Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Abstract

Infection of Zizania latifolia by the basidiomycete Ustilago esculenta enlarges the stem tissue to produce edible galls called "makomotake" in Japan. The development of stem galls in Z. latifolia may be influenced by the colonization level of U. esculenta at the seedling stage. To evaluate this possibility, it is necessary to establish a method to investigate the relationship between fungal levels in seedlings and the production of makomotake from the same seedlings in a non-destructive manner. To achieve this goal, in this study, we attempted to develop a real-time loop-mediated isothermal amplification (LAMP) method with toothpick sampling for U. esculenta detection. An U. esculenta gene-specific LAMP primer set designed for real-time LAMP showed sufficient detection sensitivity for fungal genomic DNA, which is only about 10 times lower than that of quantitative PCR. Real-time LAMP reactions following toothpick sampling detected U. esculenta efficiently in the culm part of Z. latifolia seedlings that eventually produced makomotake, but did not detect in those that failed to develop stem galls. Our data show that the combination of real-time LAMP and toothpick sampling is a simple and useful method for estimating U. esculenta levels in Z. latifolia seedlings and for studying the subsequent development of stem galls in the same individuals.