Horticulture

Prior work has typically concerned diurnal cortisol rhythms over the course of the day, within-child child care variation across a more limited span , and rather blunt indices of child care exposure . In contrast, we considered children’s resting cortisol levels at a single point in time, within-child variation over the course of many months, and variation in the actual number of hours children spent in child care per week. Despite these differences , the commonalities between this prior work and the present findings are noteworthy. Taken collectively, the findings suggest that within-child increases in child care exposure—on both short- and long-term developmental time scales—tend to be associated with within-child increases in salivary cortisol, for children from low-risk contexts. In particular, in the present study we found that—holding all other time-invariant confounds constant—fairly long-term shifts in child care exposure across spans of several months may underlie changes in HPA axis function, at least at 24 months of age. This finding with respect to developmental timing is consistent with meta-analytic data suggesting that the link between child care and children’s cortisol increases over the course of the child care day tends to be quite small in infancy yet pronounced toward early childhood .

The cause of these developmental differences remains largely unclear. On the one hand, plastic growing bag it is reasonable to conjecture that the types of experiences that occur in child care, such as exposure to peers, noise, and the divided attention of caregivers, might be particularly salient stressors during infancy. Developmentally, infants lack the internal self-regulatory control that is presumably needed to down-regulate their stress responses . On the other hand, children’s experiences of the same child care context may also be quite different at different points in development . For instance, whereas an infant and early-childhood classroom may maintain the same number of peers, children tend to be more actively engaged with their peers in early childhood . Such normative social differences may begin to explain the emergence of the childcare effect in toddlerhood. This is an important question for future work. Critically, our findings indicated an inversed within-child relation between child care hours and cortisol at 24 month of age, for children from high-risk contexts. For instance, for toddlers with cumulative risk levels that were 1 SD above the mean, increases in weekly child care hours were associated with contemporaneous decreases in children’s cortisol levels. Although this conditional association was somewhat modest—corresponding to a standardized regression coefficient of approximately .14—it is approximately one quarter of the size of the effect of time of day, a known biological mechanism underlying within person cortisol variation. Furthermore, the estimate is robust as a within-person effect, which adjusts for all possible time-invariant confounds.

These findings align with our prior work with respect to testing between-child interaction effect as predictive of children’s resting cortisol levels at 48 months. In that work we found similarly that the direction of the association between child care hours and children’s cortisol levels differed for children from low- versus high-risk contexts . For children experiencing low levels of cumulative risk, attending a greater number of child care hours—on average, between 7 and 35 months—was predictive of comparatively higher cortisol levels, whereas for high-risk children a negative relation was evident. Indeed, the consistency between our prior and present findings is rather remarkable, given the different substantive interpretations of between- versus within-person effects. The former represents one’s average child care exposure compared to other children’s average exposures; the latter represents time-specific increases in child care hours at 24 months, compared to one’s own prior child care hours at 7 and 15 months—irrespective of one’s average level. It is important to note that our prior interactive findings at 48 months and the present interactive findings between 7 and 24 months almost certainly tap distinct effects. As above, the interpretations differ. In addition, 24-month and 48-month cortisol levels are virtually orthogonal . The present findings also align with prior studies that have considered interactive effects between environmental risk and child care exposure in the context of children’s behavioral outcomes. For example, using the same sample as the present study, in prior work we have found that, for children experiencing high levels of household chaos across early childhood, greater weekly hours in child care were predictive comparatively fewer behavior problems. In particular, consistent with the idea of a buffering effect, greater child care exposure ameliorated the detrimental relation between household chaos and children’s social problems.

Similar interactive effect have been noted by others , and work conducted with predominantly lower income samples has increasingly shown that the “beneficial” relations between child care and children’s social and academic outcomes may be stronger and more consistent than those from studies of children from more affluent families . Despite some commonalities emerging across this literature, the mechanisms underlying these interactions remain largely unclear. Our results provide some indication that center based care may play a role. We found that—for children from low-risk contexts—attending greater proportions of one’s time in child care in center-based care was predictive of higher cortisol levels than spending more time on other types of non-parental care. In contrast, no such relation was evident for children from high-risk contexts. To some degree, this is may be inconsistent with prior work. For instance, on the basis of data from their heterogeneous sample, Roisman and colleagues found that greater exposure to center-based care in early childhood was predictive of a lower awakening cortisol levels in adolescence. Of note, though, is that the cortisol awakening response is typically considered to be a process that is distinct from typical HPA axis functioning throughout the day . There is also, however, some reason to suspect that our findings align with some behavioral findings. For instance, there is a good indication that greater exposure to center-based care may be linked to heightened levels of aggression in early childhood —perhaps particularly so for children from more affluent families . Meta-analytic findings indicate a positive correlation between cortisol and aggression levels in early childhood . Indeed, considering the potential secondary effects of cortisol on behaviors and cognition is an important next step we intend to take with these data. However, there is also reason to suspect that the translation of children’s cortisol levels into behavioral effects will not be a simple one. For example, in the present study we also found that children who were exposed to greater numbers of peers, on average, wholesale grow bags between 7 and 24 months, tended to have lower cortisol levels. This is consistent with the extant data , yet it is somewhat inconsistent with the idea that the link between center-based care and heightened levels of aggression is explained by its effects on heightened HPA axis activity: Peer problems may well underlie the effects of center-based care on aggression . Suffice it to say, there is much left to clarify. Interestingly, prior work has been suggested that the negative relation between greater peer play and lower daily cortisol levels may reflect the fact children who are less physically aroused may “select into” greater amounts of peer play . Of course, this may well be the case, yet it is noteworthy that our finding with regard to the number of children in child care—presumably reflecting peer exposure that is considerably less child driven—shows similar negative relation. Although it remains a conjecture to be tested directly, ongoing practice negotiating the social world of one’s peers may, in fact, play a “steeling” or “inoculating” role in the way that children respond physiologically to subsequent peer interactions. Evidence from nonhuman primates suggests that such steeling effects may be common and play an important role in resiliency .As such, our present findings suggest that child care type and peer exposure—at least when considered cumulatively between ages 7 and 24 months—are linked with HPA axis functioning across this period.

However, it is important to note that they do not appear to explain the effects of childcare quantity. Child care quantity, type, and peer exposure were additively and independently predictive of children’s cortisol levels, and no high-order interactions were evident. Like our prior work with children’s 48-month cortisol levels, caregiver responsivity was not related with children’s cortisol levels, between or within persons. Thus, as noted by others in prior work the mechanism underlying the child care quantity effect remains largely unclear. One possibility is that, for children facing the confluence of environmental challenges of poverty, child care may be comparatively less physiologically stressing than their experiences outside of child care. Indeed, the limited available evidence suggests that— unlike their more affluent peers—children from higher risk contexts tend to show a normative diurnal cortisol decline in their cortisol levels across the child care day . If this is the case, then the “beneficial” relation between greater child care exposure and cortisol observed for children from high-risk families may partially reflect a dose–response relation, whereby greater child care exposure leads to less exposure to the pronounced environmental stressors outside of child care. This, of course, remains a question to be addressed directly. Indeed, there are several potentially important mechanisms that were not addressed in the present study—such as the quality of children’s peer relations , teacher–child relationship quality , and degree of classroom chaos —that remain critical areas inquiry, with regard to clarifying the relation between child care exposure and HPA axis functioning.Several of our findings are robust to within-child analyses that accounted for all time invariant confound or between-child analyses that adjusted for several observed control covariates. In fact, with regard to within-child effects, the conservative nature of these estimates may underlie the fact that there were several null within-person relations . On the one hand, these specifications may strengthen the internal validity of our inferences. On the other hand, to the extent that the causal relation between these variables is contingent on cumulative exposure they would be missed by our within-person specification. They allow us to pick up cumulative, traitlike variability in these child care measures—variability that is likely quite meaningful. However, our inferences are only causally unbiased to the extent to which we have accounted for all sources of potential endogeneity . This assumption is typically dubious. As such, we make no causal claims. Nontrivially, though, these potential endogeneity problems would have to explain both selection into cumulative risk and selection into child care, as well as why the direction of the child care effects vary as a function of cumulative risk.As is common with large, comprehensive epidemiological deigns, trade-offs must often be made between breadth and depth. For instance, we were unable to collect multiple saliva samples over the course of a day or across several consecutive days. This would have strengthened the reliability of our cortisol measures and made our findings more directly informative to the extant literature concerning children’s diurnal cortisol patterns . Our saliva samples were collected modally around 10:00 a.m., when the child was at rest, after he or she had had time acclimate to the RAs in the home. There was little evidence that children were particularly stressed by the RAs’ visits. The vast majority of mothers rated their children’s behavior during the visit as being typical. Of the minority who indicated that their children were behaving differently, it was typically due to external reasons . For the few who ascribed differences as being due to the RAs’ visit, they were evenly split between those showing emotions that were comparatively more negative and comparatively more positive than usual. This is consistent with the extant laboratory-based literature that suggests that exposure to novel adults typically fails to evoke an acute stress response—even when it is intended to do so . That being said, we cannot rule out the possibility that our cortisol measures may partially tap reactivity to the RAs’ presence. Finally, by using terms like beneficial to describe, for instance, the negative relation between child care hours and cortisol, we imply that lower cortisol levels may be substantively “better” than higher cortisol levels. Other interpretations are possible. For instance, high levels of cumulative risk could make these children especially vulnerable to child care effects that cause the HPA axis to be underactive . Underactive HPA axis functioning is also a known risk factor for less optimal cognitive functioning and behavior problems . We cannot rule this interpretation out; however, we find it less plausible.

Bayesian analysis—The dataset was analyzed with Bayesian inference using the program MrBayes version 3.2.7a. Sequence data were subjected to a general time reversal model including the estimation of invariant sites and assuming a discrete gamma distribution with six rate categories . The relative fit of various models of nucleotide substitution for the ITS region, chloroplast regions, and combined data set to identify the best model was examined. The best model was selected based on the Akaike Information Criterion . The Markov Chain Monte Carlo sampling, starting from random trees and priors, was run for 1,000,000 generations and every 100th tree was sampled. Four MCMC chains comprising three heated chains and a single cold chain were used in the analyses. Majority rule consensus trees and posterior probabilities for nodes were assembled from all post-burn-in sampled trees. Phylogenetic reconstructions were estimated after a couple of independent runs to confirm that they converged on similar stationary parameter estimates. For the combined data set, plastic nursery plant pot the data for each region were merged and aligned using muscle in MEGA before running on MrBayes.

Maximum Likelihood—For Maximum Likelihood estimation, different regions used different models. For the nuclear region, ITS 4 and ITS 5, sequence alignment was performed using the muscle tool included in the MEGA 10 suite, and the Kimura-2 parameter model was applied. For the chloroplast region involving trnHpsbA primer, alignment was also performed using muscle, but the best model was the Hasegawa-Kishino model. For the combined data, alignments were performed using muscle while the best model was the Tamura 3-parameter model. The bootstrap consensus tree inferred from 1000 replicates in Maximum Likelihood, was taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches in the trees generated.After extraction of DNA, poor bands were obtained during PCR for many of the species studied. This could be due to the old nature of the herbarium specimens used. Several primers were tried for the chloroplast region but for many of them, only very few specimens were amplified during PCR. The best result for chloroplast region was obtained from trnH-psbA. When samples were sent for sequencing, most of the results obtained for trnH-psbA were good for one direction, and rarely did we get good results for both directions. To protect the integrity of the results, only directions with clean DNA sequences were used for alignment and analysis. This problem with trnH-psbA that is reported here was also encountered by [2]. A higher number of successful PCR reactions were obtained for the nuclear ITS region than for the chloroplast regions, unlike the result reported by [2]. Sequences obtained for the ITS region ranged from 500 to 550 bp, while those for the chloroplast region were mostly below 500 bp. Although more sequences amplified for ITS, the tree generated for both nuclear and chloroplast regions were not significantly different, so the data were combined.

Clean reads that are suitable for analysis were obtained for the two primer sets used for this study, but more samples were amplified for the nuclear region. To combine the data, only samples that we got sequences for both regions were used. This is to ensure that none of the regions will influence the topology of the tree more than the other. Also, for ease of comparison of clades obtained from the combined dataset to clades from the separate regions.Tree from combined dataset is shown below, Figure 2. The values of the posterior probabilities of the branches obtained are used as measures of branch support. Values below 0.95 are considered to have very low support. All the trees obtained from Bayesian inference and Maximum likelihood show a close relationship between Englerophytum and Synsepalum. Trees from separate analyses are available from corresponding author.Neither Synsepalum nor Englerophytum were not resolved as monophyletic and some of the clades in the polytomy corresponded to the previously recognized genera by Aubréville. Three of the species previously grouped under the genus Afrosersalisia were used in this study namely S. afzelli, S. cerasifera, and S. kassneri. As seen in Figure 2, they belong to the same clade,—clade C. For the Vincentella genus, out of the four species, data were successfully obtained for three species. These include S. muelleri, S. passargei, and S. revoluta. None of the herbaria we loaned specimens from had S. brenanii. Just like the report for Afrosersalisia, the species are in the same clade, -clade A in Figure 2. The case is slightly different for the genus Pachystella. Out of the three species in Pachystella, two species are in the same clade, clade C, separating them from S. subverticillatum, which was found in clade A. For the genus Synsepalum sensu stricto, three species were previously recognized. These include S. dulcificum—the type species for the genus, S. stipulatum, and S. subcordatum. All three species were included in this study. In this group, all the species were in one clade, clade D.

