Evaluation of Coffee (Coffea arabica L. var. Catuaí) Tolerance to Leaf Rust (Hemileia vastatrix) Using Inoculation of Leaf Discs Under Controlled Conditions

Abstract

Coffee leaf rust (CLR), caused by the obligate biotrophic fungus Hemileia vastatrix, is considered one of the most devastating diseases of Arabica coffee. The use of leaf rust resistant or tolerant coffee varieties is a critical component for effective management of this disease at the farm level. Conventional breeding of Arabica coffee for leaf rust resistance requires many years of breeding and field-testing. Induced mutagenesis is an effective tool to increase genetic variability and generate new alleles with potential benefit for addressing abiotic and biotic stresses such as leaf rust in Arabica coffee. Efficient screening methods are required to evaluate coffee germplasm or mutant populations for resistance to H. vastatrix. Here, we present a screening method that uses inoculation of leaf discs in a controlled environment. The method was evaluated using M₁V₁ and M₂ plants derived from chemically mutagenized Arabica coffee cell suspensions. In this method, the first rust symptoms appear on the leaf discs approximately 29 days after inoculation while the disease severity and incidence can be scored about 47 days after inoculation. Our results show that the methodology is simple, efficient and suitable to rapidly screen large mutant populations in a small area.

1 Introduction

Coffee (Coffea arabica L.) is one of the most important beverages in the world and the second most important commercial product exported by developing countries (Alemayehu 2017). Coffee leaf rust (CLR), caused by the biotrophic fungus Hemileia vastatrix Berk. and Broome, is one of the main limiting factors of Arabica coffee production worldwide (Waller et al. 2007). The disease can reduce global coffee production by 20 to 25%, with losses of over $ 1 billion annually (McCook 2006; Talhinhas et al. 2017).

The application of fungicides has been the most widely used method to control CLR, even when the development of varieties with genetic resistance is the best alternative (Zambolim 2016). The quest for natural resistance to CLR by traditional breeding has been the focus of research for decades (Melese Ashebre 2016; Mishra and Slater 2012). However, conventional genetic control of CLR has been hampered by the prodigious pathological diversity and rapid genetic evolution of the fungus overcoming the plant resistance genes deployed so far (Cabral et al. 2016; Lima et al. 2020). The induction of genetic variability in Arabica coffee through mutagenesis provides an important complementary tool for crop improvement programs, since a range of variants can be generated (Dhumal and Bolbhat 2012; Vargas-Segura et al. 2019).

Chemical mutagens such as sodium azide (NaN₃) and ethyl methanesulfonate (EMS), have been used in crop breeding for developing mutants (Bolívar-González et al. 2018; Laskar et al. 2018). These chemical mutagens induce a broad variation of morphological and yield-related traits. Other authors reported cases of crops treated with chemical mutagens and improved for fungal resistance or tolerance, for example, powdery mildew-resistant barley (Khan et al. 2010) and wheat resistant to leaf rust Puccinia sp. (Mago et al. 2017).

Genetic studies related to H. vastatrix and coffee genotypes pursuing resistance, require periodic inoculation of different uredospores of the fungus into the host. A safe and efficient way to evaluate the resistance to different H. vastatrix races is carried out by infection in situ, using detached leaves or leaf discs under controlled conditions of humidity, light, and temperature that stimulate the development of the pathogen (Cabral et al. 2016; Eskes 1982).

This chapter presents a Coffee leaf rust resistance screening method based on inoculation of leaf discs under controlled conditions. The method was evaluated using M₁V₁ mutant plants obtained from M₀ embryogenic callus treated with NaN₃ and EMS and the resulting M₂ population. The method proved suitable to rapidly screen large coffee populations for CLR resistance.

2 Materials

2.1 Plant Material

• 1. Coffea arabica. var. Catuaí plants M₁V₁ (see Note 1).

• 2. Coffea arabica var. Catuaí seeds M₂ (see Note 2).

• 3. C. arabica var. Obatá (or any other CLR resistant variety).

• 4. C. arabica var. Caturra (or any other CLR susceptible variety).

• 5. C. canephora (or any other CLR resistant species).

2.2 Other Biological Materials

• 1. Coffee leaves with rust spores.

• 2. Healthy coffee leaves (in greenhouse).

2.3 Consumables and Minor Equipment

• 1. Black polyethylene bags (6 × 8 in).

• 2. Calibrated scoops for fertilizer application.

• 3. Commercial potting soil.

• 4. Cylindrical punch (10 mm-diameter) (e.g., Korff model 06940-5/16).

• 5. Falcon tube (50 ml).

• 6. Latex gloves.

• 7. Microcentrifuge tubes (1.5 ml).

• 8. Mix of screened compost.

• 9. Napkins.

• 10. Permanent markers.

• 11. Plant labels (with progenies number, plant number).

• 12. Plastic boxes (12 × 10 × 3.5 cm).

