Month: December 2024

Based on the above results, it could be hypothesized that the NAC-NOR gene is specifically expressed during tomato ripening and induced by exogenous ethylene treatment. This is consistent with a role for NAC-NOR in tomato fruit ripening, but this hypothesis requires further functional verification.Mature fruit of nor mutant produces no ethylene burst, undergoes very little change in carotenoid content, and almost completely fails to ripen . Based on these results, NAC-NOR has long been considered one of the core TFs regulating the initiation and progression of tomato fruit ripening . However, the fruit of homozygotes with NAC-NOR gene mutations induced by CRISPR/Cas9 technology shows a much less severe phenotype than the nor mutant, and the fruit undergoes significant ripening . The ripening of CR-NOR tomato fruit was partially inhibited, the breaker stage of CR-NOR fruit was delayed by only 3 d compared with that of WT fruit. Ethylene and carotenoids were still synthesized, and fruit softening was initiated in CR-NOR fruit . The color of CR-NOR tomato fruit was close to that of WT fruit 30 d after the color break stage, but the mature phenotype was obviously different . From these observations, it can be inferred that NAC-NOR gene editing delays the initiation of tomato fruit ripening. Recent studies have found that the RIN–MC fusion protein of the rin mutant, rather than being a loss of function mutation, plant pot with drainage is a gain-of-function TF that regulates tomato fruit ripening by transcriptional inhibition . This new evidence shows that the RIN gene is not a necessary element for ripening initiation but is required for the development of full ripening attributes .

The question arises as to whether there is a similar explanation for the mechanism of action of the nor mutation.The de Maagd laboratory at Wageningen University and our laboratory generated CR-NOR mutants in WT tomato by CRISPR/Cas9 technology and showed that the ripening of CR-NOR tomato fruit was partially inhibited . The color change in CR-NOR fruit after color break was slower than that in WT fruit, and CR-NOR fruit became orange, rather than red, 9 d after color break, which is obviously different from the almost completely inhibited ripening phenotype of nor mutant fruit . Wang et al. further edited NOR186 in the nor mutant to obtain a phenotype similar to that of CR-NOR in the WT background. They concluded that the mutant nor protein is a dominant-negative protein. They also speculated that the truncated protein in the nor mutant still has the ability to interact with other NAC proteins and to bind DNA without transcriptionally activating its targets . Here, we provide further experimental evidence for the hypothesis that NOR186 is localized in the nucleus and is capable of binding to the promoters of SlACS2, SlPL, and SlGgpps2 target genes, but is incapable of activating them . This contrasts with the behavior of NOR#19, produced by CRISPR-Cas9, which does not enter the nucleus, does not bind to and cannot activate the promoters of SlACS2, SlPL, and SlGgpps2 . Furthermore, over expression of NAC-NOR in the nor background could not completely restore the ripening quality attributes to the level of WT fruits, as exemplified by their inability to turn completely red 15 d after color break . This phenotype was explained by co-expressing CaMV35S-NOR with CaMV35S-NOR186; the activation effect of SlACS2, SlGgpps2, and SlPL promoters was inhibited compared with the WT NOR protein present alone .

Based on our above results, and combining the hypothesis of Wang et al., we constructed a model of the NOR and nor mutants’ functions in tomato fruit . In WT tomato, WT NOR protein interacts with other fruit ripening associated TFs including other NAC TFs, binds to the NACRS, and activates ripening-associated target genes such as SlACS2, SlPL, and SlGgpps2 to regulate tomato fruit ripening. Besides, other NAC TFs that are not interact with NOR protein can also bind to the NACRS and activate the same genes at the following tomato ripening stages . In nor mutant fruit, NOR186 lacks the TRR, but retains the DNA binding region, and the protein complex of NOR is still present and can bind to the promoters of the target genes, but is unable to activate them. In addition, NOR186 can play a space-occupying role and stop other NAC TFs from binding the same NACRS site of the same target genes such as SlACS2, SlPL, and SlGgpps2 at the following tomato ripening stages . However, the specific mechanism of nor functional transformation in the mutants is unclear, and needs further research.The ripening process in tomato fruits is regulated by many ripening-related TFs, some of which play a positive role, such as RIN , TDR4 , and TAGL1 , whereas others play a negative role, such as AP2a and SlMADS1 . Here, we found that the ripening process of CR-NOR fruit is delayed and inhibited, while the ripening process of OE-NOR fruit is significantly accelerated . Physiological analysis of the materials revealed a significant decrease in ethylene production, carotenoid accumulation, and fruit softening in CR-NOR fruit and a significant increase in OE-NOR fruit . Sequencing results showed that the expression levels of genes related to these three pathways also changed accordingly .

All of the data indicated that NAC-NOR is a positive regulator of tomato fruit ripening. It has been reported that four NAC family members, SlNAC1, SlNAC4, NOR-like1, and NAC-NOR, participate in tomato fruit ripening . There are examples where different members of the same gene family of TFs, such as RIN and TDR4, participate in the regulation of target genes by forming oligomers . However, it is still unclear whether the four NAC gene-coding proteins interact with each other. NAC-NOR and our previously reported NOR-like1 belong to the same evolutionary clade. They have a close relationship and have 62.84% amino acid sequence identity . However, the ripening phenotypes of the fruit in which each of these genes has been edited are significantly different. Compared with WT, CR-NOR fruit had only a 3 d delay in color break and the accumulation of pigments was slower than in WT . Ethylene production occurred, but at a reduced rate, and softening also occurred, but the mature fruit phenotype was orange-red rather than red . In contrast, compared with the control fruit, CR-NOR-like1 fruit had a delay in color break of at least 14 d . After color break, the production of pigments, ethylene biosynthesis, and softening were also significantly inhibited. Thus, NOR and NOR-like1 have some similar functions but also some obvious differences in the development and maturation of tomato fruits. NOR-like1 appears to be more important for fruit ripening initiation, whereas NAC-NOR has a stronger influence on carotenoid accumulation. There may not be an absolute separation of functions, however, since over expression of NAC-NOR does affect fruit development and the timing of ripening.Plants produce a wide variety of human health-benefiting compounds. Numerous studies have shown that plants contain rich and complex profiles of phytochemicals, including anthocyanins and other polyphenols. The bio-active functionalities of these compounds, such as antioxidants, anti-inflammatory, anti-carcinogen, and maltase inhibition, may deter or prevent chronic diseases such as cancer, cardiovascular diseases, and diabetes. Therefore, there is an increasing demand for the integration of these bio-active compounds with food. The challenges, however, pots with drainage holes lie in the complex chemical profile of the plant-based materials and the lack of stability under processing and storage conditions of bio-active compounds. Several extraction techniques have been developed for separating these polyphenolic compounds from plant materials, such as absorption and ion-exchange technologies with microporous resins, liquid–liquid extraction, and membrane filtration. Some of the key limitations of these approaches include inefficient extraction of a large diversity of polyphenolic compounds, labor extensive processes using high volumes of organic solvent, and limited protections for the sensitive compounds against degradation after extraction. Thus, there is a significant need to develop environmentally and economically friendly approaches to efficiently separate and stabilize the high-value bio-active compounds from plant sources and deliver their health-promoting functionalities. Furthermore, these solutions may also need to address the sensory challenges with some of the plant bio-actives.

Microencapsulation processes have been applied to concentrate, protect, and facilitate the incorporation of the polyphenolic compounds from plant extracts into food and pharmaceutical matrices. By definition, microencapsulation refers to technologies of formulating solids, liquids, or gaseous materials into microparticles or dispersion, with diameters typically ranging between 0.1 and 1000 µm. The industrial applications of the microencapsulation process offer a wide array of advantages in delivering polyphenolics, such as: protecting encapsulated phyto-active compounds from degradation during processing and storage; controlling and targeting the release of the encapsulated compounds; and tailoring the undesirable physical characteristics of the polyphenolic compounds such as solubility, smell, and taste, etc. The most common coating materials are polymers, which include natural polymers and synthetic polymers , poly , and copolymers. The shell for these microparticles is often formed using both physical and chemical processes such as spray drying and coacervation. Encapsulation systems can provide protection for the bio-active compounds using a combination of exogenous preservatives and coating materials. However, conventional encapsulation systems lack mechanisms to selectively bind phytochemicals from plant extracts and to protect these health-promoting phytochemicals, often without exogenous preservatives. Biological microscale structures, such as microbial cells, have emerged as promising encapsulation carriers for bio-active compounds. The results of these prior studies illustrate that microbial cells, such as yeast cells, can bind and encapsulate purified phytochemicals and protect them from oxidative and thermal stresses. Thus, the key advantages of microbial cell-based encapsulation systems are these pre-existing cell based microstructures eliminate the need for expensive processes used for creating these microstructures from biopolymers; eliminate the exposure of phytochemicals to heat, oxygen, and other physical factors that may deteriorate the encapsulated compounds, and reduce/eliminate the need of exogenous preservatives and antioxidants. The current studies using microbial cells have focused on purified plant-derived compounds, and to the best of our knowledge, no study has evaluated the role of microbial carriers for binding and encapsulation of diverse phytochemicals from plant juices or concentrations. Furthermore, most of the studies using microbial cells have focused on yeast cells as a model system with limited emphasis on bacterial cells for the binding and encapsulation of complex polyphenolic compounds. Lactic acid bacteria are widely recognized for their role in food fermentation and are increasingly evaluated for their probiotic functionality, thus having a significant potential to impact food systems and human health. Despite this potential, there is limited evaluation of the potential of lactic acid bacteria to encapsulate diverse bio-actives. In addition, the unique structural and compositional features of lactic acid bacteria, including cell wall bio-polymer composition and structure, significantly high single-cell protein content, bio-affinity to interact with the gut, and high levels of antioxidant activity due to various small molecules, peptides, proteins, or enzymes makes these bacterial cells a preferred encapsulation matrix for plant-based bio-actives. Thus, the focus of this study was to evaluate the binding and encapsulation of phytochemicals from a model fruit juice using inactivated probiotic bacteria Lactobacillus casei. Muscadine grapes juice was selected in this study since these are popular and highly valued fruits with rich phytochemical profiles and antioxidant properties. Inactivated probiotic bacteria L. casei was selected as it is a widely used probiotic strain from a Lactobacillus family and thus is widely accepted as a beneficial ingredient in food systems. Heat-inactivated cells were selected for the encapsulation to limit the metabolism of the encapsulated compounds as well as to increase the permeability of the cells for the fruit phytochemicals. Furthermore, inactivated probiotic cells retain some of the beneficial probiotic functions, as illustrated by recent studies. To develop a simple approach that can be adapted by other researchers, a simple incubation process was utilized to bind and encapsulate phytochemicals from the juice matrix of MG using bacterial cells in this study. The anthocyanin content and antioxidant properties of the juice matrix before and after incubation was selected as an overall measure of encapsulation efficiency of complex phytochemicals from juice matrix. To further characterize the binding and localization of phytochemicals to bacterial cell matrix, multispectral fluorescence confocal imaging data was acquired. The binding and encapsulation yield of key selected phytochemical compounds from MG was also quantified using a high-performance liquid chromatography .

Flowers with a greater probability of transferring pollen to these high-quality recipients will have greater reproductive success as males, and because pollen transfer can only occur between flowers open at the same time, this advantage falls to first flowers . The optimal sex allocation of first flowers is therefore more male than that of last flowers, unless the female gain curve decelerates much more rapidly than the male . This scenario is similar to our numerical model, but where Brunet and Charlesworth assume an equal probability of fruit-set across flowers regardless of resource status, we instead assume that fruit-set probability decreases with resource status, and where Brunet and Charlesworth assume no variation in flowering onset, we allow onset to vary. We discuss these differences below. In some species, declining fruit-set within plants appears to be an outcome of decreasingly female sex allocation optima . In many others, resource shortage at least contributes to the low fruit set of last flowers . When we assumed that fruit-set probability decreases with resource status, we found that the drop in expected female reproductive success from first to last flowers outpaced the drop in expected male success . The average functional gender of first flowers on plants is therefore more female than that of last flowers , vertical aeroponic tower garden leading us to expect that optimal sex allocation is least female in the last flowers. This expectation contrasts with Brunet & Charlesworth’s prediction for optimal allocation under declining resources with constant fruit set probability.

