NE-103 Project Desription - Procedures

Objective 1. To evaluate postharvest requirements of new and existing temperate fruit varieties.

As mentioned in the Justification, the critical need to identify new cultivars of all fruit types that are optimally suited to regional climate and marketing demands will be addressed, with a focus on apple fruit. First we will perform postharvest evaluations to complement the existing NE-183 regional project, which conducts production-related evaluations of new apple cultivars, and second, plans are to continue evaluation of existing cultivars in order to establish optimal handling and storage conditions.

Complementing the NE-183 project is our major collaborative and interdependent work plan under this objective. Currently, 26 new cultivars and selections are planted in controlled field trials in over 15 sites across the US. An additional cycle of plantings is planned. In the upcoming season, most of the trees from the first planting will be producing their first full crop. Storage performance is an essential element in evaluating these cultivars/selections. The involvement of the NE-103 Technical Committee members will create a continuum in evaluation of these fruit extending from the field through the storage period and into retail sales, where the consumer is directly affected. Early postharvest evaluation should help avoid the discovery of limiting characteristics after significant investment and planting by the apple industry.

Five stations (MA, MI, NC, NY-I and PA) will evaluate cultivars from various NE-183 sites within their region. MA, MI and NC have single sites available at the respective horticultural experiment stations. Two sites are also available at USDA sites in WV. In NY, three sites are available and evaluations will be made at those other than Ithaca when appropriate. In PA there are two NE-183 sites (Biglerville and State College).

Each station will utilize the same standard protocols for evaluating maturity and storage ability of new cultivars. These protocols will be discussed with members of the NE-183 to ensure that our objectives complement theirs, and where applicable, stations that do not have a current member of the NE-103 group will be invited to participate in our project. At present, basic harvest maturity parameters such as flesh firmness, soluble solids and starch index ratings are made weekly by NE-183 project members. For the purposes of optimizing fruit storage, these data will be complemented with internal ethylene measurements and evaluations of performance under air storage in terms of firmness retention, palatability and disorder incidence. Because of the number of selections, evaluations will be scheduled to ensure that manageable numbers of fruit are assessed each year. At least four selections will be studied in detail each year. As fruit numbers allow, CA storage trials will be initiated. In CA storage trials, quality retention as a function of maturity and condition at harvest and storage atmosphere and duration, will be evaluated. Storage studies that require large numbers of CA storage chambers will be conducted in PA due to the high capacity of their excellent facilities.

Measurements of specific physiological characteristics such as aroma volatile emissions, skin resistance, respiration, and tolerance limits for O2 and CO2 will be assigned to specific stations with the appropriate expertise and equipment (MI, NC, NY-I, USDA-MD, WA). NE-103 members have developed and actively shared expertise and protocols for most of the postharvest evaluation techniques needed to address this objective. In situations where regional environmental conditions could influence the outcome of the study but appropriate analytical capability is not available at specific locations, fruit samples will be exchanged among stations. Results obtained from this objective will provide information about the importance of preharvest factors on tolerances of fruit to elevated CO2 and low O2, and is interdependent with Objective 2, in which manipulation of the levels of these two gases will be evaluated for its potential as an alternative to chemical control of diseases, pests and physiological disorders, and Objective 3, in which the underlying biological principles of fruit responses to these gases are to be investigated. The information derived from this work will prevent large plantings of inappropriate cultivars, and identify best-management practices for worthy new cultivars.

Analyses to determine the composition and amount of volatile compounds will be performed using headspace and purge-and-trap techniques developed by members of the group (MI, WA, USDA-WA). These data will be useful to ascertain the capacity for aroma/flavor regeneration after long-term storage and may assist in the non-destructive detection of physiological stress (related to Objectives 2 and 3).

NY-I and MI will carry out analyses of fruit density and skin resistance on fruit from the NE-103 plantings. These data will be important to quickly identify cultivars that may be more sensitive to low O2 or elevated CO2 concentrations. Integrated with this line of research, MI will perform storage tests using a modified atmosphere packaging approach that will determine tolerance limits to O2 and CO2 as well as measure rates of gas exchange. Tolerance limits to low O2 and elevated CO2 will also be established by non-destructive measurements of chlorophyll fluorescence and ethanol production (NS).

Collectively, this information is needed to identify the suitability of various cultivars for mixing in CA storage rooms. Cultivars that differ markedly in their low O2 tolerance may require separate storage facilities, or a commitment by commercial producers to create special handling techniques. Data on respiration are needed to estimate cooling demand and relative rates of deterioration. These data may also be useful for modified atmosphere packaging applications in the marketplace.

