Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.     

Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.     

Phylogeny of bovine species based on AFLP fingerprinting

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.     

SENSORY PROFILE DESCRIPTION OF MOZZARELLA CHEESE AND ITS RELATIONSHIP WITH CONSUMER PREFERENCE

The aim of this work was to set up the sensory profile and consumer preference test of three different Mozzarella cheeses (from full-fat cow milk, from low-fat cow milk and from full-fat water buffalo milk). The most remarkable difference in composition was the fat content. Nine commercial Mozzarella cheeses (three for each type) were evaluated in triplicate by a trained panel consisting of 10 assessors. Six Mozzarella cheeses (two for each type) out of the nine samples were selected and used for the preference test by 105 consumers. The three different Mozzarella cheeses were sensorially well differentiated. A good discrimination between the Mozzarella cheeses was obtained using the internal preference map method. The combination of cluster analysis and internal preference mapping has lead to a deeper understanding of consumer liking for Mozzarella cheeses.     

High genetic diversity vs. low genetic differentiation in Nouelia insignis (Asteraceae), a narrowly distributed and endemic species in China, revealed by ISSR fingerprinting

BACKGROUND AND AIMS: Nouelia insignis Franch., a monotypic genus of the Asteraceae, is an endangered species endemic in Yunnan and Sichuan Provinces of China. Most of the populations are seriously threatened. Some of them are even at the brink of extinction. In this study, the genetic diversity and differentiation between populations of this species were examined in two drainage areas. METHODS: DNA fingerprinting based on inter-simple sequence repeat polymorphisms was employed to detect the genetic variation and population structure in the species. KEY RESULTS: Genetic diversity at species level was high with P=65.05% (percentage of polymorphic loci) and Ht=0.2248 (total genetic diversity). The coefficient of genetic differentiation among populations, Gst, which was estimated by partitioning the total gene diversity, was 0.2529; whereas, the genetic differentiation between populations in the Jinsha and Nanpan drainage areas was unexpectedly low (Gst=0.0702). CONCLUSIONS: Based on the genetic analyses of the DNA fingerprinting, recent habitat fragmentation may not have led to genetic differentiation or the loss of genetic diversity in the rare species. Spatial apportionment of fingerprinting polymorphisms provides a footprint of historical migration across geographical barriers. The high diversity detected in this study holds promise for conservation and restoration efforts to save the endangered species from extinction.     

Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism

AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown. Previous attempts to fingerprint E. amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium. Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen. METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension. PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains. Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns. CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E. amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen. The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination. This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E. amylovora strains.     

Genetic diversity among Geotrichum candidum strains from various substrates studied using RAM and RAPD-PCR

AIMS: Assessment of genetic diversity within the species Geotrichum candidum and development of tools to trace the strains that play an important role in the agro food industry. METHODS AND RESULTS: RAM-PCR and RAPD-PCR techniques were assessed for their ability to discriminate 57 strains of various morphotypes, substrates and geographical origin. The techniques were complementary and, when combined, allowed us to discriminate isolates. Moreover, we established a link between a taxon and its occupation of an ecological niche, which should not be confused with the substrate of isolation. CONCLUSIONS: We observed a high degree of diversity, which could be linked to the variety of the ecological niches chosen and to the high degree of morphological polymorphism encountered within the species. SIGNIFICANCE AND IMPACT OF THE STUDY: Used in combination, RAM-PCR and RAPD-PCR permit traceability and monitoring systems for G. candidum strains during food processing.     

Sheep's and goat's dairy products in Italy: Technological,chemical, microbiological,and sensory aspects

Dairy sheep and goat breeding in Europe is most common around the Mediterranean basin, particularly in France, Greece, Italy, and Spain. The milk of sheep and goat is mainly reserved for cheese making that is usually conducted at farm level or in small local dairies. A large part of the production, principally for sheep milk cheese, is protected by the European Community (EC) regulations: the Protected Designation of Origin (PDO) and the Protected Geographic Indication (PGI). These designations constitute an element for the protection of the biodiversity, i.e. territory, animals, microbes, practices, and production systems. Microbiological features and diversity of sheep and goats milk compared to the cow's milk, give peculiar attributes to the cheeses. In several cases, rennet paste, produced from the abomasa of the suckling lambs or kids is used, strongly influencing chemical and sensory characteristics of the cheeses. In this review the technological, microbiological, chemical, and sensory aspects of the PDO and PGI Italian sheep and goat dairy products are reported.     

