Horticulture

These results agreed with previous studies on the relationships between grapevine water status and yield components . However, leaf area and crown porosity were not affected by applied water amount treatments in 2020. This might havebeen resulted from the remarkably high air temperature at the experimental site, diminishing the grapevine vegetative growth despite the water compensation from irrigation . Consequently, berry quality parameters were slightly affected by irrigation treatments, with only TSS being higher with greater water stress in the harvest of the first season due to berry dehydration and potential promotion in sugar accumulation .There were two flavonoid classes monitored in this study, anthocyanins and flavonols. They are highly sensitive towards environmental conditions . This study evidenced that SH increased berry skin anthocyanin and flavonol concentrations compared to the other trellis systems over the two seasons. SH might have had more advancement in berry development due to more efficient leaf area to fruit ratio achieved in this trellis system . Furthermore, low round pots the crown porosity of SH was ranging from 0.20 to 0.30, a window of inferred solar radiation exposure identified in previous works for `Cabernet Sauvignon´.

As for VSPs, anthocyanin degradation was unlikely to be the reason why VSPs had lower anthocyanin concentration since the greater leaf area could have provided berries some degree of protection from receiving excessive solar radiation . This can be confirmed by the fact that TSS and berry skin anthocyanin concentration were still synchronized in 2020. However, there was a decoupling of TSS and berry skin anthocyanin concentration in 2021, where the VSPs had higher TSS but lower skin anthocyanin content. Unlike the first season, the leaf area and canopy crown porosity showed no difference among the trellises, but the effective leaf area that can provide protection against excessive solar radiation might differ between SH and HQ from the other trellis systems. Hence, even with similar leaf areas, the VSPs still exposed clusters to the environmental stresses, which promoted TSS accumulation due to dehydration but greater anthocyanin degradation, similar to what was observed in previous studies . Although the TSS levels in this study were not at the level for reaching the tipping point of anthocyanin degradation as previously reported , compared to the SH and HQ with greater height from the vineyard floor, the VSPs might have been more easily affected by the solar radiation and heat reflected from soil surface, causing hotter and drier canopy microclimate and inevitably lead to greater anthocyanin degradation .

Additionally, some previous studies have shown negative relationships between yield and berry composition . Similar observations in this study might be due to source organs of the VSPs were not distributed widely enough to be as efficient as those of the SH and HQ, resulting in lower photosynthetic capacity in their canopies, which further reduced the translocation of photosynthates flowing into berries to promote TSS accumulation and flavonoid biosynthesis. As for flavonols, previous studies have shown that flavonols are very sensitive to solar radiation, especially UV radiation, where more light will often increase flavonol concentration in berry skins . The results from this work corroborated previous observations that less leaf area with more crown porosity would increase solar radiation inside the canopy, and further increase flavonol concentrations in berry skins . Additionally, SH and HQ showed greater concentrations in di-hydroxylated flavonols as well as some tri-hydroxylated flavonol derivatives. Water deficits, achieved by manipulating applied water amounts through irrigation, can significantly improve flavonoid concentrations in grape berries . Similar results were observed in our findings as well, where 25% ETc was able to increase anthocyanin and flavonol concentrations in grape berries. One previous study at the same experimental site showed that 25% ETc could potentially increase the possibility for flavonoid degradation and decrease the wine antioxidant capacity .

However, we did not see such effects in this study. This might be because berry sugar accumulation was not affected among the three applied water amounts, and the overall TSS levels did not exceed the tipping point . It was repeatedly been noticed that, beyond this TSS level, skin anthocyanins and even flavonols would start to significantly degrade in a hot climate . Hence, in our study, all the treatments might have ended up having similar advancements in berry flavonoid accumulation because of the similar levels of TSS without any promoted accumulation or degradation among the three irrigation strategies , but 25% ETc was able to decrease berry weights, which resulted in higher concentrations in anthocyanins and flavonols.Positive relationships between flavonols and solar radiation, especially UV-B, have been consistently observed in previous research, clearly indicating that more solar radiation penetrating into the canopy interior promotes flavonol concentration in berry skins . Further, flavonol content and derivative proportions exhibited strong relationships with solar radiation , which was confirmed in this study, where quercetin proportion and both total flavonol concentration correlated strongly with leaf area and crown porosity especially with VSP types. When the high air temperature or drought conditions became extreme, flavonoids in berry skins started to degrade . For all six trellis systems in 2020, the relationships between flavonols and canopy architecture were strong. These relationships between leaf area/crown porosity and flavonols can provide a feasible way of assessing canopy architecture in terms of the canopy’s contribution towards berry composition and vice versa. This approach is not limited only to red cultivars and can also be applied to white cultivars since flavonols are still synthesized in their skin tissues . Also, for quercetin specifically, it is the most abundant flavonol derivative in grape berry skins. Hence, the compound would be unchallenging to isolate and extract, offering an easy assessment of the effects of solar radiation on berry flavonol profiles. Interestingly, in this study, the VSPs did not result in higher quercetin or total flavonol concentrations, indicating that these trellis systems might not be suitable for accumulating or maintaining flavonoids in berry skins in a hot climate regardless of TSS levels compared to other trellis systems. Although the relationships between canopy architecture and flavonols were strong in this study and align with previous reports, the influence of canopy structure imposed by trellis system on berry chemical development needs more investigation to understand the contributions of trellis systems to canopy architecture and canopy microclimate.Commerce via global trade and transport provides a mechanism for introduction of invasive species to new territories, extending pest habitats outside of their native regions . Invasive species threaten biodiversity, habitat, nutritious food, clean water, resilient environments, sustainable economies, and human health . Agricultural production systems are continuously challenged by invasive species that attack high-value crops, plastic pots 30 liters thereby significantly hampering the ability of food industries to maintain profitability . The geographic range of agricultural crops provides the potential for invasive species to colonize regions on a global scale . Factors that aid expansion include short life cycle, fast growth rate, high plasticity, and resiliency to a wide range of environmental conditions . Such factors are drivers of rapid evolutionary change, population increase, and global colonization . Practitioners and stakeholders should aim to implement new strategies to manage such new invasive species in agricultural production . Drosophila suzukii Matsumura is an invasive species native to Southeast Asia. Passive transportation is the main reason of the dispersal of this species .

It was first detected in North America and Europe in 2008 , and later in South America in 2013 , and Northern Africa in 2017 . The long-serrated ovipositor of D. suzukii enables it to oviposit inside fresh fruit, which creates a challenging management problem . Emerged larvae burrow within fruit pulp rendering fruit unmarketable . When D. suzukii became established in the U.S. during 2008, the total annual revenue losses for the West Coast berry and cherry industries were estimated at over $500 million . Currently the situation is not changed in term of economic impact . This particular insect is challenging to manage due to its high dispersal potential, ability to survive and adapt to harsh environmental conditions, and ability to attack a wide host range. For these reasons, D. suzukii is a key pest of these fruit industries worldwide. In the last decade, conventional insecticide uses on affected crops significantly increased to manage D. suzukii fruit damage. Typically used insecticides include spinosyns, pyrethroids, and organophosphates . Intensive use of insecticides poses a tremendous risk to non-target organisms such as pollinators, natural enemies, and humans . In addition, frequent insecticide applications likely resulted in resistance development . These factors require development of an IPM program that includes alternatives to conventional insecticides for managing D. suzukii. Non-insecticidal control methods including cladding, irrigation, netting, mulching, pruning, monitoring and mass trapping have been implemented against D. suzukii . While each method provides some relief to D. suzukii pressure, they provide limited reductions in crop damage . Behavioral control of D. suzukii on susceptible fruit indicated promise for industry adoption. The food grade gum makes use of physical properties to mimic fruit, resulting in D. suzukii laying their eggs in a soft gel-like substrate, instead of the fruit itself. The food grade gum is a mixture of food-grade ingredients which is highly attractive to D. suzukii and competes with the ripening fruit throughout the season . To the best of our knowledge, the food-grade gum modifies various D. suzukii behaviors, ultimately resulting in a significant decrease in fruit damage. The product diverts D. suzukii away from ripening fruit, which results in significant retention of the pest, keeping it away from fruit. Third, the food-grade gum acts as an egg sink. Since the D. suzukii eggs laid in this medium cannot develop, this translates in a substantial reduction of the pest population growth . The aim of this work was to determine the potential of the food grade gum to reduce D. suzukii damage in large-scale commercial open-field and screen house fruit production units on blueberry, cherry, raspberry, blackberry, and wine grape. The hypothesis was that food-grade gum would reduce D. suzukii damage in small fruit, tree fruit and grapes under semi-field and small-scale field conditions. These studies were conducted during 2019 and 2020 in California and Oregon in the western United States.In all field trials, GUM dispensers were placed at least 27 meters away from untreated control plots to minimize volatile plume interaction between treatments. In the current study, cotton pads were used to apply ~1.8 g of GUM on each dispenser at the rate of 124 dispensers per hectare under commercial production conditions . Cotton pads were placed directly on the ground close to irrigation drippers to provide adequate daily moisture. Earlier work illustrated that dispensers have a field longevity of 21 days and for this reason, dispensers were therefore deployed 1 to 4 times depending on the duration of crop ripening and susceptibility. In three trials , egg laying data were collected in buffer plots that were located between UTC and GUM plots to determine the active range of released volatiles beyond treated areas. This design was implemented based on the assumption that volatiles from treatment plots may be blown or diffuse beyond treatment plots. Berries were brought to the laboratory to determine number of eggs in fruit for each of the plots using a dissecting microscope. All soft or damaged fruits were excluded when assessing presence of eggs. In some cases, at first fruit color, laboratory-reared D. suzukii flies were released in each plot with the intent to create a relatively even pest pressure in all plots. Colonies of D. suzukii used in field studies consisted of seasonally collected wild adults from multiple field sites in the Willamette Valley, Oregon, and Oxnard, California. Collected adults were released into plastic cages and reared at 24°Cand 70% relative humidity, with a 16:8 h photo period before being released in the respective field trials. Flies were constantly provided with water and artificial diet that served as both a food source and an oviposition medium. Before their use in experiments, all flies were allowed to mate for 8 d in mixed-sex cages. Some small fruit varieties were numbered since this information is proprietary.A replicated field trial on drip-irrigated Pinot noir winegrape was conducted in Yamhill County, Oregon, USA from 10 to 18 October 2019 on ~2.6 hectares. Vines were spaced at 1.5 by 5 m, and trellised on a standard four wire trellis system, supporting a ~2 m canopy.

Although the TCM use of goji berry also includes the leaves and bark of the plant, this review will discuss the potential benefits of the fruit on eye health. In addition to a robust amount of Z, goji berries contain modest amounts of β-cryptoxanthin, β- carotene, neoxanthin and L. The Z and L content among different varieties of dried goji berries cultivated in Ningxia province ranged from 25 to 152 mg/100g, and 0.3 to 1.9 mg/100g, respectively. According to the United States Department of Agriculture food database, one serving of goji berries is 28 g, which would provide up to 42.6 mg of L + Z, depending on the cultivar. Moreover, the predominant form of Z in goji berries is a dipalmitate, found with a diester linkage. The ratio of Z dipalmitate to total carotenoids was up to 55% and 88%, in fresh and dried goji berry fruit, respectively. This esterified form of Z showed a significantly higher intestinal absorption than monoester and free Z due to the high efficacy of hydrolysis, blueberry grow pot mainly by carboxyl ester lipase. Plasma Z was significantly increased in individuals consuming 15g goji berries daily for 28 days in comparison to those on a habitual diet. 

Participants consuming 5 mg of Z dipalmitate extracted from goji berries showed a higher plasma Z concentration than when they consumed the same amount as unesterified Z over a nine to 24 hour period. The high Z content of goji berries has been proposed as a dietary source to reduce the risk of AMD, although studies are limited.150 In one study, circulating Z levels were significantly higher in healthy older individuals who consumed 10 mg of Z extract from goji berries daily for 90 days. No change in macular pigmentation or soft drusen was observed, but MPOD was not measured. In an uncontrolled trial, individuals with early stage AMD who consume a beverage containing 12 mg of L and 2 mg of Z derived from marigold flower and goji berry, respectively, daily for five months, showed higher circulating levels of L and Z, lower intraocular pressures, and better best-corrected visual acuity scores. Unfortunately, the study lacked a control group, did not test the effect of Z separately, and did not clarify whether the form of Z extracted from goji berry was the dipalmitate. Another study investigating the effects of an herbal formula among healthy adults with dry eyes noted that those chewing tablets containing L , Z , extracts from blackcurrant, chrysanthemum, and goji berry showed dose-dependent reductions in eye fatigue symptoms, improved tear secretion as well as MPOD, compared to placebo. 

