This document was developed to provide a horizontal method for enumeration and genera\/species determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance with the EU Fertilising Products Regulation.<\/p>\n
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| 2<\/td>\n | undefined <\/td>\n<\/tr>\n | ||||||
| 9<\/td>\n | 1 Scope 2 Normative references 3 Terms and definitions <\/td>\n<\/tr>\n | ||||||
| 11<\/td>\n | 4 Methods for the quantification of mycorrhiza 4.1 General <\/td>\n<\/tr>\n | ||||||
| 13<\/td>\n | 4.2 How to prepare the initial sample 4.2.1 General 4.2.2 Liquid \u2013 water-based formulations 4.2.3 Liquid \u2013 oil-based (emulsifiable concentrate EC) formulations 4.2.4 Solid \u2013 wettable powder (WP) formulations 4.2.5 Solid \u2013 water dispersible granules (WDG) formulations <\/td>\n<\/tr>\n | ||||||
| 14<\/td>\n | 4.2.6 Solid \u2013 pellets, granules, microgranules (slow release) formulations 4.2.7 Solid \u2013 substrate 4.3 Enumeration methods 4.3.1 General 4.3.2 Method N\u00b0 1: Spore isolation and counting MTT 4.3.2.1 Procedure for enumeration of spores <\/td>\n<\/tr>\n | ||||||
| 15<\/td>\n | 4.3.2.2 Viability of spores <\/td>\n<\/tr>\n | ||||||
| 16<\/td>\n | 4.3.3 Method N\u00b0 2: Procedure for clearing and staining root samples and enumeration of vesicles in the stained root samples 4.3.3.1 Materials and equipment <\/td>\n<\/tr>\n | ||||||
| 17<\/td>\n | 4.3.3.2 Procedure <\/td>\n<\/tr>\n | ||||||
| 19<\/td>\n | 4.3.4 Enumeration of the total number of UPM in the product using Method N\u00b01 + Method N\u00b02 4.3.5 Method N\u00b03: Endomycorrhiza bioassay 4.3.5.1 Principle of the MPN (Most Probable Number of mycorrhizal propagule) <\/td>\n<\/tr>\n | ||||||
| 24<\/td>\n | 4.3.5.2 Protocols and examples <\/td>\n<\/tr>\n | ||||||
| 27<\/td>\n | 4.3.6 Method N\u00b04: Ectomycorrhiza and ericoid count 4.3.6.1 General 4.3.6.2 Sample preparation 4.3.6.3 Serial dilution 4.3.6.4 Plate counts of ectomycorrhiza in sterile diluent <\/td>\n<\/tr>\n | ||||||
| 28<\/td>\n | 4.3.6.5 Spread-plate counting with modified Melin Norkrans Medium (MNM) agar 4.3.6.6 Calculation <\/td>\n<\/tr>\n | ||||||
| 31<\/td>\n | 5 Molecular characterization and identification of mycorrhiza isolates 5.1 General 5.2 Materials and equipment 5.3 Method for the molecular characterization and identification of mycorrhiza isolates 5.3.1 Spores cleaning <\/td>\n<\/tr>\n | ||||||
| 32<\/td>\n | 5.3.2 DNA extraction <\/td>\n<\/tr>\n | ||||||
| 33<\/td>\n | 5.3.3 Preparation for PCR 5.3.3.1 Primer sequences 5.3.3.2 Master mix preparation <\/td>\n<\/tr>\n | ||||||
| 34<\/td>\n | 5.3.3.3 Procedure for running PCR 5.3.3.4 PCR program for each primer set <\/td>\n<\/tr>\n | ||||||
| 35<\/td>\n | 5.3.4 Preparation for gel-electrophoresis 5.3.4.1 Materials and equipment 5.3.4.2 Procedure <\/td>\n<\/tr>\n | ||||||
| 36<\/td>\n | 5.3.5 Direct sequencing (outsourced sequencing lab) 6 Method of molecular characterization and identification for ectomycorrhiza and ericoid 6.1 General <\/td>\n<\/tr>\n | ||||||
| 37<\/td>\n | 6.2 Materials 6.2.1 Fungal material 6.2.2 Molecular biology kits\/chemicals <\/td>\n<\/tr>\n | ||||||
| 38<\/td>\n | 6.2.3 Equipment 6.3 Detailed description of method 6.3.1 Material preparation <\/td>\n<\/tr>\n | ||||||
| 39<\/td>\n | 6.3.2 DNA extraction and quality check 6.3.3 PCR amplification of ITS sequences <\/td>\n<\/tr>\n | ||||||
| 40<\/td>\n | 6.3.4 Gel electrophoresis and PCR product visualization <\/td>\n<\/tr>\n<\/table>\n","protected":false},"excerpt":{"rendered":" Plant biostimulants. Determination of mycorrhizal fungi<\/b><\/p>\n |