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Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting
Abstract
We have used the techniques of DNA fingerprinting and polymerase chain reaction (PCR) with probes specific for hypervariable repetitive DNA sequences (mini‐ and microsatellite DNAs) to analyze 36 yeast strains belonging to 10 species and 2 genera. Using (GTG) 5 , (GACA) 4 , phage M13 DNA and the M13 sequence GAGGGTGGCGGTTCT as probes and primers, respectively, we obtained DNA polymorphisms which allowed us to discriminate 23 biotechnologically important strains of the yeast Saccaromyces cerevisiae and to distinguish them from strains of S. pastorianus, S. bayanus and S. willianus .
Our results demonstrate that both DNA and PCR fingerprinting are suitable tools for an easy, fast and reliable molecular typing of yeasts. The DNA fingerprinting method seems to be more sensitive than PCR fingerprinting with respect to the individualization of strains. Nevertheless, using the PCR fingerprinting technique we were able to unambigously dicriminate between yeast genotypes of different species. Therefore, PCR fingerprinting might become a useful tool in the classification of yeasts on the basis of phylogenetic relatedness.
... The primer (GTG) 5 is used for the amplification of microsatellite regions, as described in [21]. This PCR is used to group fungi that have similar morphotypes, while considering coloration, border shape, filament type, and texture. ...
... The primers BT2a and BT2b are used for partial amplification of the β-tubulin gene as well as for the rapid identification of fungal species by PCR [37]. 21. The sequences of the elongation factor 1α have been used for phylogenetic analysis. ...
Plants harbor a large reservoir of fungal diversity, encompassing endophytic, epiphytic, phytopathogenic, and rhizosphere-associated fungi. Despite this diversity, relatively few fungal species have been characterized as sources of bioactive secondary metabolites. The role of secondary metabolites is still not fully understood; however, it is suggested that these metabolites play important roles in defense mechanisms and fungal interactions with other organisms. Hence, fungal secondary metabolites have potential biotechnological applications as prototype molecules for the development of therapeutic drugs. In this chapter, we describe the main methods used for routine fungi isolation, production of crude fungal extracts, and chemical characterization of bioactive compounds. In addition, explicative notes about the steps described are provided to explore the diversity of the endophytic, phytopathogenic, epiphytic, and rhizosphere fungi and to evaluate the biotechnological potential of each group.
... Oui Oui Méthode moléculaire Fellgett, 1951Holland et al ., 1996 Non Oui IR-TF MADI-TOF-MS mt-RFLP Non Liu et al ., 1997 T-RFLP Caryotypage RISA DGGE CE-SSCP Lieckfeldt et al ., 1993 MSP-PCR ISS-PCR ERIC-PCR Sharples et Lloyd, 1990de Barros Lopes et al ., 1996 Microsatellites Ness et al ., 1993-Legras et Karst, 2003Field et Wills, 1998 RAPD Williams et al ., 1990 PCR-Interdelta Dubourdieu et al ., 1987Carle et Olson, 1985Stern et al ., 1984Vos et al ., 1995Guillamón et al ., 1998Fisher et Triplett, 1999Fischer et Lerman, 1983 Niveau d'identification déductif après analyse (Naumann et al., 1991). L'absorption des différents composants cellulaires (acides gras, protéines, peptides, glucides et composés phosphatés) génère un spectre unique qui ensuite sera comparé avec une base de données. ...
... La MSP-PCR (Microsatellite/minisatellite Primed) est une méthode basée sur l'amplification de régions du génome à l'aide de séquences « microsatellites ou minisatellites », comme par exemple le primer (GTG)5. A l'inverse des analyses de microsatellites ou de minisatellites, cette méthode analyse les régions localisées entre les cibles d'hybridation (Lieckfeldt et al., 1993) (Ramírez-Castrillón et al., 2014). (Kish et al., 1983 ;Heard et Fleet, 1986) ; levures non-Saccharomyces : milieu Lysine agar (Heard et Fleet, 1986) ; bactéries lactiques : milieu MRS (deMan, Rogosa, Sharpe) (de Man et al., 1960), milieu Lac (Bae et al., 2006 ;Simonin et al., 2018) D'autres méthodes peuvent être utilisées pour quantifier les microorganismes présents dans un échantillon, telles que la cytométrie en flux (Malacrinò et al., 2001) ou les techniques RT-q-PCR (Real Time quantitative-PCR) (Hierro et al., 2006) Granchi et al., 1999 ;Nisiotou et al., 2007 ;Wang et al., 2015 ;Simonin et al., 2018). ...
La biodiversité fongique interspécifique (Illumina Mi-Seq) et la dynamique des espèces Saccharomyces cerevisiae et Brettanomyces bruxellensis ont été étudiées au sein d’une nouvelle cuverie et/ou dans 3 caves d’élevage, plus particulièrement sur le sol, les murs, le matériel vinaire et l’extérieur des fûts. Dans la nouvelle cuverie, un consortium fongique (levures et moisissures) de départ est déjà présent sur tous les environnments étudiés avant l’arrivée de la première vendange. Ce consortium est constitué de genres tels que Aureobasidium, Alternaria, Didymella et Filobasidium. Ces genres qui persistent pendant deux millésimes, ne sont pas spécifiques de l’environnement de la cuverie et semblent être adaptés à tous les environnements naturels ou anthropiques au regard de leur caractère ubiquiste. Le consortium de départ est enrichi par des genres œnologiques (exemple : Hanseniaspora, Saccharomyces) qui sont introduits dans la cuverie soit par les vendanges, soit par des transferts potentiels entre les différents environnements de la cuverie. Cependant, ces genres ne semblent pas persister ou s’implanter probablement dû à leur faible adaptation aux conditions stressantes de l’environnement de la cuverie. La dynamique de la flore indigène S. cerevisiae dans la nouvelle cuverie a été également étudiée. Aucun isolat appartenant à cette espèce n’a été retrouvé avant l'arrivée de la première vendange confirmant que cette espèce n’est pas spécifique de l'environnement de la cuverie et que sa présence est en lien avec l'activité des fermentations alcooliques. Cependant, les résultats obtenus suggèrent une colonisation potentielle de l’environnement de la nouvelle cuverie par certaines souches de S. cerevisiae. Ces souches dites « colonisatrices » ont présenté une capacité plus élevée à former des biofilms comparée à celle de souches non implantées. Cette étude met en évidence l’importance de l’environnement de la cuverie qui constitue une véritable niche écologique pour les populations fongiques capables de s’implanter au cours du processus de vinification. Dans l’environnement des 3 caves d’élevage, le matériel vinaire et l’extérieur des fûts (en contact direct avec le vin) sont les environnements qui semblent favorables au développement et à l’installation des populations microbiennes cultivables (levures totales et bactéries lactiques) et des microorganismes d’altération (bactéries acétiques et B. bruxellensis), contrairement au sol et aux murs où des populations faibles ont été trouvées. Des souches récurrentes de B. bruxellensis ont été retrouvées sur le matériel et sur l’extérieur des fûts et pourraient être à l’origine de la contamination de vins au cours de l’élevage. Ces souches récurrentes présentent des capacités de formation de biofilms et de résistance plus importantes qui pourraient expliquer la persistance de B. bruxellensis dans des caves d’élevage. Ces résultats soulignent l’importance du nettoyage du matériel vinaire et du suivi microbien régulier des vins au cours de l’élevage afin de limiter les contaminations.
... Th e number and position of the delta elements are diff erent for each yeast strain, allowing discrimination (Ness et al., 1993). PCR fi ngerprinting with primer M13 is a method that has already long been used for the diff erentiation of yeasts (Lieckfeldt et al., 1993;Andrighett o et al., 2000;Urso et al., 2008). Th e primer corresponds to the nuclear sequence of the phage M13 (Huey and Hall, 1989), which is specifi c to minisatellite DNA sequences in the yeast genome (Lieckfeldt et al., 1993). ...
... PCR fi ngerprinting with primer M13 is a method that has already long been used for the diff erentiation of yeasts (Lieckfeldt et al., 1993;Andrighett o et al., 2000;Urso et al., 2008). Th e primer corresponds to the nuclear sequence of the phage M13 (Huey and Hall, 1989), which is specifi c to minisatellite DNA sequences in the yeast genome (Lieckfeldt et al., 1993). Th e use of yeasts to underline the regional typicity plays an increasing role in wine and apple/pear must production. ...
In recent years an increasing trend towards terroir-emphasizing yeasts has emerged, and with that a need to distinguish local strains from commercial available wine yeasts. Therefore, in the present study, a database was set up with the genetic profi les of commercial wine yeasts used in Austria. This is an important tool for the selection and verifi cation of autochthonous wine yeasts from vineyards, wineries or must productions. Three diff erent molecular markers, namely microsatellites, interdelta analysis and M13 fingerprinting, were tested for their ability to differentiate 75 commercial wine yeast strains in a single approach and in combination. It turned out that at least two of the methods should be used for a meaningful comparison and distinct discrimination. Four autochthonous yeasts could be differentiated from their commercially propagated relatives. If a quick initial assessment has to be done, then microsatellite analysis is certainly the favored method.
Differenzierung von kommerziellen Weinhefestämmen mittels molekularer Marker.
In den letzten Jahren hat sich ein zunehmender Trend zu terroir-betonten Hefen herausgebildet, mit dem die Notwendigkeit verbunden ist, lokale Stämme von kommerziell erhältlichen Weinhefen zu unterscheiden. In der vorliegenden Studie wurde daher eine Datenbank mit den genetischen Profilen der in Österreich verwendeten kommerziellen Weinhefen erstellt. Dies ist ein wichtiges Instrument für die Auswahl und Überprüfung von autochthonen Weinhefen aus Weinbergen, Weingütern oder Mostproduktionen. Drei verschiedene molekulare Marker, nämlich Mikrosatelliten, Interdelta-Analyse und M13-Fingerprinting, wurden auf ihre Fähigkeit getestet, 75 kommerzielle Weinhefestämme in einem einzigen Ansatz und in Kombination zu unterscheiden. Es stellte sich heraus, dass mindestens zwei der Methoden für einen aussagekräft igen Vergleich und eine eindeutige Unterscheidung verwendet werden sollten. Vier autochthone Hefen konnten von ihren kommerziell vermehrten Verwandten unterschieden werden. Wenn eine schnelle Ersteinschätzung durchgeführt werden soll, ist die Mikrosatellitenanalyse mit Sicherheit die zu bevorzugende Methode.
... Tsuchiya et al (1994) have 316 combined the specific PCR with temperature gradient gel electrophoresis to identify lactic acid bacteria. RAPD-PCR technology can also be used for the identification of non-brewing yeast without changes to the procedure (Lieckfeldt et al 1993). ...
