First Report of Penicillium carneum Causing Blue Mold on Stored Apples in Pennsylvania.

Blue mold decay occurs during long term storage of apples and is predominantly caused by Penicillium expansum Link. Apples harvested in 2010 were stored in a controlled atmosphere at a commercial Pennsylvania apple packing and storage facility, and were examined for occurrence of decay in May 2011. Several decayed apples from different cultivars, exhibiting blue mold symptoms with a sporulating fungus were collected. One isolate recovered from a decayed 'Golden Delicious' apple fruit was identified as P. carneum Frisvad. Genomic DNA was isolated, 800 bp of the 3' end of the ß-tubulin locus was amplified using gene specific primers and sequenced (4). The recovered nucleotide sequence (GenBank Accession No. JX127312) indicated 99% sequence identity with P. carneum strain IBT 3472 (GenBank Accession No. JF302650) (3). The P. carneum colonies strongly sporulated and had a blue green color on potato dextrose agar (PDA), Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) media at 25°C after 7 days. The colonies also had a beige color on plate reverse on CYA and YES media. The species tested positive for the production of alkaloids, as indicated by a violet reaction for the Ehrlich test, and grew on CYA at 30°C and on Czapek with 1,000 ppm propionic acid agar at 25°C; all of which are diagnostic characters of this species (2). The conidiophores were hyaline and tetraverticillate with a finely rough stipe. Conida were produced in long columns, blue green, globose, and averaged 2.9 µm in diameter. To prove pathogenicity, Koch's postulates were conducted using 20 'Golden Delicious' apple fruits. Fruits were washed, surface sterilized with 70% ethanol, and placed onto fruit trays. Using a nail, 3-mm wounds were created and inoculated with 50 µl of a 106/ml conidial suspension or water only as a negative control. The fruit trays were placed into boxes and were stored in the laboratory at 20°C for 7 days. The inoculated fruit developed soft watery lesions, with hard defined edges 37 ± 4 mm in diameter. The sporulating fungus was reisolated from infected tissue of all conidia inoculated apples and confirmed to be P. carneum by polymerase chain reaction (PCR) using the ß-tubulin locus as described. Water inoculated control apples were symptomless. Originally grouped with P. roqueforti, P. carneum was reclassified in 1996 as a separate species (1). P. carneum is typically associated with meat products, beverages, and bread spoilage and produces patulin, which is not produced by P. roqueforti (1,2). Our isolate of P. carneum was susceptible to the thiabendazole (TBZ) fungicide at 250 ppm, which is below the recommended labeled application rate of 600 ppm. The susceptibility to TBZ suggests that this P. carneum isolate has been recently introduced because resistance to TBZ has evolved rapidly in P. expansum (4). To the best of our knowledge, P. carneum has not previously been described on apple, and this is the first report of P. carneum causing postharvest decay on apple fruits obtained from storage in Pennsylvania. References: (1) M. Boyson et al. Microbiology 142:541, 1996. (2) J. C. Frisvad and R. A. Samson. Stud. Mycol. 49:1, 2004. (3) B. G. Hansen et al. BMC Microbiology 11:202, 2011. (4) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.

Fresh-cut lettuce in modified atmosphere packages stored at improper temperatures supports enterohemorrhagic E. coli isolates to survive gastric acid challenge.

Incidences of foodborne outbreaks involving enterohemorrhagic Escherichia coli strains with mutations in a key regulatory gene, rpoS, have been reported. Incentives, if any, for losing this regulatory function are not clear since the RpoS regulator is required for the expression of several environmental stress tolerance genes. RpoS also positively regulates 2 of the 3 acid-resistance systems of E. coli under aerobic growth conditions and enables the pathogen to survive gastric acid challenge. We selected 7 enterohemorrhagic E. coli isolates, 6 of which are known to carry defective rpoS gene, and then analyzed resistance to synthetic gastric juice after the strains were inoculated on fresh-cut lettuce and stored under modified atmosphere packaging (MAP) conditions. Subatmospheric oxygen partial pressures in MAP enabled all 6 rpoS-defective isolates to induce acid resistance over the 8-d storage period if the temperature was >or= 15 degrees C. No acid resistance was induced for MAP-stored lettuce left at temperatures

Temperature-dependent autoxidation of conjugated trienols from apple peel yields 6-methyl-5-hepten-2-one, a volatile implicated in induction of scald.

