As fruit grow, early feeding by avocado thrips becomes apparent as scabby or leathery brown scars that expand across the skin and is sometimes referred to as “alligator skin” . Avocado thrips damage is affected by practices that increase or decrease the abundance of succulent foliage during set and growth of young fruit. Thrips move to young fruit when leaves harden after the growth flush has finished and the most damage occurs when fruit are 5.1 to 15.2 mm long . Although Hass fruit are susceptible to feeding until they reach about 51 mm in length, thrips feeding rarely causes scars on fruit larger than about 19.1 mm. This scarring on young fruit may not become obvious until fruit enlarge. In severe cases, all fruit on a tree can have their entire fruit surface scarred by avocado thrips, causing some packinghouses to sell such fruit with the box marked “papacado.” The California Avocado Commission estimated a $50 million dollar crop lost in the 2006 due to avocado thrips scarring and the costs of control . Sticky card and beating tray sampling are research methods used for these two insects but are rarely used by growers or pest control advisors . Both PCAs and researchers monitor citrus thrips by counting the percent of fruit infested with immature thrips and the number of immature thrips per fruit is also indicative of the severity of the infestation. Thresholds in use in the San Joaquin Valley are 20% of Valencia oranges or 10% of navel oranges infested with immature thrips until the fruit reaches 20 mm in diameter or more. Avocado thrips are monitored by counting the number of immature thrips per leaf prior to fruit set or the number of thrips per fruit.

No firm economic threshold has yet been developed for avocado thrips but PCAs typically treat at 3-5 immature thrips per leaf prior to bloom in San Diego County due to restrictions on use of abamectin during bloom. The major documented citrus thrips predator is the phytoseiid mite, E. tularensis , although Jones and Morse questioned the importance of this predator. Avocado thrips are frequently preyed upon by Franklinothrips orizabensis Johansen and Chrysoperla carnea and is parasitized by the larval parasitoid Ceranisus menes . Franklinothrips vespiformis , black hunter thrips , and several banded-wing thrips also feed on avocado thrips . In many years,square pots natural enemies are unable to suppress avocado and citrus thrips populations below economic thresholds and chemical control is needed to reduce fruit scarring. By the time damage is noticed on ripening fruit, the thrips that caused the injury are often absent from the fruit. A variety of pesticides are registered for thrips control in different cropping systems . After a number of years of use, pesticides like dimethoate , formetante hydrochloride , cyfluthrin , and fenpropathrin resulted in failures in citrus thrips control in some regions, along with an increase in resistance confirmed with both laboratory and field bio-assays. Also, these materials are detrimental to natural enemies such as Aphytis melinus DeBach and other biological control agents important to citrus pest control. Since it was registered in 1998, spinosad has been the main material used for control of citrus thrips and a related and more effective material, spinetoram , was registered late in 2007 and will soon replace spinosad once MRL issues are resolved with export countries. Abamectin is the main material used for avocado thrips control with occasionally rotation with sabadilla . Resistance to sabadilla has been shown with avocado thrips and a similar pattern of resistance development with abamectin is of concern due to the persistence of this material in leaf tissue. To date, citrus thrips resistance to spinosad has not been documented but there is concern that resistance to it or spinetoram may appear soon. With a limited number of pesticides available for control and the frequency of resistance shown by thrips such as citrus thrips, it is wise to monitor population levels carefully, limit treatments to population levels of concern, and time treatments optimally .

Appropriate cultural practices and conservation of natural enemies should be practiced in concert with the use of pesticides only on an as-needed basis. Thus, the search continues for effective biological and chemical controls useful in citrus and avocado thrips management. For both species of thrips, some pupation occurs on the tree in cracks and in crevices, however, about three-fourths of avocado thrips drop as late second instars from trees to pupate in the upper layer of dry leaf litter . Propupae and pupae are rarely seen, move only if disturbed, and do not feed. This phenomenon of dropping down to the leaf-litter or soil surface for pupation may create the ideal interface for control using the entomopathogenic fungi B. bassiana. Adding coarse organic mulch beneath trees and maintaining a mulch layer may reduce survival of thrips that drop from trees to pupate below the tree, especially in avocados, because this is common practice by many growers as a method of Phytophthora management. The effectiveness of mulching to control thrips is uncertain and labor costs of adding mulch may not be justified solely for thrips control. However, applying coarse organic material such as composted yard waste beneath trees may help control weeds, and thrips reduction might be an additional benefit, particularly for blueberries. The deep mulch layer that is standard practice with blueberry culture in the San Joaquin Valley may also provide an ideal habitat for B. bassiana. It is possible that as citrus thrips are adapted to and evolved in a hot, dry climate, they may be more susceptible to B. bassiana, whereas avocado thrips has adapted to and evolved in a wet and cool climate and may be less susceptible to or even tolerant to B. bassiana. There is increasing pressure in the United States to move away from broadspectrum insecticides and focus on alternative methods of control, e.g., geneticallymodified crop plants expressing Bt toxins, use of entomopathogens, biorational insecticides. Implementation of such methods on avocado and citrus are difficult due to the relatively primitive methods available for thrips sampling, which are labor intensive and rely on experienced and intuitive pest control advisors. The goal of the work described here is to examine alternatives to traditional insecticides such as Bt proteins and entomopathogenic fungi to control avocado and citrus thrips, with the ultimate target of utilizing entomopathogens to aid in field control, evaluate the insecticides registered for avocado thrips management on the native predaceous mite Euseius hibisci, assess citrus thrips oviposition on blueberry varieties, and determine whether citrus thrips is actually a complex of species.

Citrus thrips, Scirtothrips citri , is a plant-feeding pest most widely recognized for the damage it causes to citrus and mango fruits and has been recognized as a major pest of California citrus since the 1890s . Recently, its known host range has broadened and they have become a significant pest of a relatively new crop planted in the San Joaquin Valley of California, highbush blueberries . Citrus thrips feed on blueberry foliage during the middle and late portions of the season causing distorted, discolored, and stunted flush growth and poor development of fruiting wood required to obtain the subsequent crop . High numbers of thrips on blueberries , coupled with repeated pesticide applications of the few effective and registered pesticides, poses a concern regarding pesticide resistance management . Currently, there are no integrated pest management plans available for control of citrus thrips in blueberry. This is primarily due to the recent nature of this crop-pest association. Avocado thrips, Scirtothrips perseae Nakahara, is a relatively new pest of avocados in California. It appeared in the state in 1996, and, at the time, was a species new to science . By 1998, crop damage reduced industry revenues by 12% . Avocado thrips adults can feed on over 11 plant species’, however, larvae have been found only on avocados in the field in both California and Mexico, suggesting that S. perseae has a highly restricted host range . Although it has little effect on tree health, avocado thrips feed directly on immature fruit , and obvious feeding scars cause severe downgrading and culling of damaged fruit . With a limited number of pesticides available for thrips control and the propensity with which economically important thrips develop insecticide resistance, it is wise to monitor population levels carefully,large plastic pots limit treatments to population levels of economic concern and time treatments optimally . Appropriate cultural practices and conservation of natural enemies should be practiced in concert with the use of pesticides only on an as-needed basis. Thus, continuing the search for effective biological and chemical controls useful in citrus and avocado thrips management is important. For both species of thrips, some pupation occurs on the tree in cracks and in crevices’, however, the majority of both species drop as late second instars from trees to pupate in the upper layer of the leaf litter under trees . Propupae and pupae are rarely seen, move only if disturbed, and do not feed. Thus, pupation in the upper layers of the soil surface may create the ideal interface for control using the entomopathogenic fungi Beauveria bassiana . Coarse organic mulch beneath trees and the maintenance of a mulch layer, a common practice by many growers as a method of Phytophthora spp. management in avocados , may reduce survival of thrips that drop from trees to pupate below the tree. The effectiveness of mulching to control thrips is uncertain and labor costs are required to add mulch may not be justified solely for thrips control. There is increasing pressure in the U.S. to move away from broad-spectrum insecticides and focus on alternative methods of control, e.g., genetically modified crop plants expressing Bacillus thuringiensis toxins , use of entomopathogens, and similar approaches. Applications of B. bassiana have been reported to decrease populations of thrips in greenhouse cucumbers, chrysanthemums, gerbera daisies, roses, and carnations .

Microbial insecticides containing δ-endotoxins from Bt have been used as alternatives to conventional chemical insecticides for almost 70 years . Bt produces insecticidal proteins during the sporulation phase as parasporal crystals. These crystals are primarily comprised of one or more proteins, i.e. Crystal and Cytolitic toxins, also called δ-endotoxins. From a practical perspective, Cry proteins are parasporal inclusion proteins from Bt that exhibit experimentally verifiable toxic effects to a target organism or have significant sequence similarity to a known Cry protein . Similarly, Cyt proteins are parasporal inclusion proteins from Bt that exhibit hemolytic activity or has obvious sequence similarity to a known Cyt protein. These toxins are highly specific to their target insect, are innocuous to humans, vertebrates and plants, are regarded as environmentally friendly, are completely biodegradable, and show little adverse effect on non-target species . The Cyt proteins are significantly different both in their structure and their biological activities from the Cryproteins. However, Cyt proteins have shown toxicity to non-dipterous insects . In fact, Cyt proteins in some cases can extend activity to other Bacillus spp. for mosquitoes that lack the proper receptor . Many studies with thrips involving Bt proteins have typically evaluated Cry toxins in transgenic crops targeted mainly toward lepidopterous pests and there are no published studies we know of representing the impact of Cyt proteins on thrips. Due to the synergism seen between these two Bt proteins and the method of thrips feeding, commonly described as ‘punch and suck’ , whereby leaf tissue is macerated prior to ingestion, we hypothesized that Cry or Cyt proteins could potentially be useful against thrips pests. The goal of this investigation was to determine if Cry or Cyt proteins or B. bassiana could be used effectively to manage citrus and avocado thrips. Field management of both thrips species is the ultimate goal with these biopesticides but field studies are laborious and expensive. Thus, we evaluated these materials in the laboratory to determine which were sufficiently efficacious to warrant follow-up field studies. Leaves of both avocado and citrus for all bio-assays were chosen in observably identical states; young and soft but fully expanded leaves were used as these are the type on which both species of thrips prefer to feed and large leaves were needed to fit in the Munger cell bio-assay units that confined the thrips on treated leaves . Briefly, Munger cells were constructed by using a Plexiglas sandwich; the middle cell layer was drilled with 3.2-cm diameter bit to provide a circular test arena .

Two lot numbers of RMBD representing separately prepared batches of each diet were selected for analysis in order to assess for consistency of any ingredient contamination between the 2 batches. One canine and one feline veterinary prescription extensively hydrolyzed poultry feather-based diet were used as negative controls. Of the 9 species of animal DNA tested, 8 species, including pork, chicken, duck, rabbit, lamb, beef, salmon, and turkey, were detected in at least 1 sample of the canine and feline RMBD tested. Only kangaroo DNA was not detected in any of the RMBD. The 2 extensively hydrolyzed poultry feather-protein based diets contained either trace amounts of chicken DNA or no detectable DNA . In the canine RMBD, DNA of 1 or more animal species not indicated on the label was identified in 9 out of 9 diets, in either 1 or both of the tested batches . Of the 18 batches tested, 89% tested positive for unlisted animal-source DNA. An average of 4.4 unlisted proteins was detected in each diet. A total of 2 batches were found to contain DNA consistent with the stated label ingredients. The diet with the greatest number of unlisted proteins was a single batch , labeled as containing turkey and sardine. It contained a total of 6 unlisted proteins . The unlisted DNA most frequently detected was lamb . Discrepancy in the unlisted DNA between batches was noted in 78% of batches. In the feline RMBD, DNA of 1 or more animal species not indicated on the label was identified in 7 of 9 diets, in either 1 or both of the tested batches . Of the 18 batches tested, 61% were positive for unlisted animal-source DNA. An average of 2.6 unlisted proteins was detected in each diet. A total of 7 batches were found to contain DNA consistent with the stated label ingredients. The diet with the highest number of unlisted proteins was a single batch ,snap clamp labeled as containing chicken and salmon. It contained 5 unlisted proteins . The unlisted DNA most frequently detected was turkey .

Discrepancy between batches was noted in 56% of batches. All of the canine and feline RMBD included in the analysis were found to contain the proteins listed on their labels. sources of oil did not contain cod or sardine DNA as potential allergens. In general, plant- and animal-based oils are not considered allergenic when highly purified . The commercial RMBD tested were obtained in California due to the proximity to the testing facility . Since kangaroo meat is not used by any of the manufacturers in their RMBD, this finding serves as an additional negative control for this study to validate that no DNA of kangaroo origin was detected in the analysis, as would be expected. Analysis of the 2 extensively hydrolyzed poultry feather based diets revealed no detection of unlisted animal DNA. This is consistent with previous reports that contaminants are detected less commonly and in lower numbers in hydrolyzed diets . The extensive hydrolysis of poultry feather proteins into component amino acids or very short oligopeptides is intended to avoid inducing IgE-mediated mast cell activation that can occur with proteins 10 kDa in size or greater . Extensive hydrolysis to reduce poultry allergenicity has been validated in both serum IgE and feeding trials to show the clinical benefits for CAFR . These negative control diets were selected because of the rigorous quality control methods undertaken by the manufacturer to ensure cross-contamination does not occur before market release . While the canine diet did test positive for chicken DNA, the manufacturer does list the feathers of chicken, turkey, and duck as their sourced raw materials . The target gene of the analysis for chicken DNA, transforming growth factor beta 3, is a protein expressed in chicken feathers . Additionally, the manufacturer is aware that cross-contamination needs to be avoided in a therapeutic diet and has developed and clinically validated calibration curves to prevent contamination .

