Tag: agriculture

Moore & Goodson followed children with ASD from two to four years old and found that overall rates of RRBs reduced, with low level RRBs significantly reducing, yet different RRB subtypes persisted in a more complex form. Whereas Honey, et al. examined preschoolers and found within a year there was a significant decrease in the severity of RRBs observed. However, a more recent study, which utilized observational coding of RRBs in a preschool aged sample, found there was no significant change in any of the RRB subtypes coded across 13 months and three assessment time points . Further, Richler, et al. used the ADI-R to track change in RRBs over a period of 9 years and found that low level sensory motor RRBs actually remained high. Taken together, these findings suggest that the developmental progression and transformation of RRBs overtime within individuals remain unclear, with the biggest influence being the sample population and measurement tools used .The general consensus within the field is that children with more severe adaptive and cognitive impairments exhibit higher frequency, more intense, and more persistent RRBs . More specifically, children with lower cognitive capacity exhibit the most frequent and severe low-level RSM RRBs, whereas children with higher cognitive capacity exhibit significantly less of the RSM behaviors . However, these findings are nuanced, as there is not a singular and linear relationship between IQ and RRBs, and consideration must be given to the types of RRBs being measured. For example, grow bucket in a study examining 830 children with ASD between 15 months and 12 years old with an average age of 5 years old, found that for many RRBs, a significant interaction effect was found between nonverbal IQ and age .

Specifically, in older children, NVIQ was strongly related to low-level RRBs such as hand and finger mannerisms. However, high-level RRBs like circumscribed interests were positively related to NVIQ. Bishop, Richler, & Lord used the ADI-R to examine the total as well as the individual types of RRBs, which included 13 types of behaviors considered to fall under the RRB umbrella. Another interesting finding from this study was the relationship between NVIQ and RRBs actually became stronger with increasing age, where children under the age of 3 showed no relationship between RRBs and NVIQ. Similarly, Kim and Lord found no association between NVIQ and RRBs in toddlers under two years old. Taken together, these findings further evidence the importance of measuring subtypes individually, as there are clear differences across RRB types in the relationship between cognitive ability and RRB presentation. Findings across studies highlight the importance of examining specific subtypes of RRBs and the traits associated with them, as these traits may significantly impact the persistence or the possible reduction of RRBs overtime. For example, Ray-Subramaian and Weismer found that not only were receptive and expressive language skills significantly lower among children with higher rates of RRBs, but they also concluded that higher scores in both language domainsin 2-3 year olds could significantly predict a reduced rate of RRBs. Again, consumers of this area of ASD research must take into account the number of participants and age span included across studies. Most recently, a study examined children at three time points to determine if RRB presentation at 1-2 years old, and/or 3-5 years old can predict cognitive functioning, adaptive skills and ASD symptomology at 8-10 years old . Results showed that increased severity of low-level RRBs were significant predictors of lower cognitive and adaptive skills as well as a greater ASD symptom severity at age 8-10 years. This relationship was not found when examining whether RRBs in the first two years of life could significantly predict the same school-aged outcomes .

Despite the nuanced findings among studies regarding the specific relationship and influence RRBs have on cognitive performance, it is clear that future studies must consider and control for cognitive functioning when examining the relationship between RRBs and other clinical characteristics .Children with ASD exhibit significant impairment in adaptive functioning skills that extend beyond their cognitive deficits . It is important to note that adaptive functioning skills measure the ability of an individual to successfully function within their given environment, and studies have demonstrated greater deficits in adaptive functioning for children with ASD compared to age and IQ matched peers . Several studies have examined the relationship between adaptive skills and RRB presentation in individuals with ASD . Similar to the relationship between RRBs and IQ, results have varied based on the measures used and age of participants; however, it can be deduced that in general, higher rates of RRBs are associated with lower adaptive functioning skills . However, this finding has varied across studies based on the age and IQ of the participant . The relationship between adaptive skills and RRB presentation in ASD is complex and has varied results based on the age and IQ of participants as well as the measures employed. For example, Liss, et al. found that the relationship between adaptive functioning and RRBs was dependent on the severity of adaptive skill impairment. Specifically, there was no significant relationship between RRBs and adaptive functioning in lower functioning children with ASD; yet the high functioning group exhibited a significant correlation between adaptive behaviors and RRBs. Few studies have examined adaptive functioning and RRBs using measures other than parent report. However, in young children, self-regulation has been observed in interactions between parent and child. As shown by Wetherby and Prizant , children with ASD exhibited lower proportions of well-regulated behavior bouts and higher incidences of RRBs during parent child interactions. Theoretically, some have suggested that specific RRBs may be a result of an emotional trigger for children with ASD. However, Militerni, et al. found that most of the low-level RRBs observed in 2-7 year olds were not reactive to a particular emotional trigger. The remaining 29% of RRBs deemed to be reactive in nature consisted of high intensity sensory behaviors, including self-injurious behaviors, motor RRBs and sensory stimulation, which were all more common the younger participants . This notion of an emotional trigger also highlights a theory that RRBs serve as a coping strategy to regulate their state of arousal; however, there are currently not enough results or data to full endorse this theory and further examination is needed .

There are a number of common developmental and neuropsychiatric disorders that overlap in symptom presentation, and in some cases are determined to co-occur in children with ASD. Some of the most common are attention deficit hyperactivity disorder and anxiety disorders . There is limited knowledge about how these ASD-related disorders vary across the population and what impact the co-occurring conditions impact RRB manifestation as well as the impact on overall development, adaptive skills and other child characteristics. Attention deficit hyperactivity disorder . Attention deficit hyperactivity disorder is a neurodevelopmental disorder characterized by symptoms of inattention, impulsivity, and/or hyperactivity exhibited to a degree substantially beyond what is expected for developmental level . ADHD and ASD share overlapping symptoms such as issues with communication problems, issues with attention and the presence of restricted behaviors . Although the last version of the Diagnostic and Statistical Manual of the American Psychiatric Association prohibited a dual diagnosis of ASD and ADHD, dutch bucket for tomatoes preliminary evidence suggests that when these two disorders co-occur, the risk for increased severity of psychosocial issues intensifies . Such findings are in conjunction with a growing number of researchers reporting children who meet criteria for both disorders are evidence to suggest they can co-occur .The need for research to examine the dual presence of clinically significant ADHD symptoms in individuals with ASD has begun to be addressed . This study compared school-aged children 4 to 8 years old that included younger siblings of children with ASD , children with ASD , low-risk controls and children with language delay to include reference points for functioning and skill level across groups. Results indicated that children with comorbid ASD and ADHD diagnoses had lower cognitive functioning, more severe social impairments, and greater delays in adaptive functioning than children with ASD only . There is a great need for continued exploration of the impact of co-occurring ASD and ADHD symptomology on children with ASD; specifically, how elevated levels of hyperactivity influence the presence and severity of RRBs in children with ASD. Anxiety. The role of anxiety for individuals with ASD has been proposed to play a key role in the severity of RRBs, as the function of engaging in specific RRBs has been hypothesized to serve as a coping mechanism to reduce feelings of anxiety . However, it should be noted that there is insufficient evidence currently to support this theory . Scientific evidence illustrating links between anxiety, ASD and RRBs is limited. However, there have been studies that indicate high levels of anxiety in the ASD population and even links to symptom severity increasing . The link between anxiety and ASD symptom severity is logical considering the need for routine, sameness and consistency to a severe degree. Interruption of those may result in increased levels of anxiety and intense stress often accompanied by outbursts when self-control is impaired. The theories accounting for the popular notion that anxiety and arousal states significantly contribute to increased RRB severity for individuals with ASD still needs to be explored .The present study aimed to explore the phenotypic presentation of RRBs and associated characteristics for individuals with ASD between 4 and 18 years old. Previous studies have been limited by measurement tools, limited age included, as well as limited statistical power due to smaller sample sizes; therefore, this study aimed to examine the forms of RRBs across age and IQ, and to examine the impact of hyperactivity, anxiety, coping skills, and ASD severity on RRB presentation in a large, well-characterized sample of individuals with ASD. The first aim was to define the specific RRB subtypes derived from a factor analysis of the Repetitive Behavior Scale- Revised . Secondly, the RRB subtypes derived from the factor analysis were used to cluster participants based on type and severity of co-occurring RRB subtypes into phenotypic profiles. The final aim of this study was to explore the role of clinical , cognitive and adaptive skills in predicting phenotypic profile group membership. Researchers have yet to uncover the specific function/s of RRBs; therefore, examination of the predictive power of individual clinical characteristics on RRB phenotypes contributes to this area of research.The first aim of this study was to examine the factor structure of the Repetitive Behavior Scale- Revised to determine how many unique RRB forms are measured. The first step in determining the factor structure was to run an exploratory factor analysis of the 43 RBS-R items using Mplus Version 7 , an oblique CF-quartimax rotation and a weighted least- squares with mean and variance adjustment to account for the ordinal nature of the data . EFA assumes that each variable, in this case each question on the RBS-R, may be associated with any other factor without an a priori hypothesis about factors or variables . To determine the optimal number of factors, a combination of model fit statistics and examination of factor loadings were used. The chi-square value is typically an informative model fit statistic; however, the chi-square test is sensitive to sample size, such that large samples often result in statistically significant chi-square values . Given the number of participants in the current dataset, the chi-square values were analyzed with caution. Additional model fit statistics included root mean square error of approximation , the Standardized Root Mean Square Residual , the Comparative Fit Index and the Tucker Lewis Index . The RMSEA is a measure of model fit that is not as sensitive to sample size and values below .06 indicate an acceptable model fit . The SRMR is another descriptive model fit statistic in which lower values indicate better model fit, with a suggested cut-off of .08 or below . Lastly, both the CFI and TLI are typically presented together in EFAs and both serve as measures of model fit, ranging from 0 to 1 with higher values indicating better fit and cutoff scores of .90 .Determining Factor Structure. As items were permitted to load on only one factor for the CFA, items that loaded significantly >.30 on more than one factor were evaluated to determine the ideal factor pattern.

CBF1 transcript abundance is very sensitive to chilling temperatures; it is a master regulator of the cold regulon and improves plant cold tolerance . PIF7 binds to the promoter of CBF1, inhibiting CBF1 transcript abundance, linking phytochrome, the circadian clock and CBF1 expression. Our data are consistent with this model; transcript abundance of PIF7 was higher and CBF1 transcript abundance was lower in BOD berry skins than RNO berry skins .ABA concentrations in plants increase in response to dehydration and ABA triggers a major signaling pathway involved in osmotic stress responses and seed development. ABA concentrations only increase in the seed embryo near the end of seed development when the embryo dehydrates and goes into dormancy. ABA concentrations remain high to inhibit seed germination. The transcript abundance of ABA signaling genes such as ABF2 and SnRK2 kinases increase after application of ABA to cell culture and in response to dehydration in leaves of Cabernet Sauvignon. The data in this study are consistent with the hypothesis that BOD berries are riper at lower sugar levels. The ABA signaling genes in the berry skins had higher transcript abundance in BOD berries indicating that ABA concentrations were higher in BOD than RNO berries even though RNO berries were exposed to drier conditions . ABA concentrations may be higher in the BOD berry skins based upon the higher transcript abundance of important ABA signaling and biosynthesis genes encoding ABF2, SnRK2 kinases and NCED6. We hypothesize that this would be seed derived ABA since water deficits were not apparent in BOD with the recent rainfall and high humidity. In contrast, NCED3 and NCED5 had higher transcript abundance in RNO berry skins, nft growing system which might occur as the result of the very low humidity and large vapor pressure deficit .

The lower expression of NCED6 in RNO berry skins may indicate that the seeds in the berry were more immature than the BOD berries. The higher expression of other seed development and dormancy genes in the berry skins support the argument that BOD berries matured at a lower sugar level than the RNO berries. The ABA concentrations in the berry skins are a function of biosynthesis, catabolism, conjugation and transport. ABA in seeds increase as the seed matures and some of this ABA may be transported to the skin. In fact, a number of ABCG40 genes, which encode ABA transporters, had higher transcript abundance in BOD berry skins than that in RNO . Part of the ABA in skins may be transported from the seed and part of it might be derived from biosynthesis in the skins. NCED6 transcript abundance in the skins was higher in BOD berries. Perhaps the transcript abundance of NCED6 in the skin is regulated by the same signals as the embryo and reflects an increase in seed maturity. AtNCED6 transcript abundance is not responsive to water deficit in Arabidopsis, but AtNCED3 and AtNCED5 are. This is consistent with the higher NCED3, NCED5 and BAM1 transcript abundance in RNO berries . Thus, there are complex responses of ABA metabolism and signaling. It would appear that there may be two different ABA pathways affecting ABA concentrations and signaling: one involved with embryo development and one involved with the water status in the skins. Auxin is also involved with ABA signaling during the late stages of embryo development in the seeds. Auxin signaling responses are complex. ABF5 is an auxin receptor that degrades Aux/IAA proteins, which are repressors of ARF transcriptional activity. Thus, a rise in auxin concentration releases Aux/IAA repression of ARF transcription factors, activating auxin signaling. In the berry skins, there was a diversity of transcriptional responses of Aux/IAA and ARF genes in the two locations, some with increased transcript abundance and others with decreased transcript abundance.

