Horticulture

These chemical relationships are represented as a chemical tree that can be visualized in the context of sample metadata and molecular annotations obtained from spectral matching and in silico annotation tools. We show that such a chemical tree representation enables the application of various tree-based tools, originally developed for analyzing DNA sequencing data, for exploring mass-spectrometry data. Here, we introduce Qemistree software that constructs a chemical tree based on predicted molecular fingerprints from MS/MS fragmentation spectra. Molecular fingerprints are vectors where each position encodes a substructural property of the molecule, and recent methods allow us to predict molecular fingerprints from tandem mass spectra. In Qemistree, we use SIRIUS and CSI:FingerID to obtain predicted molecular fingerprints. Users can first perform feature detection to generate a list of observed ions with associated peak areas and MS/MS fragmentation spectra, referred to as chemical features henceforth, raspberry container size to be analyzed by Qemistree . Only chemical features with MS/MS data are included; features with only MS1 are not considered.

SIRIUS then determines the molecular formula of each feature using the isotope and fragmentation patterns and estimates the best fragmentation tree explaining the fragmentation spectrum. Subsequently, CSI:FingerID operates on the fragmentation trees using kernel support vector machines to predict molecular properties . We use these molecular fingerprints to calculate pairwise distances between chemical features and hierarchically cluster the fingerprint vectors to generate a tree representing their chemical structural relationships. Although alternative approaches to hierarchically cluster features based on cosine similarity of fragmentation spectra exist , we use molecular fingerprints predicted by CSI:FingerID for this. Previous work has shown that CSI:FingerID outperforms other tools for automatic in silico structural annotation. Therefore, we leverage it to search molecular structural databases to provide complementary insights into structures when no match is obtained against spectral libraries. Subsequently, we use ClassyFire to assign a 5-level chemical taxonomy to all molecules annotated via spectral library matching and in silico prediction . Phylogenetic tools such as iTOL can be used to visualize Qemistree trees interactively in the context of sample information and feature annotations for easy data exploration. The outputs of Qemistree can also be plugged into other workflows in QIIME 2 or in R, Python, etc. for system-wide metabolomic data analyses.

In this study, we apply Qemistree to perform chemically informed comparisons of samples in the presence of technical variation such as chromatographic shifts that commonly affect mass spectrometry data analysis. Additionally, we exemplify the use of a tree-based representation to visualize and explore chemical diversity using a heterogeneous collection of food products. Qemistree can be used iteratively to incorporate multiple datasets without the need for cumbersome reprocessing , allowing for large-scale dataset comparisons. Qemistree is available to the microbiome community as a QIIME 2 plugin and the metabolomics community as a workflow on GNPS2 . Thechemical tree from the GNPS workflow can be explored interactively using the QemistreeGNPS dashboard. To verify that molecular fingerprint-based trees correctly capture the chemical relationships between molecules, we designed an evaluation dataset using four distinct biological specimens: two human fecal samples, a tomato seedling sample, and a human serum sample. Samples were prepared by combining them in binary, tertiary, and quaternary mixtures in various proportions to generate a set of diverse but related metabolite profiles . Untargeted tandem mass spectrometry was used to analyze the chemical composition of these samples and obtain fragmentation spectra.

The mass spectrometry experiments were performed twice using different chromatographic elution gradients, causing a retention time shift between the two runs . Processing the data of these two experiments with traditional LC-MS-based pipelines leads to the same molecules being detected as different chemical features in downstream analysis. Figure 1 shows the analysis of pure samples to demonstrate this. In Extended Data Figure 4, we highlight how these technical variations make the same samples appear chemically disjointed. Using Qemistree, we mapped each of the spectra in the two chromatographic conditions to a molecular fingerprint, and organized these in a tree structure . Because molecular fingerprints are independent of retention time shifts, spectra are clustered based on their chemical similarity. It is noteworthy that the structural information from chemical features with spectral library matches or other forms of annotation could also be used to compare the chemical composition of samples across different mass spectrometry runs. Qemistree improves upon this by enabling the use of all MS/MS spectra with molecular fingerprints for downstream comparative analyses, by not constraining analysis to the chemical features with spectral matches only. This tree structure can be decorated using sample type descriptions, chromatographic conditions, spectral matches obtained from molecular networking in GNPS , and any other chemical annotations23,28. Figure 1 shows that similar chemical features were detected exclusively in one of the two batches. However, based on the molecular fingerprints, these chemical features were arranged as neighboring tips in the tree regardless of the retention time shifts. This result shows how Qemistree can reconcile and facilitate the comparison of datasets acquired on different chromatographic gradients.Having demonstrated Qemistree’s practical utility on biologically inspired synthetic datasets, we now turned to a conceptual example illustrating the general principle. We demonstrated an application of a chemical hierarchy in performing chemically informed comparisons of metabolomics profiles. In standard metabolomic statistical analyses, eachmolecule is assumed unrelated to the other molecules in the dataset. Some of the pitfalls of this assumption are highlighted in Figure 2a. Consider a scenario where we want to compare samples 1–3. An analysis schema that does not account for the chemical relationships among the molecules in these samples , will assume that the sugars in samples 2 and 3 are as chemically related to the lipids in sample 1 as they are to each other. This would lead to the naive conclusion that samples 1 and 2, and samples 2 and 3 are equally distinct, yet from a chemical perspective they are not. On the other hand, if we account for the fact that sugar molecules are more chemically related to one another than they are to lipids, we can obtain a chemically informed sample-to-sample comparison. The chemical structural compositional similarity metric29 was developed to compute pairwise sample-to-sample comparison by considering cosine similarity of MS/MS spectra from molecular networking. Here, we utilize a tree-based approach to account for chemical relationships, which allows us to adopt phylogeny-based tools for metabolomics analyses . Specifically, we first constructed a tree of chemical similarities by hierarchical clustering molecular fingerprints from CSI:FingerID . This tree is analogous to phylogenetic trees used in ecology, such that the tips of the tree are molecules . In Figure 2a, we show that by using a tree of chemical relationships between molecules in samples 1–3, we can visualize that sample 1 is chemically very distinct from samples 2 and 3. Returning to our evaluation dataset, raspberry plant container we can highlight the importance of comparing samples by accounting for their molecular relatedness. Principal coordinates analysis of the evaluation dataset that ignores the tree structure performs far worse than the Qemistree PCoA that uses the tree .

With the structural context provided by Qemistree, the differences between replicates across batches are comparable to the within-batch differences . The retention time shift in this dataset leads to a strong signal due to chromatography conditions that obscures the biological relationships among the samples . We observed and remediated a similar pattern originating from plate-to-plate variation in a recently published study investigating the metabolome and microbiome of captive cheetahs . In this study, placing the molecules in a tree using Qemistree reduced the observed technical variation , and highlighted the dietary effect that was expected . These results show how systematic and spurious molecular differences can be mitigated in an unsupervised manner using chemically informed distance measures based on a tree structure.As a case study demonstrating the utility of Qemistree on a set of biological specimens, we used the platform to explore chemical diversity in food samples collected in the GlobalFoodOmics initiative . Understanding the chemical relationships between different foods is challenging because most molecules within foods are unannotated. We selected a diverse range of food ingredients to represent animal, plant, and fungal groupings. We first performed feature-based molecular networking using MZmine to obtain spectral library matches for a subset of the chemical features . Using Qemistree, we collated GNPS spectral library matches and in silico predictions from CSI:FingerID to annotate ~91% of the chemical fingerprints with molecular structures. We also retrieved chemical taxonomy assignments for structures that were classified by ClassyFire; the remaining are in the queue to be processed on the ClassyFire server for taxonomy assignment . Labeling annotations allowed us to retrieve subtrees of distinct chemical classes such as flavonoids, alkaloids, phospholipids, acyl-carnitines, and Oglycosyl compounds in food products. We propagated ClassyFire annotations of chemical features to each internal node of the tree and labeled the nodes by pie charts depicting the distribution in chemical superclasses and classes of its tips. The molecular fingerprint-based hierarchy of chemical features agreed well with ClassyFire taxonomy assignment, further demonstrating that molecular fingerprints can meaningfully capture structural relationships among molecules in a hierarchical manner. Furthermore, Qemistree coupled the chemical tree to sample metadata, revealing distinct chemical classes expected for each sample type. Branches representing acyl-carnitines were exclusively found in animal products . In contrast, honey, although categorized as an animal product, shared most of its chemical space with plant products, reflective of the plant nectar and pollen-based diet of honey bees. We observed a clade of flavonoids in both plant products and honey , but no other animal-based foods. While it is expected that a complex food such as blueberry kefir contains molecules from both blueberries and dairy, we can now visualize how individual ingredients and food preparation contribute to the chemical composition of complex foods. We noted that metabolite signatures that stem directly from particular ingredients, such as phosphoethanolamine from eggs, are present in egg scramble , but not in the other two foods highlighted . We can also observe the addition of ingredients in foods that were not listed as present in the initial set of ingredients. We were able to retrieve that there is black pepper in the egg scramble with chorizo and orange chicken, but that this signal is absent from the blueberry kefir .We show that our tree-based approach coherently captures chemical ontologies and relationships among molecules and samples in various publicly available datasets. Qemistree depends on representing chemical features as molecular fingerprints, and does share limitations with the underlying fingerprint prediction tool CSI:FingerID. For example, fingerprint prediction depends on the quality and coverage of MS/MS spectral databases available for training the predictive models, and these will improve as databases are enriched with more compound classes. Nevertheless, the use of CSI:FingerID-predicted molecularfingerprints is highly advantageous. While annotations from spectral matches may be more accurate, their coverage is too low to adequately summarize the chemical content of complex samples. Qemistree is also applicable in negative ionization mode; however, fewer molecular fingerprints can be confidently predicted due to fewer publicly available reference spectra, resulting in less-extensive trees. A key contribution of this work is to introduce the concept of building chemical hierarchies that can be used to leverage phylogeny-based tools , for metabolomics data exploration. Hierarchical relationships have provided a powerful framework to understand the relatedness of organisms. These techniques form a cornerstone for the interpretation of genomics data with phylogenetics and phylogenomics, and even taxonomy. The suite of tools and algorithms that have been developed over the past few decades in these fields, which utilize hierarchical structures, potentially have general relevance to the investigation of mass spectrometry data. Using Qemistree we can begin to explore the applicability of other methods, such as Faith’s Phylogenetic Diversity to understand within-sample complexity, or phylogeneticindependent contrasts with a metabolomics-inspired topology as these representations enter regular use. We showed that a hierarchical representation could be used to infer chemically informed relationships between samples . While we used molecular fingerprints predicted by CSI:FingerID to build chemical hierarchies here, this approach can be extended to incorporate other strategies to compare molecules for building chemical trees. For example, chemical relationships based on assigned chemical classes, spectral motifs, shared biosynthetic origin or other structural comparison methods could also be used as a basis for such a tree. These approaches will result in different tree topologies capturing complementary chemical information for subsequent analyses.

Most pollinators are generalist foragers that can, in some contexts, switch between plant species within a single foraging bout . When pollinators are promiscuous within a single foraging bout, they may transfer heterospecific pollen to floral stigmas, which can have negative effects on both male and female elements of plant reproduction . While heterospecific pollen deposition is highly variable in nature , it can represent a substantial percentage of total pollen on a stigma, often more than 50% of grains . Second, there is the extreme example of an antagonistic interaction from pollinators wherein the visitors do not visit the flower “legitimately” but rather pierce holes in a flower’s corolla to access the nectar rewards without ever touching the reproductive parts of the flower and therefore not acting as a pollinator . Some researchers suggest that most all flowering plants with accommodating floral architecture will experience some degree of nectar robbing . Furthermore, if a pollinator possesses mouth parts capable of robbing, they are likely to act as both legitimate pollinators on some plant species and primary or secondary robbers on others . On the other side of the interaction, blueberry plant pot some plants produce chemicals in pollen or nectar that that can be harmful to the development of the bees that visit their flowers, reducing bee fitness .

It has long been recognized that exploitation of mutualisms is commonplace and can have substantial impacts on the evolutionary persistence of mutualisms . While our understanding of the extent to which antagonistic interactions between plants and their pollinators is not complete, the examples listed above are common enough that the inclusion of such demonstrated negative interactions on network dynamics and how they might impact the robustness of interactions to extinctions warrants exploration. Recent network studies have begun to explore interactions in a continuous, rather than binary positive-or-not framework . While such studies do not take the potential for negative interactions into account, these models allow for some pollinators to be “better” than others in the services they provide or, in the case of Vieira and Neto , to vary the amount of dependence that the mutualistic partners have on one another. Here, we build on binary network simulation modeling approaches to asses show negative interactions impact the effects of pollinator species losses on plant species persistence, i.e. network robustness. In previous simulations where all network interactions are considered positive, the removal of a given pollinator species could result only in the loss of one or more plants. By contrast, after incorporating negative interactions, extinction cascades, also become possible , who produce extinction cascades in an all positive framework by relaxing the assumption that extinctions only take place after all partners are lost). In other words, if the removal of a pollinator species causes plant extinctions, those losses can tip the balance of interactions toward the negative for remaining pollinator species, which can then go extinct, in turn potentially leading to further plant species losses, thus an extinction cascade.

