RESUMO
Mangoes (Mangifera indica L.) are one of the most important export fruits in Peru and anthracnose, caused by several species in the Colletotrichum gloeosporioides species complex (CGSC), is one of their main postharvest diseases (Alvarez et al. 2020). Balsas is the major mango producing district in the Amazonas department, where farmers practice intercropping in orchards mostly of less than 5 ha (Cabezudo Cerpa 2022). In July 2021, mango fruits cv. Kent with anthracnose were detected at an incidence of 55 to 80% during postharvest in Balsas. Symptoms included sunken dark brown lesions with appearance of orange conidiomata at advanced stages of the disease. We collected two samples of infected mangoes from a farm located at 6°51'01" S, 77°59'48" W (1088 m.a.s.l.). One axenic culture (INDES-AM1) was obtained from a hyphal tip of a monosporic colony and cultivated on PDA medium at 25 °C in the dark. The growing rate of the colony was 8.1 mm.day-1. Conidia were hyaline, guttulate, unicellular and cylindrical with narrowing center, with dimensions of 15.8 to 23.5 × 4.5 to 7.6 µm (mean = 18.6 ± 0.03 × 6.0 ± 0.02 µm, SE, n = 50), consistent to the CGSC (Weir et al. 2012). Appressoria were dark brown, and ovoid to slightly irregular in shape, ranging from 5.3 to 10.1 × 4.7 to 8.3 µm (mean = 7.9 ± 0.02 × 6.0 ± 0.02 µm, SE, n = 50). Koch's postulates were fulfilled on mature mango fruits of the same cultivar and from the same district. Mangoes were washed with detergent and left to dry before inoculation. PDA-mycelial plugs of 0.5 cm wide were transferred on two different locations of two fruits, with four replicates. One location was previously wounded with five needle punctures of 3 mm depth. The inoculated fruits were maintained in a moist chamber at ambient light and temperature (18.9 ± 0.5 °C, SE). Symptoms appeared three-to-five days post inoculation (dpi), and the superficial diameter of the lesions were 8.3 ± 1.5 and 3.6 ± 2 mm with the invasive and the superficial inoculation approaches, respectively, at five dpi. Lesions were very similar to original symptoms. Macro and micromorphological characteristics of the re-isolated fungal colonies were the same as isolate INDES-AM1. Molecular identification of the pathogen was carried out following Weir et al. (2012). Total DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, Wisconsin) and the ribosomal internal transcribed spacer (ITS), and partial sequences of the chitin synthase (CHS1), actin (ACT), ß-tubulin 2 (TUB2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear genes were sequenced (Accession numbers: OP425395, OP440444, OP440442, OP440443, OP555062, OP555063). ITS, CHS1, ACT, TUB2, CAL, and GAPDH sequences were 98.6, 100, 100, 99.5, 100, and 99.08% identical to Colletotrichum asianum type strain ICMP 18580 sequences, respectively. Additionally, a bootstrapped maximum likelihood midpoint-rooted phylogeny with a multilocus dataset with the six sequences from reference strains of C. asianum and closely related species within the CGSC revealed that strain INDES-AM1 is C. asianum. This species has been found causing anthracnose on M. indica in at least 15 different countries in Africa, America, Asia, and Oceania (Weir et al. 2012). It was originally described from coffee and has multiple other hosts (Prihastuti et al. 2009; Lima et al. 2015). To the best of our knowledge, this is the first report of C. asianum infecting mangoes in Peru.
RESUMO
Peru is the ninth exporter of coffee (Coffea arabica) in the world, and Amazonas is among its most important producing departments (INIA 2019). In July 2021, in the nursery of the "Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva", in Huambo district (6° 26' 11.19'' S; 77° 31' 24.18'' W), four-month-old coffee seedlings cv. Catimor with 0.5-2.0 cm brown concentric leaf spots and rotten stems, bearing white mycelial tufts and black sporodochia, were observed at 30% incidence. Infected seedlings were collected. Foliar sections of 2-3 mm with infected tissue were surfaced disinfected in 2% NaClO and transferred onto Petri plates containing potato dextrose agar medium (PDA). The plates were incubated at 25° C for 7 days. We obtained three isolates (INDES-AFHP61, INDES-AFHP62 and INDES-AFHP66) with similar morphology from different seedlings. Colonies (16-17 mm diam.) formed concentric rings with white aerial mycelium, giving rise to viscous and olivaceous dark green sporodochial conidiomata. Conidia (4.82-5.77 × 1.34-1.65 µm; n = 30) were cylindric, hyaline, smooth, and aseptate. These morphological features correspond to Paramyrothecium spp. (Lombard et al. 2016). The DNA of isolates was extracted using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin), and the internal transcribed spacer 1 and 2 intervening the 5.8S subunit rDNA region (Accession numbers: OM892830 to OM892832), and part of the second-largest subunit of the RNA polymerase II, the calmodulin and the ß-tubulin genes (OM919453 to OM919461) were sequenced following Lombard et al. (2016). All sequences had a percent identity greater than or equal to 99% to corresponding sequences of the P. roridum type specimen (CBS 357.89). Additionally, a multilocus Maximum Likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al. 2016; Pinruan et al. 2022) grouped our three isolates together with the type and other specimens of P. roridum in a strongly supported clade, confirming the species identification. To evaluate pathogenicity, four-month-old coffee seedlings cv. Catimor were sprayed with 10 mL of conidial suspensions at 1 x 106 /mL. A set of control seedlings were inoculated with sterile water. Seedlings were maintained in a humidity chamber at 25 °C. After 15 days brown concentric foliar spots, stem rotting, mycelial tufts and sporodochia (same symptoms and signs observed originally at the nursery) arose in the non-control seedlings. The pathogen was re-isolated on PDA, confirming P. roridum was the causal agent of leaf spot and stem rot diseases of coffee. Paramyrothecium roridum has wide geographic distribution and host range (Lombard et al. 2016). This pathogen was reported to infect C. arabica in Mexico and Coffea sp. in Colombia (Pelayo-Sánchez et al. 2017; Lombard et al. 2016; Huaman et al. 2021). It was also reported in Africa infecting soybeans (Haudenshield et al. 2018), in Brazil infecting Tectona grandis (Borges et al. 2018), in Egypt infecting strawberries (Soliman 2020), and in Malaysia infecting Eichhornia crassipes (Hassan et al. 2021). To the best of our knowledge, this is the first time P. roridum is reported on coffee in Peru.
