Month: January 2025

A Micasense RedEdge-M multispectral camera was mounted to a DJI Matrice 100 drone to collect field imagery. Flights occurred at solar noon the day prior to destructive harvests, and images were captured automatically using DJI Ground Station Pro. The drone was flown at an altitude of 15 m with 80 percent overlap between images. Raw images from each flight were built into field-scale orthomosaics and digital surface models using Pix4Dmapper version 4.3.31. The processed orthomosaics were loaded into QGIS version 3.22.1 for extraction of data at the plot level. First a grid layer was generated using the create grid function of QGIS overlaying each plot . Next a threshold layer was generated from the NDVI orthomosaic to differentiate between plants and soil. This threshold layer was then used to partition the DSM into two separate layers, a canopy DSM and a soil DSM that contained elevation information for each pixel. The zonal statistics plugin was then used to calculate the percent canopy area, mean canopy elevation, and mean soil elevation for each plot. From these statistics we calculated plant height by subtracting the average soil elevation from the average canopy elevation and a canopy volume index by multiplying the average plant height by the percent canopy area.All analyses were performed using R statistical software . A simple linear model was used to analyze alfalfa plant height from both ground measurements and remote sensing estimates in the 2018 dormancy trial. Least squares means for each entry in the trial were calculated using the emmeans package by Lenth . The Pearson correlation coefficient was then used to compare the two forms of data collection.

In the remaining trials, square plastic plant pots the Pearson correlation coefficient was again used to compare ground measured fresh weight with remote sensing estimates for biomass volume.We have flight data from a single harvest in the grass trial and alfalfa dormancy trial, from six harvests in the large sown alfalfa trial and from eleven harvests in the transplanted mini-sward alfalfa trial. The accuracy of drone-based remote sensing varied across the range of plot types and forage species, but overall, a high correlation between the two forms of data collection was observed . The Pearson correlation between the drone estimated volume index and ground measured plot fresh weight was greatest in the transplanted family rows used in the 2018 alfalfa fall dormancy trial and was the lowest in the transplanted mini-sward plots used in the 2020 genomic selection trial . In the large sown plots, there was a greater correlation in the grass trial than the alfalfa trial , although remote sensing was highly correlated with the ground measurements in both. The relationship between remote sensing and ground data appears to be linear in all instances except for the transplanted mini-sward plots. In this trial, differences in plots with high fresh weight were not as well identified by the drone as they were in plots with low fresh weight or in the other types of plots. In addition, there appears to be several instances of high biomass plots registering low volumes from the drone imagery, likely due to lodging.The process of phenotyping in plant breeding is expensive and laborious. In perennial forage breeding programs, which often have limited resources, evaluating large breeding populations is challenging due to repeated harvests across multiple years. Drone-based remote sensing offers a fast and effective method of assessing a large amount of material with little increase in labor. Incorporation of such technology in a breeding program enables breeders to increase selection intensity by increasing the scale of breeding trials and thus improving the rate of genetic gain or to replace manual measurements for laborious phenotyping tasks.

Drone estimated biomass volume serves as an effective proxy for biomass yield across a range of perennial forage breeding plot types as demonstrated in this study. Optimizing trials for the collection of remote sensing data and improving the high throughput phenotyping data analysis and curation pipelines could make the incorporation of this technology into breeding programs routine and could help to address the low rate of genetic gain observed in most perennial forage crops. Fall dormancy is a crucial trait in alfalfa that provides growers with information related to the potential of a cultivar to perform in specific environments. Alfalfa breeders must be aware of the degree of fall dormancy in their experimental populations to ensure their cultivars fit the target environments. The standard test for fall dormancy in alfalfa has a significant labor requirement and necessitates growing a dedicated trial that only provides dormancy data. Remote sensing estimates of plant height align very closely with ground-based plant height measurements for a single location and season and may be a more accurate way to assess plant height due to the large number of data points for each plant. When creating an orthomosaic from drone images, the digital surface model includes height information for each pixel; with the camera used in this trial and at a height of 15 m, each plant was represented by hundreds of data points per plant rather than a single measurement by hand of the tallest point of the plant. This will result in a more representative reading for the height of each plant and subsequently a better estimation of fall dormancy. We have previously shown that alfalfa fall dormancy characterization in sward plots is equivalent to the spaced plants used in the standard test .

Our results here suggest that modifying the fall dormancy standard test to enable remote-sensing height data collected as a matter of course in variety trials could be feasible, saving plant breeders time and money in evaluating dormancy response and providing growers with a more precise dormancy estimate. Although remote sensing has significant potential in forage breeding programs as they currently exist, a number of design and management modifications could be made to future breeding trials to maximize the quality of data being collected. First, anything that affects the soil level or plant height will be reflected in the data. Lodging was the most significant problem that we encountered over the course of this study and is a major issue concerning plant height or volume estimates from multi-spectral aerial imagery as it results in the underestimation of true biomass. Selecting a harvest interval that minimizes lodging is one method to mitigate this issue. Additionally, breeders can adjust flight schedules in anticipation of weather events that may result in lodging, such as high winds or heavy rain. As well as causing lodging, wind can influence the quality of aerial images. Plants that are moving in the wind may cause anomalies when stitching the raw images into an orthomosaic; therefore, weather monitoring is an important consideration in remote sensing as well as having a flexible flight schedule. Machinery traffic and mammalian pests are the main causeof soil level issues that we encountered. Wheel ruts from machinery may lead to overestimation of height and biomass, square pot plastic while mounding from mammalian pests such as gophers and ground squirrels will have the opposite effect. Thus, controlling mammalian pests and avoiding traffic on trials when the ground is soft will result in better remote sensing estimates. The method of data collection used in this study requires some reference to ground level within each plot and designing trials to enable clear ground level identification will improve the data collection process. Large gaps between plots will ensure that a ground reference can be found throughout the trial. A system that we recommend is that used in our grass trial, where alleys were mown between plots prior to flying. This has the advantage of maintaining the selection pressure from competition that plants would experience in a commercial field, while also providing a solid baseline for ground level. It also has the additional benefits of limiting the impact of traffic on the soil level and controlling weeds surrounding the plots. Finally, although not essential, ensuring trials are arrayed on a regular grid with straight lines and even plot sizes will streamline the data extraction process. Overlaying a grid in QGIS requires significant manual adjustment for trial layouts that are not uniform so additional care when planning and planting a trial, or ideally, using a GPS equipped planter will greatly simplify the pipeline. Though beyond the scope of this paper, a host of alternative applications for remote sensing data beyond plant height and volume estimation can be imagined. The non-destructive nature of remote sensing means that a breeder could measure the entire growth cycle of forage crops to make better informed selection decisions. There are wide range of vegetation indices other than NDVI to highlight various properties of vegetation that are not observable to the naked eye.

Forage quality is another key trait that breeders must evaluate for which different vegetation indices may prove useful. Remote sensing imagery also serves as a digital archive of trials that can be revisited to better understand trends in trial development over time.Despite the substantial range of applications for remote sensing data in perennial forage breeding, there are a number of limitations. High throughput data collection, storage, and processing require hardware and software investment and the knowledge to develop an analytical pipeline to extract actionable information. Also, weather plays a substantial role in data collection . Finally depending on the camera and drone, there may be a large initial investment to get set up. Remote sensing offers a promising method to reduce the costs of phenotyping in perennial forage breeding programs with the accurate estimation of important traits. The results from this study suggest that breeders could increase the size of breeding trials without a proportional increase in labor and consequently increase the rate of genetic gain for forage yield Breeders also have a new method of assessing fall dormancy in alfalfa that requires significantly less labor than traditional phenotyping. Ground-measurements supplemented with remote sensing data opens the door for smaller, resource limited breeding programs to adopt new methodology such as genomic selection to help bridge the gap in genetic gain between perennial forages and alternative crops thus ensuring continued inclusion in crop rotations.By value, tomato is the fourth most important commercial crop globally . Tomato is a rich source of minerals, vitamins, and phytochemicals. Post harvest deterioration is among the major challenges for fruit industry, accounting for up to 50% of harvested losses . The primary cause of post harvest deterioration is fruit softening, which decreases fruit shelf-life and increases susceptibility to pathogens . Theoretically, regulating the rate of softening would extend shelf-life and increase pathogen resistance and be an effective strategy to reduce post harvest losses . Fruit softening is result of destruction of the fruits wall’s structural polysaccharides and reduction in intercellular cell wall adhesion . The main components of the cell wall include cellulose, hemicellulose, pectin, and a small amount of protein . Due to the complex composition and structure, many enzymes have been reported to catalyse the fruit softening. The role of polygalacturonase , pectin methyl esterase , bgalactanase, expansin, and pectate lyases regulating fruit texture has been well investigated. Downregulation of the PG and PME genes does not affect tomato fruit softening . Silencing the PL gene in tomato delayed fruits softening and reduced susceptibility to grey mould, implying prolonging fruit shelf-life by genetic modification of cell wall-modifying enzymes is a potential approach . Ascorbic acid , vitamin C, a crucial compound is present in most living organisms . In higher plants, AsA functions as an antioxidant and enzymatic cofactor, playing a crucial role in multiple physiological processes including photoprotection, cell expansion and division, ethylene biosynthesis and abiotic stress responses . As a result of these critical functions in plants and its benefits to human health, AsA biosynthesis, recycling, and accumulation in plants have been extensively investigated. The current consensus is that the L-galactose pathway is the primary pathway for AsA accumulation in higher plants. The structural genes have been identified. L-galactose 1-phosphate phosphatase catalyses the conversion of L-galactose 1-phosphate to Lgalactose in AsA synthesis . It has been reported that the expression patterns of GPP are associated with AsA content in apple and tomato plants under abiotic stress . In Arabidopsis, the VTC4 gene encodes an enzyme catalysing the similarreaction with the GPP enzyme in AsA biosynthesis .

