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.

Outbreak of Leaf Spot and Fruit Rot in Florida Strawberry Caused by Neopestalotiopsis spp.

Pestalotiopsis-like species have been reported affecting strawberry worldwide. Recently, severe and unprecedented outbreaks have been reported in Florida commercial fields where leaf, fruit, petiole, crown, and root symptoms were observed, and yield was severely affected. The taxonomic status of the fungus is confusing because it has gone through multiple reclassifications over the years. Morphological characteristics, phylogenetic analyses, and pathogenicity tests were evaluated for strawberry isolates recovered from diseased plants in Florida. Phylogenetic analyses derived from the combined internal transcribed spacer, ß-tub, and tef1 regions demonstrated that although there was low genetic diversity among the strawberry isolates, there was a clear separation of the isolates in two groups. The first group included isolates recovered over a period of several years, which was identified as Neopestalotiopsis rosae. Most isolates recovered during the recent outbreaks were genetically different and may belong to a new species. On potato dextrose agar, both groups produced white, circular, and cottony colonies. From the bottom, colonies were white to pale yellow for Neopestalotiopsis sp. and pale luteous to orange for N. rosae. Spores for both groups were five-celled with three median versicolored cells. Mycelial growth and spore production were higher for the new Neopestalotiopsis sp. isolates. Isolates from both groups were pathogenic to strawberry roots and crowns. However, the new Neopestalotiopsis sp. proved more aggressive in fruit and leaf inoculation tests, confirming observations from the recent outbreaks in commercial strawberry fields in Florida.

Anthracnose Fruit and Root Necrosis of Strawberry Are Caused by a Dominant Species Within the Colletotrichum acutatum Species Complex in the United States.

Strawberry anthracnose fruit rot and root necrosis, caused by Colletotrichum acutatum, are primary limiting factors in fruit production fields in the United States. Recent research focusing on the phenotypic and genetic characteristics of this species has shed light on the diversity of the C. acutatum species complex. In this study, we performed multilocus sequence analysis of four genetic loci to characterize 217 C. acutatum isolates collected over a 23-year period from symptomatic plant tissues of strawberry from six different states. The results revealed two Colletotrichum spp. (C. nymphaeae and C. fioriniae), with 97.7% of the isolate collection (212 of 217) belonging to C. nymphaeae as a dominant clonal linage, regardless of the isolation source. No correlation between species groups and geographical origins of the isolates was observed. Further sequence comparison between historical and contemporary isolates showed the same populations being widely distributed throughout the strawberry nurseries and production fields in the United States and Canada. Subsequently, a subset of 12 isolates representing different quinone-outside inhibitor fungicide resistance profiles from root or fruit tissue of strawberry was selected for comparison of pathogenicity on strawberry. In this test, isolates of different resistance groups or different isolation sources exhibited a similar degree of aggressiveness and caused indistinguishable symptoms on strawberry crowns (P = 0.9555 and 0.7873, respectively) and fruit (P = 0.1638 and 0.1141, respectively), although a significant difference among individual isolates was observed in detached-fruit assays (P = 0.0123). Separate pathogenicity tests using isolates of the two species revealed C. nymphaeae being more aggressive than C. fioriniae in infecting strawberry roots and crowns (P = 0.0073). Therefore, given the occurrence and pathogenicity of C. nymphaeae, this species is likely the sole cause responsible for strawberry anthracnose in the United States.

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.

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.

Effect of Inoculum Concentration and Interrupted Wetness Duration on the Development of Anthracnose Fruit Rot of Strawberry.

Anthracnose fruit rot (AFR) of strawberry, caused by Colletotrichum acutatum, greatly affects production if not controlled. Application of fungicides in addition to the use of less susceptible cultivars are important tools for AFR control. The effects of interrupted wetness duration and inoculum concentration on the development of AFR were evaluated on strawberry cultivars with different levels of susceptibility. Fruit rot and flower blight incidence generally increased with increasing inoculum concentration. 'Camarosa' and 'Treasure' were more susceptible than 'Strawberry Festival' and flowers were more susceptible than immature fruit for most cultivars. Interruption in wetness periods had a significant effect on disease incidence when fruit where exposed to dry periods of 6 h or more compared with 24 h of continuous wetness. The results of this study will be used to refine a disease-warning system to predict AFR outbreaks in Florida strawberry production fields.

Resistance in Strawberry Isolates of Colletotrichum acutatum from Florida to Quinone-Outside Inhibitor Fungicides.

Anthracnose fruit rot of strawberry, caused by Colletotrichum acutatum, is a major disease in Florida and frequent quinone-outside inhibitor (QoI) fungicide applications are needed for disease control. From 1994 to 2014, 181 C. acutatum isolates were collected from multiple strawberry fields in Florida with or without QoI spray history. Sensitivity to azoxystrobin and pyraclostrobin was tested based upon mycelial growth and germ tube elongation inhibition. Mean effective concentration where growth was reduced by 50% (EC50) values for isolates collected prior to 2013 based upon mycelial growth were 0.22 and 0.013 µg/ml and upon germ tube elongation were 0.57 and 0.03 µg/ml for azoxystrobin and pyraclostrobin, respectively. Mycelial growth and germ tube elongation of 48 isolates collected in 2013 and 2014 were not inhibited with azoxystrobin at 3 µg/ml and pyraclostrobin at 0.110 µg/ml. A fungicide discriminatory dose assay indicated that 43 of the 48 isolates had EC50 values higher than 100 and 10 µg/ml for azoxystrobin and pyraclostrobin, respectively. Azoxystrobin and pyraclostrobin sprayed preventively on strawberry fruit inoculated with C. acutatum failed to control resistant isolates. Sequencing of the cytochrome b gene of sensitive and resistant isolates showed that QoI-resistant isolates contained either G143A or F129L amino acid substitutions.