Molecular Microbiology Research 2024, Vol.14, No.6, 259-270 http://microbescipublisher.com/index.php/mmr 262 Figure 3 Phylogenetic tree based on maximum likelihood analysis. Bar: The estimated nucleotide substitutions per site are 0.02 2.4 Pathogenicity assays The pathogenicity of these six isolates was identified by wound inoculation. Three days after inoculation, tiny, round, and necrotic spots appeared on the leaves. These spots gradually expanded by 7 dpi (days post inoculation) and were similar to those found under field conditions (Figure 4). Furthermore, all fungal isolates were re-isolated from these diseased leaves while no Alternaria spp. were isolated from control leaves. All the leaves that were inoculated with the isolates of A. alternata were infected, and the average incidence was 100%. ANOVA results indicated no significant differences (P=0.238) in pathogenicity among the different isolates (Figure S2). 2.5 Effects of melatonin treatment on the inhibition of the growth of A. alternata Melatonin is a natural molecule that plays an important role in plant defense against fungal pathogens. We tested the direct antifungal properties of melatonin on A. alternata. The direct effect of melatonin on mycelial growth was evaluated. We applied different concentrations of melatonin to the PDA medium. As shown in Figure 5, the colony diameter gradually decreased with increasing melatonin concentration. Seven days after treatment, DMSO also demonstrated a minor inhibitory effect on mycelial growth. However, the inhibition property was limited, and there was no significant difference in the effect of DMSO and double-distilled water (ddH2O) 14 d after treatment. Furthermore, treating A. alternata with 5 mM to 10 mM of melatonin significantly affected the growth of ES4-2 after 14 d. Therefore, melatonin can effectively restrict the growth of A. alternata.
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