• 13. Plastic dropper.

• 14. Plastic grid.

• 15. Plastic pots (1–2 L capacity).

• 16. Rice husk.

• 17. Scalpels blades.

• 18. Scalpels.

• 19. Shovels.

• 20. Slow-release fertilizer.

• 21. Soil.

• 22. Wheelbarrow.

2.4 Reagents and Agrochemicals

• 1. Bayfolan forte (10 ml/L) (Bayer S.A, Amatitlán, Guatemala).

• 2. Distilled water.

• 3. Fungicide Vitavax 40 WP (Chemtura Corporation, Middlebury, USA).

• 4. Osmocote Pro, 19:9:10 + 2MgO + TE.

• 5. Tween 20 (Research Products International, Illinois, USA).

2.5 Equipment

• 1. LED light lamps (Heliospectra, model LX 602).

• 2. Microscope.

• 3. Neubauer chamber or Haemocytometer slide.

• 4. Stereo microscope.

• 5. Incubator with light, humidity, and temperature control (e.g., BIOBASE brand, model BJPX-L400.Shandong, China).

• 6. Electronic Digital Vernier Caliper (e.g., TOTAL, model TMT322001).

3 Methods

3.1 Germination of M₂ Seeds

• 1. Collect ripe cherries from M₁V₁ plants in the field and place the fruits in labeled paper bags.

• 2. Remove the pulp and the mucilage, wash and let dry for 12 days without full sun exposure (see Note 3).

• 3. Select normal-shaped seeds that are free of visible disease and insects.

• 4. Treat and cure the seeds with the fungicide Vitavax 40 WP (1 g/Kg) (see Note 4).

• 5. Label the plastic pots, keeping the same code obtained from their original fruit.

• 6. Place 10 cm of a substrate in plastic pots and sow the seeds.

• 7. Add a 1 cm layer of substrate over the sown seeds (approximately 20 and 30 seeds per pot).

• 8. Place the pots under controlled conditions; humidity greater than 90%; 25–30 °C, with a photoperiod of 12 h.

• 9. Record the date of planting.

• 10. Estimate the duration of seed germination and the percentage of germination per progeny.

3.2 Planting Seedlings M₂

• 1. Once the plants have their cotyledonary leaves (see Fig. 1a), transfer the plantlets to polyethylene bags (6 × 8 in) containing substrate (soil, mix of screened compost, and rice husk at 2:1:1).

  Fig. 1

  

  M₂ plant development stages. a Seedbed preparation and seed germination. b Transplanting and planting of seedlings. c Establishment of plants under controlled conditions in a greenhouse or growth chamber

• 2. Sow 2 seedlings of the same height and tap root per bag (see Fig. 1b and Note 5).

• 3. Give a permanent and unique identification code to each plant after planting.

• 4. Prepare a field map to indicate full details of plant identification and location.

• 5. Maintain the pots under controlled conditions in a greenhouse or growth chamber with 12 h light LED photoperiod at 28 ± 2 ºC, and (see Fig. 1c).

• 6. Fertilize with slow-release fertilizer (5 g/plant) (e.g., Osmocote Pro, 19:9:10 + 2MgO + TE) and weekly applications of Bayfolan forte (10 ml/L) to support growth and development.

3.3 Preparation of Coffee Leaf Rust Inoculum

• 1. Select coffee leaves with abundant Hemileia vastatrix spores, without the presence of Lecanicillium lecanii (see Fig. 2a).

  Fig. 2

  

  Sample collection for Hemileia vastatrix inoculum, a leaves with CLR spores, b sporulated lesions, c uredospores

• 2. Place the leaves in plastic bags and label (sample number, location, and coffee variety from which it was collected).

• 3. In the laboratory, using a stereo microscope and a sterile scalpel, scrape the sporulated lesions (only intense orange lesions) (see Fig. 2b, c).

• 4. Collect spores in sterile 1.5 ml microcentrifuge tubes.

• 5. Pour 30 ml of distilled water, 0.1 ml of Tween 20, and the uredospores into a sterile tube and shake the suspension of spores (see Fig. 3).

  Fig. 3

  

  Preparation of the CLR inoculum and counting cells

• 6. Determine the uredospore concentration of the spore suspension using a haemocytometer and a microscope at 10× magnification. Examine five quadrants (4 at the ends and 1 in the center) (see Fig. 3).

• 7. Count three 50 μl drops of the spore suspension (count only orange-colored spores).

• 8. Calculate the uredospore density as following: N × 10⁴ × f cell/ml, where “N” is the total counted cells, and “f” is the dilution factor (see Note 6).

• 9. Adjust inoculum to a concentration of approximately 2.3 × 10⁵ spores/ml.

3.4 Inoculation of the Coffee Leaf Discs with CLR

• 1. Collect healthy full-grown M₁V₁ leaves in the greenhouse and keep in a plastic bag on sterilized foam moistened with water (see Note 7).

