The study of the microscopic structure of tissues is called histology. In histology, advanced imaging techniques, such as electron microscopy or light microscopy, are used to analyze and identify the tissue and the structures present. In histology, samples can be specially processed and prepared for visualization of the structure and the disease. In, Figure 1.7 histology of the arterial vessel wall shows the intima, the media and the adventitia along with IEL and smooth muscle cells. Generally, there are different techniques in the processing of the tissue for histology. The tissue processing methods include plastic and paraffin histology along with different staining techniques such as hematoxylin and eosin , Movat’s Pentachrome and Elastin Trichrome staining to analyze and visualize the tissue structure. In each process, the tissue is fixed using dehydrated techniques using alcohol. Then the tissue is embedded in either plastic or paraffin resin. Each sample then is sectioned using a grinding method or cutting method using a sharp blade. In plastic histology processing each section can be cut in 19 to 90 microns vs. in paraffin histology the tissue embedded in paraffin, which is similar in density to tissue can be sectioned at anywhere from 3 to 10 microns. Analysis can be performed after staining methods under a microscope. Usually H&E staining can be used to examine cellular type and quantity and fibrin deposition, while Trichrome Elastin staining can be used for observing any type of injury in the lumen of a vessel, garden pots square the media, or EIL, EEL and other structures. At the beginning of this chapter the limitation in the current stents for use in rapid growing children was discussed, and a great interest among pediatric community in stents that can grow with an artery or be resorbed.

Coarctation of the aorta is a congenital disease in children that can potentially benefit from self-growing stents. Stenting as a superior solution compared to balloon angioplasty and surgery for fixation of CoA was discussed. The properties of Nitinol, an alloy that can be used for self-expanding stents due to some of its unique characteristic properties such as super elasticity, and biased stiffness was discussed. Also, the vascular injury and its remodeling process after the injury, such as negative and positive remodeling was reviewed. In addition, histology and several types of processing and staining that are utilized in the science of histology for microscopic evaluation of the tissue structure was mentioned. Currently, there is little information available on the effect of stent radial force on the rapid growing arteries in pediatric patients. However, there are a good number of studies focusing on the adult abdominal stent grafts, coronary and peripheral artery stents, exhibiting the effect of stent and stent grafts, and their radial forces on the vascular biology. Nevertheless, none of the investigators looked extensively at the large growth of small crimp profile bare metal stents and, particularly, did not design a stent that can grow with the small rapid growing arteries for use in the pediatric endovascular applications. An extensive literature search was performed and the findings from a few key sources are summarized in two categories below. Siegenthaler et al., evaluated the growth and the effect of the stent grafts covered with polyester on the thoracic aorta in young piglets. The authors concluded that the stent graft may inhibit growth of the nonatherosclerotic normal aorta and lead to intimal hyperplasia and focal fibrosis in the inner media adjacent to the stent. Siegenthaler et al., proposed several reasons for their finding, including vascular hemodynamics and the change in pressure profile on the arterial wall due to the polyester covers on the stent.

Polyester covers can absorb the most mechanical forces on the arterial lumen, leading the change in the wall stress and less pressure contact and reduce the pulstality exposure of the aortic wall. Another problem with the stent graft is the potential to cover the side branch vessels during deployment, usually the subclavian artery ostium in CoA. In conclusion, the authors suggested that more study should be conducted to evaluate stent and stents grafts in growing aorta. Cheung et al., reported on the early and the intermediate-term follow-up results of Wallstent a self-expandable stent implanted in children with congenital heart disease. The Wallstent has been widely used by interventionalist in Europe for adult patients with the iliac and femoral arterial stenosis. In two different centers, from 1993 to 1997, Cheung et al., implanted Wallstents in 20 children with average age of 10 years old and an average weight of 30.5 kg. The results showed immediate expansion of the stents and reduction of the pressure gradient in the patients. However, the authors observed migration in two of the optimally positioned stents within 24 hours of implantation, along with significant neointimal in growth in 28% of the patient at the mean follow-up duration of 8.1 months, which contrasts with the experience of patients with Palmaz stents where the significant restenosis is at 3%. Cheung et al., suggested the thrombogenicity of the stent could be due to the design of the stent, woven mesh with expanding radial force, versus Palmaz’s rigid slotted tubes with smooth and even surface. The authors also reported the stent did not pace with the growth of the vessel, therefore limits it use in young children. Hong et al., performed an experimental study with CardioCoil , a self-expanding stent in the coronary artery of pigs, for a duration of six months. The authors performed angiographic and histologic analyses to evaluate the deployment characteristics, patency rates, and neointimal response. The neointimal responses in this study were not significant and the stents were patent through the survival period up to 6 months.

The stents expanded over time; the diameter of the stents at the time of implant was 2.85 ± 0.78 and at the follow-up showed to be 3.24 ± 097mm. Hong et al., observed penetration of most of the stent’s struts into the adventitia. The authors concluded that the self-expanding stent is related with favorable deployment characteristics and potency rates, although appropriate sizing is more crucial than with balloon-expandable stents. More importantly, Hong et al., concluded that, unlike balloon-expandable stents, there is a dissociation between major vessel injury by the chronic strut expansion process and the neointimal reaction. Freeman et al., explored the effect of the stent forces in vascular stenosis and remodeling by placing stainless steel stents with three chronic outward forces —3.4N, 16.4N and 19.4N—in the iliac arteries of juvenile porcine models for a duration of 30 days to explore and develop an equation for identifying the optimal stent force. The results of the authors’ investigation revealed a significant increase in the total thickness and neointimal hyperplasia in the stents with higher COF than the lower ones, which corresponds with several other similar findings. Freeman et al., concluded that the geometry, structure, and mechanics of the target vessel need to be considered when a stent is designed and, in order to achieve maximum dilation, stents should not produce stress in the vessel wall greater than the end of the transitional domain of the vessel’s stress-strain curve. The authors suggested that their findings could be extremely useful in the vascular stent developments. In a 180-day study, Zhao et al., explored late stent expansion and neointimal proliferation of over-expanded Nitinol stents in the peripheral arteries. The authors used Nitinol selfexpanding stents with a maximum diameter of 8 mm and length of 28mm. Zhao et al., implanted the stents into the iliofemoral arteries of Yucatan swine. Due to variations in target artery size, the stent-to-artery ration ranged from 1.2:1 to 1.9:1 and the effect of stretching investigated. the authors observed high stent diameter-to-artery ratio, which resulted in overstretching of the arterial wall. Finally, Zhao et al., reported that the overstretching of an artery can lead to medial injury, square pots and medial injury will cause a profound long-term histological response, including significant neointimal proliferation. Saguner et al., found that stents constrained by their target artery at implantation expanded over time to near their nominal diameter within five months. Like the previous study, severe oversizing determined as an oversizing ratio resulted in significant neointimal proliferation and in-stent restenosis. Barth et al., performed a side-by side comparison of three current stents in the market that are substantially different in their physical characteristics: Palmaz stent, Strecker stent and Wallstents. Palmaz is the most rigid stent and has a very high resistive outward force in vitro in comparison to the Wallstent. The Strecker is made of tantalum, has the lowest resistive force of the three, and is very flexible and maneuverable. Among the three, the Palmaz stent is the nonelastic one with a lower profile, the Wallstent is fully elastic with a higher profile, and the Strecker stent is elastic to a lesser degree with a higher profile. All stents were implanted into canine external iliac and the flexing portion of the proximal femoral artery of dogs. Angiographic images of mid-stent luminal diameters instantly after placement of the stent and at follow-up, as well as mid-stent cross-sectional areas of neointima were compared by the investigators for significant differences. Barth et al., concluded that the Strecker stent with a high profile and low resistive force is affected by the vascular wall recoil and caused the formation of a greater amount of neointima in comparison to the lower profile high resistive force Palmaz stent and Wallstent. Medial atrophy is pronounced outside the latter two stents. The authors found that in the flexing arteries, the rigid stent can penetrate through the vascular wall. Sakakoa et al., studied the vascular response of bare Nitinol stent in porcine femoral and femoropopliteal arteries. The authors performed quantitative angiography and histopathology at one and three months to evaluate and assess the biological response to the two devices. Sakakoa et al., observed an increase in the neointimal area in FPA in comparison to FA and late lumen loss in FPA than in FA. The authors concluded that repetitive interaction between the stent and the vessel wall during dynamic vessel motion could affect vascular responses. Several clinical studies reported the use of different types of stents for fixation of the CoA. We reviewed a few of them and some of them are summarized here.

Haji-Zeinali et al., used currently in the market self-expandable Nitinol aortic stents in eight hypertensive patients with coarctation of the aorta.48 The authors showed that after implantation of the stents, the mean systolic gradient decreased significantly. Haji-Zeinali et al., also reported that Nitinol stents were easier to deploy and conformed better to the aortic anatomy compared to balloon expandable stents. Finally, the authors found that Nitinol stents could be used to treat the coarctation of the aorta safely and effectively; these types of stents had similar efficacy in reducing coarctation of the aorta as surgical repair. Although Haji-Zeinali used these stents in adult patients, we believe the application of Nitinol self-expanding stents can be extended to the pediatric applications and especially neonatal applications for the reduction of CoA. Bugeja et al., used a stent in neonatal for fixation of the coarctation of the aorta. They reported a case of a severely-ill newborn with complex coarctation, multiorgan failure, disseminated intravascular coagulation and oedema, who had to go through an emergency stenting procedure on the tenth day of her life. Since there are no designed stents for neonates, the authors used an off-label used bare metal adult coronary stent . With a fast pace of growth in the neonates, Bugeja et al., placed the stent temporarily and planned a surgical procedure to remove the stent and fix the coarctation surgically. This study clearly demonstrated the need for a stent that can be placed in patients and grow with them to eliminate or reduce the future interventions. Prior to designing the stent for this investigation, the most used stents in the congenital heart disease field was reviewed. Stents can be categorized based on their delivery method: balloon-expandable or self-expandable stents.2 Balloon expandable stents are inflated with a balloon and their size is determined by the diameter of the balloon that they are inflated with. These stents are mostly rigid with high external outward force.

Farmers who wish to remain eligible for some USDA program benefits must obtain catastrophic insurance or higher levels of coverage. Given the relatively few government programs available for specialty crop growers, this ranking may be associated with the specialty crop growers who have diversified into field crops. However, it is worth mentioning that not even one-quarter of potential respondents provided the rank for the reason for purchasing crop insurance except for “crop loss,” which was chosen by more than three-quarters of the insurance buyers. This indicated that many felt that any reason other than crop loss was remotely related.Reasons for not purchasing crop insurance and their mean ranking are presented in Figure F2. “Never lost enough production” and “premium is too high” ranked highest among the choices offered except “other.” This reflected the relatively low degree of yield variability in many specialty crops grown in California. “Lack of availability for my crop” was next. Particularly among vegetable growers, lack of availability was ranked as the primary reason for not purchasing crop insurance, with a mean rank of 1.6 . Further, “major source of risk is not an insured cause of loss” and “do not understand the program” were not trivial. Finally, for almost all crop categories, “other” ranked as the primary reason for not insuring. This may imply that there is substantial “catch up” to be done for both growers and insurance providers—that more efforts are needed to inform growers about crop insurance and for authorities to learn the unique reasons why growers of particular crops do not purchase insurance. Table F3 provides the average ranking of suggestions to improve crop insurance. Suggestions listed were mostly related to compensation schemes.

For fruit/nut and vegetable farmers, “raising the yield guarantee,” “compensating for revenue or profit,” and “guaranteeing cash production costs” ranked high, grow bag for tomato while for ornamental growers, “compensating for revenue or profit” and “guaranteeing placement costs of an inventory” ranked high. For fruit/nut farmers, guaranteeing the cost of establishing an orchard was not as preferred as compensation of cash production costs, and a compensation scheme for ornamentals needs to be devised to accommodate their production systems because traditional yield-based production is not relevant to them. Overall, it was clear that specialty crop growers were more concerned with revenue and profit variability than they were with yield variability. This attitude is common among farmers in California’s irrigated agricultural industry. Recent research on crop insurance has consistently identified some level of demand, but that demand has been influenced by numerous factors . A decade ago, research focused primarily on yield risk as the key determinant of demand for crop insurance. Studies of that period focusing on specialty crops found that growers’ reluctance to insure was based on the fact that price variance was often more significant than yield variance . This prompted the first assessments of revenue insurance as an alternative . In recent years, revenue insurance has received wide attention. However, the few studies of specialty crop producers’ demand for revenue insurance have shown a need for more detailed, crop specific analyses of market and grower factors .The final section of analysis focuses on four financial variables: off-farm income share, gross agricultural sales, assets, and debts . Previous research has shown that these factors have a significant influence on farmers’ risk attitudes and, thus, on their risk management practices. For example, off-farm income supports most farms in the United States . The cushion from off-farm income makes many of those farms less sensitive to income risk , thus decreasing the demand for risk management tools .

