Month: November 2024

The four others were an autophagy gene and constitutively activated cell death 1 , which function in autophagy, lytic pore formation, and HRs , Kinesin-like 5C , which encodes a microtubule motor protein , and an ARF-GAP encoding ADP-ribosylation factor GTPase-activating protein domain 15, which helps efficiently load vesicles and remodel the actin cytoskeleton . Several additional general functional categories were present among the 32 genes that exhibited conserved responses to GLRaVs . Genes encoding phenylalanine ammonia-lyase and cinnamate 4-hydroxylase , which catalyse the first two steps of the phenylpropanoid pathway, two genes encoding UDP glucosyltransferases , which conjugate sugars, and SWEET17, encoding a sugar transporter, were upregulated, as was a gene encoding 3-isopropylmalate dehydratase, an enzyme in the leucine biosynthetic pathway. Two genes, encoding an LRR receptor-like kinase called ERECTA and nicotianamine synthase , were downregulated. ERECTA participates in organ development and resistance to bacterial and fungal pathogens . NAS expression increases Fe and Zn abundance in rice . Generally, these genes and their changes in expression suggest that a common response to GLRaVs in Cabernet Franc berries during ripening includes the modulation of pathogen-detecting genes, an increase in ABA transport and signalling, a decrease in ROS-related signalling, and an enhancement of cytoskeleton remodelling, vesicle trafficking, phenylpropanoid metabolism, sugar transport and conjugation, and leucine biosynthesis.The same berry samples used for RNA-Seq were used to measure the levels of three hormones associated with ripening and/or stress, including SA, JA, and ABA, and additional metabolites, square plastic plant pot including xanthoxin, a precursor to ABA, and ABA glucose ester , a conjugate of ABA implicated in its long-distance transport .

The mean levels of SA and JA were significantly influenced by year and/or by interactions between year, rootstock, and GLRaV at prevéraison , but no significant differences were observed between individual groups . In contrast, year alone had a significant impact on the levels of ABA and related metabolites measured at each developmental stage, but largely did not interact with rootstock or GLRaV infection type to affect the abundance of ABA and related metabolites . In addition, the effect of GLRaV infection on ABA and ABA-GE content significantly differed based on rootstock . Significant differences in ABA and ABA-GE content were observed between rootstocks in plants with identical infection status and between plants with different GLRaV status grafted to the same rootstock . Such differences were scarcely observed for xanthoxin, a precursor to ABA . Significant differences between rootstock genotypes in the abundance of these metabolites were observed most at prevéraison and in GLRaV , GLRaV-3 , and dual infections . In GLRaV and most single infection conditions, the levels of all three metabolites tended to be higher in berries from plants grafted to MGT 101-14 than in berries from plants grafted to Kober 5BB. The opposite tended to be true when two GLRaVs were present. With one exception, significant changes in the abundance of ABA and ABA-GE in GLRaV versus GLRaV were typicallyto cluster separately from other Kober 5BB-grafted plants. Nonetheless, the effects of GLRaVs differed between rootstocks for many of these genes . One of these, encoding an ABC transporter , is also included in Figure 3; significant increases in its expression were observed in both years, in both rootstock conditions, and for several GLRaV infections. All other significant changes in GLRaV versus GLRaV that were reproduced in both years occurred in only one rootstock or the other.

These changes were sparse. However, significant differences between rootstocks in identical GLRaV were reproduced in both years for 9 out of these 19 genes. Significant differences between rootstocks in at least one year were observed for 16 out of these 19 genes. On average, three genes encoding 9-cis-epoxycarotenoid dioxygenases , both ABA 8′-hydroxylase genes, three ABC transporter genes, one gene encoding PP2C, and PYL/RCAR were upregulated in berries from plants infected with GLRaV in both rootstock conditions. One ABC transporter gene was downregulated in both rootstock conditions. Of the remaining eight genes, most were downregulated across development only in berries from MGT 101-14-grafted plants.Differential expression analysis identified 1,809 genes that were differentially expressed in at least 1 year, that were differentially expressed in only one rootstock condition and more than one GLRaV infection type versus GLRaV , and/or for which the effects of more than one GLRaV infection significantly differed between rootstocks . RNA-Seq, hormone, and metabolite data from ripening Cabernet Franc berries were integrated in a multiple factor analysis to relate these variables and distinguish the effects of GLRaVs given different rootstocks . As input for the MFA, all genes differentially expressed between GLRaV and GLRaV or between rootstocks were used, plus all hormones and metabolites measured. Overall, the rootstocks were distinct at each developmental stage . Some of the GLRaV conditions could be distinguished from others at prevéraison, véraison, and harvest. At prevéraison, GLRaV-1,2 differed overall from GLRaV-1 . At véraison, GLRaV-1,3 differed from every other GLRaV condition except GLRaV-1,2 . At harvest, the two dual infections were different than one another and GLRaV-1,2 differed from GLRaV-1 . Next, we identified which variables were best correlated with each rootstock-differentiating MFA dimension. At eachdevelopmental stage, ABA and/or ABA-related metabolites were correlated with at least one of the first two MFA dimensions .

The rootstock-dependent disparity in ABA levels and ABA-related gene expression is consistent with the observation that ABA and related metabolites tended to be highly correlated with rootstock-differentiating MFA dimensions over time and that ripening initiates earliest in Kober 5BB plants with dual infections in terms of TSS . There were 548 genes that shared high correlation to rootstock-differentiating MFA dimensions with hormones or hormone-related metabolites. Most of these genes had shared positive or negative correlations to the same dimensions as ABA, xanthoxin, and/or ABA-GE . Categories of genes with functionally relevant relationships to each hormone or hormone related metabolite were over-represented among the genes that shared high correlation to rootstock-differentiating MFA dimensions with each hormone. ABA signalling, starch biosynthesis and catabolism, and C2C2-DOF transcription factor-encoding genes were over-represented among the genes correlated to the same dimensions as ABA and xanthoxin. The latter two categories were significantly over-represented among the genes correlated with the same dimensions as ABA-GE. Genes related to heat shock protein -mediated protein folding, chaperone-mediated protein folding, the cation channel-forming HSP-70, channels and pores, the reductive carboxylate cycle, and carbon fixation were over-represented among those correlated with the same MFA dimensions as SA. Similarly, most ripening-related metabolites measured were correlated with the same MFA dimensions as ABA, xanthoxin, and/or ABA-GE . Overall, the effects of GLRaVs differ between rootstocks primarily in terms of ABA and related metabolites. This finding is especially salient because of the role that ABA plays as a ripening promoternear véraison, in root–scion communication, and in plant stress. ABA, metabolites, and genes that were well correlated to rootstock differentiating MFA dimensions and were differentially expressed were scrutinized more closely.There were 548 genes in 85 functional categories that were well correlated with rootstock-differentiating MFA dimensions and differentially expressed between GLRaV grafted to different rootstocks or in GLRaV versus GLRaV in only one rootstock condition . These functional categories were generally related to hormone and other types of signalling, amino acid and other metabolic pathways, transcription factors, transport, and cellular organization and bio-genesis . Most of these genes coincided with ABA and related metabolites along rootstock-differentiating MFA dimensions . The distribution of expression for four transcription factor families differed significantly between rootstocks at all four developmental stages . This included bHLH, C2C2-DOF, FHA, 25 liter sqaure pot and homeobox domain transcription factors. The distribution of expression of 39 hormone signalling related genes differed significantly between rootstocks . This was true at each developmental stage for ABA, gibberellin , and auxin signalling genes and at three developmental stages for JA/SA, cytokinin , and ethylene signalling genes . Genes related to all of these hormone families had similar roles in MFA and were associated with ABA, including the JA/SA signalling genes .

This may reflect interactions between hormone signalling pathways. In addition, the effects of GLRaV infections on histone H1 expression were not equal in plants grafted to both rootstocks . Linker histone H1 contributes to higherorder chromatin structure . There were seven ABA signalling pathway genes that differentiated GLRaV effects in plants grafted to different rootstocks . This included SOS2, KEG, three PP2C genes , and two genes encoding ABA-responsive element -binding proteins . SOS2 is a kinase appreciated for its role in the salt stress response, seed germination, GA signalling , and ABA signal transduction via its interaction with ABI2 and ABI5 . SOS2 was upregulated in both rootstock conditions before and at véraison and downregulated after véraison. KEG is a negative regulator of ABA signalling; it maintains low levels of ABI5 in the absence of stress by ubiquitination and degradation and helps regulate endocytic trafficking and the formation of signalling complexes on vesicles during stress . KEG was downregulated in Kober 5BB and downregulated in MGT 101-14 at véraison and mid-ripening. In the presence of ABA, ABA receptors bind PP2Cs like HAB1 and AHG3 to inhibit their phosphatase activity. As a result, ABA signal transduction is permitted via SnRK2 phosphorylation of ABRE-binding proteins . ABI5 and AREB2 are bZIP transcription factors that bind to ABREs to drive ABA signalling and ABI5 can integrate signals across hormone signalling pathways . The effects of GLRaVs on these genes in Kober 5BB were consistent with an enhancement of ABA signalling during ripening. In Kober 5BB, HAB1, AHG3, AREB2, and ABI5 were upregulated. In MGT 101-14, the PP2Cs were downregulated at two or more developmental stages; AREB2 and ABI5 were upregulated at and after véraison.In addition to analysing hormones and hormone-related metabolites, we analysed metabolites associated with the shikimate, phenylpropanoid, and flavonoid pathways and their biosynthetic and regulatory genes in Cabernet Franc berries during ripening . Significant differences in expression versus GLRaV were detected, as well as significant differences in the effects of GLRaV infection between different rootstock conditions . The effects of GLRaV infection on the genes associated with this pathway were generally consistent with the change in abundance of corresponding metabolites . Overall, these genes tended to be upregulated in GLRaV at véraison . After véraison, the amount of upregulation tended to decrease, or genes were downregulated . The three amino acids examined tended to be less abundant in GLRaV across the developmental stages and the largest decreases were observed at harvest . Mixed effects of GLRaVs were observed on the abundance of hydroxycinnamic acids , t-resveratrol, and anthocyanins. Significant changes versus GLRaV tended to occur in only 1 year. During ripening, these were significantly more abundant in Kober 5BB GLRaV-1,2 , Kober 5BB GLRaV-4 , and/or MGT 101-13 GLRaV-3 . Significant decreases were observed for GLRaV-1 and GLRaV-1,3 . Though nonsignificant, the size of the downward effect of some GLRaV infections on these metabolites tended to increase towards harvest. Finally, flavanols and flavonol glycosides tended to be elevated in GLRaV . The size of this effect tended to be greatest before and at véraison and decreased towards harvest. Significant differences between rootstocks were observed for GLRaV-1,2 in both years and for GLRaV-1 , GLRaV-1,3 , and GLRaV-3 in individual years;the increase in flavonols and flavanols tended to be greater in berries from Kober 5BB GLRaV than in those from MGT 101- 14 GLRaV .GLRaVs affect viticulture on nearly every continent and can a have considerable economic impact on a major crop. The presence and severity of symptoms in GLRaV infected grapevines is influenced by host genotype, rootstock, which GLRaV is present, and environmental conditions. In addition to the assembly and annotation of the Cabernet Franc genome, a valuable resource that might be applied for the larger purpose of understanding grapevine genomic diversity and evolution, the dedicated experimental vineyard used in this study is a tremendous asset for the study of GLRaV infections over time in a common environment. This work identified responses to GLRaVs in grape berries during ripening, including those that are conserved across experimental conditions and responses that differ based on the rootstock present. We propose which hormones and signalling pathways at least partially govern the responses observed and likely influence leafroll disease symptoms. The effects of dual infections, particularly GLRaV-1,2 , were most distinctive.

