Abstract
Early detection and identification of plant pathogens is one of the most important strategies for sustainable plant disease management. Fast, sensitive, and accurate methods that are cost-effective are crucial for plant disease control decision-making processes. Coffee leaf rust (CLR) caused by Hemileia vastatrix is a devastating worldwide fungal disease which causes serious yield losses of coffee, especially relevant for Coffea arabica. A rapid PCR assay for detecting and characterizing H. vastatrix with high specificity, high sensitivity and simple operation has been developed based on specific amplification of the Internal Transcribed Spacer (ITS) region of ribosomal genes. The specificity of the primers was determined using isolates DNA of H. vastatrix, Coleosporium plumeriae, and other fungal species that infect coffee plants and are common in coffee leaves, such as Lecanicillium sp (the H. vastatrix hyperparasite fungi), Cercospora coffeicola, Colletotrichum gloeosporioides, amongst others. Results showed specific amplification of a 396-bp band from H. vastatrix DNA with a detection limit of 10 pg/μl of pure genomic DNA of the pathogen. The PCR assay described in the current chapter allows to detect H. vastatrix rapidly and reliably in naturally infected coffee tissues, vital for the early detection and diagnostics of H. vastatrix and CLR epidemiology.
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Keywords
- Coffee
- Hemileia vastatrix
- Early molecular detection
- Polymerase chain reaction
- Internal transcribed spacer
- Specificity and sensitivity
1 Introduction
Accurate identification and diagnosis of plant diseases are vital for prevention of the spread of invasive pathogens (Balodi et al. 2017). So far, advances in the development of molecular methods have provided diagnostic laboratories with powerful tools for the detection and identification of phytopathogens, among which polymerase chain reaction (PCR) and other DNA-based techniques proved to be rapid and highly suitable approaches to improve the accuracy and efficiency of plant pathogen detection and characterization (Lévesque et al. 1998; Haudenshield et al. 2017). Detection protocols used for the diagnosis or quarantine measures should be reproducible and cost effective, time saving and simple in procedure (Elnifro et al. 2000; Hayden et al. 2008; Tomkowiak et al. 2019). In addition, sensitivity to pathogen concentration, and specificity to genetic variability within a target pathogen population are also high priorities for molecular detection (Balodi et al. 2017).
The Internal Transcribed Spacer (ITS) of the ribosomal DNA show high inter-species variability and intra-species stability and conservation, and hence is considered a reliable DNA marker to identify and classify the pathogenic fungi (Glynn et al. 2010). PCR assays based on the ITS region have been widely used for the detection of fungal pathogens in different crops such as sunflower, tobacco, soybean, cedar trees, miscanthus and others (Guglielmo et al. 2007; Chen et al. 2008; Torres-Calzada et al. 2011; Capote et al. 2012), relating to the pathogens of Phytophthora (Grünwald et al. 2012; Patel et al. 2016), Puccinia (Guo et al. 2016), Verticillium spp. (Nazar et al. 1991), Pleurotus spp. (Ma and Luo 2002), Pyricularia and anthracnose (Sugawara et al. 2009), Saccharomyces saccharum (Anggraini et al. 2019), Podosphaera xanthii (Tsay et al. 2011) and Golovinomyces cichoracearum (Troisi et al. 2010). This technique was applied to differentiate two pathotypes of Verticillium alboatrum infecting hop, to distinguish 11 taxons of wood decay fungi infecting hardwood trees, and to differentiate multiple Phytophthora species from plant material and environmental samples (Shamim et al. 2017; Belete and Boyraz 2019).
Coffee leaf rust (CLR), a major disease of Arabica coffee (Coffea arabica L.), is caused by the obligate biotrophic fungus Hemileia vastatrix Berkeley and Broome (Talhinhas et al. 2017). The infection of coffee leaves by H. vastatrix starts with urediniospore germination, appressorium formation over stomata, penetration, and inter- and intracellular colonization without any visible symptoms in the early stages of the infection in the field conditions < 10 days (Talhinhas et al. 2017; Silva et al. 2018). In field conditions, the visible rust spores can be observed about 20 days after the first infection of H. vastatrix (Schieber 1972). So far, the traditional method for detecting and characterizing CLR was time-consuming and laborious, and relied on conventional morphological examination requiring professional taxonomic knowledge and extensive experience (McCartney et al. 2003; Silva et al. 2012). Hence, rapid and high-throughput identification and detection methods for H. vastatrix are required to recognize the infection as early as possible before the appearance and spread of CLR spores in the leaf surface. Early detection methods can facilitate implementing proper management approaches to prevent the development and spread of the coffee leaf rust pathogen (Sankaran et al. 2010).
