Tag: horticulture

One factor was already mentioned: the number of OTUs in the sample. In this sense, adding another OTU would affect evenness if the sample contained few OTUs. The other factor is the number of individuals in the OTU added relative to the numbers of individuals in the other OTUs in the sample. If the new OTU contains many more members than the existing ones, then its addition will decrease the evenness and therefore decrease the overall diversity, as we observed in the effect of E. coli spike-in on the low-biomass samples in the preliminary experiments. If the new OTU has fewer members than the existing ones, its addition may not affect evenness much at all, as in the case of E. coli spike-ins relative to the preliminary cultures. In either case, rarefaction to a large enough depth would help retain biological differences, in other words, differences inherent to the samples and not as consequences of mathematical manipulations. As already pointed out in literature, a major drawback of rarefaction would be losing rarer OTUs because of the lack of sampling depth, i.e. discarding rarer sequences as large samples are reduced to smaller ones. Fortunately, that was not a major concern in the preliminary experiments, as we did not design the procedure as a way to discover rare microbes or uncover whether our methods support the growth of as-of-yet uncultivated organisms. Our goals, plastic potting pots combined with the culturing methodology as well as the rarefaction threshold we chose, meant that, ultimately, we did not lose exclude crucial or even important information from the data set.It is interesting to note that streptococcal species tend to attach to a surface first and provide a suitable colonization environment for other species.

Previous studies have shown that several streptococcal species produce adhesins that preferentially bind to different substrates in human saliva and cells. For instance, S. salivarius, an OTU prevalent in our model, expresses amylase-binding proteins that interact with both salivary amylase and surface lectins that bind to extra parotid glycoproteins. S. salivarius also expresses antigen C fibrillar glycoprotein that binds to the epithelial cells on the human cheek, and fibrillar antigen B that binds to Veillonella parvula. Similarly, S. oralis expresses surface lectins and antigens that bind to salivary glycoproteins. These surface proteins facilitate streptococcal attachment to the enamel surface on one hand, and on the other help bacterial cells from other phyla immobilize in preferred environmental niches and subsequently proliferate. These proteins are doubtless a large part of the reason that streptococcal cells act as early colonizers of the dental surface. The results from the preliminary experiments indirectly support this colonization order, by the relative abundances of Streptococcus OTUs in the cultures . Incidentally, the Streptococcus OTUs were only the second most abundant in the preliminary cultures; members from the Veillonella OTUs were the most abundant. Not as much research has been done for the attachment processes of Veillonella, though members of this genus are known to be early colonizers in dental plaque as well, coaggregating with streptococcal species and using the lactic acid produced by Streptococcus oralis. Clearly, our results indicate that the methods in this phase of the project properly support some best known early colonizers in the human dental plaque bacterial community.In this phase, three volunteer hosts were used as sources of dental plaque.

Sample collection took place under protocols 3-18-0189 and 3-19-0119, approved by the UCSB Human Subjects Committee. Prior to plaque collection, hosts abstained from food, nonwater liquids, and dental hygiene for 12 hours. At the time of collection, supragingival plaque of five molar teeth was obtained from each host using a sterilized Gracey curette. Collected plaque was immediately suspended in sterile centrifuged SHI medium, gently mixed, and divided equally among wells in a sterile surface modified 24-well plate such that each well received 1.98mL of the mixture. Prior to receiving inoculated medium, wells were conditioned with an artificial pellicle formed by clarified human saliva , which was supplied as frozen fractions pooled from healthy human volunteers. Saliva was stored at -20°C until clarification, at which point we defrosted the saliva on ice. Clarification of defrosted saliva was performed on site by centrifuging at 6,000 x g for 3 minutes at 4°C, mixing with 1X PBS in a 1:1 ratio, and passing the mixture through 0.2µm filters. Unused clarified saliva was stored at 4°C for no more than 3 days before being used or discarded. The artificial pellicle in each well was formed by adding 150µL clarified saliva to the bottom of the well and air drying at 37°C for 60 minutes. The plate was then sterilized with short-wave UV light for 60 minutes. At this point, wells were considered conditioned and ready for media. For this set of experiments, cultures derived from each host received a separate 24- well plate. We divided the plates into sections to facilitate harvesting at the specific time points of 12, 24, 48, 96, and 168 hours, in order to investigate the effects of increased incubation time on culture composition.

Each plate was divided into 6 sections of 4 wells , consisting of 2 controls in the top row with the medium and pellicle and 2 cultures in the bottom row with host-plaque-inoculated medium and pellicle. After receiving sterile or inoculated medium, each well also received 20µL of 0.5% sucrose. Plates were then incubated in a sealed vessel at 37°C in an anaerobic atmosphere composed of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide. At designated feeding times , all wells were supplemented with 5µL of 0.5% sucrose and specified volumes of SHI such that a constant well volume of 2.0mL was kept throughout the incubation process. If the feed time coincided with the harvest time for any particular section of wells, then feeding did not occur. At the five designated times, we harvested the designated section of wells by first aspirating the liquid, and then mixing the sedimented cells gently but well. We pipetted 900µL of properly mixed sedimented cultures into sterile microfuge tubes and added 100µL of 10% diluted E. coli cultures with OD600 values of 0.8. The resulting mixtures were pelleted, flash-frozen in liquid nitrogen, and kept at -80°C until further processing at the UC Davis Host-Microbe Systems Biology Core . To compare the culture composition with original host plaque and track the temporal development, we pelleted and froze plaque samples from all hosts at the start of incubation. In addition, we pelleted and froze 500µL aliquots of all E. coli cultures used for spike-ins at the times of the spike-ins, with the intentions of observing OTUs in pure E. coli cultures and retaining the ability to remove these OTUs from spiked cultures. In this phase of the project, we elected to keep the E. coli spike-in step such that we could continue checking the biomass of the controls as well as gain an understanding of the biomass in cultures and the potential differences in biomass across the cultures derived from different hosts.DNA extraction and sequencing were performed at the UC Davis Host Microbe Systems Biology Core , in a manner similar to the sequencing procedures from the preliminary experiments . Sequencing of properly amplified and diluted libraries was performed on the Illumina MiSeq platform using the paired-end method with a length of 253bp. Quality control of the raw sequences was first performed in QIIME by HMSB , and then formally reperformed in R with the mothur software. Briefly, 4,298,748 contigs were constructed from raw reads that were size-selected to be in the range of 240 to 275 bp. Constructed contigs were trimmed to eliminate ambiguous reads, and the resulting reads were screened for homopolymers with an upper threshold of 8 and pre-clustered. Chimeric sequences were then removed with VSearch, and non-bacterial sequences were removed based on the full-length SILVA database . The resulting 3,132,678 contigs were clustered into operational taxonomic units based on the full-length SILVA reference database at 97% level of sequence identity, approximating species-level taxa. These OTUs were constructed into a dense BIOM table for bio-informatics analysis.Bio-informatics analysis was first performed in QIIME at HMSB, and then formally reperformed in R with version 1.38.0 of the phyloseq software package. In this analysis, raspberry container growing we examined the sequencing depth, the number of OTUs, and the inverse Simpson’s index values of the samples.

As in the preliminary experiments, we considered the prevalence of organisms at the phylum level to gain an understanding of the general community structures. Then, we examined the correlation between sequencing depth and diversity using rarefaction curves and plots of read counts vs. diversity indices. To standardize sample sizes and minimize the presence of potentially spurious OTUs, we rarefied samples to a depth 30,000 and reexamined the number of OTUs and inverse Simpson’s index values. We also studied the absolute counts and relative abundances of the controls and cultures, inferring the biomass in controls and cultures relative to the E. coli spikes, to verify that there was minimal contamination throughout the culturing, extraction, amplification, and sequencing processes. After characterizing the samples as a whole and confirming minimal contamination, we focused on the culture and plaque samples. We removed the most prominent spike-in OTU reads from the rarefied read counts, converted read counts to relative abundances, and then examined the 12 most prominent OTUs in the plaque and culture samples. With the relative abundances, we performed Principal Coordinate Analysis using the Bray-Curtis dissimilarity metric, to investigate patterns in sample clustering according to incubation times and sample types. From the clustering patterns, we identified the OTUs that likely played the most important roles in sample differences. Lastly, we used the stats package for the relative abundances and the mixOmics package for the centered-log-ratio-transformed relative abundances to perform Principal Component Analysis .Sequencing of the 16S rRNA V4 region yielded a total of 4,298,748 raw contigs. Using the mothur software and release 132 of the full-length SILVA database, we trimmed and filtered out ambiguous bases, retaining 3,144,467 of the raw reads. From these, 122,617 unique sequences were found. Screening for homopolymers led to the retention of 99.6% and 98.1% of total reads and unique sequences, respectively. Pre-clustering to remove likely pyrosequencing errors with a tolerance of two mismatches resulted in 3,132,678 reads and 26,757 unique sequences, and chimera removal using VSearch led to the retention of 98.3% and 76.7% of total reads and unique sequences, respectively, from the previous step. Subsequent temoval of non-16S-rRNA sequences resulted in 20,508 unique sequences and 3,077,896 total reads. With the same full-length SILVA database, we then generated OTUs based on 97% sequence similarity and constructed the BIOM table from these OTUs.Temporal samples with spike-ins yielded high read counts across sample types and incubation times, except for one that failed to sequence well . Fewer than 200 OTUs were found in all samples regardless of rarefaction . As expected, negative controls and pure E. coli samples contained the fewest OTUs out of all sample types; the number of OTUs for negative controls and that for pure E. coli were comparable to each other. Interestingly, 4 out of 6 plaque samples contained fewer OTUs than many cultures while 2 plaque samples clearly yielded many more OTUs than all other samples. This difference deserves a much closer look, which we provide later in this section. A quick scan of the prevalence at the phylum level shows that Firmicutes and Proteobacteria were the most prevalent and the most abundant taxa, followed by Actinobacteria, Bacteriodetes, and Epsilonbacteraeota. These five taxa numbered among the most prevalent phyla in the preliminary experiments as well, indicating that the conditions we used favored members of these phyla and that the conditions were appropriate for promoting the growth of bacteria from key phyla of the oral microbiome. We then examined the rarefaction curves and plots of sequencing depth vs. diversity indices. The rarefaction plot of plaque and cultures shows that the number of OTUs discovered begins leveling around 30,000 reads for all samples. In other words, below 30,000 reads, new OTUs would still be discovered with increasing sampling depth, so rarefaction thresholds below 30,000 reads run the risk of artificially reducing true diversity in the samples. Furthermore, plots of read counts vs. diversity indices show that a somewhat weak but statistically significant negative linear correlation exists between sequencing depth and the Shannon index , and between sequencing depth and the inverse Simpson’s index , though the correlations are not immediately apparent on the plots.

The fact that longan, a cold-sensitive species, showed low expression levels for these three genes, suggests a possible strategy for genetic improvement of cold tolerance in this crop. Cold storage of apples is often used to extend post-harvest storage; however, it leads to superficial scald development, which is a major physiological disorder characterized by necrosis of the hypodermal cortical tissue. Karagiannis et al. applied a multiomics systems approach and created regulatory module networks to compare scald-affected and healthy apple phenotypes. Individual and combinatorial treatments with ozone , which induced scald symptoms, and 1-methylcyclopropene , which reversed O3-stimulated scald effect, were used to identify pathways and gene-to-protein-to-metabolite networks involved in scald prevention and sensitivity. Importantly, 1-MCP-induced scald tolerance correlated with the expression of genes involved in photosynthesis, stress responses, flavonoid biosynthesis, and ethylene signaling in apple peel and key TFs that may control some of these processes. This study represents an important contribution for future functional studies to develop improved apple cultivars to superficial scald. The acquisition of cold tolerance under conditions of varying light quality is essential for plants growing in regions with seasonal variation in both temperature and light . Photoinhibition, i.e., the downregulation of the electron transport chain, reduces plant productivity, but safeguards the photosynthetic apparatus during cold and light stress . Wang et al. investigated the role of light quality, specifically, low red to far-red ratios , nursery pots on photoprotection during cold stress in tomato. They showed that L-R/FR activated two pathways associated with cyclic electron flow : the PGR5/PGRL1A- and NDHdependent complexes, respectively.

