bacterias/php) under accession numbers 2813 and 3028.

O crestamento bacteriano comum do feijoeiro é uma doença amplamente distribuída no mundo e os sintomas comumente observados sáo manchas nas folhas, hastes, vagens e sementes. Em dezembro de 2009, plantas de feijoeiro cv. Uirapuru com sintomas de murcha similares aos produzidos por Curtobacterium flaccumfaciens pv. flaccumfaciens foram observadas em campos comerciais localizados no município de Itararé, estado de São Paulo, Brasil. As plantas encontravam-se no final do ciclo, com as vagens já formadas. Os sintomas foram notados em quase a totalidade da área cultivada. Observações ao microscópio óptico de fragmentos de tecido do sistema vascular das hastes de plantas doentes evidenciaram intenso fluxo bacteriano, confirmando tratar-se de doença bacteriana. Isolamentos realizados em meio nutriente ágar produziram colônias de coloração amarelada, brilhantes, convexas, lisas. A bactéria agente causal era Gram-negativa e produtora de pigmento marrom escuro em meio de cultura. Testes bioquímicos, culturais e fisiológicos confirmaram sua identidade como Xanthomonas fuscans subsp. fuscans (sin. Xanthomonas campestris pv. phaseoli var. fuscans"). A patogenicidade dos isolados foi confirmada por inoculações artificiais em mudas de feijão cv. Carioca e os reisolamentos efetuados resultaram em colônias semelhantes às originais. Embora descrita na literatura, a infecção sistêmica não é usualmente observada nos plantios de feijoeiro em nosso país. Linhagens bacterianas encontram-se depositadas na Coleção de Culturas do Instituto Biológico (IBSBF) sob os números 2813 e 3028.

Xanthomonas fuscans subsp. fuscans causing wilt symptoms in bean plants (Phaseolus vulgaris) in Brazil / Xanthomonas fuscans subsp. fuscans causando sintomas de murcha em feijoeiro (Phaseolus vulgaris) no Brasil

Common and fuscous blights of bean are diseases widely distributed in the world. The most commonly observed symptoms are spots on leaves, stems, pods and seeds. In December 2009, bean plants cv. Uirapuru showing symptoms of wilt similar to those induced by Curtobacterium flaccumfaciens pv. flaccumfaciens were observed in a commercial crop located in the county of Itarar;, State of S;o Paulo, Brazil. The plants were at the last cycle stage with mature pods and these symptoms were noted in the majority of the growing area. Optical microscopic observations of discolored vascular tissue from diseased stems revealed the presence of bacterial masses oozed from infected tissue, indicating that the disease was caused by bacterial pathogen. Isolations on nutrient agar showed circular, convex, yellow colonies with smooth edges. The causal bacterium was Gramnegative and produced a dark brown pigment in culture medium. Biochemical, cultural and physiological tests confirmed its identity as Xanthomonas fuscans subsp. fuscans (syn. Xanthomonas campestris pv. phaseoli 0;var. fuscans"). The pathogenicity of the isolates was confirmed by artificial inoculations. Systemic infection has been reported in the literature but these kinds of symptoms are not currently observed in Brazilian fields. Bacterial strains were deposited on the Phytobacteria Culture Collection of Instituto Biológico (IBSBF - www.biologico.sp.gov.br/bacterias/php) under accession numbers 2813 and 3028.(AU)

O crestamento bacteriano comum do feijoeiro é uma doença amplamente distribuída no mundo e os sintomas comumente observados sáo manchas nas folhas, hastes, vagens e sementes. Em dezembro de 2009, plantas de feijoeiro cv. Uirapuru com sintomas de murcha similares aos produzidos por Curtobacterium flaccumfaciens pv.flaccumfaciens foram observadas em campos comerciais localizados no município de Itararé, estado de São Paulo, Brasil. As plantas encontravam-se no final do ciclo, com as vagens já formadas. Os sintomas foram notados em quase a totalidade da área cultivada. Observações ao microscópio óptico de fragmentos de tecido do sistema vascular das hastes de plantas doentes evidenciaram intenso fluxo bacteriano, confirmando tratar-se de doença bacteriana. Isolamentos realizados em meio nutriente ágar produziram colônias de coloração amarelada, brilhantes, convexas, lisas. A bactéria agente causal era Gram-negativa e produtora de pigmento marrom escuro em meio de cultura. Testes bioquímicos, culturais e fisiológicos confirmaram sua identidade como Xanthomonas fuscans subsp.fuscans (sin. Xanthomonas campestris pv. phaseoli var. fuscans"). A patogenicidade dos isolados foi confirmada por inoculações artificiais em mudas de feijão cv. Carioca e os reisolamentos efetuados resultaram em colônias semelhantes às originais. Embora descrita na literatura, a infecção sistêmica não é usualmente observada nos plantios de feijoeiro em nosso país. Linhagens bacterianas encontram-se depositadas na Coleção de Culturas do Instituto Biológico (IBSBF) sob os números 2813 e 3028.(AU)