Characterization of a calcium/calmodulin-regulated SR/CAMTA gene family during tomato fruit development and ripening.

BACKGROUND: Fruit ripening is a complicated development process affected by a variety of external and internal cues. It is well established that calcium treatment delays fruit ripening and senescence. However, the underlying molecular mechanisms remain unclear. RESULTS: Previous studies have shown that calcium/calmodulin-regulated SR/CAMTAs are important for modulation of disease resistance, cold sensitivity and wounding response in vegetative tissues. To study the possible roles of this gene family in fruit development and ripening, we cloned seven SR/CAMTAs, designated as SlSRs, from tomato, a model fruit-bearing crop. All seven genes encode polypeptides with a conserved DNA-binding domain and a calmodulin-binding site. Calmodulin specifically binds to the putative targeting site in a calcium-dependent manner. All SlSRs were highly yet differentially expressed during fruit development and ripening. Most notably, the expression of SlSR2 was scarcely detected at the mature green and breaker stages, two critical stages of fruit development and ripening; and SlSR3L and SlSR4 were expressed exclusively in fruit tissues. During the developmental span from 10 to 50 days post anthesis, the expression profiles of all seven SlSRs were dramatically altered in ripening mutant rin compared with wildtype fruit. By contrast, only minor alterations were noted for ripening mutant nor and Nr fruit. In addition, ethylene treatment of mature green wildtype fruit transiently stimulated expression of all SlSRs within one to two hours. CONCLUSIONS: This study indicates that SlSR expression is influenced by both the Rin-mediated developmental network and ethylene signaling. The results suggest that calcium signaling is involved in the regulation of fruit development and ripening through calcium/calmodulin/SlSR interactions.

Penicillium solitum produces a polygalacturonase isozyme in decayed Anjou pear fruit capable of macerating host tissue in vitro.

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5-6, with an optimum at pH 4.5. PG activity was detectable 0-70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca(2+), Mg(2+), Mn(2+) and Fe(2+) and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.

Purification and biochemical characterization of polygalacturonase produced by penicillium expansum during postharvest decay of 'Anjou' pear.

A polygalacturonase (PG) was extracted and purified from decayed tissue of 'Anjou' pear fruit inoculated with Penicillium expansum. Ammonium sulfate precipitation, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. PG enzyme activity from healthy and wounded pear tissue was undetectable, which supports the claim that the purified PG is of fungal origin. The purified enzyme had a molecular mass of 41 kDa and a pI of 7.8. Activity of the PG was not associated with a glycosylated protein. The enzyme was active over a broad pH range from 3 to 6, with optimal activity at 4.5 in sodium citrate and sodium acetate buffers. The optimal temperature for activity was 37 degrees C but the enzyme was also active at 0, 5, 10, 20, and 50 degrees C. Thin-layer chromatographic analysis of PG hydrolysis products showed that the enzyme exhibits endo- and exo-activity. The purified enzyme macerated tissue in vitro causing approximately 30% reduction in mass of pear plugs compared with approximately 17% reduction for apple. Additionally, it produced 1.5-fold more soluble polyuronides on pear than apple tissue. This work shows for the first time the production of a PG by P. expansum during postharvest decay of pear fruit is different from the previously described PG produced in decayed apple fruit by the same pathogen.

Fate of Escherichia coli O157:H7 in the presence of indigenous microorganisms on commercially packaged baby spinach, as impacted by storage temperature and time.

