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The Sargassum phenomenon is currently affecting the Caribbean in several ways; one of them is the increase of greenhouse gases due to the decomposition process of this macroalgae; these processes also produce large amounts of pollutant leachates, in which several microbial communities are involved. To understand these processes, we conducted a 150-day study on the Sargassum spp environmental degradation under outdoor conditions, during which leachates were collected at 0, 30, 90, and 150 days. Subsequently, a metagenomic study of the microorganisms found in the leachates was carried out, in which changes in the microbial community were observed over time. The results showed that anaerobic bacterial genera such as Thermofilum and Methanopyrus were predominant at the beginning of this study (0 and 30 days), degrading sugars of sulfur polymers such as fucoidan, but throughout the experiment, the microbial communities were changed also, with the genera Fischerella and Dolichospermum being the most predominant at days 90 and 150, respectively. A principal component analysis (PCA) indicated, with 94% variance, that genera were positively correlated at 30 and 90 days, but not with initial populations, indicating changes in community structure due to sargassum degradation were present. Finally, at 150 days, the leachate volume decreased by almost 50% and there was a higher abundance of the genera Desulfobacter and Dolichospemum. This is the first work carried out to understand the degradation of Sargassum spp, which will serve, together with other works, to understand and provide a solution to this serious environmental problem in the Caribbean., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Large volumes of pelagic Sargassum spp. have stranded periodically on the Mexican Caribbean shoreline. The aim of this research was to study the mobility of metals through the leachates released into the environment during the natural decomposition process of Sargassum spp. Fresh Sargassum samples were placed in cone-bed reactors: under laboratory and local environmental conditions. The leachate generated naturally by decomposition in both conditions was recovered periodically and analyses of pH, volume, and metal content were carried out. Sargassum biomass was monitored by electron microscopy, FT-IR, and CHNS analysis. The Sargassum biomass studied presented a C: N ratio of 24.39, making it a potential raw feedstock for biofuels and other value-added products. Calculations performed on leachate production allowed inferring that each ton of fresh Sargassum that decomposes at a controlled temperature of 27 °C can produce 316 L of leachate. This leachate can contain 5.67 g of As and other potentially toxic metals (e.g., B, Al, Cu). At the end of both experiments, the biomass that was incubated for 30 days presented a C: N ratio of 28.86, so it can still be used as raw material for biofuels; however, the Sargassum biomass that remained 180 days in incubation decreased its C:N ratio at 8.45 at this point, it can be considered a waste. The leachate generated during the natural decomposition process of Sargassum on beaches or disposal sites represents a high risk of contamination of the Yucatan Peninsula water system due to the high content of arsenic and the presence of potentially toxic metals., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The constant golden tides of Sargassum spp., identified to be a mixture of Sargassum natans and Sargassum fluitans , observed recently in the Mexican Caribbean have affected the marine ecosystem and the local economy and have created the need for solutions for their management and use. The Sargassum arrivals have thus been considered as third-generation feedstock for biofuel. Their potential for energetic conversion to biomethane was investigated, with hydrolysis as the limiting step due to its complex composition; therefore, in the present study, different physical, chemical, and enzymatic pretreatments and a combination of them have been evaluated, with the additional use of granular activated carbon, to determine the best yield and methane quality. The combined pretreatments of 2.5% hydrogen peroxide, followed by an enzymatic pretreatment (enzymatic extract from Trametes hirsuta isolated from decomposing wood in the Yucatán Peninsula-Mexico), was the best option, reaching a biodegradability of 95% and maximum methane yield of 387 ± 3.09 L CH 4 /kg volatile solid. The use of a conductive material, such as granular activated carbon, did not generate significant changes in performance and methane concentration.