Conjugated triene (CT) oxidation products of alpha-farnesene have long been thought to be involved in development of superficial scald in apple fruit. Early studies found that CT hydroperoxides and the volatile 6-methyl-5-hepten-2-one (MHO) are major in vitro autoxidation products of alpha-farnesene. However, it was recently shown that > or =99% of the oxidation products of alpha-farnesene that accumulate in apple peel are conjugated trienols (CTols), isomers of 2,6,10-trimethyldodeca-2,7,9,11-tetraene-6-ol. HPLC-purified CTols from fruit of two scald-susceptible cultivars, Granny Smith (GS) and Red Delicious (RD), were used to study autoxidation of these compounds in vitro. Incubation of CTols in sealed glass vials under air resulted in accumulation of MHO. Oxygen enrichment did not increase the amount of MHO produced. Regardless of which cultivar CTols were derived from, at 0 degrees C autoxidation yielding MHO was quite slow and linear, whereas at 20 degrees C MHO production was much more rapid, and after several hours the rate increased abruptly. However, CTols isolated from GS and RD fruit differed in the duration of the initial lag phase and the overall level of MHO generated at 20 degrees C. The sharp increase in MHO production occurred after 3 h with GS CTols and at about 12 h with RD CTols. Also, the yield of MHO from GS CTols after 6 h at 20 degrees C was nearly 6-fold greater than that from RD CTols after 20 h at 20 degrees C. The antioxidants butylated hydroxytoluene and diphenyamine reduced the yield of MHO by about 97%. Recent work has shown that MHO can induce scald-like symptoms in apple peel and that tissue sensitivity increases with time in storage. This may explain the correlation between high CTol levels and scald development, and why symptoms rapidly intensify when fruits are removed from cold storage.

A new 1-aminocyclopropane-1-carboxylic acid-conjugating activity in tomato fruit.

A new conjugate, 1-(gamma-L-glutamylamino)cyclopropane-1-carboxylic acid (GACC), of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is identified. The only previously identified conjugate of ACC is 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). GACC, not MACC, was the major conjugate formed by crude protein extracts of tomato (Lycopersicon esculentum Mill cv Ailsa Craig) fruit pericarp and seeds incubated with [14C]ACC. GACC was resolved from [14C]ACC and [14C]MACC by reversed-phase C18 thin-layer chromatography and subsequently detected and quantified using a radioisotope-imaging system. Proteins precipitated from crude extracts failed to catalyze formation of GACC unless the supernatant was added back. Reduced glutathione, but not other reducing agents, replaced the crude supernatant. When [35S-cysteine]glutathione and [3H-2-glycine]glutathione were used as substrates, neither radiolabeled glycine nor cysteine from the glutathione tripeptide was incorporated into GACC. Oxidized glutathione, S-substituted glutathione, and di- and tripeptides having an N-terminal gamma-L-glutamic acid, but lacking cysteine and glycine, also served as substrates for GACC formation. Peptides lacking the N-terminal gamma-L-glutamic acid did not serve as substrates. Acid hydrolysis of GACC yielded ACC, suggesting that GACC is an amide-linked conjugate of ACC. Taken together, these results indicate that GACC is 1-(gamma-glutamylamino)cyclopropane-1-carboxylic acid and that its formation is catalyzed by a gamma-glutamyltranspeptidase. Gas chromatography-mass spectrometry analysis of the N-acetyl dimethyl ester of GACC confirmed this structure.

Purification and Characterization of 1-Aminocyclopropane-1-Carboxylic Acid N-Malonyltransferase from Tomato Fruit.

