Bacteriocin production inAgrobacterium tumefaciens

Agrobacterium tumefaciens C58 forms “plaques” during layer cultivation. The “plaques” were shown not to be caused by the presence of a temperate bacteriophage or by random contamination. The “plaques” and their central microcolonies were used to repeatedly isolate cultures producing an antibiotic substance against the original strainA. tumefaciens C58, other nopaline strains, some octopine strains ofA. tumefaciens and some strains of the related genusRhizobium. The substance is thus a bacteriocin; in analogy to agrocins 84 and D286 it was named agrocin C58. The agrocin is not inactivated by trypsin. Its production by strain C58 was found only on cultivation on solid but not liquid media. The producing isolate ofA. tumefaciens C58 (strain C58i2) contains neither plasmid pTiC58 nor the plasmid analogous to pAgK84 which controls the production of agrocin 84 inA. radiobacter K84.     

Assortative Mating and Lack of Temporality Between Corn and Rice Strains of Spodoptera frugiperda (Lepidoptera,Noctuidae) from Central Colombia

Spodoptera frugiperda is a Neotropical moth that has diverged into “corn” and “rice” strains. In the US, prezygotic isolation studies have shown that both populations mate assortatively. In addition, recent studies have demonstrated that mating by the same strain individuals is enhanced by an allochronic shift in mating activity and male pheromones are important during courtship. In Colombia, studies on mate choice have never been performed previously, although earlier analyses made in populations from Central Colombia showed no significant differences in time of first mating and copula duration between the strains. Here, we performed multiple choice experiments using a tetrad design composed of individuals of the corn and the rice strains. We found that corn strain females rarely mate with rice strain males, but rice strain females mate with both strains of males. In addition, no temporal isolation was found. A ML (maximum likelihood) approach was used to discriminate between mating propensity and mate choice behaviors in S. frugiperda strains. This approach showed that mating propensity of corn strain males is three times greater than rice strain males. In contrast, in females, the propensity of mating was slightly higher for the corn strain. Finally, the isolation index between the corn and the rice strains from Colombia produced a value of I?=?0.33. Our results suggest that prezygotic isolation at the behavioral and temporal levels differ between the US and Colombia. Moreover, in the US temporal isolation appears to have an important role in behavioral isolation but in Colombia temporality is not necessary to reduce encounters between S. frugiperda strains.     

Fatty acids,resin acids and phenols in Pinus muricata

Acetone-soluble extractives of “blue” and “green” strain Pinus muricata D. Don were found to consist of free and “combined” fatty acids, resin acids, and phenols. The composition of the extractives from the two strains was similar though “green” strain P. muricata contained more Δ^8(9),15 isopimaric acid than “blue” strain. This difference may be used to identify these muricata strains if the age of the wood precludes a monoterpene examination.     

The growth,toxicity and genetic characterization of seven strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated during the 2009 summer outbreak in southern Chile

The dinoflagellate Alexandrium catenella causes recurrent harmful algal blooms in southern Chile. This species belongs to the “Alexandrium tamarense/catenella/fundyense species complex” (the “tamarensis complex”), defined by morphological attributes. Ribosomal sequences serve to differentiate five evolutionary lineages (clades) in this species complex. These distinctions reflect the geographic distribution and toxicity of the populations rather than their morphological designations. Despite the social and economic impact that harmful blooms produce in Chile, few strains of A. catenella have been isolated. Moreover, physiological and/or genetic studies of the group are scarce. The aim of this work was to examine possible physiological and genetic variability among populations of A. catenella having different geographical origins but isolated from the same toxic event. Seven strains of A. catenella were isolated and established from phytoplankton samples collected in the Aysén and Los Lagos regions of southern Chile during a recent outbreak (February–March 2009). Growth, toxicity, and ITS sequences were compared among these strains. All the strains included in this study were grouped with strains belonging to the previously described “North America” clade. The genetic diversity detected among Chilean strains was 3%, a much higher value than those reported for comparisons among strains from other parts of the world. In addition, a remarkable variability of growth parameters and toxicity was detected among strains. Strain PFB45 showed the highest PSP toxin content, whereas strain PFB41 had the lowest value of this parameter but had the highest maximum cell density. In strains PFB38, PFB42, and PFB37, more than 98% of the total PSP toxin content occurred in the form of gonyautoxins (primarily GTX-4,1 and GTX-3,2). In strains PFB39, PFB36, and PFB45, neoSTX, and STX toxins were detected. These results demonstrated remarkable variability at the genetic and physiological level among strains of A. catenella isolated from the same outbreak. No correlations were found between the phenotypic traits (growth and toxicity) and the genetic affiliation of the strains studied.     

