Category: hydroponic growing

IBtolerant cultivars belonging to the ‘Smooth Cayenne’ group develop symptoms at a slower rate than do the susceptible types, and the browning is primarily seen in the F region. Pineapples classified as IB-resistant show no TS or IB symptom but may display damage externally , also called “common chilling injury” .IB in pineapple is a result of the metabolic dysfunction from chilling-induced membrane damage . Loss of organellar structure permits the normally plastidic polyphenol oxidase access to phenolic compounds that are usually sequestered to the vacuole . The o-quinones intermediates produced by PPO act on the phenolic compounds, and are then converted to the polymers responsible for the internal browning associated with PCI . Chilling injury in pineapple is also associated with the production of hydrogen peroxide , which ages the cells . Therefore, higher PPO, phenolics, and H2O2 production are common biochemical markers of IB initiation . Although these metabolic changes have been well documented, our current understanding of chilling injury of pineapple is incomplete. This may be due in part to the lack of knowledge regarding the precise site of IB initiation in pineapple fruit. IB may occur at fruit vascular bundles in pineapple. This is plausible even though there are few existing data concerning pineapple fruit anatomy in relation to IB . However, data from tomato , mango , feijoa , vertical hydroponic nft system and especially banana and avocado support a relationship between VBs and IB. In avocado, fruit VBs separate and become stringy after seven weeks storage at 5.5 ◦C or four weeks storage at 0.5 ◦C .

The exudate leached from the chilling-damaged VB strands was presumed to cause tissue browning . In banana, after storage at 6 ◦C for 3 days, chilling-induced damage to fruit peel VBs was detected by histological staining . The ruptured VBs were also associated with blackening of the surrounding tissue . In pineapple fruit, the F/C region, where IB symptoms are initiated, is the VB-rich region. Thus, investigating the occurrence ofthe early events of IB development, i.e., TS atthe VBs of pineapple fruit, was the aim of this study. Four pineapple cultivars belonging to two groups, ‘Queen’ and ‘Smooth Cayenne’, with three levels of IB sensitivity were used to determine: if IB initiation, i.e., the appearance of translucency symptoms occurs randomly in any living fruit cell or specifi- cally at the VBs, if the VBs are the sites of biochemical activity associated with TS, and if different IB-sensitive cultivars show differences in VB structure and organization.The fruit were harvested at the mature green stage, approximately 18 weeks , or 21 weeks after floral induction. Fruit were harvested early in the morning and transported to the laboratory within 8 h. On arrival, the fruit were sorted by size and maturity. The uniformity of fruit maturation was determined by visual assessment of the outer exocarp, i.e., the opening stage of the fruit lets or eyes at the stem end of the fruit. Only fruit of which all the fruit lets were totally green were selected for further testing . At this stage, flesh color was light yellow, and soluble solids content was greater than 11◦Brix. Dirt and insects on the fruit were removed using an air blower. All leaves atthe stem end were removed and the peduncle was re-cut to remain one inch long. Forty-eight fruit of uniform maturity for each cultivar were selected for chilling injury determinations.

Fruit were either examined immediately after harvest or stored at 10 ◦C and 85% relative humidity for 7, 14, and 21 days to induce translucency symptoms or chilling injury , or stored at 25 ◦C continuously for the same period .The PCI markers, H2O2 and PPO, were not detected by stereomicroscopic analysis of non-chilled fruit , whereas the presence of phenolic compounds was observed . After the TST pineapple fruit was exposed to 10 ◦C for two weeks, TS were observed on approximately 9% of the total surface area of the fruit. These spots only appeared in the F/C, notin the C or the F regions. Tissues with these localized translucent areas at the F/C region were carefully sectioned and further investigated. Higher contents of phenolic compounds, PPO activity, and H2O2 were detected in the translucent tissue as the development of an intense brown color using our diagnostic histological staining assays . These areas of TS served as an indicator of IB initiation and were further examined under a light microscopy with higher magnification . It was clearly evident that the coloration was more concentrated in the translucent tissue . Furthermore, PPO activity and H2O2 production in the F/C tissue were not detected in non-VB tissue . In this paper, we used a non-specific stain as an indicator of PPO activity, which in turn acted as a proxy for IB. Our assay does not allow us to definitely detect PPO activity, but instead, may be a good indicator of the spatio-temporal occurrence of IB. The staining appears only in chilled TST fruit and is absent from tissues with no manifestation of IB, i.e., non-chilled and senescing tissue . Furthermore, no staining was visible in MD2, a cultivar with no known PPO increase after chilling, and no IB symptoms . These data indicate that for the purpose of this study, the catechol staining was an adequate marker for IB.

VB anatomy at the F/Cregionof control, senesced, and chilled tissue of the IB-susceptible TST pineapple fruit were further investigated under a scanning electron microscope . The VBs consisted of xylem elements, phloem, and VB caps of sclerenchyma fiber. The xylem elements were composed of lignified cells and occupied the center of the VBs. The phloem consisted of thin-walled cells that were small in size and were located adjacent to the xylem elements. A zone of small parenchymal cells surrounding the phloem tissues was modified into sclerenchyma fiber . Such cells were less abundant or lacking around the VBs, resulting in the small size of the VBs in the F region . There were no obvious differences in the non-VB parenchymal cells taken from the F/C region in control compared to chilled fruit . However, there was damage at the phloem tissue in chilled fruit, which resulted in the collapse and flattening of these areas . This region was examined in detail. Unlike chilled fruit, neither TS nor IB was present in senesced fruit , and the tissues of the VB in non-chilled and senesced fruit were similarly healthy with no damage to the phloem in any of the sections examined . There was a high co-occurrence of TS and phloem collapse, nft hydroponic system regardless of tissue location . In most cases, the xylem elements and sclerenchyma fiber around the VB were unaffected by chilling. The specificity of phloem deterioration was clear:it occurred only in chilled fruit, neither in non-chilled nor in senesced tissue. The early changes in phloem integrity that precede the appearance of TS in cold-stored TST were investigated. In this experiment, TST pineapple fruit kept at 10 ◦C for 21 days showed 10% TS, whereas the fruit stored for less than 21 days did not show TS . Then, the degree of phloem damage during this chilled period was categorized as follows: “None Detectable” A), “Mild,” i.e., loss of phloem cell integrity but no collapse B–C), and “Severe,” i.e., collapse of at least 50% of the phloem tissue D). The result showed that, at day 0 and in the senesced tissue , only 25% of all VBs showed mild damage of the phloem. After 7 days at 10 ◦C, the percentage of VBs that showed mild damage increased from 25 to 90%, with the remainder starting to show severe damage. After 14 days storage, severe phloem damage increased from 10 to 40%, even though no TS were observed. After 21 days at 10 ◦C, TS were visible at the base of the fruit and, in this region, all VB showed phloem damage with more than 70% classified as severe. In addition, when the non-affected tissue adjacent to the TS region was observed, a similar proportion of phloem collapse was found . These results clearly suggest that phloem collapse occurs before TS, and that the phloem is the site of chilling injury.Identifying the site of IB occurrence due to low temperature storage might help to better understand the mechanism of PCI in pineapple fruit and other similarly affected species. The present study uniquely demonstrates that IB in pineapple occurred at the phloem and different IB-sensitive cultivars showed differences in VB structure and organization. Similar results were obtained when the entire experiment was repeated 2–3 times.The current results clearly showed that the VB tissue of pineapple fruit was the site of H2O2 accumulation.

There were also higher levels of phenolic compounds and PPO activity specifically due to chilling stress . In addition, phloem damage was found only in the chilling-stress-induced translucent tissue . There may be three explanations as to why TS selectively occurs at the phloem and why phloem collapse leads to TS. The first involves phloem properties,the second, phloem ultrastructure, and the third, changes in cell wall degradation of phloem tissue.The cells in the phloem tissue may contain different cellular properties, e.g., membrane composition or a different antioxidant system, compared to the cells in other tissues. To our knowledge, the cellular properties of phloem tissue have not yet been studied. However, some data supporting this idea was found in Paspalum dilatum Poir. After 3 days at 10 ◦C, ultrastructural change of the chloroplasts in the phloem parenchyma preceded that of the chloroplasts in the lower mesophyll. This different rate of change was also associated with the severity of damage of the upper mesophyll .The phloem tissue contains sieve elements, companion cells, parenchyma cells, and in some cases includes: fibers, sclereids, and laticifers . Transportation through the sieve tube occurs by pores in the sieve plates or sieve areas between the sieve tube elements. These pores allow the plasma membrane of a sieve tube element to be continuous with that of its neighboring sieve tube element. Chilling induces the loss of cell turgor, vacuolization of the cytoplasm, and swelling disintegration of cell organelles . This will lead to leakage of the substrate needed by PPO for the synthesis of the brown substances as proposed by Woolf et al. for avocado. These browning-related substances could be conducted throughout the fruit via the continuous sieve tube connection. The collapse of the phloem would lead to the exudates from the VBs filling up the intercellular air spaces of the surrounding tissue, resulting in translucent spots , and eventually, browning, as proposed for other species .After 21 days at 10 ◦C, paradoxically,the non-TS and adjacentTS regions had a similar proportion of phloem collapse. This may be explained by variation in the number of layers of sclerenchyma cells at the lateral side of the VBs in these regions. Non-TS regions have more fiber layers, which effectively restrict phloem exudates to the surrounding parenchyma tissue, preventing the intercellular air spaces from filling with liquid. The opposite would be true for the TS-regions, which have fewer layers, allowing for a greater flow of exudates to the intercellular spaces and the rapid appearance of TS. Only after a longer period of chilling stress, would the protective function of the sclerenchyma fibers in the non-TS tissue become less effective, leading eventually to TS.Degradation of the cell wall might play a role in phloem collapse upon IB induction. The walls are non-lignified and are weaker in structure compared to the tracheary elements of the xylem . Each sieve tube element is interconnected with the metabolically active companion cells by plasmodesmata, which act as cytoplasmic bridges . In addition, around the plasmodesmata, there is a deposition of callose . As maturation of sieve element progresses, the callose and the middle lamella cell wall material in this region are degraded to form a plate structure with widened pores . In citrus, chilling causes pitting and browning of the flavedo and induces the activity of  -1, 3-glucanases . The capability of this enzyme in degrading callose, which is deposited in the plasmodesmata in sieve element, might play a role in the phloem collapse upon chilling in pineapple. Alternatively, it is possible that the non-VB parenchymal cells located next to the VB tissue might be the sites of IB initiation .

Water used for blanching, post-blanching cooling, and cooking of fruits and vegetables can, in general, be collected and reused for the initial washing of incoming products without treatment . Reuse of flume water. Instead of discharging flume water to the wastewater stream, it can be recovered, filtered, and reused continuously in fluming applications. Alternatively, flume water can be recovered and recycled for use in equipment pre-rinsing and pre-soaking applications elsewhere in the facility . Reuse of compressor cooling water. Cooling water from compressors can be reused as seal water in vacuum pumps instead of fresh water, or as secondary water for other purposes, such as equipment pre-soaking . Warm cooling water can also be stored in insulated tanks for later use in facility cleaning, pre-soaking, and equipment pre-rinsing applications .The U.S. fruit and vegetable processing industry spent nearly $810 million on purchased fuels and electricity in 2002, making energy a significant cost driver for the industry. Energy efficiency improvement is an important way to reduce these costs and to increase predictable earnings in the face of ongoing energy price volatility. Considering the negative impacts of the 2001 spike in U.S. natural gas prices on the industry’s operating costs, stackable planters as well as more recent sharp increases in natural gas prices across the nation, energy efficiency improvements are needed today more than ever.

Many companies in the U.S. fruit and vegetable processing industry have already accepted the challenge to improve their energy efficiency in the face of high energy costs and have begun to reap the rewards of energy efficiency investments. This Energy Guide has summarized a large number of energy-efficient technologies and practices that are proven, cost-effective, and available for implementation today. Energy efficiency improvement opportunities have been discussed that are applicable at the component, process, facility, and organizational levels. Preliminary estimates of savings in energy and energy-related costs have been provided for many energy efficiency measures, based on case study data from real-world industrial applications. Additionally, typical investment payback periods and references to further information in the technical literature have been provided, when available. A key first step in any energy improvement initiative is to establish a focused and strategic energy management program, as depicted in Figure 6.1, which will help to identify and implement energy efficiency measures and practices across and organization and ensure continuous improvement. Tables 5.1 to 5.3 summarize the energy efficiency measures presented in this Energy Guide. While the expected savings associated with some of the individual measures in Tables 5.1 to 5.3 may be relatively small, the cumulative effect of these measures across an entire plant may potentially be quite large. Many of the measures in Tables 5.1 to 5.3 have relatively short payback periods and are therefore attractive economic investments on their own merit.

