Tag Archives: hydroponic

The chamber was positioned on terminal leaflets such that the midvein was not within the measured area

AtbZIP11 transcript levels are upregulated by both light and sugars, which contribute to photoassimilates availability as a result of photosynthesis. When carbohydratesupply is sufficient, sucrose-mediated repression of AtbZIP11 translation would be initiated. In this way, AtbZIP11 activity could keep carbohydrate homeostasis in plant . In contrast, the expression levels of AtbZIP1 and AtbZIP53 were induced after extended night treatment and repressed by sugars application . In strawberry, bZIP11 was induced by red and blue light, while bZIP53 homologue was depressed. In addition, sucrose treatment did not significantly affect bZIP53 at transcriptional level . These findings indicated that bZIP S1 members differentiated in response to some factors. All of bZIP S1 transcriptional factors have the SIRT-responsive uORFs, so researchers proposed a novel SIRT-bZIP technology to enhance sweetness especially for some plant species rich in sucrose . In our study, strawberry bZIP11 overexpressing in tomato indeed increased the TSS and SS content and SS/TA ratio, which provided an applicable method for improvement of strawberry and other fruit quality in the future. However, constitutive overexpression of FvbZIP11 caused a growth impairment, which have been observed in tobacco , Arabidopsis , banana . An explanation of this phenotype was that bZIP11 presumably severely affected carbohydrate partitioning via a mechanism that might include direct regulation to cell-wall invertase and sucrose transporter expression . To avoid growth impairment, Sagor et al. overexpressed SlbZIP1 and SlbZIP2 under the control of the fruit-specific E8 promoter. The growth and morphology of the resulting transgenic tomato plants were comparable to those of wildtype plants. Most fruit-specific promoters currently available have been isolated from tomato, but these promoters probably are inappropriate to be used in non-climacteric fruits, like strawberry . Hence, round planter pot identification of a suitable promoter could facilitate the function analysis of a specific gene involving fruit development and help to specifically improve fruit quality.

The rise of agriculture c. 7000 BC ensured a stable food supply, allowing human civilizations to develop and populations to grow . The challenge of feeding a growing population is exacerbated by climate unpredictability, with drought and temperature increases, leading to decreased crop yield . Tomato is by far the most widely grown vegetable crop worldwide . The narrow genetic base of most crops, combined with selection for performance under optimal conditions, has reduced the genetic variability in environmental stress responses, and the modern cultivars of tomato are no exception . The wild relatives of tomato have the genetic ability to adapt to extreme habitats, and many heirloom cultivars also retain this ability as a result of directed breeding with wild species, and less selection for commercially valuable traits . Heirloom tomatoes are defined as varieties, which have been passed down through multiple generations of a family . Improvement in tomato has focused on flowering, fruit traits, and disease resistance probably as a result of a perceived negative correlation between fruit size and sugar content . Thus, potential impacts of other factors on yield and fruit quality are relatively ignored . In a previous study by Chitwood et al. , a meta-analysis on a set of introgression lines linked leaf complexity and leaflet shape in tomato to fruit sugar content measured on the same lines by other researchers . This correlation showed that plants with complex and rounder leaflets also had increased fruit sugar content . Because leaves are the primary site of photosynthesis, it is possible that leaf shape changes may impact photosynthetic capacity and therefore result in different sugar content and yield in fruits. In addition to photosynthesis, sugar transport, and distribution to sinks are other potential sites of regulation in leaf function as source tissue. While sugar transport in plants is well described, distribution among different sink tissues is not fully understood .

We analyzed tomato cultivars with varied yield and fruit quality, photosynthetic capacity, leaflet shape, and other vegetative traits and found that leaflet shape was strongly correlated to overall fruit quality assessed as a composite measure of BRIX and yield , with rounder leaflets positively correlated with higher BY values. Photosynthesis, on the other hand, had a negative correlation with yield. Based on our analysis, leaf shape seems to play an important role in the distribution of photo assimilates. Additionally, we performed phylogenetic network analysis on 23 cultivars, including eight identified as having the rounder Potato Leaf Morph , known to be caused by a mutation in the C-locus , to determine their breeding histories and identify any potential selection for this trait.Eighteen heirloom tomato varieties identified as having a range of fruit types, including cherry and beefsteak tomatoes, and several intermediate types, were analyzed. These tomato varieties also differed in fruit production timing from early to late, and the type of leaf morphology. These cultivars were selected based on leaf shape as described in Tatiana’s TOMATO base and The Heirloom Tomato . Tomato seeds were treated, germinated, and field planted as previously described . In both the 2014 and 2015 seasons, plants were laid out in a randomized block design and were planted and grown in soil, with furrow irrigation once weekly.Gas exchange measurements were done in the field on attached leaves after the plants had recovered from transplanting. Measurements were made weekly from week 10 to week 15 , on week 17 , and weeks 18– 21 , on c. 60 plants each week, on three plants per cultivar wk–1 . Measurements were made on leaves from the upper and lower portions of the plants to eliminate positional bias within the plant, and measured for three leaves per plant. The A , gst , transpiration, and ɸPS2 of a 6 cm2 area of the leaflet were measured using the LI-6400 XT infrared gas exchange system , and a fluorescence head .

Light within the chamber was provided by the fluorescence head at 1500 µmol m2 s 1 photosynthetically active radiation , and the chamber air flow volume was 400 µmols s 1 with the chamber atmosphere mixed by a fan. CO2 concentration within the chamber was set at 400 µmols mol 1 . Humidity, leaf and chamber temperature were allowed to adjust to ambient conditions; however, the chamber block temperature was not allowed to exceed 36°C. Measured leaflets were allowed to equilibrate for 2–3 min before measurements were taken, allowing sufficient time for photosynthetic rates to stabilize with only marginal variation. The amount of intercepted PAR was measured in four orientations per plant and an average PARi calculated. PARi was measured by placing a Line Quantum Sensor onto a base made from ¼” PVC piping, and a Quantum Sensor approximately 1 m above the plant on the PVC rig. Measurements from both sensors were taken simultaneously for each sample using a Light Sensor Logger . This allowed variation in overall light intensities such as cloud movement to be measured and accounted for in the total PARi.After gas exchange measurements, three plants per cultivar were destructively harvested each week. The final yield and fresh vegetative weight of each plant harvested was measured using a hanging scale in the field. Five leaves were collected at random from the bottom and top of the plant to capture all canopy levels, round pot for plants and approximately nine fruit were collected for BRIX measurements. FW was used owing to the large number of plants and measurements being done in situ in the field setting. All measurements were made in kg. To measure the BRIX value of the tomatoes, the collected fruit was taken to the laboratory where the juice was collected and measured on a refractometer . The yield and BRIX for each plant were multiplied together to get the BRIX 9 yield index , which gives an overall fruit quality measure, accounting for variations and extreme values in either measurement. It should be noted that while BRIX is used as a standard quality measure, BY is a composite value that folds in yield to assess weight of soluble solids per plant and is being used to measure commercial quality and not consumer quality . BY measurements were done for both the 2014 and the more detailed 2015 fields. These data were compared to test for reproducibility of results .The leaf complexity measures included all leaflets present on the leaf. Subsequently, primary leaflets were used for imaging and analysis of shape and size as previously described , and the images then processed in IMAGEJ . The images were cropped to individual leaflets maintaining the exact pixel ratio of the original image, and then cropped again to only include the single leaflet using a custom Java script written for FIJI . Single leaflet images converted to a binary image as black on a white background, and smoothed to allow for the exclusion of any particulates in the image were then processed in R using MOMOCS, a shape analysis package. Leaflet images were imported and then aligned along their axes so that all images faced the same direction. They were then processed using elliptical Fourier analysis based on the calculated number of harmonics from the MOMOCS package. Principal component analysis was performed on the resulting eFourier analysis and the principal components were used for subsequent analysis. Traditional shape measures such as leaflet area, circularity, solidity, and roundness were done with the area measurement based on pixel density. These measures were compared with the PCs to determine the characteristics captured by each PC. The PC values were used for all subsequent leaflet shape and size analyses. Total leaf area for each plant was measured by imaging the whole plant and a 4 cm2 red square and then processed in the EASY LEAF AREA software .Five plants per line were used to analyze leaflet sugar content. The plants were grown under the same conditions as field plants with the following exceptions. Plants remained in the glasshouse after transfer to 1 gallon pots. All plants were watered with nutrient solution and grown until mature leaves could be sampled. Using a hole punch, a disk with an area of 0.28 cm2 was taken from the leaflets and extracted from the disks using a modi- fied extraction method from the Ainsworth laboratory . Leaf disks were placed in 2 mM HEPES in 80% EtOH and heated to 80°C for 20 min and the liquid collected and stored at placed in 2 mM HEPES in 50% EtOH and heated, collecting the 20°C. The entire process was repeated twice. They were then liquid and storing at 20°C followed by another 2 mM HEPES in 80% treatment. The collected liquid was then used to measure the amount of sugar present per area of disk. To measure leaf sugar content a working solution of 100 mM HEPES , 6.3 mM MgCl2 , and 3 mM ATP and NADP at pH 7 was prepared. From the working solution, an assay buffer was made adding 50 U of glucose-6-phosphate dehydrogenase , and 295 or 280 µl of the working solution was added to a 96-well plate for sucrose standards or samples, respectively. Standards were added at a 60-fold dilution and samples were added at a 15-fold dilution. Then 0.5 U of hexokinase , 0.21 U of phosphoglucoisomerase , and 20 U of invertase were added to each well and the plates allowed to sit overnight to reach equilibrium. The plates were measured on a UV spectrometer at 340 nm, followed by analysis in JMP .All statistical analyses were performed using JMP software. To determine statistical significance, measurements were modeled using general linear regression model and tested by a one-way ANOVA followed by Tukey’s honestly significant difference, if necessary. These modeled data for all measured values were compiled into a table and used to create a model using partial least-squares path modeling in SMARTPLS 3.0 . Modeled data were used for the statistical analyses as many measurement types varied in number of data points, and therefore a set of generated predicted values of equal size was used to make an equal data matrix . Partial least squares-PM was used to explore the cause-and-effect relationships between the measured variables through latent values. PLS-PM is effective in both exploring unknown relationships and combining large-scale data, such as field, physiological, and morphological data, that otherwise are not well described together .

Previous reports have shown ethylene levels to be very low or even undetectable in the ripening mutants

Our analysis of ripening-related gene expression in Cnr showed striking similarities to WT in the number and functions of genes changing between stages. Moreover, 69.5% of ripening related DEGs in Cnr were shared with WT . These results further support the hypothesis that Cnr is not exclusively a ripening mutant. Instead, Cnr fruit undergoes gene expression changes consistent with WT “ripening.” However, the ripening related changes in gene expression that occur in Cnr are not enough to compensate for the large defects accumulated in the fruit during growth and maturation. In a recent report, a knockout mutation to the gene body of CNR yielded little visible effects on fruit development and ripening , which suggests that the Cnr mutant phenotype may result from more than just a reduced expression of the CNR gene as previously reported . It has also been demonstrated that Cnr fruit have genome-wide methylation changes that inhibit ripening-related gene expression . The developmental defects observed in Cnr are likely caused by these methylation changes, directly or indirectly caused by the Cnr mutation . Thus, to better understand the Cnr mutation, more physiological data at earlier stages of development needs to be analyzed and complemented with more in-depth functional analysis of gene expression alterations at the corresponding stages. In addition, further molecular and genetic studies need to be performed and compared against complete CNR knockout mutants. Our data support that the mutants never produce a burst in ethylene production, even at the OR stage where more ripening phenotypes are observed . The orange-red pigmentation in nor OR fruit and the similarities of rin OR fruit in texture and taste-related attributes to WT RR fruit occur independently of an ethylene burst. These observations evidence that other regulatory mechanisms exist to initiate ripening events outside of ethylene .

Unlike previous reports, our data consistently showed that Cnr presented increased ethylene levels at the MG stage compared to WT . Interestingly, Cnr fruit produced more of the ethylene precursor ACC than WT at the RR stage. Also, rin made equivalent levels to WT fruit. Ethylene biosynthesis is divided into two programs: System 1 produces basal levels of the hormone during development, black plastic plant pots and System 2 generates the climacteric rise in ethylene during ripening . Each of these systems is catalyzed by a different set of ethylene biosynthetic enzymes . It is clear that all mutants show defects to System 2 of ethylene biosynthesis, but they also appear to have alterations specific to System 1. For example, we observed that SlACO3, a System 1- specific ACC oxidase, was higher expressed in Cnr fruit than WT .The role of ABA in climacteric ripening is not as well explored but has been reported to be complementary to ethylene . Previous reports in WT fruit have shown that ABA increases until the breaker stage, just before the ethylene burst . ABA has also been shown to induce ethylene production and linked to the NOR transcription factor . We found that nor and rin fruit did not show decreases in ABA concentration during ripening like WT did . For nor, the constant levels of ABA between MG and RR stages are another example of how fruit ripening events are delayed or inhibited. RIN and ABA have been demonstrated to have an inverse relationship where RIN expression is repressed with the induction of ABA . The significant increase of ABA accumulation in rin during ripening suggests that ABA biosynthesis and metabolism are misregulated in this mutant. rin fruit appear to present a delayed peak in ABA levels compared to WT fruit. Our results support the indirect interaction between the TFs and ABA during ripening. More developmental stages, genetic manipulations, and exogenous hormone treatments are needed to investigate further the trends of ABA accumulation seen in the ripening mutants.

