Effects of geographical origin and post-harvesting processing on the bioactive compounds and sensory quality of Brazilian specialty coffee beans.

Specialty coffee beans are those produced, processed, and characterized following the highest quality standards, toward delivering a superior final product. Environmental, climatic, genetic, and processing factors greatly influence the green beans' chemical profile, which reflects on the quality and pricing. The present study focuses on the assessment of eight major health-beneficial bioactive compounds in green coffee beans aiming to underscore the influence of the geographical origin and post-harvesting processing on the quality of the final beverage. For that, we examined the non-volatile chemical profile of specialty Coffea arabica beans from Minas Gerais state, Brazil. It included samples from Cerrado (Savannah), and Matas de Minas and Sul de Minas (Atlantic Forest) regions, produced by two post-harvesting processing practices. Trigonelline, theobromine, theophylline, chlorogenic acid derivatives, caffeine, caffeic acid, ferulic acid, and p-coumaric acid were quantified in the green beans by high-performance liquid chromatography with diode array detection. Additionally, all samples were roasted and subjected to sensory analysis for coffee grading. Principal component analysis suggested that Cerrado samples tended to set apart from the other geographical locations. Those samples also exhibited higher levels of trigonelline as confirmed by two-way ANOVA analysis. Samples subjected to de-pulping processing showed improved chemical composition and sensory score. Those pulped coffees displayed 5.8% more chlorogenic acid derivatives, with an enhancement of 1.5% in the sensory score compared to unprocessed counterparts. Multivariate logistic regression analysis pointed out altitude, ferulic acid, p-coumaric acid, sweetness, and acidity as predictors distinguishing specialty coffee beans obtained by the two post-harvest processing. These findings demonstrate the influence of regional growth conditions and post-harvest treatments on the chemical and sensory quality of coffee. In summary, the present study underscores the value of integrating target metabolite analysis with statistical tools to augment the characterization of specialty coffee beans, offering novel insights for quality assessment with a focus on their bioactive compounds.

Physicochemical Characterization, Antioxidant and Tyrosinase Inhibitory Activities of Coffea Robusta Monofloral Honey from Dak Lak Province, Vietnam.

Robusta coffee blossom honey stands as a key regional product in Dak Lak province, Vietnam. Despite its significance, there exists a dearth of scientific data for assessing its quality. This study aims to fill this gap by characterizing the physicochemical properties and biological activities of coffee blossom honeys from three distinct sub-regions within Dak Lak province, Vietnam. These activities include ferric reducing power (FRP), DPPH and ABTS radical scavenging, as well as tyrosinase inhibitory activities. Moreover, the study compares these honey samples with other popular varieties in Vietnam, such as Lychee and Longan honeys. The physicochemical parameters of the honey samples meet the standards set by Codex Alimentarius 2001. Through UPLC analysis, eleven compounds were identified, with caffeine serving as a marker for coffee honey. Furthermore, by employing multiple factor analysis (MFA), it was observed that certain physicochemical properties correlate positively with tyrosinase inhibitory, DPPH, ABTS free radicals scavenging activities, and FRP. Notably, tyrosinase inhibitory activity exhibited a positive correlation with antioxidant activity. These findings underscore the high quality of Coffea robusta honey, showcasing its potent antioxidant and tyrosinase inhibitory activities.

First report of large-berry coffee (Coffea liberica) anthracnose caused by Colletotrichum kahawae in China.

