Michelle Souza Oliveira

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.

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.

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.

During the last three seasons, approximately 1% of strawberry (Fragaria x ananassa) with symptoms of fruit softening and discoloration were observed in three fields in Florida. Fruit eventually became black and mummified with pycnidia embedded in the tissue that produced hyaline, aseptate conidia. Eleven isolates recovered from infected tissue were purified by growing them on 2% water agar (WA) with sterilized pine needles (Pavlic et al. 2004), and transferring a single conidium to a new 2% WA plate, by spreading a conidial suspension over the medium and selecting a single germinating conidium 16 h later. DNA of the 11 isolates was extracted from 2-week-old colonies and PCR was performed following the conditions of White et al. (1990) and Carbone and Kohn (1999). Two sets of universal primers, ITS1F/ITS4 and EF1-728F/EF1-986R, were used to amplify and sequence the internal transcriber spacer (ITS) and partial translation elongation factor 1-alpha (EF1-α) regions, respectively. Sequences compared to the Ge...

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.

Phytophthora crown rot is an important disease of strawberry (Fragaria x ananassa) worldwide. In Florida, Phytophthora cactorum is the most common causal agent. However, during the 2015-16 and 2016-17 seasons, strawberry plants with wilt symptoms and dark reddish-brown discoloration of crowns were observed at three commercial farms in west-central Florida. Isolations were made on P5ARP selective medium and incubated at 25°C. Phytophthora colonies with hyaline mycelium forming numerous sporangia and no oogonia were observed after four days. Colonies were transferred to V8 and PDA media to evaluate morphological features. On V8 agar, cultures formed white cottony aerial mycelium and lacked rosette colony shape, usually observed in P. cactorum. On PDA, colonies had an arachnoid shape with irregular margins and grew slower than P. cactorum. Mycelium transferred to pond water produced sporangia ranging in size from 66 to 124 μm wide and 99 to 162 μm long (n= 25), conforming to previous descriptions of P. nicotianae (Erwin and Ribeiro, 1996). When combined with P. nicotianae isolates of known mating type on V8 agar, resulting oogonia measured from 5.2 to 8.9 μm in diameter (n= 25). DNA extracted from three isolates using a FastDNA kit (MP biomedicals, Solon, OH) was used to confirm the morphological identification by multiplex PCR distinguishing P. cactorum from P. nicotianae (Li et al. 2011). The ribosomal internal transcribed spacer (ITS) regions one and two (White et al. 1990) and Ypt1 gene (Li et al. 2011) were amplified and sequenced. The ITS and Ypt1 regions from all three isolates were identical to each other and to P. nicotianae accession Nos. KJ567642 and KJ494902 in GenBank. The sequences were deposited in GenBank (Accession Nos MF590089, MF590090, MF590091, MF590092, MF590093 and MF590094). The isolates were tested for pathogenicity on Winterstar^TM ’FL 05-107’ strawberry. The roots of seven frigo plants per isolate were dipped for one hour in a zoospore suspension (10⁴ ml^-1), potted, and grown in the greenhouse under intermittent mist sprays. The experiment was repeated once. All three isolates caused greater than 85% mortality within two weeks, whereas non-inoculated controls remained healthy after four weeks. The pathogen was reisolated from symptomatic crown and confirmed to be P. nicotianae by morphological evaluation and multiplex PCR. Phytophthora crown rot caused by P. nicotianae has been reported in Japan (Li et al. 2011) and South Korea (Hee, 1998). It has also been reported causing fruit rot (leather rot), but to our knowledge this is the first report of P. nicotianae causing a crown rot of strawberries in the United States. Additional studies are needed to determine whether P. nicotianae differs from P. cactorum with respect to host cultivar resistance, fungicide sensitivity, and disease management.

Botrytis fruit rot (BFR), also known as gray mold, is caused by the fungus Botrytis cinerea and is one of the most important diseases of strawberry in Florida and worldwide. This 4-page document describes the symptoms and management of BFR among strawberry plants. Written by J. C. Mertely, M. S. Oliveira, and N. A. Peres and published by the UF/IFAS Department of Plant Pathology, February 2018. http://edis.ifas.ufl.edu/pp152

Botrytis cinerea, the causal agent of Botrytis fruit rot, is annually introduced into Florida strawberry fields by infected transplants. The disease can be managed by fungicide sprays throughout the season; however, previous studies have demonstrated that several chemical classes are no longer effective owing to B. cinerea resistance. The objectives of this study were to evaluate the effectiveness of preplant fungicide-dip treatments of strawberry transplants on reducing B. cinerea colonization and to evaluate fungicide sensitivity of surviving B. cinerea isolates. Transplants of two strawberry cultivars, Winterstar ‘FL 05-107’ and ‘Florida Radiance’, were dipped in 11 different fungicides. After the plant establishment period, eight leaves per plot were collected before and 14 days after treatment to evaluate B. cinerea incidence. Isolates (n = 139) obtained from the transplants were tested for fungicide sensitivity. Plant diameter was measured 47 days after planting. All treatments, including the controls, reduced B. cinerea incidence by at least 77% compared with predip incidence. However, fungicide-resistant isolates were recovered from all treatments tested.

Strawberry transplants produced in nurseries across Canada, northern United States, and California are shipped annually to other strawberry-growing regions, including Florida. Botrytis cinerea, the causal agent of gray mold, causes latent infections on transplants which are suggested as a potential source of primary inoculum in strawberry fields. In this study, we investigated the survival of B. cinerea isolates over the summer in Florida, the presence of B. cinerea in transplants from 14 nurseries from Canada and the United States in 2011, 2012, and 2013, and the sensitivity of nursery population to several botryticides. Botrytis cinerea was detected on dead strawberry plants sampled from commercial strawberry fields between March and June but not in July and August, suggesting that the fungus does not over-summer in strawberry fields in Florida. Nursery transplants surveyed in 2011, 2012, and 2013 showed B. cinerea incidences of 20 to 37, 20 to 83, and 2.5 to 92.5%, respectively. In total, 409 isolates were tested for sensitivity to pyraclostrobin, boscalid, pyrimethanil, fenhexamid, iprodione, penthiopyrad, fluopyram, and fludioxonil. Overall, respective resistance frequencies were 91.7, 79.3, 33.2, 20.7, 2.4, 0.2, 0.2, and 0.0%. A majority of isolates tested were resistant to either 3 or 4 fungicides simultaneously. These findings reinforce the need for an integrated approach between strawberry nurseries and production fields to improve gray mold management and mitigate future risks of resistance development in B. cinerea.