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 Physiology and Pathology of Tree Fruits Research: Wenatchee, WA
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ARS Home Pacific West Area Wenatchee, Washington Physiology and Pathology of Tree Fruits Research Research





- Rudell, David
- Honaas, Loren
- Leisso, Rachel
- 2023
- 2022
- 2021
- 2020
- Publications
- Plant Diseases (303)
- Product Quality and New Uses (306)

Research Project: Enhancement of Apple, Pear, and Sweet Cherry Quality **Location:
                            Physiology and Pathology of Tree Fruits Research** 2023 Annual Report **Objectives**
                                

The long-term goal of this project is to develop tools for quality management of deciduous tree fruits. Specifically, during the next five years we will focus on the following objectives.

Objective 1:  Resolve production and post-harvest environmental and genetic regulation of apple, pear, and sweet cherry quality.  [NP306, C1, PS1A]  

Sub-objective 1A:  Identify if interactions among storage temperature protocol, controlled atmosphere establishment date, and inhibition of ethylene action impact apple and pear fruit market quality and development of physiological disorders.

Sub-objective 1B: Determine if dynamic control of controlled atmosphere (CA) oxygen concentration can enhance apple fruit post storage volatile production without fruit quality loss.

Sub-objective 1C:  Determine relationship between sweet cherry cutin composition, gene expression, and surface defects caused by sun stress following harvest.

Objective 2:  Use of biomarkers to enhance/assist commercial apple and pear management strategies.  [NP306, C1, PS1A]

Sub-objective 2A:  Determine metabolic and genomic changes related to apple and pear fruit maturation and postharvest chilling.

Sub-objective 2B:  Identify metabolic and genomic changes linked with fruit quality loss and physiological disorder development during cold storage.

Sub-objective 2C:  Comparative analyses of fruit microbiome during pre- and post-harvest treatments throughout the cold chain to identify best management practices and abiotic/biotic conditions that improve/maintain fruit quality by influencing key pathogen communities. **Approach**
                                

Fruit from commercial orchards will be harvested then stored at ARS-Wenatchee in controlled atmosphere (CA) chambers. Fruit quality, metabolites, and RNA will be characterized at harvest and after storage using standard methods. Hypothesis 1A:  The manner in which postharvest technologies are imposed results in additive, synergistic, and antagonistic effects on fruit quality and disorder development.  Apple and pear fruit from commercial orchards will be stored continuously at 0.5°C or at 10°C for 7 days, then at 0.5°C.  Fruit will be exposed to 0 or 1 μL L-1 1-MCP for 16 hours, then in air or a CA initiated 1 or 9 days after harvest.  Following 6 to 8 months, fruit will be removed from storage and held 7 days at 20°C to allow post-storage quality loss and disorder development to occur. Hypothesis 1B: The manner in which postharvest technologies are imposed results in additive, synergistic, and antagonistic effects on apple firmness and related gene expression.  Tissues from ‘Gala’ apples stored at various temperatures, in air or CA, with or without 1-MCP treatment will be collected over time. Fruit firmness will be assessed at the same time as tissue harvest for transcriptome analysis plus after 7 at 20°C. Hypothesis 1C: Reciprocity is valid for mean oxygen concentration during CA storage and apple fruit post-storage volatile production and quality.  Honeycrisp’ apples will be CA stored continuously at 5.1% O2 as well as at 2.5% O2 with repeated periods in air to average 5.1% O2 over the storage period. Control fruit will also be held continuously in air. Fruit removed from storage after 4 and 8 months will be assessed for external disorders on the day of removal, then will be held 7 days at 20°C in air. On day 7, volatile compounds emitted from intact fruit will be collected and analyzed. Hypothesis 1D: Cuticle composition and gene expression will be impacted by sunlight exposure.  Sweet cherries will be harvested from the tree periphery and interior or following shading with black cloth.  Fruit peel will be assessed for microcracks using microscopy.  RNA will be collected from peel sections with contrasting cutin deposition patterns using laser microdissection.  Hypothesis 2A: Changes in molecular phenotypes (metabolites and gene activity) can be linked with progression of fruit maturation and low temperatures during storage. Apple and pear cultivars will be harvested over 12 weeks bracketing horticultural maturity.  Fruit maturity, metabolite and RNA content will be assessed using standard methods. Maturity dependent responses to abiotic stress will consist of holding fruit at 20 or 1°C for 48 hours. Hypothesis 2B: Specific changes in apple and pear cell membrane components are provoked by high carbon dioxide, low oxygen CA leading to higher risk of developing carbon dioxide related storage disorders. Apples treated or not with DPA will be stored in CA at 0.5% O2 and up to 5% CO2. Fruit quality and disorders will be assessed after storage as will fruit metabolites and RNA. **Progress Report**
                                

