The cardiometabolic benefits from regular consumption of nuts or berries are widely reported and include improved vascular function, reduction of cardiovascular disease risk factors, improved insulin sensitivity, and reduced risk of type 2 diabetes mellitus. Antioxidant and anti-inflammatory capacity and activity have also been noted. Metabolic outcomes may be context-specific and related to the physiologic state of the individual and host microbiome composition, among other factors. Examples include findings of ellagitannin and ellagic acid rich foods resulting in differential responses in healthy individuals compared to those with prediabetes, who are dependent on gut microbial-derived metabolite profiles. Many factors contribute to interindividual variability in response to diet that can extend to context-specific aspects influencing the magnitude of health benefits and reinforces the importance for further research aimed at advancing discoveries in precision nutrition. Additional health outcomes related to nut or berry intake are outlined below.Adding nuts or berries to the daily diet may be advantageous for weight management for several physiological reasons. One is that these foods produce feelings of satiety, helping to reduce the desire to consume calorie-rich snacks that are low in vitamins, minerals, and fibers, ultimately improving body composition over time. A second possibility is due to urolithins, secondary metabolites produced from ellagitannins in nuts and berries. Urolithins increase the activation of the adenosine monophosphate-activated protein kinase pathway, resulting in anti-obesogenic properties in vitro and in animal models. AMPK increases fatty acid oxidation and decreases triglyceride accumulation. Phosphorylation of AMPK may also decrease cholesterol synthesis and lipogenesis by downregulating 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol regulatory-element binding protein expression. In clinical studies exploring the relationship between food and body composition, raspberry grow in pots the incorporation of nuts and berries into the diet was associated with weight loss or maintenance.

Regular consumption of nuts or berries has been reported to support brain health and cognitive function, motor control, mood, and executive function at physiologically relevant intakes. Middle-aged and older adults experienced improvements in balance, gait, and memory, and children experienced higher executive function and positive affect after acute and regular intake of both strawberries and blueberries. These beneficial effects may be the result of direct effects on brain signaling or indirect effects through oxidant defense and anti-inflammatory properties of polyphenols and other bioactive compounds in nuts and berry foods. The gut-brain axis is an emerging area of research. Most studies are preclinical in nature using animal models but are suggestive of a significant role of gut microbial-derived ellagitannin metabolites on brain health and neuroprotection. Regular intake of almonds, a good source of fatty acids and polyphenols, has been associated with a significant decrease in facial hyperpigmentation and wrinkle severity. A walnut protein hydrolysate administered to rats exposed to ultraviolet radiation significantly reduced skin photoaging and enhanced skin elasticity. Supplementation with ellagic acid, a compound found in many berries, prevented ultraviolet B -related inflammation and collagen degradation related to skin wrinkling and aging in a murine model. More human studies, using objective measures of skin wrinkles, skin elasticity and response to low-dose UVB radiation exposure are warranted. Monitoring skin responses to a UVB radiation challenge has been used as a marker of whole-body antioxidant status in response to almond consumption. The response to a UVB challenge has also been used to monitor oxidant defenses and changes in skin microbiome following the intake of pomegranate juice.Age-related macular degeneration is the third leading cause of vision loss worldwide .

Anthocyanins, carotenoids, flavonoids, and vitamins C and E, found in many berries, have been shown to reduce risk of eye-related diseases. Goji berries, containing the highest amount of zeaxanthin of any known food, hold particular promise since this compound binds to receptors in the macula to offer protection from blue and ultraviolet light. Regular supplementation with 28 g/d of goji berries for 3 mo increased macular pigment optical density, a biomarker for AMD, as well as the skin carotenoid index. Nuts may also be protective against AMD since they are a rich source of vitamin E and essential fatty acids. Regular intake of nuts has been associated with a reduced risk and slower progression of AMD in 2 epidemiological studies, thought to be due to the beneficial role of polyunsaturated fatty acids.Identification of new cultivars with traits desirable for growers, processors, and consumers is a continuous effort. As researchers continue to produce new varieties by both conventional and molecular-driven approaches, assessing these varieties for nutritional value is a challenge. A combination of broad targeted and untargeted metabolomic approaches, along with defined functional phenotyping could be used for rapid screening and defining of mechanistic pathways associated with health. However, consumer preferences for new cultivars are often driven by size and appearance of the berry or nut and flavor, rather than its nutritional value . This would further confirm the need to balance improvements to nutritional profiles with enhancement of consumer-driven traits, maintaining the marketable nature of the berries and nuts.Biomedical research, particularly for clinical studies, is expensive and resource intensive. Although the USDA competitive grants program offers funding for outstanding research projects, budget limitations favor animal or in vitro study proposals. Compelling pilot data is needed to be competitive for clinical studies funded by the USDA or NIH, so many researchers submit their initial ideas to commodity groups representing specific nuts or berries. Commodity groups represent farmers, processors, and distributors and have been instrumental in supporting fundamental and applied research focused on their specific berry or nut.

The perception that studies funded by nut and berry commodity groups are inherently biased in favor of the test food is an issue sometimes raised by critics, journalists, and the general public. As in all nutrition research, ethical considerations regarding the structure of research questions, hypotheses, study design, outcome measures, interpretation of data, and conclusions must be rigorously considered. The food and beverage industries have played a key role in providing funds and supporting nutrition research on individual foods and beverages, including berries and nuts. Although this draws scrutiny regarding scientific integrity and data reporting, collaboration between academia and industry compared to exclusive corporate funding may help offset some of these concerns. For example, in multiple reported studies, matching funds were also provided by non-industry sources, including institutional and federal agencies. In other cases, while the food industry provided the test agents, key research personnel and staff were not supported by the same funding source. The academia-industry collaboration has also led to the formation of scientific advisory committees that evaluate and recommend proposals for funding, a peer review process that helps ensure rigorous study designs, data reporting, and dissemination of results. Human studies of sufficient statistical power are expensive, labor-intensive efforts requiring sophisticated and costly laboratory equipment and supplies. In order for research proposals to be competitive for funding from the USDA or NIH, pilot data is required, and for nuts and berries, the only realistic source of funding for these exploratory trials is from industry sources. Critics of industry support for nutrition research have yet to propose realistic alternatives for funding needed to generate initial data. Further, 30 planter pot ongoing industry funding of nuts and berries research has yielded important insights into the molecular and physiological understanding of mechanisms of action. Without industry support, provided in an ethical and transparent manner, advances in our understanding of the role of nuts and berries in a healthy dietary pattern would be limited. A risk-of-bias study of 5675 journal articles used in systematic reviews published between 1930 and 2015, representing a wide variety of nutrition topics, concluded that ROB domains started to significantly decrease after 1990, and particularly after 2000. Another study examined the incidence of favorable outcomes reported in studies funded by the food industry in the 10 most-cited nutrition and dietetics journals in 2018. Of the 1461 articles included in the analysis, 196 reported industry support, with processed food and dietary supplement manufacturers supporting 68% of the studies included. Studies supported by any nut or berry commodity group were not considered due to an incidence lower than 3% of qualifying articles. Studies with food industry support reported favorable results in 56% of their articles, compared to 10% of articles with no industry involvement. The authors offer a number of suggestions to help minimize real or perceived bias, calling on research institutions to enforce strict, regularly updated, and transparent oversight of all research projects involving industry.

Suggestions in support of research transparency and integrity have also been advanced from guidelines adapted from the International Life Sciences Institute North America. This served as the basis for the development of consensus guiding principles for public-private partnerships developed by a group of representatives from academia, scientific societies and organizations, industry scientists, and the USDA, NIH, US Centers for Disease Control, and the US Food and Drug Administration. These provisions include full disclosure of funding and confirmation of no direct industry involvement in the study design, data and statistical analyses, and interpretation of the results and only minimal, if any, involvement of industry coauthor, often given as a courtesy to acknowledge funding and logistical support by the investigators with no intellectual involvement by the study sponsor. This is in contrast to industry-initiated research, where the industry office or commodity group sets predetermined research objectives, provides intellectual collaboration, and often has input on the study design, interpretation of results, and decisions regarding publication. Although some critics may argue that repeated industry funding in support of research groups that report favorable results on a particular nut or berry shows a bias toward positive outcomes, other interpretations are also possible. First, few labs have the infrastructure, detailed methodology and analytical equipment, and trained personnel to conduct clinical studies in an efficient and timely manner. Industry funded studies conducted at major universities have layers of review and accountability within their organizations to guard against malfeasance, and while these layers may not focus directly on precise elements of research design and interpretation of results, faculty members at such institutions generally have a level of integrity and accountability, knowing that administrative review exists. Calls for industry-funded research are often broad in scope, which allows researchers to generate proposals, research questions, and hypotheses that do not have preconceived outcomes. A third consideration is that the nuts or berries under study may simply have sufficient bio-activity to produce favorable outcomes, independent of potential researcher bias.This is not the case for two dimensional systems. Those readers with any exposure to introductory physics have likely encountered parallel plate capacitors; these are highly idealized systems composed of a pair of infinitely thin conducting sheets separated by a small insulating space of consistent thickness. When a voltage is applied to one of these sheets with the other connected to a reservoir of mobile electrons, a uniform charge density per unit area appears on both sheets . Of course, in real metallic capacitors the charge density per unit volume is often still not microscopically uniform because the sheets are not actually infinitely thin, so electrons can redistribute themselves in the out-of-plane direction. To achieve true uniformity one of the plates of the capacitor must be atomically thin, so that electrons simply cannot redistribute themselves in the out-of-plane direction in response to the local electric field. An efficient technique for preparing atomically thin pieces of crystalline graphite was discovered in 2004 by Dr. Andre Geim and Dr. Konstantin Novoselov, an achievement for which they shared the Nobel prize in physics in 2010. The technique involves encapsulating a crystal within a piece of scotch tape and repeatedly ripping the tape apart; it works because the out-of-plane bonds in graphite are much weaker than the in-plane bonds. Graphite represents something of an extreme example of this condition, but it is satisfied to varying extents by a large class of other materials, and as a result the technique was rapidly generalized to produce a variety of other two-dimensional crystals. By constructing a capacitor with one gate replaced with one of these two dimensional crystals, as shown in Fig. 1.1D, researchers can easily access electron density as an independent variable in a condensed matter system. These systems also facilitate an additional degree of control, with no real analogue in three dimensional systems. By placing capacitor plates on both sides of the two dimensional crystal and applying opposite voltages to the opposing gates, researchers can apply out-of-plane electric fields to these systems .

Our results in the second year corroborated those of the first year, showing that the separation in both plant water status and leaf gas exchange between the two zones were consistent. Leaf gas exchange was closely related to plant water status, and this relationship was shown in previous research . The relationships between leaf gas exchange and plant water status were evident in our study, where a higher 9 stem would promote a greater stomatal conductance to increase carbon assimilation capacity and decrease intrinsic water use efficiency. In our study, the lowest 9 stem we observed were around harvest with 9 stem of -1.6 MPa and gs of around 50 mmol H2O m−2 ·s −1 , which were not severe enough to impair berry ripening although the photosynthetic activities were still affected. Overall, the gs and AN reached the maximum values at veraison and declined with decreasing plant water status and leaf age toward the end of the season. This further affirmed that the continuous water deficits during the growing season, especially being more pronounced after irrigation was ended after veraison, would reduce stomatal conductance. The water deficits would act as passive hydraulic signals or active hormonal signals with the upregulation in abscisic acid synthesis to limit plant photosynthetic activities, hence lower gs and AN values .According to the previous research, components of yield may be affected by plant water status, where higher water deficits would result in reductions of yield, berry skin weight, and berry weight . In our study, blueberry production we observed constant separation in plant water status after veraison. However, there was no difference shown in cluster number, yield, berry number, or pruning weight.

The only difference measured in yield components was that berry skin weight was higher in Zone 1 in the second season. Early season water deficit irrigation had higher probability to decrease yield than later season water deficit irrigation . However, a season-long water deficit irrigation would have the lowest yield even despite the season-long water deficit irrigation regime applying double amount of water than the other regimes . Some other studies did not have the same results, as early water deficit irrigation did not show significant influences on yield compared to late water deficit irrigation . Another possible explanation was that Zone 1 had greater water amount held in the soil due to the higher clay content. The clay soil with higher water-holding capacity had a better water status at the early season compared to Zone 2, even though the sandy soil in Zone 2 would benefit the plant growth with irrigation when the season progressed . The later season water deficit was exacerbated in Zone 1 due to its higher clay content, causing Zone 1 lost the benefits from the high water status in the early season, and eventually had similar yield components with Zone 2 at harvest. In our work, we did not see any evidence of Ravaz index being affected by spatial variability of plant water status. These results were corroborated by Terry and Kurtural when grapevine cultivar ‘Syrah’ was exposed to post-veraison water deficits in comparable severity of -1.4 MPa .Water deficits affect advancement of grape berry maturity, they promote TSS accumulation and TA degradation in grape berries . Two factors contributed to these differences between the two zones. First, a greater water deficit advanced the berry maturation, leading to a higher TSS and lower TA . Second, berry dehydration may have occurred and the TSS concentration increased in the berries. In our study, smaller berries were observed in Zone 1, which can confirm the berry dehydration could have led to higher TSS in Zone 1. As for berry TA, one study showed that grape organic acids biodegradation would be faster with more solar radiation and higher temperature .

