Temporal Dynamics of the Soil Metabolome and Microbiome During Simulated Anaerobic Soil Disinfestation.

Significant interest exists in engineering the soil microbiome to attain suppression of soil-borne plant diseases. Anaerobic soil disinfestation (ASD) has potential as a biologically regulated disease control method; however, the role of specific metabolites and microbial community dynamics contributing to ASD mediated disease control is mostly uncharacterized. Understanding the trajectory of co-evolutionary processes leading to syntrophic generation of functional metabolites during ASD is a necessary prelude to the predictive utilization of this disease management approach. Consequently, metabolic and microbial community profiling were used to generate highly dimensional datasets and network analysis to identify sequential transformations through aerobic, facultatively anaerobic, and anaerobic soil phases of the ASD process and distinct groups of metabolites and microorganisms linked with those stages. Transient alterations in abundance of specific microbial groups, not consistently accounted for in previous studies of the ASD process, were documented in this time-course study. Such events initially were associated with increases and subsequent diminution in highly labile metabolites conferred by the carbon input. Proliferation and dynamic compositional changes in the Firmicutes community continued throughout the anaerobic phase and was linked to temporal changes in metabolite abundance including accumulation of small chain organic acids, methyl sulfide compounds, hydrocarbons, and p-cresol with antimicrobial properties. Novel potential modes of disease control during ASD were identified and the importance of the amendment and "community metabolism" for temporally supplying specific classes of labile compounds were revealed.

Canopy attachment position influences metabolism and peel constituency of European pear fruit.

BACKGROUND: Inconsistent pear fruit ripening resulting from variable harvest maturity within tree canopies can contribute to postharvest losses through senescence and spoilage that would otherwise be effectively managed using crop protectant and storage regimes. Because those inconsistencies are likely based on metabolic differences, non-targeted metabolic profiling peel of 'd'Anjou' pears harvested from the external or internal canopy was used to determine the breadth of difference and link metabolites with canopy position during long-term controlled atmosphere storage. RESULTS: Differences were widespread, encompassing everything from expected distinctions in flavonol glycoside levels between peel of fruit from external and internal canopy positions to increased aroma volatile production and sucrose hydrolysis with ripening. Some of the most substantial differences were in levels of triterpene and phenolic peel cuticle components among which acyl esters of ursolic acid and fatty acyl esters of p-coumaryl alcohol were higher in the cuticle of fruit from external tree positions, and acyl esters of α-amyrin were elevated in peel of fruit from internal positions. Possibly the most substantial dissimilarities were those that were directly related to fruit quality. Phytosterol conjugates and sesquiterpenes related to elevated superficial scald risk were higher in pears from external positions which were to be potentially rendered unmarketable by superficial scald. Other metabolites associated with fruit aroma and flavor became more prevalent in external fruit peel as ripening progressed and, likewise, with differential soluble solids and ethylene levels, suggesting the final product not only ripens differentially but the final fruit quality following ripening is actually different based on the tree position. CONCLUSIONS: Given the impact tree position appears to have on the most intrinsic aspects of ripening and quality, every supply chain management strategy would likely lead to diverse storage outcomes among fruit from most orchards, especially those with large canopies. Metabolites consistently associated with peel of fruit from a particular canopy position may provide targets for non-destructive pre-storage sorting used to reduce losses contributed by this inconsistency.

Differential Partitioning of Triterpenes and Triterpene Esters in Apple Peel.

Apple peel is a rich source of secondary metabolites, and several studies have outlined the dietary health benefits of ursane-type triterpenes in apple. Changes in triterpene metabolism have also been associated with the development of superficial scald, a postharvest apple peel browning disorder, and postharvest applications of diphenylamine and 1-methylcyclopropene. Previously, studies have generated metabolite profiles for whole apple peel or apple wax. In this study, we report separate metabolic analyses of isolated wax fractions and peel epidermis to investigate the spatial distribution of secondary metabolites in peel. In addition to examining previously reported triterpenes, we identified several unreported fatty acid esters of ursane-type triterpenes (C14-C22). All free pentacyclic triterpenes and triterpenic acids, with the exception of ß-amyrin, were localized in the wax layer, along with esters of ursolic acid and uvaol. All sterols, sterol derivatives and α-amyrin esters were localized in the dewaxed peel epidermis.

Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis.

