Efficacy of Single- and Multi-Site Fungicides Against Neopestalotiopsis spp. of Strawberry.

Recently, the Florida strawberry industry faced unprecedented outbreaks of an emerging disease caused by the fungus Neopestalotipsis spp. Currently, there are no fungicides labeled to control this disease in the United States and the efficacy of single- and multisite fungicides is unknown. Therefore, this study aimed to determine the in vitro sensitivity of Neopestalotiopsis spp. isolates to fungicides with different modes of action and to evaluate the efficacy of these products on detached fruit and in the field. In preliminary in vitro tests, 30 commercially available fungicides were screened using discriminatory doses. The effective concentration that inhibited mycelial growth by 50% was determined for the most effective single-site fungicides. Four field experiments were conducted during the 2019-20, 2020-21, and 2021-22 seasons to determine product efficacy in managing the disease. The single-site fungicides fludioxonil, fluazinam, and sterol demethylation inhibitors, and the multisite fungicides captan, thiram, and chlorothalonil were the most effective in inhibiting pathogen growth and suppressing disease development. Conversely, products in Fungicide Resistance Action Committee (FRAC) groups 1 (methyl benzimidazole carbamate) and 7 (succinate-dehydrogenase inhibitors), except for benzovindiflupyr, were not effective against Neopestalotiopsis spp. Resistance to fungicides from FRAC group 11 (e.g., azoxystrobin) was confirmed by the presence of the G143A mutation in the cytochrome b gene together with inoculation tests and field trials. Our results provide information to support or discourage the registration of fungicides to manage Neopestalotiopsis fruit rot and leaf spot in strawberry production.

Sensitivity of Colletotrichum acutatum Species Complex from Strawberry to Fungicide Alternatives to Quinone-Outside Inhibitors.

Colletotrichum acutatum is a species complex that causes anthracnose fruit rot and root necrosis on strawberry. The major and minor species within the complex that affect strawberry production are C. nymphaeae and C. fioriniae, respectively. The disease can significantly reduce yield under conducive weather, and its management has greatly relied on quinone-outside inhibitor fungicides (QoI). However, due to the emergence of resistant isolates, such products are no longer effective. Therefore, alternative fungicides were investigated. C. nymphaeae and C. fioriniae isolates were collected from multiple strawberry fields in the United States from 1995 to 2017. The sensitivity of benzovindiflupyr, penthiopyrad, pydiflumetofen, fluazinam, fludioxonil, and cyprodinil was assessed by in vitro and in vivo assays. Both Colletotrichum species were sensitive to benzovindiflupyr, penthiopyrad, fluazinam, and fludioxonil based on mycelial growth assays. Interestingly, of these products, only penthiopyrad did not inhibit conidial germination at 100 µg/ml. For cyprodinil, C. nymphaeae was sensitive based on the mycelial growth, whereas C. fioriniae was not inhibited. Neither species was inhibited by pydiflumetofen in mycelial growth, conidial germination, nor detached fruit assays. The prepackaged mixtures fludioxonil + cyprodinil and fludioxonil + pydiflumetofen were effective in a field trial; however, their use should be carefully considered because of the lack of efficacy of one of the compounds in the mixture. This study sheds light on the potential registration of products alternative to QoIs, such as benzovindiflupyr and fluazinam, which could improve the management of strawberry anthracnose.

Validation of a Decision Support System for Blueberry Anthracnose and Fungicide Sensitivity of Colletotrichum gloeosporioides Isolates.

