Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffea liberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.

Biological activities of the fermentation extract of the endophytic fungus alternaria alternata isolated from coffea arabica L / Atividades biológicas do extrato da fermentação do fungo endofítico alternaria alternata isoladas de coffea arabica L

A total of 22 endophytic fungi isolated from coffee (Coffea arabica L.) were cultivated in vitro and their crude extracts tested. The screening was carried out using the agar diffusion method against Staphylococcus aureus, Escherichia coli and Candida albicans. The most effective isolate was Alternaria alternata, and subsequently, its extract was assayed. The total phenolic content was 3.44 μg GAE/mg of the crude extract. For the antibacterial and antifungal activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal and fungicidal concentrations (MBC and MFC) were determined. The ranges of MIC values were 50-100 μg/mL for S. aureus and 400-800 μg/mL for E. coli. The extract did not show activity in the tested concentrations for C. albicans. The fungal crude extract was assayed for antioxidant activities. Its ability to scavenge DPPH radicals and antioxidant activity by β-carotene/linoleic acid system oxidation was not significant. In addition, antitumor activity was studied using the MTT assay. At a dilution of 400 μg/mL, the extract displayed a cytotoxic activity of approximately 50 percent towards HeLa cells in vitro. The results indicate that endophytic fungi could be a promising source of bioactive compounds and warrant further study.

Total de 22 fungos endofíticos isolados de café (Coffea arabica L.) foi cultivado in vitro e seus extratos testados. A triagem foi conduzida pelo método de difusão em agar contra bactérias Gram-positiva, Gram-negativa e uma levedura. O isolado mais efetivo foi Alternaria alternata e, subsequentemente, seu extrato foi analisado. O conteúdo de fenólicos totais do extrato bruto foi de 3,44 μg EAG/mg de extrato. Para os testes de atividade antimicrobiana, a concentração inibitória mínima (CIM) e concentração bactericida e fungicida mínima (CBM e CFM) contra Staphylococcus aureus, Escherichia coli e Candida albicans foram determinadas. Resultados da CIM variaram entre 50-100 μg/mL para S. aureus e 400-800 μg/mL para E. coli. O extrato bruto não apresentou atividade nas concentrações testadas para C. albicans. Foram analisadas as atividades antioxidantes do extrato bruto. Sua habilidade para seqüestrar radicais DPPH e a atividade antioxidante pela oxidação do sistema β-caroteno/ácido linoléico não foram significativas. Além disso, a atividade antitumoral foi estudada pelo teste do MTT. À diluição de 400 μg/mL, o extrato apresentou atividade de aproximadamente 50 por cento sobre as células HeLa in vitro. Os resultados indicam que fungos endófitos poderiam ser uma fonte promissora de compostos bioativos necessitando de estudos futuros.

Incidence and distribution of filamentous fungi during fermentation, drying and storage of coffee (Coffea arabica L. ) beans / Incidência e distribuição de fungos filamentosos durante a fermentação, secagem e armazenamento de frutos e grãos de café (Coffea arabica L. )

The objective of this work was to isolate and characterize filamentous fungi present in different stages of harvest, fermentation, drying and storage of coffee beans processed by natural method. The cherries were hand-picked and then placed on a cement drying platform where they remained until reached 11 percent of humidity. Microbial counts were found in all samples during fermentation and drying of the coffee beans. Counts of fungi in the coffee cherries collected from the tree (time 0) were around 1.5 x 10³ CFU/g. This number increased slowly during the fermentation and drying reaching values of 2 x 10(5) CFU/g within 22 days of processing. Two hundred and sixty three isolates of filamentous fungi were identified. The distribution of species during fermentation and drying was very varied while there was a predominance of Aspergillus species during storage period. The genera found were Pestalotia (4), Paecelomyces (4), Cladosporium (26), Fusarium (34), Penicillium (81) and Aspergillus (112) and comprised 38 different species.

O objetivo deste estudo foi isolar e caracterizar fungos filamentosos presentes em diferentes estágios de beneficiamento de café processado pelo método natural, incluindo: colheita, fermentação, secagem e armazenamento. O café cereja foi colhido manualmente e então colocado em uma plataforma de cimento, onde permaneceu até atingir 11 por cento de umidade. A contagem microbiana foi realizada em todas as amostras durante a fermentação e secagem do café. A população de fungos filamentosos no café cereja ainda nos pés (tempo 0) foi em torno de 1,5 x 10³ UFC/g. Este número aumentou vagarosamente durante a fermentação e secagem, alcançando valores de 2 x 10(5) UFC/g em 22 dias do processamento. Duzentos e sessenta e três isolados de fungos filamentosos foram identificados. A distribuição das espécies durante fermentação e secagem foi bastante variada, mas no armazenamento dos grãos ocorreu o predomínio de espécies de Aspergillus. Foram encontradas 38 espécies de fungos distribuídas nos seguintes gêneros: Pestalotia (4), Paecelomyces (4), Cladosporium (26), Fusarium (34), Penicillium (81) e Aspergillus (112).

