Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Discrimination of Iranian honeybee populations (Apis mellifera meda) from commercial subspecies of Apis mellifera L. using morphometric and genetic methods

The morphological characters of honeybees have an important role for discriminating honeybee subspecies. In the present research, Iranian populations of honeybee (Apis mellifera) were collected from 19 areas in Iran. The samples were collected from stationary beekeeping sites. Moreover, pictures of honeybee forewings held in the Bee Data Bank in Oberursel were compared with Iranian honeybee populations. 19 morphological characters were measured for each forewing of worker honeybee to evaluate differentiation of Iranian honeybee populations from the commercial honeybee subspecies A. m. mellifera, A. m. carnica, A. m. caucasica and A. m. ligustica. Additionally, part of the tRNA^leu gene, an intergenic region and part of COII was used to confirm differentiation of the commercial subspecies from Iranian honeybee populations. Results of the cluster analyses showed that 19 morphological characters of forewings differentiated Iranian populations from the commercial subspecies. Moreover, the phylogenetic tree of part of the tRNA^leu gene, an intergenic region and part of COII differentiated the commercial subspecies from Iranian honeybee populations. Results of the discriminant function analyses (DFA) indicated that the references samples of A. m. meda overlapped with Iranian populations.     

The Complex Biogeography of the Plant Pathogen Xylella fastidiosa: Genetic Evidence of Introductions and Subspecific Introgression in Central America

The bacterium Xylella fastidiosa is a plant pathogen with a history of economically damaging introductions of subspecies to regions where its other subspecies are native. Genetic evidence is presented demonstrating the introduction of two new taxa into Central America and their introgression into the native subspecies, X. fastidiosa subsp. fastidiosa. The data are from 10 genetic outliers detected by multilocus sequence typing (MLST) of isolates from Costa Rica. Six (five from oleander, one from coffee) defined a new sequence type (ST53) that carried alleles at six of the eight loci sequenced (five of the seven MLST loci) diagnostic of the South American subspecies Xylella fastidiosa subsp. pauca which causes two economically damaging plant diseases, citrus variegated chlorosis and coffee leaf scorch. The two remaining loci of ST53 carried alleles from what appears to be a new South American form of X. fastidiosa. Four isolates, classified as X. fastidiosa subsp. fastidiosa, showed a low level of introgression of non-native DNA. One grapevine isolate showed introgression of an allele from X. fastidiosa subsp. pauca while the other three (from citrus and coffee) showed introgression of an allele with similar ancestry to the alleles of unknown origin in ST53. The presence of X. fastidiosa subsp. pauca in Central America is troubling given its disease potential, and establishes another route for the introduction of this economically damaging subspecies into the US or elsewhere, a threat potentially compounded by the presence of a previously unknown form of X. fastidiosa.     

Genetic differentiation among local populations of Asian elephant

Electrohoretically detectable enetic variation for 29 kinds of blood protein encoded by 33 loci was analyzed for 78 Asian eletants (Elephas maximus) which were collected from its four local populations: Sri Lanka, Souti India, Thailand and Nepal. Elehants in Sri Lanka are classified into the subspecies E.m. maximus, and those from the other tlree localities into the subspecies E. m. indicus. Six variable loci were detected, and one of them, the tetrazolium oxidase locus, was observed to show a complete allele substitution between the subspecies. Average heterozgosity within local populations were in a range of 0.0152 ? 0.0303. Whereas the Nei's genetic distance among three local populations of the subspecies indicus were 0.0013 ? 0.0031, the distance between the subspecies indicus and maximus were 0.0328 ? 0.0370, indicating that the two subspecies were well differentiated genetically.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Molecular phylogeny of the salmonellae: relationships among Salmonella species and subspecies determined from four housekeeping genes and evidence of lateral gene transfer events

The salmonellae are a diverse group of bacteria within the family Enterobacteriaceae that includes two species, Salmonella enterica and Salmonella bongori. In order to characterize the phylogenetic relationships of the species and subspecies of Salmonella, we analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongori were included in this study. We present a robust phylogeny of the Salmonella species and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of intersubspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.     

