Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora.
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Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU. (+info)
Modeling of combined processing steps for reducing Escherichia coli O157:H7 populations in apple cider.
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Probabilistic models were used as a systematic approach to describe the response of Escherichia coli O157:H7 populations to combinations of commonly used preservation methods in unpasteurized apple cider. Using a complete factorial experimental design, the effect of pH (3. 1 to 4.3), storage temperature and time (5 to 35 degrees C for 0 to 6 h or 12 h), preservatives (0, 0.05, or 0.1% potassium sorbate or sodium benzoate), and freeze-thaw (F-T; -20 degrees C, 48 h and 4 degrees C, 4 h) treatment combinations (a total of 1,600 treatments) on the probability of achieving a 5-log(10)-unit reduction in a three-strain E. coli O157:H7 mixture in cider was determined. Using logistic regression techniques, pH, temperature, time, and concentration were modeled in separate segments of the data set, resulting in prediction equations for: (i) no preservatives, before F-T; (ii) no preservatives, after F-T; (iii) sorbate, before F-T; (iv) sorbate, after F-T; (v) benzoate, before F-T; and (vi) benzoate, after F-T. Statistical analysis revealed a highly significant (P < 0.0001) effect of all four variables, with cider pH being the most important, followed by temperature and time, and finally by preservative concentration. All models predicted 92 to 99% of the responses correctly. To ensure safety, use of the models is most appropriate at a 0.9 probability level, where the percentage of false positives, i.e., falsely predicting a 5-log(10)-unit reduction, is the lowest (0 to 4.4%). The present study demonstrates the applicability of logistic regression approaches to describing the effectiveness of multiple treatment combinations in pathogen control in cider making. The resulting models can serve as valuable tools in designing safe apple cider processes. (+info)
Transcript lifetime is balanced between stabilizing stem-loop structures and degradation-promoting polyadenylation in plant mitochondria.
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To determine the influence of posttranscriptional modifications on 3' end processing and RNA stability in plant mitochondria, pea atp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3' nonencoded nucleotides. A 3' rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3' ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3' poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3' nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3' processed and stable transcripts. (+info)
Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa.
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Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level. (+info)
Extrafloral nectar production of the ant-associated plant, Macaranga tanarius, is an induced, indirect, defensive response elicited by jasmonic acid.
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Plant species in at least 66 families produce extrafloral nectar (EFN) on their leaves or shoots and therewith attract predators and parasitoids, such as ants and wasps, which in turn defend them against herbivores. We investigated whether EFN secretion is induced by herbivory and/or artificial damage, and thus can be regarded as an induced defensive response. In addition, we studied the underlying signaling pathway. EFN secretion by field-grown Macaranga tanarius increased after herbivory, artificial leaf damage, and exogenous jasmonic acid (JA) application. Artificial damage strongly enhanced endogenous JA concentrations. The response in EFN production to artificial damage was much less pronounced in those leaves that were treated with phenidone to inhibit endogenous JA synthesis. Quantitative dose-response relations were found between the increase in nectar production and both the intensity of leaf damage and the amounts of exogenously applied JA. The amount of endogenously produced JA was positively correlated with the intensity of leaf damage. Increased numbers of defending insects and decreased numbers of herbivores were observed on leaves after inducing EFN production by exogenous JA treatment. Over 6 weeks, repeatedly applied JA or artificial damage resulted in a ten-fold reduction in herbivory. These results demonstrate that EFN production represents an alternative mechanism for induced, indirect defensive plant responses that are mediated via the octadecanoid signal transduction cascade. (+info)
Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor.
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Fruit development in higher plants normally requires pollination and fertilization to stimulate cell division of specific floral tissues. In some cases, parthenocarpic fruit development proceeds without either pollination or fertilization. Parthenocarpic fruit without seed has higher commercial value than seeded fruit. Several apple (Malus domestica) mutants (Rae Ime, Spencer Seedless and Wellington Bloomless) are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruit. Through genetics, a single recessive gene has been identified to control this trait in apple. Flower phenotypes of these apple mutants are strikingly similar to those of the Arabidopsis mutant pistillata (pi), which produces flowers where petals are transformed to sepals and stamens to carpels. In this study, we have cloned the apple PI homolog (MdPI) that shows 64% amino acid sequence identity and closely conserved intron positions and mRNA expression patterns to the Arabidopsis PI. We have identified that in the apetalous mutants MdPI has been mutated by a retrotransposon insertion in intron 4 in the case of Rae Ime and in intron 6 in the case of Spencer Seedless and Wellington Bloomless. The insertion apparently abolishes the normal expression of the MdPI gene. We conclude that the loss of function mutation in the MdPI MADS-box transcription factor confers parthenocarpic fruit development in these apple varieties and demonstrates another function for the MADS- box gene family. The knowledge generated here could be used to produce parthenocarpic fruit cultivars through genetic engineering. (+info)
Binding affinity of the methyl ester of AK-toxin I to membrane fractions from Japanese pear leaves.
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The binding site of AK-toxin, a host-specific toxin against Japanese pear, was searched for in the membrane fractions of the pear leaves, using 3H-labeled AK-toxin I methyl ester. Binding activity, which was displaceable by the unlabeled ligand, was observed for microsomal fraction from a toxin-susceptible cultivar, Nijisseiki. However, the binding was also observed for those from toxin-resistant cultivars, Kosui and Hosui. Detection of the specific binding failed for the plasma membrane fraction which was prepared from microsomal fraction of the toxin-susceptible cultivar by aqueous two-phase separation, and the hitherto presumed model of the AK-toxin receptor in the plasma membrane could not be verified. (+info)
Medicinal foodstuffs. XXI. Structures of new cucurbitane-type triterpene glycosides, goyaglycosides-a, -b, -c, -d, -e, -f, -g, and -h, and new oleanane-type triterpene saponins, goyasaponins I, II, and III, from the fresh fruit of Japanese Momordica charantia L.
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Eight cucurbitane-type triterpene glycosides called goyaglycosides-a, -b, -c, -d, -e, -f, -g, and -h and three oleanane-type triterpene saponins termed goyasaponins I, II, and III were isolated from the fresh fruit of Japanese Momordica charantia L. (Cucurbitaceae) together with five known cucurbitane-type triterpene glycosides momordicosides A, C, F1, I, and K. The structures of goyaglycosides and goyasaponins were elucidated on the basis of chemical and physicochemical evidence. (+info)