Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo.

Cranberry crude extracts, in various vehicles, have shown inhibitory effects on the formation of oral biofilms in vitro. The presence of proanthocyanidins (PAC) in cranberry extracts has been linked to biological activities against specific virulence attributes of Streptococcus mutans, e.g. the inhibition of glucosyltransferase (Gtf) activity. The aim of the present study was to determine the influence of a highly purified and chemically defined cranberry PAC fraction on S. mutans biofilm formation on saliva-coated hydroxyapatite surface, and on dental caries development in Sprague-Dawley rats. In addition, we examined the ability of specific PAC (ranging from low-molecular-weight monomers and dimers to high-molecular-weight oligomers/polymers) to inhibit GtfB activity and glycolytic pH drop by S. mutans cells, in an attempt to identify specific bioactive compounds. Topical applications (60-second exposure, twice daily) with PAC (1.5 mg/ml) during biofilm formation resulted in less biomass and fewer insoluble polysaccharides than the biofilms treated with vehicle control had (10% ethanol, v/v; p < 0.05). The incidence of smooth-surface caries in rats was significantly reduced by PAC treatment (twice daily), and resulted in less severe carious lesions compared to the vehicle control group (p < 0.05); the animals treated with PAC also showed significantly less caries severity on sulcal surfaces (p < 0.05). Furthermore, specific A-type PAC oligomers (dimers to dodecamers; 0.1 mg/ml) effectively diminished the synthesis of insoluble glucans by GtfB adsorbed on a saliva-coated hydroxyapatite surface, and also affected bacterial glycolysis. Our data show that cranberry PAC reduced the formation of biofilms by S. mutans in vitro and dental caries development in vivo, which may be attributed to the presence of specific bioactive A-type dimers and oligomers.

Favorable glycemic response of type 2 diabetics to low-calorie cranberry juice.

Fruit and vegetable intake is typically low for type 2 diabetics, possibly due to a perceived adverse effect on glycemic control. Cranberry juice (CBJ) may represent an attractive means for increasing fruit intake and simultaneously affording positive health benefits. This single cross-over design compared metabolic responses of type 2 diabetics (n= 12) to unsweetened low-calorie CBJ (LCCBJ; 19 Cal/240 mL), carbohydrate sweetened normal calorie CBJ (NCCBJ; 120 Cal/240 mL), isocaloric low-calorie sugar water control (LCC), and isocaloric normal calorie sugar water control (NCC) interventions. CBJ flavonols and anthocyanins, and proanthocyanidins were quantified with HPLC, LC-MS, and MALDI-TOF that includes an original characterization of several large oligomeric proanthocyanidins. Blood glucose peaked 30 min postingestion after NCCBJ and NCC at 13.3 +/- 0.5 and 12.8 +/- 0.9 (mmol/L), and these responses were significantly greater than the LCCBJ and LCC peaks of 8.1 +/- 0.5 and 8.7 +/- 0.5, respectively. Differences in glycemic response remained significant 60 min, but not 120 min postingestion. Plasma insulin values 60 min postingestion for NCCBJ and NCC interventions were 140 +/- 19 and 151 +/- 18 (pmol/L), respectively, and significantly greater than the LCCBJ and LCC values of 56 +/- 10 and 54 +/- 10; differences were not significant 120 min postingestion. Metabolic responses within the 2 high and 2 low-calorie beverages were virtually identical; however, exposure to potentially beneficial nutrients was greater with CBJ. Relative to conventionally sweetened preparation, LCCBJ provides a favorable metabolic response and should be useful for promoting increased fruit consumption among type 2 diabetics or others wishing to limit carbohydrate intake.

Isothiocyanate NB7M causes selective cytotoxicity, pro-apoptotic signalling and cell-cycle regression in ovarian cancer cells.

The present report identifies indole-3-ethyl isothiocyanate NB7M as a potent cytotoxic agent with selective activity against cell lines derived from various tumour types. Ovarian cancer cell lines showed sensitivity to NB7M (60-70% cytotoxicity at 2.5 microM), in contrast to control cells (TCL-1 and HTR-8; IC(50) approximately 15 microM). In a screen performed by the National Cancer Institute (NCI) (NCI(60) cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC(50) between 0.1 and 10 microM depending on the cell line studied. Using SKOV-3 ovarian cancer cells as a model, mechanisms of cytotoxicity were analysed. NB7M caused hallmarks of apoptosis such as PARP-1 deactivation, chromatin condensation, DNA nicks, activation of caspases-9, -8, -3, loss of mitochondrial transmembrane depolarisation potential and upregulation of pro-apoptotic mitogen activated protein kinases (p38, SAP/JNK). NB7M downregulated phosphorylation of prosurvival kinases (PI-3K, AKT, IKK alpha), transcription factor NF-kappaB, and expression of DNA-Pk and AXL receptor tyrosine kinase. Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression. The present report suggests that NB7M is a selective cytotoxic agent in vitro for cell lines derived from ovarian and certain other tumours. In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer.

