Rapid and accurate disease diagnosis is a prerequisite for an effective disease management program in strawberry production. In Florida, Colletotrichum spp., Phytophthora spp, and Macrophomina phaseolina are the primary microorganisms causing strawberry crown rot. Even though the diseases can be caused by different pathogens, symptoms are indistinguishable and equally devastating. To timely inform strawberry growers of diagnostic results for effective deployment of chemical control practices, we developed a multiplex high-resolution melting (HRM) assay to rapidly and accurately detect the above-mentioned pathogens. The multiplex HRM assays using three pre-designed primer pairs showed high specificity for individual species by generating specific melting peaks without cross-reaction between primers or with other common strawberry pathogens. The amplification limit of the assay was 1 pg of Colletotrichum and Phytophthora and 100 pg of M. phaseolina DNA per 10 μl reaction. However, the presence of different melting peaks was observed in mixed DNA samples and was concentration- and target DNA-dependent. A crude DNA extraction protocol was developed to allow high-throughput screening by minimizing the inhibitory effects. Moreover, we applied the HRM assay to 522 plant samples and found high correlations between conventional pathogen isolation and HRM and between singleplex and multiplex assays. Altogether, this multiplex HRM assay is specific, cost-effective, and reliable for the timely detection of strawberry crown rot pathogens.
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 β-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.
Blueberry is an increasingly important crop in Florida. Anthracnose fruit rot (AFR), mostly caused by Colletotrichum gloeosporioides, is favored by long wetness periods and temperatures from 15 to 27oC. 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 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 following the model to 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 a reduced number of 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 for fungicide resistance. Our results indicate that the model used in StAS could be used in a blueberry decision-support system to aid Florida growers to manage AFR.
Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum sensu lato and C. gloeosporioides sensu lato, is an important disease in the humid tropics of the American continent. PFD mainly affects flowers, on which typical symptoms are characterized by orange-brown lesions with presence of acervuli. The disease has a sporadic occurrence, but preventative fungicide sprays are applied every season. The objective of this study was to evaluate the effectiveness of a fungicide spray strategy for PFD based on a predictive model of C. acutatum conidium germination linked to weather conditions. Fungicide sprays were performed when the model predicted pre-established thresholds of 10, 15, 20, and 25% of germinated spores (T10, T15, T20, and T25, respectively). Five experiments were conducted in two different seasons in the state of São Paulo, Brazil. PFD control efficacy of the threshold-based treatments was compared with a nontreated control and to a calendar-based spray system. Additionally, an economic analysis was performed to assess the gross income revenues of the fungicide spraying strategies. Disease control in plots treated at T10, T15, and T20 was as effective as the calendar-based strategy. The number of fungicide applications was reduced by 33 to 71% when sprays were applied at T15 and T20, and gross income increased or was comparable to that of the other treatments. Therefore, using a conidium germination model with a threshold of 15 or 20% is recommended as a spraying strategy for PFD management in Brazil.
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