RESUMEN
Winterberries (Ilex verticillata and hybrids) are deciduous species of holly whose branches bearing colorful fruit are cut in late Fall to be used for seasonal decorations. The annual wholesale value of the woody cuts is $1.5 million nationally (NASS, 2019). In June 2021, approximately 80% of the 45 Ilex verticillata 'Maryland Beauty' potted plants, which were maintained in a container yard at The Ohio State University research farm in Columbus, OH, presented leaves with irregular necrotic lesions surrounded by a chlorotic halo. No other symptoms were present on the plants. Bacterial streaming was observed from the lesions using a compound microscope and isolations were performed after surface disinfesting small sections of leaf tissue from the border of the lesions by soaking in 10% bleach for 30 sec, rinsing twice in sterile water, macerating in sterile water, and streaking the suspension on nutrient broth yeast extract agar. Creamy white, circular, smooth, and convex colonies were recovered after incubation at 28°C for 48 h. Bacterial identification of one representative isolate was initially pursued from single colonies of a purified culture using five discriminative phenotypic tests (i.e., LOPAT: "L", levan production; "O", oxidase activity; "P", pectinolytic activity; "A", arginine dehydrolase production; "T", tobacco hypersensitive reaction), which resulted in the L+ O- P- A- T+ profile consistent with the description of Pseudomonas syringae (Lelliott et al. 1996). Molecular identification was performed based on rpoD marker amplification and sequencing using primers PsrpoD FNP1/PsrpoDnprpcr1 (Parkison et al. 2011). NCBI GenBank BLASTn comparison of the rpoD sequence (GenBank Acc. No. OP221440) shared 99.12% identity to P. syringae pv. passiflorae (AB163366.1). Whole genome sequence analysis was conducted to strengthen the classification of the isolate species. To this extent, DNA was sequenced with an iSeq 100 Illumina benchtop sequencer using Illumina DNA Prep kit and iSeq 100 i1 Reagent v2 (Illumina, Inc, REF: 20060060 and 20031371). Illumina Local Run Manager software was used for base calling, demultiplexing, and trimming of the raw reads. Unicycler v0.5.0 was used for de novo assembly of the genome (Wick et al. 2017). The assembled genome size was 5.9 Mb with 959 contigs and 10× coverage (NCBI GenBank Biosample No. SAMN30281368; Acc. No. JANQCB010000000). Average nucleotide identity (ANI) analysis was performed on the server MiGA online (Rodriguez-R et al. 2018). Subgroup identification was inconclusive (p>0.05), positioning this isolate between P. syringae pv. actinidiae (96.45% ANI) and pv. viburni (96.65% ANI) (Rodriguez-R & Konstantinidis, 2016). Both these pathovars cause leaf spots on woody plants such as kiwi and viburnum (Donati et al. 2020; Garibaldi et al. 2005). To confirm pathogenicity, three separate branches on each of two I. verticillata 'Maryland Beauty' potted plants were selected, and 5-7 individual young leaves (>2 weeks from emergence) on each branch were infiltrated with a bacterial suspension (108 CFU/mL) in sterile water (SW) using a needleless syringe by delivering 30-50 µL of suspension per infiltration point. One additional branch per plant was infiltrated with SW to serve as control. Plants were covered with a plastic bag for two days post-inoculation (DPI) and maintained in the laboratory at an average of 23°C. All inoculated leaves showed necrotic lesions two DPI while control leaves remained asymptomatic. To fulfill Koch's postulates, the bacterium was re-isolated from the symptomatic leaves six DPI and confirmed to be identical to the original isolate based on rpoD gene sequencing. To the best of our knowledge, this report signifies the first instance of P. syringae causing bacterial leaf spot on winterberry worldwide. Ornamental plant sales are based primarily on visual appeal; therefore, identification and monitoring of emerging pathogens is essential to ensure the health of the industry.