Presence of alternate hosts of plants is a great threat to the agriculture industry. Plants from several species growing in the papaya orchards affected by papaya sticky disease were examined for Papaya meleira virus (PMeV) infection causing this disease. The viral dsRNA was already detected in some plants from the family Poaceae or in watermelon. To identify new hosts of PMeV, we have collected 38 plant species belonging to 15 families of common weed species found in papaya-growing areas in México and used reverse-transcription PCR (RT-PCR) or quantitative real-time RT-PCR (RT-qPCR) for virus detection. We have detected the viral RNA in 11 species belonging to the families Acanthaceae, Fabaceae and Poaceae. Under experimental conditions, PMeV-Mx in Panicum hirsutum and Ruellia nudiflora inoculated weed species, showed that PMeV-Mx is able to replicate in plant cells of these species and spread in a systemic way. These results highlight the importance of weed species as potential virus reservoirs for PMeV-Mx Keywords: Papaya meleira virus; papaya sticky disease; Carica papaya; RT-PCR; TaqMan.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater has been reported as a result of fecal shedding of infected individuals. In this study, the occurrence of SARS-CoV-2 RNA was explored in primary-treated wastewater from two municipal wastewater treatment plants in Quintana Roo, Mexico, along with groundwater from sinkholes, a household well, and submarine groundwater discharges. Physicochemical variables were obtained in situ, and coliphage densities were determined. Three virus concentration methods based on adsorption-elution and sequential filtration were used followed by RNA isolation. Quantification of SARS-CoV-2 was done by RT-qPCR using the CDC 2020 assay, 2019-nCoV_N1 and 2019-nCoV_N2. The Pepper mild mottle virus, one of the most abundant RNA viruses in wastewater was quantified by RT-qPCR and compared to SARS-CoV-2 concentrations. The use of three combined virus concentration methods together with two qPCR assays allowed the detection of SARS-CoV-2 RNA in 58% of the wastewater samples analyzed, whereas none of the groundwater samples were positive for SARS-CoV-2 RNA. Concentrations of SARS-CoV-2 in wastewater were from 1.8 × 10 3 to 7.5 × 10 3 genome copies per liter (GC l -1 ), using the N1 RT-qPCR assay, and from 2.4 × 10 2 to 5.9 × 10 3 GC l -1 using the N2 RT-qPCR assay. Based on PMMoV prevalence detected in all wastewater and groundwater samples tested, the three viral concentration methods used could be successfully applied for SARS-CoV-2 RNA detection in further studies. This study represents the first detection of SARS-CoV-2 RNA in wastewater in southeast Mexico and provides a baseline for developing a wastewater-based epidemiology approach in the area., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The decolorization of dye and textile effluent by Trametes hirsuta was studied in both induced and non-induced media. A removal of 70-100% of the color was achieved through adsorption and the action of laccases. Laccase activity was increased significantly with the addition of grapefruit peel (4000 U/mL) and effluent with grapefruit peel (16,000 U/mL) in comparison with the basal medium (50 U/mL). Analysis of the expression of laccase isoenzymes lac-B and lac-T revealed clear differences in the expression of these genes. The low levels of expression of lac-B in all media suggest a basal or constitutive gene expression, whereas lac-T was over-expressed in the media with effluent, and showed an up/down regulation depending on culture conditions and time. The results obtained suggest that the lac-T gene of T. hirsuta is involved in the decolorization of dyes.

Micromycetes from unexplored sources represent an opportunity to discover novel natural products to control insect pests. With this aim, a strain of Acremonium masseei CICY026 isolated from a tropical sinkhole was identified, cultured on fermented rice, and its ethyl acetate extract (EAE) was evaluated against three serious phytophagous insects ( Bemisia tabaci, Myzus persicae , and Rhopalosiphum padi ). DNA from A. masseei CICY026 was used to confirm its identity. EAE caused settling inhibition (SI) of M. persicae and R. padi (67.5% and 75.3%, respectively). Bioassay-guided fractionation of the active EAE led to the isolation of a novel metabolite, named hexahydroacremonintriol ( 1 ), and of acremonin A glucoside ( 2 ). The structures of 1 and 2 were determined using IR, one- and two-dimensional NMR, HRMS, and confirmed by theoretical data. The aphid M. persicae was noticeably sensitive to 1 and 2 (SI: 55.6% and 67.2%, respectively), whereas R. padi was only slightly affected by 1 (SI: 59%). This new knowledge about mycobiota from these special sinkhole ecosystems will inform the development of new biorational pesticides.

Consolidated bioprocessing (CBP), which integrates biological pretreatment, enzyme production, saccharification, and fermentation, is a promising operational strategy for cost-effective ethanol production from biomass. In this study, the use of a native strain of Trametes hirsuta (Bm-2) was evaluated for bioethanol production from Brosimum alicastrum in a CBP. The raw seed flour obtained from the ramon tree contained 61% of starch, indicating its potential as a raw material for bioethanol production. Quantitative assays revealed that the Bm-2 strain produced the amylase enzyme with activity of 193.85 U/mL. The Bm-2 strain showed high tolerance to ethanol stress and was capable of directly producing ethanol from raw flour at a concentration of 13 g/L, with a production yield of 123.4 mL/kg flour. This study demonstrates the potential of T. hirsuta Bm-2 for starch-based ethanol production in a consolidated bioprocess to be implemented in the biofuel industry. The residual biomass after fermentation showed an average protein content of 22.5%, suggesting that it could also be considered as a valuable biorefinery co-product for animal feeding., Competing Interests: The authors declare no conflict of interest.