Our study examines the overall robustness to extinctions in two ways R, or, the area under each extinction curve and extinction cascade length –i.e. higher order extinctions that occur beyond the induced pollinator knockout and the resulting plant extinction. To our knowledge there is only one network study that incorporates the possibility for negative interactions between plants and pollinators while examining the robustness of the network when faced with extinctions This model classifies interactions as either mutually beneficial or beneficial for one species and detrimental to the other. This is in contrast to our model that examines robustness of networks with all positive interactions to those that incorporate negative interactions . Importantly the Campbell model focuses on hypothetical networks and does not directly evaluate the role of assigned negative interactions in determining the robustness of the network to extinctions and furthermore does not compare networks with and without negative interactions. Our method of incorporating negative interactions in to empirical networks will allow for higher order extinction cascades, giving us a more realistic impression of what might happen to a network after pollinator extinctions. This is not the case in extinction simulations that simply allow for asymmetric positive interactions , as noted above. We examined the effects of two factors on network robustness: the proportion of negative interactions in the network, including an all-interactions positive control; and the order of extinction, random pollinator losses vs. specialist-to-generalist vs. generalist-to-specialist.

Removing specialists first could be the most probable extinction sequence as specialist pollinators also tend to be the rarest species , but see who show that loss of specialists can accelerate the rate of species loss. By contrast, generalists are thought to be the “backbone” of networks and when highly connected nodes are lost, networks are expected to collapse rather quickly . While losing generalist pollinators first from a network may seem unlikely, we have seen rapid declines and range contractions in several highly generalist bumble bee species which had previously been abundant .First, we hypothesized that increasing the proportion of negative interactions would lead to both a decrease in the robustness of the network and greater number of extinction cascades. Second, we hypothesized that inclusion of negative interactions would not change the effects of extinction order relative to all-interactions-positive networks, in which specialist-to generalist pollinator species removals had the least impact on plant extinctions, generalist-to-specialist removals had the most, and random removals intermediate between the two .Following previous binary network assessments of robustness , we used empirical networks to conduct our robustness assessments. We selected three plant-pollinator networks of varying size and connectance that represent a range of natural plant-pollinator interactions; as in previous assessments, this selection is not meant to be exhaustive . 1) The Clemens and Long network was collected on Pikes Peak, Rocky Mountains, Colorado USA. This is by far the largest with 97 plant species forming 918 unique pairwise interactions with 275 pollinator species. Data were collected in various subalpine habitats at 2500 m elevation over 11 years . 2) The Arroyo et al network data were collected at an elevation between 2200m and 2600m between 1980 and 1981 in the alpine zone of Cordon del Cepo in Central Chile. The network is medium in size with 87 plant species forming 372 unique pairwise interactions with 98 pollinator species. 3) The Dupont network is the smallest as data were collected on the sub-alpine desert above 2000 m on the island of Tenerife, Canary Islands. Data were collect between May 7 and June 7, 2001. The network consists of 11 plant species forming 109 unique pairwise interactions with 38 pollinator species. These networks were retrieved from the NCEAS Interaction Web Database .We simulated extinctions by sequentially removing pollinator species one at a time and recording the number of plant species that were left with a positive sum of pollinator interactions. Plant species left with an interaction sum less than or equal to zero were then considered extinct and removed from the network due to assumed failure to sexually reproduce. Next, we evaluated if the secondary removal of those plant species left a pollinator species with an interaction sum less than or equal to zero. If yes, plastic gardening pots they were then considered extinct and removed from the network . This cycle continued until all plant and pollinator species were left with an interaction sum greater than 0 at which point the simulation moved on to the next pollinator species knockout. We carried out the extinction simulations separately for each of the aforementioned 600 network configurations and each of the three extinction orders.We evaluated network robustness via two response variables: R – this is a quantitative measure of robustness of a network following a species knockout . R is a simple calculation of the sum of the remaining plant species at each time step along the extinction simulation. R is standardized by its maximum value which equals the starting number of plants * the starting number of pollinators . R was calculated for each of the 50 simulations per order and proportion negative for all three networks. 

Extinction cascades – the number of higher order extinction cycles that take place after a single pollinator species knockout.Cascade length is defined as the higher order extinctions that occurred beyond the induced pollinator knockout and the resulting plant extinction. A cascade of length 1 results when such plant extinctions lead directly to subsequent pollinator extinction, while a cascade of length 2 indicates additional subsequent plant extinction. We calculated the total number of cascade events that occurred across the entire knockout sequence for each of the 50 replicate network configurations for each set of starting networks using GLMs with Poisson errors and a log link function. This analysis directly mirrors the modeling approach for robustness discussed above, comprising two sets of models. The first set compared the effect of negative interactions, extinction order, and their interaction within each network , while the second set of models compared the effect of negative interactions, network ID, and their interaction, within each extinction order .Our study examined how both the incorporation of negative interactions into networks as well as order of extinction impacted robustness and total number of extinction cascades in three empirical networks. As expected, we found that incorporating negative interactions leads to lower network robustness via an increased rate of plant species loss in all three of the networks. Furthermore, when compared to random extinction order, simulations with generalist removed first show the lowest overall robustness whereas the removal of specialists first has the least impact on lowering network robustness. This is true for all three networks and followed our expectations based on previous network extinction simulations . While some of the results from our simulations were predictable or even mathematical inevitabilities , not all of our results could be predicted a priori. We did not expect the networks to behave idiosyncratically with respect to how negative interactions and extinction order impacted both R and the total number of extinction cascades. Specifically, we found that in the smallest of the networks , extinction order did not impact the magnitude of the effect of negative interactions on network robustness. The effect of removing generalist-first from the two larger networks seemed to supersede the impact of negative interactions, leading us to conclude that the impact of losing generalists can,in some cases, be so detrimental so as to make inclusion of negative interactions irrelevant. While extinction cascades can only take place in networks that incorporate negative interactions, the impact of negative interactions on total cascade length was unpredictable. Neither increasing negative interactions nor extinction order were a good predictor for how many extinction cascades networks would take place in the simulations. Total number of extinction cascades is likely relate to structural properties unique to each network and warrant further exploration. It difficult to draw conclusions as to why these distinct differences between networks were seen, as this study did not include an exhaustive exploration of other network properties that may influence network robustness and total extinction cascades. Future studies should focus on selecting networks with systematically varying properties such as; network size, nestedness, and connectance. These properties each have the potential to influence the impact that negative interactions have on network robustness. Nestedness is a network property that has been identified in nearly all empirical networks. It is the tendency of specialists to interact with generalists in the network . We would expect that an increase in nestedness could make the networks more robust to extinctions due to the redundancy in the number of pollinators available per plant. Still, Campbell et al. found that high values of nestedness actually can have the opposite effect, and can decrease robustness after the loss of a single species, in some extreme cases lead to the total collapse of the highly nested community. In our simulations, networks with more generalists will be inherently more vulnerable because true specialists were excluded from assignment of negative interactions . While we do not expect this limitation to impact our results dramatically—as true specialists that only interact with one or two plant species are uncommon—to fully explore the role of nestedness one would need to develop a method of assigning negative interactions that distributes the negatives evenly among the specialists and generalists. Connectance is simply the proportion of realized links in a network . In this study we, by chance, selected a range of network size and connectance.

An alkane series was run under the same chromatographic conditions to determine retention indices. Confirmation of identification was performed by comparing the mass spectra and retention indices with those of standards when possible or literature values when standards were not accessible. Relative response was calculated by normalizing peak area for each compound to the internal standard peak area, and relative peak area was calculated using the relative response of a compound divided by the total peak area of a sample.The phenolic compounds were measured in fresh and dry elderflowers of S. nigra ssp. cerulea, both as whole and as homogenized flowers. The treatments used for this study were chosen to reflect the common ways that elderflowers are used in food and beverage applications and to provide more information on how to best extract the phenolic compounds from the flowers. The moisture content of the elderflowers was determined as 75.6 ± 1.7%. To achieve a consistent dry weight used in extractions, square planter pots either 1.00 g of fresh flowers or 0.25 g of dry flowers were used.

The extraction solvent was optimized to increase extraction efficiency of the main phenolic acids, flavonols and flavan-3-ols which included chlorogenic acid, IR, rutin, and -catechin . While chlorogenic acid, rutin, and catechin could be extracted in either 50:50% ethanol:water or 25:75% ethanol:water for maximum concentrations, the levels of IR increased with increasing amounts of water in the solvent system. However, in solvents containing ≥ 75% water, the flowers turned brown in color suggesting extensive oxidation. Therefore, it was determined that 50:50% ethanol:water was the optimal solvent for the extraction of the range of phenolic compounds in elderflowers without excess oxidation. A recent study of the effect of organicsolvents on the extraction of phytochemicals from butterfly pea flowers also found that 50:50% ethanol:water had optimal extraction properties for the phenolic compounds in flowers. These results differ from a study on the extract of phenolic compounds from dry, powdered European elderflower, which found water to be the optimal extraction solvent, specifically at 100 °C for 30 mins, as compared to 80:20% ethanol:water or 80:20% methanol:water . Elderflowers are used in products as either fresh or, more commonly, as dry flowers and as either whole or homogenized flowers . Therefore, each of these parameters were evaluated resulting in the following types of samples: fresh whole flowers ; dry whole flowers ; fresh homogenized flowers ; dry homogenized flowers .

Phenolic compounds were quantified using HPLC-DAD-FLD and information regarding the standard curves can be found in Table 1. Significantly more phenolic compounds were extracted from FHF compared with FWF, DWF and DHF indicating that phenolic compounds are released more readily from the vacuoles during homogenization while the flower is still fresh. There was no significant difference in the sum of all measured phenolic compounds between FWF, DWF, or DHF; however, levels of most phenolics were slightly higher in the DHF, suggesting that homogenization also increases the extraction efficiency in dry flowers. Furthermore, a statistically significant interaction was found between the fresh and dry flowers and homogenization of the sample for most phenolic compounds, due to the uniquely high levels present in the FHF and the absence of an equally high increase in DHF . This trend can also be seen in the totals of each phenolic class , as the FHF were significantly higher than all other sample types, with the exception of total flavan-3-ols in DHF .The levels of IR were significantly higher in FHF, with a maximum concentration of 78.73 ± 4.84 mg g-1 . This is a significant difference as compared with the European and American subspecies, in which rutin is the predominant phenolic compound in flowers and at much lower concentrations. Levels of IR in European elderflower levels range from about 0.200 to 0.900 mg g-1 fresh weight, though higher levels were found in elderflower tea, ranging from 4.260 to 13.500 mg g -1 . This key difference in the flowers of the blue elderberry provides an opportunity to create unique products for consumers looking for high levels of bio-active phenolic compounds, as studies have shown that IR can induce apoptosis in cancer cells. 

The other flavonol glycosides found in the flowers include rutin, kaempferol-3-Orutinoside, and isorhamnetin-3-O-glucoside. Quercetin was the only flavonol aglycone identified in the flower extracts and was low relative to the flavonol glycosides. Though this compound may be due to the degradation of a quercetin glycoside, quercetin aglycone has been measured in other elderflower studies, and our results are similar to those reported by Viapiana et al. . The flavan-3-ols monomers found in the flowers include -catechin and -epicatechin, highest in the FHF at 1.110 ± 0.30 and 1.24 ± 0.19 mg g-1 , respectively . -Epicatechin, but not -catechin, had an interaction between the fresh and dried and homogenization of the sample, as it was significantly higher in FHF. Proanthocyanin B type was also tentatively identified via HPLC-MS/MS analysis in the flowers, and was present in relatively low quantities in all samples . A procyanidin trimer was identified in elderflowers extracts and beverages by Mikulic-Petkovsek et al. . Chlorogenic acid was identified as the main phenolic acid in the flowers of the blue elderberry, like the flowers of the American elderberry, whereas the predominant phenolic acid in the flowers of the European elderberry is neochlorogenic acid . Neochlorogenic acid and other caffeoylquinic acid isomers were also present in the elderflowers of S. nigra ssp. cerulea . Two isomers of 5-caffeoylquinic acid in addition to 3- and 4-caffeoylquinic acid have been identified in elderflower products. Evaluation of the phenolic content of elder plants grown in different locations and altitudes indicate, in general, square pot that plant material from shrubs at higher altitudes had higher levels of hydroxycinnamic acids and flavonols. The authors postulated that the stress of harsher climates at higher altitudes may have led to the increase in hydroxycinnamic acids and flavonols to cope with the increase in UV radiation. They also reasoned that the high amounts of sun and cool nights may increase the metabolism of phenolic compounds. The flowers in the present study experience hot, dry summers with cool breezes from the Sacramento-San Joaquin Delta at night, and these conditions may contribute to the unique phenolic profile in this flower. The average day/night temperatures for Davis, California while the flowers were growing were 24/7 °C in April 2021, 28/12 °C in May 2021, and 31/13 °C in June 2021, with less than 3 inches of rain during that time span. A phenolic compound unique to the blue elderflower was identified as 5-hydroxypyrogallol hexoside . This compound has also been identified in the berries of this subspecies grown in the same locations. The concentrations of 5-HPG ranged from 1.26 ± 0.20 mg g-1 in DWF to 2.00 ± 0.72 mg g-1 in FHF . Because no commercial standards exist for this compound, the tentative identification of this compound was determined by high resolution QTOF-MS/MS data. The mass spectrum shows the molecular ion [M-H]- at m/z 303.0728 and fragment ion showing the loss of the sugar molecule [M-hexose-H]- at m/z 141.0199. Although the biological properties of this unique phenolic compound have not yet been investigated, 5-HPG hexoside can serve as a marker for S. nigra ssp. cerulea, especially since it is present in relatively high levels in the flower and berry and is not identified in other elderberry subspecies.Elderflower tea is one of the most traditional and simplest ways that the flowers are used in the preparation of beverages. To make elderflower tea, the flowers are infused in hot water to extract the flavor and biologically active phenolic compounds from the flowers.