Genetic Divergence of Orangutan Subspecies (Pongo pygmaeus)

Microsatellites and mitochondrial DNA sequences were studied for the two subspecies of orangutans (Pongo pygmaeus), which are located in Borneo (P. p. pygmaeus) and Sumatra (P. p. abelii), respectively. Both subspecies possess marked genetic diversity. Genetic subdivision was identified within the Sumatran orangutans. The genetic differentiation between the two subspecies is highly significant for ND5 region but not significant for 16s rRNA or microsatellite data by exact tests, although F _ST estimates are highly significant for these markers. Divergence time between the two subspecies is approximately 2.3 ± 0.5 million years ago (MYA) estimated from our data, much earlier than the isolation of their geological distribution. Neither subspecies underwent a recent bottleneck, though the Sumatran subspecies might have experienced expansion approximately 82,000 years ago. The estimated effective population sizes for both subspecies are on the order of 10⁴. Our results contribute additional information that may be interpreted in the context of orangutan conservation efforts. Received: 13 June 2000 / Accepted: 30 January 2000     

Assessment of Daucus carota L. (Apiaceae) subspecies by chemotaxonomic and DNA content analyses

For a clearer distinction between the four subspecies of Daucus carota native from Portugal (subsp. carota, subsp. maximus, subsp. gummifer and subsp. halophilus), morphological features of the fruits, DNA content analyses by flow cytometry, and chemical characterization of the essential oils were undertaken.We found chemotaxonomic evidences to consider D. carota subsp. maximus as a separate species rather than a subspecies of D. carota. This separation is based on the morphometric analysis of the fruits and in the high levels of asarone present only in the essential oil of the subsp. maximus. The remaining subspecies are difficult to distinguish from each other based on the morphology of the fruits and in DNA content. However, based on the essential oils, it was possible to distinguish the subspecies halophilus from the other two (subsp. gummifer and subsp. carota) because of its high content of elemicin, with the other two having high levels of geranyl acetate.Based on these results, the subspecies maximus is proposed as a different species (Daucus maximus Desf.) and the taxonomic status of other three subspecies (subsp. carota, subsp. gummifer and subsp. halophilus) is maintained. Still, the latter three taxa need to be further studied for a more precise taxonomic characterization.     

Isolation and characterization of microsatellite loci for the analysis of genetic diversity in Whitmania pigra

The objective of this study was to isolate microsatellite loci to analyze the genetic diversity of Whitmania pigra. Four new microsatellite markers of W. pigra were developed from an enriched library and ten from a modified SAMPL assay. A total of 127 alleles were detected, with an average of 9.1 alleles per locus. The expected heterozygosity (He) of each microsatellite locus varied from 0.451 to 0.857, with an average of 0.688. The polymorphism information content (PIC) of each microsatellite locus ranged from 0.361 to 0.838, with an average of 0.640. Analysis of molecular variance showed that the main variation component existed within the populations (81.64%) rather than among the populations (18.36%). Phylogenetic tree for 15 populations of Hirudo using the NJ method by MEGA 5.1 software were divided into two major clusters. These microsatellite markers will contribute to research on the individual identification, genetic diversity, population structure, genome mapping and conservation biology of Hirudo.     

Molecular genetic relationships and some issues of systematics of rock lizards of the genus Darevskia (Squamata: Lacertidae) based on locus analysis of SINE-type repeats (Squam1)

To study the molecular genetic relationships and correlate them with the taxonomy within the complex of lacertid lizards of the genus Darevskia, the locus analysis of the copies of the SINE-type repeat (Squam1) specific for the order Squamata was used. It was demonstrated that one of the loci (No. 34) contained the Squam1 copy insert in all species and subspecies of the examined genus. SINE allelic copies in some of the loci contained large indels and specific sets of mutations. The allelic variant M (medium, about 340 bp) was found most frequently; it was detected in all subspecies of D. saxicola (saxicola, darevskii, szczerbaki, lindholmi) and in most of the other species of the genus. Two species, D. derjugini and D. praticola, differed from the other species in the presence of long (L) and short (S) alleles. The longest allele was characteristic of the D. derjugini population from the Northern Caucasus (L, 379 bp, ssp. silvatica), while the shortest allele (97 bp) united the derjugini and barani subspecies. The second allele S (279 bp) characterizes the subspecies D. praticola praticola, some individuals of which also carry allele M. The second subspecies, D. p. pontica, contains allele L2, which differs from all other medium alleles in the presence of strictly specific short indel. In addition to apomorphic indels, the specificity and mutation distribution patterns among the Squam1 alleles were also examined. An analysis of the NJ tree indicated the concordance between morphological and molecular genetic characters of the species derjugini, praticola, and saxicola. Furthermore, four subspecies of D. saxicola were much closer to each other than the subspecies within the first two species; D. d. silvatica and the group of D. d. derjugini + barani were clearly separated. It cannot be excluded that populations from Azerbaijan and Serbia can be treated as the independent subspecies of D. praticola.     