These calibration curves correspond to a known DNA level that was clinically tolerated based on Global Skin Scores in feeding trials in order to set a tolerance level for ancillary proteins known as the NPPI , which is strictly monitored in each diet prior to allowing market release . Based on the manufacturer’s quality control data, 72.3% of these extensively hydrolyzed diets contain DNA below the limit of detection , and 25.7% may have DNA above the LOD but below a safety threshold of 1.2 g/g . However, no diet released to market will exceed the established cut-offs of the NPPI based on the pre-established calibration curves . Therefore, trace copies of chicken DNA may be expected on PCR in some of the diets released to market, as was found in our study. The DNA in the feline extensively hydrolyzed diet in this study was below the LOD, and no animal DNA was detected in the assay. Based on the sensitivity of qPCR, it can be argued that these assays, being sensitive enough to detect as few as 10 copies of the target gene, are of greater sensitivity than that required to detect clinically meaningful contamination that would trigger CAFR. There is no established maximum tolerable level of a contaminating protein that may elicit a pruritic reaction in a food sensitized pet. In humans, soy protein concentration as low as 10 ppm may evoke a reaction in a soy-sensitized individual . Additionally, dose distribution has been demonstrated to vary between different food allergens in sensitized humans, showing that a tolerance range may exist for different food antigens themselves . The additional concern for CAFR pets is that, as opposed to most humans, pets are often fed a specific commercial diet with daily regularity, increasing their risk of chronic re-exposure to a food antigen contained therein. As a result, even small amounts of unknown allergens may lead to a cumulative reaction in a CAFR-affected pet and skew the clinical impression of their response to a particular ED.

These reactions may even be sporadic if there is significant variation of the protein constituents of the diet between batches. The need to validate food allergic threshold distributions in canine and feline CAFR is an important area for future research. Until such time, rigorous quality control using protein analysis methods such as qPCR or enzymelinked immunosorbent assay remains a sensitive method to confirm such contaminants are not detected in therapeutic diets fed for the purpose of the clinical diagnosis of CAFR. This remains the industry standard for quality control of commercially produced diets, including extensively hydrolyzed diets. In conclusion, this study confirms that commercial RMBD should not be considered appropriate for selection as ED in the diagnosis of CAFR as a result of their tendency to include unlisted protein ingredients, which can differ from batch to batch. A clinician should use caution when interpreting the results of an owner-directed ED trial using RMBD to exclude CAFR as a cause of their pet’s pruritic dermatopathy,growing blueberries and veterinarian-guided elimination diet oversight is still recommended. Until further evidence is presented, an elimination diet and provocation trial with a patient-appropriate prescription-based diet subjected to applicable quality control or a home-prepared novel protein diet remain the current diagnostic standard for CAFR.In vineyard production systems, canopy management practices are usually employed to control the source-sink balance and improve the cluster microclimate leading to an improved grape composition and resultant wines . Canopy density is usually controlled during the dormant season thought the winter pruning. Additional canopy management practices may be applied during berry development. Fruit-zone leaf removal and especially, shoot thinning have been widely used in order to increase the cluster exposure to solar radiation, reduce crop load as well as decreasing the pest pressures , increasing flavonoid content and diminishing herbaceous aromas . Nevertheless, when high air temperature and excessive radiation combine, detrimental effects on berry acidity and flavonoid content have been reported in warm climate regions . Leaf removal consists of removing basal leaves around the clusters in the east or north side during grape development increasing the cluster exposure to solar radiation. It is well known that an early leaf removal increased total soluble solids, anthocyanins, and flavonols . However, some authors reported increases in titratable acidity in Sangiovese and Teran cultivars while other authors found decreases in acidity with basal leaf removal on Tempranillo . Conversely, Sivilotti et al. reported a positive effect of leaf removal applied after flowering on Merlot grapevine by improving cluster integrity by reducing incidence of Botrytis, and lower herbaceous aromas without affecting yield and cluster mass. Contrariwise, Pastore et al. reported that defoliation at veraison reduced the anthocyanin content and increased the impact of sunburn. In fact, these authors found that leaf removal induced a general delay in the transcriptional ripening program, which was particularly apparent for structural and regulatory genes involved in the anthocyanin biosynthesis.

Clearly, vineyard location, cultivar , timing of leaf removal , method , and degree of leaf removal , the growing season , among others, are all factors influencing how leaf removal affects grapevine berry composition and integrity. On the other hand, shoot thinning has been related to increased cluster and berry mass and the number of berries per cluster, with a reduction on yield . Conversely, Wang et al. observed that shoot thinning had relatively minor impacts on yield components because of a compensatory effect due to the lower cluster number with concomitant increase in cluster mass. Contrarily, shoot thinning practices on grapevine did not show a great impact on berry primary metabolism , however, secondary metabolites were affected by them . In fact, we recently reported an increase of two-fold in the flavonol content of Merlot berries when leaf or shoot removal was applied mainly by increasing the proportion of quercetin and kaempferol derivatives in detriment of the myricetin derivatives . Berry composition is dependent on a complex balance between compounds derived from primary and secondary metabolism. Between secondary metabolites, flavonoids play an important role in the quality and the antioxidant properties of grapes and are very responsive to environmental factors such as solar exposure . Anthocyanin compounds are responsive of the berry color, and flavonols act as a UV shields, contribute to the wine antioxidant capacity, color stability, and hue through copigmentation with anthocyanins . On the other hand, the methoxypyrazines are wine key odorants contributing to their herbaceous characteristics and have been related to unripe berries and poor-quality wines when these are not part of the wine typicity . Since they can be present in grape berry and wines at high levels, they may have an important sensorial impact on wine quality . Among methoxypyrazines, the 3-isobutyl-2- methoxypyrazine is considered the most relevant to wine flavor due to its correlation with the intensity of the bell pepper character of wines and its content at harvest seems to be dependent of the solar exposure . The differences found in the literature about the effect of manipulating the canopy architecture on the flavonoid and aromatic content due to different solar exposure of berries in warm climates opens an important field of research. Therefore, we aimed to find the optimal ranges of berry solar exposure estimated as percent of kaempferol for flavonoid synthesis up regulation and the thresholds for their degradation, and to evaluate how canopy management practices such as leaf removal, shoot thinning and a combination of both affect the grapevine yield components, berry composition, flavonoid profile, and herbaceous aromas.An experiment was performed in 2017 on 7-year Cabernet Sauvignon vines grafted onto 110 Richter root stock with NW-SE row orientation and a vine spacing of 2 m × 2.4 m in a commercial vineyard in Oakville, CA . Individual berries were sampled at harvest according to their position in the canopy and overexposure based on visual appearance. Each independent replicate was a sample of 75 berries collected from up to 50 plants each , these plants being potentially the same for all exposures. From each sample, 55 berries were used for must analyses and berry mass, and the remaining 20 berries were storedat −20°C for analyses of flavonoids. Thus, four observational treatments with four replicates consisted in two rows of 25 vines each were established: non-exposed berries collected from interior clusters ; exposed but free of signs of overexposure, collected from northeast exposed clusters ; exposed and with mild signs of sunburn, collected from southwest exposed clusters ; and exposed and with severe signs of sunburn with signs of damage collected from southwest exposed clusters .

The top three diseases that compose this data set are Bacterial Spot, Haunglongbing, and Yellow Leaf Curl. Bacterial Spot, which composes 10% of the data, is a bacterial disease that affects many crops by causing their leaves to develop yellow spots that turn brown in the middle. It also causes crops to develop black or brown spots of rot on their fruits. Haunglongbing composes 10% of the data set. This bacterial disease affects citrus trees causing their fruits to stay green and fall to the ground early before becoming ripe. This disease is common for citrus, but keep in mind that this data set only has images of this disease affecting Oranges. Yellow Leaf Curl composes 9.9% of the data set and is a Viral infection that only affects Tomatoes. “Yellow leaf curl virus is undoubtedly one of the most damaging pathogens of tomatoes, and it limits the production of tomatoes in many tropical and subtropical areas of the world. It is also a problem in many countries that have a Mediterranean climate, such as California. Thus, the spread of the virus throughout California must be considered a serious potential threat to the tomato industry” . Note that the total percentage of diseased images contributing to this data set is 72.2% because the other 27.8% of this data set is healthy crop images. See Table 2.4 for a comparison of the amount of diseased and healthy crop images in this data set. The crops that contributed to the diseased images are Apple, Bell Pepper, Cherry, Grape, Maize, Orange, Peach, Potato, Strawberry, Squash, and Tomato. The crops that contributed to the healthy images are Apple, Bell Pepper, Blueberry, Cherry, Grape, Maize, Peach, Potato, Raspberry, Strawberry, and Tomato. For a visual representation of what the diseased and healthy crop images look like, see Figure 2.1 for nine different crop images.

The crop images have their classification labels above them to identify the crop name and disease or healthy. The images in Figure 2.1 are crop images of classification labels Apple-Apple Scab, Apple-Healthy, Peach-Healthy, GrapeHealthy, Raspberry-Healthy, Soybean-Healthy, Grape-Black Rot, and Peach-Bacterial Spot. While the Transformer architecture has become the de-facto standard for natural language processing tasks,square plastic pot its applications to computer vision remain limited. This was the motivation for Dosovitskiy et al. to look into the implementations of the transformer model for image classification tasks, and the vision transformer was created. The transformer architecture for natural language processing tasks works similarly to a vision transformer. In natural language processing, sentences are broken down into words. Then each word is treated as a sub-token of the original sentence. Similarly, the vision transformer breaks down an image into smaller patches, each patch representing a small sub-section of the original image. To visually see how sentences are broken down into word tokens and images broken down into patches, see Figure 2.2 from. Keep in mind that the position of the image patch is very important. If the image patches are out of order, then the original image will also be out of order. The vision transformer is designed to start with an extra learnable class embedding that is equivilent to 0. Which represents the start of the image and sequence of patches to come. The extra learnable class embedding allows the model to learn embeddings specific to each classification label. “The pre-training function of vision transformer is based solely on the classification label given; therefore, the learnable class embedding is even more important to successfully pre-training the vision transformed model”. Without the learnable class embedding, the transformer will not understand the classification labels that are attached to each image.

To keep the order of the sequence of patches that make up the image, the patches are instilled with positional embeddings. “For the vision transformer, these positional embeddings are learned vectors with the same dimensionality as our patch embeddings. After creating the patch embeddings and pre-pending the classification label embedding, it is then summed with the positional embeddings”. Finally, the summed embeddings are shown to the transformer encoder. After the entirety of the image is shown to the transformer encoder, the model has then learned that image under the given classification label. For a more understandable visual example, see Figure 2.4 from . It visually demonstrates the architecture of how the image patches are linearly projected and linearly embedded, how the patches receive a learnable class embedding, and then finally showing the image to the transformer encoder and the model learning the given image for the given classification label. Dosovitskiy and et al. also found that “ViT attains excellent results when pre-trained at sufficient scale and transferred to tasks with fewer data points. When pre-trained on the public ImageNet-21k data set or the in-house JFT-300M data set, ViT approaches or beats state of the art on multiple image recognition benchmarks”. A vision transformer model that has been pre-trained on large data sets such as ImageNet-21k are able to help in making a model more effective [DBK21].This project will implement the vision transformer developed by Dosovitskiy et al. and mentioned in their paper. The framework of their ViT model will be used and accessed through the Hugging Face platform and their package transforms using Python. The vision transformer model comes pre-train on the ImageNet-21k, a benchmark data set consisting of 14 million images and 21k classes. The vision transformer model has been pre-trained on images with pixel size 224 × 224. Therefore, any data that is to be further trained on this model must also be of pixel size 224 × 224.“Data augmentation is the process of transforming images to create new ones for training machine learning models” . “Data augmentation increases the number of examples in the training set while also introducing more variety in what the model sees and learns from. Both these aspects make it more difficult for the model to memorize mappings while also encouraging the model to learn general patterns.

