First report of Colletotrichum fructicola causing leaf spot on macadamia in Mauritius

Macadamia (Macadamia tetraphylla, Protaceae) is classified amongst the most lucrative crops in the world. Globally, the macadamia industry is growing exponentially and the market size was US$1.37 billion in 2020 (Bouarakia et al., 2023). In Mauritius, macadamia cultivation has been encouraged by the government, both to expand the agricultural sector and to compensate for contraction in the sugarcane industry . In October 2021 and August 2023, Macadamia tetraphylla leaves exhibiting leaf spots, resembling anthracnose, were collected from Réduit, Mauritius (20°13'55.986''S 57°29'53.368''E). The symptoms appeared mainly on the edge and the tip of the leaves, and the lesions were water-soaked, irregular, black to brown to tawny, and surrounded by light yellow haloes (Fig. 1). Small pieces of infected tissue (0.5 × 0.5 cm) were excised from four diseased leaves, which were then surface disinfected with 1% sodium hypochlorite for three minutes and rinsed twice with sterilised distilled water. The air-dried infected tissues were plated on potato dextrose agar (PDA) and incubated at 25°C for about seven days in a 12-hour dark-light cycle. Pure cultures were achieved by transferring hyphal tips to new PDA plates. Four isolates were recovered (M1BR, M4BR and M7A4R from three leaf samples collected in 2021, and MC2 from a leaf sample in 2023). The colonies were white to grey-white and turned grey in the centre upon ageing and the aerial mycelium was grey-white with a dense cottony texture (Fig. 2). A representative isolate (M7A4R) was selected for morphological and molecular characterisation. Growth rate was 10.4 mm day−1 on PDA. Conidia were cylindrical with rounded ends (Fig. 3) and had a mean length of 13.7 ±1.1 μm and a mean width of 3.7 ±0.4 μm (n = 30). The morphological features matched the description of Colletotrichum fructicola (Prihastuti et al., 2009). The morphological identification were confirmed by PCR amplification and sequencing of three genomic loci, the ITS region, GAPDH and TUB2 genes, which were amplified by PCR using primer pairs ITS4/ITS5 (White et al., 1990), GDF/GDR and T1/Bt2b (Gan et al., 2016), respectively. The ITS gene sequence confirmed that the isolate was Colletotrichum with 100% identity to GenBank Accession No. HM146134. BLASTn searches showed that the GAPDH (261 bp) and TUB2 (736 bp) gene sequences had 100% identity with C. fructicola (KU743270 and OP966689, respectively). All sequences were deposited in GenBank: OP412459 (ITS), OR297985 (GAPDH) and PP061452 (TUB2). A maximum likelihood phylogram of the combined dataset (ITS, GAPDH and TUB2; 1210–1553 bp) identified isolate M7A4R as C. fructicola, isolate M7A4R clustering with reference GenBank sequences with a high bootstrap support (Fig. 5). The pathogenicity of the isolate was confirmed by spraying potted, disease-free M. tetraphylla seedlings with a conidial suspension (1 × 102 spores/ ml) prepared from a seven-day-old colony of isolate M7A4R. Healthy macadamia plants inoculated with sterile distilled water served as a control. The plants were covered with transparent plastic bags to maintain a humid environment and placed in natural dark-light cycle at room temperature. Two weeks after inoculation, the inoculated plants developed anthracnose symptoms while the control plant remained symptomless (Fig. 4 ). The original isolate M7A4R was recovered from the diseased plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of C. fructicola causing leaf spots on macadamia in Mauritius. In 2023, C. fructicola was reported on Macadamia ternifolia in China (Li et al., 2023). Colletotrichum fructicola from the C. gloeosporioides species complex is a polyphagous fungus which causes anthracnose in more than 90 plant species (Bragard et al., 2021). This study will promote awareness about the disease affecting macadamia plants among local farmers, allowing them to adopt proper disease control strategies. This study was funded by the University of Mauritius and the European Union under the project "Enhancing climate resilience in agriculture for improved food and nutrition security through research, innovation and training in the Republic of Mauritius" under contract number (FOOD 2019/406 182).