As with ABA signaling, there may be multiple auxin signaling pathways operating simultaneously. One pathway appears to involve seed dormancy. ARF2 had a higher transcript abundance in BOD berries. ARF2 promotes dormancy through the ABA signaling pathway. This is consistent with the hypothesis that BOD berries reach maturity at a lower sugar level than RNO berries.Grapevines have very dynamic gene expression responses to pathogens. The top 150 DEGs for BOD berries were highly enriched with biotic stress genes. The BOD vineyard site had a higher rainfall and higher relative humidity than RNO and these conditions are likely to be more suitable for fungi to grow. We detected a much higher transcript abundance of powdery mildew-responsive genes in BOD berries and this may be connected to a higher transcript abundance of ethylene and phenylpropanoid genes as part of a defense response. The transcript abundance profiles of some of these genes are remarkably similar. Increased ethylene signaling in grapevines has been associated with powdery mildew infection and phenylpropanoid metabolism and appears to provide plant protection against the fungus. Genes involved with phenylpropanoid metabolism, especially PAL and STS genes, appear to be quite sensitive to multiple stresses in the environment. In Arabidopsis there are four PAL genes. These PAL genes appear to be involved with flavonoid biosynthesis and pathogen resistance in Arabidopsis. Ten different PAL1 and two PAL2 orthologs had higher transcript abundance in BOD berry skins; many STS genes also had a higher transcript abundance in BOD berry skins . Stilbenes are phytoalexins and provide pathogen resistance in grapes and STS genes are strongly induced by pathogens. Thus, the higher transcript abundance of powdery mildew genes may be associated with the higher transcript abundance of genes in the ethylene and phenylpropanoid pathways.

The transcript abundance of a number of iron homeostasis genes were significantly different in the two locations and there was a difference in soil available iron concentrations in the two locations. However, iron uptake and transport in plants is complicated depending on multiple factors, such as pH, soil redox state, organic matter composition, solubility in the phloem, etc. Thus, it is impossible to predict iron concentrations in the berry without direct measurements. The roles of these genes in iron homeostasis and plant physiological functions are diverse. Iron supply can affect anthocyanin concentrations and the transcript abundance of genes in the phenylpropanoid pathway in Cabernet Sauvignon berry skins. One of the DEGs, SIA1, is located in the chloroplast in Arabidopsis and appears to function in plastoglobule formation and iron homeostasis signaling in concert with ATH13. Another DEG, YSL3, is involved in iron transport. It acts in the SA signaling pathway and appears to be involved in defense responses to pathogens. It also functions in iron transport into seeds. FER1 is one of a family of ferritins found in Arabidopsis. VIT1 and NRAMP3 are vacuolar iron transporters and are also involved in iron storage in seeds. Other DEGs are also responsive to iron supply. IREG3 appears to be involved in iron transport in plastids; its transcript abundance increases with increasing iron concentrations. ABCI8 is an iron-stimulated ATPase located in the chloroplast that functions in iron homeostasis. It is unclear what specific roles these iron homeostasis genes are playing in grape berry skins, but they appear to be involved in iron storage in seeds and protection against oxidative stress responses. One possible explanation for the transcript abundance profiles in the BOD and RNO berry skins is that ferritins are known to bind iron and are thought to reduce the free iron concentrations in the chloroplast, thus, reducing ROS production that is caused by the Fenton reaction. As chloroplasts senesce during berry ripening, iron concentrations mayrise as a result of the catabolism of iron-containing proteins in the thylakoid membranes; thus, berry skins may need higher concentrations of ferritins to keep free iron concentrations low. This might explain the increase in ferritin transcript abundance with increasing sugar levels. Most soils contain 2 to 5% iron including available and unavailable iron; soils with 15 and 25 μg g− 1 of available iron are considered moderate for grapevines, but soils with higher concentrations are not considered toxic. Therefore, vertical hydroponic nft system for both soils in this study, iron concentrations can be considered to be very high but not toxic. The higher available iron concentrations in the BOD vineyard may be associated with the wetter conditions and the lower soil pH.Other researchers using Omics approaches have identified environmental factors that influence grape berry transcript abundance and metabolites. One study investigated the differences in transcript abundance in berries of Corvina in 11 different vineyards within the same region over 3 years. They determined that approximately 18% of the berry transcript abundance was affected by the environment. Phenylpropanoid metabolism was very sensitive to the environment and PAL transcript abundance was associated with STS transcript abundance. In another study of a white grape cultivar, Garganega, berries were analyzed by transcriptomic and metabolomic approaches.

Berries were selected from vineyards at different altitudes and soil types. Again, phenylpropanoid metabolism was strongly influenced by the environment. Carotenoid and terpenoid metabolism were influenced as well. Two studies investigated the grape berry transcriptomes during the ripening phase in two different regions of China, a dry region in Western China and a wet region in Eastern China. These two locations mirror some of the differences in our conditions in our study, namely moisture, light and elevation, although the dry China western region has higher night temperatures and more rainfall than the very dry RNO location. In the Cabernet Sauvignon study, they compared the berry transcriptomes from the two regions at three different stages: pea size, veraison and maturity. The TSS at maturity was slightly below 20°Brix. Similar to our study, the response to stimulus, phenylpropanoid and diterpenoid metabolism GO categories were highly enriched in mature berries between the two locations. Differences in the transcript abundance of NCED and PR proteins were also noted. Like in our study, the authors associated the transcript abundance of these proteins to the dry and wet locations, respectively. In the second study comparing these two regions in China, the effects of the environment on the metabolome and transcriptome of Muscat Blanc à Petits Grains berries were investigated over two seasons; specifically, terpenoid metabolism was targeted. Like in our study, the transcripts in terpenoid were in higher abundance in the wetter location. The transcript abundances were correlated with terpenoid concentrations and a coexpression network was constructed. A specific set of candidate regulatory genes were identified including some terpene synthases , glycosyl transferases and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase . We examined the transcript abundance of some of these candidate genes in our own data but did not find significant differences between our two locations. The contrasting results between our study and Wen et al. could be for a variety of reasons such as different cultivar responses, berry versus skin samples, or different environmental conditions that affect terpenoid production. Terpenoid metabolism is influenced by the microclimate and is involved in plant defense responses to pathogens and insects. Light exposure to Sauvignon Blanc grapes was manipulated by removing adjacent leaves without any detectable differences in berry temperatures. Increased light exposure increased specific carotenoid and terpene concentrations in the berry. The responses of carotenoid and terpenoid production to temperature are less clear. Some effect of temperature was associated with carotenoid and terpenoid production, but to a lesser extent than light. Higher concentrations of rotundone, a sesquiterpene, have been associated with cooler temperatures. Water deficit can also alter carotenoid and terpenoid metabolism in grapes. Terpenes can act as signals for insect attacks and attract insect predators. Thus, terpenoid metabolism is highly sensitive to the environment and influenced by many factors. In contrast to these studies, excess light and heat can affect transcript abundance and damage berry quality. In addition to a higher rate of malate catabolism, anthocyanin concentrations and some of the transcript abundances associated with them are decreased as well.BOD berries reached maturity at a lower °Brix level than RNO berries; the cause is likely to be the warmer days and cooler nights in RNO. Higher day temperature may increase photosynthesis and sugar transport and cooler night temperatures may reduce fruit respiration. °Brix or TSS approximates the % sugar in a berry and is a reliable marker of berry maturity in any given location; however, TSS is an unreliable marker of berry maturity when comparing grapes from very different climates. The differences in TSS between BOD and RNO are consistent with other studies on the temperature effects on berry development. Indirect studies have associated gradual warming over the last century to accelerated phenology and increased sugar concentrations in the grape berries.

The Berry phase reveals geometric information of quantum wave functions via their phases acquired after an adiabatic cyclic process, and its concept has laid the foundation for understanding many topological properties of materials. The theory of Berry phase is built on pure quantum states. For example, the ground state fits the description as the limit of a statistical ensemble at zero temperature. At finite temperatures, the density matrix describes thermal properties of a quantum system by associating a thermal distribution to all the states of the system. Therefore, it is an important task to generalize the Berry phase to the realm of mixed quantum states. There have been several approaches to address this problem, among which the Uhlmann phase has attracted much attention recently since it has been shown to exhibit topological phase transitions at finite temperatures in several 1D, 2D, and spin-j systems. A key feature of those systems is the discontinuous jumps of the Uhlmann phase at the critical temperatures, signifying the changes of the underlying Uhlmann holonomy as the system traverses a loop in the parameter space. However, due to the complexity of the mathematical structure and physical interpretation, the knowledge of the Uhlmann phase is far less than that of the Berry phase in the literature. Moreover, only a handful of models allow analytical results of the Uhlmann phase to be obtained. The Berry phase is purely geometric in the sense that it does not depend on any dynamical effect during the time evolution of the quantum system of interest . Therefore, the theory of the Berry phase can be constructed in a purely mathematical manner. As a generalization, hydroponic bucket the Uhlmann phase of density matrices was built in an almost parallel way from a mathematical point of view and shares many geometric properties with the Berry phase.

We will first summarize both the Berry and Uhlmann phases using a fiber-bundle language to highlight their geometric properties. Next, we will present the analytic expressions of the Uhlmann phases of bosonic and fermionic coherent states and show that their values approach the corresponding Berry phases as temperature approaches zero. Both types of coherent states are useful in the construction of path integrals of quantum fields. While any number of bosons are allowed in a single state, the Pauli exclusion principle restricts the fermion number of a single state to be zero or one. Therefore, complex numbers are used in the bosonic coherent states while Grassmann numbers are used in the fermionic coherent states. Moreover, the Berry phases of coherent states can be found in the literature, and we summarize the results in Appendix A. Our exact results of the Uhlmann phases of bosonic and fermionic coherent states suggest that they indeed carry geometric information, as expected by the concept of holonomy and analogy to the Berry phase. We will show that the Uhlmann phases of both cases decrease smoothly with temperature without a finite-temperature transition, in contrast to some examples with finite-temperature transitions in previous studies. As temperature drops to zero, the Uhlmann phases of bosonic and fermionic coherent state approach the corresponding Berry phases. Our results of the coherent states, along with earlier observations, suggest the Uhlmann phase reduce to the corresponding Berry phase in the zero-temperature limit.

The correspondence is nontrivial because the Uhlmann phase requires full-rank density matrices, which cannot be satisfied only by the ground state at zero temperature. Moreover, the fiber bundle for density matrices in Uhlmann’s theory is a trivial one, but the fiber bundle for wavevfunctions in the theory of Berry phase needs not be trivial. A similar question on why the Uhlmann phase agrees with the Berry phase in certain systems as temperature approaches zero was asked in Ref. without an answer. In the last part of the paper, we present a detailed analysis of the Uhlmann phase at low temperatures to search for direct relevance with the Berry phase. With the clues from the previous examples, we present a conditional proof of the correspondence by focusing on systems allowing analytic treatments of the path-ordering operations. Before showing the results, we present a brief comparison between the Uhlmann phase and another frequently mentioned geometrical phase for mixed quantum states proposed in Refs. , which was originally introduced for unitary evolution but later extended to non-unitary evolution. This geometrical phase was inspired by a generalization of the Mach-Zehnder interferometry in optics and was named accordingly as the interferometric phase. It has a different formalism with a more intuitive physical picture and has been measured in experiments. In general situations, the interferometric phase can be expressed as the argument of a weighted sum of the Berry phase factors from each individual eigenstate. Thus, its relation to the Berry phase is obvious. However, the concise topological meaning of the interferometric phase is less transparent since it is not directly connected to the holonomy of the underlying bundle as the Uhlmann phase does. The reason has been discussed in a previous comparison between the two geometrical phases. The interferometric phase relies solely on the evolution of the system state while the Uhlmann phase is influenced by the changes of both the system and ancilla, which result in the Uhlmann holonomy. Although Uhlmann’s approach can be cast into a formalism parallel to that of the Berry phase as we will explain shortly, its exact connection to the Berry phase is still unclear. The Uhlmann-Berry correspondence discussed below will offer an insight into this challenging problem. The rest of the paper is organized as follows. In Sec. II, we first present concise frameworks based on geometry for the Berry and Uhlmann phases, using a fiber-bundle language. In Sec. III, we derive the analytic expressions of the Uhlmann phases of bosonic and fermionic coherent states and analyze their temperature dependence. Additionally, the Uhlmann phase of a three-level system is also presented. Importantly, the Uhlmann phases of both types of coherent states and the three-level system are shown to approach the respective Berry phases as temperature approaches zero. In Sec. IV, we propose the generality of the correspondence between the Uhlmann and Berry phases in the zerotemperature limit and give a conditional proof. In Sec. V, we discuss experimental implications and propose a protocol for simulating and measuring the Uhlmann phase of bosonic coherent states. Sec. VI concludes out work. The Berry phases of bosonic and fermionic coherent sates and the special cases with a 1D Hilbert space are summarized in the Appendix.The classical approach to the central limit theorem and the accuracy of approximations for independent random variables rely heavily on Fourier transform methods. However, the use of Fourier methods is highly limited without an independence structure, which makes it far less possible to capture the explicit bounds for the accuracy of approximations. In 1972, Charles Stein introduced a novel technique, now known as Stein’s method, for normal approximation. The method works for both independent and dependent random variables. The method also provides bounds of approximation accuracy. Extensive applications of Stein’s method to obtain uniform and non-uniform Berry–Esseen-type bounds for independent and dependent random variables can be found in, for example, Diaconis , Baldi et al. , Barbour , Dembo and Rinott , Goldstein and Reinert , Chen and Shao , Chatterjee , Nourdin and Peccati and Chen and Fang . In addition to the traditional study of Berry–Esseen bounds, new developments to Stein’s method have triggered a series of research on Cramér-type moderate deviations, stackable planters which address the relative error of two tail probabilities. See, for example, Raič , Chen et al. and Shao and Zhou , among others.