Although the acid degradation was not related to water deficits, like mentioned above, water deficits would limit the grapevines’ ability to regulate temperature . Thus, waterdeficits could promote the organic acid degradation and this effect was observed in this study.Mild water deficits increased the flavonoid content and concentration of red-skinned grape berry due to the upregulation in flavonoid synthesis and the advancement of berry dehydration during growing season . A positive relationship was noticed between soil bulk EC and total skin anthocyanins in 2017 at both depths of soil bulk EC measurements. A more prolonged severe water deficit would lead to deleterious stomatal and temperature regulation and eventually resulted in flavonoid degradation, specifically anthocyanins . This was a plausible explanation for the non-significant relationship between soil bulk EC and total skin anthocyanins in 2016, wherein harvest took place at higher soluble solids and Zone 1 berry skin anthocyanins were presumably in decline. Furthermore, the berry weights were higher in Zone 2, which was similar to the observations in our previous work , indicating there was less berry dehydration. Thus, the higher anthocyanins in Zone 2 was mainly due to the upregulation in anthocyanins other than anthocyanins degradation. These effects were also observed in the wines of 2016, where Zone 2 had higher anthocyanin concentrations. However, in the second season, the differences in berry skin anthocyanins at harvest did not carry over into the wines. We contributed this to the more advanced berry maturity levels at harvest in the first season, the skin cell walls could have become more porous during ripening and increased the extractability of flavonoid compounds . With relatively greater amounts of flavonoids extracted, there was a higher chance to pass on the separations of anthocyanins from the berries to the wines.

Grape berry skin proanthocyanidins are less sensitive toward water deficits than anthocyanins . Nevertheless, their biosynthesis and concentration may be modified by water deficits . In 2016, wine total proanthocyanidins and all the subunits were greater in Zone 2. These differences were not observed in the second season. We attributed this lack of consistency in proanthocyanidin disparities between the two zones to the more advanced maturity of the berries were harvested in 2016 than in 2017. We suggest that similar to skin anthocyanins, the more advanced berry maturity in 2016 could have promoted the proanthocyanidin extractability in the skin tissues , which may augment the separations in the concentration of all the subunits between the two zones.Monitoring and handling live tissues and cell cultures as well as analyzing their secreted contents are essential tasks in experimental biology and bio-medicine. Advances in microscopy have revolutionized biological studies, allowing scientists to perform observations of cellular processes and organisms’ development and behaviors. Imaging has been pivotal to uncovering cellular mechanisms behind biological processes. Several options exist on the market to perform longitudinal imaging of biological materials. These range from super-resolution microscopes, that allow the imaging of individual bio-molecules, to conventional benchtop microscopes, which are common in academic research, industrial, and teaching laboratories. When deciding between the different technologies for longitudinal live tissue imaging, several factors need to be considered in the experimental design. The image acquisition speed of the microscope should be sufficient for the phenomenon being studied. The microscope should be able to acquire images without damaging or disturbing the specimen, such as photo bleaching. The microscope should be capable of imaging in the environmental conditions needed for the desired experiment, including temperature, light, and humidity. The resolution of the microscope should be sufficient to view the phenomenon being studied. When scaling to simultaneous multi-well longitudinal tissue imaging it is also important that the apparatus not be bulky or expensive. It has been challenging to meet all of these criteria. The use of open-source technology, including 3D printers, laser cutters, blueberry in container and low-cost computer hardware, has democratized access to rapid prototyping tools and dramatically increased the repertoire of biomedical equipment available to laboratories around the world. Through rapid prototyping and the use of open-source platforms, the technology can be replicated and quickly improved. 3D printer technology has been applied to several fields in bio-medicine, including biotechnology, bioengineering, and medical applications including fabrication of tissues and organs, casts, implants, and prostheses. Existing 3D printed microscopes range in complexity from simple low-cost systems with pre-loaded imaging modules to portable confocal microscopes capable of imaging individual molecules and even 3D printed microfluidic bioreactors. The majority of low-cost 3D printed microscopes are not intended for longitudinal imaging of simultaneous biological cultures . They usually have a single imaging unit or perform confocal, and even light-sheet imaging. Other systems have taken advantage of one camera attached to a gantry system to perform imaging of multiple experimental replicates. Few 3D-printed microscopes have been developed that perform multi-well imaging with medium throughput.

Several biological applications exist that would greatly benefit from multi-well, multi-week simultaneous imaging, as it allows for concurrent interrogation of different experimental conditions and the inclusion of biological replicates. These include cell culture applications, in which 2D and 3D culture models can be tracked over multi-week periods, as well as developmental and behavioral biology experiments in which multi-week tracking could be performed on whole organisms. Here, we report a simultaneous multi-well imaging system , which features a low-cost per well and performs longitudinal bright field z-stack imaging of 24-well cell culture plates. Images are uploaded to a server as they are captured allowing the users to view the results in near real time. We used this system to longitudinally track different animal models of development and regeneration, including Xenopus tropicalis , Danio rerio , and planaria worms. Finally, we demonstrate this system’s versatility by imaging human embryonic stem cells and 3D cortical organoids inside a standard tissue culture incubator. We demonstrate that the Picroscope is a robust low-cost, versatile multi-well imaging system for longitudinal live imaging biological studies.System design. The Picroscope is a programmable, data rich, sensor-per-well simultaneous imaging system for longitudinal bright field imaging to automate microscopy . The system simultaneously images in each one of the 24 wells multiple focal planes several times every hour for weeks, a frequency impractical to perform manually. The instrument is made using off-the-shelf components , and 3D printed Polylactic acid components with 100% infill . Cost comparison with other open-source microscope projects can be found in Table 1. A cost breakdown of the materials required can be found in Table 2. The Picroscope has been used to image Planaria worms , Xenopus tropicalis , as well as zebrafish . The system was developed to be operated remotely through the internet. Users can set and change the device settings to modify experiments on the fly. Images captured by the system are uploaded to a server where they become visible on a viewer website. We have also created several image analyses scripts that can directly access images on the server, allowing us to generate timelapse videos and composite images in an automated fashion. While the system receives commands and transmits results through the Internet, it is also capable of running on a Local Area Network if internet access is not available. Figure 2 shows the basic workflow from control console to image viewer. Further details about the software and network architecture developed to implement these features can be found in .Diffused illumination from below results in images that show contours and surface features, this is particularly useful when the sample is opaque. Illumination from above typically works best for samples that are sufficiently translucent and can show internal structures as the light can pass through the sample. The flexibility of using different illumination techniques emulates commercial bright field microscopes. The difference from over and under light is best shown in Supplementary Fig. 1. The 3D printed plate holder supports the biological sample during an experiment. For easy alignment, the holder is attached to a xy sliding stage that consists of two interconnected linear stages . The inner stage translating along the y-axis uses 8 leaf springs to connect a central piece holding the 24-well plate with four rigid elements surrounding it. The outer stage translating along the x-axis uses 8 additional leaf springs to connect the inner stage with the outside 4 rigid elements, two of them being connected to the Picroscope frame using 4 screws . While each stage is flexible along one axis , together they can slide along both, x and y axes. Each stage is actuated by two adjustment screws depicted as gray arrows in Fig. 3f.

Here we show that combining Iso-Seq with Illumina sequencing at high coverage enables expression profiling and sequence error correction of IsoSeq reads, particularly those derived from low-expression genes. The clustering analysis of the SMRT link pipeline discarded  18.5% of the FLNC reads, likely caused by low sequence accuracy. To overcome this technical issue, we applied a hybrid error correction pipeline consisting in performing the error correction of the unclustered FLNC reads, followed by an additional clustering step of both to resolve redundancies. Error correction with Illumina reads recovered a significant amount of Iso-Seq reads that would have otherwise been removed by the standard Iso-Seq pipeline, highlighting the importance of integrating multiple sequencing technologies with complementary features . Transcriptome reconstruction has been widely used to develop references for genome-wide expression profiling in the absence of an annotated genome assembly . Though a genome reference is available for grape, transcriptome reconstruction overcomes the limitations of a cultivar-specific reference that lacks the gene content of other cultivars. Although cultivar-specific genes appear nonessential for berry development, those private genes could contribute to cultivar characteristics. For example, the wine grape Tannat accumulates unusually high levels of polyphenols in the berry; its cultivar specific genes account for more than 80% of the expression of phenolic and polyphenolic compound biosynthetic enzymes . De novo transcriptome assembly from short RNA-seq reads has been used to explore the gene content diversity in Tannat , Corvina , and Nebbiolo . Iso-Seq identified 1,501 Cabernet Sauvignon transcripts expressed during berry development that were found in neither the genome of PN40024 nor the transcriptomes of Tannat, Nebbiolo and Corvina. Some private Cabernet Sauvignon transcripts have functions potentially associated with traits characteristic of Cabernet Sauvignon grapes and wines like their color and sugar content.

These transcripts included three sugar transporter-coding genes, big plastic pots which could be involved in the accumulation of glucose and fructose during berry ripening , and a chalcone synthase, a flavanone 3-hydroxylase, and a flavonoid 39-hydroxylase, all involved in the flavonoid pathway. Chalcone synthases catalyze the first committed step of the flavonoid biosynthesis pathway , which produces different classes of metabolites in grape berry, including flavonols , flavan-3-ols and proanthocyanidins , and anthocyanins . In addition, products of the flavonoid 39-hydroxylase can lead to the synthesis of cyanidin-3-glucoside, a red anthocyanin . The analysis of the gene space in the genome assembly showed that private Cabernet Sauvignon genes identified using Iso-Seq are only a fraction of the private Cabernet Sauvignon transcriptome. As in other transcriptome reconstruction methods, Iso-Seq can only identify transcripts that are expressed in the organs and developmental stages used for RNA sequencing. Obtaining the full set of private transcripts without genome assembly would require sequencing additional organs and developmental stages. In addition, it is challenging to differentiate isoforms derived from close paralogous genes, alleles of the same gene, and alternative splicing variants, in any transcriptome obtained by RNA sequencing ; this potentially leads to an overestimation of the genes in the final transcriptome reference. This study could not resolve isoform redundancy in the final transcriptome for about 37% of the gene loci in the Cabernet Sauvignon genome. This is a limitation of Iso-Seq as well as of all transcriptome references that cannot be overcome without a complete genome assembly. In this study, we tested whether the transcriptome reconstructed using Iso-Seq can be used for expression profiling. Only an approximately 3% difference in read alignment between ISNT and the genome reference was observed, implying that at high coverage, ISNT detects almost all genes expressed during berry development.

The slight difference in mapping rate between the two references can be explained by either the absence of some low-expression transcripts in the ISNT or the residual error rate in isoform sequences. Gene expression analysis using the ISNT as reference showed similar results compared to the Cabernet Sauvignon genome assembly, with a very high correlation of expression level and differential gene expression, and with similar global transcriptomic changes. However, we observed differences in the number of expressed and differentially expressed features that depend on the reference used. Those differences could be explained by the diploid phasing of the Cabernet Sauvignon genome assembly and that multiple ISNT transcripts might correspond to a single gene locus. Nonetheless, similar relative amounts of Biological Process GO terms were found among the differentially expressed genes, confirming that the transcriptome obtained using Iso-Seq captured the transcriptional reprogramming underlying the main physiological and biochemical changes during grape berry development. In addition, gene expression analysis revealed that some private isoforms are significantly modulated during berry development, indicating that in addition to identifying the private gene space, the ISNT reference makesit possible to observe its expression. In conclusion, this study demonstrates that Iso-Seq data can be used to create and refine a comprehensive reference transcriptome that represents most genes expressed in a tissue undergoing extensive transcriptional reprogramming during development. In grapes, this approach can aid developing transcriptome references and is particularly valuable given diverse cultivars with private transcripts and accessions that are genetically distant from available genome references, like the non-vinifera Vitis species used as rootstocks or for breeding. The pipeline described here can be useful in efforts to reconstruct the gene space in plant species with large and complex genomes still unresolved.

