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Late leaf spot (LLS) caused by the Ascomycete Nothopassalora personata (N.p.) (Syn. Cercosporidium personatum) is the main foliar disease of peanuts in Argentina and in peanut producing areas of the world, causing up to 70% yield losses. The extremely slow growth of this fungus in culture, that takes around one month to form a 1 cm colony (0.45 mm/day), and the lack of adequate young tissues from where to extract nucleic acids, have hindered genetic studies of this pathogen. Here, we report the first genome sequence of a N. personata isolate from South America, as well as genetic variants on its conserved genes, and the complete sequence of its mating-type locus MAT1-2 idiomorph. The N. personata isolate IPAVE 0302 was obtained from peanut leaves in Córdoba, Argentina. The whole genome sequencing of IPAVE 0302 was performed as paired end 150 bp NovaSeq 6000 and de novo assembled. Clean reads were mapped to the reference genome for this species NRRL 64463 and the genetic variants on highly conserved genes and throughout the genome were analyzed. Sequencing data were submitted to NCBI GenBank Bioproject PRJNA948451, accession number SRR23957761. Additional Fasta files are available from Harvard Dataverse (https://doi.org/10.7910/DVN/9AGPMG and https://doi.org/10.7910/DVN/YDO3V6). The data reported here will be the basis for the analysis of genetic diversity of the LLS pathogen of peanut in Argentina, information that is critical to make decisions on management strategies.
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Campomanesia guazumifolia is a native tree that produces fruit that can be consumed fresh or used by industry (Donadio et al., 2002). In February 2022, in the experimental area of the Universidade Tecnológica Federal do Paraná - Brazil, disease was observed in 22 trees, with 50% to 80% severity in crown leaves. Symptoms were small, irregular, or circular-shaped, dark-brown lesions with yellow halos (Figure S1). As the disease progressed, the lesions increased in size, without distinction between mature and young tissues, causing complete leaf wilting. Twenty symptomatic leaves from 11 trees grown in the same orchard line were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water, and dried on sterile filter paper. Five fragments of diseased leaf tissue were placed on a potato dextrose agar medium. The morphological characteristics of the colony, such as filamentous mycelium and golden yellow on the upper part, with the presence of circular to ovoid and multicellular conidia (mean 21.00 µm x 24.45 µm, n = 30) of the nine isolates, coincided with the description of the fungus of the genus Epicoccum (Valenzuela-Lopez et al., 2018). Further identification of one of these nine isolates was confirmed by amplifying and sequencing three loci (ITS, ß-tubulin, and RPB2) using the ITS1/ITS4, Bt2a/Bt2b, and 5F2/7cR primer pairs, respectively (White et al., 1990, Glass and Donaldson, 1995, O'Donnell et al., 2007). A single representative isolate (Cgen01) was analyzed and submitted to GenBank (OR020968, OR079879, and OR079878). The Bayesian Inference was used to reconstruct the phylogenetic trees (Figure S2), starting from random trees for 5,000,000 generations, using MrBayes v. 3.2.1 (Ronquist et al., 2012). The isolate clustered together with the isolate of Epicoccum nigrum (Chen et al., 2017) with a high posterior probability (0.98). For the pathogenicity tests, four young, healthy branches containing 20 leaves were spray-inoculated with 1.5 mL of conidia suspension of Cgen01 (106 conidia mL-1), covered with perforated transparent plastic bags, and moistened with distilled water in the orchard. The air temperature ranged from 14ºC to 25ºC. Sterile distilled water was used as a control. Three replicates (pathogen and control) on different trees were evaluated. After five days, the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of Cgen01. Control branches did not show fungal growth. The inoculation test was conducted twice and similar symptoms were observed. This is the first report of leaf spots caused by E. nigrum on C. guazumifolia in Brazil. E. nigrum, an endophytic fungus described as a mycoparasite, showed phytopathogenic behavior in this study, causing spots and loss of leaves in C. guazumifolia, drastically reducing the production of photoassimilates and affecting the quality of the fruits.
