Horticulture

Ethylene increases the susceptibility of tomato to B. cinerea by inducing ripening; however, its functions during strawberry infections are yet to be fully characterized. ABA, IAA and ethylene accumulation are altered by polyamine levels, which are positively correlated with fruit susceptibility to B. cinerea during strawberry ripening . Other hormones, such as brassinosteroids and jasmonic acid are present at lower levels during strawberry ripening. BR positively regulates vitamin C levels, sugar and anthocyanin biosynthesis during ripening, while negatively regulating acidity and concentration of other phenolic compounds . JA acts synergistically with ethylene by activating its biosynthesis in strawberries . Endogenous JA levels are modulated by methyl jasmonate and the JA carboxyl methyltransferase that lead to high levels in white fruit and a decline during ripening, antagonistically to ABA . In strawberry, JA appears to be involved in defence responses against B. cinerea. For example, gallon pot strawberries treated with MeJA had a delayed and much slower progression of B. cinerea infections .

As indicated previously, B. cinerea releases enzymes and metabolites that act as virulence factors but may also induce plant responses that are beneficial for fungal infection . A relevant example of the manipulation of physiological processes in the host by B. cinerea is the interference with specific developmental processes. In tomato plants, B. cinerea infections modified host gene expression to increase susceptibility, such as the induction of senescence in leaves . Moreover, infected unripe tomato fruit show premature expression of genes involved in ethylene synthesis during tomato ripening . These findings suggest that B. cinerea could initiate ethylene production and thereby stimulate early ripening. As strawberries are non-climacteric fruit, ethylene production of B. cinerea may not have substantial effects on strawberry ripening; however, the fungus was also shown to induce genes involved in the biosynthesis of other plant hormones such as ABA. Moreover, B. cinerea can synthesize and secrete ABA that functions as a virulence factor . Besides hormones, increased oxidative reactions caused by the pathogen may influence ripening progression .Defence mechanisms can be divided into preformed and induced defences. In strawberries, preformed defence compounds are especially abundant in the unripe stage, as reviewed in the section on quiescence of B. cinerea.

Even though plants accumulate defence compounds, B. cinerea has mechanisms to cope with these metabolites by efflux and detoxification of inhibitory substances. ATP-binding cassette transporters are used by B. cinerea to facilitate the efflux of anti-fungal compounds, such as stilbenes . B. cinerea is capable of detoxifying inhibitory substances, like epicatechin by secretion of laccases . Active B. cinerea infections can result in a reduction of specific secondary metabolites. It has been reported that levels of flavan-3-ol, benzoic acid and phenylpropanoids drop in B. cinerea-infected strawberries . Strawberries respond to B. cinerea infection by triggering defences. In some cases, preformed and induced defences can overlap such as in the case of PGIPs. An endogenous PGIP appears to be constitutively expressed in fruit from various strawberry cultivars . However, this PGIP and six additional ones show higher expression levels upon infection with B. cinerea . Overexpression of FaPGIP1a and FaPGIP2a in cisgenic plants conferred enhanced resistance to grey mould . Other enzymes induced by B. cinerea infections are chitinases. Expression of the chitinases FaChi2-1 and FaChi2-2 peaked 16 hpi in B. cinerea-infected strawberries . Furthermore, heterologous expression of Phaseolus vulgaris chitinase cH5B in strawberry resulted in higher resistance to infection . Another study demonstrated that application of heat-inactivated cells of the yeast Aureobasidium pullulans promoted tolerance to B. cinerea in strawberries . This primed resistance is probably due to the fruit’s perception of chitin from the yeast leading to induction of chitinases or other plant immune responses. Moreover, fruit defence responses may be primed using mechanical stimulation as it was reported for strawberry leaves .

Induced defences include accumulation of secondary metabolites and ROS. For instance, strawberries accumulate proanthocyanins around infection zones possibly to restrict fungal growth . The surroundings of infection sites generally display higher ROS production . ROS can serve as an effective defence against pathogens but also can lead to cell death, which is considered beneficial for necrotrophic fungi . B. cinerea itself produces ROS to induce host cell death, deplete plant antioxidants and increase lipid peroxidation . It is therefore interesting that, in unripe tomato fruit ROS production leads to resistance against B. cinerea, whereas in ripe fruit it seems to promote susceptibility . Future research will likely shed more light on the role of ROS in induced defences of strawberry fruit. Basal immunity is activated upon fungal infection. Degradation of fruit cell wall pectins can produce demethylated oligogalacturonides that trigger basal immune responses . Expression of the F. x ananassa pectin methylesterase 1 FaPE1 in Fragaria vesca resulted in reduced methyl-esterification of oligogalacturonides in fruit. This reduced esterification activated basal defences via the salicylic acid signalling pathway that led to a higher resistance to B. cinerea . Involvement of SA signalling in responses against B. cinerea was previously suggested when strawberry plants and fruit treated with SA showed decreased post harvest decay . B. cinerea can suppress the expression of plant defence responses by hijacking the host sRNA regulatory pathways . In strawberry fruits, B. cinerea infections can alter the expression of microRNAs involved in the regulation of defence genes, including the plant intracellular Ras group-related LRR protein 9-like gene . Interestingly, B. cinerea can also take up plant sRNAs during its interaction with the host. For instance, transgenic plants expressing sRNA that targets B. cinerea DCL1 and DCL2 show significantly reduced fungal growth in strawberries . The suppression of fungal growth via host sRNA is not well understood, and it is yet to be demonstrated that this mechanism of defence naturally occurs in plants.The diverse arsenal of infection mechanisms employed by B. cinerea explains its extremely wide-host range. It is therefore not surprising that entirely resistant strawberry genotypes do not exist . Several authors have analysed field resistance of strawberries to B. cinerea by quantifying disease development without artificial inoculation. A multi-year study of three strawberry cultivars found a significant effect of year, cultivar and cultivar by year interaction on the incidence of B. cinerea infections . Moreover, there was a positive correlation between row density and disease. Other studies investigated field resistance in annual winter production systems and found that variation of B. cinerea incidence between years was larger than genotype differences within years . Even though field resistance assessments investigate conditions similar to commercial production, considerable variability between environmental conditions and years can interfere with the detection of genotype differences.Due to the confounding effects of different non-genetic variables in field studies, gallon nursery pot assessment of post harvest resistance to B. cinerea infections has been pursued to determine genotype differences between strawberry cultivars or species. A large study of grey mould development during post harvest storage of non-inoculated fruit reported variation in disease incidence and speed of progression amongst cultivars, but no complete resistance was observed . Another approach to reducing environmental effects in disease tests is to inoculate fruit with B. cinerea conidia suspensions. Bestfleisch et al. tested quantitative resistance in 107 accessions of wild and cultivated strawberry. In this study, two wild ecotypes of F. virginiana showed high resistance to B. cinerea infections and slow disease progression. Such high tolerance in wild species was also reported in B. cinerea-inoculated leaves and fruit of F. chiloensis accessions from Chile . In these wild accessions, B. cinerea grew much slower. Comparative studies of disease progression indicated that fruit from the cultivar Chandler developed lesions at 24 hpi, while fruit from an F. chiloensis ecotype developed symptoms at 72 hpi . Fruit were entirely covered with mould at 6 days post-infection for the cultivar Chandler and at 9 dpi for the F. chiloensis ecotype. Considering that some accessions, particularly wild ecotypes, show reduced grey mould incidence and progression, there might be genetic sources of resistance against B. cinerea that could be used to increase resistance in strawberry. However, information about resistance mechanisms is mostly based on assumptions or empirical data. Differences in ripening patterns have been suggested as a potential explanation for resistance. For instance, some strawberries ripen from inside to outside, leaving the skin, which is the entry point of infections, unripe and thus resistant for a longer time .

Some more tolerant cultivars remain white or unripe around the calyx , which is where many B. cinerea infections tend to initiate. Another mechanism of resistance could be the presence of fungal inhibitors or the induction of PR proteins. FcPR5 and FcPR10 are highly induced in resistant F. chiloensis accessions when compared to commercial F. x ananassa cultivars . Based on sequence homology, FcPR5 probably possesses antifungal activity, and FcPR10 is likely a ribonuclease. These findings reflect that even though efforts have been made to explore resistance mechanisms of strawberry to B. cinerea, very little is known. Therefore, more research is necessary to better understand the biology of strawberry interactions with B. cinerea infections using diverse germplasm accessions.Many disease management strategies have been implemented for the control of B. cinerea in strawberry as further described below. However, even combined approaches are only capable of reducing disease incidence and severity but cannot completely prevent or eliminate grey mould in strawberries .Historically, B. cinerea infections in strawberry production have been managed by agronomic and horticultural practices, such as removal of senescent plant material to avoid inoculum buildup . Preventing contact of fruit with soil is another common practice to avoid B. cinerea infections, as most of the inoculum is present on the ground and soil moisture promotes conidia germination . Selecting the right irrigation system could help reduce grey mould incidence; mainly, the use of drip irrigation and micro-sprinklers results in limited inoculum spread and reduction of water films on the fruit . As canopy characteristics influence microclimates , nitrogen fertilization can lead to dense canopies and favour grey mould . Similarly, shorter plant spacings promote higher incidence of B. cinerea in the field . Additionally, plastic tunnels can avoid airborne inoculum and B. cinerea incidence is lower in non-fungicide treated tunnels than in fungicide treated fields , but tunnels favour powdery mildew and complicate harvest. In summary, cultural practices are essential to limit preharvest B. cinerea infections of strawberries, especially in organic agriculture.In modern production, pesticide applications are the most common management practice for B. cinerea control . In the previous two decades, the main pesticides used in strawberry production against B. cinerea belonged to the Fungicide Resistance Action Committee Groups 1 and 2, as well as captan . However, due to increasing fungicide resistance and new legal restrictions, producers have been forced to diversify their fungicide regimen . One application of fenhexamid at anthesis can be as efficient as multiple weekly applications . Additionally, alternation and combination of different fungicides with different modes of action are recommended . Resistance of B. cinerea to fungicides is a real challenge in horticulture and fungicide resistance profiles can shift considerably even within a single season . A screen of 13 B. cinerea isolates in Louisiana showed that all were partial to full resistance to FRAC 1 fungicides, and several of the isolates also had different levels of resistance to FRAC 2 fungicides . A larger survey of 1890 B. cinerea isolates revealed that 7 isolates from different locations were resistant to all single-action site FRAC fungicides groups that are registered for B. cinerea control . B. cinerea resistance to fungicides is usually associated with over expression of efflux transporters or with modification of fungicide targets. These resistance mechanisms are acquired via mutations and recombination that occur frequently in B. cinerea due to heterokaryosis, sexual reproduction and the presence of abundant transposable elements in its genome . Efflux of fungicides or accumulation of altered fungicide targets has also been shown to lead to multi-resistances . The presence of resistant isolates against the most common multi-action site fungicides reinforces the need for innovative management practices. A new generation of RNA-based fungicides has been proposed, which relies on the application of sRNA or dsRNAs that target B. cinerea virulence genes to reduce fungal infections in strawberries . However, these RNA-based fungicides remain far from commercialization, which is why fungicide resistance management such as mixture and rotation of different fungicides or testing local isolates for resistance is necessary .

Source fruits were then placed on top of the target fruits so that the inoculated stem end region would be in contact with the equatorial region of the target. Using the whole fruits was the most effective way to contact-inoculate oranges. Source fruits with visible mycelium and with visibly macerated tissue radially spread from the initial wound but no mycelium were placed on top of the equatorial side of three target oranges, with each infection site of the source orange in direct contact with one of the target oranges. The contact point between the source and target oranges was pre-wetted by spraying sterile water containing 0.01% Tween-20. Successful infections occurred when using oranges that did not have visible mycelium, while those with external mycelium failed to infect the target fruit. For tomatoes and apples, the contact inoculation procedure failed when using whole fruits due to leakage and accumulation of juices from the source fruits during incubation, leading to off-target infections in noncontact points. We then decided to use tissue sections instead of whole fruits to ensure a successful and uniform inoculation process. For tomatoes, a 3.5×3.5 cm, black plastic pots for plants square shaped pericarp section containing healthy/asymptomatic and symptomatic tissue in a 1:1 ratio was used.

Similarly, for apples, a cross-cut 1.5 cm thick was performed below and above the limit between symptomatic and asymptomatic tissue. The resulting disc was then cut into four 3×3×5.5 cm triangles containing decayed and macerated tissue in the middle and asymptomatic tissue on the sides. Source tissue sections of tomato and apple were placed on plastic boats, with the endocarp facing upwards for the tomato. Individual target tomatoes and apples with the equator side as the contact point were placed on top of their respective source tissue sections.The contact inoculation time was determined as the minimum time needed for successful disease development in the target fruits once the source fruits were detached. For control oranges and fungicide-treated oranges , the contact time was reduced to two days when using P. italicum WT and 1.5 days when using P. digitatum WT due to their advanced infection rates and aggressiveness on control and fungicide treated oranges. In all cases, contact inoculation was performed at room temperature, and target and source fruits were stored in high-humidity chambers .

