First Report of Alternaria tenuissima Causing Leaf Spot on Calotropis procera in Pakistan
Calotropis procera is a medicinal shrub native to the Middle East and Southwest Asia. It is widely distributed in arid and semi-arid regions and is known for its remarkable tolerance to drought and salinity stresses (Kaur et al. 2021). In September 2023, leaf spot symptoms were observed on C. procera in Punjab Province, Pakistan (District Bahawalpur; 29°22′42″N, 71°45′53″E). Initial symptoms showed round to irregular patches with brownish-red borders ranging in diameter from 3 to 7 mm. Mature leaves showed widespread necrotic spots with concentric rings. Approximately 65% disease incidence were observed on eighty-five plants having 80% disease severity. Symptomatic leaves were collected from ten randomly selected plants, placed in sterile polyethylene bags, and transported to the laboratory for further analyses. These leaves were disinfected with 1% NaOCl for 30s and rinsed thrice with d3H2O (Ahmed et al., 2023). These leaves were cut into 5x5 mm tissue pieces put on Petri plates containing PDA and incubated at 26± 2 oC for 7 days. Seven isolates having same morphological characters were obtained. Isolate showing maximum severity in pathogenicity was used for molecular identification of the pathogen. At early-stage pure colonies showed fluffy and off-white appearance that changed to a dusky neutral grey with an off-white border at maturity and reached 85 mm diameter after 7 days on PDA at 25°C. Conidia were ovoid to ellipsoid, muriform, possessing 1–6 transverse and 0–2 longitudinal septa and a short conical beak (5–15 µm). Phylogenetic analyses based on concatenated ITS, GAPDH, HIS3, and Alt a1 sequences placed the isolate within the A. tenuissima clade with strong bootstrap support. The results of sequence alignment revealed that ITS (OQ566746), GAPDH (OQ910481), HIS (PV794358), Alt a1 (PV794357) sequence of isolated fungus exhibited 100% identical to sequences from A. tenuissima accession MT487771 and OR018285 (542 bp), MK451972 and OP168374 (595 bp), AF404622 (521 bp), MZ330655 and PP662498 (504 bp), respectively. To fulfill Koch's postulates, pathogenicity assays were conducted on healthy detached leaves. Three unwounded leaves were inoculated with 5-mm mycelial plug taken from the actively growing margin of a fungal colony was placed onto the surface of each leaf and the experiment was repeated thrice. Control leaves were treated similarly with a sterile agar plug. All leaves were then incubated at 28 ± 2°C and 70% relative humidity. Pure cultures were reisolated from diseased leaf tissues used for pathogenicity and identified as A. tenuissima. To the best of our knowledge, this is the first report of A. tenuissima causing leaf spot disease on C. procera in Pakistan, expanding its host range, which underscores the need to monitor and manage this emerging disease to mitigate its impact.