Cultures cluster close to one another while liquid samples show large inter-sample variation both before rarefaction and after . While rarefaction changed both the total and coordinate-specific amounts of variation, it did not do so to a remarkable extent – 97.2% to 97.5% and 89.4% to 88.5% for total variation in spiked and unspiked, respectively, and below 5% in all cases for individual coordinate axes in all cases. On the other hand, removing the spike-in OTU from the read counts did substantially change the clustering as well as the x/y positioning of both the liquids and cultures. Even a cursory comparison of the left column in Figure 15 to the right column in the same figure shows that the E. coli OTU affected the apparent similarities of the samples shown by PCoA. A few finer points should be made here about removing this process. First, removing this OTU pulled the liquids and cultures together; whereas the first coordinate spanned from -0.5 to 0.6 before removal, it spans from about -0.3 to 0.6 afterwards, indicating a tighter grouping for all samples. Second, as might be expected, removing this OTU allowed the compositional variations in the liquid samples to surface , whereas the liquids were clustering according to whether they had received the spike in before removal . The liquid samples were clearly more compositionally diverse than the cultures, evidence by the wider horizontal spread of the triangles in Figures 15 b and d. Third, removing the E. coli reads did not greatly affect the grouping of the cultures. Culture samples still fall within 0.2 units of one another in both coordinate axes, with one exception of a culture spiked by 100µL of E. coli. Fourth, after removal of the E. coli OTU, blueberries in pots neither liquids nor cultures cluster with visibly discernible patterns that group with spike volumes anymore, whereas the grouping of samples with spike volumes was apparent before removal .

Overall, these clusters and their disappearances fall well within expectation, considering that the spike-ins were much higher in biomass than the liquids but much lower than the cultures. The difference in biomass between the liquids and cultures inevitably led to the increased propensity of the liquids to similarity/dissimilarity influences from the spike-in. For both liquids and cultures in the preliminary experiments, it seems that as expected, no distinct groupings based on inherent compositional dissimilarities can be observed. It is also not clear whether the variations in the liquid samples come, in large part, from the low numbers of read counts after E. coli removal, as even a few reads in a low-read-count sample could lead to seemingly large inter-sample differences. In any case, the total percentage of variation accounted by PCoA here falls between 89% and 98%, indicating that two axes were sufficient for this set of samples. Interestingly, removing the E. coli OTU decreased the total percentage of variation accounted for by more than 8%, once again underscoring the sway that the spike-in had on liquid samples. From the compositional analyses, we see that cultures in the preliminary experiments yielded sufficient biomass and contained dental plaque bacteria, without exhibiting unexpected similarities or dissimilarity with themselves or with the liquids above the sedimented cultures. As to what the principal coordinates represent, i.e. what underlying biological differences may have led to two coordinates being sufficient, we would need to adopt a different analytical approach, which we do in the next stage of the project.In these preliminary experiments, we established a culturing procedure that minimizes external contamination while producing high numbers of viable cells from the human oral/dental bacterial community. Compositional analysis of the cultures showed that OTUs with the highest relative abundances belong to the Neisseria, Streptococcus, and Veillonella genera, two of which have been shown to be early and middle colonizers of the oral microbiome and all three of which have been shown as core genera in the supragingival plaque community.

The prevalence of OTUs from commonly occurring oral bacterial genera confirms that the culturing conditions support the growth and proliferation of anaerobic oral microbes without resorting to traditional, closed-form anaerobic culturing techniques such as anaerobic agar. Not many members from the group of later colonizers were present in the cultures in the preliminary experiments, though an Eikenella OTU was cultivated in the in vitro oral community to the extent of having a visible relative abundance value . Previous research has show that members of this OTU belong to groups of later colonizers that also include Actinomyces spp., Capnocytophaga ochracea, Propionibacterium acnes, and Haemophilus parainfluenzae. In this case, the absence or low abundance of later colonizers is not surprising, given that the cultures were incubated for less than 24 hours and not replenished with fresh host plaque. The short incubation time and lack of re-inoculation are part of the widely known scientific truth that in vitro conditions frequently select for organisms that can survive without the rich and complex environment of the original host. For bacteria that come from humans, this truth holds even more weight because it is unfeasible to replicate the human oral cavity. The complexity of host-microbe interactions simply defies reproduction in the lab. Another aspect to consider regarding the lack of later colonizers in these cultures is that membership of the oral bacterial community can vary greatly across different hosts. Kolenbrander and coworkers presented a larger picture of all the organisms that generally colonize earlier or later, with results that implicated trends, in other words, approximate orders of succession of oral/dental bacteria instead of definite lines of succession, and their work is far from the only instance for which human microbiome compositions have shown such great inter-host variations. The oral microbiome is no exception to such variation, but there exists a core community of major genera, and our methods have captured members of these major genera .

However, the factors already mentioned as having possibly detracted from organismal diversity in the in vitro cultures can be mitigated in future experiments by periodic re-inoculation of the cultures, longer incubation times, and/or variable nutrient sources and concentrations. These changes may help meet nutrient and signaling requirements of more fastidious bacteria, as well as increase the density of cells from certain OTUs to beyond their threshold values, such that proliferation becomes possible. Some of the culturing conditions that seemed appropriate for a proof-of-concept, such as this set of experiments was intended to be, including surface hydrophobicity of the scaffold, sampling with consideration of the growth phase of the bacteria, and bacterial attachment. The results seemed to indicate a promising protocol to establish an in vitro plaque community. An aspect that deserves some special, detailed consideration is the formulation of the culturing medium, SHI, based on the work from Tian and coworkers . As we used this medium in the preliminary experiments, it provided adequate nourishment, particularly in terms of pH and ionic strength. A potential disadvantage of SHI is that it is considered an undefined culture medium because the major carbon source in SHI is porcine stomach mucin. This glycoprotein is supplied in a partially purified form, square plant pots and because the glycosyl modifi- cations on glycoproteins can varied greatly depending on the conditions in the source organism, the composition of this protein cannot be guaranteed to be entirely biochemically identical across batches. Interestingly, the undefined nature of this medium has not yet been reported to be a major obstacle. On the contrary, research has shown some evidence that this medium may outperform more defined medium. A study that com-pared the effects of two media, DMM vs. BMM , on dental plaque microcosms grown in an artificial mouth system showed that plaque growth was slower in the chemically defined DMM, which contained higher concentrations of choline, citrate, uric acid, haemin, pyridoxine, biotin, and cyanocobalamin but lower concentrations of inositol, menadione, niacin, pantothenic acid, thiamine, and riboflavin. Furthermore, enzymatic activity for DMM was lower or in some cases undetectable. The results of our preliminary experiments indirectly affirm those from the comparative media experiment – we saw fast growth with the SHI medium, which contains major components from BMM as well as supplements such as menadione. However, we did not test the enzymatic activity of the cells in the culture to ensure that it is at least detectable, and we may need to do so. Another potential improvement might be to make the medium more defined for the sake of repeatability in our lab and reproducibility in the community. There has been some evidence that an artificial saliva may substitute human saliva in the growth of streptococcal species, and the composition of this artificial saliva may be a good starting point for a defined medium that would also be nutritionally sufficient. With regards to the attempt at establishing an internal standard with a known E. coli strain, we found that it was not feasible to seek correlations between read counts and OD600 values or CFU/mL under the conditions in the preliminary experiments. Finding such correlations mathematically would require quantifying and optimizing additional steps in the sequencing process.

The key steps to optimize here would include setting a concentration of E. coli DNA to be spiked into the samples to be sequenced, rather than using cells as spike-ins; understanding the efficiency of DNA extraction and mitigating the somewhat common bias of extraction processes to preferentially yield more DNA from Gram-negative bacteria; quantifying and optimizing the efficiency of PCR for the 16S rRNA of samples, which may involve some minor primer modifications; quantifying the composition of the library to be sequenced, potentially using genus-specific primers;quantifying how the fixed sequencing depths of HTS platforms affect the read counts and apparent compositions of samples, especially when samples do not have the same biomass; and so on. The quantification and optimization of these steps, including a detailed understanding of how systematic errors mathematically affect the results and how the number of discarded low-quality sequences affect the apparent compositions, may then enable us to find empirical relationships between number of cells in the cultures and read counts from sequencing. Gaining such a great degree of control over the whole process was not feasible at the time but would be a worthwhile venture for a future project. If we can establish a facile and rapid approach to quantification and optimization, we may be able to propagate the approach to developing many such numerical, analytical protocols. The bio-informatics process used for the preliminary experiment seemed to have served its intended purposes. With this process, we were able to perform quality control on the reads and cluster reads into OTUs at a reasonable level of sequence identity . More importantly, this procedure did not produce apparent artifacts that affected the processing and interpretation of data. The results obtained from using this bio-informatics pipeline met expectations formed from existing research on the human oral microbiome, though an aspect that may merit further consideration is the standardization of sample size by rarefaction. While there is some evidence that rarefaction helps reduce false discovery rates, there is equally reasonable evidence that rarefaction omits valid data and may bias against rare OTUs. For the purposes of these preliminary experiments, we have shown that rarefaction to 20,000 reads does not produce obvious artifacts or detectably reduce features observed in non-rarefied samples. Given that one of the major goals of the bio-informatics analysis in these experiments was to establish a procedure capable of distinguishing between biologically distinct samples without introducing much bias, rarefaction was clearly a defensible part of our approach. As for PCoA, the observation that rarefaction increases the percentage of variation accounted for is an expected result because of the nature of rarefaction. Rarefaction is a standardization procedure that simultaneously equalizes sample sizes and reduces the inter-sample variation, especially for samples with high numbers of rarer OTUs. To understand this point, we need to consider the two foundational concepts of diversity: richness and evenness. In terms of richness, adding a single OTU to a sample increases richness by one, which would only change diversity greatly in samples with low numbers of OTU. As for evenness, the addition of one OTU to a sample may or may not lead to a dramatic change in diversity, depending on two major factors.

The Results section covers the performance comparison of XGBoost with CNN and the deployment system performance evaluation. Finally, we conclude our work and discuss possible future directions in the Conclusions section.The study of brain activity using electroencephalogram typically involves extracting information from signals associated with certain activities. In recent years, machine learning techniques have been applied to the classification of mTBI because it enables the extraction of complex and typically nonlinear patterns from the EEG data. Most of the work surveyed used rule-based techniques, such as k-Nearest Neighbors . Previous investigations have studied a variety of classification techniques, including classical machine learning such as SVM and deep learning such as Convolutional Neural Networks . These techniques have been shown to perform TBI classification with more than 80% accuracy. However, in most investigations we reviewed that implement machine learning for TBI detection, the primary focus was the study of classification techniques and performance of classification models rather than portable deployment. A few systems used a small, portable computer for deployment in some form. The Neuroberry platform used a Raspberry Pi 2 device to capture EEG signals but the focus was on enabling EEG signal availability on the Internet of Things domain. The Acute Ischemic Stroke Identification System utilized an Analog to Digital Converter front end with Raspberry Pi 3 to capture physical EEG signals. However, plastic plants pots this system transferred the captured data to an HPC running MATLAB for signal analysis and processing and did not focus on signal classification.

Zgallai et al. described a Raspberry Pi-based system that used deep learning to perform EEG signal classification. It was designed to identify a subject’s intended movement direction from a multichannel EEG signal to control wheel-chair movement in a closed-loop robotic system rather than as a general system for identification, analysis, and monitoring of a physiological condition such as mTBI. Bruno et al. highlighted challenges with existing medical diagnosis techniques and described a classification system from the perspective of real-time TBI diagnosis, but their work was focused on the algorithm to perform TBI diagnosis and not on the implementation of a deployment system. In our previous work, we developed and described a CNN based model to perform automated sleep stage scoring and mTBI classification. In addition, we did a limited deployment of the CNN model on a Raspberry Pi 4 system. In that work, the focus was on describing the CNN model configuration, evaluating its performance, and showcasing that deployment to RPi was feasible rather than designing a complete, portable classification system. We have reused the previously developed CNN model in the current work to provide a baseline performance comparison with a new XGBoost model developed for this work. Further, the two models enable us to demonstrate the versatility of the current system to operate with multiple types of predictive models. To the best of our knowledge, no standalone, portable system has yet been created using Raspberry Pi that can capture real-time EEG signals, detect the presence of mTBI, and classify mTBI sleep/wake epoch states.A previously published dataset as described in [3] was used to train and evaluate deployed models. This dataset was collected as part of a study involving 11 adult male mice subjects divided into two groups—mTBI and Sham. FPI procedure was used to induce mTBI in 5 subjects and the remaining 6 mice were used as Sham subjects.