Pinpointing the precise sex allocation optima of flower classes under declining fruit-set probability requires testing in an analytical ESS model. The numerical model con- firmed that declining fruit-set creates conditions that could lead to selection for variable pre-fertilization sex allocation within plants. An ESS model would help predict the long-term outcome of this selection. When flowering onset is variable and fruit-set declines, a shift occurs in the quality of male mating opportunity available to early- versus late-flowering plants. Consequently, expected male success decreases among plants across the season , and functional femaleness increases . Others have speculated that variation in the mating environment experienced by early- versus late-flowering plants in dichogamous species leads to variation in their optimal sex allocation. We similarly suggest that optimal sex allocation may be increasingly female from early- to late-flowering plants in adichogamous species whose fruit-set declines. This prediction again requires testing in an ESS model. The key finding of the numerical model was that declining fruit-set can drive temporal variation in functional gender both within and among plants. Within plants, the model revealed that the effect of declining resources among flower classes on optimal sex allocation may depend on whether resource status does or does not affect fruit-set probability. Among plants, the model presented here suggests that heterogeneity in the mating environment may lead to among-plant variation in sex allocation optima. This contrasts with past work emphasizing effects of heterogeneity in the physical environment .

These two factors may lead to mechanistically different responses: whereas heterogeneity in the physical environment selects for plasticity in sex allocation , temporal heterogeneity in the mating environment resulting from declining fruit-set might instead give rise to correlational selection on flowering time and sex allocation.Temporal trends in phenotypic femaleness in B. rapa con- flicted with trends in functional gender predicted by the numerical model. Before examining this discrepancy, we need to consider the relationship between phenotypic gender and sex allocation. Inclusion of equivalence factor E in calculating Gp means that Gp is influenced by the population, and is not an inherent property of a plant or flower . It therefore cannot be directly read as sex allocation. Unlike some other formulations of phenotypic gender , however, ours did not allow E to vary over time. Thus, the same inverse relationship between Gp and the pollen-to-ovule ratio holds for all flowers in our study, meaningGp is interpretable as an indicator of sex allocation. Varying costs per pollen grain and costs per ovule across species can complicate interpretation of p:o as sex allocation , but this concern does not apply when evaluating directions of change in sex allocation among flowers and plants of a single species. The expectation that our indicator of sex allocation, Gp, would mirror trends in functional gender predicted by the model rests on the assumption that variation in functional gender causes variation in sex allocation optima. As stated above, this assumption requires testing in an analytical ESS model.

Even if the assumption is correct, however, observed trends in phenotypic femaleness in B. rapa could oppose expectations for several reasons. We first consider our experimental design. Our indicator of sex allocation includes pre-fertilization investment only, but female investment continues through seed and fruit maturation . We restricted our focus to pre-fertilization investment because declining fruit-set cannot be both the cause and the consequence of among-flower variation in sex allocation optima, and because variation in pre-fertilization allocation often matches predicted variation in optima . Also related to the experiment, plants may have plastically adjusted their allocation to the relatively constant glasshouse environment, perhaps masking temporal variation that would be expressed in the field. Discrepancy in within-plant temporal trends in sex allocation across glasshouse and field environments in some Clarkia taxa lends credence to this possibility . Second, optimal allocation in Brassica rapa might be influenced by factors not considered in the model, such as prefertilization decline in resource status, unequal male and female gain curves, and competition among related pollen grains and seeds . Moreover, model results were sensitive to self-incompatibility, the relationship between flowers displayed and pollen export, and flower longevity . The first of these factors certainly applies to B. rapa, and the other two may. Because these factors affect mate availability, they interact with declining fruitset to shape the sex allocation optima of flowers and plants. Interaction between factors is supported by Brunet’s finding that when fruit-set declines, the estimated male success and functional gender of first versus last flowers depends on whether calculations take floral dichogamy into account. Finally, some model assumptions might not apply to the study population. For example, the model assumed resource limitation of fruit production. Most species exhibit pollen limitation in at least some populations in some years . If pollen were limiting, fruit-set probability might not decrease within plants. Predicted temporal trends in functional gender may therefore occur only in populations or years where female fitness is resource-limited. The model also assumed that flowers varied in their quality as pollen recipients, but plants did not. Selection tends to favour early flowering through female fitness , suggesting that among-plant variation in female quality may alter male mating opportunity. TheB. rapa data reported here are, to our knowledge, one of just three datasets reporting temporalvariation in pollen and ovule production both within and among plants, and the only such dataset for an adichogamous species . Data describing within-plant temporal trends are, however, available for some other adichogamous species. These reveal increasing , decreasing and constant p:o from first to last flowers, suggesting multiple influences on sex allocation optima. Further data on temporal trends in adichogamous species, particularly at the among-plant level, might help resolve the potential role of declining fruit-set in shaping allocation optima.Citrus is a genus, which is evergreen trees, that belongs to the Rutaceae family, and is grown in tropical and subtropical countries in a belt from 40 0 North Latitude to 40 0 South Latitude . Citrus is generally thought to have originated in South Asia’s tropical and subtropical areas, including China, India, and the Malay Archipelago. After domestication, it was distributed from these regions to the rest of the world as well . Before giving a scientific name to Citrus or before Linnaeus’s classification, it was named “Kedros.” The word “Citrus” emanates from “Kedros,” the Latin form. Kedros is originally a Greek word for fragrant trees such as cedar, cypress, and pine. Citrus leaves and fruits had their specific fragrance, vertical gardening in greenhouse and the smell of Citrus leaves and fruits resembled cedar. For this reason, Citrus fruits were named “Kedros” . Although the history and geographical origin of Citrus remain unclear, there are different opinions about that .

The earliest known reference about the Citrus origin is the myth of Hesperides’s Golden Apples in Greek mythology. According to the legend, golden-colored apples were hidden in Hesperides’ garden. The son of Heracles, Greek Herakles, Roman Hercules, Zeus , and the mortal Alcmene had to labor to take three golden apples from the Hesperides’ garden. A dragon guarded this garden. Hercules killed the dragon and took the apples. It was believed that if anyone ate these apples, they could give immortal life to the person. There was a rumor that Gaia gavethese apples or Citrus fruits at Hera and Zeus’s wedding. Due to the Hesperides’ golden apples legend, the Greek botanical name of Citruses is “πορτοκάλι,” which is “Hesperidoeidē.” Therefore, there was doubt whether these golden apples represent today’s apples or other fruits. In those times, the word “apple” used to be given as a name to all fruits except for berries. Although there is a rumor that these apples were quinces, most opinions are that these mentioned golden apples may be Citrus fruits . Even if its origin is not known exactly, Citrus fruits are one of the most grown fruits in the world today, with production exceeding 113 million tons year -1 from about 10 million ha in 2020 . Sweet oranges Osb., mandarins , satsumas Marc, Clementines , lemons Burn and grapefruits are cultivars that have commercial importance in the world ; Ma et. al, 2020, FAOSTAT, 2020. China, Brazil, and the US are generally among the leading Citrus-producing countries , but this differs slightly when we look at the Citrus species separately in terms of their production quantity. According to FAOSTAT values, sweet oranges and sour oranges were categorized within the orange group by indicating the production amount of orange. The most produced Citrus fruits in the world, measured by quantity, were oranges with 75 million tons. Mandarins are the second most-produced among Citrus cultivars following orange production, with 38 million tons.Tangerine, mandarin, Clementine, and Satsuma were evaluated in mandarin groups. Brazil, India, and China are the leader countries in total orange production. China, Spain, and Turkey are the places where the most mandarin is produced around the world. It is seen that the production amount of mandarin has increased since 2016. Even though the mandarin production area decreased in 2020 compared to the previous year , there was an increase in mandarin production in 2020. For the last five years, China, Spain, and Turkey have maintained their leadership in mandarin production, respectively . Thanks to the taste and aroma that Citrus fruits have, Citrus fruits are consumed as food such as fresh fruit, juice, and jam. Furthermore, Citrus varieties are used in food, cosmetics, perfumery, and chemoprophylactic drug production in the pharmaceutical industry. In addition, Citrus species are beneficial for human health due to their phytochemicals and nutrients. The peel and pulp of Citrus cultivars contain ascorbic acid , alkaloids, coumarins, limonoids, carotenoids, and flavonoids such as anthocyanins. These phytochemicals with biological activities are essential for human health, and they help to prevent human degenerative diseases, including cancer, aging, cardiovascular diseases, inflammation, and diabetes II . Citrus fruits are among the most consumed fruits in the world due to their beneficial properties for health and their taste and aroma components. For this reason, it is important to develop new varieties with higher fruit quality and meet consumer demands with Citrus breeding. One of the most important goals of Citrus breeding is to produce and improve Citrus species with desired properties for global marketing. To obtain the selected properties, fruit quality characteristics must be improved. In Citrus fruits, especially mandarins, fruit size, taste, seedlessness, peelability, and color are critical fruit quality traits. These fruit characteristics are complex, and they can be affected by genetics , environment , genetics and environment interaction . To develop new varieties with desired properties for mandarin breeding, the genetic basis of fruit quality characteristics should be well understood. Modern genetics and modern biology tools such as molecular markers, linkage mapping, and quantitative trait locus analyses are performed for genetic studies in mandarins .

We detected no directional selection on flowering time when averaged across all treatments , suggesting that treatment differences in selection intensity cancelled each other out. On average, western genotypes produced 0.75 more fruits than eastern—a 27% increase . In addition, genetic differences between populations within subspecies contributed 20.4% of the variance in fecundity . However, the lack of subspecies × treatment and genotype × treatment interactions shows that genetic effects on performance were not microbiota-dependent. Finally, fitness was consistent across microbial treatments , indicating no net effect of different microbiomes on fecundity . In contrast, we found no evidence that different sterilized field soils affect flowering time selection differentials or gradients . However, these treatments affected overall fecundity , indicating strong differences in soil quality. Western genotypes produced 69% more fruits than eastern genotypes on average , but this advantage was consistent across all soils . We detected no net selection gradient on flowering time across all field soils ; however, we did find a significant selection differential on flowering time .Sampled soil microbiomes differed between sites within year but not between years within site . These results are supported by principal coordinates analysis, in which samples clustered mainly by site .

The first three PCo cumulatively explained 69.8% of prokaryotic community variation . Because the MAH and JAM soils, square plastic pot which had the slowest and fastest flowering times respectively in our experiment, were separated primarily on the PCo2 axis , PCo2 became our candidate microbiome component to test for effects on flowering time. Because eastern genotypes appeared insensitive to microbes , we used flowering time residuals of western genotypes as the response variable. We did not find a significant relationship between mean flowering time in each treatment and the mean PCo2 score of soil samples from the corresponding site . Likewise, none of the individual OTUs with high PCo2 scores predicted flowering time ; for instance, abundance of OTU_96997 appeared to correlate with phenology but the relationship was non-significant after correction for multiple comparisons . The phyla Proteobacteria and Crenarchaeota were more abundant, and Acidobacteria were less abundant, in slow-flowering MAH compared to fast-flowering JAM soil communities . Within phyla, several families—including Koribacteraceae, Solibacteraceae, Opitutaceae, Verrucomicrobiaceae, Solirubrobacteraceae, and Mycobacteriaceae—differed in relative abundance between MAH and JAM soil communities . In addition, Verrucomicrobia and Gemmatimonadetes were enriched in the 5% of OTUs with the highest loadings on PCo2 compared to the full natural communities , indicating that these phyla contribute disproportionately to the microbiome variation summarized by PCo2.

These ‘candidate taxa’ are promising organisms for further study of microbial influences on flowering time.Our analysis of native plant genotypes and soil microbial communities from four undisturbed environments suggests that soil microbiomes contribute to the ecology and evolution of flowering phenology in Boechera stricta. First, we showed that soil microbiota influenced phenotypic plasticity of flowering time. Second, soil microbiota altered the strength and direction of selection on flowering time. Finally, we showed how this type of experiment could be combined with quantitative descriptions of soil community composition to search for microbial species that affect important phenotypes. Our results show that experimental dissection of complex environments can reveal the ecological interactions shaping phenotypic expression and natural selection. Our finding that both soil microbes and soil chemistry cause plasticity of flowering time agrees with previous reports, although to our knowledge only one other study has tested the effects of both biotic and abiotic components of the same soil. Lau & Lennon found that Brassica rapa flowered faster in dry conditions, and that a soil microbial community with a history of drought stress accelerated flowering compared to the wet-adapted replicate of the same community. Other soil properties reported to delay flowering include heavy metal concentration , nutrient depletion , high salinity , and a history of invasive plant growth . Notably, some species’ phenology may be more robust than others’ in response to soil heterogeneity . In general, such phenotypic plasticity has important ecological and evolutionary consequences . For B. stricta in particular, phenological plasticity affects the plants’ ability to time reproduction for optimal seed set . In fact, the exclusion of natural soil microbiomes from growth chamber replicates of that experiment might be partially responsible for the low genetic correlation of flowering time in the field and in the lab .

