First Report of <i>Nigrospora sphaerica</i> Causing Leaf Spot Disease of <i>Aloe vera</i> in Sri Lanka

In Sri Lanka, Aloe vera is commonly used in traditional herbal medicine, as well as in the manufacture of cosmetic products and the production of food and beverages (Thangarajah and Emmanuel 2024). A leaf spot disease was observed to be prevalent among A. vera plants across three fields in the Ilavalai region of Jaffna during late 2022 and early 2023. Affected leaves showed small, sunken, brown to black spots which enlarged and coalesced into larger black lesions with grey to light brown halos, accompanied by leaf yellowing (Figure 1). These lesions led to a loss of leaf succulency, and those extending to the leaf margins caused progressive tissue loss, reducing the economic value of the affected leaves. The incidence of the disease was c. 80%. Pure fungal cultures were obtained by isolating fungi from 18 diseased leaves and subculturing on potato dextrose agar. Isolates formed flat, circular, floccose to filiform colonies with entire margins, covering 90 mm Petri dishes within 10 days. Colonies had a greyish-green upper surface and bright brown lower surface with black patches (Figure 2). Conidiogenous cells were discrete, terminal, hyaline to pale brown, ampulliform, measuring 6.05 ±1.26 × 5.13 ± 0.74 µm (n = 30), each producing a single conidium. Conidia were black, solitary, sub-globose to spherical, smooth-walled, aseptate and 9.54 ± 1.17 µm in diameter (n = 30) (Figure 3). Morphological characteristics matched those described by Hao et al. (2020), identifying the isolates as Nigrospora spp. Total DNA was extracted from three isolates (IL, IL3 and IL4) followed by PCR amplification and sequencing of the ITS, translation elongation factor 1-α (TEF1) and β-tubulin (TUB2) regions. The generated sequences were submitted to GenBank (Accession Nos. for ITS: PP549983, PV799929 and PV799930; TEF1: PX433286, PX433287 and PX433288; and TUB2: PX433283, PX433284 and PX433285, for IL, IL3 and IL4, respectively). BLASTn analysis of ITS sequences revealed 96.33% identity with Nigrospora sphaerica strain LC 7298 (KX985937), while the TEF1 and TUB2 sequences showed 100% identity with those of the same strain (KY019401 and KY019606 for TEF1 and TUB2, respectively). A multi-locus phylogenetic tree, constructed using Maximum Likelihood and Bayesian Inference methods, showed that all three isolates clustered into a single clade with N. sphaerica strain LC 7298, supported by a 99% bootstrap value and a posterior probability of 1, confirming their identification as N. sphaerica (Figure 4). Pathogenicity was confirmed by inoculating 20 µL of a N. sphaerica conidial suspension (1 × 105 conidia/mL) onto needle-jab wounds created on a single healthy leaf of 6-month-old, disease-free seedlings, as well as on detached, surface-disinfected leaves. Controls received sterile distilled water. Inoculated seedlings were maintained in a greenhouse at 27°C, while detached leaves were incubated at 27 ± 2°C under a 16-h photoperiod. The experiment was conducted three times. Symptoms appeared after 4 days on both the inoculated seedlings and detached leaves, initially as dark brown to black sunken spots, which expanded and were accompanied by smaller surrounding lesions 14 days post-inoculation. Fungi re-isolated from symptomatic leaves were identical to the original isolates. No symptoms developed on the controls (Figure 5). N. sphaerica has been reported as the causal agent of leaf spot, twig and shoot blight in blueberries (Wright et al. 2008) and leaf blight in tea (Dutta et al. 2015). In A. vera, N. oryzae has been linked to leaf spot (Zhai et al. 2013), while Lasiodiplodia was recently identified as the cause of leaf spot and tip necrosis in Sri Lanka (Thangarajah and Emmanuel 2024). To our knowledge, this is the first report of N. sphaerica infecting A. vera and its first occurrence as a plant pathogen in Sri Lanka. The work was supported by funds obtained from the University Research Grant (URG/2019/SEIT/24), University of Jaffna, Sri Lanka.