Application of Loop-Mediated Isothermal Amplification in an Early Warning System for Epidemics of an Externally Sourced Plant Virus
Abstract
:1. Introduction
2. Results
2.1. GPA-Specific LAMP Protocol Development and Validation
2.2. LAMP Protocol Field Validation
2.3. Aphid Numbers, LAMP Detection and Virus Incidence
2.3.1. 2017 Sites
2.3.2. 2018 Sites
2.4. Predicting TuYV Epidemics
3. Discussion
4. Materials and Methods
4.1. Crude, Total RNA and Total DNA Extraction
4.2. LAMP
4.3. GPA-Specific LAMP Protocol Development and Validation
4.3.1. Primer Design
4.3.2. Specificity
4.3.3. Sensitivity
4.4. RT-PCR
4.5. ELISA
4.6. LAMP Protocol Field Validation
4.6.1. Site Location
4.6.2. Aphid Trapping and Testing
4.6.3. Green-Bridge Host and Canola Crop Testing
4.7. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Dilution | Positives | Mean Time to Positive (min) |
---|---|---|
1/1 a | 4/4 b | 19.6 ± 0.9 c |
1/10 | 4/4 | 19.2 ± 0.7 |
1/20 | 4/4 | 17.3 ± 0.6 |
1/50 | 4/4 | 19.1 ± 0.5 |
1/100 | 4/4 | 21.5 ± 0.7 |
0/100 | 4/4 | - |
Negative | 0/4 | - |
Positive | 4/4 | 17.7 ± 0.5 |
Location | Year | Zone a | TuYV Detection in Broad-Leaf Weeds b | Mean Aphids Per Trap Side Pre-Emergence to GS15 c | Trap Sides with TuYV-Carrying Aphids Pre-Emergence to GS15 (%) | Trap Sides with GPA Pre-Emergence to GS15 (%) | Crop TuYV Incidence at GS30 (%) d | Crop TuYV Incidence at GS75 (%) |
---|---|---|---|---|---|---|---|---|
Irish Town | 2017 | 1 | 100% VC | 5 | 32 | 32 | 60 | 100 |
Kojaneerup | 2017 | 2 | Nil | 2 | 18 | 25 | 27 | 47 |
Coomalbidgup | 2017 | 3 | Nil | 8 | 10 | 21 | 5 | 83 |
Jerramungup | 2017 | 2 | Nil | 2 | 0 | 22 | 4 | 38 |
Kendenup | 2017 | 2 | Nil | 2 | 0 | 9 | 4 | 27 |
Wongamine | 2017 | 1 | Nil | 2 | 0 | 19 | 1 | 41 |
Munglinup | 2017 | 3 | Nil | 4 | 0 | 25 | 1 | 27 |
South Stirlings | 2017 | 2 | <1% WR | 2 | 4 | 0 | 1 | 25 |
Mount Barker | 2017 | 2 | Nil | 5 | 0 | 29 | 1 | 23 |
Esperance Downs | 2017 | 3 | Nil | 9 | 0 | 8 | 1 | 12 |
Nunile | 2017 | 1 | <1% WR | 2 | 0 | 6 | 1 | 11 |
Gairdner | 2017 | 2 | Nil | 3 | 0 | 0 | 0 | 75 |
Wellstead | 2017 | 2 | Nil | 2 | 4 | 0 | 0 | 43 |
Gibson | 2017 | 3 | Nil | 5 | 0 | 10 | 0 | 20 |
Esperance Downs | 2018 | 3 | Nil | 7 | 32 | 39 | 88 | 100 |
Munglinup | 2018 | 3 | Nil | 10 | 43 | 32 | 87 | 100 |
Gibson | 2018 | 3 | <1% SC | 14 | 62 | 33 | 83 | 100 |
Dalyup | 2018 | 3 | - | 11 | 58 | 33 | 83 | 100 |
Coomalbidgup | 2018 | 3 | Nil | 11 | 67 | 14 | 79 | 100 |
Grass Patch | 2018 | 3 | Nil | 6 | 54 | 29 | 62 | 100 |
Jerramungup | 2018 | 2 | Nil | 1 | 15 | 15 | 6 | 63 |
Gairdner | 2018 | 2 | Nil | 0 | 0 | 0 | 0 | - |
Nunile | 2018 | 1 | Nil | 2 | 0 | 8 | 0 | 4 |
