Inheritance of Resistance to Bacterial Halo Blight and Bacterial Leaf Spot in Arabica Coffee.

Developing coffee cultivars resistant to multiple diseases by combining resistance genes is a top priority in breeding programs. To create cultivars resistant to diseases and nematodes, we transferred genes for resistance to bacterial infections caused by Pseudomonas coronafaciens pv. garcae, which causes bacterial halo blight (BHB), and P. amygdali pv. tabaci, which causes bacterial leaf spots (BLS), into Arabica coffee. Genetic analyses were conducted on breeding populations to estimate the number and function of genes that confer resistance to BHB and BLS. In total, 2,109 plants in the F2 generation and reciprocal backcrosses were inoculated with P. coronafaciens pv. garcae, while 1,996 plants were inoculated with P. amygdali pv. tabaci. Results showed that resistance to both pathogens had a heritability of 0.99, and the segregations of resistance indicated that each disease was controlled by a single dominant gene. The analyses also revealed that the resistance genes for BHB and BLS were linked, with an average distance of 10.75 cM between them on the same chromosome.

The Rhizosphere Microbiomes of Five Species of Coffee Trees.

Coffee is one of the most important commodities in the global market. Of the 130 species of Coffea, only Coffea arabica and Coffea canephora are actually cultivated on a large scale. Despite the economic and social importance of coffee, little research has been done on the coffee tree microbiome. To assess the structure and function of the rhizosphere microbiome, we performed a deep shotgun metagenomic sequencing of the rhizospheres of five different species, C. arabica, C. canephora, Coffea stenophylla, Coffea racemosa, and Coffea liberica. Our findings indicated that C. arabica and C. stenophylla have different microbiomes, while no differences were detected between the other Coffea species. The core rhizosphere microbiome comprises genera such as Streptomyces, Mycobacterium, Bradyrhizobium, Burkholderia, Sphingomonas, Penicillium, Trichoderma, and Rhizophagus, several of which are potential plant-beneficial microbes. Streptomyces and mycorrhizal fungi dominate the microbial communities. The concentration of sucrose in the rhizosphere seems to influence fungal communities, and the concentration of caffeine/theobromine has little effect on the microbiome. We also detected a possible relationship between drought tolerance in Coffea and known growth-promoting microorganisms. The results provide important information to guide future studies of the coffee tree microbiome to improve plant production and health. IMPORTANCE The microbiome has been identified as a fundamental factor for the maintenance of plant health, helping plants to fight diseases and the deleterious effects of abiotic stresses. Despite this, in-depth studies of the microbiome have been limited to a few species, generally with a short life cycle, and perennial species have mostly been neglected. The coffee tree microbiome, on the other hand, has gained interest in recent years as Coffea trees are perennial tropical species of enormous importance, especially for developing countries. A better understanding of the microorganisms associated with coffee trees can help to mitigate the deleterious effects of climate change on the crop, improving plant health and making the system more sustainable.

A metabolomic platform to identify and quantify polyphenols in coffee and related species using liquid chromatography mass spectrometry.

Introduction: Products of plant secondary metabolism, such as phenolic compounds, flavonoids, alkaloids, and hormones, play an important role in plant growth, development, stress resistance. The plant family Rubiaceae is extremely diverse and abundant in Central America and contains several economically important genera, e.g. Coffea and other medicinal plants. These are known for the production of bioactive polyphenols (e.g. caffeine and quinine), which have had major impacts on human society. The overall goal of this study was to develop a high-throughput workflow to identify and quantify plant polyphenols. Methods: First, a method was optimized to extract over 40 families of phytochemicals. Then, a high-throughput metabolomic platform has been developed to identify and quantify 184 polyphenols in 15 min. Results: The current metabolomics study of secondary metabolites was conducted on leaves from one commercial coffee variety and two wild species that also belong to the Rubiaceae family. Global profiling was performed using liquid chromatography high-resolution time-of-flight mass spectrometry. Features whose abundance was significantly different between coffee species were discriminated using statistical analysis and annotated using spectral databases. The identified features were validated by commercially available standards using our newly developed liquid chromatography tandem mass spectrometry method. Discussion: Caffeine, trigonelline and theobromine were highly abundant in coffee leaves, as expected. Interestingly, wild Rubiaceae leaves had a higher diversity of phytochemicals in comparison to commercial coffee: defense-related molecules, such as phenylpropanoids (e.g., cinnamic acid), the terpenoid gibberellic acid, and the monolignol sinapaldehyde were found more abundantly in wild Rubiaceae leaves.