These CEF complexes help to reduce cold-induced photo damage of the photosynthetic machinery by accelerating the thermal dissipation of excess energy, enhancing ROS scavenging, and reducing the hyperreduction of the electron transport chain. This work therefore provides a better understanding of the mechanistic relationship between varying light quality and low temperature in plant photosynthetic performance in temperate climates when seasonal variation induces these conditions.Spring frosts cause important economic losses in many fruit-producing areas of the world, and there is interest in identifying feasible approaches to mitigate these risks. Ethylene controls fruit ripening in climacteric species but it also plays an important role in plant stress responses . Published literature on the use of ethylene or ethylene-based compounds for protecting temperate fruit orchards against frost damage was reviewed . Experimental evidence of ethylene modulation of bud dormancy and blooming were presented and discussed. It was suggested that ethylene-delayed bloom and the associated frost protection may result from either the slowing down of floral bud responsiveness to seasonal temperature changes, an antagonistic interaction with other hormones such as abscisic acid or gibberellins, plant sensing of exogenous ethylene as a stress signal leading to longer dormancy, or ethylene-enhanced ROS accumulation resulting in extended bus dormancy. Because chilling stress in plants often leads to ROS accumulation, the questions arises whether improving the antioxidant capacity of tissues by the exogenous application of antioxidant treatments may help improve tolerance to cold as well as to other types of abiotic stress.

To this purpose, Tang et al. treated low bush blueberry seedlings with hydrogen sulfide , and found that treated plantlets performed better under low temperatures than the untreated controls, as shown by the alleviation of membrane peroxidation, the reduction of chlorophyll and carotenoid degradation, and the lessening of photosystem I and II photoinhibition. Conversely, the application of hypotaurine, a H2S scavenger, aggravated the oxidative symptoms under cold stress. Brassinolide is an important plant stress hormone shown to promote plant resistance to low-temperature environments. Zhang et al. investigated the effects of exogenous BR on the photosynthetic characteristics, leaf anatomical structure, and chloroplast ultrastructure of two species of tung tree seedlings under different temperature conditions. The results suggested that long-term low temperatures significantly reduced the photosynthetic efficiency of tung tree seedlings, affecting the formation of the internal structure of plant leaves and destroying the integrity and function of the chloroplast. To prevent this, external application of BR to tung tree seedlings could enhance the photosynthetic potential of tung trees by maintaining the stability of the leaf structure and morphology and alleviating the damage caused by cold injury. In summary, the papers in this collection illustrated the breadth of research aimed at understanding chilling responses in horticultural crops, but more importantly provided new insights that will further our future basic and applied research in this area.The human oral microbiome has been the subject of modern research for a considerably long time, from W. D. Miller’s works in the late 19th century to the exponentially increasing literature starting around 2002. The earliest published work in this area in the PubMed database goes back to 1958, and this work cites earlier works on the antibacterial characteristics of human saliva.

In addition to the anatomical components of the oral cavity and their respective roles in human health, the presence and activity of microbes in the oral cavity make this part of the body particularly interesting and difficult to study, as the relatively open nature and frequent use of the oral cavity make it a ready repository for microbes as well as a direct gateway into the body, especially into the upper respiratory system. In the last thirty years, research has begun to reveal the prevalence and diverse roles of the microbes, including strong correlations between some periodontal and respiratory diseases and bacterial members from certain genera such as Porphyromonas and Streptococcus. Despite the progress made in this field, details of the interactions among the microbes and between microbes and the host body are complex and remain difficult to elucidate. While many microbes in the oral cavity are commensal organisms that benefit from the host without harming the host, some microbes are pathogenic or opportunistically pathogenic organisms that take advantage of the weakening of the host immune system. Furthermore, the disruption of oral health can have broad and lasting consequences for the entire body. For example, even slight dental discomfort drastically decreases the range of nutrient intake and efficiency of food processing. On the microbial level, it is hardly surprising that dysbiosis of the oral microbial community can be readily linked to systemic diseases. On the other hand, this link can enable noninvasive as-sessments of the body by examination of salivary markers that are bacterial in origin. Clearly, the intricate and intimate relationships between host health and oral microbial communities, or the oral microbiome, merit deep delving, as the oral microbiome not only can act as an agent in but also serve as an indicator of human health and disease.Earlier research on the human oral microbiome faced considerable challenges due to difficulties in reliable cultivation of fastidious organisms and accurate identification of microbes. Though the effort to isolate and cultivate more oral bacterial phenotypes has been ongoing, cultivation of organisms remains a substantial obstacle. As of 2017, 32% of the 700 prokaryotic species identified in the oral cavity are known as uncultivated phenotypes. Part of the difficulty in cultivability stems from low prevalence and/or slow growth; other parts of uncultivability come from the nutritional and environmental requirements of certain strains that cannot be supplied in vitro, including resources supplied by other species and by the host system. For instance, the attachment of P. gingivalis to most strains of streptococcal cells except for the S. sanguis Challis strain is inhibited by saliva, and it extremely difficult to mimic the in vivo conditions of host salivary defense in laboratory cocultures of P. gingivalis and S. sanguis Challis strain. Another example of community-dependence is the parasitic relationship between certain microbial species, such as obligate epibiontic members of the candidate phylum TM7 parasitically preying upon an Actinomyces odontolyticus strain, which would make isolation of TM7 members impossible without the successful isolation, characterization, large pots plastic and cultivation of that particular Actinomyces odontolyticus strain. These commensal, symbiotic, or parasitic interactions among oral microbes are, in large part, stimulated by the physical proximity and biological efficiency conferred by biofilm structures in the oral microbiome, so failure to form biofilms in vitro or even thelack of a suitable surface for biofilm formation would severely interfere with the success of laboratory cultivations of more dependent members. For instance, dental plaque biofilms can exhibit a redox potential of -127mV during development at the enamel surface. This potential could be the key to the survival and proliferation of certain members of the dental plaque microbiome, but would not be possible without a proper biofilm structure.

The challenge of creating or replicating suitable environments in vitro for isolation and cultivation of bacteria is an active part of oral microbiology research, because comprehensive characterization of bacteria, including their virulence, relies heavily upon pure cultures. Ongoing efforts to develop and adapt cultivation methods for uncultivable microbes include simulating the natural environment of the oral microbiomes, supplementing with specific compounds, extending cultivation times, using assistive strains, and depleting environmental factors that contribute to the growth of other microbes. Other techniques applied to isolate and cultivate various species in other microbial communities include dilution to a smaller number of or a single cell, conditioning cultures with spent culture supernatants or cell-free extracts of helper strains, and adding signaling molecules to the cultures. These particular techniques, as-yet-untried in the cultivation of oral bacteria, are worthy of investigation for the purposes of not only isolating and cultivating yet uncultivable members of the oral community, but also enriching and expanding existing in vitro oral microbiome models. The other major challenge of oral microbiome research arises not from biological barriers to growing bacteria in vitro, but in the lack of proper methodology to distinguish among phenotypically identical but genotypically distinct species or strains. Many different methods have been used to achieve this goal. Visual identification with microscopy was widely used for some years. Confocal laser scanning microscopy has been used to identify streptococci bacteria in the early stages of dental plaque formation, determine viability, and study structure formation. Fluorescent microscopy with target-specific oligonucleotide probes has also been a common method of identificationand visualization. Non-visual methods include polymerase chain reaction to amplify 16S rRNA , PCR-based denaturing gradient gel electrophoresis, and DNA-DNA hybridization. These nucleic-acid-based techniques formed the earlier foundations of microbiome research, including the study of physical structures of the resident oral microbiome. However, they suffered from limitations such as lack of penetrative powers into the cell, incorrect hybridization of probes to targets, and PCR bias. In more recent years, the advent of high-throughput sequencing has allowed for more facile and in-depth studies of both the memberships and the abundances of the oral microbiome. In particular, the identification of microorganisms by genetic marker sequencing, especially the variable regions of the bacterial 16S rRNA, has exploded in popularity because of its versatility, ease of operation, and cost efficiency. High-throughput sequencing has deep roots in PCR, DNA microarrays, and fluorescence detection. The four most popular commercial HTS technologies rely on DNA synthesis and nucleic acid adapter ligation onto the template, and PCR lies at the heart of three of the four technologies. The Oxford Nanopore technology takes advantage of a porous bilayer through which a motor protein threads the growing DNA sequence. As the synthesized sequence proceeds through the bilayer, the base being added onto the growing strand is measured by the change in the current. This technique allows both strands of the template DNA to be sequenced, and can be done without PCR amplification. The sequencing technology from Pacific Biosciences relies on ligating hairpin adapters onto the template and using strand-displacing polymerase to sequence the template multiple times. Detection on the PacBio platform is achieved by reading signals from phosphate-labeled nucleotides in a zero-mode waveguide well. The third HTS platform comes from Life Technologies/ThermoFisher and relies on the concept of pyrosequencing. The beads are placed into microwells, and the immobilized DNA is amplified by emulsion-PCR. The nucleotide being incorporated is detected by measuring pH changes from hydrogen ion release, achieved by Ion Torrent’s semiconductor technology. These pH changes are converted to a voltage signal proportional to the number of bases incorporated. These three platforms have different applications, but are generally sub-optimal for fast identification of microorganisms. The average read lengths of the first two platforms are in the thousands of base pairs, far longer than the sufficient lengths for microbial identification, and the sequencing depth per run is limited to under 100,000 reads, which is too low for microbial communities with a large number of members.

That there are wide cultivar differences in these relationships leaves open the possibility our chosen cultivar, Cabernet Sauvignon, has naturally low concentrations.Despite the expectation that the products of membrane lysis would track with its causative reactive oxygen species, MDA concentrations did not commence at cell death onset or follow H2O2 concentrations and similarly saw no differences caused by either irrigation pulses. Like H2O2, MDA is a signal and damaging reactive compound subject to regulatory networks addressing biotic and abiotic stress, thus, accounting for its presence is not straightforward. In its most reactive state and at high levels, MDA can damage proteins, nucleic acids, and photosynthesis-related proteins, while at lower levels it can serve as a signal activating alcohol dehydrogenase . This activation helps achieve redox homeostasis by producing NADPH, which in turn oxidizes aldehydic compounds like MDA to prevent their build up to toxic levels . Post veraison, cellular conditions continue to be more favorable for the proliferation of ROS as scavenging and antioxidant capacity diminishes, whereas MDA may be more favored for degradation as ADH is more active then due to increased ethanolic fermentation in hypoxic conditions. Considering these processes we would have expected our MDA values to spike during cell death and then decline as hypoxic conditions maintained, drainage gutter but they ended the experiment at roughly the initial levels .Our experiment attempted to establish significant differences in water stress before and during cell death onset to test whether late-season-dehydration-related processes respond to irrigation pulses at those timepoints.

