It is important to note that diphacinone is a first-generation anticoagulant that requires multiple feedings to give the target species a toxic dose. As such, it is essential that you maintain a constant supply of bait in the stations throughout the duration of the baiting process. You can deploy bait stations without bait for a few days at first to allow rodents to become acclimatized. After this initial period, keep replenishing the bait for around 4 weeks or until consumption stops. After cessation of a baiting program, you can keep the bait stations in place, but filled with non-toxic oats, in order to assess re-invasion by nearby roof rats or deer mice. If you do see evidence of re-invasion, you can continue baiting until the start of the growing season.Bait stations may also be deployed at ground level, but previous studies have found that elevated baits are more effective against roof rats than bait placed at ground level . Additionally, bait stations placed at ground level increase the likelihood that non-target animals will fall prey to the bait. Re-invasion rates can be high for many rodent species, with immigration occurring as soon as two months after control if adjacent areas are home to large populations . As such, it is important to consider rat and mouse outbreaks and invasions at a broader, landscape level. Coordinated control programs implemented by neighboring landowners should provide the best results against damaging rat and mouse populations.In wet and humid conditions, the bait can absorb moisture. If this occurs, replace it with fresh, dry bait to maintain efficacy. Bait stations were designed to minimize spillage of bait during rodent feeding, but you still need to monitor for bait spillage and immediately clean up any spilled bait.

Deer mice have been known to nest in bait stations. Deer mice are known reservoirs of the Sin Nombre virus, which causes the potentially fatal Hantavirus Pulmonary Syndrome . This virus is transmitted through the inhalation or ingestion of aerosolized saliva, feces, or urine of deer mice. Because of this, it is important to make sure that any wind is behind the bait application technician when he or she opens the bait stations, so that any aerosolized particles will be carried away, downwind. Although concerns of secondary toxicity are typically quite low with first-generation anticoagulants , they still hold some inherent risk. As such, the survey of pest carcasses is always an important part of implementing a baiting program using anticoagulants. During a baiting program, most rodents die below ground but a few will be found above ground. Check for above ground pest carcasses once or twice a day. Remove the dead rodents and dispose of them by burying or burning . Alternatively, you can wrap carcasses in newspaper or double-bag them and dispose of them in the trash. Latex or nitrile gloves should be worn when handling dead rodents to prevent potential infection from the various diseases or parasites they may carry. Initial start-up costs for an effective baiting program will vary depending on which pest species are present, since deer mice require more bait stations per acre than roof rats alone. At the time of this writing, the individual bait stations cost $4.72 each, and 1 pound of the 0.005% diphacinone oat bait cost $1.75. Estimated start-up costs, then, using the 164-foot spacing, would be approximately $624.07 per 80-acre orchard . Initial costs for deer mouse management would cost considerably more: $1,794.17 per 80-acre orchard . These estimates are based on initial placement of one cup of bait per bait station. If rates of infestation are high, you may need to place additional bait. Also, these estimates do not include tie-up or labor costs, which will vary considerably depending on the tying method you use to fasten bait stations to tree branches.

