First report of Diaporthe tulliensis and D. pseudomangiferae causing cacao pod rot

Authors: Serrato Diaz, Luz; Ayala Silva, Tomas; Goenaga, Ricardo

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/6/2022
Publication Date: 2/11/2022
Citation: Serrato Diaz, L.M., Ayala Silva, T., Goenaga, R.J. 2022. First report of Diaporthe tulliensis and D. pseudomangiferae causing cacao pod rot. Plant Disease. https://doi.org/10.1094/PDIS-12-21-2634-PDN.
DOI: https://doi.org/10.1094/PDIS-12-21-2634-PDN

Interpretive Summary: Cacao pod rot is a devastating disease worldwide. Infected pods turn black and necrotic causing yield losses of up to 30%. From July 2020 to August 2021, cacao pod rot disease was observed in 142 of the 196 accessions (73.9% disease incidence) at the USDA-ARS cacao germplasm collection in Mayaguez, PR. This disease was observed at different stages of pod development (small, green, mature, and dry mummified large pods). Four isolates of the fungi Diaporthe tulliensis (Dt) and three isolates of Diaporthe pseudomangiferae (Dp) were identified using taxonomic keys and sequencing of three nuclear genes. Cacao pods were inoculated with Dt and Dp on five healthy detached green, yellow and red pods of the following varieties: TARS27, ICS16, ICS1, GS29, UF601, SIAL56, Amelonado, SIAL98, EET94, ICS129 and GNV58. Eight days after inoculation (DAI) with Dt and Dp, all cacao pods turned dark brown, and 14 DAI white mycelia of Dt and Dp were observed on cacao pods. Untreated controls had no symptoms of pod rot, and no fungi were isolated from tissue. To our knowledge, this is the first report of Dt and Dp causing cacao pod rot. Cacao pod rot disease is caused by a complex of pathogens where Dp and Dt are now included. 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.

Technical Abstract: Worldwide, cacao pod rot is a devastating disease, causing infected cacao pods to turn necrotic and reducing yield up to 30%. From July 2020 to August 2021, a survey was conducted at the USDA-ARS cacao germplasm collection located in Mayaguez, PR. Cacao pod rot was observed in 142 of the 196 accessions sampled, with a disease incidence of 73.9%. 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 variety were surface disinfested and plated on potato dextrose agar (PDA) amended with 250 mg/liter ampicillin and 60 mg/liter streptomycin. Four isolates (Phomcac16, Phomcac17, Phomcac18 and Phomcac21) of Diaporthe tulliensis (Dt) and three isolates (Phomocac8P1, Phomcac8P4 and Phomcac8P3) of Diaporthe pseudomangiferae (Dp) were identified using taxonomic keys and sequencing of the internal transcribed spacer (ITS), sections of ß-tubulin (BT) and translation elongation factor 1 alpha (EF1-a). Sequences were compared using the BLASTn tool with Diaporthe spp. deposited in NCBI GenBank. In PDA, colonies of Dt were white with immerse mycelia. Erumpent black pycnidia exuded cream droplets from ostioles and were observed aggregated in small groups. Smooth aseptate, hyaline alpha conidia (n=50) were cylindrical, rounded at the apex, averaged 5.1 µm long by 2.5 µm wide. Beta conidia were not seen. In PDA, colonies of Dp were dirty white with immerse mycelia. Erumpent disperse black pycnidia exuded beige droplets from ostioles. Smooth, aseptate, hyaline alpha conidia (n=50), were cylindrical rounded at the apex, averaged 7.3 µm long by 2.50 µm wide. Smooth, aseptate, hyaline beta conidia (n = 50) were spindle-shaped, averaged 23 µm long by 1.5 µm wide. GenBank accession numbers are OL353698, OL353699, OL353700 and OL353701 (Dt), OL353702, OL353703 and OL353704 (Dp) for ITS; OL412430, OL412431, OL412432 and OL412433 (Dt), OL412434, OL412435 and OL412436 (Dp) for BT; OL412437, OL412438, OL412439 and OL412440 (Dt), OL412441, OL412442 and OL412443 (Dp) for EF1-a. Pathogenicity tests were conducted 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 using a 1mm-diameter needle and inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in PDA of each isolate were placed on pods 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 (DAI) with Dt and Dp, all cacao pods turned dark brown, and 14 DAI white mycelia of Dt and Dp colonized cacao pods. Untreated controls showed no symptoms of pod rot, and no fungi were isolated from tissue. Both species, Dt and Dp were reisolated from their respective diseased tissues fulfilling Koch’s postulates. Diaporthe tulliensis has been reported from rotted stem end of cacao pods in Australia, whereas Dp was reported in a shipment of cacao seed pods in California, pathogenicity tests were not conducted at neither location. In California Dp is considered a quarantine pathogen with a temporary Q rating. To our knowledge, this is the first report of Dt and Dp causing cacao pod rot.