First Report of Lasiodiplodia theobromae Causing Inflorescence Blight of Mango.

Mango (Mangifera indica L.) is an important tropical fruit crop in Puerto Rico. During a disease survey from 2008 to 2010, inflorescence blight was observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz as a rotting of the rachis (main axis of the inflorescence), rachilla (lateral axis), and flowers. Diseased inflorescences from cultivars 'Haden' and 'Irwin' were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Two isolates of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. were isolated from symptomatic tissue and identified morphologically using a Botryosphaeriaceae taxonomic key (3). In APDA, colonies of L. theobromae had initial greenish gray aerial mycelia that turned dark brown with age. Pycnidia were uniloculate and dark brown to black in color. Conidiogenous cells were hyaline, cylindrical, and holoblastic. Immature conidia were subovoid to 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 26.88 µm long by 12.98 µm wide. Genomic DNA was extracted from pure cultures using a Qiagen DNeasy Plant Mini Kit. PCR amplification of three genes was used to support morphological identification. DNA analysis of the ITS1-5.8S-ITS2 region, and fragments of both ß-tubulin and elongation factor 1 alpha (EF1-α) genes were sequenced and compared using BLASTN with sequences available in GenBank. Accession numbers of gene sequences of L. theobromae from Puerto Rico submitted to GenBank were: KC631659 and KC631660 for ITS region; KC631651 and KC631652 for ß-tubulin; and KC631655 and KC631656 for EF1α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of L. theobromae reported in GenBank. Pathogenicity tests were conducted on six random healthy non-detached mango inflorescences from cultivars Haden and Irwin. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA and kept in a humid chamber using plastic bags for 8 days under field conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. For both cultivars, isolates of L. theobromae caused inflorescence (rachis, rachilla, and flowers) blight, 8 days after inoculation. Inflorescences turned brown and profuse mycelial growth was observed on the inflorescences. Untreated controls were disease-free and no fungi were reisolated from tissue. L. theobromae was reisolated from diseased inflorescences, fulfilling Koch's postulates. Fungi in the family Botryosphaeriaceae have been associated with stem-end rot, fruit rot, branch dieback, blossom blight, and cankers on mango (1,2,4). Worldwide, L. theobromae has only been reported causing dieback, stem end rot and fruit rot in mango (1,2). To our knowledge, this is the first report of L. theobromae causing inflorescence blight in mango. References: (1) N. I. Hui-Fang et al. Botanical Stud. 53:467, 2012. (2) A. M. Ismail et al. Australas. Plant Pathol. 41:649, 2012. (3) A. J. L. Phillips. Key to the various lineages in "Botryosphaeria" Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 6 August 2013. (4) B. Slippers et al. Mycologia 97:99, 2005.

First Report of Neofusicoccum parvum Causing Rachis Necrosis of Mango (Mangifera indica) in Puerto Rico.

Mango is an important tropical fruit crop in Puerto Rico that has been grown in the island for centuries. One of the major disease issues in mango production is rotting of the rachis (main axis stem of the inflorescence). During a disease survey from 2008 to 2010, rachis and flower necrosis were observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz. Diseased inflorescences from cultivars Haden and Irwin were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile, deionized, double-distilled water, and transferred to acidified potato dextrose agar (APDA). Two isolates, 91LY and K15C, of Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. were purified and identified morphologically using taxonomic keys (1,4) and DNA sequence comparisons. In APDA, colonies of N. parvum were whitish grey with aerial mycelia turning dark gray with age. Pycnidia were uni- or multilocular and dark brown to black in color. Conidiogenous cells were hyaline and holoblastic. Conidia were hyaline, ellipsoid, smooth, and one-celled with sub-obtuse apex and truncate base. Conidia (n = 50) were 16.75 µm long by 5.5 µm wide. PCR amplification of three genes was used to support morphological identification. DNA analysis of ITS1-5.8S-ITS2 region, and fragments of both ß-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and compared using BLASTn with other sequences of N. parvum submitted to the NCBI GenBank. Accession numbers of gene sequences of N. parvum submitted to GenBank were: KC631661 and KC631662 for ITS region; KC631653 and KC631654 for ß-tubulin; and KC631657 and KC631658 for EF1-α. For all genes used, sequences were 99 to 100% identical to ex-type specimen CMW9081 of N. parvum reported in GenBank. Pathogenicity tests were conducted on mango trees using six random healthy non-detached mango inflorescences for both Haden and Irwin cultivars and for both isolates. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA and kept in a humid chamber using plastic bags for 8 days under field temperature, light, and other environmental conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. For both cultivars, at 8 days after inoculation, isolates of N. parvum caused rachis necrosis ranging from 20 to 35 mm in rachis length. On cultivar Irwin, inflorescences turned brown and the necrosis was extended from the rachis to the flowers. On cultivar Haden, inflorescences turned brown and only rachis necrosis was observed. Untreated controls showed no symptoms and no fungi were reisolated from tissue. N. parvum was reisolated from diseased inflorescences, fulfilling Koch's postulates. Worldwide, N. parvum has been associated with stem-end rot, branch dieback, blossom blight, and cankers on mango (2,3). To our knowledge, this is the first report of N. parvum causing rachis necrosis on mango in Puerto Rico. References: (1) A. J. L. Phillips. Key to the various lineages in "Botryosphaeria" Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 6 August 2013. (2) G. I. Johnson et al. Ann. Appl. Biol. 120:225, 1992. (3) B. Slippers et al. Mycologia 97:99, 2005. (4) P. W. Crous et al. Stud. Mycol. 55:235, 2006.