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Myrcianthes pungens is a tree fruit native to Brazil, unknown to a large part of the population, with fruit consumed only locally. In October 2022, at the experimental area at Universidade Tecnológica Federal do Paraná (UTFPR) in the Dois Vizinhos city, Paraná State, Brazil, symptoms of the disease were observed on mature leaves and fruits of 17 trees. Fungal fructifications were observed in the form of bright yellow uredinia containing a large mass of urediniospores on the surface and on the leaves and fruits that resembled the structures typical of a Myrtaceae rust pathogen. Leaves colonized by the fungus showed deformations, turning dark and rapidly causing senescence. In the orchard, the fungus affected 80% of the trees, with a severity of 40 to 45%. Diseased fruits (10) and leaves (10) (from each tree) were collected from 17 trees from different positions in the orchard. The observed structures (optical microscope) were hyaline and globose urediniospores (n = 30) which had pointed echinulate ornaments throughout their surface (Cummins & Hiratsuka, 2003), (n = 30, 14.84 µm × 21.1 µm). These characteristics were similar to the morphological characteristics of the genus Austropuccinia previously described by Young et al. (2019). A strain was selected as a representative for molecular characterization and pathogenicity tests (accession no. APM001). For molecular identification, the internal transcribed spacer (ITS) region (Kroop et al., 1995), b-tubulin (TUB2), and translation elongation factor 1-alpha (TEF) (Machado et al., 2015) were amplified by PCR and sequenced. The sequences were deposited in GenBank (accession nos. ITS: OQ442638, TUB2: OQ506543, and TEF: OQ506542). Phylogenetic analyses using Bayesian inference grouped the isolate with the type species of Austropuccinia psidii with a high posterior probability (1.0). Pathogenicity tests used conidial suspensions (1x105urediniospores/ml). Four branches containing twenty leaves and two young asymptomatic fruits were individually inoculated with 1.5 mL of urediniospore suspension using a bottle with a spray nozzle cap. The branches were protected with perforated transparent plastic bags moistened with distilled water and incubated at room temperature (18 ºC to 25 ºC). Three replicates (pathogen and control) spread on different trees in the orchard were used in this experiment. After seven days, symptoms of rust appeared on the leaves and on the tenth day of the fruits, with morphological characteristics similar to those previously reported. Control branches showed no fungal growth. The inoculation test was repeated, confirming the symptoms. This is the first report of the incidence of rust caused by A. psidii on leaves and fruits of M. pungens in Paraná State. The importance of the disease is due to the high percentage of fruit loss due to rapid rot and drop caused by the pathogen attack.
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Campomanesia guazumifolia is a Brazilian fruit tree that has ecological importance and the potential to be explored by the food and medical industries (Lima et al., 2011). In February 2019, in the experimental orchard at the Universidade Tecnológica Federal do Paraná, Dois Vizinhos city, Paraná State - Brazil, disease symptoms were observed on leaves, stems, and fruits of 22 C. guazumifolia trees. Yellow uredinia were observed on upper side of the leaves, stems, and flowers, which resembled typical uredinia of Myrtaceae rust. The pustules occurred mainly on young shoots, and on flowers, they infected their sepals. Over time, tissues colonized by the pathogen exhibited deformations and mummification occurred in infected fruits. In the orchard, the fungus affected 80% yield. Twenty diseased plant parts (from each of the eleven trees) were collected at different positions in the orchard. One strain were selected as a representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The structures observed were epiphyllous uredinia (leaves), united in small groups with hyaline and obovoid or obpyriform urediniospores, which presented echinulate ornaments, germinated pores in the subequatorial and inordinate positions (Cummins; Hiratsuka, 2003) (n = 30, 14.84 x 21.12 µm). The morphology of uredinia and urediniospores was similar to the morphological characteristics of the genus Austropuccinia previously described in Young (2019). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region (White et al. 1990), ß-tubulin (TUB2) and translation elongation factor 1-alpha (TEF) (Machado et al. 2012) were amplified by PCR, and sequenced. Bayesian inference was used to reconstruct a phylogenetic tree, using MrBayes v. 3.2.1 (Ronquist et al., 2012). The multilocus phylogenetic analysis clearly distinguished the isolate APCG001 as Austropuccinia psidii separating it from all other species. The sequences were deposited in GenBank (accessions nos. ITS: ON003418, TUB2: ON568196, and TEF: ON437601). For pathogenicity tests, four healthy branches (20 leaves each) were sprayed with 2.5 mL of (APCG001) uredospore suspension (105 mL-1) and covered with a plastic bag in the orchard. The air temperature ranged from 16ºC to 25ºC. Sterile distilled water was used as a control. Three replications (pathogen and control) were performed on different trees. After 6 days, symptoms of rust appeared on the plants. Control branches did not show fungal growth. The inoculation test was repeated again, confirming the initial results. This is the first report of infection by A. psidii in C. guazumifolia trees in Brazil, causing rust, necrosis, and early senescence in fruits, leaves, and stems. Myrtaceae rust reduces the C. guazumifolia leaf area, affecting photosynthetic production and reducing fruit quality.
