Month: March 2025

Whenever possible, individuals located more than 2m from trails and fuel breaks were selected to avoid any edge effects. All individuals were sampled once between April and September 2019. Two branchlets , each containing necrotic lesions and adjacent asymptomatic wood tissue, were clipped per individual using sterile techniques, for a total of 600 samples. Samples were retrieved from approximately breast height and opposite sides of the shrub, whenever possible. All individuals had at least two necrotic lesions, even if no significant dieback was observed, allowing these methods to be carried out across all 300 individuals. Samples were then placed in labeled plastic bags, stored on ice, and brought back to the lab and placed in a 4o C refrigerator.Samples were rinsed of dirt and debris and surfaced sterilized using 100% ethyl alcohol, 0.5% bleach, and a 70% ethyl alcohol rinse. Cross sections between 1-2 mm were isolated from the advancing canker margin and plated onto half-strength potato dextrose agar amended with streptomycin antibiotic. Cultures were incubated at room temperature until fungal colonies developed , and isolates of hyphal tips near the advancing margin were then re-plated onto half-strength PDA-strep to obtain pure cultures. From pure culture, any samples identified to have morphological characteristics consistent with those of Bot. fungi  were selected for PCR. A few isolates from cultures inconsistent with Bot. characteristics were randomly selected from each site to amplify and sequence to verify our morphotyping method. The internal transcribed spacer region 1 and alpha-elongation factor-1 genes were amplified using PCR primer pairs ITS1F and ITS4, 25 liter pot and EF1-728F and EF1-986R, respectively, using methods modified from White et al., and Slippers et al .

Successfully amplified samples were sequenced using the UC Berkeley Sequencing Facility .The severity of Bot. infection was calculated as the isolation frequency per site. Data were square-root transformed when necessary to meet the assumptions of normality. Differences in mean Bot. infection severity between elevation categories were calculated using one-way ANOVA with Tukey’s HSD for post-hoc analysis with R Statistical Software . Correlations between actual elevation and Bot. infection severity were assessed using simple linear regression and ANOVA to test for significance . Generalized linear models were developed to identify patterns of dieback, with dieback severity values as the response variable, and elevation , Bot. infection severity , and aspect as possible explanatory variables. If multiple models received substantial support , the best model was confirmed by calculating the relative importance of each term based on the sum of their Akaike weights . The proportion of variance explained by the models was calculated by measuring the adjusted D2 value .This study provides definitive support for the hypothesis that shrub dieback, during a recent drought, and pathogen infection are strongly related in a wild shrubland setting. This is the first known quantitative support for the hypothesis that in A. glauca, an ecologically important shrub species in the study region, dieback is related to pathogen infection occurring along an elevational gradient. As expected, N. australe and B. dothidea were the two most frequently retrieved pathogens across all sites, however N. australe, the introduced pathogen, had almost twice the abundance of B. dothidea. N. australe is driving the correlation between elevation and Bot. infection, as the frequency was greater at lower elevations compared to upper elevations, while B. dothidea abundance did not change significantly across elevations. Level of Bot. infection was confirmed to be a significant predictor of stand-level dieback severity. The data also confirm that stand dieback severity is generally greater at lower elevations, which in this region experience higher temperatures and lower annual rainfall than the higher elevations sampled.

While the presence of Bot. species has been reported previously in Santa Barbara County, this study represents the first effort to understand the abundance and distribution of Bots occurring in natural shrublands, and the first wildland shrub survey of Bots across a climate gradient. The high frequency and wide distribution of Bots retrieved from our study sites support the hypothesis that Bot. species are widespread across a natural landscape, and likely contributing to the extensive dieback resulting from the recent drought. Bot. fungi were retrieved from nearly every site in this study . We could not determine Bots. presence from three sites due to contamination issues. The broad extent of the study area suggests that infection is widespread in the region, and likely extends beyond the range of our study. While both N. australe and B. dothidea together made up the most frequently retrieved pathogens, our data show that N. australe has a larger distribution and occurs in greater abundance across the study region than B. dothidea . This trend was consistent across all elevations, but particularly at lower elevations . One possible explanation for this is that N. australe, being a recently introduced pathogen, spreads more rapidly as an exotic species in A. glauca compared to B. dothidea, which has been established in California for over 150 years . This hypothesis is consistent with previous studies that have shown variations in Bot. species abundance and virulence in Myrtaceous hosts occurring in native versus introduced ranges . However, it is difficult to evaluate the incidence of B. dothidea and N. australe in the present study in relation to historical documentation since many species in the Bot. complex have, until recently, been mischaracterized . Only with the recent development of molecular tools have researchers begun to accurately trace phylogenetic and geographic origins of Bot. species. Such studies are beginning to elucidate the complex existence of Bot. fungi as both endophytes and pathogens around the world, and much more research is needed to understand their pathogenicity in various hosts under different conditions. Nevertheless, it remains clear from our study that Bot. species, particularly N. australe, are both abundant and widely distributed in this region, and are important pathogens in A. glauca shrubs.Because Bot. taxa were the most frequently retrieved pathogens and were significantly correlated with dieback, we believe that they drive A. glauca dieback. Further, stand dieback severity increased significantly with Bot. infection. This is not to say that other pathogens do not also contribute to disease symptoms, but we found no evidence of any other pathogens occurring in such high incidence as Bot. species. While Brooks and Ferrin identified B. dothidea as a likely contributor to disease and dieback in dozens of native chaparral species during an earlier drought event in southern California, and Swiecki and Bernhardt found B. dothidea in association with a dieback event in stands of Arctostaphylos myrtifolia in northern California, our study yields the most extensive results of Bot. infection and related dieback in a chaparral shrub species across a landscape. Further, our study resolves species identity within the Bot. clade and highlights the role of the recently introduced pathogen, N. australe.A significant finding in this study was the relationship of Bot. infection and dieback with elevation. Bot. abundance and dieback were both found to be greatest at lower elevations, raspberry cultivation pot which was driven mostly by the high frequency of N. australe retrieved at these sites. This represents the first quantitative evidence supporting that A. glauca vulnerability to fungal infection is influenced by stress levels along an elevation gradient.

