First Report of Diaporthe tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico.

Abstract

Worldwide cacao pod rot is a devastating disease of Theobroma cacao, infected cacao pods turn necrotic reducing yield up to 30%. From July 2020 to August 2021, a survey was conducted at the USDA-ARS cacao germplasm collection located at Mayaguez, Puerto Rico. Incidence of cacao pod rot was 73.9%, observed in 142 of the 196 accessions sampled. The disease was observed at different stages of pod development (small, green, mature pods, and dry mummified large pods). Diseased tissue from three cacao pods (1 mm2) per each cacao accession was surface disinfested by immersion in 70% ethanol for one minute, rinsed with sterile-distilled-water and plated on potato dextrose agar (PDA) amended with 250 mg/L ampicillin and 60 mg/L streptomycin. After 30 days of incubation at 25°C, seven isolates developing white fast-growing colonies with black-globose pycnidia were observed. All isolates produced hyaline, one-celled, biguttulate, and cylindrical and rounded at the apex α conidia of 5.1 to 7.3 µm × 2.5 to 3.0 µm in size and were identified as Diaporthe spp. (Gomes et al. 2013; Crous et al. 2015). To determine the species identity, seven isolates were sequenced of the internal transcribed spacer (ITS), sections of β-tubulin (BT) and translation elongation factor 1 alpha (EF1-α) and compared using the BLASTn with Diaporthe spp. type specimens deposited in NCBI GenBank. ITS, BT and EF1-α sequences of Phomocac16, Phomcac17, Phomcac18 and Phomcac21 isolates (GenBank accession nos. OL353698 to OL353701, OL412430 to OL412433, and OL412437 to OL412440 for ITS, BT and EF1-α, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. The other three isolates (Phomcac8P1, Phomcac8P3 and Phomcac8P4) were grouped to the ex-type (CBS 101339) of Diaporthe pseudomangiferae R.R. Gomes, Glienke & Crous, ITS, BT and EF1-α sequences of (GenBank accessions nos. OL353702 to OL353704, OL412434 to OL412436, and OL412441 to OL412443, for ITS, BT and EF1-α, respectively). Pathogenicity tests were conducted using isolates Phomocac16, Phomcac17, Phomcac18 and Phomcac21 of D. tulliensis and isolates Phomcac8P1, Phomcac 8P3 and Phomcac8P4 of D. pseudomangiferae on five healthy detached green, yellow and red pods of the following cacao varieties: TARS27, ICS16, ICS1, GS29, UF601, SIAL56, Amelonado, SIAL98, EET94, ICS129 and GNV58. Cacao pods were wounded and inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on PDA of each isolate and wrapped with parafilm. Untreated controls were inoculated with PDA disks only. Fruits were kept in a humid chamber for 8 days at 25°C. Tests were repeated twice. Eight days after inoculation with D. tulliensis and D. pseudomangiferae, all cacao pods turned dark brown, untreated controls showed no symptoms of pod rot, and no fungi were isolated from tissue. Both species, D. tulliensis and D. pseudomangiferae were reisolated from their respective diseased tissues fulfilling Koch's postulates. Diaporthe tulliensis has been reported from rotted stem ends of cacao pods in Australia (Crous et al. 2015), and D. pseudomangiferae was reported in a shipment of cacao seed pods in California; however, pathogenicity tests were not conducted at either location. In California D. pseudomangiferae is considered a quarantine pathogen with a temporary Q rating (Chitambar 2017). To our knowledge, this is the first report of D. tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico. Knowing the identity and incidence of these new cacao pathogens is the first step for developing specific control measures and potential sources for resistance to cacao pod rot caused by Diaporthe spp.