Phoma herbarum Causing Leaf Spot of Largehead Atractylodes in Central China

Abstract

Largehead Atractylodes (LA) is a perennial herbaceous plant (family Asteraceae) native to China. The rhizome of LA is the most commonly used Chinese herbal medicine to prevent early pregnancy loss due to miscarriage (You et al. 2013). From 2014 to spring 2017, a foliar disease with symptoms of leaf was observed on LA in a Huazhong medicinal plant garden, Enshi, Hubei Province, China. At temperatures between 23 and 28°C, 85% of plants were infected. The first symptoms of the disease appeared as round or irregular dark brown spots 1 to 4.5 mm in diameter with a yellow halo at the edge, which appeared on the leaves at an early stage of infection. The spots were brown to gray in the center and dark brown; in the end, scattered black specks appeared in the center of the spot. Severely infected leaves became chlorotic and abscised. Stems and flowers were not affected by the disease. Five isolates were obtained from symptomatic leaf samples after they were surface sterilized with 0.1% mercuric chloride solution and plated onto potato dextrose agar at 22°C for 1 week. All the isolates were similar in cultural and morphological characteristics, and one isolate (BZYB-1) was selected for further characterization. The isolate produced green to gray colonies with sparse aerial hyphae, and the reverse appeared brown to brown red in color. Black pycnidia were globose to elongate, 55.3 to 66.7 × 125.5 to 137.4 μm (n = 50) in diameter. Conidia were hyaline, elliptical, and smooth, 4.3 to 7.2 × 1.5 to 3.6 μm (n = 50). These characteristics are consistent with Phoma herbarum (Boerema et al. 2004), which was further confirmed by sequencing analysis of the internal transcribed spacer (ITS) region of rDNA. Primers ITS1 and ITS4 were used for polymerase chain reaction (PCR) (White et al. 1990), and the ITS rDNA sequence was deposited in GenBank (accession no. KY780194). BLAST analysis of the PCR product showed 99.9% identity to that of P. herbarum (KY849801) (Zhang et al. 2017). The pathogen was further identified to the species level as P. herbarum using gene sequences from 18S ribosomal RNA (MG757513) (Faisal et al. 2007). Pathogenicity was tested by spraying the foliage of 30 1-year-old plants with a suspension of 10⁶ conidia/ml in sterile distilled water. Each plant received 30 ml of the inoculum. Ten healthy potted plants were inoculated with sterilized water as a control. All plants were covered with plastic bags for 5 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 22°C. The first foliar lesions developed on leaves 7 days after inoculation and were similar to those observed in the field. No symptoms developed on the control plants. The pathogenicity test was completed twice. P. herbarum was successfully reisolated from all artificially inoculated plants and confirmed by morphological characters, which completed Koch’s postulates. The fungus was identified as P. herbarum based on both morphological and molecular characteristics. To our knowledge, this is the first report of P. herbarum causing leaf spots and lesions on LA in China. Given the recent tremendous expansion of cultivation area for this herb in China, the economic importance of this disease is likely to increase with the greater prevalence of this host species.