Agriculture plays an important role in economic growth, and improving crop yield is of great concern in Vietnam. On the one hand, insect pesticides can affect the metabolic processes of crops to degrade crop yield and quality. On the other hand, fruit flies are known to cause 50 to 100% crop loss unless timely interventions are implemented. There are just a small number of fruit fly species that have been discovered, namely Bactrocera dorsalis, B. correcta, B. cucurbitae, B. tau, B. latifrons, B. zonata, B. tuberculata, B. moroides and B. albistriga, while some species remain unidentified. The species which are harmful to fruits are of the common fruit fly species, namely B. cucurbitae and B. tau. To optimize crop yields, agricultural workers tend to use a pesticide scheduler rather than consider the likelihood of pests’ presence in the crop. Thus, this not only causes many pesticide residues in agricultural commodities but also brings great pressure to the ecological environment. The overuse of pesticides is partly because information about pest species and densities cannot be provided in a timely and accurate way. In contrast, if the information is provided in atimely fashion, it could be possible to take proper prevention steps and adopt suitable pest management strategies including the rational use of pesticides . Traditionally, the information about the environment and pest species is acquired mainly through handcrafted feature engineering such that workers manually use sensors and compare a pest’s shape, color, texture, and other characteristics with justification from the domain experts. Likewise, counting is typically time-consuming, labor intensive, and error-prone. Therefore, it is urgent and significant to establish an autonomous and accurate pest identification system. There is a growing tendency of utilizing machine vision technology to solve these problems with promising performance in the agriculture research field. In this work, growing berries in containers we focus on developing a solution to detect oriental yellow flies which usually harm citrus fruits such as oranges and grapefruits. We implement and evaluate the object detection models by applying the models with test sets simulating potential disturbances occurring in real scenario. Additionally, the work presented in this paper will not only focus on the use of different types of object detection algorithms but also apply the TFLITE format of the models compatible to edge device system such as TPU processors. This direction of study is to develop real-time detection application with the emerging edge computing technology to enhance the performance of the system in terms of detection accuracy, power efficiency, and latency reduction with the purpose of detecting the living fruit flies beside the stuck and dead ones on the trap. Moreover, the article will describe the hardware implementation so that the work can be reproduced and further developed. Our contributions are: We constructed, developed, and provided a more in-depth discussion of the end-to-end camera-equipped trap, named AlertTrap with installation of a Lynfield-inspired sticky trap, to instantly detect fruit flies and the solar-energy powering system controlled by a separate Raspberry Pi. We evaluate three different compact and fast object detection deep learning models, namely SSDMobileNetV1, SSD-MobileNetV2, and the Yolov4-tiny. Nevertheless, we introduce artificial disturbances imitating inference effects which may compromise the detection performance in real-time scenario. Moreover, we also evaluate the SSD-MobileNetV1 and SSD-MobileNetV2 models with their TFLITE format versions on a TPU device. With the results, we compare not only their ability to accurately detect and localize the fruit flies which we had trained them to predict, but also the increase in processing speed as well as the power saving factor.Insect detection techniques can be classified into three system types, namely manual, automatic, or semi-automatic systems.

Manual insect detection techniques are known as a process in which trained workers count the trapped flies on a daily basis. These turn out to be error-prone, time consuming, and labor-intensive, while semi-automatic and automatic systems can address the disadvantages with the replacement of highly accurate and autonomous emerging technological software and hardware. Specifically, the remaining two types of insect detection systems are often called e-traps as they are fueled by electronic components with extensive computer algorithms such as a center-controlled unit connecting with a camera and the trap actuators. Thus, they are also known as vision based insect traps. As suggested in the names, the automatic insect detection systems are fully autonomous, whereas the semi-automatic ones involve human interaction in the loop. For example, in [24], the images of insect body parts are classified to aid humans to better categorize the insects. Generally, the e-traps are equipped with a wide range of post-processing techniques to detect and classify trapped insects. These techniques are recognized by the sensor type that is used to capture the existence of insects in the trap. Particularly, they are image based, spectroscopy-based, and optoacoustic techniques, which correspond respectively to the visible-light camera, the near-infrared camera, and the ultrasound sensor. The image-based techniques consist of three sub-domain techniques, namely deep learning and shallow learning, which both are sub-domains in the machine learning field, and image processing techniques. Shallow learning-wise, Kaya et al. created a machine learning-based classifier that can differentiate between 14 butterfly species. The texture and color characteristics are extracted by the writers. A three-layer neural network is used to process the extracted features. The categorization accuracy achieved is 92.85 percent. The detection approach is based on image processing as described in. While image-processing techniques are simpler than deep learning techniques, their accuracy is reasonable and the system is wired with the illumination environment. However, extensive feature engineering must take place prior to the classification. By using auto-brightness adjustment, the algorithm first reduces the effect of changing lighting and weather conditions. Then, using a coordinate logic filter improves the edges by amplifying the difference between the dark bug and the bright background. Finally, the technique uses a circular Hough transform followed by a noise reduction filter to identify the trap’s limits. The achieved accuracy rate is 75%. In [14], it was reported that a Wireless Sensor Network was created for detecting pests in greenhouses. The image processing technique first removes the effect of light changes from the photos, then denoises them, and finally recognizes the blobs. In [15], it was suggested that insect image processing, segmentation, and sorting algorithms could be used as insect “soup” images. In insect “soup” photos, the insects float on the liquid surface. The method was evaluated on 19 soup images by the authors, and it worked well for many of them. Using McPhail traps, a WSN was developed to detect the olive fruit fly and medfly in the field. WSNs are sensor networks that gather data and may be built to process information and transfer it to humans. WSNs may also have actuators that respond to specific events. The template comparison algorithm is the detection algorithm. The identification is based on the detection of specific anatomical, patterning, and color characteristics. Near Infrared Spectroscopy was used to identify infested olives in harvested crops. The Genetic Algorithm extracts the features from the collected full spectral data. The retrieved features serve as the input for the classifier.

The treatments strongly affected anthocyanin compounds in both seasons. In 2019, at harvest, 50 and 100% ETc increased the proportion of peonidins and 25% ETc had a significantly higher proportion of petunidin derivatives. Flavonol composition was only affected by irrigation treatments during the 2020 growing season at harvest . Myricetin and quercetin derivatives were the main flavonols found in Cabernet Sauvignon berry skins and both accounted for about 75% of the total amount. The most restrictive applied water treatment increased proportion quercetins and kaempferols, while 100% ETc increased myricetins and syringetins.This study evaluated the effect of applied water amounts based on the replacement of fractions of the ETc for maintaining berry quality while minimizing yield losses due to the environmental impact . Results covered two seasons that strongly differed in the precipitation supply. Compared with the average total amount of precipitation received by the area in the last decade , 2019 growing season was a rainy period with 970.3 mm precipitation, while 2020 was a hyperarid growing season with only 234.2 mm of precipitation. In spite of the differences in total precipitation, the response of Cabernet Sauvignon grapevines to water deficits was consistent across both seasons and our results corroborated that deficit irrigation may mitigate the effects of water scarcity . The results achieved in this study indicated that 25% and 50% ETc treatments were effective in improving iWUE compared with previous studies reporting a compilation of data from Cabernet Sauvignon and other cultivars . The iWUE decreased when the fractions of appliedETc increased as previously reported by Keller et al. in a 3-year field experiment conducted on the same Cabernet Sauvignon clone as used in this study. Likewise, planting blueberries in containers WUEc calculated as the ratio between yield and water applied was also enhanced with the decreased water supply.

The berry must δ 13C enhanced under stronger water deficits conditions corroborating previous studies with different grapevine cultivars . The iWUE and the berry must δ 13C also indicated a linear relationship in accordance to previous research . Previous work indicated that δ 13C of grape must is a reliable indicator of plant water status and leaf gas exchange in vineyard systems, which in turn, are crucial for the identification of plant water status zones leading to better irrigation decisions and informed management . The present study also provided evidence that δ 13C is a convenient tool without intensive labor and time inputs for the assessment of environmental impacts of deficit irrigation strategies. Increased applied water amounts led to greater canopy size and yields . There was a strong negative relationship between the berry must δ 13C and grapevine vegetative growth measured as LAI. Previous studies reported a linear relationship between the δ 13C and the carbon assimilation rates and consequently with vegetative growth estimated as pruning mass . Yield achieved in this experiment ranged from 4.8 to 10.4 kg · vine−1 in accordance to a previous study conducted in a vineyard at a similar density . Thus, 100% ETc may double the yield compared with the 25% ETc as previously reported by Keller et al. . This suggested that the effect of applied water on yield components is consistent in spite of the climate difference, planting space, and grapevine age. Under our experimental conditions, primary metabolites were affected by applied water amounts in the second season, where 100% ETc accounted for lower TSS but higher pH. Increased water content in berries was associated with a lower concentration of sugars due to a dilution effect . Conversely, the lower pH in 25% ETc grapevines was related to exacerbated organic acid degradation under high temperatures by water deficit . Berry skin flavonol and anthocyanin contents decreased with the 100% ETc in 2019 but not in 2020.

Although several studies reported increases in berry flavonoid content under mild or moderate water deficit , field research conducted in California resulted in contradictory results when severe water deficits were combined with a long hang time . In general, 100% ETc irrigation treatment reduced the proportion of petunidin derivatives and increased the proportion of peonidin derivatives leading to a decreased ratio between tri-hydroxylated and di-hydroxylated anthocyanins, which was suggested to be less chemically stable for winemaking purposes . Likewise, previous studies have reported an increase in the ratio between tri-hydroxylated and dihydroxylated anthocyanins when grapevines were subjected towater deficits given the upregulation of the relevant anthocyanin biosynthetic genes . In addition, these forms were more persistent through hang time, making trihydroxylated flavonoids more abundant as maturity progressed . Flavonol composition was modified by applied water amounts in 2020 growing, where proportions of myricetin and syringetin derivatives increased and quercetin and kaempferol derivatives decreased with 100% ETc. Given that quercetin and kaempferol are important antioxidants in red wines, this shift in the composition may impact the antioxidant properties of wine . In previous work, it was reported that 100% ETc irrigation increased the net carbon assimilation and improved the grapevine water status, leading to higher soluble sugar and starch contents in leaves with the highest yields, and vegetative biomasses . However, the greatest leaf area to fruit ratios measured in this treatment showed a clear sign of disproportionate leaf biomass growth, which presumably impacted berry metabolism. Thus, both studies highlighted the importance of management of water deficits to ensure grape berry composition optimization, improving water use sustainability by rewarding quality over quantity in arid and semiarid regions .Decreasing irrigation amounts increased AMF colonization in accordance with previous studies .

The symbiotic relationship of AMF with grapevines provided several adaptive advantages, such as improved abiotic and biotic stress resistance, enhanced nutrient uptake, and grapevine growth . Previous research suggested that these effects might be related to the altered regulation of nutrient transport, cell wall-related, phenylpropanoid, and stilbene biosynthesis genes driven by AMF colonization . Additionally, it was recently reported that AMF may enhance the content of flavonoids in berries , leading to improved berry composition and antioxidant properties in spite of the lack of effect on petiole nutrient contents . However, vineyard management practices may affect the soil structure and the composition of the rhizosphere-living microbiota , as well as the microbiota associated with grapevine roots, which is mainly composed by Rhizophagus and Glomus genus , likely affecting the effectiveness of the symbiosis. The relationship between AMF and berry must δ 13C suggested that productivity of high quality grapes could still be sustained in this region with less water input because the root system of the grapevines may perform more efficiently due to greater AMF colonization. The totalWF measured in this study ranged between 484.3 and 1237.7 m3 ·tonne −1 across treatments and growing seasons, in accordance with previous studies assessing the WF of grapevine cultivation . This variation in the totalWF was related to the amount applied and the differences in precipitation between the two seasons . Indeed, the previous research speculated that changes in temperature and precipitation may affect the proportional contribution of blue and green WF to the totalWF . Previous studies reported that vineyards accounted for a higher WF compared with other crops such as olives, wheat, and other fruit trees . Wine grape growers require appropriate irrigation schedules that reduce blueWF and increase greenWF leading to a decreased totalWF for increasing sustainability of vineyards. Under our experimental conditions, 25% ETc strongly decreased the blueWF, however, container growing raspberries this came with a dramatic increase in the grayWF component, which led to increased totalWF. Conversely, 100% ETc decreased totalWF to lower values than those reported in Zotou and Tsihrintzis , presumably because of the differences of standard yields recorded in Mesogeia area , where the authors conducted their research, and Napa Valley, CA, USA where this study was performed. A recent study reported that the current values of blueWF and gray WF are unsustainable . The actual runoff of the surface water is not sufficient to satisfy the irrigation requirements and/or dilute the pollutant load associated with the diffuse and point sources to reduce it below the maximum acceptable concentration . These results highlighted that the management of natural resources, specifically water management, is paramount for the sustainability of the wine industry under future constraints . Thus, our data suggested that values ranging between 600 and 1000 m3 · tonne −1 of the totalWF may ensure a high iWUE of grapevines , optimum LAI, and profitable yields, which maintained the balance between vegetative and reproductive growths . Nevertheless, it is noteworthy to address that WF assessment also presents some limitations given that the water consumed by an irrigated crop is often a mix of residual soil moisture from previous precipitation and irrigation events and that the reference ET is strongly dependent on the local climate .Ants benefit plants . Humans have known this for quite a long time. In fact, ants were described as biological control agents in China around 304 AD . Many plants have also evolved to promote the activity of ants on their tissues. Some plants provide domatia as ant housing structures, while others attract ants to their tissues with extra-floral nectaries. Some plants are hosts to honeydew-producing hemipterans that excrete honeydew, a sugary substance consumed by ants. Still other plants are simply substrates for ant foraging.