Various extensions of Stein’s idea have been applied to many other probability approximations, most notably to Poisson, Poisson process, compound Poisson, binomial approximations and more recently to multivariate, combinatorial and discretized normal approximations. Stein’s method has also found diverse applications in a wide range of fields, see for example,Arratia et al. , Barbour et al. and Chen . Expositions of Stein’s method and its applications in normal and other distributional approximations can be found in Diaconis and Holmes , Barbour and Chen . We also refer to Chen et al. a thorough coverage of the method’s fundamentals and recent developments in both theory and applications. The paper is organized as follows. In the next section, we give a brief review on recent developments on Stein’s method. In Section 3, we present the main results in this paper, the Berry–Esseen bounds and Cramér type moderate deviations for Studentized nonlinear statistics. Applications to Studentized U-statistics and L-statistics are discussed in Section 4. The proofs of the main results are in Section 5, while other technical proofs are postponed to Appendix.A sea change in Colorado politics has vaulted the Democratic Party to unprecedented majorities in the state legislature and a stranglehold on statewide elected office. Democratic dominance to this degree appeared unlikely at the turn of the century when Republicans held majorities in both chambers of the General Assembly, a 4-2 advantage in the state’s U.S. House delegation, both U.S. Senate seats, and the governorship. In the razor-thin 2000 presidential election, Colorado cast its eight electoral votes for Texas Governor George W. Bush who comfortably carried the state by eight percentage points. Republican preeminence in state and federal electoral politics disappeared in less than a generation as Colorado became a solidly blue state with an adrift Republican party unable to wage competitive statewide campaigns. Near supermajority status in the General Assembly and firm control over all statewide executive offices has positioned Colorado Democrats with exceptional political power. While the negative economic effects of the COVID-19 pandemic continue to linger, the Colorado economy has generally rebounded from the great upheaval more rapidly than most other states . Economic growth has propelled increases in revenue; however, the Taxpayer’s Bill of Rights imposes substantial constraints on the total amount of funds available for policymakers to distribute. Spending commitments, such as mandatory increases in K-12 education funding as required by Amendment 23, further cut into the total amount available to appropriate, which creates difficult choices for members of the Joint Budget Committee. Ratified into Article X, Section 20 of the state constitution by voters in 1992, TABOR imposes restrictions on both revenue and spending. Because TABOR limits revenue collections to the prior year’s amount plus population growth and inflation, Colorado taxpayers have received $8.2 billion in TABOR refunds since its enactment including $525.5 million in 2021 and a record $3.7 billion in tax refunds in 2022 . Although it is difficult to amend the state constitution, Colorado voters have considered ballot measures proposing TABOR reform in nearly every election cycle since its adoption. Few have succeeded. The approval of just 11 of the 36 ballot measures to amend TABOR corresponds to a failure rate of nearly 70% . As a result of this unsuccessful track record to modify or repeal TABOR, its shadow continues to loom large over budgetary politics in the Centennial state. An exception to the general inability of reformers to modify TABOR occurred in 2005 when voters narrowly approved referendum C with 52% voting yes. This notable exemption to TABOR permitted the state to spend all revenue collected across the next five fiscal years, which resulted in nearly $3.6 billion in spending that would have otherwise returned to taxpayers during this time frame . Beginning in fiscal year 2010, referendum C permits the General Assembly to retain and spend all funds collected up to the “Referendum C cap.” The passage of referendum C provided greater opportunities for financial investment in areas such as health care, education, and transportation, as well as greater support for police, fire fighters, and other first responders. In retrospect, the successful passage of this reform in an off-year election was anomalistic as voters have since rejected several ballot measures to modify TABOR spending limits . Voters have also opposed an array of tax increases on a dozen occasions including proposals to fund public schools and transportation . Sensing an opportunity to capitalize on the public’s desire for property tax relief, Democrats unsuccessfully sought to connect a reduction in the property tax rate with further erosion of TABOR in 2023. Despite slowing population growth, property values across Colorado continue to soar.

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, hydroponic nft system 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. The conductance through a small number of delocalized quantum states is quite generally equal to e 2 h per quantum channel, and so the conductance between two electronic reservoirs in contact with the same edge of a Chern magnet is equal to C e 2 h . These facts together make the conductance and not just the conductivity an intrinsic property of a Chern magnet. This is a remarkable fact; indeed, as long as a researcher has access to a tool for measuring electrical resistance and a Chern magnet, they can directly measure a combination of the fundamental constants e and h through a simple electronic transport experiment- they don’t need to know anything about the geometry or band structure of the Chern magnet. This phenomenon is known as the quantized anomalous Hall effect. Chern magnets often support magnetic hysteresis, just like trivial magnetic insulators. Because the chirality of the edge state is determined by the sign of the Chern number, and the sign of the Chern number is determined by the bulk magnetization, quantized anomalous Hall effects usually exhibit magnetic hysteresis. The quantized anomalous Hall effect is so unique to Chern mangets that that it is sometimes used as to define the entire class of systems; i.e., researchers historically have described these magnets as ‘QAH materials,’ or ‘QAH magnets.’ At finite temperature, electrons occupying Bloch states in metals can dissipate energy by scattering off of phonons, other electrons, or defects into different nearby Bloch states.

This is possible because at every position in real space and momentum space there is a near-continuum of available quantum states available for an electron to scatter into with arbitrarily similar momentum and en-ergy. This is not the case for electrons in chiral edge states of Chern magnets, which do not have available quantum states in the bulk. As a result, electrons that enter chiral edge state wave functions do not dissipate energy. There is a dissipative cost for getting electrons into these wave functionsthis was discussed in the previous paragraph- but this energetic cost is independent of all details of the shape and environment of the chiral edge state, even at finite temperature. This is why the Hall resistance Rxy in a Chern magnet is so precisely quantized; it must take on a value of C 1 e h 2 , and processes that would modify the resistance in other materials are strictly forbidden in Chern magnets. All bands have finite degeneracy- that is, they can only accommodate a certain number of electrons per unit area or volume of crystal. If electrons are forced into a crystal after a particular band is full, they will end up in a different band, generally the band that is next lowest in energy. This degeneracy depends only on the properties of the crystal. Chern bands have electronic degeneracies that change in response to an applied magnetic field; that is to say, when Chern magnets are exposed to an external magnetic field, their electronic bands will change to accommodate more electrons.Simple theoretical models that produce quantized anomalous Hall effects have been known for decades. The challenge, then, lay in realizing real materials with all of the ingredients necessary to produce a Chern magnet. These are, in short: high Berry curvature, a two-dimensional or nearly two-dimensional crystal, and an interaction-driven gap coupled to magnetic order.

It turns out that a variety of material systems with high Berry curvature are known in three dimensions; three dimensional topological insulators satisfy the first criterion, and are relatively straightforward to produce and deposit in thin film form using molecular beam epitaxy, satisfying the second. These systems do not, however, have magnetic order. Researchers attempted to induce magnetic order in these materials with the addition of magnetic dopants. It was hoped that by peppering the lattice with ions with large magnetic moments and strong exchange interactions that magnetic order could be induced in the band structure of the material, as illustrated in Fig. 3.11. This approach ultimately succeeded in producing the first material ever shown to support a quantized anomalous Hall effect. An image of a film of this material and associated electronic transport data are shown in Fig. 3.12. We now have all of the tools we need to begin discussing real examples of Chern magnets on moir´e superlattices. Our discussion will begin with twisted bilayer graphene. We have already discussed the notion that moir´e superlattices can support electronic bands, and that we can expect these bands to accommodate far fewer electrons per unit area than bands in atomic lattices . This was pointed out in 2011 by Rafi Bistritzer and Allan MacDonald, but they also made another interesting observation: the band structure of the moir´e superlattice is highly sensitive to the relative twist angle of the two lattices, and the bandwidth of the resulting moir´e bands can be finely tuned using the relative twist angle as a variational parameter. It turns out twisted bilayer graphene moir´e superlattice bands can be made to have vanishingly small bandwidth by tuning the twist angle to the so-called ‘magic angle.’ The magic angle is around 1.10- 1.15◦ and a schematic of magic angle twisted bilayer graphene is shown in Fig. 4.2A. The computed band structure of twisted bilayer graphene is illustrated in Fig. 4.2B for a few different twist angles, nft channel including the magic angle. The other bands are grayed out at the magic angle to illustrate the low bandwidth of the moir´e superlattice bands. The low bandwidth of the moir´e superlattice bands combined with the low electronic density required to fill them makes them especially appealing targets for electrostatic gating experiments. The system isrelatively easy to prepare; twisted bilayer graphene devices are produced by ripping a monolayer of graphene in half, rotating one crystal relative to the other using a mechanical goniometer, and then overlaying it on the other. The ‘flatness’ of the band also makes this system especially likely to support interaction-driven electronic phases like magnetism or superconductivity .It is also worth mentioning that it is extremely easy to identify situations in which interactions produce gaps in these systems. Because gaps appear when the moir´e superlattice bands are completely filled with electrons or with holes, and we already know that the moir´e superlattice bands are fourfold degenerate, we can expect any interaction-driven insulating phase to appear as an insulating phase at precisely 1/4, 1/2, or 3/4 of the electron density required to reach full filling of the moir´e superlattice band. These are sometimes called ‘filling factors’ of 1, 2, and 3, respectively, referencing the number of electrons per moir´e unit cell. This argument is presented in schematic form in Fig. 4.2 in the context of experimental data.

Interaction-driven gaps were first discovered in 2018, and this discovery was quickly followed by the dramatic discovery of superconductivity in twisted bilayer graphene. Other researchers predicted that breaking inversion symmetry in graphene would open a gap nearcharge neutrality with strong Berry curvature at the band edges. The graphene heterostructures we make in this field are almost always encapsulated in the two dimensional crystal hBN, which has a lattice constant quite close to that of graphene. The presence of this two dimensional crystal technically always does break inversion symmetry for graphene crystals, but this effect is averaged out over many graphene unit cells whenever the lattices of hBN and graphene are not aligned with each other. Therefore the simplest way to break inversion symmetry in graphene systems is to align the graphene lattice with the lattice of one of its encapsulating hBN crystals. Experiments on such a device indeed realized a large valley hall effect, an analogue for the valley degree of freedom of the spin Hall effect discussed in the previous chapter, a tantalizing clue that the researchers had indeed produced high Berry curvature bands in graphene. Twisted bilayer graphene aligned to hBN thus has all of the ingredients necessary for realizing an intrinsic Chern magnet: it has flat bands for realizing a magnetic insulator, it has strong Berry curvature, and it is highly gate tunable so that we can easily reach the Fermi level at which an interaction-driven gap is realized. Magnetism with a strong anomalous Hall effect was first realized in hBN-aligned twisted bilayer graphene in 2019 . Some basic properties of this phase are illustrated in Fig. 4.3. This system was clearly a magnet with strong Berry curvature; it was not gapped and thus did not realize a quantized anomalous Hall effect, but it was unknown whether this was because of disorder or because the system did not have strong enough interactions or small enough bandwidth to realize a gap. The stage was set for the discovery of a quantized anomalous Hall effect in an intrinsic Chern magnet in hBN-aligned twisted bilayer graphene.An optical microscope image of the tBLG device discussed here is shown in Fig. 4.4A. The device is made using the “tear-and-stack” technique, in which one half of a graphene monolayer is torn off, rotated by a precise relative alignment angle , and then placed on top of the other half of the monolayer. The tBLG layer is sandwiched between two hBN flakes with thickness 40 and 70 nm, as shown in Fig. 4.4B. A few-layer-thick graphite flake is used as the bottom gate of the device, which has been shown to produce devices with low charge disorder.The stack rests on a Si/SiO2 wafer, which is also used to gate the contact regions of the device. The stack was assembled at 60◦ C using a dry-transfer technique with a poly film on top of a polydimethylsiloxane stamp. In an exfoliated heterostructure, the orientation of the crystal lattice relative to the edges of the flake can often be determined by investigating the natural cleavage planes of the flake. Graphene and hBN, being hexagonal lattices, have two easy cleavage planes – zigzag and armchair, each with six-fold symmetry, that together produce cleavage planes for every 30◦ relative rotation of the lattice. We tentatively identify crystallographic directions by finding edges of the flakes with relative anglesof 30◦ . From the optical image we find that the cleavage planes of the tBLG layer and the top hBN are aligned. Four-terminal resistance measurements were carried out in a liquid helium cryostat with a 1 K pot and a base temperature of 1.6 K. The measurement was done using AC current excitations of 0.1 – 20 nA at 0.5 – 5.55 Hz using a DL 1211 current preamplifier, SR560 voltage preamplifier, and SR830 and SR860 lock-in amplifiers.

