Genomic and phenotypic insight into Xanthomonas vesicatoria strains with different aggressiveness on tomato.

Xanthomonas vesicatoria is one of the causal agents of bacterial spot, a disease that seriously affects the production of tomato (Solanum lycopersicum) and pepper (Capsicum annum) worldwide. In Argentina, bacterial spot is found in all tomato producing areas, with X. vesicatoria being one of the main species detected in the fields. Previously, we isolated three X. vesicatoria strains BNM 208, BNM 214, and BNM 216 from tomato plants with bacterial spot, and found they differed in their ability to form biofilm and in their degree of aggressiveness. Here, the likely causes of those differences were explored through genotypic and phenotypic studies. The genomes of the three strains were sequenced and assembled, and then compared with each other and also with 12 other publicly available X. vesicatoria genomes. Phenotypic characteristics (mainly linked to biofilm formation and virulence) were studied in vitro. Our results show that the differences observed earlier between BNM 208, BNM 214, and BNM 216 may be related to the structural characteristics of the xanthan gum produced by each strain, their repertoire of type III effectors (T3Es), the presence of certain genes associated with c-di-GMP metabolism and type IV pili (T4P). These findings on the pathogenicity mechanisms of X. vesicatoria could be useful for developing bacterial spot control strategies aimed at interfering with the infection processes.

Revisiting the production of L( +)-lactic acid from vine shoots: bioconversion improvements by employing thermotolerant bacteria.

Vine shoots (Vitis vinifera L.) constitute an abundant lignocellulosic source which is frequently underutilised. Alkaline and acidic pretreatments (with and without washing steps) were compared and optimised to release fermentable sugars from vine shoots. An acidic pretreatment using 1.72% H2SO4 at 134 °C for 17 min (with 10% w/w solid biomass), followed by an enzymatic hydrolysis, offered the most cost-effective results, releasing 40.21 g/L sugars. Three thermotolerant strains, namely, Bacillus coagulans DSM 2314, Geobacillus stearothermophilus DSM 2313, and G. stearothermophilus DSM 494, were assessed to produce lactic acid from vine-shoot hydrolysates under aerobic and non-sterile conditions, without the need of detoxification steps. In addition, wine lees were satisfactorily employed as nitrogen sources for the fermentation, providing similar results to yeast extract and being the only nutrient added to vine-shoot hydrolysates. Under optimal conditions, B. coagulans DSM 2314 produced 29.21 ± 0.23 g/L lactic acid in 24 h, with a sugar consumption of 98.74 ± 0.07% and a yield of 96.38 ± 0.76%, when supplemented with red wine lees. The purity of the isomer L( +) reached 97.59 ± 1.35% of the total lactic acid produced. Although G. stearothermophilus was able to transform the hexoses from vine-shoot hydrolysates into lactic acid, it proved to be inefficient for metabolising pentoses, thus obtaining lower lactic acid values (16-18 g/L).

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain.

Liberibacter is a bacterial group causing different diseases and disorders in plants. Among liberibacters, Candidatus Liberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future works.

Xanthomonas citri subsp. citri and Xanthomonas arboricola pv. pruni: Comparative analysis of two pathogens producing similar symptoms in different host plants.

Comparative studies in Xanthomonas have provided a vast amount of data that enabled to deepen in the knowledge of those factors associated with virulence and Xanthomonas plant interaction. The species of this genus present a wide range of host plants and a large number of studies have been focused to elucidate which mechanism are involved in this characteristic. In this study, comparative genomic and phenotypic analysis were performed between X. citri subsp. citri (Xcc), one of the most studied pathogens within Xanthomonas, and X. arboricola pv. pruni (Xap), a pathogen which has aroused great interest in recent time. The work was aimed to find those elements that contribute to their host divergence despite the convergence in the symptoms that each species cause on Citrus spp. and Prunus spp., respectively. This study reveals a set of genes that could be putatively associated with the adaptation of these pathogens to their hosts, being the most remarkable those involved in environmental sensing systems such as the case of the TonB-dependent transporters, the sensors of the two-component system and the methyl accepting chemotaxis proteins. Other important variants were found in processes related to the decomposition of the cell wall as could be appreciated by their dissimilar set of cell-wall degrading enzymes. Type three effectors, as one of the most important factors in delineating the host specificity in Xanthomonas, also showed a different array when comparing both species, being some of them unique to each pathogen. On the other hand, only small variations could be connected to other features such as the motility appendages and surface adhesion proteins, but these differences were accompanied by a dissimilar capacity to attach on host and non-host leaf surface. The molecular factors found in this work provide the basis to perform a more in-depth functional analyses that unveil those actual factors associated with pathogenesis and host specificity in Xcc and Xap.

