Coffee Dieback Caused by Lasiodiplodia pseudotheobromae in Brazil

Abstract

Coffea arabica is the most important coffee species cultivated worldwide. Although many diseases are known to affect coffee plantations, few pathogens have been reported to cause the death of plants. In 2019, in two growing areas (0.3 and 1 ha) of C. arabica cv. Catuai 144, in Brejao, Pernambuco, Brazil (8°57′28″S; 36°32′00″W; altitude 750 m), 2% of 3-year-old plants showed dieback symptoms. Infected stems had necrotic lesions and black pycnidia on the surface. Three single-spore cultures were obtained directly from three infected stems and deposited in the Culture Collection of Phytopathogenic Fungi Prof. Maria Menezes (CMM) at the Universidade Federal Rural de Pernambuco (Recife, Pernambuco, Brazil) (codes CMM4875 to CMM4877). The colony grown on potato dextrose agar (PDA) medium showed fast growth, with abundant aerial mycelium that was initially white and became grayish. Conidia were initially hyaline, unicellular, and ellipsoidal, 21.4 to 30.7 × 11.4 to 15.1 µm (n = 30). Mature conidia showed a dark brown pigmentation, with a central septum and longitudinal striations. Morphological characteristics were compared with previous descriptions (Alves et al. 2008; Phillips et al. 2013), and the isolates were identified as Lasiodiplodia sp. To complete the identification to species level, sequences of the region of ribosomal DNA (ITS-rDNA) and translation elongation factor 1-α (TEF1-α) were successfully amplified and sequenced, and consensus sequences were deposited in GenBank database (accession nos. MN122134 to MN122136 for ITS and MN103350 to MN103352 for TEF1-α). The BLASTn search of ITS-rDNA and TEF1-α sequences showed 100 and 99% identity, respectively, with reference sequences of the type strain Lasiodiplodia pseudotheobromae CBS116459 (accession nos. EF622077 and EF622057, respectively). A combined phylogenetic tree obtained by Bayesian inference, using MrBayes version 3.1.1 (Ronquist and Huelsenbeck 2003), grouped the isolates within the L. pseudotheobromae clade, confirming the identification. Therefore, based on morphological and molecular analyses, the fungal pathogen was identified as L. pseudotheobromae. The pathogenicity of the three isolates was evaluated in five plants (6 months old) and in 15 detached leaves of C. arabica cv. Catuai 144. Unwounded and superficially wounded (with a sterilized needle) stems and leaves were inoculated by depositing mycelial plugs (5 mm) of 3-day-old colonies grown on PDA medium. As a control, PDA plugs were used. Inoculated plants were maintained in a moist chamber for 2 days and subsequently transferred to a greenhouse under a 12-h photoperiod at 25°C. Inoculated leaves were maintained in a moist chamber at 25°C for 10 days. Only wounded plants and leaves showed symptoms, whereas unwounded and control plants and leaves remained healthy. Necrotic lesions were observed in the plants at 48 h after inoculation (hai), which advanced on the stem and leaves, leading to the death of the top at 10 days after inoculation (dai). Detached leaves showed necrotic lesions at 24 hai, and most tissues were necrotized at 5 dai. Black pycnidia were found on necrotic tissues at 10 dai. The fungus was successfully recovered from inoculated symptomatic tissues, confirming Koch’s postulates. Although L. pseudotheobromae has been reported in C. arabica in Thailand, its pathogenicity has not been confirmed (Trakunyingcharoen et al. 2015). To our knowledge, this is the first report of this species causing dieback on C. arabica in Brazil and in the world. Thus, this finding highlights the occurrence of a new pathogen that can cause losses in coffee cultivation and may contribute to future studies involving disease management in this crop.