To capture the EEG signal, three ball-tipped electrodes were placed in the skull of each subject, two frontal and one in the parieto-occipital region. In this work, we proposed and demonstrated an RPi based EEG acquisition, processing, and classification system for early mTBI detection. This system was implemented using a single channel EEG data obtained from mice. This system was demonstrated to operate in a portable, real-time, and standalone configuration and perform classification of real-time EEG epochs into four target classes . As shown in Table 1, the accuracy, precision, and recall results were identical across RPi and HPC. This confirmed that the predictive model behavior did not change when the training and deployment systems involved different system architectures, i.e., x64 based MacOS/Windows HPC for training vs. ARM-based RPi for deployment and prediction. Hence, it is possible to train a predictive model on a more powerful computer and deploy it to an embedded device such as RPi that has limited memory and processing resources. This is especially applicable to multilayered neural networks like CNN that typically have long training times on an HPC, and the training times would be prohibitively long on an embedded device like RPi. We calculated the epoch processing time on RPi by varying the number of epochs, as shown in Figure 4 and described in Table 2. While it was expected that the processing time would increase as the number of processed epochs is increased, the key inference was that the processing time was considerably smaller than the time required to collect the EEG epochs. At 256 Hz sampling rate and 64 s epoch size, the processing time ranged from 0.01% to 0.03% of the epoch collection time. Hence, we concluded that the system had ample time to process previously captured EEG epochs while new epochs were captured at practical EEG signal sampling rates. We employed two different approaches for supervised learning models used in this system, the CNN model developed in our previous work, and an XGBoost predictive model created in the current work. We compared classification metrics and performance of the XGBoost and CNN models on the deployment system as well as an HPC.

We observed that the XGBoost model exhibited better performance in terms of accuracy and inference time compared to the CNN based predictive model. In the case of XGBoost, the variation of inference time remained roughly within 2 µs between HPC and RPi. A low inference time was critical for the real-time operation of the classification system. One possible reason for the better accuracy performance in the case of XGBoost compared to CNN was that the classification model for XGBoost was created using hand-crafted features which enabled learning differentiating patterns for the four target classes better than the CNN model that automatically extracted the differentiating features. These results, however, were data-dependent, so they should be validated on different datasets to verify the generality of the model. We found that overall, XGBoost was better suited for deployment on RPi because of its faster inference time and better performance than CNN. By using two different predictive models for classification, we demonstrated the flexibility of the system to deploy improved classification models in the future. In this system, we used a DAC to generate EEG signal waveform form European data format files. This provided a reliable way to generate an EEG signal waveform without requiring an actual subject to capture the EEG signal from. We verified that the EEG waveform generated using the DAC on RPi was consistent with the EEG data stored in the EDF file. The verification was done by calculating MSE across the stored and generated signal, which was found to be 0.26, a small value indicating that the generated signal represented the stored signal accurately. Synthesizing EEG signals to replicate the complex and typically nonlinear signal patterns is challenging and the ability to generate EEG signals from an actual recording data file using a DAC simplifies the setup that is required to test an EEG classification deployment system hardware and software chain. It enables the use of several available open-access EEG data files to train classification models and test the deployment system. For future use, the signal generation capability of this system can be simplified for ease of use and expanded to work with a variety of EEG data file types. This can help accelerate mTBI related future research pertaining to portable classification systems that are often constrained by the lack of readily available live EEG signals to test a hardware classification system. In addition to early mTBI detection, blueberry pot the capability of the system to perform live classification on input EEG signals can be extended to cover mTBI related health and sleep monitoring applications in the future. Typically, after the initial diagnosis, TBI patients undergo EEG sleep monitoring in a hospital setup. A portable EEG sleep monitoring system, such as the one described in this work, can enable a subject to self-monitor in home settings and greatly enhances the accuracy, efficiency, and efficacy. The classification system developed in the current work can also provide a replacement of the labor-intensive manual sleep-stage scoring of EEG signals by human experts with an online and automated system with the capability to perform fast sleep staging. Further, our technical approaches can be extended to several other EEG applications, including detection of the onset of epileptic seizures, strokes, and other neurological conditions.

In this work, we used a relatively simple hardware system to capture and digitize EEG signals, which could be improved. Because we generated EEG signals from a datafile containing clean EEG data, this hardware did not include amplification and filtering stages. A practical system designed for field use would require additional hardware and software capabilities to capture and process EEG signals in real-time. We also used a relatively simple metric for comparison of generated and stored EEG signals. While we only used MSE as a metric for this system, for cases where components in the signal path could potentially cause phase changes in the signal, MSE should be coupled another metric such as cross-correlation to verify signal integrity. In terms of hardware, such a system would require amplification, preprocessing, and filtering stages. In software, decimation, normalization, Independent Components Analysis , physiological artifact removal , and filtering stages can be implemented. Further, we used an 8-bit ADC for this proof-of-concept system, but for devices designed for practical use, ADCs typically vary from 16-bit to 24-bit resolution. For example, the OpenBCI Cyton Biosensing system for sampling EEG and other physiological signals uses a 24-bit ADC. We will note that higher resolution ADCs also involve a relatively higher cost and have lower sampling rates as the number of resolution bits increases. In addition, the system in this work was designed for single-channel EEG generation and capture, which limits its use for multichannel EEG applications. It does not directly provide connectivity to wireless EEG headsets. However, several “hardware attached on top” devices are available for RPi, for example, the brain HAT, that makes it possible to connect wireless headsets seamlessly and we anticipate the system in this work to function as intended with the actual streaming EEG data outside the particulars of EEG headset interfacing.Horticultural crops have high economic, and enrich our lives through their aesthetic and nutritional value. Many horticultural species originate from tropical regions and are sensitive to cold at every stage of their lifecycle. Cold stress leads to lower productivity and post-harvest losses in these species, with poor economic and environmental outcomes. Better understanding of the protective mechanisms mediated by hormonal and other signaling pathways may offer solutions to reduce cold-stress induced losses. The papers included in this collection illustrate this concept, examining natural cold-tolerance mechanisms and practical ways for growers to alleviate chilling stress and to reduce crop losses. The studies were remarkably diverse in terms of the species studied , plant organs examined , and approaches used . The papers encompassed the use of basic science, aimed at identifying key genes and their roles in cold signal transduction and protective pathways in fruit and photosynthetic tissues; reverse genetics for proof-of-concept on the hypothesized role of a cold-tolerance transcription factor cloned from an understudied species; and emerging technologies, by using exogenous hormones and signaling compounds to mitigate the harmful effects of chilling. These studies are described below.C-repeat binding factor proteins constitute a transcription factor subfamily known to play a key role in plants against different types of abiotic stress including cold, heat, salinity or dehydration, and thus have been extensively studied. Overexpression of CBFs has been used for the development of genetically modified plants with enhanced stress tolerance and for the investigation of the molecular mechanisms underlying plant stress responses. Using this approach, Yang et al. found that overexpression of three newly identified longan CBF genes enhanced cold tolerance in Arabidopsis by increasing the content of the osmoprotectant proline, reducing the accumulation of reactive oxygen species , and stimulating the expression of cold-responsive genes.

The mixture was centrifuged at 20,000 g for 15 minutes, after which 0.4 ml of supernatant was extracted, mixed with 0.4 ml of 2:1 chloroform:methanol mixture, and centrifuged at 12,000 g for 5 minutes. 50ul of supernatant was combined with 50ul of Amplex®Red working solution, which was prepared according to manufacturer instructions, in a black 96-well plate with a transparent flat bottom. Absorbance was read using an Epoch-2 microplate reader at 560nm. For H2O2 quantification, a standard curve was generated following the manufacturer’s instructions. Frozen grape powder was homogenized for 10min in 0.1% trichloroacetic acid and then centrifuged at 10,000 g for 5 minutes. 1mL of the supernatant was combined with 4mL 20% trichloroacetic acid containing 0.5% thiobarbituric acid, then the mixture was heated for 15 minutes at 95°C then immediately cooled in an ice bath for 5 minutes. After centrifugation at 10,000g for 10 minutes, 100 of supernatant was plated into a Corning® black-walled, clear, flat-bottomed 96-well plate and absorbance were read at 532 and 600 nm. Concentration was calculated using the MDA molar extinction coefficient 155,000 M-1 cm-1 with the following equation: MDA concentration = •1010, accounting for aliquot volumes and dilution factors.The effect of the irrigation treatments on indicators of vine water stress, hydrogen peroxide/lipid oxidation products, and osmotic potential in the berry was examined through one-way analysis of variance for each sample date. We determined the onset and rate of berry cell death, shrivel, and ROS accumulation by fitting piece wise linear regression models between time and percent cell vitality, the berry shrivel index, and H2O2 concentrations. The onset is the date these variables transitioned from relatively constant to rapidly changing, defined as the fitted breakpoints of the piece wise regression models, plastic plants pots and the rate is the slope of these variables over time after onset. We tested for significant irrigation treatment effects on onset and rate by examining their 95% confidence intervals for overlap.

Furthermore, Correlation between shrivel and % living tissue and peroxide and % living tissue was determined through the Standardized Major Axis Tests and Routines package in R using the sma function. We also tested for significant treatment differences between treatments by one-way analysis of variance for TSS, TA, and pH at harvest, when they are most relevant to growers. Because 7/25 was not used for perimeter analysis, nor was 8/1 for cell vitality, both sets of data were omitted in order to achieve a date-by-date correlation. Out of 540 berries sampled, 248 cross sections were viable for image analysis due to damage incurred in the mesocarp from the razor blade hitting and dislodging seeds. Measurements of percent living tissue and perimeter/total area were taken on different yet mostly overlapping subsets of images due to a fraction of images lacking a smooth perimeter or consistent dye stain. For cell vitality analysis, n = 68, 65, and 72 for treatments 1, 2, and 3 respectively; and for the shrivel index analysis, n = 74, 65, and 78 for treatments 1, 2, and 3 respectively. In tracing representative areas of the berry for cell vitality analysis, the selected area routinely excluded the pedicel residue, which did not typically take up the stain. Due to the violations of equal variance caused by these asymmetries, the non-parametric One-Way test in R was used for both percent living tissue and shrivel index on the last sampling date, and, likewise, the Games-Howell test was used as a posthoc test to compare treatment groups.Our augmented irrigation regimen commencing on the estimated date of onset, i.e. the late treatment, was effective in significantly reducing the rate but not the onset of cell death. Onset was defined as the fitted break point of the piece wise linear regression models, when the rate of change in cell vitality significantly increased. The onset of cell death occurred 92 ± 1 DAA for the control, 89 ± 8 DAA for the early treatment, and 83 ± 4 DAA for the late treatment . The 95% confidence intervals for these estimates overlapped, indicating that onset was not significantly different between treatments. The fitted slope after the break point indicates the rate of cell death after onset. Post-onset slopes were -2.06 ± 0.22 for the control, -1.21 ± 0.53 for the early treatment, and -0.90 ± 0.11 for the late treatment .

The significance of the late treatment’s effect on the rate of cell death is demonstrated by the non-overlapping 95% confidence intervals between the control and late treatment. There were no final treatment differences found on cell vitality for the last sampling date. Figure 6 displays the cell vitality means while Figure 7 shows the segmented fit over the means.Our late irrigation treatment, a 40% augmented pulse starting at the beginning of cell death, generated two significant effects in our experiment: it reduced the rate of cell death in the berry mesocarp tissue and produced less shriveled, more turgid berries at harvest. We also hypothesized that the early treatment would preemptively reduce water stress and postpone cell death. We correctly anticipated the date of cell death onset , yet despite the optimal timing of our treatments the early irrigation regime was not successful in reducing water stress or cell death. While the late pulse reduced the rate of cell death, its effect on shrivel at harvest was not present for the rest of the experiment. This asymmetry is highlighted by the lack of overall correlation between shrivel and cell death for the late pulse, and the lack of a steady decline in turgidity . It was only for the control that significant correlation was observed, which may be explained by the lower error observed in the piece wise linear regression model for the control than the late or early treatments . Unexpectedly, the greater turgidity in the late treatment berries was not reflected in the osmotic potential, °Brix, or TA measurements at harvest, when we would have expected solutes to be more dilute than treatments with greater shrivel . While significant, difference in shrivel were not great enough to affect solute concentrations. The cell death onset and rates we found were largely within the range observed in other studies, blueberry pot but it can be difficult to draw definitive conclusions from comparing the onset and rate of cell death among experiments with many changing variables . Cabernet Sauvignon has only been measured in one other study with cell death beginning at 100 DAA and 40 days after veraison for field-grown vines in Napa Valley , compared to 92 DAA and 34 DAV for our control vines in Davis .