Data augmentation can be a good substitute when resources are constrained” because it artificially creates more of your data when it is not possible to get more data.In the case of this project, the function being used to perform the data augmentations isset transfrom from the Hugging Face Datasets package in Python. This function performs data transformations only when the model training begins. Therefore, transformations can be done on the fly and save on computational resources. Then at each epoch, the transformations are applied to every image given to the model, so the amount of training data stays constant, but variation is added to the original data through transformations. This does not increase the number of training images as other data augmentation packages would, this artificially augments with transformations and variation .Data augmentation is an important step when training machine learning models because they can perform very powerfully if the given data sets for training are too small to train with. These models can start to over-fit, which is a problem because then the model will memorize mappings between the inputs and expected outputs. There are 54,306images in this data set, which may seem like a lot of images,square pot but for a machine learning model, it is not that much. That is why data augmentation is being implemented as a step to reduce possible model over fitting.See Table 3.1, for training results of the pre-trained ViT Image classification model trained on the Plant Village data set. The table displays the model’s Training Loss, Validation Loss, Precision, Recall, model F1 score, and model Accuracy over the 10 epochs. Model training loss indicates how well the model is fitting the training data. Model validation loss indicates how well the model fits new data. The best model that was chosen was epoch 10, which is bold in Table 3.1. The model from epoch 10 has a training loss of 0.088 with a validation loss of 0.073, the lowest pair out of all the epochs. For this model, Precision, Recall, the model F1 Score, and Accuracy all have the value of 1.00. These results indicate that the model has a high positive identification rate. The model seems to have reached a convergence in values between epochs 8 to 10. The training results for epoch 10 showed that the Evaluation of Samples per Second for the model is 50.29. This indicates that number of samples the machine learning model can process and make predictions of in one second is 50.29. Also, the Evaluation Steps per Second is 1.58, which indicates the number of iterations that the machine learning model can complete in one second. Model testing was done with the 15% of the data that was reserved for testing and never shown to the model during training. See Table 3.2, in order to see the overall testing results of the model in a table. The model has an Accuracy of 99%. Meaning the model has the ability to guess the classification label 99% of the time correctly. Total Time in Seconds is the amount of time it took for the image classification model to be tested with the test data set, which is 1460.88 seconds also 24.34 minutes. Samples per Second is 1.15, which is the amount of data samples or instances that the image classification model can process and make predictions on in one second. Latency in Seconds is 0.86, which refers to the amount of time it takes for a single prediction to be made by the image classification model in oneIn order to see how the image classification model can classify an image, see Figure 3.2 and Table 3.3.

Figure 3.2 shows an example image of which the image classification model was tested on. The image has its true classification label above it, which is Apple-Apple Scab. Table 3.3 has the scores and labels of five different classification label predictions for what it thinks the image in Figure 3.2 could be. The score is on a scale from 0 to 1, with 1 meaning 100% confidence in the classification label that the model is predicting. The value of the score is split across the five predictions, meaning when we add up all of the score values for all five predictions, the value will add up to 1. The model’s top prediction with a score of 0.91% is the classification label Apple-Apple Scab, which is the true classification label of the image in Figure 3.2.The overall performance of the pre-trained ViT image classification model with data augmentation shows good promise. The best epoch provided that F1, Accuracy, Precision, and Recall, were all equal to 1.0. This is not the best situation but shows there is room for improvement. Given the previous epochs from 1 to 8, has values for their F1 Score and Accuracy. It shows that there is possible room to improve the model with more fine-tuning and possibly an early stop in training to not have the model over-train to get the result of 1.0 for F1 Score and Accuracy. Another possible reason for the result could be the large imbalance of images between classification labels and that the Plant Village data set is considered to be a small data set. These types of results are good but not realistic for model prediction power. Even though these values are incredibly high, the training and validation loss is still fairly low and slowly converging to 0. This means that the model is learning over time, which is a good sign and shows room for improvement. A possible future improvement for this project would be to find another data set with more specific plant disease information to train the model on. Efforts will be made to look for data that includes more plant pests. That variation would be beneficial because the data set for this project mainly contains crop disease images. The disease Spider Mite, is labeled as a disease in this data set but in reality, is not a disease. It is the only classification label that has pest-inflected crop images in this data set. Having more data on crop pests would be beneficial because crop pests also cause a significant amount of crop loss and damage as well.

The enzyme UFGT catalyzes the final step of anthocyanin biosynthesis, therefore UFGT has been considered by many authors to be a critical enzyme in anthocyanin biosynthesis . Temporary stimulation of gene transcription is believed to be related to a decrease in S-ABA concentration over time. In ‘Crimson Seedless’ grapes, a constant decrease in S-ABA levels with a half-life time of 14.7 days was observed in treated grape berries . The natural decrease in ABA concentration, along with the decrease in S-ABA levels, may, therefore, lead to decreased activity of some genes, depending on the S-ABA concentration in the plant. Expression of the UFGT gene increased considerably 7 days after S-ABA application in ‘Crimson Seedless’ grapes but decreased 3 weeks after treatment, becoming similar to the control . In “Cabernet Sauvignon” grapes treated with ±cis, trans-ABA, expression analysis of anthocyanin biosynthetic genes revealed that the maximum expression levels were only reached 10–17 days after application and that they then rapidly decreased . ABA cis– and trans-isomers differ in the orientation of the carboxyl group at carbon 2. Only the ABA cis-isomer is biologically active, and it accounts for almost all of the ABA produced in plant tissues. However, unlike the S and R enantiomers, the cis– and trans-isomers can be interconverted in plant tissue . Most of the studies on S-ABA involved V. vinifera cultivars were done in temperate zones and testing a single application . In this study, we evaluated the response of a new V. vinifera × V. labrusca hybrid grape cultivar grown in a subtropical area to multiple S-ABA applications. This hybrid often shows lack of color development; therefore,25 liter round pot our results confirm the effectiveness of S-ABA to improve the color of ripening berries, even under warm climate conditions.

The application of S-ABA to berries of the seedless grape Selection 21 increased the total anthocyanin concentration, changed the proportion of individual anthocyanins, improved their color attributes, and increased the expression of transcription factors and anthocyanin biosynthetic genes. Two applications of 400 mg/L S-ABA, at 7 and 21 DAV, resulted in the best results in terms of color increment and total anthocyanin concentration, favored the accumulation of trihydroxylated anthocyanins, and increased the expression of transcription factors and of the genes F3H and UFGT. These results not only show that S-ABA is a valuable tool for improving the color of red grapes in warm areas, where color deficiency is frequently observed, but also suggest that S-ABA may be useful in grape breeding programs by permitting the selection and release of new cultivars with natural poor color, but other desirable characteristics such as high yield and resistance to common diseases. The iCT in Hemodialysis Centers initiative is governed by a Steering Committee consisting of a health professional , a new Faculty member , a health care administrator , 2 patients , and a health researcher . The Steering Committee takes overall responsibility for all aspects of the initiative, including the workshop. Aligned with the vision of CIHR’s SPOR, the grant activities are undertaken in full partnership with persons on hemodialysis , as well as health care providers and health administrators.Guided by their priorities in every stage of the research process, needed and meaningful clinical trials in hemodialysis can be implemented for successful knowledge translation and uptake in care settings.As many clinical decisions in hemodialysis care are made at the program/unit level , rather than from individual clinician-patient discussions, the trials in this initiative use cluster-randomization of entire hemodialysis units to assess the benefits and risks of intervention. With these considerations, pragmatic trials can test the effectiveness of promising interventions in real-world hemodialysis settings and broad patient groups.

Furthermore, critically needed trials can be conducted with the same quality as traditional trials with individual randomization, but with less time and at a fraction of the cost. The knowledge gained from these new trials is ultimately expected to improve the health and care of those undergoing hemodialysis. A key objective of the iCT in Hemodialysis Centers initiative is to support the development of at least 2 new promising interventions so that by the end of the 4-year grant period , they are ready for large-scale trial implementation in hemodialysis centers. With the involvement of patients, health care providers, and health care administrators, trial planning will be advanced in a manner that builds consensus on research priorities and considers the challenges to implementing different trial concepts. To fulfill this objective, a stakeholder engagement and research development workshop was held in Toronto, Ontario on June 2 and 3, 2018. The workshop emcee was Dr Amit Garg, nephrologist, Program Lead of the ICES Kidney, Dialysis and Transplantation Ontario provincial program , and the nominated principal investigator of the funded iCT CIHR SPOR grant. Through structured presentations, facilitated group discussions, and expert panel feedback , workshop attendees shared knowledge and opportunities to develop and collaborate on innovative, pragmatic, cluster-randomized registry trials embedded in hemodialysis care.In the last decade there has been a rise in adoption of sustainable soil management practices that reduce soil erosion and bolster soil organic matter to counter the impacts of climate change on agricultural soils . Traditionally, vineyard rows were kept bare, with the use of herbicides and tillage. However, there is disagreement on the utility of this practice due to the detrimental effects of tillage on air quality and soil physical, chemical, and biological properties . Thus, the adoption of cover crops and reduced tillage is considered a sustainable alternative to traditional management of vineyard floors . Furthermore, environmental regulations and public perception serve as additional incentive to adopt climate-smart practices . The benefits of cover crops on the properties of soils are well documented.

They can increase soil organic matter , improve water infiltration and aggregate stability, reduce soil erosion and greenhouse gas emissions , and increase vineyard biodiversity . Nevertheless, the adoption of cover crops in vineyards is limited by the concern of excessive competition between the cover crop and grapevine for water and nutrients . Many studies have sought to quantify the effects of cover cropping on the grapevine, yet the influence of cover crop adoption on grapevine physiology and production remains elusive. There is agreement in literature that cover crops may reduce vegetative growth, with more pronounced yield losses in warmer regions. However some studies have found no effect . Grapevine physiological responses to cover cropping were also documented, and minimal effects on leaf gas exchange have been found. The presence of a cover crop is generally reported to detrimentally affect grapevine water status . Despite wide acceptance of this particular effect, results are inconsistent as some studies have shown that cover crops may improve early season water status, yet others have concluded that cover cropped vineyards do not display better water status compared to those with bare soil . Ultimately, previous works agree that changes in grapevine physiological response to cover crop adoption are largely driven by the climatic conditions and irrigation regime at a given site . Likewise, it was assumed that the competition between the grapevine and cover crop for water and nutrients resulted in yield decreases, but this effect was also not consistent . Yield reductions and/or no effect on yield are the most common results from such work. However, cover crop species appears to be a more influential factor than merely the presence of a cover crop. Yield increases have even been reported in some vineyards planted with annual species such as oats or legumes . Consequently, any changes to berry composition as a result of cover crop adoption is closely associated with changes in yield, such as smaller berry size and purportedly greater content berry flavonoids . The adoption of reduced or no-till management preserves SOM, reduces soil erosion, improves soil structure, and is considered integral to reducing GHG emissions from the agriculture sector . The influence of tillage on soil properties, while not entirely understood, is more studied than the impact on crops themselves, particularly in permanent cropping systems such as vineyards. However,25 liter pot few reports have investigated the influence of tillage on grapevine physiology under the presence of a cover crop. While differences in leaf gas exchange have been reported between grapevines grown under conventional tillage compared to those under permanent cover crops, there is little evidence of tillage having a direct influence on grapevine stomatal conductance and net photosynthesis . Although no-till practices are often promoted for their positive influence on soil infiltration and conservation of soil water, few studies have found that this effect translated to ameliorated plant water status in grapevine . Previous works indicated that vegetative growth is greater under conventional tillage while yield reductions are typically associated with no-till management, despite research that indicated no effect on production . Furthermore, while some studies have reported increased berry skin anthocyanin content under no-till management, overall there is limited impact of tillage on berry composition . Cover cropping and reduced tillage management are two practices that directly alter the growing environment of the grapevines. Thus, the selection of appropriate vineyard floor management practices is critical in order to maximize benefits to the soil while minimizing the impact on grapevine function and productivity.

This selection involves decisions in space , type , and time including perennial vs. annual species selection and timing of termination . Factors such as cultivar, vineyard age, macroclimate, soil physiochemical characteristics, and the overall goals for the use of the selected cover crop and tillage system must also be considered. These elements have been shown to contribute to the effect of the practices on grapevine functioning and production . The objective of this work was to investigate the effects of cover cropping and tillage on whole grapevine physiology in two contrasting production regions in California, USA. Specifically, we studied the interactive effects of tillage and cover crops on grapevine water status, leaf gas exchange, components of yield, berry composition and resulting water footprint in two contrasting production regions of California during arid seasons. At both experimental sites, the experiments were arranged as a split-plot 3 × 2 factorial arrangement of treatments with four and three replications . Each treatment-replicate consisted of 15 grapevines. Three grapevines in the middle of each replicate were used for measurements and the distal plants on either end served as buffer plants. Treatments included tillage as the main plot [conventional tillage and no-till ] and the sub-plot was randomly applied within the main plots as i) Perennial grass ; ii) Annual grass ; iii) Resident vegetation . The cover crop seed was drilled in a 1.5 m wide strip according to seed manufacturer’s recommended rate prior to receiving fall/winter rains in 2019 and 2020 at a rate of 605 kg/ha and 84 kg/ha for the perennial grass and annual grass treatments, respectively. Resident vegetation was allowed to grow within the 1.5 m strip and mowed at the vineyard manager’s discretion. All other cultural practices were conducted according to University of California Cooperative Extension guidelines .At harvest, fifty berries were randomly collected from the three middle grapevines within each replicate and immediately processed. Berries were weighed and gently pressed by hand to squeeze the juice. Total soluble solids were determined using a temperature compensating digital refractometer . Must pH and titratable acidity were determined with an autotritrator . TA was determined by titrating with 0.1 N sodium hydroxide to an end point of 8.3 pH and reported as g/L of tartaric acid. Berry skin anthocyanin content was determined at harvest from 20 berries randomly collected from each treatment-replicate. Berries were gently peeled, skins were freeze-dried . Freeze-dried tissue was ground with a tissue lyser . Fifty milligrams of the resultant powder was extracted in methanol: water: 7 M hydrochloric acid to determine anthocyanin content. Extracts were filtered using a 0.45 µm filter and analyzed using an Agilent 1260 series reversed-phase high performance liquid chromatography system coupled to a diode array detector. Separation was performed on a reversed-phase C18 column LiChrospher 100, 250 mm × 4 mm with a 5 µm particle size and a 4 mm guard column of the same material at 25 ℃ with elution at 0.5 mL per minute. The mobile phase consisted of a constant 5% of acetic acid and the following gradient of acetonitrile in water: 0 min 8%, at 25 min 12.2%, at 35 min 16.9%, at 70 min 35.7%, 65% between 70–75 min, and 8% between 80–90 min.