In recent years, halotolerant biofilms have become a subject of interest for its application in Bioelectrochemical systems for wastewater treatment. To determine if the polarization potential affects the microbial community of a halotolerant bioanode, four bioanodes were poised at potentials of +0.34 V/SHE and - 0.16 V/SHE and the 16S rRNA gene was analyzed through a MiSeq (Ilumina) system. Oceanospirillum, Halomonas and Marinobacterium were the most predominant genus; no previous studies have reported the presence of Oceanospirillum in anodic biofilms. The fitness with the dataset for +0.34 V/SHE with a modified Butler Volmer Monod model, gives a value of K 1 was 0.0002 (2.64 A m -2 and 38% coulombic efficiency), indicating the fastest electrochemical reaction. Whereas that -0.16 V/SHE case, the high value of K 1 (12.2 with 1.82 A m -2 and 10% coulombic efficiency) indicated that the electron transfer was far from being reversible (Nernstian)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Papaya meleira virus (PMeV) causes sticky disease in Carica papaya in Brazil and Mexico. Despite its economic importance and the need for effective phytosanitary control, it remains unknown whether any insect is the vector of this virus. The aim of this work was to identify potential insect vectors of the PMeV-Mexican variant (PMeV-Mx) and determine whether these potential vectors are capable of transmitting the virus. Adult insects were collected in papaya fields in the south-southeast region of Mexico and were identified morphologically and molecularly. Their abundance and frequency were determined, and quantitative reverse transcription polymerase chain reaction was performed to establish if they carried PMeV-Mx. The Cicadellidae family (Hemiptera) was the most diverse and abundant, and Empoasca papayae was the most abundant species and had the highest virus titers. PMeV-Mx transmission assays were conducted under controlled conditions using E. papayae on C. papaya 'Maradol'. E. papayae was a carrier of PMeV-Mx at 6 h after exposure, and its viral titer increased with time, peaking at 2.125 pg/μl of PMeV-Mx RNA from 20 ng/µl of cDNA, 5 days after exposure (dae). From 14 days after plants were exposed to insects, PMeV-Mx was detected and quantified in 100% of the evaluated papaya plants, whose viral RNA titer increased from 0.06 (21 dae) to 26.6 pg/μl of PMeV-Mx RNA (60 dae) from 20 ng/µl of cDNA. Three months later, these plants developed sticky disease symptoms, demonstrating that E. papayae is capable of transmitting PMeV-Mx to C. papaya 'Maradol'.

The increasing incidence of Candida albicans infections and resistance to current antifungal therapies has led to the search for new and more effective antifungal compounds. Actinobacterial species from the Streptomyces genus are recognized as some of the major producers of antimicrobial compounds. Therefore, the aims of this study were: (1) the identification of Streptomyces strains isolated from Mexican tropical acidic soils, (2) the evaluation of their antifungal activity on C. albicans , and (3) the exploration of the presence of polyketide synthase genes in their genome and antifungal secondary metabolites in their extracts. Four actinobacterial strains, isolated from previously unexplored soils with antibacterial antecedents, were selected. These strains were identified as Streptomyces angustmyceticus S6A-03, Streptomyces manipurensis S3A-05 and S3A-09, and Streptomyces parvisporogenes S2A-04, according to their molecular analyses. The ethanol extract of the lyophilized supernatant of S. parvisporogenes displayed the most interesting antifungal activity against C. albicans, with a minimum inhibitory concentration (MIC) of 0.5 mg/mL. Type I polyketide synthase (PKS-I) and non-ribosomal peptide synthase (NRPS) genes were detected in all strains. In addition, type II PKS genes (PKS-II) were also found in S. manipurensis S3A-05 and S. parvisporogenes . LC-UV-HRMS analysis of the active organic extract of S. parvisporogenes indicated the presence of the known antifungal compound carbazomycin G as the major component.

