First report of Colletotrichum asianum causing mango anthracnose in Puerto Rico

Anthracnose caused by Colletotrichum spp., is a devastating fungal disease that affects mango production and shelf life. During a disease survey at the USDA-ARS mango backup germplasm collection in Isabela, Puerto Rico, necrotic spots on leaves were found in 18 Mangifera indica varieties. Diseased pieces (1 mm 2 ) of mango leaf tissues were surface sterilized and plated on potato dextrose agar (PDA). Three isolates (MFANT6, MLANT2 and MLANT4) of Colletotrichum asianum (Ca) were identified using taxonomic keys (Weir et al. 2012). Colonies of Ca grown on PDA were grayish green with aerial mycelial growth and conidial masses. Conidia (n = 50) were 13.8 × 3.6 μm in length, single-celled, smooth-walled, hyaline, cylindrical and with obtuse ends. Acervuli and perithecia were absent in culture. PCR amplifications using universal fungal primers of the internal transcribed spacer (ITS), portions of the β-tubulin (BT), actin (ACT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were used to support morphological identification. PCR products were purified using QIA Quick PCR Kit (Qiagen) and the sequenced regions were deposited in GenBank accession numbers: PQ811864, PQ811865 and PQ811866 for the ITS region; PQ815301, PQ815302 and PQ815303 for BT; PQ815278, PQ815279 and PQ815280 for ACT; and PQ815324, PQ815325 and PQ815326 for GADPH. A maximum likelihood tree was constructed with sequences of the three isolates mentioned above and sequences of the ex-type isolates of the C. gloeosporioides complex. All three isolates were clustered (Bootstrap = 99) to Ca ex-types ICMP18580 and ICMP18696 (Prihastuti et al. 2009). Pathogenicity tests were conducted on non-detached leaves and on detached mango fruits using a cocktail of the three isolates. Controls were inoculated with sterilized distilled water only. For non-detached leaves, two 15-year-old trees of the 18 varieties mentioned above, were used in the experiments. Non-detached twigs containing at least 10 healthy leaves were inoculated with 10 ml of a suspension of 4.5 x 10 5 conidia/ml from 8-day-old pure cultures of Ca. Twigs were covered with plastic bags for 30 days to maintain humidity. Thirty days after inoculations (DAI), necrotic spots on leaves were observed in all inoculated varieties. For detached fruit, 12 healthy and surface-disinfested mango fruit of commercial varieties Keitt and Nam Doc Mai were inoculated with 5 ml of a suspension of 4 x 10 5 conidia/ml from 8-day-old pure cultures of Ca. Inoculated fruits were placed in a plastic box for 8 days at room temperature. After the incubation period, fruits were placed on tables to evaluate disease progression. Anthracnose severity was evaluated at 8, 15, 20 and 25 DAI. Eight DAI, anthracnose sunken lesions were observed on Nam Doc Mai while lesions on Keitt were first observed on 15 DAI. Controls from both experiments showed no symptoms of anthracnose, and no fungi were isolated from the tissue. Ca was reisolated from both leaf and fruit tissue and sequences of isolates recovered from the pathogenicity test were grouped with the original isolate of Ca, fulfilling Koch’s postulates. Ca has been reported causing mango anthracnose in 17 countries (USDA fungal database, 2025). To our knowledge, this is the first report of Ca causing anthracnose in Puerto Rico. Identifying C. asianum is important to establish adequate control measures for anthracnose disease in Puerto Rico.