Keywords

1 Introduction

Coffee is the second most commercialized product worldwide. It is produced in over 50 countries and secures livelihoods for millions of farmers (Vega et al. 2003; ICAFE 2017). Hemileia vastatrix Berk. & Broome, the causal agent of coffee leaf rust, is one of the biotic factors that affects coffee, causing significant economic losses due to the defoliation, subsequent harvest losses, and renewal needs due to severe damage caused in plants (Barquero 2013; ICAFE 2013).

This disease caused a widespread impact in 2012 in Central America, mainly due to the susceptibility of planted varieties of Coffea arabica such as Caturra and Catuaí (Avelino and Rivas 2013). Due to the origin, domestication process, reproduction and evolution of the genome, Arabica varieties are characterized by a low genetic diversity (Hendre et al. 2008; Prakash et al. 2002). As a result of a natural hybridization process between C. eugenioides and C. canephora, C. arabica is the only tetraploid species of the genus Coffea (2n = 4x = 44). This generates a limitation for genetic improvement of resistance genes due to the homogeneity of the varieties (Jefuka et al. 2010; Naranjo Zúñiga 2018).

Hemileia vastatrix is a biotrophic fungus that penetrates the plant through the stomata located on the abaxial side of the leaf. Its taxonomic classification is as follows: Phylum: Basidiomycota, Class:Pucciniomycetes, Order: Pucciniales, Family: Zaghouaniaceae, Genus: Hemileia (gbif 2022).

H. vastatrix is a hemicyclic fungus producing urediniospores, teliospores and basidiospores, but only the dikaryotic urediniospores, which form the asexual part of the cycle, reinfect coffee leaves successively and are responsible for the disease (as revised by Talhinhas et al. 2017). As a first symptom, small yellow chlorotic spots are observed in the foliage that subsequently, as the infection progresses, produce masses of orange urediniosporic sori (see Fig. 1) (Arauz Cavallini 2011). An epidemic of coffee leaf rust can be divided into two stages: stage of the production of the initial inoculum, whose main source is the residual inoculum, and a second stage that comprises the production of the secondary inoculum, which is the result of the successive repetition of the infection process on the same leaf (Avelino and Rivas 2013, Naranjo Zúñiga 2018).

Fig. 1
A photograph of a coffee leaf with circular spots.

Coffee leaf rust symptoms and signs, chlorotic spots and urediniosporic sori on the lower leaf surface

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The disease cycle of coffee leaf rust consists of five stages which can be affected by factors such as fruit load, plant resistance, microclimate and plant nutrition (Avelino 2004; Rhiney et al. 2021).

These stages include:

  • Dissemination: It occurs in three stages, the release of urediniospores, dispersion through factors such as rain, wind and people and deposition in plant tissue.

  • Germination: Once the urediniospores are deposited in the leaves, 4–6 germinative tubes are emitted with an appressorium necessary to force stomatal entry. The optimal conditions are 22 ºC temperature, 24 h of darkness and free water until the penetration stage (Silva et al. 1999; Naranjo Zúñiga 2018).

  • Penetration: The presence of well-formed stomata is necessary to be able to enter the leaf, so that the age of the leaf influences the receptivity to infection.

  • Colonization: This phase requires the growth of hyphae of the fungus in the intercellular spaces of the spongy parenchyma and haustoria within the cells of the palisade parenchyma and even the upper epidermis to give rise to the first symptoms, a macroscopic chlorosis (McCain and Hennen, 1984).

  • Sporulation: Once hyphae invade the substomatal chamber, they differentiate to form protosori. Later urediniosporic sori protrude through the stomata (Silva et al. 1999 and references therein).

Genetic improvement is an attractive approach that enables solving production constraints caused by pests and diseases. Induced mutagenesis is one of the tools that can be used to increase genetic diversity (ICAFE 2011; Novak and Brunner 1992; Shu et al. 2011). Gamma rays have proven to be an efficient tool to improve traits of agronomic importance such as resistance to pests and diseases (Borzouei et al. 2010; Yadav and Singh 2013; Shu et al. 2011). An efficient screening protocol is therefore required for evaluation of mutant populations developed via induced mutagenesis.

This protocol describes the procedures for the inoculation and evaluation of the defense response of the coffee genetic material infected with Hemileia vastatrix under laboratory conditions, or the biological efficacy of molecules for the control of the disease. This protocol is based on Eskes and Toma-Braghini (1982), with modifications made by the Costa Rican Coffee Institute-Coffee Research Center, Phytoprotection Laboratory.

2 Materials

2.1 Preparation of Rust Inoculum

  1. 1.

    Sterile scalpel.

  2. 2.

    50 µL centrifuge tubes.

  3. 3.

    Falcon tubes of the necessary size.

  4. 4.

    Distilled water.

  5. 5.

    Neubauer chamber.

  6. 6.

    Microscope.