1-Aminocyclopropane-1-carboxylic acid (ACC) can be oxidized to ethylene or diverted to the conjugate 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by an ACC N-malonyltransferase. We developed a facile assay for the ACC N-malonyltransferase that resolved [14C]MACC from [14C]ACC by thin-layer chromatography and detected and quantified them using a radioisotope-imaging system. Using this assay, we showed that ACC N-malonyltransferase activity has developmental and tissue-specific patterns of expression in tomato (Lycopersicon esculentum) fruit. In the pericarp, activity was elevated for several days postanthesis, subsequently declined to a basal level, increased 3-fold at the onset of ripening, and again declined in overripe fruit. In the seed, activity increased throughout embryogenesis, maturation, and desiccation. Treatment of fruit with ethylene increased activity 50- to 100-fold in the pericarp. ACC N-malonyltransferase was purified 22,000-fold to a specific activity of 22,000 nmol min-1 mg-1 protein using ammonium sulfate precipitation, DyeMatrex Green A affinity, anion-exchange, Cibacron Blue 3GA affinity, hydrophobic interaction, and molecular filtration chromatography. Native and sodium dodecyl sulfate-denatured enzyme showed molecular masses of 38 kD, indicating that the enzyme exists as a monomer. The enzyme exhibited a Km for ACC of 500 [mu]M, was not inhibited by D- or L-amino acids, and did not conjugate [alpha]-aminoisobutyric acid or L-amino acids.

Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A.

Using anther-derived rice (Oryza sativa L.) cell-suspension cultures, we have identified an 18-kD protein that is posttranslationally modified by spermidine and is influenced by endogenous polyamine levels. The posttranslationally modified residue has been identified as the unusual amino acid hypusine [N[epsilon]-(4-amino-2-hydroxybutyl)lysine] by reverse-phase high-performance liquid chromatography and gas chromatography-mass-spectrometry analyses. Differential labeling of the protein with labeled amines provided evidence that the butylamine moiety of spermidine is the immediate precursor of the hypusine residue in the protein. The eukaryotic translation initiation factor 5A (eIF-5A) is the only known mammalian protein that undergoes a similar posttranslational modification with hypusine. The purified 18-kD protein co-electrophoreses with human translational initiation factor eIF-5A in both isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. The purified protein from rice stimulated methionyl-puromycin synthesis in vitro, indicating its functional similarity to mammalian eIF-5A. The results presented provide evidence that the posttranslationally modified 18-kD protein from rice containing hypusine is eIF-5A and suggest the conservation of hypusine-containing translation initiation factor eIF-5A in eukaryotes.

Translational modification of an 18 kilodalton polypeptide by spermidine in rice cell suspension cultures.

When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-(3)H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis.

Polyamine levels and tomato fruit development: possible interaction with ethylene.

Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life.

Transport and Compartmentation of 1-Aminocyclopropane-1-Carboxylic Acid and Its Structural Analog, alpha-Aminoisobutyric Acid, in Tomato Pericarp Slices.

The uptakes of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene, and its structural analog, alpha-aminoisobutyric acid (alphaAIB) by tomato pericarp slices were investigated. Both uptakes show a biphasic (saturable-linear) dependence on external concentration of the transported amino acid. At low concentrations, ACC uptake is competitively inhibited by alphaAIB and vice versa. Both uptakes also are inhibited by other neutral amino acids but not by acidic or basic amino acids. ACC and alphaAIB uptakes are metabolically dependent and are increased with time of tissue incubation. alphaAIB efflux patterns from pericarp slices indicated three distinct alphaAIB compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole. The bulk of the alphaAIB taken up by pericarp tissue is sequestered into the vacuole. The ability of pericarp tissue to accumulate alphaAIB in the vacuole declines with fruit development.

Quantitative determination of indole-3-acetic Acid in sugarbeet leaves using a double standard isotope dilution gas chromatographic assay.

Quantitative levels of indole-3-acetic acid (IAA) were determined in leaf blades of two sugarbeet cultivars by a double standard isotope dilution assay using column chromatography followed by reverse phase C(18) high performance liquid chromatography and gas-liquid chromatography with nitrogen thermionic detection. The double standard method was validated as a quantitative tool by gas chromatography/selected ion monitoring mass spectrometry using 2,',4',5',6',7'-d(5)-IAA as the internal standard. Progenies of one breeding line that had been selected for a high taproot to leaf weight ratio were used to correlate IAA levels with varying leaf and plant size at day 31 from germination. In spite of size differences, no significant difference in IAA levels per unit leaf weight could be found. The possible relationship between day 31 leaves and IAA content at an earlier stage of development is discussed in the text. A second analysis used four developmental leaf stages, classified as expanding, recently mature, aging, and senescing leaves. Expanding leaves contained the most IAA, senescing leaves contained the least IAA, with recently mature leaves and aging leaves containing intermediate amounts. The DNA content of each of the four developmental leaf stages was determined and DNA levels per gram fresh weight were found to be constant at all developmental stages.

