Category: hydroponic growing

Internal consistency reliability across the items was high . Higher scores reflect higher levels of cumulative risk. We treated cumulative risk as a strictly between-child or time-invariant variable, given the limed within-person variability in cumulative risk over time .Informed by prior studies , we adopted a variety of child and family covariates in an attempt to adjust for potential confounding factors that could bias our estimates. As we describe in detail in the online supporting material, our control covariates included infant gender , race , temperament, primary caregiver reading ability, age at birth, marital status, maternal education, maternal hours worked per week, and research site . These control covariates were measured when the target child was 2 months of age, with the exception of temperament, which was measured at 7 months. Within- and between-person variation in saliva collection times were also controlled in all models.As indicated in prior work , there is little evidence of selective attrition in the sample through early childhood. Missing data were modest. For the cortisol measures missingness ranged between 13% and 26%, the latter representing children’s cortisol levels at 24 months. There was no clear pattern to the missingness; missingnesss was not associated with maternal education, income, or partnered status. Missing data with regard to child care quantity ranged between 3% and 9%. Of those who had a spell of child care , growing bags missing data for estimated caregiver responsivity was 19%, and missing data for those reporting center-based versus non–center-based care ranged from 23% to 31%. To adjust for biases emerging due to missing data, we fitted all models using full information likelihood estimation .

FIML estimation helps to reduce biases to the extent to which missingness is missing at random —that is, conditionally random after adjusting for the observed variables included in the likelihood function . As a robustness check, we fitted an identical taxonomy of models in which only children with 10 or more hours of child care were included in the observed covariance matrix . As the findings were substantively identical and because zero hours represents a “real” value on the childcare hours distribution, we report findings based on the less restricted sample.To address our questions, we fitted a taxonomy of multilevel models . We present a simplified exemplar model in the equations given below for parsimony and ease of explication. Here, the model tests the extent to which the respective within person effects of child care hours, age, and a within-person interaction between child care hours and age varies as function of between-person cumulative risk. We, however, tested identical series of models for non-parental caregiver responsivity and type and peer exposure. In all models, we first tested the main effects of cumulative risk and the given child care predictor. We then added the respective two- and three-way interaction terms to the model in distinct steps. In a final step, we integrated all of the child care predictors into a final model to test their unique relations. Only interaction terms that survived Benjamini-Hochberg corrections were integrated into this final specification. Sometimes called a hybrid model , this multilevel specification is more efficient than typical child-fixed effect models common to the econometrics literature while still retaining the advantages of child-fixed effect models; specifically, the estimates for the Level-1 predictors are identical those obtained using a child-fixed effects specification, such that they account for all potential observed and unobserved time-invariant confounds.

The hybrid approach also affords the ability to simultaneously model between-person relations at Level 2. Importantly, for our purposes, they also allow one to estimate cross-level interactions; specifically, as shown by parameter γ31, we tested the three-way cross-level interaction addressing our questions: Does the within-person effect of child care exposure moderate the association between cumulative risk and cortisol, and does this interactive effect vary as a function of age? As displayed, the model for the residual variances includes only within-person, time-specific residual and a random intercept —implicitly constraining the within-person effects to be identical across children. This parsimonious specification is tested and relaxed, as appropriate.Child care quantity—As displayed in Column A of Table 2, preliminary main effect models indicated that, when considered in aggregate, within-child changes in child care hours were not associated with contemporaneous changes in children’s cortisol levels. However, as evidenced by the statistically significant three-way interaction in Column B , this null main effect was explained by the fact that direction of the within-person child care effect was conditional on level of cumulative environmental risk experienced by the child, as well as child age. We display these conditional relations in Panels A through C in Figure 1; the values of all other predictors in the model were held at their respective means. Irrespective of cumulative environmental risk, no within-person child care effects were evident at 7 or 15 months of age; none of the displayed simple slopes for child care hours differed statistically from zero. Instead, the interactive effect grew over time, becoming more pronounced and statistically significant by 24 months of age. For children from high-risk contexts, increases in child care exposure at 24 months were associated with contemporaneous decreases in children’s resting cortisol levels. In contrast, for children from low-risk contexts, increases in children’s child care exposure at 24 months were associated with contemporaneous increases in children’s resting cortisol levels Tests of model constraints indicated that the absolute magnitude of the simple slopes displayed in Panel C of Figure 1 were statistically identical . In absolute terms, these relations corresponded to absolute standardized simple slopes of approximately .

Albeit somewhat modest, it is worth noting that in relative terms these simple slopes are approximately two thirds the size of the standardized within-person effect for time of day —one of the most well-understood biological mechanisms underlying normative within-person variation in cortisol. Between-child differences in children’s average weekly child care hours were predictive of only children’s cortisol growth rates , and neither this relation nor the relation between average child care hours and children’s 7-month cortisol levels were moderated by children’s level of cumulative environmental risk. Children who attended greater hours in child care on average tended to show less negative declines in their cortisol levels over, irrespective level of cumulative environmental risk . We display this relation in Figure 2 for prototypical children experiencing low versus high levels of child care exposure per week on average across infancy and toddlerhood; all other predictors are held constant at their respective means. Whereas children attending 40 hours of child care per week were estimated to maintain their 7-month cortisol levels over time , those attending 10 hours a week were predicted to show a statistically significant decline their cortisol levels between 7 and 24 months of age . Note that this decline was rather modest, approximating a 0.02-SD decrease, per month. Child care type—There was no evidence of a within-person effect with respect to child care type ; that is, nursery grow bag time-specific shifts to center-based or non–center-based child care were not associated with contemporaneous changes in cortisol. There was, however, some evidence that the positive between–person relation between attending comparatively more center-based care between 7 and 24 months and children’s cortisol levels differed for children from higher versus lower levels of cumulative risk . This two-way interaction did not vary as a function of age—that is, it was not predictive of cortisol growth. There was, however, evidence that attending greater proportions of time in center-based child care was predictive of more rapid cortisol decreases over this period . In the former case of the interaction, it is also worth noting that the raw pvalue for this estimate, .01, dropped to marginal levels of statistical significance after adjusting family-wise false discovery rate. We nonetheless display the simple slopes derived from this interaction in Figure 3, with this caveat in mind.As shown in Figure 3, we found evidence that, for children experiencing low levels of environmental risk, those who attended comparatively greater proportions of their time in center-based child care tended to have higher cortisol levels . This is most clearly illustrated by considering the vertical distance between the dashed and non dashed gray trajectories at 7 months in Figure 3; it reflects the 7-month simple slope for those attending 100% of their time in child care in center-based care, compared to those who spent 100% of their time in non–center-based child care. This conditional relation is noteworthy, approximating a standardized association of .41. In contrast, child care type was unrelated to children’s 7-month cortisol levels, for children from high-risk contexts .

As indicated by the divergence of the dashed and non dashed black trajectories over time, there was a descriptive indication that, for those from high-risk contexts, attending predominantly non–center-based care was predictive of comparatively less rapid cortisol declines between 7 and 24 months. However, the conditional effect of child care type at 24 months only approached statistical significance . Considered another way, despite the descriptive differences in the estimated cortisol values plotted at 24 months of age in Figure 3, these values were statistically identical. non-parental caregiver responsivity—Within-person changes in caregiver responsivity between 7 and 24 months were not associated with contemporaneous changes in children’s cortisol levels, irrespective of child age or level of cumulative risk. Similarly null relations were evident with respect to between-person differences in children’s average levels of caregiver sensitivity across this period. Peer exposure—Within-child changes in peer exposure were not associated with changes in children’s cortisol levels over time. However, there was some indication that between-child differences in children’s average levels peer exposure were predictive of children’s overall cortisol levels. Specifically, on average, children who typically attended child care with greater numbers of peers tended to evince lower cortisol levels . This corresponded to a moderate standardized coefficient of approximately. Adjusting for false-discovery rate, this relation did not vary over time or as a function of children’s levels of cumulative risk.There is good evidence that children’s child care experiences affect HPA axis functioning . However, it is increasingly clear that these relations may be quite different for children growing up in high-risk environmental contexts. The aims of the present study were twofold. The first aim was to test the extent to which within- and between-child differences in children’s child care experiences—quantity, type, caregiver responsivity, and peer exposure —across infancy and toddlerhood were associated with children’s cortisol levels over this period. The second aim was to consider whether these relations differed for children experiencing higher levels cumulative contextual risk outside of child care.Our results provide some support for such interactive effects. As hypothesized, for children from low-risk contexts, within-child increases in weekly child care hours were associated with contemporaneous increases in children’s resting cortisol levels. The inverse was evident for children from high-risk contexts; increases in weekly child care hours were associated with contemporaneous cortisol decreases. The magnitude of this cross-level interaction strengthened over time, reaching statistical significant between the ages of 15 and 24 months. Interactive effects between cumulative risk and attending center-based care were also evident; however, they extended to only between-child differences in overall centerbased exposure between 7 and 24 months of age . Controlling for child care hours, caregiver responsivity, and peer exposure, children from low-risk families who spent greater proportions of their time in center-based child care tended to have higher cortisol levels than their peers who attended more non– center-based childcare. In contrast, for children from high-risk contexts attending center based care was unassociated with children’s cortisol levels. With respect to children’s exposure to peers in child care, there was evidence that children exposed to greater numbers of peers between 7 and 24 months tended to show comparatively lower cortisol levels. This relation did not vary as a function of cumulative risk or age. Within-person changes in peer exposure were unrelated to changes in cortisol, irrespective of age or level of cumulative risk.Prior work conducted with lower risk samples has shown that children’s cortisol levels tend to increase over the course of the day on days when they attend child care yet show normative diurnal declines on days in which they do not attend child care . Nontrivial differences between this prior literature and the present study make direct comparisons impossible.

Since birds do not eat coffee cherries, bio-control by birds would only occur during the brief dispersal period when CBB are vulnerable. There is a rich bird community during this period of time as both resident and migratory birds are present . Neotropical migrants are potentially more abundant on coffee farms than resident species that may prefer forest habitat due to higher prey abundances . Many migratory warbler species of the Setophaga genus that frequent coffee farms have been confirmed as CBB predators, as have resident bird species such as the rufous-capped warbler and common tody flycatcher   This positive density-dependent relationship between population growth and density is an Allee effect , and escape from predation is one mechanism for this phenomenon . In general, predator-driven Allee effects can occur when predators are the main driver of prey dynamics and when predators are generalists as are insectivorous Neotropical migrants . Additionally, predators can exert strong pressure when prey availability is not temporally or spatially limited—a potential limiting factor in the coffee system, since CBB are only available to birds during dispersal. The degree to which birds exert an Allee effect on CBB might depend on the starting population size of the pest. Variation in starting population size is likely dependent on how recently CBB have colonized in an area, timing of trapping , the size of the farm , and the extent to which farmers used control measures the previous year . We found that only under very low initial population sizes of CBB could birds be expected to suppress pest numbers by 50%. We note that earlier, nursery pots stronger CBB suppression by birds would lead to lower infestation numbers later in the coffee season, but this might require selective foraging by birds, depending on relative abundances of other prey species.

In conclusion, our models suggest that birds can control CBB under some circumstances, depending on the relative size of the starting CBB population and existing local bird density. To put this idea into practice it is important to remember that managing farms for bird habitat does not always result in pest reduction. Birds may not prey on the pest of interest or birds might cause pest numbers in increase by preying on insect predators that normally regulates the pest population . Aside from predators, pest species are also impacted by the agricultural environment directly . In fact, on coffee farms where bird densities are higher in shade, CBB infestations are also higher , possibly because CBB native range is in humid, shade forests of Africa . It is important that future modeling include such habitat-specific factors to understand Our research helps quantify the densities under which birds have the potential to control CBB populations. Putting these numbers into practice will require understanding how management practices affect both bird and CBB densities.Synsepalum Daniell and Englerophytum K. Krause are two closely related genera of the sub-family Chrysophylloideae in the family Sapotaceae. These two genera comprise 35 and 19 recognized species respectively and are predominantly distributed across West-Central tropical Africa. Both genera share the frequent presence of stipules, usually 5-merous flower with the irregular presence of small staminodes, similar seeds, and embryos. They are however considered to be different genera due to the consistent striate brochidodromous venation and strong fusion of the filaments into a staminal tube found in species of Englerophytum, whereas in species of Synsepalum leaf venation tends to be eucamptodromous and the filaments are free. In their study of the SynsepalumEnglerophytum complex, reported six lineages from combined data from nuclear DNA, chloroplast DNA and morphology analyzed using parsimony. The views of [2] are however different from the previously obtained results from in which the two genera formed a single heterogeneous clade where species of Synsepalum genus were grouped within species of Englerophytum. The differences between the two genera are a call for concernas the decision to either merge the two genera or separate them is yet to be reached.