The aim of this study was to identify and characterize laccase genes produced by Trametes hirsuta Bm-2 in a liquid medium, both with and without induction. The amplification of 5'and 3'regions of laccase sequences was obtained by the RACE-PCR method, and these were assembled to obtain a cDNA of total length. Two new laccase genes were isolated from basal medium ( lac - B ) and lignocellulosic grapefruit substrate ( lac - T ), both encoding open reading frames of 2566 bp. Both laccase-predicted proteins consisted of 521 amino acids, four copper-binding regions, a signal peptide, and five potential glycosilation sites (Asn-Xaa-Ser/Tre). Moreover, the deduced amino acid sequences share about 76-85% identity with other laccases of WRF. Sequence comparison showed 47 synonymous point mutations between lac - B and lac - T . In addition, 5' untranslated regions (UTR) of laccase genes lac - B and lac - T showed differences in length and number of regulatory elements that may affect transcriptional or translational expression of these genes., Competing Interests: Compliance with ethical standardsThe authors declare that there is no conflict of interest regarding publication of this paper.

Looking for a biotechnical potential, aqueous extracts of leaves of 12 native species used in the Mayan traditional medicine of the coastal dune and mangrove of Yucatan (Mexico) were selected to evaluate their biological activities. Rhizophora mangle and Manilkara zapota showed the highest free radical scavenging activity (3.94 ± 0.19 and 6.42 ± 0.32 μg/mL, respectively), and the highest antihypertensive activity was obtained from Solanum donianum (0.38 μg/mL). The anti-hyperglycemic activity of these species was also tested; the highest activities were registered with R. mangle. The antimicrobial activity of Malvaviscus arboreus, S. donianum, M. zapota, and R. mangle at 10% (w/v) was positive against six human pathogenic bacteria and Bonellia macrocarpa against one pathogenic fungus. Solanum donianum, M. zapota, B. macrocarpa, and R. mangle were positive against two pathogenic plant fungi. These results show that the aqueous extracts of five native plants of the Yucatan coast have potential as antioxidants, ACE inhibitors, α-amylase and α-glucosidase inhibitors, and as antimicrobials, which make their exploration for utilization in the agricultural and pharmaceutical industries a possibility., Competing Interests: The authors have declared that no competing interests exist.

Mexico is one of the five largest producers of papaya worldwide, but losses caused by pathogens, mainly fungus, at the pre- and post-harvest stages are often more than 50% of the crop. Papaya anthracnose, caused by three different species of the Colletotrichum genus in Mexico, occupies a preponderant place in this problem. Although two of these species, C. gloeosporiodes and C. truncatum, have been characterized morphologically and genotypically, this has not occurred with C. magnum, the third species involved, about which there is very little information. Because of this, it is vital to know its genetic characterization, much more so considering that the studies carried out on the other two species reveal a wide genetic diversity, differences in pathogenicity and in the response to fungicides of the different strains characterized. In this work, Colletotrichum spp. isolates were collected at different papaya orchards in the south-southeast of Mexico. C. magnum isolates identified by species-specific primers were characterized by morphological and molecular approaches. Differences in colony characteristics resulted in five morphological groups. AP-PCR, DAMD and ISSR markers were found to be very efficient for revealing the interspecific variability of this species. The high genetic variability found in the accessions of C. magnum was linked to the geographical area where they were collected. Isolates from Chiapas State were the most variable, showing point mutations in the ITS1-ITS2 region. These results will enable a better phytosanitary management of anthracnose in papaya in this region of Mexico.

Lignin is a source for obtaining natural phenols with high commercial value that can act as redox mediators enhancing effects in dye decolorization. In this study Trametes hirsuta Bm-2 was grown on wheat bran to produce laccases and phenol extracts (PE). Ultrafiltered phenols obtained at different times were evaluated in their potential as redox mediators of laccase activity and indigo carmin decolorization. Laccase activity (L) on ABTS increased up to 12.4 times with L/PE72 compared with laccase alone and L/PE48 showed the highest level of dye decolorization (97%) compared with laccase (12%). The chromatographic analysis by HPLC showed variation in the profile and concentration of phenols at different times of culture. Stability of the laccase mediator system (LMs) in dye decolorization was maintained over 3 months. Our results suggest the use of natural mediators as a strategy for improving efficiency in dye biodegradation by laccase-producing fungi., Competing Interests: Compliance with ethical standardsThe authors declare that there is no conflict of interest regarding publication of this paper.

