Florida Strawberry Research Articles

Effect of fumigants on inoculum of Neopestalotiopsis spp. in strawberry crowns and soil.

Florida's strawberry industry has been facing an emerging threat after several outbreaks of an aggressive Neopestalotiopsis sp. affecting fruit, leaf, and crown caused severe yield losses. Our studies found Neopestalotiopsis sp. can survive from one production season to the next on crop debris in soil, which led us to evaluate the effect of fumigants on resident inoculum in strawberry crowns and soil. Different rates of 1,3-dichloropropene, chloropicrin, and metam potassium were applied to pasteurized soil inside glass jars containing infected strawberry crowns. Soil samples were taken after broadcast and bed fumigation treatments with a combination of 1,3-dichloropropene/chloropicrin at ratios of 63:35 were applied at four commercial fields. Crowns and soil samples were processed and plated on a semi-selective medium for Neopestalotiopsis spp. and colony-forming units (CFU) were counted 5 days after plating. CFU counts in crowns treated with 1,3-dichloropropene, chloropicrin, and metam potassium decreased significantly as rates increased and were described by exponential decay models. CFUs were not recovered in most of the soil samples from fumigated strawberry beds after broadcast or bed fumigation. However, CFUs were found in non-fumigated row middles between fumigated beds which can serve as a source of inoculum to start new epidemics. Chloropicrin, 1,3-dichloropropene, and metam potassium were effective on reducing Neopestalotiopsis spp. inoculum in strawberry crowns and soil, providing new evidence on the fungicidal activity of 1,3-dichloropropene. Broadcast fumigation with 1,3-dichloropropene/chloropicrin at ratios of 63:35 could potentially be used to reduce inoculum of Neopestalotiopsis sp. in severely infested fields.

Banker Plant Efficacy to Boost Natural Predators for Management of Field Populations of Scirtothrips dorsalis Hood (Thysanoptera Thripidae) in Strawberries.

Since 2015, Scirtothrips dorsalis Hood has emerged as the main pest of strawberries in Florida. Given the limited management options, there is a recognized need to expand on the management strategies for this pest. Therefore, we explored the possibility of using banker plants to recruit naturally occurring predators of thrips into strawberry fields to suppress S. dorsalis. The study began in the 2021-2022 strawberry season where five banker plants were screened to determine which ones could consistently attract thrips predators by flowering throughout the strawberry season. Capsicum annum L. (ornamental pepper) and Lobularia maritima L. (sweet alyssum) were selected for further evaluation. In the 2022-2023 strawberry season, using a randomized complete block design we assessed the capability of these banker plants to attract thrips predators into the strawberry field. In addition, we examined how the banker plant distance from the strawberry plants influenced the S. dorsalis pest suppression. Our results showed that strawberries located within 3.7 m of ornamental pepper plants had less leaf damage from S. dorsalis compared with those farther away, which may result from the repellent effect of the ornamental peppers. Additionally, Geocoris spp. and Orius spp. were identified as the main thrips predators in the system, although in relatively low numbers. Therefore, these results highlight the potential of incorporating ornamental pepper as a banker plant in strawberry production. Additional applications of this research are explored below.

Marker-assisted Pyramiding of Charcoal Rot Resistance Loci in Strawberry

Macrophomina phaseolina, the causal agent of charcoal rot, is one of the most destructive soil-borne pathogens that affect the global strawberry industry. Resistant cultivars are critical for ensuring the profitability of strawberry production without the protection historically provided by methyl bromide. Previously, three loci, namely, FaRMp1, FaRMp2, and FaRMp3, associated with quantitative resistance to Macrophomina phaseolina have been identified and validated across diverse populations and environments. Among those, the locus with the largest effect, FaRMp3, was initially detected in crosses with an exotic Fragaria ×ananassa selection. We introgressed the favorable FaRMp3 allele into elite germplasm in the University of Florida strawberry breeding program already segregating for FaRMp1 and FaRMp2 and confirmed its phenotypic effects across various genetic backgrounds. Subsequently, we developed a high-throughput genotyping assay to facilitate the transfer and selection of FaRMp3 in breeding populations via marker-assisted selection. Given that three quantitative trait loci (QTL) contribute to partial resistance to Macrophomina phaseolina, stacking them within a single genotype presents a potential strategy for enhancing resistance. We screened 564 individuals that segregate for favorable alleles at all three QTL to assess their effects singly and in combination across multiple genetic backgrounds and production seasons. Inoculated field trials revealed that the three QTL cumulatively enhanced resistance levels, and that two-way QTL combinations including FaRMp3 provide increased protection against the pathogen. Pyramiding all three loci achieved the strongest resistance and could provide substantial economic value to the strawberry industry.