... The major advantage of using RAPD-PCR technology is that a pure single colony can be identified by its fingerprint pattern in less than one day. In the fermentation industry, this procedure has been useful for the characterization of different isolates of yeast (Lieckfeldt et al 1993 andGrando et al 1994), the identification of Obesumbacterium proteus (Savard et al 1994), the investigation of barley variants (Ko and Henry, 1994), and the identification of hopleaf tissue (Abbott and Fedele, 1994). Obesumbacterium proteus has also been differentiated using a PCR technique called enteric repetitive intergenic consensus-PCR (ERIC-PCR) (Prest et al 1994). ...
... The supernatant was collected and used for PCR, using an Applied Biosystems thermal cycler, with M13 (5′GAGGGTGGCGGTTCT3′) and (GTG)5 (5′GTGGTGGTGGTGGTG3′) primers. The amplification conditions and sequences for M13 and (GTG)5 are those described previously by Huey and Hall and Lieckfeldt et al. [41,42], respectively. The fingerprinting profiles were compared by the Pearson's correlation coefficient and unweighted pair group with arithmetical average (UPGMA) as a ...
... The supernatant was collected and used for PCR, using an Applied Biosystems thermal cycler, with M13 (5 GAGGGTGGCGGTTCT3 ) and (GTG) 5 (5 GTGGTGGTGGTGGTG3 ) primers. The amplification conditions and sequences for M13 and (GTG) 5 are those described previously by Huey and Hall and Lieckfeldt et al. [41,42], respectively. The fingerprinting profiles were compared by the Pearson's correlation coefficient and unweighted pair group with arithmetical average (UPGMA) as a clustering method using the BioNumerics software (version 5.0, Applied Maths, Sint-Martens-Laterat, Belgium). ...
Some non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have been proposed as selected starters due to their contribution for the overall aroma and chemical profiles of wines. In this work, we aimed to evaluate the genetic and phenotypic diversity of Metschnikowia pulcherrima strains isolated from different locations of Douro Wine Region, and to explore their potential as co-adjuncts of S. cerevisiae in alcoholic fermentation. For that purpose, a set of 64 M. pulcherrima isolates were used. Polymerase chain reaction (PCR) fingerprinting with M13 primers demonstrated to be an efficient tool in intraspecific discrimination of M. pulcherrima strains. No significant associations were found between genotypic profiles and either geographical origin or winery. The isolates were screened for their stress resistance ability (ethanol, SO2, chitosan, copper, H2O2, and Grape Juice Medium), aroma-related activities (resistance to 5, 5′, 5′′-trifluor-d, l-leucine and cerulenin and β-glycosidase, β-lyase and sulfite-reductase activities) as well as other relevant technological proprieties (protease activity and biogenic amines production). M. pulcherrima response to the different enological traits evaluated was greatly strain-dependent. The most discriminant features were the ability of the strains to grow in Grape-Juice Medium (GJM) and sulfite-reductase, and their β-lyase and protease activities. The enological potential of a selected M. pulcherrima strain in mixed-culture with S. cerevisiae was also assessed in natural grape-juice of a local variety, under two nitrogen regimes. M. pulcherrima proved to be promising for future industrial application as a co-starter, lowering ethanol, acetic acid and, reported here for the first time, lowering hydrogen sulfide levels in the wines.
... Total DNA extraction followed the protocol of Rosa et al. (2009). Fungi were grouped according to the morphotypes of their colonies and the banding patterns of microsatellite regions amplified via fingerprinting (PCR-MST) using the oligonucleotide (GTG) 5 (Lieckfeldt et al. 1993). For filamentous fungi, the internal transcribed Fig. 1 The location of Boeckella Lake, Hope Bay, north-eastern Antarctic Peninsula. ...
This study characterized cultivable fungi present in sediments obtained from Boeckella Lake, Hope Bay, in the north-east of the Antarctic Peninsula, and evaluated their production of enzymes and biosurfactants of potential industrial interest. A total of 116 fungal isolates were obtained, which were classified into 16 genera within the phyla Ascomycota, Basidiomycota and Mortierellomycota, in rank. The most abundant genera of filamentous fungi included Pseudogymnoascus, Pseudeurotium and Antarctomyces; for yeasts, Thelebolales and Naganishia taxa were dominant. Overall, the lake sediments exhibited high fungal diversity and moderate richness and dominance. The enzymes esterase, cellulase and protease were the most abundantly produced by these fungi. Ramgea cf. ozimecii, Holtermanniella wattica, Leucosporidium creatinivorum, Leucosporidium sp., Mrakia blollopis, Naganishia sp. and Phenoliferia sp. displayed enzymatic index > 2. Fourteen isolates of filamentous fungi demonstrated an Emulsification Index 24% (EI24%) ≥ 50%; among them, three isolates of A. psychrotrophicus showed an EI24% > 80%. Boeckella Lake itself is in the process of drying out due to the impact of regional climate change, and may be lost completely in approaching decades, therefore hosts a threatened community of cultivable fungi that produce important biomolecules with potential application in biotechnological processes.
... Total DNA extraction followed the protocol of Rosa et al. (2009). Fungi were grouped according to the morphotypes of their colonies and the banding patterns of microsatellite regions amplified via fingerprinting (PCR-MST) using the oligonucleotide (GTG) 5 (Lieckfeldt et al. 1993). For filamentous fungi, the internal transcribed spacer region (ITS1-5.8S-ITS2) of the ribosomal DNA gene was amplified using the primers ITS1 and ITS4 (White et al. 1990), along with the β-tubulin genes, using BT2a and BT2b primers (Glass and Donaldson 1995), and RNA polymerase II using primers RBP2 5F and RPB2 7R (Malkus et al. 2006). ...
We studied the culturable fungal community recovered from deep marine sediments in the maritime Antarctic, and assessed their capabilities to produce exoenzymes, emulsifiers and metabolites with phytotoxic activity. Sixty-eight Ascomycota fungal isolates were recovered and identified. The most abundant taxon recovered was the yeast Meyerozyma guilliermondii, followed by the filamentous fungi Penicillium chrysogenum, P. cf. palitans, Pseudeurotium cf. bakeri, Thelebolus balaustiformis, Antarctomyces psychrotrophicus and Cladosporium sp. Diversity indices displayed low values overall, with the highest values obtained at shallow depth, decreasing to the deepest location sampled. Only M. guilliermondii and P. cf. palitans were detected in the sediments at all depths sampled, and were the most abundant taxa at all sample sites. The most abundant enzymes detected were proteases, followed by invertases, cellulases, lipases, carrageenases, agarases, pectinases and esterases. Four isolates showed good biosurfactant activity, particularly the endemic species A. psychrotrophicus. Twenty-four isolates of P. cf. palitans displayed strong phytotoxic activities against the models Lactuca sativa and Allium schoenoprasum. The cultivable fungi recovered demonstrated good biosynthetic activity in the production of hydrolytic exoenzymes, biosurfactant molecules and metabolites with phytotoxic activity, reinforcing the importance of documenting the taxonomic, ecological and biotechnological properties of fungi present in deep oceanic sediments of the Southern Ocean.
... RAPD-PCR analysis was initially used for clustering the isolates, employing the primer M13 (5′-GAGGGTGGCGGTTCT-3′), according to the protocol of Lieckfeldt et al. (1993). PCR amplification was conducted in 20 μL final reaction volumes, containing 5 μL of One Taq Quick-Load Reaction Buffer (New England Biolabs, USA), 1 U of One Taq Quick-Load DNA Polymerase (New England Biolabs, USA), 100 μM of dNTP's (10 mM), 10 μM of M13 oligonucleotide primer and 20 ng of template DNA. ...
The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H 2 S, β-glucosidase production and resistance to the antimicrobial agent; SO 2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta-PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed. CITATION Tzamourani AP, Taliadouros V, Paraskevopoulos I and Dimopoulou M (2023) Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.
... For assessment of the global distribution, ITS barcode sequences of the four isolates were used in a BLAST search in the GlobalFungi Database (Větrovský et al. 2020). For genetic fingerprinting of the isolates, microsatellite-primed PCRs (MSP-PCR) were conducted using the primers (ATG) 5 , (GTG) 5 (Lieckfeldt et al. 1993;Vuyst et al. 2008), and (GAC) 5 (Baleiras Couto et al. 1996). The amplicon fingerprints of the different samples were compared by agarose gel electrophoresis with standardized conditions. ...
Heat resistance is the ability to survive short, extreme temperature stresses, exceeding the own growth temperature by far. Despite their occurrence in natural substrates and their relevance for the food and healthcare industry, the diversity of fungi with heat resistance abilities is poorly studied. Sampling of boreal forest soils in Canada in combination with a heat-shock treatment (75 °C, 30 min) yielded, among others, four heat resistant, mesophilic fungal isolates. Based on rDNA barcode sequences, the novel isolates were assigned to Basidiomycota . In this study, we use macromorphological and micromorphological observations, cultivation assays and comparative genomics for physiological characterization, interspecific differentiation, and phylogenetic placement of these isolates. A phylogeny of 38 single-copy orthologous genes, an orthology analysis, and septal pore type analysis revealed the isolates as representatives of two new basidiomycetous species of the novel class Peribolosporomycetes , a sister lineage to all other members of Ustilaginomycotina . Further genomic and phenotypic data support two different species ( Peribolospora kevripleyi , Peribolospora baueri ), which are heat resistant and osmotolerant.
... Rep-PCR with primer (GTG) 5 was employed according to the protocol of [24], modified by [25,26]. PCR amplification was conducted in a Thermal Cycler 2720 (Applied Biosystems, Foster City, CA, USA) with the following program for bacterial DNA: 5 min of denaturation at 94 • C, 30 cycles of 1 min at 95 • C, 1 min at 40 • C, and 8 min at 72 • C, then 16 min at 72 • C for the final elongation. ...
Kombucha is a fermented tea with a long history of production and consumption. It has been gaining popularity thanks to its refreshing taste and assumed beneficial properties. The microbial community responsible for tea fermentation—acetic acid bacteria (AAB), yeasts, and lactic acid bacteria (LAB)—is mainly found embedded in an extracellular cellulosic matrix located at the liquid–air interphase. To optimize the production process and investigate the contribution of individual strains, a collection of 26 unique strains was established from an artisanal-scale kombucha production; it included 13 AAB, 12 yeasts, and one LAB. Among these, distinctive strains, namely Novacetimonas hansenii T7SS-4G1, Brettanomyces bruxellensis T7SB-5W6, and Zygosaccharomyces parabailii T7SS-4W1, were used in mono- and co-culture fermentations. The monocultures highlighted important species-specific differences in the metabolism of sugars and organic acids, while binary co-cultures demonstrated the roles played by bacteria and yeasts in the production of cellulose and typical volatile acidity. Aroma complexity and sensory perception were comparable between reconstructed (with the three strains) and native microbial consortia. This study provided a broad picture of the strains’ metabolic signatures, facilitating the standardization of kombucha production in order to obtain a product with desired characteristics by modulating strains presence or abundance.