The second major work plan concerns the postharvest evaluation of existing cultivars. Some overlap of these tasks with those described above exists, as some of the cultivars being evaluated by NE-183 have already been planted extensively throughout North America. Our focus here, however, is to address specific quality issues limiting the storability of several popular cultivars. Again, as the knowledge base expands, information obtained here is expected to integrate with knowledge derived from the fundamentally basic research planned under Objective 3.

Examination of flavor characteristics of Gala apples will be performed as an expansion of previous efforts by NE-103 members. Loss of flavor is a limiting factor for storage of Gala, so a cooperative project between PA and USDA-MD has been established to determine optimum storage conditions to maintain flavor in fruit of this cultivar. Volatile compounds contributing to Gala aroma are being identified in a cooperative project between USDA-W and OSU-Corvallis. USDA-W is also evaluating volatile production and quality retention in Gala fruit following dynamic CA regimes.

Another important project will be reduction of watercore in Fuji apples, as fruit of this cultivar grown in northern North America frequently exhibit this disorder at harvest. Evidence indicates that preharvest temperatures may affect the propensity of fruit to develop watercore. For example, Fuji apples grown in Washington are very susceptible to watercore, whereas those grown in California show only minor watercore, even though they are harvested at a more advanced maturity. Development of protocols to minimize the risk of internal breakdown due to watercore will focus on predictions based on preharvest temperatures (CA, MA, MI, NC, NY-I, PA, WA ), and on delay of CA establishment, CA conditions (atmosphere and temperature) and storage duration (WA). Because Fuji is one of the cultivars grown in the NE -183 trial, fruit will be available from a variety of growing locations for assessment of the effects of preharvest temperatures on watercore development of this cultivar. Temperature and watercore development data will be collected from all stations at which Fuji are grown (CA, MA, MI, NC, NY-I, PA, WA) and will subsequently be used to develop a preharvest temperature watercore-prediction model using expertise developed by MA for prediction of superficial scald (see Objective 2). Collaboration of this type enables us to obtain data on a wide range of temperature conditions quickly for more accurate and rapid model development.

Improving methods to estimate fruit maturity forms the final project under this work plan. The Streif-index [firmness/(starch content x soluble solids)], which has been used successfully in Europe, will be evaluated for its capacity to accurately estimate the optimum harvest window for various cultivars (NS). Two years of commercial evaluation on McIntosh, Cortland and Jonagold in NS has shown it to be a simple, cheap and accurate method to predict final harvest date for each orchard. It is not known if this index will be universally applicable across all climates represented by locations of NE-103 members, but since, under this objective, the requisite data are continuously acquired for many cultivars of apples, its predictive value at different locations should be easy to assess. In addition, MI is developing a promising new method for assessing apple maturity, the 'dip-stick' ELISA for ACC oxidase, which is an immunochromatographic assay similar to the pregnancy test. Prototypes of the ELISA should be available for testing by scientists in other states by the 1998 season. Extensive inter-regional collaboration is expected in evaluating this technology in relation to other maturity indices.

Additional benefits to be realized from progress under this research objective lie in the close coordination with efforts under Objectives 2 and 3. Examination of many different cultivars will facilitate comparative studies of disorder mechanisms, volatiles associated with disorders, composition and regeneration of flavor compounds, molecular biology, and mechanisms of ripening and softening.

Objective 2. To develop sustainable alternatives to chemical control of physiological disorders, diseases, and pests.

Within this objective, three avenues of investigation with be undertaken with goals of developing control or management mechanisms for the storage problems of apple and pear scald, fungal decay and insect pests. The focus will be to develop techniques that minimally impact the environment, have the potential to reduce agricultural chemical residues, or provide consumer-acceptable alternatives. Efforts are to be coordinated such that one control strategy does not preclude the use of another. Research for this objective will be coordinated by NE-103 committee members who are recognized leaders the indicated research areas.

Physiological disorders: Control of superficial scald in apple and pear. We have categorized the means for scald control as targeting either scald avoidance or scald reduction. In the proposed project, the priority is to focus on the storage disorder of apples and pears known as superficial scald. Promising scald control mechanisms will be integrated with traditional storage protocols and subjected to a systems evaluation. The criteria for evaluation will be ease of transition, profitability, and sustainability of storage strategies.