Influence of cranberry phenolics on glucan synthesis by glucosyltransferases and Streptococcus mutans acidogenicity.

AIMS: To investigate the influence of several phenolic compounds isolated from cranberry fruit (Vaccinium macrocarpon) on some of the virulence properties of Streptococcus mutans associated with glucan synthesis and acidogenicity. METHODS AND RESULTS: Individual phenolic acids, flavonols and proanthocyanidins were isolated by semi-preparative high-performance liquid chromatography from fresh cranberry fruit. Flavonols and proanthocyanidins (at 500 micromol l(-1)) moderately inhibited the activity of surface-adsorbed glucosyltransferases (GTFs) B and C and F-ATPases (15-35% inhibition; P < 0.05), and also disrupted acid production by S. mutans cells without affecting bacterial viability. Phenolic acids displayed minimal biological effects. Quercetin-3-arabinofuranoside, myricetin and procyanidin A2 displayed the most inhibition of S. mutans virulence traits; a combination of these compounds displayed enhanced effects. CONCLUSIONS: Specific flavonoids from cranberries exhibit statistically significant but moderate biological activity against S. mutans. The biological activity of cranberry extracts may be a result from the complex mixture of flavonoids rather than a single active compound. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study to identify the bioactive constituents in cranberry against an oral bacterium using highly purified isolated compounds. The combined effects of specific flavonols and proanthocyanidins from cranberry on GTFs activity, acid production and acid tolerance of S. mutans make them attractive compounds to fully explore for their anti-biofilm and cariostatic properties.

Intraspecific variation in aphid resistance and constitutive phenolics exhibited by the wild blueberry Vaccinium darrowi.

Illinoia pepperi (MacGillivray) infests cultivated highbush blueberries, Vaccinium corymbosum L., in the Northeastern United States. Allopatric resistance to I. pepperi was examined in Vaccinium darrowi Camp, which evolved in the absence of I. pepperi in the Southeastern U.S. V. corymbosum cv. "Elliott", was used as a susceptible control. Between population variability in I. pepperi resistance was assessed by measuring length of the prereproductive period, fecundity, and survivorship on 14 V. darrowi accessions representing 11 discrete wild populations. Length of I. pepperi's prereproductive period and survivorship were not significantly affected. However, differences were detected in fecundity and the intrinsic rate of increase (r ( m )). Within population variability in resistance was measured by confining first instars to 24 accessions from a single wild population of V. darrowi (NJ88-06). Significant differences in the mean total number of aphids occurring after 20 d were only detected between 2 of the 24 V. darrowi accessions. A greater degree of diversity in I. pepperi resistance exists between populations of V. darrowi compared to within a population. Constitutive leaf and stem polyphenolics were identified by HPLC-MS and quantified from 14 of the V. darrowi accessions. The accessions varied in concentrations of five phenolic acids and seven flavonol glycosides, but a correlation was not found between individual or total phenolics and aphid performance. Overall, screening within and between populations of V. darrowi identified promising sources of aphid resistance, but phenolic acid and flavonol glycoside profiles did not predict resistance levels. The mechanism of resistance remains to be identified.

Transmission of a cyclical translocation in two cranberry cultivars.

The occurrence of cyclical translocation involving three non-homologous chromosomes and affecting pollen stainability has been observed in two cranberry cultivars: Howes, and Wilcox, a progeny of Howes. These cultivars were crossed with six normal cranberry cultivars to study the transmission of the cyclical translocation to their progeny. The translocational progeny were determined by pollen tetrad analysis. A total of 102 individuals (6 crosses) were analyzed in the progeny of Wilcox and 116 individuals (5 crosses) in the progeny of Howes. The ratios observed in the progeny of Wilcox and Howes were 71 translocated: 31 normal, and 79 translocated: 37 normal, respectively. The segregations deviated from the expected 1 translocated: 1 normal progeny ratio, but fit either a 3:1 or 2:1 ratio. The altered segregations may indicate the presence of a balanced lethal system located in the translocated segments of both Howes and Wilcox. Sterile individuals were found in the progeny of WilcoxxHowes, which could indicate that the two parents have non-identical translocations. The translocated progeny of both cultivars had a normal distribution for pollen stainability, which indicated that both the occurrence of crossing over in the interstitial region and the segregation of chromosomes are under polygenic control.