It is important to note, however, that these three species were not the only species found in the clade, other species formed the same clade with them.Two principal issues are addressed in this work. One is the merging of the small genera recognized by [7], while the second issue is the divergent view of some authors on the Synsepalum–Englerophytum complex. The previously recognized small genera Afrosersalisia, Vincentella, Pachystela, Synsepalum, Tulesta. After the revision of Sapotaceae by Pennington in 1991, 19 more species have been added to the genus Synsepalum bringing the total to 35 species currently recognized. Some authors including have called for the separation of Synsepalum from the previous small genera that were combined. As seen in the combined data tree generated, some of the clades corresponded to the previous genera that were recognized by Aubréville. Data analysis using Bayesian inference and Maximum likelihood for ITS grouped all three species of Synsepalum sensu stricto used in this study into one clade. A dichotomous tree having species of Synsepalum in one clade and species of Englerophytum in another clade would have supported the monophyly of Synsepalum, however, the tree obtained from the nuclear, chloroplast region and combined dataset resulted in a phylogenetic separation of subgroups of species belonging to both genera. In the combined tree reported here, clades of Englerophytum are nested within Synsepalum. This consistent nesting of Englerophytum within Synsepalum further suggests that the genera are closely related. Leaf venation, presence or absence of stipules, inflorescence, fusion of sepals, stamens insertion, anther position, and number of ovaries tend to overlap in the previously merged genera. The overlapping morphological character and the molecular evidence seen in tree suggest very strongly that Synsepalum sensu lato should be reversed into small genera.Molecular evidence obtained from this study and all other phylogenetic studies involving the species merged by Pennington and the newly described ones for both Synsepalum and Englerophytum shows that the two genera are not phylogenetically distinct from each other. Thus, the trees obtained in this research do not agree with the circumscription of the genera as defined by Pennington. All the trees obtained using MrBayes for Bayesian inference and MEGA X for Maximum likelihood show that Englerophytum is nested within Synsepalum. It is important to state here that only a few morphological characters were used by [7] to separate Englerophytum from Synsepalum. Synsepalum has an eucamptodromous venation and Englerophytum has a brochidodromous venation and a strong fusion of thefilament into a staminal tube. Although it is taxonomically correct, in some cases, seedling starter pot to use few morphological characters to separate genera, this is not just the case with the EnglerophytumSynsepalum complex. The nesting of Englerophytum within Synsepalum in trees generated for both nuclear chloroplast regions indicates very strongly that the group do not merit distinct generic status.In this study of the phylogenetic relationships of the Synsepalum–Englerophytum complex, neither of the two genera resolved as monophyletic.

Although resolution of relationships among the major clades were only weakly to moderately supported, the clades of Englerophytum are nested within Synsepalum as seen in the combined tree. This shows that the morphological circumscription of the two genera does not align with the molecular evidence. We think that morphological character states that distinguish Synsepalum might be ancestral while the character states that distinguish Englerophytum may be independently derived synapomorphies for each subclade. The polytomy observed in the genus Synsepalum shows that the genus is not a single lineage. Some of the clades in the phylogenetic tree correspond to some of the small genera merged by Pennington. The need to resurrect some of the previously merged genera, like Vincentella, is undeniably evident in the result obtained in this work and that of [2]. The Synsepalum–Englerophytum complex certainly requires some formal taxonomic changes to align with molecular evidence, but such changes cannot be made until a broader sampling of all species currently recognized in the genera is collected for comprehensive morphological as well as molecular studies. The complete genome of Synsepalum has been published. If the complete genome of Englerophytum is published, it would serve as additional information relevant to making definitive and comprehensive conclusions on taxonomic circumscriptions within the Englerophytum–Synsepalum complex.After zinc, boron is the second mostimportant micronutrient deficiency problem in California vineyards. Boron deficiencies are most common in the old flood plains and alluvial fans of the Stanislaus, Merced, San Joaquin, Kings and Kaweah rivers; the Sierra Nevada foothills; and North Coast sites with basaltic soils subject to high rainfall. Vineyard boron deficiencies are mostly associated with soils derived from basaltic and granitic parent material of the Sierra Nevada and North Coast ranges. Low boron is also associated with higher rainfall areas and soils irrigated with snowmelt water originating from the Sierra Nevada. In contrast, boron levels are typically higher and can even be toxic in soils originating from marine sedimentary material, such as in the Central Coast range. Grapevine reproductive tissues are most sensitive to boron deficiency, which results in reduced fruit-set, small “shot berries” that are round to pumpkin-shaped, and flower and fruit cluster necrosis. Boron deficiency can have a drastic effect on fruit quality and yield, even when there are only mild-to-moderate foliar symptoms. At the same time, the over-application of boron can result in plant phytotoxicity. Phytotoxicity begins as a necrosis of the leaf margins that can cause a downward cupping of the young leaves. Most commonly, grape growers have applied boron to the soil by hand or as a direct soil spray, sometimes in combination with an herbicide application . However, such applications must be carefully timed to allow for winter rainfall or irrigation to move the boron into the root zone. Boron applications by foliar spray and drip irrigation are of increased interest for their convenience and the potential for faster vine response. Foliar boron application has been studied in tree crops such as pears, prunes, cherries and almonds, and the application timing was found to A boron-deficient Thompson Seedless cluster in the trial vineyard shows reduced fruit-set, the presence of numerous pumpkin-shaped “shot berries” and necrosis of some branching. Fewer than 10% of the berries are normal size and shape. influence fruit-set and development . However, there is limited research on vine uptake and response to foliar boron and the potential for toxicity. We conducted several studies on the timing of boron foliar-spray applications in an eastern Fresno County vineyard with mild-to-moderate boron deficiency symptoms.The studies were conducted in 1998 and 1999 in an own-rooted, furrow irrigated ‘Thompson Seedless’ grape vineyard on Delhi loamy sand. The vineyard was irrigated with canal water and about 25% supplementation from well water. The extremely low boron content of canal water can contribute to low boron availability in sandy vineyard soils. A preliminary study was conducted in 1998 to determine the influence of foliar sprays on boron concentrations in vegetative and reproductive parts of the vine at bloom. Spray treatment was applied at 2 1/2 weeks pre-bloom on May 6, 1998. The two comparative treat-ments were an untreated control and a foliar boron spray at 1 pound per acre applied as Solubor at 100 gallons per acre . The trial design was a randomized complete block with four-vine plots, replicated 10 times. Vine tissue samples were taken at bloom on May 23, 1998; triple-rinsed with distilled water and oven-dried; and analyzed for boron at the ANR Analytical Laboratory at UC Davis.

The consequence of such a relation in 3D is discussed. The organization of this chapter is as follows. We introduce the cubic lattice Dirac model and a transformation on the low energy Dirac fermions to bring it into a ‘normal’ form in Sec. 3.2.2, from which one can easily read off the orders that lead to an energy gap. The chiral topological insulator is identified, and a microscopic model with hopping along the body diagonals of the cube, is shown to lead to this phase. An intuitive picture in terms of a quasi 2D starting point is developed in Sec. 3.3.2, which directly demonstrates the existence of surface Dirac states. Here, we also show how a Z2 topological insulator protected by TRS with bulk Dirac nodes at TRIM can be understood within this picture. In Sec. 3.4 the magnetoelectric coefficient ‘θ’ of such an insulator is argued to be quantized , and is calculated to be θ = π for the spinless fermion model we discuss. Introducing spin, and studying gapped superconducting states, we show in Sec. 3.6 that only a pair of singlet superconductors is allowed, which, in addition to the regular onsite s-wave paired state, bucket flower includes a singlet topological superconductor, with pairing along the body diagonals. Sec. 3.7 describes an attempt to move towards a more physical realization of the cTI phase, utilizing a layered honeycomb lattice structure.

Finally, in Sec. 3.8, we explore some topological properties of the 3D Dirac fermion system by studying its physics in the presence of point topological defects in its order parameters, and deriving the Berry’s phase terms that determine quantum interference of different orders.In many ways, a chiral topological insulator can be viewed as a close cousin of the known topological states in 3D, such as a Z2 topological insulator: A hallmark of both of these states is an appearance of non-trivial surface modes when topological bulk states are terminated by a boundary. However, for a chiral topological insulator, an arbitrary number of flavors of Dirac fermions can appear at the surface and be stable. We discussed a physically transparent picture of the chiral topological insulator, which explains the appearance of surface Dirac fermion states, and their stability in the presence of chiral symmetry. A similar picture also explains the stability of TRS Z2 topological insulators whose bulk Dirac nodes are centred at TRIM. It is shown that the θ = π axion electrodynamics can also be realized in chiral topological insulators, in addition to the known realization in Z2 topological insulators. We should also stress that chiral symmetry, which is realized in the models discussed here as sublattice symmetry, is likely broken in any realistic systems. Nevertheless, as far as breaking of chiral symmetry is sufficiently weak, θ is expected to be close to π, and can It is also worth while mentioning that chiral symmetry need not to be realized only as sublattice symmetry: class AIII symmetry can be realized in the BdG Hamiltonians for Szconserving superconductors. Thus, chiral symmetry when realized in this way is much more robust than sublattice symmetry.

Furthermore, in the 3D π-flux lattice model with inclusion of spin degree of freedom, we found a spin-chiral topological insulator , and also a singlet topological superconductor . The latter is stable as long as the physical symmetries of SU spin rotation and time reversal are present. Finally, utilizing the proximity to a Dirac state, we derived an interesting correlation, or “duality”, between the singlet topological superconductor and Neel order. These order parameters are dual in the sense that a topological defect in either one of these phases carry complementary quantum numbers: e.g.. a defect in the Neel vector can carry electric charge. We also find many such 6-tuplets of order parameters, including a six component vector consists of three Neel order and three VBS order parameters. These dualities are a natural extension of those discussed in 1D and 2D quantum spin models, the latter in the context of deconfined criticality. While in this chapter we have studied the properties of these topological defects at single particle level, and hence the topological defects are static objects, we cannot resist contemplating more interesting situation where they are dynamical entities. In particular, it is interesting to ask if there is a counterpart of the non Landau-Ginzburg transition, realized in two dimensions, can exist, possibly in the presence of strong electron correlations in three dimensions. This is left for future study. P.H. would like to thank Shinsei Ryu and Ashvin Vishwanath for the collaboration in this work. We wish to thank Ying Ran and Tarun Grover for insightful discussions, and the Center for Condensed Matter Theory at University of California, Berkeley and NSFDMR-0645691 for support. This work is published in Physical Review B, 81, 045120 .In the last chapter, we saw interesting physics emerge when appropriate sets of phases were combined together to form three component order parameters, and topological defects were created in them. A natural question to ask is whether there are other topologically non-trivial ways of combining the phases which give rise to unconventional phenomena. The strong topological insulator is a good candidate for being involved in some novel physics, since rich phenomena have already been predicted to arise when this phase is combined with conventional orders such as magnetism, crystalline order and superconductivity.

The last is particularly interesting. Superconductivity induced on the surface of a TI was predicted to have vortices harboring Majorana zero modes. These are of interest to quantum information processing, since they are intrinsically robust against errors. Recently, superconductivity was discovered in a doped TI, which could be used to induce surface superconductivity. Below we discuss a new theoretical approach to studying this remarkable superconducting phase, which provides different insights and directions for experiments. In this case, the low energy description of the system is entirely in terms of bosonic coordinates , much as the Landau Ginzburg order parameter theory describes superconductors at energies below the gap. Can fermions ever emerge is such a theory? While it is easy to imagine obtaining bosons from a fermionic theory, the reverse is harder to imagine. However, it has been shown in principle that bosonic theories that contain additional Berry’s phase terms, can accomplish this transmutation of statistics. We show that this indeed occurs in the superconductor-TI system; the order parameter theory contains a Berry phase term which implies that a particular configuration of fields – the Hopf soliton – carries fermionic statistics. While such statistics transmutation is common in one dimension, it is a rare phenomenon in higher dimensions. In the condensed matter context, an physically realizable example exists in two dimensions: solitons of quantum Hall ferromagnets are fermionic and charged, and have been observed. However, the superconductor-TI system is, to our knowledge, the first explicit condensed matter realization of this phenomenon in three dimensions. The organization of this chapter is as follows. First, we introduce our simplified model of a topological insulator with surface superconductivity, cut flower bucket and review properties of the Hopftexture. We then discuss evidence from numerical calculations on a lattice model, that demonstrate the Hopfions are fermions. We also discuss the connection between fermionic Hopfions and 3D non-abelian statistics of [128]. A simplified two dimensional example, where skyrmions are fermionic, is also discussed. Next, we provide a field theoretical derivation of the same result in the continuum, and introduce the necessary theoretical tools to compute a topological term, that leads to the fermionic statistics. Finally, we mention physical consequences, for tunneling experiments as well as for Josephson junctions.In the previous chapters, the focus has largely been on bulk topological phases and especially on the bulk of the topological insulator. Although the Majorana zero mode discussed in the previous chapter was localized on the surface, its existence was governed by the bulk topological phase. In contrast, in this chapter we focus only on the surface states of the strong topological insulator . In their simplest incarnation, the SSs of TIs correspond to the dispersion of a single Dirac particle, which cannot be realized in a purely two dimensional band structure with time reversal invariance. This dispersion is endowed with the property of spin-momentum locking, i.e., for each momentum there is a unique spin direction of the electron.