Also, [5] in their multi-gene phylogeny study, found support for the monophyly of the Synsepalum–Englerophytum clade but did not sample either genus extensively, only very limited sampling of the two genera.Synsepalum has undergone several taxonomic changes throughout history as new species have been discovered. It is comprised of trees and shrubs native to tropical lowland areas of Africa. It was described in 1852 and currently consists of about 35 species, including the very popular miracle berry plant, S. dulcificum Daniell which is the type species on which the genus is based. Like the genus Diploon Cronquist, Synsepalum has glabrous staminodes and imbricate to valvate corolla lobes. A very common feature in the genus is their fused sepals, a character that gave the name to the genus. Synsepalum can also be characterized by its long spreading corolla lobes and large stipules. The current 35 recognized species in the genus are a combination of species from previously recognized smaller genera, including Afrosersalisia A.Chev., Pachystela Radlk, Vincentella Pierre, Synsepalum, and Tulestea Aubrév. & Pellegr. Previous generic classifications were considered unsatisfactory and therefore the genera were united under Synsepalum. The small genera were merged using overlapping characters to form the currently recognized genus. The combination of the following characters was used to describe Synsepalum: frequent occurrence of large stipules, eucamptodromous venation, 5-merous flowers, corolla nearly always rotate, cyathiform or shortly tubular with wide-spreading lobes, corolla lobe aestivation imbricate or induplicate valvate, stamens fixed at or near the top of the corolla tube, exserted with well-developed filaments. The seed is broad and not laterally compressed, with a broad adaxial scar that sometimes extends to cover most of the surface. The embryo has plano-convex cotyledons and endosperm is known to be generally absent in the genus. Due to the inconsistency in the characters of the small genera that were merged, species in the genus are often individually very distinct.

This has complicated the taxonomic revision of the genus and caused many synonyms to have emerged. With the emergence of molecular technique, the lumping of these genera to form the genus Synsepalum has been disputed by many authors as the conclusion was purely based on morphological characters.Englerophytum K. Krause was described as a genus, with Englerophytum stelechanthum as the type species. Five species were added to the genus, two of which were newly described while the other three were products of new combinations of species previously classified in different genera. As opposed to the views of [11], who advocated for the distinct status of the genera Englerophytum, Wildemaniodoxa Aubrév. & Pellegr. and Zeyherella Aubrév. & Pellegr, united the genera based on the fusion of their filaments and the number of floral parts. Although considered Synsepalum to be closely related to Englerophytum because of the frequent presence of stipules, usually 5-merous flowers, irregular presence of small staminodes, and similar structure of seeds and embryo, he considered Synsepalum distinct genus from Englerophytum.The Sapotaceae classification of was purely morphological as that was the standard used then in reaching taxonomic conclusions. Nuclear DNA and plastid trnH-psbA were used by to estimate phylogeny within the Synsepalum–Englerophytum clade. Their results do not support the classification by Pennington, and the species of the two focal genera of this study that they analyzed were resolved in a polytomy of six clades: two comprising the species of Englerophytum and four of Synsepalum. However, plastic planters their result cannot be considered final due to incomplete sampling, as only 11 out of the 35 accepted species of Synsepalum and 8 out of the 19 species of Englerophytum were used for the study. They also recommended that more work is required before a comprehensive taxonomic conclusion about the clade can be reached. Aside from the work of [2], there are no published reports on phylogenetic relationships within the Synsepalum-Englerophytum clade. In their studies of the SynsepalumEnglerophytum complex, reported that four of the six lineages comprised Synsepalum species, and three out of the four lineages of Synsepalum corresponded to the smaller genera of the earlier generic classification by [9]. There are, however, some concerns with the lineages reported. Some of the lineages had just a single species, which was not the type species of the small, segregated genus . More species need to be investigated to better understand the phylogenetic relationships among species currently classified in Synsepalum and Englerophytum and to determine the number, names, and circumscriptions of genera that should be recognized in a phylogenetically based classification.In general, plant phylogenetic studies provide a framework for understanding the fundamental processes of evolution and help in organizing the diverse plants of the earth in a way that will make sense to all. In the genus Synsepalum, although the presence of stipules and 5-merous flowers has been suggested as diagnostic characters for the genus, the presence of stipules is not consistent. They are missing in some species, these may represent secondary losses, however. Phylogenetic analyses based on molecular data should make it possible to evaluate relationships among species in the group and compare them with the ancient generic concept. Moreover, not much has been done in resolving the divergent views of researchers on the merging of the small genera by Pennington to form Synsepalum sensu lato. This research proffers a solution to taxonomic problems in the Synsepalum–Eglerophytum complex.

It is generally believed that fresh materials from the field are more reliable for DNA extraction but due to the outbreak of the Covid-19 pandemic, getting to the field to sample materials was not an option to be explored for this study. Thus, materials for both Synsepalum and Engleropytum were mostly accessed from herbarium material. Materials were obtained as loans through the University of California Davis Herbarium . Samples were collected from Missouri Botanical Gardens , New York Botanical Gardens , Harvard University Herbarium and The Conservatory and Botanical Garden of the city of Geneva . A few other samples were collected in silica gel from people who grow them in their gardens. Leaf material sufficient for use in extracting DNA was removed from the herbarium samples.To avoid the destructive removal of leaf samples, leaves already placed in the fragmented packet in the herbarium sheets were first used. Where there were no leaves in the fragment packet, a single leaf was removed and used for the experiment. A total of 103 leaf samples were used for this study, comprising 43 from different herbaria in the United States , 56 from Switzerland and France , and four were fresh samples, see Supplementary Materials, Table S1.The amplified fragments for both regions were controlled for their quality by electrophoresis. 1.8 g of powdered agarose gel was added to 100 mL of 1X TAE buffer. The mixture was shaken vigorously to ensure the agarose gel was completely immersed in the 1X TAE buffer. After heating, 1 µL of Sybrsafe DNA gel stain is added to the beaker containing the agarose gel, which is placed in a bath containing water for a few seconds until the beaker is cool enough to be handled with the hand using hand gloves. The gel solution was poured into a tray fitted with combs and allowed to stay for 20 min until it solidified. After solidification, the comb is removed, and the wells are loaded with PCR. The chamber containing the loaded DNA is connected to power at 76 KVA and allowed to run for 1 h. The gel is then visualized under UV light. Wells that produce bands are considered successful. The bands are excised using a razor blade. DNA was extracted from the bands and purified by application of a QIA quick PCR purification kit from Qiagen .To obtain DNA sequences, extracted purified DNA from the gel was sent to the UC Davis sequencing center. For each direction of the primer, six micro-liters were used. Raw data from the facility were opened on Sequencher 5.4.6 which was used to assemble contigs and edit the sequences. The first nucleotides of each end of the sequences were trimmed until readable bases were obtained. After trimming up the sequences, BLAST searches were performed to ensure the results obtained were that of Sapotaceae. In cases of contamination, blast results give different plant families and in some cases insects. Whenever contamination was observed the experiment was repeated to be sure the right species was used for the research. Alignment was done using muscle in MEGA X. For the GenBank codes of sequences used in our phylogenetic analyses, see Supplementary Materials, Table S2.The evolutionary history was inferred using the Bayesian Inference and Maximum Likelihood methods.

Analogous to exchanging charges between Gaussian surfaces, changing the topological invariant necessarily requires two sets of Brillouin zone wave functions or equivalently, two bands to touch and exchange quanta of the invariant. Thus, the system becomes gapless as it is tuned across a topological phase transition. Table 1.1 contains a list of analogies between Gauss’s law in electrostatics and topological phenomena in non-interacting band structures. An important consequence of the gaplessness of band structures as they are tuned across a topological phase transition is the existence of protected gapless states on the surface of a topological insulator or superconductor. If a topological insulator or superconductor is placed next to a topologically trivial one, including vacuum, the phases on the two sides of the interface are topologically distinct. Thus, gapless states exist at the interface and survive in the presence of weak perturbations as long as certain basic symmetries of the system are preserved by the perturbations. The number of such protected surface states is intimately tied to the value of the bulk topological invariant. These surface states are very important from a practical point of view as well as they are more easily accessible to many experimental probes than the bulk states and hence, help in identifying the topology of the bulk bands without requiring the bulk bands to be probed directly. Moreover, the surface states fully determine the low energy physics in the presence of a bulk gap and hence, are the only ones relevant for transport. And finally, black flower buckets the surface states of many topological phases display various unusual phenomena that ordinary quasiparticle states do not.

As mentioned earlier, the precise form of the topological invariant in terms of the Bloch functions depends on the symmetry properties of the system of interest. Table 1.2 lists the classes of non-interacting Hamiltonians classified using their symmetry properties, and the sets of topologically distinct gapped phases each class of Hamiltonians may have in one, two and three spatial dimensions. The only physical symmetries used in the classification process are time-reversal symmetry and particle-hole symmetry , since these are the only ones generic to random, disordered systems. Thus, crystal symmetries, which are in general absent in disordered systems, are not considered. The sublattice symmetry in Table 1.2 is defined as the product of TRS and PHS. The ten classes are obtained as follows: both time-reversal and particle-hole conjugation are anti-unitary operations and hence, can either be absent, or be present and square to either +1 or −1. This gives 9 classes of Hamiltonians. The tenth class, AIII is one where both TRS and PHS are broken but their product, the SLS, is preserved.The second way in which topology appears in band Hamiltonians is in the form of topological defects in momentum space. These defects occur at points where the band structure becomes gapless and are analogous to real space topological defects such as domain walls, vortices and hedgehogs, where an order parameter has a non-trivial winding around the defect and vanishes at the defect. Like real space topological defects, topological defects in momentum space can be combined by moving them in momentum space while keeping their total topological quantum number conserved. On a finite system, band structure singularities result in peculiar surface states. Whereas gapped topological phases carry dispersing surface states, gapless topological phases host dispersionless or flat bands on their surface. The flatness of these bands is protected by the topological nature of the bulk band structure and cannot be removed as long as the topological objects in the bulk band structure survive.

One crucial manner in which the topological nature of gapless band structures is different from that of gapped ones is that the former is not immune arbitrary disorder. Since the topological entities occur in momentum space, it is vital that momentum be a good quantum number. Thus, perturbations that break translational symmetry can potentially gap out the topological defects. We now briefly review two common instances of band structure singularities and their associated flat surface bands.The Fermi surface of a Weyl semimetal on a slab consists of unusual states known as Fermi arcs. The Fermi arcs are like a two dimensional Fermi surface, except that the two dimensional Fermi surface is broken into two parts and one part is localized on the top surface while the other is localized on the bottom surface. On each surface, the Fermi arc connects the projection of the bulk Weyl nodes onto the surface, as shown in Figure 1.2.2. A simple way to understand the presence of Fermi arcs is by recalling that the Weyl points are sources of Chern flux. Each two dimensional slice in momentum space perpendicular to the line joining the Weyl nodes can be thought of as a Chern insulator, and the Chern numbers of two slices on either side of a Weyl node differ by one. Thus, if the slices in the region far away from the Weyl nodes have Chern number 0, the Chern number of the slices between the Weyl nodes must be 1 and so on. The Fermi arcs, then, are simply the edge states of the Chern insulators. In summary, there are two broad ways in which topology manifests itself in the spectra of non-interacting band Hamiltonians. For gapped Hamiltonians, a topological phase exists when the Bloch wave functions of the occupied bands form non-trivial topological textures across the Brillouin zone. The exact class of textures that is stable against perturbations depends on the symmetries and the dimensionality of the system. Gapless Hamiltonians, on the other hand, can be characterized by topological invariants by focusing only on the states near the gapless region. The latter manifestation of topology in band structures is, in some sense, less stable than the former because it requires translational invariance and is thus notoblivious to disorder. Both classes of topological phases are associated with unconventional states on the surface of a finite system – dispersing Dirac, Weyl or Majorana modes for gapped phases and flat bands for gapless phases.

