Antitumor and antibacterial activity of metabolites of endophytic Colletotrichum siamense isolated from coffee (Coffea arabica L. cv IAPAR-59).

Endophytic fungi produce a range of known metabolites and several others, not yet explored, which present important biological activities from the pharmaceutical and industrial perspective. Several studies have reported the diversity of endophytes in Coffea arabica plants, although few have been described in organic cultures. In the current paper, we describe the chemical profile of specialized metabolites in the ethyl acetate phase in a strain of the endophytic fungus Colletotrichum siamense associated with coffee (Coffea arabica L.) (Rubiaceae) and its potential against tumor cells and bacteria of medical and food importance. Cytotoxicity assays in tumor cells MCF-7 and HepG2/C3A were performed by MTT and microdilution in broth to evaluate the antibacterial action of metabolic extract. The antiproliferative assay showed promising results after 24 h of treatment, with 50% injunction concentrations for the two cell types. UHPLC-MS/MS analyses with an electrospray ionization source were used to analyze the extracts and identify compounds of species Colletotrichum siamense, which is still little explored as a source of active metabolites. Many of these compounds observed in the endophytic need to be chemically synthesized in industry, at high costs, while production by the fungus becomes a chemically and economically more viable alternative. Pyrocatechol, gentisyl alcohol, and alpha-linolenic acid, associated with different mechanisms of action against tumor cells, were detected among the main compounds. The extract of the endophytic fungus Colletotrichum siamense presented several compounds with pharmacological potential and antibacterial activity, corroborating its potential in biotechnological applications.

Nocardia coffeae sp. nov., an endophytic actinobacterium isolated from the root of Coffea arabica (L.).

The polyphasic taxonomic study of a novel endophytic actinobacterium strain (CA2R105T) was carried out. The strain formed fragmented substrate mycelium and showed chemotaxonomic properties typical of members of the genus Nocardia, i.e. the presence of mycolic acid and MK-8 (H4ω-cycl) in its cells. Strain CA2R105T exhibited the highest 16S rRNA gene sequence similarity to Nocardia jiangxiensis NBRC 101359T (99.2%). The genome-based taxonomic analysis revealed low average nucleotide identity-blast and digital DNA-DNA hybridization values (<93.7, and <65.2%, respectively) to its closest relative. Moreover, many different phenotypic characteristics were observed between strain CA2R105T and all related Nocardia-type strains. This taxonomic evidence suggested that strain CA2R105T should be judged as representing a novel species of the genus Nocardia and the name, Nocardia coffeae sp. nov. is proposed. The type strain is CA2R105T (=TBRC 11247T=NBRC 114292T).

Streptomyces coffeae sp. nov., an endophytic actinomycete isolated from the root of Coffea arabica (L.).

An endophytic actinobacterium, designated strain CA1R205T, was isolated from the surface-sterilized root of Coffea arabica L. collected from Ratchaburi province, Thailand. The taxonomic position of this strain was evaluated using a polyphasic approach. The strain produced light yellowish brown to dark brownish black substrate mycelium and greyish white aerial mycelium. The spiral spore chains were produced directly on aerial mycelium. CA1R205T was found to have ll-diaminopimelic acid in the cell peptidoglycan, galactose, glucose, mannose and ribose as whole-cell reducing sugars, MK-10(H4), MK-9(H6), MK-10(H2), MK-9(H4), MK-10(H6) and MK-10(H8) as menaquinones and iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and C16 : 0 as major fatty acids. Diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were detected in the cells. These characteristics were consistent the typical chemotaxonomic properties of members the genus Streptomyces. The taxonomic affiliation at the genus level of this strain could be confirmed using its 16S rRNA gene sequence data. CA1R205T showed the highest 16S rRNA gene sequence similarity value to Streptomyces rapamycinicus NRRL B-5491T (98.9 %), followed by Streptomyces iranensis HM 35T (98.8 %). Digital DNA-DNA hybridization and average nucleotide identity-by blast (ANIb) values between CA1R205T and S. rapamycinicus NRRL B-5491T were 27.2 and 81.5 %, respectively. The DNA G+C content of genomic DNA was 70.7 mol%. Due to the differences in physiological, biochemical and genotypic data, CA1R205T could be discriminated from its closest neighbour. Thus, CA1R205T should be recognized as representing a novel species of the genus Streptomyces, for which the name Streptomyces coffeae sp. nov. is proposed. The type strain is CA1R205T (=TBRC 11244T=NBRC 114295T).