It is important to note that subsequent costs for bait programs go down dramatically, since the initial cost of bait stations accounts for the bulk of the expense of a start-up baiting program. The bait stations described are very durable and can be used across multiple fields and multiple years, further reducing the long-term cost of baiting programs. Ultimately, we believe this baiting strategy will allow for effective, economical management of roof rats and deer mice in nut and tree fruit orchards in most areas where these species are found. This approach also substantially reduces the potential for negative impacts from rodenticide bait on non-target species, thereby minimizing the hazard to the natural environment.There is strong evidence that the majority of wild and cultivated plant species benefit from or rely entirely on the transportation of pollen grains by bees, other insects, birds and mammals . Furthermore, at the within-plant or whole plant scales, many crops exhibit decreased crop production in response to decreases in the number and types of pollinator , and these results have been extrapolated to field and landscape scales , as well as regional and global scales . In natural populations, pollination may also strongly limit reproduction, particularly in fragmented landscapes . While pollination clearly affects reproductive yields of many wild and crop plants , the degree to which it regulates yield in real-world cropping systems is debated . The emphasis on the role of pollination in determining yield has been criticised on the basis that it does not account for possible post-pollination processes affecting the amount and quality of fruit maturation . For example, water and nutrient limitation can strongly affect early fruit abortion and variation in losses to pests and diseases may ultimately be more important than pollination in determining realized yields . Furthermore, the interactions between pollination and post-pollination processes are rarely considered for wild plants and are almost unstudied in crop plants . Given widespread concerns about colony losses of Apis mellifera L., a major crop pollinator worldwide , as well as evidence for declines of other pollinators at landscape and regional scales , a greater understanding of the importance of pollination processes for reproduction and yield is critically needed. To date, studies of pollination limitation in tree crops have examined only a subset of flowers per plant or invokepollinator limitation without supplementary hand-cross pollination of the flowers . Instead, square plastic planter yields should be analysed at the whole plant scale to avoid the confounding effects of resource allocation among individual flowers or branches . Thus, experiments are needed that compare hand-pollinated yields to those in which pollinators are totally excluded , with and without resource applications. Furthermore, high fruit yield may come at the expense of the trees’ vegetative performance, such as quantity of foliage. The indirect influences of pollination in combination with plant resource variation on vegetative features of crops such as leaf quality and quantity is not yet investigated. Commercial almond production is an excellent model system for testing effects of pollination on crop production and foliage variables in different water and nutrient applications because results are likely to be applicable to many fruiting trees, such as apples, cherries, pears, peaches and plums, all with main varieties depending on insect pollination. Almond trees are generally considered to be drought-tolerant , but growers are highly dependent on irrigation and nutrient inputs to produce high yields of top quality . Moreover, almond production in California is highly dependent on honeybee management to set a commercial crop, but declines in honeybee colonies and an increase in production acreage have resulted in honeybees becoming a limiting resource for almond in the USA .

Furthermore, California, as one of the largest irrigated agricultural areas in the world, is facing water shortages, and almond growers may be forced to reduce their annual water use . Almond was therefore selected to test experimentally the effect of pollination on fruit yield and foliage while taking into account the interactions of pollination, water and nutrient applications in a full-factorial design.Almond [Prunus dulcis D.A. Webb] is a subtropical obligate outcrossing tree that is intensively grown in large monocultures in California, where the climate is similar to its place of origin in the Middle East . Almond flowers must be pollinated by a compatible variety to produce a fruit . Bees, and possibly other insects, are the principal vectors for transferring the sticky pollen between flowers of different varieties, and there is little to no transfer by wind . The mature fruit consists of one or occasionally two kernels per fruit, surrounded by a shell nested inside a hull. The most popular variety for nut production in the USA is Nonpareil, which comprises around one-third of all marketable nuts produced in California . Compatible pollen donor varieties for Nonpareil include Padre, Mission, Wood Colony and Carrion. Almond trees flower from early February to mid-March in California, and fruits ripen from July to September, depending on variety, weather and age of the trees.The experiment was carried out from January to August 2008. The site was located in the Sacramento Valley, the northern portion of California’s agriculturally intensive Central Valley, in Colusa County, near the border with Yolo County . Colusa County produces around 5% of California’s marketable almonds. Precipitation in this area is low, with an annual amount of 268.7 mm . Rainfall occurs primarily from October through May, with a peak in January; no precipitation typically occurs from June to September. We selected young, productive trees for this experiment, because kernel quantity and quality of the whole tree can be measured, and accumulated resources are limited in smaller trees . The 3.2-ha study site consisted of Nonpareil trees that were grafted onto peach rootstock [Prunus persica Batsch] in 2005 and were planted in 2006 . The trees were approximately 1.75-m tall and had not been harvested prior to this study. The trees were planted 4-m apart within rows and 6.4 m between rows. Several ‘polleniser’ varieties compatible with Nonpareil were available in surrounding orchards located 100–300 m away from the experimental trees. These included a 16-year-old, 4.7- ha orchard with Mission and Carrion varieties and a 13-yearold orchard with Nonpareil and Wood Colony. The eight hives closest to the experimental trees were 300–350 m away and were part of the commercial pollination management system used by the grower. Padre pollen was placed every second day at 10:00 and 14:00 h at the nest entrance of the honeybee hives to maximise the amount of compatible pollen transported by honeybee workers to Nonpareil trees.

