IBA and melatonin increase trigonelline and caffeine during the induction and initiation of adventitious roots in Coffea arabica L. cuttings.

Caffeine and trigonelline are found in Coffea arabica, and show antioxidant roles and growth and development functions. However, there are no reports on trigonelline and caffeine in relation to coffee rooting. The aim was to evaluate the impact of application of indole-3-butyric acid (IBA) and melatonin on caffeine and trigonelline at different stages of adventitious rooting in cuttings. In addition, to study the correlation between these metabolites and H2O2, phenols, and antioxidant enzymes. Four treatments (Control, melatonin 21 µM (M21), melatonin 43 µM (M43), and IBA 7380 µM (IBA)) were used, with four replications. The growth and biochemical parameters of the antioxidant system were performed in induction, initiation, and extension rooting stages. Higher concentrations of trigonelline and caffeine quantified in the induction and initiation stages were positively correlated with higher percentage of rooted cuttings. Trigonelline and caffeine were positively correlated with H2O2 in all stages of development of adventitious roots. The correlations of trigoneline and caffeine with phenols and antioxidant enzymes reveal different profiles, depending on the phases. The results indicate that IBA and melatonin increase trigonelline and caffeine during the induction and initiation of adventitious roots in Coffea arabica cuttings, which is correlated with a higher percentage of rooted cuttings.

Potassium Phosphite Activates Components Associated with Constitutive Defense Responses in Coffea arabica Cultivars.

Phosphites have been used as inducers of resistance, activating the defense of plants and increasing its ability to respond to the invasion of the pathogen. However, the mode of action of phosphites in defense responses has not yet been fully elucidated. The objective of this study was to evaluate the effect of potassium phosphite (KPhi) in coffee cultivars with different levels of resistance to rust to clarify the mechanism by which KPhi activates the constitutive defense of plants. To this end, we studied the expression of genes and the activity of enzymes involved in the defense pathway of salicylic acid (SA) and reactive oxygen species (ROS), in addition to the levels of total soluble phenolic compounds and soluble lignin. Treatment with KPhi induced constitutive defense responses in cultivars resistant and susceptible to rust. The results suggest that KPhi acts in two parallel defense pathways, SA and ROS, which are essential for the induction of systemic acquired resistance (SAR) when activated simultaneously. The activation of the mechanisms associated with defense routes demonstrates that KPhi is a potential inducer of resistance in coffee plants.

Potential of rust-resistant arabica coffee cultivars for specialty coffee production

Rust is the main disease affecting Coffea arabica, the most economically important coffee species. The objective of this study was to analyze C. arabicacultivars with different levels of rust resistance, including bean size, raw bean appearance, finalsensory scores (FSS), and aromaand taste nuances of the coffee cup. The experiment was designed in randomized blocks (RBD) with three replications and 20 treatments (cultivars), totaling 60 experimental plots. The rust-susceptible cultivars IPR 100, Rubi MG 1192, and Topázio MG 1190 were compared with 17 rust-resistant cultivars.Cultivars IPR 103, MGS Aranãs, and SaíraII presented the highest percentages of high sieves, highest scores of raw bean appearance, and low percentages of mocha-type beans. All cultivars had FSS above 82 and were classified as specialty coffees. The cultivars with the highest FSS (Arara and Catiguá MG2) showed a greater diversity of coffee cup aroma and flavor nuances. Rust-resistant Arabica coffee cultivars are promising for the physical quality of beans and have potential for the specialty coffee market.

Genome-wide association study for resistance to Pseudomonas syringae pv. garcae in Coffea arabica.

Bacteria halo blight (BHB), a coffee plant disease caused by Pseudomonas syringae pv. garcae, has been gaining importance in producing mountain regions and mild temperatures areas as well as in coffee nurseries. Most Coffea arabica cultivars are susceptible to this disease. In contrast, a great source of genetic diversity and resistance to BHB are found in C. arabica Ethiopian accessions. Aiming to identify quantitative trait nucleotides (QTNs) associated with resistance to BHB and the influence of these genomic regions during the domestication of C. arabica, we conducted an analysis of population structure and a Genome-Wide Association Study (GWAS). For this, we used genotyping by sequencing (GBS) and phenotyping for resistance to BHB of a panel with 120 C. arabica Ethiopian accessions from a historical FAO collection, 11 C. arabica cultivars, and the BA-10 genotype. Population structure analysis based on single-nucleotide polymorphisms (SNPs) markers showed that the 132 accessions are divided into 3 clusters: most wild Ethiopian accessions, domesticated Ethiopian accessions, and cultivars. GWAS, using the single-locus model MLM and the multi-locus models mrMLM, FASTmrMLM, FASTmrEMMA, and ISIS EM-BLASSO, identified 11 QTNs associated with resistance to BHB. Among these QTNs, the four with the highest values of association for resistance to BHB are linked to g000 (Chr_0_434_435) and g010741 genes, which are predicted to encode a serine/threonine-kinase protein and a nucleotide binding site leucine-rich repeat (NBS-LRR), respectively. These genes displayed a similar transcriptional downregulation profile in a C. arabica susceptible cultivar and in a C. arabica cultivar with quantitative resistance, when infected with P. syringae pv. garcae. However, peaks of upregulation were observed in a C. arabica cultivar with qualitative resistance, for both genes. Our results provide SNPs that have potential for application in Marker Assisted Selection (MAS) and expand our understanding about the complex genetic control of the resistance to BHB in C. arabica. In addition, the findings contribute to increasing understanding of the C. arabica domestication history.