Following the contact inoculation, the source whole fruits or tissue sections were removed, and target fruits were stored at 20 °C under high relative humidity until mycelium reached the equatorial region, or until evaluations were completed. Target oranges inoculated with P. italicum were stored at 20 °C for four days and at 15 °C for 12 days; while oranges inoculated with P. digitatum were stored at 15 °C for 15 days. Negative control samples were included for each trial. Control source fruits underwent the same steps as inoculated samples but were not inoculated with a fungal spore suspension.After contact inoculation, disease incidence, and severity were measured daily for tomatoes and every two days for apples and oranges. Disease incidence was calculated as the percentage of fruits displaying visual signs of tissue maceration or soft rot. Disease severity was obtained by measuring lesion area from pictures taken at each time point using a Nikon D5100 DSLR Camera with 18–55 mm f/3.5–5.6 and a custom-made macro in the ImageJ software.High-resolution multispectral images were taken using a VideometerLab 4 and processed with the VideometerLab software version 3.22.29. This equipment includes a sphere that uses strobe light-emitting diode technology to provide uniform and diffuse illumination. Reflectance images were taken at 19 wavelengths , including the long pass filters for a total of 50 spectral bands of the electromagnetic spectrum.

Multispectral images for all target fruits were taken with the stem end pointed to the side and the equator in the center before and after contact-based inoculation. For image analysis, pixels representing healthy and infected tissues were collected from a subset of fruit images from the total oranges. A normalized canonical discriminant analysis transformation based on the reflectance of each pixel was created to minimize the distance within classes and to maximize the distance among classes. A region of interest was obtained from all images by applying a mask to segment the fruits from the background. All fruits were collected in a blob database, and the healthy and infected areas were extracted based on the previously created nCDA transformation. Shape and spectral features were extracted from individual blobs, including area and tristimulus components of color, such as hue and saturation. The SpectralMean feature extracts the reflectance mean of each fruit for the 50 spectral bands. Region MSI_Mean calculates a trimmed mean of transformed pixel values within the blob , and MSIThreshold measures the percentage or area of the blob region with a transformation value higher than the threshold, based on the nCDA model .After choosing the appropriate source inoculum type and incubation periods, we tested the reliability and reproducibility of the contact-based inoculation method using different fungal species and strains on target fruits with and without fungicide treatments . In all the tests, the fungi spread from the contact points between the source and target fruit, causing tissue maceration and, in some cases, extensive mycelium growth . No symptoms were evident when non-infected fruits or tissues were used as source inoculum to control for secondary or unintended infections. Although we noticed some variation across different fruit commodities, fruit treatments, and fungal species and strains, most of our trials yielded disease incidence values of at least 80%, indicating the high performance of the contact-based inoculation method . Additionally, we observed that established lesions in the target fruits continued to expand over time, confirming that the fungal pathogens tested in this study could colonize and complete their life cycle once they penetrated the fruits . Wild-type strains of P. italicum and P. digitatum reached a disease incidence of 80% and 96.7% after 10 and 8 dpci, respectively, in untreated target oranges. In contrast, when using fungicide-treated oranges, a low overall disease incidence was observed for P. italicum and P. digitatum WT strains after 8 dpci and 14 dpci, respectively . On the other hand, disease incidence in fungicide and wax treated oranges contactinoculated with a P. digitatum fungicide-resistant strain was found to be 95.5%, which was similar to the untreated control oranges infected with P. digitatum WT . Lesion expansion in oranges varied depending on the target orange treatment and fungal isolate used . Overall, although lesion sizes were smaller for the FT oranges when using WT strains, the increase in lesion size across time points shows that the fungal pathogens tested in this study can colonize and complete their life cycle once they penetrate the target fruit. Field-grown tomatoes of the ‘Celebrity’ hybrid cultivar showed a high disease incidence, drainage pot reaching 90% across all evaluated time points when contact-inoculated with a B. cinerea WT strain . Meanwhile, other field grown tomatoes from the ‘Rutgers’ variety and ‘Shady Lady’ hybrid cultivar achieved the maximum disease incidence at 4 dpci and 6 dpci, with 80% and 94.4%, respectively. Commercial, greenhouse-grown, hybrid ‘Beefsteak’ tomatoes showed 100% disease incidence at 2 dpci when contact-inoculated with a B. cinerea FR strain. Tomatoes that did not show any disease incidence remained uninfected throughout the trial. Similarly to oranges, lesion size development varied across tomato cultivars and strains used, but shows the fungal strains used can colonize the target fruits . In apples, P. expansum FR infected 72.5% of the apples at 4 dpci and 90% at 6 dpci. The number of infected target apples remained unchanged until the last recorded time. Seven out of 40 apples never showed infection or lesion development from P. expansum and were considered as not infected during this study .

For the infected apples, lesion sizes steadily increased throughout the duration of the evaluation period .In oranges, Penicillium spp. growth showed minimal visual progression and seemed limited to the contact point until 12 dpci . However, using multispectral imaging we detected disease progression on the surface of target fruits, which was not apparent to the naked eye. Changes in the reflectance profiles of contact-inoculated oranges show that lesions appeared as early as 8 dpci and continued to expand even when mature lesions and visible mycelium were only obvious until 14 dpci and 18 dpci, respectively . On the other hand, practically no changes in the reflectance profile were observed for control fruits . Furthermore, normalized canonical discriminant analysis transformation, which combines all wavelengths, could detect changes in areas where lesions were to be developed in earlier time points. These areas were calculated using a threshold in the nCDA transformed scale ranging from healthy tissues to infected tissues . The highest separation potential in the nCDA transformation was obtained when combining about ten wavelengths with minor gain with additional wavelengths . Using MSI to monitor pathogen growth revealed the early onset of infection in oranges. It also can be used for sensitive quantification of lesion area before the disease is visible to the naked eye.Consistent and reliable inoculation methods that mimic natural conditions and industry scenarios are key for the study of plant-pathogen interactions and the development of post harvest control methods. Here, we established a non-wounding, contact inoculation protocol that recreates the infections naturally occurring in the post harvest supply chain through nesting. We produced whole fruits or tissues that were infected and served as inoculum sources for spreading the disease to healthy fruit. In all fruits, except for fungicide-treated oranges contact inoculated with wild-type pathogen strains, disease incidence rates of 80% or higher were observed by the final evaluation time point, showing the effectiveness of the proposed methodology. Disease severity measurements were used to evaluate disease progression and growth behavior of fungal pathogens, confirming successful infections beyond the initial contact point in each fruit commodity. Even though disease incidence was lower in fungicide-treated oranges contact-inoculated with fungicide-sensitive pathogens, having successful infections in fungicide-treated oranges showed that the pathogens were capable of causing disease through this protocol, even in disadvantageous conditions. Furthermore, visualizing the fruits using MSI allowed us to confirm that, although lesions were not visible to the naked eye until 14 days post-contact inoculation with P. digitatum, the fungus was established in the fruit tissue and growing by 8 dpci. While successful B. cinerea infections were observed in all tomato trials, differences in disease incidence and severity values were observed across varieties. ‘Rutgers’ tomatoes exhibited greater variation between infection rates and a lower disease incidence than other tomato varieties. These differences could be due to surface characteristics, such as cuticle thickness and permeability, which influence the generation of pathogen-induced signals that activate defense responses. The ‘Rutgers’ tomatoes are more similar to processing types. Furthermore, thicker fruit cuticles provide higher resistance to initial B. cinerea infections. Also, differences in pathogen behavior were observed in this study, particularly between Penicillium spp. when contact inoculated in oranges and apples. P. italicum WT showed an earlier mycelium appearance as compared to P. digitatum WT, although it was the latter that covered the fruits completely in mycelium first. In apples, P. expansum showed a slower disease incidence and severity progression than the other commodities. This could be due to differences in infection mechanisms between fungal species, as well as due to the fact that blue mold develops better at cooler temperatures compared to green mold. For all three fruit commodities tested, a percentage of the fruits did not get infected, even after several weeks of monitoring. This could be because the fruit was able to halt pathogen infection at the contact point, preventing it from spreading throughout the tissues. Another possibility is that, despite homogeneous incubation conditions, fruit-specific microclimates were not always conducive to disease development in all fruits, even though we ensured consistency of the technical aspects of the protocol. The overall results of this study confirm that the non-wounding, contact-based inoculation method was effective in all fruit-pathogen interactions tested. Although an initial step of wound-inoculating the source fruit material is required, spread of the disease to healthy target fruits is done solely through contact between the tissues, and the target fruits remain unwounded throughout the entire procedure. This method holds promise for further application in other pathosystems by focusing on several key aspects. First, it is crucial to recognize the importance of the homogeneity of the fruits used as source inoculum and the initial 24 h during contact inoculation, as both play a significant role in the establishment and spread of fungal infections. For example, it is recommended that, if possible, fruits should come from the same location and supplier, and transportation-storage conditions should remain constant. Also, the incubation should be done at high humidity with some level of gas exchange , especially after the first day of contact. Second, investigating the contact time between source and target fruits is essential to ensure the accuracy and reproducibility of the inoculation method. Third, exploring the position of infected fruits or tissues, which serves as the source of inoculation, will help identify the most favorable conditions for efficient pathogen transfer between fruits through contact.

A strong positive relationship between crop size and the number of seeds dispersed and in the number of seeds being dispersed long distance is expected in abiotic dispersal modes such as anemochory and hydrochory and in the biotic dispersal mode epizoochory . However, the expectation is less clear with endozoochory, synzoochoryand myrmecochory, where animal dispersers make foraging decisions in resource-heterogeneous environments where these dispersers can be satiated or prematurely leave feeding trees in order to mix diets with complementary resources . Nonetheless, expected patterns with respect to crop size have been proposed for endozoochory. Howe and Estabrook developed two models based on specialized versus opportunistic frugivore/seed dispersal systems. They suggested that the number of seeds dispersed should increase with fruit availability for both types of species, although the number dispersed should plateau for model 1 plant species that depend on specialized dispersers that tend to involve relatively few species and become satiated. They further predicted that the effect of crop size on the proportion of the available seeds dispersed would differ for model 1 and model 2 species.

For model 1 species, pot with drainage holes the proportion of seeds dispersed was expected to initially increase with crop size but would reach a peak at some intermediate crop size due to disperser satiation and then drop with ever larger fruit crops. In contrast, for model 2 species they predicted that the proportion of the seed crop dispersed would increase with increasing crop size, perhaps stabilizing at a constant proportion at larger crop sizes, but not decreasing. However, the dichotomy between specialized and opportunistic dispersal systems is not generally accepted at this point , leading other authors to propose a general expectation that the number of seeds dispersed should increase with increasing crop size. In fact, this is considered one major driver of the development of frugivory hubs, where hub individuals in the network receive more dispersal services than expected, leaving non-hub individuals with little dispersal services . This last prediction appears to be supported by studies mostly of endozoochory that demonstrate that as crop size increases, visitation rate by avian and mammalian dispersers increases, which translates into an increased quantity of seeds dispersed . For example, Prunus mahaleb fruit crop size explained 80 % of seeds dispersed in a population in southern Spain . With respect to the proportion of seeds dispersed, results to date show no consistent relationship . These patterns suggesting a general increase in the number but not the proportion of seeds dispersed with increasing crop size are supported by a meta-analysis that found positive bird-mediated selection on fruit crop sizes as measured by both visitation rate and the quantity of seeds dispersed, but no selection on the proportion of seeds dispersed .

Crop size can also affect the quality component of SDE and the probability of LDD. Increasing crop size lifts the entire dispersal kernel, resulting in more seeds in the tail of the distribution and thus more LDD and increased population spread and gene flow . Increasing crop size also results in more seeds dispersing farther in a local dispersal context, increasing the chances of reaching suitable sites and surviving distance- and density-dependent mortality . Although empirical evidence is limited, crop size can also affect the quality of endozoochorous dispersal by altering disperser behaviour and disperser assemblages of individual plants. For example, as Vassobia breviflora crop size increased, disperser residence time in the canopy decreased, increasing the probability of seed dispersal away from the parent rather than seeds processed in situ . Increasing crop size also increased fruit consumption by legitimate dispersers without affecting consumption by pulp consumers, altering the realized dispersal assemblage and increasing overall dispersal quality . Lastly, with a population-wide increase in Fagus sylvatica crop size , there was an increase in survival of seeds cached by Apodemus flavicollis, a clear increase in the quality of dispersal . On the other hand, seed survival in caches can be lower under trees that produced large seed crops ; thus, spatial variation in crop size might have different effects than temporal variation .