The basis of this formula was derived from TCM, so the multi-component formulation could not directly inform the role of any single ingredient. A study in patients with early AMD reported that the MPOD was significantly higher in those consuming 25 g/day of goji berries for 90 days, compared to their baseline levels and to a habitual diet control group. The BCVA was also significantly improved in the goji berry group compared to their baseline values. We recently reported that MPOD and skin carotenoid scores were increased in healthy middle-aged individuals consuming 28 g/day of goji berries five times a day for 90 days compared to a group taking a supplement with 6 mg of L and 4 mg of Z. These results illustrate that MPOD levels can increase in healthy individuals even without early signs of AMD. While these results are encouraging, longer intervention periods with a larger number of participants are necessary. In addition to AMD, goji berries have been studied as a therapy for retinitis pigmentosa, an inherited retinal disease. Patients who consume 0.35 g/d of LBP for 12 months showed a significant improvement in visual acuity and macular thickness, compared to control subjects who did not consume L or Z. Examples of human studies that evaluated the effects of supplements containing goji berries on retinal health are shown in Table 1. Based on preclinical evidence, potential benefits of goji berry intake on glaucoma and diabetic retinopathy may also exist. Goji berry extract ameliorated the high glucose-induced blood-retinal barrier disruption in human retinal pigment epithelial cells.

Studies reported that LBP showed significant neuroprotective effects over retinal ganglion cells in male C57BL/6N mice and Sprague-Dawley rats with ocular hypertension. In db/db mice, goji berry extract restored the thickness of the retina, the ganglion cell number, and the integrity of RPE after daily intake over eight weeks. Although research on the upper limit of goji berry intake is scarce, goji berry allergy risk has been associated with the existence of cross-reactivity to nonspecific lipid transfer proteins from peaches, tomatoes, tobacco, tree nuts, and select pollens. In addition, bleeding symptoms after consuming goji berry juice, tea, or wine have been described in case reports among patients taking warfarin, an anticoagulant medicine. Although the potential value of foods high in L and Z during pregnancy and lactation has been discussed above, the utilization of goji berry products during these unique periods in a woman’s life needs special caution. Clinical studies of goji berries on eye health have been conducted primarily in Asia, with emerging research reported from Italy, Lithuania, and Switzerland. Potential gene-nutrient interactions must be considered when comparing results from Asian with Caucasian populations. Over the past decade, the number of H-2A guest workers employed on California farms increased more than tenfold, so that almost 44,000 farm jobs were certified to be filled by H-2A workers in fiscal year 2022 . During FY2020, two-thirds of the H-2A jobs certified in California were in crop support services. Most crop support jobs were with farm labor contractors , but one sixth were hired directly by fruit producers. Almost 10% of the H-2A workers were hired directly by vegetable producers . Until the 2008–2009 recession, most H-2A workers were in southeastern states such as Florida. However, the slowdown in migration of undocumented individuals after 2008–2009, hydroponic bucket combined with a stable demand for farm workers and the aging and settling of undocumented workers who arrived before 2008–2009, contributed to the rapid growth in the H-2A program in the three Pacific Coast states that employ half of U.S. farm workers, a third of whom work in California.This paper analyzes agricultural employment data from the California Employment Development Department and the Quarterly Census of Employment and Wages to understand changing patterns of farm worker employment in the 21st century. The data show that seasonality is declining in most regions and commodities, primarily because of higher employment during the winter months, which may reflect more winter pruning jobs and fewer summer harvesting jobs. Second, the data emphasize the increasing importance of non-farm crop support employers, mostly labor contractors, who bring workers to farms to perform specific tasks. More farms appear to be developing a year-round workforce that is hired directly and supplemented when needed with workers brought to farms by labor contractors, including H-2A guest workers . California requires all employers who pay $100 or more in wages to enroll in the state’s unemployment insurance system and pay taxes of 1.5% to 6.2% on the first $7,000 of each employee’s wages to cover the cost of unemployment benefits for laid-off workers . Employers also report their employment for the payroll period that includes the 12th of the month. Summing these monthly employment totals and dividing by 12 months generates average employment, also referred to as year-round equivalent jobs.

The monthly employment measures allow us to determine the peak and trough employment months. Agricultural employment, as defined by the North American Industry Classification System , peaked at 470,000 in May 2020 and was 346,000 in March 2020, generating a peak-trough ratio of 1.4. More than 470,000 workers are employed on California farms sometime during the year. Workers who are employed only in payroll periods that do not include the 12th of the month, such as those who work only during the first, third, or fourth weeks of the month, are excluded from average employment. In 2016, when California’s agricultural employment averaged 425,000, almost a million unique Social Security Numbers were reported by the state’s agricultural employers, suggesting 2.3 unique workers for each year-round equivalent job . California became the leading farm state in terms of sales in 1949, when Los Angeles County led the United States in farm sales . The state’s population doubled between 1950 and 1970, from 10 million to 20 million, and agricultural sales grew fastest in the San Joaquin Valley after water projects allowed more acres to be irrigated and suburbanization reduced the availability of farmland in coastal areas. Citrus and dairy farms in Southern California migrated north to SJV, while tree fruit farms moved from the urbanizing Bay Area to the San Joaquin and Sacramento valleys . Three SJV counties — Fresno, Kern and Tulare — accounted for 20% of California farm sales in 1949, a third in 2000, and almost half of the state’s farm sales in 2020 . Some crops that were already concentrated in the Sacramento and San Joaquin valleys expanded in acreage. For example, there were 90,000 bearing acres of almonds in 1950, almost 150,000 acres in 1970, 500,000 acres by 2000, and 1.3 million acres in 2022. Most of this additional almond acreage was in the San Joaquin Valley. New orchards and dairies in the San Joaquin and Sacramento valleys were often larger and more efficient than the coastal farms they replaced, and their higher productivity was reflected in rising yields. Average yields of many fruits and vegetables doubled and tripled over the past three decades; bell peppers and cantaloupes are examples. Yield rose over 50% to 33 tons an acre for strawberries . The major change in California crop farming over the past half-century has been the rising share of high value fruits, nuts, vegetables, and melons, as well as horticultural specialties such as flowers and plants, in the state’s farm sales. In 1960, the value of fruit, vegetable, and horticultural commodities was two-thirds of the total value of California crops; since 2000, FVH commodities have accounted for over 90% of the value of California crops, reflecting growing consumer demand for fresh produce and nursery plants . Cotton was California’s most valuable crop in 1950; by 2000, cotton was the sixth most valuable crop, and by 2020 cotton was no longer among the state’s top 20 crops. The demand for FVH commodities rises with income, and rising farmland prices encouraged individuals and investors to buy farmland as a hedge against inflation in the 1970s, a decade in which the value of California farmland more than doubled . Higher interest rates in the 1980s led to a farm financial crisis that was more severe in midwestern states than in California, but encouraged some oil firms and conglomerates to sell their California farmland. The data in table 1 show that California’s farm sales almost tripled in three decades, and that fruit and nut sales almost quintupled. The value of the state’s vegetables and melons doubled, as did the value of greenhouse and nursery crops. The state’s farm sales were $17.8 billion in 1990, including $4.4 billion worth of fruits and nuts and $3.9 billion worth of vegetables. Farm sales were $27.2 billion in 2000, including $7.3 billion worth of fruits and nuts, $6.2 billion worth of vegetables, and $2.8 billion worth of greenhouse and nursery commodities. This rose to $37.5 billion in 2010, including $13.5 billion worth of fruits and nuts, $6.7 billion worth of vegetables, and $3.8 billion worth of greenhouse and nursery commodities. In 2020, farm sales were $49.1 billion, including $20.6 billion worth of fruits and nuts, $7.8 billion worth of vegetables, and $6.3 billion worth of greenhouse and nursery commodities. In real or inflation-adjusted terms, California farm sales rose by 40% over 30 years, and fruit and nut sales by 140%, while vegetable and nursery sales were little changed.Many FVH commodities are labor intensive, so expanding production increases the employment of farm workers. These are non-farm businesses that bring workers to farms to accomplish specific tasks. For example, farmers may rely on labor contractors to bring crews of workers for a few weeks to prune, thin, or harvest their crops. Contractors may be the sole employers of the workers they bring to farms under some labor laws, such as unemployment insurance and workers’ compensation, and joint employers with farms under others, such as the Agricultural Labor Relations Act.

Once a complete set of sensors were developed and tested, we implemented a basic remote control application to verify the functionality of the Max32 as well as the AGV platform. Details on the AGV are presented in Chapter 7. We designed the OSAVC with a few goals in mind. The most important of which being that the controller be vehicle agnostic—that is, adaptable to a large class of vehicle types. This translates into the hardware supporting as wide a variety of interface and output protocols as practical. Fig. 3.2 shows a more detailed view of the controller. The firmware architecture was designed to be as simple and as temporally deterministic as possible. To this end the dynamic control algorithm and the state estimation algorithms are modular. This allows these algorithms to be designed and tested in control-specific software and ported out as C functions which can then be compiled with the vehicle firmware directly2 . We demonstrate this approach in Chapter 5. We also required the sensor drivers to be non-blocking and the control loop to operate using a hardware timer to minimize timing jitter. The last goal is to enable as large a community as possible. To support this goal the hardware design and firmware are open source and hosted online.

The sensors we selected are ones typically used in resource-constrained autonomous vehicles. Possibly the most common one is the inertial measurement unit, or IMU. This sensor consists of three triaxial sensors in a single package: gyroscopes to measure angular rotation rates, nft hydroponic system accelerometers to measure specific force, and magnetometers to measure the earth’s magnetic field strength. The gyroscopes are used to compute the dynamics of the vehicle, the accelerometers and magnetometers are used to determine the vehicles attitude relative to known inertial vectors—gravity and the magnetic field. Almost as ubiquitous as the IMU is the GPS receiver. This sensor calculates the vehicle’s location in global coordinates by measuring the range of the sensor to four or more satellites at known orbital locations and computing the optimal fit of those ranges. GPS sensors can also provide other useful data, such as the current time, vehicle heading and speed in inertial coordinates. Another commonly used sensor is an angular encoder. This device measures the angle of rotation around an axis. In the AGV it is used to calculate the speed of the motors as well as the angle of the steering servo. In other areas of robotics they measure joint or actuator angles. The microcontroller itself has built in analog to digital converters . Some sensors communicate their output using analog signals.

The last sensor we selected is a LiDAR sensor . LiDAR is commonly used to detect local obstacles in the environment. It operates by measuring the time of flight of a laser beam on a round trip path from the vehicle to an obstacle. In addition to the sensors decribed above, there are several peripherals commonly needed for autonomous vehicles. Electronic speed controllers are used to provide control signals to brushless DC motors . ESCs require a specific form of PWM signals to command the motor velocity. Servo motors use the same PWM signal but instead control the angle of a motor and are used for various types of actuators, such as robot arms or steering systems. Finally, it is useful to have onboard data storage for vehicle parameter information. A common inexpensive non-volatile memory device is the EEPROM , typically available in the Kbit range. Many autonomous systems rely on remote manual control through serial radio receivers. Other forms of radio control are possible but most modern systems utilize serial data streams. Other radios are used to communicate from the vehicle to ground control systems. These radios typically use a serial protocol. Another form of communication is supported through a serial to USB converter. This can be used as a debug port or to transmit data to an external computer. Finally, the microprocessor is programmed using the Microchip PICkit 3/4 serial programmer.

