First Report of Leaf Black Spot on White Chrysanthemum (Chrysanthemum morifolium) Caused by Phoma bellidis in China

White chrysanthemum (Chrysanthemum morifolium) is one of the most important Chinese medicinal plants in Zhejiang province. In Tongxiang city of Zhejiang province, which produces 60% of white chrysanthemum in China, leaf black spot (LBS) disease of C. morifolium has become more and more problematic. In 2016, LBS disease incidence on C. morifolium in Tongxiang city was more than 50%. Symptoms on leaves were initially round or oval puce spots, which then coalesced into larger black lesions. A total of 47 infected leaves collected from nine different fields in Tongxiang city were surface sterilized with 1.5% sodium hypochlorite for 2.5 min, rinsed twice in sterilized water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark. Colonies of the fungus on PDA were initially yellowish and later became grayish-white. After 25 days on 3% oatmeal agar, black spherical pycnidia produced light pink cirrhi containing single-celled, colorless, long oval conidia (3.3 ± 0.3 × 1.9 ± 0.2 µm) for 35 tested isolates. These characteristics were consistent with Phoma bellidis. To confirm the identification based on morphological traits, the ribosomal internal transcribed spacers (ITS), large subunit (LSU), and tub2 of three isolates were amplified from DNA extracted from mycelium produced on PDA and analyzed (Pearce et al. 2016), respectively, with the primers ITS1/ITS4, LR0R (5′-GTACCCGCTGAACTTAAGC-3′)/LR7 (5′-TACTACCACCAAGATCT-3′) and Btub2Fd (5′-GTBCACCTYCARACCGGYCARTG-3′)/Btub4Rd (5′-CCRGAYTGRCCRAARACRAAGTTGTC-3′). The sequences were deposited in NCBI with accession numbers of ITS (MK419331, MK419332, and MK419333), LSU (MK419337, MK419338, and MK419339), and tub2 (MK572949, MK572950, and MK572951). BLAST analysis of the ITS (525-bp) segments showed 99% identity with the ITS sequence of P. bellidis isolate 346 (GenBank accession no. KM507775.1). The LSU (1,046-bp) segments showed 99% identity with that of P. bellidis isolate CBS 714.85 (GU238046.1), and the tub2 (499-bp) gene segments showed 95% identity with that of P. bellidis (KR818909.1). Results of multilocus phylogenetic analyses based on ITS, LSU, and tub2 suggested the pathogen was P. bellidis. Uninfected healthy plants of C. morifolium were sprayed either with a conidial suspension of 5 × 10⁶ conidia/ml in distilled water as the inoculum or with distilled water only to provide an uninoculated control and were incubated in pots (4 cm diameter by 6 cm) in growth chambers at 25°C under an 8-h/16-h day/night regime with 90% relative humidity for 24 h. For each isolate, 20 whole plants were inoculated. After 10 days, 24 of 25 isolates caused lesions on all inoculated plants, whereas no symptoms developed on the noninoculated controls. Cultures reisolated from lesions and cultured on PDA exhibited morphological characteristics identical to those of P. bellidis (Chen et al. 2015; Lv et al. 2011), confirming Koch’s postulates. Inoculation tests were conducted twice. To our knowledge, this is the first report of LBS disease in white chrysanthemum, which is a major factor limiting its production. Corresponding measures must be adopted to manage this disease in a timely manner.