First report of Lasiodiplodia pseudotheobromae and Colletotrichum siamense causing cacao pod rot, and first report of C. tropicale causing cacao pod rot in Puerto Rico

Authors: SERRATO-DIAZ, LUZ - University Of Puerto Rico; MARIÑO, YOBANA - University Of Puerto Rico; GUADALUPE, ILANDRA - University Of Puerto Rico; BAYMAN, PAUL - University Of Puerto Rico; Goenaga, Ricardo

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/28/2019
Publication Date: 12/4/2019
Citation: Serrato-Diaz, L.M., Mariño, Y.A., Guadalupe, I., Bayman, P., Goenaga, R.J. 2019. First report of Lasiodiplodia pseudotheobromae and Colletotrichum siamense causing cacao pod rot, and first report of C. tropicale causing cacao pod rot in Puerto Rico. Plant Disease. https://doi.org/10.1094/PDIS-06-19-1333-PDN.
DOI: https://doi.org/10.1094/PDIS-06-19-1333-PDN

Interpretive Summary: Pod rot is the most economically important and widespread disease of cacao, Theobroma cacao L. Total losses due to pod rot exceed $400 million worldwide. Symptoms resembling those of pod rot disease were observed at the National Plant Germplasm Collection System of cacao at the USDA-ARS, Tropical Agriculture Research Station (TARS) in Mayaguez, Puerto Rico. In an effort to assess pod rot disease problems in this collection, a disease survey was conducted from June to September of 2017. Two isolates of Lasiodiplodia pseudotheobromae (Lps), two isolates of Colletotrichum siamense (Cos) and one isolate of Colletotrichum tropicale (Ctr) were identified using taxonomic keys and PCR amplification of the internally transcribed spacer sequences (ITS). Pathogenicity tests were conducted on five healthy detached and superficially sterilized cacao pods per isolate. Eight days after inoculation (DAI) with Lps, all cacao pods turned dark brown and fourteen DAI, gray mycelia and pycnidia of Lps were observed on cacao pods. Fourteen DAI with Cos and Ctr, all fruits turned brown, and acervuli were produced on cacao pods. Untreated controls had no symptoms of pod rot and no fungi were isolated from tissue. Colletotrichum spp. and L. pseudotheobromae were reisolated from their respective diseased tissue, fulfilling Koch's postulates. USDA-ARS-TARS is the official site for the cacao germplasm collection, thus, a detailed integrated pest management plan that includes the evaluation for resistance, sanitation measures, and use of fungicides to reduce disease levels has been implemented. To our knowledge, this is the first report of L. pseudotheobromae, C. tropicale and C. siamense causing pod rot of cacao. Knowledge of the identity of these plant pathogens is the first step to assess their impact on production of trees and to develop control measures.

Technical Abstract: Pod rot of cacao (Theobroma cacao L.) is the most important disease in cacao production worldwide. During a disease survey from June to September of 2017, cacao pod rot was observed at the USDA - ARS cacao germplasm collection in Mayagüez Puerto Rico. Infected cacao tissue was disinfested and plated on PDA amended with 0.250 gr/L of Ampicillin and streptomycin. Two isolates (I41 and I46) of Lasiodiplodia pseudotheobromae (Lps), two isolates (K21 and K51) of Colletotrichum siamense (Cos) and one isolate (K07) of Colletotrichum tropicale (Ctr) were identified using taxonomic keys and PCR amplification of the ITS rDNA, fragments of ß tubulin (BT), translation elongation 1 alpha (EF1-a) for Lasiodiplodia spp. and ITS, BT, chitin synthase (CHS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes for Colletotrichum spp. Sequences were compared using BLASTn tool with Colletotrichum spp. and Lasiodiplodia spp. submitted to NCBI GenBank. In PDA, colonies of Lps had initial greenish gray aerial mycelia that turned dark brown with age. Pycnidia were uniloculate and dark brown to black in color. Immature conidia were ellipsoid, apex rounded, truncate at the base, thick walled, hyaline and one-celled, becoming dark brown two-celled with irregular longitudinal striations when mature. Conidia (n = 50) averaged 25.4 µm long by 13.58 µm wide. In PDA, colonies of Cos were grayish white with aerial mycelial growth and conidial masses on culture, conidia (n=50) averaged 15.1 x 4.5 µm in length, one-celled, smooth walled, hyaline, cylindrical to oblong, and rounded at both ends. Apressoria were 4.5 x 5 µm diameter, perithecia were absent. In PDA, colonies of Ctr were gray with aerial mycelial growth and conidia (n=50) were 15 × 4.5 µm in length, cylindrical, hyaline, straight, and rounded at both ends. Appressoria were 7–10 µm diameter and perithecia were absent. Sequences submitted to GenBank included accession Nos MK693210 and MK693211 (Lps), MK693213 and MK693214 (Cos) and MK693212 (Ctr) for ITS MK693701 and MK693702 (Lps), MK693704 and MK693705 (Cos) and MK693703 (Ctr) for BT; MK693706 and MK693707 (Lps) for EF1-a; MK693712 and MK693713 (Cos) and MK693711 (Ctr) for CHS; and MK693709 and MK693710 (Cos) and MK693708 (Ctr) for GADPH. Pathogenicity tests were conducted on five healthy detached and superficially sterilized cacao pods per isolate. Pods were inoculated with 5 mm mycelial disks from 8-day-old pure cultures grown in PDA. Untreated controls were inoculated with PDA disks only. Fruits were kept in a humid chamber for 14 days at 25°C. The test was repeated twice. Eight days after inoculation (DAI) with Lps, all cacao pods turned dark brown and fourteen DAI, gray mycelia and pycnidia of Lps were observed on cacao pods. Fourteen DAI with Cos and Ctr, all fruits turned brown, and acervuli were produced on cacao pods. Untreated controls had no symptoms of pod rot and no fungi were isolated from tissue. Colletotrichum spp. and L. pseudotheobromae were reisolated from their respective diseased tissue, fulfilling Koch's postulates. Colletotrichum tropicale and C. siamense have been reported in asymptomatic cacao tissues in Panama and Australia (James et al. 2014; Rojas et al. 2010). To our knowledge, this is the first report of L. pseudotheobromae, C. tropicale and C. siamense causing pod rot of cacao.