Given Davis is a warmer climate than Oakville, this may explain why our vines exhibited cell death onset earlier. These differences could also reflect a lack of precision in estimating onset dates, since fluctuations in %living tissue measurements can weaken trends with time, as in this study . This could also reflect differences in growing conditions or typical variation within cultivars. The reported ranges of cell death onset dates for any given cultivar vary by as much as 12 days. Syrah onset tends to fall between 87-96 DAA after which tissue vitality sharply declines while Chardonnay’s cell death much slower at an approximately constant rate. . Our rates of cell death fell within the reported range of living tissue analysis performed thus far on grape berries. The Cabernet in Krasnow et al. , between 100-150DAA, lost cellvitality at a rate of approximately -0.83 %LT/DAA while our control and early treatments for Cabernet had a rate of -1.21 and -2.06 respectively. Metabolic processes sensitive to temperature have been connected to cell death, however Xiao et al. and Bonada et al. have both observed no impact of elevated temperature on cell death rate. For Bonada et al. , within the range of 80-120DAA, Syrah lost living tissue at a rate of -0.62 %LT/DAA and Chardonnay at -0.53. The greatest rate of cell death was reported by Xiao et al with living tissue in Syrah diminishing at rates as high as -5.17 %LT/DAA. Interestingly, Xiao et al. found that irrigation postponed cell death onset much later than the non-irrigated vines, yet the irrigated vines crashed twice as fast as the non-irrigated vines , which may have developed more extensive root systems with greater access to water. Conversely, our late treatment vines exhibited the slowest cell death and the earliest onset, highlighting the efficacy in slowing cell death. Our results for cell vitality and shrivel analysis accord with the reported role that xylem backflow plays in the relationship between cell death and LSD. Cell death occurs prior to LSD by reducing turgor pressure and accelerating water loss through xylem backflow, thus a more negative ψx would enhance the pulling force for water out of the berry . Since ψmd and berry osmotic potentials were nearly the same across treatments, the observed differences in cell death rate and shrivel index cannot be attributed to differences in xylem water potential or the berry solute concentrations. In the late treatment berries cell death was slower and cell vitality remained greater from 92DAA onwards , which indicates more water retention and less backflow. Less water loss in berries from cell death supports our finding that the late treatment berries maintained the greatest turgidity for almost all dates during that same period, from 92DAA to harvest. Another factor to consider affecting xylem backflow is pedicel hydraulic conductivity.

Xylem blockages that are known to develop post-veraison in grapevines could hinder pedicel hydraulic conductivity and slow down backflow out of the berry , though there is no basis for such a physiological response to our late irrigation pulse. There are established differences in pedicel hydraulic conductivity between grape cultivars post-veraison and it has been reported that grapevines produce tylose blockages in response to pruning wounds and bacterial infection . Yet, there is no reported evidence of grapevines producing xylem gelblockages in relation to water stress during our period of ripening. For future studies, it may be of interest to track phloem inflow to the berries in response to extra water availability during cell death. Post-veraison the phloem remains the main source of water influx into the berry, remaining functional until 25-26 Brix, a range our berries did not exceed during the late treatment pulse, which suggests the phloem likely remained functional . Our results have shown vines receiving additional irrigation during the onset of cell death, despite having an earlier onset of cell death, retained water better than vines with later onset. Since phloem functionality declines steadily during ripening , an earlier onset of cell death comes at a time of potentially greater phloem hydraulic conductivity, rendering an irrigation pulse more effective at providing more phloem inflow to compensate for xylem backflow. Greater sap influx into the berry would introduce more dissolved oxygen and alleviate hypoxic stress due to respiration and ethanolic fermentation, though this has not been experimentally determined.We expected that H2O2 levels to begin to rise at cell death and continue throughout the experiment. Our H2O2 concentrations saw an uptick during the onset of cell death and, with exception of one outlying date, steadily increased throughout cell death proliferation at similar levels for all treatments . Across the three treatments cell death onset coincided with the onset of H2O2 accumulation at 88 DAA . This is evidenced by the significant correlation found between the two variables for the late treatment and the control. It should be noted that it is only coincidental that those same two treatment groups were significantly different in our cell death rate analysis—matching significant H2O2 correlations and cell death rates would not be causally related findings. Whether the concentrations found in our study, between 1-3 nmol/g FW, are great enough to cause the degree of cell death we observed is not clear. Gowder measured H2O2 in combined skin and pulp samples finding concentrations in the range of 4-10 nmol/gFW in Chardonnay, 12-9 in Grenache, and 15-40 nmol/gFW in Syrah between 90-120 DAA with similar percentages of living mesocarp tissue to this experiment. We would expect to find lower values in the mesocarp only given that the skin is known to contain higher levels of H2O2, yet Pilati et al. found values in range of 18-27 nmol/gFW for the mesocarp only of Pinot Noir berries between 70-84 DAA. Interestingly, also in Gowder the three cultivars expressed no clear relationships between H2O2 accumulation and cell death, indicating there is much greater complexity to be addressed in the sources of H2O2, its scavenging, and alternative causes of cell death such as lipoxygenases.

To determine if the effects of PhylloStart bacteria on plant reproductive success would be seen in an environment with more potential sources of phyllosphere bacteria and/or whether early inoculation of plants changed subsequent microbiome assembly in the field, we included a field component in the third trial experiment. After inoculation, we transferred both PhylloStart-inoculated and control plants into the field. These plants were sampled concurrently with the plants from the same cohort that remained in the greenhouse , and their phyllosphere communities were sequenced. We analyzed greenhouse and field locations separately and found a significant effect of PhylloStart inoculation density on bacterial abundances in the greenhouse =10.17, p=0.006, but no effect in the field =4.07, p=0.13. A Dunn Post-Hoc test showed that PhylloStart High treated plants had significantly higher bacterial abundance than the control plants in the greenhouse . Further, while we see that community composition is influenced by PhylloStart treatment in the greenhouse, we see no such effect in the field grown plants. When looking at a PCoA of bacterial community similarity using Bray-Curtis distance we see that the PhylloStart-treated greenhouse plants clearly separate out from the control plants, with the plants treated with high concentrations of PhylloStart distinct from the controls, and the plants treated with low concentrations of PhylloStart falling between the controls and the high inoculation. Meanwhile, the communities associated with the field grown plants differ from those grown in the greenhouse, plant pot with drainage but do not otherwise separate by PhylloStart treatment. When analyzing dissimilarity using an Adonis PERMANOVA, we see a significant effect of PhylloStart treatment , location; ie. field vs. greenhouse , as well as a significant PhylloStart by location interaction .

When analyzing each location separately with a pairwise Adonis, we see that there are significant differences between PhylloStart high and control treated plants in the greenhouse , but no difference between PhylloStart high and low , or PhylloStart low and control in the greenhouse. The plant microbiome is increasingly recognized for its role in shaping plant health, but most work to date has focused on below ground associations between plant-microbe interactions. Thus far,most evidence for an impact of the above-ground, phyllosphere microbiome on their hosts has focused on disease or herbivore resistance . Our initial experiment established that greenhouse-grown plants develop a significantly more abundant microbial community when inoculated with a synthetic microbial community . When inoculated onto plants early in development, we found that our taxa represent the dominant members of the phyllosphere and that overall bacterial densities were far higher in amended plants relative to controls . With two additional greenhouse studies, we determined that these microbial associations lead to a significant increase in the total number of fruit produced by greenhouse-grown tomato plants . We also found that, in a growth chamber trial, the bacterial community provided nutrient status dependent protection from P. syringae establishment. In contrast, plants that were transplanted into a field environment did not appear to benefit from the initial inoculation of PhylloStart bacteria . Given the minimal development of the phyllosphere community in non-treated greenhouse control plants , our findings suggest that greenhouses present an ideal location to study the effect of microbial amendments on agriculturally relevant plant traits, and support previous work finding that greenhouse-grown plants develop bacterial communities distinct from outdoor environments .

The extraordinarily low background levels of bacterial colonization allowed us to examine the importance of phyllosphere bacteria to plant fitness, where we found that the application of PhylloStart bacteria was associated with increased total fruit production . Further, that we do not see a fruit number/size tradeoff in this study suggests that the microbial amendment is increasing investment in above-ground biomass, rather than simply redirecting resources from fruit size to number, as is commonly observed in seeds for example . Our results add to a body of work describing how fruit yield can be affected by both local nutrient conditions and microbial associations but extend the latter to the above ground tissues. In contrast to the greenhouse, we did not see evidence for either establishment or impact of PhylloStart amendment in the field. In this case, PhylloStart bacteria were not found at significant abundances on these plants after a month in the field , and their initial community structure did not seem to shape the future composition of the phyllosphere communities . While this would seem to contradict results finding initial colonizers dominate plant microbiome assembly , priority effects often depend on the identities of the earlycolonizing species and their environments. For example, when wood disks were pre-colonized with fungi and placed in a field for six months, retention of initial colonizers in the eventual community varied between ascomycetes and basidiomycetes, and from season to season . In the tomato plant phyllosphere, PhylloStart bacteria may have been overwhelmed by dispersal from neighboring plants , or from other sources. Further, the field conditions during which we ran our trials may have differed from those under which we initially quantified the phyllosphere composition to design PhylloStart. Previous work has shown that community composition will depend on both plant host genotype as well as local environmental conditions .

It remains possible that ‘local’ or otherwise well-adapted isolates may have yielded better performance. Further field trials under a broad spectrum of conditions and locations, as well as with larger sample sizes, are needed to determine whether bacterial amendments to the phyllosphere can potentially confer benefits to commercial field tomato production. There are various mechanisms by which the phyllosphere community might provide the observed benefits to its host. These include: 1) altering plant hormone signaling, either directly by producing phytohormones or indirectly through the elicitation of a plant response; 2) by increasing the nutrients available to the plant either through enhanced nutrient fixation or availability; and 3) through reducing stress, either environmental or due to pathogen pressure . While our study does not seek to explain the mechanism underlying observed biostimulant effects, it likely relies on a combination of these factors. However, that the effects of Azomite fertilization and PhylloStart inoculation acted primarily in an additive fashion suggests that altered nutrient acquisition is not a particularly dominant force. Like many phyllosphere microbiome studies, our experimental design did not specifically exclude the possible movement of bacteria to the soil , and it thus remains possible that some fraction of the inoculated bacteria colonized the below-ground compartment. However, recent studies have found that phyllosphereand rhizosphere-associated bacteria are predominately adapted for survival in their respective niches , and, when paired with our 16S rRNA sequencing results showing robust and long-term establishment of the community on the leaf surfaces, we think it is more likely that the effect is mediated through phyllosphere interactions directly. With this in mind, future work exploring the mechanisms of phyllosphere associated growth promotion should specifically differentiate any effects impacting above versus below ground responses to the PhylloStart bacteria, either through reciprocal translocation of the species or by physically preventing inoculation of or migration to the soil. One specific potential mechanism for the increased reproductive success is linked to the phytohormone auxin , which is a major regulator of plant growth, is commonly produced by bacteria inhabiting the phyllosphere , and has been linked to increased biomass accumulation in rice and corn . In this context, increased fruit yield could be mediated by the action of auxin in decreasing flower abscission , potentially leaving more flowers available to set. Of note, we did not observe a significant change in flower number across the first trial. Using BLAST to search the genomes of the PhylloStart bacteria, we found that several members have matches for idpC , a key protein in auxin production . Future research is needed to confirm that these bacteria can produce auxin in planta, growing blueberries in pots and if this may explain some of their plant-beneficial effects. It is also possible that the PhylloStart bacteria alter the plant’s response to environmental cues, allowing the plant to better optimize its growth strategy and invest more resources in reproduction.