Virulence factor genotyping of Helicobacter pylori isolated from Costa Rican dyspeptic patients.

BACKGROUND AND AIMS: Costa Rica is one of the countries with the highest incidence and mortality rates for gastric cancer. Helicobacter pylori infection rates are high in the whole country. We have previously shown that H. pylori CagA+ is significantly associated with atrophic gastritis (AG) of the antrum in a dyspeptic population. The aim of this work is to determine if other H. pylori virulence factors (vacA, dupA, oipA, iceA and babA2) are associated with atrophic gastritis (AG) or duodenal ulcer (DU). METHODS: The presence of virulence genes in Costa Rican H. pylori isolates was analyzed by PCR in 151 cultured strains from patients with dyspeptic symptoms. Endoscopic and histopathological diagnoses were available. Odds-ratio and 95% confidence intervals for AG patients vs. non-atrophic gastritis (NAG) or DU patients vs. no duodenal ulcer (NDU) patients were calculated. RESULTS: Amongst the studied isolates, 82% had the cagA+, 76.2% had the vacA s1m1, 97.0% had the oipA+, 21.0% had the icea1, 79.0% had the iceA2, 44.0% had the babA2+ and 76.0% the dupA+ genotypes. Infection with H pylori cagA+, dupA+, oipA+, iceA, babA2+, and vacA s1m1 genotypes was not associated with AG risk. The frequency of the dupA gene was 78.7 and 60.9% in isolates from patients with NDU and DU, respectively, and its presence was significantly associated with decreased risk of duodenal ulcer [odds-ratio: 0.33, p = 0.024, confidence interval 95% (0.11-0.85)]. CONCLUSION: H. pylori dupA genotype is inversely associated with DU risk in this population.

Biobutanol production from coffee silverskin.

BACKGROUND: Coffee silverskin, a by-product from coffee roasting industries, was evaluated as a feedstock for biobutanol production by acetone-butanol-ethanol fermentation. This lignocellulosic biomass contained approximately 30% total carbohydrates and 30% lignin. Coffee silverskin was subjected to autohydrolysis at 170 °C during 20 min, with a biomass-to-solvent ratio of 20%, and a subsequent enzymatic hydrolysis with commercial enzymes in order to release simple sugars. The fermentability of the hydrolysate was assessed with four solventogenic strains from the genus Clostridium. In addition, fermentation conditions were optimised via response surface methodology to improve butanol concentration in the final broth. RESULTS: The coffee silverskin hydrolysate contained 34.39 ± 2.61 g/L total sugars, which represents a sugar recovery of 34 ± 3%. It was verified that this hydrolysate was fermentable without the need of any detoxification method and that C. beijerinckii CECT 508 was the most efficient strain for butanol production, attaining final values of 4.14 ± 0.21 g/L acetone, 7.02 ± 0.27 g/L butanol and 0.25 ± 0.01 g/L ethanol, consuming 76.5 ± 0.8% sugars and reaching a butanol yield of 0.269 ± 0.008 gB/gS under optimal conditions. CONCLUSIONS: Coffee silverskin could be an adequate feedstock for butanol production in biorefineries. When working with complex matrices like lignocellulosic biomass, it is essential to select an adequate bacterial strain and to optimize its fermentation conditions (such as pH, temperature or CaCO3 concentration).

Xanthomonas prunicola sp. nov., a novel pathogen that affects nectarine (Prunus persica var. nectarina) trees.