Fruit and seed size variation is likely the second most widely studied driver of inter individual variation in seed dispersal. Fruit and seed size vary within individuals, but also among individuals, years and populations . In a study of 39 species from 46 populations, on average 62 % of seed size variation was within individuals while 38 % was among individuals, though individual species varied substantially . Thus, fruit and seed size variation can influence animal disperser decisions regarding which plants to forage in and then which fruits to consume . Furthermore, mean fruit size of individuals can be highly heritable, indicating potential selection response . Many studies have demonstrated size-based fruit or seed selection by dispersers, suggesting a potentially important role for fruit/seed size in driving inter individual variation in the quantity component of SDE, although actual patterns of selection are not consistent and appear to depend on the plant and animal species involved . It is generally thought that fruit/seed size-based selection is a function not so much of fruit/seed size, but rather by the fruit/ seed size relative to the disperser size. For example, for endozoochorous birds that swallow fruits whole, it is widely believed that fruit selection is driven by fruit diameter and bird gape width . Similarly, it is thought that seed size selection by synzoochorous seed dispersers is related to the ratio of seed to disperser size . The extent to which fruit/seed size selection contributes to inter individual differences in the quantity of seeds dispersed is unclear. Dispersers may select among individual plants based on mean traits or among individual fruits independent of the mother plant. While some studies demonstrate that frugivores select among fruiting plants based on mean fruit or seed size , others demonstrate that at the population level, individual fruits are selected based on their sizes . Thus, even strong selection of fruits based on size need not lead to differential selection of individual plants based on fruit size. For example, dispersers of P. mahaleb strongly selected fruits based on size, but this was almost entirely driven by selection of smaller fruits within an individual plant’s fruit crop, while there was inconsistent and weak selection among individual plants based on fruit size . Because multiple traits associated with selection by dispersers may be correlated with seed size, the degree to which selection is driven by fruit or seed size, rather than a correlated trait is unclear. For example, the four main avian dispersers of Rubus ulmifolius in southern Spain differed in the distribution of seed sizes dispersed, but seed size, seed number, pulp/seed ratio and percent pulp co-varied, making it difficult to determine which trait or traits were being selected . Fruit and seed size can also drive intraspecific variation in the quantity and quality of dispersal in other ways. For gape-limited, endozoochorous birds, large pot with drainage intra- and inter individual variation in fruit diameter can affect the proportion of a plant’s fruit crop that a disperser can swallow. For instance, in a Myrtus communis population in southern Spain, some individuals produced large fruits that only Turdus merula and T. philomelos could swallow and disperse. However, other individuals in the same population produced smaller fruits that were completely available to these species and partially available to Sylvia atricapilla, Erithacus rubecula and, in the case of one individual, the smallest disperser, S. melanocephala . Thus, the realized disperser assemblages of individual plants varied from two to five species. Moreover, realized disperser assemblages of individual plants varied across years due to changes in fruit size.

Such among-individual and among-year variation in realized disperser assemblages can affect inter individual variation in dispersal outcomes. First, variation in the number of animal species feeding on an individual plant likely affects the quantity of seeds dispersed . Second, inter individual variation in realized disperser assemblages is expected to drive inter individual variation in LDD, gene flow and the quality of dispersal because disperser species differ in their dispersal kernels, treatment in the mouth and gut, and microhabitat destination of seeds . Species-specific preferences in microhabitat and fruit/seed size can also result in microhabitats accumulating different seed size distributions .Lastly, seed size affects whether a seed is swallowed and passed through the digestive system versus being dropped, spat out or regurgitated, which affects both treatment in the mouth and gut and dispersal distances . In general, larger seeds tend to be dispersed more rapidly and farther, and are more likely to be cached than smaller seeds; in contrast, no obvious pattern links seed size and the probability of surviving in a cache. The actual outcome of the interaction may be more related to the ratio of seed to disperser size rather than seed size alone . However, three species of rodents varying 4-fold in mass all preferentially selected and dispersed larger fruits of the Chilean desert shrub Myrcianthes coquimbensis . While there is abundant evidence that synzoochorous dispersers select and handle individual seeds based on size, there are fewer studies documenting dispersers selecting on mean seed size among individual plants. The large Japanese wood mouse preferentially dispersed seeds of individual Q. serrata trees with larger mean acorn size . Similarly, Apodemus spp. disproportionately dispersed and cached seeds from Pinus armandii individuals with larger mean seed mass , although the greater probability of their seeds being consumed cancelled the benefits of increased dispersal. By contrast, mean seed size of the Queen palm had no influence on tree selection by squirrels , and Garrulus glandarius preferentially fed on Quercus ilex trees with smaller acorns . Thus, although evidence is limited, there is potential for seed size to contribute to inter individual variation in the quantity and quality of seed dispersal by synzoochorous dispersers. Although the consequences of within-individual variation in plant traits have not been considered frequently in ecology , in addition to selection based on individual or mean fruit/seed size, mutualistic dispersers may select among individual plants based on the extent of intra individual variation in fruit or seed size. In a latitudinal study of Crataegus monogyna seed dispersal by Turdus spp. in Europe, birds selected against intra individual fruit size variation in populations with lower variation and selected for intra individual fruit size variation in populations with higher variation . Similarly, A. speciosus not only selected individual Q. serrata trees with larger acorns, but also selected individual trees with a greater variability in acorn weight . Seed size also affects abiotic seed dispersal. In the seagrass Zostera marina, settling rate increases with seed size, suggesting smaller seeds disperse farther . When grazed by the specialist herbivore Ophraella communa, the riparian weed Ambrosia artemisiifolia produces lighter, more buoyant seeds, demonstrating a clear mechanism for inter individual variation in dispersal . In ballistically dispersed species, both seed and fruit size can affect patterns of seed dispersal. In Oxalis acetosella and O. corniculata , dispersal distances increased with seed mass, while in Mercurialis annua, dispersal distances decreased with increasing seed mass . In the only study on fruit size and ballistic dispersal of which we are aware, dispersal distance increased with fruit length in Erodium cicutarium . It is generally believed that dispersal distances of anemochoric species will decrease as seed mass increases, and this expectation appears to be well-supported, although the variance explained is generally low. This general pattern has been reported in both tropical and temperate environments as well as across trees, shrubs and herbaceous species , although there are exceptions . Given that seed mass varies both among and within individuals , seed mass variation may contribute to inter individual variation in dispersal distances. For example, under highly competitive conditions, plants of the wind-dispersed desert annual Dithyrea californica produce smaller, lighter seeds that are dispersed farther . Given the typical heterogeneous distribution of individuals in populations, it is likely that D. californica individuals vary continuously in competitive environments and thus potentially in dispersal ability. Finally, the actual pattern of wind dispersal is driven not simply by seed mass, but by the relationship between seed mass and the dispersal structure . Interspecific studies of the effect of plant height on seed dispersal suggest plant height is a major correlate of dispersal distances and is considerably more important than seed size .

The enlarged biding sites were then mapped to the CLV3 genomic sequence, tolerating up to 2 mis-matches in the flanking regions. In order to account for the presence of transcription factors whose cis-motifs are not currently known, MEME analysis were employed to identify motifs shared between genes that are co-expressed with CLV3.Overall, 231 potential cis-motifs and transcription factor binding sites were identified. Most were randomly distributed over the entire CLV3 genomic sequence, but irregular clusters could be recognized near the coding region. The largest cluster occurred in the upstream 500bp of the 5’ promoter, while up to three smaller clusters occurred in the 3’ enhancer region . The list of potential factors was then filtered to include those found inside the previously identified CLV3 regulatory regions, which left just 157 predictions . Many of the remaining predictions were found to have overlappingsequences, though it is unclear how well this might predict their actual function in-vivo. One notable example of this phenomenon is a predicted MYB-like binding site located at -155bp, square pot which was predicted by four different databases. In other cases, two structurally different transcription factors were predicted to have overlapping cis-motifs, such as the bZIP/homeodomain pair Opaque-2/ALFIN-1 in the 3’ enhancer region. Interestingly, the data also revealed four partial miR414 targets, three of which overlapped with the DNA/Mariner family transposable element At2gTE50670 in the 3’ enhancer , and the fourth occurred in the 3rd exon.

In an alternative approach to identify unknown cis-motifs, phylogenetic footprinting was used to compare CLV3 orthologous sequences from different species. In this method, functional regulatory structures can be identified by their conservation over evolutionary time, which often requires little more than performing a sequence alignment. The method is also quite robust, as previous studies found that the identified footprints matched 80 and 85% of known transcription factor binding sites. To begin this analysis, three CLV3 orthologs were identified by their syntenic relationships within the Brassicaceae using the tools in the Brassica Genome.org database. Their cDNA sequences were aligned with 27 CLE family paralogs identified in A. thaliana  in order to identify features that were unique to CLV3 orthologs, before expanding the search to additional species. This analysis revealed three potentially unique traits that might be used to distinguish orthologs from the multitudes of closely related CLE genes. These included three consecutive histidines at the C terminal end of the CLE motif, a C-terminal oligo extension, and a 3-exon gene structure, all of which had been previously identified in the CLV3 sub-group. Additional orthologs werethen identified using tBlastn searches against the AtCLV3 protein, for which nine species which met the criteria described above: Brachypodium distachyon, Oryza sativa, Ricinus communis, Glycine max, A. thaliana, Arabidopsis lyrata and Brassica rapa, Capsella grandiflora, and Camelina sativa. No AtCLV3 orthologs were identified in the gymnosperms, basal angiosperms, or the Asteriids using these search parameters.

The Euphorbiaceae and Fabaceae each contributed one species in the closely related Eurosiids I, while the monocots are represented by two species in the Poaceae. As a result, this sampling is heavily biased towards the Brassicaceae family , which provide more than half of the total number of species. In order to footprint the promoter regions, initial sequence alignments were performed using 8kb genomic fragments, containing up to 5kb of upstream and downstream sequences on either side of the coding region. However, little or no homology was found when all nine orthologs were aligned simultaneously. This was not improved by removing monocot clade, as the two grass orthologs failed to align with each other. Repeating this pattern, both R.communis and G. max also failed to alignment with each other, or with any of the remaining orthologs. In contrast, conserved regions became clearly visible when the five Brassicaceae species were aligned separately . This result appears to reflect the optimum degree of sequence divergence for this gene, as previous studies have found that orthologs outside of the Brassicaceae were less informative due excessive divergence, whereas sequences obtained entirely within the Brassicaceae have been found to have too little divergence. Three of the remaining species had complete genomic sequences, while the other two consisted of two contigs separated by a gap of unknown size. In the B. rapa ortholog, the gap was located in the 3’ region, and was flanked by 256 and 452 base pair sequences that did not align with any of the other Brasssicaceae orthologs, despite strong sequence conservation in the surrounding regions.

This indicates the recent insertion of a large DNA fragment, potentially >700bp in size. Attempts to locate the source of the two end-fragment sequences in the B. rapa genome with BLAST searches, unexpectedly found that each was present in multiple copies, and were distributed across several different chromosomes. No evidence of transposable element sequences were found, so the flanking regions were here interpreted to be contaminating scaffold sequences from the original genome assembly. A similar gap of unknown size occurred in C. grandiflora, where one contig aligned with the CDS and 3’ UTR, while the entire 5’ upstream contig failed to align with any other ortholog. In both cases, the non-aligning sequences were removed from the analysis, providing a final alignment consisting of four orthologs in the 5’ promoter region, and five orthologs spanning the CDS and 3’ UTR. Overall, the five orthologs shared between 27% and 65% sequence similarity, and grouped into two closely related pairs. One pair contained C. grandiflora and C. sativa, and the othercontained A. thaliana and A. lyrata. In contrast, B. rapa was found to be distinct from all other Brassicaceae orthologs, which accurately recapitulates its predicted evolutionary relationship with the rest of the family. Upon closer inspection, the coding regions were found to be 79-93% similar, which dropped to just 14-34% in regions with no significant alignments. The initial alignment was considerably fragmented, with many insertions, deletions, and isolated nucleotides. In many cases, the position of these features varied with the settings in the alignment software, and were here interpreted to be artifacts of the alignment procedure. To correct such artifacts, isolated nucleotides were manually adjusted left or right to maximize local sequence alignments within ±5bp. Where variation in the length of tandem repeats was apparent, gaps were introduced into one or more ortholog sequences to accommodate the largest number of repeats present. Conserved regions were then identified by using a 5bp sliding window to identify regions with more than 60% identity. This window is unusually small compared to previous studies that have used 15-50bp sliding windows, but was chosen here to more accurately reflect the minimum size of known transcription factor binding sites. Where large contiguous conserved regions were found, drainage collection pot the presence of small 1-3bp indels within their sequences were used to break them into smaller fragments, as disruption of these sites indicates that they do not contain functional cis-motifs. scattered in the 3’ UTR. Several predicted transcription factor sites were found within the coding regions, but these were interpreted to be non-functional, as previous GUS-reporter systems did not reveal any significant regulatory elements within this region. Among other notable features was a predicted signal peptide in the first exon, identified with signal P 4.0, which was almost entirely conserved and is consistent with the secretion of the mature CLV3 oligopeptide. In addition, the second exon was found to be completely conserved with no In all, 42 conserved regions were identified, ranging in size from 5 to over 111bp long. Fourteen footprints were found in the coding sequence, of which nine of were clustered around the three exons.