Cloning, sequencing, and promoter identification of Blueberry red ringspot virus, a member of the family Caulimoviridae with similarities to the "Soybean chlorotic mottle-like" genus.

The double-stranded DNA genome of Blueberry red ringspot virus (BRRV), a member of the family Caulimoviridae, was cloned and sequenced. The genome organization and relationships of the 8303 nt sequence revealed BRRV to be a tentative member of the genus that has been provisionally named "Soybean chlorotic mottle-like viruses", rather than a member of the genus Caulimovirus, in which it had been placed previously. Insertion of the putative 35S promoter homolog of BRRV into promoterless constructs carrying the UidA (beta-glucuronidase) gene resulted in high-level transient expression from cranberry and stable expression from transgenic tobacco. Sequences of 5'-RACE clones derived from transcripts from transgenic tobacco were consistent with the map position of the promoter.

Resistance of Diploid Vaccinium spp. to the Fruit Rot Stage of Mummy Berry Disease.

Mummy berry disease caused by Monilinia vaccinii-corymbosi is the most widespread economically important problem of cultivated blueberry in North America. In an attempt to identify new sources of resistance to the fruit rot (mummification) phase of mummy berry, 140 accessions from a total of 21 populations from seven wild diploid species of blueberry were evaluated for resistance under greenhouse conditions. Six isolates of M. vaccinii-corymbosi from three states were used as inoculum. A highly resistant response to mummy berry fruit rot was exhibited by all accessions of Vaccinium boreale, V. myrtilloides, V. pallidum, and V. tenellum, and by most accessions of V. darrowi. Most of the V. corymbosum and V. elliottii accessions were moderately to highly susceptible. Introgression of the resistance found in the wild diploid species into horticulturally desirable cultivars could significantly improve available resistance.

Allozyme evidence for genetic autopolyploidy and high genetic diversity in tetraploid cranberry, Vaccinium oxycoccos (Ericaceae).

Polyploidy has been important in the evolution of angiosperms and may significantly affect population genetic diversity and structure. Nineteen isoenzyme loci were studied in diploid and tetraploid populations of Vaccinium oxycoccos (Ericaceae), and the results are compared with data previously reported for the related V. macrocarpon. Diploid V. oxycoccos and V. macrocarpon were readily discriminated based on their allozymic variation. No evidence for fixed heterozygosity was found in tetraploid V. oxycoccos. In contrast, all polymorphic loci exhibited both balanced and unbalanced heterozygotes, with some individuals exhibiting a pattern consistent with the presence of three alleles. These results support an autopolyploid origin for tetraploid V. oxycoccos. However, tetraploid V. oxycoccos possessed a suite of alleles not found in diploid V. oxycoccos; half of these alleles were shared with V. macrocarpon. This suggests that autotetraploid V. oxycoccos may have undergone hybridization with V. macrocarpon or that the autotetraploid retained the genetic variation present in an ancestral diploid species. Following theoretical expectations, proportion of polymorphic loci, mean number of alleles, and observed heterozygosity were significantly higher for the autotetraploid than for the diploid. Mean inbreeding (F(IS)) was similar for diploid and tetraploid V. oxycoccos. The latter exhibited population differentiation (F(ST)) exceeding both diploid species.

A-Type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli.

Three proanthocyanidin trimers possessing A-type interflavanoid linkages, epicatechin-(4beta-->6)-epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin (4), epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin-(4beta-->8)-epicatechin (5), and epicatechin-(4beta-->8)-epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin (6), were isolated from the ripe fruits of Vaccinium macrocarpon (cranberry) and prevented adherence of P-fimbriated Escherichia coli isolates from the urinary tract to cellular surfaces containing alpha-Gal(1-->4)beta-Gal receptor sequences similar to those on uroepithelial cells. The structure of 4 was elucidated by a combination of spectroscopic methods and acid-catalyzed degradation with phloroglucinol. Also isolated were the weakly active epicatechin-(4beta-->8, 2beta-->O-->7)-epicatechin (procyanidin A2) (3) and the inactive monomer epicatechin (1) and the inactive dimer epicatechin-(4beta-->8)-epicatechin (procyanidin B2) (2).

The structure of cranberry proanthocyanidins which inhibit adherence of uropathogenic P-fimbriated Escherichia coli in vitro.