Pseudomonas fragariae sp. nov., a novel bacterial species causing leaf spots on strawberry (Fragaria×ananassa).

In Florida, angular leaf spot, caused by Xanthomonas fragariae, was the only known bacterial disease in strawberry, which is sporadic and affects the foliage and calyx. However, from the 2019-2020 to 2023-2024 Florida strawberry seasons, unusual bacterial-like symptoms were observed in commercial farms, with reports of up to 30 % disease incidence. Typical lesions were water-soaked and angular in early stages that later became necrotic with a circular-ellipsoidal purple halo, and consistently yielded colonies resembling Pseudomonas on culture media. Strains were pathogenic on strawberry, fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco, and lacked pectolytic activity. Although phenotypic assays, such as fatty acid methyl profiles and Biolog protocols, placed the strains into the Pseudomonas group, there was a low similarity at the species level. Further analysis using 16S rRNA genes, housekeeping genes, and whole genome sequencing showed that the strains cluster into the Pseudomonas group but do not share more than 95 % average nucleotide identity compared to representative members. Therefore, the genomic and phenotypic analysis confirm that the strains causing bacterial spot in strawberry represent a new plant pathogenic bacterial species for which we propose the name Pseudomonas fragariae sp. nov. with 20-417T (17T=LMG 32456T=DSM 113340 T) as the type strain, in relation to Fragaria×ananassa, the plant species from which the pathogen was first isolated. Future work is needed to assess the epidemiology, cultivar susceptibility, chemical sensitivity, and disease management of this possible new emerging strawberry pathogen.

A Semi-Selective Medium to Evaluate Over-Summering Survival of Neopestalotiopsis sp. in Florida Strawberry Fields.

Devastating disease outbreaks with leaf spot symptoms and fruit rot caused by a fungus identified as a cryptic species of Neopestalotiopsis have generated concern in the Florida strawberry industry. Some Neopestalotiopsis species are saprobes in soil and plant debris, but the ability of the new Neopestalotiopsis sp. to survive in strawberry debris was unknown. Therefore, the objectives of this study were to develop a semiselective medium for isolation of Neopestalotiopsis spp. and to evaluate Neopestalotiopsis sp. survival in soil and strawberry plant debris over the summer in Florida. The Neopestalotiopsis semiselective medium (NSM) inhibited growth of most fungal species pathogenic to strawberry in Florida, except Neopestalotiopsis rosae, Neopestalotiopsis sp., and Phomopsis obscurans, in addition to Penicillium spp., which are nonpathogenic. However, Neopestalotiopsis species, P. obscurans, and Penicillium spp. could be distinguished in the medium through morphological characteristics. Soil samples arbitrarily collected from six commercial fields toward the end of the season (May) and before (July) and after (September) preplant soil fumigation in the following season were processed with NSM, and Neopestalotiopsis spp. populations were calculated as CFU per gram of soil. CFU ranged from 48.3 to 2,410.8 at the end of the season and from 1.7 to 630.8 before soil fumigation, but Neopestalotiopsis spp. were not recovered after soil treatment. However, 1.7 to 25 CFU were obtained from the nontreated areas in the row middles. Neopestalotiopsis sp. survival was also evaluated on diseased strawberry plants (leaves and crowns) exposed to the environment for 17 months. On leaves, Neopestalotiopsis spp. were recovered for 6 months before leaf decomposition, whereas the number of CFU on crowns declined monthly, but colonies were still recovered during the final evaluation months. A high-resolution melting analysis confirmed most of the colonies from soil and crowns were the new Neopestalotiopsis sp. Our results show Neopestalotiopsis sp. can survive in soil and strawberry debris under Florida summer conditions and may serve as a source of inoculum for the subsequent season.