Interestingly, our data hint that West genotypes may be more sensitive to soil microbiome than East genotypes , suggesting intraspecific genetic variation for microbe-induced flowering time plasticity. In contrast, the two subspecies show very similar sensitivities to abiotic soil properties , indicating that plasticity to these two stimuli may have different genetic bases. Typically, selection analyses allow us to infer and compare the adaptive value of particular traits in particular environments, but do not tell us why differential selection exists. Here we identified the soil microbiome as an agent of selection on flowering time in B. stricta . The evolutionary relevance of this finding is best illustrated by comparison with Anderson et al. , who measured a decrease in fecundity of 0.06 fruits/day to flowering in a typical B. stricta habitat. Our results suggest that all else remaining constant, a change in soil microbiome could increase or decrease that selection differential by up to 0.07 fruits/day, or >120% . The effects of the soil microbiome on both flowering time and its adaptive value may prove especially important in the context of conservation and adaptation to global change, given that bothmicrobial communities and plant phenology are sensitive to climate . Although we are not the first to report that soil biotas affect flowering time or selection on flowering time , our experiment adds to previous work in several unique ways. First, we used deep 16S rDNA sequencing methods to greatly enhance our resolution of the microbiome . Second, we described how this type of microbiome data can be combined with experimental phenotype data to determine which microbial taxa influence traits of interest. Third, including a diverse set of genotypes in the experiment allowed us to test for intraspecific genetic variation for flowering time plasticity in response to biotic and abiotic soil attributes, and link this ecological finding to the field of evolutionary biology. Finally, we are the first to use microbial communities from several undisturbed field sites to confirm that naturally occurring variation in soil microbes affects phenology and its adaptive value. We identified several taxa that are enriched or depleted in soils associated with fast flowering compared to slow flowering ; these groups are promising targets for future study of the microbe-flowering time relationship. As evolutionary biologists, a fundamental goal is to measure selection and trait expression in the field because the genotype-phenotype-fitness relationship is frequently contextdependent . In particular, the true relationships between plants and microbial communities may depend on neighboring plants or on other soil properties . For instance, Lau and Lennon found that the interaction between microbes and soil moisture synergistically affects fitness in Brassica rapa. Factorial application of a wider sampling of microbiomes and environmental variables—or, eventually, direct manipulation of these variables in the field —would be especially informative for understanding the ecological mechanisms of plantmicrobe interactions such as the flowering time effect we observed in B. stricta. Nonetheless, we demonstrate here that greenhouse experiments can reveal ecological interactions that may have been extremely difficult to detect directly in the field: all else remaining equal, natural differences in the soil microbiome can influence plant phenology and patterns of selection. This discovery required the isolation of soil microbiota from the larger, 25 liter pot more complex natural habitat. Further environmental simplification—the reduction of the microbiome into principal coordinates and then specific OTUs—potentially could reveal even more details of the relationship between genotype, phenotype, and the microbial environment . Although in this experiment we lacked power to fully utilize this method, it holds promise for future studies on the phenotypic effects of the environmental microbiome. This approach is generally applicable to search for microbial community members that alter biological characteristics of interest.Table grapes that meet minimum maturity standards, including sugar and acid content, and the ratio of sugar to acid, are harvested by hand and typically marketed as entire or partial clusters. The quality and value of the grapes are strongly affected by the size, texture, and color of the individual berries, and the overall appearance of the cluster . These quality attributes are commonly achieved, in part, through the use various plant growth regulators , agrochemicals with plant hormones or hormone-like compounds as active ingredients . For example, gibberellic acid is used to thin and size berries, forchlorfenuron is used to increase berry size, and firmness , and ethephon and abscisic acid may also be used to improve the color of red grapes .

Though the vast majority of table grapes are sold as entire or partial clusters, there is growing interest in marketing stemless fresh-cut grapes . However, destemming may damage grape berries, stimulating decay, and diminishing quality . Mechanical damage associated with destemming might be minimized through the use of abscission agents, PGRs which reduce fruit detachment force and promote the development of a dry stem scar, an abscission layer between the berry and pedicel . Research on the potential use of abscission agents as mechanical harvest aids for wine or raisin grapes have shown that 1-aminocyclopropane-1-carboxylic acid , coronatine, ethephon, and methyl jasmonate stimulate abscission of mature grape berries . Of those, ethephon is the only compound registered for use on grapes, though the registrations are for improving the color of red and black fruited grapes, or hastening grape maturity, both at considerably lower use rates than what is required to stimulate berry abscission. Ethephon is an ethylene-releasing molecule. Stable in a low pH solution, it hydrolyses in the higher pH of plant tissues releasing ethylene, a gaseous plant growth regulator . Ethephon’s chemical characteristics enable growers to apply it to grapes and other plants in the field with commercial spray equipment, and thereby stimulate ethylenedependent reactions. Ethephon absorption by plant tissues is influenced by temperature, relative humidity, and pH of the surface on which the spray droplets are deposited . Hedberg and Goodwin suggested that ethephon absorption by plant tissues is predominantly cuticular rather than stomatal and Nir and Lavee found that the thickness and composition of cuticle layers play an important role in penetration. How the molecule diffuses within the plant is not yet well understood. Studies conducted with the 2- chloroethylphosphoric acid marked with the 14C showed limited and mainly basipetal mobility . Ethylene regulates many aspects of fruit development including maturation, senescence, and abscission . Grape is considered non climacteric but an ethylene peak detected at veraison, the onset of ripening, may be higher than the physiological threshold for metabolic activities , and Giovannoni reported some aspects of non-climacteric ripening are probably associated with ethylene responses. Likewise, Chervin et al. reported that ethylene seems required for the increase in berry diameter, decrease in berry acidity and anthocyanins accumulation that occurs after veraison. Regardless of the endogenous role of ethylene in grape berry development, ethephon has well-established commercial uses in viticulture to promote fruit maturationrelated processes, including the synthesis and accumulation of anthocyanins in berries and the accumulation of soluble solids , and grape berries, which generally don’t abscise naturally, can be induced to abscise with exogenous application of ethephon or other compounds that stimulate ethylene production by grape berries . The potential for ethephon as an abscission agent for table grapes is a relatively new concept that has been little studied . If ethephon is to ever be registered for that use, the potential for excessive residues will have to be considered. This is especially important since relatively high rates of ethephon are needed to stimulate grape berry abscission, the process occurs quickly , and berries are consumed whole, without peeling. Therefore, the present study aimed to verify the effects of ethephon on the abscission of grape berries of two globally important seedless table grape Vitis vinifera cultivars, and on the residual concentration of ethephon in the berry in order to evaluate its potential for aiding in the production of fresh-cut fruit.Experiments were carried out in 2012 in Thompson Seedless and Crimson Seedless table grape vineyards located in the countryside of Adelfia and Francavilla Fontana , respectively. Both Thompson Seedless and Crimson Seedless were grafted onto 140 Ru and trained to an overhead trellis system , with the first spaced 2.8 m between rows and 2.5 m within rows and the latter 3.0 m between rows and 2.5 m within rows.

Compared to the placebo group, participants consuming the antioxidants plus zinc and copper showed a 28% reduction in progression to advanced AMD after five years. Subsequently, the AREDS2 was conducted with a newer formulation that included vitamins C and E, either 10 mg of L plus 2 mg of Z, and either 350 mg of docosahexaenoic acid plus 650 mg of eicosapentaenoic acid , or both. Patients were also given either 25 or 80 mg of zinc, each with 2 mg of copper. Beta-carotene was eliminated from the supplement due to a potential increased risk of lung cancer among smokers, who were already at high risk for AMD. Primary analyses of the AREDS2 formula found no additional benefit in reducing progression to advanced AMD, in comparison to the original AREDS formula. However, in a secondary analysis of combined data from AREDS and AREDS2, the progression risk in those receiving L and Z was significantly lower than in other groups. Neither formulation reduced the progression from early to intermediate AMD. These clinical trials did not monitor the MPOD status over time, thus limiting our understanding of the link between L and Z intake, retinal accumulation, and AMD development or progression. To date, dutch buckets the AREDS2 formula remains the standard of care for management of patients with intermediate AMD.The accumulation of L and Z in the macula starts in utero in primates and plays a critical role in visual development and maturation later in life.

Lutein and Z were detected as early as 20 weeks of gestation in macular tissue from human fetuses inspected at autopsy. Unlike fully matured human eyes, L is the dominant macular pigment in infants under the age of two regardless of eccentricities. The retina is less mature at birth compared to other eye structures, with complete differentiation requiring four to five years. The maturation of the macula is associated with a change in the L:Z ratio over the first four years of life, which correlates with the development of cone photoreceptors. Studies in premature infants illustrate the importance of these L and Z in visual development. In preterm human neonates, extremely low levels of serum L and Z are associated with an undetectable MPOD.79 When a carotenoid-fortified formula containing 211 µg/L of combined L and Z was given to preterm infants, plasma carotenoid levels became comparable to breastfed preterm infants, and were significantly higher than those fed formulas without L or Z fortification. In a small study that monitored the concentration of L and Z in various infant formulas and breast milk from different mothers, Z was not detected in any formula but was present in all breast milk samples, while L was consistently higher in breast milk. Serum L was also noted to be six-fold higher in breastfed infants compared to those fed with a formula devoid of L. Further studies are warranted to assess the prospective effects of L- and Z-fortified formula on MPOD and visual development in infants as they enter adulthood. Lutein and Z may also protect against oxidative damage in premature infants, especially those with retinopathy of prematurity . Premature infants with ROP usually have poor visual acuity, even after laser treatment or intravitreal injection of anti-vascular endothelial growth factor agents.

In a model of oxygen-induced retinopathy, mouse pups given L showed less vessel leakage and lower avascular area compared to those given a L-free control. The authors suggested that the anti-oxidant properties of L may have contributed to these results, although ROS levels were not measured. Studies that investigate L and Z supplementation in ROP babies have produced inconsistent results. In a multi-center, double-blind, randomized controlled trial of very-low-birth-weight infants, no difference was found in the incidence of ROP between those supplemented with daily oral L and Z or placebo. However, the progression rate of threshold ROP showed a lower trend in the supplemented group. No adverse events were noted with L and Z supplementation, suggesting that they were well-tolerated. Another study examining the effects of daily oral L and Z supplementation in preterm infants from the seventh day after birth until 40 weeks of age or until hospital discharge found no change in the rate or severity of ROP compared to placebo. Further, a meta-analysis of three randomized controlled trials also found no protective association between L and Z supplementation and the risk of ROP. Additional studies are needed to assess the role of prenatal L and Z supplementation in pregnant women at risk for premature delivery.During development, L and Z are not interchangeable. Serum Z in newborns and in their mothers is strongly correlated with the MPOD of the babies, but no relationship was noted for either maternal or infant levels of L.

During delivery, a high maternal plasma Z, but not plasma L, was significantly associated with a lower risk of visual acuity problems in children at three years of age. Further investigations that can accurately distinguish and quantify dietary and plasma Z from L are needed to better understand the role of these two carotenoids in visual performance during development. The L and Z in human milk is particularly important for infant eye and brain development, and may provide long term benefits to vision and cognition. Since humans cannot synthesize carotenoids, the fetus and breastfed infants must obtain these compounds from the mother through the placenta and the breast milk. During gestation, maternal lipoprotein synthesis increases, which accelerates the transport of carotenoids to the fetus. This transfer may deplete maternal stores if the dietary intake of carotenoids in general, and L and Z specifically, is inadequate to maintain body stores. Low maternal skin and serum carotenoid levels have been reported in mothers of newborn infants. The prevalence of AMD is higher in women than in men, even though on a global basis more men smoke. At the same time MPOD levels are lower in females. The potential reasons for an increased lifetime risk for AMD in women are complex and multi-factorial in nature and may include maternal depletion of L and Z during pregnancy and lactation . Importantly, the average dietary consumption of L and Z among females in the US is far below the amount of 10 mg/d known to increase MPOD. Therefore, either the intake of supplements containing L and Z, or increased intake of foods rich in these two carotenoids for the duration of pregnancy and lactation may be of value.The concentration of β-carotene, lycopene, L and Z, the main carotenoids in breast milk are associated with maternal dietary intake over the first six months of lactation. Daily maternal supplementation of either 6 mg of L with 96 µg of Z, or 12 mg of L with 192 µg of Z, over six weeks resulted in a dose-dependent increase in L and Z levels in the breast milk and of the mothers and their infants when assessed three to four months postpartum. Another study reported that more carotenes were present than xanthophylls in maternal plasma, whereas more xanthophylls such as L and Z were presented in breast milk, in comparison to carotenes. These findings support the notion that maternal-infant transfer of carotenoids may occur, possibly at the expense of the mother. Future studies are needed to clarify if breastfeeding or L and Z intake may impact their AMD risk. The L-ZIP supplementation trial is currently exploring whether prenatal supplementation of 10 mg of L and 2 mg of Z will maintain maternal body stores, prevent potential macular pigment depletion during pregnancy, or enhance systemic and ocular carotenoid stores for both mothers and infants. Clinical trials on the long-term effects of perinatal L and Z intake on MPOD changes among mothers and infants are also warranted.100 Unfortunately, longitudinal studies on AMD in females often do not include breast-feeding history. A useful study design would be to investigate MPOD levels and relative risks of AMD between multiparous and nulliparous women, and in mothers practicing breastfeeding compared to formula feeding. Dietary intake of L and Z would be important to assess. Recognizing that such a study would take decades, shorter term studies could be conducted in non-human primates. Another challenge in retrospective studies is that breastfeeding history may not be accurate. Therefore, studies on the maternal transfer of L and Z during pregnancy and lactation with MPOD changes in infants and throughout the lifespan, grow bucket could be important but difficult to conduct. Future research should also focus on the measurement of L and Z status and MPOD in mother-infant pairs of twins or short birth intervals. Last, when accessing AMD risk in women, reproductive hormone status may be a confounding factor.