In other words, off-farm income substitutes for other risk management tools to some extent. Figure G1 presents the distribution and mean of off farm income shares by crop category. The “share” refers to the percentage of total household income that comes from off-farm sources. The mean share for the entire survey was 63 percent . In general, there seemed to be a common pattern in the distribution for each crop category. Each distribution showed relatively heavy densities at the 1 to 10 percent range and then in the mid-range at 41 to 50 percent. The density started to increase at the 71 to 80 percent range. Note that the 91 to 100 percent range showed the highest density among all ranges for both fruits/nuts and ornamentals . However, the distribution of farms in the vegetable category deviated from the other two categories. The distribution of vegetable farmers showed greater density in the ranges with relatively low off-farm income shares, indicating that vegetable growers tend to spend less time on off-farm activities and get more of their income from farming than do fruit/nut or ornamental growers. Table G1 provides average values of gross agricultural sales, assets, and debts. Along with mean dollar figures, the table also reports the standard deviations in parentheses. There were substantial differences across crop categories. Consistent with the earlier findings on mean acreage, vegetable growers’ mean gross sales were much higher than those of other categories—nearly three times that of fruits/nuts and one and a half times that of ornamentals. The standard deviations of the mean gross sales were relatively large, indicating substantial variation in sales figures across farms. Nevertheless, judging from the values of the coefficients of variation, it was possible to infer that the variation in gross sales was less severe for vegetable farms. Vegetable operations also had the highest mean values for assets and debts. 

The reported mean values of assets and debts gave debt/asset ratios of 0.42 for fruits/ nuts and 0.50 for vegetables. More importantly, when viewing assets and debts as financial inputs necessary to generate revenue, the ratio of gross sales revenue to the sum of assets and debts was highest for vegetables and lowest for fruits/nuts. This implies that one unit of financial inputs is associated with a higher level of revenue for vegetables than for fruits/ nuts, or equivalently, one unit of revenue is associated with a lower level of financial inputs for vegetables than for fruits/nuts. This cursory observation may be linked to the relatively high intensiveness of financial inputs required, or the relatively low performance of financial inputs in fruit/nut production. The mean gross sales by region varied substantially. Gross sales data by crop category and by region indicated that the lowest gross sales were in the Far North region for both the fruit/nut and the vegetable categories, as expected because of those region’s lack of suitability for such crops . The highest mean sales for the fruit/nut category were the Central Coast – North’s $0.6 million ; for the vegetable category, the highest mean sales were the Sacramento Valley’s $1.8 million. Figure G2 provides the distribution of gross agricultural sales by crop category. The median and mean gross sales diverged considerably; the median was only about one-tenth of the mean value due to inclusion of some extremely high sales values for a few very large scale operations combined with the large number of small-scale farms. In the vegetable category, there were relatively higher proportions of farmers in higher sales ranges. The proportions of farmers with more than $1 million in sales were 6 percent for fruits/nuts, 29 percent for vegetables, and 13 percent for ornamentals. Figures G3 and G4 provide the mean gross sales by off-farm income share and by acreage class, respectively. Mean gross agricultural sales were negatively correlated Figure G2. Distribution of Gross Agricultural Sales Fruits and Nuts 5,001 and greater 35% 15% 10% 5% 0% 0–10 11–50 51–100 101–500 501–1,000 1,001– 2,000 2,001– 5,000 14% 17% 5% 26% 3% 2% 1% 25% 30% 20% 33% with off-farm income share and positively correlated with acreage, grow bag for blueberry plants confirming our expectation that higher agricultural revenues were generated by farms with larger acreage and farmers with less off-farm work. However, when sales revenue was computed as per-acre revenue, Figure G4 suggests that revenue per acre decreases as acreage increases. This is not counter-intuitive, given that specialty crops vary widely in unit value and the survey results indicated that smaller sized farms were, in general, associated with higher crop values.The main purpose of this report was to provide detailed and unique survey-based information on the fruit/nut, vegetable, and ornamental crop industries of California. The main findings from these survey data are as follows. California has fewer vegetable farms but, measured by gross sales and other dimensions, they are larger operations than fruit/nut farms are. Diversification increases with farm size, measured by acres. Fruit/nut farms are, on average, less diversified than vegetable farms, and when fruit/nut farmers diversify, they tend to use similar crops. About 6 percent of fruit/nut and vegetable farms have some organic land. These organic farmers represent 6 percent of fruit/nut farms, 14 percent of vegetable farms, and 4 percent of ornamental crop farms. Many of these farms also engage in conventional farming, and they devote, on average, about one-third of their primary crop land to organic farming. California farms tend to grow produce for either processing or fresh use but not for both. About 71 percent of the sampled fruit/nut farms produced mainly for processing use. About 67 percent of sampled vegetable farms produced mainly for fresh use. Contracts play a major role in marketing for specialty/ horticultural crops. They are particularly important in markets for crops designated for processing. Nearly 60 percent of fruit/nut farmers and 90 percent of vegetable farmers marketed their processing commodities through contract arrangements. The majority of these contracts provided for a predetermined price.

About 13 percent of vegetable farms but only 3 percent of orchard farms are grower/shippers. These farms tend to be larger than average and supply to mass merchandisers. Among the various channels, “directly to consumers” was used by the largest share of farms , but the farms tended to be smaller than average. Yield variability is an important risk factor for growers. Orchard and vineyard crop yields tend to fluctuate more than vegetable yields. Orchard and vineyard crop yields deviated an average of 15 percent for the five-year moving-average yield, compared to an average of 8 percent for vegetable crop yields. Despite considerable yield variation from year to year for these California crops, price variability is listed by growers as the most important risk source. Growers list price declines due to industry-wide overproduction as the number one concern. Growers use diversification and some marketing channels to manage risk. Crop insurance is less available for vegetable crops than it is for fruit, vine, and nut crops. Vegetable producers view crop insurance as a “less preferred” risk management tool. When asked about crop insurance programs, many farmers suggested that a “higher yield guarantee” would improve crop insurance. Further, most farmers strongly suggested the need for crop insurance that compensates in value terms, but they expressed no strong preference among compensations based on gross sales, profit, or production costs.The information provided in this study and the data set that underlies it will prove useful to agricultural business firms, including individual farms, as well as to government policy advisors and program designers. The study results provide a benchmark to industries that allows them to compare operations to the averages and medians for specific crops or locations. It also allows agricultural marketing and other service and supply firms to better understand their own potential supply and customer base for planning and product development. Such detailed data have not been available previously. The data are being used in risk management education efforts for growers and in summary form to provide objective data about grower operations and attitudes.The data and results also have implications for public policy and implementation of public policy, especially relative to risk management. Some examples are provided here. We find that many growers use crop diversification to smooth their revenue streams, but some growers find diversification more difficult or costly. Even if more diversified farms tend to have less variability in farm income, the degree and form of diversification affects the probability and magnitude of losses. The importance of diversification and its variation across specific industries points to the conditions under which yield insurance may be of interest and where it is less important to a farm’s annual revenue and thus less appealing as a risk management tool. The covariance between price and individual farm yield is another crucial piece of information in assessing farm revenue risk related to either price or yield variability. USDA’s Risk Management Agency has been developing whole-farm revenue insurance products.

Since 1990, UCCE researchers have used a farm budget software program to analyze the data and present results in several formats detailing costs for cultural and harvest practices, monthly cash costs and business and investment overhead costs. The studies also include an analysis estimating net returns to growers for several yield and price scenarios. Representative costs for food safety and environmental quality programs have been incorporated into more recent studies as they have evolved to become standard business practices. The resulting production and economic information is specifically designed to assist growers, bankers, researchers and government agencies with business and policy decisions. The first economic analysis of fresh market strawberry production for Santa Cruz and Monterey counties was performed in 1969; at least one subsequent analysis has been conducted every decade since then. Though the level of detail and data included in each study has changed over time, some interesting trends can be noted. Annual land rent climbed from $150 per acre in 1969 to $2,700 in 2014, representing 2.5% and 5.5% of total production costs, respectively. The cost of soil fumigation for conventional strawberry production increased from $350 per acre in 1969 to $3,302 in 2010, representing 5.5% and 6.9% of total production costs, respectively. Production year water use gradually decreased from 80 acre-inches per acre in 1969 to 36 acre-inches by 1996 as drip irrigation became the standard. The amount of water used to bring a crop to harvest has remained roughly the same since that time; however, growers and researchers continue to investigate methods to increase water use efficiency even further. In some areas, grow bag soil types and fields, growers have been able to reduce per acre water use by several acre-inches more .

When the above costs and water usage are assessed on a per ton rather than a per acre basis, production practice cost increases are less notable, and water savings even greater. Labor-intensive practices such as hand weeding and harvest are consistently shown as costly line items relative to other operations. Representative yields for conventionally produced fresh market strawberries rose from 20 tons per acre in the 1969 study to 30 tons in 2010, an increase of 50%. Even higher yields are discussed for some varieties and production conditions; county production statistics confirm that higher yields are indeed possible . Representative yields for organic strawberries, studied over a much shorter time period, rose from 15 tons per acre in 2006 to 17 tons in 2014, an increase of 13%. As more research is directed towards organic agriculture in general and strawberries in particular, yields will likely increase even more with time. Recent efforts include improvements in cultivar breeding, cultural practices and disease management, especially soil pathogen management. The most recent economic analyses for conventional, second year conventional and organic strawberry production were performed in 2010, 2011 and 2014, respectively. Second year conventional strawberries, or those producing a crop for a second year after having produced the first without replanting, represent about 15% of the total strawberry acreage in the area. Similarities and differences in total, cultural and pest management costs for the three management approaches are shown in figures 1 to 3. Total costs for conventional strawberries were $47,882 per acre and include expenses for all practices from land preparation to harvest . For the second year conventional strawberry crop, total costs were lower at $32,798 per acre, reflecting a reduction in expenditures for land preparation and reduced harvest costs because of lower yield. For organic strawberries, total costs were $49,044 per acre, slightly higher than for conventional production, mostly due to higher soil fertility input costs.

Harvest, a labor-intensive practice, clearly represents the lion’s share of total costs, at 58% in organic production, 60% in conventional production and 67% in second year conventional berries. Cultural costs represent 26% of total costs in the conventional and organic systems, but only 15% for second year strawberries because there were no associated planting costs, and because pest management costs were lower . Looking more closely at pest management, soil fumigation is the highest cost category for conventional production at $3,302 per acre, with weed control, another labor-intensive practice, the highest cost in second year and organic strawberries at $1,212 and $2,506 per acre, respectively . However, for organic strawberries the cost to control insects ran a close second at $2,488 per acre, which was dominated by control for lygus bug with a bug vacuum, and two-spotted spider mite with the release of predatory mites. By comparison, estimated costs for insect control in conventional strawberries were lower at $702 per acre and still lower at $579 in second year conventional berries. Raspberry and blackberry production were not routinely studied in years prior to 2003. Since then, several primocane-bearing raspberry and floricane-bearing blackberry cost and return analyses have been performed, with the most recent studies conducted in 2012 and 2013, respectively. Both studies detail establishment and first year production and harvest costs for not-yet-fully-mature crops. For raspberries, first year of production includes a $12,460 per acre construction, management and investment cost for protective tunnels. Costs for a mature raspberry crop are analyzed in the second production year and total $48,210 per acre . For blackberries, costs for a mature crop are shown for the second through fifth production years, and total $43,406 per acre per year. Harvest costs again represent the vast majority of total costs, at 81% and 71% of total costs for raspberries and blackberries, respectively.