Recommended steeping times can vary widely, however there are no studies investigating the impact of steep time on the extraction of phenolic compounds in the elderflower tea. To address this, the impact of time on the extraction of phenolic compounds from teas made from dried flowers was evaluated. The profile of phenolic compounds extracted in hot water infusions was similar to the profile obtained in ethanol/water extracts, however the concentrations were lower in the hot water extracts . Phenolic compounds were quantified at 5, 10, 15, and 20 minutes of steep time. Over time, the concentrations of total measured phenolic compounds increased 47% from five minutes . These results suggest that longer infusion times are beneficial for extracting the highest level of compounds. The overall levels of phenolic compounds in elderflower of S. nigra ssp. cerulea are comparable to the levels in the European flowers. However, variability between studies due to post-harvest conditions, extraction solvent, and analytical method make it challenging to make direct comparisons. For example, in this study, FWF, DWF, and DHF all had about 50-60mg g -1 dry weight, while the FHF had ~120 mg g -1 dry weight. The unique composition of phenolic compounds in these flowers provides an opportunity to make different products for the market, especially nutraceutical or functional food products that take advantage of the high levels of IR. Based on our results, it would be best to use fresh flowers and blend or homogenize the flowers to extract the highest levels of phenolic compounds.Before analyzing samples, method parameters were evaluated to find the optimal equilibrium time, temperature, and the extraction time for headspace VOCs. The optimal parameters for flower samples were determined to be 40 °C, 20 min equilibration , and 30 min extraction with a SPME fiber. The optimized conditions for the tea samples were 40 °C, 30 min equilibration, and 30 min extraction. The profiles of volatile compounds were evaluated in fresh whole, dry ground, and teas made from whole fresh and dry flowers by HS-SPME/GC-MS, and relative levels of identified volatile compounds were calculated. The fresh flowers were evaluated as whole flowers because homogenizing them can cause oxidation and create artifacts in the volatile headspace profiles. This led to higher variability in the relative peak areas of compounds, but it is believed to be truer to the real headspace VOC profile as compared to a homogenized fresh flower sample. Overall, 25 compounds in the fresh whole flowers, 44 compounds in the dry ground flowers, and 18 compounds in the tea preparations were identified. Table 1S contains the m/z ion of the base peak and the average match factor for each of the compounds identified in the headspace of samples. In the headspace of fresh flower, the most concentrated compounds were pentadecane > methyl eugenol > cis-3-hexenyl acetate > α-farnesene > and cis-3-hexenyl-α- methylbutyrate . The contribution of each compound’s odor to the overall aroma of these elderflowers cannot be determined from the concentration alone, as each compound has its own odor activity and threshold. However, it can be useful to know the characteristic odors of these compounds as a way to understand what comprised the general aroma. These compounds are described to have odors such as waxy; clove, spice; fresh, green, sweet, fruity, apple, pear, melon;wood, sweet; ; fruity, sweet, minty, fresh, and green apple, respectively. In addition to pentadecane, several straight chain hydrocarbons were also present, which may be released from the cuticle of the petal or peduncle of the flower. These include 1-pentadecene, heptadecane, 8- heptadecene and 6,9-heptadcadiene. Flowers also contain 4.6% methyl salicylate a compound with a sweet, minty odor80 that is frequently used as an analgesic in liniments to relieve pain. Methyl salicylate has been identified in several other studies on the volatile profile of elderflowers. The profile of headspace VOCs in elderflowers of S. nigra ssp. cerulea differ significantly from the European elderflowers as linalool oxides and other derivatives predominate in the European flowers, and are absent in the present study. Furthermore, the present study indicates a unique headspace VOC profile in the blue elderflowers because pentadecane and methyl eugenol have been identified as major contributors to the headspace VOC profile. Pentadecane has been identified at trace levels in some European elderflower extracts, however methyl eugenol has not been identified in European elderflowers. Methyl eugenol, which has a clove-like aroma , appears to be unique to the S. nigra ssp. cerulea elderflower, and could be a unique volatile marker for this subspecies. Tea made with fresh whole elderflowers presented a slightly different headspace VOC profile as compared to fresh flowers.

This design was implemented based on the assumption that volatiles from treatment plots may be blown or diffuse beyond treatment plots. Berries were brought to the laboratory to determine number of eggs in fruit for each of the plots using a dissecting microscope. All soft or damaged fruits were excluded when assessing presence of eggs. In some cases, at first fruit color, laboratory-reared D. suzukii flies were released in each plot with the intent to create a relatively even pest pressure in all plots. Colonies of D. suzukii used in field studies consisted of seasonally collected wild adults from multiple field sites in the Willamette Valley, Oregon, and Oxnard, California. Collected adults were released into plastic cages and reared at 24°Cand 70% relative humidity, with a 16:8 h photo period before being released in the respective field trials. Flies were constantly provided with water and artificial diet that served as both a food source and an oviposition medium. Before their use in experiments, blueberry container all flies were allowed to mate for 8 d in mixed-sex cages. Some small fruit varieties were numbered since this information is proprietary.

A replicated field trial on drip-irrigated Pinot noir winegrape was conducted in Yamhill County, Oregon, USA from 10 to 18 October 2019 on ~2.6 hectares. Vines were spaced at 1.5 by 5 m, and trellised on a standard four wire trellis system, supporting a ~2 m canopy. Rows were oriented along a north-south direction on an east facing slope. Three treatments , were included with ~0.056 ha plots. No pesticides were applied during the experimental period. Here, there were 28 GUM and buffer plots each and 18 UTC plots. GUM dispensers were applied on 10 October and ten berries were collected from each plot on this date. Sampling dates were 11, 14, and 18 October 2019.Trials were conducted in a commercial sweet cherry orchard located at the Mid-Columbia Agricultural Research and Extension Center , Hood River, Oregon, USA. A 1.12-hectare orchard was divided into twelve plots . UTC, buffer, and GUM plots were replicated four times. The GUM dispensers were deployed on day 0 . No insecticides were applied to the orchard for the duration of the experiment. Here, an additional 200 mated 8- to 12-day-old D. suzukii were released in the center of each plot on a weekly basis on 23 June, and 1, 8, and 15 July 2020 . Data were collected for 35 days from 16 June through 22 July 2019. Because of relatively large canopy size of cherry trees compared to the other crops, ten cherries were collected from the lower , middle , and upper portions of the central two trees in each plot weekly.

The trial was conducted in an organic highbush blueberry planting on 23.76 hectares in Independence, Oregon, USA . The experiment began on 9 July and continued through 11 September 2020. There were three treatments: UTC , grower standard , and GUM. Grower standard applications targeting D. suzukii at the registered field rate included spinosad , peroxyacetic acid and Chromobacterium subtsugae . Each treatment had 12 plots, and each plot was ~0.66 hectare. GUM dispensers were deployed on 9 and 13 July , and on 9 and 22 August 2019. Blueberries were collected every two days for the duration of the experiment. One fruit sample was collected in each of the respective 36 plots. Collected samples were at least 20 m from the edge of the crop and each sample contained 10 blueberries collected from the interior of the bush at ~0.75 m from the ground.Trials were conducted in 1.8 hectare of highbush blueberry plants . The experiment ran from 6 October to 15 October 2020. There were three treatment levels i.e., UTC , buffer , and GUM. The GUM plots were located directly next to the buffer, followed by UTC plots of equal size. Plots were each ~0.05 hectares . Spinosad was applied on 6 October on the UTC and buffer areas. Insecticide application and GUM deployment occurred only on 6 October. On 8, 10, 13 and 15 October, one fruit sample consisting of 10 berries was collected from each of the 36 plots. Samples were collected at least 20 m from the edge of the crop and at ~0.75 m above the ground.This trial was conducted in Oxnard, California, USA on highbush blueberry plots during 2020. Plants were irrigated with three drip stakes per plot ten times a day for ten-minute intervals delivering 1.1 liters of water per hour. Screenhouses were fully enclosed with screen material to prevent insects from entering.

There were three 70 m x 5 m screenhouses with GUM or UTC treatment randomly assigned to the north or south end of each screenhouse for a total of 6 plots. Within each screenhouse, treatment plots contained twelve plants in two rows, and plots were separated by 45 m. one-hundred flies were released in each plot four times, once per week. Three GUM deployment plots were compared with three UTC plots. GUM dispensers were installed in every other plant with irrigation stakes placed directly through the pads. The GUM application was completed on 14 April. Plots were sampled every seven days from 14 April to 12 May. One sample consisted of 50 berries.Ten field trials were conducted from September to November 2020 across multiple coastal production regions in California, USA , at different ranches and on multiple varieties being grown under high tunnels. Each location was a replicate consisting of two plots and were randomly assigned at each ranch to GUM or to UTC. Plots within a ranch received similar irrigation, fertilizer, and insecticides. Each plot received a minimum of four spinosad sprays timed 7-10 days apart during the cropping period and based on monitoring trends from fruit collections. Additional peroxyacetic acid applications were applied at 2-3 day intervals after each spinosad application, followed by a C. subtsugae application 1-2 d after each peroxyacetic acid application. Throughout the experimental periods, GUM dispensers were distributed evenly throughout each plot and replaced every 21 days. GUM dispensers were staked directly under the drip line in soil plots, and irrigation stakes were placed directly through the dispenser in substrate plantings. Six fruit samples were collected from each treatment plot every week for 4 to 12 weeks. Samples were collected at least 2 m from each edge of the tunnel as well as from the center of the tunnel approximately 20- 30 m from the edge of the tunnel and at ~0.75 m from the ground. Each sample consisted of 50 berries. Sample berries were incubated at room temperature for 2-4 days to allow for larval growth and facilitate detection. Samples were evaluated by crushing fruit and submerging them in a saltwater solution . The crushed fruit solution was then poured into a tray where D. suzukii larvae subsequently floated to the top of the solution and were counted. A trial was conducted in a 4.85 ha blackberry field with cultivar Prime-Ark® 45, in Salinas, California, USA in 2020 . Each treatment consisted of 7 plots: UTC, , GUM, or GS + GUM. Each plot was approximately 0.23 hectares. Two insecticide treatments were applied on 19 and 25 September in the GS and GS + GUM plots. The GUM dispensers were deployed on 21 September 2020, three days before the first sampling, and reapplied on 8 October 2020 in the GUM and GS + GUM treatments. The gum was placed under drip emitters. Blackberry samples were collected twice per week with one pre-treatment collection occurring before the insecticide treatments and gum deployment. Each sample consisted of 300 blackberries for each plot. Biweekly collections continued for 5 weeks after the pretreatment count. Samples were evaluated 3 to 4 days after collection to allow larvae to develop and the number of D. suzukii eggs and larvae were counted under a stereo-microscope .The current study supports findings from previous laboratory and small-scale field cage trials. Here we show through field collected and modeled data that food-grade gum use can reduce D. suzukii fruit damage . The aim of this work was to acquire detailed knowledge about limitations of food-grade gum in a range of commercial cropping systems including growing blueberries in container, blackberry, cherry, raspberry, strawberry, and winegrape. These studies were conducted in two key production regions i.e., California and Oregon in the USA.