The same procedure executed for the 2018 cultivar experiment was used to calculate and re-inoculate the pots. Field grafting experiment. Field experiments were performed in a commercially-owned field south of Bakersfield, Kern County, CA. The field has historically been under consistent tomato production and typically experiences southern blight. The soil was a sandy clay loam with a pH of 6.37 and 2.19% organic matter. In 2017, the preliminary field experiment evaluated two cultivars under two graft treatments that were mechanically transplanted on May 15, 2017. Plots were 165 m long with 30.4 cm plant spacing and were arranged in a randomized complete block with 7 replications. Plants were irrigated with a buried drip system at a depth of 26 cm. The field experiment in 2018 and 2019 evaluated the same treatment structure as the 2018 and 2019 greenhouse experiments. The field experiments consisted of treatments arranged in a randomized complete block design with 6 replications with plots that measured 34 m long in 2018 and 30.5 m long in 2019. In both the 2018 and 2019 field trials, the plants were mechanically transplanted at a spacing of 60.9 cm in single row beds. Transplants were established with a towed water tank or sprinklers , then irrigation was switched to drip. Although not located within an active production field, the experiments were maintained by the commercial grower using standard practices for processing tomato in the southern San Joaquin Valley. Data Collection. In both the cultivar and grafting greenhouse experiments, Southern blight severity was rated using the following 0 to 7 ordinal rating scale: 0 = no disease symptoms; 1 = chlorosis of the older leaflets; 2 = wilting of the older chlorotic leaflets; 3 = wilting of the older leaves with a wilted apex; 4 = necrotic older leaflets with a wilted apex, black plastic plant pots and apex leaflets showing chlorosis; 5 = all leaflets are dry; 6 = all leaflets are wilted and dry with a chlorotic stem; and 7 = a dead plant that is completely wilted and dry .

Data was collected weekly after southern blight symptoms began to develop for the greenhouse cultivar experiments in 2018 and 2019. For the grafting greenhouse experiments, disease severity was rated every 2 weeks after southern blight symptoms began to develop for the 2017 and weekly for the greenhouse graft study in 2018 and 2019. For the field trials of objective , in 2017 data was collected weekly beginning six weeks after planting. Strike counts, defined as plants observable as infected or not infected, were collected from four 15.2 m sections per plot in the 2017 field trial. In the 2018 and 2019 trials, data collection began two and five weeks after transplanting after transplanting, respectively, and approximately every one to two weeks thereafter. In these trials, the status of each plant was individually recorded on each rating date. Plants that were wilting, collapsed, and lime-colored were rated as exhibiting southern blight symptoms . Other diseases were also observed in these trials. Plants with crisp leaves that roll or curl upwards with or without appearing stunted were rated as symptomatic of curly top, and plants with crinkled leaves having interveinal yellowing and typically with stunted growth were rated as symptomatic of unknown virus. Plants completely brown and dry were rated as dead. When a dead plant was observed, it was marked with a flag to ensure it would be counted on subsequent rating dates. Yield data was collected from 165 m long plots from the 2017 field trial on September 18, 2017. Yield data was not collected in 2018 due to quick collapse of plants ending in poor fruit quality for harvest. Yield data was not collected in 2019. Data Analysis. For the 2018 and 2019 cultivar and grafting trials in the greenhouse, the influence of experimental factors on southern blight severity was analyzed with generalized linear mixed models with PROC GLIMMIX in SAS 9.4 using the multinomial distribution and the cumulative logit link function.

The cultivar trial was analyzed as a nested model, with inoculum as a main effect and inoculum × cultivar as an interaction effect, because only a small set of the cultivars were evaluated in noninoculated control plots. The grafting trial was analyzed as a factorial. For both trials, rating date was included separately as an additional main effect and not included as an interaction to reduce complexity of attempting to model ordinal data. Block was included as a random effect for both trials. When interactions were significant, the effect of cultivar within inoculum and the effect of graft within cultivar were examined with the slice statement for the cultivar and grafting trials, respectively. Levels of significant main effects or interactions were separated by obtaining odds ratios for all pairwise comparisons with the model statement. Due to the large number of treatments in the cultivar study, odds ratios were summarized with the lsmeans statement in PROC PLM with the Tukey-Kramer adjustment for multiple comparisons. In the cultivar trials, the effect of cultivar among inoculated plants was analyzed using a dataset with noninoculated cultivars removed because odds ratios cannot be determined for interaction terms. Initial analysis of the greenhouse grafting trials did not detect statistical evidence for an effect of inoculum despite a total lack of symptoms in control pots, therefore all non-inoculated pots were removed for analysis. In addition, initial analysis of the 2019 cultivar trial did not find statistical evidence for separation of cultivars despite clear variation in the raw data. Therefore, three cultivars which possessed all 0 ratings on all dates were excluded from analysis. For the 2018 and 2019 field trials, individual plant status data was first subjected to quality control. In some cases, the same plant was rated with more than one disease over the course of each trial. This was generally due to lack of clarity of the symptoms when they are first observed or a secondary disease affecting plants following the first.

Quality control consisted of assigned the true or primary pathogen retroactively to all symptomatic ratings. Then, for dead plants, the cause of death was determined from ratings on previous dates when the plant was symptomatic but alive. Following quality control, ratings were summarized at the plot level. The total number of plants in each plot with a given rating was determined, and southern blight incidence was determined by adding the number of plants exhibiting southern blight symptoms and the number of plants dead due to southern blight. The influence of cultivar, graft, rating date, and all interactions on southern blight incidence in the 2018 and 2019 field trials was analyzed with a generalized linear mixed model in PROC GLIMMIX with the binomial distribution and the logit link function. Block was included as a random effect. The effect of graft within significant cultivar × graft interactions was examined with the slice statement. Means of significant main or sliced effects were separated using the least significant difference test with Tukey-Kramer adjustment for multiple comparisons with the lsmeans statement. For the 2017 field trial, yield and strike count data were analyzed with PROC GLIMMIX procedure in SAS v9.4 using the log normal and binomial distributions, respectively. The 2017 greenhouse experiments were analyzed as relative treatment effects with repeated measures using the nparLD package v2.1 in R v3.3.2 . An analysis of variance -type statistic was used to determine the effect of treatment, black plastic planting pots and means will be separated using 95% confidence intervals calculated from the nparLD package.Cultivar greenhouse experiment. By the end of the experiments, some inoculated plants from almost all cultivars had died from southern blight, but many plants did not develop any symptoms . The raw data showed that cultivars differed primarily in the number that did not develop any symptoms and that most died after exhibiting disease symptoms. One cultivar in 2018 and three in 2019 did not develop any symptoms. No symptoms were observed in any non-inoculated plants. In both trial years, analysis of fixed effects showed that the interaction of cultivar and inoculum had a significant effect on disease severity, and slicing these interactions showed inoculum of 10 sclerotia per 100 cm3 of soil had an effect on disease severity . There were few differences among cultivars in the multiple comparison analyses in both years, and relative differences among cultivars varied between years . In 2018, HZ 4707 had the lowest risk of developing disease, but was not different from SUN 6366, HZ 1428, and N 6428. In 2019, risk of both HZ 4707 and HZ 1428 was relatively low but was similar to several commercial cultivars and Texas A&M breeding lines. In contrast, N 6428 had the highest risk in 2019 but was not significantly different from six other cultivars. Cultivar N 6416 had the highest risk in 2018 and relatively high risk in 2019, but was not different from 9 or 12 other cultivars, respectively. Although Maxifort and Multifort were included as positive controls in 2018, their risk of developing southern blight was similar to all but 1 and 4 of the remaining cultivars,respectively. Of the Texas A&M breeding lines that were not excluded from analysis in 2019, 5635M and 5913M exhibited the least risk, but were not significantly different from the two remaining breeding lines and 8 commercial cultivars.

Grafting greenhouse experiments. The preliminary 2017 study showed under moderate inoculum pressure, disease severity was significantly higher in non-grafted HZ 5608 compared to HZ 5608 grafted to Maxifort, but was similar for H 8504 grafted and non-grafted . Disease severity was low in both 2018 and 2019 experiments. The Type III analysis of fixed effects detected a significant effect of grafting on disease severity in 2018 and 2019 . However, odds ratio estimates and confidence intervals of the pairwise comparisons control-standard and standard-tall were not sensical , and the control-tall comparison suggested that control had significantly greater odds to develop disease in 2018 but significantly lower odds in 2019. HZ 5608 had numerically higher incidence of southern blight compared to HZ 8504 for both replicate trials in inoculated pots across all grafted treatments , however a statistical effect of cultivar was not detected in either experiment. Field grafting experiment. Disease incidence was significantly lower on four of five rating dates in grafted plots compared to non-grafted in 2017 . On the final rating date, southern blight incidence was 52% and 58% lower in grafted compared to non-grafted plots. A significant effect of grafting was observed on yield, in which yield was 30.0% higher in grafted plots compared to non-grafted . In 2018 and 2019, disease severity was moderate to high . The Type III analysis of fixed effects on the 2018 field trial showed a significant interaction of cultivar and grafting on disease incidence, whereas in 2019 only the main effect of grafting was significant . For both cultivars in 2018 and in 2019, disease incidence was significantly lower in grafted plots regardless of height when compared to the non-grafted control. Mean incidence in non-grafted plots was approximately 7.5 and 11.5 times higher in 2018 and 2019, respectively, when averaged over cultivar and height of the graft union. Additionally, for HZ 8504 in 2018, incidence in tall grafted plots was significantly lower than incidence in standard plots . This numeric trend was also observed for HZ 5608 in 2018 and in 2019, but the difference was not significant.This study presents options for the management of southern blight of processing tomato in California. We found that grafting to resistant rootstocks dramatically reduced southern blight in processing tomato. Our finding agrees with previous literature on the benefit of grafting for management of southern blight and other diseases. In addition, our results suggest that raising the height of the graft union may reduce southern blight incidence. Finally, we observed variation in susceptibility to southern blight among commercial cultivars currently planted in California.While our findings in processing tomato agree with previous research in fresh market tomato, the utility of grafting to processing tomato production may be lower due to the relative costs and returns between the two systems. Although we did not perform a comprehensive economic analysis of production using grafted transplants, the current cost of F1 hybrid seed and the grafting operation exceeds returns under reasonable price and yield scenarios.

Maturation of coffee cherries is slow, with immature green cherries taking up to 240 days to develop into red, ripe fruit that is ready for harvest in mid-October through January . After harvest, coffee plants are left to recuperate until flowering is initiated again the following year by the next onset of rain.Following the coffee flowering period and initiation of cherry growth, adult female CBB emerge and disperse via flight in search of new cherries to colonize . Timing of emergence appears to be driven primarily by relative humidity and temperature, with dispersal peaks occurring around the end of the coffee harvest, from December through March . Females begin ovipositing in chambers carved out of the coffee endosperm roughly 120–150 days after coffee flowering, when the dry content of the seed is 20% or higher . It is this dispersal period, and subsequent drilling into the coffee cherry, when CBB are vulnerable to predation by birds, as the remainder of the CBB life cycle occurs within the coffee cherry. There are five main CBB developmental stages: egg, larva, pupa, juvenile, and adult. Females can oviposit daily for up to 40 days, averaging 1–2 eggs per day . After a week, eggs hatch and larva take 17 days to develop into pupa. Following pupation , juveniles emerge and reach sexual maturity after about 4 days . The length of the CBB life cycle can be slowed and accelerated depending on average temperature ; the developmental times used here are based on 25 C rearing conditions . Offspring sex ratio is skewed toward females, ranging from 1:5 to 1:494 . Since males are flightless, mating occurs between siblings within the natal cherry.