For raspberries, cultural costs represented a much smaller share of total costs at $4,656 per acre, roughly half of which was for trellis and tunnel management. Blackberry cultural costs totaled $5,709 per acre, of which over half was for pruning and training canes. Each study also includes an analysis of potential net returns to growers above operating, cash and total costs for a range of yields and prices. When evaluating net returns above total costs, gains are shown for higher yield and price points; losses are also documented at many lower yields and prices . Farms with productive soils, experienced managers, optimal production conditions and robust market plans generally realize higher net returns. In contrast, farms with less-than-optimal production conditions, reduced yields, poor fruit quality or inexperienced managers may contribute to lower net returns. Results from the strawberry analyses show that on a per acre basis, organic strawberries tend to be more profitable than conventional berries, even with lower yields. Organic price premiums explain the result; in this example price per tray for organic strawberries ranged from $12 to $18, while price per tray for conventional berries ranged from $7.30 to $11.30. Prices for second year conventional strawberries were slightly lower still to account for a portion of the crop that was diverted to the freezer market. Net returns for both caneberries were mostly positive. Other noteworthy entries in all recent berry studies include per acre costs for pest control advisers , management of invasive pests and food safety and regulatory programs for water and air quality. Though each alone represents a relatively small portion of total costs, they provide readers with insights into the changing nature of berry production activities and costs over time.Cultural practices in the berry industry have evolved to address changes in soil, water and pest management needs. New varieties have been developed to enhance yield and quality attributes. Based on historical trends, and to meet both industry needs and consumer demands, grow bag gardening we expect to see new varieties continually developed over time. Businesses have responded to consumer and market demands for fresh, safe and organic products by implementing food safety programs and/or transitioning more lands to organic production. Water and air quality programs have been developed to comply with state regulatory requirements. In the past, growers customarily hired those with expertise in financial and market management; they now also enlist the support of experts in food safety, organic agriculture and environmental quality to assist with farm management. But challenges remain, and management of key agricultural risks — including those for production, finances, marketing, legal and human resources — have become increasingly important. Invasive pests pose significant management and regulatory constraints and increase production, financial and market risks. Two recent examples are light brown apple moth and spotted wing drosophila . LBAM infestations can lead to loss of part or all of the crop because of field closure from regulatory actions, increasing production and financial risk. SWD presents substantial market risk to growers in that its larvae can infest fruit and render the crop unsaleable. Growers minimize the risk of loss from these two organisms with the routine use of PCAs. PCAs monitor fields more frequently than growers alone would be able to do, identify pests and recommend actions, for example, the use of pheromone mating disruption for LBAM and field sanitation for SWD. Since their introduction, the soil fumigants CP and MB have unquestionably contributed to the expansion of the berry industry.

However, the full phaseout of MB as a pest management tool — it will no longer be available for use in berry production after 2016 — presents both production and financial risks. While a substantial research commitment has been made to finding alternatives to MB, nothing has yet come close to offering the same level of protection from the large-scale loss to soil pathogens or the gains in productivity associated with the application of CP and MB as synergistic preplant fumigants. We anticipate that the berry industry will adapt to the MB phaseout by using alternative fumigants and preplant soil treatments, but these are likely to carry a higher level of risk for berry production in the short term and may lead to a decrease in planted acreage and production. However, this may also stimulate an even more robust research agenda directed towards soilborne diseases and plant health to minimize disruption to the industry. Reliance on fumigants as the primary strategy for pest management is almost certainly a thing of the past. Instead, adoption of integrated approaches, including alternatives to fumigants, to manage diseases, weeds and other pests will be key to sustaining berry production over the longer term . Social and demographic changes in Mexico — the source of a majority of the area’s agricultural labor — have resulted in markedly lower immigration rates into the United States, a shrinking labor pool and upward competition and wage pressures for the agricultural workers who remain . In recent years, growers have reported difficulty in securing and retaining sufficient numbers of workers to ensure timely and effective farm operations. The lower production figures seen in strawberries in 2014 may in part have been the result of an insufficient labor pool from which to draw . However, no known regional employment or wage data are available to specifically document this. Some growers minimize labor risk by paying higher wages and providing year-round employment when possible. However, these strategies can be difficult for some businesses to justify economically. Arguably, the area’s berry industry, and agriculture more generally, increasingly face political risk. Immigration legislation that may assist with the current labor challenge languishes at the federal level, with major policy changes unlikely before 2017 . Farming practices are under ever more scrutiny by consumers, local municipalities and state and federal agencies. Soil fumigants and pesticide use have been the focus of many intense debates and discussions, especially in Santa Cruz and Monterey counties. At the time of this writing, several new regulations related to pesticide application notifications, pesticide and fumigant application buffer zones and worker safety have been proposed by the California Department of Pesticide Regulation or the U.S. Environmental Protection Agency but have not yet been finalized. It is anticipated that implementation will begin in 2017, with full compliance required in 2018. And, as California struggles through a fifth year of drought, water use, quality and cost has become a more robust part of the local, state and federal discourse, with directives issued and new legislation proposed. Compliance with each new directive or regulation presents production and logistical challenges for growers and can be costly to manage. Although it is unlikely that regulatory pressures will lessen in the future, there is every expectation that growers will continue to adjust business practices to meet or exceed any new requirements or standards.

In East Oakland’s Fruitvale District, the Coalition for Healthy Communities and Environmental Justice joined with PUEBLO, the Center for Environmental Health, and Greenaction, and triumphed in 2001 after a four-year battle to shut down the Integrated Environmental Systems medical waste incinerator which had been polluting since the early 1980s . While the movement against these industries was sometimes fractious due to disagreements over potential job loss, the coalitions were ultimately strong Unlike the Black Panther Party’s Food Program, nothing about the EJ movement spoke directly to the issue of food access. What the EJ movement did provide, however, was training in the trenches for a generation of activists. It mobilized community members to act; victories cultivated a sense of empowerment and reclaimed a political voice that had been silenced by decades of flatlands devaluation, while failures underscored the importance of ongoing resistance. The EJ movement also drew attention to the flatlands and to the injustices that have produced them as a social and ecological space. Importantly, the movement fostered and galvanized alliances between policy and research intermediaries and community-based organizations and neighborhood residents. Alliances such as these would be central to the success of the urban agriculture and food justice movement that was slowly beginning to coalesce in the flatlands at the same time. A pivotal moment connecting EJ to what would become the food justice movement occurred around the same time and involved a theoretical shift in the way that struggles over race, poverty, and environment were framed.

A new “spatial justice” framework helped to highlight the interrelations between racial and economic segregation, built environment, square black flower bucket and access to entitlements such as healthy food, clean air and water, and open space. This new theoretical framing was forged in large part through the efforts of Carl Anthony and Karl Linn. By the early 1990s Anthony had become a prominent voice in the Bay Area EJ movement. Like other EJ activists, he attempted to shift the attention of the mainstream environmental movement towards urban areas, and fought to overcome what he termed the “apartheid of consciousness”—the belief that social and environmental issues were somehow distinct—keeping inner-city people of color and white suburban environmentalists from joining forces to tackle environmental issues. Studying architecture at Columbia University in the 1960s while working as a civil rights activist on the side, Anthony began to think about the relationships between social justice and the built environment. He later became involved in the “community design” and “advocacy planning” movements, both of which emphasized moving the process of urban planning and design out of the hands of technocrats and into those of lowincome communities . In the late 1980s, Carl Anthony reconnected with Karl Linn, a landscape architect who had led a long and productive life as a farmer, psychologist, landscape architect, and educator on three continent. The two were old friends, having met in North Philadelphia in the early 1960s when Linn was teaching landscape architecture at the University of Pennsylvania. Through his “community design-and-build service education program” Linn and his students worked with community members in ramshackle neighborhoods and vacant lots throughout the city. He was later instrumental in the community gardening movement of the 1970s and was a founding member of the American Community Gardening Association . Anthony credits Linn with giving him “some sense that you could actually put together a social agenda and an environmental design agenda” . When Linn moved to Berkeley in 1986 upon his retirement, the two joined forces to expand awareness within the white environmental world of the issues of social, racial, and economic justice that were at the forefront of concern for people of color.

The underlying structural conditions of the flatlands—the demarcated devaluation I described in the previous chapter—proved fertile ground in which a productive synthesis of the theories and activism of the two men could take root. Until this point, environmental groups, many of them located in the Bay Area, focused primarily on struggles to conserve wilderness areas at all costs, often conflating subsistence resource use by indigenous peoples with large-scale capitalist resource extraction. Linn urged Anthony to connect with David Brower and other white environmentalists, some of whom were supporting social justice struggles in the Global South. On Linn’s urging Anthony joined the board of Brower’s Earth Island Institute, provided that that he “could create a program that would really address the environmental issues from the perspective of social justice” . In a 2003 oral history, Anthony remembers, “What we found was that every environmental issue was also a social justice issue. As we began to get into it, we could see the connections … We had to have more of a sense that these issues have to be together” . In 1989 Urban Habitat was born. Most of the justice-oriented urban agriculture efforts cropping up at the time were concentrated in flatlands of southwest Berkeley just across the city limits from Oakland. The majority of these projects pushed the boundaries of conventional community gardening by emphasizing youth employment and food security. These efforts, which predominantly employed young African Americans, helped to increase the involvement of people of color in urban agriculture. Shyaam Shabaka, a PCGN member and co-founder of EBUG, and Melody Ermachild Chavis, a white neighborhood activist, founded Strong Roots in 1994. Shabaka had spent time working on a horticulture project in Mali and hoped to reconnect African Americans with “the lost agricultural heritage that’s rightfully ours” . The Strong Roots motto was “Gardening for Survival” and employed fourteen youth at six gardens throughout Berkeley, including at a vacant lot at the corner of Sacramento Ave. and Woolsey St. that was home to drug deals and drive-by shootings. Funding came in part from the federal Summer Youth Employment and Training Program before it was axed by the 1995 budget under Newt Gingrich’s Contract for America. Other funding came from a federal substance abuse prevention program . A host of similar programs cropped up at the same time, focusing on youth employment and training.

Berkeley Youth Alternatives Director Niculia Williams and UC Berkeley Landscape Architecture student Laura Lawson started the BYA Garden Patch as an alternative to the fast food breakfasts that most of the children attending BYA’s programs were eating. In 1994 the garden was established with the labor of community members, AmeriCorps and East Bay Conservation Corps volunteers, and UC Berkeley students. Through the ‘90s it grew to include community garden plots and a Youth Market Garden that provides youth with employment and on-the-job training and the organization with revenue. By 1998 the Youth Market Garden had earned more than $10,000 in sales. Cut flower sales added to revenue, as did a twenty-five member sliding scale CSA . In 1993, the same year as the BYA Garden Patch was planned, Spiral Gardens was created “by a handful of individuals dedicated to urban greening, innovative organic farming methods, food security, and environmental justice issues” on Sacramento Avenue in South Berkeley, across the street from the Strong Roots garden . A project of the Agape Foundation for Nonviolent Social Change, the organization grew vegetables, herbs, and native plants for sale, in addition to offering community gardening plots and horticulture workshops. One of the founders, Daniel Miller, also ran the Urban Gardening Institute, a garden based job training and microenterprise program for people enrolled in a drug rehabilitation program and transitioning from homelessness. The program was run through Building Opportunities through Self-Sufficiency at several homeless shelters, residential hotels, and community gardens. The two programs merged in 1997 and in 2004 became a 501 nonprofit called the Spiral Gardens Community Food Security Project . Berkeley’s justice-oriented urban agriculture activists also gained inspiration and material support from a growing national movement that brought together anti-hunger, square black flower bucket wholesale sustainable agriculture, farm labor, environmental, and health and nutrition activists . In the summer of 1994, the Community Food Security Coalition formed and drafted their equity-based vision for integration into the Farm Bill. While most of their recommendations failed under a Republican-controlled Congress, the 1996 Farm Bill included a provision to provide annual funding for projects that would “meet the needs of low-income people, increase the self-reliance of communities in providing for their own needs; and promote comprehensive responses to local food, farm, and nutrition issues” . These Community Food Project Grants would play a role in the East Bay over the next decade, some destined for school gardens, others to developing local community food security gardens. Alliances with CFSC activists also helped to galvanize the fledgling justice oriented urban agriculture movement by linking activists in the East Bay to a larger national network that shared ideas, information, and other resources through newsletters, conferences, working papers, small grants, and email list-serves, once again opening up new spaces of engagement to defend spaces of dependence, first in Berkeley’s flatlands and later in Oakland. Berkeley essentially served as a hub of urban agriculture innovation, attracting activists and organizations that were, in turn, able to marshal public and private funding necessary to sustain the equity-oriented urban agriculture activity.

