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.

Colletotrichum caladii sp. nov. causing anthracnose leaf spot of Caladium × hortulanum (Araceae) in Florida, USA.

Caladium (Caladium × hortulanum) is an ornamental plant popular for its variable and colorful foliage. In 2020, plants showing leaf spots and blight, typical of anthracnose, were found in a field trial at the University of Florida's Gulf Coast Research and Education Center (UF/GCREC) in Wimauma, FL, USA. Leaf samples consistently yielded a Colletotrichum-like species with curved conidia and abundant setae production in the acervuli. The internal transcribed spacer (ITS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene (gapdh), actin gene (act), chitin synthase 1 gene (chs-1), beta-tubulin gene (tub2), and histone3 gene (his3) were amplified and sequenced. Blastn searches in the NCBI GenBank database revealed similarities to species of the Colletotrichum truncatum species complex. Phylogenetic analyses using multi-locus sequence data supports a distinct species within this complex, with the closest related species being C. curcumae. Based on morphological and phylogenetic analyses, a new species of Colletotrichum, named C. caladii, is reported. Pathogenicity assays and subsequent isolation confirmed that this species was the causal agent of the disease.

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 ß-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.

First Report of Leaf Rust on Blackberry (Rubus spp.) Caused by Kuehneola uredinis in Florida.

During the fall of 2020 and summer of 2021, symptoms of leaf rust were observed on blackberry plants of 'Kiowa', and breeding line 1734 (progeny of 'Natchez' and Arapaho') in a field trial at the University of Florida, Wimauma, FL. Symptoms consisted of small chlorotic spots (1 to 3 mm) on the upper side of the leaf, while the underside had yellow-orange pustules. Disease incidence was up to 100% on both 'Kiowa' and the breeding line 1734, and severity was up to 20% with most of the symptoms observed on older leaves. Two isolates were collected from 'Kiowa' and one from the breeding line 1734 for further investigation. Isolates were maintained and multiplied on healthy 'Kiowa' plants in growth chambers (25 ºC and 12-12 h photoperiod). Uredinia (n=30) were erumpent and ranged from 90 to 320 µm (Average=285 µm, SD=5.3 µm) in diameter. Urediniospores (n=50) were obovoid, yellow, and ranged from 24 to 36 µm long (Average=32 µm, SD=3.2 µm) and 22 to 30 µm wide (Average=28 µm, SD=2.5 µm). Based on morphology and literature, the pathogen was tentatively identified as Kuehneola uredinis (Link) Arth (Arthur 1906; Shands et al., 2018). Spores from a single uredinium of each isolate were collected with a needle and suspended in 50 µL of molecular biology-grade water yielding a final concentration of approximately 5 x 104 spores/mL. Two µL of each spore suspension was used for the PCR reactions. Two DNA fragments were amplified using the primers Rust2inv and LR6, and Rust18S-R and NS1 for the 5.8S-ITS2-28S gene region of rDNA (1,755 bp) and partial 18S gene region of rDNA (2,684 bp), respectively. The amplified products of the partial 28S gene region were sequenced with the primers LR3 and LR0R, and the 18S gene region with NS5, NS6, and NS4 (Aime 2006). DNA sequences were deposited in GenBank (accession nos. OK509845 - OK509848). BLASTn searches revealed that the isolates were 100% identical to K. uredinis reported causing leaf rust on blackberry in California (1044/1044bp, and 1540/1540bp for accession numbers MF158087, and MF158088, respectively). To test for pathogenicity, blackberry cultivars Kiowa, Natchez, Osage, Ouachita, Ponca, Prime-Ark® 45, Prime-Ark® Freedom, Prime-Ark® Traveler, and Prime-Ark® Horizon were inoculated. Five plants of each cultivar were inoculated with a mixture of spores of the three isolates, and two plants of each cultivar were used as controls. Spores were washed from leaves of 'Kiowa' exhibiting sporulation using a suspension of 1% Tween 20 in deionized water. The final concentration of the inoculum was adjusted to 104 spores/mL. Plants were inoculated in the greenhouse with a spray bottle until run-off and kept inside clear plastic boxes for 48 h. Controls were sprayed with sterile deionized water. Plants were watered by mists of 3 s every 10 min twice a week. Disease incidence and severity were evaluated weekly on five leaves per plant that had been tagged before inoculation. The experiment was repeated once. Symptoms identical to the original were only observed in 'Kiowa' and 'Prime-Ark® Freedom'. One week after inoculation, disease incidence was already 100% in both cultivars, with at least one pustule on all the inoculated leaves, and six weeks later disease severity was up to 50% (Average= 35%, SD=2.4%). To our knowledge, this is the first report of K. uredinis causing leaf rust on blackberry in Florida. This disease was reported on Rubus spp. in several U.S. states, and recently in California on Rubus ursinus (Farr and Rossman 2021; Shands et al. 2018). Blackberry is an emerging crop in Florida and efforts should be implemented to monitor the occurrence and spread of leaf rust considering that urediniospores disperse long distances by wind, especially if growers choose the susceptible cultivars 'Kiowa' and 'Prime-Ark Freedom'. The apparent resistance observed in other commercial cultivars such as 'Osage', 'Ouachita', and 'Ponca' may serve as valuable breeding parents for developing new blackberry cultivars with resistance to leaf rust.