Fertilized females then disperse to colonize other cherries, though multi-generational oviposition within the natal cherry is possible. The prolonged maturation of the coffee crop allows continual reproduction, drainage planter pot with 2–8 CBB generations feasible in a single season if environmental conditions and food availability be favorable . With the removal of cherries during harvest, adult CBB will enter diapause in coffee cherries that remain on the plant or fall to the ground .Since birds do not eat coffee cherries, bio-control by birds would only occur during the brief dispersal period when CBB are vulnerable. There is a rich bird community during this period of time as both resident and migratory birds are present . Neotropical migrants are potentially more abundant on coffee farms than resident species that may prefer forest habitat due to higher prey abundances . Many migratory warbler species of the Setophaga genus that frequent coffee farms have been confirmed as CBB predators, as have resident bird species such as the rufous-capped warbler and common tody flycatcher  Overall, insectivorous birds are the most abundant on coffee farms and hold great potential as bio-control of many insect pests . Details on bird densities on Costa Rica coffee farms used in the model are expanded on below .We created a deterministic Leslie matrix for coffee berry borers with one-day time steps using data reported by Mariño et al. for an artificially infested coffee farm. Mariño et al. estimated the amount of time in each life stage , each of which had a narrow window, and calculated transition probabilities between stages. We converted each of the stage transition probabilities into daily transition probabilities as Gi 1 d, where d = the number of days in a life stage.

We assumed that mortality was evenly distributed across days within each life stage. Similarly, fecundity estimated by Mariño et al. for a seven-day period was converted to daily egg laying rate, assuming eggs are produced at a constant rate. To account for the female skewed sex ratio, the new daily Fi was multiplied by 0.9 to model a conservative 10:1 F:M sex ratio. Lab experiments show fecundity decreases when multiple ovipositing females cohabitate . However, it is rare to find a cherry bored by more than one female, likely due to the high abundance of coffee cherries in the field. Therefore, we assumed density-independent growth. Since all developmental stages of CBB occur within the coffee cherry and are assumed to be protected from predation by birds, we added a dispersal life stage to the population growth matrix, and limited bird-related mortality to this life-stage. The dispersal life stage includes the time a gravid adult female emerges from the natal cherry, disperses via flight, and the initial stages of boring into a new cherry to oviposit, while part of its body is still exposed, outside the cherry. Coffee berry borers are weak flyers and boring into the cherry and reaching the endosperm can take 2– 8 h . Consequently, we estimated the disperser life stage to last 1 day.To our knowledge, there is little information about population densities of CBB in coffee plantations at the start of the growing season. The start of CBB reproduction commenced 120 days after coffee flowering and continued until 305 days after flowering, yielding a 185-day CBB breeding season. We confirmed CBB reproduction was possible within this period for Central Valley Costa Rica using degree day calculations from Jaramillo et al. based on CBB thermal tolerance.

We then calculated how much the dispersing adult survival rate would have to be reduced to cause a 50% reduction in adult female borer population size on day 185. To determine how many CBB would need to be consumed by birds to achieve this goal, we found the difference between daily borer population sizes of unsuppressed and suppressed populations and summed the differences across the CBB reproductive season. We used sensitivity analysis to estimate the degree to which changes in each vital rate affects population growth rate . All models were implemented using the popbio package in R . R code for all analyses is provided in the Supporting Information . We also wanted our model to project CBB population growth that represented “low” and “high” infestations observed in the field. To start, we estimated probable CBB densities using data on the number of dispersing females collected in alcohol-lure traps. At peak dispersal, CBB numbers have been recorded as high as 1000–6120 CBB/trap/week to as low as 50–105 CBB/trap/week . Using these trap counts, we calculated potential CBB densities per hectare via reported trap densities and converted weekly capture estimates to the number of daily dispersers to complement our daily population model. We used a density independent model, a standard first step in many population models. However, note that we would need to divide CBB numbers by plant density to evaluate the impacts of CBB population growth on yield. We also would need empirical data on how the demography of CBB populations change with coffee-plant density to implement a revised model, and we are unaware of published data on this. Consequently, this analysis is beyond the scope of this paper . Using data from Aristizabal et al. , we selected a high peak dispersal count from farms with large infestations and a low peak dispersal count from farms with small infestations to represent peak dispersal on Day 185 in our model. We then back calculated the initial population sizes that would yield those ultimate densities. We used our calculated values of 269 and 5 as our “high” and “low” initial population sizes of gravid females at the start of the coffee season and used 100 CBB to represent “medium” initial population size.The mass, in dry weight, of a female adult CBB was determined from the weighted average of CBB using midpoint values for weight ranges from Moore et al. . We estimated the caloric content of a single CBB using the average energy value of Coleoptera species in the adult stage . Using our estimated CBB caloric content, plant pot with drainage we calculated the number of CBB required to make up 5% and 10% of an average bird’s daily diet . We calculated daily energy requirements for birds under field conditions as M =  2.5, where W is the weight of an average insectivorous bird on coffee farms . We calculated the weight of an average insectivorous bird by averaging body masses of 33 insectivorous resident and migrant bird species reported to consume CBB on Jamaican and Costa Rican coffee farms , or predicted to consume CBB based on morphology and diet breadth . Sherry et al. found that CBB made up 5%–10% of the diet of three Neotropical migratory warblers by number of individuals consumed; we used these percentages to estimate how many calories, and therefore how many CBB, birds potentially eat. Avian consumption rate of CBB was constant, with even effort across the coffee season. For avian densities, we used estimates from Karp et al. of 3 to 14 birds per ha, because these densities include known CBB predators on coffee farms in Costa Rica.Parameters for our Leslie matrix for coffee berry borers are broadly consistent with expectations and general knowledge . For example, our conversion of fecundity to a daily value, F1 = 1.341, is consistent with published literature stating that 1–2 eggs are laid per day by CBB .

Model projections showed that across a 185-day CBB breeding period starting at the point of first ovipositing, an initial population size of 100 female dispersers would produce 1.3 million offspring, resulting in a new adult population of 70,245 females . Assuming
99% of colonizing females successfully bore and oviposit in a coffee cherry on Day 0, the first generation of new dispersing females does not appear until day 37. At Day 38, the adult population begins to increase, and continues to do so exponentially.The daily growth rate of this population converged on 1.042. Sensitivity analysis revealed that survival of adult females had the largest impact on overall population growth , followed by daily survival of pupa , juveniles , eggs and larvae and dispersing females . In addition to modeling growth with 100 initial colonists , we projected the population growth of low and high starting populations calculated from observed weekly alcohol-lure trap catches during peak dispersal . Comparing the three population projections, peak number of dispersers at Day 185 varied considerably, with 162, 3259, and 8768 daily dispersers for low, medium, and high colonizing populations, respectively. In the high population projection, the adult population toward the end of the growing season reached over 18,800 individuals. Note that because these are density-independent models, the number of CBB does not depend on plant density. However, the impacts of the CBB population on yield would depend on coffee plant density. To reduce the final adult population by 50%, the daily survival rate of dispersing females would have to be reduced from 0.99602 to 0.83202. This change represents a 16.4% reduction in daily survival when dispersing. The number of CBB that birds need to eat to reduce the adult population at this rate was driven by the initial population size as a straight line, y = 79.23 N0 . At medium starting population , birds need to consume 7628 CBB during the borer breeding season, while at high starting population , about 20,500 dispersing CBB must be consumed by birds. Daily consumption rates by birds would have to increase over time as the CBB population grows and could vary from 15 to 750 CBB being consumed a day, depending on starting population size . Overall, we calculated that for every female CBB in the initial colonization, birds need to consume 79 CBB to reduce the end of season population by half.We estimated that the caloric content of a 195 μg adult CBB to be 1.09 calories per gram dry weight, or 0.00109 kcal. At 5%–10% of a bird’s daily diet based on number of prey items, birds would consume <7 CBB per day. This represents 0.03%–0.05% of daily caloric requirements of our average insectivorous bird. At these feeding rates, our models suggest that by the time of peak dispersal, 4, 88, and 236 birds are required at low, medium, and high starting population sizes, respectively, to reduce CBB populations by 50% on day 185 .Our model suggests that avian predation is likely to be effective at reducing CBB populations by 50% only during small infestations , or during the early stages of larger infestations . Birds appear unable to successfully suppress medium and large infestations because the number of CBB that need to be eaten in a season requires higher bird densities than are reported in the literature.

Therefore, a comparison of the geometric effects reflected by the generalizations of the Berry phase of purified states or thermal vacua is expected to be achievable in future experiments on quantum computers or quantum simulators. For example, one may consider two identical composite quantum systems of Example V.1 of the generalized Berry phase and then apply a partial transposition to one of the composite systems. As a consequence, the composite system with a partial transposition corresponds to a purified state while the one without partial transposition may be viewed as a thermal vacuum. By applying parallel transport that involves the ancilla to both composite systems and extract their generalized Berry phase after a cycle, a π-phase difference is expected between the two composite systems. Given the large phase difference between them after a cycle, the result is robust against small perturbations or noise from the hardware and offers another demonstration of geometrical protection of information. We have presented two generalizations of the Berry phase, the thermal Berry phase and generalized Berry phase, for distinguishing the two state-vector representations of mixed states via the purified state and thermal vacuum. From the geometrical and physical points of view, pots with drainage holes the generalized Berry phase has more desirable properties since the thermal Berry phase is generated by a temperature-dependent thermal Hamiltonian and may carry non-geometrical information.

We caution that while the transformations can be on the system, ancilla, or both in the construction of the generalized Berry phase, an operation on the ancilla is necessary if we want to differentiate the purified state and thermal vacuum.The two state-vector representations of mixed states via purified states or thermal vacua have been developed in different branches of physics, but both have been realized on quantum computers [32, 33]. We have pointed out that their difference lies in a partial transposition of the ancilla, which has its origin in the Hilbert-Schmidt product. Available physical quantities, including previously studied geometric phases, cannot differentiate the two representations. By analogue of the adiabatic process of pure states, the thermal Berry phase has been constructed and shown to differentiate a purified state froma thermal vacuum. However, the thermal Berry phase may include non-geometrical information. The generalized Berry phase is then constructed by generalizing the parallel-transport condition to properly include the system and ancilla, and only geometrical contributions are included. Depending on the protocol and setup, the generalized Berry phase may also differentiate the purified state and thermal vacuum. Future demonstrations of the interplay between geometric effects and partial transposition of state-vector representations of mixed states on quantum computers or simulators will advance our understanding of quantum systems at finite temperatures.Magnesium has great potentials to serve as next-generation bio-resorbable implants for medical applications due to their excellent mechanical properties, biodegradability, and bio-compatibility . Biodegradability of Mg-based implants and interactions with relevant cells have been studied in vitro for orthopedic and urological applications. Most of the in vitro studies on the degradation of Mg-based implants were performed by immersion in physiologically relevant fluids at the body temperature of 37°C to represent the chemical and thermal environment in vivo.