Biodiversity studies in Phaseolus species by DNA barcoding

The potential of DNA barcoding was tested as a system for studying genetic diversity and genetic traceability in bean germplasm. This technique was applied to several pure lines of Phaseolus vulgaris L. belonging to wild, domesticated, and cultivated common beans, along with some accessions of Phaseolus coccineus L., Phaseolus lunatus L., and Vigna unguiculata (L.) Walp. A multilocus approach was exploited using three chloroplast genic regions (rbcL, trnL, and matK), four intergenic spacers (rpoB-trnC, atpBrbcL, trnT-trnL, and psbA-trnH), and nuclear ITS1 and ITS2 rDNA sequences. Our main goals were to identify the markers and SNPs that show the best discriminant power at the variety level in common bean germplasm, to examine two methods (tree based versus character based) for biodiversity analysis and traceability assays, and to evaluate the overall utility of chloroplast DNA barcodes for reconstructing the origins of modern Italian varieties. Our results indicate that the neighbor-joining method is a powerful approach for comparing genetic diversity within plant species, but it is relatively uninformative for the genetic traceability of plant varieties. In contrast, the character-based method was able to identify several distinct haplotypes over all target regions corresponding to Mesoamerican or Andean accessions; Italian accessions originated from both gene pools. On the whole, our findings raise some concerns about the use of DNA barcoding for intraspecific genetic diversity studies in common beans and highlights its limitations for resolving genetic relationships between landraces and varieties.     

Independent maternal origin of Chinese swamp buffalo (Bubalus bubalis)

To obtain more knowledge on the origin and genetic diversity of the swamp buffalo (Bubalus bubalis) in China, the complete mitochondrial D-loop sequences of 119 samples representing seven native types were compared. Two mitochondrial DNA (mtDNA) lineages (lineages A and B) were determined for the Chinese swamp buffalo. Examination of the diversity patterns suggest that lineage A has undergone a population expansion event. Divergence of lineages A and B was estimated at 18,000 years ago. Combined analyses of mtDNA sequences from Chinese, Indian, Brazilian/Italian and Southeast Asian/Australian buffalo samples showed independent domestication events in the swamp buffalo from China and the river buffalo from the India subcontinent. The spread of swamp and river buffalo from China and India respectively to mainland Southeast Asia suggests that Southeast Asia is a hybrid zone for buffalo. Our data support the hypothesis of the evolution of domesticated swamp and river buffalo from ancestral swamp-like animals. These ancestral animals were extensively distributed across mainland Asia and most likely are represented today by the wild Asian buffalo (Bubalus arnee).     

糙皮侧耳(Pleurotus ostreatus)的AFLP指纹图谱分析

在对糙皮侧耳(Pleurotus ostreatus)的AFLP分析条件进行优化的基础上,利用该技术建立了14株产自不同地区的糙皮侧耳：DNA指纹图谱,并进行了数据分析。结果表明,在合成的14条引物的不同组合中,引物对E-3／M-3可以产生较多的DNA多态片段,E-AGC／M-CAT引物对的扩增效果最好,共获得184条DNA扩增带,其中多态性条带i101条,占54．89％。利用UPGMA法对所获数据进行聚类分析,计算得到糙皮侧耳菌株之间的遗传距离,发现不同品种间遗传距离差异较大,从0．192到0．754,说明糙皮侧耳的遗传多样性比较丰富。绘制的指纹分析树状图表明,14个糙皮侧耳菌株被分为6个组群,其中P17和杂3的相似性系数最高,达到了81．2％,而侧5与其他菌株的亲缘关系相对最远。     