The degree of implementation of these measures will vary by plant and end use; continuous evaluation of these measures will help to identify further cost savings in ongoing energy management programs. In recognition of the importance of water as a resource in the U.S. fruit and vegetable processing industry, as well as its rising costs, this Energy Guide has also provided information on basic, proven measures for improving plant-level water efficiency, which are summarized in Table 5.4. For all energy and water efficiency measures presented in this Energy Guide, individual plants should pursue further research on the economics of the measures, as well as on the applicability of different measures to their own unique production practices, in order to assess the feasibility of measure implementation.This work was supported by the Climate Protection Partnerships Division of the U.S. Environmental Protection Agency as part of its ENERGY STAR program through the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Many people within and outside of the fruit and vegetable processing industry provided valuable insights in the preparation of this Energy Guide. The authors are particularly grateful to the following people for their helpful comments and advice during the development of this Energy Guide: John Batura of Campbell’s Soup Company, Joseph Benevides of Ocean Spray Cranberries, Austin H. Bonnett , Alan Christie of the J.R. Simplot Company, Elizabeth Dutrow of the U.S. Environmental Protection Agency, Dan Fonner of Heinz North America, Paul Halberstadt of ConAgra Foods, Al Halvorsen of Frito-Lay, Don Hertkorn of ICF Consulting, Ted Jones and Ilene Mason of the Consortium for Energy Efficiency, John Malinowski of Baldor Electric Company, Aimee McKane of Lawrence Berkeley National Laboratory, Leland McPherrin of Escalon Premier Brands, Rob Neenan of the California League of Food Processors, Richard Pate of the Pacific Gas and Electric Company, Linda Raynes of the Electrical Apparatus Service Association, Jon Russett of General Mills, and Terry Young of ConAgra Foods.

Any remaining errors in this Energy Guide are the responsibility of the authors. The views expressed in this Energy Guide do not necessarily reflect those of the U.S. Environmental Protection Agency, the U.S. Department of Energy, or the U.S. Government. We discovered that there are only a few categories of interest in a highly domesticated breeding population and that a small number of features are that ordinal shape categories are highly heritable and that the features needed for accurate classification are also heritable.The data released with this article contain digital images of 6,874 strawberry fruit from 572 hybrids originating from the University of California, Davis, Strawberry Breeding Program. The data for this article, including pre-processed images , processed images , and extracted features , are available on Zenodo. The pre-processed images typically contained multiple berries per image along with a data matrix bar code indicating the genotype ID and other elements of the experiment design. The processed images are 1,000 × 1,000 pixels-scaled binary images of individual fruit. The extracted features data set is provided as a CSV file. Additionally, snapshots of the code and data supporting this work are available in the GigaScience repository, GigaDB. We hope that the release of these data assists others in developing novel morphometric approaches to better understand the genetic, developmental, and environmental control of fruit shape in strawberry, and more broadly in other fruits, vegetables, and specialty crops.k-Means clustering rapidly detects patterns in large, multidimensional data sets used for clustering, decision making, and dimension reduction. It is an iterative algorithm that partitions a data set into a pre-defined number of non-overlapping clusters, k, by minimizing the sum of squared distances from each data point to the cluster centroid. A centroid corresponds to the mean of all points assigned to the cluster. Here, we used k-means to cluster flattened binary images . Individual fruits were segmented from the image background as a binary mask, normalized by the major axis, resized to 100 × 100 pixels, and flattened into a vector . We represented each image as a 10,000-element vector containing binary pixel values. We were able to rapidly and reliably assign images to classes using k-means clustering. In this experiment, we allowed k, the number of permitted categories, to range from 2 to 10. This range was chosen because we anticipate that a human-based classification system would not have the speed or reliability needed for this task, particularly for larger values of k.As high-throughput phenotyping for external fruit characteristics becomes of interest to specialty crop researchers, we expect that this work will have various applications in both applied and basic plant research, stacking pots intellectual property protection and documentation, and waste reduction. Our study showed that strawberry fruit shapes could be robustly quantified and accurately classified from digital images. Most importantly, our analyses yielded quantitative phenotypic variables that describe fruit shape , arise from continuous distributions, and are moderately to highly heritable . We accomplished this by translating 2D, digital images of fruit into categorical and continuous phenotypic variables using unsupervised machine learning and morphometrics. We found that mathematical approaches developed for human face recognition were powerful for strawberry fruit shape phenotyping , that unsupervised shape clustering was robust to sample size deviations, and that only a few quantitative features are needed to accurately classify shapes from images , indicating a paradigm appropriate for genetic dissection.

Digital plant phenotyping is able to empower quantitative genetic analyses by providing heritable and biologically relevant, latent phenotypes in a cost-effective manner. In many cases, these latent traits are derived from PCA, multidimensional scaling , structured equation modeling , persistent homology , or auto-encoding convolutional neural networks, which can be exceedingly abstract and difficult to interpret biologically but may also reveal unexpected patterns of phenotypic and genetic variation. Many of the features described in this study, along with those reported by Turner et al. and Gage et al. , had high heritability and are exciting targets for future quantitative genetic analyses, including GWAS and genomic prediction, which have been shown to be successful for shape features in recent work in rice, apple, and pear. However, the H2 of 1 selected feature in this study, EigenFruitPC3, was estimated to be 0.00. Similar results were reported in carrot for pixel-based root and shoot features, apple for elliptical Fourier leaf shape features , and corn for pixel-based shoot features. Turner et al. attributed the null H2 of root shape characteristics to low phenotypic variation between the inbred parents and genotype × environment interactions. This pattern, while seemingly present, was not discussed in detail by either Migicovsy et al. or Gage et al.. While there may be many drivers for this pattern, we hypothesize that the null estimate may arise from the pixel-based descriptors describing more complex aspects of fruit or root shape. If the non-genetic component of a multivariate phenotype is large, then performing PCA on that multivariate trait could produce leading PCs that describe mostly non-genetic variance . However, there are too few reports to adequately determine the likelihood and causal source of this phenomenon. We empirically derived the shape progression produced in the present study through the application of a new method, PPKC, and used these mathematical categories to interpret the extracted shape features . Ordinal categorical traits are commonplace in quantitative genetic studies, a current standard for phenotyping external fruit characteristics, and enable understanding and explanation of complex, latent space plant phenotypes . PPKC specifically considers the relationship between a cluster at k and all clusters for values <k as a covariance matrix and projects this k-dimensional space to 1 dimension using eigen decomposition. Ordination using dimension reduction techniques, including PCA, correspondence analysis, and MDS has been previously proposed and used in community ecology. Theoretically, the eigen decomposition step of PPKC could be replaced with another technique. However, unlike methods using eigen decomposition, which progressively subdivides variation such that the position on the leading axis is fixed regard-less of the number of axes examined, the position of samples on MDS axes may change when different dimensions are extracted, making MDS axes arbitrary and without meaning other than a convenient reference. PPKC identified 4 exemplary strawberry shape categories in the population that we studied, which were characterized by a progression from ”longer-than wide” to ”wider-than-long”. This ordinal scale can be used in breeding and research programs as traits of interest, or it can be used to organize and interpret more abstract quantitative features, such as EigenFruitPCs or SEM latent variables, through supervised machine learning algorithms. Critically, this gradient agreed with the arbitrarily defined progressions in previous reports. However, unlike previous studies, which suggested using 9 ordinal or11 nominal shape categories, our work presented empirical evidence for a smaller number of mathematically defined shape categories. We determined that k = 4 was the appropriate level of complexity on the basis of the visual appearance of the discovered clusters , high H2 estimates , and the information criteria calculated for the k-means models. Interestingly, PPKC can determine a visually, reasonable phenotypic gradient up to k = 8 despite strong evidence of overfitting for k > 4. We extrapolate that PPKC should continue to work beyond k = 9 so long as new clusters are distinct and do not arise as an artifact of overfitting k. The specific genetic factors that give rise to variation in fruit shape in octoploid, garden strawberry are currently unclear or understudied. The selective pressure exerted on fruit shape in strawberry could have affected large-effect loci, in which case ordinal phenotypic scores are likely to be sufficient for identifying genetic factors affecting fruit shape. Loss- and gain-of function mutations have played an essential role in identifying genes affecting fruit shape in tomato, a model that has been highly instructive and important for understanding the genetics of fruit shape and enlargement in plants.

The U.S. EPA’s ENERGY STAR Building Upgrade Manual recommends a stepwise approach to recommissioning, in which a series of strategically-ordered building “tune up” strategies are pursued in order. First, lighting and supplemental loads should be assessed, then the building envelope, then controls, then testing, adjusting and balancing, then heat exchange equipment, and finally heating and cooling systems. Most of these steps relate to HVAC system components or factors that will directly affect HVAC system energy consumption . For more information, the U.S. EPA’s ENERGY STAR Building Upgrade Manual should be consulted . Energy monitoring and control systems. An energy monitoring and control system supports the efficient operation of HVAC systems by monitoring, controlling, and tracking system energy consumption. Such systems continuously manage and optimize HVAC system energy consumption while also providing building engineers and energy managers with a valuable diagnostic tool for tracking energy consumption and identifying potential HVAC system problems. Several industrial case studies from the United States indicate that the average payback period for HVAC control systems is about 1.3 years .Non-production hours set-back temperatures. Setting back building temperatures during periods of non-use, such as weekends or non-production times, nft channel can lead to significant savings in HVAC energy consumption. Duct leakage repair. 

Duct leakage can waste significant amounts of energy in HVAC systems. Measures for reducing duct leakage include installing duct insulation and performing regular duct inspection and maintenance, including ongoing leak detection and repair. According to studies by Lawrence Berkeley National Laboratory, repairing duct leaks in industrial and commercial spaces could reduce HVAC energy consumption by up to 30% . One commercial building in Apple Valley, California, adopted a technique called the mobile aerosol-sealant injection system to reduce duct leakage. The application of MASIS resulted in a reduction in overall duct leakage from 582 cfm to 74 cfm, leading to a 34% increase in the overall efficiency of the building’s HVAC system . Variable-air-volume systems. Variable-air-volume systems adjust the rate of air flow into a room or space based on the current air flow requirements of that room or space. Variableair-volume systems therefore work to more closely match HVAC load to heating and cooling demands, which reduces energy use. Adjustable-speed drives . Adjustable speed drives can be installed on variable volume air handlers, as well as recirculation fans, to match the flow and pressure requirements of air handling systems precisely. Energy consumed by fans can be lowered considerably since they are not constantly running at full speed. Adjustable-speed drives can also be used on chiller pumps and water systems pumps to minimize power consumption based on system demand.

Heat recovery systems. Heat recovery systems reduce the energy required to heat or cool facility intake air by harnessing the thermal energy of the facility’s exhaust air. Common heat recovery systems include heat recovery wheels, heat pipes, and run-around loops. The efficiency of heat pipes is in the 45% to 65% range , while the efficiency of run-around loops can be slightly higher, in the 55% to 65% range . Fan modification. Changing the size or shape of the sheaves of a fan can help to optimize fan efficiency and airflow, thereby reducing energy consumption. In a case study from the automotive industry, a Toyota plant optimized the sheaves of its fans in lieu of installing ASDs on fans. Toyota found better savings and payback periods with sheave modification than they anticipated to experience from ASDs . Efficient exhaust fans. Exhaust fans are standard components in any HVAC system. Mixed flow impeller exhaust fans offer an efficient alternative to traditional centrifugal exhaust fans. Mixed flow impeller fans are typically 25% more efficient than centrifugal fans, and can also be cheaper to install and maintain. The expected payback period for this measure is around two years . Use of ventilation fans. Ventilation fans installed in the ceilings of work areas can help destratify the workspace air, leading to better circulation of cool air in summer and warm air in winter, and more even distributions of temperature from floor to ceiling. Such fans can help to reduce the load on building heating systems by helping to “push down” warm air that rises to the ceiling during facility heating months.

Yasama Corporation U.S.A., a manufacturer of soy sauce, installed new high bay ceiling fans to improve air circulation at its Salem, Oregon, facility in 2004. Previously, to provide heat during the winter, the company operated ceiling-mounted heaters with 15 hp fans in its production area. However, the fans didn’t de-stratify the air in the production area’s tall ceilings, nor take advantage of the heat given off by process equipment. Furthermore, to provide ventilation in the summer, the company ran the heater fans in “fan only” mode in conjunction with six 3 hp exhaust fans to remove hot air. The new high-bay ceiling fans were operated using only 1.5 hp motors, which were expected to lead to electrical energy savings of 48,000 kWh per year and electricity cost savings of $2,500 . Furthermore, the company expected to save significant amounts of natural gas in heating months through reduced operation of the heaters. Cooling water recovery. If available, secondary cooling water from municipal sources can be leveraged to reduce chiller energy consumption. In Washington, Boeing partnered with Puget Sound Power and Light and the King County Department of Metropolitan Services to recycle secondary treated cooling water into its chiller system. By doing so, Boeing reduced its water consumption by 48 million gallons per year, leading to projected savings of 20% in its cooling energy consumption . As an additional benefit, Boeing also expected to save on refrigerants and treatment chemicals for its cooling tower water. Solar air heating. Solar air heating systems, such as Solarwall® , use conventional steel siding painted black to absorb solar radiation for insulation. Fresh air enters the bottom of the panels where it is heated as it passes over the warm absorber. Fans distribute the air. Using this technology, Ford Motor Company’s Chicago Stamping plant turned the south wall of its plant into a huge solar collector . Energy savings were estimated to be over $300,000 per year compared to conventional gas air systems. Capital costs were $863,000 resulting in a payback period of less than three years. In addition to energy savings, the system was said to provide clean fresh air for employees, even out hot and cold spots in the plant, and reduce emissions. However, this measure is only of interest for buildings in cold climates, and the potential benefits should be analyzed based on the local conditions of each site. Building reflection. Use of a reflective coating on the roof of buildings in sunny, hot climates can save on air conditioning costs inside. Two medical offices in Northern California used reflective roofs on their buildings; one reduced air conditioning demand by 8%, hydroponic nft the other reduced air conditioning demand by 12% . For colder climates, heat lost due to cool roofs also needs to be taken into account, and often negates savings. In addition to location and weather, other primary factors influence energy savings, such as roof insulation, air conditioning efficiency, and building age. Reflective roof materials are available in different forms and colors. Roof gardens on a flat roof improve the insulation of buildings against both hot and cold by providing both heat and air conditioning . In winter, green roofs can freeze, so they carry a slight heating penalty but often still yield net energy savings . In addition, a roof garden can increase the lifetime of the roof, provide and reduce runoff, and reduce air pollution and dust. Today, Germany installs over 10 million ft2 of green roofs a year, helped in part by economic incentives . The Gap Headquarters in San Bruno installed green roofs in 1997 . In addition to saving energy and lasting longer than traditional roofs, a roof garden absorbs rain, slowing run-off to local storm drains. Other simple options for decreasing building HVAC energy use exist for certain conditions. Shade trees reduce cooling for hot climates. Shade trees should be deciduous trees and planted on the west and southwest sides of the building . Trees planted on the north side of the building in cold climates can reduce heating in winter by shielding the building from the wind. Vines can provide both shade and wind shielding. Building insulation. 

