First report of Colletotrichum fioriniae causing anthracnose fruit rot on Vaccinium corymbosum in Chile.

Abstract

Vaccinium corymbosum L. is the most cultivated blueberry species in Chile. Chilean fruits typically take up to 50 days to reach oversea markets; therefore, controlling post-harvest pathogens is of outmost importance to maintain international food safety and quality standards. In February 2019, the Microbial Genetic Resources Bank at INIA received fruits of V. corymbosum cv. 'Brigitta Blue' from Mariquina (-39.567869, -72.992461), located in the southern Chilean blueberry production zone, for post-harvest disease diagnosis. Asymptomatic fruits were incubated in moist-chambers at 25 °C with light/darkness cycles of 12 h. After 5 d, some fruits showed sunken areas and small surface wounds that exudated orange masses of conidia; under the epidermis, gray acervuli were also detected. After 15d, these fruits became dehydrated, mummified, and covered by mycelia, all characteristic symptoms of anthracnose (Wharton and Schilder 2008). In Chile, Colletotrichum gloeosporioides has, thus far, been the only causal agent of anthracnose reported in blueberry (Lara et al. 2003). Conidia exudated from the diseased fruit were inoculated on potato-dextrose agar (PDA) and incubated at 25 °C for 7 d. The resulting colony was predominantly cottony with gray aerial mycelium, displaying masses of pale orange conidia; on the reverse side, the colony was a pink-reddish color. Under a microscope, conidia were hyaline, fusiform to elliptic in shape, and displaying guttulate of 12.2±1.2 × 4.17±0.3 μm (n=30), characteristics coinciding with those described for Colletotrichum fioriniae (Pennycook 2017; Shivas and Tan 2009) (Supplementary Figure 1). The isolate was deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) as RGM 3330. Genomic DNA extraction of RGM 3330 and phylogenetic analyses were carried out according to Cisterna-Oyarce et al. (2022). A multi-locus sequencing analysis was carried out using five genetic markers. The internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (gapdh), actin (act), and chitin synthase 1 (chs-1) were PCR-amplified following Damm et al. (2012) and beta-tubulin (tub) following Glass and Donaldson (1995). Sequences were deposited in GenBank (ON364141 for ITS and ON369167-70 for tub, act, chs-1, and gapdh, respectively) (Sayers et al. 2021). A BLAST analysis carried out in SequenceServer (Priyam et al. 2019), using a custom database of sequences retrieved from Damm et al. (2012) and Liu et al. (2020), showed that all genetic markers were 100% identical to those of C. fioriniae CBS 128517T (ITS (540/540 identities), gapdh (249/249), act (245/245), and chs-1 (274/274)), except for tub, which shared 99.8% of its identities (416/417) with this species. Maximum likelihood phylogenetic estimation clustered RGM 3330 with C. fioriniae strains CBS 128517T and CBS 126526 with 100% bootstrap support (Supplementary Figure 1). Koch’s postulates were carried out with asymptomatic fruits of V. corymbosum cv. 'Brigitta Blue'. Prior to inoculation, fruits were surface-sterilized for 10 s in 70% ethanol, 3 s in 1% NaOCl, 10 s in 70% ethanol, rinsed three times with sterile distilled water, and subsequently placed in moist-chambers. Two groups of three repetitions of 20 fruits each were sprayed with 9 × 106 conidia/mL of RGM 3330 for the first group and with sterile distilled water for the control. After 5 d at 25 °C with light/darkness cycles of 12 h, only fruits sprayed with the conidial solution developed symptoms of anthracnose and the re-isolated fungi were identical in morphology to RGM 3330. This is the first report of C. fioriniae in blueberry in Chile.