First report of cladode spot caused by <i>Alternaria alternata</i> on <i>Opuntia robusta</i> in South Africa

Opuntia robusta is a cactus species cultivated for both fodder and fruit production. In November 2024, cladode spot symptoms were observed on approximately 60% of O. robusta plants cultivated at an experimental farm in Bloemfontein (29.1035° E, 26.1740° S), South Africa. Symptoms were characterized by light brown circular spots, with alternating rings around the areoles that coalesced to form a scab (Fig. 1A). The infection was confined to the epidermal layer. Ten symptomatic cladodes were randomly collected, surface disinfested, washed three times with sterile distilled water, and lesions (5mm2) were plated on potato dextrose agar and incubated at 28°C for 5 days in the dark. Fungal isolate HF1 had the highest isolation frequency, and was chosen for further study. The septate mycelium of HF1 was fluffy gray to black and produced branching septate conidiophores and chained conidia (19.8µm long and 10µm wide) with both longitudinal and transverse septa, consistent with the description of Alternaria sp. (Lawrence et al. 2016; Fig. 2A-D). For confirmation, genomic DNA was extracted from the isolate. The internal transcribed spacer region (ITS) region and three protein-coding genes - translation elongation factor 1-alpha (TEF 1-α), RNA polymerase II second largest subunit gene (RPB2) and Alternaria major allergen (Alt a 1) (Woudenberg et al., 2014) – were amplified using primers ITS1/ITS4 (White et al., 1990), fRPB2-5F/fRPB2-7cR (Liu et al., 1999), (O'Donnell et al., 1998) and A21F/ A21R (Gabriel et al., 2015), respectively. Sequences were deposited in GenBank under accession numbers ITS (PX112669), RPB2 (PX136066), Alt a1 (PX136067), and TEF 1-α (PX136068). BLASTn searches showed high sequence identity to Alternaria alternata with alignments of 99.8%, 100%, 99.9%, and 97.2% to ITS (MT453271.1), TEF 1-α (PV963120.1), RPB2 (PP783614.1), and Alt a1 (XM_018532577.1), respectively. In the multilocus phylogenetic tree constructed using maximum likelihood (ML) analysis of concatenated ITS-TEF1-RPB2-ALT a1 sequences, isolate HF1 clustered with reference A. alternata strains with 95% bootstrap support (Fig. 3). For pathogenicity testing, sterile toothpicks pre-colonized with isolate HF1 mycelium were used to inoculate 1-year-old O. robusta cladodes. Four inoculation points per cladode were made, with sterile toothpicks as controls. Each treatment was replicated 8 times in each of two independent trials. The cladodes were incubated in the glasshouse under natural light conditions at 25°C and 45% relative humidity and watered daily. In the first trial, 5 weeks after inoculation, 100% of inoculated cladodes had developed lesions averaging 16.4 mm in diameter, whereas no lesions were observed on the controls (Fig. 4). The second trial showed the same response pattern. In both trials, symptoms observed were consistent with those recorded under field conditions (Fig. 5). Fluffy gray to black mycelium fungi was isolated from the artificially inoculated cladodes and was confirmed as A. alternata using morphological and molecular methods as previously described, fulfilling Koch’s postulates. To our knowledge, this is the first report of A. alternata causing cladode spot on O. robusta worldwide. This finding expands the known host range of A. alternata and provides a basis for early disease diagnosis and the implementation of management strategies, including sanitation and timely fungicide applications, to reduce disease spread and protect cladodes.