Publications

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    Primera Edición edited by Mario Orozco Santos, 04/2013; INIFAP., ISBN: 978-607-37-0019-1
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    ABSTRACT: The hemibiotrophic fungus Mycosphaerella fijiensis is the causal agent of black Sigatoka disease (BS), the most devastating foliar disease in banana (Musa spp.) worldwide. Little is known about genes that are important during M. fijiensis-Musa sp. interaction. The fungal cell wall is an attractive area of study because is essential for maintenance of cellular homeostasis and it is the most external structure in the fungal cell and therefore mediates the interaction of the pathogen with the host. In this manuscript we describe the in silico identification of glycosyl phosphatidylinositol-protein (GPI) family in M. fijiensis, and the analysis of two β-1,3-glucanosyltransferases (Gas), selected by homology with fungal pathogenicity factors. Potential roles in pathogenesis were evaluated through analyzing expression during different stages of black Sigatoka disease, comparing expression data with BS symptoms and fungal biomass inside leaves. Real-time quantitative RT-PCR showed nearly constant expression of MfGAS1 with slightly increases (about threefold) in conidia and at speck-necrotrophic stage during banana-pathogen interaction. Conversely, MfGAS2 expression was increased during biotrophy (about 7 times), and then reached a maximum at speck (about 23 times) followed by a progressive decrease in next stages, suggesting an active role in M. fijiensis pathogenesis.
    Mycologia 09/2012; · 2.11 Impact Factor
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    ABSTRACT: Sigatoka disease is the most important threat for banana production worldwide. Many species of Mycosphaerella have been described from banana but, to date, the three species Mycosphaerella fijiensis, M. musicola and M. eumusae are the only species found to be pathogenic to banana. Reliable identification by classical methods requires expertise because these fungi produce similar symptoms and they are morphologically similar. For studies of ecology, genetic diversity and epidemiology their differentiation is crucial. Several laboratories have developed molecular protocols to differentiate these fungi. Currently, a number of primers targeting ribosomal sequences, actin, tubulin and histone 3 genes are available for diagnosis of the Sigatoka complex. In the present work, we report a direct colony-polymerase chain reaction (DC-PCR) approach to rapidly distinguish M. fijiensis and M. musicola strains in multiplex PCR reactions. This is the most economical and the fastest procedure reported so far for diagnosis of these two Mycosphaerella species, which are distributed in banana-growing regions in the world; the DC-PCR technique was also found to be amenable for the identification of mating type of M. fijiensis isolates. This DC-PCR may also be applicable to prepare DNA templates for basic PCR-based analyses in other fungi.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 05/2012; 11:8172-8180. · 0.57 Impact Factor
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    ABSTRACT: The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.
    Plant Physiology and Biochemistry 02/2011; 49(6):572-8. · 2.78 Impact Factor
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    ABSTRACT: Two microbial fuel cells (MCC) type PEM (proton exchange membrane) with different configuration at the anode were compared. The respective microbial samples which were isolated in order to identify the types of bacteria by amplification 16S gene. Sequencing of two clones showed the presence of alpha and beta proteobacteria as exoelectrogens. Such bacteria have been previously identified in activated sludge. The systems were operated in batch and mesophilic temperature intervals. CCM1 and CCM2 used carbon paper and granular graphite anodes, respectively. The substrate used was synthetic wastewater (ARS), containing glucose as carbon source, as well as using a mixed inoculum as biocatalyst; in the cathode chamber an O2 saturated aqueous solution was used. The power density in CCM1 was 6W/m3, with a COD (chemical O2 demand) removal of 70%, while in CCM2 the observed average power density was ~48W/m3, with a 95% COD removal. Both systems were evaluated during 120 days, the organic load was 4.7kg DQO/m3 per day and the HRT (hydraulic retention time) was of 24h.
    Interciencia 01/2010; 35(1):19-25. · 0.28 Impact Factor
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    ABSTRACT: The ER fraction from red beet taproot was purified on sucrose gradient and giant liposomes, suitable for patch clamping, were formed by dehydration-rehydration of the lipid film. Single-channel recordings on excised and attached patches revealed a large conductance (165 pS) cation (P(Cl-)/P(K+) < 0.03) channel with equal conductance and relative permeability for Na+ and K+. This non-selective cation channel was also highly permeable for Ca2+. We failed to detect any single-channel currents activated by a direct application of d-myo-inositol 1,4,5 trisphosphate, despite the fact that the ER membranes were native.
