A new species of <Emphasis Type="Italic">Nilssoniocladus</Emphasis> Kimura et Sekido from the Lower Cretaceous of the Markovsky peninsula (Southern Primorye)

An imprint of shoot Nilssoniocladus Kimura et Sekido bearing leaves Nilssonia Brongniart was found for the first time in the Lower Cretaceous deposits of the Markovsky peninsula near the city of Vladivostok. A new species, Nilssoniocladus anatolii, is identified. The finds of such shoots in organic connection with leaves are rare, and new data supplement our knowledge on the diversity, stratigraphic and geographic distribution of the genus Nilssoniocladus.     

An endophytic fungus from <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. that produces azadirachtin

Azadirachtin A and its structural analogues are a well-known class of natural insecticides having antifeedant and insect growth-regulating properties. These compounds are exclusive to the neem tree, Azadirachta indica A. Juss, from where they are currently sourced. Here we report for the first time, the isolation and characterization of a novel endophytic fungus from A. indica, which produces azadirachtin A and B in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified as Eupenicillium parvum by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Azadirachtin A and B were identified and quantified by LC-HRMS and LC-HRMS², and by comparison with the authentic reference standards. The biosynthesis of azadirachtin A and B by the cultured endophyte, which is also produced by the host neem plant, provides an exciting platform for further scientific exploration within both the ecological and biochemical contexts.     

Diagnostic properties of three conventional selective plating media for selection of <Emphasis Type="Italic">Bacillus cereus</Emphasis>, <Emphasis Type="Italic">B. thuringiensis</Emphasis> and <Emphasis Type="Italic">B. weihenstephanensis</Emphasis>

The aim of this study was to assess the diagnostic properties of the two selective plating media and a chromogenic medium for identification of Bacillus cereus. The 324 isolates were B. cereus (37%), Bacillus weihenstephanensis (45%) or Bacillus thuringiensis (18%), as identified by a new combination of techniques. All isolates were growing on mannitol–egg yolk–polymyxin agar (MYP), and they did not form acid from mannitol. However, a significant lower number of B. thuringiensis isolates did not show lecithinase activity. All isolates were also growing on polymyxin–egg yolk–mannitol–bromothymol blue agar (PEMBA); however, 11% isolates indicated that they did produce acid from mannitol, and 15% isolates did not show any lecithinase activity. Five of the isolates did not grow at all on the chromogenic agar, and 14 of the growing isolates were β-glucosidase negative. It is concluded that the two recommended selective plating media MYP and PEMBA for detection of B. cereus group bacteria both have their limitations for identification of some B. cereus, B. weihenstephanensis or B. thuringiensis. However, MYP is preferable compared to PEMBA. The chromogenic medium has its own advantages and limitations, and some of the limitations seem to be solved by incubation at 30°C instead of the recommended 37°C.     

Typification,taxonomy, and worldwide distribution of Braunia secunda (Hook.) Bruch & Schimp. (Hedwigiaceae)

Diagnosis and illustrations are given for Braunia secunda (Hook.) Bruch & Schimp., and a Humboldt and Bonpland specimen (BM) is selected as the lectotype. Also a collection by Arséne (BM) is designated as the lectotype for B. secunda var. crassiretis Thér. This variety is retained as synonym of B. secunda. The species concept of B. secunda is revised and a provisional key is provided to help in the identification of 23 species of Braunia, based on examination of herbarium specimens worldwide. About half of specimens from Mexico actually represent another species, B. andrieuxii Lorentz. All material examined from India, previously identified as B. secunda belongs to B. macropelma (Müll.Hal.) A.Jaeger, whereas collections from Africa are actually B. rupestris (Mitt.) A.Jaeger, B. entodonticarpa Müll.Hal., or B. diaphana (Müll.Hal.) A.Jaeger. These and other species should no longer be considered synonyms of B. secunda. The worldwide distribution of the species is documented for the United States, Mexico, Guatemala, Dominican Republic, Haiti, Venezuela, and Bolivia.     

Coleifolides A and B,Two New Sesterterpenoids from the Aerial Parts of Scutellaria coleifolia H.Lév.