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Opportunistic pathogenic fungi arise in agricultural crops as well as in surrounding human daily life. The recent increase in antifungal-resistant strains has created the need for new effective antifungals, particularly those based on plant secondary metabolites, such as capsaicinoids and capsinoids produced by Capsicum species. The use of such natural compounds is well-aligned with the One Health approach, which tries to find an equilibrium among people, animals, and the environment. Considering this, the main objective of the present work is to review the antifungal potential of capsaicinoids and capsinoids, and to evaluate the environmental and health impacts of biofungicides based on these compounds. Overall, capsaicinoids and their analogues can be used to control pathogenic fungi growth in plant crops, as eco-friendly alternatives to pest management, and assist in the conservation and long-term storage of agrifood products. Their application in different stages of the agricultural and food production chains improves food safety, nutritional value, and overcomes antimicrobial resistance, with a lower associated risk to humans, animals, and the environment than that of synthetic fungicides and pesticides. Nevertheless, research on the effect of these compounds on bee-like beneficial insects and the development of new preservatives and packaging materials is still necessary.
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Berry fruits of Capsicum annuum L. cv. "Cacho de Cabra" are used for the manufacture of a traditional pepper powder known as Merkén. In the present study, aflatoxins (AFs) and ochratoxin A (OTA) contamination in berry fruits of C. annuum was determined at harvest, drying, and smoking stages of Merkén production, in cumin and coriander seeds used as Merkén ingredients, and in the final packaged Merkén produced by local farmers. Additionally, Merkén samples from local markets in the region of La Araucanía (Chile) were also evaluated. Chromatographic analysis was based on a qualitative method. AFs and OTA were not detected on pepper pods and seeds. There was no detection of AFs and OTA on cultured Aspergillus and Penicillium strains isolated from pepper pods, cumin and coriander seeds and Merkén. The lack of AFs/OTA-producers among the isolated fungal species can explain and support the absence of contamination in pepper pods. In contrast, the AFB1 was detected in 75% of Merkén obtained from farmers and 46% of Merkén samples purchased from local markets; while OTA was detected in 100% of Merkén samples obtained from farmers and local markets. In the Merkén production chain, the harvest and post-harvest are key stages for fungal growth while the commercialization stage is highly susceptible to AFs and OTA contamination.
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The Eugenia myrcianthes fruit can be consumed in natural or processed form (jellies and juices) (Infante et al., 2016). In 2019, in the UTFPR, Dois Vizinhos city, Paraná State - Brazil, yellow uredinias epiphyllous were observed on the tissue surface (leaves, stems, flowers, and fruit) of twenty-one trees of E. myrcianthes, which resembled structures typical of Myrtaceae rust. All colonized tissues showed necrotic lesions that varied in size and shape, causing death, especially in fruit. In the orchard, the fungus affects 50% to 95% yield. Fruit (10) and leaves (20) with symptoms were collected from 11 trees from different positions in the orchard. Infected tissues were incubated (25°C and 12-hour photoperiod) for 7 days to induce sporulation. The epiphyllous uredinia, united in small groups with hyaline and globose urediniospores were observed and presented equinulate ornaments and germinated pores in the subequatorial and inordinate positions (De Pieri, 2012; Cummins; Hiratsuka, 2003) (mean 14.00 µm × 21.12 µm, n = 30) similar to the morphological characteristics of the Austropuccinia genus described by Young (2019). The identification of 10 samples (fruit and leaf) of the pathogen taken from infected parts of the trees was confirmed. For molecular identification, the internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (White et al. 1990) and sequenced. Phylogenetic analyses using Bayesian inference grouped the strain from Eugenia myrcianthes with the epitype species of Austropuccinia psidii (Beenken, 2017), with a high posterior probability (0.99). The sequences of one representative strain (Emg1) were submitted to GenBank (OM948983). For pathogenicity tests, three healthy branches containing 20 leaves were sprayed with 3.0 mL of urediniospores suspension (105) of Emg1 and covered with a plastic bag in the orchard (25ºC). Sterile distilled water was used as a control. Three replications (pathogen and control) were performed on different trees. After 6 days, symptoms appeared and their morphological features were similar to those previously reported. Control branches did not present fungal growth. The inoculation test was repeated again, confirming symptoms such as uredinia and urediniospores, characteristic of the disease. This is the first report of the incidence of A. psidii infection in E. myrcianthes trees in Brazil, causing rust, necrosis, and senescence in fruit, leaves, flowers, and stems. The rust on E. myrcianthes causes destructive damage to yield, as the pathogen causes fruit to rot and drop prematurely.