In the next chapter, the physics of a famous gapped topological phase – the strong topological insulator – will be briefly introduced and reviewed. Some novel phenomena associated with this phase as well as some material realizations will be mentioned.In the last half of a decade, the buzzword that has perhaps stolen the greatest amount of limelight in condensed matter physics is topological insulators. Two examples of insulators with non-trivial band topology, hence topological insulators according to the definition, were touched upon in the previous chapter. This section does a brief review of the phase that is colloquially called the topological insulator and is the one that has actually captured most of the attention. This is the three-dimensional insulator with time-reversal symmetry – class AII according to Table 1.2 – which has a non-trivial Z2 topological invariant. Henceforth, unless otherwise mentioned, the term ‘topological insulator’ will be used to refer to this particular realization of an insulating topological phase. It is closely related to the quantum spin Hall state; thus, the latter is often also termed the ‘two-dimensional topological insulator’.Having reviewed the main ideas in band structure topology and the basics of topological insulators, we now investigate other possible gapped phases of band Hamiltonians in three dimensions. A convenient starting point is a three Dirac dispersion, french flower bucket since it is proximate to a variety of orders, which when established, lead to an energy gap. In the context of graphene, charge density wave and valence bond solid order as well as antiferromagnetism are known to induce a gap, and lead to an insulating state. Several years back Haldane pointed out that the integer quantum Hall state could be realized starting from the graphene semimetal, in the absence of external magnetic fields. A valuable outcome of this Dirac proximity approach, was the discovery of an entirely new phase of matter, the Z2 Quantum Spin Hall insulator, obtained in theory by perturbing the graphene Dirac dispersion. By analogy, here we study three dimensional Dirac fermions, and their proximate gapped phases, on a cubic lattice. In three dimensions, Dirac points naturally occur in some heavy materials like bismuth and antimony, with strong spin orbit interactions. A three dimensional version of the quantum spin Hall state – the Z2 topological insulator , can be realized by appropriately perturbing such a state, as demonstrated in [38], in a toy model on the diamond lattice. According to recent experiments, this phase is believed to be realized by several Bibased materials including Bi0.9Sb0.1, Bi2Se3 and Bi2Te3. Both the Z2 quantum spin Hall and the Z2 topological insulator phases require time-reversal symmetry to be preserved. The Z2 index represents the fact that only an odd number of edge or surface Dirac nodes are stable in these phases. In contrast, in this chapter we study a toy model on the cubic lattice, with π flux through the faces, which realizes three dimensional Dirac fermions, and identify the proximate states.

To begin with, we consider insulating phases of spin polarized electrons. In addition to conventional insulators, e.g., with charge or bond order, we also find an additional novel topological insulator phase within this model, the chiral Topological Insulator . This provides a concrete realization of this phase, which was recently predicted on the basis of a general topological classification of three dimensional insulators in different symmetry classes. This phase is distinct from the spin-orbit Z2 topological insulators in two main respects. First, it is realized in the absence of time-reversal symmetry. Instead, it relies on another discrete symmetry called the chiral symmetry. Second, these insulators also host protected Dirac nodes at their surface, but any integer number of Dirac nodes is stable on its surface. Thus, it has a Z rather than Z2 character. In an insulator, chiral symmetry restricts us to Hamiltonians with only hopping terms between opposite sublattices. Clearly, this is not a physical symmetry, and hence such insulators are less robust than topological insulators protected by time reversal symmetry. However our results will be relevant if such symmetry breaking terms are weak, or in engineered band structures in lattice cold atom systems . With spin, an interesting gapped state that can be reached from the Dirac limit is the singlet topological superconductor , also first discussed in [119]. This state also possesses protected Dirac surface states. The stability of these states is guaranteed, as long as time reversal symmetry and SU spin symmetry, both physical symmetries, are preserved. The Dirac limit allows for an easy calculation of the charge response of the cTI , and provides an intuitive picture of these phases. For example, the cTI can be understood as arising from a quasi 2D limit of layered Dirac semi-metals, with a particular pattern of node pairings, leading to a bulk gap, but protected surface states. It is hoped that this intuition will help in the search for realistic examples of these phases. Additionally, this picture helps in understanding Z2 topological insulators protected by TRS whose bulk Dirac nodes are at time-reversal-invariant momenta , such as Bi2Se3, Bi2Te3 and Sb2Se3. Finally, we utilize the Dirac starting point to derive relations between different gapped phases. We show that there is a duality between Neel and VBS phases: point defects of the Neel order are found to carry quantum numbers of the VBS state and vice versa. This is done by studying the midgap states induced by these defects, and the results agree with spin model calculations that are appropriate deep in the insulating limit. Thus, the Dirac approach is a convenient way to capture universal properties of the gapped phases in its vicinity. These results are also derived following a technique applied to the one and two dimensional cases, by integrating out the Dirac fermions and deriving an effective action for a set of orders. In particular we focus on the Berry’s phase term which, when present, implies non-trivial quantum interference between them. Such sets of ‘quantum competing’ orders can be readily identified within this formalism. We show that in addition to Neel and VBS orders, interestingly, Neel order and the singlet topological superconductor also share such a relation.

There is precedent in the human cognitive neuroscience literature suggesting noisier representations in aging underlie performance decrements. Using fMRI, Samanez-Larkin et al. revealed that in older adults, greater temporal variability in nucleus accumbens BOLD response was directly related with suboptimal performance on a financial risk-taking task. Variability has been shown to be reproducible within subject across time and tasks , and has been linked to individual differences in dopamine . In aging, declines in D1 receptors have been associated with increased intraindividual variability in reaction times during an executive function task . Generally, there is consensus that intraindividual variability in neural and behavioral responses is a valuable measure for understanding the underlying neural basis of impaired performance , though it is important to note that in some cases neural variability may be beneficial . One central component of this line of research will be to distinguish between variability that is driven by imprecision in signaling versus variability that simply reflects a healthy dynamic range in neural responses that are not muted by, for example, disease processes. Important research by Garrett, Kovacevic, McIntosh, and Grady has demonstrated that, overall, procona buckets older adults show less variability in BOLD signal than younger adults, and that this reduced variability is associated with poorer cognitive performance.

Interestingly, they found subcortical structures including hippocampus and regions in the striatum that were more variable in older adults than young adults . Together, these findings suggest variability in subcortical responses may be an important, age-sensitive measure in human imaging studies that warrants further investigation to establish possible relationships with dopamine. For example, future studies could test whether individuals with highly variable cortical responses also have highly variable subcortical response, or whether these are dissociable measures potentially reflecting different underlying sources.PET imaging provides spatial information that allows for the assessment of regional differences in dopamine function. This provides unique opportunities to test hypotheses about the differential influence of region-specific measures of dopamine on discrete cognitive operations. For example, PET can be useful for specifying a role of PFC dopamine signaling in decision-making. Spatial information may be particularly relevant for studying dopaminergic mechanisms of decision-making in aging as there is growing evidence that dopamine receptor densities may decline at different rates across the brain . Here, we describe ways in which the spatial information afforded by PET imaging can be leveraged to probe the role of dopamine in agerelated changes in reliance on striatal versus extrastriatal brain regions during decision making. Evolving research has developed our understanding of complementary learning mechanisms that may trade off or interact with model-free reinforcement learning processes to affect decision-making.

These include, but are not limited to, processes for the building of deliberative internal models to guide choices , as well as processes for learning and planning that more heavily rely on prefrontal processes including working memory or medial temporal lobe episodic memory . The degree to which these processes interact, and the nature of these interactions is an area of active research . However, the multiprocess view of decision-making incorporates roles for multiple neural systems that include frontoparietal, medial temporal lobe, and limbic structures. There is general agreement that most value-based decision-making tasks accommodate multiple strategies. In some cases, these strategies can be distinguished from one another using computational modeling approaches . These lines of research have revealed profound individual differences in the extent to which people adopt one strategy over another. Age differences in the adoption of task strategy has been identified as a critical factor in considering discrepant findings in the aging literature. For example, older adults may show better or worse performance than young adults depending on whether a given task favors win-stay lose-shift strategies . Research examining how reliance on specific strategies is affected by regional differences in dopamine function is only beginning, but holds promise for informing findings in aging. Influential learning models have distinguished between model-free processes and model based processes, which can be dissociated from one another computationally using the two state Markov decision task . Briefly, this task involves two decision phases . In the first step, participants choose between two stimuli, and this selection determines a second set of choices with differing reward probabilities. Similar to single phase tasks, learning can occur slowly and incrementally via model-free mechanisms. Task performance may also rely on goal-directed, model-based strategies, for which subjects develop an internal model of the task structure. At the first decision phase, subjects may prospectively consider future reward probabilities that would occur after the second phase.

This strategy is more computationally demanding than model-free strategies, but supports rapid and flexible learning. While links between model-free reinforcement learning and striatal dopamine have been long established, model-based strategies are also powerfully modulated by dopamine. Pharmacological manipulations provide general evidence that elevating dopamine tone increases reliance on model-based processes, though do not give information about the spatial specificity of dopamine’s role. Enhancing dopamine in young adults shifts bias in task strategy toward model-based processes . Consistent with this, Parkinson’s disease patients tested off medication show selective impairment in model-based learning that is remediated by dopaminergic medication . Complementing these findings in Parkinson’s patients, model-based processing is reduced in people with disorders characterized by alteration in dopamine function such as addiction , obsessive compulsive disorder , and schizophrenia . To date, there has been only limited investigation of the spatial specificity of dopamine’s influence on model-based learning. Dopamine may modulate PFC via direct inputs from the ventral tegmental area or by indirect effects in striatum where dopamine affects PFC function via fronto-striato-thalamic loops . Pointing to a role of striatal dopamine, PET evidence in healthy young subjects demonstrated greater striatal dopamine synthesis capacity was associated with preferential reliance on model-based learning and was correlated with fMRI activation in PFC . Pointing to a role of PFC dopamine, people with genetically inferred reductions in COMT enzymatic activity show greater reliance on model-based processes . Additional research is needed to understand how direct dopamine signaling in PFC may influence model-based strategies.Though model-based processing has been linked to striatal activity , it is also associated with increased reliance on prefrontal systems. Supporting this view, higher working memory capacity is associated with greater propensity to engage model-based strategies . Further, manipulations that increase cognitive load , stress , or perturb PFC function via rTMS reduce reliance on model-based processing, particularly in participants with low working memory capacity. In aging, there is evidence of reduced reliance on model-based processing . These findings are generally consistent with age-related changes in PFC function though, somewhat perplexingly, propensity to adopt model-based rather than model-free appears to be dissociated from working memory capacity . Other studies have identified subgroups of older adults demonstrating an over reliance on model-free learning . However, the extent to which age-related shifts from model-based to model-free learning mechanisms are explained by alterations in PFC dopamine function is unknown. Accounting for alteration in dopamine function in PFC and striatum may be valuable for understanding the neural basis of shifts away from model-based processes in aging. In aging, loss of PFC D2/3 dopamine receptors may outpace losses in ventral striatum . Further, rates of D1 receptor losses may differ between nigrostriatal pathways that innervate dorsal striatum versus mesocortical/mesolimbic pathways that innervate PFC and ventral aspects of striatum, respectively . Together, such changes may alter the weighting of PFC versus striatal influences on task performance and the coherence of dorsal versus ventral striatal contributions to processes for learning, procona florida container updating and integration that support value-based decision-making. Future studies pairing task performance with neurochemical PET measures could directly test how individual differences in receptor densities and dopamine release in PFC versus ventral striatum influence variability on the adoption of model-based versus model-free strategies in aging.

Such studies may identify those subgroups of older adults most reliant on model-free processes are those with most marked reductions in PFC dopamine measures. Such lines of research are ripe for cross-species comparisons which could assess relationships between age and performance while providing critical information as to the temporal dynamics of dopamine signaling in PFC versus striatum using microdialysis techniques and voltammetry. Together, such studies hold promise for promoting our basic understanding of PFC dopamine’s involvement in goal-directed decision-making while characterizing age differences in the recruitment of distinct dopamine pathways underlying specific task strategies.Models of complex decision-making support the view that multiple systems for learning, memory, and attention interact to shape our choices. We have argued that in vivo dopamine imaging is a powerful tool for understanding the neural basis of individual differences in performance in aging. This line of research can illuminate the extent to which neurochemical traits affect decision-making. However, it is important to also consider how neurochemical influences on behavior interact with other attentional or state measures to affect decision making. A recent study by Kircanski et al. illustrates the mutual influence of these factors on value-based choices. First, they modified the monetary incentive delay task by including trials in which participants unexpectedly gained or lost relatively large amounts of money . They found monetary loss and gain modifications induced negative and positive affective arousal respectively, which they assessed with self-report rating scales. Following the affective arousal manipulation, participants performed a separate decision-making task in which they chose whether or not to purchase items with misleading advertisements. They found deficits in choice performance following both arousal conditions relative to a neutral condition. Together, these findings demonstrate the capacity for financial reward and punishment manipulations to impact self-reported affective state, which in turn modulates the quality of subsequent value-based decisions. This study found no evidence for differences in the induction of arousal between young and older adults or in the detrimental effect of arousal on later choice performance. Other studies have shown differential age effects such that following positive affect induction , older adults, but not young adults, are more likely to make risky decisions than when in neutral states . In the following section, we describe possible systematic changes in affective attention over the life span that may influence decision-making performance. We consider how these attentional changes may interact with influences of dopamine on performance, and suggest strategies for empirically examining these interactions. Older adults report reduced levels of negative affective experience as they age, but preserved levels of positive experience . These observations have been linked specifically to memory and attention where older adults are more likely to remember positive events than young adults . It is possible that there are age-related changes in neurophysiology and neurochemistry that drive these effects, though there has been little direct investigation of this possibility model first described by Mather, Clewett, Sakaki, & Harley, 2016). Functionally, there appears to be relative preservation of the networks supporting emotional processing in aging , though in some cases the engagement of these networks by older adults may be more effortful . The dominant interpretation is that at the end of life there is a shift in motivational goals derived from changes in the perception of time horizons . This view is supported by evidence that positivity effects have been reported in young people diagnosed with terminal illness, and people on death row, where systematic neurophysiologic and neurochemical changes mirroring those that occur in aging are unlikely . Further, positivity can be enhanced in young subjects experimentally in task scenarios in which they are encouraged to think about a limited future . The positivity effect has been implicated in fMRI findings that responses associated with reward anticipation are intact in aging, but that responses associated with the anticipation of monetary losses in the insula are muted in older adults . Other studies have examined relationships between subjective ratings of emotional stimuli and fMRI responses in PFC and amygdala. Behaviorally, older adults report lower arousal for negative stimuli than young adults , report less unpleasantness for “lowarousal” negative stimuli, and greater pleasantness for “low-arousal” positive stimuli in a study that manipulated the levels of stimulus valence . These behavioral differences are accompanied by age-group differences in amygdala responsivity. Older adults consistently show greater enhancement of amygdala activation for positive relative to negative stimuli . Further, there is evidence that the relative suppression of amygdala activation for negatively valenced stimuli in aging is associated with greater recruitment of rostral anterior cingulate cortex when viewing unpleasant stimuli .