At the time I started my PhD, experiments in condensed matter physics had already begun a rapid expansion in their ability to conduct in situ modifications of the electronic structure of crystals, and in particular the electron density within crystals. The number of electrons per unit volume is an extremely important property of a crystal; it determines which quantum states within the crystal are filled and which are empty, and thus whether the system is metallic, insulating, or even something more exotic. Almost all of the properties of a crystal are impacted by electron density. Historically the number of electrons in a crystalline system has primarily been modified by adding dopants, i.e., additional atoms with more or fewer conduction electrons than the rest of the crystal .One can achieve dramatic changes in charge density using this technique, but that comes with a heavy cost- the crystal is no longer uniform, as every dopant contributes to disorder, and at high doping levels the band structure itself can be modified by the dopant atoms. More important than all of this for the purposes of experimental physics, however, is that under most circumstances the dopant concentration within a crystal can only be modified through laborious chemical treatments of a particular sample. Materials scientists working under these constraints who wish to explore electron density as an independent variable must either find ingenious material-specific techniques for modifying the dopant concentration in situ , or else they must make a separate sample for each data point they would like to present in their experiment. This is an incredibly labor-intensive process, hydroponic indoor growing system and it also comes with another significant downside: comparing the properties of two different samples with different doping densities exposes results to systematic differences in sample geometry and imperfections in protocol repeatability, and it is difficult to deconvolute these from the effects of changes in electron density.

For these reasons electron density has generally been an awkward and labor intensive independent variable to manipulate. I have found that there are a few ideas that occur naturally to newcomers and outsiders to the field that insiders know enough to immediately discount, and I’d like to discuss one of those ideas here. Chemical doping to manipulate electron density is an ingenious and important technique, but suppose we tried something much sillier- suppose we simply forcefully deposit electrons onto a crystal using some mechanical or electrical process. Would this not achieve our goal? In fact this does indeed work, we have machines that can do this- van de Graff generators can deposit charge onto a piece of metal mechanically, and a variety of other machines can mimic this behavior electronically. So why aren’t condensed matter physicists going around gluing interesting crystals to van de Graff generators so that we can controllably charge them up and measure their responses to changes in charge density? There are a few reasons, but the most important one is that there is a fundamental issue with manipulating charge density this way in three dimensions: this process does not produce a uniform distribution of electron density within the crystal we’d like to study. In three dimensional systems subjected to this treatment, as illustrated in Fig. 1.1B, excess charge accumulates on the surfaces of the crystal, and although we can force additional electrons into acrystal this way we do not ultimately get a system with a modified but still uniform electron density for us to study. This is not the case for two dimensional systems.

Those readers with any exposure to introductory physics have likely encountered parallel plate capacitors; these are highly idealized systems composed of a pair of infinitely thin conducting sheets separated by a small insulating space of consistent thickness. When a voltage is applied to one of these sheets with the other connected to a reservoir of mobile electrons, a uniform charge density per unit area appears on both sheets . Of course, in real metallic capacitors the charge density per unit volume is often still not microscopically uniform because the sheets are not actually infinitely thin, so electrons can redistribute themselves in the out-of-plane direction. To achieve true uniformity one of the plates of the capacitor must be atomically thin, so that electrons simply cannot redistribute themselves in the out-of-plane direction in response to the local electric field. An efficient technique for preparing atomically thin pieces of crystalline graphite was discovered in 2004 by Dr. Andre Geim and Dr. Konstantin Novoselov, an achievement for which they shared the Nobel prize in physics in 2010. The technique involves encapsulating a crystal within a piece of scotch tape and repeatedly ripping the tape apart; it works because the out-of-plane bonds in graphite are much weaker than the in-plane bonds. Graphite represents something of an extreme example of this condition, but it is satisfied to varying extents by a large class of other materials, and as a result the technique was rapidly generalized to produce a variety of other two-dimensional crystals. By constructing a capacitor with one gate replaced with one of these two dimensional crystals, as shown in Fig. 1.1D, researchers can easily access electron density as an independent variable in a condensed matter system.

These systems also facilitate an additional degree of control, with no real analogue in three dimensional systems. By placing capacitor plates on both sides of the two dimensional crystal and applying opposite voltages to the opposing gates, researchers can apply out-of-plane electric fields to these systems . A semiclassical model- in which electrons within the system redistribute themselves in the out-of-plane direction to screen this electric field- does not apply; instead, the wave functions hosted by the two dimensional crystal are themselves deformed in response to the applied electric field . This changes the electronic band structure of the crystal directly, without affecting the electron density. So to summarize, when a two dimensional crystal is encapsulated with gates to produce a three-layer capacitor, researchers can tune both the electron density and the band structure of the crystal at their pleasure. In the first case, this represents a degree of control that would require the creation of many separate samples to replicate in a three dimensional system. The second effect cannot be replicated in three dimensional systems with any known technique.There is a temptation to focus on the exotic phenomena that these techniques for manipulating the electronic structure of two dimensional crystals have allowed us to discover, and there will be plenty of time for that. I’d first like to take a moment to impress upon the reader the remarkable degree of control and extent of theoretical understanding these technologies have allowed us to achieve over those condensed matter systems that are known not to host any new physics. I’ve included several figures from a publication produced by Andrea’s lab with which I was completely uninvolved. It contains precise calculations of the compressibility of a particular allotrope of trilayer graphene as a function of electron density and out-of-plane electric field based on the band structure of the system . It also contains a measurement of compressibility as a function of electron density and out-of-plane electric field, performed using the techniques discussed above . The details of the physics discussed in that publication aren’t important for my point here; the observation I’d like to focus on is the fact that, for this particular condensed matter system, quantitatively accurate agreement between the predictions of our models and the real behavior of the system has been achieved. I remember sitting in a group meeting early in my time working with Andrea’s lab, long before I understood much about Chern magnets or any of the other ideas that would come to define my graduate research work, and marvelling at that fact. Experimental condensed matter physics necessarily involves the study of systems with an enormous number of degrees of freedom and countless opportunities for disorder and complexity to contaminate results. Too often work in this field feels uncomfortably close to gluing wires to rocks and then arguing about how to interpret the results, grow hydroponic with no real hope of achieving full understanding, or closure, or even agreement about the conclusions we can extract from our experiments.

Within the field of exfoliated heterostructures, it is now clear that we really can hope to pursue true quantitative accuracy in calculations of the properties of condensed matter systems. Rich datasets like these, with a variety of impactful independent variables, produce extremely strong limits on theories. They allow us to be precise in our comparisons of theory to experiment, and as a result they have allowed us to bring models based on band structure theory to new heights of predictive power. But most importantly, under these conditions we can easily identify deviations from our expectations with interesting new phenomena- in particular, situations in which electronic interactions produce even subtle deviations from the predictions of single particle band structure theory.This is more or less how I would explain the explosion of interest in the physics of two dimensional crystalline systems within experimental condensed matter physics over the past decade. If you ask a theorist if two dimensional physical systems have any special properties, they will tell you that they do. They might say that the magnetic phase transitions in a Heisenberg model on a two dimensional lattice differ dramatically from those on a three dimensional one. They might say thathigh Tc superconductivity is apparently a two dimensional phenomenon. They might note that two dimensional electronic systems can support quantum Hall effects and even be Chern magnets , while three dimensional systems cannot. But it is easy to miss the forest for the trees here, and I would argue that interest in these particular physical phenomena is not behind the recent explosion in the popularity of the study of exfoliated two dimensional crystals in condensed matter physics. Instead, much more basic technical considerations are largely responsible- it is simply much easier for us to use charge density and band structure as independent variables in two dimensional crystals than in three dimensional crystals, and that capability has facilitated rapid progress in our understanding of these systems. The techniques described above still have some limitations, and chief among them is a limited range of electronic densities that they can reach. Of course, the gold standard of electron density modulation is the ability to completely fill or deplete an electronic band, which requires about one electron per unit cell in the lattice. Chemical doping can achieve enormous offsets in charge density, sometimes as high as one electron per unit cell. This limitation isn’t fundamental and there are some ideas in the community for ways to improve it, but for now it remains true that electrostatic gates can modify electron densities only slightly relative to the total electron densities of real two dimensional crystals. As it stands, electrostatic gating can only substantially modify the properties of a crystal if the crystal happens to have large variations in the number and nature of available quantum states near charge neutrality. For many crystals this is not the case; thankfully it is for graphene, and for a wide variety of synthetic crystals we will discuss shortly. Electrostatic gating of two dimensional crystals was rapidly becoming a mature technology by the time I started my PhD. So where does nanoSQUID magnetometry fit into all of this? A variety of other techniques exist for microscopic imaging of magnetic fields; the most capable of these other technologies recently developed the sensitivity and spatial resolution necessary to image stray magnetic fields from a fully polarized two dimensional magnet, with a magnetization of about one electron spin per crystalline unit cell, and this was widely viewed within the community as a remarkable achievement. We will shortly be discussing several ferromagnets composed entirely of electrons we have added to a two dimensional crystal using electrostatic gates. Because of the a fore-mentioned limitations of electrostatic gating as a technology, this necessarily means that these will be extremely low density magnets with vanishingly small magnetizations, at least 100 times smaller than those produced by a fully polarized two dimensional magnet like the one in the reference above. It is difficult to summarize performance metrics for magnetometers, especially those used for microscopy.

The basic dynamics are illustrated in cartoon form in figure 8b. To the extent there exists evidence to support the ideas of chaos and basin or boundary collision as represented in figure 8, we display some data in figure 9. The expectation from a chaotic approach to a basin boundary collision is complicated and requires a far more dense data set than we currently have, but what is available certainly supports the idea that, as the system approaches the refuge, it undergoes complicated and relatively unpredictable behavior within a hysteretic zone. A complicating issue is the fact that the scale insect population density is the main determinant of the probability of infection, and that density increases as the refuge is approached. As the refuge is approached, we expect an increase in total scale population and an increase in the fungal infection rate. With the increase in total scale population we expect an increase in susceptible scales as the refuge is approached. Within the refuge, we expect the signal of flickering , which, in this case, would result in a bimodal distribution of bushes, many with no infection and many with high infection with few intermediates. That is precisely the pattern suggested by the data presented in figure 9. At the periphery of the refuge , we generally have very few scales and low levels of disease incidence. Moving further into the refuge area, we see a gradual buildup of disease incidence , culminating in bimodality when we arrive at a distance of 0–1 meters. What is not immediately obvious from this analysis is that there are really three qualitatively distinct outcomes at a local level, which is to say on a particular coffee bush. If the bush is very close to the Azteca nest, large round pot the ants forage vigorously and, therefore, the scale insects are highly protected from the beetle predator. Consequently, they build up very high local populations, and are subjected, eventually, to the white halo fungus disease, effectively eliminating the entire population .

