Horticulture

Overall, including a practical amount of red raspberry in the diet regularly is a low-calorie dietary strategy that improves gut microbiota composition and function in individuals with prediabetes and insulin resistance resulting in improvements in metabolic health. With a sustained emphasis on the role of gut microbiota in nutrition research, advances in our understanding of food-gut dynamics will provide new insights about the role of nuts and berries in human health and performance. Although research on a specific nut or berry provides insight into bio-activity and potential mechanisms of action, such focus also creates the potential for fragmentation because the search for overall dietary patterns is not addressed. The composition of fruits and nuts differ at the molecular level, and a broader view assessing similarities in chemistry and health benefits is critical for translational research as well as for messaging purposes. For example, blueberries, strawberries, pomegranate, walnuts, and grapes all have reported benefits for cardiovascular health, driven largely by the presence of similar polyphenols, which are present at varying quantities in each of these foods . Although health professionals and consumers often hear messaging on a single berry or nut, snap clamps ABS pvc pipe clip the potential benefits of increasing consumption of the broader category may be obscured or lost.

This challenges the ability to maintain consistent messaging and align better with translatable dietary guidance. Future interventions that combine nuts and berries with one or more other foods within a food matrix at dietary achievable doses and in more diverse populations are warranted. To date, multi-omics technologies have provided valuable insights into exposure-disease relationships. Coupled with artificial intelligence, predictive modeling and continuous, personalized monitoring, these data-intensive outcomes can provide further insights about the health benefits associated with regular intake of nuts or berries. One of the challenges of similar foods being studied in differing formats and by various research groups is the utility of the data as a combined set. Differences in test materials and experimental designs make integration of data difficult. The proper curation of combined data, whether physiologic, metabolomic, or genomic, is critical to ensure that combined datasets provide synergy, statistical power, and enhanced usefulness.The cardiometabolic benefits from regular consumption of nuts or berries are widely reported and include improved vascular function, reduction of cardiovascular disease risk factors, improved insulin sensitivity, and reduced risk of type 2 diabetes mellitus. Antioxidant and anti-inflammatory capacity and activity have also been noted. Metabolic outcomes may be context-specific and related to the physiologic state of the individual and host microbiome composition, among other factors.

Examples include findings of ellagitannin and ellagic acid rich foods resulting in differential responses in healthy individuals compared to those with prediabetes, who are dependent on gut microbial-derived metabolite profiles. Many factors contribute to inter individual variability in response to diet that can extend to context-specific aspects influencing the magnitude of health benefits and reinforces the importance for further research aimed at advancing discoveries in precision nutrition. Additional health outcomes related to nut or berry intake are outlined below.Adding nuts or berries to the daily diet may be advantageous for weight management for several physiological reasons. One is that these foods produce feelings of satiety, helping to reduce the desire to consume calorie-rich snacks that are low in vitamins, minerals, and fibers, ultimately improving body composition over time. A second possibility is due to urolithins, secondary metabolites produced from ellagitannins in nuts and berries. Urolithins increase the activation of the adenosine monophosphate-activated protein kinase pathway, resulting in anti-obesogenic properties in vitro and in animal models. AMPK increases fatty acid oxidation and decreases triglyceride accumulation. Phosphorylation of AMPK may also decrease cholesterol synthesis and lipogenesis by down regulating 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and sterol regulatory-element binding protein expression.

In clinical studies exploring the relationship between food and body composition, the incorporation of nuts and berries into the diet was associated with weight loss or maintenance.Regular consumption of nuts or berries has been reported to support brain health and cognitive function, motor control, mood, and executive function at physiologically relevant intakes. Middle-aged and older adults experienced improvements in balance, gait, and memory, and children experienced higher executive function and positive affect after acute and regular intake of both strawberries and blueberries. These beneficial effects may be the result of direct effects on brain signaling or indirect effects through oxidant defense and anti-inflammatory properties of polyphenols and other bioactive compounds in nuts and berry foods. The gut-brain axis is an emerging area of research. Most studies are preclinical in nature using animal models but are suggestive of a significant role of gut microbial-derived ellagitannin metabolites on brain health and neuroprotection.The influence of nuts and berries on skin health and appearance is an emerging area of research. Regular intake of almonds, a good source of fatty acids and polyphenols, has been associated with a significant decrease in facial hyperpigmentation and wrinkle severity. A walnut protein hydrolysate administered to rats exposed to ultraviolet radiation significantly reduced skin photoaging and enhanced skin elasticity. Supplementation with ellagic acid, a compound found in many berries, prevented ultraviolet B -related inflammation and collagen degradation related to skin wrinkling and aging in a murine model. More human studies, using objective measures of skin wrinkles, skin elasticity and response to low-dose UVB radiation exposure are warranted. Monitoring skin responses to a UVB radiation challenge has been used as a marker of whole-body antioxidant status in response to almond consumption. The response to a UVB challenge has also been used to monitor oxidant defenses and changes in skin microbiome following the intake of pomegranate juice.Age-related macular degeneration is the third leading cause of vision loss worldwide. Anthocyanins, carotenoids, flavonoids, and vitamins C and E, found in many berries, have been shown to reduce risk of eye-related diseases. Goji berries, containing the highest amount of zeaxanthin of any known food, hold particular promise since this compound binds to receptors in the macula to offer protection from blue and ultraviolet light. Regular supplementation with 28 g/d of goji berries for 3 mo increased macular pigment optical density, a biomarker for AMD, as well as the skin carotenoid index. Nuts may also be protective against AMD since they are a rich source of vitamin E and essential fatty acids. Regular intake of nuts has been associated with a reduced risk and slower progression of AMD in 2 epidemiological studies, thought to be due to the beneficial role of polyunsaturated fatty acids.Identification of new cultivars with traits desirable for growers, processors, and consumers is a continuous effort. As researchers continue to produce new varieties by both conventional and molecular-driven approaches, assessing these varieties for nutritional value is a challenge. A combination of broad targeted and untargeted metabolomic approaches, along with defined functional phenotyping could be used for rapid screening and defining of mechanistic pathways associated with health. However, consumer preferences for new cultivars are often driven by size and appearance of the berry or nut and flavor, greenhouse snap clamps rather than its nutritional value. This would further confirm the need to balance improvements to nutritional profiles with enhancement of consumer-driven traits, maintaining the marketable nature of the berries and nuts.

Biomedical research, particularly for clinical studies, is expensive and resource intensive. Although the USDA competitive grants program offers funding for outstanding research projects, budget limitations favor animal or in vitro study proposals. Compelling pilot data is needed to be competitive for clinical studies funded by the USDA or NIH, so many researchers submit their initial ideas to commodity groups representing specific nuts or berries. Commodity groups represent farmers, processors, and distributors and have been instrumental in supporting fundamental and applied research focused on their specific berry or nut. The perception that studies funded by nut and berry commodity groups are inherently biased in favor of the test food is an issue sometimes raised by critics, journalists, and the general public. As in all nutrition research, ethical considerations regarding the structure of research questions, hypotheses, study design, outcome measures, interpretation of data, and conclusions must be rigorously considered. The food and beverage industries have played a key role in providing funds and supporting nutrition research on individual foods and beverages, including berries and nuts. Although this draws scrutiny regarding scientific integrity and data reporting, collaboration between academia and industry compared to exclusive corporate funding may help offset some of these concerns. For example, in multiple reported studies, matching funds were also provided by non-industry sources, including institutional and federal agencies. In other cases, while the food industry provided the test agents, key research personnel and staff were not supported by the same funding source. The academia-industry collaboration has also led to the formation of scientific advisory committees that evaluate and recommend proposals for funding, a peer review process that helps ensure rigorous study designs, data reporting, and dissemination of results. Human studies of sufficient statistical power are expensive, labor-intensive efforts requiring sophisticated and costly laboratory equipment and supplies. In order for research proposals to be competitive for funding from the USDA or NIH, pilot data is required, and for nuts and berries, the only realistic source of funding for these exploratory trials is from industry sources. Critics of industry support for nutrition research have yet to propose realistic alternatives for funding needed to generate initial data. Further, ongoing industry funding of nuts and berries research has yielded important insights into the molecular and physiological understanding of mechanisms of action. Without industry support, provided in an ethical and transparent manner, advances in our understanding of the role of nuts and berries in a healthy dietary pattern would be limited. A risk-of-bias study of 5675 journal articles used in systematic reviews published between 1930 and 2015, representing a wide variety of nutrition topics, concluded that ROB domains started to significantly decrease after 1990, and particularly after 2000. Another study examined the incidence of favorable outcomes reported in studies funded by the food industry in the 10 most-cited nutrition and dietetics journals in 2018. Of the 1461 articles included in the analysis, 196 reported industry support, with processed food and dietary supplement manufacturers supporting 68% of the studies included. Studies supported by any nut or berry commodity group were not considered due to an incidence lower than 3% of qualifying articles. Studies with food industry support reported favorable results in 56% of their articles, compared to 10% of articles with no industry involvement. The authors offer a number of suggestions to help minimize real or perceived bias, calling on research institutions to enforce strict, regularly updated, and transparent oversight of all research projects involving industry. Suggestions in support of research transparency and integrity have also been advanced from guidelines adapted from the International Life Sciences Institute North America. This served as the basis for the development of consensus guiding principles for public-private partnerships developed by a group of representatives from academia, scientific societies and organizations, industry scientists, and the USDA, NIH, US Centers for Disease Control, and the US Food and Drug Administration. These provisions include full disclosure of funding and confirmation of no direct industry involvement in the study design, data and statistical analyses, and interpretation of the results and only minimal, if any, involvement of industry coauthor, often given as a courtesy to acknowledge funding and logistical support by the investigators with no intellectual involvement by the study sponsor. This is in contrast to industry-initiated research, where the industry office or commodity group sets predetermined research objectives, provides intellectual collaboration, and often has input on the study design, interpretation of results, and decisions regarding publication. Although some critics may argue that repeated industry funding in support of research groups that report favorable results on a particular nut or berry shows a bias toward positive outcomes, other interpretations are also possible. First, few labs have the infrastructure, detailed methodology and analytical equipment, and trained personnel to conduct clinical studies in an efficient and timely manner. Industry funded studies conducted at major universities have layers of review and accountability within their organizations to guard against malfeasance, and while these layers may not focus directly on precise elements of research design and interpretation of results, faculty members at such institutions generally have a level of integrity and accountability, knowing that administrative review exists. Calls for industry-funded research are often broad in scope, which allows researchers to generate proposals, research questions, and hypotheses that do not have preconceived outcomes.

Some volatiles have been lost during domestication and breeding as a combined result of negative selection and linkage drag in tomato and watermelon . Likewise, gain and loss of terpene compounds during strawberry domestication and its genetic causes have been investigated . Recent advances in sequencing technology and analytical approaches have opened new opportunities to understand the chemistry and genetics of fruit flavor. Genome-wide association studies have revealed loci for flavor in a variety of fruit crops . Meanwhile, genomes-wide expression quantitative trait loci studies have the capability to bridge the gaps between GWAS signals and their underlying causative genes. Integration of GWAS and eQTL studies has led to discovery of a master metabolite regulator in tomato and a flesh-color-determining gene in melon . Long-read sequencing now allows assembly of genomes with high contiguity, and when coupled with parental short-read data , the two haplotypes of a heterozygous individual can be fully resolved. Phased assemblies have improved variant discovery, plant pot with drainage especially for large structural variants . The extent, diversity and impact of SVs increasingly are being studied in horticultural crops and have been shown to alter fruit flavor, fruit shape and sex determination .

