Horticulture

Another gap in the oral microbiome model research is the effect of preservation on oral microbiomes and laboratory oral cultures. The lack of research in this area is perhaps unsurprising given the newness of this subfield, though the paucity by no means implies that preservation of microbiomes is by unimportant, especially when we consider the potential of preserving oral communities for inoculating in vitro cultures or the potential of preserving oral samples for therapeutic applications. While preservation experiments have been extensively performed on single bacteria species, preservation of microbiome samples has only been studied recently, with the primary emphasis on the gut microbiome. Research on the gut microbiome has provided evidence that storing samples at various temperatures with different preservatives does not significantly impact the structures of the bacterial communities, though sampling method matters; that cryopreservation, especially using glycerol and inulin, greatly helps maintain the composition and functionality of artificial gut microbiota; that cryopreservation is less detrimental to sensitive strains than lyophilization; that intra- and inter-subject variability outweighs the technical variability, blueberries in containers including variations between sequencing runs and as a consequence of long-term storage; and that after long term storage, freeze-dried fecal samples could still be used for transplantation treatment of C. difficile infections.

However, there has not been much research on the effects of preserving an oral bacterial community and even less on oral bacterial communities generated in vitro. The importance of studying the effects of preserving an in vitro oral bacterial community is two-fold: First, to examine the effects of preservation on an in vitro oral bacterial community; and second, to investigate the feasibility of preserving a universal inoculum for seeding in vitro oral microbiome cultures, wherein the inoculum is extracted from as simple of a model as possible. A universal oral microbiome inoculum would help bring a great degree of control into the generation of in vitro oral microbial models, potentially in oral disease research where a healthy baseline oral community needs to be established.As mentioned above, what would benefit the investigation of oral microbiome members and their roles in health and disease – specifically, the correlations and relationships of the organisms, their effects on one another during development in both healthy and disease, and the effects of various conditions and agents on the community – is the ability to reliably study reproducible versions of this community of organisms. This ability necessitates the development of a complex, stable, and generalizable in vitro model community.

While the construction of lab models of the bacterial oral microbiota has received a fair amount of attention, and some such models have produced communities that resemble the human oral microbiome at least in membership if not in relative abundances, most such models still require specialized incubators and fastidious surfaces and exhibit somewhat poor repeatability across both technical replicates and biological replicates. There is clear a need for a model of the human oral bacterial microbiome that is relatively simple to cultivate, cost-effective, easy to maintain for longer growth, and reproducible to the extent allowable by statistical margins of error from technical replicates. To this end, we intended for our work to contribute to the foundation for this model, by constructing and characterizing an in vitro culture that encompasses the initial stages of the oral community development. We began by focusing on the dental plaque community, as it requires the least fastidious surface and can help refine the methodological details needed for more complex in vitro oral models. The goals of this project were sequential in nature. First, we investigated the feasibility of generating an oral bacterial community in vitro with the resources available to our particular group. Then, we examined the temporal behavior of the community to see how closely it adhered to the time evolution of other models and to the development of the human oral community in vivo.

Thirdly, we determined whether the community would be fitting to serve as the basis for the preservation experiment. Lastly, we developed an approach that would help elucidate the apparent correlations and covariances discovered among the microorganisms during the preservation experiment. It was our hope that at its conclusion, the project would have provided us with not only data on how a minimally complex multi-organism in vitro oral microbiome performs under basic culturing conditions, but also data on how such a community responds to preservation and subsequent propagation. This data would help determine the degree of representation and generalizability of our model, and help create a “baseline” inoculum for in vitro oral bacterial microbiome models.To investigate the feasibility of the project and establish foundational procedures, we performed preliminary experiments to cultivate a complex in vitro supragingival dental microbiome. The community requires plaque from healthy hosts to serve as inoculum; the model is based on previously published research on in vitro dental communities, specifically, communities generated in 24-well culture plates without the use of additional substrata, such as removable hydroxyapatite disks that partially mimic dental enamel. We chose this basis for the model for its versatility in the initial stages of community formation, facile operation, and low cost. The goal for this phase of the project was to test whether the on-site facilities and conditions would allow us to generate a community that reasonably resembles the initial colonization stages of the host oral community. Should the community exhibit reasonable resemblance, it could then serve as the basis upon which to build subsequent parts of the model. In addition to investigating the feasibility and quality of the procedure to generate a simplified community, we also aimed to assemble and test reliable tools for this project, including DNA extraction for sequencing, potential quantitative determination of different types of cells by spike-ins, and bio-informatics analysis. While DNA extraction in this phase of the project was ultimately performed by the sequencing center at UC Davis , we decided to examine the extraction efficiency of two different commercial kits that were most commonly used in microbiome research at the time, with the goal of bringing this part of the process into the lab in later phases of the project. In terms of quantitative determination of the number of cells, we used E. coli as “doping” or “spiking-in” cells. We chose an E. coli strain that was easy to grow and manipulate, such that we could roughly specify the number of live cells – approximated by colony-forming units – for spiking the dental plaque cultures after incubation. It was our goal to use the known number of E. coli cells to estimate the number of sequencing reads, and then to use the number of E. coli reads, the number of reads from other OTUs, and 16S copy numbers for all OTUs from sequencing to estimate the number or concentration of cells of dental plaque bacteria in the model communities. To achieve these goals, we needed a solid bio-informatics foundation using either the QIIME or mothur pipeline, planting blueberries in pots so we also examined the results from both pipelines to determine which one better suited our purposes.A single healthy volunteer host was used for dental plaque sample collection during this phase of the project.

The host was a 29-year-old female in good health with no systemic diseases or family history of systemic diseases. The host routinely practiced good dental hygiene and had no periodontal diseases, but did have two fillings on two back molar teeth. Sample collection took place under Protocols 3-18-0189 and 3-19-0119, approved by the UCSB Human Subjects Committee. Sample collection took place as follows: Supragingival plaque of molar teeth, cheek side, was obtained using a sterilized Gracey curette; on average, plaque was collected from 5 molar teeth. Prior to plaque collection, hosts had abstained from food, non-water liquids, and dental hygiene for 12 hours. Collected plaque was immediately suspended in sterile SHI medium, gently mixed, and divided equally among wells in a sterile 24-well plate such that each well received 1.98mL of the mixture. Prior to receiving sterile or inoculated medium, wells were preconditioned with clarified human saliva , which was supplied as frozen pooled fractions from healthy human volunteers and clarified in the lab. Clarification consisted of centrifuging defrosted saliva at 6,000 x g for 3 minutes, mixing with 1X PBS in a 1:1 ratio, and passing the mixture through 0.2µm filters. Conditioning was performed by adding 150µL clarified saliva to each well, allowing saliva to air dry without the plate lid at 37°C for 60 minutes, and sterilizing with short-wave UV light for 60 minutes.After sterile and inoculated medium had been dispensed into the wells, each well received 20µL of 0.5% sucrose prior to being covered with the well-plate lid. Controls and cultures were incubated in triplicates or quadruplicates to test for repeatability. Lidded well plates were place in a closed chamber , and the chamber was flushed three times with an anaerobic gas mixture consisting of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide, and then filled to positive pressure with the same gas mixture. During flushing and filling, gas from the cylinder was passed to the chamber through a 0.2µm filter to minimize potential microbial contamination. The chamber was then placed in an incubator set at 37°C and allowed to incubate for 20 to 24 hours. Some cultures were also incubated for four days to test the efficacy of commercial DNA extraction kits. The approximately one-day cultures were not fed again during incubation; each well of the four-day cultures was fed with 20µL of 0.5% sucrose every 24 hours before harvesting, with reestablishment of the anaerobic atmosphere after each feeding. After incubation, we observed for bacterial cell presence by looking for sedimented cells in the wells. From the wells with clear presence of cells , we harvested the wells by first aspirating 1.0mL of the liquid above the sedimented cultures into sterile microfuge tubes, and then mixing and pipetting the rest of the well into separate sterile microfuge tubes. For wells with no substantial cell presence , we gently and thoroughly mixed the wells and aspirated 1.0mL of the well contents into sterile microfuge tubes. Three samples from each type – controls, liquid above, and sedimented culture – were kept free of E. coli cells. We will henceforth refer to the E. coli cells as spike-ins and refer to the mixing with E. coli cells as “spiking”. Other samples received spike-ins at specified volumetric ratios, detailed in the E. coli Standard Curve and Spike-In subsection below. For controls and liquids with no spike-ins, the aforementioned 1.0mL aliquot in microfuge tubes was flash-frozen in liquid nitrogen and kept at -80°C until processing at the UC Davis Host Microbe Systems Biology Core . For sedimented cultures with no spike-ins, the 1.0mL of the culture was pelleted by centrifugation at 13,000 x g, the supernatant discarded, and the pellet flash-frozen and kept at -80°C until processing at the HMSB. Constitution and processing of spiked samples are described in the subsection titled E.coli Standard Curve and Spike-In.To observe the layers of sedimented cells, we reserved wells for microscopy without disturbing the layers, by carefully aspirating the liquid above and adding a commercial stain, which consisted of 200µL of a SYTO 9 and propidium iodide mixture from the FilmTracer LIVE/DEAD Biofilm Viability Kit . Dyes were allowed to penetrate the cells at room temperature for 30 minutes. To determine whether the surface of the bottoms of the wells affected the apparent viability of the cultured cells, we incubated cells in two different types of culture plates: standard tissue-culture-treated and surface-modified . Standard tissue-culture-treated plates have a net-negative surface charge while surface modified plates contain a mixture of positive and negative surface charges. Two plates of each type were used for this part of the preliminary experiments. Controls and cultures in both types of plates received identical treatment in terms of the volume of media, concentration of nutrients, and duration of incubation time. Results were visualized by staining and fluorescence microscopy procedures as described above.To determine the membership and abundances of the bacteria in these preliminary cultures using HTS, we attempted to develop an internal cell-counting standard using the E. coli K12 ER2738 strain . To do so, we first constructed a standard curve of colony-forming units vs. optical density at 600nm for ER2738 cells in the growth phase. For this curve, we grew overnight cultures of ER2738 and measured their optical density.

We maintain the assumption of two applications of grass herbicides, although we increase the treated acreage to 80 and 60 percent with one application composed of 40 percent molinate and 40 percent thiobencarb and the other composed of propanil on 60 percent of the acreage. Broadleaf control was adjusted to one application of triclopyr on 45 percent of the total rice acreage. Material and application costs of the herbicides are updated as well using information provided by UCCE . Finally, all cash operations are assumed to be financed at a nominal interest rate of 10.51 percent in accordance with the UCCE sample-costs document . As such, any change in the cost structure directly affects interest on operating capital, though the magnitude tends to be small. Overall, these updates result in a per-acre cost increase of $17.69 over the 2001 cost study. To more accurately represent the current world rice market , we assume the market price per cwt at harvest is the average price from 1986 through 2002 of $6.50 with average yields at 80 cwt per planted acre.

Government payments are divided into two components: direct payments and counter cyclical income-support payments as described by USDA, ERS . In accordance with the 2002 Farm Bill, square pots direct payments are calculated at 85 percent of average yields at $2.35 per cwt. Williams et al. estimate that growers of approximately 95 percent of planted acres have received this payment in the past, so the total direct payments are multipliedby 0.95. Counter cyclical income-support payments are calculated using the ERS formula, which we can summarize as 85 percent of average yields at $1.65 per cwt. Incorporation of these changes results in a $28.01 increase in gross revenue per acre over the 2001 UCCE sample-costs study. The original and adjusted costs and returns per acre are reported in Table 2. Given the public nature of experimental data on LibertyLink® rice grown in California and the full cooperation of Bayer CropScience through phone interviews and email correspondence, we use this transgenic variety as the basis for our analysis . We assume a price for Liberty® herbicide of $60 per gallon16 and an application rate of 0.446 pounds of AI per acre [500 grams AI per hectare ] in accordance with the company’s projected label recommendations .

To fully represent the fact that weed infestations will differ across plots, scenarios for transgenic cultivation are presented for both one and two applications of the herbicide on 100 percent of the acreage.The latter result is a direct consequence of the cost differential between ground and air applications of herbicides; ground applications of glufosinate cost approximately $12 per acre while air applications range from $6 to $7.25 per acre . The savings in chemical costs, however, drive the overall cost savings associated with transgenic rice and are explained using the information provided in Table 3. While the price of glufosinate per pound of AI is greater than all of the chemicals under consideration with the exception of triclopyr, the application rate per pound of AI is only 6 to 13 percent of the average herbicide control system. This decreases the cost of herbicide materials per acre by almost 62 percent as shown in the last column Table 2. When these results are combined, net returns over operating costs increase in the range of $45.89 to $74.90 per acre depending on the herbicide application rate, or $0.57 to $0.94 per cwt. Thus, this baseline scenario, which assumes perfect substitutability between medium-grain transgenic LibertyLink® rice and conventional varieties in terms of market acceptance and yields, predicts considerable economic incentives for rice growers to adopt transgenic varieties with similar characteristics due to their increased profitability. It is important to recognize, however, that these results are based on average costs over the entire Sacramento Valley ricegrowing region and utilize aggregate data to estimate the conventional herbicide weed-management regime.

