Abstract
Didymella pinodella is the major pathogen of the pea root rot complex in Europe. This wide host range pathogen often asymptomatically colonizes its hosts, making the control strategies challenging. We developed a real-time PCR assay for the detection and quantification of D. pinodella based on the TEF-1 alpha gene sequence alignments. The assay was tested for specificity on a 54-isolate panel representing 35 fungal species and further validated in symptomatic and asymptomatic pea and wheat roots from greenhouse tests. The assay was highly consistent across separate qPCR reactions and had a quantification/detection limit of 3.1 pg of target DNA per reaction in plant tissue. Cross-reactions were observed with DNA extracts of five Didymella species. The risk of cross contamination, however, is low as the non-targets have not been associated with pea previously and they were amplified with at least 1000-fold lower sensitivity. Greenhouse inoculation tests revealed a high correlation between the pathogen DNA quantities in pea roots and pea root rot severity and biomass reduction. The assay also detected D. pinodella in asymptomatic wheat roots, which, despite the absence of visible root rot symptoms, caused wheat biomass reduction. This study provides new insights into the complex life style of D. pinodella and can assist in better understanding the pathogen survival and spread in the environment.
Highlights
Introduction published maps and institutional affilRoot rot of pea is one of the main factors contributing to the decline of cultivated area of this important crop worldwide
The primer pair DpinodellaTef targeting TEF-1 alpha gene selected for the insertion of locked nucleic acid (LNA) probe (Table 2) demonstrated high specificity to D. pinodella and no cross reactivity with D. pinodes and D. lethalis when evaluated on a 3 isolates/3 species exclusion panel (Table 3)
When DpinodellaTef was tested against the genomic DNAs from 35 non-target organisms cross-reactivity with DNA extracts of five Didymella species, namely D. heteroderae, D. aurea, D. microchlamydospora, D. protuberans and D. americana was observed
Summary
Introduction published maps and institutional affilRoot rot of pea is one of the main factors contributing to the decline of cultivated area of this important crop worldwide. Besides Fusarium spp., Aphanomyces euteiches has been recognized as a devastating pathogen in North America and France, when pea is grown in short rotations and wet soils [3,6]. Mycosphaerella pinodes), Rhizoctona solani, Pythium spp., and Sclerotinia sclerotiorum are an important part of the disease complex [2,7,8,9,10] Their prevalence and dominance may vary greatly depending on the geographical region and pedo-climatic conditions
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