First Report of Diaporthe eres Leaf Spot on Photinia × fraseri ‘Red Robin’ in Qingdao, China

Abstract

Photinia × fraseri ‘Red Robin’ is a hybrid between P. glabra and P. serrulata. Owing to its flower-like red leaves and its ability to tolerate stressful environments, Red Robin is widely cultured as an ornamental shrub in China. It is becoming more widely planted owing to its economic and ecological value (Zhu and Wei 2010). In March 2018, on a street in Qingdao, China (119°30′∼121°00′ E, 35°35′∼37°09′ N), approximately 10% of Red Robin leaves were observed to have leaf spots. Approximately four shrubs were affected in this planting. The spots were brown, oval to irregular, with or without yellow haloes, and half of the spots had perforations. Small pieces of tissue taken from the edge of leaf spots were surface sterilized in 70% alcohol for 1 min, rinsed in sterile distilled water once, and then placed on potato dextrose agar (PDA). Eight out of 10 strains isolated from infected leaves were morphologically identical, whereas the other two strains were Alternaria sp. Thus, two strains were taken from the eight morphologically identical isolates for further identification. After 5 days of incubation at 25°C in the dark, fungal colonies showed white, aerial mycelium with dark pigmentation developing in the center. On PDA, pycnidia formed within black stomata after 7 to 10 days incubation, and they oozed yellowish or cream-white liquid drops. Alpha conidia were 4.8 to 7.4 × 1.6 to 2.8 μm (mean 6.2 × 2.3 μm, n = 100), unicellular, fusiform, elongated, and mostly biguttulate. Beta conidia were 18.9 to 23.1 × 0.9 to 1.4 μm (mean 20.8 × 1.2 μm, n = 100), hyaline, unicellular, filiform, and curved or hamate. Colony morphology and conidial characteristics were consistent with those of Diaporthe sp. The internal transcribed spacer (ITS) region of rDNA, β-tubulin, and a histone gene were amplified from DNA extracted from two single-spore isolates using the primers ITS1/ITS4, BT2a/BT2b, and HIS1F/HISR (GenBank accession nos. MH204101, MH230806, and MH230807) (Gao et al. 2017; Gomes et al. 2013). These sequences had 99% nucleotide identity with the corresponding sequences (KY569368.1 and KX866914.1; KJ420785.1 and KJ420822.1; and KY569367.1 and KC343564.1, respectively) of Diaporthe eres in GenBank. Single-gene phylogenetic analysis of rDNA, β-tubulin, and a histone gene was performed using the neighbor-joining method with 1,000 bootstrap replications and a p-distance substitution model. Based on the morphological and molecular data, the two isolates were identified as D. eres. Pathogenicity tests were performed using a conidial suspension (10⁷ conidia/ml) in sterile distilled water. Twenty microliters of the conidial suspension was placed on the right side of detached, prewounded Red Robin leaves (15 per isolate), and 20 μl of sterile distilled water was placed on the left side as a control treatment. Inoculated leaves were incubated at 25°C and 90% relative humidity with a 12-h photoperiod per day for 7 days and then 25°C and 20% relative humidity for 1 day. After 8 days, D. eres caused leaf spots similar to those previously observed on infected leaves in the field, whereas the control did not. The fungus was reisolated from the symptomatic tissues, fulfilling Koch’s postulates. To our knowledge, this is the first report of D. eres causing leaf spots on P. × fraseri in the world. Leaf spots caused by D. eres on P. × fraseri would greatly affect its value; understanding the cause of disease is crucial to disease management.