Microsatellite markers for two species of dampwood termites in the genus Zootermopsis (Isoptera: Termopsidae)

Dampwood termites in the genus Zootermopsis inhabit forested areas in western North America. To better understand the colony composition and breeding structure of Zootermopsis, we identified polymorphic microsatellite loci to use in population analysis. Microsatellite loci were isolated from Zootermopsis nevadensis nevadensis (Hagen); however, all primers amplified homologous loci in Zootermopsis angusticollis (Hagen) and Zootermopsis nevadensis nuttingi (Hagen). Twelve loci were polymorphic in one or more of the above subspecies and species. The number of alleles per locus ranged from one to six, with some allelic differences among subspecies and species. We are currently utilizing the microsatellite markers to investigate the population genetics of Zootermopsis.     

Molecular phylogenetics and anti-<Emphasis Type="Italic">Pythium</Emphasis> activity of endophytes from rhizomes of wild ginger congener, <Emphasis Type="Italic">Zingiber zerumbet</Emphasis> Smith

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50 %. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

Novel polymorphic microsatellite loci for distinguishing rock bass (<Emphasis Type="Italic">Ambloplites rupestris</Emphasis>), Roanoke bass (<Emphasis Type="Italic">Ambloplites cavifrons</Emphasis>), and their hybrids

The rock bass (Ambloplites rupestris) is a popular sport-fish native to the Mississippi and Great Lakes basins of North America. The species has been widely introduced outside its native range, including into Atlantic-slope streams of Virginia where it may hybridize with an imperiled, similar-looking congener, the Roanoke bass (Ambloplites cavifrons). In this study, we identified and evaluated novel molecular markers to facilitate identification of these species and study the extent of hybridization. Using molecular libraries developed from A. rupestris, we identified a suite of candidate nuclear microsatellite loci, synthesized primer sets, and tested these markers for amplification and polymorphism in populations of both species. We then calculated standard diversity statistics within and differentiation statistics between species, the latter providing an indication of marker power for distinguishing the species and their hybrids. Additionally, we evaluated our efficiency for identifying hybrids by classifying simulated genotypes of known ancestry. Eleven loci were polymorphic (2–22 alleles per locus) and reliably amplified in both species. Multilocus genetic differentiation between A. cavifrons and A. rupestris was quite high (F _ST  = 0.66; D _LR  = 19.3), indicating the high statistical power of this marker set for species and hybrid identification. Analyses of simulated data suggested these markers reliably distinguish between hybrids and non-hybrids, as well as between F1 hybrids and backcrossed individuals. This panel of 11 loci should prove useful for understanding patterns of hybridization between A. rupestris and A. cavifrons. As the first microsatellite markers developed for Ambloplites, these markers also should prove broadly useful for population genetic studies of this genus.     

Early characterization of somatic hybrids from symmetric protoplast electrofusion of <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> Dun. and <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Variability of 31 somatic hybrids of Solanum pinnatisectum Dun. with Solanum tuberosum L. for leaf morphology, plant vigor, resistance to Phytophthora infestans, ploidy level, and cytoplasm type was evaluated in vitro. The composition of these somatic hybrids was as follows: [S. pinnatisectum Dun. (2n = 2x = 24; cytoplasmic type Wγ) + S. tuberosum L. (2n = 4x = 48; cytoplasmic type Tß)]. Based on leaf morphology and plant growth vigor, plants were divided into three groups, including plants close to tbr parent with unlobed leaves, small plants with scarcely dissected leaves, and vigorous plants with asymmetrically and pinnately lobed leaves. Nine of the somatic hybrids were found to be highly resistant to P. infestans. Somatic hybrids were either tetraploid or hexaploid, with hexaploids being predominant. The cytoplasm of somatic hybrids was either Tßγ or Wßγ, with Tßγ being more common. Overall, in contrast to leaf morphology and growth vigor, level of resistance to P. infestans was not related to either ploidy level or type of cytoplasm. These findings demonstrate that early in vitro selection of promising hybrids can be useful in breeding programs.     