Ethyl acetate extracts of Sephadex LH20-purified proanthocyanidins of American cranberry (Vaccinium macrocarpon Ait.) exhibited potent biological activity by inhibiting adherence of uropathogenic isolates of P-fimbriated Escherichia coli bacteria to cellular surfaces containing alpha-Gal(1-->4)beta-Gal receptor sequences similar to those on epithelial cells in the urinary tract. The chemical structures of the proanthocyanidins were determined by 13C NMR, electrospray mass spectrometry, matrix-assisted laser absorption time-of-flight mass spectrometry and by acid catalyzed degradation with phloroglucinol. The proanthocyanidin molecules consisted predominantly of epicatechin units with mainly DP of 4 and 5 containing at least one A-type linkage. The procyanidin A2 was the most common terminating unit occurring about four times as frequently as the epicatechin monomer.

Evidence for RAPD heteroduplex formation in cranberry: implications for pedigree and genetic-relatedness studies and a source of co-dominant RAPD markers.

Silver-stained random amplified polymorphic DNA (ssRAPD) markers have been identified that are always jointly present or absent in the ssRAPD profiles of cranberry varieties. On the basis of segregation data and the ability to re-create these "associated ssRAPDs" through the intermixing of amplified DNA from individuals lacking them, five of the six pairs of associated ssRAPDs analyzed were shown to be consistent with heteroduplex molecules. Heteroduplexes are "hybrid"" double-stranded DNAs that are formed following the polymerase chain reaction (PCR) amplification of two DNA segments that have a high degree of homology to one another, yet differ in their nucleotide sequences as a result of base pair deletions, additions, or substitutions. Three of the five putative heteroduplex systems identified are consistent with a one locus, two-allele heteroduplex model. The remaining two systems appeared to be multi-allelic, involving interactions among three and four alleles, respectively. RAPD heteroduplex formation has the potential to confound genetic relatedness and pedigree studies. Heterozygous individuals exhibit heteroduplex RAPDs not seen in either of the two homozygote classes. Genetic estimates under such a circumstance would "inflate" the differences between the heterozygote and the homozygote classes. Heteroduplex formation is also a mechanism for the presence of non-parental RAPDs in progeny of parents homozygous for alternate alleles. While this class of molecular markers can confound RAPD analyses, they also offer a source of co-dominant RAPD markers, which are of value in genetic relatedness estimates and as markers for studying breeding behavior.

RAPDs identify varietal misclassification and regional divergence in cranberry [Vaccinium macrocarpon (Ait.) Pursh].

The majority of cultivated cranberry varieties were selected from native populations in the 1800s and early 1900s from sites in Massachusetts, New Jersey, and Wisconsin. Since their initial selections 100-150 years ago, varietal identities have become increasingly confused; primarily the result of there being a paucity of qualitative markers to effectively distinguish among varieties. Random amplified polymorphic DNA (RAPD) technology has the potential for allowing a more definitive classification of varieties and was used in this study to characterize 22 cranberry varieties. Twenty-two decamer primers amplified 162 scorable DNA fragments, of which 66 (41%) were polymorphic. On the basis of these 66 silver-stained RAPDs (ssRAPDs), 17 unique profiles were identified rather than the expected 22. Fourteen varieties had unique ssRAPD profiles, while the remaining 8 were represented by 3 ssRAPD profiles. Permuational analyses of the data suggest that the observed ssRAPD profile duplications are examples of varietal misclassification. Further analyses identified 2 ssRAPD markers that were found only in Eastern varieties (from Mass. and N. J.) and not in Wisconsin varieties. With varieties differing on average by 22 bands, ssRAPDs are shown to be effective in varietal identification and the assessment of genetic diversity in cranberry.

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry.

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r(2)=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that "conventional polyploid evolution" has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.

Occurrence of unreduced pollen in diploid blueberry species, Vaccinium sect. Cyanococcus.

A total of 1475 individuals belonging to 43 natural populations of seven diploid (2x) blueberry species (Vaccinium section Cyanococcus) and two natural interspecific 2x hybrid populations were evaluated for unreduced pollen production. Significant differences were found in the frequency of unreduced pollen producers between species and within and between populations of the same species. Individuals with 1% or more unreduced pollen were considered unreduced pollen producers. The average frequency of unreduced pollen producers in these diploid species was 13.5%, ranging from 7.4% (V. corymbosum) to 18.4% (V. darrowii). The frequency of unreduced pollen grains in individual clones varied from <-1% to 28.6%. The production of unreduced pollen was not associated with male fertility. The widespread occurrence of unreduced pollen in the diploid species should allow the introgression of this germ plasm to the tetraploid level via unilateral sexual polyploidization.