Biocontrol of grey mould by Ulocladium atrum applied at different flower and fruit stages of strawberry

Grey mould is an important disease ofstrawberries resulting from flower and fruitinfection by Botrytis cinerea Pers. Thesaprophytic fungus Ulocladium atrumPreuss is a promising biological controlagent for control of B. cinerea instrawberry and other crops. The objective ofthis research was to determine the efficacy ofU. atrum to control grey mould by asingle application of a spore suspension (2 ×10⁶ conidia/ml) at different flowerand fruit development stages. Four experimentswere carried out in 1999, two under natural andtwo under enhanced inoculum levels of B.cinerea. In each experiment, flowers and youngfruits in six distinct stages of developmentwere sprayed with either water or U.atrum suspension. U. atrum suppressedB. cinerea sporulation on petals by 15 to54%. One to four days after spraying, U.atrum was present on less than 30% of stamensand did not affect the incidence of B.cinerea on these flower parts. The efficacy ofthe U. atrum sprays in controlling greymould was low to moderate, and resulted onaverage in a reduction of 21% in diseaseincidence on ripe fruits. Low control efficacywas probably due to poor coverage with orcolonisation of stamens by U. atrumspores, and a relatively low level ofsuppression of the colonisation of flower partsby B. cinerea. Significant reductions ofgrey mould in comparison to the control(p 0.10; on average 41% reduction) werefound most frequently when the antagonist wasintroduced at late flowering or early fruitstages. Therefore, these are the most suitablestages to apply U. atrum. Further studiesare needed to improve the spray coverage andpersistence of U. atrum inoculum.     

Genetic diversity in natural and anthropogenic inland populations of salt-tolerant plants: random amplified polymorphic DNA analyses of Aster tripolium L. (Compositae) and Salicornia ramosissima Woods (Chenopodiaceae)

Eight populations of Aster tripolium (Compositae) and six of Salicornia ramosissima (Chenopodiaceae) from inland, naturally salt-contaminated habitats and anthropogenic salt-polluted sites in central Germany (Thuringia, Anhalt-Saxony) were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the patterns of genetic variation. In both species, the genetic diversity observed in the younger, anthropogenic sites caused by potash mines during the last century was found to be not significantly lower than in the older, naturally salt-contaminated habitats. Therefore, it is speculated that the loss of genetic diversity caused by founder effects on the anthropogenic habitats was balanced by successive colonization events, actual gene flow between populations, or the rapid growth of populations on the secondary habitats after colonization. Analyses of molecular variance (amova) of the RAPD markers, neighbour-joining clustering of populations based on Reynolds' co-ancestry distances, and Mantel tests indicate that: (i) anthropogenic habitats were colonized independently; (ii) genetic differentiation among populations of S. ramosissima is more pronounced than in A. tripolium, which is considered to be mainly due to biological differences between the two species; and (iii) the geographical pattern of genetic diversity was considerably modulated by historical events and/or population genetic effects.     

Bradykinin and muscarine induce Ca(2+)-dependent oscillations of membrane potential in rat glioma cells indicating a rhythmic Ca2+ release from internal stores: thapsigargin and 2,5-di(tert-butyl)-1, 4-benzohydroquinone deplete InsP3-sensitive Ca2+ stores in glioma and in neuroblastoma-glioma hybrid cells.

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 nM) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-gamma-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 nM to 2 microM, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca2+, the substances thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHA, which are known to block the Ca2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca2+ response to bradykinin which is known to be due to release of Ca2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP3-sensitive Ca2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to "quantal" Ca2+ release from stores with different inositol 1,4,5-triphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca2+ seems to be due to a lack of replenishment of the intracellular stores with Ca2+, which comes from the extracellular compartment.     

Activity of some enzymes in Physarum polycephalum

Summary The activity levels of seven enzymes were studied in growing plasmodia of Physarum polycephalum. The enzymes were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Six of the enzymes showed a continuous increase in activity during the mitotic cycle; glutamate dehydrogenase exhibited a stepwise increase about 5 h after mitosis. Cycloheximide immediately inhibited the activity of all enzymes. Actinomycin D was ineffective in inhibiting enzyme activity until after one mitotic cycle had been completed; this indicates that mRNA was stable for all of these enzymes during the G2 period. Attempts to induce enzyme activity were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf von sieben Enzymen wurde in wachsenden Plasmodien von Physarum polycephalum untersucht. Es handelte sich um die Enzyme: Isocitrat-Dehydrogenase, Glucose-6-Phosphat-Dehydrogenase, Glutamat-Dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Sechs dieser Enzyme wiesen einen kontinuierlichen Aktivitätsanstieg während des Mitosecyclus auf; Glutamat-Dehydrogenase zeigte einen stufenförmigen Anstieg etwa 5 Std nach der Kernteilung. Cycloheximid hemmte sofort die Aktivität der Enzyme, während Actinomycin D erst nach Ablauf eines halben Teilungscyclus inhibierend wirkte. Dies deutet auf eine relativ stabile mRNA hin. Versuche, die Aktivität zu induzieren, schlugen fehl.

