Introduction: perspectives for economically important wild species and neglected crops in the Mediterranean

The Mediterranean region is one of the major centres of origin and diversification of cultivated plants and many crop wild relatives are found there. In addition, many native species are still widely harvested from the wild for food, medicine and other uses and some of these have potential for development as alternative crop especially in marginal zones. While there have been several recent initiatives that address the cataloguing and conservation of these species, such as the Network on Identification, Conservation and Use of Wild Plants in the Mediterranean Region (MEDUSA and the Bioversity International (IPGRI) studies on Underutilized Mediterranean Species (VMS), no comprehensive assessment has yet been made and little work undertaken on their agricultural potential. It has been confidently predicted that consequences of global change in the Mediterranean region - population movements and migrations, changes in disturbance regimes, and climate change - will be serious. One the one hand, this will affect the survival prospects of many of these underutilized species and on the other hand it will enhance their importance as the source of potential new crop germplasm. The conservation and availability of genetic diversity of both crops and underutilized species is essential if we are to be able to meet the increasing demand for food and other crops that will be adapted to the new ecoclimatic envelopes that will develop in the region as a consequence of global change. The rapid rate of climatic and other change that is expected adds urgency to the task of assessing, conserving and sustainably using this rich diversity of wild species of economic value in the region but new strategies will be need to be developed to achieve this. The Mediterranean region has the potential of becoming a major source of new crop development in the coming decades.

Retention time prediction and protein identification

Proteins are commonly identified through enzymatic digestion and generation of short sequence tags or fingerprints of peptide masses by mass spectrometry. Separation methods, such as liquid chromatography and electrophoresis, are often used to fractionate complex protein or peptide mixtures and these separations also provide information on the different species, such as molecular weight and isoelectric point from electrophoresis and hydrophobicity in reversed-phase chromatography. These are also properties that can be predicted from amino acid sequences derived from genomic sequences and used in protein identification. This chapter reviews recently introduced methods based on retention time prediction to extract information from chromatographic separations and the applications to protein identification in organisms with small and large genomes. Novel data on retention time prediction of posttranslationally modified peptides is also presented.

Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover

We have identified two mutations in the ace1 gene of Aphis gossypii that are associated with insensitivity of acetylcholinesterase (AChE) to carbamate and organophosphate insecticides. The first of these, S431F (equivalent to F331 in Torpedo californica), is associated with insensitivity to the carbamate insecticide pirimicarb in a range of A. gossypii clones. The S431F mutation is also found in the peach-potato aphid, Myzus persicae (Sulzer), and a rapid RFLP diagnostic allows the identification of individuals of both aphid species with a resistant genotype. This diagnostic further revealed the presence of S431 in several other pirimicarb-susceptible aphid species. The serine at this position in the wild-type enzyme has only been reported for aphids and provides a molecular explanation of why pirimicarb has a specific aphicidal action. A less specific insensitivity to a wide range of carbamates and organophosphates is associated with a second mutation, A302S (A201 in T. californica).

Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium, bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Isolation of culturable yeasts from market wines and evaluation of the 5 center dot 8S-ITS rDNA sequence analysis for identification purposes

Aims: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. Methods and Results: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. Conclusions: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. Significance and Impact of the Study: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.

Molecular identification and anti-pathogenic activities of putative probiotic bacteria isolated from faeces of healthy elderly individuals

One hundred and nine lactic acid bacterial strains (56 bifidobacteria-like and 53 lactobacilli-like) were isolated from faecal samples donated by healthy elderly individuals (>65 years old). Isolates were identified to species level by phenotypic analysis (by API) and by 16S rDNA sequencing. Eleven species of Lactobacillus and six species of Bifidobacterium were identified. The most frequently isolated lactobacillus was L. fermentum and the most frequently isolated bifidobacterium was closely related to B. infantis by 16S rDNA sequence alignment. The isolates were characterized for their antimicrobial activity against Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC) and Campylobacter jejuni. The lactobacilli displayed variations in their antimicrobial activity with few strains showing inhibitory activity against all pathogens. The bifidobacteria displayed higher levels of inhibitory activity against C. jejuni and Cl. difficile than against the E. coli strains. Keywords: Lactobacillus, Bifidobacterium, elderly, gastrointestinal microbiota, inhibition, Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC), Campylobacter jejuni.

Artificial keys for botanical identification using a multilayer perceptron neural network (MLP)

In this paper, practical generation of identification keys for biological taxa using a multilayer perceptron neural network is described. Unlike conventional expert systems, this method does not require an expert for key generation, but is merely based on recordings of observed character states. Like a human taxonomist, its judgement is based on experience, and it is therefore capable of generalized identification of taxa. An initial study involving identification of three species of Iris with greater than 90% confidence is presented here. In addition, the horticulturally significant genus Lithops (Aizoaceae/Mesembryanthemaceae), popular with enthusiasts of succulent plants, is used as a more practical example, because of the difficulty of generation of a conventional key to species, and the existence of a relatively recent monograph. It is demonstrated that such an Artificial Neural Network Key (ANNKEY) can identify more than half (52.9%) of the species in this genus, after training with representative data, even though data for one character is completely missing.