Effect of plant hormones on sucrose uptake by sugar beet root tissue discs.

Abscisic acid (ABA), auxins, cytokinins, gibberellic acid, alone or in combination were tested for their effects on short-term sucrose uptake in sugar beet (Beta vulgaris cv USH-20) roots. The effect of ABA on active sucrose uptake varied from no effect to the more generally observed 1.4-to 3.0-fold stimulation. A racemic mixture of ABA and its trans isomer were more stimulatory than ABA alone. Pretreating and/or simultaneously treating the tissue with K(+) or IAA prevented the ABA response while cytokinins and gibberellic acid did not. While the variable sensitivities of beet root to ABA may somehow be related to the auxin and alkali cation status of the tissue, tissue sensitivity to ABA was not correlated with ABA uptake, accumulation, or metabolic patterns. In contrast to ABA, indoleacetic acid (IAA) and other auxins strongly inhibited active sucrose uptake in beet roots. Cytokinins enhanced the auxin-induced inhibition of sucrose uptake but ABA and gibberellic acid did not modify or counteract the auxin effect. Trans-zeatin, benzyladenine, kinetin, and gibberellins had no effect on active sucrose uptake. None of the hormones or hormone mixtures tested had any significant effect on passive sucrose uptake. The effects of IAA and ABA on sucrose uptake were detectable within 1 h suggesting a rather close relationship between the physiological activities of IAA and ABA and the operation of the active transport system.

Sucrose uptake and compartmentation in sugar beet taproot tissue.

Active sucrose uptake by discs of mature sugar beet (Beta vulgaris L. cv GW-D2 and USH-20) root tissue shows a biphasic dependence on external sucrose. At concentrations up to 20 millimolar sucrose, the active uptake mechanism appears to approach saturation, with an apparent K(m) of 3.6 millimolar. At higher external sucrose concentrations, a linear dependence becomes obvious indicating the probable presence of a nonsaturable, metabolically dependent uptake component. Active transport was not observed at external sucrose concentrations that caused tissue plasmolysis. Passive sucrose uptake in unplasmolyzed tissue showed a linear dependence on external sucrose concentration. The mitochondrial and/or suspected vacuolar ATPase inhibitors oligomycin, diethylstilbestrol, and N,N-dicyclohexylcarbodiimide strongly inhibited active sucrose uptake, whereas the putative plasmalemma-specific ATPase inhibitor orthovanadate was without effect.Sucrose efflux patterns from root discs indicated three distinct sucrose compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole with the vacuole being the slowest releasing compartment. The sucrose contents and volumes of the compartments indicated that sucrose uptake into the vacuole was against a concentration gradient. Combined sucrose uptake/efflux analyses indicated that sucrose uptake into the vacuole is primarily an active transport process while transport into the cytoplasm is apparently passive, at least at external sucrose concentrations above 20 millimolar. We discuss the possibility that active sucrose uptake into the vacuoles of sugar beet storage cells is rate limited by passive sucrose transport to the active uptake site.

Reduction in Sink-Mobilizing Ability following Periods of High Carbon Flux.

Sink tissues may play a significant role in determining photosynthetic rates through their ability to mobilize assimilates. The objective in this study was to determine if the mobilizing ability of taproot sink tissues of sugarbeet (Beta vulgaris) could become limiting when assimilate supply was maintained at a high level for an extended period of time. Assimilate supply was either enhanced by CO(2) enrichment or reduced by shading.Field-grown sugarbeet plants were exposed to ambient CO(2) and one of five photosynthetically active radiation (PAR) durations: 10-hours PAR; 6-hours PAR; 3-hours PAR; 1-hour PAR; and continuous 80% shade conditions or 1,000 microliter per liter CO(2) and 10-hour PAR. Taproots were harvested at 1600 hours on the day following the initiation of the treatments. The sucrose-uptake capacity of excised tissue discs was determined in 30 millimolar morpholinopropane sulfonic acid (pH 7.0) containing 40 millimolar [(14)C]sucrose.Rates of sucrose uptake were inversely related to the supply of photosynthate during the preceding light period. CO(2) enrichment reduced uptake capacity relative to the control. In contrast, reducing the duration of PAR increased uptake over the control. Leaf starch accumulation was correlated with reduced uptake capacity. The results indicate that, under the conditions employed here, the mobilizing ability of sinks may limit carbon flux from source and to sink during periods of high photosynthetic rates.