A summary of the basic sensors and peripherals supported by the OSAVC and their respective hardware interfaces is found in Table 3.1. Every sensor has a physical connector and there are nearly as many connector types as there are sensor types. After a review of the most common connectors we selected the Molex Picoblade for the OSAVC. It is a good compromise between footprint and mechanical reliability. It has 1mm pitch between conductors to minimize size and uses through hole pins for connection to the PCB, making it more reliable than surface mount equivalents, and easier to assemble. Because this research is primarily open source, the tools used for development need to be open source as well; we used the KiCad electrical design automation suite3 for the electrical and PCB layout design. The first design decision before embarking on the electrical design was the choice of microcontroller. we selected a microcontroller that satisfied the requirements listed in the Chapter 2, the Microchip PIC32MX795 series microcontroller, a 32 bit MIPSbased processor. This device includes a wide suite of hardware peripherals including numerous UARTs, SPI ports, I2C ports, IC blocks, OC blocks, and timers. It allows for CAN bus integration with the inclusion of an external CAN transceiver. It operates at a reasonably fast 80 MHz clock frequency, has 128 Kb SRAM and 512 Kb flash memory, is a mature design, and is well supported by documentation and a functional integrated development environment called MPLabX. The PIC32MX795 was offered in a development board which we acquired to begin developing the code base for the controller and for use as a reference design for the OSAVC. The basic requirements for the microcontroller operation are decoupling capacitors on all the voltage input and reference pins, a reset connection to the MCLR pin, connections to the programming interface, and the oscillator pins. Although the microcontroller may operate without an external oscillator for precise and stable timing it is recommended to use a crystal oscillator. We selected an 8 MHz oscillator with 30 pF capacitors, and a 0 Ohm resistor to allow for attenuation of the oscillator signal if required. To reset the microcontroller, we implemented a manual push button switch, as well as following the guidelines for the programmer reset function. This is shown in Fig. 3.3. In addition to the basic requirements an FTDI serial to USB converter chip is connected to the first UART port of the microcontroller to allow for communication to an external device. This feature is primarily used for debugging purposes during development, as well as communication to a single board computer or ground control station during normal operation. This is detailed in the microprocessor sheet of the schematics4 . The microcontroller requires a stable 3.3V power supply, provided by the Texas Instruments LP38690DT-3.3 low dropout voltage regulator. Many sensors and peripherals require 5V operation, and the LP38690DTX-5.0 LDO regulator provides this capability. Because the OSAVC may provide power to an external SBC as well as some high current devices such as servo motors, a Microchip 29300-5.0 5V/3A LDO regulator is used. Finally, the TI PTN78020W switching regulator accepts battery volt-ages in the range of 7-36V and regulates down to 7V/6A, and feeds the two 5V LDO regulators. The switching regular is used because it is very efficient—above 90%—and allows for reducing high battery voltages without wasting energy in the form of heat. The output of the LP38690DTX-5 in turn provides power to the 3.3V LDO.

Alternatively, hydroponic nft system the USB port can power the LP38690DTX-5 and LP38690DT-3.3, typically used when developing and testing firmware. This is switched by a P-channel MOSFET transistor using a voltage divider and comparator circuit when USB power is present detailed in Fig. 3.4. It can also be found in the power section of the schematics. The peripherals supported by the OSAVC are listed in Table 3.1. LEDs indicating communication were placed on signal lines for most sensors5 as well as to indicate the operation of the various voltage sources. A jumper is provided to power the OC modules with the 29300-5.0 and the SBC with up to 3A current at 5V when operated with a battery. The GPIO pins have Schottky diodes to protect the microcontroller from electrostatic discharge events. The detailed design is located in the I/O sheet of the schematics. The guiding principles for the PCB layout balanced phsyical size with manufacturability. Because the design is open source as opposed to a commercial product, we cannot guarantee that an adopter will have access to a reflow oven for soldering components to the PCB. Therefore the design has to support manual soldering while also minimizing board area. Thus, we selected surface mount devices —in order to save board space—that were still possible to solder by hand. As a result, the smallest discrete devices in the design come in the 0603 package, with a footprint of approximately 1.55 by 0.85 mm. For the microcontroller, we selected the TQFP-100 package, which has leads on a 0.5 mm pitch and occupies a 14 mm by 14 mm footprint. we made similar choices for the other components. To ease the assembly all the components are located on the top side of the PCB. For the PCB itself, we also balanced cost vs size by choosing a four layer board. The PCB has two signal planes , an internal ground plane, and an internal 3.3V plane. This design provides some noise immunity by having these mostly complete internal copper planes.we chose to mount the switching power supply on the PCB despite its large size in order to minimize the mechanical complexity. The board could have been made much smaller by placing the power supply separately, but we deemed the convenience of a single PCB worth the tradeoff. The top signal plane and silkscreen of the PCB is shown in Fig. 3.5. For the following discussion refer to the physical board of Fig. 3.6 where the silkscreen designations are easier to see. For the component layout, we chose to have the battery connection enter the PCB from a pair of mounting holes sized for 16 AWG wires on the lower left hand side . Mounted directly adjacent is the PTN78020W . The three LDOs are located along the top and right hand sides of the PTN78020W , and a USB-C power output is located on the upper left hand corner of the PCB . Also on the left hand edge are the motor outputs and GPIO pins located on a 19 by 3 row header. On the top and bottom edges of the PCB are the sensor and peripheral connectors . The microcontroller is located near the center of the PCB . The oscillator is located adjacent to the microcontroller to keep the signal traces short and of nearly equal path length. The EEPROM is located on the lower right of the PCB . On the upper right is the FTDI serial-USB converter which connects to the outside world on the right hand edge of the PCB . The reset button and the in-circuit serial programming port are also located on the right hand edge of the PCB. Refer to the schematics for the location of the various discrete components. For most signals we selected 0.25 mm trace widths matching the lead width of the microcontroller. For higher current carrying traces, such as those from the voltage regulators or battery, we chose traces of at least 1 mm in width—well above the minimum width recommended by the KiCad calculator—in order to minimize resistive losses and to keep the traces as cool as possible. Signal path crossings between the top signal and bottom signal layers were kept as close to perpendicular as was possible to eliminate coupling between signals. There are several tests to perform prior to attempting to load code onto the OSAVC. These tests ensure that the assembly was performed correctly. The first test is to plug a USB-B cable from a computer to the matching receptacle on the OSAVC. This should provide power to the board and the LED from the 3.3V LDO should light up. This indicates that the USB power is succesfully delivered to the regulator and therefore the USB connnector is connected.

In almost all viral diseases occurring in the vineyard, the virus is distributed systemically throughout the grapevine. Once introduced in the host, viral particles move rapidly within the vascular tissue towards sink tissues and establish infections . Although we detected the presence of GRBaV in vegetative and berry tissues during growing and harvest seasons, symptoms of red blotch were only evident after véraison, which suggests that the disease onset is mostly dependent on grapevine phenology and not necessarily linked to viral accumulation. Similar observations have been made during grapevine leaf roll disease, supporting the hypothesis that the appearance of viral disease symptoms in the vineyard may result from the interaction between pathogen and host cellular factors at specific phenological stages . Whether GRBaV is able to modulate its infection strategy as a function of plant development and/ or grapevines have distinct responses to red blotch throughout the season remains to be resolved. GRBaV shares several similarities with geminiviruses, stacking pots including a small single stranded DNA genome that encodes six potential proteins .

Because of their limited protein-coding capacities, geminiviruses rely heavily on host cellular machinery and interact with an assortment of plant proteins and pathways to promote infection . We confirmed the expression of five GRBaV genes in ripening red-skinned berries. Although three of these genes have been assigned putative functions in viral DNA replication and coat formation , their specific functions in pathogenesis are yet to be elucidated. Our results indicated that the developmental stage of the berries may not influence the expression of GRBaV viral genes and that host factors could play a more critical role in the establishment of successful infections. Red blotch symptoms in grape berries include abnormal chemical composition and asynchronous ripening in the clusters , both reflecting alterations in host metabolic homeostasis and developmental processes. Interestingly, we determined that GRBaV infections induced some processes associated with early fruit development in berries at late stages of ripening, while repressing pathways involved in fruit ripening . Abiotic stress responses to hypoxia and temperature were also suppressed by red blotch in grape berries. These observations may imply that the virus, as a bio-trophic pathogen, could redirect host metabolic processes to sustain higher energy demands due to viral replication and at the same time suppressing ripening related events and responses to stress , potentially counterproductive to viral infections. However, further evidence using infected berries from different grape cultivars and environmental conditions will be necessary to understand how both grapevine genotype and environment may influence the disease outcome.

It is also important to consider that the effects of GRBaV infections on berries may not be comparable with those occurring in vegetative tissues; for instance, photosynthetic pathways are generally inhibited during viral infections of grape leaves . GRBaV infections restricted the biosynthesis and accumulation of phenylpropanoids and derivatives, which preferentially accumulate after véraison in red-skinned berries. These secondary metabolites function as antioxidants and phytoalexins to protect the berries against a variety of stresses, and are important contributors of berry quality parameters, such as color, flavor, and aroma . The inhibition of phenylpropanoid metabolism appears to be a hallmark of viral infections in red-skinned berries, as has previously been reported in leaf roll-affected berries . In particular, the anthocyanin biosynthetic pathway was greatly impaired as a result of leaf roll and red blotch, correlating with the reduced coloration observed in GRBaVinfected berries from several red-skinned cultivars . In this study, we demonstrated that GRBaV infections compromised the regulation of ripening by: suppressing specific ripening events; altering the expression patterns of transcription factors that control the transition from the growth to ripening phases and the activation of ripening pathways ; and causing hormonal imbalances. Most of the alterations in the ripening regulatory networks occurred in infected berries at véraison, the same developmental time when red blotch symptoms initiated, suggesting a link between mechanisms involved in the control of grapevine responses to viruses and berry development. Similar observations were made in leaf roll-affected berries, which presented lower expression levels of MYB transcription factors at late stages of ripening .

Plant hormones modulate ripening processes and stress responses in grape berries. Red blotch had a profound impact on ABA, ethylene, and auxin pathways. ABA is considered the triggering signal of berry ripening, since its accumulation coincides with véraison and ABA responsive transcription factors have been implicated in the activation of ripening processes . Particularly, the induction of anthocyanin biosynthesis in a variety of grape cultivars has been linked to ABA-mediated signaling pathways . Our results indicate that alterations of ripening events in the GRBaV-infected berries, including the reduced anthocyanin content at late stages of ripening, could be a direct result of lower ABA levels at véraison. ABA has been shown to stimulate ethylene-mediated pathways in berries, and together both hormones appear to regulate the progression of ripening . GRBaV infections also affected genes involved in ethylene biosynthesis and responses , which could further account for the antagonistic effect of red blotch on ripening events and immune responses. In contrast, red blotch promoted auxin-mediated pathways, known to suppress berry ripening. Auxins play crucial roles in the early development of grape berries and are known to inhibit ripening processes by delaying ABA-triggered processes . The mechanisms by which grapevine viruses cause disease in ripening berries need to be investigated. GRBaV may actively interfere with the regulation of berry ripening by hijacking the plant post-transcriptional control. We identified elements of the host post-transcriptional machinery that were misregulated in GRBaV-infected berries. Post-transcriptional regulation intersects plant immune responses, developmental transitions, and hormone signaling . Plant viruses, in particular geminiviruses, encode multiple silencing suppressors that interfere with host siRNA production and alter plant DNA methylation and miRNA pathways, causing developmental defects . A previous study on leaf roll indicated that the viral infection caused up-regulation of Dicer-like genes in ripening berries . Therefore, the relationship between the viral-induced repression of host RNA silencing processes and the post-transcriptional regulation of ripening should be addressed from the perspective of compatible grapevine– virus interactions.Shriveled berries on ripening clusters are not uncommon in California vineyards. They usually occur in only a small proportion of a vineyard’s fruit , nft hydroponic but in particular vineyards and years, shriveling can affect more than half of the crop . Most shrivel disorders make the fruit less desirable for winemaking, with subsequent yield and production losses. Before taking steps to reduce the incidence of fruit shriveling in vineyards, it is necessary to differentiate between shrivel disorders. We describe four common causes of fruit shriveling and detail compositional differences between normally developing fruit and that affected by sugar accumulation disorder .Fruit exposed to direct sunlight for all or part of the day, especially in the heat of the afternoon, can be damaged by sunburn, which may be caused by high temperature, ultraviolet radiation or a combination of the two . The physical appearance of sunburned fruit depends on the grape variety and stage of development — white grapes and red grapes exposed before pigment accumulation begins develop brown discoloration, which varies depending on severity.Veraison and early postveraison red varieties with sunburn often exhibit poor color development, and may remain pink for the remainder of the season. Post-veraison sunburn leads to fruit with less color and a shiny appearance. Sunburned berries often crack, presumably due to damaged epidermal tissues. Extreme sunburn leads to complete berry desiccation and the formation of raisins in both red and white varieties. Sunburn only affects berries that are directly exposed to sunlight. If a cluster is fully exposed to the sun on both sides, or if the rachis itself is damaged, then the cluster may be completely affected.