All systems with size-controlling rootstocks were irrigated and fertigated using sub-surface drip to maintain a soil moisture between -20 and -60 cbar throughout the growing season. Microsprinklers were used for irrigation and fertigation in the Nema Quad system. The soil at the site is a well-drained Hanford, fine sandy loam. Weeds were controlled by mowing the row middles and applying herbicides to maintain a 1.5m wide weed-free strip down the tree rows. All systems received a light summer pruning and heavy dormant pruning to establish desired structure and improve light interception. Approximately a week before harvest, total canopy light interception using a ACCUPAR LP- 80 meter was measured in each plot. Harvest occurred on two or three separate days, depending on rootstock and cultivar, during the growing season due to variance in fruit maturity, as is common in stone fruit production. Each data tree was harvested individually, total quantity of fruit produced, and total fruit weight data were recorded which enabled calculation of mean fruit weight per tree. Harvest data were collected for growing seasons 2017-2019. A linear model was created in R markdown for each season and cultivar’s harvest. With each linear model, an ANOVA test was conducted using a 95% confidence level and Dunnett’s method adjustments to identify significant differences among the four orchard systems for both scion cultivars. A true significant difference was concluded if the comparison between two systems had a p-value less than 0.05, a t-ratio greater than 1.68 , black plant pots plastic and a confidence level range that did not include 0.During the 2017 harvest season for the June Flame cultivar, trees in all systems produced commercially acceptable mean fruit size, >200g per individual fruit .

The C-6 Quad system produced significantly larger fruit when compared to the Nema Quad system . In the June Flame 2018 harvest, mean individual fruit weights in all systems were again > 200g, . Although the C-9 Quad system produced large enough fruit for fresh market sale, the mean individual fruit weight was significantly less compared to fruit in the Nema Quad system . The harvest season of 2019 for June Flame had individual fruit weight above 200g in all systems . However, it should be noted that the C-6 V and C-9 Quad systems produced significantly smaller fruit compared to the Nema Quad system . In all three seasons for the June Flame cultivar, the C-6 Quad system produced fruit of equal or larger size than the Nema Quad system. In the 2017 harvest season for the August Flame cultivars, all systems with size-controlling rootstocks produced significantly larger fruit than the Nema Quad system . In a few cases, fruit size exceeded 300g per fruit in systems with size controlling rootstocks, >50% larger than the minimum requirement for large sizing in the fresh market . The harvest season of 2018 for August Flame may have been the most productive of all years for both cultivars, all systems exceeded 250g in mean individual fruit weight . The C-6 Quad system had significantly larger fruit than the Nema Quad system while C-9 Quad had significantly smaller fruit . In the 2019 harvest season all systems produced fruit sizes above 200g but were smaller than fruit from previous seasons . Although the C-6 V and C-9 Quad systemsdid not differ significantly from the Nema Quad system, the C-6 Quad system produced significantly smaller fruit compared to the Nema Quad system .During the 2017 harvest season of the June Flame cultivar, the C-6 V and C-9 Quad systems produced significantly fewer fruit per hectare compared to the Nema Quad system.

There was no significant difference between the C-6 Quad and Nema Quad systems . For June Flame in 2018 there were no significant differences in yield per hectare among the C-6 V, C-6 Quad and Nema Quad systems. The C-9 Quad system produced significantly fewer fruit per hectare compared to the Nema Quad system . The 2019 June Flame harvest had close to identical fruit count per hectare between the C-6 Quad and Nema Quad systems .Once again, the C-9 Quad was the only system that produced significantly fewer fruit per hectare compared to the Nema Quad system . In August Flame’s harvest of 2017 there were no significant differences among systems using size-controlling rootstocks and the Nema Quad system for fruit produced per hectare . In the 2018 harvest season for August Flame, the C-9 Quad system produced significantly fewer fruit per hectare compared to the Nema Quad system . Meanwhile the C-6 V and C-6 Quad systems maintained similar fruit counts per hectare as the Nema Quad system. In the 2019 harvest for August Flame the C-6 V system produced significantly fewer fruit per hectare than the Nema Quad system. The C-6 Quad and C-9 Quad systems did not differ significantly for fruit count per hectare compared to the Nema Quad system . It should be noted that during the 2019 harvest season some trees displayed signs of water stress in the field which may have hindered production and skewed results for that season.During the 2017 harvest of the June flame cultivar, there was a significant difference in the slope of the relationship between fruit size and fruit per hectare among the C-6 V and the Nema Quad systems . Data from all systems fit a linear model that had a negative correlation between fruit size and fruit per hectare. Although a negative correlation was visible between fruit size and fruit per hectare in the C-6 V system, its magnitude was not as steep as with other systems in the same season . The following season, 2018, for June Flame there were significant differences in the fruit size vs. fruit per hectare relationship among systems. . Even though no significant differences were detected with the ANOVA analysis, linear models were weak at representing the relationship between fruit size and fruit per hectare and all systems using size-controlling rootstocks had an R-squared value <0.15 .

Continuing the trend from the previous season, in 2019 for June Flame, there were no significant differences in the slope of fruit size vs fruit per hectare relationship for any of the systems . The contrast between the C-6 Quad system and Nema Quad system did have a t.ratio with a greater absolute value than 1.68, however the P.value for the same comparison was still greater than the designated alpha, > 0.05. In this same season the C-6 Quad system had the best fit for the linear model showing a negative correlation between fruit size and fruit per hectare. All other systems fit the model poorly and also did not indicate a clear negative correlation between fruit size and fruit count per hectare . For the August Flame harvest of 2017, data from all systems fit linear models that showed a negative correlation between fruit size and fruit per hectare . Values for the t. ratio between the C-9 Quad and Nema Quad systems were beyond the absolute limit but had a P. value greater than the declared alpha, thus no significant differences were confirmed .For the 2018 harvest of August Flame there were no significant differences in the fruit size vs. fruit per hectare relationships detected among systems . Linear models fit 2018 August Flame data better than other years and showed a clear negative correlation between fruit size and fruit per hectare . In 2019 there was a wide spread of mean fruit sizes per tree in the August Flame data and no significant differences occurred among systems for the relationship between fruit size and fruit per hectare . Although the ANOVA analysis did not indicate differences among systems, linear models indicated a weak negative correlation between fruit size and fruit per hectare with all systems having near horizontal models accompanied by Rsquared values <0.1 .Although R-squared values for the linear models representing the relationship between fruit size and fruit per hectare were identical to those for fruit size and fruit per tree , large plastic pots for plants there were differences detected in the contrast analysis for slopes . Data for the June Flame 2017 and 2018 harvest seasons indicated no significant differences in the relationship for fruit size vs fruit count per tree among any of the systems . In 2019 there was a significant difference in the data for the June Flame cultivar between the C-6 Quad system and the Nema Quad system . In the 2017 harvest data of August Flame there was a significant difference in the fruit size vs. fruit per tree relationship among C-6 V and Nema Quad systems . The difference in 2017 data was visually apparent in the steeper slope indicated in the C-6 V system but that might be a result of the narrow range of fruit loads per tree in that system . No significant differences in the fruit size vs. crop load per tree relationship were detected in the harvest season of 2018, however both, C-6 Quad and C-6 V systems, had t. ratios indicating one may exist, but p-values remained above alpha, therefore a difference was not conclusive . Data for the 2019 harvest of August Flame indicated no significant differences in this relationship between systems, and in fact, the fruit size vs. crop load per tree relationship were most similar among systems in this year compared to other years .