Only one footprint was found entirely within in the 5’ UTR, and the remaining four were intervening gaps. The second exon also completely overlapped with several predicted transcription factors, including HOX2a, as well as cytokinin and gibberellic acid responsive motifs. This suggests as-yet unrecognized functional role for the second exon, which might explain why it has been retained in a family that consists largely of single exon genes. The 3rd exon was also highly conserved, although curiously the most conserved region only partially overlapped with the CLE motif and instead included part of the C-terminal extension. In the 3’ UTR, the footprints were found to overlap with potential zinc-finger and MYB binding sites, as well as a cytokinin responsive ARR10 site. In the upstream regulatory region, the 5’ promoter contained ten conserved footprints, eight of which formed a large and nearly contiguous block near the TSS. The two isolated footprints were located at -204bp and -167bp upstream, corresponding to the palindromic Motif#2 and the redundantly predicted MYB binding site, respectively. In the remaining footprints, additional predictions were found for an overlapping AGL15/CBF site, an auxin response element, overlapping GT1 and AGAMOUS sites, and one prediction for a TATA-less promoter. The latter may be related to the position of the only recognizable TATA box-like sequence, which at – 68bp upstream, which is more than double the usual 25-35bp described for other TATA-based promoters. In contrast, the 3’ enhancer region contained seventeen footprints arranged in roughly three clusters, spanning a region nearly 600 bp long. Two of these clusters closely corresponded with the previously noted clusters of predicted transcription factor sites, while the third was distinctly isolated and had no predicted transcription factors. Together, the footprints contained one of the three known WUS binding sites , two predicted AtHB1 binding sites, a cytokinin responsive element , several bZIP motifs, a KNOX-like site, and a predicted cis-motif forNPR1. Strikingly, the majority of the footprints also overlapped with a DNA transposable element in A. thaliana, At2TE50665 . It has previously been implied that WUS controls CLV3 expression in a concentration dependent manner, which is consistent with the close proximity of two demonstrated WUS binding sites . The region around these two sites also contains several other TAAT cores within a single stretch about 100bp long, much of which is represented by four conserved footprints, which together might form a WUS binding site cluster. However, only the +970 WUS binding site was found to be perfectly conserved, while the other TAAT cores displayed mutations or were interrupted by indel sequences in one or more orthologs. Instead, when the region around the known WUS binding sites was examined in more detail with a 5bp sliding window, a strikingly periodic pattern was observed, where four different conserved motifs were found to be regularly spaced about 15 bp apart. In order from 5’-3’, these motifs were identified as CCGTTGGG, AGTAC, TTGTCAA, and TAATTAATGG , the latter two of which correspond to a predicted W-box motif, and the +970 WUS binding site. In addition, a perfectly conserved sequence was found just 25-36 bp downstream in all orthologs, which consisted almost entirely of tandem repeats containing ATG. The ATG repeats also overlapped with a predicted ALFIN-1 homeodomain/Opaque-2 binding site, suggesting that this sequence may actually represent a modified bZIP motif, or perhaps an atypical homeodomain binding site containing a TGAT core motif. It is not clear how many potential binding sites are present in these ATG repeats, but in consideration of the size of the conserved region, it seems likely that they could accommodate up to three transcription factor proteins simultaneously. The potential functional role of the TGAT motifs is further supported by the observation that they are 4x over-represented in the surrounding 124 bp conserved region, while the TAAT cores actually are 5x under-represented. In addition, pair-wise distance measurements between the two cores revealed a skewed distribution, where few sites were found closer together than the median value of 5bp. When several median-length pairs were aligned, this corresponded to the 13bp motif TAATnnWnnTGAT. When this motif was subjected to Patmatch searches of the A. thaliana genome, it was found to be 26x over represented among the genes directly targeted by WUS. Multiple copies of the 13bp motif were also found in several target genes, including two in the 3’ enhancer of AtCLV3. Together, this evidence suggests the presence of a larger cis-regulatory module that may include up to five other transcription factors besides WUS. Slightly further downstream, another conspicuous feature of the 3’ enhancer is the presence of two large and perfectly conserved sequence blocks, spanning 42 and 32 bp, respectively . Both the size and the degree of sequence conservation in these two regions were exceptional in that they exceeded those found in the coding region of AtCLV3.

Alternate bearing is also perhaps one of the oldest known plant behaviors, as written descriptions of it can be found going back over several millennia. Most often these references are simple observations of trees that flower in one year but not the next , or as one early account of cider production in England elegantly described it, the trees would “. . . provide a full and complete blessing every second year” . Other written accounts offer advice on how to get sterile trees back into production, though it is not always clear if these early authors were aware of the full two-year cycle. Nevertheless, the prescribed regimes of girdling, branch twisting, and pruning are well-matched to its existence nonetheless: by removing a lot of branches or inflicting specific types of damage, this advice would stimulate the trees to produce a dense crop of new branches, which go on to bear fruit in the following year. Other accounts instead try to explain the biennial pattern by speculating that the trees require a period of “rest” before or after a large crop. Alternatively, vertical tower for strawberries the cycle has been suggested to be the result of rainfall, variation in cross-pollination efficiency, and/or total the flower numbers.

Not everyone was inclined to such speculation though, as Japanese farmers reportedly accepted the cycle as nature’s way, without trying to intervene at all.Despite the long history of this tree behavior, there have been relatively few attempts to learn how or why the plants produced the cycle. Even when descriptions of such experiments have survived, they tend to be hearsay accounts published by 3rd parties, making it difficult to determine what exactly transpired. For example, one horticultural manual from the 1800’s recognized that an unequal growth trade-off between the fruits and branches was a significant symptom of alternate bearing trees, and recommended that farmers artificially restore that balance by cutting off excess vegetative growth. In another example twenty five years later, a reporter for a popular horticultural magazine visited a nearby orchard and held a walking interview with the owner. In the process, we learn that the proprietor had performed an experiment some years earlier, in which he had removed all the flowers from a single tree one season, and found an abundance of fruit in the next. In contrast, academic interest in alternate bearing was essentially non-existent until the late 1800’s, when the subject apparently received a major boost from the establishment of state sponsored agricultural experiment stations. This was apparently a global trend, as England adopted a private research station that began as early as 1843, Japan obtained one by official decree in 1871, and United States began building its research stations following the Hatch Act in 1887.

By the turn of the century, these and other experiment stations had begun to produce a large number of agricultural publications, quickly establishing the foundation of crop science as we know it today. Research in the alternate bearing field lagged behind somewhat, perhaps by the need to establish experimental orchards, but began to produce a burst of new research starting around 1900. Thus began a roughly 30 year period of clever experimental designs and careful scholarship, ultimately producing the tools and basic concepts of alternate bearing research. Beginning with the development of chemical sprays in the 1940’s, the focus of this research began to shift away from basic inquiries and instead frequently focused on practical farming issues. Over the next few decades, this work made slow but steady progress towards understanding the physiological mechanism that propagates the cycle, while also identifying a broad array of species with similar physiological symptoms. The past few decades were also an extremely productive time for alternate bearing research, as this student estimates that the field has accumulated more than 1200 papers that mention the cycle by name, as well as another 3- 4000 papers in closely related subjects, with less obvious key words. In contrast, work to incorporate modern genetic tools is a much more recent phenomenon, beginning with just a handful of papers in the early 2000’s , then escalating rapidly by the end of the decade.

Generally such genetic work has attempted to observe when and where key developmental genes respond to the cycle, because it is not yet possible to make an entirely predictive model of alternate bearing, based on known developmental pathways from other species. Such knowledge is however, is expected to help farmers precisely control the behavior in popular tree crops, either through selective breeding , transgenic modification, or predictive mathematical models. A genetic understanding may even provide a way to clarify current ideas about the ecology and evolution of the cycle, while also identifying key developmental differences that separate annual and perennial species. In many cases alternate bearing is described largely in terms of fruit production, but close observations have shown that the actual phenotype is much more complicated. Similar two-year cycles have also been found in leaf area, flower numbers, branch lengths, and trunk diameter, just to name a few . Several physiological parameters have also been found to change in phase with the cycle, including mineral nutrients and carbohydrates, which tend to accumulate in the fastest growing organ, and are depleted in slower growing organs. Plant hormones have also been correlated with specific parts of the cycle, and are important regulators of floral induction, dormancy, fruit ripening, and abscission. Collectively, these findings outline an inverse relationship between fruit and vegetative growth that is broadly applicable to all species: When fruits are abundant, the vegetative growth is reduced. Conversely, when fruit are rare, the vegetative growth is abundant. This pattern is commonly known as a growth trade-off. Such trade-offs have been known to plant specialists for almost two centuries, and there are numerous documented examples where the fruits are negatively correlated with growth elsewhere in the plant, container vertical farming and occasionally with tissue death, especially of the meristem region. The existence of a growth trade-off however, does not immediately explain how this contributes to a biennial cycle. Most temperate trees shed their fruits in the fall, and there is usually nothing left to affect growth at the start of the second season. With the exception of more tropical trees like Avocados, this suggests the plants actually retain a “memory” of the fruit load from the previous season, a hypothesis that immediately raises questions about how that information is stored and transmitted over time. While the idea that plants may retain that memory in terms of their gene expression patterns has never been tested, the available evidence instead suggests that this information is recorded in the plant’s physical anatomy. For example, most trees produce flowers from their axillary buds a few months to a year after the buds are first produced. The number of such buds is proportional to the vegetative growth in the previous season, and since vegetative growth is often coeval with immature fruit, it is easy to see how a growth trade off might be recorded directly by the number of axillary buds. In addition, immature flowers and inflorescences typically begin growing while still inside the buds many months before they burst open, so the growth trade-off has the potential to affect the size of the inflorescence at a very early stage in their development.

Any initial differences in size incurred in this way are then likely magnified in the second season, when both inflorescence and vegetative growth resumes at an accelerated rate. Together the combination of inflorescence size and number can substantially change the number of flowers in subsequent season, and thus have an indirect control over number of subsequent fruits. In fact, when branch lengths and flower numbers are mapped to a generic branch structure, it is possible to derive a reasonably descriptive model of alternate bearing anatomy, summarized in Figure 1.0. In the “ON” year, the presence of a large number of fruit is thought to cause the reduction in vegetative branch length and leaf numbers, providing fewer axillary buds for the return bloom in the next season. In the following “OFF” year, the axillary buds produce fewer flowers and fruit, allowing the vegetative longer branches to grow longer and bear more leaves. The cycle then repeats itself when the axillary buds produce a large number of flowers in the 3rd season, creating yet another “ON” year. Once started, this mechanism is thought to be sufficient to propagate the biennial cycle indefinitely, barring intervention by the environment or anthropological factors. The variation in branch length is also permanently recorded in the plant anatomy, and with a few caveats, several years worth of cycles can be seen simply by noting the distance between successive sets of bud scars, or the remnants of old cones or inflorescences. The simplicity of this model is perhaps its greatest selling point, as it can readily incorporate information from several other areas of research. Following the discovery of plant hormones for example, it was soon found that gibberellic acid was an important regulator of flower numbers in many species, either as an activator or a repressor, and has its strongest effect during the period of floral induction. The response to auxin apparently changes over the course of the season, as mid-season auxin treatments can increase fruit abscission rates, while applications to immature inflorescences and mature fruit tend to prevent abscission. Ethylene seems to have a dual function, enhancing the number of flowers in some cases, while stimulating abscission of young fruits in others. The role of abscisic acid is not quite as clear, but the concentration of this hormone is known to fluctuate with the cycle in citrus trees, yet it was not correlated with abscission of immature pistachios nuts. In addition to the hormones, the distribution of starches, soluble carbohydrates and even inorganic mineral content can be mapped to anatomically different phases of the cycle. The result is a comprehensive physiological model of alternate bearing, which can be used to make reliably accurate predictions for both practical and theoretical applications. The basic physiological model is also surprisingly compatible with more recent concepts of plant development. For example, most vegetative branches do not grow continuously, but are instead produce in regular bursts commonly known as a “flush”. Also variously known as a “growth unit” “iterative growth” or even polycyclic growth, a flush typically occurs once per year in temperate climates, but tropical trees can produce as many as 3-4 flushes per year. Each flush is composed of at least two discrete phases of growth, known as the juvenile and adult phases. Confusingly, these two terms are also used to describe growth patterns related to the age of the plant in years, so for the sake of clarity it is necessary to borrow a slightly different terminology and refer to the variation along a single branch as “seasonal heterophylly”. The first leaves of the flush are distinguished from the later leaves by subtle-to-significant differences in trichome density, color, size, and leaf morphology, and typically, the first leaves are also abscised while still immature. Typically a flush is thought to begin and end with SAM dormancy, and as a result, alternate bearing anatomy can easily be outlined as the sum of two consecutive flushes, attached end to end.The flowers in contrast, are born on a completely different type of flush, usually called an inflorescence. These are typically produced from dormant axillary buds, though some plants also use the SAM as well. All such buds have the option of producing vegetative or reproductive structures, and the deciding factor is determined by a combination of information derived from the external and internal environments. Once the correct combination for each species is recognized though, the buds are said to be “induced”, irrevocably committing them to a reproductive fate. The resulting Inflorescence Meristem then produces the branches, bracts and flowers of the mature inflorescence. While the flowers are an obvious indication of reproductive growth, the inflorescence branches may also be distinguished from vegetative branches by their unique patterns in color, diameter, and trichomes. In addition, the inflorescence is also a determinate structure that senesces at maturity. The resulting abscission zone or boundary with dead tissue then provides a clear indication of which tissues were reproductive and which were vegetative .