The domestication of cultivated strawberry has followed a path different from that of other horticulturally important species, many of which were domesticated over millennia and traced to early civilizations, e.g., apple , olive , and wine grape . Although the octoploid progenitors were cultivated before the emergence of F.  ananassa, the full extent of their cultivation is unclear and neither appears to have been intensely domesticated; e.g., Hardigan et al. did not observe changes in the genetic structure between land races and wild ecotypes of F. chiloensis, a species cultivated in Chile for at least 1,000 years . With less than 300 years of breeding, pedigrees for thousands of F. ananassa individuals have been recorded, albeit in disparate sources. To delve more deeply into the domestication history of cultivated strawberry, we assembled pedigree records from hundreds of sources and reconstructed the genealogy as deeply as possible. One of our initial motives for reconstructing the genealogy of cultivated strawberry was to identify historically important and genetically prominent ancestors of domesticated populations, in large part to guide the selection of individuals for whole-genome shotgun sequencing and DNA variant discovery, inform the development of single-nucleotide polymorphism genotyping platforms populated with octoploid genome-anchored subgenome-specific assays, and identify individuals for inclusion in genome-wide studies of biodiversity and population structure . The genetic relationships and genetic contributions of ancestors uncovered in the genealogy study described here guided the selection of individuals for downstream genomic studies that shed light on genetic variation and the genetic structure of domesticated populations worldwide .

Our other early motive for reconstructing the genealogy of strawberry was to support the curation and stewardship of a historically and commercially important germplasm collection preserved at the University of California, Davis ,black plastic plant pots with accessions tracing to the early origins of the strawberry breeding program at the University of California, Berkeley , in the 1920s . We sought to develop a complete picture of genetic relationships among living and extinct individuals in the California and worldwide populations, in part to assess how extinct individuals relate to living individuals preserved in public germplasm collections. Because 80% or more of the individuals we documented in the genealogy appear to be extinct, they could only be connected to living individuals through their pedigrees. One of the ways we explored ancestral interconnections between extinct and living individuals was through multivariate analyses of a combined pedigree–genomic relationship matrix estimated from genotyped and ungenotyped individuals . The holdings and history of the UCD Strawberry Germplasm Collection were shrouded in mystery when our study was initiated in 2015. The immediate challenge we faced in reconstructing the genealogy was the absence of pedigree records for 96% of the 1,287 accessions preserved in the collection, which is hereafter identified as the “California” population. To solve this problem, authenticate pedigrees, and fully reconstruct the genealogy of the California population, we applied an exclusion analysis in combination with high-density SNP genotyping . Here, we demonstrate the exceptional accuracy of diploid paternity analysis methods when applied to individuals in an allooctoploid organism genotyped with subgenome-specific SNPs on high-density arrays .

Several thousand SNP markers common to the three arrays were integrated to develop a SNP profile database for the parentage analyses described here. SNPs on the 50-K and 850-K arrays are uniformly distributed across the octoploid genome and informative in octoploid populations worldwide . The 50-K SNP array harbors 1 SNP/16,200 bp, whereas the 850-K array harbors 1 SNP/953 bp, telomere-to-telomere across the 0.81-Gb octoploid genome. The genealogies of domesticated plants, especially those with long-lived individuals, overlapping generations, and extensive migration and admixture, can be challenging to visualize and comprehend . We used Helium to visualize smaller targeted pedigrees; however, the strawberry pedigree networks we constructed and investigated were too large and mathematically complex to be effectively visualized and analyzed with Helium and other traditional hierarchical pedigree visualization approaches. Hierarchical methods often produce comprehensible insights and graphs when applied to pedigrees of individuals or small groups but yield exceedingly complex,labyrinthine graphs that are difficult to interpret when the genealogy contains a large number of individuals and lineages. We turned to social network analysis to explore alternative approaches to search for patterns and extract information from the complex genealogy of strawberry. The pedigree networks of plants and animals share many of the features of social networks with nodes connected to one another through edges relationships. We used SNA methods, in combination with classic population genetic methods, to analyze the genealogy and develop deeper insights into the domestication history of strawberry . SNA approaches have been applied in diverse fields of study but have apparently not been applied to the problem of analyzing and characterizing pedigree networks . With SNA, narrative data are translated into relational data and summary statistics and visualized as sociograms . Here, we report insights gained from genealogical studies of domesticated strawberry populations worldwide.

Our studies shed light on the complex wild ancestry of F. ananassa, the diversity of founders of domesticated populations of cultivated strawberry that have emerged over the past 300 years, and genetic relationships among extinct and extant ancestors in demographically unique domesticated populations tracing to the earliest ancestors and interspecific hybrids .To develop a SNP profile database for DNA forensic and population genetic analyses , we recalled and reanalyzed SNP marker genotypes for 1,495 individuals, including 1,235 UCD and 260 USDA accessions previously genotyped by Hardigan et al. with the iStraw35 SNP array . SNP marker genotypes were automatically called with the Affymetrix Axiom Analysis Suite . DNA samples with > 6% missing data were dropped from our analyses. This yielded 14,650 high-confidence codominant SNP markers for paternity–maternity analyses. While SNP markers are codominant by definition, a certain percentage of the SNP markers assayed in a population produce genotypic clusters lacking one of the homozygous genotypic clusters. These so-called no minor homozygote SNP markers were excluded from our analyses.For a second DNA forensic analysis, 1,561 UCD individuals were genotyped with 50-K or 850-K SNP arrays . This study population included 560 hybrid offspring from crosses among 27 elite UCD parents, the F.  ananassa cultivar “Puget Reliance,” and the F. chiloensis subsp. lucida ecotypes “Del Norte” and “Oso Flaco.” Hardigan et al. included 16,554 SNP markers from the iStraw35 and iStraw90 SNP arrays on the 850-K SNP array. To build a SNP profile database for the second paternity–maternity analysis, we identified 2,615 SNP markers that were common to the three arrays and produced well-separated codominant genotypic clusters with high confidence scores . We subdivided the global population into “California” and “Cosmopolitan” populations, in addition to continent-, region-, or country-specific populations, for different statistical analyses. These subdivisions are documented in the pedigree database . The California population included 100% of the UCD individuals from the global population, in addition to 262 non-California individuals that were ascendants of UCD individuals. The Cosmopolitan population included 100% of the non-California individuals ,black plastic planting pots in addition to 160 California individuals that were ascendants of non-California individuals. We subdivided individuals in the US population into Midwestern, Northeastern, Southern, and Western US populations. The Western US population included only those UCD individuals that were ascendants in the pedigrees of Western US individuals. The country-specific subdivisions were Australia, China, Japan, South Korea, Belgium, Czechoslovakia, Denmark, England, Finland, France, Germany, Israel, Italy, the Netherlands, Norway, Poland, Russia, Scotland, Spain, Sweden, and Canada.DTR and TTR statistics were estimated from equations and using custom R code that we developed and provided as supplemental material . DTR estimates for PO duos and TTR estimates for PPO trios were compared to empirically estimate statistical thresholds to exclude parents. With a perfect dataset , TTR ¼ 0 when both parents in a trio are correctly identified. When estimated from a real-world dataset , TTR ¼ 0 even when both parents in the trio are correctly identified. However, TTR estimates for correctly identified parents are typically exceedingly small and approach zero when genotyping errors are small and DNA profiles are informative . The probability of a type I error depends on the genetic relatedness of individuals in the DNA profile database and the number, informativeness, and genotyping error rates of the DNA markers . A false-positive error occurs when an individual that is not a parent is declared to be a parent , whereas a false negative error occurs when an individual that is a parent is excluded . DTR and TTR thresholds for excluding parents were empirically estimated by bootstrapping .

We drew 50,000 bootstrap samples from a population of 1,002 individuals with known pedigrees by replacing one or both parents in the known PPO trio with a randomly selected individual from the population. We built empirical DTR and TTR distributions from the 50,000 estimates and ascertained the statistical thresholds needed to accurately identify parents, exclude non-parents, and minimize false-positive errors. The bootstrap estimated DTR threshold of DTR  0:0016 yielded a false-positive probability of zero and a false-negative probability of 5%, whereas the bootstrap-estimated TTR threshold of TTR  0:01 yielded false-positive and false-negative probabilities of zero. Thesethresholds were estimated by summing transgression scores summed over 14,650 SNP marker loci. To increase the computational speed and efficiency, DTR statistics were estimated for every PO combination, whereas TTR statistics were only estimated for PPO combinations where the DTR estimates for both parents were less than the empirical threshold . This was done because the number of PPO combinations was prohibitively large and most PPO combinations could be unequivocally excluded using DTR estimates.We reconstructed the genealogy of cultivated strawberry as deeply as possible from wild founders to modern cultivars . To build the database, pedigree records for 8,851 individuals were assembled from more than 800 documents, including scientific and popular press articles, laboratory notebooks, garden catalogs, cultivar releases, plant patent databases, and germplasm repository databases . The database holds pedigree records and passport data for 2,656 F. ananassa cultivars, of which approximately 310 were private sector cultivars with pedigree records in public patent databases . The parents of the private sector cultivars, however, were nearly always identified by cryptic alphanumeric codes, and thus could not be integrated into the “giant component” of the sociogram . Our computer forensic search did not recover pedigree records for 220 individuals in the California population; however,we suspected that their parents might be present in the SNP profile database. Using duo and trio exclusion analyses, we identified both parents for 214 of these individuals and one parent each for the other six individuals. Hence, using a combination of computer and DNA forensic approaches, 2,222 out of 2,470 possible parents of 1,235 individuals in the California population were identified and documented in the pedigree database . The parents declared in pedigree records , identified by DNA forensic methods , or both are documented in the pedigree database . Despite their historic and economic importance, the pedigrees of individuals preserved in the UCD Strawberry Germplasm Collection had not been previously documented. Besides reconstructing the genealogy of the California population, previously undocumented pedigrees of extinct and extant individuals were discovered in the laboratory notebooks of Harold E. Thomas, Royce S. Bringhurst, and others , and integrated into the pedigree database . To further validate the accuracy of DNA forensic approaches for parent identification in strawberry, we applied an exclusion analysis to a population of 560 hybrid individuals developed from crosses among 30 UCD individuals . The pedigrees of the parents and hybrids were known. The parents and hybrids and 1,561 additional UCD individuals were genotyped with 50-K or 850-K SNP arrays . The 50-K array was developed with SNP markers from the 850-K array , which included a subset of 16,554 legacy SNP markers from the iStraw35 and iStraw90 arrays . We developed an integrated SNP profile database using 2,615 SNP markers common to the three arrays. Using PPO trios, we discovered that the SNP profile for one of the parents was a mismatch, whereas the SNP profiles of the other 29 parents perfectly matched their pedigree records. We discovered that the parent stated for 11C151P008 was correct, but that the DNA sample and associated SNP marker profile were incorrect. Hence, the DNA sample mismatch was traced by trio exclusion analysis to a single easily corrected laboratory error. This analysis empirically demonstrated the utility of exclusion analysis for authenticating pedigrees and curating germplasm collections, and showed that parents can be accurately identified with substantially smaller numbers of DNA markers than those applied in our initial study.

The effect of dietary fiber has long been proposed to contribute to human health through prebiotic enhancement of certain beneficial microbes that produce butyrate,absorb bile acids,decrease colon pH,112 and promote GI motility via shortening of the mean transit time.However, not all dietary fibers have the same effect, which is dependent on their physicochemical characteristics.The prebiotic effect of indigestible polysaccharides on gut microbiota has previously been broadly discussed. Several human dietary intervention studies have shown that intake of certain types of dietary fibers can significantly modify the gut microbiota observed in feces. For example, consumption of whole-grain breakfast cereal for 3 weeks significantly increased fecal bifidobacteria and lactobacilli compared to wheat bran alone; however, no difference was observed in fecal short-chain fatty acids .Introduction of barley β-glycan in the diet elevated fecal Bifidobacterium and Bacteroides counts in older healthy human subjects , whereas only the Clostridium perfringens count increased in the younger group.Typically, consumption of nondigestible carbohydrates such as wheat bran arabinoxylan oligosaccharides, short-chain fructooligosaccharides , and soybean oligosaccharides and galactooligosaccharides induces enrichment of human fecal bifidobacteria.Inulin has been shown to stimulate the growth of Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium bifidum and the butyrateproducing bacteria Faecalibacterium prausnitzii and Roseburia inulinivorans. Similarly, Bifidobacterium spp. levels significantly increased upon consumption of biscuits containing partially hydrolyzed guar gum and FOS for 21 days, whereas Bacteroides spp., Clostridium spp., and Lactobacillus−Enterococcus spp. remained at similar levels.