In Mexico, the corn tortilla is a food of great economic importance. Corn tortilla production generates about 1500-2000 m 3 of wastewater per 600 tons of processed corn. Although this wastewater (also known as nejayote) has a high organic matter content, few studies in Mexico have analyzed its treatment. This study presents fresh data on the potential methane production capacity of nejayote in a two-phase anaerobic digestion system using an Anaerobic-Packed Column Reactor (APCR) to optimize the acidogenic phase and an up-flow anaerobic sludge blanket (UASB) reactor to enhance the methanogenic process. Results indicate that day 8 was ideal to couple the APCR to the UASB reactor. This allowed for a 19-day treatment that yielded 96% COD removal and generated a biogas containing 84% methane. The methane yield was 282 L kg -1 of COD removed . Thus, two-phase anaerobic digestion is an efficient process to treat nejayote; furthermore, this study demonstrated the possibility of using an industrial application by coupling the APCR to the UASB reactor system, in order to assess its feasibility for biomethane generation as a sustainable bioenergy source.

Sticky disease, which is caused by Papaya meleira virus (PMeV), is a significant papaya disease in Brazil and Mexico, where it has caused severe economic losses, and it seems to have spread to Central and South America. Studies assessing the pathogen-host interaction at the nano-histological level are needed to better understand the mechanisms that underlie natural resistance. In this study, the topography and mechanical properties of the leaf midribs and latex of healthy and PMeV-infected papaya plants were observed by atomic force microscopy and scanning electron microscopy. Healthy plants displayed a smooth surface with practically no roughness of the leaf midribs and the latex and a higher adhesion force than infected plants. PMeV promotes changes in the leaf midribs and latex, making them more fragile and susceptible to breakage. These changes, which are associated with increased water uptake and internal pressure in laticifers, causes cell disruption that leads to spontaneous exudation of the latex and facilitates the spread of PMeV to other laticifers. These results provide new insights into the papaya-PMeV interaction that could be helpful for controlling papaya sticky disease.

Anthracnose, caused by Colletotrichum truncatum (syn. C. capsici), has become a common disease of tropical crops, severely affecting the quantity and quality of fruit and seed and, therefore, reducing their market value. For years, chemical control has been extensively used for managing this disease. However, the appearance of isolates that are resistant to the most commonly employed fungicides is increasingly widespread. Twenty C. truncatum isolates from pepper, papaya, and physic nut were tested in vitro against four fungicides to determine their sensitivity. All evaluated isolates were resistant to azoxystrobin and thiabendazole and susceptible to cyprodinil + fludioxonil and mancozeb. To determine the molecular mechanism conferring thiabendazole resistance, the TUB-2 gene was characterized, revealing a glutamic acid to alanine substitution at position 198 in 6 of the 20 isolates that were tested. This work confirms the emergence of benzimidazole-based fungicide resistance in C. truncatum populations and highlights the need for monitoring fungicide sensitivity as an essential activity for the development of effective control schemes.

Vinasse is the dark-colored wastewater that is generated by bioethanol distilleries from feedstock molasses. The vinasse that is generated from molasses contains high amounts of pollutants, including phenolic compounds and melanoindin. The goal of this work was to study the expression of laccase genes in the Trametes hirsuta strain Bm-2, isolated in Yucatan, Mexico, in the presence of phenolic compounds, as well as its effectiveness in removing colorants from vinasse. In the presence of all phenolic compounds tested (guaiacol, ferulic acid, and vanillic acid), increased levels of laccase-encoding mRNA were observed. Transcript levels in the presence of guaiacol were 40 times higher than those in the control. The lcc1 and lcc2 genes of T. hirsuta were differentially expressed; guaiacol and vanillin induced the expression of both genes, whereas ferulic acid only induced the expression of lcc2. The discoloration of vinasse was concomitant with the increase in laccase activity. The highest value of enzyme activity (2543.7 U/mL) was obtained in 10% (v/v) vinasse, which corresponded to a 69.2% increase in discoloration. This study demonstrates the potential of the Bm-2 strain of T. hirsuta for the biodegradation of vinasse.

Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease, which is characterized by a spontaneous exudation of fluid and aqueous latex from the papaya fruit and leaves. The latex oxidizes after atmospheric exposure, resulting in a sticky feature on the fruit from which the name of the disease originates. PMeV is an isometric virus particle with a double-stranded RNA (dsRNA) genome of approximately 12 Kb. Unusual for a plant virus, PMeV particles are localized on and linked to the polymers present in the latex. The ability of the PMeV to inhabit such a hostile environment demonstrates an intriguing interaction of the virus with the papaya. A hypersensitivity response is triggered against PMeV infection, and there is a reduction in the proteolytic activity of papaya latex during sticky disease. In papaya leaf tissues, stress responsive proteins, mostly calreticulin and proteasome-related proteins, are up regulated and proteins related to metabolism are down-regulated. Additionally, PMeV modifies the transcription of several miRNAs involved in the modulation of genes related to the ubiquitin-proteasome system. Until now, no PMeV resistant papaya genotype has been identified and roguing is the only viral control strategy available. However, a single inoculation of papaya plants with PMeV dsRNA delayed the progress of viral infection.