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Anthracnose, caused by Colletotrichum spp., is the most important fungal disease of papaya (Carica papaya L.) worldwide. In March 2020, mature papaya fruit (cv. Maradol) showing typical symptoms of anthracnose were observed in an orchard located in Pinotepa Nacional, Oaxaca, Mexico. Disease incidence of 100 papaya plants surveyed in the orchard was estimated at about 45%. Initially, small and water-soaked lesions appeared on the fruit surface, which later enlarged to circular sunken lesions with translucent light brown margins. On advanced infections, salmon-pink masses of spores were observed on the lesions. Twenty Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium at 25°C in the dark for 6 days and 10 monoconidial isolates were obtained. An isolate was selected as representative for further characterization. The isolate was deposited as CPM-H4 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. On PDA, the colonies were initially light grey then later became dark grey with orange conidial masses after incubation for 7 days. Conidia (n= 50) were hyaline, aseptate, cylindrical with rounded ends, and measured 10.2 to 13.6 × 4.1 to 5.3 µm. Appressoria (n= 20) were mostly simple, solitary and smooth-walled, dark brown, and clavate, measuring 6.8 to 14.8 × 5.5 to 7.7 µm. Based on morphology, the isolate was tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes were amplified (Weir et al. 2012), and sequenced. The sequences were deposited in GenBank (accessions nos. OM965612 (ITS), OM959540 (ACT), ON065005 (TUB2), ON065003 (CHS-1), ON065004 (GAPDH). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, GAPDH, and CHS-1 sequence dataset for Colletotrichum spp. was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CPM-H4 as Colletotrichum chrysophilum. Pathogenicity of the fungus was verified on 10 healthy papaya fruits (cv. Maradol) without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on three locations on each fruit. Ten control fruit were treated in the same way but with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated papaya fruits developed sunken necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Colletotrichum chrysophilum has been previously reported to cause anthracnose on mango (Fuentes-Aragón et al. 2020a), avocado (Fuentes-Aragón et al. 2020b), and banana (Fuentes-Aragón et al. 2021) in Mexico; however, to our knowledge, this is the first report of C. chrysophilum causing papaya anthracnose in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species associated with papaya in Mexico through subsequent phylogenetic studies as well as to monitor the possible movement and distribution of this pathogen into other Mexican regions.
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Lanthanide-based beta-tricalcium phosphate (ß-TCP) upconversion nanoparticles are exploited as a non-viral vector for imaging guided-gene therapy by virtue of their unique optical properties and multi-modality imaging ability, high transfection efficiency, high biocompatibility, dispersibility, simplicity of synthesis and surface modification. Ytterbium and thulium-doped ß-TCP nanoparticles (ßTCPYbTm) are synthesized via co-precipitation method, coated with polyethylenimine (PEI) and functionalized with a nuclear-targeting peptide (TAT). Further, in vitro studies revealed that the nanotheranostic carriers are able to transfect cells with the plasmid eGFP at a high efficiency, with approximately 60% of total cells producing the fluorescent green protein. The optimized protocol developed comprises the most efficient ßTCPYbTm/PEI configuration, the amount and the order of assembly of ßTCPYbTm:PEI, TAT, plasmid DNA and the culturing conditions. With having excellent dispersibility and high chemical affinity toward nucleic acid, calcium ions released from ßTCPYbTm:PEI nanoparticles can participate in delivering nucleic acids and other therapeutic molecules, overcoming the nuclear barriers and improving the transfection efficacy. Equally important, the feasibility of the upconversion multifunctional nanovector to serve as an effective contrast agent for imaging modality, capable of converting low-energy light to higher-energy photons via a multi-photons mechanism, endowing greater unique luminescent properties, was successfully demonstrated.
