First report of leaf spot disease caused by Alternaria alternata in Tilia miqueliana in Jiangsu Province, China.

Abstract

Tilia miqueliana Maxim., a tall deciduous tree in the Malvaceae family, is native to china and is cultivated in the Jiangsu, Zhejiang, Anhui, and Jiangxi provinces as an ornamental plant. T. miqueliana has currently received increased attention because of its value as timber, a source plant for honey, and in formal landscape architecture (Wang et al. 2022). In the last three years, symptoms of leaf spot disease were observed in T. miqueliana fields （9-year-old tree）and a breeding nursery _(1-year-old saplings) in Nanjing, Jiangsu Province, China. Field surveys showed that, the disease incidence was approximately 10% and 40% in the fields and breeding nursery, respectively. Symptoms of leaf spot disease on T. miqueliana appeared as small, circular, or near-circular/irregular black lesions on the upper surfaces of the leaves. As the disease progressed, the spots expanded into irregular shapes with the center turning yellow to black and leaves wilted from margins to centers. In severe cases, the yellow necrotic area extended to the margin, which ultimately led to leaf curling and death. To analyze the presence of the pathogenic microorganism, pieces of leaf at the junction of diseased and healthy tissue (about 3x3 mm) were taken from symptomatic plants, sterilized with 75% ethanol for 1 min and 2% sodium hypochlorite (NaClO) for 2 min, and washed 3 times with ddH2O. The leaf pieces were transferred onto Potato Dextrose Agar (PDA) plates and incubated at 28°C for 3 days in the dark. Colonies were observed and transferred to fresh PDA plates. Sixteen fungal isolates were obtained and one (named D4-2) of them was verified using Koch's assumption. Single spores of D4-2 were cultured in PDA medium, while its initial colony morphology was fluffy white colonies, which then gradually turned dark yellow from the centre to the edge. To further analyze the form of the spores, a hypha was incubated on fresh synthetic low-nutrient agar (SNA) using cellophane and inverted cultivation was performed for 20 days in the dark. Obclavate or obpyriform conidia (10 to 30 × 2 to 18 µm, n=5) were produced in chains and were pale brown to brown in color. The morphology was consistent with that of Alternaria sp.. The molecular identification of the isolated representative fungus D4-2 was conducted via the amplification of the internal transcribed spacer (ITS) (White et al. 1990),translation elongation factor 1 alpha (TEF) (Carbone and Kohn. 1999), glyceraldehyde-3-dehydrogenase (GAPDH) (Berbee et al. 1999) and RNA polymerase II beta subunit (RPB2) using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R, GPD1/GPD2, and RPB2-5F2/fRPB2-7cR (Sung et al. 2007) respectively. All these sequences were deposited in GenBank under accession numbers OP108438 (ITS), OP168372 (RPB2), OQ473880 (TEF), and OP168374 (GAPDH). Based on the GAPDH, RPB2, and TEF regions of D4-2, MEGA-11 was used to construct a neighbor-joining tree (1000 replications) with 31 other known species that had a relatively close evolutionary relationship with species from the Alternaria genus (Woudenberg et al. 2013). The phylogenetic tree indicated 100% bootstrap support between D4-2 and A. alternata. The branches showing the distribution of other species were exactly the same as that of the consensus tree based on the Bayesian 50 % majority rule that represented the Alternaria-complex (Woudenberg et al. 2013). The initial Koch's Postulates was verified using PDA slices with mycelium (pure PDA slices were used as control) that were inverted on the leaves at 25℃ for 3 days before removing slices and observing the phenotype after 7 days. Only leaves infected with D4-2 showed the same symptoms. Moreover, the spore suspension (at a concentration of 107 spores/mL) of D4-2 was applied to the leaves of T. miqueliana seedlings. After 15 days of incubation at 26 ℃ , the leaves showed the same symptoms. Subsequently, the pathogen was reisolated and cultured from these invaded leaves using the same method described above. Morphological and molecular identifications were consistent with those of D4-2. Above all, the pathogenic fungus (D4-2) isolated from T. miqueliana leaves was confirmed to be A. alternata. T. miqueliana is considered to be an endangered species due to the deep dormancy of its seeds that result in a low natural germination rate (Wu and Shen. 2021). At present, research on T. miqueliana mainly focuses on seed germination and breeding technology, and there is hardly any research on the diseases occurring in T. miqueliana. Therefore, we believe that this is the first report of leaf spot disease caused by A. alternata on T. miqueliana in China.