A multiplex qPCR to detect and differentiate A. alternaria, A. tenuissima and A. arborescens collected from potato brown spot lesions - CLICK FOR VIDEO
Historically, brown leaf spot of potato (Solanum tuberosum L.) was thought to be caused by Alternaria alternata. However, recent literature has indicated that brown spot in Northwest US is caused by several small spored Alternaria spp. Our laboratory has collected putative A. alternata sensu lato isolates from several states in US for over 20 years and from Canada in 2020. An NCBI BLAST nucleotide match from OPA gene sequences of these isolates identified three species of small spore Alternaria (SSA), A. alternata sensu stricto, A. tenuissima and A. arborescens. These three species are similar morphologically, displaying overlapping and variable characteristics, and have indistinguishable disease symptomatology. Moreover, preliminary data from greenhouse evaluations suggests these three species may differ in sensitivity to SDHI fungicides registered on potato. This prompted us to develop a reliable molecular method to differentiate the three SSA species, which can in turn help in developing and incorporating appropriate disease management strategies. A multiplex Taqman real-time PCR was developed for detecting A. alternata (E=98.4%, R2 value=0.98), A. tenuissima (E=102.3%, R2 value=0.98) and A. arborescens (E=94.9%, R2 value=0.98). Multiplex real-time PCR differentiation was 99% successful when compared to sequencing the OPA gene. The real-time PCR detection system developed to differentiate SSA was highly specific and did not amplify other closely-related phytopathogenic fungi. Preliminary results indicate that all three species cause leaf spot in the US and Canada. In both populations, A. alternata represented greater than 60% of isolates recovered. Isolates of all three species were found as early as 2000 in the US. A real time primer-probe set (OPAF4/R4/P4) targeting a 180 bp region in OPA was also developed to distinguish between A. solani and SSA without solely relying on the morphological characteristics. These assays will facilitate accurate characterization of the potato foliar pathogen complex, aiding in disease management.