It is desirable to include physiological loading as one of the key contributing factors when studying in vitro degradation of Mgbased metals for medical implant applications, because mechanical stress could increase the corrosion rates of Mg-based alloys and composites. For example, cyclic loading significantly increased the corrosion rates of high purity magnesium , binary Mg-1Ca, and ternary Mg–2Zn–0.2Ca alloys in simulated body fluid. Li et al. reported that the degradation of Mg/Poly wires was accelerated under a dynamic compressive stress of 0.9 MPa at a frequency of 2.5 Hz. Mg-based alloys are known to be susceptible to stress corrosion cracking ; and Mg-based implants may degrade faster and experience sudden fracture under load, especially in a humid environment such as inside the body. Mechanical behaviors of Mg have been investigated using a slow rate test method in modified simulated body fluid, and Mg did show a lower elongation and ultimate tensile strength due to SSC. Thus, it is important to study the degradation behaviors of Mg-based implants under load for a long period of time, preferably weeks to months, to understand the properties of these implants as they degrade. Although in vivo studies in animal models can provide complimentary information about the performance of Mg-based implants under load, the load in small animal models, such as rats, cannot be directly translated to the human study due to the significant differences in musculoskeletal structures between small mammals and human. Before clinical studies, large animal models, such as sheep and dogs, are often recommended for evaluating orthopedic implants because they have similar loading conditions as human. However, long-term studies in large animal models are always costly and involving sacrifice of many animals.

Therefore, the objective of this study was to develop and build a novel loading device to simulate the human-like physiological loading conditions in vitro for studying biodegradable implants in a long period of time from weeks to months. The degradation behaviors of Mg rods under applied loads of 500 N were investigated for up to two weeks using this loading device. Mg rods were cut into 15 mm × 6 mm using a handsaw, and then polished using silicon carbide papers from 600 grit to 1200 grit. The polished samples were degreased and cleaned in acetone for 30 min and in 100% ethanol for 30 min respectively, using an ultrasonic cleaner . Before immersion, all of the Mg samples were weighed using an analytical balance , and the mass of each sample was recorded as the initial mass . The well design of the loading chamber for housing the Mg samples is shown in Figure 3. To prevent the galvanic corrosion between the Mg samples and the piston, the wells and the caps on the pistons were machined out of Teflon to avoid the metal to metal contact. The Mg rod samples were placed into Teflon wells and immersed in 2.5 mL of revised simulated body fluid that has the same ionic composition as human blood plasma. A load of 500 N was applied on each Mg rod at room temperature until the prescribed immersion time point is reached. The Mg rod controls were also placed in the Teflon wells respectively and immersed in 2.5 mL of rSBF but without load. The Mg rod samples were immersed in rSBF for 3 days, 1 week, and 2 weeks. The rSBF was replenished every other day. The immersion degradation experiment was run in triplicate concurrently. After each immersion period, the rSBF was collected from the wells and the Mg rod samples were dried in a vacuum at room temperature. The macroscopic images of the dried Mg rod samples that were tested with or without 500 N of load were taken using a camera . The dried Mg samples were also weighed using an analytical balance to determine the final mass after immersion. The mass change of Mg samples at different time points was then calculated following the equation /Mo, drainage pot where Mf is the final mass and Mo is the initial mass. The pH of the collected rSBF was measured using a pH meter . The Mg2+ ion concentrations were quantified using inductively coupled plasma – optical emission spectrometry . Briefly, the collected solutions from each well were diluted with deionized water by a factor of 1:100 into a total volume of 10 mL. Mg2+ ion concentrations were then quantified based on the calibration curves generated using Mg2+ standards serially diluted to a concentration of 0.5, 1, 2, and 5 mg/L. The characterization process was repeated for each time point.The macroscopic images of the Mg under load and Mg controls without load showed different surface morphologies after 14 days of immersion in rSBF . Generally, all the Mg samples showed deposition of degradation products after 3 days of immersion. The white degradation products increased as the time increased during the immersion. Mg rods under load, however, had a less degradation products on the surface than that of Mg controls, especially at 7 days and 14 days. Figure 4b shows the mass change of the Mg under load and Mg controls after 14 days of immersion in rSBF. Statistically significant difference was found among the Mg-based samples during the 11 days of immersion [F=175.7, p<0.0001]. All the Mg samples had a significant mass decrease after the immersion. At the 3 days of immersion, all the Mg samples had a mass increase due to the deposition of the degradation products. The Mg under load showed a higher mass increase than the Mg controls. Starting at 7 days of immersion, all the Mg samples had a significant mass decrease, where the Mg under load showed a significantly higher mass loss than the Mg controls. At 14 days of immersion, the mass of Mg controls had no significant change in comparison with the previous time point.

The mass of Mg under load, however, showed a mass loss which was significantly lower than the Mg controls.Figure 4c displays the pH of the rSBF for the Mg under load and Mg controls after 14 days of immersion. Statistically significant difference was found among the Mg-based samples during the 11 days of immersion [F=10.86, p=0.004]. Generally, the pH of Mg-based samples showed an increasing trend as time increased during the immersion. When comparing the Mg under load and Mg controls without load, the pH of Mg controls was higher than that of Mg under load at 3 days and 7 days of immersion. The pH of Mg controls at 14 days, however, showed a lower pH than the previous time point, possibly because the continuous deposition of degradation products slowed the degradation of Mg samples. At 14 days of immersion, the pH of Mg controls was significantly lower than that of Mg under load. From Figure 4d, the Mg2+ ion concentration of the rSBF for the Mg rods under load and Mg controls showed a significant increase during the 14 days of immersion. Statistically significant difference was found among the Mg-based samples during the 11 days of immersion F=55.82, p<0.0001]. The Mg2+ ion concentrations of Mg-based samples showed an increasing trend as time increased during the immersion. The Mg under load showed a higher Mg2+ ion concentration in average than that of Mg controls at all-time points during the immersion. Statistical difference was found at 3 days of immersion and 14 days of immersion.Engineering the loading device for studying Mg-based bio-materials in vitro involves three major challenges, that is, automating, powering, and down scaling in size. Although the current version of our loading device meets the critical design criteria and functional requirements for studying Mg degradation under load, further improvements in the following aspects are still recommended to make the device more user friendly and more robust for repeated experiments. First, automating operation of the loading device can greatly improve the repeatability of experimental results and benefits the users especially in the long-term studies that span from weeks to months. The current pneumatic pistons are powered through an air compressor, which needs to be manually adjusted by the user due to natural accruing air leaks in the system. To improve this, the pneumatic powered pistons can be replaced with electrically powered pistons, and a feedback or closed loop control system can be added for autonomous regulation. One piston type of interest would be hydraulic pistons as they are small, and able to output large forces that would be required for testing various medical implant material. Using these electrical powered pistons, a feedback loop can be created through various means, such as using electrical components that read the output force of the electrical powered pistons, and with that data the device could self correct itself to the desired load output without the need of a user. To implement this, a self adjusting controller such as a PID controller, would be able to provide the device with both versatility and higher accuracy. Second, the device should simulate the body conditions more closely during the in vitro experiments in addition to applying a load onto the implant material. Specifically, it is beneficial to conduct the experiments under standard cell culture conditions inside an incubator, i.e., a sterile, 37°C, 5% CO2/95% air, humidified environment, because such environment resembles the conditions inside the body.

Compounds were identified based on retention time and spectral comparisons with standards. Information about the linear equations and lower limits of detection and quantitation can be found in Table S1 in the supplementary material. The LLOD was calculated as 3.3 times the standard deviation of the y-intercept of the curve divided the slope, while the LLOQ was calculated as 10 times those values.Several peaks appeared in the HPLC chromatograms that could not be identified using the above parameters. Chromatographic eluents of these peaks were collected individually and dried under vacuum. These extracts were reconstituted with mobile phase A, and 5 µL were injected into the HPLC- QTOF-MS/MS for accurate mass analysis . A Poroshell 120 EC-C18 column was used at 35 °C. Mobile phase A was 1% formic acid in distilled water, and mobile phase B was 1% formic acid in acetonitrile. The gradient used was 0 min 3% B, 30 min 50% B, 31-32 min 95% B, 33-38 min 3% B. The mass spectrometer was used in negative mode, and the mass range for MS was 100 to 1000 m/z while the range for MS/MS was 20-700 m/z. Collision energies at 10, 20, and 40 V were applied. The drying gas was set to a flow of 12 L/min at 250 °C, while the sheath gas was set to 11 L/min at 350 °C. The nebulizer was set to 40 psig, the capillary voltage was 3500 V, the nozzle was set to 500 V, vertical gardening in greenhouse and the fragmentor was set to 100 V. Data was analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 .

Tentative identification was achieved by comparing the mass to charge ratio of the precursor and fragment ions to online libraries of compounds as well as using formula generation for the peaks in the spectra.The composition of blue elderberries is presented for the first time, which is key to understanding how this subspecies of Sambucus nigra compares to commercialized elderberry subspecies, S. nigra ssp. nigra and S. nigra ssp. canadensis. These data help to establish the blue elderberry grown in hedgerows in California as a viable source of berries and bio-active compounds. Data for the compositional assays is presented for the 2018 and 2019 harvest years as the average of all shrubs sampled in Table 2. The average moisture for the blue elderberries was 79.5 ± 1.5% in 2018 and 79.5 ± 1.6% in 2019, which is very similar to the levels found in wild elderberries in Spain 95. The average soluble solids found in blue elderberry ranged from 11.94 ± 2.08 to 14.95 ± 1.02 g per 100 g FW in 2018 and from 12.64 ± 1.86 to 17.09 ± 1.60 g per 100 g FW in 2019. These values are slightly higher than the soluble solids found in S. nigra ssp. cerulea grown in Slovenia29 and American elderberries grown in Ohio52. Compared to European and American elderberries evaluated in other studies, blue elderberries have similar levels of soluble solids 8,18,29,49,50,95. In the present study, the overall average content of soluble solids was significantly different between years, as blue elderberries harvested in 2019 had significantly higher average soluble solids than the elderberries harvested in 2018 . The pH in the blue elderberry ranged from 3.44 to 3.86 in 2018 and from 3.46 to 3.79 in 2019, with no significant difference found between harvest years.

These values are slightly lower than the values found in European elderberry, which ranged from 3.9 ± 0.06 to 4.1 ± 0.04 with an average pH of 3.9 ± 0.2, and American elderberry, which ranged from 3.9 ± 0.04 to 4.5 ± 0.03 with an average pH of 4.2 ± 0.2 49 Another evaluation of pH in American elderberries had a range of 4.5 ± 0.08 to 4.9 ±0.12,higher than those found in the blue elderberry.52 The higher sugar and lower pH levels in blue elderberry could potentially impact taste and performance in food and beverages as compared with the European and American species. The average titratable acidity in blue elderberries ranged from 0.45 ± 0.08 to 0.77 ± 0.03 g citric acid per 100 g FW in 2018 and from 0.54 ± 0.06 to 0.77 ± 0.11 g citric acid per 100 g FW in 2019 with no significant difference found between harvest years. These values are lower than the total acids found by Mikulic-Petkovsek et al. 29 in S. nigra ssp. cerulea , but they are similar to the levels found in European elderberry 8,18,49,50 .Anthocyanins are a class of phenolics that contribute red, purple, and blue hues to fruits and vegetables, act as attractants for pollinators, and are potent antioxidants. European and American elderberries are well-known for containing high levels of anthocyanins 8,18,49. The anthocyanin content of elderberries strongly correlates to the antioxidant potential of the fruit, which may confer health-promoting properties 50,89, which is one reason why elderberries are used in supplements and value-added products. Elderberry is also used as a source of natural food colorants due to the levels of anthocyanins35. Understanding the levels of anthocyanins in the blueelderberry grown in hedgerows is critical towards establishing this native fruit as an additional and more sustainable elderberry. TMA was variable between hedgerows in both years of harvest, with relative standard deviation values between 16% and 30%, yet there was not a significant difference in the overall average TMA between 2018 and 2019 . Furthermore, most hedgerows were not significantly different from the other hedgerows harvested that year despite significant differences in TMA values found between farms in both years .