Indeed, the food security and youth employment projects in South Berkeley were mere blocks from the boundary of North Oakland. Many of the young activists involved in urban agriculture at the time actually lived in Oakland where rent was cheaper. One former activist working in one of the South Berkeley gardens blames changes in rent control in Berkeley for his move to West Oakland in the mid ‘90s; in 1995 the passage of a state law, AB 1164, allowed landlords in Berkeley to raise rents when units became vacant and many young activists were simply priced out of Berkeley.By the early 1990s several school gardens had sprouted up. A few of these were in Oakland, but like the community gardens, the nexus of school gardening activity in the Bay Area was in Berkeley. Ground was broken at Willard Middle and LeConte and Malcolm X Elementary Schools. These new gardens were by no means the first in Berkeley’s history. A 1918 history of Berkeley’s public schools dedicates a short chapter to the school gardens that were used to “provid[e] vital contact with the facts and forces of nature” and “to teach children order, industry, respect for labor, and thrift, besides a love and sympathy for the wonderful and beautiful” . 82 While the emphasis three-quarters of a century later was perhaps less about industry, labor, and thrift, fostering a love for nature was surely still on the agenda. Perhaps new to the garden-based curriculum was an emphasis on nutrition. In March 1997, another CUESA conference helped to galvanize the importance of urban gardens in the East Bay as well as draw national attention—and funding—to the area’s fledgling school garden initiatives. Like the previous conference that helped bring an emphasis on social justice into the urban agriculture discourse, this event helped to emphasize the linkages between urban agriculture and nutrition. Held at MLK Middle School, “A Garden in Every School: Cultivating a Sense of Season and Place” was intended to cultivate a vision of fresh and nutritious food for all school children, and brought school system officials, teachers, planners, and gardeners under the same roof. CUESA Director Sibella Kraus recalls, “The thinking was that a high end farmers market in San Francisco is making a difference to some people, but not to others…. We thought we’d maybe get thirty people or fifty, but we got 900 people! It completely sold out. People were just really ready for it to happen” . The event coincided with the establishment of the Edible Schoolyard at the school. Founded by Chez Panisse’s owner Alice Waters, the Edible Schoolyard incorporates garden- and cooking-based education, connecting fresh food to healthy lunches. The program has been widely lauded and replicated nationally, and has become a model for revamping the school food system. The parents of school children were also central to the expansion of school gardens, and urban agriculture more broadly. Beebo Turman, a pre-school teacher, parent, and backyard gardener met with Alice Waters and “six or eight other parents” at a Parent-Teacher Association meeting at King Middle School in 1993 and began organizing, writing grants, and fundraising to get the Edible Schoolyard up and running.

In an often-cited example, the expansion of capitalist agriculture in Europe and North America led to a soil fertility crisis during the 19th century. A mad dash for new sources of fertility ensued, notably for South American guano and saltpeter, and a nascent synthetic fertilizer production industry developed. The scramble to locate new sources of fertility drove imperialist expansionism which ultimately displaced the metabolic rift elsewhere . As Engels explained in the late 19th century, each technological triumph over nature leads to other crises: “For each such victory takes its revenge on us. Each victory, it is true, in the first place brings about the results we expected, but in the second and third places it has quite different, unforeseen effects which only too often cancel the first” . These short-term technological fixes inevitably generate new metabolic rifts, amounting to “a shell game with the environmental problems [capitalism] generates, moving them around rather than addressing the root causes” . However, this shell game is not just a matter of space, but also a matter of scale. While a rift in a particular metabolic process occurs at a particular scale, social metabolism of nature continues at new spatial and temporal scales as production is relocated or becomes dependent on new inputs. Capitalist rationalization of agriculture arose from the pursuit of new markets and from the need to avert crises of production, such as falling rates of profit due to competition, a decline in availability of raw materials, flower buckets wholesale or environmental pollution and declining worker health resulting from production practices . These shifts in production severed particular metabolic interactions.

The separation of animal and crop production in industrial farming systems, for example, ruptured cycling of nutrients at the farm scale, leading to an increased reliance on off-farm inputs, such as fertilizers and feed shipped in from other regions. This rift in nutrient cycling therefore resulted in a rescaling of social metabolism; put simply, the inputs necessary to sustain human life under this new production system came from farther and farther away. Sustaining social metabolism under a food production system that depletes rather than regenerates the resource base depends not only on such spatial rescaling, but also on temporal rescaling. Rescaling requires what ecologists refer to as spatial and temporal “subsidies” to the food web , inputs that are produced on a different geographic and/or time scales. Since a subsidy is cross-scalar , its incorporation into a metabolic system inherently creates a new ecological rift as it is depleted; it is impossible to close the loop between the source and sink of such a cross-scalar subsidy. During the aforementioned crisis in soil fertility, for example, guano and nitrates were mined from decades- and centuries-old deposits from Peru and Chile, then transported across oceans to Europe and America . Replenishing these stocks would have been impossible within the span of a single cropping season, much less within the span of a human life. Once guano stocks were exhausted, agribusiness interests turned to synthetic fertilizers. The natural gas and petroleum needed to produce synthetic fertilizer and power tractors is millions of years-old, drawn from gas fields and oil wells around the globe and shipped to factories and refineries before being used thousands of miles from the point of extraction.

It becomes easy to see how ecological rift scales up, making social metabolism a global affair, dependent on millions-year-old subsidies from tens of thousands of miles away. If, as Huber argues, fossil fuel use is “an internal and necessary basis to the capitalist mode of production,” ecological rift and the resulting spatiotemporal rescaling of social metabolism is internal and integral to the contemporary agri-food system. Relocalizing these nutrient cycles and reducing dependence on petroleum-based food production lie at the heart of urban agriculture’s potential to mitigate metabolic rift. British agronomist Sir Albert Howard , concerned that organic wastes were rarely cycled back to their point of origin in large-scale agriculture, plaintively pondered, “Can anything be done at this late hour by way of reform? Can Mother Nature secure even a partial restitution of her manurial rights?” . While unclear if he was aware of Marx’s views on social metabolism , Howard echoed the concerns of Liebig, Marx, and Engels. Noting that “the Chinese have maintained soil fertility on small holdings for forty centuries” and inspired by the traditional farming practices he witnessed around him in the colonies, Howard championed compost use over chemical fertilizers and pondered a possible transformation of the industrial model where waste would be cycled back to farmland. In this same tradition, mending ecological rift via the recycling of organic waste is central to urban agriculture across the globe. This concept of returning nutrients to agricultural soils in the form of urban waste is vital to overcoming the “antithesis between town and country” and is fundamental to a “restitutive” agriculture. While few urban planners and mainstream development practitioners likely look towards Marx and Engels for inspiration, these obscure passages describing metabolic rift are particularly prescient, relevant not only to the development of sustainable agriculture, but also to urban waste management and the impending environmental crises of mega-urbanization . For millennia, farmers worldwide have maintained soil fertility on small plots through the application of organic waste; urban farmers are no exception. Adapting to the rising cost of chemical fertilizers and stagnant market prices for their produce, urban farmers in many parts of the South rely on intensive applications of manure from urban and peri-urban livestock production, ash, and composted garbage as a free or low-cost fertilizer and soil conditioner.

Periurban livestock producers, in addition to tapping rising urban demand for meat, dairy, and eggs, sell manure to urban market gardeners and to large-scale vegetable farms in the urban outskirts. To profit from compost’s fertilizing potential, farmers frequently cultivate the peripheries of garbage dumps or establish illicit contracts with garbage truck or cart drivers to obtain compost for their fields, paying them to simply dump a load of garbage in their fields while en route to central collection facilities. Advocates argue that redirecting the organic fraction of waste streams to agricultural production in urban areas and their hinterlands will help to boost soil fertility, as well as reduce soil and water pollution arising from heavy agrochemical use and large concentrations of waste deposited in landfills, dumps, and waterways . Yet to truly close the nutrient cycle and diminish the impacts of this ecological rift, human waste from urban consumers would need to be returned to the crops’ fields of origin. Every day, on average, every human produces 1 to 1 ” kg of nutrient-rich feces. Human waste, or “night soil”, is a common source of organic fertilizer in urban and peri-urban agriculture, though less commonly promoted due to cultural biases and to the higher public health risks associated with its application. Despite the social stigma, foul odor, and contamination risk of its use, there is stiff competition among farmers for access to night soil. Inone study, two-thirds of farmers surveyed in two peri-urban zones in northern Ghana used human waste in their fields . In China, in particular, flower harvest buckets application of human waste to farmland has been central to both urban waste management and agricultural production, but has been diminishing as rapid industrialization and urbanization transform agricultural production at the urban edge . While such forms of restitutive soil fertility management In the Global South generally arise from creative exploitation of limited resources and adaptation to limited access to land, fertilizer, and credit, they have been celebrated by urban farming advocates worldwide as fundamentally sustainable practices. In North America and Europe, where the discourse of ecological sustainability generally informs urban agriculture practice, the age-old nutrient cycling practices used in the Global South are a cornerstone of urban agriculture advocacy. Practices such as compost application, planting of nitrogen-fixing cover crops, and incorporation of crop residues are presented as a sustainable way to close the nutrient cycle and reduce urban ecological footprints. Indeed, application of compost to urban soils can also provide other environmental services, such as reducing erosion, improving drainage and water holding capacity, controlling pathogens, and immobilizing heavy metals. For commercial growers in peri-urban areas, a growing consumer demand for local and organic food often drives the transition to more ecologically-sound farming practices. A growing number of municipalities collect green waste for composting. Much of the compost is sold at low cost or provided for free to local farmers, landscapers, and gardeners.

Infrastructure for the collection, composting, and distribution of compost seems to be the greatest hurdle preventing urban agriculture’s ability to minimize ecological rift in nutrient cycling. Nevertheless, development workers and planners are optimistic about its role and argue that with improved waste management technology, access to land, and policies favoring agricultural production in urban areas, urban agriculture can contribute significantly to feeding the world’s cities and mending ecological rift by restoring “Nature’s manurial rights”, rescaling production to a more local level, and relying less on petroleum-based inputs and other crossscalar subsidies.Understanding this social rift is not only essential to explaining urbanization, but to elucidating the linkages between urbanization and the agri-food system. The rise of large- and industrial-scale farming has entailed the consolidation of land and expansion of mechanization and other new farming technologies, both of which reduce the demand for agricultural labor. This was evident in Europe at the dawn of the capitalist era, in the US during the latter half of the 20th century , and more recently in China where as many as 70 million farmers were dispossessed by expanding land markets in the last decade of the 20th century . In the Global South, a host of pressures—structural adjustment programs, land consolidation, drought, war, expansion of natural resource extraction and biofuels plantations—has dispossessed rural populations over the last several decades and fueled the growth of megacities and their slums across the globe . Indeed, as Marx predicted, “Part of the agricultural population is therefore constantly on the point of passing over into an urban or manufacturing proletariat” . Social rift is a central driver of urban agriculture in the Global South, where production of food is often a subsistence activity. Between 70 and 75 percent of farmers in a survey of urban agriculture in Nairobi, for example, produced for household consumption, citing hunger and the need for food as their principal motivation . Similar rates have been found in other parts of Africa, with lower rates in Asia, and Latin America . A recent FAO study revealed that over 30 percent of households in 11 of the 15 countries studied engage in some form of urban agriculture. The results also showed the urban poor are more likely to practice urban agriculture than wealthier city dwellers . Many must therefore improvise new means of survival, particularly in those cities where social services were gutted under structural adjustment during the 1980s and ‘90s. Many embark on small-scale agriculture on marginal plots of land tucked in between housing, industry, and infrastructure, within the city itself or in its immediate hinterlands, in order to buffer themselves from the socio-economic upheaval of dispossession from their land and from the lack of formal employment opportunities in the city and its peripheral slums. The slashing of government jobs under structural adjustment in many parts of the Global South also drove members of the urban professional class to embark on urban agriculture projects to augment their diets, and for those selling on informal local markets, to supplement their income. According to Guyer subsistence and small-scale urban food production, along with the informal food economy to which it contributes, often undermine the expansion of more formal markets. At the same time, however, self-provisioning effectively subsidizes the cost of social reproduction within the larger capitalist economy ; in short, wages can stay lower if workers are feeding themselves, ultimately facilitating the accumulation of capital.29 Urban agriculture therefore exists in tension with capital, arising as a strategic response to social rift on one level by exploiting underutilized land and buttressing against the expansion of commercial agri-food markets in poor areas, while subsidizing ongoing accumulation on a more macro-level.

A few isolates from cultures inconsistent with Bot. characteristics were randomly selected from each site to amplify and sequence to verify our morphotyping method. The internal transcribed spacer region 1 and alpha-elongation factor-1 genes were amplified using PCR primer pairs ITS1F and ITS4, and EF1-728F and EF1-986R, respectively, using methods modified from White et al., and Slippers et al . Successfully amplified samples were sequenced using the UC Berkeley Sequencing Facility .The severity of Bot. infection was calculated as the isolation frequency per site. Data were square-root transformed when necessary to meet the assumptions of normality. Differences in mean Bot. infection severity between elevation categories were calculated using one-way ANOVA with Tukey’s HSD for post-hoc analysis with R Statistical Software . Correlations between actual elevation and Bot. infection severity were assessed using simple linear regression and ANOVA to test for significance . Generalized linear models were developed to identify patterns of dieback, with dieback severity values as the response variable, and elevation , Bot. infection severity , and aspect as possible explanatory variables. If multiple models received substantial support , the best model was confirmed by calculating the relative importance of each term based on the sum of their Akaike weights . This study provides definitive support for the hypothesis that shrub dieback, during a recent drought, and pathogen infection are strongly related in a wild shrubland setting. This is the first known quantitative support for the hypothesis that in A. glauca, an ecologically important shrub species in the study region, wholesale plant containers dieback is related to pathogen infection occurring along an elevational gradient.