The interactions between the CNR, NOR, and RIN in ripening have been debated in the literature . The TF RIN directly interacts with NOR and CNR, binding to their respective promoters, and therefore has been proposed to be the most upstream TF among the three regulators . Here we provided evidence that the three TFs display at least indirect effects on each other. We have argued that the Cnr mutant shows a wide breadth of defects across fruit development before ripening begins, and thus, we propose the Cnr mutation is acting before NOR or RIN. This further supports the hypothesis made in Wang et al. that Cnr acts epistatically to nor and rin. The gene expression patterns of CNR, NOR, and RIN across ripening stages were decreased or delayed in each of the single ripening mutants. The most substantial variation in gene expression was the downregulation of NOR and RIN expression across all stages in the Cnr mutant . We present for the first time double ripening mutants, homozygous for both loci, that can be used to see the combined effects of each mutation on fruit development and quality traits. We successfully generated the double mutants by establishing reliable and high throughput genotyping protocols for each mutation and evaluating segregation of the mutant phenotypes in field trials across multiple growing seasons. We obtained double mutants from both reciprocal crosses but saw no fruit phenotypic differences between them, suggesting that the ripening mutations are not influenced by maternal or paternal effects . Because the nor and rin mutants look so similar, it was hard to visually determine the individual effects of each mutation on the appearance of rin/nor fruit. However, when specific fruit traits were measured, we could detect additive or intermediate fruit phenotypes in this double mutant, supporting the proposed relationship in Wang et al. . Thus, nor and rin appear to influence similar fruit traits and act in coordination.

The Cnr mutation had a significant effect on the Cnr/nor and Cnr/rin mutants resulting in fruit with similar appearance and ethylene production to the Cnr fruit . When analyzing the gene expression profiles of the Cnr/nor fruit, we also observed multiple similarities to the Cnr parent, but also several deviations . Surprisingly, Cnr/nor was also reminiscent of nor, as it displayed few ripening-related gene expression changes, suggesting the inhibition or delay of specific ripening events in nor carried over to the double mutant. Here, we proposed that the Cnr mutation causes defects throughout fruit development while the nor mutation causes defects predominantly in ripening. However, the Cnr/nor double mutant showed additional phenotypic and transcriptional defects before ripening than both mutant parents . These observations indicate that in combination with Cnr, nor may contribute to alterations in early fruit development and the inhibition of ripening progression.Fruit breeders actively selected several morphological and quality phenotypes during the domestication of the garden strawberry , an allo-octoploid of hybrid origin. F. × ananassa was created in the early 1700s by interspecific hybridization between ecotypes of wild octoploid species , multiple subsequent introgressions of genetic diversity from F. virginiana and F. chiloensis subspecies in subsequent generations, and arti-ficial selection for horticulturally important traits among interspecific hybrid descendants. Domestication and breeding have altered the fruit morphology, development, and metabolome of the garden strawberry, distancing modern cultivars from their wild progenitors. Approximately 300 years of breeding in the admixed hybrid population has led to the emergence of high yielding cultivars with large, firm, visually appealing, long shelf life fruit that can withstand the rigors of harvest, handling, storage, and long-distance shipping. Fruit shape is an essential trait of agricultural products, particularly those of specialty crops, owing to perceived and realized relationships with the quality and value of the products. Image-based fruit phenotyping has the potential to increase scope, throughput, and accuracy in quantitative genetic studies by reducing the effects of user bias, enabling the analysis of larger sample sizes, and more accurate partitioning of genetic variance from environments, management, and other non-genetic sources of variation. Many fruit phenotyping approaches rely on the human eye to sort fruit into discrete, descriptive categories for planar shapes. Categories are either nominal, existing in name only, or ordinal, referring to a position in an ordered series or on a gradient. Classification into categories is often labor-intensive and prone to human bias, black plastic garden pots which can increase with task complexity and time requirements. Alternative scoring approaches rely on morphometrics and machine learning to automate classification; e.g., sorting fruit into shape categories in both tomato and strawberry. Unsupervised machine learning methods , unlike supervised methods, are useful for pattern detection and clustering, while supervised machine learning methods are useful for prediction and classification.

Unsupervised clustering enables the calculation of several measures of model performance and overfitting to balance compression and accuracy. However, the categories derived from these techniques are without order, resulting in the need for a suitable transformation to an ordinal scale more appropriate for quantitative genetic analyses. In this context, ordinal categories give the interpretation of relationship with, or distance from, other shape categories in a series. To enable this interpretation, we developed a method for asserting the progression through fruit shape categories derived from unsupervised machine learning methods. The Principal Progression of k Clusters allowed us to nonarbitrarily determine the appropriate shape gradient for statistical analyses using empirical data. The advantages of PPKC, relative to a manually determined ordinal scale, are that it does not require arbitrary, a priori decisions and is unsupervised, which obviates additional operator bias. Here, we describe approaches for translating digital images of strawberries into computationally defined phenotypic variables for identifying and classifying fruit shapes. Fruit shape and anatomy are complex, multi-dimensional, and, potentially, abstract phenotypes that are often not completely or intuitively described by planar descriptors and individual qualitative or quantitative variables. Beyond the qualitative definitions used in plant systematics, references to fruit shape encompass a wide variety of mathematical parameters and geometric indices that establish quantitative measurements of plant organs . Much like human faces or grain yield, fruit shape and anatomy are products of the underlying genetic and non-genetic determinants of phenotypic variability in a population. Quantitative phenotypic measurements have allowed researchers to uncover some of the genetic basis of fruit shape in tomato, pepper, pear, melon, potato, and strawberry. However, the major genetic determinants of fruit shape remain unclear, or understudied, in octoploid strawberry, in part because researchers have not yet translated fruit shape attributes into holistic, quantitative variables, which may empower the identification of underlying genes or quantitative trait loci through genome-wide association studies and other quantitative genetic approaches. Quantitative features often rely on linear metrics of distance and are generally modified into compound descriptors that remove the effects of size. However, compound linear descriptors often have limited resolution compared to more comprehensive, multivariate descriptors. Elliptical Fourier analysis quantifies fruit shape from a closed outline by converting a closed contour into a weighted sum of harmonic functions . Generalized Procrustes analysis quantifies the distance between sets of biologically homologous, or mathematically similar, landmarks on the surface of an object. Fruit shape can also be described using linear combinations of pixel intensities from digital images extrapolating from analyses generally used to quantify color patterns and facial recognition. Similar pixel-based descriptors have recently been referred to as ”latent space phenotypes” and arise from unsupervised analyses that allow a computer to produce novel, independently distributed features directly from images. Here, we generate a dictionary of 68 quantitative features, including linear-, outline-, landmark-, and pixel-based descriptors to investigate the quality of different features in preparation for quantitative genetic analyses. The ultimate goal of our study was to develop heritable phenotypic variables for describing fruit shape, which could then be used to identify the genetic factors underlying phenotypic differences in fruit shape. The phenotyping and analytic workflow for this study are summarized in Figs 1 and 2. We first describe and demonstrate the application of PPKC, which transforms categories discovered from unsupervised machine learning methods to a more convenient and analytically tractable ordinal scale. We then explore the relationship between machine acquired categories and 68 quantitative features extracted from digital images. Next, we apply random forest regression to select critical sets of quantitative features for classification and use supervised machine learning methods, including support vector regression and linear discriminant analysis , to determine the accuracy of shape classification.

Strawberry also shares common volatiles with a variety of fruit crops

Finetuning of metabolomic traits such as amylose content in rice and sugar content in wild strawberry recently were made possible via CRISPR-Cas9 gene-editing technology. Similar approaches can be taken in cultivated strawberry for flavor improvement, but not before the biosynthetic genes responsible for metabolites production and their regulatory elements are identified. Our pipeline has proven to be effective in identification of novel causal mutations for flavor genes responsible for natural variation in volatile content and can be further applied to various metabolomic and morphological aspects of strawberry fruit such as anthocyanin biosynthesis , sugar content and fruit firmness. These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Specific esters are shared with apple , certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, plastic grow pots challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication .

Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits.

Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.In a fruit tree orchard system, individual trees are composed of two genetically different genotypes, one being the rootstock which includes the mass of the tree below the soil surface to a graft union about midway up the trunk. Rootstocks can be selected for pest resistance or tolerance towards adverse soil conditions, and they can also influence vigor and cropping . The second portion of the tree is referred to as the scion and accounts for most of the above-ground mass, usually chosen for fruit production traits . Over the last 40+ years, the University of California has had a peach rootstock development program that has identified several promising size controlling rootstocks which allow for the establishment of new commercially viable orchard systems . New dwarfing rootstocks for peach must be graft compatible, reduce vigor, and not diminish marketable fruit production by reducing fruit size or quality . Previous peach rootstock trials monitored vigor control and grafting compatibility in conventional planting systems however, yield parameters such as fruit size and quantity have not been as thoroughly evaluated using these rootstocks in pedestrian orchard systems . Fruit size is paramount in peach production as larger fruit, free of cosmetic imperfections, have a higher market demand and therefore higher market value . It has been reported that peach fruit produced on trees with size-controlling rootstocks can tend to be smaller in size than fruit on trees with more vigorous rootstocks .Vascular tissue known as xylem is responsible for the movement of water and nutrients in all trees. In trees, every year a new ring of xylem forms surrounding the previous year’s growth and water conduction in the xylem often occurs only in this outermost annual ring . It has been reported that size-controlling peach rootstocks contain a higher proportion of narrow diameter xylem vessels and fewer larger vessels when compared to more vigorous rootstocks in addition to having an increased axial diameter .

Both characteristics create a reduction of hydraulic conductance in the size-controlling peach rootstocks compared to traditional, vigorous rootstocks. Reduced hydraulic conductance, as demonstrated by and , can cause reductions in stem water potential during mid-day hours that can lead to a reduction in vegetative growth .An mean peach fruit’s fresh weight is composed of over 80% water . Thus, it is reasonable to assume a reduced hydraulic conductance created by size controlling rootstocks could hinder fruit size. However, the relationship between fruit growth and water availability is dynamic and depends on the developmental stage of the fruit, the severity of water limitations, and the component of growth being considered . It has been reported that mild water stress applied during the intermediate developmental period of slow fruit growth has no effect on crop yields but significantly reduces vegetative growth in peach . Fruit developmental stages may differ in time of initiation and duration among peach varieties, an example of this would be an early vs. late harvested cultivar as demonstrated by . Fruit growth occurs in stages from fruit set to harvest, in all cultivars, and during the final growth phase of peach fruit is when 65% of a fruit’s dry weight and 80% of a fruit’s fresh weight are accumulated . Available water varies throughout the growing season, including diurnal fluctuations brought on by daily temperature fluctuations , day-to-day changes brought on by a shift in evapotranspiration , and possible seasonal changes brought on by the formation of new xylem . Water conduction in the tree is largely dependent on newly formed xylem each spring and the new xylem cells are smaller in size-controlling rootstocks. It is thought that the spring flush of vegetative growth is limited in trees on size controlling rootstocks compared to growth on vigorous rootstocks because of temporary reductions in root hydraulic conductance caused by smaller xylem vessels. A question that arises from these findings, does the reduction of water conductance in dwarfed peach trees also limit fruit growth?In peach production, fruit size is often manipulated with the use of a management practice known as fruit thinning. With fruit thinning, shortly after fruit set, a portion of immature fruit is removed from the tree to reduce carbohydrate competition among those remaining. It is widely recognized that fruit size is largely influenced by crop load, with larger fruit size obtained as the crop load is reduced . Quality of fruit may also be affected by crop load, low-cropped trees have been shown to produce larger and firmer fruit than those from heavily cropped trees . Although minor in comparison tocarbohydrate demand, big plastic pots fruit size may also be diminished by inducing higher water stress with larger crop loads. An experiment by found that larger crop loads were responsible for reducing midday stem water potential in nectarines.

