Selection and identification of autochthonous yeasts in Slovakian wine samples using a rapid and reliable three-step approach

Aims: The investigation of yeast microflora during the must fermentation of two wine varieties (Frankovka modra – Blaufränkisch and Veltlinske zelene – Grüner Veltliner) from two consecutive vintages was performed using a three‐step approach. Methods and Results: The investigation strategy consisted of the combination of yeast cultivation, selection of the isolated yeasts based on the amplification of internal transcribed spacer 2 using a fluorescence‐labelled primer (f‐ITS‐PCR) and a final identification step based on amplification and sequencing of the ITS1‐5.8S rDNA‐ITS2 region of the selected yeasts. By this three‐step approach, it was possible to screen 433 yeasts isolates that belonged to 13 different species. Conclusions: The f‐ITS‐PCR allowed the unambiguous differentiation of all isolated yeast species that produced their typical f‐ITS‐PCR profile. Significance and Impact of the Study: This is one of few reports that treat the yeast diversity in Slovakian wines and in two varieties largely cultivated in Central Europe. The three‐step approach permitted the rapid and reliable identification of isolated yeasts. The f‐ITS‐PCR with its good discrimination power can represent a suitable molecular tool for the selection of yeast members recovered from food or other environments.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.     

Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

ITS1: a DNA barcode better than ITS2 in eukaryotes?

A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer (ITS/ITS2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS1 and ITS2 regions. Here, a large‐scale meta‐analysis was carried out to compare ITS1 and ITS2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity‐based methods, we calculated species discrimination efficiencies for ITS1 and ITS2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS1 has significantly higher efficiencies than ITS2 in 17 of the 47 families and 20 of the 49 genera, which are sample‐rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS1 primers was found superior to that of ITS2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS1 for sequencing. In summary, ITS1 represents a better DNA barcode than ITS2 for eukaryotic species.     

Specific PCR-based detection of Phomopsis heveicola the cause of leaf blight of Coffee (Coffea arabica L.) in China

Coffee (Coffea arabica L.) is currently grown in many tropical and subtropical areas countries and is a major traded commodity for the developing world. Coffee leaf blight, caused by Phomopsis heveicola, is one of the most important fungal diseases dangerous to coffee crops in China. This study aimed to develop a PCR-based diagnostic method for detecting P. heveicola in planta. Specific primers (CPHF/CPHR) were designed based on sequence data of region of internal transcribed spacer (ITS1 and ITS4) of P. heveicola. The efficiency and specificity of CPHF/CPHR were established by PCR analysis of DNA from P. heveicola strains isolated from China and fungal isolates of other genera. A single amplification product of 318 bp was detected from DNA P. heveicola isolates. No amplification product was observed with any of the other fungal isolates tested. The specific primers designed and employed in PCR detected P. heveicola up to 3 pg from DNA isolated. This is the first report on the development of a species-specific PCR assay for identification and detection of P. heveicola. Thus, the PCR-based assay developed was very specific, rapid and sensitive tool for the detection of pathogen P. heveicola.     

Internal transcribed spacer 2 amplicon as a molecular marker for identification of Peronospora parasitica (crucifer downy mildew)

AIMS: The purpose of the study was to characterize the internal transcribed spacer (ITS) regions of Peronospora parasitica (crucifer downy mildew) in order to evaluate their potential as molecular markers for pathogen identification. METHODS AND RESULTS: PCR amplification of ribosomal RNA gene block (rDNA) spacers (ITS1 and ITS2) performed in 44 P. parasitica isolates from different Brassica oleracea cultivars and distinct geographic origins, revealed no length polymorphisms. ITS restriction analysis with three endonucleases, confirmed by sequencing, showed no fragment length polymorphisms among isolates. Furthermore, ITS amplification with DNA isolated from infected host tissues also allowed the detection of the fungus in incompatible interactions. The combination of the universal ITS4 and ITS5 primers, for amplification of full ITS, with a new specific forward internal primer for ITS2 (PpITS2F), originates a P. parasitica specific amplicon, suitable for diagnosis. CONCLUSIONS: As ITS2 regions of P. parasitica, B. oleracea, other B. oleracea fungal pathogens and other Peronospora species are clearly distinct, a fast and reliable molecular identification method based on multiplex PCR amplification of full ITS and P. parasitica ITS2 is proposed for the diagnosis of crucifer downy mildew. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be applied to diagnose the disease in the absence of fungal reproductive structures, thus being useful to detect nonsporulating interactions, early stages of infection on seedlings, and infected young leaves packed in sealed plastic bags. Screening of seed stocks in sanitary control is also a major application of this diagnostic method.     