This study investigated the effect of storage temperature and time on the survival and growth of Escherichia coli O157:H7, the growth of indigenous microorganisms, and the changes in product quality of packaged baby spinach. Commercial packages of spinach within 2 days of processing were cut open at one end, sprayed with fine mists of E. coli O157:H7 inoculum, resealed, and then stored at 1, 5, 8, and 12 degrees C for 12 days until their labeled best-if-used-by dates. Microbial enumeration and product quality evaluation were conducted on day(s) 0, 3, 6, 9, and 12 postinoculation. Spinach held at 12 degrees C supported significant (P < 0.001) E. coli O157:H7 growth, with a 1.0-log CFU/g increase within 3 days postinoculation, which was followed by additional growth during continued storage. E. coli O157:H7 grew slowly when held at 8 degrees C, with a significant (P < 0.01) level of growth reached after 6 days of storage. However, on products held at 1 and 5 degrees C, E. coli O157:H7 populations declined significantly (P < 0.01 and P < 0.001, respectively) within 3 days of storage. Aerobic mesophilic bacteria, psychrotrophic bacteria, and yeast and mold populations increased significantly at all storage temperatures, with more growth on products held at elevated temperatures. Product quality scores remained high within the first 6 days of storage, with a sharp decline noted on samples held at 12 degrees C on day 9. Results suggest that E. coli O157:H7 can grow significantly on commercially packaged spinach held at 8 degrees C or above before significant product quality deterioration occurs.

Effectiveness of bacteriophages in reducing Escherichia coli O157:H7 on fresh-cut cantaloupes and lettucet.

Consumption of produce contaminated with Escherichia coli O157:H7 has resulted in cases of foodborne illness. We determined the efficacy of a mixture of three E. coli O157:H7-specific bacteriophages (ECP-100) in reducing the number of viable E. coli O157:H7 on contaminated fresh-cut iceberg lettuce and cantaloupe. E. coli O157:H7 was spot inoculated on lettuce pieces (9 cm2) with a population of 3.76 log CFU/cm2, allowed to dry, and then sprayed with a control (phosphate-buffered saline) or ECP-100 to deliver 7.98 log PFU/cm2 to lettuce stored for 2 days at 4 degrees C. Cut pieces of cantaloupe were spot inoculated with E. coli O157:H7 (4.55 log CFU/ml) and treated with the control or ECP-100 (6.69 log PFU/ml), and then stored at 4 or 20 degrees C for up to 7 days. On days 0, 2, 5, and 7, cantaloupe samples were homogenized, and populations of E. coli O157:H7 were enumerated. Populations of E. coli O157:H7 on lettuce treated with ECP-100 on 0, 1, and 2 days (0.72, <0.22, and 0.58 log CFU/cm2 of lettuce) and stored at 4 degrees C were significantly (P < 0.05) lower than those treated with the control (2.64, 1.79, and 2.22 log CFU/cm2), respectively. Populations on cut cantaloupes treated with ECP-100 on days 2, 5, and 7 (0.77, 1.28, and 0.96 log CFU/ml) and stored at 4 degrees C were significantly lower than those cut cantaloupes treated with the control (3.34, 3.23, and 4.09 log CFU/ml), respectively. This study is the first to show the effectiveness of bacteriophages to reduce E. coli O157:H7 on fresh-cut lettuce and cantaloupes.

Isolation, purification, and characterization of a polygalacturonase produced in Penicillium solitum-decayed 'Golden Delicious' apple fruit.

Polygalacturonase (PG) was extracted and purified from decayed 'Golden Delicious' apple fruit inoculated with Penicillium solitum. Ammonium sulfate, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. The purified PG most likely originates from the fungus because PG activity from healthy and wounded apple tissue was undetectable. Analysis of cation exchange-purified material using sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 50-kDa band. The enzyme was active over a broad pH range (3 to 7), with optimal activity between pH 4 and 5. PG was highly active at 20 and 37 degrees C but was also detectable at 2, 50, and 75 degrees C. Divalent cations affected PG enzyme activity; Mg and Fe increased, whereas Ca and Mn reduced activity in vitro. Thin-layer chromatographic separation of hydrolysis products and data from a PG plate activity assay based on staining with ruthenium red showed that the enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit.

Biocontrol of the food-borne pathogens Listeria monocytogenes and Salmonella enterica serovar Poona on fresh-cut apples with naturally occurring bacterial and yeast antagonists.