Recent work has focused on the phenomenon of microbiome-dependent ontogenic timing , by which the presence of certain bacterial species acts as essential cues in the developmental timing of their host organism . For example, the composition of the Boechera stricta soil-associated bacterial community has been found to significantly alter the timing and duration of flowering . Further research is needed to assess the potential role that host-associated microbes play in developmental timing. Throughout these trials we saw no evidence of an interaction between the nutrient status of the plant and the effect of the PhylloStart bacteria; instead, the bacterial and Azomite treatments additively increased the total yield. Given these observations, we were curious if the PhylloStart community would show the nutrient dependent pathogen protection found in our lab’s previous work . Indeed, we found, in a growth chamber experiment, that the addition of this community limited the growth of the pathogen P. syringae in nutrient-limited plants, but that this effect disappeared when organic phosphorus fertilizer was added . These results are in line with the stress gradient hypothesis, which posits that inter-species interactions should become more facilitative under adverse conditions , and highlight the important role that phyllosphere bacteria play in stress response. Indeed, previous work in this system has shown that the PhylloStart bacteria up-regulate defense responses in Arabidopsis and subsequently reducing pathogen colonization, . In summary, we find that the presence of phyllosphere-associated bacteria benefit their plant host when grown in a microbially depauperate greenhouse environment, through an increase in reproductive success as measured by total fruit production, with further evidence for pathogen resistance. These results are important for understanding the role of microbial communities in host outcomes and are broadly relevant in an agricultural context where, for example, 32% of domestic and 56% of imported tomatoes in the United States are grown in greenhouses that may not provide adequate colonization of phyllosphere bacteria . Further, we show that bacterial inoculation provides an additive increase in fruit production when applied with a common supplement containing micronutrients, opening avenues for further optimization of agricultural production by harnessing the biostimulant properties of phyllosphere microbes.The detachment of a grape berry from its pedicel generally damages the berry because the vascular tissues and associated parenchyma, collectively known as “the brush”, remain attached to the pedicel and are pulled out of the berry on detachment, leaving an open wound sometimes called a “wet” stem scar on the berry’s stem-end. Berry detachment may also remove pieces of skin or cause the whole berry to rupture. Such mechanical damage can reduce the yield and quality of machine-harvested grapes for wine or raisins. Stem-end picking damage also limits the quality and storage life of stemless table grapes. Certain plant growth regulators known as “abscission agents” activate an abscission zone at the pedicel-fruit boundary. The activation of this abscission zone reduces fruit detachment force and promotes the development of dry stem scars . Abscission agents could reduce picking damage and thereby serve as harvest aides if treated grapes can be harvested after the abscission zone is activated, but before the fruit abscises. However, once the abscission zone has been activated, development proceeds quickly and may lead to excessive preharvest fruit drop .The first compound tested as an abscission agent for grape was ethephon, a phosphonic-acid compound that decomposes to release the gaseous plant hormone ethylene. Ethephon can induce the abscission of mature grape berries within 7 to 14 days after treatment, but high dosages are needed. The use of ethephon as an abscission agent for grapes would require an application dosage which is higher, and a preharvest interval that is shorter, than those for the current registered use of ethephon on grapes in order to enhance berry color. Such changes could be expected to increase ethephon residues on treated fruit, and it seems unlikely that regulatory agencies would approve a use that could increase ethephon residues on grapes since existing residues are already a concern. However, it should be noted that Ferrara et al. found that effective dosages of ethephon did not result in excessive residues. Jasmonates, including methyl jasmonate, a natural product, have also been shown to induce the abscission of various fleshy fruits, including blueberry , orange , and tomato.

A direct one-to-one comparison was made between the transcriptomes of the samples S and LS at the same time of cold storage, given the notion that this analysis would outperform the general profile comparison to identify the candidates to be involved in tolerance/susceptibility to cold . Figure 2A shows how the number of differentially expressed genes at each time decreased with storage time , thus confirming PCA results . Functional enrichment analysis showed that by 1 week of cold storage, the transcripts with higher levels of expression in fruits CS1-LS were preferentially related to energy production, RNA translation and protein assembly, the antioxidant system, structure maintenance, and genes with unknown functions . As 1 week cold storage is critical timing i.e. when maximum differences were shown when later transferring fruits to shelf life ripening , these functions may play a prominent role in the tolerant/sensitive character of fruits . By 2 weeks of cold exposure, only the genes with unknown functions were over represented in the tolerant pool , whereas a significant enrichment was noted for the genes linked to amino acid metabolism, pyruvate, signal transduction and transport in the genes at higher levels in CS2S. Interestingly, most of the genes expressed at higher levels in S fruits by 2 weeks had already reached this state by 1-week of cold storage . As two weeks of cold exposure results in mealiness upon shelf life in both S and LS fruits , square plastic pot but with large differences in MI severity, high levels of these genes may correlate negatively with the tolerant character of fruits.

After 3 weeks in the cold, only the highly expressed genes in tolerant fruits showed signal transduction as an over represented class . In this case, the genes differed from those identified as being over represented at 1 and 2 weeks . At this time, both S and LS developed mealy fruits with MI 1.0 and MI 0.8, respectively , but S was probably much more severely affected or underwent other downstream processes. In order to analyze if the transcript program in the cold may have a direct effect on eventual mealiness development during shelf life, a Pearson correlation analysis was conducted between the gene expression values and the projected MI will be achieved when subjected to shelf life ripening after cold exposure . This ‘‘projected MI’’ correlation analysis resulted in 113 directly correlated genes and 159 inversely correlated genes according to their pattern of expression in the cold . The functional enrichment analysis indicated that genes directly correlated to projected MI were enriched in RNA transcription and RNA posttranscriptional regulation. A further inspection revealed genes related to RNA bio-genesis and processing, splicing, RNA transcription machinery and the transcription factors . In addition, genes correlated positively with the projected MI were also enriched in transport category , that includes transporters for auxin, anthocyanin, amino acid, peptides, sulfate, carbohydrates and metal-ions . No functional enrichment was observed for those genes which correlated negatively with projected MI . However, a detailed inspection indicated that this set of genes contained calcium-related genes, including a transcription factor of the CAMTA family, and genes related to antioxidant systems which could participate in the regulation of this transient tolerance mechanism.The possibility that, in addition to cold-inducible mechanisms, some sort of tolerance mechanism may already be partly preprogrammed in tolerant fruits was investigated. The direct comparison between S and LS fruits at mature stage resulted in 63 differentially expressed genes .

Out of them, 13 genes we high expressed in fruits T and some have to do with flavonoid metabolism , structure protection and that forms part of a cycle that generates asparagine for more energyeconomical nitrogen remobilization under darkness and stress conditions. Several cell wall modifying activities were also differentially expressed between fruits S and LS . As no differences at the maturity stage were between pools , it is likely that differences in the expression levels of these genes at harvest may protect fruits and/or contribute to develop the tolerance program at least in the early stages of the cold response. HCA of samples M, R and CS showed that genes differentially expressed between fruits S and LS at harvest qualified in fruits LS as ripening genes . Notwithstanding, it is most interesting to note these genes were characterized by continuing the ripening program during cold storage , which did not happen so clearly in fruits S . However and as expected this behavior of the differential M genes is the exception rather than the rule for ripening genes. As seen in Figure 3B, a similar analysis with a set of 862 ripening genes showed that although cold affect the expression many of ripening genes, is quite effective stopping the molecular ripening program in fruits LS. This result is in agreement with the findings from PC2 . The main expression differences between LS and S fruits involved changes occurring in the same direction in R and cold stored fruits. In fruits LS, the expression of several ripening genes during cold storage remained at the same or higher level that they were in the M stage, but achieved similar expression levels to fruits R in the sensitive backgrounds . Apart from the delayed or attenuated ripening program in the fruits LS during cold storage, these fruits also showed specific ripening processes that became activated during cold storage , which is in agreement with the findings for genes differentially expressed at harvest . A more detailed analysis of shelf life ripening conditions and mealiness will be addressed in a future manuscript .In this section we wanted to see if there were similarities between the adaptation mechanisms operating in peach fruits stored in cold and darkness and those well-characterized in the cold acclimation of Arabidopsis plants grown in day/night regimes.

We wanted to see if the patterns of gene expression for the peach homologues of Arabidopsis genes in cold/dehydration regulons were consistent with the differential cold responses in S and LS peaches. First we analyzed the overlap between the response of cold stored peach fruits and those to various stimuli, including abiotic/ biotic stresses and hormones . Gene-bygene comparisons revealed that the vast majority of the cold regulated genes in our peach cold storage experiment have Arabidopsis orthologs, which have been described as being regulated by cold , or by ABA . Similarly to Arabidopsis, approximately 30% of peach cold-regulated genes were found to be associated with drought and/or salinity treatments . More strikingly however, approximately 35% of the cold-responsive genes in peach were known pathogen responsive genes or have been postulated to play a role in pathogen resistance . Furthermore, the genes described as being regulated by darkness in Arabidopsis account for up to 3.7% of peach cold-regulated genes ,square pot indicating that, although its contribution to all cold-regulated genes was less than those also involved in other stresses, dark stress could contribute to the differences observed in the cold response between peach fruits and Arabidopsis plants . Second, a list of Arabidopsis genes reported in cold regulons and dehydration regulons was used to identify homologous peach genes that were present on Chillpeach microarray . In total, 163 Chillpeach unigenes corresponded to the genes found in at least in one of the previously defined cold and/or dehydration Arabidopsis regulons . The expression profiles of these genes in response to cold storage were compared to those described for Arabidopsis and scored as matching when they behave similarly. More than 60% of the genes associated to the regulons CBF, HOS9, ICE and DREB2 correlated well with both the known Arabidopsis WT cold response pattern and the Arabidopsis mutant expression . That is, the ortologs genes to those up-regulated in Arabidopsis in response to cold showed higher expression levels in LS peach fruits than in high sensitive ones, while the genes down-regulated in Arabidopsis had higher levels in high sensitive peach fruits than in low sensitive ones. In contrast, most of the genes in HOS15, ZAT12, ESK, AREB, MYB, ZF/HD-NAC presented low correlation levels . Therefore, these latter are less likely to contribute to the differences in response to cold between the S and LS pools of fruits. The individual participation of each regulon to the differential response to cold between fruits S and LS was assessed by studying their contribution to the traits/trends observed in the global dataset analysis. For this purpose, we performed both PCA and 2D-HCA using the gene expression values for all the genes in each regulon as input datasets and quantitatively evaluate the importance of each regulon to discriminate samples S from LS and to separate the samples that would eventually became mealy, or not, by assessing by the number of genes well correlated with Arabidopsis in the gene expression models . The importance to discriminate samples S from samples LS was calculated by multiplying the number of genes that correlated well by the variance explained by PC2.

The importance of an operon to separate the samples that would eventually become mealy, or not , was quantified by dividing the number of genes in that operon that correlated well by the weight of the nearest node to CS1-LS. Both PCA and 2D-HCA revealed that regulon ICE1 was the one most contributing to discriminate samples LS and S, as to separate samples CS1-LS from the rest of cold-stored fruits that developed mealiness when submitted to shelf life ripening . Furthermore, this analysis also indicated that the regulon CBF1 was the next major regulon in discriminating between samples LS and S , while emphasized the relevance of HOS9 to separate CS1-LS from the remaining samples . The rest of the cold operons produced no such separation between CS1 S and LS, or did so but to a lesser extent . The expression pattern of the subsets the genes appertaining to the regulons ICE1 , CBF and HOS9 across the different samples showed that although extended exposure to cold debilitated the response of ICE-CBF regulated genes, fruits LS were able to maintain a longer and greater response for many of the genes in the regulon in the cold. In the case of HOS9 regulon, many of its members were up-regulated or without change in LS fruits as compared to M fruits .The same bulked samples used in this microarray experiment were used to validate the results by using medium-throughput qRT-PCR over a set of genes selected because they 1) contributed to separate samples S from samples LS at 1 week of cold storage , 2) showed a differential expression in, both, the M stage and 1-week of cold storage , and 3) showed differences at harvest . In order to examine at the single sibling level the reliability of the differential gene expression patterns obtained from the pools, the analysis was performed also on 15 individual genotypes of the pop-DG population . The qRTPCR results obtained from the pools and from the individual lines making up this pools indicate that 72.5% of the genes had the same expression pattern in the microarray experiment as in the qRT-PCR experiment . However, the magnitude of expression varied slightly in many of the genes and samples tested . Furthermore qRT-PCR experiments conducted on individual pop-DG siblings revealed that 42 out of the 50 genes validated in the pools were consistent with the expected patterns for which they were selected . These results support the validity of our approach and indicate that the genes selected from the microarray analysis could be either involved in chilling tolerance and/or be associated with the differential response to chilling response, and for some of them could even prove to general enough to hold true in individual fruits/plants.Since cold induced mealiness is not observed until the cold stored fruit are allowed to ripen, the chilling sensitivity phenotype of each fruit in the cold was estimated from the protracted mealiness incidence observed for equivalent fruit samples after shelf life ripening . Although mealiness, probably, a downstream effect of cold stress in peach fruits , it is the best phenotyping tool to assess the effect of cold on peach fruit, and has be used successfully to identify CI QTLs in peach. For BSGA we use Chillpeach microarray, interrogating part of peach genome. This provides only an incomplete picture of the genes behind the process; that is partially compensated by Chillpeach microarray being enriched in fruit-specific and cold responsive genes.