RESUMEN
Dracaena trifasciata (Prain) Mabb. is a popular houseplant in the United States. In September 2021, two diseased samples from two Ohio homeowners were received by the Ornamental Pathology Laboratory at The Ohio State University. Each sample included one or two detached leaves displaying circular gray water-soaked lesions scattered throughout the lamina and blighted areas with concentric rings bearing brown to black acervuli. Lesions covered between 25 and 50% of the leaf surface. Isolations were made by excising small portions of leaf tissue from the margin of the lesions, surface-disinfesting in 10% bleach for 45 s, rinsing in sterile water, and plating on potato dextrose agar (PDA). Plates were incubated at 23°C for one week. Two representative isolates, one per sample (FPH2021-5 and -6), were obtained by transferring hyphal tips to fresh PDA plates. Mycelia of both isolates were aerial, cottony, grayish-white, producing spores in a gelatinous orange matrix, and appeared gray to olivaceous-gray on the plate underside. Conidia produced by both isolates were cylindrical, single-celled, hyaline, measuring 12.02 to 18.11 (15.51) × 5.03 to 7.29 (6.14) µm (FPH2021-5; n=50) and 15.58 to 20.90 (18.39) × 5.63 to 8.27 (7.05) µm (FPH2021-6; n=50). Appressoria were globose to subglobose, single-celled, dark brown to sepia, measuring 6.62 to 13.98 (8.97) × 5.05 to 6.58 (6.58) µm (FPH2021-5; n=50), and 6.54 to 11.32 (8.63) × 4.54 to 8.94 (7.09) µm (FPH2021-6; n=50). Genomic DNA (gDNA) samples were extracted from both isolates and the internal transcribed spacer (ITS) region was amplified using primers ITS1F/ITS4 (Gardes and Bruns, 1993; White et al. 1990). GenBank BLAST sequence analysis resulted in 99.83% (FPH2021-5; GenBank Acc. No. OP410918.1) and 100% (FPH2021-6; OP410917.1) identity with 100% query coverage to the type strain of Colletotrichum sansevieriae Miho Nakam. & Ohzono MAFF239721 or Sa-1-2 (NR_152313.1; Nakamura et al. 2006). Whole genome sequencing was conducted for FPH2021-6 and the assembly was deposited in GenBank (JAOQIF000000000.1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin (ß-tub) regions were either extracted from the genome of FPH2021-6 (OP414603.1 and OP414601.1, respectively) or amplified from FPH2021-5 gDNA using primers GDF/GDR (OP414604.1) and Bt-2b/T1 (OP414602.1), respectively (Templeton et al. 1992; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997). A multilocus partitioned analysis (Chernomor et al. 2016) based on concatenated sequences of ITS, GAPDH, and ß-tub using ModelFinder (Kalyaanamoorthy et al. 2017) was performed to build a maximum likelihood tree (IQ-TREE v2.0.3; Nguyen et al. 2015), suggesting that these two isolates are phylogenetically closer to the type strain from Japan than to a previously reported isolate 1047 from Florida (Palmateer et al. 2012). To fulfill Koch's postulates, two parallel leaf sections from one 10-inch D. trifasciata 'Laurentii' plant maintained in a 1.3-liter container were selected. Three wounds were made in each section using a sterile syringe needle. A 10-µl drop of either a 1×106 conidia/ml suspension of isolate FPH2021-6 or sterile water was placed on each wound. The plant was covered with a plastic bag for two days post-inoculation (DPI) and maintained in a greenhouse at 25°C with a 12- h photoperiod. The experiment was conducted twice. Grayish water-soaked lesions, acervuli, and leaf blight were observed on the inoculated sections 3, 10, and 14 DPI, respectively, while no symptoms appeared on the sections treated with sterile water. C. sansevieriae was re-isolated from the lesions and confirmed to be identical to the original isolate based on ITS sequencing and morphological examinations. To the best of our knowledge, this is the first report of C. sansevieriae on D. trifasciata in Ohio and the first genome draft of an isolate from the United States. Availability of whole-genome sequence data is paramount for resolving species identification in this highly diverse fungal genus, and a powerful tool to conduct comparative genomic analyses in the future.
RESUMEN
Ohio is one of the top five floriculture producers in the United States, grossing over $200 million annually (NASS 2019). Within the international floriculture trade, gladiolus cut flowers represent the fifth highest grossing crop (Ahmed et al. 2002). In September 2021, the Ornamental Crops Pathology Lab at the Ohio State University received a gladiolus (Gladiolus spp.) sample of an unknown cultivar from a home garden in Franklin Co., OH where several plants had failed to grow from planted corms or were stunted and displaying symptoms of disease. Bleached, water-soaked spots with necrotic margins along the flowering stems, stunted flowers with partial necrosis, and necrotic bracts were observed on the submitted sample. Bacterial isolations were performed by surface disinfesting small sections of bract tissue from the border of a lesion by soaking in 10% bleach for 30 sec and rinsing twice in sterile water, macerating the tissue in sterile water, and streaking the suspension on nutrient agar (NA) plates. Plates were incubated at 28°C for 48 hours and the resulting colonies were purified by re-streaking a single colony on NA twice. Bacterial colony morphology on NA presented as cream-colored and shiny with an irregular form and undulate margin. Five in vitro tests were performed using one representative isolate to identify the bacterium to the genus level: (1) confirmed levan production, (2) confirmed pectinolytic activity, (3) confirmed ability to grow at 40°C, (4) inability to grow under anaerobic conditions, and (5) a negative oxidase test (Schaad et al. 2000). All test results identified the genus as Burkholderia. To identify to species level, gyrase subunit B (gyrB) and RNA polymerase subunit D (rpoD) markers were PCR amplified and sequenced using primers UP1-E/AprU, and 70F2/70R2, respectively (Maeda et al. 2006). NCBI GenBank BLASTn comparison showed that the gyrB sequence shared 99.33% identity to the type strain of B. gladioli (CP009323.1), while the rpoD sequence showed 99.53% identity (CP009322.1). Sequences were deposited in GenBank under accession numbers ON597852 (gyrB) and ON597853 (rpoD). To confirm pathogenicity, each of two Gladiolus communis 'Mini Elvira' potted plants were inoculated with two bacterial and two control treatments (3 leaves/treatment/plant) as follows: leaf infiltration with 1 mL of either (i) a distilled water-Tween 20 (0.03% v/v) bacterial suspension (106 cfu/mL) or (ii) a sterile water-Tween 20 suspension using a needle-less syringe; foliar spray with either (iii) the bacterial suspension or (iv) water-Tween suspension until run-off. Following inoculation, plants were covered for 24 hours with a plastic bag to increase humidity and favor infection and maintained in a greenhouse at an average temperature of 23°C. After 3 days, water-soaked, necrotic lesions were observed on the inoculated plants regardless of inoculation method, while control leaves remained asymptomatic. To fulfill Koch's postulates, bacteria were re-isolated from the lesions 7 days post-inoculation and confirmed to be identical to the original isolate based on rpoD gene sequencing. Bacterial scab of gladiolus was reported in Ohio in the late 1900s as caused by Pseudomonas gladioli (syn. P. marginata; Ellett, 1989). To the best of our knowledge, this report represents the first molecular identification of the causal agent as Burkholderia gladioli. In Ohio, the pathogen has also been observed causing slippery skin on onion but not officially reported in the peer-reviewed literature. Additionally, B. gladioli has been reported in other parts of the United States on orchid, corn, and rice (Keith et al. 2005; Lu et al. 2007; Nandakumar et al. 2009). Given the significant role of gladiolus within Ohio's floricultural trade, as well as the ability of this pathogen to infect other regional crops, monitoring of bacterial scab is important for floriculture and field crop growers alike.
