A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of toxigenic Fusarium temperatum in maize stalks and kernels

Highlights

• F. temperatum is an emerging pathogen and represents a toxicological risk to maize from pre-harvest to storage stage.

• A visualized loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of F. temperatum was developed.

• The LAMP reaction conditions were optimized.

• Early detection of F. temperatum in infected maize stalks and kernels was demonstrated.

Abstract

Fusarium temperatum is an emerging maize pathogen that causes maize ear and stalk rot diseases and produces various mycotoxins including moniliformin, beauvericin, enniatins and fumonisin B1, which poses a potential risk to the human food or animal feed supply chains. Early detection of F. temperatum is crucial to prevent its derived mycotoxins from entering the food chain, and is also a useful tool in disease management practices. Here, we describe a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of F. temperatum. The 28S ribosomal DNA sequences (28S rDNA) of F. temperatum were used to design a set of six primers. The reaction conditions were optimized for developing a fast assay with high specificity and sensitivity, and were able to detect the presence of less than 10 pg of target DNA per reaction within 60 min. Furthermore, the resulting amplicons were visualized by adding SYBR Green I to the reaction tubes. Suspected F. temperatum infected maize stalk samples collected from Yunnan province, China were identified using the developed LAMP assay. In conclusion, the method not only provides a rapid and specific screening for the existence of F. temperatum in a bulk of maize samples without using sophisticated equipment, but also is potentially useful for other agriculturally important toxigenic fungi.

Introduction

Fusarium is a large and diverse genus of fungi with significant agricultural and economic importance, including a large number of species that are important pathogens of maize and other cereal crops (Ma et al., 2013). Fusarium spp. also produce a wide range of biologically active secondary metabolites in infected plants or in stored grains, including mycotoxins which are harmful to animals and humans consuming the grain (Desjardins and Proctor, 2007). In China, many Fusarium species are associated with ear and stalk rot diseases of maize, which resulted in significant yield loss and mycotoxin contamination problems in the past (Zhang et al., 2015). The most abundant Fusarium species isolated from maize are Fusarium verticillioides, F. graminearum, F. meridionale and F. temperatum (Duan et al., 2016; J.H. Wang et al., 2014). F. temperatum is an important maize pathogen and has been described as a new species by Scauflaire et al. (2011). Until now, it is widespread in Europe, Asia as well as North and South America (Boutigny et al., 2017; Fumero et al., 2015; Lanza et al., 2016; Robles-Barrios et al., 2015; Shin et al., 2014; Varela et al., 2013; Venturini et al., 2016; J.H. Wang et al., 2014). F. temperatum is able to produce various mycotoxins including moniliformin, beauvericin, enniatins and fumonisin B1 (Fumero et al., 2015; Scauflaire et al., 2012). Due to the production of a variety of mycotoxins, F. temperatum could represent a toxicological risk for maize production from pre-harvest to storage stage. Therefore, new diagnostic techniques for the rapid identification of F. temperatum would be valuable for use in the food and feed industry.

Conventionally, identification of Fusarium species has been mainly based on morphological characteristics and differences in DNA sequences of housekeeping genes. However, morphological identification to the species level is very difficult, time-consuming and needs in-depth knowledge of taxonomy. In addition, PCR-based amplification methods are still expensive because they require dedicated laboratory equipment and well-trained staff. These factors impede the use of PCR-based analysis in on-site applications e.g. agriculture, food and feed production (Niessen and Vogel, 2010). As an alternative approach, loop-mediated isothermal amplification (LAMP) is a novel method of nucleic acid amplification that is catalyzed by Bst DNA polymerase with strand displacement activity and occurs under isothermal condition at a temperature range from 60 to 65 °C (Notomi et al., 2000). A simple heating block is sufficient to fulfill the requirements for a LAMP assay, eliminating the need for costly equipment such as thermal cyclers. Furthermore, the temperature can be maintained by an exothermal reaction linked to specifically engineered phase-change materials to perform the analysis without any instruments (Sema et al., 2015). In addition, the LAMP assay can be easily monitored by the following ways: (i) agarose gel electrophoresis; (ii) measuring turbidity of magnesium pyrophosphate in the LAMP reactions; (iii) measuring fluorescence using SYBR green or calcein; and (iv) by color change using a metal ion-binding indicator dye such as hydroxynaphthol blue (HNB) (Das et al., 2012). In this study, we aimed to develop an effective, sensitive, accurate, reliable and rapid diagnostic method for F. temperatum, to be used in the field or in storage facilities. The developed visualization method for LAMP products, in the reaction tube, demonstrates the potential for application to improve quality management in a cost-effective manner in the cereal industry.