Geographic patterns of genetic variation in nuclear and chloroplast genomes of two related oaks (<Emphasis Type="Italic">Quercus aliena</Emphasis> and <Emphasis Type="Italic">Q. serrata</Emphasis>) in Japan: implications for seed and seedling transfer

In this study, we assessed geographic patterns of genetic variations in nuclear and chloroplast genomes of two related native oaks in Japan, Quercus aliena and Q. serrata, in order to facilitate development of genetic guidelines for transfer of planting stocks for each species. A total of 12 populations of Q. aliena and 44 populations of Q. serrata were analyzed in this study. Genotyping of nuclear microsatellites in Q. aliena was done with only nine populations (n = 212) due to limited numbers of individuals in two populations, while all 12 populations (n = 89) were used in sequencing chloroplast DNA (cpDNA). In Q. serrata, 43 populations (n = 1032) were genotyped by nuclear microsatellite markers, while cpDNA of 44 populations (n = 350) was sequenced. As anticipated, geographic patterns detected in the variations of Q. aliena’s nuclear genome and its chloroplast haplotype distribution clearly distinguished northern and southern groups of populations. However, those of Q. serrata were inconsistent. The geographic distribution of its chloroplast haplotypes tends to show the predicted differentiation between northern and southern lineages, but geographic signals in the genetic structure of its nuclear microsatellites are weak. Therefore, treating northern and southern regions of Japan as genetically distinct transferrable zones for planting stocks is highly warranted for Q. aliena. For Q. serrata, the strong NE-SW geographic structure of cpDNA should be considered.     

Genetic composition of interspecific potato somatic hybrids and autofused 4<Emphasis Type="Italic">x</Emphasis> plants evaluated by DArT and cytoplasmic DNA markers

Key message

Using DArT analysis, we demonstrated that all Solanum × michoacanum (+) S. tuberosum somatic hybrids contained all parental chromosomes. However, from 13.9 to 29.6 % of the markers from both parents were lost in the hybrids.

Abstract

Somatic hybrids are an interesting material for research of nucleus-cytoplasm interaction and sources of new nuclear and cytoplasmic combinations. Analyses of genomes of somatic hybrids are essential for studies on genome compatibility between species, its evolution and are important for their efficient exploitation. Diversity array technology (DArT) permits analysis of the composition of nuclear DNA of somatic hybrids. The nuclear genome compositions of 97 Solanum × michoacanum (+) S. tuberosum [mch (+) tbr] somatic hybrids from five fusion combinations and 11 autofused 4x mch were analyzed for the first time based on DArT markers. Out of 5358 DArT markers generated in a single assay, greater than 2000 markers were polymorphic between parents, of which more than 1500 have a known chromosomal location on potato genetic or physical map. DArT markers were distributed along the entire length of 12 chromosomes. We noticed elimination of markers of wild and tbr fusion components. The nuclear genome of individual somatic hybrids was diversified. Mch is a source of resistance to Phytophthora infestans. From 97 mch (+) tbr somatic hybrids, two hybrids and all 11 autofused 4x mch were resistant to P. infestans. The analysis of the structure of particular hybrids’ chromosomes indicated the presence of markers from both parental genomes as well as missing markers spread along the full length of the chromosome. Markers specific to chloroplast DNA and mitochondrial DNA were used for analysis of changes within the organellar genomes of somatic hybrids. Random and non-random segregations of organellar DNA were noted.

     

Simple Sequence Repeat and <Emphasis Type="Italic">S</Emphasis>-locus Genotyping to Explore Genetic Variability in Polyploid <Emphasis Type="Italic">Prunus spinosa</Emphasis> and <Emphasis Type="Italic">P. insititia</Emphasis>

Polyploid Prunus spinosa (2n = 4×) and P. insititia (2n = 6×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programmes. In Hungary, 17 cultivar candidates were selected from wild-growing populations including 10 P. spinosa, 4 P. insititia and three P. spinosa × P. domestica hybrids (2n = 5×). Their taxonomic classification was based on their phenotypic characteristics. Six simple sequence repeats (SSRs) and the multiallelic S-locus genotyping were used to characterize genetic variability and reliable identification of the tested accessions. A total of 98 SSR alleles were identified, which presents 19.5 average allele number per locus, and each of the 17 genotypes could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified. The complete and partial S-genotype was determined for 8 and 9 accessions, respectively. The identification of a cross-incompatible pair of cultivar candidates and several semi-compatible combinations help maximize fruit set in commercial orchards. Our results indicate that the S-allele pools of wild-growing P. spinosa and P. insititia are overlapping in Hungary. A phylogenetic and principal component analysis confirmed the high level of diversity and genetic differentiation present within the analysed genotypes and helped clarify doubtful taxonomic identities. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species. The analysed accessions represent huge genetic potential that can be exploited in commercial cultivation.     