Wongamine | 2018 | 1 | Nil | 1 | 0 | 0 | 0 | 4 |
South Stirlings | 2018 | 2 | Nil | 1 | 0 | 0 | 0 | 3 |
Bejoording | 2018 | 1 | Nil | 1 | 0 | 4 | 0 | 2 |
Mount Barker | 2018 | 2 | Nil | 0 | 0 | 0 | 0 | 2 |
Coondle | 2018 | 1 | - | 0 | 0 | 0 | 0 | 1 |
Tenterdon | 2018 | 2 | Nil | 0 | 0 | 0 | 0 | 1 |
Kendenup | 2018 | 2 | Nil | 0 | 0 | 0 | 0 | 0 |
Primer | Type | Position on Genome a | Length (nt) | Sequence 5′-3′ |
---|---|---|---|---|
F3 | Forward outer | 897-914 | 18 | TGATGTCACCCTCCTCCG |
B3 | Backward outer | 1084-1102 | 19 | AGTGTCCTCCTTCCGTGTG |
FIP | Forward inner | 970-991 and 926-945 | 42 | TGCATTTTGCTAGGTTGGCAGCATTGGGAAGGACTGTTAGGC |
BIP | Backward inner | 1019-1040 and 1064-1083 | 42 | ATGGCTGGGTTAGCGGTTATGCGCTCAGGACCATAACATCGG |
LF2 | Loop forward outer | 946-964 | 19 | TGACGTTGGCCGCTTTACA |
LB2 | Loop backward outer | 1041-1062 | 22 | CGAGATTGTAGGCTCAGAAGGT |
Primer | Type | Position on Gene a | Length (nt) | Sequence 5′-3′ |
---|---|---|---|---|
F3 | Forward outer | 339-356 | 18 | TACAGCCGTCAGCAAGGA |
B3 | Backward outer | 534-553 | 20 | CAGTCTGATCAGAAGGCGAG |
FIP | Forward inner | 406-426 and 362-281 | 41 | TAAGTTACGGCCGGTGTCCGTCCAGGGATTTCATGGCAGTG |
BIP | Backward inner | 435-455 and 495-514 | 41 | CGATGTTACCAAGTGGCCCGCGTACCAAAGCCAATCCTCGG |
LF2 | Loop forward outer | 382-403 | 22 | GATCCCTGACTACATCTGGGAA |
LB2 | Loop backward outer | 456-479 | 24 | AAAGCTGTTGCAATACAATGTGCC |
Year | Zone a | Location | February to April Rainfall (mm) | Cultivar | Neonicotinoid Seed Treatment | Sowing Date | Date Aphid Trap First Deployed b | Green-Bridge Species Tested (Number of Plants) c |
---|---|---|---|---|---|---|---|---|
2017 | 1 | Irish Town | 123 | ATR Bonito | No | 1-May | 8-Apr | VC (20) |
2017 | 1 | Nunile | 123 | InVigor T4510 | Yes | 16-May | 8-Apr | AM (100), BN (100), CG (100), WR (100) |
2017 | 1 | Wongamine | 123 | Pioneer 43Y23 | Yes | 12-May | 8-Apr | AM (100), CG (100), VC (100), WR (100) |
2017 | 2 | Gairdner | 160 | ATR Bonito | No | 24-Apr | 23-Mar | AM (100), BN (100), SC (100), Se (100), VC (100), WR (100) |
2017 | 2 | Jerramungup | 131 | ATR Bonito | Yes | 20-Apr | 23-Mar | BN (100), CG (100), SC (100), VC (100), WR (100) |
2017 | 2 | Kendenup | 102 | ATR Mako | Yes | 8-May | 23-Mar | BN (100), FF (100) |
2017 | 2 | Kojaneerup | 137 | ATR Mako | No | 13-Jun | 23-Mar | AM (100), BN (100), CG (100), SC (100), Se (100), VC (100), WR (100) |
2017 | 2 | Mount Barker | 185 | ATR Mako | Yes | 1-May | 23-Mar | Nil |
2017 | 2 | Wellstead | 243 | Thumper TT | Yes | 20-Apr | 23-Mar | BN (100), CG (100), Se (100), SC (100) |
2017 | 2 | South Stirlings | 137 | Nuseed GT-53 | Yes | 22-Apr | 23-Mar | WR (100) |
