Occurrence of <i>Colletotrichum chlorophyti</i> and <i>C. sojae</i> Causing Soybean Leaf Anthracnose in Iowa

In August 2023, soybean (<i>Glycine max</i> L.) leaf anthracnose (SLA) symptoms, predominantly at leaf margins, were observed in variety trials and commercial fields in Iowa (<1% incidence field on Newton and Story counties). Acervuli covered with conidial masses were consistently observed, primarily at leaf edges. Symptomatic leaves were collected, surfaced-disinfested (70% ethanol for 30 s, followed by 0.75% NaClO for 1 min, and rinsed twice in sterile water), and placed in a humid chamber at 20°C with a diurnal cycle until sporulation. Conidial masses developed on foliar lesions were plated onto potato dextrose agar (PDA). Six isolates were obtained and subcultured as monoconidial cultures. Initially, fungal growth appeared pinkish but darkened after three days. Isolates were categorized into two groups based on phenotypic features. Group 1: (C03S23, C04S23, C05S23, C07S23): colonies brown-pinkish, conidia cylindrical, aseptate, and hyaline, of 12.3-20.6 × 4.9-6.2 μm (n = 70). Group 2: (C09S23, C10S23): colonies blackish, conidia falcate, aseptate, and hyaline, of 18.01-22.09 × 3.4-5.2 μm (n = 70). The phenotypic features were similar to those of <i>Colletotrichum</i> (Sutton 1980). For species identification, DNA was extracted, and the ACT, GAPDH, ITS, and TUB2 genes were amplified and sequenced using the PCR primers described by Weir et al. (2012). Sequences were deposited in GenBank (accession nos. PQ592521-PQ592531, PQ615274-PQ615279) and concatenated, and analyzed with representative <i>Colletotrichum</i> sequences from previous studies for a multilocus phylogenetic analysis. The morphological and phylogenetic analyses indicated that two species were found: <i>Colletotrichum sojae</i> and <i>C. chlorophyti</i>. Pathogenicity was verified on soybean cv. Williams 82 plants at the V1-V2 growth stage. Isolates were grown on 1/3-strength PDA for 14 days at 25°C under a diurnal cycle. Seeds were disinfected with 70% ethanol for 1 min, followed by 2.25% NaClO for 6 min, and rinsed twice in sterile water, then planted in trays with sterile perlite at 25°C under a diurnal cycle. Plants were inoculated with approx. 100 ml conidial suspension (1×10⁴ conidia/ml) with 0.25% surfactant Tween 20 until runoff on unwounded leaves from each isolate. Controls were inoculated using the same approach with just sterile water. Plants were maintained in a humid chamber in darkness at 25°C for 24 h, and settings were changed to 25°C under a diurnal cycle (25% light intensity). Trifoliate leaves developed symptoms three days after inoculation, whereas control remained symptomless. The symptoms produced by each fungal species were identical. The fungi were consistently re-isolated and identified culturally and morphologically on PDA, fulfilling Koch´s postulates. Each <i>Colletotrichum</i> species was phenotypically as described by Takaki et al. (2024), Hassan et al. (2022), and confirmed molecularly with our phylogenetic tree. <i>Colletotrichum sojae</i> has been reported causing SLA in Brazil and the USA (Damm et al. 2019; Rogério et al. 2020). Similarly, <i>C. chlorophyti</i> has been identified causing SLA and infecting seed in Brazil (Takaki et al. 2024) and Arkansas, USA (Yang et al. 2013). This study presents the first report of <i>C. sojae</i> and <i>C. chlorophyti</i> causing SLA in Iowa. The potential risk of finding this complex species as seedborne that affect seed emergence and germination, seed quality and foliage is still unknown in Iowa.