Real-Time PCR and LAMP Assays for the Detection of Spores of Alternaria solani and Sporangia of Phytophthora infestans to Inform Disease Risk Forecasting

Abstract

Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, Phytophthora infestans, and spores of the main causal pathogen of early blight, Alternaria solani, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for P. infestans and a newly developed real-time PCR assay for A. solani. Primers were designed for real-time LAMP of P. infestans and A. solani. The specificity of the P. infestans real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of P. infestans was consistently amplified as was DNA of the taxonomically closely related species Phytophthora mirabilis, Phytophthora phaseoli, and Phytophthora ipomoea; no amplification of DNA from the potato pathogens Phytophthora erythroseptica or Phytophthora nicotianae occurred. Real-time LAMP and PCR assays were developed for A. solani, and the specificity was compared with an existing conventional PCR assay. Importantly, the A. solani real-time LAMP and PCR assays did not amplify the species Alternaria alternata. However, cross-reactivity with Alternaria dauci was observed with the real-time PCR assay and Alternaria brassicae with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The P. infestans real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of A. solani, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with P. infestans, and the other infected with A. solani. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of P. infestans and A. solani with both real-time PCR and LAMP was assessed.

Comparative evaluation of lateral flow immunoassays, LAMP, and quantitative PCR for diagnosis of fire blight in apple orchards

“…5). The sensitivity of LAMP and qPCR were identified to be higher than conventional d i a g n o s t i c m e t h o d s f o r A l t e r n a r i a s o l a n i a n d Phytophthora infestans detection from potato, tomato, and other related host plants (Okiro et al 2019;Khan et al 2018;Lees et al 2019). The real-time PCR has previously also been identified as the most sensitive method, followed by LAMP assay, to detect the Xylella fastidiosa pathogen from blueberry (Waliullah et al 2019).…”

Use of LAMP for Assessing Botrytis cinerea Colonization of Bunch Trash and Latent Infection of Berries in Grapevines

“…LAMP methods have been developed to enable early on-site detection of plant pathogens from crude extract samples, including the detection of latent infections of Plasmopara viticola in grape leaves [65], grapevine phytoplasmas in crude leaf-vein homogenate [66], Peronospora effusa in symptomless spinach leaves [67], Spiroplasma citri in citrus leaves [68], and Phytophthora infestans in potato leaves [54], as well as the detection of plant viruses and viroids [69]. When coupled with spore trap systems, LAMP assays have been used for the detection and quantification of airborne pathogen inoculum, including the conidia of Alternaria solani and sporangia of P. infestans [70], and the conidia of Magnaporthe oryzae [56]. LAMP assays have been also developed for the quantification of airborne Erysiphe necator inoculum and have been evaluated for potential implementation by vine growers for the initiation of fungicide programmes [55,71].…”

A Review of the Most Common and Economically Important Diseases That Undermine the Cultivation of Tomato Crop in the Mediterranean Basin

“…Efficient disease management can be achieved through the use of sensitive and sophisticate detection methods. Thus, in-field loop-mediated isothermal amplification (LAMP), hyperspectral measurements, and ensemble machine learning methods have been recently developed, which are suitable for early and mass detection [75,76]. In addition, species-specific polymerase chain reaction (PCR) and multilocus sequencing are highly recommended techniques to differentiate A. solani from other Alternaria species infecting tomato [66,77].…”