A relationship between light interception and yield was most apparent in the June Flame cultivar with the C-6 Quad and C-9 Quad systems which produced data that fit linear models with the highest R-squared values. The linear model for the Nema Quad system had the steepest slope but not a very strong R-squared value. Data from the C-6 V system had a poor fit with a linear model. Interestingly the systems with data that had a poor fit to the model also had the highest % light interception, often >50% . August Flame cultivars showed a similar pattern for the relationship between amount of light intercepted and yield. Data from the Nema Quad and C-6 V systems had poor fits to the linear models but also had the highest light interception. Data from the C-9 Quad system had a moderate correlation between PAR and yield, fit the model best. The C-6 Quad system is an apparent outlier, having a value of almost 5 Kg/m2 yield with only about 40% light interception, and a very slight negative correlation between the two parameters . Both of the C-6 V systems with the June and August flame cultivars had trends as shown in previous research, higher density systems were able to intercept a higher proportion of light during earlier years because the trees fill their allotted space more quickly, .The mean fruit size for the June Flame cultivar in 2017 was similar among all systems, most likely a result of consistent thinning resulting in the desired crop loads per tree. In 2018 the mean fruit size for June Flame systems was exceptionally large, especially for an early bearing cultivar. Considering that the C-6 Quad and C-6 V systems had some of the largest fruit sizes provides strong evidence that size-controlling rootstocks are not always associated with reductions in fruit size. The C-9 system had poor performance in the trial but, with its success in previous studies and how well systems with the more size controlling rootstocks performed in this trial, it is likely not due to the reduced hydraulic conductance associated with size controlling rootstocks . June Flame systems in 2019 closely mirrored fruit sizes from the previous season, providing more confidence that any reduction in fruit size compared to the Nema Quad system is unlikely a result of size controlling rootstocks. The results from the June Flame cultivar are most promising because there were concerns that the size-controlling rootstocks may have the potential to have negative effects on fruit size in early maturing cultivars. With how quickly early bearing cultivars must set and mature fruit during the spring flush growth, there was concern that reduced hydraulic conductance associated with undeveloped xylem would influence fruit size . However, this trial did not provide evidence that early maturing cultivars on the size-controlling rootstocks produce smaller sized fruit compared to those on more vigorous rootstocks. With the August Flame cultivar, systems using size-controlling rootstocks also were not found to diminish fruit size in this later maturing cultivar. In 2017 all dwarfing systems performed beyond expectations.

Furthermore, Burkholderia seminalis strain ASB21 was found to be able to produce the plant hormone auxin, promote rice seedling growth, and reduce aluminum toxicity symptoms in host plants. Similarly, a Burkholderia seminalis strain isolated from Bangalore, India can produce indole acetic acid and enhance tomato seedling growth. Although it is known that Burkholderia seminalis belongs to the plant-growth-promoting rhizobacteria , only limited strains and their promoting abilities are well characterized. In this study, we examined the amounts of IAA produced by B. seminalis strain 869T2 in various growth conditions, detected the strain’s siderophore synthesis and phosphate solubilization abilities, and demonstrated its growth-promoting abilities in several leafy vegetables, including pak choi, lettuce, and amaranth.Various growth parameters of different plant species were measured at selected days, ranging from 14 to 80 days, after inoculation with strain 869T2. The fresh weight, dry weight, and length of leaves and roots as well as the width, number, and surface area of leaves were measured in harvested pak choi, lettuce, and Chinese amaranth as described previously. The fresh weight, length, number, and color of fruits of hot pepper and okra were recorded following previously described methods . Chlorophyll was extracted from the leaves with N, N-Dimethylformamide for 1 hour in the dark, square plant pot and chlorophyll a and b concentrations were calculated from the absorbance of the crude extract at 647 and 664 nm. Anthocyanin concentrations were determined using a published acidified methanol method. Hot pepper fruits were first ground with liquid nitrogen.

Acidified methanol was then mixed with the ground materials for 10 min in darkness with shaking. These crude extracts were subsequently mixed with an extraction solvent containing 1:1 chloroform:water to isolate anthocyanins. After centrifugation, the absorbance of the supernatant was read at 530 and 657 nm by the spectrophotometer, and anthocyanin contents were calculated from these values.The effects of pH were also examined by culturing strain 869T2 in LB media at 30 ◦C over a pH range of 4 to 9. Strain 869T2 was able to grow over this entire pH range . The results shown in Figure 1D demonstrate that IAA production was at a similar level when bacteria were grown at pH 6 to 9, whereas the IAA amount decreased 44.0% when bacteria were grown at pH 4. Additionally, three different sugars, glucose, fructose, and sucrose, were used in the minimal medium to examine the effects of different carbon sources on IAA production. Strain 869T2 grew similarly in the M9 salt media with different kinds of sugars . The results shown in Figure 1F indicate that when strain 869T2 was grown in the media with two kinds of monosaccharide, glucose and fructose, the IAA amounts were higher than for the bacteria grown in the media with sucrose. We further investigated whether strain 869T2 had other plant-growth-promoting traits, including siderophore production and phosphate solubilization abilities, with agar plate assays. Supplementary Materials Figure S1A shows that the strain 869T2 colonies exposed to CAS agarose turned yellow, indicating the siderophore production ability of strain 869T2. Furthermore, Figure S1B reveals that the formation of halos around the strain 869T2 colonies grown in Pikovskaya’s agar medium with 0.5% tricalcium phosphate suggests that strain 869T2 may have the ability to solubilize phosphate.Seedlings of ching chiang pak choi and pak choi from the Brassica genus were also inoculated with strain 869T2 to examine its effects on plant growth.

At 27, 33, and 40 days after inoculation with strain 869T2, the average fresh weight and dry weight of above ground leaves of ching chiang pak choi were higher than those of the control plants . Furthermore, the average leaf length and width, petiole length and width, number of leaves per plant, total leaf area per plant, and leaf area per leaf were greater in the 869T2-inoculated ching chiang pak choi compared to the control plants . The results shown in Figure 3J,K demonstratethat the average plant height and width of the 869T2-inoculated ching chiang pak choi were also greater compared to the control plants. Similarly, after the ching chiang pak choi was inoculated with strain 869T2, the average values of root fresh weight, dry weight, and length were higher in comparison to control plants . Figure 3O–Q indicate that both the aerial and below ground parts of ching chiang pak choi were larger after inoculation with strain 869T2. Figure 3R also shows that the ching chiang pak choi inoculated with strain 869T2 grew faster and flowered earlier than control plants 53 days after inoculation. Similarly, after inoculation with strain 869T2, the pak choi grew larger, including larger and more numerous leaves, larger aerial parts overall, and longer and heavier roots . These data indicate that inoculation of strain 869T2 in two vegetables from the Brassicaceae family significantly improved their growth.Because B. seminalis strain 869T2 successfully colonized Arabidopsis and two types of plants from the Brassicaceae family and promoted their growth, we further examined whether strain 869T2 could promote the growth of plants from the Asteraceae and Amaranthaceae families. At 35, 43, 50, and 56 days after inoculation with strain 869T2, the fresh weight of the aerial parts of inoculated loose-leaf lettuce plants increased 12.7- to 46.6-fold compared to the 0-day post-inoculation plants . By comparison, in the mock-inoculated control plants, the fresh weight increased 8.0- to 36.0-fold over the same period .

Similarly, the dry weight of the inoculated loose-leaf lettuce increased more than that of the control plants at 35, 43, 50, and 56 days after inoculation . These data indicate that inoculation of the loose-leaf lettuce with strain 869T2 significantly enhanced plant growth. The weight increases of the inoculated loose-leaf lettuce plants were due to increases in average leaf width and length , the number of leaves per plant , total leaf area per plant and per leaf , and plant height and width . Furthermore, the root fresh weight of the inoculated loose-leaf lettuce plants increased 4.5- to 12.4-fold at 35, 43, 50, and 56 days after inoculation compared with the 0-day post-inoculation plants ; in contrast, that of the mock-inoculated control only increased 2.5- to 8.5-fold compared with the 0-day post-inoculation plants . Additionally, the root dry weight and length increased more in the inoculated loose-leaf lettuce plants than in the control plants . As seen in Figure 4M–O, overall plant size and leaf size increased after inoculation with strain 869T2, suggesting that strain 869T2 improves loose-leaf lettuce growth.We also inoculated strain 869T2 into romaine lettuce and red leaf lettuce. The results shown in Figures S4 and S5 demonstrate that both kinds of lettuce grew taller and wider, had more and larger leaves, and had heavier aerial and below ground tissues after inoculation with strain 869T2 compared with the control plants. The chlorophyll contents of red leaf lettuce leaves were also higher in the 869T2-inoculated plants than the control plants . These data collectively indicate that the three evaluated kinds of lettuce can grow significantly better after inoculation with strain 869T2. We also selected Chinese amaranth of the Amaranthaceae family to test the effect of strain 869T2 on its growth. At 36, 43, and 50 days after inoculation, the fresh weight of the 869T2-inoculated Chinese amaranth exhibited a 20.0- to 56.6-fold increase when compared to the 0-day post-inoculation plants, square pot whereas the control plants only showed an 8.3- to 33.5-fold increase when compared to the 0-day post-inoculation plants . Other plant growth parameters of the 869T2-inoculated and control plants were also examined 36, 43, and 50 days after inoculation . Figure 5 illustrates that the 869T2-inoculated Chinese amaranth individuals had more and larger leaves, were taller and wider, and had heavier and longer roots than the control plants. These data show that inoculating strain 869T2 into Chinese amaranth promoted its growth.Because B. seminalis strain 869T2 promoted the growth of several leafy vegetables, we next tested the effects of the strain 869T2 on the flowering and fruit production of hot pepper and okra . Hot pepper plants, from the Solanaceae family, were inoculated with strain 869T2 but we did not observe significant growth promotion effects on the aerial and root parts of the plants. However, we did observe that the 869T2-inoculated hot pepper plants flowered 20 days after inoculation; the number of flowers continually increased and had more than a 7-fold increase at 37 days after inoculation . In the mock-inoculated control plants, we observed flowering 21 days after inoculation, and the number of flowers had only increased 5-fold at 37 days after inoculation . The average number of fruits on the 869T2-inoculated plants was higher than that on the control plants at 30, 37, 44, and 51 days after inoculation . The average numbers of flower buds, flowers, and fruits per plant were higher in the 869T2-inoculated plants than in the control plants beginning 21 days post-inoculation . Furthermore, the percentages of hot pepper fruits with red and green/yellow coloring were higher in the 869T2-inoculated plants than in the control plants 59, 66, 73, and 80 days after inoculation . Similarly, the average anthocyanin contents of the 869T2-inoculated plants were significantly higher than those of the control plants at 66, 73, and 80 days after inoculation . However, the average length, width, and fresh weight of the fruits were not significantly different between the inoculated and control plants .