Regarding the age of the elderberry shrub, hedgerows 2 and 14 had two of the three highest concentrations of TMA in 2019 . This suggests that blue elderberries can be harvested from plants as young as two years without a significant loss of TMA concentrations. TMA values for the blue elderberries are lower than those found in other elderberry subspecies. In European elderberries, TMA levels range from 170 ± 12 to 343 ± 11 with an average of 239 ± 94 mg CGE per 100 g FW 49. A study of American elderberry grown in Ohio showed a range from 354 ± 59 to 595 ± 26 mg CGE per 100 g FW. In the present study, bare root prerooted cuttings of American elderberries were planted, along with blue elderberries, on Farm 1 in 2018, and three shrubs were harvested in 2019. These American elderberries had an average TMA value of 263 ± 5.4 mg CGE per 100 g FW, which is more similar to what has been observed in other studies on this subspecies. This suggests it is a subspecies difference contributing to the lower anthocyanin concentration in the blue elderberry and not the difference in growing conditions. Compared to other berries, blue elderberries have similar levels of anthocyanins as raspberries, but lower levels than blueberries and blackberries . The lower concentration of anthocyanins in theblue elderberry may require adjustment of levels used in supplements, food and beverages for optimal performance or health benefit, or as natural coloring agents.In addition to anthocyanins, elderberries contain other phenolic compounds, such as flavonols and phenolic acids, which also contribute to the health promoting properties of elderberry. Phenolic compounds are responsible for organoleptic properties and can help protect foods against lipid oxidation. Therefore, TPC can be useful for making approximate comparisons, for example, greenhouse vertical farming between varieties of the same fruit, between similar fruits or in the evaluation of a processing step . It is important to note that the TPC assay is a non-selective assay and is easily impacted by extraction conditions and interfering substances, such as ascorbic acid and reducing sugars. Although there is no evidence that the beneficial effects of polyphenol-rich foods can be attributed to the TPC of a food, it can be a useful measure for making general comparisons with other studies in the literature which reported these values but should be supported by quantitative HPLC data. Herein, the range of TPC measured in the blue elderberries was from 514 ± 41 to 791 ± 34 mg GAE per 100 g FW in 2018 and from 459 ± 50 to 695 ± 41 mg GAE per 100 g FW in 2019 . TPC in the blue elderberries was significantly higher in 2018 than in 2019 . While there were significant differences found between the farms in both years , most hedgerows were not significantly different than most other hedgerows in the given year when evaluated together . Although the farms in this study were near each other and experience similar climates, there can still be differences in growing conditions for each hedgerow, such as water availability, which has been shown to influence the levels of phenolics in blueberries 101 and strawberries 102 . Hedgerows 2 and 14 were not significantly different from other hedgerows in 2019, indicating that the blue elderberries can be harvested earlyin the plant’s lifetime, which allows farmers to earn an early return on the investment of establishing hedgerows.

These comparisons show that blue elderberries from hedgerows are a rich source of phenolic compounds.Phenolic compounds were identified and quantified in the blue elderberry based upon retention time, absorbance spectra and authentic standards when available. Concentrations for samples from 2018 are presented in Table 4, while samples from 2019 are presented in Table 5. Two peaks with significant area were observed in the HPLC chromatograms at 6.96 min and 11.70 min that did not correlate to standards or library matching. Both compounds eluted between the retention time of gallic acid and protocatechuic acid. The first eluting compound had a maximum absorbance at 300 nm while the second compound had a maximum absorbance at 280 nm. These peaks were collected individually and further evaluated by accurate mass quadrupole time-of-flight tandem mass spectrometry . TOF acquires mass spectral data by pulsing ions entering the flight tube in an orthogonal beam, therefore full spectra are collected. The data captured is accurate enough to determine the elemental composition therefore allowing identification without standards. The two compounds were tentatively identified using high mass accuracy as 5-hydroxypyrogallol hexoside, a tetrahydroxybenzene , and protocatechuic acid dihexoside . Accurate mass was especially helpful since commercial standards for these compounds are not available. 5- HPG hexoside was identified by its fragmentation pattern , showing a precursor ion [MH]- at m/z 303.0723 and product ion [M-hexose-H]- at m/z 141.0186 . This compound was one of the most abundant phenolic compounds in the blue elderberry. While no evidence of5-HPG glycoside was found in the literature, the aglycone has shown to have a high radical scavenging activity compared to other simple phenols.Like other elderberry species, rutin was the predominant flavonol and overall had the highest concentration of any of the flavonols measured, with an average of 57.01 ± 17.42 mg per 100 g FW in 2018 and 51.89 ± 25.53 mg per 100 g FW in 2019. These values fall within the range of what has been found in European elderberry. Other flavonols identified include isoquercetin , kaempferol-3-rutinoside, and isorhamnetin-3-rutinoside, which was also a major phenolic compound in the berry. Isorhamnetin- 3-rutinoside averaged 28.30 ± 14.03 mg per 100 g FW in 2018 and 24.71 ± 14.83 mg per 100 g FW in 2019, which is higher than what has been found in other subspecies. Overall, the blue elderberry analyzed in the present study has much higher levels of total flavonols as compared to European elderberry. In the American elderberry, the main flavonols are rutin followed by isorhamnetin-3-rutinoside whereas in European elderberries, the main flavonols are rutin followed by isoquercetin. In blue elderberry grown in Slovenia, rutin and isoquercetin were the two predominant flavonols, though the total flavonols in found for the subspecies was similar to the levels found in this study 59 . The predominant anthocyanin present in the blue elderberry is cyanidin-3-sambubioside, like the European subspecies. The average concentration in 2018 was 32.70 ± 10.18 mg per 100 g FW and 29.66 ± 16.81 mg per 100 g FW in 2019.

During the growing seasons, from April to September, the site received 23.2% of the total precipitation in 2020 and only 2.1% in 2021 . In addition, there was minimal precipitation during data collection of this study from June to September, where only 2 mm and 1.2 mm of precipitation were received in 2020 and 2021. As for the air temperature during the growing seasons, the average maximum air temperature was slightly higher in July, August, and September in 2020 compared to 2021, but lower in March and April. The average minimum air temperature was constantly higher in 2020 compared to 2021 from March until harvest in September, except July. Similarly, the average air temperature was generally higher in 2020 than 2021 except both Julys which had the same average air temperature. As for GDD accumulation , the two seasons were slightly different. In 2020, there was 1525.4°C GDD accumulated when the berries reached 23.9°Brix on average; in 2021, there was 1292.3°C GDD accumulated when the berries reached 22.6°Brix on average. Thus, 2020 was a slightly drier and hotter season than 2021.LAI and crown porosity were assessed in both seasons, and leaf areas were calculated based on the unit ground area and LAI . In 2020, VSP80 had the most leaf area among the six trellis systems, VSP60 and GY had similar leaf areas, 25 liter pot followed by VSP . SH and HQ had the lowest leaf areas as the canopies in these two trellises still had gaps.

This was also confirmed with the fact that SH and HQ had the highest crown porosities among the six trellis systems . The other trellis systems had similar lower crown porosities than SH and HQ. There was no difference in canopy architecture among the three irrigation regimes in the first season . In 2021, all the trellis systems had similar leaf areas . HQ had higher crown porosity than VSP60, but the other trellis systems had similar crown porosities to either HQ or VSP60 . These effects were not modified by the irrigation treatments and no significant interactions between factors were found. For applied water amounts, 50% ETc had higher leaf area than 25% ETc, but there was no difference between 100% ETc with either 25% or 50% . However, 50% ETc still had the highest crown porosity compared to 100% ETc, and 25% ETc did not show any difference with the other two irrigation treatments .Grapevine leaf gas exchange was monitored throughout both seasons, and their integrals were calculated to represent the season-long plant response of grapevines for net carbon assimilation rate , stomatal conductance , and intrinsic water use efficiency . In 2020, there were no differences in gs and An among the six trellis systems . However, HQ had the highest WUEi, whereas VSP, VSP60, and SH had lower WUEi .

Regarding the irrigation treatments, there was no difference in gs integrals . However, a linear response to water amounts were observed for An and WUEi, with 100% ETc having the highest values of both gas exchange variable monitored .In 2021, there were no differences in gs, An, and WUEi among the six trellis systems . Nevertheless, a linear response to water amounts was recorded, with 100% ETc showing the highest An and gs, followed by 50% ETc, and 25% ETc which accounted for a higher WUEi in 25% ETc with 50% treatments compared with 100% ETc . The analysis of the gas exchange recorded at each measurement day indicated that in 2020, despite starting with the highest gs, SH had lower gs over the season . Contrarily, HQ trellis system showed higher gs in July and August which was connected with higher An over the season . On the other hand, GY and VSP80 systems enhanced An during some periods over the season. Regarding WUEi, VSP60 and HQ had the highest values while SH decreased it in the early season and increased it in early August . However, all these differences tended to diminish at the end of the season. For irrigation treatments, a constant effect of water amount was observed with 100% ETc increasing gs and An and decreasing WUEi . In 2021, GY and VSP60 showed higher gs and An values in general . HQ showed lower gs values and VSP had lower An values compared to the other trellis systems throughout the season. HQ increased WUEi throughout the whole season . Although GY had higher WUEi in the early season, it showed constantly lower WUEi values after 23 June 2021.

Besides GY, VSP showed lower WUEi in July and August. A similar effect of irrigation treatments was observed over the second season, with a linear response for increased gs and An and decreased WUEi when the irrigation water amount was increased .Yield components and berry quality parameters were assessed at harvest in both seasons . SH and HQ had the smallest berries among the six trellis systems in the two seasons. In 2020, SH and VSP increased the cluster number, while VSP80 and GY decreased it whereas, in 2021, SH and HQ accounted for increased the cluster number. VSP, VSP60, VSP80, and GY increased the cluster weight compared to SH in 2020. In 2021, SH showed the lowest cluster weight and skin weight. Regarding yield, differences were only significant in 2020 where SH enhanced vine yield compared to the other trellis systems. On the other hand, 100% ETc enhanced berry weight, cluster weight, and yield over the two seasons with no difference on leaf area to fruit ratio. Regarding berry quality parameters, SH had the highest TSS among and the lowest pH in 2020, whereas in 2021, VSPs and GY enhanced the TSS and the pH. Results also showed that irrigation treatments had little effect on the berry quality parameters over the two seasons with only TSS being increased in the 25% ETc treatment in the harvest of 2020.Different trellis systems affected not only the total anthocyanin concentration but also modified the anthocyanin composition, leading to modifications in the profile stability. In both seasons, SH had the highest concentrations in all the anthocyanin derivatives besides di- and tri-hydroxylated anthocyanins among the six trellis systems. In 2021, HQ also notably increased most of the anthocyanin derivatives, tri-hydroxylated, di-hydroxylated, and total anthocyanins compared to the VSP trellis systems. On the other hand, VSP trellis systems tended to decrease the anthocyanin concentrations. Regarding the irrigation treatments, 25% ETc generally showed the higher concentrations in petunidins, di- and tri-hydroxylated anthocyanins, and total anthocyanins in 2020 compared to 100% ETc. In 2021, 25% ETc increase most of the anthocyanin concentration in berries. 50% ETc performed similarly in 2021 and showed higher concentrations in malvidins, trihydroxylated anthocyanins, and total anthocyanins. In parallel with anthocyanin assessments, berry skin flavonols were measured at harvest in both seasons . In 2020, SH showed the highest concentration in myricetins. SH and HQ showed the highest concentrations in quercetins, isorhamnetins and kaempferols in both seasons. SH and HQ also showed the highest concentration in tri- and di-hydroxylated as well as total flavonols in both seasons. In 2020, there were no differences among the six trellis systems in laricetins and syringetins. While in 2021, VSPs enhanced syringetin concentration. Regarding applied water amounts, raspberry cultivation pot little effects of irrigation treatments were shown in 2020. However, in 2021, 25% ETc increased most of the flavonol derivatives except laricetins and syringetins compared to the other two treatments.The relationships between berry skin flavonol concentrations and canopy architecture were investigated in both seasons. In 2020, crown porosity had positive and significant correlations with quercetin , total flavonol concentration , R2 = 0.248, p< 0.0001, Figure 5Aa-2), and total flavonol concentration . Leaf area was also correlated with these variables, but the correlations were negative with quercetin , total flavonol concentration . In 2021, the correlations were similar but not as significant as 2020. Crown porosity still had significant and positive relationships with quercetin and total flavonols concentration . However, the relationship between crown porosity and total flavonol concentration did not persist, as was observed in 2020. The relationships between leaf area and quercetin and total flavonol concentration were significant, although not as strong . Leaf areas were negatively correlated with these two variables . The significant correlation between leaf area and total flavonol concentration did not hold in 2020 as compared to 2020 .