RESUMEN
Japanese apple rust, caused by the heteroecious and demicyclic rust fungus Gymnosporangium yamadae Miyabe ex G. Yamada, can affect juniper (Juniperus spp.), where the telial stage of this disease occurs, and apple or crabapple (Malus spp.), where the aecial stage occurs (Yun, 2010). Leaf samples displaying symptoms and signs of rust disease were collected in August 2020 from 14 different crabapple cultivars ('Amerspirzam' [American Spirit®], 'Amsalzam' [American Salute™], 'Excazam' [Excalibur™], 'Guinzam' [Guinevere®], 'Hargozam' [Harvest Gold®], 'Mary Potter', 'Orange Crush', 'Prairie Maid', 'Professor Sprenger', 'Pumpkin Pie', 'Rawhide', 'Select A' [Firebird®], 'Shotizam' [Show Time™], 'Sinai Fire') in the crabapple research plot of Secrest Arboretum (Crablandia) in Wooster, OH. Samples displayed adaxial leaf lesions with brown necrotic centers surrounded by a red-yellow coloration, corresponding on the abaxial side to lesions containing brown-orange aecia, producing aeciospores, surrounded by a dark red-orange coloration (Supplemental Figure 1). One to multiple lesions were present per symptomatic leaf. DNA was extracted from symptomatic leaf tissue containing fungal material on all 14 cultivars using the DNeasy Plant Mini Kit (QIagen) and the D1/D2 region of the 28S rDNA was amplified using primers NL1 and NL4 (O'Donnell 1993) according to Dagar et al. (2011). GenBank BLAST sequence analysis of all 14 sequences resulted in 99.83-100% sequence identity to G. yamadae with with 99% query coverage (MN605735). Sequences from all samples were deposited in GenBank under Accession Nos. MW131119.2-131125.2 and MW131127.2-131132.2. Morphological features were characterized for the three representative cultivars 'Amerspirzam' (American Spirit®), 'Orange Crush' and 'Pumpkin Pie' (Supplemental Figure 2). Aecia were hypophyllous, roestelioid, with cornute, yellow-brown, peridia with lacerate sides. Peridial cells appeared yellow and were long-linear rhomboid, verrucose with long papillae, smooth outer walls and echinulate inner walls, measuring 45 - 78 × 16 - 27 µm (average 65 × 21 µm), 51 - 82 × 16 - 30 µm (average 66 × 23 µm), and 47 - 93 × 14 - 31 µm (average 64 × 24 µm), respectively (n=50 per cultivar). Aeciospores were globose, 20 - 26 × 18 - 24 µm (average 23 µm × 20 µm), 21 - 28 µm × 19 - 24 µm (average 24 µm × 21 µm), and 21 - 27 µm × 18 - 23 µm (average 23 µm × 21 µm), respectively, with a slightly coronate surface and dark yellow walls 1.6 - 2.7 µm (average 2 µm), 1.4 - 2.4 µm (average 2 µm), and 1.3 - 2.5 µm (average 1.8 µm) thick, respectively (n=50 per cultivar). The telia, known to occur on Juniperus spp., were not observed. Specimens from these three cultivars were deposited into the U.S. National Fungus Collections (BPI 923889, 923888, 923887). Japanese apple rust has been officially reported in parts of Eastern Asia and the Eastern United States and is also known to be present in parts of Far East Russia and Ontario, Canada (Yun et al., 2009; CAB International, 2008). This report constitutes the first confirmed instance of G. yamadae causing Japanese apple rust in Ohio. Because infected trees tend to be highly symptomatic, this disease poses a significant threat to the nursery and landscape industries as it can decrease the market value of ornamental varieties and affect yield and crop quality in varieties used for fruit production.