At the other extreme, when the coffee bush is far removed from the ant nest, the scale insect is constantly attacked by the adult beetles and never is able to build up a substantial population. Therefore, it would appear that either very close to an Azteca nest or very far away from an Azteca nest thegreen coffee scale is kept under control. However, in the real world, there is no such thing as only far away or very near; many coffee bushes are neither. The result is a complex system in which the Azteca ant forms a reaction or diffusion Turing-like pattern-forming complex that acts as a pilot structure, driving the spatially dependent direct control system. The ant exerts a behavioral restriction on the beetle , but the phorid exerts a behavioral restriction on the ant , causing what has been referred to as a trait-mediated cascade of effects and imposing a hypergraph-like structure on the system . The concentration of ants creates refuges within which the adult beetle predator is restricted from active predation but within which the pest, the scale insect, builds up very dense local populations, the consequence of which is a high attack rate of the white halo fungus disease. Regulation of this herbivore is therefore effected through a complex system involving a Turing process, nonlinear indirect interactions, critical transitions, hysteresis, chaos, basin or boundary collisions, and a hypergraph, all elements of the burgeoning field of complex systems. The elements of the system are illustrated in figure 10. Note the central role of the Azteca ants. It is worth emphasizing that these ants are obligate tree nesters, which suggests that the trend to eliminate the shade trees in the system, thought to be a modernizing effort, completely breaks down this complex system. An interesting complication emerges as we understand the importance of these two obvious natural enemies of the scale insect . We began by proposing a predator–prey driven Turing mechanism to generate the clustered distribution of ant nests, a clear application of what has previously been noted; predator–prey systems distributed in space can generate Turing-like patterns.

The predator was the phorid parasitoid, and the prey was the Azteca ant. However, the spirit of the Turing mechanism involves only generalized reaction and diffusion terms, wherein the reaction is thought to be a coupled positive or negative effect. And in the present example, there are two clear negative effects on the ant through the attack on its food. Both the predatory beetle A. orbigera and the white halo fungus are enemies of the scale insect, the main food of the Azteca, and therefore both constitute a negative effect. It has been independently suggested that either the beetle or the fungus could be the repression agent that generates the Turing-like pattern. If the beetle is the cause of the pattern formation, it is an especially interesting situation in that the beetle population itself is dependent on the existence of the spatial pattern for its own survival but is the cause of the formation of that pattern in the first place .Perhaps the most directly obvious of the three pests is the infamous coffee berry borer, because of its habit of drilling directly into the seed, which is the basic commodity that goes to market. It emerged as a major pest in the 1980s and is regarded as far more important, on most farms, than the green coffee scale. The literature on the coffee berry borer is now enormous because of its sometimes devastating effect . A variety of natural enemies have been reported, including the fungus Beauveria bassiana , anole lizards , birds , possibly bats , and parasitic Hymenoptera . But, by far, the most obvious natural enemies are ants. There is now a substantial literature documenting the general category of ants as major predators on this seed-eating herbivore . As in the case of the predacious beetle on the scale insect, some rather casual observations can easilyconvince one that, in particular, the Azteca ants are major predators to the coffee berry borer, and a variety of detailed studies support that conviction . However, further examination reveals another major ant predator, Pheidole synanthropica, a rather large-body species that nests in the ground but forages vigorously both on the ground and in the coffee bushes. Detailed observations established that this species is a major predator of the berry borer. It takes the berry borer approximately 1–2 hours to completely burrow into the fruit , which means it is unprotected and unable to escape the predacious activity of the ants for that period of time. Both Azteca and P. synanthropica, if they encounter a berry borer trying to burrow into a seed, grab the borer by its posterior end and pull it out of the fruit. Azteca tends to simply throw the borer off the tree , whereas P. synanthropica almost inevitably takes the borer back to its nest.

Therefore, we might say that Azteca is mainly a general antagonist to the berry borer, whereas P. synanthropica is definitely a predator . The consequences of this difference are quite important. Although Azteca provides some protection to the coffee from the ravages of the berry borer, when the borer is simply thrown to the ground it can easily climb back up and try again to bore into the seed. Indeed, there is some reason to suspect that the borer actually prefers to burrow in seeds that are protected by ants, presumably taking advantage of the ant’s mutualistic behavior toward the scale insects and protecting it from other predators that may attempt to enter the seed in which it is eating endosperm and creating the new generation of berry borers. However, this strategy is compromised by other species of predators that can directly prey on the borers when they are thrown to the ground, one of which is P. synanthropica. And this species is highly aggressive, big round plant pot limiting the activity of the smaller species . A number of these smaller species of ants are also known predators of the coffee berryborer . These species offer considerable regulatory potential because they are capable of entering the coffee seed through the hole that the borer makes . One group is the twig-nesting complex, including the genus Pseudomyrmex , and Procryptocerus scabriusculus, all adept at entering hollow arboreal structures because they normally nest in hollow twigs . Other small arboreal ants capable of entering the hole made by the berry borer include the arboreally nesting Solenopsis picea, which nests in superficial structures, such as moss, surrounding the branches of the coffee bushes . On the ground, a variety of ground foraging ants, including Pheidole protensa , and a variety of other species in that same genus are small enough to enter the borer’s hole. Of particular interest is the well-known Wasmannia auropunctata , which nests and forages on both the ground and arboreally . There are many other potential ant predators in the system, but these are the ones we have studied in particular. Azteca clearly dominates over P. synanthropica, and both of them dominate over the smaller species in the system, reducing their nest density significantly . In summary, there are at least six species of ants that are predators on the coffee berry borer, suggesting that ants represent an excellent natural enemy to regulate the coffee berry borer. However, the foregoing natural history suggests that the system is not so simple. Although several of the smaller arboreal species could be effective predators on adults, larvae and pupae of the berry borer within the fruit on the bush, they are effectively unable to engage in such predation if Azteca or P. synanthropica ants are around. Fruits that are not harvested tend to dry out and fall to the ground, providing a refuge for the beetles during the dry season but also being exposed to the potential predation from the smaller ants . However, those smaller ants have dramatically reduced populations if they are forced to compete with P. synanthropica, which, because of its larger size, is unable to penetrate the borer hole in the fruit. In other words, the whole system seems to be operating in a complicated fashion with potential predators interfering with one another but perhaps acting in an emergent fashion to at least partially regulate this key herbivore, the coffee berry borer.Adding to this complication is the phorid fly. As was noted earlier, this fly has an important trait-mediated effect on the Azteca ants, the foundation of the Turing patterns we seem to see at a large scale and effectively contributes to the maintenance of the major predator of the green coffee scale . However, because, as all evidence suggests, the Azteca ants so dominate the coffee bushes where they forage that the smaller ants are unable to persist there, we might expect the same sort of trait-mediated cascade we saw with the control of the green coffee scale. Indeed, in controlled laboratory settings, the coffee berry borer has its success rate of penetrating coffee fruits reduced in the presence of the phorid flies. The importance of this effect is in the addition of what has been referred to as vertical biodiversity to the system , the smaller ants who had been effecting control over the berry borer were restricted from doing so by the action of the Azteca, but when the phorids were introduced, the smaller ants again became effective predators. Although the Azteca ants reduced the effectiveness of berry borer predation from these smaller ants , the phorids reduced the effectiveness of the Azteca in reducing the effectiveness of the smaller ants in their effectiveness in controlling the berry borers, a similar trait-mediated cascade that we saw for the green scale control. All of this is summarized in figure 12. It is tempting to conclude something like “the more ants the better.” However, cascading indirect effects sometimes can have unexpected consequences, meaning that such a conclusion ought to be tempered with more careful analysis. Although we acknowledge this system as complex, it makes some sense to try and simplify it a little to perhaps gain some deeper insight into its operation.

Previous works show berry sunlight exposure to alter the composition of anthocyanins, such as the proportion of acetylated and coumarylated forms . Modulation of acylated, methylated, and hydroxylated forms of anthocyanins result from the synergistic effect of solar radiation exposure and the coupled increases in berry temperature . Generally, high berry temperatures resulting from increased solar exposure results in increased acylated anthocyanins in the grape berry, particularly coumarylated forms . Also, high temperatures result in accumulation of highly methylated anthocyanins such as malvidin derivatives, as these compounds are less likely to degrade than their counterparts . In 2020, D1 and D5 wines demonstrated highest concentrations of acetylates, coumarylates, and methylated anthocyanins compared to C0 wines. While D1 and D5 treatments demonstrated cluster temperatures less than those from C0 treatments , the concomitant thermal degradation of total anthocyanins in C0 treatments proved to negate any modulation towards acylated or methylated forms in resultant wines. Similarly in 2021, C0, D1 and D5 wines exhibited reduced acylation compared to D4 wines. Again, while D4 consistently exhibited less intense cluster temperatures, square plastic plant pots the thermal degradation in more exposed treatments eclipsed any identifiable acylation modulation from hot growing conditions. Acylated anthocyanins are more stable compounds and provide color stability and increase blueness in wine . However, an increase in methylated anthocyanins will lead to redder hues in wine .

Therefore, the improvement in acylated and methylated anthocyanin content due to partial solar radiation exclusion may enhance color perception in young red wines through color stabilization and alteration of wine hue. Likewise, anthocyanin hydroxylation is also directly influenced by temperature and solar radiation exposure. Previous studies on berry exposure utilizing UV selective shade nets as well as leaf removal, demonstrated anthocyanin tri-hydroxylation increases with increasing berry temperature . Increases in trihydroxylation are driven by accumulation of malvidin derivatives and the temperature sensitivity of F3’H, the catalyzing enzyme for 3’- hydroxylated anthocyanin biosynthesis . The highest ratio of tri- to di-hydroxylated anthocyanins in 2020 C0 wines were driven by higher concentrations of 3-pcoumaroyl-glucoside derivatives of delphinidin, petunidin and malvidin, despite the ratio of tri- to di-hydroxylated anthocyanins being unaffected at harvest in the grape berry in 2020 . Among shade film treatments in 2020, the reduction of UV light exposure, was the determining factor in anthocyanin hydroxylation patterns rather than berry temperature. Previous shade net studies at the experimental site showed a reduction in UV radiation with black-40% and blue-40% shade nets led to higher anthocyanin tri-hydroxylation in the grape berry compared to control vines at harvest . With the reduction of UVB and UVC radiation in D4 and D5 vines, anthocyanin trihydroxylation was reduced, regardless of temperature. Ultimately, the upregulation of F3’H from sun exposure could be negated by the reduced catalytic activity of this enzyme under high temperatures experienced in 2020.

In the cooler 2021 vintage, the ratio of tri- to dihydroxylated anthocyanins was unaffected, due to non-significant effect of shade films on acetylated anthocyanins. Ultimately, increased tri-hydroxylation in young red wines will also impact wine hue, resulting in more purple wines . Flavonols in the grape berry skin act as a photoprotectant and are strongly induced by ultraviolet radiation . Flavonol composition in the grape berry can be used to determine overexposure, specifically by quantifying the molar abundance of kaempferol. C0 berries in this study were shown to be overexposed by surpassing the previously described threshold of approximately 7% molar abundance of kaempferol . In both years of the study, flavonol composition in grape berries was maximized in C0 fruit, but D4 and D5 fruit contained the most flavonols across the shade films with minimal thermal degradation of the compounds on the vine . Likewise in both wine vintages, flavonol concentration was modulated by UV radiation exposure, proportional to the amount of UV radiation transmitted to the grapevine. Of the wines produced from shade films treatments, D4 allowed for the most UV transmission while subsequently reducing near infrared transmission by approximately 15%. These light conditions ultimately optimized flavonol content in D4 wines compared to the other shade treatments from both wine vintages. As such, this demonstrated the transmissibility of berry composition under shade treatments to directly improve wine flavonoid profiles. For hot viticulture regions, photoselective solar radiation exclusion provides a strategy to improve not only flavonoid profile but also wine color intensity through copigmentation with anthocyanins.These compounds are derived from microbial mediated cleavage of the C-C double bonds in linoleic and linolenic acids, by lipoxygenase and alcohol dehydrogenate enzymes in yeast . Compounds such as 1-hexanol and -2-hexen-1-ol are associated with aromas such as cut grass, green, fat and herbaceous aromas and their OAV thresholds are 8000 and 400 ug/ L, respectively . The effect of shade films on C6-alcohols was evident in both years; however, there was a yearly effect on which alcohol was altered by the treatment. In 2020, -2- hexen-1-ol was the lowest in D4. In 2021, -2-hexen-1-ol was unaffected by shade films, while 1-hexanol was highest in C0, D4 and D5. Although there was a statistical difference in C6 alcohols, the differences were not large enough between C0 and treatments to crossthe OAV thresholds for these compounds. Increases of C6-alcohols in C0, D4, and D5 wines may be explained by solar radiation overexposure in the treated clusters. L. He et al. reported higher linoleic and linolenic acid biosynthesis with leaf removal at veraison. Subsequently, fruit exposed to increased solar radiation had elevated precursors for C6- alcohol production during yeast metabolism. Additionally, L. He et al. showed higher initial concentration of C6-alcohols in grape berries from leaf removal treatments due to modulation of the volatile compound metabolome and transcriptome in grape berries exposed to sunlight under dry-hot conditions. Therefore, in our experiment which has similar climatic conditions to L. He et al. , fruit from shade films with higher percentages of UV radiation may have both an increase in linoleic and linolenic acids to act as C6-alcohols aromas precursors and increased C6-alcohols in the exposed grape berries. Ultimately, overexposure of the grape berry led to more green and grassy aromas in wine, which may lead to an unripe perception of these wines. Higher alcohols are also produced during fermentation from yeast metabolism of amino acids. These compounds are generally pleasant aromas including mushroom, roses, honey, candy and fruity notes. Of these compounds, shade treatments increased isoamyl alcohol concentration in 2020 and benzyl alcohol concentration in wines from both vintages. Isoamyl alcohol is associated with solvent and cheese aromas and, while benzyl alcohol is characterized as being citrusy and sweet . The odor active thresholds for these compounds are 30000 mg/L and 10000 mg/L, respectively . In 2020, C0 had the lowest concentration of isoamyl alcohol in wines. The effect of shading on the concentration of isoamyl alcohol in wines varies in literature . In hot growing regions, 75% of total solar radiation exclusion with black polyethylene canopy side shade nets resulted in wines with reduced isoamyl alcohol compared to the uncovered control vines .