Adding insulation to a facility will nearly always result in the reduction of utility bills. Older buildings are likely to use more energy than newer ones, leading to very high heating and air conditioning bills. Even for a new building, adding insulation may save enough through reduced utility bills to pay for itself within a few years . Various states have regulations and guidelines for building insulation, for example, California’s Energy Efficiency Standards for Residential and Nonresidential Buildings . Going beyond regulated insulation levels may be economically beneficial and should be considered as part of the design of a new building, as well as for reconstruction of existing buildings. For refrigerated warehouses, much higher levels of insulation are preferred. Low emittance windows. Low emittance windows are another effective strategy for improving building insulation. Low emittance windows can lower the heat transmitted into a building and therefore increase its insulating ability. There are two types of Low-E glass, high solar transmitting and low solar transmitting . The U.S. DOE supports the development of new window and glazing technology, while ENERGY STAR provides a selection of rated Low-E windows. New window and glazing technology is being developed continuously around the world.22Turning off lights in unoccupied areas. An easy and effective measure is to encourage personnel to turn off lights in unoccupied building spaces. An energy management program that aims to improve the awareness of personnel with regard to energy use can help staff get in the habit of switching off lights and other equipment when not in use. Lighting controls. Lights can be shut off during non-working hours by automatic controls, such as occupancy sensors that turn off lights when a space becomes unoccupied. Occupancy sensors can save up to 10% to 20% of facility lighting energy use . Numerous case studies throughout the United States suggest that the average payback period for occupancy sensors is approximately 1 year . In a case study from the pharmaceutical industry, at the Merck office and storage building in Rahway, New Jersey, lighting panels were programmed to turn off automatically during expected periods of building non-use . Annual savings amounted to 1,310 MBtu per year, which corresponded to avoided energy-related carbon dioxide emissions of nearly 260 tons per year . Manual controls can be used in conjunction with automatic controls to save additional energy in smaller areas. Other lighting controls include daylight controls for indoor and outdoor lights, which adjust the intensity of electrical lighting based on the availability of daylight. An example of energy-efficient lighting control is illustrated by Figure 11.1, which depicts five rows of overhead lights in a workspace. During the brightest part of the day, ample daylight is provided by the window and thus only row C would need to be turned on. At times when daylight levels drop, all B rows would be turned on and row C would be turned off. Only at night or on very dark days would it be necessary to have both rows A and B turned on . These methods can also be used as a control strategy on a retrofit by adapting the luminaries already present. Exit signs. Energy costs can be reduced by switching from incandescent lamps to light emitting diodes or radium strips in exit sign lighting. An incandescent exit sign uses about 40 W, while LED signs may use only about 4W to 8 W, reducing electricity use by 80% to 90%. A 1998 Lighting Research Center survey found that about 80% of exit signs being sold use LEDs . The lifetime of an LED exit sign is about 10 years, compared to 1 year for incandescent signs, which can reduce exit sign maintenance costs considerably. In addition to exit signs, LEDs are increasingly being used for path marking and emergency way finding systems. Their long life and cool operation allows them to be embedded in plastic materials, which makes them well suited for such applications .

These leaks can reduce the heat transferred to the steam and increase pumping requirements. However, such leaks are often easily repaired, saving 2% to 5% of the energy formerly used by the boiler . This measure differs from flue gas monitoring in that it consists of a periodic repair based on visual inspection. The savings from this measure and from flue gas monitoring are not cumulative, as they both address the same losses. Reduction of excess air. When too much excess air is used to burn fuel, energy is wasted because excessive heat is transferred to the air rather than to the steam. Air slightly in excess of the ideal stochiometric fuel/-to-air ratio is required for safety and to reduce emissions of nitrogen oxides , but approximately 15% excess air is generally adequate . Most industrial boilers already operate at 15% excess air or lower, and thus this measure may not be widely applicable . However, if a boiler is using too much excess air, numerous industrial case studies indicate that the payback period for this measure is less than 1 year . For example, at a U.S. DOE sponsored energy audit of a Land O’Lakes dairy facility in Tulare, California, it was estimated that by reducing excess oxygen from 4.5% to 3.0%, the facility would reduce its natural gas costs by $113,000 per year while still meeting stringent NOX emissions limits . As a rule of thumb, the Canadian Industry Program for Energy Conservation estimates that for every 1% reduction in flue gas oxygen, boiler efficiency is increased by 2.5% .

Properly sized boiler systems. Designing the boiler system to operate at the proper steam pressure can save energy by reducing stack temperature, round plant pot reducing piping radiation losses, and reducing leaks in steam traps. This measure is particularly important in fruit and vegetable processing facilities, where due to the seasonality of production, large boilers can often be run at low capacity during the off season, which can result in significant energy losses. In a study done in Canada on 30 boiler plants, savings from this measure ranged from 3% to 8% of total boiler fuel consumption . Savings were greatest when steam pressures were reduced below 70 pounds per square inch . One industrial case study has shown that correct boiler sizing led to savings of $150,000 at a payback period of only 2.4 months . However, costs and savings will depend heavily on the current boiler system utilization at individual plants. Improved boiler insulation. It is possible to use new materials, such as ceramic fibers, that both insulate better and have a lower heat capacity . Savings of 6% to 26% can be achieved if improved insulation is combined with improved heater circuit controls. Due to the lower heat capacity of new materials, the output temperature of boilers can be more vulnerable to temperature fluctuations in the heating elements . Improved boiler process control is therefore often required in tandem with new insulation to maintain the desired output temperature range. At a U.S. DOE sponsored assessment of a Land O’Lakes dairy facility in Tulare, California, it was found that by improving insulation on the facility’s steam header, boiler economizer, and process hot water tank, the company could save nearly $35,000 per year in reduced boiler fuel costs . Boiler maintenance. 

A simple maintenance program to ensure that all components of a boiler are operating at peak performance can result in substantial savings. In the absence of a good maintenance system, burners and condensate return systems can wear or get out of adjustment. These factors can end up costing a steam system up to 30% of initial efficiency over two to three years . On average, the energy savings associated with improved boiler maintenance are estimated at 10%. Improved maintenance may also reduce the emission of criteria air pollutants. Fouling on the fire side of boiler tubes or scaling on the water side of boilers should also be controlled. Fouling and scaling are more of a problem with coal-fed boilers than natural gas or oil-fed boilers . Tests reported by CIPEC show that a fire side soot layer of 0.03 inches reduces heat transfer by 9.5%, while a 0.18 inch soot layer reduces heat transfer by 69% . For water side scaling, 0.04 inches of buildup can increase fuel consumption by 2% .Flue gas heat recovery. Heat recovery from flue gas is often the best opportunity for heat recovery in steam systems . Heat from flue gas can be used to preheat boiler feed water in an economizer. While this measure is fairly common in large boilers, there is often still room for more heat recovery. The limiting factor for flue gas heat recovery is that one must ensure that the economizer wall temperature does not drop below the dew point of acids contained in the flue gas . Traditionally, this has been done by keeping the flue gases exiting the economizer at a temperature significantly above the acid dew point. In fact, the economizer wall temperature is much more dependent on feed water temperature than on flue gas temperature because of the high heat transfer coefficient of water. As a result, it makes more sense to preheat feed water to close to the acid dew point before it enters the economizer.

This approach allows the economizer to be designed so that exiting flue gas is just above the acid dew point. Typically, one percent of fuel use is saved for every 45°F reduction in exhaust gas temperature . At the Odwalla Juice Company’s facility in Dinuva, California, the installation of an economizer was expected to save over $21,000 per year in energy costs and over 4,000 MBtu of boiler fuel per year . Odwalla’s expected payback period for the economizer was just 10 months. McCain Foods, a major producer of frozen French fried potatoes, installed an economizer at its Scarborough, England, facility as part of a plant-level heat recovery project in 1995. The new economizer saved the facility 67 therms of natural gas per hour, leading to energy savings of £67,000 per year with a simple payback period of 2.5 years . Similar results were expected at Schneider Foods, a packaged and frozen meats company in Ontario, Canada. In 2005, the company installed a dual-stage economizer, which heats both boiler feed water and boiler makeup water with heat recovered from flue gas, leading to savings of about $225,000 per year and a payback period of less than two years . Condensate return. Reusing hot condensate in boilers saves energy, reduces the need for treated boiler feed water, and reclaims water at up to 100°C of sensible heat. Typically, fresh feed water must be treated to remove solids that might accumulate in the boiler; however, returning condensate to a boiler can substantially reduce the amount of purchased chemical required to accomplish this treatment. The fact that this measure can save substantial energy costs and purchased chemicals costs often makes building a return piping system attractive. A 2005 study of seven different fresh fruit and vegetable processing plants in California estimated a payback period for this measure ranging from approximately two to three years . Blow down steam recovery. When water is blown from a high-pressure boiler tank, the pressure reduction often produces substantial amounts of steam. This steam is typically low grade, but can be used for space heating and feed water preheating. The recovery of blow down steam can save around 1% of boiler fuel use in small boilers . In addition to energy savings, blow down steam recovery may reduce the potential for corrosion damage in steam system piping.Green Giant of Canada, a manufacturer of frozen and canned vegetables, installed a shell and tube heat exchanger to recover heat from boiler blow down. This measure led to annual energy savings of roughly $1,500 with a payback of approximately 2 years . Boiler replacement. Substantial efficiency gains can often be realized by replacing old boilers with new, round garden pot higher efficiency models. In particular, the replacement of inefficient coal fired boilers with natural gas-fired boilers is a sound strategy for reducing boiler fuel costs while also reducing emissions of air pollutants. Valley Fig, a manufacturer of fig pastes and concentrates in Fresno, California, replaced their old and inefficient 300 boiler horsepower fire tube boiler in 2004 in order to meet stringent NOX emissions limits. The 300 bhp boiler was replaced with two smaller and more efficient 100 bhp boilers, which not only allowed them to meet the facility’s steam demands while lowering NOX emissions, but also reduced their natural gas costs by 8% to 10% . Additionally, Valley Fig received a $16,000 rebate check from Pacific Gas & Electric for improved fuel efficiency. Direct contact water heating. In direct contact water heaters, water is sprayed downward through a vertical chamber that serves as a flue for combustion gases.

Because the hot combustion gases heat the water directly, this water heating system is more efficient than traditional boilers. Hot water is collected in a storage tank while the combustion gases exit the system at near-ambient temperatures. Since water does not contact the burner flames, complete combustion occurs before the gases heat the water. Thus, water quality is maintained to a level that is appropriate for food processing operations . Additionally, direct-contact water heaters can operate at atmospheric pressure, which avoids the safety hazards and insurance premiums that can come with pressurized boiler operation. One commercially-available direct-contact water heater by Kemco Systems, Inc., offers water heating efficiencies of up to 99.7%, which is a significant improvement compared to the 60% to 75% efficiencies achievable with traditional water heating technologies . Approximately 3,000 Kemco direct-contact water heaters are said to be in operation worldwide, with average payback periods ranging from one to two years. Another commercially-available direct-contact water heating system by QuickWater was installed at Golden Temple, a natural foods manufacturing company based in Oregon, in 2003. Golden Temple’s annual energy savings for water heating were estimated at 22%, with annual energy cost savings totaling around $2,300 . Additionally, the direct-contact water heater was said to offer a smaller footprint than traditional systems as well as a longer life .Steam and hot water distribution systems are often quite extensive and can be major contributors to energy losses within a fruit and vegetable processing plant. Energy efficiency improvements to steam distribution systems are primarily focused on reducing heat losses throughout the system and recovering useful heat from the system wherever feasible. The following measures are some of the most significant opportunities for saving energy in industrial steam distribution systems. Improved distribution system insulation. Using more insulating material or using the best insulation material for the application can save energy in steam systems. Crucial factors in choosing insulating material include low thermal conductivity, dimensional stability under temperature change, resistance to water absorption, and resistance to combustion. Other characteristics of insulating material may also be important depending on the application, such as tolerance of large temperature variations, tolerance of system vibrations, and adequate compressive strength where the insulation is load bearing . Industrial case studies indicate that the payback period for improved insulation is typically about one year . The S. Martinelli Company, an apple juice manufacturer in Watsonville, California, found that insulating steam distribution lines not only led to energy savings, but also reduced the amount of heat inadvertently released to interior spaces . Insulation maintenance. It is often found that after heat distribution systems have undergone some form of repair, the insulation is not replaced. In addition, some types of insulation can become brittle or rot over time. As a result, a regular inspection and maintenance system for insulation can also save energy . Steam trap improvement. Using modern thermostatic element steam traps can reduce energy use while also improving reliability. The main efficiency advantages offered by these traps are that they open when the temperature is very close to that of saturated steam , purge non-condensable gases after each opening, and are open on startup to allow a fast steam system warm-up. These traps also have the advantage of being highly reliable and useable for a wide variety of steam pressures . Steam trap maintenance. A simple program of checking steam traps to ensure that they are operating properly can save significant amounts of energy for very little money.

