Abstract
Phytopathogenic microorganisms belonging to the genus Phytophthora have been recognized many times as causal agents of diseases that lower the yield of many plants important for agriculture. Meanwhile, Phytophthora cactorum causes crown rot and leather rot of berry fruits, mainly strawberries. However, widely-applied culture-based methods used for the detection of pathogens are time-consuming and often inaccurate. What is more, molecular techniques require costly equipment. Here we show a rapid and effective detection method for the aforementioned targets, deploying a simple molecular biology technique, Loop-Mediated Isothermal Amplification (LAMP). We optimized assays to amplify the translation elongation factor 1-α (EF1a) gene for two targets: Phytophthora spp. And Phytophthora cactorum. We optimized the LAMP on pure strains of the pathogens, isolated from organic plantations of strawberry, and successfully validated the assay on biological material from the environment including soil samples, rhizosphere, shoots and roots of strawberry, and with SYBR Green. Our results demonstrate that a simple and reliable molecular detection method, that requires only a thermoblock and simple DNA isolation kit, can be successfully applied to detect pathogens that are difficult to separate from the field. We anticipate our findings to be a starting point for developing easier and faster modifications of the isothermal detection methods and which can be applied directly in the plantation, in particular with the use of freeze-dried reagents and chemistry, allowing observation of the results with the naked eye.
Highlights
The reduction in harvest during the production of fruits, caused by pathogenic microorganisms and the diseases they bring to the plantation, is a severe obstacle in agriculture
The results suggest that the detection limit for the optimized reaction is 3 pg/μL for Phytophthora spp. and 300 fg/μL for Phytophthora cactorum, regardless of the tested DNA isolation method
Traditional identification methods based on the observation of microstructures of pathogens do not offer sufficient certainty when it comes to valid identification, as opposed to molecular techniques [45]
Summary
The reduction in harvest during the production of fruits, caused by pathogenic microorganisms and the diseases they bring to the plantation, is a severe obstacle in agriculture. The number of species recognized inside the genus and their hosts is constantly increasing [7,8], whereas P. cactorum has been reported as a soil-borne pathogen causing dieback mainly of the strawberry (Fragaria × ananassa) by both crown rot and leather rot of fruits [9]. The Phytophthora spp. are not host-specific and can attack many plants, causing their dieback [1]. These four facts increase the severity of the infestation of fields with these pathogenic microorganisms. Assays deploying molecular biology techniques offer reliable and immediate detection in comparison to traditional identification methods based on the morphological attributes of the pure strains [11,12,13,14]
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