Enzyme Production by Induratia spp. Isolated from Coffee Plants in Brazil

Abstract Endophytic fungi belonging to the genus Muscodor now transferred to Induratia are known producers of bioactive volatile organic compounds (VOCs) with many industrial applications. However, the members of this genus have rarely been reported to produce non-volatile metabolites including enzyme. Enzymes of the endophytes are degraders of the polysaccharides available in the host plants and the knowledge of enzyme production by Induratia spp. may provide insights into their possible biotechnological applications. The aim of this study was to evaluate the activity of amylase, cellulase, lipase, pectinase, phytase, protease, endo β-1,4 glucanase and exo β-1,4 glucanase enzymes produced by fungi of the species Induratia coffeana, Induratia yucatanensis and Induratia sp. isolated from organic coffee plants. All Induratia spp. were able to produce the extracellular enzymes cellulase, pectinase, protease, and phytase. Eight fungi were able to produce lipase and four produced amylase. The specific activity of endo β-1, 4 glucanase and exo β-1,4 glucanase enzymes were detected for 9 and 8 endophytic fungi, respectively. This work demonstrated for the first time, the array of enzymes produced by Induratia spp. isolated from Coffea arabica in organic systems in Brazil.

Inhibition of growth and ochratoxin A production in Aspergillus species by fungi isolated from coffee beans.

Ochratoxin A (OTA) is a mycotoxin found in several agricultural commodities. Produced by Aspergillus spp., it is nephrotoxic and hepatotoxic and can be carcinogenic. Preventive measures are preventing fungal growth and OTA production. In this study, fungal strains (Rhizopus oryzae, Lichtheimia ramosa, Aspergillus westerdijkiae, Aspergillus niger, Aspergillus tamarii, Aspergillus sp., and Aspergillus fumigatus) isolated from coffee beans were identified for their abilities to inhibit the growth of Aspergillus ochraceus, Aspergillus westerdijkiae, Aspergillus carbonarius, and Aspergillus niger, and OTA production. All fungi strains tested were able to inhibit growth of the four Aspergillus species and OTA production, where A. niger showed the best results in both tests. L. ramosa showed the lowest growth-reducing potential, while the other fungal strains had a growth-reducing potential higher than 70% against all Aspergillus species tested. Regarding OTA production, L. ramosa and Aspergillus sp. completely inhibited the mycotoxin production by A. ochraceus and non-toxigenic strain A. niger completely inhibited OTA production by A. niger. Our findings indicate that the strains tested can be used as an alternative means to control growth of OTA-producing fungi and production of the mycotoxin in coffee beans.

Bioactivity of Fermented Green Coffee Bean Extract Containing High Chlorogenic Acid and Surfactin.

Chlorogenic acid (CGA) is a major component of green coffee beans. Surfactin, a cyclic lipopeptide, is produced and secreted by Bacillus subtilis strains. In this study, bioactivities of fermented green coffee bean extract (FGCBE) and the individual compounds, CGA and surfactin. were compared in HepG2 cells. The concentration of surfactin and CGA in the FGCBE and non-fermented green coffee bean extract (NFGCBE) were determined to be 9.2 and 7.33 and 0.72 and 0.53 mg·mL-1, respectively. The FGCBE contained about 20% and 26% more CGA and surfactin than the NFGCBE. Although CGA and surfactin exhibited cytotoxicity at concentrations more than 100 and 20 µg respectively, the FGCBE 50 containing CGA (460 µg·mL-1) and surfactin (720 µg·mL-1) effectively prevented cell death by oxidative stress and also strongly activated the proliferation of cells incubated with under 50 µM H2O2. The CGA and surfactin in FGCBE were 9.2 and 72 times higher than the CGA and surfactin compounds (50 and 10 µg·mL-1). The relative proliferation of the FGCBE-treated cells also was 3.3 and 8.8 times higher than the CGA and surfactin compounds treated the oxidative stressed cells with 50 µM H2O2. These results suggest that the single compounds such as CGA and surfactin generally have cytotoxicity at low concentration of them but FGCBE contained them acted as strong antioxidants, activators of cell proliferation, inhibitors of cell apoptosis. Various bioactive compounds in fermented coffee bean also seem to help cell proliferation and decreasing of cytotoxicity by CGA and surfactin in coffee bean.