Mapping and pedigree analysis of the gene that controls the easy peel pellicle trait in Japanese chestnut (Castanea crenata Sieb. et Zucc.)

The Japanese chestnut (Castanea crenata Sieb. et Zucc.) has a pellicle that is difficult to peel, which increases the labor and cost for removing the pellicle from the nut during processing. Thus, a pellicle that is easier to peel has been an important objective of Japanese chestnut breeding programs. A newly released cultivar (“Porotan”) exhibits a unique, easily peeled pellicle. A previous study indicated that this trait is controlled by recessive gene p, and that several of the ancestors of Porotan (e.g., “Tanzawa” and 550-40) were P/p heterozygotes. Two F₁ populations from intra-specific crosses of Japanese chestnut, Tanzawa (P/p) × Porotan (p/p) and 550-40 (P/p) × Tanzawa (P/p), were used for genetic mapping of the gene that controls this characteristic. A total of 11 simple sequence repeat (SSR) markers were obtained that showed significant linkages to the p gene, and genetic linkage maps for the region around the p gene were established. Pedigree analysis was conducted for eight ancestors of Porotan around the pellicle-peeling locus using graphical genotypes based on the 11 SSR loci. The two recessive p alleles and surrounding haplotypes of Porotan were inherited through different intermediate cultivars: one allele was derived from “Otomune” (P/p) via Tanzawa and the other was derived from Otomune via Tanzawa, “Kunimi” (P/p), and breeding line 550-40. A recombination event was found in the flanking region close to the p gene in Kunimi. Molecular identification of the easy peel pellicle trait will lead to marker-assisted selection and will greatly improve Japanese chestnut breeding.     

Trypanosoma brucei: influence of host strain and parasite antigenic type on infections in mice

Inbred mice were infected with cloned populations of Trypanosoma brucei brucei Lister S42 under carefully controlled conditions. The course of infection was found to depend both on host strain and the antigenic type of the infecting organisms. The two antigenic types used, “NIM2” and “NIM6” had differing virulence for (CBA/H × C57BL/6)F₁ mice, and when mice were infected with parasites of one clone, trypanosomes of the other type frequently appeared after the initial population had been eliminated. Different mouse strains had varying susceptibility to clone NIM6. In most cases there was an inverse relation between the survival time and the parasite load during the first peak of parasitemia. The differences in resistance to T. brucei were unrelated to H-2 haplotype: C57BL/6 and (CBA/H × C57BL/6)F₁ were most resistant, CBA/H, BALB/c and DBA/2 less so, and C3H/He most susceptible.     

Occurrence of islands in genomes of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> native isolates

Genomes of 184 Sinorhizobium meliloti native isolates were studied to test the occurence of islands Sme21T, Sme19T, and Sme80S previously described in the model strain Rm1021. This analysis was conducted using PCR methodology involving specific primers. It was demonstrated that, in the examined geographically distinct populations of S. meliloti from the Northern Caucasus (NCG) and the Aral Sea region (PAG), the strains containing genomic islands were observed with similar frequency (0.55 and 0.57, respectively). Island Sme80S, denoted as an island of “environmental adaptivity,” was identified predominantly (frequency of 0.38) in genomes of strains which exhibited a lower level of salt tolerance and was isolated in PAG, a modern center of introgressive hybridization of alfalfa subjected to salinity. Island Sme21T designated as “ancestral” was observed in genomes of strains isolated in NCG, the primary center of host-plant biodiversity, 10-fold more often than in strains from PAG. An island Sme19T, which predominantly carries genes encoding transposases, was observed in genomes of strains in both populations with average frequency of 0.10. The analysis of linkage disequilibrium (LD) based on the assessment of probability for detection of different islands combinations in genomes revealed an independent inheritance of islands in salt-sensitive strains of various geographic origin. In contrast, the absence of this trend was noted in the majority of the examined combinations of salt-tolerant strains. It was concluded that the structure of chromosome in PAG strains which predominantly possessed a salt-sensitive phenotype was subjected to active recombinant processes, which could predetermine the intensity of microevolutionary processes in bacterial populations and facilitate an adaptation of bacteria in adverse environmental effect.     

