First report of a Herbaspirillum sp. causing leaf spots on Boston Fern (Nephrolepis exaltata) in Florida.

Boston fern (Nephrolepis exaltata) samples were submitted by a nursery operation in Florida separately to the University of Florida Plant Diagnostic Center (UFPDC, Gainesville, FL) and to the North Carolina State University Plant and Pest Diagnostic Lab (NCSU PPDL, Raleigh, NC) in October 2021. Symptoms included tan spots on pinnules, some of which progressed into pinnule blight (Fig. S1). Bacterial streaming was noted from samples in both labs. Leaf spot margins were excised, macerated in sterile tap water, then streaked onto nutrient agar (NA) plates and incubated for 48 h at 27°C. Individual representative colonies that were opaque, creamy white, mucoid, and round with smooth margins were transferred and streaked onto additional NA plates. One strain from each lab (G21-1742, UFPDC and NC40101, NCSU PPDL) was selected for subsequent characterization. A suspension of each strain was adjusted to 108 CFU/mL and infiltrated into tobacco and tomato leaves, and confluent necrosis was observed 72 h after infiltration. The isolates were Gram-negative, oxidase-positive, HR-positive on tomato and tobacco, aerobic, not pectolytic, and nonfluorescent on King's Medium B. DNA was extracted from G21-1742 using Qiagen Stool kit (Qiagen cat#51604) and the 16S rRNA gene from strain G21-1742 was amplified using 16SrRNA universal primers UP1 (5'-TACGTGCCAGCAGCCGCGGTAATA-3') and UP2 (5'-AGTAAGGAGGGTATCCAACCGCA-3') (Kuppusamy et al. 2014). The amplicon was sequenced and submitted to NCBI (Genbank Accession No. OR004801). BLASTn analysis of 16S rRNA of G21-1742 resulted in 99.7% sequence identity to the type strain of Herbaspirillum huttiense subsp. huttiense ATCC 14670T (Genbank Accession NR_024698). The 16S rRNA sequence of NC40101 was identical to that of G21-1742. To determine if the G21-1742 strain was pathogenic, Boston fern plants were inoculated by suspending bacterial cells in tap water from a 24h culture grown on NA, adjusting the suspension to 108 CFU/mL and spraying the suspension on one three-week old frond from each of three healthy Boston fern plants. A second frond from each plant was sprayed with sterile tap water. Each treated frond was individually sealed in a clear plastic bag for 24h at approximately 25°C. Inoculated plants remained on the greenhouse bench after the plastic bags were removed. The inoculation experiment was repeated once. After 4 days, tan spots were observed on pinnules of inoculated plants that were identical to the original submitted samples, while no symptoms developed on water-inoculated plants. Bacterial strains were reisolated from symptomatic plants and were morphologically identical to G21-1742. The 16S rRNA sequence of the reisolated strain was identical to G21-1742. Additionally, we conducted MLSA analysis using 12 housekeeping genes (See Table S2 for housekeeping genes and accession numbers) from the fern strains and the corresponding housekeeping genes for the type strains of 13 Herbaspirillum species, which placed the fern strains most closely with H. huttiense (see Fig. S2). This is the first known report of a Herbaspirillum sp. on Boston fern, an important ornamental crop, that renders the plants aesthetically unsaleable. Previously, a Herbaspirillum sp. was reported in Florida to cause a leaf spot and blight on greenhouse grown tomato seedlings (Obradovic et al. 2007).

First report of Bipolaris sorokiniana leaf spot disease on watermelon (Citrullus lanatus) in Florida.