Three isolates obtained from symptomatic nectarine trees (Prunus persica var. nectarina) cultivated in Murcia, Spain, which showed yellow and mucoid colonies similar to Xanthomonas arboricola pv. pruni, were negative after serological and real-time PCR analyses for this pathogen. For that reason, these isolates were characterized following a polyphasic approach that included both phenotypic and genomic methods. By sequence analysis of the 16S rRNA gene, these novel strains were identified as members of the genus Xanthomonas, and by multilocus sequence analysis (MLSA) they were clustered together in a distinct group that showed similarity values below 95 % with the rest of the species of this genus. Whole-genome comparisons of the average nucleotide identity (ANI) of genomes of the strains showed less than 91 % average nucleotide identity with all other species of the genus Xanthomonas. Additionally, phenotypic characterization based on API 20 NE, API 50 CH and BIOLOG tests differentiated the strains from the species of the genus Xanthomonas described previously. Moreover, the three strains were confirmed to be pathogenic on peach (Prunus persica), causing necrotic lesions on leaves. On the basis of these results, the novel strains represent a novel species of the genus Xanthomonas, for which the name Xanthomonas prunicola is proposed. The type strain is CFBP 8353 (=CECT 9404=IVIA 3287.1).

Xanthomonas arboricola pv. pruni, causal agent of bacterial spot of stone fruits and almond: its genomic and phenotypic characteristics in the X. arboricola species context.

BACKGROUND: Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of stone fruits and almond, an important disease that may reduce the yield and vigour of the trees, as well as the marketability of affected fruits. Xap lies within the Xanthomonas genus, which has been intensively studied because of its strain specialization and host range complexity. Here, we summarize the recent advances in our understanding of the complexities of Xap, including studies of the molecular features that result after comparative phenotypic and genomic analyses, in order to obtain a clearer overview of the bacterial behaviour and infection mechanism in the context of the X. arboricola species. TAXONOMIC STATUS: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species X. arboricola; Pathovar pruni. HOST RANGE AND SYMPTOMS: Xap infects most Prunus species, including apricot, peach, nectarine, plum and almond, and occasionally cherry. Symptoms are found on leaves, fruits, twigs and branches or trunks. In severe infections, defoliation and fruit dropping may occur. DISTRIBUTION: Bacterial spot of stone fruits and almond is worldwide in distribution, with Xap being isolated in Africa, North and South America, Asia, Europe and Oceania. It is a common disease in geographical areas in which stone fruits and almonds are grown. Xap is listed as a quarantine organism in several areas of the world. GENOME: The genomes of six isolates from Xap have been publicly released. The genome consists of a single chromosome of around 5 000 000 bp with 65 mol% GC content and an extrachromosomal plasmid element of around 41 000 bp with 62 mol% GC content. Genomic comparative studies in X. arboricola have allowed the identification of putative virulence components associated with the infection process of bacterial spot of stone fruits and almond. DISEASE CONTROL: Management of bacterial spot of stone fruits and almond is based on an integrated approach that comprises essential measures to avoid Xap introduction in a production zone, as well as the use of tolerant or resistant plant material and chemical treatments, mainly based on copper compounds. Management programmes also include the use of appropriate cultivation practices when the disease is already established. Finally, for the effective control of the disease, appropriate detection and characterization methods are needed for use in symptomatic or asymptomatic samples as a first approach for pathogen exclusion. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTPR; http://www.cost.eu/COST_Actions/ca/CA16107; http://www.xanthomonas.org.

Yeast screening and cell immobilization on inert supports for ethanol production from cheese whey permeate with high lactose loads.

Eight yeast strains of the genera Saccharomyces and Kluyveromyces were screened to ferment high lactose-load cheese whey permeate (CWP) (>130 g/L lactose) without nutrient supplementation. The fermentation conditions (temperature, pH and time) were optimized to maximize the fermentation performance (ethanol titer, ethanol yield and lactose consumption) for the two preselected strains, K. marxianus DSM 5422 and S. cerevisiae Ethanol Red, using a response surface methodology (RSM). Under optimized conditions, K. marxianus DSM 5422 attained ethanol titers of 6% (v/v) in only 44 h. Moreover, the feasibility of immobilizing this strain on four different inorganic supports (plastic, glass and Tygon silicone Raschig rings and alumina beads) was assessed. Glass Raschig rings and alumina beads showed a more stable performance over time, yielding ethanol titers of 60 g/L during 1,000 hours, which remarkably reduces yeast cultivation costs. Results demonstrate the feasibility of using CWP for successful ethanol production in a simple and economical process, which represents an attractive alternative for waste treatment in dairy industries.

Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors.

Xanthomonas arboricola is a plant-associated bacterial species that causes diseases on several plant hosts. One of the most virulent pathovars within this species is X. arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruit trees and almond. Recently, a non-virulent Xap-look-a-like strain isolated from Prunus was characterized and its genome compared to pathogenic strains of Xap, revealing differences in the profile of virulence factors, such as the genes related to the type III secretion system (T3SS) and type III effectors (T3Es). The existence of this atypical strain arouses several questions associated with the abundance, the pathogenicity, and the evolutionary context of X. arboricola on Prunus hosts. After an initial characterization of a collection of Xanthomonas strains isolated from Prunus bacterial spot outbreaks in Spain during the past decade, six Xap-look-a-like strains, that did not clustered with the pathogenic strains of Xap according to a multi locus sequence analysis, were identified. Pathogenicity of these strains was analyzed and the genome sequences of two Xap-look-a-like strains, CITA 14 and CITA 124, non-virulent to Prunus spp., were obtained and compared to those available genomes of X. arboricola associated with this host plant. Differences were found among the genomes of the virulent and the Prunus non-virulent strains in several characters related to the pathogenesis process. Additionally, a pan-genomic analysis that included the available genomes of X. arboricola, revealed that the atypical strains associated with Prunus were related to a group of non-virulent or low virulent strains isolated from a wide host range. The repertoire of the genes related to T3SS and T3Es varied among the strains of this cluster and those strains related to the most virulent pathovars of the species, corylina, juglandis, and pruni. This variability provides information about the potential evolutionary process associated to the acquisition of pathogenicity and host specificity in X. arboricola. Finally, based in the genomic differences observed between the virulent and the non-virulent strains isolated from Prunus, a sensitive and specific real-time PCR protocol was designed to detect and identify Xap strains. This method avoids miss-identifications due to atypical strains of X. arboricola that can cohabit Prunus.

Draft Genome Sequence of Two Strains of Xanthomonas arboricola Isolated from Prunus persica Which Are Dissimilar to Strains That Cause Bacterial Spot Disease on Prunus spp.

The draft genome sequences of two strains of Xanthomonas arboricola, isolated from asymptomatic peach trees in Spain, are reported here. These strains are avirulent and do not belong to the same phylogroup as X. arboricola pv. pruni, a causal agent of bacterial spot disease of stone fruits and almonds.

Comparative Genomic and Phenotypic Characterization of Pathogenic and Non-Pathogenic Strains of Xanthomonas arboricola Reveals Insights into the Infection Process of Bacterial Spot Disease of Stone Fruits.

Xanthomonas arboricola pv. pruni is the causal agent of bacterial spot disease of stone fruits, a quarantinable pathogen in several areas worldwide, including the European Union. In order to develop efficient control methods for this disease, it is necessary to improve the understanding of the key determinants associated with host restriction, colonization and the development of pathogenesis. After an initial characterization, by multilocus sequence analysis, of 15 strains of X. arboricola isolated from Prunus, one strain did not group into the pathovar pruni or into other pathovars of this species and therefore it was identified and defined as a X. arboricola pv. pruni look-a-like. This non-pathogenic strain and two typical strains of X. arboricola pv. pruni were selected for a whole genome and phenotype comparative analysis in features associated with the pathogenesis process in Xanthomonas. Comparative analysis among these bacterial strains isolated from Prunus spp. and the inclusion of 15 publicly available genome sequences from other pathogenic and non-pathogenic strains of X. arboricola revealed variations in the phenotype associated with variations in the profiles of TonB-dependent transporters, sensors of the two-component regulatory system, methyl accepting chemotaxis proteins, components of the flagella and the type IV pilus, as well as in the repertoire of cell-wall degrading enzymes and the components of the type III secretion system and related effectors. These variations provide a global overview of those mechanisms that could be associated with the development of bacterial spot disease. Additionally, it pointed out some features that might influence the host specificity and the variable virulence observed in X. arboricola.

Draft genome sequence for virulent and avirulent strains of Xanthomonas arboricola isolated from Prunus spp. in Spain.