Large-berry coffee (Coffea liberica) is one of the three cultivated coffee species and a precious breeding germplasm in China (Yan et al, 2019). Anthracnose is a damaging epidemic disease on coffee worldwide (Mohammed et al. 2015). Between June and September 2022, anthracnose was observed on coffee plants in Puer area, Yunnan, China and disease incidence (% plants diseased) of 8.5%-28.2% was recorded in the field. The disease symptoms were observed at all growth stages. Lesions on leaves were circular or oval, with a white to gray central zone outlined by a brown margin and surrounded by a chlorotic halo, Φ5.1-18.5 mm; some lesions extended and coalesced later to form large, blighted areas, leading to complete leaf senescence, defoliation and bare blighted branches on heavily infected trees. The spots on coffee berries were oval or fusiform, sunken and brown-black; diseased berries became gray-black and dried-out but remained on the tree. Leaves with typical anthracnose lesions were collected from fields in Simao ( 22.07°E,100.98°N) to isolate the pathogen. Leaf pieces (5×5mm) from the lesion margin were cut, surface-sterilized with 75% ethanol and 2% NaClO, and cultured on PDA at 25°C. Three isolates with the same colony morphology were obtained by hyphal tip purification. Detached and intact leaves of 6-month coffee seedlings were inoculated with Φ5mm mycelial discs of the isolates. Anthracnose lesions developed on the inoculated leaves, with all 3 isolates, 7d after incubation in a growth chamber (25°C, > 90% RH and lighting 8 h/d at 11000 lux). Pathogens with the same colony morphology as those of the original isolates were re-isolated from the infected tissues of inoculated leaves, thus fulfilling Koch's Postulates. The ITS sequence (PP550861) for the isolate was PCR-amplified and Blast-n analyses showed 100 % (554/554bp) identity to Colletotrichum kahawae LWTJ01; so they were the same population and coded as KFTJ02. The actin (ACT), calmodulin(CAL), glyceraldehydes-3-phosphate dehydrogenase (GAPHD) and histone 3 (HIS3) genes (Qiu et al. 2020) were amplified from one of KFTJ02 isolates, sequenced and deposited in NCBI GenBank (OR842543, OR842544, OR842545 & OR842546). A phylogenetic tree was generated based on the concatenated sequences of the four genes and those of related Colletotrichum spp. using MEGA 6.0 and KFTJ02 clustered in the same clade with C. kahawae IMI319418 on the tree (Bootstrap sup.=88%). When cultured at 25°C on PDA for 7 days, its colonies were near round or ovoid, gray-white, contoured, Φ73.2-80.1 (76.2±2.3)mm or growth rate 10.2-11.1(8.1) mm/d (n=10). The hyphae were hyaline, septated, branching at near right angles. Conidial masses formed 14 days after incubation. The conidia were elliptical, hyaline, monocellular, 10.2-15.5 (12.7±1.06)×3.8-5.2 (4.3±0.52) µm (n=50). The appressoria were black-brown, oval or irregular, 7.8-9.3 (8.5±0.81)µm (n= 50). These morphological characteristics were consistent with those of C. kahawae (Bridge et al, 2008). Therefore, KFTJ02 was identified as C. kahawae, which has been found to infect Camellia oleifera, Areca catechu and Ficus microcarpa (Wei et al, 2023; Zhang et al, 2020; Lin 2023). The coffee berry disease pathogen (C. kahawae) is a quarantine species which has not been recorded and so it is first reported on coffee crops in China. Results of the present study provide important references for further studies on this disease.

Synthesis and toxicity assessment of Coffea arabica extract-derived gold nanoparticles loaded with doxorubicin in lung cancer cell cultures.

Cancer is the second leading cause of death worldwide, despite the many treatments available, cancer patients face side effects that reduce their quality of life. Therefore, there is a need to develop novel strategies to increase the efficacy of treatments. In this study, gold nanoparticles obtained by green synthesis with Coffea arabica green bean extract were loaded with Doxorubicin, (a highly effective but non-specific drug) by direct interaction and using commercial organic ligands that allow colloidal dispersion at physiological and tumor pH. Conjugation of these components resulted in stable nanohybrids at physiological pH and a tumor pH release dependent, with a particle size less than 40 nm despite having the ligands and Doxorubicin loaded on their surface, which gave them greater specificity and cytotoxicity in H69 tumor cells.

Coffee Extract as a Natural Antioxidant in Fresh Pork Sausage-A Model Approach.