Great opportunity exists to coherently integrate these multi-omics resources for the discovery of flavor genes. Garden strawberry is an allo-octoploid species with highly palatable non-climacteric fruit . It increasingly has been utilized as a model for Rosaceae fruit crops genomics and flavor research as a result of its short generation time, wide cultivation and high value. Through exploration of spatiotemporal changes in gene expression and homolog search, several flavor genes have been cloned and validated, including an alcohol dehydrogenase and several alcohol acyltransferases for esters, a nerolidol synthase 1 for terpenes and a quinone oxidoreductase for furaneol. Recently, QTL studies and transcriptome data analyses for strawberry volatiles using biparental crosses have detected QTL and causative genes for mesifurane and gamma-decalactone . Nevertheless, low mapping resolution and a lack of subgenome-specific markers have hampered further characterization of causal genes underlying other QTL. This problem recently was addressed by the development of 50K Fana SNP array using probe DNA sequences physically anchored to the octoploid ‘Camarosa’ genome . High heterozygosity combined with an allopolyploid genome presents difficulties for resolving causative genes and their haplotypes. To further the goal of discovering causative genes affecting flavor in strawberry, association studies with larger sample sizes and additional genetic resources such as eQTL and additional genomes are required.

Furthermore, these resources must span the breadth of natural variation in breeding germplasm. Here we present multi-omics resources consisting of an eQTL study representing the genetic diversity of strawberry breeding programs in the US, phased genome assemblies of a highly- flavored University of Florida breeding selection, a structural variation map in octoploid strawberry and a volatile GWAS of 305 individuals. These are combined to leverage the extensive metabolomic, genomic and regulatory complexity in strawberry for the discovery of natural variation in genes affecting flavor. Ultimately, the functional alleles identified will be selected in breeding to achieve superior flavor.The eQTL population consisted of 196 genotypes including 133 newly sequenced accessions . The University of Florida genotypes were grown at GCREC and collected in the spring of 2020 and 2021. The University of California-Davis collection of diverse selections from multiple breeding programs were grown at either Santa Maria CA or Oxnard CA, for day-neutral and short-day accessions, respectively, and collected in the spring of 2021. Four UC genotypes were collected at both sites to ensure sequencing and SNP quality. Total RNA was extracted from a bulk of three fully ripe fruits using a Spectrum™ Plant Total RNA Kit , after flash freezing in liquid nitrogen. Illumina 150-bp pair-end sequencing was performed on the Illumina NovoSeq platform by Novogene Co. . On average, 6.9 Gb of sequence data were obtained for each sample. Raw RNA-Seq data of 63 samples from previous published studies were retrieved from the NCBI SRA database . In order to quantify gene expression, short reads were trimmed for adapter sequences and low-quality reads with TRIMMOMATIC v.0.39 and aligned against the reference genome using STAR v.2.7.6a in the two-pass mode .

Only unique aligned reads were scored by HTSEQ v.0.11.2 in the union mode with the ‘–nonunique none’ flag supplied with the latest Fragaria_ananassa_v1.0.a2 annotation . All count files were compiled in R and normalized with the DESEQ package . To generate the marker dataset for eQTL mapping, SNPs and InDels were called using the mpileup and call commands. Markers were further hard-filtered using BCFTOOLS with the following steps: individual calls with lower than sequencing depth of three were set to missing using + setGT plugin; marker sites with quality < 30, missing rate > 0.3, heterozygous call rate > 0.98, minor allele frequency < 0.05, or number of alternative alleles > 1 were purged; the filtered markers were imported and analyzed in R, and only markers showing more than three matched calls in four duplicated sample pairs were retained. A total of 491 896 markers passed the three stages of filtering. The missing calls were imputed, and all calls were phased using BEAGLE v.5.2 using the default settings . The eQTL mapping was performed for 62 181 fruit expressed genes using the filtered markers. Linear mixed models implemented in GEMMA were used for association analysis . The relationship matrix was computed in GEMMA and supplied to explain relationship within populations, and the top five principal components with a total of 25.0% variance explained were imported as covariates to reduce effects from population stratification to signify the genetic variance underlying the target traits. The Bonferroni corrected 5% significance threshold was used, determined the by number of LD-pruned markers . The approach to define an eQTL was similar to that used in previous studies . Briefly, we first clustered all significant markers with distance < 100 kb and purged clusters with fewer than three markers. The lead marker with lowest P-value was used to identify the eQTL, and boundaries of eQTL were defined as the furthest flanking significant markers. Clusters in LD were merged and boundaries were updated. The longest distance between cis-eQTL boundaries and eGene boundaries was limitedto 500 kb. Because a substantial number of regulatory elements were found for fruit-expressed genes, a structural variant map would greatly facilitate the identification of potential causative SVs underlying the regulatory elements. To construct an SV map, pot with drainage holes we first assembled a phased genome of an UF accession. The genome of FL 15.89-25 was assembled into 1480 and 672 phased contigs with N50 of 12.8 and 12.4 Mb, respectively , with similar contiguity to other recent high-quality octoploid strawberry genomes . A Kmer-based approach revealed 97.1% and 99.2% completeness for the haploid assemblies based on parental Illumina short reads, which were corroborated by 98.1% and 98% completeness of the BUSCO eudicots odb10 genes . Phasing quality was evaluated by parent-specific Kmers; the average switching error and hamming error were 0.19% and 0.18% for the F12 haploid assembly , comparable to phased genomes in other species . The phased contigs were scaffolded into pseudochromosomes based on alignment to the ‘Camarosa’ reference genome, with 96.0% and 92.8% of phased contigs placed on 28 pseudochromosomes for the respective F12 and Bea haploid assemblies , consistent withprevious flow cytometry estimations . There were only 88 and 79 gaps in the final scaffolds, averaging 3.14 and 2.82 per chromosome for the respective F12 and Bea assemblies . Scaffolding quality was evaluated by a linkage map and public Hi-C data . High collinearity was observed between haplotypes . The FL 15.89-25 assemblies and three additional haploid assemblies were utilized to explore SV diversity in garden strawberry. These geographically and genetically diverse accessions empowered the discovery of SVs across all chromosomes except for a large portion of Chr 4B which may be under strong purifying selection .

Individual haplotypes had between 31 574 and 60 453 SVs relative to the PHASE1 assembly of ‘Royal Royce’ , with the WONG haplotype harboring the most SVs, consistent with the larger genetic distance of Asian populations to North American populations . Insertions and deletions were the most common SV types, together consisting of 88.3– 94.1% of SVs. All SVs across haplotypes werethen merged into a nonredundant set of SVs . In total, 56 342 deletions, 60 983 insertions, 12 016 translocations, 166 interspersed duplications, 236 tandem duplications and 137 inversions were identified. Unlike the SV composition of a tomato population in which the majority of SVs were singletons , an average of 62.6% strawberry SVs were shared by at least two haplotypes . We observed a gradually reduced number of new SVs every time a new haplotype was merged , suggesting this SV map surveys a substantial portion of SV diversity in cultivated strawberry. The majority of SVs were < 1 kb , whereas only 3.3% were > 10 kb . Structural variations were present extensively in exons , introns and promoter regions . Transposable elements were rich resources of SVs. We identified 34 379 deletions overlapped with TEs, especially inverted tandem repeats and long terminal repeats , consisting of 61.0% of total deletions, significantly higher than the genome-wide TE percentage of 38.42% .In order to investigate the genetic control of fruit volatiles, we performed volatile phenotyping and SNP array genotyping with 49 330 markers on a panel of 305 accessions from the UF strawberry breeding program, with 59 individuals overlapped with the eQTL panel . A total of 97 volatiles including esters, terpenes, aldehydes, alcohols, acids, ketones and lactones were quantified . Based on relationships among volatiles, we identified at least five clusters belonging to the same chemical class or biosynthetic pathway, including clusters of eight aldehydes, three ethyl esters, three hexanoic acid derivatives, seven medium-chain esters and three terpenes . Generally high narrow-sense heritability was observed across volatiles , ranging from 0.212 to 0.916, with a mean of 0.660. The highest value of h2 was found for mesifurane and the lowest for octanoic acid, ethyl ester . The lead SNP effects varied from 0.27 to 2.44 , with the largest effect for methyl anthranilate . Two hotspots which contained multiple signals of volatiles belonging to the same class or pathway were found for medium chain esters and for terpenes , which also were detected to in previous studies and reflected in chemical relationships . Our GWAS results confirmed previous homoeologous group assignments for these volatile QTL and clarified their subgenome and physical positions. The SNP AX-166515537 was the lead SNP for three esters, and a 14 Mb region on Chr 6A shared signals for six medium-chain esters. An LD analysis revealed three linkage blocks . The distal region of Chr 3C was associated with six volatiles including five terpenes . This 3.1-Mb region did not display clear LD block separation . Two significant markers for medium-chain ester hotspot and methyl thiolacetate were tested for their predictability of flavor characteristics . Some abundant volatiles including: 2-hexenal, -; butanoic acid, 2-methyl-; and pentanal were associated with multiple DNA variants , suggesting polygenic inheritance. Pentanal was associated with threeIn this study we leveraged eQTL, GWAS and haplotype-resolved genome assemblies of a heterozygous octoploid to identify allelic variation in flavor genes and their regulatory elements. Finetuning of metabolomic traits such as amylose content in rice and sugar content in wild strawberry recently were made possible via CRISPR-Cas9 gene-editing technology. Similar approaches can be taken in cultivated strawberry for flavor improvement, but not before the biosynthetic genes responsible for metabolites production and their regulatory elements are identified. Our pipeline has proven to be effective in identification of novel causal mutations for flavor genes responsible for natural variation in volatile content and can be further applied to various metabolomic and morphological aspects of strawberry fruit such as anthocyanin biosynthesis , sugar content and fruit firmness. These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Strawberry also shares common volatiles with a variety of fruit crops.

Limited prior research that has looked at the effects of multiple soil management practices indicates that metrics for soil health are a product of both inherent soil properties and dynamic soil properties . Whether available soil indicators could translate these soil properties and processes when management systems are complex remains unclear. As an added layer of complexity, field variability is hard to distinguish from management-induced changes in soil properties . To address this challenge, prior studies have suggested increasing samples, the number of sites, and sampling strategies that account for spatial and temporal variability ; however, as farmers themselves expressed in this study, such an approach requires additional time and resources, and may not increase their utility—at least to farmers—in the end. In this sense, farmer knowledge may serve as an important mechanism for ground-truthing soil health assessments, particularly when management is synergistic and does not rely heavily on organic fertilizers. As emphasized by our results above, farmer involvement in soil health assessment studies is imperative to better converge soil indicators with farmer knowledge of their soil. Lastly, our results also highlight the utility of incorporating information about nitrogen-based fertilizer application on sampled field sites, round plastic pot particularly when assessing soil indicators on working farms with a large variation in the quantity of N-based fertilizers applied .

Farms on the low end of additional organic fertilizer application showed minimal differences between farmer selected fields for soil fertility, particularly in terms of soil inorganic nitrogen —which suggests that differences in soil fertility in fields with more circular nutrient use may be less detectable using commonly available soil indicators. This cursory finding here corroborated farmer observations touched on in the previous section above, and requires further investigation to see if similar trends extend to other organic systems. Here, we have identified several gaps in the utility of commonly available indicators for soil fertility among a unique group of organic farmers in Yolo County, California using interviews with farmers and field surveys. Our study highlights that if available soil indicators are to be considered effective by farmers, they must be grounded in farmers’ realities. Moving forward, working in collaboration with farmers to close this continued gap in soil health research will be essential in order to ground widely available soil indicators in real working farms with unique management systems and variable, local soil conditions. This approach is particularly needed among organic farms that do not rely extensively on nitrogen-based organic fertilizers and additional nutrient input to supply their fertility, as available soil indicators do not adequately reflect farmers’ descriptive metrics for soil fertility.

