Analysis of somatic hybrids between two sterile dihaploid Solanum tuberosum L. breeding lines. Restoration of fertility and complementation of G. pallida Pa2 and Pa3 resistance

Fourteen somatic hybrids generated by electrofusion of mesophyll protoplasts from a non-flowering dihaploid S. tuberosum clone, DHAK-11, and a male-sterile dihaploid clone S. tuberosum, DHAK-33, were grown in the greenhouse and subjected to morphological assessments and tests for fertility and resistance to the white potato cyst nematode Globodera pallida pathotypes Pa2 and Pa3. The ploidy level of the hybrids ranged from 38 to 63 chromosomes. All hybrids developed flowers with violet petals except for one, hy-56, that possessed red petals. The colour of the tuber skin was purple in all hybrids except in hy-56 where the tuber skin was red. All of the hybrids were female fertile and generated viable seeds. Near-tetraploid hybrids produced the highest number of seeds per fruit and these seeds had a normal size. Hybrids with 58 or more chromosomes produced smaller seeds and less seeds per fruit. The germination frequency of the seeds was not influenced by the chromosome number of the hybrids. Pollen viability was determined and the male fertility of three hybrids was tested. Pollination with these three hybrids gave rise to fruit development, but only one produced viable seeds. The hybrids were tested for resistance to G. pallida pathotypes Pa2 and Pa3. A high level of resistance to Pa3, inherited from one parental clone, DHAK-11, and a high level of resistance to Pa2, inherited from the other parental clone, DHAK-33, was combined in four hybrids. These results demonstrate, that protoplast fusion is an efficient method for restoring the fertility of somatic hybrids generated from sterile parent clones, and is a powerful procedure for the complementation of multigenetic disease resistance traits in potato breeding lines.     

Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.     

Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×10⁸ N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.     

Transformation of Phycomyces with a bacterial gene for kanamycin resistance

Summary Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.     

Expression of a plant cystatin confers partial resistance to Globodera,full resistance is achieved by pyramiding a cystatin with natural resistance

Constructs based on a cysteine proteinase inhibitor (cystatin) from sunflower and a protein engineered variant of a rice cystatin (Oc-ID86) conferred similar levels of resistance on potato plants to Globodera spp as chicken egg white cystatin (CEWC) under the control of CaMV35S. The level of resistance on challenge by 6.5 viable eggs Globodera spp g^–1 soil in a small scale field trial was similar to that provided by the natural partial resistance of cv. Sante. PCR of an internal transcribed spacer region of the ribosomal cistron of the nematode genome established that the challenging population was a mixture of G. pallida and G. rostochiensis with no evidence of a differential effect of the transgenic resistance on the reproductive success of the two species. Transformation of Sante and the South American cultivar Maria Huanca with CaMV35S/CEWC raised the status of both cultivars from partial to full resistance in this study. The results establish the potential of plant cystatins and demonstrate an additive effect of expressing CEWC with natural resistance genes.     

Controlled gene expression utilising Lambda phage regulatory signals in a cyanobacterium host

Summary This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.Abbreviations AP^r ampicillin resistance - Cm^r chloramphenicol resistance - CmActase chloramphenicol acetyltransferase - Km^r Kanamycine resistance - [ ] indicates plasmid carrier state     

Chronic herbivory negatively impacts cone and seed production,seed quality and seedling growth of susceptible pinyon pines

Although herbivory often reduces the reproduction of attacked trees, few studies have examined how naturally occurring insect-resistant and susceptible trees differ in their reproduction, nor have these effects been experimentally examined through long-term herbivore removals. In addition, few studies have examined the effects of herbivory on the quality of seeds produced and the implications of reduced seed quality on seedling establishment. We evaluated the impact of chronic herbivory by the stem-boring moth, Dioryctria albovittella, on cone and seed production of the pinyon pine (Pinus edulis) during two mast years. Three patterns emerged. First, moth herbivory was associated with reductions in cone production, viable seed production and seed mass. Specifically, pinyons susceptible to moth attack had 93–95% lower cone production, and surviving cones produced 31–37% fewer viable seeds, resulting in a 96–97% reduction in whole tree viable seed production. In addition, surviving seeds from susceptible trees had 18% lower mass than resistant trees. Second, long-term experimental removal of the herbivore resulted in increased rates of cone and seed production and quality, indicating that moth herbivory was the driver of these reductions. Third, seed size was positively associated with seed germination and seedling biomass and height, suggesting that trees suffering chronic herbivory produce poorer quality offspring. Thus, the resistance traits of pinyons can affect the quality of offspring, which in turn may affect subsequent seedling establishment and population dynamics.     