Consumers are increasingly looking for healthy foods without the addition of synthetic additives. The aim of this study was to evaluate the efficiency of coffee extracts as a natural antioxidant in fresh pork sausage. Firstly, the conditions for obtaining coffee green extracts were optimized (Central Composite Rotatable Design 23, variables: extraction time, ethanol-water ratio, and sample-solvent ratio) in an ultrasound bath (70 °C). The response variables were the bioactive compounds levels and antioxidant activity. Valid models were obtained (p ≤ 0.05, R2 > 0.751), with higher bioactive content and antioxidant activity in the central point region. Extracts of Robusta and Arabica coffee green (RG and AG) and medium roast (RR and AR) obtained, and central point (10 min, an ethanol concentration of 30%, and a sample-solvent ratio of 10 g/100 mL) and optimized (14.2 min, 34.2%, and 5.8 g/100 mL) parameters were characterized. The RG presented a significantly (p ≤ 0.05) higher content of caffeine (3114.8 ± 50.0 and 3148.1 ± 13.5 mg/100 g) and 5-CQA (6417.1 ± 22.0 and 6706.4 ± 23.5 mg/100 g) in both extraction conditions, respectively. The RG and RR coffee presented the highest antioxidant activity. Two concentrations of RG and RR coffee extracts were tested in fresh pork sausage. The Robusta coffee extract presented the highest antioxidant activity in both roasted and green states. However, when applied to a meat product, the extract prepared with RG coffee showed better results, with efficiency in replacing synthetic antioxidants (content of malonaldehyde/kg of sample below 0.696 ± 0.059 in 20 days of storage), without altering the sensory attributes of the product (average scores above 7.16 ± 1.43 for all attributes evaluated). Therefore, the RG coffee extract was a suitable alternative as a natural antioxidant applied to fresh pork sausage.

Perceptions of Deficiencies in the Basic Conditions for Farm Management and Quality of Life in Coffee-Growing Households: A Panel Analysis of a Rural Community in Eastern Uganda.

In the present study, information collected from 360 coffee-cultivating households (HHs) is used to investigate perceptions of deficiencies in three sub-counties in Eastern Uganda and to study changes in these perceptions between two survey rounds. The results of an explorative principal components analysis identify five factors affecting farmers' perceptions. Whereas perceptions of deficiencies in the preconditions for farm management activities differ significantly between the three sub-counties investigated, indicators of deficiencies in general life quality are distributed more equally. Deteriorations are explained mainly by perceived changes in weather conditions. On the one hand, it can be assumed that the high constraint level will continue to increase in the future due to climate change and its impacts on life quality and the basic conditions required for farm management. On the other hand, access to resources such as water taps but also increased competition between buyers, have improved the situation. Results further indicate that if activities such as the expansion of information access and improvement of road conditions (after land registration) are implemented on a larger scale, these negative trends can be partly counteracted to help farmers maintain the conditions for effective farm management and improve their quality of life in the future.

Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee.

BACKGROUND: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. RESULTS: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME-GC-MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. CONCLUSIONS: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

Transcriptomic Analysis of Alternative Splicing Events during Different Fruit Ripening Stages of Coffea arabica L.

To date, genomic and transcriptomic data on Coffea arabica L. in public databases are very limited, and there has been no comprehensive integrated investigation conducted on alternative splicing (AS). Previously, we have constructed and sequenced eighteen RNA-seq libraries of C. arabica at different ripening stages of fruit development. From this dataset, a total of 3824, 2445, 2564, 2990, and 3162 DSGs were identified in a comparison of different fruit ripening stages. The largest proportion of DSGs, approximately 65%, were of the skipped exon (SE) type. Biologically, 9 and 29 differentially expressed DSGs in the spliceosome pathway and carbon metabolism pathway, respectively, were identified. These DSGs exhibited significant variations, primarily in S1 vs. S2 and S5 vs. S6, and they involve many aspects of organ development, hormone transduction, and the synthesis of flavor components. Through the examination of research findings regarding the biological functions and biochemical pathways associated with DSGs and DEGs, it was observed that six DSGs significantly enriched in ABC transporters, namely, LOC113712394, LOC113726618, LOC113739972, LOC113725240, LOC113730214, and LOC113707447, were continually down-regulated at the fruit ripening stage. In contrast, a total of four genes, which were LOC113732777, LOC113727880, LOC113690566, and LOC113711936, including those enriched in the cysteine and methionine metabolism, were continually up-regulated. Collectively, our findings may contribute to the exploration of alternative splicing mechanisms for focused investigations of potential genes associated with the ripening of fruits in C. arabica.

Drought Stress Responses in Arabica Coffee Genotypes: Physiological and Metabolic Insights.

