Randolph M. Beaudry

Effect of O2 and CO2 partial pressure on selected phenomena affecting fruit and vegetable quality

Abstract It is likely that from the time of the Roman Empire and perhaps before, people involved in the storage of plant material as food recognized that atmospheric modification can provide some benefit in improving storability. However, active, commercial modification of the atmosphere for the preservation of fresh fruit and vegetables dates to the early part of this century. Early successes with apple fruit has lead to the attempt to apply modified atmospheres to a wide range of commodities. Responses to atmospheric modification are found to vary dramatically among plant species, organ type and developmental stage and include both unwanted and beneficial physiological responses. Desirable responses include a reduction in respiration, a reduction in oxidative tissue damage or discoloration, a reduction in the rate of chlorophyll degradation and a reduction in ethylene sensitivity with the concomitant reduction in the rate of ripening and other ethylene-mediated phenomena. Undesirable responses have included the induction of fermentation, the development of disagreeable flavors, a reduction in aroma biosynthesis, the induction of tissue injury and an alteration in the makeup of microbial fauna. The physiological bases for some of these responses to elevated CO2 and reduced O2 are discussed.

The Tomato odorless-2 Mutant Is Defective in Trichome-Based Production of Diverse Specialized Metabolites and Broad-Spectrum Resistance to Insect Herbivores

Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) produce a wide array of volatile and nonvolatile specialized metabolites. Many of these compounds contribute to the characteristic aroma of tomato foliage and constitute a key part of the language by which plants communicate with other organisms in natural environments. Here, we describe a novel recessive mutation called odorless-2 (od-2) that was identified on the basis of an altered leaf-aroma phenotype. od-2 plants exhibit pleiotrophic phenotypes, including alterations in the morphology, density, and chemical composition of glandular trichomes. Type VI glandular trichomes isolated from od-2 leaves accumulate only trace levels of monoterpenes, sesquiterpenes, and flavonoids. Other foliar defensive compounds, including acyl sugars, glycoalkaloids, and jasmonate-regulated proteinase inhibitors, are produced in od-2 leaves. Growth of od-2 plants under natural field conditions showed that the mutant is highly susceptible to attack by an indigenous flea beetle, Epitrix cucumeris, and the Colorado potato beetle, Leptinotarsa decemlineata. The increased susceptibility of od-2 plants to Colorado potato beetle larvae and to the solanaceous specialist Manduca sexta was verified in no-choice bioassays. These findings indicate that Od-2 is essential for the synthesis of diverse trichome-borne compounds and further suggest that these compounds influence host plant selection and herbivore community composition under natural conditions.

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Gα) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA)activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis [33]. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus [34]. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor-1::GUS reporter) and TJYP1-22(fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. The fadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/ PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.

Effect of carbon dioxide partial pressure on blueberry fruit respiration and respiratory quotient

Blueberry (Vaccinium corymbosum L. cvs. “Bluecrop” and “Elliot”) fruit were sealed in 0.00254 cm (1 mil) thick low density polyethylene packages and stored in gas-tight containers at 15°C. The chambers were continually purged with gas mixtures containing levels of CO2 sufficient to produce approximately 5, 20, 40 or 60 kPa CO2 in the pouches. Sampling the gas composition of the enclosed package permitted measurement of O2 uptake and CO2 production rates and calculation of the respiratory quotient (RQ) despite the high background CO2 level. O2 uptake was minimally affected by the CO2 treatments. RQ was dependent on both O2 and CO2 levels, increasing as O2 declined and as CO2 increased. As O2 declined, the O2 level at which RQ began to increase (RQ breakpoint) increased with CO2 partial pressure. The lower O2 limit was estimated to be 6, 7, 11 and 23 kPa O2 for the 5, 20, 40 and 60 kPa CO2 treatments, respectively.

Determination of the low oxygen limit for several commercial apple cultivars by respiratory quotient breakpoint

Abstract Low oxygen limits for apple ( Malus domestica , Borkh) fruit were determined using modified-atmosphere packaging (MAP) techniques. Fruits were sealed in low density polyethylene (LDPE) packages and placed at 0°C until steady-state respiration was reached. Steady state O 2 and CO 2 partial pressures were varied by altering package thickness, package surface area, and the total fruit weight within the package. Based on measured permeabilities of the LDPE packages to O 2 and CO 2 , package partial pressures were used to determine the gas flux for O 2 and CO 2 for each package, with gas flux rates representing respiratory rates. The effect of O 2 partial pressure on O 2 uptake, CO 2 production, and the respiratory quotient (RQ) was then determined. As the O 2 partial pressure decreased to approximately 2 kPa, RQ remained relatively constant. A marked increase in the RQ (the RQ breakpoint) occurred below 2 kPa O 2 , which was associated with elevated ethanol concentrations. The low O 2 tolerance limit was then estimated for each cultivar as the O 2 level at the RQ breakpoint; these values ranged from 0.7 kPa O 2 for cultivars “Red Delicious” and 0.8 kPa for “Law Rome” to approximately 1.9 kPa for “McIntosh”.

Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

BackgroundFilamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes.ResultsVolatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation.Conclusions1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and β-oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.

Ethylene modulates development and toxin biosynthesis in aspergillus possibly via an ethylene sensor-mediated signaling pathway.

