The activities of suaveolol and other compounds from Hyptis suaveolens and Momordica charantia against the aetiological agents of African trypanosomiasis, leishmaniasis and malaria.

African trypanosomiasis and malaria are among the most severe health challenges to humans and livestock in Africa and new drugs are needed. Leaves of Hyptis suaveolens Kuntze (Lamiaceae) and Momordica charantia L. (Cucurbitaceae) were extracted with hexane, ethyl acetate, and then methanol, and subjected to silica gel column chromatography. Structures of six isolated compounds were elucidated through NMR and HR-EIMS spectrometry. Callistrisic acid, dehydroabietinol, suaveolic acid, suaveolol, and a mixture of suaveolol and suaveolic acid (SSA) were obtained from H. suaveolens, while karavilagenin D and momordicin I acetate were obtained from M. charantia. The isolated biomolecules were tested against trypomastigotes of Trypanosoma brucei brucei and T. congolense, and against Plasmodium falciparum. The most promising EC50 values were obtained for the purified suaveolol fraction, at 2.71 ± 0.36 µg/mL, and SSA, exhibiting an EC50 of 1.56 ± 0.17 µg/mL against T. b. brucei trypomastigotes. Suaveolic acid had low activity against T. b. brucei but displayed moderate activity against T. congolense trypomastigotes at 11.1 ± 0.5 µg/mL. Suaveolol and SSA were also tested against T. evansi, T. equiperdum, Leishmania major and L. mexicana but the antileishmanial activity was low. Neither of the active compounds, nor the mixture of the two, displayed any cytotoxic effect on human foreskin fibroblast (HFF) cells at even the highest concentration tested, being 200 µg/mL. We conclude that suaveolol and its mixture possessed significant and selective trypanocidal activity.

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

The Trypanosoma cruzi TcrNT2 Nucleoside Transporter Is a Conduit for the Uptake of 5-F-2'-Deoxyuridine and Tubercidin Analogues.

Among the scarce validated drug targets against Chagas disease (CD), caused by Trypanosoma cruzi, the parasite's nucleoside salvage system has recently attracted considerable attention. Although the trypanocidal activity of tubercidin (7-deazapurine) has long been known, the identification of a class of 7-substituted tubercidin analogs with potent in vitro and in vivo activity and much-enhanced selectivity has made nucleoside analogs among the most promising lead compounds against CD. Here, we investigate the recently identified TcrNT2 nucleoside transporter and its potential role in antimetabolite chemotherapy. TcrNT2, expressed in a Leishmania mexicana cell line lacking the NT1 nucleoside transporter locus, displayed very high selectivity and affinity for thymidine with a Km of 0.26 ± 0.05 µM. The selectivity was explained by interactions of 2-oxo, 4-oxo, 5-Me, 3'-hydroxy and 5'-hydroxy with the transporter binding pocket, whereas a hydroxy group at the 2' position was deleterious to binding. This made 5-halogenated 2'-deoxyuridine analogues good substrates but 5-F-2'-deoxyuridine displayed disappointing activity against T. cruzi trypomastigotes. By comparing the EC50 values of tubercidin and its 7-substituted analogues against L. mexicana Cas9, Cas9ΔNT1 and Cas9ΔNT1+TcrNT2 it was shown that TcrNT2 can take up tubercidin and, at a minimum, a subset of the analogs.

Role of B-Type Natriuretic Peptide in Predicting In-Hospital Outcomes in Acute Exacerbation of Chronic Obstructive Pulmonary Disease With Preserved Left Ventricular Function: A 5-Year Retrospective Analysis.

