First report of Colletotrichum siamense Causing anthracnose on Cinnamomum camphora in Malaysia

Cinnamomum camphora (Lauraceae), commonly known as camphor tree, is widely grown as an ornamental and is used as a source of camphor in Malaysia. In June 2021, leaves of three camphor trees with anthracnose symptoms were collected from a park (6°02'00.8"N, 116°07'18.5"E) at the Universiti Malaysia Sabah in Sabah province. The average disease severity across diseased plants was about 60% with 30% incidence on 10 surveyed plants. The disease severity on disease area of 10 leaves from each three diseased plants was estimated using ImageJ software. The disease incidence was determined based on Sharma et al. (2017). Gray spots were observed primarily on the surface of the leaves. After a week, the spots coalesced into larger patches, and anthracnose developed. Small pieces (5 x 5 mm) of symptomatic leaf tissue from three camphor trees were excised from the margin between healthy and symptomatic tissue. The pieces were surface-sterilized with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before plating in three Petri dishes with Potato dextrose agar, and incubated for 7 days at 25°C in the dark. After 7 days, all the PDA plates had abundant gray-white fluffy hyphae. Mycelium was dark brown when observed from the underside of the plate. The isolates UMS02, UMS04 and UMS05 were characterized morphologically and molecularly. The conidia were one-celled, cylindrical, hyaline, and smooth, with blunt ends, and ranged in size from 13.9 to 16.3 x 3.8 to 6.1 μm (n = 20). Appressoria were round to irregular in shape and dark brown in color, with size ranging from 7.8 to 9.8 μm x 5.3 to 6.8 μm (n= 20). Genomic DNA was extracted from fresh mycelium of the isolates based on Khoo et al. (2022a). Amplification of the internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of the isolate was performed using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 (Weir et al. 2012). PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. Sequences of the isolates were deposited in GenBank as OK448747, OM501094, OM501095 (ITS), OL953034, OM513908, OM513909 (CAL), OL953031, OM513910, OM513911 (ACT), OL953037, OM513912, OM513913 (CHS-1), and OL953040, OM513914, OM513915 (GAPDH). They were 100% identical to ITS (MN296082), CAL (MN525840), ACT (MW341257, MN525819), CHS-1 (MT210318), and GAPDH (MT682399, MN525882) sequences of Colletotrichum siamense. Phylogenetic analysis using maximum likelihood on the concatenated ITS, CAL, ACT, CHS-1 and GAPDH sequences indicated that the isolates formed a clade (82% bootstrap support) to C. siamense. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2022). Koch's postulates were performed by spraying a spore suspension (106 spores/ml) on leaves of three healthy two-month-old camphor trees, while water was sprayed on three additional camphor trees which served as control. The inoculated camphor trees were covered with plastics for 48 h at 25°C in the dark, and then placed in the greenhouse. Monitoring and incubation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). Symptoms similar to those observed in the field occurred 8 days post-inoculation. No symptoms occurred on controls. The experiment was repeated two more times. C. siamense has been reported causing anthracnose on camphor tree in China (Liu et al. 2022), Citrus spp. in Mexico (Pérez-Mora et al. 2021), and Crinum asiaticum and eggplant in Malaysia (Khoo et al. 2022b, 2022c). To our knowledge, this is the first report of C. siamense causing anthracnose on C. camphora in Malaysia. Our findings expand the geographic range of C. siamense and indicate it could be a potential threat limiting the camphor production of C. camphora in Malaysia.