Morphological, Molecular, and Pathogenic Characterization of Alternaria alternata Isolates from Apple

Abstract

Apple (Malus domestica Borkh.) production is increasingly threatened by fungal diseases under conditions of intensive horticulture and ongoing climate change. In southeastern Kazakhstan, symptoms associated with Alternaria spp. have become more frequent in commercial orchards; however, molecularly confirmed data on the pathogen associated with these symptoms remain limited. This study aimed to identify, using multigene molecular phylogenetic analysis, an Alternaria isolate obtained from infected apple leaves and fruits, to confirm its pathogenicity experimentally, and to compare the susceptibility of apple cultivars to this pathogen. For molecular identification, nucleotide sequences of four genetic markers—the ITS region of rDNA, SSU, tef1-α, and RPB2—were obtained by PCR and sequencing. The sequences were compared with reference data from the GenBank database using BLASTn, and phylogenetic relationships were inferred using the maximum likelihood method based on a concatenated dataset of the four loci. The isolate KZ17 clustered within the A. alternata clade, with the broader node uniting this group with A. gossypina and A. longipes receiving a bootstrap value of 78%. A total of 20 fungal isolates were obtained from 112 symptomatic apple leaf and fruit samples. Among them, one representative isolate, KZ17, was selected for multilocus molecular identification and pathogenicity testing. The pathogenicity of isolate KZ17 was confirmed in inoculation experiments on apple microshoots under controlled conditions. Artificial inoculation of detached leaves and ripe fruits of 14 apple cultivars revealed significant cultivar-dependent differences in susceptibility. Lesion diameters ranged from 12.3 ± 0.20 to 19.2 ± 0.35 mm on detached leaves and from 15.0 ± 1.2 to 30.0 ± 2.4 mm on ripe fruits. The least susceptible cultivars were ‘Granny Smith’, ‘Zaman’, and ‘Maksat’, whereas ‘Voskhod’, ‘Saltanat’, and ‘Kamila’ showed the greatest susceptibility. These differences were statistically significant (p < 0.001). This study provides the first multigene-based confirmation of the association of A. alternata with apple leaf and fruit lesions in southeastern Kazakhstan and demonstrates cultivar-dependent differences in susceptibility to this pathogen. These findings contribute to improved pathogen diagnostics, germplasm screening for resistance, and the development of plant protection strategies for commercial apple orchards.