MacFayden et al., concluded that an increased crop load also increased the fruit water deficit which may reduce fruit growth in peach. According to another study by , rootstocks also influenced the crop load’s effect on fruit size, and more vigorous rootstocks had larger fruits at specific crop loads. The fore mentioned findings relay the importance of better understanding the relationship between fruit size and crop load among vigorous and reduced-vigor rootstocks.While crop load per tree is controlled by thinning, crop load per area is most influenced by planting density. The reduced vigor and overall size of trees on size-controlling rootstocks facilitates the establishment of high-density plantings . The primary principle in establishing an appropriate planting density for an orchard using trees on size controlling rootstocks is that total tree dry matter production and crop yield are related to total light interception . This principle holds for essentially all crops . However, although higher light interception often leads to higher yields, yield may also vary significantly with other environmental stressors such as available water, nutrients, temperature, and amount of time the fruit has for growth . Orchard systems with increased planting densities have also been shown to reach maximum yield capacity earlier than conventional plantings since the trees are able to fill out their allotted space more quickly . In a small trial using the ‘Summer Bright” nectarine cultivar, trees that were pruned to a standard height of 12 to 13 feet or limited to heights of 8 or 9 feet produced similar sized fruit and crop yields. The reasoning for this was that, despite the height difference, both tree shapes had equal planar volume and therefore intercepted similar amounts of photosynthetically active radiation .The goal of this study was to address three production characteristics and their relationship with four different orchard systems. 1) Fruit size: can peach orchard systems using trees on size controlling rootstocks produce fruit of equal size compared to orchard systems with trees on vigorous rootstocks? 2) Fruit count: if crop load per area is similar among size-controlling and vigorous systems is fruit size also similar? 3) PAR interception and yield: is there a difference in the relationship of fruit production vs light interception among orchard systems with vigorous rootstocks and those with size-controlling rootstocks? A better understanding of production capabilities will allow researchers and growers to better estimate the potential of an orchard system on size-controlling rootstocks as a commercially viable option.In April 2015, an orchard system trial was established at the University of California Kearney Agricultural Center, Parlier, CA. The research block consisted of two peach [Prunus persica Batsch] scion cultivars, June Flame and August Flame grafted onto three different rootstock genotypes: HBOK 27 , P-30-135 , and Nemaguard . Controller 6 was used in two of the four training systems . The C-6 V was a high-density planting system with an in-row spacing of 1.2m and trained to the KAC-V perpendicular V pruning system . The C-6 Quad system was pruned to a Quad V where four main scaffolds are selected in each tree and pruned to resemble an open vase, the system also had a larger in-row spacing of 2.4 m . The Controller 9 Quad system was identical to the C-6 Quad system with the only difference being the rootstock. Between-row spacing was 4.6m in all systems using size controlling rootstocks. Nemaguard was used as the commercial standard rootstock with a planting density of 2.4m in-row spacing and 5.5m between-row spacing . Shortly after harvest, orchard systems using size-controlling rootstocks were topped to a height of 2.5m while systems using the Nemaguard rootstock were topped at 3.5m. The four systems were divided into three replications for each of the two scion cultivars making a total of eight unique orchard systems. Each replication consisted of four rows of trees with the northern and southern most rows used as guard rows, the first and last two trees in each data row were also considered guard trees making nine trees in each of the two inner rows the sample size per replication . In total, each cultivar was represented by approximately 54 data trees .

The survival of the pathogens varied amongst the different types of dried fruits

Tertiary models are established based on primary and secondary models and use predicted values of growth parameters from secondary models to predict changes in pathogen density at times and levels of independent variables that have not been tested or used in the model development .The survival of all three pathogens was longest in high-moisture and low-moisture dates at refrigerated temperature. The combination of their high pH values and low aw compared to the other dried fruits may be reasons why the dates have larger D-value than the other dried fruits. Juneja et al found that L. monocytogenes, E. coli O157:H7, and Salmonella spp. were able to survive on dates for 32 days when stored at 4 °C. Furthermore they found that there was no significant difference in any of the pathogens when the dates were treated with antimicrobial washes of peracetic acid or with ethanol . Because of this strong ability for pathogens to survive, Medjool dates should be further explored from a microbial safety viewpoint. The storage temperature had the biggest influence on decimal reduction time in Salmonella. While having the highest D values of the three pathogens in refrigerated dried fruit, Salmonella had the lowest D values in the dried fruit stored at ambient temperature . This shows that temperature has a large influence on the survival time of Salmonella. While thermal death time does increase for all three pathogens when put in colder conditions, the difference in Salmonella is the starkest. For example, according to the models made, the time is would take to reduce Salmonella by 90% in high moisture dates at refrigerated temperature would be 396 days, whereas at ambient temperature it would take 21 days . These results suggest that Salmonella has particularly increased survival at lower temperatures compared to other pathogens. While the specific mechanisms that allow for this survival in low moisture environments are not completely clear, garden pots square temperature most likely has an influence on those mechanisms. Andino and Hanning suggest one possible mechanism that Salmonella spp. may use to enhance its survival at lowered temperatures is cold shock proteins .

Upregulation of these proteins allow Salmonella to adapt to colder environments as temperatures drop, leading to better survival of the pathogen. Looking at the dried peaches , the decimal reduction time of the pathogens were higher when the peaches were inoculated with the dry carrier versus the wet carrier. This suggests that pathogens are more persistent when using a dry carrier to simulate a dry environment. However, a factor that might have influenced the lower D value in the wet inoculated peach is that the initial inoculation strength is several logs higher with a wet carrier than a dry carrier. Due to the higher initial microbial load in the wet inoculated peaches compared to the dry inoculated, there is difficulty in comparing the true impact that the wet and dry carrier had on the decimal reduction time.Dried fruit were inoculated with Salmonella spp., Escherichia coli, or L. monocytogenes to determine how they would survive in two storage conditions. All three pathogens were able to survive in dried fruits and should be taken into consideration when looking at the safety of dried fruit processing. Salmonella had the longest survival potential among all three tested pathogens. This observation is expected as Salmonella is known for its ability to survive in low moisture conditions. The condition that allowed for longer pathogens survival was storage at refrigerated temperature rather than ambient temperature. This is important because many dried fruit processors store their dried fruits at refrigerated temperatures if not being sold immediately. This allows for a longer shelf life of the dried fruit compared to ambient storage, but increases the ability for bacterial survival. This may be due to the intrinsic factors of the dried fruit: pH, aw, and available nutrient. Based on the current data, survival was the longest in the dried fruits that had the highest relative pH and the lowest relative aw. Salmonella inoculated in Medjool dates survived to the very end of the 180-day survival study.

The Medjool dates had the highest pH of all the dried fruits and had some of the lowest aw of the dried fruits. Other intrinsic factors that were not measured could have also played a role in the long survival time in the dates. For instance, dates are known to have a high sugar content, which may have played a role in pathogen survival. Measuring various compounds in the dried fruits might give more insight on why certain dried fruits allowed for longer survival than others. Regardless the reason, pathogen survival was long in Medjool dates, and should be something that those who produce dates consider. Since dates are not dried the same way other dried fruits are, the steps in the date harvesting process should be looked at carefully. While conducting this research, a new potential outbreak associated with Medjool dates was reported . Twenty-eight people in England were infected with Hepatitis A in 2021 and is suspected to be from Medjool dates . The dates have since been recalled due to their possible contamination . In 2018 there was another outbreak of Hepatitis A in Denmark and was believed to be from dates from Iran . There has been no evidence to show that those dates were contaminated with the virus . That potentially makes this 2021 outbreak the first to be associated with a dried fruit not part of a mixed product. With the occurrence of this outbreak, it makes it all the more important to understand when and where potential contamination of pathogens in dried fruits can occur. Although the data generated from our study is based on bacteria, we did see that the survival of pathogens in Medjool dates is longer compared to other dried fruits. Additional research will be necessary to better understand the survival of foodborne virus on dried fruits. As discussed earlier in this thesis, there are many pre- and post-drying treatments that can be applied to fresh or dried fruits. When looking into the available literature, the efficacy of these treatments has not been systematically evaluated. One on-going project in the lab is to summarize the current knowledge about these treatments and their efficacies and develop a study that fills in the knowledge gaps. In the meantime, identifying a surrogate for testing the different pre- and post-drying treatments as well as different drying methods is needed.

Enterococcus faecium NRRL B-2354 has been validated and approved for being used as a surrogate for almond thermal processing validation . However, whether this strain can be as a surrogate for dried fruit related studies or not still needs further evaluation. One on-going test project in the lab is go evaluate the survival of E. faecium NRRL B-2354 in dried peaches and apricots. In this first test trial, E. faecium was inoculated onto two types of dried fruits and its survival at ambient and refrigerated temperatures is being monitored. In addition, the highest temperatures that can be achieved by various dry methods are being monitored and recorded. The efficacy of different pre-drying treatments is also being tested in the lab by using Salmonella-inoculated peaches and E. faecium-inoculated peaches. In summary, the microbial safety of dried fruits is important and needs more research attention. The survivability of common foodborne pathogens on different types of dried fruits and the recalls and outbreaks associated with dried fruits highlight the importance of the validation of pre- and post-drying treatments as well as different drying methods. The findings of this study, square pots along with future work, hopes to provide the foundation needed for the development of food safety plans for dried fruits.Under global warming and climate change, cultivated plants are encountering increased biotic and abiotic stresses, which lead to reductions of plant growth and reproduction and consequently economic losses. The use of plant endophytic bacteria to promote plant growth and increase tolerance of environmental stresses has provided an alternative to standard agricultural practices that has fewer safety concerns. Endophytic bacteria can be defined as non-pathogenic bacteria that colonize the interior of host plants and can be isolated from surface-sterilized plant tissues. These bacteria can obtain a constant nutrient supply from host plants by living inside the plants and having close contact with plant cells. The endophytic bacteria colonization process is usually initiated at wounds and cracks in the roots by a rhizospheric population of the bacteria in the soil. After entering the plant roots, endophytic bacteria can systemically colonize the above ground parts of plants, including stems and leaves.A wide diversity of endophytic bacteria has been discovered in several plant species. Endophytic bacteria communities include five main phyla. Proteobacteria is the most dominant phylum isolated from host plants, which includes α-, β-, and γ-Proteobacteria. Actinobacteria, Planctomycetes, Verrucomicrobia, and Acidobacteria are also commonly identified. The most frequently isolated bacteria genera are Bacillus, Burkholderia, Microbacterium, Micrococcus, Pantoea, Pseudomonas, and Stenotrophomonas, with the two major genera being Bacillus and Pseudomonas. Several factors affect the composition of endophytic bacteria populations, including plant growth conditions, plant age, types of analyzed plant tissues, soil contents, and other environmental factors. Endophytic bacteria can have several beneficial effects on host plants, such as promotion of plant growth and yield, increased resistance to plant pathogens, enhanced tolerance to abiotic stresses, elimination of soil pollutants through the facilitation of phytoremediation, and production of various metabolites with potential applications in agriculture, medicine, and industry. Some endophytic bacteria help host plants acquire increased amounts of limited resources from the environment. This can include enhancing the uptake of nitrogen, phosphorous, or iron by expressing nitrogenase, solubilizing precipitated phosphates, or producing iron-chelating agents in bacteria, respectively. Some endophytic plant-growth-promoting bacteria can increase host plants’ metabolism and nutrient accumulation by providing or regulating various plant hormones, including auxin, cytokinin, gibberellins, or ethylene. Auxin and ethylene are the two major hormones that affect plant growth and development and that are involved in plant-endophytic bacteria interactions. In addition to these four hormones, several endophytes can utilize signaling pathways mediated by salicylic acid, jasmonic acid, and ethylene to initiate induced systemic resistance and protect host plants from phytopathogen infection. A number of endophytic bacteria can also produce various antibiotics, toxins, hydrolytic enzymes, and antimicrobial volatile organic compounds to limit pathogen infection. We previously isolated a plant endophytic bacterium, Burkholderia sp. strain 869T2, from surface-sterilized root tissues of vetiver grass. Strain 869T2 can also live within banana plants, in which it promoted growth and reduced Fusarium wilt disease occurrence. Genomic sequences of the strain 869T2 contain the gene for 1-aminocyclopropane-1-carboxylate deaminase, which may modulate host plant ethylene levels. Strain 869T2 also has genes related to the synthesis of pyrrolnitrin, which may function as a broad-spectrum anti-fungal agent, as well as dioxin-degradation-related genes. Furthermore, strain 869T2 can degrade the toxic dioxin congener 2,3,7,8-tetrachlorinated dibenzo-p-dioxin , mainly via its 2-haloacid dehalogenase. A recent study compared the genome sequences of 31 Burkholderia spp. and reclassified Burkholderia cenocepacia strain 869T2 as Burkholderia seminalis. We also compared the genome sequences of the strain 869T2 with those of five published B. seminalis strains: FL-5-4-10-S1-D7, FL-5-5-10-S1-D0, Bp9022, Bp8988, and TC3.4.2R3. The strain 869T2 shared 93–95% of its genome with the other five B. seminalis strains. Furthermore, strain 869T2 lacked several genetic loci that are important for human virulence. Based on the results of our analysis of the core genome phylogeny and whole-genome average nucleotide identity , strain 869T2 was classified as B. seminalis. B. seminalis is a member of the Burkholderia cepacia complex , which is a group of Gram-negative, aerobic, non-sporulating, rod-shaped bacteria. Bcc consists of opportunistic human pathogens that exist in patients suffering from cystic fibrosis as well as pathogens of many vegetables and fruits, such as onion and banana. Contrary to the pathogenic traits that led to their original discovery, some Bcc bacteria have ecologically beneficial interactions with host plants. The plant endophytic bacterium B. seminalis strain TC3.4.2R3, isolated from sugarcane, can serve as a biocontrol agent to reduce infections with Fusarium oxysporum and the cacao pathogens Moniliophthora perniciosa , Phytophthora citrophtora, P. capsici, and P. palmivora as well as orchid necrosiscaused by Burkholderia gladioli through the production of pyochelin, a rhamnolipid, and other unidentified diffusible metabolites. Another strain of Burkholderia seminalis, strain R456 isolated from rice rhizosphere soils, decreased the occurrence of rice sheath blight disease caused by Rhizoctonia solani.