Our metagenomic data analysis predicts genes in GB-CYN1 involved in the Calvin cycle, TCA cycle, and photosystems I and II . Sequence identities for these genes in GB-CYN1 and published Chroococcales genomes were high, ranging from 75 to 99% . The observation of photosystem II genes in GB-CYN1 indicates the metabolic potential for oxygenic photosynthesis, unlike UCYN-A which lacks photosystem II, RuBisCo and the TCA cycle. These findings confirm that GB-CYN1 resembles free-living Chroococcales of the clades B and C rather than the metabolically streamlined, endosymbiotic UCYN-A clade . Related Chroococcales species are known to produce copious quantities of exopolysaccharides , and EPS produced by Crocosphaera watsonii has been linked to the formation of transparent exopolymeric particles in pelagic environments . A cassette of genes required for EPS production has been identified by comparative genomic analysis of related Crocosphaera watsonii strains ; however, we did not recover homologs of these genes in our metagenomic sequence . We conclude that the absence of these sequences is most likely due to our incomplete sampling of the GB-CYN1 genome, though it could also indicate either an unknown pathway for EPS synthesis in GB-CYN1, or an alternate source of the green berry exopolymeric matrix . Consistent with the dynamic oxygen conditions within the green berries, plastic plant pot we found sequences in the metagenomic data that suggest the metabolic potential for aerobic and anaerobic respiration, and anaerobic fermentative pathways assigned to several different phyla of bacteria .

Abundant fixed nitrogen from diazotrophy and transient anoxia presents an ecological opportunity for dissimilatory nitrogen metabolism, a process that could cause concomitant nitrogen fixation and loss over rapid spatiotemporal scales. Indeed, previous studies of diazotrophic Nodularia spumigena aggregates measured significant rates of both nitrification and denitrification . We specifically investigated potential marker genes for dissimilatory nitrogen metabolism within the green berries. Metagenomic sequences homologous to the nitrite reductase gene nirK were found in six unassembled reads and on a corresponding 753 bp long contig . A single read homologous to nitrous oxide reductase nosZ was also found suggesting the presence of a denitrification pathway . The top database matches to these nirK and nosZ sequences belonged to marine phytoplankton epiphytes from the Alphaproteobacteria and Flavobacterales . In the case of the nirK metagenomic sequences, the best match was to Roseibium sp. TrichSKD4, an alphaproteobacterial species isolated from a nitrogen-fixing Trichodesmium aggregate in the Atlantic Ocean . Homologs to the napA periplasmic nitrate reductase and the nirB nitrite reductase were identified from six and five unassembled reads, respectively, and were most similar to database sequences from marine heterotrophs in the Alphaproteobacteria and Gammaproteobacteria . We did not detect sequences supporting the presence of nitrifying bacteria or archaea, though given our limited sequence depth, this could be a function of missing data.

Cyanobacterial nitrogen fixation in the global oceans is frequently aggregate-associated, as with Trichodesmium sp. colonies and rafts , filamentous heterocystous cyanobacterial colonies , or Crocosphaera watsonii associated with TEP . The green berries of the Sippewissett Salt Marsh are nitrogen-fixing macroscopic consortia of unicellular cyanobacteria , diatoms and heterotrophic bacteria. While nitrogen and carbon fixation mediated by the green berries is unlikely to play a major role in the overall marsh ecosystem due to their low abundance and patchy distribution , these consortia provide an interesting comparative system to investigate the dynamics nitrogen flux within diazotrophic cyanobacterial aggregates. More broadly, studies of coastal marine estuarine sediments have indicated complex dynamics and close spatial coupling of co-occurring nitrogen fixation and denitrification processes . In other oceanic diazotrophic cyanobacterial aggregates, similarly rapid respiratory rates create transient anoxic zones within the aggregate core , and create a heterogeneous microenvironment where both aerobic and anaerobic metabolisms co-exist. This parallel is not merely conceptual: the green berry heterotrophic bacteria were similar, both phylogenetically and in their metabolic marker genes, to those observed to colonize other marine phytoplankton aggregates. The recovery of denitrification marker genesequences in metagenome suggests that there might be heterotrophic denitrifiers in the green berries with the potential to exploit this suboxic niche. However, further studies including rate measurements and better genome reconstructions are needed to clarify the importance of denitrification in the berries. The presence of such a pathway for nitrogen loss in the green berries is speculative, given the fragmented metagenome and absence of activity measurements, but remains an interesting direction for future work.

The existence of closely coupled nitrogen cycling within diazotrophic cyanobacterial aggregates has been explored previously, though initial studies demonstrating the association of heterocystous cyanobacterial aggregates with key bacterial species and marker genes for nitrification and denitrification measured only low to negligible rates . However, more recent investigations of in vitro rates and in situ marker gene transcription indicates that denitrification within anoxic cyanobacterial aggregates could contribute significantly to nitrogen loss in hypoxic waters, where bulk oxygen concentrations would normally inhibit such activity . In the light of modeling studies showing tight spatiotemporal coupling of nitrogen fixation and denitrification processes in the global ocean , we postulate that this coupling may be occurring at the microscale within ephemeral, aggregate associated niches. The potential for such interactions emphasizes the need to examine bio-geochemical cycles from the microbial perspective in the spatially heterogeneous marine environment.Green berries were sampled in June and July of 2010 or July and August 2014 from a single intertidal pool formed in the Little Sippewissett Salt Marsh, Falmouth, MA United States . These aggregates, however, have also been observed at other locations throughout Little and Great Sippewissett marshes. Berries were collected from the sediment-water interface by sieving and were washed three times in 0.2 µm filter sterilized marsh water. Imaging of the berries was conducted using a Zeiss Axio IMAGER MZ epifluorescence microscope equipped with a color camera and a Zeiss LSM 710 spectral confocal scanning laser microscope .The MG-RAST 3.3 pipeline was used to classify unassembled metagenomic sequence reads as ribosomal RNA and protein coding sequences using the M5RNA and M5NR databases to provide a diversity description shown in Figure 2 . Metagenomic sequence data was mined using MG-RAST. Functions of interest were mined using the hierarchical function assignment and were assigned to GB-CYN1 when the best hits were to sequenced Chroococcales genomes. Marker genes were further investigated via profile HMM and BLASTX searches . PCR amplified SSU rRNA gene sequences were aligned to the SILVA 115 database using SINA and curated using ARB . 16S rRNA phylogenies were inferred using the GTRGAMMA rate approximation. For functional genes of interest that were found to have frame shift sequencing errors , the sequences were corrected using Framebot . The metagenomic nifH amino acid sequence was aligned to references sequences from the nifH database available from the Zehr research group using the ARB software package . ProtTest3 was used to select the fixed WAG model of amino acid evolution with an inverse gamma rate approximation for the nifH phylogeny, plastic planter pot though similar topologies were recovered using related models. Phylogenetic reconstruction for both 16S rRNA and nifH genes were conducted as follows: maximum likelihood phylogeny was constructed with RAxML 7.2.8 with 1000 rapid bootstrap inferences and Bayesian phylogeny with MrBayes 3.3 . For Bayesian tree inferences, MCMC was run with default parameters and convergence was assessed when the reported average standard deviation of split frequencies fell below 0.01. Hidden Markov model profiles of 40 phylogenetic marker genes for Bacteria and Archaea were used to search the green berry metagenomic peptide sequence database using the trusted cutoffs. For each of the 34 markers that have green berry hits, green berry peptide sequences were aligned with all the bacterial and archaeal references sequences by hmmalign in HMMER3 . A maximum likelihood tree was built by Fastree2 for each alignments, and the alignments and trees were examined. As a result, 33 green berry peptide sequences were selected for further analysis because they branched with Cyanobacteria with good alignments. These sequences were further filtered to include only those 29 phyeco gene families with a single copy, cyanobacterial green berry hit. Single-copied reference sequences from 126 Cyanobacteria genomes of the 29 markers and the green berry cyanobacterial metagenomic sequences were retrieved from the alignments built in the previous step and were concatenated into a large alignment. A maximum likelihood tree was built using PHYML3.0 with the LG substitution model .

Tree topology and branch lengths were optimized by the program and SH statistics was used for branch support estimation.Whole-aggregate in vivo acetylene reduction assays were conducted on two 30 mL serum bottles of five washed green berries each. Acetylene, generated by the hydration of calcium carbide in an evacuated 150 mL serum-vial, was added to 10% of the headspace in a 30 mL serum-vial containing the berries in 5 mL of anoxic 0.2 µm filtered in situ marsh water under an 90% N2-CO2 atmosphere . Experiments were incubated on a 14 h light, 10 h dark cycle with full spectrum illumination at 30◦C for 2 days. Acetylene and ethylene were quantified using a Varian 2400 series gas chromatograph with an H2 flame ionization detector, as described previously . Rates were calculated over the 2 days incubation period with the assumption that nitrogen fixation was restricted to the dark hours of incubation, as previously described for unicellular cyanobacteria .Surface mulches are widely used in the production of strawberries and certain high value vegetable crops. Polyethylene mulch is used on virtually all tomato and strawberry production in Florida and is also widely used in the production of other crops such as peppers, eggplant, and melons throughout much of the southern United States. Researchers at the University of Florida estimate that more than 100,000 acres of vegetable crops in that state currently use plastic mulches annually, making Florida the national leader in this production system . In California, the majority of strawberry and staked tomato production uses polyethylene mulch. Peppers, eggplant, and melons also use mulches in certain situations, especially when earliness is desired. Field management and research related to plastic mulches in these production regions is now quite developed. Potential benefits as well as drawbacks of polyethylene mulches for vegetable crop production are given in table 1. The use of inter crop cover crop residues as surface mulches is a more recent and far less widely used production practice. It has recently gained considerable interest in a number of commercial vegetable crop production regions in the United States. The use of organic mulches in smaller-scale gardening contexts has had a very long history. Potential advantages and disadvantages of this vegetable crop production technique are summarized in table 2. Reflective plastic and some cover crop mulches share similar features relative to crop production: insect and disease management, weed management, fertilizer availability, and water conservation. In order for production practices using either polyethylene or cover crop mulches to be successfully adopted in California, specific production goals must be carefully matched with specialized management know how and experience.Plastic mulches have been commonly used for commercial vegetable crop production for more than 30years. Excellent sources of information on specific characteristics and applications of plastic mulches are available on the World Wide Web, and several of these are provided in the references to this publication. Most plastic mulch materials are made of either high- or low-density polyethylene, ranging from 0.30 to 0.79 inches in thickness, are 5 to 6 feet wide, and are available on rolls 555 to 1,338 yards long, depending on the thickness of the mulch . Black, white, and clear plastic mulches are most commonly used in commercial production, with black being the dominant color used for vegetables. Black plastic mulch is typically used for spring-seeded crops because it increases soil temperatures about 5ºF at a depth of 2 inches and 3ºF at 4 inches , compared to those of bare soil . Recently, a mulch has been introduced that consists of a strip of black plastic 30 cm wide down the center that is flanked on either side by metalized reflective plastic. This plastic combines the advantages of black plastic over the seed row, to help heat the soil, with the reflective characteristics of metalized plastic for insect and disease management. Black mulches have also recently been shown to reduce weed growth.

