Qualitative and quantitative monitoring of mycotoxigenic Fusarium species in Danish maize samples using Real - Time PCR and Microarrays.

Index I

Index

3.1.1 Taxonomy and phylogenesis ...13

3.1.2 Pathogenicity ...23

3.1.3 Factors affecting Fusarium spp. development and geographical distribution ...28

3.1.4 Toxicity: Fusarium mycotoxins ...30

3.2 Maize: one of the main Fusarium hosts...40

3.3 Molecular methods for the monitoring of mycotoxinogenic species ...43

3.3.1 Quantitative Real-Time PCR (QPCR) ...43

3.3.2 Microarray ...53

4 Experimental background ...60

5 Material and methods ...63

5.1 Plant Material ...63

5.2 Fungal isolates ...63

5.3 Total DNA Extraction ...69

5.3.1 DNA Extraction ...69

5.3.2 DNA Purification ...72

5.3.3 Gel electrophoresis ...73

5.4 Real-Time PCR (QPCR) ...74

Index

II

5.4.2 SYBR® Green analysis ...74

5.4.3 Validation of CTAB DNA extraction method ...80

5.5 DNA Cloning and sequencing ...81

5.6 Microarray ...84

5.6.1 Plant material and fungal isolates ...84

5.6.2 DNA Extraction ...84

5.6.3 PCR amplification ...84

5.6.4 Labelling of hybridization probe ...85

5.6.5 Capture probes ...85

5.6.6 Array production ...87

5.6.7 Microarray hybridization and washing ...88

5.6.8 Microarray Scanning ...90

6 Results...92

6.1 DNA extraction and gel electrophoresis ...92

6.2 Real-Time PCR ...93

6.2.1 SYBR® Green Analyses ...93

6.2.2 Validation of CTAB DNA extraction method ...119

6.3 Correlation between mycotoxins amount and Fusarium species contamination ...123 6.4 Microarray ...126 6.4.1 PCR amplification...126 6.4.2 Microarray scanning...127 7 Conclusions ...131 8 Appendix ...140 9 References ...149 10 Acknowledgments – Ringraziamenti...167