First Report of <i>Phytophthora palmivora</i> Causing Black Pod Rot in Cacao (<i>Theobroma cacao</i>) in Three Municipalities of Tabasco and One Municipality of Chiapas, Mexico

Cacao (Theobroma cacao) is a highly important crop for the production of chocolate, which is in high international demand (Zhang & Motilal, 2016). Black pod disease, caused by Phytophthora species, is one of the main limiting factors in cacao production, leading to yield losses of up to 30% annually worldwide. In Mexico, national production surpasses 28 thousand t, cultivated on 53 thousand hectares. The states of Tabasco and Chiapas are the main producers (SIAP, 2023). Phytophthora tropicalis has been reported as the causal agent of black pod rot (BPR), affecting cacao production each year (Chávez-Ramírez et al. 2021). From October 2023 to June 2024, 69 cacao pods were collected, as they are the main affected organ, that exhibited symptoms typical of BPR (circular dark brown lesions with a well-defined border) in Tabasco and Chiapas. The pods were disinfected according to Erwin & Ribeiro 1996. Four 5×5 mm fragments from the interior of the rind were obtained, and then three sections were obtained from the edges of the lesions. The fragments were placed in V8 agar medium and incubated at 24 ± 1 °C for 5 d. Morpho-cultural characteristics were recorded. The colonies of six strains were atypical, with a star-shaped growth pattern, sparse mycelia, and smooth margins. The microscopic characteristics were recorded with 50 measurements of each structure. The sporangia, arranged in simple sympodium, were papillate, ovoid, averaging 47 µm long and 27 µm wide, with short pedicels (3-5 µm). Terminal and intercalary chlamydospores were globose, with a diameter of 35 µm. These characteristics coincide with the descriptions of P. palmivora (Abad et al. 2023; Erwin & Ribeiro, 1996; Stamps et al. 1990). For molecular identification, DNA was extracted from lyophilized mycelia using the Qiagen DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. Four genomic regions of a representative strain (CPTABP149) were amplified and sequenced: internal transcript spacer (ITS rDNA) and protein related to ras (Ypt1) (Abad et al. 2023), beta-tubulin (β-TUB) (Blair et al. 2008) and mitochondrial cytochrome c oxidase subunit 1 (COI) (Robideau et al. 2011) with the primer pairs PHY.OO.18S.F/PHY.OO.28S.R, YPH4BF/YPh9R, F-βtub1/F-βtub4r andCOIR-1 FM85Mod/COIF-1, respectively. The BLAST analysis of the sequences obtained from the strain CPTABP149 (PQ869733) for ITS and (PQ875189) for COI revealed 100% identity with different isolates of P. palmivora available in GenBank (PP06980, MT7554523, GU111650) and (OP477330, MH760230, MT032128), respectively. The other two sequences with accession numbers PQ875187 and PQ875188 for β-TUB 99 showed 99.7% similarity with the isolate MT074255 and, for ypt1, 99.3% similarity with MK058413. Based on the molecular characteristics of the CPTABP149 strain, it is P. palmivora. Pathogenicity tests were conducted on physiologically mature cacao pods disinfected with 3% sodium hypochlorite (Erwin & Ribeiro, 1996). Three 0.5 mm mycelial plugs of strain CPTABP149 were inoculated onto the pod epidermis. Control pods were inoculated with sterile V8 discs. All the pods were incubated at 25 °C with a photoperiod of 12 h for 5 d. Dark brown circular spots like those observed on the pods in the field developed; control pods remained asymptomatic. P. palmivora was reisolated from the symptomatic lesions, thereby fulfilling Koch's postulates. This is the first report of P. palmivora causing black pod rot in Comalcalco, Huimanguillo, and Teapa, Tabasco, and Tecpatán Chiapas, Mexico. This pathogen is considered a quarantine species, as it has not been officially reported in the country.