Blueberry is an increasingly important crop in Florida. Anthracnose fruit rot (AFR), caused mostly by Colletotrichum gloeosporioides, is favored by long wetness periods and temperatures from 15 to 27°C. Currently, the model in the Strawberry Advisory System (StAS) guides fungicide applications targeting strawberry AFR. Given the similarity between blueberry and strawberry AFR, we hypothesized that the model used in StAS could be used in a decision support system (DSS) built for blueberry AFR. There is no information on inhibition posed by fungicides on C. gloeosporioides isolates from blueberry. Our objectives were to demonstrate that the model used in the StAS could be used for blueberry AFR management in Florida and to assess the sensitivity of isolates to fungicides. Four trials were undertaken in blueberry fields in Florida during two seasons to compare the effectiveness of fungicide applications according to the model with that of the growers' standard calendar. Sensitivity of blueberry C. gloeosporioides isolates to azoxystrobin, benzovindiflupyr, penthiopyrad, pydiflumetofen, boscalid, thiophanate-methyl, fluazinam, and fludioxonil was evaluated. AFR incidence and yield were compared between treatments. Following recommendations from the model resulted in disease control as effective as the standard program and in some cases with fewer applications. All isolates were sensitive to benzovindiflupyr, penthiopyrad, fluazinam, and fludioxonil. Low frequency of in vitro inhibition of isolates by azoxystrobin, pydiflumetofen, boscalid, and thiophanate-methyl should raise concern about fungicide resistance. Our results indicate that the model used in StAS could be used in a DSS to help Florida growers to manage AFR in blueberry.

Sensitivity of Colletotrichum acutatum Isolates from Citrus to Carbendazim, Difenoconazole, Tebuconazole, and Trifloxystrobin.

Postbloom fruit drop (PFD) of citrus is caused by the Colletotrichum acutatum and C. gloeosporioides species complexes. The disease is important when frequent rainfall occurs during the flowering period of citrus trees. In Brazil, until 2012, PFD was mainly controlled by preventive applications of the methyl-benzimidazole carbamate (MBC) carbendazim and demethylation-inhibitor (DMI) fungicides such as difenoconazole. Since then, mixtures containing the DMI tebuconazole and the quinone-outside inhibitor (QoI) trifloxystrobin have been commonly used. Fungicides are often applied preventively, sometimes even when conditions are not conducive for PFD development. Excessive fungicide applications may favor the selection of resistant populations of Colletotrichum spp. In this study, we assessed the fungicide sensitivity of C. acutatum isolates collected during the two distinct periods of PFD management in Brazil: before and after the trifloxystrobin and tebuconazole mixture became widely employed. The sensitivity of 254 C. acutatum isolates to carbendazim and difenoconazole and of 164 isolates to tebuconazole and trifloxystrobin was assessed. Mycelial growth inhibition of these isolates was evaluated for all the fungicides using either serial dilution of fungicide rates or the spiral gradient dilution method. In addition, inhibition of conidial germination was also assessed for trifloxystrobin. Analysis of partial ß-tub, cytb, and cyp51b gene sequences did not reveal any mutations related to resistance to MBCs, QoIs, and DMIs, respectively. In mycelial growth assays, mean EC50 values were 0.14, 0.11, and 0.21 µg/ml for difenoconazole, tebuconazole, and trifloxystrobin, respectively. The conidial germination inhibition by trifloxystrobin was similar among the tested isolates, and the mean EC50 value was 0.002 µg/ml. All isolates had similar mean mycelial growth inhibition for carbendazim, regardless of the fungicide concentrations. Therefore, based on similar EC50 values and molecular analyses, no shift in the sensitivity of isolates has been observed to the fungicides commonly used in different citrus-producing areas in Brazil.

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.

Fitness, Competitive Ability, and Mutation Stability of Isolates of Colletotrichum acutatum from Strawberry Resistant to QoI Fungicides.