Collectively, these data suggest that the inoculation of hot pepper with strain 869T2 could increase flowering and fruiting in hot pepper plants and accelerate fruit maturation.We subsequently examined the effects of strain 869T2 on okra, which belongs to the Malvaceae family. The overall plant size and weight were not significantly different between the 869T2-inoculated and control okra plants. We observed, however, that the number of nodes of the first flower was smaller in the 869T2-inoculated okra than in the control plants, suggesting that the 869T2-inoculated okra plants flowered earlier than the control plants . In addition, the average fresh weight and diameter of the fruits from the 869T2-inoculated plants were greater than those of the control plants , although the average fruit lengths were similar. These data demonstrate that the okra fruits became heavier and wider after inoculation with strain 869T2. In summary, inoculation of strain 869T2 into hot pepper and okra plants could cause plants to flower at earlier growth stages.The members of the genus Burkholderia belong to the class β-proteobacteria and have a broad distribution, residing universally in soil, water, and in association with plants, fungi, animals, and humans. Some Burkholderia species are plant pathogens in many vegetables and fruits, while others have been reported as opportunistic pathogens of humans and other animals. However, many other Burkholderia species are beneficial to plants, suppressing plant diseases and promoting plant growth by various processes, including the production of antibiotics, secretion of allelochemicals, induction of pathogen resistance in plants, nitrogen fixation, or enhancing nutrient uptake by host plants. These beneficial Burkholderia species are free-living or endophytic and form mutualistic associations with their host plants. Burkholderia species’ high versatility and adaptability to different ecological niches rely on the high genomic plasticity of their large multichromosome genomes and the production of various bacteria secondary metabolites. In this study, we characterized the endophytic bacterium Burkholderia seminalis strain 869T2 isolated from vetiver grass, which was recently described and included in the Burkholderia cepacia complex . We have documented the IAA production, siderophore synthesis, and phosphate solubilization abilities of B. seminalis strain 869T2. Inoculations of strain 869T2 into tested plants demonstrated the plant growth promotion ability of this bacterium in several plant species from the Brassicaceae, Asteraceae, and Amaranthaceae families. Plant endophytic bacteria can increase the nutrient uptake and biomass accumulation of host plants through the production or regulation of various plant hormones, such as auxin, cytokinin, gibberellins, and ethylene. Indole acetic acid is a naturally occurring auxin produced by several endophytic bacterial species through the L-tryptophan metabolism pathway. Tryptophan can exist in the exudates of plants and is utilized by the bacteria to synthesize auxin, which enhances the growth of host plants. Auxin is the major plant hormone that regulates various aspects of plant growth and development, such as root initiation and development, leaf formation, fruit development, floral initiation and patterning, phototropism, and embryogenesis. Several plant-growth promoting bacteria can synthesize IAA, including Bacillus, Burkholderia, and Pseudomonas species. In this study, Burkholderia seminalis strain 869T2 was able to synthesize approximately 2.0 to 2.2 µg mL−1 IAA in the presence of tryptophan and increased both the above ground and below ground biomass of tested plant tissues.

The extracted phytochemicals created inhibition zones against Bacillus subtilis, Staphylococcus aureus, E. coli, and Salmonella spp. of 13, 15, 9, and 11 mm respectively . These results suggest that the phytochemicals found in dried fruits could play a role in the survival of pathogens. Dry fruit related outbreaks. While not many, there have been several foodborne outbreaks associated with low moisture foods . In 2020, an outbreak of the hepatitis A virus associated with dates occurred in the United Kingdom . Twenty eight people were infected and the dates, which were imported from Jordan, were subsequently voluntarily recalled. Another outbreak of hepatitis A occurred in England from semi-dried tomatoes, which infected two people . An outbreak of salmonellosis in Norway was associated with consumption of a Salmonella Agbenicontaminated dried fruit and nut mix . In this outbreak, 39 people were infected. Another outbreak was linked to Salmonella Phage type 13a in a dried vegetable spice mix, in which 108 people were infected in Sweden . Objectives. As discussed above, dried fruits are of great economic importance to California and different processors follow distinct protocols to prepare their dried fruits. In addition to the various drying methods used, pre- and post-drying treatments can also be applied. Unfortunately, there has not been a systematic evaluation of the antimicrobial efficacy of different drying methods or pre- and post-drying treatments or a combination of them.Pathogen contamination of the final products can happen at any processing point. Once happened, it is critical to better understand the behavior of these pathogens in dried fruits. The goal of this study is to fill in the current knowledge gaps associated with microbial safety risks of dried fruit by conducting a challenge study. The three pathogens selected include Salmonella, E. coli O157:H7, and L. monocytogenes. Dried fruits, including peaches, pluots, tomatoes, blueberry grow bag and dates were purchased from local farmers markets for this study.When conducting challenge studies and preparing artificially contaminated food items, there are different carriers that can be used.

The currently available inoculation methods can be grouped into three major categories: a liquid-carrier method, a dry-carrier method, and a no-carrier method. Examples of liquid carriers include peptone water, saline buffers, and ultrapure water. Cocoa butter oil has also been used to carry out inoculation . Dry carriers that have been tested include sand, chalk, and talc . The no-carrier method utilizes freeze- or vacuum-dried cultures or cell pellets and directly applies them to products . Choosing the correct inoculation carrier is critical. Although liquid carriers, such as buffered peptone water or ultrapure water, have been more widely used for delivering pathogens onto product surfaces, the addition of liquid or the introduction of additional moisture into the products changes the moisture content and aw of a dry substrate and may requires additional or extended drying steps . As indicated by Palipane and Driscoll , moisture adsorption/desorption isotherms are inherently non-equivalent, the aw of the product after an additional drying step may not be the same as the original food. Beuchat and Mann used two different methods for inoculating dried cranberries, raisins, and strawberries and date paste. No difference in Salmonella behavior was observed between two inoculation methods . Similar observation was made by Blessington et al. , in which no difference in Salmonella decline was observed between dry-inoculated and wet-inoculated nut kernels. Both studies indicated that when choosing the proper carriers fordried products, the key features or changes that need to be monitored are the physical or chemical properties. In addition, inoculation methods should try to mimic real life contamination scenarios to give the most accurate representation of survival after a contamination event. It is a water-based salt solution containing sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate. It helps maintain the osmolarity balance of bacteria when being used as the carrier for inoculation. However, since another key function if this buffer is to help maintain a constant pH, there is a concern over the use of PBS for inoculation and sample homogenization when measuring the pH of inoculated dried fruits. In this case, before the long-term survival study, the impact of PBS on the pH measurement needs to be studied. An efficient recovery method that can release and recover target bacteria from food surfaces, is the foundation for accurate detection and enumeration.

Stomaching, shaking, rubbing, sonication, pummeling, pulsing, as well as blending have been tested and used for releasing and recovering bacteria from food or environmental samples . Based on the various physical and chemical properties of the samples, the efficacy of these cell recovery method change. As shown by Kim et al. , bacterial populations recovered from pummeled and pulsed iceberg lettuce, perilla leaves, cucumber, and green pepper samples were higher than those recovered from sonicated and hand-shaken samples. However, this trend was not observed on cherry tomatoes. Thus, which bacteria recovery method is more appropriate for sand-inoculated dried fruit needs to be determined.Dried fruits. Dried fruits used for this survival study were purchased from local farmers markets. Fruits used included sundried tomatoes, peaches, peaches processed with sulfur, pluots processed with sulfur, low-moisture Medjool dates, and high-moisture Medjool dates . High-moisture dates are harvested directly from the tree; low-moisture dates are dates allowed to continue drying in nets after falling off the tree and have a harder texture compared to the high-moisture dates. Tomatoes and the sulfured peaches and pluots were sun dried. The peaches that were not processed with sulfur were dried using a dehydrator. Once purchased, the dried fruits were stored at room temperature for up to 1 week prior to use in experiments. Inoculation with wet and dry carriers. Dried fruits were combined with either water or sand by the following methods. Briefly, ultrapure water was added to every 100 g of each of dried fruits and massaged by hand for 1 min. The dried fruits were then transferred to plastic containers with drying racks lined with filter paper . The lids of the containers were taped down slightly ajar with a piece of mesh to cover the opening . The containers of fruit were set out to dry at room temperature for 48 h.