Obvious signs of sunburn may only occur on the exposed portions of individual berries. Sunburn can be avoided by reducing the fruit’s exposure to direct sunlight, especially in the afternoon. While leaves are removed in the fruit zone in many growing regions to increase cluster exposure to indirect light, in north-south row orientations leaves are removed on the east side of the canopy to reduce direct exposure in the afternoon and the probability of sunburn. This practice does not completely eliminate the risk of sunburn, however, because morning sun can also cause damage.Natural dehydration is another type of shrivel that may affect berries late in ripening but prior to commercial harvest. These berries appear similar to fruit with bunch stem necrosis, but the rachis look green and healthy. For this type of shrivel, which is especially pronounced in Syrah , berries lose weight due to water loss, and sugars are concentrated . Both increased transpiration and decreased phloem influx have been suggested as causes for late-season dehydration, but recent studies provide evidence that several varieties of grape berries remain hydraulically connected to the parent vine and therefore may lose water back to the parent plant late in ripening as well as to dry, ambient air .Clusters affected by bunch stem necrosis are identified by necrotic rachis tissue, with shriveled berries distal to the necrotic tissue. The visible symptoms of bunch stem necrosis begin as small black spots on pedicels , and progress to the lateral stem structure and rachis . Usually, necrosis symptoms are not noted until the rachis is affected. bunch stem necrosis may affect an entire cluster as well as the wings and tips of otherwise healthy clusters . It can occur in many varieties, but is especially prevalent in Cabernet Sauvignon on California’s North Coast. It has been described in the literature in many different countries, with descriptive terms that include waterberry , bunch stem dieback , shanking , stiellähme , palo negro , desséchement de la rafl e and dessichimiento della rachide . No specifi c cause of bunch stem necrosis has been identifi ed, despite many years of research. In some cases, varietal differences in susceptibility have been correlated to xylem structure, specifically a reduction on the area of xylem distal to branch points in the peduncle . The incidence of bunch stem necrosis has also been correlated to various concentrations or ratios of mineral nutrients, including magnesium, calcium, potassium and nitrogen . Work in Chile and Australia has shown that the amino acid metabolite putrescine is associated with bunch stem necrosis. More light in the canopy can also reduce bunch stem necrosis . bunch stem necrosis can appear very early in fruit development or after veraison. The terms “inflorescence necrosis” and “early bunch stem necrosis” have been used to describe bunch stem necrosis around bloom . The composition of such fruit varies depending on when during fruit development the rachis becomes necrotic. Presumably, the necrosis prevents both sugar and water transport to the berry. Hence, if the rachis becomes necrotic early in the ripening period before the berry has accumulated much sugar, fruit will have low Brix . On the other hand, if the rachis becomes necrotic after the berries have accumulated appreciable sugar, subsequent shriveling can concentrate the sugars. bunch stem necrosis in Cabernet Sauvignon on the North Coast is usually the latter type. Fruit with bunch stem necrosis can have a Brix as high as 42 .Another disorder with symptoms that occur during the ripening period has been called “berry shrivel”; we recently proposed that it be called “sugar accumulation disorder” . This disorder was first described in Emperor table grapes from California’s San Joaquin Valley and is characterized by poor coloration and low sugar accumulation. Sugar accumulation disorder has been found in a number of varieties and is present in many areas of California. In general, it affects only a small proportion of clusters in a vineyard, though in certain years and vineyards up to 50% of the fruit can be affected. Regardless of the variety or location, fruit affected by sugar accumulation disorder has lower pH, berry weight and Brix compared with normally developing fruit . When multiple rachises and fruit with sugar accumulation disorder were tested for minerals, the only consistent difference from normally developing fruit or rachises was increased calcium in the rachis tissue . To test the hypothesis that fruit exhibiting sugar accumulation disorder may have altered nitrogen metabolism, we measured the amounts of nitrogenous compounds at harvest in fruit with the disorder compared to normally developing fruit.

Previous efforts to develop rough allometric woody biomass equations for vines notwithstanding, there is still a need to improve our precision in estimating of how biomass changes with different parameters. Because the present analysis was conducted for 15 year old Cabernet vines, there is now a need for calibrating how vine C varies with age, varietal and training system. There is also uncertainty around the influence of grafting onto root stock on C accumulation in vines. As mentioned in the methods, the vines in this study were not grafted—an artifact of the root-limiting duripan approximately 50 cm below the soil surface. The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites perse, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, hydroponic nft they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility.

The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, plant health and fruit production, as well as for those concerned with C storage. The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.Grapes are the most valuable fruit crop in the United States, valued at over $6.5 billion annually , but climate change is projected to reduce grape production and quality . Climate affects grape quality by impacting the concentration of sugars, organic acids, and secondary compounds .

The climatic conditions producing the highest quality wine cause the berries to reach optimal ratios between sugar and acid concentrations and maximum concentrations of pigment, aroma, and flavor compounds simultaneously . Hot temperatures accelerate sugar accumulation, forcing growers to harvest earlier, before berries reach optimal flavor development, to avoid the high alcohol content and insipid wine flavor from excessive sugar to acid ratios . Harvest dates have shifted earlier historically, and climate models predict further acceleration of ripening . Growers can partly compensate through management practices, such as trimming canopies or using shade clothes to reduce the ratio of sugar supply to demand , though these practices are costly and increasingly ineffective in the face of climate change . Planting existing cultivars or developing new cultivars with slower sugar accumulation are promising alternative strategies to mitigate these climate change impacts, but these efforts have been hindered by uncertainty around the plant traits controlling sugar accumulation . Grape cultivars vary in berry maturation and sugar accumulation rates, and in their response to abiotic stress, but the main anatomical and physiological mechanisms driving these differences remain unknown .

Multiple physiological processes influence berry sugar accumulation and its responses to climate, including photosynthesis, long-distance sugar transport, and local transport and metabolism in the berries . However, the relative importance of these factors in regulating sugar concentrations and fruit growth is debated . Photosynthetic responses to heat and water stress could impact cultivar differences in accumulation rates by affecting the sugar supply for ripening . Further, sugar is transported from the photosynthesizing leaves to the berries through the sugar-conducting vascular tissue – the phloem. At the onset of ripening , the berries significantly accelerate sugar accumulation by initiating active sugar unloading from the phloem, making the phloem the primary pathway for water and resource influx into the berries . The importance of phloem transport to ripening suggests that phloem traits could be important drivers of cultivar differences in sugar accumulation, and that modifying phloem traits to slow sugar accumulation under hot conditions could help mitigate the impacts of climate change on wine quality. However, the main traits controlling sugar accumulation in grape remain unclear . The rate of phloem transport is determined by both the hydraulic resistance to the flow of sugar sap, and the activity and kinetics of water and sugar transporters in the sources, sinks, and along the transport pathway . Modeling studies suggest that increasing the hydraulic resistance of the phloem reduces sugar export to the sinks . Therefore, selecting grape cultivars with lower total phloem conductance could decelerate sugar accumulation and improve the synchronization of sugar accumulation with flavor development under hotter conditions. However, a higher hydraulic resistance can make the phloem more susceptible to declines or even complete failures in transport under severe water stress . Thus, we expect cultivars that produce high-quality wine in hot, dry conditions to exhibit phloem hydraulic resistances that slow berry sugar accumulation while avoiding phloem failure. The phloem transport pathway is composed of individual sugar-conducting cells with porous end walls stacked to form conduits . The anatomy of the transport pathway, including the total cross-sectional area of sieve tubes in plant organs, lumen area of individual sieve tubes, and porosity of the sieve plates, significantly impacts pathway resistance . Plants with a greater cross-sectional area dedicated to phloem , sieve tubes with wider lumen areas , and larger and more abundant pores in the sieve plates are expected to have a lower hydraulic resistance . Total phloem cross-sectional area in the shoots has been found to vary between several grape cultivars , hydroponic channel and a greater cross-sectional phloem area has been linked to faster sugar accumulation in the fruit in other crop species . However, the variation of phloem structural traits across cultivars adapted to a diverse range of climatic conditions and the relationship of these traits to sugar accumulation is largely unknown for grapevines. Establishing these anatomical links could allow breeders to modify sugar accumulation by selecting for phloem traits, instead of management practices that can negatively impact the fruit zone environment or yield . In this study, we used a common garden experiment to evaluate the links between phloem anatomy and sugar accumulation across 18 winegrape cultivars typically grown in climatically diverse grape growing regions. We assessed phloem and xylem vascular anatomy in leaf petioles and midveins and berry pedicels, to capture hydraulic resistance along the long-distance transport pathway. We also measured maximum berry sugar accumulation rates in the post-veraison ripening period to capture the greatest capacity for sugar transport . We predicted that traits that reduce hydraulic resistance, including larger total cross-sectional phloem areas, larger mean lumen areas for individual sieve tubes, and more porous sieve plates would increase maximum sugar accumulation rates. We also predicted that cultivars typically grown in hotter wine regions would havetraits that increase hydraulic resistance, as an adaptation to increase wine quality by reducing the rate of sugar accumulation. In addition, we measured photosynthesis and vine water stress to compare the impacts of phloem anatomy, vine carbon supply, and vine water status on sugar accumulation rates. Overall, our goals were to determine the most influential traits for sugar accumulation in grape berries and evaluate the role of phloem anatomy in adapting grape cultivars to a wide range of different climates.Berry chemistry, anatomy and physiology were measured in summer 2020 for 18 grape cultivars established in an experimental vineyard on the University of California, Davis campus . There were 13 red-fruited and 5 white-fruited cultivars. Further, 9 cultivars were classified as hot-climate, 7 as warm-climate and 2 as temperate climate, using the definitions from Anderson & Nelgen . Anderson & Nelgen sorted the major wine-growing regions worldwide into climate categories based on mean temperature over the growing season . Cultivars were then placed into their respective climate category based upon the highest proportion of bearing area grown in a particular climate category as of 2020. This proportion of land area devoted to growing a particular cultivar worldwide was taken as a thermal requirement, genotypically driven, to match sugar accumulation with a region’s climate. Plants were growing as mature vines, grafted to the same rootstock , and trained to a bilateral, spur-pruned, vertical shoot-positioned trellising system with a North-South row orientation . Davis is considered a hot, dry site for wine growing, with campus weather stations reporting a decadal average mean annual precipitation of 436 mm and mean growing season temperature of 19.8°C . Our study period was exceptionally hot, with mean daily and mean maximum daily temperatures ranging from 22.8-24.5°C and 32.6-34.4°C, respectively, partly due to the anomalous August 16 – 18 heatwave . Over the study period, vines were drip-irrigated weekly at 50% replacement of vineyard evapotranspiration, which was estimated from the reference evapotranspiration reported by the campus weather status and published crop coefficients for this trellising system and vine × row spacing .Leaves and berries were sampled to measure petiole, midvein, and pedicel anatomy in the morning on three days at the end of the growing season . Two berries and leaves per vine were excised with a razor blade. Leaf position was standardized as the 6th leaf from the shoot apex, to capture the most photosynthetically active leaves. Two leaf and one berry sample per vine were then prepared for light microscopy, and the other berry sample was prepared for scanning electron microscopy. For light microscopy, a 1-cm segment of leaf petiole and lamina and the entire pedicel of the berry were immediately excised and placed into a vial of chilled Formalin-Acetic Acid . Vials were put on ice and refrigerated at 4°C for at least 24 hours before further processing. For scanning electron microscopy, pedicels were immediately flash-frozen and immersed in liquid nitrogen for 1-min and placed into a chilled micro-centrifuge tube of 100% ethanol, then the tube was immersed in liquid nitrogen until the ethanol congealed . Samples were then immediately placed on ice and stored in a -20°C freezer for at least 24 hours before further processing.After 7 days in FAA, the light microscopy samples were soaked in 50% ethanol for 5 mins and then stored in 70% ethanol in preparation for paraffin embedding. Samples were first infiltrated with paraffin by using an Autotechnicon Tissue Processor to treat samples with the following sequence of solutions: 70%, 85%, 95%, 100% ethanol, 1 ethanol:1 toluene, 100% toluene , and paraffin wax , each for 1 hour. The infiltrated samples were then embedded into paraffin blocks with a Leica Histo-Embedder , and allowed to cool. A rotary microtome was then used to make 7μm-thick cross-sections for leaf laminas, petioles, and berry pedicels. Pedicel cross-sections were sampled from the receptacle and petiole and midvein cross-sections were sampled near the interface of the lamina and petiole. After the cross-sections were imaged, pedicels for four cultivars were remelted from their wax molds, oriented longitudinally and sectioned again at 7μm to obtain sieve element lengths. Sections were stained using a 1% aniline blue and 1% safranin solution following a modified staining procedure . Sections were then viewed under bright field or florescence microscopy using a Leica DM4000B microscope and a DFC7000T digital camera .The pedicel electron microscopy samples were processed following Mullendore .

These practices include use of vegetative shoots to shade the fruit, using shade cloths to reduce berry radiative heating, spraying berries with a natural “sunscreen” such as a clay compound, water misting with sprinklers and, when slope permits, changing the row orientation to reduce or balance solar radiation exposure. Shade cloths, which are the focus of this work, reduce temperature by controlling the transmission of incoming radiation to the fruit zone,without completely blocking all incident radiation, which could lead to a reduction in grape quality and an increase in disease risk. Shade cloths can be used to cover the entire canopy and thus affect a large-scale change in vineyard microclimate, or they can be applied directly to the fruiting zone to localize their impacts to berry microclimate. Previous studies have reported that shade cloths are effective in reducing maximum berry temperatures, though they have used different methods for quantifying these effects. Mart´ınez-Luscher et al. ¨ reported that 40% black shade cloths covering the fruiting zone of the canopy reduced cluster temperature by 3.7◦C during the warmest time of the day, large round plant pots while Greer found that 70% shade cloths covering the grapevines reduced canopy temperature by an average of 4.6◦C throughout the day.