Understanding the impact of drought stress on Arabica coffee physiology and metabolism is essential in the pursuit of developing drought-resistant varieties. In this study, we explored the physiological and metabolite changes in coffee genotypes exhibiting varying degrees of tolerance to drought-namely, the relatively tolerant Ca74110 and Ca74112, and the sensitive Ca754 and CaJ-19 genotypes-under well-watered conditions and during terminal drought stress periods at two time points (0 and 60 days following the onset of stress). The metabolite profiling uncovered significant associations between the growth and the physiological characteristics of coffee genotypes with distinct drought tolerance behaviors. Initially, no marked differences were observed among the genotypes or treatments. However, at the 60-day post-drought onset time point, notably higher shoot growth, biomass, CO2 assimilation, pigments, and various physiological parameters were evident, particularly in the relatively tolerant genotypes. The metabolite profiling revealed elevations in glucose, maltose, amino acids, and organic acids, and decreases in other metabolites. These alterations were more pronounced in the drought-tolerant genotypes, indicating a correlation between enhanced compatible solutes and energy-associated metabolites crucial for drought tolerance mechanisms. This research introduces GC-MS-based metabolome profiling to the study of Ethiopian coffee, shedding light on its intricate responses to drought stress and paving the way for the potential development of drought-resistant coffee seedlings in intensified agro-ecological zones.

Leaf surface microbiota transplantation confers resistance to the coffee leaf rust in susceptible Coffea arabica.

Coffee leaf rust, caused by the fungus Hemileia vastatrix, became a major concern for coffee-producing countries. Additionally, there is an increase in the resistance of certain races of the fungus to fungicides and breeding cultivars, making producers to use alternative control methods. In this work, we transplanted the leaf surface microbiota of rust-resistant coffee species (Coffea racemosa and Coffea stenophylla) to Coffea arabica and tested whether the new microbiota would be able to minimize the damage caused by H. vastatrix. It was seen that the transplant was successful in controlling rust, especially from C. stenophylla, but the protection depended on the concentration of the microbiota. Certain fungi such as Acrocalymma, Bipolaris, Didymella, Nigrospora, Setophaeosphaeria, Simplicillium, Stagonospora, Torula and bacteria such as Chryseobacterium, Sphingobium and especially Enterobacter and have their populations increased and may be related to the antagonism seen against H. vastatrix. Interestingly, relative population of bacteria from genera Pantoea, Methylobacterium and Sphingomonas decreased after transplant, suggesting a positive interaction between them and H. vastatrix development. Our findings may help to better understand the role of the microbiota in coffee leaf rust as well as help to optimize the development of biocontrol agents.

Assessing scale-dependency of climate risks in coffee-based agroforestry systems.

Agroforestry is a management strategy for mitigating the negative impacts of climate and adapting to sustainable farming systems. The successful implementation of agroforestry strategies requires that climate risks are appropriately assessed. The spatial scale, a critical determinant influencing climate impact assessments and, subsequently, agroforestry strategies, has been an overlooked dimension in the literature. In this study, climate risk impacts on robusta coffee production were investigated at different spatial scales in coffee-based agroforestry systems across India. Data from 314 coffee farms distributed across the districts of Chikmagalur and Coorg (Karnataka state) and Wayanad (Kerala state) were collected during the 2015/2016 to 2017/2018 coffee seasons and were used to quantify the key climate drivers of coffee yield. Projected climate data for two scenarios of change in global climate corresponding to (1) current baseline conditions (1985-2015) and (2) global mean temperatures 2 °C above preindustrial levels were then used to assess impacts on robusta coffee yield. Results indicated that at the district scale rainfall variability predominantly constrained coffee productivity, while at a broader regional scale, maximum temperature was the most important factor. Under a 2 °C global warming scenario relative to the baseline (1985-2015) climatic conditions, the changes in coffee yield exhibited spatial-scale dependent disparities. Whilst modest increases in yield (up to 5%) were projected from district-scale models, at the regional scale, reductions in coffee yield by 10-20% on average were found. These divergent impacts of climate risks underscore the imperative for coffee-based agroforestry systems to develop strategies that operate effectively at various scales to ensure better resilience to the changing climate.

