TY - JOUR
T1 - TaqMan qPCR for quantification of clonostachys rosea used as a biological control agent against fusarium graminearum
AU - Gimeno, Alejandro
AU - Sohlberg, Elina
AU - Pakula, Tiina
AU - Limnell, Jenni
AU - Keller, Beat
AU - Laitila, Arja
AU - Vogelgsang, Susanne
N1 - We thank Irene Bänziger and Andreas Kägi at Agroscope as well as several interns for their valuable help in preparation of the experimental samples from Switzerland. We would also like to thank Jürgen Köhl from the Wageningen University for providing the fungal strain 016, Claudio Altomare from the Italian National Research Council for providing the fungal strain ITEM908 (VTT D-161648), William Brown from the Adjuvants Plus Inc. for providing the fungal strain ACM941 (VTT D-161647) and Florian Freimoser at Agroscope for providing the fungal strains NBB2.9 (CCOS 1865) and SHA77.3 (CCOS 1864).
Publisher Copyright:
Copyright © 2019 Gimeno, Sohlberg, Pakula, Limnell, Keller, Laitila and Vogelgsang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - Biological control agent
KW - Clonostachys rosea
KW - Fusarium graminearum
KW - Fusarium head blight
KW - MycoKey
KW - QPCR
UR - http://www.scopus.com/inward/record.url?scp=85069527025&partnerID=8YFLogxK
U2 - 10.3389/fmicb.2019.01627
DO - 10.3389/fmicb.2019.01627
M3 - Article
C2 - 31379780
AN - SCOPUS:85069527025
SN - 1664-302X
VL - 10
SP - 1627
JO - Frontiers in Microbiology
JF - Frontiers in Microbiology
IS - JULY
M1 - 1627
ER -