Estrogen has been shown to reduce oxidative stress and inflammation in RPE cells as well as systemically. Lifetime estrogen exposure such as the number of pregnancies, menopause, reproductive period, oral contraceptive use, and hormone replacement therapy may all influence the risk of developing AMD. Current evidence regarding estrogen exposure and risk of AMD is inconsistent. One study reported that postmenopausal hormone use decreased the risk of neovascular AMD but increased the risk of early AMD, while parous women showed a reduced risk of early AMD but not neovascular AMD. Two nationwide studies from South Korea among postmenopausal women noted that exogenous estrogen exposure was not a protective factor for AMD. A cohort study found that hormone replacement therapy and a longer reproductive period was associated with an increased risk of neovascular AMD. A cross sectional study showed that oral contraceptive use was associated with an increased risk of late AMD.106 In addition, a review summarizing the effect of estrogen exposure and the risk of all age-related eye diseases concluded that HRT, or the use of oral contraceptives, could be either positively or negatively associated with the risk of AMD.103 In contrast, some studies have reported that a longer duration of breastfeeding may be protective from late but not early AMD, even when the estrogen level was low during lactation. Future studies on the interaction of different reproductive and estrogen exposure histories and AMD risk are needed.Humans cannot synthesize carotenoids, and the best dietary sources are fruits, vegetables, egg yolks, and dairy products. Consuming a diet rich in green leafy vegetables and fish is recommended by the National Eye Institute for the high carotenoid and DHA and EPA contents. Nevertheless, in the carotenoid group, L and Z are not yet considered essential, or even conditionally essential, so no dietary reference intakes for these two compounds exists. The US intake of L and Z has been decreasing. According to the U.S. National Health and Nutrition Examination Survey , the average intakes of L plus Z were 2.15 mg/d in males and 2.21 mg/d in females in 1987, and 2.15 mg/d in males and 1.86 mg/d in females in 1992. In NHANES 2013-2014, the average intakes of L and Z in males and females was 1.58 mg/d and 1.76 mg/d, respectively. Moreover, based on data from NHANES 2003-2004, the reported intake of L was significantly higher than Z in all age groups and ethnicities. Importantly, the Z to L ratio was also lower in females than males older than 31 years of age, which may result in a higher risk of AMD in women than in men. However, due to difficulties in analyzing dietary L and Z separately, most studies analyze both carotenoids together. Since the amount of L in most foods is significantly greater than Z, precise quantification of Z has been a challenge. The amount of L and Z in foods and dietary supplements appears to be safe.112 No adverse events were found in clinical trials giving L at 30 mg/d for 120 days or 40 mg/d for 63 days. The only reported adverse effect after a daily supplementation of 15 mg L in a 20-week trial was a single case of self-reported carotenodermia, a reversible condition of orange skin color. Although a higher amount has been used in human studies, after assessing the potential risks, the observed upper safety level for L has been proposed as 20 mg/d. The European Food Safety Authority concluded safe upper limits for L and Z for use in dietary supplements were 1 mg/kg body weight/d and 0.75 mg/kg body weight/d, respectively. In primate models, rhesus monkeys fed a xanthophyll-free diet for 3 to 6.5 years developed extremely deficient or absent macular yellow pigment and drusen-like bodies. 

Larger roots were left in the oven for 4 days. Stumps were considered part of the root system for this analysis.In vineyard ecosystems, annual C is represented by fruit, leaves and canes, and is either removed from the system and/or incorporated into the soil C pools, which was not considered further. Structures whose tissues remain in the plant were considered perennial C. Woody biomass volumes were measured and used for perennial C estimates. Cordon and trunk diameters were measured using a digital caliper at four locations per piece and averaged, and lengths were measured with a calibrated tape. Sixty vines were used for the analysis; twelve vines were omitted due to missing values in one or more vine fractions. All statistical estimates were conducted in R.The present study provides results for an assessment of vineyard biomass that is comparable with data from previous studies, as well as estimates of below ground biomass that are more precise than previous reports. While most studies on C sequestration in vineyards have focused on soil C, some have quantified above ground biomass and C stocks. For example, a study of grapevines in California found net primary productivity values between 5.5 and 11 Mg C ha−1—figures that are comparable to our mean estimate of 12.4 Mg C ha−1 .

For pruned biomass, hydroponic nft channel our estimate of 1.1 Mg C ha−1 were comparable to two assessments that estimated 2.5 Mg of pruned biomass ha−1 for both almonds and vineyards. Researchers reported that mature orchard crops in California allocated, on average, one third of their NPP to harvestable biomass, and mature vines allocated 35–50% of that year’s production to grape clusters. Our estimate of 50% of annual biomass C allocated to harvested clusters represent the fraction of the structures grown during the season . Furthermore, if woody annual increments were considered this proportion would be even lower. Likewise the observed 1.7 Mg ha−1 in fruit represents ~14% of total biomass , which is within 10% of other studies in the region at similar vine densities. More importantly, this study reports the fraction of C that could be recovered from winemaking and returned to the soil for potential long term storage. However, this study is restricted to the agronomic and environmental conditions of the site, and the methodology would require validation and potential adjustment in other locations and conditions. Few studies have conducted a thorough evaluation of below ground vine biomass in vineyards, although Elder field did estimate that fine roots contributed 20–30% of total NPP and that C was responsible for 45% of that dry matter. More recently, Brunori et al. studied the capability of grapevines to efficiently store C throughout the growing season and found that root systems contributed to between 9 and 26% of the total vine C fixation in a model Vitis vinifera sativa L. cv Merlot/berlandieri rupestris vineyard.

The results of our study provide a utilitarian analysis of C storage in mature wine grape vines, including above and below ground fractions and annual vs. perennial allocations. Such information constitutes the basic unit of measurement from which one can then estimate the contribution of wine grapes to C budgets at multiple scales— fruit, plant or vineyard level—and by region, sector, or in mixed crop analyses. Our study builds on earlier research that focused on the basic physiology, development and allocation of biomassin vines. Previous research has also examined vineyard-level carbon at the landscape level with coarser estimates of the absolute C storage capacity of vines of different ages, as well as the relative contribution of vines and woody biomass in natural vegetation in mixed vineyard-wildland landscapes. The combination of findings from those studies, together with the more precise and complete carbon-by-vine structure assessment provided here, mean that managers now have access to methods and analytical tools that allow precise and detailed C estimates from the individual vine to whole-farm scales. As carbon accounting in vineyard landscapes becomes more sophisticated, widespread and economically relevant, such vineyard-level analyses will become increasingly important for informing management decisions. The greater vine-level measuring precision that this study affords should also translate into improved scaled-up C assessments . In California alone, for example, there are more than 230,000 ha are planted in vines. Given that for many, if not most of those hectares, the exact number of individual vines is known, it is easy to see how improvements in vine-level measuring accuracy can have benefits from the individual farmer to the entire sector.

Previous efforts to develop rough allometric woody biomass equations for vines notwithstanding, there is still a need to improve our precision in estimating of how biomass changes with different parameters. Because the present analysis was conducted for 15 year old Cabernet vines, there is now a need for calibrating how vine C varies with age, varietal and training system. There is also uncertainty around the influence of grafting onto rootstock on C accumulation in vines. As mentioned in the methods, the vines in this study were not grafted—an artifact of the root-limiting duripan approximately 50 cm below the soil surface. 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, 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.In plants, a fruit is the seed-containing section, which is formed from the ovary after flowering. Fruits have their vivid colors due to the presence of phytochemicals as pigments, which are natural compounds that protect against threats and insults such as insects and ultraviolet sunlight. Bright colors of fruits also attract animals and human beings for seed dispersing purposes. For instance, cocoa beans and blueberries have been used traditionally as therapies among indigenous people in North America. The fruit, leaves, seed, nft growing system and bark of the mango plant have been used as traditional medicine in Southeast Asia, Oceania, Africa, and Central America. Goji berries have been used as traditional Chinese medicine for two thousand years. Phytochemicals can be classified primarily as terpenoids, phenolics, alkaloids, nitrogen containing plant constituents, and organosulfur compounds. Examples of major phytochemical groups that are abundant from dietary sources and related to human health include carotenoids and polyphenols. Carotenoids are a type of terpenoid. Carotenoids can be classified as carotenes and xanthophylls. Phenolics can be classified as phenolic acids and polyphenols. Two primary subclasses of phenolic acids are hydroxybenzoic acid and hydroxycinnamic acid. Polyphenols include flavonoids, tannins, stilbenes, lignans, and xanthones. As one of the most studied categories of polyphenols, subclasses of flavonoids can be categorized to flavanones, flavones, anthocyanins, flavanols , chalcones, flavonols, and isoflavonoids.6Among the thousands of phytochemicals that have been identified in plants, both health promoting and toxic compounds exist.

For instance, some tannins decreased the activity of digestive enzymes or the bio-availability of protein or minerals and have been considered as antinutrients. Phytochemicals that exist in plant-based dietary sources and have value in human health maintenance and prevention of diseases are defined as phytonutrients. Fruits, vegetables, legumes, spices, nuts, wine, cocoa, tea, and olive oil are examples of foods rich in bioactive phytonutrients. The consumption of these dietary components has been related to decreased risk of developing chronic diseases, including cardiovascular diseases , age-related eye diseases, type II diabetes, cancers, and all-cause mortality. Observational studies also have reported that the total dietary polyphenol intake was inversely associated with the risk of hypertension, hypercholesterolemia, and cardiovascular events. Polyphenols under different categories may play various roles in reducing CVD risk. In the United States, the estimated flavonoid intake is 345 mg/day, with flavanols as the most abundant source. The three most consumed flavanols are catechin, epicatechin, and their polymers. Subanalyses of a cohort study indicated that dietary intakes of flavanols along with lignans, dihydrochalcones, and hydroxybenzoic acids showed a stronger inverse association with the risk of overall CVD events than other phenolic compounds. Another cohort study reported that the dietary intakes of anthocyanins, dihydrochalcones, dihydroflavonols, proanthocyanidins, catechins, flavonols, hydroxybenzoic acids, and stilbenes were significantly associated with decreased risks of total CVD. Blueberries and cranberries contain high amount of anthocyanin and proanthocyanidin, respectively, with moderate concentration of flavonoids. Cocoa is rich in flavanols,especially epicatechin and catechin. Mango, as the fourth leading fruit crop worldwide, is high in carotenoids, phenolic acids, and mangiferin, a polyphenol classified as a xanthonoid. Carotenes exist in dietary sources primarily as α-carotene, β-carotene, and lycopene. Major xanthophylls that exist in dietary sources include lutein , zeaxanthin , and β-cryptoxanthin. Epidemiological studies report inconsistent results on the relationship between dietary L and Z intakes and the risk of age-related macular degeneration. However, clinical studies have shown that the supplementation of L and Z was able to increase the level of these compounds in the retina, suggesting their protection against age-related macular degeneration . A major dietary source of L and particularly of Z is goji berry, which also have other in carotenoids, as well as phenolic acids, and flavonoids. While many examples of fruits used traditionally for health promotion exist, this literature review focuses on the evidence of mango, cocoa, blueberries, and cranberries in cardiovascular health, and goji berries in eye health. The application of modern scientific methods to assess traditional remedies is important because evidence-based data is necessary to transfer historical stories and ancient wisdom to contemporary life and advancement of health and human performance.Cocoa is the dried and fully fermented product obtained from the seeds of Theobroma cacao L. and is the main ingredient in chocolate products. While used traditionally in a number of cultures, one of the best examples of its medicinal use is from the Kuna Indians who have lived for centuries on remote islands off of the Caribbean coast of Panama. This group of indigenouspeople is famous the lack of hypertension, an infrequent prevalence of CVD, diabetes, and cancer, and a longer lifespan, compared to Panamanians living on the mainland. However, when these people migrate to an urban environment, the incidence of hypertension and vascular diseases increased significantly. Nutritional assessments showed that the consumption of total fruit, fish, and cocoa-containing beverages were significantly higher among Kuna Indians living on the island compared to those residing in Panama City, even though the overall dietary intake of added sugars and salt was higher in the indigenous group. 