Development of a Multiplex High-Throughput Diagnostic Assay for the Detection of Strawberry Crown Rot Diseases Using High-Resolution Melting Analysis.

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 inform strawberry growers in a timely fashion 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 abovementioned pathogens. The multiplex HRM assays using three predesigned 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.

First Report of Diaporthe phaseolorum Causing Stem Canker of Hemp (Cannabis sativa).

Hemp is an annual herbaceous plant that is used for its fiber and oil in a variety of commercial and industrial products. In Florida, it is currently being explored as a new specialty crop. During a field trial from October to January 2019 in Wimauma, FL, a stem canker was observed on up to 60% of three-month-old plants of 'Eletta Campana', 'Carmagnola Selezionata', and 'Tygra'. Symptoms started on the main stems with light-to-dark brown lesions of different sizes and shapes. Over time, the lesions coalesced into large necrotic areas and bore pycnidia. Isolations were made from diseased stem tissues on General Isolation medium (Amiri et al. 2018) after surface disinfestation (Marin et al. 2020). The plates were placed in a growth chamber at 25°C under a 12/12 photoperiod. A fungus with white, floccose, aerial mycelium and pycnidia producing alpha and beta conidia was consistently isolated. Three single spore isolates were chosen for identification and pathogenicity tests. Pycnidia on PDA were globose to irregular and ranged from 170 to 250 µm long (210 ± 2.5, n = 50) and 140 to 220 µm wide (180 ± 2.7, n = 50). The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform and ranged from 5.3 to 7.7 µm long (6.5 ± 1.6, n = 50) and 1.5 to 4.6 µm wide (2.8 ± 1.8, n = 50). The beta conidia were hyaline, elongated, filiform, straight or curved and ranged from 10.2 to 17.7 µm long (16.1 ± 2.2, n = 50) and 0.5 to 1.8 µm wide (0.8 ± 0.2, n = 50). Perithecia were not observed. Based on morphological features, the fungus was similar to anamorphs of Diaporthe spp. (Santos et al. 2011; Udayanga et al. 2015). DNA from the same three isolates was extracted using the FastDNA kit, and the ribosomal internal transcribed spacer (ITS), ß-tubulin (TUB), and calmodulin (CAL) regions were amplified following Udayanga et al. (2014), and Sanger sequenced by Genewiz. Sequences were deposited in GenBank (accession no. MT497039 to MT497047 for ITS, TUB, and CAL). BLASTn searches revealed isolates 20-58, 20-59, and 20-60 were 96.34% identical to the epitype isolate D. phaseolorum AR4203 for ITS (KJ590738.1, 527 bp out of 547 bp), 100% for TUB (KJ610893.1, 459 bp out of 459 bp), and 100% for CAL (KJ612135.1, 522 bp out of 522 bp) (Udayanga et al. 2015). Their identity was confirmed by phylogenetic analyses using maximum likelihood and Bayesian inference methods. To complete Koch's postulates, pycnidia of the same three isolates were harvested and crushed in 2 mL Eppendorf tubes containing 0.01% Tween 20. Conidia suspensions were adjusted to 106 spores/mL. Three 5-week-old potted plants of 'Eletta Campana' and 'Carmagnola Selezionata' per isolate were inoculated using a 1 mL syringe with a needle by injecting 200 µL of the suspension into the stem. Plants were placed inside clear plastic bags for 48 h and maintained in the greenhouse. Control plants were injected with sterile deionized water and kept under the same conditions. The pathogenicity test was repeated once. Four weeks after inoculation, inoculated plants developed stem cankers from which the same pathogen was isolated, whereas controls remained healthy. To our knowledge, this is the first report of D. phaseolorum causing stem canker on hemp. This pathogen has been reported causing canker on sunflower and Phaseolus spp. (Gomzhina and Gannibal 2018; Udayanga et al. 2015; Vrandecic et al. 2009). This discovery may help shape future research into disease epidemiology and management for a crop in which very limited disease information is available at the moment.

