First report of the new Neopestalotiopsis species causing strawberry leaf spot and fruit rot in Georgia.

In April 2023, symptomatic strawberry (Fragaria × ananassa) plants (cv. 'Camarosa' and 'Florida Brilliance') were observed at a commercial farm in Worth County, GA (USA). Symptoms included foliar, irregularly distributed, and different-sized spots (dark brown with light brown centers) and dark brown V-shaped necrotic areas starting at the leaf edge. By the time of sample collection, ~50% incidence was reported in the field. Leaf samples were collected and shipped overnight to the laboratory. Black acervuli were observed readily on old necrotic foliar lesions. Conidial morphology was consistent with that observed with Neopestalotiopsis species (Maharachchikumbura et al. 2014). Conidia were ellipsoid to fusiform, five-celled, with three light brown median cells and one hyaline apical and basal cell. Apical cells had two-to-four flexuous appendages, and the basal cell had one non-flexuous appendage (Fig 1). The average (n = 20) conidia length, not including the appendages, was 26.6 µm (SD: 2.8), and width was 6.3 µm (SD: 0.94). Fungal isolation was conducted on acidified PDA and incubated at 25°C for 6 days. Dense, white mycelia were observed on the upper plate surface, while a pale pink/orange coloration was observed on the underside (Fig 1). Black acervuli formed on the surface of the white mycelial mat. Six isolates were purified and selected to confirm the species identity. DNA was extracted from 6-day-old cultures and PCR was conducted following Kaur et al. (2023). Amplified DNA was digested with the restriction enzyme BsaWI and two bands were clearly visualized (~130 and ~290 bp), along with a faint band of 20-bp (Fig 2). Four of the six isolates were selected for sequencing of the ß-tubulin gene. BLAST queries using the consensus sequence showed that all isolates had 100% identity to strain N21002 from Florida (FL), characterized as Neopestalotiopsis sp. (Kaur et al. 2023). One representative isolate (AJ07-2023) was deposited in GenBank (accession No. PP316103). Pathogenicity tests were performed on 27-day-old transplants of Sensation 'Florida127' provided by Natalia Peres from the UF. Plants were grown in 10.5 cm pots in the greenhouse. Isolate AJ07-2023 was grown on PDA for 30 days at 25°C, and the spore suspension was adjusted at 106 spore/ml. Five strawberry plants were sprayed with 5 ml of inoculum using a Preval sprayer with a CO2 canister, and 5 plants were sprayed with sterile distilled water. Plants were placed in a growth chamber for 6 days and covered with plastic bags after the sixth day to maintain ~85% relative humidity and 25°C. Foliar symptoms, including dark-brown circular lesions occurring towards the edge of leaves with light-brown center and light-yellow halo, developed 13 days after inoculation. No symptoms were observed on control plants. Neopestalotiopsis sp. was reisolated from inoculated plants as described above. Colony, conidial morphology, and PCR results were consistent with the original isolates. Neopestalotiopsis disease has been reported on strawberry in FL (Baggio et al. 2021), OH (Rotondo et al. 2022) and IN (Guan et al. 2023). Although the disease has been observed sporadically in GA since 2020 (Brannen, personal communication), to our knowledge, this is the first official report of the new Neopestalotiopsis sp. in GA. It has been reported that this new strain is more aggressive on fruits and leaves than other Neopestalotiopsis spp. (Baggio et al. 2021), therefore, accurate identification is critical for proper management.

First report of areolate mildew of cotton, caused by Ramulariopsis pseudoglycines in Mississippi.