Spatial and temporal distribution of Scirtothrips dorsalis (Thysanoptera: Thripidae) and their natural enemies in Florida strawberry fields.

Given the recent invasion of Scirtothrips dorsalis Hood in North America, there is limited information regarding their distribution and population dynamics in cultivated small fruit crops. Therefore, we investigated the spatial and temporal distribution of S. dorsalis and their natural enemies in commercially produced strawberry fields in Florida. During 2 consecutive strawberry production seasons, 4 and 6 geographically separated strawberry fields were sampled and were divided into grids with 30-40 sampling points per field. At each sampling point, 4-5 leaf and flower samples were collected, and sticky traps were deployed. We quantified the occurrence of S. dorsalis as well as potential natural enemies, including Orius spp., Geocoris spp., and other predators such as long-legged flies. During both years, most of the S. dorsalis and natural enemies were found on field borders, and counts progressively diminished further into the interiors of plots and away from field edges. Cluster and outlier analysis revealed that S. dorsalis formed statistically significant clusters and that these "hot spots" remained in the same general locations throughout the season. There was a strong relationship between the occurrence of natural enemies and the presence of S. dorsalis, but the number of natural enemies was generally low compared to S. dorsalis. Our results indicate that targeting field borders for chemical control or planting strawberries away from natural areas containing potential alternative hosts for thrips may be an effective strategy for reducing agricultural inputs; however, future field assessments are needed to determine if these methods could replace the treatment of entire fields.

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.

Morphological and Molecular Analysis of Two Mycophagous Nematodes, Aphelenchoides bicaudatus and A. rutgersi (Nematoda: Aphelenchoididae) from Florida Strawberry.

From 2016 to 2021, nematode surveys in Florida strawberry fields revealed several species of foliar nematodes (Aphelenchoides spp.). Aphelenchoides besseyi sensu stricto was detected only in 2016 and 2017 on photosynthetic strawberry leaves/buds, but other not well characterized populations of Aphelenchoides sp. were found on declining/dessicated leaves. Morphological analyses showed that these samples of Aphelenchoides sp. consisted of A. bicaudatus, a species detected in Florida for the first time, and A. rutgersi, a species previously reported in Florida from the citrus rhizosphere. These two species differed from A. besseyi in the shape of their tail terminus: bifurcate in A. bicaudatus; mucronate with a ventral thin mucro in A. rutgersi; and stellate in A. besseyi. One population each of these species was used for morphological and molecular analyses after being reared on Monilinia fructicola. Body and tail length differences were observed among Florida A. bicaudatus and other populations from the Far East and South Africa. Phylogenetic analyses of the rRNA gene sequences showed that Florida A. bicaudatus grouped with those of species from South Korea, Taiwan, and the Netherlands and several other populations listed as Aphelenchoides sp. from Brazil, Costa Rica, and Japan, which were considered as representatives of A. bicaudatus in this study. Similarly, sequences of Florida A. rutgersi grouped with those from environmental samples in Japan and North Carolina, which were listed as Aphelenchoides sp. and were considered as representatives of A. rutgersi in this study. Photosynthetic strawberry leaf samples were free from both A. bicaudatus and A. rutgersi, indicating that these two species did not damage strawberry. They were associated with desiccated leaves and/or propagative stolons, usually infected by fungi, confirming that they are mycetophagous under field conditions in this study. Results of soybean leaf inoculation on moist filter paper containing A. bicaudatus specimens showed that this species could become phytophagous under artificial conditions. Nematodes penetrated the leaf epidermis and migrated into the mesophyll causing leaf tissue discoloration/necrosis, which remained localized within the infested area. Soybean leaf damage was almost negligible, and no nematode reproduction was observed in the inoculated soybean areas.

