Horticulture

Across two training phases participants learned about categories A, B, C, and D in Table 1 via inference or classification. Eye tracking was used throughout to monitor participants’ attention to the three feature dimensions and the category label. A test phase examined classification performance and attention profiles as people made novel category contrasts. From prior research we expected classification subjects to learn to ignore the irrelevant dimensions during training; this attention optimization should lead to a difficulty in making novel classifications. In contrast, prior research has demonstrated a tendency for inference learners to acquire within-category information, suggesting a general motivation to learn about all the dimensions in the inference task. Such motivation can potentially produce flexible category representations—that is—ones that support novel contrasts. Measuring eye movements during training will help explain differences in concept flexibility between groups. In contrast to previous studies comparing inference and classification, black plastic nursery pots a change was introduced to our inference training procedure: One of the dimensions, the contrast dimension 3, was never queried .

This change was made to better equate the two tasks; allowing inference participants to ignore task-irrelevant dimensions just like classification learners could. This allowed a test of whether inference learners are in fact generally motivated to learn about category features, or whether the demands of the task, i.e., querying the features, is what draws learners’ attention.Learning AB and CD training performance are shown in Figure 2. The figure shows average classification performance for the classification group and relevant cue inference performance. Both classification and inference groups improved over training blocks, but classification training was easier than inference training, with a higher proportion correct over blocks. The inference learners performed above chance levels in predicting the valid cue, t = 4.46, p < .01, but were marginally lower than the classification group, t = 1.81, p < .10 on the last AB training block. The CD training blocks were similar. Fixations A crucial question was whether inference learners fixated the non-queried dimension during learning. If inference is a more natural learning task than classification, it should motivate a general interest in learning about the category dimensions; fixations should be distributed to all dimensions, regardless of whether those dimensions are queried. However, if it is the attentional demands of the inference task that drive learning about dimensions , then fixations should shift away from the non-queried dimension, since it is no longer immediately relevant for the task.

The latter result would suggest that differences in what is learned via inference and classification are from different attentional requirements, and not motivational factors. Eye fixations will be used to distinguish between these two possibilities. Figure 3 shows proportion of fixations to category label and dimensions over AB and CD blocks, as a function of task. Replicating our earlier work, at the beginning of learning, the average classification learner fixated dimensions about equally. We also observed the expected shift in fixations from irrelevant to relevant dimensions, until irrelevant dimensions were fixated rarely or not at all. At the onset of CD training in block six, there is uneven attention distribution resulting from the learned fixation patterns from AB training, so that in the first trial of CD training, classification learners were not fixating the contrast dimension or the CD relevant dimension. A second attention optimization obtained for classification subjects. Recall that the contrast dimension was never queried. If inference motivates a general interest in the category features, we should observe continued fixations to the contrast dimension, in spite of it now being task-irrelevant. However, Figure 3 shows that throughout learning, inference learners largely ignored the contrast dimension. Although attention to dimensions 1 and 2 remained high throughout learning, even in the first learning block inference learners largely ignored the contrast dimension.

In fact, in the first block of learning, the amount of time fixating the contrast dimension was already significantly less than that of fixating the other two dimensions and the category label . Apparently, inference learners do in fact optimize their attention away from task-irrelevant cues. Attention optimization in the inference task contradicts the idea that inference motivates a general interest in the category features beyond what is strictly necessary. Rather, the results of Figure 3 support the idea that what distinguishes classification from inference is the attentional demand it places on the learner. Learners fixate dimensions because the task requires it and not because of motivational factors. Any motivation there may have been to learn about all of the category features extinguished quickly . Switch-trial performance Eye fixation data have ruled out that inference motivates general interest in category features. By not querying the contrast dimension in the inference condition, we allowed inference learners the opportunity to optimize their attention, just as the classification learners could. In fact, inference learners optimized their attention to just those queried dimensions, ignoring the never-queried contrast dimension. As a result of this manipulation, the inference learners may now struggle to include the contrast dimension, since they largely ignored it during training. On the other hand, although the inference learners never directed their attention to the contrast dimension, because it was not part of the task, they never had to learn to direct their attention away from that dimension either. Rather, the task focused their attention more on the two queried cues, and inference subject learned which dimensions were task-relevant. It is this fact that may still allow inference learning to nevertheless produce flexible attention allocation. By not learning to ignore the contrast dimension, inference learners may be free to use it during the switch trials. Blocks 10 and 11 of Figure 2 show proportion correct for switch-classification. In spite of not deploying significant fixations to the contrast dimension during training, the inference condition nevertheless showed an advantage during the switch trials. In the first block of switch trials, the inference group outperformed the classification group , t = 1.95, p = .064. Likewise, during the second block of switch trials, the inference group outperformed the classification group , t = 2.26, p < .05. Spending a large amount of time fixating a dimension during learning does not seem necessary for using that dimension later in a flexible way. Whatever inference subjects learned during training allowed them to perform well during switch trials. The eye movement results from training showed that classification and inference learners both largely ignored the contrast dimension. It makes sense then that classification learners should fail to use the contrast dimension during the switch trials, 30 plant pot but what allowed inference learners to have more flexible category representations than the classification group? Figure 4 shows learners’ attention allocation to the contrast dimension as a function of trial for the first block of AB and CD training . The figure shows that at the trial level, the largest attentional difference between the two conditions was that the classification learners allocated more attention to the contrast dimension early in learning. The different patterns of attention reflect different reasons the two groups probably ignored the contrast dimension. Inference learners ignored it because the task directed their attention to those dimensions being queried. Since the contrast dimension was never queried, their attention was never allocated to it. Classification learners were in a different position. From their perspective, any or all dimensions could have been important for getting the answer right, so they had to learn to ignore the contrast dimension, as they gradually discovered that the contrast dimension didn’t help them classify As from Bs or Cs from Ds.

We suspect that this is why there is an initial increase in fixations to the contrast dimension in the first CD block, because classification learners attended to it, and then learned that it was useless in classifying Cs and Ds. Classification learners’ fixation results reflected a learned inattention to the contrast dimension, which probably caused their difficulty in attending to the contrast dimension during the switch trials.We began with the observation that real-life categorizers can make novel category contrasts and that information learned about one set of categories transfers to another without difficulty. This observation seemed to be at odds with the robust finding that people in classification experiments tend to optimize their attention to the fewest necessary dimensions. Such optimization would necessarily force learners to reallocate attention when previously irrelevant dimensions at once become relevant. To resolve the contradiction that people can make novel category contrasts on one hand and but also tend to optimize attention on the other, we looked to other types of learning tasks they may produce classification performance that is less optimal but more flexible overall. Inference training seemed like the best candidate. There were two reasons for this. The first was based on evidence that inference yields a special type of processing in humans; although the exact source of this special processing was until now not entirely clear, classification learning has been found to cause humans to attend to diagnostic information and inference learning can cause learners to focus on within category correlations and prototypical features. We imagined that such differences may reflect that inference is a more typical learning task than classifying, and it isn’t hard to imagine how familiarity in the learning task can lead to greater ease and flexibility in using the acquired information. Our second hypothesis for how inference learning could yield flexible category representations was based on differences in attentional demands of inference and classification. Whereas most classification tasks allow learners to ignore some of the irrelevant dimensions, in the typical inference learning experiment, all of the dimensions are queried several times throughout training. Focusing people’s attention on all of the dimensions in this way may cause people to look at all dimensions on every trial, in order to prepare for future queries. In fact, the eye tracking results from this study show that never querying one of the dimensions allows the inference learner to optimize their attention to only those task-relevant dimensions, i.e., those dimensions that are sometimes queried. As it turned out, our initial hypotheses about inference learning were not exactly right. Our data showed that inference subjects very quickly ignored the never-queried dimension. Significant differences in fixations to the contrast dimension were found within the first learning block. Apparently, attending to the contrast dimension during training was not necessary for creating flexible category representations. Rather, what gave subjects the advantage in switch-classification trials is that they never had to learn to ignore the contrast dimension, as the classification subjects did, as evidence by the much larger drop in attention to the contrast dimension from the beginning to the end of training in the classification condition. In other words, classification subjects were harmed in their task by their learned attention profiles, but the inference subjects were not. Such a finding is in fact consistent with theories of attention and category learning. Several models, for example, RASHNL Kruschke and Johansen , and EXIT Kruschke , which are based on Macintosh’s theory of learned attention, propose that attention weights are learned for a given set of inputs. In these models, if feature inputs are irrelevant, or if for other reasons the features increase the number of classification errors committed, the attention system will direct attention away from those features in favor of others. These attention mechanisms help the models explain a large array of blocking and highlighting phenomena in addition to benchmark category learning data. They also explain why itis that our classification subjects failed to redirect attention to the contrast dimension during the switch trials. Beyond supporting certain theories of categorization and attention, our results underscore an important difference between the attention profiles acquired through trial and error learning and those that arise out of task goals. It seems that ignoring features as a result of discovering that they are statistically irrelevant over numerous trials is qualitatively different than cues that are never queried, and are thus irrelevant for the task. Thus, how the learner acquires an attention profile is as important as the attention profile itself.The 2020–2025 Dietary Guidelines for Americans encourages the intake of a variety of plant-based foods including nuts and berries. With the goal of increasing current knowledge on nuts and berries, as well as addressing research challenges and opportunities, the Nuts and Berries Conference: Pathways to Oxidant Defense, Vascular Function, and Gut Microbiome Changes was held on 5 to 6 May, 2022 at the University of California, Davis. Tree nuts and berries were selected as the focus of the conference for their unique composition, bioactivity, and multitude of associated health-promoting qualities.

The soil was formed in place from the weathering of softly to moderately consolidated granitic sediments. The particle size distribution of the surface soil is 63% sand, 25% silt and 12% clay. At the onset of the experiment, boron tissue levels were in the adequate range, 40 ppm. In both experiments, drip irrigations during the season were based on a schedule using historical evapotranspiration and developed for raisin vineyards in the San Joaquin Valley . The irrigation source was high-quality pump water with a boron concentration less than 0.1 ppm. The experimental design and methods were identical in both vineyards, except that the 1/16-poundper-acre boron treatment was omitted in the second vineyard. The Fresno County trial was discontinued after tissue samples were taken at bloom and veraison in 1998.At both the Tulare and Fresno county sites, boron uptake was rapid when fertilizer was applied in the spring. In both vineyards, applying boron at 2/3 or 1 pound per acre increased the boron concentration in blades by bloom, 3 weeks after application. Boron increased further in blades by veraison . In the Tulare County vineyard, boron in bloom tissue increased from a questionable deficiency range to adequate; at the Fresno location, raspberry container size boron in bloom tissue increased from 40 ppm to 54 ppm, a dramatic increase considering boron fertilizer was applied just 3 weeks prior. This indicates that boron uptake is rapid.

None of the fertigation treatments resulted in either symptoms of boron toxicity or deficiency. Applying boron at 1/3 pound per acre or less did not significantly increase boron tissue levels by bloom or veraison at either site the first year. Fertigation over consecutive years was evaluated at the Tulare County location. Boron in grapevine tissue continued to increase with consecutive years of application. At the higher fertilizer rate , boron levels in blades increased from 35 ppm in control vines to about 60 ppm. We speculate that continuing with annual applications of 1 pound boron per acre would result in toxicity within 4 to 5 years. The 1/3-pound-per-acre rate significantly elevated boron in blades by veraison of the second year to adequate levels . There were no visual signs of toxicity in any of the fertilizer treatments, even when boron was applied at 2 pounds per acre in a single application. Boron levels in tissue remained unchanged 2 years after fertilization was discontinued at the Tulare County location . Rainfall during this experiment was below normal, which helped minimize leaching. Also, well-managed drip irrigation minimizes leaching. Under drip irrigation, salts tend to accumulate near the soil surface and 2 to 3 feet away from the drip line, with minimal water and salt movement below the root zone when irrigations are accurately scheduled . Boron concentrated more in the blades than in the petioles in response to fertilization.

At the onset of the Tulare County experiment, boron concentrations in petioles and blades were similar at 31 ppm and 34 ppm, respectively. Fertilizing with 1 pound boron per acre for 2 consecutive years resulted in a 25% increase of boron in petioles but a 76% increase in blades . Allfertilizer treatments increased boron in blades more than in petioles, indicating that blades should be sampled when monitoring the vines’ boron status following fertilization. Potential boron toxicity values at the time of sampling during the bloom period are 80 ppm for petioles and 120 ppm for blades, and in mid- to late summer are 100 ppm for petioles and 300 ppm for blades.Annual boron fertigation at 1/3 pound per acre elevated grapevine tissue levels from questionable to the adequate range within 2 years . In addition, tissue boron levels remained unchanged 2 years after fertilization was discontinued. This is probably because leaching was reduced by two factors: below-normal rainfall and accurately scheduled drip irrigations. After fertilization, boron was concentrated more in blades than in petioles, indicating that blades are the best choice for monitoring toxicity. Blade samples should be monitored on a routine basis and fertilizer amounts should be adjusted accordingly to avoid boron toxicity or deficiency. The results of this research can be applied to other drip-irrigated vineyards in the San Joaquin Valley under similar conditions: rapidly drained soils, high quality irrigation water, and low boron content in soil, water and vine tissue. In other regions of the state where winter rainfall is much higher, there would presumably be more leaching of boron fertilizer during winter months and less carryover time after fertilization is discontinued. In contrast, less leaching and greater carryover of boron would be expected in areas of less rainfall or on soils with finer texture and higher water-holding capacity.