Arguably the most momentous shift in cultural practices for strawberries was the introduction of preplant soil fumigants, beginning with chloropicrin in the 1950s and methyl bromide in the 1960s. Fumigation is a soil disinfestation practice that improves plant productivity and helps with the management of arthropods, nematodes, weeds, soilborne fungi and other plant pathogens. Some of the most difficult to control pathogens include Verticillium dahliae, Fusarium spp. and Macrophomina phaesolina. Without soil fumigation, these pathogens have the potential to completely destroy strawberry plantings. Early on, when CP and MB were mixed and applied together, the synergistic effects allowed strawberries to be produced as an annual rather than a biennial crop, and to be grown continuously on the same land without rotation to another crop, resulting in an increase in annual strawberry acreage. The use of fumigants also led to higher and more predictable yields and fruit quality, blueberry grow pot and further enabled the development of more stable markets for strawberries . Yields for strawberries statewide increased from a range of 2 to 4 tons per acre prior to the introduction of soil fumigants to 16 tons per acre by 1969 . Additional cultural improvements included the development of both UC and proprietary strawberry varieties uniquely adapted to coastal production conditions. Varieties were bred, for example, for disease resistance, yield and market potential.

Notable UC-bred strawberry varieties include Tufts , Pajaro, Douglas, Chandler, and Selva , Camarosa and Seascape , and Aromas, Albion and Monterey . Irrigation practices also evolved, shifting from furrow irrigation in the 1960s to drip irrigation in the 1980s, which led to further improvements in plant disease management and greater water use efficiency. These and other enhancements meant that by 2012, yields could exceed 35 tons per acre . More recently, the strawberry industry has focused on “fine-tuning” fertility and water management for more efficient resource use, along with additional yield and fruit quality improvements . The Santa Cruz–Monterey area is also recognized for its early experience with conversion of conventional strawberry production to organic management . Organic strawberry production was shown to result in lower yields, which, when offset by premium prices could potentially offer higher net returns to growers. The importance of crop rotation for disease management was not addressed in the initial study by Gliessman et al. but has since been the focus of additional research, as have more complete analyses of the economics of organic strawberry production . Growers and area researchers continue to collaborate and advance organic strawberry production techniques. Most notably, a long-term research commitment has been made to determine organically acceptable disease management practices such as anaerobic soil disinfestation , the use of commercially available soil-applied biological organisms and the incorporation of soil amendments such as mustard seed and its derivatives.

The area is now seen as a global leader in organic strawberry research, and in 2012 the first organic strawberry production manual was published by UC Agriculture and Natural Resources . Statistics documenting expansion of the organic strawberry industry over time are not available on a county-by-county basis, but statistics for California show prodigious growth in acreage and value of production: from $9.7 million in 2000 to $93.6 million in 2012, a 621% increase in real dollars . Caneberries Like strawberries, raspberries and blackberries have benefitted from enhancements in cultural practices. When well-managed, both types of caneberries can produce crops for up to 20 years. However, to maintain acceptable quality and yield Central Coast growers typically manage raspberries and blackberries so that they produce two and five crops, respectively, prior to removal and replanting. In Santa Cruz County, raspberry production was relatively flat in the 1960s and 1970s, but began to increase substantially in the 1980s . This can be explained by a shift from floricane, or spring-bearing varieties, to the then newly developed proprietary primocane, or fall-bearing varieties, that do not carry the productivity constraints associated with the inadequate chill requirements along the Central Coast. Primocane-bearing varieties allow growers to successfully produce a high quality raspberry crop in low- or no-chill coastal locations, and further manipulate time to harvest and yield with pruning and other management practices .

Between 1990 and 2014, the number of acres planted to Santa Cruz and Monterey area raspberries almost tripled, tons produced increased by about 350% and the value of production was up by over 400% in real dollars . Santa Cruz County raspberry growers began to experiment with and adopt field-scale semi-permanent protective structures or tunnels in the 1990s and 2000s . Initially developed in Europe, field-scale tunnels allow growers to extend their production seasons, enhance yield and fruit quality, and capture high off-season prices for fresh market fruit . The controlled environment, and resulting security of production, also allows for greater market stability. This shift away from open-field production to protected cropping, along with breeding improvements, has had lasting impacts on the raspberry industry and its expansion. Cultural improvements geared towards fresh market blackberry production are more recent and include advances in breeding for thornless varieties and quality attributes . In 2011, a public primocane-bearing blackberry variety became commercially available for the first time and is now being planted in the area. Since that time, additional public and proprietary primocane-bearing varieties have been in development; some have already become available. Open-field production was the norm until recently, but to ensure marketable fruit of high quality, and as growers have shifted additional acreage to primocane-bearing varieties, tunnel culture has been more widely adopted and, based on discussions with growers, is now estimated at roughly 80% of the acreage. Like organic strawberries, remarkable growth in the statewide production of organic raspberries and blackberries was documented between 2000 and 2012 . Acreage climbed by over 500% in both organic berry categories. Value of production was up over 3,000% in real dollars for organic raspberries and up by almost the same percentage for organic blackberries. It is important to note that although the organic raspberry and blackberry categories have demonstrated extraordinary growth, they still represent a relatively small percentage of all berry production in the area.Research points to several factors that have spurred consumer demand for all berries. Berries contain bio-active compounds, including essential vitamins, minerals, fiber and antioxidants that contribute to healthy diets, and that help to reduce the risks associated with some chronic diseases and cancers . This information has been widely shared with consumers through, for example, government programs promoting healthy eating , and more generic berry promotion programs . Per capita consumption of fresh strawberries in the United States almost doubled from 1994 to 2014, increasing from 4.1 to 8.0 pounds . U.S. per capita consumption of fresh raspberries was small by comparison, at just 0.5 pounds in 2014. Similar consumption data are not available for blackberries, but Cook notes that consumers generally view berries as complementary, and that sales for all berries have increased. Indeed, in 2014, berry sales increased 5.8% over 2013; berries were the number one produce category for U.S. grocery retailers, at $5.7 billion in annual sales . Some berry operations also benefit from their proximity to the area’s urban centers, which have sizeable cohorts of educated, high-income consumers who generally demonstrate an interest in health and wellness, hydroponic bucket local agriculture and fresh and organic products. In addition to the more traditional grower shipper and direct marketing channels, new technology-driven food marketing companies — virtual food hubs — have evolved to cater to this demographic. They promote the values of sustainable communities, local food economies and business integrity and transparency, all important attributes for new 21st century consumers . These companies form relationships with local growers, provide some technical and market support, and enhance sales and engagement with consumers. It is not yet clear what impacts these still-niche marketing businesses may have on the industry in total. However, growers have responded to the various health and market signals by ramping up production of both conventional and organic products, berries included.

Specialists and farm advisors with UC Cooperative Extension have performed economic analyses for Santa Cruz and Monterey county fresh market berry crops for decades . The studies estimate production costs for a representative enterprise based on characteristics common to the area’s farms. Data are collected from established growers, input suppliers and other industry experts so that a diversity of operations and practices are taken into account. Since 1990, UCCE researchers have used a farm budget software program to analyze the data and present results in several formats detailing costs for cultural and harvest practices, monthly cash costs and business and investment overhead costs. The studies also include an analysis estimating net returns to growers for several yield and price scenarios. Representative costs for food safety and environmental quality programs have been incorporated into more recent studies as they have evolved to become standard business practices. The resulting production and economic information is specifically designed to assist growers, bankers, researchers and government agencies with business and policy decisions.The first economic analysis of fresh market strawberry production for Santa Cruz and Monterey counties was performed in 1969; at least one subsequent analysis has been conducted every decade since then. Though the level of detail and data included in each study has changed over time, some interesting trends can be noted. Annual land rent climbed from $150 per acre in 1969 to $2,700 in 2014, representing 2.5% and 5.5% of total production costs, respectively. The cost of soil fumigation for conventional strawberry production increased from $350 per acre in 1969 to $3,302 in 2010, representing 5.5% and 6.9% of total production costs, respectively. Production year water use gradually decreased from 80 acre-inches per acre in 1969 to 36 acre-inches by 1996 as drip irrigation became the standard. The amount of water used to bring a crop to harvest has remained roughly the same since that time; however, growers and researchers continue to investigate methods to increase water use efficiency even further. In some areas, soil types and fields, growers have been able to reduce per acre water use by several acre-inches more . When the above costs and water usage are assessed on a per ton rather than a per acre basis, production practice cost increases are less notable, and water savings even greater. Labor-intensive practices such as hand weeding and harvest are consistently shown as costly line items relative to other operations. Representative yields for conventionally produced fresh market strawberries rose from 20 tons per acre in the 1969 study to 30 tons in 2010, an increase of 50%. Even higher yields are discussed for some varieties and production conditions; county production statistics confirm that higher yields are indeed possible . Representative yields for organic strawberries, studied over a much shorter time period, rose from 15 tons per acre in 2006 to 17 tons in 2014, an increase of 13%. As more research is directed towards organic agriculture in general and strawberries in particular, yields will likely increase even more with time. Recent efforts include improvements in cultivar breeding, cultural practices and disease management, especially soil pathogen management. The most recent economic analyses for conventional, second year conventional and organic strawberry production were performed in 2010, 2011 and 2014, respectively. Second year conventional strawberries, or those producing a crop for a second year after having produced the first without replanting, represent about 15% of the total strawberry acreage in the area. Similarities and differences in total, cultural and pest management costs for the three management approaches are shown in figures 1 to 3. Total costs for conventional strawberries were $47,882 per acre and include expenses for all practices from land preparation to harvest . For the second year conventional strawberry crop, total costs were lower at $32,798 per acre, reflecting a reduction in expenditures for land preparation and reduced harvest costs because of lower yield. For organic strawberries, total costs were $49,044 per acre, slightly higher than for conventional production, mostly due to higher soil fertility input costs. Harvest, a labor-intensive practice, clearly represents the lion’s share of total costs, at 58% in organic production, 60% in conventional production and 67% in second year conventional berries. Cultural costs represent 26% of total costs in the conventional and organic systems, but only 15% for second year strawberries because there were no associated planting costs, and because pest management costs were lower .

Alternatively, shrub stress and dieback may be dependent on a wider variety of variables, particularly in a landscape as heterogeneous as this. Additionally, we predicted that dieback severity and individual shrub death would increase over time in lower elevations and exposed slopes compared to upper elevations and more mesic slopes.The specific area chosen for this study is located in the Santa Ynez mountains of Los Padres National Forest . Stands of A. glauca occur from approximately 400m to 1200m elevation, and are frequently mixed with other co-dominate woody evergreen shrub species including Adenostoma fasciculatum, Ceanothus megacarpus and, at lower elevations, Malosma laurina. The landscape of this region is extremely heterogeneous, with unstable terrain composed largely of sandstone rock outcroppings and sandstone-derived soils , and steep slopes and ridges that are interrupted by deep canyons . These dramatic features, while common habitat for chaparral plant communities, were a limitation in our ability to choose field sites. Thus, we relied heavily on accessibility by road and trail in finding sites. The climate in this region is of a mediterranean-type, with cool moist winters and a hot, dry summer season. The majority of rainfall typically occurs from November to April, and mean annual rainfall, based on a 120-year average, is 47cm . 2.2 Precipitation data Three weather stations, equipped with real-time, self-recording data loggers and maintained by the Santa Barbara County Public Works Hydrology Division, nft hydroponic system were chosen to retrieve precipitation data during the drought based on proximity and similar elevation to study sites. The Trout Club , San Marcos Pass , and El Deseo Ranch stations represented low, intermediate, and high elevations, respectively.