Cardinium endosymbionts are widespread in synanthropic mite species(Acari: Astigmata)

‘Candidatus Cardinium’ is an intracellular endosymbiont or parasite frequently occurring in invertebrates including mites and ticks. In this work we report Cardinium bacteria in Astigmata mites and explore their incidence in synanthropic species. Amplification of a 776 bp bacterial 16S rRNA gene fragment, using specific primers, enabled identification of closely related Cardinium sequences in 13 laboratory-reared populations of mites. In addition, Cardinium sequences were identified in three wild mite populations. Large scale screening of these populations showed 100% prevalence of Cardinium, representing the highest incidence compared to other major Chelicerate groups.

Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database (http://pgrc.ipk-gatersleben.de/poci).

Classification of seed storage behaviour of 67 Amazonian tree species

Information was collated on the seed storage behaviour of 67 tree species native to the Amazon rainforest of Brazil; 38 appeared to show orthodox, 23 recalcitrant and six intermediate seed storage behaviour. A double-criteria key based on thousand-seed weight and seed moisture content at shedding to estimate likely seed storage behaviour, developed previously, showed good agreement with the above classifications. The key can aid seed storage behaviour identification considerably.

Global transcriptome analysis and identification of differentially expressed genes after infection of Fragaria vesca with powdery mildew (Podosphaera aphanis)

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry

Effectiveness of mobile apps in teaching field-based identification skills

It has been suggested that few students graduate with the skills required for many ecological careers, as field-based learning is said to be in decline in academic institutions. Here, we asked if mobile technology could improve field-based learning, using ability to identify birds as the study metric. We divided a class of ninety-one undergraduate students into two groups for field-based sessions where they were taught bird identification skills. The first group has access to a traditional identification book and the second group were provided with an identification app. We found no difference between the groups in the ability of students to identify birds after three field sessions. Furthermore, we found that students using the traditional book were significantly more likely to identify novel species. Therefore, we find no evidence that mobile technology improved students’ ability to retain what they experienced in the field; indeed, there is evidence that traditional field guides were more useful to students as they attempted to identify new species. Nevertheless, students felt positively about using their own smartphone devices for learning, highlighting that while apps did not lead to an improvement in bird identification ability, they gave greater accessibility to relevant information outside allocated teaching times.

Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

Taxonomic and Phylogenetic Relationships Between Species of the Genus Culex (Diptera: Culicidae) From Brazil Inferred From the Cytochrome c Oxidase I Mitochondrial Gene

Species of the genus Culex Linnaeus have been incriminated as the main vectors of lymphatic filariases and are important vectors of arboviruses, including West Nile virus. Sequences corresponding to a fragment of 478 bp of the cytochrome c oxidase subunit I gene, which includes part of the barcode region, of 37 individuals of 17 species of genus Culex were generated to establish relationships among five subgenera, Culex, Phenacomyia, Melanoconion, Microculex, and Carrollia, and one species of the genus Lutzia that occurs in Brazil. Bayesian methods were employed for the phylogenetic analyses. Results of sequence comparisons showed that individuals identified as Culex dolosus, Culex mollis, and Culex imitator possess high intraspecific divergence (3.1, 2.3, and 3.5%, respectively) when using the Kimura two parameters model. These differences were associated either with distinct morphological characteristics of the male genitalia or larval and pupal stages, suggesting that these may represent species complexes. The Bayesian topology suggested that the genus and subgenus Culex are paraphyletic relative to Lutzia and Phenacomyia, respectively. The cytochrome c oxidase subunit I sequences may be a useful tool to both estimate phylogenetic relationships and identify morphologically similar species of the genus Culex.

Resurrection of Two Species From Synonymy of Anopheles (Nyssorhynchus) strodei Root, and Characterization of a Distinct Morphological Form From the Strodei Complex (Diptera: Culicidae)

Anopheles albertoi Unti and Anopheles arthuri Unti are revived from the synonymy with Anopheles strodei Root, and a distinct morphological form (designated in this study as Anopheles CP Form) from the Strodei Complex of Anopheles (Nyssorhynchus) is characterized. The male genitalia of An. arthuri and An. albertoi are described and illustrated for the first time. An. strodei, An. arthuri, and An. albertoi were first distinguished based on scanning electron microphotos of the eggs, and then each egg type was associated with diagnostic characters of the male genitalia. Identification of Anopheles CP Form was based on morphological characters of the male genitalia, characterized and illustrated in this study. Molecular phylogenetic analysis was most clear when an outgroup was not included, in which case using the nuclear white gene, or the white gene in combination with the mitochondrial cytochrome c oxidase subunit I (COI) gene, clearly separated these four taxa. When Anopheles quadrimaculatus Say and Anopheles stephensi Liston were included as an outgroup, combined white and COI data resolved An. strodei and An. albertoi, whereas An. arthuri was not well resolved. The single sequence of Anopheles CP Form was recovered well separated from other groups in all analyses.