Detection and Control of Fusarium oxysporum from Soft Rot in Dendrobium officinale by Loop-Mediated Isothermal Amplification Assays

Abstract

Soft rot causing Fusarium oxysporum is one of the most destructive diseases of Dendrobium officinale Kimura et Migo in China that reduces D. officinale yield and quality. A key challenge for an integrated management strategy for this disease is the rapid and accurate detection of F. oxysporum on D. officinale. Therefore, a new loop-mediated isothermal amplification (LAMP) assay was developed for this purpose. In this study, the primers were selected and designed using the translation elongation factor-1α (TEF-1α) gene region as the target DNA sequence in order to screen the best system of reaction of LAMP to detect F. oxysporum through optimizing different conditions of the LAMP reaction, including time, temperature, concentrations of MgSO4, and concentrations of inner and outer primers. The optimized system was able to efficiently amplify the target gene at 62 °C for 60 min with 1.2 μM internal primers, 0.4 μM external primers, 7 mM Mg2+, and 5 fg/µL minimum detection concentration of DNA for F. oxysporum. The amplified products could be detected with the naked eye after completion of the reaction with SYBR green I. We were better able to control the effect of soft rot in D. officinale using fungicides following a positive test result. Additionally, the control effect of synergism combinations against soft rot was higher than 75%. Thus, LAMP assays could detect F. oxysporum in infected tissues of D. officinale and soils in field, allowing for early diagnosis of the disease.