... The advancement in the understanding of the genetic information of microorganisms has been fundamental for the development of molecular diagnosis techniques of pathogenic fungi. These techniques include PCR -restriction fragment length polymorphism analysis (Dendis et al., 2003), random amplified polymorphic DNA analysis (Brandt et al., 1998;Mobasherizadeh et al., 2016), hybridization with genus/species specific DNA or RNA probes (Sandhu et al., 1995;Lindsley et al., 2001), PCR-fingerprinting (Lieckfeldt et al., 1993), species-specific PCR assays (Martin et al., 2000;Kulik et al., 2004), real-time PCR (Klingspor and Jalal, 2006), and increasingly DNA sequencing (Pryce et al., 2003;Romanelli et al., 2010). DNA sequencing introduced by Sanger et al. (1977) dominated the sequencing technology for 20 years, being recognized as firstgeneration sequencing. ...
Identification of the causative infectious agent is essential in the management of infectious diseases, with the ideal diagnostic method being rapid, accurate, and informative, while remaining cost-effective. Traditional diagnostic techniques rely on culturing and cell propagation to isolate and identify the causative pathogen. These techniques are limited by the ability and the time required to grow or propagate an agent in vitro and the facts that identification based on morphological traits are non-specific, insensitive, and reliant on technical expertise. The evolution of next-generation sequencing has revolutionized genomic studies to generate more data at a cheaper cost. These are divided into short- and long-read sequencing technologies, depending on the length of reads generated during sequencing runs. Long-read sequencing also called third-generation sequencing emerged commercially through the instruments released by Pacific Biosciences and Oxford Nanopore Technologies, although relying on different sequencing chemistries, with the first one being more accurate both platforms can generate ultra-long sequence reads. Long-read sequencing is capable of entirely spanning previously established genomic identification regions or potentially small whole genomes, drastically improving the accuracy of the identification of pathogens directly from clinical samples. Long-read sequencing may also provide additional important clinical information, such as antimicrobial resistance profiles and epidemiological data from a single sequencing run. While initial applications of long-read sequencing in clinical diagnosis showed that it could be a promising diagnostic technique, it also has highlighted the need for further optimization. In this review, we show the potential long-read sequencing has in clinical diagnosis of fungal infections and discuss the pros and cons of its implementation.
... The SSR microsatellite markers were first described in plants by Condit & Hubbell [43], who detected the repeats (AC)n and (AG)n. The application of microsatellites as PCR primers was first described by Lieckfeldt et al. [44] and Meyer et al. [45]. The genome of eukaryotic species is densely populated by different classes, with repeated sequences and little complexity. ...
... Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was carried out for fingerprinting the isolates by random M-13 primer previously described with the sequence of 5`-GAGGGTGGCGGTTCT -3` [17]. The thermocycler device (MJ mini BioRad, USA) program for this method was as follows: initial denaturation was first performed at 94°C for 3 minutes, then 35 cycles were performed at a denaturation temperature of 94°C for 1 minute, 45°C annealing temperature for 45 seconds, and extension was carried out at 72°C for 1 minute. ...
The present study was conducted to compare the S. aureus isolates from different sources in the basis of resistance phenotypic and genotypic features and phylogenetic differences. Total of 70 S. aureus isolates (including 25 human, 25 raw milk and 20 pet animal isolates) were subjected to the antimicrobial susceptibility testing, polymerase chain reaction (PCR) detection of the resistance genes and DNA fingerprinting using random amplification of polymorphic DNA–PCR (RAPD-PCR) to survey the variability of the isolates. Among 70 S. aureus, 55 (78.5%) isolates were MRSA. The isolates showed the highest antibiotic resistance to methicillin, ampicillin and penicillin (78.5%) and showed the lowest resistance to ciprofloxacin (12.8%). ErmB and tetM resistance genes were present in all isolates and the vanA gene was not detected in any of the isolates. Thirteen distinct clusters were identified in RAPD-PCR fingerprinting. Statistical analysis showed that the isolates without resistance to antibiotics were significantly in associated with raw milk origin (P<0.05). According to the results of the study, S. aureus strains with pets and raw milk origin are significant sources of antibiotic-resistant isolates such as MRSA. They are also carriers of resistance genes that can be transmit to human isolates and cause drug resistance in human infections. Identifying the source of these infections is possible with a reliable genotyping method such as RAPD-PCR.
... Yeast DNA was then extracted according to protocols previously described [27]. Phenotypic groupings were confirmed by PCR fingerprinting using (GTG) 5 [28,29]. Yeast strains with identical PCR fingerprinting patterns were grouped and putatively considered to belong to the same species. ...
Honey bee colony losses worldwide call for a more in-depth understanding of the pathogenic and mutualistic components of the honey bee microbiota and their relation with the environment. In this descriptive study, we characterized the yeast and bacterial communities that arise from six substrates associated with honey bees: corbicular pollen, beebread, hive debris, intestinal contents, body surface of nurses and forager bees, comparing two different landscapes, Minas Gerais, Brazil and Maryland, United States. The sampling of five hives in Brazil and four in the USA yielded 217 yeast and 284 bacterial isolates. Whereas the yeast community, accounted for 47 species from 29 genera, was dominated in Brazil by Aureobasidium sp. and Candida orthopsilosis, the major yeast recovered from the USA was Debaryomyces hansenii. The bacterial community was more diverse, encompassing 65 species distributed across 31 genera. Overall, most isolates belonged to Firmicutes, genus Bacillus. Among LAB, species from Lactobacillus were the most prevalent. Cluster analysis evidenced high structuration of the microbial communities, with two distinguished microbial groups between Brazil and the United States. In general, the higher difference among sites and substrates were dependents on the turnover effect (~ 93% of the beta diversity), with a more pronounced effect of nestedness (~ 28%) observed from Brazil microbiota change. The relative abundance of yeasts and bacteria also showed the dissimilarity of the microbial communities between both environments. These results provide a comprehensive view of microorganisms associated with A. mellifera, highlighting the importance of the environment in the establishment of the microbiota associated with honey bees.
... Then, alternatively, molecular methods have been developed for typing microorganisms such as (GTG)5 allowing the differentiation inter-and intra-specific based on the amplification of regions between GTG and CAC microsatellites in a fast, safe and relatively easy way (da Silva-Filho et al., 2005;Illeghems et al., 2012;Englezos et al., 2015). It is based on the amplification of regions of high polymorphism for intraspecific characterization (Lieckfeldt et al., 1993), showing a great potential for the grouping of yeasts in processes of industrial dynamics of populations such as in wine productions (Englezos et al., 2015), sugar cane (da Silva-Filho et al., 2005;Basílio et al., 2008;Nova et al., 2009) and cocoa fermentation (Crafack et al., 2013; Brazilian Journal of Development, Curitiba, v.7, n.6, p. 60739-60759 jun. 2021 et al., Pereira et al., 2017) also being used in environmental yeasts (Silva-Bedoya et al., 2014). ...
Cocoa is a fruit of great economic importance, being the main raw material in the manufacture of chocolate. Among the stages of pre-processing, the main and most important is the spontaneous fermentation of the cocoa pulp by microorganisms, especially the yeasts, which initiate the process and contribute to the death of the germ of the seed, releasing compounds that directly influence the quality of the final product (flavor and aroma). Poorly fermented almonds confer bitter and astringent taste on chocolate, so it is advantageous to select autochthonous yeasts with better performance in the fermentation (producing enzymes of interest in the process) to be used as inoculum starter when added in the spontaneous fermentation, where they can accelerate the fermentation and contribute to raising the quality of the product. Therefore, the objective of this work was to qualitatively determine the production of enzymes of biotechnological interest by yeasts for the fermentation of cocoa through the cup plate method, in order to select a candidate yeast inoculum and use molecular typing technique to evaluate the diversity. Many promising yeasts were identified for use as inoculum among the diverse yeast groups found.
... This enabled a differentiation of morphologically similar isolates by obtaining a genetic fingerprint for distinct species, without a barcode sequencing of each culture (Supplementary Table S2). Therefore, the (GTG) 5 (Lieckfeldt et al. 1993;De Vuyst et al. 2008) primer was used in a PCR reaction containing 6.25 μl GoTaq G2 Hot Start Colorless Master Mix (Promega), 5.5 μl ddH 2 O, 0.25 μl (GTG) 5 (10 μM), and 0.5 μl DNA extract in a total volume of 12.5 μl. The PCR program started with an initial denaturation at 95°C (2 min), followed by 35 cycles of denaturation at 95°C (30 s), annealing at 55°C (40 s), and elongation at 72°C (90 s), and a final elongation at 72°C (7 min). ...
Thermophilic, thermotolerant and heat-resistant fungi developed different physiological traits, enabling them to sustain or even flourish under elevated temperatures, which are life-hostile for most other eukaryotes. With the growing demand of heat-stable molecules in biotechnology and industry, the awareness of heat-adapted fungi as a promising source of respective enzymes and biomolecules is still increasing. The aim of this study was to test two different strategies for the efficient isolation and identification of distinctly heat-adapted fungi from easily accessible substrates and locations. Eight compost piles and ten soil sites were sampled in combination with different culture-dependent approaches to describe suitable strategies for the isolation and selection of thermophilous fungi. Additionally, an approach with a heat-shock treatment, but without elevated temperature incubation led to the isolation of heat-resistant mesophilic species. The cultures were identified based on morphology, DNA barcodes, and microsatellite fingerprinting. In total, 191 obtained isolates were assigned to 31 fungal species, from which half are truly thermophilic or thermotolerant, while the other half are heat-resistant fungi. A numerous amount of heat-adapted fungi was isolated from both compost and soil samples, indicating the suitability of the used approaches and that the richness and availability of those organisms in such environments are substantially high.
... Filamentous fungal isolates were grouped into diferent morphospecies according to their characteristics of the culture: colony color and texture, border type, and radial growth rate on PDA agar (Fröhlich et al. 2000). Isolates with identical morphological characteristics were grouped together and subjected to PCR ingerprinting using the microsatelliteprimed PCR technique (GTG) 5 by Lieckfeldt et al. (1993). Based on the electrophoretic profile of PCR-amplified products with primer (GTG) 5 , an isolated among those who showed the same pattern of bands was selected for sequencing of the nuclear ribosomal RNA gene. ...