Although shape shows a negative relationship with biomass, this influence is minimal when compared with photosynthesis . However, leaf shape shows the largest influence on both yield and fruit BRIX, with photosynthesis second, and is the only positive contributor to yield . This positive correlation is from rounder, Potato Leaf Morph-like leaves, while narrower leaves have the opposite effect based on the PC contributions to leaf shape. The negative effect of photosynthesis on tomato fruit yield and the strong contribution of leaf shape to yield and BRIX are novel findings that run counter to the interpretation of fruit quality improvement, as increased photo assimilate should result in more available sucrose to stronger sinks such as fruit . To test the model performance we used PLSPREDICT on the entire heirloom dataset used to build the structural model. Table S8 shows the mean absolute percentage error and Q2 value for the complete model. We also used part of the dataset that included ABC Potato Leaf and Aunt Ginny’s Purple in a similar analysis . The complete model has c. 20–30% error for each LV, which is expected given the diversity of genotypes in the dataset, with fruit weight giving the highest MAPE, at 93.2% . The Q2 value for most variables is positive and shows that they have relevance in the predictive performance, with the exception of leaf sugar, which is slightly negative . In the case of ABC Potato Leaf and Aunt Ginny’s Purple, two lines selected randomly to test the model on individual cultivars, round plant pot a significant increase in Q2 and decrease in MAPE is seen for all LVs except leaf sugar . This indicates that the model is substantially stronger in predictive performance for individual cultivars, but also predicts well with the complete model.

To evaluate the predictive performance of our model on additional datasets, we used data from two other cultivars grown in the same field, M82 and Lukullus, that were not used to construct the model. PLSPREDICT was used in SMARTPLS 3.0, along with the structural model constructed using the heirloom cultivars, to test the model performance by use of training sets and hold out samples, both taken from the M82/Lukullus dataset. By using the leaf shape PC values, we were able to compare the predicted mean values for the remaining MVs, or the predicted measured values, against the actual measured values and evaluate the relative performance of the model. Tables 2 and 3 show the results for M82 and Lukullus, respectively. PC values for leaf shape are not included as they are input variables and used for predicting the other values. For M82 the predicted median values compared with the actual median values showed under 1% difference for all except leaf complexity, which had a percentage difference of 8.42% . This indicates that the model was underpredicting the leaf complexity of M82 by c. 8%. Lukullus-predicted values were also under 1% different, except for leaf complexity and stomatal conductance which varied by 2.56% and 1.31%, respectively . In addition to the predicted values PLSPREDICT also tests the model performance and reports the root mean square error, mean absolute error, and MAPE for each of the MVs tested . The MAPE shows the accuracy of the predictions, with lower percentages representing better performance. Leaf complexity for both cultivars showed the largest MAPE values, 201.2% and 26.5% in M82 and Lukullus, respectively .

The M82 MAPE indicates that the model does not predict leaf complexity well for mid-level complexities such as 18 but does improve at high-end leaf complexities near 40 . Most heirloom cultivars had low leaf complexities , potentially explaining the poor performance in predicting leaf complexity for M82. Contrary to previous findings , we found that leaf complexity does not impact yield or BRIX, and only impacts vegetative biomass, so this inaccuracy would only impact vegetative output predictions by the model. Lukullus has indeterminate growth like the heirlooms analyzed here, but M82 is determinate; however, the predictive accuracy of the model was still good, indicating its usefulness in assessing field performance of other tomato cultivars.The primary focus of crop improvement has been on fruit traits and photosynthesis , with some studies focusing on how sugars are moved from source to sink. Despite heirloom varieties with the Potato Leaf Morph being prized for fruit quality by the gardening community, vegetative traits such as leaf shape have been relatively ignored in breeding efforts. In this study we investigated the role of leaf shape on fruit quality by measuring both input traits and output traits for 18 heirloom cultivars. All these cultivars were classified as Potato Leaf, but varied greatly in their leaf shapes, development, and fruit quality . We found that these lines do not vary significantly in overall photosynthetic capacity, or their usage of light when available , suggesting that the variation in BY among these cultivars was not a result of improved/decreased photosynthetic capacity. While our measurements for photosynthesis do not show significant difference when PAR is available, the PARi differed between cultivars based on their growth patterns . All cultivars exceeded 1200 lmols m 2 s 1 of PARi but varied in the later weeks between 1200 and 2000 lmols m 2 s 1 . Combining multiple complex physiological and morphological measurements into informative relationships has proven difficult and has limited our understanding of how these different traits impact each other .

Focusing on any one part, such as photosynthesis or fruit sink strength, while providing improvements , occurs at the expense of a comprehensive understanding of the overall relationships between these traits. Analyzing the individual PCs revealed significant differences in leaf shape among the heirloom cultivars, with several having stronger Potato Leaf Morphs and higher BY values , with some correlation between these traits. Potential epidermal shape changes that could arise from leaf shape changes and that could influence yield would relate to stomatal number. A previous study in 2002 analyzed several tomato cultivars developmentally and histologically and found no real differences between these cultivars. This and another study in 2010 suggest that there are no gross anatomical differences between these tomato cultivars. We used PLS-PM to combine all these measured traits, using the modeled final harvest data as input to find causative relationships . Strong relationships among gas exchange, light, and photosynthesis were expected, along with a strong positive effect of photosynthesis on vegetative biomass . Photosynthesis has a strong positive effect on fruit BRIX, both directly and indirectly . Increased photosynthesis results in lowered leaf sugar content, and a concomitant increase in fruit BRIX. It is possible that increased sugar production from photosynthesis results in higher rates of transport of sugars out of the leaves and into sinks. The mechanisms that regulate source–sink relations and sugar distribution are still not fully understood on a whole-plant physiological level ; however, based on our model, increased photosynthesis negatively impacts total yield . While photosynthesis does lead to increased sugar production and is shown in our model to drive higher sugar content within existing fruit, it does not provide a means to increase yield. Leaf shape, specifically rounder, less lobed leaves, round garden pot has a positive effect on both fruit BRIX and yield . Of all the factors measured here, only leaf shape positively influenced yield, with other paths having negative influences . Rounder leaves still drive slightly increased photosynthesis indicated by the thin arrow , which results in increased fruit BRIX. This path should also result in decreased yield. However, leaf shape has a strong positive and direct correlation with yield that overcomes the negative impact of photosynthesis and leads to increased yield as well as BRIX . Conversely, with narrow leaflets there is a small negative impact on photosynthesis which should result in increased yield, but narrow leaves have a direct negative impact on yield which is stronger than the photosynthetic pathway . The strong causative relationship among leaf shape, fruit BRIX, and yield suggests that leaf shape impacts both highfruit BRIX and increased number of fruits, probably by modulating sugar distribution, therefore bypassing the direct impacts of photosynthesis itself . How leaf shape affects this distribution is unclear, as it does not act directly through leaf sugar content, or through strong regulation of photosynthesis to improve yield . A recent study looked at the diversity of leaf shape in sweet potato . Any correlations between leaf shape and yield traits in this species would be of interest and help to establish general principles. The whole-genome phylogenetic analysis of 23 tomato cultivars showed many of the Potato Leaf Morph cultivars were closely related to each other, with the exception of Brandywine, though it did not show the origin of the C-locus mutation .

To address this and identify if this morphology was selected for in breeding, we performed PHYLONETWORKS analysis . This analysis showed several hybridizations between Potato Leaf Morph and nonPotato Leaf Morph cultivars, and probably a unique incidence of the C-locus mutation in Prudens Purple . PHYLONETWORKS analysis of chromosome 1, 6, and 12 specific common SNPs each showed unique hybridization events, distinct from those seen in the WGS analysis . The PHYLONETWORKS analysis suggests multiple hybridization events with Potato Leaf Morph-containing cultivars. Potato leaf cultivars have been suggested to increase disease resistance compared with regular leaf varieties and may have been selected for this reason or for other as yet-unknown benefits present. We have shown that leaf shape strongly impacts the overall fruit quality in tomato, with rounder, less lobed leaves giving rise to higher yield and higher fruit BRIX. Photosynthesis, surprisingly, has a negative impact on yield while still positively contributing to fruit BRIX. Using data from cultivars not included in making our path model, we also showed that the model has a strong predictive performance for linking leaf shape to BY and could be used to potentially predict the outputs of a cultivar using leaf shape data . Our work shows the importance of leaf shape to yield and BRIX across a wide array of genetic backgrounds, implicating leaf morphology in playing a significant and previously unidentified role in tomato fruit quality.The absence of leaves in winter provides a clear view of the framework of the tree and the opportunity to thin or head any branches. The amount of dormant pruning will be less if trees receive appropriate summer training and pruning. For pest and disease control, prune out any dead, diseased, crossed, or broken branches as well as water sprouts and root suckers. Remove and destroy all diseased wood. Periodically disinfect cutting tools with a sanitizing liquid or a 1:1 bleach and water solution during pruning and at any time tools come into direct contact with diseased tissue. Oil shears immediately after use to avoid corrosion. Paint the trunk and lower branches of young trees that are exposed to hot afternoon sun with a 1:1 mixture of white interior latex paint and water to prevent sunburn injury and reduce borer infestations. Apply the paint mixture from 2 inches below the soil line to 2 feet above.Sanitation is necessary to prevent disease and pests and reduce the need for sprays. If possible, rake and dispose of all leaves after they drop and before the first rains. Remove and destroy over-wintering fruit in the tree and on the ground to eliminate sources of insects and diseases the next season.Oils used at this time of year include insecticidal oils, narrow range, supreme, or superiortype oils. A supreme or superior-type oil spray, applied during the delayed dormant period just as eggs are about to hatch, should keep European red and brown mites below damaging levels if predators are not disrupted by sprays for other pests. Supremeor superior-type oils will kill overwintering eggs of aphids on fruit trees if applied as a delayed dormant application just as eggs are beginning to hatch in early spring. These treatments will not give complete control of aphids and are probably not justified for aphid control alone. Dormant oil sprays alone DO NOT control oriental fruit moth, navel orange worm, peach twig borer, or two-spotted and Pacific spider mites. Dormant disease control applications use materials such as copper, lime sulfur , Bordeaux , or a synthetic fungicide to limit infection and prevent the spread of certain fungal diseases like leaf curl, shot hole, powdery mildew, and scab.A dormant spray may not be warranted every year in the backyard orchard except where peach leaf curl is consistently a problem.

The VTC4 gene has been reported to be a bifunctional enzyme, also catalysing conversion of D-myoinositol 3-phosphate to myoinositol in myoinositol biosynthesis . The myoinositol can be converted to UDP-glucuronic acid , which is a common but cell wall-specific biochemical precursor for cell wall bio-genesis . The bifunctional VTC4 enzyme facilitates formation of AsA and cell walls . Therefore, the VTC4 may be a candidate gene for enhancing nutrition and delaying softening via influencing of the AsA production and cell wall formation in tomato fruit. Tomato has three VTC4 homologue genes SlIMP1, SlIMP2, and SlIMP3 . The SlIMP3, which has the highest expression level in tomato fruit, was selected for this investigation as it also has bifunctional enzyme activity, similar to that of the VTC4 in Arabidopsis. Overexpression of SlIMP3 in tomato increases the AsA content in multiple tissues. Overexpression of SlIMP3 increased the myoinositol accumulation, cell wall thickness, and altered cell-wall composition. Overexpression of SlIMP3 markedly delayed fruit softening and enhanced fruit resistance to Botrytis cinerea. The results demonstrate a critical role for SlIMP3 in AsA biosynthesis and cell wall bio-genesis and provide new method of delaying fruit softening and extending shelf-life of tomato.Three IMP genes have been identified in tomato . Alignment and sequence analysis revealed that the IMP amino acid sequences contained signature motifs . A phylogenetic analysis of tomato IMP sequences, together with Arabidopsis and tobacco IMP-related genes, large plastic planting pots was carried out using the neighbour-joining method on mega6. The results indicated that SlIMP3 was most closely related to NtIMP3 and clustered with SlIMP1, NtIMP1, and AtVTC4 into one subfamily .