The overall results supported initial findings and provided additional evidence that this tool can reduce D. suzukii crop damage especially when applied together with the grower standard. Both field-collected data and model simulations indicates that there is a synergistic effect of food-grade gum when used in combination with a conventional insecticide. For most of the experiments , field plots receiving the food-grade gum resulted in either numerical or statistical differences in D. suzukii damage compared to untreated control plots. This was not recorded for the cherry, strawberry, and blackberry trials. Reasonable hypothesis about these data are discussed below. In trials where D. suzukii infestations were measured in buffer plots , there was evidence of a reduction in damage, but not at the same level as in plots treated by the food-grade gum. Overall, considering all the trials, crop damage was reduced up to 78% over a period of up to 21 days post application of the food-grade gum. The results from the current study indicate that the food-grade gum can be used in combination with standard insecticides , and in some cases as a stand-alone treatment to reduce the infestation level of D. suzukii. Similar reductions in D. suzukii damage were reported under laboratory and controlled semi-field conditions , suggesting that the food-grade gum resulted in lower damage due to oviposition. These findings support earlier results where the effects of semiochemical volatiles emanating from the food-grade gum resulted in significant behavioral changes . In several trials, data lower oviposition and fruit infestation in the presence of the food-grade gum under field conditions. Reasons of why in multiple trials a statistical difference was not reached, can be explained by multiple parameters observed by scientists and growers such as animals removing the cottons pads, water-irrigation issues, and wind. These factors are addressed in a future publication . In the Hood River cherry trial, constant windy conditions may have resulted in dispersion of volatiles, ultimately resulting in impacts that were less pronounced. There is little doubt that efficiency of the food-grade gum can vary depending on production conditions and crop . Host preference of D.suzukii was ranked 4th for cherry, followed by blueberry and winegrape . Such differences in host preference should be considered when applying food-grade gum. Synthetic blends can be less attractive compared to the actual fruit; thus, additional adjustments may be required to minimize egg-laying in the fruit. Results showed that the application of the food-grade gum in grape shows clear impacts to protect berries from D. suzukii attack. Considering the vulnerability of several winegrape cultivars towards D. suzukii and the encouraging results collected, we have reasons to believe that the food-grade gum can be a useful tool for the winegrape production. For the food-grade gum applications in blueberry in open field experiments, the infestation rate for the food-grade gum and grower standard were 70% and 85% lower than that for untreated control respectively, with the food-grade gum treatment resulted in a significantly lower infestation rate compared with the control. Open and semi-field experiments conducted in California provide similar outcomes to those in Oregon. Blueberry experiments conducted in California within a screenhouse provided 45.5% egg reduction. There were sequential applications with differing timing and the results indicated that early applications resulted in lower egg reductions . A potential hypothesis for this phenomenon could be related to environmental conditions including temperature and humidity that could significantly change the emission of plant volatiles . Egg reduction in raspberry and blackberry varied from 42-90% and 24-70% respectively. Two cultivars of raspberry have been subjected to the trial and in both cases there was reduction in egg infestation. Results were consistent between the different locations. For strawberry, in several cases results showed numerically increased larval levels compared in the food-grade gum treatments. A potential hypothesis for this phenomenon could be related to either unreported production practices or environmental conditions that could significantly change the emission of plant volatiles or the food-grade gum. Other reasons that can justify the negative results, range from lack of irrigation to rodents removing food-grade gum within a day of placement .

The five-year project is part of an effort funded by the U.S. Department of Agriculture to improve scientific knowledge about organic systems and to strengthen the Central Coast network of organic farmers and agricultural researchers . Dan Schmida of Sandpiper Farms is the study’s grower cooperator. Other UCSC researchers involved in the project include Center director Carol Shennan, Environmental Studies professor Steve Gliessman, and specialist Sean Swezey, along with Steve Koike, plant pathologist with UC Cooperative Extension. The researchers are testing the effects of several ecological farming practices, including suppressing disease with “biofumigation,” an alternative to fumigation with synthetic chemicals such as methyl bromide. Broccoli residues and mustard crops are incorporated into the soil priorto vegetable and strawberry plantings. As the broccoli and mustard break down they release naturally occurring chemicals thought to reduce the levels of soilborne diseases such as Verticillium dahliae, large round garden pots which causes wilt in strawberries. Other practices include rotating strawberries with broccoli and spinach crops, which do not host Verticillium; using strawberry cultivars less susceptible to disease; and incorporating compost to enhance biological activity and increase levels of organic matter.

Learning to manage nutrients efficiently is another of the project’s goals. “Strawberries are a tricky plant—they need nitrogen at the right time and in the right amount,” says Muramoto. “We’re looking at the best ways to adjust fertilizer and compost levels and application timing in order to meet crop demands throughout the season while decreasing the loss of nitrogen from the system.” Nitrogen that leaches into groundwater or runs off the fields can pollute adjacent waterways—including the ecologically sensitive Elkhorn Slough—and cost growers money in the form of lost resources. “The weather pattern here on the Central Coast makes nutrient management challenging,” says Muramoto. Most of the season’s rainfall occurs from late November through April, when the shallow root systems of strawberry plants are not fully developed. Although applying a plastic mulch over the beds prior to heavy rains helped decrease the loss of nitrogen from the system, there was still a significant level of nitrogen in storm runoff from the fields following the season’s first rains. “We found that surface runoff, rather than leaching, is the main way that nitrogen is carried out of the fields,” says Muramoto. “If there is a high level of inorganic nitrogen in the soil when the first heavy rains hit, a significant portion of that nitrogen can be lost.” Muramoto notes that growers should work to reduce the basal nitrogen level in the soil by minimizing pre-plant compost applications, especially if a broccoli residue has been incorporated prior to planting the strawberry crop. “Growers can take advantage of the nitrogen that the broccoli residue provides and save money by reducing the use of other inputs,” he says.

Monitoring for Verticillium dahliae has shown that incorporating broccoli residue prior to planting strawberries consistently reduced the numbers of Verticillium propagules in the soil. In contrast, incorporating mustard did not reduce Verticillium numbers. Although it was thought that the chemicals released by both broccoli and mustard should supress Verticillium populations, studies suggest that another factor may be at work. “Broccoli residues may increase the population of bacteria that break down both broccoli and Verticillium, which have similar cellular structures [chitin], whereas mustard may not stimulate populations of these bacteria,” says Muramoto. Despite the fact that the mustard did not lower the populations of Verticillium, no wilting effect was seen in the strawberry crop. According to Muramoto, other researchers suggest that mustard residues may stimulate competition among microbes for entry points into the strawberry roots, thus blocking some of Verticillium’s ability to infest the crop. However, Muramoto noted that it’s difficult to tease out the specific effects of mustard and broccoli in this study since the crops are planted in sequence. In addition, Muramoto learned that Capsella bursa-pastoris, or shepherd’s purse, a common weed in the region, also hosts the Verticillum dahliae fungus and can increase its presence in the soil. Based on this finding, plant pathologist Steve Koike suggests that weed management should be integrated with soil-borne disease management. The research will continue for another season, during which all of the fields will be planted with strawberries. “This fifth year of the study will tell us the most about the various rotations’ effect on strawberry production and plant health,” says Muramoto.Controlling soil-borne diseases without synthetic chemical fumigants such as methyl bromide is one of the biggest challenges facing organic growers. This is particularly true for organic strawberry growers, whose delicate crop is subject to a range of soil-borne fungal diseases.

Verticillium wilt, caused by the fungus Verticillium dahliae, canbe particularly vexing—growers are often forced to find “clean” ground every year in order to avoid planting strawberries into areas where levels of Verticillium have built up over time. As housing and other development projects eat away at the amount of available farmland in areas such as California’s Central Coast region, finding new ground is becoming more difficult. One promising technique for controlling a number of plant pathogens across a range of crops comes from the Netherlands and Japan. The technique involves growing and incorporating a cover crop, watering the area, and then sealing it with an oxygen-impermeable tarp to create anaerobic conditions that kill the fungal pathogens. Japanese use wheat bran in place of a cover crop for a carbon source. In the summer of 2004, Center director Carol Shennan, researcher Joji Muramoto, and UCSC Farm manager Jim Leap conducted a trial of the anaerobic residue digestion technique in a field that had experienced an outbreak of Verticillium wilt and Phytophthora root rot in strawberries. The average population of Verticillium dahliae in the plot’s topsoil was 3 micro-sclerotias per gram soil; only 1 micro-sclerotia per gram of soil is needed to cause Verticillium wilt in strawberry plants. The randomized, complete block design compared two types of cover crops , with or without tarping. The cover crops were incorporated on July 13, 2004; following incorporation, a soil sample densely infested by V. dahliae was buried as an inoculant in each plot. Leap then applied 50 millimeters of water to the plots overnight; a plastic tarp was applied on July 14 and kept in place for 12 weeks, during which Eh and temperature were measured in the plots. Regardless of cover crop type, large round pots the number of V. dahliae microsclerotia in recovered inoculants decreased to 0.3 per gram in the tarped plots , while the number in the non-tarped plots increased to an average of 23 per gram. Eh levels indicated that weak anaerobic condition developed during the first two weeks of tarping. On November 18, 2004, strawberries were planted in all plots. Disease symptom of strawberry plants, V. dahliae population in the soil, and strawberry fruit yield are being monitored through the growing season, with initial findings showing a lower level of Verticillium wilt symptoms in the tarped plots. Based on the encouraging results from the 2004 study, a new tarping trial was initiated this summer at the UCSC Farm. The researchers are comparing tarping periods of 3, 6, and 12 weeks. The study will also compare covering the plots with clear versus black tarps to see whether differences in temperature affect levels of V. dahliae. The plots will be planted with strawberries in the fall of 2005.A new greenhouse, a national conference on sustainable agriculture education, scholarships for apprentices, a farm to-cafeteria project, and a stock-free demonstration field project at the UCSC Farm—these are a few of the things that new grants and gifts will help fund in the coming year. Along with key funding for the Apprenticeship training program, a total of $135,000 has been raised so far for 2006 education-related projects.

We are grateful to the following funders for their support – The True North Foundation has granted $30,000 for the new Farm to Cafeteria project along with the continuation of the Community Supported Agriculture Education and Outreach program. The new funding will allow Apprenticeship staff to work with the Campus Food Systems group at UC Santa Cruz to bring organic produce to the campus cafeterias and restaurant while doing outreach and education on campus about sustainable agriculture and local food systems. A $25,000 grant from the AT&T Pebble Beach Charities will go toward the construction of a new educational greenhouse at the UCSC Farm. We are grateful for this grant brought in with the help of the Monterey Peninsula Foundation, which will help us leverage further funding for the new greenhouse. Newman’s Own Organics, with a $30,000 grant, and Nan McEvoy, with a $5,000 gift, have provided ever-important core support for the Farm & Garden Apprenticeship training in organic farming and gardening. A new grant of $5,000 from the Foundation for Sustainability and Innovation will allow Apprenticeship staff to do more extensive outreach and recruitment than ever, with a goal of increasing the number and the diversity of applicants to the six-month Apprenticeship training program. An anonymous donor gifted $25,000 to create the “Wild- flower Fund” for Apprenticeship scholarships, a fund that could support as many as seven apprentices over the next seasons of the Apprenticeship course. A new demonstration field at the UCSC Farm will be managed without any inputs from animal sources, thanks to a $4,000 grant from the Nalith Foundation. This “Stock-free” or vegan organic farming demonstration site will provide information on cover crop and plant-based composts as alternatives to animal manures and other animal products such as blood meal and bonemeal.Farm Foundation has pledged $10,000 for the coordination of the first-ever National Sustainable Agriculture Educator’s Conference being planned for January 2006 . This conference will bring together instructors from colleges, universities, and other educational organizations and will be put on in conjunction with the Ecological Farming Conference at Asilomar, California, on January 24–25 . The California Agriculture Teachers Association and the Kellogg Foundation, through the California Food, Fibers, and Futures project, granted $3,000 each to make possible the creation of a sustainable agriculture course curriculum for the community college level as well as a one day agriculture teachers’ in-service training hosted by the Center in June. Program organizer Albie Miles, the Center’s curriculum specialist, noted that colleges throughout the state are developing sustainable or organic agriculture courses at their institutions. The in-service training program for college and university instructors focused on the way that innovative production and marketing strategies used in organic agriculture are influencing the way that food and fiber are produced and sold.The Center recently produced the sixth title in its Research Briefs series. Participatory Action Research and Support for Community Development and Conservation: Examples from Shade Coffee Landscapes in Nicaragua and El Salvador describes a form of research that links both social and ecological questions while generating information that can be used to foster environmental and social change. Based on their experiences conducting research in coffee-growing areas of El Salvador and Nicaragua, Ernesto Mendez and Chris Bacon report and reflect on the opportunities and challenges involved in conducting participatory action research . Both Mendez and Bacon recently completed their doctoral work in Environmental Studies at UC Santa Cruz. The Research Brief uses examples from Mendez and Bacon’s studies in Tecuba, El Salvador, and Matagalpa, Nicaragua to illustrate the process of PAR. Like many coffee-growing communities, coffee growers in these areas are suffering severe economic hardships due to steep declines in coffee prices. Mendez’s work in El Salvador originally focused on biodiversity conservation and farmer livelihoods on shade coffee farms. As the coffee crisis deepened, his “action research” grew to include action-oriented activities such as farmer trainings in organic growing techniques, development of alternative markets, and diversification on the farm. Bacon’s experience in Nicaragua includes work with coffee cooperatives tied to the Fair Trade movement. His dissertation focused on the livelihoods of households linked to cooperatives selling into different coffee trade networks. Among the myriad requirements for successful rose growing is picking the “right” spot in the garden. By and large, roses require a minimum of 6–8 hours a day of full sun during the growing season . This intriguing class of roses, developed in the early 20th century, is capable of growing and blooming prolifically in moderate shade .