The present study was undertaken with the objective of early detection of H. vastatrix based on the PCR amplification of a specific ITS region in the rDNA of H. vastatrix. A simple, accurate and rapid PCR-based assay for CLR is presented as a reliable technique to monitor H. vastatrix in the early stages of the infection, as well as to provide scientific basis for the prevention and control of CLR.
2 Materials
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1.
ddH2O.
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2.
1 X TE buffer (pH 8.0).
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3.
CTAB.
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4.
KAc.
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5.
Chloroform.
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6.
Isoamyl alcohol.
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7.
Isopropanol.
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8.
75% ethanol.
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9.
Anhydrous ethanol.
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10.
Phenol.
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11.
Na2Ac.
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12.
rTaq (Dalian TaKaRa Co., Ltd., 5 U/µl).
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13.
10X PCR Buffer (Mg2+ plus).
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14.
dNTPs (2.5 mM).
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15.
Biowest regular agarose G-10 (CB005-100G).
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16.
Tris/borate electrophoresis buffer.
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17.
Microwave.
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18.
GoldView II Nuclear Staining Dyes (5,000×) (Solarbio® LIFE SCIENCES).
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19.
Electrophoresis tank.
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20.
DL 2000 Marker (Dalian TaKaRa Co., Ltd.).
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21.
RNAse A solution (Solarbio® LIFE SCIENCES, 10 mg/ml).
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22.
Water bath.
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Specific primers (see Fig. 1).
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Genomic DNA of the pathogen (see Fig. 2).
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Ice.
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Ice machine.
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Autoclave.
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Mortar.
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Measuring cylinder (100 ml).
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30.
Scissors.
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Liquid nitrogen.
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32.
Micropipette (1,000, 200, 10, 2.5 μl).
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Centrifuge tube (1.5, 2 ml).
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34.
NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA).
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35.
PTC-100™ Programmable Thermal Controller (MJ Research Inc, USA).
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36.
BIO-RAD GelDoc 2000 GelDoc 2000™.
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37.
Power/PAC300.
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38.
PCR tubes (0.2 ml).
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39.
Tips (1,000, 200, 10 µl).
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40.
Absolute alcohol.
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41.
Refrigerated Centrifuge Sigma 3k15.
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42.
SCILOGEX_D2012_Centrifuge.
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43.
Refrigerator.
3 Methods
3.1 Designing the Specific Primers for Hemileia vastatrix
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1.
The primers Hv-ITS-F/R were designed to specifically amplify the ITS2 region of H. vastatrix. The sequence of the forward primer Hv-ITS-F is 5’-GGTACACCTGTTTGAGAGTATG-3’, and the sequence of the reverse primer is Hv-ITS-R is 5’-CAAAATATGTCATACCTCTCATTCT-3 (see Fig. 1).
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2.
Primer sequences of Hv-ITS-F and Hv-ITS-R were used as inputs for a BLAST search against the NCBI database to confirm the specificity. The primers were synthesized by Invitrogen Biotechnology (Shanghai) Co., Ltd.
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3.
Upon delivery, dilute lyophilized primers to the concentration of 10 µM by adding 0.1 X TE buffer. Store at − 20 °C for later use.
3.2 Total DNA Extraction from Suspected Diseased Leaves or Typical Diseased Samples
The CTAB method (Siegel et al. 2017) was used to extract DNA from diseased leaves.
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1.
Preheat the CTAB extraction buffer to 65 °C in a water bath.
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Grind approximately 1 g of diseased leaf tissue into a fine powder in a mortar using liquid nitrogen (see Note 1).
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Add 15 ml of pre-heated CTAB buffer into each tube. Mix well and incubate at 65 °C for 30 min. Turn the tubes upside down every 10 min to resuspend the samples in the buffer (see Note 2).
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Add 3 ml of 5 M KAc to the tube containing the lysate and let it stand on ice for 20 min.
