Author: IGNACIO RODRIGO ISLAS FLORES
IGNACIO RODRIGO ISLAS FLORES (2010)
Morphological characterization of habanero pepper (Capsicum
chinense Jacq.) showed that chilli fruit occasionally contains a small
internal fruit (SIF) developing inside the main fruit. SIF morphology
showed a normal fruit with pericarp, two locules and small
reminiscent ovules joined to placental-like tissues. Histological
analysis showed that SIFs maintain physical connexion with the
placental tissues of the main fruit through vascular tissues. A HardyWeinberg
analysis suggested that the frequency of SIF formation in
the Capsicum chinense population studied is not the result of natural
selection. This is the first report on this morphological abnormality
in Capsicum chinense and constitutes an initial attempt to explain
SIF formation frequency in this chilli pepper.
IGNACIO RODRIGO ISLAS FLORES (2011)
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.
Pathogenic microorganisms cause great losses annually and are a constant threat to agriculture and food production. The strategies used to control pathogen microorganisms' population such as spraying of fungicides, bactericides or insecticides are becoming ineffective as pathogens have being developing resistance against many of these compounds. Today, in agriculture there are serious concerns regarding the increasing volumes of pesticides that must be applied to control plant pathogens, and the environmental contamination. The development of safer and more efficient compounds to control plant pathogens is a demand that guarantees food production with the absence of residual pesticides. An opportunity that fulfills these criteria is represented by the antimicrobial peptides (AMPs), a class of small rich cysteine peptides with biological activities to kill fungi and bacteria. Sources for AMPs have been studied in animals and plants. However, it is clear that plants are an accessible and cheaper source for this kind of compounds. Many AMPs are produced in organs that are regarded as waste after plants' fruits or seeds have been harvested. AMPs from Chili pepper (Capsicum sp) have been extracted from leaves and seeds. The genes encoding AMP are being expressed in heterologous systems to explore the potential of these genes to protect the host against pathogens. In the present study, we carried out a review to highlight the work related with the production and cloning of AMPs from chili pepper. We also included our findings regarding the cloning of a defensin gene from habanero pepper leaves (Capsicum chinense Jacq) and the antimicrobial activity of some of their AMPs isolated from seeds.
Pathogens are able to deliver small-secreted, cysteine-rich proteins into plant cells to enable infection. The computational prediction of effector proteins remains one of the most challenging areas in the study of plant fungi interactions. At present, there are several bioinformatic programs that can help in the identification of these proteins; however, in most cases, these programs are managed independently. Here, we present EffHunter, an easy and fast bioinformatics tool for the identification of effectors. This predictor was used to identify putative effectors in 88 proteomes using characteristics such as size, cysteine residue content, secretion signal and transmembrane domains.
COMPUTATIONAL PREDICTION HOST-PATHOGEN INTERACTION EFFECTOR PROTEINS FUNGAL SECRETOME BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR BIOLOGÍA MOLECULAR DE PLANTAS BIOLOGÍA MOLECULAR DE PLANTAS
As our interest is focused in obtaining a betler knowledge of the plant phosphate metabolism and its interactions with Al ion, we used an in vitro coffee cellline (Coffia arabica L.) tolerant to allllIlimun ion to purify a protein with phosphate hydrolysing activity with the airn to analyze whether the enzyrne can be contributing to the Al-tolerance in this coffee cellline. The protein was purified at least 138-fold; silver stain on a 10% SDS-PAGE detected a partially purified 30 kDa polypeptide which showed ability to hydrolyse phosphate either in native gels or soluble assays. The semipurified protein is able to hydrolyse sodiurn pyrophosphate (PPi-Na) and adenosine triphosphate (ATP) very efficiently, a1lhough P-serine (P-ser), P-Threonine (P-thr), P-Tyrosine (P-Tyr), Phytate, D-Myo-lnositol-lP (D-Myo Inos-lP) and lhe synlhetic p-nyliophenyl phosphate (P-NPP) could also be used as substrates. Enzyrnatic activity ofthe EDTA-inactivated enzyrne can be restored by Mg2+, as well as other divalent cations such as Fe2+, Co2+, Cu2+, Zn2+, Ca2+ and Mn2+. In contrast, Al3+ could only partially reactivate the phosphate hydrolysing activity, which suggests that it is not a cofactor for this enzyrne. Vv'hen Al3 + was added to the Mg2+ -enzyrne complex, it strongly inhibited the enzyrnatic activity either, exerting a negative effect over the enzyrne or the substrate. Between a munber of phosphohydrolase inhibitors, only KN03 was able to decrease the activity suggesting that this enzyme should be related with the V-ATPase protein family or with plant phosphatases.
