DOI: 10.5061/dryad.4j0zpc8d6
title: Screening coffee genotypes for brown eye spot resistance in Brazil

description: Several researchers have attempted to develop coffee plants that are
 resistant to brown eye spot (BES); however, no coffee cultivars are
 resistant to the disease. In the present study, a blend of strains from
 Cercospora coffeicola was inoculated into 19 Brazilian commercial
 cultivars and 41 accessions from the Germplasm Collection of Minas Gerais
 to evaluate the genetic resistance ability within the population and
 select superior genotypes for the breeding program. After predicting the
 genotypic values of the studied material, the evaluations number necessary
 for selecting genotypes with accuracy and efficiency was determined based
 on the data of severity to BES. The action of defense mechanisms plant was
 also investigated by assessing the levels of total soluble phenolic
 compounds and soluble lignin in contrasting genotypes for disease
 susceptibility. Based on the results, the accession MG 1207 Sumatra, had
 an intrinsic genetic capacity to maintain low levels of severity to BES.
  The genotype MG 1207 Sumatra can substantially contribute to the
 development of new cultivars, which may lead to the reduced use of
 pesticides. According to the accuracy and efficiency results obtained,
 four evaluations BES severity are sufficient to achieve accuracy,
 providing expressive genetic gains. Finally, the levels of lignin and
 phenolic compounds were not found to be associated with the resistance of
 coffee genotypes to BES.
description: <b>BES severity
 </b> The experiment was carried out under
 greenhouse conditions at the Department of Phytopathology of the
 Universidade Federal de Lavras - UFLA, Brazil. Coffee seedlings of 19
 commercial cultivars and 41 accessions from the Germplasm collection of
 the Agricultural Research Corporation of the State of Minas Gerais
 (EPAMIG) in Patrocínio, MG, were evaluated to determine their resistance
 to BES (Table S1). The accessions were selected according to their
 characteristics of yield, drink quality, and/or resistance to other
 diseases of economic importance. Seeds were sown in 5 L
 plastic trays containing autoclaved sand. The germination chamber was
 adjusted to 30 ° C and 80% relative humidity. The seedlings were
 transplanted into punched-black polyethylene pots (0.11 x 0.20 m) at the
 phase of cotyledons. The substrate consisted of 300 L of cattle manure and
 700 L of soil mix extracted from the 0.4 to 0.8 m layer of Dystrophic Red
 Latosol and fertilized with 5 kg of simple superphosphate and 500 g of
 potassium chloride. The seedlings were inoculated at
 the stage of three pairs of true leaves. The inoculum consisted of a blend
 of different isolates of <em>C. coffeicola</em> obtained from
 coffee leaves with symptoms of BES collected in the municipalities of
 Marechal Floriano (ES), Cachoeirinha, Ervália, Lavras, and Patrocínio
 (MG). Different isolates were employed to include a higher variability of
 the fungus [6, 11]. The sporulation of the isolates was
 performed as described by Souza et al. [14], with adaptations. Eight
 mycelial discs (6 mm in diameter) were removed from the colony borders (on
 day 15 of growth) of different isolates of <i>C.
 coffeicola</i>. Discs were macerated in 400 µL of sterile distilled
 water. The macerated mycelium from each isolate was placed in Erlenmeyer
 flasks containing 20 mL of liquid V8 culture medium (100 mL of V8 in 900mL
 of distilled water) under shaking at 100 rpm for 12 d at room temperature.
 The liquid containing the mycelium was transferred to plates containing a
 water-agar medium. The plates were kept in a BOD incubator (Bio-Oxygen
 Demand), with a photoperiod of 12 h at 25 °C. After culture medium
 dehydration (approximately 5 days of incubation), 10 mL of sterile water
 was added to each plate and the conidia were removed with a Drigalski
 spatula. The suspension was filtered with sterile gauze to remove residues
 and the conidia were subsequently quantified in a Neubauer chamber. The
 suspension used for inoculation was adjusted to 5 x
 10<sup>4</sup> conidia·mL<sup>-1</sup> and sprayed
 on the abaxial side of the leaves of all seedlings using a manual sprayer.