As expected, N. australe and B. dothidea were the two most frequently retrieved pathogens across all sites, however N. australe, the introduced pathogen, had almost twice the abundance of B. dothidea. N. australe is driving the correlation between elevation and Bot. infection, as the frequency was greater at lower elevations compared to upper elevations, while B. dothidea abundance did not change significantly across elevations. Level of Bot. infection was confirmed to be a significant predictor of stand-level dieback severity. The data also confirm that stand dieback severity is generally greater at lower elevations, which in this region experience higher temperatures and lower annual rainfall than the higher elevations sampled. While the presence of Bot. species has been reported previously in Santa Barbara County, this study represents the first effort to understand the abundance and distribution of Bots occurring in natural shrublands, and the first wildland shrub survey of Bots across a climate gradient. The high frequency and wide distribution of Bots retrieved from our study sites support the hypothesis that Bot. species are widespread across a natural landscape, and likely contributing to the extensive dieback resulting from the recent drought. Bot. fungi were retrieved from nearly every site in this study . We could not determine Bots. presence from three sites due to contamination issues. The broad extent of the study area suggests that infection is widespread in the region, and likely extends beyond the range of our study. While both N. australe and B. dothidea together made up the most frequently retrieved pathogens, our data show that N. australe has a larger distribution and occurs in greater abundance across the study region than B. dothidea . This trend was consistent across all elevations, but particularly at lower elevations . One possible explanation for this is that N. australe, being a recently introduced pathogen, spreads more rapidly as an exotic species in A. glauca compared to B. dothidea, which has been established in California for over 150 years .

This hypothesis is consistent with previous studies that have shown variations in Bot. species abundance and virulence in Myrtaceous hosts occurring in native versus introduced ranges . However, it is difficult to evaluate the incidence of B. dothidea and N. australe in the present study in relation to historical documentation since many species in the Bot. complex have, until recently, been mischaracterized . Only with the recent development of molecular tools have researchers begun to accurately trace phylogenetic and geographic origins of Bot. species. Such studies are beginning to elucidate the complex existence of Bot. fungi as both endophytes and pathogens around the world, and much more research is needed to understand their pathogenicity in various hosts under different conditions. Nevertheless, it remains clear from our study that Bot. species, particularly N. australe, are both abundant and widely distributed in this region, and are important pathogens in A. glauca shrubs.Because Bot. taxa were the most frequently retrieved pathogens and were significantly correlated with dieback, we believe that they drive A. glauca dieback. Further, stand dieback severity increased significantly with Bot. infection. This is not to say that other pathogens do not also contribute to disease symptoms, but we found no evidence of any other pathogens occurring in such high incidence as Bot. species. While Brooks and Ferrin identified B. dothidea as a likely contributor to disease and dieback in dozens of native chaparral species during an earlier drought event in southern California, and Swiecki and Bernhardt found B. dothidea in association with a dieback event in stands of Arctostaphylos myrtifolia in northern California, our study yields the most extensive results of Bot. infection and related dieback in a chaparral shrub species across a landscape.

Further, our study resolves species identity within the Bot. clade and highlights the role of the recently introduced pathogen, N. australe.A significant finding in this study was the relationship of Bot. infection and dieback with elevation. Bot. abundance and dieback were both found to be greatest at lower elevations, which was driven mostly by the high frequency of N. australe retrieved at these sites. This represents the first quantitative evidence supporting that A. glauca vulnerability to fungal infection is influenced by stress levels along an elevation gradient. A similar pattern was observed in northern California by Swiecki and Bernhardt , who suggested that dieback in Ione manzanita infected with B. dothidea was greater in drier sites compared to more mesic ones, although no comparison of infection rates between sites was conducted in their study. The elevation gradient in our study was used a proxy for stress levels because annual precipitation decreases with decreasing elevation within our study region . Higher temperatures, which are associated with lower elevations, are also known to play an important role in drought-related mortality, as water loss from evapotranspiration is increased . Furthermore, unpublished data for dry season predawn xylem pressure potentials on a subset of sites along the same elevational gradient revealed more negative water potentials in A. glauca at lower elevations compared to upper elevations as spring and summer drought sets in . Thus, there is evidence that shrubs at low elevations indeed experienced the greatest water stress during the 2011-2018 drought, which predisposed them to higher levels of Bot. infection and enhanced dieback compared to upper elevation sites. More in-depth studies on the microbial communities and fungal loads of healthy and diseased shrubs throughout the region would help elucidate such trends. Another possibility for the higher incidence of Bot. infection at lower elevations is that the lower ranges of A. glauca populations in Santa Barbara are often located adjacent or in close proximity to agricultural orchards, ranches, and urban settings, which are common sources of plant pathogens, including Bots . Eucalyptus, avocado, and grapevines, which are abundant in these areas, are particularly well-known Bot. hosts and potential facilitators of Bot. introduction . Therefore, sources of inoculum from nearby populations of agricultural and horticultural hosts could be responsible for continual transmission Bots in wildland A. glauca populations, and would likely result in greater rates of infection at lower elevations. Furthermore, many of the lower sites in the survey were located near roads and/or trails, which are often subjected to additional stress from human activity like pruning and trail clearing; activities that are known to spread and promote infection by Bot. pathogens . While we avoided sites that showed signs of such activities in our survey, we cannot rule out the potential contributions of proximity to human encroachment to the overall higher rates of Bot. infection across the lower elevation zone. It is worth noting that while our study revealed a trend of increased dieback in lower elevations, some upper elevation sites also exhibited high levels of dieback, and Bot. fungi were retrieved from many of these sites. Upper elevations also experienced significant stress during the 2011-2018 drought, plastic pot manufacturers and water-related microsite variables outside the scope of this study like slope, solar incidence, soil composition, and summer fog patterns factors likely contributed to increased stress and subsequent dieback. Additionally, N. luteum, N. parvum, and D. sarmentorum were isolated primarily from upper sites. Host plants in these sites may serve as potential reservoirs for disease because the milder climate conditions promote greater host survival and thus pathogen persistence as endophytes. This serves as an important reminder that continued global change-type drought may eventually jeopardize susceptible species populations even at the upper boundary of their range.Our results are consistent with well-known theoretical models describing the relationship between environmental stress and biotic infection, which generally ascribe extreme drought stress as a mechanism for plant predisposition to disease .

These frameworks illustrate dynamic interactions between environmental stress, plant hydraulic functioning and carbon balance, and biotic attack, and a growing body of research has focused on understanding the roles of these factors in driving plant mortality, especially during extreme drought . While the data collected in this study do not directly address the specific mechanisms leading to Bot. infection and dieback in A. glauca, our results can be discussed in the context of how life histories and physiological adaptions elicit differential responses to drought in woody plants, particularly in chaparral shrubs. For example, shallow-rooted, obligate seeder shrubs like A. glauca have been shown to be more susceptible to drought-induced mortality during acute, high intensity drought than deep-rooted, resprouter shrubs . This supports our observations of pronounced A. glauca decline during an historic California drought compared to nearby resprouter species like chamise , and laurel sumac . Additionally, physiological mechanisms related to drought tolerance may further explain predisposition to disease in A. glauca. For example, high resistance to cavitation is a common trait associated with more dehydration-tolerant species like A. glauca that maintain hydraulic conductivity during seasonal drought . While cavitation resistance is thought to assist in the continuation of photosynthetic activity even at very low seasonal water potentials , it has also been associated with greater mortality rates during high intensity drought in a variety of woody plant systems including mediterranean shrublands , temperate deciduous forests and eucalyptus forests . High resistance to cavitation requires heavy carbon investment for stronger and denser stem xylem tissue , which can result in limited carbon for investment in defense against pathogens like Bot. fungi. Furthermore, colonization of pathogens during drought may further disrupt the carbon balance of plants as it influences defense and repair, creating a feedback loop that can drive plants toward a mortality tipping point . Thus, while dehydration tolerance may be important during typical seasonal drought conditions ), it may be a much riskier strategy and lead to greater mortality during global-change type drought, especially in the presence of pathogens. These frameworks are consistent with our findings and provide further evidence that A. glauca experiencing acute levels of drought stress are highly predisposed to Bot. infection particularly at lower elevations that experience heightened levels of water stress. The results of this study provide strong evidence that A. glauca in the study region are vulnerable to Bot. disease and dieback, and possibly eventual mortality, related to acute drought. This is consistent with Venturas et al. , who found that acute drought in 2014 led to reduced abundance in A. glauca and other obligate seeder chaparral species and even type-conversion in the Santa Monica Mountains of southern California, USA. A review by Jacobsen and Pratt found similar consistencies among shallow-rooted, obligated seeding shrubs. Clearly, there is strong support that A. glauca populations are at risk for future dieback, and thus should be the focus of more intense studies aimed at understanding the possible mechanisms driving such events. Manzanita are important members of the chaparral ecosystem and large-scale dieback and mortality of this species could reduce resource availability for wildlife , as well as increase the risk of more intense, fires in an ecosystem already associated with increasingly frequent fire activity.

Prevalence of STEC in domestic pigs reared outdoors on diversified small-scale farms from Chapter 1 was lower than this current study, but had a similar sample size . Samples were collected in 2018 for Chapter 3 and 2015-16 for Chapter 1. Differences in STEC prevalence between 2015-2016 and 2018 may be due to different laboratory processing methods or environmental factors. Both study periods were drought years in California; however, 2017 was a very wet year, which may have affected 2018. Three farms participated in both studies and all three farms saw increases in STEC prevalence between 2015-16 and 2018: Farm 1 had a 5.13% STEC prevalence in 2015-16 compared to 20.00% in 2018, Farm 2: 0% STEC prevalence increased to 83.33% and Farm 3: 11.11% to 66.67% . However, a smaller number of samples and animals for Farm 2 and 3 accounts for some of this seemingly large increase between studies. A 2018 study conducted in Georgia reported 62.5% STEC in organic “free ranging” domestic swine, but reported a small sample size of eight. Differences between STEC prevalence may be due to different study designs, laboratory tests, environmental factors or farm management practices, such as the density of pigs raised in each paddock. The scarcity of data regarding STEC in swine raised outdoors indicates a need for future studies. Studies measuring the prevalence of STEC in feral pig populations in the US are infrequent, unlike European studies. A 2006 US study sampled swine necropsy and fecal samples and reported 0 – 23.4% prevalence E. coli O157:H7 in feral pigs. A 2018 study conducted in Georgia detected an overall STEC prevalence of 19.5% in feral swine and they identified a higher prevalence of STEC in feral pigs sampled in agricultural counties.

Feral pigs are attracted to agricultural areas because of resource availability , grow raspberries in a pot and their direct or indirect contact with livestock may create a risk of food borne pathogen transmission. The risk of pathogen sharing between feral pigs and domestic swine has been studied, but only a small subset of these studies investigated the risks to outdoor based pigs, even though there have been multiple cases of feral pigs transmitting pathogens, such as Brucella suis, to domestic swine raised outdoors. Wyckoff et al concluded that increasing populations of feral swine are a risk for the reintroduction of eradicated diseases as well as emerging TBD, especially for backyard operations that allow domestic swine outdoor access, because male feral pigs are attracted to female pens. In a Corsica study that focused on traditional pig farms that raise their animals outdoors, the authors determined that a significant risk factor for the spread of diseases between wild boars and domestic swine was interactions between these two swine groups. Our study results indicated that 45.45% of farm participants had seen evidence of feral pig presence on their farms. Schembri et al conducted a questionnaire of backyard and small-scale swine producers in Australia and found that a third of producers, both indoor and outdoor, had seen feral pigs on their farms. Understanding the prevalence of STEC in feral pigs, combined with the aforementioned study results indicating that these animals reside near resource-rich farms, highlights the need for further studies to address the risk of disease transmission associated with feral pig presence near operations that raise swine outdoors. Serotypes identified in this study that can cause severe human illness included E. coli O157:H7 , O26:H11 and O103:H11 . The serogroups O26:H11 and O103:H11 contained only the stx1 gene, not stx2.

The only O103:H11 serotype contained both eae and ehxA and all the O26:H11 isolates contained the eae gene, with five O26:H11serotypes also containing the ehxA gene. A study by Cha et al also found O26 with stx1 and eae in commercial swine raised indoors in Ohio, US. A study conducted in finishing swine, measured 6.9% of positive samples were O26 and 2.4% contained O103. In 2017, the US Food Safety and Inspection Service conducted a Raw Pork Baseline Study to determine the prevalence of STEC in various types of pork products at slaughterhouses and processing facilities and measured a prevalence of 0.2% STEC, mostly in comminuted pork products. However, this study only looked for the top seven STEC serogroups, even though 309 other samples were positive for key virulence factors like stx and eae genes. Additionally, on-farm or slaughterhouse swine samples may reflect different prevalence ranges than meat products. Considering most studies identified E. coli O157:H7 and non-O157 STEC serotypes that cause human illness in swine samples, pigs should be considered an important reservoir of STEC, and mitigation strategies established to prevent the spread of food borne pathogens from farm to consumer. Significant risk factors associated with the presence of STEC in fecal samples collected during this study included distance from the nearest surface water and whether domestic swine had access to wild areas, such as forest or wetlands. These variables were measured as a proxy for suitable feral pig habitat that borders farms. Feral pigs are reservoirs of STEC, and surface water and/or wild areas provide habitat for these animals to exist near OPO. For instance, a study by Rutten et al predicted suitable habitat for wild boar in Belgium and identified forest , as a significant predictor. Additionally, Wu et al reported distance from a forest to be a significant risk factor for contact with wild boars in Switzerland, especially those domestic pigs less than 500 meters from a forest. A 2017 study reported that distance to water affects feral pigmovement mostly in states where water is scarce versus states where water is more prevalent .