In August of 2022, cotton (Gossypium hirsutum L.) growing in several north central Mississippi counties was observed to exhibit yellowish lesions on the adaxial leaf surface with white powdery fungal growth on the corresponding abaxial surface. By the end of the 2022 growing season, 19 Mississippi counties were observed to have infected cotton. Symptomatic leaves were collected from affected plants, sealed in plastic freezer bags, stored on ice in a cooler, and transported to the laboratory. Prior to isolation, the pathogen was microscopically examined and determied to be morphologically similar to the description of Ramulariopsis spp. (Ehrlich and Wolf 1932). Using a sterile needle, conidia were transferred to V8 medium amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter) and incubated in the dark at 25°C. After 14 days, the colony diameter was measured, and morphological characteristics were consistent with previous descriptions (Videira et al. 2016; Volponi et al. 2014). On V8 medium, the 7 mm diameter colonies grew raised, lumpy, and lobed, with iron-grey coloration. The mycelia were hyaline, septate, branched, and 1 to 3 µm in diameter. Conidia ranged from 2.8 to 25.6 µm in length and 1.0 to 4.9 µm in width (x = 12.8 × 3.1 µm; n = 20). Pure cultures were obtained on V8 medium, and DNA was extracted from a 14-day-old-culture. TW098-22, a representative isolate, was amplified, and sequenced targeting the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF 1-α), and actin (ACT) genes as described by Videira et al. (2016). The consensus sequences were deposited in GenBank (accession no. OQ653427, OR157986, OR157987). BLASTn query of the NCBI GenBank showed 100% identity of the 483-bp (ITS) and 706-bp TEF 1-α sequences from TW098-22 with Ramulariopsis pseudoglycines CPC 18242 (type culture; Videira et al. 2016). Koch's postulates were performed after multiplying individual colonies by streaking on V8 medium as above. Culture plates were subsequently incubated at 25°C for 14 days in the dark. Colonies were aseptically transferred to 50 ml centrifuge tubes containing 50 ml of autoclaved reverse osmosis (RO) water amended with Tween 20 (0.01%). The resulting inoculum suspension was adjusted to 13.5 × 105 conidia/ml using a hemocytometer. The foliage of five, 25-day-old cotton plants were sprayed with 10 ml of the suspension and a plastic bag was placed over each plant to maintain humidity for 30 days. Five plants were sprayed with sterilized RO water to serve as controls. Plants were incubated in a growth chamber at 25°C and ~70% relative humidity with 16:8 h of light:dark. Thirty days post-inoculation, foliar symptoms and signs were observed on all inoculated plants including small necrotic lesions and white powdery growth. Control plants remained asymptomatic. The trial was repeated. When reisolated, the colony and conidia morphology and DNA sequence (ITS) were consistent with the description of the original field isolate. Areolate mildew of cotton can be caused by two species of Ramulariopsis: R. gossypii and R. pseudoglycines (Videira et al. 2016). The two species have been reported in Brazil (Mathioni et al. 2021); however, this is the first report of R. pseudoglycines in the United States. In addition, even though areolate mildew has previously been reported from much of the southeastern U.S. (Anonymous 1960), the report herein serves as the first description of R. pseudoglycines in Mississippi and U.S. cotton.

First Report of Neocosmospora Stem Rot of Soybean Caused by Fusarium neocosmosporiellum in Mississippi.