For sand inoculation, 20 g of sand was added to 100 g of each dried fruit and massaged and shaken for 1 min. The dried fruits were then transferred to gallon storage zipper bags and stored at room temperature. For the water-inoculated samples, the pH and aw was measured before and right after the inoculation. After 48 h, the pH and aw of both the wet and dry inoculated dried fruits were both measured. A pH meter and water activity meter were used to take the measurements. Effect of phosphate buffered saline on pH measurement of dried fruits. Phosphate buffered saline was chosen as the wet carrier with which to inoculate the dried fruits. Since PBS is a buffer solution, the impact of PBS on the pH measurement of dried fruits was tested. To do so, dried fruits were combined with either ultrapure water or PBS and massaged and shaken by hand for 1 min. The pH of the sample was measured before the addition of the liquid, immediately after massaging, and after 48 h of drying. To measure the pH, blueberry grow bag size each fruit sample was combined with water or PBS equal to 40% of the sample mass and then stomached for 1 min at the fast setting to homogenize. The pH meter was used to take the measurements. Bacterial cultures and inoculum preparation. The strains of bacteria used for this study were provided courtesy of Dr. Linda J. Harris at the University of California, Davis. Five strains of rifampicin-resistant Salmonella were used. The strains are summarized in Table 1.1.Individual frozen stock cultures were streaked onto tryptic soy agar , Sparks, MD, supplemented with 50 µg/mL of rifampicin , and incubated at 37 °C overnight. Each isolated colony was transferred into 10 mL of tryptic soy agar supplemented with rifampin at 50 µg/mL , and then incubated at 37 °C overnight. One 10-µL loopful of the overnight culture was transferred to 10 ml of fresh TSBR and incubated at 37 °C for another 24 h. The newly inoculated broth was spread onto TSAR plates, 250 µL per plate, one plate per strain, and incubated for 24 h at 37 °C. To recover bacterial lawns from plates, 1 mL of phosphate-buffered saline was pipetted onto each plate, and an L-shaped plastic cell spreader was used to loosen and scrape the lawn. The re-suspended cells were then pipetted into a 15- mL Falcon™ tube . The addition of PBS and lawn scraping was repeated two more times for each plate, for a total of 3 mL of PBS used per plate. Approximately 2.5 mL of culture was recovered from each plate. Once all plates were scraped, 15 mL of the recovered culture from each strain were combined to make the 5-strain cocktail of Salmonella. The cocktail was diluted and plated onto TSAR for calculating the inoculum level. Evaluation of homogenization methods for recovering pathogenic cells from inoculated sand. Salmonella-inoculated sand was used to test the recovery method used for dried fruit . Twenty grams of sand was inoculated with 1 mL of the 5- strain Salmonella cocktail and they were mixed together by hand for 1 min. Samples of the inoculated sand were sampled immediately after mixing and after 48 h of drying. The drying process was done at 40 °C for 48 h in a gravity oven . At each sampling point, three 10-g sub-samples were taken for the analysis of Salmonellacounts.

Each 10-g inoculated sand sample was divided into two portions . These two portions were added to two 24-oz filter bags together with 95 mL of PBS in each bag. One bag was stomached for 1 min using a smasher , while the other bag was shaken by hand for 1 min. The contents were then serially diluted appropriately with PBS and two 100 µL suspensions from each dilution were spirally plated onto TSA with rifampicin and Xylose Lysine Tergitol 4 agar with 50 µg/mL rifampicin . After 24 h of incubation at 37 °C, colonies were counted and the populations determined. Statistical Analysis. One trial was conducted for every test performed in this section. At each sampling point, three samples were analyzed . Means comparison were performed using Excel to determine whether carrier type had a significant impact on pH and water activity of dried fruits as well as if homogenization methods for inoculated sand had a significant impact on recovery of pathogenic cells. Differences between mean values were considered significant at P < 0.05.Inoculation with a wet or dry carrier. Table 1.2 shows the pH and water activity of dried fruits before and after the addition of either water as a wet-carrier or sand as a dry carrier. In low-moisture dates, the initial pH was 5.83 ± 0.06. With both wet and dry-inoculation, the change in pH was statically significant, dropping to 5.24 ± 0.05 and 5.59 ± 0.04 respectively. The initial aw of the low-moisture dates was 0.62 ± 0.03 and showed no significant change after either wet or dry-inoculation, with their values being 0.64 ± 0.00 and 0.61 ± 0.00, respectively. In high-moisture dates the initial pH was 5.59 ± 0.04. The pH did not change significantly afterwet-inoculation. The pH dropped after the dry-inoculation to 5.39 ± 0.04. The initial aw of the high-moisture dates was 0.55 ± 0.02. No significant change was observed after either wet or dry inoculation. The initial pH of the dried peaches was 3.94 ± 0.07, and neither the wet nor the dry inoculation generated significant change on the pH. Additional loss of moisture might have occurred during the 48-h of drying after wet inoculation, which might be the reason why there was differences between the aw before and after wet-inoculation. A similar observation was made in dried peaches made with sulfur treatment, as the aw decreased after wet-inoculation. Both the wet- and dry-inoculation slightly reduced the pH value of the products, changing from 3.59 to 3.51 and 3.48 respectively. In dried pluots, although neither wet- nor dry-inoculation generated any impact on aw, dry-inoculation reduced the pH of the products . For sundried tomatoes, inoculation had no impact on pH but the dry inoculation significantly reduced the aw of the products .

In addition to ABA, other hormone biosynthesis genes exhibited a similar expression such as auxin and cytokinin, both of which have been implicated in non-climacteric fruit maturation and ripening . Thus, pistachio should be considered a non-climacteric fruit.While ethylene was not important for ripening in pistachio, ethylene may be more critical prior to kernel development at the end of Stage II where a rise in biosynthesis occurs . The exact function of the hormone at this stage of development is unknown but has previously been suggested to be involved in bud abscission in alternate bearing years and needs further investigation . JA-related genes were also elevated during Stage II and were among the highest expressed hormone-related genes. JA is best known as a stress hormone involved in many responses to abiotic and biotic stress but can also function in fruit development . JA biosynthesis genes were previously shown to be differentially expressed in pistachio vegetative tissues that underwent a salinity treatment, compared to the control . However, there were no known stresses occurring during our samplings to explain elevated expression. This suggests JA is a critical hormone in pistachio fruit Stage II development prior to kernel initiation, or other events occurring concurrently. As pistachio is known to be tolerant to environmental stress, JA and ethylene levels may play a role evolutionarily to adapt to these extreme environments.The hull functions as a protective tissue encapsulating the shell and kernel. The breakdown of the hull caused by senescence can lead to a lower quality commodity; for example, plastic grow bag the shell becomes more vulnerable to staining from the hull and the kernel becomes more accessible to pests. The hull is rich in volatile compounds mainly composed of terpenes . We examined the dynamic changes of volatile compounds in the hull during Stage III and IV to gain insight into the events leading to hull ripening and senescence.

We saw a rise of volatile compounds at the onset of ripening that may offer a signal of developmental changes occurring We observed limonene and alpha-terpinolene to be the highest produced monoterpenes consistently across years. Alpha-terpinolene had also been found in high proportions of Tunisian pistachio variety but did not have as high of concentrations of limonene, which could be due to varietal differences . Limonene has been shown to accumulatein orange peel with ripening in order to attract insects and pathogens . This relationship between limonene and other organisms was proposed to have evolved to facilitate seed dispersal, opening up the fruit to expose the seeds. This could also be the case for pistachio, in which the volatile production at the start of ripening signals that the kernel is mature and attracts seed dispersing organisms. Volatile signals further define the events leading to ripening in pistachio and have additional implications for management practices to time treatments against insects.Like canonical fleshy fruit, pistachio fruit quality is determined during fruit ripening. Ripening changes in the hull coincide with important quality traits and can be used to anticipate the best harvest time. Therefore, understanding the timing and relationship between the hull and kernel during Stage IV allows for increased quality. Our study integrates multiple approaches, including physiology, biochemistry, and genomics, to provide the most thorough understanding of pistachio fruit development to date. Fruit ripening in pistachio to our knowledge has not been previously explored. The hull undergoes changes in composition preceding harvest consistent with fruit ripening including, softening and color change which we define as an additional stage, Stage IV . These changes are important attributes that help determine harvest time and maximize fruit quality. For example, hull softening allows fruits to be detached from the tree, however, if overripe the hull senesces and the degradation can cause shell stain and make kernels vulnerable to pests and disease decreasing its nutritional and market value.