Despite a 40% increase in volume in subsequent two-week periods, vines under different irrigation regimes showed no treatment effects across all water stress parameters. The only difference was that the late pulse vines began the experiment with significantly higher stomatal conductance and photosynthesis rates , yet by the time the late pulse occurred these differences were erased. Other experiments investigating the relationship between water stress and cell death maintained consistent differences in watering throughout the growing season and found significant effects on both ψmd and cell death dynamics . We sought a strategy fit for implementing late in the season when root systems and canopies are fixed, and irrigation treatments would only affect post-veraison processes related to cell death. Root growth after veraison is largely inactive as the vine expends more energy into reproductive growth, thus our water treatments would not have spurred much new root growth but rather provided more water availability to fixed root systems. This water would be largely available because of the sizable portion of clay in our soils, though soil moisture probes would be needed to confirm the late treatment had significantly more water available. The low-vigor rootstock used in this study, 420A, could have limited the efficacy of our treatments due to its lower root hydraulic conductivity and aquaporin activity, whereas high-vigor, drought-avoidant rootstocks such as 140Ru, could have rendered the treatments more effective .

For example, after irrigating minimally pre-veraison, Netzer et al. imposed an augmented irrigation regime of .55in/week during a 45 day period post veraison for Cabernet Sauvignon on 140Ru in a Mediterranean climate with sandy soils and found continuously improving ψmd. A higher-vigor rootstock could have enabled our vines to transpire all of the water that was available to them, reduce ψmd, potentially prompting a different response. Given Netzer et al.’s success with alleviating water stress in late ripening, to achieve clarity on the effectiveness of late season irrigation pulses on water stress and cell death, it may be advantageous to use a high vigor rootstock under stronger drought conditions pre-veraison before irrigation is augmented. Of additional consideration regarding the similarity of ψmd across our treatments is that the vines used in this study were pruned by students, not an experienced crew, which does not guarantee uniformity in sap flow or canopy environment. Pruning without respect to sap flow—that is, year-after-year pruning cuts are made adjacent to each other to minimize the redirection of sap flow—has been shown to effect negative changes in xylem hydraulic conductivity in grapevines . The strong relationship found between xylem hydraulic conductivity and photosynthetic capacity of the canopy indicates this would have also affected our gas exchange measurements . Without a consistent approach to pruning among our vines in this respect, variance in xylem hydraulic conductivity could have created enough statistical noise to mute differences in gas exchange and water potential.This study demonstrated that increasing irrigation at the onset of cell death in winegrapes can delay the rate of cell death and maintain greater membrane integrity. When cell death is reduced, xylem backflow, which is difficult to compensate for as cell death progresses, is held off and turgidity is better preserved. Our results also confirm both the approximate range of onset date for Cabernet Sauvignon and the direct relationship between reactive oxygen species and cell death.

The exact mechanism of cell death remains opaque and many questions remain as to how cell death ought to be managed, especially in drought conditions. It is ultimately unclear why the late treatment’s cell death rate was positively affected by the late pulse, as we would expectxylem backflow to be too strong to overcome once cell lysis proliferates, considering there was no difference in water stress across treatments. Nonetheless, we believe this opens the door to more paths of inquiry in the mechanism of cell death. To investigate why a pulse of irrigation near cell death can effectively delay cell death without altering gas exchange or stem water potentials will require monitoring additional parameters, such as xylem/phloem conductivity in the pedicel or peduncle, dissolved oxygen ingress into the berry, berry respiration/fermentation rate, and soil moisture. On the other hand, establishing firm differences in water stress prior to onset would clarify whether the activity of programmed-cell-death-related genes does in fact respond to increased water stress and account for increased ROS and cell death. Establishing such differences would also clarify how far in advance a preemptive transcriptional response occurs, which would inform the timing of proposed irrigation strategies to postpone or delay cell death onset. A framework for an efficient irrigation regime that postpones cell death onset is needed to address future water scarcity, and our treatments and the levels of irrigation applied throughout this experiment are far from optimal to this end.Increasing chemical use, in conjunction with growing weed resistance and limited options for chemical weed control, has raised costs and depleted the bottom line for many rice producers in California. Many of the restrictions on farm chemical use can be traced to growing recognition of environmental externalities from chemicals used on the land and political pressure from environmental groups. For example, a recent district-court ruling banned the application of 38 pesticides along Northwest salmon streams, plastic gutter and estimates of the economic impact of the decision vary wildly . Environmental groups such as Greenpeace oppose the adoption and diffusion of genetically modified food crops such as GM2 rice. This opposition is largely based on the uncertainty of potentially adverse health and environmental impacts of GM rice and the lack of labeling requirements for GM foods. This is a potentially ironic position for environmental groups to take, given the possible environmental advantages of GM crops over more conventional varieties that depend heavily on the use of multiple chemicals and applications that may prove more damaging than the corresponding GM regime. This issue is critical in California, where agriculture is intensive and a relatively heavy user of chemicals. The economic impact on growers from chemical use regulations depends critically on the number of substitution possibilities available for cost-effective weed control. The more options individual rice growers have to control weeds, the less severe will be the adverse impact of the regulations on grower profits. However, environmental activists, regulators, and the courts view a wide range of available chemicals that have varied environmental risks as undesirable. In recent years, widespread adoption of GM crops such as herbicide-tolerant soybeans and canola and pest-resistant [e.g., Bacillus thuringiensis ] corn and cotton has provided growers with new production alternatives that reduce chemical usage. But the new technologies are not without controversy as some consumers have expressed resistance to purchasing foods made from transgenic materials. In California, environmental groups and organic-rice farmers are also opposed to any cultivation of GM rice in the state. This report examines these issues in the context of California rice production. In particular, we estimate the potential economic impacts of one alternative weed-management strategy, namely, cultivation of HT transgenic rice. Potential grower benefits, measured by net returns over operating costs per acre of first-year adoption, are calculated using a partial-budgeting approach3 based on a representative cost structure.

Sensitivity analysis is then utilized to account for the heterogeneity in growing conditions across the state as well as uncertainty regarding yields, technology fees, and government assessments on transgenic seed. To augment these results, the partial-budgeting approach is applied to data from an independent three-year field trial designed to evaluate alternative herbicide regimes, including one transgenic rice cultivar. The report proceeds as follows: The next section reviews available information on transgenic rice and describes the potential impacts of grower adoption in California, including market-acceptance issues. We then describe our methodology and present results for a typical Californiarice producer. Next, a range of estimated impacts based on alternative yield differentials and technology fees is presented, followed by a Monte Carlo analysis. The subsequent section provides an economic analysis corresponding to the three-year field study. Environmental regulations for rice production and potential environmental impacts of the new technology are then evaluated, and the final section discusses the limitations of our analysis and concludes.In 2003, California rice growers harvested 495,000 acres of rice, which yielded 39.6 million hundredweight , constituting about 16.5 percent of acreage and 20 percent of total rice production in the United States . The vast majority of California’s rice is of the medium-grain variety while the southern U.S. states primarily produce long-grain varieties. Over the last several years, there has been no discernible trend in California acreage planted or in total volume of production. World rice prices, on average, have been on a decreasing trend4 and, simultaneously, California growers have faced increasing production costs, especially in the area of weed management [U.S. Department of Agriculture , Economic Research Service 2002]. The top three weeds in California rice production are barnyardgrass, watergrass, and sprangletop while various other broadleaf plants, grasses, sedges, and cattails affect production [Gianessi et al.; California Rice Commission 2003]. Interestingly, red rice, a weed of the same genus and species as domesticated rice, is not a major problem in California despite being the number one weed in Louisiana, Arkansas, and Missouri . The combined effect of lower prices and higher production costs has put downward pressure on California rice grower returns and led to considerable research efforts to improve overall weed management through cultural, chemical, and other management means. In California, both chemical and non-chemical techniques are used for weed control . Recently, however, California rice production has experienced what has been called an “epidemic” of herbicide resistance, especially from watergrass, which has resulted in herbicide costs increasing to close to $200 per acre for some growers .5As such, technologies that allow for a small number of applications of chemicals where efficacy is not affected by the resistance problem, as would most likely be the case for HT rice, have the potential to significantly lower this component of rice production costs. There are currently no commercialized GM rice varieties anywhere in the world. However, many transgenic varieties are in the “development pipeline,” including HT, insect resistant , bacterial and fungal resistant, and nutrient-enhancing “Golden Rice,” which produces beta-carotene, a substance that the body can convert to Vitamin A. A non-transgenic but genetically altered variety called Clearfield® IMI by BASF, a mutated HT variety, was released in the United States in 2002 . Approximately 200,000 acres of Clearfield® were planted across the Southeast in the 2003 growing season, accounting for about 8 percent of the seeded area in that region . Countries that are major rice producers and consumers, including China and Japan, are rapidly developing and testing GM rice varieties . For instance, China has approved for environmental release three insect-resistant rice varieties and four disease-resistant varieties and is developing HT, salt-tolerant, and nitrogen-fixing cultivars . Many of these varieties have the potential to be of value to producers through reduced disease or pest-control costs and to the environment through reduced use of chemicals, thereby reducing runoff and water pollution.

Moreover, the Environmental Protection Agency of the United States ruled that MeJA was exempted from the requirement of a tolerance for residues in or on all food commodities when applied pre-harvest , a ruling that could facilitate the development of jasmonates as active ingredients in agrichemicals. A screening trial confirmed that MeJA and coronatine, a jasmonate mimic, also induce abscission in grape. The physiological basis for jasmonate-induced fruit abscission appears to involve ethylene. The exogenous application of jasmonates stimulates ethylene production in several fruits, including apple , orange, grape, strawberry , and tomato. The work carried out on orange and grape showed that the application of MeJA stimulated ethylene production by the fruit which was followed by fruit abscission. Malladi et al. provided indirect evidence that MeJA stimulates the abscission of blueberry fruits at least partly via ethylene action, as the co-application of MeJA with aminoethoxyvinylglycine , an ethylene biosynthesis inhibitor, attenuated MeJA effects on abscission. However, MeJA still induced some abscission in blueberry, even when co-applied with AVG which suggests that MeJA may initiate some abscission processes independently of ethylene. Moreover,grape berries treated with MeJA or 1-aminocyclopropane-1-carboxylic acid , the direct precursor of ethylene, produced similar levels of ethylene in the first 2 days after treatment , but thereafter, berries treated with MeJA produced less ethylene than berries treated with ACC, even though the MeJA-treated grapes generally had lower FDF, greater abscission, drainage gutter and a higher proportion of dry stem scars than the grapes treated with ACC. Together, these findings suggest that ethylene and jasmonic acid can promote fruit abscission via independent pathways and interact to promote abscission.

The potential for synergistic effects has sustained interest in research on the coapplication of jasmonates and ethylene-promoting compounds. This is especially important because of the relatively high dosages of MeJA needed for consistent efficacy when applied alone and because MeJA is much more expensive than ethephon. Because of the high rate of ethephon needed for abscission activity, the short time between abscission zone activation and fruit drop, and concerns about ethephon residues, alternatives to ethephon are desired. 1-Aminocyclopropane-1-carboxylic acid is not particularly effective at stimulating abscission on its own, but the co-application of MeJA with ACC improved efficacy in such a way that lower dosages of MeJA could be used. Recent improvements in jasmonic acid biosynthesis have the potential to make JA and its metabolite MeJA more available and affordable than they are now. The effects of JA on the abscission of fleshy fruits has not been tested, and the relative efficacy of JA versus MeJA with respect to grape berry abscission is unknown. Therefore, two studies were conducted to compare the efficacy of JA and MeJA at inducing abscission of Thompson Seedless grapes and to determine if JA interacts with ACC to promote abscission.Methyl jasmonate at 2 mM was ineffective, whereas 4 mM and 8 mM MeJA were equally effective at inducing preharvest abscission, reducing fruit detachment force , and increasing the proportion of detached berries with dry stem scars . Jasmonic acid was as effective or more effective than MeJA at inducing the abscission of Thompson Seedless grapes. Compared with MeJA, JA induced a similar or higher preharvest berry abscission, a similar or lower fruit detachment force, and a similar or higher percentage of detached berries with dry stem scars after treatment with JA, versus MeJA . The most effective treatment overall was 4 mM JA, which induced the highest level of preharvest abscission and percentage of detached berries with dry stem scars .

