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 minutes, fruit was triple washed with sterile water and air dried. Five fruits per species were pin-pricked and inoculated with 10µL of conidial suspension (7 x 106 to 1 x 107 conidia/mL) prepared with sterile water containing Tween 20 (1µL/mL H2O) from 6-day-old cultures grown on PDA. Control fruit was pin-pricked and mock-inoculated with sterile water containing Tween 20 (1µL/mL H2O). All fruit was incubated in moist chambers at 25°C for 7 days. The three Colletotrichum species produced anthracnose symptoms on inoculated fruit whereas no symptoms were observed on control fruit (Supplementary Fig. 3). Each one of the species was successfully re-isolated from symptomatic tissue and identified using the methods described above, fulfilling Koch's postulates. While Cf and Cs have been reported from several hosts and countries to date (Weir et al. 2012), Cp has only been found from avocado in Israel (Sharma et al. 2017) and grape in Japan (Yokosawa et al. 2020). Although a number of species from the C. gloeosporioides species complex, i.e. C. alienum, C. aotearoa, C. cigarro, and C. gloeosporioides have been previously associated with avocado diseases in New Zealand, the detections of Cf, Cp, and Cs represent first records. In this study, eight Colletotrichum species were associated with the "tannin stain" fruit symptoms in New Zealand avocado orchards. The individual contribution of the newly recorded pathogens Cf, Cp, and Cs to the observed disease symptoms was not determined, but their detection highlights the importance of sequence-based identification of Colletotrichum species, as morphology is insufficiently robust to separate cryptic species. Accurate identification of pathogens provides knowledge of species biodiversity that may be useful in biosecurity decision making. Since it has been reported that fungicide treatment efficiencies differ for some closely related Colletotrichum species on grape (Yokosawa et al. 2020), accurate identification might also contribute to establishing effective management strategies.

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