This report documents fiscal year (FY) 2023 progress for project 2094-43000-008-000D, titled, “Enhancement of Apple, Pear, and Sweet Cherry Quality”.

In support of Sub-objective 1A, Hypothesis 1A, ARS researchers in Wenatchee, Washington, used ‘d’Anjou’ pears to determine new storage management protocols that reduce ripening during storage while still managing browning disorders of the peel and flesh tissue. These were harvested at optimal commercial maturity. Pears were stored for up to eight months under ultra-low oxygen (ULO); (0.5% oxygen (O2)) and conventional CA; (1 and 1.5% O2) conditions at three different temperatures: 31, 33, and 37 degrees F. ARS researchers sought to determine if pears could be stored under ULO conditions, which can cause internal browning at 31 degrees F, at higher temperatures without causing internal browning. Quality and disorder assessment, as well as sampling of peel and flesh tissue for metabolic profiling, was completed at 0, 1, 3, 6, and 8 months. Pears will be stored in air at 0.6 degrees C for up to two months following the 8-month storage removal to simulate a prolonged post-storage cold chain with the goal of providing producers a means to supply the market with this cultivar year-round. Pears from one of the orchards developed internal browning of the flesh, only at 0.6 degrees C, while pears from other orchards developed low incidences of pithy brown core, a mild core disorder at multiple temperatures. Metabolic analysis and simulated cold chain evaluations are ongoing. 1-Methylcyclopropene (1-MCP) is widely used in conventional apple cold chains to reduce the rate of ripening and better preserve quality. However, when applied at harvest on pear, this crop protectant often is too effective, resulting in pears that never fully ripen, impacting quality and consumer acceptance. Extended post-storage cold chains in addition to improved CA storage protocols are required to meet the expected year-round availability of most pear cultivars. With appropriate CA storage conditions, post-storage 1-MCP treatment may allow for ripening control, without loss of ripening capacity over an extended post-storage cold chain. To test this, ‘d’Anjou’ pears were harvested at commercial maturity and stored for up to eight months in 0.5, 1, and 1.5% O2 CA storage (31 degrees F). Pears were treated with 1-MCP at 0, 0.5, 1, and 2 months and placed back in storage. Others were treated upon removal from CA at eight months. Disorder incidence and fruit quality were evaluated during a two month simulated cold chain (33 degrees F air storage). 1-MCP treatment less effectively reduced ripening as the treatment delay increased. Quality and disorder evaluation is ongoing. ‘Gem’ is a new bi-color European pear cultivar with good disease resistance. Although research initially indicated ‘Gem’ could be stored in air at 30 degrees F for five months with good retention of quality, in practice ‘Gem’ storage has proved more challenging, due to internal browning that can develop as early as two months after harvest, either in air or CA storage conditions. Internal browning has also occasionally been noted in-orchard. In response to stakeholder request, we initiated research to address postharvest internal browning which is ongoing. For Hypothesis 1B, ARS researchers discovered gene expression patterns that were related to changes in fruit texture during storage for ‘Gala’ apple fruit. Subsequent validation tests showed that these putative biomarker gene transcripts have predictive value for ‘Gala’ fruit texture during storage, even among seasons and orchards. These markers are also under evaluation using a related cultivar, ‘Scilate’ (see below). A manuscript reporting ‘Gala’ biomarkers has been submitted for publication. ‘Scilate’ storage trials were expanded to include biomarkers for apple fruit texture during the postharvest period. Previous seasons’ activities primarily examined storage parameters/treatments that impact internal browning, including developing and validating new mitigation strategies. This data by-and-large have been collected, and the final physiological evaluations are ongoing. The expanded work is a parallel experiment with largely the same parameters. Additional samples were taken to assess gene activity accompanying each physiological attribute to grow the transcriptome data set for the fruit texture biomarker project described below. This experiment was designed to match the ‘Gala’ biomarker experiments described below because ‘Gala’ is a parent of ‘Scilate.’ This will allow us to explore differences in storage patterns to the shared and unique genetics of these two important cultivars. 