Grapes that were treated with 4 mM JA also measured among the lowest FDF values .MeJA can stimulate the abscission of many fruits and endogenous JA is known to promote abscission of floral organs and fruits, but the data presented here may be the first report of an exogenous application of JA stimulating the abscission of a mature fruit. Moreover, JA appears to be at least as effective, and possibly more effective, than MeJA at stimulating grape berry abscission. Interestingly, this is in contrast with a recent study that showed MeJA was more effective than JA at inducing abscission in lupine flowers. Improved efficacy at lower dosages could facilitate the commercial development of jasmonate-based abscission agents for grapes and other fleshy fruits because these natural products are expensive. In the first study, 4 mM JA was more effective than 8 mM JA; however, the opposite appeared to be the case in the second study. Therefore, additional research is needed to clarify the lowest dosage of JA that is consistently effective. The general range of effective dosages agrees with previous research that used MeJA as the active ingredient. Abscission zone activation reduces FDF and promotes the development of dry stem scars, both of which could help minimize picking damage and possibly improve the quality of destemmed table grapes. However, the final stage of AZ activation is undesirable unless catchment systems can be employed. Previous studies with MeJA suggested that harvest should occur within 3 days after treatment. Jasmonic acid also stimulates rapid abscission zone activation, with 14 to 25% abscission observed 2 days after treatment, and 52% to 60% by 3 DAT.

Without catchment systems, it may be necessary to harvest treated fruit within 2 days to avoid excess crop loss. This could be logistically difficult, and data are lacking to determine whether the abscission zone would be sufficiently developed by 1 or 2 DAT to provide the potential quality benefits that are desired from abscission agents. Another outcome that needs to be determined is whether the abscission zone could be activated preharvest and develop during postharvest storage. If so, this could be a way to achieve fruit quality benefits for stemless table grapes while minimizing the risk of preharvest fruit drop. Lavee demonstrated that preharvest applications of plant growth regulators can affect the postharvest abscission of grapes. Specifically, the preharvest application of 1- naphthaleneacetic acid and some other synthetic auxins reduced the postharvest abscission of “Muscat of Alexandria” that were held at room temperature for three days before entering cold storage. However, the abscission of grapes that were placed into cold storage immediately after picking was greatly suppressed, regardless of whether the grapes were pretreated with auxins or not. This suggests that the effect of plant growth regulators on postharvest abscission may depend on postharvest storage conditions. If abscission zone activation and development could both occur postharvest, it may be possible to treat the fruit in a packing house which should enable more efficient application of active ingredients, in addition to preventing crop loss due to preharvest abscission. The results of the second experiment also demonstrate a benefit in applying ACC with JA, as previously shown with MeJA. The co-application of 500 ppm ACC was sufficient to make 4 mM JA as effective as 8 mM JA in promoting dry stem scars, one of the most important treatment effects with respect to the quality of detached grapes. Because 500 ppm was the lowest dosage of ACC tested, it is unknown if a lower dosage might be as effective, but this should be tested in future research since the 500 ppm and 1000 ppm ACC treatments had similar interaction effects with JA. Likewise, a lower dosage of JA could be effective, especially if combined with ACC. Thus, additional work should be carried out to determine if combination treatments could enable lower dosages of JA and ACC to be reliably effective. In conclusion, plastic gutter the exogenous application of JA activates the pedicel-fruit abscission zone of Thompson Seedless grapes, rapidly reducing FDF, increasing the proportion of berries with dry stem scars after detachment, and leading to significant preharvest abscission within 2 days. Treatment effects require more than 2 mM and less than 8 mM JA if applied alone, but possibly less if co-applied with ACC. Additional work is needed to determine if harvest within 2 days after treatment is sufficient to reduce FDF and increase dry stem scar incidence while reducing preharvest abscission. Work should also be carried out to determine if preharvest or postharvest treatments are effective at inducing abscission postharvest.The experiments were conducted in September 2020 with own-rooted Vitis vinifera cv. Thompson Seedless grapevines and supported by an overhead-arbor trellis in a vineyard at the University of California Kearney Agricultural Research and Extension Center, in Parlier, CA. The vines were planted in 1995, trained to quadrilateral-cordons, and canepruned, leaving approximately 6 canes per vine, and 15 nodes per cane. Vines were spaced approximately 1.83 m within rows, and 3.65 m between rows which were oriented east to west.

All vines were subjected to cultural practices considered normal and ordinary for dry-on-vine raisin grapes in the San Joaquin Valley, except that the canes were not severed, and raisins were not made. Each of the two experiments used some methods comparable to those employed in a previous study. Clusters on individual vines were considered treatment replicates and each vine was considered a block. Since there were two experiments, each replicated six times, two groups of six adjacent vines were identified, with one group assigned to the first experiment, and the second group to the second experiment. There were seven treatments in the first experiment: an untreated control; 2 mM, 4 mM, and 8 mM MeJA ; and 2 mM, 4 mM, and 8 mM JA . Unique tags were made for each replicate and treatment and placed in different bags, according to their block. Available clusters were randomly assigned to each treatment by pulling the tags out of a bag and tying them to the peduncle of each cluster. The morning after labelling each cluster, solutions were prepared with the proper amount of MeJA or JA in water, with 0.05% of Latron-B1956 spreader-sticker . Control treatments consisted of water with Latron-B1956. Each solution was placed into a spray bottle , agitated well, and then applied to an appropriately labelled cluster until runoff occurred. Polyethylene shields were used to prevent overspray from contacting other clusters. After the clusters dried, they were enclosed in polypropylene mesh bags to catch any berries at risk of abscission. The bags had a re-sealable flap at the bottom from which abscised berries could be collected and weighed. In the first experiment the berries were collected once, 3 days after treatment. The abscised berries were weighed and discarded, and then each cluster was harvested and taken to a laboratory in their mesh bags where FDF and dry stem scar measurements were made. At the laboratory, clusters were gently removed from their bags, and small shears were used to sever 10 berries from the top, middle, and bottom part of each cluster, retaining the pedicel and a short section of rachis with each berry. Each berry was then placed in a jig attached to a force gauge , and force parallel to the fruit axis was applied to the rachis until it detached from the berry and peak force was recorded. After each berry was detached, the stem end was observed and assigned to one of two classes: dry or wet stem scar. The remaining berries on each cluster were then removed, combined with the ten used for FDF and dry stem scar measurements, and weighed. The total weight of each cluster was determined by adding the weight of any berries that had abscised prior to harvest, and the percentage of preharvest fruit drop was calculated based on the cumulative weight of the abscised berries and the weight of the berries remaining at harvest. The second experiment had nine treatment combinations: three levels of JA in a factorial design. The ACC was provided by Valent BioSciences . Cluster selection, solution preparation , treatment application, and bagging, were similar to the procedures employed in the first experiment. In the second experiment, cumulative preharvest abscission was determined daily, by weight. After 3 DAT, all the clusters were taken to the laboratory for determination of FDF, dry stem scar development, and cluster weight determination. All data were subjected to analysis of variance using the GLM procedure of SAS . In the first experiment, a randomized complete block design was used, with seven treatments. Means were compared by Duncan’s new multiple range test .

Our study differs from prior peach transcriptomic analyses in two ways. First, we are using samples from pools of genetically related siblings with contrasting sensitivity to chilling injury subjected or not to cold storage. Thus we expect to reveal genes whose expression patterns are linked to the different cold sensitivity, while leveraging transcript differences associated with other phenotypic traits, as it would be the case when comparing only two peach cultivars that have different chilling susceptibilities in addition to other phenotypic differences. Second, by medium throughput qRT PCR we extended our microarray results derived from the comparison of the contrasting pools to a relatively large number of 15 individual lines from the same population differing in the mealiness sensibility and the gene expression results of the selected genes were consistent with their individual sensitivity level.Orthologs of several transcription factors found upregulated similarly in S and LS cold-treated fruits have been previously reported as being up-regulated during cold acclimation in Arabidopsis and some of them also were described as belonging to a given cold acclimation regulon. This suggests the activation of a cold response program in peach fruits in part similar to those described for Arabidopsis cold acclimation. Despite observing similarities some genes exhibited an opposite trend compared to Arabidopsis which may partially reflect the sensitive character of peach fruit to cold . Several studies have associated cold tolerance and cold acclimation the transcriptional activation of genes encoding heat-shock proteins , chaperonins, LEA proteins, antioxidant/scavenging systems and related to protein synthesis.

Genes in these functional categories were generally down-regulated by cold storage in both LS and S fruits, square plastic planter what correlates well with their sensitivity to cold. Further, the orthologs of HSF4B and HSP21 were up-regulated peach fruits, whilst were down-regulated in Arabidopsis. This is particularly interesting as these genes are highly up-regulated in Arabidopsis chilling sensitive mutants upon chilling treatment. It should be noted that we are comparing the transcriptomes of different species and tissues at various physiological and growth stages, and it is likely that some differences in strategies to cope with exposure to low temperatures operate in each case. The basic question is: why do LS PopGG siblings tolerate better cold storage than S? Our results indicate that during cold storage fruits LS maintain higher levels of expression for a series of components of the antioxidant system, structure maintenance proteins and protein synthesis at least during the first week of storage . In addition, the orthologs of some TF with a higher expression levels in tolerant peach fruits have been reported to be upregulated by cold and/or other biotic or abiotic stresses in Arabidopsis . In this sense, our data indicated that the peach orthologs for genes in ICE1, CBF and HOS9 regulons may be implicated in the tolerance of fruits LS. The central role played by the ICE1-CBF cold response pathway in cold acclimation and cold tolerance is well-established in plants and has been demonstrated to exist in a wide range of plants, although, there are differences in the regulation or the size of their CBF regulons. The existence of ICE-CBF pathway has been also confirmed in fruits. Further, LeCBF1 expression levels correlates positively with cold tolerance in tomato fruits. We found that genes in the regulons ICE1and CBF were the most contributing to discriminate samples S from LS, and/or to separate samples that will become mealy, or not .

Moreover, PCA analysis identified CBF1 as the second gene that contribute the most to separate the S and LS series and qRT PCR analysis showed that the expression levels of CBF1 correlate well with the tolerance/ sensitivity of the individual pop-DG siblings . Thus, confirming ICE-CBF as important actors in the differential response to chilling between peaches S and LS. In the case of the genes in regulon HOS9 our results suggest that it is more likely related with the ability to up-regulate or to maintain similar expression levels to those observed in M fruits . Zhu et al. concluded that HOS9 must be important for both the constitutive expression and cold-induced expression of the genes that may be required for full tolerance to freezing stress. These results are consistent with peaches having the basic components of a cold response pathway, but additional studies will be required to elucidate their size and how they are regulated. In normal commercial fruit operations cold storage, involves also complete darkness. Gene by gene comparisons has revealed that around 3% of our cold regulated genes in peaches could be related to darkness . Moreover, we identify some genes whose orthologs have been described in the regulation or in response to light . Several, light sinaling elements among which were GI, DFL2, PHYA and FYPP3 were repressed by cold storage in both LS and S , consistently with the storage in darkness conditions. In addition, genes differentially expressed between fruits S and T include a number of regulators involved in light response that indicates we should take into account this factor as contributing the differential response observed in peach fruits. In Arabidopsis light is required for cold induction of several genes involved in cold acclimation, including CBFs and some light signaling mutants have impaired cold acclimation. Thus the differential response to cold storage of fruits S and LS probably have to do fruits’ ability to deal with cold and darkness. However, further experiments are required to determine in more detail the nature of the interaction between the cold and the darkness during storage.Despite no visible mealiness symptoms are observed during cold storage, the BSGA indicated dramatic changes in the peach transcriptome in response to the exposure to mealiness-inducing temperatures in a manner that these changes could be useful to predict future mealiness development .