The dietary intake of Z is lower than L in all age groups and ethnicities in the U.S.. Dietary intakes of L and Z are strongly associated with their serum levels, as well as with MPOD. Previous studies have shown that high intakes of these carotenoids from dietary sources or supplements can increase plasma L and Z, and MPOD. Once early AMD has progressed to the intermediate stage, dietary supplements are indicated, but no clinical evidence yet exists for interventions thatcan address the prevention of small-intermediate drusen with pigmentary changes, the initial clinical signs of macular disruption . Goji berry , also termed wolfberry or Go Chi Zi, has been used in traditional Chinese medicine for more than 2000 years. The bright red berry contains the highest amount of Z among all known dietary sources and is mainly present in a dipalmitate form. The intake of zeaxanthin dipalmitate extracts from goji berry increases plasma Z to a greater extent than non-esterified Z supplementation. The berries also contain unique carbohydrates that are present as conjugates with peptides or proteins, which are often referred to L. barbarum polysaccharides . These have shown anti-inflammatory and neuroprotective effects in animal and cell culture studies. The typical adult human eye has approximately 2.4 times more Z than L in the central fovea of the macula, plastic pots large making goji berry intake a prime candidate for increasing MPOD.

Nevertheless, there is a paucity of clinical evidence on goji berry and MPOD particularly for the prevention or delay of progression from early to intermediate AMD. In individuals from China with signs of early AMD, 25 g of daily consumption of goji berries for 90 days significantly increased both serum Z and MPOD. However, this study had a broad age range , some participants smoked, and others had certain preexisting medical conditions. Additionally, the authors only reported central MPOD values up to 0.5 retinal eccentricity , whereas macular pathology and visual dysfunction in AMD may extend beyond that central region. Therefore, to provide a more complete understanding of the influence of goji berry intake on the progression AMD, data is needed on for different population groups that measures MPOD at eccentricities over the entirety of the macula. In the current study, we prospectively evaluated if the daily intake of 28 g of goji berries or a commercially available supplement providing 6 mg of L and 4 mg for 90 days can improve MPOD and skin carotenoid levels, an index of total carotenoid intake, among healthy middle-aged adults, 45 to 65 years old, with no signs of drusen or early AMD.Eighty-eight volunteers, ages from 45 to 65 years old, were recruited from an online website and public advertisements in the area of greater Sacramento, California. Participants provided informed consent and were screened with a questionnaire. Inclusion criteria were being generally healthy , having a normal macular condition as verified by an optometrist, and if relevant, being prescribed the same medication regimen for at least 6 months that was not related to carotenoid metabolism and was approved by the study physician.

Exclusion criteria were a dislike of, or allergy to goji berries, diseases of the eye, malabsorption problems, substance or alcohol abuse, smoking, drugs for management of lipids, glucose, or blood pressure, use of dietary supplements other than multivitamins and minerals that provided greater than 100% of the U.S. Dietary Reference Intake, or any supplement containing L or Z. The intervention was registered on ClinicalTrials.gov , with the first posted date of 6 December 2019, complied with the tenets of the Declaration of Helsinki, was approved by the Institutional Review Board of the University of California , Davis and was conducted at the UC Davis Ragle Human Nutrition Research Center.Twenty-eight grams of goji berries is considered a single serving size. The berries in this study were USDA-certified organic goji berries grown in the Ningxia region of northern China and provided by Navitas Organics, Novato CA, USA. The goji berries were portioned into clean, single-serving plastic bags and provided in 45-day allotments. The commercially available supplements were purchased online, contained 6 mg of L and 4 mg of Z per serving and were repackaged into 45-day supplies in clean plastic bottles. Compliance was monitored by a self-administered log. Habitual dietary information was collected with the Automated Self-Administered 24 h dietary assessment web-based tool once between day 0 and 45, and once again between day 45 and 90. The MPOD was assessed by the psychophysical method of customized heterochromatic flicker photometry using a macular densitometer . After participants viewed a 5-minute video detailing the measurement procedures, they were dark-adapted for 7 minutes and then began the test. The light intensity of each relevant wavelength was calibrated with a photodiode.

The flicker frequency was selected based on a preliminary test of the participant’s sensitivity. The task was to eliminate or minimize the flicker in the visual field three times by turning a dial that changed the intensity of a 460 nm light. Each participant performed the test while looking directly at the flickering light at 0.25, 0.5, 1, and 1.75 RE degrees, representing the MPOD level from the center to the periphery of the macula. Skin carotenoid content was measured by reflection spectroscopy . After cleaning, the tip of the right index finger was inserted into the spectrophotometer and three measurements were collected. A skin carotenoid score was calculated by the system software. Carotenoids that exist in human plasma, including β-carotene, lycopene, L, Z, and their isomers have been successfully detected in toto and quantified by this device, which has been validated to reflect fruit and vegetable consumption.Sample size was based on a study that assessed the impact of a Z supplement on MPOD in 24 healthy people. Statistical analyses were performed with JMP version 16 . Two-tailed t-tests evaluated potential between group differences at baseline. The MPOD and skin carotenoid data were analyzed with mixed-effects models using time and treatment as the main factors, with age and sex as the covariates, and participant ID as the random effect. For main effects, student t-tests determined significance within group pairs. p-Values of 0.05 or less were considered statistically significant. Correlation coefficients between the outcome measures were determined via Spearman’s method. The mean values of the dietary intake data were compared by two-tailed t-tests, which were log-transformed when necessary, and presented as the mean ± S.E.M. or the back-transformed mean with 95% confidence intervals .Thirty-one healthy, middle-aged adult males and females met the inclusion criteria between May 2019 and Jan 2020. The participants consumed either goji berries or the LZ supplement 5 days per week for 90 days. Twenty-eight individuals completed the intervention, after which two in the goji berry and one from the LZ group were excluded from the data analysis due to measurement errors. Furthermore, data from one was subsequently removed after learning of a major change in dietary patterns that included a low intake of macronutrients between days 45 and 90 .Ninety days of 28 g of goji berry intake significantly increased the optical biomarker MPOD in healthy adults at 0.25 and 1.75 REs. These results suggest that even in a healthy population with no evidence of small drusen or early AMD, goji berry intake can improve eye health. Our results are consistent with data of improved MPOD after a similar amount and intake period of goji berry in a Chinese population at risk for intermediate AMD. Moreover, our trial is consistent with reports of protection against macular hypopigmentation and drusen development in a population of generally healthy and older individuals who were provided Z at approximately a third of the amount of Z provided in the current trial. Our findings suggest that a higher intake of Z relative to L may be useful in reducing the risk of AMD. This is consistent with increased MPOD levels after 4 months of supplementation with 20 mg Z or 26 mg Z with 8 mg L plus 190 mg of mixed omega-3 fatty acids by young healthy adults. Interestingly, black plastic nursery pots we observed a significant increase in MPOD at 1.75 RE, but not at 0.5 or 1 RE, in the goji berry group. A possible explanation for this trend is the relatively low macular pigment at 1.75 RE compared to the other REs, which may increase the potential for improved MPOD in this peripheral area of the macula.

Our results are also consistent with data from 11 randomized controlled trials where supplementation with at least 10 mg of the macular carotenoids was effective at increasing MPOD. Significant correlations were observed between the overall skin carotenoid score and MPOD, which is consistent with clinical results of carotenoid supplementation. Further analysis demonstrated that L and Z, but not goji berry intake, was significantly influencing this trend. Previous work has shown an association between serum L and Z in skin and blood with macular pigment carotenoid accumulatio. Data from the current trial are consistent with this observation as goji berry intake was significantly associated with the skin carotenoid score. However, in contrast to data with L and Z supplements, MPOD score was not correlated with changes in skin carotenoids with goji berry intake. The skin photometer detects overall carotenoid content, and as goji berries are also rich in β-carotene, neoxanthin, and cryptoxanthin, these carotenoids likely influenced the skin measurements, and would not reflect the selective carotenoid accumulation of L and Z in the macula. Other goji berry components such as taurine, vitamin C, zinc, and LBP may influence the results by lowering oxidant stress and improving eye health. For example, studies in animals and cell lines suggest that LBP can protect against AMD by reducing oxidative stress and cell apoptosis in retinal pigment epithelium. Taken together, under the conditions tested, it is reasonable that MPOD may not fully correlate with skin carotenoids in the goji berry group. To our knowledge, the impact of goji berry intake on MPOD in healthy middle-aged people has not been previously reported. While others have noted improved MPOD after LZ supplementation among people with low MPOD baseline levels, our findings suggest that even in populations with normal MPOD values, a significant increase can be detected after goji berry consumption at the most central part of the macula . A meta-analysis regarding the effects of L, Z, and meso-Z supplementation noted that the MPOD at baseline was inversely associated with macular responses, suggesting individuals with a relatively lower macular pigment status may receive more benefit with higher amounts of L or Z. The Age-Related Eye Disease Study 2 trial assessed the impact of dietary supplements containing 10 mg of L, 2 mg of Z, 500 mg of vitamin C, 400 IU of vitamin E, 80 or 25 mg of zinc, 2 mg of copper, and/or 350 mg of docosahexaenoic acid plus 650 mg of eicosapentaenoic acid. The results showed a significantly reduced rate of progression from intermediate- to late-stage AMD after 5 years. Secondary analyses of the study indicated protective roles of L and Z. We did not use the AREDS2 supplement for the comparison group because this formula has only been shown to be effective for those with intermediate AMD, and no clinical evidence exists for its efficacy in our study population of healthy people. In addition, we note that 80 mg of zinc in the AREDS2 supplement is twice the upper limit of recommended daily intakes for zinc. In epidemiological studies, L and Z intakes have been inversely associated with the development of AMD. In the current study, the reported dietary intake of L plus Z, not including the berries or supplement, was 3.1 and 1.9 mg/d in the goji berry and supplement groups, respectively, which is higher than the typical estimated intakes in the US of 1.6–1.86 mg/d. Three to five mg/d of L and Z have been recommended to help support normal macular function, although no recommended dietary allowance values yet exist. Daily consumption of one Hass avocado containing 0.5 mg of L over 6 months was associated with a significant increase in MPOD in healthy adults. In contrast, no increase in MPOD was observed after consuming one Hass avocado daily for 3 months. Daily consumption of egg yolks providing 1.38 mg L and 0.21 mg Z resulted in a significant increase in MPOD and other measures of visual acuity in older adults with signs of early stage AMD after 12 months.

The tool could also be effective in performing model-based vineyard designs in which the optimal design is determined under a set of constraints such as slope aspect or minimum row spacing. Although this means that the optimal design is likely to be case-specific, the tool was used to examine general trade-offs in various designs, which are summarized below. While the N-S row orientation on flat terrain was effective in balancing daily berry light interception and heat accumulation between opposing sides of the vine, it is also susceptible to temporally localized berry temperature spikes on the west side that could be managed by applying dense shade cloth. For cases with no shade cloth, the NE-SW orientation was likely the best compromise between berry temperature reduction and balance between opposing sides of the vine, although it still had significant imbalance in heat accumulation and extreme berry temperatures. Addition of sloped terrain tended to exacerbate berry temperature extremes and imbalance when the slope was facing south or west, which in several cases could not be well managed using shade cloth. The shade cloths were more effective in reducing berry temperatures in cases with greater row spacing relative to plant height because adjacent rows could potentially provide their own shade. The simulation experiment in this work used the new modeling tool to examine general trends in berry temperature and light interception as vineyard architecture and shade cloth density were varied.

Because of the challenge in concisely presenting results of a very large number of simulated cases, growing raspberries in container results are limited to a limited number of architectures, a narrow time period, a single latitude, and site-specific weather conditions. As such, care should be taken in direct application of the simulated values for vineyard design, as they may change for a certain site or design. However, the model itself provides a tool that could be used to provide quantitative guidance for vineyard design or management at a specific site.Together, Chapters 2-4 motivated and carried out the development of a modeling tool that can be used to identify strategies for mitigating the effect of excess sunlight and unfavorable temperatures on grape berries. The tool was then used to study how vineyard design and management strategies related to berry shading interacted to influence berry temperature and light interception. Chapter 2 evaluated widely used assumptions when modeling solar radiation interception in plant canopies, namely assumptions of vegetation homogeneity and isotropy. Because of their simple, tractable form, one-dimensional turbid medium models of radiation interception that assume homogeneity or isotropy are used across a broad range of disciplines. However, it is relatively well-known that with varying levels of vegetation sparseness and preferential leaf orientations , the implicit assumptions of vegetation homogeneity and isotropy in this simple class of 1D models are frequently violated. Yet it is not well-known how this assumption violation translates into model errors in a given situation.

