A qPCR-based method for detection and quantification of <em>Polystigma amygdalinum</em>, the cause of red leaf blotch of almond

Abstract

Red leaf blotch of almond, caused by the fungus Polystigma amygdalinum, results in early defoliation of trees and decreases fruit production in many Mediterranean and Middle Eastern countries. A latent period of 30–40 d has been reported for this pathogen before disease symptoms are expressed, which makes targeted fungicide control difficult. A quantitative real-time PCR detection method was developed to detect and quantify the fungus in biological samples, to assist early detection. A primer pair was designed based on the ITS region of the fungal rDNA, and this was highly specific and sensitive, enabling detection of a minimum of 12 pg of P. amygdalinum DNA and seven ascospores in artificially-prepared ascospore suspensions. A protocol was also developed to quantify ascospores on plastic tapes which are commonly used in volumetric air samplers. The detection limit for these samples was the same as for ascospore quantification in aqueous suspensions. The pathogen was also quantified in naturally infected leaves showing different stages of disease development, including early stages of leaf infection with doubtful visual identifications. Future practical applications of the method developed here are discussed in view of improving the management of red leaf blotch of almond.