However, this experimental site was in a region that received approximately 704.5° C less growing degree days than the present experimental site in the hotter 2020 season, and 514.1°C growing degree days less than the cooler 2021 season. In the study by Lu et al., 2021, reduced solar radiation exposure in a cooler growing region may have resulted in reduced isoamyl alcohol in shaded fruit. When cluster temperatures exceed 42°C in exposed vines, there is a reduction in isoamyl alcohol in resultant wines compared to wines produced from fruit under red and black shade nets . With cluster temperatures of C0 fruit exceeding 42°C, excessive cluster temperatures may be prompting the reduction in isoamyl alcohol and overall wine fruitiness from those produced from overexposed clusters. However, square pot plastic while there was a statistical difference in isoamyl alcohol concentrations between C0 and treatment wines, the effect was not large enough to exceed the OAV threshold for this compound . Shade films affected the ester composition predominantly in 2020 wines. Pleasant esters in red wines include ethyl acetate which has a OAV threshold of 12264 µg•L-1 and is described as fruity and balsamic , as well as isoamyl acetate, described as banana aroma with a OAV threshold of 30 µg•L-1 . In 2020, ethyl acetate was reduced in C0 and D5 wines, shading and reduced cluster temperatures preserved isoamyl acetate aromas in D1, D3 and D5 wines. When compared to wines from 2021, cooler vintage conditions did not result in ester compositional changes in exposed and shaded wines. Similarly, fatty acid esters were preserved in shaded wines, while 2020 C0 wines consistently had the lowest concentration of all measured fatty acid ethyl esters and various esters, all of which are associated with fruity and candy-like aromas . Concentrations of ethyl octanoate and ethyl decanoate remained beneath the reported perception threshold, thus observed shifts in composition with shading may be undetectable in Cabernet Sauvignon wines. However, ethyl hexanoate and ethyl isovalerate have remarkably low OAV thresholds of 5 µg•L-1 and 1 µg•L-1, respectively . In the present study, all wines were above these thresholds, indicating that reductions in fruitiness may be perceived. This overall decrease in fruity aromas with cluster exposure and excess temperatures may negatively impact the marketability of Cabernet Sauvignon wines from hot viticulture regions with increasingly more frequent heat wave events associated with climate change. Unpleasant and rancid aromas include isobutyric acid which imparts a cheese aroma and benzaldehyde which is associated with almond aroma in red wines . In this study, isobutyric acid concentrations were only affected in 2020, with D4 having the highest isobutyric acid concentration. The detection threshold for this aroma compound is 2300µg•L-1 . Concentrations detected in the experimental wines were substantially below this threshold, indicating that this slight increase in rancid aromas in D4 wines may not negatively impact overall wine perception. Given that D4 wines also exhibited enhanced fruitiness in with improved ester composition, the trade-off of slight increases in rancid aromas may be offset by the net benefit from increased fruity aromas in the wine aroma profile. While terpenes are often critical in white wines, these compounds when present in red wines have a large effect on wine aromas as their OAV thresholds are relatively low . The OAV threshold for a-terpinene, cis-rose-oxide and linalool are 250 µg•L-1 , 0.2 µg•L-1 and 25.2 µg•L-1 , respectively . The In 2020, aterpinene, cis-rose-oxide and linalool were all reduced in C0 wines compared D4 and D5 wines, however concentrations of these compounds did not exceed the OAV threshold. These compounds produce odors such as peach, citrus, rose, and floral aromas in red wines . Previous work indicated an increase in terpenoids, particularly linalool in wines produced from fruit under black and red shade nets . It was demonstrated that heat treatment will down-regulate genes encoding key enzymes in terpenoid metabolism in Cabernet Sauvignon grapevines . Thus, increases in terpenoid content in shade film wines in 2020 may be due to reduced cluster temperature in a growing season with frequent heat wave events. In 2021, C0 wines exhibited the highest concentration aterpinene, while cis-rose-oxide concentrations remained low in C0, and linalool was unaffected. In 2021, a cooler growing season with fewer days above 38°C may have resulted in less variation in terpenoid composition and net accumulation of terpenoids in exposed fruit . Ultimately, climatic shifts towards more frequent heat wave events will reduce floral and citrus aromas in wines produced from overexposed clusters. However, the year-to-year weather variation will enhance the unpredictability of the development of these compounds, leading to challenges for wine producers looking to produce a consistent product.As carotenoid breakdown products, C-13 norisoprenoids like bdamascenone often described by sweet and floral aromas and has an OAV threshold of 0.05 µg•L-1 . C-13 norisoprenoids have been shown to have a positive linear relationship with sunlight exposure to the grape cluster . Under extreme light intensity and temperature conditions, there are decreases in carotenoid concentration in the berry, thus reducing C-13 norisoprenoid precursors. In the present study, b-damascenone was highest in C0, D4 and D5 wines in 2020, while b-damascenone was highest in C0 and D1 wines in 2021, contrary to previous findings in hot viticultural areas. Lee et al. reported that grape clusters without leaf removal and inner canopy clusters contained more b-damascenone than south-facing clusters exposed to solar radiation by leaf removal. Likewise, black cloth and red shade net enhanced b-damascenone concentration compared to uncovered control .

We note that different grape varieties were used as recipient test vines in the field and laboratory studies, which limits the direct comparison of the two studies. In addition, plants used in the laboratory study were only tested for GLRaV-3 ; it is possible that interactions among virus species could have influenced vector transmission and pathogen establishment. The physiological status of mature field vines compared to greenhouse cuttings that were several weeks old at the time of inoculation may also influence virus transmission. Despite these relevant caveats, the overall finding is suggestive that laboratory studies may overestimate vector transmission efficiency. For example, Hooks et al. also found a higher transmission efficiency of Banana bunchy top virus by aphids based on laboratory experiments compared to commercial agricultural conditions. Another study that included transmission of Cucumber mosaic virus and Zucchini yellow mosaic virus by multiple aphid species found that the relative transmission rates between field and laboratory conditions depended both on aphid and virus species . We propose that this question should be addressed in more detail in future studies due to its epidemiological relevance. While our findings are informative, similar studies should be performed with other mealybug species, genetically distinct variants of GLRaV-3, grape cultivars, and in different climatic conditions to evaluate the general applicability of the results . For example, black plant pots plastic berry quality of white wine varieties is affected by GLRaV-3 infection, yet resulting disease symptoms are not readily identifiable .

Another open question with respect to disease progression is the amount of time that passes after graft-mediated infections before symptoms can be identified or diagnostic tools can detect new GLRaV-3 infections in the recipient vine. Even though spread of grapevine leafroll disease was documented 25 years ago , many parameters that contribute to spread and progression remain poorly understood. In Napa Valley, Ps. maritimus produces two synchronous generations per year that do not overlap, and our inoculation study coincided with the emergence of the second generation in July . Differing climatic conditions during the first generation, which usually emerges in March, could potentially lead to different transmission efficiency and different timing of disease development. Varied climatic conditions could lead to variation in host traits and resulting host susceptibility, variation in the virus life cycle in response to varying environmental conditions within host and vector, and variation in mealybug activity that could lead to higher or lower transmission efficiencies. There is a need for understanding how the interactions between host, vector, and pathogen are affected by climate and seasonality. Other mealybug species in California produce higher numbers of overlapping generations; therefore vines may be vulnerable to new infections throughout the year . For example, Pl. ficus produces four to seven overlapping generations per year, and is not yet present in most areas of Napa Valley, CA, where our field study was conducted . If the invasive Pl. ficus establishes more widespread populations in Napa Valley and other areas of Northern California at the higher densities typically associated with this species, spread of GLRaV-3 could dramatically increase. In summary, the information provided by our study regarding key biological traits of GLRaV-3 can inform sound management practices. For example, when certified virus-free material has been used for planting, newly symptomatic vines can be used to infer that the infection resulted from insect-borne inoculations made during the previous growing season, and that the newly symptomatic vines can be an efficient source for further disease spread.

Decline in crop quality can be expected during the same growing season in which symptoms first appear, which may influence roguing strategies based on economic models .There is natural spatial variability present in vineyards due to the variations in soil characteristics and topography . Soil characteristics are too complex to be thoroughly surveyed effortlessly. With traditional destructive methods, it is difficult to obtain enough comprehensive information from the soil pits at the field scale. These soil characteristics may directly affect the water availability for grapevines, which eventually determine the physiological performance of the plants . However, there is no variable management practices currently available to accommodate the natural spatial variability. Thus, the spatial variability derived from vineyard soils will inevitably be expressed in the whole plant physiology at the cost of homogeneity of vineyard productivity and quality. We previously reported the spatial variation of midday stem water potential affecting grapevine carbon assimilation and stomatal conductance of grapevine . The resultant variations in whole plant physiology were associated to flavonoid composition and concentration at the farm gate. However, there is a lack of information about the effects on the chemical composition in the final wine, which would ultimately determine wine quality as perceived by consumers. Georeferenced proximal sensing tools can capture the spatial and temporal variability in vineyards, making it possible to supervise and manage variations at the field scale . Previous studies showed that soil bulk electrical conductivity may be used to evaluate many soil attributes, including soil moisture content, salinity, and texture . Soil electromagnetic induction sensing has been used in precision agriculture to acquire soil bulk EC at the field scale due to its non-invasive and prompt attributes .

Although research had been conducted on the relationships between soil electrical properties with plant water status, they were mostly point measurements and the results were rarely interpolated to whole fields. There were only a few studies that investigated the EMI sensing and soil-plant water relationships over a vineyard . Previous research suggested that the connection between soil water content and soil bulk EC could have relied on specific soil profiles, and needed to include soil physical and chemical properties to complete this connection . Nevertheless, there is evidence that soil bulk EC may still be useful not only to identify the variability in soil, but also in the plant response affected by vineyard soils such as yield, plant physiology, and grape berry chemistry . Plant available water is a determinant factor on grapevine physiology, together with nitrogen availability in semi-arid regions . Wine grapes are usually grown under a moderate degree of water deficits as yields were optimized at 80% of crop evapotranspiration demand with sustained deficit irrigation . Water deficits would limit leaf stomatal conductance and carbon assimilation rate that sustain grapevines’ vegetative and reproductive growth and development . When grapevines are under water deficits, carbohydrates repartitioned into the smaller berries would enhance berry soluble solids content . Sucrose and fructose, which are the major components of total soluble solids in grape berry, can act as a signaling factor to stimulate anthocyanin accumulation . The effects on grapevine physiology and berry composition also depend on the phenological stages they occur and how severe and prolonged the water deficits are . Flavonoids are the most critical compounds dictating many qualitative traits in both grape berries and wine . The variations in environmental factors could alter the concentration and biosynthesis of flavonoids and can be extrapolated spatially within the same vineyard, including water deficits , solar radiation , and air temperature . Among flavonoid compounds, anthocyanins are responsible for the color of berry skin as well as wine . Moderate water deficits during growing season can increase anthocyanin concentration in berry skin and wine . However, water deficits can impair plant temperature regulation through evaporative cooling . They may also inhibit berry growth by limiting berry size and altering berry skin weight . Thus, in some cases it may be uncertain if water deficit promotes anthocyanins biosynthesis or reduces berry growth, or contributes to anthocyanin degradation . Applying water deficit on grapevines can contribute to greater proportion in tri-hydroxylated over dihydroxylated anthocyanins due to the up-regulation of F30 5 0 H. Another major class in flavonoids, proanthocyanidins, are polymers of flavan-3-ol monomers and they contributes mainly toward astringency or bitterness in wine . Compared to anthocyanins, water deficits showed mild effects on proanthocyanidins . However, water deficits with great severity can still alter the concentration and composition of proanthocyanidins in both berries and wine . Selective harvest is one of the targeted management strategies to minimize the spatial variation in berry chemistry in vineyards . By differentially harvesting or segregating the fruits into batches prior to vinification, large plastic pots for plants the berry composition can be artificially set at a more uniform stage with minimal variations . In our previous work, we reported the use of plant water status to determine the spatial variation of grape berry flavonoids . The goal of this study was to deduce if the spatial variability of soil bulk EC and differences in soil texture can be related to plant physiology and grape and wine composition.

