LAMP‐based detection of Monilinia fructigena, Monilinia polystroma and Monilinia fructicola in latently infected apple fruit

Abstract

AbstractBrown rot in apples is caused by fungi belonging to the genus Monilinia and occurs annually in orchards with varying severity. The symptoms of the disease on apple fruits may occur during harvest and can also develop much later in storage. Therefore, from a practical point of view, it is very important to be able to test a sample of asymptomatic apples before putting them in storage or sending them to distant markets. Thus, there is a need to develop a quick and sensitive method of detecting disease causal agents in asymptomatically infected fruit. For this purpose, a procedure for DNA isolation from the skin of apples was developed, and a set of primers for the loop‐mediated isothermal amplification (LAMP) reaction was designed based on a fragment of the hsp60 gene encoding heat shock protein 60 of Monilinia fungi that are pathogenic to apple: M. fructigena, M. fructicola and M. polystroma. The method was validated by reactions with DNA from infected apple fruit exhibiting brown rot symptoms and DNA from infected apple fruit without visible brown rot symptoms (latent infection). To increase the sensitivity of detection before the LAMP reaction, preamplification of the target DNA was carried out. The sensitivity of detection in the LAMP assay for the DNA of all three tested species of fungi was 2 pg. The DNA preamplification significantly increased the sensitivity of Monilinia DNA detection to 0.1 pg. This study shows that the developed LAMP method can be used to detect Monilinia fungi in asymptomatically infected apples.