The amount of boron fertigation used in a maintenance program will vary with leaching potential. These variables underscore the importance of monitoring boron in tissue when developing a long-term fertilization program.Stroke recovery is an exhausting, isolating, and expensive process. Physical therapy to recover limb function and neural pathways is the most expensive part of this process due to the need for frequent one-on-one appointments with physical therapists over the course of months or even years that may not be covered by insurance . In addition, many stroke patients prefer athome rehabilitation whenever possible to allow for more schedule flexibility and to avoid the need to find transportation to what are often extremely distant specialist centers . The need for frequent one-on-one appointments can be reduced through robot-assisted therapy , which improves the quality of both group therapy and at-home physical therapy .Many previous studies have utilized common stroke rehabilitation techniques to create robots to retrain motor movement in the upper limbs by utilizing TST and repetitive motions similar to what a physical therapist might assign as an exercise for a patient . TST can lead to muscle pain and fatigue, however RAT and non-RAT TST have been shown to result in the same level of pain and fatigue for patients . In addition, RAT can include systems to monitor fatigue and pain. Patients also rate higher levels of enjoyment and interest in physical therapy when utilizing these robots, so the only cons in these robots come from their common design traits . For stabilization and ease of calibration, these systems are often large and heavy so the position of the exoskeleton or robotic interface is easily known . This leads to increased costs for consumers in both purchasing the additional bulky casing around the robot as well as creating a space for such personalized gym equipment. In addition, these heavy systems are incredibly difficult for patients to set up at home and can be very confusing . Robotic physical therapy systems also usually deal with a single joint to simplify their control schemes . This means most RAT TST devices only utilize specific muscle groups in a specific part of the body. However, raspberry plant container most people in stroke recovery are attempting to strengthen neural pathways in entire limbs, so RAT for multiple muscle groups can take up a huge amount of space and too much money for at-home usage throughout all of recovery to be feasible. All of these considerations have led to a market where only physical therapy centers and the few who can afford to buy, store, and replace such customized devices are able to utilize them.Drawing therapy is effective because it requires the coordinated and deliberate use of several muscle groups together. In fact, the steadiness, active range of motion, and spatial awareness required to draw a circle have led to the process being used as a common metric of stroke rehabilitation . Increased circle size can be linked to increased active range of motion, as multiple muscle groups must be fully engaged to create a large shape . The roundness of the circle is connected to both hand stability and coordination of muscle groups, as uncoordinated muscles lead to very eccentric ellipses . Creating a robot that can automate this test would allow assessments to be conducted at home or in group settings, reducing the frequency of physical therapy sessions, allowing check-ins to be conducted online, and creating more data for physical therapists to monitor recovery between sessions.

All of these effects would greatly reduce the costs of physical therapy for patients and create better quality remote health-care. Drawing therapy can also help with the psychological issues that can come with a stroke by aiding patients in creative expression and teaching new skills . Many stroke patients are frustrated with their sudden loss of fine motor control, so providing an avenue to gradually regain control where patients can see visible progress helps ease their concerns. Stroke patients often feel isolated and have identity issues due to their loss of motor function and difficulty with the coordination necessary to engage in old hobbies . Drawing and other forms of expression in a group setting can help mitigate these symptoms by providing community and helping the patient develop new hobbies. These hobbies in turn can help motor function as the repetitive and precise motor movements necessary to create art can improve coordination and aROM. Additionally, many artists and professionals who want to draw steady straight lines or curves without using software in a digital medium may benefit from a device that would aid in this task.There are no commercially available assistive devices designed to aid in drawing therapy due to the complexity of predicting both motion and the physical location of the hand to ensure images are drawn as intended, however, it has been proposed that this could be resolved by utilizing surface electromyography sensing, internal measurement units, or similar sensors . Similarly, there are no commercially available robots designed to collect data during circle drawing tests, a common way to determine the active range of motion and recovery of stroke patients . Through our complex controls scheme, we hope to continue to develop a robot with a high enough prediction accuracy, at a marketable cost, to eventually create a robot with the potential of aiding physical therapy centers, stroke rehabilitation patients, professional artists, hobbyists with unsteady hands, and professional craftsmen. We propose a design for a robot that combines sEMG sensing, internal measurement units, and drawing therapy techniques to assist in physical therapy of stroke patients with upper limb weakness. This robotic physical therapy will strengthen neural pathways for motion of the entire arm, providing a way for circle drawing tests to be accurately conducted and drawing therapy to be less frustrating at home. Our design aims to be compact, affordable, and accessible for at-home usage, allowing users to draw with assisted-as-needed technology that will incorporate feedback on their task-specific training , while they attempt to improve their active range of motion or quantify their recovery process.In 2021, we created a series of Arduino robot prototypes as proof of concept for this idea. As part of a course, these initial prototypes were created under extreme budget and time constraints. However, this also means that a final product will likely be affordable for the average consumer; we calculated that our final prototype’s components only totalled a cost of $69, with the potential for a total of $34.39 each in small batch production. Arduino prototypes are on display in Figure 1.Due to the initial budget constraints, our initial prototypes were created with Arduino components and 3D printed parts, as we had access to these from prior coursework. The robot consisted of three main sections: the circuit board, the servo motors, and the 3D printed arm. Our initial circuit board was an Arduino Uno, chosen due to its versatility with hobbyist parts, its utilization of the C++ programming language which we were familiar with, and the fact that we already had access to it. We 3D printed a case for the board to protect it and act as a weight to ensure users could not easily knock the arm over. The purpose of this component was to store code, process the input from our sensor scheme, and anchor the rest of the robot. In addition, we added an internal power supply to this prototype to increase portability. The servo motors are the method through which the robot stabilizes the user’s motion. We used two metal sg90 servos with analog feedback, as this servo model is very cheap, hardy, and can read position while allowing for a large range of motion.

The Kruskal–Wallis analysis of variance, a non-parametric equivalent of ANOVA, was used to compare groups across species. When significant, this test was followed by pairwise Mann–Whitney U-tests, and the p-values were corrected for multiple comparisons using the Bonferroni technique. In this and subsequent analyses, the Bonferroni adjustment multiplies the p-values we obtained by the number of comparisons performed . For the morphological comparisons, we tested the following models: number of bristles ¼ f and ovipositor area ¼ f. The Kruskal –Wallis analysis of variance was used to compare groups across strains. When significant, this test was followed by pairwise Mann–Whitney U-tests, and the p-values were corrected for multiple comparisons using the Bonferroni technique.We sought to determine the susceptibility of four types of fruit to each of the four species of flies. They included a fruit with relatively soft skin and fruits with much tougher skin . Our aim was to discover whether females of each species were capable of penetrating the intact skin of ripening fruit. We were aware, however, that the failure of females of a given species to lay eggs in a fruit could be a sign of either their inability to puncture the skin or a general aversion to fruit of that variety.

To distinguish between these possibilities, we counted separately the number of eggs in the exposed region of the fruit, blueberry plant pot which included the pit and any regions where the skin had naturally broken, and the undamaged fruit body. The absence of eggs in both the easily accessible exposed region and the unexposed area would indicate an aversion to the fruit, while the presence of eggs in the exposed area, and their absence wherever the skin was intact, would be consistent with the hypothesis that the skin was acting as a barrier. We found that all four species of flies laid eggs in the exposed portion of the four fruit varieties , indicating that none of the species had a strong aversion to using these fruit as larval food sources. The eggs that were laid in this region were found in a number of positions. The egg was sometimes embedded in the fruit with the respiratory filaments pointing outwards, but it could also be found in the opposite orientation or lying sideways . In the raspberry assay, a significantly larger number of eggs were laid in the exposed region by D. mimetica and D. biarmipes females when compared with D. suzukii and D. subpulchrella . This may be a consequence of the fact that the latter two species appear to have had no difficulty puncturing intact raspberry skin , and therefore did not gravitate to the far smaller exposed area in the pit.

These results contrast with the data from both varieties of grapes, where all four species laid eggs in the exposed portion, but D. suzukii laid over three times as many as the other three species. In the case of cherries, both In the raspberry and cherry assays, only D. suzukii and D. subpulchrella laid eggs in areas of the fruit where the skin was intact . There was no significant difference between these two species in the number of eggs laid in either fruit. We also found cases of punctures that lacked egg filaments , a possible indication that an egg-laying attempt was unsuccessful. These were relatively rare in both the raspberry and cherry assays , suggesting that neither species had much difficulty laying their eggs in these fruits. The finding that D. subpulchrella flies are capable of puncturing the skin of cherries and raspberries raises the question of whether this species, like D. suzukii, could be a threat to soft fruit industries.We therefore endeavoured to determine whether D. subpulchrella larvae could survive and develop in the pulp of these fruits. For the cherry assays, we observed an average of 19.9 living larvae in each fruit 5 days after the assays were set up . For the raspberry assays, we observed the bottles 14 days after they were set up and found that an average of seven adult flies had emerged from pupae. This indicates that larvae can survive in both of these fruits and, at least in raspberries, can develop to adulthood. No punctures of any form were found in the intact regions of either raspberries or cherries exposed to D. mimetica and D. biarmipes. In conjunction with the results of §3a, the findings are consistent with the hypothesis that these species rarely or never puncture intact skin even in soft-skinned fruits and that the propensity to do so evolved in the ancestor of D. suzukii and D. subpulchrella .

The results of the two grape assays differed strongly from the other fruits. Only D. suzukii laid eggs through the intact skin of any grapes, but these were relatively rare in the Thompson grapes, and entirely absent from the red grapes. However, grapes exposed to D. suzukii showed numerous punctures without the telltale sign of egg filaments . The egg-free punctures were often found in clusters.We determined, in a separate experiment , that punctures without protruding egg filaments were considerably smaller than those with filaments . These findings are consistent with the hypothesis that D. suzukii females tried to lay their eggs in the grape bodies and often succeeded in making small holes in the skin, but in most cases failed to insert an egg. However, from a pest-management perspective, it is important to note that punctures without filaments can be sites of secondary infections. By contrast, the intact body regions of grapes exposed to flies of other species never showed punctures. However, in a few cases, we found evidence of ‘slashes’ in the bodies of grapes in bottles with either D. subpulchrella or D. suzukii . The slashes, which were not found in control fruits that were not exposed to these flies, may represent unsuccessful attempts to puncture the skin. Our results demonstrate that while all species have an affinity for grapes and will lay their eggs in exposed regions of the fruit, they rarely penetrate the intact skin of the two varieties we tested. The evidence of egg-laying attempts, plastic gardening pots especially in D. suzukii, suggests that a property of the skin may make the insertion of eggs difficult. The D. suzukii findings are consistent with the results of previous research.The fruit susceptibility experiments suggest that the ability to puncture fruit skin first evolved in, or at least was strongly enhanced in, the ancestor of D. suzukii and D. subpulchrella . We tested whether this capacity was accompanied by concomitant phenotypic changes in the structure of the ovipositor. We separated, mounted and imaged ovipositor plates from two strains of each of the four species and compared them on a number of traits. One categorical distinction between ovipositors in the D. suzukii and D. subpulchrella clade, on the one hand, and D. mimetica and D. biarmipes, on the other, is the presence of thick, pigmented bristles on the former, explaining why this type of ovipositor has been referred to as ‘serrated’ . These modified bristles are found close to the distal tip of the ovipositor , which comes into contact with fruit, while the more proximal bristles are unmodified and resemble the homologous bristles present on the ovipositors of other species, referred to in the literature as ‘thorn bristles’.