The specific objective of the study was to determine if the spatial variability of proximally sensed vineyard soil bulk EC would affect plantwater status, and if this relation would affect leaf gas exchange, components of yield, berry composition, and flavonoids in both berries and wine.Soil bulk EC was assessed with EM38 in 2016 when the vineyard soil was at field capacity condition. Both vertical dipole mode and horizontal dipole mode were used to assess EC at two depths, including deep soil and shallow soil . The instrument was calibrated according to manufacturer instructions. The device was placed on a PVC sled and driven through the vineyard with an allterrain vehicle along the inter-rows. A distance of approximately 0.5 m from the vehicle to the device was maintained to avoid interference with the vehicle. A stratified grid was used to collect soil samples corresponding to the two depths at which we measured soil bulk EC. Soil texture was assessed according to the soil analysis method: hydrometer analysis in the North American Proficiency Testing program.Geostatistical analysis was performed in the R language by using package “gstat” 1.1-6 . The bulk EC data were filtered by Tukey’s rule to remove outliers either below the first quartile by 1.5 inter-quartile range or above the third quartile by 1.5 inter-quartile range. To further remove the outliers, the data were filtered by the speed that the vehicle was driving, which was between 3.2 km per hour to 8.0 km per hour. Variograms were assessed by “automap” package 1.0-14 , and fitted to perform kriging. The soil bulk EC values were extracted from the location of each experimental unit, these values were further used to perform regression analysis. Kriging and k-means clustering on plant physiology variables were performed with the R packages “gstat” and “NbClust,” v3.0 . Universal kriging was utilized on plant water status because of the existing trend in longitude and latitude. Variograms were assessed by “automap” package 1.0-14 , and fitted to perform universal kriging. The vineyard was delineated into two clusters by k-means clustering, including Zone 1 with higher water deficit and Zone 2 with lower water deficits. The separation described 78.1% in 2017 of the variability in the plant water status according to the result of between sum of squares/total sum of squares. The resulting maps were organized and displayed by using QGIS software . Cluster comparison was analyzed by “raster” package reported as Pearson’s Correlation between two cluster maps . Data were tested for normality by using Shapiro-Wilk’s test, and subjected to mean separation by using two-way ANOVA with the package “stats” in RStudio . Significant statistical differences were determined when p values acquired from ANOVA were <0.05, and the zones were classified according to Tukey’s honestly significant difference test. Regression analysis was performed by SigmaPlot 13.0 . Correlation coefficient between variables were calculated in by Pearson’s correlation analysis, and p-values were acquired to present the significances of the linear fittings.Berry skin anthocyanins were different between the two zones in 2016 . Total delphinidins, petunidins, malvidins, and the sum of them as trihydroxylated anthocyanins were all higher in Zone 2 than Zone 1 . Total cyanidins, peonidins, and the sum of them as di-hydroxylated anthocyanins were greater in Zone 2 on 23 August, 15 September, and at harvest . Total skin anthocyanins were 2.2 mg per g of berry fresh weight in Zone 2 which was higher than the 1.85 mg measured in Zone 1 . In 2017, there were no differences between the two zones in delphinidin, cyanidin, petunidin, or peonidin at harvest . Zone 1 had higher malvidins from 24 August until harvest, and tri-hydroxylated anthocyanins, total anthocyanins from 7 September until harvest . Conversely, total malvidins, tri-hydroxylated anthocyanins, and total anthocyanins were higher in Zone 1 at harvest .

We excluded hospitalizations that included liver transplantation , and used the cohort of those who did not receive liver transplantation as the primary cohort. The primary outcome was ODS at any point during hospitalization . Patient age, sex, race, etiology of liver disease, general medical comorbidities, complications of cirrhosis, and hospital outcomes including discharge disposition, length of stay, and inflation-adjusted total cost were also extracted as described in depth elsewhere. In brief, we identified specific complications of cirrhosis using discharge diagnosis codes ; we identified specific patient comorbidities such as congestive heart failure using the Clinical Classification Software; and identified paracenteses and thoracenteses with procedure codes. Charlson Comorbidity Index was used as a marker of general illness severity/degree of comorbidities, stratified into three groups: mild , moderate ,severe . Baveno IV consensus criteria was used as a marker of cirrhosis illness severity, where those with Stages 3 , and 4 represented decompensated cirrhosis. For descriptive statistics we presented categorical variables as percentages and continuous variables as medians with respective interquartile ranges . To compare characteristics between patients with vs. without ODS, we used Pearson chi-square test for dichotomous variables; non-parametric Kruskal-Wallis to compare categorical variables; and Wilcoxon rank-sum for continuous variables. We used univariable logistic regression to assess unadjusted odds ratios associated with ODS, square plant pot and used stepwise backward selection to determine the final multi-variable logistic model. Statistical analysis were performed using Stata .

In this investigation of the National Inpatient Sample, 2009-2013, we found that the prevalence of ODS in hospitalized patients with cirrhosis was extremely rare, and much lower than the prevalence reported in patients undergoing liver transplantation.2-6,8 Alcohol related cirrhosis, younger age, and female gender were associated with an inpatient diagnosis of ODS. Notably, markers for cirrhotic decompensation and severity of comorbid illness were not found to be associated with ODS. This included no evidence for an association with ascites, which ran counter to our hypothesis that those with portal hypertension might be at higher risk for ODS due to labile serum sodium levels during hospitalization. Our findings that those with vs. without ODS experienced longer hospital stays, higher hospitalizations costs, and increased chance of receiving a discharge disposition to somewhere other than home help to quantify the burden of ODS on the health care system. Furthermore, the increased health care burden and poorer outcomes we found in those with vs. without ODS provide additional evidence that we appropriately identified pronounced cases of ODS with our selection methods. We acknowledge our study’s limitations. As with all large database investigations, our results are susceptible to case ascertainment and measurement biases. While ICD-9 codes have been well-validated for the selected measures of cirrhosis and overall disease,7 ICD-9 codes for ODS have not been systematically validated. In particular, we cannot know whether subtler cases of ODS might have gone unrecognized in the hospital setting only to be diagnosed at outpatient follow-up upon review of MRI imaging. Our low prevalence estimate likely reflects this, underestimating the total prevalence of ODS by failing to detect these subtler cases. That being said, we aimed to capture clinically-apparent cases of ODS, for which ICD-9 coding would be most specific. Finally, our study was limited by a paucity of sodium level data.

Unfortunately, the NIS does not contain laboratory values, so we couldn’t associate serum sodium changes with ODS. Additionally, hyponatremia as detected by ICD9 coding has been demonstrated to be variable and often lacking, representing perhaps only one third of inpatients experiencing hyponatremia.9 Because of this, and because hyponatremia is already known as a major precipitant of ODS, we elected to focus our research questions on other risk factors beyond it.1 In conclusion, our investigation of a large nationwide database demonstrates that ODS is extremely rare, occurring in 0.02% of hospitalized patients with cirrhosis. ODS is associated with alcohol-related cirrhosis, younger age, and female gender. ODS is not associated with specific cirrhosis complications including ascites, nor with overall liver disease severity or general comorbid disease severity. These data may help inform management of hyponatremia in patients with cirrhosis by reassuring providers of the rarity of ODS, while reinforcing the consideration of a broad range of differential diagnoses in cirrhosis patients exhibiting altered mental status after hyponatremia correction.Cobalt monosilicide crystalizes in a chiral structure in the P213 space group inset. It has been intensely studied as a potential thermoelectric material due to its large power factor at room temperature. Recent theoretical works have found that CoSi and its isostructural siblings possess a chiral double sixfold-degenerate spin-1 Weyl fermion at R point and a fourfold-degenerate chiral fermion at Γ point in their Brillouin zone. These “new fermions” with large topological charges are connected by long, robust Fermi arcs on the surface which have been later confirmed by angle-resolved photoemission spectroscopy experiments. However, no transport properties of CoSi directly related to its topological nature have been reported until now. As far as we are aware, previous reported CoSi single crystals manifest relatively low carrier mobilities and no quantum oscillations have been observed in their electrical properties under magnetic field. It is difficult to bridge the transport properties with its topological band structure due to low sample quality.

Here we report our crystal growth and a survey of the electrical transport properties of single-crystalline CoSi. Tellurium was found to be an appropriate metal flux for the growth of CoSi which yields high-quality single crystals with large magneto-resistance and carrier mobilities. Although there is plenty of research work on the thermopower of CoSi, few of them have paid attention to its magneto-Seebeck and Nernst effect. Combining the high quality of our Te-flux grown samples and the sensitivity of thermoelectricity measurement, we are able to observe, for the first time, QOs in the thermoelectric signals of CoSi. By analyzing the QOs in magneto-Seebeck and Nernst signals at different temperatures and magnetic field directions, we reveal two spherical Fermi surfaces around the BZ corner R point, which is consistent with our density functional theory calculations. The extracted Berry phases of electron orbit equal zero, agreeing well with the scenario of −2 chiral charge at R point. We also found that the spin-orbit coupling induced band-splitting is lessthan 2 meV near the Fermi level and this result is one order smaller than our DFT calculations. We also report a large Nernst effect in CoSi due to the combination of high mobility and phonon-drag contribution at intermediate temperatures. As a consequence, a relatively large Nernst-Ettinshausen figure of merit of around 0.03 is achieved at 42 K in 14 T.Grape berries undergo a series of complex physiological and biochemical changes during their development that determine their characteristics at harvest . Genome-wide expression studies using microarray and, more recently, RNA sequencing revealed that berry development involves the expression and modulation of approximately 23,000 genes and that the ripening transition is associated with a major transcriptome shift . Transcriptomic studies characterized the ripening program across grapevine cultivars , identifying key ripening-related genes and determining the impact of stress and viticultural practices on ripening . This knowledge increases the possibility of exerting control over the ripening process, improving fruit composition under suboptimal or adverse conditions, and enhancing desirable traits in a crop with outstanding cultural and commercial significance . These genome-wide expression analyses were possible because a highly contiguous assembly for the species was produced ;this first effort used a grape line created by several rounds of back crossing to reduce heterozygosity, facilitating genome assembly . Though poor by current standards, this pioneering, square pot chromosome-resolved assembly served as the basis for numerous publications. However, the structural diversity of grape genomes makes using a single one-size-fits-all reference genome inappropriate . There is substantial unshared gene content between cultivars, with 8–10% of the genes missing when two cultivars are compared . Although many of these genes are not essential for plant survival, they can account for 80% of the expression within their respective families and expand key gene families possibly associated with cultivar-specific traits . Assembling genome references for all interesting cultivars is impractical, in part because its cost remains prohibitive and because of genomic features that impede the development of high-quality genome assemblies for any grape cultivar. Although the V. vinifera genome is relatively small and as repetitive as other plant genomes of similar size , it is highly heterozygous . Most domesticated grape cultivars are crosses between distantly related parents; this and clonal propagation cause the high heterozygosity observed in the species . Earlier attempts using short reads struggled to resolve complex, highly heterozygous genomes . A limited ability to call consensus polymorphic regions yields highly fragmented assemblies where structural ambiguity occurs and alternative alleles at heterozygous sites are excluded altogether . Single Molecule Real Time DNA sequencing has emerged as the leading technology for reconstructing highly contiguous, diploid assemblies of long, repetitive genomes that include phased information about heterozygous sites . Recently, we used Vitis vinifera cv. Cabernet Sauvignon to test the ability of SMRT reads and the FALCON-Unzip assembly pipeline to resolve both alleles at heterozygous sites in the genome .