Electrophoretic study of isozyme patterns in some wild populations of Aubrieta columnae Guss. (Cruciferae)

Aubrieta columnae Guss. is currently divided into three subspecies: A. columnae subsp. columnae, A. columnae subsp. italica, both found as isolated and fragmented populations in rocky habitats of Central and Southern Apennines (Italy), and A. columnae subsp. croatica found in the Balkan region. In order to gain information about the degree of genetic variability and to clarify taxonomic relationships among these taxa, we studied the isozyme patterns at 8 marker loci of 376 individuals from 8 populations by means of starch gel electrophoresis. Data analysis by using Wright's F-statistics and UPGMA clustering method was performed. The results show: 1) a general deviation from Hardy–Weinberg equilibrium within each subspecies; 2) a lesser genetic variation in populations occurring in habitats characterized by milder climatic conditions and relatively small seasonal variations; 3) a relatively high degree of differentiation between the three subspecies; 4) the possible common transadriatic origin of A. columnae subsp. italica and A. columnae subsp. croatica; 5) the possible origin of A. columnae subsp. columnae from A. columnae subsp. italica; and 6) that the current taxonomic status of A. columnae may be substantially confirmed, even if the findings are from a limited number of loci explored.     

Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.     

The genetic status of naturally occurring black-nosed impala from northern South Africa

The impala (Aepyceros melampus ssp.) is a widespread antelope species occurring in sub-Saharan Africa. The two recognized subspecies have non-overlapping distribution ranges, with no known natural mixture of these subspecies until human interference. A number of common impala individuals (A. m. melampus) displaying phenotypic characteristics commonly observed in the black-faced impala (A. m. petersi), namely black facial markings, were seen on a farm in the Limpopo Province, South Africa. This farm falls outside the natural distribution range of A. m. petersi. We therefore aimed to identify the taxonomic placement of these individuals (i.e. A. m. melampus or A. m. petersi) through phylogenetic and molecular clock analyses using D-loop and cytochrome subunit b sequence data. Our results showed that these black-nosed impala from Limpopo are in fact A. m. melampus individuals. The existence of the black-nose phenotype in common impala could be more widespread than previously estimated. The occurrence of introgression between the two subspecies in this region could, however, not be fully excluded, and can only be fully assessed through the use of nuclear DNA analysis.     

Genetic Analysis of Cold Tolerance at the Germination and Booting Stages in Rice by Association Mapping

Low temperature affects the rice plants at all stages of growth. It can cause severe seedling injury and male sterility resulting in severe yield losses. Using a mini core collection of 174 Chinese rice accessions and 273 SSR markers we investigated cold tolerance at the germination and booting stages, as well as the underlying genetic bases, by association mapping. Two distinct populations, corresponding to subspecies indica and japonica showed evident differences in cold tolerance and its genetic basis. Both subspecies were sensitive to cold stress at both growth stages. However, japonica was more tolerant than indica at all stages as measured by seedling survival and seed setting. There was a low correlation in cold tolerance between the germination and booting stages. Fifty one quantitative trait loci (QTLs) for cold tolerance were dispersed across all 12 chromosomes; 22 detected at the germination stage and 33 at the booting stage. Eight QTLs were identified by at least two of four measures. About 46% of the QTLs represented new loci. The only QTL shared between indica and japonica for the same measure was qLTSSvR6-2 for SSvR. This implied a complicated mechanism of old tolerance between the two subspecies. According to the relative genotypic effect (RGE) of each genotype for each QTL, we detected 18 positive genotypes and 21 negative genotypes in indica, and 19 positive genotypes and 24 negative genotypes in japonica. In general, the negative effects were much stronger than the positive effects in both subspecies. Markers for QTL with positive effects in one subspecies were shown to be effective for selection of cold tolerance in that subspecies, but not in the other subspecies. QTL with strong negative effects on cold tolerance should be avoided during MAS breeding so as to not cancel the effect of favorable QTL at other loci.     

Comparison of acetate differential agar with sellers citrate-mannitol-agar

A total of 245 isolates of Escherichia coli (paracolons) were employed in comparing the use of acetate differential agar (ADA) with the simple test described by Sellers to eliminate these organisms from consideration as enteric pathogens. At 18 hr after the media were inoculated, 83% of the isolates produced a positive citrate reaction on Sellers citrate-mannitol-agar (SCM) and 7% gave a positive reaction on ADA. At 24 hr, 92% of the cultures were positive on SCM versus 42% on ADA. At 48 hr, 244 (99.6%) of the isolates were positive on SCM, whereas a 72-hr incubation period was required for equal results with ADA. One isolate failed to grow on either medium.     

