First Report of Internal Black Rot Caused by Neoscytalidium dimidiatum on Hylocereus undatus (Pitahaya) Fruit in Israel

Abstract

Pitahaya (Hylocereus undatus [Haw.] Britton & Rose) was introduced to Israel in 1994, and is grown throughout the country. In the summer of 2009, fruit with internal black rot was collected from a field in central Israel. Symptomatic tissue from the black rot was placed on potato dextrose agar (PDA) plates amended with 12 μg/ml tetracycline and incubated at 25°C for 3 days. A dark, gray to black, fast-growing fungus was isolated from all samples (10 fruits). For identification, single-spore cultures were grown on PDA at 25°C for 5 days, and colonies with gray to black, wooly mycelium were formed. The mycelia were branched and septate (4 to 8 μm wide). The arthroconidia were dark brown, thick-walled, and one-celled, 6.3 to 14.2 × 2.0 to 4.5 μm (n = 5), and ovate to rectangular. Based on these characteristics, the fungus was identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (1). The internal transcribed spacer (ITS) region of rDNA and β-tubulin gene were amplified using ITS1 and ITS4, T121 (2), and Bt1b (3) primers, respectively, and then sequenced (GenBank Accessions KF000372 and KF020895, respectively). Both sequences were identical to sequences previously deposited in GenBank. The ITS (561 bp) and β-tubulin (488 bp) sequences exhibited 99% and 100% identity, and 100% and 84% coverage, respectively, to N. dimidiatum (JX524168 and FM211185, respectively). Thus, the results of the molecular identifications confirmed the morphological characterization. To establish fungal pathogenicity and the mechanism of infection, 60 flowers in a disease-free orchard were marked to form three different treatments (15 flowers per treatment): inoculations of the flower tube by inserting PDA plugs (0.5 × 0.5 cm) from a 5-day-old culture to the base of the flower, inoculations of the flower stigma by placing the fungus plug on intact, or pre-wounded flower stigma. The wounds were made by scratching the stigma with a sterile scalpel. For each treatment, five additional flowers were used as negative controls in which the PDA plugs did not contain any fungus. All flowers were hand-pollinated and left to grow for a month until the fruit had ripened. Only flowers inoculated by insertion of the fungus into the flower tube developed black rot in the fruit (8 of 15 fruit) 3 to 4 weeks post inoculation, suggesting involvement of the flower tube in the mechanism of infection. All other treatments and controls failed to develop any detectable disease symptoms. N. dimidiatum was reisolated from the rot, fulfilling Koch's postulates. Flowers with wounded stigma developed significantly smaller fruit. Interestingly, diseased fruit changed color about a week before ripening from the flower opening downwards, whereas healthy fruit changed color from the attachment point to the stem upwards. These results indicate that N. dimidiatum is the pathogen of pitahaya internal black rot disease. Recently, this pathogen was reported to cause brown spot disease and stem canker disease of pitahaya in China (4) and Taiwan (5), respectively. To date, the disease can be detected in all orchards in Israel, with up to 50% of the fruit being infected. Since the disease symptoms of the Israeli isolate are located in the fruit, the commercial loss due to pathogen attack is significant. To our knowledge, this is the first report of internal black rot caused by N. dimidiatum on pitahaya fruit in Israel. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylo, Evol. 7:103, 1997. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microiol. 61:1323, 1995. (4) G. B. Lan and Z. F. He. Plant Dis. 96:1702, 2012. (5) M. F. Chuang et al. Plant Dis. 96:906, 2012.