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Elementos da Série dos Lantanídeos , Nanopartículas , Fosfatos de Cálcio , Terapia Genética/métodos , Células HeLa , Humanos , Nanopartículas/química , Medicina de PrecisãoRESUMO
Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by eight pathogenic species of the Sporothrix genus. Different ex situ preservation methods are used around the world to maintain the survival, morphophysiological and genetic traits of fungal strains isolated from patients with sporotrichosis for long terms. The main aim of the present study was to evaluate the survival, phenotypic and genotypic stability of Sporothrix strains after preservation on PDA slant stored at 4 °C, sterile water and cryopreservation at -80 °C, for a period of time of 6, 12, 18 and 24 months of storage. Eight clinical Sporothrix isolates were identified based on a polyphasic approach consisting of classical macro- and micro-morphological traits, biochemical assays, proteomic profiles by MALDI-TOF MS and molecular biology. According to the final identification, one strain was identified as S. schenckii (CMRVS 40428) and seven strains were re-identified as S. brasiliensis (CMRVS 40421, CMRVS 40423, CMRVS 40424, CMRVS 40425, CMRVS 40426, CMRVS 40427 and CMRVS 40433). In addition, it was observed that the isolates survived after the different time points of storage in distilled water, PDA slant and cryopreservation at -80 °C. For fungi preserved in water, low polymorphisms were detected by the partial sequencing of ß-tubulin. Cryopreservation at -80 °C induced morphological changes in one single isolate. The proteomic profiles obtained by MALDI-TOF MS after preservation showed differences among the methods. In conclusion, preservation on agar slant stored at 4 °C was the most effective method to preserve the eight clinical Sporothrix strains. This method produced less change in the phenotypic traits and kept the genetic integrity of all strains. Agar slant stored at 4 °C is a simple and inexpensive method and can be especially used in culture collections of limited funding and resources.
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Abstract One of the texts in the "Biodiversity in the State of São Paulo" series, within the FAPESP-Biota Program, was dedicated to the Infrastructure for Biodiversity Conservation, with a focus on Biological Collections and Conservation Units. From the early 1960s, when FAPESP was established, to the present day, financial resources have been invested in the preservation of the biodiversity of the national genetic heritage, besides other fields. History of years of advances in scientific knowledge was built, which can be portrayed through the projects that resulted in high-quality data of national and international impact. Microbiological collections are centers that generate technology and specialized human resources, and act (among other things) as living repositories preserving reference material and as witnesses to the history of microbial biodiversity because they preserve what may no longer exist. They have enormous potential to promote the global bioeconomy and address problems that have resulted from the misuse of natural resources. This reading brings everyone the history, advances, and future perspectives of culture collections, within the efforts of 60-year scientific activities in Brazil.
Resumo Um dos textos da série "Biodiversidade do Estado de São Paulo", dentro do Programa FAPESP-Biota, foi dedicado à Infraestrutura para Conservação da Biodiversidade, com foco nas coleções biológicas e nas unidades de conservação. Do início dos anos 60, quando a FAPESP foi criada, até os dias atuais muito foi investido em pesquisa nas mais diversas áreas, incluindo a preservação da biodiversidade do patrimônio genético nacional. Uma história de longos anos de avanços no conhecimento científico foi construída, a qual pode ser retratada através dos projetos que resultaram em dados de alta qualidade com impacto nacional e internacional. As coleções microbiológicas são centros geradores de tecnologia e recursos humanos especializados, que atuam (dentre outros) como repositórios vivos, preservando material de referência, e como testemunho da história da biodiversidade microbiana, preservando o que pode não mais existir. Possuem enorme potencial para alavancar a bioeconomia global e tratar de problemas que resultaram do mau uso dos recursos naturais. Essa leitura traz a todos o histórico, os avanços e as perspectivas futuras das coleções de microrganismos, dentro dos esforços de 60 anos de atividades científicas no Brasil.