Similarly, thin shade cloths and plastic films covering the grapevines were shown by Rana et al. to reduce midday berry temperatures by 2◦C and 6◦C below air temperature, respectively. While it is clear that shade cloth can be effective in reducing overall berry temperature, a higher degree of control of berry temperature may be desired. In certain instances, it may be beneficial to reduce berry temperature by a defined margin to avoid negative trade-offs, while also balancing temperatures between opposing sides of the vine. However, many interacting variables are likely to influence the efficacy of shade cloth, such as row orientation, row spacing, trellis type, and topography. Due to the large number of important variables affecting berry temperature, it can be difficult to generalize the relatively small number of experimental results that are only able to explore a few variable combinations in order to predict the effect of shade cloth for a given vineyard system. Crop models provide the potential for generalizing the results of field experiments to predict the outcomes of proposed management strategies for a specific site or climate scenario. Such models could allow for optimization of the design or management of vineyards to mitigate elevated berry temperatures under current or future climates, given that a large number of simulations can be efficiently performed to cover a wide parameter space.

Previous work has developed models of spherical fruit temperature, including one study that simulated the effect of hail nets on apple temperature. In a recent study, a 3D model was developed and validated that accurately simulated the spatial and temporal temperature fluctuations of grape berries in vineyards with different climates, topographies, and trellises.However, the current version of this model is not able to represent the effects of shade cloth on canopy and berry temperature. The overall goal of this work was to enable model-based evaluation and optimization of strategies for grape berry temperature control using fruit zone shade cloth. With this goal in mind, specific objectives of this study were to: 1) develop a physically-based 3D model of grape berry temperature that incorporates the effect of shade cloth, 2) generate an experimental data set against which the model can be validated, and 3) quantify the interacting effects of different strategies for excessive berry temperature mitigation such as altered row orientation, row spacing, topography, and shade cloth density.The model of grape berry temperature was based on the 3D model described in Ponce de Leon´ and Bailey, and modified to include the effects of shade cloth. A brief description of the overall model is provided below, with a focus on novel additions associated with shade cloth. The model was developed within the Helios modeling framework and has been validated based on field measurements of berry temperature between veraison to harvest. The computational domain consists of 3D geometric elements that fully resolve the spatial structure of the plants, berries, and shade cloth .

The berries were represented by 3D tessellated spheres composed of triangular elements, the ground surface by a planar grid of rectangular elements, the woody tissues by a cylindrical mesh of triangular elements, and the leaves by planar rectangles masked to the shape of leaves using the transparency channel of a PNG image. The shade cloth was formed by rectangles masked to the shape of a grid using the transparency channel of a PNG image, where “holes” in the shade cloth were created based on a grid of transparent pixels.Radiation transfer was simulated using a backward-ray-tracing approach that ensures each geometric element is adequately sampled for both short-wave and long-wave radiation. The model launches a large number of rays from each geometric element to simulate the various modes of radiation transfer, including emission, reflection and transmission based on the radiative properties of each element. When a ray encounters a transparent pixel on an element masked by a PNG image, the ray continues with no interaction. This allows for an efficient fully-resolved representation of shade cloth with a large number of holes. To eliminate domain edge effects, periodic lateral boundaries can be enabled that effectively creates an infinitely repeating vine geometry in the horizontal. For berries, the latent flux term was assumed to be zero since latent cooling is typically considered negligible after veraison. For this study, heat released from metabolic activity ´ within the fruit was assumed to be low enough to be neglected. The heat transfer coefficient from fruit, ground, and leaf surfaces to the atmosphere, h, are specified as described in Ponce de Leon and Baile. Previous field tests suggested an important role of berry heat storage in accurately representing temperature dynamics, and laboratory and field tests have shown that the chosen values for Cp and rhoA result in berry dynamic temperature responses in close agreement with measurements.To evaluate the model accuracy, the time series of experimental measurements and simulated temperatures for berries furthest west were compared. Model agreement with field measurements was quantified using the error metrics normalized root mean squared error – normalized by the difference between maximum and minimum measured data, the coefficient of determination , plant pots round and the index of agreement. To analyze the modeled berry temperature results in the “simulation experiment”, we took the average temperature of five berries in each of five exterior clusters on both sides of the vine. For comparison purposes, the total canopy and cluster daily light interception was calculated by integrating the light interception fluxes over the day. The daily light interception was calculated per vine area for the canopy and per berry surface area for the clusters To compare the effect of shade cloth density on berry temperatures in the simulated vineyard designs, we used indices based on the duration of elevated temperature and the intensity . The 35◦C temperature threshold was chosen because it has been reported to correspond to the approximate onset of unfavorable effects of elevated temperature on grape composition. Furthermore, to identify conditions that effectively balance berry temperature between opposing sides of the vine, we constructed a conditional inference tree with the variables listed in Table 4.2. The conditional inference tree was constructed using the “party” package of R statistical software. The conditional inference tree is a tree-based classification algorithm that performs binary recursive partitioning of data into groups containing observations with similar values. Conditional inference trees are similar to decision trees, the main difference being that each node in the conditional inference trees uses a significance test of independence to select a predictor variable rather than selecting the predictor variable that maximizes the information measure. In the conditional inference tree, predictor variables are circled and ranked and the ones at the top have the highest correlation with the response variable.

The algorithm stops if the null hypothesis of independence is not rejected, however, for visualization purposes, the maximum depth of the decision tree was set to 4.The magnitude of the wind speed varied across the sampling period, but overall Unilateral tended to have greater wind speeds compared to Goblet . During the day, the wind in the fruiting zone tended to come from the east while at night the wind tended to come from the west for both trellis systems . Since the row orientation of Goblet and Unilateral was N-S and the vines were on an east-facing slope, the wind coming from the east tended to be of greater magnitude. For both Goblet and Unilateral, the relative humidity significantly decreased on 25 Sept. 2019, likely due to the increase in air temperature. Overall, during the sampling period, Unilateral tended to be slightly less humid compared to Goblet. This can be explained by the wider row spacing in Unilateral that enhanced mixing of the canopy air compared to Goblet .During the sampling period, average daily maximum temperatures of the west-facing berries under the shade cloths were 1.6◦C and 2.7◦C higher than the daily maximum air temperature in Goblet and Unilateral, respectively. The control west-facing berries with no shade cloth were up to 8.2◦C and 5.9◦C above the daily maximum air temperature in Goblet and Unilateral, respectively . The relative reduction in maximum berry temperatures due to the shade cloth in Goblet and Unilateral was consistent across the sampling period, which included a wide range of ambient air temperatures . Pulp temperatures of more than 40◦C were measured on 25 Sept. 2019 for unshaded berries in both trellis systems. For both Goblet and Unilateral, the berry temperature under the shade cloth was close to the air temperature in the morning, while air temperature differences between berries under the shade cloths increased in the afternoon. The peak in measured elevated temperatures happened at different times in Goblet and Unilateral , primarily due to the influence of the ratio between plant height and row spacing on berry exposure. In the evening, under low-light conditions, the berry temperatures were similar to the air temperatures, while at night, radiative cooling likely caused the pulp temperature to fall below the air temperatures. Under low light and at night, the temperature differences among treatments were small .The model was validated by using the experimental measurements to determine whether the modeled berry temperatures appropriately responded to the shade cloth relative to the control . The model reproduced the magnitude of the berry temperature increase over air temperature reasonably well for both Goblet and Unilateral. The largest source of error appeared to be due to the transition period when berry sun exposure began, where the time of the simulated maximum temperature increase tended to happen earlier than that of the measurement . This mismatch could be due to slight inaccuracies in determining the position of each berry and leaf.Figures 4.7, 4.8, and 4.6 summarize the results of the simulation experiment in which the effect of different row orientations, row spacing, and slope aspects on berry temperature were evaluated. Orienting vineyards N-S on a flat terrain allowed for uniform canopy and cluster exposure to solar radiation, since radiation is approximately symmetric about the N-S axis . However, the berry temperature on the west side of the vine significantly increased in the afternoon compared to the east side because hysteresis in air temperature causes asymmetry about solar noon. On average, berry temperature on the west side of the vines was greater than 35◦C for about 1-2.5 hours longer than the east side . Interestingly, although there was temporal asymmetry due to air temperature and temperature extremes, the net daily accumulation of berry growing degree hours was virtually identical between each side of the vine . On flat terrain, rows oriented NW-SE increased light interception and fruit overexposure in the afternoon and E-W reduced light interception and fruit overexposure in the afternoon . The high exposure to direct sunlight in NW-SE oriented rows resulted in simulated berry temperatures up to 7.8◦C higher on the SE side compared to the NW side . Compared to rows oriented NE-SW, rows oriented NW-SE had an additional 3 hours of canopy light interception above 200 W m−2 between 14:00 and 17:00 and berry temperatures greater than 35◦C for 2 additional hours .

In both 2020 and 2021, there were HWs that occurred mid to late August. Looking at anthocyanin concentration, there was more variation in concentrations among treatments in 2020. Additionally, concentrations were higher in 2021 among the three treatments. The higher anthocyanin concentrations may be due to less extreme HW temperatures, thus less anthocyanin degradation. Looking at Figure 5, in 2020, there were no significant differences between the treatments during HW2, but looking at HW3, the 60% ET treatment suffered the most significant losses. At the start of HW4 the 120% ET treatment had significantly higher flavonol concentrations than the 60% or the 180% treatments. Nevertheless, at harvest there were no significant differences between the three treatments. In 2021 as seen in Figure 5, there were no significant differences in flavonol concentration until HW2 where there were differences between the 60% and the 120% ET treatments. Interestingly, there was a major decline in concentration for the 120% ET treatment at the start of HW2, and by commercial harvest, plant plastic pots this treatment has the lowest concentration. This may be due to differences in light exposure because flavonols develop when exposed to light.

The 120% treatment may have had the lowest flavonol concentration at harvest because these vines had the most water applied during the growing season, so there was likely more foliage covering the berries. Similar to anthocyanin concentration, flavonol concentrations reached higher peaks in the 2021 growing season than in the 2020 growing season. Flavonol concentration went beyond 0.20 mg/B for the 60% ET treatment in 2021, but did not exceed 0.15 mg/B in 2020. This may be due to less extreme HW temperatures in 2021 and more direct sunlight exposure that allowed for increased flavonol development. As seen in Figure 6, the 2020 results show that, following HW2, there was a significant difference between the 60% and 120% treatments. This is also seen at the start HW3, and all treatments saw a marked decrease in PAs leading to no significance between them for the rest of the season until at harvest where there was a difference between the 60% and 180% treatments. Also seen in Figure 6, in 2021, there was a significant difference between the 90% and 120% ET treatments at the start of HW1 and at the start of HW2. After HW2, significant differences were seen between the 60% and the other two treatments. Overall, there were more significant differences between the treatments during the 2021 growing season compared to the 2020 growing season. In 2020, PA concentrations were on a similar trend among the three treatments. The larger variations in PA concentrations between treatments in 2021 may be attributed to the severity of the HWs and the impact it had on the grapevines.

During the 2021 growing season, the berries got sunburnt due to high exposure, and therefore more irrigation was applied in order to preserve quality. Table 1 shows the summary statistics of the RNA sequencing. Sample 1_NW_B3R2 was an outlier and removed from the analysis due to having a higher number of reads compared to the other samples. For each irrigation treatment, the berry transcriptomes of two consecutive dates of collection were compared to evaluate the number of differentially expressed genes . The purple represents the 60% ET treatment, the teal represents the 120% ET treatment, and the yellow represents the 180% ET treatment. As seen in the figure, there were significantly more genes differentially expressed between the HW vs. pre-HW dates than the HW and post-HW dates for the 60% ET and 120% ET treatments. Interestingly, it was the opposite for the 180% ET treatment. For each pairwise comparison of collection dates, the DEGs detected for the three irrigation treatments were compared . Looking at the top two diagrams, there is a larger number of shared genes unique to the 60% and the 120% treatments compared to those uniquely shared with the 180% treatment. Specifically, there were 550 up-regulated genes shared only between the 60% and 120% treatments versus the 96 and 38 genes only shared with the 180% treatment. Looking at the bottom two diagrams, there is an overall decrease in the number of genes being differentially expressed when comparing the post-HW date relative to the HW date. There is also a higher number of genes being differentially expressed for the 180% treatment with 478 being up-regulated and 605 being down-regulated. In terms of berry temperature, significant differences among the treatments were only observed on August 13th, which is the pre-HW date for the August 2020 HW. Measurements recorded throughout the remaining HW dates showed that berry temperature remained similar among the three treatments. These results may be due to the berries being in the shade. In a study done by Ponce de Leon and Bailey , they found that shaded berries tended to have similar temperatures to the ambient air temperature and could reach over 10 °C above ambient temperature when in direct sunlight. There is currently a lack of published literature that explores the effects of HWs and irrigation on berry temperature, and the results from this study suggest that the two factors do not play a significant role in berry temperature. There were major declines in anthocyanin concentration in 2020 and 2021 for the 60% ET treatment, which suggests a suppression of anthocyanin synthesis and promotion of degradation at such high temperatures. This finding remains consistent with published literature, such as the work done in Yan et al. . In their study, three experiments were conducted on Vitis vinifera L. cv. Merlot, cl. 347 in which the grapevines were exposed to three different temperature regimes.

