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Fingerprinting techniques as tools towards molecular quality control of Pseudozyma flocculosa
Sophie J Caron1, Tyler J Avis, Teun Boekhout
1Département de Phytologie, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Quebec G1K 7P4, Canada.
Abstract:
In an effort to meet the stringent requirements towards registration of microorganisms as biopesticides, several molecular techniques were tested as part of a strategy to develop a quality control system for Pseudozyma flocculosa, the active ingredient of Sporodex, a biofungicide used for the control of powdery mildew fungi. In the first approach, multiplex PCR fingerprints generated by three sets of primers allowed differentiation of several isolates of P. flocculosa from closely related species, or genera such as Tilletiopsis. The same set of primers was used in quality control experiments and revealed fungal contamination that was otherwise not observed by standard culture and microscopy techniques. In addition, the use of random amplified microsatellites generated by (GT)n and (CCA)n primers was applied as a measure of possible genetic variation over 65 repeated subcultures of P. flocculosa. Finally, a novel technique was developed and named reverse intron PCR (RIP), based on the presence of an intron revealed by partial sequencing of mtLSU of P. flocculosa. RIP allowed not only differentiation of P. flocculosa from other species but also separated the isolates of different origins within P. flocculosa. This new technique could find useful applications in phylogenetic studies of closely related fungal species and isolates.
Insights
Molecular techniques like multiplex PCR and reverse intron PCR (RIP) were developed to ensure the quality of Pseudozyma flocculosa, a biofungicide. These methods effectively identify the fungus and detect contamination, aiding biopesticide registration.
Area of Science:
- Agricultural Microbiology
- Molecular Biology
- Mycology
Background:
- Biopesticide registration requires stringent quality control for microbial agents.
- Pseudozyma flocculosa is the active ingredient in Sporodex, a biofungicide for powdery mildew control.
- Developing reliable molecular methods is crucial for quality assurance.
Purpose of the Study:
- To develop a quality control system for Pseudozyma flocculosa using molecular techniques.
- To differentiate P. flocculosa isolates from related species and detect contamination.
- To assess genetic variation within P. flocculosa and develop novel identification methods.
Main Methods:
- Multiplex PCR with specific primers for P. flocculosa identification and contamination detection.
- Random Amplified Microsatellites (RAMS) using (GT)n and (CCA)n primers to assess genetic variation.
- Development of Reverse Intron PCR (RIP) based on mtLSU sequencing for isolate differentiation.
Main Results:
- Multiplex PCR differentiated P. flocculosa from Tilletiopsis and revealed hidden fungal contamination.
- RAMS indicated genetic stability over 65 subcultures of P. flocculosa.
- RIP successfully distinguished P. flocculosa from other species and separated isolates of different origins.
Conclusions:
- Molecular techniques, particularly multiplex PCR and RIP, are effective for P. flocculosa quality control.
- These methods enhance biopesticide registration by ensuring purity and identity.
- RIP offers a novel approach for fungal phylogenetic studies and isolate discrimination.