The association between pollen size and Renner complex in Oenothera villaricae and O. picensis ssp. picensis and their hybrids: evidence for preanthesis pollen competition

In both Oenothera villaricae Dietrich and O. picensis ssp. picensis, chromosomes are transmitted as two Renner complexes. Reciprocal combinations of the Renner complexes produce eight different F₁ hybrids, but only seven are viable. Each species, and each F₁ hybrid, produces three sizes of pollen, approximately 50% small sterile grains, 15% medium-sized viable grains and 35% large viable grains. Medium- and large-sized grains were separated manually and subjected to random amplified polymorphic DNAs (RAPDs) analysis. A pattern of RAPD amplifications was obtained which indicates that, for each species and F₁ hybrid, one specific Renner complex characterizes the medium- and another the large-sized viable pollen. The results indicate that pollen size is determined in part by the pollen genotype and in part by the genotype of the other pollen grains developing within the same anther.     

Comparison of pH and Bile Resistance of Lactobacillus acidophilus Strains Isolated from Rat, Pig, Chicken, and Human Sources

Summary To provide a useful screening method for selecting probiotics, we compared the pH and bile resistance of four strains of Lactobacillus acidophilus, KCTC3140, KCTC3146, KCTC3154, and KCTC3179, isolated from a rat, pig, chicken, and human, respectively. When we compared the pH resistance of these strains at pH 2, 3, 4, 5 and 7, we found that L. acidophilus isolated from the rat, chicken, and pig showed little or no decrease in viable cell numbers, except at 240 min, whereas the numbers of L. acidophilus KCTC3179 from the human decreased significantly. All four strains were slightly suppressed over time and showed bile resistance, even at 3%. At 5% oxgall, the number of KCTC3179 rapidly decreased at 30 min. These results indicate that lactic acid bacteria selected for probiotic use should be screened at pH 2 for 120 min and/or at an oxgall concentration of 5% for 30 min.     

Propylene glycol and contact-lens solutions containing this diol induce pseudocyst formation in acanthamoebae

Propylene glycol used as an ophthalmic demulcent in certain contact-lens care systems has been included recently among factors responsible for increasing Acanthamoeba keratitis. In this study, we provide evidence that propylene glycol as well as examined contact-lens solutions containing it induce rapid differentiation of acanthamoebae into pseudocysts. The partial resistance of the pseudocysts and their reversibility to viable trophozoites even after 24-h exposure to the contact-lens solutions indicate a potential risk of infection to contact-lens users.     

Influence of acaricide resistance on cattle-fever tick (Boophilus spp.) infestations in semi-arid thornshrublands: a simulation approach

Cattle-fever tick (Boophilus microplus and B. annulatus) populations that develop acaricide resistance become more difficult to control or eradicate. We used a simulation model to assess the direct and indirect effects of interactions among season, habitat type, grazing strategy, and acaricide resistance on the ability to eradicate Boophilus infestations in semi-arid thornshrublands of Texas, USA. Season of infestation appeared to have the strongest effect, with infestations begun on 27 September (autumn) tending to die out sooner than those begun on 1 March (spring) and to remain undetected. Habitat type had the next strongest effect, with infestations surviving much longer as canopy cover increased from uncanopied buffelgrass (Cenchrus ciliaris) habitats to mesquite (Prosopis glandulosa)-canopied grass habitats. Acaricide resistance had a moderate effect; as expected, highly resistant tick populations survived longer than those with no acaricide resistance. The importance of grazing strategy varied with changes in habitat type: as canopy cover increased, infestation duration increased faster under continuous grazing than under rotational grazing strategies. Importance of grazing strategy also varied with acaricide resistance: detected tick populations with no and slight acaricide resistance subjected to acaricide treatments tended to survive longer under rotational grazing than continuous grazing, due to reduced contact with a treated host. Populations with moderate and high resistance behaved more like untreated populations, tending to survive longer under continuous, rather than rotational, grazing, because they experienced less mortality on a treated host. Assuming acaricide treatments at 2-week intervals and maintenance of cattle in infested pastures, results indicate that, for each habitat type, infesting ticks have a threshold of acaricide resistance below which one can eradicate them faster with continuous grazing than with rotational grazing. As canopy cover increases, this threshold appears to shift from high resistance (in grass) to slight resistance (in mesquite).     

Somaclonal variation in celery: screening for resistance to Fusarium oxysporum f. sp. apii

Summary Two methods were used to screen putative Fusarium-resistant celery (Apium graveolens L.) plantlets from cell culture: placing plantlets on a mycelial mat for one month or planting them directly in Fusarium-infested soil. Resistant phenotypes were identified with both methods, but the plants grown on the mycelial mat died before they reached reproductive maturity. Four plants, K, T-2, T-3, and R-R₁ from the soil screen, survived and produced viable seed. Tests of self-pollinated progeny, in field and greenhouse conditions, showed that T-2, T-3, and R-R₁ were superior to the original cultivar, 5270R, with respect to disease resistance, as measured by vascular discoloration and plant height. Chi-square analysis of progeny scores for root and crown decay showed that the new variation was heritable and appeared to be conditioned by more than one locus.     