Individual growers, of course, will most likely differ in regime from these averages depending on the characteristics of the specific operation. Those growers with “superior” land, as defined by lower aggregate weed-management costs, would benefit the least from adoption of transgenic rice while those with marginal land or serious weed-resistance problems tend to benefit more from the herbicide-management cost savings offered by the transgenic system and are hence most likely to adopt. To further investigate these issues, the assumption of perfect chemical substitutability, which essentially drives the assumption of identical yields, can be relaxed. A severely infested plot with a large, resistant seed bank of watergrass or some other weed would likely experience yield increases with adoption of a transgenic control system. Such yield gains have been observed in practice for HT soybeans and HT canola in the range of 0 to 20 percent . However, yields are not necessarily guaranteed to increase for all plots. Under generally ideal conditions, a yield drag of between 5 and 10 percent for medium-grain cultivars of LibertyLink® rice relative to conventional varieties has been observed in California rice field trials. This is consistent with similar field trials of HT soybeans. Such losses would decrease revenues and would thus reduce the increased profitability of adoption of this new technology. Yield drag should not be an issue with most growers given the advanced, widespread state of weed resistance to currently licensed chemicals for rice weed control in the Sacramento Valley. However, it is important to note that, in the short run, a few producers could actually experience a slight yield drag if the new technology was adopted; this is not expected to persist in the long run. A fall in demand for California rice due to consumer concerns, coupled with increased supply as a result of productivity gains, could cause rice prices to decline over time and decreasing net returns in the presence of yield changes. Similarly, a price premium for non-transgenic rice varieties could erode net-returns differences between traditional and HT cultivars but benefit conventional rice producers. Decreased yields or prices for transgenic rice, ceteris paribus, would reduce the gross rents from the technology. Furthermore, the seller of the transgenic seed is likely to charge a premium of up to 60 percent of total per-acre seed costs, depending on the pricing structure of the technology. Roundup Ready® and Bt seed for commercially produced transgenic crops has historically been priced from 30 to 60 percent higher than non-transgenic varieties, and price premia for LibertyLink® corn seed range from 0 to 30 percent, although average chemical costs per acre are typically greater . Furthermore, growers will likely pay at least part of the burden of the fees assessed by the CRCA. Assuming that these effects are constant per cwt of output, they can all be represented as a unit increase in costs in terms of net returns. Increased unit costs of this form, ceteris paribus, would alter the distribution of the rents between stakeholders but not dissipate gross rents. As points of reference, base assumptions on price and yields are $6.50 per cwt and 80 cwt per acre, large plastic pots so gross revenues from sales of rice output are assumed to be $520. A price premium of $0.25 per cwt for conventional rice as compared to transgenic rice with no associated change in yields would thus have the equivalent effect on net returns to the grower of a fee of about $20 per acre. Note that changing output prices does not affect the cost structure of the average farm operation and, thus, there is a direct, linear relationship between net returns and price. To calculate the impact of these effects, simple subtraction of the product of the price change and yield from the baseline scenario is appropriate. On the other hand, both a technology fee and the CRCA assessments directly enter the cost structure and, as such, affect interest costs as well. Tables 4 and 5 lay out these effects.

A 30 to 60 percent technology fee, assuming a seeding rate of 1.5 cwt per acre and price of conventional seed of $14 per cwt, is equivalent to $6.30 to $12.60 per acre. Total fees assessed as a result of the CRCA would currently be $8.50 per acre at identical seeding rates and yields of 80 cwt per acre, although it is unlikely that 100 percent of these assessments would be passed to the grower. Table 4 assumes no pass-through to growers of the legislated fees while Table 5 assumes the maximum pass-through, thus bounding the estimates. Both conservatively assume two applications of glufosinate per growing season. Without the CRCA legislation, adoption of LibertyLink® rice is profitable for a technology fee of $6.30 regardless of any realistic yield assumptions and profitable at a technology fee of $12.60 per acre so long as yield drag is no greater than 8.9 percent . With zero yield gains, net returns per acre in this range of seed price premium increase by between 21 and 25 percent over conventional rice returns with even greater benefits for those experiencing positive yield gains. If we assume a small price premium of, say, $0.25 per cwt, the technology is profitable for either yield losses of 7 percent with no technology fee or no yield change with an unrealistic $25.89 technology fee. This highlights the importance of yield and price assumptions on the calculation of net benefits. However, it is clear that, even with a small output price premium and a seed price premium at the upper end of the observed range, the most likely adopters will benefit from increased returns over costs. Allocation of maximum CRCA assessments to the grower slightly changes the per-acre benefits but does not affect the qualitative conclusions . Net returns over the baseline scenario with a $6.30 technology fee are no longer positive with an 8.6 percent yield drag nor for a $12.60 technology fee and a 6.7 percent yield drag. However, identical yields still result in net benefits of between $24.50 and $30.80 per acre, more than enough to cover a $0.25 price premium for conventional rice. To bound the per-acre benefits, we assume a lower bound of $0.25 per cwt price premium and an upper bound of no price premium with no CRCA pass-through. Under these assumptions, we conclude that the per-acre benefits of the transgenic HT technology are between –$7.22 and $58.10 for any given California rice grower with a midpoint estimate of $21.90. However, if we restrict attention to those producers most likely to adopt, as defined by at least zero difference in net returns, yield drag at the lower end of the range can be as high as 1.2 percent and they will still adopt.The preceding deterministic sensitivity analysis accounts for heterogeneity in land, weed infestation, and management ability as well as for the distribution of the rents generated by the technology. However, the magnitude of these rents is determined primarily through assumptions regarding yield and the price of rice as well as base assumptions on the price of alternative herbicide systems. While these point estimates are based on the best information available, another approach is to parameterize the distributions of those variables, which can be perceived as stochastic, and use Monte Carlo simulations to estimate the distribution of the surplus benefits of the transgenic rice technology. We take the specification in the equation and estimate distributions for a transgenic yield premium, the transgenic-rice price, and a conventional-rice price premium. Yields for the HT cultivar are assumed to vary according to a symmetric triangular distribution centered around 80 cwt per acre with a minimum value of 72 cwt and a maximum value of 88 cwt . This distribution allows for the possibility of yield gains and losses and, with symmetry, tends to be very conservative given the state of weed infestation and resistance across the state. Prices for California rice are essentially determined on the world market and thus are not influenced by the individual producer. Using historical data from USDA for 1986 through 2002, we assume a lognormal distribution for output price with a mean of $6.50 per cwt and a standard deviation of 1.67. Finally, the price premium for conventional rice is assumed to be distributed as a skewed triangular with a most-likely value of $0.25 , a minimum value of zero, and a maximum value of $0.52 or about 8 percent. These values are consistent with experience with corn, soybeans, and canola cited previously .

The system is divided into two parts. The first part is focused on storage and transmission of the EEG data for two different events: when the subject’s eyes are open and when eyes are closed. From researching brain waves, it was discovered that brain activity would be different between the two events. After careful study and testing, the conclusion was drawn that when the eyes are open, the Alpha and Beta wave features which are generated from the raw EEG signal are high. When the eyes are closed, Alpha and Beta features were low. However, these features were not enough to give reliable and appropriate results for the classification of these two events. Therefore, we tried to use different features, adding more features to the system to increase the reliability of the system. After working with one feature and two features, we extended the system to work with four different brain wavefeatures in the range of 0.5 to 30 with a sampling rate of 512. These features are Delta, Theta, Alpha and Beta.An Edge class is a powerful WuClass that has internal data storage and time series data processing capability. Like every WuClass, an edge class should be assigned a unique WuClass ID, which should be refered to in WuKong Standard Library.xml in master. In the EEGEdge Class, square pots the ID is explicitly defined by the WuClass annotation, so that the object instance of EEGEdgeClass can be discovered by the master.

According to the property declaration of WuClass in WuKong Standard Library.xml, a developer needs to define inputs and outputs for the edge class, and use the annotations for theedge framework to help developers to declare what kind of data structure they want to use to temporarily store upcoming time series data. The input property is declared as a buffer , whose data ring capacity is 2000 data points, index ring capacity is 30 units, and index is built every 1000 miliseconds. Therefore, the buffer will hold data in a time window of 30 seconds; it will at most keep 2000 data points. The buffer will store raw signal from EEG Device from which time series operators will fetch data and generate features. For every output property, an Edge class needs to define the corresponding set function. So, that a written WKPF property message will be generated and sent out when its value is updated. In the example, output is a property of output, whose property is 1. After defining properties as inputs and ouputs of the intelligent component, we implement two important methods of Edge Class. The register extension function will return a list of extensions, each of which is a handler of a specific stage in the data processing pipeline. In the EEG Edge Class, we registered EEG Feature Extractin Extension and EEG Execution Extension, both of which will be introduced in detail later. Beside providing data processing pipeline, the edge server also provides the communication pub/sub capability, so that an offline model can be initialized and updated through a pub/sub topic. model is loaded locally, so that there is no topic needed.Since models are offline trained in the EEG study, we ignore the online learning extension, and only focus on how to use models to do online classfication on the features extracted. Here, the EEG Execution Extension implements both Executable Number and Initiable.

Within the init function, we firstly load the model from local file system. The execute function accepts features in a list as the first parameters, and execution context as second parameter. Within the function, we use the model to classify whether we should label the features as eyes close or eyes open. Once we can know the probability of both eye close and eye open, we set 0.88 as the probability threshold to really trigger eyes close action by setting the output value. We tested the application on real physical devices.The importance of emotions lies in every-day-human-to-human communication and interaction. Understanding and recognizing human emotional status play an important role in human communication. Human-computer interface can play the same role of the human being to understand and recognize human emotions, and adjust its setting to fit with their human emotions. There are different approaches detecting and recognizing human emotions. First, facial expression is one of the earliest techniques used to detect human emotions and voice recognition based on the voice tone can detect emotions. However, these techniques are susceptible to deception, and vary from one situation to another. Second, Physiological signals are also used to detect emotion such as electrocardiogram and respiration. This approach provides more complex information than what is needed to detect emotion. Third, brain wave signal is used to detect emotion such as electroencephalogram , Electrocorticography and Functional magnetic resonance imaging . The advantages of using brain wave are its strong relevance for emotions and that it is not proneto deception.There is a spectrum of emotions which can differ from one person to another. There are different models presented by the research community to model human emotions. One model presents the basic emotions as happiness and sadness, another model presents the basic emotions as fear, anger, depression and satisfaction. Another model uses multiple dimensions or scales for emotion categorization. In this model emotion characterize by two main dimensions, valence and arousal.

Valence emotions range from positive to negative whereas arousal emotions range from low to high. For instance, fear can be defined as a negative valence and high arousal, whereas excitement can be defined as a positive valence, and high arousal. Figure shows the dimensional model of emotions.There are different type of stimuli to trigger emotion: self-eliciting, recalling, and using external stimulus. In order to stimulate the subject’s emotion in this thesis, I used excerpt videos from Liris-accede library. LIRIS-ACCEDE library has around 9800 good quality video excerpts used to induce different types of human emotions. This thesis used the library videos in the first trial and in the second trial. The third trial used a different resource to trigger emotions through videos that has marketing background. Only two emotions captured in the third trial which are High valence-High arousal, Low-valence and Low arousal. Figure shows some images from these different video excerpts.The aim of this project is to investigate the ability of a low-quality cheap commercial EEG Headset to classify different mental tasks. The EEG signal in this thesis was acquired using a single electrode placed on the forehead. The EEG signal was sent to the computer via Bluetooth. In order to integrate the EEG headset with the Wukong framework, large plastic pots the EEG Server Wuclass has been designed and built for the first two applications. Also, in order to build edge classification, the EEG edge WuClass has been designed and built for the third application.In the first two applications, this thesis intends to integrate the EEG headset with the Internet of Things framework ”WuKong” to allow the user to control external applications, such as turning lights on/off, playing music, etc. Two applications is built one application aimed to assist old people to do different tasks and the second application aimed to read a mind in an office. In the Old People Assistant Application, the system was built using two sensors. The first sensor is the camera, which captures eye blinking using the Haar algorithm from OpenCV library to classify and detect the eye blinking. The second sensor is the EEG headset which capture eSense signals, defined as attention and meditation signals and their values ranging from 0 to 100. Different patterns are built based on the attention or meditation signal values. In order to change from one state to another, or to keep on a certain state, the number of eye blinks has to be detected by the system. The result are shown in Chapter 3 for switching between 2 LEDs, but they can be extended to play different types of music and switch different LEDs, as shown in Figure 3.2. The second application is designed to show the mind state of a person inside in an office. In this application two WuClass have been built,the first is the EEG server WuClass which receives EEG signals, builds different patterns, and generates different index number for different pattern. The second Wulclass is the LED strips WuClass which receives the index number and plays the patterns that corresponds to the index number. In this application both eSense signals send to two EEG server in WuKong,then the WuKong framework has the ability to receive two different signals, attention and meditation.The third application is called classification eye states on the edge. In order to do this, the first step is to capture the raw EEG signal from EEG headset which done using a python code. The second step to build this application was the preprocessing steps that are applied to the collected EEG signals, including Fast Fourier Transform, removing EMG and EOG artifacts, and segment the EEG to one second segments.