Hybridization rate and genotypic diversity of apomictic hybrids between native (<Emphasis Type="Italic">Taraxacum japonicum</Emphasis>) and introduced (<Emphasis Type="Italic">T. officinale</Emphasis>) dandelions in western Japan

Hybridization between the introduced and native plants may enhance invasiveness, especially in asexually reproducing species. Hybrid apomictic dandelions between native (Taraxacum platycarpum and T. japonicum) and exotic (T. officinale) species are distributed widely throughout Japan. To estimate the origin(s) and dispersal of the hybrids, we investigated the hybridization rate and genotypic diversity in mixed populations of T. japonicum, T. officinale and their hybrids at two green parks in western Japan. Among the plants identified as exotics from flower morphology, 86–96% were hybrids by genetic analysis. Genetic data with simple sequence repeat markers revealed a high clonal diversity of the hybrid both within and between populations, indicating multiple origins. A hybrid seed was found from among the 1891 seeds collected from T. japonicum in the parks, indicating ongoing hybridization in the field. T. officinale and hybrids were genetically differentiated between the two parks independent of the ploidy level; the allele frequency of T. officinale and tri- and tetraploid hybrids were similar within each park but different between the two parks. This suggests that the origins of hybrids were similar within the park but different between the parks. Overall, our results suggest that hybridization, including backcross, is an ongoing process, and that genetically diverse hybrids with various origins have been spreading in western Japan, probably because hybridization enhanced invasiveness at native habitat.     

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> isolated from raw milk and unpasteurized traditional cheeses in Romania

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla _SHV , bla _CMY , bla _TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n = 13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n = 8). A high percent (33.3%; n = 9) revealed the beta-lactamase (bla _TEM ) resistance gene, and none of the samples tested positive for bla _CMY and bla _SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n = 4) and 7.4% (n = 2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.     

Review of the genus <Emphasis Type="Italic">Banjos</Emphasis> (Perciformes: Banjosidae) with descriptions of two new species and a new subspecies

A taxonomic review of the genus Banjos (Perciformes: Banjosidae), previously restricted to a single species, Banjos banjos (Richardson 1846), recorded from the northwestern Pacific Ocean from the South China Sea north to Japan, as well as Lombok (Indonesia), New Caledonia and Australia, resulted in the recognition of three species, including B. banjos (northwestern Pacific Ocean, Indonesia and western Australia), Banjos aculeatus sp. nov. (eastern Australia) and Banjos peregrinus sp. nov. [northern Australia (Timor Sea)]. Records of B. banjos from New Caledonia probably also represent B. aculeatus, which is clearly distinct from other congeners in having a relatively long, strongly serrated spine at the posteroventral angle of the preopercle and an entirely dusky membrane on the spinous dorsal fin in juveniles < ca. 70 mm SL, in addition to slightly longer first and second dorsal-fin spines. Banjos peregrinus is characterized by a relatively greater head length, orbit diameter, postorbital length and pre-pelvic-fin length, as well as poorly developed serration of the exposed margin of the cleithrum. Within B. banjos, a population from the southeastern Indian Ocean, including Indonesia and western Australia, is regarded as a distinct subspecies (Banjos banjos brevispinis ssp. nov.), distinguishable from B. b. banjos from the northwestern Pacific Ocean by a relatively narrow least interorbital width, and shorter second and eighth dorsal-fin spines. Ontogenetic morphological changes within the genus and the status of the holotype of Anoplus banjos Richardson 1846 are discussed in detail.     

Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.     