Moreover, our research elevates concerns that currently available soil indicators used in soil health and fertility assessments may not fully capture the complex plant-microbe-soil interactions that regulate soil fertility, particularly on organic farms that use minimal organic fertilizer application. Moving forward, additional studies that pursue a deeper dive into nutrient dynamics across a gradient of management and varying nitrogen-based fertilizer input is needed. Overall, the strong overlap between farmer knowledge in this study and ongoing soil health research speaks to the opportunity to further engage with farmers in developing useful indicators for soil health and fertility that are better calibrated to local contexts and draw on local farmer knowledge. A deeper investigation of farmers knowledge systems, in particular farmer understanding of soil function in connection with crop productivity, soil health, and soil fertility, represents a critical path forward for this research arena. Additionally, we recommend placing greater emphasis on developing descriptive indicators for soil health and fertility in collaboration with farmers that are better integrated with ongoing qualitative soil health and fertility metrics. These descriptive indicators should not be developed in isolation to ongoing research on soil health and fertility assessment, but rather as an integrated research process among scientists, farmers, and extension agents—importantly, with scientists as listeners working toward a shared language. Ants benefit plants . Humans have known this for quite a long time. In fact, ants were described as biological control agents in China around 304 AD . Surveys of tropical forests show that up to one third of all woody plants have evolved ant-attracting rewards .

Some plants provide domatia as ant housing structures, while others attract ants to their tissues with extra-floral nectaries. Some plants are hosts to honeydew-producing hemipterans that excrete honeydew, a sugary substance consumed by ants. Still other plants are simply substrates for ant foraging. The majority of studies conducted across these ant–plant groups show that ants benefit plants by removal of herbivores . Nonetheless, in many agroecosystems, the benefits of pest control services by ants are not recognized. Agricultural managers often view them as pests or annoyances to agricultural production because some ants tend honeydew-producing insects that can damage crops . However, a review of the literature on ant-hemipteran associations suggests that even these associations benefit plants indirectly because ants remove other, more damaging herbivores . Regardless, the literature lacks studies investigating ant–plant interactions in agroecosystems. Here, we broadly survey the pest control services provided by a suite of ant species to better understand the role of ant defense of coffee. Coffee is a tropical crop that occurs as an understory shrub in its native range, and coffee plants are therefore often grown under a canopy of shade trees in agroforestry systems in some parts of the world . This canopy layer provides plantatsions with a forest-like vegetation structure that can help maintain biodiversity . Ant biodiversity is high in many coffee plantations and ants attack and prey on many coffee pests, including the coffee berry borer . For example, Azteca instabilis F. Smith is a competitively dominant ant that aggressively patrols arboreal territories in high densities and previous research has found that it impacts the CBB . Some laboratory and observational field studies have found that Pseudomyrmex spp., Procryptocerus hylaeus Kempf, and Pheidole spp. may limit the CBB . However, other field experiments have not found ants to be biological control agents of the CBB . Further, round pot the pest control effects of many ant species on the CBB have not yet been evaluated and it could be that previously documented effects are specific to only a few species. Natural ant pest control of the CBB is particularly important because chemical insecticides used to control CBB are not always effective. This lack of effectiveness is in part because the CBB lifecycle takes place largely hidden within coffee berries and also because the CBB has developed insecticide resistance . Several of the stages of the CBB life cycle make it vulnerable to attack by ants . First, the CBB hatches from eggs within the coffee berry, where it consumes the seeds . Small ants may enter the berry through the beetle entrance hole and predate the larvae and adults inside . Second, old berries infested with the CBB may not be harvested because they often turn black and remain on the coffee branches or may fall to the ground . These old infested berries may act as a population reservoir of borer populations and ant predation at this stage could be very important for limiting CBB populations in the next season. Third, as adult borers disperse to colonize new berries, ants may prevent them from entering new berries . To date, no field experiment has specifically investigated how coffee-foraging ants limit CBB colonization of berries. Here, we studied the abilities of eight ant species to prevent colonization of berries by the CBB. We hypothesized that only species with high activity on branches would limit CBB colonization of berries. We show that six of eight ant species limit CBB colonization of berries and that the effect of ants is independent of ant activity on branches. This study is the first field experiment to provide evidence that a diverse group of ant species limits the CBB from colonizing coffee berries.Our goal was to capture a broad survey of the ant species that occupy the coffee vegetation in the coffee plantation.

Within the plantation, five Crematogaster spp. forage in the coffee, however field identification at the time was not reliable therefore taxonomic resolution for Crematogaster spp. remained at the genus level. For P. simplex and P. ejectus it was not always possible to find occupied bushes by observation of ant foraging. Instead, for P. simplex and P. ejectus, we determined occupation by removing all dead twigs on the coffee bush and searching these for ant nests within the hollow branches . We reattached the nested hollow branch to a living branch with thin wire and treated these bushes as bushes occupied by P. simplex or P. ejectus. To test the effects of each ant on CBB colonization of berries, we performed an ant exclusion experiment. We surveyed bushes occupied by one of the eight target ant species. We excluded coffee bushes with few branches to control for the size of the foraging area of each ant species. On each bush, we searched for two branches of equal age and position and roughly the same number of coffee berries . On each branch, we removed all berries that had CBB entrance holes. We then removed all ants from one branch and applied tangle foot to the base of the branch near the coffee trunk. On the second branch, we left ants to forage freely . To estimate ant activity, we counted the total number of ants foraging on the stem, leaves, and berries of each branch for 1-min including those that travelled onto the branch during the 1-min survey. We also counted ants on exclusion branches after the experiment and if a branch had more than one ant individual present, we excluded the bush from analysis . To release CBB onto control and treatment branches, we created a leaf platform to aid their chances of encountering berries. The leaf was wedged between the branch stem and a cluster of berries to create a platform surrounding the cluster . A coffee leaf was used as a platform because artificial structures attract attention from many ant species. After waiting several minutes to ensure normal ant activity, we released 20 CBBs on the leaf platforms of the control and exclusion branches. After 24 h, we counted the number of berries per branch that had CBBs inside entrance holes. We did not count partially bored holes in berries, nor CBBs that had bored into twigs and leaves. Multiple bored entrance holes per berry were only counted as one bored berry. We modified the experiment slightly for P. simplex and P. ejectus because of the difficulty in locating these species within a bush using visual cues . For these two species, we used the living branch to which the nest was attached to as the control branch . This was done because we wanted to make sure that ants were actively foraging on control branches after the disturbance of removing nests. To statistically analyze experimental data, we opted to use linear mixed models instead of paired t tests because mixed models allow inclusions of experimental non-independencies through the incorporation of covariates. We included bush as a random effect in the model to pair control and exclusion branches within each bush. Ant species and treatment and the species 9 treatment interaction were included as fixed effects in the model. To control for differences between each branch and bush, we included the number of berries per branch, the number of berries in contact with the leaf platform, and the number of worker ants per branch as covariates in the model. We performed type III F tests of significance for main effects with maximum likelihood to estimate the fixed effect parameters and variance of random effects . We removed non-significant factors from models and compared nested and null models with likelihood ratio tests to determine the best-fit model. We also compared ant activity across different species to determine if this factor might correlate with berries bored and vary across ant species. To determine if ant activity correlated with the number of coffee berries bored, we limited the dataset to only control branches and used a generalized linear model with a Poisson log-link function because data did not meet the assumptions of normality. To determine if ant activity varied by species, we again limited the dataset to only control branches and used ANOVA with Tukey’s HSD analysis.

Farm Type I consistently showed the highest values for total soil N, total organic C, POXC, and soil protein, which suggests sites in this farm type had higher soil quality compared to Farm Type II and III; similarly, Farm Type II consistently showed intermediate values for all four indicators for soil organic matter. Lastly, Farm Type III consistently showed the lowest values across all four indicators, which suggests sites in this latter farm type had lower soil quality compared to the other two farm types. These initial results highlight the usefulness of establishing farm typologies based on indicators for soil organic matter as a novel approach to study gradients in soil quality on organic farms. The three farm types generated based on soil organic matter levels served as a key starting point for further analysis of the role of management in relation to soil quality. Accordingly, not only were the three farm types identified in this study significantly different based on indicators for soil organic matter levels, but the farm types also aligned with general trends in management among sites, 10 liter drainage collection pot which indicated a link between soil organic matter levels and management.

In particular, as the four indicators for soil organic matter collectively serve as a proxy for soil quality, our results suggest that soil quality indicators may show responsiveness to the impacts of short-term management. In our study, crop diversity, crop rotational complexity, and tillage emerged as the strongest drivers of farm type differences, as shown by LDA coefficients . These results also coincided with average values for management variables compared across all three farm types , though variables for ICLS and cover crop application overlapped considerably across all three farms. These cursory findings extend results from ongoing work from others , including a recent 4-year study by Sprunger et al. —which focused on organic corn systems in the Midwest. Sprunger et al. likewise reported strong links between soil metrics such as total N, total C, soil protein, and POXC—and on-farm management practices, such as crop rotation patterns, manure and cover crop application, and tillage. While extensive work has been done on organic corn and grain systems in the midwestern region of the US, our study provides new insight on the applicability of these common soil metrics in entirely different organic farming systems and climate regions—specifically on high-value vegetable farms operating in the dry, hot Mediterranean climates of northern California.

Our results also underscore the usefulness of on-farm interviews in developing management variables that are potentially linked to soil indicators . Whereas most previous studies have frequently utilized mail-in surveys that rely on binary responses from farmers to understand management , our study, following Guthman and others, highlights the uneven gradient in management practices that exists among organic farms and the importance of in-depth interviews . For example, rather than simply noting the presence or absence of tillage at a field site, our study accounted for the number of tillage passes per season that a farmer implemented on a particular field site, which required soliciting a range of responses from each farmer to create a congruent metric across all field sites. As displayed in Table 6, the mean values for frequency of tillage and crop abundance differed across the three farm types in our study; these management variables strongly separated Farm Type I from the other two farm types and weakly correlated with soil quality. On the other hand, crop rotational complexity generally separated all three farm types, but did not correlate with increasing soil quality. These results suggest that while certain management practices may increase soil organic matter pools as frequency decreases, some management practices may require finding a “sweet spot” to achieve higher soil organic matter levels.