Differences between the Leaf Mycobiome of Coffea arabica and Wild Coffee Species and Their Modulation by Caffeine/Chlorogenic Acid Content.

The study of microbes associated with the coffee tree has been gaining strength in recent years. In this work, we compared the leaf mycobiome of the traditional crop Coffea arabica with wild species Coffea racemosa and Coffea stenophylla using ITS sequencing for qualitative information and real-time PCR for quantitative information, seeking to relate the mycobiomes with the content of caffeine and chlorogenic acid in leaves. Dothideomycetes, Wallemiomycetes, and Tremellomycetes are the dominant classes of fungi. The core leaf mycobiome among the three Coffea species is formed by Hannaella, Cladosporium, Cryptococcus, Erythrobasidium, and Alternaria. A network analysis showed that Phoma, an important C. arabica pathogen, is negatively related to six fungal species present in C. racemosa and C. stenophylla and absent in C. arabica. Finally, C. arabica have more than 35 times the concentration of caffeine and 2.5 times the concentration of chlorogenic acid than C. stenophylla and C. racemosa. The relationship between caffeine/chlorogenic acid content, the leaf mycobiome, and genotype pathogen resistance is discussed.

Multiple resistance to bacterial halo blight and bacterial leaf spot in Coffea spp / Resistência múltipla à mancha aureolada e à mancha foliar bacteriana em Coffea spp

Breeding for genetic resistance is an important method of crop disease management, due to the numerous benefits and low cost of establishment. In this study, progenies of 11 Coffea species and 16 wild C. arabica accessions were tested for their response to Pseudomonas syringae pv. garcae, the causal agent of bacterial halo blight, a widespread disease in the main coffee-producing regions of Brazil and considered a limiting factor for cultivation in pathogen-favorable areas; and also to P. syringae pv. tabaci, causal agent of bacterial leaf spot, a highly aggressive disease recently detected in Brazil. Separate experiments for each disease were carried out in a greenhouse, with artificial pathogen inoculations and ideal moisture conditions for disease development. The results showed that C. canephora, C. congensis, C. eugenioides, C. stenophylla, and C. salvatrix progenies, the wild C. arabica accessions Dilla & Alghe and Palido Viridis, and cultivar IPR 102 contain satisfactory levels of simultaneous resistance against bacterial halo blight and bacterial leaf spot. These results are useful in breeding programs for durable resistance to multiple biotic agents, providing new combinations of resistance alleles by hybridization, as well as for phytopathological studies, to identify infraspecific variability of the pathogens.(AU)

O melhoramento de plantas para resistência genética é um método importante para o manejo de doenças, pelos inúmeros benefícios e baixo custo de implementação. No presente estudo, progênies de 11 espécies de Coffea e 16 acessos selvagens de C. arabica foram testados quanto à resposta a Pseudomonas syringae pv. garcae, agente causal da mancha aureolada, doença disseminada nas principais regiões produtoras de café do Brasil e considerada fator limitante para o cultivo em áreas favoráveis a patógenos; e também para P. syringae pv. tabaci, agente causal da mancha foliar bacteriana, doença altamente agressiva detectada recentemente no Brasil. Experimentos separados para cada doença foram realizados em estufa, por meio da inoculação artificial dos patógenos em condições ideais de umidade para o desenvolvimento das doenças. Os resultados mostraram que as progênies Coffea canephora, C. congensis, C. eugenioides, C. stenophylla e C. salvatrix, além dos acessos selvagens de C. arabica Dilla & Alghe e Palido Viridis e da cultivar IPR 102, possuem níveis satisfatórios de resistência simultânea contra mancha aureolada e mancha foliar bacteriana. Os resultados descritos são úteis em programas de melhoramento para resistência duradoura a múltiplos agentes bióticos, fornecendo novas combinações de alelos de resistência por hibridização, bem como para estudos fitopatológicos, para identificar a variabilidade infraespecífica dos patógenos.(AU)