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Herpes simplex virus is among the most prevalent sexually transmitted infections. Acyclovir is a potent, selective inhibitor of herpes viruses and it is indicated for the treatment and management of recurrent cold sores on the lips and face, genital herpes, among other diseases. The problem of the oral bioavailability of acyclovir is limited because of the low permeability across the gastrointestinal membrane. The use of nanoparticles of pseudoboehmite as a drug delivery system in vitro assays is a promising approach to further the permeability of acyclovir release. Here we report the synthesis of high purity pseudoboehmite from aluminium nitrate and ammonium hydroxide containing nanoparticles, using the sol-gel method, as a drug delivery system to improve the systemic bioavailability of acyclovir. The presence of pseudoboehmite nanoparticles were verified by infrared spectroscopy, transmission electron microscopy, and X-ray diffraction techniques. In vivo tests were performed with Wistar rats to compare the release of acyclovir, with and without the addition of pseudoboehmite. The administration of acyclovir with the addition of pseudoboehmite increased the drug content by 4.6 times in the plasma of Wistar rats after 4 h administration. We determined that the toxicity of pseudoboehmite is low up to 10 mg/mL, in gel and the dried pseudoboehmite nanoparticles.
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Aciclovir/administração & dosagem , Hidróxido de Alumínio/química , Óxido de Alumínio/química , Antivirais/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nanogéis/química , Aciclovir/sangue , Aciclovir/farmacocinética , Administração Oral , Hidróxido de Alumínio/farmacologia , Óxido de Alumínio/farmacologia , Animais , Antivirais/sangue , Antivirais/farmacocinética , Disponibilidade Biológica , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Herpes Simples/tratamento farmacológico , Herpes Simples/virologia , Humanos , Modelos Animais , Ratos , Ratos Wistar , Simplexvirus/efeitos dos fármacosRESUMO
Gallbladder cancer (GBC) is one of the most common sites for biliary tract cancers. It has a worldwide distribution being endemic in South America and Southern Asia. These high GBC rates have previously been linked to the determinants of health such as nutrition, genetics, lifestyle, and environment. Exposure to aflatoxin B1 (AFB1), a human carcinogen, is suggested to be involved with GBC development. This work aims to analyse the interplay of social, lifestyle, and genetic predisposing factors to GBC. AFB1 plays a pivotal role in carcinogenic onset by genetic and epigenetic modifications. AFB1 can induce molecular changes involved in the GBC pathogenesis, such as overexpression of UCHL1 gene, mutagenesis of TP53 gene, abnormal expression of oncogenes BCL-2, and aberrantly methylation of ERBB family receptors. However, a large-scale scientific cooperation is needed to confirm these molecular links through which AFB1 may increase the GBC risk. For that, monitoring AFB1 exposure through AF-albumin and AFB1-lysine will clarify the level of exposure of the population to AFB1 in the GBC hotspot. Further, analyses of AFB1-adduct concentrations in GBC cases (fatal and non-fatal) are needed to understanding if AF contamination can trigger gallbladder cancer.
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Aflatoxina B1/efeitos adversos , Exposição Ambiental/efeitos adversos , Neoplasias da Vesícula Biliar/etiologia , Neoplasias da Vesícula Biliar/genética , Carcinogênese/induzido quimicamente , Carcinógenos/análise , Predisposição Genética para Doença , HumanosRESUMO
Guava (Psidium guajava L.) is a small tree belonging to the Myrtaceae family and it is distributed worldwide in the tropical and subtropical areas. During the summer of 2019, symptoms of fruit anthracnose were observed on approx. 90% of 250 guava trees located in backyards in Juan Jose Rios, Sinaloa, Mexico. Lesions on guava fruit were irregular, necrotic, and sunken. On advanced infections, acervuli containing salmon-pink masses of spores were observed on the lesions. Twenty fruits were collected from 10 trees (2 fruits per tree). Colletotrichum-like colonies were consistently isolated on PDA medium and 20 monoconidial isolates were obtained. Four isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of El Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF205-FAVF208). Colonies on PDA medium were flat with an entire margin, with abundant felty and white aerial mycelium, with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with ends rounded, and measuring 14.8 to 18.1 × 4.4 to 5.3 µm. Based on morphological features, the isolates were tentatively allocated in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), as well as partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), chitin synthase (CHS-1) and glutamine synthetase (GS) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference and including published ITS, GAPDH, TUB2, ACT, CHS-1, and GS data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the four isolates FAVF205-FAVF208 as C. siamense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accessions nos. ITS: MW598512-MW598515; GAPDH: MW595216-MW595219; TUB2: MW618012-MW618015; ACT: MW595208-MW595211; CHS-1: MW595212-MW595215; and GS: MW618008-MW618011). Pathogenicity of the four isolates was verified on 40 healthy guava fruits. Twenty fruits were wounded with a sterile toothpick (2 mm in depth) and a mycelial plug (6 mm of diameter) was placed on each wound. Ten fruits inoculated with a PDA plug without mycelial growth served as controls. The fruit was kept in a moist plastic chamber at 25°C for 7 days. Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. The experiments were repeated twice with similar results. All inoculated fruits developed sunken necrotic lesions 4 days after inoculation, whereas no symptoms were observed on the control fruits. The fungi were consistently re-isolated only from the diseased fruits, fulfilling Koch´s postulates. Colletotrichum siamense has been previously reported on guava fruit in India (Sharma et al. 2015). However, to our best knowledge, this is the first report of C. siamense causing fruit anthracnose on guava in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species on guava in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.
