DNA extraction ITS amplication and sequencing ITS identifies to species level Ceratocystis fagacearum Ceratocystis virescens Melampsora medusae (only to species) Mycosphaerella laricis-leptolepidis Phaeoramularia angolensis Phytophthora kernoviae Puccinia horiana Puccinia pittieriana Stenocarpella macrospora Stenocarpella maydis Thecaphora solani ACT, CAL, COI, TEF or TUB2 required for species level Ceratocystis fimbriata f. sp. platani - TEF required Davidiella (Mycosphaerella) populorum - TUB2 required Didymella ligulicola - ACT required Monilinia fructicola - TEF required Mycosphaerella dearnessii - TUB2 required Mycosphaerella gibsonii - TUB2 required Mycosphaerella pini - TEF required Phoma andigena - ACT required Phoma tracheiphila - ACT required Phyllosticta citricarpa - TEF required Phytophthora fragariae var. fragariae - COI required Phytophthora fragariae var. rubi - COI required Phytophthora lateralis - COI required Phytophthora ramorum - COI required Septoria lycopersici var. malagutii - TUB2 required Verticillium albo-atrum (only to species) - CAL required Verticillium dahliae (only to species) - CAL required ITS protocol ACT protocol TEF protocols CAL protocol TUB2 protocol COI protocol High quality version of picture and protocols
Fungi DNA extraction Extraction protocols for DNA isolation from fungal mycelium Points to consider Mycelium of pure cultures is removed from the agar surface (approximately 2 cm2) using a sterile scalpel or micro pestle and used as starting material for the DNA extraction. DNA is extracted using the UltraClean Microbial DNA Isolation Kit (MoBio) or DNeasy Plant Mini Kit (Qiagen) following the manufacturer s instructions. Particular care should be given to ensure the sample is adequately homogenised in the initial step. Micro pestles can be used to grind fungal tissue but specialist equipment can be used when high throughput is required (e.g. Retsch Mixer Mill MM301 or Precellys tissue homogenizer). DNA is eluted in 50 µl elution buffer (provided in the isolation kits). After DNA extraction, no DNA clean-up is required. Either use extracted DNA immediately or store it at 20 o C or below until use.
Fungi ITS amplification and sequencing PCR for 550-1,700 bp of the internal transcribed spacer regions of the nrrna genes for species allocation of majority of EU regulated fungi PCR-Sequencing approx. 550-1,700 bp of the ITS region ITS4 TCC TCC GCT TAT TGA TAT GC X X ITS5 GGA AGT AAA AGT CGT AAC AAG G X X V9G TTA CGT CCC TGC CCT TTG TA (X) LR5 TCC TGA GGG AAA CTT CG (X) Remarks: 550 (ITS5/ITS4) -1700 (V9G/LR5) bp. 5 min 94 C, 40x (45 sec 94 C, 30 sec 52 C, 90 sec 72 C), 6 min 72 C, quick cooling to room. The annealing can be decreased from 52 C to 48 C if problems with ITS amplification is encountered using a different DNA Polymerase. Cycle sequencing reactions are performed using the primers ITS5 and ITS4 in separate reactions. Although the combination of ITS5 / ITS4 works for many fungi, the combination of ITS5 / LR5 or V9G / LR5 could be useful alternatives. When these alternative amplification primers are used, ITS5 and ITS4 are still used as sequencing primers. Master mix per reaction (µl) 6 reactions final concentration DNase and RNase free water 14,73 88,38 µl 5x Colorless GoTaq Flexi buffer (Promega) 5,0 30,0 µl 1x MgC l 2 (25 mm, Promega) 2,0 12,0 µl 2 mm dntp's (10 mm each) 0,15 0,90 µl 0.06 mm each ITS5 (10 µm) 0,5 3,0 µl 0.2 µm ITS4 (10 µm) 0,5 3,0 µl 0.2 µm GoTaq DNA polymerase (5 U/µl, Promega) 0,12 0,72 µl 0.6 Unit subtotal 23,0 138,0 µl template 2,0 total 25,0