Can the leaf age influence the susceptibility to bacterial-leaf-spot and bacterial-halo-blight on coffee seedlings? / A idade das folhas pode influenciar na suscetibilidade de cafeeiros à macha-foliar-bacteriana e à mancha-aureolada?

Pseudomonas syringae van Hall, 1902, causes yield losses in innumerous economic important crops. On coffee trees, P. syringae pv. garcae causes the bacterial-halo-blight (BHB) and P. syringae pv. tabaci the bacterial-leaf-spot (BLS). Recently, these diseases incidence has increase in occurrence areas and aggressiveness in Brazil. Although leaf age plays a role in the severity response of BHB, it is not known yet if this phenomenon also occurs in coffee-BLS interaction, and with highly virulent strains. So, we examined differences in the diseases severity by inoculation of P. syringae pv. garcae and P. syringae pv. tabaci strains on coffee leaves with different ages, to compare this aspect with coffee-BLS interaction. Our results showed that, for both pathovars, the severity was greater at the first internodes leaves, although for the most aggressive strains it was quite similar on any leaf age.(AU)

Bactérias da espécie Pseudomonas syringae van Hall, 1902, causam perdas na produção em inúmeras culturas de importância econômica. Em cafeeiros, P. syringae pv. garcae provoca a mancha-aureolada e P. syringae pv. tabaci ocasiona a mancha-foliar-bacteriana, doenças cuja ocorrência e agressividade têm aumentado nos últimos anos no Brasil. Embora a idade das folhas influencie na expressão da severidade de mancha-aureolada, não se sabe ainda se essa influência se mantém em plantas infectadas por estirpes altamente virulentas da bactéria. Desse modo, o presente estudo foi realizado com a finalidade de examinar diferenças na severidade de mancha-aureolada em folhas de cafeeiro com diferentes idades, bem como estudar comparativamente tais aspectos na interação entre cafeeiro e mancha-foliar-bacteriana, empregando-se isolados altamente virulentos. Os resultados evidenciaram que, assim como a mancha-aureolada, a severidade da mancha-bacteriana também é maior em folhas jovens do primeiro internódio, entretanto, as estirpes mais agressivas de P. syringae pv. garcae e P. syringae pv. tabaci provocaram danos de magnitude semelhantes em folhas de diferentes idades, do primeiro ao quinto internódio.(AU)

Unsaponifiable matter from oil of green coffee beans: cosmetic properties and safety evaluation.

CONTEXT: Unsaponifiable matter (UM), a fraction of green coffee oil (GCO) contains functional compounds responsible for desirable cosmetic properties such as UV-B absorption. OBJECTIVES: To evaluate oil content and sun protection factor (SPF) variability of the two most important species of coffee and, the toxic and cytotoxic effects, as well as cosmetic properties, including antioxidant and antimicrobial activities of UM obtained from green Coffea arabica seed oil. MATERIALS AND METHODS: The safety and potential cosmetic properties of UM extracted from green coffee oil (GCO) were evaluated by the brine shrimp viability and the MTT cytotoxicity assays. The SPF and antioxidant activity were evaluated using in vitro methods. RESULTS: Relevant cytotoxicity was found against keratinocytes for concentrations ≥25 µg/mL and in the brine shrimp assay (LC50 24 µg/mL). Antimicrobial and antioxidant activities (IC50 1448 µg/mL) were low in UM but SPF was 10 times higher than in GCO. CONCLUSION: UM is a novel potential UV-B absorbent but its use as a cosmetic ingredient should be better considered due to the considerable cytotoxicity shown in the experimental conditions described.

Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner-toward the identification of candidate genes for marker assisted-selection.