Pruning mature open center fruit trees involves keeping the center free of vigorous upright shoots, reducing tree height, and thinning out branches to reduce crowding. For peaches and nectarines, select 1-year-old lateral fruiting branches that originate close to main branches. Thin these fruiting branches and head them by one-third if they are longer than about 1 to 2 feet . Remove or cut back 2-year-old fruiting shoots or cut them back to 1- year-old shoots. On other species, old fruiting spurs should be renewed periodically. Table 1 shows the location of fruiting buds, the longevity of spurs, and the desired severity of pruning of selected species. When dormant pruning, become familiar with the plump fruiting buds and prune accordingly to ensure adequate flowering the following spring. Pruning of mature almonds involves few or no heading cuts; simply thin out branches—often fairly large branches—to prevent crowding.Central leader training is often used for apples and sometimes for pears, Asian pears, pecans, and quince. These trees tend to have dominant central leaders, a characteristic that lends itself to the central leader training method. However, because many apple and pear varieties are susceptible to fire blight, plastic pots 30 liters open center or other multiple-leader methods are often preferred so that if a major limb is lost the tree can be more easily redeveloped.

Central leader training involves keeping trees shaped somewhat like Christmas trees, with lateral branches arranged in separate layers, or “tiers,” separated by open areas of canopy, and branches in lower tiers wider than those in upper ones. Instead of sunlight reaching lower fruiting branches through the center, as with the open center method, it reaches them from the sides and between branches. For young central leader trees, the goal is to create three or four tiers of lateral branches, with about four branches per tier. This is done preferably during spring and summer by heading back or bending down any vigorous shoots that grow upright and compete with the central leader. Create the first tier of four lateral branches by tying or staking branches outward at an angle just above horizontal after they have grown 2 to 3 feet long. When the central leader has reached about 21⁄2 to 3 feet past the first tier, usually in the first dormant season after planting, head it just below this point and train a second tier of four branches outward from the cut leader. These branches should be offset vertically from those of the first tier. Then create the third tier in a similar manner.

Avoid bending one branch directly over another; also, maintain the tree’s pyramidal shape by keeping lower branches longer than upper branches. The entire process will take 3 to 4 years, depending on the tree’s vigor. Some side branching of these main lateral branches should be encouraged. Vigorous upright shoots should be removed or headed back during the growing season to only three to six buds, although some shoots should be left longer if fruit are exposed to hot afternoon sun. Heading these shoots a couple of times during the growing season creates fruit-bearing spurs if the tree is not too vigorous. Heading may also encourage the growth of another set of vigorous shoots; simply remove these shoots or prune them back to create more spurs.With walnuts, the first main branch originates higher than on most trees, about 5 to 8 feet , and the spacing between branches is about 3 to 5 feet . Pruning of mature walnut trees is not essential, but thinning of branches will keep trees healthy and productive. Persimmons bear on current-season shoots that originate from buds produced near the ends of 1-year-old lateral branches. For this reason, once the tree is developed, avoid heading these shoots and be sure that sunlight reaches lower shoots, or they will become less productive or die. Also, each limb must be kept strong from the start by ensuring adequate sunlight and shortening branches if necessary by cutting back to upward- and outward-growing lateral branches.

Avoid heading cuts except to stimulate branching, such as on young trees; such heading cuts are best made in late spring and summer to reduce strong upright growth of “whip” branches. Persimmons often grow taller than is desired for picking fruit. Trees that are allowed to grow unchecked can make large, beautiful trees; however, branches often break with the weight of fruit. If they are headed at a given height each year, the subsequent vigorous shoots will shade lower shoots by midsummer, so these shoots must be thinned by summer pruning. Where possible, cut to lateral branches rather than making heading cuts.“Fruit bushes” are standard trees or, preferably, trees on dwarfing rootstock that are kept small by periodic summer pruning. This method can work for nearly all fruit species. The beauty of this system is its simplicity and ease of management. Pruning begins in about late April or early May of the first growing season, when new growth is about 2 feet long. At this time, cut the new growth in half, aiming for a uniform, bushy appearance. Hedge-trimming shears may be useful in this. In about late June, cut the subsequent new growth in half. If new growth is vigorous, it may need to be cut once more during the season. These heading cuts promote an excess of branches, so thin them by removing some shoots to allow sunlight to reach the lower branches. If needed, thin out additional crowding branches in the dormant season when they are more visible. In the second year, continue cutting new growth in this manner until the trees reach 5 to 7 feet tall, a height at which you can easily prune the top. Pruning in subsequent years involves cutting off any shoots above the tree’s permanenth eight two to three times per year. Also, thin crowding branches, especially at the top of the tree, and remove unproductive fruiting wood in early spring when branches without flowers are visible.No matter which training method you choose, do some pruning in the spring and summer to train young trees and shorten the time to full fruit production. When necessary, bend and stake shoots of young trees during the spring and summer so they will grow in the desired direction. Bending branches in this manner develops the scaffold structure faster than heading them and waiting for new lateral branches to form. On mature trees, summer pruning mainly involves removing vigorous upright shoots that are not needed as permanent branches and heading or thinning shoots to control tree height and develop branches. If trees receive appropriate summer training and pruning, far less dormant pruning is necessary. However, the absence of leaves in winter provides a clear view of the framework of the tree. At that time, thin or head any branches that were not adequately pruned during the growing season. One notable feature about apricots is that they are susceptible to infection by the branch-killing disease eutypa dieback, round plastic pots which is a particularly serious problem in Northern California around the San Francisco Bay Area. Infection occurs on wounds made in fall or early winter, causing severe gumming at pruning wounds and branch dieback.

Therefore, it is best to prune apricots either in late summer, so that at least 6 weeks of rain-free weather follow the pruning, or late in the dormant season , by which time far fewer viable spores are present. If pruned in summer, be sure to prevent sunburn by leaving enough foliage to cover limbs or by painting exposed limbs white with a 50-50 mixture of interior white latex paint and water.Refrigeration is indisputably the most effective strategy to prolong shelf-life, preserve quality and delay the deterioration of many fruits and vegetables . However, in cold sensitive commodities such as tomato , storage at temperatures between 0-12°C induces the onset of molecular, biochemical, and physiological alterations known as post harvest chilling injury , which are manifested when fruit are rewarmed to room temperature . PCI is a complex and multilayered phenomenon. Its early stages are temperature-dependent and are mediated by physical changes in cellular membranes . Loss of membrane stability triggers the activation of a signal transduction cascade that transmits the cold stimulus downstream through a series of molecular players, e.g., second messengers, eliciting symptoms characteristic of this disorder . These symptoms include modifications in respiration and ethylene production, disruption in the synthesis of aroma volatiles, accumulation of reactive oxygen species , lipid peroxidation, and DNA and protein damage . These molecular and cellular processes ultimately lead to failures in fruit ripening, the development of surface pitting, seed browning and higher susceptibility to post harvest decay . Most of what is known about the cold signal transduction pathway in plants, comes from studies of Arabidopsis thaliana, which is able to cold-acclimate and endure freezing temperatures . The C-Binding Factor gene family of transcription factors are positive regulators of the cold response that interact with the cis-elements of downstream cold-responsive genes . These target genes, also known as the CBFregulon, encode protective proteins and enzymes, and are involved in the synthesis of metabolites that enhance the plant’s fitness during cold stress . In tomato fruit, SlCBF1-3 genes are also induced by cold , but the size and types of genes comprising the CBF regulon are not the same as in Arabidopsis . This might partly explain tomato’s inability to cold acclimate . Different members of the Arabidopsis CBF gene family have been ectopically expressed in tomato plants under the control of the constitutive CaMV35S promoter, resulting in increased stress tolerance, but with concomitant growth reduction and flowering delay . This, due to CBF’s involvement in gibberellin repression, and DELLA protein accumulation . A transgenic phenotype over expressing AtCBF1 in tomato fruit was characterized, and revealed this gene influenced ripening as well as fruit’s response to post harvest cold stress . In this work, we hypothesized that CBF1 over expression in tomato cv. Micro-Tom fruit during post harvest chilling would enhance fruit tolerance to cold stress and reduce the incidence of PCI. We cloned this gene from two sources: cultivated tomato and the wild tomato relative Solanum habrochaites . S. habrochaites has been extensively studied due to its tolerance to cold stress . ShCBF1 has been cloned and expressed into Arabidopsis plants conferring tolerance to freezing and salinity but displaying phenotypic abnormalities . In this study, both ShCBF1 and SlCBF1 genes were driven by the stress-inducible promoter RD29A . Our goal was to specifically induce CBF1 expression in harvested fruit stored in the cold, as well as minimize pleiotropic effects caused by constitutive over expression. To test our hypothesis, transgenic fruit were cold-stored, which elicited ectopic CBF expression, and their post harvest performance was examined and compared to wild-type fruit under the same conditions. To broaden the scope of traditional studies of chilling injury beyond fruit post harvest, we also tested if the photosynthetic responses of CBF1-overexpressing seedlings would be affected by cold stress. The goal was to understand the physiological effects of additional CBF1 transcripts at different phases of the plant life cycle. Finally, we evaluated the influence of post harvest fruit chilling on seeds and seedling traits that were measured under control or cold conditions, to explore the concept of transgenerational adaptive mechanisms transmitted from fruit to progeny.Fruit were cold-stored and transferred to 20°C for three additional days to induce PCI. The presence of surface lesions or pitting and deterioration in the form of decay, were recorded. Wild-type fruit consistently had the lowest CII scores among all genotypes, maintaining levels below 30% after 2 or 3 weeks of cold storage, and rewarming . In contrast, transgenic fruit developed pits after just one week at 2.5°C, especially in Sh-36 and Sl-2. There was a slight decrease in CII between weeks 2 and 3, which was linked to the pitted lesions becoming ‘swollen’ in appearance . The highest incidence of decay was observed in Sh-36, Sl-2, and Sl-12 . A myriad of symptoms was recorded, i.e., severe discoloration, wrinkles around the stem scar, and surface ‘translucency’ , but they were absent or minimized in the WT. Degradation of RNA samples obtained from the transgenic fruit was observed and verified during agarose gel electrophoresis and may be connected to the phenotypic deterioration induced by CBF1 over expression. Based on their contrasting PCI phenotype, further analyses were conducted on Sl-2 and Sh-13 fruit in addition to the WT.

Daily cycles of light and darkness also promoted maintenance of glucosinolate content during post harvest storage of cabbage . Total glucosinolate content in the cabbage leaf disks stored under light/dark cycles remained stable with no significant fluctuation in levels over the 21 days of analysis . In comparison to total glucosinolate levels in light/dark-stored cabbage, the glucosinolate levels were significantly lower by 7 days when cabbage leaf disks were stored under constant darkness and by 21 days when stored under constant light . Total glucosinolate levels declined by 70% and 88%, respectively, in cabbage disks stored at 22°C under constant light or constant darkness. Remarkably, glucosinolate levels of the cabbage leaf disks stored at 4°C also showed a significant decrease by 21 days, with a loss of 50% of the initial glucosinolate levels , indicating that storage under cycle of light/darkness led to enhanced retention of this valuable phytochemical even relative to refrigeration.In this work, hydroponic bucket we examined whether kale, cabbage, lettuce and spinach leaf tissue maintain the ability to respond to light/dark cycles during post harvest storage and whether under these conditions that better mimic the natural light cycles of the environment tissue deterioration would be reduced.

Our goal was to expose plant tissues to diurnal conditions known to maintain the functioning of the circadian clock and thereby capitalize on physiological enhancements conferred by robust circadian rhythms. Plants grown under light/dark cycles that match the endogenous cycling of their internal circadian clock have a growth and reproductive advantage over plants exposed to light/dark cycles that do not match their internal oscillator . Furthermore, phasing of circadian rhythms so as to be synchronized with the external environment promotes biotic stress resistance. We found that storing green leafy vegetables in cycles of 12 hours of light followed by 12 hours of darkness improved several post harvest performance markers compared to post harvest storage of the leaf tissues under constant light or constant darkness. Similarly, a modest reduction in senescence was noted for post-harvest broccoli stored under natural light/dark cycles. Perhaps surprisingly, we found that storage in light/dark cycles resulted in several aspects of post harvest performance being comparable to storage under refrigeration, a commonly practiced method of post harvest storage thought to slow down cellular breakdown. The longevity of kale and lettuce leaf color, chlorophyll levels, and tissue integrity, which are important contributors to the appeal of green leafy vegetables to consumers, were largely indistinguishable whether the kale and lettuce leaf samples were stored at 22°C under light/dark cycles or were stored under refrigeration in constant darkness .

Spinach leaf samples also maintained green coloration and chlorophyll levels under light/dark cycles at 22°C as well as when refrigerated, but refrigeration was more successful at preventing spinach leaf tissue breakdown. Significant improvement of green coloration and chlorophyll content was seen when cabbage leaves were stored under light/dark cycles at 22°C compared to refrigeration, demonstrating that light may not only be important for clock entrainment but also can provide the additional benefit of promoting continued photosynthesis during post harvest storage. Promotion of photosynthesis and/or chlorophyll levels was previously observed in post-harvest crops stored under light. However, constant light during post harvest storage can also cause detrimental physiological activity, such as respiration leading to browning and transpiration contributing to weight loss. Therefore, cycling of light treatment with darkness periods may not only maintain clock function but may also avoid physiological damage that may occur in plant tissues under too much light. In addition to improvement of green leafy vegetable appearance by post harvest storage under light/dark cycles, we found that this post harvest storage treatment of plant crops may improve human health benefits through maintenance of phytochemical content .

