First report of leaf spot disease of Tabernaemontana divaricata caused by Colletotrichum coffeanum in India
Year: 2023
Type: article
Abstract: Tabernaemontana divaricata (crape jasmine, pinwheel flower) is an ornamental, flowering, evergreen shrub in the family Apocynaceae (Jena et al., 2019). It is native to India and other tropical regions in Asia. The different parts of the plant are used in traditional medicine for a range of pharmacological activities. In September 2023, black spot-like symptoms were observed on the leaves of T. divaricata near Gauhati University (26°9'14.8“N, 91°39'38.1”E), Guwahati Assam, India. At the early stage of infection, disease symptoms appeared as small, circular and yellow-halo spots and developed into angular brown spots in the later stages of infection. As the disease progressed, the leaf tissues around the spots became yellow and the lesions enlarged. Gradually adjacent lesions merged, became necrotic and shot-hole symptoms appeared (Figure 1). Twenty diseased leaves were collected from ten plants and washed in running tap water to remove soil and associated debris. The samples were surface sterilised using 70% ethanol solution, followed by 0.2% NaOCl solution, for one minute each, and were subsequently rinsed three times with distilled water (Omar et al., 2018). The surface-sterilised infected leaves were cut into small fragments (5 × 5 mm2) using a sterile scalpel. Each fragment was transferred onto potato dextrose agar (PDA) plates and incubated at 28±2°C for seven days. Hyphal tips growing from the fragments were sub-cultured on PDA. Pure cultures were produced by transferring the tips of fungal hyphae onto PDA plates. Colonies were cottony, white to pale orange, whereas the reverse side formed a pale orange to a central salmon colour with white patches towards the margin (Figure 2). Although acervuli were not observed, conidia were produced from hyphae on simple, short, hyaline conidiophores, measuring 22.5−31.5 × 1.0−1.8 μm in size. Conidia were unicellular, hyaline, non-septate, smooth-walled, cylindrical sometimes slightly ovoid, with rounded ends and measured 12.0−18.75 × 2.5−4.8 μm (n = 20) (Figure 3). Appressoria were dark-brown, irregular in size, ranging from 9.5−12.0 × 5.5−6.8 μm (n = 10), subglobose or elliptical, simple with an appressorial pore. Morphological characteristics were consistent with Colletotrichum sp. as described by Cannon et al. (2012). Molecular characterisation was done using internal transcribed spacer (ITS) rDNA sequence analysis. Genomic DNA was extracted and the ITS region was amplified and sequenced using ITS1/ITS4 primers (White et al., 1990). The consensus sequence obtained was submitted to Genbank (Accession No. OR633330). BLASTn analysis showed that the sequence had closest identity (99.14%) with Colletotrichum coffeanum isolate MEBP0133 (MT580975.1). A phylogenetic tree was constructed using the maximum likelihood method and the Kimura 2-parameter model using MEGA11 software (Tamura et al., 2021). The phylogenetic tree showed that isolate TDCK1 clustered with C. coffeanum (Figure 4). Pathogenicity assays were performed on detached leaves with petioles. Healthy leaves were surface sterilised with 70% ethanol, washed with sterile distilled water three times, and dried using sterilised filter paper. Wounds were made (c. 1 cm long) on the surface of the leaves using a sterile scalpel and 20 μL of a conidial suspension (1×107 conidia/ml) was placed on wounded and unwounded leaf surfaces using a micropipette. Sterile distilled water was used as a control (Omar et al., 2018). Leaves were kept in a sterile Petri plate on sterile tissue paper soaked in sterile distilled water to maintain relative humidity and then incubated at 28±2°C for three days. The experiment was repeated in triplicate. After six to eight days, leaves inoculated with conidial suspensions showed symptoms on both unwounded and wounded leaves confirming isolate TDCK 1 as a pathogen (Figure 5). The pathogen was re-isolated from the symptomatic parts of the inoculated leaves and had similar morphological characteristics to those described above fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. coffeanum causing leaf spot disease on T. divaricata in India and globally. This report will enable proper management strategies to be developed to control leaf spot disease on this host. The authors are grateful to the Head, Department of Botany, Gauhati University for providing necessary facilities to conduct this work.
Source: New Disease Reports
DOI: 10.1002/ndr2.12232
Institution: Gauhati University
Language: English
Subfield: Plant Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: diamond
OpenAlex ID: W4388909017