It was evident that when crown porosity was greater, there was greater flavonol accumulation as well greater molar percentage of quercetin.A trellis system selected in grapevine vineyard is usually aimed at optimizing canopy architecture to further maximize canopy photosynthetic activity and improve canopy microclimate, which can yield desirable production and berry composition . In historically cooler regions according to Winkler’s Index, VSPtrellis system is widely used as it offers relatively higher compatibility with mechanization and is suitable for the regional production goals . However, with the warming trend in air temperature getting more pronounced, VSPs have been showing greater chances of getting cluster overexposure, resulting in sunburnt berries with yield loss and color degradation . Under our experimental conditions, HQ trellis system showed less leaf area and greater crown porosity than the other trellis systems in 2020 in accordance with previous studies, where split trellis designs might allow more solar radiation to penetrate the canopy interior . Conversely, SH had similar leaf area but lower crown porosity in 2020. However, the differences in leaf areas and crown porosities were not as noticeable in 2021. This could be attributed to the fact that the HQ and SH might have still been filling up spaces with new growth compared to the VSPs, which might have already had relatively more established canopy architectures. In addition, the differences in leaf areas and crown porosities could be minimized by arid growing season in 2021, despite the supplemental irrigation applied to them, accounting for diminished leaf areas as shown in 2021 than 2020. Furthermore, precipitation received at the vineyard prior to bud break , as well as precipitation received immediately prior to flowering in semi-arid regions were deemed key determinants of canopy response for latter parts of the growing season.In this study, yield per vine was not constantly determined by the trellis systems in both years, although similar bud densities at pruning were achieved. Furthermore, more leaf area did not account for more yield at harvest, despite it was well established that a sufficient leaf area would support fruit development , and in contrast with some previous studies . This could be attributed to not only the total amount leaf area but also how the leaves were distributed within the canopy. Commonly, HQ would have more open space to distribute more exposed and photosynthetically active leaves to the sunlight to optimize production . Previous studies have shown that greater leaf area can also contribute to higher TSS accumulation , which was not observed in this study. On the contrary, more leaf area resulted in less TSS accumulation. This might be explained by the fact that the leaf area to fruit ratio, which represented the source-sink balance within the grapevine, might have a greater influence on the berry TSS accumulation. In this study, even though no statistical differences were observed in leaf area to fruit ratio, SH in 2020 showed relatively higher values with higher TSS accumulated at harvest. A similar situation was observed during the second season, where VSP80 showed relatively higher leaf area to fruit ratio and subsequently higher TSS at harvest. When crown porosity was considered, higher porosity resulted in greater TSS accumulation in berries, which could be attributed to the higher potential of berry exposure to the hot environment, causing the berries to experience greater dehydration . This relationship was not observed in 2021, and it might be derived from the relatively higher crop load and lower leaf area to fruit ratio in 2021 compared to 2020, especially by SH and HQ . SH and HQ did not have a similar source-sink balance as 2020, which lowered their capacity to translocate photosynthates into the berries, this might have been the reason why they had a more reduced TSS at harvest compared to the other trellis systems. Regarding the applied water amounts, the results were clear and consistent, with increased water status in grapevines irrigated with higher water amounts, and consequently, greater berry weight, cluster weight, and yield.

In a multi-center, double-blind, randomized controlled trial of very-low-birth-weight infants, no difference was found in the incidence of ROP between those supplemented with daily oral L and Z or placebo. However, the progression rate of threshold ROP showed a lower trend in the supplemented group. No adverse events were noted with L and Z supplementation, suggesting that they were well-tolerated. Another study examining the effects of daily oral L and Z supplementation in preterm infants from the seventh day after birth until 40 weeks of age or until hospital discharge found no change in the rate or severity of ROP compared to placebo. Further, a meta-analysis of three randomized controlled trials also found no protective association between L and Z supplementation and the risk of ROP. Additional studies are needed to assess the role of prenatal L and Z supplementation in pregnant women at risk for premature delivery. During development, L and Z are not interchangeable. Serum Z in newborns and in their mothers is strongly correlated with the MPOD of the babies, but no relationship was noted for either maternal or infant levels of L. During delivery, a high maternal plasma Z, but not plasma L, was significantly associated with a lower risk of visual acuity problems in children at three years of age. Further investigations that can accurately distinguish and quantify dietary and plasma Z from L are needed to better understand the role of these two carotenoids in visual performance during development.

The L and Z in human milk is particularly important for infant eye and brain development, grow bucket and may provide long term benefits to vision and cognition. Since humans cannot synthesize carotenoids, the fetus and breastfed infants must obtain these compounds from the mother through the placenta and the breast milk. During gestation, maternal lipoprotein synthesis increases, which accelerates the transport of carotenoids to the fetus. This transfer may deplete maternal stores if the dietary intake of carotenoids in general, and L and Z specifically, is inadequate to maintain body stores. The prevalence of AMD is higher in women than in men, even though on a global basis more men smoke. At the same time MPOD levels are lower in females. The potential reasons for an increased lifetime risk for AMD in women are complex and multifactorial in nature and may include maternal depletion of L and Z during pregnancy and lactation . Importantly, the average dietary consumption of L and Z among females in the US is far below the amount of 10 mg/d known to increase MPOD. Therefore, either the intake of supplements containing L and Z, or increased intake of foods rich in these two carotenoids for the duration of pregnancy and lactation may be of value. The concentration of β-carotene, lycopene, L and Z, the main carotenoids in breast milk are associated with maternal dietary intake over the first six months of lactation. Daily maternal supplementation of either 6 mg of L with 96 µg of Z, or 12 mg of L with 192 µg of Z, over six weeks resulted in a dose-dependent increase in L and Z levels in the breastmilk and of the mothers and their infants when assessed three to four months postpartum. 

Another study reported that more carotenes were present than xanthophylls in maternal plasma, whereas more xanthophylls such as L and Z were presented in breast milk, in comparison to carotenes. These findings support the notion that maternal-infant transfer of carotenoids may occur, possibly at the expense of the mother. Future studies are needed to clarify if breastfeeding or L and Z intake may impact their AMD risk. The L-ZIP supplementation trial is currently exploring whether prenatal supplementation of 10 mg of L and 2 mg of Z will maintain maternal body stores, prevent potential macular pigment depletion during pregnancy, or enhance systemic and ocular carotenoid stores for both mothers and infants. Clinical trials on the long-term effects of perinatal L and Z intake on MPOD changes among mothers and infants are also warranted.100 Unfortunately, longitudinal studies on AMD in females often do not include breast-feeding history. A useful study design would be to investigate MPOD levels and relative risks of AMD between multi-parous and nulliparous women, and in mothers practicing breastfeeding compared to formula feeding. Dietary intake of L and Z would be important to assess. Recognizing that such a study would take decades, shorter term studies could be conducted in non-human primates. Another challenge in retrospective studies is that breastfeeding history may not be accurate. Therefore, studies on the maternal transfer of L and Z during pregnancy and lactation with MPOD changes in infants and throughout the lifespan, could be important but difficult to conduct.

Future research should also focus on the measurement of L and Z status and MPOD in mother-infant pairs of twins or short birth intervals. Last, when accessing AMD risk in women, reproductive hormone status may be a confounding factor. The pathogenesis of AMD involves oxidative stress and immune dysregulation.Estrogen has been shown to reduce oxidative stress and inflammation in RPE cells as well as systemically. Lifetime estrogen exposure such as the number of pregnancies, menopause, reproductive period, oral contraceptive use, and hormone replacement therapy may all influence the risk of developing AMD. Current evidence regarding estrogen exposure and risk of AMD is inconsistent. One study reported that postmenopausal hormone use decreased the risk of neovascular AMD but increased the risk of early AMD, while parous women showed a reduced risk of early AMD but not neovascular AMD. Two nationwide studies from South Korea among postmenopausal women noted that exogenous estrogen exposure was not a protective factor for AMD. A cohort study found that hormone replacement therapy and a longer reproductive period was associated with an increased risk of neovascular AMD. A cross sectional study showed that oral contraceptive use was associated with an increased risk of late AMD.106 In addition, a review summarizing the effect of estrogen exposure and the risk of all age-related eye diseases concluded that HRT, or the use of oral contraceptives, could be either positively or negatively associated with the risk of AMD.103 In contrast, some studies have reported that a longer duration of breastfeeding may be protective from late but not early AMD,106,107 even when the estrogen level was low during lactation. Future studies on the interaction of different reproductive and estrogen exposure histories and AMD risk are needed. Humans cannot synthesize carotenoids, and the best dietary sources are fruits, vegetables, egg yolks, and dairy products. Consuming a diet rich in green leafy vegetables and fish is recommended by the National Eye Institute for the high carotenoid and DHA and EPA contents. Nevertheless, in the carotenoid group, L and Z are not yet considered essential, or even conditionally essential, so no dietary reference intakes for these two compounds exists. The US intake of L and Z has been decreasing. According to the U.S. National Health and Nutrition Examination Survey , the average intakes of L plus Z were 2.15 mg/d in males and 2.21 mg/d in females in 1987, and 2.15 mg/d in males and 1.86 mg/d in females in 1992. In NHANES 2013-2014, dutch bucket for tomatoes the average intakes of L and Z in males and females was 1.58 mg/d and 1.76 mg/d, respectively. Moreover, based on data from NHANES 2003-2004, the reported intake of L was significantly higher than Z in all age groups and ethnicities. Importantly, the Z to L ratio was also lower in females than males older than 31 years of age, which may result in a higher risk of AMD in women than in men. However, due to difficulties in analyzing dietary L and Z separately, most studies analyze both carotenoids together. Since the amount of L in most foods is significantly greater than Z, precise quantification of Z has been a challenge. The amount of L and Z in foods and dietary supplements appears to be safe. No adverse events were found in clinical trials giving L at 30 mg/d for 120 days or 40 mg/d for 63 days. The only reported adverse effect after a daily supplementation of 15 mg L in a 20-week trial was a single case of self-reported carotenodermia, a reversible condition of orange skin color. Although a higher amount has been used in human studies, after assessing the potential risks, the observed upper safety level for L has been proposed as 20 mg/d. 