The α-glucosidase assay is a method used to measure the activity of the enzyme α-glucosidase, which catalyzes the hydrolysis of disaccharides into glucose. This assay involves incubating the enzyme with a substrate like p-nitrophenyl-α-D-glucopyranoside and then measuring the release of p-nitro phenol, typically using spectrophotometry, as an indicator of enzymatic activity. The phenolic aqueous extract was used to determine the percentage of α-glucosidase enzyme inhibitory activity.Wild-type D. melanogaster Harwich strain flies were reared using the method described by Oyeniran et al. in the Drosophila Laboratory unit of FUTA. This method indicates the temperature, humidity, and nutrition conditions to preserve the wild-type strain. For best health and reproduction, we used suitable containers with standard Drosophila food medium, controlling the population density and providing fresh medium on a regular basis.The choice of the eggplant flour concentration in the formulated food was based on a preliminary study on fly survival rate where various concentrations between 0.1 and 2% were used. Flies 3–5 days old were separated into 8 groups, with 40 flies per vial. The experimental groups and design are shown in Table 2. The incubator was set at 25 ± 1 ◦C. The fruit flies were sacrificed, and the obtained tissue homogenates were extracted. The tissue extracts were used to carry out in vivo assays for the determination of protein content, α-amylase activity, α-glucosidase activity, glucose level—using the Sigma Randox kit and expressing it in mg/dL—ROS level, MDA content, total thiol, CAT activity, GST activity, and SOD activity.As shown in Table 1 and Supplementary Figures S2 and S3, it was found that the ripe fruits contained considerable amounts of phenolic compounds such as phenol, cinnamic acid, quercetin, chlorogenic acid, and myricetin. All the polyphenols found in the ripe fruits were also found in the unripe fruits.

The ripe fruits had a total phenolic content of 2205.43 mg/100 g, while the unripe fruits contained 2354.94 mg/100 g of phenolic compounds. This suggests that the ripe fruits could be as useful as the unripe fruits and should not be discarded. This clearly shows how useful nutrients which could be utilized in food products’ development are wasted in farms due to lack of information.This study compared the effects of ripe and unripe fruit diets on diabetes in order to evaluate their biological potential; however, our main focus was on the effects of ripe fruits, which are not always reported in studies because ripe fruits are considered waste. Due to ripening, about 60% of indigenous eggplant Solanum anguivi lam fruits in Nigeria are left to waste in farms, because the ripe fruit is given little or no importance.Meanwhile, this unwanted fruit is rich in viable phytochemicals, as reviewed in [36], which explained the benefits of phyto-compounds such as hesperidin—known as a bioflavonoid with anti-inflammatory, skin-lightening, antimicrobial, and wound healing properties—and naringin—which was found to have broad pharmacological potential. In addition, myricetin was previously reported to have neuroprotective, analgesic, antihypertensive, antidiabetic. and hypolipemic potential. This clearly shows that many beneficial bio-active compounds from nature are wasted yearly due to the early ripening of fruits and vegetables as well as inadequate/poor storage facilities. This present study showed that the ripe fruit contains more vitamins and minerals than the unripe one, which are essential for the proper functioning of the body. Vitamins are essential nutrients that are needed in the body and are involved in cell regulation, body metabolism, growth, and development, physiological processes, such as iron absorption, and the immune response. We found that the levels of vitamins A, C, E, and carotenoids and of some minerals increased with ripening, which confirmed a previous report; the change in coloration of the Solanum anguivi lam fruit could be due to the synthesis of alpha-carotene and beta-carotene, precursors of vitamin A.