2.2 Rust Inoculation

  1. 1.

    Scissors.

  2. 2.

    Plastic boxes.

  3. 3.

    Foams.

  4. 4.

    Plastic grid.

  5. 5.

    Water.

  6. 6.

    Micropipette (50 µl graduation).

  7. 7.

    Micropipette tips.

  8. 8.

    Ceramic spoon.

  9. 9.

    Adhesive plastic.

2.3 Rust Evaluation

  1. 1.

    Magnifying glass.

  2. 2.

    Light source.

3 Methods

3.1 Preparation of Rust Inocula

  1. 1.

    Collect coffee leaves with abundant rust spores (race II, or the most important for the region or country) from the field or greenhouse-grown susceptible varieties (Caturra or Villa Sarchí) (see Note 1).

  2. 2.

    Scrape the spores with a sterile scalpel and store in 50 µL centrifuge tubes.

  3. 3.

    Prepare a suspension of spores in distilled water and determine the concentration of urediniospores using a Neubauer chamber. Count the spores with orange coloration located in the corners and the center of the central sub-chamber (see Fig. 2).

    Fig. 2
    A Neubauer chamber grid contains 4 circles at the corners of the intersection region and a circle at the center.

    Urediniospores count points in Neubauer chamber

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  4. 4.

    Determine the concentration of urediniospores applying the formula (see Note 2):

    $$Urediniospore\,concentration/ml = urediniospores\,counted\, \times 5 \times 1e10^{4}$$

3.2 Rust Inoculation

  1. 1.

    Collect healthy leaves from the second node of branches from the middle stratum of the plants selected for rust resistance evaluation (see Fig. 3 and Note 3).

    Fig. 3
    A flow diagram with a set of photographs. First stage. Preparation of rust inoculums with photographs of a leaf, tubes with sample, and microscope. The second stage is rust inoculation. The leaf is cut into square-shaped pieces. The third stage is the evaluation of segments with diseases or treatments.

    A general description of the preparation stage of the inoculum and of the plant material, of the process for the germination and incubation of the pathogen and of the quantification of the presence or absence of the disease

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  2. 2.

    Cut 2 × 2 cm square segments from collected leaves.

  3. 3.

    Prepare humid chambers that consist of a plastic box with a wet foam in the background and a plastic grid on the foam.

  4. 4.

    Place the leaf segments on the foam and inoculate with 50 µl of suspension by placing a drop in the center and spreading it with a ceramic spoon.

  5. 5.

    Cover the wet chamber with clear adhesive plastic, ensuring it is airtight.

  6. 6.

    Incubate in darkness at room temperature for 3 days (see Note 4).

  7. 7.

    Transfer into a room with a photoperiod of 12 h light /12 h dark and 22 ± 1 °C for 28 days.

3.3 Rust Evaluation

  1. 1.

    Uncover the humid chambers and place in a location with enough light.

  2. 2.

    Remove necrotic segments.

  3. 3.

    Count number of segments inoculated and number of segments with rust.

  4. 4.

    The incidence rate of the disease is determined by the formula:

    $$\frac{{\% \,{\text{incidence}} = {\text{Number of segments with presence of uredospores}}\, \times {1}00}}{{\text{total segments inoculated}}}$$
  1. 5.

    Determine the presence and abundance of signs and symptoms (Table 1).

    Table 1 Scale used to measure the severity of rust in coffee segments (Eskes and Toma-Braghini 1982) (see Note 5)
    Full size table

This inoculation technique can also be used to evaluate the response of different natural or chemically synthesized molecules for the defense of plants susceptible to the disease. To do this, the use of Table 1 allows us to understand the mechanism of action of the products according to the incubation and latency periods of the pathogen.

4 Notes

  1. 1.

    When selecting leaves to collect rust spores, it is necessary to check whether the fungus Lecanicillium lecanii, a hyperparasite of H. vastatrix, is not present. Application of fungicides should be avoided two months prior to collection of leaves.

  2. 2.

    The concentration of the spore suspension should be approximately 1 × 105 urediniospores/ml.

  3. 3.

    It may be important to evaluate different leaf ages of individual mutant plants with putative resistance to the disease.

  4. 4.

    Some protocols (in greenhouse and laboratory conditions) indicate that 24 h of incubation is enough. At this time, the fungus concludes the germination, appressoria differentiation, and penetrates the host tissues. In our laboratory, the sporulation is more abundant and successful when dark conditions remain for three days. Therefore, it is recommended that this step is being tested and adjusted to the conditions of the laboratory in which the evaluation is going to be performed.

  5. 5.

    In this protocol, in addition to the rust incidence the scale described in Table1 is being used. The inoculation technique presented in this chapter can also be used to evaluate the response of different natural or chemically synthesized molecules for the defense of plants susceptible to the disease. To do this, the use of Table 1 allows us to understand the mechanism of action of the products according to the incubation and latency periods of the pathogen.