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1.
Aims:  Some fungi present on the surface of grapes may have a negative effect on the quality of wine. The aim of this study was to evaluate PCR-denaturing gradient gel electrophoresis (PCR-DGGE), for the establishment of fungal community profiles from grapes, in order to monitor fungi potentially involved in wine defects.
Methods and Results:  A fragment of the β-tubulin gene was amplified from filamentous fungi and yeasts described from grapes and analysed using two different denaturing gradient gels to constitute a reference database. The use of β-tubulin sequences instead of ITS rDNA in PCR-DGGE showed a progress in the discrimination of these fungal species but comigration problems were still observed. The technique was then applied on grape samples. The profiles counted up to 10 bands of which half corresponded to species which were not recorded in the reference database.
Conclusion:  PCR-DGGE represents a useful tool to compare environmental samples for the study of the dynamics of fungal communities, but comigrations represent a limit in its use to describe the species present.
Significance and Impact of the Study:  A better knowledge of the fungal diversity on grapes, particularly species responsible for wine defect, is necessary to develop accurate molecular detection tools.  相似文献   

2.
AIMS: The mycoflora of healthy grapes (i.e. without visible symptoms of rot) for wine production in Portuguese wine-making regions was assessed and its potential for ochratoxin A (OTA) production evaluated. The OTA content of grapes was also determined. METHODS AND RESULTS: A total of 386 fungal strains were isolated by plating methods. The most frequent genera found in grapes were non-ochratoxigenic species: Cladosporium (28%), Penicillium (24%), Botrytis (13%) and Aspergillus (9%). Two OTA-producing strains were isolated, belonging to the species Aspergillus carbonarius and Aspergillus ochraceus. OTA was detected in three of four grape samples, up to 116 ng l(-1). CONCLUSIONS: OTA is being produced in healthy berries by Aspergillus species, namely A. carbonarius, at levels below the maximum recommended limit of 2,000 ng l(-1) in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The OTA concentration detected in healthy Portuguese grapes does not represent a risk to wine regarding the legal limit established.  相似文献   

3.
Aims: To describe a new molecular technique for the assessment of fungal diversity in the air. Methods and Results: Air samples were collected every week in a henhouse in France during a 15‐week period. After air sampling, the collecting membrane was diluted, and the liquid was used for subsequent cultivation and molecular analysis: PCR‐temperature temporal gradient electrophoresis (TTGE), which has already been used for the identification of fungal species in air samples and PCR‐denaturing high‐performance liquid chromatography (D‐HPLC), a new technique for the analysis of complex microbial populations. D‐HPLC profiles were reproducible from run‐to‐run, and several fungal organisms could be identified at the species level by sequencing. Conclusions: PCR‐D‐HPLC enabled the identification of fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The new technique allowed the detection of more fungal species than did the PCR‐TTGE technique. However, some fungal species were detected only by PCR‐TTGE, suggesting that PCR‐D‐HPLC and PCR‐TTGE are complementary. Significance and Impact of the Study: PCR‐D‐HPLC represents a considerable saving in time over currently available procedures for detection and identification of fungal organisms in air. However, the fungal diversity detected by PCR‐D‐HPLC or by PCR‐TTGE was lower than that revealed by culture.  相似文献   

4.
Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.  相似文献   

5.
AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.  相似文献   

6.
Mycotoxin production from fungi isolated from grapes   总被引:6,自引:0,他引:6  
AIMS: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. METHODS AND RESULTS: The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. CONCLUSION: Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.  相似文献   

7.
Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and STAPHYLOCOCCUS: The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.  相似文献   

8.
AIMS: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. METHODS AND RESULTS: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8-357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. CONCLUSION: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.  相似文献   

