New Genotypes of Phytophthora nicotianae Found on Strawberry in Florida.

Phytophthora cactorum is the most common causal agent of Phytophthora crown rot and leather rot of strawberry, but P. nicotianae is also responsible for the disease in Florida. Studies of P. nicotianae populations have suggested that different groups of genotypes are associated with different hosts; however, it is not yet clear how many lineages exist globally and how they are related to different production systems. The aim of this study was to determine the genetic relationships of P. nicotianae isolates from Florida strawberry with genotypes reported from other hosts, quantify the genetic variation on strawberry, and test for an association with nursery source. A total of 49 isolates of P. nicotianae were collected from strawberry plants originating from multiple nursery sources during six seasons of commercial fruit production in Florida. Microsatellite genotyping identified 28 multilocus genotypes on strawberry that were distinct among 208 isolates originating from various hosts and locations. Based on STRUCTURE analysis, two genetic groups were identified: one consisting of isolates from strawberry, and the other comprising samples from different hosts. Multilocus genotypes were shared among nursery sources, and populations defined by nursery were not differentiated. Both mating types were found among the isolates from North Carolina- and California-origin plants and in most strawberry seasons; however, a predominance of A1 was observed, and regular sexual reproduction was not supported by the data. This study reveals a unique genetic population of P. nicotianae associated with strawberry and emphasizes the vital role of nursery monitoring in mitigating disease spread.

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. × ananassa).

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

Pseudocercospora pancratii Causing Leaf Spots on Commercial Blackberry (Rubus sp.) in Florida.

Blackberry (Rubus L. subgenus Rubus Watson) is a deciduous berry crop that is the fourth most economically important berry crop, and its production is expanding in the southeastern United States. However, since most commercially available cultivars were bred under temperate conditions, they are not always well adapted and could be threatened by new pathogen populations inhabiting subtropical areas. In 2017, plants showing purple or brown leaf spots and angular-to-irregular lesions on both leaf surfaces, with clusters of black conidiophores at the center, were observed in a field trial at the University of Florida's Gulf Coast Research and Education Center (UF/GCREC) in Wimauma, FL. A fungus resembling Cercospora/Pseudocercospora was isolated from the lesions. The ribosomal DNA internal transcribed spacers, the translation elongation factor 1-alpha, and the actin genes were amplified and sequenced. Based on the phylogenetic analysis, the closest related species was Pseudocercospora pancratii. Pathogenicity assays and subsequent reisolation confirmed that this species is the causal agent of the disease. Among eight cultivars screened, no complete resistance was found. However, 'Osage' was the least susceptible, and 'Kiowa' was the most susceptible. This study is the first report of P. pancratii causing leaf spots on blackberry worldwide, and it may help shape future research into disease epidemiology and management for a crop that is rapidly expanding but has very limited disease information currently available for Florida growers.

Resistance of Strawberry Cultivars and the Effects of Plant Ontogenesis on Phytophthora cactorum and P. nicotianae Causing Crown Rot.

Phytophthora crown rot (PhCR) is an important disease of strawberry worldwide. Phytophthora cactorum is the most common causal agent, however, P. nicotianae was also recently reported causing PhCR in the U.S. Therefore, the goals of this study were to evaluate the resistance of strawberry cultivars from Florida and California, and to study the etiology of the two Phytophthora species causing PhCR. Sixteen strawberry cultivars were evaluated over three Florida seasons for susceptibility to P. cactorum, and P. nicotianae. Inoculations at different days after transplanting (DAT) were also carried out to evaluate the ability of both species to cause PhCR at different phenological stages of the plant. Plant wilting and mortality were assessed weekly, and disease incidence, and the area under the disease progress curve were calculated. Cultivars Sensation 'Florida127', 'Winterstar FL 05-107', and 'Florida Radiance' were susceptible, whereas 'Florida Elyana', 'Camarosa', 'Fronteras', 'Sweet Charlie', and 'Strawberry Festival' were highly resistant to both Phytophthora species. However, some cultivars exhibited stronger resistance to one species over the other. P. cactorum caused more PhCR when plants were inoculated at transplanting, 45, and 60 DAT, whereas P. nicotianae only caused disease when inoculated at transplanting. These results emphasize the importance of screening for disease resistance to guide management recommendations in commercial strawberry production as well as the need for proper pathogen identification since cultivar susceptibility might differ. Varying susceptibility to P. cactorum and P. nicotianae at different growth stages emphasizes the importance of considering both plant and pathogen biology when making management recommendations.

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.

Phosphite Is More Effective Against Phytophthora Crown Rot and Leather Rot Caused by Phytophthora cactorum than P. nicotianae.

