METABOLOMIC AND MICROBIAL PROFILING OF TROPICAL/SUBTROPICAL FRUITS AND SMALL FRUITS FOR QUALITY FACTORS AND MICROBIAL STABILITY

Objective 1: quality of new breeding lines of citrus (mandarin/tangerine and orange), citrus x poncirus hybrids, strawberry, tomato and blueberry was evaluated each year of the project using sensory taste panels and chemical analysis (volatiles, sugars acids, flavonoids and limonoids for citrus, carotenoids for tomato). Volatile profiles of fruits from citrus breeding populations were used to classify hybrids from sweet oranges or mandarins. In collaborative work with University of Florida, this team showed that the carotenoid and terpenoid pathways in citrus may be divergent, leading to cultivars with either high carotenoids and low valencene/sesquiterpenes (tangerines) or the opposite (such as in oranges) . Composition and maturity of hybrids issued from bud irradiation of citrus (�Fallglo� and �U.S. Earlypride�) or from the same crosses varied in maturity or chemical composition. Fruit of a sibling of �Ambersweet� orange hybrid developed delayed bitterness when juiced and stored, making it unsuitable for processing, in spite of excellent flavor otherwise. The effect of field nutritional sprays on the quality of orange juice made with oranges from trees affected by the Huanglongbing disease was studied during five years. A positive effect appeared around the second year, but was not maintained and was dependent on the season, and levels of sugars, acids and other components in the juice. Off flavor being more intense in early-season Hamlin juice from symptomatic fruit. A separate study evaluated Citrus x Poncirus hybrids for fruit quality and revealed the potential of using such hybrids as alternatives to oranges with tolerance to Huanglongbing. The effect of Huanglongbing disease on the composition of orange peel oil was investigated. Overall, Huanglongbing decreased the amount of peel oil extracted from affected fruit. Eight compounds were detected only in the oil from Huanglongbing-affected fruit; they were terpene and non- terpene hydrocarbons. However, only small differences were detected by gas chromatography and olfactometry, and no differences were detected by sensory evaluation smelling the whole oil samples. In the course of analyzing deoxyribonucleic acid (DNA) from Candidatus liberibacter asiaticus in orange juice, the bacterium responsible for Huanglongbing disease, DNA of Diplodia natalensis, a fruit pathogen that normally affects postharvest fruit decay, was found associated with Candidatus liberibacter asiaticus DNA 50 to 60% of the time. It was hypothesized that Diplodia also contributes to Huanglongbing-associated pre-harvest fruit drop. Commercial fungicides were applied to citrus groves and reduced fruit drop, but not in all cultivars. Two strawberry cultivars were released by University of Florida during the life of the project, Winterstar�, 2011, and Sensation�, 2013, with ARS contributing to the flavor and sensory data. A procedure was developed in collaboration with University of Florida scientists to identify genes controlling volatiles contributing to strawberry flavor. The gene for gamma-decalactone was identified; work is continuing to determine the genetic inheritance of methyl anthranilate, another fruity volatile in European strawberries. Blueberry hybrids from UF were selected with the �crisp� trait and tested in sensory trials, showing that this change in texture was perceived by panelists. The storage potential of these hybrids is being pursued. Evaluation of alternative crops for the Florida agriculture was initiated, providing an initial database of chemical and sensory descriptors for winter melon (Benincasa hispida) and avocado (Persea Americana). The feasibility and acceptability of wintermelon juice alone or incomination with orange juice was demonstrated. Objective 2: Relating fruit chemical composition with sensory qualities and disease resistance was established for citrus and tomatoes. The two bitter limonoids, limonin and nomilin, induced by Huanglongbing disease were further studied and thresholds were established in orange juice. It was also found that the polymethoxylated flavones chemical group contributes to the harshness/astringency found in juice from Huanglongbing-symptomatic fruit. The amount of juice from Huanglongbing symptomatic fruit that could be blended with juice from healthy or non- symptomatic fruit before panelists could detect bitterness and off flavor was established at 25%. Volatiles that increased perception of sweetness or decreased perception of sourness or bitterness in orange juice and tomato genotypes were identified using multiple regression models. The model was tested in citrus by spiking 21 of these volatiles in an orange juice matrix and evaluated by consumer and trained panels. These volatiles, added to deodorized orange juice, did not significantly increase sweetness or decrease bitterness; however, they increased fruitiness and decreased �stale�, �green� and �burning� descriptors typical of Huanglongbing juice. In a separate study, non-volatile compounds were isolated from orange juice or peel molasses, and alone or in combination, were spiked to orange juice with typical Huanglongbing off-flavor. Some compounds reduced bitterness without completely masking it. Further, in analyzing hundreds of orange juice samples from trees with various degrees of Huanglongbing disease, the polymerase chain reaction (PCR) method to detect and quantify the DNA of Candidatus liberibacter asiaticus was optimized. A strong correlation was found between the amount of Candidatus liberibacter asiaticus DNA and off flavor in juice, making this a method for screening juice from infected groves and to predict off-flavor of juice in processing lines. A patent application was