This study characterized Clavispora lusitaniae strains isolated from different stages of the processing and early fermentation of a henequen (Agave fourcroydes) spirit produced in Yucatan, Mexico using a molecular technique. Sixteen strains identified based on morphological features, obtained from different substrates, were typed molecularly. Nine different versions of the divergent D1/D2 domain of the large-subunit ribosomal DNA sequence were identified among the C. lusitaniae strains. The greatest degree of polymorphism was found in the 90-bp structural motif of the D2 domain. The MSP-PCR technique was able to differentiate 100% of the isolates. This study provides significant insight into the genetic diversity of the mycobiota present during the henequen fermentation process, especially that of C. lusitaniae, for which only a few studies in plants have been published. The applied MSP-PCR markers were very efficient in revealing olymorphisms between isolates of this species.

Background: C. gloeosporioides sensu lato is one of the most economically important post-harvest diseases affecting papaya production worldwide. There is currently no information concerning the genetic structure or demographic history of this pathogen in any of the affected countries. Knowledge of molecular demographic parameters for different populations will improve our understanding of the biogeographic history as well as the evolutionary and adaptive potential of these pathogens. In this study, sequence data for ACT, GPDH, β-TUB and ITS gene regions were analyzed for C. gloeosporioides sensu lato and C. truncatum isolates infecting papaya in Trinidad and Mexico in order to determine the genetic structure and demographic history of these populations., Results: The data indicated that Mexico is the ancestral C. gloeosporioides sensu lato population with asymmetrical migration to Trinidad. Mexico also had the larger effective population size but, both Mexico and Trinidad populations exhibited population expansion. Mexico also had greater nucleotide diversity and high levels of diversity for each gene. There was significant sub-division of the Trinidad and Mexico populations and low levels of genetic divergence among populations for three of the four gene regions; β-TUB was shown to be under positive selection. There were also dissimilar haplotype characteristics for both populations. Mutation may play a role in shaping the population structure of C. gloeosporioides sensu lato isolates from Trinidad and from Mexico, especially with respect to the ACT and GPDH gene regions. There was no evidence of gene flow between the C. truncatum populations and it is possible that the Mexico and Trinidad populations emerged independently of each other., Conclusions: The study revealed relevant information based on the genetic structure as well as the demographic history of two fungal pathogens infecting papaya, C. gloeosporioides sensu lato and C. truncatum, in Trinidad and Mexico. Understanding the genetic structure of pathogen populations will assist in determining the evolutionary potential of the pathogen and in identifying which evolutionary forces may have the greatest impact on durability of resistance. Intervention strategies that target these evolutionary forces would prove to be the most practical.

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.

The molecular characterization of 14 strains of Kluyveromyces marxianus isolated from Agave fourcroydes (Lem.) in Yucatan, Mexico, was performed by AP-PCR analysis, PCR-RFLP of 5.8S-ITS, and complete NTS regions. A sequence analysis of the D1/D2 domain of the 26S rDNA was also carried out in six selected strains. The AP-PCR approach had the highest discrimination power for the molecular characterization of new henequen K. marxianus strains. PCR-RFLP of 5.8S-ITS regions did not reveal polymorphisms in this group of strains. The restriction enzyme digestion analysis of NTS region enables the separation among strains which coincides with ascospore shape groups. The molecular tools used in this article may be useful to confirm a preliminary screen of yeasts isolated from henequen without the use of growth characteristics or morpho-physiological tests.

A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A(260)/A(280)) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.

Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A260/A280) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The quality of the DNA was suitable for PCR amplification.

An aluminium (Al)-tolerant cell line (LAMt) of coffee (Coffea arabica L.) was obtained from a cell suspension culture and biochemically and molecularly characterized in an MS medium at half ionic strength and low pH. LAMt grew 30% more than the control line (susceptible to Al) in the presence of different concentrations of Al, showed a lower free Al concentration in the medium and had higher phospholipase C specific activity (80%). Membrane integrity of the LAMt was 50% greater than the control line when both were incubated in the presence of different Al concentrations (measured by Evans Blue uptake). Finally, the use of microsatellite primers revealed no difference in the DNA pattern of both cell lines.