The expression pattern of three SlIMPs in vegetative and reproductive tissues was carried out using the online TomExpression platform. The SlIMPs had ubiquitous expression in all tested tissues, including roots, shoots, leaves, flowers, and fruits. It was interesting that SlIMP3 gene had highest expression levels during fruit development and ripening . A qRT-PCR test was performed to confirm the expression patterns of SlIMPs in tomato plants. The results were consistent with the TomExpression data, with the highest expression levels of SlIMP3 in fruits . The expression pattern of SlIMP3 was also explored through a transgenic tomato plant in which GUS reporter gene was driven by the SlIMP3 promoter. Consistent with the qRT-PCR results, the GUS staining revealed the ubiquitous expression pattern of SlIMP3 in leaves, stems, buds, flowers, and fruits at different developmental stages, with strong expression in immature green fruit. The expression of SlIMP3 was also decreased in the ripening stages, though weakly expression in orange fruits .It is established that the VTC4 gene encodes a bifunctional enzyme that influences myoinositol and ascorbate biosynthesis . We expressed and purified recombinant SlIMP3 to analyse the catalytic character. The open reading frame of the SlIMP3 gene was cloned into a pGEX-4T-1 vector to generate translational fusion with glutathione-S-transferases . The recombinant protein was expressed in E. coli and purified with affinity chromatography. The molecular mass of the GST-SlIMP3 fusion protein was estimated to be 55 kD , similar to the predicted molecular mass. Mg2+ is necessary for myoinositol monophosphatase activity.

Optimal SlIMP3 activity was obtained by examining the optimum MgCl2 concentration for enzyme activity from 1 to 30 mM of MgCl2. The 3.5 mM of MgCl2 concentration was the most effective at a pH of 7.0 . The Arabidopsis VTC4 enzyme has been reported to use L-Gal 1-P and D-Ins 3-P as substrates . Therefore, the catalytic properties of SlIMP3 enzyme were analysed using L-Gal 1-P and D-Ins 3-P as substrates. In the reaction mixtures of 3.5 mM of MgCl2, pH 7.0, and 2 mg of enzyme, the apparent Km for L-Gal 1-P was 0.29 mM and that for D-Ins 3-P was 0.28 mM . The apparent Vmax values of SlIMP3 for L-Gal 1-P and D-Ins 3-P calculated were 6.0 and 8.0 units, respectively. LiCl was an important inhibitor, which inhibited the catalytic effect of IMPs on the substrate. In previous studies, LiCl inhibited the catalytic activity of VTC4 for D-Ins 3-P, and the half-maximal inhibitory concentration was 0.08 to 0.1 mM . The inhibition of SlIMP3 for D-Ins 3-P by LiCl was also tested . The IC50 of SlIMP3 with LiCl was 0.03 to 0.05 mM when the reaction contained 0.5 mM of substrate. The relationship between AsA biosynthesis and SlIMP3 gene function, transgenic lines expressing either sense or anti-sense SlIMP3 constructs under the control of the cauliflower mosaic virus 35S promote were further characterized. A qRT-PCR was conducted to analyse the expression levels. The independent overexpressed or anti-sense lines, which displayed substantial altered gene expression by comparison with the wild type plants, were used for further analysis . Total AsA and reduced AsA content quantification were conducted using these transgenic plants. Interestingly that altered SlIMP3 expression led to dramatic AsA content change in different tissues of the transgenic lines .

The AsA content quantification in leaf, stem, and different fruit developmental stages indicated that SlIMP3-overexpressed fruits accumulated higher amounts of AsA than the WT plants, whereas the downregulated fruits had lower AsA content than the WT plants. In addition, qRT-PCR results indicated that SlPGI, SlGMP1, SlGMP3, SlGGP1, SlGGP2, and SlGalLDH were significantly upregulated in SlIMP3-overexpressed fruits . We also found that SlGalLDH was significantly down-regulated in downregulated fruits . These results indicated that the SlIMP3 gene regulated AsA biosynthesis in tomato plants.Postharvest fruit storage was conducted to test the influence of SlIMP3 overexpression on fruit softening. The WT and SlIMP3- downregulated fruits were shrivelled after 26 d of storage versus the well maintained pericarp quality of the SlIMP3-overexpressed fruits . The fruit water loss of the SlIMP3-overexpressed fruits was less than that of WT and SlIMP3-downregulated fruits . The SlIMP3-overexpressed fruits also were firmer throughout fruit development versus the downregulated fruits and WT fruits, which were again similar . Ethylene production in SlIMP3-overexpression and SlIMP3-downregulated fruits did not change significantly compared with the WT fruits . Overexpression of SlIMP3 had no effect on fruit yield and weight . In addition, both the up-regulation and down-regulation of SlIMP3 did not affect the fruit development and maturation . Transmission electron microscopy was used to examine the cell wall thickness to determine the effects of altered SlIMP3 expression on cell walls. The SlIMP3-overexpression fruits had thicker cell walls than those of WT fruit but not of those of SlIMP3-downregulation fruits . At the same time, we measured the expressions of cell wall metabolism-related genes as previously described . qRT-PCR results indicated that SlEXP1, SlPG2, SlPL, SlTBG4, SlXYL1, and SlXTH5 were significantly down-regulated in SlIMP3-overexpressed fruits . Moreover, like fruits, the cell wall in SlIMP3- overexpression leaves and stems were also increased . Collectively, these results suggested overexpression of SlIMP3 increased the cell wall thickness, delayed the fruit softening, and enhanced tomato shelf-life.To gain more insight into the mechanism by which cell wall thickness was impacted in SlIMP3-overexpressed plants, biochemicals related to cell wall bio-genesis was analysed. Measurement demonstrated overexpression of the SlIMP3 gene increased myoinositol in mature green and red fruits versus no effect of the downregulation of SlIMP3 gene . Uronic acid content was measured in the cell wall of mature green and red fruits by high-performance liquid chromatography . The uronic acid content increased markedly in SlIMP3-overexpressed red fruits but was unchanged in SlIMP3-downregulated fruits and WT fruits . Various neutral sugars, rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose were also measured. The rhamnose, xylose, mannose, black plastic planting pots and galactose content of SlIMP3-overexpressed red fruits were significantly higher than that of WT . Collectively, these results suggested that overexpression of SlIMP3 improved cell wall bio-genesis in tomato fruit.Because myoinositol is related to cell wall bio-genesis, the WT tomato plants were treated with 10 mg/L, 100 mg/L, and 1 g/L of myoinositol to examine the role myoinositol in cell wall bio-genesis and fruit softening. When treated with 10 mg/L of myoinositol, the fruit cell wall thickness increased slightly, but the fruit softening and water loss were unchanged versus the control.

When treated with 100 mg/L, myoinositol fruit softening was delayed, cell wall thickness increased, and water loss decreased significantly . However, after treatment with 1 g/L of myoinositol, fruit cell wall thickness, storage life, and water loss were unaffected . The 10 mg/L and 100 mg/L of myoinositol treatments sharply increased fruit AsA concentration, but 1 g/L of myoinositol did not modulate fruit AsA production . These experimental results demonstrated exogenous myoinositol application 100 mg/L enhanced the cell wall bio-genesis and delayed softening in tomato fruits. A B. cinerea spore suspension surface applied to injured tomato transgenic tomato fruits produced significantly smaller lesion diameters in SlIMP3-overexpressed fruits versus those in WT and SlIMP3-downregulated fruits, which were approximately equal . Similarly, the biomass of B. cinerea detected with qRT-PCR was significantly lower in the SlIMP3-overexpressed fruits versus the WT and downregulated fruit, which were again approximately equal . Moreover, the expression levels of pathogen-related genes were significantly up-regulated in SlIMP3-overexpressed fruits but were unchanged in SlIMP3-downregulated fruits and WT fruits. This demonstrated overexpression SlIMP3 in tomato significantly improved the tolerance to B. cinerea.In Arabidopsis, the VTC4 gene encodes a bifunctional enzyme that catalyses conversion of L-Gal 1-P to L-galactose in AsA biosynthesis and catalyses conversion of the D-Ins 3-P to myoinositol. In tomato, three SlIMP isoforms are lithium-sensitive enzymes that catalyse the myoinositol biosynthesis from myoinositol monophosphate. In this study, the SlIMP3 gene that had highest expression level among the SlIMP genes was expressed and the SlIMP3 protein purified. The SlIMP3 showed high affinity with the L-Gal 1-P and D-Ins 3-P, and was sensitive to lithium, consistent with enzymatic properties of the Arabidopsis VTC4 . Overexpression of the SlIMP3 gene increased the AsA content, while downregulation of the SlIMP3 gene decreased the AsA content in tomato .Overexpression of the SlIMP3 gene also increased the myoinositol content in tomato fruit . Our data indicated the SlIMP3, which functions like the VTC4, is involved in the biosynthesis of AsA and myoinositol in tomato. But downregulation of the SlIMP3 did not decrease the myoinositol in tomato fruit , which may indicate a redundancy in myoinositol biosynthesis, and suggests the SlIMP1 and SlIMP2 may supplement the enzymatic activity of SlIMP3 in tomato. Interestingly, we found that the expression level of SlIMP2 was significantly up-regulated in SlIMP3-downregulated fruits, which may compensate myoinositol biosynthesis. However, the relationship between SlIMP2 and SlIMP3 remains unknown. Perhaps, in future studies, a tertiary mutant of SlIMPs can be generated using CRISPR/Cas9 methods and used to study their precise function in the biosynthesis of AsA and myoinositol in tomato. In Arabidopsis, overexpression of the myoinositol oxygenase increased AsA content, suggesting that myoinositol can act as a precursor for ascorbate biosynthesis. However, it has also been reported that the MIOS controls the myoinositol level, but does not contribute to AsA biosynthesis . In this study, treating fruits with 100 mg/l of myoinositol not only increased the cell wall bio-genesis, but also increased the fruit’s AsA content . The myoinositol was converted into D-Glucuronic acid by MIOS and the D-Glucuronic acid was catalysed into L-gulonate, which can be converted into the AsA by a multistep process in animals. Our results support that the hypothesis that the myoinositol oxidation pathway contributes to AsA biosynthesis in tomato fruit. Perhaps rigorous radiotracer experiments could elucidate the pathway of myoinositol conversion into AsA in tomato fruits.Myoinositol has been reported to play an important role in cell wall formation . Myoinositol is catalysed by inositol oxidase to D-glucuronic acid, a precursor of pectin and hemicellulose in cell wall biosynthesis . Radioactive myoinositol injected into ripening strawberry fruits was converted to the Dgalacturonosyl residue of pectin and D-xylosyl residues of hemicellulose . In this study, overexpression of SlIMP3 markedly increased the myoinositol, uronic acid and neutral sugar content, and fruit cell wall thickness . Simultaneously, treating fruits with 100 mg/L of myoinositol produced a similar phenotype as that of SlIMP3-overexpressed fruits . The data presented here proved that myoinositol participates in tomato fruit cell wall bio-genesis. Silencing of the PL gene reinforced the tricellular junction in the fruit cell wall and delayed the tomato fruit softening . Mutations in the GA2-oxidase gene improves cutin and wax biosynthesis and increases tomato fruit firmness and shelf-life . In this study, overexpression of SlIMP3 gene increased cell-wall thickness and fruit firmness, delayed fruit softening, and prolonged tomato fruit shelf-life . These results support that improving cell wall bio-genesis is an effective strategy for delaying fruit softening and extending fruit shelf-life.

With fruit sizes reaching almost 300 grams, it is likely that thinning may have been excessive and crop load per tree could have been increased while still reaching above minimum fresh market size requirements. The strong yield for high density plantings of August Flame during the 2017 harvest supports reports of higher density plantings reaching full cropping sooner than low density systems . By 2018 the Nema Quad systems were able to produce fruit of similar size compared to systems with size-controlling rootstocks, but fruit were still not the largest. Large fruit sizes indicate that the amount of thinning could, again, have been reduced. In 2019 there was noticeable water stress in the field due to some irrigation problems, but the magnitude of the problem was not documented. It is likely the water stress was a reason for some of the smaller fruit sizes compared to previous years. Most interesting about the 2019 season was the performance of the C-9 Quad system and how after producing significantly smaller and fewer fruit in both previous seasons, it now had the largest mean fruit size. Overall, systems with size-controlling rootstocks performed well and on par compared to the Nema Quad system giving confidence that reduced hydraulic conductance associated with size-controlling rootstocks does not necessarily reduce fruit size in either early or late bearing cultivars such as June and August Flame.In addition to fruit size, plastic pot plant containers number of fruits produced was not diminished in systems using size controlling rootstocks compared to the Nema Quad system.

The 2017 harvest for the June Flame cultivar was the only harvest that the Nema Quad system produced significantly more fruit per hectare than all other systems. These results differ with previous studies where KAC_V plantings reached full cropping at the same time as trees on vigorous rootstocks but, systems with size-controlling rootstocks pruned to an open-vase lagged behind more vigorous rootstocks . By 2018 the C-6 Quad and C-6 V systems produced more fruit per hectare than the Nema Quad system while the C-9 system had a substantially reduced yield compared to all other systems. Fruit count could have been increased had thinning been more consistently managed but since fruit sizes were also similar, results would not likely have changed in terms of differences between systems. 2019 was by far the most productive harvest for June Flame, with strong yields in the C-6 Quad, C-6 V, and Nema Quad systems while the C-9 Quad was less productive. Due to the lack of significant differences among systems there is no evidence that a reduction in either fruit size or fruit count would be expected in an orchard system using size-controlling rootstocks compared to a system with more vigorous rootstocks, when using appropriate management practices and planting densities adjusted for the reduced tree size. Results from the 2017 harvest of August Flame were much more aligned with previous studies where systems with high-density plantings reached maximum yield capacity earlier than in low-density systems . It is possible that if the amount of thinning in the C-6 Quad and C-6 V systems had not been as severe, they could have produced significantly more fruit than the Nema Quad system. The C-9 Quad system had the lowest fruit count but, with such a large fruit size, could have potentially produced a fruit count similar to the Nema Quad system if thinning had been done more precisely. In 2018 the fruit count was similar in the C-6 Quad, C-6 V, and Nema Quad systems while the C-9 Quad system had half the crop load as the other systems.