Ethylene, a biologically active natural compound, inhibited aflatoxin accumulation by Aspergillus parasiticus on a solid growth medium in a dose-dependent manner at concentrations of 0.1 to 150 ppm. The activity of the nor-1 promoter (an early aflatoxin gene) was reduced to nondetectable levels by similar quantities of ethylene, suggesting that the inhibitory effect on toxin synthesis occurred, at least in part, at the level of transcription. The inhibitory effect of ethylene on aflatoxin accumulation was also observed when A. parasiticus was grown on raw peanuts. Under similar growth conditions and doses, ethylene strongly inhibited development of asci and ascospores in Aspergillus nidulans, with no detectable effect on Hülle cell formation, conidiation, or sterigmatocystin accumulation. During early growth, A. parasiticus and A. nidulans produced ethylene with approximately twofold higher quantities measured in continuous light than in the dark. 1-Methylcyclopropene (an inhibitor of ethylene receptors in plants), light, CO2, temperature, and growth medium composition altered the effect of ethylene on A. nidulans and A. parasiticus. These observations are consistent with the existence of an ethylene sensor molecule that mediates the function of an ethylene-responsive signaling pathway(s) in Aspergillus.

Aspergillus Volatiles Regulate Aflatoxin Synthesis and Asexual Sporulation in Aspergillus parasiticus

ABSTRACT Aspergillus parasiticus is one primary source of aflatoxin contamination in economically important crops. To prevent the potential health and economic impacts of aflatoxin contamination, our goal is to develop practical strategies to reduce aflatoxin synthesis on susceptible crops. One focus is to identify biological and environmental factors that regulate aflatoxin synthesis and to manipulate these factors to control aflatoxin biosynthesis in the field or during crop storage. In the current study, we analyzed the effects of aspergillus volatiles on growth, development, aflatoxin biosynthesis, and promoter activity in the filamentous fungus A. parasiticus. When colonies of Aspergillus nidulans and A. parasiticus were incubated in the same growth chamber, we observed a significant reduction in aflatoxin synthesis and asexual sporulation by A. parasiticus. Analysis of the headspace gases demonstrated that A. nidulans produced much larger quantities of 2-buten-1-ol (CA) and 2-ethyl-1-hexanol (EH) than A. parasiticus. In its pure form, EH inhibited growth and increased aflatoxin accumulation in A. parasiticus at all doses tested; EH also stimulated aflatoxin transcript accumulation. In contrast, CA exerted dose-dependent up-regulatory or down-regulatory effects on aflatoxin accumulation, conidiation, and aflatoxin transcript accumulation. Experiments with reporter strains carrying nor-1 promoter deletions and mutations suggested that the differential effects of CA were mediated through separate regulatory regions in the nor-1 promoter. The potential efficacy of CA as a tool for analysis of transcriptional regulation of aflatoxin biosynthesis is discussed. We also identify a novel, rapid, and reliable method to assess norsolorinic acid accumulation in solid culture using a Chroma Meter CR-300 apparatus.

Willow volatiles influence growth, development, and secondary metabolism in Aspergillus parasiticus

Aflatoxin is a mycotoxin and the most potent naturally occurring carcinogen in many animals. Aflatoxin contamination of food and feed crops causes a significant global burden on human and animal health. However, available methods to eliminate aflatoxin from food and feed are not fully effective. Our goal is to discover novel, efficient, and practical methods to control aflatoxin contamination in crops during storage. In the present study, we tested the effect of volatiles produced by willow (Salix acutifolia and Salix babylonica) and maple (Acer saccharinum) bark on fungal growth, development, and aflatoxin production by the fungus Aspergillus parasiticus, one economically important aflatoxin producer. S. acutifolia bark volatiles nearly eliminated aflatoxin accumulation (>90% reduction) by A. parasiticus grown on a minimal agar medium. The decrease in aflatoxin accumulation correlated with a twofold reduction in ver-1 (encodes a middle aflatoxin pathway enzyme) transcript level. Expression data also indicate that one histone H4 acetyltransferase, MYST3, may play a role in epigenetic control of aflatoxin gene transcription in response to volatile exposure. Volatiles derived from wood bark samples also increased fungal growth up to 20% and/or enhanced conidiospore development. Solid-phase microextraction–gas chromatographic–mass spectrometric analysis of bark samples identified sets of shared and unique volatile compounds that may mediate the observed regulatory effects on growth, development, and aflatoxin synthesis. This work provides an experimental basis for the use of willow industry by-products to control aflatoxin contamination in food and feed crops.

Estimates of Mechanization Effects on Fresh Blueberry Quality

Fresh market blueberries are usually hand picked so that they will possess high quality, long shelf life, and good consumer appeal. Continuing expansion of U.S. production and markets for fresh blueberries requires that mechanical harvesting methods be developed that provide berries having high quality. In 1992 tests, ‘Bluecrop’ berries that were commercially hand picked had an average of 77% of the berries either damage free or with slight internal bruising after sorting. Conventional mechanical harvesters had an average of 22% of the berries that were damage free or slightly bruised. In 1993 tests, an experimental harvester, that required the bushes to be divided into a V-shape during the shaking operation and handled the berries gently, had an average of 68% of the berries that were damage free or slightly bruised.