We integrated gene expression data with the observed physical changes to inform the events occurring leading to fruit ripening. Color change is a characteristic of ripening and provides a visual indication of when fruit are ready for harvest. Previous studies have identified anthocyanin, carotenoids, and chlorophyll compounds in pistachio hulls . However, the proportion of these compounds present depended on the stage sampled and variety, with measure-ments limited to pistachio green hulls prior to ripening. Thus, it is unclear which specific compounds lead to the pink colorations in the hull during ripening. Our gene expression analysis indicated that the flavonoid pathways were active in the hull, however there was not a strong expression of anthocyanin reductase genes, the critical final step for anthocyanin production, giving the compounds their pigmentation. It is clear that the fruit produce some anthocyanins because they have been identified in the purple colored seed coat surrounding the kernel . We found stronger gene expression of carotenoid biosynthesis. Among the pigments identified in Grace et al., lutelin was the highest measured in pistachio hulls . We observed high expression of ctrZ which is annotated to act in the step leading to lutein biosynthesis. The change in green coloration to yellow at the end of Stage III also indicates chlorophyll degradation may be occurring. We identified several chlorophyll degradation genes expressed in modules with ripening patterns, such as STAY GREEN , a chlorophyllide reductase that regulates chlorophyll protein degradation . Thus, from our analysis we hypothesize that hull color becomes yellow from chlorophyll degradation and shades of pink from carotenoids. It is well known that fruit softening is mediated by cell wall degrading enzymes in both climacteric and non-climacteric fruits, such as tomato and strawberry . Cell wall degrading enzymes acting on the backbone of pectin molecules, such as polygalacturonase and pectin lyase, are highly expressed in other fruit and exhibit a ripening-specific gene expression pattern . Further, α-LAFase is highly expressed and acts as a catalyst with other coexpressed cell wall degrading enzymes . Thus, we were interested if these enzymes were expressed in a ripening-specific pattern. Pectate lyase was among the highest expressed CWDEs annotated from the CAZy database in pistachio and began to rise in expression at Stage IV .

Consistent with this, pectins have been measured in pistachio hulls and were proposed as a potential source of commercial pectins . The presence of a large proportion of pectins in green hulls and the high expression of pectin-degrading enzymes suggest these enzymes promote pectin degradation and softening of the tissue. The mechanisms involved in this still need to be explored. Overall, knowing how and when hull softening occurs during the growing season can help advise the optimal time for harvest.Kernel growth during Stage III leads to the maturity of the seed and ripening of the hull. Understanding when the kernel is most desirable for consumption and when the fruit is ready for harvest can improve management practices and fruit quality. Maturity can be observed when kernels reach their maximum size and fat content at the start of Stage IV, as ripening progresses . From our gene expression and metabolite data we see that fatty acid biosynthesis occurs early on in kernel development and is primarily composed of unsaturated fatty acids, with much of the production reaching its maximum during ripening . Further, pe grow bag the kernel shows hormonal indications of seed maturity with an increase of GA at the start of Stage IV and ABA increasing throughout Stage IV. Pistachio kernels are consumed for their unique flavor and nutritional benefits. Kernels are made up of primarily unsaturated fats, including both poly- and mono- unsaturated fatty acids . Although PUFAs provide essential fruitrients to the human diet, they make kernels more vulnerable to rancidity, reducing their shelf life . Thus, the ratios of PUFA and MUFA are important for considering nutritional benefits and shelf life. We identified important enzymes in our gene expression data explaining the accumulation of specific unsaturated fats . Interestingly, our study showed fluctuations in the composition of unsaturated fatty acids through kernel development similar to a previous study . The mono-unsaturated fatty acid oleic acid increases through time while the poly-unstaturated linoleic acid decreases. These fluctuations were not completely explainable with our expression data, but are likely caused by other fluxes in the fatty acid metabolism downstream of these compounds.Calcium is an essential plant nutrient required for proper plasma membrane function, in storage organelles to counterbalance anionic charges, in the cytosol for cellular signalling responses, and in the apoplast for cell wall structure . Ca2+ deficiency disorders in fruit have been attributed to lower total tissue Ca2+ content, as well as abnormal regulation of cellular Ca2+ partitioning and distribution . The symptoms of Ca2+ deficiency disorders in fruit start with cell plasmolysis and the water-soaked appearance of blossom-end tissues that eventually becomesdark brown as cells die . Although Ca2+ is believed to move in the plant exclusively through the xylem vascular tissue , the mechanisms regulating Ca2+ partitioning and allocation in tomato plants and fruit remain poorly understood. Consistent with xylem sap flow, the direction and rate of xylemic Ca2+ flow in the plant should be determined by water potential gradients in response to different tissue transpiration and growth rates . In that case, higher transpiration and growth rates can reduce water potential and increase tissue strength as sinks for xylemic Ca2+. Therefore, the partitioning of Ca2+ flowing from the roots toward leaves and fruit will depend on the xylem sap Ca2+ concentration, as well as leaf and fruit transpiration and growth rates. Accordingly, leaves have much higher transpiration rates than fruit, which results in much higher Ca2+ content in the leaves than in the fruit .

Previous studies have shown that specifically reducing leaf transpiration by decreasing atmospheric vapour pressure deficit or treating tomato plants with abscisic acid can potentially decrease xylemic Ca2+ movement into the leaves, and increase its movement into the fruit . However, direct measurements of xylemic Ca2+ concentration and xylem sap flow rates into leaves and fruit in response to reduced leaf transpiration rates have not been reported. Spraying whole plants with ABA increases fruit total tissue and apoplastic Ca2+ concentrations, and reduced fruit cell membrane leakage and the incidence of blossom-end rot . These studies suggest that ABA may affect not only total fruit tissue Ca2+ concentration but also the regulation of cellular Ca2+ distribution, which could affect fruit susceptibility to Ca2+ deficiency disorders such as BER . Since these studies were based on whole-plant ABA sprays, the results cannot be specifically attributed to whole plant or fruit responses to ABA . Fruit-specific ABA studies are still needed to understand if the prevention of BER development is a whole-plant, a fruit specific, or a combination response to ABA. The objectives of this study were to determine Ca2+ partitioning and allocation in tomato plants and fruit in response to whole-plant and fruit-specific ABA treatments, as well as to analyse the effect of changes in Ca2+ partitioning and allocation on fruit susceptibility to BER under water stress conditions. The HRM was developed to measure low net sap flow rates that can take place in either direction in the vascular tissue , but for the current study only the xylem sap flow rate was determined by heat girdling the middle leaf pedicel or fruit peduncle. The heat girdling was accomplished by passing an electrical signal for 20 s across a constant an wire with 0.8mm diameter looped twice around the pedicel or peduncle 1 cm upstream of each heat sensor before starting the sap flow measurements . Heat girdling destroys the phloem cells, obstructing phloem sap movement, while the xylem sap flow remains intact and functional due to its non-living cells. This technique has been used to isolate and quantify phloem and xylem sap flow rates . After heat girdling, sap flow measurements were made over a 24h period. After xylem sap flow measurements, zero sap flow readings were determined by cutting the middle leaf pedicel or fruit peduncle 1 cm downstream of each sensor. The zero xylem sap flow readings were used to determine the baseline accurately for each sap flow sensor after sap flow measurements. After determining the zero sap flow rate, the middle leaf pedicel or fruit peduncle was cut at the heat sensor region to measure the diameter of the xylem vascular tissue, which was used to calculate the volume of xylem sap moving into the leaf and fruit over time. One fully expanded top leaf and one tagged fruit on each plant replication were used for the sap flow analysis.

Fruit are rich in essential nutrients, yet most people do not consume the recommended amount of fresh produce to sustain healthy diets and reduce disease risks ERS — Food Availability and Consumption. To promote consumption, expand access, and reduce waste, fruit quality and shelf life need to be in- creased through breeding and biotechnology, alongside adequate harvest practices, transportation logistics, and post harvest treatments. Commercial fruit crops are primarily bred for high yield and extended shelf life to meet the expectations for mass production and global markets, however, recently, there has been a shift of focus toward developing new crop varieties that meet consumer demands for better flavor and nutrition. Balancing shelf life with consumer-based quality traits is perhaps the biggest challenge breeders and researchers face in the quest for better quality fruit, mainly because these attributes appear to have negative genetic correlations in many crops . Quality peaks when fruit reach their optimum ripeness. Many studies on fruit ripening of various plant species have emerged in the past decade, helping to identify genetic pathways and molecular regulators that can be manipulated for crop improvement . Moreover, biotechnology advances have provided access to high- quality genomic resources and tools, supporting breeding strategies, genetic modification, and gene editing in traditional and nontraditional fruit crops. Here, we review current knowledge of the genetics of fruit traits and argue that manipulating transcription factors is a promising approach to enhance fruit quality. We discuss how pleiotropic effects could potentially be avoided by targeting TFs that exclusively regulate specific pathways instead of master regulators. However, ripening master regulators may remain useful if their effects on gene expression can be fine-tuned . Similarly, plastic square flower bucket the timing and coordination of regulators need to be considered to achieve desired effects on fruit traits .

