Biochemical and molecular characterization of Zasmidium fructicola associated with citrus greasy leaf spot and its implications for disease management

Adnan Kashif, Luqman Amrao, Sajid Aleem Khan, Ahmad Nawaz

Abstract


Citrus Greasy Leaf Spot (CGLS) is an emerging foliar disease that threatens citrus productivity in major growing regions of Pakistan. This study aimed to characterize the causal pathogen Zasmidium fructicola, assess its pathogenic variability, and evaluate host biochemical responses along with potential management strategies. Field surveys revealed that the highest disease severity index (41.13%) and disease incidence (56.01%) occurred in Sargodha, while the lowest values were recorded in Faisalabad. Grapefruit and mandarin were the most susceptible cultivars. Morphological characterization of fifteen fungal isolates confirmed their identity as Z. fructicola, exhibiting typical colony morphology and septate, pigmented conidia consistent with Mycosphaerellaceae. Molecular identification using ITS and TEF-1α gene regions showed 98-99% sequence similarity with reference strains, and phylogenetic analysis grouped all isolates into a well-supported clade, confirming species identity. Pathogenicity assays demonstrated significant variability among isolates, with SAK-1 and AKM-1 exhibiting the highest virulence on grapefruit, mandarin, and sweet orange. Biochemical analyses revealed that resistant cultivars showed higher activities of antioxidant enzymes (SOD, POD, CAT), phenolics, proteins, and soluble sugars, whereas susceptible cultivars exhibited reduced defense-related metabolites following infection. In vitro assays indicated that carbendazim and tebuconazole significantly inhibited mycelial growth, with up to 95.7% and 92.16% inhibition, respectively. Among botanical extracts, Syzygium aromaticum showed the highest antifungal activity, achieving up to 97% growth inhibition. In conclusion, Z. fructicola was confirmed as the causal agent of CGLS, exhibiting notable pathogenic variability and wide geographic distribution. Integrated management using resistant cultivars, fungicides, and botanical extracts offers an effective strategy for its sustainable control in citrus orchards.


Keywords


Zasmidium fructicola, Citrus greasy leaf spot (CGLS), Molecular characterization, Biochemical defense responses, Integrated disease management

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DOI: https://doi.org/10.33804/pp.010.02.6145

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