ABSTRACT
Nance fruit [Byrsonima crassifolia (L.) HBK] is a native crop widely distributed in Mexico and South America (Medina-Torres et al. 2018). It has been reported that nance is a good source of active compounds with anti-inflammatory, neuropharmacological and antioxidant effects. In 2022, the annual production of nance fruit in Mexico was of 7,713.13 tons and average yield of 5.64 t/ha with economic value of 51,952.66 million pesos (SIAP, 2022). This production generated significant economic income for the communities at a local, regional, and national level. In January 2023, irregular necrotic spots were observed on leaves and fruit of nance in an orchard of 50 nance trees located in San Sebastián Nopalera (16°54'52.73"N; 97°47'50.35"W), Oaxaca, Mexico. The incidence of the disease ranged from 50 to 60% of the trees. Infected fruit first showed dark-brown lesions with defined borders that coalesced to form large necrotic area. Isolates were purified by single spore isolation method (Choi et al. 1999).Colletotrichum strains were grown in PDA medium and five monoconidial isolates were obtained. A representative sample was selected (CNC-NP3) and deposited in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. Colony on PDA was white with sparse aerial mycelium, and the center was dark grey with abundant acervuli. Conidia (n = 100) were hyaline, aseptate, cylindrical with rounded apex, 13.5 to 15.2 × 4.3 to 5.1 µm. Appressoria (n = 20) were terminal or lateral, obovoid to clavate and some with slightly lobed, 9.9 to 11.6 × 5.3 to 6.6 µm. Based on the morphology, the isolate was identified as belonging to the Colletotrichum gloeosporioides species complex (Jayawardena et al. 2016). The representative isolate CNC-NP3 was identified by multilocus phylogenetic analysis using nucleotide sequences of internal transcribed spacer (ITS), actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Jayawardena et al. 2016). The sequences were deposited in GenBank (accessions nos. OQ861102 (ITS), OQ870548 (ACT), OQ870549 (TUB2), OQ870550 (GAPDH). The phylogenetic analysis was carried out by Maximum likelihood method using concatenated sequences of ITS, ACT, TUB2 and GAPDH genes (Kozlov et al. 2019). The multilocus phylogenetic analysis revelated clearly the isolate CNC-NP3 as Colletotrichum chrysophilum. To confirm pathogenicity of CNC-NP3, 30 healthy fruits were inoculated. Fifteen disinfected nance with wounds and fifteen nance without wounds were inoculate with 10 µL of conidial suspension (1×105 spores/mL) from 7-day old culture. And controls were inoculated using sterile distilled water. Fruits were placed in a moist chamber covered with plastic bag at 25 °C for 48 h to maintain high humidity. After 4 days the inoculation sites development symptoms that were identical to those initially observed in the field, whereas the control group remained symptomless. The pathogenicity test was performed twice, with the same results. The pathogen was re-isolated from the lesion to fulfill Koch's postulates. Currently, Colletotrichum chrysophilum has been reported causing anthracnose disease in several crops: apple in New York (Khodadadi et al. 2020), papaya in Mexico (Pacheco- Esteva et al.2022), Blueberry (Soares et al. 2022) and banana in Brazil (Astolfi et al. 2022). To our knowledge, this is the first report of anthracnose in Byrsonima crassifolia caused by Colletotrichum chrysophilum in Oaxaca, Mexico.
ABSTRACT
Anthracnose, caused by Colletotrichum spp., is the most important fungal disease of papaya (Carica papaya L.) worldwide. In March 2020, mature papaya fruit (cv. Maradol) showing typical symptoms of anthracnose were observed in an orchard located in Pinotepa Nacional, Oaxaca, Mexico. Disease incidence of 100 papaya plants surveyed in the orchard was estimated at about 45%. Initially, small and water-soaked lesions appeared on the fruit surface, which later enlarged to circular sunken lesions with translucent light brown margins. On advanced infections, salmon-pink masses of spores were observed on the lesions. Twenty Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium at 25°C in the dark for 6 days and 10 monoconidial isolates were obtained. An isolate was selected as representative for further characterization. The isolate was deposited as CPM-H4 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. On PDA, the colonies were initially light grey then later became dark grey with orange conidial masses after incubation for 7 days. Conidia (n= 50) were hyaline, aseptate, cylindrical with rounded ends, and measured 10.2 to 13.6 × 4.1 to 5.3 µm. Appressoria (n= 20) were mostly simple, solitary and smooth-walled, dark brown, and clavate, measuring 6.8 to 14.8 × 5.5 to 7.7 µm. Based on morphology, the isolate was tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes were amplified (Weir et al. 2012), and sequenced. The sequences were deposited in GenBank (accessions nos. OM965612 (ITS), OM959540 (ACT), ON065005 (TUB2), ON065003 (CHS-1), ON065004 (GAPDH). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, GAPDH, and CHS-1 sequence dataset for Colletotrichum spp. was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CPM-H4 as Colletotrichum chrysophilum. Pathogenicity of the fungus was verified on 10 healthy papaya fruits (cv. Maradol) without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on three locations on each fruit. Ten control fruit were treated in the same way but with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated papaya fruits developed sunken necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Colletotrichum chrysophilum has been previously reported to cause anthracnose on mango (Fuentes-Aragón et al. 2020a), avocado (Fuentes-Aragón et al. 2020b), and banana (Fuentes-Aragón et al. 2021) in Mexico; however, to our knowledge, this is the first report of C. chrysophilum causing papaya anthracnose in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species associated with papaya in Mexico through subsequent phylogenetic studies as well as to monitor the possible movement and distribution of this pathogen into other Mexican regions.