Since several materials were theoretically predicted to be in this phase, most of the experimental focus on TIs so far has been towards trying to directly observe these exotic SSs in real or momentum space, in tunneling and photo emission experiments, respectively, and establish their special topological nature. However, there has so far been a dearth of experiments which study the response of these materials to external perturbations, such as an external electromagnetic field. In order to fill this gap, we calculate here the response of TI surfaces to circularly polarized light. Since photons in CP light have a well-defined angular momentum, CP light can couple to the spin of the surface electrons. Then, because of the spin-momentum-locking feature of the SSs, this coupling can result in dc transport which is sensitive to the helicity of the incident light. This phenomenon is known as the circular photogalvanic effect . In this work, we derive general expressions for the direct current on a TI surface as a result of the CPGE at normal incidence within a two-band model and estimate its size for the surface of Bi2Se3, an established TI, and find it to be well within measurable limits. Since bulk Bi2Se3 has inversion symmetry and the CPGE, which is a second-order non-linear effect, is forbidden for inversion symmetric systems, this current can only come from the surface.We find, remarkably, that the dominant contribution to the current is controlled by the Berry curvature of the electron bands and grows linearly with time. In practice this growth is cut-off by a scattering event which resets the current to zero. At the microscopic level, this part of the current involves the absorption of a photon to promote an electron from the valence to the conduction band. The total current contains two other terms – both time-independent – one again involving an interband transition and the other resulting from intraband dynamics of electrons. However, for clean samples at low temperatures, the scattering or relaxation time is expected to be large, and these contributions will be eclipsed by the linear-in-time one. Hence, this experiment can also be used to measure the relaxation time for TI SSs. Historically, the Berry curvature has been associated with fascinating phenomena such as the anomalous Hall effect and the integer quantum Hall effect and therefore, it is exciting that it appears in the response here. Its main implication here is that is gives us a simple rule, in addition to the requirement of the right symmetries, for identifying the perturbations that can give a linear-in-time CPGE at normal incidence: we look for perturbations that result in a non-zero Berry curvature. Put another way, we can identify perturbations that have the right symmetries but still do not give this current because the Berry curvature vanishes for these perturbations. Importantly, for TI SSs, the requirement of a non-zero Berry curvature amounts to the simple physical condition that the spin-direction of the electrons have all three components non-zero. In other words, if the electron spin in the SSs is completely in-plane, the Berry curvature is zero and no linear-in-time CPGE is expected. The spins must somehow be tipped slightly out of the plane, as shown in Figure 6.1.1a, in order to get such a response. Thus, a pure Dirac dispersion, for which the spins are planar, cannot give this response; deviations from linearity, such as the hexagonal warping on the surface of Bi2Te3 , are essential for tilting the spins out of the plane. CPGE has been observed in the past in GaAs, SiGe and HgTe/CdHgTe quantum wells – all systems with strong spin-orbit coupling. The effect in these systems can be understood within a four-band model consisting of two spin-orbit split valence bands and two spin-degenerate conduction bands. In contrast, TI SSs can be faithfully treated within a two-band model. The simplicity of the latter system makes it more convenient for studying theoretically compared to semiconductor quantum wells, and hence, enables us to determine a connection between the CPGE and the Berry curvature.

Older adults with greatest engagement of rostral anterior cingulate, a prime regulator of amygdala activity , self-reported less unpleasantness for lowarousal negative stimuli. Together, these findings provide insight into the neural basis of agerelated changes in affective experience by which older adults preferentially enhance positive information, preferentially suppress negative information, or both. Given the recent reinvigoration of research establishing roles of the amygdala in reinforcement learning and decision-making , future studies should pay specific attention to the amygdala as a possible hub where emotional affective processes and dopaminergic reward-based processes intersect. The positivity effect may also influence decision-making through its modulation of medial temporal lobe memory systems. If positive information has privileged access to memory in aging , future choice behavior may be preferentially biased by positive experiences rather than negative experiences . There is some evidence to suggest that in the context of reward-based learning paradigms, older adults show intact episodic memory performance for the visual cues that predicted reward .

Intact subsequent memory for positive cues is particularly notable because it occurred despite reduced levels of reward-based probabilistic learning in the same subjects . How the strength of episodic memory for reward-related information is related to individual differences in the positivity effect is an open question, procona London container but represents one mechanism by which age-related changes in affective attention may shape decision-making. There are important boundary conditions that affect the occurrence and presentation of the positivity effect that are relevant for understanding how it may impact decision-making in aging. The positivity effect in aging is diminished in scenarios in which older adults are given explicit instructions on how to behave, and which stimuli to attend to . Therefore, it is more likely to impact performance on tasks that permit multiple strategies. Supporting this view, in dynamic learning environments that support exploratory behavior, older adults preferentially bias decision-making on recent positive feedback . The positivity effect is also sensitive to cognitive load. Specifically, enhancement of attention and memory for positively valenced information is absent or reversed relative to young adults when cognitive load is increased .

Therefore, the positivity effect is more likely to impact decision-making performance on tasks that support multiple strategies and are low in their computational requirements. To illustrate how the age-related positivity effect may influence decision-making performance, we use PST as an example. The PST, which does not have explicit instructions guiding choice strategies and is relatively low on working memory demands, is potentially sensitive to the age-related positivity effect. For young adults in which the positivity effect is largely absent, performance may be primarily predicted from measures of dopamine function. For example, D1 BPND would be positively correlated with choice selection to approach reward . For older adults in which the positivity effect is present, performance may be shifted from predictions based solely on measures of dopamine function . Reduced D1 BPND in aging would predict a general reduction in choice selection to approach reward relative to young participants. However, socioemotional selectivity accounts of PST performance in aging would predict greater choice selection driven by motivation to approach reward, especially if memory for the reward-predicting cue is particularly salient . Here, we suggest that the positivity effect can shift choice bias in directions that are opposite to what would be predicted based on dopamine effects alone. Such shifts may rely on the strength of the positivity effect within individuals, but nonetheless likely reflects a factor contributing to mixed and null effects in studies examining decision-making in aging.

Lines of future research should empirically test the possibility that dopaminergic decline and enhancement of positive affective processing counteract one another in laboratory-based decision-making tasks. This could be achieved by generating a normative measure of memory for positive, negative and neutral images in a large group of young adults from which to measure the magnitude of biases in individual older adults. Lang, Bradley, and Cuthbert have developed a catalogue of images with affective normative ratings, which could support these efforts . Assessment of the degree to which affective bias measures are related to variability in reward-based and punishment based decision-making would be an important contribution. Future studies could also test the extent to which individual differences in amygdala reactivity and hippocampal engagement during learning positive versus negative associations are predicted by individual differences in the positivity effect.There is an increasing number of innovative new studies examining the neural mechanisms underlying age-related changes in decision-making. The integration of in vivo imaging measures such as PET and neuromelanin-sensitive MR with computational models is a promising avenue for resolving the basic neurochemical drivers of individual differences in performance. This approach will be essential for disentangling age effects of dopamine from age effects of affective attentional bias that contribute to biases in decision-making. This approach will also be useful for defining the nature of changes in reward processing in psychological and psychiatric disorders associated with altered dopamine function and dys regulated affective processing such as schizophrenia, addiction, and depression. Critical steps for future research will be to determine the degree to which findings of laboratory-based assessments of decision-making and financial risk taking can be extended to real-world scenarios. To date, the relatively few cognitive neuroscience studies that have tested these relationships in aging have shown general agreement between performance on financial reward tasks and real-world financial measures . Future studies may focus on predicting the conditions under which older adults are vulnerable to suboptimal decision making. If changes in affective focus in aging partly counteract the detrimental effects of dopamine loss, older adults may be differentially impacted in decisions that occur in conditions in which the positivity effect is suppressed. For example, the positivity effect may be diminished for decisions made in high cognitive load conditions, where there is excessive or irrelevant information presented.Grapes are profitable fruit crop that are widely grown in the state of California, with an increasing need to accomplish cultural tasks mechanically , 2020; Kurtural and Fidelibus, 2021. However, there are many factors that are currently challenging the productivity, quality, and sustainability in wine grape vineyards, one being the increasingly significant global warming trend affecting California and the whole world , where more frequent heat waves and continued warming of air temperature imposes great threats to vineyard yield, berry and wine composition . Grape berry and wine quality are determined by the composition and concentration of secondary metabolites accumulated in berries. Flavonoids are the most abundant secondary metabolites and contribute to many quality determining traits, including color, mouthfeel, cut flower transport bucket and aging potential of wine . There are generally three classes of flavonoids in wine grapes, including anthocyanins, flavonols, and proanthocyanidins. Anthocyanins are responsible for grape berry and wine color, and they are sensitive to external environmental conditions when clusters are exposed to solar radiation and heat, with overexposure resulting in anthocyanin degradation . On the other hand, flavonols tend to be positively related to solar radiation . Solar radiation, especially UV-B, can often up-regulate flavonols’ biosynthesis, resulting in more flavonols accumulated in berry skins. However, excessive solar radiation received and heat accumulated in California would accelerate the degradation of not only anthocyanins, but also flavonols, which will cause a decline in the antioxidant capacity of resultant wine and a possible reduction in wine aging potential . In viticulture, trellis system selection is a critical aspect grower needs to consider when establishing a vineyard. An ideal trellis can promote grapevines’ photosynthetic capacity through optimizing light interception by the grapevine canopy.

Most importantly, a suitable trellis can optimize canopy microclimate by providing sufficient solar penetration into canopies since solar radiation is necessary to enhance the berry composition without excessive exposure of clusters to direct sunlight to avoid flavonoid degradation . There is evidence that grape clusters over-exposed to solar radiation are prone to occur with some of the widely used trellis systems. For example, vertical shoot position , a traditional and commonly used trellis system in viticulture production, has been found to produce canopies with high porosity which increases vulnerability of clusters to over-exposure , causing overly enhanced maturity and considerable degradation in berry anthocyanins . However, there is a lack of evaluations of the performance among various trellis systems in relation to the warming climate trends, and how their specific architectures contribute to variations in berry chemical profiles. In warm climates such as California, viticulture relies on irrigation for maintaining production, and previous work in the area showed that the application of different amounts of crop evapotranspiration can significantly modify polyphenolic and aromatic profiles in wine . Due to the increasingly frequent drought condition in many wine grape growing regions, recent studies have been focusing on the grapevine physiological and berry chemical responses towards specific levels of water deficits imposed by different ETc replacements, where water deficits are affective in manipulating grapevine water status, leaf gas exchange, components of yield, and berry composition: often, more water deficits applied to the grapevines would diminish photosynthetic capacities, but promote berry maturity . In some extremely drought conditions, however, severe water deficit might lower flavonoid concentration due to encouraged chemical degradation . Moreover, these effects resulted from different irrigation regimes can be modified by the canopy architecture as functions of trellis system since trellis systems can directly determine canopy sizes, hence resulting in different water demands from grapevines accordingly . On the other hand, over extraction of ground water to irrigate permanent crops have recently been questioned and legislation has been enacted in the state of California called the ‘Sustainable Groundwater Management Act’ . As a result, in some regions such as Napa Valley of California, grape growers will only be allowed to irrigate 120 mm per year. However, there is a lack of information on how the existing vineyards will cope with this water limitation in terms of irrigation scheduling. Therefore, the objectives of this study were to evaluate and compare 6 different trellis systems in combination with 3 irrigation strategies to understand the impact of trellis system and applied water amount on canopy architecture, grapevine physiology and berry composition. We hypothesized that traditional VSP systems would not be as efficient as the other trellis systems in terms of yield production and flavonoid accumulation, leading to greater berry flavonoid degradation and overall lower flavonoid concentrations.The second subset of 20 berries was used for the determination of skin flavonoids from each individual treatment-replicate. Skins were manually removed from the subset of 20 berries and subsequently lyophilized . After lyophilization, dry skin weights were recorded and then, the dried skins were ground into finepowder with a mixing mill . 50 mg of the freeze-dried berry skin powder were collected, and the skin flavonoids were extracted with 1 mL of methanol:water:7M hydrochloric acid to simultaneously determine flavonol and anthocyanin concentration and profile as previously described by MartınezLüscher et al. . The extracts were stored overnight in a refrigerator at 4°C. In the next day, the extracts were centrifuged at 30,000 g for 15 minutes, and the supernatants were separated from the solids and transferred into HPLC vials after being filtered by PTFE membrane filters . Then, the samples were injected into HPLC for chromatographic analysis.Anthocyanin and flavonol concentrations in berry skin tissues were analyzed with a reversed-phase HPLC with the use of two mobile phases: 5.5% formic acid in water and 5.5% formic acid in acetonitrile. The specific method used for this study required a C18 reversed-phase HPLC column for the analysis . The flow rate of the mobile phase was 0.5 mL min- 1 and the flow gradient started with 91.5% A with 8.5% B, 87% A with 13% B at 25 min, 82% A with 18% B at 35 min, 62% A with 38% B at 70 min, 50% A with 50% B at 70.01 min, 30% A with 70% B at 75 min, 91.5% A with 8.5% B from 75.01 min to 90 min. The column temperature was maintained at 25°C on both left and right sides of the column. All chromatographic solvents were of high-performance liquid chromatography grade, including acetonitrile, methanol, hydrochloric acid, formic acid. These solvents were purchased from Thermo-Fisher Scientific . Detection of flavonols and anthocyanins was recorded by the diode array detector at 365 and 520 nm, respectively. Investigated anthocyanin derivatives included di-hydroxylated forms: cyanidin and peonidin, and trihydroxylated forms: delphinidin, petunidin, and malvidin; investigated flavonols included a mono-hydroxylated form: kaempferol, di-hydroxylated forms: quercetin and isorhamnetin, and tri-hydroxylated forms: myricetin, laricitin, and syrigintin.