Quinone-outside inhibitor (QoI) fungicides are used to manage anthracnose of strawberry, caused by Colletotrichum acutatum. However, selection for resistance to QoI fungicides was first reported in 2013 in Florida and, subsequently, in strawberry nurseries and production areas across the United States and Canada. C. acutatum resistance to QoIs is associated with the G143A point mutation in the cytochrome b gene. This mutation is known to be associated with field resistance even at high rates of QoI. In this study, we investigated the relative fitness and competitive ability of QoI-resistant and -sensitive C. acutatum isolates. A fitness comparison did not indicate any difference between resistant and sensitive isolates in aggressiveness, spore production, and mycelial growth at different temperatures. Additionally, in the absence of selection pressure, resistant and sensitive isolates were equally competitive. Cultivation of QoI-resistant and QoI-sensitive isolates for four culture cycles in vitro in the absence of azoxystrobin showed that QoI resistance was stable. The observed lack of fitness penalties and stability of the G143A mutation in QoI-resistant C. acutatum populations suggest that the interruption and further reintroduction of QoI fungicides might not be an option for strawberry nurseries and fruit production areas. Further investigation of alternative chemical and nonchemical C. acutatum control practices, in addition to the integration of multisite fungicides, is needed to reduce the occurrence and distribution of QoI-resistant populations in strawberry fields.

A prospective randomized study comparing partially covered metal stent versus plastic multistent in the endoscopic management of patients with postoperative benign bile duct strictures: a follow-up above 5 years.

BACKGROUND: Self-expandable metal stents (SEMS) are often used for biliary drainage due to obstruction of neoplastic etiology, with better results than plastic stents, but seldom for biliary drainage of benign etiology. OBJECTIVE: Primary end point: Verify bile duct patency after placement of self-expandable metal and (multiple) plastic stents in patients with benign strictures resulting from bile duct surgery with follow-up above five years. Secondary end point: Evaluate long-term complication rate in patients with metal self-expandable and plastic stents. RESULTS: Self-expandable stents were placed in 15 patients and multiple plastic stents in 16. Mean age was 45 years with prevalence of female patients in both groups and no statistical difference between groups was observed (SEMS: 66.7%; plastic multistent: 62.5%; p=0.8). Complication rate resulting from stents occurred more often in the metal stent group (40% versus 25%; p=0.37). In the metal stent group, most frequent complications were hemorrhage (n=3; 20%), stent migration (n=2; 13.3%) and perforation (n=1; 6.7%). In plastic multistent group, most frequent complications were perforation (n:2; 12.5%), hemorrhage (n=1; 6.2%) and ulcer (n=1; 6.2%). Long-term patency rate after biliary drainage was higher in the self-expandable metal stent group, SEMS: 81.67% ± 2.56; CI: 76.47-86.54; plastic multistent: 71.88% ± 2.93; CI: 66.08-77.27. CONCLUSION: The temporary passage of partially covered self-expandable metal stents is a feasible option for patients with benign bile duct strictures.

Un estudio prospectivo y randomizado comparando el uso de stents de metal parcialmente cubiertos de plastico versus multistents en el manejo endoscópico de los pacientes con estenosis benignas postoperatorias del ducto biliar en un seguimiento de más de 5 años / A prospective randomized study comparing partially covered metal stent versus plastic multistent in the endoscopic management of patients with postoperative benign bile duct strictures: a follow-up above 5 years

