First Report of Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde causing anthracnose disease in Santalum album Linn. in India.

Abstract

Indian sandalwood (Santalum album), valued for its medicinal properties, is an indigenous species of India. Circular or irregular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the centre of the lesion were observed on leaves of sandalwood seedlings in a nursery located in Karnataka, with a disease incidence of 75% (n = 100 investigated plants) during July 2020. The disease prevailed in monsoon followed by winter season (July 2020 - January 2021); summer was less supportive for the disease incidence. As the disease progressed, lesions expanded and merged, causing necrosis of the whole leaf. Isolation of the pathogen involved excision of small sections of diseased tissues from the lesions followed by surface sterilization with 70% ethanol for 30 s and in 1% NaClO for 1 min. Sections were rinsed in sterile distilled water, placed on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Ten isolates of Colletotrichum ssp. were obtained with an isolation frequency = 10/12×100 = 83%. One representative single-spore isolate (CSSA-1) was used for further study. Initially, pure cultures exhibited a white mycelium which later turned gray with time, and had orange conidial masses in a concentric ring pattern with the aggregation of black acervuli at the center of the culture Conidia were single celled, hyaline, and cylindrical having smooth rounded ends and the size ranged from 12.6 to 18.5 µm in length, and 3.5 to 5.6 µm in width (n = 100). The morphological characteristics were in agreement with the species description of fungi in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm the species designation of the isolate CSSA-1, a multilocus phylogenetic analysis was performed using six genomic loci (Weir et al. 2012; Marin-Felix et al. 2017). The internal transcribed spacer (ITS) region of rDNA and a partial sequence of the beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and glutamine synthetase (GS) genes were amplified using ITS1/ITS4, BT2F/BT4R, GAPDHF/R, 79F/354R, 512F/783R and GSF/GSR primers, respectively. The ITS (OK254122), TUB2 (OL462863), GAPDH (OL462859), CHS-1 (OL462860), actin (OL462861), and glutamine synthetase (OL961822) sequences of representative isolate CSSA-1 showed 99 to 100% identity with sequences MZ148628, MK967339, MN525882, MW192791, MT263504, MF111030, MH370542 and KX578767, respectively to the holotype isolate of Colletotrichum siamense (Prihastuti et al. 2009). The sequences were analysed with representative sequences of Colletotrichum and a multilocus Bayesian inference phylogenetic tree with ITS-GAPDH-ACT-CHS1-GS-TUB2 concatenated data sets (concatenated with Sequence Matrix v.1.8 (Vaidya et al. 2011)) was constructed using Beast version 1.8.4 to confirm the isolate identification (Drummond et al. 2012; Hyde et al. 2014; Weir et al. 2012). Isolate CSSA-1 clustered with C. siamense isolates. To complete Koch's postulates, for the characterized isolate CSSA-1, a pathogenicity test was conducted on 3-month-old sandalwood seedlings by spore spray inoculation. Ten plants were inoculated with a conidial suspension (106 conidia/ml) and control plants inoculated with sterilized water then kept in a glass house at 25°C and >85% relative humidity with a 12-h photoperiod. Humidity was maintained by spraying the plants with water in the morning and evening to enhance the infection. Typical symptoms of anthracnose disease similar to naturally infected leaves were observed, which included circular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the center of the lesion 7 days after inoculation, while the control plants remained unaffected. The pathogen was reisolated from infected leaves and its identity was confirmed as C. siamense based on morphological characteristics. Previously, C. siamense was identified causing disease on chili in India (Gunjan and Shenoy, 2014), but to our knowledge this is the first report of leaf anthracnose caused by C. siamense on Indian sandalwood in India or globally. This study documents crucial information, paving way for epidemiologic studies and design of control strategies to combat this newly emerging disease.