The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare,Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463^T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36^T, genetic group 3 isolate 90-106^T, and genetic group 4 isolate Costa Rica 04-02-TN^T shared less than <98.8 % sequence identity to F. columnare ATCC 23463^T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463^T, genetic group 2 isolate AL-02-36^T, genetic group 3 isolate 90-106^T, and genetic group 4 isolate Costa Rica 04-02-TN^T were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36^T), F. davisii sp. nov. (90-106^T), and F. oreochromis sp. nov. (Costa Rica 04-02-TN^T) are proposed to represent genetic groups 2, 3, and 4, respectively.     

基于细胞色素b基因的中国岩羊不同地理种群遗传差异分析

为了揭示中国岩羊不同地理种群的遗传差异,探讨岩羊亚种分化的分子机制,采用中国岩羊不同地理种群的细胞色素b(Cyt b)基因的全序列,分析了碱基变异情况、遗传距离以及核苷酸序列差异。用最大似然法和贝叶斯法构建分子系统树并对获得的拓扑结构进行分析。结果发现,西藏亚种与四川亚种Cyt b基因平均序列差异为4.2%(±0.007),处于偶蹄目亚种的序列差异范围内,支持了目前对岩羊西藏亚种的分类地位。四川亚种内部各地理种群之间的遗传距离(0.033±0.0 111)与它们分别到西藏种群的遗传距离(0.042±0.007)差异不显著(t=1.824,P=0.084),说明四川亚种内部各地理种群间已经发生较显著的遗传分化。其中,四川、甘肃和青海种群亲缘关系较近,并与四川亚种内部的其它种群已产生了显著的遗传分化。因此认为四川亚种内部各地理种群的种下分化需要深入研究。     

Natural breeding with bulls experimentally infected with Neospora caninum failed to induce seroconversion in dams

Four bulls and 56 heifers seronegative to Neospora caninum were used to determine the feasibility of venereal transmission in bovine neosporosis under natural conditions. Bulls were experimentally infected with 10⁸ live N. caninum tachyzoites. Two of them with the Nc-1 isolate and the other two with the Nc-Spain-7 isolate. After 13 months of initial infection, each bull was re-infected with the same isolate and dose. The experiments were carried out from March to September during 2006 and 2007 where groups of cyclic heifers were naturally mated by the experimentally infected bulls. In year 2006, two bulls infected with different N. caninum isolate serviced 12 heifers each. In year 2007, the same bulls serviced the same heifers a second time (now primiparous) and six new heifers were also added to each group. In addition, the other two bulls serviced 10 additional heifers each. Experimental animals were monitored for 30 weeks and serum samples were collected weekly and fortnightly in years 2006 and 2007, respectively to evaluate the presence of specific antibodies to N. caninum. Experimentally infected bulls showed a significant increase of specific IgG antibodies from 13 (Nc-SP-7) and 21 (Nc-1) days post-infection. Serum IgG antibody responses of individual animals were similar in kinetics but slightly different in magnitude. Serum samples from heifers were all negative. Pregnant rates were 100% in heifers and 91% in primiparous animals. Calves did not show precolostral specific antibodies to N. caninum. Venereal transmission of bovine neosporosis under natural grazing conditions is unlikely to occur.     

Infraspecific classification reflects genetic differentiation in the widespread Petunia axillaris complex: A comparison among morphological,ecological, and genetic patterns of geographic variation

Plants are subjected to natural selection during expansion of their geographic range or when facing changes in environmental conditions, which in turn may affect phenotypic diversity. Studies on geographic phenotypic variation provide insight into the evolutionary processes and have long contributed to better understanding of diversification within and among species. Petunia axillaris is widely distributed in temperate South America, occurring throughout the Pampas region. The current taxonomy of this species recognizes three allopatric subspecies, which occupy nearly adjacent territories, according to floral morphology: P. axillaris subsp. axillaris, P. axillaris subsp. parodii, and P. axillaris subsp. subandina. In this study, we sampled the three P. axillaris subspecies and used both molecular markers (from the plastid and nuclear genomes) and morphological measurements to investigate how genetic diversity and morphological variation correlate to ecological variables and the geographic context. We used different forms of Mantel tests (partial and correlograms) to investigate the geographic distribution patterns in distinct types of similarity/dissimilarity among the populations and their relationships. We also modeled the morphological variation in P. axillaris as a function of the genetic marker frequencies in the populations. We found that the morphological differences leading to the recognition of different subspecies of P. axillaris reflect historical processes of isolation and that adaptation to different ecological conditions faced by each lineage is perhaps not merely a consequence of phenotypic plasticity. These findings suggest that differences between subspecies could represent an incipient speciation stage.