The assembly produced was significantly more contiguous than the original PN40024 assembly and provided the first phased sequences of the diploid V. vinifera genome . Despite recent advances in genome reconstruction methodologies, assembling a complex plant genome is still costly. Transcriptome reconstruction is the only alternative strategy to depict known and unknown gene content information . De novo assembly of RNA-seq reads is widely used for this purpose . SMRT technology was recently deployed to investigate expressed gene isoforms in a variety of organisms, including a handful of plant species . Long reads delivered by this methodology report full-length transcripts sequenced from their 59-ends to polyadenylated tails , making Iso-Seq an ideal technology for reconstructing a transcriptome without a reference genome sequence and without assembling fragments to resolve the complete isoform sequence . Moreover, alternative transcripts that contribute to the gene space complexity and vary with cell type , developmental stage , and stress cannot be definitively characterized without full-length transcript information. The objective of this study was to test whether full-length cDNA sequencing with Iso-Seq technology is a suitable alternative to traditional genome sequencing, assembly, and annotation for reconstructing a grape transcriptome reference for transcriptional profiling. We compared how Cabernet Sauvignon’s Iso-Seq transcriptome fares as a reference for RNA-seq analysis vs. its annotated genome. We sequenced the full-length transcripts of ripening berries with Iso-Seq and Illumina RNA-seq reads. The high-coverage short-read data were used to profile gene expression and to error-correct low-expression isoforms that would have been otherwise lost by the standard Iso-Seq pipeline. The transcriptome reference built with Iso-Seq data represented most of the expressed genes in the grape berries and included cultivarspecific or “private” genes. When used as the reference for RNAseq, Iso-Seq generated transcriptome profiles quantitatively similar to those obtained by mapping on a complete genome reference. These results support using Iso-Seq to capture the gene space of a plant and build a comprehensive reference for transcriptional pro- filing without a pre-defined reference genome.Grape berries from Cabernet Sauvignon FPS clone 08 were collected in Summer 2016 from vines grown in the Foundation Plant Services Classic Foundation Vineyard . Between 10 and 15 berries were sampled at pre-véraison, véraison, post-véraison, and at commercial maturity. Table S1 provides weather information for the sampling days. The ripening stages were visually assessed based on color development and confirmed by measurements of soluble solids . On the day of sampling, berries were deseeded, frozen in liquid nitrogen, and ground to powder . Total RNA was isolated using a Cetyltrimethyl Ammonium Bromide -based extraction protocol as described in Blanco-Ulate et al. . RNA purity was evaluated with a Nanodrop 2000 spectrophotometer . RNA was quantified with a Qubit 2.0 Fluorometer using the RNA broad range kit . RNA integrity was assessed using electrophoresis and an Agilent 2100 Bioanalyzer . Only RNA with integrity number greater than 8.0 was used for SMRTbell library preparation. One mg of the pooled RNA was used for cDNA synthesis and for SMRTbell library construction using the SMARTer PCR cDNA synthesis kit . First-strand cDNA synthesis was performed using the SMRTScribe Reverse Transcriptase . Each developmental stage was individually barcoded . To minimize artifacts during largescale amplification, a cycle optimization step was performed by collecting five 5 ml aliquots at 10, 12, 14, 16, and 18 PCR cycles.

Given the later fruit development of grapevine and the grape chemistry requirements for red wine making , the length of the growing season is often a limitation for achieving the desired ripening and vintage quality in cool climates . Thus, yield is often sacrificed to balance source-to-sink ratio in favor of accelerated fruit ripening or to mitigate the effects of early fall frosts . Although the initial control of crop level comes during pruning , the number of dormant buds retained at pruning time is maintained constant through the years in warm climate regions. Cluster thinning is a management practice fine-tuned each year to achieve vine balance . This effect could be exacerbated with high nitrogen amounts inhibiting anthocyanin biosynthesis , the absence of water stress, or changes of cluster microclimate due to mutual shading , and thus, not by the under cropping itself. Therefore, grapevine canopy development is managed through the control of inputs, vine spacing, irrigation, rootstocks, pruning, leaf removal, hedging, or cover crops, among others. A great part of the carbon assimilated through the growing season is incorporated into cellulose or lignin in roots, trunks, and shoots . However, blueberry grow bag resumption of a new season’s growth depends on the carbon stored as nonstructural carbohydrates, majorly in the form of starch, but also soluble carbohydrates such as sucrose, glucose, and fructose .

Roots are the greatest sink of non-structural carbohydrates and root-derived carbohydrates constitute the principal reserve source for annual resumption of growth in the spring. The grapevine’s capacity for replenishment of these carbohydrate reserves increases at mid-ripening, when canopies are at their maximum and fruit demand slows down sugar accumulation in perennial parts . Therefore, the loss of photosynthetically active leaf area or excessive number of clusters may impair the reconstitution of reserves . In addition, high crop levels may delay fruit maturation and shorten the post-harvest period and subsequently reduce the time needed to accumulate reserve carbohydrates. Grape growing systems based on high yields are typically in warm to hot regions, relying on early harvest to replenish these reserves. However, it is common that excessive yields lead to a reduction in yields the following season . Loss of photosynthetically active leaf area or excessive number of clusters may deplete these reserves. High crop levels may reduce the reserve carbohydrate accumulation and delayed fruit maturation and may shorten the postharvest period. Therefore, the grapevine may not have sufficient time to accumulate carbohydrates for the following season in cool climates. Conversely, there is not consensus in literature regarding the effect of high cropping levels on storage reserves . This was explained by sink limitation as the grapevine was able to maintain equilibrium by adjusting physiological processes . In addition to the modulation of berry ripening and storage reserves, other compensatory mechanisms have been described in response to over and under cropping. Components of yield, which include clusters per vine, berries per cluster, and berry mass, are susceptible to change together with berry ripening in compensation of each other . Although grapevine pruning, canopy, and crop load management are the most frequently reported case of study for source-to-sink ratios, most studies may not offer direct observations , enough combinations, duration of the study, or range of source-to-sink ratios to respond to some fundamental questions.

The aim of this study was to determine the in-season and carryover effects of carbon source and sink imbalances in grapevine. Specifically, we investigated the combined effects of defoliation and fruit removal on components of yield, canopy area, and seasonal integrals of leaf gas exchange, shifts in phenology, carbohydrate, and soluble sugar concentration in the roots.The experimental design was a factorial arrangement of treatments. There were three levels of manual defoliation by three levels of manual fruit removal applied . The treatments were applied as follows. Leaves were removed on every shoot in an alternating pattern. For instance, 66% of leaf treatments retained leaves in positions 1st, 2nd, 4th, 5th, 7th, 8th etc. while 33% of leaf treatments kept leaves in positions 1st, 4th, 7th, etc. in every shoot . The fruit removal treatments retained a percentage of clusters after standardizing the cluster numbers in each year. Each treatment combination was replicated four times and each treatment-replicate consisted of three experimental units. In 2017, all vines were standardized at fruit set to 20 shoots and 30 clusters per vine, and laterals were removed prior to defoliation and fruit removal treatments. In 2018, all vines were standardized to 24 shoots and 45 clusters and laterals were removed prior to treatment application. Treatments were applied at pepper-corn size . In 2019, after two seasons of growth under the nine combinations of treatments, the carryover effects were studied by leaving all vines untreated . For each experimental unit one vine was shoot thinned to 24 shoots, and others were left unmanaged . All clusters at pepper-corn size in all treatment-replicates were dipped in a 5.5% kaolin solution to provide protection from the afternoon sun due to the row orientation of the vineyard in every year of the experiment.Percentage of bud break , flowering , veraison , and leaf senescence per plant were recorded at time intervals of either 1, 2, or 3 times a week depending on weather and phenology events . A leaf was considered senescent when 50% of its area was yellow.

Measurements started soon after the application of treatments in 2017 until leaf senescence 2019. In 2019, only grapevines thinned to 24 shoots per vine were followed.Leaf gas exchange was measured bi-weekly in all years of the experiment with an infra-red gas analyzer . Three sun-exposed leaves were selected from the main shoot axis in each experimental unit, and three readings were taken from each leaf. Gas exchange measurements were taken when the sunlight conditions were close to saturating levels in all instances. The relative humidity was set at 40%, the reference CO2 concentration was set at 400 µmol CO2 mol−1 as the standard environmental condition setting in CIRAS-3. Net carbon assimilation rate and stomatal conductance were obtained. To express the season-long response of AN, and gs , their integrals were calculated by using natural cubic splines for plant water status and gas exchange measurements to assess the cumulative values for these parameters over the whole experiment period during the growing season. Then, these cumulative values were normalized as divided by the number of days elapsed between the first measurement date and the last measurement date to make the data comparable to each individual measurement.After harvest, leaves from one vine per replicate were collected, weighted, and dried in a forced-air oven at 80◦C for 3 days. Dry leaf weights were converted into area by measuring the area of a subsample of 50 random leaves with a leaf area meter as reported previously . On 12 December 2018, after the second season of treatments, one vine per experimental unit of the most extreme treatments were pruned, coppiced, blueberry grow bag size and the root systems were removed with a back-hoe. The sectioned grapevine portions were weighed on a top loading scale, and dried in a forced-air oven at 60◦C until no weight change of tissue was detected. At harvest , clusters were removed, counted, and weighed for each plant in the experiment. Total soluble solids were measured from 55 berries collected randomly at harvest point. The berries were crushed by hand and filtered to obtain must. A digital refractometer was then used to measure total soluble solids of must.Soon after the harvest of 2017 was completed, root tissues were sampled every 2 months. The top layer of soil was removed until the roots were visible. Each grapevine root zone was divided into four quadrants and on each date and one single quadrant was sampled, leaving the other 11 quadrants undisturbed. Roots were gently cleaned with water, freeze-dried, and ground to a fine powder with a tissue lyser . Thirty milligrams of the resultant powder were extracted in 80:20 ethanol solution. A 1.5 mL aliquot of the extract was then placed in a 90◦C water bath for 10 min, then centrifuged at 10,000 rpm for 1 min. The supernatant was collected for total soluble sugars determination. The same procedure was repeated for starch determination, in which the pellet was collected for its determination.

Total soluble sugars in the roots were determined as reported elsewhere by Torres et al. . Briefly, the 1.5 mL sample was filtered by PTFE membrane filters and transferred into high performance liquid chromatography vials. Equipment consisted of a reversed-phase HPLC system Agilent 1100 coupled to a diode array detector and an Agilent Infinity Refractive Index Detector . The reversed-phase column was Luna Omega Sugar with a guard column of 5 mm. The temperature of the column compartment was maintained at 40◦C and the RID flow cell was kept at 35◦C. The mobile phase system consisted in an isocratic elution with acetonitrile:water at a flow rate of 1.0 mL•min−1 with a run time of 22 min. Standard solutions of 10 mg/L of D-glucose, D-fructose, D-sucrose, and D-raffinose were injected to obtain the retention time for each compound, and detection was conducted by RID. Sugar standards were purchased from VWR . Sugar concentration of each sample was determined by comparison of the peak area and retention time with standard sample curves. Starch content of roots was measured using the Starch Assay Kit SA-20 following the manufacturer’s instructions. Briefly, pellets of root tissues were dissolved in 1 mL DMSO, and incubated for 5 min in a water bath at 100◦C. Starch digestion was started by adding 10 µL α- amylase and incubated in boiling water for another 5 min. then, the ddH2O was added to a total volume of 5 mL. Then, 500 µL of the above sample and 500 µL of starch assay reagent were mixed and incubated for 15 min at 60◦C. Negative controls with the starch assay reagent blank, sample blank, and glucose assay reagent blank and positive controls with starch from wheat and corn were performed. Reaction started with the incubation of 500 µL of each sample and 1 ml of glucose assay reagent at 37◦C and was stopped with the addition of 1 mL of 6 M Sulfuric acid after 30 min. Reaction was followed with analytical measurements with a Cary 100 Series UV-Vis Spectrophotometer and starch content expressed as mg of starch per tissue dried weight.In our experiment the results indicated that there was an interaction of year and defoliation on cluster weight, berries per cluster and yield per vine . When we analyzed the data by year, the effect of defoliation was clearer. In both experimental years , there was a strong linear trend of defoliationon all components of yield except for cluster number; which was only affected by the fruit removal treatments. In 2017 defoliating 66% of the leaves resulted in an 8% decrease in berry weight. The differences were exacerbated in cluster weight , berries/cluster , and yield when 66% of the leaf area was removed. In 2018, the effect of defoliation was evident with a 12% decrease in berry weight. As in the previous year, we saw a diminution in cluster weight, berries/cluster, and yield. However, the decline in yield in 2018 was 56% when 66% of the leaves were defoliated. Fruit removal was effective in modulating the cluster number and thus the yield in both experimental years as expected . Furthermore, we measured a strong linear decrease in cluster weight in 2017. However, the same response was not evident in 2018. Removing 66% of the cluster resulted in a 55 and 60% decrease in yield of Cabernet Sauvignon in 2017 and 2018, respectively. Surprisingly, we did not measure an interaction of defoliation and fruit removal on components of year in either of the experimental years . The carry-over effects of source-sink adjustments on components of yield in 2019 were strongly evident ; even though no defoliation or fruit removal treatments were applied. Berry weight, cluster number per vine, cluster weight, and yield per vine were all affected by the carry over effects of defoliation from the previous 2 years. They all declined linearly with the 33% defoliation treatment. Conversely, we did not measure a carryover effect of fruit removal in 2019 in the majority of components of yield monitored. There was an interaction of defoliation and fruit removal in 2019 on the number of berries per cluster.