Fresh-cut apples contaminated with either Listeria monocytogenes or Salmonella enterica serovar Poona, using strains implicated in outbreaks, were treated with one of 17 antagonists originally selected for their ability to inhibit fungal postharvest decay on fruit. While most of the antagonists increased the growth of the food-borne pathogens, four of them, including Gluconobacter asaii (T1-D1), a Candida sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcherrima (T1-E2), proved effective in preventing the growth or survival of food-borne human pathogens on fresh-cut apple tissue. The contaminated apple tissue plugs were stored for up to 7 days at two different temperatures. The four antagonists survived or grew on the apple tissue at 10 or 25 degrees C. These four antagonists reduced the Listeria monocytogenes populations and except for the Candida sp. (T4-E4), also reduced the S. enterica serovar Poona populations. The reduction was higher at 25 degrees C than at 10 degrees C, and the growth of the antagonists, as well as pathogens, increased at the higher temperature.

Physicochemical properties and bioactivity of fungal chitin and chitosan.

Chitinous material was extracted from mycelia of Aspergillus niger and Mucor rouxii grown in yeast peptone dextrose broth for 15 and 21 days, respectively. The extracted material was characterized for purity, degree of acetylation, and crystallinity and tested for antibacterial and eliciting properties. The maximum glucosamine level determined in the mycelium of A. niger was 11.10% dw and in the mycelium of M. rouxii was 20.13% dw. On the basis of the stepwise extraction of freeze-dried mycelia, it appeared that M. rouxii mycelia contained both chitin and chitosan, whereas A. niger contained only chitin. The yields of crude chitin from A. niger and M. rouxii were 24.01 and 13.25%, respectively, and the yield of chitosan from M. rouxii was 12.49%. Significant amounts (7.42-39.81%) of glucan were associated with chitinous compounds from both species and could not be eliminated by the extraction method used. The degrees of acetylation were determined to be 76.53 and 50.07% for chitin from A. niger and M. rouxii, respectively, and 19.5% for M. rouxii chitosan. The crystallinity of fungal chitin and chitosan was estimated to be less intense than in corresponding materials from shrimp shells. The extracted chitin and chitosan in a concentration of 0.1% reduced Salmonella Typhimurium DT104 2576 counts by 0.5-1.5 logs during a 4 day incubation in tryptic soy broth at 25 degrees C. Furthermore, all tested chitinous materials from fungal sources significantly reduced lesions caused by Botrytis cinerea and Penicillium expansum in harvested apples.

Optimizing concentration and timing of a phage spray application to reduce Listeria monocytogenes on honeydew melon tissue.

A phage cocktail was applied to honeydew melon pieces 1, 0.5, and 0 h before contamination with Listeria monocytogenes strain LCDC 81-861 and 0.5, 1, 2, and 4 h after contamination. The phage application was most effective when applied 1, 0.5, or 0 h before contamination with L. monocytogenes, reducing pathogen populations by up to 6.8 log units after 7 days of storage. This indicates that under commercial conditions, if contamination occurs at the time of cutting, phage would have to be applied as soon as possible after cutting the produce. However, all phage applications from 1 h before to 4 h after contamination and all phage concentrations ranging from 10(4) to 10(8) PFU/ml reduced bacterial populations on honeydew melon pieces. Higher phage concentrations were more effective in reducing pathogen populations. A phage concentration of approximately 10(8) PFU/ml was necessary to reduce the pathogen populations to nondetectable levels immediately after treatment, and pathogen growth was suppressed by phage concentrations of 10(6) through 10(8) throughout the storage period of 7 days at 10 degrees C. In an attempt to enhance the effectiveness of the phage cocktail on low pH fruit, such as apples, the phage was applied in combination with MnCl2. This combination, however, did not enhance the effectiveness of the phage on apple tissue. The results from this study indicate that the effectiveness of the phage application on honeydew melon pieces can be optimized by using a phage concentration of at least 10(8) PFU/ml applied up to 1 h after processing of the honeydew melons.

Relationship Between Host Acidification and Virulence of Penicillium spp. on Apple and Citrus Fruit.