Normas e indices DRIS para la evaluación nutricional del cafeto / DRIS norms and indexs for coffee nutritional evaluaction

El sistema integral de diagnóstico y recomendación (DRIS), es un método alternativo para evaluar el estado nutricional de diversos cultivos. Para evaluar nutricionalmente al cafeto en Venezuela mediante el DRIS, se obtuvo información sobre su producción y sobre los niveles foliares de N, P, K, Ca, Mg, Zn, Fe, Cu, Mn y B, procedentes de fincas productoras de Lara, Portuguesa, Trujillo y Yaracuy, a través de un muestreo aleatorio estratificado. Con las medias de los niveles foliares obtenidos, se generaron las normas DRIS para el B (55,05 mg kg-1) y sus interrelaciones con los nutrientes N, P, K, Ca, Mg, Fe, Cu, Mn y Zn. Utilizando cuatro diferentes métodos de cálculo reportados en la literatura, se desarrollaron índices DRIS (IN-DRIS) e índices de balance de nutrientes DRIS (IBN-DRIS) y ambos fueron evaluados contra el rendimiento. Pudo determinarse la existencia de una relación positiva y significativa entre el contenido foliar de los nutrientes del cafeto y los IN-DRIS, independientemente del método de cálculo utilizado para el mismo. Los diferentes métodos de obtener los IN-DRIS, alteraron muy poco los resultados y coincidieron en determinar al K como el nutriente más deficiente y más limitante de la producción.

The diagnosis and recommendation integrated system (DRIS), is an alternative method to evaluate the nutritional status of diverse crops. To nutritionally evaluate the coffee plantations in Venezuela using DRIS, information regarding yields and foliar levels of N, P, K, Ca, Mg, Zn, Fe, Cu, Mn and B, of farms from Lara, Portuguesa, Trujillo and Yaracuy states was obtained through a randomized stratified sampling process. With the obtained means foliar levels, B DRIS norms (55,05 mg kg-1) as well as those among B and its relationship with N, P, K, Ca, Mg, Fe, Cu, Mn and Zn were developed. DRIS indexes (IN-DRIS) and nutrient balance indexes (NBI-DRIS) were developed through four different reported estimation methods and then evaluated against yield. A positive and significant relationship between foliar nutrient content and the IN-DRIS was found, independently of the procedure of calculation used for to obtain them. The different methods of IN-DRIS development altered very little the results and coincided in determining K as the most deficient and most yield limiting nutrient.

Cambios morfoanatómicos y de elementos químicos como posibles marcadores del proceso de embriogénesis somática en café (Coffea arabica L) / Morphoanatomical and chemical changes as possible markers of the process in somatic embryogenesis in coffee (Coffea arabica L)

El objetivo fundamental de este trabajo fue describir los cambios morfoanatómicos y/o de elementos químicos que ocurren en explantes foliares de Coffea arabica L. cv. Catuai, inducidos a producir embriones somáticos. Los explantes se cultivaron en el medio de Murashige y Skoog con algunos suplementos. Se tomaron y fijaron muestras de los explantes en FAA o glutaraldehido con el fin de realizar estudios bajo microscopía óptica, epifluorescencia y electrónica (barrido y transmisión). También, se hizo un microanálisis de rayos X. Se logró embriogénesis somática directa e indirecta. Se describe el origen y se caracterizan morfoanatómicamente los embriones producidos. Entre los indicadores de inducción embriogénica pueden considerarse: deposición de calosa y cutina en las paredes celulares, deposición de almidón, presencia de material fibrilar o membranoso sobre callos embriogénicos. En relación a elementos químicos indicadores de embriogénesis en el cultivar de café estudiado, el calcio pareció jugar un papel importante, al igual que los iones cloro y sodio.

The objective of this research was to describe cultivated in Murashige and Skoog’s culture medium. Samples of theexplants were placed in FAA or glutaraldehyde buffer with to be studied under optical, epifluorescence and electronic microscope (SEM and TEM). Aditionally X-rays microanalysis were made. Direct and indirect somatic embryogenesis was obtained. The origin of produced embryos was described and characterized from the morphoanatomical point of view. Among the markers of embryogenic induction can be regarded the following aspects: callose deposition and cutine on the wall cell, starch deposition and the presence of either fibrillar or membranous material on embryogenic calluses. Calcium seems to play an important role during embryogenesis as a chemical marker just as chlorine and sodium ions.