We characterized the diversity, distribution, systematic colonization, and xerophilic capabilities of fungi associated with the Antarctic angiosperms Colobanthus quitensis and Deschampsia antarctica collected at different sites of the South Shetlands Islands, Antarctic Peninsula. A total of 684 fungal isolates were obtained and identified into 67 taxa from 32 genera. The highest fungal diversity and richness were obtained from the rhizosphere, roots, and leaves, in order, and only 11 taxa shared between both plants. Penicillium and Pseudogymnoascus were the dominant fungal genera. However, the rarefaction curves for plant fungal assemblages did not reach a plateau, suggesting that these Antarctic plants may host more fungi in their tissues and rhizospheres. A total of 460 isolates grew at water activity (aw) = 0.95, 200 at 0.90, 110 at 0.81, and 47 at 0.66. Antarctomyces, Cladosporium, Mortierella, Leptosphaeria, Penicillium, Pseudogymnoascus, and Thelebolus taxa grew at aw = 0.81 and 0.66 and considered highly xerophilic. In addition, specific isolates of Penicillium and Thelebolus exhibited the highest mycelial growth at aw = 0.66. Our results show that the internal tissues and rhizosphere of Antarctic angiosperms host rich and diverse fungal communities dominated by cold-adapted and endemic taxa, which seem to coexist as symbionts and decomposer fungi. In addition, specific fungi are able to colonize different parts of the plant, suggesting a high ecological relationship with their hosts. Finally, different fungi living in the rhizosphere displayed remarkable xerophilic tolerance, representing promising candidates for further biotechnological studies, including identification of genes for applications in industry and agriculture.
... ease of pines, using the internal short sequence repeats-polymerase chain reaction (ISSR-PCR) technique (van der Nest et al . 2000;Burgess et al . 2001) . ISSR-PCR fragments from F. circinatum isolates MRC 7601 (Mexico) and MRC 7484 (South Africa) were produced using primers described previously by Buscot et al . (1996), Hantula et al . (1996) and Lieckfeldt et al . (1993). PCR (50 µ L) consisted of PCR buffer [10 m m Tris-HCl (pH 8.3), 1.5 m m MgCl 2 , 50 m m KCl], 50 µ m of each dNTP, 0.2 µ m primer, 0.5 U Taq DNA polymerase (Roche) and 1 µ L DNA (25 ng/ µ L). The reaction conditions were: 5 min at 94 ° C, followed by 35 cycles of 1 min at 94 ° C, 1 min at an appropriate annealing temperature (Tm) and 1 ...
Mango malformation is an economically important disease of Mangifera indica globally. A recent DNA-based study indicated that two distinct, phylogenetic lineages previously identified as Fusarium subglutinans are associated with this disease in South Africa. The objective of this study was to characterize Fusarium isolates associated with mango malformation, including the two different F. subglutinans groups, based on morphological characteristics. For this purpose we examined Fusarium strains isolated from diseased mango inflorescences from diverse geographical origins. We also used sexual compatibility tests to determine whether sexual reproduction among the strains was possible. The morphological characters considered were shape of the conidia, presence of mono- and/or polyphialides, origin of the conidiophores from the substrate, presence of chlamydospores and the presence of sterile coiled hyphae. Three unique Fusarium spp. were identified. In this paper, we provide formal descriptions for the two new taxa in the section Liseola that we have named F. mangiferae and F. sterilihyphosum. Fusarium mangiferae is conspecific with strains that were previously identified as F. subglutinans and reported to be the causal agent of malformation in mango growing areas throughout the world. Fusarium sterilihyphosum, on the other hand, has been isolated only from malformed mango tissue in South Africa.
... Following phylogenetic tree inspection, isolates that clustered in the same group and that derived from the same substrate were subjected to PCR-fingerprinting by using the micro-and mini-satellite primers (GTG)5 and M13 [31,32] to exclude duplicates from further analysis. DNA fingerprints were visualized with 1.5% agarose gel stained with 5 mL 100 mL −1 ethidium bromide while a GelPilot 1 kb plusDNA Ladder was used as a reference. ...
Nineteen fungal strains associated with the seagrass Posidonia oceanica, with the green alga Flabellia petiolata, and the brown alga Padina pavonica were collected in the Mediterranean Sea. These strains were previously identified at the family level and hypothesised to be undescribed species. Strains were examined by deep multi-loci phylogenetic and morphological analyses. Maximum-likelihood and Bayesian phylogenies proved that Parathyridariella gen. nov. is a distinct genus in the family Thyriadriaceae. Analyses based on five genetic markers revealed seven new species: Neoroussoella lignicola sp. nov., Roussoella margidorensis sp. nov., R. mediterranea sp. nov., and R. padinae sp. nov. within the family Roussellaceae, and Parathyridaria flabelliae sp. nov., P. tyrrhenica sp. nov., and Parathyridariella dematiacea gen. nov. et sp. nov. within the family Thyridariaceae
... RAPD analysis with primers RAPD24 (Baleiras Couto, Eijsma, Hofstra, Huisin't Veld, & van der Vossen, 1996) and RF2 (Paffetti et al., 1995) and microsatellite-primed PCR (MSP) with (GTG) 5 (Lieckfeldt et al., 1993) were performed with amplification programmes described in Pfliegler, Horvath, Kallai, and Sipiczki (2014). For interdelta genotyping (δ-PCR) the primers delta1, delta2 and delta12 (Legras & Karst, 2003) were applied in combinations delta1-delta2 and delta12-delta2 with parameters described in Pfliegler and Sipiczki (2016). ...
The effect of long-term laboratory maintenance on the biological diversity and certain oenolological properties of wine yeasts was investigated by the analysis of 17 strains isolated from Tokaj (Hungary) wines more than a century ago and maintained since then in culture collections. The analysis of the D1/D2 domains of the large subunit ribosomal RNA genes and the ITS sequences assigned them all to S. cerevisiae but divided them into two groups. One group showed sequence identity with wine strains occurring mainly in countries of the region. The combined results of the interdelta, RAPD, microsatellite-primed PCR (MSP), mtDNA and karyotype analyses found both groups diverse and defined each strain as unique, demonstrating that the long period of maintenance under identical conditions has not resulted in convergent evolution in their genomes. High diversity was detected also in certain phenotypic traits of oenological relevance (osmotolerance, killer phenotype, production of glycerol, ethanol, H2S and volatile acids). In the microvinification experiments all strains performed better than the laboratory strain S288c and were comparable to the industrial strain used as a control or even surpassed it in certain parameters. Thus, the oenological abilities of wine yeasts can be preserved over long periods of maintenance under laboratory conditions.
... To counter these difficulties in identification, many molecular techniques have been developed over the last 20 years. These include PCR-RFLP analysis (Dendis et al. 2003;Diguta et al. 2011), RAPD (Baires-Varguez et al. 2007Brandt et al. 1998), hybridisation with genus/species specific DNA or RNA probes (Lindsley et al. 2001;Sandhu et al. 1995), PCR fingerprinting (Hierro et al. 2004;Lieckfeldt et al. 1993), species-specific PCR assays (Kulik et al. 2004;Martin et al. 2000), real time PCR (Bergman et al. 2007;Klingspor and Jalal 2006), and, increasingly, DNA sequencing (Pryce et al. 2003;Romanelli et al. 2010). Sequence-based identification methods have proven to be more accurate than conventional methods in diagnostic clinical mycology (Balajee et al. 2007;Ciardo et al. 2006). ...
With new or emerging fungal infections, human and animal fungal pathogens are a growing threat worldwide. Current diagnostic tools are slow, non-specific at the species and subspecies levels, and require specific morphological expertise to accurately identify pathogens from pure cultures. DNA barcodes are easily amplified, universal, short species-specific DNA sequences, which enable rapid identification by comparison with a well-curated reference sequence collection. The primary fungal DNA barcode, ITS region, was introduced in 2012 and is now routinely used in diagnostic laboratories. However, the ITS region only accurately identifies around 75% of all medically relevant fungal species, which has prompted the development of a secondary barcode to increase the resolution power and suitability of DNA barcoding for fungal disease diagnostics. The translational elongation factor 1α (TEF1α) was selected in 2015 as a secondary fungal DNA barcode, but it has not been implemented into practice, due to the absence of a reference database. Here, we have established a quality-controlled reference database for the secondary barcode that together with the ISHAM-ITS database, forms the ISHAM barcode database, available online at http://its.mycologylab.org/. We encourage the mycology community for active contributions.
... PCR and sequencing Amplification and sequencing of the yeast and bacterial rDNA sequences was performed as described before, using ExTaq TM polymerase (Takara Bioinc, Japan) according to the provider's recommendations (Birmeta et al. 2004b). PCR fingerprints were done using a GTG 5 -primer (Lieckfeldt et al. 1993). Reaction conditions for the fingerprints were 5 min 95°C; 29 cycles of 30 s 94°C, 30 s 50°C, 2 min 72°C; 6 min at 72°C (final extension). ...
Enset (Ensete ventricosum) is the basis of the staple food consumed by about 20% of the Ethiopian population. Kocho is one of the food products generated from enset by spontaneous fermentation of decorticated and pulverized pseudostem and corm sections. We isolated culturable microbes associated with kocho from different stages of fermentation. Twelve yeast species, six lactic acid bacteria (LABs) species and eleven species of aerobic bacteria were identified by sequencing ITS/D1D2 regions of 26S rDNA of yeasts and 16S rDNA of bacteria, respectively. More yeast species were identified in fresh (fermented for 2–5 days) kocho, compared to long-term (7–12 months) fermented kocho, while we observed an opposite trend for LABs. In fresh kocho, the most frequently isolated yeast species were Pichia exigua, Galactomyces geotrichum, and Pichia fermentans. From mid-term (3–4 months) kocho most frequently Candida cabralensis, G. geotrichum, and Candida ethanolica were isolated. In the long-term fermentations, the most frequently isolated yeast was Saturnispora silva. Lactobacillus plantarum was the most frequently isolated LAB in both fresh and mid-term kocho. In long-term fermented kocho, Acetobacter pasteurianus and L. plantarum were most frequently isolated. L. plantarum was consistently isolated from all the three stages of fermentation. Aerobic bacteria in fresh kocho were mostly gram-negative, with Raoultella planticola and Pantoea agglomerans being the most frequently isolated species. In long-term fermented kocho, mainly gram-positive, spore-forming bacteria of the genus Bacillus were found, among them also species of the Bacillus cereus group, Bacillus anthracis and Bacillus thurigiensis.
... All yeast isolates were grouped by their molecular profiles using the PCR fingerprinting technique with the microsatellite primer (GTG) 5 (5 -GTGGTGGTGGTGGTG-3 ). 24 The PCR mixture contained 2.5 L of 10× buffer, 1 L of dNTPs 0.1 mM, 1.5 L of MgCl 2 , 2 L of the primer (GTG) 5 10 mol −1 (Invitrogen, Carlsbad, CA, USA), 0.2 L of Taq DNA polymerase 1 U and 1 L of DNA in a total volume of 25 L. The PCR reaction was performed on the Mastercycler ® Pro and showed the following conditions: initial denaturation at 94 • C for 2 min, 40 cycles of denaturation at 95 • C for 45 s, annealing at 50 • C for 1 min and extension at 72 • C for 1 min, followed by final extension at 72 • C for 6 min. One representative of each different molecular profile was identified by sequencing of the D1/D2 domains of the large subunit of rRNA gene using the primers NL1 (5 -GCATATCAATAAGCGGAGGAAAAG-3 ) and NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) according to Lachance et al. 25 The ...
