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.
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 β-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 (β-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 (β-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.
Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, Cercospora beticola, has been reported to be resistant to Quinone outside inhibitors (QoIs) and to have reduced sensitive to Demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. Isolates of C. beticola resistant to QoIs, DMIs, and both QoIs and DMIs were collected from Foxhome fields, Minnesota in 2017. Fitness of these resistant isolates was compared to that of QoI- and DMI-sensitive isolates in laboratory and greenhouse studies. In the lab, mycelial growth, spore production, and spore germination were measured. The results showed that resistant isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. In the greenhouse, six leaf-stage sugar beets were inoculated with a spore suspension made from each resistant group and then incubated in separate humidity chambers. CLS disease severity was evaluated visually at 7, 14, and 21 days after inoculation (DAI) and the areas under disease progress curve (AUDPC) were calculated. Resistant isolates had significantly smaller AUDPC but still caused high disease severity as the sensitive ones at 21 DAI. Although QoI and/or DMI resistant isolates had a relatively slower disease development, they still caused high disease severity and need to be factored in disease management practices.
Cercospora leaf spot (CLS) is the most destructive foliar disease in sugar beet (Beta vulgaris). It is caused by Cercospora beticola Sacc., a fungal pathogen that produces toxins and enzymes which affect membrane permeability and cause cell death during infection. In spite of its importance, little is known about the initial stages of leaf infection by C. beticola. Therefore, we investigated the progression of C. beticola on leaf tissues of a susceptible and resistant sugar beet varieties at 12 h intervals during the first five days after inoculation using confocal microscopy. Inoculated leaf samples were collected, and stored in DAB (3,3'-Diaminobenzidine) solution until processed. Samples were stained with Alexa Fluor 488 dye to visualize fungal structures. Fungal biomass accumulation, reactive oxygen species (ROS) production, and the area under disease progress curve were evaluated and compared. ROS production was not detected on any variety before 36 hours post inoculation (hpi). C. beticola biomass accumulation, percentage leaf cell death and disease severity were all significantly greater in the susceptible variety compared to the resistant variety (P<0.05). Conidia penetrated directly through stomata between 48- to 60-hpi and produced appressoria on stomatal guard cells at 60- to 72-hpi in susceptible and resistant varieties, respectively. Penetration of hyphae inside the parenchymatous tissues varied in accordance with time post-inoculation and varietal genotypes. Overall, this study provides a detailed account to date of events leading to CLS disease development in two contrasting varieties.
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