RESUMO
Peru is the second largest producer of organic cocoa and one of the most important suppliers of fine aroma cocoa beans in the world (Sánchez et al. 2019). The fine aroma cocoa produced by smallholder farmers in the Bagua and Utcubamba Provinces, Amazonas Department, under the name of "Cacao Amazonas Peru", is protected by the Peruvian appellation rules (Díaz-Valderrama et al. 2020). Despite this importance, native diseases of the crop (Theobroma cacao) are poorly documented due to difficulty of access in this region. In November 2020 we conducted expeditions into Imaza District (4°47'09.4"S 78°16'51.6"W), a significant producer of fine aroma cocoa in terms of number of cultivated plots (4,651 out of 6,505 total in the Bagua Province) (INEI 2012). We visited 20 farms of < 2-ha in size; in 19 of these small farms, T. cacao trees were found infected with a white fungal thread blight and rhizomorphs covering branches and leaves. Disease incidence ranged from 90 to nearly 100%, and severity exceeded 80% on the eight farms with the most deficient phytosanitary management. Heavily infected leaves were hanging on branches by mycelial threads, harboring tiny (0.5 to 5.3 mm broad) white mushrooms. These symptoms and signs correspond to the thread blight disease constellation (TBD) of cacao caused by various species of Marasmius and Marasmiellus (Amoako-Attah et al. 2020). Mushrooms lacked a collarium, and their stipes were absent or rudimentary (< 2-mm long) and eccentric, consistent with Marasmius tenuissimus (Tan et al. 2009). Axenic cultures were obtained by surface sterilization of mycelium threads with 2% NaClO, rinsed three times in sterile water, plated on potato dextrose agar medium (PDA), and incubated for 7 days at 25°C. Hyphae was non-pigmented with clamp connections, consistent with the genus Marasmius. We extracted the DNA of isolate INDES-AFHP31 using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin) and sequenced the rDNA internal transcribed spacer 1 and 2 intervening the 5.8S subunit (ITS), and the 28S subunit (LSU) (Accession numbers: OM720123 and OM720135) according to Aime and Phillips-Mora (2005). The ITS and LSU sequences were 97.92 to 98.79% and 99.07 to 99.30% identical, respectively, with published sequences from M. tenuissimus from Ghana (Amoako-Attah et al. 2020). The pathogenicity test was conducted by inoculation of ten healthy cacao leaves with 7-day-old mycelium PDA discs of isolate INDES-AFHP31. An equal number of healthy cacao leaves were inoculated with PDA discs without mycelium as control. The observation of TBD symptoms and signs in the non-control set of cacao leaves starting at 3 days post inoculation, and the re-isolation of the same fungus from infected tissue confirmed its pathogenicity on cacao. Isolate INDES-AFHP31 was deposited as a dried culture into the herbarium Kuélap of the Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas (voucher KUELAP-2251). Marasmius tenuissimus was originally reported from dead and living twigs and leaves of unidentified dicotyledonous trees from Indonesia, Brazil, and Bolivia (Singer 1976). However, it was first associated with TBD of cacao in Ghana in 2020, being the most frequently TBD-causing fungus isolated in the country (Amoako-Attah et al. 2020). Its discovery in 19 of the 20 surveyed cacao farms in Imaza District, Amazonas, Peru, reveals its importance as a cacao pathogen in the Western hemisphere.
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.