During favorable periods, growth, development and reproduction of D. suzukii life stages depend on temperature, the drying power of the air, and host quality and availability . Zerulla et al. followed via dissection of field collected females the lack of ovary development in D. suzukii during winter and the resumption of ovary development in spring . Plantamp et al. found that cold treatments had a strong impact on adult survival but had no effect on female’s fertility. The magnitude and pattern of age-specific fecundity in D. suzukii contrasts sharply with other drosophilids such as D. melanogaster that have oviposition rates an order of magnitude higher during the first 20 days of adult life, but unlike D. suzukii reproduction falls off dramatically after 40 days . Toxopeus et al. found that acclimated winter morph adults have delayed reproductive maturity, and can remain active at lower temperatures than summer morph adults. These above attributes suggest that D. suzukii is able to maintain population pressures longer and under more adverse conditions than other drosophilids . The above biology is further enhanced in temperate areas by the fly’s ability to attack a wide range of hosts, some of which are available year around . In Europe, Kenis et al. reared SWD from 84 plant species belonging to 19 families, 38 of which are non-native. While development, fecundity and mortality rates vary with hosts , modeling the dynamics of these hosts and their effects on D. suzukii is vexing. In the PBDM, round plastic plant pot we assume hosts are available for D. suzukii reproduction when temperatures are in the favorable range.The recent literature speaks to the urgent need for approaches to estimate the geographic distribution and relative abundance of native and exotic species that may experience novel climate due to range expansion or climate change.

Specifically, Sect. 4.3 by Working Group II in the assessment reports of the Intergovernmental Panel on Climate Change outlines the shortcomings of widely used standard methods based largely on the climate envelope approaches commonly used to assess the impact of climate change on ecosystem. Among the gaps identified in IPCC AR4 were: the ‘‘inability to account for species interactions, the lack of physiological mechanisms, and the inability to account for population processes’’ . Single species and multi-trophic PBDM circumvent some of these problems and often challenge assumptions on how field data should be interpreted , especially where data gaps exists . The polyphagous spotted wing Drosophila, D. suzukii, is a native of Eastern and Southeastern Asia . Phylogenetic analysis by Ometto et al. indicates that the species originated during the late Miocene when its native Asian range was characterized by extended mountain temperate forests. Kimura found that D. suzukii had one of the broadest geographic ranges among the drosophilids of Japan, ranging from Hokkaido in the north to Iriomote Island east of Taiwan. The fly has extended its range in temperate and tropical areas in North America and Europe and the Mediterranean Basin where hosts are abundant , including areas where weather may be only moderately suitable for population development. Several biological attributes appear to enhance the fly’s ability to expand its range. For example, it has a very wide host range ; unlike most drosophilid larvae that develop on relatively protein-rich rotting fruit, D. suzukii larvae uniquely develop on protein-poor, carbohydrate-rich ripening fruit ; and its thermal characteristics are conducive to its range expansion. Specifically, laboratory data on developmental rates were consistent across studies and predicted a lower thermal threshold of 5.95 C and an upper threshold of *31 C .

Ryan et al. estimated a lower threshold of 8.1C and found that no adults emerged above 30.9 C. The differences in threshold estimates likely reflect differences in experimental methods. A low threshold for immature stages enables the species to be active early in the spring and to complete development of immature stages in the fall to produce winter morph adults , while the high threshold for reproduction would appear to delay reproduction until hosts are available in spring. Reproduction potential in D. suzukii is relatively low, approximately half that of D. melanogaster . Reported differences in reproductive rates observed in European and North American populations may be due to experimental conditions or may have some genetic roots . In our analysis, we used the higher fecundity figure, noting that it has little impact on the prospective geographic range and patterns of relative abundance. The effects of temperature on D. suzukii mortality on summer adults were variable among reported studies, especially at low and high temperatures . Tochen et al. characterized the effects of relative humidity and temperature on reproduction and mortality of summer adults, and the combined effects were incorporated in the PBDM . There is general agreement in the literature that cold winter temperatures are the major limiting factor for the fly in north temperate regions. Based on laboratory and field studies, Jakobs et al. concluded that adult D. suzukii phenotypic plasticity alone is insufficient to allow D. suzukii to overwinter in northern temperate areas. Stephens et al. studied cold hardiness of winter-acclimated D. suzukii in the laboratory, and concluded that the species is chill-intolerant, and further stressed that while the winter-morph adult is the most cold-tolerant life stage, both summer and winter-morph adult forms could not overwinter in cold climates without microclimate refuges . Using available data on summer morphs survival with temperature, the PBDM predicts a wide distribution for the fly in the USA and Mexico with highest densities in warmer tropical areas of Mexico = Hawaii [Florida[coastal southern California with lower year round populations in temperate areas of Northern California , and mid- to late-summer populations developing in Oregon and Washington .

Similar mid to late summer populations develop in the north Central USA with highest densities found in woodlands relative to crop fields such as raspberry . Recent, albeit incomplete, studies on winter morph adult survival at 1 and 5C suggest phenotypic plasticity may significantly increase survivorship at low temperatures . Data from Toxopeus et al. on summer and winter morph adult survival at 0 C and after 1 h exposure to temperatures from 0 to -13 C suggest that the mortality function for winter morph adults is displaced approximately 5–6 C to lower temperatures . For example, survivorship of the winter morph at 0 C is about the same as for the summer morph at 5 C. We explored heuristically the effect of shifting mortality Eq. 2i leftward 5.5 C in the PBDM and found that this does not radically change the prospective geographic distribution of D. suzukii in the USA and Mexico, but because more adults survive the winter, densities increase in many areas such as coastal California and in northern areas such Maine and Wisconsin . In south and central Europe and the Mediterranean Basin, fly densities increase more widely to levels predicted for coastal California , and inter annual variability declines . The effect of increased winter morph survival on the density dynamics of egg production and summer and winter morph adults were explored for the cold location of Benton, WA . The results show the increase in winter survival, a modest increase in population densities, but no changes in population phenology. In colder areas such as Benton WA, the favorable season is limited by cool average temperature below *12.75 C that delay reproduction in spring and that induce the development of non-reproducing winter morph adults in fall . Data to characterize fully winter morph adult mortality are currently unavailable, but can easily be incorporated in the PBDM when they become available, and a complete analysis will be performed.Behavioral factors can also affect survival during adverse periods. For example, Tochen et al. posited that behavioral adaptation of short-distance migration enables D. suzukii adults to move towards favorable microclimates that enhance their survival and reproduction in otherwise marginal environments . This point was clearly illustrated by Harris et al. during 2011–2013 using apple cider vinegar traps to monitor D. suzukii adults in multiple crops and associated fruiting plants at the Wolfskill USDA Germplasm Repository at Winters, CA, USA. During spring and summer, D. suzukii adults were trapped in the orchards, 25 liter round pot but during November through April, high trap captures were associated only with citrus, with highest numbers of flies found around a nearby house where fruiting ornamentals such as common myrtle and fire thorn were present and had fruit until April 2013.

Had the trapping occurred only in the orchard area, the results would have suggested that D. suzukii had decreased to very low numbers during winter when in fact adults were active nearly all year around with trap catches near the house during winter being nearly twice as high as those during the season in the orchards . In a mark-recapture study, D. suzukii adults were found to take refuge in wild habitat surrounding a cultivated raspberry field before migrating back to the field when the susceptible crop was present . This information suggests that surrounding habitats with hosts, especially in sheltered areas, should be monitored for D. suzukii adult winter activity and survival . Fly movement makes accurate sampling of adult densities throughout the year vexing. The trapping records of Dalton et al. during 2010 at five sites in Oregon, Washington and California likely had unknown sampling bias. As predicted by the PBDM, D. suzukii adults were found during much of the year at Stockton , CA which is located about 40 miles south of Winters, CA where Harris et al. recorded D. suzukii year around . The climate at both locations has the moderating influence of San Francisco Bay . Specifically, the PBDM predicts that cold weather mortality [i.e., l] had little impact on D. suzukii at Stockton CA, but temperatures were often too low for reproduction . Hot weather during summer caused some suppression of D. suzukii, but the effect was not large. Predicted cold weather mortality rates at Stockton, CA were 1/10th those at Marion and Wasco Counties of Oregon and Benton County in Washington where populations grew only after mid-summer . This delayed phenology is due to cold temperatures in late fall and winter that drive D. suzukii populations to near zero despite the presence of alternate hosts in the area , allowing populations to resurge only in mid- to late-summer. The impact of cold temperatures on reproductive quiescence occurred only during fall in Oregon and Washington, while mortality due to higher temperatures during summer did not appear to have much impact at any of the four western locations studied by Dalton et al. . The delayed phenology of the fly in these colder areas suggests that, as occurs with aphids and other insects carried long distance as aeroplankton , D. suzukii adults may be carried from more favorable nearcoastal areas of the northwest to augment reinvasion of colder areas . This possibly explains the earlier phenology of D. suzukii at Marion Co, OR compared to more inland areas of Wasco Co, OR or Benton Co, WA . Similar scenarios may also occur in the northern reaches of the central USA that may be reinvaded from warmer southern areas, and in Europe into higher latitudes and elevations in mountainous areas . Climatic conditions in the Mediterranean Basin are more favorable and stable for D. suzukii where year-round survival is predicted. In contrast, D. suzukii is limited by high temperatures and low RH in hot dry areas of the southwestern USA and northern Mexico , but cold winter weather can also be a factor in parts of this area. Similarly, high temperatures and low RH would also impact D. suzukii in inland North Africa. In summary, D. suzukii is a cold intolerant invasive species that attacks a wide range of wild and domesticated berries and fruit. The cold resistant winter morph adults can suvive winters and reproduce during spring in marginal north temperate climates . The PBDM gave good predictions of the prospective distribution and relative favorability of climate for D. suzukii in North America, Europe and the Mediterranean Basin. Despite not including the effects of host plants availability and microclimate effects, the PBDM was able to predict the relative dynamics and phenology of adult trapping data at four locations ; data that were likely complicated by sampling bias and fly movement among potential hosts and possibly between sheltered habitats .

A subtler issue raised by our data is the density dependence of magnetic pinning; as shown in Fig. 5.3, Bc does not simply track 1/m across the entire density range, in particular failing to collapse with the rise in m in the Chern magnet gap. This suggests nontrivial dependence of either the pinning potential or the magnetocrystalline anisotropy energy on the realized many body state. Understanding the pinning dynamics is critical for stabilizing magnetism in tBLG and the growing class of related orbital magnets, which includes both moir´e systems as well as more traditional crystalline systems such as rhombohedral graphite. In order to understand the microscopic mechanism behind magnetic grain boundaries in the Chern magnet phase in tBLG/hBN, we used nanoSQUID magnetometry to map the local moir´e superlattice unit cell area, and thus the local twist angle, in this device, using techniques discussed in the literature. This technique involves applying a large magnetic field to the tBLG/hBN device and then using the chiral edge state magnetization of the Landau levels produced by the gap between the moir´e band and the dispersive bands to extract the electron density at which full filling of the moir´e superlattice band occurs . The strength of this Landau level’s magnetization can be mapped in real space , and the density at which maximum magnetization occurs can be processed into a local twist angle as a function of position . It was noted in that the moir´e superlattice twist angle distribution in tBLG is characterized by slow long length scale variations interspersed with thin wrinkles, black plastic plant pots across which the local twist angle changes rapidly. These are also present in the sample imaged here . The magnetic grain boundaries we extracted by observing the domain dynamics of the Chern magnet appear to correspond to a subset of these moir´e superlattice wrinkles.

It may thus be the case that these wrinkles serve a function in moir´e superlattice magnetism analogous to that of crystalline grain boundaries in more traditional transition metal magnets, pinning magnetic domain walls to structural disorder and producing Barkhausen noise in measurements of macroscopic properties.There is another way to make a moir´e superlattice. Two different 2D crystals with different lattice constants will form a moir´e superlattice without a relative twist angle; these systems are known as heterobilayers . These systems do not have ‘magic angles’ in the same sense that tBLG and tMBG do, and as a result there is no meaningful sense in which they are flat band systems, but interactions are so strong that they form interaction-driven phases at commensurate filling of the moir´e superlattice anyway. Indeed, many of the interaction-driven insulators these systems support survive to temperatures well above 100 K. The most important way in which heterobilayers differ from homobilayers, however, is in their insensitivity to twist angle disorder. In the small angle regime, the moir´e unit cell area of a heterobilayer is almost completely independent of twist angle, as illustrated in 5.17E-F. A new intrinsic Chern magnet was discovered in one of these systems, a heterobilayer moir´e superlattice formed through alignment of MoTe2 and WSe2 monolayers. The researchers who discovered this phase measured a well-quantized QAH effect in electronic transport in several devices, demonstrating much better repeatability than was observed in tBLG. The unit cell area as a function of twist angle is plotted for three moir´e superlattices that support Chern insulators in 5.17G, with the magic angle regime highlighted for the homobilayers, demonstrating greatly diminished sensitivity of unit cell area to local twist angle in the heterobilayer AB-MoTe2/WSe2. MoTe2/WSe2 does have its own sources of disorder, but it is now clear that the insensitivity of this system to twist angle disorder has solved the replication issue for Chern magnets in moir´e superlattices.

Dozens of MoTe2/WSe2 devices showing well-quantized QAH effects have now been fabricated, and these devices are all considerably larger and more uniform than the singular tBLG device that was shown to support a QAH effect, and was discussed in the previous chapters. The existence of reliable, high-yield fabrication processes for repeatably realizing uniform intrinsic Chern magnets is an important development, and this has opened the door to a wide variety of devices and measurements that would not have been feasible in tBLG/hBN.The basic physics of this electronic phase differs markedly from the systems we have so far discussed, and we will start our discussion of MoTe2/WSe2 by comparing and contrasting it with graphene moir´e superlattices. In tBLG/hBN and its cousins, valley and spin degeneracy and the absence of significant spin-orbit coupling combine to make the moir´e subbands fourfold degenerate. When inversion symmetry is broken the resulting valley subbands can have finite Chern numbers, so that when the system forms a valley ferromagnet a Chern magnet naturally appears. Spin order may be present but is not necessary to realize the Chern magnet; it need not have any meaningful relationship with the valley order, since spin-orbit coupling is absent. MoTe2/WSe2 has strong spin-orbit coupling, and as a result, the spin order is locked to the valley degree of freedom. This manifests most obviously as a reduction of the degeneracy of the moir´e subbands; these are twofold degenerate in MoTe2/WSe2 and all other TMD-based moir´e superlattices. The closest imaginable analog of the tBLG/hBN Chern magnet in this system is one in which interactions favor the formation of a valley-polarized ferromagnet, at which point the finite Chern number of the valley subbands would produce a Chern magnet. This was widely assumed to be the case at the time of the system’s discovery. There is now substantial evidence that this system instead forms a valley coherent state stabilized by its spin order, which would require a new mechanism for generating the Berry curvature necessary to produce a Chern magnet. In general I think it is fair to say that the details of the microscopic mechanism responsible for producing the Chern magnet in this system are not yet well understood.

In light of the differences between these two systems, there was no particular reason to expect the same phenomena in MoTe2/WSe2 as in tBLG/hBN. As will shortly be explained, current-switching of the magnetic order was indeed found in MoTe2/WSe2. The fact that we find current-switching of magnetic order in both the tBLG/hBN Chern magnet and the AB-MoTe2/WSe2 Chern magnet is interesting. It may suggest that the phenomenon is a simple consequence of the presence of a finite Chern number; i.e., that it is a consequence of a local torque exerted by the spin/valley Hall effect,which is itself a simple consequence of the spin Hall effect and finite Berry curvature. These ideas will be discussed in the following sections.In spin torque magnetic memories, electrically actuated spin currents are used to switch a magnetic bit. Typically, these require a multilayer geometry including both a free ferromagnetic layer and a second layer providing spin injection. For example, spin may be injected by a nonmagnetic layer exhibiting a large spin Hall effect, a phenomenon known as spin-orbit torque. Here, we demonstrate a spin-orbit torque magnetic bit in a single two-dimensional system with intrinsic magnetism and strong Berry curvature. We study AB-stacked MoTe2/WSe2, which hosts a magnetic Chern insulator at a carrier density of one hole per moir´e superlattice site. We observe hysteretic switching of the resistivity as a function of applied current. The real space pattern of domain reversals aligns with spin accumulation measured near the high Berry curvature Hubbard band edges. This suggests that intrinsic spin or valley Hall torques drive the observed current-driven magnetic switching in both MoTe2/WSe2 and other moir´e materials. The switching current density is significantly less than those reported in other platforms, suggesting moir´e heterostructures are a suitable platform for efficient control of magnetic order. To support a magnetic Chern insulator and thus exhibit a quantized anomalous Hall effect, a two dimensional electron system must host both spontaneously broken time-reversal symmetry and bands with finite Chern numbers. This makes Chern magnets ideal substrates upon which to engineer low-current magnetic switches, black plastic planting pots because the same Berry curvature responsible for the finite Chern number also produces spin or valley Hall effects that may be used to effect magnetic switching. Recently, moir´e heterostructures emerged as a versatile platform for realizing intrinsic Chern magnets. In these systems, two layers with mismatched lattices are combined, producing a long-wavelength moir´e pattern that reconstructs the single particle band structure within a reduced superlattice Brillouin zone. In certain cases, moir´e heterostructures host superlattice minibands with narrow bandwidth, placing them in a strongly interacting regime whereCoulomb repulsion may lead to one or more broken symmetries. In several such systems, the underlying bands have finite Chern numbers, setting the stage for the appearance of anomalous Hall effects when combined with time-reversal symmetry breaking.

Notably, in twisted bilayer graphene low current magnetic switching has been observed, though consensus does not exist on the underlying mechanism.We emphasize that in both twisted bilayer graphene and our current MoTe2/WSe2 heterostructure, magnetic switching arises in regimes for which doping, elevated temperature, or disorder ensure that electrical current flows in the sample bulk. Ultra-low current switching of magnetic order has also been observed in twisted bilayer graphene. In that system, where spin orbit coupling is negligible, nearly identical mechanisms may arise mediated by orbital, rather than spin, Hall effects. The bulk nature of the spin Hall torque mechanism means that similar phenomena should manifest not only in the growing class of intrinsic Chern magnets, but in all metals combining strong Berry curvature and broken time-reversal symmetry, including crystalline graphite multilayers. Research into charge-to-spin current transduction has identified a set of specific issues restricting the efficiency of spin torque switching of magnetic order. Spin current is not necessarily conserved, and as a result a wide variety of spin current sinks exist within typical spin torque devices. Extensive evidence indicates that in many spin torque systems a significant fraction of the spin current is destroyed or reflected at the spin-orbit material/magnet boundary. In addition, the transition metals used as magnetic bits in traditional spin-orbit torque devices are electrically quite conductive, and can thus shunt current around the spin-orbit material, preventing it from generating spin current. These issues are entirely circumvented here through the use of a material that combines a spin Hall effect with magnetism, and as a result of these effects this spin Hall torque device has better current-switching efficiency than any known spin torque device.We do not know the precise origin of these holes, but there are a few possibilities that we can discuss. Bubbles are some of the most common defects in stacks of two dimensional crystals, and they can form between any two layers of a stack. As presented and described in Fig. 6.7A-C, AFM imaging reveals topographic defects precisely aligned with the regions in the Chern magnet in which magnetism has been destroyed. There are two clearly distinct distributions of defects, with thicknesses that differ by about an order of magnitude. It is possible that these correspond to bubbles between two distinct pairs of layers of the stack. Another possibility is that partial oxidation or deliquescence of the MoTe2 crystal has occured. This crystal is indeed air and moisture sensitive, and degradation can happen even inside a glovebox, as illustrated for a CrI3 crystal in Fig. 6.7D-F. Whatever issue is generating this disorder, it will likely be necessary to resolve it in order to fabricate more sophisticated devices based on this Chern magnet.As in three dimensional crystals, many two dimensional crystals have multiple allotropes that are stable under different conditions. Trilayer graphene is such a material. We label multilayer grapheneallotropes using letters that refer to the relative positions of atoms of different layers, projected onto the two dimensional plane. We have already encountered ABA trilayer graphene in the introduction, and this material has atoms in the third layer aligned to atoms in the first layer. At room temperature and pressure the ABA stacking order is preferred, but trilayer graphene has a metastable allotrope, ABC trilayer graphene, that can either be prepared or found naturally occurring. In ABC trilayer graphene atoms in the third layer are aligned neither with the first nor with the second layer.

A meta-analysis of 43 studies that looked at single balloon, double balloon,spiral enteroscopy and short scope double balloon found 83% biliary cannulation success rates for spiral and single balloon methods and 95% success rates for long scope and short scope double balloon, with adverse events ranging from 0%-3%. Gastroscopes are now less commonly used, predominantly only in older patients, without entero-enteric anastomosis. However, they are still used for initial inspection and for primary visualization of anastomose-type. Patient anatomy with long afferent loops or post Roux-en-Y anastomosis who require subsequent ERCP, may require an enteroscope longer than 170 cm for forward-viewing endoscopic techniques . Besides papilla size, location, duodenal positioning, PAD, and iatrogenic patient varied anatomy, other factors that lead to swelling of the papilla also contribute to difficult cannulation. In the case of biliary malignancies, tumor infiltration of the papilla or duodenum can make the papilla difficult to find. In addition, malignancy makes the cystic tracts and vasculature more friable; this leads to more papillary edema, trauma and bleeding, with fewer cannulation attempts. Last, even in patients with normal anatomy and easily visualized papilla, multiple attempts of SBC can traumatize the papilla and extensively opacify the pancreas, these factors in themselves can distort visualization of the papilla and can make further attempts even more difficult.When SBC using the techniques described is not easily achieved, drainage planter pot it is considered a difficult cannulation. Over the years, there have been several attempts to objectively define difficult cannulation. Most definitions use a combination of a minimum number of cannulation attempts, typically 5 to 15, and a minimum time spent on standard cannulation techniques, typically greater than 5 to 20 min.

The number of inadvertent MPD injections or cannulations may also be considered part of the definition of difficult cannulation, with some studies suggesting > 4 MPD cannulations as the limit. Recently, the European Society of Gastrointestinal Endoscopy guidelines defined difficult biliary cannulation in an intact papilla as any procedure in which the duration of cannulation attempt exceeded 5 min or 5 attempts, or a procedure with more than one unintentional MPD cannulation or opacification . However, there is no uniform definition of what comprises a cannulation attempt. Friedland et al defined a cannulation attempt as any repositioning or wedging of the cannulation device while attempt SBC, while Bailey et al defined an attempt as sustained contact between the cannulation device and the papilla for five seconds or more. A 2013 study that compared the accuracy of cannulation time versus cannulation attempts asdetermined by two third party observers in 14 patients found that there was significant disagreement between observers in terms of observed number of attempts, illustrating the difficulty and variation in defining a cannulation attempt and thus difficult biliary cannulation when using number of attempts. Regardless of which definition is used, it is generally accepted that once difficult cannulation is encountered, the risk of PEP or complete failure of the procedure is dramatically increased. It is important to note that when the purpose of SBC is for pancreatic intervention only, cannulation of the minor papilla can be pursued as an alternative to the methods discussed below. Although SBC of the major papilla the most common and effective method used for management of pancreatic diseases, when access to the major papilla is difficult or impossible due to severe duct distortion or obstructive mass, cannulation of the minor papilla may be easier and safer than persistent attempts at major duct cannulation.

The minor papilla is the papilla of the accessory pancreatic duct, or sometimes, a variant duct anatomy in pancreas divisum . Access to the minor papilla enables therapeutic options for pancreatic diseases such as chronic or recurrent acute pancreatitis and pseudocysts, but not for biliary disease as the minor papilla does not connect to the CBD. Studies have shown minor papilla SBC success rates using WGC range from 74%-90% and a PEP rate of 10%-14%. SBC in the pediatric population appears to have similar success and complicationrates to the adult population when performed by an experienced advanced endoscopistbased on a number of large series. Difficult cannulation in the pediatric population is most frequently due to not having properly sized sphincterotomes designed for smaller papillae, and, in rare cases, biliary atresia . The pancreatic guide wire technique involves the placement of a guide wire into the MPD and then attempting to cannulate the biliary duct. A guide wire in the MPD helps straighten the intramural segment of the bile duct and direct the ST or other catheter into the bile duct and thus reduces the chance of accidental cannulation of the MPD. When the pancreatic guide wire method is combined with WGC, it is known as the double guide wire technique . A retrospective study involving 363 and a prospective multi-center RCT in 274 patients comparing PGT to early DGT found no difference in the success rate of cannulation or in PEP rates. However, a recent meta-analysis of 7 RCTs including 577 patients found that using DGT increased the risk of PEP when compared other techniques including standard WGC, MPD, and early pre-cut. Another technique is to place a temporary pancreatic stent and then perform WGC above the stent, called wire-guided cannulation over a pancreatic stent technique. A short 5-Fr pancreatic stent between 2 cm to 5 cm can be used, with the proximal tip not past genu to prevent duct injury. After placement of the pancreatic stent, the papilla is then cannulated using the WGC technique above the stent. The pancreatic stent all but ensures that no further accidental cannulation of the MPD can occur.

An abdominal x-ray should be performed 2 wk after the procedure to confirm spontaneous passage; if the stent has not passed, a stent removal procedure may be needed. The advantages of the WGC-PS technique is that a pancreatic stent is easy to insert, especially if a pancreatic guide wire is already in place, and has been shown to lead to a significant lower rate of PEP, with various studies showing rates reduced from as high as 23% to less than 3% after placement of a PD stent. A recent retrospective study of 177 patients compared WGC-PS to DGT found that both groups had similar cannulation rates, but the WGC-PS had lower rates of PEP . In this study however, about half of the cases that failed DGT were successfully salvaged with WGC-PS. The WGC-PS technique is also more cost effective, most likely due to the lower rates of PEP, and can be combined with other ancillary methods of cannulation such as needle-knife sphincterotomy. Due to lower rates of PEP seen with pancreatic duct stenting, the ESGE suggests a placement of a pancreatic duct stent both prior to both wire-based cannulation methods as well as and precut techniques. It is important to note, however, that pancreatic duct stenting has not been shown to reduce PEP in the pediatric population. In fact, a 2015study of 432 ERCPs in the pediatric population found that placing a prophylactic pancreatic stent was actually associated with a significantly higher rate of PEP . The cause is unclear, but the authors suggest that it may be related to physiologic differences and the smaller size of the pancreatic ducts in the pediatric population.When biliary cannulation using the techniques mentioned above fails, many endoscopists opt to create a papillotomy to access the hepatopancreatic ampulla; this may involve the sphincter of Oddi, thereby performing a sphincterotomy, or be performed staying above the sphincter, i.e., a firstulotomy. These techniques are collectively known as precut techniques to facilitate access to the biliary tree and require an intimate understanding of papillary anatomy to ensure a safe and effective procedure. The most common tool employed in precut techniques is the needle-knife, plant pot with drainage a small precision cutting tool that cuts when current is applied. The tip should not be extended beyond the catheter further than 2 to 3 mm as the tip of the needle knife cuts easily and rapidly; over-extension of the needle knife increases the risk of perforating the back wall or causing a retroduodenal perforation. Newer “hybrid-tomes” integrate the needle-knife directly into the ST and may be easier to handle than regular needle knives. If possible, a pancreatic duct stent should be placed beforehand to protect the pancreatic orifice, straighten the intramural segment of the bile duct, and position the biliary duct for easier access with the ST after the cut is complete. There is currently no standard for the naming of the various precut techniques. For this review, the naming system described by Davee et al will be used . Precut papillotomy: In precut papillotomy , the needle knife is used to dissect the major duodenal papilla to visualize and cannulate the CBD. Typically, needle-knife is placed at the 11-12 o’clock position of the papillary orifice and cut upward along the midline of the intraduodenal segment of the bile duct to expose the CBD. The biliary sphincter muscle can be recognized by its whitish onion-skin appearance. Once this muscle is exposed, the papilla can often be seen as a red dot or nipple-like structure. If examined carefully, bile may be seen flowing from the papilla.

The papilla can then be cannulated or the biliary sphincter can be transected further and then cannulation afterwards can be performed. Precut fistulotomy: In a precut fistulotomy, an incision is made using a needle knife in an area of the papilla, above the papillary orifice, that covers the intraduodenal segment of the distal CBD to create a fistula between the duodenal lumen and the CBD lumen. The incision can be extended downward towards the papillary orifice or upward, depending on the initial incision site. The precut fistulotomy technique leaves the sphincter and papillary orifice intact and creates a fistula that allows the endoscopist to directly cannulate the CBD. At least in theory and based on anecdotal evidence, this method reduces the risk of thermal injury to the pancreatic orifice and therefore the risk of PEP. A variation of this technique, the supra-papillary puncture, creates direct duodenocholedochal access using a catheter fitting with a needle to directly puncture the biliary duct under fluoroscopic guidance without cautery. When combined with EUS, this method has been shown to reduce the rate of PEP while having seemingly acceptable perforation rates. Transpancreatic precut sphincterotomy: Achieving an adequate precut papillotomy or fistulomy using a needle knife may be difficult in patients with small or difficult tolocate papilla. For such patients, the transpancreatic precut sphincterotomy may be a viable alternative method. First reported in 1995 by Goff, the TPS method uses a standard ST oriented toward the CBD at approximately 11 o’clock that is inserted superficially in the ampulla or MPD. The ST itself is then used to incise upward to perform a papillotomy. The advantages of TPS include not needing to exchange the ST for a needle-knife device and better control of the depth of incision compared to needle knife device. Although TPS alone carries a risk PEP of 9%, likely due to irritation and edema involving the MPD, placement of a PD stent after TPS has been shown to reduce the incidence of PEP to 4%.Early studies of precut techniques showed PEP rates to be as high as 15% to 20%, an alarmingly high number that is 2-3 times the PEP rate for uncomplicated SBC. However, it was unclear whether these rates were attributable to using needle knife precut techniques or due to the multiple attempts at SBC already performed. Many endoscopists now advocate for early precut techniques when difficult cannulation is predicted or recognized early on to reduce the risk of PEP. Several studies have compared the cannulation success and PEP rates of early precut techniques to persistent standard cannulation attempts. These studies were analyzed in five metaanalysis. Four of the five meta-analysis concluded there was not significant difference in SBC success rates between the two groups, with only Sundaralingam et al finding increased SBC success in the early precut group . Four of the studies noted lower PEP rates in the precut group, though the different was not significant in Navaneethan et al and Choudhary et al , and none were adequately powered to assess the difference. Two studies have compared PEP rates between early precut techniques and using pancreatic duct stents after successful SBC in difficult cannulation using persistence. A 2016 RCT of 50 early precut patients and 50 patients who underwent MPD stenting after difficult cannulation without precut techniques found similar rates of PEP of approximately 4% .

Important theorizing in dementia research suggests the crucial role of social psychological factors in determining the level and expression of functional ability . The current study expands on these ideas by showing how the social context of household care mediates individual expressions of functional decline. Studies of social support around decision-making provide a useful foundation for a social process understanding of functional decline. This research examines how family members manage the affected individual’s decisions around medical treatment, end-of-life care, finances, and moves to long-term care facilities . An important dimension of the work characterizes the level of involvement elders and caregivers have in the decision-making process and the effects that different levels of involvement have on caregivers and elders individually and on their relationship. Findings highlight the challenges families face in executing decisions that correspond with the authentic preferences of affected individuals, but do not explicate how management of the decision-making process relates to avoiding risks caused by waning functional ability more generally. Thus, this area of work examines social support around cognitive decline through a narrow strip of behavior that provides limited insight into the larger process occurring across the individual’s natural range of activities. A second, smaller collection of studies offer modest insights into the larger process, especially around familial attempts to help the individual maintain ability-based dimensions of personal and social identities. Linking identity to activity participation, a number of studies, for instance, tie the effort of promoting autonomy to the concern with preserving or protecting the individual’s identity . Perry and O’Connor , for instance, danish trolley describe how caregivers decrease the complexity of the elder’s old activities so that they may still engage in them to some extent and preserve a sense of self identity. In complementary work, Clare identifies the coping strategies that family members and affected individuals collaboratively constructed in order to maintain normality and protect a sense of self.

Relevant to the current study, she highlights how family members “struggled” to find the balance between constructively helping and “taking over in an undermining way” . Finally, the current piece also builds on work explaining how family members engage in “cognitive support work” in order to minimize confusion and disorientation in the elder’s social interactions . This piece identifies the lay health practices that family members develop to minimize the effects of symptoms on everyday life. Whereas that work focuses on attempts to lessen confusion so that the individual can effectively maintain independent lines of conduct, the current piece focuses on attempts to manage involvement in everyday activities to lessen risk of injury to self and others. While the literature offer insights into social support around ability decline, researchers still know little about how family members manage decline in an elder’s natural range of daily activities—such as cooking, driving, shopping, and bathing—over time. Though researchers have developed numerous interventions designed to increase functional autonomy at home , there is little research elucidating how families navigate functional decline within the social context of their own households. This work advances the study of functional decline in dementia by revealing how family members become autonomy management practitioners who develop their own logics of support.This is a three-wave retrospective interview study with individuals who provide caregiving to relatives diagnosed with Alzheimer’s disease. In total, we conducted 45 interviews with 15 individuals over the course of 2 years . We recruited this non-probability sample through the Alzheimer’s Disease Center at the University of California, Davis following IRB approval. Among these participants, 12 were adult-children and 3 were spouses of the affected individual. We utilized a pool of Latina caregivers assembled for a related project investigating the effects of social capital on dementia caregiving.

When the study began, participants ranged in age from 44 to 77 and care recipients ranged in age from 67 to 96. Time since diagnosis ranged from 1 to 12 years with a mean of 3.73 years. Individuals with Alzheimer’s disease about whom participants reported displayed varying degrees of disease advancement at the start of the study. Twelve of the fifteen participants lived with the individual with dementia part time or full time . In these part time living arrangements, participants either stayed part of the week at the affected individual’s home or switched off with a sibling in hosting the individual weekly.The findings were derived from data collected through three waves of in-depth, open-ended interviewing conducted over the course of 2 years. We interviewed participants three times each at six month intervals. In the first interview, we focused our questions on the period from the first signs of the disease until the moment of the interview. In the subsequent two interviews, we focused on the period since the last interview. Interviews typically lasted between an hour-and-a-half and two hours each. They were conducted by phone or in person and in Spanish or English depending on the participant’s preferences. In total, 29 were conducted by phone and 16 were conducted in person. All interviews were digitally recorded and then transcribed verbatim. Each participant received a $40 gift card for participating in each interview. We began the interview process with the understanding that current research does not explain satisfactorily how families deal with the household risks that occur due to cognitive decline. We conducted interviews using an interview guide that we constructed before each wave of interviewing. We designed each interview guide to accomplish two main tasks: to understand the nature of the participant’s relationship to the affected individual over time and to document the participant’s concrete behaviors around the elder’s daily activities over time. We accomplished the first task by asking questions about living arrangements, amount of time spent together, and the types of activities that they engaged in together.

To complete the second task, we asked questions about the participant’s perception of the affected individual’s abilities in his or her common daily activities over time and about the participant’s behaviors around the elder during these activity involvements. We asked participants to describe the step-by-step details of how specific events and situations had occurred in their households and discouraged participants from speaking in generalizations and abstractions . Because trust is a crucial interpersonal achievement when seeking to gain insight into a stigmatized health condition like Alzheimer’s, we made rapport-building a cornerstone of our research design. First, we used a multi-wave interview protocol to increase the likelihood of building trust and facilitating participant disclosure of emotionally-difficult occurrences . Second, we selected an interviewer that shared a racial-ethnic background and language fluency with our participant sample and relied on her to conduct each interview.The analysis consisted of a modified grounded theory procedure that entailed engaging directly with the research literature related to functional decline during the coding process . Each author engaged in line-byline coding of the complete set of interview transcripts unassisted by qualitative coding software. This approach helped us identify a common substantive interest and establish inter-coder reliability. The topic of autonomy management around functional decline emerged out of the first round of coding. Through this phase of analysis we learned that family members exhibited substantial concern with the risks of household injury and with how much influence they should exert over their mom, dad, or spouse in everyday activities.In a subsequent phase of coding, we identified the techniques and management practices that family members used to influence the elder’s activity involvements and placed them in typological categories. In a final phase of analysis, we examined these practices for discernible trends and turning points from the onset of the disease until the final interview. To facilitate this process, vertical aeroponic tower garden we developed summary timelines that visually depicted each participant’s shifts in management behavior over time. We compared these timelines and identified the common stages between them. This analytic technique revealed that participants began shifting from collaborative to unilateral autonomy management techniques when they detected the elder’s waning deficit awareness. The research team met regularly to discuss this ongoing analytic work and to resolve interpretive discrepancies through a consensus building process. Interpretive discrepancies typically occurred when determining if a family member’s response to an elder’s behavior fit within a category we had previously identified.The findings show that family members managed the elder’s involvement in daily activities across three stages of support: a collaborative, transition, and unilateral stage. The collaborative stage began early on in the disease trajectory when elders drew on family members for support in pursuing their decades-old activities. Support entered a transition period when family members began to perceive that the elder was exhibiting diminishing deficit awareness with a corresponding increase in risk of self-harm around everyday activities.