    Physiologia Plantarum 04/2008; 132(4):399-406. · 3.66 Impact Factor
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    ABSTRACT: The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato and it is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in banana. A fragment of approximately 550 bp was amplified, cloned and sequenced. The sequence analysis of several clones revealed 13 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value <3e-53), seven of them were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the banana Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs and also several conserved residues that are crucial for Pto function. Moreover, the phylogenetic analysis showed that the banana Pto-RGCs were clustered with Pto suggesting a common evolutionary origin with this R gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in banana.
    Molecular and General Genetics 11/2007; 278(4):443-53. · 2.88 Impact Factor
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    ABSTRACT: A bacterial artificial chromosome library of the causal agent of the Black Sigatoka leaf spot disease of banana and plantain, Mycosphaerella fijiensis, has been constructed using a non-sphaeroplasting technique and characterized using both homologous and heterologous probes. After first and a second size selection of PFGE-fractionated DNA, a ligation was obtained using a 1:4 molar ratio (insert:vector). One hundred random clones were analyzed, and the mean insert size was estimated to be 90 kb. The range of the insert sizes was between 40 and 160 kb. The highest percentage of inserts belonged to the range between 80 and 100 kb; 32% of the inserts had 2 or 3 internal NotI sites. This library consists of 1920 clones, if the genomic size is at least 35 Mb, then this represents 4.9 x genome equivalents, which was supported by hybridization results with homologous and heterologous probes.
    Molecular Biotechnology 06/2007; 36(1):64-70. · 2.26 Impact Factor
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    ABSTRACT: SUMMARY Idiomorphs mat1-1 and mat1-2 from Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana, were isolated. Degenerate oligos were used to amplify the HMG box of the mat1-2 idiomorph from M. fijiensis, showing homology with the HMG box of Mycosphaerella graminicola. Using a DNA walking strategy, anchored on the DNA lyase gene towards the HMG box, a 9-kb-long region of mat1-2 was obtained. A 5-kb fragment from the mat1-1 region was obtained by long-range PCR using primers on the flanking regions, which have close to 100% identity between both idiomorphs. High-identity (77-89%), inverted regions within both idiomorphs were found, which suggest unique inversion events, which have not been found before, and that could have been significant in the evolution of this species. The predicted genes showed the conserved introns in both idiomorphs as well as an additional intron within the alpha box. The implications for the evolution of species in the Mycosphaerella complex on banana are discussed.
    Molecular Plant Pathology 01/2007; 8(1):111-20. · 3.88 Impact Factor
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    ABSTRACT: High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.
    Molecular Biotechnology 10/2006; 34(1):45-50. · 2.26 Impact Factor
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    ABSTRACT: The electrophoretic karyotype of the filamentous fungus Mycosphaerella fijiensis was determined using contour-clamped homogenous electric field (CHEF) gel electrophoresis. Among 10 isolates from different geographical areas, the highest variation in the banding profiles was found in the chromosomes with sizes below 1.5 megabases pairs (Mbp). The minimal genome size was estimated to range from 27 to 35 Mbp. Eight to 13 chromosomal bands, ranging approximately from 0.5 to 8.6 Mbp in size, were separated. These results open perspectives for the physical mapping of specific genes, such as those associated with avirulence, pathogenicity, and fungicide resistance, and for the construction of chromosome-specific libraries.
    Canadian Journal of Plant Pathology-revue Canadienne De Phytopathologie - CAN J PLANT PATHOL. 01/2006; 28(2):236-241.
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    ABSTRACT: Geraniol 10-hydroxylase (G10H) is a P450 containing enzyme which is the first committed step in the biosynthesis of monoterpene indole alkaloids (MIAs), including the Catharanthus roseus-anticancer drugs vinblastine and vincristine. It is thought that G10H has a regulatory role in MIA production. In the present paper, we report the characterization of a polyclonal serum raised against the purified G10H polypeptide. Anti-G10H IgG was able to inhibit the G10H activity and also recognized the G10H polypeptide from C. roseus and other plants producing MIAs. These results establish the usefulness of this antiserum as a biochemical tool for the study of G10H regulation.