COX and 5-LOX metabolic products such as prostanoids and leukotrienes are implicated in degenerative diseases such as cancer progression and may represent important targets for cancer chemoprevention or treatment. From the in vitro results, the bio-active compounds present in the ripe eggplant extract could serve as dual inhibitors of inflammatory enzymes, able to block the COX and the 5-LOX metabolic pathways better than synthetic drugs . The ripe eggplant extrac bio-active compounds, as shown from the results, have either additive or synergistic effects on the two major enzymes in arachidonic acid metabolic pathways, thus inhibiting their inflammatory activities. Secondly, it could be suggested that eggplant extracts and related diet also possess another advantage, as they have small or no gastric toxicity, being natural products with a balancing effect compared with synthesized non-steroidal anti-inflammatory drugs such as sulindac and aspirin, which are commonly used but cause major side effects, such as ulcerative colitis, due to non-selective COX inhibition. This study clearly shows that the eggplant, especially if ripe, could display an enhanced anti-inflammatory potency based on polyphenol structure, which contains almost the same structural and functional groups as the above-mentioned drugs. This observation confirms the need to utilize natural products such as eggplant for non-selective dual COX/5-LOX inhibition without side effects. Some reports indicated that some plant products such as curcumin, tea, silymarin, resveratrol, and green and black tea, rich in polyphenols, are used as natural dual enzyme inhibitors, which means that eggplant extracts or and its related diets could exert beneficial, therapeutic effects through the modulation of COX and LOX metabolic pathways. In addition, it was also confirmed that chronic inflammation is implicated in the pathophysiology of diabetes, especially type-2 diabetes, and to that end, targeting inflammation may help ameliorate diabetes, preventing its sequence and other metabolic complications, which means that an eggplant-supplemented diet could exert multiple beneficial functions. Bio-active compounds such as vitamins, carotenoids, phenolic compounds, and flavonoids, which seem to be abundant in fruits, 25 liter plant pot whether ripe or unripe, could be the reason behind the low weight gain, the increased survival, and the decreased mortality rate of the flies. This study confirmed what was reported in [5], i.e., that diabetic fruit flies administered a high-sucrose diet show obesity-linked lipogenesis caused by the repartition of fatty acids toward esterification, away from fatty acid β-oxidation, possibly by increasing malonyl-CoA. Moreover, there is a relationship between movement and weight gain which is also linked to obesity, meaning that a healthy lifestyle involving exercise and nutritious food rich in phyto-compounds, minerals, vitamins, and fiber, and with a low GI could holistically enable body wellness, as suggested by Nwanna et al.. It was also observed that the ripe eggplant-inclusive diet was able to elicit positive effects better than the unripe fruit-inclusive diet, at the same time reducing the glucose,MDA, and thiol levels in the diabetic subjects compare to the untreated subject. There is connection between diabetes mellitus, oxidative stress, and reduced antioxidant activity, while the biomarkers of oxidative stress are related to reactive oxygen species, MDA, and thiols. Thiol is an organic compound containing a sulfur atom. It is also referred to as mercaptan.

Thiol acts as an antioxidant, stabilizing free radicals by accepting their unpaired electron. This means that a ripe eggplant diet, with its bio-active compounds, contains relatively more antioxidant molecules than an unripe eggplant diet. Furthermore, an increase in endogenous antioxidant enzyme activities was observed in the groups treated with the supplemented diet, and diabetes causes tissue injury in flies as a result of the increased production of ROS. The elevation in the activity of these enzymes might be a result of the scavenging ability of the bio-active compounds in the fruit diet, which eliminated the ROS species and exerted their effects by restoring the activity of antioxidative enzymes. SOD is an enzymatic antioxidant that defends against free radicals by converting superoxide anions to compounds with less damaging effects like hydrogen peroxide; it serves as the first-line defense mechanism against pro-oxidative injury in tissues, which is important for lifespan extension and survival of Drosophila melanogaster. CAT is an enzyme that converts lesser damaging compounds such as peroxides and H2O2 to water and molecular oxygen. CAT activity was reduced in the sucrose-treated and control groups; this could also be explained by the accumulation of H2O2. We found that the inclusive diet increased the activities of SOD and CAT, which suggests a quick response to fight free radicals by reducing the endogenous H2O2 that was generated and diminishing the toxic effects it could cause. GST is needed to prevent damage and serves as a defense mechanism. The ability of the Solanum anguivi lam fruits, whether ripe or unripe, to restore the damaged antioxidant enzymes status in sucrose-induced diabetic flies cannot be overemphasized. It is interesting that the fruit contains high levels of bioflavonoid compounds such as hesperdin, naringin, and myricetin, with high binding affinity for all the proteins of interest in this study; these compounds could serve as good inhibitors of the enzymes associated with diabetes and inflammation, although it was earlier reported that they have numerous biological properties such as anti-microbial, anti-lipidemic, anti-cancer, and antioxidant properties. Ripening increases the levels of some of these bio-active compounds, which further confirms the report of Nwanna et al. of the immerse benefit of the eggplant fruit especially, the wild species.Tephritid fruit flies are well-known agricultural pests, and there are approximately 4500 species worldwide . As typical herbivores, host plant expansion is an important survival strategy for tephritid flies, especially when introduced into new areas. Host plant expansion is the ability of an herbivore to use novel host plants without losing their ability to use their original hosts , which facilitates the establishment of tephritids when entering new geographic areas and expanding their damage . Therefore, understanding the mechanism of host plant expansion will be helpful for the control of tephritid pests. Host expansion is well documented in the most destructive species of the genera Anastrepha, Bactrocera, Ceratitis, Dacus, and Rhagoletis among tephritid flies because they have expanded their range worldwide . For example, the ancestral hosts of Zeugodacus cucurbitae  in India are primarily cucurbits, but it began to infest papaya in Hawaii , and it expanded its host range to include mango in Africa . The peach fruit fy Bactrocera zonata  expanded to oranges and tomatoes when introduced from southeastern Asia to Egypt . Because of the typical frugivorous pest, the tephritids spend some stages of life from eggs and larvae to pupae in the fruit of host plants. Therefore, the microenvironment of host fruits to which flies try to expand will have an important infuence on the survival and adaptation of fruit flies. Therefore, when tephritids successfully expand their host range from ancestral host fruits to new hosts, they must adapt well to the chemical and non-chemical properties of the microenvironment from the novel host fruits, including theirphytochemicals, color, and phenology. The color of the host fruit is an important cue to many fruit-infesting insects when selecting a new host .

