Abstract
Early blight (EB), caused by the pathogen Alternaria solani, is a major threat to global potato and tomato production. Early and accurate diagnosis of this disease is therefore important. In this study, we conducted a loop-mediated isothermal amplification (LAMP) assay, as well as conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (RT-qPCR) assays to determine which of these techniques was less time consuming, more sensitive, and more accurate. We based our assays on sequence-characterized amplified regions of the histidine kinase gene with an accession number (FJ424058). The LAMP assay provided more rapid and accurate results, amplifying the target pathogen in less than 60 min at 63°C, with 10-fold greater sensitivity than conventional PCR. Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR. qPCR was the most sensitive among the assays evaluated, being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration (100 fg). The LAMP assay was more sensitive than conventional PCR, but less sensitive than nested PCR and qPCR; however, it was simpler and faster than the other assays evaluated. Despite of the sensitivity, LAMP assay provided higher specificity than qPCR. The LAMP assay amplified A. solani artificially, allowing us to detect naturally infect young potato leaves, which produced early symptoms of EB. The LAMP assay also achieved positive amplification using diluted pure A. solani culture instead of genomic DNA. Hence, this technique has greater potential for developing quick and sensitive visual detection methods than do other conventional PCR strategies for detecting A. solani in infected plants and culture, permitting early prediction of disease and reducing the risk of epidemics.
Highlights
Alternaria solani (Ellis and Martin), a pathogen causing early blight (EB), is a major threat to global potato and tomato production (Song et al, 2011)
In a more recent studies, Kumar et al, 2013 conducted experiment on real time quantitative analysis of A. solani resulting in 0.5 pg sensitivity targeting β-tubulin gene, which is 10 times more sensitive than the method developed in our study, which, provided feasibility of loop-mediated isothermal amplification (LAMP) assay in the field
Real time-qPCR assays have been used to identify seed-borne fungi in previous studies, revealing that it can be a beneficial technique in seed health testing and quarantine inspections, due to its high specificity and sensitivity (Guillemette et al, 2004; Lievens et al, 2006; Alaei et al, 2009; Debode et al, 2009) but it did not show promising specificity in contrast to our findings
Summary
Alternaria solani (Ellis and Martin), a pathogen causing early blight (EB), is a major threat to global potato and tomato production (Song et al, 2011). EB produces symptoms on leaves, stems, petioles, twigs, and fruits, leading to defoliation, twig drying, and premature fruit fall, which can result in 35–78% fruit yield losses (Datar and Mayee, 1982; Grigolli et al, 2011) Control measures such as prophylactic methods, fungicide application, and the use of relatively resistant tomato. Most assays used in A. solani detection depend on visual assessment of the symptoms, including lesion diameter measurement and spore load count (Bock et al, 2010). These methods are time consuming and often cannot distinguish fine-scale differences in inoculum loads and plant resistant levels
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