White or coextruded white-on-black mulches can slightly lower surface soil temperatures by about 1ºC at 2 cm depth or 0.4ºC at 10 cm relative to bare soil because they reflect most incoming radiation . These mulches are used when lower soil temperatures may be desirable for planting vegetables in particular summer production windows. Clear mulches effectively retain much of the heat normally lost to the atmosphere by bare soil, increasing daytime soil temperatures from 4º to 8ºC at a depth of 5 cm , and 3º to 5ºC at 10 cm relative to bare soil . These clear plastics are the choice for soil solarization. Clear plastics, however, do not control weeds and require other weed management practices such as fumigation and herbicide application. Plastic mulches also influence nutrient levels and uptake. Wien and Minotti found plastic mulching increased shoot concentrations of nitrogen , nitrate , phosphorus , potassium , calcium , magnesium , copper and boron in transplanted tomatoes. Bhella , also working with tomatoes, 30 litre plant pots found higher levels of ammonium , nitrate , and magnesium in plastic mulched soils.

Hassan et al. found higher levels of nitrogen, phosphorus, potassium, and calcium in leaf tissue of chilies grown over plastic reflective mulch compared to those grown over bare soil. A wide variety of other colored plastic mulches, including red, yellow, silver, blue, gray, and orange, have been used in various efforts to achieve specific production goals. Each of these colors has distinct spectral reflectivity characteristics and thus modifies the radiation balance in and below a crop canopy. These colored mulches affect not only the microclimate around a crop but have also been shown to influence insect behavior. Yellow, for example, is generally highly attractive to insects and has been shown to increase green peach aphid and striped and spotted cucumber beetle populations compared to plants grown over bare soil . White at times repels aphids and at other times attracts them, depending on the physiological state of the insect . Orange has been shown to repel various aphids and white flies , while pink and green attract aphids . Red has been shown to attract both aphids and white flies . Highly reflective or shiny aluminum plastic mulches have been shown to repel certain aphids and thereby reduce or delay the onset of aphid-vectored mosaic viruses in zucchini squash and melons and tomato spotted wilt virus in tomatoes . These are the mulches of choice when insect and disease management are the principal objective. On balance, other than UV-metalized reflective mulches, repeated and consistent benefits in managing insects with most colored mulches have not been documented.

Colored plastic mulches have also been used in many parts of the United States to enhance yields. These mulches, however, have produced mixed results . Mahmoudpour and Stapleton noted that “the influence of mulch colour on growth and productivity has been postulated to be highly specific, and may vary with plant taxa, climate, and seasonal conditions.” As is the case with insect management, aluminum mulches have provided the most positive and consistent findings on crop production .Using cover crops as mulches is a relatively recent management strategy that is currently being refined and evaluated in a wide range of vegetable production systems. The winter annual legume hairy vetch , for example, has been used successfully as both a cover crop and as a mulch in fresh market tomato production systems in the southeastern United States. As a cover crop, the vetch fixes nitrogen, recycles nutrients, reduces soil erosion, and adds organic matter to the soil. When mowed and converted to a mulch, the vetch reduces weed emergence, lowers soil temperatures during the hot summer months, reduces water loss from the soil, and acts as a slow-release fertilizer . This system, developed by USDA ARS researchers, eliminates tillage, reduces the need for applying synthetic fertilizers and herbicides, and reportedly adapts to both large- and small-scale tomato production in a low-input, no-tillage system. Recent work in Florida by Chellemi et al. has shown that although a cover crop surface residue mulch production system had lower yields than the standard black polyethylene plastic, the overall profitability of the alternative system was actually higher.

Work in California’s Central Valley has shown that cover cropping increases water infiltration and reduced winter runoff and increases soil carbon . Additionally, cover crops, when cut and dried, have been shown to delay and reduce the incidence of aphids and white flies as well as the incidence of aphidborne viruses. Burton and Krenzer observed a reduction in green bug populations where surface residues of a previous wheat crop existed. Summers et al. found wheat straw mulch significantly delayed and reduced the incidence of alate aphids and several aphidborne cucurbit viruses in zucchini squash. The incidence of silver leaf whitefly and squash silver leaf was also significantly reduced. Similar results were obtained with cantaloupe grown over wheat straw residue. A number of other examples of the successful use of cover crop mulches have been reported in Georgia, Virginia, North Carolina, and Pennsylvania, but their potential in California’s vegetable crop production is only now beginning to be investigated, evaluated, and refined. Combining the potential benefits of surface residue cropping alternatives with those of conservation tillage is becoming increasingly attractive to row crop producers in many of California’s agricultural regions as shown by the following case studies.Beginning in 1995, a series of studies and demonstrations were initiated in the Central Valley to evaluate and develop conservation tillage and cover crop mulch production systems for tomato crop rotations. While the immediate goal of these efforts was to reduce production costs, a longer-term objective was to develop information on the potential of reduced tillage to improve soil quality, store carbon in the soil, and conserve resources. Initial studies, conducted at the University of California West Side Research and Extension Center in Five Points, at the UC Davis campus, and in commercial production fields in Tracy, Vernalis, and Le Grand, evaluated the use of winter cover crops as surface mulches, the feasibility of no-till and strip-till transplanting, and options for in-season weed management. No-till transplanting requires the use of coulters or some form of residue manager to cut surface residues ahead of the transplanter shoe. Strip-till is a form of CT in which a set of coulter or shank implements tills a narrow band of soil 15 to 20 cm wide to a depth of about 8 to 36 cm only in the line into which transplants will be placed. Results from these preliminary evaluations have indicated that planting and harvesting both processing and fresh market tomatoes is possible and that yields comparable to those attained using standard winter fallow techniques may be achieved with certain reduced-till approaches that do not result in excessive cover crop regrowth or weed competition with the tomato crop. On-farm strip trial data for demonstrations conducted in 1999 in Tracy and in 2000 in Vernalis are given in table 3. This early work, and other experiments summarized by Herrero et al. 2001b, also revealed that in-season weed control by a surface cover crop mulch alone is not adequate. The authors of this publication have subsequently investigated and refined the use of a high-residue cultivator that is able to effectively slice through residues while cultivating weeds. Cover crop mulch species selection and mulch management must be optimized if organic mulch tomato production is to expand in California. Care must be taken to avoid the use of certain cover crops such as sorghum-Sudan hybrid as mulches because they are highly allelopathic to tomatoes and several other vegetable crops . More efficient and low-risk production protocols for managing cover crop mulches in vegetable crop rotations must also be developed.Aphid-transmitted virus diseases cause significant economic losses to California’s multi-million-dollar vegetable crop industries annually.

Over the past few years, 25 liter pot plastic production of fall melons , squash , peppers , and tomatoes has been extremely difficult in certain regions of the San Joaquin Valley due to extensive virus epidemics and severe infestations of silverleaf whitefly . Spring crops, while affected to a lesser extent, have also suffered significant losses by aphid-transmitted viruses and whitefly infestations.Several plant viruses are responsible for these epidemics, and most are capable of infecting all of the crops mentioned above. Among the most important viruses are cucumber mosaic virus , zucchini yellows mosaic virus , potato virus Y , and watermelon mosaic virus . These viruses are transmitted by aphids in a styletborne, nonpersistent manner. They are acquired and transmitted in as few as 15 seconds, and are transmitted by a large number of aphid species, all of which are abundant throughout California. Due to the rapidity with which the viruses can be acquired and transmitted, insecticides are of little value in preventing virus spread and under some circumstances may actually increase the rate of virus transmission and spread. This has not, however, dissuaded a large number of growers and PCAs from attempting to control the spread of these viruses by using insecticides. UV-reflective mulches consist of a polyethylene base to which a thin coat of aluminum ions has been adhered. The mulches are collectively referred to as metalized mulches. These mulches reflect UV wavelength , which confuses and repels incoming alate aphids, adult whiteflies, and leaf hoppers , reducing their incidence of alighting on plants . UV-reflective plastic mulches have been used successfully to reduce the incidence of aphidborne virus diseases in squash and other crops . Brown et al. found silver plastic mulch superior to white, yellow, or black with yellow edges in repelling aphids in yellow crookneck summer squash. Plants grown on silver mulch produced significantly higher yields of marketable fruit than did those grown on bare soil. Mulches applied to the planting beds before seeding were effective in repelling alate aphids and delaying the onset of several virus diseases as well as the onset of silver leaf white fly colonization and the appearance of squash silver leaf in spring and fall-planted zucchini squash in California’s San Joaquin Valley . Disease symptoms in plants growing over these mulches appeared 10 to 14 days later than in plants growing on unmulched beds. In spring-seeded squash, approximately 30 percent of the plants on unmulched beds were infected with one or more viruses by the first harvest, while only 10 to 15 percent of those grown over the metalized mulches showed virus symptoms. In fall-planted trials, 100 percent of the plants grown on unmulched beds, with and without insecticide applications, were virus-infected by the first harvest. Metalized mulches were generally more effective in repelling aphids and delaying virus onset than were white-pigmented mulches . Although plants grown over the metalized mulched plots eventually became infected, they continued to produce a significantly higher percentage of marketable fruit throughout the season than did the unmulched controls. In addition, squash, cantaloupes, cucumbers, and corn grown over reflective mulch produced marketable fruit 7 to 10 days earlier that plants growing over bare soil. Stapleton and Summers also showed than cantaloupe grown over reflective mulches yielded over 500 cartons of marketable fruit per acre compared to less than 50 cartons per acre from plants grown on bare soil. A delay of 4 to 6 weeks in infection by CMV in plants growing over reflective mulch allowed them time to mature and set a good crop of melon fruit before becoming infected. Even though the plants eventually became infected, the delay in infection permitted the harvest of a highly profitable crop. Summers and Stapleton have shown that tomatoes grown over reflective mulches averaged approximately 7 percent virus-infected plants, while plants grown over bare soil averaged in excess of 50 percent infection with the same viruses. This approach is currently the only viable means of managing virus disease in these production systems. Growers are cautioned to use only metalized reflective mulches when insect and 5 ANR Publication 8129disease management is the primary objective. Other colors lack the high degree of UV reflectance necessary to repel incoming insects. Also, the mulch must be applied prior to seedling emergence. Plants may be inoculated with aphidborne viruses in the cotyledon stage, and any delay in applying the mulch could lead to an extensive infection. Some crops are more susceptible to injury when planted over metalized mulches.

Another study giving older adults two egg yolks/day for 5 weeks, followed by four egg yolks/day for 5 weeks, reported increases in MPOD, but only among those with low baseline MPOD value. The addition of either spinach or corn , or the combination, for 14 months significantly increased the MPOD among the majority of healthy individuals. Our study has some limitations. Choice of a control is always a challenge in whole food studies, since masking is an issue. A commercially available LZ supplement was used, rather than an inert capsule, since our research design was intended to compare options available to consumers and explore the role of goji berries over and above the intake of purified L and Z. The actual amount of L and Z in the supplement was not confirmed. A previous report noted that the carotenoid content of some powder-based supplements tested in 2017 did not meet label claims, while oil-based supplements did. Since L and Z are preferentially deposited at different eccentricities in the retina, 30 plant pot the different amounts of Z in the goji berries and supplement may not be ideal. Volunteers were not screened for low MPOD as an inclusion criterion.

Although the relatively modest number of participants in each group may raise some concerns, these numbers are similar to those reported by Obana et al. and are consistent with an initial probe study. Finally, although MPOD was the primary outcome measure, other ocular measurements such as contrast sensitivity and best corrected visual acuity were not assessed. Future studies on goji berry intake and eye health ideally should combine functional and anatomic measurements.Viruses are obligate intracellular pathogens that require living host cells to replicate and spread in the infected plant. During compatible interactions, viruses overcome the plant immune system and hijack host cellular processes to establish active infections . Viruses disrupt the plant cell cycle, inhibit cell death pathways, restrict macromolecular trafficking, alter cell signaling, protein turnover, and transcriptional regulation, and suppress defense mechanisms. The interference with these processes in the host leads to a wide range of plant developmental and physiological defects . Cultivated grapevines are highly susceptible to a variety of viruses and viroids, which cause significant crop losses and shorten the productive life of vineyards. More than 65 different viral species classified in at least 15 families have been reported to infect grapevines, which represents the highest number of viruses so far detected in a single cultivated plant species .