Avoidance of superficial scald will be studied primarily through the use of predictive tools. The goal is to minimize postharvest chemical usage by predicting when the use of antioxidants is necessary. Mathematical prediction models developed by the previous project (MA) require further testing and refinement at various locations throughout the U.S. A multi-year cooperative project will be initiated between the eastern and western growing areas (MA, MI, NY-I, ONT-V, PA, WA, NS) to refine these models using the Delicious cultivar grown in varied climates. At MI and ONT-V, the effects of fruit maturity will also be studied. A similar dependency of scald on preharvest temperatures will be investigated for d'Anjou pear fruit (OR). Relationships between preharvest temperature and scald incidence will be used to generate a mathematical model for scald prediction for pear. Variations in the content of scald-related compounds such as antioxidants and/or enzymes may also be used to improve the predictive model developed from the temperature data.

Reduction of scald using non-chemical techniques will be studied using approaches developed by members in the previous NE-103 Project. Continuous low O2 treatments for scald reduction in commercial conditions requires further examination due to problems with off-flavor development in fruit from some locations, especially the Northeast, and variable efficacy of treatment. In our recently published NE-103 cooperative study (Lau et al., 1998) we used 0.7% O2 + 1.0% CO2. However, Delicious apples in NS were not given the 1% CO2 treatment and scald was not reduced. Another cooperative scald study (BC, NS, ON-V, CA, OR, MI, WA, NY-I, PA, NC, MA) will be initiated to determine if a minimum level of CO2 is required for scald control. CO2 levels to be tested will range from 0 to 2% and the O2 level will be raised to 1% to avoid the induction of fermentative metabolism. The breadth of cultivars tested also will be expanded to other scald-susceptible cultivars such as Rome Beauty and Granny Smith (MI, CA, NY-I, WA).

A number of independent scald-related projects remain underway with the anticipation that these approaches will be incorporated into larger-scale cooperative projects if promising results are obtained. Important among the more recent findings is that scald control in apple can be achieved by initial low oxygen stress followed by CA storage (MI). In future work, the level and duration of initial low O2 stress and subsequent CA conditions will be optimized to safely and effectively control scald without compromising fruit quality for apple (MI, NS, NY-I). Conditions and handling prior to and after the low O2 stress will approximate commercial procedures as closely as is possible.

Decay control. We will be continuing our efforts to develop alternatives to persistent agricultural chemicals for postharvest decay control, focusing on the control of P. expansum and B. cinerea, two of the most destructive postharvest pathogens. Investigation of host/fruit-pathogen interactions will entail a pair of parallel studies using the same molecular biological approach. Work at USDA-MD and CA is examining the interaction of fungal pathogens and fruit with the aim of identifying factors that can limit the development of decay during storage. The focus of this research has been on the polygalacturonase inhibitor protein (PGIP) of apple fruit (USDA-MD) and pear and tomato fruits (CA). The proteins have been purified and the genes for all 3 PGIPs have been cloned. Tests are underway to identify factors that determine which pathogen PG isoforms are selectively inhibited by a specific PGIP. The pear PGIP has been expressed in transgenic tomato plants in order to test whether increased expression of PGIP can influence fruit susceptibility to pathogens. Researchers at USDA-MD and CA will collaborate by exchanging pathogen strains (primarily Botrytis cinerea), antibodies and gene probes, as well as information on protocols and results.

Other control measures to be examined will be confined to those with potential to be integrated into modern handling and storage strategies employing sealed storage construction or sealed packages. Investigations will focus on the use mixtures of O2 and CO2 (CA, OR, WA), antifungal volatiles (MI, PA), biological control (CA, MI) and heat (OR), separately and in combination (CA). With the exception of CO2 treatments, these control measures are relatively untested, and are rather exploratory in nature. Therefore, the research does not warrant interaction between numerous participants at this stage. If, however, a technique is considered to have special promise, we anticipate expanding the scale of work and the number of stations involved in efficacy testing.

Optimum levels of O2 and CO2 for suppressing fungal growth; fruit toxicity thresholds for the various O2 and CO2 atmospheres; and specific atmospheres which will suppress disease development and will maintain fruit quality (CA, MI). Other possible options to the use of fungicides are the use of ozone and/or chlorine dioxide gases in storage atmospheres and during fruit handling operations (WA). Other approaches using reduced or non-chemical control of biotic disorders include use of hexanal and other aldehyde vapor (MI), acetic acid fumigation to control decay of apples (PA), and organic aroma volatiles to control decay of sweet cherries (ONT-V). Decay incidence will be determined as a function of dose. The possibility of applying the gas in modified atmosphere packages will be explored (MI). Another approach will be the use of extremely high levels of (>20%) CO2 and heat treatments (CA).