Finally, we consider the current climate surrounding consumer acceptance of genetically modified and gene-edited fruit.Fruit are used as food source for macronutrients and micronutrients . Macronutrients are essential to provide energy and maintain the body’s structure and functions. Vitamins are required for various bodily functions and can only be found in food sources such as fruit. Antioxidants inhibit cell damage caused by oxidative agents. Carotenoids , and phenolics , reviewed above, are all antioxidants, as well as vitamin C . Vitamin-D deficiency is a global health problem due to few dietary sources of this vitamin. Bio-fortification of vitamin D in tomatoes has recently become possible by engineering its biosynthesis from a pre-existing pathway . Owing to partial duplication of the pathway, a single enzyme could be knocked out with CRISPR–Cas9 to convert the precursor into vitamin D without an expense to other metabolites. This discovery has further implications for other Solanaceae plants. Ascorbic acid, an important antioxidant and nutrient for immune health and wound healing, has proven to be less easily bio-fortified into fruit because increasing biosynthesis also leads to activation of catabolic and recycling pathways . Post transcriptional regulation of an ascorbic acid biosynthesis enzyme from kiwifruit has been demonstrated using tobacco leaves . Removing the upstream open reading frame that repressed translation increased ascorbic acid concentration in the leaves. Function-specific TFs can increase ascorbic acid in fruit without negative impact on quality as demonstrated in tomato. SlHZ24, a bHLH TF, regulates ascorbic acid biosynthesis and catabolism genes, and its transient over expression has been reported to increase the accumulation of this vitamin . Other TFs, SlNL33 and SlNFYA10, have been found to regulate the pathway negatively, and silencing them also increased ascorbic acid level . Understanding regulatory pathways governing nutrients can also facilitate traditional breeding programs. For example, a genome-wide association study led to the discovery and validation that alleles in SlbHLH59 determine ascorbic acid content in tomato cultivars . A less specific effect on nutrient accumulation can be achieved with hormone applications. For example, ethylene application in kiwifruit, a climacteric fruit, increased ascorbic acid and other antioxidants during ripening . Ascorbic acid also increased after nitric oxide application in sweet pepper, a non-climacteric fruit .Fruit flavor is a critical quality trait for consumer acceptance. Flavor includes all sensations experienced when eating, consisting of taste, aroma, and texture . In fruit, taste is mainly defined by a balance between sweetness and acidity but can include bitterness and umami.

Fruit aroma comes from specific classes and combinations of volatile organic compounds . For example, the unique kiwifruit flavor is associated with esters, mainly ethyl butanoate and methyl butanoate . Modulation of function-specific transcriptional and post-transcriptional regulators offers an effective solution for flavor improvement. A bHLH TF in banana activates 11 starch-degrading genes expressed during fruit ripening and is a likely candidate for increasing sugar and sweetness . In strawberry, editing the uORF of a bZIP TF that controls sucrose biosynthesis led to its translational activation and higher sugar content in fruit . Combining datasets generated through genomic, transcriptomic, metabolomic, and consumer panel studies has proven to be an effective strategy for identifying flavor-related genes and TFs. In tomato, metabolite data, associated loci, and consumer panels were analyzed across hundreds of varieties to determine key genes contributing to flavor . Coupling metabolites relevant to flavor with transcriptomic analyses can produce gene networks and identify regulatory TFs involved in flavor pathways. This approach was used in kiwifruit, where the AcNAC4 TF regulating a key gene in ester biosynthesis was validated . Homologs of these NAC TFs have been implicated in ester formation in peach and apple , suggesting that their functions are conserved across diverse families of climacteric fruit . Flavor is impacted by post harvest handling. Chilling during transportation and storage can alter fruit flavor. Epigenetic factors have been shown to regulate the suppression of VOC biosynthesis in chilled tomatoes . Most climacteric fruit are picked unripe and later treated with ethylene to induce ripening. However, this practice has been associated with poor flavor development. A study in off-vine ripened tomatoes confirmed that the fruit presented reduced VOC emission and a low sugar/acid ratio, due to alterations in gene expression and decreased methylation of their promoters . Genomic resources can help anticipate consumer preference and assist in breeding to select fruit with enhanced flavor. A population genomic study revealed that distinct consumer preferences between eastern and western countries drove selection for peach cultivars with different acidity . Fruit VOC profile data can help predict consumer liking before performing sensory panels, allowing for a more efficient selection of high-flavor fruit, as seen in tomato and blueberry . Moreover, the tomato pan-genome helped identify a rare favorable allele selected against during domestication that could be incorporated back into new cultivars to improve fruit flavor .Fruit texture is associated with freshness, flavor, and shelf-life potential. Texture involves many attributes, such as firmness, juiciness, crispiness, and meltiness . Fruit softening is mainly attributed to the remodeling and degradation of the polysaccharides in the primary cell walls .

The cuticle layer, deposited on the CWs of epidermal tissue, also contributes to fruit firmness by preventing water loss and maintaining cell turgor pressure . Traditional breeding has focused on creating firmer fruit that withstand transportation and have longer shelf life. This has been accomplished in tomato by developing hybrid lines between elite varieties and non-ripening mutants such as ripening-inhibitor and non-ripening . These mutants have defects in TFs considered master regulators of many ripening processes,plastic plant pot including the induction of cell wall-degrading enzymes and changes in cuticle composition . Other efforts to improve firmness and shelf life in climacteric fruit have taken advantage of mutations affecting ethylene biosynthesis and perception, as this hormone is also known to regulate genes encoding CWDEs, among others . However, as already discussed, modulating master regulators or hormone pathways has numerous drawbacks to other quality traits, such as color and flavor. Thus, downstream TFs controlling specific CW enzymes, such as LOB TF in tomato, may prove to be better targets . Another possibility is to leverage the availability of natural or induced allelic variants in ripening master regulators to produce a range of fruit phenotypes. For example, spontaneous or equivalent delayed fruit deterioration and CRISPR–Cas9-generated mutations in the ripening regulator NOR can extend shelf life with minimal impact on other fruit attributes , compared with the canonical mutant nor . RNAi and gene editing approaches have been used to target ripening-specific CWDEs that influence fruit firmness, such as polygalacturonase , pectate lyase , and pectin methyl esterase . The first GM fruit product, the FLAVR SAVR™ tomato, was engineered with antisense RNA against SlPG, however, it did not show a phenotype for fruit firmness . In contrast, the CRISPR–Cas9 SlPL knockout in tomato significantly improved fruit firmness and shelf life . Similar observations were previously reported in strawberry using RNAi knockdowns of FvPL and FvPME. In addition to firmer fruit, tomato and strawberry SlPL mutants have reduced fruit susceptibility to fungal disease . Breeding strategies and molecular studies have also focused on ameliorating textural defects such as fruit mealiness caused by cold storage. Mealiness, considered the opposite of juiciness, occurs when neighboring cells lose adhesion and detach while remaining intact . In peach, quantitative trait loci associated with cold-tolerant varieties have been identified to support breeding for less mealy fruit . Beyond QTLs, understanding the genetic mechanisms behind the trait provides avenues for targeting breeding and genetic modifications. For instance, peach mealiness appears to be associated with increased DNA methylation, leading to the downregulation and hypermethylation of mealy-associated genes such as PpCYP82A3 . Finding molecular approaches to avoid the deposition of methyl groups in the promoters of key ripening genes in response to cold storage can serve as a potential solution to mealiness.The FLAVR SAVR™ tomato hit the market in the early 90s promising a product with longer shelf life. However, this GM fruit had high production costs and was not well accepted by consumers, which led to its removal from the marketplace . Since then, other bioengineered crops with improved plant disease resistance or production-related traits have become available worldwide . These products have not sparked much enthusiasm mainly because they were not generated considering consumer-based traits or due to public fear of GM organisms. Recently, two fruits bio-engineered for quality attributes were approved by both US and Canadian regulatory entities and are available to consumers. These are the Pinkglow™ pineapple and the Arctic™ apple. The latter was bioengineering to reduce oxidative browning in the cut fruit . These fruit are considered novelty items because they are less available in the marketplace and significantly more expensive than traditional cultivars. Both fruits are primarily sold online and offered in limited supplies. The Pinkglow™ pineapple costs nearly ten times more than a common yellow pineapple. Despite their limitations, these fruits were developed with the consumer in mind, which may entice the public more than previous GM products.In 2022, the USDA deregulated the purple tomato developed with the expression of two snapdragon TFs. Novel fruit colors and potential higher nutrition may draw consumers to a new emerging category of bio-engineered produce. These fruit will also need to meet high consumer expectations of flavor, affordability, and food safety to ensure their success. Gene editing techniques such as CRISPR–Cas9 enable the fine-tuning of quality traits in a variety of fruit crops and may be more well received than previous GM products. For example, Japan started selling the first Cas9-edited fruit in the world in 2021, a health-promoting γ-aminobutyric acid -enriched tomato . Gene-edited fruit without foreign DNA have a more straightforward regulatory path in the United States than GM products, increasing the speed to market and reducing costs associated with authorization. However, this is not the case in other parts of the world, such as the European Union. In conclusion, researchers are armed with knowledge on fruit ripening and tools to improve fruit quality and generate greater access to fresh, flavorful, and nutritious food. Demands for better-tasting, more sustainable fruit, are in reach . We sit on the edge of an era where gene-edited and bio-engineered commodities can become a new category in the market, if the consumer will allow it.Fleshy fruit gain most of their quality traits, such as color, texture, flavor, and nutritional value, as a result of physiological and biochemical changes associated with ripening.

