First Report of Leaf Spot Caused by Phyllosticta capitalensis on Illiciumdifengpi in China.

Abstract

Illicium difengpi B. N. Changet al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initialleafsymptoms were roundspotswith gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomaticleaveswere collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affectedleaves.Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n= 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n= 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllostictacapitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 forITS, OL539429 forACT, OL539430 fortef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the correspondingITS(MT649668),ACT(MN958710), tef1-α(MN958711) andGPDH (KU716077) sequences ofP. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACTby the maximum likelihood method showed that isolate GX-2 clustered together withP. capitalensis.The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 μL spore suspension (1 × 106spores/ml). Another four sterilized leaves were inoculated with 20 μL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy.P. capitalensiswas successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensiscausedleafspotson various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report ofP. capitalensiscausingleafspoton I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology ofthis disease should be investigated in further research.