Coleifolides A and B ( 1 and 2 ), two new sesterterpenoids with a β‐methyl‐α,β‐unsaturated‐γ‐lactone moiety, were isolated from the aerial parts of Scutellaria coleifolia H.Lév . (Lamiaceae), together with three known compounds. Their structures were elucidated by NMR and MS examinations. Coleifolides A and B were concluded to be partially racemic compounds by the HPLC analysis using a chiral column or introduction of chiral derivatizing agents. The absolute configuration of the major isomer was determined by analyses of the CD spectrum as well as NMR data of (R)‐ and (S)‐2‐NMA derivatives. Coleifolides A and B are structurally similar to manoalide derivatives, previously isolated from marine sponges, and appear to be the first examples of this type of compounds being isolated from higher plants.     

Polymorphism of the <Emphasis Type="Italic">CONSTANS</Emphasis> gene in <Emphasis Type="Italic">Brassica</Emphasis> plants

Using a direct amplification of genomic DNA from two Brassica rapa forms, we obtained two homologs of the CONSTANS gene, which controls the photoperiodic induction of flowering in Arabidopsis plants. The cloned fragments of B. rapa genome were identified as members of the CONSTANS-LIKE1 class. By aligning the nucleotide sequences of the CONSTANS gene and its homologs, three classes, CONSTANS, CONSTANS-LIKE1, and CONSTANS-LIKE2, were distinctly discerned by their primary structure. The pattern of restriction fragment length polymorphisms (RFLP) of the CONSTANS homologs in B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa were genome-specific; in addition, the CONSTANS homologs were classified by plant geographic origin, and we assume that such classification is related to plant photoperiodic response.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 274–281.Original Russian Text Copyright © 2005 by Martynov, Khavkin.This revised version was published online in April 2005 with a corrected cover date.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Detection,introgression and localization of genes conferring specific resistance to <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> from <Emphasis Type="Italic">Brassica rapa</Emphasis> into <Emphasis Type="Italic">B. napus</Emphasis>

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of “new” resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.     

Reconsideration of the African species <Emphasis Type="Italic">Sesbania sudanica</Emphasis> and <Emphasis Type="Italic">S. hepperi</Emphasis> (Leguminosae subfam. Papilionoideae) as to their variation in Ethiopia

Summary  Recently collected material of Sesbania (Leguminosae subfam. Papilionoideae) from western Ethiopia shows variation and character combinations not recorded in J. B. Gillett’s revision of Sesbania from Africa and tropical Arabia from 1963 and combines three taxa in his revision, S. sudanica J. B. Gillett, S. sudanica subsp. occidentalis J. B. Gillett and S. hepperi J. B. Gillett. The three taxa are therefore united into one more variable species, S. sudanica, that can still be distinguished from other Sesbania species with no or hardly any free tips on the appendages on the standard by having the style pilose or tomentose and a long sterile rostrum on a non- or only faintly torulose pod. The amended S. sudanica is distributed throughout the Sudanian woodland zone from near the Atlantic to the foothills of the Ethiopian highlands. The distribution area agrees well with the Sudanian region in Frank White’s classification of African phytochoria.     

Chemical Composition of Ballota macedonica Vandas and Ballota nigra L. ssp. foetida (Vis.) Hayek Essential Oils – The Chemotaxonomic Approach

The essential oils isolated from fresh aerial parts of Ballota macedonica (two populations) and Ballota nigra ssp. foetida were analyzed by GC and GC/MS. Eighty five components were identified in total; 60 components in B. macedonica oil (population from the Former Yugoslav Republic of Macedonia), 34 components in B. macedonica oil (population from the Republic of Serbia), and 33 components in the oil of B. nigra ssp. foetida accounting for 93.9%, 98.4%, and 95.8% of the total oils, respectively. The most abundant components in B. macedonica oils were carotol (13.7 – 52.1%), germacrene D (8.6 – 24.6%), and (E)‐caryophyllene (6.5 – 16.5%), while B. nigra ssp. foetida oil was dominated by (E)‐phytol (56.9%), germacrene D (10.0%), and (E)‐caryophyllene (4.7%). Multivariate statistical analyses (agglomerative hierarchical cluster analysis and principal component analysis) were used to compare and discuss relationships among Ballota species examined so far based on their volatile profiles. The chemical compositions of B. macedonica essential oils are reported for the first time.     