Chloroplast DNA base substitutions: an experimental assessment

An experimental assessment was carried out to determine directly the frequency and types of spontaneous base substitutions that occur in chloroplast DNA. A target site within the chloroplast 16S rRNA gene of the green alga Chlamydomonas reinhardtii was chosen for the assay. Mutations at this site were known to confer spectinomycin resistance and simultaneously result in the loss of an AatII cleavage site. In the experiments reported here, base substitutions at any individual base occurred at a frequency in the range of 0.9–11 per 10⁹ viable cells plated. Four new mutations that confer resistance to spectinomycin were identified at the target site in the Chlamydomonas chloroplast 16S rRNA gene. When the relative rates of transition and transversion mutations were quantified, a bias toward transversions was observed. The prominence of A/T C/G transversions in the observed mutation spectrum suggests that oxidative damage may be the major cause of base substitution mutations within the chloroplast.     

Comparison of airborne spore concentrations and fungal allergen content

The exposure to spores causing health effects is usually assessed by determining the concentration of viable spores per cubic meter of air (CFU/m³).Since allergens might also be present in dead spores or smaller particles, the objective of this study was to investigate the correlation between the viable spores of Alternaria and Cladosporium at different indoor and outdoor sites and the corresponding allergen concentration detected with a specially developed ELISA (Enzyme Linked Immunosorbent Assay). In outdoor air, the results show a strong correlation between the different sampling techniques applied for viable spores (Slit-Sampler and Multistage Liquid Impinger) and between the viable spores and the allergen concentrations detected in the liquid samples of the impingers. Indoors, the number of viable spores and the allergen concentration do not correlate and the allergen load is underestimated if colony counting methods are used. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inheritance of resistance to potato y viruses in Phaseolus vulgaris L

Summary Resistance to watermelon mosaic virus-2 in Phaseolus vulgaris L. is conferred by two distinct dominant alleles at independent loci. Based on segregation data one locus is designated Wmv, the other, Hsw. The dominant allele Wmv from cv. Great Northern 1140 prevents systemic spread of the virus but viral replication occurs in inoculated tissue. In contrast, Hsw confers both local and systemic resistance to WMV-2 below 30C. At higher temperatures, plants that carry this allele in the absence of modifying or epistatic factors develop systemic veinal necrosis upon inoculation with the virus that results in rapid death. Patho-type specificity has not been demonstrated for either allele; both factors confer resistance to every isolate tested. A temperature-sensitive shift in epistasis is apparent between dominant alleles at these loci. Because Hsw is very tightly linked if not identical to the following genes for hypersensitivity to potyviruses I, (bean common mosaic virus), Bcm, (blackeye cowpea mosaic virus), Cam, (cowpea aphid-borne mosaic virus) and Hss (soybean mosaic virus), parental, reciprocal dihybrid F₁ populations, and selected F₃ families were inoculated with each of these viruses and held at 35 C. F₁ populations developed vascular necrosis completely or primarily limited to inoculated tissue, while F₃ families from WMV-2-susceptible segregates were uniformly susceptible to these viruses. The relationship between Hsw, Wmv and other genes for potyvirus resistance suggest patterns in the evolution of resistance and viral pathogenicity. Characterization of the resistance spectrum associated with each factor provides an additional criterion to distinguish genes for plant virus resistance.     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

Role of a differentially expressed cAMP phosphodiesterase in regulating the induction of resistance against oxidative damage in Leishmania donovani

Differentiation-coupled induction of resistance of Leishmania parasites to macrophage oxidative damage was shown to be associated with an increased cAMP response. This study explores the significance of the cAMP response in the parasite by identifying a differentially expressed cAMP phosphodiesterase (LdPDEA) and deciphering its role in regulating antioxidant machineries in the parasite. LdPDEA, a high K_M class I cytosolic cAMP phosphodiesterase, was expressed maximally in log-phase promastigotes, but was significantly reduced in stationary-phase promastigotes and amastigotes. Chemical inhibition or silencing of PDEA conferred enhanced resistance to pro-oxidants in these cells and this led to studies on trypanothione biosynthesis and utilization, as trypanothione is one of the major modulators of antioxidant defense in kinetoplastidae. Despite enhanced arginase and ornithine decarboxylase activity, trypanothione biosynthesis seemed to be unaffected by PDEA blockage, whereas significant elevations in the expression of tryparedoxin peroxidase, ascorbate peroxidase, and tryparedoxin were detected, suggesting a definite shift of trypanothione-pool utilization bias toward antioxidant defense. Moreover, parasites that overexpressed PDEA showed reduced resistance to oxidative damage and reduced infectivity toward activated macrophages. This study reveals the significance of a cAMP phosphodiesterase in the infectivity of Leishmania parasites.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium