First report of <i>Colletotrichum siamense</i> causing anthracnose on blueberry in Brazil

Blueberry (Vaccinium corymbosum) is a temperate-climate fruit valued for its flavor and high content of bioactive compounds. Anthracnose, caused by Colletotrichum spp., is considered one of the most important diseases limiting blueberry yields (Liu et al. 2020). In Brazil, blueberry cultivation has expanded from the southern to the southeastern region. To date, two species, C. karsti and C. chrysophilum, have previously been associated with blueberry anthracnose in the country (Rios et al. 2015, Soares et al. 2022). In November 2024, an anthracnose epidemic was observed in an organic commercial blueberry orchard of the cultivar ‘Emerald’ (Piracicaba, SP, Brazil; 22°45′02.5″S, 47°31′59″W), where approximately 80% of the 2000 plants evaluated in a 0.6 ha production area were symptomatic, showing twig blight, anthracnose fruit rot, and predominantly black leaf spots. Acervuli without setae, bearing conidia masses, were collected and microscopically observed in leaf sections slides. These structures were carefully collected with a needle and transferred to Potato Dextrose Agar (PDA) plates. A single-spore Colletotrichum sp. isolate (LFN0500) was obtained, on PDA, and 15-day-old colonies colors were grayish white, with the reverse pale orange. Conidia (n = 100) were cylindrical with rounded ends, measuring 11.13 – 13.87 × 3.63 – 4.47 µm (mean 12.25 × 4.04 µm). DNA was extracted from aerial mycelium using the Wizard® Genomic DNA purification kit (Promega), following the manufacturer’s instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin 2 (TUB2), rDNA transcribed spacer region (ITS), and actin (ACT) regions were amplified using the primer pairs GDF/GDR (Guerber et al. 2003), Bt2a/Bt2b (Glass and Donaldson 1995), ITS-1F/ITS-4 (Gardes and Bruns 1993), ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. Amplicon sequences were deposited in GenBank (accession numbers PX461595 (ITS), PX531255 (ACT), PX531256 (TUB2), PX531254 (GAPDH)) and showed highest similarity (100%) with Colletotrichum siamense, a member of the Colletotrichum gloeosporioides species complex. Phylogenetic analysis placed isolate LFN0500 with C. siamense reference strain (ICMP 18578) (posterior probability = 0.96). To confirm pathogenicity, 6-months-old blueberry potted plants (cv. Emerald, n = 4) and detached fruits (average diameter, 15 mm, n = 6) were spray inoculated with a conidial suspension (1 × 10⁶ conidia/mL), incubated at 25 ± 5 °C in the dark for 48 hours, and then transferred to a growth chamber. The experiment was repeated twice. Blueberry potted plants exhibited typical black leaf spots 13 days after inoculation, whereas the negative controls (n = 4), sprayed with distilled water, remained asymptomatic. Anthracnose rot appeared on detached fruits 3 days after inoculation, with no symptoms observed on negative controls (n = 6). The pathogen was reisolated only from the edge of symptomatic tissues originated in both fruits and leaves, but not from asymptomatic negative controls, and its identity was confirmed through a new sequencing of the same gene regions previously analyzed. To our knowledge, C. siamense has previously been associated with blueberry in the United States and China (Liu et al. 2020; Talhinhas and Baroncelli 2023). The identification of C. siamense in this study expands the known diversity of Colletotrichum spp. affecting blueberry in Brazil and provides useful information for disease management in the country.