Their results consistently showed that the low-temperature regimes and high-temperature regimes had the highest and lowest anthocyanin levels, respectively. Further evidence of anthocyanin synthesis suppression and promotion of degradation at high temperatures is found in Mori et al. . Similar to the experimental design in Yan et al. , Kyoho grape berries in this study were grown under different temperature conditions. Anthocyanin levels were consistently lower in berries grown under high temperature conditions compared to berries grown at 25 °C or berries grown at 30 °C during the day and 15 °C at night. These results were attributed to a decrease of UFGT activities in the flavonoid pathway, as previously mentioned. Particular attention should be given to the anthocyanin results seen in 2020 . By harvest, the 120% ET treatment was significantly different from the 60%, black plastic pots but not significantly different from the 180% treatment. This is important because similar results were seen in anthocyanin concentrations with much less water used for irrigation. These results further support research that has proven the beneficial aspects of deficit irrigation. As seen in Bucchetti et al. and Roby et al. , deficit irrigation consistently increased anthocyanin concentrations by reducing berry size and thus increasing content per berry. That being said, theresults from this study show that overwatering during HWs is unnecessary when trying to compensate for berry phenolics. In terms of flavonol concentration, particular attention should be given to the 120% ET treatment in the 2021 growing season . By harvest, there was a major decline in concentration for this treatment. As previously mentioned, flavonol synthesis is light-dependent, and shading can have notable effects on flavonol concentration . Since sunlight is a key influencing factor, the decline in flavonol concentration for the 120% ET treatment may be attributed to a denser canopy that provided more shading to the fruit, which inhibited flavonol synthesis. In the 2020 and 2021 growing seasons, total PA concentration was significantly lower in the 60% ET treatment than the other two treatments by harvest . Since HWs occurred in both seasons pre-veraison, the treatment differences likely happened during PA synthesis, which occurs from flowering to veraison. The pre-veraison HWs could have impacted PA synthesis in the 60% ET treatment, which was exposed to higher water and heat stress, thus decreasing PA concentration. Although studies have shown variable results on the effects of heat events on PA concentration, PAs are unlikely to degrade due to the high stability of their chemical structure . A common trend seen throughout both years is that the 180% and 120% treatments from 2020 and 2021 respectively resulted in lower phenolic concentrations than the 120% and 90% treatments. This is important to note because these results suggest that irrigation prior to HWs can be beneficial in maintaining fruit quality, but excessive watering may negate the beneficial aspects of deficit irrigation. In terms of the lower anthocyanin biosynthesis in the 3x and 2.5x treatments, it may be related to plant water status since moderate deficit irrigation has been shown to increase anthocyanin biosynthesis and promote ripening . Additionally, this may also be due to insufficient light exposure of the clusters since the canopies were more dense with foliage.

As seen in Figure 7 and Figure 8, there were significantly more genes differentially expressed between the HW vs. pre-HW dates than the HW and post-HW dates for the 60% ET and 120% ET treatments, and it was the opposite for the 180% ET treatment. This may potentially be a combined response to the heat stress and too much irrigation being applied. The RNA sequencing results showed that there were several down-regulated genes from the phenylpropanoid and flavonoid pathways shared between the pre-, during, and post-HW dates and irrigation treatments. What is potentially being observed is a general repression of core and peripheral phenylpropanoid pathways, which are normally triggered in red-skinned grape berries throughout ripening . Some of the genes that deserve particular attention include trans-cinnamate 4-monooxygenase , 4-coumarate-CoA ligase , chalcone synthase , flavonol synthase, F3’5’H, and UFGT. C4H and 4CL play key roles in the phenylpropanoid pathway. C4H catalyzes the reaction that forms p-coumaric acid, and 4CL converts p-coumaric acid to 4-Coumaroyl-CoA . The down-regulation of C4H and 4CL are important to note because 4-Coumaroyl-CoA is an important precursor for compounds produced in the flavonoid pathway, and decreased production of this precursor may lead to overall decreased levels of phenolic compounds. Chalcone synthase is the first key enzyme in the flavonoid pathway, and therefore plays a central role in initiating flavonoid biosynthesis . The down-regulation of this gene may lead to reduced flavonol levels in grape berries, which is crucial in times of excessive heat and light exposure due to their nature as UV protectants. Flavonol synthase plays an important role in converting dihydroflavonols to flavonols . F3’5’H catalyzes flavonoid hydroxylation, which leads to the formation of flavonols, anthocyanins, and PAs . UFGT converts anthocyanidins to anthocyanins . The down-regulation of these genes lead to decreased production of phenolic compounds, and such effects have been well-documented . Implementation of differential irrigation prior to and during HWs were shown to have a major impact on berry composition and gene expression. Damaging effects on berry quality were observed from underwatering and overwatering. Significant changes in berry composition occurred throughout the HWs, including a suppression of anthocyanin synthesis and promotion of degradation, and lower flavonol and PA concentrations in highly irrigated treatments. The down-regulation of key enzymes involved in the phenylpropanoid and flavonoid pathways, suggest a possible mechanism for the lower phenolic concentrations. The frequency and duration of extreme heat events predicted for the upcoming decades indicates the need for further field research looking at potential strategies to mitigate the negative effects of heat waves on berry composition and gene expression. Misting, shade cloths, cover crops, canopy management, and trellis systems that protect fruit exposure remain as options to help cope with the effects. Furthermore, studying acclimation within and across seasons of different cultivars to extreme heat events will help guide the grape and wine industry towards cultivars with better adaptations and ultimately higher quality fruit and wine. Obesity has become a major health problem worldwide and in the absence of an effective universal medical approach to tackle this epidemic, many patients use over-the-counter weight loss supplements to help them lose weight. Numerous products are available that promote weight loss and appetite suppression and are sold as fat burners. These supplements are often sold without any regulatory process or having undergone any Food and Drug Administration approved clinical trial showing safety and efficacy.

While used traditionally in a number of cultures, one of the best examples of its medicinal use is from the Kuna Indians who have lived for centuries on remote islands off of the Caribbean coast of Panama. This group of indigenous people is famous the lack of hypertension, an infrequent prevalence of CVD, diabetes, and cancer, and a longer lifespan, compared to Panamanians living on the mainland. However, when these people migrate to an urban environment, the incidence of hypertension and vascular diseases increased significantly. Nutritional assessments showed that the consumption of total fruit, fish, and cocoa-containing beverages were significantly higher among Kuna Indians living on the island compared to those residing in Panama City, even though the overall dietary intake of added sugars and salt was higher in the indigenous group. Scientists hypothesized that cocoa may be an influencing factor in the low prevalence of CVD in this population, due to its high concentration of flavonoids. The primary flavonoids in cocoa are flavanols, including monomeric catechins, epicatechin, and polymeric procyanidins. Cocoa also contains methylxanthines, i.e., theobromine and caffeine, large plastic growing pots which remain in flavanol-poor cocoa butter and cocoa solids after pressing. The effect of cocoa flavanols on vascular function was first reported more than two decades ago.

Extending the observations about cocoa flavanols to broader dietary patters, studies exploring the association between the intake of flavonoids and the risk of CVD have yielded inconsistent results, suggesting that certain sub-classes may be more effective than others in terms of cardio-protection. Some observational studies have shown that the dietary intake of flavanols was associated with a decreased risk of CVD and ischemic heart diseases. Epidemiological studies have reported that a moderate amount of chocolate consumption was associated with a decreased risk of CVD. A Cochrane meta-analysis concluded that the consumption of flavanol-rich chocolate and cocoa products had a small but statistically significant effect on reducing systolic and diastolic blood pressure, both by 1.76 mmHg. Similarly, another systematic review and meta-analysis included 42 acute and short- term randomized controlled trials concluded that chocolate, cocoa, and flavanol intake significantly improved vasodilation function as measured by flow-mediated dilatation , reduced diastolic blood pressure by 1.6 mmHg, and marginally improved the serum cholesterol profile. However, these results were strongest among individuals with moderately elevated blood pressure and untreated hypertension. The vasodilation function in healthy males, measured by FMD, was significantly increased one to four hours after taking a cocoa drink containing 917 mg of cocoa flavanols, compared to baseline values and to those consuming a control beverage with 37 mg of flavanols. 

The pattern of improvement in FMD from the flavanol-rich cocoa beverage was closely mirrored when participants also consumed 1 or 2 mg/kg of body weight of -epicatechin dissolved in water, suggesting -epicatechin and its metabolites were the main contributors of the vascular effects. Additional research from the same study found that while -epicatechin is a primary contributor to the vasodilation function, dimeric and oligomeric procyanidins that are metabolized by the gut microbiome may also contribute to the vasculo-protection. In healthy young men, daily intake of a cocoa extract containing 130 mg of -epicatechin and 560 mg of procyanidins for 30 days significantly improved FMD and reduced blood pressure and arterial stiffness as measured by pulse wave velocity , while 20 mg of -epicatechin and 540 mg procyanidins, or a control capsule, did not. However, total cholesterol was decreased after in both groups consuming cocoa, suggesting synergistic effects of -epicatechin and procyanidins, possibly through gut microbiome-mediated catabolism. Cocoa also contains methylxanthines, which are biologically active. While the intake of theobromine and caffeine alone did not result in a significant change in vascular endothelial function measures, interestingly, the combination of methylxanthines plus a high cocoa flavanol drink induced a significant improvement in FMD response than the beverage only. In addition, plasma metabolites of -epicatechin were higher after consuming the flavanol-rich cocoa drink with methylxanthines than when the flavanols were consumed alone, indicating a likely interaction between theobromine, caffeine, and cocoa flavanols on vascular function. Several molecular mechanisms regarding the effects of flavanols on blood pressure and vasodilation have been proposed. Flavanols such as -epicatechin can increase nitric oxide production directly by increasing endothelial nitric oxide synthase expression. The release of NO consequently increases intracellular cGMP which then induces a relaxation of vascular smooth muscle cells. The increased eNOS activation may also be modulated by flavanols through a calcium/calmodulin pathway by increasing the intracellular calcium concentration, or by phosphatidylinositol 3-kinase/protein kinase B -dependent eNOS phosphorylation. In addition, -epicatechin has been shown to down-regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase by inhibiting the synthesis of vasoconstrictors such as endothelin-1, and therefore increase the utilization of NO.

Flavanols may also directly inhibit angiotensin-converting enzyme activity, which increases NO production. Apart from modulating NO production, flavanols may also induce the release of endothelium-derived relaxing factors such as hydrogen peroxide and prostacyclin. Cocoa flavanols also benefit cardiovascular health by inhibiting platelet activation and adhesion. In healthy individuals, platelet aggregation induced by collagen and adenosine diphosphate, and the expression of P-selectin, was significantly decreased compared to a placebo group after the daily intake of 234 mg of cocoa flavanols and procyanidins for 28 days. Some studies have reported that the reduction in platelet aggregation was not different between flavanol-rich darkchocolate or low-flavanol dark chocolate mixed with white chocolate, suggesting potential antiadhesive effects from methylxanthines. While the exact mechanisms to explain the interaction between flavanols and platelets are still under investigation, proposed mechanisms from in vitro and ex vivo models include an inhibition in the expression of endothelial adhesion molecules , and the down-regulation of pro-inflammatory factors such as interleukin -6 and tumor necrosis factor-α, large plastic pots which also decrease the recruitment of other proinflammatory compounds. Mango originated from the Indian subcontinent and has been cultivated for thousands of years. The bark, leaves, roots, and flowers of the tree, and the peel, kernel, and pulp of the fruit, have been used in traditional medicine in tropical and sub-tropical regions throughout the world. The bark and leaves have been used for treating diarrhea and diabetes in Bangladesh, and Ghana, with the pulp and kernel used for hemorrhaging in the lungs and intestines in India.54 Mango fruit is a rich source of fiber, vitamins C and E, folate, potassium, β-carotene, and phenolic compounds. Dietary intake of vitamins C and E, and β-carotene, are associated with reduced risks for CVD. Major phenolic compounds reported in mango pulp include mangiferin, quercetin, kaempferol, myricetin, catechin, gallic acid, ferulic acid, protocatechuic acid, and chlorogenic acid. One study observed that a higher mango intake was associated with improved nutrient intakes, diet quality, and body mass index , factors known to reduce the risk of CVD. Clinical trials also suggest that mango fruit may have protective effects against the development of CVD. Daily intake of 200g of fresh-cut Ataulfo mango for 30 days decreased blood lipids and increased the plasma antioxidant capacity in healthy adults. In obese men and women aged 20-50 years, supplementation of 10 g/d freeze-dried mango pulp for 12 weeks decreased blood glucose levels but not inflammatory or any cholesterol markers. Another study reported that the daily intake of 400 g of fresh frozen mango pulp significantly decreased systolic blood pressure only in individuals with a BMI of 18-26.2 kg/m2 . In contrast, plasminogen activator inhibitor 1, IL-8, and mitochondrial pyruvate carrier-1 were significantly reduced in individuals when the BMI was partitioned as > 28.9 kg/m2 . In addition, in participants with impaired glucose sensitivity, the supplementation of 100 or 300 mg/d of mango fruit powder with 250 ml water daily for four weeks significantly increased the vasodilation of arteries as measured by the reactive hyperemia index compared to a placebo group.