BACKGROUND: Superficial scald is a physiological disorder of apple fruit characterized by sunken, necrotic lesions appearing after prolonged cold storage, although initial injury occurs much earlier in the storage period. To determine the degree to which the transition to cell death is an active process and specific metabolism involved, untargeted metabolic and transcriptomic profiling was used to follow metabolism of peel tissue over 180 d of cold storage. RESULTS: The metabolome and transcriptome of peel destined to develop scald began to diverge from peel where scald was controlled using antioxidant (diphenylamine; DPA) or rendered insensitive to ethylene using 1-methylcyclopropene (1-MCP) beginning between 30 and 60 days of storage. Overall metabolic and transcriptomic shifts, representing multiple pathways and processes, occurred alongside α-farnesene oxidation and, later, methanol production alongside symptom development. CONCLUSIONS: Results indicate this form of peel necrosis is a product of an active metabolic transition involving multiple pathways triggered by chilling temperatures at cold storage inception rather than physical injury. Among multiple other pathways, enhanced methanol and methyl ester levels alongside upregulated pectin methylesterases are unique to peel that is developing scald symptoms similar to injury resulting from mechanical stress and herbivory in other plants.

Gene expression and metabolism preceding soft scald, a chilling injury of 'Honeycrisp' apple fruit.

BACKGROUND: 'Honeycrisp' is an apple cultivar that is susceptible to soft scald, a chilling injury expressed as necrotic patches on the peel. Improved understanding of metabolism associated with the disorder would improve our understanding of soft scald and contribute to developing more effective management strategies for apple storage. It was expected that specific gene expression and specific metabolite levels in the peel would be linked with soft scald risk at harvest and/or specific time points during cold storage. RESULTS: Fruit from nine 'Honeycrisp' apple orchards that would eventually develop different incidences of soft scald between 4 and 8 weeks of cold air storage were used to contrast and determine differential transcriptomic and metabolomic changes during storage. Untargeted metabolic profiling revealed changes in a number of distinct pathways preceding and concurrent with soft scald symptom development, including elevated γ-aminobutryic acid (GABA), 1-hexanol, acylated steryl glycosides, and free p-coumaryl acyl esters. At harvest, levels of sesquiterpenoid and triterpenoid acyl esters were relatively higher in peel of fruit that did not later develop the disorder. RNA-seq driven gene expression profiling highlighted possible involvement of genes and associated metabolic processes with soft scald development. These included elevated expression of genes involved in lipid peroxidation and phenolic metabolism in fruit with soft scald, and isoprenoid/brassinosteroid metabolism in fruit that did not develop soft scald. Expression of other stress-related genes in fruit that developed soft scald included chlorophyll catabolism, cell wall loosening, and lipid transport while superoxide dismutases were up-regulated in fruit that did not develop the disorder. CONCLUSIONS: This study delineates the sequential transcriptomic and metabolomic changes preceding soft scald symptom development. Changes were differential depending on susceptibility of fruit to the disorder and could be attributed to key stress related and mediating pathways.

Chilling-related cell damage of apple (Malus × domestica Borkh.) fruit cortical tissue impacts antioxidant, lipid and phenolic metabolism.

'Soggy breakdown' (SB) is an internal flesh disorder of 'Honeycrisp' apple (Malus × domestica Borkh.) fruit that occurs during low temperature storage. The disorder is a chilling injury (CI) in which visible symptoms typically appear after several weeks of storage, but information about the underlying metabolism associated with its induction and development is lacking. The metabolic profile of flesh tissue from wholly healthy fruit and brown and healthy tissues from fruit with SB was characterized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatograph-mass spectrometry (LC-MS). Partial least squares discriminant analysis (PLS-DA) and correlation networks revealed correlation among ester volatile compounds by composition and differences in phytosterol, phenolic and putative triacylglycerides (TAGs) metabolism among the tissues. anova-simultaneous component analysis (ASCA) was used to test the significance of metabolic changes linked with tissue health status. ASCA-significant components included antioxidant compounds, TAGs, and phytosterol conjugates. Relative to entirely healthy tissues, elevated metabolite levels in symptomatic tissue included γ-amino butyric acid, glycerol, sitosteryl (6'-O-palmitoyl) ß-d-glucoside and sitosteryl (6'-O-stearate) ß-d-glucoside, and TAGs containing combinations of 16:0, 18:3, 18:2 and 18:1 fatty acids. Reduced metabolite levels in SB tissue included 5-caffeoyl quinate, ß-carotene, catechin, epicatechin, α-tocopherol, violaxanthin and sitosteryl ß-d glucoside. Pathway analysis indicated aspects of primary metabolism differed according to tissue condition, although differences in metabolites involved were more subtle than those of some secondary metabolites. The results implicate oxidative stress and membrane disruption processes in SB development and constitute a diagnostic metabolic profile for the disorder.