Additionally, feral pigs may contaminate these habitat areas, which may lead to indirect STEC transmission to swine raised outdoors, as studies have shown that STEC can be transmitted through contaminated surface water sources and the environment. A 2014 study conducted in the Central Coast of California detected E. coli O157:H7 and non-O157 in many water sources. These results indicate a need to separate domestic swine raised outdoors from wild areas to avoid direct or indirect transmission of pathogens from feral pigs. In this current study, only the juvenile age group, which included weaners, finishing and market swine , was significant when compared to adults. Many US and international studies have tested similar-aged pigs at slaughterhouses and reported a wide range of STEC prevalence. A study by Tseng et al sampled finishing pigs , which are included in our juvenile category, and determined that the highest prevalence amongst three cohorts occurred between 14-18 weeks of age. At 24 weeks, STEC prevalence in all cohorts had dropped and ranged from 0 – 6.7% in the three groups. This same study mentions that the finishing age group are most susceptible to STEC oedema, which is caused by E. coli strains carrying the stx2 gene and may be associated with detecting STEC in this juvenile age category. A longitudinal study conducted by Cha et al in commercial indoor domestic swine found that 68.3% of finishing pigs shed STEC at least once during the study period, which showcases the intermittent nature of STEC shedding in swine. The high prevalence identified in this study might be due to repeated sampling over a longer period of time than conducted in our study. Additionally, our study sampled all ages of swine only once, best grow pots which might indicate an under reporting of STEC in our results. The effect of age on STEC shedding is more frequently reported in cattle versus swine. For instance, a Raies et al study sampled beef cattle and reported that STEC prevalence was highest during the first six months of life and then decreased toward adulthood. Another study by Cho et al also detected that calves over one month old were two times more likely to shed STEC than those younger than one month, except for pre-weaned calves. If age is a risk factor for STEC shedding in swine, then targeting key age groups for STEC mitigation strategies to reduce the overall bacterial load in slaughtered swine may reduce the risk of these pathogens in the food supply. Limitations of this study included a small sample size for the total number of farm participants as well as the final number of feral pig samples collected, as we could only gather feral pig feces in three of the six targeted counties. The post-hoc power calculation results were 0.12 for feral pigs and 0.69 for OPO, which indicated that the prevalence estimates are inexact. Moreover, many of the significant variables in the final logistic regression model had wide confidence intervals, which indicates less precise estimates.

Since this was a cross-sectional study conducted only during two seasons and only one season per farm, we may have missed STEC positive farms due to seasonality of shedding or other factors that affect STEC detection in feces, including the intermittent nature of shedding in pigs. Our study participants volunteered and therefore we could not conduct random sampling; our study results contain selection bias and are not generalizable to other OPO in California or the US. Strengths of our study included measuring STEC in both feral pig and outdoor reared pigs in California. This study is an innovative approach toward evaluating areas of contact between feral and domestic pigs reared outdoors, by targeting STEC surveillance based on a risk map built in Chapter 2. Moreover, assessing STEC prevalence in feral pigs near OPO serves as a proxy for the risk of exposure and transmission of other zoonotic pathogens to domestic pigs reared outdoors. Future research studies could enhance our current study results by comparing STEC strains between the two swine groups using WGS bio-informatic analyses. Similarity of STEC isolates can be used as a biological indicator to track possible transmission of diseases between feral and outdoor-raised swine, as noted in a few recent studies.The three scientific research projects in this dissertation added important epidemiological information to the body of knowledge regarding STEC detected on DSSF in California and the risk of potential disease transmission from suitable feral pig habitat located near domestic pigs raised outdoors. Although consumers perceive small-scale farms or outdoor-raised meat as safer and more natural, these studies together demonstrate that even livestock raised outdoors on small-scale farms are reservoirs for STEC, including serogroups that cause severe illness in humans, including O157:H7, O26, O103 and O111. Interestingly, Chapter 1 and 3 models indicated that livestock raised outdoors that have access to wild areas, such as wetlands or forest, was a key risk factor for the presence of STEC. Chapter 2 results revealed that nearly 50% of domestic pigs raised outdoors are located near suitable feral pig habitat, and this overlap of feral and domestic swine could be a risk factor for potential emerging or reemerging disease transmission. Also, STEC was detected in domestic swine in both Chapter 1 and 3, even though pigs are currently considered a low risk key species for STEC outbreaks by the US FSIS. These study results indicate the need for further studies on DSSF to ascertain risk factors for food borne pathogens. The objective of Chapter 1 entailed conducting an overall assessment of prevalence and risk factors of STEC on diversified small-scale farms in California, while also describing the unique characteristics of DSSF. Temperature was a key risk factor identified in the final multilevel logistic regression model. Many food borne pathogen studies indicate season as a risk factor for STEC, however, seasons vary across the US. For instance, California summers are characterized by dry heat whereas summers in most states are humid and hot. Measuring and monitoring temperature during field sampling may be a more precise indicator of risk than season and allow for more accurate comparisons between studies. Additionally, as weather patterns shift due to climate change, assessing environmental factors, , as a risk factor for the presence of STEC on farms will be useful for stakeholders, to understand how weather affects the presence of food borne pathogens in livestock raised on DSSF. Studies elucidating whether ambient temperature affects survival of STEC in a farm environment or whether temperature affects the host animal harboring STEC will be useful, especially as extreme climate events become more common.

In comparison to mitochondrial genomes from other members of the Ericaceae, this assembly is similar to that of Vaccinium macrocarpon at 459 678 bp but considerably smaller than that of the saprophytic Hypopitys monotropa or Rhododendron simsii .Our assembly is the first sequenced genome in the genus Arctostaphylos, representing the initial step toward understanding genetics and adaptation in this highly diversified genus. In addition to enhancing ongoing phylogenetic and conservation research, this assembly will enable investigations of the genetic basis of adaptations including drought tolerance and fire resilience. These traits, which are ecological hallmarks of manzanitas, are of growing importance in the context of the increased drought and fire frequency and intensity that are occurring in California as a result of climate change. This assembly can also serve as a reference for studying the diversification and population genetics of Arctostaphylos, which may shed light on aspects of diversification in other complex groups of the CFP. Arctostaphylos is the third genus with a genome assembly in the heath family, Ericaceae, following release of assembled genome sequences of Rhododendron and Vaccinium . These two genera are of significant economic importance: many species, hybrids, and cultivars of Rhododendron, including rhododendron and azalea, are important landscape and ornamental plants, and the fruits of many Vaccinium species, container raspberries which include cranberry, blueberry, and huckleberry, are consumed by humans and other animals. Species in the Ericaceae are also notable for their ability to tolerate acidic and nutrient-poor soils that often characterize boreal forests and bogs, allowing them to thrive in habitats that are inaccessible to most species.

Their tolerance for these conditions is due in part to the formation of mutualistic associations between the roots of the plants and soil fungi of a type unique to the heath family known as ericoid mycorrhizae. Ericoid mycorrhizae are distinct from common mycorrhizal associations found in most angiosperms, and are far less well understood . Complete genome sequences from three genera in this family will provide a strong foundation for investigating the basis of this unique mutualism and its ability to promote survival in inhospitable soils. The size of the A. glauca assembly is 547Mb, which is similar to the two Rhododendron genomes and half that of V corymbosum, which is tetraploid . The tetraploid nature of V. corymbosum also explains the vastly greater number of duplicated genes in its assembly compared to the two diploid assemblies. The scaffold N50 of the A. glauca assembly is longer than R. williamsianum, and close to R. simsii and V. corymbosum, suggesting that the contiguity of Arctostaphylos is comparable to the other taxa . Analysis using RepeatModeler indicated that 57.71% of the A. glauca genome is composed of different categories of repetitive elements . In contrast, analysis using RepeatModeler identified only 26%, 47.5%, and 44.3% of the genome comprising repeat elements in R. williamsianum, R. simsii, and V. corymbosum respectively. The BUSCO completeness assessment of the A. glauca assembly is higher than R. williamsianum and close to the V. corymbosum , indicating that our final assembly is high quality . Overall, the A. glauca, R. simsii and V. corymbosum genomes are of comparably high contiguity and completeness. The lower contiguity and completeness of the R. williamsianum genome may be due to the lack of HiFi or other longread data in the assembly. This explanation is consistent with other studies demonstrating improved assembly with the inclusion of longer reads . Although the big berry manzanita is a common and widespread species, nearly half of the 60+ manzanita species are rare or threatened.

Many are now represented by only one or two populations, and are thus vulnerable to complete eradication by the increasingly common and intense wildfires experienced across California each year. Our manzanita genome sequence will help fulfill the overall goal of the CCGP, serving as a key resource to assess genetic diversity in these threatened endemics and move forward with coordinated conservation programs.The increasing number of diversified small-scale farms and outdoor-raised livestock in the United States , reflects growing consumer interest and demand for sustainably-produced or organic local foods, including animal products such as meat and eggs. However, there is a lack of research evaluating the unique agricultural management practices of DSSF and if these types of farming operations involve risk factors that affect the transmission of food borne pathogens in the food supply. California is the top agricultural production state in the US with annual sales over $50 billion from 69,900 total farms. California is also the sole producer in the US of 17 crops including figs, artichokes and almonds. Additionally, California leads organic sales, accounting for 40% of all organic crop production and 18.16% of United States Department of Agriculture certified organic farms. Many organic farms are small-scale and diversified and these type of DSSF sell food directly to consumers through marketing channels, such as farmers markets or Community Supported Agriculture programs. To suit the unique characteristics of California’s diverse, and within interior valley regions, year-round growing environment, we adjusted the USDA-ERS definition of “small-scale farm” to encompass operations that gross less than $500,000 annually and market directly to consumers through farmers markets, farm stands, CSAs, etc. DSSF are defined as those operations that grow a combination of livestock and specialty crops or raise multiple livestock species with the intent of selling sustainably-raised animal products directly to consumers. Some diversified farms integrate livestock and crop production by using their animals to graze crop residues or cover crops before planting with fresh market crops. Grazing enhances soil fertility, recycles farm nutrients and animals provide another source of revenue through fiber or food products.

Many consumers perceive small-scale farms or outdoor-raised livestock as more natural and safer than food grown on large-scale conventional farms or meat animals raised in confinement systems, but animals naturally harbor food borne pathogens that can cause severe human illness, like Salmonella spp., Campylobacter spp. and Shiga toxin-producing Escherichia coli . For instance, a study by Patterson et al calculated a 4.17% prevalence of STEC in sheep raised on a diversified organic farm in California. Animals are intermittent shedders of enteric pathogens and shedding may increase under certain conditions, such as during periods of stress or due to husbandry practices . Many food borne pathogens can exist in the soil for extended periods of time and can be transmitted to humans through direct contact with feces or animals, or indirect contact with a contaminated environment or through ingestion of produce, meat or water. STEC is consistently one of the major pathogens involved in food borne outbreaks in the US. Vegetables and fruit consumed raw, including spinach, tomatoes and melons, are especially considered high-risk foods. In the summer of 2011, a small family farm u-pick berry operation was the center of an E. coli O157:H7 outbreak when strawberries were contaminated by wild deer feces. Six of the fifteen cases were hospitalized and two people died. More recently, several nationwide outbreaks of E. coli O157:H7 have occurred through consumption of romaine lettuce, including one outbreak traced back to California farms and linked to cattle grazing upstream from the lettuce fields. STEC outbreaks associated with DSSF might be under reported, due to their smaller volume of sales compared to large farms. A study by Harvey et al identified six STEC outbreaks connected with organic agricultural operations between 1992-2014. All these food borne outbreaks underscore the need to conduct prevalence studies of food borne pathogens on DSSF. Swine are especially a livestock species of concern because they are reservoirs for zoonotic diseases like swine influenza and brucellosis and food borne pathogens like STEC and Campylobacter spp. Although most swine production in the US occurs inside buildings with high levels of bio-security, draining pots the US is currently experiencing a resurgence of outdoor-based swine operations, due to consumer demand for sustainably-raised animal products. Still primarily considered a niche production system in the US, outdoor-raised pig operations  are numerous and broadly distributed within California. The USDA National Animal Health Monitoring System swine report defined “small-enterprise operations” as those raising fewer than 100 pigs. Approximately 68.8 – 78.9% of the small operations included in this nationwide survey raised domestic pigs with some level of outside access. A challenge in raising domestic pigs outdoors is the increasing risk of their directly or indirectly interacting with wildlife like feral pigs, and a subsequent potential increase for pathogen sharing, especially as feral pig abundance and distribution grows throughout the US. Feral pigs are considered an invasive species as they only need water, food and shrub cover to survive, can double their population in four months and are difficult to eradicate., Moreover, if an area contains favorable habitat for feral pigs, then their population numbers can be maintained or increase over time. They also have the widest geographic distribution and one of the broadest habitat ranges of any large mammal except humans.