Crop-to-wild gene flow in wild coffee species: the case of Coffea canephora in the Democratic Republic of the Congo.

BACKGROUND AND AIMS: Plant breeders are increasingly turning to crop wild relatives (CWRs) to ensure food security in a rapidly changing environment. However, CWR populations are confronted with various human-induced threats, including hybridization with their nearby cultivated crops. This might be a particular problem for wild coffee species, which often occur near coffee cultivation areas. Here, we briefly review the evidence for wild Coffea arabica (cultivated as Arabica coffee) and Coffea canephora (cultivated as Robusta coffee) and then focused on C. canephora in the Yangambi region in the Democratic Republic of the Congo. There, we examined the geographical distribution of cultivated C. canephora and the incidence of hybridization between cultivated and wild individuals within the rainforest. METHODS: We collected 71 C. canephora individuals from home gardens and 12 C. canephora individuals from the tropical rainforest in the Yangambi region and genotyped them using genotyping-by-sequencing (GBS). We compared the fingerprints with existing GBS data from 388 C. canephora individuals from natural tropical rainforests and the INERA Coffee Collection, a Robusta coffee field gene bank and the most probable source of cultivated genotypes in the area. We then established robust diagnostic fingerprints that genetically differentiate cultivated from wild coffee, identified cultivated-wild hybrids and mapped their geographical position in the rainforest. KEY RESULTS: We identified cultivated genotypes and cultivated-wild hybrids in zones with clear anthropogenic activity, and where cultivated C. canephora in home gardens may serve as a source for crop-to-wild gene flow. We found relatively few hybrids and backcrosses in the rainforests. CONCLUSIONS: The cultivation of C. canephora in close proximity to its wild gene pool has led to cultivated genotypes and cultivated-wild hybrids appearing within the natural habitats of C. canephora. Yet, given the high genetic similarity between the cultivated and wild gene pool, together with the relatively low incidence of hybridization, our results indicate that the overall impact in terms of risk of introgression remains limited so far.

Taxonomic and Functional Diversity of Flower-Visiting Insects in Coffee Crops.

Despite the important role that flower-visiting insects play in agricultural production, none of the previous studies of coffee pollinators in Colombia have incorporated functional diversity into their analysis. Therefore, this study aimed to quantify the abundance, richness, and functional diversity of insects that visit flowers in coffee crops. Twenty-eight plots were selected among five sites in the north, center, and south of Colombia. In each plot, coffee flower insect visitors were collected and recorded on 90 trees at eight-minute intervals per tree, at three different times over three days. All sampling was carried out during two flowering events per year, over three years, resulting in a total of 1240 h of observations. Subsequently, the insects were taxonomically identified, and the number of individuals and species, as well as the diversity of the order q, were estimated. Functional diversity was also characterized in the bee community. The results: (a) 23,735 individuals belonging to 566 species were recorded; of them, 90 were bees, with the native species being the most abundant during 10:30 and 13:00 h; (b) bees formed five functional groups, with corbiculate and long-tongued non-corbiculate bees being the most abundant and occupying the largest regions of functional space; (c) potential pollinators in coffee crops are Apis mellifera, Nannotrigona gaboi, Tetragonisca angustula, Geotrigona cf. tellurica, and Partamona cf. peckolti. Coffee crops host a wide diversity of flower visitors, especially bees, which could be beneficial for productivity and contribute to the maintenance of plant species that accompany coffee cultivation.

Shaping the Future of Coffee: Climate Resilience, Liberica's Rise, and By-Product Innovation-Highlights from the International Coffee Convention 2023 (ICC2023).

