Molecular Characterization of Botrytis cinerea Isolates from Different Host Plants
Background: Botrytis cinerea gray mold is one of the most significant diseases that limits strawberry productivity under field and postharvest conditions. It is essential to accurately identify the pathogen and examine potential control options to develop effective disease-management strategies. Objective: This study aimed to isolate and identify Botrytis cinerea associated with gray mold of strawberry, validate representative isolates through ITS-rDNA sequencing, determine their pathogenicity, and perform an in vitro assessment of the efficacy of selected fungicides, antagonistic microorganisms (AMO) and botanical extracts. Methodology: From Dec. 2024 to Mar. 2025, symptomatic strawberry samples were collected from various growing areas of northern Iraq. Isolation of the fungal isolates was performed on potato dextrose agar and subsequently purified with the hyphal-tip technique. Identification was performed first through colony and microscopic characteristics, then confirmed with pathogenicity tests and amplifying the internal transcribed spacer region of ribosomal DNA (ITS-rDNA) using primers ITS1 and ITS4. PCR products were sequenced, compared with reference sequences in the NCBI database using BLAST and analyzed phylogenetically using MEGA. In vitro evaluations were conducted to assess the inhibitory effects of selected fungicides, microbial antagonists, and plant extracts. Data were analyzed by analysis of variance and means compared using Tukey's test at p < 0.05 when appropriate. Results: B. cinerea was confirmed in infected strawberry tissues by morphology determination and pathogenicity test. ITS-rDNA sequencing of representative isolates revealed significant homology with reference B. cinerea sequences found in GenBank, and phylogenetic analysis confirmed that the tested isolates fell within the B. cinerea group. Isolates evaluated differed in virulence. In vitro, all tested fungicides decreased fungal growth, compared to the control with dicarboximide having the lowest EC50 followed by benzimidazole and then other fungicides. Biological Control Trichoderma spp. showed the highest antagonistic activity. The botanical extracts tested also inhibited the growth of fungi, and all separated these 12 strains into three antifungal bands; Allium sativum had the maximum antifungal effect, followed by Mentha spicata and Tagetes patula, Asystasia gangetica & Syzygium cumini & Rosmarinus officinalis & Achillea millefolium. Conclusion: Based on the results obtained, the current study concluded the presence of abnormally shaped colonies through a multi-faceted approach that included pathogenesis tests and ITS-rDNA sequencing to provide accurate identification of gray mold in strawberries (Botrytis cinerea). The study demonstrated significant variability among the isolates, highlighting the importance of accurate pathogen identification before implementing control measures. The results indicate that dicarboxymides, Trichoderma species, and garlic extract (Allium sativum) were the most effective chemical, biological, and phytochemical treatments, respectively. These findings support the feasibility of integrated management approaches that combine biological, phytochemical, and conventional fungicides for controlling gray mold in strawberries.