First Report of Lasiodiplodia pseudotheobromae Causing Postharvest Decay of Strawberries in Florida

Postharvest decay of strawberry (Fragaria × ananassa Duch.) is a major factor causing fruit losses. Strawberries were obtained from various harvests at cooling facilities located in Dover and Plant City, FL during the 2018-19 and 2019-20 seasons. After the fruits were incubated at 22ºC for up to 5 days (d) to promote disease development, Lasiodiplodia decay was observed at up to 3% from some harvests, exhibiting gray mycelia on small lesions that gradually covered the whole fruit. The fungus was isolated onto potato dextrose agar (PDA). Five isolates (SBD18-14, SBD18-277, SBD18-279, SBD19-02 and SBD19-57) were characterized. Fungal mycelia were initially grayish white and then gradually changed to gray to dark gray on PDA at 25oC, and later produced black pigments (Fig. S1). Pycnidia were observed from inoculated strawberries at 14 d. Isolates shared similar conidia morphology: aseptate, hyaline, ellipsoid to ovoid, measuring L × W: 24.0-34.0 (28.3) × 13.0-16.0 (14.3) μm (n =100). Mature conidia were brown, one septate, measuring L × W: 25.0-33.0 (28.8) × 13.0-16.0 (14.5) μm (n =100). The isolates were identified as Lasiodiplodia spp. morphologically (Alves et al. 2008). DNA was extracted from fungal mycelia using an OmniPrep DNA extraction kit, and PCR amplification of ITS and EF1-α genes was performed following the conditions described by White et al. (1990) with some modifications using primers ITS1F-F/ITS4-R (Gardes and Bruns, 1993; White et al., 1990) and EF1-668-F/EF1-1251-R (Alves et al., 2008), respectively. The BLASTn in GenBank showed that the sequences obtained had 99.61 to 100% homology with those of ITS (EF622077) and EF1-α (EF622057) from L. pseudotheobromae CBS116459 (an ex-type strain) (Alves et al., 2008). Sequences of the isolates have been deposited in GenBank with accessions OP326017 to OP326021 for ITS, and OP356202 to OP356206 for EF1-α. Phylogenetic analysis showed that these isolates clustered in the same clade (bootstrap value at 64) with L. pseudotheobromae (Fig. S2). Two fungal inoculum types (mycelia and conidia), two fruit inoculation methods (injury and non-injury) and five fungal isolates were used for pathogenicity tests. Fungal mycelia (2-day-old) on PDA plug (5 mm) or 10 µL of conidial suspension (106 spores/mL) was placed onto each injury (1 x 1 mm in size) or a non-injury area on the surfaces of five strawberry fruits (cv. Florida Brilliance). PDA plug alone or water drops placed on injury or non-injury areas on fruits served as respective controls. Inoculated and control fruits were incubated in a covered plastic container with 100% RH at 22ºC. The experiment was repeated twice. Decay initially appeared as soft and lightly discolored tissue at inoculation areas 2 d post-inoculation (dpi) that extended quickly thereafter. Brown to dark lesions on both injury- and non-injury fruits inoculated with conidia or mycelia were observed at 3 dpi. Decay and gray mycelia gradually developed over the whole fruit at 6 dpi, and pycnidia were observed after 14 dpi (Fig. S1). Disease incidence of 100% was observed on all tests. Control fruits did not develop decay. The results indicate that these isolates are pathogenic to strawberries and infect fruit via both non-injured and injured fruit surfaces. The inoculated fungal isolates were re-isolated, thus, fulfilling Koch’s postulates. L. theobromae, Neofusicoccum parvum/N. ribis species complex causing strawberry fruit rot in Florida fields was reported (Oliveira et al., 2019), but not L. pseudotheobromae. To our knowledge, this is the first report of postharvest decay caused by L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous on strawberries in Florida and in the USA, and it should be considered in strawberry disease management.