The evolution of modified bristles in this clade was accompanied by a significant increase in the overall number of thorn bristles . Interestingly, the three to four marginal bristles of the D. subpulchrella ovipositor tip are generally of the thick, pigmented variety, whereas those in the equivalent region of the D. suzukii plate are almost always unmodified . However, on the lateral side of the ovipositor, D. suzukii has more modified bristles than D. subpulchrella, and the total number of modified bristles does not differ significantly between the two species . The evolution of the serrated ovipositor was accompanied by a considerable increase in the ovipositor size , with the two species with the serrated variety having ovipositor plates with approximately three to four times the area of the two other species’ plates. By contrast, comparing the same species on wing area, often used as a proxy for body size, shows an increase of only 1.6-fold to twofold in D. suzukii and D. subpulchrella . Therefore, while there has been an overall increase in body size in D. suzukii andD. subpulchrella, there has been a disproportionate increase in ovipositor area. In order to have a quantitative method of distinguishing ‘sharp’ ovipositors from ‘blunt’ ones, we measured the length towidth ratio .While this ratio does not vary significantly among D. mimetica, D. biarmipes and D. subpulchrella , it increased markedly in D. suzukii, giving the egg-laying organ a pointed, streamlined shape . We used EFA to compare the ovipositor shape among the four species, focusing on the distal half of the structure , because this is the portion of the egg-laying organ that comes into contact with the fruit during an attempted puncture. EFA produced a series of four Fourier coefficients for each of the 25 harmonics used in the analysis. Using the PCA, we calculated PCs for each of the ovipositors . The vast majority of the variation was captured by the first two PCs, which are plotted in figure 5a. Three groupings are apparent in this figure, each indicated with an ellipse: the D. mimetica and D. biarmipes strains; the D. subpulchrella strains; and the D. suzukii strains. To help to visualize what the individual PCs represent, we used the inverse Fourier transform to reconstruct ovipositor outlines on the basis of each PC . The outlines suggest that the first PC represents the difference between a sharp and blunt ovipositor. Indeed, we found that there is a strong negative Pearson product correlation between the first PC and length to width ratio. Therefore, the horizontal axis of figure 5a shows that D. biarmipes and D. mimetica, the two species that did not puncture the intact skin of any of the fruits tested, both have relatively blunt ovipositors and do not differ significantly on this variable . Drosophila subpulchrella has a sharper distal ovipositor, whereas the ovipositor of D. suzukii is the sharpest of all. A positive value for the second PC is indicative of an ovipositor with a bulb at the tip, as is seen in D. subpulchrella . Not surprisingly, there is a strong, significant difference between the D. subpulchrella strains and those of the other three species on this PC , while the latter do not vary significantly among each other on this variable . The third PC , which only explains about 4% of the variation , appears reflective of the asymmetry in the direction in which the tip is pointed. The remaining PCs each accounted for less than 1% of the variation, and the use of an omnibus Kruskal–Wallis test found no evidence of significant differences between strains. We present a model for the evolution of a modified ovipositor in figure 6 . The appearance of enlarged bristles was accompanied by a change in the shape of the ovipositor tip, which became significantly sharper. Following this event, in one species, D. subpulchrella, a distal bulb appeared, while in the second species, D. suzukii, the ovipositor became even sharper and increasingly streamlined. The latter is associated with the ability of D. suzukii to puncture the skin of the grape varieties we assayed, which was not shared by the other species in this study.While there were numerous Japanese studies of D. suzukii infestations during the first half of the twentieth century , these reports were not widely disseminated among Western researchers. When this species was first collected in California in 2008, it was not readily identified and was initially mistaken for D. biarmipes. At the time, the only known drosophilid pest in the Western Hemisphere was the distantly related Zaprionus indianus, which had first been reported in North America in 2006, 7 years after its identification in Brazil . Although Z. indianus is a formidable threat to agriculture , it can only colonize exposed fruit. Other distantly related species, however, such asthe leaf-mining fly Scaptomyza flava, possess serrated ovipositors. In this species, the ovipositor is used to puncture a leaf, and the female then feeds on its contents.

The sensitivity of B. cinerea conidia to SO2 increases two- to fourfold for every 10°C increment between 0 and 32°C, because of the effect of temperature on SO2 absorption on the fruit, fungi and surrounding packaging . SO2 has mainly been used to control gray mold disease of table grapes, but the low application rates applied as part of the total utilization technique have not prevented the emergence and spread of brown spot during cold storage . The objectives of our two-part study were to evaluate, in vitro, the effect of temperature and SO2 concentration over time applications on fungal colony growth of three Cladosporium species , and to determine the efficacy of different SO2 concentrations over time in inhibiting the growth of Cladosporium species on artificially inoculated Redglobe berries.Cladosporium species were isolated from brown spot–symptomatic berries grown in Delano, California, from 2013 to 2015. Species isolates were obtained from symptomatic tissue placed on 150 by 15 mm petri plates with 3% rose bengal potato dextrose agar amended with 500 ppm tetracycline and 300 ppm streptomycin. Cladosporium colonies were hyphal tipped after 7 days to produce pure cultures for species identification. PCR amplification of the actin gene using primers ACT-512 and ACT- 783 and DNA sequencing confirmed the identity of species .

Species isolates were maintained in Dr. W. Douglas Gubler’s laboratory, Department of Plant Pathology, UC Davis, square pot as of June 2016. Pathogenicity was demonstrated when species isolates were inoculated and re-isolated from lesions on Crimson Seedless berries following standard protocols . Asymptomatic, nonwounded berries were removed from clusters with the pedicel still intact by clipping with sterile scissors. Berries were then vigorously washed in a 0.5% potassium chloride and 0.1% Tween 20 solution to remove surface debris. Berries were surface disinfected first in a 70% ethanol solution for 30 seconds, then in a 10% bleach solution for 5 minutes, and dried in a sterile laminar flow hood . Dry berries were aseptically distributed in triplicate on sterilized polyethylene chambers on sterilized polyethylene grids at 2°C with high relative humidity . RH was obtained with paper towels moistened with sterile deionized water and placed below a plastic grid. RH was measured with a humidity sensor .To determine the effect of temperature and SO2 on fungal growth, potato dextrose agar plates were inoculated with one of three species: C. ramotenellum, C. cladosporioides or C. limoniforme. Inoculum was grown on 2% PDA at 23°C for 5 to 7 days. Plugs of colonized PDA obtained with a sterile 4-mm cork borer were placed on a 2% PDA plate, spore side down. The baseline for all measurements was 4 mm. Inoculated plates were incubated in a polyethylene chamber at 23°C for 13 hours. The petri plates were moved to a fumigation chamber and exposed to the three SO2 treatments while still lidded.

SO2 concentrations were measured using passive colorimetric dosimeter tubes and a portable SO2 detector that continuously measured SO2 concentration inside the fumigation chambers . Dosimeter tubes were taped to chamber walls opposite the SO2 flow as well as inside of a petri plate. Untreated controls were inoculated as previously described, received no SO2 exposure but were treated and incubated identically. For each species, three petri plates were inoculated and treated in triplicate and were incubated at 2°C and −2°C for up to 32 days after treatment. Radial measurements of fungal colonies were taken to assess the effect of SO2 concentration over time on mycelial growth. Colony measurements were taken from the reverse side of the lidded petri plate. Using a polyethylene ruler, two perpendicular diameter measurements were made and averaged to determine overall colony size. The diameter of the mycelium plug inoculum was included as the minimal measurement for all treatments. Measurements were taken every 6 to 8 days during incubation at 2°C and −2°C.Inoculated berries were not wounded prior to inoculation. Inoculum was prepared with 14-day-old cultures grown on PDA. Spores were suspended in 0.5% potassium chloride and 0.1% Tween 20 in sterile distilled water. Spore density was determined using a hemocytometer, and the suspension was adjusted to 1 × 107 spores/mL by the addition of sterile deionized water. The shoulder of each berry was inoculated by placing 10 μL of spore suspension within a 4-mm Vaseline ring, to prevent the inoculum from moving. Berries were positioned with the inoculated shoulder facing up. Untreated controls consisting of berries inoculated with sterile deionized water were included for each temperature and SO2 treatment. Berry lesions on unwounded berries were measured after 28 days of storage at 2°C.Twenty-four hours after berries were inoculated with one of the three Cladosporium species, they were exposed to three concentration-over-time treatments of gaseous SO2: 100 ppm-h , 200 ppm-h or 400 ppm-h . SO2 concentrations were measured as previously mentioned. Dosimeter tubes were taped to chamber walls opposite the SO2 flow. For both studies, fumigation chambers were Sterilite containers modified to allow for a rubber tube to flow SO2 into the chamber.

Chambers were placed inside a biological safety cabinet during treatment. Chamber lids were opened once a treatment was concluded and the inoculated berries on a grid/mycelial plugs on petri plates were placed into polyethylene chamber to be stored in cold storage.Radial growth of Cladosporium colonies was analyzed using a linear mixed model approach for all three species using the R package lme4 . The mixed model used a Gaussian error distribution and consisted of SO2 treatments, temperature and species as fixed effects and replicate as a random effect. We calculated the estimated marginal means and computed all pairwise comparisons using Tukey’s honestly significant difference test. For the berry study, we analyzed the proportion of infected berries with a linear mixed model approach using a Gaussian error distribution for all species, with SO2 treatments and species as fixed effects and replicate as a random effect and computed all pairwise comparisons using Tukey’s HSD test.Radial colony growth of the three Cladosporium species on petri plates with PDA was significantly reduced by the 400 ppm-h SO2 treatment, as seen in figures 2 and 3. The 400 ppm-h concentration was most effective against C. cladosporioides when petri plates were incubated at −2°C, which resulted in no radial growth. The 400 ppm-h was also effective in slowing C. cladosporioides colony growth at 2°C; radial growth grew only from 4 to 9 mm on average. Radial colony growth of C. limoniforme and C. ramotenellum was also slowed down at 400 ppm-h,growing from 4 to 5 mm and 4 to 6 mm, respectively, at −2°C. However, the same treatment was less effective at 2°C, where, on average, fungal radial growth reached 10 mm for C. limoniforme and 12 mm for C. ramotenellum after 30 days. At the lower SO2 ppm-h , there was no significant difference in radial colony growth between any of the treated species and the untreated controls by 30 days at 2°C. All species after 10 days at −2°C had reduced colony growth for all concentrations compared with those incubated at 2°C. The colony growth of C. cladosporioides at −2°C was slower than that of the other species; and it was also slower than the colony growth of C. cladosporioides incubated at 2°C, illustrating the effect of temperature on rate of growth.In the berry study, all Cladosporium species caused disease in untreated control berries: C. cladosporioides caused disease in 65% of berries, C. limoniforme in 55% berries and C. ramotenellum in 75% of berries . By contrast, SO2 treatments significantly reduced disease incidence by Cladosporium species on inoculated Redglobe berries. The 100 ppm-h treatment reduced disease incidence to less than 25% of berries for all species. The 200 ppmh treatment was the most effective, blueberries in containers in that it eliminated disease for all three Cladosporium species tested. In the 400 ppm-h treatment, there was less than 5% disease incidence in berries inoculated with C. ramotenellum and no infection occurred in berries inoculated with C. cladosporioides or C. limoniforme. Nevertheless, there was no statistically significant difference in the proportion of infected berries between the 100, 200 and 400 ppm-h SO2 treatments of the three Cladosporium species tested .

Our results demonstrate that a single application before cold storage of at least 200 ppm-h SO2 at cold storage temperatures may be an effective tool for reducing brown spot disease incidence on grape berries, although the ability of mycelium of Cladosporium species to grow on PDA growth medium in petri plates with an SO2 application was somewhat variable. We have demonstrated SO2 of 200 ppm-h and 400 ppm-h essentially prevent disease development in Redglobe berries from the three Cladosporium species tested. C. cladosporioides exhibited tolerance at 100 ppm-h, whereas 100 ppm-h was effective against C. ramotenellum and C. limoniforme. While mycelial growth on PDA was reduced with a treatment of 400 ppm-h SO2, mycelial growth was not completely eliminated. Additionally, C. ramotenellum grows faster than the other species on a petri plate with PDA, regardless of temperature or SO2 treatment. Unsurprisingly, in the berry study, inoculation of C. ramotenellum resulted in the highest disease incidence in the untreated control berries. Our study also confirmed the critical importance of maintaining table grapes below 0°C and above their freezing point to maximize their storage life potential .These results are promising for the control of brown spot during long-term storage and export of Redglobe table grapes. Although not much is known about the natural berry infection by Cladosporium, we do know that infection can occur on the surface through the epidermis and may also be a latent infection where infection starts from within the berry. Berries used in our experiments were surface sterilized before inoculation so we could be sure that infection occurred through the epidermis with our inoculum; thus, we can conclude the SO2 application prevented disease from of the Cladosporium surface inoculum. However, SO2 may not be adequate to prevent infection resulting from latent infection, causing internal rot of the berry and inoculum production, but may inhibit the resulting spread if applied after the emergence of fungal growth . This work demonstrates a potential management strategy of brown spot post-harvest. In practice, multiple factors can influence the amount of gaseous SO2 that comes into contact with the berry surface, including air flow in storage, sorption of packing materials and the amount of condensation on berries, which can absorb gasses. Additionally, a single application of SO2 is typically not as effective as multiple SO2 applications, but multiple applications may not be feasible during transport . Total utilization technique focused on gray mold control applies SO2 at 100 to 150 ppm-h, which may be too low to be effective against brown spot, especially under cold storage conditions. Future studies should be done to improve sampling and the detection of brown spot in the field prior to harvest as well as during post harvest, so this research can be applied to known compromised lots in transport and better determine the efficacy of this practice under commercial conditions.Macrotunnel production has been increasing in coastal counties of California and is poised for expansion due to its recent adoption as a standard practice by the U.S. Department of Agriculture . In high tunnel production, crops are grown within plastic-covered structures to enhance crop performance, extend production seasons and to protect crop quality. While most caneberries, some strawberries, cut flowers, herbs and leafy greens are widely grown under plastic in California, contributing $1 billion to the state’s economy, in other states small fruits, melons and nuts are also grown in high tunnel systems. This interest in plasticulture tunnels is driven by many factors: increased production due to season expansion; reduced exposure to deleterious weather events; consumer demand for fresh, local produce; and national interest in reducing transportation-related greenhouse gas emissions, amongst other concerns. Unfortunately, it comes at a time when climate-induced weather pattern changes, particularly shorter-duration, higher-frequency storm events, are expected to become the norm . The plastic covering hoop structures can reduce the available permeable surface of a field’s production area by over 90%, which increases the volume of water likely to run off a field in a storm event . During rains, water intercepted by plastic covers is channeled into post rows , accelerating soil erosion, especially on slopes, which ultimately degrades surface water quality. In California, surface water quality is regulated by the State Water Resources Control Board through the Irrigated Lands Regulatory Program .