Acaricidal and repellent effects of <Emphasis Type="Italic">Cnidium officinale</Emphasis>-derived material against <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acari: Dermanyssidae)

The acaricidal activity of a methanolic extract and fractions from the rhizome of Cnidium officinale against Dermanyssus gallinae adults was investigated. The C. officinale methanolic extract exhibited 100% acaricidal activity after 48 h of treatment at a dose of 4000 ppm. The acaricidal constituents of the plant were sequentially partitioned with several solvents and then purified using silica gel column chromatography and high-performance liquid chromatography. Gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy revealed (Z)-ligustilide as a constituent of C. officinale. Acaricidal activity was examined in three experimental tests (spray, fumigation and contact), with the spraying method being the most effective. The methanolic extract of C. officinale showed both contact and fumigant activities, though only fumigant activity was observed with (Z)-ligustilide. The fumigant effects of the methanolic extract and (Z)-ligustilide caused 86.5 and 62.6% mortality, respectively, of D. gallinae adults at 48 h. Among (Z)-ligustilide, acaricides (bifenthrin, cypermethrin and spinosad) and butylidenephthalide, bifenthrin displayed the highest acaricidal activity, and the activity of butylidenephthalide was 2.3-fold higher than that of (Z)-ligustilide. These results suggest that C. officinale-derived material can be used for the development of a control agent for D. gallinae.     

Stable,fertile somatic hybrids between <Emphasis Type="Italic">Sinapis alba</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> show resistance to <Emphasis Type="Italic">Alternaria brassicae</Emphasis> and heat stress

Wild relatives of Brassica are a rich reservoir of genes that are invaluable for the improvement of cultivated species. Sinapis alba is a close relative of crop Brassicas that possesses several desirable traits such as tolerance to Alternaria black spot disease, heat stress, insect pests and nematodes. This study is aimed at developing and characterizing hybrids between Brassica juncea and S. alba with the ultimate goal of transferring genes for tolerance to Alternaria brassicae and heat stress, the traits that are lacking in cultivated Brassica. We generated three hybrids between B. juncea and S. alba through protoplast fusion. The hybridity was confirmed through cytology and molecular markers. While two of the hybrids were symmetric, the third one was asymmetric and had greater resemblance to B. juncea. Hybrids showed some characteristic features of the parents and were fully male and female fertile and also set seeds upon back crossing with the parent species. In vitro leaf assay and field inoculation studies revealed that the hybrids are highly resistant to A. brassicae. Besides, hybrids set seeds at temperature of >?38 °C when parents failed to produce seeds indicating that hybrids possess heat tolerance. These stable hybrids provide a reliable genetic resource for transfer of genes from S. alba into cultivated Brassica species.     

Molecular cytogenetic characterization of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> chromosomes provides new insight on genome evolution of <Emphasis Type="Italic">T. zhukovskyi</Emphasis>

Triticum timopheevii (2n = 4x = 28, GGA^tA^t) is a tetraploid wheat formerly cultivated in western Georgia. The natural allopolyploid Triticum zhukovskyi is a hexaploid taxon originated from hybridization of T. timopheevii with cultivated einkorn T. monococcum (2n = 2x = 14, A^mA^m). Karyotypically T. timopheevii and T. zhukovskyi differ from other tetraploid and hexaploid wheats and were assigned to the section Timopheevii of the genus Triticum L. Triticum timopheevii and T. zhukovskyi are resistant to many fungal diseases and therefore could potentially be utilized for wheat improvement. We were aiming to precisely identify all T. timopheevii chromosomes and to trace the evolution of T. zhukovskyi. For this, we developed a set of molecular cytogenetic landmarks based on eleven DNA probes. Each chromosome can now be characterized by two to eight probes. The pTa-535 sequence allows the identification of all A^t-genome chromosomes, whereas G-genome and some A^t-genome chromosomes can be identified using (GAA/CTT) _n and pSc119.2 probes. The probes pAesp_SAT86, pAs1, Spelt-1, Spelt-52 and 5S and 45S rDNA can be applied as additional markers to discriminate particular chromosomes or chromosomal regions. The distribution of (GAA/CTT) _n , pTa-535 and pSc119.2 DNA probes on T. timopheevii chromosomes is distinct from other tetraploid wheats and can therefore be used to track individual chromosomes in introgression programs. Our study confirms the origin of T. zhukovskyi from hybridization of T. timopheevii with T. monococcum; however, we show that the emergence was accompanied by changes involving mostly A^t-genome chromosomes. This may be due to the presence of two closely related A-genomes in the T. zhukovskyi karyotype.     