A taxonomic Study of Species of Bothriocephalus Rudolphi, 1808 (Cestoda: Pseudophyllidea) from Eels in Japan: Morphological and Molecular Evidence for the Occurrence of B. claviceps (Goeze, 1782) and Confirmation of the Validity of B. Japonicus Yamaguti, 1934

A taxonomic study of specimens of Bothriocephalus from eels (Anguilla spp.) in Japan has demonstrated the occurrence of two species, B. claviceps (Goeze, 1782) and B. japonicus Yamaguti, 1934. The former species is a parasite of eels (A. anguilla and A. rostrata) in the Holarctic Region and was recently reported from A. marmorata in Japan. The conspecificity of tapeworms newly found in an eel (A. ? japonica) from Lake Biwa, central Japan, with B. claviceps has been confirmed by the great similarity of their ITS-2 gene sequences (similarity 95.3% and 95.2%). However, the sequences of worms identified as B. claviceps from A. marmorata differed considerably from those of B. claviceps from two populations of A. anguilla from Europe and the above-mentioned one from Japan (similarity 66.3%, 67.1% and 65.1 %, respectively), thus indicating that the former cestodes may have been misidentified. This assumption was confirmed by morphological evaluation of a voucher specimen from A. marmorata. The morphology of this cestode, as well as those from A. japonica from two localities in Japan (Lakes Biwa and Suwa), indicates their conspecificity with B. japonicus. The validity of this taxon has been confirmed on the basis of a re-examination of the type-specimens. The two taxa, B. japonicus and B. claviceps, differ from each other in the shape and length of the scolex (619–730 μm in B. japonicus versus 1,180–2,100 μm in B. claviceps), the relative position of the cirro-vaginal and uterine pores (opposite each other in relation to the median line of the body in B. japonicus versus tandem or slightly offset along the median line in the latter species), and the size of the eggs (41–52 × 28–35 μm in B. japonicus versus 50–70 × 31–43 μm in B. claviceps).

     

Simultaneous detection of pathogenic <Emphasis Type="Italic">B. cereus,S. aureus</Emphasis> and <Emphasis Type="Italic">L. monocytogenes</Emphasis> by multiplex PCR

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.     

Identification and Expression of a <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> Collagenase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis. A number of protein bands were present in the B. pseudomallei profile but absent or less abundant in the B. thailandensis profile. Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B. pseudomallei genome sequence database. The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B. pseudomallei and B. thailandensis. The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli. Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.     

Antiamnesic Effect of <Emphasis Type="Italic">B. monniera</Emphasis> on L-NNA Induced Amnesia Involves Calmodulin

Amnesia may result from ageing, chronic drug abuse or head injury and there are limited therapeutic strategies to such conditions. We have shown that Bacopa monniera, a memory enhancing drug can reverse both diazepam and scopolamine induced amnesia in mice. In order to understand the downstream effects of B. monniera, this study was designed to investigate how B. monniera antagonizes MK801, an NMDA receptor antagonist and N _ω-Nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor. We compared the degree of reversal B. monniera imparts on MK801 and L-NNA induced anterograde amnesia in experimental mice. Our data revealed that L-NNA induced anterograde amnesia was significantly reversed by B. monniera, however, it did not attenuate the MK 801 induced anterograde amnesia. B. monniera significantly increased calmodulin (CaM) and pCREB/CREB levels when the whole brain lysates of B. monniera pretreated amnesic mice were compared with those of L-NNA treated mice. We conclude that antiamnesic effect B. monniera on L-NNA induced amnesia may be mediated by NO pathyway involving CaM, which is required for LTP sustenance. These studies evoke interest in their future development as potential antiamnesic drugs.     