    Journal of Plant Physiology 05/2005; 162(4):393-402. · 2.70 Impact Factor
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    ABSTRACT: Los alcaloides son uno de los grupos de metabolitos secundarios más diversos encontrados en los organismos vivos. Este grupo incluye alrededor de 12,000 productos, entre los cuales se encuentran los alcaloides indólicos, alcaloides derivados del triptofano que conforman alrededor de la cuarta parte de todos ellos. Los alcaloides se han reportado en varias familias vegetales, pero principalmente en las Apocinacea, Loganiaceae y Rubiaceae, todas del orden Gentianales. Entre los alcaloides más importantes se tiene a los de tipo bisindólico como la vinblastina, utilizada en el tratamiento del mal de Hodgkin, y a la vincristina empleada en el tratamiento de la leucemia; además de los alcaloides monoterpén-indólicos ajmalicina y serpentina utilizados como agentes antihipertensivos contra las arritmias cardiacas y el mejoramiento de la circulación cerebral. La complejidad de los procesos genéticos, catalíticos y de transporte en la biosíntesis de los alcaloides monoterpén indólicos, es actualmente uno de los retos intelectuales más estimulantes en el área de los metabolitos secundarios. Si bien se requieren más de 50 pasos metabólicos para sintetizar los alcaloides más importantes producidos por C. roseus, hasta ahora solamente se han determinado y caracterizado, en algún grado, 20 de las enzimas requeridas. Faltan aún por elucidar un importante número de pasos metabólicos, para después purificar las correspondientes enzimas e intentar clonar sus genes. También es necesario elucidar los diversos aspectos de la regulación de la biosíntesis de los alcaloides, tanto en el nivel celular como en el molecular, pero sobre todo determinar cual es su función en las plantas que los producen. En esta revisión se presenta un análisis del estado actual que guarda el conocimiento en las rutas de biosíntesis de los alcaloides monoterpén-indólicos en C. roseus.
    Journal of the Mexican Chemical Society 01/2004; · 0.28 Impact Factor
  • B Canto-Canché, V M Loyola-Vargas
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    ABSTRACT: Plants produce thousands of different compounds through the secondary metabolism pathways. Since many of these products are obtained by direct extraction from plants that are cultivated in the field or some times even collected in their original habitat several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution to our understanding of secondary metabolism. Furthermore, a new alternative has arisen: transformed root cultures. Until now, hairy roots have been obtained from more than 100 different species. The products that they are able to produce range from alkaloids to aromatic compounds and dyes. These kinds of cultures have turned out to be an invaluable tool to study the biochemistry and the gene expression of the metabolic pathways in order to elucidate the intermediaries and enzymes involved in the biosynthesis of secondary metabolites.
    Advances in experimental medicine and biology 02/1999; 464:235-75. · 1.83 Impact Factor
  • Blondy B. Canto-Canché, Víctor M. Loyola-Vargas
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    ABSTRACT: NADPH-cytochrome P450 oxidoreductase (CPR, EC 1.6.2.4) is the redox partner of classical P450-monooxygenases, which have crucial roles in the metabolism of terpenes, alkaloids, flavonoids, phytoalexins, etc. It becomes evident that, contrary to animals and yeast, various CPR isoforms occur in some plants, although their specific physiological functions are largely unknown. C. roseus-CPR has been reported as encoded by a single gene and early papers concerning the C. roseus-CPR protein also reported a single CPR polypeptide. The observation of diverse CPRs during purification or by immunoblot were attributed to proteolytic degradation. We obtained CPR immunotype of C. roseus roots using two heterologous antisera directed against the CPR from Sorghum bicolor and Helianthus tuberosus, respectively. Both antisera developed the same immunogenic profile with two cross-reactive polypeptides. Further evaluation of anti-H. tuberosus CPR serum excluded non-specific binding of antiserum with C. roseus microsomal proteins. The two immuno-reactive polypeptides are probably not the result of proteolytic degradation, since increasing protease inhibitor concentration during the extraction and manipulation of the samples did not affect the occurrence of these two CPR forms. Roots from plants growing in the field showed identical CPR immunotypes with those seen in vitro, indicating that this immunoprofile actually belongs to C. roseus roots. The lectin concanavalin A was able to inhibit the CPR activity from C. roseus hairy roots; therefore, the immuno-reactive polypeptides probably result from post-translational glycosylation of the original polypeptide. Not only the roots, but also the flowers, leaves and the stem showed more than a single CPR form. The different tissues of the plant showed different immuno-reactive bands, which were reproducible even though they came from tissues of plants growing in the field. This opens the possibility of the occurrence of diverse tissue-specific CPRs.
    In Vitro Cellular & Developmental Biology - Plant 37(5):622-628. · 1.14 Impact Factor

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