Dragon fruit or pitahaya (Hylocereus spp.) is a tropical fruit belonging to the Cactaceae. It is native to Central and South America and commercially grown in the United States in southern California, south Florida and Puerto Rico. During a disease survey from April to June 2020, stem canker was observed in greenhouses and commercial orchards located in Mayaguez and San Sebastian, Puerto Rico with an incidence of 80%. Diseased cladodes (stems) of 1 mm2 tissue sections of 23 pitahaya varieties (NOI-13, NOI-14, NOI-16, N97-15, N97-17, N97-18, N97-20, N97-22, American Beauty, Cosmic Charlie, Halley's comet, Purple Haze, Alice, Bloody Mary, Dark Star, David Bowie, Delight, Makisupa, Red Jaina, Soul Kitchen, Vietnamese Jaina, Neitzel and Lisa) were disinfested with 70% ethanol, rinsed with double distilled water and plated on potato dextrose agar (PDA) amended with 60 mg/L streptomycin. Three isolates (17B-173-T3, 12C-118-T1 and 13B-131-T2) of Neoscytalidium dimidiatum (syn. N. hyalinum) were identified using taxonomic keys (Crous et al., 2006) and sequencing of the internal transcribed spacer (ITS) with primers ITS5 and ITS4 (White et al. 1990) and translation elongation factor 1 alpha (TEF1-α) with primers EF1-728F and EF1-986R (Carbone and Kohn, 1999). Sequences were compared using the BLASTn tool with N. dimidiatum deposited in NCBI GenBank. In PDA, colonies of N. dimidiatum were initially powdery white and turned grayish-black with age. Arthroconidia (n=50) were dark brown, disarticulating, truncate or cylindrical at the base, thick-walled with 0 to 1 septum, averaging 9.1 X 5.5um in length. GenBank accession numbers of N. dimidiatum DNA sequences were MT921260, MT921261 and MT921262 for ITS and MT920898, MT920899 and MT920900 for TEF1-α. Sequences were 99-100% identical with Ex-isotype CBS145.78 accession numbers KF531816 for ITS and KF531795 for TEF1-α. Pathogenicity tests were conducted on 12 healthy dragon fruit plants of 1.5 years old using three non-detached cladodes per plant. Cladodes were inoculated with 5mm mycelial plugs from 8-day-old pure cultures grown on PDA. Three healthy dragon fruit plants were used as controls and were inoculated with PDA plugs only. The experiment was repeated once. Twenty days after inoculations (DAI), isolates of N. dimidiatum caused stem canker on dragon fruit plants. For all isolates, sunken orange spots averaged 3 X 2 mm in length at 8 DAI. Necrotic blotches with chlorotic halos averaged 10 X 15 mm at 14 DAI; stem cankers with water-soaked tissue were observed at 20 DAI, and arthroconidia and black pycnidia on dry stem cankers at 30 DAI. Untreated controls had no symptoms of stem canker, and no fungi were isolated from tissue. Neoscytalidium dimidiatum has been reported to cause stem canker on Hylocereus spp. in China, Florida, Israel, Malaysia and Taiwan (Chuang et al. 2012; Lan et al., 2012; Ezra et al., 2013; Sanahuja et al., 2016). To our knowledge, this is the first report of N. dimidiatum causing stem canker on dragon fruit in Puerto Rico. References: 1. Carbone, I., and Kohn, L. 1999. Mycologia, 91:553. doi:10.2307/3761358 2. Chuang, M. F. et al. 2012. Plant Disease 96: 906. https://doi.org/10.1094/PDIS-08-11-0689-PDN. 3. Crous, P. W., et al. 2006. Stud. Mycol. 55:235. https://doi.org/10.3114/sim.55.1.235 4. Ezra et al. 2013. Plant Disease 97: 1513. https://doi.org/10.1094/PDIS-05-13-0535-PDN 5. Lan, G.B. et al. 2012. Plant Disease 96: 1702. https://doi.org/10.1094/PDIS-07-12-0632-PDN 6. Sanahuja et al. 2016. Plant Disease 100: 1499. https://doi.org/10.1094/PDIS-11-15-1319-PDN 7. White, T., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.
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.