Firms that had made investments that led to higher worker productivity should have wanted to advertise that information in order to convince workers that they should accept a lower piece rate, while firms that had not should have wanted to hide the fact. Newman and Jarvis also found that piece rates were adjusted in many packing sheds if the quantity and/or quality of the grapes being processed changed. Although strikes were legally prohibited, female workers were able to “stop” production and successfully negotiate an increase in the piece rate, and the reverse was also true. Indeed, several male shed managers said that they found it easier to negotiate a “fair” piece rate with women than to negotiate pay with men. The evidence in Newman and Jarvis suggested that women workers were both aware of the importance of firm-influenced productivity differences and able to obtain information about what firms actually provided. Thus, there was evidence that female workers operated within and were part of a highly sophisticated labor market in which firms and workers obtained and used information regarding about their heterogeneity. Neither workers nor firms thought that firms or workers were homogeneous.Each worker was asked how many months he/she would have liked to work during 1991 and 1992. The mean response by females was 11.1 months and 9.7 months, respectively. The responses were surprisingly high, round flower buckets given that most women had worked much less than this in 1991. The question did not specify a wage at which they would be employed, but the responses suggested that most women wanted to work most of the year.

To further explore workers’ preferences for seasonal as opposed to permanent employment, including a specified wage, each worker was asked to indicate her/his preference for one of three employment options: 1) 3 months employment per year at100,000 pesos per month, 2) 6 months employment at 60,000 pesos per month, and 3) 12 months employment at 35,000 pesos per month. Option 2) required twice the time to obtain an additional 60,000 pesos of annual income, as did option 3). When the options were designed, it was believed that most women preferred to work only the summer months and thus would choose the first option. In fact, 50% of workers chose permanent employment as their preferred option and an additional 25% choose the 6-month option. Only 25% choose the 3-month high-salary option, even though its 300,000-peso salary turned out to significantly exceed the mean annual earnings of workers. What do these answers mean? We believe, based on respondents’ anecdotal comments, that most female temporary agricultural laborers want to work most of the year. First, most women wanted to earn more, even if the marginal earnings associated with longer employment were low. Second, some women expressed a desire to spread their income throughout the year and thought that a permanent salary would help achieve this. They commented on how hard it was for them to adequately carry funds from one month to the next given a lack of accessible savings institutions1 and constant pressure from friends and relatives for loans and assistance. Third, many women said that they enjoyed working, particularly as it allowed them to feel productive, benefit economically, and have greater social contact with others. When asked what they liked about their work, some women responded simply that they enjoyed it. However, most provided additional insight into what the work experience meant for them. Some responded that work allowed them to financially help their families, others that they enjoyed socializing with other women, having an opportunity to talk about their problems, possible solutions, and simply their concerns, and to form personal connections that they could not otherwise make.

Others were glad simply to feel useful. Having said that women wanted additional employment, they were not idle. Combining employment and family responsibilities placed a harsh demand on women’s time and energy. Their summer work schedule was especially grueling. They worked very long hours. Processing sheds began work at 2:00 p.m. and continued until all of the grapes picked had been packed. On average, cleaners worked 9.6 hours per day and packers 10.3 hours, not counting time waiting for the sheds to open, or for coffee breaks and dinner. Work commonly finished well after midnight and, during the seasonal peak, as late as 6:00 a.m. Many females liked beginning work in the early afternoon because it gave them time to take care of family responsibilities in the morning. Women also mentioned the burden that they faced in meeting these dual responsibilities. Some women indicated that their husbands did not want them to work and a few said they had obtained agreement to work only during peak season when earnings were high. However, more women indicated that their husbands appreciated their income, recognized that it improved family welfare, and supported their working. A few women said that their husbands had accepted some household tasks to help. All of the women interviewed worked at a task that was paid on a piece rate basis most of the time. Despite the increased pressure and expenditure of effort that piece rate work required, when asked their preference for summer work, 58% of workers expressed a preference for piece rate pay when working in a packing shed during the summer. The overwhelming reason for preferring piece rate pay was that it allowed the worker to earn more. Nonetheless, a significant proportion of these workers would have preferred a salaried job during the summer and most did not want piece rate employment on a year round basis. Indeed, 61% of workers preferred a fixed wage if they could get a year around job. Roughly half said they did not want to work at the intense pace required by piece rate work on a continuing basis. Others commented that they felt that they would earn more if working for a wage, including some who said that they would earn a higher rate for overtime, while others said their earnings would be more stable. The workers interviewed understood the advantages and disadvantages of their various employment options. Their decision to accept piece rate work depended partly on a lack of other remunerative employment during the rest of the year. Their relative poverty contributed to their desire to work hard when an employment opportunity presented itself. One might expect the supply of labor for temporary agricultural work on a piece rate basis will decrease if and when other employment opportunities materialize.Many women entered the labor force to supplement the income of other family members and/or to satisfy their own special needs. However, a majority of women in our data set were the major income earner in their household. Approximately 180 women lived in households that contained no male workers. Another 25% of women were the primary earner in their households even though their households included an adult male who worked at least part of the time. We lacked data to test whether women’s acceptance of temporary work in the fruit sector had improved household income. We only observed the households in which female workers resided, not the households they resided in prior to obtaining employment, plastic flower buckets wholesale and we had no information on rural families that did not have any members working in the fruit sector. It is nonetheless instructive to see how the income earned by women in our sample affected their current households.It is often tempting, after eating a particularly good orange or avocado, to plant the seed and grow your own tree full of these delicious fruit. Trees grown from these seed, however, may produce fruit that are not edible at all, or the trees may not bear fruit for many years.

The best way to produce good-quality fruit is to grow seedlings from them and then attach, by budding or grafting, material from trees that are known to be good producers. Budding and grafting can also be used to change or add varieties to mature citrus or avocado trees, a process known as top working. This publication is a brief introduction to budding and grafting for the home gardener. For more information, consult the materials listed at the end of this publication or contact your local Cooperative Extension office.To germinate citrus or avocado seed, plant them in a shallow container such as a nursery flat or a pan with drainage holes in well-drained commercial potting mix. Plant the seed two to three times deeper than their length. For example, a citrus seed about 1 ⁄4 inch long should be planted about 1 ⁄2 to 3 ⁄4 inch deep. Keep the seed in a warm place—between 70° and 80°F —and keep the soil moist. Covering the nursery flats with clear glass or plastic will help maintain the proper humidity. Avocado seed can also be germinated by suspending them in water. Place toothpicks horizontally into the seed near the top. Suspend the wide end of the seed in a small container of water with the toothpicks resting on the edge of the container. Place it in indirect light and refresh the water at least weekly. After germination , replant the seedlings into a larger container of good-quality commercial potting mix. Good choices for containers include a cardboard milk carton cut horizontally in half or a one-gallon can. Punch drain holes in the bottom of the container. The seedling will be ready for budding or grafting when it has grown to 24 to 30 inches tall.Budding and grafting are vegetative propagation techniques in which a single bud or stem of a desired plant is attached to a rootstock plant. In budding, a single bud with its accompanying bark is used as the scion. In grafting, part of a stem or branch is used as the scion. One of the most important keys to successful budding and grafting is properly positioning the scion on the rootstock. In order for the scion and rootstock to grow together, the thin greenish plant layer just under the bark of the scion and rootstock must be aligned so that they touch each other. If they do not touch each other, the bud or graft will fail. Within 10 to 15 days, a successful bud or graft forms a hard whitish tissue where the two cambium layers grow together. Always use sharp cutting or grafting instruments and make clean, even cuts. Options include a budding knife, a sharp kitchen knife, or a single-sided razor blade. Do not allow the cut surfaces of the scion or rootstock to dry out: immerse cut scions in a pail of water, wrap them in plastic, or graft them immediately after cutting. Also, remove any leaves from scions after cutting to help keep the scions from losing water. Keep the scions in a cool place during the work.Budding and grafting are best done in the spring or fall when the bark is easily separated from the wood. It should be timed to be early enough so that warm weather will help ensure a good bud union, yet late enough so that the bud will not begin to grow and callus will not grow over the bud itself. Citrus budded or grafted in the fall must be protected from frost. Avocados are best grafted in the spring when the bark is easily separated from the wood.Budding is the standard method used to propagate citrus. Aside from being the easiest method, it allows a large number of plants to be propagated from a small amount of scion wood and is suitable for trees, rootstocks, or branches from 1 ⁄4 to 1 inch in diameter. Budwood should be taken only from high-producing, disease-free trees . The best citrus budwood is located just below the most recent flush of new growth; the best avocado budwood is located near the terminal end of shoots that have fully matured, leathery leaves.Cultivated alfalfa is a major forage crop that plays a key role in global livestock production. Alfalfa hay is a relatively cheap, high-quality forage which is high in protein and thus an ideal feed for ruminant animals . Often referred to as the ‘Queen of forages’, alfalfa is one of California’s most important crops, driving production of the state’s most significant agricultural enterprise: dairy . In 2022, 200,000 hectares of alfalfa were harvested for hay and haylage with a total value of over $1B .