Role of cytoplasmic proteins in cold shock injury of Amoeba

Strains of Amoeba have been used to study the mechanisms of cellular injury induced by rapid cooling (cold shock). Cell viability was found to depend on the time and temperature of cold exposure, on the rate of cooling and on the morphology of the cells prior to chilling. All strains underwent a granuloplasmic contraction following undercooling to ?10 °C, although its extent varied; strains most damaged by cold shock exhibited the most violent cytoplasmic contractions. Cryomicroscopy demonstrated that the cellular contraction occurred upon rewarming, not during cooling. Cells damaged by cold shock were osmotically responsive, demonstrating that irreversible damage to the plasmalemma does not account for the phenomenon.Several compounds protected Amoeba against cold shock injury, glycerol and glucose being the most effective. With glycerol an optimum rate of cooling was observed upon cooling to ?10 °C, at both faster and slower cooling rates damage increased.The state of cellular actin in control cells and following cold shock was monitored by the DNase 1 inhibition assay and by electron microscopy. A comparatively “cold shock resistant” strain of A. proteus was found to contain less total actin per unit cellular protein than the more “sensitive” Amoeba sp. strain Bor. In the Bor strain a cold-induced aggregation of cytoplasmic filaments was evident in electron micrographs, presumably a crosslinking of preexisting F-actin.     

Forest and laboratory evaluations of hybridizedApanteles melanoscelus [Hym.: Braconidae], a parasitoid ofPorthetria dispar [Lep.: Lymantriidae]

Hybrids ofApanteles melanoscelus (Ratzeburg) were produced from colonies originating from France, Yugoslavia, and Connecticut. All strains, as well as freshly collected “wild” Connecticut parasitoids of the same species were evaluated in the laboratory for developmental rate, host attack rate, and sex ratios. Development was significantly slower in all the laboratory strains compared to the progeny of forest collected Connecticut females. Progeny production was greater (almost 2X) for the “wild” females and the French-Yugoslavian-Connecticut hybrid than for the laboratory Connecticut strain. The proportion of females collected from the “wild” (Connecticut) strain was higher than that observed in any laboratory strain. A field test was conducted using the triple hybrid in 3 release plots with ca. 6000A. melanoscelus cocoons released per plot in central Connecticut, U.S.A. Weekly collections of gypsy moth larvae showed that the % parasitism was significantly higher in release plots than in the 3 check plots. These results suggest the value of inundative releases ofA. melanoscelus for reduction of sparse gypsy moth populations, but they did not show that hybridization of these strains produced a more effective parasitoid under forest conditions.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.     

Control of pathogenic PAC strains by non-pathogenic PAC strains in planta does not correlate with higher competitiveness of non-pathogenic PAC strains ex planta

Ascomycetes of the Phialocephala fortinii s.l.—Acephala applanata species complex (PAC) are frequent root endophytes of forest trees. Roots are colonized by multiple PAC genotypes that interact, and recent findings indicate that adverse effects on plant performance caused by pathogenic PAC strains are attenuated by non-pathogenic PAC strains. However, it was not known if this “self-control” works only in planta, or also ex planta, i.e., prior to infection during saprotrophic life of the PAC. Interactions between PAC strains were therefore studied in a plant-free system on malt extract agar. The mycelia of two pathogenic (A and T1) and two non-pathogenic (B and C) PAC strains were mixed pairwise 5:1, 1:1 and 1:5 (fresh weight ratios) and incubated at 15 and 25 °C. Mycelial biomass of each strain was measured after 2 and 8 weeks. The combination of strains and the mixture ratio had a significant effect on strain biomass, whereas temperature influenced only the biomass of pathogenic strain T1. Biomass production of strain T1 was inhibited by all other strains, whereas biomass production of the other pathogenic strain A was significantly stimulated by the two non-pathogenic strains. This contrasts strongly with results from a previous experiment in planta using strains A, B and C, because the two non-pathogenic PAC strains successfully inhibited the pathogenic strain, probably by space occupation or the induction of host resistance. Therefore, it is impossible to predict the outcome of PAC-PAC interactions in planta based on the results gained from interactions ex planta.     

Characterization of Growth and Metabolism of Commercial Strains of Propionic Acid Bacteria by Pressure Measurement

Dairy propionibacteria are essential starters for Emmental cheese manufacture. The behavior of three commercial strains of Propionibacterium freudenreichii subsp. shermanii (P.f. 1, P.f. 2 and P.f 3) were studied in a liquid medium under air and N₂ atmosphere using an on‐line pressure measurement technique. Growth kinetics and metabolite production were characterized under conditions usually reported as “optimal conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and also evaluated under “stressful conditions” (pH 5.2, NaCl 2 %, temperature 20 °C) simulating the cheese ripening conditions. In both cases, the effects of oxygen on growth were strain‐dependent. Under “stressful conditions”, two of the three strains were inhibited by oxygen under conditions of air atmosphere, while all three strains grew under conditions of N₂ atmosphere. In the latter case, the duration of the lag phase and the maximum rate of pressure variation were significantly different, however, no significant differences were found between the strains with regard to the total fermentation time. Under “optimal conditions” metabolite production was strain‐dependent. In an air atmosphere, all strains produced more acetate and CO₂ and less propionate than in a nitrogen atmosphere.     