Genome-Wide Identification, Characterization, and Comparative Analysis of NLR Resistance Genes in Coffea spp.

The largest family of disease resistance genes in plants are nucleotide-binding site leucine-rich repeat genes (NLRs). The products of these genes are responsible for recognizing avirulence proteins (Avr) of phytopathogens and triggering specific defense responses. Identifying NLRs in plant genomes with standard gene annotation software is challenging due to their multidomain nature, sequence diversity, and clustered genomic distribution. We present the results of a genome-wide scan and comparative analysis of NLR loci in three coffee species (Coffea canephora, Coffea eugenioides and their interspecific hybrid Coffea arabica). A total of 1311 non-redundant NLR loci were identified in C. arabica, 927 in C. canephora, and 1079 in C. eugenioides, of which 809, 562, and 695 are complete loci, respectively. The NLR-Annotator tool used in this study showed extremely high sensitivities and specificities (over 99%) and increased the detection of putative NLRs in the reference coffee genomes. The NLRs loci in coffee are distributed among all chromosomes and are organized mostly in clusters. The C. arabica genome presented a smaller number of NLR loci when compared to the sum of the parental genomes (C. canephora, and C. eugenioides). There are orthologous NLRs (orthogroups) shared between coffee, tomato, potato, and reference NLRs and those that are shared only among coffee species, which provides clues about the functionality and evolutionary history of these orthogroups. Phylogenetic analysis demonstrated orthologous NLRs shared between C. arabica and the parental genomes and those that were possibly lost. The NLR family members in coffee are subdivided into two main groups: TIR-NLR (TNL) and non-TNL. The non-TNLs seem to represent a repertoire of resistance genes that are important in coffee. These results will support functional studies and contribute to a more precise use of these genes for breeding disease-resistant coffee cultivars.

LysM receptors in Coffea arabica: Identification, characterization, and gene expression in response to Hemileia vastatrix.

Pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) localized on the host plasma membrane. These receptors activate a broad-spectrum and durable defense, which are desired characteristics for disease resistance in plant breeding programs. In this study, candidate sequences for PRRs with lysin motifs (LysM) were investigated in the Coffea arabica genome. For this, approaches based on the principle of sequence similarity, conservation of motifs and domains, phylogenetic analysis, and modulation of gene expression in response to Hemileia vastatrix were used. The candidate sequences for PRRs in C. arabica (Ca1-LYP, Ca2-LYP, Ca1-CERK1, Ca2-CERK1, Ca-LYK4, Ca1-LYK5 and Ca2-LYK5) showed high similarity with the reference PRRs used: Os-CEBiP, At-CERK1, At-LYK4 and At-LYK5. Moreover, the ectodomains of these sequences showed high identity or similarity with the reference sequences, indicating structural and functional conservation. The studied sequences are also phylogenetically related to the reference PRRs described in Arabidopsis, rice, and other plant species. All candidates for receptors had their expression induced after the inoculation with H. vastatrix, since the first time of sampling at 6 hours post-inoculation (hpi). At 24 hpi, there was a significant increase in expression, for most of the receptors evaluated, and at 48 hpi, a suppression. The results showed that the candidate sequences for PRRs in the C. arabica genome display high homology with fungal PRRs already described in the literature. Besides, they respond to pathogen inoculation and seem to be involved in the perception or signaling of fungal chitin, acting as receptors or co-receptors of this molecule. These findings represent an advance in the understanding of the basal immunity of this species.

Production and characterization of a new distilled beverage from green coffee seed residue.