BACKGROUND: The role of B-type natriuretic peptide (BNP) is less understood in the risk stratification of patients with an acute exacerbation of chronic obstructive pulmonary disease (AECOPD), especially in patients with normal left ventricular ejection fraction (LVEF). METHODS: This retrospective study from 2008 to 2012 evaluated all adult patients with AECOPD having BNP levels and available echocardiographic data demonstrating LVEF ≥40%. The patients were divided into groups 1, 2, and 3 with BNP ≤ 100, 101 to 500, and ≥501 pg/mL, respectively. A subgroup analysis was performed for patients without renal dysfunction. Outcomes included need for and duration of noninvasive ventilation (NIV) and mechanical ventilation (MV), NIV failure, reintubation at 48 hours, intensive care unit (ICU) and total length of stay (LOS), and in-hospital mortality. Two-tailed P < .05 was considered statistically significant. RESULTS: Of the total 1145 patients, 550 (48.0%) met our inclusion criteria (age 65.1 ± 12.2 years; 271 [49.3%] males). Groups 1, 2, and 3 had 214, 216, and 120 patients each, respectively, with higher comorbidities and worse biventricular function in higher categories. Higher BNP values were associated with higher MV use, NIV failure, MV duration, and ICU and total LOS. On multivariate analysis, BNP was an independent predictor of higher NIV and MV use, NIV failure, NIV and MV duration, and total LOS in groups 2 and 3 compared to group 1. B-type natriuretic peptide continued to demonstrate positive correlation with NIV and MV duration and ICU and total LOS independent of renal function in a subgroup analysis. CONCLUSION: Elevated admission BNP in patients with AECOPD and normal LVEF is associated with worse in-hospital outcomes and can be used to risk-stratify these patients.

Experimental investigation of craze morphology of isotactic polypropylene using computed tomography.

In this paper we investigated the morphology of crazes formed during the fracture process of polymeric fibres. Computed tomography technique was applied to reconstruct the image of fibrils-voids structure of crazes. This allowed us to investigate the initial stages of crazes formation during the fracture process of mechanically stretched isotactic polypropylene fibres. The density and morphology of crazes were studied at different regions inside the stretched sample. Accordingly, we are able to visualize the internal structure of the stretched fibres in three dimensions. This revealed in-depth information on the fracture process of polymeric fibres.

A novel method for identifying the order of interference using phase-shifting digital holography.

In this paper, we introduced a mathematical method for measuring the optical path length differences (OPDs), which is suitable for large OPD values where the fringes connections are difficult to detect. The proposed method is based on varying the width of the fringes, without changing the wavelength of the used coherent source. Also, in this work, we discussed the need for such method in off-axis phase-shifting digital holography. Low-resolution off-axis holograms failed to detect the correct interference order. In general, off-axis phase-shifting digital holography is limited by the resolution of the captured holograms. The results obtained using our proposed technique were compared to the results obtained using off-axis phase-shifting digital holograms and conventional two-beam interferometry. Holograms were given for illustration.

Fenugreek seed extract attenuates cisplatin-induced testicular damage in Wistar rats.

Cisplatin (CIS) provides oxidative stress and inflammations in testicular tissues. Fenugreek seed extract (FSE) is a widely used herbal medicine with potent antioxidant and anti-inflammation properties. The purpose of this study was to investigate the protective effects and the possible mechanisms of FSE against CIS-induced testicular damage in rats. Adult male Wistar rats were given vehicle, single dose of CIS alone (10 mg kg(-1)), single dose of FSE alone or single dose of CIS followed by FSE (50, 100 or 200 mg kg(-1)) every day for 5 days. On day 6, oxidative stress and apoptotic testicular toxicity were evaluated. FSE attenuated both germ cell degenerations and apoptosis in seminiferous tubules in CIS-treated rats. Furthermore, FSE counteracted CIS-induced oxidative stress in rats as assessed by the restoration of superoxide dismutase and catalase activities and reduction in the myeloperoxidase activity and malondialdehyde levels in testes. CIS increased expressions of inducible nitric oxide synthase and nuclear factor-kappa B in testicular tissues. Importantly, treatment with FSE at all doses effectively alleviated all of these inflammatory parameters in testes. Based on these results, we concluded that FSE reduces CIS-induced reproductive toxicity in rats by the suppression of testicular oxidative stress, apoptosis and inflammations.

Influence of Left Ventricular Hypertrophy on In-Hospital Outcomes in Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Left ventricular hypertrophy (LVH) is associated with worse outcomes in chronic obstructive pulmonary disease (COPD); however, its role in an acute exacerbation of COPD (AECOPD) has not been reported. This was a retrospective cohort study during 2008-2012 at an academic medical center. AECOPD patients >18 years with available echocardiographic data were included. LVH was defined as LV mass index (LVMI) >95 g/m2 (women) and >115g/m2 (men). Relative wall thickness was used to classify LVH as concentric (>0.42) or eccentric (<0.42). Outcomes included need for and duration of non-invasive ventilation (NIV) and mechanical ventilation (MV), NIV failure, intensive care unit (ICU) and total length of stay (LOS), and in-hospital mortality. Two-tailed p < 0.05 was considered statistically significant. Of 802 patients with AECOPD, 615 patients with 264 (42.9%) having LVH were included. The LVH cohort had higher LVMI (141.1 ± 39.4 g/m2 vs. 79.7 ± 19.1 g/m2; p < 0.001) and lower LV ejection fraction (44.5±21.9% vs. 50.0±21.6%; p ≤ 0.001). The LVH cohort had statistically non-significant longer ICU LOS, and higher NIV and MV use and duration. Of the 264 LVH patients, concentric LVH (198; 75.0%) was predictive of greater NIV use [82 (41.4%) vs. 16 (24.2%), p = 0.01] and duration (1.0 ± 1.9 vs. 0.6 ± 1.4 days, p = 0.01) compared to eccentric LVH. Concentric LVH remained independently associated with NIV use and duration. In-hospital outcomes in patients with AECOPD were comparable in patients with and without LVH. Patients with concentric LVH had higher NIV need and duration in comparison to eccentric LVH.

