Diversity and pathogenicity of Cladosporium species associated with alfalfa leaf spot in West Azerbaijan province, Iran

Abstract Alfalfa ( Medicago sativa ) is the most important and high-yielding forage legume cultivated worldwide. The aerial parts of the plant play a crucial role in feeding livestock, and the roots contribute to soil nutrient levels, shape microbial communities, and support overall soil improvement. During our field survey of leaf-inhabiting fungi associated with this forage crop, we observed a widespread distribution of cladosporioid leaf spot symptoms in West Azerbaijan province, Iran. However, the causal agents associated with cladosporioid leaf spots and their pathogenicity in northwest Iran remain insufficiently resolved. Accordingly, this study aimed to identify the Cladosporium species associated with alfalfa leaf spot and assess their pathogenicity in this region. Samples with leaf spot symptoms were collected from various fields in West Azerbaijan province, including the cities of Miandoab, Mahabad, and Naghadeh, and then subjected to microscopic examination, resulting in the isolation of 49 Cladosporium isolates. A three-locus phylogenetic analysis (ITS, ACT and TEF1-α ), combined with a detailed morphological examination of representative isolates, confirmed the association of six Cladosporium species with alfalfa leaf spot in Iran: C. asperulatum , C. cladosporioides , C. limoniforme , C. macrocarpum , C. submersum , and C. tenellum . Among these, C. asperulatum and C. submersum are new records for the funga of Iran. In addition, C. asperulatum , C. limoniforme , C. macrocarpum , C. submersum , and C. tenellum are reported for the first time on M. sativa in the world. Pathogenicity tests under greenhouse conditions confirmed that four species, including C. asperulatum , C. limoniforme , C. macrocarpum , and C. submersum , were pathogenic on alfalfa leaves. These findings clarify the species complex involved in alfalfa leaf spot in northwest Iran and provide an initial evidence base for disease management, with the potential to reduce economic losses.