Electrical potentials in stomatal complexes.

Guard cells of several species, but predominantly Commelina communis, were impaled by micropipette electrodes and potential differences measured that occurred between cell compartments and the flowing bathing medium. The wall developed a Donnan potential that was between -60 and -70 millivolt in 30 millimolar KCl at pH 7. The density of the fixed charges ranged from 0.3 to 0.5 molar; its dependence on pH was almost identical with the titration curve of authentic polygalacturonic acid. The vacuolar potential of guard cells of Commelina communis L., Zea mays L., Nicotiana glauca Graham, Allium cepa L., and Vicia faba L. was between -40 and -50 millivolt in 30 millimolar KCl when stomata were open and about -30 millivolt when stomata were closed. The vacuolar potential of guard cells of C. communis was almost linearly related to stomatal aperture and responded to changes in the ionic strength in the bathing medium in a Nernstian manner. No specificity for any alkali ion (except Li(+)), ammonium, or choline appeared. Lithium caused hyperpolarization. Calcium in concentrations between 1 and 100 millimolar in the medium led to stomatal closure, also caused hyperpolarization, and triggered transient oscillations in the intracellular potential. Gradients in the electrical potential existed across stomatal complexes with open pores. When stomata closed, these gradients almost disappeared or slightly reverted; all epidermal cells were then at potentials near -30 millivolt in 30 millimolar KCl.

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet.

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na(+) and K(+)) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.

Binding of (45)Ca (2+) to particulate fractions of coleoptile tissue.

Using recently developed techniques, we have investigated the binding of (45)Ca(2+) to membrane preparations from corn (Zea mays L) and oat (Avena sativa L) coleoptile tissue. Scatchard plot analysis reveals at least two Ca(2+)-binding sites in each tissue, a high affinity binding site (K m=7.7×10(-7) M, n=6.9×10(-10) mol·0.5 g f.w.(-1) in corn, K m=4.93×10(-6) M, n=2.29×10(-9) mol·0.5 g f.w.(-1) in Avena) and a low affinity binding site (K m=9.01×10(-5) M, n=5.4×10(-8) mol·0.5 g f.w.(-1) in corn; K m=1.03×10(-4) M, n=3.40×10(-8) mol·0.5 g f.w.(-1) in Avena). There is also some evidence of a third, lower affinity binding site in each tissue, especially corn.More detailed studies with corn coleoptile homogenates show that they contain a potent dialyzable inhibitor of Ca(2+) binding. Monovalent cations were observed to be ineffective as inhibitors of Ca(2+) binding in corn. However, of six divalent cations tested, all were capable of strong inhibition of Ca(2+)-binding and there appeared to be a relationship between size of the atomic radius of the ion and potency as an inhibitor of calcium binding.

Selective effects of victorin on growth and the auxin response in Avena.

Victorin, the pathotoxin from the host-specific pathogen, Helminthosporium victoriae, promotes the growth of coleoptile segments when given at concentrations that are high but which still show selective effects on susceptible and resistant tissue. The latent period in the growth response of both susceptible and resistant tissue is about 3.6 minutes compared to 11.0 minutes in the response of these tissues to auxin. The victorinpromoted rate of elongation of 8-millimeter segments is about 0.2 millimeter per hour in susceptible tissue and about 0.1 millimeter per hour in resistant tissue compared to about 0.4 millimeter per hour in response to auxin. At low concentrations, the toxin has no growth-promoting effect in either susceptible or resistant coleoptile segments. Over a wide range of concentrations, victorin inhibits the growth response of susceptible tissue to auxin completely while having no effect on the response of resistant tissue to auxin.Victorin induces solute leakage in coleoptile tissue of susceptible but not resistant varieties of Avena. In susceptible tissue, solute leakage begins about 2 minutes after the addition of toxin and continues for at least 4 hours.The rapidity and close correlation of toxin effects on membrane permeability (2 minutes) and on growth (3.6 minutes) suggest the possibility that the initial action of victorin in both cases may be at the cell surface, perhaps the plasmalemma.