Rapid Detection of Alternaria Species Involved in Pear Black Spot Using Loop-Mediated Isothermal Amplification

Abstract

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b (cyt-b) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl2 (4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri, A. solani, and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.