Data from these stations were retrieved from the Santa Barbara County Public Works Hydrology website.Average rainfall at these stations, based on 54-69 year means, increases with elevation from 68.3cm to 90.4cm . Annual rainfall data for this study are presented in “rainfall years” from November 1 of one year to October 31 of the next, to reflect the seasonal wet period preceding each sampling period. Consistent with these historical trends, annual rainfall at Trout Club was lowest between the 2014-15 and 2018-19 water years . However, during this same time period, rainfall totals were generally lower at El Deseo Ranch compared to San Marcos Pass .Data on canopy dieback for each individual were collected in fall 2016. Dieback was assessed as the actual percent of “non-green” vegetation, defined as yellow, brown, and black/gray leaves, as well as bare/defoliated stems within the canopy. Percent dieback of each canopy was estimated by two-to-three researchers viewing multiple angles of each shrub, and final estimates were determined after thorough consultation. Site dieback was then calculated as the mean of all selected shrubs within a site. Entire stand dieback was also estimated using a combination of ground-level assessments and, when available, aerial drone photographs. These were used to confirm that canopy dieback of individual shrubs were collectively representative of whole stand dieback.Data were again collected approximately every 4-6 weeks throughout the summer dry season, this time from June through October. XPP measurements were taken using the same methods as described above. Additionally, we measured daytime gas exchange to gain a better understanding of plant function throughout the dry season and as water availability declined.

Anet was measured using a Licor 6400XT . CO2 was set to 400ppm, and photosynthetically active radiation was set between 1400-1800, to reflect maximum photosynthesis conditions at peak daytime hours. All measurements were taken between 9am and 11am DST, typically on the morning after the predawn measurements were made. A single fully-expanded leaf was chosen per plant, and two readings were recorded and averaged later during data processing. Leaves were traced in the field, and tracings were brought back to the lab and scanned to calculate leaf area within the chamber using ImageJ software . Our FMS 2 system broke in August, and we were therefore unable to collect any Fv/Fm data after the July fieldwork.Differences in means between sites for plant physiology and dieback were compared using one-way ANOVA with Tukey’s HSD post hoc analysis for means separations. Dieback data were square-root transformed for normality. Linear regression models were used to identify correlations of elevation and aspect between plant drought stress and dieback. Multiple regression models were developed using landscape factors and plant physiology as predictors for canopy dieback, and AIC values were calculated for choosing the best fit model. A repeated measures ANOVA was performed to compare dieback levels between sites and across four years of the study . Statistical analyses were performed using JMP Pro Statistical Software and R Studio .This study sought to understand spatial patterns of A. glauca canopy dieback across the landscape during an unprecedented drought, and track the progression of dieback and eventual mortality in this classically drought-tolerant shrub. Our data support the hypothesis that dieback is related to water stress from drought, and that this varied across the landscape, but there was considerable variation between sites and across time. While aspect was significantly correlated with dieback across all years, we found no consistent significant effect of elevation on dieback until 2019, late in drought period. Further, while dieback generally increased across all sites from 2016 to 2019, we observed no new incidences of mortality during this study, suggesting that in this landscape A. glauca individuals are resilient to this punctuated but overall prolonged drought.

Consistent with our predictions, elevation and aspect appeared to both have significant correlations with XPP and Anet. While these correlations were weak, their significance lends support to our hypothesis that plant stress is related to these landscape variables. These relationships also provide evidence that elevation and southwestness may be used as tools for identifying areas where plant canopies are more vulnerable drought. The low correlation coefficients may be due to extreme landscape heterogeneity in the study region including heterogeneity in substrate rockiness, soil accumulation and topographic concavity, confounded by a relatively small sample size within each site. There are many microclimate variables that were not included in this study and that may contribute to variations in water stress, including site temperature, vapor pressure deficit, and fog patterns. Additionally, though we did not find plant size to be correlated with drought stress or dieback, we believe this is due to our efforts to select even-aged individuals for monitoring, while previous studies have found significant effects of plant canopy diameter on dieback and mortality . Also, because A. glauca recruit from seed after fire, individuals within a stand tend to be of similar size and age. Therefore, we recommend that future studies include greater variation in stand age so that relationships between age/size class and dieback can be better evaluated.Elevation was not shown to be highly correlated with dieback in all years of the study, hydroponic nft system and there did not appear to be a correlation between dieback annual precipitation. Yet, some important landscape patterns were revealed. Our hypothesis that dieback severity is related to landscape variables associated with water availability was most strongly supported in fall 2019, when dieback increased significantly with decreased elevation and increased southwestness. Aspect was correlated with dieback severity across all years, suggesting thiscould be a significant variable to consider when generating models for future drought risk. Moreover, it was found that the increase in dieback severity from 2016-2019 was greatest at lower elevations compared to high. In other words, while most sites experienced an overall increase in dieback, the increase at lower elevations was much more dramatic. Collectively, these results suggest that while populations of A. glauca across the landscape in Santa Barbara county are susceptible to extreme drought, those occurring on exposed, southwest-facing slopes and/or at the lower edge of their range may be less capable of recovery, particularly during prolonged drought. This is consistent with studies showing that low elevations and exposed slopes can correlate with plant water stress and mortality . Interestingly, the dieback severity was greatest in fall 2019, despite the region experiencing above-average rainfall in both the 2016-17 and 2018-19 water years . Thus, a single year of above-average rainfall was not sufficient to restore shrubs to predrought canopy health levels.

One possible explanation for this is that 2011-2016 and the 2017-18 wet seasons were extremely dry, pushing A. glauca individuals towards a threshold of drought resistance that could not be counteracted by one or two wet years. Venturas et al. found A. glauca to have high levels of hydraulic failure, dieback, and whole shrub mortality in Malibu County during the drought year of 2014, and a study by Paddock et al. from an intense drought year in California yielded similarly high mortality for this species. This supports the significance of intense drought years that preceded higher rainfall years in our study. Additionally, Gill and Mahall and Mahall et al. found evidence that some chaparral shrubs do not respond to surplus water, and thus high-rainfall years may not be accurate predictors of shrub recovery. Shallow-rooted species like A. glauca may only be able to benefit from soil water availability near the surface, and/or they may be relying on water availability in rocky outcrops that is more dependent on outcrop cavity structure than on individual rain event totals. Therefore, short-term excessive rainfall may be irrelevant for the recovery of these shrubs. Another hypothesis is that opportunistic fungi, as identified in prior work , may have played a significant role in the sudden development and continuation of dieback. Biotic agents are known to exacerbate drought effects by amplifying hydraulic failure and/or carbon starvation . A. glauca, already weakened by drought, may have been further impacted by fungal pathogens emerging from their latency between 2012 and 2016. Drake-Schultheis et al. have documented synergistic interactions of drought and latent fungal pathogens on A. glauca in a greenhouse setting. However, more research is required to identify the exact role of latent fungal pathogens in A. glauca in the field. Interestingly, Mahall et al. report detailed phenological measurements of A. glauca individuals in a nearby population in the 1980s following individual leaves for up to three years. They do not report dieback or evidence of fungal disease in the canopy of any individuals despite detailed descriptions of leaf condition. Persona communication with Mahall and Thwing likewise confirm the absence of evidence for pathogenic fungi in their study plants. Drake-Schultheis et al. have evidence for the very recent introduction of the most virulent pathogen in this system, Neofusicoccum australe. While the Mahall et al. study was conducted during the drought of the late 1980s and this drought was similar in magnitude, itmay be cumulative drought and a new fungal pathogen together that are today causing canopy dieback.A surprising result of this study was that while mortality among A. glauca was noted across the landscape at the beginning of this study, no new mortality was observed over the following four years of drought, despite some very low XPP measurements in 2016. Indeed, the XPP obtained in Fall 2016 are almost twice as negative as those measured in A. glauca shrubs in this same region during an earlier drought in the late 1980s . Furthermore, a significant number of shrubs across sites reached extreme levels of canopy dieback yet survived and in some cases even showed evidence of recovery. These observations are indicative of an impressive resilience to drought stress in this chaparral shrub species. As previously reported, A. glauca are typically identified as anisohydric, exhibiting continued gas exchange during drought and high resistance to cavitation . Our data showing the continuation of gas exchange well into the dry season further supports this strategy, although Anet did shut down in the lower elevation populations. The Venturas et al. and Paddock et al. studies also found greater mortality in A. glauca than was recorded in our study, and in other chaparral shrubs with high cavitation resistance– a relationship suggested to be the result of greater susceptibility to high intensity drought. Therefore, why high mortality was not also observed in our study is unclear. It may be a result of Santa Barbara experiencing a slightly milder climate, compared to those of the aforementioned studies, which both took place in more arid regions of southern California, including in a chaparral-desert ecotone. Summer fog, a normal occurrence in Santa BarbaraCounty, may also play an important role in reducing vapor pressure deficit, thus providing critical drought stress relief during the summer months. Furthermore, our study areas were dominated by stands of relatively large, mature plants, which have been shown to exhibit greater survival rates during drought than smaller individuals .

Modern nanochemistry has developed efficient techniques to manipulate nanoscale objects with a highly advanced degree of control. Chemically engineered nanoparticles can be synthesized with a large choice of sizes, shapes, constituent materials and surface coatings, and further assembled spatially into self-assembled structures, either spontaneously or in a directed manner. Advances in particle self-assembly and the quasi unlimited range of nanostructures with controlled architectures and functions available suggest that such assemblies may also provide a simple route to meta materials at infrared and visible length scales. Indeed, nanochemistry and self-assembly strategies are able to inexpensively produce materials whose inner structure is natively in the right range of sizes for optical and infrared applications and can provide fully three dimensional structures, thus opening the way to the fabrication of 3Dmeta material samples of finite volume of the highest importance to many applications. Such meta materials may be used, for example, to create 3D homogeneous, isotropic negative index materials , with simultaneously negative permittivity and magnetic permeability, cloaking devices or light-based circuits manipulating local optical electric fields rather than the flow of electrons. In this work we investigate certain EM properties of meta materials formed by densely arrayed clusters of plasmonic nanoparticles, big round plant pot which will be referred to as nanoclusters. Nanoclusters are formed by a number of metal nanocolloids attached to a dielectric core, as in the examples shown in Fig. 1, and can be easily realized and assembled by current state-of-the-art nanochemistry techniques.

Such a kind of structure generalizes the concept of nanorings originally proposed in [2] to realize a magnetic media at visible frequencies and has been recently shown in [3] to have the potential of providing resonant isotropic optical magnetism. An approximate model based on the single dipole approach in conjunction with the multipole expansion of the scattered field is used here to evaluate the electric and magnetic polarizabilities of the nanocluster. Then, the permittivity and permeability of the composite medium are estimated by the Maxwell Garnett homogenization model. Results obtained by this approximate method will be compared with data from full-wave simulations, focusing on the characterization of the nanocluster resonant isotropic electric and magnetic responses to anincident wave field, and the possibility to realize an isotropic NIM at optical frequencies.Extreme droughts are increasing in frequency, severity, and duration in arid and semiarid regions around the world due to climate change. As a result, plant species that are typically capable of withstanding regular drought stress are exposed to conditions outside of their normal range, rendering them susceptible to opportunistic disease-causing agents. Theoretical frameworks describing the roles of environmental and biotic stressors in driving plant mortality are well established. However, there is a lack of empirical data with which to resolve how these factors interact in vivo. Furthermore, studies that document progression of stress and die back throughout the course of a multi-year drought event in situ are rare. In this dissertation, I detail a series of studies aimed at understanding mechanisms of dieback and mortality by focusing on a severe canopy dieback event in a classically drought tolerant chaparral shrub, big berry manzanita in Santa Barbara, California, during an historic California drought.

I provide strong evidence that dieback is caused by members of the fungal Botryosphaeriaceae family in conjunction with extreme drought, and that dieback is also related to increased drought stress along an elevational gradient. By conducting a field survey, I identify Neofusiccocum australe as the most prevalent and widely distributed fungal pathogen in A. glauca., and that dieback is strongly correlated with Bot. infection. Using a full-factorial design in a greenhouse experiment, I provide evidence that extreme drought and infection by N. australe can indeed act synergistically, together driving faster and greater mortality in young A. glauca than either factor alone. Lastly, by taking measurements on water availability, dark-adapted leaf fluorescence, and photosynthesis in A. glauca shrubs across an elevational gradient, I provide evidence that landscape-level factors can contribute to localized variability in water stress and canopy dieback severity in A. glauca, and may be useful in predicting vulnerabilities during future drought. Remarkably, no new mortality was observed throughout the study, suggesting extreme resiliency in adult shrubs. However, canopy dieback alone can impact wildlife and fuel loads, even when not associated with mortality. Together, these results provide strong evidence that A. glauca dieback was caused synergistic effects between extreme drought and infection by N. australe, and that lower elevations and exposed slopes may be at greatest risk for future events. According to the most conservative estimates, global mean annual temperatures are now outside the historic range of the last 1,300 years . Simultaneously, mean annual precipitation has declined in many parts of the Northern Hemisphere, resulting in increased drought events . Extreme climatic shifts are predicted to affect, both directly and indirectly, biogeochemical cycling, energy fluxes, wildlife habitat, and ecosystem goods and services on a global scale . An important component in preparing for the effects of these events is to understand how communities will change in response to them, making this a critical topic for ecological research . For species to survive in dry climates, they must have evolved drought tolerance mechanisms .