Watermelon is an important crop in Florida, representing $88.2 million in cash receipts in 2015 (USDA/NASS 2017). In April and May 2021, the UF/IFAS Plant Diagnostic Center in Gainesville, Florida received eight diseased watermelon leaf samples from Alachua, Gilcrest, Levy, and Suwannee counties in Florida. Lesions were round to oblong, light gray to tan with reddish brown margins and white to light gray center, and some were coalescing resulting in about 15% disease severity. Symptomatic leaf tissue (0.5 cm2) was surface sterilized in 0.6% sodium hypochlorite for one minute, rinsed with sterile tap water, plated onto water agar media plates, and incubated at 27°C under 12-h light/dark cycle for 7 days. Characteristic Bipolaris conidia with gray to black brownish cottony mycelial growth were consistently found growing from plated lesions. The pathogen was isolated from two of the eight samples using a 0.5 mm diameter sterile metal needle to transfer a single conidium onto DifcoTM Potato Dextrose Agar (PDA) plates. Three isolates were designated G21-562 from Levy and G21-599a and G21-599b from Alachua County. All three isolates produced curved or straight, cylindrical, obclavate, distoseptate brownish gray conidia with 3 to 8 septa, mostly tapering towards ends with dark brownish to black hilum, that ranged from averaged 62um x 25um (n=30, SD=8 for length and 3 for width). Conidiophores were brownish, septate, smooth, and straight, single or in small groups, simple or branched, and swollen at the upper tip. Internal transcribed spacer region (ITS) and partial glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene sequences were amplified using primers ITS1/ITS4 and GPD-1/GPD-2 (Berbee et al. 1999). Reference sequences (Adhikari et al. 2020 and Manamgoda et al. 2014) were aligned using MUSCLE and trimmed to consistent length. Using concatenated sequence alignments of both loci, a maximum likelihood phylogenetic tree was constructed based on K2+G substitution model selected by BIC using Mega X (Kumar et al. 2018) with 1,000 bootstrap. The ITS and GPDG sequences of G21_599b, G21_599a and G21_562 (GenBank accessions OK614094 to 96, OP297398 to 400) showed 100% identity across 888 nucleotides across both loci to B. sorokiniana isolates CBS_110.14 and CBS_ 120.24 and were distinct from other reference isolates. To fulfill Koch's postulates, all three isolates were grown on PDA at 27°C and 12-h light/dark cycle. After a week, conidia were harvested in sterile water, and the conidial suspensions were adjusted to 105 conidia/ml using a hemocytometer. Each conidial suspension and Tween 20 water control was sprayed onto three seedlings of 'Sugar Baby' watermelon until runoff, and inoculated seedlings were sealed in a plastic bag for 24 hrs. The experiment was done in a greenhouse (20- 25°C) and repeated once. After a week of incubation, the same leaf lesion symptoms described above were observed on seedlings inoculated with conidia, whereas seedlings sprayed with the control were asymptomatic. Isolations from symptomatic tissue produced gray to black mycelia with conidia that were the same as described from field samples. To our knowledge, this is the first report of leaf spot on watermelon caused by B. sorokiniana. B. sorokiniana is a common pathogen of grasses and agronomic crops (Farr and Rossman 2020). The extent to which this emerging disease of Florida watermelon may negatively impact production is unknown and should be the subject of future observation and research.

Bacterial Gall of Loropetalum chinense caused by Pseudomonas amygdali pv. loropetali pv. nov.

In 2012, stem gall samples on Loropetalum chinense were sent to Florida diagnostic labs from Alabama and Florida nurseries. A fluorescent pseudomonad was consistently isolated from the galls. The organism was originally identified in Alabama based on 16S rRNA sequencing as Pseudomonas savastanoi, which causes a production-limiting disease of olive. The loropetalum strains and reference strains were compared using LOPAT, Biolog, fatty acid analysis, multilocus sequence analysis (MLSA), and pathogenicity tests. The LOPAT tests placed the loropetalum strains within Pseudomonas syringae. Biolog and fatty acid analysis placed the strains in various pathovars of P. syringae and P. savastanoi, respectively. MLSA of a set of housekeeping genes separated the loropetalum strains from the olive knot-inducing strains. Our work indicates there is a need to use more tests than 16S rRNA to accurately diagnose new bacterial diseases. In pathogenicity tests, the loropetalum strains produced galls only on loropetalum, but not on olive, mandevilla, or almond, indicating this strain is not a threat to the olive industry. Based on the pathogenicity assays and molecular tests, loropetalum strains represent a distinct and new pathovar, P. amygdali pv. loropetali pv. nov., for which the strain PDC13-208 (= DSMZ 105780PT) has been designated as the pathotype strain.

Plant disease diagnostic capabilities and networks.

Emerging, re-emerging and endemic plant pathogens continue to challege our ability to safeguard plant health worldwide. Further, globalization, climate change, increased human mobility, and pathogen and vector evolution have combined to increase the spread of invasive plant pathogens. Early and accurate diagnoses and pathogen surveillance on local, regional, and global scales are necessary to predict outbreaks and allow time for development and application of mitigation strategies. Plant disease diagnostic networks have developed worldwide to address the problems of efficient and effective disease diagnosis and pathogen detection, engendering cooperation of institutions and experts within countries and across national borders. Networking maximizes impact in the face of shrinking government investments in agriculture and diminishing human resource capacity in diagnostics and applied pathology. New technologies promise to improve the speed and accuracy of disease diagnostics and pathogen detection. Widespread adoption of standard operating procedures and diagnostic laboratory accreditation serve to build trust and confidence among institutions. Case studies of national, regional, and international diagnostic networks are presented.