Multilocus Phylogenetic Analyses of Colletotrichum gloeosporioides Species Complex Causing Crown Rot on Strawberry in Florida.

Colletotrichum gloeosporioides is the causal agent of Colletotrichum crown rot of strawberry in the southern United States. Recent multigene studies defined C. gloeosporioides as a complex species comprised of 37 species. In our study, we phylogenetically characterized C. gloeosporioides isolates from strawberry and other noncultivated plants around strawberry fields. One hundred fifteen strawberry isolates and 38 isolates from noncultivated hosts were sequenced for five genomic regions: internal transcribed spacer, actin, calmodulin, chitin synthase, and glyceraldehyde-3-phosphate dehydrogenase. Phylogenetic analysis using the maximum likelihood and Bayesian inference methods, based on partition-specific models, revealed that most of the isolates in Florida (86%) were closely related to C. siamense, whereas 14 isolates were closely related to C. theobromicola (syn. C. fragariae), four isolates were C. fructicola, and three isolates were C. clidemiae. However, only the first three species were pathogenic to strawberry. Morphological characteristics evaluated show that mycelial growth of all species is approximately 5 mm/day, but colony morphology varies by species and incubation conditions. In vitro mating of the isolates demonstrated that C. fructicola is homothallic whereas C. siamense and C. theobromicola isolates are heterothallic. The biological importance of these different Colletotrichum species is currently being investigated to determine whether different management strategies are needed in strawberry production fields.

Increased Oxidative Stress in Gastric Cancer Patients and Their First-Degree Relatives: A Prospective Study from Northeastern Brazil.

BACKGROUND AND AIMS: First-degree relatives of gastric cancer patients are at increased risk of developing gastric cancer. Increased oxidative stress, including lipid peroxidation, has been associated with gastric carcinogenesis. Whether first-degree relatives of gastric cancer patients have increased oxidative stress remains unknown. We aimed to compare oxidative stress in patients with gastric cancer, their first-degree relatives, and dyspeptic controls. METHODS: A total of 155 patients undergoing upper endoscopy were prospectively enrolled, including 50 with gastric cancer, 49 first-degree relatives of gastric cancer patients, and 56 controls. Serum concentrations of malondialdehyde (MDA) and glutathione) and activities of superoxide dismutase (SOD) and catalase were measured. Multivariate analysis adjusting for sex, age, smoking status, and alcohol consumption was performed. RESULTS: Lipid peroxidation, as measured by concentration of MDA (nmol/mL), was higher (p = 0.04), and glutathione levels were lower (p < 0.001) in the gastric cancer group compared to controls. There was no difference in the catalase activity among the groups. There was no difference in glutathione and MDA concentration or catalase activity between the different stages of gastric cancer based on the TNM classification. Relatives of gastric cancer patients had higher glutathione concentration (µmol/mL) compared to gastric cancer patients (262.5 vs. 144.6; p = 0.018), while there was no difference in MDA concentration. Catalase and superoxide dismutase activity were lower in the gastric cancer group (3.82 vs. 0.91; p < 0.001 and 1.04 vs. 0.6; p < 0.001) compared to their first-degree relatives. Interestingly, MDA concentration in the first-degree relative group was higher than in the control group (7.9 vs. 5.1; p = 0.03). CONCLUSIONS: In this study, similarly to gastric cancer patients, their first-degree relatives were found to have increased oxidative stress compared to controls. Further studies are warranted to validate this observation and to better understand the role of oxidative stress as a possible biomarker in this population.

Efficacy and Baseline Sensitivity of Succinate-Dehydrogenase-Inhibitor Fungicides for Management of Colletotrichum Crown Rot of Strawberry.

The current management of Colletotrichum crown rot (CCR) of strawberry, caused by Colletotrichum gloeosporioides sensu lato, relies on the use of a few fungicide classes, particularly QoI fungicides. Since resistance to QoI fungicides has recently been detected, alternative fungicide groups are needed to control this disease. Our objective was to evaluate the efficacy of succinate-dehydrogenase-inhibitor (SDHI) fungicides in managing CCR. Five SDHI fungicides, fluopyram, isofetamid, penthiopyrad, fluxapyroxad, and benzovindiflupyr, were applied 2 days before or 1 day after inoculation of cultivar Strawberry Festival. SDHI treatments were compared with the most common fungicides used for CCR management, i.e., thiophanate-methyl, pyraclostrobin, and captan. Benzovindiflupyr applied 1 day after inoculation was effective in reducing plant mortality and disease development. The baseline sensitivity of C. gloeosporioides isolates was determined in vitro using a spiral gradient dilution assay. The EC50 for benzovindiflupyr and penthiopyrad varied from 0.08 to 1.11 and 0.45 to 3.17 µg/ml, respectively, whereas the other SDHI fungicides did not inhibit fungal growth. If registered, benzovindiflupyr could serve as an alternative to manage CCR in Florida.