9.
AIMS: To explain the dominance of OTA-producing fungal species in sun-dried grapes for special wine production through an ecophysiological approach. METHODS AND RESULTS: Grapes at different ripening stages, sun-dried grapes and raisins were analysed for fungal presence, and isolates identified. Aspergillus section Nigri incidence in grapes increased with grape maturation. In the ecophysiological study five isolates (Alternaria alternata, Cladosporium herbarum, Penicillium decumbens, Aspergillus carbonarius, A. niger aggregate and A. section Nigri uniseriate) were inoculated in SNM medium at four a(w) (0.82-0.97) and incubated at 20, 30 and 40 degrees C for 18 d. Isolates were also inoculated in pairs to evaluate fungal interactions recording their growth rates and indexes of dominance. Aspergillus section Nigri grew in a wider range of temperature and a(w), had higher growth rates than the others under most of the conditions tested and showed behaviour usually dominant. CONCLUSIONS: The presence of A. section Nigri is predominant in grapes during harvesting and sun-drying period likely because of a better adaptation to hot and humid environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of the drying period should be reduced as much as possible without compromising the quality of the final product, or drying the grapes in controlled chambers with dry hot air flow.  相似文献   

10.
AIMS: To investigate the influence of a specific ecological niche, the wine grape, on the survival and development of Saccharomyces cerevisiae. METHODS AND RESULTS: A strain with a rare phenotype was sprayed onto the grape surfaces and monitored through two vintages using a specific indicative medium and analysing the internal transcribed spacer regions in the 5.8S rDNA. During the ripening process, there was a progressive colonization of the surface of the undamaged and damaged grapes by epiphytic yeasts, up to the time of harvest. The damaged wine grapes showed a much greater epiphytic yeast population. However, the inoculated S. cerevisiae strain showed a scarce persistence on both undamaged and damaged wine grapes, and the damaged grapes did not appear to improve the grape surface colonization of this strain. CONCLUSIONS: Results indicated that wine grape is not a favourable ecological niche for the development and colonization of S. cerevisiae species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this work are further evidence that S. cerevisiae is not specifically associated with natural environments such as damaged and undamaged wine grapes.  相似文献   

11.
AIMS: The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. METHODS AND RESULTS: Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5.5 and 3.5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. CONCLUSIONS: The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.  相似文献   

12.
AIMS: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must. METHODS AND RESULTS: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases beta-glucanases, lichenases, beta-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clarified grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except beta-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species. CONCLUSION: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.  相似文献   

13.
Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.  相似文献   

14.
几种主要分子生物学技术在真菌生态学研究中的应用   总被引:3,自引:0,他引:3  
李娟 《菌物研究》2005,3(1):58-62
真菌在自然界的物质循环与降解等生态过程中发挥着重要的作用,是生态系统的重要组成部分。然而约90%的真菌种类仍然未知,且大部分难于分离和培养。因此核酸杂交;核酸序列分析;DNA指纹分析等分子生物学技术被用于真菌分类、鉴定、种群结构、群落多样性研究。本文综述了这几种主要分子生物学技术的基本原理及其在真菌生态学研究中的应用现状。  相似文献   

15.
New PCR-based methods for yeast identification   总被引:2,自引:0,他引:2  
AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.  相似文献   

16.
赵鹏  段维军  刘芳  周欣  范国梅  马紫英  蔡磊 《微生物学报》2021,61(12):3806-3819
近年来,随着我国对外贸易的不断增长,口岸检测样品量巨大,外来检疫性有害生物尤其是病原菌物传入我国的风险日趋增加,正成为我国国门生物安全的重要威胁。加强外来入侵菌物的防御能力建设,有效防范其造成的生物安全威胁迫在眉睫。由于外来入侵菌物各类群的基础研究薄弱,标准参比物质缺乏、基础数据和可检索数据库缺失,大部分物种缺乏准确、高效的鉴定手段,使得现有口岸菌物检疫存在准确性较低、速度较慢、误检漏检率较高等问题。针对上述问题,以我国进境检疫对象及主要进口农林作物上的高频检出但鉴定困难、误检率高的菌物类群为对象,建立了其标准参比物质库、形态特征信息库、多基因序列数据库,并通过整合多个信息库资源实现从检疫样品中初筛到物种精准鉴定的多模块服务平台www.casbrc.org/pqfungi,并开放共享。该数据库平台的应用有望促进我国口岸检疫部门的检测便利化水平大幅提升,在“智能海关”建设、维护国门生物安全及促进农林产品安全贸易中发挥重要作用。  相似文献   