However, extreme climate events can expose species that are typically capable of withstanding regular drought stress to conditions outside of their normal range. Furthermore, physiological responses to extreme drought can also have a negative feedback on plants’ defensive abilities, rendering them susceptible to biotic attack including by insects or disease agents . Consequently, synergies between extreme climatic events and biotic attack will likely lead to more dramatic changes than would otherwise occur in historically “drought tolerant” plant communities . Future climate change is expected to exacerbate these interactions worldwide. . Widespread tree mortality from drought has been documented in forested systems around the world , and biotic attack has been associated with many of these events . However, much less focus has been given historically to understanding the consequences of extreme drought on shrubland communities like chaparral , particularly in conjunction with biotic influences. Therefore, as we face predictions of hotter, longer, and more frequent drought , it is becoming increasingly critical to hone in on the mechanisms, tipping points, and ecosystem impacts of these events. Furthermore, identifying plant mortality thresholds is of upmost importance for predicting susceptibility to extreme drought events of the future . California recently experienced a record-breaking, multi-year drought from 2012- 2018, estimated to be the most severe event in the last 1,000 years , with the 2013-2014 winter season being one of the driest on record . Drought tolerance has long been considered a common trait of shrub species in California chaparral communities where hot, rainless summers are the norm . However, round plant pot in the Santa Ynez mountain range in Santa Barbara County, the dominant and widespread big berry manzanita exhibited dramatic dieback related to multi multi-year drought along with infection by opportunistic fungal pathogens in the Botryosphaeriaceae . These observations indicate that this species may be reaching a threshold in its drought resistance capabilities. Studies have reported Arctostaphylos spp. to exhibit unusual scales of dieback during periods of extreme drought stress, , however this could be the most severe dieback event in recent history, both in terms of scale and severity. Manzanita are important members of the chaparral ecosystem, providing habitat for wildlife and food through their nectar and berries . Additionally, their structure makes them important components of historical chaparral fire regimes, and their fire-induced germination strategies contribute to post-fire successional trajectories . Large-scale mortality of this species could reduce resource availability for wildlife, as well as alter fuel composition and structure in the region, resulting in an increased risk of more intense, faster burning fires. Therefore, the potential continued dieback of A. glauca is of great concern for both ecosystem functions and human populations alike. Significant dieback of A. glauca in Santa Barbara county, California, was first observed in winter, 2014 . Preliminary observations revealed patterns of dieback occurring along an elevational gradient, with effects being most pronounced at lower elevations than at higher elevations. It was also observed that dieback was most prevalent in stands located on steep, exposed southerly-facing slopes. These observations are consistent with findings by previous studies , Since A. glauca is classically drought-tolerant and able to function at very low water potentials , it raises the question of what is driving this extreme dieback event?

Could A. glauca be reaching a tipping point as a result of extreme drought stress, presence of a fungal pathogen, or both?My dissertation research focuses broadly on the influence of drought and fungal pathogens on this classic, drought tolerant chaparral shrub species. Through a combination of methods, I explore the individual and interacting roles of water stress and opportunistic fungal pathogens in A. glauca in a major dieback event, and track the fate of individual shrubs through the progression of an historic drought. My findings are organized into three chapters based on the following questions: What are the identities and distribution of fungal pathogens associated with A. glauca dieback ; How do drought stress and fungal infection interact to promote dieback and mortality in A. glauca ; and How does A. glauca dieback progress over time during drought, and how do landscape variables and drought stress correlate with dieback ? In Chapter 2, I identify fungal pathogens in A. glauca, and discuss their distribution across the landscape in the Santa Barbara county front country region. Based on preliminary findings showing significant levels of N. australe in the field, I expected to find high incidence of this opportunistic pathogen in A. glauca across the landscape, suggesting their role in drought-related dieback. The data support this prediction, as over half of the pathogens isolated were members of the Bot. family, and the majority of these were identified as N. australe, a novel pathogen in the region. Furthermore, Bot. infection was highly correlated with dieback severity, which was greatest at lower elevations. Taken together, the results show that opportunistic Bot. pathogens, particularly N. australe, are highly associated with A. glauca dieback across the landscape, and that lower elevations may be particularly vulnerable. In Chapter 3, I address the hypothesis that extreme drought and N. australe function synergistically to promote faster and greater mortality than either factor alone. I designed a full-factorial greenhouse experiment to identify whether A. glauca dieback is driven by extreme drought, infection by the fungal pathogens, or both. The results of this experiment support my hypothesis. Young A. glauca inoculated with N. australe while simultaneously exposed to extreme water stress exhibited faster stress symptom onset, faster mortality, and overall higher morality than those subjected to either factor alone. These results provide strong evidence that the severe A. glauca dieback event observed during the 2012-2018 drought was the result of synergistic interactions between extreme drought and opportunistic pathogens, rather than the nature of the drought or particularly virulent pathogens. In Chapter 4, I explore factors that are associated with climatic stress in order to draw correlations between A. glauca stress and dieback severity. Identifying such relationships can be useful in making predictions on dieback and mortality across the landscape. By analyzing data on predawn xylem pressure potentials and net photosynthesis in shrubs along an elevational gradient, I found that patterns of water availability and physiological function both varied greatly across the landscape, and only weakly correlate with dieback severity, suggesting factors other than elevation and aspect must also be important in driving plant stress and dieback. Extreme heterogeneity across this landscape likely confounded my results, yet may also play an important role in supporting the resiliency of A. glauca populations as a whole. By measuring the progression of dieback in these same shrubs over time, I found that dieback severity throughout the drought increased most at lower elevations compared to high, providing evidence that shrubs at lower elevations may be particularly vulnerable. Unexpectedly, no new mortality was observed in surveyed shrubs as the drought progressed, even though many plants exhibited severe levels of dieback throughout the study. This result shows that high levels of dieback severity do not necessarily predict morality in A. glauca. In summary, my dissertation provides strong evidence that A. glauca dieback during the recent California drought was caused by synergistic interactions between extreme drought stress and infection by widely distributed opportunistic fungal pathogen N. australe.

Analysis in the following sections expands on previous work by including consideration of recharge water from reservoir reoperation, evaluation of recharge water sourcing, cropland characteristics and groundwater hydrology for a site-specific setting and demonstrating a hydro-economic optimization approach that simulates separate decisions for land access and water delivery in the performance of Ag-MAR.The regional-scale analysis is conducted for a semi-arid part of California, USA that has conditions fairly common for many parts of the globe. The two groundwater sub-basins in the study area are part of the much larger Central Valley groundwater system with an interfingered assemblage of alluvial and flood-basin deposits of local maximum depth exceeding 1,000 ft . Many of the sub-basin boundaries shown in Fig. 2a are arbitrarily based on surface-water features, and the southern boundary has recently been adjusted northward to accommodate governance considerations for current groundwater management efforts . The 525,000-acre study area has a mix of urban , agricultural , wetland and undeveloped rangeland land uses . Over 90% of the total water use in the study area is supplied by groundwater . Moreover, square pot plastic approximately 41% of the agricultural acreage is planted as vineyards and orchards . This investment in perennial crops hardens water demand and intensifies groundwater extraction during droughts. The spatial distribution of recent water levels indicates localized depressions from extractions far exceeding groundwater recharge . Groundwater levels have dropped as much as 60 ft over the past several decades so that surface water frequently becomes disconnected from saturated groundwater and drains into the subsurface. The lower reaches of the Cosumnes River, in the central part of the study area , are dry 85% of the time .

New regulations for sustainable groundwater management in California require that this chronic lowering of groundwater levels and depletion of storage be addressed through active measures . While restoration of surface-water base flow in the study area may not be required because impact occurred before implementation of the regulations, there is interest in maintaining, and possibly improving, groundwater support of surface-water flows . Consistent with recent analysis , local stakeholders are interested in harvesting runoff from high-precipitation events for recharging groundwater. One option is reoperation of Folsom Reservoir to release extra water in advance of significant rain events alone could achieve a potentially significant amount of aquifer recharge using some of the 140,000 ac of croplands in the study area . This work presents a planning-level analysis of what might be possible. While infrastructure construction costs are not considered, the results of this work might encourage further evaluation of necessary investments.A retrospective analysis is conducted to evaluate the range of improvements in groundwater system state that might have occurred for the study area from an Ag-MAR recharge program. Recharge water is from simulated reoperation of Folsom Reservoir with delivery through the Folsom South Canal consistent with capacity limitations over a 20-year period that covers water years 1984 through 2003 . The timing and amounts of surface water delivered to croplands for recharge application is prescribed by a linear programming model that combines available information regarding surface water and groundwater hydrology with the spatial distribution of croplands. Groundwater recharge is simulated with a groundwater/surface-water model that incorporates existing land uses, surface-water deliveries and groundwater demands over the period considered .This analysis applies a formulation of simulation-optimization to MAR.

Previous work includes Mushtaq et al. who simulated unsaturated flow from individual recharge basins and applied nonlinear programming to identify optimal loading schedules for maximizing recharge volume. Marques et al. included decisions for recharge area allocation and water volume application as part of a two stage quadratic programming analysis that maximized crop profits. Hao et al. used a genetic algorithm to maximize recharge volume while meeting constraints on groundwater elevations. To the best of the authors’ knowledge, the approach presented here is new in that it combines elements of recharge basin and groundwater hydraulics with economic considerations at a regional scale. The foundation of the linear programming approach is based on the study area hydrology which is adapted to include economic considerations regarding land use. A hydrologic formulation is presented as an explanatory step in developing the full hydro-economic formulation.The formulation objective, Eq. , maximizes the volume of water recharged over the planning horizon subject to a set of operational constraints. The total volume of water recharged in any period t cannot exceed the water available for recharge , which is derived from a reoperation of Folsom Reservoir to provide additional water during November through March each year. The reoperation is performed by maximizing reservoir releases during the aforereferenced months while maintaining expected levels of service for flood control, water supply and hydropower generation . The levels of service are maintained with a set of optimization constraints that include downstream requirements for minimum environmental flows and water supply as well as the reservoir operation rule curve. The analysis is based on a perfect foresight formulation which provides an upper bound for recharge water available from the reservoir. A static upper bound on the volume of water recharged at a particular location , is based on local infiltration capacity and field berm height through an analytical ponding and drainage model described in the Appendix. Equations and dynamically constrain the magnitude of recharge decisions as a result of a cap on groundwater elevation to avoid water-logging of soil. This constraint is tied to the buildup and redistribution of recharged water as a result of groundwater flow and is described further in the Appendix.

Negative recharge decisions are prevented with Eq. .Cropland area use for recharge as a function of funding is presented in Fig. 10a,b. These are the results of parametric analysis using Eq. . Differences between the results for hydroeconomic analyses and the reference curves occur because, as indicated by the curves for individual crop categories , some of the more expensive land is brought into use before all of the least expensive land has been used. This result is driven by variation in infiltration rate across the study area which is controlled by the shallow geology and the interconnectedness of high conductivity sediments at depth used in the ponding model of Eq. . Figure 11 shows the spatial distribution of land use for two different levels of funding. For low amounts of funding, land is brought into use where there is a combination of cheaper land and higher infiltration rates in an effort to maximize the product of decision variables RA and D. This observation is consistent with the steep slope of recharge volume as a function of funding for land use at low funding levels . Spatial distribution of the recharge water cumulative depth per year is presented for the maximum funding and land use in Fig. 13. The values are generally within a reasonable range based on currently available information on crop inundation tolerance; however, large plastic planting pots constraints could be added to control cumulative water application as necessary. Figure 14 indicates the increase in groundwater storage from recharge using all of the cropland . Recharging over the 20-year planning period used 36% of the WAR . Simulation of the optimal recharge scenario with the groundwater model indicates the most of the water remains in the groundwater system ; however, appreciable amounts exit to surface water or flow across sub-basin boundaries . Additionally,the recharge provides enough base flow to support flow in the Cosumnes River throughout the 20-year simulation except during a 5-year drought from 1987 through 1992. Table 2 presents results for a range of recharge funding levels. Volumes discharging to surface-water and flowing to other sub-basins increase with the volume recharged since head buildup from adding water to the system is more pronounced. Comparison of the recharge volume results from the hydroeconomic analysis for cost set No. 1 with reference curves from the initial capture analysis indicates the effect of including study area hydrogeology in the analysis . High infiltration rate sites are selected preferentially, even when the amount of recharge area is limited by funding, and plot on the left side of the hydro-economic curve. These sites drain quickly and the results plot above the reference curves . Only few such sites are within the footprint of the cropland and, when greater amounts of land are used for recharge, the additional sites drain slower and plot below one or both of the reference curves. The result is a recharge capture curve for the study area that is shallower in slope than the reference curves. Therefore, the spatial variability in infiltration rate magnifies the diminishing returns to scale already occurring as a results of the temporal variability of the water source. More recharge could be achieved, and the study area capture curve moved higher on the plot, if the berm heights around the cropland were increased.