Varietal Tracing of Virgin Olive Oils Based on Plastid DNA Variation Profiling

Olive oil traceability remains a challenge nowadays. DNA analysis is the preferred approach to an effective varietal identification, without any environmental influence. Specifically, olive organelle genomics is the most promising approach for setting up a suitable set of markers as they would not interfere with the pollinator variety DNA traces. Unfortunately, plastid DNA (cpDNA) variation of the cultivated olive has been reported to be low. This feature could be a limitation for the use of cpDNA polymorphisms in forensic analyses or oil traceability, but rare cpDNA haplotypes may be useful as they can help to efficiently discriminate some varieties. Recently, the sequencing of olive plastid genomes has allowed the generation of novel markers. In this study, the performance of cpDNA markers on olive oil matrices, and their applicability on commercial Protected Designation of Origin (PDO) oils were assessed. By using a combination of nine plastid loci (including multi-state microsatellites and short indels), it is possible to fingerprint six haplotypes (in 17 Spanish olive varieties), which can discriminate high-value commercialized cultivars with PDO. In particular, a rare haplotype was detected in genotypes used to produce a regional high-value commercial oil. We conclude that plastid haplotypes can help oil traceability in commercial PDO oils and set up an experimental methodology suitable for organelle polymorphism detection in the complex olive oil matrices.     

Investigating the relationship between raw milk bacterial composition, as described by intergenic transcribed spacer–PCR fingerprinting, and pasture altitude

Aims:  To assess the bacterial biodiversity level in bovine raw milk used to produce Fontina, a Protected Designation of Origin cheese manufactured at high-altitude pastures and in valleys of Valle d'Aosta region (North-western Italian Alps) without any starters. To study the relation between microbial composition and pasture altitude, in order to distinguish high-altitude milk against valley and lowland milk.
Methods and Results:  The microflora from milks sampled at different alpine pasture, valley and lowland farms were fingerprinted by PCR of the 16S–23S intergenic transcribed spacers (ITS-PCR). The resulting band patterns were analysed by generalized multivariate statistical techniques to handle discrete (band presence–absence) and continuous (altitude) information. The fingerprints featured numerous bands and marked variability indicating complex, differentiated bacterial communities. Alpine pasture milks were distinguished from lowland ones by cluster analysis, while this technique less clearly discriminated alpine pasture and valley samples. Generalized principal component analysis and clustering-after-ordination enabled a more effective distinction of alpine pasture, valley and lowland samples.
Conclusions:  Alpine raw milks for Fontina production contain highly diverse bacterial communities, the composition of which is related to the altitude of the pasture where milk was produced.
Significance and Impact of the Study:  This research may provide analytical support to the important issue represented by the authentication of the geographical origin of alpine milk productions.     

Detection of Staphylococcus aureus and enterotoxin genotype diversity in Monte Veronese, a Protected Designation of Origin Italian cheese

AIMS: To evaluate the risk associated with the load and enterotoxigenicity of Staphylococcus aureus in Monte Veronese, a PDO (Protected Designation of Origin) cheese of the Lessinia area (Verona, Italy). METHODS AND RESULTS: Staphylococcus aureus was quantified by a conventional culture method and by a nucA targeted real-time PCR assay developed in this study. Staphylococcus aureus numbers in cheese were higher than the limit tolerated by the Italian food legislation in 78% instances, according to both detection methods. Multiplex PCR tests for 17 Staph. aureus enterotoxin (SE) genes were applied to nucleic acids extracted from curds, cheeses and Staph. aureus isolates. The SE gene diversity appeared reduced after ripening. The gene encoding SED was found most frequently in dairy samples and the enterotoxin genes ser, sed, seg and sem predominated in the isolates. CONCLUSIONS: The occurrence of enterotoxigenic Staph. aureus strains with complex SE genotypes in this PDO cheese at numbers often exceeding the Italian tolerance threshold represents an important risk factor. SIGNIFICANCE AND IMPACT OF THE STUDY: The high frequency of contamination of Monte Veronese PDO cheese and, expectedly, similar typical productions from raw milk, by enterotoxigenic Staph. aureus imposes a tighter hygienic control in the earlier manufacturing phases.     