Another intrinsic factor of dried fruits that may impact pathogen survival are antimicrobial properties

There are multiple detection methods for Salmonella. Traditional cultural methods for isolation include plating on selective agars and incubating for 24 h at 35 °C . Before plating, naturally-contaminated samples are often enriched with non-selective and/or selective broths such as lactose broth since a low concentration of Salmonella is expected. Standard methods and media used to isolate Salmonella can be found in the FDA Bacteriological Analysis Manual . In conjunction with traditional cultural methods, rapid biochemical or antigen-antibody-based methods can be used for quicker isolation and identification of Salmonella . Salmonella can also be identified through testing a combination of biochemical and serological reactions. Most Salmonella will provide a positive result for glucose , lysine decarboxylase , H2S , lysine carboxylase broth, phenol red dulcitol broth, polyvalent flagellar test, polyvalent somatic test, and methyl red test; and provide a negative result for urease, potassium cyanide broth, malonate broth, indole test, phenol red lactose broth, phenol red sucrose broth, and VogesProskauer test Salmonella can be further identified through phenotyping methods such as serotyping, phage typing, biotyping, and R typing . Finally, Salmonella can be identified through genotyping by PCR or pulse-field gel electrophoresis . PFGE was a highly used method to trace outbreaks, but whole genome sequencing is now the current method used by PulseNet . Whole genome sequencing is a laboratory procedure that determines the order of bases in the genome of an organism in one process . Because millions of bases make up the WGS for every organism, grow bag for tomato it is much more detailed method than Pulse Field Gel Electrophoresis which was the former gold standard method for differentiating among pathogen isolates .

The CDC started implementing the use of WGS as its main way tracking foodborne outbreaks in 2013 . They are able to compare genomes from outbreak strains to reference genomes from public data bases such as EnteroBase . Shiga toxin producing Escherichia coli. Shiga toxin producing E. coli is a gramnegative, non-spore-forming bacteria that can cause infection in humans. Like Salmonella, it belongs to the family Enterobacteriaceae. STEC can grow in temperatures ranging from 7 °C to 45 °C but has optimal growth from 35 °C to 42 °C . It can grow in a pH range of 4-10, and requires a water activity of 0.95 or higher . STEC can be carried by many types of animals and is commonly associated with ruminants such as cattle . STEC will be passive in many of these hosts, but can cause disease in humans. Symptoms of infection by STEC include bloody diarrhea, vomiting, and in certain cases hemolytic uremic syndrome . STEC infects humans by using attachment and effacement lesions encoded for on their LEE pathogenicity island . As the name suggests, the main toxins used by STEC are Shiga toxins, which is what leads to cell death in the host. Apart from being the most known disease-causing STEC serotype, E. coli O157:H7 informs most of what is known about STEC . The serotype E. coli O157:H7 was first identified in 1982 and was well studied during that decade . The pathogen rose to infamy in 1993 when a large outbreak occurred across multiple locations of the fast food chain Jack in the Box . The consumption of the chain’s undercooked hamburgers led to illness in more than 600 people . Because of this incident, the way food safety processes are handled, especially the inspection of meat and poultry, have drastically changed . This incident is also the reason why O157:H7 has been so well-studied compared to other STEC serotypes. Among other STEC serotypes E. coli O26 is less likely to cause HUS compared to O157, even though its toxins are similar . Hemolytic uremic syndrome, or HUS, is a severe condition that damages the blood vessels of the kidneys and leads to renal failure.

Once in the body, Shiga toxin can bind to globotriaosylceramide in vascular endothelial cells, and damages those cells by inhibiting protein synthesis. If those cells are part of the kidney, it can lead to HUS . In general, E. coli O157 is more likely to cause severe symptoms than other types of STEC . STEC and may also be of concern in low-moisture foods. While the main reservoir for E. coli O157:H7 is cattle, the pathogen can easily spread through fecal contamination of water and other foods . According to the World Health Organization , this contamination can occur at many stages of growing and processing produce, which has led to increases in outbreaks of the pathogen in fruits and vegetables . Because of the recent outbreaks associated with STEC in low-moisture foods, and the various stages at which contamination can occur, it is important to explore its ability to survive in dried fruit, which can have many processing steps . The detection of STEC can also be culturable or molecular based. Selective media often used for STEC plating include MacConkey agar, violet red bile agar, and Levine’s Eosine methylene blue agar . To differentiate E. coli O157 from other E. coli, sorbitol can be added to the agar since O157 will not usually ferment sorbitol . Because the number of E. coli cells present in food is low, enrichment is very important to make sure that any cells present are detected. Common enrichments forSTEC include brain heart infusion broth, tryptic soy broth, and modified buffered peptone water with pyruvate . For identification in pure cultures, agglutination assays are useful for serotyping . The enzyme-linked immunosorbent assay is becoming more common for identifying STEC. Use of this assay has led to a better understanding of the most common serotypes of STEC. While O157:H7 is the most common STEC serotype associated with disease, there is a decrease in proportion of that serotype when using ELISA compared to culture-based methods . When screening with biochemical tests, most pathogenic E. coli will have negative test results for H2S, urease, arabinose non-fermenting, and indole .

To further determine if pathogenic E. coli is STEC specifically, real-time PCR can be used. The genes that should be targeted during PCR are stx1, stx2, and uidA, with the latter being highly conserved in O157:H7 strains . As mentioned with Salmonella, the main outbreak identification tool used by PulseNet is WGS. It has more differentiation capability than past methods used like PFGE. Abdelhamid et al. looked at a recent outbreak of E. coli O157:H7 from cattle to human and found that WGS was able to distinguish which isolates from the cattle matched the isolates in the infected patients, while the use of PFGE was unable to differentiate between all the isolates tested. Listeria monocytogenes. L. monocytogenes is a gram-positive, non-spore forming bacteria belonging to the family Listeriaceae. It can grow in a temperature range of from -0.4 to 45 °C with optimal growth from 30 to 37 °C and can grow within a pH range of 4.4-9.6, but has optimal growth at 6-8 . L. monocytogenes can grow in foods with a water activity of 0.9 or higher .L. monocytogenes is found in a variety of places, including plants, animals, soil, water, and humans . It can cause listeriosis, which can be a very serious infection in high-risk groups but is unlikely to manifest severely in other groups of people . Foodborne Listeria needs only a few cells to infect and once in the digestive tract Listeria can invade cells and use cell-to-cell transmission to spread to the rest of the body. Symptoms of listeriosis in high-risk individuals can include miscarriage, sepsis, and meningitis, grow bag for blueberry plants while in the rest of the population people may experience only mild gastroenteritis. There is some debate of whether L. monocytogenes poses a significant risk in low moisture foods. There have been no documented outbreaks of L. monocytogenes associated with low-moisture foods and the current prevalence of the pathogen in low-moisture foods is likely low . However, L. monocytogenes can survive for long periods of time in low-moisture foods and there have been recalls associated with L. monocytogenes in these foods, including in dried fruits, nuts, biscuits, and oats . L. monocytogenes is notorious for its ability to grow in cold environments. This is why outbreaks of this pathogen are often found in refrigerated, ready-to-eat foods , as they do not require heating before consumption.

Dried fruits are an RTE and are often stored at refrigerated temperatures by processors, but due to the inability of pathogens to grow at low water activities, L. monocytogenes growth should not be a concern in dried fruits. Pathogen survival is still a concern though, as L. monocytogenes has been shown to have a desiccation tolerance of up to 1 year in certain low moisture foods. For instance, Kimber et al. found that 6 log CFU/gof L. monocytogenes inoculated onto raw almonds did not decline significantly when the almonds were stored at 4 °C for 12 months. Agar used for selective plating of Listeria include Oxford, Modified Oxford, PALCAM, Chromogenic Listeria agar, and lithium chloride-phenylethanol-moxalactam . Selective enrichment can be done with buffered Listeria enrichment broth . Proper subtyping is particularly important in identifying Listeria, as many different strains can have similar phenotypic qualities . The most common serotypes of L. monocytogenes isolated from patients are type 1 and type 4 . There can also be strains that have qualities that are unusual to Listeria that make identification more difficult. The FDA BAM mentions as examples isolates of Listeria innocua that are hemolytic and L. monocytogenes and Listeria welshimeri isolates that are rhamnose negative . When trying to differentiate L. monocytogenes specifically, the species should usually test negative for mannitol and xylose, and should test positive for rhamnose, virulence, and beta hemolysis . Again, sequencing plays an important role in identification of many pathogens such as Listeria. In fact, Listeria was the first bacteria the CDC began using WGS with and has since then spread its use to other organisms including Salmonella and E. coli . Intrinsic factors influencing pathogen survival. Pathogen survival can be influenced by many factors, including aw and pH. In general, the ability of microorganisms to survive common food processes increase when aw is lowered. However, while higher aw promotes growth, high aw also enhances lethality of thermal treatments . The mechanisms for thermal resistance are not completely agreed upon but are shown to be strongly influenced by aw . The lower the aw, the more difficult it is for the number of cells present to decline . For example, Keller et al. found that Salmonella inoculated onto pumpkin seeds became increasingly resistant to thermal inactivation when the aw decreased from its original value of 0.97 to below 0.20. The pumpkin seeds began with a Salmonella population of 7.48 ± 0.57 log CFU/g and dropped to 0.68 ± 0.81 log CFU/g after 6 h or drying at 60 °C . After 6 h, the aw dropped to below 0.20 and no more significant decrease in the Salmonella population was seen during 12 more h of drying at 60 °C . Just knowing the aw alone is not enough information to understand pathogen survival, as water activity is often working in conjunction with other factors such as temperature . pH is also known to have some effect on bacterial survival. While bacteria have a specific pH range in which they can grow, they can survive outside that pH range. Thermal resistance is decreased at lower pH, so pathogens are generally easier to inactivate in more acidic food matrices . Deng et al. inoculated dry infant cereals of pH 4.0 and 6.8 with 6 log CFU/g of E. coli O157:H7. After 24 weeks of storage at 5 °C the cereal with a pH of 4.0 had 3.19 log CFU/g of E. coli, while no E. coli was detected in the cereal at pH 6.8 . The phytochemicals found in dried fruits, including alkaloids, flavonoids, and phenolic compounds, can exhibit antibacterial activity . Jagathambal et al. screened various phytochemicals from dried figs to see if they had any inhibitory effects on various bacteria. The phytochemicals extracted from dried figswere able to inhibit Salmonella spp., Klebsiella spp., Haemophilus spp., and Serratia spp. with a minimum inhibitory concentration of 1.0 mg/mL . Mainasara et al. screened phytochemical from dates to see how inhibitory they could be against pathogens.

