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

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.

Phytophthora infestans Sporangia Produced in Artificial Media and Plant Lesions Have Subtly Divergent Transcription Profiles but Equivalent Infection Potential and Aggressiveness.

Sporangia of the potato late blight agent Phytophthora infestans are often used in studies of pathogen biology and plant responses to infection. Investigations of spore biology can be challenging in oomycetes because their sporangia are physiologically active and change in response to environmental factors and aging. Whether sporangia from artificial media and plant lesions are functionally equivalent has been a topic of debate. To address these issues, we compared the transcriptomes and infection ability of sporangia from rye-sucrose media, potato and tomato leaflets, and potato tubers. Small differences were observed between the mRNA profiles of sporangia from all sources, including variation in genes encoding metabolic enzymes, cell-wall-degrading enzymes, and ABC transporters. Small differences in sporangia age also resulted in variation in the transcriptome. Taking care to use sporangia of similar maturity, we observed that those sourced from media or plant lesions had similar rates of zoospore release and cyst germination. There were also no differences in infection rates or aggressiveness on leaflets, based on single-spore inoculation assays. Such results are discordant with those of a recent publication in this journal. Nevertheless, we conclude that sporangia from plant and media cultures are functionally similar and emphasize the importance of using "best practices" in experiments with sporangia to obtain reliable results.

Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia.

The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m-3 for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m-3 to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.