Keywords

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

  1. 1.

    ddH2O.

  2. 2.

    1 X TE buffer (pH 8.0).

  3. 3.

    CTAB.

  4. 4.

    KAc.

  5. 5.

    Chloroform.

  6. 6.

    Isoamyl alcohol.

  7. 7.

    Isopropanol.

  8. 8.

    75% ethanol.

  9. 9.

    Anhydrous ethanol.

  10. 10.

    Phenol.

  11. 11.

    Na2Ac.

  12. 12.

    rTaq (Dalian TaKaRa Co., Ltd., 5 U/µl).

  13. 13.

    10X PCR Buffer (Mg2+ plus).

  14. 14.

    dNTPs (2.5 mM).

  15. 15.

    Biowest regular agarose G-10 (CB005-100G).

  16. 16.

    Tris/borate electrophoresis buffer.

  17. 17.

    Microwave.

  18. 18.

    GoldView II Nuclear Staining Dyes (5,000×) (Solarbio® LIFE SCIENCES).

  19. 19.

    Electrophoresis tank.

  20. 20.

    DL 2000 Marker (Dalian TaKaRa Co., Ltd.).

  21. 21.

    RNAse A solution (Solarbio® LIFE SCIENCES, 10 mg/ml).

  22. 22.

    Water bath.

  23. 23.

    Specific primers (see Fig. 1).

    Fig. 1
    A sequence alignment of P C R. It resembles a table. The r D N A-I T S sequences are arranged in 19 rows and 5 columns, with positions labeled from 1 to 901. The continuous nucleotide sequences are underlined in positions 351, 401, and 751.

    Primers Hv-ITS-F/R designed for H. vastatrix PCR assay based on rDNA-ITS sequences

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  24. 24.

    Genomic DNA of the pathogen (see Fig. 2).

    Fig. 2
    A result of specificity test of H v-I T S-F slash R primer sets. The gel consists of markers, and 13 lanes are loaded with primers and samples and allowed to run. The light shaded bands formed in lanes from 2 to 5 loaded with 4 strains of D N A stacks between 500 and 250 base pairs.

    Example of specificity test of Hv-ITS-F/R primer sets. The DNA of 4 strains of H. vastatrix (lanes 2–5), 8 other fungi (lanes 6–13) (see Note 5) and sterilized ddH2O as the negative control (lane 1) were amplified by PCR using Hv-ITS-F/R primers. Primers for Hv-ITS-F/R amplify a 396-bp specific band from the DNA of H. vastatrix, while no bands were observed from the DNA of other fungi. M: DL 2000 DNA marker; 1: ddH2O control; 2–5: H. vastatrix; 6: Colletotrichum gloeosporioides; 7: Lecanicillium sp.; 8: Cercospora coffeicola; 9: Coleosporium plumeriae; 10: Colletotrichum falcatum; 11: Ustilago scitaminea; 12: Leptosphaeria sacchari; 13: Aspergillus niger

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  25. 25.

    Ice.

  26. 26.

    Ice machine.

  27. 27.

    Autoclave.

  28. 28.

    Mortar.

  29. 29.

    Measuring cylinder (100 ml).

  30. 30.

    Scissors.

  31. 31.

    Liquid nitrogen.

  32. 32.

    Micropipette (1,000, 200, 10, 2.5 μl).

  33. 33.

    Centrifuge tube (1.5, 2 ml).

  34. 34.

    NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA).

  35. 35.

    PTC-100™ Programmable Thermal Controller (MJ Research Inc, USA).

  36. 36.

    BIO-RAD GelDoc 2000 GelDoc 2000™.

  37. 37.

    Power/PAC300.

  38. 38.

    PCR tubes (0.2 ml).

  39. 39.

    Tips (1,000, 200, 10 µl).

  40. 40.

    Absolute alcohol.

  41. 41.

    Refrigerated Centrifuge Sigma 3k15.

  42. 42.

    SCILOGEX_D2012_Centrifuge.

  43. 43.

    Refrigerator.

3 Methods

3.1 Designing the Specific Primers for Hemileia vastatrix

  1. 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).

  2. 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.

  3. 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.

  1. 1.

    Preheat the CTAB extraction buffer to 65 °C in a water bath.

  2. 2.

    Grind approximately 1 g of diseased leaf tissue into a fine powder in a mortar using liquid nitrogen (see Note 1).

  3. 3.

    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).

  4. 4.

    Add 3 ml of 5 M KAc to the tube containing the lysate and let it stand on ice for 20 min.

  5. 5.

    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.

  6. 6.

    Repeat step 4.

  7. 7.

    After centrifugation, transfer supernatant into a new tube.

  8. 8.

    Add 12 ml of a pre-cooled isopropanol, mix by inverting and put at  −20 °C to fully precipitate the DNA.

  9. 9.

    Centrifuge the tube at 10,000 rpm for 15 min to pellet the DNA.

  10. 10.

    Rinse the pellet twice with 75% ethanol, and once with anhydrous ethanol. Air-dry the DNA pellet and dissolve in 10 ml TE buffer.

  11. 11.

    Treat the DNA samples with 1 μl RNase (10 mg/ml) at room temperature for 1–2 h.

  12. 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.

  13. 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.

  14. 14.

    Centrifuge at 12,000 rpm for 30 min at 4 °C.

  15. 15.

    Discard the supernatant, rinse the DNA pellet with 75% ethanol and dissolve in 1 ml TE after drying.

  16. 16.

    Determine the DNA concentration by e.g., a NanoDrop 2000c.

  17. 17.

    Store the DNA at − 20 °C until further use (see Note 3).

3.3 Preparation of the PCR Reaction Mixture and PCR Amplification

  1. 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

  1. 2.

    Mix all components, spin briefly and immediately place in a thermocycler (here a gradient Mastercycler was used).

  2. 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.

  3. 4.

    Upon termination store samples at 15 °C.

3.4 Gel Electrophoresis

  1. 1.

    Prepare a 1% agarose gel by mixing 1 g of agarose and 100 ml of TBE buffer (pH 8.0).

  2. 2.

    Melt thoroughly in a microwave.

  3. 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.

  4. 4.

    Load 10 μl of PCR products and run at 120 V for 20 min.

  5. 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).

    Fig. 3
    A result of the specificity test of H v-I T S-F slash R primer sets. The gel with markers and 8 lanes is loaded with primers and samples of different concentrations. The light-shaded bands stacked in lanes from 2 to 5 denote the yield of 365 base pairs.

    Example sensitivity test of primer sets Hv-ITS-F/R. Prepare a series of DNA concentrations to determine the sensitivity of the detection system. The initial genomic DNA concentration of H. vastatrix was adjusted to 10 ng/μL, with serial tenfold dilutions to reach 10−5 ng/μl. The results showed that samples with DNA concentration of 10 pg/μL or higher yielded a clearly visible 396-bp band while samples with a lower concentration were negative. M: DL 2 000 DNA marker; 1: ddH2O control; 2: 10 ng/μl; 3: 1 ng/μl; 4: 10−1 ng/μl; 5: 10−2 ng/μl; 6: 10−3 ng/μl; 7: 10−4 ng/μl; 8: 10−5 ng/μl

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4 Notes

  1. 1.

    The leaf samples should be fully ground into a fine powder. To prevent sample cross-contamination, change gloves after finishing each sample.

  2. 2.

    All the tubes, tips and utensils should be sterilized prior to use.

  3. 3.

    To prevent cross contamination, the pipette tips must be used once after contact with samples.

  4. 4.

    The PCR reaction mix is prepared on ice in a clean environment.

  5. 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.