Sour cassava starch (Polvilho azedo) is obtained from a spontaneous fermentation conducted by microorganisms from raw materials and fermentation tanks. This product is traditionally used in the baking industry for the manufacture of biscuits and Brazilian cheese breads. However, the end of fermentation is evaluated empirically, and the process occurs without standardization, which results in products of inconsistent quality. Predominant microbiota from a cassava flour manufacturer was isolated in order to select starter cultures for the production of sour cassava starch in a pilot-scale fermentation process. Lactic acid bacteria and yeasts were isolated, enumerated and grouped by Restriction Fragment Length Polymorphism, and PCR fingerprinting, respectively. One isolate of each molecular profile was identified by sequencing of the rRNA gene. LAB were prevalent throughout the entire process. Lactobacillus brevis (21.5%), which produced the highest values of acidity, and Lactobacillus plantarum (13.9%) were among the most frequent species. Pichia scutulata (52.2%) was the prevalent yeast and showed amylolytic activity. The aforementioned species were tested as single and mixed starter cultures in a pilot-scale fermentation process for 28 days. L. plantarum exhibited better performance as a starter culture, which suggests its potential for the production of sour cassava starch.
... One of the most popular approaches based on PCR is the PCR fingerprinting technique which uses specific oligonucleotides for simple repetitive DNA sequences. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GAC) 5 , (GACA)4, and the M13 sequence has been widely applied for the identification of yeasts (Lieckfeldt et al., 1993;Couto et al., 1996aCouto et al., , 1996bCaruso et al., 2002;Martorell et al., 2005;Orlić et al., 2010). The DNA sequencing of the genes encoding the D1/D2 domain of the 26S ribosomal RNA gene and the ITS (internal transcribed spacer) region of rDNA has provided highly reliable data for yeast taxonomy, and the characterisation of new genera and species (Kurtzman and Robnett, 1998Robnett, , 2003Groenewald et al., 2011). ...
In the article by Erdem et al. published in Journal of Microbiology 2016; 54, 618–625, the figure 1 should be corrected as below.
... DNA from all isolates was used as a template for amplification by the MP-PCR technique with microsatellite primer (GTG)5 (Lieckfeldt et al. 1993). The products of these amplifications were analyzed on 1.5% agarose gel profiles analyzed in the program PyElph 1.4 (Pavel and Vasile, 2012) with the aim of grouping more similar individuals. ...
Due to widespread bacterial resistance to commercial antibiotics, the search for capable substances to combating these microorganisms became a priority. In this context, the endophytic fungi gained prominence as potential producers of bioactive substances with pharmacological interest. It is considering that endophytes are still poorly studied, especially in tropical species. The antibacterial and antifungal potential of endophytic fungi associated with the medicinal plant Casearia sylvestris were isolated and evaluated. A total of 162 strains were obtained, among these strains, 34 were selected for antimicrobial assays, after molecular sorting with oligonucleotide (GTG)5. A total of 25 isolates showed some antifungal and / or antibacterial activity against the bacteria Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, and yeasts Candida albicans and Candida tropicallis. The results show the endophytic fungi present in C. sylvestris have a high potential to produce bioactive compounds inhibiting pathogenic microorganisms.
... The variability found in these regions can be shown by PCR amplification using specific oligonucleotides, such as (GTG) 5 , (GAG) 5, (GACA) 4 or M13. The ability of these oligonucleotides to reveal polymorphisms among S. cerevisiae strains has been demonstrated by Lieckfeldt et al. (1993) by hybridization techniques. These same authors were the first to use these sequences as primers in a PCR reaction, and proved the usefulness of this technique for characterization at the strain level. ...
The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the availability of rapid and simple molecular techniques that show genetic polymorphisms at species and strain levels have enabled the study of yeast diversity during wine fermentation. This review will summarize the mechanisms involved in generating genetic polymorphisms in yeasts, the molecular methods used to unveil genetic variation, and the utility of these polymorphisms to differentiate strains, populations, and species in order to infer the evolutionary history and the adaptive evolution of wine yeasts, and to identify their influence on their biotechnological and sensorial properties.
... Strains were compared by PCR fingerprinting using microsatellite primer (GTG) 5 , which allowed differentiation of strains at subspecies level [9]. The PCR condition was as follows: an initial denaturation step of 95° C for 2 min followed by 35 cycles of 94°C for 40 sec, 55°C for 40 sec and 72°C for 2 min. ...
Glucosidase is an important enzyme for production of ethanol from lignocellulose. With hydrolytic activity on cellooligosaccharides, especially cellobiose, β-glucosidase removes product inhibitory effect on cellulases and forms fermentable sugars. In this study, β-glucosidase encoding gene (BGL1) from traditional starter yeast Saccharomycosis fibuligera BMQ908 was cloned and expressed in Pichia pastoris. BGL1 of S. fibuligera BMQ 908 shared 98% nucleotide homology with the closest GenBank sequence (M22475) but identity in amino-acid sequences of catalytic domains. Recombinant plasmid pPICZαA/BGL1 containing the sequence encoding BGL1 mature protein and α-factor secretion signal was constructed and transformed into methylotrophic yeast P. pastoris by electroporation. The recombinant strain produced single extracellular protein with molecular weight of 120 kDa and cellobiase activity of 60 IU/ml. The optimum pH of the recombinant β-glucosidase was 5.0 and the optimum temperature was 50°C.
... One of the most popular approaches based on PCR is the PCR fingerprinting technique which uses specific oligonucleotides for simple repetitive DNA sequences. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GAC) 5 , (GACA)4, and the M13 sequence has been widely applied for the identification of yeasts (Lieckfeldt et al., 1993;Couto et al., 1996aCouto et al., , 1996bCaruso et al., 2002;Martorell et al., 2005;Orlić et al., 2010). The DNA sequencing of the genes encoding the D1/D2 domain of the 26S ribosomal RNA gene and the ITS (internal transcribed spacer) region of rDNA has provided highly reliable data for yeast taxonomy, and the characterisation of new genera and species (Kurtzman and Robnett, 1998Robnett, , 2003Groenewald et al., 2011). ...
A new method based on high resolution melting (HRM) analysis was developed for the differentiation and classification of the yeast species that cause food spoilage. A total 134 strains belonging to 21 different yeast species were examined to evaluate the discriminative power of HRM analysis. Two different highly variable DNA regions on the 26 rRNA gene were targeted to produce the HRM profiles of each strain. HRM-based grouping was compared and confirmed by (GTG)5 rep-PCR fingerprinting analysis. All of the yeast species belonging to the genera Pichia, Candida, Kazachstania, Kluyveromyces, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Ustilago, and Yarrowia, which were produced as species-specific HRM profiles, allowed discrimination at species and/or strain level. The HRM analysis of both target regions provided successful discrimination that correlated with rep-PCR fingerprinting analysis. Consequently, the HRM analysis has the potential for use in the rapid and accurate classification and typing of yeast species isolated from different foods to determine their sources and routes as well as to prevent contamination.
... In order to evaluate the genetic variability of the F. verticillioides strains, PCR reactions using the ISSR primers (GTG) 5 [38] and (GACA) 4 [39] were used. Amplification reactions were performed in a final volume of 25 µL using the following contents: 20 mM Tris-HCl buffer (pH 8. Amplification reactions were performed in a Techne TC-512 thermocycler (Analitica, São Paulo, Brazil) with the following programmed cycles: an initial denaturation step at 93˝C for 5 min, 40 cycles of 20 s at 93˝C, 45 s at 55˝C and 90 s at 72˝C, followed by a final extension at 72˝C for 6 min. ...
Fusarium verticillioides is considered one of the most important global sources of fumonisins contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol).
... Thus, S. paradoxus strains from the central parts of European Russia and Ukraine, northern Europe, and regions with a warm climate (Spain, southern coastline of the Crimea, Uzbekistan, and Azerbaijan) exhibited different PCR profiles. The molecular marker (GTG) 5 with its multiple localization in the genome of Saccharomyces [18] allows the identification of S. cerevisiae, S. bayanus, and S. paradoxus, as well as the differentiation between individual strains of each of these species [17,19]. We compared the (GTG) 5 patterns of the yeasts of the near-Moscow population and of six S. paradoxus strains (CBS 432, CECT 10178, N8, 9, 12, and 17) isolated in various regions of Europe and differing in dsRNA and in PCR profiles. ...
... Die RFLP-Analyse benötigt relativ große DNA-Mengen und ist erst nach Hybridisierung mit spezifischen moderat repetitiven Sonden, wie z.B. der Ca3-und der 27A-Sonden (Sadhu et al. 1991, Scherer & Stevens 1988) oder mit " simple repeat " -Sonden wie (GGAT)4 oder (GTG)5 (Sullivan et al. 1993Bostock et al. 1993). Polymorphe DNA-Abschnitte bei Pilzen wurden sowohl mit der RAPD-Technik (Bostock et al. 1993, Niesters et al. 1993, Lehmann et al. 1992) als auch durch den Einsatz von Primern, die an Mini-oder Mikrosatelliten-DNA und andere repetitive Sequenzen anlagern , amplifiziert (Thanos et al. 1996, Lieckfeldt et al. 1993 Niesters et al. 1993, Schönian et al. 1993a ). Dieses PCR-Fingerprinting, bei dem Polymorphismen nachgewiesen werden, die zufällig über das gesamte Genom verteilt sind (Welsh et al. 1992), benötigt keine vorherige Sequenzinformation. ...