A proposed method to reconstruct the three-dimensional dispersion profile of polymeric fibres based on variable wavelength interferometry.

In this paper, we suggest a modification to the conventional variable wavelength interferometry. This modification allowed us to calculate the dispersion curve of each point inside polymeric fibres instead of calculating the mean dispersion of these fibres. This modified mathematical treatment was used to calculate the three-dimensional dispersion profile of isotactic polypropylene fibres suffering from necking deformation. The different steps of calculating the three-dimensional dispersion profile of the fibre were demonstrated. The application of this modified method revealed the variation of the fibre material dispersion before, inside and after the necking region. In addition, the birefringence profile of the necked isotactic polypropylene was determined using the proposed mathematical treatment. This allowed us to diagnose the interaction of the incident waves with necked polypropylene fibres, which gives extensive information on the orientation of the molecular chains during the formation of the necking phenomenon.

Nonray-tracing determination of the 3D refractive index profile of polymeric fibres using single-frame computed tomography and digital holographic interferometric technique.

In this paper, we present a nonray-tracing technique for evaluating the three dimensional distribution of the refractive index values inside polymeric fibres. This technique, named 'single-frame computed-tomography (SFCT)', is applied to digital holography. A comparative study between the calculated optical phase values using ordinary tomography and SFCT is carried out, and a negligible deviation is detected. The proposed technique is used to determine the three-dimensional refractive index profile of isotactic Polypropylene fibres, IPP. The variation of the optical properties is measured before, during and after the formation of the necking phenomenon. In addition, SFCT technique is applied to the online determination of the change of the optical properties of IPP fibres. Holograms are given for illustration.

A novel simultaneous photoelastic and two-beam interferometric system: I. Dynamic full-field evaluation of the elasticity modulus profile of polymeric fibres.

A novel optical setup for simultaneous capturing of photoelastic and two-beam interference patterns was designed. The designed optical setup was used to simultaneously record two types of patterns. The first pattern is two-beam interference pattern, and the second one is photoelastic interference pattern produced by objects under stress. This simultaneous capturing of the two patterns allowed us to calculate the full-field distribution of the elasticity modulus profile of fibres. A mathematical expression of the profile of the elasticity modulus was derived. This was applied to evaluate the elasticity modulus of anisotropic isotactic polypropylene fibres during stretching processes. The profile of the elasticity modulus was determined for both static and dynamic in situ cases where the propagation of different structural deformations was observed and studied using the designed optical setup. Patterns were given for illustration.

A novel double-image Fizeau system for accurate investigation of anisotropic polymer fibres.

This paper introduces a double-image multiple-beam Fizeau fringes system. The introduced system can dynamically determine the variations of the refractive indices for both parallel and perpendicular polarization simultaneously. This is achieved by the simultaneous capturing of two multiple-beam interference patterns during the mechanical processing of isotactic polypropylene fibre. This parallel determination of the refractive indices of both polarization directions allowed us to determine the full-field distribution of the stress vector, S. To accomplish this, a mathematical model was deduced to calculate the components of the stress vector, S, i.e. parallel stress component, S1, and perpendicular stress component, S2. Double-image Fizeau fringes system and the deduced mathematical model were used to investigate the variation of the refractive index and stress components of the fibre during the stretching process and propagation of necked regions.

First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Grande Comore and Anjouan.