submitted for this technology. A collaborative study with the University of Florida tomato breeding program was conducted over 7 years with 2-3 seasons per year, to understand tomato flavor. Thirty-eight genotypes were analyzed for sensory attributes �sweet�, �sour� and �overall flavor� as well as for color, sugars, acids and aroma volatiles. December-harvested fruit were generally associated with more acids and sourness perception and less sugars and sweetness perception and, therefore, lower overall flavor ratings compared to June-harvested fruit. March-harvested samples were intermediate. There were significant differences between genotypes for sensory perception and chemical profile as well. When comparing �Florida 47�, the Florida industry standard, with �Tasti-Lee�, a newly released high flavor quality cultivar, 8 out of 29 aroma compounds were higher in �Tasti-Lee� (including many fruity/floral notes) and 4 were higher in Florida 47 (C-5 and C-6 aldehydes and alcohols giving green notes). This provides a useful chemical model for 2 genotypes that differ in flavor quality that can be exploited by breeders seeking to improve flavor. Extracts of the soil fungus Trichoderma virida were tested as antimicrobial agents against the citrus canker organism (Xanthomonas citri subsp. citri) and Escherichia coli, with positive results on E. coli. Objective 3: Pre-harvest applications of a food sanitizer (�Tree-wash�) reduced postharvest small fruit decay. Likewise, field applications of fruit waxes decreased canker incidence due to Xanthomonas citri (Xcc) on grapefruit and increased pack-out. A clamshell packaging was designed with a lower opening ratio (0.05-0.5%) than commercial clamshells (2-5% opening ratio). The new clamshell reduced water loss from fruit and was efficient at storing cherries, lychees, grapes, blueberries, and cherry tomatoes. It was less efficient at controlling decay of stored strawberries, blackberries and raspberries due to increased moisture in the clamshell. A patent application has been entered for this new design. A new formulation of chlorine dioxide was developed in collaboration with a company. The most effective formulation was a slow-release of the chlorine dioxide gas (Curoxin�) placed in a sachet attached to a clamshell lid. In general, Curoxin� significantly reduced postharvest decay, as well as maintained firmness, color and overall quality of whole berries such as strawberries, blueberries and grapes. In citrus, fruit inoculated with Xanthomonas citri had their bacterial count significantly reduced after placing slow release ClO2 sachets (Curoxin�) inside the fruit container. Natural colorants were applied to fresh oranges as alternatives to Citrus Red No. 2. Annatto, paprika and paprika oleoresin were stable at 5 �C in the dark, under conditions that simulates commercial cold storage. These colorants were more stable after application of a commercial fruit was and only annatto maintained stable color at 23 �C in daylight, such as is experienced by oranges in a retail or consumer countertop environment. Studies were performed to evaluate the effect of short-term cold storage on tomato flavor volatiles. Tomatoes stored at 5 �C during 4-5 days (consumer refrigerated storage) had lower flavor due to a decrease in volatile production in comparison with tomatoes stored at room temperature. Hot water dips, methyl salicylate and methyl jasmonate treatments alleviated chilling-induced aroma loss. Similarly, grapefruits were dipped in hot water and stored at 5 �C (chilling) or 10 �C. Hot water-treated grapefruit had higher soluble solids content and were perceived as sweeter than untreated fruit, regardless of storage temperature. Plant essential oils were tested against E. coli, Penicillium digitatum and the citrus black spot organism, Guignardia citricarpa. Carvacrol, trans-cinnamaldehyde and cinnamaldehyde, had strong activity against Guignardia, Penicillium and E. coli. These essential oils were added to edible coatings (chitosan) and applied on blueberries with positive effect on firmness and microbial control including control of E. coli. This project has been replaced by a new research project #6034-41430-006- 00D. Accomplishments 01 Nutritional foliar field sprays to manage Huanglongbing partially mitigate orange juice Huanglongbing-induced off-flavor. Huanglongbing is a devastating citrus disease decreasing fruit yields and quality, giving orange juice a typical off-flavor, with high bitterness and harshness. Growers have implemented foliar nutritional treatments in the field to offset the symptoms of Huanglongbing on the trees. ARS researchers at Fort Pierce, Florida evaluated some of these treatments and found that they also improved fruit/juice quality in some seasons and years, but was not consistent. Until a solution to the disease is found, growers may sustain tree growth and production using foliar spray programs, which sometimes, if not consistently, improves fruit and juice quality. 02 A method to alleviate symptoms of internal chilling injury in tomatoes. Chilling injury occurs when tomatoes are stored at temperatures below 10-13 �C, and aroma loss usually takes place before visual symptoms of chilling injury. ARS researchers at Fort Pierce, Florida found that chilling such as 5 �C during 4-5 days extended shelf-life, did not induce visual defects but decreased aroma production including important aroma contributors in comparison with non-chilled tomatoes. Pre-chilling treatments with hot water dip or methyl salicylate fumigation substantially alleviated chilling-induced aroma loss. Application of heat treatments prior to low temperature storage is proposed to be an effective postharvest handling method to alleviate tomato chilling injury, hence offering the consumer tomatoes with better shelf-life and eating quality.