Since the most size-controlling rootstocks produced yields on par with the Nema Quad system, it is probable that the C-9 Quad system was under some stress that hindered production rather than its reduced fruit count being a result of reduced hydraulic conductance in the rootstock. It is likely in 2019 that all systems were under stress. Not only was fruit sizesignificantly smaller than previous years, fruit count per hectare was also fewer than that of even the earlier bearing cultivar for all systems except the C-9 Quad. In this study the relationship between crop load and fruit size was similar among systems with high density plantings on size-controlling rootstocks and the system with lower planting densities on a vigorous rootstock. Results were as expected, as crop load increased fruit size diminished. Although the relationship between fruit size and fruit produced per hectare was not significantly different among systems, appropriate crop load per tree and fruit size was influenced by planting density. The larger crop load that trees in the Nema Quad system could hold while maintaining similar fruit size as trees from other systems with significantly reduced crop load per tree indicate that trees with size-controlling rootstocks planted at a higher density may not be able to maintain as large of fruit size with larger crop loads compared to trees with more vigorous rootstocks at wider spaced plantings, this concurs with findings from Inglese et al., .

Results from this study also demonstrate that an increased number of trees per unit area compensate for the reduced crop load per tree, thus allowing high-density plantings on size controlling rootstocks to be a viable option for commercial production, similar findings have been reported by Webster and DeJong et al., .It is well documented that an orchard’s ability to intercept photosynthetically active radiation influences yield and that the two are linearly related up to 50% light interception . Data from this trial had a similar trend for the relationship between light interception and yield. In the June Flame cultivar, yield measurements up to 50% light interception had little deviation from the linear model, above this point yield varied significantly. Above 50% light interception, differences in yield are likely caused by other limiting factors such as water or nutrients . This would also explain the poor fit for the model of the C-6 V system. With a canopy that intercepted >50% in all three seasons, yield variation was likely a result of environmental conditions. August Flame systems had more time in the first growing season to fill out their allotted space. This resulted in most systems having >50% light interception in all three seasons. Due to the higher light interception, all models had a poor fit and did not provide confident yield predictions.Plants and their secondary metabolites have been used as one of the important sources in the field of medicines or health related issues since ancient times. The role of medicinal plants in the health care had been already mentioned in the Indian holy books like “Vedas”. Recent report of World Health Organization has been estimated that approx 45,000 plants being practiced for the medicinal purposes across the globe. Presently, around 65% of Indian population directly are dependent upon the traditional medicine for their need of primary health. Secondary metabolites of these herbal plants is an alternative source broadly used in the treatment of chronic diseases. Currently, traditional medicine is broadly used in the treatment of ulcer worldwide, and has been proven as one of the best strategies for the disease management of ulcer . Ulcer is a discontinuity or break in a bodily membrane in the form of wound or sores that are slow healing or keep returning. It impedes the organ of which that membrane is a part from continuing its normal functions . It is of many forms which occur on both, inside and outside of the human body. Currently, large plastic gardening pots different types of ulcer forms are recognized in medicine such as peptic ulcer, corneal ulcer, stomach ulcer, foot or leg ulcer etc. Ulcer causing problems in digestive system and wounds appearing in the lining of digestive track in human beings are very common. The digestive track of human beings is very sensitive and the health of digestive track can be good or bad and depends on many factors. Pepsin exposed ulcers i.e., peptic Ulcers are the most common type in the gastrointestinal tract area that result from an imbalance between stomach acid-pepsin and mucosal defence barriers and more than 4 million people affected worldwide annually. In medicine, the ulcer which occurs as mucosal lesions which penetrate the muscularis mucosae layer and form a cavity surrounded by acute and chronic inflammation is defined as peptic ulcer.Adesanwo et al.studied the anti-ulcerogenic effect of Melaleuca bracteata stem bark extract and showed thatthe extract significantly reduced gastric acid secretion. They also reported that the bark extract contains two important constituents’ betulinic acid and oleanolic acid, play major role in anti-ulcer effect. In another attempt, Agrawal et al. studied the antiulcer activity of petroleum ether, alcohol and aqueous extracts of Smithia conferta. Phytochemical analysis of petroleum ether extract found to have steroids, alcohol extract constitute isoflavonoids, alkaloids and carbohydrates whereas in the aqueous extract significant amount of amino acids, carbohydrates and flavonoids were present. However, the aqueous and alcoholic extracts showed significant reduction in ulcer index compared to petroleum ether extract.

All through in our evolution, natural products have enormous eminence in the fields of medicine and health. Natural products along being the earth friendly, they are free from any adverse effect to the human health.Plant metabolites have been the most successful source of potential drugs since ancient period. However, due to the emergence of new human diseases with the changing environment, continuous screening and validation of secondary metabolites in the form of drug identification/designing needs to be updated. Different cheminformatics approaches like target identification, active site prediction, drug likeliness properties, biological activity and molecular docking of selected phytoligands are the key features for identifying for functional aspects of any drug. Secondary metabolites of the plants have been recognized to elicit beneficial effects in virulent factors of diseases. The raw materials and pharmaceuticals needed for the preparation of essential drugs are largely obtained from the local herbal plants. The revolution of metabolic engineering and the development molecular docking algorithms approaches lead to improved molecular simulations with crucial applications in virtual high throughput screening and drug discovery. Analysis with molecular docking of interactions between protein-ligand, become an emerging tool in drug design. In case of Helicobacter pylori infected individuals, the frequencies of virulent factor IFNg cells have been increased in the antrum, which induces development of gastric ulcers. Protodioscin a secondary metabolites of Asparagus racemosus is used as medicinal compounds against several diseases. The analysis by molecular docking between the virulent factor and plant metabolites showed the interaction between structural protodioscin and interferon-g , in which all residues of interferon-g exhibited hydrophobic interactions . Although, the obtained binding energy of protodioscin- interferon-g complex revealed disruptions of interferon-g integrity. These types of interactions between the virulent factors of ulcer and plants secondary metabolites open a new door in the field of designing and discovery of a new drug in the ulcer treatment.Fleshy fruit gain most of their quality traits, such as color, texture, flavor, and nutritional value, as a result of physiological and biochemical changes associated with ripening. Fruit ripening has been studied for decades, yet there are still many unanswered questions about the timing and coordination of the biological processes related to this developmental program. Much of this research has been done in the model for fleshy fruit ripening, tomato , and has utilized the spontaneous single ripening mutants Cnr , nor , and rin . Each of these mutations produces pleiotropic defects to ripening and occur in or near genes encoding the transcription factors SPL-CNR, NACNOR, and MADS-RIN, belonging to the SQUAMOSA promoter binding protein-like , NAM, ATAF1/2, CUC2 and, MCM1, AG, DEF, SRF TF families, respectively. Each TF family functions in diverse developmental processes and have distinct spatiotemporal expression patterns . These mutants were used to study ripening under the assumption that the mutations cause a complete loss of function to the corresponding protein. Recently, it has been discovered that the nor and rin mutations produce proteins that are still functional and gain the ability to negatively regulate their targets . In nor, the two base pair deletion truncates the protein but still produces a functional DNA-binding and dimerizing NAC domain . In rin, a large deletion creates a chimeric protein with the neighboring gene MACROCALYX , producing a functional protein with suppression activity .

Several previous reports also demonstrated that low levels of IAA stimulated primary root growth. Similar to our observations, the Burkholderia sp. SSG that was isolated from boxwood leaves produced 2.9 to 4.5 µg mL−1 of IAA with tryptophan and had plant growth promotion ability in three boxwood varieties. Additionally, Burkholderia phytofirmans strain PsJN, which was isolated from onion roots, showed higher IAA production, around 12 µg mL−1 , with the addition of tryptophan and improved the growth of potato, tomato, maize, and grapevines. Other Burkholderia seminalis strains can also synthesize IAA and have been reported to increase rice and tomato seedling growth. These previous studies, along with our observations, suggest that B. seminalis strain 869T2 may be similar to other Burkholderia species and other plant-growth-promoting bacteria that utilize IAA to increase root growth, which may assist host plants in taking up nutrients from the surrounding environment and improve aerial tissue growth. Consistent with this hypothesis, we observed that plant size, height, fresh weight, dry weight, and total leaf areas of several tested plant species all significantly increased after inoculation with B. seminalis strain 869T2. It is known that the IAA can positively affect cell division, enlargement, tissue differentiation, root formation, and the control process of nutrition growth. The IAA can also function as a signal molecule to influence the expression of various genes involved in energy metabolism and other plant hormone synthesis, such as gibberellin and ethylene. Interestingly, we observed earlier flowering in the 869T2-inoculated hot pepper and okra plants, square plastic plant pots suggesting that acceleration of plant growth rates might occur in these plants.

In the future, transcriptome analysis of plant hormone response genes and energy-metabolic-related genes in the 869T2-inoculated plants might help us further decipher the possible mechanism of plant growth promotion ability of strain 869T2. From the results of our study, we observed that B. seminalis strain 869T2 had a better IAA yield at a temperature range of 25 ◦C to 37 ◦C and pH of 6 to 9. Similarly, Burkholderia pyrrocinia strain JK-SH007 reached the maximum production of IAA at 37 ◦C and pH 7.0. Several other plant-growth-promoting bacteria, including Bacillus siamensis, Bacillus megaterium, Bacillus subtilis, and Bacillus cereus, had relatively higher IAA yields at temperatures of 2–135 ◦C and pH 7–8. Three different bacteria isolated from therhizosphere of Stevia rebaudiana also exhibited greater production of IAA at a pH range of 6–9 and a temperature of 35 ◦C to 37 ◦C; these bacteria also increased the root and shoot bio-masses of wheat and mung bean. Various carbon sources are used as an energy source for IAA production and could enhance recycling of cofactors in bacterial cells. Our results revealed that IAA yields of B. seminalis strain 869T2 were slightly better when glucose and fructose were used in media. Several previous publications also indicated that the ability of plant-growth-promoting bacteria to produce IAA was different, depending on the carbon source used in the media. Results from these studies and our study demonstrated that IAA production by different plant-growth promoting bacteria can be influenced by various factors, such as temperature, pH, carbon sources, culture conditions, and bacterial species. In this study, we utilized the colorimetric method to estimate the IAA amounts of B. seminalis strain 869T2 when grown in various in vitro conditions and media.

Because the available tryptophan in the rhizosphere and root exudates of plants might be relatively lower than the tryptophan used in the media, the IAA production of B. seminalis strain 869T2 when grown in inoculated plants shall be determined with more sensitive and accurate methods, such as high-performance liquid chromatography or ultra-performance liquid chromatography systems. Apart from the IAA production ability of B. seminalis strain 869T2, this bacterium exhibited siderophore production and phosphate solubilization activities. Iron is an important element for many biological processes in plant growth and development. Most iron in soils is present in the highly insoluble ferric form, which is unavailable for plant absorption. Endophytic bacteria can yield iron-chelating agents such as siderophores, which bind ferric iron and help transport it into plant cells via root-mediated degradation of organic chelate, ligand exchange, or other mechanisms. Phosphorus is another essential macronutrient for numerous metabolism processes in plants, such as biosynthesis of macromolecules, signal transduction, photosynthesis, and respiration. Most of the phosphorus in soil is insoluble and not available for root uptake to support plant growth. In order to increase the bio-availability of phosphorus for plants, certain endophytic bacteria turn insoluble phosphate into soluble forms via the processes of chelation, ion exchange, acidification, or production of organic acids. Previous studies have also correlated siderophore production and phosphate solubilization abilities with the plant growth promotion traits of other Burkholderia species, such as the Burkholderia sp. SSG isolated from boxwood and the Burkholderia sp. MSSP isolated from root nodules of Mimosa pudica. Burkholderia cenocepacia strain CR318, which was isolated from maize roots, significantly enhanced maize plant growth by solubilizing inorganic tricalcium phosphate. Other studies have revealed that additional Burkholderia species also have the ability to solubilize inorganic phosphate to increase available phosphorous in agricultural soils and improve agricultural production.