ABA has been found to be the primary hormone involved in non-climacteric ripening

Limonene and a-terpinolene were the highest produced monoterpenes, which exhibited the strongest patterns consistently . To investigate the events leading to this accumulation of monoterpenes, we performed a Fisher’s exact test to identify enriched KEGG pathways in the modules. Terpenoid backbone biosynthesis was significantly enriched in the H-II-1 module . Figure 3.4 depicts the MEP terpene backbone biosynthesis producing GPP that leads to monoterpene biosynthesis. A flux of terpene biosynthesis occurred at the end of Stage II and the beginning of Stage III, which indicates precursors for monoterpene metabolism were being synthesized . From the WGCNA, we also identified the top 5% highest connected genes within the module network. In the H-II-1 module, the top highly connected genes included the gene encoding HDR and a limonene synthase in the terpene biosynthesis pathway.Color changes are a characteristic of fruit ripening. To further define ripening in the pistachio hull, we investigated the underlying biological cause of the change in fruit coloration from green-yellow to hues of red-pink observed in the hull during Stage IV . We found a significant correlation between red coloration increase in the hull with the H-IV-1 and H-IV-2 =0.73 modules during ripening. This was further supported by a Fisher’s exact test for enrichments of KEGG pathways in each module. The H-IV-2 module was significantly enriched for the carotenoid biosynthesis pathway. The B-carotene hydrolase was the highest expressed carotenoid gene in this module and is annotated to be involved in the production of lutelin and zeaxanthin. We examined the highest connectivity genes in this module and among them was a phytoene synthase gene with 887 connections, the rate-limiting step in the carotenoid pathway. Because pink coloration often comes from anthocyanins we also looked at anthocyanin biosynthesis in the hull. While expression was present in the phenylpropanoid and flavonoid pathways, grow bag expression was low in the steps exclusive toanthocyanin biosynthesis. We also found a high negative correlation between the hull redness and the H-III-1 module corresponding to a loss of green coloration.

A significant enrichment of photosynthesis genes in the same H-III-1 module , meaning gene expression of photosynthesis genes decreased after Stage III when fruit became less green.Pistachio kernels contain a high proportion of fatty acids and reach their maximum fat content as the kernel matures during ripening . To further understand the composition of the fat content, we measured unsaturated and saturated fatty acids across six time points during Stage III and IV of kernel development . Unsaturated fatty acids made up 87% of the total fatty acids present when the fruits were ready to be consumed . We confirmed that the unsaturated fatty acids were composed of a higher ratio of mono-unsaturated to poly-unsaturated . This ratio changed through time, such that by ripening MUFA were the predominant class of fatty acids present in the fruit. We determined alterations of metabolites within each class of fatty acid contributed to the changes in MUFA and PUFA ratios during maturation . To further understand what causes these alterations, we examined gene expression of kernel in gene modules associated with the increase in fat content. The module-trait relationships indicated that the increase in fat content was highly and significantly correlated with the K-III-1 module, along with K-IV-1, K-IV-2, and K-IV-3 . This same relationship was also evident for these same modules and the proportion of unsaturated fatty acids through time . We performed an enrichment of KEGG pathway annotations in kernel modules and found that fatty acid biosynthesis was significantly enriched in the K-III-1 module . The high expression of biosynthesis genes during Stage III indicates that fatty acids are produced early on at the start of kernel development, and taper off at the beginning of Stage IV . Within this module, 19 genes encoding fatty acid biosynthesis were found including key genes FAB2 and FAD2 which desaturates steric acid into oleic acid and oleic acid into linoleic acid, respectively . The FAB2 and FAD2 genes were the highest expressed genes in the pathway, and were among the top 5% of genes in the module. FAB2 peaked in expression with 12,500 normalized reads at 1508 GDD while FAD2 peaked with 14,700 normalized reads at 1749 GDD.

Consistent with the expression data, the metabolite data also showed that oleic and linoleic acid were the top two produced fatty acids, throughout development. Interestingly, the concentration of linoleic acid decreased over time while oleic acid increased, which was not evident in the expression data.Defining the biological events occurring during pistachio fruit development that lead to traits of interest can allow for breeding and management strategies to improve fruit quality. Further, a high-quality reference genome has been lacking, as previous genomes are incomplete and fragmented. Therefore, in order to facilitate molecular breeding and broaden the understanding of nut tree crop fruit developmental processes, we present for the first time an assembled 561 Mb reference-quality chromosome-scale genome of P. vera cv. Kerman. Based on k-mer distribution analysis with PacBio HiFi reads, the Kerman genome showed a moderate heterozygosity estimate in comparison with other outcrossing highly heterozygous crops, such as pear 1.6% and grape 1.6-1.7% . This is unexpected because the previously-reported heterozygosity levels of pistachio genome were higher, 1% and 1.72% , which was attributed to the nature of outcrossing by wind pollination and dioecy of pistachio trees . In addition, the genome size estimate of 521 Mb in our study was smaller than the first attempt for pistachio genome size estimate with 26.77 Gb whole genome sequencing data using 17-mers . However, later, the genome size estimated with the larger amount of data using 21-mers was rather similar in size to our assessment . In the final genome assembly, the Kerman genome size was larger than the estimated size but smaller than previously published genome assemblies of different pistachio cultivars, Batoury and Siirt and Bagyolu . Although the size variation of the estimated and assem-bled pistachio genome assemblies could be explained by possible genome size variation across different cultivars as documented in other plant genomes, it is likely that pseudoduplication in the assemblies, especially from the highly repetitive regions in chromosome arms in the case of pistachio, is the primary cause of assembly size variation . In 2015, Sola-Campoy and colleagues characterized massive enrichment of 180 bp repeat on one arm of 11 chromosomes in pistachio, which was also observed in the Kerman genome .

The largest region with dense distribution of 180 bp repeats reached about 9 Mb in chromosome 7, where no protein-coding gene was annotated . These extremely repetitive regions could have been the major issues of accurate pistachio genome assembly and chromosome construction. Although Omni-C reads are known to offer uni-form coverage across the genome without RE sites over represented, it was observed that the overall coverage of Omni-C reads was significantly lower in those regions in Omni-C analysis, likely due to the limitation of mapping capability . Therefore, more careful validation on those regions is needed to improve the pistachio genome. The annotation of intact and fragmented transposable elements in the Kerman assembly resulted in about 11% higher genome coverage than the estimated repetitiveness . As discussed in genome size and estimate, this is likely caused by the pseudo-duplication in the assembly or misestimation due to exceptionally repeat-dense regions in chromosome arms. Among 65% of repetitive regions, nearly 49% of the genome was composed of LTRs, which have been widely known as the dominant TE groups in plants and can play a major role in adaptation and evolution by introducing novel genetic material. The protein-coding gene annotation shows high completeness based on BUSCO assessment with almost 99% . However, minor improvements can still be made by filtering out false-positive gene models and recovering missing BUSCO genes. Macrosynteny patterns between Pistacia vera cv. Kerman, Mangifera indica , and Citrus sinensis provided evidence that P. vera has not experienced a lineage-specific whole genome duplication event , unlike the recent WGD which occurred in the mango genome as described in . The synteny between mango and pistachio genomes and the similarity between their fruit morphology and growth patterns provides an interesting evolutionary comparison within the Anacardiaceae family.During the growing season, pistachios undergo a unique asynchronous development of the kernel and maternal tissues. The hull and shell develop together in the first months, marking Stages I and II , grow bag gardening while embryo development takes place during Stages III and IV. In contrast to previous reports, we found that shell hardening continues to take place with kernel growth starting in late June at approximately 1000 GDD through late August at approximately 2000 GDD . The asynchronous developmental pattern between the fruit and embryo has not been well described in the literature for other tree crops. While peaches appear to exhibit a similar pattern in seed development, this trait does not seem to have been studied in a crop whose seed is consumed . Carbohydrate dynamics in the tree may offer some explanation of the asynchrony. Carbohydrates reserved from the prior year are utilized by the tree to produce buds and develop fruit in early spring, through Stage I . The lull in fruit growth identified as Stage II may serve as a transition between a net carbon loss and a net carbon gain in photosynthesis leading to the growth of the kernel. The RNAseq experiment assessed genetic changes through time and tissue type during fruit development. The shell and hull have the most similar gene expression patterns . This was obvious in the expression of hormone-related gene expression. The shell and hull tissues exhibited very similar expression patterns for each hormone biosynthesis pathway, while the kernel expression patterns were distinct . Interestingly, the hull and shell total gene expression became more similar over time . This contrasts with the morphology of the tissues, which early on in development are physically fused together and appear to become increasingly different through time as shells become woody and split, and the hull and shell tissues separate during ripening . The similarity in gene expression may be due to both tissues undergoing terminal developmental programs. This occurs earlier in the shell when the tissue reaches its peak firmness at the beginning of Stage IV, while in the hull this occurs at the end stages of Stage IV as ripening finishes. Shell lignification was previously reported to start as early as May-June, falling in Stage I-II . While the secondary cell walls become lignified, the shells are green and flexible at this point. However, as described above, the texture of the shell continues to change through Stage III leading to a woody tissue that then splits . The shell tissues appear to senesce and be fully lignified at around 2100 GDD, as RNA content became very low in shell tissues after this point. Our gene expres-sion analysis found a proportion of the genes involved in the phenylpropanoid pathway leading to monolignols to be expressed highest at Stage II followed by a sharp decline, marking the initial lignification . The genes exhibiting this pattern were among the highest expressed homologs; however, other copies of the genes displayed patterns with peak expression later on during Stage III or IV indicating lignin was still being produced, contributing to the increased firmness of the shell. This suggests that the lignification process does not complete until the shell reaches peak firmness, as has been described in walnuts . While continued lignification may be a factor leading to shell firmness changes, other factors such as cell wall modifications likely also contribute, but require further investigation. Overall, understanding the composition and alterations in the shell tissue will be important to ascertaining the underlying mechanisms leading to shell split for a higher quality nut.Although ripening has not previously been well explored in fruit tree crops, early reports suggest that pistachios are non-climacteric fruit . We confirmed ethylene is not produced in a climacteric pattern during ripening and remains at constant low levels, as shown through biosynthesis gene expression . In conjunction with this we found evidence that abscisic acid may be involved in regulating ripening in pistachio. NCED is the rate limiting enzyme in ABA biosynthesis . We found that a primary copy was expressed in the shell and hull tissues right before ripening changes began to occur, i.e., the transition between Stage III and Stage IV. This corresponded to an increase in ABA signaling genes such as, PYLs, PP2C, SNRK2, and ABFs, suggesting ABA is active at the onset of ripening .

We will continue to develop the predictive model as more material is evaluated and adjust accordingly

Moving forward, we intend to generate a range of populations based on both phenotypic and genomic selection from this yield evaluation trial. These will then be evaluated alongside other elite material for DMY to assess if there has been any improvement. Due to the lengthy breeding process of perennial forages, it will take several years to determine whether these methods have been successful. To improve the predictive ability of the model moving forward a combination of evaluating a greater number of families and improving the quality of phenotypic data through better modeling will be imposed. Increasing the size of the training population could be facilitated without a significant increase in costs by using modern high-throughput phenotyping tools, such as dronebased remote sensing. With decreasing costs of genotyping, improved computational software and the availability of genomic resources , genomic selection is becoming increasingly available to more resource limited breeding programs like alfalfa. There is still much research required to assess whether actual yield gain can be achieved; however, these studies provide a baseline for future studies to investigate potential yield improvement.Yield is the most important trait for profitable forage production, yet the rate of genetic gain for dry matter yield in perennial forage crops is lower than the main cereal crops and has been essentially zero in alfalfa over the past 30 years . Limited resources, low heritability, square black flower bucket significant genotype by environment interaction and long selection cycles limit the rate of genetic gain in perennial forages in comparison to many annual food and feed crops .

Improvement of perennial forages is typically carried out through recurrent phenotypic selection with or without progeny testing to accumulate desired alleles at high frequency in a population . Ideally the number of families to be evaluated is very large, particularly with the advent of modern breeding methodology such as genomic selection. In reality, breeders must strike a balance between the available resources and the size and scope of breeding trials. Phenotypic evaluation of perennial forage traits requires significant investment of land, labor, and capital. Forage DMY and dormancy in alfalfa are two crucial traits that require significant resources to phenotype. The standard test for fall dormancy in alfalfa requires height measurements for each trial entry 25-30 days after the final harvest, often across multiple environments and years . To accurately assess forage yield, experimental units must be harvested, dried,and weighed to estimate dry matter content across multiple harvests and years, resulting in up to 40 total harvests over the lifetime of a trial . Further complexity is added to the breeding of perennial forages considering the diversity of evaluations trials often used, ranging from single plant evaluations to transplanted rows, seeded rows, or solid seeded swards. The choice depends on the traits of interest, the number of genotypes or families being evaluated, seed quantity, and the capital and labor resources available to the breeder, with most programs using a combination of sown and transplanted trials . Transplanted family rows are the most common as they are a cost-effective method of evaluating large numbers of trial entries for traits with high heritability. They are commonly used to screen populations for resistance or tolerance to various pests and diseases, investigating growth habit, dormancy, flowering time, and forage quality . In the past, forage yield has often been selected indirectly based on evaluation of vigor on spaced plants or short family rows . Although a useful method for evaluating other important, highly heritable traits, a poor correlation exists between these assessment methods and yield in a commercial setting . Large sown plots are commonly used for variety trials.