GOS alone or combined with FOS are often supplemented in infant formula to favor the growth of bifidobacteria spp.,drainage planter pot and the bifidogenic response of GOS has been shown to be dosedependent.Interestingly, Rossi et al. reported that only 8 of the total 55 Bifidobacterium strains were able to grow on longchain inulin in vivo,suggesting that not all bifidobacteria species benefit in the same way from the presence of these substrates as their energy source. Indeed, the specificity of polysaccharide use by the gut microbiota supports the underlying cross-feeding interaction between gut microbiota . Various types of resistant starch demonstrate substrate specialization of the gut microbiome. For example, the impact of type 2 resistant starch is associated with enrichment of Ruminococcus bromii, whereas type 3 resistant starch elevates both E. rectale and R. bromii in healthy130 and overweight subjects.Type 4 resistant starch significantly differs from types 2 and 3 and has been shown to induce a profound phylum-level change and elevate B. adolescentis and Parabacteroides distasonis. Furthermore, the variability observed in these studies suggests that the host-specific environment affects the composition of the gut microbiome. The fermentation profile depends on the glycosidic linkage type of the dietary substrate as well as the functional capability of the gut microbiota . Metatranscriptome analysis revealed a functional enrichment of genes associated with carbohydrate uptake and metabolism in the small intestine and feces.A fecal metaproteomic analysis from three healthy subjects over a period of 6−12 months revealed a common functional core enriched in carbohydrate transport and degradation.In particular, the ability to degrade complex polysaccharides has been identified in a range of bacteria.In particular, the Bacteroides phylum contains a large repertoire of genes that exhibit broad capacities to degrade a great diversity of plant-derived polysaccharides.Microbial sequencing projects revealed that starch utilization systems are highly abundant and conserved in the phylum Bacteroidetes .

In contrast, members from the Firmicutes phylum exhibit a wide range of functionalities; for example, Ruminococcus, which is in the order of Clostridiales, can degrade cellulose and pectin,whereas E. rectale and Eubacterium eligens have fewer polysaccharide degrading enzymes and are enriched with more ATP binding cassette transporters and phosphotransferase systems than the Bacteroidetes.Although many microbes have the ability to ferment undigestible dietary components, diet-induced microbial changes seem to favor the groups that have a stronger survival advantage and perhaps specifically depend on host-derived factors such as pH and bile acid profiles.The human colon has not often been considered to be a site of fat absorption; however, the small intestine absorbs only approximately 95% of dietary lipids after consumption of a typical Western diet.Furthermore, some studies have suggested that colonic absorption of medium-chain fatty acids takes place in dogs,rats,and humans and that glycerol accumulates in the colon when fat absorption is disturbed in the small intestine.Accumulation of glycerol has been shown to alter the Lactobacillus and Enterococcus communities in the gut.A diet high in animal fat and low in dietary fiber stimulates the synthesis and enterohepatic circulation of primary bile acids.Although the majority of bile acids are recycled in the ileum, some of them escape the enterohepatic circulation in the intestine and become substrates for microbial metabolism in the colon.Bile acids restrict the growth of several microbes.Accordingly, only the microbes that are able to tolerate the physiologic concentrations of bile acids survive in the gut; thus, bile acids appear to exert strong selective pressure on gut microbial structure and function. For example, administration of cholic acid to mice induces phylum-level population shifts of the relative abundance of Firmicutes and Bacteroidetes that resembles microbial changes observed by feeding a high-fat diet alone.Gut microbiota detoxify primary bile acids via deconjugation, in which well-conserved bile salt hydrolases release the amino acids glycine and taurine.

The free primary bile acids are then converted into various types of secondary bile acids such as deoxycholic acid and lithocholic acid by the 7α- dehydroxylation reaction.A detailed list of bacteria with genes encoding bile salt hydrolase activity is reviewed by Ridlon et al.In general, the conjugated bile acid profile is heavily dependent on microbial activity. The bile acid distribution profile in multiple compartments of germ-free animals shows less diversity and is smaller in size compared with conventional counterparts.Dietary lipid composition can also modulate bile acid profile, in particular, increasing taurocholic acid that, as a consequence, promotes the growth of Bilophila wadsworthia, a Gram-negative opportunistic pathogen.B. wadsworthia utilizes taurine as a source of sulfite,which serves as the terminal electron acceptor for the respiratory chain.The concentrations of bile acids and their conjugation status to glycine or taurine between individuals may be influenced by diet, as people eating a meat-rich diet tend to have more taurine-conjugated bile acids in their bile acid pool than those eating a vegetarian diet.It has been recognized that the production of secondary bile acids is pH dependent. The proximal colon is more acidic than the distal colon,which results in an elevated activity of 7α-dehydroxylase in the cecum versus the left colon.Subjects consuming diets high in resistant starch showed a significantly decreased stool pH compared with subjects consuming a low resistant starch diet. A decrease in pH is associated with an elevated production of SCFAs, which selectively regulate the intestinal microbial community, with a tendency to suppress Bacteroides spp.and promote butyrate-producing Gram-positive bacteria such as E. rectale. Similarly, subjects on a vegan or vegetarian diet showed significantly more acidic stool pH89 and significantly lower fecal secondary bile acid production83 than omnivores. Higher consumption of animal protein is one possible explanation of higher fecal pH value in an omnivorous diet, as proteolytic putrefactive bacteria are able to increase stool pH by producing alkaline metabolites. Thus, increases in SCFAs result in a more acidic colonic pH, a decreased solubility of bile acids, an indirect increased absorption of minerals, and a reduction of ammonia absorption, which indirectly alters the composition of gut microbiota.Importantly, 50−70% of acetate taken up by the liver becomes the primary substrate for cholesterol synthesis, whereas propionate inhibits cholesterol synthesis in hepatic tissue . Therole of cholesterol on gut microbiota was first elucidated using germ-free rats. Danielsson et al. demonstrated that germ-free rats exhibit a higher serum cholesterol level than their conventional counterparts.More recently, the characterization of the gut microbiota in a hamster model of hypercholesterolemia showed that dietary intervention with grain sorghum lipid extract180 modulated the gut microbial composition, with bifidobacteria being positively associated with increases in HDL cholesterol level and the family Coriobacteriaceae being associated with non-HDL cholesterol.Together, high intake of dietary fat, in particular animal fat, and cholesterol not only changes the composition of bile acids and neutral sterols in the colon but also modifies the gut microbiota,plant pot with drainage which consequently transforms these compounds into secondary bile acids and cholesterol metabolites.Polyphenols present in a wide range of plant-based foods have received great interest owing to their antioxidant capacity and potential protective effect in reducing cardiovascular disease risk through improvement in vascular function and modulation of inflammation. The interpretation of the influence of polyphenols on cardiovascular health in dietary intervention studies can be complicated due to dynamic bio-availability during the processes of absorption, metabolism, distribution, and excretion. Generally, the absorption of dietary polyphenols is widely dependent on the type and structure of the compound and is often slow and largely incomplete in the small intestine. Therefore, significant quantities of polyphenols are retained in the colon. In addition, the nonabsorbed polyphenols are subjected to biotransformation via the activity of enzymes from various microbial groups . Consequently, the microbiota-derived metabolites of polyphenols are better absorbed in the gut,which then become an important factor in the health effect of polyphenol-containing foods. Important plant polyphenols and their microbial derivatives are listed in ref.

Many of the studies that assess bio-availability and effects of polyphenols have evaluated the balance between the enterohepatic circulating levels, residence time in plasma, and urine excretion rate of the parent phenolic compounds and their microbial-derived metabolites using metabolomic techniques.Importantly, although endogenous enzyme and transporter activities in the small intestine as well as transformation of polyphenols are subject to a wide inter individual variability, the functional capability of the gut microbiota is important to partially explain the variation of bio-availability among the population.Assessing the properties of a single dietary constituent from the polyphenol family alone without dietary fiber is difficult due to the complex dietary food matrix present in a flavonoid-rich diet. For example, regular consumption of apples increased the numbers of fecal bifidobacteria and decreased the C. perf ringens count.Similarly, the concomitant dietary presence of apple polyphenols and FOS increased SCFA production.In contrast, compared to consumption of an inulin-containing diet alone, including a grapefruit flavonoidrich extract decreased both production of SCFAs and the bifidobacteria population.Furthermore, a randomized crossover intervention study in which subjects consumed high levels of cruciferous vegetables for 14 days revealed an alteration of the fecal microbial community profile compared with a lowphytochemical, low-fiber diet, including a higher abundance of Eubacterium hallii, Phascolarctobacterium faecium, Burkholderiales spp., Alistipes putredinis and Eggerthella spp.The observed changes could also be explained by the increase in dietary fiber that is enriched in cruciferous vegetables.Overall, the direct effects of fiber blur the ability to judge the specific effects of individual dietary ingredients on gut microbiota. These dietary ingredients may act in an additive or a synergistic manner, exerting their effects on gut microbiota. Six week consumption of a wild blueberry drink that was high in polyphenols was shown to increase the proportion of Bifidobacterium spp. compared to the placebo group;however, a high interindividual variability in response to the dietary treatment was also observed.Similarly, the daily consumption of a high-cocoa flavonol drink for 4 weeks significantly enhanced the growth of fecal bifidobacterial and lactobacilli populations, but decreased the Clostridia histolyticum counts relative to those consuming a low-cocoa flavonol drink .Furthermore, unabsorbed dietary phenolics and their metabolites selectively inhibit pathogen growth and stimulate the growth of commensal bacteria. For example, grape pomace phenolic extract increases Lactobacillus acidophilus CECT 903 growth in liquid culture media.In addition, upon bacterial incubation, tea phenolics were shown to suppress the growth of potential pathogens such as Clostridium spp. and Gram-negative Bacteroides spp., whereas commensal anaerobes such as Bifidobacterium spp. and Lactobacillus spp. were less affected.Similarly, the flavanol monomer -catechin significantly increases the growth of the Clostridium coccoides−Eubacterium rectale group, Bifidobacterium spp., and E. coli and significantly inhibits the growth of C. histolyticum group in vitro.To date, there is a wide range of phenolic compounds that have been demonstrated to have antimicrobial properties,and many have been previously reviewed.Although many of the studies highlighting the beneficial role of plant polyphenols through regulation by gut microbiota appear promising, there are limitations in the results that can be drawn regarding the ability of flavonoids to influence the growth of selected intestinal bacterial groups using a batch-culture model.

To make foods safe, cyanogenic glycosides are frequently hydrolyzed through processes including grinding, pounding, boiling, steaming or during soaking or fermentation in water.Thermal treatments have also been used to effectively decrease levels of cyanogenic glycosides in foods.Commercial elderberry juice is thermally processed to inactivate enzymes, improve microbiological stability, and to degrade the potentially toxic cyanogenic glycosides found in this berry which include amygdalin, dhurrin, linamarin, and sambunigrin . The range and content of cyanogenic glycosides in the blue elderberry have not yet been identified. However, there was an occurrence of people getting sick after consuming raw blue elderberry juice at a at a religious gathering in Monterey County, California in 1983.Therefore understanding levels of cyanogenic glycosides in this subspecies is critical if it is going to be considered for use in commercial products. In European elderberry, sambunigrin is the main CNG in all parts of the plant, with the highest levels in the leaves and the lowest levels in the ripe berries .6 Sambunigrin is a diastereoisomer of prunasin, containing L-glucose instead of D-glucose.In the American subspecies, amygdalin, dhurrin, linamarin, and sambunigrin were identified in the skin, seed, and juice of the berry as well as in the stem.Juice of the American elderberry contained the lowest levels of CNGs relative to the skin, seeds and stems, averaging about 4 µg g- 1 , while the stems had the highest concentrations in the two cultivars evaluated.A recent study demonstrated that the total phenolic content of the blue elderberry is similar to the European and American subspecies,danish trolley however this subspecies has significantly lower levels of anthocyanins.

The dominant phenolic compounds identified in the blue elderberry include the flavonols rutin and isorhamnetin glucoside, the phenolic acid chlorogenic acid, a novel phenolic compound tentatively identified as 5-hydroxypyrogallol hexoside , and the anthocyanins cyanidin 3-glucoside and cyanidin 3-sambubioside .At the same time, anthocyanidins are desired by consumers for their potential health promoting bioactivity.Unfortunately, heat processing and pasteurization can lead to the degradation of anthocyanins resulting in a loss in color or a formation of brown polymers, possibly impacting the acceptability of the final product. Elderberry juice and extracts have been evaluated for their thermal stability, which have shown that anthocyanins degrade following first-order reaction kinetics.The stability of anthocyanidins can be reinforced via intra- and inter-molecular interactions with protective structures and flavonoids through a phenomenon termed copigmentation.Notably, acylated anthocyanins, such as those found in American elderberry like cyanidin 3-coumaroylsambubioside, are sometimes more stable during thermal processing due to protective properties of the coumaroyl group folding over the flavylium ion.Like the European elderberry, the blue elderberry does not contain acylated anthocyanins.The thermal stability of anthocyanidins in blue elderberry juice has not yet been evaluated. However, as elderberry juice and extracts are frequently thermally processed to make products, such as jam, syrup, or gummies, it is important to understand the thermal degradation of anthocyanidins in the juice from blue elderberry.