Chemical defences of native European coccinellid eggs against intraguild predation by the invasive Asian coccinellid, <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas)

Harmonia axyridis (Pallas) is a coccinellid of Asian origin that has recently invaded substantial parts of Europe and is suspected to affect native coccinellid populations through intraguild predation and competition for food. Previous work has shown that two species from the Calvia genus appeared to be well protected against H. axyridis predation. To deepen our understanding on chemical protection of Calvia spp. and the predation risk by H. axyridis, we tested for susceptibility and palatability of Calvia spp. and H. axyridis eggs against predation by H. axyridis neonate larvae. Results show that eggs of C. quatuordecimguttata were mostly not eaten by H. axyridis, while eggs of the congeneric C. decemguttata were found to be largely unprotected against predation by the invasive coccinellid. We also observed that H. axyridis first instars successfully cannibalized on conspecific eggs. Removing the surface chemicals from C. quatuordecimguttata eggs resulted in significantly reduced protection from being preyed upon by H. axyridis, while applying these extracts onto C. decemguttata and H. axyridis eggs resulted in increased protection against H. axyridis larvae. The importance of surface chemicals in the interactions between H. axyridis and native coccinellids was confirmed by GC–MS analysis, showing a high diversity of hydrocarbons located on the surface of C. quatuordecimguttata eggs, i.e. more than twice as many when compared to C. decemguttata. Survival of H. axyridis larvae feeding on eggs of C. quatuordecimguttata, C. decemguttata or conspecific eggs, from which surface chemicals were removed by washing them with hexane, was not different from survival on unwashed eggs.     

Growth and biochemical analysis of evergreen haloxeric tree species <Emphasis Type="Italic">Salvadora oleoides</Emphasis> and <Emphasis Type="Italic">Salvadora persica</Emphasis> under NaCl stress

In vitro growth, development, total soluble proteins and peroxidase profiles of Salvadora oleoides and Salvadora persica under NaCl stress were analysed in the present investigation. The plants are evergreen haloxeric tree species of family Salvadoraceae. Shoot apex from natural plants were initially used for screening of NaCl tolerance on MS culture medium. Shoot apex of S. oleoides and S. persica could survive optimally up to 200 and 100 mM NaCl. Axillary buds from nodal shoot segments of S. oleoides and S. persica were activated on 6 and 4 μM BAP, and were used further for extraction of total soluble proteins and peroxidases. Total soluble proteins were increased up to 150 mM NaCl in S. oleoides, but decline above 50 mM NaCl in S. persica. Peroxidase activity remained almost constant in S. oleoides at all the concentrations and duration of NaCl, but increased at 100 mM NaCl during fourth week of treatment in S. persica. Eleven peroxidase isozymes were observed in zymogram of S. oleoides. Isozymes P1, P2, P3, and P4 were slightly appeared, but P6 isozyme was lacking in S. persica. The P5 isozyme was more prominent in S. persica than S. oleoides. Isozyme P9 of S. persica was visible during the first week of NaCl treatment, but disappeared in the fourth week. Molecular biology of these plants can be useful further for the understanding of stress tolerance mechanisms for prospects.     

Short and flat grass preferred by adults of the endangered dragonfly <Emphasis Type="Italic">Sympetrum pedemontanum elatum</Emphasis> (Odonata: Libellulidae)

Sympetrum pedemontanum (Müller in Allioni) (Odonata: Libellulidae) is widely distributed across the Eurasian continent and its neighboring islands. However, the populations of its subspecies S. pedemontanum elatum (Selys) in Japan have been rapidly decreasing with the loss of habitats in rural and suburban areas since the 1970s. For the conservation of this subspecies, which is now listed as endangered in many prefectures, it is important to understand the habitat preferences of the adults. Previous studies indicate that adult males of this species tend to fly on the flat surface of rice paddy fields. Thus, we hypothesized that they preferred short and flat grass. Field experiments in the Sakasegawa River, Hyogo Prefecture, Japan, showed that adult S. p. elatum significantly preferred the trimmed grass of Phragmites japonicus to untreated shaggy grass, regardless of sex. Our results indicate the importance of grass management for the conservation of this species, not only in and around paddy fields but also in fluvial habitats, which are abundant in Japan.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.