First report of Colletotrichum fructicola, C. perseae, and C. siamense causing anthracnose disease of avocado (Persea americana) in New Zealand

In January and March 2019, an inspection of 11 commercial 'Hass' avocado orchards in mid-North and Tauranga (New Zealand) was conducted by NZ Avocado Growers Association Inc. (NZAGA) and the samples were sent to Plant Diagnostics Limited for investigation of a newly observed fruit staining symptom termed "tannin stain". Fruit symptoms consisted of areas of minute small spots which coalesced into areas of tear staining associated with water movement over the fruit's surface (Supplementary Fig. 1). Up to seven trees per orchard were sampled targeting symptomatic fruit with the aim of determining the cause of the problem. Fruit was surface disinfected for 4 minutes in 1% sodium hypochlorite solution and sections from lesions were plated on agar medium (prune extract agar) to isolate any plant pathogens. The predominant fungi isolated, represented species in the Colletotrichum acutatum, C. gloeosporioides, and C. boninense species complexes. Since the morphological characters within these complexes overlap (see Supplementary Fig. 2 for examples), the isolates were differentiated by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene and, where necessary, the calmodulin (CAL) gene and/or the Apn2-Mat1-2 intergenic spacer region (ApMat) locus (Weir et al., 2012; Rojas et al., 2010). The sequence analysis revealed eight Colletotrichum species comprising C. alienum, C. aotearoa, C. cigarro, C. fioriniae, C. fructicola, C. karstii, C. perseae, and C. siamense. This range included three species that have not previously been recorded in New Zealand: C. fructicola (Cf), C. perseae (Cp), and C. siamense (Cs). Colonies for all these three fungi were white to grey with salmon-coloured and black acervuli. Conidia were aseptate, hyaline, straight, cylindrical, with broadly rounded ends, forming on cylindrical conidiogenous cells. The respective GPDH, CAL, and/or ApMat sequences of the Cf, Cp, and Cs isolates were identical to reference sequences of representative isolates in GenBank (e.g. ApMat: Cf - KX620181, Cp - KX620177, Cs - KP703788). An isolate for each species is stored in the International Collection of Microorganisms from Plants (Cf - ICMP22409, Cp - ICMP22431, Cs - ICMP22411) and sequences are deposited in GenBank (accession numbers MT522858-MT522865). Pathogenicity of each of the newly recorded species was confirmed on freshly picked 'Hass' avocado fruit. After surface disinfection with 1% sodium hypochlorite solution for 5