In summary, both previous studies and our results suggest that the IAA synthesis, siderophore production, and phosphate solubilization abilities of B. seminalis strain 869T2 may collectively contribute to the growth enhancement observed in the several plant species tested here. We successfully inoculated and reisolated B. seminalis strain 869T2, which was originally isolated from the monocot plant vetiver grass, in several eudicot plant species of the Brassicaceae, Asteraceae, Amaranthaceae, Solanaceae, and Malvaceae families. Strain 869T2 can significantly improve the growth of both the roots and aerial parts of Arabidopsis and several leafy vegetables, including ching chiang pak choi, pak choi, loose-leaf lettuce, romaine lettuce, red leaf lettuce, and Chinese amaranth. These results suggest that the endophytic bacterium strain 869T2 may have a wide host range. A similar observation was reported for Burkholderia phytofirmans strain PsJN, first isolated from onion roots, which enhanced the growth of Arabidopsis, switch-grass, potato, tomato, maize, wheat, and grapevines. We did not observe significant growth improvement in hot pepper or okra plants after inoculation with strain 869T2; however, we did observe early flowering and better fruit development in these tested plants. These results suggest that the plant growth promotion abilities of strain 869T2might be more apparent in crops with a shorter life cycle or that the latter two tested host plant species might not be fully compatible with this bacterium. The plant colonization process and growth promotion abilities of endophytic bacteria seem to be active processes that are regulated by different characteristics of both the host plants and bacteria. In conclusion, our study revealed the potential of Burkholderia seminalis strain 869T2 for use as a bio-inoculant in agriculture to improve plant growth and production.Tropical fruit flies , such as the Mediterranean fruit fly from Africa, square pot plastic the oriental fruit fly from Asia and the Mexican fruit fly from the Americas, are recognized by entomologists as among the most destructive agricultural insect pests in the world. Because of tephritids’ economic importance, US states such as California—considered by both the US Department of Agriculture and the California Department of Food and Agriculture to be free of these pests, but with climates favourable to their establishment—invest heavily in measures to keep tephritids from becoming established. These steps include restricting importation of commodities that originate in regions with ongoing tephritid outbreaks, requiring post-harvest treatments for imported fruits and vegetables grown in areas where the pests are endemic or established, maintaining large-scale monitoring programmes for early detection, supporting preventive release programmes of sterile flies to pre-empt establishment, and launching eradication campaigns to eliminate pest populations once discovered. Indeed, 90% of the eradication projects initiated in California between 1982 and 2007 were directed against tropical fruit flies . The historical challenges posed by the fruit fly threat to California are similar to those posed by many other invasive insect species. For example, the propagule pressure of fruit flies resulting from the ever-increasing movement of people and products is an ongoing challenge posed by all invasive species. Similarly, global warming has resulted in the expansion of pest ranges worldwide. Fewer frost days, longer growing seasons, more heat waves and greater frequency of warm nights in California, combined with an abundance of suitable hosts in both urban and commercial environments, create ideal conditions for a wide range of species, particularly tropical tephritids, to successfully invade. Two aspects of California’s fruit fly invasions are unique, however. First, in most years and locations, fruit fly detections are extremely rare because of a combination of the slow population growth of newly introduced species and of population suppression from intervention programmes.

This combination of elements makes it difficult to decipher patterns in detections, because there are few ‘dots’ to connect, and small numbers of captures separated in both time and space may give the illusion that previously detected populations have been eliminated. Second, an unprecedented number of pest tephritids have been detected in California in recent decades, including a more than eight fold increase in the number of species , and thousands more flies have been captured in California than in all other US mainland states combined. We are unaware of any other single taxonomic group that consists of such a large number of economically important invasive species that are continually reappearing in the same region. Our broad goal in this paper is to bring principles of invasion biology, mapping techniques and quantitative methods to bear on detection and interception data to answer questions about the residency status of tropical fruit flies captured in California. We show that, despite the due diligence, quick responses and massive expenditures of government agencies to prevent entry and establishment of these pests, virtually all of the species against which eradication projects were directed have been reappearing; several species reappear annually, and several others every 2–5 years. The preponderance of evidence supports the hypothesis that at least five and as many as nine species are established in the state. Tephritids have been detected in nearly all regions of California where conditions are favourable for fruit fly establishment . Although the largest numbers of detections by far have been in the greater metropolitan areas of southern California, including the Los Angeles Basin and San Diego, a substantial number of flies were also detected in northern California, in the San Francisco Bay Area. Tephritids also began appearing in the state’s main agricultural growing region, the Central Valley, which includes the Sacramento and San Joaquin Valleys, and the Imperial Valley. The non-random pattern of the invasions is reflected in the fact that 100% of first records for all species were in southern California , all but one of which were found in two regions: Los Angeles and San Diego. These regions contain only around one-third of the state’s population, yet account for 100% of the tephritid first records. California was free of any tropical fruit fly species before the mid-1950s , despite the rapid growth of the fruit industry in the late nineteenth century and first half of the twentieth century, as well as relatively lax regulatory protocols at ports of entry. Two species were detected during the 1950s , followed by four more in the 1960s and 1970s. The tephritid situation in the state changed drastically in the 1980s because of: continued reappearances and spread of previously detected species in metropolitan Los Angeles and San Diego; seven new species detected, raising the total in the state from six to 13 species ; and first tephritid detections in northern California , including a massive, widespread medfly outbreak in the Bay Area. Three new tephritid species captured in the 1990s raised the total in the state to 16. The economic stakes were elevated to a new level when one of these, the olive fly , was declared established, and several previously detected species appeared in the Central Valley growing region. Even though only one new species has been captured during the past 12 years, nine previously detected species have been recaptured repeatedly over ever-expanding areas , including seven that have been captured multiple times during the past 3 years . The magnitude and geographical scope of the recurrent detections are evident in maps in figure 1b, showing the historical records of the hundreds of state-wide, regional and local detections of the four most frequently captured species.

Table 1.2 shows the pH values of dried fruits before and after using PBS or MilliQ water as the wet carrier and then homogenizing with PBS or MilliQ water. As shown in Table 1.3, no difference was observed between pH measurements taken from samples inoculated with PBS or MilliQ water and homogenizing with PBS or MilliQ water for most of the samples. The only significant difference in pH measurement was observed from dried peaches that were inoculated with PBS and dried for 48 h. When these samples were homogenized with PBS or MilliQ water for pH measurement, the pH taken from samples homogenized with MilliQ water were higher than the pH taken from samples homogenized with PBS . Evaluation of homogenization methods for recovering pathogenic cells from inoculated sand. The efficacy of recovering Salmonella from inoculated sand by stomaching or manually shaking was evaluated. Inoculated sand samples were taken right after inoculation and after 48 h of drying in the oven. The concentration of the Salmonella cocktail was 11.07 ± 0.04 log CFU/mL. Since 20 g of sand was mixed with 1 mL of liquid culture, the theoretical inoculation level in sand is 9.77 log CFU/g. As shown in Table 1.4, 9.51 ± 0.04 log CFU/g of Salmonella was recovered from freshly inoculated sand by stomaching. After 48 hours of drying, 6.93 ± 0.09 Log CFU/g of Salmonella was recovered from the inoculated sand by stomaching. Drying at 40 °C for 48 h caused an approximately 2.5 log reduction of Salmonella. The differences between counts obtained from TSAR and XLT-4R indicated the formation of injured cells during drying.

Comparing the cell numbers recovered by stomaching with the cell numbers recovered by shaking methods, blueberry box no difference in TSAR counts was observed from inoculated sand that has been dried for 48 h . When looking at the XLT-4R counts, stomaching method had higher counts on XLT-4R than shaking method . Similar trend was observed from freshly inoculated sand. Based on this result, stomaching was used in the following studies for recovering pathogens from inoculated sand or sand-inoculated dried fruits.The objective of this chapter was to determine the inoculation carriers for different dried fruits, the buffer system for pH measurement, as well as the pathogen recovery method for sand-inoculated dried fruits. Based on the results, both wet and dry inoculation changed the pH of low-moisture dates, while generating no significant impact on the aw of the samples . A similar observation of aw before and after inoculation was found in high-moisture dates. Dry inoculation reduced the pH of high-moisture dates more than the wet-inoculation. However, when looking at the physical properties of the inoculated dates, wet inoculation caused the skin to start to peel from the dates. Based on these observations, dry inoculation was chosen to inoculate dates. In addition, since dates are typically grown in sandy regions where sand storms are common and some dates are cleaned by air pressure without any contact with water before packaging . For dried peaches, the wet inoculation reduced the aw slightly compared to the noninoculated ones . This reduction in aw might be caused due to the additional 48 h drying after inoculation. As indicated by Palipane and Driscoll , moisture adsorption/desorption isotherms are inherently non-equivalent; the aw of the product after an additional drying step may not be the same as in the original food.

Dry inoculation did not impact the aw of either dried peaches. Neither dry nor wet inoculation altered the pH of dried peaches without sulfur treatment significantly. They both reduced the pH of dried peaches made with sulfur treatment. Based on the measurement, there is no strong preference between two inoculation methods. In this case, it was decided to use both wet and dry carriers to inoculate dried peaches.For dried pluots and sundried tomatoes, wet inoculation generated no impact on their pH or aw. In addition, since pluots and tomatoes are also often processed in large quantities and washed before drying like the peaches . Using a liquid carrier could help represent possible contamination from water during washing. The impact of different inoculation methods on the behavior of inoculated pathogens can be determined by the types of products. As discussed earlier, both Blessington et al. and Beuchat and Mann did not find any significant differences between wet- and dry inoculated products. Bowman et al. monitored the survival of Salmonella on black peppercorns and cumin seeds. The tested methods included wet-inoculation with Salmonella cells grown in TSB wet-inoculation with Salmonella grown on TSA biofilm inoculation , and dry inoculation with TSAgrown-Salmonella-inoculated sand. Their results showed that the biofilm inoculation method had the least Salmonella reduction. When comparing the dry inoculation method vs. the wet inoculation methods , the dry inoculation method had no difference with wet inoculation method on black peppercorn but had less reduction of Salmonella on cumin seed. One potential explanation for these differences might be the content and release of different amounts of antimicrobials from the food during wet inoculation . Deng et al. investigated the impact of aw, pH and temperature on the survival of E. coli O157:H7 in a commercial dry infant rice cereal. Results showed that much better survival was seen when the pH was at 6.8 than 4.0. In the current study, if PBS is chosen to carry out the wet inoculation, how it impacts the pH and pH measurement needs to be understood before the experiment. PBS is more often used as a carrier than MilliQ water because of its ability to prevent cells bursting due to osmosis .

Based on our results, wet inoculation with PBS did not have a significant impact on the pH of the dried fruit and using PBS for pH measurement also did not impact the result . Thus, PBS was appropriate to use as the liquid carrier for the dried fruits as changes in pH can influence pathogen survival in dried foods. In the Blessington et al. study, in which both sand and chalk were tested as dry carriers, bigger impact on the weight of the inoculated nuts was observed from the chalk inoculated nuts, especially almonds . Based on our own lab’s data, chalk and talc are lighter and tend to fly around when conducting the inoculation. When dealing with pathogens, it is not safe. Thus, sand is a better option compared with other dry carriers. Unfortunately, sand is an abrasive substance and could potential damage cells during inoculation. Sahin used sand in their study to disrupt bacterial cell walls, indicating that sand can lead to cell abrasion if used in a particular way. When comparing stomaching and shaking by hand in this study for homogenization of samples, Salmonella was recovered at significantly higher levels from stomached samples than shaken samples . When looking at the cell counts obtained from the selective agar, stomaching recovered a higher number of Salmonella from sand samples that had been dried for 48 h. The major difference observed in this study was the injured cells formed during drying as indicated by the differences between counts on TSA and XLT-4 agars. Based on these results, stomaching was chosen for recovering pathogens from sand-inoculated samples.Sulfur treatments are often used in dried fruits to preserve color . In other food applications, blueberry package sulfur is often used as an antimicrobial but it has not been evaluated in this capacity for dried fruits. Based on a recent report , dried fruits produced in the U.S. contain free SO2 ranging from 20 to 635 ppm. Among all the products tested, dried pineapple contains the least free SO2 while dried apples contain the highest levels of SO2. Dried peaches contain 355 ppm SO2 when measured in slurry . Witthuhn et al. evaluated the sulfur dioxide content in various commercial South African dried fruits and the microbial quality associated with these products. Results showed that raisins, Clingstone peaches, apricots, nectarines, and prunes contained approximately 1,302, 866, 1,318, and 806 mg/kg of SO2 respectively. However, no correlation between the SO2 content and the microbial counts was identified. On the other hand, Karabulut et al. showed that the total number of microbes present on sulphurated dried apricots were 3 logs less compared to the non-sulfured dried apricots. Türkyılmaz et al. reported a significantly lower total microbial population on dried apricots made with the sulfur dioxide treatment compared to non-sulfured dried apricots. In the Liu et al. study , the authors have shown that pathogens survived at higher levels for a longer period on dried apricots made without sulfur treatment.