17.
Fungi are a megadiverse group of organisms, they play major roles in ecosystem functioning and are important for human health, food production and nature conservation. Our knowledge on fungal diversity and fungal ecology is however still very limited, in part because surveying and identifying fungi is time demanding and requires expert knowledge. We present a method that allows anyone to generate a list of fungal species likely to occur in a region of interest, with minimal effort and without requiring taxonomical expertise. The method consists of using a cyclone sampler to acquire fungal spores directly from the air to an Eppendorf tube, and applying DNA barcoding with probabilistic species identification to generate a list of species from the sample. We tested the feasibility of the method by acquiring replicate air samples from different geographical regions within Finland. Our results show that air sampling is adequate for regional‐level surveys, with samples collected >100 km apart varying but samples collected <10 km apart not varying in their species composition. The data show marked phenology, and thus obtaining a representative species list requires aerial sampling that covers the entire fruiting season. In sum, aerial sampling combined with probabilistic molecular species identification offers a highly effective method for generating a species list of air‐dispersing fungi. The method presented here has the potential to revolutionize fungal surveys, as it provides a highly cost‐efficient way to include fungi as a part of large‐scale biodiversity assessments and monitoring programs.  相似文献   

18.
Most of fungal biodiversity on Earth remains unknown especially in the unexplored habitats. In this study, we compared fungi associated with grass (Poaceae) roots from two ecosystems: the temperate pine barrens in New Jersey, USA and tropical rain forests in Yunnan, China, using the same sampling, isolation and species identification methods. A total of 426 fungal isolates were obtained from 1600 root segments from 80 grass samples. Based on the internal transcribed spacer (ITS) sequences and morphological characteristics, a total of 85 fungal species (OTUs) belonging in 45 genera, 23 families, 16 orders, and 6 classes were identified, among which the pine barrens had 38 and Yunnan had 56 species, with only 9 species in common. The finding that grass roots in the tropical forests harbor higher fungal species diversity supports that tropical forests are fungal biodiversity hotspots. Sordariomycetes was dominant in both places but more Leotiomycetes were found in the pine barrens than Yunnan, which may play a role in the acidic and oligotrophic pine barrens ecosystem. Equal number of undescribed fungal species were discovered from the two sampled ecosystems, although the tropical Yunnan had more known fungal species. Pine barrens is a unique, unexplored ecosystem. Our finding suggests that sampling plants in such unexplored habitats will uncover novel fungi and that grass roots in pine barrens are one of the major reservoirs of novel fungi with about 47% being undescribed species.  相似文献   

19.
Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of approximately 150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.  相似文献   

20.
AIMS: To examine how UVC affects the different genera of fungi commonly isolated from grapes, with the aim of understanding changes in mycobiota during grape ripening and possible applications for preventing grape decay during storage. METHODS AND RESULTS: Spores of Aspergillus carbonarius, Aspergillus niger, Cladosporium herbarum, Penicillium janthinellum and Alternaria alternata (between 100-250 spores/plate agar) were UVC irradiated for 0 (control), 10, 20, 30, 60, 300 and 600 s. Plates were incubated at 25 degrees C and colonies were counted daily up to 7 days. Alternaria alternata and Aspergillus carbonarius were the most resistant fungi. Conidial germination in these species was reduced by approx. 25% after 10 s of exposure, compared with greater than 70% reduction for the remaining species tested. Penicillium janthinellum spores were the most susceptible at this wavelength. UVC exposures of 300 s prevented growth of all isolates studied, except for Alternaria alternata. CONCLUSIONS: UVC irradiation plays a major role in selecting for particular fungi that dominate the mycobiota of drying grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The UVC irradiation of harvested grapes could prevent germination of contaminant fungi during storage or further dehydration.  相似文献   

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