The third step was to extract the EEG features, namely the power spectrum density features. In this application three different subjects volunteer to give their EEG signal, which acquired, preprocessed, and extracted the power spectrum density features. After collecting the EEG data we applied different machine learning algorithms in order to classify between two different eye states. We Used the accuracy of the classifier as our evaluation measurement. We reach 90% accuracy to classify between two different eye states using Support Vector Machine with Radial Basis Function kernel as our classifier. In order to use this classifier online in real time, we used the progression WuKong framework which supports intelligent edge. We built an EEG Edge class which has two components: first, feature extraction extension that uses relative intensive ration operator to calculate the intensive ration of EEG features; second, execution extension that loads the model which was generated offline, and based on the on the probability of the eye events, the threshold was chosen to be 0.88 to trigger an action when the eyes are closed.The emotions application presented in Chapter 4. The emotions application was implemented in order to classify between two different types of emotion. Using video clip from LIRIS-ACCEDE library which is designed to trigger these different emotion. Six subjects participated in this experiment which lasted for 60 seconds for high valence, high arousal and another 60 seconds for low valence, low arousal. Different types of features are extracted from the collected EEG signals, included time,frequency and nonlinear features, and 24 of these features are selected in order to use them with machine learning algorithms. Different machine algorithms are applied on the collected data, and the average accuracy using Linear discernment analysis was 79.31%. The model was evaluated using confusion matrix, which showed the diagonal is darker than the others, and those implies that the classifier is a good classifier . The second method used to evaluate the model was f1-score, which show an average of 83% for the subject number 4. The last method used to evaluate the model was Receiver Operating Characteristics which show the area under the curve with 91% for subject number 4. The last method used to evaluate the model was the learning curve in which the learning curve showed a misclassification range from 12% to 17% for the subject number 4.This thesis studies and discusses the ability of low-cost headset EEG to classify different eye events, and different mantel tasks such as attention, meditation and emotions. Table 5.1 compares different research literature that applied eyes events classification, using different EEG headset ranging from 4 – 14 electrodes with a cost of $799. Different features and classifiers are used to classify eye events. The accuracy ranges from 73% to 95%. The thesis presents a different approach to classify eyes events using low cost EEG headset that does not exceed $100. The power spectrum density features extracted , and Support Vector Machine with Radial basis function is used for classification. In this thesis, an accuracy of 90% is obtained to classify between eyes open and eyes close. With 90% accuracy of this work using only one electrode, and only cost of $99 is a better solution to detect the different eye states compared to other studies shown in Table 5.1.Table 5.2 shows data from research literature that discusses emotions classification using different EEG headsets.

A new central, upright shoot is selected to continue the leader, and the lateral branches that develop will create the second tier. This pattern is repeated until the desired tree height is reached. Alternatively, lateral branches can simply be vertically and radially separated up the trunk. To prevent lateral branches from competing with the leader, bend branches outward to 45 to 60 degrees from vertical, if needed, using branch spreaders or string tie-downs. Each branch must be offset somewhat from the branch immediately below it to prevent shading. Lower branches are allowed to grow longer than those higher in the tree to encourage sunlight penetration and keep lower fruit wood alive. Tree height is usually limited to about 8 feet for dwarf trees and 10 to 12 feet for semidwarf or standard trees. Persimmon flowers grow on new shoots that originate toward the ends of 1-year-old branches; thus, early training should include heading these branches in winter to prevent structural branches from bending excessively due to the fruit weight.This method is an option for walnuts , persimmons, pistachios, and pomegranates. After the central leader is formed and five to seven lateral branches are formed radially around the trunk with at least 6 to 12 inches of vertical spacing from each other, gallon nursery pot the leader is headed back or simply allowed to develop a few competing leaders. However, the lower canopy should still remain wider than the upper canopy to allow light to reach the lower branches.

Annual pruning of walnuts involves strategically thinning branches to allow sunlight through the canopy but not creating openings large enough to result in sunburn damage to large, newly exposed limbs.Fruit bushes have become very popular due to their ease of management and the accessibility of their fruit . Fruit bush trees are developed on standard or dwarfing rootstock and are kept small by heading during the growing season. To create a fruit bush, after planting and heading the tree, shoot length is reduced by half when shoots reach about 2 to 3 feet. When the subsequent shoots reach the same length, they too are similarly shortened. Usually two such prunings are sufficient in the first year , but more vigorous trees may require such pruning three times. Since heading cuts cause several new shoots to grow, some should be thinned to prevent crowding. The process is continued in the second year, or until the permanent tree height is reached— usually 5 to 7 feet. Pruning mature trees consists of cutting off new shoots above the established height two to three times during each growing season and thinning branches and shoots to allow sunlight penetration . It may be beneficial to keep the center of the tree open to increase light in the lower canopy.Training trees on a sturdy trellis can provide a fruiting wall in a narrow space, either freestanding or against a fence or house . Apples, pears, Asian pears, and citrus are especially well adapted to espalier training.

Many ways exist to train the branches, but typically three to four horizontal wires are spaced about 11⁄2to 2 feet apart vertically, and the lateral shoots are tied along the wire running in either direction. Alternatively, lateral shoots could initially be directed upward and outward to create a fan shape, or upright branches could be created from main lateral limbs. Unwanted vigorous upright shoots should be cut back during the growing season to reduce shading and to encourage spurs along lateral branches. Lateral growth from the branches should be kept short to prevent shading of lower branches. If shoot growth is excessive, provide more space by extending the trellis outward or upward. Do not allow branches to grow through small holes in a fence or they will become girdled or break the fence.Arborists are often asked to prune large, overgrown fruit trees. After first determining the client’s goals for a tree, the arborist can then develop a pruning approach or, preferably, a long-term maintenance plan. The health of the tree should be evaluated because large, older trees have often been neglected or poorly pruned. Some may be in decline and there may be extensive branch dieback, bleeding or gumming, wood rot, root rot, or branch cankers. Sunburned or diseased branches may also secondarily be infested with shot hole borer or Pacific flat head borer. The presence of such conditions and injuries could, depending on severity, make restorative pruning pointless. Also, determine if structural limbs are present low enough to warrant lowering the tree height to an acceptable level for picking fruit. Evaluate the amount of shade cast by nearby ornamental trees, which may have grown tall and now excessively shade the fruit tree, especially if the height is to be reduced.

Therefore, a tree that is currently 22 feet tall but is desired to be 10 feet tall is cut to about 18 feet in the first year, then cut to 14 feet in the second year, and then cut to the desired height in the third year. To avoid branch diseases, prune during early spring when dry weather is predicted for an extended period. This method assumes that there are three or four healthy and stable lateral limbs well below 10 feet, which is not always the case . Cut limbs back to strong lateral branches where possible, although some heading cuts will likely be necessary. These cuts will stimulate vigorous shoot growth, so follow up by heavily thinning the new shoots in summer to allow sunlight to reach branches below the permanent height. If restorative pruning exposes limbs below the permanent height to several hours of late afternoon sun, paint the exposed surfaces of limbs on the north and east sides of the tree with a 50-50 mixture of interior white latex paint and water to prevent sunburn. In the first year, strongly head back lateral branches low in the tree to generate new growth that can be developed into permanent low branches or fruiting wood , and thin lower branches as needed. A more radical approach is to reduce the height in just 1 year . The advantage with this method is that the tree can be reformed and brought back into production very quickly. However, this may be considered a temporary solution, since heading of fairly large branches may be necessary—and the cuts, depending on size, may result in wood rot. It is preferable to cut to lower laterals that are at least one-third the diameter of the stem. Decay is also more likely to develop if the resultant shoot growth is excessively thinned or not allowed to develop near the wound site. Sufficient leaf surface area of branches originating just below the large cuts is needed to produce callus growth to cover and close the wound. However, leaf area will likely be insufficient anyway if the tree is kept small. Another disadvantage is that shoot growth will be extremely vigorous and will need to be managed until fruit production is reestablished. The best timing for making the large cuts is March or April, growing blueberries after most winter rainfall and when shoot growth is beginning. Do not apply wound dressings to pruning wounds.Development in the search for materials with topological electronic properties has rapidly progressed in the past decade. With a refined understanding of the role symmetries have on the electron wave functions Berry curvature, the experimental study of new and exotic quantum phenomena has now become widely accessible. A well-recognized example is the breaking of time-reversal symmetry in magnetic materials, which may result in producing Berry curvature fields that generate an intrinsic anomalous Hall response. Along similar lines, the breaking of inversion symmetry in non-centrosymmetric materials is understood to be essential for fostering new quantum phenomena, such as non-local gyrotropic effects, quantum nonlinear Hall effects, photogalvanic effects, accidental two-fold band degeneracies that are protected by a quantized non-zero integer Chern number, and anomalous transport. In this study, we observe that magnetic non-centrosymmetric PrAlGe hosts the emergent topological properties of Weyl fermions by photoemission-based spectroscopy and magnetotransport. In contrast to previous works on magnetic Weyl semimetal candidates Mn3Sn and Co3Sn2S2, PrAlGe is calculated to exhibit Weyl fermions in proximity to the Fermi level, making it more suitable for experimentally probing its Berry curvature properties and exploring the connection between photoemission-based band structure and transport.

In addition, because PrAlGe lacks both inversion and time-reversal symmetry it can uniquely induce quantum spin currents without a concomitant charge current. Motivating future studies on PrAlGe, we experimentally resolve the key topological properties of Weyl fermions by relying predominately on our measurements.PrAlGe crystallizes in a body-centered tetragonal Bravais lattice with space group I41md . The basis consists of two Pr, two Al and two Ge atoms, Fig. 1a inset. Along the direction, each atomic layer is comprised of one element, and the layer is shifted relative to the one below by half a lattice constant in either the x or y direction. Single crystal X-ray diffraction suggests that our samples possess the correct lattice structure and lack inversion symmetry . Measurements of magnetic susceptibility as a function of temperature were fitted to the inverse Curie-Weiss law. The obtained positive Weiss constant indicates the presence of ferromagnetic interactions, Fig. 1a. A direct measurement, to be discussed below, shows that PrAlGe is ferromagnetic with Curie temperature TC = 16 K. The ferromagnetic ground state arises from the spin-polarized f-electron states that are locally coupled and aligned along the c axis, rendering the conduction electron bands near the Fermi level spin-polarized. This is reflected in our ab initio band structure calculations without spinorbit coupling , in which it also shows that PrAlGe has a semi-metallic profile . The inclusion of SOC interactions couples the spin-up and spin-down states and slightly perturbs the electronic bands . The absence of inversion and time-reversal symmetry both contribute to band-splitting at generic crystal momenta. Kramers degeneracy splitting is linked to magnetism in the crystal.Table grapes that meet minimum maturity standards, including sugar and acid content, and the ratio of sugar to acid, are harvested by hand and typically marketed as entire or partial clusters. The quality and value of the grapes are strongly affected by the size, texture, and color of the individual berries, and the overall appearance of the cluster . These quality attributes are commonly achieved, in part, through the use various plant growth regulators , agrochemicals with plant hormones or hormone-like compounds as active ingredients . For example, gibberellic acid is used to thin and size berries, forchlorfenuron is used to increase berry size, and firmness , and ethephon and abscisic acid may also be used to improve the color of red grapes .Though the vast majority of table grapes are sold as entire or partial clusters, there is growing interest in marketing stemless fresh-cut grapes . However, destemming may damage grape berries, stimulating decay, and diminishing quality . Mechanical damage associated with destemming might be minimized through the use of abscission agents, PGRs which reduce fruit detachment force and promote the development of a dry stem scar, an abscission layer between the berry and pedicel . Research on the potential use of abscission agents as mechanical harvest aids for wine or raisin grapes have shown that 1-aminocyclopropane-1-carboxylic acid , coronatine, ethephon, and methyl jasmonate stimulate abscission of mature grape berries . Of those, ethephon is the only compound registered for use on grapes, though the registrations are for improving the color of red and black fruited grapes, or hastening grape maturity, both at considerably lower use rates than what is required to stimulate berry abscission. Ethephon is an ethylene-releasing molecule. Stable in a low pH solution, it hydrolyses in the higher pH of plant tissues releasing ethylene, a gaseous plant growth regulator . Ethephon’s chemical characteristics enable growers to apply it to grapes and other plants in the field with commercial spray equipment, and thereby stimulate ethylenedependent reactions. Ethephon absorption by plant tissues is influenced by temperature, relative humidity, and pH of the surface on which the spray droplets are deposited . Hedberg and Goodwin suggested that ethephon absorption by plant tissues is predominantly cuticular rather than stomatal and Nir and Lavee found that the thickness and composition of cuticle layers play an important role in penetration. How the molecule diffuses within the plant is not yet well understood. Studies conducted with the 2- chloroethylphosphoric acid marked with the 14C showed limited and mainly basipetal mobility .