Chlorophyll, responsible for the visual appeal of green leafy vegetables, also has beneficial impact upon human health upon ingestion. Chlorophyll can limit efficacy of carcinogens, such aflatoxin B1 and can activate Phase II detoxifying enzymes. Glucosinolates, sulfurcontaining compounds that play a major role in Brassicaceae plant herbivore defense, also underlie the human health benefits attributed to Brassicaceae vegetable consumption. For example, the glucosinolate glucoraphanin has potent anticancer activity. Previous studies have shown that glucosinolate levels can be maintained by refrigeration or exposure to radiation; here we find that post-harvest storage under light/dark cycles can also lead to sustained glucosinolate levels .Fruits are a good source of compounds with phenolic functionality including phenols, lignins, lignans, coumarins, tannins, phenolic acids, and Žavonoids which are important in the human diet. ese phenolic secondary metabolites comprise of an aromatic benzene ring with one or more hydroxyl groups that can exist as a simple monomeric phenolics or a complex polymerized polyphenolic molecules. ese molecules generally play a critical role in plant defense mechanisms including pathogen or insect attack, ultraviolet light, and mechanical damage in plants. ey also play an important role in human health, protecting against damage induced by reactive oxygen or nitrogen species. Phenolics are classified based upon their carbon skeleton and in accordance with the number of phenol units present. Phenolics arise from either phenylalanine or tyrosine amino acids that are deaminated into cinnamic acids and are primarily derived from the phenylpropanoids. Simple phenolic acids are divided into two groups: hydroxybenzoic acids and hydroxycinnamic acids. ese molecules play a vital function to protect the organism against to biotic or abiotic stress factors. Most phenolic compounds are highly reactive, are therefore not stabile, and can degrade into a wide variety of products. In the last decade, many epidemiological studies indicate a direct relationship between consumption of fruits and the prevention of diseases such as cancer and those related to inŽammation and oxidative stress. Monomeric phenolic compounds can polymerize to form more complex phenolic compounds. ese include Žavonoids, tannins, and lignin. Complex phenolics have a range of biological activities and functional characteristics and are of great interest to the food industries, cosmetic industry, and allied health fields.Flavonoids are complex phenolics that can be classified into six subclasses that include flavonols , flavanones , flavones , isoflavones , anthocyanins , and flavanols and are present principally as glycosylated, esterified, and polymerized derivative forms in fruits. Flavonols play an important role as antioxidants; for example, they protect ascorbic acid from autoxidation in juices and which can lead to juice discoloration. Although flavonoids are abundant in fruit, and fruits or beverages can be a significant source of dietary flavonoids, levels will vary depending on the varieties, environmental conditions, soil, and climatic factors. Berries are a good source of quercetin and its derivatives , stackable planters whereas the most abundant dietary flavanone glycoside is hesperetin-7-O-rutinoside present in citrus fruits. Peterson et al. reported that the most prevalent dietary flavanone aglycones are naringenin, hesperetin, isosakuranetin, and eriodictyol. ,e same authors demonstrate that a citrus fruit is also a primary source of narirutin, eriocitrin, didymin, neohesperidin, naringin, hesperidin, neoeriocitrin, and poncirin. ,e ratio of these compounds to each other can vary. For example, narirutin and naringin were detected in grapefruit in high ratios, while the levels of hesperidin and narirutin in oranges and eriocitrin in lemons were even higher. In addition, some flavanone glycosides such as 7-rutinoside are tasteless, in contrast to neohesperidin , naringin, and hesperetin which have an intense bitter taste isolated from bitter oranges and grapefruit. Apigenin is another key flavone found in fruits, vegetables, spices, and herbs and is abundant in grapefruit, beverages, some vegetables, and herbal plants such as chamomile.

Isoflavones are present in plants in the glycosylated forms but are converted to aglycone forms through the action of intestinal microflora. Isoflavones are detected commonly in legumes such as green beans, fava, and soybeans, and among them, genistein -4H-1-benzopyran-4- one and daidzein -4H-1- benzopyran-4-one are the two major forms of dietary isoflavones and are consumed in soy products. Due to the structural similarities to human hormone estrogen, isoflavones have potent estrogenic properties. Anthocyanins are another important class of flanovids that are colorful water-soluble glycosides and acylglycosides of anthocyanidins. 3-O-glycosides or 3,5-di-O-glycosides of malvidin, delphinidin, pelargonidin, cyanidin, petunidin, and peonidin are known as the most common natural anthocyanins and are classified based on the number and position of hydroxyl and methoxy groups. Anthocyanins are responsible for the brilliant colors of various plant parts including flowers and leaves and especially fruits having red, blue, purple colors, particularly strawberries, blueberries, black currants, cherries, raspberries, and red and purple grapes. Anthocyanidins are also responsible for the color of red wines. ,eir color based upon the degree of methylation and with pH is discrete from other phenolics by the range of colors each forms . Color differences of anthocyanins depend on the substitutions of the B ring, the pattern of glycosylation, and the degree and nature of esterification of the sugars with aliphatic or aromatic acids, and also on the pH, temperature, type of solvent, and the presence of copigments. Berries are a good source of anthocyanins, and 100 g of berries can provide up to 500 mg of anthocyanins. Flavan-3-ols are the core structure of condensed tannins and are the most complex subclass of flavonoids. Flavan-3-ols have been previously reported as an antioxidant, chemopreventive, and immunoregulation agents. Procyanidins exist in a wide range of foods and often exist in foods in a range of galloylated forms. Most widely used techniques for phenolics are HPLC , LC/MS, GC , GC/MS, UV-Vis spectrophotometry, mass spectroscopy, electrochemical, and fluorometric methods. Liquid chromatography mass spectrometry is used to determine phenolics in both APCI and ESI techniques, ABTS+ and DPPH.Sample preparation and extraction methods varied widely based on the nature of the sample matrix of the fruit or vegetable and based on the chemical structures of the phenolic compounds being extracted. As most samples contain a mixture of simple and complex polyphenolic compounds, such as phenolic acids, flavonoids, anthocyanins, and proanthocyanins, it is critical to choose a suitable method for sample preparation and extraction. Proteins, carbohydrates, lipids, or other elements may play negative effect to extraction of phenolics. In addition, it is not always possible to extract fresh samples, and special preparation techniques such as lyophilization, nitrogen pulverization, or drying may be needed. Particle size of extracted material and solvent-to-solute ratios need to be considered. As seen in Figure 1, there are many reliable qualitative and quantitative methods available for the measurement and characterization of the phenolic content in different natural products. Moreover, the success of these techniques will depend on the most effective sample preparation and extraction methods. Extraction efficiency is greatly influenced by solvent choice and composition and plays a critical role in the extraction yield of phenolics from fruits and vegetables. Generally, for the extraction of phenolics, water, acetone, ethyl acetate, alcohols , and their various percentages of mixtures are used. In addition to the solvent type extraction conditions, parameters such as temperature and duration also influence the yield of phenolics.Khoddami et al. previously reported that recovery of phenolics varied from one sample to another sample. It is also reported that acid- or base-catalyzed hydrolysis is also an important consideration for the stability of the phenolics in extracts. Davidov-Pardo and Marn-Arroyo reported that the extraction pH plays an important role in the extraction efficiency of phenolic compounds, and the same authors implied that catechins and their isomers are detected more efficiently in alkaline conditions as compared with acidic ones. Extraction of phenolic compounds are commonly done using either liquid-liquid or solid-liquid extraction technique. However, liquid-liquid extraction has some disadvantages because of using costly and potentially toxic solvents. For this reason, improved extraction methods such as solid-phase microextraction and solid-phase extraction techniques are used to extract phenolics from liquid samples. In general, inexpensive and simple methods such as soxhlet, reflux, and maceration processes are the more conventional procedures used to recover phenolics from solid samples. In addition, ultrasound-assisted extraction , microwave-assisted extraction , ultrasound microwave-assisted extraction , supercritical fluid extraction , subcritical water extraction , and high hydrostatic pressure processing are the methods that help us to shorten extraction times and decrease the release of toxic pollutants through reducing organic solvent consumption and are relatively simple to perform.

However, the production of H2O2 and phenolic compounds, and higher PPO activity in the translucent tissue were detected specifically in the VB tissues, not in other living non-VB cells after chilling . These data indicated that IB did not randomly occur in any living parenchyma cell but is specific to the phloem. Therefore, if the phloem is the site of IB initiation in PCI of pineapple fruit, then future studies should focus on how different traits ofthephloem, e.g.,membrane properties, antioxidant system, etc., vary among cultivars differing in IB response.In all IB-occurring cultivars studied, the VBs found at the F/C boundary contained fewer sclerenchyma fiber cells than did the VBs found at the C region, except in the PTV cultivar . The sclerenchyma fibers are composed of lignified cells, which offer protection to the VBs . When these anatomical features of the pineapple are considered, the link between VB number and the appearance of IB at the F/C is clear. This occurrence may be explained by the morphological structure of the pineapple fruit. The fruit is a composite of multiple individual fruitlets, fused to each other and to the C.

At maturity, hydroponic nft system the fruitlets become the ediblefleshly part ofthe fruit composed predominantly of parenchyma cells and some VBs containing lignified cell walls . The C of the fruit consists mainly of VBs with connections to the stem. The C region constitutes a stele of VB, one connected to the other vertically. From the C region,theVBs are distributed to the F/C of the fruit . The VBs in the F/C region form the denser network that grows both vertically and horizontally toward the F regions . These data suggested that the higher number of VBs at the F/C regions might explain why IB symptoms are easily detectable to the naked eye. However, as mentioned previously, the properties of phloem, e.g., their membrane properties and antioxidant system, at the F/C region might play a role in IB development.Consistent with published data,the IB-resistant MD2 showed no IB and TS after being stored at 10 ◦C for three weeks . It is possible to hypothesize that differences invascular structure may play a role in the observed lack of IB in resistant cultivars. Among all four cultivars studied here,the number and size of the xylem elements and phloem per VB did not correlate with varietal IB resistance . It is noteworthy,however,thatthe IB-resistantMD2hadfewerVBs but more sclerenchyma fiber cells surrounding the phloemand xylem, resulting in a larger size of VBs compared with other cultivars. In addition, the number and size of VBs and the number of sclerenchyma cells in all regions of the MD2 fruit were similar . In MD2, there were the multiple layers of the sclerenchyma cells on the lateral sides of VB. In contrast, in PTV and SV, there were no more than two layers of the sclerenchyma cells on the lateral sides ofVB, and only a single one in TST.

These special anatomical features may serve a protective purpose, preventing separation of the VBs themselves and limiting further damage to the tissue from chilling injury . Interestingly, MD2 stored at 10 ◦C for six weeks exhibited chilling injury at the exocarp . These data suggested that there are potentially different physiological and biochemical processes occurring in the fruit of the IB-resistant MD2 including: different physiological and biochemical properties of phloem cell ,fewer metabolically active phloem cells due to the lower number of VBs compared to the susceptible lines , and very low levels of PPO activity or phenolics so that any browning compounds produced were not visibly detectable .A plant-based diet is defined as the consumption of plants such as fruits, vegetables, nuts, seeds, oils, whole grains, legumes, and beans. It may or may not include small amounts of animal products such as meat, fish, sea-food, eggs, and dairy, but these foods tend to be avoided. Plant foods are rich in phytochemicals, antioxidants, vitamins, and minerals which prove beneficial to health on a micronutrition level, especially when plants are consumed in their whole, unprocessed form. When brought to the forefront of a patient’s dietary pattern, these naturally nutritious foods may translate into clinically significant improvements to patients’ quality of life and disease prognosis, and mitigate disease complications.

Historically, these foods have been avoided in patients on dialysis due to concerns of hyperkalemia and protein deficiency. However, the recent recognition of their potential benefits and improved understanding of potential pitfalls have favored the reintroduction of these foods into the diet of patients on dialysis.Traditional dietary interventions in patients with kidney disease encourage the restriction of phosphorus, potassium, sodium, and fluid as well as tailoring protein intake to the patient’s CKD stage and whether they require kidney replacement therapy. These guidelines tend to result in a diet devoid of fruits and vegetables, lacking in variety and resulting in limited satisfaction. Broadening the diet of patients with kidney disease to include these otherwise healthy foods may not only improve satisfaction and increase variety but may also carry additional benefits. A recent multinational cohort study found that higher consumption of fruits and vegetables in patients on dialysis was associated with lower all-cause and non-cardiovascular death. In this study, patients receiving long-term hemodialysis completed a food frequency questionnaire for a median follow-up of 2.7 years . Only 4% of the patients consumed at least 4 servings of fruit and vegetables per day as recommended in the general population. Plant-based diets, a fiber- and polyphenol-rich intervention, may attenuate cardiovascular disease risk markers in patients on hemodialysis. Polyphenols are a diverse group of micronutrients only consumed through plants. A systematic review and meta-analysis of 12 studies found that polyphenol-rich interventions in patients on hemodialysis improved diastolic blood pressure , triglycerides , and myeloperoxidase. Fiber supplementation was found to significantly lower total cholesterol, LDL cholesterol, and inflammatory markers – all of which are known cardiovascular risk factors. Plant fats, which are rich in unsaturated fatty acids, have shown to be beneficial in the diets of patients on hemodialysis by improving inflammatory markers. Vegetable oils are rich in polyunsaturated fatty acids, such as linoleic acid, nft channel which are known to have cardioprotective and anti-inflammatory properties. In one study, linoleic acid levels, as a proportion of plasma fatty acids, were shown to be inversely associated with IL-6 and all-cause mortality in a cross-sectional study of hemodialysis patients.Dietary fiber is a nondigestible, nonabsorbable carbohydrate polymer that may be used to treat chronic kidney disease by exploiting its ability to reduce uremic toxin production and, perhaps, postpone dialysis. Since dietary fiber is exclusively found in plants, there may be a unique advantage associated with the use of plant-based diets . Fermentable fibers are metabolized into short-chain fatty acids, especially acetate, propionate, and butyrate. Short-chain fatty acids may play a role in kidney health by modulating systemic inflammation and maintaining the integrity of the gut mucosal barrier. Today, plant-derived complex carbohydrates are recognized as prebiotics for their role in promoting the growth and metabolic activity of beneficial saccharolytic organisms like Bifidobacteria and Lactobacillus.