The wide distribution of feral swine is in part due to their ability to adapt to many ecological habitats and their opportunistic omnivore diet. California has one of the largest and widest distributions of feral pigs. Feral pigs in the US are a mix of introduced Eurasian wild boars, which are native to Asia and Europe, and domestically-raised pigs turned feral. As early as 2005, Corn et. al described the disease implications of expanding feral pig populations in the US, because they can serve as a vehicle for pathogen transmission to domestic pigs, and they could play a significant role in the transmission and maintenance of transboundary animal diseases , that may be introduced or re-introduced to North America , classical swine fever). Other studies have also reported the disease risks from the expanding distribution of feral pigs in the US, including their future role as spreaders of TBD like ASF, which was recently found in the Dominican Republic, a mere 700 miles from Florida, US. Additionally, eradicated diseases in indoor-pig herds have been documented in feral swine populations in California, for example, feral pig samples collected by the California Department of Fish and Wildlife from 1978- 2013 showed feral pigs testing positive for Brucella suis, Leptospira spp. and swine influenza virus . Contact between feral pigs and outdoor-raised pig herds increases the risk for the transmission of these diseases in domestic swine. Many studies have reported that feral pigs maintain and transmit zoonotic and food borne pathogens; however, only a small subset of these studies focused on the risk of pathogen sharing between feral pigs and outdoor raised pigs. Transmission of pathogens between feral pigs and outdoor raised pigs has been documented in the US. For example, a 2016 human brucellosis case on a NewYork State farm began with a feral pig infecting domestic pigs reared outdoors. Swine sold from this index farm led to Brucella suis positive domestic swine in nine other herds in multiple states. Additionally, one swine brucellosis case each in Texas, Iowa and Georgia in 2005 also involved domestic swine being exposed to feral pigs through inadequate bio-security or wildlife controls. Feral pigs are known to forage on farmland and some California farmers and ranchers regularly experience feral pig intrusions in their crop fields and/or contact between outdoor raised pigs and feral pigs. According to studies conducted in California and Texas, contact has been documented between feral pigs and outdoor-raised pigs. A 2012 spatial study by Wyckoff et al reported that feral pigs are attracted to agricultural habitats as food sources, which may facilitate pathogen transmission to livestock raised outdoors and humans or contaminate crops. The authors assessed habitat and movement of feral swine within 10 miles of outdoor domestic pig operations in Texas and calculated that at least 50% of these facilities were surrounded by suitable feral pig habitat. Another Wyckoff et al study assessed the disease transmission risk of feral pigs near domestic pigs facilities in Texas. This 2009 study used GPS collars to quantify contact between feral and domestic pigs and detected evidence of direct contact, as well as antibodies for the same diseases in both swine groups. They concluded that feral swine are an increasing risk for the reintroduction of eradicated diseases as well as emerging TBD, especially for operations that allowed domestic swine outdoor access, as male feral pigs are attracted to female pens. International studies have also assessed the risk of disease transmission at the wild boar-domestic pig interface.

This is consistent with the higher NCED3, NCED5 and BAM1 transcript abundance in RNO berries . Thus, there are complex responses of ABA metabolism and signaling. It would appear that there may be two different ABA pathways affecting ABA concentrations and signaling: one involved with embryo development and one involved with the water status in the skins. Auxin is also involved with ABA signaling during the late stages of embryo development in the seeds. Auxin signaling responses are complex. ABF5 is an auxin receptor that degrades Aux/IAA proteins, which are repressors of ARF transcriptional activity. Thus, a rise in auxin concentration releases Aux/IAA repression of ARF transcription factors, activating auxin signaling. In the berry skins, there was a diversity of transcriptional responses of Aux/IAA and ARF genes in the two locations, some with increased transcript abundance and others with decreased transcript abundance. As with ABA signaling, there may be multiple auxin signaling pathways operating simultaneously. One pathway appears to involve seed dormancy. ARF2 had a higher transcript abundance in BOD berries. ARF2 promotes dormancy through the ABA signaling pathway. This is consistent with the hypothesis that BOD berries reach maturity at a lower sugar level than RNO berries.Grapevines have very dynamic gene expression responses to pathogens. The top 150 DEGs for BOD berries were highly enriched with biotic stress genes. The BOD vineyard site had a higher rainfall and higher relative humidity than RNO and these conditions are likely to be more suitable for fungi to grow. We detected a much higher transcript abundance of powdery mildew-responsive genes in BOD berries and this may be connected to a higher transcript abundance of ethylene and phenylpropanoid genes as part of a defense response.

The transcript abundance profiles of some of these genes are remarkably similar. Increased ethylene signaling in grapevines has been associated with powdery mildew infection and phenylpropanoid metabolism and appears to provide plant protection against the fungus. Genes involved with phenylpropanoid metabolism, blueberry pot size especially PAL and STS genes, appear to be quite sensitive to multiple stresses in the environment . In Arabidopsis there are four PAL genes. These PAL genes appear to be involved with flavonoid biosynthesis and pathogen resistance in Arabidopsis. Ten different PAL1 and two PAL2 orthologs had higher transcript abundance in BOD berry skins; many STS genes also had a higher transcript abundance in BOD berry skins . Stilbenes are phytoalexins and provide pathogen resistance in grapes and STS genes are strongly induced by pathogens. Thus, the higher transcript abundance of powdery mildew genes may be associated with the higher transcript abundance of genes in the ethylene and phenylpropanoid pathways.The transcript abundance of a number of iron homeostasis genes were significantly different in the two locations and there was a difference in soil available iron concentrations in the two locations. However, iron uptake and transport in plants is complicated depending on multiple factors, such as pH, soil redox state, organic matter composition, solubility in the phloem, etc. Thus, it is impossible to predict iron concentrations in the berry without direct measurements. The roles of these genes in iron homeostasis and plant physiological functions are diverse. Iron supply can affect anthocyanin concentrations and the transcript abundance of genes in the phenylpropanoid pathway in Cabernet Sauvignon berry skins. One of the DEGs, SIA1, is located in the chloroplast in Arabidopsis and appears to function in plastoglobule formation and iron homeostasis signaling in concert with ATH13. Another DEG, YSL3, is involved in iron transport. It acts in the SA signaling pathway and appears to be involved in defense responses to pathogens.

It also functions in iron transport into seeds. FER1 is one of a family of ferritins found in Arabidopsis . VIT1 and NRAMP3 are vacuolar iron transporters and are also involved in iron storage in seeds. Other DEGs are also responsive to iron supply. IREG3 appears to be involved in iron transport in plastids; its transcript abundance increases with increasing iron concentrations. ABCI8 is an iron-stimulated ATPase located in the chloroplast that functions in iron homeostasis. It is unclear what specific roles these iron homeostasis genes are playing in grape berry skins, but they appear to be involved in iron storage in seeds and protection against oxidative stress responses. One possible explanation for the transcript abundance profiles in the BOD and RNO berry skins is that ferritins are known to bind iron and are thought to reduce the free iron concentrations in the chloroplast, thus, reducing ROS production that is caused by the Fenton reaction. As chloroplasts senesce during berry ripening, iron concentrations mayrise as a result of the catabolism of iron-containing proteins in the thylakoid membranes; thus, berry skins may need higher concentrations of ferritins to keep free iron concentrations low. This might explain the increase in ferritin transcript abundance with increasing sugar levels. Most soils contain 2 to 5% iron including available and unavailable iron; soils with 15 and 25 μg g− 1 of available iron are considered moderate for grapevines, but soils with higher concentrations are not considered toxic. Therefore, for both soils in this study, iron concentrations can be considered to be very high but not toxic. The higher available iron concentrations in the BOD vineyard may be associated with the wetter conditions and the lower soil pH.Other researchers using Omics approaches have identified environmental factors that influence grape berry transcript abundance and metabolites. One study investigated the differences in transcript abundance in berries of Corvina in 11 different vineyards within the same region over 3 years. They determined that approximately 18% of the berry transcript abundance was affected by the environment.

Climate had an overwhelming effect but viticultural practices were also significant. Phenylpropanoid metabolism was very sensitive to the environment and PAL transcript abundance was associated with STS transcript abundance. In another study of a white grape cultivar, Garganega, berries were analyzed by transcriptomic and metabolomic approaches. Berries were selected from vineyards at different altitudes and soil types. Again, phenylpropanoid metabolism was strongly influenced by the environment. Carotenoid and terpenoid metabolism were influenced as well. Two studies investigated the grape berry transcriptomes during the ripening phase in two different regions of China, a dry region in Western China and a wet region in Eastern China. These two locations mirror some of the differences in our conditions in our study, namely moisture, light and elevation, plant raspberry in container although the dry China western region has higher night temperatures and more rainfall than the very dry RNO location. In the Cabernet Sauvignon study, they compared the berry transcriptomes from the two regions at three different stages: pea size, veraison and maturity. The TSS at maturity was slightly below 20°Brix. Similar to our study, the response to stimulus, phenylpropanoid and diterpenoid metabolism GO categories were highly enriched in mature berries between the two locations. Differences in the transcript abundance of NCED and PR proteins were also noted. Like in our study, the authors associated the transcript abundance of these proteins to the dry and wet locations, respectively. In the second study comparing these two regions in China , the effects of the environment on the metabolome and transcriptome of Muscat Blanc à Petits Grains berries were investigated over two seasons; specifically, terpenoid metabolism was targeted. Like in our study, the transcripts in terpenoid were in higher abundance in the wetter location. The transcript abundances were correlated with terpenoid concentrations and a coexpression network was constructed. A specific set of candidate regulatory genes were identified including some terpene synthases , glycosyl transferases and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase . We examined the transcript abundance of some of these candidate genes in our own data but did not find significant differences between our two locations. The contrasting results between our study and Wen et al. could be for a variety of reasons such as different cultivar responses, berry versus skin samples, or different environmental conditions that affect terpenoid production. Terpenoid metabolism is influenced by the microclimate and is involved in plant defense responses to pathogens and insects. Light exposure to Sauvignon Blanc grapes was manipulated by removing adjacent leaves without any detectable differences in berry temperatures. Increased light exposure increased specific carotenoid and terpene concentrations in the berry. Some effect of temperature was associated with carotenoid and terpenoid production, but to a lesser extent than light. Higher concentrations of rotundone, a sesquiterpene, have been associated with cooler temperatures. Water deficit can also alter carotenoid and terpenoid metabolism in grapes.

Terpenes can act as signals for insect attacks and attract insect predators. Thus, terpenoid metabolism is highly sensitive to the environment and influenced by many factors. In contrast to these studies, excess light and heat can affect transcript abundance and damage berry quality. In addition to a higher rate of malate catabolism, anthocyanin concentrations and some of the transcript abundances associated with them are decreased as well.BOD berries reached maturity at a lower °Brix level than RNO berries; the cause is likely to be the warmer days and cooler nights in RNO. Higher day temperature may increase photosynthesis and sugar transport and cooler night temperatures may reduce fruit respiration. °Brix or TSS approximates the % sugar in a berry and is a reliable marker of berry maturity in any given location ; however, TSS is an unreliable marker of berry maturity when comparing grapes from very different climates. The differences in TSS between BOD and RNO are consistent with other studies on the temperature effects on berry development. Indirect studies have associated gradual warming over the last century to accelerated phenology and increased sugar concentrations in the grape berries . Increasing temperature can accelerate metabolism, including sugar biosynthesis and transport, but the increase in metabolism is not uniform. For example, the increase in anthocyanin concentration during the ripening phase is not affected as much as the increase in sugar concentration. These responses vary with the cultivar, complicating this kind of analysis even further. Direct studies of temperature effects on Cabernet Sauvignon berry composition also are consistent with our data. In one study, the composition of Cabernet Sauvignon berries was altered substantially for vines grown in phytotrons at 20 or 30 °C temperatures. Cooler temperatures promoted anthocyanin development and malate concentrations and higher temperatures promoted TSS and proline concentrations. In a second study, vines were grown at 20 or 30 °C day temperatures with night temperatures 5 °C cooler than the day. In this study, higher temperatures increased berry volume and veraison started earlier by about 3 to 4 weeks. The authors concluded that warmer temperatures hastened berry development. In a third study, Cabernet Sauvignon berry composition was affected in a similar manner by soil temperatures that differed by 13 °C. TSS concentrations are also affected by light and the vine water status. Light is generally not a factor because there is usually a large enough leaf area and sufficient light levels to saturate this source to sink relationship. Sun-exposed Cabernet Sauvignon berries in the vineyard had higher TSS than shaded berries. This sunlight effect was attributed largely to an increase in berry temperature rather than an increase in the fluence rate per se. A higher grapevine water status results in larger berry size and lower sugar concentrations and water deficit is known to increase sugar concentrations in Cabernet Sauvignon. However, temperature is thought to have the largest effect on sugar concentrations. Other transcriptomic data in the present study indicated that BOD berries were more mature at a lower sugar level than RNO berries. These included the transcript abundance profiles of genes involved in autophagy, auxin and ABA signaling, iron homeostasis and seed development. Many of these DEGs had an accelerated rate of change in BOD berries. While these transcripts are in the skins, they may be influenced by signals coming from the seed. In addition, there was a higher transcript abundance for most genes involved with the circadian clock in BOD berries. PHYB can regulate the circadian clock and PHYB activity is very sensitive to night temperatures ; PHYB reversion is accelerated to the inactive form at warmer temperatures . The inactivity of phytochrome promotes the expression of RVE1, which promotes auxin concentrations and seed dormancy. Thus, all things considered, it is likely that temperature and/or the temperature differentials between day and night significantly contributed to the differences in the rate of berry development and sugar accumulation in the two locations.

Grapevine berry ripening can be divided into three major stages. In stage 1, berry size increases sigmoidally. Stage 2 is known as a lag phase where there is no increase in berry size. Stage 3 is considered the ripening stage. Veraison is at the beginning of the ripening stage and is characterized by the initiation of color development, softening of the berry and rapid accumulation of the hexoses, glucose and fructose. Berry growth is sigmoidal in Stage 3 and the berries double in size. Many of the flavor compounds and volatile aromas are derived from the skin and synthesized at the end of this stage. Many grape flavor compounds are produced as glycosylated, cysteinylated and glutathionylated precursors and phenolics and many of the precursors of the flavor compounds are converted to various flavors by yeast during the fermentation process of wine. Nevertheless, there are distinct fruit flavors and aromas that are produced and can be tasted in the fruit, many of which are derived from terpenoids, fatty acids and amino acids. Terpenes are important compounds for distinguishing important cultivar fruit characteristics. There are 69 putatively functional, 20 partial and 63 partial pseudogenes in the terpene synthase family that have been identified in the Pinot Noir reference genome. Terpene synthases are multi-functional enzymes using multiple substrates and producing multiple products. More than half of the putatively functional terpene synthases in the Pinot Noir reference genome have been functionally annotated experimentally and distinct differences have been found in some of these enzymes amongst three grape varieties: Pinot Noir, Cabernet Sauvignon and Gewürztraminer. Other aromatic compounds also contribute significant cultivar characteristics. C13-norisoprenoids are flavor compounds derived from carotenoids by the action of the carotenoid cleavage dioxygenase enzymes.

Cabernet Sauvignon, Sauvignon Blanc and Cabernet Franc are characterized by specific volatile thiols and methoxypyrazines. Enzymes involved in the production of these aromas have been recently characterized. Phenolic compounds play a central role in the physical mouthfeel properties of red wine; recent work relates quality with tannin levels. While the grape genotype has a tremendous impact on tannin content, blackberries in containers the environment also plays a very large role in grape composition. The pathway for phenolic biosynthesis is well known, but the mechanisms of environmental influence are poorly understood. Ultimately, there is an interaction between molecular genetics and the environment. Flavor is influenced by climate, topography and viticultural practices. For example, water deficit alters gene expression of enzymes involved in aroma biosynthesis in grapes, which is genotype dependent, and may lead to increased levels of compounds, such as terpenes and hexyl acetate, that contribute to fruity volatile aromas. The grapevine berry can be subdivided into the skin, pulp and seeds. The skin includes the outer epidermis and inner hypodermis . A thick waxy cuticle covers the epidermis. The hypodermal cells contain chloroplasts, which lose their chlorophyll at veraison and become modified plastids; they are the sites of terpenoid biosynthesis and carotenoid catabolism. Anthocyanins and tannins accumulate in the vacuoles of hypodermal cells. Pulp cells are the main contributors to the sugar and organic acid content of the berries. Pulp cells also have a much higher set of transcripts involved in carbohydrate metabolism, but a lower set of transcripts involved in lipid, amino acid, vitamin, nitrogen and sulfur metabolism than in the skins. Hormones can influence berry development and ripening. Concentrations of auxin, cytokinins and gibberellins tend to increase in early fruit development of the first stage. At veraison, these hormone concentrations have declined concomitant with a peak in abscisic acid concentration just before veraison.

Auxin prolongs the Stage 2 lag phase and inhibits anthocyanin biosynthesis and color development in Stage 3. Grapevine, a non-climacteric fruit, is not very sensitive to ethylene; however, ethylene appears to be necessary for normal fruit ripening. Ethylene concentration is highest at anthesis, but declines to low levels upon fruit set; ethylene concentrations rise slightly thereafter and peak just before veraison then decline to low levels by maturity. Ethylene also plays a role in the ripening of another non-climacteric fruit, strawberry. ABA also appears to be important in grape berry ripening during veraison when ABA concentrations increase resulting in increased expression of anthocyanin biosynthetic genes and anthocyanin accumulation in the skin. ABA induces ABF2, a transcription factor that affects berry ripening by stimulating berry softening and phenylpropanoid accumulation. In addition, ABA affects sugar accumulation in ripening berries by stimulating acid invertase activity and the induction of sugar transporters. It is not clear whether ABA directly affects flavor volatiles , but there could be indirect effects due to competition for common precursors in the carotenoid pathway. Many grape berry ripening studies have focused on targeted sampling over a broad range of berry development stages, but generally with an emphasis around veraison, when berry ripening is considered to begin. In thisstudy, a narrower focus is taken on the late ripening stages where many berry flavors are known to develop in the skin. We show that that the abundance of transcripts involved in ethylene signaling is increased along with those associated with terpenoid and fatty acid metabolism, particularly in the skin.Cabernet Sauvignon clusters were harvested in 2008 from a commercial vineyard in Paso Robles, California at various times after veraison with a focus on targeting °Brix levels near maturity. Dates and metabolic details that establish the developmental state of the berries at each harvest are presented in Additional file 1. Berries advanced by harvest date with the typical developmental changes for Cabernet Sauvignon: decreases in titratable acidity and 2- isobutyl-3-methoxypyrazine concentrations and increases in sugar and color . Transcriptomic analysis focused on four harvest dates having average cluster °Brix levels of 22.6, 23.2, 25.0 and 36.7. Wines made in an earlier study from grapes harvested at comparable levels of sugars or total soluble solids to those in the present study showed clear sensory differences. Six biological replicates, comprising two clusters each, were separated into skins and pulp in preparation for RNA extraction and transcriptomic analysis using the NimbleGen Grape Whole-Genome Microarray. Thus, a 4 × 2 factorial experimental design was established. After standard microarray processing and data normalization, two-way ANOVA indicated that the transcript abundance of 16,280 transcripts statistically significantly changed across the °Brix levels below the adjusted p-value of 0.05 , the transcript abundance of 10,581 transcripts changed significantly across Tissue types, and the abundance of 2053 transcripts changed significantly with respect to the °Brix x Tissue interaction term p-value column: adjBrix, adjTissue or adjTissue*Brix. A note of caution must be added here. There are high similarities amongst members in certain Vitis gene families , making it very likely that cross-hybridization can occur with probes on the microarray with high similarity to other genes. We estimate approximately 13,000 genes have the potential for cross-hybridization, with at least one probe of a set of four unique probes for that gene on the microarray potentially cross-hybridizing with probes for another gene on the microarray. Genes with the potential for cross hybridization have been identified and are highlighted in light red in Additional file 2. The rationale to include them is that although individual genes can not be uniquely separated, the probe sets can identify a gene and its highly similar gene family members, thus, providing some useful information about the biological responses of the plant. An additional approach was taken, removing cross-hybridizing probes before quantitative data analysis . Many of the significant genes were unaffected by this processing, blackberry container but 3600 genes were completely removed from the analysis. Thus, it was felt that valuable information was lost using such a stringent approach.

The less stringent approach allowing for analysis of genes with potential cross hybridization was used here in the rest of the analyses. To assess the main processes affected by these treatments, the gene ontologies of significantly affected transcripts were analyzed for statistical significance using BinGO. Based on transcripts that had significant changes in abundance with °Brix level, 230 biological processes were significantly over represented in this group . The three top over represented processes were response to abiotic stress, bio-synthetic process, and response to chemical stimulus, a rather generic set of categories. Tissue differences were more revealing at the stage when flavors peak; 4865 transcripts that were significantly higher in skins compared to pulp at 23.2 °Brix were tested for over represented GO functional categories . Some of the top GO categories included photosynthesis, isoprenoid biosynthesis, and pigment biosynthesis . Some of the transcripts with the largest differences between skin and pulp at 23.2 °Brix are β-ketoacyl-CoA synthase , taxane 10-β-hydroxylase , wax synthase, a lipase, an ABC transporter, and phenylalanine ammonia-lyase . The abundance of 5716 transcripts was significantly higher in pulp than skin at 23.2 °Brix . Some of the top GO categories over represented were a variety of transport processes and small GTPase mediated signal transduction . Some of the transcripts with the largest differences in abundance with pulp greater than skin at 23.2 °Brix were polygalacturonase , flavonol synthase, stachyose synthase, an amino acid transporter, a potassium channel , and HRE2 . The transcript abundance of 2053 genes had significantly differential expression across °Brix levels and tissues . The top GOcategories over represented in this set involved photosynthesis and phenylpropanoid metabolism, both associated with the berry skin . Other flavorcentric categories of the 57 categories over represented include aromatic compound biosynthesis, fatty acid metabolism and alcohol catabolism. This transcript set was further analyzed by dividing into 10 clusters using k-means clustering . The over represented GO categories were determined for each cluster . Eight of the 10 clusters had distinct over represented GO categories; two clusters did not have any over represented GO categories, meaning that the genes in these two clusters were assigned to GO categories of expected proportions when compared to the entire NimbleGen array. Clusters 1, 8, 9 and 10 had a large number of over represented categories. Many GO categories within a cluster are subsets of others in that cluster and were grouped together. For example, cluster 4 had four over represented GO categories, oxygen transport, gas transport, heat acclimation and response to heat. The four categories could be grouped into two, as two are subsets of the others; this is how they were listed in Table 1.As we were interested in compounds associated with berry flavors as they develop or change in the late stages of berry ripening, we took a more targeted approach for analysis with this in mind. Berries at 24° Brix are known to be near-optimal for flavor, thus we took a simple approach to look for genes that were peaking around this stage. We found some significant and large increases in transcript abundance between the 22.6 and 23.2 °Brix levels. A group of VviERF6 transcription factor paralogs represented 6dance from 22.6 to 23.2 °Brix in the skin, but not in the pulp . These VviERF6 TFs were also found in Cluster 8 . This is very interesting since many flavor compounds are derived from the skin and ERF TFs are known to be responsive to ethylene, a known fruit-ripening hormone. These VviERF TFs were named ERF105 in the annotation by Grimplet et al., however they are more orthologous with AtERF6 as determined by a more comprehensive phylogenetic method using many plant species at Gramene . Annotation details of the V1 gene models of the VviAP2/ERF super family can be found in Additional file 8 including updated Vvi symbols according to its closest Arabidopsis ortholog as instructed by the Grapevine Gene Nomenclature System developed by the International Grape Genome Program Supernomenclature committee. This renaming of the AP2/ERF super family should facilitate comparative analyses and functions with other species, particularly Arabidopsis. To properly annotate the AP2/ERF super family of Vitis vinifera according to the IGGP Supernomenclature committee instructions, a phylogenetic tree was generated for the AP2/ERF super family of Arabidopsis thaliana and Vitis vinifera using the TAIR 10 and V1 gene models, respectively . The labeled family classifications were derived from the Arabidopsis naming scheme by Nakano et al.. There are 130 members in the VitisAP2/ERF super-family in the Pinot Noir reference genome. However, the six paralogs of ERF6 discussed above belong to a Vitis vinifera clade in subfamily IX and are distinctly different or separate from any Arabidopsis subfamily IX ERF TFs .