Abstract
Diaporthe species are fungal plant pathogens of many important crops. Seed decay is one of the most important diseases on soybean. It is caused by various species of the genus Diaporthe and responsible for significant economic damage. In central Europe the four species D. longicolla, D. caulivora, D. eres, and D. novem are considered the principal species of Diaporthe on soybean. Fast and accurate detection of these pathogens is of utmost importance. In this study four species-specific TaqMan primer-probe sets that can be combined into a quadruplex assay were designed based on TEF sequences. The specificity and efficiency of the primer-probe sets were tested using PCR products and genomic DNA from pure cultures of the four Diaporthe species and other soybean fungal pathogens. Our results indicate that the primer-probe sets DPCL, DPCC, DPCE, and DPCN allow discrimination of D. longicolla, D. caulivora, D. eres, and D. novem, respectively, and can be used to detect and quantify these four Diaporthe species in parallel using quadruplex real-time PCR. In addition, the quadruplex real-time PCR assay was evaluated on different plant materials including healthy and infected soybean seeds or seed lots, soybean stems, and soybean leaves. This assay is a rapid and effective method to detect and quantify Diaporthe species from samples relevant for disease control.
Highlights
Soybean (Glycine max L.) is one of the major sources of oil and protein in the world
Soybean seeds are heavily attacked by D. longicolla (Hobbs) and other species of the genus Diaporthe, which can significantly affect yield, quality, and stability of this industrial crop [2]
Single-spore isolates of ten strains of Diaporthe isolated from soybean seeds ([4] or received from Kristina Petrović (Institute of Field and Vegetable Crops, Novi Sad, Serbia), respectively) were used in this study (Table 1) for DNA preparation to test the specificity of the assay
Summary
Soybean (Glycine max L.) is one of the major sources of oil and protein in the world. Due to the expansion of soybean cultivation soybean diseases are becoming relevant in the new soybean growing areas. Among the most important pathogens of soybean, fungal species of the genus Diaporthe cause severe diseases including seed decay, pod and stem blight, and stem canker [1]. Soybean seeds are heavily attacked by D. longicolla (Hobbs) and other species of the genus Diaporthe, which can significantly affect yield, quality, and stability of this industrial crop [2]. Infected soybean seeds are smaller and lighter than healthy seeds and their germination rate is reduced, which leads to their economic devaluation.
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