Use of the rpoB gene as an alternative to the V3 gene for the identification of spoilage and pathogenic bacteria species in milk and milk products

Aim: To evaluate the rpoB gene as an alternative to the V3 gene for the identification of bacterial species in milk and milk products. Methods and Results: DNA obtained from different bacterial species strains was amplified by PCR using rpoB primers. PCR products of each bacterial species were then separated on a DGGE gel. The molecular fingerprints of the bacterial species tested were integrated into a database. The DGGE analysis shows a single band for the rpoB gene amplicons per each bacterial species. Comparison of electrophoretic profiles obtained from V3 16S rDNA amplification with those from this study obtained with rpoB showed that for some bacterial species that co‐migrated after amplification of the V3 region, distinct bands were observed on the gel with the amplification products of the rpoB region. Conclusions: The results obtained in this study show the discriminatory power of the rpoB gene, indicating that it can be used as an alternative to the V3 16S rRNA gene for the identification of bacterial species in milk and milk products. Significance and Impact of the Study: PCR‐DGGE targeting the rpoB gene is a way of discriminating the bacterial species that co‐migrated with the amplification of the V3 gene and so avoids the sequencing of the co‐migrating bands.     

Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization

Aims: The aim of this study was to explore a new PCR target gene for Vibrio parahaemolyticus, based on the histone‐like nucleoid structure (H‐NS) gene. Methods and Results: Primers for the H‐NS gene were designed for specificity to V. parahaemolyticus and incorporated into a PCR assay. The PCR assay was able to specifically detect all of the 82 V. parahaemolyticus strains tested, but did not result in amplification in the 47 other Vibrio spp. and nonVibrio spp. strains. The detection limit of the PCR assay was 0·14 pg purified genomic DNA and 1·8 × 10⁵ CFU g^?1 spiked oyster samples from V. parahaemolyticus RIMD2210633. Furthermore, a multiplex PCR assay targeting the hns, tdh and trh genes was successfully developed to detect virulent V. parahaemolyticus strains. Conclusions: The H‐NS‐based PCR assay developed in this study was sensitive and specific, with great potential for field detection of V. parahaemolyticus in seawater or seafood samples. Significance and Impact of the Study: The H‐NS gene was validated as a new specific marker gene in PCR assays for accurate detection and identification of V. parahaemolyticus, which has the potential to be applied in diagnostics and taxonomic studies.     

Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high‐resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two‐step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1‐5.8S‐ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.     

ITS as an environmental DNA barcode for fungi: an <Emphasis Type="Italic">in silico</Emphasis> approach reveals potential PCR biases

Background  

During the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates.     

Investigation on Natural Diets of Larval Marine Animals Using Peptide Nucleic Acid-Directed Polymerase Chain Reaction Clamping

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.     

Direct PCR of indigenous and invasive mosquito species: a time‐ and cost‐effective technique of mosquito barcoding

Millions of people die each year as a result of pathogens transmitted by mosquitoes. However, the morphological identification of mosquito species can be difficult even for experts. The identification of morphologically indistinguishable species, such as members of the Anopheles maculipennis complex (Diptera: Culicidae), and possible hybrids, such as Culex pipiens pipiens/Culex pipiens molestus (Diptera: Culicidae), presents a major problem. In addition, the detection and discrimination of newly introduced species can be challenging, particularly to researchers without previous experience. Because of their medical importance, the clear identification of all relevant mosquito species is essential. Using the direct polymerase chain reaction (PCR) method described here, DNA amplification without prior DNA extraction is possible and thus species identification after sequencing can be achieved. Different amounts of tissue (leg, head; larvae or adult) as well as different storage conditions (dry, ethanol, ?20 and ?80 °C) and storage times were successfully applied and showed positive results after amplification and gel electrophoresis. Overall, 28 different indigenous and non‐indigenous mosquito species were analysed using a gene fragment of the COX1 gene for species differentiation and identification by sequencing this 658‐bp fragment. Compared with standard PCR, this method is time‐ and cost‐effective and could thus improve existing surveillance and control programmes.     

Identification of clinically relevant yeasts by PCR/RFLP

For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.     

The phylogenetic relationship and non‐specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium)

To investigate the specificity of the symbiotic relationship between Cymbidium plants and their mycorrhiza fungi, thirty mycorrhiza fungi were isolated from roots of six terrestrial Cymbidium species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) with universal fungal primers ITS1/ITS4. All fungal strains isolated from natural roots of orchids were inoculated into the rhizomes of in vitro Cymbidium goeringii. Phylogenetic analysis indicated fungal isolates of different cluster could be obtained from a special terrestrial Cymbidium species. Observation of light microscope and scanning electron microscope showed that fungi entered the cortical tissue by destroying cell wall of epidermal cells, where they formed hyphal knots in the cortical cells and were digested gradually. A large number of small protuberances were visible on cross sections of the rhizome. There was no strict inter‐species specificity between the isolated mycorrhiza fungi and terrestrial Cymbidium.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background

Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.

Methods

One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.

Results

For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.

Conclusions

rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.     

Analysis of Yeast-Like Symbiote Diversity in the Brown Planthopper (BPH), Nilaparvata lugens Stål, Using a Novel Nested PCR-DGGE Protocol

Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S–ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S–ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC–ITS2, ITS1FGC–ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.