The purpose of this study was to determine the stability of the cyanogenic glycosides and main phenolic compounds in blue elderberry juice cooked at 72 °C and 95°C for two hoursto elucidate the kinetics of degradation of these important compounds. HPLC-grade methanol , and LCMS grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific . Ultrapure water was obtained from a Milli-Q water system . Prunasin was also obtained from Millipore Sigma. HPLC-grade acetonitrile , rutin , isorhamnetin 3-O-glucoside, caffeic acid, chlorogenic acid, -catechin, protocatechuic acid, ammoniumformate, and amygdalin were purchased from Sigma-Aldrich . Cyanidin 3-Osambubioside chloride and cyanidin 3-O-glucoside were purchased from ExtraSynthese Ripe berries were harvested in July 2019 from a farm in Winters, CA at latitude and longitude coordinates of 38.634884, -122.007502. Fruit was selected from all sides of the plant at a variety of heights to obtain a representative sample of berries from each plant. Only fully ripe berries . About 5 kg were harvested from each of shrub in three hedgerows. The plant material was transported to University of California, Davis on ice in plastic gallon bags within two hours of harvest and stored at -20 °C until analysis. A hot water bath was prepared using an immersion circulator , set to the desired cooking temperature . The temperature was also monitored with a thermometer in the water bath. Vials of juice randomized and place into the hot water bath and the bath was covered during cooking. Duplicate vials were removed at the following times: 15, 30, 45, 60, 75, 90, 105, and 120 min. An extended processing time was used to observe degradation of more heat-stable compounds as well as to make comparisons to other studies that processed juice for multiple hours at these temperatures. Once removed from the hot water bath, vials were placed immediately into an ice bath for 15 min.

Then from each vial, 1 mL of juice was placed in a microcentrifuge tube and centrifuged at 4 °C, 15,000 rpm for 15 min . Next, the supernatant was diluted 1:10 with 1% formic acid in water, filtered with 0.2 µm PTFE filter, and placed in an HPLC vial for analysis. Five replicate juice samples were prepared and cooked at both temperatures. Composite juice samples were prepared by combing equal aliquots of the time points 0, 15, 30, 60, and 120 minutes of thermal processing for both temperatures for each juice prepared. To extract CNGs for blue elderberry juice, 0.500 mL of juice was mixed with 2.00 mL of methanol in a 5 mL centrifuge tube, then sonicated at 30 °C for 30 min. After sonication, 1.00 mL of extract was transferred to a 1 mL microcentrifuge tube and centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatant was collected and filtered through 0.22 µm PTFE into an HPLC vial and used for analysis. Five replicate extractions were made of raw elderberry juice and triplicate extractions were made for the other time/temperature juices. CNGs were analyzed via ultra-high performance liquid chromatography with electrospray ionization and triple quadrupole tandem mass spectrometry using an Agilent 1290 Infinity HPLC and 6460 mass spectrometer . The UHPLC was equipped with a binary pump with an integrated vacuum degasser , an autosampler with thermostat , and a thermostated column compartment . CNGs were separated using a Kinetex F5 column at 40.0 °C. The mobile phase consisted of a linear gradient of 1 mM ammonium formate in water and 1 mM ammonium formate in 650:50 MeOH:ACNe as follows: 5% B, 0–5 min; 100%B 5-5.50 min, 95% A 5.60-6.50 min. The flow rate was 0.400 mL/min, and the injection volume was 5.0 μL. The CNGs were analyzed using negative ESI mode. The drying gas temperature was 300 °C and the flow rate was 8.0 L min-1 . The sheath gas temperature and flow rate were 350 °C and 11.0 L min-1 , respectively. The nebulizer gas pressure and capillary voltage were 45 psi and 3.5 kV, respectively. The fragmentor voltage was 160 V for amygdalin and 100 V for sambunigrin. The dwell time was 100 ms for amygdalin and 200 ms for sambunigrin. The collision energy was set to 12 V for amygdalin, 0 V for sambunigrin. The multiple reaction monitoring mode was utilized to analyze amygdalin and sambunigrin. Quantification of CNGs was performed using external calibration curves using standard addition at levels of 500, 100, 50, and 5 ng L-1 . For amygdalin, vertical aeroponic tower garden the area of m/z 456.2 to m/z 323.1 was measured. For sambunigrin, the area of m/z 340.3 to m/z 294.2 was measured. Half-life values of phenolic compounds were calculated by plotting the natural log of C0/C ratio vs heating time t, where C0 is the initial concentration of a compound, C is the concentration of the same compound at time t in hours. The slope calculated from the figure is k . Longitudinal Analysis of variance was performed with Tukey’s post-hoc test with p value at 0.05. Excel used for average and standard deviation and R Studio was used for ANOVA and post hoc analysis . CNG data was analyzed using MassHunter Quantitative Analysis to obtain peak areas for the targeted compounds. Microsoft Excel was used to create the calibration curves and determine CNG concentrations in samples . The concentrations of cyanogenic glycosides were quantified in raw and cooked blue elderberry juice for the first time. Results indicate that neoamygdalin , sambunigrin and prunasin are the primary CNGs in blue elderberry . Concentration of neoamygdalin were significantly higher than sambunigrin and prunasin . Neoamygdalin has been measured in raw bitters almonds in concentrations lower than amygdalin.133 In studies of American and European elderberry, sambunigrin is typically major CNG identified. Levels of total CNGs in blue elderberry are lower than American and European elderberry. European elderberry CNG levels range from 0.08 ± 0.01 to 0.77 ± 0.08 µg g-1 depending on the elevation and growing location.6 CNG levels in American elderberry juice range from 0.29 to 2.36 µg mL-1 .

Differences between the subspecies may be due to genetic variation, impact of growing environment such as altitude, or methodology used to extract and analyze CNG content in the fruit and fruit juice, including how berries were handled prior to juice and juicing method. The degradation of neoamygdalin > sambunigrin > prunasin was observed during cooking and the rate of degradation was faster at 95 °C as compared to 72 °C . However, degradation in juice at 72 °C was not linear, such that sambunigrin levels in juice cooked at significantly increased during the final timepoint measured . This may be attributed to neoamygdalin breaking down resulting in sambunigrin and a glucose molecule, an equivalent pathway to amygdalin degrading to prunasin and a glucose molecule. However, some of the resulting sambunigrin from that reaction would also have to be degrading since the decrease in concentration of neoamygdalin did not cause an equivalent increase in sambunigrin. An increase in sambunigrin at the end of the processing time was not observed in the juice cooked at 95 °C. In the juice processed at 95 °C, the combined concentration of neoamygdalin and sambunigrin decreased at each measured time point and did not increase at any time points like the juice processed at 72 °C. Prunasin levels did not significantly change in the elderberry juice cooked at 72 °C but prunasin did degrade significantly when processed at 95 °C . It appears that sambunigrin is more stable than prunasin in the elderberry juice; retaining about 70% of the original concentration in the juice heated to 95 °C. Neoamygdalin levels decreased significantly in the elderberry juice at both processing temperatures, with increased degradation at 95 °C as compared with the treatments at 72 °C. As previously mentioned, sambunigrin is the expected breakdown product from neoamygladin, but sambunigrin levels did not have concomitant increase due to thermal degradation of sambunigrin as well. Thermal processing has been seen to degrade CNGs in elderberry, flaxseed, and almond in previous studies.Furthermore, studies have seen enzymatic activity contributing to the breakdown of CNGs in nuts to reduce after exposure to heat.If the β-glucosidases for the CNGs present in blue elderberry are similar, they would also be inactivated during thermal processing at 72 and 95 °C, indicating thermal degradation is the main contribution to CNG levels decreasing in the present study. Because enzymatic degradation of CNGs was not measured during the thawing and juicing steps, the impact of the enzymes before the thermal processing cannot be evaluated here. The presence of neoamygdalin instead of amygdalin is unexpected. Amygdalin can convert to neoamygdalin with heat and in alkaline conditions. However, herein the raw elderberry juice had significantly higher levels of neoamygdalin as compared to amygdalin. In a study of amygdalin content in almond varieties, amygdalin was found to convert to neoamygdalin during extraction , but the addition of acetic acid prevented the conversion.Blue elderberry naturally contain citric and malic acids, with an average titratable acidity of 0.60 ± 0.10 to 0.65 ± 0.07 g citric acid per 100 g FW.The average pH value of the juices in the present study was 3.76 ± 0.11. Therefore, there may not be enough acid in the matrix to prevent the conversion. In contrast, another study of amygdalin and derivatives in almonds found that heat of cooking caused neoamygdalin and amygdalin amide to convert to amygdalin, which was not observed in the present study.Further analysis of conversion of amygdalin to neoamygdalin in the blue elderberry could uncover why this epimer is dominant.

Electrophoresis was run at 80 V and then the gel was visualized with a UVP High Performance Ultraviolet Transilluminator . Genomic DNA was also amplified with primer pairs pSA3_Cm_F/pSA3_CmR for the chloramphenicol resistance gene of plasmid pSA3 and gfp_qPCR_F/gfp_qPCR_R for the egfp gene of plasmid pIGSAF. Primers nifH_qPCR_F/nifH_qPCR_R for the nifH nitrogenase gene from the F. alni genome were used as a positive control for the quality of DNA extracts. Additionally, purified plasmid pDiGc from E. coli DH5α was used as a DNA template for egfp amplification as a positive control. PCR products were separated on an agarose gel and extracted as in Plasmid Synthesis, above, and sequenced by the UCDNA Sequencing Facility . Forward and reverse sequences were combined to make fulllength amplicon sequences. These were aligned with MUSCLE with published reference sequences for the egfp and camR genes. Additionally, the sequences were compared by BLAST against gene sequences obtained from the original plasmids . Confocal microscopy was used to visualize expression of egfp in F. alni hyphae and vesicles. egfp was expressed both constitutively, from plasmid pIGSAF, and differentially between N and N media under control of the F. alni nif promoter using plasmid pIGSAFnif. Cultures were imaged after on1 week of incubation from the previous transfer, 25 weeks after transformation, as described in Plasmid Maintenance on Selective Media, below. For imaging, F. alni cultures were grown either in N or N BAPP medium as in Culture Conditions, above,blueberry grow pot and immobilized on glass slides with a drop of 3% molten agarose solution maintained in a water bath at 50◦C. Slides were pre-heated on a slide warmer at 50◦C. Fifteen microliter of Frankia hyphal suspension was then pipetted onto each slide and covered with 35 µl of 3% molten agarose.

A #1.5 coverslip was added to the Frankia cells in agarose, and then the slide was allowed to cool to room temperature. The Frankia preparations were visualized on a Leica TCS SP8 STED 3X confocal microscope with either a 20X objective, or a 100X oil-immersion objective, using either bright field or fluorescence with a HyD detector, in the Advanced Imaging Facility, University of California, Davis. For fluorescence imaging, samples were excited with 488 nm light. The emission wavelengths were collected from 500 to 550 nm. Images were stored as .lif files from Leica LAS X and then viewed in FIJI . For imaging of individual hyphae and vesicles, the 100X objective lens was used and images were taken in Z-stacks with a step size of 0.1 µm. To visualize F. alni colonies at low magnification, Z-stack images were taken with the 20X objective in five steps of 2 µm each and then combined using the highest fluorescent intensity of each pixel . Three separate transformation experiments were carried out, from August 2017 through March 2019, as outlined in Table 2. Transformed cultures were maintained with weekly subculturing into fresh selective media and used in experiments over the course of several months following each transformation. Initial transformations of F. alni with plasmid pIGSAF were performed in August of 2017; the cultures were maintained by subculturing and were visualized by confocal microscopy from December 2017 through March 2018 . Transformation of F. alni with plasmid pIGSAFnif was performed in January 2018, selected as above, and imaged by confocal microscopy in April 2018 . These cultures were maintained by subculturing in selective media for RNA extraction for qPCR through July 2018 . These cultures were transformed in September 2018 and subcultured weekly in selective media through March 2019. The presence of the plasmid was confirmed by DNA extraction and PCR amplification of the egfp gene in March 2019 as described in Frankia Transformation, above.

To test the persistence of plasmid pIGSAF in transformed F. alni without selection, a time-course of growth in media without antibiotics was performed. Fresh cultures were transformed with plasmid pIGSAF and initially selected as described in Frankia Transformation, above. After this 4-week selection process, cultures were then grown in non-selective media: cells were first pelleted and re-suspended in fresh BAPP media, then subcultured without the addition of chloramphenicol into six-well plates and incubated for 1 week. A separate set of cultures from the same stock was maintained in selective media and subcultured at the same time points as a control. Each week both sets of cultures were pelleted and re-suspended in 500 µl fresh BAPP media. The suspensions were homogenized by passage through a 21G needle twice. 250 µl of each homogenate was transferred to 4 ml fresh BAPP media without chloramphenicol in each well and the remaining 250 µl was used for total genomic DNA extraction as described in DNA Extraction above. This process was repeated once per week for 4 weeks. At each sampling point, the relative amount of plasmid in each sample was quantified by qPCR, performed in duplicate for each of three biological replicates, using egfp primers GFP_qPCR_F and GFP_qPCR_R . Fold-change of plasmid between each time point was calculated using the 11 Ct method . The infC gene , amplified from the same DNA extracts with primers infC_qPCR_F and infC_qPCR_R , was used as a control to normalize the amount of DNA in each sample. To determine significant changes in plasmid abundance, twotailed Welch’s t-tests were performed in R on normalized 1 Ct values . Cultures grown with and without selection for 4 weeks were also imaged with fluorescence according to methods in Confocal Microscopy, above. After electroporation F. alni cells formed visible hyphae in culture after about 10 days. When subcultured into chloramphenicolselective media, F. alni cultures transformed with unmethylated plasmid pIGSAF were able to grow, whereas untransformed cultures and those transformed with methylated plasmid were not. When analyzed on the gel, purified plasmid pIGSAF from E. coli formed bands representing linear , circular , and supercoiled plasmid .