Objective 3. To expand fundamental knowledge to improve and create new technologies to assure high quality and wholesomeness of fruit and enhance market opportunities.

Under this objective, members of NE-103 will conduct various lines of primarily basic research aimed at elucidating the biochemical, physiological and genetic bases of postharvest storage disorders, ripening and softening, and the effects of CAs (both beneficial and detrimental) on quality attributes such as color, aroma, texture and nutritional value. Some of these studies will utilize molecular genetic techniques to address specific problems or questions, and the long-range goal of much of the research will be to introduce into fruits genetic traits which obviate the need to control decay, disorders, and deterioration of quality with chemical treatments that may pose a human health risk and restrict international sales. Research activities under Objective 3 will be distributed among three general areas as follows: 1) Stress-induced disorders and injuries; 2) Biochemical/physiological bases of the effects of O2, CO2 and ethylene on quality of intact and fresh-cut fruits; and 3) Regulation of fruit ripening and softening.

Stress-induced disorders and injuries. As indicated in Objective 2, understanding and controlling scald in apple and pear fruits will be a major focus of NE-103 participants. Scald is currently controlled by treatment with DPA and\or by storage in low O2 atmospheres. DPA treatment is costly, is considered a health risk, is environmentally unsound, and necessitates the use of a fungicide to limit decay in storage. Furthermore, all fruit will not tolerate the low level of oxygen required to prevent scald. Finally, the present control measures are not always effective; sometimes after the fruit are removed from storage scald symptoms arise. Recent research by NE-103 members supports the hypothesis that scald as well as other stress-induced injuries are mediated by active oxygen species (AOS). Also, further evidence was found that development of scald is linked with the synthesis and oxidation of the sesquiterpene -farnesene. Thus, future research efforts by the group will center on: 1) investigating the role of AOS in scald induction and of antioxidative defense mechanisms in scald resistance, and 2) elucidating the pathway of alpha-farnesene synthesis and the role of its oxidation products in development of scald symptoms.

In addition to comparison of commercially important apple cultivars that are clearly scald-susceptible (e.g. Granny Smith, Delicious, McIntosh and Cortland) or scald-resistant (e.g. Gala and Empire), this collaborative work will utilize novel susceptible and resistant hybrid lines of Rome Beauty x White Angel available to NY-I. Analyses of the levels of naturally occurring antioxidants (ascorbic acid , glutathione, tocopherols, carotenoids, phenylpropanoids, and simple phenolics) and the activities of AOS scavenging enzymes (superoxide dismutase, peroxidase, catalase, ascorbic acid peroxidase, glutathione peroxidase and glutathione reductase) in resistant and susceptible fruits will be conducted by NY-I, MI, ONT-G, MA, and USDA-MD. Molecular biology protocols will be employed by NY-I and MI to screen for the presence of antioxidant enzyme proteins (by Western blots) and their activities (on non-denaturing PAGE gels), to determine levels of antioxidant enzyme gene expression (by Northern blots using their respective cDNAs as probes) and to determine the number of copies of the various antioxidant enzyme genes (by Southern blot analysis). This approach is expected to identify the biochemical and enzymatic pathways of AOS metabolism that relate to potentiation or prevention of scald development.

The processes by which AOS are generated will be investigated by GA and ONT-G. It is now apparent that the mitochondrial electron transport chain is a major site for production of AOS. Factors that regulate production of AOS in fruit tissue will be examined by isolating mitochondria from scald-resistant and scald-susceptible fruit. These will be used to assess the components which autoxidize and the level of reduction of the component pool which reacts with molecular oxygen to produce AOS. Levels of reduced and total ubiquinones are to be determined by HPLC and production of the superoxide anion will be measured. An exchange of results and protocols with MD is anticipated, as this station plans to test the ability of new porphyrin-based superoxide radical scavengers to reduce or prevent scald development in highly susceptible Granny Smith fruit. A series of chemically-tailored spin-trapping reagents that can be used to "fix" and then identify the radicals produced in stressed tissue is being developed at WA. Further efforts will be made to develop novel chemical indicators that react with AOS enabling quantification.