Fungi ACT amplification and sequencing PCR for approximately 330 bp of the partial actin region for species allocation of Phoma (-like) species PCR-Sequencing approx. 330 bp of the partial ACT region ACT-512F ATG TGC AAG GCC GGT TTC GC X X ACT-783R TAC GAG TCC TTC TGG CCC AT X X Approximately 330 bp. 5 min 94 C, 40x (45 sec 94 C, 30 sec 52 C, 90 sec 72 C), 6 min 72 C, quick cooling to room Cycle sequencing reactions are performed using the primers ACT-512F and ACT-783R in separate reactions. DNase and RNase free water 14.73 88.38 µl 5x Colorless GoTaq Flexi buffer (Promega) 5.0 30.0 µl 1x MgCl 2 (25 mm, Promega) 2.0 12.0 µl 2 mm dntp's (10 mm each) 0.15 0.90 µl 0,06 mm each ACT-512F (10 µm) 0.5 3.0 µl 0,2 µm ACT-783R (10 µm) 0.5 3.0 µl 0,2 µm Taq DNA polymerase (5 U/µl, Promega) 0.12 0.72 µl 0,6 Unit subtotal 23.0 138.0 µl
Fungi CAL amplification and sequencing PCR for 470 bp of the partial calmodulin region for species allocation of Verticillium PCR-Sequencing approx. 470 bp of the partial CAL region CAL-228F GAG TTC AAG GAG GCC TTC TCC C X X CAL-737R CAT CTT TCT GGC CAT CAT GG X X Approximately 470 bp. 2 min 94 C, 35x (60 sec 94 C, 30 sec 50 C, 90 sec 72 C), 10 min 72 C, quick cooling to room Cycle sequencing reactions are performed using the primers CAL-228F and CAL-737R in separate reactions. DNase and RNase free water 14.73 88.38 µl 5x Colorless GoTaq Flexi buffer (Promega) 5.0 30.0 µl 1x MgCl 2 (25 mm, Promega) 2.0 12.0 µl 2 mm dntp's (10 mm each) 0.15 0.90 µl 0,06 mm each CAL-228F (10 µm) 0.5 3.0 µl 0,2 µm CAL-737R (10 µm) 0.5 3.0 µl 0,2 µm Taq DNA polymerase (5 U/µl, Promega) 0.12 0.72 µl 0,6 Unit subtotal 23.0 138.0 µl
Fungi COI amplification and sequencing PCR for 700 bp of the partial cytochrome oxidase I region for species allocation of Phytophthora PCR-Sequencing approx. 700 bp of the partial COI region OomCoxI-Levup TCA WCW MGA TGG CTT TTT TCA AC X X OomCoxI-Levlo CYT CHG GRT GWC CRA AAA ACC AAA X X Approximately 700 bp. 2 min 95 C, 35x (30 sec 95 C, 30 sec 52 C, 1 min 72 C), 7 min 72 C, quick cooling to 10 C Cycle sequencing reactions are performed using the primers OomCoxI-Levup and OomCoxI-levlo in separate reactions. DNase and RNase free water 16,20 97,20 µl 5x Colorless GoTaq Flexi buffer (Promega) 2,50 15,0 µl 1x MgCl 2 (25 mm, Promega) 1,2 7,2 µl 2 mm DMSO 1,4 8,4 µl dntp's (10 mm each) 0,50 3,00 µl 0,06 mm each OomCoxI-Levup (10 µm) 0,50 3,0 µl 0,2 µm OomCoxI-Levlo (10 µm) 0,50 3,0 µl 0,2 µm Taq DNA polymerase (5 U/µl, Promega) 0,20 1,20 µl 0,6 Unit subtotal 23,0 138,0 µl template 2,0 total 25,0