Baseline Sensitivity of Botrytis cinerea Isolates from Strawberry to Isofetamid Compared to other SDHIs.

Succinate dehydrogenase inhibitors (SDHIs) are the fungicides most commonly used to control Botrytis fruit rot on commercial strawberry in Florida. The medium-to-high risk of selection of resistance in the causal agent Botrytis cinerea is a threat to the efficacy of this fungicide group. In this study, we characterized the sensitivity of B. cinerea to the SDHI isofetamid, evaluated the SdhB gene mutation associated with resistance, and monitored resistance frequencies to five SDHI fungicides for two consecutive seasons. EC50 values of 70 isolates were obtained using the spiral gradient dilution (SGD) method and averaged 0.098 µg/ml of isofetamid. EC50 averages of 3.04 and >500.00 µg/ml were obtained for isolates with the N230I and P225F mutations indicating moderate and high resistance to isofetamid, respectively. A total of 565 B. cinerea isolates collected during 2015-2016 and 2016-2017 seasons from strawberry nurseries and Florida production fields were evaluated using conidial germination assays. Results for the first season showed resistance frequencies of 95, 33, 21, 25, and 0% to boscalid, penthiopyrad, fluopyram, benzovindiflupyr, and isofetamid, respectively. The respective resistance frequencies for the following season were 91, 95, 44, 27, and 1.3%. Only three isolates were found to be moderately resistant to isofetamid during the second season, and the mutation N230I was identified after sequence analysis. These isolates were confirmed to be resistant to isofetamid in fruit assays with disease incidence of 55.6 to 77.0%; however, the conidial production of the isolates was inhibited by an average of 83.9%. In general, isofetamid efficacy was higher than the other evaluated SDHIs, but a slight increase in resistance frequencies was observed in our study.

Development of High-Throughput SNP Genotyping Assays for Rapid Detection of Strawberry Colletotrichum Species and the G143A Mutation.

Colletotrichum spp. cause major diseases of strawberry and disease management depends on the species present. However, species identification based on symptoms and spore morphology is difficult. Therefore, development of molecular techniques for trustworthy and high-throughput identification of Colletotrichum spp. is vital for the accurate diagnosis. A high-resolution melting (HRM) assay was developed for simultaneous identification and differentiation of Colletotrichum spp. from fungal colonies or from symptomatic strawberry tissue. HRM markers were designed based on the internal transcribed spacer region of Colletotrichum acutatum and C. gloeosporioides from strawberry, and accurately identified and differentiated the two species. In addition, for the rapid detection of a single-nucleotide polymorphism (SNP) in the cytochrome b (cytb) gene of C. acutatum and C. gloeosporioides associated with resistance to quinone-outside inhibitor fungicides, an endpoint SNP genotyping analysis was developed. The HRM and endpoint SNP genotyping assays are useful methods that can be implemented in plant diagnostic clinics for the rapid and accurate identification of Colletotrichum spp. and detection of the G143A mutation in the cytb gene of C. acutatum and C. gloeosporioides.

High levels of filamentous actin and apoptosis correlate with mast cell refractoriness under alloxan-evoked diabetes.

Mast cell number and reactivity were shown to be down-regulated under diabetic conditions. Since the balance between globular and filamentous actin plays a pivotal role in the activity of secretory cells, we investigated whether an imbalance in that system could underlie the hyporesponsiveness of mast cells in diabetes. The apoptotic state was also evaluated. By means of rhodamine/phalloidine staining of F-actin, we noted that diabetic mast cells exhibited an increase in fluorescence intensity and reduction in cellular size, when compared with cells from normal animals, in parallel with elevation in the percentage of cells developing apoptosis. The levels of Bax, a pro-apoptotic member of Bcl-2 family, appeared increased at baseline in mast cells from diabetic rats compared with normal cells. These phenomena correlated with reduction in histamine and PGD2 release following antigen challenge in vitro. The steroid antagonist RU 486 abolished the reduction of histamine secretion from diabetic mast cells. We conclude that hyporesponsiveness of mast cells noted in diabetes may be accounted for by reduction in actin filament plasticity, in clear association with the rise in the percentage of cells undergoing apoptosis. In addition, the refractoriness of diabetic mast cells to antigen in vitro seems to be dependent on glucocorticoids.