Electroporation is a feasible method to introduce circularized or linearized DNA into <Emphasis Type="Italic">B. subtilis</Emphasis> chromosome

Here, we present the electroporation as a feasible and efficient method for introducing circularized and linearized DNA into Bacillus subtilis chromosome. Two integration experiments were carried out and demonstrated the feasibility and efficiency of electroporation to introduce the target DNA into the B. subtilis chromosome. By using of electroporation, a multiple-cistron contained five genes from B. subtilis biotin biosynthetic pathway was introduced into the B. subtilis chromosome efficiently and created a repeated copy in chromosome via a single crossover event. Then an ectopic promoter was introduced conveniently into the upstream of one of the repeated multiple-cistron via a double crossover event. To further demonstrate the application of electroporation in genetic research, the early sporulation gene spo0A of B. subtilis was knocked out and, consequently, the null of sporulation and logged growth was observed in this study. Thus, the electroporation as an alternative method of integration in B. subtilis is feasible and practical.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Shifts in the distributional ranges of <Emphasis Type="Italic">Boophilus</Emphasis> ticks in Tanzania: evidence that a parapatric boundary between <Emphasis Type="Italic">Boophilus microplus</Emphasis> and <Emphasis Type="Italic">B. decoloratus</Emphasis> follows climate gradients

Based on a national tick survey conducted in Tanzania between 1998 and 2001, predictive maps of tick distribution for B. microplus and B. decoloratus were computed and compared with historical maps that originated about 40 years ago. The new data collected showed that except for extremely cold and dry areas B. microplus has extended its distribution range and is now present in all the northern regions of Tanzania, and that high suitability is currently recorded for most of the previously non-occupied areas. In contrast, B. decoloratus, once abundant in Tanzania, has largely retreated to highlands in north and central Tanzania and several other high-altitude refuges. Geostatistical analysis revealed a dissimilar character of distribution for the two species at a local and regional scale, as well as an antagonistic relationship between them in a spatial context. The Tanzania data suggest that an equilibrium phase with a stable parapatric boundary that follows a climate gradient has been achieved. The zone of overlap in the distribution ranges of B. microplus and B. decoloratus is characterized by a decreased infestation rate as well as mutual exclusion on infested cattle. This finding matches studies on parapatry with hybrid zones, where the latter zones that separate species tend to rest in density troughs. Analysis of the Tanzania data shows that the critical factor for the advance of B. microplus and the retreat of B. decoloratus is associated with the 58 mm isohyet and the 22–23°C isotherm and indicates a well developed higher-temperature tolerance for B. microplus. It can be anticipated that climate changes may enhance the spread of B. microplus and consequently Babesia bovis into new areas of the African continent.     

Biodegradation of 2-Nitrotoluene by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain SMN-1

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.     

Protein profile determination of <Emphasis Type="Italic">Borrelia afzelii</Emphasis> and <Emphasis Type="Italic">Borrelia garinii</Emphasis> isolated from skin and cerebrospinal fluid

In Europe the initial skin manifestation of Lyme borreliosis (erythema migrans) is predominantly caused by Borrelia afzelii while nervous system involvement is usually associated with Borrelia garinii. The aim of our study was to compare protein profiles of B. afzelii and B. garinii isolated from skin of patients with erythema migrans and from cerebrospinal fluid (CSF) of patients with Lyme neuroborreliosis. We analyzed 187 Borrelia strains, 74 B. afzelii and 113 B. garinii, for the presence of flagellin, and outer surface proteins A, B and C. Their protein profiles were obtained by SDS–PAGE and analyzed by computer program Gel Doc. Differences in the presence of proteins were found comparing isolates according to species (B. afzelii and B. garinii) and to their origin (skin and CSF isolates); the heterogeneity regarding the presence or absence of borrelial proteins was established within particular species. Protein profiles of the analyzed strains showed differences in the number, amount and molecular mass of analyzed proteins. Distinctions in the synthesis of outer surface proteins may play a role in the dispersal of borreliae within and between animal reservoir and vector ticks, as well as in pathogenesis of Lyme borreliosis in humans.