The causal agent of Asiatic citrus canker, Xanthomonas citri pv. citri, is a bacterium of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit, leaf drop, and twig dieback on susceptible cultivars. This quarantine organism can strongly impact citrus markets so it has consequently been subjected to eradication efforts and international quarantine regulations. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region including the Mascarene and Seychelles archipelagos. In the Comoros archipelago, the disease was observed for the first time in Mohéli island in 1966 (2), but had not yet been reported in neighboring islands, Grande Comore and Anjouan. In September 2013, leaves of key lime (Citrus aurantifolia) and sweet orange (C. sinensis) showing symptoms of citrus canker were collected from Anjouan, Grande Comore, and Mohéli. Nine Xanthomonas-like strains (three from each of the three islands) were isolated using KC semi-selective medium (5) from diseased samples (LK126-3, LK127-7, LK128-2, LK131-10, LK137-1, LK141-3, LK144-5, LK145-5, LK146-2). Based on a specific PCR assay with 4/7 primers (4), all Xanthomonas-like strains were tentatively identified as X. citri pv. citri. All strains produced a 468-bp amplicon similar to X. citri pv. citri strain IAPAR 306 used as a positive control. Negative control reactions with sterile tris buffer did not produce amplicons. Multilocus sequence analysis (MLSA) targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,3) fully identified all strains from the Comoros as X. citri pv. citri. More specifically, eight strains were identified as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (3) (including all strains from the Southwest Indian Ocean region) while one of them (LK141-3 from Mohéli) was identified as a new sequence type based on a non-synonymous single nucleotide polymorphism in gyrB (accession KJ941208). All strains were inoculated by a detached leaf assay (3) onto Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), sweet orange New Hall Navel SRA 343 (C. sinensis), grapefruit Henderson SRA 336 (C. paradisi), and Ortanique tangor SRA 110 (C. reticulata × C. sinensis). All citrus species inoculated produced typical erumpent, callus-like tissue at wound sites. Xanthomonas-like yellow colonies were re-isolated from lesions produced on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (4) and produced the expected amplicon, fulfilling Koch's postulates. No lesions developed on the negative control consisting of inoculations with sterile tris buffer. This is the first report of X. citri pv. citri-A causing Asiatic citrus canker in Grande Comore and Anjouan islands confirming the wide distribution of the pathogen in Southwest Indian Ocean islands. Canker-free nurseries and grove sanitation should be implemented to decrease the prevalence of Asiatic canker in the Comoros. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) J. Brun. Fruits 26:533, 1971. (3) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (4) J. S. Hartung et al. Phytopathology 86:95, 1996. (5) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

Photodynamic therapy in cancer treatment: properties and applications in nanoparticles.

Most of the treatment strategies for tumors and other disorders is photodynamic therapy (PDT). For several years, increasing the efficiency of nanostructured treatment devices, including light therapy, has been considered in different treatment methods. Light Dynamics The use of nanomaterial in this method's production and progress. The use of nanoparticles as carriers is a promising accomplishment, since all the criteria for an ideal photodynamic therapy agent can be given with these nanomaterials. The kinds of nanoparticles that have recently been used in photodynamic therapy are mentioned in this article. Latest advancements are being explored in the use of inorganic nanoparticles and biodegradable polymer-based nanomaterial as carriers of photosynthetic agents. Photosynthetic nanoparticles, self-propagating nanoparticles, and conversion nanoparticles are among the successful photodynamic therapy nanoparticles addressed in this report.

Pyrimidine salvage in Toxoplasma gondii as a target for new treatment.

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 ± 0.8 µM, which was inhibited by uracil with high affinity (Ki = 1.15 ± 0.07 µM) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 ± 0.40 µM, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki of 2.44 ± 0.59 µM, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately -1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated, as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 ± 2.12 µM (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2'-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2'-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment.

Synthesis and Biophysical and Biological Studies of N-Phenylbenzamide Derivatives Targeting Kinetoplastid Parasites.

The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.

Imidazoline- and Benzamidine-Based Trypanosome Alternative Oxidase Inhibitors: Synthesis and Structure-Activity Relationship Studies.

The trypanosome alternative oxidase (TAO), a mitochondrial enzyme involved in the respiration of the bloodstream form trypomastigotes of Trypanosoma brucei, is a validated drug target against African trypanosomes. Earlier series of TAO inhibitors having a 2,4-dihydroxy-6-methylbenzoic acid scaffold ("head") and a triphenylphosphonium or quinolin-1-ium cation as a mitochondrion-targeting group ("tail") were shown to be nanomolar inhibitors in enzymatic and cellular assays. We investigated here the effect of different mitochondrion-targeting cations and other scaffold modifications on the in vitro activity of this class of inhibitors. Low micromolar range activities were obtained, and the structure-activity relationship studies showed that modulation of the tail region with polar substituents is generally detrimental to the enzymatic and cellular activity of TAO inhibitors.