The protective effects of gut microbiota have been widely studied in chronic kidney disease, but there is a paucity in data regarding the bidirectional gut-kidney axis in hemodialysis patients. Compared to healthy individuals, a cross-sectional study found end-stage kidney disease patients on hemodialysis to have an increased relative abundance of Proteobacteria , Actinobacteria, and Firmicutes by phylogenic microarrays. Of note, these taxa are not usually associated with uremic toxin production. Indoxyl sulfate and p-cresol are uremic toxins generated by colonic bacteria in response to food that is not absorbed in the small bowel. Uremic toxins accumulatein patients with chronic kidney disease and may contribute to the progressive loss of glomerular filtration rate by increasing the expression of transforming growth factor beta which increases fibrogenesis. A cross-sectional study of patients undergoing hemodiafiltration found that vegetarian patients had 47% lower levels of indoxyl sulfate and 67% lower levels of p-cresyl sulfate than the nonvegetarian patients. In another study of patients on hemodialysis, an increase in fiber intake was shown to significantly reduce plasma levels of indoxyl sulfate by 17% and non-significantly reduce plasma levels of p-cresol by 8% in a randomized control trial . Another cross-sectional study found that increased levels of p-cresol were associated with an increased risk of death in patients on maintenance hemodialysis. Outside of decreasing uremic toxins, fiber supplementation in patients on hemodialysis also improved lipid profiles and oxidative status and decreased systemic inflammation when compared to a placebo in a 6-week randomized control trial. The results of the trial revealed a significant decrease in total cholesterol level, high-density lipoprotein level, and TC:LCL ratio; a significant decrease in total antioxidant capacity and malondialdehyde; and a significant decrease in tumor necrosis factor-α, interleukin-6, interleukin-8, and C-reactive protein in the 10 and 20 g per day fiber supplement groups. A long term prospective cohort study of patients on peritoneal dialysis showed an independent association between fiber intake and all-cause mortality wherein each gram per day increase in fiber intake correlated with a 13% reduction in all-cause mortality. Contrarily, low dietary fiber intake has been shown to negatively impact patients on hemodialysis on multiple occasions. In a prospective cohort study of patients on dialysis , lower fiber intake was associated with an increased risk for major adverse cardiovascular events when compared to patients with higher fiber intake. Another study on maintenance hemodialysis patients found that dietary fiber level was independently correlated with advanced glycation end products and C-reactive protein levels.In hemodialysis patients, the incidence of constipation varied according to the definition of constipation, but it was noted to be 71.7% in a 2013 cross-sectional study using the Roma III criteria. As a chronic or frequently recurrent symptom, constipation impairs patients’ health-related quality of life and may result in a substantial emotional and social burden. The issue may be easily treated by emphasizing the consumption of high-fiber foods, like plants. It may be possible that the historically low consumption of fiber-rich foods of patients on hemodialysis may be to blame for the high rate of constipation in this population. In the general population, patients with constipation had 12% higher all-cause mortality, 11% higher incidence of coronary heart disease, and 19% higher incidence of ischemic stroke compared to patients without constipation. US Veterans using one or >2 types of laxatives experienced a similarly higher risk of all-cause mortality and cardiovascular and cerebrovascular burden when compared to US veterans not taking any laxatives. A recent multi-center study evaluated the prevalence and correlates of constipation in hemodialysis patients from 4 dialysis centers using a questionnaire based on the Roma IV criteria to assess constipation status and lifestyle habits along with a food frequency questionnaire focused on the main sources of dietary fiber and found that >30% of patients had constipation at a given point in time. In this study, the independent predictors of constipation symptoms included diabetes and lower frequency of fruit intake. Moreover, constipation can worsen hyperkalemia, while having soft bowel movement may be associated with a lower likelihood of hyperkalemia.Although phosphorus is an essential nutrient, it is difficult for patients to avoid states of phosphate excess in advanced CKD. Consequently, patients must be placed on phosphorus-restricted diets to reduce the risk of hyperphosphatemia, which has been associated with an increased risk of death and cardiovascular disease. Given the varying bio-availability of phosphorus from foods, dietary changes may be made to take advantage of low phosphate-containing foods. In the gut, 40–60% of phosphate in animal protein is absorbed by the intestinal tract compared to only 10–30% of phosphorus in plant protein. This is partly because the bulk of phosphorus in plants is bound to phytate, which is difficult for humans to digest due to lack of phytase to release the phosphorus. In a cross-over trial in patients with CKD, the effects of vegetarian and meat diets on phosphorus balance showed that 1 week of a vegetarian diet led to significantly lower serum phosphorus and significantly lower fibroblast growth factor-23 levels than being on a meat diet . This finding has also been seen in a small observational study by Wu et al. wherein serum phosphate levels were significantly lower in vegetarian patients than in non-vegetarian patients on hemodialysis.When diagnosed with end-stage kidney disease, traditional dietary interventions may place patients at risk for vitamin deficiencies.

There are striking examples in tomato and other plants where identified genes regulate the development of fruit shape. For example, the OVATE gene in tomato regulates the phenotypic transition from round to pear-shaped fruit . If large-effect mutations underlie differences in strawberry fruit shape, the ordinal classification system proposed here should enable the discovery of such effects. Furthermore, quantitative phenotypes were linked to genetic features that interact with large-effect genes, i.e., suppressors of OVATE , through bulk segregant analysis and quantitative trait locus mapping . In woodland strawberry , fruit size and shape are linked to the accumulation and complex interaction of auxin, gibberellic acid, and abscisic acid, mediated by the expression and activity of FveCYP707 and FveNCED, as well as other genes. Because of the high H2 estimates for several of the newly created phenotypic variables , we hypothesize that quantitative, nft hydroponic latent space phenotypes can yield a more comprehensive understanding of the underlying genetic mechanisms of fruit shape in garden strawberry through GWAS and other quantitative genetic analyses. We anticipate that the analyses in this study will enable us to discover and study the genetic determinants of fruit shape in strawberry and other specialty crops.

Over the past few decades, models of peach fruit growth and plant development have identified useful principles for assisting growers in making horticultural management decisions. For example, harvest-date prediction models are now available to aid in managing fruit crops . The unusually early harvest of California’s peach crop in 2004 — which had record high temperatures during bloom time — and attendant difficulties in attaining the fruit sizes desired by the market have increased interest in using physiological concepts to understand the effects of early-spring temperature on peach fruit growth and in anticipating fruit size at harvest. The dependence of peach fruit development on spring temperatures during the first 30 days after bloom has been established; there is a strong correlation between the sum of growing degree hours accumulated in the 30 days after bloom and the number of days between bloom and harvest for several stone-fruit cultivars . Traditionally, the California cling stone peach industry has used reference-date fruit size to predict what the fruit size potential will be for a given year, and then peach growers conduct fruit-thinning accordingly. Reference-date fruit sizes are known to vary from year to year, but the reasons for this variation were previously unclear.

Lopez and DeJong compared a 20-year cling stone-peach data set collected and archived by the California Canning Peach Association, which included full bloom date , reference date and fruit size at reference date from different locations in California. When the data — collected from orchards near the Central Valley cities of Kingsburg, Modesto and Yuba City — was correlated with seasonal weather data available through the California Irrigation Management Information System , a strong correlation was found between accumulated GDH30 and the number of days between full bloom date and reference date . The number of days between full bloom date and reference date decreased as accumulated GDH30 increased . There was also a strong correlation between the number of days from full bloom date to reference date, and fruit size at reference date. Fruit size at reference date increased with an increase in the number of days from full bloom date to reference date . This study indicated that peach trees apparently could not supply resources rapidly enough to support the potential maximum fruit growth rates when accumulated GDH30 was higher than a threshold value . Consequently, fruit size at reference date in years with very warm spring temperatures was less than in years when accumulated GDH30 was below that threshold value. Furthermore, previous research has documented that fruit growth potential unfulfilled in early spring cannot be compensated for later in the season .

Although the effects of early-spring temperature on fruit size have been quantified for cling stone peaches at reference date, and fruit size at reference date is thought to be a good indicator of fruit size at harvest, industry wide data on fruit size at harvest is not available from the canning cling stone peach industry. However, the industry wide data situation is the opposite for California fresh market peaches. This industry does not keep representative seasonal data on full bloom date or reference date, but general fruit-size data is available from industry records maintained by the California Tree Fruit Agreement . At the outset of this study, we anticipated that the environmental factors influencing fruit growth and development rates for canning cling stone peaches would be the same as for fresh-market freestone peaches. Thus we initiated a follow-up study using relationships we had established for cling stone peaches to analyze the seasonal environmental effects on industry wide data for the fruit size of freestone peach cultivars. When fresh-market peaches are packed for shipping, the fruit are separated into different size categories related to the number of fruit that will fit into a standard-size lug box. Fruit packed in a size 30 lug are larger than those in a size 40 lug, while the fruit in a size 40 lug are larger than those in a size 50 lug, and so on. The CTFA annually reports the percentage of the total number of lugs for specific fruit-size categories that are shipped for each major cultivar at harvest. Data from three different fresh market peach cultivars were used for this study and compared with full bloom and fruit reference-date data for cling stone canning peach cultivars . However, the average fruit size lug category of the fresh-market cultivars was not correlated with the number of days between full bloom date and reference date for the cling stone cultivars in a given year in the Kingsburg area . When we analyzed historical trends in fruit size, there was a clear trend toward lower average lug categories; over the 20 years of this study, the average size of the fruit packed for each cultivar increased significantly . Although improvements in cultural practices may account for some of the increases in packed fruit size, this long-term trend presumably can be attributed to marketing pressures. Consumer acceptance of California peaches has been related to soluble solid concentration, acidity or soluble solid concentration/acidity ratio, but the major quality factor is fruit appearance . Likewise, market pricing has consistently favored larger-sized fruit. The percentage distribution trends of average fruit-size categories over the 20 years were similar for the three cultivars, and there were no significant differences in the slope of the regressions between Elegant Lady and O’Henry, or between Elegant Lady and Flavorcrest. However, the slope of the response for Flavorcrest was steeper than for O’Henry .When the effect of the year on the percentages of four fruit-size lug categories at harvest was analyzed independently, different patterns were observed among the cultivars and categories . Although all the cultivars increased the percentages of larger fruit-size lug categories and decreased the percentages of the smaller fruit size lug categories , nft system the slopes of the 30s and 80s categories of Flavorcrest relationships were clearly different from those of Elegant Lady and O’Henry . The change in Flavorcrest peaches observed in figure 4 could be primarily explained by a drastic reduction in the percentage of fruit packed in the smallest fruit-size lug category . However, the change observed in Elegant Lady and O’Henry was mostly related to an increase in the percentage of fruit packed in the largest fruit-size lug category . Although industry wide data on fruit packed per acre is not available for these cultivars, one practical implication of these results is that the average yield of packed fruit has likely declined over the same 20-year period, since, on average, fruit size is generally correlated with crop load . For example, a California thinning study showed a substantial effect on yield for both O’Henry and Elegant Lady .

