Abstract
Clonostachys rosea is a biological control agent against Fusarium graminearum in small grain cereals and maize. Infections with F. graminearum do not only reduce the yield but, due to the production of mycotoxins, also affect the entire value chain of food and feed. In addition, production of other secondary metabolites such as hydrophobins, also known as gushing inducers, may cause quality challenges for the malting and brewing industry. Sustainable disease control strategies using C. rosea are treatment of infected residues of the previous crop, direct treatment of the actual cereal crop or post-harvest treatment during malting processes. Follow-up of growth and survival of biocontrol organisms during these different stages is of crucial importance. In the current study, we developed a quantitative real-time PCR detection method that amends the currently available culture-dependent techniques by using TaqMan chemistry with a highly specific primer and probe set, targeting the actin gene. We established a sensitive assay that detects the biological control agent down to 100 genome copies per reaction, with PCR efficiencies between 90 and 100%. The specificity of the assay was confirmed against a panel of 30 fungal and 3 bacterial species including 12 members of the Fusarium head blight complex and DNA of barley, maize and wheat. The DNA of C. rosea was detected in Fusarium-infected maize crop residues that were either treated in the laboratory or in the field with C. rosea and followed its DNA throughout the barley malting process to estimate its growth during grain germination. We used a standardized DNA extraction protocol and showed that C. rosea can be quantified in different sample matrices. This method will enable the monitoring of C. rosea during experiments studying the biological control of F. graminearum on cereal crop residues and on cereal grains and will thus contribute to the development of a new disease control strategy.
Highlights
Clonostachys rosea is a mycoparasitic fungus able to attack many important plant pathogens in the rhizosphere and the phyllosphere including different Fusarium species (Jensen et al, 2000; Xue, 2003, Xue et al, 2009)
For all C. rosea isolates, the amplification produced a single product with a size of 150 bp
The C. byssicola isolate CBS 364.78 was certainly differentiated by its later amplification (+10 cycles compared with C. rosea), but 10 ng of DNA per reaction were still detected within the considered cycle threshold (Ct) range
Summary
Clonostachys rosea is a mycoparasitic fungus able to attack many important plant pathogens in the rhizosphere and the phyllosphere including different Fusarium species (Jensen et al, 2000; Xue, 2003, Xue et al, 2009). Catenulata (formerly Gliocladium catenulatum) that are found in soils living as parasites and decomposers (Schroers, 2001). Both forms are extensively described as potential biological control agents (BCA). Clonostachys rosea is well recognized for its ability to antagonize the mycotoxin producing fungus Fusarium graminearum (Schöneberg et al, 2015), the predominating causal agent of Fusarium head blight (FHB) of wheat and barley (Goswami and Kistler, 2004; Dweba et al, 2017). Considering the negative impacts on growers and buyers, Wilson et al (2018) estimated the economic loss caused by FHB and DON contamination including the cost for risk mitigation solely in the United States between 2016 and 2017 at US$1.47 billion for wheat and barley
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