High Antioxidant Action and Prebiotic Activity of Hydrolyzed Spent Coffee Grounds (HSCG) in a Simulated Digestion-Fermentation Model: Toward the Development of a Novel Food Supplement.

Spent coffee grounds are a byproduct with a large production all over the world. The aim of this study was to explore the effects of a simulated digestion-fermentation treatment on hydrolyzed spent coffee grounds (HSCG) and to investigate the antioxidant properties of the digestion and fermentation products in the human hepatocellular carcinoma HepG2 cell line. The potentially bioaccessible (soluble) fractions exhibited high chemoprotective activity in HepG2 cells against oxidative stress. Structural analysis of both the indigestible (insoluble) and soluble material revealed partial hydrolysis and release of the lignin components in the potentially bioaccessible fraction following simulated digestion-fermentation. A high prebiotic activity as determined from the increase in Lactobacillus spp. and Bifidobacterium spp. and the production of short-chain fatty acids (SCFAs) following microbial fermentation of HSCG was also observed. These results pave the way toward the use of HSCG as a food supplement.

Inoculation of starter cultures in a semi-dry coffee (Coffea arabica) fermentation process.

The aim of this study was to evaluate the use of yeasts as starter cultures in coffee semi-dry processing. Arabica coffee was inoculated with one of the following starter cultures: Saccharomyces cerevisiae UFLA YCN727, S. cerevisiae UFLA YCN724, Candida parapsilosis UFLA YCN448 and Pichia guilliermondii UFLA YCN731. The control was not inoculated with a starter culture. Denaturing gradient gel electrophoresis (DGGE) was used to assess the microbial population, and organic acids and volatile compounds were quantified by HPLC and HS-SPME/GC, respectively. Sensory analyses were evaluated using the Temporal Dominance of Sensations (TDS). DGGE analysis showed that the inoculated yeasts were present throughout the fermentation. Other yeast species were also detected, including Debaryomyces hansenii, Cystofilobasidium ferigula and Trichosporon cavernicola. The bacterial population was diverse and was composed of the following genera: Weissella, Leuconostoc, Gluconobacter, Pseudomonas, Pantoea, Erwinia and Klebsiella. Butyric and propionic acids, were not detected in any treatment A total of 47 different volatiles compounds have been identified. The coffee inoculated with yeast had a caramel flavor that was not detected in the control, as assessed by TDS. The use of starter cultures during coffee fermentation is an interesting alternative for obtaining a beverage quality with distinctive flavor.

Ochratoxigenic fungi associated with green coffee beans (Coffea arabica L.) in conventional and organic cultivation in Brazil

The genera Aspergillus comprises species that produce mycotoxins such as aflatoxins, ochratoxins and patulin. These are cosmopolitan species, natural contaminants of agricultural products. In coffee grains, the most important Aspergillus species in terms of the risk of presenting mycotoxins belong to the genera Aspergillus Section Circumdati and Section Nigri. The purpose of this study was to assess the occurrence of isolated ochratoxigenic fungi of coffee grains from organic and conventional cultivation from the South of Minas Gerais, Brazil, as well as to evaluate which farming system presents higher contamination risk by ochratoxin A (OTA) produced by fungi. Thirty samples of coffee grains (Coffea arabica L.) were analysed, being 20 of them of conventional coffee grains and 10 of them organic. The microbiological analysis was done with the Direct Plating Technique in a Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. The identification was done based on the macro and micro morphological characteristics and on the toxigenic potential with the Plug Agar technique. From the 30 samples analysed, 480 filamentous fungi of the genera Aspergillus of the Circumdati and Nigri Sections were isolated. The ochratoxigenic species identified were: Aspergillus auricoumus, A. ochraceus, A. ostianus, A. niger and A. niger Aggregate. The most frequent species which produces ochratoxin A among the isolated ones was A. ochraceus, corresponding to 89.55%. There was no significant difference regarding the presence of ochratoxigenic A. ochreceus between the conventional and organic cultivation systems, which suggests that the contamination risk is similar for both cultivation systems.

