Development of a Real-Time PCR Assay for the Early Detection of the Eucalyptus Pathogen Quambalaria eucalypti
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fungal Isolates and Plant Materials
2.2. Genomic DNA Extraction and Quantification
2.3. Primer Design and cPCR Amplification
2.4. SYBR Green Real-Time PCR Optimization
2.5. Real-Time PCR Assay’s Specificity and Sensitivity
2.6. Interlaboratory Tests
3. Results
3.1. Primer Design and cPCR Amplification
3.2. SYBR Green Real-Time PCR Optimization
3.3. Real-Time PCR Assay Specificity and Sensitivity
3.4. Interlaboratory Tests
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | Isolate a | Host | Origin | Cq b Value ± SD c | Tm (°C) d |
---|---|---|---|---|---|
Quambalaria eucalypti | CBS 118844 e | Eucalyptus grandis | South Africa | 21.54 ± 0.71 | 87.2 |
CBS 119680 f | 21.30 ± 0.52 | 87.0 | |||
ITEF/1937 | 20.48 ± 0.35 | 86.1 | |||
ITEF/1999 | 20.43 ± 0.14 | 86.8 | |||
EGES-1 | Eucalyptus globulus | Brazil | 21.14 ± 0.35 | 86.1 | |
EGES-2 | 20.87 ± 0.62 | 87.1 | |||
EGES-5 | 20.91 ± 0.53 | 87.0 | |||
EGES-10 | 22.07 ± 0.26 | 86.0 | |||
EGES-11 | 22.09 ± 0.88 | 86.2 | |||
EGES-13 | 21.30 ± 0.13 | 87.0 | |||
EGES-17 | 21.58 ± 0.44 | 86.9 | |||
EGES-19 | 22.43 ± 0.46 | 86.1 | |||
EGES-20 | 22.46 ± 0.35 | 86.2 | |||
EGES-22 | 22.45 ± 0.21 | 87.2 | |||
RGS-1 | 21.41 ± 0.57 | 86.3 | |||
RGS-2 | 21.35 ± 0.22 | 87.0 | |||
RGS-3 | 21.47 ± 0.31 | 87.2 | |||
RGS-4 | 21.29 ± 0.81 | 86.2 | |||
RGS-5 | 20.93 ± 0.73 | 87.0 | |||
RGS-6 | 21.02 ± 0,54 | 86.6 | |||
SUL/01 | Uruguay | 21.27 ± 0.52 | 86.7 | ||
SUL/03 | 22.05 ± 0.54 | 86.8 | |||
UY198 | 21.24 ± 0.31 | 86.3 | |||
UY199 | 21.63 ± 0.61 | 87.0 | |||
MATY 4665 | 19.98 ± 1.07 | 86.5 | |||
MATY 4751 | 20.15 ± 0.86 | 86.5 | |||
MATY 4752 | 21.73 ± 0.24 | 86.7 | |||
MATY 5001 | 21.65 ± 0.35 | 86.6 | |||
3421-S | 19.82 ± 1.13 | 87.0 | |||
3422-T | 21.36 ± 0.52 | 87.1 | |||
Quambalaria cyanescens | CBS 876.73 | Eucalyptus pauciflora | Australia | n/a | n/a |
IMI 178848 | n/a | n/a | |||
EU-PA | n/a | n/a | |||
Quambalaria pitereka | CERC/03/08 | Eucalyptus sp. | China | n/a | n/a |
CERC/04/08 | n/a | n/a | |||
CERC/05/05 | n/a | n/a | |||
Quambalaria simpsonii | CBS 124772 f | Eucalyptus tintinnans | Australia | n/a | n/a |
EU-TI3 | n/a | n/a | |||
Quambalaria tasmaniae | CBS 145602 a | Eucalyptus sp. | Australia | n/a | n/a |
7608 | n/a | n/a | |||
Alternaria alternata | CYC-60 | Eucalyptus grandis | Uruguay | n/a | n/a |
Calonectria spathulata | CS11340 | Eucalyptus grandis | Brazil | n/a | n/a |
Colletotrichum boninense | MWJ-43 | Eucalyptus grandis | South Africa | n/a | n/a |