The connection between alpha-farnesene metabolism and AOS in the promotion of scald has been elusive, but through the efforts of NE-103 participants a breakthrough appears imminent. ONT-G is pursuing the pathway of -farnesene synthesis in apple peel tissue, with the ultimate aim of isolating and characterizing farnesene synthase. Using scald-susceptible apple fruit to clone genes that are induced by low temperature but suppressed by low O2 atmosphere may also provide a cDNA of the farnesene synthase gene (MI). This would enable the eventual testing of antisense transgenics for low alpha-farnesene production and resistance to scald. The primary oxidation products of alpha-farnesene, conjugated trienes (CTs), are much more closely correlated with scald development than alpha-farnesene itself. Therefore, USDA-MD, MD and MI are collaborating to elucidate the mechanism of alpha-farnesene oxidation and the subsequent metabolic fate of its CT products. It was recently shown that the CTs which accumulate in apple peel during storage are, surprisingly, comprised mainly of two conjugated triene-6-ol isomers, with one isomer constituting 90% (USDA-MD, MD). This raises the possibility that oxidation of alpha-farnesene is enzymatic in vivo. USDA-MD and MD plan to devise an assay system using peel tissue from scald-susceptible fruit to test this hypothesis. Should it prove to be the case, the " alpha-farnesene hydroxylase" would be another target enzyme for genetic regulation to prevent scald. MI has provided evidence for a direct role of a volatile breakdown product of alpha-farnesene, 6-methyl-5-hepten-2-one (MHO), in scald development. MHO induced scald-like symptoms in apple peel, tissue sensitivity to MHO increased with time in storage, and in vivo MHO production rose sharply after several months at 0 C in air. A joint effort by USDA-MD and MI will ascertain if MHO is a degradation product of CTs. Preliminary results confirm this, and further indicate that autoxidation of CTs is markedly temperature dependent. This could explain why scald symptoms intensify greatly when fruit are rewarmed after long-term storage. Future studies will focus on full elaboration of alpha-farnesene metabolism, the precise role of MHO in scald induction, and the effects of low O2, ethylene and low temperature on

Alpha-farnesene synthesis and oxidation. Biochemical/physiological bases of the effects of O2, CO2 and ethylene on quality of intact and fresh-cut fruits. Comparative studies to identify differences between low-O2-induced and elevated-CO2-induced fermentative metabolism, and factors affecting the shift from aerobic to anaerobic respiration in whole and fresh-cut fruits, will be carried out at CA. Also, investigation of the capacity of fruit tissue for repair following stress induced by low O2 and/or high CO2 levels will involve collaboration of CA, MD and NY-I. Studies of the roles of pH and copigmentation on stability of internal anthocyanins in strawberries under elevated CO2, as well as the effects of O2, CO2, and C2H4 on phenolic metabolism and browning of fruit tissues, are planned (CA). Other quality-related biochemical studies will include examination of atmospheric composition-time-temperature interactions on nutritional quality, especially on levels of vitamins A and C, and bioactive compounds such as carotenoids and polyphenols (CA).

A major effort will continue in the area of flavor biochemistry. It will center on understanding the metabolic processes involved in the loss of characteristic aroma volatiles in fruit kept in air or controlled atmospheres beyond a certain duration (CA, MI, WA, USDA-WA, PA, USDA-MD). Regulation of fruit ripening and softening. Control of ripening and softening by synthetic and natural bioregulants will be examined (NS, WA, CA, NY-G). The ethylene biosynthesis inhibitor aminoethoxyvinylglycine (trade name ReTain) is now approved in the U.S. to stop fruit drop and ReTain firmness in apples and pears at harvest. NS and WA will evaluate the possible benefits of ReTain on various ripening-associated changes in fruit quality (especially softening, and retention and regeneration of flavor compounds) after long-term storage and subsequent shelf time.

CA will continue to investigate the origin and mode of action of pectin-derived oligosaccharides (PDOs), which promote ripening in tomato. The role of novel pectolytic enzymes in softening-related cell wall metabolism and generation of PDOs will be studied, as well as the possibility that a recently discovered PDO-binding protein (remorin) is part of the signal transduction pathway through which PDOs stimulate ripening. This work will utilize the explanted tomato pericarp system that was devised several years ago. NY-G will take a molecular genetic approach to solving the problem of softening in storage in fruit of important apple varieties such as 'McIntosh.' This softening is the result of cell wall degrading enzymes such as polygalacturonase (PGase), which are controlled by ethylene. Ethylene production depends on the synthesis of its precursor, 1-aminocyclopropane carboxylic acid (ACC), by the enzyme ACC-synthase. Experiments are in progress to transform 'McIntosh' apple with sense or antisense versions of the ACC-synthase gene, with the aim of reducing ACC-synthase activity in the fruit, and thereby reducing ethylene production and consequent softening. A similar strategy will be utilized to achieve a reduction in PGase activity in harvested apple fruit. The overall goal of this work is to attenuate ripening and softening, and to thereby improve storability.

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