454-pyrosequencing of Coffea arabica leaves infected by the rust fungus Hemileia vastatrix reveals in planta-expressed pathogen-secreted proteins and plant functions in a late compatible plant-rust interaction.

Coffee (Coffea arabica L.), one of the key export and cash crops in tropical and subtropical countries, suffers severe losses from the rust fungus Hemileia vastatrix. The transcriptome of H. vastatrix was analysed during a compatible interaction with coffee to obtain an exhaustive repertoire of the genes expressed during infection and to identify potential effector genes. Large-scale sequencing (454-GS-FLEX Titanium) of mixed coffee and rust cDNAs obtained from 21-day rust-infected leaves generated 352 146 sequences which assembled into 22 774 contigs. In the absence of any reference genomic sequences for Coffea or Hemileia, specific trinucleotide frequencies within expressed sequence tags (ESTs) and blast homology against a set of dicots and basidiomycete genomes were used to distinguish pathogen from plant sequences. About 30% (6763) of the contigs were assigned to H. vastatrix and 61% (13 951) to C. arabica. The majority (60%) of the rust sequences did not show homology to any genomic database, indicating that they were potential novel fungal genes. In silico analyses of the 6763 H. vastatrix contigs predicted 382 secreted proteins and identified homologues of the flax rust haustorially expressed secreted proteins (HESPs) and bean rust transferred protein 1 (RTP1). These rust candidate effectors showed conserved amino-acid domains and conserved patterns of cysteine positions suggestive of conserved functions during infection of host plants. Quantitative reverse transcription-polymerase chain reaction profiling of selected rust genes revealed dynamic expression patterns during the time course of infection of coffee leaves. This study provides the first valuable genomic resource for the agriculturally important plant pathogen H. vastatrix and the first comprehensive C. arabica EST dataset.

Efeito do silício na intensidade da cercosporiose e na nutrição mineral de mudas de cafeeiro / Effect of silicon on the intensity of brown eye spot and on the mineral nutrition of coffee seedlings

O presente trabalho objetivou estudar o efeito do silício na intensidade da cercosporiose e na nutrição mineral de mudas de cafeeiro. No experimento 1, testou-se seis doses de ácido silícico (0, 0,5; 1; 2; 4 e 6 g kg­1 de solo) em mudas da cultivar Catuaí Vermelho IAC 99 inoculadas com o fungo Cercospora coffeicola. No experimento 2, foram realizadas microanálises de raios-X para a avaliação de nutrientes presentes nas folhas das mudas de cafeeiro das cultivares Topázio MG1190 e Icatu Precoce IAC 3282, inoculadas e não inoculadas com C. coffeicola, com e sem silicato de cálcio (1 g kg-1 de solo). Com o aumento das doses de ácido silícico observou-se redução na área abaixo da curva de progresso do número de lesões por folha (AACPNLF), redução linear nos teores foliares de magnésio e fósforo e aumento nos teores de enxofre e cobre. Os teores foliares de boro apresentaram comportamento quadrático, diminuindo com o aumento das doses de ácido silícico e aumentando a partir da dose de 4 g kg-1 de solo. Em microanálise de raio X, mudas de cafeeiro com cercosporiose apresentam menores picos de potássio e cálcio, independente da cultivar utilizada.

Our objective was to verify the effect of silicon on the intensity of brown eye spot and on the mineral nutrition of coffee seedlings. In the first experiment, 6 doses of silicic acid (0, 0.5, 1, 2, 4 and 6 g kg-1 soil) were tested using a complete randomized block design with 4 replicates with 6 coffee seedlings cultivar Catuaí Vermelho IAC 99 inoculated with the fungus Cercospora coffeicola. In the second experiment, X-ray microanalysis in a scanning electron microscope was performed on 2 coffee cultivars (Topazio MG1190 and Icatu Precoce IAC 3282), inoculated and non-inoculated with C. coffeicola, treated and untreated with calcium silicate (1 g kg-1 of soil). With the increase of the silicic acid doses, there was observed a reduction in the area under the disease progress curve of the number of lesions per leaf (AUPCNLL), coupled with a linear reduction in the foliar contents of magnesium and phosphorus as well as an increase in the contents of sulfur and copper. The foliar contents of boron presented a quadratic behavior, decreasing with the increase of silicic acid and increasing with the dose of 4 g kg-1 of soil. In X-ray microanalysis, coffee seedlings with brown eye spot presented lower peaks of potassium and calcium, regardless of the cultivar used.