We propose the transcript differences observed while in the cold might underlie the molecular basis of a mealiness phenotype which is still undetectable, but will be fully developed later during shelf life. This is in agreement with previous reports of the cold induction of specific target genes that are associated with the mealiness disorder. Surprisingly, our results showed that cell wall is not found among enriched categories in none of the clusters/comparisons performed on cold stored samples, suggesting that although specific changes in cell wall remodeling transcript are detected most of the changes would probably occur during shelf life. Our results reveal also that transport and signaling elements presented higher levels in S fruits, which in some cases, correlated well with the eventual mealiness phenotype. We found the orthologs of genes described as positive regulators of ABA signaling and/or osmotic stress and transporters related to Na+ and K+, sugar and nitrate homeostasis among genes high expressed in fruits S This suggests that fruits S during cold storage undergo some sort of dehydration or osmotic adjustment. It has been proposed that during cold storage, before mealiness is manifested, pectin depolymerisation but not deesterification is inhibited, square plastic plant pot what may lead to the formation of gel-forming pectins that traps free water from the surrounding tissue. As no significant differences in global water content are found between LS and S fruits it is likely that water is being lost from the cell to be trapped on the pectins of the cell wall, which still would be sensed as loss of internal water by the cell. Among genes with higher expression in sensitive fruits we identified components of auxin and ethylene signaling cascades as well the orthologs of genes involved in the biosynthesis of ABA, auxin and ethylene . We must highlight the large list of genes related to auxins among with were positive regulators of auxin responses and transporter locations . In addition, among the genes high expressed in the fruits LS at one week there were the orthologs of genes such as HAB1, PP2CA/AHG3, SAD1 and ERD15 ,which have all been described as negative regulators of ABA signaling, and IAA17/AUX3, proposed to be a negative regulator in auxin and ABA signaling. Ethylene and auxins has been described in the regulation of the ripening program of peach fruits and their involvement in the cold response has been described for Arabidopsis, tomato, apple and peach. Our results indicate that part of the ripening program probably continues during cold storage in sensitive fruits . Hence, we could expect that interactions between cold and hormones controlling the peach ripening program, which are differential between fruits S and T, impact the way fruits respond to cold and ripen afterwards during shelf life. Because the activity of most of these genes is mainly determined at post-trasncriptional level reviewed in [90], it is not possible from expression data only to infer the role of these genes during cold storage. However from our data it is clear that all three hormones may play a role in regulating the differential response of peach fruits to cold and they seem operate in association with dehydration/osmotic stress. In support of that, the orthologs of many of hormone related genes higher expressed in CS1-S fruits have been described previously either in relation to drought and osmotic stress . For example, the orthologs of SKIP, BRM and ERD1 mediate the responses or are induced by ABA, salinity and dehydration stress; CPL2 modulates auxin responses, plant growth and osmotic stress and EIN2 has been described to be an important cross-link node for the interaction of ethylene, ABA and plant response to abiotic stress. We cannot rule out that the ‘‘sensitivity’’ program is the consequence or the cause of low levels ICE1-CBF regulons. It is possible that the up-regulation of a set of common genes concomitantly with low CBF levels triggers this program. It is also feasible that among CS1 S.LS there are genes which negatively regulate the CBF response. To support this, EIN2 has been described as a negative regulator of plant response to freezing stress by negatively regulating the expression of CBF1-3 and its target genes; interestingly, CBF genes have been found to be directly repressed by IAA. Finally, it may also be possible that this program is activated to compensate efficient acclimation during cold storage. It has been described that hos9 mutants hyperactivate some cold-regulated genes through a compensating response to their increased cold sensitivity.At the mature stage specific differences at the gene expression level between the pools of fruits S and T already exist . Although our approach used pools of fruits in accordance to how they respond to cold storage, therefore minimizing differences in other aspects between genotypes, we can’t dismiss the possibility that these differences have nothing to do with adaptation to cold. Preformed mechanisms have been described in both biotic and abiotic stress tolerance and we previously identified a subset of genes differentially expressed at harvest that correlate well with CI. Cell wall metabolism has been extensively related to mealiness in peach fruits, and it has been reported that endopolygalacturonase plays a qualitative role in the mealiness expression. Our results indicate that the composition of the cell wall at harvest could play a role in the tolerance or sensitivity of peach fruits to withstand cold storage. This is in agreement with previous results. In addition the type of functional categories for the differentially expressed genes at the stage M, and the fact that most of these genes continue to show these differences during cold storage , suggest the possibility that a pre-programmed tolerance/sensitivity mechanism can be partly established previously to cold. Among the highly expressed genes in fruits LS at the mature stage, we found orthologs of genes such as CHS/TT4 and GST12/TT19 , which have been described being essential for anthocyanin and proanthocyanin accumulation. Anthocyanins have been related with browning in peaches. However, no significant differences in browning, bleeding nor in ppLDOX expression were observed between our pools. It is suggested that AtTT19 functions as a carrier to transport proanthocyanin precursors to the tonoplast to be later secreted and linked to cell wall polysaccharides. Binding that depends on the composition of the proanthocyanin. The tt19 mutation leads to the formation of aberrant PA derivatives. Thus is possible that differences in TT19 have to do with cell wall composition and chilling sensitivity. Further experiments are required to test this hypothesis. In addition, flavonoids act as negative regulators of auxin transport.

The model suggests that in colder areas, the observed late-season buildup of the fly results from low winter survival and the movement of adults from areas with phenologically earlier populations.Berry curvature is a key to understand novel physical phenomena such as anomalous Hall effect, chiral anomaly, topological Hall effect, and spin-valley Hall effect. Moreover, the BC classifies the topology of a solid via a topological number that predicts the presence of protected states at its boundary. The response of a system governed by the BC is constrained by the Onsager relation in the linear order. This stringent constraint, however, is no longer valid in the high-order responses that are proportional to the second-order or even higher orders of the driving field. The integration over higher-order fields gives a nonlinear response of the system, contributing to the optical and transport responses . This finding not only provides a methodology to explore the momentum texture of the BC of a system, but also paves a way to utilize the response even though the linear order is vanishingly weak or when a large driving field makes the higher-order response exceed the linear order. To make use of the higher-order response has great potential for applications in rectification devices , photosensitive devices , and photovoltaic devices that potentially overcome the quantum efficiency limit.Recent studies on the higher-order response have invited nonmagnetic materials with broken centrosymmetry as a new member of the Hall effect family, so-called nonlinear Hall effect , characterized by a quadratic behavior of the Hall voltage with second harmonic frequencies in the presence of a perpendicular AC driving current. In the absence of the linear Hall effect due to time reversal symmetry, 25 liter plant pot the lowest-order Hall current is driven by the Berry curvature dipole.

Since the response is proportional to the gradient of the BC, tilted anticrossing bands and Weyl points are predicted to exhibit strong BCD that can generate a nonlinear Hall angle close to 90 degrees. As a result, the momentum-dependent texture of the BC based on the electronic structures is essential to understand the NLHE in which the evolution of BC momentum texture by lattice strain, interlayer twisting, and external electric fields can lead to NLHE-based device applications. Nevertheless, while the majority of studies on the NLHE have predominantly focused on exploring its transport properties, applications, and theoretical simulations, the direct experimental confirmation and comprehensive understanding of the BCD based on electronic structures have not yet been fully attained. This achievement would provide crucial insights into the underlying mechanism and controllability of the BCD. In this paper, we report the room temperature NLHE in NbIrTe4 thin flakes, which exhibit a frequency-doubled Hall conductivity proportional to the square of the driving current. We also demonstrate that the sign change in the NLHE at 150 K is induced by the sign change of the BCD because of the chemical potential shift at high temperatures. It is unambiguously evidenced by direct observation of a chemical potential shift in the temperature-dependent band dispersion using angle-resolved photo emission spectroscopy and calculated BCDs. Investigation of the electronic structures using ARPES and density functional theory also indicates that the main contributor of BCDs is a partial occupation of spin–orbit split bands. Our findings provide important insights into the momentum texture of the Berry curvature and into controlling the Berry curvature dipole hosting the NLHE, which can be utilized for NLHE-based devices.

It is often asserted and then assumed that fundamental research from model systems, such as Arabidopsis, is valuable because this knowledge would be utilized later to elucidate practical aspects of plant function in species of agronomic importance. This has been successful in like organs and developmental processes. For example, a regulatory module involved in photoperiod control of flowering has been elucidated through extensive research in Arabidopsis , and more recently this same mechanism has been shown to control flower time in Populus trichocarpa . Unexpectedly, Bohlenius et al. revealed that the same regulatory module is involved in the control of bud dormancy. This illustrates that components of regulatory mechanisms can be conserved across disparate developmental processes that share commonalities; a dependence on daylength and action in the shoot apex in the case of flowering and bud dormancy. Less is known about the utility of extending knowledge to a class of organ not even present in a given model species. The onset of ripening marks a transition in fruit development leading to changes in physiology involving sugar metabolism, softening, and color development. Most understanding of this process arises from work with climacteric fruits in which the control of ripening is predominately by ethylene. However, there are many fruits of economic importance that are nonclimacteric, such as grape, strawberry, and citrus, where the processes controlling the onset of ripening are still poorly understood. Study has traditionally focused on ethylene in climacteric fruits and on other hormones such as sugar and abscisic acid in non-climacteric fruits. However, there are numerous studies implicating ethylene in the ripening of non-climacteric fruits , and conversely, sugar and ABA in the ripening of climacteric fruits . In addition, other hormones have been show to influence the ripening processes such as auxin and brassinosteroids .

Fruit ripening in general involves the integration of multiple hormone signals. Climacteric and non-climacteric fruits are not delimited by phylogeny and both exist even among closely related species . This suggests that ripening in all fleshy fruit may have a common foundation. Sugar and ABA function as signals in many aspects of plant development and there are numerous lines of evidence correlating increases in sugar and ABA with the onset of ripening in grape . In field-grown berries, exogenous ABA increases anthocyanin accumulation , and in our own work berries fail to synthesize anthocyanins when sugar import into the berry is disrupted via phloem girdling prior to the onset of ripening . Finally, water deficits also result in increases in anthocyanin accumulation , and have been shown to increase ABA accumulation at the onset of ripening . Many studies investigating the role of sugar and ABA in ripening have focused on color development. In viticulture, the onset of ripening is traditionally referred to as veraison and is defined by the initiation of color development. More contemporary genomic studies demonstrate that color can act as a proxy for developmental stage in that individual grape berries of like color have similar global gene expression patterns . A previous study in our laboratory found that water deficits advanced the onset of color development, but sugar and anthocyanin accumulation remained closely linked . Furthermore, the induction of core flavonoid and anthocyanin-specific genes reflected this advancement as well . Therefore, in the current study, water deficit is used as a means to manipulate the timing of the onset of ripening in the field. There are few examples of studies focused on identifying the molecular mechanisms responsible for sugar and ABA action in grape, although several recent studies have demonstrated roles for sugar and ABA in controlling sugar metabolism. ABA stimulates acid invertase activity , and expression of the grape hexose transporter VvHT1 is regulated by both ABA and hexose . Two other recent studies have focused on core sugar and ABA-signaling components. A calcium-dependent protein kinase is regulated by ABA during ripening , and a study in grape cell culture provides evidence that sugar induced increases in anthocyanin accumulation are dependent on calcium, calmodulin, and kinase activity . In this study, we begin to examine the hypothesis that orthologous sugar and ABA-signaling components, black plastic plant pots characterized across such diverse processes as seed dormancy and stomatal control, function similarly in the control of the onset of ripening in fleshy fruit. This study employs a comprehensive approach in identifying novel sugar and ABA-signaling candidates in grape.