These trials require large quantities of seed, cover a large area, and provide phenotypic data for relatively few trial entries. In a breeding program, these trials are typically used to compare advanced breeding populations to released cultivars for key traits such as stand establishment, DMY, forage quality, flowering time, and dormancy. Although useful for obtaining phenotype data that well represents a commercial forage operation, it is usually not feasible to evaluate hundreds of families in this way. Transplanted mini-sward plots provide a compromise between family rows and large sown plots. They seek to provide a better estimation of forage DMY than family rows without the need for large quantities of seed or significant land area that large sown plots require. In these trials asmall number of plants are planted close to one another to mimic the competition observed in commercial forage stands. In recent decades, remote sensing has been widely adopted in agricultural research , offering a plethora of non-destructive vegetative data with massively reduced labor requirements. Remote sensing has the potential to address the lack of yield improvement in alfalfa and increase the rate of genetic gain for yield in other perennial forages by enabling breeders to greatly increase the size of trials without the associated increase in labor costs. This is particularly important for breeding programs looking to use genomic selection, where the size of the training population is a key component of predictive ability . Remote sensing techniques have been shown to enable accurate estimation of biomass yield in alfalfa at the field level , and at the large plot level in breeding trials . However, its accuracy has not been widely reported across the range of plot types used in forage breeding or for estimating fall dormancy in alfalfa. The overall objective of this research project was to assess the accuracy of drone-based remote sensing versus traditional phenotyping for forage biomass yield and alfalfa fall dormancy across a variety of plot types used in perennial forage breeding. The goal is to give breeders the ability to evaluate a wider range of material without the associated increase in labor and costs. In addition, we aim to provide recommendations for researchers looking to incorporate similar technology into their breeding programs.

This experiment was carried out across several trials previously established as part of the UC Davis forage breeding program located on the UC Davis Plant Sciences Farm in Davis, CA on a Yolo silt clay loam . It is a Mediterranean environment with hot, dry summers, cool winters and moderate annual rainfall which falls predominantly in the cooler months from November-March . Soil tests were conducted prior to planting to adjust P, K, and pH according to soil test recommendations. The trials consist of three alfalfa breeding trials and a forage grass variety trial.The trial consisted of 72 released cultivars, experimental cultivars, germplasm populations, and eleven standard test check cultivars . Plants were germinated in 128-cell flats in the greenhouse in February before transplanting to the field in April 2018. This experiment consisted of four replications laid out in a randomized complete block design. Plots consisted of a single row of 25 plants spaced 30 cm apart with a 90 cm gap between plots and 60 cm spacing between rows. Fertilizer was applied to maintain P and K at appropriate levels for a high yielding alfalfa stand, with weeds and insect pests monitored and control measures applied when necessary. Plants were initially watered using sprinkler irrigation until fully established, following which they were flood irrigated to satisfy full evapotranspiration requirements.This trial contained 80 half-sib families of an experimental population UC2588 that had been selected for tolerance to lygus feeding. We had had sufficient seed of each family to plant solid seeded plots. This experiment was established following the NAAIC standard procedures for variety yield trials . It consisted of two replications laid out in a randomized complete block design with ten rows and twenty ranges. Plots were 1 m x 3 m and were drilled using a small plot planter at a seeding rate of 15 kg ha-1 with 1.5 m gaps between ranges. UC Impalo was sown as a border between ranges and around the exterior of the trial. As with the 2018 dormancy trial, crop nutrient demand, weeds and pests were monitored and adjusted when necessary. Sprinklers were used immediately after sowing to get the trial established, square black flower bucket wholesale followed by flood irrigation to meet water demand.This trial included a total of 198 entries of which 193 were half-sib families from two closely related elite UC Davis populations derived from various UC Davis germplasm that underwent selection for root rot and other stresses in El Centro and Davis, California. In addition, three cultivars: Highline, UC Impalo and CUF 101 were included as repeated checks and the remaining two entries were balanced bulks from each of the two populations . The trial was sown in the greenhouse in March 2020 and transplanted two months later in early May at two locations on the UC Davis research farm in Davis, California. Each site has the same layout consisting of two replicates with 7 rows and 29 ranges for a total of 203 plots per rep, 812 plots overall. Plots consisted of 24 plants laid out in a regular 4 × 6 grid with 20 cm spaces between plants. There was a 30 cm space between rows and a 110 cm space between ranges to allow room for mechanical harvesting.

This trial was managed as a high-yielding alfalfa stand, soil tests were conducted each year, with amendments made accordingly. The trial was established using sprinkler irrigation, which was switched to flood irrigation after plants were well established. Irrigation water was added to roughly match crop ET. Weeds were managed by a combination of manual removal and herbicides, and insect pests were monitored and controlled with insecticide application as necessary, primarily for alfalfa weevil control in spring.A grass variety trial containing 88 cultivars was sown in October 2020. Plots are 1.5 m x 4.5 m and were drilled using a small plot planter. Table 1 outlines the seeding rates used for each species. The trial was separated by species with two blocks of tall fescue, two blocks of orchard grass, one block of timothy and reed canary grass, and the remaining species in the final block. Each block contained four rows of plots with 14 ranges. The blocks are separated by borders of either tall fescue, timothy, or orchard grass to allow irrigation pipes to be laid across the field without lying on top of the plots. This trial was irrigated by sprinklers on a weekly to biweekly basis as needed to approximate ET demand. N, P and K levels were monitored, and fertilizer applied when necessary. N was applied at 100 kg ha-1 in spring and again after first harvest.Plant height measurements for the alfalfa fall dormancy standard test were measured following the protocol outlined by Teuber et al. . Twenty-five days after the final fall harvest, the natural plant height was measured on each of the 25 plants per plot. Natural plant height was deemed to be the distance from the soil surface to the top of the tallest stem as the plant stands in the field . The measurements were then averaged over the whole plot to generate a single data point for each plot. Biomass yield data were collected using a small self-propelled plot harvester. Harvests occurred in alfalfa when the field had reached 10% bloom with the first harvest usually occurring in late March/April and the final harvest in October. For alfalfa trials, subsamples were taken during each harvest, weighed wet, dried for at least 4d at 60C, and weighed dry to adjust moisture percentage. Several subsamples were taken from each replication as composite samples from all entries, rather than for every entry, and the average dry matter was used to adjust the wet weights. In the grass trial, harvests occurred when the most plots of tall fescue and orchard grass had reached the late boot stage, with the first harvest in April and subsequent harvests every 6-8 weeks for a total of four harvests per year. All species were harvested at the same time for logistical reasons, even though this was likely not ideal for individual species . All forage was clipped uniformly at 7.5 cm, weighted, and removed from the trial area. Subsamples were taken from every plot in the grass trials, weighed wet, dried for at least 4d at 60C, and weighed dry to adjust moisture percentage.Prior to remote sensing data collection, the borders surrounding the trial and between plots were mown. Drone flights and preliminary image processing were conducted following methods modified from Parker et al. .

School attendance reduced participation for males and females when schools were in session

Women reported working, on average, less than 10 days a month during five months compared to 15 to 21 days per month during the peak season. Although a significant proportion of the individuals surveyed lived in or close to towns, roughly 85% of the jobs reported by this sample of workers were in agriculture. Females had somewhat greater packing shed employment experience than males. Surprisingly, women had higher average daily earnings than did men. Women worked more frequently on a piece rate basis , which paid more than comparable wage employment, and women were employed primarily during the peak season, when earnings were highest.Most workers lived in households with several workers. Twenty-five percent of the females surveyed and half of the males provided more than 50% of their household’s annual income. Only a third of the females who were widows or separated were their household’s major earner . Still, interviews indicated that many women had been able to separate from their husbands and/or live apart from their parents because of income obtained as a temporary fruit laborer. Although female-headed households tended to have lower incomes than male-headed households, many female heads of households spoke with satisfaction that their work allowed them to support themselves. Worker’s household characteristics influenced the number of days employed each year. Figure 3 shows that married men worked the most, especially if they had young children, approximately 275 days per year. Single males worked much less, about 170 days. Men who were separated or widowed worked an amount intermediate between these levels. The significant affect of marriage on the number of days worked suggests that marriage affected the motivation to work and that search effort was an important determinant of employment.

Women averaged significantly fewer days worked per year than men did. Some women worked more than 220 days per year, flower buckets wholesale but no female category had such a high average. Women also showed less variation in the number of days worked with respect to their household situation, at least as here categorized, and the variation shown was directly reversed from that of men. For example, married women with young children worked the least of individuals in the sample, while single women who were not living with their parents worked the most of all female categories. There is thus evidence that married women with young children had a higher reservation wage than other workers. However, women lacking income from a husband or parents worked substantially even when they had young children.Female labor force participation varied greatly by season, declining sharply from February to May, remaining low through September, and then rising steadily to February. Labor force participation was less variable for males. Daily earnings varied seasonally more in agricultural than in non-agricultural jobs, especially for jobs held by women. Women tended to earn more than men in agricultural jobs during the peak season, but less during the slack season, while the situation was reversed for non-agricultural jobs. As agricultural wages declined, a rising proportion of workers was employed in non-agricultural jobs . While female temporary workers face greater wage variation than men and vary their labor participation more, they also suffered substantially more unemployment . The female unemployment rate exceeded 50% during five months. Male unemployment was also high, but averaged only about half as much. 4.1. Labor Market Participation Equation and Expected Earnings Jarvis and Vera Toscano explored adjustment in this market to identify whether seasonal differences in labor force participation was attributable to the existence of specific ‘barriers’ to employment, differences in preferences or differences in observed worker characteristics.

Specifically, they modeled labor force participation for male and female workers by estimating a random effects probit that allowed for unobserved heterogeneity in preferences. Table 5 reports the results. For women, the estimated coefficients on the explanatory variables were generally highly statistically significant and in line with prior expectations. Few of the estimated coefficients were statistically significant for men, a result consistent with the relatively constant male labor force participation rate.13 Women participated in the labor force less than men did. Female labor force participation increased with age. Since rising education was associated with higher daily earnings, education may have altered the preference for work versus leisure. Marriage reduced labor force participation for females, perhaps due to increased household responsibilities and/or a social-cultural bias against work, but did not affect male participation. Female labor participation declined as the number of the worker’s children aged 0-5 years increased, but this effect was reduced if another adult female lived in the household, suggesting that childcare was gender specific and indicating the importance of childcare for female labor force participation. Men and women were more likely to participate during the peak season and less during the slack season as compared to the transition months of April and October through December, a result probably linked to expected earnings. Jarvis and Vera Toscano examined the sensitivity of labor force participation decisions to changes in expected earnings using a probit equation that included the same regressors plus estimated earnings . The coefficient on expected earnings was positive and significant and the other coefficients were closely similar to those obtained cols. 1 and 2. Though labor force participation for men and women responded strongly and positively to the expected wage, the female participation rate varied substantially more because females tended to have a higher reservation wage. Still, female unemployment was generally much higher than male unemployment .

Although wages varied greatly by season, Jarvis and Vera Toscano found they did not vary sufficiently to fully equate the supply and demand of labor and achieve zero unemployment. Four factors were advanced to explain this high unemployment. First, frictional unemployment was high as a result of individuals entering and/or leaving the labor force, changing jobs, and searching for employment in a spatially dispersed market where jobs were relatively short lived and search costs relatively high. Second, many or all firms may have paid an efficiency wage or piece rate to motivate workers, thereby causing the unemployment rate to remain above zero even during periods when labor demand is high. Third, the average reported wage in agriculture lay above the average reported wage in the non-agricultural sector throughout the year. Thus, waiting for an agricultural job could easily have been the better strategy for most workers even when few agricultural jobs were available. Fourth, some workers, especially females, may incorrectly report having been in the labor force and actively seeking work. Alternatively, they may have considered themselves in the labor force, but searched only within a small, local area, where there were few jobs.The average wage rose by about 50% from the slack season to the peak season, a surprisingly large variation. To understand the determinants of changes in daily earnings over the one-year period, Jarvis and Vera Toscano estimated an earnings equation where the dependent variable was the log of average daily earnings and the regressors included both supply and demand side factors. Human capital variables such as education and experience were hypothesized to influence worker productivity and earnings, while monthly dummies reflected the net influence of seasonal fluctuations in agricultural labor supply and demand. Wages were hypothesized to vary in response to the worker’s decision to seek either piece rate or wage employment, and either non-agricultural or agricultural employment. Such choices were assumed dependent on a worker’s willingness to supply effort and preference for factors such as work environment and a shorter commute time to work. Since dummy variables were used to measure the effect of working at a piece rate as opposed to a wage, flower harvest buckets the other coefficients measured the effect of the respective independent variables on the daily wage. Consistent estimates of the earnings function were obtained using the two-step estimator proposed by Vella and Verbeek. The results for both men and women are reported in Table 6. The earnings of both men and women increased with schooling, suggesting that education significantly increased labor productivity in agricultural work, although the higher return was probably partly due to the innate ability that allowed individuals to successfully complete additional schooling. Experience had a significant positive impact on female daily earnings in jobs throughout the year; the analogous coefficient was not significant for males. The square of experience had a significant negative coefficient, indicating that rising experience had a non-linear effect.