The International Coffee Convention 2023 comprehensively addressed the contemporary challenges and advancements in the coffee industry, emphasizing sustainability, health, and innovation. This convention gathered experts and stakeholders to explore diverse aspects of coffee, ranging from the potential of underutilized species like Coffea liberica in terms of climate resilience to the innovative use of coffee by-products. The convention featured presentations and discussions, employing both empirical research and analytical reviews to explore various topics, including the health benefits of coffee, the advancements in traceability and authentication methods, and the impact of global regulatory changes on coffee production and trade. The key findings highlighted the importance of biodiversity in coffee production as a response to climate change, the significant health benefits and sustainability potential of coffee by-products, and the evolving landscape of coffee consumption patterns driven by technological innovations. The convention also stressed the need for alignment in global coffee trade regulations, particularly concerning deforestation and traceability. The 2023 convention underscored the complexity and interconnectivity of the coffee industry's challenges and opportunities. It concluded with a forward-looking perspective, emphasizing the need for continued research, sustainable practices, and collaborative efforts to shape the future of the coffee industry. The community is looking forward to furthering these discussions at the next International Coffee Convention in 2024.

The floral development of the allotetraploid Coffea arabica L. correlates with a small RNA dynamic reprogramming.

Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co-expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage-specific manner. Members of the miR482/miR2118 superfamily and their 21-nucleotide phasiRNAs originating from resistance genes show a robust co-expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage-specific manner, related to modulated hormonal responses and transcription factor expression.

SPME-GC-MS untargeted metabolomics approach to identify potential volatile compounds as markers for fraud detection in roasted and ground coffee.

Roasted ground coffee has been intentionally adulterated for economic revenue. This work aims to use an untargeted strategy to process SPME-GC-MS data coupled with chemometrics to identify volatile compounds (VOCs) as possible markers to discriminate Arabica coffee and its main adulterants (corn, barley, soybean, rice, coffee husks, and Robusta coffee). Principal Component Analysis (PCA) showed the difference between roasted ground coffee and adulterants, while the Hierarchical Clustering of Principal Components (HCPC) and heat map showed a trend of adulterants separation. The partial Least-Squares Discriminant Analysis (PLS-DA) approach confirmed the PCA results. Finally, 24 VOCs were putatively identified, and 11 VOCs are candidates for potential markers to detect coffee fraud, found exclusively in one type of adulterant: coffee husks, soybean, and rice. The results for possible markers may be suitable for evaluating the authenticity of ground-roasted coffee, thus acting as a coffee fraud control and prevention tool.

A semi-quantitative histochemical method for assessment of biochemical responses to osmotic stress in Coffea arabica leaf disks.

A simple method set for assessing biochemical changes associated with osmotic stress responses was developed using coffee (Coffea arabica L.) leaf disks. Stress was induced by polyethylene glycol (PEG) exposure. Quantitative evaluation of tissue physiological stress parameters was carried out using analytical methods to validate the conversion of classic qualitative histochemical tests for localizing lipid peroxidation, hydrogen peroxide, and total xanthine alkaloids into semi-quantitative assays. Relative electrolyte leakage (EL%) and chlorophyll content (SPAD index) were also recorded. EL% levels of treated disks were higher than those of control ones, whereas SPAD indexes were comparable. Histochemical localization indicated that levels of lipid peroxidation, H2O2, and total xanthines were also higher under osmotic stress than in control conditions. Semi-quantitative data obtained by image processing of histochemical staining consistently matched quantitative evaluations. Chromatographic analyses revealed that theophylline and caffeine concentrations increased in the presence of PEG, whereas theobromine remained constant in relation to the control. The methods herein described can be useful to rapidly acquire initial data regarding biochemical osmotic stress responses in coffee tissues based on simple staining and imaging steps. Moreover, it is likely that the same method may be applicable to other types of stresses and plant species upon minor adjustments.

First Report of Alternaria alternata Causing Leaf Spot on Coffea arabica in China.