Although these viruses are generally transmitted by plant-feeding insects or soilborne nematodes, they can also be spread through infected propagation material . Grapevine red blotch is a viral disease discovered in northern California in 2008 that has become a major economic problem for the wine industry in the USA . This disease is caused by the Grapevine red blotch-associated virus , a circular ssDNA virus with resemblance to geminiviruses, which infects wine grape cultivars with significant detrimental effects on productivity . The incidence and severity of the red blotch symptoms vary depending on the grape cultivar, environmental conditions, and cultural practices . In red-skinned varieties, GRBaV infections result in the appearance of red patches on the leaf blades, veins, and petioles; in white-skinned varieties, they manifest as irregular chlorotic regions on the leaf blades. GRBaV also affects berry physiology, causing uneven ripening, higher titratable acidity, and lower sugar and anthocyanin content, among others . Consequently, must and wine produced from infected berries present altered flavor and aroma. To date, there is limited information on how GRBaV infections affect grape metabolism. Comprehensive analyses to study specific cellular processes that GRBaV exploits to promote infections in berries are still needed, in particular those that relate to changes in berry chemical composition during fruit development. Grape berry development exhibits a double sigmoid growth pattern with three distinct phases: early fruit development, lag phase, and berry ripening.

Most metabolic pathways that promote desired quality traits in grape berries are induced during ripening. The onset of ripening is accompanied by significant changes in berry physiology and metabolism, including softening, sugar accumulation, decrease in organic acids, and synthesis of anthocyanins and other secondary metabolites that define the sensory properties of the fruit . Berry ripening is controlled by multiple regulatory pathways, and occurs in an organized and developmentally timed manner. Interactions between transcriptional regulators and plant hormones regulate the initiation and progression of ripening processes . Like other non-climacteric fruit, grape berries do not display a strong induction of ethylene production and respiration rate at véraison, and the activation of ripening events does not depend primarily on ethylene signaling. Even though the hormonal control of grape berry development is not completely understood, it is established that abscisic acid , brassinosteroids, and ethylene are positive regulators of ripening processes, while auxin delays the initiation of ripening . In the context of virus–grape berry interactions, dissecting the mechanisms that regulate ripening and plant defenses may provide new opportunities to develop vineyard management strategies to control viral diseases and ameliorate the negative effects on berry quality. In this study, we integrated genome-wide transcriptional profiling, targeted chemical and biochemical analyses, and demonstrated that grapevine red blotch disrupts ripening and metabolism of red-skinned berries. We sampled berries at different ripening stages from vines infected with GRBaV and healthy vines in two vineyards. We identified grape metabolic pathways that were altered in ripening berries because of the viral infection. We determined that GRBaV-induced pathways that are normally associated with early fruit development in berries at late stages of ripening, and suppressed secondary metabolic pathways that occur during normal berry ripening and/ or in response to stress. Using targeted metabolite profiling and enzyme activity analyses, we confirmed the impact of GRBaV on phenylpropanoid metabolism. We identified specific ripening-related processes that were disturbed in GRBaV-infected berries. Remarkably, these processes included alterations in ripening regulatory networks mediated by transcriptional factors, post-transcriptional control, and plant hormones, which lead to berry developmental defects caused by red blotch.To determine the impact of grapevine red blotch on berry physiology, we studied naturally occurring GRBaV infections in distinct wine grape-growing regions in northern California . We sampled red-skinned grape berries from two different vineyards, one in Oakville and one in Healdsburg . We used multiple vineyard sites to focus on observations consistently made across environments and, thus, to exclude factors associated with specific environmental or cultural conditions. Prior to sampling, vines were screened for the presence of GRBaV and other common grapevine viruses. The appearance of red blotch symptoms on leaves of GRBaV-positive vines and not on those of healthy controls confirmed the initial viral testing. We sampled grape berries from vines that tested positive for GRBaV and negative for other common grapevine viruses. At the same time, we also collected berries from vines that tested negative for all viruses and included them in the study as healthy controls. In order to determine the impact of the disease on berry development and metabolism, we collected GRBaV-positive and control berries at comparable developmental stages: pre-véraison , véraison , post-véraison , and harvest . This sampling strategy also aimed to limit confounding effects due to differences in the progression of ripening between berry clusters of GRBaV-positive and healthy vines. In some cases, we observed that GRBaV-positive vines presented grape clusters with evident uneven ripening . Comparisons between berries from GRBaV-positive vines and healthy controls indicated that, at equivalent stages of development, grow raspberries in a pot berries affected by red blotch had reduced soluble solids and total anthocyanins in agreement with previous reports on red-skinned wine grapes .

Sampled berries were used for genome-wide transcriptional profiling of viral and grape genes. RNAseq was performed using 3–4 biological replicates of each ripening stage, infection status, and vineyard. We first confirmed the presence of the virus in the berries of GRBaV-positive vines by qPCR amplification of viral DNA . Viral activity in the berries was also assessed by quantifying plant-derived mRNA transcripts of GRBaV genes in the RNAseq data. Plant expression of five out of the six predicted genes in the GRBaV genome was detected in all berry samples obtained from GRBaV-positive vines but not in berries collected from the control vines . The most expressed GRBaV genes in the berries corresponded to V1, which encodes a coat protein, and V3 with unknown function. Expression levels of the GRBaV genes appeared to change as berries ripened. However, we could not determine to what extent the progression of ripening or other environmental factors influenced the plant’s transcription of viral genes because their pattern of variation between ripening stages differed in the two vineyards . Expression of 25 994 grape genes was detected by RNAseq across all berry samples. Principal component analysis was carried out with the normalized read counts of all detected genes. The two major PCs, which together accounted for 42.97% of the total variability, clearly separated the samples based on ripening stage, regardless of the vineyard of origin or their infection status . These results indicated that the intervineyard variation was smaller than the ripening effect, and the overall progression of ripening was similar between berries from GRBaV-positive and control vines. Therefore, we hypothesized that GRBaV infections in berries have altered the expression of particular grape genes and/or molecular pathways, which could subsequently have led to developmental and metabolic defects.While the PCA described above indicated that overall transcriptome dynamics associated with berry ripening were not perturbed by the infection, the lower levels of soluble solids and anthocyanins in GRBaV-positive berries,particularly later in development, suggested that red blotch may affect specific primary and secondary metabolic processes. We therefore focused the RNAseq analyses to identify grape molecular pathways that were differentially regulated as a result of GRBaV infections. We identified grape genes with significant differential expression due to red blotch by comparing GRBaV-positive and GRBaV-negative berries at each ripening stage and independently for each vineyard. We then looked at the intersection of differentially expressed genes between the two vineyards to identify common responses to red blotch. A total of 932 grape DE genes were found to be consistently down- or up-regulated in infected berries in both vineyards at a given ripening stage, and were classified as GRBaV-responsive genes . On average these GRBaV-responsive genes showed 0.49 ± 0.22-fold changes compared with the healthycontrols. Comparing berries at similar ripening stages may have contributed to exclude more dramatic changes in gene expression associated with more pronounced ripening delay due to GRBaV. Key metabolic processes that were suppressed or induced as a consequence of red blotch in ripening berries were identified by enrichment analyses of the functional categories defined by Grimplet et al. in the set of GRBaV-responsive genes . Amino acid bio-synthetic pathways were repressed in GRBaV-positive berries, while amino acid catabolic pathways were induced. Changes in carbohydrate metabolism were also observed; in particular, genes involved in glycolysis/gluconeogenesis and starch metabolism had reduced expression in GRBaV-infected berries.Genes involved in nucleic acid metabolism, including RNA processing and surveillance, showed higher expression in GRBaV-infected berries. These pathways coincided at véraison with the induction of genes involved in stress responses to virus . RNA metabolic pathways are commonly altered in plants during viral infections and have been related to resistance or susceptibility depending on the particular plant–virus interaction . Red blotch also impacted the transcription of several abiotic stress response pathways. In particular, berries after véraison showed lower expression of genes encoding hypoxia responsive proteins and heat stress transcription factors, among others . A prominent feature of the GRBaV-positive berries was the transcriptional suppression of secondary metabolic pathways, in particular the biosynthesis of phenylpropanoids, stilbenoids, and lignin . Because the lower anthocyanin content observed in the GRBaV-positive berries may have resulted from reduced metabolic flux in the core phenylpropanoid pathway and alterations in flavonoid and anthocyanin biosynthesis, we pursued a deeper evaluation of these pathways using an integrated approach of transcriptional and metabolite profiling coupled to enzymatic analyses.Most enzymes involved in phenylpropanoid metabolism are encoded by large gene families. There is also high redundancy among these genes, which ensures the functional integrity and plasticity of the phenylpropanoid-related pathways . Therefore, to test the hypothesis that the red blotch-induced transcriptional changes had an actual impact on phenylpropanoid metabolism, we measured the activity of key enzymes and the abundance of compounds involved in these pathways .

Raspberries account for the majority of the production volume and the total value of this category. This California export category was valued at $157 million, and accounted for approximately 85% of total US fresh and processed raspberry, blackberry, mulberry, and loganberry exports. 84% of these exports are received by Canada, 6% by Japan, and 5% by the European Union. The presence of SWD has clearly increased production costs and caused yield losses for California raspberry producers through a variety of channels. Three previous studies have attempted to quantify the economic cost of the SWD invasion. However, these studies occurred within one or two years of the first SWD infestations in North America when information on the pest was still sparse and management techniques were rapidly evolving. We can improve on these original estimates now that much more is known about SWD biology, risks, and management. We briefly review these original studies before establishing new estimates of the economic cost of SWD in the California raspberry industry. Walsh et al. 1 and Bolda et al. are the first studies to estimate the economic cost of SWD.

These studies utilize yield loss estimates and observations for strawberries, blueberries, raspberries plant container, blackberries, and cherries in California, Oregon, and Washington in conjunction with production data to calculate revenue loss estimates for each state and crop pairing. Walsh et al. assume a yield loss of 20% for all the listed crops in these states. As a result, the study estimates a total of $511 million in potential damages annually due to SWD. Bolda et al. continue the analysis by assuming the maximum reported yield losses of 40% for blueberries, 50% for blackberries and raspberries, 33% for cherries, and 20% for processing strawberries. The study concludes that potential revenue losses across these states and crops could be as large as $421.5 million given current prices. Goodhue et al. 18 refine these estimates of lost revenue for the California raspberry and strawberry industries by including potential price responses into their estimates. This additional assumption reflects that as the production of raspberries and strawberries decreases, the prices of these products may increase in response. The interaction between production and price is quantified with the inverse own-price elasticity of demand for each crop. The elasticity predicts the percentage change in price of a good in response to a 1% increase in quantity demanded. Drawing upon elasticity estimates established in prior studies, the authors conclude that SWD-induced yield losses could decrease California raspberry and processed strawberry revenues by up to 37% and 20%, respectively.

The authors also evaluate the cost of different SWD-targeting insecticide applications and the cost of a specific conventional raspberry pest control program in California’s Central Coast region. The insecticide material and application costs are estimated to be $825.33 per hectare. However, these chemical applications may also provide incidental control of other pests. This implies that the estimate represents an upper bound of the potential chemical control costs associated with SWD.18 The revenue loss and management cost estimates in these prior studies can be substantially improved using current information about SWD-induced yield losses and management practices. Fruit losses due to SWD and SWD management costs have decreased over time as researchers and producers have developed and implemented better techniques for reducing crop losses. We can also more accurately estimate historic yield losses now that more is known about SWD biology, its spread, and the efficacy of different management techniques.Lastly, we can now incorporate increases in labor costs into these SWD management cost estimates.Prior estimates of SWD-induced revenue losses were based on the maximum observed yield losses in different industries where SWD infestations occurred. These estimates provide information about SWD’s damage potential, but do not yield an accurate estimate of actual SWD crop damage. Actual crop damage is useful for estimating revenue losses due to SWD and will differ by year and production style. This analysis incorporates field trial results and expert opinions to estimate SWD-induced revenue losses for the California raspberry industry. SWD infestations directly reduce raspberry yields in two ways. First, fruit infested by SWD decay more quickly. These yield losses are difficult to attribute to SWD because the initial infestation is difficult to detect, and the accelerated decay has a similar appearance to decay caused by fungal diseases, bacteria, and yeasts. Second, raspberry shippers that detect SWD infestations may reject the entire delivery from the grower.

