First report of causing Phomopsis stem canker on sunflower ( L.) in Minnesota.

Cultivated sunflower (Helianthus annuus L.) is a globally important oilseed crop that is grown primarily in the Northern Great Plains region of the United States. In September 2018, sunflower stems exhibiting brown stem lesions centered on the leaf axils and accompanied by pith degradation, consistent with symptoms of Phomopsis stem canker (PSC) disease, were sampled from a commercial field of approximately 520 hectares in Polk County, MN (47°50'24" N, 96.34'13" W). Incidence of PSC in this field was approximately 44%. Ten diseased stem samples were collected from plants spaced at least ten meters apart. Pieces of diseased stems 15 mm in length and 10 mm in width were excised, surface sterilized in 10% sodium hypochlorite and plated onto potato dextrose agar (PDA) containing 50 µg/ml streptomycin. Plates were incubated at 22°C for 3-5 days and hyphal tips of emerging mycelia were transferred twice to new PDA plates. Genomic DNA was extracted from each isolate and polymerase chain reaction (PCR) was carried out using primers specific to either Diaporthe helianthi or D. gulyae, the most common species causing PSC on sunflower (Elverson et al., 2020). Eight isolates were identified as D. helianthi by PCR, while the remaining two isolates, designated H48 and H49, exhibited morphologies on PDA plates distinct from those of D. helianthi or D. gulyae. The ITS region, along with portions of the EF-1α and β-tubulin (TUB) genes were amplified and sequenced, and the sequences were deposited in Genbank (H48: ITS-OP429627.1, EF-1α-OP429589.1, TUB-OP429587.1; H49: ITS-OP429628.1, EF-1α-OP429590.1, TUB-OP429588.1). BLAST analyses using the H48 and H49 ITS sequences revealed 100% identity with D. caulivora isolates including the type specimen CBS 127268 (Genbank accessions AF000567.2 and type specimen MH864501.1). Additionally, 100% identity of EF-1α and TUB sequences from H48 and H49 with those of type specimen CBS 127268 (Genbank accessions KC343771.1 and KC344013.1) were also observed. A multi-locus phylogenetic tree was constructed aligning the ITS, EF-1α, and TUB sequences of H48 and H49 with those of twenty Diaporthe species. Isolates H48 and H49 formed a clade with three other D. caulivora isolates, including the type specimen. Pathogenicity of isolate H49 on sunflower was confirmed by inoculating susceptible sunflower inbred line HA 410 using a stem wound method (Mathew et al., 2018; Thompson et al., 2011; Underwood & Misar, 2024). Plants were inoculated by creating a wound approximately 5 mm deep and 8 mm long using a scalpel and affixing a PDA plug carrying mycelium of isolate H49, D. helianthi isolate H45 known to be pathogenic, or mock inoculum of PDA with no mycelium over the wound using laboratory film. Three independent experimental runs were conducted in which two 6-week-old plants grown in 1-gallon pots under greenhouse conditions at 22 ± 3°C were inoculated with each treatment, resulting in inoculation of six total HA 410 plants per treatment. Isolate H49 formed lesions with mean length of 61.27 mm on HA 410 at 14 days-post-inoculation compared to mean lesion length of 173.05 mm for D. helianthi isolate H45 and no disease symptoms were observed upon mock inoculation. D. caulivora was re-isolated from inoculated stems and confirmed by observing morphological features on PDA plates as well as sequencing the ITS region, fulfilling Koch's postulates. D. caulivora is typically associated with stem canker of soybean and has been previously reported to cause PSC on sunflower in Argentina (Zambelli et al., 2021). Additionally, this species was identified as an endophyte on asymptomatic sunflowers in the U.S., though pathogenicity was not confirmed in this study (Dangel, 2022). To our knowledge, this is the first report of D. caulivora causing Phomopsis stem canker on sunflower in the U.S. It will likely be prudent to monitor this species as a potential causal agent of PSC on sunflower and potentially to determine if sunflower lines resistant to other Diaporthe species are also resistant to D. caulivora should a need arise to deploy resistance to this species in commercial sunflower hybrids.