Therefore, in hot climates, the time during which the fruit is harvestable and marketable is considerably shortened, and fruit decay occurs faster than in the colder regions . Hot climate has the opposite effect on color break, which requires the correct number of cold night-time to develop . Therefore, not only the fruit decay faster in hot climates, but their color does not fully develop, and in extreme cases may even remain green. One of the expected outcomes of climate change is warmer winter temperatures with shorter cold-night times . As most citrus cultivars are harvested during this season, the effect of global warming is expected to be negative on both internal and external citrus fruit quality.Sink strength is determined by the sink’s size and activity . In crop plants, it is defined in practice by yield parameters , and quality parameters, such as carbohydrate and protein levels. Fruit size is genetically controlled, but physiological parameters, such as sink position in relation to other sinks and source tissues, and the time it takes to develop, also affect sink size and therefore, its strength . In tomato, there are over 30 loci that define fruit size, with many genes acting to control cell division at various developmental stages . Practically, sink activity is defined as the rate of photo assimilate translocation and their contribution to growth and developmental processes relative to their accumulation. To simplify the discussion, we will refer here only to sugars, as the major photo assimilates in fruit in general, and in citrus fruit in particular. As with many other plant species, in citrus, sucrose is the major sugar translocated from the leaves to the fruit .

During fruit maturation, it is the major accumulated sugar, with a sucrose:glucose:fructose ratio of 2:1:1 in many cultivars and references therein. In many cases, sucrose accumulation is detected early in fruit development, indicating a higher translocation than utilization rate . As discussed further on, sucrose catabolism into hexoses within the fruit provides the central mechanism controlling sink activity, and therefore sink strength. Sucrose is hydrolyzed either to fructose and UDPglucose by sucrose synthase , a bidirectional enzyme, or to glucose and fructose by invertase, a unidirectional enzyme . Following hydrolysis, glucose and fructose are phosphorylated to glucose-6-phosphate and fructose-6-phosphate by hexose kinase and fructokinase, respectively, while UDP-glucose is phosphorylated to glucose- 1-phosphate by UDP-glucose phosphorylase. While SuSy is cytosolic, sucrose hydrolysis by invertase is performed in the apoplasm by cell-wall invertases, in the cytosol by neutral/ alkaline invertases, and in the vacuole by acidic invertases. The enzyme is modulated post-translationally by invertase inhibitor, which might act in vivo, but not necessarily in vitro . In a few plant systems, it has been shown that alteration of the activities of SuSy and various forms of invertase results in altered yield and/or carbohydrate levels, and thus altered sink strength. For example, one amino acid change in the tomato cell-wall invertase LIN5 enhanced specific activity of the enzyme, the rate of sucrose uptake and, overall, BRIX . Increased expression of cucumber SuSy induced sucrose and starch accumulation and increased fruit size .