Changes in expression at the onset of ripening occurred earlier in ED than in controls. Previous work in our laboratory also demonstrated that ED advanced the onset of ripening, apparent from an earlier onset of color development and corresponding induction of flavonoid genes, and accelerated accumulation of anthocyanins and sugars after the onset of ripening . Other studies examining the effects of water deficit have not found a temporal advancement in the onset of ripening, but did find that water deficits accelerated anthocyanin accumulation after the onset of ripening . That some studies have not found a temporal advancement in the onset of ripening may reflect differences in the nature of the applied water defi- cits. ED fruit in the current study was subject to water deficit for more than a month prior to the onset of ripening, while fruit in Castellarin et al. and Deluc et al. was subject to water deficit for only 1–2 weeks prior to the onset of ripening. Alternatively, drainage pot the weekly sampling interval used in Castellarin et al. and Deluc et al. may have missed the acceleration as the onset of ripening occurs over *7 days.

Regardless, these studies show that differences in fruit composition brought about by water deficit are established in a very short period of time surrounding the onset of ripening whether through advancement in the onset of ripening, accelerated accumulation, or both. At this time, there is no direct evidence demonstrating that changes in ripening brought about by water deficit are indeed mediated by ABA. However, several studies demonstrate a strong correlation between the timing of the onset of ripening and ABA. In this study, berry culture treatments demonstrate that ABA, in the presence of high sugar, can bring about many of the changes associated with onset of ripening in grape. Wheeler et al. brought about an advancement in the onset of ripening through the repeated application of ABA to immature field-grown Cabernet Sauvignon, and Deluc et al. demonstrated that the acceleration in ripening in response to water deficit was correlated with increases in ABA. Without more sophisticated genetic studies, the evidence for ABA’s role in mediating the effects of water deficit on fruit development remains corollary.Several lines of evidence suggest that changes in berries cultured with exogenous sucrose and ABA parallel changes in field-grown fruit at the onset of ripening. Softening, color development, and changes in gene expression associated with onset of ripening in were brought about by culture on high sucrose ? ABA, but not with low or high sucrose treatments alone. The 2 and 10% sucrose concentrations used in this study approximate sugar concentrations of berries prior to and at , the onset of ripening in the field . ABA concentrations in the culture media match or exceed peak ABA concentrations at the onset of ripening in the field .

The induction of color development in the high sucrose ? ABA treatments was not surprising given the strong evidence for sugar and ABA induction of anthocyanin biosynthesis both in model systems and in grape cell culture . Cultured berries grew and increased Brix at a rate similar to field-grown berries for the range of Brix represented by 2–10% sucrose. Similarly, the elasticity of both field-grown and cultured berries decreased from approximately 6 to 1 MPa or less from 61 to 84 DAA . The parallels in patterns of gene expression are striking. Those genes strongly up-regulated at the onset of ripening in control and ED field-grown fruit were similarly regulated between high sucrose, and high sucrose ? ABA treatments . Other genes, such as VvSUT2-1 were up-regulated at the onset of ripening in the field, while in culture they were up-regulated in response to sugar only . Furthermore, other genes constitutively expressed during ripening in field experiments exhibit the same pattern of expression in culture . Koyama et al. also demonstrated that, in culture, exogenous ABA brings about changes in gene expression analogous to the onset in ripening in the field. Decrease in elasticity in response to exogenous ABA suggests that ABA treatment resulted in decreases in mesocarp cell turgor, although turgor was not measured directly in this study. The decrease in elasticity in culture is attributable to a decrease in the DWs between the symplast and apoplast , and/or a decrease in cell wall rigidity through loosening of the cell wall, and not to desiccation because the berries grew during culture. In fruit, there is evidence that ABA plays a role in both of these processes through the stimulation of various invertases and cell wall-modifying enzymes . The large weight gain, swelling of the mesocarp tissue, and berry cracking in low sucrose ? ABA treatments, supports a role for ABA in stimulating cell wall loosening. This could be an interesting focus of future study. In the introduction, we discussed the idea that components of regulatory networks can be conserved across disparate developmental processes that share commonalities.

Sugar and ABA signals act across diverse processes . Assuming that sugar and ABA signals work to control similar physiological processes in these different contexts what common physiology underlies their action? One strong candidate is the control of turgor, which is intimately connected to germination , stomatal control, the onset of ripening , and the control of growth.Exposure of susceptible plant tissues to non-freezing temperatures below 10–12 ◦C induces a physiological disorder called Chilling Injury . There appears to be two phases in the development of CI. The first phase is initiated in the cold and could involve a change in membrane fluidity or enzyme activity . Overt symptoms develop after prolonged chilling or upon warming to non-chilling temperatures . These secondary symptoms are predicated by primordial events initiated in the cold, and include a host of metabolic and physiological changes that include increased membrane permeability , increased respiration and ethylene production , uneven ripening, disease susceptibility, water soaking and surface pitting . A technique that could detect the earliest physiological changes associated with CI would foster a better understanding of the initial events leading to this disorder, and point to more effective ameliorative action. Magnetic resonance imaging is a nondestructive imaging technique, which is increasingly used to visualize and quantify fruit physiological response to endogenous or exogenous stimuli . MRI uses the magnetic properties of nuclei and their interactions with radio frequency and applied magnetic fields to produce an image . Variations in the chemical composition and integrity of cellular structures can change the movement of water within and among tissues. These changes can be detected as modifications in the relaxation times of the protons in water, drainage planter pot which in turn alters the signals used to construct MR images . Diffusion-weighted MRI of tissues provides a quantitative measure of the apparent diffusion coefficient of water, instead of estimations of water mobility from relaxation measurement that include the influence of translational mobility, composition and other factors . In addition, a spatially resolved map of the apparent diffusion coefficient of water can be obtained, which could help to understand and quantify the development of disorders such as CI within the tissue. MRI has been used to gain insight into early phases of different post harvest physiological disorders before the manifestation of external symptoms . These include core breakdown in pear water core disorder , internal browning and mealiness in apple . There are few reports where MRI was used to detect the early stages of CI in sensitive produce. In persimmon, MR images of cold-stored fruit were distinct from those stored at ambient temperature . In zucchini squash , MRI provided enough data to act as a predictor of where water soaking would occur in the epidermis after the cold-storage.

These studies both indicated that MRI has great potential for studying CI in fruit tissues. Tomato is one of the most important horticultural crops both economically and as a genomics, molecular, biochemical, and physiological model for biological processes occurring in fleshy fruits . Like most subtropical fruit, tomato is susceptible to CI. Studies with tomato fruit could leverage existing functional genomics resources to pinpoint the molecular basis of this trait. To our knowledge, MRI has not been used to study CI in this species. We used the dwarf cultivar ‘Micro-Tom’ because it is the functional genomics model for tomato . Its high-density growth, short life cycle and concentrated fruit-set makes it possible to obtain harvests of 500 fruit or more per square meter per year . Because tomato post harvest studies can be hampered by biological variability , the availability of numerous, similarly aged fruit makes Micro-Tom a convenient experimental model for post harvest studies . Furthermore, we have previously characterized Micro-Tom fruit physiological response to a range of post harvest chilling temperature-time combinations , and used this information to design a metabolomics investigation of CI. This has established a baseline with this cultivar for the further CI studies we exploit here. The specific objective of this study was to determine if MRI could detect some of the earliest physiological changes that accompany CI in tomato fruit. Current methods of assessing the occurrence and severity of CI are: time consuming , destructive , or occur only after the activation of secondary, downstream events . These methods are time-proven and are indispensable, but there is a need for non-destructive methods with equivalent or better sensitivity to those currently used. MRI potentially offers such advantages and could be an important complementary tool for studying incipient CI. We show that MRI can provide spatio-temporal resolution of chilling induced changes in MicroTom tomato fruit prior to development of downstream symptoms.Chilled fruit were removed from 0 ◦C and allowed to slowly warm to 20 ◦C before CO2 and ethylene production were measured. Twelve fruit were evaluated per treatment by placing four fruit in each of three 500 mL glass containers. These jars were sealed for 1 h and a 1-mL sample of the head space was withdrawn using a syringe and its CO2 concentration was measured with an infrared gas analyzer as previously described . Ethylene production was measured from a 2.5 mL sample of the head-space using a Gas Chromatograph equipped with a flame ionization detector. These two samples were taken within 30 s of each other from the same jar.Each fruit was cut into four radial segments, cleaned of adhering locular tissue, washed for 5 s in running tap water, blotted dry, and one segment was placed in each sector of a 4-sectored Petri dish under aseptic conditions. The dishes were placed in plastic tubs lined with wet paper towels and loosely covered with aluminum foil. The tubs were held at 12.5 ◦C for 18 h to produce ‘aged’ tissue, i.e., to allow the tissue to overcome the wound-induced alterations in membrane permeability . After transferring to room temperature for 1 h, the four aged segments from each Petri dish were put into a 50 mL plastic centrifuge tube containing 20 mL of an aqueous solution of 0.2 M mannitol. Preliminary experiments determined that 0.2 M was isotonic for these excised radial segments The conductivity of the bathing solution was measured with an Extech Model 480 digital conductivity meter every 5 min for 30 min and then less frequently for 180 min with gently shaken between readings. After 3 h the tubes were capped, frozen at −20 ◦C and warmed to room temperature and frozen and thawed twice before the total conductivity of the solution was measured at room temperature after 1 h of shaking. Ion leakage was expressed as percent of total and plotted over time. The linear increase in ion leakage from 0.5 to 2.0 h was used to calculate the rate of ion leakage .The aim of this study was to examine the extent to which MRI could detect changes in chilled Micro-Tom tomato fruit. Based on our previous data using Micro-Tom , fruit were stored at 0 ◦C in the dark for 0 , 1, or 2 weeks, and the fruit that were chilled for 2 weeks were held for 1 week at 20 ◦C. The onset of CI was evaluated using changes in respiration, ethylene evolution and ion leakage . One week of chilling increased all ofthese indicators to levels that were maintained during an additional week of cold-storage . In fruit transferred to 20 ◦C after 2 weeks of chilling, the rate of respiration returned to that of the pre-chilling levels, ion leakage remained at the same elevated levels and ethylene production continued to increase.

Trigger the second transport lock-in off of the first one. Attach the output of one of the lock-ins to the 1/10 voltage divider, then to a contact of the sample. You can attach the voltage contacts somewhere if you want to, this is not particularly important though. It may be necessary to a apply a voltage to the gates, especially if you are working with semiconducting materials, like the transition metal dichalcogenides.Increase the voltage until you see 1 µA of current. In my experience, members of the nanoSQUID team tend to be a little too timid about applying large currents to these samples because they are very susceptible to damage through electrostatic discharge, and of course it feels pretty bad to damage a device somebody else made for you. Although it’s true that researchers doing transport measurements almost never use currents as high as 1 µA, I can tell you that we have never damaged a heterostructure with high current at all, large pot with drainage and certainly not at 1 µA. It is generally pretty safe to go as high as 100 µA, and we have gone considerably higher than that too.