Molecular ecology and polyphasic characterization of the microbiota associated with semi-dry processed coffee (Coffea arabica L.).

This work was aimed at isolating and identifying the microbiota present during the semi-dry method of coffee processing using polyphasic methods and to evaluate microbial diversity with PCR-DGGE. Samples of Coffea arabica L. were collected during different processing stages in southern Minas Gerais, Brazil. The bacterial and fungal isolates were phenotypically characterised and grouped according to the ARDRA technique, in which the 16-23S and ITS1-5.8S regions of the rDNA were sequenced for species identification. The bacterial counts varied from 3.7 to 7 log CFU g(-1). The yeast counts ranged from 3.4 to 6.9 log CFU g(-1), and the filamentous fungal population varied from 2 to 3.7 log CFU g(-1). Bacillus subtilis, Escherichia coli, Enterobacter agglomerans, Bacillus cereus and Klebsiella pneumoniae were the predominant bacteria detected during the processing of the coffee, and Pichia anomala, Torulaspora delbrueckii and Rhodotorula mucilaginosa were the dominant yeasts. All of the yeast and bacterial species detected by PCR-DGGE were isolated using culture-dependent methods, with the exception of one uncultivable bacterial species. Aspergillus was the most common genus among the filamentous fungal isolates. The use of polyphasic methods allowed a better characterization of the microbiota that is naturally present in semi-dry processed coffee.

An effective and low-cost culture medium for isolation and growth of Xylella fastidiosa from citrus and coffee plants.

Buffered charcoal-yeast extract medium (BCYE) has been used for isolation of Xylella fastidiosa from citrus (Citrus sinensis) and coffee (Coffea arabica) plants affected by citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). BCYE is composed of ACES (2-[2-amino-2oxoethyl) amino]-ethanesulfonic acid) buffer, activated charcoal, yeast extract, L-cysteine, ferric pyrophosphate, and agar. ACES buffer is costly and not always commercially available in Brazil, and the L-cysteine and ferric pyrophosphate need to be filter sterilized in 0.22-mum pore membranes before inclusion in the medium. Omission of L-cysteine, addition of magnesium sulfate, and replacements of ACES and ferric pyrophosphate for potassium phosphate and ferrous sulfate resulted in an effective, less expensive, and entirely autoclavable medium, named phosphate buffered charcoal-yeast extract medium (PCYE). The final cost of PCYE was approximately one tenth that of BCYE. Its effectiveness was tested for the isolation of X. fastidiosa from symptomatic leaves collected from 52 citrus plants affected by CVC and 43 coffee plants affected by CLS. PCYE was as effective as BCYE and has been used routinely in our and other laboratories for isolation, growth, and quantification of X. fastidiosa from plant tissues.

Diversidade de fungos filamentosos associados a Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) e suas galerias em frutos de Coffea canephora (Pierre) / Diversity of filamentous fungi associated with Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) and its galleries in berries of Coffea canephora (Pierre)

Amostragens de campo foram realizadas em Ouro Preto d'Oeste (10°45'S e 62°15'W) para avaliar a micobiota associada a Hypothenemus hampei Ferrari [cutícula, aparelho bucal, protórax (micângia), tubo digestivo e fezes] e suas galerias em frutos de Coffea canephora Pierre. Dez gêneros (201 isolados) estavam diretamente relacionados com o inseto, enquanto cinco gêneros (20 isolados) estavam relacionados com galerias do inseto nos frutos. Todos os gêneros identificados associados aos insetos estavam também presentes nas suas galerias, o que indica que o broqueamento pode significar um modo ativo de inoculação de fungos por H. hampei. Na broca, a diversidade de gêneros de fungos foi maior no aparelho bucal e protórax e menor nas fezes. Fusarium, Penicillium e Geotrichum, com abundâncias de 55,7, 24,3 e 10,8 por cento, respectivamente, foram os gêneros dominantes. Nas galerias Fusarium, Geotrichum, Trichoderma e Aspergillus com abundância de 33,3; 29,6; 18,5 e 14,8 por cento, respectivamente, foram os gêneros dominantes. A presença de Fusarium no inseto e suas galerias reforça indicações anteriores de uma interação íntima entre H. hampei-Fusarium. A presença de Aspergillus e Penicillium enfatiza a possibilidade de dispersão da ocratoxina pela broca. Este trabalho fornece o primeiro registro da micobiota associada a H. hampei em C. canephora. Entre os gêneros identificados, Cephalosporium, Geotrichum e Oidiodendrum foram registrados pela primeira vez em associação com o inseto e suas galerias em C. canephora.

