Isolation and structural characterization of a non-diketopiperazine phytotoxin from a potato pathogenic Streptomyces strain.

Two Streptomyces spp. strains responsible for potato common scab infections in Uruguay which do not produce diketopiperazines were identified through whole-genome sequencing, and the virulence factor produced by one of them was isolated and characterized. Phylogenetic analysis showed that both pathogenic strains can be identified as S. niveiscabiei, and the structure of the phytotoxin was elucidated as that of the polyketide desmethylmensacarcin using MS and NMR methods. The metabolite is produced in yields of ∼200 mg/L of culture media, induces deep necrotic lesions on potato tubers, stuns root and shoot growth in radish seedlings, and is comparatively more aggressive than thaxtomin A. This is the first time that desmethylmensacarcin, a member of a class of compounds known for their antitumor and antibiotic activity, is associated with phytotoxicity. More importantly, it represents the discovery of a new virulence factor related to potato common scab, an economically-important disease affecting potato production worldwide.

Enzymatic variability among Brazilian Pythium insidiosum isolates

Background. Pythium insidiosum is an oomycete classified in the kingdom Stramenopila. P. insidiosum hyphae are not able to initiate infection without the secretion of hydrolytic enzymes, which are considered an important factor in microbial virulence. Aims. To evaluate the extracellular enzymatic activity of 14 Brazilian P. insidiosum isolates and a standard strain (ATCC 58637) by the API-ZYM System screening method. Methods. Zoospores were grown in RPMI 1640 broth, and 65 μL of the liquid phase were inoculated in each cupule of the API-ZYM strips. Results. Differences in the enzymatic activities were observed among the isolates, although phosphohydrolases and ester hydrolases were conspicuous among all isolates. β-glucosidase was also present in most of the isolates. Enzymatic activities of β-glucosidase and chymotrypsin were not observed, differing from a previous study involving Australian isolates and intracellular enzymes. Conclusions. The discrepancy in the enzymatic profile observed among Brazilian P. insidiosum isolates reflects the phenotypic variations found in susceptibility tests

Caracterización fisiológica y molecular de aislamientos de Phytophthora infestans de la región andina central colombiana / Physiological and molecular characterization of Phytophthora infestans isolates from the Central Colombian Andean Region

Antecedentes. El tizón tardío, causado por Phytophthora infestans, es una enfermedad devastadora de la papa y el tomate a nivel mundial, y en Colombia también ataca otros cultivos como la uchuva y el tomate de árbol. El conocimiento de la población del patógeno es determinante para el diseño efectivo de estrategias de control. Objetivos. Determinar las características fisiológicas y moleculares de aislamientos colombianos de P. infestans. Métodos. El nivel de resistencia al mefenoxam y al cimoxamil fue evaluado en aislamientos de Cundinamarca y Boyacá. Se estimó su virulencia y se determinó la producción y viabilidad de oosporas en diferentes sustratos con cruces entre aislamientos A1 y el aislamiento colombiano A2. Además, se determinó la diversidad molecular en el gen de avirulencia Avr3a, el gen de la β-tubulina y otros dos genes de copia única con motivo RXLR. Resultados. Los aislamientos colombianos tuvieron la posibilidad de reproducirse sexualmente. Encontramos todos los niveles de sensibilidad al mefenoxam, con el 48% de los aislamientos resistentes. Se detectó una diversidad de razas y a nivel genético la población fue clonal. Conclusiones. Estos resultados ayudarán a optimizar el uso de fungicidas y reducir la resistencia como estrategias de control, además de contribuir al conocimiento de la diversidad de este patógeno(AU)

Background. Late blight, caused by Phytophthora infestans, is one of the most devastating diseases found in potato and tomato crops worldwide. In Colombia it also attacks other important crops: cape gooseberry and tree tomato. The knowledge of the pathogen population is determinant to effectively design control strategies. Aims. To determine the physiological and molecular characteristics of a set of Colombian P. infestans isolates. Methods. Strains isolated from Cundinamarca and Boyacá were examined for the level of resistance to mefenoxam and cymoxanil. Virulence was tested for all strains and crosses between A1 mating type, from different hosts, and the Colombian A2 mating type were tested for the production and viability of oospores in different substrates. Additionally, the molecular diversity of the avirulence gene Avr3a, the β-tubulin gene, and two single copy genes showing RxLR motif, was assessed. Results. We found all levels of mefenoxam sensitivity, with 48% of the strains resistant. A high diversity of races was detected and the population was genetically clonal. Colombian strains had the possibility of sexual reproduction. Conclusions. These results will help in optimizing the use of fungicides and deployment of resistance as control strategies and will contribute to broader studies on diversity of this pathogen(AU)

Aislamiento y caracterización de dos cepas de Fusarium oxysporum causantes de la pudrición seca en Solanum tuberosum en Colombia / Isolation and characterization of two strains of Fusarium oxysporum causing potato dry rot in Solanum tuberosum in Colombia

resumen(AU)

Background. Fusarium oxysporum has worldwide distribution and causes severe vascular wilt or root rot in many plants. Strains are classified into formae speciales based on their high degree of host specificity, of which multilocus sequence typing provides a fairly good estimate. Aims. The main aim of this study was to identify the causal agent of an infected potato tuber in Colombia. Methods. Two F. oxysporum isolates were recovered from a potato tuber showing symptoms of dry rot. Both macroscopic and microscopic morphology differences were observed between the two isolates. Koch's postulates were verified and in quantitative tuber pathogenecity trials, both isolates induced moderate dry rot. Ribosomal internal transcribed spacer (ITS) and partial intergenic spacer region (IGS) sequences were PCR-amplified, sequenced and shown to be identical for the two isolates. A maximum parsimony phylogeny was created using F. oxysporum IGS sequences available in the Genebank database, which does not include sequences from the formae speciales tuberosi. Results. Our two isolates were most closely related to a red clover (Trifolium pratense) pathogenic isolate and two non-pathogenic F. oxysporum isolates from birdsfoot trefoil (Lotus corniculatus) and Lycopersicon sp. rhyzosphere (99% identity). Conclusions. These experiments showed that our isolates are not restricted to potato and that a molecular marker is needed to differentiate the formae speciales since the IGS and EF-1alpha do not have the power to do it(AU)

A novel zinc binding system, ZevAB, is critical for survival of nontypeable Haemophilus influenzae in a murine lung infection model.

Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative bacterial pathogen that causes upper and lower respiratory infections. Factors required for pulmonary infection by NTHI are not well understood. Previously, using high-throughput insertion tracking by deep sequencing (HITS), putative lung colonization factors were identified. Also, previous research indicates that secreted disulfide-dependent factors are important for virulence of H. influenzae. In the present study, HITS data were compared with an informatics-based list of putative substrates of the periplasmic oxidoreductase DsbA to find and characterize secreted virulence factors. This analysis resulted in identification of the "zinc binding essential for virulence" (zev) locus consisting of zevA (HI1249) and zevB (HI1248). NTHI mutants of zevA and zevB grew normally in rich medium but were defective for colonization in a mouse lung model. Mutants also exhibited severe growth defects in medium containing EDTA and were rescued by supplementation with zinc. Additionally, purified recombinant ZevA was found to bind to zinc with high affinity. Together, these data demonstrate that zevAB is a novel virulence factor important for zinc utilization of H. influenzae under conditions where zinc is limiting. Furthermore, evidence presented here suggests that zinc limitation is likely an important mechanism for host defense against pathogens during lung infection.

Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli.

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application.

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.