The linear programming results obtained can help develop guidance on where such capital investment might be most valuable. Reformulating the Lagrange multiplier for Eq. in terms of the berm height indicates where and how much additional water could be recharged over the planning horizon if berms were raised from 1–2 ft . This result provides a high estimate of what might be possible since some perennial crops may be unable to accommodate the increased ponding depth; nevertheless, this information provides guidance for where efforts might be best spent increasing berm heights. The values for Lagrange multipliers based on increasing berm height by 1 ft are low in the northern portion of the study area because little cropland is present . Given the high infiltration rates of the deeper geology in the north , recharge potential would be much better for a gravel pit since it would provide additional land area and also penetrate the low hydraulic conductivity soil layer included in this analysis. Cropland present in one of the northern model elements with high-infiltration rate was used to simulate the potential effect of repurposing a gravel pit for recharge. A total of 570 ac in crop categories 2, 3 and 4 were used to simulate gravel pits by increasing the hydraulic conductivity of the soil layer to match the underlying geology and increasing the berm height to 20 ft . Figure 16a,b summarizes the results of gravel pit simulation at the maximum annual funding level. Recharging over the 20-year planning period uses 50% of the WAR . Most of the water remains in the groundwater system with amounts similar to the previously presented results exiting to surface-water and flowing across sub-basin boundaries . Allocation is skewed towards the gravel pits and provides enough base flow to support continuous flow in the Cosumnes River throughout the 20-year simulation including during the previously mentioned 5-year drought. This approach could entail representing cropland managers as individual profit maximizing agents along with the groundwater management agency charging fees for groundwater pumping and providing rebates for recharge. This approach would relax the assumption of uniform land use rents for each crop category and include a more likely dispersion of land use costs across the study area. It is unclear if the aggregate effect of net metering with modest pumping fees would significantly differ from the work presented here since the influence on rational profit maximizers of a net rebate, rather than a payment for using land for recharge, may be similar. However, the effect of net metering combined with a cash flow constraint applied to water management operations could impose limits on a program for improving groundwater system conditions. Given the regulatory requirement for improved groundwater system state, these changes could drive pumping fees higher and influence the behaviors of profit maximizing land managers. It may also be possible to explore improving groundwater conditions through water banking operations where capital investments and operations costs would be paid by a client, or clients, external to the sub-basins. Management policy questions would include: how much water would be left in-place to benefit the groundwater system and the longevity of withdrawal rights . Details of the policy decisions would likely have implications for the amount of infrastructure investment a water banking client might be willing to make. Either the cash flow or water banking approach might be modified to encourage recharge in areas where it is most needed.

Drawing upon Bar-Tal , we further define farmer values as a farmer’s worldview on farming – a set of social values or belief system that a farmer aspires to institute on their farm . In our study, examples of social-ecological mechanisms for farmer knowledge formation among these farmers included direct observation, personal experience, on-farm experimentation, and inherited wisdom from other local farmers. Similar to Boons’ conceptual guide, our results suggest that social-ecological mechanisms may play a central role in producing a farmer’s values and in integrating ecological knowledge into their farm operation. At the same time, results also highlight that social-ecological mechanisms may contribute to a farmer’s local ecological knowledge base, and importantly, place limits on the incorporation of social values in practice on farms. It is possible that social-ecological mechanisms may also provide the lens through which farmer values and ecological knowledge are reevaluated over time. Moreover, farmer values may also mutually inform ecological knowledge – and vice versa – in a dynamic, dialectical process as individual farmers apply their values or ecological knowledge in practice on their farm. Social-ecological mechanisms may also be key in translating abstract information into concrete knowledge among farmers interviewed. For example, experimentation may codify direct observations to generate farmer knowledge that is both concrete and transferable; or, to a lesser degree, personal experience may enhance farmer knowledge and may guide the process of experimentation. In general, large plastic pots for plants we found that farmers interviewed tended to rely less on abstract, “basic” science and more on concrete, “applied” science that is based on their specific local contexts and environment .

This finding underscores that for these farmers, their theory of farming is embedded in their practice of farming, and that these farmers tend to derive theoretical claims from their land.For example, the farmers who possessed a stewardship ethos viewed themselves as caretakers of their land; one farmer described his role as “a liaison between this piece of land and the human environment.” Farmers that self identified as stewards or caretakers of their land tended to rely most heavily on direct observation and personal experience to learn about their local ecosystems and develop their local ecological knowledge. This acquired ecological knowledge in turn directly informed how farmers approached management of their farms and the types of management practices and regimes they applied. That said, farmer values from this study did not always align with farming practices applied day-to-day due to both social and ecological limits of their environment. For example, one farmer, who considered himself a caretaker of his land expressed that cover crops were central to his management regime and that “we’ve underestimated how much benefit we can get from cover crops.” This same farmer admitted he had not been able to grow cover crops the last few seasons due to early rains, the heavy clay present in his soil, and the need to have crops ready for early summer markets. In another example, several of the farmers learned about variations in their soil type by directly observing how soil “behaved” using cover crop growth patterns. These farmers discussed that they learned about patchy locations in their fields, including issues with drainage, prior management history, soil type, and other field characteristics, through observation of cover crop growth in their fields.

Repeated observations over space and time helped to transform disparate observations into formalized knowledge. As observations accumulated over space and time, they informed knowledge formation across scales, from specific features of farmers’ fields to larger ecological patterns and phenomena. More broadly, using cover crop growth patterns to assess soil health and productivity allowed several farmers to make key decisions that influenced the long-term resilience of their farm operation . This specific adaptive management technique was developed independently by several farmers over the course of a decade of farming through long term observation and experimentation – and, at the time, was not codified in mainstream farming guidebooks, policy recommendations, or the scientific literature . For these farmers, growing a cover crop on new land or land with challenging soils is now formally part of their farm management program and central to their soil management. While some of the farmers considered this process “trial and error,” in actuality, all farmers in this study engaged in a structured, iterative process of robust decision-making in the face of constant uncertainty, similar to the process of adaptative management in the natural resource literature . This critical link between farmer knowledge formation and adaptive mangement is important to consider in the broader context of resilience thinking, wherein adaptive management is a tool in the face of shifting climate and changing landscape regimes . The underlying social and ecological mechanisms for farmer knowledge formation discussed here may have a role in informing adaptive management and pathways toward more resilient agriculture . In this sense, farmer knowledge represents an overlooked source for informing innovation in farming alternatively.

Farmer knowledge provides an extension to scientific and policy knowledge bases, in that farmers develop new dimensions of knowledge and alternative ways of thinking about aspects of farming previously unexplored in the scientific literature. Farmers offer a key source of and process for making abstract knowledge more concrete and better grounded in practice, which is at the heart of agriculture that is resilient to increased planetary uncertainties . Most of the farmers considered themselves separate from scientific knowledge production and though scientific knowledge did at times inform their own knowledge production, they still ultimately relied on their own direct observation and personal experiences to inform their knowledge base and make decisions. This finding underscores the importance of embedding theory in practice in alternative agriculture. Without grounding theoretical scientific findings or policy recommendations in practice, whether that be day-to-day practices or long-term management applied, farmers cannot readily incorporate such “outsider” knowledge into their farm operations. Farmers in alternative agriculture thus may provide an important node in the research and policy making process, whereby they assess if scientific findings or policy recommendations may or may not apply to their specific farming context – through direct observation, personal experience, and experimentation.Similar to Sūmane et al. , we found that the process for farmer knowledge formation, or precisely how farmers learn, is systematic and iterative in approach. In this study, farmer ecological knowledge was developed over time based on continuous systematic observation, personal experiences, and/or experimentation. This systematic approach that relies on iterative feedback to learning applied among these organic farmers is akin in approach to examples of adaptive management in agriculture . As highlighted in the results, it is possible for a farmer to acquire expert knowledge even as a first- or second-generation farmer. Documenting this farmer knowledge within the scientific literature – specifically farmer knowledge in the context of relatively new alternative farmers in the US – represents a key way forward for widening agricultural knowledge both in theory and in practice . This study provides one example for documenting this farmer knowledge in a particularly unique site for alternative agriculture. Future studies may expand on this approach in order to document other sites with recent but practical agricultural knowledge on alternative farms.Farmers in this study tended to think holistically about their farm management. For example, when the farmers were asked to talk about soil management specifically, several of the farmers struggled with this format of question, plastic pot plant containers because they expressed that they do not necessarily think about soil management specifically but tend to manage for multiple aspects of their farm ecosystem simultaneously. This result aligns with similar findings from Sūmane et al. across a case study of 10 different farming contexts in Europe, and suggests that farmers tend to have a bird’s eye view of their farming systems. Such an approach allows farmers to make connections across diverse and disparate elements of their farm operation and integrate these connections to both widen and deepen their ecological knowledge base.For most farmers in this study, maintaining ideal soil structure was the foundation for healthy soil. The farmers emphasized that ideal soil structure was delicately maintained by only working ground at appropriate windows of soil moistures. Determining this window of ideal soil moisture represented a learned skill that each individual farmer developed through an iterative learning process.

This knowledge making process was informed by both social mechanisms gained through inherited wisdom and informal conversations and ecological mechanisms through direct observation, personal experiences, and experimentation .As these farmers developed their ecological knowledge of the appropriate windows of soil moisture, their values around soil management often shifted. In this way, over time , farmers in this study learned that no amount of nutrient addition, reduced tillage, cover cropping, or other inputs, could make up for damaged soil structure. Destroying soil structure was relatively easy but had lasting consequences and often took years, in some cases even a decade, to rebuild. This key soil health practice voiced by a majority of farmers interviewed was distinct from messaging about soil health vis-a-vis extension institutions ), where soil health principles focus on keeping ground covered, minimizing soil disturbance, maximizing plant diversity, keeping live roots in the soil, and integrating livestock for holistic management . While these five key principles of soil health were mentioned by farmers and were deemed significant, for most farmers interviewed in this study, the foundation and starting point for good soil health was maintaining appropriate soil structure. The results of this study emphasize that the most successful entry point for engaging farmers around soil health is context specific, informed directly by local knowledge. Among farmers in Yolo County – a significant geographical node of the organic farming movement – soil structure is a prevalent concept; however, in another farming context, this entry point may significantly diverge for social, ecological, economic, or other reasons. Each farming context therefore necessitates careful inquiry and direct conversation with local farmers to determine this entry point for engagement on soil health. For this reason, in some cases it may be more relevant to tailor soil health outreach to the local context rather than applying a one-size-fits all model.The capacity to learn and pass on that learning are essential for farms that practice alternative agriculture to be able to adapt to everchanging social and ecological changes ahead . Across all farmers interviewed, including both first and second-generation farmers, farmers stressed the steep learning curves associated with learning to farm alternatively and/or organically. While these farmers represent a case study for building a successful, organic farm within one generations, the results of this study beg the question: What advancements in farm management and soil management could be possible with multiple generations of farmer knowledge transfer on the same land? Rather than re-learning the ins and outs of farming every generation or two, as new farmers arrive on new land, farmers could have the opportunity to build on existing knowledge from a direct line of farmers before them, and in this way, potentially contribute to breakthroughs in alternative farming. In this sense, moving forward, agriculture in the US has a lot to learn from agroecological farming approaches with a deep multi-generational history . To this end, in most interviews – particularly among older farmers – there was a deep concern over the future of their farm operation beyond their lifetime. Many farmers lamented that no family or individual is slated to take over their farm operation and that all the knowledge they had accumulated would not pass on; there exists a need to fill this gap in knowledge transfer between shifting generations of farmers, safeguard farmer knowledge, and promote adaptations in alternative agriculture into the future. As Calo and others point out, technical knowledge dissemination alone will not resolve this ongoing challenge of farm succession, as larger structural barriers are also at play – most notably, related to land access, transfer, and tenure .Most studies often speak to the scalability of approach or generalizability of the information presented. While aspects of this study are generalizable particularly to similar farming systems, the farmer knowledge presented in this study may or may not be generalizable or scalable to other regions in the US. To access farmer knowledge, relationship building with individual farmers leading up to interviews as well as the in-depth interviews themselves required considerable time and effort.