First report of Didymella glomerata causing leaf spot on Lonicera caerulea L. in Heilongjiang province, China.

Abstract

China has the largest blue honeysuckle (Lonicera caerulea L.) cultivation area globally. In June 2022, leaf spots were observed on approximately 10% of blue honeysuckle (cv. 'Lanjingling') leaves in a 0.03-ha field in Harbin (127.66°E, 45.61°N), Heilongjiang Province, China. The leaves of the affected plants displayed chlorotic to tan dieback with a darker brown margin along the leaftip and leave margins. Cross-sectional segments of approximately 3 mm were cut from 50 typical infected plant leaves. Their surfaces were sterilized with 75% ethanol for 30 s followed by 3 min in 5% sodium hypochlorite (NaOCl), rinsed three times with sterile water, and transferred to 9-cm Petri dishes containing 15 ml of sterile PDA growth medium. Five purified cultures with similar culture characteristics were finally obtained and their colonies were dark brown on the PDA plates. The pycnidia were subglobular and deep black and measured avg. 215.48 (135.30-331.20) μm × avg. 170.28 (99.90-282.90) μm (n = 50) (Chen et al., 2015; Huang et al., 2018). Conidia were single-celled, hyaline, and ellipsoidal and measured avg. 6.22 (5.40-7.20) µm × avg. 3.42 (2.70-3.90) µm (n = 50). For molecular verification, genomic DNA was extracted from a representative isolate, LD-75. The internal transcribed spacer region (ITS), the second-largest subunit of RNA polymerase II (rpb2), the partial 28S large subunit rDNA (LSU), beta-tubulin (TUB), and actin (ACT) genes were amplified with the primers ITS1/ITS4, RPB2f/RPB2r, LROR/LR7, TUB2Fd/TUB4Rd, and ACT512f/ACT783R, respectively (White et al. 1990; Carbone and Kohn, 1999; Staats et al., 2005; de Gruyter et al., 2009; Chen et al., 2015). BLAST results indicated that the genes of LD-75 (GenBank OP218870, OP264863, OQ561448, OQ597233, and OQ597232) shared 99%-100% identity with those of Didymella glomerata (OK485138, GU371781, EU754185, MZ073910, and MW963190, respectively). Therefore, based on morphological characteristics and molecular phylogeny, LD-75 was identified as D. glomerata. Six two-year-old healthy plants from the 'Lanjingling' cultivar were selected for a pathogenicity test. The leaves were surface disinfested with 75% ethanol and then wiped with sterilized water three times. All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle. Whole plants sprayed with conidial suspension of isolate LD-75 (106 spores/mL) (n = 3) displayed leaf spot symptoms after 14 d, while no symptoms were detected on whole plants sprayed with sterile water (n = 3). The same isolate, reisolated from infected leaves and with the same morphological and molecular traits, was also identified as D. glomerata, confirming Koch's postulate. The fungus was previously reported in Cornus officinalis in Nanyang City, China (Huang et al., 2018). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by D. glomerata in China. Reducing blue honeysuckle production losses caused by leaf spots is crucial for growers, and we hope that researchers will develop efficient control strategies for managing this emerging plant disease.