 Thereafter, the seedlings were placed in a humid chamber for 72
 h. Temperature and relative humidity data were
 collected over the experimental period with a Datalogger HT-500,
 Instrutherm®. Weekly assessments of disease severity were performed on the
 first two pairs of true leaves throughout the five weeks, starting from
 the onset of symptoms (approximately 15 d after inoculation). The severity
 of BES in different coffee genotypes was quantified using a diagrammatic
 scale with six classes of the proportion in the infected area by BES [15]:
 class 1: 0.1-3.0%; class 2: 3.1-6.0%; class 3: 6.1-12.0%; class 4:
 12.1-18.0%, class 5: 18.01-30.0%, and class 6: 30.1-50.0%. The experiment
 was repeated twice. The distribution of the phenotypic segregation was
 evaluated in each genotype within the six classes described. Based on this
 assessment, the resistance level was determined: class 1- resistant (R);
 class 2- partially resistant (PR); class 3- moderately susceptible (MS);
 class 4- susceptible (S); classes 5 and 6- highly susceptible
 (AS). The severity dates were used to calculated the
 area under disease progress curve (AUDPC), as previously proposed by
 Shaner and Finney [16].   <i>AUDPC</i><i>= </i><sub><i>i</i><i>=1</i></sub><sup><i>n</i><i>-1</i></sup><i>si</i><i>+</i><i>si</i><i>+1</i><i>2</i><i>ti</i><i>+1-</i><i>ti</i>                                (1)                                                                                                                                 where AUDPC is area under the disease progress curve; Si is disease severity in the time of evaluation, and it is the time of evaluation i.   <b>Total soluble phenolic compounds and soluble lignin</b> Both total soluble phenolic compounds and soluble lignin levels were quantified in the leaves of MG 1207 accession (presenting low disease severity in the present study) and MG 0291 and Catuaí Vermelho IAC 144 genotypes (presenting high disease severity). The Catuaí Vermelho IAC 144 genotype was used as a control of susceptibility, according to Patricio et al. [10] and Botelho et al. [11]. The samples consisted of 2<sup>nd</sup> and 3<sup>rd</sup> pairs of fully expanded leaves, collected at 24, 120, 240, 480, and 720 h after inoculation (hai) of <i>C. coffeicola</i>. Samples of non-inoculated plants with the pathogen were also collected at 24 and 720 h to confirm that the inoculation influences the levels of total soluble phenolic compounds and soluble lignin. After collection, the samples were immediately stored in liquid nitrogen and keep an ultra-freezer until sample processing. The macerated samples were lyophilized and approximately 30 mg of the material was homogenized in 80% methanol. The solution was centrifuged at room temperature for 5 min at 14000 rpm. The supernatant and the precipitate were used to quantify total soluble phenolic compounds and soluble lignin, respectively. The levels of total soluble phenolic compounds were determined as described by Spanos and Wrolstad [17], with modifications. The supernatant was homogenized with 0.25 N Folin-Ciocalteau reagent, 1 M Na<sub>2</sub>CO<sub>3,</sub> and distilled water. The reaction was standardized in 200 μL and quantified using a spectrophotometer at 725 nm. Based on the standard curve of chlorogenic acid, the levels of total soluble phenolic compounds were calculated. Lignin was quantified as proposed by Doster and Bostock [18]. The precipitate was homogenized in 80% methanol and centrifuged as described for phenolic compounds. The contents were evaporated in an oven at 45 °C overnight and mixed with thioglycolic acid and 2 M HCl (ratio 1:10) in a water bath at 100 °C for 4 h. After centrifugation and solubilization in 0.5 M NaOH, the supernatant was homogenized with HCl P.A., and kept at 4 °C for 4 h before centrifugation. The precipitate was homogenized in 0.5 M NaOH. A 200-μL aliquot of this solution was used for the reaction, and the absorbance was assessed in a Power Wave XS microplate spectrophotometer (Biotek®) at 280 nm. Based on a standard curve of lignin, the soluble lignin content was subsequently estimated. The quantification of total soluble phenolic compounds and soluble lignin contents was performed in triplicate.   <b>Statistical analysis</b> The experiment BES severity were conducted in a randomized complete block design, with 60 treatments (genotypes), eight replicates, and five evaluations. The experimental plot comprised two coffee plants. A mixed model analysis was accomplished using the Selegen REML/BLUP software [19]. The following equation was used:  Y= Xm + Zg+Tp +e where <i>y</i> is the data vector; <i>m</i> is scalar referring to the general average of fixed effect; <i>g</i> is the vector of random genetic effects; <i>p</i> is the vector of the random-effects of blocks within replications and experiment; and <i>e</i> is the vector of random errors. X, Z, and T are incidence matrices of referred effects. The variance components were subjected to the likelihood ratio test at 5% probability. The experiment of quantification of total soluble phenolic compounds and soluble lignin in coffee genotypes, were conducted in a randomized complete block design with three replicates and two plants per plot. Analysis of variance was conducted in a 3 x 5 factorial scheme, with three evaluated genotypes and five collection times (24, 120, 240, 480, and 720 h) after inoculation (hai) of <i>C. coffeicola</i>. The data were subjected to analysis of variance and the means were compared by Tukey’s test at 5% probability.