Fresh fruit are held to rigorous quality standards. The risk of rejection of an entire delivery incentivizes growers to eliminate all visible defects in harvested fruit. SWD infestations are more prevalent late in the year as the population grows until winter weather reduces the population. Further, raspberry production is fairly concentrated geographically and the leftover, overripe fruit from nearby fields’ summer harvest acts as a breeding ground for SWD. SWD infestations are also more prevalent in fruit destined for the processing market, where the price is lower than in the fresh market. Fruit intended for processing are harvested later in the season, tend to be riper because they are harvested less frequently, and receive less frequent pesticide treatments. SWD damage rates could change significantly in the future due to pesticide resistance development and the introduction of new SWD management practices, including introducing biological control agents. Recent studies in the US and Europe found that indigenous parasitoids had limited effect on SWD populations. However, in Asia, where SWD originates, several endemic parasitoids attack and develop from SWD. We begin by examining SWD-induced yield losses in California’s conventional raspberry industry. The original reports of SWD damage in the raspberry industry indicated that as much as 50% of production could be lost if SWD was left unmanaged. Yield losses of this magnitude occurred as raspberry producers first learned how to manage SWD, but are now uncommon due to implementation of extensive academic research and industry experience. According to private communications with conventional raspberry producers, they have managed to reduce SWD induced yield losses to less than 3% of production. In recently published reports, conventional raspberry producers that employ effective chemical management programs face virtually no yield losses due to SWD. This substantial reduction in yield losses is primarily attributable to two factors. First, conventional raspberry producers have access to cheap and effective chemical management options. Second, these producers are harvesting their crop more frequently in order to reduce the amount of time raspberries are susceptible to infestation. These observations of actual SWD-induced yield losses are consistent with field trial observations as well. Entomologists Kelly Hamby and Frank Zalom monitored traps and evaluated fruit samples for damage between October 2010 and December 2012 in both organically- and conventionally-managed raspberry sites. Analyzing the 40-fruit samples collected from these fields resulted in estimated yield loss observations for raspberry producers employing standard management practices at the time. SWD-induced yield losses for conventional producers in the study were estimated to be approximately 10% of production in 2011 and less than 1% in 2012. These estimated yield losses are consistent with those observed by De Ros et al. in Italy between 2011 and 2013. De Ros et al. estimated container raspberry losses of 11.5% prior to i and 3.24% after the implementation of an integrated strategy. The yield losses observed in the UC Davis study were concentrated in the fall harvest. The summer harvest is hypothesized to experience less SWD pressure because the population grows throughout the year until cold weather arrives and lack of host fruit in the winter significantly reduces population levels. SWD biology and infestation intensity is affected by climatic conditions and the availability of host fruit, implying that different climatic conditions and influences of neighboring crops could significantly impact SWD-related yield losses. On the other hand, organic raspberry producers still face significant SWD-induced yield losses. Private communications with raspberry producers indicate that these producers experience yield losses between 5% and 15% of production due to a lack of efficacious chemical treatments approved for organic use, and the efficacy and high cost of other labor-intensive SWD management practices.

Once again, these field observations are consistent with the yield losses measured in field trials. SWD-induced yield losses for organic raspberry producers in the study were estimated to be approximately 12% of production in both 2011 and 2012. We calculate yearly estimates of industry-level revenue losses using these observed yield losses due to SWD and a procedure similar to Goodhue et al. . First, we assume an ownprice elasticity of demand for raspberries of -1.66. This elasticity value is the value estimated for fresh raspberries by Sobekova, Thomsen, and Ahrendsen . Second, we assume that actual yield losses in the California raspberry industry correspond to the yield losses observed in the field trials. Specifically, we assume that SWD-induced yield losses between 2009 and 2011 correspond to the yield losses observed in 2011, and that losses after 2011 correspond to the yield losses observed in 2012. Raspberry production and price data are obtained from the U.S. Table 2 provides the resulting revenue loss estimates organized by production practice and year grouping. California’s conventional raspberry producers faced a total of $36.1 million in revenue losses due to SWD between 2009 and 2011. These estimated revenue losses are equivalent to 4.62% of realized revenues over the same period. After 2011, effective SWD management techniques in conventional production eliminated virtually all revenue losses. Revenue losses due to SWD between 2011 and 2014 are estimated to be $277 thousand, which is less than 1% of realized revenues over the same period. In total, California’s conventional raspberry producers faced $36.4 million in revenue losses due to SWD between 2009 and 2014. California’s organic raspberry producers faced a total of $3.43 million in revenue losses due to SWD between 2009 and 2014. These estimated revenue losses are equivalent to 5.74% of realized revenues over the same period. Revenue losses of this magnitude are expected to continue in organic raspberry production until more effective chemical, cultural, or biological management programs are discovered. Furthermore, revenue losses incurred by organic raspberry producers could potentially increase dramatically if SWD populations develop greater resistance to the current, limited set of chemical controls approved for organic use.SWD management is multifaceted. In addition to yield losses, managing SWD has significantly increased production costs for raspberry producers. Raspberry growers increase the number of insecticide applications and use additional labor to harvest their crop in response to SWD infestation pressure. These necessary insecticide applications require additional chemical purchases and access to sprayers and specialized equipment through custom application or purchase. Overuse of pesticides can lead to rejections of shipments if residues exceed legal tolerances for the chemicals; however, producers who adhere to mandatory label rates should, theoretically, never encounter this problem. Conventional raspberry producers have access to a variety of insecticides that provide excellent control for SWD populations at present. Raspberry growers observed in the UC Davis study discussed earlier applied SWD-targeting insecticides four to six times for both the fall and spring harvests. The most commonly used insecticides for this purpose were spinetoram, zetacypermethrin, and malathion. Assuming these chemicals are applied at their maximum label rates and with generic purchase prices observed in 2015, the per hectare material costs of these insecticide applications are $179.40, $7.22, and $29.78, respectively. Using a conventional raspberry grower observed in the UC Davis study as a point of reference, an example chemical management program included two applications of spinetoram and a combined application of zeta-cypermethrin and malathion in both the fall and spring harvest seasons. Each application is estimated to have labor and equipment costs of $61.78 per hectare. In 2015, such a program would cost an estimated $581.14 per hectare in both the fall and spring harvests for a total cost of $1,161.28 per hectare for a single planting. This is consistent with the per hectare treatment program cost of $825.33 observed in Goodhue et al in 2011.Even though conventional raspberry producers have developed effective chemical management programs that virtually eliminate fruit losses due to SWD, organic producers still experience non-trivial yield losses due to more expensive and less effective insecticide options. Most California organic raspberry producers used only two SWD-targeting insecticides, spinosad and pyrethrin, during the time of this study. Of these two insecticides, only the organic formulation of spinosad has efficacy comparable to conventional insecticides. 

Cases with relatively high ground cover fractions and uniformly arranged plants showed good agreement between the 1D and 3D models regardless of whether the assumption of leaf isotropy was made. As the canopies became more heterogeneous in space, agreement between the models generally declined. Although Potato-Uniform and Potato-Row had identical leaf area indices and leaf angle distributions, the regular distribution of plants within the canopy in Potato Uniform resulted in better agreement between the 1D and 3D models compared to Potato-Row. Despite all cases having highly anisotropic leaf inclination distributions, the assumption of leaf anisotropy had relatively small impact for all cases except for the Grape E-W case. Any effects of heterogeneity or anisotropy tended to decrease as the day of year became further away from the summer solstice. Toward the end of the year , the 1D and 3D models were in fairly good agreement for all canopy cases.The diurnal flux of radiation intercepted by the canopy for an hourly time step on Julian day 153 is shown in Fig. 2.6, plastic gardening pots with corresponding fractions of total radiation intercepted by the canopy shown in Fig. 2.7. The fraction of total radiation intercepted on Julian day 253 and 305 are shown in Figs. 2.8 and 2.9, respectively.

For the homogeneous canopy cases, the assumptions of vegetation homogeneity and isotropy were closely satisfied, and therefore, the 1D model was in very good agreement with the 3D model regardless of leaf density . For the crop canopy cases, the 1D model consistently over estimated light interception as compared to the 3D model, except for Grape E-W and Potato-Row on Julian day 305. For all but the grape cases, eliminating the isotropic assumption resulted in little improvement of agreement between the 1D and 3D models, indicating that errors arose primarily from heterogeneity in these cases. For the Grape N-S, Almond, and Potato cases, errors between the 1D and 3D models were largest near midday when sunlight could most readily reach the ground by penetrating through gaps in vegetation. For Grape E-W, the largest discrepancies occurred at early and late times of the day. The effect of row orientation on diurnal interception patterns for the grape cases was dramatic, as this completely changed the character of interception at different times of the year . The potato cases also illustrated the pronounced effect of heterogeneity in planting pattern on diurnal interception patterns.Figure 2.10 depicts vertical profiles of the absorbed radiation flux at 8:00, 10:00, and 12:00 hours on Julian day 153 for Grape N-S, Grape E-W, Almond, and Corn. Errors in absorbed fluxes for Grape N-S were relatively consistent with height, where errors at a given height were most closely related to the magnitude of the absorbed flux at that height. This was also roughly the case for Almond, except that there was the potential for some under estimation of absorption in the lower canopy when the 1D model was used, which was most pronounced for larger solar zenith angles.

For Grape E-W, the 1D model tended to shift the peak in the absorbed flux deeper into the canopy, which was most pronounced for larger solar zenith angles. In the corn canopy, the vertical pattern in radiation interception differed significantly between the 1D and 3D models. There were up to 50% differences between 1D and 3D fluxes at a given vertical level, with irregular patterns of over or under estimation. In the lower canopy, there was a peak in absorption in the 3D model, which was largely absent in the 1D model, leading to under prediction of absorption by the 1D model in the lower canopy. This is likely due to the substantial over prediction of absorption by the 1D model in the upper canopy, which removes the necessary energy needed to produce a secondary peak in absorption in the lower canopy.If leaf azimuth is uniformly distributed, this effectively reduces the impact of anisotropy in leaf inclination on the projected area fraction G. Since a leaf with a certain elevation angle could be parallel to the sun at one azimuth and perpendicular to the sun at another, an integration over all azimuths can smear out the effects of leaf inclination alone. As in the virtual canopies of this study, field measurements have shown that leaf inclination distributions are usually highly anisotropic. The azimuthal distribution of leaves may be strongly anisotropic within a single plant, but for relatively dense canopies, the azimuthal distribution is often fairly isotropic. In these cases, the assumption of leaf isotropy is likely to result in minimal errors. However, sparse, row-oriented crops such as vineyards may have highly anisotropic azimuthal distributions, in which case it may be necessary to explicitly calculate G based on measurements.

These types of canopies are becoming increasingly prevalent in agricultural applications, due in part to the improved access to mechanical harvesters that a trellised or hedgerow canopy provides.Plant spacing and the resulting heterogeneity had the most pronounced effect on errors resulting from the use of Beer’s law. For the Grape N-S case, the assumption of heterogeneity resulted in an overestimation of the total daily absorbed radiation by 28%, 30%, and 36% on Julian days153, 232, and 305, respectively, with larger instantaneous over estimation near midday. For the Grape E-W case, the assumption of heterogeneity also resulted in overestimating the total daily absorbed radiation by 74%, 51%, and 5% on Julian days 153, 232, and 305, respectively. This was not simply an effect related to L, as was illustrated by the two potato cases. By simply rearranging the potato plants from a uniformly spaced into a row-oriented configuration, errors in the 1D model increased substantially. It is possible that the effect of horizontal heterogeneity can vary in the vertical direction, which appeared to be the case with the Corn canopy. This significantly altered the performance of the 1D model at any given height, although the canopy was dense enough overall that the 1D model performed well when predicting whole-canopy radiation absorption. This could have important implications if the radiation model is coupled with other biophysical models such as a photosynthesis model. The response of photosynthesis to light is nonlinear and asymptotic, so although whole-canopy absorption may be well-represented in some cases by a 1D horizontally homogeneous model, it is unclear if that will result in significant errors in total photosynthetic production given the non-linearity of its response to light. A limitation of this study is that results are only applicable under clear sky conditions. However, results can provide some insight regarding diffuse sky conditions by simultaneously considering all canopy geometries and simulated sun angles. Under a uniformly overcast sky, equal energy originates from all directions. A particular combination of sun angle and leaf orientation bias was required in order to observe a pronounced effect of leaf anisotropy. Thus, for diffuse solar conditions, it is speculated that the impact of leaf anisotropy will be decreased. Sun angle had an important effect on the instantaneous impact of leaf heterogeneity, blueberry pot size and most commonly it was observed that low sun angles resulted in a decreased impact of heterogeneity. Therefore, it is likely that highly diffuse conditions will reduce the impact of heterogeneity near midday because a significant fraction of incoming radiation will originate from directions nearer to the horizon. Estimating light interception with Beer’s law is based on the assumption that canopies are homogeneous. This inherently means that the rate of radiation attenuation along a given path is linearly related to the flux at that location. As the canopy becomes sparse, there are pathways for radiation propagation that allow radiation to penetrate the entire canopy without any probability of interception, which fundamentally violates the assumptions behind Beer’s law or a turbid medium. Therefore, the non-random leaf dispersion in canopies limits the ability of Beer’s law to link light interception to simple bulk measures of plant architecture.