Data were analyzed using a 3-way ANOVA with density of seed , application of B. bassiana , and date as factors . Unrecovered insects were counted as missing data and were not included in the analysis. The commercial blueberry test site selected was located north of Bakersfield in Delano, CA. The trial began in August of 2008 and was conducted post blueberry harvest. The V. corymbosum varieties contained within the test area were, ‘Santa Fe’, ‘Jewel’, and ‘Star’. The most susceptible variety of blueberry to citrus thrips damage grown at the test site was the ‘Star’ variety and ‘Star’ was used consistently for evaluation of thrips numbers for all aspects of the trial . Our cooperator was interested in alternatives to traditional pesticides as the farm regularly was dealing with extremely high citrus thrips populations. For example, in 2008 the grower sprayed 5-10 times per field , rotating with traditional chemicals to reduce thrips impact on the subsequent year’s fruit set. Irrigation in all fields took place via drip irrigation with one water delivery emitter per line at each plant base , plastic pot black but additionally, one portion of the blueberry field was equipped with 360° overhead sprinklers.

This irrigation setup provided the ideal situation to test B. bassiana under two watering regimes. The commercially available GHA strain is formulated to be mixed with water and for application via chemigation or as a foliar spray. The label states that no surfactant is needed to keep the spores in suspension. However, agitation alone in the 1,892.7 L holding tank was not sufficient to keep the material from precipitating, therefore 312.3 ml of Silwet L-77 was added to the tank mix. Mycotrol O® was applied directly to the soil surface with a gas-powered sprayer with a hand spray gun equipped with an adjustable flow meter. The dimensions of the plots were used to calculate the amount of material needed for both B. bassiana formulations . Plants in the test field were spaced every 0.92 m down each row, 3.35 m between each row, and each row was about 165 meters in length. Our studies were conducted inan 18-row section of a 4.04 ha field. The overhead sprinklers were spaced every 7 meters in the row and were located every other row for 12 rows. We chose to investigate the effectiveness of the B. bassiana colonized millet seed versus a Mycotrol O® soil application under two watering regimes, drip-line alone versus drip-line with overhead sprinkler, because B. bassiana conidia are highly subject to desiccation. Comparing the soil drench in both irrigation types with the colonized millet elucidated the effectiveness of the treatments when compared to the control.

The blocks were laid out in a 3 x 2 factorial design, with each block consisting of most of five rows of blueberries , each being 27.4 m long . The berm used to grow blueberries at the commercial farm was 1.21 meters wide and each plot was 27.4 meters long. The spacing between adjacent rows was 3.35 m, while the spacing between the plants down a row was approximately 0.92 m with 30 plants per treatment plot . These dimensions result in 0.157 ha treated with raw spores but because the top of the berm was where thrips activity was evident and would be sampled, only 36% of the soil surface area was treated. The Mycotrol O® label states that the maximum field rate is 6.9 L/ha mixed in 935.3 L/ha water. We therefore chose to apply the entire 6.9 L of Mycotrol O® in 378.5 L of water per ha directly to the berm with no application between the rows, which resulted in 100% of the per ha rate of product being applied to 36% of the area and allowed the maximum amount of active ingredient to be applied to the area that would have almost all thripsactivity . Our field trial was intended to determine the extent to which B. bassiana might fit into a program projected to both control citrus thrips effectively and provide rotation among available chemistries so as to reduce thrips resistance evolution.

Thus, we felt it was important to operate under the best possible conditions for thrips infection by Mycotrol O® , regardless of financial considerations, i.e. application of product at the maximum label rate in the area where thrips were most likely to be active. The amount of millet seed used in the field trail was calculated based on the area of the berm to be treated and likewise with the Mycotrol O® treatment, only 36% of the total field area was treated. The amount of seed used was one colonized seed/ 2 cm2 over an area of 576 m2 ; the fact that 0.45 kg of seed was needed per 840 cm2 resulted in the application of 3.40 kg of colonized millet seed for the 8 treated plots . Every other plant within the middle ten plants of the middle row of each plot were sampled with pupation emergence cages . These cages were placed tight against the base of each set of canes on the east side . With 5 cages per block and 4 replicate blocks per treatment, a total of 20 cages sampled thrips pupation per treatment over two sample periods, i.e. for two consecutive 3-day periods after the Mycotrol O® soil drench. The treatments were: no B. bassiana with and without overhead sprinkler; colonized millet seed with and without overhead sprinkler; and a soil drench of Mycotrol O® with and without overhead sprinkler . In total, data were collected from 240 emergence cages over the duration of the trial . The colonized millet seed was set to imbibe water and allowed to sporulate for three days before application and was applied using a hand fertilizer applicator . Four days post application of the millet seed, the soil drench of Mycotrol O® was applied and pupation emergence cages were placed in the field and left out for 3 days . After three days, the sticky cards from each emergence cage were collected and replaced with new cards and the traps were switched to the next plant on the east side. These traps were left in the field to sample thrips for another 3 days . Because the traps were placed out every other plant, this ensured that all of the middle ten plants were sampled over the two, 3-day sampling periods . For two weeks before through two weeks after the applications of B. bassiana , plastic growers pots counts were taken of thrips levels on plants twice per week. Beat samples were taken by beating random canes of flush foliage such that the thrips would fall onto a 12 x 12 cm black acrylic beat tray. The numbers of thrips on the beat trays were counted quickly in the field. The counts were taken twice per week from each of the 10 central “data plants” from the ‘Star’ variety of each of the 24 test plots. The new green flush growth was measured on three dates to record the amount of growth since the beginning of the fungal treatment applications to determine if there were differences based on the treatments and amount of water applied to the different plots. Measurements were made of the average cm of new shoot growth over the 6-week trial period. Due to the complex nature of the experimental design, i.e. treatments nested in a 5-way ANOVA , beat count data were analyzed using PROC MIXED and means were separated using Tukey’s test .Location of citrus thrips pupation in potted blueberries. Figure 3-2 shows the location of late second instar citrus thrips at death in the greenhouse study as well as those that located pupal refuges on the plant. Based on where they dropped off the plant, data indicated that more than 92% of the thrips would have pupated off the plant, likely in the soil near the base of the plant.

Numbers did not vary significantly by location over the seven sample dates of this study; therefore data were pooled . A key result was that the proportion of second instar thrips crawling down the base of the plant was higher than the proportion dropping off the plant at distances measured past the base the plant . Field sampling of thrips pupation sites. The four emergence cages placed under the field blueberry plants in each cardinal direction provided a means of sampling late second instar thrips moving towards the soil to pupae versus adults emerging out of the soil following pupation . Total numbers of thrips collected were pooled for the four traps in each direction at each respective location to determine which cardinal direction showed the most activity, and therefore was the most appropriate location to sample for citrus thrips in the field trial. Emergence cage data were summarized in two ways; the number of thrips moving off the plant to pupate in the leaf litter and the number of thrips emerging from the leaf . Data from the nested ANOVA generated p-values for direction , as well as distance grouping from the base of the plant . The cage closest to the base of the plant had significantly higher numbers of thrips emerging from the soil . Numbers of thrips trapped from the eastern cardinal direction were significantly higher for both mean numbers of thrips moving to and from the soil , indicating that for the field trial, emergence cages should be placed directly next to the base of the plant on the eastern side to sample the location that would have the most thrips activity.Fungal formulation and timing of application. Not only did the second instar thrips not avoid the GHA colonized millet seed, they were observed actively walking through it. Mortality was 100% in millet seed treatments across all trials compared to the untreated checks, which ranged from 0% to 8% mortality across these trials. Thrips avoidance of colonized millet seed. Of the proportion of thrips not finding pupal refuge on the plants with colonized seed , 100% infection was seen with each of the different quantities of seed, i.e. each of 0.5, 1, or 2 seeds/cm2 was a sufficient density to infect and kill all late second instar thrips in the greenhouse study. No thrips were infected in the control treatment. There were insufficient data to conduct a 3-way ANOVA because all recovered thrips were infected with the fungus. Because all three densities tested were effective, we chose to utilize the most economical density in the field trial, i.e. 0.5 seeds/cm2 . Field evaluation of B. bassiana for citrus thrips management. In the split-plot design model, the whole plot factor was water and the split-plot factor was fungus treatment in a type three analysis of variance . Water, time and treatment were the main effects in the full model. Thrips levels measured on pupation traps at 3 days after treatment were lowest with colonized millet seed, intermediate with Mycotrol O ® , and highest in the untreated control . Additionally, there was fewer thrips counted in the colonized millet seed treatment than in Mycotrol O® treated plots . However, at time two , thrips levels with Mycotrol O® were no longer significantly reduced in relation to the control . While thrips levels measured using pupation traps were significantly less than observed in the control in all plots , thrips levels on plants measured using beat samples did not show a significant decrease , although comparing data with no overhead water that with overhead water, there appeared to be fewer numbers of thrips in the overhead sprinkler plots . The measurements from the new green flush growth in the overhead sprinkler treatments showed that those plants had longer growth than those without overhead sprinklers , but thrips numbers were not significantly lower on those plants. At none of the times when foliar beat counts were taken were there significant differences in thrips numbers across any of the three treatments .The ultimate goal of this work was to determine if the GHA strain of Beauveria bassiana could be used effectively as an alternative to traditional insecticides in commercial blueberries in California.

Many studies have revealed that dormancy plays a crucial role in assisting insects in responding to various phenological environments, including the phenology of different host fruits . The dormant state of tephritids was determined by the rate of growth and development. Therefore, genes associated with development are crucial factors that regulate the adaptation of phenology of various hosts. For example, genes related to sensing daylength or photoperiodism and the central nervous system regulate chronic adaptation . Under the regulation of related genes, diapause may involve the deceleration of the developmental progress of tephritids to synchronize the phenological environment . R. pomonella of tephritids is a typical case. The ancestral host of R. pomonell is the hawthorn Crataegus mollis, but its species host expanded to the domestic apple Malus domestica and subsequently formed a new apple race . Apple fruits ripen earlier than hawthorn. The flies that infest apples and hawthorns must differentially time their life rhythms to match the differences in ripening times of their respective hosts .

To realize this process, tall plastic pots the flies of the two host races varied their time of overwintering pupal diapause. Under the pressure of different host fruit phenologies, many development-related genes are involved in regulating the adaptation to the different phenologies of two host plant fruits . Functional genes associated with cell/tissue development , metabolism , translation , and cell division are highly enriched . By increasing the expression levels of these genes, the CNS development of apple flies was elevated during their diapausing period compared to that of hawthorn flies. Adult emergence-associated genes, including key hormone signaling genes, the ecdysone receptor partner usp, the ecdysone biosynthesis protein ecd, cell cycling genes Myb and rbf, genes coding Mediator complex proteins, and various genes in the Wnt signaling pathway , etc., were enriched to regulate adult fly eclosion to match their host fruit ripeness .Genes coding for ribosomal proteins are often associated with protein translation by stably expressing ‘housekeeping’ genes. This type of gene is involved in many basic biological processes, such as digestion, detoxifcation, growth, and development, in most organisms . Therefore, ribosomal genes may also be involved in the host plant expansion of tephritids after receiving chemical and nonchemical stimuli.

As mentioned above, ribosomal genes increased their expression level to regulate the growth of R. pomonella in response to the different phenology of its new host apple . The role of ribosomal genes involved in host expansion and new host adaptation of insects, including tephritid flies, is mainly related to the response of ribosome-inactivating proteins in host plants . RIPs have been found to have insecticidal functions in many insects, including beetles, mosquitoes, and moths . Ribosome genes can help insects such as tephritids realize host shifting by regulating their expression levels to counteract the RIPs of various host plants . In addition, ribosome genes interact with some epigenetic factors, which leads to chromatin remodeling to change gene expression and regulate different biological processes, including host plant adaptation . In response to different secondary chemicals, ribosomal genes were also involved in host detoxification of different species of the R.pomonella complex. R. zephyria and R. pomonella are sister species in the R. pomonella complex that specialize in snowberry and domestic apple plants, respectively . In reciprocal transplant tests of these two Rhagoletis taxa, microarray data indicated signifcant enrichment of mitochondrial ribosomal proteins when the two fly species fed on their new hosts, which contain different complements of phenolic and glycosidic in laboratory studies . Several studies on lepidopteran species revealed the role of ribosomal genes in response to host expansion . For example, ribosomal genes were downregulated in C. suppressalis when extended to the novel host water oats , which may be a more suitable host for C. suppressalis than its native host, rice. In contrast, ribosomal genes were upregulated in H. armigera when shifting to unsuitable novel hosts .The role of genes associated with the oxidative phosphorylation pathway is primarily involved in energy metabolism and provides energy in the form of ATP for most organisms and most biological actions .