A dummy variable was also used to measure the earnings effect of working on a piece rate basis. A piece rate system was frequently used to motivate and remunerate temporary agricultural workers in the fruit sector and a substantial theoretical literature indicates that the piece rate system should increase worker’s productivity and workers’ incomes . There have been few empirical studies. The estimated coefficient on the piece rate dummy indicates that piece rate jobs in this case earned a daily premium of about 12 percent relative to wage jobs. A dummy variable was also used to measure the effect of working in the agricultural as opposed to the non-agricultural sector. Agricultural work paid substantially more, particularly for women . Men’s wages in this sample were about 18 percent higher when working in agriculture, while women’s wages were about 37 percent higher. Agricultural jobs were probably even more attractive than shown for women since there were few piece rate jobs available in non-agricultural work. As earlier noted, women’s average daily earnings were higher than men’s average daily earnings . Women working as temporary agricultural laborers were thought to earn relatively high wages in the Chilean fruit sector , and the results in Jarvis and Vera-Toscano supported that view. Nonetheless, women earned substantially less than men did in wage employment once earnings were adjusted for observed and unobserved characteristics. The estimated gender wage differential was about 25 percent. Although females had higher average daily earnings than men, women earned less than men when working for a wage, but not when working for a piece rate. Jarvis and Vera-Toscano suggested that these results indicated discrimination in the wage market. There may be less possibility of discrimination when workers are employed at piece rate since pay is directly linked to productivity. The large magnitude of the gender wage differential suggests an area for further analysis.Newman and Jarvis found that women were highly informed about many aspects of the packing shed jobs that they accepted, e.g., shed-related characteristics that affected workers’ productivity, fringe benefits, and the expected duration of the job. Women’s willingness to accept work at a specific piece rate was strongly influenced by these characteristics. Piece rates for the same tasks were found to vary by as much as 100%among different packing sheds and these differentials were well explained econometrically by the observed heterogeneity among workers and firms. For example, most processing sheds provided workers with some combination of fringe benefits that included meals, snacks, transportation to and from work, childcare, interest-free loans, and higher quality bathrooms. Supervisors and managers in different sheds treated created different quality work environments. According to the theory of equalizing wage differentials, sheds that provide more and better fringe benefits and/or a better work environment should have paid lower piece rates. This hypothesis was supported by the data. Similarly, Newman and Jarvis hypothesized that firms’ investments in technology, improved plant organization, or the ability to process grapes that were in better condition would raise worker productivity. Further, so long as workers were aware of firm-influenced productivity differences, such higher productivity should lead to lower, not higher piece rates. To the extent that firms possessed improved technology that allowed their workers to achieve higher productivity or were better organized and could provide a constant flow of good quality grapes to workers, allowing workers to process more boxes per time period, the firm should pay a lower piece rate. This followed from the assumption that each worker should earn an income consonant with her opportunity cost in equilibrium. If a firm’s characteristics allowed its workers to produce more output, ceteris paribus, worker competition for the jobs at the firm should have caused the piece rate to decline until its workers’ incomes were equal to what they would earn elsewhere. This hypothesis was also supported by the econometric results. Workers could easily ascertain the piece rates paid by different firms, but the effect of firm characteristics on a worker’s productivity should have been harder topredict.

We are assuming that all damaged fruits had a third of the average number of seeds per fruit removed

Plants employ a large array of strategies, both direct and indirect, to minimize the effect of seed predators. These include alternative strategies of regeneration, seed banks, abundant seed crops, altering phenology, spatial and temporal variation in seed productions and increasing plant defenses . It has also been demonstrated that pre-dispersal predation can facilitate the stratification of the seeds facilitating germination . A hybrid-derived lineage that has already successfully invaded a new habitat represents a useful model because, when combined with its progenitors, it allows for a replicated study of the invasion process. Here, I focus on the hybrid-derived California wild radish, which is an invasive lineage in western North America . Its progenitors, the cultivated radish Raphanus sativus and the wild radish R. raphanistrum, were introduced in western United States around mid 1800 . These two lineages naturally hybridize, and there is evidence for the hybridderived lineage to have originated from interspecific bidirectional hybridization between them . Genetically based differences between both progenitor lineages and a partial and temporary reproductive isolation during the first generations of hybrids results from a single reciprocal translocation . The polymorphism in fruit, flower color and shape, root morphology, chemical and structural defenses found in Raphanus lineages has been the focus of numerous studies in ecology, evolution, genetics and agricultural and food chemistry . Anecdotal observations have reported that bird pre-dispersal predation of seeds can be extensive in all three Raphanus lineages and birds are arguably the primary consumers of pre-dispersed seeds over invertebrate granivory . When granivory is excluded, the hybrid-derived California wild radish exhibits superior fitness compared to its parental lineages in common garden experiments across its Californian distribution . However, black plastic plant pots wholesale the hybrid’s relative fitness and that of one of the progenitors in the presence and absence of granivores, is unknown.

Our aim was to answer the following questions: what species of bird granivore is the main consumer of radish seeds? how much does the bird granivore affect relative fitness and relative potential fitness? are the variables likely to affect the birds’ selection of individual plants such as days to germination, plant final weight, total fruit production and potential reproduction, correlated with fruit damage? and are there viable seeds in the debris due to granivory resulting from the bird foraging behavior under damaged plants? In addition, the comparison among lineages allowed us to better understand novel biotic interactions in a successful invasive hybrid-derived lineage and to propose a mechanism that led to the replacement of both progenitors .Seed sources – The seeds used to breed the mother plants in the present study came from plants reared in a common garden during Spring 2005 and Winter 2006. The seed sources for the first generation of maternal plants are described in table 2.1. The second generation seeds are the result of natural open pollination in common gardens at the Agricultural Operations fields at the University of California-Riverside . More details on how the first generation plants were grown can be found in Ridley and Ellstrand . Common garden and experiment design – The common garden experiment took place during Spring and Summer seasons of 2010 at AgOps-UCR. Three replicate sites, each one consisting of two plots of 7 m by 7 m, were planted with 36 plants placed in a 6 x 6 grid with 1 m spacing in rows and columns. One of the plots at each of the three sites was covered with 3/4″ x 3/4″ orchard mesh to exclude above ground vertebrate damage while the other plot remained unprotected. These two conditions created two different treatments for the plants to grow in: protected from vertebrate seed predators and unprotected to vertebrate seed predators. In both cases the plants were exposed to open pollination, invertebrates and potentially underground vertebrates. All plots were oriented in the same North-South direction. We selected at random 8 seeds from 4 different mothers within each of the 9 above-mentioned populations for a total of 288 seeds. These seeds were divided in groups of 36, such that all mothers were represented in those 8 groups by 2 seeds. That is, each population had 4 seeds, for a total of 12 seeds per lineage.

These 8 groups of seeds were germinated in Petri dishes at the beginning of March and transplanted into seed starting trays filled with sterilized UC Soil Mix III at a climate controlled greenhouse. Once the seedlings had attained a three-leaf stage, 6 of the 36- grouped seeds were transplanted to the pre-water and plowed field plots. The two additional groups of seeds were used to replace any seed that did not germinate or any seedlings that did not survive the transplanting process. The plants were watered once daily for 10 min with a sprinkler system until most of the plants had started to flower. To maintain favorable abiotic conditions for the plants that flowered later, the watering persisted only every-other-day for 5 min. Granivores – We visited the sites at AgOps at least every two days to ensure that the experimental conditions were kept consistent during the entire length of the study. During those visits I also spent time observing the foraging behavior of the birds that began when fruits had attained a fully formed size. Once I became familiar with the birds foraging patterns, I spend an afternoon filming their behavior. Videos were captured with a digital video camera on a tripod. Videos are available as supplemental information.Fruit damage, fecundity, fitness related values and debris due to granivory – Variables related to morphology, damage and fitness were recorded before planting, during the experiment, and after the surviving plants were collected. All seeds were weighed to within 0.01 mg with an analytical balance . The germination and growth of seeds in the dish was recorded daily. At the end of the experiment when the plants were dry and had senesced, I recorded the final plant weight to within 0.001 g. To calculate fecundity and fruit damage, I counted total number of: damaged fruits that included all fruits with clear signs of missing or damaged sections, whole dropped fruits that were found detached from the dry plant, and whole attached fruits. With these variables I calculated fruit damage and fecundity. We counted total numbers of: flower buds, flowers, whole empty pedicels , and broken or pedicel scars on the stems. We also collected the fruit material or debris accumulated under heavily damaged plants, herein referred to as “debris due to granivory”, to discern what was discarded during the birds foraging behavior.

Potential seed viability was determined by visually inspecting the seed coat and by putting pressure on each seed between the thumb and the index fingers; when unviable, seeds had black and/or wrinkled seed coats and crumbled easily. All the previously described values and those in table 2 allow us to calculate relative fecundity and relative potential fecundity of plants in unprotected and protected plots. Because I did not count number of seed per fruits, I calculated the number of seeds based on the average number of seeds per fruit per populations. These average values,listed in table 2, were obtained from a previous study where I counted total number of seeds from 884 fruits that belonged to the same populations represented here . We consider these values appropriate to extrapolate the number of seed in our study because: the plants that produced them developed from pure lineage seeds from the same populations represented in our study listed in table 2.1, the plants were grown under similar conditions to the present study, and the plants were exposed to open pollination . Total number of seeds were extrapolated for a given plant by: multiplying total number of whole fruits per plant by the average value of seeds in table 2.2 according to the population of origin, black plastic plant pots bulk followed by multiplying total number of damaged fruits by the 2/3 of the average number of seeds according to the population of origin in table 2.2, and finally by adding the numbers obtained for whole and damaged fruits. Fecundity and female fitness values were calculated as follows. The average number of extrapolated seeds per population was calculated by dividing the total number of extrapolated seeds divided by the total number of fruits per population. Relative fecundity is the average number of extrapolated seeds divided by the highest average number of extrapolated seeds. Potential reproduction was calculated by adding flower buds, flowers, whole empty pedicels broken to whole, damaged and dropped fruits for a given plant. The average potential reproduction was calculated by adding the potential reproduction for a given population or lineage and dividing by the total number of plants and multiplied by 100. Finally, the percentage of the relative potential fecundity was calculated by dividing a given average potential reproduction to the highest average one among for populations and lineages separately and then multiplying by 100. Our fitness values did not explicitly include male fitness. Nevertheless I know based on prior studies in plants of the Raphanus lineage that male fertility is highly influenced by environmental factors and weakly correlated with female fertility values . Data analysis – Data were normalized as needed either with log-normal or Box Cox transformations using functions in R . Significant P values were adjusted a posteriori with sequential Bonferroni test to adjust for type I error . We used one-way analysis of variance to tests the effects of treatments and lineages on total fruit production. Variables related with fruit damage and with fitness were compared in pairs among lineages and between treatments with Wilcoxon tests. The effect of the treatments on relative fecundity and relative potential fecundity as well as average number of fruits and seeds were tested for significance with Fisher exact tests. These tests were performed to individually compare CAwr values to its progenitors. We also compared fecundity values to the highest ones with chi-square tests. Total number of fruit damaged was correlated using Spearman correlation coefficients and covariance to variables possibly related to final fruit production and general performance. Those variables included: days to germination, final plant weight as well as total number of fruits and total potential reproduction. In this case each lineage was tested independently.Flower buds, flowers and pedicels – No differences were found between lineages and treatments in average number of flower buds and open flowers . With respect to the average number of pedicels, values for CAwr from protected and unprotected plots are significantly different and higher than both progenitors under protected treatment as well as for the cultivar under unprotected treatment, respectively . Fruits with and with no damage – We only found damaged fruits in plants that were collected in unprotected plots . Consistent with our previous results, average numbers of fruits with damage are significantly different among lineages and treatments as revealed by Wilcoxon tests . Whole undamaged fruits were categorized as either attached to the dry plant or detached and on the ground. The cultivar Rs differs significantly from CAwr and Rr on lower average number of whole dropped fruits, whereas both wild lineages, CAwr and Rr, are comparable . No differences are found in the average number of whole attached fruits among lineages with the exception of CAwr and Rs from protected plots . The average proportions of damage, calculated as total number of damaged fruits over the total fruits produced for each lineage and population, are listed in Table 2.3. When the damage is estimated based on seeds removed, calculations of damage per population are reduced by at least 33 % and at most by 60% relative to the damage calculated based on fruit damaged. Damage, based on seeds removed, was calculated as the total number of seed removed divided by total number of seeds produced. As mentioned earlier, I did not count the total number of seeds per fruits during this experiment. Fruits from Cst-CAwr suffer higher damage than interior populations . Fruit production – Total fruit production does not differ under protected or unprotected treatments but does differ among lineages . The cultivated Rs lineage produced fewer fruits in protected treatments relative to both wild lineages, significantly differing from both CAwr and Rr . However, under unprotected conditions, Rs only substantially differs from unprotected and protected CAwr fruit production . Fecundity and fitness related values – CAwr has significantly higher fecundity in protected plots than in unprotected ones, as shown in table 2.4.