Für die Identifizierung bzw. Differenzierung von Candida- Spezies und -Stämmen sowie für die Bestimmung der genetischen und epidemiologischen Verwandtschaft von Stämmen der gleichen Spezies wurde eine PCR-Fingerprint-Technik und eine RFLP-Analyse der amplifizierten ITS-Region angewandt. Das PCR-Fingerprinting amplifiziert anonyme Sequenzen in der chromosomalen DNA, die über das gesamte Genom verteilt sind. Die ITS-Region ist Bestandteil des ribosomalen Operons, welches in ca. 50-100 Kopien/Zelle vorhanden ist. 1.a. Beide molekularbiologischen Verfahren wurden zur Unterscheidung von routinemäßig schwer differenzierbaren klinischen Candida famata und Candida guilliermondii-Isolaten genutzt. Von insgesamt 37 fraglichen Stämmen konnten 31 als C. guilliermondii und 3 als C. famata identifiziert werden, die drei verbliebenen Stämme waren mit diesen Techniken nicht identifizierbar. Mit der Biochemotypie gelang nur die Zuordnung eines der 3 C. famata-Isolate sowie von 23 der 31 C. guilliermondii-Isolate. 14 Isolate wurden mit den konventionellen Methoden gar nicht oder falsch identifiziert. 1b. Mit dem PCR-Fingerprinting wurde auch die Spezieszugehörigkeit phänotypisch veränderter Candida albicans-Isolate überprüft. Alle atypischen Stämme, bei denen solche für C.albicans charakteristischen Merkmale wie die Bildung von Chlamydosporen, die Verwertung der Aminozucker Glukosamin und N-Acetylglukosamin sowie die Assimilation von 2-Ketogluconat und Xylose nicht ausgeprägt waren, wiesen die für C. albicans typischen Fingerprintmuster auf. Unsere Studie zeigte, daß die biochemische Typisierung an Grenzen stößt, wenn typische Stoffwechselreaktionen nicht nachgewiesen werden können. 2. Bei 6 verschiedenen C. albicans-Populationen aus Angola, Madagaskar, Deutschland und Portugal wurde die Variabilität phänotypischer und genotypischer Merkmale untersucht, wobei in diese Analyse auch atypische Stämme miteinbezogen wurden. Während die phänotypischen Eigenschaften, bis auf die der atypischen Stämme, kaum variierten, wurden für die insgesamt 212 C. albicans-Isolate 87 unterschiedliche PCR-Fingerprint-Genotypen nachgewiesen. Eine Analyse der Beziehungen zwischen den Fingerprint-Genotypen wurde mit der UPGMA-Distanz-Methode durchgeführt. 3. Weiterhin wurden Candida-Vaginalisolate von Patientinnen mit rezidivierenden Episoden von Candida-Vaginitis mit Stämmen verglichen, die aus anderen Körperregionen stammten bzw. bei ihren Partnern isoliert wurden. Es konnte gezeigt werden, daß Stammaustausche zwischen den Partnern vorkommen, ein Stamm ohne oder mit geringfügigen genotypischen Veränderungen trotz Therapie persistieren kann und daß eine Reinfektion auch durch einen neuen Stamm möglich ist. Das Problem der Erregeridentifizierung in der mykologischen Labordiagnostik ist sowohl klinisch als auch epidemiologisch relevant. Molekularbiologische Methoden sollen gut funktionierende konventionelle Methoden zur Erregeridentifizierung nicht ersetzen, können aber bei Problemfällen eine wertvolle Ergänzung für die mykologische Diagnostik vorzugsweise in fachständigen Referenzlaboratorien darstellen.
... L'utilisation initiale de marqueurs microsatellites individuels avait mis en évidence l'utilité de cette approche pour différencier des souches de S. cerevisiae (Lieckfeldt et al., 1993 ;Gallego et al., 1998 ;Perez et al., 2001a). L'association de plusieurs de ces marqueurs génétiques s'est avérée très utile pour la discrimination entre souches de S. cerevisiae puisqu'un grand polymorphisme intraspécifique a été observé (Field and Wills, 1998 ;Hennequin et al, 2001 ;. ...
We explored in this study the biodiversity of the fermenting indigenous Lebanese flora
of Saccharomyces cerevisiae by collecting samples of natural fermentations of grape musts
from various localities dispersed within the Lebanese territory. A great molecular diversity of
the Lebanese flora was found in the natural fermentations, as well as in the wineries as a
whole and between different geographic localities. However, cases of dominance and
perenniallity were also recorded in wineries. Besides, fermentations in the same wineries
seemed to involve lineages of related strains. Relatedness of strains was also seen in narrow
geographical areas while it was lost as the areas enlarged. Despite its diversity, the Lebanese
flora could nevertheless be interrelated, which property was observed when this flora was
compared to other oenological floras of diverse origins. Finally, it seemed that the effect of
the thriving media was greater on the lineages of fermenting floras than the effect of the
geographical distance.
We also evaluated the usefulness of the molecular typing methods used for assessing the
biodiversity of the S. cerevisiae floras. Two of them, interdelta fingerprinting and
microsatellites analysis, were previously developed, while we tested a new method, the
MLST. The first two methods were very useful for typing due to the great variability of their
markers and thus to their great discriminatory power, while MLST showed a lower
discriminatory power. But our MLST scheme could probably be improved by using more
variable loci. MLST was nonetheless more useful in inferring strains relatedness, especially
for poorly related isolates.
We further examined the phenotypic diversity that accompanies the molecular one in
order to initiate a selection scheme for some oenological strains and also to verify the
correspondence between the molecular and the phenotypic diversity. The studied strains of S.
cerevisiae presented different fermenting capacities which could be exploited to push further
the selection scheme for potential commercial uses, in particular in the Lebanese winemaking
industry. On the other hand, a certain congruence between the molecular and the phenotypic
diversities was observed and it was mainly due to a correlation between high acetaldehyde
productions and particular molecular profiles. If this path is studied further, this congruence
might help predict acetaldehyde production for particular strains
Medicinal plants represent a promising reservoir of diverse endophytic fungi, including taxa that are able to produce bioactive metabolites. In Brazil, the genus Copaifera includes species that are well known in folk medicine mainly due to their ability to produce oleoresin. In this study, we characterized the endophytic fungal communities associated with Copaifera langsdorffii and Copaifera pubiflora and investigated their ability to produce antimicrobial agents. We obtained 668 fungal isolates from the leaves, stems, and seeds of both plants, which were later classified into 64 taxa and 22 genera. Diaporthe sp. 6, Xylariaceae sp. 1, Diaporthales sp. 1, and Diaporthales sp. 2 were the most abundant taxa in C. langsdorffii, while Phyllosticta sp., Diaporthe sp. 7, Diaporthales sp. 3, and Diaporthe miriciae were the most abundant taxa in C. pubiflora. Diaporthe sp. 4, Phyllosticta sp., Diaporthe sp. 1, Diaporthe sp. 7, and Neopestalotiopsis sp. were the only taxa common between the two plants. Both plants were found to have high fungal diversity, especially C. langsdorffii. Six extracts displayed antibacterial, being Alternaria sp., Diaporthe sp. 1, D. miriciae, and Diaporthe sp. 14. Our results showed that different tissues of the ethnomedicinal plants C. langsdorffii and C. pubiflora are systematically colonized by rich and diverse endophytic fungal communities, and that some of the fungi are able to produce antimicrobial compounds, which may be explored in further studies as potential candidates for the development of new drugs.
Kombucha is a drink produced by spontaneous fermentation, and several studies have been conducted to unveil its microbiological and physicochemical aspects with numerous human health claims. The integration of these results is fundamental to understand and discuss the biological activities attributed to kombucha. In the present study, we isolated bacteria and yeasts involved in the fermentation of kombucha produced with green (GK) and black (BK) teas, as well as the amplicon metagenomic of the microbial communities (16S and ITS) during 0, 3, 5, 10, and 15 days of fermentation, at 28 °C. Microbial communities were linked to key biochemical parameters monitored during fermentation such as pH, total titratable acidity, total reducing sugars, polyphenols, acetic acid, and ethanol production. Moreover, ordination analysis (principal component analysis, PCA) revealed clear GK and BK separation groups during the fermentation process. Caffein, gallic acid, and chlorogenic acids majorly influenced the separation of GK and BK. Furthermore, the presence of Komagataeibacter spp. and catechins exerted selective pressure against microbial contamination. This study essentially contributes to the knowledge about the effects of integrated microbiota to the chemical results of the kombucha fermented in GK and BK teas.
Dry edible beans are a vital food source in Mozambique, East Africa—one that alleviates hunger and malnutrition and adds value to the economy. In recent years, root/crown rot (RCR) pathogens have emerged as limiting constraints in dry bean production. Not much has been characterized concerning the causal agents of RCR in Mozambique. The purpose of this study was to identify the primary pathogen(s) associated with RCR dry bean samples collected at breeder nursery sites and farmer fields in Mozambique using molecular sequencing and culture-based methods. Sequencing revealed, not surprisingly, an increased diversity of fungal/oomycete operational taxonomic units when compared to culture-based methods oof diversity. Species of Fusarium, mainly F. oxysporum, were the dominant taxa detected in RCR dry beans through sequencing the ITS rDNA region and partial EF-1α gene. Collectively, 333 fungi and/or Oomycetes were isolated in culture during the 2014–2015 growing seasons and tested for pathogenicity on healthy bean seedlings. Fusarium species were identified by both morphological and molecular characters. At least 60% of the isolates inoculated on common bean were recognized as potentially pathogenic. From both isolation frequency and pathogenicity testing, F. oxysporum and related species play an important role in the bean RCR complex. We found similar results from dry beans grown in the two main bean-growing regions of Mozambique. These findings will allow breeders to screen for resistance to F. oxysporum in greenhouse grown bean plants as well as within field grown bean cultivars.
Trichoderma is an important genus known for the past nearly 200 years. Till recently, Trichoderma and Hypocrea were treated as separate genera, with several species linked as asexual (anamorph) and sexual (teleomorph) morphs, respectively. As per the revised International Code of Nomenclature for Algae, Fungi and Plants (ICN) any fungi would no longer bear more than one name. Under this new provision of ICN, Trichoderma became valid and supersedes teleomorphic Hypocrea. Biotechnological applications of species of Trichoderma have seen tremendous changes in recent years, which has drawn serious attention toward fundamental taxonomy and systematics. The purpose of this chapter is to compile important information on current status of taxonomy, especially related to morphology, molecular and phylogeny of important species. Considering immense biotechnological importance of several species of this genus, it is pertinent to discuss importance of conservation of its species as it is largely ignored. Biological Resource Centres (BRCs)/Culture Collections play an important role in conserving the mycological resources. In order to reflect biodiversity, selected species of Trichoderma isolated from different natural sources and geographical locations, and already deposited at National Fungal Culture Collection of India (NFCCI) and a few newly isolated ones were reexamined morphologically as well as sequencing of recommended gene regions and their phylogenetic analysis were conducted.
Alcoholic fermentation and the production of wine has accompanied humanity for more than 10000 years. However, it has been only in the last 50 years when the winemakers have had the tools to manage and control the process. The methodology to analyze and monitor the succession of the microorganisms that participate in the process along with the effective use of antimicrobial compounds (for instance sulfur dioxide), the control of the temperature and, above all, the use of cellar-friendly fermentation starters (mostly as Active Dry Wine Yeast) have provided the appropriate conditions for that control. However, the use of a limited number of commercial presentations of the starters has generated an unwanted uniformity of the wines produced. Furthermore, new tendencies in wine making with limited or no human intervention have considered these tolls as a negative aspect in the wine quality, although most of these concerns are only philosophical, without clear scientific evidence. We present a revision of the present state of the art in these methodologies where our research group has been working for the last 25 years.
