The First Report of Lasiodiplodia theobromae Causing Leaf Spot on Illicium verum in China

Abstract

Star anise (Illicium verum) production is an important industry in parts of southern China (Wang et al. 2011). The production of star anise (cultivar Dahong) was seriously affected by a leaf spot disease during the summer of 2020 in Rong County, Guangxi province, China. Approximately 20% to 30% of the trees (n = 200, 15-16 years old) had obvious leaf spots on more than 80% of the leaves. Local growers had to cut down the seriously diseased trees. Symptomatic samples were collected and sent to us at the end of August 2021. A single brown-edged round gray spot appeared on each leaf. The spot was ~20 mm in diameter. The margin was cut into 5 mm pieces, then disinfested with 3% NaOCl for 30 s, 75% ethanol for 30 s and sterile deionized water for 1 min before dried and placed on potato dextrose agar (PDA) medium at 25°C in the dark. A total of 25 fungal isolates were obtained (isolation rate 82%). Genomic DNAs was extracted from the mycelia of these isolates and three diagnostic regions including ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995) were amplified. The colony morphology on PDA and the sequences of the five isolates BJ20-1, BJ20-4, BJ20-5, BJ20-7, BJ20-8 were identical. Fungal colonies had light gray mycelium and black pigment on PDA. The average colony growth rate was 4.25±0.31 cm per day and no spores were produced. Sequences of the representative isolate BJ20-1 were deposited in GenBank (Accession nos. OK483326, OL547596 and OL547597). BLASTn search indicated high identity 99.6%, 98.58% and 100% to ITS (AY640255), EF1-α (AY640258), and β-tubulin (KU887532) of Lasiodiplodia theobromae, respectively. Combined phylogenetic analysis using MEGAX (Kumar et al. 2018) clustered BJ20-1 and L. theobromae CBS164.96 in one clade. To test pathogenicity, 2-years-old healthy I. verum trees (cultivar Dahong) maintained in a greenhouse were inoculated. Leaves were surface sterilized with 70% ethanol. One PDA plug (5mm in diameter) was placed on each wound acupunctured with a sterile needle pick. Ten PDA plugs with mycelial of BJ20-1 growing 7 days on it were inoculated on five leaves. Four sterile PDA plugs placed on two leaves served as controls. All the plugs were removed from the leaves the day after inoculation. The experiment was repeated three times. At 1 day post-inoculation (DPI), brown expanding lesions were observed on the inoculated leaves. At 7 DPI, a mature ellipse of necrosis formed with 18±4 mm in diameter with black pycnidia in the center. Conidia were observed in the pycnidia. The immature conidia were thick-walled, hyaline, aseptate and ellipsoid, measuring 20 - 25.2 × 11 - 13 μm (n = 25). The mature conidia were dark brown with one central septum, measuring 24.3 - 27 × 13 - 14 μm (n = 25). At 10 DPI, the control leaves remained asymptomatic. Re-isolation was successful from the spot on the inoculated leaves. The colony morphology and molecular identification of the re-isolations were all the same as that of BJ20-1. In conclusion, the morphological and molecular evidence consistently indicated these isolates were L. theobromae. Koch’s postulates were fulfilled that L. theobromae was pathogenic on star anise. Although L. theobromae has been reported to cause leaf spot disease on Camellia sinensis (Bao et al, 2021), Kadsura longipedunculata (Fan et al, 2020) and Broussonetia papyrifera (Luo et al, 2020), etc., this is the first report of L. theobromae causing leaf spot on I. verum in China. Due to the leaf spot disease resulting in serious yield reduction on star anise, accurate pathogen identification in this study would significantly improve the control of the leaf spot disease on star anise.