Conversely, in under cropping, where there is excessive vigor or reduced crop level, this is not necessarily deleterious for speed of ripening . However, it may be a wasteful management of resources if there is not a trade-off with farm-gate prices. Given the later fruit development of grapevine and the grape chemistry requirements for red wine making , the length of the growing season is often a limitation for achieving the desired ripening and vintage quality in cool climates . Thus, yield is often sacrificed to balance source-to-sink ratio in favor of accelerated fruit ripening or to mitigate the effects of early fall frosts . Although the initial control of crop level comes during pruning , the number of dormant buds retained at pruning time is maintained constant through the years in warm climate regions. Cluster thinning is a management practice fine-tuned each year to achieve vine balance . Excess vine vigor was linked to deleterious effects on berry flavonoids . This effect could be exacerbated with high nitrogen amounts inhibiting anthocyanin biosynthesis , the absence of water stress, or changes of cluster microclimate due to mutual shading , and thus, not by the under cropping itself. Therefore, growing blueberries in containers grapevine canopy development is managed through the control of inputs, vine spacing, irrigation, rootstocks, pruning, leaf removal, hedging, or cover crops, among others.

A great part of the carbon assimilated through the growing season is incorporated into cellulose or lignin in roots, trunks, and shoots . However, resumption of a new season’s growth depends on the carbon stored as nonstructural carbohydrates, majorly in the form of starch, but also soluble carbohydrates such as sucrose, glucose, and fructose . Roots are the greatest sink of non-structural carbohydrates and root-derived carbohydrates constitute the principal reserve source for annual resumption of growth in the spring. The grapevine’s capacity for replenishment of these carbohydrate reserves increases at mid-ripening, when canopies are at their maximum and fruit demand slows down sugar accumulation in perennial parts . Therefore, the loss of photosynthetically active leaf area or excessive number of clusters may impair the reconstitution of reserves . In addition, high crop levels may delay fruit maturation and shorten the post-harvest period and subsequently reduce the time needed to accumulate reserve carbohydrates. Grape growing systems based on high yields are typically in warm to hot regions, relying on early harvest to replenish these reserves. However, it is common that excessive yields lead to a reduction in yields the following season . Loss of photosynthetically active leaf area or excessive number of clusters may deplete these reserves. High crop levels may reduce the reserve carbohydrate accumulation and delayed fruit maturation and may shorten the post harvest period. Therefore, the grapevine may not have sufficient time to accumulate carbohydrates for the following season in cool climates. Conversely, there is not consensus in literature regarding the effect of high cropping levels on storage reserves .

This was explained by sink limitation as the grapevine was able to maintain equilibrium by adjusting physiological processes . In addition to the modulation of berry ripening and storage reserves, other compensatory mechanisms have been described in response to over and under cropping. Components of yield, which include clusters per vine, berries per cluster, and berry mass, are susceptible to change together with berry ripening in compensation of each other . Although grapevine pruning, canopy, and crop load management are the most frequently reported case of study for source-to-sink ratios, most studies may not offer direct observations , enough combinations, duration of the study, or range of source-to-sink ratios to respond to some fundamental questions. The aim of this study was to determine the in-season and carryover effects of carbon source and sink imbalances in grapevine. Specifically, we investigated the combined effects of defoliation and fruit removal on components of yield, canopy area, and seasonal integrals of leaf gas exchange, shifts in phenology, carbohydrate, and soluble sugar concentration in the roots.The experiment was conducted at the University of California Davis, Oakville Experimental Vineyard from 2017 to 2019 over three growing seasons. Eight-year-old Vitis vinifera “Cabernet Sauvignon” Clone FPS08 grafted on 110 Richter rootstock were used. Plants were trained to a bilateral cordon, manually pruned to 24 buds. The shoots were vertically shoot positioned. Row and vine spacing was 2.4 × 2.0 m, respectively, and rows were oriented Northwest to Southeast. The plants were drip-irrigated with two pressure compensating emitters per plant delivering 2.0 L/h each. The plants were irrigated from fruit-set to end of harvest at 0.5 of crop evapotranspiration replacement as previously reported .The experimental design was a factorial arrangement of treatments. There were three levels of manual defoliation by three levels of manual fruit removal applied . The treatments were applied as follows. Leaves were removed on every shoot in an alternating pattern.

For instance, 66% of leaf treatments retained leaves in positions 1st, 2nd, 4th, 5th, 7th, 8th etc. while 33% of leaf treatments kept leaves in positions 1st, 4th, 7th, etc. in every shoot . The fruit removal treatments retained a percentage of clusters after standardizing the cluster numbers in each year. Each treatment combination was replicated four times and each treatment-replicate consisted of three experimental units. In 2017, all vines were standardized at fruit set to 20 shoots and 30 clusters per vine, and laterals were removed prior to defoliation and fruit removal treatments. In 2018, all vines were standardized to 24 shoots and 45 clusters and laterals were removed prior to treatment application. Treatments were applied at pepper-corn size . In 2019, after two seasons of growth under the nine combinations of treatments, the carryover effects were studied by leaving all vines untreated . For each experimental unit one vine was shoot thinned to 24 shoots, and others were left unmanaged . All clusters at pepper-corn size in all treatment-replicates were dipped in a 5.5% kaolin solution to provide protection from the afternoon sun due to the row orientation of the vineyard in every year of the experiment.Leaf gas exchange was measured bi-weekly in all years of the experiment with an infra-red gas analyzer . Three sun-exposed leaves were selected from the main shoot axis in each experimental unit, and three readings were taken from each leaf. Gas exchange measurements were taken when the sunlight conditions were close to saturating levels in all instances. The relative humidity was set at 40%, the reference CO2 concentration was set at 400 µmol CO2 mol−1 as the standard environmental condition setting in CIRAS-3. Net carbon assimilation rate and stomatal conductance were obtained. To express the season-long response of AN, and gs , their integrals were calculated by using natural cubic splines for plant water status and gas exchange measurements to assess the cumulative values for these parameters over the whole experiment period during the growing season. Then, these cumulative values were normalized as divided by the number of days elapsed between the first measurement date and the last measurement date to make the data comparable to each individual measurement.After harvest, leaves from one vine per replicate were collected, weighted, square pots and dried in a forced-air oven at 80◦C for 3 days. Dry leaf weights were converted into area by measuring the area of a subsample of 50 random leaves with a leaf area meter as reported previously . On 12 December 2018, after the second season of treatments, one vine per experimental unit of the most extreme treatments were pruned, coppiced, and the root systems were removed with a back-hoe. The sectioned grapevine portions were weighed on a top loading scale, and dried in a forced-air oven at 60◦C until no weight change of tissue was detected. At harvest , clusters were removed, counted, and weighed for each plant in the experiment. Total soluble solids were measured from 55 berries collected randomly at harvest point. A digital refractometer was then used to measure total soluble solids of must.Soon after the harvest of 2017 was completed, root tissues were sampled every 2 months.

The top layer of soil was removed until the roots were visible. Each grapevine root zone was divided into four quadrants and on each date and one single quadrant was sampled, leaving the other 11 quadrants undisturbed. Roots were gently cleaned with water, freeze-dried, and ground to a fine powder with a tissue lyser . Thirty milligrams of the resultant powder were extracted in 80:20 ethanol solution. A 1.5 mL aliquot of the extract was then placed in a 90◦C water bath for 10 min, then centrifuged at 10,000 rpm for 1 min. The supernatant was collected for total soluble sugars determination. The same procedure was repeated for starch determination, in which the pellet was collected for its determination. Total soluble sugars in the roots were determined as reported elsewhere by Torres et al. . Briefly, the 1.5 mL sample was filtered by PTFE membrane filters and transferred into high performance liquid chromatography vials. Equipment consisted of a reversed-phase HPLC system Agilent 1100 coupled to a diode array detector and an Agilent Infinity Refractive Index Detector . The reversed-phase column was Luna Omega Sugar with a guard column of 5 mm. The temperature of the column compartment was maintained at 40◦C and the RID flow cell was kept at 35◦C. The mobile phase system consisted in an isocratic elution with acetonitrile:water at a flow rate of 1.0 mL•min−1 with a run time of 22 min. Standard solutions of 10 mg/L of D-glucose, D-fructose, D-sucrose, and D-raffinose were injected to obtain the retention time for each compound, and detection was conducted by RID. Sugar standards were purchased from VWR . Sugar concentration of each sample was determined by comparison of the peak area and retention time with standard sample curves. Starch content of roots was measured using the Starch Assay Kit SA-20 following the manufacturer’s instructions. Briefly, pellets of root tissues were dissolved in 1 mL DMSO, and incubated for 5 min in a water bath at 100◦C. Starch digestion was started by adding 10 µL α- amylase and incubated in boiling water for another 5 min. then, the ddH2O was added to a total volume of 5 mL. Then, 500 µL of the above sample and 500 µL of starch assay reagent were mixed and incubated for 15 min at 60◦C. Negative controls with the starch assay reagent blank, sample blank, and glucose assay reagent blank and positive controls with starch from wheat and corn were performed. Reaction started with the incubation of 500 µL of each sample and 1 ml of glucose assay reagent at 37◦C and was stopped with the addition of 1 mL of 6 M Sulfuric acid after 30 min. Reaction was followed with analytical measurements with a Cary 100 Series UV-Vis Spectrophotometer and starch content expressed as mg of starch per tissue dried weight.The same grapevines were measured on each date throughout the execution of the experiment. Season-long measurements of root starch and soluble sugars, and phenology were analyzed separately for each year via three-way ANOVA for a date × defoliation × fruit removal design using PROC MIXED procedure of SAS using REPEATED option for measurement dates. Measurements of season-long leaf gas exchange integrals, grapevine vegetative growth, yield and yield components, and total soluble solids variables were analyzed via three-way ANOVA for year × defoliation × fruit removal using the same procedure of SAS. Whenever the year and treatment interactions were significant the analyses was conducted by year. Post-hoc analyses were conducted using Tukey’s HSD at p < 0.05. The trend analysis was carried to the quadratic level and was conducted with planned orthogonal contrasts using PROC GLM procedure of SAS. Certain variables were log transformed based on most-likelihood analysis.In our experiment the results indicated that there was an interaction of year and defoliation on cluster weight, berries per cluster and yield per vine . When we analyzed the data by year, the effect of defoliation was clearer. In both experimental years , there was a strong linear trend of defoliationon all components of yield except for cluster number; which was only affected by the fruit removal treatments. In 2017 defoliating 66% of the leaves resulted in an 8% decrease in berry weight.

All of the leafroll viruses selected for this study belong to the Closteroviridae family and all but one belong to the Ampelovirus genus; GLRaV-2 belongs to the genus Closterovirus. All of the GLRaVs used in this study contain a conserved replication gene block but are diverse outside of the RGB . In addition to host genotype and environment, the sequencespowdery and downy mildew in grapevine . The abundance of these genes varies among Vitis species and are particularly dense at resistance loci . The HR to viruses is mediated by resistance genes. HR is a means of prohibiting pathogen spread and can confer resistance when a corresponding dominant avirulence protein is produced by the pathogen . However, GLRaV infections are systemic and persist over time, and SA does not seem to play a preeminent role in the response to GLRaV infections. In a previous study of GLRaV-3 infections in Cabernet Sauvignon and Carmenère, the authors also remarked on the induction of expression of defence genes but their inability to impede systemic infection . Both SA and ABA can participate in the response to viruses, gallon pot though considerably less is understood about the role of ABA and its relationship to NBS-LRRs.