Genetic diversity and population structure of the Italian fungi belonging to the taxa Pleurotus eryngii (DC.:Fr.) Quèl and P. ferulae (DC.:Fr.) Quèl

A study using allozymes and PCR fingerprinting was conducted to estimate the genetic diversity of Italian populations of two economically important cultivated fungal taxa, Pleurotus eryngii and P. ferulae. Very little is known about the genetic diversity distribution pattern of these taxa. Heterozygote deficiency was observed at few loci; in fact the inbreeding coefficients were not high, which demonstrates that mechanisms restrain the inbreeding act at the local level. Estimates of genetic differentiation indicated a pattern of greater variation within, rather than between, populations. These results were supported by AMOVA analysis, which attributed a low proportion of the total genetic variation to large geographical scale divergence, and indicated that most of the genetic diversity was because of differences within populations. This distribution pattern of genetic variation of P. eryngii and P. ferulae populations seems to be the result of high gene flow, by efficient basidiospore dispersal, and outcrossing mechanisms, which restrain inbreeding within populations.     

Differential pathogenicity and genetic diversity among Pectobacterium carotovorum ssp. carotovorum isolates from monocot and dicot hosts support early genomic divergence within this taxon

The capability of Pectobacterium carotovorum isolates to infect monocotyledonous plants has been previously reported; however, no full consideration was given to characterize the association between such isolates and their monocot hosts. To assess differences in aggressiveness among P. carotovorum ssp. carotovorum isolates originating from monocotyledonous or dicotyledonous plants, we used as model plants two susceptible monocot hosts, the ornamentals Zantedeschia aethiopica and Ornithogalum dubium, as well as two common dicot hosts, Solanum tuberosum and Brassica oleracea. Using virulence assays and different genetic analyses we characterized P. carotovorum ssp. carotovorum isolates from diverse geographical locations which originated from plants belonging to four unrelated orders of monocots and five orders of dicots. Invariably, isolates originating from monocots exhibited higher virulence towards the tested monocot plants than dicot isolates, independently of their geographical source. Moreover, monocot and dicot isolates were clearly differentiated by various genetic analyses, such as 16S rRNA sequence clustering, intergenic transcribed spacer-PCR (ITS-PCR) banding pattern and amplified fragment length polymorphism (AFLP). We propose that the observed relationship between pathogenicity and genetic diversity among P. carotovorum ssp. carotovorum isolates reveals a co-evolutionary specialization trend in the interaction between this pathogen and its hosts.     

Integration of Retrotransposons-Based Markers in a Linkage Map of Barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Population structure of African buffalo inferred from mtDNA sequences and microsatellite loci: high variation but low differentiation

The African buffalo ( Syncerus caffer ) is widespread throughout sub-Saharan Africa and is found in most major vegetation types, wherever permanent sources of water are available, making it physically able to disperse through a wide range of habitats. Despite this, the buffalo has been assumed to be strongly philopatric and to form large aggregations that remain within separate home ranges with little interchange between units, but the level of differentiation within the species is unknown. Genetic differences between populations were assessed using mitochondrial DNA (control region) sequence data and analysis of variation at six microsatellite loci among 11 localities in eastern and southern Africa. High levels of genetic variability were found, suggesting that reported severe population bottlenecks due to outbreak of rinderpest during the last century did not strongly reduce the genetic variability within the species. The high level of genetic variation within the species was found to be evenly distributed among populations and only at the continental level were we able to consistently detect significant differentiation, contrasting with the assumed philopatric behaviour of the buffalo. Results of mtDNA and microsatellite data were found to be congruent, disagreeing with the alleged male-biased dispersal. We propose that the observed pattern of the distribution of genetic variation between buffalo populations at the regional level can be caused by fragmentation of a previous panmictic population due to human activity, and at the continental level, reflects an effect of geographical distance between populations.     

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish

AIMS: To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare. METHODS AND RESULTS: Genetic variability among Fl. columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP) fingerprinting. Thirty Fl. columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study. Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II. Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates. The maximum resolution was provided by AFLP fingerprinting, as up to 22 AFLP profiles could be defined within the species. CONCLUSIONS: We confirmed the division of Fl. columnare isolates from cultured fish into different genogroups. We showed that both genomovars I and II are present in channel catfish from the US. We described a unique genetic group represented by four Fl. columnare isolates from tilapia in Brazil which appears to be related to both genomovars. We were able to further subdivide the species by analysing the ISR. Finally, the use of AFLP allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl. columnare.