ABSTRACT Penicillium expansum, P. digitatum, and P. italicum acidify the ambient environments of apple and citrus fruit during decay development. They use two mechanisms for this: the production of organic acids, mainly citric and gluconic, and NH(4)(+) utilization associated with H(+) efflux. Exposure of P. expansum and P. digitatum hyphae to pH 5.0 increased their citric acid production, compared with the production of organic acids at acidic ambient pH. In decayed fruit, both pathogens produced significant amounts of citric and gluconic acids in the decayed tissue and reduced the host pH by 0.5 to 1.0 units. Ammonium depletion from the growth medium or from the fruit tissue was directly related to ambient pH reduction. Analysis of transcripts encoding the endopolygalacturonase gene, pepg1, from P. expansum accumulated under acidic culture conditions from pH 3.5 to 5.0, suggesting that the acidification process is a pathogenicity enhancing factor of Penicillium spp. This hypothesis was supported by the finding that cultivars with lower pH and citric acid treatments to reduce tissue pH increased P. expansum development, presumably by increasing local pH. However, organic acid treatment could not enhance decay development in naturally acidic apples. Conversely, local alkalinization with NaHCO(3) reduced decay development. The present results further suggest that ambient pH is a regulatory cue for processes linked to pathogenicity of postharvest pathogens, and that specific genes are expressed as a result of the modified host pH created by the pathogens.

Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin.

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.

Osmotic dehydration of apple slices with CaCl2 and sucrose limits decay caused by Penicillium expansum, Colletotrichum acutatum, and Botrytis cinerea and does not promote Listeria monocytogenes or total aerobic population growth.

The interaction of Penicillium expansum Link, Colletotrichum acutatum, and Botrytis cinerea Pers.:Fr. with Listeria monocytogenes on osmotically dehydrated apple slices was evaluated. In mineral analyses of the slices, the calcium content of the peel and flesh tissues increased by 4- and 11-fold, respectively, when processed in 2% CaCl2. These slices also exhibited less decay by P. expansum, C. acutatum, and B. cinerea. Inoculation of slices with P. expansum resulted in a decrease in the pH of the flesh tissue at the infection site, while the pHs of slices infected with C. acutatum and B. cinerea increased and remained stable, respectively. Total mold population increased in wounds inoculated with P. expansum or C. acutatum. The presence of L. monocytogenes in the wounds did not significantly affect mold growth. The association of P. expansum and L. monocytogenes on apple slices resulted in a decrease in the bacterial population, whereas L. monocytogenes survived when slices were inoculated with C. acutatum. When associated with B. cinerea, there was a fourfold decrease in the L. monocytogenes population when slices were treated with 2% CaCl2. The total aerobic population was not significantly affected by the type of microorganism added to the wounds or by the osmotic treatment. These data show that osmotic dehydration with 2% CaCl2 combined with 20% sucrose limits decay of apple slices and does not promote bacterial or total aerobic population growth.

Survival and Growth of Listeria monocytogenes on Fresh-Cut Apple Slices and Its Interaction with Glomerella cingulata and Penicillium expansum.

The food-borne human pathogen Listeria monocytogenes survived and its populations increased on cv. Delicious apple slices at 10 or 20°C in air or controlled atmosphere of 0.5% O2 and 15% CO2, but did not grow at 5°C. Controlled atmosphere had no significant effect on the survival or growth of L. monocytogenes. The pathogen populations declined over time when grown in various concentrations of apple juice and the decline was greater as the concentration of the juice decreased. Populations of L. monocytogenes inoculated into decayed apple tissue continually increased on fruit decayed by Glomerella cingulata but did not survive after 5 days on fruit decayed by Penicillium expansum. The pH of the decayed area declined from pH 4.7 to 3.7 in the case of P. expansum, but in the case of G. cingulata the pH increased from pH 4.7 to 7.0. This pH modification may be responsible for affecting the growth of the food-borne pathogen. Storage temperature, as well as the absence of postharvest pathogens such as G. cingulata, is important for maintaining the safety of fresh-cut apples.