FUNDAMENTO: Los Stent de metal autoexpandibles se utilizan a menudo para el drenaje biliar en obstrucción de origen neoplásico, con mejores resultados que los stents plásticos, pero son rara vez utilizados en el drenaje biliar en estenosis de etiología benigna. OBJETIVO: El objetivo final primario: Es verificar la permeabilidad de la vía biliar después de la colocación de los stents auto-expandible de metal y (múltiples) prótesis plásticas en pacientes con estenosis benignas derivadas de la cirugía del conducto biliar con un seguimiento superior a cinco años. Punto final secundario: Evaluar a largo plazo la tasa de complicaciones en pacientes con stents metálicos autoexpandibles y plástico. RESULTADOS: Los stents autoexpandibles de metal se colocaron en 15 pacientes y múltiples stents de plástico en 16. La edad media fue de 45 años, con prevalencia de pacientes de sexo femenino en ambos grupos y no hubo diferencia estadística entre los grupos se observó (Stent metálico: 66,7%; plástico multistent: 62,5%, p = 0,8). La tasa de complicaciones resultantes de los stents fueron más frecuentes en el grupo de stent de metal (40% versus 25%, p = 0,37). En el grupo de stent de metal, las complicaciones más frecuentes fueron hemorragia (n = 3; 20%), la migración del stent (n = 2; 13,3%) y perforación (n = 1; 6,7%). En el grupo multistent plástica, las complicaciones más frecuentes fueron la (n: 2, un 12,5%), hemorragia (n = 1; 6,2%) y la úlcera (n = 1; 6,2%). A largo plazo la tasa de permeabilidad tras el drenaje biliar fue mayor en el grupo de metal autoexpandible, SEMS: 81,67% ± 2,56, IC: 76,47-86,54; plástico multistent: 71,88% ± 2,93, IC: 66.08-77.27. CONCLUSIÓN: El paso temporal de los stents metálicos cubiertos parcialmente auto-expandible es una opción viable para los pacientes con estenosis benignas del conducto biliar.

BACKGROUND: Self-expandable metal stents (SEMS) are often used for biliary drainage due to obstruction of neoplastic etiology, with better results than plastic stents, but seldom for biliary drainage of benign etiology. OBJECTIVE: Primary end point: Verify bile duct patency after placement of self-expandable metal and (multiple) plastic stents in patients with benign strictures resulting from bile duct surgery with follow-up above five years. Secondary end point: Evaluate long-term complication rate in patients with metal self-expandable and plastic stents. RESULTS: Self-expandable stents were placed in 15 patients and multiple plastic stents in 16. Mean age was 45 years with prevalence of female patients in both groups and no statistical difference between groups was observed (SEMS: 66.7%; plastic multistent: 62.5%; p=0.8). Complication rate resulting from stents occurred more often in the metal stent group (40% versus 25%; p=0.37). In the metal stent group, most frequent complications were hemorrhage (n=3; 20%), stent migration (n=2; 13.3%) and perforation (n=1; 6.7%). In plastic multistent group, most frequent complications were perforation (n: 2; 12.5%), hemorrhage (n=1; 6.2%) and ulcer (n=1; 6.2%). Long-term patency rate after biliary drainage was higher in the self-expandable metal stent group, SEMS: 81.67% ± 2.56; CI: 76.47-86.54; plastic multistent: 71.88% ± 2.93; CI: 66.08-77.27. CONCLUSION: The temporary passage of partially covered self-expandable metal stents is a feasible option for patients with benign bile duct strictures.

A One-step Procedure by Using Linear Echoendoscope to Perform EUS-guided Choledochoduodenostomy and Duodenal Stenting in Patients with Irresectable Periampullary Cancer.

OBJECTIVE: Endoscopic ultrasound-guided choledochoduodenostomy (EUS-CD) has become an alternative method after unsuccessful endoscopic retrograde cholangiopancreatography (ERCP) treatment. We present a case series study and its feasibility by using only a linear therapeutic channel echoendoscope to create both a biliary-enteral fistula and anatomic enteral recanalization. METHODS: We presented seven cases of unresectable periampullary cancer with both biliary and duodenal obstruction. In these cases, the EUS-guided technique might be an alternative to double stenting (biliary and enteral) in the same procedure and equipment. RESULTS: In all cases, the location of the biliary obstruction was in the distal common bile duct (CBD) and the grade of proximal dilation diameter varied from 15 mm to 20 mm. Two patients had type I (28.6%) and five had type II (71.4%) duodenal obstruction. Technical success of EUS-CD, by the stent placement, occurred in 100% of the cases. There were no early complications. Biliary drainage was effective clinically as well as in laboratory in 6 cases (6/7), by relieving obstructive jaundice and decreasing bilirubin levels. CONCLUSION: EUS equipment may offer an alternative to double stenting in the same procedure and with palliative propose.