In September 2021, diseased soybean [Glycine max (L.) Merr.] plants were observed in a commercial field in Lamar County, Mississippi (MS). Foliar symptoms included mild interveinal chlorosis and necrosis in the affected plants (Fig. 1a). Stems and roots exhibited orange to black lesions and pith discoloration (Fig. 1b). Signs of the fungus included orange-to-red spherical perithecia, observed in clusters on crowns and roots. Perithecia were surface-sterilized with 70% ethanol for 1 min. Subsequently they were gently pressed in a droplet of 5 µl of sterilized reverse osmosis (RO) water and the suspension was streaked onto potato dextrose agar (PDA) amended with chloramphenicol (75 mg/liter) and streptomycin sulfate (125 mg/liter). Plates were incubated in the dark at 25°C. White to light pink and floccose mycelia developed after four days of incubation and hyaline, one celled, cylindrical to oblong-ellipsoidal microconidia were observed (Fig. 2b; ×400). Conidia measured 8.8 to 19 × 2.8 to 5 µm (n=15). Abundant orange to brown perithecia developed on PDA after three weeks of incubation (Fig. 2a). The asci within the perithecia were cylindrical, eight-spored, thin walled and measured 94.6 to 123.6 µm × 10 to 14 µm (n=10). Ascospores were uniseriate, globose to ellipsoidal, hyaline to brown and measured 11.6 to 16 µm × 7 to 10 µm (n=10; Fig. 2c). The morphological characteristics were consistent with those of Fusarium neocosmosporiellum O'Donnell & Geiser (≅ Neocosmospora vasinfecta E. F. Sm.; Geiser et al. 2013). Genomic DNA was extracted from isolate TW068-21 from a 3-week-old culture plate. The internal transcribed spacer region (ITS), elongation factor 1-alpha (EF1-alpha) and calmodulin (cmdA) gene were amplified, and consensus sequences deposited in GenBank (OM640625, OM681343, OM681344). ITS and EF1-alpha sequence comparison using NCBI BLAST, showed > 99.2% similarity with N. vasinfecta JL2210 while cmdA sequence was 99.8% similar to strain CBS 517.71. A pathogenicity test was performed on 2-week-old Asgrow 46X6 soybean seedlings grown in 10.2 cm pots in a growth chamber. Isolate TW068-21 was grown on antibiotic-amended PDA for 4-weeks and inoculum suspension was prepared with sterilized RO water and adjusted to 2 × 105 ascospores/ml. Soybean seedlings (n=8) were removed from pots and roots were dipped into the inoculum suspension for 20 min. Four control plants were dipped in autoclaved RO water. Plants were re-planted in potting mix, and the inoculated plants were immediately drenched with 20 ml of the inoculum and placed in a growth chamber (25°C; 14 h light). The experiment was repeated once. Inoculated plants presented dark brown discoloration at the base of the stem after 3 weeks of incubation, but no foliar lesions were observed. Control plants remained asymptomatic. Symptomatic stems were placed in a moist chamber (≈23°C; 12 h:h light:dark) and light brown perithecia developed after 1 week. Fusarium neocosmosporiellum was re-isolated from perithecia and stems and colony and spore morphology were similar as described above. To our knowledge, this is the first report of F. neocosmosporiellum in MS. Signs and symptoms of this disease resemble red crown rot. Consequently, careful morphological and molecular assessments should be used for confirmation. Neocosmospora stem rot has been previously reported in Alabama (Gray et al. 1980), Arkansas (Greer et al. 2015), and Georgia (Phillips 1972). Yield losses due to this pathogen in MS are currently unknown.

Specific Environmental Temperature and Relative Humidity Conditions and Grafting Affect the Persistence and Dissemination of Salmonella enterica subsp. enterica Serotype Typhimurium in Tomato Plant Tissues.

Little is known about the abiotic factors contributing to the preharvest persistence of Salmonella in tomato tissues. Therefore, we investigated the effects of specific environmental conditions and contamination methods on the persistence and dissemination of Salmonella enterica subsp. enterica serotype Typhimurium (JSG626) in tomato plants. When plants were sprayed on the leaves with a JSG626-contaminated solution, JSG626 persistence in the phyllosphere (bacteria located on the surface of the inoculated foliage and stem tissues) was lower at higher temperatures (30°C day/25°C night) than at lower temperatures (20°C day/15°C night). However, wounding cotyledons with contaminated tools improved JSG626 persistence and the internalization rate (2.27%) in planta compared to spray inoculation (0.004%). The systemic dissemination of JSG626 to other tissues increased when contaminated plants were grown under low relative humidity (<40%); however, JSG626 was only detected in the root systems at later sampling times (between 21 and 98 days postinoculation [dpi]). Further, after tomato scions were grafted onto rootstocks using contaminated cutting tools, dissemination of JSG626 was preferentially basipetal and occasionally acropetal in the plants, with higher persistence rates and loads of JSG626 in root systems compared to foliar tissues. JSG626 was detected in the grafting point and root systems up to 242 dpi; however, none of the fruits harvested from contaminated plants between 90 and 137 dpi were positive for JSG626. This study demonstrates that environmental temperature and relative humidity could be good indicators for estimating the persistence of Salmonella enterica in tomato plants. Further, root systems may represent a risk for long-term persistence of Salmonella enterica in tomato plants.IMPORTANCE Tomatoes are one of the most widely produced vegetables around the world; however, fresh tomatoes have been connected to multiple wide-scale salmonellosis outbreaks over the past decades. Salmonella is commonly found in the environment and can persist in hostile conditions for several weeks before being internalized into plant tissues, where it is protected from conventional sanitation methods. In addition to biotic factors (host, inoculum size, and phytobiome), abiotic factors (environmental conditions) may affect the persistence of Salmonella in crop production. This study demonstrates that specific environmental conditions, the inoculation method, and the inoculum density affect the persistence and dissemination of JSG626 in tomato plant tissues. Our findings enhance the understanding of interactions between Salmonella enterica and fresh produce and may lead to the development of novel management practices on farms.