Yunnan Province is the major region for coffee (Coffea arabica) cultivation in China, contributing to over 98% of the national yield and total production value (Ma et al. 2022). In May 2023, brown spot symptoms were observed only on the leaves of coffee plants in a field located in Baoshan City (98°52'37.988400"E, 24°58'17.673600"N), Yunnan Province. Notably, brown and irregularly shaped spots initially started on the leaf bases. The spots enlarged and developed concentric rings with dark brown margins, which are often surrounded by yellow halos. Finally, the necrotic spots spread across the entire leaf and caused the leaf to curl and fall off. The incidence of the disease was approximately 3% of the coffee plants (n = 600). The symptomatic leaves collected from 10 plants were sectioned (5 × 5 mm), subjected to surface sterilization with 70% ethanol for 40 s, rinsed with sterile distilled water, air-dried, and transferred to potato dextrose agar (PDA). Fungi with grayish-white, cotton-like aerial mycelia grew after 7 days at 28°C. The older mycelia of these isolates displayed dark gray pigmentation. Single conidia were cultivated on PDA, and 15 morphologically similar monosporic isolates were ultimately obtained. Microscopic observation revealed that these isolates produced branched, septate, transparent and amber mycelium. Brown, elliptical or pear-shaped conidia with 2 to 4 transversal septa and 0 to 3 longitudinal septa, measuring 9.6 to 33.3 long × 6.0 to 15.0 µm wide (n = 30), were observed on potato carrot agar (PCA). Molecular identification of multiple genes, such as ITS (Schoch et al. 2012), RPB2 (O'Donnell et al. 2010) and GAPDH (Berbee et al. 1999), indicated consistent 100% identity among these isolates. Sequences of the representative isolates CFSY1-CFSY5 were deposited in GenBank (acc. nos.: OR351112, PP188577, PP188578, PP294863, PP294864, OR509742, PP215341-PP215344, OR509740 and PP239378-PP239381), revealing 98.35% - 100% homology with distinct Alternaria alternata strains previously deposited in GenBank (acc. nos.: PP110780, MN649031 and OR485338). The multigene phylogenetic analysis positioned isolates CFSY1-CFSY5 within a distinct cluster, alongside diverse A. alternata isolates. Based on morphological and molecular characterizations, the pathogen was identified as A. alternata. To verify its pathogenicity, a conidial suspension (1×106 conidia/mL) of isolate CFSY1 was sprayed on six leaves of three healthy one-year-old C. arabica seedlings. Subsequently, the inoculated seedlings were covered with plastic bags and placed in a growth chamber under controlled conditions (a 14 h daylight period and a 10 h dark period at 28°C). The experiment was repeated three times. After 20 days, typical brown spot symptoms analogous to those originally observed in the field appeared on the leaves in all inoculated plants. Reisolation, morphology identification and DNA sequencing substantiated Koch's postulates. In contrast, control plants treated with sterilized water remained asymptomatic, and no pathogen was reisolated from them. Significantly, A. alternata has been previously reported as the causal agent for leaf spot disease in a diverse variety of woody plant species in China, including Prunus avium (Ahmad et al. 2020), Magnolia grandiflora (Liu et al. 2019) and citrus (Wang et al. 2010). This study represents the first report of brown leaf spot caused by A. alternata specifically on C. arabica in China, enriching the contents of fungal pathogens under Chinese coffee cultivation conditions.

Mozambican Coffea accessions from Ibo and Quirimba Islands: identification and geographical distribution.

Mozambique does not have a tradition of farming Coffea arabica or Coffea canephora, the two species that dominate the worldwide coffee market. However, native coffee plants have been growing spontaneously and in some cases cultivated in the Ibo and Quirimba islands in the north of the country and Inhambane province in the south. Historically there has been confusion over the precise taxonomic classification of these indigenous coffee plants, with different botanists identifying the species as C. racemosa, C. zanguebariae or various synonyms of both. The present research aims to clarify the subject and provide new information on these little-described coffee species which may prove valuable as new breeding material for future cultivars, something that is sorely needed to face the present and future challenges of coffee production. Leaf samples were collected from 40 accessions from Ibo Island, Quirimba Island and Inhambane province. The samples were sequenced by whole-genome technology and WGS reads were filtered to identify relevant SNP variants. Diversity among the samples was assessed by PCA, and a phylogenetic tree including several Coffea species was built using additional data available in public databases. Experimental data confirm the presence of C. zanguebariae as the only coffee species present in both Ibo and Quirimba Islands, while it appears that C. racemosa is exclusive to the southern Inhambane province. The present research provides the most detailed analysis so far on the genetic identity of the traditional Mozambican coffee crops. This is the prerequisite for undertaking further scientific studies on these almost unknown coffee species and for starting agronomic development programs for the economic revival of Ibo and Quirimba islands based on coffee cultivation. Furthermore, these species could provide much-needed genetic material for the breeding of new hybrids with the two main commercial coffee species.