First Report of Sclerotinia sclerotiorum Causing Strawberry Fruit Rot in Florida.

During the 2019-2020 Florida strawberry season (October to April), a strawberry (Fragaria × ananassa) fruit rot was observed in two fields (Plant City and Wimauma, FL) with up to 5% incidence on short-day cultivars SensationTM Florida127, and Florida Brilliance. Symptoms on pink and ripe fruit consisted of softening, discoloration, watery rot with white fuzzy mycelium, and initial sclerotium formation. Diseased tissue was placed on General Isolation (GI) medium (Amiri et al. 2018) and incubated at 25°C under a 12-h photoperiod. A fungus producing spreading cottony white colonies with dark sclerotia near the outer edges of the plates was consistently isolated. One isolate from each cultivar field (20-51 and 20-55) was selected for identification and pathogenicity tests. Apothecial production was induced following the protocol of Li and Rollins (2009), and apothecia (n=30) had an average diameter of 4.5 (3.5 to 7.2) mm. Sclerotia were 3.6 (2.5 to 6.2) mm by 4.5 (3.1 to 5.9) mm (n=30) in size. Dimensions of asci were 130.2 (115.1 to 160.5) µm by 8.5 (6.2 to 13.1) µm (n=30), and those of ascospores were 12.2 (10.8 to 14.6) µm by 6.8 (5.7 to 8.1) µm (n=30). Based on these morphological features, the pathogen was tentatively identified as Sclerotinia sclerotiorum (Lib.) de Bary (Maas 1998). DNA was extracted from the same two isolates using the FastDNA kit (MP Biomedicals, Solon, OH), and the ribosomal internal transcribed spacer (ITS) region was amplified using the primers ITS1 and ITS4 (White et al. 1990). Sequences were deposited in GenBank (accession nos. MT378215 and MT378216). BLASTn searches revealed that isolates 20-51 and 20-55 were 99.62% identical (526 / 528 bp) to S. sclerotiorum CBS 499.50 (MH856725.1). Immature pink fruit of SensationTM 'Florida127' were harvested, surface disinfested in bleach solution (0.08% NaClO) for 90 sec, rinsed twice with deionized water, then placed into styrofoam egg cartons inside clean plastic boxes (30x24x7 cm) containing 150 ml of sterile deionized water to maintain moisture, and kept at 25°C with a 12-h photoperiod. Ascospores and sclerotia were used for inoculation tests with three repetitions in an egg carton containing 12 fruit each per isolate and inoculation method. The experiment was repeated once. Fruit were inoculated by placing 20 µl of a 1 × 106 ascospores/mL suspension or a single sclerotium on the upper half of the fruit. Controls were included, by placing 20 µl of sterile DI water or fruit with no sclerotia. Evaluations were done 6, 10, and 15 days after inoculation (DAI). Control fruit remained healthy, while inoculated fruit developed symptoms of softening and discoloration. For ascospore inoculation, disease incidence was 55 (± 5) and 78% (± 4), for 6 and 15 DAI, respectively, whereas for sclerotia inoculation incidence was 100% 6 DAI. Morphologically identical fungi to the original isolates were re-isolated from the diseased fruit. No other fruit decay fungi were observed. S. sclerotiorum has been previously reported causing strawberry fruit rot in Washington state in the United States, England, Israel, and Scotland (Alcorn 1966; Maas 1998; McLean 1957). It has also been listed in the indices of plant diseases from Florida, North Carolina, and California as causing crown rot (Farr and Rossman 2020). To our knowledge, this is the first report of S. sclerotiorum causing strawberry fruit rot in Florida. The pathogen is an aggressive necrotroph with prolonged survival and affects several vegetable crops grown in Florida (Paret et al. 2018). Because only the strawberry beds are fumigated, sclerotia remaining in the alleys could serve as inoculum sources. Currently, the disease is rare and of minor significance to strawberry production. However, efforts should be implemented to monitor its occurrence and spread.