The inoculation of Saccharomyces cerevisiae starter cultures in grape musts is a common practice in wineries worldwide; however, native non-Saccharomyces yeast species are increasingly investigated as co-starters to augment the complexity and regionality of wine. In this study, an extensive collection of non-Saccharomyces yeasts from high-sugar matrices was created and screened with the aim to discover new strains with potentially positive oenological traits. After mining >400 yeasts from 167 samples collected across multiple Italian regions, the isolates were identified based on RAPD-PCR analysis and ITS sequencing. About one quarter of them, belonging to the genera Starmerella, Lachancea and Metschnikowia, were picked up for an in-depth molecular and physiological characterization, since these yeasts were well strewed and have a good oenological reputation. Following the genotyping, stress tolerance assays, enzymatic activity trials and single inoculum fermentations, a huge diversity was acknowledged within and between the species. Strains of S. bacillaris showed a high tolerance to ethanol and increased glycerol production, L. thermotolerans reduced volatile acidity while increasing total acidity with lactic acid, and Metschnikowia spp. exhibited remarkable aroma-related enzymatic activities, which are all prized features in winemaking. Since most of the characteristics analyzed were species and strain dependent, the obtained results are valuable for the selection of a new generation of co-starters for attempting mixed fermentation strategies aimed to improve the overall quality of regional wine.
Isolates of Glomus mosseae from international collections were compared using two molecular techniques: PCR-fingerprinting of genomic DNA with direct amplification of microsatellite regions, and sequencing of the small subunit (SSU) rDNA. Numerical analyses of these data using parsimony models were used to calculate phylogenetic topologies. The phylogenetic tree from SSU rDNA sequences was similar to the phylogenetic trees obtained from genomic fingerprinting using the amplification of microsatellite regions, except for one G. mosseae isolate, DAOM221475. Another isolate was not grouped with the other G. mosseae isolates by either method and was found to be Glomus sp. a posteriori. Both analyses showed considerable genetic variation within the species G. mosseae. We suggest that molecular data such as SSU rDNA sequences be used in the description of Glomales species, and PCR-fingerprinting could be used to study diversity within species of Glomales.
The endophytic fungal community associated with the ethnomedicinal plant Echinacea purpurea was investigated as well as its potential for providing antifungal compounds against plant pathogenic fungi. A total of 233 endophytic fungal isolates were obtained and classified into 42 different taxa of 16 genera, being Alternaria alternata, Colletotrichum dematium and Stagonosporopsis sp. 2 the most frequent colonisers. The extracts of 29 endophytic fungi displayed activities against important phytopathogenic fungi. Eight antifungal extracts were selected for chemical analysis. Forty fatty acids were identified by GC-FID analysis. The compounds (-)-5-methylmellein and (-) (3R)-8-hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin were isolated from Biscogniauxia mediterranea EPU38CA crude extract. (-)-5-Methylmellein showed weak activity against P. obscurans, P. viticola and F. oxysporum, and caused growth stimulation of C. fragariae, C. acutatum, C. gloeosporioides and B. cinerea. (-) (3R)-8-Hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin appeared slightly more active in the microtiter environment than 5-methylmellein. Our results indicate that E. purpurea lives symbiotically with different endophytic fungi, which are able to produce bioactive fatty acids and aromatic compounds active against important phytopathogenic fungi. The detection of the different fatty acids and aromatic compounds produced by the endophytic community associated with wild E. purpurea suggests that it may have intrinsic mutualistic resistance against phytopathogen attacks in its natural environment. This article is protected by copyright. All rights reserved.
Yeast populations used in industrial production of fuel-ethanol may vary according to the plant process conditions and to the environmental stresses imposed on yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as the starter strain instead of less adapted commercial strains. This work reports the use of a PCR-fingerprinting method based on microsatellite primer (GTG)(5) to characterize the yeast population dynamics during the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominated the yeast population and were more commonly present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from the fuel-ethanol producing process.
Mycorrhizal fungi interact symbiotically with the roots of about 90% of land plants forming different types of mycorrhiza. The total number of soil fungi involved in this symbiosis is still unknown, but in order to evaluate their biodiversity it is first necessary to make an identification at species and isolate level. This goal has received substantial impetus thanks to the development of molecular techniques based on the polymerase chain reaction (PCR), which allow the characterization of nucleic acids amplified from minute amounts of fungal samples. PCR-based techniques have already been applied to those endo- and ectomycorrhizal fungi where morphological characters are in conflict, ambiguous or missing. This approach has allowed the development of molecular tools for their identification (Henrion et al. 1992; Gardes and Bruns 1993; Lanfranco et al. 1993, 1995a, b; Wyss and Bonfante 1993). PCR-based techniques include a wide range of protocols, among which the most commonly used are: amplification of variable regions in the ribosomal genes (ITS or IGS), restriction fragment length polymorphism (RFLP) of PCR-generated fragments, amplification of short repeated sequences (microsatellites) and random amplification of polymorphic DNA (RAPD) (Erlich et al. 1991). These techniques provide a different degree of resolution in the study of genetic polymorphisms. For example, amplified ITS regions usually reveal interspecific variations, although differences in fragment length may not be found among closely related species and may require further restriction analysis.
Molecular analyses of nucleic acid polymorphisms have proved to be very helpful in avoiding taxonomic ambiguities and simplifying yeast identification schemes. PCR mediated typing assays have the advantage of a rapid amplification of yeast target DNA in the laboratory thus enabling rapid analysis of DNA polymorphisms on different levels. We used two approaches - restriction analysis of amplified rDNA fragments (RFLPs of 2550 bp long 18S-ITS1-5.8S-ITS2 and 3350 bp long 25S rDNA amplicons) and AP-PCR fingerprinting of yeast DNA with six non-specific oligonucleotide primers for the characterization of Hanseniaspora (anamorph Kloeckera) yeasts. Species-specific restriction patterns were derived from type strains analysis. Two restriction analyses of 18S-ITS1-5.8S-ITS2 rDNA and/or 25S rDNA fragment were sufficient for species delineation among Hanseniaspora/Kloeckera yeasts. They were used for rapid species identification of thirthy-three Hanseniaspora/Kloeckera strains isolated from grapes and musts at the start of spontaneous fermentation in two geographically distinct wine-producing subregions in Slovenia. All identified strains were H. uvarum/K. apiculata. AP-PCR fingerprinting revealed great heterogeneity of the isolates at the intraspecies level. No correlation between genetic similarity, calculated from AP-PCR fingerprints, and the geographical origin of H. uvarum/K. apiculata strains was evidenced.
Commercially available oligonucleotide primers (10-mers) were used in a random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) to screen and characterize potential beer-contaminating bacteria. The species identification of Lactobacilli, Pediococci, Leuconostoc, and others was achieved using four different primers in the RAPD-PCR. For each bacterial species, each primer produced a unique and characteristic "core" fingerprint pattern. Using the core patterns as a guide, we were able to distinguish and recognize unknown contaminants obtained during regular testing by our quality control laboratories. The procedure is faster than traditional characterization techniques - the identification of a single colony was typically achieved in less than 10 hr.
This chapter discusses molecular identification and characterization of wine yeasts. Yeast species can be identified by comparison of nucleotide sequences from rDNA regions. The two most commonly used regions are the D1 and D2 regions at the 5' end of the genes encoding the 26S and 18S ribosomal subunits. The availability of sequences in DNA databases, particularly for the D1/D2 region of the 26S gene, makes this technique particularly useful for assigning unknown yeast to a specific species when the homology of the sequences is greater than 99%. Molecular characterization of commercial yeast strains is necessary for two reasons. First, it is needed for quality-control purposes to confirm that the obtained yeast is the one that was originally selected and not a contaminant, and, second, to detect fraud. Wine is a highly appropriate culture medium for the growth of a large number of microorganisms, in part due to its richness in organic acids, amino acids, residual sugars, growth factors, and mineral salts. Techniques for the detection of spoilage yeasts are essential. These techniques must be very sensitive and allow quantification of the number of microorganisms present. They must also be rapid to allow the application of corrective measures on the production line prior to release of the products onto the market.
El uso combinado del análisis de restricción del ADN mitocondrial y del RAPD-PCR permite diferenciar levaduras de jamón ibérico con alta producción de compuestos volátiles implicados en el aroma a curado. En la especie Debaryomyces hansenii se detectan los biotipos más productores, los cuales han sido seleccionados como cultivos iniciadores. Se han encontrado biotipos de levaduras exclusivos de lugar y etapa de maduración.
Experimental conditions for the separation of chromosomes from the model zygomycete Absidia glauca by rotating field electrophoresis were established. The sexually compatible strains of the mating type pair A. glauca CBS 100.48 (+) and CBS 101.48 (-) showed considerable differences in their electrophoretic karyotype. By Southern hybridization with homologous probes we have mapped the chromosomal locations for rDNA, the repetitive element rAg1, and the genes for actin and the elongation factor EF1. For the mapping of ubiquitin information we used a heterologous probe from U. maydis. The combination of electrophoretic karyotyping and Southern mapping proved to be a useful tool for characterizing mutant genotypes, which were induced by integrative transformation.
Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between strains of almost
any organism. We applied the technique to the mouse (Mus musculus). The characteristic differences In the AP-PCR genomic fingerprints between strains will be of value in strain identification
and verification. Using one primer, we genetically mapped four polymorphisms in a set of C57BL/6J × DBA/2J recombinant Inbreds.
One of these polymorphisms is a length variant. The method will allow rapid genetic mapping of DNA polymorphisms without Southern
blotting.
When used to probe EcoRI-digested Candida albicans DNA, the moderately repetitive sequence Ca3 generated a Southern blot hybridization pattern which included 15 to 25 bands, depending upon the strain. The pattern was stable through 400 generations in each of three independent strains but variable between most of 46 unrelated tester strains, making it a very effective probe for discrimination between strains. Computer-assisted methods (Dendron) were developed for storage of Ca3 patterns in data files, calculation of similarity (SAB) values between strains based upon band positions and intensities, and generation of histograms and dendrograms based on SAB values for all strains or any subset of strains in large epidemiological studies. In testing the effectiveness of the system, it was found that (i) multiple isolates from different body locations of the same healthy individual could represent either the same strain or different strains, (ii) isolates from oral lesions of a husband and wife represented the same strain, (iii) strains isolated from the mouths of 10 healthy individuals on the same day and in the same geographical location were as dissimilar on average as the 46 unrelated tester strains, and (iv) strains isolated from seven immunocompromised patients hospitalized over a 2.5-month period in the same hospital were highly similar, indicating nosocomial origin. The apparent effectiveness of these fingerprinting methods and the Dendron program suggests that interlaboratory procedures for fingerprinting should be standardized and all patterns should be analyzed and stored in a common and accessible data base for broad epidemiological analysis.
Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase
chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two
cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by
comparing polymorphisms in genomic fingerprints. The generality of the method Is demonstrated by application to twenty four
strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).
The primary structure of the small ribosomal subunit RNA (srRNA) molecule of the type strains of the ascosporogenous yeasts Debaryomyces hansenii, Pichia anomala (synonym: Hansenula anomala), Pichia membranaefaciens, Schizosaccharomyces pombe, Zygosaccharomyces rouxii and Dekkera bruxellensis was determined. The srRNA sequences were aligned with previously published sequences from fungi, including those of 5 Candida species, and an evolutionary tree was inferred.
