Leaf Spot Disease of Michelia alba Caused by Alternaria alternata in China

The perennial deciduous tree Michelia alba is a widely cultivated street plant in China. In June 2021 and March 2022, M. alba trees with leaf spots were found in the green belt of the approximately 200,000 m2 community (32.62°N, 116.98°E) of Tianjia'an District, Huainan, Anhui, China, where approximately half of the M. alba trees had brown leaf spots surrounded with irregular yellow halos ranging from 2 to 6 mm in diameter (Fig S1A). The leaves of M. alba trees with multiple lesions became blighted. To isolate the potential pathogens causing leaf spot symptoms in M. alba trees, twenty fragments (2 cm2) were excised from the margin of the necrosis on symptomatic leaves, immersed in 1% sodium hypochlorite for 45 s, and then washed three times in sterile water. The fragments were plated and incubated on potato dextrose agar (PDA) at 25 °C and 15 dark green fungal colonies were obtained 5 days later. Single-spore isolates of the fungal colonies plated on potato carrot agar (PCA, Simmons 2007) produced gray, floccose colonies, which reached 71 mm after 7 days at 25 °C were obtained 5 days later (Fig S1C). Optical microscopy analysis showed that single-spore isolates formed sparsely branched chains with pale brown conidiophores on PCA after incubation at 25 °C in darkness for 7 days. The conidia were ellipsoidal, inverted rod, or ovoid, light brown, and 10.0 to 52.5 × 4.5 to 22.7 μm, with zero to four longitudinal or oblique and zero to eight transverse septa (n = 50). Partial conidia are 2.5 to 27.5 × 0.6 to 3.7 μm with cylindrical light brown beaks (n = 50) (Fig S1D, E). The cultural and morphological characteristics of the isolated fungi were consistent with the description of Alternaria alternata (Woudenberg et al. 2015). To further characterize the isolated fungi, the genomic DNA of three representative strains (BYL-1, BYL-2 and BYL-3) were extracted from their mycelia, respectively. ITS region and housekeeping genes GPD, and TEF, were amplified and sequenced using ITS4/ITS5 (White et al. 1990), Gpd1/Gpd2 (Berbee et al. 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), primer pairs, respectively. BLAST analysis showed that the isolates BYL-1 (GenBank accession nos. OP325693, OP405008, and OP405009), BYL-2 (GenBank accession nos. PP057859, PP138442, and PP138444), and BYL-3 (GenBank accession nos. PP057860, PP138443, and PP138445) shared 99 to 100% identity with Alternaria alternata (GenBank accession nos. AF347032.1, AY278809.1, KC584693.1), which suggested that all the three isolates belong to A. alternata. The identifications were further confirmed by phylogenetic analysis based on combined DNA sequences data of ITS, GPD, and TEF. As showed in Fig S2, the strains of BYL-1 , BYL-2 and BYL-3 formed a robust clade with A. alternata CBS918.96. Taken together, the morphology and molecular assays suggest that strain BYL-1 is A. alternata. To test pathogenicity, the isolate BYL-1 was cultured on PCA for 7 days to prepare conidial suspensions, and the spore concentration was adjust to a final concentration of 105 spores/ml. The leaves of 3-5-leaf stage of six 5-years-old natural planting M. alba plants were sprayed with conidial suspensions and sterile distilled water, respectively. The petiole of each inoculated leaves of M. alba were secured with sterile wet cotton, and covered with plastic bags to prevent moisture evaporation after incubation. After a 3- to 5- day of inoculation, necrotic lesions appeared on the leaves inoculated with conidial suspensions, whereas no necrotic lesion was observed in the control leaves inoculated with sterile distilled water (Fig S1B). To fulfill the Koch,s postulates, fungi were re-isolated from the margin of necrotic lesions and identified as A. alternata by DNA sequencing the ITS gene. To our knowledge, this is the first report of A. alternata causing leaf spot on M. alba. Because the disease could cause damage to the foliage influencing the greening and ornamental effects of these trees, control measures may need to be implemented during daily management.