The OXPHOS pathway is coupled with the mitochondrial electron transport chain, and mitochondria are major sites of reactive oxygen species production in the majority of eukaryotic cells . The level of mitochondrial oxygen fow through the OXPHOS pathway influences ROS homeostasis and regulates the energy supply in different biological processes . OXPHOS genes can take part in many biological activities, and therefore they may also be important in the regulation of the response to host plant expansion of tephritid flies. Research on Bactrocera tau reared on two native cucurbit hosts and a novel host showed a large number of upregulated NADH genes in the OXPHOS pathway in transcript data of B. tau when feeding on banana. These results suggest that OXPHOS genes play an important role in the process of novel host fruit use in B. tau . OXPHOS was also involved in the host expansion of R. pomonella in response to the different phenologies of various hosts, as mentioned above. Certain genes in the fat bodies of tephritids are also involved in the energy supply for many biological processes, including digestion, detoxifcation, development, and immunity . Differentially expressed genes, such as the lipase gene, ATP synthase gene, and alpha-amylase genes , were documented in the tephritids B. dorsalis and P. utilis in response to different secondary chemical environments .The various types of genes summarized above led to multilevel responses in tephritids, including nervous-, behavioral-, chemical-, and physical-level responses, when the flies faced different host environments. These multilevel responses to host expansion result in multilevel adaptations in flies, which lead to successful expansion to a novel host . Adaptation to a novel host is a complex process. Multilevel adaptation in fruit flies results from multigene regulation rather than a single gene or several genes performing various regulatory roles. The transcriptome data revealed that olfactory-, digestion- and detoxifcation-related genes and ribosomal genes were all involved in novel host adaptation in R. pomonella . Laboratory strains of B. tau also had activated OXPHOS genes and digestive and detoxifcation genes when the fly responded to a novel host environment . The multiple-gene regulation mechanism during host expansion to a novel host was also documented in other insects. For example, C. suppressalis launched three types of genes simultaneously to regulate adaptation to the new novel host water oat . S. yangi differentially expressed genes related to digestion, detoxification, oxidation–reduction, stress response, water deprivation, and osmoregulation during adaptation to the new host Ephedra lepidosperma . Various genes also regulate the adaptation of tephritids to new hosts via multiple mechanisms. As summarized above, large plastic pots the alteration of gene expression levels, gene family expansion, and the use of various gene types or subfamilies are the major mechanisms involved in novel host adaptation.Many tephritid species attack economically important crops, including vegetables and fruits. The economic losses caused by tephritids reach over US$2 billion annually . Control strategies for tephritids primarily involve chemical use inmany countries, which may be harmful to the environment and human health. Therefore, more environmentally friendly control methods should be sought and recommended when possible. RNA interference is an effective method to safely control tephritid flies. Therefore, selecting the target genes to be ‘silenced’ is a key step in the RNAi control method . Some target genes are associated with functions such as temperature sensitivity and sex determination .

For tephritid species, including Anastrepha suspense , Anastrepha fraterculus , B. dorsalis , B. minax , Bactrocera tryoni , and C. capitata , effective RNAi controls have been developed based on the suppression of functional genes associated with eye pigmentation, embryonic segmentation regulation, postembryonic growth/development, reproduction, embryonic temperaturesensitive lethality and sex determination . Based on these target genes, RNAi can be applied in pest control not only for tephritid species but also for some Coleopterans and Lepidoptera insects by foliar spays, ingested dsRNA or sterile insect technique application . However, functional genes related to host plant adaptation are also target genes in RNAi control methods for tephritids. For example, the vision-related gene R6 or gustation gene GR59f of B. minax , digestion-related genes try1, try2, try4, and try5 of B. dorsalis , olfactory Orco gene of B. oleae , CSP2 gene of B. dorsalis , and detoxification genes CYP6A41 and CYP6EK1 of B. dorsalis are associated with host adaptation functional genes, and all of these genes possess an exploitable potential as target genes to control fruit flies. More target genes related to host plant expansion for tephritids need to be identified for their major functions and implemented in pest management. Although RNAi is an effective and tractable genetic tool, other novel gene tools, such as clustered regularly interspaced short palindromic repeats and the CRISPR-associated protein 9 gene editing system, can also provide scalable pest control strategies . Compared with traditional RNAi, CRISPR‒Cas9 can knock down or modifily the target gene precisely instead of just suppressing the expression of target gene . The target genes edited by the CRISPR‒Cas9 system can create stable and heritable strains, which can be applied in actual tephritid control. Applying the CRISPR‒Cas9-mediated editing system, some target genes in tephritid flies have been evaluated for their potential for functional application, such as the eye pigmentation gene we , embryonic segmentation gene prd , sex-determination gene Astra-2 , tra2 , and pupae color gene wp . CRISPR/Cas9-mediated precise editing is a process in whichCas9 endonuclease recognizes a specific genomic region under the leading of chimeric single guide RNA . The CRISPR/Cas9 system editing the functional target gene shibire, tsl in B. tryoni and the white pupae gene wt in B. dorsalis, C. capitate, and Z. cucurbitae have been applied in the development of genetic sexing strain application in SIT control. This gene tool also has broad application prospects in tephritid management based on host plant adaptation-related genes in the future. Regulation of host adaptation would be an important mechanism to target because this adaptation allows tephritids to expand in new habitats and change to new biotypes. Therefore, developing suitable novel host adaptation functional genes as target genes in genetic disruption control strategies could help prevent tephritids in an environmentally friendly manner.Thrips are members of the order Thysanoptera. This order is subdivided into two suborders, the Terebrantia and the Tubulifera, with about 5,500 described species in nine families . The Terebrantia consist of seven families, six of which are present in North America. All members of the Terebrantia have the following in common: the last abdominal segment is rounded or conical, females possess an ovipositor, forewings have veins and setae, fringed cilia of the forewing arise from the basal sockets, and the wing surface typically has numerous microtrichia . The Tubulifera consist of two families, but only one of them is present in North America . The Tubulifera may be distinguished from the Terebrantia by the following characteristics: both males and females have a tubular last abdominal segment, females lack an ovipositor, forewings lack veins and setae except at the base, the fringe cilia lack basal sockets, and the wing surface is bare of microtrichia . The families within the Terebrantia are separated by antennal characters, mainly the number of segments and type of sensoria on the third and fourth segments . Thrips are tiny, slender, and soft-bodied insects that are 0.5 to 5.0 mm in length. When wings are present, there are four very long and narrow wings that are fringed with long hairs. This fringe, or tassel, gives the order its name, thysano, the Greek word for tassel and ptera meaning wing. The mouthparts of thrips are unusual and of the sucking/piercing type. There are two principle structures. The first structure consists of the left mandible, which is modified into a tough, sharp, piercing organ that is hollow but lacking an aperture; the right mandible is reduced and vestigial . The second structure is composed of the paired styliform lacineae of the maxillae, which are interlocked to form a single feeding tube . These structures are contained inside the proboscis that is located opistignagthically on the ventral surface of the head.

For those less familiar with the Apprenticeship, it is a six month, full-time organic training for up to 38 apprentices from around the world. The roots of the program extend to the arrival of Alan Chadwick at UCSC 40 years ago, when he and a group of student “apprentices” created the Student Garden Project . Apprentices have traditionally been allowed to live on the farm in their own tents for the six months, sharing cooking and other chores in the Farm Center kitchen and dining room. As a past apprentice and staff member, Boyce-Abel said that for her the planned apprentice housing demonstrated a new level of support from UCSC and a new, more permanent status for the Apprenticeship. When the campus approved the apprentice housing project, Garden Manager Orin Martin said, “This represents staying power.” The apprentice tent cabins, planned as “light-on-the-land,” appropriate technology structures tucked in behind the plum orchard, will give the program a solid foundation. Olivia’s challenge inspired a $20,000 gift from 1978-79 apprentice Meg Cadoux Hirshberg and her husband Gary Hirshberg , whose gift amount allows them to name one of the cabins. Phyllis Anderson, a 1967-70 apprentice and a Friends member, large square planting pots pledged $5,000 which entitles her to name one room of a cabin, as will Jeannine Bonstelle Bassett’s $5,000 gift through the Silent Gong Fund.

Many other apprenticeship graduates and Friends of the Farm & Garden members have also supported the project with gifts and pledges. With these gifts, added to the Center facilities funds set aside for this purpose and funds brought in through this summer’s Back 40 event, more than half the needed $487,000 has been raised. Should you be inspired to donate now, please send a check to the above address made out to the UCSC Foundation with “Apprentice Housing Project” in the memo line.To subsist as an herbivore, an organism must overcome substantial barriers in the form of physical or chemical plant defenses and less than optimal nutrient availability. In some instances, the plant defenses interact directly with nutrient availability by decreasing uptake or impeding digestion , although myriad mechanisms have been described for how herbivores adapt to or avoid defenses. In addition to these deterrents, plant tissues typically maintain high carbon to nitrogen ratios, and plant fluids are depleted in many essential amino acids, making it more difficult for herbivores to acquire nitrogen-based nutrients. To overcome these dietary limitations, herbivores evolved partnerships with bacteria that facilitated transitions to new feeding niches, e.g., on phloem or xylem, or otherwise augmented plant palatability by attenuating defenses. Symbioses can fail, however, when symbiont genomes degrade or limit host range . Thus, there is likely selection pressure to either replace symbionts with more efficient ones, or to evolve novel feeding strategies to avoid symbiont dependence. Indeed, several hemipteran lineages, including leaf hoppers and the Phylloxeridae , have transitioned to novel plant-feeding strategies and lost their obligate symbiont associations.

The transitions in and out of symbioses have left genomic signatures such as reduced genome structure and function for many obligate symbionts, although the effects of symbiosis on herbivore genomes with or without symbionts is unknown.The metabolic coordination in amino acid synthesis and usage between bacteria and host requires amino acid transporters that function in transporting amino acids across the insect/symbiont interface, membranes that separate the cytoplasm of symbionts from insect hemolymph. Two types of AATs mediate this transport: the amino acid polyamine organocation transporter superfamily and the amino acid/auxin permease transporters family . Although both groups belong to the APC superfamily, members of the AAAP family have relatively divergent amino acid sequences, varying substrate specificities, and 11 transmembrane domains, compared to other transporters of the APC family. Expression profiles of these two families of AAT genes for several herbivorous species and their bacterial endosymbionts support a role for these transporters in the evolution of nutritional endosymbiosis. A growing body of evidence has demonstrated that insects induce nutrient sinks in plants in the form of galls that abundantly supply minerals, carbohydrates, and free amino acids. Given that numerous insect taxa form galls, an intriguing question arises: how does the accessibility of gall-enriched nutritive compounds influence the evolution of insect hosts and/or their symbionts? Among the Sternorrhyncha, few lineages secondarily lost endosymbionts concurrent with a shift to parenchyma feeding, and some taxa, such as the Phylloxeridae also induce galls. Insects within the Phylloxeridae are considered sister to the aphids and adelgids , groups that also retain galling and free-living species.