Relatedly, the implementation of ICLS did not appear to be as strong of a source of differentiation among the three farm types. One reason for this weak link between soil organic matter levels and ICLS may be due to the lack of a temporal component in the development of this soil metric. For example, some farms may have recently rotated livestock on their fields, while other farms may not have rotated livestock for several years on that particular field; our metric does not capture such spatial and temporal differences. Though limited studies on organic systems in California currently exist, previous studies in the midwestern US have found that the integration of livestock does increase organic matter levels on-farm ; however, based on our results, crop diversity, crop rotational complexity, and frequency of tillage present stronger influences than cover crop application and ICLS in differentiating working organic farms—at least in this particular context. While management is undoubtedly an important driver of soil organic matter levels, our findings also suggest that soil texture may play a more significant role than management in determining levels of SOM than originally considered. Though management explained 18% of the variance among the three farm types, further analysis showed that soil textural class was the more dominant factor as shown in Figure 5; in fact, soil texture class was 44% greater than management in explaining the three farm types. This important result from our study complements parallel findings from Sprunger et al. , who also determined that soil textural class, rather than management, explained the largest amount of variation among the soil indicators they measured on their midwestern US-based organic corn systems . Our combined findings provide an initial indication that regardless of the organic system— ie, crop, climate, and/or geography—soil texture is the more dominant determinant of soil indicators for soil quality rather than the diverse management practices applied to these systems .This broader finding is significant because it supports emergent research that suggests that while management certainly contributes to soil quality, inherent characteristics of the soil in a given field may place limits on achievable organic matter levels on organic farms . Based on our findings, it is evident that even along minimal gradients in soil texture class, organic matter levels strongly differ. Soil texture is known to be a strong control on soil organic matter dynamics across diverse ecological systems—not just agricultural systems—in part because organic compounds, particularly those derived from soil microbes, are among those capable of stabilization by physical and chemical mechanisms, including aggregation, sorption on mineral surfaces, and entrapment within fine pores . At a fundamental level, 10 liter drainage pot soils with greater amounts of clay tend to stabilize SOM on surfaces more than soils with high sand and/or silt content , as clay particles provide greater surface area through organo-mineral associations than other particle sizes . For example, it has been shown in numerous previous studies that as clay content increases, the relative abundance of total soil N also increases . Further other studies have shown that soil texture and structure can influence SOM chemistry, and therefore, SOM stabilization . Our study takes previous research in agricultural contexts one step further to show that while management is important to consider, soil texture may be the more dominant factor; however, based on our results, it is still unclear which direction soil texture may be driving SOM. Nonetheless, our results highlight that contextualizing management in the native soil texture is essential to understand the limits of management imposed by pre-existing constraints of the soil. In practice, current emphasis in on-farm soil health research and quality assessments tends to focus on the importance of changing management to build healthy soils and improve soil quality without explicit consideration for soil texture .

In this study, the gradient of soil textures across the farm fields sites was relatively limited and even so—soil texture still explained a significant component of the variance observed compared to management. Given this outcome, our findings here reinforce the importance of using soil texture as a starting point for evaluating soil quality. Knowing the soil textural class of different fields may help farmers determine the management practices that have greatest potential for improving soil quality on farms with even small variances in soil textures; soil texture class may also help farmers better contextualize results of their soil health tests. Our study suggests that moving forward, soil texture should be more explicitly considered when making management recommendations to improve soil quality on organic farms. That said, understanding the interactive effects between management and soil texture continues to be a gap in on-farm research and soil health assessment. Future studies might build on our approach and examine whether applying a similar suite of indicators to capture soil organic matter levels may yield similar connections with management in other organic farming contexts in California—and elsewhere in the US. Our study provides a potentially widely applicable method for developing a functional understanding of soil organic matter in complex agricultural landscapes. In this sense, the overall significance of the results of the cluster analysis highlights the efficacy of developing typologies to provide a useful tool for understanding the complexity of working agricultural landscapes. Importantly, the development of farm typologies allowed for additional analysis of other soil indicators for N cycling an availability—by using the farm types as a central tool for further investigation.Though the range of gross N cycling rates from this study are comparable to N cycling values reported from previous studies in organic agricultural systems , we found that farm types did not have significantly different gross N mineralization and nitrification rates—contrary to our initial hypothesis and despite that farm types strongly differentiated based on soil organic matter levels. These hypotheses were in part based on prior work with organic farms in this region that reported instances where inorganic N pools were low—well below established soil nitrate threshold sufficiency values—but that the crops themselves showed high production of, and sufficient N . Fields in which this trend was observed had the highest levels of soil C, and so in this previous study, it was hypothesized that higher rates of N production explained this observed trend. However, nitrogen bio-availability for crops is not just a function of the gross production of inorganic N by microbes but is also influenced by physical soil characteristics within the rhizosphere, such as the local soil structure and mineralogy, plant root structure and associated mycorrhizal pathways, as well as accessibility of water to plants and soil microbes . These variable conditions in the rhizosphere are not captured by measuring N cycling rates but still directly influence bio-availability of N. For these reasons, the N cycling results of this study may not follow prior findings from Bowles et al. . Still, we did observe an influence of soil organic matter levels on N cycling, particularly in terms of gross nitrification rates. As shown in the Linear Mixed Model results in Table 12, SOM indicators do appear to have an influence in predicting gross nitrification rates , even as the proportion of variation explained is modest . This slight trend is also evident in the boxplots . The weak but significant link between soil organic matter levels and gross nitrification rates is important to highlight because these results suggest that building soil organic matter presents one way to increase nitrification rates and potentially crop N availability. Because the plant-soil-microbe N cycling system is strongly influenced by soil water content and soil structure, it is possible that gross N cycling indicators lack the responsiveness that SOM indicators exhbiti especially in scenarios where improved soil quality allows for crops to continue accessing soil microsites with available N . Similarly, crops with more abundant and active mycorrhizal community associations can extend into smaller Ncontaining aggregates that may be otherwise locked up for crops with less root proliferation and hyphal associations. Additionally, it is also possible that changing microbial community composition in the soil may lead to greater immobilization of N, locking up available N but not necessarily impacting gross production of N. These plant-soil-microbe interactions that control availability of N may not be detectable solely by measuring gross N flows. While not significant, SOM indicators were also selected in the development of the LMM for gross mineralization rates as well. These results are congruent with previous research looking across ecosystem types that reported a relationship between N cycling rates and SOM indicators. For example, a meta-analysis published by Booth et al. that examined woody, grass, and agricultural ecosystems found a strong positive relationship between indicators for SOM and gross N mineralization.

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 , vertical vegetable tower 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 elder flower levels range from about 0.200 to 0.900 mg g-1 fresh weight, though higher levels were found in elder flower 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 bioactive 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. Rutin ranged from 3.20 ± 0.395 µg g-1 in FWF to 10.01 ± 0.97 mg g-1 in FHF . The concentrations of rutin vary greatly across studies of European elder flowers. For example, levels are reported as 1.65490 ± 0.10951 mg g-1 fresh weight,6 1.8877 ± 0.2691 mg g-1 fresh weight, between 15.70 and 23.90 mg g-1 in dried elder flower tea which varied with cultivar,and 4.14443 ± 0.11469 mg g-1 in a traditional beverage called sabesa which is made from fresh flowers. Herein, kaempferol-3-O-rutinoside ranged from 1.24 ± 0.03 to 3.010 ± 0.37 mg g-1 dry weight, and isorhamnetin-3-O-glucoside ranged from 0.77 ± 0.05 to 1.42 ± 0.40 mg g-1 dry weight . 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 elder flower 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 elder flowers 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 elder flowers of S. nigra ssp. cerulea . Two isomers of 5-caffeoylquinic acid in addition to 3- and 4-caffeoylquinic acid have been identified in elder flower products. Evaluation of the phenolic content of elder plants grown in different locations and altitudes indicate, in general, 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 elder flower 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 elder flower 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 elder flower 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 elder flower of S. nigra ssp. cerulea are comparable to the levels in the European flowers. However, vertical farm tower 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 elder flowers 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 elder flowers. The profile of headspace VOCs in elder flowers of S. nigra ssp. cerulea differ significantly from the European elder flowers 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 elder flowers 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 elder flower extracts, however methyl eugenol has not been identified in European elder flowers. Methyl eugenol, which has a clove-like aroma19 , appears to be unique to the S. nigra ssp. cerulea elder flower, and could be a unique volatile marker for this subspecies. Tea made with fresh whole elder flowers presented a slightly different headspace VOC profile as compared to fresh flowers. Although methyl eugenol was still a prominent compound in the headspace , there were also two ketones in relatively high concentrations: 2,2,6-trimethyl-4H-1,3-dioxin-4-one and 4-methyl-2-heptanone . Aroma descriptors were not found for these compounds. In addition, two aldehydes including heptanal and nonanal were also present in the headspace of the tea but not the fresh flowers. Heptanal odor is described to be fresh, fatty, green, and herbal, whereas nonanal is described to have waxy, rose, orange peel or fatty notes. Methyl salicylate comprised only 1.27 ± 0.29% of the headspace volatiles in the tea made from fresh flowers. In the headspace of dry elder flowers, the most concentrated compounds were -3-hexen-1-ol > 1-penten-3-ol > 3-methyl-butanal > heptanal, > isocyanato-methane . In general, the dry flowers contain a wider range of volatiles than the fresh flowers, including more aldehydes, alcohols, alkanes, and other hydrocarbons. Other notable volatiles identified include methyl salicylate , dihydroedulan , which is a driver of typical elderberry aroma, and linalyl acetate which is the only linalool derivate identified any preparations of the elder flowers of S. nigra ssp. cerulea, unlike European elder flowers which typically have high concentrations of linalool derivatives. In tea prepared from dried flowers, the headspace aroma less complex than the dried flowers, but many of the aldehydes were still identified, including nonanal , heptanal , and hexanal .

The most significant impacts of the vineyard floor treatments were of the cover-crop treatments on soil parameters in the middles. Soil organic matter in cover-cropped middles was higher than in bare middles each year . Cover crops affected key soil nutrients in the middles; for instance, cover crops greatly reduced nitrate-nitrogen , and to a lesser extent, extractable phosphorus , which may be beneficial in reducing loss of these nutrients in runoff during winter storms, but which also may have reduced the phosphorus content in the vines. In addition, cover crops in the middles also significantly reduced soil boron , extractable sodium and pH , and increased chloride and zinc when compared to bare soil.Soil microbial biomass. Microbial biomass varied as a result of both the cover-crop and weed control treatments. In both the middles and vine rows, microbial biomass was higher in rye cover-crop plots compared to bare plots . These results confirm earlier observations by Ingels et al. that microbial biomass carbon was higher in cover-cropped middles compared to bare middles. In the vine rows, microbial biomass was greater in plots adjacent to rye cover-cropped plots compared to bare plots. The effect of cover crops grown in the middles on soil in the vine rows may be due to cover-crop roots or tops extending into the vine rows and their subsequent decomposition, providing a food source for soil microbes.