Notably, however, ABA deficiency is associated with an increase in R gene efficacy in incompatible interactions with Pseudomonas syringae and in a manner independent of SA . Hormones have been implicated in mediating defence- and development-related networks and are over-represented at network hubs . Hormones like ABA, SA, and JA act as important signalling molecules during ripening and defence. The pathways engaged under stress are often tailored to particular pathogens. This entails coordination between hormone pathways . Interestingly, the effects of GLRaVs on gene expression in several hormone signalling pathways differed between rootstocks. A subsequent effort could be made to measure the abundances of hormones not quantified here, like cytokinins, GAs, and ethylene. Of the hormones considered in this study, however, the abundance of ABA and ABA-GE tended to increase in GLRaV and this was influenced by rootstock. ABA can antagonize SA and JA signalling pathways and suppress ROS signalling . WRKY transcription factors regulate and/or are regulated by ABA, SA, and JA . SA and ABA both can interact with RNAi, which is a fundamental component of antiviral defence. AGO1 expression is positively correlated with ABA levels and the expression of miR168a, which regulates AGO1, and contains ABREs in its promoter . Levels of ABA and ABA-GE increase in tobacco mosaic virus-infected leaves. One way in which ABA might aid plant defence is by increasing callose deposition to impair virus movement ; a gene encoding callose synthase is upregulated in the leaves of grapevine virus B-infected plants .

Relatively more is known about ABA’s function as a ripening promoter , in response to drought stress , and in transmitting long-distance signals from roots to aerial organs and vice versa . In a study of the impact of GLRaV-3 infection, drought stress, and a combination of both on grapevine plantlets in vitro, individual stresses both induced increases in ABA levels . Drought stress increases ABA levels and induces the flavonoid pathway in both tea plants and grapes . Our findings, in which ABA abundance tends to increase in GLRaV , are different than that observed for red blotch virus-infected berries, in which ABA abundance and NCED expression decrease in infected fruits . The results of our analysis of metabolites associated with the phenylpropanoid and flavonoid pathways are mixed in their consistency with previous work. Though non-significant decreases in anthocyanin levels were observed, anthocyanin levels significantly increased in several GLRaV conditions, albeit usually in individual years. These findings differed from others; some observed significant decreases in anthocyanin levels in fruits from GLRaV-infected plants and others observed no significant changes in anthocyanin at harvest . In agreement with the results by Vega et al. , flavonol levels were elevated in GLRaV and the largest differences versus GLRaV occurred at the first two stages, FLS expression was downregulated in GLRaV at harvest, and CHS and MYBPA1 were upregulated at véraison and generally downregulated at harvest. In the present study, changes in the abundance of ABA and related metabolites distinguished the effects of GLRaVs between rootstocks. The parentage of the two rootstocks used in this study,Kober 5BB and MGT 101-14, includes Vitis riparia. The other parents of Kober 5BB and MGT 101-14 are Vitis berlandieri and Vitis rupestris, respectively.

These rootstocks were developed at different times. MGT 101-14 originated in France in 1882 and Kober 5BB originated in Austria in 1930 . Rootstocks are chosen for the advantages they confer to the scion given a particular set of circumstances, often having to do with resistance to Phylloxera, nematodes, scion vigour, soil type, and abiotic stress tolerance . V. riparia, V. rupestris, and V. berlandieri are asymptomatic hosts of GLRaVs . Yet, the particularly severe response to GLRaV-1,2 was not entirely unexpected in Kober 5BB-grafted vines . It would be interesting to determine whether differences in viral titre exist between Kober 5BB, MGT 101-14, and Cabernet Franc and between different infection conditions and whether such differences, if they exist, influence the severity of leafroll disease. Differences in wood abnormalities given different rootstocks have been observed for particular isolates causing grapevine rugose wood disease . Notably, nutritional deficiencies in phosphorus, magnesium, and potassium produce symptoms that resemble those typically observed in GLRaV-infected plants . Magnesium deficiency tolerance , the impact of phosphorus deficiency on canopy growth , and potassium uptake and channels are influenced by rootstock . Potassium uptake, channel activity, and related gene expression are also regulated by ABA , and the application of ABA to tomato roots by drip irrigation affects fruit mineral composition . Furthermore, elevated levels of potassium are observed in leafroll virus-infected Burger and Sultana fruits and in leaf petioles but potassium levels are lower in leaf blades . Perhaps potassium deficient and GLRaV phenotypes are similarly governed by ABA and fine-tuned by rootstocks. If some portion of scion ABA originates in roots and/or if rootstock can influence scion ABA levels and signalling genes, as observed here and by others , then perhaps this partially accounts for the variation in response observed between rootstocks. This experiment did not include a comprehensive survey of phytohormones, which would be beneficial, but ABA’s function in root–shoot communication, its role in ripening, and the results here make it a good candidate around which to study the basis of leafroll disease symptom variability going forward. In addition, the transport of RNAs across the graft junction may perform some function that affects scion disease severity, but this remains to be seen in the particular case of GLRaV . Together, these data support several conclusions. The majority of genes differentially expressed as a consequence of infection or between GLRaV plants with different rootstocks were yearspecific. A small subset of effects was consistently observed across experimental conditions and in both years. These shared changes in expression involved genes associated with pathogen detection, ABA signalling and transport, ROS-related signalling, gallon nursery pot cytoskeleton remodelling, vesicle trafficking, phenylpropanoid metabolism, sugar transport and conjugation, and leucine biosynthesis. The impacts of GLRaV-1,2 dual infection on Kober 5BB-grafted vines were the most distinctive and severe. Though there was variation between GLRaV infections observed, only the effects of GLRaV-1,2 were distinguishable overall from those of other infections. The particular effects of GLRaVs in plants grafted to different rootstocks were distinguishable overall at every developmental stage. ABA-related variables were among those that best distinguished the responses to GLRaVs in different rootstock conditions. This included the abundance of ABA, the abundance of ABA-GE, and the expression of genes associated with ABA and other hormone signalling pathways.

Finally, this work alone is insufficient to recommend the use of one rootstock or another, but the disparity in sensitivity and symptom severity observed in berries from Cabernet Franc vines grafted to different rootstocks suggests that rootstock selection should be further explored as a strategy to mitigate some of the negative consequences of leafroll virus infections, should vectors of the virus encroach upon a vineyard.The experimental vineyard used in this study was established in 2010 and consists of Cabernet Franc clone 04 grapevines grafted on different rootstocks and infected with zero, individual, or pairs of GLRaVs. The rootstock portion of these plants was inoculated with chip buds carrying each virus in 2009 . All rootstocks and Cabernet Franc scions were tested for grapevine pathogens. Total nucleic acid extracts were prepared from all rootstocks and Cabernet Franc scions as described by Al Rwahnih et al. . Extracted TNA samples were analysed by reverse transcription quantitative PCR using TaqMan probes on the QuantStudio 6 Flex Real-Time PCR System as described previously . The samples were screened for the following pathogens: GLRaV-1, GLRaV-3, GLRaV-4 ; GLRaV-2 ; GLRaV-7 ; grapevine fleck virus ; grapevine rupestris vein feathering virus ; grapevine fanleaf virus, tobacco ringspot virus, and tomato ringspot virus ; grapevine virus A, grapevine virus B, grapevine virus D, grapevine virus E, and grapevine virus F ; grapevine red blotch virus ; and grapevine rupestris stem pitting-associated virus , phytoplasmas, and Xylella fastidiosa. In autumn 2008, Cabernet Franc grapevines were bench-grafted onto rootstocks, including MGT 101-14 and Kober 5BB. These plants were subsequently grown in a greenhouse. Between 2009 and 2011, the rootstock portions of these plants were inoculated with two chip buds from single leaf roll-infected plants. Infected plants used for chip buds were reconfirmed by RT-qPCR. Plants infected with a single species of GLRaV received two identical chip buds. Plants infected with two species of GLRaVs also received two chip buds, each carrying a single virus. Plants infected with GLRaV-1, GLRaV-2, and/or GLRaV-3 were inoculated with two or more isolates of each species of GLRaV. The inoculated plants were kept in a greenhouse for approximately 1 month, acclimatized, and then planted in the field. Healthy controls included nonchip budded plants and plants chip budded from a healthy source. The vines were planted in a randomized complete block design, with 7 feet between vines and 9 feet between rows. One group of five vines was planted per rootstock × infection condition in each of three blocks. Healthy vines were distributed throughout each block to monitor the spread of viruses, and experimental vines were sampled yearly to test and reaffirm the vines’ infection status. A buffer zone of healthy vines was planted as a barrier between the leafroll vineyard and other vineyards in the area. Vines were trained with a bilateral cordon and spur pruned.High-quality genomic DNA was isolated from grape leaves using the method described in Chin et al. . SMRTbell libraries were prepared for Cabernet Franc clone 04 as described by Massonnet et al. . Final libraries were evaluated for quantity and quality using a Bioanalyzer 2100 and sequenced on a PacBio RS II . De novo assembly of Cabernet Franc clone 04 was performed using FALCON-Unzip as described in Minio et al. . Repetitive sequences were masked before and after read error correction using the TANmask and REPmask modules in Damasker . Contigs were polished with Quiver . The primary assembly was scaffolded to reduce sequence fragmentation. First, primary contigs were scaffolded with SSPACE-LongRead v. 1.1; . Junctions supported by at least 20 reads were allowed. Hi-C data and the proprietary HiRise software were used for hybrid scaffolding. A Dovetail Hi-C library was prepared by Dovetail Genomics as described in Lieberman-Aiden et al. and sequenced on an Illumina platform, generating 2 × 150-bp pairedend reads. The repeat and gene annotation were performed as reported in Vondras et al. . Briefly, RepeatMasker and a custom V. vinifera repeat library were applied to identify repetitive elements in the genome. Publicly available data sets were used as evidence for gene prediction. Transcriptional evidence included Vitis expressed sequence tags, Cabernet Sauvignon corrected Iso-Seq reads , Tannat , Corvina , and Cabernet Sauvignon transcriptomes , and previously published RNA-Seq data . Swissprot Viridiplantae data and Vitis data were used as experimental evidence. Each RNA-Seq sample was trimmed with Trimmomatic v. 0.36 , assembled with Stringtie v. 1.3.3 , and mapped onto the genome using Exonerate v. 2.2.0 and PASA v. 2.1.0 . Alignments and ab initio predictions generated with SNAP v. 2006-07-28 , Augustus v. 3.0.3 , and GeneMark-ES v. 4.32 were used as input for EVidenceModeler v. 1.1.1 . EVidenceModeler was used to identify consensus gene structures.

Grafting can increase overall plant vigor and yield in the absence of disease or other stress, but studies in California focusing on grafting for increasing yield have found both increases and occasional decreases in yield when grafted to fresh market tomatoes . Therefore, absent developments that reduce the costs of these inputs, grafting is currently not economically feasible for processing tomato production in California. In addition to economic challenges with grafting, we encountered cultural issues with grafting in our experiments. Notably, we observed that the grafted and the tall grafted plants can outgrow the scion. The plants in the greenhouse and the field developed shoot growth from the rootstock, particularly in the tall grafted plants, likely due to the higher number of nodes on its rootstock stem compared to the standard grafted plants. This overgrowth was especially prevalent in 2019, in which a month after planting in the field, outgrowths of Maxifort were observed to emerge from the soil in between the grafted transplants. The different procedure used by the nursery to produce the transplants in 2019 compared to 2018 may explain the greater incidence of overgrowing the scion. Thus, black plastic pots for plants while the grafted plants reduce southern blight incidence, overgrown plants develop an undesirable taller vegetative canopy and produce unwanted fruit of the rootstock.

Although statistical and numeric trends suggest that increasing the height of the graft union may reduce southern blight incidence, the magnitude of this difference is small and not likely to make a practical difference in commercial production. The tall grafted plants did not develop as much southern blight as the non-grafted plants, but there are additional cultural issues with using the resistant rootstock in a processing tomato field such the risk of transplants breaking at their union while being transplanted or quickly after transplanting. Therefore, increasing the height of the graft union is likely not an economical approach for processing tomato production. We found a range of variation in susceptibility to southern blight among commercial cultivars. One Heinz cultivar was promising in 2018, although it developed disease in 2019. In 2019 one Harris Moran cultivar showed promise in its resistance to southern blight, but it was not evaluated in 2018 due to poor germination. We found the breeding lines reported by Leeper et al. 1992 to also perform well in one replicate year in greenhouse conditions, therefore these breeding lines may be beneficial for southern blight resistance breeding for California processing tomatoes. However, three of the breeding lines performed similarly to two Heinz and one Nunhmens cultivars that are already planted in the southern San Joaquin Valley. The cultivars that performed well in our experiments may have lower susceptibility to southern blight compared to other cultivars and thus may be options for growers to plant in fields with a history of southern blight.While results from the cultivar experiments were promising, cultivars could not be separated easily when evaluating their risk of developing disease.