Transcriptomic events associated with internal browning of apple during postharvest storage.

BACKGROUND: Postharvest ripening of apple (Malus x domestica) can be slowed down by low temperatures, and a combination of low O2 and high CO2 levels. While this maintains the quality of most fruit, occasionally storage disorders such as flesh browning can occur. This study aimed to explore changes in the apple transcriptome associated with a flesh browning disorder related to controlled atmosphere storage using RNA-sequencing techniques. Samples from a browning-susceptible cultivar ('Braeburn') were stored for four months under controlled atmosphere. Based on a visual browning index, the inner and outer cortex of the stored apples was classified as healthy or affected tissue. RESULTS: Over 600 million short single-end reads were mapped onto the Malus consensus coding sequence set, and differences in the expression profiles between healthy and affected tissues were assessed to identify candidate genes associated with internal browning in a tissue-specific manner. Genes involved in lipid metabolism, secondary metabolism, and cell wall modifications were highly modified in the affected inner cortex, while energy-related and stress-related genes were mostly altered in the outer cortex. The expression levels of several of them were confirmed using qRT-PCR. Additionally, a set of novel browning-specific differentially expressed genes, including pyruvate dehydrogenase and 1-aminocyclopropane-1-carboxylate oxidase, was validated in apples stored for various periods at different controlled atmosphere conditions, giving rise to potential biomarkers associated with high risk of browning development. CONCLUSIONS: The gene expression data presented in this study will help elucidate the molecular mechanism of browning development in apples at controlled atmosphere storage. A conceptual model, including energy-related (linked to the tricarboxylic acid cycle and the electron transport chain) and lipid-related genes (related to membrane alterations, and fatty acid oxidation), for browning development in apple is proposed, which may be relevant for future studies towards improving the postharvest life of apple.

'Scarlett Spur Red Delicious' apple volatile production accompanying physiological disorder development during low pO2 controlled atmosphere storage.

Apple (Malus domestica Borkh.) fruit volatile production is regulated by a variety of factors including low oxygen storage conditions. This study examined the impact of low pO2 controlled atmospheres on 'Scarlett Spur Red Delicious' apple volatile production and disorder development. Accumulation of apple volatile compounds was characterized during long-term cold storage at 0.5 °C in air or low pO2 (0.3, 0.8, or 1.5 kPa) with 1 kPa CO2. Volatile accumulation differed quantitatively with pO2 as acetaldehyde, ethanol, and ethyl ester accumulation increased with decreased pO2 during the first weeks in storage. Differences in volatile accumulation among atmospheres were evident through 6 months. The rate of ethanol accumulation increased with decreased pO2 and could potentially be used to monitor low O2 stress. Incidence of low oxygen disorders after 9 months was highest in fruit held at the lowest pO2. The sesquiterpene α-farnesene was not detected throughout the storage period.

Biomarker development for external CO2 injury prediction in apples through exploration of both transcriptome and DNA methylation changes.

Several apple cultivars are susceptible to CO2 injury, a physiological disorder that can be expressed either externally or internally, and which can cause major losses of fruit during controlled atmosphere (CA) storage. Disorder development can also be enhanced using SmartFresh™ technology, based on the inhibition of ethylene perception by 1-methylcyclopropene (1-MCP). Injury development is associated with less mature fruit with lower ethylene production, but the aetiology of the disorder is poorly understood. Here we report on the progress made using mRNAseq approaches to explore the transcriptome during the development of external CO2 injury. Next-generation sequencing was used to mine the apple transcriptome for gene expression changes that are associated with the development of external CO2 injury. 'Empire' apples from a single orchard were treated with either 1 µL L(-1) 1-MCP or 1 g L(-1) diphenylamine or left untreated, and then stored in a CA of 5 kPa CO2 and 2 kPa O2. In addition, susceptibility to the disorder in the 'Empire' apples from five different orchards was investigated and the methylation state of the ACS1 promoter investigated using McrBC endonuclease digestion and real-time quantitative polymerase chain reaction. Expression of over 30 000 genes, aligned to the apple genome, was monitored, with clear divergence of expression among treatments after 1 day of CA storage. Symptom development, internal ethylene concentrations (IECs) and methylation state of the ACS1 promoter were different for each of five orchards. With transcriptomic changes affected by treatment, this dataset will be useful in discovering biomarkers that assess disorder susceptibility. An inverse correlation between the frequency of this disorder and the IEC was detected in a multiple orchard trial. Differential methylation state of the ACS1 promoter correlated with both IEC and injury occurrence, indicating epigenetic regulation of ethylene biosynthesis and possibly events leading to disorder development.