Microbial biomass varied between the weed treatments in the vine rows but not middles . In the vine rows, stackable planters microbial biomass was significantly higher in the cultivation plots compared to the pre-emergence weed control plots . The most likely explanation is the incorporation of greater amounts of weed-derived carbon into the surface soil of the cultivated plots. Mycorrhizae. AMF can benefit grapevines by improving the nutritional 35 30 25 20 15 10 5 0 Cultivation Post-emergence Pre-emergence herbicide herbicide Bare ground Merced rye Trios 102 status of the plant and producing a highly branched root system. We quantified AMF reproductive structures in grapevine roots to determine if the weed control treatments in the rows and/or cover-crop treatments in the middles had significant effects on mycorrhizal colonization from 2003 through 2005. Based on ANOVA, the effects of weed control on colonization were not consistent among covercrop treatments . Grapevines adjacent to ‘Merced’ rye had higher colonization compared to those adjacent to ‘Trios 102’ triticale or bare ground, in both the cultivation and pre-emergence treatments . In contrast, grapevines in the post emergence treatment had the lowest colonization when adjacent to ‘Merced’ rye. These findings were consistent in each study year, based on the absence of significant main or interactive effects of time . It is possible that low colonization of grapevines in the post-emergence-by-‘Merced’ rye treatment is associated with this treatment’s weed community. Indeed, weed species vary in their ability to host AMF , so their presence or absence may affect mycorrhizal colonization of grapevines. Indeed, reports on the influence of plant community composition on AMF suggest that plant diversity has a strong effect on AMF diversity , and this may affect the colonization of individual plant species. All yield, fruit quality and vine growth parameters varied by year, and this was the only significant effect for these parameters, with the exception of berry weight and titratable acidity . No differences in crop yield or fruit composition were observed from 2001 to 2005 due to weed control treatments . Cover-crop treatments also had no significant effect on yield or fruit composition, although in 2001 and 2004, there was a reduction in berry size in the ‘Trios 102’ triticale treatment. Weed control treatments also had no effect on vine growth , based on shoot counts and pruning weights taken at dormancy. Cover-crop treatments had no significant effect on vine growth when averaged over 5 years, although in 2001 and 2005 the ‘Trios 102’ triticale treatment significantly reduced pruning weights. The trend for lower pruning weights may be related to the greater decline in soil moisture in the middles where this cover crop was used. It appears that vine growth, yield and grape quality are more significantly affected by annual precipitation than by vineyard floor management practices. In low rainfall areas the choice of cover crop is critical because of its effect on available soil moisture. We observed that late-maturing ‘Trios 102’ used more soil moisture during the vine growing season; if irrigation water does not compensate for water used by the cover crop, reduced vine growth and yield losses may result. The clear benefits of cover crops were increased organic matter in the middles and reduced sediment loss. Microbial biomass was increased in cover-cropped middles and there were indications that this effect extended to under the vines. Although there were no negative impacts of weed control treatments on vine productivity, we observed increased compaction over time from the use of cultivation. This study indicated that the choice of weed control strategy and cover crop must be carefully considered to maximize the benefits and minimize negative impacts of the practices. The benefits of cover crops are concentrated in the middles,and future research should focus on evaluating practices that improve the quality of soil under the vines.Elderberry is a part of the Viburnaceae family and grows all over the world, including Europe, North America, and Asia. Due to the vast geographic and morphological variety within Sambucus, there have historically been many species within the genus. However, a reorganization by Bolli reclassified some of the most common species under Sambucus into subspecies of S. nigra. More recently, elderberry was moved out of the Adoxoaceae family, which had already been changed before when elderberry was taken out of the Caprifoliaceae family. These changes have impacted the three subspecies most of interest in this work: the European elderberry S. nigra ssp. nigra; the American elderberry S. nigra ssp. canadensis; and the blue elderberry S. nigra ssp. cerulea . However, due to wide acceptance of this naming scheme for the subspecies, it will be used through this work to align with the current naming, but previous works cited may use the former species names. Furthermore, some sources refer to the entire plant as an “elder”, while others refer to the plant as “elderberry”, which is also used to denote the fruit of the plant. In this work, “elderberry” is used to discuss the plant as well as the fruit. “Elderflower” is used to refer to the blossoms of the plant. European elderberry is the most well-studied and widely used subspecies of elderberry in the market. This subspecies grows throughout the European continent, including countries such as Slovenia, Portugal, and Austria. The fruit and flower have been studied for decades for their composition and bio-activity, and while elderberry and elder flower are not new ingredients to the market, they have garnered more attention in the last several years as consumers look for more natural remedies and supplements to support their health. This has been especially true during the COVID-19 pandemic, in which elderberry became a popular ingredient in immunity-supporting supplements. Thus, investigating other elderberry subspecies like the blue elderberry, the focus of future chapters, allows for farmers in the United States to capitalize on this demand, but more information is needed on this particular plant if it is going to be used in consumer products.There is a long, rich history of the use of different parts of the elderberry plant by many cultures. For example, the wood has been used for kindling and musical instruments. Indeed, stackable flower pots the name of the plant is derived from various ancient words related to instruments. The flowers and berries have been used in a variety of beverages, foods, and other herbal supplements. Folklore has many stories about the healing power of the elderberry and elder flower. The plant itself has been revered by many cultures, with a story about the “Elder Mother” living within the plant would protect those near the plant. It was even expected to ask the Elder Mother for the berries or flowers before taking them; without permission, she may seek revenge. The leaves, branches, flowers, and berries were believed to have protective powers for a home and the leaves were also used during burial rituals by some Celtic people. The personification and deep reverence for the elderberry show the importance of the plant through generations. Hippocrates and Pliny the Elder both wrote about elderberry and its medicinal properties. Tribes indigenous to North America used flowers and fruit for medicinal and beverages. Berries were also used as a natural dye for baskets and branches were used to make musical instruments. Elderberry is a perennial, deciduous plant native to many regions of the northern hemisphere. Elderberry plants are neither tree nor bush, as the plant sends new canes up each season, which without pruning, can lead to a large, shrub-like plant that can be several meters tall and wide. They prefer to grow in sunny, riparian climates with moist, well-drained soil, though subspecies in North America can be drought-tolerant. While pruning even down to the ground level of the elderberry can improve yield and accessibility for harvesting, there is a limitation on pruning of the blue elderberry in the Central Valley of California. Due to the threatened status of the Valley Elderberry Longhorn Beetle , which lives only in the elderberry, branches larger than one inch in protected areas should not be pruned or removed from a growing site. Elderberry shrubs typically produce small white flowers with five petals in the spring, though the elder flowers of the blue elderberry are a creamy yellow color. Small, dark blue-purple berries ripen in the summer in large clusters called umbels or cymes, though there are examples of subspecies that have some variation to these morphologies, such as the blue elderberry that has a white bloom on the berries, causing the berries to look blue, and S. racemosa, which are red. Variation can also occur within a subspecies due to growing conditions, such as soil type, precipitation, and temperatures, as well as a key differentiation tool: cultivars or genotypes. There are established cultivars or genotypes of the European subspecies , such as Sambu or Haschberg, as well as of the American subspecies , like Bob Gordon or Wylewood. Cultivars can have more consistent growing patterns, such as blooming or ripening all at once, and desired chemical compositions, such as increased anthocyanins, thus are more desirable to use in large scale growing of elderberry for commercial use. Blue elderberry does not have any established genotypes to date. If commercial interest in this subspecies continues to expand, effort should be made to develop cultivars with consistent quality and improved harvest ability, which is hampered right now due to flowers and berries ripening throughout a season, instead of a smaller window of time like the American and European subspecies. Indeed, starting this work can help increase the commercial interest viability of the blue elderberry. A primary driver in interest in the composition of elderberry and elder flower is for their potential health benefits. Several reviews have recently been published on this topic; thus, it will not be explored in depth here. European elderberry has been studied for its antioxidant, antimicrobial, anti-inflammatory, anticancer, immunomodulatory, and antidiabetic properties, as well as neuroprotection and cardiovascular protection in vitro and in vivo. These activities have been mainly attributed to the phenolic compounds like cyanidin 3-glucoside and cyanidin 3- sambubioside, but some other compounds have been shown to be bioactive as well, including terpenes, lectins, pectin, peptides, and malic acid. Using data from randomized, controlled clinical trials, a recent review found that elderberry could reduce symptoms from upper respiratory viral infections, providing support for the use of elderberry supplements by consumers to combat colds and flus without the use of antibiotic medicine. In a more unique application, elderberry and elder flower extracts have both shown to be effect in combatting gingival inflammation using a topical herbal patch and elder flower tea, respectively. A study of the mechanism of cyanidin 3-glucoside to treat against the influenza virus showed that elderberry extract had some inhibitory effect during the early stages of virus cycle with stronger impacts during post-infection.

Because of their limited protein-coding capacities, geminiviruses rely heavily on host cellular machinery and interact with an assortment of plant proteins and pathways to promote infection . Although three of these genes have been assigned putative functions in viral DNA replication and coat formation , their specific functions in pathogenesis are yet to be elucidated. Our results indicated that the developmental stage of the berries may not influence the expression of GRBaV viral genes and that host factors could play a more critical role in the establishment of successful infections. Red blotch symptoms in grape berries include abnormal chemical composition and asynchronous ripening in the clusters , both reflecting alterations in host metabolic homeostasis and developmental processes. Interestingly, we determined that GRBaV infections induced some processes associated with early fruit development in berries at late stages of ripening, strawberry gutter system while repressing pathways involved in fruit ripening . Abiotic stress responses to hypoxia and temperature were also suppressed by red blotch in grape berries.

These observations may imply that the virus, as a biotrophic pathogen, could redirect host metabolic processes to sustain higher energy demands due to viral replication and at the same time suppressing ripening related events and responses to stress , potentially counterproductive to viral infections. However, further evidence using infected berries from different grape cultivars and environmental conditions will be necessary to understand how both grapevine genotype and environment may influence the disease outcome. It is also important to consider that the effects of GRBaV infections on berries may not be comparable with those occurring in vegetative tissues; for instance, photosynthetic pathways are generally inhibited during viral infections of grape leaves . GRBaV infections restricted the biosynthesis and accumulation of phenylpropanoids and derivatives, which preferentially accumulate after véraison in red-skinned berries. These secondary metabolites function as antioxidants and phytoalexins to protect the berries against a variety of stresses, and are important contributors of berry quality parameters, such as color, flavor, and aroma . The inhibition of phenylpropanoid metabolism appears to be a hallmark of viral infections in red-skinned berries, as has previously been reported in leaf roll-affected berries . In particular, the anthocyanin biosynthetic pathway was greatly impaired as a result of leafroll and red blotch, correlating with the reduced coloration observed in GRBaVinfected berries from several red-skinned cultivars .

In this study, we demonstrated that GRBaV infections compromised the regulation of ripening by: suppressing specific ripening events; altering the expression patterns of transcription factors that control the transition from the growth to ripening phases and the activation of ripening pathways ; and causing hormonal imbalances. Most of the alterations in the ripening regulatory networks occurred in infected berries at véraison, the same developmental time when red blotch symptoms initiated, suggesting a link between mechanisms involved in the control of grapevine responses to viruses and berry development. Similar observations were made in leaf roll-affected berries, which presented lower expression levels of MYB transcription factors at late stages of ripening . Plant hormones modulate ripening processes and stress responses in grape berries. Red blotch had a profound impact on ABA, ethylene, and auxin pathways. ABA is considered the triggering signal of berry ripening, since its accumulation coincides with véraison and ABA responsive transcription factors have been implicated in the activation of ripening processes . Particularly, the induction of anthocyanin biosynthesis in a variety of grape cultivars has been linked to ABA-mediated signaling pathways . Our results indicate that alterations of ripening events in the GRBaV-infected berries, including the reduced anthocyanin content at late stages of ripening, could be a direct result of lower ABA levels at véraison. ABA has been shown to stimulate ethylene-mediated pathways in berries, and together both hormones appear to regulate the progression of ripening . GRBaV infections also affected genes involved in ethylene biosynthesis and responses , which could further account for the antagonistic effect of red blotch on ripening events and immune responses. In contrast, red blotch promoted auxin-mediated pathways, known to suppress berry ripening. Auxins play crucial roles in the early development of grape berries and are known to inhibit ripening processes by delaying ABA-triggered processes .