With a loss of confidence in the elder’s ability to regulate his or her activities to avoid these risks, family members employed unilateral practices to manage the elder’s autonomy around his or her activity involvements. In sum, all participants had at least entered the transition period and five had entered strictly into the unilateral stage of support.Family members reported that as elders lost cognitive ability, interest in decades-old activities and routines did not wane substantially. A majority of elders from this study’s sample continued to display pre-symptomatic activity preferences, sometimes for many years after diagnosis. One man, for instance, continued to drive his truck around an adjacent orchard and frequently expressed a desire to drive it in the nearby town as he had done for many decades even though he had gotten lost a number of times in recent months and had a minor accident. A woman continued to prepare food in her kitchen even though her creations were seen as sometimes un-edible by her family members. Another woman took regular walks to the grocery store though she frequently showed signs of confusion in everyday tasks and her daughter had taken to monitoring her walks from the front yard. These cases conform to a larger pattern of family member reports indicating that as mental acuity declined for elders, desire to engage in familiar activities largely remained.Early on in the disease trajectory, family members reported that elders exhibited awareness of their own ability decline through direct acknowledgment of their deficits or indirectly through their efforts to work collaboratively to avoid the problems they could cause. One family member noted that her mother showed awareness of her cognitive decline by drawing on a light bulb as a metaphor for describing her intermittent cognitive failings.This study charts the way families attempted to manage an individual’s growing incompetence in daily activities across the disease trajectory. It shows the role family members have in shaping the form that functional decline takes and thus, makes the larger point that dementia’s symptoms do not have a pure form that exists outside of social context. Rather, dementia’s expression as a disease entity always takes shape within a particular social context that constrains individuals to see it in a particular light and respond to it in particular ways. Early on in the disease progression, elders continued to seek engagement in decades-old activity preferences despite growing impairment . At this stage, elders were generally perceived as having awareness of their growing deficits, working collaboratively with family members to maintain functional ability in certain activities. As time passed, family members began to notice the elder’s declining awareness of these deficits. Through lay awareness tests and deliberation with other family members, they determined that the elder sometimes reverted back to a pre-disease conception of his/her own abilities and sought to engage in activities that he or she could no longer manage independently. Perception of declining deficit awareness by family members acted as a key turning point in how they related to elders and managed the disease, spurring them to modify their conception of the elder as a self-directed agent and develop a greater vigilance around the elder’s activity choices. Family members developed their own idiosyncratic techniques that assisted and restricted the elder’s autonomy to minimize risks. We describe the effort to use these techniques as autonomy management work. Within the larger effort to manage symptoms, family members treated autonomy management work as a last line of defense against the symptoms of declining competence and deficit awareness. When these symptoms occurred despite using some combination of pharmacological and lay efforts to manage them, family members relied on autonomy management techniques to minimize the dangerous impact that they could have. In this way, autonomy management techniques complemented other symptom management measures, providing a buffer between elders and unsafe activities when symptom control proved impossible.

It could also be that the region’s slightly more mesic climate offers a climatic buffer that prevented shrubs from reaching their mortality thresholds. More research is needed to identify these exact mechanisms and thresholds in A. glauca. Collectively, the results of this dissertation work provide valuable knowledge on the severe dieback of an important chaparral shrub during an historic drought, with the potential for ecologically and economically costly consequences. Additionally, the data I present provide insight into the scale and progression of A. glauca dieback in a chaparral system, and potential patterns of future dieback in the face of predicted climate change. Future research that seeks to further resolve landscape and environmental variables contributing to plant stress would help in identifying these patterns.Heterogeneity and rugged topography across the landscape, while likely beneficial for the resilience of regional A. glauca populations during drought, presents significant challenges for on-the-ground monitoring. Out of necessity for safe access , many of the plants surveyed were located on the outer boundaries of stands, where edge effects may have been a factor. Monitoring intact, undisturbed stands using drones would yield valuable additional insight into the extent of disease deeper into stands and in stands on steep terrain or that are outside of normal visual range. The challenges of working in rugged landscapes covered in impenetrable vegetation highlight the need for using and refining remote sensing technologies, such as drone imaging, Light Detection and Ranging , and hyperspectral imaging as monitoring tools. Large-scale, long-term monitoring using these tools would allow researchers to retrieve data in areas that have previously been inaccessible, blueberry grow pot while also gaining a larger scale understanding of drought impacts. They ultimately will enable future studies to reveal more nuanced patterns across the landscape and between years of varying climatic conditions. Outbreaks can alter ecosystem structure and function, often with substantial consequences .

Over the past 200 years, pest/disease outbreaks have increased due to mass exchange of biological materials from global trade and a rise in unusual climate events resulting from global climate change . Prolonged climate irregularities can subject plants to environmental stress outside of their normal resistance thresholds and make them susceptible to pests and pathogens . For example, the increase in extreme droughts, defined here as greater in intensity and duration than historical drought regimes, has been directly linked to enhanced mortality in woody plant systems worldwide, often in association with pest/pathogen outbreaks . Plant disease outbreaks are often economically costly , and can result in loss of ecosystem services in natural ecosystems. With global trade continuing to spread pests and pathogens, and global change-type drought events predicted to increase , incidences of plant disease outbreaks are expected to increase. Understanding the role of drought and pathogens in plant dieback and mortality is therefore of critical importance. Latent fungal pathogens are of particular concern for natural ecosystems yet their ecological roles remain poorly understood. These pathogens can live as asymptomatic endophytes within their hosts and remain undetected for long periods of time . The Botryosphaeriaceae fungi, a groupthat causes considerable damage to hundreds of agricultural, ornamental, and naturally occurring host species around the world , includes many latent fungal pathogens that are difficult to detect in wild plant populations. Members of this diverse family can occur as endophytes, pathogens, and saprophytes on diverse woody hosts . They are best known as pathogens that cause leaf spots, cankers, severe branch dieback, and death in economically important hosts such as grapevines , avocado , and eucalyptus . While Bot. fungi are rapidly becoming one of the most important agents of disease in agricultural plant hosts , relatively few studies have been conducted on these pathogens in natural systems .

The Bot. fungi have a long history of taxonomic confusion, in part due to indistinctive morphological characteristics among species and from other fungal taxa, as well as historically poor and inconsistent descriptions early on in their discovery . Furthermore, Bot. host specificity and pathogenicity can vary widely among species and across geographical regions, complicating our understanding of their influence in various host species and across systems . While advances in molecular sequencing and data basing have added clarity in this area , challenges remain in understanding the diversity and pathogenicity of Bot. species among hosts and across regions. As a result, there is a dearth of knowledge on their ecological roles, particularly in native ecosystems.One consistent finding is that disease outbreaks from Bot. fungi in agriculture are often associated with environmental stress, such as extreme heat fluctuations and drought . Furthermore, studies have shown latent pathogens like Bots cause more damage to water-stressed hosts , and some Bot. species have been shown to grow well in water potentials much lower than what their plant hosts can tolerate , suggesting drought conditions increase virulence by these pathogens. Therefore, regions that have historically dry climates or experience periodic extreme drought may be especially vulnerable to disease outbreaks from latent pathogens as they are predicted to experience an increase in drought events due to climate change . Mediterranean-type climate areas are projected to be global change “hot spots” , and dry shrublands are predicted to experience some of the most rapid increases in mean temperatures . Indeed, recent drought-related morality in California’s semi-arid Mediterranean climate shrublands has provided support for these predictions . Furthermore, the combination of dense human settlement and agricultural lands in close proximity to many natural shrubland habitats in southern California creates a likely pathway for exotic pathogen introductions and movement of pathogens from agricultural settings into wildland species. Not surprisingly, Bot. species have been retrieved on a variety of native chaparral shrub species in California, including Ceanothus spp. , Malosma laurina , and other species of Arctostaphylos . Understanding the response of native species and these pathogens to extreme weather conditions will help to predict future vegetation change and potential species losses . From 2011-2018, southern California experienced one of the most severe droughts in recorded history, with 2014 being the driest in the past 1,200 years .

Field observations in winter 2014 identified high levels of branch dieback, and in some cases mortality, in a common ecologically important shrub, Arctostaphylos glauca in coastal California. Two well-known Bot. species were isolated from the symptomatic shrubs . Like other members of the Bot. family, both N. australe and B. dothidea infect a broad range of hosts, and are known to be responsible for disease outbreaks associated environmental stress in agricultural species . While B. dothidea is well established in California, with over 35 different host species having been identified , phylogenic evidence suggests N. australe may be more recently introduced . Its impact on shrublands of California has not been quantified. Preliminary observations suggested high levels of branch dieback, and in some cases mortality, at lower elevation sites and along exposed ridges compared to higher elevations in coastal montane settings. We hypothesized that identifiable patterns would exist in the distribution of B. dothidea and N. australe across these landscapes that correlate with branch dieback and environmental variables associated with drought stress. Manzanita dieback has previously been causally associated with Bot. infection . A greenhouse experiment by Drake-Schultheis et al. , revealed that drought enhances onset of stress symptoms and mortality in young A. glauca inoculated with N. australe compared to shrubs subjected to drought or inoculation alone. However, to the authors’ knowledge no previous quantitative studies exist on the distribution of Bot. species in California shrubland environments with Mediterranean climates. To better understand the occurrence, distribution, and severity of Bot. infections in chaparral shrublands, we surveyed infection in A. glauca between April and September 2019. We also collected data on site elevation, aspect, square plastic pot and average percent canopy dieback at each site sampled for infection. While a variety of landscape variables are likely to influence plant stress at any given site , we focused on elevation because A. glauca already tends to occur mostly on xeric and rocky soils of exposed slopes, and therefore elevation was presumed to be the most significant factor influencing precipitation and water availability in this setting. Also, other studies have used elevation as a proxy for climate variation . We also recorded aspect of each sampled shrub since it influences sun exposure, temperature, and water stress.

To test our hypothesis that Bot. fungi and level of stress each played a role in extensive canopy dieback in A. glauca, the following questions were addressed: What is the distribution of Bot. infection in A. glauca stands across the chaparral landscape in coastal Santa Barbara County? How do levels of infection by the two Bot. fungi, N. australe and B. dothidea, compare across elevation? and How do stand-level infection and elevation correlate with dieback severity? We predicted N. australe and B. dothidea to be present across all sites and elevations, but also that N. australe, having been previously isolated with high frequency in the area , would likewise have the greatest incidence in this study. Furthermore, we expected levels of Bot. infection and dieback severity to be greater at lower elevations compared to higher elevations, because lower sites typically receive less annual rainfall, thus exacerbating drought stress. This study presents the first quantitative survey summarizing the severity and distribution of Bot. fungi in natural shrublands, and seeks to identify important patterns of infection and dieback in A. glauca to predict future vulnerabilities across the landscape.The study sites were located on the generally south-facing coastal slopes of the Santa Ynez Mountains in Santa Barbara, California, USA . The sites range from a lower elevation of ~550m to an upper elevation of ~1145m, and cover an area of ~47km2 . This region is characterized by a Mediterranean climate, with wet winters and hot, dry summers. Mean annual precipitation ranges from 68.4cm at lower elevations to 90.6cm at upper elevations . During the 2013-2014 wet season, which was two years into a multiyear drought and one of the driest years on record in California , these areas received only 24.8cm and 31.6cm precipitation, respectively .Sites were initially randomly generated from polygons drawn in the field around relatively pure stands of A. glauca , and Drake-Schultheis, unpublished data. Polygons were then categorized according to elevation , and numbered within their respective elevation categories. Ten sites per elevation zone were randomly selected using random number generator for a total of 30 sites. When necessary, some randomly generated sites were substituted with nearby stands that were more accessible. Furthermore, any randomly selected sites that were discovered to be in recent fire scars were exchanged for nearby stands that contained intact, mature A. glauca.Elevation data were collected in situ using Altimeter GPS Pro and corroborated using Google Earth . Aspect was recorded in situ in degrees, then converted to radians and transformed to linear data for analysis of “southwestness” using cos according to Beers et al. . This yielded aspect values ranging from -1 to 1 , which were then used for modeling the effects of aspect on shrub dieback and Bot. infection. The total percent dieback was assessed at each site as a measure of canopy health. Sites were demarcated by >50% A. glauca cover within a stand, as determined by visual on the-ground assessments where the tops of the canopies could be viewed. Stand dieback was then visually estimated by two-to-three people as the percent of “non-green” vegetation compared to live, green vegetation within the defined boundaries of a site . Categories of NGV included yellow, brown, and black leaves, and bare/defoliated canopy, and percentages were summed to reflect total NGV within a site. Total canopy cover was thus the sum of percent GV and NGV, and dieback was calculated as the total percent NGV to reflect the severity of canopy-level symptoms across each site.Ten individuals within each of the 30 sites were randomly selected for sampling using the random points generator feature in ArcMap , for a total of 300 shrubs. Individuals were located in the field using a combination of a 1m resolution NAIP imagery base map , a GPS device, a laser range finder, and transect tape. For stands not located within a polygon, individuals were selected either using a transect tape and a point intercept method , or haphazardly selected within the accessible confines of the stand to provide an even distribution of sampling throughout the stand.