Xanthomonas arboricola is a species in genus Xanthomonas which is mainly comprised of plant pathogens. Among the members of this taxon, X. arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruits and almond, is distributed worldwide although it is considered a quarantine pathogen in the European Union. Herein, we report the draft genome sequence, the classification, the annotation and the sequence analyses of a virulent strain, IVIA 2626.1, and an avirulent strain, CITA 44, of X. arboricola associated with Prunus spp. The draft genome sequence of IVIA 2626.1 consists of 5,027,671 bp, 4,720 protein coding genes and 50 RNA encoding genes. The draft genome sequence of strain CITA 44 consists of 4,760,482 bp, 4,250 protein coding genes and 56 RNA coding genes. Initial comparative analyses reveals differences in the presence of structural and regulatory components of the type IV pilus, the type III secretion system, the type III effectors as well as variations in the number of the type IV secretion systems. The genome sequence data for these strains will facilitate the development of molecular diagnostics protocols that differentiate virulent and avirulent strains. In addition, comparative genome analysis will provide insights into the plant-pathogen interaction during the bacterial spot disease process.

[Hemipteran diversity (Cicadellidae and Clastopteridae) in three coffee production zones affected by Xylella fastidiosa (Wells et al.) in Costa Rica]. / Diversidad de cicadélidos y clastoptéridos (Hemiptera) en tres zonas productoras de café afectadas por Xylella fastidiosa Wells et al. en Costa Rica.

A survey was conducted during 2002, 2003 and 2004 to determine the leafhopper species composition, abundance, richness, diversity, evenness, occurrence and flight activity among three coffee production zones of Costa Rica. Yellow sticky traps were used to qualify and quantify the number of aerial leafhoppers during the sampling period. A total of 82,500 individuals, belonging to 139 species within nine leafhopper subfamilies, were trapped. San Isidro de León Cortés site presented the highest diversity from the three surveyed sites. Twenty five species were frequently trapped at least in one of the studied zones, and only Coelidiana sp.1, Osbornellus sp.1, Scaphytopius sp.1 and Empoasca sp. were trapped throughout the sampling period. The flight activity of the taxa that contain the main vectors of Xylella fastidiosa Wells et al. showed differences among the sampling zones.

Diversidad de cicadélidos y clastoptéridos (Hemiptera) en tres zonas productoras de café afectadas por Xylella fastidiosa Wells et al. en Costa Rica / Hemipteran diversity (Cicadellidae and Clastopteridae) in three coffee production zones affected by Xylella fastidiosa (Wells et al.) in Costa Rica

Se realizó un estudio durante los años 2002, 2003 y 2004, con el fin de determinar la composición, la abundancia, riqueza, diversidad, equidad, ocurrencia y fluctuación temporal de las especies de saltahojas en tres zonas productoras de café de Costa Rica. Se utilizaron trampas amarillas pegajosas para clasificar y cuantificar el número de saltahojas adultos durante el período de muestreo. Se encontró un total de 82.500 individuos, pertenecientes a 139 especies ubicadas dentro de nueve subfamilias. San Isidro de León Cortés presentó la mayor diversidad entre los sitios estudiados. Veinticinco especies se encontraron frecuentemente en al menos uno de los sitios muestreados, y solamente Coelidiana sp.1, Osbornellus sp.1, Scaphytopius sp.1 y Empoasca sp. se capturaron a lo largo de todo el período de estudio. La fluctuación temporal de los taxa que comprenden los principales vectores de X. fastidiosa Wells et al. mostró diferencias entre las zonas estudiadas.

A survey was conducted during 2002, 2003 and 2004 to determine the leafhopper species composition, abundance, richness, diversity, evenness, occurrence and flight activity among three coffee production zones of Costa Rica. Yellow sticky traps were used to qualify and quantify the number of aerial leafhoppers during the sampling period. A total of 82,500 individuals, belonging to 139 species within nine leafhopper subfamilies, were trapped. San Isidro de León Cortés site presented the highest diversity from the three surveyed sites. Twenty five species were frequently trapped at least in one of the studied zones, and only Coelidiana sp.1, Osbornellus sp.1, Scaphytopius sp.1 and Empoasca sp. were trapped throughout the sampling period. The flight activity of the taxa that contain the main vectors of Xylella fastidiosa Wells et al. showed differences among the sampling zones.