First Report of Leaf Spot Disease Caused by Colletotrichum Siamense on Turmeric in India

Turmeric (Curcuma longa) is a valuable crop plant, being used as a spice and having various pharmacological properties. A leaf spot disease was recorded on C. longa plants in the experimental field of the Department of Botany, University of Lucknow (26.858773 N 80.934664 E), Lucknow, India from September to November, 2022. Symptoms were observed in the form of small spots over the upper surface of leaves. Gradually, leaves become yellowish with brown irregular patches (Figs. 1, 2). One hundred and twenty plants were surveyed and the disease incidence was 80%. Thirty diseased leaves were collected from fifteen plants and washed in running tap water to remove soil and associated debris. Samples were surface sterilised with 70% ethanol and 0.1% sodium hypochlorite for one minute each. After that the samples were washed twice with distilled water and air dried. Leaf pieces (5 × 5 mm) were transferred onto potato dextrose agar (PDA) plates and incubated at 25±2°C. Pure cultures were obtained by transferring hyphal tips to fresh PDA plates. The colony was whitish to greyish in colour with cottony mycelia. Conidia were single celled, straight, cylindrical, obtuse ends (oblong) and measured 12.0- 15.12 × 4.2-5.9 μm (n = 15) in size (Fig. 3). Morphological characteristics were consistent with Colletotrichum siamense as described by Prihastuti et al. (2009) and Prasad et al. (2017). The internal transcribed spacer (ITS) region and glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene from the genomic DNA were amplified using the ITS1/ITS4 (White et al., 1990) and GDF/GDR (Weir et al. 2012) primer pairs, respectively. Sequences of the amplified region were submitted to Genbank with Accession Nos OQ086954 and OQ991912 for ITS and the GAPDH gene, respectively. A phylogenetic tree was constructed using concatenated sequences of ITS and the GAPDH gene using MEGA11 software (Tamura et al., 2021). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1) and isolate CRPL-1 clustered with C. siamense (type strain ICMP:18578) (Fig. 4). A pathogenicity testing was conducted in triplicate using two-month-old plants grown in pots (Fig. 5). Three healthy leaves of each plant were inoculated with 10 μl of a conidial suspension (1×106 conidia/ml) from the isolated culture or with distilled water as a control. All pots were covered with a sterile polythene bag for two days to keep the environment humid and were then transferred to a glasshouse. Leaves inoculated with the conidial suspension showed similar symptoms after fifteen days to those observed in the field, while control leaves remained healthy. Colletotrichum siamense was reisolated from the symptomatic parts of the inoculated leaves and no fungus was isolated from the healthy leaves, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. siamense causing a leaf spot disease on C. longa in India and globally. This report will enable proper management strategies to be developed to control leaf spot diseases of C. longa. The authors would like to thank the Council of Scientific and Industrial Research- (National Chemical Laboratory, Pune, India for providing ITS sequencing services.