Cryphonectria havanensis | GSEG_95 | Eucalyptus grandis | Brazil | n/a | n/a |
Cylindrocladium candelabrum | 11356 | Eucalyptus sp. | Brazil | n/a | n/a |
Cylindrocladium pteridis | IMI 354530 | Eucalyptus grandis | Brazil | n/a | n/a |
Neopestalotiopsis eucalyptorum | 912/88 | Eucalyptus globulus | Portugal | n/a | n/a |
Phomopsis arnoldiae | CR 345-96 | Eucalyptus grandis | Uruguay | n/a | n/a |
Pseudocercospora eucalyptorum | BBR 5689 | Eucalyptus globulus | Spain | n/a | n/a |
Isolate | Disease Severity | Tm (°C) | Cq a Value ± SD b | DNA Conc. (fg) ± SD b |
---|---|---|---|---|
CBS 118844 | 0 | 87.2 | 33.10 ± 0.02 | 16.48 ± 2.40 |
1 | 86.9 | 27.70 ± 0.05 | 329.66 ± 5.24 | |
2 | 87.3 | 26.75 ± 0.03 | 558.45 ± 11.07 | |
3 | 87.0 | 24.89 ± 0.02 | 1567.40 ± 14.85 | |
EGES-5 | 0 | 87.3 | 32.87 ± 0.18 | 18.72 ± 0.65 |
1 | 86.8 | 27.43 ± 0.27 | 382.94 ± 5.18 | |
2 | 87.1 | 26.73 ± 0.02 | 564.69 ± 11.67 | |
3 | 86.9 | 24.95 ± 0.03 | 1516.08 ± 12.36 | |
UY198 | 0 | 87.0 | 32.84 ± 0.26 | 19.03 ± 0.15 |
1 | 87.0 | 27.53 ± 0.02 | 362.27 ± 4.73 | |
2 | 86.9 | 26.71 ± 0.04 | 570.99 ± 10.45 | |
3 | 87.1 | 24.81 ± 0.06 | 1638.54 ± 21.74 |
DNA Conc. (pg) | Cq a Value ± SD b (Intra-Assay) | Cq a Value ± SD b (Inter-Assay) | Coefficient of Variance (%) | |||
---|---|---|---|---|---|---|
Operator 1 | Operator 2 | Operator 3 | Intra-Assay | Inter-Assay | ||
10 | 21.49 ± 0.15 | 21.40 ± 0.54 | 20.98 ± 0.66 | 20.78 ± 0.15 | 0.3 | 0.9 |
1 | 25.50 ± 0.11 | 25.18 ± 0.36 | 25.34 ± 0.12 | 26.03 ± 0.24 | 0.4 | 1.5 |
0.1 | 30.01 ± 0.12 | 29.87 ± 0.21 | 29.91 ± 0.15 | 30.05 ± 0.45 | 0.6 | 1.3 |
0.01 | 34.01 ± 0.13 | 33.85 ± 0.18 | 34.13 ± 0.11 | 33.97 ± 0.21 | 0.8 | 2.4 |
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Faedda, R.; Silva, G.B. Development of a Real-Time PCR Assay for the Early Detection of the Eucalyptus Pathogen Quambalaria eucalypti. Forests 2024, 15, 375. https://doi.org/10.3390/f15020375
Faedda R, Silva GB. Development of a Real-Time PCR Assay for the Early Detection of the Eucalyptus Pathogen Quambalaria eucalypti. Forests. 2024; 15(2):375. https://doi.org/10.3390/f15020375
Chicago/Turabian StyleFaedda, Roberto, and Gabriela B. Silva. 2024. "Development of a Real-Time PCR Assay for the Early Detection of the Eucalyptus Pathogen Quambalaria eucalypti" Forests 15, no. 2: 375. https://doi.org/10.3390/f15020375
APA StyleFaedda, R., & Silva, G. B. (2024). Development of a Real-Time PCR Assay for the Early Detection of the Eucalyptus Pathogen Quambalaria eucalypti. Forests, 15(2), 375. https://doi.org/10.3390/f15020375