Results of this work provided quantitative guidance for when a simple 1D model can be appropriately used to estimate light interception. For complex canopies like grapevines, the results highlighted the need to use a 3D radiation model because these models can represent the vertical and horizontal variability in the canopy and their effect on light interception accurately. Chapter 3 developed and validated a three-dimensional model for grape berry temperature to understand whether the effect of excess sunlight on grape production can be mitigated by designing and managing vineyards in a way that effectively creates a favorable berry microcli-mate. For the first time, a 3D vine-resolving structural model was coupled with a high-resolution energy balance model for providing accurate and spatially-explicit predictions of berry temperature dynamics. The developed 3D model accurately simulated the spatial and temporal temperature fluctuations of grape berries in vineyards with different climate, topographies, and trellises. Chapter 4 explored different scenarios to mitigate the effect of excess sunlight and temperature. Previously developed models for grape temperature have not been used to evaluate the interacting effects of different management strategies to reduce grape berry temperature. This chapter measured the effect of vineyard design and shade cloth on berry temperature and quantified trade-offs between the different management strategies to maintain optimal berry heat balance and reduce the elevated berry temperatures. Altering light interception by using shade cloths affected the reduction of elevated berry temperatures depending on the vineyard design. A great effect of the shade cloths was found in clusters exposed to direct sunlight for prolonged hours, which tended to be in cases where the vines had greater plant spacing relative to plant height, and where rows were oriented NS and NW-SE in either a flat or sloped terrain facing W or SW, respectively.

While changing the row orientation to NE-SW can be an effective long-term practice to reduce the effect of elevated temperature, for cases where changing the vineyard design is not possible, the shade cloths presented an alternative to reduce unfavorable berry temperatures in many cases. The variables that determine cluster exposure to direct sunlight, such as topography, trellis systems, row orientation, and shade cloths should be considered carefully to develop management strategies for optimizing grape quality. The primary novel outcome of this dissertation that advances the current state-of-the-art is the development of a new tool that could be used to address the role of agricultural management in climate adaptation. Technologies that increase the rate of adaptation to climate change are of great value to farmers, the wine industry, policymakers, and the scientific community. In future studies, the 3D model could be used to evaluate the effect of vineyard designs on berry temperature under changing environmental conditions, to assess the effect of different irrigation strategies on grape production, and to relate the spatial and temporal variability in berry temperature to variations in berry quality traits.Drosophila suzukii , also known as the spotted wing drosophila , is a vinegar fly originating from Southeast Asia. SWD was first detected in North America in August 2008 in Santa Cruz County, California, where it was observed infesting strawberries and caneberries.1,2 In 2009, SWD was detected in Washington, Oregon, and Florida. By 2010, SWD was detected in Utah, Mississippi, North Carolina, South Carolina, Wisconsin, and Michigan in the United States, and Alberta, Manitoba, Ontario, and Quebec in Canada.3 Recent trapping indicates that SWD can be found in virtually any region of North America where host fruit are available. A coincidental invasion of SWD with a genetically distinct population has also been observed in Europe, with initial detections in both Spain and Italy in 2008, followed by its spread throughout the continent.2,4,5 In North America, SWD is primarily a pest of berries and cherries. In Europe, it is reported to also damage a number of stone fruits and grapes. Unlike native vinegar flies in North America and Europe, female SWD possess a serrated ovipositor that can pierce the skin of healthy, soft-skinned fruits to lay eggs. These eggs quickly develop into larvae, which consume the fruit and render it unmarketable. The only other Drosophila species known to oviposit in sound, marketable fruit is Drosophila pulchrella Tan. This species is native to Japan.1 Growers have attempted to mitigate crop damage risk by applying additional insecticide, harvesting more frequently, performing field sanitation, and implementing trapping programs to detect SWD populations. These management practices are costly and many growers still face significant yield losses from SWD infestations. We examine the economic impact of SWD infestations in the California raspberry industry. Raspberry producers are perhaps the most affected by SWD’s invasion amongCalifornia commodities, although producers of blueberries and cherries have experienced substantial losses too. Strawberry producers have experienced lower damage rates and primarily on the lower-value fruit produced for processing. SWD-related losses in these industries vary by year and crop depending on management practices, weather conditions, time of the year, raspberry container size and geographic location. A primary motivation for focusing on the California raspberry industry is that California accounts for the majority of raspberry production in the U.S. and the raspberry industry accounts for the majority of economic losses due to SWD among berry crops. 6 A second motivation is the magnitude of change in pest management practices; few of the SWD control practices used by raspberry producers were needed to prevent injury from other pests prior to its establishment. Economic losses in the California raspberry industry include the cost of managing SWD and the value of the fruit lost due to SWD infestations despite management efforts. First, we compute the cost of the chemical management programs and the labor-intensive sanitation practices implemented to mitigate SWD-related yield losses. Second, we calculate the industry level yield losses due to infestation. These components form an estimate of the full economic cost of SWD’s invasion into California raspberry production.

In 2013, raspberries were estimated to be the twenty-seventh largest crop in California by value of production. California accounted for 74% of all raspberry production in the United States. The United States is the third largest producer of raspberries in the world, producing 91,300 tonnes, after the Russian Federation and Poland, which produce 143,000 and 121,040 tonnes,respectively. Across all counties, California’s raspberry production was worth an estimated $239 million according to the United States Department of Agriculture’s National Agricultural Statistics Service , and $437 million according to California County Agricultural Commissioners’ Reports.  The difference in these estimates reflects that the NASS data report cash receipts to producers while the Agricultural Commissioners’ Reports estimate the total value of production. Figures 1, 2, 3, and 4 plot California raspberry hectares, production, yield per hectare, price per kilogram, and the total cash receipts between 2004 and 2013. Note that raspberry hectares multiplied by yield per hectare is equivalent to production, and production multiplied by price per kilogram is equivalent to total cash receipts. Four counties account for virtually all commercial raspberry production in California: Ventura, Santa Cruz, Santa Barbara, and Monterey. In 2014, Ventura County produced approximately 52% of California’s raspberry crop by value, $241 million, on 1,873 hectares. Raspberries are the third most valuable crop in Ventura County. Santa Cruz County produced approximately 28% of California’s raspberry crop by value, $131 million, on 979 hectares. Raspberries are the second most valuable crop in Santa Cruz County. Santa Barbara County produced approximately 10% of California’s raspberry crop by value, $45.2 million, on 591 hectares. Raspberries are the ninth most valuable crop in Santa Barbara County. Monterey County produced approximately 10% of California’s raspberry crop by value, $45 million, on 316 hectares. Raspberries are the sixteenth most valuable crop in Monterey County. Table 1 summarizes California raspberry production by county. Counties are listed from north to south along the Pacific Coast. Figure 5 identifies these berry-producing regions with a stylized map of California.Most commercial raspberry plantings in California have had an 18-month lifespan. The crop is planted in the winter and then harvested twice, first in the fall following planting and then in the subsequent summer. Both harvest seasons last approximately three months, with crews harvesting fruit every three days on average. Variations in harvest frequency depend on yields and pest management activities. Yields are low at the beginning and end of a harvest season, and peak near the middle of a season. Pesticide applications may require an interval of time, depending on the particular pesticide, before normal harvesting activities can resume. This period is known as the pre-harvest interval , and it is determined by the U.S. Environmental Protection Agency. Occasionally, low yields are realized during the harvest season due to crop damage resulting from weather, pest activity, or other external factors. The summer harvest is typically larger than the fall harvest. Organically produced raspberries represent a significant share of total California raspberry production. In 2008 and 2011, California’s organic raspberry production was valued at $11.4 million and $8.98 million, respectively, according to the USDA-NASS. In 2012, 408 hectares of California raspberries were organically managed according to the University of California Agricultural Issues Center. Raspberry prices vary throughout the year, but on average organic raspberries are sold at a price premium. In 2015, the national average retail price of organic raspberries over the entire year was $3.52 per six ounce tray according to the USDA Agricultural Marketing Service. The average retail price of conventional raspberries over the same period was $2.55 per tray. The average California terminal market prices for organic and conventional raspberries were $3.29 and $1.97 per tray, respectively. California raspberries are a major export crop. In 2013, the combined category of raspberry, blackberry, mulberry, and loganberry exports was the twentieth largest export crop category by value in California.

These results highlighted the need to use a 3D radiation model to account for complex canopies because these models can represent the vertical and horizontal variability in the canopy and its effect on light interception accurately. Chapter 3 develops a 3D model that can accurately resolve spatial and temporal heterogeneity in berry temperature. The spatially-explicit nature of the model allows for robust representation of varying canopy architectures and their effect on berry temperature. The high model complexity is afforded by performing calculations in parallel on the computer’s graphic processing unit . This ability to resolve the geometry of the vineyard is critical in this particular study because it means the model is robust to changes in row spacing, trellis system, row height, etc. To generate data for validation of the 3D grape berry temperature model, field and laboratory experiments were conducted. Validation results demonstrated that by accurately representing the 3D vine structure, the model was able to closely replicate measured spatial and temporal fluctuations in berry temperature. Chapter 4 aims to explore whether elevated berry temperature can be mitigated by designing and managing vineyards in a way that effectively creates a favorable microclimate for berry development. Identifying strategies that have the potential to reduce elevated temperatures in a warming climate is of great interest to grape growers. However, raspberry container growing given the extremely large number of interacting variables that determine berry temperature it is not feasible to independently vary all of these parameters in field experiments. Thus, to study the interactions between these variables that might yield favorable results, Chapter 4 expands the model developed and validated in Chapter 3 by incorporating the effects of shade cloth on berry temperature.

The model was used to ultimately predict the efficacy of potential mitigation strategies for high berry temperature. The results of this study provided new insights into the effect of fruit zone shading to control berry temperature for the establishment of new vineyards and targeting the management of existing vineyards.Light interception in plant canopies is most commonly estimated using a simple one dimensional turbid medium model . Inherent in this class of models are assumptions that vegetation is uniformly distributed in space and in many cases that vegetation orientation is uniformly distributed . It is known that these assumptions are violated in a wide range of canopies, as real canopies commonly have heterogeneity at multiple scales and almost always have highly anisotropic leaf angle distributions. However, it is not quantitatively known under what conditions these assumptions become problematic given the difficulty of robustly evaluating model results for a range of canopy architectures. In this study, assumptions of vegetation homogeneity and isotropy were evaluated under clear sky conditions for a range of virtually-generated crop canopies with the aid of a detailed three-dimensional, leaf-resolving radiation model. Results showed that Beer’s law consistently over predicted light interception for all canopy configurations. For canopies where the plant spacing was comparable to the plant height, Beer’s law performed poorly, and over predicted daily intercepted sunlight by up to 115%. For vegetation with a highly anisotropic leaf inclination distribution but a relatively isotropic leaf azimuth distribution, the assumption of canopy isotropy resulted in relatively small errors. However, if leaf elevation and azimuth were both highly anisotropic, the assumption of canopy isotropy could introduce significant errors depending on the orientation of the azimuthal anisotropy with respect to the sun’s path.

Solar radiation is a primary driver of most plant biophysical processes, including energy transfer, turbulent transport, evapotranspiration, photosynthesis, and phenology. Fluxes of absorbed radiation in plant canopies have strong gradients in the vertical direction, and potentially in horizontal directions in the case of heterogeneous canopies. Capturing these high gradients through direct measurement is often challenging, and therefore models are frequently used to predict absorbed radiative flux distributions. For practical purposes, relatively simple models are frequently used to estimate light interception in plant canopies. For example, crop models have become important tools for studying agricultural system, yet they commonly utilize relatively simple models for light interception given the frequent lack of detailed architectural inputs. The most commonly used approach for estimating light interception treats the canopy as a homogeneous medium of unresolved vegetation , which allows for the use of a simple exponential model for radiation attenuation commonly know as Beer’s law, Beer-Lambert law, or Beer-Lambert-Bouguer law. Beer’s law calculates the probability of radiation interception as an exponentially increasing function of the leaf area projected in the direction of radiation propagation and the distance travelled through the canopy.The form of Beer’s law given in Eq. 2.1 functions under two main assumptions. The first assumption is that leaves are randomly distributed both vertically and horizontally in a continuous medium where leaves are relatively small. The second assumption is that leaves absorb all incident radiation, which may be reasonable for photosynthetically active radiation bands where leaves absorb roughly 90% of incident radiation, but is likely a poor assumption in other bands such as the near-infrared where absorption is low.