Comparative rearing parameters for bisexual and genetic sexing strains of Zeugodacus cucurbitae and Bactrocera dorsalis (Diptera: Tephritidae) on an artificial diet

The Sterile Insect Technique (SIT) is an important component of area wide programs to control invading or established populations of pestiferous tephritids. The SIT involves the production, sterilization, and release of large numbers of the target species, with the goal of obtaining sterile male x wild female matings, which yield infertile eggs. A major advance in SIT involved sex-linked, genetic manipulations that allowed the production and release of male-only strains (also termed genetic sexing strains, GSS). The use of GSS avoids matings between sterile males and females, which may divert males from seeking and mating with wild females, and studies show that male-only releases result in greater suppression of wild populations than standard bisexual releases (i.e., those including both males and females). GSS based on sex-linked pupal color exist for Zeugodacus cucurbitae (Coquillett) and Bactrocera dorsalis (Hendel), two important agricultural pest species, but their rearing characteristics have not been documented in detail. The goal of the present study was to compare the pupal color sexing and bisexual strains for each of these species with respect to important rearing parameters, including egg production and eclosion of larvae from eggs (egg hatch), pupal recovery, and weight, emergence rate, and flight ability. In both species, most of these parameters were significantly greater for the bisexual strain than the GSS, and, for a given number of eggs, the production of flight-capable adults was approximately 2 times greater in the bisexual strains of both species. The potential usefulness of GSS in SIT against Z. cucurbitae and B. dorsalis is assessed based on these findings.     

Haemonchus contortus: New data on its genetic constitution

Studies on the genetic constitution of single smooth-bodied females, appearing from time to time in the homozygous (aa) linguiform strain “C,” revealed that these worms have the constitution of the linguiform (aa) morph but the character “flap” in them has failed to develop because of the undetermined effect of some additional factors. Experiments were also performed to check the dominance to recessiveness ratios of the characters “flap,” “knob,” and “smooth.” With the Haemonchus types present in Bulgaria these ratios have persisted over seven or eight years. It is stated that they are the same in populations whose morphologic types in female specimens show different percentage distributions.     

Microbial characterisation of fermented meat products from the Sicilian swine breed “Suino Nero Dei Nebrodi”

Two traditional sausage products (“salsiccia” and “salame”) processed from the raw meat of the Black Sicilian swine “Suino Nero dei Nebrodi” were microbiologically investigated during the manufacturing and ripening stages. Both products were dominated by lactic acid bacteria (LAB), especially rod-shaped types. The concentration of enterococci was consistent in salame. Coagulase-negative cocci increased slower than LAB. Yeasts showed an increasing trend during the ripening of both products. Enterobacteriaceae were counted at a constant level of about 10⁵ CFU/g in both products, while pseudomonads diminished during ripening. Coagulase-positive staphylococci, Listeria monocytogenes and Salmonella spp. were not detected at the end of the ripening process. Characterisation of LAB at the strain and species level revealed that Lactococcus lactis was found only in the meat mixture, while Lactobacillus sakei and various enterococci persisted during the monitoring period. Some LAB strains isolated from sausages were also identified on the surface of the factory equipment. Two strains (Lactobacillus sakei SS106A and Enterococcus faecalis SS91) were characterised by their anti-Listeria properties due to bacteriocin-like inhibitory substance production. A multiple strain starter composed of Lactobacillus sakei and enterococci has been proposed to maintain the typical characteristics of the two fermented meat products microbiologically investigated in this study.     

Differences in photoprotective pigment production between Japanese and Australian strains of Chattonella marina (Raphidophyceae)

Previous studies have shown that isolates of Chattonella marina from Australia and Japan exhibit differences in tolerance to high intensities of visible light. Here we show that the Australian strain of C. marina produces around five times more UV-absorbing mycosporine amino acids (MAAs) than the Japanese strain. This corresponds with 66% increased growth by the Australian strain under UVB exposure compared to no UV exposure. The MAA mycosporine-glycine, which reportedly acts as an antioxidant, was found in high quantity (110 fg cell^-1) in the Australian but was absent in the Japanese strain. In contrast, changes in the concentration of violaxanthin and zeaxanthin per cell were 4.7-4.8 times greater in the Japanese relative to the Australian strain suggesting that the Japanese strain uses a xanthophyll cycle to moderate inhibition by high photosynthetically active radiation (PAR) irradiance. Increased MAA production under high irradiance was also observed in other Australian strains of Chattonella, but not noted in other Japanese strains suggesting ecophenotypic adaptation due to differing environmental conditions.     