In contrast with aphids and adelgids that harbor symbionts in bacteriocytes, Phylloxeridae species lack stable intracellular symbionts. Further, Phylloxeridae comprises numerous life history strategies, including galling and free-living species that allow a phylogenetically controlled comparison to understand how these strategies arose with respect to their nutrient acquisition and metabolism. As an important grape pest worldwide, the grape phylloxera is capable of making leaf and root galls and its interaction with plant hosts has been the most investigated among the Phylloxeridae. Studies showed that infestation of D. vitifoliae reprograms plant metabolism, leading to the accumulation of nutrients such as carbohydrates and free amino acids. Recently, D. vitifoliae AATs were compared to paralogs in aphids to help pinpoint which transporters underlie the maintenance of nutrient symbiosis between aphids and Buchnera with an emerging conclusion that ecological context may contribute to AAT gene copy number and evolution. To expand the understanding of amino acid metabolism associated with herbivorous insects, we compared species that manipulate plant host amino acid content by gall forming to free-living species, and among species with and without stable nutritional endosymbionts. We sequenced the transcriptomes of nine Phylloxeridae species including D. vitifoliae and eight from the genus Phylloxera. Oak phylloxera has a free-living life history and thus was compared to other galling phylloxerid species regarding AAT evolution whereas two aphid species whose genomes are sequenced were compared to four other galling aphids. Our results indicated that galling insects, in Phylloxeridae and among aphidomorphs, experienced increased constraints on the evolution of AATs likely because of their ability to manipulate plant host metabolism.We collected nine known species within the Phylloxeridae for RNA sequencing. Phylloxeridae is a sister family of Adelgidae under the superfamily Phylloxeroidea, which is sister to the Aphidoidea: Aphididae all within the suborder Sternorrhyncha . These nine species include three that gall stems/petioles , one that feeds across hosts causing crinkles/folds in leaf veins , three that form spheres on leaves , one freely living , and one that galls both roots and leaves of Vitis species . In contrast to P. quercus that lives freely on oak trees , all the other Phylloxera species induce galls on different hickory species and/or tissues . Although the description of P. foveata places it on C. cordiformis and the individuals collected in this study came from C. glabra, we are considering the insects to be the same species for this study because of similarity in the induced phenotype. The Phylloxeridae represents an unresolved taxon , where ongoing research is delineating species. All Carya originating phylloxerids were collected from the Arnold Arboretum of Harvard University, Boston, Massachusetts. Phylloxera quercus was collected from Quercus sp. at a horticultural nursery in Bellevue, WA. Daktulosphaira vitifoliae was collected from native grapes near Madera Canyon, Arizona. Collected insect samples were stored in RNAlater solution at room temperature initially, transferred to 4 °C within eight hours for temporal storage , plastic square planter pots and later kept at −80 °C until RNA isolation. Insects at all stages were collected initially but separated to include only juveniles and adults for sequence analysis.Amino acid uptake, transport, and catabolism underlie the success of herbivory as a life history strategy. Here we present the first multigene tree for members within the Phylloxeridae; a family with both galling and free-living herbivores. We also present transcript profiles across fluid feeding herbivores that support plant host constraint on insect amino acid transporter evolution. Galling sap-feeding insects show fewer AAT transcripts than free-living species within the same insect families and within the same genus of Phylloxera. The ability of galling insects to manipulate plant nutrient content likely altered selection to retain or duplicate the number of functioning AATs within the insect.

Previous research suggests some AATs facilitate the evolution of endosymbioses but also that ecological context may interact with nutrient transporter evolution to shape adaptive duplication or loss. Our data advance this idea by highlighting how complex the selective environment is and suggest specialized interactions with plants play a large role in determining the evolution of herbivore genomes, especially when nutrient manipulating strategies are involved. Previous research on some AATs correlates gene expression and presence with the maintenance of endosymbioses; however, phylloxerids lack stable endosymbionts and still retain many of these same AATs. We found members of the Phylloxeridae family retain at least one copy of each APC found among other aphidomorphs with the exception of two clades that show duplications. Otherwise, phylloxerids retained at least one APC similar to many other Sternorrhyncha insects and D. melanogaster. Interestingly, free-living P. quercus often showed multiple AAT copies within clades where galling phylloxerids possessed only one copy . Free-living aphids also show a similar pattern compared to galling aphids for many clades . This increase within clades suggests that these paralogs may function generally to support nutrient transport when feeding on host parenchyma, atissue where nutrients are lower than when feeding on gall tissue where nutrients can be enriched by and for the galling insect. We hypothesize then that host nutrient manipulation altered the selection environment to maintain certain AATs. In support of this we identified fewer AATs in galling insects than free-living relatives. In some instances, galling phylloxerids did not accumulate specific AAT transcripts; however, lack of accumulation may result from a missing gene or lack of conditions under which expression occurs. While we recognize the limitations of transcriptome information to resolve this, the use of the D. vitifoliae and M. persicae genomes suggests all phylloxerid and nearly all aphid genes were accounted for, and that variation in AATs among genera occurs within aphidomorphs. Some Phylloxera species show accumulation of AATs absent from the D. vitifoliae genome whereas galling Phylloxera species show different numbers of AAT transcripts across clades. Similarly, some galling aphids also lack transcripts for some clades where free-living aphids retain one if not more transcripts. This provides support that differences in the nutrient environments across plant hosts differentially alter selection to retain certain AATs. Little information exists for comparing extensive metabolite profiles of hosts across galled taxa, but the diversity in morphology, color, and specialized tissues that are induced in plant hosts by galling insects suggests nutrient pools that insects feed upon differ widely. Future studies examining metabolite pools among closely related taxa will help resolve what limitations, if any, are present in induced plant phenotypes, and provide additional tests of the role of host nutrient manipulation in the evolution of insect AATs. The microbial community plays a fundamental role in animal nutrient acquisition from food, especially for sapfeeding insects where coevolution with endosymbiotic bacteria alleviates low amino acid content provided by phloem diets. Galling or less apparent manipulation of host nutrients increases nutrient flux to feeding sites, potentially altering selection on the stability of endosymbiotic relationships. Our data and previous transcriptome profiles support increases in paralogs for two free-living aphids, but no galling aphids share these increases. This pattern suggests lineage specificity; however, until more insects are profiled in a way that controls for phylogeny while spanning the range of plant nutrient manipulation, either host manipulation, lineage specific evolution, or both may alter selection on AAT gene evolution. Prior transcriptome assessments correlated transcript abundance and presence with maintaining endosymbiosis. By examining more phylloxerids, we increased resolution of slimfast gene evolution, providing support for previous data that all aphidomorphs experienced slimfast duplication . Thus, slimfast expansions likely occurred in the ancestral aphidomorph. Because the ancestral state of phylloxerids is unresolved without a phylogeny, it is possible that the ancestor lost AATs with the evolution of galling or gained AATs with the transition away from galling. Either scenario would link this clade to nutrient acquisition strategies. Although phylloxerids lack stable nutritional endosymbionts, numerous microbial partners exist within the gut microbiome .

Jardineand Sampson were quickly elevated from “special agents” to permanent positions in the Forest Service, becoming “the two pioneers in national forest-range research” . Jardine was promoted to Inspector of Grazing in charge of the new Office of Grazing Studies in 1910, overseeing range research and directing the monumental task of “range reconnaissance” throughout the national forests over the following decade . He later served as chief of the Office of Experiment Stations and Director of Research for the USDA. Sampson was appointed to head the new Utah Experiment Station on the Manti National Forest, where he worked until 1922, when he accepted a newly created academic post in range science at the University of California-Berkeley. Both authored reports in 1919 that rank among the most influential in the history of US rangelands: Jardine’s “Range Management on the National Forests” was the first comprehensive statement of the policies and principles guiding forest rangeland management. It was still in use at his retirement in 1945—having been “three times reprinted without change”—when he was described as having “brought out the principles on which are founded the standards of good grazing practice over the whole western range country” .

Sampson’s “Plant Succession in Relation to Range Management” established range science on the basis of Clementsian ecological theory. For governmental and scientific purposes, blueberry packaging box respectively, Jardine and Sampson became the principal architects of the dominant paradigm affecting US rangelands in the 20th century. It relied on fencing and predator control not only to remake the physical landscape in favor of livestock production, but also to modifly the social landscape, reducing livestock owners’ dependence on herders to tend and protect their animals. In this realm and many others, the policies of the Forest Service benefited some people at the expense of others, and the agency relied heavily on “science,” howsoever flawed, to buttress its legitimacy . With the creation of the Office of Grazing Studies within the Forest Service, moreover, a critical bureaucratic shift took place, apparently without comment or resistance. The scientific challenge of studying grasses, grazing, and rangelands passed from the Bureau of Plant Industry into the hands of the Forest Service, first under the umbrella of the Branch of Grazing and, after 1926, the Branch of Research. There is no indication that Coville objected to this transfer of research authority, and perhaps he could not have foreseen its longer-term consequences. Foremost among these consequences was the permanent subordination of range research to the Forest Service’s core mandate, timber production, and to its corollary imperative of fire suppression. What this meant, in practice, was that range researchers would be discouraged, if not prohibited outright, from investigating the possible benefits of fire to grasslands, savannas and forests throughout the West for most of the 20th century.

It is impossible to know what might have happened had range research remained in the portfolio of the BPI, but there is reason to believe that fire might well have been viewed in a more favorable light, at least in certain parts of the country such as the Southwest .The effects of the Coyote-Proof Pasture Experiment extend down to the present and across literally hundreds of millions of acres of rangelands in the US and elsewhere. It contributed directly to policies of fencing the land into pastures and eradicating predators of all kinds in the belief that these measures would benefit both livestock production and rangeland conditions. Fencing has since become ubiquitous and almost unquestioned as a basic tool of ranching and rangeland management, subsidized by US government agencies and aggressively promoted in pastoral development projects overseas; Netz , who acknowledges that the US West was where barbed wire fencing found its first widespread use, goes so far as to view it as fundamental to the “ecology of modernity.” Many predators continue to be persecuted by the BBS’s descendent agency, Wildlife Services; even those that are not, persist only at much reduced numbers. Perhaps most importantly, the Wallowa experiment contributed indirectly to institutionalizing range research in an agency whose primary mission lay elsewhere, inhibiting scientific recognition of the ecological importance of recurrent fires. The ecological effects of fencing probably cannot be disentangled from the other factors it enables or accompanies, such as water development, reduced herd mobility, and land tenure rationalization; suffice to say that fragmentation is considered a major threat to rangelands worldwide .

The magnitude of the influence of predators on ecosystem processes is controversial , but there is wide agreement that the consequences of long-term fire suppression are profound .Critical physical geography reveals the concatenation of particular events that influenced the Coyote-Proof Pasture Experiment, while simultaneously illuminating the institutional and political conditions that enabled it to have such widespread effects. We can then ask new questions about present-day issues and debates concerning rangeland conservation and management. In recent decades, scientists and environmentalists have challenged many predator control programs, and some extirpated predators, such as wolves, have been protected and/or reintroduced in portions of their former species ranges. But fencing is rarely challenged. In view of the history recounted here, one has to wonder if restoring predators can only be compatible with continued range livestock production if herding, too, is restored—in which case fences may no longer be necessary.Bronson’s words proved prophetic: the Student Garden Project that Alan Chadwick and his student apprentices brought to life at UC Santa Cruz starting in 1967—and all that has grown from it—have indeed had a major impact on food and farming systems over the past four decades. Chadwick and his apprentices not only created a vibrant organic garden—they set a precedent for forty years of groundbreaking work in sustainable food and agriculture education, research, and public service at UCSC. The garden sparked the growth of a 25-acre organic campus farm and an internationally known apprentice training course, as well as cutting edge programs in food systems and organic farming research and extension, national and international work in agroecology, a model farm-to-college program, an award-winning children’s garden, and much more. “We are small but mighty—an incubator for innovation,” says Sheldon Kamieniecki, dean of the Division of Social Sciences at UCSC. “Our influence proves you don’t have to be a big ag school to have major impacts on farming and food distribution.” In this article we’ll take a brief look back at the Farm & Garden’s history and examine some of the innovative ways that the Center for Agroecology & Sustainable Food Systems continues to stay at the cutting edge of sustainable food and agriculture research, education, and public service.Beginning in 1967, long before sustainability became part of the vernacular, students at UC Santa Cruz were practicing organic gardening under Chadwick’s exacting direction. Chadwick had been brought aboard at the fledgling campus to start a garden project that would help give students a “sense of place” amidst the chaos of construction at the newest of the University of California campuses. Chadwick’s students formed the core of an informal student “apprenticeship,” working alongside him to transform a chaparral-covered slope in what was then the heart of the growing campus into a lush, vibrant organic garden. This apprenticeship approach to teaching—in which instructors worked side-by-side with the students, gradually giving them increased responsibility—would become a hallmark of the training approach used at UC Santa Cruz. Inspired by the garden’s success, students lobbied for a larger plot of ground on which to put Chadwick’s organic practices to work. In 1971, seventeen acres on the lower campus were set aside for an organic campus farm. Later expanded to twenty-five acres, blueberry packaging containers the UCSC Farm became a demonstration and teaching site for small- and medium-scale organic farming techniques. Faculty and student involvement in the garden and farm grew in the 1970s with courses in organic horticulture and agriculture offered as “practicums” through the Environmental Studies Department, as well as appropriate technology and natural history classes based at the farm. Students took advantage of opportunities provided by the farm and garden to design thesis projects and learn through independent studies. Students and staff planted orchards, windbreaks, and perennial borders, creating a diversified organic farm on the growing campus.