The mechanisms by which grapevine viruses cause disease in ripening berries need to be investigated. GRBaV may actively interfere with the regulation of berry ripening by hijacking the plant post-transcriptional control. We identified elements of the host post-transcriptional machinery that were misregulated in GRBaV-infected berries. Post-transcriptional regulation intersects plant immune responses, developmental transitions, and hormone signaling . Plant viruses, in particular geminiviruses, encode multiple silencing suppressors that interfere with host siRNA production and alter plant DNA methylation and miRNA pathways, causing developmental defects . A previous study on leaf roll indicated that the viral infection caused up-regulation of Dicer-like genes in ripening berries . Therefore, the relationship between the viral-induced repression of host RNA silencing processes and the post-transcriptional regulation of ripening should be addressed from the perspective of compatible grapevine– virus interactions.Shriveled berries on ripening clusters are not uncommon in California vineyards. They usually occur in only a small proportion of a vineyard’s fruit , but in particular vineyards and years, shriveling can affect more than half of the crop . Most shrivel disorders make the fruit less desirable for winemaking, with subsequent yield and production losses. Before taking steps to reduce the incidence of fruit shriveling in vineyards, it is necessary to differentiate between shrivel disorders. We describe four common causes of fruit shriveling and detail compositional differences between normally developing fruit and that affected by sugar accumulation disorder .Fruit exposed to direct sunlight for all or part of the day, especially in the heat of the afternoon, can be damaged by sunburn, which may be caused by high temperature, ultraviolet radiation or a combination of the two . The physical appearance of sunburned fruit depends on the grape variety and stage of development — white grapes and red grapes exposed before pigment accumulation begins develop brown discoloration, which varies depending on severity.Veraison and early postveraison red varieties with sunburn often exhibit poor color development, and may remain pink for the remainder of the season. Post-veraison sunburn leads to fruit with less color and a shiny appearance. Sunburned berries often crack, presumably due to damaged epidermal tissues. Extreme sunburn leads to complete berry desiccation and the formation of raisins in both red and white varieties. Sunburn only affects berries that are directly exposed to sunlight. The non-exposed side of a sunburned cluster often develops normally. If a cluster is fully exposed to the sun on both sides, hydroponic nft gully or if the rach is itself is damaged, then the cluster may be completely affected. Obvious signs of sunburn may only occur on the exposed portions of individual berries. Sunburn can be avoided by reducing the fruit’s exposure to direct sunlight, especially in the afternoon. While leaves are removed in the fruit zone in many growing regions to increase cluster exposure to indirect light, in north-south row orientations leaves are removed on the east side of the canopy to reduce direct exposure in the afternoon and the probability of sunburn. This practice does not completely eliminate the risk of sunburn, however, because morning sun can also cause damage.Natural dehydration is another type of shrivel that may affect berries late in ripening but prior to commercial harvest. These berries appear similar to fruit with bunchstem necrosis, but the rachis look green and healthy. For this type of shrivel, which is especially pronounced in Syrah , berries lose weight due to water loss, and sugars are concentrated . Both increased transpiration and decreased phloem infl ux have been suggested as causes for late-season dehydration, but recent studies provide evidence that several varieties of grape berries remain hydraulically connected to the parent vine and therefore may lose water back to the parent plant late in ripening as well as to dry, ambient air .Clusters affected by bunch stem necrosis are identifi ed by necrotic rachis tissue, with shriveled berries distal to the necrotic tissue. The visible symptoms of bunch stem necrosis begin as small black spots on pedicels , and progress to the lateral stem structure and rachis .

Usually, necrosis symptoms are not noted until the rachis is affected. Bunchstem necrosis may affect an entire cluster as well as the wings and tips of otherwise healthy clusters . It can occur in many varieties, but is especially prevalent in Cabernet Sauvignon on California’s North Coast. It has been described in the literature in many different countries, with descriptive terms that include waterberry , bunchstem dieback , shanking , stiellähme , palo negro , desséchement de la rafl e and dessichimiento della rachide . No specifi c cause of bunchstem necrosis has been identified, despite many years of research. In some cases, varietal differences in susceptibility have been correlated to xylem structure, specifically a reduction on the area of xylem distal to branch points in the peduncle . The incidence of bunchstem necrosis has also been correlated to various concentrations or ratios of mineral nutrients, including magnesium, calcium, potassium and nitrogen . Work in Chile and Australia has shown that the amino acid metabolite putrescine is associated with bunchstem necrosis. More light in the canopy can also reduce bunchstem necrosis . Bunchstem necrosis can appear very early in fruit development or after veraison. The terms “inflorescence necrosis” and “early bunchstem necrosis” have been used to describe bunchstem necrosis around bloom . The composition of such fruit varies depending on when during fruit development the rachis becomes necrotic. Presumably, the necrosis prevents both sugar and water transport to the berry. Hence, if the rachis becomes necrotic early in the ripening period before the berry has accumulated much sugar, fruit will have low Brix . On the other hand, if the rachis becomes necrotic after the berries have accumulated appreciable sugar, subsequent shriveling can concentrate the sugars. Bunchstem necrosis in Cabernet Sauvignon on the North Coast is usually the latter type. Fruit with bunchstem necrosis can have a Brix as high as 42 .Another disorder with symptoms that occur during the ripening period has been called “berry shrivel”; we recently proposed that it be called “sugar accumulation disorder” . This disorder was first described in Emperor table grapes from California’s San Joaquin Valley and is characterized by poor coloration and low sugar accumulation. Sugar accumulation disorder has been found in a number of varieties and is present in many areas of California. In general, it affects only a small proportion of clusters in a vineyard, though in certain years and vineyards up to 50% of the fruit can be affected. Regardless of the variety or location, fruit affected by sugar accumulation disorder has lower pH, berry weight and Brix compared with normally developing fruit . When multiple rachises and fruit with sugar accumulation disorder were tested for minerals, the only consistent difference from normally developing fruit or rachises was increased calcium in the rachis tissue . To test the hypothesis that fruit exhibiting sugar accumulation disorder may have altered nitrogen metabolism, we measured the amounts of nitrogenous compounds at harvest in fruit with the disorder compared to normally developing fruit. The vines were located at the UC Oakville Experimental Vineyard in the Napa Valley. Samples were taken at harvest on Oct. 21, 2005. Berries with sugar accumulation disorder came from clusters on six vines that historically exhibited the disorder and showed symptoms in 2005 . Normally developing berries came from clusters on three nearby vines that had no history of sugar accumulation disorder and did not display symptoms at harvest. Two berries were sampled from each cluster and eight to 10 berries were pooled to ensure enough material for analysis. Berries were peeled, their seeds removed and flesh homogenized. One milliliter of the homogenate was used for the analysis of nitrogenous compounds. Individual amino acids in three samples of berries with sugar accumulation disorder and normally developing berries were measured at the UC Davis Molecular Structure Facility . Briefly, juice samples were acidified with sulfosalicylic acid to precipitate any intact protein before analysis. Free amino acids were separated using a Li-citrate buffer system with ion exchange chromatography on a Hitachi L-8900 amino acid analyzer. Amino acids were quantified by a postcolumn ninhydrin-reaction detection system. Amino acid concentrations were quantified from peak areas using standard curves. Data was analyzed by ANOVA .

The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites per se, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility. The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, hydroponic channel plant health and fruit production, as well as for those concerned with C storage.

The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.Grapes are the most valuable fruit crop in the United States, valued at over $6.5 billion annually , but climate change is projected to reduce grape production and quality . Climate affects grape quality by impacting the concentration of sugars, organic acids, and secondary compounds . The climatic conditions producing the highest quality wine cause the berries to reach optimal ratios between sugar and acid concentrations and maximum concentrations of pigment, aroma, and flavor compounds simultaneously . Hot temperatures accelerate sugar accumulation, forcing growers to harvest earlier, before berries reach optimal flavor development, to avoid the high alcohol content and insipid wine flavor from excessive sugar to acid ratios . Harvest dates have shifted earlier historically, and climate models predict further acceleration of ripening .

Growers can partly compensate through management practices, such as trimming canopies or using shade clothes to reduce the ratio of sugar supply to demand , though these practices are costly and increasingly ineffective in the face of climate change . Planting existing cultivars or developing new cultivars with slower sugar accumulation are promising alternative strategies to mitigate these climate change impacts, but these efforts have been hindered by uncertainty around the plant traits controlling sugar accumulation . Grape cultivars vary in berry maturation and sugar accumulation rates, and in their response to abiotic stress, but the main anatomical and physiological mechanisms driving these differences remain unknown . Multiple physiological processes influence berry sugar accumulation and its responses to climate, including photosynthesis, long-distance sugar transport, and local transport and metabolism in the berries . However, the relative importance of these factors in regulating sugar concentrations and fruit growth is debated . Photosynthetic responses to heat and water stress could impact cultivar differences in accumulation rates by affecting the sugar supply for ripening . Further, sugar is transported from the photosynthesizing leaves to the berries through the sugar-conducting vascular tissue – the phloem. At the onset of ripening , the berries significantly accelerate sugar accumulation by initiating active sugar unloading from the phloem, making the phloem the primary pathway for water and resource influx into the berries . The importance of phloem transport to ripening suggests that phloem traits could be important drivers of cultivar differences in sugar accumulation, and that modifying phloem traits to slow sugar accumulation under hot conditions could help mitigate the impacts of climate change on wine quality.

However, the main traits controlling sugar accumulation in grape remain unclear . The rate of phloem transport is determined by both the hydraulic resistance to the flow of sugar sap, and the activity and kinetics of water and sugar transporters in the sources, sinks, and along the transport pathway . Modeling studies suggest that increasing the hydraulic resistance of the phloem reduces sugar export to the sinks . Therefore, selecting grape cultivars with lower total phloem conductance could decelerate sugar accumulation and improve the synchronization of sugar accumulation with flavor development under hotter conditions. However, a higher hydraulic resistance can make the phloem more susceptible to declines or even complete failures in transport under severe water stress . Thus, we expect cultivars that produce high-quality wine in hot, dry conditions to exhibit phloem hydraulic resistances that slow berry sugar accumulation while avoiding phloem failure. The phloem transport pathway is composed of individual sugar-conducting cells with porous end walls stacked to form conduits . The anatomy of the transport pathway, including the total cross-sectional area of sieve tubes in plant organs, lumen area of individual sieve tubes, and porosity of the sieve plates, significantly impacts pathway resistance . Plants with a greater cross-sectional area dedicated to phloem , sieve tubes with wider lumen areas , and larger and more abundant pores in the sieve plates are expected to have a lower hydraulic resistance . Total phloem cross-sectional area in the shoots has been found to vary between several grape cultivars , and a greater cross-sectional phloem area has been linked to faster sugar accumulation in the fruit in other crop species . However, the variation of phloem structural traits across cultivars adapted to a diverse range of climatic conditions and the relationship of these traits to sugar accumulation is largely unknown for grapevines. Establishing these anatomical links could allow breeders to modify sugar accumulation by selecting for phloem traits, instead of management practices that can negatively impact the fruit zone environment or yield . In this study, hydroponic dutch buckets we used a common garden experiment to evaluate the links between phloem anatomy and sugar accumulation across 18 winegrape cultivars typically grown in climatically diverse grapegrowing regions. We assessed phloem and xylem vascular anatomy in leaf petioles and midveins and berry pedicels, to capture hydraulic resistance along the long-distance transport pathway. We also measured maximum berry sugar accumulation rates in the post-veraison ripening period to capture the greatest capacity for sugar transport . We predicted that traits that reduce hydraulic resistance, including larger total cross-sectional phloem areas, larger mean lumen areas for individual sieve tubes, and more porous sieve plates would increase maximum sugar accumulation rates. We also predicted that cultivars typically grown in hotter wine regions would havetraits that increase hydraulic resistance, as an adaptation to increase wine quality by reducing the rate of sugar accumulation. In addition, we measured photosynthesis and vine water stress to compare the impacts of phloem anatomy, vine carbon supply, and vine water status on sugar accumulation rates. Overall, our goals were to determine the most influential traits for sugar accumulation in grape berries and evaluate the role of phloem anatomy in adapting grape cultivars to a wide range of different climates.Leaves and berries were sampled to measure petiole, midvein, and pedicel anatomy in the morning on three days at the end of the growing season . Two berries and leaves per vine were excised with a razor blade.