The srRNA results were compared with chemotaxonomic criteria, e. g. the coenzyme Q system. The heterogeneity of the genera Candida and Pichia is clearly reflected by the srRNA analysis.
Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species. S. cerevisiae and S. bayanus strains had only 22% of their genomes in common. S. pastorianus, with intermediate hybridization values between S. cerevisiae and S. bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species. S. pastorianus replaces S. carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904. These data do not agree with the results of traditional physiological tests.
Deoxyribonucleic acid (DNA) base composition and DNA base sequence relatedness comparisons were used for species delineation in the genus Kluyveromyces. Base composition values separated the members of the genus into three groups. The groups were further subdivided by comparing base sequences by using DNAIDNA renaturation experiments. Two DNA homology groups were identified. The first group included Kluyveromyces marxianus, Kluyveromyces fragilis, Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces wikenii, and three anamorphs (Candida kefyr, Candida pseudotropicalis, and Torula cremoris); the members of this group exhibited 290 % DNA base sequence complementarity. The second group consisted of Kluyveromyces lactis, Kluyveromyces vanudenii, Kluyveromyces drosophilarum, and Kluyveromyces phaseolosporus; various pairs of these yeasts shared 64 to 98% of their DNA sequences. The two groups were only distantly related to each other (115% DNA base sequence complementarity). The other Kluyveromyces species appear to be unique, not being closely related to either of the two homology groups or to one another. Relationships deduced from comparisons of DNAs agreed well with those deduced by other workers from immunological comparisons of exo-P-glucanases and from isoenzyme analysis but were only in partial agreement with a taxonomic arrangement made on the basis of mating studies. We propose recognition of the following species: Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces blattue, Kluyveromyces delphensis, Kluyveromyces dobzhanskii, K. lactis (syn. K. drosophilarum, K. phaseolosporus, and K. vanudenii), Kluyveromyces lodderi, K. marxianus (syn. K. bulgaricus, K. cicerisporus, K. fragilis, and K. wikenifi, Kluyveromyces phafii, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Kluyveromyces waltii, and Kluyveromyces wickerhamii.
In this paper we describe two complementary approaches to a molecular taxonomy for the yeasts which exploit hybridisation to restriction fragments containing the repetitive sequence poly[dGdT.dCdA] (polyGT). This method will be of particular use to industrial users of the yeasts, whether they are brewers, bakers or other biotechnologists, since it has the potential to supplant the array of biochemical and physiological tests currently employed.
We have analyzed nine different species of the filamentous fungus Trichoderma and three strains of T. reesei for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides [(CT)8, (GTG)5, and (GACA)4]. On the basis of the DNA-fingerprints obtained, the Trichoderma aggregate is re-classified into five groups: I (T. reesei, T. todica), II (T. polysporum, T. longibrachiatum, T. koningii, and T. pseudokoningii), III (T. virgatum), IV (T. saturnisporum) and V (T. harzianum). These results contradict the claim that T. reesei is a subspecies of T. longibrachiatum. Furthermore, hybridization with (CA)8 allowed a subdivision of group II, wherein T. pseudokoningii formed a subgroup, IIb, which is highly homologous with, but distinct from subgroup IIa. The results show that RFLP analysis may be used to re-classify the Trichoderma aggregate.
We used the polymerase chain reaction (PCR) to amplify specific regions of the nuclear and mitochondrial genomes of fungi using DNA extracted from pure cultures as well as that directly from ectomycorrhizal rootlets. The internal transcribed spacer (ITS) of the nuclear ribosomal repeat unit and a portion of the mitochondrial large subunit ribosomal RNA gene were chosen as target sequences because both exist in high copy number and amplification primers for both discriminate between plant and fungal DNAs. These features provided a sensitivity and specificity sufficient for detection and analysis of a single mycorrhizal rootlet. We evaluated the variations in the amplified products with regard to the length, restriction endonuclease sites, and primary sequence for use in identification of genera, species, and strains of ectomycorrhizal fungi, with particular attention to selected Laccaria species. Accidental contamination of jack pine seedlings by Telephora terrestris was easily recognized. Amplification an...
Thirteen independent populations of Saccharomyces cerevisiae (nine haploid and four diploid) were maintained in continuous culture for up to approximately 1000 generations, with growth limited by the concentration of organic phosphates in medium buffered at pH 6. Analysis of clones isolated from these populations showed that a number (17) of large-scale chromosomal-length variants and rearrangements were present in the populations at their termination. Nine of the 16 yeast chromosomes were involved in such changes. Few of the changes could be explained by copy-number increases in the structural loci for acid phosphatase. Several considerations concerning the nature and frequency of the chromosome-length variants observed lead us to conclude that they are selectively advantageous.
Wine yeast strains are characterized by a high chromosomal DNA polymorphism. This can be explained partly by a size difference of different variants of specific chromosomes. This difference can reach up to 45% of the size of the chromosome in question. Two strains, SB1 and Eg8, have a very complex chromosomal pattern and show one band hybridizing with probes from two different chromosomes derived from a reference strain. This is an indication of the presence of "hybrid" chromosomes in these wine strains. The most astonishing result concerns chromosome VIII, frequently present in wine strains in two variant forms. The first normal form has a size of about 580 kb while the second is around 1000 kb. These two forms segregate at meiosis and recombine with a normal chromosome VIII from a laboratory strain. Wine yeasts are thus very different from haploid laboratory strains.
DNAs of several species of domestic animals digested with the restriction endonucleases HinfI, AluI and HaeIII were hybridized with different synthetic probes. DNA fingerprint patterns were found in each investigated species by at least two of these probes. Furthermore, two probes gave sex-specific banding patterns in the chicken. Some applications of DNA fingerprinting in domestic animals are discussed.
Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.
We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.
We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)8, (GTG)5 and (GACA)4, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)4 produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.
Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms
(RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random
DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which
amplify from one parent but not the other, are inherited in a Mendellan fashion and can be used to construct genetic maps
in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.
The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate "fingerprinting" probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of "DNA fingerprinting" was also introduced into the analysis and characterization of plant genomes, initially by using human or M13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra- and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety-specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy-to-perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin-based labeling techniques proved to be of similar quality.
The analysis of mitochondrial DNA (mtDNA) from several strains of Candida parapsilosis and Candida rhagii by restriction endonucleases enabled us to discriminate between several groups within the C. parapsilosis species and to allocate laboratory strains to one of these. The mtDNAs isolated ranged in size from 20 to 31 kb. The mtDNA isolated from group 1 C. parapsilosis hybridises with both ATPase subunit 6 and 8 gene probes, the same restriction fragment hybridising with both probes.
Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.
Simple tandemly organized GATCA sequences occurred in all eukaryotic genomes investigated. The amount and organization of individual GATCA sequences or derivatives thereof vary considerably in animal DNAs and can be assessed by simple but specific hybridization procedures with chemically pure oligonucleotide probes. In several animal species, including humans, GATCA sequences show extensive polymorphism, thus allowing individual-specific "DNA fingerprints." In selected rodents the sex-chromosomal organization of GATCA sequences is being studied extensively, revealing rapid evolutionary changes. In addition, insight can be expected into the sequences involved in obligatory meiotic crossing over between the X and Y chromosomes, into unequal crossing-over events, and into the linkage of GATCA elements to male-specific as well as to male-determining genes on the Y chromosome. The exact provenance of GATCA sequences in present-day eukaryotes cannot be pinpointed, but evolutionary conservation and several modes of de novo generation are discussed. Among these are unequal recombination, slipped strand mispairing, and other unspecified mechanisms. The latter include inherent properties that are responsible for the "selfish" or "ignorant" nature of simple repeats. Expression, if any, of GATCA sequences is critical to the overall significance of these ubiquitously interspersed simple repeats.
Over the past twenty years, several techniques from biochemical and molecular genetics, such as enzyme electrophoresis and isoelectric focusing, have been widely and successfully applied to the study of population differentiation and evolution. However, they have been less applicable to demographic problems such as assigning parentage to individuals within a population. This stems from a general weakness of data derived from enzyme loci: allele frequencies at polymorphic loci are sufficiently skewed that the majority of individuals are of one or two genotypes. Many enzyme systems can only be examined post mortem, so that the loci are of little use if the animals are to be studied in the wild. The search for new and more sensitive techniques for detecting genetic variation has continued, and recently a major discovery has come from molecular biology. Jeffreys et al. have reported the detection of a type of hypervariable 'minisatellite' DNA that is extraordinarily polymorphic in human populations. We have applied their technique to several bird species and particularly to a population of house sparrows (Passer domesticus) near Nottingham. We report here that one of the human minisatellite clones is a suitable probe for sparrow DNA and that it reveals variation as extensive as that found in man. These results suggest that analysis of minisatellite DNA will be a powerful tool in the study of demographic population genetics.
The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli. Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite. A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis.
SUMMARY The DNA-RNA hybrid technique of Gillespie & Spiegelman (1965) was used to measure the genetic relatedness among ten different species within the genus Candida and among three of these species and their counterparts within perfect genera. Close relationships were found between Candida albicans (guanine + cytosine content (YoGC), 35-1), C. claussenii (%GC, 34.9) and C. stellatoidea (% GC, 35-7), which showed a relative homology of 63 to 73 yo, and between C. brumptii (yoGC, 54-1) and C. catenulata (% GC, 549, relative homology 75 yo. Very close relationships were seen between the three pairs of perfect and imperfect counterparts, C. pelliculosa (yo GC, 36.8) and Hansenula anomala (yo GC, 36-6), relative homology 8 I Yo, C. melinii (% GC, 40'9) and H. wingei (yo GC, 41 -2), relative homology 97 %, and C. pseudotropicalis (% GC, 41 '3) and Kluyveromyces fragilis (%GC, 41.6), relative homology 92 %. Some degree of homology (32 %) was also found between C. pelliculosa and C. pseudotropicalis. The relatedness between C. albicans and the seven other Candida species (yo GC, 36.6 to 57.6) examined was low, relative homology 4 to 13 %. This also applies to C. tropicalis (% GC, 34.9).
Yeasts: Characteristics and Identification, First Edition Yeasts: Characteristics and Identification, Second Edition The taxonomy of the genus Saccharomyces MEYEN ex REESS: a short review for non-taxonomists
- J A Barnett
- R W Payne
- D Yarrow
- Usa
- J A Barnett
- R W Payne
- D Yarrow
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Nachweis hypervariabler repetitiver DNA-Sequenzen bei Pilzen mittels DNA-Fingerprinting
- W Meyer
Homologe und heterologe Transformation von Trichoderma reesei mit den Orotidin-5′-Phosphat-Decarboxylase Genen als Selektionsmarker
- F Gruber