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Add the same volume of a chloroform:iso-amyl alcohol (24:1) mixture to the tube, mix well and centrifuge at 12,000 rpm at 4 °C for 15 min.
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Repeat step 4.
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After centrifugation, transfer supernatant into a new tube.
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Add 12 ml of a pre-cooled isopropanol, mix by inverting and put at −20 °C to fully precipitate the DNA.
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Centrifuge the tube at 10,000 rpm for 15 min to pellet the DNA.
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Rinse the pellet twice with 75% ethanol, and once with anhydrous ethanol. Air-dry the DNA pellet and dissolve in 10 ml TE buffer.
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11.
Treat the DNA samples with 1 μl RNase (10 mg/ml) at room temperature for 1–2 h.
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12.
Add the same volume of phenol: chloroform: isoamyl alcohol (25:24:1), mix well and then centrifuge at 12,000 rpm at 4 °C for 15 min.
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13.
Transfer the supernatant to a new tube, mix with 1 ml ice-cold 3 M Na2Ac, and 20 ml of anhydrous ethanol, and place at − 20 °C overnight.
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14.
Centrifuge at 12,000 rpm for 30 min at 4 °C.
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15.
Discard the supernatant, rinse the DNA pellet with 75% ethanol and dissolve in 1 ml TE after drying.
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16.
Determine the DNA concentration by e.g., a NanoDrop 2000c.
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17.
Store the DNA at − 20 °C until further use (see Note 3).
3.3 Preparation of the PCR Reaction Mixture and PCR Amplification
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1.
Prepare a 20 µl PCR reaction mix as follows (see Note 4):
10X PCR Buffer | 2 µl |
dNTPs (2.5 mM) | 1.6 µl |
Forward Primer (10 µM) | 1 µl |
Reverse Primer (10 µM) | 1 µl |
rTaq (5 U/µl) | 0.1 µl |
DNA template | 1 µl |
ddH2O | 14.2 µl |
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2.
Mix all components, spin briefly and immediately place in a thermocycler (here a gradient Mastercycler was used).
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3.
Set the thermocycler conditions as following: initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; final extension time at 72 °C for 5 min.
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4.
Upon termination store samples at 15 °C.
3.4 Gel Electrophoresis
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1.
Prepare a 1% agarose gel by mixing 1 g of agarose and 100 ml of TBE buffer (pH 8.0).
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2.
Melt thoroughly in a microwave.
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3.
Allow the mixture to cool down to 40 °C, add 1 µl GoldView DNA dye solution (1 µl/100 ml gel) and mix. Pour the gel and allow to solidify.
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4.
Load 10 μl of PCR products and run at 120 V for 20 min.
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5.
View the gel under the UV light. The H. vastatrix positive samples are defined as the ones that show a specific single band of 396-bp (see Figs. 2 and 3).
4 Notes
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1.
The leaf samples should be fully ground into a fine powder. To prevent sample cross-contamination, change gloves after finishing each sample.
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2.
All the tubes, tips and utensils should be sterilized prior to use.
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3.
To prevent cross contamination, the pipette tips must be used once after contact with samples.
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4.
The PCR reaction mix is prepared on ice in a clean environment.
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5.
The urediniospores of Coleosporium plumeriae and the other fungal isolates were extracted using a Fungal DNA kit (E.Z.N.A.TM Fungal DNA Kit, Omega, Bio-tek, USA) according to the manufacturer’s protocol.
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Acknowledgements
Funding for this work was provided by the National Key R&D Program of China (2018YFD0201100), the IAEA Collaborative Research Project D22005 (No. 20380), the International Exchange and Cooperation Project funded by the Agricultural Ministry ‘Construction of Tropical Agriculture Foreign Cooperation Test Station and Training of Foreign Managers in Agricultural Going-Out Enterprises’ (SYZ2019-08) and the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630042017021).
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Wu, W. et al. (2023). A PCR-Based Assay for Early Diagnosis of the Coffee Leaf Rust Pathogen Hemileia vastatrix. In: Ingelbrecht, I.L., Silva, M.d.C.L.d., Jankowicz-Cieslak, J. (eds) Mutation Breeding in Coffee with Special Reference to Leaf Rust. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-67273-0_18
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DOI: https://doi.org/10.1007/978-3-662-67273-0_18
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