First report of anthracnose caused by Colletotrichum magnum on lima bean in South Carolina

Anthracnose of lima beans (Phaseolus lunatus) in South Carolina (SC) has been exclusively attributed to Colletotrichum truncatum, which causes reddish brown lesions on aerial plant tissues. Here we report C. magnum as an additional causal agent of this disease. In October 2023, anthracnose symptoms were observed on the pods and leaves of lima beans in a ~1000 sq. ft plot at a market farm in Charleston County, SC, with all foliage and ~10% of pods affected. Pod lesion margins were excised and surface disinfested by briefly agitating in 0.1% Tween, soaking in 70% ethanol for 30 s and 1% sodium hypochlorite for 1 min, and blotting dry. The lesions were incubated on 1:10 potato dextrose agar (PDA) amended with tetracycline (10 µg/mL) for 4 days at room temperature. Putative Colletotrichum spp. were plated onto full-strength PDA and incubated for 5 days at room temperature. Three isolates were obtained via single hyphal tipping. Isolate PL7 exhibited growth and sporulation on PDA at 20°C under 1550 lm LED lights. Colonies exhibited cottony, filamentous growth that was slightly raised from the colony surface. Mycelia were white in appearance and septate. Acervuli were dark brown to black and produced salmon mucilage when sporulating. Conidia were cylindrical with an average length and width of 19.21 µm and 5.52 µm, respectively (n = 10). Species-level identification was performed by amplifying the internal transcribed spacer (ITS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene regions using primer pairs ITS1F/ITS4 (Glass and Donaldson 1995; White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), and GDF/GDR (Templeton et al. 1992), respectively. These loci were selected based on their established utility for species-level identification within the Colletotrichum genus (Talhinhas and Baroncelli 2021). The sequences shared 99.83% (ITS), 99.62% (ACT), and 100% (GAPDH) similarities with Colletotrichum magnum (CBS 519.97) when analyzed with BLASTn. The resulting sequences were deposited into GenBank (ITS, PZ433468; ACT, PZ445365; GAPDH, PZ445309) and aligned to 214 Colletotrichum species reference sequences (Talhinhas and Baroncelli 2021) using MAFFT. A maximum-likelihood phylogenetic tree was constructed in IQtree (Minh et al. 2020) and visualized with iTOL (Letunic and Bork 2024). PL7 clustered with Colletotrichum magnum (CBS 519.97) within the magnum species complex. Koch’s postulates were repeated twice by spray inoculating three 2-week-old lima bean plants (cv. Jackson Wonder) with a PL7 conidial suspension of 1.0 x 10 ⁶ conidia/mL; three control plants received sterile deionized (DI) water. High humidity was maintained by daily misting with DI water. Characteristic anthracnose symptoms appeared on the abaxial side of leaves after 7 days. Control plants did not develop symptoms in any experiment. C. magnum was reisolated from the leaves of each inoculated plant. The reisolates had identical morphology and ITS, GAPDH, and ACT sequences to the initial isolate, fulfilling Koch’s postulates. Disease symptoms were notably milder than those of C. truncatum. C. magnum is known to cause disease in Cucurbitaceae (Damm et al. 2019). To our knowledge, this is the first report of Colletotrichum magnum causing anthracnose of lima bean in SC. The presence of an additional Colletotrichum species causing lima bean anthracnose in SC underscores the importance of accurate species-level identification for effective disease management.