Chemical investigation of different extracts and essential oil from the tubers of (Tunisian)Cyperus rotundus. Correlation with their antiradical and antimutagenic properties

The mutagenic potential of aqueous, Total Oligomers Flavonoids (TOF), ethyl acetate, and methanol extracts as well as essential oil (EO) obtained from tubers ofCyperus rotundus L. was assessed by “Ames assay”, usingSalmonella tester strains TA98 and TA100, and “SOS chromotest” usingEscherichia coli PQ37 strain with and without an exogenous metabolic activation system (S9). None of the different extracts showed a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed thatC. rotundus extracts have antimutagenic effects withSalmonella typhimurium TA98 and TA100 strains towards the mutagen Aflatoxin B1 (AFB1), as well as withE. coli PQ37 strain against AFB1 and nifuroxazide mutagens. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers ofC. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. These extracts showed IC50 values of respectively 5, 20 and 65 μg/ml. The beneficial effects of TOF, ethyl acetate, methanol and essential oil extracts ofC. rotundus have been assessed by antioxidant and antimutagenic activities.     

Genetic variation and Wolbachia infection status of the lycaenid butterfly Zizina emelina (Lepidoptera,Lycaenidae) from Korean populations

The lycaenid butterfly Zizina emelina emelina (de l’Orza) (previously Zizina otis emelina; Lepidoptera, Lycaenidae) has populations distributed across Japan and Korea. Zizina emelina is listed on Japan’s Red List as an endangered species, owing to the loss of its principal plant food and habitat. Researchers previously investigated the genetic diversity and structure in Japanese populations by using mitochondrial and nuclear DNA analyses alongside their Wolbachia infection status to consider the conservation strategy. In this study, we investigated the genetic variation and Wolbachia infection status of Korean populations and compared our findings with these traits in the Japanese populations. Four novel mitochondrial haplotypes were found in the Korean populations, whereas nuclear DNA analysis revealed one new haplotype and another that matched the one reported previously in Japan. The two known strains of Wolbachia in the Japanese populations were also found in the Korean ones. Mitochondrial DNA analysis demonstrated that no gene flow had occurred between the Japanese and Korean populations. The fact that they share the same nuclear haplotype and Wolbachia strains, however, indicates a low level of population differentiation between the two.     

TLC screening of thraustochytrid strains for squalene production

Screenings of thraustochytrids (Labyrinthulomycetes) have been conducted for 176 strains isolated from various sites in the Asian region to investigate what type of species and strains accumulate high levels of squalene. Thin layer chromatography (TLC) screening for squalene production revealed that 38 strains were rated as “+” (high), 29 as “±” (medium), and 109 as “?” (low). Further, high performance liquid chromatography analysis strongly supported the TLC screening results. Besides the 18W-13a strain of Aurantiochytrium sp., which was previously recognized as a squalene-rich strain, several strains produced squalene at approximately 1 g L^?1 of culture volume. Squalene production was strongly related to locality, colony color, and phylogenetic clade. Most strains with “+” squalene spots were isolated from Okinawa, a subtropical region of Japan, while the strains with “±” and “?” squalene spots were isolated from wide geographical regions from tropical to subarctic. Approximately half the strains with orange colonies on GTY medium plates produced a high amount of squalene, whereas the other strains with different colors showed less or no squalene spots on TLC. All the squalene-rich strains were assigned to the Aurantiochytrium clade. Overall, our results suggest that (1) the thraustochytrids show tendentious locality in terms of squalene production, (2) a relationship exists between the metabolic synthesis of carotenoid pigments and squalene production, and (3) the Aurantiochytrium clade may have evolved to accumulate squalene.     

Three New Freshwater Cochliopodium Species (Himatismenida,Amoebozoa) from the Southeastern United States

Cochliopodium is a lens‐shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often‐used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum‐likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, “crystal UK‐YT2” (Cochliopodium crystalli n. sp.) and “crystal‐like UK‐YT3” (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical‐shaped crystals. The “Marrs Spring UK‐YT4” isolate, C. marrii n. sp., was 100% identical to “Cochliopodium sp. SG‐2014 KJ569724 .” These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.