In 1975, the loosely organized apprenticeship that began under Chadwick’s direction was formalized into a full time, year-round program offered through UC Santa Cruz Extension. The Apprenticeship Program was the first university-based program to offer students intensive academic and experiential training in organic gardening and farming techniques. With a dedicated work force, the original Garden Project expanded and the farm grew to include tractor-cultivated row crops, as well as hand-worked garden beds, generating enough produce to support a small direct marketing and wholesale effort, and establishing one of California’s first organic “farmers’ markets.”The late 1970s saw increasing concern over the environmental and social effects of conventional farming, from pesticide pollution and soil erosion to the impoverished status of farm workers. The Environmental Studies faculty recognized that the UCSC Farm & Garden projects held potential for wider academic and research applications that could address such issues. To help develop that potential, Steve Gliessman was hired in 1980 to create the Agroecology Program—this was the first University of California program to focus specifically on what would come to be called “sustainable” agriculture. Recognizing that sustainability required social as well as environmental changes, the Agroecology Program hired social scientist Patricia Allen in 1984. Allen initiated the nation’s first work on social issues in sustainable agriculture, addressing topics such as labor, gender, and access to food. Allen also organized and spearheaded the first University of California systemwide conference on agricultural sustainability in 1985. In addition to its work with academic groups, the Agroecology Program also created ties with local, small-scale growers. In 1989 the program hired entomologist Sean Swezey and began a Farm Extension effort to serve growers interested in organic farming techniques. This was the first public organic extension service offered in the U.S. Program researchers worked with local growers on their farms on studies examining the transition from conventional to organic management in crops such as strawberries, apples, artichokes, and cotton. This work took a unique “whole systems” approach that included research on soil fertility, pest control, and economic impacts. In 1994 the Agroecology Program was renamed the Center for Agroecology & Sustainable Food Systems, to better reflect its blend of natural and social sciences work. In 1997, agroecologist Carol Shennan was hired as the center’s director. Shennan brought an interest in agroecosystems and landscape ecology and developed a focus on intersections among agroecology, environment, and community. In 2007 Patricia Allen was appointed director, and continues her work to address various social issues in sustainable agriculture .Over the past decade the growing interest in organic food and alternative food initiatives, such as farmers’ markets, community supported agriculture , farm-to-college programs, and school gardens has heightened the need for research and education in these areas. Building on its history of innovations, CASFS has expanded its work to respond to these needs. Center members provide training materials and expertise to college programs around the country; conduct research to analyze and improve organic farming practices and minimize farming’s impact on natural resources; and analyze the latest trends in consumer interests, food equity issues, and alternative food systems. The growing Farm-to-College effort at UCSC is creating new ways for CASFS to work with students, staff, and faculty to develop a food system that is environmentally sound and socially just. Some of the highlights of work now taking place at CASFS include: High school students in a new youth empowerment program based at the UCSC Farm, with flowers from their farm plot; these were donated to a Santa Cruz family shelter. Trend-setting research and scholarship on social issues in the agrifood system, with current fields of study including perceptions of and priorities for social justice in the agrifood system, farm-to-institution programs, food-system localization efforts, gender issues in agrifood systems, priorities and pedagogies in sustainable agriculture education, and consumer interests and preferences. Basic and applied natural science research on ways to conserve nutrients on organic farms, minimize the impacts of farming on surrounding ecosystems, and manage pests and diseases with organically acceptable techniques, including an innovative approach to minimizing pest damage in strawberries .

Due to the low concentration of resveratrol specified within the Japanese knotweed rhizome used to simulate the base case model, approximately 6,000 kg of Japanese knotweed rhizomes is needed per batch. While ethanol is widely used across the simulated facility, a large quantity of ethanol at an approximate 1:1 ratio is required during the extraction process, largely attributing to the high cost for ethanol. To demonstrate the relationship between resveratrol concentration and the quantity and cost of ethanol in the process, we calculated ethanol consumption as we increased the concentration. Here, Figure 4.2 illustrates the change in the total cost of ethanol and amount of ethanol used when the concentration of resveratrol per knotweed rhizome is increased up to 3 mg/g.The increase of resveratrol found within the rhizomes led to an exponential decrease in the quantity of ethanol needed for processing. In comparison to the base case model, there’s a reduction of $1.6 million to the overall cost of ethanol when the concentration of resveratrol is increased an order of magnitude to 1mg/g. When resveratrol concentration is as high as 3 mg/g, ethanol cost decrease by $2.1 million, a 53% decrease from the base case model results. When the cost of total ethanol used is compared between 1 mg/g and 3 mg/g models, wholesale grow bags the difference is only about $575 thousand or a 23% decrease.

A simple method to evaluate the effect of the concentration in rhizomes, we measured the mass of knotweed rhizomes needed for processing as concentration was varied. As expected, an increase in knotweed concentration results in fewer knotweed rhizomes needed for processing. Figure 4.3 shows the change in mass of knotweed rhizomes needed and ethanol consumption per increasing concentration of resveratrol within the knotweed.Notably, ethanol is a commodity which has experienced some volatility in price during the last few years. A commodity tracker provided by trading economics.com tracks the cost of ethanol per gallon in USD daily . Trading Economics demonstrates that the price of ethanol per gallon reached a low of about $0.95 in April of 2020 and a high of $3.43 on November of 2021, with a current price of about $2.65 per gallon during the time this report was written 2 . The initial jump in ethanol price was over 260% in only a time span of one year and seven months. The price has since dropped about 23% since its peak in a span of 4 months with financial analysts at Trading Economics forecasting a further reduction in price in the near future. This fluctuation in price ultimately limits the ability to effectively assess the effect that the cost of ethanol has towards resveratrol production and similar bio-manufacturing facilities utilizing large quantities of ethanol.

In effort to assess the effect the ethanol price has on the OPEX, the price of ethanol was varied by increments of $0.50 from $1.00 to $3.00. The corresponding OPEX values for each scenario is demonstrated in Figure 4.4 below.In the scenario where ethanol was priced towards its low price of $1.00 per gallon, the annual operating cost to produce 100 MT of resveratrol is reported to be $13.2 and $9.2 million, with and without depreciation, respectively. In the same simulation file, where ethanol is now priced at $3.0 per gallon, the annual operating cost increases to $17.0 and $13.2 million, with and without depreciation, respectively. The base case scenario was performed using a price of $2.00 per gallon of ethanol. The annual operating cost for the base case model is $15.0 and $11.2 million, with and without depreciation, respectively. The difference in annual operating cost is roughly 16% when compared to the $1.00 per gallon scenario and 11.5% when compared to the $3.00. With each increment of $0.50, the annual operating costs steadily increases an average of 6.8% from the last. To assess the cost of ethanol to the COGS, the COGS values was plotted against the change in ethanol price, shown below in Figure 4.5.As expected, the COGS was shown in increase in a linear fashion, in a similar trend to that shown in the relationship between OPEX and ethanol price. An incremental increase of $0.50 from $1.0 to $3.0 per gallon of ethanol increased the COGS an average value of $10.

The COGS value for the optimistic case of $1.0 per gallon case is reported to be $131 and $92 including and not including depreciation, respectively. The variation in price is about $20 for both COGS values and a percentage difference of 13.5% and 18.1%, includingand not including depreciation, respectively. While ethanol is understood to change in price due to many unprecedented factors, it is recommended that the price of ethanol be discussed and be agreed upon for long periods of time with commercial supplies in efforts to hedge against the variation in price in the global market.Another commodity deemed essential to produce 100 MT of resveratrol is the cellulase enzymes used for hydrolysis in the process. Patents released by three resveratrol manufacturers in China detail steps on how to utilize ß-glucosidase for hydrolyzing polydatin into resveratrol to increase production . Here, the simulation for the base case model was designed in a similar fashion to incorporate the utilization of ß-glucosidase found in cellulase. As a result, the annual cost of cellulase enzymes was expected to be $71,436 or 1.3% of the annual operating cost. However, the price of cellulase enzymes used within the model was retrieved using literature values derived from a techno-economic analysis on enzymes costs for bio-fuel production3 . This value was not discussed or confirmed with a large-scale commercial manufacturer of industrial enzymes. The use of industrial enzymes remains a challenge as prices remain inconsistent due to enzymes being reported in terms of dollars per gallon of biofuels3,4. These prices often account for factors beyond the cost of enzymes themselves, such as overall bio-fuel yield, feedstock choice, and enzyme loading 3 . Consequently, a wide range of prices for industrial cellulase enzymes exists. Notably, an analysis performed by scientists at the United States National Renewable Energy Laboratory mention retrieving a Multi-Year Program Plan from the Office of the Biomass Program, Energy Efficiency and Renewable Energy, U.S. Department of Energy where the price of cellulase enzymes was anticipated to be within the range of $0.35/gal in 2007 and $0.12/gal by 2012. The same authors at the NREL performed a techno-economic analysis on thedesign and economics for conversion of lignocellulosic biomass to ethanol and concluded they were able to retrieve a price of $0.34/gal when using their own on-site enzyme production section, aligning their cost with the expectations of the DOE. Novozymes, an industry leader in industrial enzyme production, released a press release titled “New enzymes turn waste into fuel” in February of 2010 mentioning they can offer cellulase enzyme at a competitive price of $0.50 per gallon of cellulosic ethanol5 . Using the pricing information retrieved by the techno-economic analysis on cellulase enzymes for industrial applications mentioned above3 , grow bags for gardening a range of prices for cellulase enzymes per gallon of ethanol can be found to be between $0.68-$1.47. The difference in price is understood to be attributed to using the maximum theoretical yields of sugar consumption and if yields were based on saccharification and fermentation yields found in literature3 .

Since the price of enzymes are another variable cost which attribute to the cost of production, a sensitivity analysis was performed to assess the impact of such a large spread between enzyme cost. A scatter plot demonstrating the relationship between the COGS and enzymes cost is shown below in Figure 4.6.The range of ethanol used for this analysis were chosen from the values retrieved during our search for enzymes costs and a case where the enzymes are supplied at no additional cost. The list is as follows, $0.00/gal, $0.12/gal, $0.35/gal, $0.50/gal, $0.68/gal, $0.85/gal, and $1.47/gal. A large change in COGS values was not seen. The largest change in COGS when comparing to the base case occurred when the price of enzymes increased an order of magnitude to $1.47/gal. Here, the change in total cost was 188%, or a price increase of $134,000 a year when compared to the base case model. The low cost of enzymes for resveratrol production can be expected since the amount of enzymes being loaded to the reaction vessel is relatively small compared to the total mass also entering the reactor . To ensure the model was appropriately modeled, the percentage of enzyme costs was measured as the enzyme cost was varied, shown in Figure 4.7. As expected, as the cost of enzymes increase, as did the percentage of total enzymes cost to the total raw material costs.An alternative to single use enzymes highlighted in other Rsv production patents and scientific literature is the use of fermentation. Rather than purchasing and mixing pure enzymes with plant tissue, this approach mixes plant tissue with microbial cultures, utilizing the enzymes secreted within the solution, ultimately reducing operating costs and raw material cost associated to the addition of water for mixing. This method’s feasibility has already been demonstrated by Wang, H. et al., who effectively compares hydrolyzing P. cuspidatum herbs using fermented fungi versus using acid hydrolysis was performed and dubbed using fungi as an effective and feasible alternative6 . Arguably, applying this method for large scale production might not be practical, as one patent reports the time for fermentation ranges from 10 to 15 days7 , significantly reducing processing time and annual production throughput. Additionally, other researchers argue that the activity of the β-glucosidase enzyme responsible for converting polydatin to Rsv does not perform optimally under fermentation conditions 8 . Another alternative which can be utilized to address high enzyme costs was demonstrated in the analysis performed by the U.S. NREL. Design a bio-processing facility with its own on-site enzyme production section4 . This approach is one that has already been utilized in Rsv literature where the feasibility of fermenting Aspergillus oryzae and separating the β-glucosidase enzyme from solution to hydrolyze P. cuspidatum plant tissue was demonstrated8 . The use of on-site enzyme production is expected to reduce purchasing costs and provide a consistent supply of enzymes available for industrial use.As described above, the most utilized approach when extracting resveratrol from Japanese knotweed is the use of an enzymatic hydrolysis step to convert any existing polydatin to resveratrol. However, data surrounding large scale processing of Japanese knotweed is limited, therefore, bioprocessing parameters such as percent conversion was retrieved using literature on laboratory scale experiments. It should be noted that literature describes an efficient process where conversions can yield values as high as 100%9 . Rather than initializing 100% conversion within the simulation, another conservative approach was taken, and 90% conversion was specified within the base case model. To evaluate the impact that the percent conversion had on the CAPEX and COGS, the percentage was varied from 90 to 100 by increments of two, shown in Figure 4.8.Increasing the enzymatic conversion led to a reduction in both CAPEX and COGS. Utilizing the 90 and 100 percent conversion results for comparison, the difference in COGS is $3.1 and a $200 thousand difference in CAPEX. The largest drop in CAPEX among the different percent conversions occurred between 92 and 94 percent conversion, yielding a decrease in CAPEX of 55 thousand . Here, the reduction in price is attributed to 3 factors: the reduction in equipment size, the reduction of units needed for processing knotweed, and lastly, the reduction in raw materials such as water and ethanol entering the process at a 1:1 mass ratio with the mass of knotweed. The relationship between Japanese knotweed and COGS to increasing enzymatic conversion percentages is shown in Figure 4.9. One specific example where the reduction in CAPEX is seen is the reduction of reactor size needed to perform the enzymatic hydrolysis. The size of the reactor in the base case model is 13,621 L but the reactor is resized to 12,925 L when the conversion was increased to 94%. While there is a price decrease in both the CAPEX and COGS when 100% conversion is initialized, the author would advise against expecting to replicate similar values as 100 percent conversion may not be practical at large scale.