It is well-known that this heterogeneity or “clumping” of vegetation usually results in decreased radiation interception as compared with an equivalent homogeneous canopy. A common means of dealing with this problem without significantly increasing model complexity is to add a “clumping coefficient” W to the argument of the exponential function in Beer’s law. While this is a simple and practical means of reducing the amount of radiation attenuation predicted by Beer’s law, the challenge in applying the clumping coefficient approach is that W is a complex function of nearly every applicable variable, and thus is it is difficult to mechanistically specify. Another approach is to use a model that explicitly resolves plant-level heterogeneity, as it may not be necessary to explicitly resolve every leaf if within-plant heterogeneity is small.Row orientation played an important role when estimating light interception from Beer’s law, particularly when the rows were widely spaced. For sparse, row-oriented canopies, the effective path length of the sun’s rays through vegetation can change dramatically with changes in sun azimuth. For East-West rows, absorption is significantly reduced early and late in the day because the rows are close to parallel with the sun’s rays, whereas North-South rows are perpendicular to the sun at this time. As the day of year progresses further from the summer solstice, the sun spends more time closer to the horizon and thus the impact of heterogeneity in an East-West row orientation increased. For the East-West row configuration, G and light interception were surprisingly constant throughout much of the day, which resulted in 41% and 36% less absorption on Julian days 153 and 232, respectively, compared to North-South rows. In some climates, it may be desirable to maximize sunlight interception, whereas in others it may be desirable to mitigate effects of excess sunlight to reduce temperatures and water use.Despite the simplified assumptions in Beer’s law regarding scattering, there was good agreement between predicted radiation interception using the 1D and 3D models in the PAR band. Scattering did not significantly influence light interception in this band because most of the incident radiation received by individual leaves was absorbed. However, in the NIR band, scattering introduced significant over estimation of absorption using the standard 1D model, since leaves are poor absorbers in this band. Using an ad hoc correction to account for reflection only reduced this over estimation of absorption. An additional correction to account for both reflection and transmission resulted in over correction, and a net under prediction of total radiation absorption.The objective of this work was to evaluate common assumptions used in estimating radiation absorption in plant canopies, namely assumptions of homogeneity or isotropy of vegetation. Our results demonstrated that for relatively dense canopies with azimuthally symmetric leaves, a 1D model that assumes homogeneity and isotropy of vegetation generally produced relatively small errors. As plant spacing became large, the assumptions of homogeneity break down and model errors became large. In the case of a vineyard with rows oriented in the East-West direction, errors in daily intercepted radiation were up to ⇠ 70% due to heterogeneity alone, with much larger instantaneous errors occurring during the day. If leaves were highly anisotropic in the azimuthal direction, there was also the potential for large errors resulting from the assumption of vegetation isotropy which had the potential to increase errors above 100%. Day of year had an impact on model errors, which was that overall errors tended to decline with time from the summer solstice. In cases of canopies where the plant spacing starts to approach the plant height, it is likely necessary to use a plant-resolving radiation model in order to avoid substantial over prediction of absorbed radiative fluxes. Additionally, if vegetation is highly anisotropic in terms of both elevation and azimuthal angle distributions, it is also likely necessary to explicitly calculate the projected area fraction G based on measurements and the instantaneous position of the sun.Recent shifts in climatic patterns have influenced the frequency, timing, and severity of heat waves in many wine grape growing regions, which has introduced challenges for viticulturists. Growing the same varieties under these altered climatic conditions often requires mitigation strategies, but quantitative, generalized understanding of the impacts of such strategies can be difficult or time consuming to determine through field trials. This work developed and validated a detailed three-dimensional model of grape berry temperature that could fully resolve spatial and temporal heterogeneity in berry temperature, and ultimately predict the impacts of potential high berry temperature mitigation strategies such as the use of alternative trellis systems.

We created a multiplexed probe-based quantitative PCR assay able to identify whether a sample contains T. absoluta, K. lycopersicella, or P. operculella DNA based on single nucleotide polymorphisms between species. In addition, we created RPA-Cas12a assays to detect the presence of T. absoluta DNA, using either a fluorescent reader or a simple UV illuminator paired with a cell phone camera. This will allow for more rapid field detection of T. absoluta.We assessed 21 single nucleotide polymorphism markers differentiating T. absoluta, K. lycopersicella and P. operculella identified in Tabuloc et al. 2019 for qPCR genotyping potential using RealTimeDesign software . Our analysis resulted in seven potential qPCR assays, each containing a set of universal amplifying primers and two distinguishing fluorescent oligonucleotide probes. We used a serial dilution qPCR on all seven sets of amplifying primers on all three species to assess primer efficiency. Reactions were performed with 300nM of forward and reverse primers with SsoAdvanced Universal SYBR Green Supermix on a CFX96 real time PCR machine . We checked for off-target products when multiplexing pairs of assay primers using melt curve analysis as well as gel electrophoresis. From the seven loci tested, square pot we selected two to develop assays and ordered single-strand BHQplus probes unique to each of the four alleles .

Each probe was tagged with a distinct 5′ fluorophore and 3’ quencher. Assay 1 distinguishes T. absoluta from K. lycopersicella and P. operculella, while assay 2 distinguishes P. operculella from K. lycopersicella and T. absoluta.When considering which method is best suited for T. absoluta identification, a key consideration is sensitivity. Based on our analyses, we find that our qPCR diagnostics can reliably identify species down to 0.02 ng of input DNA, while our RPA-Cas12a assay was capable down to 0.1 ng input DNA. Assuming a haploid genome size of 564Mb from flow cytometry estimates , this represents approximately 35 copies of target DNA for qPCR detection and approximately 170 copies of target DNA for RPA-Cas12a detection. Both should be sufficient for scenarios where DNA is extracted from a single insect or small pool of insects. However, in situations where target DNA concentrations are extremely low, such as the case would be with eDNA collections, the more sensitive qPCR method is preferred.signal in the RPA-Cas12a assay as measuring fluorescence with a qPCR machine. Compared to the qPCR diagnostic, the RPA-Cas12a diagnostic does require additional user time, as amplification and detection occur in two distinct steps, although a one-pot reaction variant has been described . While we used an oligonucleotide with a Cal Red 610 fluorophore paired with the appropriate quencher as the substrate for Cas12a-mediated ssDNAse activity, the fluorophore/quencher molecules can easily be swapped for other fluorophores that absorb and emit at different wavelengths, making this protocol adaptable to the equipment available to the user.

It is possible in the future to replace the fluorometric detection method with a colorimetric detection by using biotin and FAM-labeled oligonucleotides applied to a lateral flow assay. This flexibility has been implemented in other detection assays and lets Cas12a-based detection systems stand out from qPCR methods, despite the slight reduction in sensitivity . While T. absoluta has not yet been detected in North America, demographic modeling suggests low to moderate levels of invasion are possible in Mexico, the California Central Valley, and the southeastern USA . Rapid reliable detection of an invasive pest is one of the key components of a successful eradication program, meaning it is crucial that molecular diagnostics are available . Even if eradication is not possible, molecular diagnostics reduce the need for expert entomologists to hand identify specimens, allowing detection to be done faster, cheaper, and more accurately . This is especially relevant for T. absoluta testing as tomato crops in the United States already contain the presence of P. operculella and K. lycopersicella, which are both nearly identical morphologically. Once detected, agencies can decide between strategies including eradication, quarantine, or continued monitoring . We expect that the molecular diagnostics presented here will add to the toolkit available to institutions to rapidly monitor for the appearance of T. absoluta.Tomato fruit are highly perishable, and sensitive to handling techniques and storage conditions after harvest .

Post harvest losses in quantity and quality commonly occur in fresh market tomatoes . The irreversible and continuous changes after fruit harvest leads to cell death and senescence, which makes some post harvest losses unavoidable . However, some losses are caused by improper post harvest practices, such as mechanical injury or pathological breakdown . Appropriately applying post harvest techniques could slow fruit senescence and therefore, maintain fruit quality from vine to consumers. While the specific recommendations for post harvest handling are dependent on the desired fruit shelf-life and vary among the produced and consumed regions of tomatoes, some examples of commonly applied tomato post harvest practices are discussed below.Fruit maturity at harvest plays an important role in its post harvest quality. Tomato fruit could be harvested between the stages of mature green and fully ripe, depending on the demand for fruit shelf-life. For the tomato produced for local markets, producers harvest fruit close to the fully ripe stage. Tomato destined for sale in distant markets, may be harvested earlier , followed by off-the-vine ripening to effectively satisfy the market need for an extended shelf-life. Tomato fruit that is harvested early, i.e., ‘Mature green’ fruit is able to fully ripen, but it loses its carbon and energy source from the mother plant, resulting in quality loss . Reduced fruit quality including loss of sugar, a lower ratio of TSS/TA, and changes in volatiles have been reported in off-the-vine ripening fruit compared with optimal harvest .Storage of the fresh market tomato after harvest provides stability to the supply chain. Precise temperature management is powerful in fruit storage, and the prescribed temperature may differ by fruit cultivar, harvesting stage, expected storage period, or, transportation . Before storage, precooling of freshly harvest fruit is used to remove ‘field heat’, ameliorating the rise in metabolic activities in fruit . For short-periods of storage, tomato fruit can be placed at room temperature under good ventilation, while, for longer storage time, early harvesting followed by low-temperature storage is a possible strategy, but this will negatively reduce the fruit flavor . When sensitive produce are exposed to low, non-freezing temperature for long periods, post harvest chilling injury may occur . PCI symptoms in tomato fruit include a failure to ripen,uneven ripening, surface pitting, decay, and high rates of ethylene and respiration upon rewarming . There are other approaches to calibrate the rate of fruit ripening during storage. Applying controlled and modified atmospheres could delay fruit ripening during post harvest storage. Controlling relative humidity , and modifying the relative proportion of oxygen and carbon dioxide in the atmosphere can extend the shelf-life of tomato fruit without a significant loss of flavor . For greater flexibility, ripening can be further controlled by regulating post harvest ethylene. When fruit are exposed to ethylene, ripening would speed up . Conversely, reducing ethylene production will maintain fruit quality and prolong shelf-life, blueberries in containers and several commonly examples are as following: precooling or heat treatment is efficient in slowing down ethylene production during storage ; high rates of CO2 is able to inhibit autocatalytic ethylene biosynthesis ; applying 1-Methylcyclopropene would delay fruit ripening by binding to the ethylene receptors , however use of these chemicals may affect fruit quality ; calcium chloride is successfully used in inhibiting ethylene production after harvest ; ethylene biosynthesis and sensitivity are affected by low-temperature post harvest storage .Consumers have been complaining about the quality of store-bought, conventional varieties of tomatoes over the last 30 years .

Understanding the physiological basis of fruit quality has therefore become the main focus of tomato researchers, as this information would support the breeding of new tomato cultivar with better quality . Fruit quality characteristics depend on the fruit developmental and physiological pathways which are dynamically regulated in the ripening process . The quality of the tomato fruit is mainly determined by its color, texture and flavor. Tomato fruit color is not only an appealing eating quality for consumers, but also a commonly used indicator of the fruit ripening stages. During ripening in most tomato cultivars, chloroplasts are converted to chromoplasts accompanied by chlorophyll breakdown and carotenoids accumulation, resulting in fruit color transition from green to red . The color of the ripe fruit is red primarily due to lycopene and the accumulation of a smaller amount of the orange or yellowish β-carotene . The dynamic interplay of carotenoid and chlorophyll metabolism dictates tomato fruit color .Fruit texture is a sensory, multi-parameter characteristic that can be perceived through the consumer’s senses, i.e., vision, touch, and taste . Tomato fruit texture is commonly described by the attributes of firmness, juiciness, and mealiness . There are various methods of measuring fruit texture, in which puncture and compression are commonly used destructive methods, and non-destructive approaches may include ultrasonic and optical techniques . Firmness of tomato fruit is considered as the main determinant of texture, and the decline of firmness leads to the fruit softness . Fruit softening is a natural phenomenon in ripening, and critical to seed dispersal and attracting animals . A certain degree of fruit softening is desirable in fresh market tomatoes, but too much softening is unwanted , as it limits fruit shelf-life and increases susceptibility to decay. Tomato fruit softening is associated with disassembly of the cell wall matrix, involving depolymerization and solubilization of the cell wall polysaccharides components, i.e., cellulose, hemicellulose and pectin . This event is mainly controlled by the activity of many cell wall degrading enzymes, such as polygalacturonase, pectin methylesterase , galactanase, or remodeling proteins, like expansin . Tomato fruit flavor is determined by a complex interaction of many biochemical compounds, including sugars, acids, amino acids, with aroma volatiles . Tomato aroma volatiles are often complex secondary metabolites, and they are derived from carotenoids and simpler primary metabolites precursors such as amino and fatty acids . There are more than 400 volatiles discovered in tomato, but only a limited proportion influences tomato flavor . The non-volatile compounds determine fruit taste. Sugar, mainly the reducing sugars glucose and fructose, makes the largest contribution to tomato taste. There are also organic acids in tomato fruit, predominantly citric and malic acids. It is well established that tomato flavor is highly interdependent on the relative levels of sugars, acids, and key volatiles, and no single chemical controls quality .Carbohydrate metabolism is important in determining tomato fruit post harvest quality . Fruit are “sink” tissues , that require carbohydrates supplied by photosynthetic “source” tissues . The chloroplasts in green tomato fruit have active photosynthetic ability , thus, green tomato fruit is able to fix carbon . The carbon dioxide from respiration is the major source of green fruit photosynthesis . Fruit fixed carbon accounts for 10-15% of that used by the fruit, and may be critical for seed development , and maximal carbohydrate and carotenoid accumulation . The carbohydrate metabolic pathway in developing tomato fruit is mediated by the activities of numerous enzymes and sugar transporters . Sucrose, the main translocated assimilate, is taken up by tomato fruit . In early fruit development, starch synthesis is the main flux; fruit starch level reaches its peak at the mature green stage, and it is then degraded during ripening . Starch synthesis and breakdown coexists throughout fruit development, with net synthesis occurring prior to the mature green stage, and net degradation to sugars occurring during ripening . Starch degradation therefore contributes to sugar accumulation in ripening fruit .Epigenomic modifications, especially DNA methylation of gene promoter regions, are considered to play a critical role in triggering tomato fruit ripening . DNA methylation is characterized by the addition of a methyl group to the 5’ position of cytosine. The cytosines that are methylated occur in either a symmetrical CHG and CG, or an asymmetrical CHH context . Symmetrical and asymmetrical DNA methylation are regulated by different genes . DNA demethylases are able to remove methyl groups from cytosines, a process termed DNA demethylation. In plants, DEMETER-Like demethylases are a group of enzymes that can activate gene promoter demethylation, by the activity of DNA Glycosylase-Lyases . There are four DML isoforms in tomato, of which SlDML2 is most important in fruit .

