First Report of Branch Decay Caused by Diaporthe nobilis on Punica granatum ‘Taishanhong’

Punica granatum (Punicaceae, Myrtales, Rosidae) is a perennial deciduous fruit tree that originates from the Central Asia. It is widely cultivated in the entire tropical, subtropical, and warm regions on account of its edible, medicinal, and ornamental value. In June 2017, branch decay on pomegranate was observed on Taishanhong pomegranate cultivar in Zaozhuang, Shandong, China. The percentage of symptomatic plants was 5 to 12% in the investigated four orchards. The diseased epidermises on branch became dry and showed puce with lots of dark spots. Occasionally, the rotten tissue was covered with fungal mycelia in a humid environment. Soon afterward, rifts appeared between the healthy and diseased tissues, and then diseased epidermises fell off with their fracture and embossing. Finally, the upper branch of infected parts died. Eight single-spore isolates were obtained from three different branches by tissue isolation on potato dextrose agar medium with 0.02% streptomycin sulfate. The growth rate was 10.8 mm/12 h at 25 and 28°C. The black pycnidia were obtained after inoculation for 14 days at 25°C under a 12-h photoperiod. In pycnidia, two types of single-cell conidia were observed. Alpha conidia were oval to spherical, two-guttulate, 5.8 to 7.6 × 3.3 to 4.2 µm; and beta conidia were filiform, straight but sometimes with a curved hook, 20.5 to 28.1 × 0.7 to 1.1 µm. The molecular identification was carried out by three-locus sequence analysis including internal transcribed spacer (ITS), beta-tubulin gene (TUB2), and translation elongation factor 1-alpha (TEF1). Three sequences (MG736062, MG736064, and MD736063) obtained were deposited in GenBank after polymerase chain reaction amplification and sequencing. The BLAST searches showed that they were identical with 99% (KJ609011), 98% (KX016113), and 99% (KJ623307) for the ITS, TUB2, and TEF1 of Diaporthe nobilis, respectively. Based on the morphological and molecular data, the fungus was identified as D. nobilis (Li et al. 2017). The pathogenicity tests proceeded on three 5-year-old pomegranate plants with four branches per plant according to Koch’s postulates. After spraying the spore suspension (10⁵ conidia/ml), the branches of the pomegranate seedlings were covered by wet gauze. The seedlings were moved to a stainless steel pallet, covered with polyethylene film, and incubated at 25°C in an illumination incubator under a 12-h photoperiod. All branches were sprayed with the sterile distilled water every day. The diseased symptoms on the inoculated branches were similar to those observed in the pomegranate orchard after 20 to 30 days of inoculation. Water-inoculated controls showed no symptoms. The pathogen was only reisolated from the inoculated pomegranate branches. D. nobilis has been reported on pepper (Zhang et al. 2016) and tea (Li et al. 2017) in China. To our knowledge, this is the first report of D. nobilis causing branch rot on pomegranate in China or in the world.