TaqMan qPCR for Quantification of Clonostachys rosea Used as a Biological Control Agent Against Fusarium graminearum

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.

Biocontrol of potato early blight and suppression of Alternaria grandis sporulation by Clonostachys spp.

“…Field applications of C . rosea on crop debris before the next sowing season may reduce the inoculum of pathogens (Gimeno et al, 2019); this practice may be enhanced by addition of cover crops. In fact, field spraying of fungal biocontrol agents for reduction of pathogen inoculum on necrotic and dead plant tissues may be more easily achieved than plant protection against infection.…”

Microbiota succession during aerobic stability of maize silage inoculated with Lentilactobacillus buchneri NCIMB 40788 and Lentilactobacillus hilgardii CNCM‐I‐4785

“…Bionectriaceae has rarely been reported in corn silage but has been observed in sorghum before ensiling (Gonda et al, 2019). Interestingly, in this family, the Clonostachys related‐OTU is an epiphytic mycoparasitic fungus that can be used for biocontrol of Fusarium graminearum (Gimeno et al, 2019), and also has the enzymatic potential to degrade zearalenone (Ogunade et al, 2018). The heterogeneity of the fungal population in the LB + LH samples may be due to the number of fungal counts below the threshold of detection (<log 10 of 2.0) for the molds.…”

A versatile microfluidic platform measures hyphal interactions between Fusarium graminearum and Clonostachys rosea in real-time

“…Transformation by plasmid-mediated homologous integration facilitated the expression 44 , 45 . The antagonistic fungus Clonostachys rosea strain 016 was previously identified as a BCA against F. graminearum 25 , 46 , 47 . The fungus Trichoderma rossicum strain NEU135 was isolated from soil and showed positive interaction with soil-dwelling bacteria by facilitating their migration on hyphae 48 .…”