In support of Sub-objective 2A, ARS researchers completed fine-scale harvest fruit quality and gene expression evaluation for ‘Granny Smith’ and ‘Delicious’, ‘Gala’, and ‘WA 38’ apples. Putative maturity biomarkers were identified and validated in follow-up tests in new cultivars and orchards as well as subsequent seasons. The putative markers have a high success rate in our experiments to assess harvest maturity based solely on gene activity patterns. Importantly, this was accurate for cultivars, orchards, and season that were not part of the discovery phase, indicating broad potential applicability as a new maturity index. Similar maturity marker experiments were completed for ‘d’Anjou’ and ‘Bartlett’ pears. The fruit quality evaluations for these experiments have been completed and partially analyzed, showing that we captured critical differences in fruit ripening capacity. This is a central issue for European pear growers because tests that assess future ripening capacity at harvest and would be a significant improvement for assessment of harvest maturity to optimize storability and ripening capacity. Gene expression analysis is ongoing. ARS researchers selected a 30 plant genomes to construct a Rosaceae gene classification scaffold to be implemented via a recently published software, and deployed at the Genome Database for Rosaceae in collaboration with Washington State University. This tool will allow for fast and efficient comparison of genes among apple and pear cultivars and rosaceous species. This solves a long-standing problem in comparative genomics of tree fruit. ARS researchers can now readily look across commodities and learn about the genes that influence rosaceous tree fruit quality despite extensive polymorphism and complex evolutionary and domestication histories that have confounded such efforts in the past. This is expected to accelerate risk assessment biomarker discovery in apple and pear. Research progress continued for Sub-objective 2B. ARS researchers in Wenatchee, Washington, discovered that elevated levels of carbon dioxide (CO2) during cold storage can trigger apple peel and flesh browning disorders in sensitive apple cultivars. They evaluated levels of metabolites linked in previous seasons with CO2 sensitivity in ‘Fuji’ apple, a sensitive cultivar, at 33 degrees F under stepwise increases of CO2 levels ranging from 0 to 5 % CO2 in ULO (0.5% O2) conditions that accentuate CO2 sensitivity. Disorder incidence and severity were evaluated, and flesh sampled at 0, 1, 2, 4, 6, and 8 weeks. Levels of over 500 metabolites were assessed in the flesh samples. Researchers identified a relationship among a class of cellular membrane components, phytosterol conjugates, that appear to be indicative of symptoms that are related to CO2 sensitivity. Other membrane components were similarly linked with disorder risk. Their investigation of whether monitoring phytosterol conjugate levels would be a valuable procedure for assessing risk of multiple common disorders of apple and pear is ongoing. ‘Honeycrisp’ apples are highly susceptible to develop bitter pit, soft scald/soggy breakdown, and CO2-related flesh browning, all postharvest disorders that render fruit unsalable. Conditioning fruit at higher temperatures (50 degrees F) before cold storage can reduce or eliminate soft scald, although while promoting bitter pit. A new practice developed in our earlier project establishes CA during conditioning, thereby controlling both disorders, albeit while promoting CO2-related flesh browning. As CO2 can be removed from the atmosphere, establishing what CO2 levels put fruit at risk and indicating if fruit are at risk may be means to reduce this disorder. ‘Honeycrisp’ apples were harvested and stored at increasing CO2 levels. Flesh was sampled during the conditioning period and evaluating appearance and quality up to 6 months. Five percent CO2 resulted in internal browning in fruit from one of the orchards. Apples did not develop bitter pit or soft scald/soggy breakdown. Metabolic analysis is ongoing. For apples, evaluating starch clearing via iodine staining is common industry practice to estimate fruit maturity for harvest management and to indicate storability. This method is not reliable for pear, perhaps due to differing starch ratios of amylose and amylopectin, and their relative propensities to react with tincture of iodine. Also, for high temperatures can reduce starch synthesis in other crop plants. To enable assessment of both the ratio of starch molecules in pear as well as potential preharvest climate effects on postharvest storability, we are developing protocols for tissue processing and evaluating a spectrophotometric method for amylose and amylopectin in pear tissues. **Accomplishments**
                                

