| 摘要 |
[Objectives] To develop methods for the early and rapid detection of tomato gray mold. [Methods] Utilizing the ACTIN gene of Botrytis cinerea as the target, a set of specific primers for loop-mediated isothermal amplification (LAMP) was designed and screened. Subsequently, the reaction system and conditions were optimized to achieve rapid isothermal amplification of B. cinerea. [Results] Through agarose gel electrophoresis and SYBR Green I visualization analysis, the optimal dosages of Bst II DNA polymerase and dNTPs, as well as the optimal ratio of internal to external primers, were determined to be 0.6 U/μL, 1.25 mmol/L, and 2：1, respectively. The specific detection of B. cinerea was accomplished at 61 ℃ for 40 min, achieving a sensitivity of 100 ag/μL, which is 106 times greater than that of conventional PCR detection. When this method was applied to the detection of tomato diseases, the detection limit for B. cinerea spores reached 20 spores/mL. Furthermore, the pathogen was detectable in tomato leaves that had been infected for 4 d, despite the absence of visible phenotypic symptoms of gray mold. [Conclusions] This method is suitable for the early, rapid, sensitive, and visual detection of tomato gray mold, thereby offering technical support for its early diagnosis, prevention, and control. |
