First report of Lasiodiplodia mahajangana causing canker of mango (Mangifera indica) in Puerto Rico

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.