• 2. In the laboratory, carefully wash the leaves with water and dry them for 1 h at 24 °C (see Note 8).

• 3. Clearly mark the humidity chambers (each plastic box) with the plant accession number.

• 4. Using a 10 mm diameter cylindrical punch, cut out circular leaf discs (see Fig. 4a) without midribs that do not contain stomata (CLR entry point) (Eskes 1982).

  Fig. 4

  

  Preparation of the humidity chambers, a cylindrical punch to cut circular (10mm) leaf discs, b moist chambers, and c inoculum in each leaf disc

• 5. Ten leaf discs per plant are needed for the detection of resistance. Include susceptible (e.g., C. arabica L. var. Caturra) and resistant plants (e.g., C. canephora, C. arabica var. Obatá) as controls.

• 6. The discs are moistened and placed upside down in the moist chambers (see Fig. 4b and Note 9).

• 7. Inoculate each leaf disc with one droplet of approximately 50 μl of the spore suspensions (1 mg spores per mL) (see Fig. 4c).

• 8. Close the boxes with a transparent lid and keep them at 22.5 ± 1.5 °C in the incubator in the darkness for 36 hours and, a relative humidity greater than 90%, to allow rust germination.

• 9. After this period, the humidity chambers are uncovered to allow the suspension to dry for 3 h, allowing the evaporation of the inoculation droplets.

• 10. Incubate at approximately 2,000 lux intensity of artificial light, with 12 h light period and temperature 22.5 ± 1.5° C, with a relative humidity greater than 90% for 15 days.

• 11. After this period, keep at 23 ± 1.5 °C and relative humidity of 85% under natural light for approximately 10 h and 14 h of darkness for a total of 22 days.

3.5 Evaluation of Plant Resistance Against CLR

• 1. Record weekly until the appearance of symptoms (chlorotic lesions according to the scale degree 1; see Table 1).

  Table 1 Disease severity-rating scale used to record symptoms caused by Hemileia vastatrix in coffee plants (Rozo-Peña and Cristancho-Ardila 2011).

• 2. After observing the first symptoms, the incidence and severity are evaluated every 72 hr for 26 days.

• 3. Record symptoms and disease severity rate from 29 to 47 days after inoculation on a scale from 0 to 7 using the disease severity rating shown in Table 1 (0 = resistant and 7 = highly susceptible).

• 4. Calculate disease incidence as follows: [(no. of diseased discs/no. of inoculated discs) * 100] (Rozo-Peña and Cristancho-Ardila 2011).

• 5. The disease severity is calculated based on the scale shown in Table 1, as follows: [Σ (scale grade * frequency) / (total units observed) * 100] (Rozo-Peña and Cristancho-Ardila 2011; Leguizamón et al. 1998).

• 6. From infection, calculate the Incubation Period (IP: number of days from inoculation to appearance of chlorosis) and the Latency Period (LP: number of days to appearance of sporulated lesions i.e., uredospores) (Rozo-Peña and Cristancho-Ardila 2011; Leguizamón et al. 1998).

• 7. Select the plantlets that show CLR resistance (see Note 10).

4 Notes

• 1. M₁V₁ coffee plants (Coffea arabica var. Catuaí) were obtained after treatment of embryogenic callus (M₀) with the mutagenic agents NaN₃ (5 mM for 15 min) and EMS (185.2 mM for 120 min) according to Bolívar-González et al. (2018).

• 2. M₂ coffee seeds of 81 different progenies were obtained after treatment of seeds (M₀) with NaN₃ (50 mM for 8h) according to Vargas-Segura et al. (2019).

• 3. Coffee beans rapidly lose their viability when dried (their humidity content cannot be less than 10%).

• 4. Curing the seed before sowing is the first step to obtain healthy plants; it allows to eliminate pathogens and prevents possible diseases originating from the soil.

• 5. Young plantlets are highly susceptible to diseases. The best size of the plantlet is 8–10 cm with a tap root of 6–8 cm.

• 6. To count cells using a haemocytometer or a Neubauer chamber add 15–20 μl of the cell suspension between the haemocytometer and a cover glass. Count the number of cells in all five quadrants (4 at the ends and 1 in the center) and divide by five (see Fig. 3 step 5). The number of cells per square ×10⁵ = the number of spores/ml of the suspension.

• 7. It is convenient to collect samples of leaves with rust from different locations and coffee varieties to have greater variability of the pathogen.

• 8. It is recommended to carefully clean the coffee leaves before cutting the discs.

• 9. The chambers consist of clear sterile plastic boxes (12 × 10 × 3.5 cm) with foam on the bottom. Place a plastic tray with 20 1.2 cm diameter depressions to hold the leaf discs (see Fig. 4b).

• 10. The procedure proves to be reliable and very sensitive; it is not time-consuming, requiring small amounts of inoculum and plant tissue.