Mango originated in the Indo-Burmese region (Alphonse de Candolle, 1885). In the Caribbean, Puerto Rico currently produces and exports mangoes to the United States and Europe. Globally, an important disease affecting mango production is dieback, caused by fungi belonging to Botryosphaeriaceae family. During a one-year survey from 2019 to 2020, conducted at the mango germplasm collection of the Agricultural Experiment Station of the University of Puerto Rico, located at Juana Díaz, PR, symptoms of dieback were observed in shoots, descending towards the woody part, and vascular necrosis. We sampled bimonthly, 35 Keitt trees for one year. At the end of the evaluation, we detected that a 74% disease incidence was caused by Botryosphaeriaceae. Lasiodiplodia mahajangana (syn. L. caatinguensis) was associated with 4% disease incidence. In addition, we identified other Botryosphaeriaceae species causing 70% of disease incidence. To identify the causal agent, sections of symptomatic tissue (4mm2) were surface disinfected by immersion in 70% ethanol, 10% sodium hypochlorite and rinsed with sterile-distilled water for 1 minute at each solution. Sections were transferred to petri dishes containing potato dextrose agar acidified with 85% lactic acid (aPDA). Ten fungal isolates were obtained with similar morphological characteristics such as colony color and texture, after 12 days. Of these, one representative (isolate 17) was selected and identified as L. mahajangana (Lm) using morphological parameters and sequences of four nuclear genes (Zhang, W. et al., 2021). In aPDA, Lm colonies showed sparse and slow-growing aerial mycelium with dark gray-greenish color at the center and light gray edges. Black pycnidia were observed after 15 days of incubation at 28°C and dark conditions. Hyaline, ovoid to ellipsoid immature conidia (n=40) with average size of 22 µm long and 12 µm wide were observed. Mature bicellular pigmented conidia (n=40) had longitudinal striate and its average size was 23 µm long and 12 µm wide. Internal transcribed spacer (ITS), β-tubulin (βtub), elongation factor 1-alpha (EF1-α) and large ribosomal subunit (LSU) genetic regions were amplified by PCR from the original and pathogenicity test recovered isolates. Sequences of PCR products were compared with NCBI database BLAST tool with other Lm sequences. Sequence accession numbers of the four genetic regions of Lm are as follows: OL375401 and OL375402 for the ITS region; OL405579 and OL405580 for β-tubulin; OL455922 and OL455923 for EF1-α; and OL375648 and OL375649 for LSU. All the sequences were grouped with the ex-type CMM1325 of Lm (BS=84). Pathogenicity tests were performed on 6-month-old mango trees of cv. Keitt. Three healthy trees were inoculated with 5 mm mycelial disks of Lm, on stems, with and without wounds. Controls were inoculated with aPDA disks only. Inoculated trees were covered for 3 days with plastic bags, keeping them in conditions of high relative humidity with constant irrigation, temperature of 28°C, and 12 hours of light and 12 hours of darkness for 12 days. Twelve days after inoculation, Lm isolates caused stem necrosis and canker, with differences in lesion severity from 2 to 17 mm2 with wound, and 0 to 6 mm2 without wound. Untreated controls showed no symptoms of canker. Lasiodiplodia mahajangana was re-isolated from diseased stems fulfilling Koch's postulates, and a sequence of the recovered isolate from the pathogenicity test was compared and included in the phylogenetic analysis. Lasiodiplodia mahajangana has been reported to cause stem-end rot of mango in Malaysia (Li, L. et. al., 2021). To our knowledge, this is the first report of Lm causing canker of mango in Puerto Rico. Knowing L. mahajangana as a new pathogen that causes canker of mango is important to establish an adequate and effective control management of this disease in mango producing countries worldwide.
In Puerto Rico, the agricultural production of pineapple (Ananas comosus (L.) Merr.) comprises nearly 5,000 tons harvested annually from over 250 ha (USDA 2018). With an annual income of approximately $3 million USD, pineapple ranks fourth in importance among Puerto Rican crops (USDA 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 (SEA 2015), because this cultivar produces fruit more than once during a single growing season. In August 2018 (when the rainy season normally starts in Puerto Rico), soft rot symptoms appeared at commercial fields in Manatí (WGS 84 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 ranged from 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 (5mm2) were placed in nutrient agar. Bacterial colony-forming units (CFU) 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 one-and-a-half-year-old pineapple seedlings in humid chambers. Bacteria were grown on sterile nutrient agar for 3 days at 25 ± 2°C. Inoculation assays (three replications) were performed using 1X108 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 observed in the field. A partial DNA sequence of the 16S rRNA gene of the bacterial isolate and the re-isolated 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 database of the National Center for Biotechnology Information (NCBI). This partial 16S rRNA sequence of the bacterial isolate was deposited in GenBank® at NCBI (Accession no. 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 (GenBank accession nos. 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) resolved the Puerto Rican isolate as belonging to a clade broadly ascribable to D. zeae, most closely related to strains isolated from earlier Hawaiian pineapple bacterial heart rot outbreaks. Dickeya 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; Sueno et al. 2014; Ramachandran et al. 2015). D. zeae had not previously been reported as causing bacterial heart rot in pineapples in Puerto Rico 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.