Leaf position was standardized as the 6th leaf from the shoot apex, to capture the most photosynthetically active leaves. Two leaf and one berry sample per vine were then prepared for light microscopy, and the other berry sample was prepared for scanning electron microscopy. For light microscopy, a 1-cm segment of leaf petiole and lamina and the entire pedicel of the berry were immediately excised and placed into a vial of chilled Formalin-Acetic Acid . For scanning electron microscopy, pedicels were immediately flash-frozen and immersed in liquid nitrogen for 1-min and placed into a chilled micro-centrifuge tube of 100% ethanol, then the tube was immersed in liquid nitrogen until the ethanol congealed . Samples were then immediately placed on ice and stored in a -20°C freezer for at least 24 hours before further processing.After 7 days in FAA, the light microscopy samples were soaked in 50% ethanol for 5 mins and then stored in 70% ethanol in preparation for paraffin embedding. Samples were first infiltrated with paraffin by using an Autotechnicon Tissue Processor to treat samples with the following sequence of solutions: 70%, 85%, 95%, 100% ethanol, 1 ethanol:1 toluene, 100% toluene , and paraffin wax , each for 1 hour. The infiltrated samples were then embedded into paraffin blocks with a Leica Histo-Embedder , and allowed to cool. A rotary microtome was then used to make 7μm-thick cross-sections for leaf laminas, petioles, and berry pedicels. Pedicel cross-sections were sampled from the receptacle and petiole and midvein cross-sections were sampled near the interface of the lamina and petiole. After the cross-sections were imaged, pedicels for four cultivars were remelted from their wax molds, oriented longitudinally and sectioned again at 7μm to obtain sieve element lengths. Sections were stained using a 1% aniline blue and 1% safranin solution following a modified staining procedure . Sections were then viewed under bright field or florescence microscopy using a Leica DM4000B microscope and a DFC7000T digital camera . Each pedicel , midvein , and petiole section was then measured for total phloem and xylem cross-sectional area using ImageJ software, by manually selecting relevant tissue areas. Vascular tissue in longitudinal sections and cross sections was identified by cell size and/or stain color. Safranin stained the secondary cell walls of the xylem red and phloem cell walls were stained blue by aniline blue. The phloem area measurements included sieve tubes and phloem fibers and parenchyma , and xylem area measurements included xylem vessels, fibers, and parenchyma. Xylem and phloem rays greater than 4 cell layers thick were excluded. The pedicel electron microscopy samples were processed following Mullendore . Briefly, samples were thawed at room temperature, washed in DI water, and cut into 1 mm cross sections with a fresh double-sided razor blade. Sections were then transferred to 1.5 ml of 0.15% Proteinase K solution and mixed at 55°C and 300 RPM rotation for 14 days with an Eppendorf Thermomixer . Samples were then washed in DI water and placed into an 0.1% amylase solution for 24 hours at 50°C. Samples were then washed in DI water again, lyophilized overnight, mounted on aluminum stubs, and viewed under a Field Emission Scanning Electron Microscope . Sieve plates were viewed under low vacuum , 20- KV of accelerating voltage and a spot size of 2.5.Phloem anatomy was a stronger predictor of maximum sugar accumulation rates than vine carbon gain or water stress. Maximum sugar accumulation rates were not significantly correlated with photosynthesis or midday leaf water potentials . Including photosynthesis and midday leaf water potential as additional predictors also did not substantively improve the relationships between maximum sugar accumulation rates and petiole or pedicel cross-sectional phloem areas. Akaike Information Criterion corrected for small sample size values were higher for the larger models than the univariate models predicting maximum accumulation rates from petiole or pedicel phloem area alone, indicating that accounting for vine carbon gain and water stress did not improve predictive capacity for sugar accumulation . In addition, only one correlation was found between phloem petiole area and minimum mid-day water potential , while other average photosynthesis and water potential variables did not correlate with the phloem anatomical parameters. Finally, a previous dataset measuring leaf area for each cultivar did not find any significant correlations with °Brix accumulation, or other parameters measured .

The models all use community fixed effects and, for simplicity, have farm size entering linearly.The estimated coefficients from models 1 – 5 are largely consistent. They indicate a strong inverse relationship between farm size and frontier TFP and that the frontier is increasing over time, reflecting positive technical change. The coefficients on inputs are positive and stable across specifications, with family labor and purchased intermediate inputs being significant. The variance of the inefficiency term 2 is roughly double the size of the variance of the noise 2 in all models, and lambda – the ratio of the two variances – indicates that estimation of a stochastic frontier is appropriate with the MxFLS data. The models indicate an inverse relationship between farm size and productivity at the technological frontier of the same order of magnitude as the farm size-TFP relationship estimated in the preceding analysis of the average production function. The coefficients on survey year dummies in Table 2.12 are all positive and significant, u plangting gutter indicating that the frontier is increasing over time. Thus, in contrast to the results from the average production function analysis where evidence of declining average TFP over time was found, here we find evidence of positive technical change at the frontier.

The interaction between farm size and the survey year dummies in model 5 identifies a positive and significant relationship between farm size and technical change, suggesting that technical change has been biased towards larger farms and that the inverse relationship along the frontier became less steep over time.Models 1 through 4 show that, while the variance of the inefficiency distribution increased over time, there is no relationship between farm size and inefficiency. The inclusion of interactions between farm size and survey year dummy variables in model 5, however, reveals a more nuanced dynamic relationship between farm size and technical inefficiency. Larger farms were indeed more efficient than smaller farms in 2002 but inefficiency is increasing faster for larger farms. These differential changes in inefficiency across the farm size distribution have caused the farm size – inefficiency relationship to disappear in the latter waves of the MxFLS.20 Model 5 reveals that rising technical inefficiency has accompanied technological change, suggesting that the majority of farms have been unable to keep up with the TFP growth of the most productive farms. This is particularly true for larger farms, who have experienced faster growth in both frontier productivity and technical inefficiency. Having secondary or college education reduces the variance of the one-sided inefficiency term when education is included in the inefficiency equation. When education of the household head is included in the frontier specifications but not in the explanation of inefficiency , having secondary education or a college education is positively associated with higher levels of productivity among frontier producers.

When education is included in both the frontier and inefficiency equations , almost none of the education dummies are significant as the model appears to struggle to identify the separate relationships with education. In models not shown here, we estimate a stochastic frontier including the household controls from Table 2.8 as explanatory variables of the inefficiency term. In addition to educational attainment of the household head, technical inefficiency is lower among Procampo participants and higher among farms practicing monocropping. When interacted with farm size, none of the interaction terms are statistically significant, suggesting that they do not fundamentally change the relationships observed in Table 2.12. The analysis of Mexican data reveals an inverse and time-invariant relationship between farm size and TFP. Underlying this IR is a negative relationship between farm size and frontier productivity that has diminished over time and a positive relationship between farm size and technical efficiency that disappeared over the sample period. This evidence suggests that, in the wake of NAFTA era reforms, the IR is weakening for the most productive farms along the production frontier but that this change is not widespread. Although frontier productivity is increasing most rapidly for larger farms, the higher growth of inefficiency for large farms leaves the farm size – TFP relationship unchanged over the period.

The evolving relationships between farm size and frontier productivity and technical efficiency cast doubt on the ability to exploit the existing inverse relationship between farm size and TFP to generate productivity gains. These results are complemented by previous work on the farm size – productivity relationship in Brazil. Whereas the Brazilian experience suggests a dynamic farm size –TFP relationship, with an inverse relationship in traditional agriculture becoming flat and potentially positive with modernization, we observe no such dynamics in the Mexican sample. The relationship observed in the MxFLS is time invariant and persistently negative, contrasting with the emerging U-shaped relationship observed in the modernizing regions of Brazil. It is quite similar, however, to the more traditional agricultural regions in Brazil that display a persistent inverse relationship between farm size and TFP. The lack of corporate-run commercial farms is one limitation of using the MxFLS data, inhibiting analysis of the farm size-productivity relationship across all sectors of Mexican agriculture. This is especially true in light of findings that, in Brazil, larger commercial farms exhibit distinct advantages in achieving productivity growth . The frontier analysis using MxFLS data finds that technical change has been biased towards larger farms, weakening the farm size – productivity relationship at the frontier. This indicates that if inefficiency had not increased, the average inverse relationship between farm size and productivity would have weakened with modernization of the agricultural sector. This analysis indicates the potential for larger farms to be the key drivers of future productivity growth in Mexico. Policies geared towards smaller family farms may not have large returns in terms of increasing overall agricultural productivity, but they are likely very important for poverty reduction. Even if small farms generate an increasingly smaller share of agricultural output, they are likely here to stay because of their roles in generating livelihoods for rural households. Increasing their productivity remains an important component of facilitating poverty reduction in rural areas. These findings are largely consistent with earlier empirical work by Kagin et al. , who estimate both an average production function and a stochastic production frontier using a different panel of Mexican family farms. They find that both technical change and technical inefficiency increased over time and, as with the current analysis, planting gutter their fixed effects estimates show inverse relationships between farm size and both TFP and frontier productivity. Similarly, they find that smaller farms are more efficient than larger farms. In addition to highlighting the non-linearity in the farm size – TFP relationship, we provide evidence of a more nuanced and dynamic relationship between farm size and technical inefficiency and between farm size and productivity at the frontier. Larger farms have both more rapidly growing frontier productivity and technical inefficiency than their smaller counterparts, and these considerations are important for effective policy. We find evidence of declining average TFP over the period of analysis for the MxFLS sample of family farms. This appears to be driven by increasing average technical inefficiency offsetting the positive technical change and expansion of the productivity frontier. The largest farms in the sample and their relatively rapidly growing technical inefficiency are an important factor here, indicating a growing advantage for some large farms in harnessing more modern agricultural practices that has not been widespread enough to translate into sector-wide average TFP growth. Policies enabling broader inclusion in the benefits from technical change would both increase average TFP and likely further diminish the IR. Whereas policies promoting technical change are more relevant for smaller farms, policies improving technical efficiency, such as extension services, are exceptionally important for larger farms.

The growing technical inefficiency observed in Mexico indicates the potential for policies designed to promote and support the adoption and efficient use of best practices to achieve gains in agricultural productivity. The finding of declining average TFP over time is a curious result, running counter to both the body of long-run country-level analyses and the micro-level analysis of Kagin et al. over similar time periods. One important caveat is the MxFLS sample does not include corporate run commercial farms as do national-level studies such as an agricultural census. To the extent that such farms have more effectively harnessed the gains from technological change, as with larger family farms on the frontier, the potentially heightened productivity of such large farms is not included in the current evaluation of the farm size – TFP relationship in Mexican agriculture or growth in average TFP over time. This has important policy implications for the development impacts of agriculture productivity gains – if these gains are experienced primarily by corporate-run commercial farms and not by family-run farms, the potential impacts on poverty and broader rural economic development will not be fully realized. Productivity gains for smaller family farms not only reduce poverty directly but are also likely to contribute more to local development because of how they interact with the local economy. To be most effective, policy directed at spurring development and poverty reduction through agricultural productivity gains should be inclusive of smaller family farms. The lack of commercial farms does not, however, reconcile this finding with that of Kagin et al. , who find rising average TFP over a similar period in a different sample of rural households. One difference is the MxFLS includes more larger family farms, and these farms are experiencing the most rapid increase in technical inefficiency. The inclusion of more large family farms may be the source of this result. One possible explanation of the finding of declining average TFP over the first decade of the 21st century is that the productivity of Mexican family farms has declined in the wake of the NAFTA era reforms. This interpretation is consistent with claims that NAFTA era reforms were insufficient for generating positive change in Mexico’s agricultural sector, and that these reforms may have been detrimental to some segments of Mexican agriculture. Participation in Procampo and increased education are found to be positively correlated with the agricultural productivity of Mexican family farms, whereas the practices of monocropping and operating as a subsistence farm are found to be negatively correlated with TFP. We are tentative in drawing stronger conclusions about the causal impact of these variables, as they are likely endogenous. However, the frontier analysis suggests how these controls relate to productivity. Education appears to increase the efficiency with which inputs are used on family farms, and monocropping is found to be an inefficient use of inputs. In this light, farmer education – particularly in methods such as intercropping – is expected to increase technical efficiency on family farms. Procampois primarily an income support program it is unclear how participation would affect agricultural productivity. On the one hand, participation may relax income constraints and allow for adopting more productive methods because payments are distributed prior to planting season. This would suggest an emphasis on improving access to credit to improve the efficiency of Mexico’s family farms. On the other hand, the historical production requirements of Procampo participation may mean that participants are simply more experienced producers. Policies to ensure that farms have the necessary documentation could potentially help provide farms with the opportunity to keep abreast of technical change, as documented property rights are an important condition for accessing credit and thus facilitating adoption. This is especially true for ejido farms transitioning into participation with private credit and land markets. Nevertheless, we find no correlation here between agricultural TFP and property right documentation, access to credit, or ejido status, as we would have expected. Working with a sample of family farms from the Mexican Family Life Survey , we document a persistent inverse relationship between farm size and land productivity over the period 2002 to 2009. Similarly, when estimating an average production function we find a time-invariant inverse relationship between farm size andTFP, driven by the relatively high productivity of the smallest farms relative to those in the middle, and relatively low productivity of the largest farms. This is complemented by a stochastic frontier analysis, allowing for estimation of the relationship between farm size and frontier productivity and between farm size and technical inefficiency.

