First report of Colletotrichum tropical causing leaf spot on Cariniana legalis in Brazil.

Cariniana legalis (Mart.) Kuntze (Lecythidaceae), commonly known as "Jequitibá Rosa," an endemic tree from Brazil, is one of the largest trees that grows in the Atlantic Rain Forest. Jequitibá Rosa has been extensively exploited due to the excellent quality of its wood, which has resulted in the species being placed as "vulnerable" on the IUCN red list of threatened species (IUCN, 2021). Thus, conservation of the species is paramount. In October 2018, in the municipality of Ilhéus, Bahia (14º 79' 67" S, 39º 17' 32" W), trees of C. legalis were observed with approximately 80% of leaves presenting light to dark brown necrotic lesions with yellow edges at the tips and margins of the leaves; the lesions gradually expanded through the leaves and coalesced to form wider lesions, which resulted in death of the leaves and weakening of the trees. The symptoms indicate infection caused by a Colletotrichum spp., important pathogens known to cause diseases in several economically important plants. Symptomatic leaves were collected and lesions examined under a stereomicroscope at 20x magnification (Supplementary Figure S1. A). Conidia were transferred directly and aseptically from the leaf spots to potato-dextrose-agar (PDA) and incubated in the dark at 25 ± 1 ºC for 72 h. Subsequently, pure cultures were obtained by monosporic isolation. All fungal structures were measured using a Leica DM500 microscope at 1000x magnification. A pathogenicity test was performed both on hosts in the field and using detached leaves by inoculation with a conidial suspension 106 conidia/mL prepared from a culture grown on PDA incubated in the dark at 25 ± 1 ºC for 10 days. Six healthy and fully developed detached leaves were inoculated with two 10 µL drops of conidial suspension on the adaxial surface. Six leaves were inoculated with two drops of sterile water were used as controls. The inoculated leaves were kept in a humid chamber at 25º C with a 12 h photoperiod. Four trees of C. legalis growing in the field were selected and 120 healthy leaves (30 per tree) sprayed with a conidial suspension and covered with transparent polyethylene plastic bags, with a piece of wet cotton inside, to maintain moisture for 72 h. A further 30 leaves per tree were sprayed with sterile water as controls. DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method following the protocol described by Oliveira et al. (2016). Internal transcribed spacer (ITS), partial histone3 (HIS3), and partial glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene sequences were amplified by polymerase chain reaction (PCR) using ITS1/ITS4 (White et al. 1990), CYLH3F/CYLH3R (Crous et al. 2004), and GDF1/GDR1 (Guerber et al. 2003) primers. The PCR for the ITS amplification were performed according to Oliveira et al. (2014), and for the HIS3 and GAPDH according Damm et al. (2012). Maximum likelihood analysis (with support estimated by a bootstrap analysis with 1,000 replicates) was performed using PhyML 3.0 (Guindon and Gascuel 2003) and launched from Topali 2.5. The colony produced abundant white to light gray aerial mycelium, and an orange viscous mass of conidia (Supplementary Figure S1. B). Conidia were single-celled, smooth, hyaline and sub-cylindrical with rounded ends, 13 (11-15) × 3.5 (3-4) µm (Supplementary Figure S1. C); appressoria were aseptate, brown, subglobose to clavate, 11 (10-12) × 7 (6-8) µm (Supplementary Figure S1. D). BLASTn analysis revealed that the partial gene sequences of ITS (URM 8381 ‒ MZ158701), GAPDH (URM 8381 ‒ MZ189259) and HIS3 (URM 8381 ‒ MZ189260) were 100% identical to Colletotrichum tropicale (CPO 27.830 ‒MN744296/CBS 129983 ‒ MH865615), (CMM 4071 ‒ KC517181/CPO 27.719 ‒MN737355) and (CBS 124949 ‒ KY856395). A maximum likelihood phylogenetic tree was generated by combining all sequenced loci. The phylogenetic tree revealed that sequences of the isolate URM8381 formed a clade with the sequences from the type species of C. tropicale E.I. Rojas, S.A. Rehner & Samuels (CBS 124949) with a high support value (95), which is distinct from other related species (Supplementary Figure S1. E). The sequences from the isolate were deposited in GenBank under the following accession numbers: ITS: MZ158701; GAPDH: MZ189259 and HIS3: MZ189260. C. tropicale was reisolated from the inoculated leaves, and had the same cultural and morphological characteristics as the original isolate. Both detached leaves and leaves on trees were inoculated in the field and presented leaf spot symptoms (Supplementary Figure S1.F), at 6 and 8 days after inoculation, respectively, which further confirms C.tropicale as the causal agent of the symptoms observed on the leaves of C. legalis. The controls did not develop any symptoms (Supplementary Figure S1. G). C. tropicale belongs to the C. gloeosporioides species complex. In Brazil, C. tropicale causes anthracnose on various hosts, including Annona muricata L. (soursop) and Myrciaria dubia (Kunth) McVaugh ("camu-camu") (Costa et al. 2019; Matos et al. 2020). To our knowledge, this is the first report of C. tropicale causing leaf spot on C. legalis. References: Costa, J. F. O., et al. 2019. Eur. J. Plant Pathol. 153:1119. Crous, P. W., et al. 2004. Stud. Mycol. 50:415. Damm, U., et al. 2012. Stud. Mycol. 73:1. Guerber, J. C., et al. 2003. Mycol. 95:87. Guindon, S.; Gascuel, O. 2003. Syst Biol 52(5): 696-704. https://doi.org/10.1080/10635150390235520. IUCN. 2021. http://www.iucnredlist.org. Matos, K. S., et al. 2020. Plant Dis. 104. https://doi.org/10.1094/PDIS-04-19- 0882-PDN Oliveira R. J. V., et al. 2016. N. Hedw. 103:185. White, T. J., et al. 1990. In: Innis, M.A., et al. (Eds.) Academic Press, San Diego, pp. 315-322. http://dx.doi.org/10.1016/b978-0-12-372180-8.50042-1.