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BACKGROUND: Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. RESULTS: During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. CONCLUSIONS: Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.
Assuntos
Cacau/microbiologia , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Cacau/química , Cacau/metabolismo , Chocolate/análise , Chocolate/microbiologia , Fermentação , Aromatizantes/química , Aromatizantes/metabolismo , Microbiologia de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Sementes/química , Sementes/metabolismo , Sementes/microbiologia , Paladar , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
Citrus anthracnose, caused by Colletotrichum spp., is a major disease in many citrus-growing regions of the world. During the spring of 2019, symptoms of petal necrosis and necrotic lesions on fruits were detected on Mexican lime (Citrus aurantifolia), sweet orange (Citrus sinensis), and grapefruit (Citrus paradisi) trees in three commercial orchards distributed in northern Sinaloa (El Fuerte and Ahome municipalities), Mexico. Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium from symptomatic petals and fruits, and 30 monoconidial isolates (10 per orchard) were obtained. Five isolates were selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were designated as FAVF355-FAVF359 and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agronomy of El Fuerte Valley at the Autonomous University of Sinaloa (Mexico). Colonies grown on PDA at 25ºC were cottony, dense, with grayish white aerial mycelium and with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, 13.7 to 18.8 × 4.3 to 5.8 µm, with both ends rounded. Based on morphological features, the five isolates were tentatively identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin (TUB2) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference for species belonging to the C. gloeosporioides species complex was constructed. The multilocus phylogenetic analysis distinguished the isolates FAVF355-FAVF357 as C. gloeosporioides sensu stricto and the isolates FAVF358-FAVF359 as C. siamense. The sequences were deposited in GenBank (accession numbers ITS: MT850050-MT850054; ACT: MT834528-MT834532; GAPDH: MT855979-MT855982; TUB2: MT834533-MT834536). Pathogenicity of the five isolates was verified on healthy fruits of their original host species. Five fruits per isolate were inoculated using the colonized agar plug method. Fruits were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) removed from the margin of a 6-days-old culture were placed onto three wound sites in each fruit. Non-colonized agar plugs were placed on the wounds of 10 fruits used as the control. The fruits were kept in a moist chamber at 25°C for 8 days. The experiment was repeated twice. All inoculated fruits developed circular and necrotic lesions 6 days after inoculation, whereas the control fruits remained symptomless. The fungi were consistently re-isolated from the diseased fruits and were morphologically identical to that originally inoculated, fulfilling Koch´s postulates. To date, only C. gloeosporioides sensu lato and C. acutatum sensu lato has been associated with sweet orange and Mexican lime in Mexico (Farr and Rossman 2020), whereas C. gloeosporioides sensu stricto has been recently recorded in a different area (Iguala, Guerrero) of Mexico (Cruz-Lagunas et al. 2020). To our knowledge, this is the first report of C. gloeosporioides sensu stricto causing anthracnose on sweet orange, and of C. siamense on Mexican lime in Mexico, as well as C. gloeosporioides s. s. causing disease on grapefruit in Sinaloa, Mexico.