Presence of plasmid pIGSAF in transformed cultures was verified by a band present in DNA extracts corresponding to the linear form of plasmid pIGSAF that was not present in extracts from wild-type F. alni . PCR amplification and sequencing of the egfp gene from DNA extracts of transformed F. alni cultures confirmed the identity of the plasmid . Absolute copy number quantification with qPCR estimated the plasmid was present at 12.5 copies of plasmid per molecule of genomic DNA; relative quantification gave a similar estimate of 11.8 copies per genome . Each experiment was performed on cultures maintained with routine weekly subculture for 15–32 weeks post-transformation, as described in the Methods. When imaged under 488 nm wavelength of excitation in the confocal microscope, green fluorescence typical of GFP was observed in hyphae as shown in Figure 4. Wild-type F. alni hyphae displayed autofluorescence around 575 nm as observed by Hahn et al. , but no autofluorescence was observed in the 500–550 nm range . When grown in N culture, transformants carrying the pIGSAFnif plasmid showed significant up-regulation of the egfp gene conjugated to the nif cluster promoter, approximately 100- fold relative to N culture, or approximately 8.5-fold per copy of plasmid . The F. alni nifH gene , used as a positive control for nitrogen fixation, was similarly significantly up-regulated approximately 8.5-fold in N media compared with N cultures. Expression of the F. alni rpoD housekeeping gene , used as a negative control, was not significantly different between N and N cultures . F. alni containing pIGSAFnif grown in N media fluoresced predominantly in the vesicles,square plastic pot observed at 100X magnification . Little to no fluorescence was observed in hyphae. Fluorescence in the vesicles was present both in the spherical portion as well as in the stalk connecting the vesicle to the hyphae. No fluorescence was observed in hyphae or vesicles of wild-type F. alni grown in N media . Due to the step size of 0.1 µm bright fluorescence was only observed when vesicles were in the plane of focus . Few vesicles were present in images likely since the cells were observed after 1 week of culture in N medium. We have shown that F. alni can be stably transformed with an unmethylated replicating plasmid introduced by electroporation. The methods presented here circumvent two major transformation barriers in Frankia. First, the lack of methylation avoids restriction of the plasmid by type IV methyl-directed restriction enzymes. Second, the use of a plasmid replicated and maintained outside the genome does not rely on the reduced homologous recombination rate in actinobacteria . Plasmids pIGSAF and pIGSAFnif were stably maintained in F. alni culture and used to perform routine experiments. Three independent transformations were performed over the course of 2 years and the resulting transformants were maintained on selective media by repeated subculture for at least 7 months .

The presence and stability of the plasmids were confirmed with gels of whole genome DNA extracts , PCR and qPCR amplification of plasmid-bound genes , and visualization of GFP expressed from plasmids . The copy number of plasmid pIGSAF per genome in F. alni was determined to be about 12 copies per cell , in line with findings for other plasmids derived from plasmid pIP501 that have been estimated to be maintained at approximately 10 copies per cell . In addition, plasmid pIGSAF was determined to be stable in non-selective media for a period of at least 3 weeks . Of the restriction enzymes identified in Frankia genomes our analysis indicated that the type IV enzymes posed the most likely barrier to transformation. Types I and II enzymes recognize specific sequence motifs of generally six to eight nucleotides and hence are statistically highly unlikely to be a broad-range barrier to transformation or horizontal transfer . Additionally, in the genome of F. alni ACN14a the majority of types I and II genes showed very low expression in both N-culture and symbiosis . A type IV homolog of an mrr type methyladenine-targeting restriction gene was highly expressed in F. alni in culture , suggesting that DNA with methylated adenine bases is degraded in this organism. Actinobacteria, especially Frankia, express type IV methyl-directed restriction enzyme genes more highly in culture than do proteobacteria and firmicutes , a finding that correlates with previous reports of higher transformation efficiencies with unmethylated plasmids than methylated in Corynebacterium and Streptomyces spp. . Genomes of the majority of actinobacteria are missing homologs of the dam methyltransferase gene whose product is used to mark parent DNA strands during replication, and mutS and mutL that form a complex for the removal and repair of mismatched bases on the daughter strand determined by the methylation of adenine residues . Together, these factors suggest a preference for unmethylated over methylated DNA among most of the actinobacteria. Type IV restriction enzymes have been suggested to have evolved as a counter to phage methylation systems that themselves evolved to evade host restriction systems through the methylation of restriction target sites . Phage genomes adopt the methylation patterns of their previous host thus increasing the likelihood of digestion by actinobacterial enzymes if replicated in a dam + host. The expression of type IV restriction enzymes in actinobacteria therefore could represent an adaptation to prevent infection by phages based on the methylation state of their genomes. Differences in methylation patterns between actinobacteria and other bacterial phyla potentially constitute a barrier to horizontal gene transfer between these groups, including phage-mediated gene transfer. Of particular interest to the evolution of root nodule symbioses is the possibility of transfer of relevant genes between Frankia and the rhizobia, and vice versa. It has been suggested that the nodA gene involved in Nod factor biosynthesis evolved in the actinobacteria, including some Frankia, and was then horizontally transferred to the rhizobia . If type IV restriction enzymes create a barrier to horizontal transfer into actinobacteria from dam + bacteria including proteobacteria, it would seem that horizontal transfer from actinobacteria to other phyla would be more likely than the reverse. However, F. alni was observed to down-regulate its type IV mrr gene substantially in symbiosis . As roots contain much lower concentrations of bacteriophage than the surrounding soil this could represent a decreased necessity for restriction enzymes as a defense mechanism during symbiosis. A potential side-effect of this down-regulation, however, is that the barrier to horizontal transfer posed by type IV enzymes is likely lowered during symbiosis. In plants, the endophytic compartment is dominated by actinobacteria, with specific taxa of other phyla including proteobacteria and bacteroidetes .

Similarly, Mohaisen and Ma developed and simulated asolar assisted liquid dehumidification air conditioning system using LiCl solution on Matlab Simulink platform. Their result has been validated with experimental results by Fumo and Goswami. The simulation results based on three consecutive sunny summer days in Sydney show daily COP of 0.5-0.55 by the proposed system and 73.4% of thermal energy required for thermal regeneration was provided by the solar collectors. Croffot and Harrisson experimentally evaluated the performance of a solar driven system installed in Canada. The system includes a low-flow parallel plate liquid desiccant air conditioner, and a 95m2 evacuated tube solar collector array. Results from five test days have shown an overall solar collector efficiency of 56%, solar fraction of 63% and a thermal COP of 0.47. The average total cooling was 12.3kW and average latent cooling was 13.2kW. Peng et al.studied a solar air pre-treatment collector/regenerator. Their simulation results showed that the storage capacity of proposed system could be improved by 50%, when the regeneration temperature was 60℃, and the inlet air specific humidity was 2.33kg/kg. In another study, Lychnos and Davies performed experimental and theoretical simulation for a solar powered liquid desiccant system using MgCl2. The theoretical model was developed and verified with the experimental results. Compared with conventional evaporative cooler,dutch buckets system the proposed system could further lower the average daily maximum temperature by 5.5-7.5oC. Alizadeh fields tested implementing polymer plate heat exchanger into LDAC in Australia summer weather condition. Lithium chloride with 43% was used in the experiments and a scavenger air regenerator concentrates the dilute solution from the dehumidifier using hot water from flat plate solar. Alizadeh experimentally analyzed the effects of various air flow rate and desiccant flow rate on the system performance.

The experimentally obtained data was compared with a developed model. The comparison showed a good agreement between the experiments and model predictions. The results showed that the proposed system could provide cooling capacity up to 20kW with dehumidification efficiency up to 72%. Qi et al.simulated yearly system performance of solar assisted LDAC for commercial application in five cities . In this study, the effects of various solar collector areas and monthly solar radiations, ambient air conditions on the electricity consumption saving and monthly average COP were theoretically analyzed.Results shows that for buildings located in humid areas with low sensible-total heat ratio, the electricity energy reduction of the system was high and about 450MWh in Houston and Singapore and payback was approximately 7 years. For building in dry climate, although the total cooling load was low, up to 45% of electricity of AC can be saved because of significant improve in chiller COP during more than 70% of operation time and the payback was around 22 years. However, for the buildings with mild outdoor humidity, such as those in Beijing and Los Angeles the electricity energy saved only around 100MWh and the cost payback period was more than 30 years, and the application of LDAC was not that suitable. Li and Zhang investigated a solar energy driven hollow fiber membrane-based desalination system. The system consists of a membrane-based humidifier, a fin-and-tube type dehumidifier and a solar heating unit, which consists of a U-tube evacuated solar collector and a heat storage tank. The hollow fiber membrane-based humidifier is similar to a shell-and-tube heat mass exchanger. Through numerical modelling and experimental tests, they found that the COP of the system can reach about 0.75 and electric COP of 36.13 is achievable, which means electrical energy consumption is much less due to solar energy reclamation. Results show that solar accounts for 92.0% of the electric energy consumption by the entire system.

Sensible heat losses account for most of the energy losses from the system. Chen et al. proposes solar assisted liquid desiccant dehumidifier and regenerative indirect evaporative cooling system for fresh air treatment. The hot and humid fresh air is firstly dehumidified by LDD and then sensibly cooled by RIEC. The thermal energy captured by solar collectors is used for desiccant solution regeneration. In this study, they have looked into the influences of solar collector area and inlet air conditions and optimizing the extraction air ratio of RIEC. The energy saving ratio is quantitatively evaluated with respect to a conventional A/C system. Results shows that the energy saving ratio ranges from 22.4% to 53.2% under various inlet air conditions. This characteristic of liquid desiccant dehumidification system that the dilute liquid desiccant can be regenerated by low grade heat makes it attractive option for integrating with thermal sources. Many studies have been done on integration of liquid desiccant system with solar, vapor compression, heat pump, and CHPs. But only two research has been done integrating liquid desiccant with SOFCs. The typical SOFC system exhaust temperature matches very well with the required temperature for liquid dehumidification. Elmer has looked into design, development and proof of concept combined generation of power, cooling and heating based on SOFC and Liquid desiccant air conditioning technology for building application. A 1.5kW SOFC integrated with liquid desiccant has been proposed for providing heating, cooling and electricity to low carbon buildings. Elmer et al.used empirical SOFC and liquid desiccant component data for an energetic, economic and environmental analysis. They have used a simple 0D model in Engineering Equation Solver platform for regeneration, dehumidifier and fuel cell. The model is based on dimensionless vapor pressure and temperature difference ratio designed for packed bed liquid desiccant. The results show the moisture removal of the supply rate is mainly controlled by desiccant temperature and cooling water temperature in constant flow rates. 

The air inlet condition has a large effect on cooling output performance and the unit performs better in a hot and humid climate. By increasing the regeneration heat source temperature more water vapor vaporizes from the weak solution. The model they used have limitation such as not including the effectiveness of heat and mass exchanger effectiveness, geometry of the contactor, and desiccant carry over. For desiccant air conditioning system Elmer developed an Integrated Desiccant air Conditioning System . This system combines regenerator, dehumidifier and evaporative inter-cooler into a single membrane-based heat and mass exchanger to reduce the size, complexity and leakage. The IDCS operates with a KCOOH desiccant working fluid. The liquid desiccant is sprays through nozzle from the top where regenerator is located and flows downward due to gravity. In this design instead of preheating desiccant before regeneration and precooling it before dehumidification, thermal energy is supplied to the regenerator through airstream and an evaporative inter-cooler is designed. This is because all desiccant flow is contained within one complete Heat and Mass Exchanger the solution cannot be extracted for prior heating and cooling. This feature is weakness of this design as precooling and preheating of liquid desiccant increases the performance of the system.Their design was supposed to significantly reduces the number of heat exchangers, pipes, and ducting in liquid desiccant air conditioning systems. The main issue with this integrated system was imbalance between moisture removal rate in the dehumidifier and moisture addition rate in the regenerator. This mass imbalance is primarily due to the available surface area for heat and mass exchange in the regenerator being too small and an insufficient vapor pressure differential between the air and desiccant solution. In order to regenerate the desiccant solution back to its original condition following the dehumidification process,dutch buckets the regenerator needs to operate for extended time periods. Across the variables investigated there is a greater instantaneous moisture removal rate in the dehumidifier than moisture addition rate in the regenerator. Desiccant solution leakage/carry-over on the dehumidifier side of the HMX has been noticed during the experiment as well. In response to the highlighted shortcomings of the novel IDCS Theo Elmer developed a Separated liquid Desiccant air Conditioning System for trigeneration system integration. SDCS consist of three separate cores including dehumidifier, regenerator, and an evaporative cooler. The SDCS uses a semi-permeable, microporousmembrane-based cross flow contactor, operating with KCOOH desiccant solution. The solution channel consists of polyethylene sheet, with fiber membrane attached on either side to allow diffusion of water but prevent the liquid desiccant entering the air side. In evaporative cooler, air and water come into contact in a cross flow HMX. Before liquid desiccant entering the dehumidifier, a plate heat exchanger is used to precool the desiccant to increase its potential. After dehumidifying the air, the weak solution enters the dehumidifier tank.