Sulfur dioxide is considered by the FDA as generally recognized as safe . The FDA does not set limits on the amount of sulfur dioxide permitted in foods. However, proper labeling is required for foods containing levels of sulfur dioxide that exceeds 10 ppm. In dried fruits, less than 100 ppm is typically found . However, when inhaled or ingested by sensitive groups, it can induce asthma, even in low concentrations . The California Office of Environmental Health Hazard Assessment has developed a proposed MADL for SO2 of 220 µg/day . Taking dried peaches as an example and assuming 26 grams of dried peaches are consumed each day, the estimated exposure to SO2 is 191 µg/day .Bacterial strains and preparation. The strains of bacteria used for this survival study were provided courtesy of Dr. Linda J. Harris at the University of California, Davis. Five strains each of rifampicin-resistant Salmonella spp., Listeria, and E. coli O157:H7 were used. The strains are summarized in Table 2.1. Preparation of inocula. Individual frozen stock cultures were streaked onto TSAR and incubated at 37 °C overnight. Each isolated colony was transferred into 10 ml of TSBR, and then incubated at 37 °C overnight. One 10-µL loopful of the overnight culture was transferred to 10 mL of fresh TSBR and incubated at 37 °C for another 24 h. The newly inoculated broth was spread onto TSAR plates, 250 µL per plate, 6 plates per strain, and incubated for 24 h at 37 °C. To recover bacterial lawns from plates, 1 mL of phosphate-buffered saline was pipetted onto each plate, and an L-shaped plastic cell spreader was used to loosen and scrape the lawn. The re-suspended cells were then pipetted into a 15-ml Falcon™ tube. The addition of PBS and lawn scraping was repeated 2 more times for each plate, for a total of 3 ml of PBS used per plate. Approximately 2.5 ml of culture was recovered from each plate and a total of 15 mL cell suspension was recovered for each strain. Once all plates were scraped, 15 mL of the recovered culture from each strain were combined to make the 5-strain cocktail. A 5-strain cocktail was made for each of the pathogens and was then used to inoculate dried fruits. Each cocktail was diluted and plated onto TSAR for calculating the inoculum levels. Inoculation of dried peaches with wet or dry carriers. Before inoculation, 3 samples of uninoculated dried peaches were homogenized with 100 mL of PBS. One hundred microliters of each of the homogenates were plated on to TSAR to check for the presence of rifampicin-resistant bacteria. This was done to ensure that any bacteria present on plates during the study were from the rifampicin-resistant inoculum used and not from background microbes. Once the 5-strain cocktails were prepared, they were used to inoculate the dried peaches or sand. For sand inoculation, 1 mL of each 5-strain cocktail was added for every 20 g of fine white sand and mixed thoroughly. The inoculated sand was then spread as thinly as possible across a sheet of filter paper in a metal tray. The tray was placed in a gravity oven at 40 °C to dry for 48 h. Once dry, 160 g of the inoculated sand was used to inoculate 800 g of dried peaches and massaged into the fruit for 1 min by hand. The dry inoculated peaches were transferred into Ziploc bags and placed in a plastic container for storage at ambient or refrigerated temperature for 6 months. For wet inoculation, the remaining 5-strain cocktail was added to enough PBS to make a 1:10 dilution.Dry-inoculation, due the cell loss during the drying of inoculated sand, had lower initial inoculation levels. On Day 0 , there were 7.15 ± 0.11 log CFU/g of Salmonella on dried peaches. Storage temperatures directly impacted the die-off pattern of Salmonella.

All sensor signals were logged at 20min intervals and averaged every hour. The results presented are the averages of four replications. Xylem sap uptake into the fruit was determined based on the daily average xylem sap flow measurements from 15 and 30 DAP.Extractions were accomplished at the end of the irrigation cycle, before 08:00h. Soil solution was extracted by adding 450ml of the nutrient solution, without Ca2+, to each plant pot, and collecting the drained leachate. After collecting the soil leachate, plants were decapitated 15 cm above the soil level and the pots containing the roots plus stem stump were used to extract the stem xylem sap. Two fruit and two middle leaves were harvested at the end of the irrigation cycle from each replicate plant with the entire peduncle and pedicel attached and kept in a sealed plastic bag for xylem sap extraction. Xylem sap extraction was accomplished by placing the plant pot, fruit, or leaf blade inside a pressure chamber , while the cut end of the stem, peduncle, or pedicel was exposed to the outside of the chamber through a seal. After sealing, the pressure inside the chamber was increased up to 0.8MPa with N2. The initial xylem sap moving out of the stem, peduncle, or pedicel cut end was blotted dry to reduce Ca2+ contamination from the cut. The following 100 μl was collected over a period of 5min and used to determine the Ca2+ concentration in the xylem sap . The plant pots were pressurized in a custom-built chamber large enough to hold a 9.5 litre pot, with a two-part lid in order to allow assembly around the stem of an intact plant .

The custom-built chamber was also used to pressurize the roots of whole plants to induce guttation on leaf blades, growing bags which were collected for Ca2+ quantification. The guttation samples represent the xylem sap extracted from leaf blades without any contamination from a cut surface. Apoplastic water-soluble Ca2+ was extracted from the blossom-end pericarp tissue of tomato fruit as previously described by De Freitas et al. .The Ca2+ concentration in the soil solution, xylem sap, and apoplastic solution was determined with an Ultra-M micro Ca2+-selective electrode . A standard Ca2+ calibration curve was used to determine the Ca2+ concentration in the samples. The Ca2+ concentration in leaf and fruit tissues was determined in freeze-dried leaf blades, as well as pericarp tissues manually cut from the peduncle and blossom regions of the fruit. Freeze-dried samples were subjected to microwave acid digestion and analysed by inductively coupled plasma atomic emission spectrometry . Calcium accumulation was quantified by subtracting the total middle leaf and fruit Ca2+ contents observed at 15 DAP from the total middle leaf and fruit Ca2+ contents observed at 30 DAP. Calcium accumulation was also estimated by multiplying the quantified xylem sap Ca2+ concentration observed in the middle leaf pedicel and fruit peduncle by its respective daily average xylem sap flow rate observed at 15 and 30 DAP.BER was completely suppressed by spraying the whole plants weekly with ABA during fruit growth and development, compared with water-sprayed fruit that reached a 36% incidence of BER at 30 DAP . Dipping the fruit in solutions containing ABA prevented BER development at 15 DAP, but ABA-dipped fruit reached a 16% incidence of BER at 30 DAP. Control fruit dipped in water had a 39% incidence of BER at 30 DAP. The electrolyte leakage of fruit pericarp tissue was lower in response to whole-plant and fruit-specific ABA treatments at 15 DAP . At 30 DAP, only the whole-plant ABA treatment had lower electrolyte leakage in fruit pericarp tissue. SWP was less negative in response to whole-plant ABA treatment at 15 and 30 DAP compared with all other treatments .

Leaf stomatal conductance progressively increased from the base, middle, to the top regions of the plant, and was lower in the whole-plant ABA treatment at 15 and 30 DAP compared with all other treatments . Whole-plant water spraying, as well as water and ABA dip treatments, had similar stomatal conductance for the basal, middle, and top leaves. Based on the stomatal conductance analysis, the plant ABA uptake was considered high with whole-plant ABA treatment, and no significant ABA movement from the fruit into the plant was observed based on changes in stomatal conductance in response to fruit-specific ABA dip treatment . Plant water loss was reduced by the whole-plant ABA treatment at 15 and 30 DAP , but was similar in all other treatments, including the ABA fruit dip. Whole-plantat 15 and 30 DAP . The water spray treatment had the highest sap flow during most of the daylight period at 15 and 30 DAP . The average flow of xylem sap moving into the fruit during a 24h irrigation cycle was substantially higher on plants sprayed with ABA, compared with all other treatments , with the same diurnal pattern as seen in the leaves . At 15 DAP, fruit on water-sprayed plants, as well as water- and ABA-dipped fruit had a reverse flow of xylemic sap from the fruit back to the plant, starting in the late afternoon until the next irrigation cycle in the morning . The ABA-dipped treatment had a slightly higher sap flow to the fruit than the two water treatments at 15 and at 30 DAP . Fruit on plants sprayed with ABA had no reverse xylemic sap flow throughout the irrigation cycle at 15 DAP, but had the same diurnal pattern, with the lowest flows occurring during the night period . The diurnal pattern of fruit xylem flow at 30 DAP was similar to that at 15 DAP, but the magnitude was substantially reduced, again with no discernible reverse flow . Spraying tomato plants with ABA resulted in higher total fruit water uptake used for growth and lower fruit water uptake through the phloem from 15 to 30 DAP .

The estimated phloem sap solute concentration uptake into the fruit from 15 to 30 DAP was higher in ABA-sprayed plants than in non-sprayed plants .The Ca2+ concentrations in the soil solution and in the main stem xylem sap were similar among all treatments at 15 and 30 DAP. The average Ca2+ concentration in the soil solution among treatments was 1.41±0.09 mM at 15 DAP and 1.08±0.14 mM at 30 DAP. The average Ca2+ concentration in the main stem xylem sap was 0.72±0.04 mM at 15 DAP and 0.63±0.05 mM at 30 DAP. There was no statistical difference among treatments in Ca2+ concentrations in the xylem sap of basal, middle, or top leaves at 15 or 30 DAP. The same results were obtained when Ca2+ was determined on an independent set of plants under the same treatments using the leaf guttation method . The average xylem sap Ca2+ concentrations were 0.71±0.06 mM and 0.86±0.02 mM in top leaves, 0.77±0.03 mM and 0.87±0.02 mM in middle leaves, and 0.64±0.03 mM and 0.81±0.02 mM in basal leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in the peduncle xylem sap was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, nursery grow bag compared with all other treatments , and water-soluble apoplastic Ca2+ was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, compared with all other treatments . Fruit dipped in ABA solution had slightly higher water-soluble apoplastic Ca2+ than fruit dipped in water and fruit from plants sprayed with water at 15 DAP. The Ca2+ concentration in the top and middle leaves was statistically lower in response to whole-plant ABA treatment compared with all other treatments at 15 and 30 DAP . The Ca2+ concentrations in ABA-sprayed plants were 8.7±0.21 and 8.1±0.09mg g DW–1 in top leaves and 17.5±0.52 and 16.1±0.63mg g DW–1 in middle leaves at 15 and 30 DAP, respectively. The Ca2+ concentrations in all other non-ABA-sprayed plants were 13.0±0.36 and 13.0±0.15mg g DW–1 in top leaves and 25.1±0.96 and 23.9±0.81mg g DW–1 in middle leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in basal leaves was similar in all treatments at 15 DAP , and statistically lower in plants sprayed with ABA than all other treatments at 30 DAP. The Ca2+ concentration in fruit tissue collected at thepeduncle and blossom ends of the fruit was higher in ABAsprayed plants at 15 and 30 DAP . Fruit dipped in ABA had a higher Ca2+ concentration at the blossom-end tissue at 15 DAP, compared with water-dipped fruit and fruit of water-sprayed plants . Ca2+ accumulation was lower in the leaf and higher in the fruit of ABA-sprayed plants than in the other treated plants and fruit from 15 to 30 DAP . Ca2+ accumulation in leaf and fruit quantified by tissue analysis was similar to the estimated Ca2+ accumulation based on the Ca2+ concentration in the xylem sap and xylem sap flow rates into leaf and fruit tissues . The average relative humidity and air temperature from 15 to 30 DAP inside the greenhouse, where the tomato plants were grown, oscillated from 58.2% and 27.8 °C during the day up to 77.8% and 18.2 °C during the night, respectively . The VPD increased from 0.5 kPa at 05:30h to 1.6 kPa at 14:30h, decreasing thereafter . The number of Safranin-O-stained vascular bundles in the placenta and pericarp tissues at the peduncle and blossomend regions of the fruit was higher in response to whole-plant and fruit-specific ABA treatments at 15 DAP . The number of stained vascular bundles decreased in all treatments from 15 to 30 DAP, and all treatments showed a similar number of stained vascular bundles in the placenta and pericarp tissues at the peduncle and blossom-end regions of the fruit at 30 DAP . The fruit growth rate was higher in ABA-sprayed plants compared with all other treatments at 15 and 30 DAP . All treatments showed a positive fruit growth rate during a 24h period at 15 and 30 DAP . For all treatments, the fruit growth rate was higher at 15 DAP than at 30 DAP . The average fruit weight was also higher in ABA-sprayed plants at 15 and 30 DAP . Fruit Ca2+ uptake, both directly quantified and estimated based on the product of fruit xylem sap uptake and fruit peduncle xylem sap Ca2+ concentration, was 6-fold higher in ABA-sprayed plants compared with water-sprayed controls . A much smaller increase in Ca2+ uptake was found in ABA-dipped fruit, but, again, this was consistent for both directly quantified and estimated values . The sprayed and dipped ABA/water ratios for fruit growth rate were 1.41 and 1.15, respectively .Previous studies showed that weekly spraying of tomato plants with ABA prevented BER development in the fruit, while water-sprayed plants reached a 30–45% incidence of BER at 40–45 DAP . At that time, possible mechanisms through which ABA increased fruit Ca2+ concentration and reduced fruit susceptibility to BER were suggested based on estimations of fruit xylem sap uptake and Ca2+ concentration in the xylem sap .Water uptake in leaves comes exclusively from xylem vessels, while water uptake into the fruit comes from both phloem and xylem vascular tissues . Treating the whole plant with ABA reduced stomatal conductance, which resulted in lower plant water loss, lower soil water uptake and xylemic water movement into the leaves, as well as higher SWP and increased xylemic water movement into the fruit. Considering that Ca2+ concentrations in the soil solution and stem xylem sap were similar among all treatments, the observed lower Ca2+ accumulation in ABA-sprayed plants was due to lower soil solution uptake triggered by lower leaf transpiration rates . Our results also estimate a higher solute concentration in the phloem sap moving into the fruit of ABA-sprayed plants . Although ABA reduced stomatal conductance and this would be expected to decrease leaf photosynthesis , the improved plant water status associated with ABA application may have caused compensatory physiological effects in other areas, such as reduced carbon partitioning to roots and/or improved carbon transport rates, resulting in higher solute concentration in the phloem sap, compared with the other treatments. The non-ABA-sprayed plants had an average fruit phloem sap uptake of 1.04ml fruit–1 d–1 and an average phloem sap solute concentration of 144.3mg ml–1 .