Occurrence and characterization of Alternaria alternata causing leaf spot of soybean (Glycine max) in Pakistan

Soybean (Glycine max) as an oilseed crop plays a vital role in agro-based industries. However, its sustainable production is challenged by biotic and abiotic factors. Fungal pathogens infecting soybean are a major concern. But, their extent in Pakistan is less explored. During crop season 2019-20, soybean plants showing leaf spots were observed in a research trial. Infected samples were assayed for isolation of pathogen on potato dextrose agar. Initial mycelial growth was purified on fresh nutrient media. Based on growth pattern and microscopic observations, the fungus was identified as Alternaria alternata. Koch postulates also confirmed by inoculating spore suspension of A. alternata in soybean plants under controlled environmental conditions. Moreover, phenotypic features of fungus were validated through amplification of internal transcribed spacer (ITS) regions using ITS-1and ITS-4 primers. Amplicon size of 570-bp was sequenced and NCBI BLASTn results confirmed fungus as A. alternata. Representative sequences of isolates (NBG1-4) were submitted to public database NCBI with accession numbers MZ769364-67. Phylogenetic study also inferred their relationship with previously reported isolates of A. alternata. Morphological feature of fungus, its pathogenicity and molecular characterization revealed the association of A. alternata with leaf spot in soybean. These results confirmed the incidence of leaf spot of soybean caused by A. alternata for the first time from Pakistan.