First Report of Bacterial Pineapple Heart Rot Caused by Dickeya zeae in Puerto Rico

In Puerto Rico (PR), the agricultural production of pineapple (Ananas comosus [L.] Merr.) comprises nearly 5,000 t harvested annually from over 250 ha (USDA NASS 2018). With an annual income of ∼US$3 million, pineapple ranks fourth in importance among Puerto Rican crops (USDA NASS 2018). Recently, the pineapple industry on the island underwent a change from growing a local cultivar known as Cabezona to cultivar MD2, introduced from Hawaii around 1996 (Servicio de Extension Agricola 2015), because this cultivar produces fruit more than once during a single growing season. In August 2018 (when the rainy season normally starts in PR), soft rot symptoms appeared at commercial fields in Manatí (WGS84, lat 18.42694, lng −66.44779) and persisted through 2019. Symptoms observed in the field included foliar water-soaked lesions with gas-filled blisters, especially at the base of the leaf. Leaves exhibited brown discoloration and a fetid odor (rot) at the basal portion of the plant. Finally, leaves collapsed at the center of the pineapple crown, effectively killing the apex and preventing the fruit from developing. Disease incidence was 25 to 40% depending on the weather and season; when there was more rain, there was higher disease incidence. Symptomatic leaves were collected in February 2019, disinfected with 70% ethanol, and rinsed with sterile distilled water. Tissue sections (5 mm²) were placed in nutrient agar (NA). Bacterial colony-forming units were a translucent cream color, circular, with a flat convex surface and wavy edge. Biochemical analysis showed that bacteria were gram-negative, oxidase positive, catalase positive, and facultatively anaerobic. Pathogenicity was tested on leaves of 1.5-year-old pineapple seedlings in humid chambers. Bacteria were grown on sterile NA for 3 days at 25 ± 2°C. Inoculation assays (three replications) were performed using 1 × 10⁸ CFU/ml of bacteria suspended in sterile water and applied with a cotton swab to leaves wounded with a needle. The inoculated tissue was incubated at 28°C and kept in a dark environment. Negative controls were inoculated with sterile water. Five days after inoculation, foliar water-soaked lesions were observed, followed by the formation of brown leaf tissue and gas-filled blisters, the same symptoms as in the field. A partial DNA sequence of the 16S rRNA gene of the bacterial isolate and the reisolated bacteria were amplified using primers 27F and 1492R (Lane et al. 1985) and sequenced. The isolate was determined to the genus Dickeya through a BLAST search against sequences available in the NCBI database. This partial 16S rRNA sequence of the bacterial isolate was deposited in GenBank (MT672704). To determine the identity of the Dickeya species, we sequenced the genes dnaA, gyrB, dnaX, and recN (Marrero et al. 2013) for the bacterial isolate (OM276852, OM276853, OM276854, and OM276855) and conducted a multilocus sequence analysis including reference Dickeya sequences of Marrero et al. (2013). The phylogenetic analysis (using WinClada [Nixon 2002]) resolved the Puerto Rican isolate as belonging to a clade broadly ascribable to Dickeya zeae, most closely related to strains isolated from earlier Hawaiian pineapple bacterial heart rot outbreaks. D. zeae was responsible for bacterial heart rot of pineapple in Malaysia and was later reported as the causal agent for outbreaks in Costa Rica and Hawaii (Kaneshiro et al. 2008; Ramachandran et al. 2015; Sueno et al. 2014). D. zeae had not previously been reported as causing bacterial heart rot in pineapples in PR, and this study points to a close relationship with strains first detected in Hawaii and which should further be explored to determine the precise nature of this relationship. This information should facilitate the adoption of effective control measures for this disease on the island, promote more effective methods of preventing future introductions of pathogens, and encourage further investigations into the occurrence of D. zeae on the island.