Gibberellin and cytokinin-related genes were mostly downregulated in symptomatic fruits

Glucose-1-phosphate adenylyltransferase was highly down-regulated in symptomatic fruits while invertase was clearly up-regulated in the peel of apparently healthy and symptomatic fruits from the infected orchard. Sucrose symporter was up-regulated in infected, symptomatic fruits. The terpenoid pathway was affected by HLB disease, as shown by the down-regulation of terpene synthase cyclase . Induction of lipid transfer protein was observed in the absence of symptoms. The expression of other genes such as acidic cellulase and methyltransferase2 was diminished in infected fruits.Huanglongbing, a highly destructive disease of citrus, threatens citrus-producing areas worldwide. Previous studies monitored transcriptional changes in leaves on a large scale using microarrays to investigate host responses and reveal the mechanisms underlying disease development. The present work focuses on transcriptional regulation in fruit peel to analyze the response to CaLas infection at different disease stages. Next-generation sequencing technology , as used here, can find differential expression of an increased number of transcripts, many of which may not be present in EST databases or represented in microarrays. NGS data can be used for specific transcriptome assemblies that become resource datasets for annotation of genomes and annotation of differentially regulated genes and proteins analyzed using any ‘‘omic’’ technique. However, 30 planter pot the absence of a completed genome sequence limited the advantages of RNA-Seq technologies. The aim of this work was to provide data using NGS technology for a comprehensive analysis of metabolic changes in fruit induced by HLB disease.

These findings will uncover the fruit disorder mechanisms and facilitate development of short-term therapeutic strategies for already-infected trees. Toward this end, the experimental design included four types of fruit: healthy control fruit from an HLB-free orchard, apparently healthy fruit from non-symptomatic trees in an orchard affected by HLB, and asymptomatic and symptomatic fruit from infected symptomatic trees in the affected orchard. This design made it possible to identify differentially expressed genes at different disease stages. A comparison between apparently healthy and asymptomatic fruit revealed genes induced early in disease development. Comparing asymptomatic and symptomatic fruit identified genes involved in the host response during disease progression. Comparing symptomatic and apparently healthy fruit revealed host response genes related to the presence of HLB symptoms. Healthy fruit from the HLB-free location were also compared to the three sample types from the infected location. Differences in gene expression in these comparisons are likely to result from environmental and agronomic variability due to the difference in location, in addition to the effects of HLB. A range of 1154 to 1762 differentially regulated genes were found using RNA-Seq comparing the three categories of fruits from the infected orchard with those taken from a location free of HLB . In qRTPCR analysis, 31 of 33 differentially regulated genes confirmed the pattern of expression found by RNA-Seq. Comparisons among the three fruit categories in the infected orchard resulted in fewer differentially regulated genes . This was expected, since samples within the same orchard grew under similar environmental and agronomic conditions. A similar contrast between samples from the infected and uninfected orchards is seen in principal component analysis. The transcripts that most strongly characterize the asymptomatic and apparently healthy samples are common between the two samples.

The symptomatic and uninfected orchard samples are both distinct groups and widely separated in the biplot . A visual summary outlines the most important transcriptional changes in the networks among genes, pathways, and cell functions in the fruit peel . Gene set enrichment analysis identified several pathways significantly affected by HLB as symptoms appear, considering both within-tree and between-tree comparisons, such as those for phenylpropanoids, starch and sucrose metabolism, carbon fixation, ascorbate, and alpha-linolenic acid . Other pathways were also affected by HLB in the two comparisons within the same orchard. Transcripts encoding different subunits of the photosystem II reaction center and cytochrome b6-f complex subunit were more abundant in symptomatic HLB-infected fruit than in healthy fruit. Photosynthesis is central to all aspects of plant biology, since it provides energy for growth and reproduction, but its regulation by biotic and abiotic stresses is still unclear. The induction of photosynthetic light reactions in the fruit is consistent with the observation that symptomatic HLB-infected fruit often remains green. The retention of green color and increase of photosynthesis reactions is probably linked to the lower amount of ethylene detected insymptomatic fruits. Photosynthesis is usually downregulated by pathogen attacks. The upregulation of photosynthetic reactions in the fruit does not contradict this common consideration because CaLas infections typically occur in young leaves. The transcriptomic changes observed in the fruit are probably linked to the source-sink disruption caused by leaf infections. Protein degradation and modification pathways were significantly changed by CaLas infection, as shown by the upregulation of genes such as C3HC4-type ring finger proteins involved in ubiquitin-mediated degradation. Interestingly, heat shock proteins HSP82 and HSP70, highly interactive proteins in the PPI network inferred in citrus, were down-regulated at different stages of the disease. Heat shock proteins are highly conserved proteins induced in cells subjected to elevated temperatures or other environmental stresses.

These proteins act as molecular chaperones to stabilize, reduce misfolding, or facilitate refolding of proteins that have been denatured during stress events. In plant cells, HSP70 and HSP90 are involved in signal transduction leading to plant defense responses. Both proteins interact with a salicylic acid-induced protein kinase and their silencing affected the hypersensitive response in Nicotiana benthamiana while inducing non-host resistance to Phytophthora infestans. HSP90 also modulates the innate immune responses involving gene-for-gene specific interactions, acting as a scaffold protein in a complex that mediates signal transduction. Based on these findings, we speculate that down-regulation of heat shock proteins observed at different stages of HLB disease might increase protein misfolding in the fruit peel. Genes encoding ATP synthase gamma and delta chains were also induced in symptomatic fruit, supporting the idea that CaLas may act as an energy parasite by scavenging ATP from its host with a pathogen-specific ATP/ADP translocase. Indeed, the recently sequenced genome of CaLas revealed the presence of an ATP/ADP translocase in addition to ATP synthase. ATP scavenging may be a possible mechanism of pathogenicity, affecting the fruit peduncle, columella, and seed coat. Photosynthesis regulation in infected fruit peel may also reduce transport of sugars and ions such as nitrate, sulfate, and potassium. In this study, several differentially expressed genes were associated with transport of ions, including ammonium, sulfate, and phosphate . Inorganic ions can modify sugar metabolism and photosynthesis. Polarized vesicle trafficking, transport, and secretion of plant materials are associated primarily with non-specific resistance during host-pathogen interactions. ABC transporters play important roles in this process, since they are also involved in virulence, host range, and symptom elicitation. Citrus proteins related to sugar and PDR/ABC transporters implicated in secretion of antimicrobial terpenoids were specifically induced by HLB at the symptomatic stage . Interestingly, among the genes identified in the CaLas genome were those for phosphate and zinc uptake into the cell. This implies that mineral uptake by the pathogen may be enhanced due to induction of endogenous genes as well as host genes. However, plastic growers pots phloem necrosis induced by CaLas contributes to the impaired nutritional transport functions and source-sink communication observed in this study. Starch accumulation in leaf chloroplasts of sweet orange trees infected with CaLas has previously been demonstrated and is characteristic for HLB. Genes encoding the large subunit of ADP-glucose pyrophosphorylase, the key enzyme catalyzing the first and limiting step in starch biosynthesis, were up-regulated in infected leaves. Interestingly, a gene encoding the large subunit of glucose-1-phosphate adenylyltransferase was downregulated in infected and symptomatic fruit in the present study. Also of interest are contrasting patterns of expression for different isoforms of starch synthase and starch cleavage genes, leaving the dynamics of starch accumulation in the fruit unclear . This is in contrast to experimental observation of starch accumulation in infected leaf tissues. In leaves, this accumulation was linked to up-regulation of genes involved in starch biosynthesis and down-regulation of its conversion to maltose. It has been hypothesized that starch accumulation is an effect of phloem plugging/necrosis during HLB infection, although it usually occurs before these symptoms are visible. Differences in starch accumulation are probably linked to the different types of organs: mature leaves are ‘‘source’’ and fruit are ‘‘sink’’. However, further analysis of fruit starch accumulation must be performed to validate this hypothesis.

An increased abundance of transcripts for genes involved in the first steps of glycolysis and sucrose metabolism was observed in fruits from infected trees. In the cytosol, an invertase gene was upregulated in asymptomatic and symptomatic fruit, affecting the sugar balance and communication between sink and source tissues. We speculate that this leads to increased glucose and fructose and decreased sucrose in fruit cells, which should be further validated with carbohydrate analysis. It is important to note that elevated glucose and fructose has been demonstrated in citrus leaves infected with CaLas. Interestingly, different types of invertases were up-regulated in leaves and fruits . Over-expression of yeast invertase in the cell wall of transgenic tobacco disrupted sucrose export, allowing soluble sugars and starch to accumulate, which consequently inhibited photosynthesis and resulted in stunted growth and bleached or necrotic leaf areas. As previously suggested for leaves, it is possible that the differential expression of key genes involved in sucrose and starch metabolism, as observed in CaLas-infected citrus fruit, might affect the osmotic potential and induce plasmolysis, thus altering the ripening process and producing typical HLB symptoms. However, further analysis of sugar concentrations will be necessary to clarify the causes and effects of disease symptoms in the fruit. Other studies on CaLas-infected leaves have shown increased sucrose and glucose, but not fructose. In fruit, it is possible that altered fructose and glucose concentrations might be responsible for physiological disorders and affect source-sink relationships with leaves. The gene set enrichment analysis confirmed that sucrose and starch metabolism were highly affected by the disease. Integrated analysis of leaf and fruit data indicates that sugar and starch metabolism play a key role in the metabolic dysfunction induced by HLB disease. However, few studies have been conducted to address the effects of altering sugar metabolism on resistance to pathogen infections. Invertase plays a key role in the activation of stress responses and may function as an extracellular indicator for pathogen infection. Indeed, transgenic plants overexpressing sugar metabolism enzymes such as a heterologous invertase from yeast have helped clarify source-sink relationships. The expression of different viral movement proteins in transgenic plants and the resulting effects on photosynthesis, carbohydrate accumulation, and partitioning emphasize the importance of sugars in activating defense responses against biotic attacks. Sink metabolism may be essential to satisfy the energy requirements of activating the cascade of defense responses. Interestingly, sucrose synthase was also more abundant in symptomatic fruit while sucrose transporter genes were downregulated by HLB. The Genevestigator database, indicated that these proteins may be down-regulated by hormones such as ethylene, methyl-jasmonates, and indol-3-acetic acid. The concept of sucrose metabolism regulators as a potential target for HLB therapeutics is intriguing. Increasing evidence indicates an extensive cross-talk between sugar, hormone, and light signal transduction networks in plants. Hormone pathways were significantly altered in fruit peel inresponse to CaLas infection. Two genes involved in auxin synthesis, GH3.1 and GH3.4, were induced in affected fruit. Gibberellin regulation has been observed in other fruit disorders such as albedo breakdown disorder and applications of GA3 before fruit color break can reduce the occurrence of some fruit disorders. It is possible that sugar metabolism changes observed in the fruit might be linked with the down-regulation of gibberellins that regulate energy and carbohydrate metabolism. Previous studies have demonstrated that regulation of gibberellic acid-induced gene expression is affected by sugar and hormone signaling. That cytokinins play a role in sugar regulation has been demonstrated. Therapeutic approaches using small-molecule hormones such as cytokinins and gibberellins may allow modification of fruit metabolism to mitigate the negative impact of HLB on fruit quality and productivity.Ethylene regulates a variety of developmental processes and stress responses in plants, including seed germination, cell elongation, senescence, fruit ripening, and defense. Nonetheless, ethylene can promote either disease resistance or susceptibility, depending on the host–pathogen interaction. In our study, considerable changes were observed in the transcriptional profiles of genes related to ethylene biosynthesis and signal transduction. ACC synthase and ACC oxidase play pivotal roles in ethylene biosynthesis and their expression is often affected by pathogen attack. It was unclear how ethylene concentration changes in fruit in response to HLB.