Ten gene families, containing sugar and ABA-signaling components elucidated in model systems, were considered including; the putative sucrose sensor SUT2, core G-protein signaling components GPA1 and RGS1, hexokinases , PP2C protein phosphatases, Snf1-related kinases , and the sugar-related WRKY, and ABA-related homeodomain–leucine zipper, or homeobox , ABRE-binding factor , and AP2 transcription factors . Their orthologous families in grape were identified, and the expression of individual genes were characterized in control- and deficit-irrigated, field-grown Cabernet Sauvignon. The onset of ripening was induced in cultured immature berries with sugar and ABA treatments. Finally, the effect of exogenous sugar and ABA on the expression of several key orthologs was investigated in berry culture.The field experiment was the same as presented in Castellarin et al. . In short, experiments were conducted during 2006 in a commercial vineyard of R.H. Phillips Winery using Vitis vinifera ‘‘Cabernet Sauvignon’’ clone 337 grafted onto rootstock 140R. Two irrigation treatments were established. Irrigation was applied to control vines in order to maintain midday leaf water potential between -0.9 and -1.2 MPa. Irrigation was cut off to early deficit vines at fruit set until 77 days after anthesis ; then vines were irrigated at the C rate till 133 DAA. Plant water status was monitored weekly by measuring midday leaf water potential according to Matthews et al. . Each treatment was replicated four times in 0.5-ha plots dispersed over a 36-ha vineyard according to a randomized complete block design. Samples were collected randomly from ten vines located in the central row of the plots. Berries were sampled six times between 27 June and 5 October , when the grapes were harvested. Twenty berries were collected from each plot for use in studies of gene expression. During the onset of ripening, samples of green and red berries were collected separately from the same clusters and analyzed separately.Cabernet Sauvignon clusters were collected from fieldgrown vines at 61 DAA. Clusters were surfaced sterilized for 10 s in a 70% ethanol solution, 10 min in a 1% bleach solution, and then rinsed three times with sterilized water. Berries were removed from the clusters by cutting the stem at the peduncle level, and the peduncle was removed from the rest of the fruit with a scalpel. Berries were weighed and individually cultured for 23 days in 20 mL glass vials filled with 7 mL of six different 1% agarose media 2% sucrose, 10% sucrose, 200 lM ABA alone, 2% sucrose 200 lM ABA, 10% sucrose 10 lM ABA, 10% sucrose 200 lM ABA. The 2 and 10% sucrose concentrations used in this study approximate sugar concentrations of berries prior to and at , the onset of ripening in the field . Each treatment was replicated ten times. Berry color changes were visually analyzed twice, 10 days after T0 and at the end of the experiment. Elasticity was measured at T0 and at the end of the experiment. In short, berry elasticity was determined non-destructively using a custom-fabricated instrument, which measured force and displacement during berry compression. As described in Thomas et al. , the observed force/displacement relation was fit using SAS PROC NLIN to that expected for compression of a perfectly elastic sphere . 23 days after T0, berries were collected from the vials, weighed, and stored at -80 C till the tissues were processed for the RNA extractions. Three berries from the 2% sucrose, 10% sucrose, 10% sucrose 10 lM ABA, 10% sucrose 200 lM ABA treatments were peeled and total RNA was extracted from each individual skin following the procedure described above.Utilizing the sequenced grape genome , 67 genes were identified representing orthologs of sugar and ABA-related genes in 10 gene families . The genomic complexity of these families varied from small families with just one or few genes to large families made up of nearly 100 genes . Small gene families were comprehensively characterized for all grape orthologs, while only those subgroups containing genes specifically shown to be involved in sugar and ABA signaling were included for large gene families. Given the scope of this work, we focused primarily on those gene families encoding the following well characterized orthologs the Arabidopsis Class I HB transcription factors, the Arabidopsis ABA-insensitive mutants, abi1 and abi2, encoding PP2C protein phosphatases, and the WRKY transcription factors SUSIBA2, AtWRKY4, and AtWRKY34. Other gene families are summarized and referenced as supplemental material when pertinent. The homeodomain–leucine zipper, or homeobox transcription factors represent a medium-sized gene family with approximately 47 members in Arabidopsis.

The Chern number is just a property of a band and does not come with an energy scale, so there is no reason to expect to encounter Chern bands only at low temperatures. Indeed, bands with finite Chern numbers have been shown to support quantized Hall effects in graphene quantum Hall devices at room temperature and high magnetic fields, as illustrated in Fig. 8.5A,B. The energy scale in a Chern magnet is set by the band gap produced by magnetic interactions. So if we’d like to know what the maximum temperature at which we can expect to find Chern magnets is, we need to think about the energy scales of known magnets. Magnetism is an interaction-driven electronic phase, and interaction-driven phases almost always melt at sufficiently high temperatures. However, among interaction-driven electronic phases ferromagnetism is particularly stable. Many common transition metals, including iron, cobalt, and nickel, support ferromagnetism into the range 600-1200 K, and all of these have found applications in a variety of electronic technologies as a result. These are of course all three dimensional crystals, and Chern magnets are two dimensional crystals. So the next question we can ask is: do two dimensional magnets exist with Curie temperatures as high as room temperature? The answer turns out to be yes, as illustrated in Fig. 8.5C,D. This magnetic system appears not to be a Chern magnet, unfortunately, but the point is that there is nothing in particular stopping a Chern magnet with a Curie temperature above 300 K from existing. The first intrinsic two dimensional ferromagnets were discovered in 2017, so I think it’s safe to say that our field hasn’t yet come particularly close to identifying all possible two dimensional magnets. It’s hard to do an accurate accounting of all of the so-far discovered two dimensional magnets, drainage pot and it is certainly the case that many of these are are not Chern magnets.

But of the two dimensional magnets we have found, a surprisingly large fraction are intrinsic Chern magnets. We know of eight intrinsic Chern magnets stable in the absence of an applied magnetic field in the published literature so far. These are presented, along with a few of their basic properties, in Table 8.1.Over the course of my PhD, four nanoSQUID microscopes were proposed, and construction began in some form on all of them. By the time I left we had finished three of these microscopes. The first nanoSQUID microscope we completed was inserted into a bath of liquid helium and could operate at 4 K. The second nanoSQUID microscope had a pumped He-4 evaporative cooling pot, and could reach temperatures of 1.5 K. The tBLG/hBN Chern magnet transport measurements, the tBLG/hBN Chern magnet imaging measurements, and the AB-MoTe2/WeSe2 Chern magnet imaging measurements were all performed in this system. The third nanoSQUID microscope had a closed cycle He-3 sorption pump cooling system, and could reach 300 mK. The ABC trilayer orbital magnet imaging measurements were performed in this system. The fourth and final microscope remains under construction, and is designed to operate inside of a dilution refrigerator. Pictures of several of these microscopes are shown in Fig. 8.6. Acoustic isolation chambers and the 300 mK system are not shown. All nanoSQUIDs have liquid He-4 baths for primary stage cooling, and all are mounted on several thousand pound vibration isolation tables floating on air legs to protect the nanoSQUID sensors from mechanical and acoustic shocks close to the surface. The nanoSQUID sensor circuit is fairly simple, with only one important non-standard circuit element in it, other than the nanoSQUID itself of course. This is the series SQUID array amplifier. Current is forced into the nanoSQUID sensor in parallel with a shunt resistor of comparable resistance to the nanoSQUID sensor in the voltage state, which is generally a few Ohms.

Current through the nanoSQUID side of the circuit is inductively coupled to a series of identical SQUIDs. These SQUIDs in series generate a large voltage, which is detected at room temperature. Current is forced through a feedback coil to maintain constant flux through the SQUIDs in series. This allows the circuit to maintain sensitivity over a wide range of currents . This current amplification circuit has good current sensitivity and enormous dynamics range, easily able to accommodate the several hundred µA necessary to reach the critical current of the nanoSQUID sensor.There are a lot of things that make scanning probe microscopy tough relative to other techniques for performing microscopy. One particularly challenging issue is navigation of the sensor to the sample. Those experienced with optical imaging might be spoiled by a contrast mechanism that is sensitive to a ton of different phenomena- the nanoSQUID can only see local gradients in magnetic field and temperature, and those are rare unless you have intentionally built structures and devices that generate them for use in navigation. In particular, large thermal gradients and variations in local magnetic field aren’t general properties of surfaces, so it’s very easy to blunder a nanoSQUID sensor into a surface without ever seeing it coming! Experiments are thus much safer and more expedient if we can provide the nanoSQUID sensor with topographic feedback- i.e., some way of detecting surfaces without crashing into them and destroying the sensor. We did this using shear force microscopy, which is a form of atomic force microscopy, or AFM. There is nothing particularly atomic about this contrast mechanism in the nanoSQUID microscope- we don’t have nearly that much resolution- but it is incredibly useful for navigation because it allows us to safely and reliably detect surfaces without destroying the SQUID. Researchers and companies building scanning tunneling microscopes will often accomplish this by gluing their sensor, which is a microscopic metallic wire, onto a piezoelectric tuning fork and then exciting the tuning fork at its resonant frequency. This is a good strategy, but it must be modified for use with the nanoSQUID sensor, because the nanoSQUID sensor is considerably more massive than scanning tunneling microscope wires, so it cannot be glued onto the tuning fork without destroying its quality factor. We preserve the tuning fork’s quality factor by instead pressing a piezoelectric tuning fork against the side of the nanoSQUID sensor and performing shear force microscopy instead of tapping mode microscopy. The glass micropipettes serving as substrates for the nanoSQUID sensors are so thin that they bend easily when pressed agains the tuning fork, and this keeps them in mechanical contact with the fork. An optical microscope image of a nanoSQUID sensor pressed against a tuning fork is shown in Fig. 8.8A, and the resonant frequency of the piezo electrically driven tuning fork is shown in Fig. 8.8B, with a fit to a Butter worth Van-Dyke model. A phase-locked loop and PID feedback system together allow us to approach the surface with the nanoSQUID sensor, detect it without crashing into it and destroying the tip, and maintain feedback while scanning. Schematics of this assembly are shown in Fig. 8.9. A calibration of the scan range and height of the nanoSQUID AFM is shown in Fig. 8.10, with a comparison to a Bruker Icon AFM displayed as well.By far the most common experimental campaign for the nanoSQUID microscope during my time in Andrea’s lab involved being handed a sample fabricated primarily for transport or capacitance measurements, with little consideration afforded to the viability or ease of a scanning probe microscopy campaign on the sample. I think this is fairly common in scanning probe microscopy, and it often means that we need to get sensors to samples without much in the way of navigation infrastructure. For this reason the vast majority of nanoSQUID microscopy campaigns start with thermal navigation. Before cooling down the nanoSQUID microscope, drainage planter pot an attempt is made to align the nanoSQUID sensor with the heterostructure under an optical microscope, but the nanoSQUID sensor often still starts several hundred microns away from the sample. Once the system is cold, we generally proceed by injecting a few mBar of helium gas into the sample chamber.