First Report of Geotrichum candidum Causing Rubbery Rot on Potato in Arizona.

In January 2023, stand loss due to seed decay (<3% incidence) was reported in a commercial seed potato field in Cochise County, Arizona. Several rotted 'Ciklamen' tubers with wilting shoots were submitted to the University of Arizona Extension Plant Pathology Laboratory for diagnosis. External symptoms included sunken black-colored lesions on affected tubers; white fungal-like growth was also present on the surface of the more severely affected tubers. Internal tissues were watery, rubbery-textured, and spongy with slight gray discoloration. Isolation from the necrotic tubers on PDA yielded yeast-like fungal colonies. Two derived fungal isolates were subcultured on PDA at room temperature for three days, and colonies had a white, dense, and creamy appearance. Hyphae were hyaline, septate, dichotomously branched, and broken into chains of cylindrical to oval-shaped, single-celled arthrospores ranging in size from 3.8-5.3 μm × 5.4-12.5 μm (n = 20). These morphological characteristics matched those of Geotrichum candidum (Carmichael, 1957). Other common potato pathogens, including Pythium, Phytophthora, and Fusarium, were not detected in the samples. For molecular identification, genomic DNA was extracted from the mycelia of two isolates using the DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Partial DNA sequences of the internal transcribed spacer (ITS) region of rDNA and elongation factor 1-alpha (EF-1α) gene were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-1567R (Carbone and Kohn 1999) and EF1-1018/EF1-1620R (Stielow et al., 2015). The resulting sequences showed no difference between the isolates and were deposited under the GenBank Accession Numbers OQ690039 and OQ981192. Based on BLASTn analysis, ITS sequence OQ690039 (369-bp) had a 99.46% match with Geotrichum candidum accessions MN861070 and KY103456. EF-1α sequence OQ981192 (843-bp) showed 100% identity with G. candidum accessions MT346368 of the G. candidum isolates responsible for sour rot of strawberries in Florida (Alonzo et al., 2020). A pathogenicity test was conducted twice on ten healthy 'Ciklamen' tubers, surface sterilized using 10% sodium hypochlorite solution, and rinsed copiously in sterilized water. A conidial suspension (106 conidia/ml) of the isolated fungus was sprayed onto the surface of the tubers. The control consisted of ten tubers that were mock-sprayed with sterilized water. All tubers were incubated in separate sterilized plastic bags in the dark at room temperature for four weeks and examined daily for symptom development. Symptoms of rubbery texture and fluid leaking when cut, and a distinctive smell, were produced on the inoculated tubers. In contrast, the controls did not show any symptoms. These symptoms were similar to those observed on naturally infected tubers. The fungus was re-isolated from symptomatic tissues of inoculated tubers and identified by PCR as G. candidum, thus fulfilling Koch's postulates. To our knowledge, this is the first report of G. candidum as a potato pathogen in Arizona. Rubbery rot of potatoes by G. candidum has been reported previously in Idaho (Duellman et al., 2020) and Michigan (Willbur et al., 2021). Arizona annually grows approximately 1,000 hectares of seed potatoes shipped to various states. Therefore, growers must obtain a correct diagnosis to avoid spreading the disease despite the low incidence of disease.

Delayed spinetoram application is useful in managing Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) in Florida strawberry.

Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is an invasive, early-season pest of strawberry in Florida, causing feeding injury to young foliage that results in stunted plant growth and yield loss. Spinetoram, an effective insecticide for thrips pests with up to 3 applications per season permitted in strawberry, is often applied repeatedly during the early-season (Oct-Nov) to manage S. dorsalis, leaving few or no applications for flower thrips pests later in the season (Dec-Mar). Therefore, new strategies are needed to manage S. dorsalis with less insecticide, with the hypothesis that the first insecticide application can be delayed because young strawberry plants can compensate for minor feeding injury without compromising strawberry yield. Experiments conducted in strawberry field plots in Balm, FL, during 2018 and 2019 showed that delaying a spinetoram application for 14 days after infesting a plant with zero, 5, 10, or 20 S. dorsalis adults did not reduce the plant vigor and yield compared to spinetoram application after 4 days. Furthermore, young plants recovered from injury (10-30% bronzing injury on leaf veins and petioles) due to 1 or 2 S. dorsalis adults or larvae per trifoliate. A strategy of delaying the first spinetoram application when plants have 4-5 trifoliates should help reduce the number of insecticide applications needed for S. dorsalis management and reserve spinetoram applications for later in the season. Lower input costs in Florida strawberry without compromising yields due to thrips damage will improve the economics and sustainability of production systems.

Effect of sweet alyssum on efficacy and sustainability of Neoseiulus californicus (Acari: Phytoseiidae) for biological control of spider mites in multiple cultivars of organic strawberries

The twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is one of the major pests of strawberry in the United States, causing losses of photosynthetic efficiency of up to 30% in strawberries. The predatory mite, Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) is one of the primary biological control agents used against T. urticae in strawberries in Florida. We hypothesized that intercropping sweet alyssum (Lobularia maritima (L.)) would provide pollen as an alternative food source for N. californicus, allowing populations to survive longer when their preferred prey, spider mites, is not available. To test this hypothesis, three treatments including sweet alyssum in combination with N. californicus mites, N. californicus mites (only), and a miticide regimen were evaluated in in a field study. We found that N. californicus mites consistently controlled the spider mite population when populations were high compared to a miticide regimen (control). Furthermore, there was no evidence that the presence of sweet alyssum affected the efficacy or sustainability of N. californicus. The implications of these results are discussed for organic growers who favor banker planting.

Object-Detection from Multi-View remote sensing Images: A case study of fruit and flower detection and counting on a central Florida strawberry farm

Object-Detection from Multi-View remote sensing Images: A case study of fruit and flower detection and counting on a central Florida strawberry farm

Fertilization of Strawberries in Florida

Strawberry is an important crop in Florida, produced as an annual crop on plastic-mulched beds with drip irrigation. The main production area is in west-central Florida, including Hillsborough, Polk, and Manatee counties. Fertilizer is becoming a more significant portion of strawberry production costs, hence, proper fertilizer management is important for minimizing production costs and maximizing yield and fruit quality, while minimizing potential negative impacts on the environment caused by leaching or runoff of excess fertilizer nutrients. This guide summarizes the recent history of strawberry fertilization research and presents the updated research-based fertilizer management recommendations for strawberry production in Florida.

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 U.S. and the efficacy of single- and multi-site 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% (EC50) 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, the sterol de-methylation inhibitors (DMIs), and the multi-sites captan, thiram, and chlorothalonil were the most effective in inhibiting pathogen growth and suppressing disease development. Conversely, products in FRAC groups 1 (MBC) and 7 (SDHI), 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. .

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Development of a Molecular Tool for Identification of a New Neopestalotiopsis sp. Associated with Disease Outbreaks on Strawberry.

A new Neopestalotiopsis sp. was recently reported causing outbreaks of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best, and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida; isolates of N. rosae, N. honoluluana, N. ellipsopora, N. saprophytica, N. samarangensis, and P. rhododendri; and isolates from South Carolina suspected to be the new Neopestalotiopsis sp. were included in this study. This method is based on PCR amplification of a β-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice, yielding fragments of 290 base pairs (bp) and 130 and 20 bp in size, whereas fragments from other species are only cut once, yielding fragments of 420 and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry.