This study evaluated green coffee seed residue (GCSR) as an alternative substrate for producing distilled beverages. Two proportions of GCSR, 10% and 20% (w/v), were fermented and distilled in a copper alembic still. The spirits were characterized by GC-FID, HS-SPME GC-MS, and sensory analysis by trained panelists. Most of the 62 identified volatile compounds were affected by the GCSR concentration. Total terpenes, higher alcohols, and acetals showed the highest concentrations in the 10% GCSR spirit. Esters, acetates, and aldehydes were most abundant in the 20% GCSR. In the sensory analysis, the 10% GCSR spirit was characterized by floral, dairy, and almond aromas, while the 20% GCSR spirit was embodied coffee, vegetable, hazelnut, cooked cabbage, and nut descriptors. The results demonstrate the potential of GCSR as a substrate for producing coffee spirits with chemical and sensory qualities, with the 10% GCSR being the better option for fermentation.

Constitutive Defense Strategy of Coffee Under Field Conditions: A Comparative Assessment of Resistant and Susceptible Cultivars to Rust.

Coffea arabica is the most economically important coffee species worldwide. However, its production is severely limited by diseases such as rust. The mechanisms underlying constitutive defense responses in coffee are still poorly understood, compared with induced defense mechanisms. We aimed to characterize constitutive defense responses of thirteen cultivars of C. arabica. Cultivars were classified under field conditions according to the level of resistance to rust: resistant (R), moderately resistant (MR), and susceptible (S). Based on this classification, the stability of eight reference genes (RGs) was evaluated. The most stable RGs were EF1α, APT1, and 24S. We also evaluated the expression of CaWRKY1, CaPAL1, CaCAD1, and CaPOX1, and activities of PAL, CAD, and POX, which are involved in lignin biosynthesis, and leaf content of total phenolic compounds and lignin. Gene expression and enzymatic activity were not correlated with defense metabolites in the R cultivar group but showed a negative correlation with phenolic compounds in MR cultivars. Cultivar S showed positive correlations of gene expression and enzyme activity with phenolic compounds. These results may assist coffee breeding programs regarding selection of genotypes and in optimization of rust resistance.

Chemical Composition, Production of Secondary Metabolites and Antioxidant Activity in Coffee Cultivars Susceptible and Partially Resistant to Bacterial Halo Blight.

Coffee production is one of the main agricultural activities in Brazil, and several coffee cultivars with disease resistance have already been developed. The secondary metabolites produced by plants are closely associated with defense strategies, and the resistance of coffee cultivars to bacterial halo blight (BHB) can be related to these compounds. Therefore, this study aims to compare a partially resistant coffee cultivar (Iapar-59) and a susceptible cultivar (Mundo Novo 376/4) to BHB (Pseudomonas syringae pv. garcae) in relation to the chemical composition and antioxidant activity of the leaf extracts. In addition, this study determined the total phenolic and flavonoid contents and phenolic profiles of the Iapar-59 leaf extracts of plants inoculated with P. syringae pv. garcae. The Iapar-59 extract showed a higher content of phenolic compounds and flavonoids than the Mundo Novo 376/4 extract. Both cultivars contained gallic, chlorogenic and caffeic acids; however, the highest contents were quantified in the Iapar-59 cultivar. The leaf extracts from the Iapar-59 cultivar exhibited higher antioxidant activity. Higher concentrations of gallic, caffeic and chlorogenic acids and the presence of vanillin were detected in the extract of cultivar Iapar-59 inoculated with P. syringae pv. garcae.

Production and characterization of a new distillate obtained from fermentation of wet processing coffee by-products.

Coffee is one of the most important commodities worldwide. The industrial processing of coffee cherries generates a considerable volume of by-products such as wastewater, coffee pulp, mucilage, and husk. These by-products have sugars and nutrients that can be converted into value-added products via microbial action. In this study, for the first time, we evaluated the potential of coffee pulp and coffee wastewater as substrate for alcoholic fermentation produce a distilled beverage. The must composed by dry or wet coffee pulp and coffee wastewater added of commercial sucrose or sugarcane molasses was fermented by S. cerevisiae. After a screening step, a larger fermentation was carried out with the wet pulp added of sucrose due to its higher alcoholic fermentation efficiency. The distilled beverage contained 38% (v/v) ethanol and 0.2 g/L of acetic acid. The contaminants furfural, hydroxymethylfurfural and ethyl carbamate were below detection level. Among the 48 volatile compounds detected, the majority (21) were ethyl esters usually associated with floral and sweet aromas. Ethyl decanoate (996.88 µg/L) and ethyl dodecanoate (1088.09 µg/L) were the most abundant esters. Coffee spirit presented taste acceptance of 80% and sugarcane spirit, 70%. The tasters indicated an aroma acceptance of 86% for the coffee spirit and 78% for the sugarcane spirit. The results of this work demonstrate the potential for using coffee by-products to produce a good quality distilled beverage. Considering our results, especially sensorial analysis, we can infer that the produced coffee beverage represents a new alternative for adding value to the coffee production chain.

Development of a transformation system for Crinipellis perniciosa, the causal agent of witches' broom in cocoa plants.

Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment. Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.