Equation 2.1 also requires specification of G, which is most commonly set to be equal to 0.5 based on the assumption that leaves are isotropic. Leaf area density typically varies sharply in the vertical direction. Many natural plant canopies have considerable horizontal heterogeneity such as savannas, or heterogeneity due to natural or man-made disturbances. Crop canopies also commonly have a sparse, row-oriented configuration that creates high heterogeneity in light interception. Furthermore, it is rare to find canopies with isotropic leaf angle distributions, as this is typically not the most efficient configuration for light interception. Despite the known limitations of Beer’s law in the above cases, it is still frequently applied in these systems due to its simple, tractable form. However, there is a general lack of quantitative understanding of the errors resulting from the application of these simplified models in various canopy architectures, primarily because it is difficult to quantify light interception from field measurements for a range of architectures. The objective of this study is to better understand and quantify errors in modeled radiation absorption under assumptions of vegetation heterogene-ity or isotropy for various canopy configurations. The authors’ hypothesis is that Beer’s law will perform well for relatively dense, closed canopies provided that G is specified appropriately. For sparse canopies, it is hypothesized that assumptions of vegetation homogeneity will result in significant model errors, thus necessitating a more complicated model. Since accurately measuring the distribution of absorbed radiation in space and time is often unfeasible using traditional experimental approaches, we used a sophisticated 3D radiation model along with virtually-generated canopies to evaluate Beer’s law under different simplifying assumptions. Virtual canopies with varying levels of heterogeneity, sparseness, and leaf orientation distributions were generated to evaluate assumptions of vegetation homogeneity or isotropy in terms of absorption of direct solar radiation. A considerable advantage of using virtually generated canopies is that the input parameters in Eq. 2.1 can be calculated exactly from the virtual canopies. When combined with a detailed 3D radiation model, this resulted in a robust means for evaluating the performance of simplified models for a range of canopy architectures.For simulating plant light interactions, blueberry plant pot detailed 3D geometric models were used to describe the architecture of the canopy. Agricultural crops were chosen for the plant types because: many 3D models are readily available, they have sufficient yet regular heterogeneity that limited the degrees of freedom when generating the canopies, and they represent an economically important practical application of the use of Beer’s law. The chosen crop canopies were grape, almond, potato, and corn, which were represented in the 3D model using a mesh of rectangular and triangular elements.

To minimize the number of elements needed to describe their complex geometries, images with a transparency channel could be overlaid on these basic elements, where the transparency channel is used to remove a portion of the element’s surface. Virtually-generated plants were either read from a polygon file , or created using the procedural plant generator described by Weber and Penn .Parameters used to quantify canopy architecture are given in Table 2.1. The procedural model used to generate the grape and almond plants had a random component to each architectural parameter, making each plant unique. Each corn and potato plant was identical, therefore a random azimuthal rotation was applied to each plant to decrease regularity of the canopy. Plants were placed in a marked row structure to form a canopy. Two grape canopy cases were considered: one with a North-South row orientation and one with an East-West row orientation . Two potato canopies were considered in which plants were arranged in either a East-West row-oriented pattern , or a uniformly spaced planting pattern . In all cases, the size of the 3D scene was chosen such that further increasing the total number of plants did not have an impact on results. To test the model in the case of homogeneous and isotropic vegetation, a set of canopies were created with uniformly distributed leaves in space with three different leaf area index values: L =1.5, which consisted of 100,000 leaves; L =3.1, which consisted of 200,000 leaves; and L =6.2, which consisted of 400,000 leaves. The surface area of each leaf was 0.006 m2. Each leaf angle was set by randomly drawing from a spherical distribution. To characterize the plant geometry, L and the leaf inclination angle probability density function were calculated for all five generated canopies, and the leaf azimuthal angle PDF was calculated for the Grape N-S and Grape E-W cases . The L was calculated by summing the one-sided area of all leaves in the canopy and dividing by the total canopy footprint area. The leaf inclination angle and leaf azimuthal angle were calculated for each of the elements from the surface normal of the leaf, and a PDF was formed by weighting each element’s contribution to the PDF by its surface area, then normalizing such that the PDF integrates to unity. The corn model had predominantly vertically oriented leaves, while the almond and potato models had leaves closer to horizontal on average . Grape leaf inclination skewed toward vertical, and leaf azimuth tended to be oriented parallel with the row , which is supported by previous manual and LiDAR measurements. The gap fraction was calculated from the 3D models by computing the fraction of direct sunlight not intercepted when qs = 0 . Gap fraction values ranged from 80% in the grape canopy cases down to 21% for the corn canopy case. Although both potato canopy cases had the same L, their gap fractions were 22% for uniformly spaced plants and 36% for row-oriented plants.The incoming radiation data used to drive the radiation absorption simulations in this study was generated following the REST-2 model of Gueymard. The hourly incoming radiation was calculated based on the assumed virtual site longitude , latitude , offset from UTC , atmospheric pressure , air temperature near ground level , atmospheric turbidity coefficient , relative humidity , and Julian day of the year . It is noted that the precipitable water in the REST-2 model was specified using the model of Viswanadham. The direction of the sun for any time of day at the virtual site was calculated following the approach outlined by Iqbal. In cases where scattering was included, two radiative bands were considered – one character-istic of efficient absorption by leaves such as the photo synthetically active band , and another characteristic of high scattering such as the solar near-infrared band . The total incoming solar flux was partitioned as 47% in the PAR band and 53% in the NIR band. In the PAR band, r was set to 0.056 and t to 0.042, while in the NIR band, r was set to 0.425 and t to 0.334.Results for the daily total light interception on Julian days 153, 232, and 305 are listed in Table 2.2, and shown graphically in Fig. 2.5. For the homogeneous canopy cases, very close agreement was found between the 1D and 3D models regardless of L, which indicated that the approach used to compare the 1D and 3D models was consistent and that leaf-scale heterogeneity created by discrete leaf surfaces did not create significant errors.

Several genes were captured in this interval, including NADH:Ubiquinone oxidoreductase subunit A8 ; mucin-5AC-like, a gene putatively related to human mucin-5AC, and two hemomucin genes involved in hemocyte adhesion and innate immunity in insects. The PBS hotspot on contig 15 contained cryptochrome-2 , encoding a key component of the circadian clock . Finally, the large hotspot on contig 22 contained 69 genes, including multiple copies of juvenile hormone binding protein, ribosomal proteins, gustatory response genes, rhodopsins, andacetylcholinesterase , a gene implicated in organophosphate insecticide resistance . Interestingly, based on alignment data it appears the Central and Andes populations may have two copies of ache, while North populations only have one. We looked for known mutations conferring organophosphate resistance and found moderate frequencies in all three clusters as well .Using whole genome sequencing data, we found that Tuta absoluta samples collected from 11 locations in Latin America clustered into three basic regions, comprised of a North, Andes, and Central group. In addition, we see that Spanish populations likely originated from a Central Chilean source based on their low level of Fst with the Andes and location on the ML tree. Previous analyses with mitochondrial sequences were unable to differentiate populations ; however, large pots plastic analyses using microsatellite data was able to identify these same three clusters and suggest a Central Chilean source for the European migration as well .

In agreement with this conclusion, looking at fresh tomato export data we see Chile is a worldwide exporter, shipping 12 tons of tomatoes an average distance of 11,700 km in 2018, while most other countries in South America tend to export within the continent . The Andes Mountains represent an obvious geographic barrier that would separate the Central population from the North and Andes populations. Population structure between Andes and North populations may be due to factors related to the changing latitude, including temperature and daylength. While PCA groups our Ecuador samples with Andes populations, admixture analysis and Treemix both provided some evidence that Ecuador may represent an admixture zone between the two regions. The weighted Fst between the North and Andes is also lower than between North and Central, suggesting that North and Andes are indeed more closely related. This general Fst pattern was also observed based on microsatellite analyses . Sequencing of more samples fromPeru and Ecuador might be needed to further elucidate the extent of an admixture zone between these clusters. While Tuta absoluta was first discovered in Peru in 1917, its native range is not well established. One hypothesis is that Tuta absoluta migrated out of the Andes region and across South America through the 1960s-80s because of human transport by agricultural shipping. This aligns with the surge in domestic tomato agriculture in South America at the same time . However, based on the similar nucleotide diversity levels between clusters, as well as high levels of Fst, we hypothesized it might be more likely that this migration across South America may have happened prior to tomato commercialization, with populations of Tuta absoluta later adapting to the appearance of commercial tomato agriculture.

Based on our simple 3 population model, it appears the ancestral population diverged twice tens of thousands of years ago. Relative to the ancestral population size, the combined effective population size is roughly three times larger, although this is heavily weighted toward a very large Andes population, relative to the North and Central regions. The fact that the estimated Andes population size is nearly 10 times larger than that of Central or North populations, ,as well as its slightly higher level of genetic diversity, could suggest that the Andes cluster represents the ancestral population range. Given that the wild ancestors of tomatoes and potatoes are also native to the Andes region , the Andes region would be an ideal place to search for native parasitoids for biocontrol. To date, biocontrol methods in South America have relied on non-natives or generalist parasites, with only a handful of native specialists identified in the literature and none that are commercially available . Thus, knowledge of Tuta absoluta’s native range may help focus efforts to identify more natural parasitoids. Using the PBS, we found multiple genomic windows under apparent selective forces. Not surprisingly, one of the highest PBS windows contained the para gene, which encodes a sodium ion channel that is targeted by pyrethroids. The extremely low allele diversity in the Andes and Central populations relativeto the North suggest a hard selective sweep occurred here. Heavy pyrethroid use in Brazil led to the appearance of resistant strains starting in the 1990s . We found that Central and Andes populations were completely fixed for the L1014F mutation, one of the most common causes of knock-down-resistance to pyrethroids , while the North had an intermediate frequency. A study looking at Brazilian populations found a similar pattern of fixed L1014 , while another study looking at multiple populations in South America also found the same pattern of L1014F fixation in Central and Andes populations but not in the North .

While both studies also found M918T and T929I at elevated frequencies in all populations, we additionally detected the resistance allele V1016G in the North. We also found L925M in Central and I254T in Andes. While these have not been characterized as resistance alleles, mutations at these same positions have been shown to confer resistance in Drosophila melanogaster and Bemisia tabaci . Other regions under selection were less obvious. We found one region in contig 2 containing Ndufa8 and several hemomucin/mucin genes with a similar low genetic diversity in the Central and Andes populations and high PBS in North, indicating a hard selective sweep. Ndufa8 produces a nuclearencoded subunit of the NADH dehydrogenase complex I, part of the electron transport chain in the mitochondria used to generate ATP. Mutations here are known to cause mitochondrial complex I deficiency in humans , although a few studies have found evidence of positive selection occurring in other species, potentially related to metabolism . The hemomucin genes are a component of the insect immune system that are involved in endocytosis . Selection here could be in response to the increased use of parasitoids and predators such as Trichogramma evanescens and Nesidiocoris tenuis as an biological control alternative to insecticides .The elevated PBS region in contig 22 was relatively large at approximately 1Mb in size, containing over 60 genes. Interestingly, we noticed two copies of ache contained within this window, which codes for acetylcholinesterase, a gene which encodes a protein which degrades the neurotransmitter acetylcholine . As this enzyme is the main target of organophosphates and carbamate insecticides, alleles in ache have been documented to confer resistance. Using the amino acid numbering scheme based on T. california , the resistance allele A201S has previously been reported to be present in European populations , and we found this allele to be at moderate to high frequency in all three regions. We also found moderate frequencies of the mutation F290V and F290N. The F290V resistance allele has been documented in mosquitoes and moths , while other mutations such as F290Y have been documented in Drosophila and M. domestica . Interestingly, based on mapping read depth the North population only contained a single copy of ache. As duplication of ache has been implicated in improved organophosphate resistance , square planter pots this large structural duplication may be the reason for elevated PBS levels across such a large interval. Follow-up work with long-read methods or higher sequencing coverage will be needed to confirm the presence of structural duplication at this locus. We expect that addition of a new contiguous genome assembly with annotations will be of benefit to the Tuta absoluta and Lepidopteran research community. Previous studies have worked to develop potential RNA interference strategies to use as an alternative to traditional pesticides . Work is also being conducted to develop Cas9 gene-editing techniques for Tuta absoluta to facilitate future genetics studies . These developments in combination with an accurate assembly and gene annotations will allow for accelerated research towards understanding Tuta absoluta biology and methods to contain its economic impacts and spread.The tomato plant represents a massive economic industry worldwide, with an estimated 252 million metric tons of tomatoes harvested in 2020 .