The disease risk ranking of some cultivars changed drastically between the two experimental replications: a few cultivars with low risk in 2018 developed more disease in 2019 and vice versa, but several cultivars were in the highest risk tier in both years. Although cultivars were difficult to separate, the study in 2019 developed more disease than the study in 2018. This may have been due to excessively high irrigation pressure in two blocks of the 2018 study that is suspected to have washed the inoculum out of pots, resulting in minimal disease development. Further evaluation of these cultivars in the greenhouse and the field is needed to confirm these findings. Disease incidence was low overall in the greenhouse grafting studies, especially in HZ 8504 compared to HZ 5608. This may be due to our experimental setup in which plants were trellised because of space constraints on the greenhouse bench. When plants were trellised, the stem did not branch near the soil line, and vegetative tissues did not touch inoculated soil, while in the field the vegetative canopy is at risk of coming into contact with sclerotia in the soil. The utility of greenhouse experiments for evaluating the effect of grafting on southern blight may be limited. These studies showed promising results for the management of southern blight in California. Based on our studies, the approach of grafting for management of southern blight may not be the best application. The use of resistant cultivars is a better and accessible approach for California processing tomato growers.

We recommend the development of field studies to evaluate the promising cultivars in the greenhouse under natural conditions.Raspberry fruit are produced on a perennial shrub belonging to the vastly diversified Rubus genera of the Rosaceae family. There are mainly two types of raspberries: red and black. Yellow raspberries are a mutated version of red raspberries that prevents the formation of red color . Purple raspberries are a hybrid between black and red raspberries. Raspberries are an aggregate fruit, a combination of individual drupelets that stay together with the help of an invisible hair-like structure. Each small drupelet is developed from a carpel, a significant characteristic of Rubus, where the mesocarps become fleshy and the endocarps become hard and form a tiny pit that encloses a single seed within each drupelet. . The drupelets separate from the receptacle at harvest, creating a hollow, thimble-shaped fruit.The primary planting material for raspberries is the cane. Cane traditionally refers to aerial raspberry shoots but also often refers to rooted suckers . Raspberry plants generally start fruiting in the second year and can continue up to 15 years if properly managed. However, the canes are biennial. Usually, the cane grows in one year and fruits the next year. Canes sprouting in the first year are called primocanes , and in the second year are called floricanes . Both canes are present during the growing season . Primocane-fruiting types can produce two crops per year; onein the summer from the floricanes and the other in the fall from the primocanes. With the onset of cold temperatures, floricanes often go into a period of dormancy. Six weeks or more at 4℃ or lower is required to break dormancy. After fruiting, the entire cane senesces and dies. When second-year canes are flowering, first-year canes are growing from the crown or roots. Like other brambles, raspberry is a self-pollinated species . Fruit development occurs for 30-36 days in most cultivars . The best yields take place under sunny, cool summer conditions . Recently, the use of the high tunnels has extended the berry cultivation season both at the beginning and the end. This extension enables growers to gain more profit from the market as berry prices are usually higher during early and late seasons . Like all crops, water is very important for raspberry production. According to Prange and DeEll , lack of water can affect the production and quality of berry fruit after harvest. However, excessive water, mostly due to high rainfall during the fruit growing period, drainage pot made raspberries more susceptible to mechanical damage during transportation and storage .The red raspberry is believed to have originated in Turkey’s Ide mountains. The Romans may have expanded raspberry cultivation throughout Europe. However, the British improved and popularized raspberries throughout the middle ages and had the plant exported to New York by 1771 . In the early 1900s, raspberry cultivation got its momentum, and in 1920, New York State growers harvested more than 10,000 acres of raspberries. In 2018, there were three major raspberry production regions: Russia, Europe , and the Pacific Coast of North America . World raspberry production has grown 80% over the last 10 years. From 2010 to 2019, production increased from 373,000 tons to 684,000 tons. . In 2020, the total area under raspberry cultivation in the United States was 16,900 acres producing 111,000 tons of raspberries valued at US$469 million. In California, there were 8,000 acres, valued at US$395 million for fresh raspberries only. Canada is the biggest importer of US raspberries, and in 2021, Canada imported a total of 24,400 tons of fresh raspberries valued at $154 million from the US .

A fruit’s quality is generally assessed based on its visual appearance, texture, flavor, and nutritional compounds . Raspberry quality and shelf life can be adversely affected by a variety of pre-harvest and post harvest factors. Pre-harvest factors include genetics, environment, and cultural practices. Postharvest factors such as handling, transportation, storage temperature, condition and duration, relative humidity also play important role on maintaining raspberry fruit quality. Raspberry is renowned for its aroma and flavor. Raspberry is a non-climacteric fruit and their taste and flavor mostly develop while they are ripening on the plant. The ratio of sugar and organic acids determines raspberry taste . Soluble solids from 9 to 10% and titratable acidity from 1.5 to 1.8% constitute good raspberry taste according to De Ancos . Wang et al. evaluated raspberry fruit harvested at 5%, 20%, 50%, 80% and 100% ripe and they concluded that berries that were 50% to 80% ripe developed the same degree of SS, TA, and sugars as berries that were 100% ripe, whereas berries that were 5 to 20% ripe neverattained those properties. There are many volatile compounds, notably, α and β-ionone, linalool, α and β-pinene, caryophyllene and citral, contributing to raspberry flavor . Berry purchases are linked to several factors, the most important being freshness and origin, while price does not play a significant role . Visual quality is also very important for raspberry and a good indicator of shelf life. Brighter color without any visible decay and leakiness is perceived as fresher. Krüger et al. categorized raspberries in three groups based on their ripening stage; semi-ripe, ripe and over-ripe. They concluded that semiripe raspberries were potentially more suitable for shipping while maintaining acceptable sensory quality .Consumers have always been concerned about food quality and appearance, in general. However, consumer preference has been shifting toward fruit flavor and nutritional qualities, including their composition and level of bioactive compounds, such as vitamins, minerals, fiber, and even phenolic compounds . Raspberry fruit are 85- 90% water, ~9% SS and the remaining are insoluble solids. Raspberry fruit contain 13.6–31.1 mg/100 g Vitamin C and 0.2–83.6 mg/100 g anthocyanins . These compounds vary by cultivar, harvest time, cultural practices, environment and weather conditions . Raspberry fruit also contain a broad range of polyphenolic compounds; phenolic acids, flavanols, anthocyanins, proanthocyanidins, and ellagitannins . These compounds have been extensively studied for their antioxidant capacity and impact on human health . High antioxidant capacity is believed to contribute to health benefits by ameliorating the detrimental effects of reactive oxygen species generated in the body through oxygen metabolism . Berry polyphenols also have been shown to protect against ROS-induced neurological diseases such as Alzheimer’s , and red raspberries have been reported to have a neuroprotective effect . However, the impact of raspberry fruit’s high antioxidant capacity might be limited by very low uptake into the bloodstream from dietary intake . Nonetheless, they could have beneficial effects on the gastrointestinal tract as they pass along the digestive system, thus preventing oxidation from foods already in the stomach and GIT or by affecting food digestion, glucose levels, and calorie usage . In addition, raspberry contains a significant amount of ellagitannins; a large group of polyphenols that are beneficial to fight cervical cancer, cardiovascular disease, and diabetics .Raspberries have a short shelf life of 2 to 5 days because of their natural soft texture and sensitivity to mold and other pathogens. Postharvest handling and storage conditions, including packaging, relative humidity, temperature, and light, can affect the bioactive compounds in brambles . Low temperatures slow pathogen growth and reduce the rate of deterioration of freshly harvested commodities, thus extending shelf life and the marketing period . The recommended temperature for raspberry storage is 0-1℃ , but it is challenging to maintain this recommended temperature during transportation and marketing. Although low storage temperatures can slow the development of Botrytis cinerea infections, they do not provide adequate control when pathogen inoculum loadsare high . Acidity and SS as well as pigment compounds such as anthocyanins play an important role in berry marketability after storage , and the presence of light and temperature during storage might affect anthocyanin compound stability .Modification of storage or transport atmospheres help maintain raspberry shelf life and quality.

Karp et al. estimated 4–12 birds/ha of species that are confirmed or suspected CBB predators. Flocks of migratory birds on coffee farms are estimated at 19/ha and 24/ha , but these values are also short of our estimates of necessary densities for suppressing larger CBB outbreaks. One caveat to our conclusions is that our calculations were based on CBB accounting for 5%–10% of a bird’s daily diet . This assumption meant birds would only eat a set maximum of 7 CBB per day. Sherry et al. reported up to 116 CBB in the stomach contents of a single warbler, suggesting under certain circumstances in the field, birds eat more CBB. Generalist insectivores, particularly Neotropical migrants, have flexible foraging preferences and would likely feed opportunistically on CBB in response to dramatic dispersal peaks. Therefore, birds might be expected to increase feeding rates as CBB disperser abundances increase, though it may depend on the relative abundances of other prey. Better data on CBB consumption rates by birds under different circumstances would improve our estimates of the circumstances under which birds can control CBB populations.

A second caveat is that bird densities used in the model may not represent the potential for CBB control because bird densities depend on the structure of the agricultural landscape, pot with drainage holes which the current model does not consider. On coffee farms, birds are more abundant when native tree cover is highest and natural forests are close by . Across tropical and temperate regions, the propensity for birds to forage on farms, and thus exert pressure on agricultural pests, is correlated with the physical complexity and diversity of the agroecosystem . For example, birds make more frequent foraging trips to apple orchards with high native tree coverage . In alfalfa fields, edge habitat complexity supports greater avian richness leading to lower pest abundances . Under some circumstances, the density of birds foraging in certain areas may be higher than average densities would imply, leading to greater control potential than our models suggest. More generally, our CBB population model is density independent and assumes environmental conditions and sufficient resources to allow CBB populations to increase without restriction. As a result, our model is limited, as it does not consider localized effects of weather and temperature fluctuations on CBB developmental time , nor characteristics of coffee farms that influence both CBB infestation and bird density. We assumed maximal capacity for CBB population growth and used estimates of bird densities from the literature that only included birds known to consume CBB, perhaps underestimating the potential for avian control.

Models are an important tool for estimating population dynamics, but as with any species, the growth potential for CBB and availability of its predators, is context dependent. Our study echoes Kendall et al.’s conclusion that, even though errors in model construction are common, these seldom change qualitative conclusions. From our population matrix, CBB daily growth rate converged on λdaily = 1.042 around day 124, with an observed rate of population change across the entire coffee-growing season of 705 . Our λdaily is higher than Mariño et al.’s reported lambda of 1.32 over
50– 56 days, which corresponds to λdaily ≈ 1.006 . Part of this discrepancy may come from the fact that Marino et al. combined vital rates across life stages with different time steps. Nonetheless, both models are consistent in predicting rapidly growing populations. Observed CBB population growth rates are similar to ours: Baker, Barrera, & Rivas, calculated a 1.067 growth rate in wild populations and RuizCardenas and Baker reported 1.047 in CBB reared in laboratory settings. In their sensitivity analysis, Mariño et al. reported that adult female survival, and transitions from larva to pupa and pupa to juvenile had high sensitivity in contributing to population growth rate, with adult survival the highest . We found a similar peak sensitivity value for female adult survival in our matrix , supporting the idea that CBB population growth is most sensitive to adult survival rate. Interestingly, dispersal survival from our matrix was estimated to have low impact on population growth , even though this life stage is when CBB are vulnerable to bird predation.

Thus, our analysis superficially suggests that population control once CBB are established should focus on reducing adult survival rather than on trapping dispersing females , if the same impact on numbers could be achieved. However, dispersing females are much more accessible to control methods like spraying fungal bio-insecticide than are adult females, which are inside the coffee cherries, so despite the tremendous difference in sensitivity values, management of an established population is likely to be more cost effective by continuing to focus on dispersing females . Population models specific to CBB have been criticized for not being representative of wild populations, since more generations are estimated through modeling than are observed in field studies . We analyzed CBB population growth using a deterministic model, with an even distribution of dispersal and a fixed predation pressure. While CBB dispersal is continuous, there can be dramatic intraseasonal peaks in numbers that were not captured by our model . In addition, reported longevity of female CBB varies widely from 55 to 380 days, though some studies looked at CBB reared on artificial diet . Refinements of survival in natural settings would, therefore, improve models of CBB population growth, and the potential for control by birds. If field data on CBB vital rate stochasticity become available, and bird densities opportunistically increase during CBB peak numbers, it could affect our conclusions about the capacity of birds to control larger CBB outbreaks. Based on our analyses, there is a population density of CBB above which their capacity to produce more adults exceeds the ability of birds to control their numbers, at least to limit the population size by 50%. This positive density-dependent relationship between population growth and density is an Allee effect , and escape from predation is one mechanism for this phenomenon . In general, predator-driven Allee effects can occur when predators are the main driver of prey dynamics and when predators are generalists as are insectivorous Neotropical migrants . Additionally, large pot with drainage predators can exert strong pressure when prey availability is not temporally or spatially limited—a potential limiting factor in the coffee system, since CBB are only available to birds during dispersal. The degree to which birds exert an Allee effect on CBB might depend on the starting population size of the pest. Variation in starting population size is likely dependent on how recently CBB have colonized in an area, timing of trapping , the size of the farm , and the extent to which farmers used control measures the previous year . We found that only under very low initial population sizes of CBB could birds be expected to suppress pest numbers by 50%. We note that earlier, stronger CBB suppression by birds would lead to lower infestation numbers later in the coffee season, but this might require selective foraging by birds, depending on relative abundances of other prey species. In conclusion, our models suggest that birds can control CBB under some circumstances, depending on the relative size of the starting CBB population and existing local bird density. To put this idea into practice it is important to remember that managing farms for bird habitat does not always result in pest reduction. Birds may not prey on the pest of interest or birds might cause pest numbers in increase by preying on insect predators that normally regulates the pest population . Aside from predators, pest species are also impacted by the agricultural environment directly . In fact, on coffee farms where bird densities are higher in shade, CBB infestations are also higher , possibly because CBB native range is in humid, shade forests of Africa .