This facilitates thermal transport between the nanoSQUID sensor and the sample. We then run an AC current through the sample, heating it and generating an AC temperature distribution. The nanoSQUID sensors are excellent thermometers as well as magnetometers, so we can use this thermal gradient to navigate to the sample. An image of the resulting distribution of temperature over the device is shown in Fig. 8.13A. Some of the details are described in a later section, but in summary this technique works surprisingly well- we can usually find samples even several millimeters away from the nanoSQUID sensor using this technique. Once the nanoSQUID is reasonably close to the sample, it is usually necessary to pump out the heat exchange gas before attempting magnetic imaging, since thermal contrast can produce large backgrounds. After the heat exchange gas is removed, further navigation must proceed by imaging the magnetic fields produced by applied current through the Biot-Savart effect, as illustrated in Fig. 8.13B.Thermal navigation does not work for all systems. In the simplest case in which other techniques are necessary, current cannot be driven through magnetic insulators, so if you want to find them with the nanoSQUID you must arrange for some navigation technique other than flowing current through the sample. There are a variety of solutions to this problem, and perhaps the simplest is fabricating an additional device adjacent to the one you’d like to investigate and running current through that instead. There are reasons you might want to avoid this- some samples are so unstable in air and moisture that it makes sense to avoid photolithography on heterostructures entirely- and for these situations, I’m going to discuss ferromagnetic navigation.There are a few engineering challenges associated with fabricating nanoSQUID sensors. I will briefly describe a particularly challenging one in this section. Many of the best elemental superconductors are soft, heavy metals with low melting points like lead and indium. As any person who has spent some time in an experimental physics laboratory knows, solder doesn’t wet too many materials well, and it certainly doesn’t wet glass, so these metals tend to form droplets when deposited onto glass substrates. To form a uniform film, the superconducting metal must freeze instantly upon landing on the glass micropipette. To make sure this occurs, we must cryogencially cool the glass micropipettes while evaporating the superconducting metal onto them. This process involves specialized machinery that is covered in great depth in other documents and publications, so I won’t discuss it here. However, I do want to discuss the nature of the failure modes of this process. When liquids don’t wet surfaces well, they dewet into droplets, and these droplets tend to get more spherical and less film-like the worse they wet the surface. If this process is allowed to proceed to its conclusion before deposited metal solidifies, the resulting films won’t be connected at all, and your nanoSQUID circuit will be open. If the substrate is cold enough, the resulting film will at least be continuous, and it is likely that you will get a nanoSQUID. However, the formation of droplets is impossible to completely stop, especially near the edges of films and on the oblique surfaces of the nanoSQUID sensor . These droplets generally won’t short the sensor, but the nanoSQUID sensor is so small that electrons can reach these droplets through tunneling processes. Whenever droplets form between the two superconducting contacts on the nanoSQUID sensor electrons can tunnel between the contacts through the droplet, with the droplet functioning as a quantum dot. The resulting Coulomb blockade phenomenon gives nanoSQUID sensors a very slight electric field sensitivity. Gating exfoliated heterostructures tends to produce large electric fields, and these are detectable as variations in the current through the nanoSQUID as a result of Coulomb blockade in parallel with the SQUID on the tip. Droplets functioning as quantum dots and producing a parasitic Coulomb blockage are so common that we observe them on nearly every nanoSQUID sensor, and we almost always have finite electric field sensitivity . This can be useful for finding the edges of devices in the absence of magnetism, but it is important to remember that not all nanoSQUID signals can be understood as local magnetic fields. Other parasitic contrast mechanisms do exist, but they are rarely dominant over magnetic or electric field sensitivity.

A subsequent 2017 multicenter RCT of 100 patients in the same groups also found similar pancreatitis rates but a 29.3% higher cost in the pancreatic duct stenting group, suggesting that early precut may be more cost-effective. The relationship between endoscopist experience and the success rates of pre-cut techniques were explored in six studies. Three found a positive correlation between cannulation success rates and endoscopist experience while the other three did not . In the meta-analysis by Sundaralingam et al, it was noted that only studies that involved expert endoscopists and not trainees showed a significant reduction in the risk of pancreatitis in the precut group compared with standard technique. Taken together, these studies suggest that in the hands of experienced endoscopists, early pre-cut techniques may facilitate SBC with lower PEP rates when compared to repeated attempts at standard cannulation. The TPS technique warrants additional discussion as this technique was not specifically evaluated in the studies included in the meta-analysis mentioned above. Through 5 studies, biliary cannulation success rates using TPS ranged from 85% to 100% and adverse events ranging from 3.5% to 20.5% and PEP rates ranging from 3.5% to 22.4% of cases. More recently, a meta-analysis comparing TPS to needle knife PP found significantly a lower success rate and a higher rate of bleeding complication for the needle-knife group but no difference in PEP, perforation, or total complication rates when compared to TPS. The authors conclude that TPS may have a higher SBC success rate with less bleeding but does not reduce the risk of PEP.Another type of rendezvous technique is the percutaneous rendezvous technique , pots with drainage holes in which access to the bile duct is achieved percutaneously , after which a guide wire is threaded anterograde through the needle into the bile duct and out through papilla.

This technique has been used in cases of difficult anatomy, e.g., patients with large, infiltrative tumors involving the papilla or cases of post-operative anatomy, such as Roux-en-Y anastomosis, Billroth II gastrectomy, where the location of the papilla may be difficult to access conventionally or via EUS. Case reports combining the Perc-RV technique with the placement of a hydrophilic guiding angiocatheter at the papilla to facilitate entry of the ST and/or percutaneous balloon dilation of the papilla via the anterograde direction prior to SBC have also been described. A retrospective analysis that looked at 24 patients with SBC failure due to many reasons found a 96% success rate with the perc-RV technique with a 2.4% complication rate . Perc-RV involves increased technical difficulty and training required to obtain percutaneous access to the biliaryducts, however, it allows a percutaneous transhepatic biliary drain to be inserted to alleviate biliary obstruction if other methods of obtaining biliary access fails . There are other methods of performing rendezvous-based SBC such as enteroscopy-assisted EUS or Perc-RV techniques for Roux-en-Y or Billroth II anatomy or intraoperative rendezvous technique where a guide wire is surgically inserted into the biliary ducts. These techniques are beyond the scope of this review, are generally reserved for advanced cases, and are typically performed in specialized endoscopy centers by endoscopists who have had extensive training and experience with difficult cannulation.ERCP has rapidly grown in its therapeutic abilities since its advent 50 years ago. One aspect that has stayed constant that both the first endoscopist to successfully cannulate the papilla, and most endoscopists today, would likely both agree is that successful ERCP hinges on adequate training, careful preparation in selecting the right patient for procedure, ensuring proper positioning and deciding initial techniques. Even with the ideal patient, up to 20% of SBC still fail when using conventional methods of contrast-assisted or WGC and are labelled difficult cannulation.

Although definitions vary, cannulation attempts of a duration over 5-10 min, over 5 attempts, or more than one unintentional MPD cannulation or opacification are the most widely used definitions for difficult cannulation. In this review, we have described steps to be considered and employed when difficult SBC is encountered. If the MPD has been cannulated, then a PD stent or CBD cannulation can be considered to reduce the risk of PEP. When the anatomy of the papilla is unfavorable or a difficult cannulation is anticipated, endoscopists can choose to perform a variety of early precut or rendezvous techniques, the choice of which depends on the experience of the endoscopist, disease being treated, and anatomy of the patient. The decision of which technique to pursue when difficult cannulation is encountered should include consideration of endoscopist experience, patient anatomy, and the disorder being treated, among other factors.Bunch grapes , notably European , are considered among the major fruit crops worldwide, producing roughly 70–80 million tons each year . Cultivars of V. vinifera L. are used for wine, juice, and table grape production. Grape berries are classified as nonclimacteric fruits, exhibiting a double-sigmoid developmental pattern with two rapid growth phases: the berry formation and the ripening phase , separated by an intermediate lag phase called the green plateau . The exponential increase in berry size characterizes both growth stages , but not the lag one . During phases and , also known as immature stages, organic acids, mainly tartrate and malate, accumulate leading to induction of acidity levels . At the end of the lag phase, a step-change point takes place known as veraison, where acidity starts to decline while sugars, mostly glucose and fructose, as well as anthocyanins in colored varieties, increase. Of particular interest are phenolic compounds, which are major and ubiquitous plant secondary metabolites derived from the shikimate/phenylpropanoid and polyketide pathways, with three utmost categories: proanthocyanidins , also known as condensed tannins, the gallo- and ellagitannins , and the phlorotannins . Such diversity of polyphenols, with more than 8000 structural variants, bestows them a wide range of biological functions ranging from growth, development, and protection inside the plant to, to some extent, human-related issues . In grapevines, the accumulation pattern of phenolic compounds, along with the aforementioned berry attributes, distinguishes each of the berry phases throughout berry development . Indeed, berry quality and sensory characteristics are notably defined by its polyphenol content . Remarkably, astringency is among the hardest sensory traits to depict and interpret as many intricate processes underpinning its perception . For instance, a sensory characterisation of the astringency of 11 varietals of Italian red wine revealed that neither total phenols nor PAs can predict how all astringency subtleties will be perceived . It is worth noting that the amounts, compositions, and proportions of polyphenols in a given species may vary widely depending on several factors, such as genotypic variations, developmental stages, and environmental circumstances . Scarlet Royal is a mid-season ripening table grape variety, producing seedless, red-skinned, oval-shaped, firm, and moderate to large berries with a sweet to neutral flavor .

In the San Joaquin Valley, California, it typically ripens in mid to late August, filling the harvest window between Flame Seedless and Crimson Seedless, and has thus become a very popular red table grape variety in California. However, an undesirable astringent taste has been observed occasionally in some cases. In fact, the economic value of grapevines depends substantially on the environmental conditions, including climate, soil, cultural practices, cultivar, and rootstock. Hence, the term “terroir” is used in viticulture to describe the effect of such an interactive ecosystem on grapevine and wine quality . The current study aimed to understand the underlying mechanism of astringency development in Scarlet Royal berries at two contrasting vineyards . The first location produces well-colored, non-astringent berries; however, drainage pot the second site yields astringent taste, poorly colored berries . The data showed a large variation in berry astringency within the same vineyard and from year to year. The data illustrated that the divergence in berry astringency stemmed from alterations in its polyphenol composition , most notably tannins. Additionally, the ripening stage was the most distinguishing platform for such variation between both vineyards. We were able to determine the tannins’ threshold level that causes the Scarlet Royal astringency taste to be ~ 400 mg/L. Given the changes in the levels of polyphenols during berry ripening, the question was raised: what is the mechanism governing the distinctive tannins accumulation pattern between V7-berries and V9-berries, and hence astringency diversity? To answer this question, RNA-seq data generated at one ripening time point was associated to the changes in polyphenolic levels using a systems biology approach, WGCNA . The module-trait association analysis positively correlated the key flavonoid/PAs biosynthetic genes with the accumulation of tannins, catechin, and quercetin glycosides exclusively in V9-berries. The modulation of the berry’s transcriptomic profile is concomitant with its polyphenols’ composition, which finally disturbs berry quality, including astringency levels.Five-year-old V. vinifera cv. Scarlet Royal grafted on Freedom rootstock was chosen for its berry astringency diversity at two commercial vineyards located in Delano, San Joaquin Valley, California, USA. Vineyards were located at a close distance of 10 km, and the local weather conditions during the two seasons were collected from the Delano CIMIS weather station . Both vineyards were planted at the spacing of 2.44 and 3.66 m in an open gable trellis supporting system with East-West row orientation. Vines were pruned in a Quadrilateral cordon training with 7–8 spurs left on each cordon during the winter pruning. In addition, general UC guidelines practices were applied in both vineyard. Starting from veraison and until the end of the season, during two consecutive years . During the first year, sampling dates were July 8th , August 1st , August 10th , September 9th , September 15th , and October 19th ; and for the second year, sampling dates were: July 15th , August 10th , August 25th , September 10th , September 29th , and October 21st . Sampling dates varied from the first to the second year due to the vineyard’s accessibility. At each sampling point, two sets of fifty berries were collected periodically. The first set was used to measure the berry weight, and then these berries were macerated in an electric blender, filtered through a paper towel, and an aliquot of juice was used to determine soluble solids , pH, and titratable acidity . Soluble solids were determined using a tabletop Milwaukee MA871-BOX digital refractometer . The TA and pH were determined by titrating a 40 mL aliquot of juice with 0.1 N NaOH to a pH of 8.2 using an automatic titrator Excellence T5 . Another random 50 berries from each replicate were collected for color, tannins, and phenolic compounds and sent immediately in a cooler to EST laboratories. At harvest, which was during the month of September, an extra set of samples was collected and promptly frozen in liquid nitrogen and stored at −80°C for subsequent analysis, including RNA extraction and gene expression studies. Harvest time was determined by the growers, and the marketable clusters were picked based on the color, and yield was determined from the three harvest dates.At bloom, fifty leaves from each replicate were collected, resulting in a total of 200 leaves from each vineyard, for nutrient analysis. The leaf positioned at the front of the cluster was specifically selected, and the petiole was immediately separated from the blade. The petioles were transported to the laboratory, where they were triple-washed with distilled water to remove any impurities before being sent to a private laboratory for nutrient analysis. In the winter, soil samples were collected at a depth of 30 cm and at a distance of 30 cm from the vine. These samples were transported immediately to the laboratory for analysis. The nutrient content was determined using the methods described in US Salinity Laboratory Staff .The taste panel evaluation of Scarlet Royal table grapes was conducted with the participation of twelve nonprofessional panelists. Astringent taste perception was assessed using a scale ranging from one, representing an extremely low level of astringency, to seven, indicating an extremely high level of astringency. The taste evaluation was performed on 24 clusters from each vineyard. Phenolic compounds analysis. Total phenolic analysis was performed on 250 grams of whole berries by ETS laboratory using a reversed-phase HPLC method adapted from Price et al. .Total RNA was isolated from whole berry samples following the protocol described by Boss et al. . To remove any residual DNA, RNase-free RQI treatment was performed according to the manufacturer’s instructions , and the samples were further purified using the RNeasy mini kit .