Currents greater than 1 µA will saturate the lock-in input, but you can still increase the voltage if you need to . Alternatively you can use the Ithacos adjustable transimpedance amplifier as the sink. If you do so, be careful not to adjust any of the knobs on this device while it is hooked up to the heterostructure, because adjusting those knobs can produce pulses of current large enough to damage devices. While you’re increasing the voltage, keep an eye on the SQUID signal. Increase the time constant if it helps you see the signal . Once you see a signal on the nanoSQUID channel of the Zurich, set up a scan. Check that the auxiliary outputs from the Zurich are going to the right ADC inputs, the right ADC inputs are correctly labelled in the scan window, the right auxiliary outputs are set up in the Zurich ‘Aux’ window and are sampling the right channels, and the right channels are set up and activated in the Zurich window. You should definitely see a thermal gradient if the signal is 3-5x the noise floor. If you don’t, I’d recommend investing some time into making sure the measurement is set up correctlyyou don’t want to just keep increasing current through the sample in response to not seeing features on a scan that isn’t set up right! If you get really frustrated and want a sanity check, click “Set Position” to each of the corners of the scan range and watch the signal on the Zurich control panelit should change if everything is working. There are a lot of issues that can affect scanning, and it isn’t really possible to cover all of them in this document, so you will have to rely on accumulated experience.

Some problems will become obvious if you just sit and think about them- for example, if the thermal gradient is precisely along the x-axis and coarse positioner navigation is failing to find a strong local maximum it likely means that the y-axis scanner is disconnected or damaged. In Andrea’s lab, the basic circuits on the 1.5K and 300 mK systems as currently set up should be pretty close to working, so if there’s a problem I’d recommend observing the relevant circuits and thinking about the situation for at least a few minutes before making big changes. The scanners as currently installed on the 1.5K system do not constitute a healthy right-handed coordinate system, so to navigate you will need a lookup table translating scanner axes into coarse positioner axes. I think this issue is resolved on the 300 mK system, but this is the kind of thing that can get scrambled by upgrades and repair campaigns. In all of our note taking Powerpoints and EndNotes, we have a little blue matrix that relates the scan axes to the coarse positioner axes. Use this to determine and write down the direction you need to move in the coarse positioner axes in your notes. You now have an initial direction in which you can start travelling. We will next perform long distance thermal navigation, at a height of 150 µm above the surface. Retract 150 µm using axis 3 of the coarse positioners. I’d recommend doing this in one or two big steps, because the coarse positioner can slide in response to small excursions. Verify that you can still see the thermal signal on the SQUID. It is Ok if it’s faint or close to the noise floor; it will increase in size, and you know which directions to start travelling. If the resistive encoders are working , then use them to move in 100 um steps, checking the SQUID signal in between movements. There is no need to ground the SQUID in between coarse positioner steps, there will be crosstalk but this is not hazardous for the nanoSQUID. If the resistive encoders are not working, click the Step+ button repeatedly until the SQUID signal increases to a maximum.

This might take a few minutes or so of clicking. You can work on a software solution instead if you like , but remember that there is always a simple, safe solution available! Once the signal is at a maximum, take another scan to verify that you’re centered above the device. You should see a local maximum in the temperature in the middle of your scan region. Ground the SQUID. Ramp the current through the device down to zero. Zero and ground any gates you have applied voltages to. Ground the sample. Make sure the SQUID is grounded to the breakout box by a BNC . Hook up the second little red turbo pump to the sample chamber through a plastic clamp and o-ring, and turn it on. Slowly, over 10-20 minutes, drainage collection pot open the valve to the sample chamber and pump it out. Make sure the sand buckets for vibration isolation are set up and the bellows aren’t touching the ground. If there are vibration issues you can often feel them on the bellows and on the table with your hand. Repeat the setup for approaching to contact, and approach to contact. Definitely watch the first few rounds of this approach! You can even watch the whole thing- it’ll take 30-45 mintues, but if you’ve messed something up then the approach will destroy both the SQUID and the device, because you’ve carefully aligned the SQUID with the device! Once you’ve reached the surface, you will set up the SQUID circuit. Attach the preamplifier to one of the SMA connectors at the top of the insert. Attach its output to the input of the feedback box. This output goes through the ground breaker that is clamped to the table in Andrea’s lab; all of these analog electronic circuits are susceptible to noise and ringing, so I’m sure there will be different idiosyncracies in other laboratories with other electromagnetic environments. Attach the output of the feedback box to the BNC labelled FEEDBACK . This is the BNC that should get a resistor in series if you wanted to increase the transfer function. We generally use resistors between 1 kΩ and 10 kΩ for this. To start with, just using nothing is fine . Plug the preamp and feedback box into fresh batteries . Turn the preamp on. Turn the feedback OFF. Hook up the SQUID bias wires to SQUID A and SQUID B. You can tell which they are because of the chunky low pass filters on the end, but of course they are also labelled. Make sure both sides of the SQUID are grounded while hooking it up- there is a BNC T there for a grounding cap for this purpose. Hook up Output 2 of the Zurich to signal input on the feedback box. Apply 1 V to signal input. There’s a good chance you just used this same output and cable to apply avoltage to the device, so be careful not to skip this step and apply this voltage to the device itself! You should see the SQUID array transfer function on the oscilloscope . Turn the rheostat/potentiometer on the preamp until this pattern has maximum amplitude. Turn the Offset rheostat/potentiometer on the feedback box until this passes through zero . There is a more sophisticated procedure for minimizing noise in the SQUID array; this is covered in great detail by documents Martin Huber has provided to the lab. But if you are a beginner this simple procedure will work fine. Flip the On switch on the feedback box, and watch the interference pattern vanish, replaced by a line near V = 0. Turn off the AC voltage going to signal input. You are now ready to characterize the SQUID, although you’ll need to unground it. That includes removing the BNC grounding caps from the T’s downstream of the SQUID bias filters and also flipping the BNC switch on the top of the rack. Click ‘preliminary sweep’ on the nSOT characterizer window. Sweep from 0 to 0.1. If you see a linear slope, a ton of stuff is working!

The SQUID bias circuit, the SQUID array, the feedback electronics, all the cryogenics- that’s a really good sign. If you see no signal, don’t panic. Once again, there’s a lot of stuff involved in this circuit and a ton of mistakes you can make. Go back through the list and check everything, then check to make sure the SQUID bias isn’t grounded somewhere. Increase the sweep range until you see a critical current or you get above 3.3 V, which is where the feedback box will fail. If you don’t see a critical current, you have a SHOVET but not a SQUID. If you see a critical current, close the window, switch to the nSOT characterizer, and characterize the SQUID. At this point, you are at the surface and over the device with a working SQUID, and you can begin your imaging campaign, so what comes next is up to you!Anthocyanins constitute a large family of plant polyphenols and are responsible for many of the fruit and floral colors observed in nature. Anthocyanins are water-soluble pigments located in the grape skin vacuoles that, during the fermentation process, are released into the wine. It has been demonstrated that determining the amount of pigments present in the berries is not enough to estimate the concentration of anthocyanin in the final product. This lack of correlation is mainly attributed to the interaction between the pigments and the skin cell walls during the extraction process. Additionally, the adsorption of phenolics to solids in the fermentor after being released, such as grape skins and yeast hulls, has previously been demonstrated. Previous studies have shown that the interaction between polyphenols and skin cell walls is dependent on the composition of the latter suggesting that specific cell wall constituents show different adsorption capacities for polyphenols. In the case of anthocyanins, the cellulose content and the degree of methylation of the pectin have shown positive correlations with the adsorption capacity. Moreover, some studies suggest that other components of the cell walls, such as proteins, can occupy binding sites resulting in overall lower anthocyanin adsorption.Another factor that greatly influences the extraction of phenolics during wine fermentation has been shown to be the temperature at which the fermentation is performed. Previous research has shown that elevated fermentation temperatures produce finished wines that are more highly colored and have greater concentrations of pigmented polymers. The increase in extracted phenolics at elevated temperatures has been accredited to two temperature related effects: an increased permeability of the hypodermal cells of the grape skins and an increase in the solubility of phenolics at higher temperature. It has also been shown that changes on the temperature can impact the physical structure of the cell wall material . It has been postulated that, at high temperatures, the cellulose structure opens up, potentially creating new sites and a faster exchange between the molecules. Additionally, an increase in temperature can disrupt hydrogen bonds between the cell wall and the phenolics increasing its concentration in solution. A second fermentation factor that is also likely to have a significant effect is the production of ethanol during fermentation.

The DEGs obtained from the two pipelines were assigned to both M21 and M30, yielding 604 and 1362 genes, respectively . Interestingly, the number of DEGs in each module, M21 and M30, was roughly equal to the down- and upregulated genes, respectively . To identify flavonoids/tannins-related genes that might result in such astringency diversity between V7-berries and V9-berries, hub genes were searched in the DEGs list of both modules . Only 8 hub genes were identified based on their transcript abundances in V9- berries and predicted functions. However, based on our previous work , we found another 11 genes that are significantly expressed but with a log2FoldChange less than 1.5, and they were included in our further analysis . The enrichment analysis of GO showed considerable enrichment in the BP GO terms for secondary metabolite bio-synthetic process , flavonoid bio-synthetic/metabolic process , L-phenylalanine metabolic/ catabolic process , phenylpropanoid metabolic process , phenylpropanoid biosynthetic process , chorismate biosynthetic/metabolic process , cinnamic acid biosynthetic/ metabolic process , anthocyanin containing compound bio-synthetic/metabolic process . The KEGG pathway analysis confirmed the BP GO terms, exhibiting enrichment for the biosynthesis of secondary metabolites , phenylpropanoid biosynthesis , flavonoid biosynthesis , and glutathione metabolism .imilarly, the upstream structural genes in the phenylpropanoids pathway, drainage collection pot including phenylalanine ammonia lyase , trans-4- coumarate biosynthesis , and 4-coumaroyl:CoAligase 2 , were significantly induced by approximately 2- to 9-fold in V9-berries.

Regarding flavonoids/PAs biosynthesis, chalcone synthase is considered a key enzyme in this pathway, converting p-coumaroyl-CoA to naringenin chalcone, which is later turned into naringenin by chalcone isomerase . Both genes were highly expressed in V9-berries . Naringenin is subsequently converted by flavonoid 3’-monooxygenase to dihydromyricetin and dihydroquercetin, which are further transformed by dihydroflavonol 4-reductase into leucodelphinidin and leucocyanidin, respectively . The expression levels of F3H and DFR also showed a commensurate induction with the upstream genes in V9-berries relative to V7-berries. Subsequently, leucoanthocyanidin dioxygenase and leucoanthocyanidin reductase catalyse the conversion of leucodelphinidin to delphinidin and -gallocatechin, respectively, as well as leucocyanidin to cyanidin and catechin, respectively. These three genes also exhibited a significant increase in V9-berries. Finally, the expression of genes encoding glutathione Stransferases , one of the most essential anthocyanin transporters, was significantly higher in V9-berries compared to V7-berries, with approximately 3- to 9.2-fold changes . Our data showed that the expression of flavonoids/PAs related-genes was highly increased in V9-berries at the third harvest time compared to V7- berries, resulting in the accumulation of more PAs in V9-berries.Developing and producing table grapes with high quality is of utmost importance for the success of grapevine breeding programs.

Scarlet Royal table grape , variety , is one such success story, producing premium fruit quality and becoming one of the major red varieties in California. However, under certain unknown circumstances, the berry quality of Scarlet Royal grapes can be affected by undesirable astringent taste, which can negatively impact marketability and consumer acceptance. Research on the relationship between astringency and phenolic composition in table grapes is still scarce, especially on the transcriptomic level. In this study, we aimed to understand the molecular events involved in the development of berry astringency, which is a complex set of sensations resulting from the shrinking, drying, drawing, or puckering of the mouth epithelium . We focused on Scarlet Royal berries from two different vineyards with contrasting astringency and analyzed the changes in phenolic-related compounds at six different time points from veraison until the last harvesting time. Our panel test revealed that the V9-berries were perceived as more astringent, a characteristic that could be attributed to their elevated levels of tannins . Differences observed between the two vineyards under study indicate that V7 vines yield more compared to V9. This difference may be attributed to the lower cluster count in V9, a factor known to potentially contribute to astringency, as suggested by Cañon et al., 2014 in wine grapes. Additionally, petiole analysis revealed higher levels of nitrogen and potassium in V9 vines compared to V7. These factors may also contribute to the higher levels of tannins detected in V9 berries; however, further research is needed to confirm this theory. It’s worth noting that weather conditions can play a role in inducing astringency. Nevertheless, the two vineyards are located in close proximity to each other, and weather data collected from the same station in the Delano area indicates similar conditions. Therefore, it is unlikely that astringency or higher phenolic compounds are induced by weather factors. In fact, several studies, mainly in wine, have pointed to PAs as a determining factor for astringency intensity .