A similar pattern was observed in northern California by Swiecki and Bernhardt , who suggested that dieback in Ione manzanita infected with B. dothidea was greater in drier sites compared to more mesic ones, although no comparison of infection rates between sites was conducted in their study. The elevation gradient in our study was used a proxy for stress levels because annual precipitation decreases with decreasing elevation within our study region . Higher temperatures, which are associated with lower elevations, are also known to play an important role in drought-related mortality, as water loss from evapotranspiration is increased . Furthermore, unpublished data for dry season predawn xylem pressure potentials on a subset of sites along the same elevational gradient revealed more negative water potentials in A. glauca at lower elevations compared to upper elevations as spring and summer drought sets in . Thus, there is evidence that shrubs at low elevations indeed experienced the greatest water stress during the 2011-2018 drought, which predisposed them to higher levels of Bot. infection and enhanced dieback compared to upper elevation sites. More in-depth studies on the microbial communities and fungal loads of healthy and diseased shrubs throughout the region would help elucidate such trends. Another possibility for the higher incidence of Bot. infection at lower elevations is that the lower ranges of A. glauca populations in Santa Barbara are often located adjacent orin close proximity to agricultural orchards, ranches, and urban settings, which are common sources of plant pathogens, including Bots . Eucalyptus, avocado, and grapevines, which are abundant in these areas, are particularly well-known Bot. hosts and potential facilitators of Bot. introduction . Therefore, sources of inoculum from nearby populations of agricultural and horticultural hosts could be responsible for continual transmission Bots in wildland A. glauca populations, and would likely result in greater rates of infection at lower elevations. Furthermore, many of the lower sites in the survey were located near roads and/or trails, which are often subjected to additional stress from human activity like pruning and trail clearing; activities that are known to spread and promote infection by Bot. pathogens . While we avoided sites that showed signs of such activities in our survey, we cannot rule out the potential contributions of proximity to human encroachment to the overall higher rates of Bot. infection across the lower elevation zone. It is worth noting that while our study revealed a trend of increased dieback in lower elevations, some upper elevation sites also exhibited high levels of dieback, and Bot. fungi were retrieved from many of these sites. Upper elevations also experienced significant stress during the 2011-2018 drought, and water-related microsite variables outside the scope of this study like slope, solar incidence, soil composition, and summer fog patterns factors likely contributed to increased stress and subsequent dieback. Additionally, N. luteum, N. parvum, and D. sarmentorum were isolated primarily from upper sites. Host plants in these sites may serve as potential reservoirs for disease because the milder climate conditions promote greater host survival and thus pathogen persistence asendophytes. This serves as an important reminder that continued global change-type drought may eventually jeopardize susceptible species populations even at the upper boundary of their range.Our results are consistent with well-known theoretical models describing the relationship between environmental stress and biotic infection, which generally ascribe extreme drought stress as a mechanism for plant predisposition to disease . These frameworks illustrate dynamic interactions between environmental stress, plant hydraulic functioning and carbon balance, and biotic attack, and a growing body of research has focused on understanding the roles of these factors in driving plant mortality, especially during extreme drought . While the data collected in this study do not directly address the specific mechanisms leading to Bot. infection and dieback in A. glauca, our results can be discussed in the context of how life histories and physiological adaptions elicit differential responses to drought in woody plants, particularly in chaparral shrubs. For example, shallow-rooted, obligate seeder shrubs like A. glauca have been shown to be more susceptible to drought-induced mortality during acute, high intensity drought than deep-rooted, resprouter shrubs . This supports our observations of pronounced A. glauca decline during an historic California drought compared to nearby resprouter species like chamise , and laurel sumac .Additionally, physiological mechanisms related to drought tolerance may further explain predisposition to disease in A. glauca. For example, high resistance to cavitation is a common trait associated with more dehydration-tolerant species like A. glauca that maintain hydraulic conductivity during seasonal drought . While cavitation resistance is thought to assist in the continuation of photosynthetic activity even at very low seasonal water potentials , it has also been associated with greater mortality rates during high intensity drought in a variety of woody plant systems including mediterranean shrublands , temperate deciduous forests and eucalyptus forests . High resistance to cavitation requires heavy carbon investment for stronger and denser stem xylem tissue , which can result in limited carbon for investment in defense against pathogens like Bot. fungi.

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.