1. Improved apple harvest maturity analysis. Evaluation bias, training, and rate of analysis reduce the precision and efficiency of starch clearing evaluation, the most common apple harvest maturity test. ARS researchers in Wenatchee, Washington, developed an image-based analytics software program for research labs and a packing house that reduces user bias, enhances data granularity, and facilitates data continuity for starch clearing tests. Producers are already using or have requested this software, and collaborative projects are underway. **Review Publications**
                            *Zhang, H., Wafula, E., Eilers, J.R., Harkess, A., Ralph, P., Timilsena, P., Depamphilis, C., Waite, J.M., Honaas, L.A. 2022. Building a foundation for gene family analysis in Rosaceae genomes with a novel workflow: a case study in Pyrus architecture genes. Frontiers in Plant Science. 13. Article 975942. https://doi.org/10.3389/fpls.2022.975942.*
                                *Garg, S., Leisso, R.S., Kim, S., Mayhew, E., Song, M., Jarrett, B., Kuo, W. 2022. Market potential and value-added opportunities of cold-hardy berries and small fruits in the Intermountain West, USA. Journal of Food Science. 88(2):860-876. https://doi.org/10.1111/1750-3841.16426.*
                                *Yoo, J., Sepulveda, G., Rudell Jr, D.R., Torres, C. 2022. Comparative analysis of metabolic differences between sunburn and sunscald disorder on 'Packham’s triumph’ pear. Postharvest Biology and Technology. 195. Article 112153. https://doi.org/10.1016/j.postharvbio.2022.112153.*
                                *Sheick, R., Serra, S., Rudell Jr, D.R., Musacchi, S. 2022. Investigations of multiple approaches to reduce green spot incidence in ‘WA 38’ apple. Agronomy. 12(11). Article 2822. https://doi.org/10.3390/agronomy12112822.*
                                *Lwin, H., Leisso, R.S., Lee, J. 2023. Pre-storage temperature conditioning reduces cortex browning and cavity and alters organic, amino, and fatty acid metabolism in cold-stored ‘Chuhwangbae’ pears. Scientia Horticulturae. 315. Article 111989. https://doi.org/10.1016/j.scienta.2023.111989.*
                                *Sheick, R., Serra, S., Musacchi, S., Rudell Jr, D.R. 2023. Metabolic fingerprint of ‘WA 38’ green spot symptoms reveals increased production of epicuticular metabolites by parenchyma. Scientia Horticulturae. 321. Article 112257. https://doi.org/10.1016/j.scienta.2023.112257.*
                                *Wafula, E., Zhang, H., Von Kuster, G., Leebens-Mack, J.H., Honaas, L.A., dePamphilis, C.W. 2023. PlantTribes2: tools for comparative gene family analysis in plant genomics. Frontiers in Plant Science. 13. Article 1011199. https://doi.org/10.3389/fpls.2022.1011199.*
                                *Argenta, L., Wood, R., Mattheis, J.P., Thewes, F., Nesi, C., Neuwald, D. 2023. Factors affecting development of disorders expressed after storage of ‘Gala’ apple fruit. Postharvest Biology and Technology. 204. Article 112439. https://doi.org/10.1016/j.postharvbio.2023.112439.*


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