Elucidating the process by which family members managed growing incompetence sheds light on the important role of awareness perception in household dementia care. Currently, there exists an extensive literature examining clinical dimensions of declining deficit awareness or anosognosia, including its detection and measurement, especially for diagnostic purposes , cognitive correlates and the nature of its expression, including its fluctuations and effects on self . Despite these research gains, there are currently few studies that address the impact that perceived anosognosia has on family care giving and none that elucidate the social process by which it occurs. Related work, for instance, shows that anosognosia is associated with increased caregiver burden . The current study extends this area of research by showing the central role that lay perceptions of awareness play in efforts to manage growing incompetence in everyday activities. It explains the social mechanism by which the perception of declining awareness transforms care from a collaborative pursuit with the declining individual into a unilateral pursuit utilizing an array of tricks and ruses to influence the individual’s activity choices. The study’s naturalistic design helps show the importance of the interpretive process in assessing the household impact of a clinically-defined symptom, suggesting one reason why the clinical presence of anosognosia would not always translate into caregiver burden. This study’s insight about the interpretive challenges that families encounter around deficit awareness holds important epistemological implications for a common clinical approach to identifying and measuring anosognosia. The approach measures deficit awareness using discrepancy scores generated by comparing the elder’s self-rated scores on daily functional abilities with the scores rated by a caregiver . In this approach, vertical gardening in greenhouse the caregiver’s rating is treated as an accurate assessment and its discrepancy with the elder’s score reveals to what extent he or she is not aware of his or her own ability decline.

The current study however, suggests that there can be problems with treating the caregiver’s report as fully reliable. The findings show that caregivers may sometimes have difficulty grasping an individual’s level of decline. First, family members themselves may suffer from a kind of courtesy anosognosia, a state in which they are unwilling or unable to admit to the extent of a loved one’s decline. Multiple family members in this study, for instance, reported that the individual’s decline was denied by others in the household when met with arguments or plausible evidence to the contrary. Second, family members also signaled interpretive troubles by describing moments when elders employed a likely cover-up tactic to hide small errors in ability. They attempted to hide errors by taking issue with or showing nonchalance around what others defined as a “correct” procedure for accomplishing a task. In this way, affected individuals attempted to resist or redefine normative measures of successful activity engagement. One family member, for instance, suspected this maneuvering when her mom served her dad cold soup and claimed that he liked it that way. Another participant reported her dad’s nonchalance or apparent lack of care after she pointed out that he did not have to have coffee with the grounds in the coffee cup.Facing a disease that is deeply stigmatized, individuals with Alzheimer’s have social and psychological reasons as to why they would hide their awareness of errors in ability from others . Efforts taken to avoid acknowledging the disease’s effects, for instance, may signal the elder’s effort to protect him or herself from social and/or psychological injury . While these interpretive obstacles pose challenges to using caregiver reports of ability level, this study suggests that to better understand the interpretive challenges that observers face and thus the reliability of observer reports, researchers must better understand the social process in which the interpretations are made. These findings call for more research around the interpretive challenges that proxy raters may encounter. While researchers have highlighted the issue of duplicity in care giving and the kinds of ethical dilemmas it can create , the current study identifies the turning point in the social process of care when family members began using duplicitous techniques.

The evidence shows that they began using these techniques as a self-conscious strategy when the elder’s awareness ability was thought to have declined severely enough to make transparent negotiation difficult. At this point in the progression of declining deficit awareness, family members found that elders were less capable of heeding their warnings and following their instructions for avoiding activities that had become too dangerous to pursue independently. At this stage, family members resorted to techniques that function as ruses to fool or trick elders away from risky activities. This study has two important limitations. First, the participant sample is almost entirely made up of adult-children. It is likely that adult-children will experience a more protracted and potentially more risky transition period between collaborative and unilateral autonomy management work in contrast to spousal caregivers. Adult-children commonly must make decisions in concert with other siblings or family members. Negotiating with multiple members may raise the likelihood of disagreement and conflict that can delay a decision to curtail an elder’s independence when it is urgently needed. In addition, spouse caregivers likely will have spent many more years in close proximity with the affected individual and be better suited to notice minute, yet critical changes in the individual’s awareness level that can lead to timely changes in their autonomy management practices. Second, the participant sample only includes family members dealing with individuals who have one type of dementia: Alzheimer’s. While there are important differences in symptom behaviors between dementia sub-types , we believe that these results would extend to families dealing with vascular, Lewy body, and frontotemporal dementias. Future research can test the extent to which features of the autonomy management process identified within the Alzheimer’s context can effectively explain features of the support process around other forms of dementia. This study reveals the social contextual factors that influence how family members identify and respond to growing signs of risk in an elder’s decades-old activities. Across theautonomy management process, family members attempted to balance their concerns of reducing risk with their concerns of permitting enough autonomy to promote wellbeing. In limiting an elder’s activities too much, for instance, they feared they could reduce his or her chances of finding spontaneous sources of meaningful engagement and potentially cause friction within their relationship. Yet in limiting their activities too conservatively, family members feared they could inadvertently permit elders to injure themselves and possibly others. Autonomy management provides a useful conceptual framework for understanding how family members adjusted their measures to deal with this dynamic as the disease advanced.The fresh market berry industry in Santa Cruz and Monterey counties is an excellent example of transformation in the business of agriculture over the last 50 years. Located along the Central Coast of California, the two counties span the fertile Pajaro and Salinas valleys, and are well known for their amenable climate and production conditions, their diverse crop mix and grower demographics, and their developed agricultural infrastructure and support industries. The majority of the berry sector is comprised of strawberries , raspberries and blackberries , with blueberries and other miscellaneous berries produced on a much more limited basis. Substantial research-based literature and historical information is available for Central Coast strawberries; however, despite the area’s move towards greater production of raspberries and blackberries, less information exists for these crops. We seek here to provide a more complete portrayal and historical context for the berry industry in the Santa Cruz and Monterey area, greenhouse vertical farming which is the origin of the berry industry in California. While the berry industry has been very successful in recent decades, it now faces new challenges, such as invasive pests and the phaseout of the soil fumigant methyl bromide. This article draws on previous and more recent research to discuss some of the influences that have contributed to the berry industry’s dramatic expansion in Santa Cruz and Monterey counties, including selected innovations in agricultural practices and heightened consumer demand.During the 1960s and 1970s, the number of acres planted to berries, tons produced and value of production fluctuated.

The fluctuations can be partly explained by farm management: in the past growers often rotated berry and vegetable crops to assist with soil and pest management, thereby influencing these statistics. However, annual crop reports from the county agricultural commissioners show that since the 1980s, berries have become increasingly important to each county’s overall value of production, and by 2014 accounted for 64% and 17% of the total value of all agricultural products in Santa Cruz and Monterey counties, respectively . The industry’s growth can be explained by a shift of some acreage out of tree fruits and field crops , among others, into berries, and by additional acreage put into agricultural production.Strawberries are the undisputed leader in the berry sector and in 2014 represented 58% and 94% of the value of all berry production in Santa Cruz and Monterey counties, respectively , and 50% and 93% of all berry acreage . Table 2 documents the remarkable expansion of the strawberry industry over time in both counties with respect to acreage, tons produced and value of production. Between 1960 and 2014, acreage more than tripled and production increased tenfold. The value of production, in real dollars, increased by 424% in Monterey County and by 593% in Santa Cruz County, reaching an astonishing combined value of nearly $1 billion in both 2010 and 2014. The gains in all statistical categories in Monterey County were enabled in part by an expansion of production into the southern reaches of the county where more and larger blocks of farmland are available, and where land rents are lower than in Santa Cruz and northern Monterey counties. However, from 2010 to 2014 Monterey County’s tonnage and production values declined, possibly because the area has recently experienced a shortage of labor to harvest fresh market crops. Tonnage was also lower in Santa Cruz County, but production values increased. This may be because of the county’s greater emphasis on local agriculture, organic production and direct market sales, which are often associated with higher crop values. For raspberries, the acreage, tons produced and value of production grew steadily and most strikingly in Santa Cruz County , where production conditions for caneberries are optimal. For example, caneberry fields in Santa Cruz County are situated in areas that have well-drained soils and are protected from damaging winds. Also, fields are planted to take advantage of the growth and yield gains associated with southern exposures. Moreover, field-to-cooler travel distances are shorter in Santa Cruz County, which is critical for safeguarding the quality and marketability of these highly perishable crops. By 2014, raspberries represented 33% of the county’s total value of production for all berries. In contrast, Monterey County raspberry production accounted for only 6% of the county’s total berry value. Blackberries have not been consistently reported as a separate category in archived statistical analyses, but instead were often included under the terms “bush- or miscellaneous berries”. Therefore, similar data for blackberry acreage and value of production cannot be reported here. However, between 1990 and 2010, Santa Cruz County agricultural commissioner crop reports reported an upward trend for the broad category with respect to acreage planted and value of production . In 2010, blackberries were promoted to a position of prominence in the report and shown as a separate statistic; at the same time, the miscellaneous berry category was shown to be very small indeed. Between 2010 and 2014, however, blackberry acreage and value of production leveled off and have shown only modest gains . This may be because there has been less emphasis on production and market research and promotion for blackberries than for strawberries or raspberries. No comparable data are available for Monterey County. The two counties have contributed significantly to California’s total berry sector: in 2014, area strawberry acreage represented 35% of the statewide total, 37% of the total tons produced and 38% of the total value of production . Area raspberry acreage represented 43% of the statewide total, 42% of the total tons produced and 39% of the total value of production. Comparable statewide statistics are not available for blackberries. County agricultural commissioners’ reports show that the majority of all berries produced in the two counties — up to 98% — are sold as fresh market fruit .

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

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

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

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