The majority of studies conducted across these ant–plant groups show that ants benefit plants by removal of herbivores . Nonetheless, in many agroecosystems, the benefits of pest control services by ants are not recognized. Agricultural managers often view them as pests or annoyances to agricultural production because some ants tend honeydew-producing insects that can damage crops . However, a review of the literature on ant-hemipteran associations suggests that even these associations benefit plants indirectly because ants remove other, more damaging herbivores . Regardless, the literature lacks studies investigating ant–plant interactions in agroecosystems. Here, we broadly survey the pest control services provided by a suite of ant species to better understand the role of ant defense of coffee. Coffee is a tropical crop that occurs as an understory shrub in its native range, and coffee plants are therefore often grown under a canopy of shade trees in agroforestry systems in some parts of the world . This canopy layer provides plantatsions with a forest-like vegetation structure that can help maintain biodiversity . Ant biodiversity is high in many coffee plantations and ants attack and prey on many coffee pests, including the coffee berry borer . For example, Azteca instabilis F. Smith is a competitively dominant ant that aggressively patrols arboreal territories in high densities and previous research has found that it impacts the CBB . Some laboratory and observational field studies have found that Pseudomyrmex spp., Procryptocerus hylaeus Kempf, and Pheidole spp. may limit the CBB . However, other field experiments have not found ants to be biological control agents of the CBB . Further, the pest control effects of many ant species on the CBB have not yet been evaluated and it could be that previously documented effects are specific to only a few species. Natural ant pest control of the CBB is particularly important because chemical insecticides used to control CBB are not always effective. This lack of effectiveness is in part because the CBB lifecycle takes place largely hidden within coffee berries and also because the CBB has developed insecticide resistance . Several of the stages of the CBB life cycle make it vulnerable to attack by ants . First, the CBB hatches from eggs within the coffee berry, where it consumes the seeds . Small ants may enter the berry through the beetle entrance hole and predate the larvae and adults inside . Second, old berries infested with the CBB may not be harvested because they often turn black and remain on the coffee branches or may fall to the ground . These old infested berries may act as a population reservoir of borer populations and ant predation at this stage could be very important for limiting CBB populations in the next season. Third, as adult borers disperse to colonize new berries, ants may prevent them from entering new berries . To date, no field experiment has specifically investigated how coffee-foraging ants limit CBB colonization of berries. Here, we studied the abilities of eight ant species to prevent colonization of berries by the CBB. We hypothesized that only species with high activity on branches would limit CBB colonization of berries.

The SN65LVDT41 chip is configured to use low-voltage differential signaling to reduce the effects of noise and electromagnetic interference and allow increased cable length. However, the Raspberry Pi communicates using complementary metal-oxide-semiconductor level logic. To translate between the two signal types, the expansion shield uses the SN65LVDT41 chip from Texas Instruments. The SN65LVDT41 chip has four LVDS line drivers and one LVDS line receiver to control data lines required to communicate with the Intan chip over its Serial Peripheral Interface . Connection to electrodes—Electrodes are connected to the Intan RHD 32-channel recording head stage. For experiments reported here, we created a connection to a commercially available 6-well multi-electrode array plate from Axion Bio-systems. However, any other electrode system fitting an Omnetics 32-pin connector is compatible. The design can be adapted to custom and commercial MEAs of different form factors using adapter boards. The Axion electrode plate mates its bottom contacts to spring finger pins on our designed adapter board. The parts are aligned using a custom holder consisting of a plastic interior surrounded by aluminum plates and compressed together by screws on four corners. The aluminum plate casing prevents warping of the plastic and ensures even pressure compressing the plate and connector on both sides. The compressing holder provides consistent mating of spring finger pins to electrode contacts on the plate.The Piphys system runs custom software to perform: communication with the Intan RHD2132 bio-amplifier chip, buffering and file storage of recorded voltage data locally, near real-time data streaming and plotting on the online dashboard, plant pots with drainage and experiment control from the dashboard. In order to stream data, interact with data being recorded, and control the device, we deployed Redis, Amazon IoT, and S3, as described in Methods.

To perform an electrophysiology recording, the user can configure the sampling rate and start the experiment from the dashboard. Once started, the neural cell activity is firstly digitized and sampled by the Intan RHD2132 bio-amplifier chip in 32 channels. Raspberry Pi stores the data on local memory and also streams it to Redis for near real-time visualization on the online dashboard. Since the Raspberry Pi computer has a 10 second streaming buffer, the data visualized on the dashboard is offset by 10 seconds from the data recorded. Therefore the data streaming is “near real-time”. During a recording, raw data is saved in chunks of 5 minutes to local memory and streamed in chunks of 10 seconds to Redis. Once the recording ends, all local data files are uploaded to S3 for permanent storage, and data is further backed up to Amazon Glacier for long-term archiving. Local data files on the Pi auto-erase every 14 days to release memory. To view a dated recording, the user can select and pull the data files from S3 to the dashboard for display . The Raspberry Pi has 4 CPU cores, and allows multicore and multi-threading. According to resource monitoring with “htop”, the Piphys program runs on two cores. The software uses four threads to record and stream to the cloud simultaneously. One thread is used for hardware interfacing with the Intan chip; a second thread is for cloud streaming, the third one is for local saving and another is for experiment control that gets MQTT messages. Communication with hardware—Communication between Raspberry Pi and Intan RHD2132 bio-amplifier chip uses Serial Peripheral Interface . SPI is a fast and synchronous interface that is widely used in embedded systems for short-distance data streaming. It is a full-duplex master-slave-based interface where both master and slave can transmit data at the same time. The protocol for both Raspberry Pi and Intan RHD2132 bio-amplifier chip is a four-wire interface: Clock , Chip select CS , Master-OutSlave-In , and Master-In-Slave-Out . In Piphys, the Raspberry Pi acts as the master device and generates a clock signal and recording commands to configure the Intan RHD2132 bio-amplifier chip through MOSI.

The Intan chip responds as a slave and sends the digitized data back by MISO. The chip allows the configuration of sampling rate and bandwidth of the low-noise amplifiers. The 32 channels on the chip are sampled sequentially with available sampling rate options from 2 kHz to 15 kHz per channel. The SPI clock is divided from the core clock on Raspberry Pi. Performance and error of the SPI clock are discussed in the Supplementary Material. The amplifiers give 46 dB midband gain with lower bandwidth from 0.1 Hz to 500 Hz, and upper bandwidth from 100 Hz to 20 kHz.Online dashboard—Users interact with Piphys devices through a web browser application, referred to as the Graphical User Interface . The GUI allows a user to initiate a recorded experiment and monitor electrical activity on each channel. Programatically, the GUI mimics an IoT device that sends messages to other devices and listens to their corresponding data streams in a high-performance Redis database service. The Piphys device produces a single data stream to Redis, and many users can view the stream from the Redis server. Therefore, many users can monitor and interact with a particular Piphys device without additional overhead placed on that device. Users can be located anywhere on the Internet without concern for where the physical Piphys device is or which network it is on. We routinely perform electrophysiology experiments from Santa Cruz on a Piphys-connected device that is located 90 miles away in San Francisco. When a new user opens the browser GUI, the web application queries the AWS IoT service for online Piphys devices to populate a device dropdown list. When the user selects a device from the dropdown, an MQTT ‘ping’ message is sent to the relevant device every 30 seconds, indicating that a user is actively monitoring data from that device. As long as the Piphys device receives these pings, the Piphys device will continue to send its raw data stream to the central Redis service. When the Piphys device has not received any user messages for at least a minute, it will cease sending its raw data stream. This protocol ensures the proper decoupling of users from devices. The Piphys device is not dependent on a user gracefully shutting down. While the Piphys device feeds raw data to the Redis service, data transformations are applied downstream by other IoT-connected processes. For example, the Piphys Control Panel displays a threshold spike sorted transformation of the raw data.

This data transformation is an independent process that listens for MQTT requests for the raw data stream and transforms the raw stream into a stream containing the past ten spike events detected per channel. For channels with no detected spikes, a random sample of the channel is saved to the stream every 30 seconds to provide a sampling of the channel’s activity.We tested the Piphys system for long-term recordings of human primary neurons. The goal of this work is to compare the neural signal recorded by the proposed apparatus to commercially available systems. Therefore, as a reference we choose two neural recording devices: Axion Maestro Edge by Axion Biosystems, as one of the leading commercial instruments for neural recording and Intan RHD interface board as one of the leading commercial open source neural recording instruments. These neurons were cultured in an Axion CytoView MEA 6-well plate ‡ . We designed a set of adapters , plastic plants pots which allowed the same culture plate to be used by Piphys and Intan RHD interface board. As mentioned in the Discussion section, the proposed system can interface with any type of neural recording electrodes using the Omnetics connector. After recording, the raw data was ingested to SpyKING CIRCUS software on a personal computer for analysis. SpyKING CIRCUS is a semi-automatic spike sorting software that uses thresholding, clustering, and greedy template match approaches to detect single cell action potentials. Here, we show two types of results, first for single neuron recordings and second for a bursting neural network.After 14 days in culture, primary neurons were recorded with the Piphys system and two commercially available systems: the Intan RHD USB interface board and the Axion Maestro Edge. After recording, all three datasets were filtered with bandpass filtering from 300 Hz to 6000 Hz and spike sorted with a threshold of ± 6 μV. Figure 5 shows a ten-second spike train from Piphys with dots highlighting detected spikes in the raw data. To further demonstrate the applicability of Piphys to primary neuron recording, we compare the shape of the detected action potential and quality metrics such as amplitude distribution, interspike interval distribution, and firing rate to commercially available systems . The data was recorded from the same channel in the same well of neurons by Piphys, Intan, and Axion systems in sequential order on the same day. The data recorded on Piphys corresponds to the data obtained from both commercial systems, with high similarity to Intan and overall consistency with Axion across metrics in Figure 6. The mean spike waveform, shown in the first column of Figure 6, was determined by averaging the voltage in a 3 ms window centered around the point where the voltage crossed the spike threshold. Differences in Axion’s waveform shape are a flatter starting point and a higher upstroke before settling to resting state. The amplitudes for the mean waveform are −24.67 ± 3.92 μV for Piphys, −26.92 ± 4.96 μV for Intan, and −24.50 ± 1.69 μV for Axion. Axion has a smaller deviation than Piphys and Intan, showing lower noise in the recording system. The amplitudes of the detected spikes over time, shown in the middle column of Figure 6 are more sparse for Axion than for Intan and Piphys.

Firing rates in events per second over the recording period shown are 8.05 for Piphys, 8.44 for Intan, and 6.86 for Axion. The interspike interval histograms, shown in the middle column of Figure 6, have similar longer-tail distributions for Piphys and Intan centered at 122.79 ms and 118.15 ms, and a tighter distribution for Axion centered at 145.57 ms. However, the interspike interval means for all three systems are significantly close together.On day 42 of culture, we recorded from the neurons with Piphys and found the primary neurons displayed synchronized network bursts, consistent with previous observations. Figure 7 shows the synchronous activity captured across four channels. After spike sorting, most detected spikes were arranged in short intervals with periods of silence in between. The spikes inside the bursts align among the channels, indicating that synchronized activity was present through the network. Quantitatively, the bursting has a general population rate of 0.13 bursts each second, with each burst lasting around 1 second. Within one burst, the number of spikes is 55 ± 17.58.The variation between Piphys and Axion shown in Figure 6 could be attributed to physical differences in the circuity and possible advanced filtering performed by Axion’s proprietary BioCore v4 chip §. The filtering could account for the smoothness and low variability of the signal , resulting in a smaller number of identified firing events with a tighter distribution. Piphys and Intan systems both use the same amplifier chips , where the optional on-chip filtering was disabled during recording ∥. The raw signal, therefore, has a larger noise margin , which may create more false-positive firing events. The tail of the amplitude distributions in Intan and Piphys is skewed towards lower-amplitude events, closer to the noise floor. The interspike intervals for Intan and Piphys register several events with near-zero intervals, likely suggesting false-positive spikes from noise contamination. Contamination from noise, which is likely symmetrical, could affect the shape of the mean waveform calculated by overlaying and averaging all registered spikes. Overall, these results demonstrate that Piphys can record neural activity in a manner comparable to commercially available hardware and software.Comparison to other platforms—Comparing electrophysiology platforms side by side is challenging because each system fits a specific niche and requirements for a particular workflow. Different platforms are targeted to particular problems and, therefore, have specific challenges and user needs. Piphys is intended to integration with other IoT sensors, and flexible recording equipment that can be used in a fleet for longitudinal study of many in vitro replicates. It should be noted that that the system proposed in this paper has an average signal to noise ratio of 4.35 dB above the baseline noise in recording neuron burst, which is comparable with other similar systems. Table 1 summarizes electrophysiology systems comparable to Piphys.

In an effort to improve wine quality, many smaller high-end wineries employ laborers to hand sort individual berries after destemming to remove unwanted material such as raisins, diseased berries, unripe berries, and materials other than grapes such as leaves and stems. This can be costly, labor intensive, and it can slow down the process line. To reduce costs and increase throughput, many wineries have adopted optical sorting technology. Using this technology, MOG can be removed more efficiently, and parameters such as color, shape, and size can be used to sort individual berries. Depending on the type of sorter, processing speeds can range between 2 and 15 tons per hour. Furthermore, fewer workers are needed to operate an optical sorter than to hand sort the respective amount of fruit. In addition to saving time and money, optical sorters have the potential to decrease the impact of inconsistent ripening in grapes. One study successfully sorted Carlos Muscadine grapes into four different ripeness levels using light at two different wavelengths in the visible spectrum. The researchers found that with successive sorting levels, there was an increase in Brix and pH, along with a decrease in titratable acidity in grape samples. In the wines, an increase in tannin and pH and a decrease in titratable acidity was found with increasing sorting. In sensory analysis, the first and fourth sorting levels were found to be inferior compared to the middle two treatments. Even though this study used outdated equipment compared to today’s standards, growing raspberries in pots it shows that white grapes can be sorted into different ripeness levels and this can affect the quality of the wine produced. A recent study used visible near-infrared spectroscopy to classify table grapes into different groups based on soluble solid and phenolic content.