Mangiferin is a unique compound in mango that has been studied for its vasculo protective effects . A mango bark extract with a high concentration of mangiferin decreased cholesterol in plasma and liver, and reduced oxidative stress in mice. A subsequent human study showed that the daily intake of 900 mg of Vimang® for 90 days reduced a measure of serum oxidative stress compared to a control group among older individuals. In both the animal and human studies, the marker of oxidative stress, while considered valid at the time of the study, is now viewed with limitations. In overweight hyperlipidemic individuals, the daily intake of 150 mg of mangiferin for 12 weeks significantly improved lipid profiles and glucose homeostasis. In hyperuricemic rats, mangiferin intake significantly reduced SBP, serum uric acid and inflammatory markers, and increased the expression of eNOS. One potential mechanism to help explain the vasodilatory effect of mangiferin may be due to the increased expression of eNOS, and therefore enhanced the production of NO. Reports have shown that the composition of phenolic compounds in mango varied significantly among different varieties. Mango varieties with high polyphenol content, such as Ataulfo, may play a more prominent role in cardiovascular health, but the interaction between the polyphenols, carotenoids and other bio-active compounds in mango must be considered. Further studies may also focus on the potential effects of mango by-products on metabolic health, since the total concentration of phenolic compounds is higher in the kernel, peel, leaves, and bark compared to the edible fruit. Such explorations may be useful in processing what is considered as agricultural waste into useful extracts. Low-bush blueberry and high-bush blueberry are two common species originally grown in North America. Native Americans have a long folklore history of using both types of blueberry plants to treat rheumatism and infection. Anthocyanins are responsible for the red, blue, and purple color in ripe berries. Blueberries are one of the most abundant sources of anthocyanins in commonly consumed fruits. The total anthocyanin level in fresh blueberries is significant, reaching up to 487 mg/100g. Blueberries also contain appreciable amounts of proanthocyanidins and hydroxycinnamic acids , along with vitamins and minerals, fiber, and small quantities of flavonols and flavanols. Epidemiological studies suggest that a higher dietary anthocyanin intake is associated with a lower risk of hypertension, and reduced arterial stiffness in women, though these studies do not specify blueberries as the sole source of these bio-actives. A meta-analysis of 19 cohort studies reported that the dietary intake of anthocyanins was associated with a decreased risk of coronary heart disease and CVD mortality, but not myocardial infarction, stroke, or total CVD risk. The effects of blueberries on markers of CVD risk have been studied. In obese postmenopausal women with pre- and stage I-hypertension, daily consumption of 22 g of freeze-dried blueberry powder containing 469 mg of anthocyanins for eight weeks significantly reduced systolic and diastolic blood pressure by 7 mmHg and 5 mmHg, respectively, and arterial stiffness measured by brachial-ankle PWV, compared to their baseline values or to a placebo group. In healthy males, FMD was significantly increased one, two, and six hours after the intake of 34, 57, and 80 g of blueberry powder mixed in water , compared to a control drink. However, no changes were seen in arterial stiffness measures. In addition, the increase in polyphenol metabolites and decrease in neutrophil NADPH oxidase in plasma were correlated to FMD, suggesting that the phenolic metabolites after blueberry powder consumption effectively improved vasodilation functions by elevating the bio-availability of NO through inhibition of NADPH oxidase. Later the research group identified that the FMD improvements were mainly due to anthocyanin metabolites. The blood pressures of overweight and obese smokers who consumed 250 g of blueberries for three weeks showed no significant changes from baseline values . Among mid-aged women who were at risk for type II diabetes, daily consumption of 240 ml of wild blueberry juice with 314 mg of anthocyanins for seven days significantly improved serum nitrates and nitrites, but no change were noted for in glucose metabolism parameters, cholesterols, inflammatory markers, platelet adhesion molecules, vasodilation, or blood pressure, compared to baseline and the placebo group. Taken together, the above results suggest that clinical trials with blueberries may need require a few weeks or longer of regular intake in order to observe clinically significant changes. Similar to blueberries, the American-cranberry is also a plant that is native to North America and has a long history of botanical uses by indigenous people, such as for urinary tract disorders and diarrhea.

Sugars were measured at 25% using a Refractometer PAL-1 . The C content of sugar was calculated at 42% using the formula of sucrose. Below ground biomass was measured by pneumatically excavating the root system with compressed air applied at 0.7 Mpa for three of the 12 sampling blocks, exposing two vines each in 8 m3 pits. The soil was prewetted prior to excavation to facilitate removal and minimize root damage. A root restricting duripan, common in this soil, provided an effective rooting depth of about 40 cm at this site with only 5–10 fine and small roots able to penetrate below this depth in each plot. Roots were washed, cut into smaller segments and separated into four size classes , oven-dried at 60 °C for 48 h and weighed. Larger roots were left in the oven for 4 days. Stumps were considered part of the root system for this analysis.In vineyard ecosystems, annual C is represented by fruit, leaves and canes, and is either removed from the system and/or incorporated into the soil C pools, square pots for plants which was not considered further. Structures whose tissues remain in the plant were considered perennial C.

Woody biomass volumes were measured and used for perennial C estimates. Cordon and trunk diameters were measured using a digital caliper at four locations per piece and averaged, and lengths were measured with a calibrated tape. Sixty vines were used for the analysis; twelve vines were omitted due to missing values in one or more vine fractions. All statistical estimates were conducted in R.An earth moving machine was used to uproot vines and gather them together to form mounds. Twenty-six mounds consisting of trunks plus cordons and canes were measured across this vineyard block . The mounds represented comparable spatial footprints within the vineyard area . Mound C stocks were estimated using their biomass contribution areas, physical size, density and either a semi-ovoid or hemispherical model.The present study provides results for an assessment of vineyard biomass that is comparable with data from previous studies, as well as estimates of below ground biomass that are more precise than previous reports. While most studies on C sequestration in vineyards have focused on soil C, some have quantified above ground biomass and C stocks. For example, a study of grapevines in California found net primary productivity values between 5.5 and 11 Mg C ha−1 —figures that are comparable to our mean estimate of 12.4 Mg C ha−1 . For pruned biomass, our estimate of 1.1 Mg C ha−1 were comparable to two assessments that estimated 2.5 Mg of pruned biomass ha−1 for both almonds and vineyards. Researchers reported that mature orchard crops in California allocated, on average, one third of their NPP to harvestable biomass, and mature vines allocated 35–50% of that year’s production to grape clusters.

Our estimate of 50% of annual biomass C allocated to harvested clusters represent the fraction of the structures grown during the season . Furthermore, if woody annual increments were considered this proportion would be even lower. Likewise the observed 1.7 Mg ha−1 in fruit represents ~14% of total biomass , which is within 10% of other studies in the region at similar vine densities. More importantly, this study reports the fraction of C that could be recovered from winemaking and returned to the soil for potential long term storage. However, this study is restricted to the agronomic and environmental conditions of the site, and the methodology would require validation and potential adjustment in other locations and conditions. Few studies have conducted a thorough evaluation of belowground vine biomass in vineyards, although Elderfield did estimate that fine roots contributed 20–30% of total NPP and that C was responsible for 45% of that dry matter. More recently, Brunori et al. studied the capability of grapevines to efficiently store C throughout the growing season and found that root systems contributed to between 9 and 26% of the total vine C fixation in a model Vitis vinifera sativa L. cv Merlot/berlandieri rupestris vineyard. The results of our study provide a utilitarian analysis of C storage in mature wine grape vines, including above and below ground fractions and annual vs. perennial allocations. Such information constitutes the basic unit of measurement from which one can then estimate the contribution of wine grapes to C budgets at multiple scales— fruit, plant or vineyard level—and by region, sector, or in mixed crop analyses. Our study builds on earlier research that focused on the basic physiology, development and allocation of biomass in vines.

Previous research has also examined vineyard-level carbon at the landscape level with coarser estimates of the absolute C storage capacity of vines of different ages, as well as the relative contribution of vines and woody biomass in natural vegetation in mixed vineyard-wildland landscapes. The combination of findings from those studies, together with the more precise and complete carbon-by-vine structure assessment provided here, mean that managers now have access to methods and analytical tools that allow precise and detailed C estimates from the individual vine to whole-farm scales. As carbon accounting in vineyard landscapes becomes more sophisticated, widespread and economically relevant, such vineyard-level analyses will become increasingly important for informing management decisions. The greater vine-level measuring precision that this study affords should also translate into improved scaled-up C assessments . In California alone, for example, there are more than 230,000 ha are planted in vines. Given that for many, if not most of those hectares, the exact number of individual vines is known, it is easy to see how improvements in vine-level measuring accuracy can have benefits from the individual farmer to the entire sector. Previous efforts to develop rough allometric woody biomass equations for vines notwithstanding, there is still a need to improve our precision in estimating of how biomass changes with different parameters. Because the present analysis was conducted for 15 year old Cabernet vines, there is now a need for calibrating how vine C varies with age, varietal and training system. There is also uncertainty around the influence of grafting onto rootstock on C accumulation in vines. As mentioned in the methods, the vines in this study were not grafted—an artifact of the root-limiting duripan approximately 50 cm below the soil surface. The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites per se, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, large square plant pots they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility. The current study was also designed to complement a growing body of research focusing on soil-vine interactions . Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, plant health and fruit production, as well as for those concerned with C storage. The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al.estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons . With the contribution of this study, the magnitude of the below ground reservoir can now be updated.California’s Mediterranean climate, albeit highly variable with frequent periods of drought and floods, provided the foundation for a diverse and vibrant agricultural industry to grow in response to the availability of low-cost labor and water supplies. Starting in the middle of the 19th century field crops–grains, forages, and cotton–dominated California crop landscapes, if not value of production, for a hundred years.

Toward the beginning of the 20th century, though, California agriculture began its move toward intensive cropping of vegetables and fruits. Railroads helped expand produce markets and low-wage immigrant labor. Later, migrants from the Dust Bowl, and then from Mexico, kept labor costs on fruit and vegetable farms competitive . Importantly, irrigation infrastructure and regulation—particularly water pumping, storage, transport, and rules of use—allowed cultivation of water-intensive summer crops where no rain fell for 6 mo each year. The Great Depression catalyzed massive surface water infrastructure developments such as the Colorado River Project and, in the late 1930s, the Central Valley Project by the US Bureau of Reclamation . Further growth of infrastructure in the postwar era included the State Water Project serving mostly cities and some agricultural lands in central and southern California. Infrastructure development over this period created one of the largest and most engineered irrigated agricultural systems in the world. The water supply network bridged the gap of hundreds of kilometers between the water-rich north—with mountains and heavy precipitation in the winter—and the low-precipitation Mediterranean climate central and south that plays host to most of California agriculture production and population. For nearly two centuries California farms have prospered through technological adoption, innovation investments, and on-farm management improvements. Yet with a changing climate coupled with increased concerns over the environment and sustainability, the landscape of California agriculture is changing. Over the past two decades, noticeably less land and water has been devoted to extensive field crops, as farms shifted to vegetables and tree and vine crops. These specialty crops generally produce higher revenues per unit of land and water . Expectations of higher returns have contributed to more than half of the state’s irrigated agricultural croplands growing fruits, nuts, and vegetables, which comprise roughly 80% of the farm revenue and employment . The degree to which these changes and concerns significantly reduce agriculture’s presence and productivity will depend on how Californians, including its growers and policymakers, respond.The mosaic of agriculture in California is driven by a variety of natural and human-created conditions . California’s terrain, climate, and soil heterogeneity are instrumental to California’s diverse array of agricultural commodities. The irrigated crop footprint alone is nearly 3.8 million hectares . Land in farms spans more than 10 million ha, producing over 400 crop and livestock commodities that annually generate around $50 billion in cash receipts and support 420,000 jobs in 2021 . The food and beverage processing sector, which primarily relies on local crop and animal supplies, supports an additional 250,000 jobs. Agriculture contributes significant shares of the income and employment in areas such as the CV, where labor, capital, irrigation water, management, and downstream sectors in livestock and food processing are closely linked. We briefly describe agriculture in three regions comprising the largest areas of irrigated acreage and commodity value: the CV, the Southern California Region, and the Coastal California. Agriculture in California’s foothills and mountain areas provide nearly 5 million ha of pasture and hay for cattle, along with the winter snow pack that historically stores nearly a third of California’s runoff that supplies CV irrigation as well as urban water use. CV. The nearly 52,000 km2 CV accounts for more than two thirds of California’s irrigated agriculture, encompassing a few major cities and dozens of moderate-sized rural communities. The northern part of the CV contains the Sacramento Valley and the Sacramento River basin, which averages 890 mm/y. of precipitation and is close to the snow pack-heavy northern mountains.