Ripening, storage temperature, ethylene action, and oxidative stress alter apple peel phytosterol metabolism.

The chilling conditions of apple cold storage can provoke an economically significant necrotic peel disorder called superficial scald (scald) in susceptible cultivars. Disorder development can be reduced by inhibiting ethylene action or oxidative stress as well as intermittent warming. It was previously demonstrated that scald is preceded by a metabolomic shift that results in altered levels of various classes of triterpenoids, including metabolites with mass spectral features similar to ß-sitosterol. In this study, a key class of phytosterol metabolites was identified. Changes in peel tissue levels of conjugates of ß-sitosterol and campesterol, including acylated steryl glycosides (ASG), steryl glycosides (SG) and steryl esters (SE), as well as free sterols (FS), were determined during the period of scald development. Responses to pre-storage treatment with the ethylene action inhibitor, 1-methylcyclopropene, or an antioxidant (diphenylamine), rapid temperature elevation, and cold acclimation using intermittent warming treatments were evaluated. Diphenylamine, 1-MCP, and intermittent warming all reduced or prevented scald development. ASG levels increased and SE levels decreased in untreated control fruit during storage. Removing fruit from cold storage to ambient temperature induced rapid shifts in ASG and SE fatty acyl moieties from unsaturated to saturated. FS and SG levels remained relatively stable during storage but SG levels increased following a temperature increase after storage. ASG, SE, and SG levels did not increase during 6 months cold storage in fruit subjected to intermittent warming treatment. Overall, the results show that apple peel phytosteryl conjugate metabolism is influenced by storage duration, oxidative stress, ethylene action/ripening, and storage temperature.

Metabolomic change precedes apple superficial scald symptoms.

Untargeted metabolic profiling was employed to characterize metabolomic changes associated with 'Granny Smith' apple superficial scald development following 1-MCP or DPA treatment. Partial least-squares discriminant analyses were used to link metabolites with scald, postharvest treatments, and storage duration. Models revealed metabolomic differentiation between untreated controls and fruit treated with DPA or 1-MCP within 1 week following storage initiation. Metabolic divergence between controls and DPA-treated fruit after 4 weeks of storage preceded scald symptom development by 2 months. alpha-Farnesene oxidation products with known associations to scald, including conjugated trienols, 6-methyl-5-hepten-2-one, and 6-methyl-5-hepten-2-ol, were associated with presymptomatic as well as scalded control fruit. Likewise, a large group of putative triterpenoids with mass spectral features similar to those of ursolic acid and beta-sitosterol were associated with control fruit and scald. Results demonstrate that extensive metabolomic changes associated with scald precede actual symptom development.

Diphenylamine metabolism in 'braeburn' apples stored under conditions conducive to the development of internal browning.

Diphenylamine metabolism and ethylene action were evaluated as factors influencing the development of 'Braeburn' apple internal browning and cavitation during cold storage. Apples treated with the antioxidant diphenylamine (DPA) and/or the ethylene action inhibitor 1-methylcyclopropene (1-MCP) were held at 1 degrees C for up to 6 months in air or a controlled atmosphere (CA) containing 1 kPa of O2 and 3 kPa of CO2. Cortex tissues from fruit without disorders as well as from symptomatic and asymptomatic areas of fruit with disorders were analyzed for DPA and DPA derivative content. Internal browning and cavities developed in control and 1-MCP-treated fruit stored in CA, whereas air-stored and CA fruit treated with DPA or with DPA and 1-MCP prior to storage did not develop disorders. Depending on the storage regimen and duration, less DPA was detected in 1-MCP-treated fruit. The 4-hydroxydiphenylamine (4OHDPA) content of control fruit decreased during air storage duration but increased between 2 and 4 months in CA storage. 4OHDPA content in 1-MCP-treated fruit increased with storage duration in CA but not air. N-Nitrosodiphenylamine (NODPA) was detected after 2 months in control fruit stored in air or CA and in 1-MCP-treated fruit stored in CA, and NODPA content in control fruit was higher compared to that in 1-MCP-treated fruit. Accumulation of 4-methoxydiphenylamine (4MeODPA) in control fruit stored in air increased with storage duration, but 4MeODPA content did not change in 1-MCP-treated fruit stored in air or CA. 2-Nitrodiphenylamine content was reduced by prestorage treatment with 1-MCP, but storage environment and duration had no effect on its accumulation. The results indicate that CA storage increases the risk of disorder development in 'Braeburn' apples, that DPA can prevent disorder development, and that the content of DPA and DPA derivatives is influenced by storage environment and ethylene action. A clear relationship between DPA derivative formation and storage conditions that promote internal browning was not apparent.