For example, Vidal et al. reported that the total amount of tannins is the most plausible factor for sensory astringency, with flavan-3-ols dimers, trimers, and non-galloylated tetramers contributing to the astringency sensation. The PAs are a group of oligomers and polymers of flavan-3-ols and are the naturally occurring and predominant type of tannins in grapes and wine . Another study on aronia berry juice confirmed PAs as the key astringent compounds using sensory evaluation and phenolic profile approaches along with in-vitro models . The study found that PAs with higher degrees of polymerization were responsible for the strong astringent mouthfeel . The composition of phenolic substances, especially PAs, seems to play a crucial role in determining berry astringency, and further exploration of this relationship is warranted in fresh fruits of different species and cultivars. Understanding the molecular basis of astringency development in Scarlet Royal berries can provide valuable insights for improving grape breeding programs and enhancing the overall quality of table grapes.The relationship between astringency and the berry polyphenols content has not been explored yet in table grape at the molecular level. To the best of our knowledge, the present study provides the first transcriptome profiling along with the changes of polyphenols in grape berries of the same variety but having astringency diversity . The transcriptome profile of both V7-berries and V9-berries underlined the remarkable transcriptional shift during berry ripening at different vineyards . Commonly, berry transcriptome profiles may widely vary based on many factors, including genotypic variations among varieties/species and developmental stages , as well as environmental circumstances. In our case of study, stemmed differences from the developmental stages and genotypic variations were eliminated, andhence the difference of the vineyard locations was the main source of variance with 97% of variance . The identified DEGs output of V9-berries compared to V7-berries is highly explanatory, including polyphenolic-related genes that are robustly expressed and co-regulated with astringency development, particularly in the V9-berries. The enrichment of the up-regulated genes with BP GO terms related to the biosynthesis of secondary metabolites, phenylpropanoid, and nitrogen compound metabolic process , commensurate with the higher amounts of N found in V9- berries . In fact, not only the levels of N fertilization but also its different forms highly affected the composition of phenolic compounds in leaves and wine . However, the synergistic/antagonistic effects of other macro- and micro-nutrients should also be considered. Our results highlighted the negative impact of above-normal amounts of macronutrients, mainly N, and to a lesser extent P and K, on the desirable attributes of grape berries. Otherwise, the positive effect of Ca, Mg, and Mn were achieved as their levels were maintained within the normal range . These data should be also seen in the light of the highly enriched BP GO terms in the downregulated genes . Particularly, those for hormonal signaling pathways such as auxin, abscisic acid, strigolactones, 10 liter pot as well as the KEGG pathway for the carotenoid biosynthetic pathway .The transcriptome profiling identified the common and unique molecular events featuring the development of tannins/astringency in grape berries. It is well-documented that the synthesis of PAs in grapevines is achieved via three sequential pathways: the shikimate pathway, the phenylpropanoid pathway, and ultimately the flavonoid pathway . Our results revealed that the expression levels of flavonoids/PAs-related genes were highly induced in V9- berries at the third harvesting time compared to V7-berries. The 19 selected genes were involved in the three pathways: the shikimate pathway, phenylpropanoids pathway, and flavonoids pathway. The shikimate pathway is an alternative route to produce aromatic compounds, including phenylalanine, tyrosine, and tryptophan, which serve as precursors for various metabolites, such as phenolic compounds .

The up-regulation of genes like chorismate synthase and chorismate mutase in V9-berries may lead to the accumulation of phenylalanine, which is a critical precursor for the phenylpropanoid pathway. The latter pathway is responsible for synthesizing several end products, including PAs, anthocyanins, lignin, lignans, hydroxycinnamic acid esters, and hydroxycinnamic acid amides . Under the conditions of the V9 vineyard, several PAs/flavonoids structural genes such as PAL, C4H, 4CL, CHS, CHI, F3H, LDOX, LAR, and ANR were induced in V9-berries, leading to the accumulation of PAs in the berries . This process is facilitated by GSTs and transported by multidrug and toxic compound extrusion transporters. The activation of the PAs biosynthetic pathway in V9- berries may lead to a reduction in the necessary substrates for anthocyanin synthesis, resulting in low red color intensity in V9- berries compared to V7-berries. Additionally, the accumulation of PAs is associated with the development of astringency taste in V9-berries. Our study provides valuable insights into the molecular events underlying astringency development in Scarlet Royal berries. By integrating transcriptome profiling with polyphenolic composition analysis, the research shed light on the co-regulation of genes involved in the shikimate, phenylpropanoid, and flavonoid pathways, leading to the synthesis of PAs and ultimately influencing astringency. The findings from this research have implications for grapevine breeding programs and the production of high-quality table grapes. Understanding the molecular mechanisms underlying astringency development can help breeders in selecting and developing grape varieties with desirable attributes. Additionally, the knowledge gained from this study can inform vineyard management practices, such as nutrient fertilization, to optimize polyphenolic composition and berry quality. In conclusion, the data presented in this study indicates that berry astringency is strongly correlated with a high tannin content, likely resulting from the activation of nineteen genes within the phenylpropanoid pathway. The activation of these genes shifts the flavonoid biosynthesis pathway towards proanthocyanins, leading to increased tannin accumulation in the berries. It appears that triggering these events is associated with nutritional imbalances and a lower number of clusters per vine, as confirmed by petiole nutrient levels and the observed lower berry soluble solids and higher titratable acidity levels. The identification of these genes holds significant value for table grape genetic improvement programs. The nutrient imbalance theory derived from this research could be applied worldwide to optimize grapevine fertilization programs. Furthermore, it paves the way for further research in this area, with a particular focus on vine nutrients, crop load management, and berry astringency, thereby contributing to advancements in the field of table grape cultivation.It is important to remember that these quantum states are just as real as Bloch states, and apart from the short list of differences discussed above, they can be analyzed and understood using many of the same tools. For example, in a metallic system, the Fermi level can be raised by exposing a crystal to a large population of free electrons and using an electrostatic gate to draw electrons into the crystal. This process populates previously empty Bloch states with electrons. These Bloch states have a fixed set of allowed momenta associated with their energies, and experiments that probe the momenta of electrons in a crystal will subsequently detect the presence of electrons in newly populated momentum eigenstates. Similarly, attaching a Chern magnet to a reservoir of electronsand using an electrostatic gate to draw electrons into the magnet will populate additional chiral edge states. Properties that depend on the number of electrons occupying these special quantum states will change accordingly. In all of these systems, conductivity strongly depends on the number of quantum states available at the Fermi level. For metallic systems, the number of Bloch states available at any particular energy depends on details of the band structure. The total conductance between any two points within the crystal depends on the relative positions of the two points and the geometry of the crystal. Thus conductivity is an intrinsic property of a metal, but conductance is an extrinsic property of a metal, and both are challenging to compute precisely from first principles. When the Fermi level is in the gap of a Chern magnet, there exists a number of quantum states at the Fermi level exactly equal to the Chern number.

Harvest, a labor-intensive practice, clearly represents the lion’s share of total costs, at 58% in organic production, 60% in conventional production and 67% in second year conventional berries. Cultural costs represent 26% of total costs in the conventional and organic systems, but only 15% for second year strawberries because there were no associated planting costs, and because pest management costs were lower . Looking more closely at pest management, soil fumigation is the highest cost category for conventional production at $3,302 per acre, with weed control, another labor-intensive practice, the highest cost in second year and organic strawberries at $1,212 and $2,506 per acre, respectively . However, for organic strawberries the cost to control insects ran a close second at $2,488 per acre, which was dominated by control for lygus bug with a bug vacuum, and two-spotted spider mite with the release of predatory mites. By comparison, container vertical farming estimated costs for insect control in conventional strawberries were lower at $702 per acre and still lower at $579 in second year conventional berries.

Raspberry and blackberry production were not routinely studied in years prior to 2003. Since then, several primocane-bearing raspberry and floricane-bearing blackberry cost and return analyses have been performed, with the most recent studies conducted in 2012 and 2013, respectively. Both studies detail establishment and first year production and harvest costs for not-yet-fully-mature crops. For raspberries, first year of production includes a $12,460 per acre construction, management and investment cost for protective tunnels. Costs for a mature raspberry crop are analyzed in the second production year and total $48,210 per acre . For blackberries, costs for a mature crop are shown for the second through fifth production years, and total $43,406 per acre per year. Harvest costs again represent the vast majority of total costs, at 81% and 71% of total costs for raspberries and blackberries, respectively. For raspberries, cultural costs represented a much smaller share of total costs at $4,656 per acre, roughly half of which was for trellis and tunnel management. Blackberry cultural costs totaled $5,709 per acre, of which over half was for pruning and training canes.Each study also includes an analysis of potential net returns to growers above operating, cash and total costs for a range of yields and prices. When evaluating net returns above total costs, gains are shown for higher yield and price points; losses are also documented at many lower yields and prices .

Farms with productive soils, experienced managers, optimal production conditions and robust market plans generally realize higher net returns. In contrast, farms with less-than-optimal production conditions, reduced yields, poor fruit quality or inexperienced managers may contribute to lower net returns. Results from the strawberry analyses show that on a per acre basis, organic strawberries tend to be more profitable than conventional berries, even with lower yields. Organic price premiums explain the result; in this example price per tray for organic strawberries ranged from $12 to $18, while price per tray for conventional berries ranged from $7.30 to $11.30. Prices for second year conventional strawberries were slightly lower still to account for a portion of the crop that was diverted to the freezer market. Net returns for both caneberries were mostly positive. Other noteworthy entries in all recent berry studies include per acre costs for pest control advisers , management of invasive pests and food safety and regulatory programs for water and air quality. Though each alone represents a relatively small portion of total costs, they provide readers with insights into the changing nature of berry production activities and costs over time.Cultural practices in the berry industry have evolved to address changes in soil, water and pest management needs. Based on historical trends, and to meet both industry needs and consumer demands, we expect to see new varieties continually developed over time. Businesses have responded to consumer and market demands for fresh, safe and organic products by implementing food safety programs and/or transitioning more lands to organic production. Water and air quality programs have been developed to comply with state regulatory requirements. In the past, growers customarily hired those with expertise in financial and market management; they now also enlist the support of experts in food safety, organic agriculture and environmental quality to assist with farm management. But challenges remain, and management of key agricultural risks — including those for production, finances, marketing, legal and human resources — have become increasingly important. Invasive pests pose significant management and regulatory constraints and increase production, financial and market risks. Two recent examples are light brown apple moth and spotted wing drosophila .

LBAM infestations can lead to loss of part or all of the crop because of field closure from regulatory actions, increasing production and financial risk. SWD presents substantial market risk to growers in that its larvae can infest fruit and render the crop unsaleable. Growers minimize the risk of loss from these two organisms with the routine use of PCAs. PCAs monitor fields more frequently than growers alone would be able to do, identify pests and recommend actions, for example, the use of pheromone mating disruption for LBAM and field sanitation for SWD. Since their introduction, the soil fumigants CP and MB have unquestionably contributed to the expansion of the berry industry. However, the full phaseout of MB as a pest management tool — it will no longer be available for use in berry production after 2016 — presents both production and financial risks. While a substantial research commitment has been made to finding alternatives to MB, nothing has yet come close to offering the same level of protection from the large-scale loss to soil pathogens or the gains in productivity associated with the application of CP and MB as synergistic preplant fumigants. We anticipate that the berry industry will adapt to the MB phaseout by using alternative fumigants and preplant soil treatments, but these are likely to carry a higher level of risk for berry production in the short term and may lead to a decrease in planted acreage and production. However, this may also stimulate an even more robust research agenda directed towards soil borne diseases and plant health to minimize disruption to the industry. Reliance on fumigants as the primary strategy for pest management is almost certainly a thing of the past. Instead, adoption of integrated approaches, including alternatives to fumigants, to manage diseases, weeds and other pests will be key to sustaining berry production over the longer term . Labor is also a current and significant challenge for growers of berry crops. Social and demographic changes in Mexico — the source of a majority of the area’s agricultural labor — have resulted in markedly lower immigration rates into the United States, a shrinking labor pool and upward competition and wage pressures for the agricultural workers who remain . In recent years, hydroponic vertical garden growers have reported difficulty in securing and retaining sufficient numbers of workers to ensure timely and effective farm operations. The lower production figures seen in strawberries in 2014 may in part have been the result of an insufficient labor pool from which to draw . However, no known regional employment or wage data are available to specifically document this. Some growers minimize labor risk by paying higher wages and providing year-round employment when possible. However, these strategies can be difficult for some businesses to justify economically. Arguably, the area’s berry industry, and agriculture more generally, increasingly face political risk. Immigration legislation that may assist with the current labor challenge languishes at the federal level, with major policy changes unlikely before 2017 . Farming practices are under ever more scrutiny by consumers, local municipalities and state and federal agencies. Soil fumigants and pesticide use have been the focus of many intense debates and discussions, especially in Santa Cruz and Monterey counties. At the time of this writing, several new regulations related to pesticide application notifications, pesticide and fumigant application buffer zones and worker safety have been proposed by the California Department of Pesticide Regulation or the U.S. Environmental Protection Agency but have not yet been finalized. It is anticipated that implementation will begin in 2017, with full compliance required in 2018. And, as California struggles through a fifth year of drought, water use, quality and cost has become a more robust part of the local, state and federal discourse, with directives issued and new legislation proposed. Compliance with each new directive or regulation presents production and logistical challenges for growers and can be costly to manage.