Additionally, the induction of defense genes in the RR core response could be ineffective if their expression levels were too weak compared with those seen in resistant MG fruit. We evaluated the levels of gene expression in inoculated RR fruit via a differential expression comparison to inoculated MG fruit. Of all the RR core defense genes identified above, 269/302 were expressed at equal or greater levels in inoculated RR fruit compared with inoculated MG fruit for all three pathogens. Conversely, 33/302 of these defense genes were expressed at higher levels in MG fruit compared with RR fruit for at least one of the three pathogens . These genes are diverse, representing 15 different defense categories. Prominent genes in this category include TAP1 and TAP2 , two peroxidases associated with defensive suberization in tomato ; CHI3 and CHI17 , two chitinases associated with C. fulvum infection ; and the JA biosynthesis gene OPR3 . While it is possible that resistance may be determined by these genes, growing blueberries in containers these results indicated that the differences in immune responses observed between MG and RR fruit are not likely solely responsible for differences in susceptibility, and, therefore, we considered the alternate hypothesis.

We explored the possibility that the increase in susceptibility to fungal pathogens is heavily influenced by a decline of preformed defenses and accumulation of susceptibility factors that occur during fruit ripening prior to pathogen challenge. Due to the complexity of the ripening program, we utilized isogenic non-ripening tomato mutants as tools to identify specific developmental features that are integral to fruit resistance or susceptibility. The Cnr, rin, and nor mutants produce fruit that lack most of the characteristic changes associated with normal ripening, such as color, texture, acidity, sugar accumulation, and ethylene production, but yet are phenotypically different from one another . All three mutant lines likely result from spontaneous gain-of-function mutations in transcription factors with key roles in the regulation of ripening . We inoculated fruit of these mutant genotypes at comparable stages to MG and RR wild-type fruit with B. cinerea, F. acuminatum, and R. stolonifer and measured disease incidence and severity up to 3 dpi . For all three pathogens at both MG-like and RR-like stages, only nor fruit were consistently resistant to infection. MG-like fruit of Cnr were the only unripe fruit susceptible to any pathogen, with both B. cinerea and F. acuminatum able to produce lesions on a significant number of these fruit. Consistent with this, Cnr RR-like were more susceptible than wild-type RR fruit to B. cinerea, with average disease severity nearly twice as great at 3 dpi .

The fruit of rin at both MG-like and RR-like stages showed similar or slightly lower susceptibility to all pathogens when compared with wild-type, with the exception of a significant reduction in disease incidence to F. acuminatum at the RR-like stage. Because some ripening processes may promote susceptibility, others may maintain resistance, and others may have no impact, we hypothesized that the Cnr, rin, and nor mutations differentially affect ripening-associated genes or pathways that are critical to tip the balance towards either susceptibility or resistance. We sequenced mRNA from B. cinerea-inoculated and healthy fruit from the non-ripening mutants at MG-like and RR-like stages at 1 dpi. We chose B. cinerea inoculations because this pathogen showed the clearest differences in susceptibility phenotypes between these genotypes. We first characterized transcriptional responses of mutant fruit to pathogen challenge by using enrichment analysis of defense-related processes to determine if differences in immune responses could explain the distinct susceptibility phenotypes . In most cases, the mutant fruit exhibited similar patterns of defense classification enrichments as wild-type fruit in both stages, with some notable exceptions. Compared with the other genotype–stage combinations, Cnr MG-like responses were deficient in the expression of genes from several prominent defense classifications, including chitin catabolic process , the plant–pathogen interaction and glutathione metabolism pathways, ERF and WRKY transcription factors, and RLK and CAMK genes.

Given that Cnr fruit were the only genotype at the MG-like stage to display susceptibility to B. cinerea infection, it can be suggested that these defense processes may be necessary for resistance in unripe fruit. However, these processes were enriched in the susceptible RR-like fruit of Cnr and rin, as well as wild-type RR fruit, which clearly indicates that they are not sufficient to result in a resistant outcome.For example, the responses of resistant nor fruit in both MG-like and RR-like fruit were noticeably less enriched in ethylene-associated pathways and more enriched in JA-associated pathways. These results suggest that JA-mediated defenses may contribute to tomato fruit resistance in the absence of ethylene, and that the nor mutation may activate JA-associated resistance. In support of this observation, levels of JA in healthy fruit appeared to be linked to resistance: they were highest in RR-like nor fruit, and only nor fruit experienced an increase in JA in the transition from MG-like to RR-like . Ethylene levels increase dramatically during ripening in wild-type fruit, but they remain low in all three non-ripening mutants . However, both Cnr and rin mutants produce ethylene in response to B. cinerea inoculation, with ethylene production in inoculated Cnr MG-like fruit reaching levels nearly three times greater than wild-type MG fruit by 3 dpi. Moreover, ethylene signaling/response genes are highly enriched in Cnr MG-like fruit responses . In contrast, healthy nor fruit did not produce substantial amounts of ethylene at either stage, and inoculation in nor fruit did not appear to induce significant ethylene production as in rin and Cnr fruit. These results indicate that high levels of ethylene are not required for B. cinerea resistance and most likely promote susceptibility. Regardless, the combination of hormone activity and defense gene enrichment suggests that, with the exception of Cnr MG-like fruit, resistance or susceptibility in the non-ripening mutants cannot be merely explained by the presence and/or magnitude of immune responses.

To identify genes that are involved in resistance or susceptibility that change during tomato fruit ripening, we used a differential expression analysis comparing healthy RR/RR-like to healthy MG/MG-like fruit for each wildtype and mutant line. In wild-type fruit, 6574 genes were significantly down-regulated in RR fruit compared with MG, while 5674 genes were significantly up-regulated . We used the susceptibility phenotypes and the transcriptional profiles of the mutant fruit to filter these ripening-associated genes and identify critical preformed defense mechanisms or susceptibility factors. Of the four genotypes, square pots all except nor experience an increase in susceptibility in the transition from MG/MG-like to RR/RR-like fruit. Thus, we selected ripening-associated genes that showed the same expression pattern in wild-type, Cnr, and/or rin, but not nor. This filtering resulted in 2893 down-regulated and 2003 up-regulated genes, respectively. We assumed that effective preformed defenses will decrease during ripening. Thus, the set of filtered down-regulated genes, being those that are highly expressed in healthy MG fruit compared with healthy RR fruit, should contain key genes related to preformed defenses. The filtered down-regulated genes contained 251 defense genes, while up-regulated genes included only 171 defense genes, indicating a net loss of about 80 genes in the transition from MG/MG-like to RR/RR-like susceptible fruit. Furthermore, the 251 defense genes from the filtered down-regulated set were over-represented by functional categories involved in reactive oxygen species response and detoxification, proteolysis, and the biosynthesis of secondary metabolites . These down-regulated ROS-related genes spanned several subfamilies including thioredoxins, glutaredoxins, glutathione S-transferases, and peroxidases. Among the down-regulated proteolytic genes were several subtilisin-like proteases, including SBT3 . Lastly, in addition to several genes involved in the methylerythritol 4-phosphate pathway of terpenoid biosynthesis, two copies of the lignin biosynthesis gene CCoAOMT were also among the filtered down-regulated class, suggesting that cell wall fortification could be inhibited upon infection. These results indicate that ripening involves a loss of multiple defense genes, and that the pre-existing levels of genes involved in ROS regulation, proteolysis, and secondary metabolite biosynthesis may be critical for resistance. Finally, we evaluated filtered up-regulated genes that are highly expressed in healthy RR fruit compared with healthy MG fruit, as they may include potential susceptibility factors. Since there is little scientific literature on classes of genes that may constitute susceptibility factors in plants, we focused on the up-regulated genes that were highly expressed in the RR/ RR-like fruit of the susceptible genotypes. Such genes may have disproportionate impacts on susceptibility due to their high expression. To identify these genes, we calculated average normalized read count values for each gene across WT, Cnr, and rin RR/RR-like fruit. The distribution of these values over the filtered up-regulated genes is a notably long-tailed one with a range of 2.43 to 179 649.29 and an average of 1295.

We identified genes with abnormally high expression values by selecting outliers from a log10-transformed distribution of the data. This resulted in a list of 16 genes . They include several genes previously discovered to be active during tomato fruit ripening, including the flavor volatile biosynthesis gene ADH2 , the carotenoid biosynthesis gene Z-ISO , the pectin-degrading enzymes PG2a and PL , and the ethylene receptor ETR4 , among other genes involved in carbohydrate metabolism. While any of these genes has the potential to impact susceptibility, genes for cell wall-degrading enzymes, such as PL and PG2a, which facilitate fruit softening during ripening, represent especially good candidates given both the importance of cell wall integrity in defense against fungal pathogens and previous research on RNAi-developed mutants in tomato . To validate the impact of PG2a and PL expression in wild-type RR fruit on susceptibility to B. cinerea, we utilized CRISPR-based mutants in each of these genes . RR fruit from these lines are similar in regards to soluble solids content, titratable acidity, and juice pH, but CRISPR-PL fruit are nearly 30% firmer than fruit from the CRISPR-PG2a and azygous WT control lines . In conjunction with these firmness differences, RR fruit of the CRISPR-PL line, but not the CRISPR-PG2a line, demonstrated reduced susceptibility to B. cinerea compared with the azygous line . At 3 dpi, disease incidence in the CRISPR-PL fruit was 56% lower than that in azygous fruit. We conclude that the ripeningassociated pectate lyase enzyme is a major susceptibility factor for B. cinerea infection in tomato fruit.During ripening, fruit may gradually lose either the ability to activate or the effectiveness of components of the plant immune system, defensive hormone production and signaling, and downstream transcriptional responses. Alternatively, ripening processes such as cell wall breakdown, simple sugar accumulation, changes in pH and secondary metabolite composition, and, in climacteric fruit, increased levels of ethylene may impact the fruit’s capability to resist fungal attack . The widespread nature of this phenomenon in diverse fruit pathosystems suggests that ripening-associated susceptibility is likely to be mediated by combinations of the above factors. In tomato, ripening-associated susceptibility has been demonstrated not only for the model necrotrophic pathogen B. cinerea, but for other fungal pathogens including Colletotrichum gloeosporioides , R. stolonifer, and F. acuminatum . Here, for the first time, we identified specific host responses in both resistant unripe and susceptible ripe fruit that are common to multiple pathogens and thus represent core responses to fungal infection. Most prominently, these core responses featured RLKs, WRKY and ERF transcription factors, JA biosynthesis, and chitin catabolism . Some genes that appear in both the MG core and the RR core responses were previously studied components of plant immunity in tomato, including the JA biosynthesis gene LoxD , the subtilisin-like protease SBT3 , the peroxidase CEVI-1 , and the chitinase CHI9 . Though response to inoculation overlaps somewhat with response to wounding in MG fruit , transcriptional profiles , and ethylene measurements indicate that the bulk of inoculation responses are a direct result of fungal attack. This is also evident by the presence of a necrotic ring only in inoculated MG fruit and not in the wounded controls or the inoculated RR fruit, indicating that the unripe fruit is capable of inducing an oxidative burst in response to the pathogen presence . However, most defense genes uncovered were found solely in the RR core response. These included several well-known defense genes that were only expressed in RR fruit, such as WRKY33 , the ERF PTI5 , the RLK TPK1b , and the MAP kinase MPK3 . While the MG core response did contain some defense genes that were not present in the RR core response , expression of most of these genes was also identified in the RR response to one or two pathogens.