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J Fungi (Basel). 2019 May 14;5(2). pii: E39. doi: 10.3390/jof5020039.

A Universally Primed-Polymerase Chain Reaction (UP-PCR) Marker to Discriminate Clonostachys rosea ACM941 from Related Strains.

Author information

1
Aquatic and Crop Resource Development, National Research Council Canada, Ottawa, ON K1A 0R6, Canada. Zerihun.demissie@nrc.ca.
2
Adjuvants Plus Inc., 1755 Division Road N., Kingsville, ON N9Y 2Y8, Canada. bill@adjuvantsplus.com.
3
Aquatic and Crop Resource Development, National Research Council Canada, Ottawa, ON K1A 0R6, Canada. michele.loewen@nrc.ca.
4
Department of Biomedical and Molecular Sciences, Queens University, Kingston, ON K7L 3N6, Canada. michele.loewen@nrc.ca.
5
Department of Chemistry and Biomolecular Sciences, University of Ottawa, 75 Laurier Ave E, Ottawa, ON K1N 6N5, Canada. michele.loewen@nrc.ca.

Abstract

Clonostachys rosea strain ACM941 is an effective biocontrol agent against several crop diseases including Fusarium head blight. In anticipation of its increased relevance going forward, the development of a reliable DNA-based molecular marker to track it is essential. Universally primed-PCR (UP-PCR) has been used successfully to differentiate other C. rosea strains. Herein, the development of a UP-PCR marker for ACM941 is described. A combination of two primers (AS15 and L45) produced a ~450 bp fragment that was unique to ACM941 compared to other commercial biocontrol agents. Primers subsequently designed based on the obtained fragment also produced a similarly unique band from ACM941 alone. BLAST analysis of the amplified sequence did not yield any homologous sequence in available online databases or within the closely related C. rosea IK726 and CBS125111 strains' genomes. The specificity of this marker for ACM941 was validated against ten additional C. rosea strains isolated from Canada, with ACM941 producing the brightest band. Taken together, these results imply that the UP-PCR primers AS15 and L45 and the amplified fragment can be used to detect and monitor the ACM941 strain after its release into the environment.

KEYWORDS:

Clonostachys rosea; UP-PCR; bio-fertilizer; biocontrol agent; genetic marker; sequence characterized amplified regions (SCAR), universal primers

PMID:
31091661
DOI:
10.3390/jof5020039
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