The European Food Safety Authority concluded safe upper limits for L and Z for use in dietary supplements were 1 mg/kg body weight/d and 0.75 mg/kg body weight/d, respectively. In primate models, rhesus monkeys fed a xanthophyll-free diet for 3 to 6.5 years developed extremely deficient or absent macular yellow pigment and drusen-like bodies. When 3.9 µmol/kg per day of L or Z for 24 to 101 weeks were supplemented, the rhesus monkeys showed significant increases in their corresponding serum, retinal, and adipose tissue concentrations. In their retina samples, L and meso-Z, but not Z, appeared in the L-supplemented group, while only Z was found in the group supplemented with Z. In humans, a study investigating the serum and macular responses of L, Z, and meso-Z from dietary supplements found that 13.13 mg/d for 12 weeks provided maximum MPOD improvement, whereas 7.44 mg/d was the amount that increased serum levels at the highest efficacy. These and other studies support the need for dietary recommendations for L and Z, particularly as conditionally essential nutrients due to their protective effects on eye health. Lutein and Z fulfill many criteria as essential nutrients, including high concentrations in select tissues, biological plausibility for eye health, depletion outcomes such as vision impairment in primates, and inverse associations with certain diseases. In addition to their role in eye health, L and Z are involved in cognitive function at all stages of life. A randomized controlled trial reported that L and Z supplementation improved neural efficiency and learning performance by increasing the interaction of numerous brain regions in older adults. Other reports have demonstrated an association between L intake or circulating levels and preserving age-related cognitive decline, reducing the risks of certain cancers, coronary heart disease, stroke, metabolic syndrome, and achieving higher levels of physical activity. A systematic review of in vivo, ex vivo, and in vitro studies concluded that L may benefit vascular health by improving endothelial function, reducing inflammation, regulating favorable lipid profiles, and maintaining glucose homeostasis. Systematic reviews summarizing the amount of L needed for cognitive functions and enhancement of gray matter volume estimated that at least 10 mg/d for 12 months could be beneficial. Age must be considered when creating DRI values, since MPOD values are lower in older compared to younger individuals. Whether the proposed intake of L and Z should be based on the amount that can reduce AMD risk, benefit visual maturation in newborns, protect cognitive health, or reduce the risk of other diseases requires further consideration. Sex differences in AMD prevalence must also be considered, especially in relation to pregnancy and lactation, as discussed above. Nevertheless, L and Z are not included for DRI consideration due to inadequate details from food databases, limited large-scale dietary intake studies, and insufficient knowledge regarding their metabolism and biological functions. Many continue to advocate for a DRI for L, since it satisfies all nine criteria for bio-active compounds. A rich dietary source of L and Z is goji berry, also called wolf berry or Gou Qi Zi. The bright orange-red colored oval fruit, has been used for millennia in traditional Chinese medicine for its role in visual health, to provide immunoregulatory, neuro-protective, and anti-inflammatory benefits, and to help regulate liver and kidney meridians . Commercially-available goji berries and their products come primarily from the Ningxia and Xinjiang autonomous regions in western China. Goji berry is known for its high amount of carotenoids, with the Z content higher than any other known food. In addition to carotenoids, other bioactive compounds found in goji berries include Lycium barbarum polysaccharides , flavonoids, vitamins, minerals, betaine, cerebrosides, phenolic acids, and certain amino acids which may also support the overall health of the eye, particularly when working synergistically. 

All the architectures they review are modular with the low level control functions deployed on a microcontroller and higher level tasks performed by various systems—either ROS or a custom-designed middleware. The real-time control in two cases use a HAL, the others do not. Within the Autonomous Systems Laboratory there is an important related work called the Santa Cruz Low-Cost UAV GNC Subsystem, which was an early autopilot intended for UAVs. It was programmed using the embedded code generation capability of Matlab and Simulink. This project, though successful, was never widely adopted. One reason is that Matlab and Simulink are expensive commercial software packages and come with a significant learning curve. Another is that the project wasn’t open source limiting access to vehicle developers or potential contributors. These limitations motivate this work which is open source, both hardware and firmware.This dissertation is organized as follows. Chapter 2 is a review of the requirements for the real-time controller. Chapter 3 details the hardware development process, architecture, electrical design, and the final controller. Chapter 4 details the software design, architecture, and development process. In Chapter 5 we propose a benchmark algorithm to quantify the performance of the real-time controllers. Chapter 6 shows the results of using the benchmark on four different processors, three real-time systems using micro-controllers and one SBC running Debian Linux. Chapter 7 details the development of an AGV from the chassis to autonomous navigation used as the main test platform for the controller. Chapter 8 covers three new cases for different autonomous systems that use this architecture or are in the process of deploying it. Chapter 9 demonstrates several areas where this work can be extended. Finally, Chapter 10 provides the conclusions.

There are two supplementary appendices.Appendix A is a derivation of the complementary filter, nft growing system the basic algorithm behind the benchmark. Appendix B details the application of the autoregression with external inputs method for system identification using a simple motor model as an example. The primary motivation for this work is to provide a means for an autonomous vehicle developer to rapidly prototype the real-time guidance, navigation and control system of a new vehicle. To enable this we decided at the inception of the project to make the design and firmware open source and named it the Open Source Autonomous Vehicle Controller, or OSAVC for short. The advantages provided by open source methodologies to a would-be vehicle developer are that the designs and algorithms are freely accessible, they are easily modifiable , and can be sourced from numerous vendors. From the project side open sourcing allows contributors from all over the globe to add new functionality, to test and provide feedback, and iterate on the designs. Indeed, during the development of this project we were able to participate in the Google Summer of Code program which hires student interns to contribute to open source projects. Over the course of two summer sessions, the OSAVC project received over 45 applications from students all over the globe, and was able to provide six paid internships. Additionally, the OSAVC prototype was used by two other graduate researchers for an ASV and a quadcopter which was an early demonstration of the capability and modularity of the project. The OSAVC provides real-time control of the vehicle actuators and measurement of the sensors. In the context of control systems, real-time refers to a fixed output period of the controller, i.e., the period between updating the vehicle actuators is deterministic and constant. All modern control systems are digital, that is they do not provide continuous outputs, instead they update the outputs on a fixed clock cycle. The systems under control, however, exist in the continuous real world.

If the controller clock cycle, τc, is short compared to the dynamics of the vehicle, then the digital controller approximates a continuous controller. In other words, if the rise time1 of a system in response to a step change in input is τv, then the digital control system approximates the continuous case when τc << τv. In the frequency domain a usual figure of merit is that the update rate is 1/20th-1/30th the bandwidth of the system. A finite clock cycle introduces a lag in the response of the controlled system. In addition to having as small a lag as is practical, it is also important to minimize the variation in the clock cycle. Small variations are inevitable but larger variations can lead to poor control or, in the worst case, instability. Not all tasks in an autonomous system require real-time control. For example, logging of telemetry data for post-mission use does not require a fixed clock—the system can buffer the data until it is convenient to store it. Thus, when designing a distributed control system it is natural to divide tasks into real-time and non-real-time categories. Real-time tasks are the domain of the real-time controller and all others belong to a processor with an OS. Note that some control systems attempt to handle all tasks within the same processor by using an RTOS. We discuss the tradeoffs of this type of OS in Section 2.6.1. Strictly real-time tasks are ones that support the control of the vehicle. These comprise the measurement of all the inputs and computation of the algorithms necessary to provide the actuator output. These include any sensor that is used to estimate the vehicle’s state . A non-comprehensive list of these sensors include wheel encoders, airspeed sensors, GPS, gyroscopes, accelerometers, magnetometers, barometers, etc. Accordingly, the navigation tasks, which include state estimation, are also real-time as they provide the input to the controller.

Although mapping of the environment isn’t a strictly real-time task, simultaneous localization and mapping is, as it provides vehicle state information . Communications generally do not have real-time requirements with the exception of remote control, when the system is operated manually using a radio. Finally, the controller itself must operate on a fixed cycle. All other tasks are non-real-time. Note that this doesn’t imply that they are not time-sensitive. For example, a range sensor might be used for obstacle avoidance, or a camera may be used to identify landmarks in the environment. In both cases, these data are needed to support guidance of the vehicle. Guidance is the process of determining the vehicle trajectory and therefore must be done quickly, but not necessarily on a fixed clock. Generally, guidance tasks are not real-time but are typically time sensitive. Other tasks, such as logging of data, measurement of sensors not used for state estimation, and general communications are non-real-time. A generic taxonomy of the tasks of an autonomous system is summarized in Table 2.1 showing the classification of real-time versus non-real-time as discussed above.From a high level, the OSAVC should support the main hardware and communication interfaces used by commercially-available components. The components needed for a vehicle will include sensors, radios , and actuators . Additionally, as a real-time subsystem, vertical hydroponic nft system the OSAVC module should communicate efficiently and quickly to the larger vehicle control system. For example, a vehicle may have an onboard guidance and navigation SBC that requires periodic communication of the vehicle state variables in order to plan a route. The module should support the real-time requirements of latency and speed for a vehicle. That is, it should be able to operate as quickly as needed by the vehicle and meet the deterministic latency requirement for the control algorithms. A final requirement is that the OSAVC provide power, signal conditioning, and power management for the sensors and processor, and optionally for any external peripherals or modules . The most common hardware interfaces are the inter-integrated circuit interface, the serial peripheral interface , the universal serial bus , and the universal asynchronous receiver-transmitter —commonly known as a serial port. Less common but used extensively in automobiles and robotics is the controller area network bus . Other peripherals needed for the OSAVC are hardware timers, input capture modules —used for decoding incoming digital signals, and output compare modules used for generating pulse-width modulated signals needed for motor and servo actuator control. Finally, general purpose input-output pins are useful for turning on LEDs, setting switches, and other requirements. The OSAVC should have most or all of these hardware peripherals in order to support the broadest range of applications; while tradeoffs can be made with respect to capability versus size, weight, and power, we typically fall on the side of greater capability in this work. The firmware is written entirely in the C programming language for speed and efficiency. The most important requirement of the firmware is modularity. Each sensor driver, estimation algorithm, and control algorithm is modular. Each module has a header file which provides the interfaces to public methods and a source file containing the code; each source file has a built-in test harness to allow for module troubleshooting outside of an application.

To maximize efficient utilization of the processor each sensor module is non-blocking. This requirement forces the use of vectored interrupts and each sensor has a unique interrupt priority assigned to it. The firmware library contains a selection of commonly used sensors needed for vehicle autonomy both to provide a minimum of functionality for a new developer as well as a template for new sensor drivers. The application itself should be as simple as possible to achieve the desired control. An example application for an AGV will be provided as a template for other applications. The application is a bare metal application consisting of an infinite loop with a hardware timer for task scheduling. This avoids the complexity and overhead of an RTOS. This requirement merit some discussion and is covered in Section 2.6.1. An RTOS is a piece of software that brings in real-time and non-real-time tasks into the same controller. It uses a HAL that allows for programmers with no expertisein embedded programming to communicate with various sensors without any understanding of the underlying hardware. The difficulty in using an RTOS lies in modifying the real-time tasks. Even with well-structured code, debugging the latency of a new realtime algorithm can be challenging. Autopilot firmware running on an RTOS typically implements the algorithms with tuneable parameters . This may work well for many cases, but modifying an algorithm for faster operation or for different parameters is challenging. Developing a new algorithm is even more difficult. Of course any OS increases the complexity of an application as well using more computational resources. A bare metal application, on the other hand, allows for strict control over the sensing and control elements of an autonomous system. Measuring the latency is straightforward using a hardware timer of the processor, as is managing processor bandwidth. Debugging the function is similarly simplified. The challenge in programming on bare metal is the need for an understanding of the hardware in order to configure it properly, although device manufacturers often offer configuration apps or software development kits to ease this process. Another benefit of bare metal programming is efficiency due to the much lower overhead. The main downside for bare metal applications is the challenge in duplicating the common features of a standard OS. For this reason, a bare metal application is suited better for a distributed architecture where the real-time and non-real-time tasks are strictly segregated between the real-time core and the SBC as discussed earlier. The design of the OSAVC PCB consisted of two development efforts. As we had no prior experience designing a PCB we decided to start with a ‘daughter board’—a simpler I/O PCB that could interface with a commercially available development board1 . The first daughter board was hand-wired to determine an initial layout and to kick start the development process. This allowed us to develop PCB layout and assembly skills and at the same time acted as a platform to test sensor driver modules. The I/O board—a simple two layer PCB—required two revisions before its performance was satisfactory. We also selected a vehicle platform for the AGV test bed on which to validate OSAVC. To complement the AGV we purchased a suite of sensors that are widely used for small autonomous vehicles and proceeded to develop a library of sensor drivers and integrated the sensors onto the vehicle. The discussion of the software design is found in Chapter 4.