BACKGROUND: A successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach. RESULTS: The microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars. CONCLUSIONS: Our results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation.

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Atributos fenológicos e agronômicos em cultivares de cafeeiro arábica / Phenological and agronomic attributes in arabica cultivars of coffee tree

O objetivo deste trabalho foi avaliar o efeito do ano agrícola sobre os atributos fenológicos e agronômicos em diferentes cultivares de cafeeiro arábica. Os atributos fenológicos determinados foram estádios de frutificação, de gema dormente até fruto seco e duração do ciclo. Como atributos agronômicos, foram avaliados o rendimento, a produtividade, os tipos e o tamanho de grãos. O período entre antese até fase chumbinho não variou entre as cultivares para o ano agrícola 2004/2005, já para o ano agrícola seguinte a cultivar 'Icatu Precoce' apresentou antecipação da fase chumbinho, e a cultivar 'Obatã' atrasou o início do ciclo fenológico em relação às demais cultivares estudadas. A duração do ciclo variou com o ano agrícola, o que possibilitou a confirmação da influência dos efeitos ambientais nessa característica. Os atributos agronômicos (produtividade, rendimento, tipo e tamanho de grãos), independentemente das cultivares, também variaram com o ano agrícola.

The aim of this study was to evaluate cultivars of Coffea arabica on the basic of phenology and agronomic attributes a function of the year of production. The phenological attributes were evaluated regarding phenology and percentage of cherry fruits at harvesting time. Agronomic traits evaluated included productivity and outturn, type of seeds and grain size. The period between the blooms till the fruits at the beginning of growth did not vary among the coffee trees in the year of production 2004/2005. While in the following year the Icatu Precoce cultivar presented anticipation of the fruits at the beginning of growth and the 'Obatã' cultivar delayed the beginning of the phenological cycle due to the other cultivars studied. The duration of each cycle varied with the year of production and then all the coffee trees in this study could be differentiated on the timing of maturation (early, middle and late). The agronomic attributes (productivity, outturn, type and size of grains), regardless the different cultivars of coffee also varied with the year of production.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

Phenol contents, oxidase activities, and the resistance of coffee to the leaf miner Leucoptera coffeella.

We examined the role of phenolic compounds, and the enzymes peroxidase and polyphenol oxidase, in the expression of resistance of coffee plants to Leucoptera coffeella (Lepidoptera: Lyonetiidae). The concentrations of total soluble phenols and chlorogenic acid (5-caffeoylquinic acid), and the activities of the oxidative enzymes peroxidase (POD) and polyphenol oxidase (PPO), were estimated in leaves of Coffea arabica, C. racemosa, and progenies of crosses between these species, which have different levels of resistance, before and after attack by this insect. The results indicate that phenols do not play a central role in resistance to the coffee leaf miner. Differences were detected between the parental species in terms of total soluble phenol concentrations and activities of the oxidative enzymes. However, resistant and susceptible hybrid plants did not differ in any of these characteristics. Significant induction of chlorogenic acid and PPO was only found in C. racemosa, the parental donator of the resistance genes against L. coffeella. High-performance liquid chromatography (HPLC) analysis also showed qualitative similarity between hybrids and the susceptible C. arabica. These results suggest that the phenolic content and activities of POD and PPO in response to the attack by the leaf miner may not be a strong evidence of their participation in direct defensive mechanisms.

Caffeine and resistance of coffee to the berry borer Hypothenemus hampei (Coleoptera: Scolytidae).

The role of caffeine as a chemical defense of coffee against the berry borer Hypothenemus hampei was investigated. No positive correlation was observed between resistance and caffeine content in experiments in which seeds from several coffee species presenting genetic variability for the alkaloid were exposed to adult insects. The same was observed in an experiment with coffee seeds that had their caffeine content doubled by imbibition with caffeine aqueous solutions. Other experiments showed that the attractiveness to insects was not related to the caffeine content of mature fruits. These results indicate that H. hampei has evolved an adaptation to handle the toxic effects of caffeine.