Molecular Microbiology Research 2024, Vol.14, No.6, 259-270 http://microbescipublisher.com/index.php/mmr 261 Figure 1 Symptoms of leaf spot disease onG. jasminoides Ellis. Bar, 1 cm 2.2 Fungal isolates and morphological characterization A total of six Alternaria isolates were obtained from infected leaf samples and named ES1-1, ES2-1, ES2-2, ES2-3, ES4-2, and ES4-3. The six isolates were morphologically consistent with Alternaria alternata (Fonseca-Guerra et al., 2023). The colonies of these isolates on PDA varied. ES1-1 (Figure 2a) and ES4-2 (Figure 2f) displayed an olive-green color, and colony margins were white with a feathery texture. Moreover, the growth rates of these two isolates were slower than those of the other four species. The whole mycelium of the other four isolates had a dense, cottony texture, and the color was dark olive. Furthermore, the surface of all six colonies exhibited concentric rings. The conidia were ovoid to obclavate in shape and ranged from 14.6 μm to 34.9 μm×7.0 μm to 16.4 μm (length×width) in size (n=15). All types of conidia were muriformly septate with 1 to 6 transverse septa and 0 to 5 longitudinal septa. The beak size varied from 1.3 μm to 9.8 μm (n=15). Therefore, based on these characteristics, the isolates were identified as A. alternata. There were no significant differences in the length (P=0.532) and width (P = 0.166) of the conidial body or length (P = 0.665) of the beak among the isolates (Figure S1). Figure 2 Colony appearance and conidia morphology of six isolates Image caption: a~f: Colonies of six isolates, ES1-1, ES2-1, ES2-2, ES2-3, ES4-2, and ES4-3, respectively, cultured on PDA plates for 7 d at 25 ℃ in the dark; g~l: Conidia of six isolates, ES1-1, ES2-1, ES2-2, ES2-3, ES4-2, and ES4-3, respectively 2.3 Phylogenetic analysis of PCR-generated DNA sequences The Sanger sequencing results showed that the fragments of ITS, gapdh, rpb2, and Alt a 1 were 514 bp to 515 bp, 572 bp to 579 bp, 752 bp, and 444 bp to 453 bp, respectively. When the 24 sequences of genes of the Alternaria isolates were searched using the blast tool in GenBank of the National Center for Biotechnology Information (NCBI), the isolates were 99% to 100% similar to other A. alternata sequences reported in the GenBank. Further phylogenetic trees were constructed based on maximum likelihood analysis with these sequences, and all these isolates were identified as A. alternata (Figure 3), which was consistent with previous morphological characterization. Furthermore, ES2-2, ES2-3, and ES2-1 were grouped into the same subclade; ES4-2, ES4-3, and ES1-1 formed another subclade. Sequences of Alternaria alternantherae (CBS 124392; HSAUP2798) were used as outgroups (Woudenberg et al., 2015) (Figure 3).
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