For O’Henry, the change in lug size from 1985 to 2004 required average fruit weights to increase from 0.48pound to 0.56 pound . To obtain these fruit weights, a typical tree would need to be thinned to 976 and 523 fruit in 1985 and 2004, respectively. Thus, yields would have dropped from 464 pounds per tree to 291 pounds per tree , a 37% decrease over the 20 years. The results for Elegant Lady were similar. However, the profitability of early cultivars such as Flavorcrest was probably affected more by market pressures than Elegant Lady and O’Henry, since decreases in the amount of fruit in small size categories were not offset as much by increases in fruit packed in the large-size categories . When the data in figure 4 was used to normalize the fruit-size lug data to account for the long-term general trend, the deviation of a given year’s average fruit-size lug category from the long term trend was clearly related to spring weather patterns. The average, long term, trend-adjusted fruit-size lug category for the fresh-market cultivars in a given year decreased, with an increase in the number of days of fruit growth between full bloom and reference date recorded in the same year for cling stone peaches . Although the variability in this relationship among the different cultivars could have been related to the inherent variability in sources of fruit that were packed, the similarity of the slopes of the relationships for the three cultivars indicates that the conditions driving the relationships were likely similar for all three cultivars .Previous research with cling stone peaches, combined with this analysis of fresh-market peach data, indicate that early fruit development rates are clearly related to heat accumulation, and that high early-spring temperatures tend to decrease the average size of fruit packed in a given year. This is apparently because in especially warm springs, the tree cannot supply resources rapidly enough to support the potential fruit growth rates associated with high rates of phenological development. The relationships between fruit developmental patterns, fruit growth potentials and spring temperatures are even more important in light of the clear long-term marketing trends toward packing larger-sized fruit. Grower success will depend upon the ability to anticipate yearly fruit-sizing potential for individual cultivars and make the appropriate, cost-effective adjustments in cultural practices. It is well documented that early and heavy fruit thinning can increase average fruit size, but may cost more and/or reduce overall yields . However, these practices may be particularly useful in difficult fruit sizing years in light of the increasing market pressure for large-size fruits.California’s coastal districts, where 86% of the nation’s strawberries are produced on 38,600 acres, are the most productive strawberry-growing areas in the United States . To achieve this level of productivity, California strawberry producers need effective soil disinfestation, productive varieties and cultural practices such as polyethylene mulch and drip irrigation . Strawberries are very sensitive to soil pathogens, and growers with these highly productive systems have become dependent on preplant fumigation. Traditionally, they used methyl bromide plus chloropicrin as the basis for soil pest control. Fumigation with these chemicals controls soilborne pathogens such as Verticillium dahliae, Phytophthora species, Pythium species, Rhizoctonia species, Fusarium oxysporum and Cylindrocarpon species, as well as nematodes, soilborne insects and weed seeds in the soil seedbank . In 1992 methyl bromide was classified as a Class I stratospheric ozone-depleting chemical. Since 2005, under the Montreal Protocol, the use of methyl bromide for fumigation in the United States has been permitted only through critical use exemption . The methyl bromide phase-out and other regulatory limitations make research on alternative pest control measures essential. Currently, some California strawberry fields can still be treated with methyl bromide under the critical use exemption, which is subject to annual review by the parties of the Montreal Protocol.However, methyl bromide costs have been increasing, and its use in strawberry production has been decreasing . In traditional fumigation of California strawberry fields, beginning in the 1960s and continuing until recently, growers applied methyl bromide plus chloropicrin to the total field area. This process was called flat fumigation; the entire field was covered with polyethylene film to hold the fumigant at the concentration needed to kill soil pests . In the last decade, a sizable portion of the strawberry acreage has been treated with fumigants applied to the strawberry bed by drip fumigation .

New LED exit signs are inexpensive, with prices typically starting at around $20. The U.S. EPA’s ENERGY STAR program website provides a list of suppliers of LED exit signs. Tritium exit signs are an alternative to LED exit signs. The advertised lifetime of these signs is around 10 years and prices typically start at around $150 per sign. Electronic ballasts. A ballast regulates the amount of electricity required to start a lighting fixture and maintain a steady output of light. Electronic ballasts can require 12% to 30% less power than their magnetic predecessors . New electronic ballasts have smooth and silent dimming capabilities, in addition to longer lives , faster run-up times, and cooler operation than magnetic ballasts . New electronic ballasts also have automatic switch-off capabilities for faulty or end-of-life lamps. Replacement of T-12 tubes with T-8 tubes. In many industrial facilities, it is common to find T-12 lighting tubes in use. T-12 lighting tubes are 12/8 inches in diameter . T-12 tubes consume significant amounts of electricity, hydroponic channel and also have extremely poor efficacy, lamp life, lumen depreciation, and color rendering index.

Because of this, the maintenance and energy costs of T-12 tubes are high. T-8 lighting tubes have around twice the efficacy of T-12 tubes, and can last up to 60% longer, which leads to savings in maintenance costs. Typical energy savings from the replacement of a T-12 lamp by a T-8 lamp are around 30% . Replacement of mercury lights. Where color rendition is critical, metal halide lamps can replace mercury or fluorescent lamps with energy savings of up to 50%. At a Basic American Foods facility in Shelley, Idaho, the production area lighting system was upgraded using metal halide lamps. According to the company, the improved color rendition and increased light levels offered by the metal halide lamps helped production workers better detect deffects in the plant’s potato products. Plant sanitation was also improved, because staff could better see debris on the equipment and floors . Where color rendition is not critical, high-pressure sodium lamps offer energy savings of 50% to 60% compared to mercury lamps . High-intensity discharge voltage reduction. Reducing lighting system voltage can also save energy. A Toyota production facility installed reduced-voltage HID lights and realized a 30% reduction in lighting energy consumption .

Commercial products are available that attach to a central panel switch and constrict the flow of electricity to lighting fixtures, thereby reducing voltage and saving energy, with an imperceptible loss of light. Voltage controllers work with both HID and fluorescent lighting systems and are available from multiple vendors. High-intensity fluorescent lights. Traditional HID lighting can be replaced with high intensity fluorescent lighting systems, which incorporate high-efficiency fluorescent lamps, electronic ballasts, and high-efficacy fixtures that maximize output to work areas. These systems have lower energy consumption, lower lumen depreciation over the lifetime of the lamp, better dimming options, faster startup and re-strike capabilities, better color rendition, higher pupil lumens ratings, and less glare than traditional HID systems . Daylighting. Daylighting involves the efficient use of natural light in order to minimize the need for artificial lighting in buildings. Increasing levels of daylight within rooms can reduce electrical lighting loads by up to 70% . Unlike conventional skylights, an efficient daylighting system may provide evenly dispersed light without creating heat gains, which can reduce the need for cooling compared to skylights.

Daylighting differs from other energy efficiency measures because its features are integral to the architecture of a building; therefore, it is applied primarily to new buildings and incorporated at the design stage. However, existing buildings can sometimes be cost effectively refitted with daylighting systems. Daylighting can be combined with lighting controls to maximize its benefits. Because of its variability, daylighting is almost always combined with artificial lighting to provide the necessary illumination on cloudy days or after dark . Daylighting technologies include properly placed and shaded windows, atria, clerestories, light shelves,and light ducts. Clerestories, light shelves, and light ducts can accommodate various angles of the sun and redirect daylight using walls or reflectors. More information on daylighting can be found at the website of the Daylighting Collaborative led by the Energy Center of Wisconsin .The use of on-site electricity generation appears to be quite limited in the U.S. fruit and vegetable processing industry. In 2002, only 5% of the industry’s electricity was generated at individual facilities . The use of on-site generation was confined almost exclusively to the fruit and vegetable canning sub-sector, where the extensive use of steam in blanching, evaporating, pasteurizing, and sterilizing applications makes combined heat and power systems particularly attractive. Self generation can be an attractive option for many facilities for reducing the energy intensity of utilities services. This chapter provides a brief overview of several self-generation measures applicable to the U.S. fruit and vegetable processing industry. Combined heat and power . For industries like fruit and vegetable processing that have simultaneous requirements for process heat, steam, and electricity, the use of CHP systems may be able to save energy and reduce pollution. Combined heat and power plants are significantly more efficient than standard power plants because they take advantage of waste heat. In addition, electricity transmission losses are minimized when CHP systems are located at or near the facility. Often, utility companies will work with individual companies to develop CHP systems for their facilities. In many cases, the utility company will own and operate the facility’s CHP system, allowing fruit and vegetable processors to avoid the capital expenditures associated with CHP projects while reaping the benefits of a more energy-efficient source of heat and electricity. In addition to energy savings, CHP systems also have comparable or better availability of service than utility generation. In the automobile industry, for example, typical CHP units are reported to function successfully for 95% to 98% of planned operating hours . Many large-scale CHP systems use steam turbines. Switching to natural gas-based systems is likely to improve the power output and efficiency of the CHP system, due to increased power production capability. Although the overall system efficiency of a steam turbine-based CHP system is higher than that of a gas turbine-based CHP system , the electrical efficiency of a gas turbine-based CHP system is superior . Furthermore, modern gas-based CHP systems have low maintenance costs and will reduce emissions of NOx, SO2, CO2, and particulate matter from power generation considerably, especially when replacing a coal-fired boiler . In general, the energy savings of replacing a traditional system with a standard gas turbine-based CHP unit is estimated at 20%-30% . However, hydroponic dutch buckets savings may be greater when replacing older or less maintained boilers.Additional advantages are that the amounts of power and thermal energy produced by the turbine can be adjusted to meet current power and thermal energy loads. If steam loads are reduced, the steam can then be used for power generation, increasing output and efficiency . Drawbacks include the additional complexity of the turbine’s design. The economics of a CHP system depend strongly on the local situation, including power demand, heat demand, power purchasing and selling prices, natural gas prices, as well as interconnection standards and charges, and utility charges for backup power. In some states, programs may offer support for installation of CHP systems .

Tri-generation. Many new CHP systems offer the option of tri-generation, which provides cooling in addition to electricity and heat. Cooling can be provided using either absorption or adsorption technologies, which both operate using recovered heat from the co-generation process. Absorption cooling systems take advantage of the fact that ammonia is extremely soluble in cold water and much less so in hot water. Thus, if a water-ammonia solution is heated, it expels its ammonia. In the first stage of the absorption process, a water-ammonia solution is exposed to waste heat from the co-generation process, whereby ammonia gas is expelled. After dissipating the heat, the ammonia gas—still under high pressure—liquefies. The liquid ammonia flows into a section of the absorption unit where it comes into contact with hydrogen gas. The hydrogen gas absorbs the ammonia gas with a cooling effect. The hydrogen-ammonia mixture then meets a surface of cold water, which absorbs the ammonia again, closing the cycle. One food company that has successfully implemented absorption technology is the Ghirardelli Chocolate Company, a California based manufacturer of chocolate products. Ghirardelli’s manufacturing facility in San Leandro, California, uses an on-site electricity generating system, which is powered by four 350 kW natural gas-fired reciprocating engines. In 2003, the company installed a single-stage 145 ton absorption chiller that runs entirely on heat from the engines’ exhaust and jacket water. According to the company, the combined area of the buildings being cooled by the absorption chiller is approximately 35,000 square feet . In contrast to absorption cooling, adsorption cooling utilizes the capacity of certain substances to adsorb water on their surface, from where it can be separated again with the application of heat. Adsorption units use hot water from the co-generation unit. These systems do not use ammonia or corrosive salts, but use silica gel . Adsorption units were originally developed in Japan and are now also marketed in the United States. The thermal performance of absorption and adsorption systems is similar, with a coefficient of performance between 0.68 and 0.75. The capital costs of both systems are also comparable. However, the reliability of an adsorption unit is expected to be superior and its maintenance costs are expected to be lower . Back pressure turbines. At many facilities, steam is produced at a higher pressure than is demanded by process requirements. Often, steam pressure is reduced for process use by passing steam through pressure reducing valves, essentially wasting thermal energy. A back pressure steam turbine can perform the needed pressure reduction while converting this otherwise wasted thermal energy to electricity for use throughout the facility. According to the U.S. DOE, back pressure turbines can be considered wherever a pressure reducing valve has constant steam flow of at least 3,000 pounds per hour and when the steam pressure drop is at least 100 psi . Morning Star Packing Company, a manufacturer of tomato paste and other canned tomato products located in Williams, California, uses back pressure turbines to generate 100% of facility electricity needs . In the mid- to late- 1990s, the company installed three 1 MW back pressure turbines at a cost of around $847,000, including capital costs and installation expenses. Reported electricity cost savings have totaled nearly $500,000 per year. The company projected that over the 20-year lifetime of the back pressure turbines, they expect to save almost $9 million in total energy bills and realize a compound annual rate of return of more than 60% .Photovoltaic panels. Photovoltaic panels convert sunlight directly into electricity and can provide a reliable and renewable source of electricity to facilities with ample sunlight. Photovoltaic panels, which are typically mounted on the roof of a facility, convert electricity to DC current, which is subsequently sent through an inverter and transformer and converted into AC power. The AC power can be fed directly into a facility’s power supply. While the capital and installation costs of photovoltaic systems are currently somewhat high , manufacturers can often receive substantial rebates and tax credits from state and federal agencies that can help make photovoltaic investments more economically attractive. Inverters typically last 10 to 20 years, while photovoltaic panels can typically generate power for 25 to 40 years . Kettle Foods, a producer of all natural snacks based in Salem, Oregon, installed a 114 kW photovoltaic power system on the roof of its processing plant and headquarters in 2003. Reportedly, the system saves the company $8,400 in energy costs each year, while also avoiding around 2,500 tons of CO2 emissions. The initial capital and installation costs totaled $675,000, but the company received over $400,000 in clean energy incentives, Oregon energy tax credits, and U.S. federal energy tax credits, which helped to make the project more economically viable . Over the 40-year life of the system, the company estimated a 7% average rate of return and a net present value of $55,000. However, the project has also helped reinforce Kettle Foods’ image as an environmental steward and has reportedly led to good corporate publicity.