Susceptibility of seven strawberry cultivars to Belonolaimus longicaudatus and interaction with Phytophthora cactorum

Summary Belonolaimus longicaudatus and Phytophthora cactorum are pathogens threatening Florida strawberry production. The interaction among these and cultivar susceptibility were assessed in this study. Cultivars ‘Sensation® Florida127’, ‘Florida Brilliance’, ‘Florida Radiance’, ‘Winterstar™ FL 05-107’, ‘Florida Elyana’, ‘Strawberry Festival’ and ‘Florida Beauty’ were used to conduct one trial using plug transplants and a second trial using bare-root transplants, which was repeated twice. A factorial combination of P. cactorum (+/−) and B. longicaudatus (+/−) inoculation was applied to evaluate strawberry cultivar resistance to P. cactorum and B. longicaudatus as well as their interaction. The effect of P. cactorum on the canopy, root length, and root and shoot weight was dependent on cultivars. For the plug transplant trial, the overall average of plant mortality among all tested strawberry cultivars was 0.1%, suggesting higher tolerance against P. cactorum when vigorous transplants were used. Conversely, the repeated bare-root transplant trial demonstrated different levels of susceptibility in strawberry cultivars when transplants were inoculated with P. cactorum. ‘Sweet Sensation’ ‘Florida127’, ‘Florida Brilliance’, ‘Florida Radiance’ and ‘Winterstar FL 05-107’ were highly susceptible, whereas ‘Florida Elyana’ and ‘Strawberry Festival’ were resistant. Moreover, ‘Florida Beauty’ was moderately resistant to P. cactorum. Our findings demonstrated the importance of using vigorous transplants to reduce P. cactorum damage. Sting nematode reproduction was suppressed when strawberry transplants were inoculated with P. cactorum, suggesting an antagonistic interaction between the pathogens. None of the cultivars was tolerant to B. longicaudatus as this nematode reproduced from 13.4- to 23.8-fold and caused root damage under glasshouse conditions.

First Report of Neopestalotiopsis Disease in Ohio caused by an emerging and novel species of Neopestalotiopsis on Strawberry.

In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 - GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~104 spore/ml) four-week-old 'Cabrillo' strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~104 spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.

First Report of Meloidogyne javanica Infecting Strawberry (Fragaria × ananassa) in the United States.

Strawberry (Fragaria × ananassa) is native to temperate regions. However, it has been produced in tropical areas, as a seasonal crop including in Florida, USA during the winter months. In March 2022, root galls resembling those induced by root-knot nematodes (Meloidogyne spp.) were observed in declining strawberry plants 'WinterstarTM FL 05-107' growing in an organic-certified research site in Hillsborough County, Florida, USA. To our knowledge, M. hapla is the only root-knot species reported to infect strawberry in Florida. Preliminary molecular analyses, including newly synthesized DNA sequences (TW81/AB28 = OQ469833 - OQ469836; D2A/D3B= OQ473043 - OQ473047) using extracted nematode females from the strawberry roots, initially identified the RKN as M. javanica. Nematode species confirmation was further performed using the morphology of the female perineal patterns and isozyme analysis, mainly esterase (EST) and malate dehydrogenase (MDH), DNA sequencing, (NAD5-F/NAD5-R) and the SCAR primer set (Fjav/Rjav), species-specific for M. javanica. Isozyme analyses, EST= J3, which is specific for M. javanica and MDH=N1, as well as the morphology of female perineal patterns, agreed with data previously reported for M. javanica. A pathogenicity test on strawberry 'WinterstarTM FL 05-107' transplants was performed using 10,000 eggs of the original M. javanica population, which induced galls on strawberry plants (Gall index, GI = 4.1) with egg masses clearly visible outside of the roots, producing an average of 1,344 eggs/gram of fresh root and 9,201 ± 4,206 eggs/root system. No galls or egg masses were observed on non-inoculated plants. Tomato 'HM 1823' was used as a control for the viability of the inoculum and showed numerous galls and egg masses (GI=5.0;). The newly obtained DNA sequences using NAD5-F/NAD5-R (OQ474970 - OQ474972) were compared with other sequences available in the GenBank and were shown to be 100% identical to five M. javanica populations from Polk County, Florida, USA (OM418745 - OM418749) and the complete mitochondrion genome of M. javanica (NC026556). To our knowledge, this is the first report of M. javanica infecting strawberry in the United States.