The researchers were able to differentiate berries of different classes with accuracy ranging from 77% to 94%. Another study found that wine made from optically sorted Chardonnay grapes had higher residual sugar, pH, and total phenols than the unsorted control. The wines were analyzed sensorially with descriptive analysis and the judges scored the sorted wines significantly higher in tropical fruit and sweetness. However, with only two significant attributes out of twenty, the wines were determined to be similar in character. Another study investigating the effect of mechanical harvesting and optical berry sorting on Pinot noir grapes found that, in general, wines made from optically sorted fruit were significantly lower in total phenol and tannin, potentially due to the removal of MOG during sorting. In sensory analysis only two significant attributes out of eighteen were found and it was concluded that the wines were similar in character. A study published in 2014 used an optical sorter on Riesling, Müller-Thurgau, and Pinot gris grapes infected with Botrytis cinerea to investigate the effect of optical sorting on sulfur binding compounds in the finished wine. The researchers found that wine made from optically sorted fruit contained significantly less 2-oxoglutaric acid and pyruvic acid . They concluded that optical sorting is an effective method for reducing the amount of sulfur dioxide needed in the winemaking process using these varieties. There is a lack of published research investigating the impact of optical berry sorting on wine composition and only a few cultivars of Vitis vinifera have been tested. The objective of the current study was to provide more information on the effect of optical berry sorting on different varieties and investigate the capabilities of today’s optical sorters to sort for different ripeness levels using red grapes and using color as a sorting parameter. The current study found that although optical sorting can efficiently replace hand sorting, the overall impact on wine sensory attributes was minimal. Therefore, in general, the study supported the findings of previous researchers.Three varieties were tested in 2016: Barbera , Cabernet Sauvignon , and Grenache .

BA was harvested on 19 August 2016, CS was harvested 30 August 30 2016, and GN was harvested 8 September 2016. All three varieties were hand harvested early in the morning from UC Davis campus vineyards and processed the same day. Fruit condition was good with seemingly little variation, although GN fruit showed more variation in color than the other cultivars. Half-ton bins were dumped by a forklift into a receiving hoper. Clusters were carried by a Delta TR elevator into a Delta E2 destemmer . Destemmed berries fell onto a moving belt and were carried onto a ChromaxHD Berrytek Optical Sorter . Rejection parameters were established by capturing color profiles of optimal berries, suboptimal berries , and MOG. These parameters were optimized with the assistance of a WECO technician for removing suboptimal berries and MOG while rejecting as few optimal berries as possible. This process was repeated, and parameters were adjusted for each variety. The must was pumped directly into 200 L stainless steel research fermentors, which were filled incrementally to reduce vineyard variation. The rejected material was collected in buckets and transferred into research fermentors. The grapes were processed in three treatments, control , sort , and reject . The rejection rates were 14.9%, 3.9%, and 1.5% for GN, BA, and CS, respectively. Juice collected in trays from the rolling belts during processing operations was added back to each treatment in proportional amounts. This was done to maintain a consistent solid-to-juice ratio in the must among treatments. Wines were made in the UC Davis Teaching and Research Winery using 200 L stainless steel research fermentors. Duplicate fermentations were used for the reject treatment wines because only a small amount of reject material was obtained during grape processing.

Fermentation replications were kept separate through the entire experiment. Juice samples were taken from each fermentation vessel after mixing. Fifty milligrams per liter of SO2 was added to the must after processing using a 15% potassium metabisulfite solution. Yeast assimilable nitrogen was adjusted to 250 mg/L using diammonium phosphate , titratable acidity was adjusted to 6 g/L using tartaric acid. The must was heated to 25 ◦C before inoculation with Lalvin EC1118 yeast using the manufacturers rehydration procedure. One tank volume was pumped over twice per day using automated pump overs for all wines except for the reject treatment for CS. The volume in these tanks was too low for the automated pumps to create suction, therefore, the wines were punched down manually once per day during the fermentation. Once wines were dry, they were pressed using a basket press and allowed to settle for 5 days before being racked and transferred to a temperature-controlled room held at 20 ◦C. The wines were then inoculated with Viniflora CH16 Oenococcus oeni bacteria by Chr. Hansen . Upon finishing the malolactic fermentation, 50 mg/L SO2 was added to the wines and they were held in a 9 ◦C cold room until bottling. Free SO2 was adjusted to 30 mg/L before bottling. All samples for basic wine chemical analyses were taken at the time of bottling. Ethanol was measured using an Alcolyzer and pH was measured using an Orion 5-star pH meter . Titratable acidity and free SO2 were measured using a Mettler Toledo DL50 auto titrator . Residual sugar, malic acid, and volatile acidity were measured using a Thermo Fisher Scientific Gallery automated analyzer . Wines were sterile filtered using 0.45 µm membrane filters prior to bottling using green Bordeaux style bottles and screw cap closures with Saranex liners by Amcor . Samples for phenolic analysis were taken from bottles at the time of sensory analysis. The modified Adams-Harbertson assay was used to determine levels of anthocyanin , tannin , and total iron-reactive phenolics. A Genesis 10S UV-Vis spectrophotometer was used for this assay.An external calibration was used for the quantification of phenolic compounds and curves were made for gallic acid, -catechin , -epicatechin , caffeic acid , quercetin-3-rhamnoside, plant pot with drainage and malvidin-3- glucoside. Caftaric acid was quantified as caffeic acid equivalents, quercetin-glycosides as quercetin-3-O-rhamnoside units and all pigments as malvidin-3-glucoside units. Bottle duplicates for each fermentation replication were analyzed and the sequence was randomized. Wine aroma compounds were analyzed using head-space solid-phase microextraction gas chromatography mass spectrometry . The method used was adapted from a previous study. Samples used for wine volatile analysis were taken at the time of sensory analysis and stored at 4 ◦C for no more than one month. Identified volatile peaks are normalized against an internal standard and the obtained data is thus semiquantitative only. Twenty mL amber glass headspace vials were used, containing 10 mL milliliters of wine sample, 3 g of NaCl salt and 50 µL of a 10 mg/L solution of 2-undecanone . Twenty millimeter green magnetic caps with 3 mm PTFE silicone septa were crimped onto the vials and the samples were mixed by vortexing. The analysis was done using an Agilent Technologies 7890A GC system with a Gerstel MPS2 multipurpose sampler . The mass analyzer was an Agilent Technologies 5975C inert XL EI/CI MSD. The column used was an Agilent Technologies DB-Waxetr with a temperature range of 30 ◦C to 260 ◦C. The dimensions of the column were 30 m, 0.250 mm, and 0.25 µm. Maestro software was used to control the instrument and data were collected using ChemStation software . During the analysis, the oven was held at 40 ◦C for 5 min and then increased 3 ◦C/min to 180 ◦C, followed by 30 ◦C/min to 250 ◦C and held for 7.67 min. The MSD interface was kept at 260 ◦C. HS-SPME-GC-MS conditions were as previously described. Shortly, samples were heated to 30 ◦C for five minutes while agitating with a speed of 500 rpm prior to exposing the fiber to the sample for 45 min at 30 ◦C with agitation at 250 rpm. The SPME fiber was desorbed in split mode with a 10:1 split ratio and the inlet temperature was kept at 260 ◦C. Bottle duplicates were analyzed in triplicate for each treatment. Compound details are provided in Table S1. Wines were analyzed sensorially using descriptive analysis in the J. Lohr Wine Sensory Room, University of California, Davis, CA. GN, BA, and CS wines were analyzed approximately two, three, and four months, respectively, after bottling. Three separate descriptive analysis panels were utilized, one for each variety. Eleven panelists were recruited for GN, and ten each for BA and CS.

The panelists were offered $30 gift certificates for completion of the study. The study was approved by the International Review Board and all participants reviewed and agreed to the terms of the experiment. None of the panelists knew details of the experiment. Two fermentation replicates were selected from each treatment totaling six wines for each descriptive analysis study. There were six training sessions and three evaluation sessions. The panelists were given 30 mL of each wine sample for both the training sessions and the evaluation sessions. The wines were presented blind using black wine glasses and the order was randomized for each session. In the first training session, panelists generated descriptors used for differentiating the wines. In subsequent sessions, the reference standards for each descriptor were optimized through panel discussions until there was general agreement. The list of descriptors was narrowed down until therewere twenty descriptors for GN , twenty-six for BA , and twenty-two for CS . Panelists were asked to rate the intensity of each attribute using an unmarked line scale. Reference standards were given as an anchor for the high end of the intensity scale of each attribute. In addition to these attributes, panelists also analyzed color by matching each wine with a color chart . Panelists were given 30 mL of wine in a clear glass and instructed to hold the glass at arm’s length with a white background and match with the closest color on the poster. Scores were reported by assigning number values to each color on the poster. Perceived color differences from sensory analysis were compared to wine colors determined using a CR-400 Chroma Meter using the CIELAB color space.Wines were analyzed by the panelists in triplicate using a randomized block design over a one-week period. All analyses were completed in isolated booths with positive air flow and red lighting. Randomized three-digit codes were assigned to the wines to eliminate biases. Panelists were given breaks in between each wine and were encouraged to drink water and eat an unsalted cracker as a pallet cleanser. All samples were expectorated. Data were collected using FIZZ software .All statistical analyses were carried out using XLSTAT .

These raspberry drupelets were held in a –80℃ freezer until use. Raspberry volatiles were analyzed using a method modified from Forney et al. . Frozen raspberry drupelets were removed from the -80℃ freezer and 5 g were added to 100 g NaCl saturated H2O, and homogenized in a blender for 1 min. Five mL of the homogenate was transferred to a 20 mL headspace vial . Headspace volatiles were then analyzed by solid phase micro extraction gas chromatography mass spectroscopy . Vials were incubated at 50℃ for 10 min, and then the headspace was exposed to a gray SPME fiber for 10 min with agitation. The fiber was desorbed at250℃ for 15 min onto a BD-WAX UI column held at 35℃ for 5 min, then ramped to 240℃ at 0.167℃/s and held at 240℃ for 4.5 minutes. Helium was the carrier gas at a flow of 16.7 ml/s. Peaks were initially identified through comparison with NIST Mass spectral library . Retention indices of these compounds were used to further verify identity by comparison against standard compounds and relatively quantified with GC using a BD-FATWAX UI column. The same method was followed except the sample amount was 1 mL and nitrogen was used as the carrier gas. The samples’ peak area was multiplied by the reference standard concentration, drainage collection pot and the result was divided by the peak area of the reference standard.A descriptive sensory analysis was performed with 12 panelists who were trained ahead of the sensory evaluations to align their sensory perception. There were four one-hour training sessions over two weeks. During the training, panelists were provided with references for each attribute to compare against the training samples.

The sensory evaluations took place in the UC Davis Department of Plant Sciences Sensory Lab, equipped with five separate evaluation booths with individual computers with sensory analysis software . Samples were prepared the morning of evaluation and stored at 5℃. The samples were brought to room temperature before being tasted by the panel. One sample included 3-4 raspberries and was provided to the panelists in sealed sensory tasting cups. Each sample was blinded with random 3-digit codes generated by the software .Panelists tasted three replications of raspberries at harvest , and again for each treatment after 5, 10, and 13-days in atmosphere storage and evaluated their taste, texture, and flavor. The panelists were instructed to cleanse their palates with crackers and water in between samples. On day 10, there was only one replication of the air treatment and two replications each of 15 kPa and 5 kPa atmospheres appropriate for sensory evaluation due to decay growth. On day 13, there were no samples of the air treatment, 5 kPa treatment had two replications, 8 kPa treatment had thee replications and 15 kPa treatment had two replications. The panelists measured the intensity of sensory attributes of the raspberry samples and marked their score for each given attribute on a 10 cm straight line anchored with less and more using sensory evaluation software . This software transmuted the markings for each attribute into a numerical value ranging from 1 to 10 units, where 1 was less and 10 was more intensity. The tasted attributes were sweetness, acidity/tartness, firmness , juiciness, raspberry flavor, and off-flavor. The tasting lexicons were decided and agreed upon during the training.Data were analyzed using R statistical program . A total of 4 treatments and 3 replications across the four evaluation dates were analyzed for instrumental and sensory qualities of the raspberries. Data were assessed through ANOVA followed by Fishers Least Significance Difference test to reveal significant differences among treatments and evaluation times.

