Biofilm formation and virulence of uropathogenic Escherichia coli in urine after consumption of cranberry-lingonberry juice.

Cranberry-lingonberry juice (CLJ) was effective in preventing urinary tract infections (UTIs) in our earlier randomized clinical trial. We aimed to test whether consumption of CLJ at a similar dose to earlier reduces the biofilm formation and virulence of uropathogenic Escherichia coli in urine. Twenty healthy women drank 100 ml of CLJ daily for two weeks. Urine samples were obtained 2-4 hours after the last dose. Control samples were taken after a one-week period without berry consumption. Biofilm formation of 20 E. coli strains was measured at 72 hours by the polystyrene microtitre plate method. Quantitative real-time PCR analyses were performed for selected genes. Four of the 20 clinical strains produced more biofilm in urine after CLJ consumption (P < 0.05) and one produced less. Expression levels of the pga, cpxA, fimA and papF genes did not differ between bacteria grown in control urine and urine obtained after CLJ consumption, except for pga gene expression, which was reduced in one strain after CLJ (P = 0.04). It appears that the effect of CLJ in preventing UTIs is not explained by mechanisms that reduce biofilm formation or the expression of selected virulence genes of Escherichia coli in urine.

Phylogenetic background of Orange lily (Lilium bulbiferum s.l.) cultivars from a genetically isolated environment.

Domesticated Orange lily ( LILIUM BULBIFERUM s.l.) cultivars do not typically produce seeds, and Orange lily is not native to Finland. Therefore, back crossing of the cultivars with wild species has not been possible. Genetic variability and genuineness of eight Finnish traditionally-grown Orange lily cultivars was studied. RAPD patterns were compared between the cultivars and genuine Orange lily ( LILIUM BULBIFERUM L.), and a related Dauricum group. The results showed partition of tested genotypes into four groups, L. CANADENSE as the outgroup. The cultivars were divided into two subgroups where the trait to form bulbils was characteristic to subgroup I. The cultivated strains differed from each other as much as from the seedling strains, but were genetically closer to genuine Orange lily than the Dauricum group. This indicates that the cultivars are genuine forms of Orange lily species. The special morphological features of the cultivars have likely been formed during centuries-long genetic isolation from natural populations.

Two endophytic fungi in different tissues of scots pine buds (Pinus sylvestris L.).

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.

Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit.

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.

Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization.

Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified as Methylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.

A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings.

We isolated a rapidly growing, pigment-producing mycobacterium from senescent tissue cultures derived from mature Scots pine (Pinus sylvestris L.). The bacterium was found in three senescent suspension cultures and in a senescent protoplast culture. Growth of Scots pine cells had ceased in all of these cultures. Exogenous contamination was eliminated by rigorous surface sterilization of the buds with hypochlorite before aseptic removal of the bud scales. Based on biochemical and physiological properties and DNA sequence comparisons, the isolated mycobacterium did not belong to any known species. Its sequence most closely resembled those of Mycobacterium obuense (97%) and M. aichiense (96%). Tissue browning was frequently observed in callus or suspension culture of Scots pine. Because the effect of the mycobacterium on growth of undifferentiated tissues that were browning was difficult to evaluate, we applied the bacterium to Scots pine seeds in aseptic conditions. Seedlings grown in the presence of the mycobacterium had shorter hypocotyls than control seedlings and seedlings cocultivated with a Pseudomonas strain known to be harmless to plants. However, hypocotyl growth of seedlings cocultivated with another mycobacterium, M. chlorophenolicum, was similar to that observed in the presence of the isolated mycobacterium. Phenylalanine ammonia-lyase activity of seedlings cocultivated with the mycobacterium isolate was significantly higher than that of control seedlings or seedlings cocultivated with M. chlorophenolicum or Pseudomotnas fluorescens. We believe that this is the first report of the isolation of mycobacteria from tissue cultures of a tree. Our finding that the mycobacterium may interfere with the growth of Scots pine in vitro warrants further study.

Light-regulated and organ-specific expression of types 1, 2, and 3 light-harvesting complex b mRNAs in Ginkgo biloba.

In a prior study (E. Chinn and J. Silverthorne [1993] Plant Physiol 103: 727-732) we showed that the gymnosperm Ginkgo biloba was completely dependent on light for chlorophyll synthesis and chloroplast development and that expression of light-harvesting complex b (Lhcb) mRNAs was substantially increased by light. However, dark-grown seedlings that were transferred to constant white light took significantly longer than angiosperm seedlings to initiate a program of photomorphogenesis and the stems failed to green completely. We have prepared type-specific probes for mRNAs encoding major polypeptides of light-harvesting complex II (Lhcb1, Lhcb2, and Lhcb3) and have used these to analyze the expression of individual Lhcb mRNAs during greening. All three sequences accumulated in the top portions of dark-grown seedlings transferred to light, but, as was seen previously for total Lhcb mRNAs, there was a transient, reproducible decline in the levels of all three mRNAs after 4 d in the light. This transient decrease in Lhcb mRNA levels was not paralleled by a decrease in Chl accumulation. By contrast, there were significantly lower levels of all three Lhcb mRNAs in the lower portions of greening dark-grown stems as well as lower Chl levels. We conclude that although the tops of the plants have the capacity to etiolate and green, Gingko seedling stems continue a program of development into woody tissue in darkness that precludes greening when the seedlings are transferred to the light.

Seasonal changes in the transient expression of a 35S CaMV-GUS gene construct introduced into Scots pine buds.

Seasonal changes in the transient expression of Beta-glucuronidase gene (GUS) driven by a constitutive 35S CaMV-promoter in Scots pine (Pinus sylvestris L.) buds were studied by the microprojectile DNA-delivery method. Buds were collected from 5-, 15- and 50-year-old trees. In buds from all age groups the amount of transient expression was dependent on the season; the highest values were found in March, and values were lowest both at the beginning and at the end of the growing season. Pretreatment with growth regulators increased both the amount of transient GUS expression and arginine decarboxylase (ADC) activity in buds indicating an increase in metabolic activity. These results confirm that the genetic transformation technique can be used to study seasonally dependent regulation in mature Scots pine tissues.