First Report of Thread Blight Caused by Marasmius tenuissimus on Cacao (Theobroma cacao) in Peru

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 the 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 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% NaOCl, rinsed three times in sterile water, plated on potato dextrose agar medium (PDA), and incubated for 7 days at 25°C. Hyphae were nonpigmented with clamp connections, consistent with the genus Marasmius. We extracted the DNA of isolate INDES-AFHP31 using the Wizard Purification Kit (Promega, Madison, WI) and sequenced the rDNA internal transcribed spacer 1 and 2 intervening the 5.8S subunit (ITS), and the 28S subunit (LSU) (accession nos. 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 of M. tenuissimus from Ghana (Amoako-Attah et al. 2020). A pathogenicity test was conducted by inoculation of 10 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 controls. The observation of TBD symptoms and signs in the noncontrol set of cacao leaves starting at 3 days postinoculation, and the reisolation 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 frequent 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.