A correlation analysis was also conducted to investigate the relationship between volatile compounds and sensory attributes. The sensory data was analyzed using principal component analysis using R and R Studio software andPCA plots are presented for 5- and 10-day evaluations. The sensory data on day 13 was insufficient for analysis due to decay.Storing raspberries under high CO2 atmospheres reduced fruit softening in a concentration dependent manner . Raspberries stored in 15 kPa atmosphere did not soften until day 13, and only slightly. Raspberries stored in 8 and 5 kPa CO2 had intermediate firmness throughout storage, and softened gradually, while raspberries stored in 0.03 kPa atmosphere lost firmness quickly during storage and had the lowest firmness among all the atmosphere treatments at each evaluation. Across all evaluation’s times, raspberries stored in 15 kPa atmosphere were most firm, raspberries stored in air were least firm, and raspberries stored in 5 or 8 kPa atmosphere were intermediate and not different from each other .A total of 14 volatile compounds were detected in the raspberry fruit . There were five terpenes , three alcohols , two aldehydes and one each of ester , ketone , alkyne and carboxylic acid . The aromatic volatiles limonene, linalool, hexanoic acid and α-terpineol increased in concentration over time, particularly in 0.03 kPa atmosphere stored raspberries . However, storage in elevated CO2 atmospheres resulted in significantly lower concentrations of these volatiles as well as α-ionone . Raspberries held in 15 kPa atmosphere exhibited a sharp and significant increase in the fermentative volatiles, acetaldehyde and ethanol, on day 5 and had higher concentrations than raspberries stored in other atmosphere treatments . Acetaldehyde, ethyl acetate, and ethanol all increased in concentration overtime, particularly on the last day of storage .

Raspberry fruit mouthfeel firmness, raspberry flavor and TSS decreased over time and juiciness and off-flavor increased over time . Firmness scores, both hand and mouthfeel, were significantly higher in raspberries stored in 15 kPa atmosphere followed by fruit held in 8 kPa atmosphere. The trend was opposite for juiciness and sweetness, where raspberries held in 0.03 kPa or 5 kPa atmosphere had the highest juiciness scores, and fruit held in 15 kPa atmosphere had a lower sweetness score than fruit held in air atmosphere. Tartness score was higher in fruit held in 8 kPa or 15 kPa atmosphere than fruit held in 0.03 kPa atmosphere. Raspberry flavor was significantly higher in fruit held in 8 kPa atmosphere than in 5 kPa atmosphere . After five days of storage, the PCA biplot showed that raspberry firmness was strongly associated with the 15 kPa atmosphere treatment, and less so with the 8 kPa atmosphere treatment . Fermentative volatiles: acetaldehyde, ethyl acetate, and ethanol were also clustered with the 15 kPa atmosphere treatment, and less so with the 8 kPa atmosphere treatment. Raspberry flavor was most closely associated with sweetness andtetradecane at the bottom of the biplot. No treatments were closely associated. Tartness, TA, TSS, heptanol, α-terpineol and limonene were associated with each other and the 5 kPa atmosphere treatment at the top of the bi-plot. Sweetness, off-flavor, and juiciness were clustered with each other and the 0.03 kPa atmosphere treatment. Most of the aromatic volatiles were associated closely with the 0.03 kPa and 5 kPa atmosphere treatments, and on the opposite side of the bi-plot from firmness and 8 kPa and 15 kPa atmosphere treatments. After ten days, firmness remained associated with treatments with high CO2 concentrations . Raspberry flavor, TSS, TA, and tartness were associated with each other and the 8 kPa atmosphere treatment, and raspberry flavor shifted to the top of the bi-plot. Juiciness, sweetness, tetradecane and heptanone were clustered on the top left with the 5 kPa atmosphere treatment. At the bottom of the bi-plot, ethanol and ethyl acetate were associated with the 15 kPa and 0.03 kPa atmosphere treatments, respectively, and acetaldehyde was in between 15 kPa and 0.03 kPa atmosphere. Aromatic volatiles maintained their association with lower CO2 atmosphere treatments, and were also associated with off-flavor as at ten days .Across evaluation days, total soluble solids and juiciness were positively correlated . Sweetness was negatively correlated with hand firmness and tartness, and mouthfeel firmness and juiciness were negatively correlated. Acetaldehyde and ethanol were negatively correlated and tetradecane was positively correlated with juiciness and TSS . 2-Heptanol was positively correlated with juiciness. Hexanal, hexanoic acid, α-ionone, linalool, and α-terpineolwere negatively correlated with hand firmness, and all but hexanal were positively correlated with sweetness. Limonene was the only volatile significantly correlated with off-flavor , and α-terpineol was the only volatile correlated with tartness .Raspberry firmness remained stable or decreased more slowly with increasing CO2 concentration in storage, with the highest firmness in 15 kPa CO2. In agreement with our results, Haffner et al. , found that an atmosphere of 15% CO2, 10% O2 maintained the firmness of five raspberry cultivars stored for seven days at 1℃.Strawberries exposed to high CO2 exhibited changes in apoplastic pH, square plastic pot which may have induced cell to cell adhesion by precipitation of soluble pectin . This may explain why high CO2 stored raspberries were perceived as firmer by our sensory panelists. However, firmer fruit tasted less sweet to the sensory panelists. Stec et al. reported that firmer kiwifruit tasted less sweet than softer ones. This finding aligned with the general notion that softer fruit have more ripe fruit characteristics such as sweetness, juiciness and higher aroma intensity .

This can explain the negative correlation of juiciness with firmness in our experiment. In addition, storage under high CO2 atmospheres also might have inhibited further ripening of the fruit which would inhibit fruit softening. High CO2 atmospheres also reduced development of leakiness and color darkening. Exposure to CO2 atmospheres can induce fermentative metabolism , likely due to its capacity to disrupt enzyme systems . Elevated CO2 has been reported to induce development of alcoholic flavors in fruit if the concentration is too high for longer times . High CO2 enhances the activity of pyruvate decarboxylase and alcohol dehydrogenase, but reduces activity of alcohol acetyltransferase. As a result, acetaldehyde and ethanol accumulate and this trigger further production of ethyl esters and reduction of other esters, thus, enhancing alcoholic flavor . Larsen suggested that accumulation of ethyl acetate was linked to development of off-flavor in raspberries in some cultivars. High CO2 atmospheres can impact the lipoxygenase pathway which is involved in the formation of aromatic volatile compounds through effects on enzymes or by limiting substrates due to production of fermentative volatiles . In our study, fermentative volatiles were strongly associated with storage in the higher CO2 atmospheres early in storage, but after 10 days of storage, raspberries stored in air also accumulated fermentative volatiles, likely due to over-ripening. In addition, very low O2 atmospheres can contribute to off-flavors. Joles et al. reported that raspberries stored in 3% O2 developed off-flavor because fermentative respiration occurs when O2 levels drop below this critical level . However, this might not be the case for our experiment, because our lowest O2 level was ≥ 6 kPa. The one exception might be the 15 kPa atmosphere. The combination of 15 kPa CO2 with 6 kPa O2 could have resulted in additional impacts on fruit metabolism given the relatively low O2 concentration, resulting in a stronger impact of the 15 kPa atmosphere on fruit quality. The concentration of individual fermentative volatiles was as much as 1000-fold higher than the aromatic volatiles, and the fermentative volatiles were 4 to 300-fold higher in raspberries stored under 15 kPa CO2 compared to fruit stored in air. Accumulation of acetaldehyde, ethanol and ethyl acetate can contribute to objectionable changes in taste . However, in our experiment, the off flavor sensory score was lower in fruit stored in 15 or 8 kPa atmosphere than in 5 kPa atmosphere, and was moreclosely clustered on the bi-plot with aromatic volatiles than fermentative volatiles. Limonene was most highly correlated with off-flavor. The concentrations of ethanol detected in our raspberry samples appear to be below the corresponding odor threshold of 990 µl /L, . This may explain why our sensory panelists did not sense any off-flavor in raspberries stored in 15 kPa atmosphere, even when fermentative volatile concentrations were significantly higher in those fruit than in fruit stored in lower CO2 atmospheres. The odor threshold is the lowest concentration of a volatile that can be smelled and can vary as much as 106 to 108 among volatiles in fruit . Therefore, the most abundant volatile is not necessarily always the dominant fruit aroma. Alcohols usually have considerably higher threshold values, near 990 µl /L , and therefore contribute less to aroma building than their corresponding aldehydes .

The treatments at ranch 1 were applied during the winter, which in Ventura county is marked by cool ambient temperatures , lower average relative humidity, and increased chance of rain and clouds. In contrast, ambient conditions at the end of summer when treatments were applied at ranch 2 are marked by warm temperatures , consistently moderate relative humidity , no rain, little cloud cover, and calm winds. Actual conditions during 2018 generally reflected these long-term averages. Ambient temperature was generally lower during the ranch 1 experiments compared to the ranch 2 experimental period after treatment application . Relative humidity was much lower for ranch 1 compared to ranch 2 between about 30 and 10 days before treatment application, but was not different between the ranches on the day of treatment application or after. A total of 2.6 cm rain over two days fell in the area within one week of treatment application at ranch 1. There are two possible ways that ambient weather conditions differentially influenced cane Botrytis development between the ranches. One way is that the favorability to disease development of ambient weather conditions may combine with other factors, such as wounding, to influence colonization and symptom expression. However, conditions were slightly more favorable for ranch 1 after treatments were applied due to lower temperatures and rain, therefore wounding from leaf removal treatments would have been expected to increase disease severity. Another way is that the higher relative humidity before treatment application at ranch 2 could have led to higher levels of inoculum present in the environment, which in turn could lead to higher disease. Thirdly, conditions for ranch 2 may have been more favorable for infection on the days of treatments application and shortly afterward. While relative humidity, round plastic plant pot daily maximum temperature, precipitation, and wind were similar between the two ranches, daily minimum temperature was higher by about 10o C at ranch 2.

If it is assumed that the optimal temperature of 20°C for infection of strawberry flowers also applies to raspberry canes, then conditions at ranch 2 were more favorable than ranch 1 inthe days after treatment application, which could have led to higher incidence of cane Botrytis. We hypothesized the removal of lower leaves would shift environmental conditions within the canopy to be less favorable for cane Botrytis development. Unexpectedly, in our study leaf removal treatments appeared to increase relative humidity within the raspberry canopy, especially within dense plantings. However, this effect was not consistent across experiments. In Northwest Washington, minimum air temperature, night air temperature, cumulative rain, leaf wetness, and duration of leaf wetness was correlated with B. cinerea colonization of processing raspberry fruit . In strawberry, incidence of flower infections by B. cinerea has been reported to be correlated at relative humidity >80% and >90% . Given that leaf wetness typically forms at night and that leaf wetness is important in development of many diseases caused by B. cinerea, we would expect disease to be more severe in 3-row treatments that had higher relative humidity at night. In our study, however, relative humidity was higher at night for only two of the treatments at ranch 2 but disease was more severe compared to the control for all three treatments. Therefore, relative humidity may not be a significant factor for cane Botrytis severity. Leaf removal treatments also influenced temperature, but the effect was more consistent than relative humidity across experiments. Temperature was higher in treated canopies than the control during the day but were lower than the control at night. This suggests that the leaves in the raspberry canopy serve to moderate temperatures, or reduce variability, with respect to daily fluctuations in ambient temperature. This assertion is supported by the generally greater time per day of significant change from control in manual treatments, given that the manual treatment removed the most material from the lower canopy. Because the effect was generally consistent across experiments and treatments yet few significant differences in disease incidence were observed, the influence of canopy on temperature is may not an important factor for cane Botrytis incidence.

We found few significant differences in cane Botrytis incidence among treatments despite finding a strong influence of leaf removal on disease severity. However, a non-significant trend of higher incidence in the manual and twine treatments versus blade removal and the control was observed in all three experiments on the first rating date. Because the manual and twine treatments removed petioles from the cane whereas petioles remained attached to the cane in the control and blade treatments, the wounding caused by petiole removal may increases disease incidence early in the season. By the last rating date, however, this trend had dissipated or reversed at ranch 1, but remained a trend at ranch 2, especially between the blade and twine treatments. Taken together, this suggests leaf removal treatments may have an impact on disease incidence right after leaf removal or early in the season but that the impact may diminish as the season progresses. Furthermore, the length of time the trend was observed supports our suggestion above that the cultivars used in our experiment differ in susceptibility or response to wounding. While this suggests that primocane yield is not influenced by cane Botrytis, itis unknown if receptacle counts are representative of marketable yield. Even if actual marketable fruit could have been collected, collecting yield data from small plots of raspberry is known to be logistically challenging and may not be a reliable measure of yield or representative of the potential raspberry yield. Though there were many infected canes that developed sclerotia near the end of each experiment, the impact of the sclerotia on the floricane is unknown. Additional studies are needed to understand the effects of sclerotia on bud break and bud elongation in fresh market raspberry. Because we did not perform a true replication of any of our experimental conditions, future research is needed to confirm the influence of leaf removal on incidence and severity of cane Botrytis in different production conditions. Currently, in California fresh market raspberry production there are no management practices commonly used to manage cane Botrytis.