Genetic and Pathological Diversity Among Xanthomonas Strains Responsible for Bacterial Spot on Tomato and Pepper in the Southwest Indian Ocean Region.

Bacterial spot of tomato and pepper, a major problem in tropical climates, can be caused by several Xanthomonas genospecies. We examined the genetic and pathological diversity of a collection of 72 strains from the southwest Indian Ocean region as part of a regional research and development program to update inventories of agricultural pests and pathogens. Xanthomonas euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria were identified in our strain collection. The identification of strains at the species level was consistently achieved by amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA). Overall, X. euvesicatoria was the species recovered prevalently. MLSA data based on four housekeeping genes identified two to three sequence types per genospecies. It suggested that sequence variations primarily consisted of synonymous mutations, although a recombination event spanning several hundred nucleotides was detected for some strains of X. euvesicatoria on the atpD gene coding for the F1-F0-ATPase ß subunit. The pathogenicity of strains was consistent with data found in the literature. Some pathological variations were primarily observed among strains identified as X. euvesicatoria. This study provides the first ever comprehensive description of the status of Xanthomonas species that cause bacterial spot of tomato and pepper in the southwest Indian Ocean region.

A New Type of Strain of Xanthomonas euvesicatoria Causing Bacterial Spot of Tomato and Pepper in Grenada.

Bacterial spot of tomato and pepper (BSTP) can be caused by several Xanthomonas genospecies (2). BSTP is a major disease in Grenada where A and B phenotypic groups (Xanthomonas euvesicatoria and X. vesicatoria, respectively, [2]) have been reported (3). There is no previous report of group A strains, which are strongly amylolytic and pectolytic, in Grenada. In March 2007, tomato and pepper leaves with lesions typical of BSTP were collected in Saint David and Saint Andrew parishes of Grenada. Bacterial isolations were performed on KC semiselective agar medium (4), resulting in isolation of five yellow-pigmented, Xanthomonas-like strains. Three strains isolated from tomato or pepper in Saint David were negative for starch hydrolysis and pectate degradation, two tests that were found useful for strain identification in the 1990s (2). Two strains isolated from pepper in Saint David were strongly amylolytic and degraded pectate. Amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) assays targeting atpD, dnaK, efp, and gyrB were performed on the five strains from Grenada together with a type strain of each of X. euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria as well as other reference strains of X. euvesicatoria and X. perforans as described previously (1). All strains from Grenada were identified as X. euvesicatoria regardless of the typing technique. On the basis of AFLP assays, the two strains with phenotypic features not reported in Grenada were closely related (distances of ≤0.002 nucleotide substitutions per site [1]) to a group of strains from India (ICMP 3381, LMG 907, LMG 908, and LMG 918). These two strains were also identical to the Indian strains based on MLSA, but differed from the X. euvesicatoria type strain by at least one nucleotide substitution in all loci examined. The three strains from Grenada that were negative for starch hydrolysis and pectate degradation had sequences identical to that of the type strain. Young leaves of tomato plants of cv. Marmande and pepper plants of cvs. Yolo Wonder and Aiguille were infiltrated (six inoculation sites per leaf, three replicate plants per cultivar per experiment, and the experiment was replicated once) using inoculum of each of the five strains from Grenada made from suspensions in Tris buffer containing approximately 1 × 105 CFU/ml. Two reference strains of X. euvesicatoria (NCPPB 2968 and LMG 922) were also inoculated as positive control treatments. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical water-soaked lesions that developed into necrotic spots were observed 3 to 8 days after inoculation (dai) for all strains on all cultivars, except NCPPB 2968, which was not pathogenic on pepper cv. Aiguille. Xanthomonas population sizes from lesions plated onto KC agar medium (4) 25 dai ranged from 3 × 106 to 5 × 107, 8 × 107 to 2 × 108, and 9 × 106 to 2 × 108 CFU/lesion on tomato cv. Marmande and pepper cvs. Yolo Wonder and Aiguille, respectively. The epidemiological importance of this previously unreported group of X. euvesicatoria strains in Grenada needs to be assessed. References: (1) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (2) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (3) L. W. O'Garro. Plant Dis. 82:864, 1998. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.