RESUMEN
In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 µm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and ß-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (ß-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (ß-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.
RESUMEN
Hemp (Cannabis sativa L.) is grown for medicinal and industrial uses. Symptomatic hemp (Mountain Mango) seedlings were received from a grower's greenhouse in Towner County (48.7486° N, 99.2761° W), North Dakota (ND), USA in July 2020. Seedlings had brown to blackish root tips, thread-like hypocotyl rot and seedling collapse, with about 8 to 10% disease incidence. Roots were surface disinfested in 1% sodium hypochlorite for 1 min, rinsed thrice with sterile distilled water, and blotted dry. About 1-cm sectioned root tips were plated on water agar (WA) and acidified potato dextrose agar (PDA, pH: 4.8) media and incubated under fluorescent light with a 12-h photoperiod at 25° C. After 7 days, single spores were isolated and sub-cultured on PDA and carnation leaf agar for morphological observations (Dhingra and Sinclair, 1995). Colonies had uniform appearances and produced white, thick and floccose mycelium. Conidiophores produced from lateral hyphae were simple to branched. Phialides were slender, smooth, hyaline and septate. Macro-conidia were 12.5 to 30.2 x 2.2 to 3.6 µm, septate (3-5), thick walled, hyaline and moderately curved shaped. Micro-conidia were oval to ellipsoid, smooth walled, no septa and measured 3.4 to 8.8 µm and 1.3 to 4.3 µm. Chlamydospores were round shaped, thick-walled, and produced singly or in pairs. Based on morphological characteristics, isolates were identified as Fusarium solani (Mart.) (Carbone and Kohn 1999; Leslie and Summerell 2006). For molecular identification, genomic DNA of three representative fungal isolates were extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR was done using both the primers (EF1/EF2) of the translation elongation factor (TEF-1α) and primers 5F2/7cR and 7cF/11aR of the RNA polymerase II (RPB 2) for Fusarium species (O'Donnell et al. 2009 and 2010). All isolates had identical PCR product sequences for the respective primer sets. The DNA sequences were deposited to NCBI GenBank with accession No. OK880264 (TEF-1α), OK880266 (RBP 5F2/7cR), and OK880265 (RBP 7cF/11aR). The NCBI Megablast search of the OK880264, OK880266, and OK880265 showed 100% similarity with respective homologue sequences from F. solani species complex (GU170628, KC808344, and EU329608). Similar results were obtained by BLASTN search in the FUSARIUM ID database (Geiser et al. 2004). For pathogenicity testing, 200 µl conidial suspension (1 x 106 conidia/ml) was pipetted, without wounding roots, onto the soil around the base of four plants individually potted in peat mix (Sunshine mix 1, Sun Gro Horticulture Ltd.; Alberta, Canada) and maintained in the greenhouse with 12 h photoperiod and temperature of 23 ± 2°C (Argus Control Systems Ltd.; British Columbia, Canada). Four plants inoculated with distilled water served as control. The test was conducted twice. At 10 days post inoculation (dpi), yellowing of leaves and damping off were observed in all inoculated plants. Re-isolated fungi from infected plant samples were morphologically identical to the isolate used for root inoculation. F. solani has been reported to cause damping off and root rot in several states in the U.S., Canada and Italy (Gauthier et al. 2019; Iriarte et al. 2021; Sorrentino et al. 2019). This is the first report of F. solani causing seedling damping off and root rot on hemp in ND. Hemp acreage has decreased in ND because of diseases (Buetow et al. 2020). Information on identification and management of diseases affecting hemp will be useful to producers.
RESUMEN
In May 2019, sugar beet (Beta vulgaris L.) seedlings with symptoms of wilting and root tip discoloration and necrosis were found in Moorhead (46.5507° N, 96.4208° W), Minnesota, USA. Roots of infected seedlings were surface sterilized with 10% bleach for 15 seconds, rinsed with sterile distilled water and cultured on water agar (MA Mooragar®, Inc, CA) for 3 days at 23 ± 2°C. Isolates were transferred to carnation leaf agar (CLA) and incubated at room temperature (22°C) under fluorescent light for 14 days. Abundant macroconidia were produced in sporodochia. Macroconidia were 5- to 7-septate, slightly curved at the apex, and ranged from 35 to 110 ×1.2 to 3.8 µm. No microconidia were produced. Chlamydospores with thick, roughened walls were observed in chains or in clumps, and were ellipsoidal or subglobose. Single spore was transferred from CLA to potato dextrose agar (HIMEDIA Laboratories, India) produced abundant white mycelium and was pale brown where the colony was in contact with the media. The morphological features of the isolates were consistent with Fusarium equiseti (Corda) Sacc. (Leslie and Summerell 2006, Li et al. 2015). Genomic DNAs (NORGEN BIOTEK CORP, Fungi DNA Isolation Kit #26200) of two representative isolates were used for polymerase chain reaction (PCR). The second largest subunit of RNA polymerase (RPB2) was amplified by PCR with primers 5f2/7cr (O'Donnell et al. 2010). The amplified PCR product was sequenced and deposited in GenBank (accession number MW048778). A BLAST search in Genbank and the Fusarium MLST database showed 100% sequence alignment to F. equiseti with accession MK077037.1 and NRRL 25795, respectively. Pathogenicity testing was done using three sugar beet seedlings (Hilleshög proprietary material, Hilleshög Seed, LLC, Halsey, OR 97348) at cotyledonary stage grown in a pot (4Ë×4Ë×6Ë) with six replicates. Seedlings were inoculated with F. equiseti conidial suspension (104 conidia ml-1 for 8 minutes) by the root dip method (Hanson, 2006). Mock inoculated plants were dipped in sterile water. Inoculated and control plants were placed in the greenhouse at 25 ± 2°C, and 75 to 85% relative humidity. One week later, inoculated seedlings showed root tip tissue discoloration similar to those observed in the field and non-inoculated seedlings were symptomless. This study was repeated. The fungus was re-isolated from diseased roots and confirmed to be F. equiseti based on morphological characters. Fusarium equiseti was reported on freshly harvested and stored beet in Europe but was not found to be pathogenic (Christ et al. 2011). Strausbaugh and Gillen (2009) reported the association of F. equiseti and root rot of sugar beet but did not report pathogenicity. This pathogen is reported in several crops including edible beans that is grown in rotation with sugar beet in several production areas (Jacobs et al. 2018). The most important Fusarium species reported to cause significant economic damage to sugar beet include F. oxysporum and F. secorum (Secor et al. 2014, Webb et. al. 2012). The presence of another pathogenic Fusarium species in sugar beet will require monitoring to determine how widespread it is and whether current commercial cultivars are resistant. To our knowledge, this is the first report of F. equiseti causing disease on sugar beet seedlings in Minnesota, USA.
