Rapid Detection of Monilinia fructicola and Monilinia laxa on Peach and Nectarine using Loop-Mediated Isothermal Amplification

Abstract

Monilinia laxa and M. fructicola are two causal agents of brown rot, one of the most important diseases in stone fruit. Two species cause blight on blossoms and twigs and brown rot on fruit in pre- and postharvest. Both species are distributed worldwide in North and South America, Australia, and Japan. In Europe, M. laxa is endemic, while M. fructicola was introduced in 2001 and it is now widespread in several countries. Currently, both species coexist in European stone fruit orchards. Monilinia spp. overwinter in cankers and mummified fruit. Mummy monitoring during winter permits growers to understand which species of Monilinia will be prevalent in an orchard during the following season, permitting planning of an appropriate crop protection. Traditionally, the identification has been carried out using morphological features and even with polymerase chain reaction (PCR)-based assays that requires time and well-equipped laboratories. In this study, two isothermal-based methods were designed to identify these pathogens in a faster way than using traditional methods. The loop-mediated amplification (LAMP) assays were validated on some isolates of Monilinia spp. coming from the mummy monitoring according to the international European and Mediterranean Plant Protection Organization standard (PM7/98), taking into account specificity, sensitivity, repeatability, and reproducibility. The sensitivity of both assays was checked by monitoring (at different time points) two nectarine varieties artificially inoculated and stored at two different temperatures. The reliability of both LAMP assays against the quantification of the inoculum was compared with previously published quantitative PCR assays. Both LAMP methods were able to detect a low number of cells. These LAMP methods could be a useful tool for monitoring brown rot causal agents in the field and during postharvest.

New Molecular Tool for a Quick and Easy Detection of Apple Scab in the Field

“…The tests performed showed a level of sensitivity of some pg of DNA comparable with other LAMP assays previously developed [36,38,40,45,47,55,56]. On the other hand, the limit of detection using infected leaf samples varied in the function of the DNA extraction.…”

“…Alkaline DNA extraction was used to extract the DNA from plugs of the artificially inoculated apple leaves (1.5 cm diameter plugs) and from naturally infected symptomatic and asymptomatic apple leaves (1.5 cm diameter plugs), to perform a crude extraction method, as described by Franco Ortega et al [46,47]. The extraction included one 7/16" stainless steel 316 GD ball (Spheric Trafalgar Ltd., Worthing, UK) and 1 mL of pH 13-13.5 PEG buffer (50 g/L PEG average Mn 4600; 20 mmol/L KOH; pH 13.5) in a 5 mL tube homogenized by vigorous manual shaking for one minute.…”

“…The LAMP reactions for V. inaequalis and for COX were prepared on 25 µL reaction including 0.2 µmol/L of the external primers (F3 and B3), 2 µmol/L of each internal primer (FIP and BIP), 1 µmol/L of each loop primer and 1× Isothermal Mastermix ISO-004 ® (OptiGene Ltd.,Horsham, UK). LAMP assay was performed as reported by Franco Ortega et al [47] with both a Genie II ® instrument and a StepOne instrument (Applied Biosystem, Loughborough, UK) set to perform an amplification at 65 • C for 45 min and measurement of annealing temperature.…”

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

“…Despite, the end-point PCR assays showed a high specificity of 82.0% with an LOD of 110 fg/µL of DNA. In terms of sensitivity, the LAMP assay designed here, showed a limit of detection of 100-999 pg/µL of DNA, like the limit of detection previously described in other assays [66,67]. However, Niessen et al [50] also developed a LAMP assay that focused on the detection of aflatoxigenic strains of Aspergillus section Flavi, with primers designed on the nor1 gene, with a lower detection limit of 9.03 pg of genomic DNA per reaction and based on the detection of the positive results by a colorimetric change or by a fluorescence dye.…”

DNA sequencing, genomes and genetic markers of microbes on fruits and vegetables

“…The inter‐simple sequence repeat (ISSR), RAPD (Fazekas et al , 2014) and HRM (Papavasileiou et al , 2016) methods have been used for interspecies identification. Specific regions of the cytochrome b gene (Ortega et al , 2019), laccase‐2 gene (Wang et al , 2018b), β‐tubulin gene (Fan et al , 2014), ITS (Guinet et al , 2016), MO368‐1 , Laxa (Cote et al , 2004) and small subunit ( SSU ) rDNA (18S) gene (Fulton and Brown, 1997) have been reported as markers for PCR‐based detection.…”