Phytophthora crown rot (PhCR) and leather rot (LR) caused by Phytophthora spp. are major threats to strawberry production worldwide. In the United States, these diseases are mainly caused by Phytophthora cactorum; however, P. nicotianae has also been recently reported causing PhCR. Growers have relied on three different chemical products (i.e., mefenoxam and phosphites for PhCR and LR, and azoxystrobin for LR). Because resistance to mefenoxam and azoxystrobin has been reported, this study aimed to assess the in vitro sensitivity of Phytophthora spp. isolates from strawberry to phosphites and investigate its efficacy on in vivo assays. In vitro sensitivity of P. cactorum (n = 128) and P. nicotianae (n = 24) isolates collected from 1997 to 2018 was assessed for phosphite at 10, 50, 100, 150, and 300 µg/ml. Regardless of the Phytophthora sp. and isolation organ, most of the isolates (75% for P. cactorum and 54.2% for P. nicotianae) had effective concentration that inhibits pathogen growth by 50% (EC50) values ranging from 50 to 100 µg/ml. In vivo tests with strawberry fruit and plants revealed that commercial formulations of phosphite applied at the highest field rate controlled P. cactorum isolates but failed to control PhCR and LR caused by some isolates of P. nicotianae. In this study, EC50 results from in vitro assay did not truly translate the efficacy of phosphites on controlling LR and PhCR caused by P. cactorum and P. nicotianae. Our findings support the hypothesis that the product acts in a dual way: direct on the pathogen and stimulating the plant immune system. Moreover, this has important implications for disease management, highlighting the importance of a correct diagnosis before phosphite recommendations, because its efficacy varies within Phytophthora spp.

Detection and Characterization of Quinone Outside Inhibitor-Resistant Phytophthora cactorum and P. nicotianae Causing Leather Rot in Florida Strawberry.

Phytophthora cactorum and P. nicotianae cause leather rot (LR) of fruit and Phytophthora crown rot (PhCR) in strawberry. LR occurs sporadically but can cause up to 70% fruit loss when weather is conducive. In Florida's annual strawberry winter production system, PhCR can be severe, resulting in plant stunting, mortality, and severe yield loss. Azoxystrobin is labeled for control of LR but not for PhCR. The aims of this research were to determine the sensitivity of P. cactorum and P. nicotianae isolates from strawberry to azoxystrobin and to investigate mechanisms of quinone-outside-inhibitor resistance present in P. cactorum and P. nicotianae based on the known point mutations within the cytochrome b (cytb) gene. Isolates of both Phytophthora spp. causing LR and PhCR were collected from multiple strawberry fields in Florida between 1997 and 2020. Isolates were tested for sensitivity to azoxystrobin at 0, 0.01, 0.1, 1.0, 10, and 50 µg/ml on potato dextrose agar amended with salicylhydroxamic acid (100 µg/ml). Isolates were separated into two groups - sensitive isolates with the 50% effective concentration (EC50) values <1.0 µg/ml, and resistant isolates having EC50 values >50 µg/ml. P. cactorum and P. nicotianae resistance to azoxystrobin was found for isolates collected after 2010. The first 450 nucleotides of the mitochondrial cytb gene were sequenced from a selection of resistant and sensitive isolates of both species. The G143A mutation reported to confer resistance to azoxystrobin was found in all resistant P. cactorum isolates. However, in P. nicotianae, qualitative resistance was observed, but the isolates lacked all the known mutations in the cytb gene. This is the first report of resistance to azoxystrobin in P. cactorum and P. nicotianae.

Phytophthora Crown Rot of Florida Strawberry: Inoculum Sources and Thermotherapy of Transplants for Disease Management.

Phytophthora crown rot, caused mainly by Phytophthora cactorum but also by P. nicotianae, reported in 2018, is an important disease in the Florida strawberry annual production system. Mefenoxam is the most effective and widely used fungicide to manage this disease. However, because of pathogen resistance, alternatives to chemical control are needed. Phytophthora spp. were rarely recovered during the summer from soil of commercial farms where the disease was observed during the season. In a more detailed survey on research plots, neither of the two species was recovered 1 month after the crop was terminated and water was shut off. Therefore, Phytophthora spp. does not seem to survive in the soil over summer in Florida. In a field trial, asymptomatic nursery transplants harboring quiescent infections were confirmed as the major source of inoculum for these pathogens in Florida. Heat treatment of P. cactorum zoospores at 44°C for as little as 5 min was effective in inhibiting germination and colony formation; however, oospore germination was not inhibited by any of the tested temperatures in vitro. In the field, thermotherapy treatment of inoculated plants was shown to have great potential to serve as a nonchemical approach for managing Phytophthora crown rot in production fields and reducing mefenoxam-resistant populations in nursery transplants.

Resistance to Mefenoxam of Phytophthora cactorum and Phytophthora nicotianae Causing Crown and Leather Rot in Florida Strawberry.

Phytophthora cactorum and Phytophthora nicotianae cause leather rot on fruit and crown rot (PhCR) of strawberry plants. Leather rot is not a common disease in Florida; however, up to 50% yield loss has been reported in harvests after intense rainfall events. PhCR is an important disease worldwide and is characterized by a sudden wilting and collapse of plants. Mefenoxam is the most effective and widely used fungicide to control both diseases. P. cactorum and P. nicotianae isolates from leather rot and PhCR have been collected from multiple strawberry fields in Florida since 1997, and the sensitivity of 185 isolates was tested at 0, 0.05, 0.5, 5, and 100 µg/ml. The 50% effective concentration (EC50) values of sensitive isolates ranged from 0.05 to 1 µg/ml. Resistance to mefenoxam (EC50 values >100 µg/ml) was found among P. cactorum isolates collected after 2015, but no resistance was found in P. nicotianae isolates. During the 2015 to 2016, 2016 to 2017, 2017 to 2018, and 2018 to 2019 seasons, resistance was detected on 9, 10, 21, and 23% of the isolates collected, respectively. Mefenoxam-resistant isolates originated from 3 of the 24 strawberry nurseries monitored. This is the first report of the occurrence of P. cactorum resistance to mefenoxam in Florida, suggesting that alternative control strategies are needed to avoid the increase of mefenoxam-resistant populations of P. cactorum in Florida fields.