Our data showed that the expression of flavonoids/PAs related-genes was highly increased in V9-berries at the third harvest time compared to V7- berries, resulting in the accumulation of more PAs in V9-berries.Developing and producing table grapes with high quality is of utmost importance for the success of grapevine breeding programs. Scarlet Royal table grape , variety , is one such success story, producing premium fruit quality and becoming one of the major red varieties in California. However, under certain unknown circumstances, the berry quality of Scarlet Royal grapes can be affected by undesirable astringent taste, which can negatively impact marketability and consumer acceptance. Research on the relationship between astringency and phenolic composition in table grapes is still scarce, especially on the transcriptomic level. In this study, we aimed to understand the molecular events involved in the development of berry astringency, which is a complex set of sensations resulting from the shrinking, drying, drawing, or puckering of the mouth epithelium . We focused on Scarlet Royal berries from two different vineyards with contrasting astringency and analyzed the changes in phenolic-related compounds at six different time points from veraison until the last harvesting time.

Our panel test revealed that the V9-berries were perceived as more astringent, blueberry packaging a characteristic that could be attributed to their elevated levels of tannins . Differences observed between the two vineyards under study indicate that V7 vines yield more compared to V9. This difference may be attributed to the lower cluster count in V9, a factor known to potentially contribute to astringency, as suggested by Cañon et al., 2014 in wine grapes. Additionally, petiole analysis revealed higher levels of nitrogen and potassium in V9 vines compared to V7. Vine nutrient levels contribute to the final berry quality at harvest . These factors may also contribute to the higher levels of tannins detected in V9 berries; however, further research is needed to confirm this theory. Nevertheless, the two vineyards are located in close proximity to each other, and weather data collected from the same station in the Delano area indicates similar conditions. Therefore, it is unlikely that astringency or higher phenolic compounds are induced by weather factors. In fact, several studies, mainly in wine, have pointed to PAs as a determining factor for astringency intensity . For example, Vidal et al. reported that the total amount of tannins is the most plausible factor for sensory astringency, with flavan-3-ols dimers, trimers, and non-galloylated tetramers contributing to the astringency sensation. The PAs are a group of oligomers and polymers of flavan-3-ols and are the naturally occurring and predominant type of tannins in grapes and wine . Another study on aronia berry juice confirmed PAs as the key astringent compounds using sensory evaluation and phenolic profile approaches along with in-vitro models .

The study found that PAs with higher degrees of polymerization were responsible for the strong astringent mouthfeel . The composition of phenolic substances, especially PAs, seems to play a crucial role in determining berry astringency, and further exploration of this relationship is warranted in fresh fruits of different species and cultivars. Understanding the molecular basis of astringency development in Scarlet Royal berries can provide valuable insights for improving grape breeding programs and enhancing the overall quality of table grapes.The relationship between astringency and the berry polyphenols content has not been explored yet in table grape at the molecular level. To the best of our knowledge, the present study provides the first transcriptome profiling along with the changes of polyphenols in grape berries of the same variety but having astringency diversity . The transcriptome profile of both V7-berries and V9-berries underlined the remarkable transcriptional shift during berry ripening at different vineyards . Commonly, berry transcriptome profiles may widely vary based on many factors, including genotypic variations among varieties/species and developmental stages , as well as environmental circumstances. In our case of study, stemmed differences from the developmental stages and genotypic variations were eliminated, and hence the difference of the vineyard locations was the main source of variance with 97% of variance . The identified DEGs output of V9-berries compared to V7-berries is highly explanatory, including polyphenolic-related genes that are robustly expressed and co-regulated with astringency development, particularly in the V9-berries. The enrichment of the up-regulated genes with BP GO terms related to the biosynthesis of secondary metabolites, phenylpropanoid, and nitrogen compound metabolic process , commensurate with the higher amounts of N found in V9- berries . In fact, not only the levels of N fertilization but also its different forms highly affected the composition of phenolic compounds in leaves and wine . However, the synergistic/antagonistic effects of other macro- and micro-nutrients should also be considered. Our results highlighted the negative impact of above-normal amounts of macro-nutrients, mainly N, and to a lesser extent P and K, on the desirable attributes of grape berries.

Otherwise, the positive effect of Ca, Mg, and Mn were achieved as their levels were maintained within the normal range . These data should be also seen in the light of the highly enriched BP GO terms in the down regulated genes . Particularly, those for hormonal signaling pathways such as auxin, abscisic acid, strigolactones, as well as the KEGG pathway for the carotenoid biosynthetic pathway .The transcriptome profiling identified the common and unique molecular events featuring the development of tannins/astringency in grape berries. It is well-documented that the synthesis of PAs in grapevines is achieved via three sequential pathways: the shikimate pathway, the phenylpropanoid pathway, and ultimately the flavonoid pathway . Our results revealed that the expression levels of flavonoids/PAs-related genes were highly induced in V9- berries at the third harvesting time compared to V7-berries. The 19 selected genes were involved in the three pathways: the shikimate pathway, phenylpropanoids pathway, and flavonoids pathway. The shikimate pathway is an alternative route to produce aromatic compounds, including phenylalanine, tyrosine, and tryptophan, which serve as precursors for various metabolites, such as phenolic compounds . The up-regulation of genes like chorismate synthase and chorismate mutase in V9-berries may lead to the accumulation of phenylalanine, which is a critical precursor for the phenylpropanoid pathway. The latter pathway is responsible for synthesizing several end products, including PAs, anthocyanins, lignin, lignans, hydroxycinnamic acid esters, and hydroxycinnamic acid amides . Under the conditions of the V9 vineyard, several PAs/flavonoids structural genes such as PAL, C4H, 4CL, CHS, CHI, F3H, LDOX, LAR, and ANR were induced in V9-berries, leading to the accumulation of PAs in the berries . This process is facilitated by GSTs and transported by multidrug and toxic compound extrusion transporters. The activation of the PAs biosynthetic pathway in V9- berries may lead to a reduction in the necessary substrates for anthocyanin synthesis, resulting in low red color intensity in V9- berries compared to V7-berries. Additionally, the accumulation of PAs is associated with the development of astringency taste in V9-berries. Our study provides valuable insights into the molecular events underlying astringency development in Scarlet Royal berries. By integrating transcriptome profiling with polyphenolic composition analysis, the research shed light on the co-regulation of genes involved in the shikimate, phenylpropanoid, and flavonoid pathways, leading to the synthesis of PAs and ultimately influencing astringency. The findings from this research have implications for grapevine breeding programs and the production of high-quality table grapes. Understanding the molecular mechanisms underlying astringency development can help breeders in selecting and developing grape varieties with desirable attributes. Additionally, the knowledge gained from this study can inform vineyard management practices, such as nutrient fertilization, to optimize polyphenolic composition and berry quality. In conclusion, the data presented in this study indicates that berry astringency is strongly correlated with a high tannin content, blueberry packaging box likely resulting from the activation of nineteen genes within the phenylpropanoid pathway. The activation of these genes shifts the flavonoid biosynthesis pathway towards proanthocyanins, leading to increased tannin accumulation in the berries. It appears that triggering these events is associated with nutritional imbalances and a lower number of clusters per vine, as confirmed by petiole nutrient levels and the observed lower berry soluble solids and higher titratable acidity levels. The identification of these genes holds significant value for table grape genetic improvement programs. The nutrient imbalance theory derived from this research could be applied worldwide to optimize grapevine fertilization programs. Furthermore, it paves the way for further research in this area, with a particular focus on vine nutrients, crop load management, and berry astringency, thereby contributing to advancements in the field of table grape cultivation.The cultivated garden strawberry , an allo-octoploid , has a unique natural and domestication history, originating as an interspecific hybrid between wild octoploid progenitor species approximately 300 years before present1 . The genomes of the progenitor species, Fragaria virginiana and Fragaria chiloensis, are the products of polyploid evolution: they were formed by the fusion of and interactions among genomes from four diploid progenitor species approximately 1 million years before present.

Whereas two of the diploid progenitor species have been identified, the other two diploid progenitor species have remained unknown. Moreover, the history of events leading to the formation of the octoploid lineage and the evolutionary dynamics among the four sub-genomes that restabilized cellular processes after ‘genomic shock’ in allopolyploids remain poorly understood. Here, we present what is, to our knowledge, the first chromosome-scale assembly of an octoploid strawberry genome, the identities of the extant diploid progenitor species of each sub-genome, and novel insights into the collective evolutionary processes involved in establishing a dominant sub-genome in this highly polyploid species. The Rosaceae are a large eudicot family including a rich diversity of crops with major economic importance worldwide, such as nuts , ornamentals , pome fruits , stone fruits , and berries. Strawberries are prized by consumers, largely because of their complex array of flavors and aromas. The genus Fragaria was named by the botanist Carl Linnaeus, on the basis of the Latin word ‘fragrans’, meaning ‘sweet scented’, describing its striking, highly aromatic fruit . A total of 22 wild species of Fragaria have been described, ranging from diploid to decaploid. The genus Fragaria is highly interfertile between and within ploidy levels, thus leading to the natural formation of higher-polyploid species. Polyploid events, also known as whole-genome duplications, have been an important recurrent process throughout the evolutionary history of eukaryotes and have probably contributed to novel and varied phenotypes. Polyploids are grouped into two main categories: autopolyploids and allopolyploids, involving either a single or multiple diploid progenitor species, respectively. Many crop species are allopolyploids, thus contributing to the emergence of important agronomic traits such as spinnable fibers in cotton, diversified morphotypes in Brassica, and varied aroma and flavor profiles in strawberry. Allopolyploids face the challenge of organizing distinct parental sub-genomes—each with a unique genetic and epigenetic makeup shaped by independent evolutionary histories—residing within a single nucleus. Previous studies have proposed, as part of the ‘sub-genome dominance’ hypothesis, that the establishment of a single dominant sub-genome may resolve various genetic conflicts in allopolyploids. However, understanding of the underlying mechanisms and ultimate consequences of sub-genome dominance remains largely incomplete. sub-genome-level analyses in most allopolyploid systems are greatly hindered by the inability to confidently assign parental gene copies to each sub-genome, owing to both large-scale chromosomal changes and homoeologous exchanges that shuffle and replace homoeologs among parental chromosomes. Octoploid strawberry still has a complete set of homoeologous chromosomes from all four parental sub-genomes, thus greatly simplifying homoeolog assignment. Furthermore, gene sequences from extant relatives of the diploid progenitor species, which probably still exist for octoploid strawberry, can be used to accurately assign homoeologs to each parental sub-genome. However, a highquality reference genome for the octoploid is needed to fully exploit strawberry as a model system for studying allopolyploidy as well as to provide a platform for identifying biologically and agriculturally important genes and applying genomic-enabled breeding approaches. The assembly of the octoploid strawberry genome, with an estimated genome size of 813.4 Mb, has been particularly challenging because of its high heterozygosity and ploidy level. For example, the most recently published version of the octoploid strawberry genome is highly fragmented, with more than 625,000 scaffolds, and largely incomplete, with less than 660 Mb assembled after removal of the numerous gaps. Thus, that version of the genome, owing to its overall highly fragmented nature, has not been a useful resource for genome-wide analyses including the discovery of molecular markers for breeding.Our goal was to obtain a high-quality reference genome for the Fragaria×ananassa cultivar ‘Camarosa’, one of the most historically important and widely grown strawberry cultivars worldwide.