The twine treatment is the current experimental method of leaf removal practiced by growers in Ventura County due to its labor efficiency. Because it is an aggressive practice, we hypothesized that a practice that causes less wounding would improve cane Botrytis management. The method we examined was the manual removal treatment, which was designed as an equivalent canopy treatment but with less wounding. Across all three experiments there were no significant differences in cane Botrytis severity between the twine and manual treatments. The use of high powered blowers to remove leaves in the lower raspberry canopy would be beneficial to evaluate for novel management practices of cane Botrytis. Our study indicates that wounding by twine leaf removal is not important for disease severity. Our experiments have shown leaf removal methods can be applied for certain cultivars and row spacings.California agriculture is in a time of opportunity to adopt sustainable practices for the management of challenging issues such as soilborne disease. In the previous decades, soilborne diseases were commonly managed with the use of chemical fumigation, 25 liter round pot but the widening restrictions on the use of fumigants in the San Joaquin Valley of California has posed a challenge for growers. One of the affected crops in California is processing tomato , which averaged 1.1 billion dollars in value from 2013 to 2017, and accounted for 93% of the production in the United States in 2017 . Despite the total value of processing tomato statewide, growers face challenges due to the low market return on a per area basis. Southern blight is a disease of processing tomato that has long been an economic concern in the San Joaquin Valley, and recently caused a widespread epidemic in both the San Joaquin and Sacramento Valleys . The threat of southern blight has caused reductions in acres planted with processing tomato in the southern San Joaquin Valley . Southern blight is caused by the soilborne fungus Athelia rolfsii C.C. Tu & Kimbr. that has a host range of over 500 different plant species . The fungus produces a white, typically fan-shaped mycelial mat and distinctive tan to reddish brown sclerotia with walls are composed of chitin and laminarin . The sclerotia survive and germinate at soil depths of 0 to 8 cm and are commonly dispersed by the movement of infested soil or plant material . Initial infections in the field most commonly occur on plant tissues that are in contact with the soil surface where sclerotia are stimulated to germinate by drying and remoistening . Following germination from sclerotia, mycelia of A. rolfsii colonizes aboveground plant tissue and releases cell wall degrading enzymes. The enzymes disintegrate host tissues and when colonizing stem tissue form a lesion around the stem near the soil line that advances rapidly to the point of girdling the stem . The pathogen is most damaging when it infects stems or crowns, in which it causes wilting, cankers, rot, or whole-plant necrosis on various crops . In processing tomato, the most common symptom of southern blight is rapid wilt of vegetation above the ground. The temperature range for mycelial growth is from 8 to 40ºC, and the optimal temperatures for sclerotia formation is from 27 to 30ºC . Overall, temperatures 25 to 35o C are most conducive to disease development . Sclerotia form on mycelial mats that are 5 to 6 days old . When mature, sclerotia can persist in the soil for many years . Additionally, A. rolfsii can persist in the soil saprophytically as mycelium on plant debris . If management practices are not adopted, within a season or two a single infected plant can produce thousands of sclerotia, potentially resulting in serious yield loss due to southern Blight . There are several methods to reduce losses caused by A. rolfsii, but their uses are limited in processing tomato production in California.

These include rotation with nonhost crops, minimizing soil moisture on the soil surface, and deep plowing to bury the sclerotia. Rotating with crops that are non-hosts including corn, barley, wheat, and small grains has been shown to reduce sclerotia density in subsequent years . However, these rotation crops are not economically viable as regular rotation partners in the San Joaquin Valley of California, and few alternatives are available due to the wide host range of the pathogen. Although reducing soil moisture has been shown to reduce southern blight , the use of subsurface drip irrigation has already become the standard practice in California. The drip lines are buried at a depth of 25.4 cm to 30.48 cm for weed management, water efficiency, and to reduce A. rolfsii inoculum accumulating in the furrow . Athelia rolfsii sclerotia cannot survive long periods under anaerobic conditions, thus deep plowing of infected plant tissue and sclerotia to at least 20 cm depth has shown to reduce inoculum . However, this approach is not feasible in California production systems because the drip lines remain buried for two to three consecutive crop seasons and could be subject to damage from deep plowing. Taken together, agronomic methods have little potential to further improve the management of southern blight.Fumigation with metam sodium or metam potassium was traditionally relied upon to effectively manage A. rolfsii in processing tomato in the San Joaquin Valley. For effective control the product must be applied through sprinklers, however, sprinkler application is restricted. The alternative method to sprinkler application is shanking the product into the soil, but this approach does not allow for effective dispersion of metam sodium into the soil, therefore it is not effective . There are effective fungicides such as flutolanil, penthiopyrad, and tebuconazole available to manage southern blight in vegetable crops . However, processing tomato cultivars are determinant, not trellised, and the canopy is often full late in the season when the pathogen is most active. These characteristics prevent chemical application to the vulnerable stem tissue at or above the soil line and is the main reason why chemical management of southern blight has proven ineffective in processing tomato . The processing tomato industry in the San Joaquin Valley of California would benefit from having new efficient and sustainable approaches to manage southern blight.

The berry firmness was then determined . In the 2015 season, three grape clusters of uniform size and at the same phenological stage were identified in each plot, and two berries were collected randomly from each bunch at each sampling time. The berry skins were removed, frozen, kept at −80◦C, and transferred to the Department of Viticulture and Enology of the University of California, Davis, CA, United States, for further analyses. The skins were then placed in liquid nitrogen and ground using a TissueLyser II . RNA was extracted using 0.5 g of ground tissue based on the protocol described by Blanco-Ulate et al. . RNA concentration and purity were determined using a NanoDrop 2000c Spectrophotometer , and RNA integrity was checked by electrophoresis on 1.5% agarose gel. Reverse transcription was performed using 1 µg of RNA and M-MLV Reverse Transcriptase according to the manufacturer’s instructions. qRT-PCRs were performed using the SYBRR Green PCR Master Mix kit . The PCR program consisted of 70◦C for 10 min, 36 cycles at 42◦C for 2 s, and 37◦C for 50 min. VvActin was used as the reference gene and processed in parallel with the genes of interest. Gene sequences used for primer design were obtained from the GenBank of the National Center for Biotechnology Information using Primer-BLAST software . The relative levels of target gene expression were calculated using the formula 2 . The linearized values correspond to the relative gene expression within a given sample and are comparable across genes. Four biological replicates of S-ABA treated and control grape berries were used to obtain the relative gene expression data.Application of abscisic acid increased the total anthocyanin concentration in berry skins of the seedless grapeSelection 21 during the 2013 and 2014 seasons, regardless of the S-ABA concentration and time of application .

However, berries that received 400 mg/L of S-ABA at 7 and 21 DAV had significantly higher, almost two to three times more, 25 liter pot anthocyanin concentrations than other treatments. According to the CIRG, berries from control treatments had a green to a yellow color in both seasons . In 2013, berries treated with one or two applications of 200 mg/L S-ABA or one application of 400 mg/L S-ABA at 7 DAV, and those in the 2014 season that were treated with one application of 200 mg/L S-ABA developed a pink color . Remarkably, berries of the 2013 season treated with two applications of 400 mg/L S-ABA and berries of the 2014 season treated with one or two 400 mg/L S-ABA applications, developed a stronger red color . For both the 2013 and 2014 seasons, color coverage was lowest in control grapes and highest in grapes treated with two applications of 400 mg/L S-ABA. Increase in total polyphenols was evident in grapes subjected to two 400 mg/L S-ABA applications during the 2013 and 2014 seasons. These berries also presented the lowest mean berry firmness . Importantly, the increased softening due to S-ABA application did not result in higher frequency of cracked berries in any of the studied seasons. Qualitative assessment of berry cracking was performed visually. Further analyses of the effect of 400 mg/L S-ABA treatments on CIRG, total and individual anthocyanins concentrations, and gene expression of transcription factors and biosynthetic enzymes were performed with grape berries collected from the 2015 trial. Measurements of CIRG confirmed previous results obtained during the 2013 and 2014 seasons, at the time of harvest , grapes treated with two S-ABA applications had the highest CIRG values . Grape bunches from the control treatment presented pink berries , whereas those treated with one or two applications of S-ABA had red berries . As determined in the previous seasons, berries treated with 400 mg/L S-ABA also presented higher total anthocyanin content than the control at 14 and 28 DAV . At 28 DAV, grapes treated with one or two applications of 400 mg/L S-ABA presented total anthocyanin concentrations almost three times higher than the control.

Even 3 weeks after the first application , berries treated with only one S-ABA application showed a total anthocyanin content similar to those treated with two S-ABA applications. Nonetheless, the second application of 400 mg/L S-ABA significantly affected the total anthocyanin accumulation at the time of harvest . With the exception of petunidin- 3-glucoside, S-ABA application significantly improved the concentrations of all the measured anthocyanins. Cyanidin-3- glucoside and peonidin-3-glucoside levels increased at 14 DAV, 1 week after the first S-ABA application. The second S-ABA application stimulated the accumulation of the anthocyanin delphinidin-3-glucoside at 28 DAV, yielding differences relative to both the control and to the samples treated with only one S-ABA application. At 28 DAV, the concentrations of peonidin-3-glucoside and malvidin-3-glucoside increased after exogenous S-ABA application but were not further increased by the second application. At the time of harvest , peonidin-3-glucoside and cyanidin-3-glucoside were the dominant pigments present after all treatments. Delphinidin- 3-glucoside, cyanidin-3-glucoside, and peonidin-3-glucoside presented higher accumulation following the second application of 400 mg/L S-ABA, but the number of applications did not affect the accumulation of malvidin-3-glucoside. As presented in Figure 4, treatment with 400 mg/L S-ABA significantly increased the expression of the transcription factors VvMYBA1 and VvMYBA2 and the expression of the biosynthetic genes CHI, F3H, DFR, and UFGT 1 week after the first application . Three weeks after the first S-ABA application , expression of CHI, F3H, and DFR genes remained high, but this was not observed for the transcription factors VvMYBA1 and VvMYBA2 or the UFGT gene. Four weeks after the first S-ABA application , no significant differences were observed in the expression of genes or transcription factors between berries that received one S-ABA application and those that received the control treatment.

The two applications of 400 mg/L S-ABA induced expression of the genes CHI, F3H, DFR, and UFGT and the transcription factors VvMYBA1 and VvMYBA2 at 14 and 28 DAV . F3H expression was the most affected by S-ABA application, displaying higher levels than the control until the final stages of berry maturation at 35 DAV,whereas the remaining genes presented no differences from the control at harvest. Overall, the gene expression results indicate that a second S-ABA application contributed to the maintenance of the expression of the transcription factors VvMYBA1 and VvMYBA2 and the genes F3H and UFGT at higher levels than in the control for an extended period of time.Exogenous application of S-ABA improves the color of table grapes by stimulating the anthocyanin synthesis and accumulation in the grape skin . Our results are similar to those reported previously for “Flame Seedless” grapes, in which applications of 300 mg/L ABA during or after véraison were more effective at increasing anthocyanin concentrations than application before véraison . In ‘Crimson Seedless’ grapes, anthocyanin concentrations increased with application of 400 mg/L S-ABA at 17 DAV, but the response varied widely between seasons depending on S-ABA dosage and time of application . Exogenous S-ABA application is thought to simulate plant stress responses and accelerate ripening processes . High ABA concentrations are believed to be perceived by grapevines as a drought stress signal . Subsequently, water stress leads to changes in grape secondary metabolism, significantly increasing flavonoid levels and, especially, anthocyanin biosynthesis . In our study, in addition to increasing anthocyanin concentration, exogenous S-ABA improved both berry color intensity and uniformity. This is important because the visual assessment of berry color characteristics determine, in part, the commercial value of table grapes . Grape clusters with more intense and uniform berry color have higher consumer acceptance. Improved color characteristics such as increased color coverage of grape berries, and uniformity of berry color within a cluster, were also observed in “Benitaka” and “Rubi” table grapes following application of 400 mg/L S-ABA at 7 DAV and 15 days before harvest . In our experiments, berries from the seedless grape Selection 21 treated with one or two applications of 400 mg/L S-ABA presented higher CIRG values than the controls. Besides, 25 liter plant pot improving cluster color and attractiveness, exogenous S-ABA can potentially decrease the time to harvest, a feature that is very advantageous for grape commercialization . The observed increase in anthocyanin concentration resulting from S-ABA application does not necessarily result in an increase in total polyphenol content; polyphenols include phenolic acids, stilbenes, coumarins, tannins, and flavonoids , as reported in “Alachua” muscadine grapes and in “Isabel” grapes . Environmental factors such as temperature, rainfall, and altitude could also influence berry polyphenol concentrations. In addition, the berry ripening stage is directly correlated to the concentration and proportion of several polyphenols that impact the organoleptic properties, nutritional value, and antioxidant capacities of the grapes . S-ABA application can negatively affect berry firmness, an important characteristic for the successful postharvest handling of grapes for the fresh fruit market because it influences transportability and shelf life . ABA application is known to cause loosening and decreased rigidity of the cell wall, resulting in fruit softening and a higher probability of berry cracking .