Transgenic down regulation of tomato SuSy resulted in reduced sucrose uptake early in fruit development, reduced fruit set, reduced fruit number and reduced fruit size . Similarly, reduced expression of acid invertase and SuSy in muskmelon and cucumber, respectively, reduced fruit size and sucrose level . Phenotypes associated with reduced sink strength were also demonstrated in carrot roots by down regulating vacuolar and cell-wall invertases as well as SuSy . Taken together, these studies demonstrated the importance of invertases and SuSy for sink strength and supported the notion that sink strength is controlled, at least in part, within the sink cells and/or at translocation points, i.e., zones of phloem unloading.Sugar transport from the leaf to the collecting phloem is defined as sugar or phloem loading, and its release from the transport system, the releasing phloem, into the sink cell is defined as sugar or phloem unloading . Movement of photo assimilates from the leaves to the sink through the stem via the transport phloem is a complex process. Although a major driving force is the concentration gradient between source and sink according to the pressure flow hypothesis , long-distance movement requires in and out movement of solutes from the transport system to the surrounding tissue, temporal accumulation, and energy investment . The mechanism of sugar unloading has been investigated in a number of fruit and other sink organs, such as tomato, grapeberry, cucumber, apple, walnut and potato tuber, by microscopy, fluorescent dyes, immunolocalization of sugar transporters, and use of transporter inhibitors . Unloading is operated by two major mechanisms, symplasmic, in which sugar transport occurs through the plasmodesmata connecting the transport cells and the sink cells, and apoplasmic, where sink cells are not connected symplasmically to the transport cells, and transport must therefore cross membranes through the apoplasm, usually using facilitated transport mechanisms . In some fruit, such as cucumber, apple, and kiwifruit, unloading is apoplasmic throughout fruit development . In other cases, such as tomato fruit and grape berry, there is a shift between symplasmic and apoplasmic unloading , whereas in the potato tuber, the shift is from apoplasmic to symplasmic . In jujube, two shifts occur during fruit development; apoplasmic unloading early in fruit development shifts to symplasmic unloading, which then shifts back to apoplasmic unloading close to ripening . The two types of unloading occur simultaneously during walnut fruit and seed development, symplasmic in the seed and apoplasmic in the fruit . Overall, symplasmic unloading is considered faster than apoplasmic unloading . Therefore, the mechanism is dependent on the rates of sink development and photo assimilate utilization versus the rate and form of their accumulation, in order to avoid an increase in osmoactive molecules in the cytosol. For instance, drainage pot in tomato fruit and grape berry, fruit development and sugar utilization are rapid during the first half of their development, requiring symplasmic unloading. Later, when the fruit shifts from a utilizing to accumulating sink, the unloading rate is reduced by shifting it to apoplasmic. During the first half of potato tuber development, it accumulates soluble, osmoactive sugars, and therefore apoplasmic unloading is required, but during later stages, starch is accumulated, allowing a faster unloading. Jujube fruit is characterized by rapid growth during the middle stages of its development, and therefore apoplasmic unloading is interrupted by symplasmic unloading during this stage.As phloem unloading has never been investigated directly in citrus fruit, the mechanisms in the various fruit tissues are unclear . Nevertheless, potential mechanisms can be discussed based on the following: the kinetics of sugar transport into the various fruit tissues, vascular bundles, segment epidermis, stalk of the juice sac, and juice sac, as determined using photo assimilate distribution by 14CO2-feeding of leaves, through either continuous labeling or pulse-chase experiments , the steady state distribution of sucrose and hexoses, as well as the activities of sugar-metabolizing enzymes and their protein levels in the various fruit tissues, especially during intensive sugar uptake . While the activities of sugar-metabolizing enzymes have been well-studied and characterized, understanding their physiological role during the various stages of fruit development is more challenging.