2017 | 3 | Coomalbidgup | 256 | ATR Wahoo | Yes | 25-Apr | 30-Mar | BN (100), CG (100), VC (100), SC (100) |
2017 | 3 | Esperance Downs | 221 | ATR Mako | Yes | 30-Apr | 30-Mar | Se (100) |
2017 | 3 | Gibson | 221 | ATR Bonito | Yes | 28-Apr | 30-Mar | WR (100) |
2017 | 3 | Munglinup | 256 | Hyola 559TT | Yes | 18-Apr | 30-Mar | BN (100), WR (100) |
2018 | 1 | Bejoording | 16 | Nuseed GT-53 | Yes | 21-Apr | 20-Mar | AM (3), WR (62) |
2018 | 1 | Coondle | 16 | ATR Bonito | Yes | 14-May | 10-Jul | - |
2018 | 1 | Nunile | 16 | Pioneer 44Y27 | Yes | 26-Apr | 20-Mar | CST (10), So (100), WR (30) |
2018 | 1 | Wongamine | 16 | Pioneer 44Y27 | Yes | 25-Apr | 20-Mar | So (50), WR (6) |
2018 | 2 | Gairdner | 38 | ATR Mako | No | 10-Apr | 27-Mar | Nil |
2018 | 2 | Jerramungup | 42 | ATR Bonito | No | 10-Apr | 27-Mar | AM (6), BN (2), CG (2), CST (6), FF (2), MM (15), SC (2), WR (8) |
2018 | 2 | Kendenup | 39 | InVigor T4510 | Yes | 23-May | 27-Mar | AM (10), WR (15) |
2018 | 2 | Mount Barker | 39 | InVigor T4510 | Yes | 11-May | 27-Mar | AM (10), SC (5), VC (20), WR (20) |
2018 | 2 | Tenterdon | 36 | InVigor T4510 | Yes | 3-May | 27-Mar | Nil |
2018 | 2 | South Stirlings | 44 | Pioneer 45Y25 | Yes | 16-Apr | 27-Mar | Nil |
2018 | 3 | Coomalbidgup | 78 | Hyola 404RR | Yes | 5-May | 27-Mar | AM (5), BN (3), CG (100), CST (16), WR (45) |
2018 | 3 | Dalyup | 78 | Pioneer 44Y27 | Yes | 15-May | 19-Jun | - |
2018 | 3 | Esperance Downs | 146 | ATR Bonito | Yes | 21-May | 27-Mar | AM (4), SC (20), WR (7), VC (7) |
2018 | 3 | Gibson | 146 | ATR Bonito | Yes | 28-Apr | 27-Mar | MM (30), SC (65), WR (30), |
2018 | 3 | Grass Patch | 186 | ATR Bonito | No | 7-Apr | 27-Mar | MM (10), WR (18) |
2018 | 3 | Munglinup | 78 | Hyola 559TT | Yes | 20-Apr | 27-Mar | CST (10), VC (130), WR (95) |
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Congdon, B.; Matson, P.; Begum, F.; Kehoe, M.; Coutts, B. Application of Loop-Mediated Isothermal Amplification in an Early Warning System for Epidemics of an Externally Sourced Plant Virus. Plants 2019, 8, 139. https://doi.org/10.3390/plants8050139
Congdon B, Matson P, Begum F, Kehoe M, Coutts B. Application of Loop-Mediated Isothermal Amplification in an Early Warning System for Epidemics of an Externally Sourced Plant Virus. Plants. 2019; 8(5):139. https://doi.org/10.3390/plants8050139
Chicago/Turabian StyleCongdon, Benjamin, Paul Matson, Farhana Begum, Monica Kehoe, and Brenda Coutts. 2019. "Application of Loop-Mediated Isothermal Amplification in an Early Warning System for Epidemics of an Externally Sourced Plant Virus" Plants 8, no. 5: 139. https://doi.org/10.3390/plants8050139