Fungi Ceratocystis TEF amplification and sequencing PCR for 430 bp of the partial translation elongation factor 1-alpha region as additional check for species allocation of Ceratocystis fimbriata f. sp. platani and Ceratocystis virescens. PCR-Sequencing approx. 430 bp of the partial TEF region EFCF1 AGT GCG GTG GTA TCG ACA AG X X EFCF6 CAT GTC ACG GAC GGC GAA AC (X) EFCF2 TGC TCA CGG GTC TGG CCA T X X Remarks: 430 bp. 5 min 94 C, 40x (45 sec 94 C, 30 sec 52 C, 120 sec 72 C), 6 min 72 C, quick cooling to room Cycle sequencing reactions are performed using the primers EFCF1 and EFCF2 in separate reactions. Dimethyl sulfoxide for molecular biology >99.9% ( DMSO, Sigma) could be required as supplement if other DNA Polymerases (e.g. Bioline) are used - substitute 1.4 µl of MGW with 1.4 µl DMSO in a 25 µl total reaction volume DNase and RNase free water 14.73 88.38 µl 5x colorless GoTaq Flexi buffer (Promega) 5.0 30.0 µl 1x MgCl 2 (25 mm, Promega) 2.0 12.0 µl 2 mm dntp's (10 mm each) 0.15 0.90 µl 0.06 mm each EFCF1 (10 µm) 0.5 3.0 µl 0.2 µm EFCF2 (10 µm) or EFCF6 (10 µm) 0.5 3.0 µl 0.2 µm GoTaq DNA polymerase (5 U/µl, Promega) 0.12 0.72 µl 0.6 Unit subtotal 23.0 138.0 µl
Fungi Monilinia, Mycosphaerella and Phyllosticta TEF amplification and sequencing Q-bank, protocol version January 2015 PCR for 400 bp of the partial translation elongation factor 1-alpha region for species allocation of Scirrhia pini (=Mycosphaerella pini, with anamorph Dothistroma septosporum), Dothistroma pini, as well as Monilinia fructicola and Phyllosticta citricarpa. PCR-Sequencing approx. 400 bp of the partial TEF region EF1-728F CAT CGA GAA GTT CGA GAA GG X X EF-2 GGA RGT ACC AGT SAT CAT GTT X X EF1-986R TAC TTG AAG GAA CCC TTA CC (X) (X) Remarks: 400 bp. 5 min 94 C, 40x (45 sec 94 C, 30 sec 52 C, 90 sec 72 C), 6 min 72 C, quick cooling to room Cycle sequencing reactions are performed using the primers EF1-728F and EF-2 in separate reactions. Dimethyl sulfoxide for molecular biology >99.9% ( DMSO, Sigma) could be required as supplement if other DNA Polymerases (e.g. Bioline) are used - substitute 1.4 µl of MGW with 1.4 µl DMSO in a 25 µl total reaction volume. As an alternative reverse primer, EF1-986R can be used but will result in a shorter fragment. DNase and RNase free water 14.73 88.38 µl 5x Colorless GoTaq Flexi buffer (Promega) 5.0 30.0 µl 1x MgCl 2 (25 mm, Promega) 2.0 12.0 µl 2 mm dntp's (10 mm each) 0.15 0.90 µl 0,06 mm each EF1-728F (10 µm) 0.5 3.0 µl 0,2 µm EF-2 (10 µm) 0.5 3.0 µl 0,2 µm Taq DNA polymerase (5 U/µl, Promega) 0.12 0.72 µl 0,6 Unit subtotal 23.0 138.0 µl
Fungi TUB2 amplification and sequencing PCR for 400 bp of the partial beta-tubulin region for species allocation of Mycosphaerella gibsonii and Septoria lycopersici var. malagutii PCR-Sequencing approx. 400 bp of the partial TUB region TUB2Fd GTB CAC CTY CAR ACC GGY CAR TG X X TUB4Rd CCR GAY TGR CCR AAR ACR AAG TTG TC X X 400 bp. 5 min 94 C, 40x (45 sec 94 C, 30 sec 52 C, 90 sec 72 C), 6 min 72 C, quick cooling to room Cycle sequencing reactions are performed using the primers TUB2Fd and TUB4Rd, in separate reactions DNase and RNase free water 14.73 44.19 µl per reaction (µl) 3 reactions final concentration 5x Colorless GoTaq Flexi buffer (Promega) 5.0 15.0 µl 1x MgCl 2 (25 mm, Promega) 2.0 6.0 µl 2 mm dntp's (10 mm) 0.15 0.50 µl 0.06 mm TUB2Fd (10 µm) 0.5 1.5 µl 0.2 µm TUB4Rd (10 µm) 0.5 1.5 µl 0.2 µm GoTaq DNA polymerase (5 U/µl, Promega) 0.12 0.36 µl 0.6 Unit subtotal 23.0 69.1 µl