In tomato, for instance, about 20 days postanthesis, SuSy activity decreased and the activity of an apoplasmic invertase, eventually identified as LIN5, was induced . This shift was associated with the well-studied shift from symplasmic to apoplasmic unloading and with the conversion of the fruit from utilizing to accumulating sink . It might be concluded, therefore, that in tomato fruit, SuSy activity is required to maintain a high rate of sucrose utilization, whereas invertase activity is associated with hexose accumulation. Clearly, the equivalent information is still missing in citrus fruit. In the following, photo assimilate movement and distribution, as well as the activities of sugar metabolizing enzymes and their protein levels are described for the various fruit tissues . In most of the studies, the activities of the various forms of invertases are defined by their pH optima and solubility. Herein, alkaline/neutral-soluble invertase is referred to as cytosolic invertase, acid-soluble invertase as vacuolar invertase, and acid-insoluble invertase as cell-wall invertase .The vascular bundle terminates near the segment epidermis. However, albedo cells are present between the bundle and the segment epidermis, and therefore phloem unloading is expected to occur primarily into albedo cells before sugar reaches the pulptissue . In young fruitlets and fruit, the albedo is the major tissue; cell division terminates within 4–5 weeks post-anthesis, and fruit growth during stage II is achieved by pulp expansion . Therefore, it might be assumed that most of the sugar in the albedo is transported, while only a minor part of it is required for albedo cell metabolism and development. Pulse-chase experiments demonstrated that most of the radiolabel remains in the post-phloem compartment, i.e., albedo cells, for about 24 h before reaching the pulp tissues, juice sac and segment epidermis . This slowing of sugar movement could indicate that most of the transport is via the apoplasmic path. The presence of insoluble acid invertase activity could provide an indication for this type of unloading; such activity has been detected in albedo cells, although at a lower level than in other tissues—the vascular bundle, segment epidermis, and juice sacs . In addition, the albedo contained considerable activities of SuSy, vacuolar invertase and cytosolic invertase ; in fact, the activity of vacuolar invertase was strongest in the albedo compared to other fruit tissues, indicating active storage of sucrose/hexoses in the albedo after unloading. Obviously, the presence of plasmodesmata and a symplasmic pathway between the vascular bundle and albedo cells cannot be ruled out at this stage.As already noted, the segment epidermis provides a continuous layer with the juice sac epidermis . It is considered part of the transport tissues, and therefore enzymatic activities are sometimes reported for the vascular bundle and segment epidermis together , although in other cases they are separated . A considerable percentage, about 30%, of the total radiolabel could be recovered in the epidermis, with maximal accumulation between 24 and 48 h after feeding in a pulse-chase experiment using grapefruit . With continuous labeling, about 50% of the total radiolabel was recovered in the segment epidermis within 24 h. However, when radiolabeled sugars were quantified in Satsuma mandarin after 48 h of feeding with 14CO2, the segment epidermis displayed the lowest amount per fresh weight or per fruit . These discrepancies could be due to different experimental designs or reflect cultivar differences. Regardless, the segment epidermis provides a strong sink, and movement of photo assimilates from this sink to the juice sac cells cannot be ruled out. Sucrose hydrolysis in the segment epidermis was mediated by relatively high activities of SuSy and soluble invertase .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Previous efforts to develop rough allometric woody biomass equations for vines notwithstanding, there is still a need to improve our precision in estimating of how biomass changes with different parameters. Because the present analysis was conducted for 15 year old Cabernet vines, there is now a need for calibrating how vine C varies with age, varietal and training system. There is also uncertainty around the influence of grafting onto rootstock on C accumulation in vines. As mentioned in the methods, the vines in this study were not grafted—an artifact of the root-limiting duripan approximately 50 cm below the soil surface. The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites per se, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility. The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, plant health and fruit production, as well as for those concerned with C storage. The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.In plants, a fruit is the seed-containing section, which is formed from the ovary after flowering. Fruits have their vivid colors due to the presence of phytochemicals as pigments, which are natural compounds that protect against threats and insults such as insects and ultraviolet sunlight. Bright colors of fruits also attract animals and human beings for seed dispersing purposes. For instance, cocoa beans and blueberries have been used traditionally as therapies among indigenous people in North America. The fruit, leaves, seed, nft growing system and bark of the mango plant have been used as traditional medicine in Southeast Asia, Oceania, Africa, and Central America. Goji berries have been used as traditional Chinese medicine for two thousand years. Phytochemicals can be classified primarily as terpenoids, phenolics, alkaloids, nitrogen containing plant constituents, and organosulfur compounds. Examples of major phytochemical groups that are abundant from dietary sources and related to human health include carotenoids and polyphenols. Carotenoids are a type of terpenoid. Carotenoids can be classified as carotenes and xanthophylls. Phenolics can be classified as phenolic acids and polyphenols. Two primary subclasses of phenolic acids are hydroxybenzoic acid and hydroxycinnamic acid. Polyphenols include flavonoids, tannins, stilbenes, lignans, and xanthones. As one of the most studied categories of polyphenols, subclasses of flavonoids can be categorized to flavanones, flavones, anthocyanins, flavanols , chalcones, flavonols, and isoflavonoids.6Among the thousands of phytochemicals that have been identified in plants, both health promoting and toxic compounds exist.

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

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

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

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

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

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

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

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

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