Plant morphology is more than an attribute affecting plant organization, it is also dynamic. Developmentally, morphology reveals itself over the lifetime of a plant through varying rates of cell division, cell expansion, and anisotropic growth . Response to changes in environmental conditions further modulate the above mentioned parameters. Development is genetically programmed and driven by biochemical processes that are responsible for physical forces that change the observed patterning and growth of organs . In addition, external physical forces affect plant development, such as heterogeneous soil densities altering root growth or flows of air, water, or gravity modulating the bending of branches and leaves . Inherited modifications of development over generations results in the evolution of plant morphology . Development and evolution set the constraints for how the morphology of a plant arises, regardless of whether in a systematic, ecological, physiological, or genetic context . In 1790, Johann Wolfgang von Goethe pioneered a perspective that transformed the way mathematicians think about plant morphology: the idea that the essence of plant morphology is an underlying repetitive process of transformation .

The modern challenge that Goethe’s paradigm presents is to quantitatively describe transformations resulting from differences in the underlying genetic, developmental, dutch bucket hydroponic and environmental cues. From a mathematical perspective, the challenge is how to define shape descriptors to compare plant morphology with topological and geometrical techniques and how to integrate these shape descriptors into simulations of plant development.Several areas of mathematics can be used to extract quantitative measures of plant shape and morphology. One intuitive representation of the plant form relies on the use of skeletal descriptors that reduce the branching morphology of plants to a set of intersecting lines or curve segments, constituting a mathematical graph. These skeleton-based mathematical graphs can be derived from manual measurement or imaging data . Such skeletal descriptions can be used to derive quantitative measurements of lengths, diameters, and angles in tree crowns and roots, at a single time point or over time to capture growth dynamics . Having a skeletal description in place allows the definition of orders, in a biological and mathematical sense, to enable morphological analysis from a topological perspective .

Topological analyses can be used to compare shape characteristics independently of events that transform plant shape geometrically, providing a framework by which plant morphology can be modeled. The relationships between orders, such as degree of self similarity or self-nestedness are used to quantitatively summarize patterns of plant morphology. Persistent homology , an extension of Morse theory , transforms a given plant shape gradually to define self-similarity and morphological properties on the basis of topological event statistics. In the example in Figure 2B, topological events are represented by the geodesic distance at which branches are “born” and “die” along the length of the structure. In the 1980s, David Kendall defined an elegant statistical framework to compare shapes . His idea was to compare the outline of shapes in a transformation-invariant fashion. This concept infused rapidly as morphometrics into biology and is increasingly carried out using machine vision techniques . Kendall’s idea inspired the development of methods such as elliptical Fourier descriptors and new trends employing the Laplace Beltrami operator , both relying on the spectral decompositions of shapes . Beyond the organ level, such morphometric descriptors were used to analyze cellular expansion rates of rapidly deforming primordia into mature organ morphologies . From a geometric perspective, developmental processes construct surfaces in a three-dimensional space. Yet, the embedding of developing plant morphologies into a three dimensional space imposes constraints on plant forms. Awareness of such constraints has led to new interpretations of plant morphology that might provide avenues to explain symmetry and asymmetry in plant organs or the occurrence of plasticity as a morphological response to environmental changes .

Computer simulations use principles from graph theory, such as graph rewriting, to model plant morphology over developmental time by successively augmenting a graph with vertices and edges as plant development unfolds. These rules unravel the differences between observed plant morphologies across plant species and are capable of modeling fractal descriptions that reflect the repetitive and modular appearance of branching structures . Recent developments in functional-structural modeling abstract the genetic mechanisms driving the developmental program of tree crown morphology into a computational framework . Similarly, functional-structural modeling techniques are utilized in root biology to simulate the efficiency of nutrient and water uptake following developmental programs . Alan Turing, a pioneering figure in 20th-century science, had a longstanding interest in phyllotactic patterns. Turing’s approach to the problem was twofold: first, a detailed geometrical analysis of the patterns , and second, an application of his theory of morphogenesis through local activation and long range inhibition , which defined the first reaction diffusion system for morphological modeling. Combining physical experiments with computer simulations, Douady and Coudert subsequently modeled a diffusible chemical signal produced by a developing primordium that would inhibit the initiation of nearby primordia, successfully recapitulating known phyllotactic patterns in the shoot apical meristem , the number of floral organs , the regular spacing of root hairs , and the establishment of specific vascular patterns .A true synthesis of plant morphology, which comprehensively models observed biological phenomena and incorporates a mathematical perspective, remains elusive. In this section, we highlight current focuses in the study of plant morphology, including the technical limits of acquiring morphological data, phenotype prediction, responses of plants to the environment, models across biological scales, and the integration of complex phenomena, such as fluid dynamics, into plant morphological models. There are several technological limits to acquiring plant morphological data that must be overcome to move this field forward. One such limitation is the acquisition of quantitative plant images. Many acquisition systems do not provide morphological data with measurable units. Approaches that rely on the reflection of waves from the plant surface can provide quantitative measurements for morphological analyses. Time of flight scanners, dutch buckets system such as terrestrial laser scanning, overcome unitless measurement systems by recording the round-trip time of hundreds of thousands of laser beams sent at different angles from the scanner to the first plant surface within the line of sight . Leveraging the speed of light allows calculation of the distance between a point on the plant surface and the laser scanner. Laser scanning and the complementary, yet unitless, approach of stereovision both produce surface samples or point clouds as output. However, both approaches face algorithmic challenges encountered when plant parts occlude each other, since both rely on the reflection of waves from the plant surface . Radar provides another non-invasive technique to study individual tree and forest structures over wide areas. Radar pulses can either penetrate or reflect from foliage, depending on the selected wavelength . Although more compact and agile systems are being developed for precision forestry above- and below ground , their resolution is too low to acquire the detail in morphology needed to apply hierarchy or similarity oriented mathematical analysis strategies. Image acquisition that resolves occlusions by penetrating plant tissue is possible with X-ray and magnetic resonance imaging . While both technologies resolve occlusions and can even penetrate soil, their limitation is the requirement of a closed imaging volume.

Thus, although useful for a wide array of purposes, MRI and X-ray are potentially destructive if applied to mature plant organs such as roots in the field or tree crowns that are larger than the imaging volume . Interior plant anatomy can be imaged destructively using confocal microscopy and laser ablation or nano- or micro-CT tomography techniques, that are limited to small pot volumes, to investigate the first days of plant growth. One of the outstanding challenges in plant biology is to link the inheritance and activity of genes with observed phenotypes. This is particularly challenging for the study of plant morphology, as both the genetic landscape and morphospaces are complex: modeling each of these phenomena alone is difficult, let alone trying to model morphology as a result of genetic phenomena . Although classic examples exist in which plant morphology is radically altered by the effects of a few genes , many morphological traits have a polygenic basis . Quantitative trait locus analyses can identify the polygenic basis for morphological traits that span scales from the cellular to the whole organ level. At the cellular level, root cortex cell number , the cellular basis of carpel size , and epidermal cell area and number have been analyzed. The genetic basis of cellular morphology ultimately affects organ morphology, and quantitative genetic bases for fruit shape , root morphology , shoot apical meristem shape , leaf shape , and tree branching have been described. Natural variation in cell, tissue, or organ morphology ultimately impacts plant physiology, and vice versa. For example, formation of root cortical aerenchyma was linked to better plant growth under conditions of suboptimal availability of water and nutrients , possibly because aerenchyma reduces the metabolic costs of soil exploration. Maize genotypes with greater root cortical cell size or reduced root cortical cell file number reach greater depths to increase water capture under drought conditions, possibly because those cellular traits reduce metabolic costs of root growth and maintenance . The control of root angle that results in greater water capture in rice as water tables recede was linked to the control of auxin distribution . Similarly, in shoots, natural variation can be exploited to find genetic loci that control shoot morphology, e.g., leaf erectness . High-throughput phenotyping techniques are increasingly used to reveal the genetic basis of natural variation . In doing so, phenotyping techniques complement classic approaches of reverse genetics and often lead to novel insights, even in a well-studied species like Arabidopsis thaliana. Phenotyping techniques have revealed a genetic basis for dynamic processes such as root growth and traits that determine plant height . Similarly, high-resolution sampling of root gravitropism has led to an unprecedented understanding of the dynamics of the genetic basis of plasticity .Phenotypic plasticity is defined as the ability of one genotype to produce different phenotypes based on environmental differences and adds to the phenotypic complexity created by genetics and development. Trait variation in response to the environment has been analyzed classically using ‘reaction norms,’ where the phenotypic value of a certain trait is plotted for two different environments . If the trait is not plastic, the slope of the line connecting the points will be zero; if the reaction norm varies across the environment the trait is plastic and the slope of the reaction norm line will be a measure of the plasticity. As most of the responses of plants to their environment are nonlinear, more insight into phenotypic plasticity can be obtained by analyzing dose-response curves or dose-response surfaces . Seminal work by Clausen et al. demonstrated using several clonal species in a series of reciprocal transplants that, although heredity exerts the most measureable effects on plant morphology, environment is also a major source of phenotypic variability. Research continues to explore the range of phenotypic variation expressed by a given genotype in the context of different environments, which has important implications for many fields, including conservation, evolution, and agriculture . Many studies examine phenotypes across latitudinal or altitudinal gradients, or other environmental clines, to characterize the range of possible variation and its relationship to the process of local adaptation . Below-ground, plants encounter diverse sources of environmental variability, including water availability, soil chemistry, and physical properties like soil hardness and movement. These factors vary between individual plants and within an individual root system, where plants respond at spatio-temporal levels to very different granularity . Plasticity at a microenvironmental scale has been linked to developmental and molecular mechanisms . The scientific challenge here is to integrate these effects at a whole root system level and use different scales of information to understand the optimal acquisition in resource limited conditions . Since it is extremely difficult to examine complex interdependent processes occurring at multiple spatio-temporal scales, mathematical modeling can be used as a complementary tool with which to disentangle component processes and investigate how their coupling may lead to emergent patterns at a systems level .