Viruses disrupt the plant cell cycle, inhibit cell death pathways, restrict macromolecular trafficking, alter cell signaling, protein turnover, and transcriptional regulation, and suppress defense mechanisms. The interference with these processes in the host leads to a wide range of plant developmental and physiological defects . Cultivated grapevines are highly susceptible to a variety of viruses and viroids, which cause significant crop losses and shorten the productive life of vineyards. More than 65 different viral species classified in at least 15 families have been reported to infect grapevines, which represents the highest number of viruses so far detected in a single cultivated plant species . Although these viruses are generally transmitted by plant-feeding insects or soilborne nematodes, they can also be spread through infected propagation material . Grapevine red blotch is a viral disease discovered in northern California in 2008 that has become a major economic problem for the wine industry in the USA . This disease is caused by the Grapevine red blotch-associated virus , a circular ssDNA virus with resemblance to geminiviruses, which infects wine grape cultivars with significant detrimental effects on productivity . The incidence and severity of the red blotch symptoms vary depending on the grape cultivar, environmental conditions, and cultural practices . In red-skinned varieties, GRBaV infections result in the appearance of red patches on the leaf blades, veins, and petioles; in white-skinned varieties, hydroponic bucket they manifest as irregular chlorotic regions on the leaf blades.

GRBaV also affects berry physiology, causing uneven ripening, higher titratable acidity, and lower sugar and anthocyanin content, among others . Consequently, must and wine produced from infected berries present altered flavor and aroma. To date, there is limited information on how GRBaV infections affect grape metabolism. Comprehensive analyses to study specific cellular processes that GRBaV exploits to promote infections in berries are still needed, in particular those that relate to changes in berry chemical composition during fruit development. Grape berry development exhibits a double sigmoid growth pattern with three distinct phases: early fruit development, lag phase, and berry ripening. Most metabolic pathways that promote desired quality traits in grape berries are induced during ripening. The onset of ripening is accompanied by significant changes in berry physiology and metabolism, including softening, sugar accumulation, decrease in organic acids, and synthesis of anthocyanins and other secondary metabolites that define the sensory properties of the fruit . Berry ripening is controlled by multiple regulatory pathways, and occurs in an organized and developmentally timed manner. Interactions between transcriptional regulators and plant hormones regulate the initiation and progression of ripening processes . Like other non-climacteric fruit, grape berries do not display a strong induction of ethylene production and respiration rate at véraison, and the activation of ripening events does not depend primarily on ethylene signaling.

Even though the hormonal control of grape berry development is not completely understood, it is established that abscisic acid , brassinosteroids, and ethylene are positive regulators of ripening processes, while auxin delays the initiation of ripening . In the context of virus–grape berry interactions, dissecting the mechanisms that regulate ripening and plant defenses may provide new opportunities to develop vineyard management strategies to control viral diseases and ameliorate the negative effects on berry quality. In this study, we integrated genome-wide transcriptional profiling, targeted chemical and biochemical analyses, and demonstrated that grapevine red blotch disrupts ripening and metabolism of red-skinned berries. We sampled berries at different ripening stages from vines infected with GRBaV and healthy vines in two vineyards. We identified grape metabolic pathways that were altered in ripening berries because of the viral infection. We determined that GRBaV-induced pathways that are normally associated with early fruit development in berries at late stages of ripening, and suppressed secondary metabolic pathways that occur during normal berry ripening and/ or in response to stress. Using targeted metabolite profiling and enzyme activity analyses, we confirmed the impact of GRBaV on phenylpropanoid metabolism. We identified specific ripening-related processes that were disturbed in GRBaV-infected berries. Remarkably, these processes included alterations in ripening regulatory networks mediated by transcriptional factors, post-transcriptional control, and plant hormones, which lead to berry developmental defects caused by red blotch.

To determine the impact of grapevine red blotch on berry physiology, we studied naturally occurring GRBaV infections in distinct wine grape-growing regions in northern California . We sampled red-skinned grape berries from two different vineyards, one in Oakville and one in Healdsburg . We used multiple vineyard sites to focus on observations consistently made across environments and, thus, to exclude factors associated with specific environmental or cultural conditions. Prior to sampling, vines were screened for the presence of GRBaV and other common grapevine viruses. The appearance of red blotch symptoms on leaves of GRBaV-positive vines and not on those of healthy controls confirmed the initial viral testing. We sampled grape berries from vines that tested positive for GRBaV and negative for other common grapevine viruses. At the same time, we also collected berries from vines that tested negative for all viruses and included them in the study as healthy controls. In order to determine the impact of the disease on berry development and metabolism, we collected GRBaV-positive and control berries at comparable developmental stages: pre-véraison , véraison , post-véraison , and harvest . This sampling strategy also aimed to limit confounding effects due to differences in the progression of ripening between berry clusters of GRBaV-positive and healthy vines. In some cases, we observed that GRBaV-positive vines presented grape clusters with evident uneven ripening . Comparisons between berries from GRBaV-positive vines and healthy controls indicated that, at equivalent stages of development, berries affected by red blotch had reduced soluble solids and total anthocyanins in agreement with previous reports on red-skinned wine grapes . Sampled berries were used for genome-wide transcriptional profiling of viral and grape genes. RNAseq was performed using 3–4 biological replicates of each ripening stage, infection status, and vineyard. We first confirmed the presence of the virus in the berries of GRBaV-positive vines by qPCR amplification of viral DNA . Viral activity in the berries was also assessed by quantifying plant-derived mRNA transcripts of GRBaV genes in the RNAseq data. Plant expression of five out of the six predicted genes in the GRBaV genome was detected in all berry samples obtained from GRBaV-positive vines but not in berries collected from the control vines . The most expressed GRBaV genes in the berries corresponded to V1, which encodes a coat protein, and V3 with unknown function. Expression levels of the GRBaV genes appeared to change as berries ripened. However, we could not determine to what extent the progression of ripening or other environmental factors influenced the plant’s transcription of viral genes because their pattern of variation between ripening stages differed in the two vineyards . Expression of 25 994 grape genes was detected by RNAseq across all berry samples. Principal component analysis was carried out with the normalized read counts of all detected genes. The two major PCs, which together accounted for 42.97% of the total variability, clearly separated the samples based on ripening stage, regardless of the vineyard of origin or their infection status . These results indicated that the inter vineyard variation was smaller than the ripening effect, stackable planters and the overall progression of ripening was similar between berries from GRBaV-positive and control vines. Therefore, we hypothesized that GRBaV infections in berries have altered the expression of particular grape genes and/or molecular pathways, which could subsequently have led to developmental and metabolic defects.While the PCA described above indicated that overall transcriptome dynamics associated with berry ripening were not perturbed by the infection, the lower levels of soluble solids and anthocyanins in GRBaV-positive berries, particularly later in development, suggested that red blotch may affect specific primary and secondary metabolic processes. We therefore focused the RNAseq analyses to identify grape molecular pathways that were differentially regulated as a result of GRBaV infections. We identified grape genes with significant differential expression due to red blotch by comparing GRBaV-positive and GRBaV-negative berries at each ripening stage and independently for each vineyard.

We then looked at the intersection of differentially expressed genes between the two vineyards to identify common responses to red blotch. A total of 932 grape DE genes were found to be consistently down- or up-regulated in infected berries in both vineyards at a given ripening stage, and were classified as GRBaV-responsive genes . On average these GRBaV-responsive genes showed 0.49 ± 0.22-fold changes compared with the healthy controls. Comparing berries at similar ripening stages may have contributed to exclude more dramatic changes in gene expression associated with more pronounced ripening delay due to GRBaV. Key metabolic processes that were suppressed or induced as a consequence of red blotch in ripening berries were identified by enrichment analyses of the functional categories defined by Grimplet et al. in the set of GRBaV-responsive genes . Amino acid biosynthetic pathways were repressed in GRBaV-positive berries, while amino acid catabolic pathways were induced. Changes in carbohydrate metabolism were also observed; in particular, genes involved in glycolysis/gluconeogenesis and starch metabolism had reduced expression in GRBaV-infected berries. Most enzymes involved in phenylpropanoid metabolism are encoded by large gene families. There is also high redundancy among these genes, which ensures the functional integrity and plasticity of the phenylpropanoid-related pathways . Therefore, to test the hypothesis that the red blotch-induced transcriptional changes had an actual impact on phenylpropanoid metabolism, we measured the activity of key enzymes and the abundance of compounds involved in these pathways . We detected significant reductions in activity of seven enzymes that catalyze important steps in the core phenylpropanoid, stilbene, flavonoid, and anthocyanin biosynthetic pathways due to GRBaV infections of berries at three ripening stages . In addition, the first enzyme committed to flavone and flavonol biosynthesis, flavonol synthase , had significantly lower activity at post-véraison and harvest stages . Red blotch altered the accumulation of 17 compounds that result from the phenylpropanoid metabolism and two compounds upstream of this pathway, shikimic acid and gallic acid . Most of these compounds showed significantly lower abundance in the GRBaV-positive berries compared with the controls at later stages of ripening. The main anthocyanins present in grape berries: malvidin-3-O-glucoside, petunidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargodin-3-O-glucoside, and cyanidin-3-O-glucoside, were significantly reduced by red blotch at harvest. Gallic acid, sinapic acid, and quercetin also showed lower abundance in infected berries. Few exceptions to this general suppression of phenolic accumulation were the accumulation of the precursor shikimic acid, which significantly increased in infected berries at harvest, and of resveratrol that showed significantly greater accumulation at véraison and postvéraison. Additional experiments are necessary to understand the accumulation of these two metabolites in the presence of GRBaV: preliminarily, we can hypothesize that the higher abundance of resveratrol is due either to a restriction of subsequent enzymatic steps in stilbene metabolism for which this compound is a substrate, or to the enzymatic hydrolysis of resveratrol glycosides or stilbenoid dimers previously synthesized. The integrated analysis of transcriptomic, metabolite, and enzyme activity data supported a general repression of the core and peripheral phenylpropanoid pathways, which are normally triggered in red-skinned berries throughout ripening and in response to stress . These results suggest that GRBaV infections disrupt secondary metabolic pathways by altering the regulation of berry ripening processes and/or signaling mechanisms related to plant defense. Interestingly, GRBaV infections seemed to have a more a pronounced impact on enzymatic activities and metabolite accumulation than on the expression levels of the genes in the pathway, which in general displayed small fold change differences between healthy and infected samples. This observation further confirms the importance of evaluating metabolic perturbations at multiple regulatory levels.Understanding how plants respond to external stimuli in the field is crucial to improve agricultural traits under naturally fluctuating conditions. Most studies on plant–pathogen interactions are performed with model organisms in the greenhouse or laboratory, which reduce the confounding effects of the environment, but also challenge the reproducibility of the results in the field . Compatible plant–virus interactions in perennial woody crops are complex due to the presence of multiple and systemic infections, tissue and developmental stage-specific responses, differences between species and cultivars, and the combination of biotic and abiotic factors during the crop season . The application of a system biology approach to study red blotch under multiple vineyard conditions allowed us to explore grapevine responses to GRBaV infections in real agronomic settings and to characterize the influence of viral activity on berry physiology.