Desiccant is preheated before entering the dehumidifier. The strong desiccant flows to regenerator tank after regeneration. In this design, because the entire SDCS is placed within the environmental, the water for the evaporative cooler and desiccant for dehumidifier are at the ambient temperature which has an impact on moisture absorption capacity of the desiccant and the sensible cooling. This design causes little to no sensible cooling achievable. Also, the evaporative cooler only provides around 80–150W of cooling over their study range. As a result, the evaporative cooling provided is not enough to produce a sufficient solution temperature decrease and to provide sensible cooling to the supply air in the dehumidifier. Elmer et al. experimental results on SDCS show effective instantaneous balancing of the dehumidifier and regenerator across a range of environmental and operational values, operation of the dehumidifier is dictated, to some degree by the available SOFC thermal exhaust, COP values in the range of 0.4–0.66 are achievable with a low-grade thermal input typical of an SOFC CHP system and potential for non-synchronous operation in a tri-generation system context, bringing about improvements to peak cooling output and total system efficiency. Elmer et al. used empirical SOFC and liquid desiccant component data for an energetic, economic and environmental analysis. The 1.5kW BlueGen SOFC systems is electrically connected to the grid to import or export as required. The waste heat recovery circuit is connected to a home’s 300L hot water cylinder which also includes an auxiliary gas boiler. Experimental results shows that the electrical efficiency of SOFC system increases from 14% at 200W to maximum of 60% at 1500W and then decreases to 56% as 2000W capacity. Thermal output from fuel cells varies linearly from 320Wth at 200We to 540Wth at 1500W. The calculated water heat recovery temperature for 2l/min flow based varies between 47 C at 100We to 52℃ at 2000We. Be Power Tech, Inc. developed BeCool, a natural gas-powered air-conditioning system that produced electricity as by product on the site. The innovative idea shifts some grid powered electricity for cooling demand to natural gas which would substantially reduce peak summer power demand and help to reduce the need for costly peaking power plants. Their studies showed that their 5ton BeCool unit eliminates ~ 10kW of peak electricity demand, generates 43MWh/yr electricity and saves ~ 10MWh/yr compared to conventional air conditioning. They build a prototype and the test results showed that the electrical power efficiency was 45% to 60%. The primary potential energy saving is 4.1 Quads, and the technology has the potential to reduce 222 million metric tons of CO2. Their analysis for California climate 7 and 14 shows 53% CO2 reduction in a 50% electrical efficiency for fuel cell and 90% combined heat and power efficient fuel cell system is used. BeCool uses exhaust heat from the fuel cell and heat from the burner to increase the concentration of LiCl to 42% mass fraction in the regeneration process. Be Power Tech designed and built an experimental prototype BeCool system integrating a 2.5kW SOFC system , a boost natural gas burner, and a custom-made liquid desiccant air conditioner designed to produce from 2.5-tons to 4-tons of cooling. The system was tested under multiple outdoor air conditions. Their study showed that, in all cases the heat supplied by the fuel cell was not sufficient to provide the heat required for the air conditioning process, since these isolated tests could not rely on storage from daily continuous regeneration. For this reason, the supplemental natural gas burner was used to supply the heat required [80]. Integrated fuel cell dehumidification systems have never previously been studied for data center application. The only two studies that have looked at integration of SOFC with dehumidification systems were focused on building applications of the technology.

White or coextruded white-on-black mulches can slightly lower surface soil temperatures by about 1ºC at 2 cm depth or 0.4ºC at 10 cm relative to bare soil because they reflect most incoming radiation . These mulches are used when lower soil temperatures may be desirable for planting vegetables in particular summer production windows. Clear mulches effectively retain much of the heat normally lost to the atmosphere by bare soil, increasing daytime soil temperatures from 4º to 8ºC at a depth of 5 cm , and 3º to 5ºC at 10 cm relative to bare soil . These clear plastics are the choice for soil solarization. Clear plastics, however, do not control weeds and require other weed management practices such as fumigation and herbicide application. Plastic mulches also influence nutrient levels and uptake. Wien and Minotti found plastic mulching increased shoot concentrations of nitrogen , nitrate , phosphorus , potassium , calcium , magnesium , copper and boron in transplanted tomatoes. Bhella , also working with tomatoes, found higher levels of ammonium , nitrate , and magnesium in plastic mulched soils. Hassan et al. found higher levels of nitrogen, phosphorus, potassium, and calcium in leaf tissue of chilies grown over plastic reflective mulch compared to those grown over bare soil. A wide variety of other colored plastic mulches, including red, yellow, silver, blue, gray, and orange, have been used in various efforts to achieve specific production goals. Each of these colors has distinct spectral reflectivity characteristics and thus modifies the radiation balance in and below a crop canopy. These colored mulches affect not only the microclimate around a crop but have also been shown to influence insect behavior. Yellow, for example, is generally highly attractive to insects and has been shown to increase green peach aphid and striped and spotted cucumber beetle populations compared to plants grown over bare soil .

White at times repels aphids and at other times attracts them, depending on the physiological state of the insect . Orange has been shown to repel various aphids and whiteflies , while pink and green attract aphids . Red has been shown to attract both aphids and whiteflies . Blue has been shown to be repellent in some studies and attractive in others . Highly reflective or shiny aluminum plastic mulches have been shown to repel certain aphids and thereby reduce or delay the onset of aphid-vectored mosaic viruses in zucchini squash and melons and tomato spotted wilt virus in tomatoes . These are the mulches of choice when insect and disease management are the principal objective. On balance, other than UV-metalized reflective mulches, repeated and consistent benefits in managing insects with most colored mulches have not been documented. These mulches, however, have produced mixed results . Mahmoudpour and Stapleton noted that “the influence of mulch colour on growth and productivity has been postulated to be highly specific, and may vary with plant taxa, climate, and seasonal conditions.” As is the case with insect management, aluminum mulches have provided the most positive and consistent findings on crop production .Using cover crops as mulches is a relatively recent management strategy that is currently being refined and evaluated in a wide range of vegetable production systems. The winter annual legume hairy vetch , for example, has been used successfully as both a cover crop and as a mulch in fresh market tomato production systems in the southeastern United States. As a cover crop, the vetch fixes nitrogen, recycles nutrients, reduces soil erosion, and adds organic matter to the soil. When mowed and converted to a mulch, the vetch reduces weed emergence,grow strawberry in containers lowers soil temperatures during the hot summer months, reduces water loss from the soil, and acts as a slow-release fertilizer .

This system, developed by USDA ARS researchers, eliminates tillage, reduces the need for applying synthetic fertilizers and herbicides, and reportedly adapts to both large- and small-scale tomato production in a low-input, no-tillage system. Recent work in Florida by Chellemi et al. has shown that although a cover crop surface residue mulch production system had lower yields than the standard black polyethylene plastic, the overall profitability of the alternative system was actually higher. Work in California’s Central Valley has shown that cover cropping increases water infiltration and reduced winter runoff and increases soil carbon . Additionally, cover crops, when cut and dried, have been shown to delay and reduce the incidence of aphids and whiteflies as well as the incidence of aphidborne viruses. Burton and Krenzer observed a reduction in green bug populations where surface residues of a previous wheat crop existed. Summers et al. found wheat straw mulch significantly delayed and reduced the incidence of alate aphids and several aphidborne cucurbit viruses in zucchini squash. The incidence of silver leaf whitefly and squash silver leaf was also significantly reduced. Similar results were obtained with cantaloupe grown over wheat straw residue. A number of other examples of the successful use of cover crop mulches have been reported in Georgia, Virginia, North Carolina, and Pennsylvania, but their potential in California’s vegetable crop production is only now beginning to be investigated, evaluated, and refined. Combining the potential benefits of surface residue cropping alternatives with those of conservation tillage is becoming increasingly attractive to row crop producers in many of California’s agricultural regions as shown by the following case studies.Beginning in 1995, a series of studies and demonstrations were initiated in the Central Valley to evaluate and develop conservation tillage and cover crop mulch production systems for tomato crop rotations.

While the immediate goal of these efforts was to reduce production costs, a longer-term objective was to develop information on the potential of reduced tillage to improve soil quality, store carbon in the soil, and conserve resources. Initial studies, conducted at the University of California West Side Research and Extension Center in Five Points, at the UC Davis campus, and in commercial production fields in Tracy, Vernalis, and Le Grand, evaluated the use of winter cover crops as surface mulches, the feasibility of no-till and strip-till transplanting, and options for in-season weed management. No-till transplanting requires the use of coulters or some form of residue manager to cut surface residues ahead of the transplanter shoe. Strip-till is a form of CT in which a set of coulter or shank implements tills a narrow band of soil 15 to 20 cm wide to a depth of about 8 to 36 cm only in the line into which transplants will be placed. Results from these preliminary evaluations have indicated that planting and harvesting both processing and fresh market tomatoes is possible and that yields comparable to those attained using standard winter fallow techniques may be achieved with certain reduced-till approaches that do not result in excessive cover crop regrowth or weed competition with the tomato crop. On-farm strip trial data for demonstrations conducted in 1999 in Tracy and in 2000 in Vernalis are given in table 3. This early work, and other experiments summarized by Herrero et al. 2001b, also revealed that in-season weed control by a surface cover crop mulch alone is not adequate. The authors of this publication have subsequently investigated and refined the use of a high-residue cultivator that is able to effectively slice through residues while cultivating weeds. Cover crop mulch species selection and mulch management must be optimized if organic mulch tomato production is to expand in California. Care must be taken to avoid the use of certain cover crops such as sorghum-Sudan hybrid as mulches because they are highly allelopathic to tomatoes and several other vegetable crops . More efficient and low-risk production protocols for managing cover crop mulches in vegetable crop rotations must also be developed.Aphid-transmitted virus diseases cause significant economic losses to California’s multi-million-dollar vegetable crop industries annually. Over the past few years, production of fall melons , squash , peppers , and tomatoes has been extremely difficult in certain regions of the San Joaquin Valley due to extensive virus epidemics and severe infestations of silver leaf whitefly . Spring crops, while affected to a lesser extent, have also suffered significant losses by aphid-transmitted viruses and whitefly infestations.Several plant viruses are responsible for these epidemics,hydroponic nft channel and most are capable of infecting all of the crops mentioned above. Among the most important viruses are cucumber mosaic virus , zucchini yellows mosaic virus , potato virus Y , and watermelon mosaic virus . These viruses are transmitted by aphids in a stylet borne, non-persistent manner. They are acquired and transmitted in as few as 15 seconds, and are transmitted by a large number of aphid species, all of which are abundant throughout California. Due to the rapidity with which the viruses can be acquired and transmitted, insecticides are of little value in preventing virus spread and under some circumstances may actually increase the rate of virus transmission and spread. This has not, however, dissuaded a large number of growers and PCAs from attempting to control the spread of these viruses by using insecticides. UV-reflective mulches consist of a polyethylene base to which a thin coat of aluminum ions has been adhered. The mulches are collectively referred to as metalized mulches. These mulches reflect UV wavelength , which confuses and repels incoming alate aphids, adult whiteflies, and leaf hoppers , reducing their incidence of alighting on plants .

UV-reflective plastic mulches have been used successfully to reduce the incidence of aphidborne virus diseases in squash and other crops . Brown et al. found silver plastic mulch superior to white, yellow, or black with yellow edges in repelling aphids in yellow crookneck summer squash. Plants grown on silver mulch produced significantly higher yields of marketable fruit than did those grown on bare soil. Mulches applied to the planting beds before seeding were effective in repelling alate aphids and delaying the onset of several virus diseases as well as the onset of silver leaf whitefly colonization and the appearance of squash silver leaf in spring and fall-planted zucchini squash in California’s San Joaquin Valley . Disease symptoms in plants growing over these mulches appeared 10 to 14 days later than in plants growing on unmulched beds. In spring-seeded squash, approximately 30 percent of the plants on unmulched beds were infected with one or more viruses by the first harvest, while only 10 to 15 percent of those grown over the metalized mulches showed virus symptoms. In fall-planted trials, 100 percent of the plants grown on unmulched beds, with and without insecticide applications, were virus-infected by the first harvest. Metalized mulches were generally more effective in repelling aphids and delaying virus onset than were white-pigmented mulches . Although plants grown over the metalized mulched plots eventually became infected, they continued to produce a significantly higher percentage of marketable fruit throughout the season than did the unmulched controls. In addition, squash, cantaloupes, cucumbers, and corn grown over reflective mulch produced marketable fruit 7 to 10 days earlier that plants growing over bare soil. Stapleton and Summers also showed than cantaloupe grown over reflective mulches yielded over 500 cartons of marketable fruit per acre compared to less than 50 cartons per acre from plants grown on bare soil. A delay of 4 to 6 weeks in infection by CMV in plants growing over reflective mulch allowed them time to mature and set a good crop of melon fruit before becoming infected. Even though the plants eventually became infected, the delay in infection permitted the harvest of a highly profitable crop. Summers and Stapleton have shown that tomatoes grown over reflective mulches averaged approximately 7 percent virus-infected plants, while plants grown over bare soil averaged in excess of 50 percent infection with the same viruses. This approach is currently the only viable means of managing virus disease in these production systems. Growers are cautioned to use only metalized reflective mulches when insect and disease management is the primary objective. Other colors lack the high degree of UV reflectance necessary to repel incoming insects. Also, the mulch must be applied prior to seedling emergence. Plants may be inoculated with aphidborne viruses in the cotyledon stage, and any delay in applying the mulch could lead to an extensive infection. Some crops are more susceptible to injury when planted over metalized mulches.