Although it is unlikely that regulatory pressures will lessen in the future, there is every expectation that growers will continue to adjust business practices to meet or exceed any new requirements or standards. The economic sustainability of individual farming operations and the area’s berry industry in total will ultimately be impacted by and continue to evolve with the ever changing business environment, and by an array of risks and challenges.Angle-resolved photo emission spectroscopy is used to directly measure the band structure of solids and is an essential experimental tool for solid state physics research. In addition to the band structure, ARPES provides information on other aspects of the electronic structure. For example, ARPES with a spin detector can be used to obtain spin information of the initial states . Polarization dependent experiments can provide symmetry information on the initial states; initial states from, for example, px and py orbitals can show dramatically different ARPES intensities depending on the polarization of the incident light. In recent years, there has been much interest in using circular dichroism in ARPES as a way to measure some aspects of initial states, such as the orbital angular momentum or the Berry curvature. It is well understood that OAM plays an important role in spin-split phenomena in systems without inversion symmetry, such as surfaces of solids and monolayer transition metal dichalcogenides. CD-ARPES has been utilized to obtain the crucial information on the electronic structures of such systems. While the final state of the photo emission process certainly has an effect on the CD-ARPES intensities, experimental results show that CD-ARPES is a rough measure of the OAM of the initial state if the photon energy is not too low. Exploiting this feature in CD-ARPES measurements, information on the OAM and hidden Berry curvature of 2H-WSe2 was recently obtained using CD-ARPES. An important aspect of this research was that the Berry curvature contribution to the CD-ARPES intensity could be isolated by decomposing the CD-ARPES intensity map into symmetric and antisymmetric components about the experimental mirror plane, which is perpendicular with respect to the crystal mirror plane of 2H-WSe2. Te symmetric component was attributed to the OAM or Berry curvature contribution, since the electronic structure should be symmetric about the chosen experimental mirror plane set along K–Ŵ–K′ in momentum space.Experimental geometry, including single crystal orientation, is especially important in this experiment. The crystal structure of the top atomic layer or ML of 2H-WSe2 is a hexagonal lattice, as shown in Fig. 1a; there is a unique mirror plane in the crystal structure, as indicated in the figure. Te experimental mirror plane is defined by the plane defined by the normal of the sample surface and the direction of incident light. The experimental mirror plane was set to be the same as the crystal mirror plane. Two experimental geometries are possible, according to the direction of incident light, as indicated by blue and red arrows in Fig. 1a. The experimental geometries using incident light described by blue and red arrows are regarded as geometry-A and geometry-B for convention, respectively. Notably, the signals from the top layer of bulk 2H-WSe2 dominate the CD-ARPES data due to the surface sensitivity of ARPES; the corresponding momentum space view is shown in Fig. 1b. The mirror plane is oriented along the M–Ŵ–M direction, and the direction of incident light is indicated by blue and red arrows on the mirror plane in Fig. 1b. This experimental geometry differs from that used in previous work, in which the experimental mirror plane was rotated by 30° with respect to the crystal mirror plane, such that the experimental mirror plane is oriented along the K–Ŵ–K′ direction. We expanded on our previous CD-ARPES work on 2H-WSe2 by focusing on a different mirror plane. Here, we report our CD-ARPES studies on 2H-WSe2 with the experimental mirror plane parallel to the crystal mirror plane or along the M–Ŵ–M direction in momentum space .

To confirm Koch’s postulates, the standard criteria to determine the agent causing a disease , we reisolated fungi from stem tissue at least 2-cm above the point of inoculation in harvested plants, amplified using primer pairs ITS1F/ITS4 for the ITS and EF1-728F/986R for alpha-elongation factor-1 . They were sequenced using the protocol described by Schultheis et al.. Daily temperatures were maintained between 18–30°C during the day and 10–15°C at night. Humidity maintained at 50%. Photosynthetically active radiation lights were set to provide 14 h of daylight per day and a maximum of 2000 µmol. Plant positions were randomized weekly using a random number sequence generator to eliminate any microclimate effects in the greenhouse.Physiological stress due to drought and pathogen infection was inferred from weekly measurements of net photosynthesis and dark-adapted chlorophyll fluorescence using a LI-COR 6400XT and Hansatech FMS2 system fluorometer , respectively. Leaves were dark-adapted using leaf clips for 20–30 min before measuring fluorescence. One healthy , fully expanded leaf per plant, round plastic pots or on one healthy and one stressed/diseased leaf per plant if symptom onset had begun . All data were collected between 10:00 hours and 16:00 hours to capture peak values for the day, with the majority of measurements taken between 10:00 hours and 12:00 hours.

Due to mechanical issues, chlorophyll fluorescence was not measured on 4 and 11 November and on 20 December 2016. Net photosynthesis and dark-adapted fluorescence were chosen as proxies for plant health, as lower values correlate strongly with higher levels of drought stress .Soil moisture, plant structure, physiological data, and disease severity were statistically compared using ANOVA in JMP, version 14 Pro , and post hoc analyses of means were performed using Wilcoxon signed rank test. Two-way factorial ANOVAs were conducted on the influence of watering regime , inoculation treatment , and interaction effects between watering regime and inoculation treatment on plant Anet, Fv/Fm, and disease severity. Correlations between disease severity and physiological stress responses were also examined in JMP using a linear regression analysis to determine maximum fit. Survivorship of each treatment group was estimated using the Kaplan–Meier survival analysis with the survival package in R v. 3.5.1 . A Cox proportional-hazards model was followed by a Peto and Peto post hoc test to test for statistical significance of Kaplan–Meier survivorship. Due to the small sample size of individuals available for the experiment, all reported results for survival were based on a 90% confidence level, and P-values above 0.05 but below 0.1 were considered significant trends. All other tests were conducted using a 95% confidence level for significance.The results of this study support the hypothesis that drought stress reduces resistance to pathogens in A. glauca, and fungal infection enhances plant mortality compared to drought alone.

As predicted, both physiological metrics showed declines as drought and disease progressed, suggesting rapid plant responses to both stresses. Furthermore, a strong correlation was found between declining physiological function and increases in stress severity index, suggesting that visible signs of stress may be used to assess physiological decline and reveal more severe underlying problems in the field. Finally,although mortality rates for inoculated groups were similar, drought-stressed A. glauca shrubs infected with N. australe trended toward faster and greater mortality than in any other treatment group.The results of this experiment, along with the identification of N. australe and other Bot. species in the region , suggest that the severe canopy dieback of A. glauca observed in Santa Barbara County between 2012 and 2016 is likely the result of global-change-type drought combined with the presence of opportunistic fungal pathogens like N. australe. While there is evidence to suggest that acute drought alone may cause some mortality in A. glauca , the presence of N. australe and other pathogens likely exacerbates stress and accelerates mortality in these hosts. Furthermore, N. australe has long been reported in avocado orchards in Santa Barbara County ; however, there are no known reports or indications of major disease and dieback of A. glauca in surrounding chaparral shrubland system until recently, during the especially dry winters of 2013 and 2014 . Thus, we suspect that while Naustrale has likely been present on A. glauca hosts , the drought of 2011–2018 was the most severe in the region in the past 1200 years and may have been significant enough to push adult A. glauca past a tipping point of defensibility against N. australe.

It should be noted that results of experiments on young plants, which may be highly susceptible to drought and drought-related mortality due to limited carbon reserves, may not scale directly to large, mature individuals in the field . This study showed high mortality in 2-year-old A. glauca exposed to a fungal pathogen with and without drought, in contrast with field observations of diseased, large adults exhibit severe canopy dieback and are ridden with fungal cankers, yet still survive . Previous studies have yielded similar results: for instance, photosynthesis was shown to be greatly reduced in oak seedlings compared to adults in drought years compared to wet years , and He et al. reported that responses of red maple and paper birch saplings to a 1995 drought were significantly different than those of mature adults. Similarly, since hosts are often able to allocate carbon reserves to compartmentalize canker-causing agents like N. australe within carbon-rich barriers , larger individuals with more biomass and greater carbon stores are able to utilize and direct more resources to defense than younger, smaller individuals. Thus, mature plants can better persist through biotic attack during environmental stress than their younger counterparts and experience various levels of canopy dieback rather than full mortality. Arctostaphylos glauca are obligate seeders, meaning they are killed by fire and must maintain populations by individuals recruiting from seed rather thanresprouting from their base. Therefore, young, small individuals may be of greatest concern for future populations of this species. Because current research is predicting more frequent and extreme drought events , more exotic pathogens , and more frequent fire in these southern California shrublands , populations of A. glauca could decline because small individuals may be highly susceptible to disease and mortality. A valuable next step for understanding these risks and predicting future shifts in vulnerable chaparral communities would be to monitor young recruiting populations of A. glauca for N. australe for signs of stress, infection, and mortality in the wild.In the face of rapid climate change, it is increasingly important to understand the abiotic and biotic mechanisms driving ecological landscape change. Large plant dieback events can produce major ecological consequences, hydroponic bucket including changes in vegetation cover , increased fire risk , and changes in hydrology , all of which affect ecosystem structure and functioning . Furthermore, the loss of even a few species can trigger effects on the local food web structure , and increase risk of invasion . The results of this study suggest that small individuals of A. glauca, one of the most common and widespread species the southern California chaparral community, are at high risk of disease and dieback due to opportunistic pathogens and extreme drought. The potential for dieback of Arctostaphylos spp., which provide food for animals such as mice, rabbits, and coyotes and are an important component of post-fire woody regeneration in chaparral, raises concerns regarding changes to ecosystem structure and functioning in the coming decades. Many ecosystems today are facing unprecedented drought ; yet, the interactions of drought and pathogens in wild land settings are difficult to study because the multitude of confounding variables and the challenges of manipulating both the pathogens themselves and climate. Thus, greenhouse studies such as this one are increasingly essential to understand the influences of drought and pathogens as they relate to dieback events, as well as to understand the relationship between stress and shrub/tree ontogeny . Critical questions remain regarding the relative tipping points for large-scale dieback among historically drought-tolerant species such as A. glauca that today are facing the combination of extreme drought and novel pathogens. These pathogens may not express themselves until there is drought, highlighting the need for broader field surveys and long-term monitoring of wildland ecosystems. An important step to understanding the role of disease in contributing to vegetation change is also to isolate pathogens and test their pathogenicity under varying controlled conditions.