This production is concentrated in a few major producing countries, with 70% of the world’s production currently accounted for by China, the European Union, India, the USA, and Turkey . However, this industry is quickly becoming threatened by the invasive gelechiid moth Tuta absoluta Meyrick, commonly known as the tomato leafminer. T. absoluta is an agricultural pest of the nightshade family, including peppers, eggplants, and potatoes, but primarily represents a serious threat to tomatoes. Left untreated, the larvae will consume leaf tissue, bore into flower buds, or burrow into fruit with infestations causing crop losses as high as 80 to 100% . While T. absoluta was first identified in Peru in 1917 , it was not considered an agricultural pest until the 1960s when T. absoluta was detected in farms in Argentina, causing significant crop loss . From there it rapidly spread throughout South America, causing severe agricultural losses everywhere it was found. In 2006, T. absoluta was identified in a greenhouse in Spain, marking the first intercontinental migration of the pest, likely due to anthropological transportation . From Spain, it rapidly spread throughout Europe, SubSaharan Africa, the Middle East, and most recently into Asia . Without rapid identification and quarantine strategies, T. absoluta will likely continue to spread to North America. Preventing initial establishment of an invasive species in a risk ecosystem can be an effective strategy but requires vigilant monitoring of imported products as well as an ability to rapidly detect the presence of the target . In the United States, policies such as federal orders issued by the Animal and Plant Health Inspection Service have been used to prevent accidental introduction of T.absoluta by regulating import of tomato fruit or propagation material from infested countries . Correctly detecting and identifying the insect, however, remains challenging due to the presence of other gelechiid moths such as P. operculella and Keiferia lycopersicella in the United States, which look nearly identical to T. absoluta and are also commonly found on tomato and potato crops . Distinguishing these species morphologically requires dissection of adult male genitalia, which requires entomological expertise and increases waiting times for identification. Molecular diagnostics, on the other hand, would allow for accurate and rapid identification without entomological expertise. To date, several molecular diagnostics have been developed, all using Polymerase Chain Reaction to amplify target DNA presence before detecting signal either by fluorescence, gel electrophoresis, or mass spectrometry . A potential limitation of these PCR-based diagnostics is the requirement for a thermocycler, as well as specialized equipment to visualize results. For field detection, a system that requires minimal specialized equipment would be ideal. The CRISPR-Cas system has recently become an attractive option for molecular diagnostics . CRISPR-Cas originated as a bacterial immunity system, in which the Cas protein complexes with a CRISPR RNA that is complementary to a target DNA sequence . When the Cas-crRNA complex binds to its target it cleaves it, causing a double-strand break in the DNA. While Cas9 is the most commonly used Cas protein for its genomic editing capabilities, the Cas12a enzyme was discovered to exhibit indiscriminate single-stranded DNA nuclease activity upon binding to its target DNA . If a single-strand oligonucleotide probe modified with a fluorophore and quencher is present when Cas12a-crRNA binds its DNA target, Cas12a will proceed to also cleave the single-strand probe, separating the fluorophore from the quencher and allowing a fluorescent signal to be measured. As ssDNAse activity is proportional to the number of target DNAmolecules and can occur isothermally between 15℃ to 50℃, using an isothermal DNA amplification method such as Recombinase Polymerase Amplification to amplify target DNA allows for sensitive detection with Cas12a at constant temperature, eliminating the need for an expensive thermocycler. Additionally, detection of probe cleavage is flexible as several methods have been published on alternative visualization methods, including using flow strip assays, gold-conjugated DNA probes, and naked-eye fluorescence detection . In this study, we leveraged prior sequencing data to develop two molecular diagnostic assays.

Even if honey bees displace wild insects at the flower scale , this is unlikely to scale up to the field, as indicated by our data, if mass flowering crops provide floral resources in excess of what can be exploited by local pollinator populations. Therefore, although insect pollinators appear not to be limited by crop floral resources, yield was commonly pollen limited, as crops set more fruit in fields with more visitation by pollinators . Contrary to the fourth prediction, fruit set increased with flower visitor richness independently of honey bee visitation . Correspondingly, the CVs of fruit set decreased with richness; in contrast, evenness did not affect the mean or CV of fruit set . Visitation by wild insects increased strongly with richness , and improved model fit , even when richness was included in the model . However, richness did not enhance model fit when added to a model with wild-insect visitation , suggesting that the effects of richness on fruit set reflect increased wild-insect visitation . Like wild-insect visitation , richness did not correlate with honey bee visitation . Previous studies have shown that agricultural intensification reduces both species richness of pollinator assemblages and wild-insect visitation . Our results for multiple crop systems further demonstrate that fields with fewer species experience less visitation by wild insects and reduced fruit set, independent of species evenness or honey bee visitation. Globally, plastic gutter wild-insect visitation signals both species richness and pollination services, and is a relatively inexpensive indicator that can be standardized easily among observers in field samples .

Large, active colonies of honey bees provide abundant pollinators that can be moved as needed, hence their appeal for pollination management in most animal-pollinated crops . By comparison, methods for maintaining diverse wild insects for crop pollination are less developed, and research on such pollination services is more recent . Although honey bees are generally viewed as a substitute for wild pollinators , our results demonstrate that they neither maximize pollination, nor fully replace the contributions of diverse, wild-insect assemblages to fruit set for a broad range of crops and agricultural practices on all continents with farmland. These conclusions hold even for crops stocked routinely with high densities of honey bees for pollination, such as almond, blueberry or watermelon . Dependence on a single species for crop pollination also carries the risks associated with predator, parasite and pathogen development . Our results support integrated management policies that include pollination by both wild insects as ecosystem service providers, and managed species, such as honey bees, bumble bees , leaf cutter bees , mason bees , and sting less bees as agricultural inputs . Such policies should include conservation or restoration of natural or semi-natural areas within croplands, promotion of land use heterogeneity , addition of diverse floral and nesting resources, and consideration of pollinator safety as it relates to pesticide application . Some of these recommendations entail financial and opportunity costs, but the benefits of implementing them transcend the supply of pollination services alone and extend to, for example, mitigation against soil erosion, and improved pest control, nutrient cycling and water use efficiency .

Without such changes, the on-going loss of wild insects is destined to compromise agricultural yields worldwide.Half of all fruit and vegetables produced globally are lost each year . While the causes of losses vary by region and commodity, fungal phytopathogens have a widespread role, as 20–25% of all harvested fruit and vegetables are lost to rotting caused by such fungi . In fleshy fruits, this issue is exacerbated because, in general, fruit become more susceptible to fungal pathogens as they ripen . Ripening-associated susceptibility has been demonstrated in multiple commodities including climacteric fruits such as tomato, stone fruit, banana, apple, and pear, as well as non-climacteric fruits such as strawberry, cantaloupe, citrus, and pineapple . The most devastating post harvest pathogens in fruit are those with necrotrophic lifestyles, which deliberately kill host tissue, resulting in rotting. Example pathogens include the model necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum as well as Monilinia spp., Alternaria spp., Rhizopus spp., Penicillium spp., and Fusarium spp. . Plant immune responses against necrotrophic fungi are multilayered, involving recognition of pathogen-associated molecular patterns, such as chitin or chitosan, by pattern recognition receptors, intracellular signaling through mitogen-activated protein kinase cascades, induction of downstream defenses by coordinated activity of phytohormones, particularly ethylene and jasmonic acid , cell wall fortifications, and production of various secondary metabolites and antifungal proteins . However, most defense strategies have been studied in leaves, and their utilization and effectiveness in fruit have been assessed only with single pathogens .

The outcome of any fruit–necrotroph interaction relies on the balance between the presence or induction of defenses and the contributions of susceptibility factors. Though induced defenses are heavily studied in plant immunity, the impact of preformed defenses and susceptibility factors are less researched . Preformed defenses include structural barriers, such as the cell wall and cuticle, and the accumulation of secondary metabolites , while susceptibility factors include the abundance of simple sugars and organic acids or activity of host cell wall modifying proteins . A sufficient understanding of ripening-associated susceptibility requires a characterization of the ripening program’s impact on the ability of the host to express necessary defense genes upon pathogen challenge, the integrity of preformed defenses, and the abundance of susceptibility factors. In this study, we first applied a transcriptomic approach to characterize core tomato fruit responses to three fungal pathogens and changes in gene expression that occur during ripening to promote susceptibility. To identify core responses that are not merely pathogen-specific, we used three pathogens with necrotrophic infection strategies: B. cinerea, Rhizopus stolonifer, and Fusarium acuminatum. Using well-established defense gene classifications, we developed profiles of host defense gene expression responses in unripe and ripe fruit. We then determined the susceptibility phenotypes of three non-ripening mutants: Colorless non-ripening , ripening inhibitor , and non-ripening , which have unique defects in ripening features . After demonstrating that each mutant has distinct susceptibility to disease, we identified ripening genes whose expression changes may impact the disease outcome. By integrating our transcriptomic data and mutant analyses, we found preformed defenses and susceptibility factor candidates associated with B. cinerea infections. Using CRISPR-based mutants, we established that one candidate, the pectin-degrading enzyme pectate lyase, is indeed a disease susceptibility factor in ripe tomato fruit.To characterize tomato fruit responses to fungal infection at unripe and ripe stages, we inoculated fruit with B. cinerea, F. acuminatum, or R. stolonifer spores. Each pathogen successfully infected RR fruit, producing visible water-soaked lesions and mycelial growth by 3 dpi, whereas MG fruit remained resistant and, except in samples inoculated with R. stolonifer, had a dark, necrotic ring around the inoculation sites , a feature of the pathogen response that did not appear in wounded fruit. Thus, MG fruit resistance and RR fruit susceptibility are a feature common to multiple necrotrophic infections. We hypothesized that these susceptibility phenotypes are the result of differences in immune responses at each ripening stage and developmental processes during ripening that alter the levels of preformed defenses and susceptibility factors. First, we assumed that, compared with arobust immune response in MG fruit, RR fruit have a weaker response, consisting of fewer genes induced, less diverse functionality, and absent expression of critical genes. Additionally, blueberry container we predicted that ripening may decrease the expression of preformed defenses and increase the expression of susceptibility factors, which create a more favorable environment for infection.

To test if immune responses to fungal pathogens are compromised in RR compared with MG fruit, we sequenced mRNA from B. cinerea-, F. acuminatum-, and R. stolonifer-inoculated fruit at 1 dpi, an early time point at which either a resistant or a susceptible phenotype becomes apparent. A principal component analysis of the mapped normalized reads for all tomato genes revealed that the major driver separating sample data was the ripening stage , while inoculation status accounted for less of the separation . The one exception to this pattern was the R. stolonifer-inoculated MG samples, which clustered with the healthy and wounded MG samples, suggesting that unripe fruit did not display strong responses to this pathogen and yet remained resistant. However, quantification of normalized pathogen reads confirmed that all three pathogens were detectable at 1 dpi even in MG samples. To identify the responses for each ripening stage common to all three pathogens, we performed a differential expression analysis between inoculated and healthy samples for MG and RR fruit. We chose the healthy samples as controls for these comparisons in order to capture responses to necrotrophic infection, which may share features with mechanical wounding. Of all 34 075 protein-coding genes found in the tomato transcriptome, 9366 were found to be differentially expressed in response to inoculation in fruit at 1 dpi in at least one comparison . Of these, 475 genes were significantly up-regulated in MG fruit in response to all three pathogens, corresponding to the MG core response , whereas 1538 genes formed the RR core response . The MG core response overlapped substantially with the wounding response in MG fruit , which suggests that unripe fruit activate similar functions when responding to pathogen attack and mechanical damage. However, this large overlap is also due to the similarity between the gene expression profiles of wounded and R. stolonifer-inoculated samples as seen in the PCA . In contrast, the lack of a strong wounding response in RR fruit indicates that nearly all RR core response genes were strictly pathogen-related . Downregulated genes in response to infection were largely unique to each pathogen, with only 57 and 225 down-regulated across all three pathogens in MG and RR fruit, respectively, and thus we decided to continue our analysis only on the up-regulated core response genes. Complete lists of gene set intersections of up-regulated and down-regulated genes are in given in Supplementary Table S5. We then assessed the MG and RR core responses for the presence of various well-established gene classifications relatedto pathogen defense, including selected GO terms, KEGG pathways, transcription factor families, hormone biosynthesis, signaling and response genes, and receptor-like kinase genes . For each category, we performed enrichment analyses to identify classifications of particular importance in both MG and RR core responses. A total of 70 defense genes were identified in the MG core response. Interestingly, these were enriched in only two categories: chitin catabolic process and RLK genes. The RR core response was enriched in 13 defense categories, including the plant–pathogen interaction and MAP kinase signaling pathways , secondary metabolite biosynthesis pathways , WRKY and ethylene responsive factor transcription factors, RLKs, and JA biosynthesis. Altogether, 302 defense genes were identified among the RR core response. Thus, in contrast to their respective susceptibility phenotypes, RR fruit appear to mount a more robust and diverse immune response than MG fruit early during inoculation, demonstrating that, contrary to our initial hypothesis, weakened immune responses in RR fruit are not a contributor to ripening-associated susceptibility. However, it is possible that tomato fruit resistance to necrotrophs could be determined by a small number of genes that were exclusive to the MG core response. Out of the 70 defense genes in the MG core response, 27 were not found in the RR core response . These 27 genes are heterogeneous, representing 12 different defense categories. Notable genes in this category include a three-gene cluster of PR-10 family proteins , a chitinase previously identified during infections of tomato with Cladiosporum fulvum , and an ERF active at the onset of ripening . Although these 27 genes were not in the RR core response, most of them were induced during RR infections by one or two of the pathogens studied. Only seven were not up-regulated by any of the three pathogens in RR fruit, including the ERF mentioned above , as well as three RLK genes, two glutaredoxin genes involved in the response to oxidative stress, and a cysteine protease. Given that each of these genes belongs to a large family of genes whose members are often functionally redundant, and their average expression levels in infected MG fruit were fairly low , we consider it unlikely that the lack of these genes in the RR core response contributes heavily to susceptibility.