Habanero chili pepper (Capsicum chinense) is widely consumed as a fresh vegetable, although its extremely high capsaicin content has led to other uses (e.g., medicine and self-defense). Recently described antimicrobial peptides from C. annuum were very efficient in inhibiting growth in human and plant pathogenic bacteria and fungi. In order to explore the potential use of Capsicum chinenseseeds as a source of antimicrobial peptides, in the present study a peptide fraction from C. chinense pepper seeds, denominated G10P1, was enriched, partially purified and its antimicrobial activitytested against the plant and human pathogens Xanthomonas campestris, Pseudomonas syringae, Pseudomonas aeruginosa, Erwinia carotovora, Agrobacterium sp., Shigella flexnerii, Escherichia coli, Staphyllococcus aureus and Bacillus subtilis. The minimum inhibitory concentration of the G10P1 peptide fraction against X. campestris was 12.5 μg mL-1. Electrophoresis of the G10P1 in a denaturant 15% polyacrylamide gel showed it to be composed of ~7.57 and ~5.6 kDa polypeptides, both associated with an area of strong antibacterial activity. The sequencing of 18 amino acids from the N-terminal of the ~7.57 peptides and 12 from the ~5.6 kDa peptides showed no clear association with previously described antimicrobial peptides. However, the ~5.6 kDa peptides were related to the NAC and WRKY transcription factors, both involved in direct regulation of the plant defense response against pathogen attack and the ~7.57 kDa peptides had low homology with a 3-oxo-[acyl-carrier-protein] synthase from Capsicum chinense.
The objective of this study was to validate the use of banana leaf sections as a technique to study the molecular interaction between Mycosphaerella fijiensis and Musa spp. without the interference of biotic and abiotic factors that commonly occur under field conditions. The growth of M. fijiensis in banana leaf sections was evaluated and compared with the growth of the fungus in leaves under field conditions. Growth comparison was carried out through the absolute quantification by real-time PCR of a segment of the β-tubulin gene of M. fijiensis. Validation of the banana leaf sections technique consisted in monitoring M. fijiensis MfAvr4 gene expression and its relative quantification by real-time PCR in banana leaf sections. With this technique, it was shown that the growth of M. fijiensis and MfAvr4 gene expression were similar to those observed in infected leaves in the field. These quantitative real-time PCR results support the suitability of using banana leaf sections for molecular studies of gene expression in M. fijiensis-Musa spp. interactions.
Black Sigatoka is a disease that occurs in banana plantations worldwide. This disease is caused by the hemibiotrophic fungus Pseudocercospora fijiensis, whose infection results in a significant reduction in both product quality and yield. Therefore, detection and identification in the early stages of this pathogen in plants could help minimize losses, as well as prevent the spread of the disease to neighboring cultures. To achieve this, a highly sensitive SPR immunosensor was developed to detect P. fijiensis in real samples of leaf extracts in early stages of the disease. A polyclonal antibody (anti-HF1), produced against HF1 (cell wall protein of P. fijiensis) was covalently immobilized on a gold-coated chip via a mixed self-assembled monolayer (SAM) of alkanethiols using the EDC/NHS method. The analytical parameters of the biosensor were established, obtaining a limit of detection of 11.7 µg mL−1, a sensitivity of 0.0021 units of reflectance per ng mL−1 and a linear response range for the antigen from 39.1 to 122 µg mL−1. No matrix effects were observed during the measurements of real leaf banana extracts by the immunosensor. To the best of our knowledge, this is the first research into the development of an SPR biosensor for the detection of P. fijiensis, which demonstrates its potential as an alternative analytical tool for in-field monitoring of black Sigatoka disease.
ROBERTO CARLOS VAZQUEZ EUAN ROSA GRIJALVA ARANGO BARTOLOME HUMBERTO CHI MANZANERO MIGUEL ALONSO TZEC SIMA IGNACIO RODRIGO ISLAS FLORES CECILIA MONICA RODRIGUEZ GARCIA LETICIA PERAZA ECHEVERRIA ANDREW CHRISTOPHER JAMES GILBERTO MANZO SANCHEZ BLONDY BEATRIZ CANTO CANCHE (2012)
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.
BRISIA ALEJANDRA AGUILAR BARRAGAN OLGA ELIZABETH ODRIOZOLA CASAS GLORIA MARGARITA MACEDO RAYGOZA TETSUYA OGURA GILBERTO MANZO SANCHEZ ANDREW CHRISTOPHER JAMES KAY IGNACIO RODRIGO ISLAS FLORES MIGUEL JUAN BELTRAN GARCIA (2014)
Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.