Screening coffee genotypes for Cercospora coffeicola resistance in Brazil

Several efforts have been made by many researchers worldwide to develop coffee plants resistant to different Cercospora species; however, studies concerning C. coffeicola, specifically, are still incipient. In the present study, a blend of strains from this pathogen was inoculated into 18 Brazilian commercial cultivars, a coffee clone of Arabica, as well as into 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 genotypic values of the evaluated material, the most efficient way to select genotypes based on the data of severity to brown eye spot (BES) was also examined. Moreover, the action of defense mechanisms against C. coffeicola attacks was investigated by assessing the levels of total soluble phenolic compounds and soluble lignin in contrasting genotypes regarding disease susceptibility. Based on the results, the accession MG 1207 Sumatra demonstrated an intrinsic genetic capacity to maintain low levels of severity to brown eye spot. This genotype can thus substantially contribute to the development of new cultivars, which may lead to reduced use of pesticides. This study also evidenced that four evaluations of severity is enough to reach accuracy and efficiency for the severity of BES, thus providing expressive genetic gains. Finally, it is suggested that the levels of lignin and phenolic compounds are not associated with the resistance of coffee genotypes to brown eye spot.


Introduction
heritability of the phenotypic variation. This strategy may not be effective in crop 80 breeding programs that aim at developing materials resistant to BES as it does not 81 assess the potential effects of selection on the studied characters. 82 To defend themselves against pathogen attacks, plants have a set of defense 83 mechanisms, which can be either constitutively structural or biochemical; as well as a 84 defense system responsive to infection occurrences [8]. Among the induced 85 mechanisms, the production of phenolic compounds and lignin is commonly observed 86 during defense reactions, which are produced via metabolism of phenylpropanoids [9], 87 thereby providing higher resistance to the plant cell wall during pathogen attack. Hence, 88 understanding how C. coffeicola affects biochemical aspects of the plant cell can base a 89 further mechanism to control brown eye spot. 90 The first objective of this study was to select superior Coffea arabica genotypes 91 concerning resistance to BES. The possibility of selecting coffee plants with higher 92 resistance to this disease is extremely important, but the success of this strategy depends 93 on the existence of genetic variation within studied characteristics, as well as a high 94 heritability rate. The second objective was to better understand the action of defense 95 mechanisms against the attack of the pathogen C. coffeicola, by quantifying total 96 soluble phenolic compounds and soluble lignin in contrasting genotypes regarding 97 disease susceptibility. It is worth mentioning that no studies were found relating the 98 concentration of such compounds in coffee genotypes with resistance to brown eye spot.  (Table S1). Accessions were selected according to their 108 characteristics of yield, drink quality and/or resistance to other diseases of economic 109 importance.

110
Seeds were sown in 5L plastic trays containing autoclaved sand. The  of incubation), 10 ml of sterile water was added to each plate and the conidia were 133 removed with a Drigalski spatula. The suspension was filtered with sterile gauze to 134 remove residues and the conidia were subsequently quantified in a Neubauer chamber.

135
The suspension used for inoculation was adjusted to 5 x 10 4 conidia mL -1 and sprayed 136 on the abaxial side of the leaves of all seedlings, by using a manual sprayer. Afterward, 137 the seedlings were placed in a humid chamber for 72 h.

138
Temperature and relative humidity data were collected over the experiment with a 139 Datalogger HT-500, Instrutherm®. Weekly assessments concerning disease severity 140 were performed on the first two pairs of true leaves during five weeks, starting from the

157
The samples consisted of 2nd and 3rd pairs of fully expanded leaves, collected at 158 24, 120, 240, 480, and 720 h after inoculation (hai) of C. coffeicola. Samples of non-159 inoculated plants with the pathogen were also collected at t 24 and 720h to confirm 160 whether the inoculation influences the levels of total soluble phenolic compounds and 161 soluble lignin. After collection, the samples were immediately stored in liquid nitrogen 162 and then in an ultra-freezer until sample processing.

163
The macerated samples were lyophilized and approximately 30 mg of the 164 material were homogenized in 80% methanol. The solution was centrifuged at room 6 165 temperature at 14000 rpm for 5 min. The supernatant and the precipitate were used to 166 quantify total soluble phenolic compounds and soluble lignin, respectively.

167
The levels of total soluble phenolic compounds were determined as described by    (Table S1).

224
The studied population presented a high genotypic variability for the severity of  (Table S1).

228
Based on the analysis of frequency-severity, it was observed that the genotypes 229 were clustered mainly into 4, 5 and 6 classes (Table 1)   were selected for quantification of total soluble phenolic compounds and soluble lignin.

283
They were MG 1207, as low severity, and MG 0291, which presented a high severity, 284 being thus placed alongside the control (Catuaí Vermelho IAC 144).

285
There were no significant differences in total soluble phenolic compounds and

291
Phenolic compounds content showed variation among the three genotypes over 292 time (Fig 3)    most efficient way to select the severity data for brown eye spot was investigated.

347
Given the rejection of the null hypothesis for progeny variance ( selection gain of 70% to reduce the severity (Fig 1) produced as a physical and chemical defense mechanism against pathogen attacks [27, 417 28, 29], it was expected that the inoculated genotypes would present higher levels of 418 these metabolites as compared to non-inoculated ones.

419
The evidence that the levels of metabolites are not associated with coffee tree 420 resistance to brown eye spot may be associated with phenylpropanoid pathway processes that negatively affect plant growth and reproduction [29]. Thus, in this study, 431 plants may have triggered other physiological processes rather than the accumulation of 432 phenolic compounds and lignin (plant defense). As lignification is a process tightly 433 controlled by various regulatory levels, it will only occur at the appropriate time and 434 location of lignin deposition [32,31].

435
Leaf tissue necrosis results from the oxidation and polymerization of o-436 diphenols [33,34]. In the present study, decreases in the content of phenolic compounds characterized as a generic response in response to insects, fungi, or bacteria attacks [35].

442
Decreases in phenol content followed by successive increases were demonstrated 443 elsewhere with the resistance of olive cultivars to Verticillium dahliae [36].

444
Lignin, on the other hand, plays an important role in plant growth and 445 development, as it promotes increased cell wall rigidity and its metabolism is involved 446 in the response to different biotic and abiotic stresses [37]. In this sense, environmental