Field sampling was carried out in Ouro Preto d'Oeste Rondônia (10°45'S and 62°15'W) to evaluate the mycobiota associated with Hypothenemus hampei Ferrari [cuticle, mouth, prothorax (mycangia), gut and feces] and its galleries on berries of Coffea canephora Pierre. Ten genera (201 isolates) were directly related with the insect while five genera (20 isolates) were related with galleries on berries. All the genera identified in the insects were also present in their galleries, what indicates that boring may be an active way of fungi inoculation by H. hampei. The fungi genera were more diverse in the mouth and prothorax of borers, and lower in feces. Fusarium, Penicillium and Geotrichum, with abundance of 55.7, 24.3 and 10.8 percent, respectively, were dominant genera. In the galleries Fusarium, Geotrichum, Trichoderma and Aspergillus with abundance of 33.3, 29.6, 18.5 and 14.8 percent, respectively, were dominant genera. The overall presence of Fusarium in coffee berry borer and its galleries) reinforces previous indications of a close interaction between H. hampei-Fusarium. The presence of Aspergillus and Penicillium emphasizes the possibility of "ochratoxin dispersion" by the borer. This work provides the first record of the mycobiota associated with H. hampei in C. canephora. Among the identified genera, Cephalosporium, Geotrichum and Oidiodendrum were recorded for the first time in association with H. hampei and its galleries in C. canephora.

Effect of the frequency of the mixing of coffee cherries put out for drying on the kinetics of drying and the relationship to ochratoxin A production.

The effect of the frequency of the mixing of coffee cherries put out for sun drying on the kinetics of the drying, fungal growth and kinetics of ochratoxin A production was evaluated. The results showed that the more coffee cherries were mixed, the quicker they dried. This rapidity of drying led to a reduction of fungal development. Indeed, coffee cherries mixed eight and ten times a day, dried quickly and were free inside of fungi. However, infection by fungi gives little indication of ochratoxin A production in coffee cherries. Indeed, although coffee cherries mixed twice a day were more contaminated by fungi, the analysis of ochratoxin A content showed they were free of this mycotoxin. The coffee cherries that were more contaminated by ochratoxin A were those mixed four times a day (containing 0.35-5.46 microg kg(-1) ochratoxin A). Ochratoxin A contamination was essentially due to the presence of Aspergillus species capable of producing ochratoxin A inside the coffee cherries.

PCR method for the detection of potential ochratoxin-producing Aspergillus species in coffee beans.

Ochratoxin A (OA) is a nephrotoxic and carcinogenic mycotoxin that has been found in cereal and food commodities. Currently, Aspergillus carbonarius, A. niger and A. ochraceus have been recognized as the species responsible for OA in coffee beans. With the aim of developing multiplex-PCR for detection of these species in coffee bean samples, we first developed specific primers for A. niger detection. The primer designed (OPX7(372)) provided an amplicon of 372 pb in all A. niger stricto sensu isolates. The PCR assay developed for A. niger identification in pure culture was also successfully used for detecting this species in coffee beans. No cross-reaction was observed using DNA from coffee beans inoculated with closely related black aspergilli species. A multiplex-PCR method for detection of A. carbonarius, A. niger and A. ochraceus species in coffee beans was developed. From a single assay this method detected the amplicons of 809, 372, and 260 pb that represent the three most ochratoxigenic species found in coffee bean samples, i.e., A. carbonarius, A. niger and A. ochraceus, respectively.

A molecular method for detection of Aspergillus carbonarius in coffee beans.

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7809, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F809 and OPX7R809) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.