Treatment of grapes with exogenous S-ABA can result in changes in the regulation of proline-rich cell wall proteins and in the induction of cell wall degrading genes, such as polygalacturonases that promote pectin solubilization and depolymerization . The effect of S-ABA on berry firmness was also observed in “Flame Seedless” grapes, where it caused softening similar to that caused by ethephon application , as well as in ‘Crimson Seedless’ and “Red Globe” grapes . Therefore, it is still necessary to evaluate if the benefits of applying exogenous ABA to improve berry color can outweigh a potential reduction in the shelf life of treated grapes.Multiple applications of exogenous ABA can promote anthocyanin accumulation for longer periods of time . It is possible that more than one ABA application could induce a milder response at later grape phenological stages or that the effects of a second application could take more time to be evident. In this study, the second application of 400 mg/L S-ABA significantly increased the total anthocyanin content at the time of harvest, which supported the latter hypothesis and confirmed that two S-ABA applications had a more pronounced effect than only one application. The higher total anthocyanin concentration observed with S-ABA application appeared to result from a transient effect of S-ABA, because the anthocyanin concentration of grapes that received only one S-ABA application remained essentially constant between 28 and 35 DAV. It may, therefore, be inferred that the action of S-ABA decreases over time and that its levels increase with a second application, allowing maintenance of its activity. Three applications of 400 mg/L S-ABA at 1-week intervals prior to véraison resulted in an earlier accumulation of anthocyanin in “Cabernet Sauvignon” grapes, but no differences in anthocyanin concentration at harvest were observed in grapes that received different treatments . The increase in endogenous ABA concentration in grape berries occurs at the beginning of véraison and extends until the establishment of maturation when endogenous ABA concentrations peak. ABA concentration then decreases until harvest, the period over which the decrease occurs ranges from 13 to 20 days depending on the cultivar . Application of exogenous S-ABA close to véraison, when ABA naturally reaches its highest concentration in berries, was shown to be more effective in increasing anthocyanin accumulation than application at other times . S-ABA application significantly increased endogenous ABA levels 7 days after application in “Carménère” grapes, 40 days later, the ABA levels in the treated berries remained higher than those from control .dihydroxylated precursors of red anthocyanins in grape skin, whereas delphinidin, petunidin, and malvidin are trihydroxylated precursors of blue and purple anthocyanins . Accumulation of individual anthocyanins in grapes may be induced by S-ABA application and varies with the cultivar. In “Noble” and “Alachua” muscadine grapes, application of S-ABA during véraison and again at 8 DAV for “Noble” or again at 13 DAV for “Alachua” resulted in higher levels of accumulation of all evaluated anthocyanins in “Noble” grapes but not in “Alachua” grapes, which only presented higher accumulation of peonidin-3-diglucoside compared to the control . Changes in the proportions of individual anthocyanins resulting from S-ABA application were also observed in “Cabernet Sauvignon” grapes, both in berries and in wine . In “Isabel” grapes, application of S-ABA increased the accumulation of individual anthocyanins both in must and in processed whole juice .

Unfortunately, heat processing and pasteurization can lead to the degradation of anthocyanins resulting in a loss in color or a formation of brown polymers, possibly impacting the acceptability of the final product.Elderberry juice and extracts have been evaluated for their thermal stability, which have shown that anthocyanins degrade following first-order reaction kinetics.138 The stability of anthocyanidins can be reinforced via intra- and inter-molecular interactions with protective structures and flavonoids through a phenomenon termed copigmentation. Notably, acylated anthocyanins, such as those found in American elderberry like cyanidin 3-coumaroylsambubioside, are sometimes more stable during thermal processing due to protective properties of the coumaroyl group folding over the flavylium ion. Like the European elderberry, the blue elderberry does not contain acylated anthocyanins. The thermal stability of anthocyanidins in blue elderberry juice has not yet been evaluated. However, as elderberry juice and extracts are frequently thermally processed to make products, such as jam, syrup, or gummies, it is important to understand the thermal degradation of anthocyanidins in the juice from blue elderberry. The purpose of this study was to determine the stability of the cyanogenic glycosides and main phenolic compounds in blue elderberry juice cooked at 72 °C and 95°C for two hoursto elucidate the kinetics of degradation of these important compounds.HPLC-grade methanol , and LCMS grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific . Ultrapure water was obtained from a Milli-Q water system . Prunasin was also obtained from Millipore Sigma. HPLC-grade acetonitrile , rutin , isorhamnetin 3-O-glucoside, caffeic acid, chlorogenic acid, -catechin, protocatechuic acid, ammoniumformate, and amygdalin were purchased from Sigma-Aldrich .

Cyanidin 3-Osambubioside chloride and cyanidin 3-O-glucoside were purchased from ExtraSynthese .Ripe berries were harvested in July 2019 from a farm in Winters, vertical plant tower CA at latitude and longitude coordinates of 38.634884, -122.007502. Fruit was selected from all sides of the plant at a variety of heights to obtain a representative sample of berries from each plant. Only fully ripe berries . About 5 kg were harvested from each of shrub in three hedgerows. The plant material was transported to University of California, Davis on ice in plastic gallon bags within two hours of harvest and stored at -20 °C until analysis.Elderberry juice was prepared from 300 g previously frozen berries, thawed at room temperature for one hour in a mesh bag in a metal bowl. Thawed berries were juiced with a manual fruit press . The juice obtained was then aliquoted into glass vials and sealed with a screw cap to avoid evaporation . A time-zero aliquot of juice was immediately placed on ice for analysis. An aliquot was also analyzed for Brix and pH measurements .A hot water bath was prepared using an immersion circulator , set to the desired cooking temperature . The temperature was also monitored with a thermometer in the water bath. Duplicate vials were removed at the following times: 15, 30, 45, 60, 75, 90, 105, and 120 min. An extended processing time was used to observe degradation of more heat-stable compounds as well as to make comparisons to other studies that processed juice for multiple hours at these temperatures. Once removed from the hot water bath, vials were placed immediately into an ice bath for 15 min. Then from each vial, 1 mL of juice was placed in a microcentrifuge tube and centrifuged at 4 °C, 15,000 rpm for 15 min . Next, the supernatant was diluted 1:10 with 1% formic acid in water, filtered with 0.2 µm PTFE filter, and placed in an HPLC vial for analysis. Five replicate juice samples were prepared and cooked at both temperatures.Composite juice samples were prepared by combing equal aliquots of the time points 0, 15, 30, 60, and 120 minutes of thermal processing for both temperatures for each juice prepared.

To extract CNGs for blue elderberry juice, 0.500 mL of juice was mixed with 2.00 mL of methanol in a 5 mL centrifuge tube, then sonicated at 30 °C for 30 min. After sonication, 1.00 mL of extract was transferred to a 1 mL microcentrifuge tube and centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatant was collected and filtered through 0.22 µm PTFE into an HPLC vial and used for analysis. Five replicate extractions were made of raw elderberry juice and triplicate extractions were made for the other time/temperature juices.CNGs were analyzed via ultra-high performance liquid chromatography with electrospray ionization and triple quadrupole tandem mass spectrometry using an Agilent 1290 Infinity HPLC and 6460 mass spectrometer . The UHPLC was equipped with a binary pump with an integrated vacuum degasser , an autosampler with thermostat , and a thermostated column compartment . CNGs were separated using a Kinetex F5 column at 40.0 °C. The mobile phase consisted of a linear gradient of 1 mM ammonium formate in water and 1 mM ammonium formate in 650:50 MeOH:ACNe as follows: 5% B, 0–5 min; 100%B 5-5.50 min, 95% A 5.60-6.50 min. The flow rate was 0.400 mL/min, and the injection volume was 5.0 μL. The CNGs were analyzed using negative ESI mode. The drying gas temperature was 300 °C and the flow rate was 8.0 L min-1 . The sheath gas temperature and flow rate were 350 °C and 11.0 L min-1 , respectively. The nebulizer gas pressure and capillary voltage were 45 psi and 3.5 kV, respectively. The fragmentor voltage was 160 V for amygdalin and 100 V for sambunigrin. The dwell time was 100 ms for amygdalin and 200 ms for sambunigrin. The collision energy was set to 12 V for amygdalin, 0 V for sambunigrin. The multiple reaction monitoring mode was utilized to analyze amygdalin and sambunigrin. Quantification of CNGs was performed using external calibration curves using standard addition at levels of 500, 100, 50, and 5 ng L-1 . For amygdalin, the area of m/z 456.2 to m/z 323.1 was measured. For sambunigrin, the area of m/z 340.3 to m/z 294.2 was measured.Half-life values of phenolic compounds were calculated by plotting the natural log of C0/C ratio vs heating time t, where C0 is the initial concentration of a compound, C is the concentration of the same compound at time t in hours. The slope calculated from the figure is k .

Longitudinal Analysis of variance was performed with Tukey’s post-hoc test with p value at 0.05. Excel used for average and standard deviation and R Studio was used for ANOVA and post hoc analysis . CNG data was analyzed using MassHunter Quantitative Analysis to obtain peak areas for the targeted compounds. Microsoft Excel was used to create the calibration curves and determine CNG concentrations in samples .The concentrations of cyanogenic glycosides were quantified in raw and cooked blue elderberry juice for the first time. Results indicate that neoamygdalin , sambunigrin and prunasin are the primary CNGs in blue elderberry . Concentration of neoamygdalin were significantly higher than sambunigrin and prunasin . Neoamygdalin has been measured in raw bitters almonds in concentrations lower than amygdalin. In studies of American and European elderberry, sambunigrin is typically major CNG identified. Levels of total CNGs in blue elderberry are lower than American and European elderberry. European elderberry CNG levels range from 0.08 ± 0.01 to 0.77 ± 0.08 µg g-1 depending on the elevation and growing location. CNG levels in American elderberry juice range from 0.29 to 2.36 µg mL-1 . Differences between the subspecies may be due to genetic variation, impact of growing environment such as altitude, or methodology used to extract and analyze CNG content in the fruit and fruit juice, growing strawberries vertically including how berries were handled prior to juice and juicing method.The degradation of neoamygdalin > sambunigrin > prunasin was observed during cooking and the rate of degradation was faster at 95 °C as compared to 72 °C . However, degradation in juice at 72 °C was not linear, such that sambunigrin levels in juice cooked at significantly increased during the final timepoint measured . This may be attributed to neoamygdalin breaking down resulting in sambunigrin and a glucose molecule, an equivalent pathway to amygdalin degrading to prunasin and a glucose molecule. However, some of the resulting sambunigrin from that reaction would also have to be degrading since the decrease in concentration of neoamygdalindid not cause an equivalent increase in sambunigrin. An increase in sambunigrin at the end of the processing time was not observed in the juice cooked at 95 °C. In the juice processed at 95 °C, the combined concentration of neoamygdalin and sambunigrin decreased at each measured time point and did not increase at any time points like the juice processed at 72 °C. Prunasin levels did not significantly change in the elderberry juice cooked at 72 °C but prunasin did degrade significantly when processed at 95 °C . It appears that sambunigrin is more stable than prunasin in the elderberry juice; retaining about 70% of the original concentration in the juice heated to 95 °C. Neoamygdalin levels decreased significantly in the elderberry juice at both processing temperatures, with increased degradation at 95 °C as compared with the treatments at 72 °C. As previously mentioned, sambunigrin is the expected breakdown product from neoamygladin, but sambunigrin levels did not have concomitant increase due to thermal degradation of sambunigrin as well. Thermal processing has been seen to degrade CNGs in elderberry, flaxseed, and almond in previous studies. Furthermore, studies have seen enzymatic activity contributing to the breakdown of CNGs in nuts to reduce after exposure to heat. If the β-glucosidases for the CNGs present in blue elderberry are similar, they would also be inactivated during thermal processing at 72 and 95 °C, indicating thermal degradation is the main contribution to CNG levels decreasing in the present study. Because enzymatic degradation of CNGs was not measured during the thawing and juicing steps, the impact of the enzymes before the thermal processing cannot be evaluated here. The presence of neoamygdalin instead of amygdalin is unexpected. Amygdalin can convert to neoamygdalin with heat and in alkaline conditions. However, herein the raw elderberry juice hadsignificantly higher levels of neoamygdalin as compared to amygdalin. In a study of amygdalin content in almond varieties, amygdalin was found to convert to neoamygdalin during extraction , but the addition of acetic acid prevented the conversion. Blue elderberry naturally contain citric and malic acids, with an average titratable acidity of 0.60 ± 0.10 to 0.65 ± 0.07 g citric acid per 100 g FW. The average pH value of the juices in the present study was 3.76 ± 0.11. Therefore, there may not be enough acid in the matrix to prevent the conversion. In contrast, another study of amygdalin and derivatives in almonds found that heat of cooking caused neoamygdalin and amygdalin amide to convert to amygdalin, which was not observed in the present study. Further analysis of conversion of amygdalin to neoamygdalin in the blue elderberry could uncover why this epimer is dominant. The total levels of CNGs measured here are much lower than CNG concentrations found in European or American elderberry. In a study of European elderberries evaluated at various growing locations and altitudes found that sambunigrin levels range from 0.08 ± 0.01 to 0.77 ± 0.08 µg g-1 . A nearly 10-fold difference in concentrations between elderberry samples highlights the variation on CNG levels due to differences in growing conditions and environmental factors like sun exposure and temperature fluctuations. Furthermore, evidence of CNGs degrading with thermal processing has been evaluated in European elderberry products: when sambunigrin levels were measured in raw and cooked elderberry juice and other products, heating of elderberry juice reduced the level of sambunigrin, from 18.8 ± 4.3 mg kg-1 to 10.6 ± 0.7 mg kg-1 . Liqueur, tea, and spread also had significantly lower CNG concentrations as compared to the raw and cooked juice. American elderberry was evaluated for concentration of CNGs in the seeds, juice, skin, and stem of two genotypes: Ozone and Ozark. Elderberry juice was prepared by thawingpreviously frozen berries in a plastic bag and gently pressing to release juice. The juice of these elderberries contained amygdalin, dhurrin, prunasin/sambunigrin , and linamarin. Total concentrations of these four CNGs was 4.01 µg g-1 in Ozone and 3.66 µg g-1 in Ozark elderberries.