It is important that future modeling include such habitat-specific factors to understand Our research helps quantify the densities under which birds have the potential to control CBB populations. Putting these numbers into practice will require understanding how manag Raspberries are an important crop for California, where it is among the top 20 commodities with an average annual value of $448 million from 2015 to 2017 . Maintaining a high yielding, disease-free crop has been difficult due to low availability of farm workers to harvest and maintain the fresh market raspberry canopies. As a method to adjust to their limited field workers, some growers in California began experimenting with new cultural practices, such as pruning leaves near the base of the canes. In Ventura County, the common pruning practice is to remove mature and senescent leaves using the twine that is part of the trellis. The influence of these experimental cultural practices on incidence and severity of cane Botrytis is not known. Managing B. cinerea cane and fruit infections during this early period can be important for reducing severe epidemics during the rest of the crop. The first chapter focuses on our studies that evaluate the influence of pruning leaves on incidence and severity of cane Botrytis. In the previous decades, soil borne diseases were commonly managed with the use of chemical fumigation, but the widening restrictions on the use of fumigants in the San Joaquin Valley of California has posed a challenge for growers. The California processing tomato industry averaged $1.1 billion in value from 2013 to 2017, and accounted for 93% of the production in the United States in 2017 . Southern blight is a soil borne disease of processing tomato that has long been an economic concern in the San Joaquin Valley, and recently caused a widespread epidemic in both the San Joaquin and Sacramento Valleys . The threat of southern blight has caused reductions in acres planted with processing tomato in the southern San Joaquin Valley . The objectives of the second chapter were to: evaluate susceptibility of commercial processing tomato cultivars to southern blight; and evaluate grafting and increased height of the graft union with the resistant rootstock Maxifort for southern blight management in processing tomato.Raspberries are an important crop for California, where it is among the top 20 commodities with an average annual value of $448 million from 2015 to 2017 . This represented 82% to 88% of the domestic raspberry production. The four California counties where raspberry is produced are Ventura, Santa Cruz, Santa Barbara, and Monterey. Specifically in Ventura and Santa Cruz counties, raspberries are among the top commodities . On the West Coast of the United States, raspberry is typically produced in two stages from a single planting that is grown for a maximum of two years. In the primocane stage or first year cycle, harvest generally begins four months after planting of bare root transplants and continues for approximately three to five months. After harvest, the primocane growth is pruned near the last fruiting lateral or is mown at the soil line. The growth that follows this pruning begins the floricane stage or second cycle, which has a harvest period that generally begins three to four months after pruning and can last approximately four months. In Ventura County, a crop can be planted during four periods throughout the year: early spring, late spring, mid-summer, or late summer. The ascomycete fungal pathogen Botrytis cinerea Pers.:Fr causes multiple diseases of raspberry. The first is grey mold or Botrytis fruit rot, a widespread and damaging disease of fruits and flowers of many hosts including raspberry . The second is cane Botrytis, a disease of the raspberry cane that was first reported in England in 1931 . Cane Botrytis is reportedly more severe on red raspberry canes compared to other Rubus spp. . Cane botrytis is known to more commonly affect the floricane stage in the U.S. Pacific Northwest , but in coastal California and in eastern Canada B. cinerea causes disease primarily to the primocane stage of raspberry . Managing B. cinerea cane and fruit infections during this early period can be important for reducing severe epidemics during the rest of the crop. For example, a study evaluating B. cinerea conidia dispersal in raspberries showed locally-produced inoculum was important for controlling fruit infections . New raspberry plantings are often established next to older plantings where mummified fruits, damaged canes, and dead plant debris potentially colonized by B. cinerea sclerotia are prevalent . These infected debris from older plantings are an important source for primary spore inoculum . Resting mycelia and sclerotia can also be found on weeds .

The power system of our current device, i.e., outlet, is not ideal for most standard incubators, because it requires cords coming out of the incubator and causes a concern on maintaining the environmental parameters inside an incubator. To address these issues, the loading device could have its own built-in source of power such as a battery. The battery should be able to provide enough power that allows the device to run for the full duration of the study that could last from weeks to months. Considering that the standard environment inside an incubator involves heat and humidity, a proper shield system would have to be incorporated to protect the battery and electrical components. Third, down scaling the overall size of the device is another important aspect of future improvement, because smaller size would allow the device to be more portable and easier to be placed inside a standard incubator. As aforementioned, large pot with drainage the hydraulic pistons and the battery can significantly reduce the overall size of the device due to the smaller size of each component and elimination of using an air compressor.

Other electrical components, such as the Arduino Uno and Raspberry Pi, can be integrated into one to reduce the overall size as well. The possible drawback of these improvements is the increased cost for the device. The current first-generation loading device was designed and built with a small budget of a few hundred dollars, and demonstrated its functionality successfully in studying the degradation of Mg rods in rSBF under load.Agricultural intensification simplifies ecosystems through management practices such as increases in agrochemical use, decreases in habitat complexity, and decreases in crop and vegetation diversity . Agricultural intensification alters functional biodiversity;in particular, reductions in habitat complexity impact the arthropod community composition, decrease arthropod diversity and reduce pest control services. Notably, biological pest control is likely the ecosystem service most affected by biodiversity loss at the local scale. In coffee agroecosystems, management intensification alters habitat complexity by impacting vegetation connectivity and structure.

The management intensification gradient ranges in coffee systems from the least intensive traditional shaded “rustic system”, in which coffee grows under a diverse closed canopy of native forest, to the most intensive “sun monoculture”, which refers to rows of open unshaded coffee monoculture, that require high inputs of agrochemicals. On the shaded end of the intensification gradient, shade coffee habitats are naturally vegetatively complex, with diverse and dense shade canopies and vines and weeds that form connections between the shade trees and the coffee plants. This vegetation connectivity is an important aspect of habitat complexity that impacts species interactions at the local scale. However, while progressing along the management intensification gradient, reductions in habitat complexity, driven by decreases in shade trees, increases in herbicide use, and the clearing of vegetation between coffee plants, reduce vegetation connectivity and alter species interactions within ecological communities and the ecosystem services that they provide. Connectivity is one physical component of habitats that has a profound impact on arboreal insects and ant community structure. In the absence of connectivity, trees are insular habitats with crown isolation that inhibits the movement of some taxa .

Connectivity in the form of lianas and nylon ropes shape the local community structure of arboreal ants, with higher ant species richness often occurring in trees that are connected artificially or vegetatively as compared with trees without these physical connections, and higher ant species coexistence occurring in trees with higher levels of naturally occurring canopy connectivity. These results also reflect the nature of ants as highly efficient foragers, known to use branches and lianas as “opportunist walkways” that provide the quickest foraging routes by allowing for faster traveling speeds through avoiding obstacles on the ground, even if these routes are not necessarily the shortest distance. The variation in texture of natural walkways, characterized as “surface roughness”, further impacts both arboreal and ground ant running speeds and foraging efficiency. Physical connections between trees are thus important structures that facilitate not only arboreal ant mobility but also their foraging success, resource recruitment efficiency, and ant-provided ecosystem services, including pest removal. Ants play an important role in the control of the coffee berry borer , the most damaging insect pest of coffee. In particular, the aggressive arboreal ant Azteca sericeasur nests in shade trees, forages on coffee shrubs, and is a keystone predator that controls the CBB. Like many arboreal ants, A. sericeasur prefers walking on branches and vegetation to avoid traveling on the ground. Given the role of A. sericeasur as a biological control agent, understanding how connectivity at the local scale impacts these ants has potential implications for coffee agroecosystem management. In Chiapas, Mexico, Jiménez-Soto et al. found that artificially increasing connectivity between A. sericeasur nests and coffee plants by tying jute string between ant nest trees and coffee plants increased the capacity for A. sericeasur to remove the CBB by throwing them off the coffee plants. These results suggest that naturally occurring vegetation connectivity might have a similar effect as that of artificial string connectivity on A. sericeasur activity and their associated pest removal services. Our study tests and expands on this hypothesis by examining the impact of both artificial connectivity and naturally occurring vegetation connectivity on A. sericeasur activity, its ability to recruit to resources, and its removal of the CBB with a manipulative experiment. Specifically, we tested the following hypotheses: We predicted that the coffee plants with vegetation or artificial connections to the ant nest tree have higher A. sericeasur activity than that of the isolated control plants; A. sericeasur ants recruit to resources more efficiently on coffee plants with vegetation or artificial connections to the nest tree; coffee plants with vegetation or artificial connections to the nest tree have greater CBB removal rates by A. sericeasur ants; and A. sericeasur activity, resource recruitment rates, and CBB removal rates decrease with increased distance from A. sericeasur nests.This study was conducted in the Soconusco region of Chiapas, Mexico at Finca Irlanda, a shaded, 300-hectare commercial polyculture coffee plantation. The plantation is located in the Sierra Madre de Chiapas Mountains at an elevation of 1100 m.a.s.l. The average canopy cover throughout the farm is 75 percent and the majority of the plantation shade trees are of the genus Inga. The climate is semi-tropical, square pot with the rainy season occurring between May and October. Vegetation management at Finca Irlanda frequently includes “chaporreo”, in which farm workers periodically use machetes to clear the weeds and epiphytes that grow between coffee plants. This management practice facilitates farm worker movement between coffee plants and reduces competition between weeds and coffee plants, but in the process inadvertently eliminates vegetation connections between the coffee plants and A. sericeasur nest trees.

We collected data between June and August in the summer of 2022. Within the 300 hectares of Finca Irlanda, we selected 17 trees with active A. sericeasur nests as study sites. Each site was located at least 10 m away from any other active A. sericeasur nests to prevent overlapping ant activity, following the methodology used by Jimenez-Soto et al..We chose six coffee plants within a 5 m radius of each A. sericeasur nesting tree for a total of 102 coffee study plants. At each nesting tree site, we selected two coffee plants for the natural vegetation connectivity treatment, two for the artificial connectivity treatment, and two as isolated control plants . For the vegetation connectivity treatment, we selected two coffee plants with existing vegetation connections. The vegetation connections were either coffee branches directly touching the A. sericeasur nest tree or coffee branches touching a secondary plant, such as a vine or epiphyte that was touching the nest tree. We selected two coffee plants for the artificial connectivity treatment, in which we tied jute strings between the point of the nest tree trunk with the most active ant foraging trail and the central trunk of each coffee plant. We ensured that there were no existing vegetation connections on these plants and that the string was the only point of connection between each coffee plant and the nest tree. For the control treatment, we selected two isolated coffee plants with no connections between the coffee plants and the nest tree. We measured the distance between the central trunk of each study coffee plant and the ant nest tree.At each site, we quantified the ant activity on the coffee plants by counting the number of A. sericeasur that passed a central point on the central trunk of each coffee plant during 1 min . The observations took place between 7:30 AM and 2 PM before the afternoon rainy period. The observations were stopped if it began to rain, as rain significantly reduces ant activity. After setting up the strings, we returned to each site between 7 and 13 days after the initial setup and re-measured ant activity on the coffee plants.To assess the impact of artificial and vegetation connectivity on prey removal by A. sericeasur, we placed five dead adult female CBB on white index cards on the centraltrunk of each coffee plant . We monitored A. sericeasur interactions on the cards for one hour, ensuring that only A. sericeasur were responsible for removing CBB, and counted the number of CBB removed. Because it has already been well-documented that A. sericeasur remove live CBB from coffee plants, we used dead prey to avoid the possibility of live CBB escaping during a longer observation period. The CBB were collected from infested coffee berries in the field, then frozen for up to 5 days before use.Recruitment is understood to be an integral component of trail-following in which ant workers follow chemical foraging trails to a food source, then re-apply chemical trails until that food source is exhausted. Tuna baiting is an effective and widely used method of assessing ant recruitment in coffee agroecosystems. To assess the impact of connectivity on ant resource recruitment efficiency, we placed 1 g of canned tuna on the central trunk of each coffee plant 1 m above ground and recorded the number of A. sericeasur that recruited to each tuna bait after 20 min.To test for statistical differences in ant activity, resource recruitment efficiency, and CBB removal between the control, string, and vegetation treatment coffee plants over the 5-week experiment, we fit our data with generalized linear mixed models using the lme4 package in R. For each response variable , we included the time , the treatment method , the distance between the coffee plant and the ant nest tree , the interaction between the treatment and the time, and the interaction between the time and the distance as fixed effects. As random effects, we modeled the coffee plant identity nested within the site to control for site variation and spatial non-independence. To assess count data , we originally fit each model to a Poisson distribution. However, to correct for observed over-dispersion, we instead modified each model to a negative binomial distribution.We observed A. sericeasur using the artificial string connections at 12 of the 17 sites and on 20 of the 33 strings placed . A. sericeasur were the only ants observed using the strings. Out of 33 vegetation treatment plants , 20 plants included primary connections , and 13 plants were connected by secondary connections . Ant activity was higher on the vegetation treatment coffee plants than on both the control treatment and the string treatment . There was no difference in ant activity between the string and control treatments . There was a significant effect of time on ant activity for the string treatment, indicating an increase in ant activity on the string plants after connecting the strings . There was no effect of time on ant activity for either the control or vegetation treatments, indicating that there was no significant change in ant activity for those treatments over the 5-week experiment .Treatment, time, and distance all impacted the ant recruitment to tuna baits. More ants recruited to the tuna baits on the vegetation treatment plants than on the strings, and more ants recruited to the bait on string plants as compared to the control plants . The overall number of ants that recruited to the baits decreased with time poststring placement on both the control and vegetation plants, but there was no significant change in the number of ants recruiting to the baits on the string treatment . The number of ants recruiting to the baits on the control and string plants declined with distance from the nest tree, but was consistent over all distances for vegetation treatment plants .