This study provides one such step for what appears to now be a widespread, opportunistic introduced pathogen in an important native California chaparral shrub.Extreme drought events from climate change have produced immediate and dramatic effects in recent years, with costs often exceeding $1 billion due to their widespread economic and ecological impacts . Among the ecological consequences is widespread tree mortality, , event within plant systems that have historically been considered drought-tolerant . While seasonal droughts are known to be a natural and regular occurrence in arid and semi-arid regions, the increased frequency, duration, and intensity with which they have occurred in recent years is highly unusual . Such extreme droughts, referred to as “global-change type drought” , are predicted to continue, and even become the norm, as a result of human-induced climate change . Consequently, species that are typically capable of withstanding regular drought stress may be susceptible to canopy dieback, and mortality, as a result of shifts in drought regimes . One such plant community that may be vulnerable to extreme climatic change is chaparral. Chaparral shrublands, which occupy approximately 7 million acres throughout California , are a dominant vegetation community in southern California, composed primarily of evergreen, drought tolerant shrubs and subshrub species including manzanita , ceanothus , and chamise . These species are well adapted to the seasonal variations in temperature and precipitation typical of mediterranean climates where hot, rainless summers are the norm . However, mediterranean-type regions like southern California are predicted to experience rapid increases in temperature , and increased drought occurrence and severity ; IPCC, 2013, resulting from human-caused climate change. These regions have thus been designated as worldwide global change “hot spots” . Indeed, recent studies have reported extensive mortality of chaparral shrub species resulting from global-change type drought throughout southern California . Thus, climate change represents a significant threat to native plant community persistence in this region. A critical topic for ecological research is understanding where, how, and to what extent plant communities will change as a result of increased drought . Studies aimed at understanding the physiological mechanisms behind drought-related plant mortality – and why some plants suffer mortality from drought while others survive – have elucidated a variety of complex mechanisms of plant mortality . These include loss of hydraulic conductance , exhausted carbon reserves , and susceptibility to pests and pathogens due to being in a weakened state from drought . Measuring xylem pressure potential can be a useful index of soil water availability , and dark-adapted fluorescence can be a quick and accurate indicator of plant stress, as values drop significantly in water-stressed plants,. Together, these may be useful tools for predicting plant vulnerabilities to drought and biotic invasion. Landscape variables such as elevation, slope, and aspect have also been shown to correlate with plant water stress and mortality , and can be useful for predicting vulnerabilities during drought. However, major knowledge gaps still remain, and studies combining field mortality patterns with physiological data on plant water stress are rare . Plants employ a variety of complex strategies to cope with drought stress, but generally fall along a continuum of “drought avoiders” or “drought tolerators”. Drought avoidance, also known as “isohydry”, refers to plants that regulate stomatal conductance to maintain high minimum water potentials as soil dries out . While this strategy reduces the risk of xylem cavitation and subsequent hydraulic failure, it may increase the likelihood of carbon starvation, as C assimilation is greatly reduced . Conversely, drought tolerant plants maintain higher Gs, even at very low water potentials, which allows for continued C assimilation but with greater risk of xylem cavitation . These different strategies can have significant implications for ecosystem level consequences of severe drought ; indeed, recent studies have linked anisohydry with greater levels of mortality in chaparral systems . An historic drought in southern California provided an opportunity to simultaneously measure physiological stress and dieback severity along an elevational gradient in aclassically drought-tolerant evergreen chaparral shrub, big berry manzanita . A. glauca is one of the largest and most widely-spread members in a genus consisting of nearly 100 species.

Therefore, it is recommended that multiple modalities be used to capture and characterize RRBs to gain a better understanding of how they evolve over time, across contexts and as a result of individual characteristics.Historically, the two most universally agreed upon characteristics found to significantly relate to RRB presentation have been age and IQ . It has even been noted that due to methodological limitations and specific study aims, “untangling relationships between sub-scale scores and age and IQ has not been possible in the majority of RBS-R studies to date” . Taking a novel approach to address this issue in this study has helped to clarify some of the previous findings, and subsequent theories, regarding the relationship between age, IQ and RRBs. The basic correlational relationships between RRBs, age, and IQ have been replicated across studies; however, a basic correlation analysis is a missed opportunity to ask the more informative research question of how they are related. In other words, instead of addressing IF RRBs, age, and IQ are related to one another, it should be HOW are they related. More specifically, how much does one variable account for or predict the others. Surprisingly, stackable planters as the current study showed, although IQ may be an important predictor of the presence of any RRBs, it is not the most important factor in determining a specific RRB phenotype.

Lastly, it should be noted that coping was significant in the MLR model, yet with a very distinct influence on cluster prediction in comparison to anxiety and hyperactivity. That is, each of the odds ratios were less than one indicating less of a chance that they would belong to any of the clusters in comparison to Cluster 2 . This finding is consistent with our understanding of maladaptive behavior and RRBs, as an increase in coping behaviors would mean better adaptive skills such as managing emotionally arousing situations, and exerting selfregulatory control, thus reducing likelihood of engaging in RRBs. The association between phenotypic patterns of RRB expression in ASD and the behavioral proxies for common comorbid symptomology in ASD has not yet been clearly defined. However, given the results of Rao & Landa on the additive effect of presenting behavioral phenotypes spanning across both diagnostic categories of ADHD and ASD on the severity of RRB presentation, it is not surprising that hyperactivity was the strongest predictor of RRB cluster membership. Further, the finding that non-ASD specific traits were able to significantly predict variations in half of the diagnostic dyad of behaviors warrants continued Investigation.Results from the current study should be interpreted and applied somewhat cautiously for several reasons. First, this study examined data from a racially homogeneous sample, which is not reflective of the general population.

Future studies should include a more diverse sample with more variety in the economic status of participating families as well as a more accurate sampling and balanced representation of racial groups. Finally, the sample size included in this study is the largest to date compared to similar studies. However, without analyzing longitudinal data, it’s impossible to statistically or methodologically conclude how RRBs will change overtime within individuals, which prohibits the current study to be able to conclude how these phenotypic profiles change overtime within individuals. Specifically, relationships described by this study’s findings may also shift over time along with the relationship between RRBs and influence those shifts will have on other developmental and clinical characteristics for individuals with ASD.Repetitive behaviors can present significant barriers for individuals with ASD and their families. Therefore, it is important to continue to examine the ways in which this set of heterogeneous behaviors relate to specific developmental or behavioral characteristics; particularly those which have not been examined in previous studies. Results find that symptoms of hyperactivity and anxiety are better predictors of RRB subtypes than are IQ and age. Moreover, coping strategies are also important in the expression of RRBs. The ultimate goal in this field of research will be to understand the function that RRBs serve, related traits, and finally to use this information to inform effective intervention strategies. Therefore, researchers should continue to explore RRB phenotypic profiles and identify related characteristics to continue to discover the interaction effects of individual traits and RRB presentation This study provides a significant foundation in understanding the complex nature of this diverse behavioral phenotype.The San Joaquin Valley of California is a major wine grape growing region of the United States. In 2019, it produced 48% of the total wine grapes crushed in the state of California.

The average grower return for Merlot wine grapes from the SJV was only USD 310 per ton, whereas the state average for that variety was USD 826 per ton. Red wine grapes from this region are generally priced lower than similar grapes from cooler growing regions because its climate, specifically the high growing season temperatures and rapid growing degree day accumulation, favor high yields of fruit with relatively low berry flavonoid concertation at harvest. Hence, wines made of SJV grapes are usually marketed as high volume, low-cost wines. The economics of this industry favor the development of production practices that minimize grape and wine production cost while maintaining or improving grape and wine quality. For example, mechanization of canopy management practices and the implementation of optimal irrigation practices can minimize labor costs and improve grape berry flavonoid concentration .Flavonoids are critical in determining the color, flavor, and mouthfeel of red wine and thus directly affect wine quality. They are also the primary antioxidants that help plants cope with environmental stresses. Their biosynthesis and concentration respond to environmental cues, including water deficits, solar radiation exposure, and temperature. Previous studies observed that moderately increasing the severity of water deficits and solar radiation increased the content of two major flavonoid classes, anthocyanins and flavonols, in berries . However, excessive exposure of grape berries to sunlight, high air temperatures, and water deficits, reduces these compounds at harvest. Another major class of flavonoids, proanthocyanidins, determine wine astringency and help stabilize wine color via copigmentation. They are less sensitive to environmental stresses compared to other flavonoids. However, under relatively severe environmental stress, even proanthocyanidin composition and concentrations may be altered, in a manner similar to the other flavonoids. Leaf removal and deficit irrigation are the two cultural practices most commonly used to manage canopy structure and plant water status, other than dormant pruning. Leaf removal in the fruiting zone can directly affect canopy microclimate, and thereby affect berry flavonoid accumulation. Leafing may affect grapevine source-sink relations, which would also contribute to the changes in berry development . Removal of leaves around clusters of grapes at different developmental stages were investigated to help growers understand the various benefits selective leaf removal can provide. When leaves were removed early , berry set, and therefore yield, were reduced in cool climate vineyards. Studies in warm and hot climates deduced that early leaf removal increased berry total soluble solids , stacking pots and berry skin flavonoid concentration without adversely affecting yield. When leaves were removed later in the season in cool climates, the total proanthcoyanidin content was increased in berries, but decreased in wine. Other studies suggested that late leaf removal could enhance berry anthocyanins. Water is a critical environmental factor for grapevine physiological development. Water deficits reduce grapevine vegetative growth and berry weight. Severe water deficits might inhibit photosynthesis and promote berry maturity and vine dormancy by stimulating abscisic acid biosynthesis, Mild to moderate water deficits improve berry chemical composition due, in part, to suppressing grapevine vegetative growth, and thereby increasing the sink strength of berries. Moreover, water deficits increase berry flavonoid concentrations, and the increases in TSS and flavonoid concentrations can be attributed to the alteration of bio-synthetic pathways, or simply the reduction in berry weight due to water loss . Imposing water deficits on grapevines at different developmental stages can result in different effects. The SJV in California is a semiarid region and growers typically replace 70% to 80% of crop evapotranspiration from bud-break to harvest.

Preveraison water deficits increased berry anthocyanin concentration whereas post veraison water deficits promoted TSS concentration. Castellarin et al. reported that preveraison water deficits hastened sugar accumulation and anthocyanin biosynthesis, where the genes related to anthocyanin biosynthesis, including flavonoid 3-hydroxylase , dihydroflavonol 4-reductase , UDP-glucose: flavonoid 3-O-glucosyl-transferase and glutathione S-transferase were upregulated. There was also evidence showing that both pre and postveraison water deficits can enhance anthocyanin biosynthesis. Sometimes water deficits may increase the total anthocyanin content while the extractable anthocyanins might be lower. Nevertheless, berry dehydration due to water deficits can overrule the metabolomic regulation and directly determine the flavonoid concentration in wine. We previously studied the effects of mechanical leaf removal and water deficits on the anthocyanin content and profile of grape berries. In this study, we subjected grapevines in a hot climate to mechanical canopy management treatments and water deficits in anattempt to promote flavonoid accumulation in grapes and wine. We hypothesized that the leaf removal and water deficit would improve the berry and wine flavonoid profile without adversely affecting yield. Overall, the objective of this study was to investigate the physiological and chemical impacts of mechanical leaf removal and water deficits on berry and wine flavonoid concentration of Merlot in a hot climate.The concentration of berry skin flavonoids was measured in 2014 and 2015 . In 2014, berries from vines subjected to PBLR had less delphinidin, cyanidin, and petunidin compared to berries from vines subject to the other two leaf removal treatments. The di-hydroxylated anthocyanins were the highest in berries from vines subjected to PFLR. In 2015, however, PFLR obtained the highest concentrations of malvidin and tri-hydroxylated anthocyanins. It also obtained the highest concentrations of quercetin, myricetin, and total flavonols in the second season. In 2014, RDI increased delphinidin, cyanidin, petunidin, tri-hydroxylated, and total anthocyanin. However, there was no difference in either anthocyanins or flavonols between SDI and RDI in 2015. When comparing the two years, the flavonoid concentrations in the second year were generally lower than the first.Wine flavonoids were measured in 2014 and 2015, and differences observed in berry skins did not transfer into wine with leaf removal treatments . In 2014 and 2015, there was no differences observed with leaf removal treatments in any of the anthocyanin derivatives. However, the differences in flavonols from leaf removal treatments were significant enough to be observed in wine, where PFLR had higher quercetin, myricetin, and total flavonols although there was no separation between PBLR and PFLR in 2015. As for irrigation treatments, in 2014, SDI increased the concentrations of cyanidin and petunidin, and also increased quercetin and total flavonols in both seasons. Like berry skin flavonoid concentrations, the concentrations of most of the wine anthocyanin and flavonol derivatives were lower in the second season than the first one.The wine proanthocyanidin concentration and composition were measured in 2014 and 2015 . There were no significant differences in any of the proanthocyanidin subunits due to either leaf removal or deficit irrigation treatments, except epicatechin terminal subunits, which were reduced by PBLR in 2014. The general concentrations, including the total proanthocyanidins, were lower in the second season compared to the first season, and the mDP was higher.The effects of various timings of leaf removal on berry development and composition were previously investigated. Late leaf removal can affect grapevine yield and berry composition, but prebloom leaf removal has been shown to be more effective in modifying yield and berry composition than post-fruit-set leaf removal. In our study, yield was reduced by PFLR in 2014, and by PBLR in 2015. This inconsistency might be due to treatment effects on LAI. Vines subjected to PFLR had the lowest LAI in 2014, perhaps sufficiently to reduce yield capacity compared to vines with larger canopies, as typically found in SJV. Previous studies reported that berry weight may be affected by leaf removal, especially when the leaf removal was conducted early in the season. Skin weight was affected by leaf removal, where altered canopy microclimate by leaf removal could be the direct factor to manipulate berry skin weight. In our study, berry skin weight was reduced with PFLR in 2014. This might be because that late leaf removal diminished the growth of berry skin, as witnessed in previous studies. Previous studies showed that leaf removal increased berry TSS concentration. Leaf removal could increase berry TSS by dehydration sunlight, or increased carbohydrate accumulation and partitioning to the fruits. However, in our study, treatments had few and small effects on TSS.