RESUMO
This work aims to provide the first study on the mycobiota present in Chilean pepper Capsicum annuum L. cv. "Cacho de Cabra" throughout the early production stages. Two hundred and forty berry fruits were sampled: 1) at the ripe fruits harvest day; 2) during drying; and 3) smoking processes. A total of 192 strains, encompassing 11 genera and 44 species, were identified through analysis of ß-tubulin (benA) gene and internal transcribed spacer of ribosomal DNA (ITS) region. All collection points showed samples with high fungal contamination, but the mycobiota composition varied as a result of different environmental conditions. Alternaria spp. and Fusarium spp. were predominantly isolated from fresh fruits of C. annuum. Penicillium spp. was the most frequent genus in all analysed points. Penicillium brevicompactum and P. crustosum were the most abundant species. Among Aspergillus, A. niger and A. flavus were dominant after the drying phase. In our study, none of the analysed strains of Penicillium (113) and Aspergillus (35) produced Ochratoxin A at detectable levels. The broad characterization of the fungal community of C. annuum carried out in this study, could be a guideline for future mycotoxin analyses performed directly on the pod. Understanding the role and dynamics of mycobiota and its relationship with the toxins present in this substrate, will be useful to establish and improve control measures considering the specificities of each point in the C. annuum production chain.
Assuntos
Capsicum/microbiologia , Microbiologia de Alimentos/métodos , Alimentos em Conserva/microbiologia , Micobioma , Chile , DNA Espaçador Ribossômico/genética , Frutas/microbiologia , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Micotoxinas/análise , Tubulina (Proteína)/genéticaRESUMO
Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 µm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.
RESUMO
Identification of filamentous fungi by conventional phenotypic methods are time-consuming, and a correct identification at the species level is prone to errors. Therefore, a more accurate and faster time-to-results, and cost-effective technique, is required, such as the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). In this study, we describe the development of an in-house spectra library for the identification of filamentous fungi frequently isolated from patients with infections. An in-house spectra library was constructed using 14 reference strains grown in solid medium. Clinical isolates were identified either by the in-house spectra library or the Biotyper commercial library from Bruker Daltonics. Fungal identification was carried following the Biotyper's established scores: ≤1.699: not reliably identified (NRI); 1.700-1.999: genus-level; ≥2.000: species-level. Clinical isolates were identified, with the in-house library, at species- and genus-level at 88.70% (55) and 3.22% (2), respectively. While 4.80% (3) was NRI and 3.22% (2) was discrepant concerning sequencing. On the contrary, identification up to species and genus-level with the commercial library was 44.44% (16) and 22.22% (8), respectively. NRI and the discrepancy was 30.55% (11) and 2.77% (1), respectively. For the reaming 26 isolates, 16 from Neoscytalidium dimidiatum and 10 from Sporothrix spp., respectively, the absence of spectrum and the specific spectra within the Sporothrix complex in the commercial library resulted in the inability to obtain an identification. In conclusion, the current results advocate the importance that each clinical microbiological laboratory needs to develop an ad hoc library associated with the MALDI-TOF MS fungal identification to overcome the limitations of the available commercial libraries.
RESUMO
OBJECTIVE: The aims of this study were: 1) to evaluate the effect of sintering temperature on microstructure, density and flexural strength of a 3Y-TZP/TiO2 composite containing 12.5 wt% of TiO2 compared to 3Y-TZP specimens (control); 2) to compare 3Y-TZP with the experimental 3Y-TZP/TiO2 composite, both sintered at 1400 °C, with respect to the following parameters: optical properties, characteristic strength, Weibull modulus, fatigue behavior, induction of osteoblasts proliferation and differentiation (mineralization nodules formation). METHODS: The 3Y-TZP and 3Y-TZP/TiO2 powders were uniaxially pressed and sintered at 1200 °C, 1300 °C, 1400 °C or 1500 °C for one hour in a furnace. The microstructural analysis consisted of X-ray diffraction and scanning electron microscopy. The density was measured by the Archimedes' principle and the flexural strength was obtained by the biaxial flexure test. The optical properties were measured using a spectrophotometer operating in the visible light wavelength range. The step-stress accelerated life testing was performed by the pneumatic mechanical cycler and the biological behavior achieved by using osteoblast-like cells (Osteo-1 cell line). RESULTS: Tetragonal zirconia was identified in all groups and cubic zirconia was identified only at 3Y-TZP group. The addition of TiO2 decreased the values of density and flexural strength of the composite 3Y-TZP/TiO2 in relation to 3Y-TZP regardless of the sintering temperature. The color difference between the two materials was not significant regarding L*a*b* parameters. The composite showed higher probability of failure, and induced higher proliferation and differentiation than control. SIGNIFICANCE: The composite developed have good aesthetic and biologics properties. However, its microstructure and mechanical properties need to be improved for future dental implant applications.