Cyanidin-3,5-diglucoside was the next most concentrated anthocyanin, averaging 20.11 ± 5.63 mg per 100 g FW in 2018 and 19.80 ± 6.92 mg per 100 g FW in 2019. This is unlike European elderberries, in which cyanidin-3-glucoside is typically the second most prominent anthocyanin, except for the Ljubostinja cultivar which has more cyanidin-3,5-diglucoside than cyanidin-glucoside42. Cyanidin-3-sambubioside-5-glucoside and cyanidin-3-glucoside were also quantified in the berries. Cyanidin-3,5-diglucoside and cyanidin-3-sambubioside-5-glucoside were not detected in blue elderberries grown in Slovenia,suggesting the growing location impacts the profile of phenolic compounds or perhaps the two samples going by the same name are not, in fact, related 109. There were no acylated anthocyanins identified in the blue elderberry, like those abundant in the American elderberry. Overall, total anthocyanin concentrations averaged 61.54 ± 16.70 mg per 100 g FW in 2018 and 58.58 ± 22.18 mg per 100 g FW in 2019. The total concentration of anthocyanins in the berries was much lower compared to the other subspecies. Analysis of European elderberries that measured cyanidin-based anthocyanins found an average of 863.8 ± 49.9 mg per 100 g FW 8 .

European elderberries grown in different locations at different altitudes had a range of 289.74 ± 66.18 to 792.66 ± 27.97 mg per 100 g FW 6 . In studies on American elderberries, one had an average of 265 ± 74 mg per 100 g FW 49, another had average of 248 ± 83 mg per 100 g FW 18, and a third had an average of 242.7 ± 91.0 mg per 100 g FW 50 . The flavan-3-ols catechin and epicatechin were measured in the elderberry, nft vertical farming with epicatechin typically present in higher concentrations. The concentrations found in the present study are similar to those found in others, even across subspecies. Blue elderberry grown in Slovenia had 4.40 ± 0.26 mg per 100 g FW of catechin and 8.49 ± 0.37 mg per 100 g FW of epicatechin. The same study found no catechin present in S. nigra ssp. nigra, but 6.37 ± 0.28 mg per 100 g FW of epicatechin. In a study of European berries growing in different locations at different altitudes, total flavanol concentrations ranged from 1.93 ± 0.22 to 9.67 ± 0.66 mg per 100 g FW 6 . The variability in phenolic and anthocyanin content observed in this study is not surprising, as multiple other studies have shown significant variability in other commercialized elderberry subspecies, even with clonally propagated cultivars. For example, Lee and Finn 49 saw an average of 45% higher anthocyanins in their second harvest of American elderberries grown inOregon as compared to their first harvest, though the total phenolics only increased an average of 20%. Johnson et al. 54 observed significant changes between subsequent years in anthocyanin and phenolic compound concentrations in juices prepared from American elderberry grown in two locations in Missouri.For example, in the Adams II sample grown in one Missouri location, the quercetin 3-rutinoside content was 298 ± 48 mg L-1 in 2012, 792 ± 143 mg L-1 in 2013, and 47 ± 13 mg L-1 in 2014 54.

In a study of 107 wild American elderberries samples grown in five regions of the eastern United States by Mudge et al. 110 high variability was found in selected flavonoid compounds with an average RSD of 55.3% across samples. Overall, there is a body of evidence demonstrating that elderberry composition can vary year to year or by growing conditions even in clonally-propagated cultivars; therefore, it may be necessary to use standardization techniques for bioactive compounds in order to maintain consistent quality in elderberry products. Blue elderberry grown in California hedgerows has similar levels of sugar, organic acids, and TPC to the European and American elderberry subspecies. Furthermore, the phenolic profile of blue elderberry is similar to European elderberry, in that chlorogenic acid, rutin, and cyanidin-3-sambubioside are the predominant hydroxycinnamic acid, flavonol, and anthocyanin, respectively. However, anthocyanin levels are significantly lower in the blue elderberry compared to European and American subspecies, yet the levels of total flavonols appears to be much higher than the other subspecies. 5-Hydroxypyrogallol hexoside and protocatechuic acid dihexoside were identified for the first time in elderberry, which could potentially serve as markers of this subspecies in products that use blue elderberry. There was considerable variation within and between hedgerows in both harvest years, but this appears to be a common attribute for the elderberry species. Blueelderberries have many ecological benefits for farms when planted in hedgerows, grow well in challenging environments, are not killed by wildfires and can therefore, serve as a sustainable source of an increasingly popular fruit.The elderberry is a deciduous, multi-stemmed shrub or small tree. 

It can grow several meters high and in diameter and produces hundreds of clusters of aromatic flowers in the spring, that mature into small berries in summer. The plant grows well in a variety of soils and climates, and is a native of Northern America, Europe, and parts of Asia. While there are many subspecies within Sambucus nigra, the primary subspecies widely grown and commercially cultivated include S. nigra ssp. nigra found across Europe, and the “American” subspecies S. nigra ssp. canadensis, which is native to the eastern regions of North America.56 The blue elderberry , is a drought-tolerant subspecies native to the western region of North America. The blue elderberry grows in riparian ecosystems from southern British Columbia, Canada to northwest Mexico. In California, there have been efforts for more than a decade to increase the levels of blue elderberry planted in hedgerows on farms because of its environmental benefits, such as improving the air, water, and soil quality, as well as providing food and shelter for pollinators. It is now recognized that these mature hedgerow plants can be a source of locally grown elderberries and elderflowers to increase income and sustainability for the farm. However, to date there is no data on the concentration of the aroma or phenolic compounds in the flowers from this hardy heat-tolerant subspecies. The berries, flowers and bark of the elderberry plant have a long history of use by humans as both food and traditional medicine. Seeds have been found in archeological sites that date to the late stone age and their medicinal use is documented in the writings of Theophrastus , Pedanius Dioscorides and Gaius Plinius Secundus . Elderflowers are frequently used in medicinal and herbal teas, tonics, liqueurs, lemonades, and sparkling waters for their subtle and unique floral, fruity, and green aromas and medicinal properties. Infusions of elderflowers have been used in many cultures for the treatment of inflammation, colds, fever, and respiratory illness and for their diuretic and antidiabetic effects. Some studies have found evidence to support their use, such as antimicrobial activity of elderflower extract against Gram-positive bacteria and high vitro antioxidant activity. Much of the interest for using elderflower in health-promoting applications is based on the high content of biologically active phenolic compounds in the flowers. European and American elderflowers contain an array of phenolic compounds, such as phenolic acids , flavonols , flavonol glycosides [isorhamnetin-3-O-rutinoside , rutin ], flavan-3-ols [-catechin, -epicatechin], and flavanones. In European-grown elderflowers, the dominant phenolic acid and flavonol glycoside include chlorogenic acid and rutin, although isoquercetin, vertical tower for strawberries isorhamnetin-3-rutinoside and kaempferol-3-rutinoside are also present. For example, in a study of European elderflowers grown in different locations and altitudes, the dominant class of phenolic compounds were the flavonols, namely rutin , whereas chlorogenic acid levels were lower . This study also found that the flowers contain four times more chlorogenic acid than the leaves or berries. The predominant phenolic compounds identified in elderflower syrup, a traditional herbal beverage, include chlorogenic acid and rutin . There has been only one study on the phenolic profile of the flowers of S. nigra ssp. canadensis which appears to be similar to the European subspecies, in that rutin and chlorogenic acid are the primary flavonol and phenolic acid identified, respectively. 

The aroma of the elderflower is derived from the volatile organic compounds in the flower and is an important characteristic to understand for consumer acceptance in applications.To date, only the VOCs of elderflowers from the European subspecies have been studied. The American subspecies S. nigra ssp. canadensis has not yet been investigated. As fresh flowers are highly perishable, many commercial products rely on dry, and in some cases, frozen flowers. Thus, it is important to understand how the organoleptic properties of elderflowers change in response to processing. The VOC profile of tea made with elderflowers of three European cultivars using dynamic headspace sampling revealed compounds important to the characteristic aroma to be linalool, hotrienol, and cis– and trans-rose oxide. Similarly, studies indicate that in fresh and dried flowers analyzed by headspace solid phase microextraction coupled with gas chromatography mass spectrometry , linalool oxides are the main aroma compounds. Linalool oxide has a floral, herbal, earthy, green odor. In hexane extracts of dry elderflowers analyzed via HS-SPME/GC-MS, cis-linalool oxide and 2-hexanone were the primary volatiles. The compound 2-hexanone has a fruity, fungal, meaty, and buttery odor.80 In syrups made from elderflowers, terpene alcohols and oxides were identified as the primary aroma compounds. Studies of the impact of drying on volatiles in the flowers demonstrate that nearly all types of drying change the volatile profile significantly. The aim of this study was to characterize the composition of phenolic compounds and VOCs in flowers of the blue elderberry , and to determine how these compounds change in response to drying and in the preparation of teas. Understanding how the aroma and phenolic compounds compare with current commercially available European and American subspecies will help to establish a role for blue elderflowers in commercial applications such as herbal teas and as a flavoring for beverages, as well as identify unique compositional qualities of this native and underutilized flower.An aqueous mixture of ethanol was used to extract the phenolic compounds from flowers. The optimal mixture of ethanol to water was determined by extracting flowers in 0, 25, 50, 75, and 100% ethanol. Solvents also contained 0.1% HCl and 0.1% ascorbic acid . For each extraction, 0.25 g dry flower material and 25 mL solvent were added to 50 mL Eppendorf tubes. The dry flowers with solvent were homogenized for 1 min at 7000 rpm with a 19 mm diameter probe head in the 50 mL tubes. Homogenized extracts were refrigerated overnight at 4 °C, then centrifuged at 4000 rpm for 7 min . The supernatant was filtered through 0.45 µm PTFE, then diluted 50% with 1.5% phosphoric acid before analysis. Three replicates were made for each extraction condition . Hence, four types of samples were made: fresh whole flowers , dry whole flowers , fresh homogenized flowers , and dry homogenized flowers . Flowers were mixed with the determined optimal extraction solvent and followed the same extraction process as described above, except whole flower samples were not homogenized and instead placed directly into the refrigerator to extract overnight.All sample extracts were analyzed via high performance liquid chromatography using an Agilent 1200 system with diode array detection and fluorescence detection . Separation of phenolic compounds was performed on an Agilent PLRP-S column at 35 °C, using a previously published method. Mobile phase A was 1.5% phosphoric acid in water and mobile phase B was 80% acetonitrile, 20% mobile phase A . The flow was set at 1.00 mL min-1 . The gradient used was as follows: 0 min, 6% B, 73 min, 31% B, 78-86 min, 62% B, 90-105 min 6% B. Most phenolic compounds were detected using a at 280 nm , 320 nm , and 360 nm . Flavan-3-ols were detected using a fluorescence detector . Compounds were quantified using external standard curves employing surrogate standards for each group of phenolic compounds [-catechin for flavan-3- ols, chlorogenic acid for phenolic acids and simple phenols, quercetin for flavonol aglycones, and IR for flavonols]. Standards were prepared at concentrations of 200, 100, 50, 10, and 5 mg L -1 , except IR which included an additional concentration of 500 mg L -1 . Triplicate analyses of each concentration were performed .Compounds were separated using HPLC-DAD-FLD as described above and identified using authentic standards to check retention time and absorption spectra. Several peaks in the chromatograms did not match tR or spectra of authentic standards. Therefore, fractions of these peaks were collected. Fractions were dried and reconstituted in 1% formic acid in water.