     

Thermoanaerobacter ethanolicus gen. nov., spec. nov., a new,extreme thermophilic,anaerobic bacterium

Two strains, JW 200 and JW 201, of an extreme thermophilic, non-spore-forming anaerobic bacterium were isolated from alkaline and slightly acidic hot springs located in Yellowstone National Park. Both strains were peritrichously flagellated rods. Cell size varied from 0.5–0.8 by 4–100 m; coccoid-shaped cells of about 1 m in diameter frequently occurred. Division was often unequal. Spheroplast-like forms were visible at the late logarithmic growth phase. The Gram reaction was variable. The DNA base composition of the two strains was between 37 and 39 mol% guanine plus cytosine as determined by buoyant density measurements and approximately 32% by the thermal denaturation method. The main fermentation products from hexoses were ethanol and CO₂. Growth occurred between 37 and 78°C and from pH 4.4 to 9.8. The name Thermoanaerobacter ethanolicus gen. nov., spec. nov. was proposed for the two, new isolates. Strain JW 200 was designated as the type strain.A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Los Angeles, CA, 1979 (J. Wiegel and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol., 1979, 163, p. 105) and at the 27th IUPAC Congress Helsinki, 1979 (L. G. Ljungdahl and J. Wiegel, Abstracts p. 546)     

Ectothiorhodospira vacuolata sp. nov., a new phototrophic bacterium from soda lakes

A new phototrophic bacterium was isolated from Jordanian and Kenyan alkaline salt lakes. Cells are rod shaped, 1.5 m wide and 2–4 m long, and motile by polar flagella. They divide by binary fission, and possess photosynthetic membranes as lamellar stacks similar to those in the other species of the genus Ectothiorhodospira and the brown colored Rhodospirillum species. The presence of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series is indicated by the absorption spectra of living cells. Under certain growth conditions the cells form gas vacuoles, may become immotile and float to the top of the culture medium. Sulfide and thiosulfate are used as photosynthetic electron donors. During the oxidation of sulfide to sulfate, elemental sulfur is formed, which is accumulated outside the cells. The organisms are strictly anaerobic, do not require vitamins, are moderately halophilic and need alkaline pH-values for growth. The new species Ectothiorhodospira vacuolata is proposed.     

Specific regulation of SOD isoforms by NaCl and osmotic stress in leaves of the C3 halophyte Suaeda salsa L

The halophyte Suaeda salsa L., exposed to different NaCl concentrations (100 and 400 mmol/L) and polyethylene glycol (isoosomotic to 100 mmol/L NaCl) containing nutrient solutions under normal or K+-deficient conditions for 7 days, was used to study effects of NaCl salinity and osmotic stress on chlorophyll content, chlorophyll fluorescence characteristics, malonedialdehyde (MDA) content, and superoxide dismutase (SOD) isoform activities. Photosynthetic capacity was not decreased by NaCl treatment, indicating that S. salsa possesses an effective antioxidative response system for avoiding oxidative damage. Seven SOD activity bands were detected in S. salsa leaf extracts, including an Mn-SOD and several isoforms of Fe-SOD and CuZn-SOD. It turned out that NaCl salinity and osmotic stress lead to a differential regulation of distinct SOD isoenzymes. This differential regulation is suggested to play a major role in stress tolerance of S. salsa.     

Functional consequences of single:double ring transitions in chaperonins: life in the cold

The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5 degrees C to -13.7 degrees C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266-1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)(7) was the active oligomer at 4-10 degrees C, whereas at > 10 degrees C, this complex was converted to (O.Cpn60)(14). The dissociation reduces the ATP consumption (energy-saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double-ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild-type chaperonin was 24-28 degrees C and 4-18 degrees C, whereas that for the mutants was 45-55 degrees C and 14-36 degrees C respectively. The temperature inducing unfolding (T(M)) increased from 45 degrees C to more than 65 degrees C. In contrast, a single ring mutant, O.Cpn60(SR), with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10 degrees C. Above 10 degrees C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4 degrees C as the single ring variant. We demonstrated that expression of O.Cpn60(WT) and O.Cpn60(SR) leads to a higher growth of E. coli at 4 degrees C ( micro (max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15 degrees C, only E. coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn). These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation. Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37 degrees C to 4 degrees C.