Prestorage ultraviolet-white light irradiation alters apple peel metabolome.

Global metabolic profiling of 'Granny Smith' apple peel was employed for evaluating metabolomic alterations resulting from prestorage UV-white light irradiation. Apples were bagged midseason to restrict sunlight, harvested at the preclimacteric stage prior to bag removal, treated with fluorescent UV-white light for 0-48.5 h, and stored for 6 months at 0 degrees C. Trimethylsilyl (oxime) derivatized or underivatized aliquots of methanolic extracts from peel samples collected immediately after irradiation or following cold storage were evaluated using GC-MS and LC-UV/vis-MS, respectively. The profile, including more than 200 components, 78 of which were identified, revealed changes in the metabolome provoked by UV-white light irradiation and cold storage. Analyses of individual components selected using principal component analysis (PCA) models showed distinct temporal changes, before and after cold storage, related to prestorage irradiation in a diverse set of primary and secondary metabolic pathways. The results demonstrate metabolic pathways associated with ethylene synthesis, acid metabolism, flavonoid pigment synthesis, and fruit texture, are altered by prestorage irradiation, and many of the alterations are detectable after 6 months of cold storage in air.

Influence of ethylene action, storage atmosphere, and storage duration on diphenylamine and diphenylamine derivative content of Granny Smith apple peel.

The application of diphenylamine (DPA) to prevent the apple peel disorder superficial scald can result in accumulation of a number of DPA derivatives resulting from C-nitration, C-hydroxylation, O-methylation, and N-nitrosation during fruit storage. As the presence of these compounds may be indicative of metabolic processes leading to superficial scald development, the contents of DPA and DPA derivatives were determined in fruits treated at harvest with DPA or DPA plus the ethylene action inhibitor 1-methylcyclopropene (1-MCP), which also prevents scald development. Influences of fruit maturity, storage environment, storage duration, and a 14 day poststorage ripening period on accumulation of DPA metabolites were also assessed. Poststorage ripening, 1-MCP treatment, and controlled atmosphere storage had varied effects on DPA derivative contents suggesting that reactive oxygen and nitrogen species, such as *OH, *NO, and *NO2, or enzyme-catalyzed reactions may be present during certain ripening and senescence-related physiological processes. Definitive correlations between superficial scald incidence and contents of specific derivatives were not observed.

Relationship of superficial scald development and alpha-farnesene oxidation to reactions of diphenylamine and diphenylamine derivatives in Cv. Granny Smith apple peel.

Cv. Granny Smith apple fruit, treated at harvest with aqueous emulsions containing diphenylamine (DPA) and DPA derivatives, were evaluated for the peel disorder superficial scald (scald) after 6 months of cold storage at 1 degrees C plus 0 or 7 days at 20 degrees C. Metabolism of these derivatives and alpha-farnesene oxidation were also evaluated after 6 months. Derivatives substituted at the para position prevented scald, but scald developed on fruit treated with derivatives substituted in the amino, ortho, or meta positions. The extent of scald control was also dependent on the chemical nature of the functional group used to derivatize DPA. Hydroxylation of DPA and DPA derivatives during storage was not associated with scald control. Methoxylated DPA derivatives produced during storage resulted from O-methylation of C-hydroxylated derivatives rather than C-methoxylation of DPA. N-Nitrosodiphenylamine provided partial scald control, possibly resulting from its degradation to DPA, indicating that the amino hydrogen of DPA may be crucial for scald control. Results suggest that functional group position and chemical properties both contribute to the efficacy of DPA derivatives for scald control.

Evaluation of diphenylamine derivatives in apple peel using gradient reversed-phase liquid chromatography with ultraviolet-visible absorption and atmospheric pressure chemical ionization mass selective detection.

A method was developed for extracting, identifying, and quantifying diphenylamine (DPA) derivatives in the peel of DPA-treated apples using gradient reversed-phase liquid chromatography with ultraviolet-visible absorption and atmospheric pressure chemical ionization detection (LC-UV-vis-APCI-MS). Compounds routinely analyzed using this method included hydroxylated, nitrosated, nitrated, and methoxylated diphenylamine derivatives. Analysis of peel treated with 0-8 g L(-1) DPA showed that peel DPA content was a limiting factor in derivative production and that recovery of most compounds over this range was linear.