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

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.

Clonostachys rosea ‘omics profiling: identification of putative metabolite-gene associations mediating its in vitro antagonism against Fusarium graminearum

“…6 A – genome alignment). This was in agreement with previous reports where the genetic similarity between ACM941 and 88–710 was described using different molecular methods [ 4 , 5 ]. In particular, previous reports showed that the ACM941 genome harbours genes encoding one unique NRPS, one unique hybrid and two unique PKS genes which were missing in 88–710, while the 88–710 genome encoded two unique PKS homologs [ 5 ].…”

“…C. rosea strain 88–710 was patented in both the United States of America and Canada for its benefits in promoting plant vigor, health, growth and yield [ 3 ]. Despite the differences in their patented applications, the two strains are genetically closely related [ 4 , 5 ] and their beneficial properties display strong overlap. C. rosea strain 88–710 is well regarded for its strong PGP ability but is also noted to induce disease resistance against economically important fungal pathogens in different crops including cereals, vegetables, peas, etc.…”

“…Consistent with their functional similarities, the genomes of the C. rosea strains ACM941 and 88–710 are highly conserved [ 4 , 5 ]. Thus, the subtle differences in relative mycoparasitism and PGP activities between these strains are likely the result of fairly limited variation in their encoded genetic material, which in turn manifests as differential metabolite accumulation.…”

Molecular Study on Cladosporium Species Isolated From Air of Cairo, Using Universally Primed-PCR (Up) and Internal Transcribed Spacer (Its) PCR Techniques

“…Some of the advantages of UP-PCR are the use of relatively high annealing temperatures, fast ramping and relatively long primers, features that seem to enhance the reproducibility which many have found to be problematic with RAPDs (Lubeck et al, 2005). Other advantages are the resulting banding profiles which consist of higher numbers of bands than most RAPDs, facilitating identification of specific markers, and at the same time showing species conservative bands (Demissie et al, 2019). A variant of the UP-PCR technique is UP-PCR product cross hybridization assay that facilitates investigation of sequence similarity (homology) of UP-PCR products.…”