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 .

To date, quantum anomalous Hall effects have been observed at band fillings ν = 1 and ν = 3 in various graphene heterostructures, where ν = An corresponds to the number of electrons per unit cell area A with n the carrier density. Although orbital magnetism is generally expected theoretically in twisted bilayer graphene, no direct experimental probes of magnetism have been reported because of the relative scarcity of magnetic samples, their small size, and the low expected magnetization density they are predicted to have.We perform spatially resolved magnetometry to image the submicron magnetic structure of the same sample presented in the previous chapter and in Ref. , which consists of a twisted graphene bilayer aligned to one of the hexagonal boron nitride encapsulating layers. Figure 5.1B shows a schematic representation of our experimental setup. We use a superconducting quantum interference device fabricated on the tip of a quartz tube from cryogenically deposited indium with a magnetic field sensitivity of approximately 15 nT/Hz1/2 at select outof-plane magnetic fields of less than 50 mT. The SQUID is mounted to a quartz tuning fork and rastered in a 2D plane parallel to, and at a fixed height above, the tBLG heterostructure. Here n is the nominal density inferred from a parallel plate capacitor model, with the capacitance determined from the lowfield Hall density . Images are acquiredin the same background magnetic field B = 22 mT but on opposite branches of the hysteresis loop shown in Fig. 5.1A. As demonstrated in Fig. 8.16, and explained in more detail in the appendix, raspberry container the measured BT F contains contributions from magnetic signals as well as other effects arising from electric fields or thermal gradients. To isolate the magnetic structure that gives rise to the observed hysteretic transport, we subtract data from Figs. 5.1, C and D, from each other.

The result is shown in Fig. 5.1E, which depicts the gradient magnetometry signal associated with the fully polarized orbital ferromagnet. To reconstruct the static out-of-plane magnetic field, Bz, we integrate BT F along ˆa from the lower and left boundaries of the image . We infer the total magnetization density m from the Bz data using standard Fourier domain techniques as shown in Fig. 5.1G. The algorithm used for this process is covered in depth in the associatd publication. Figures 5.1, H and I, show a comparison of BT F and m plotted along the contours indicated in Figs. 5.1, E and G. The shaded regions in Fig. 5.1I denote confidence intervals, whose absolute error bounds form the dominant systematic uncertainty in |ˆa|. Our measurements are taken close to ν = 3, equivalent to a single hole per unit cell relative to the nonmagnetic state at ν = 4 that corresponds to full filling of the lowest energy bands. We find that the magnetization density is considerably larger than 1 µB per unit cell area A ≈ 130/nm2 , where we have taken g = 2 as appropriate for graphene, in which spin orbit coupling is negligible. Without any assumptions about the nature of the broken symmetries, this state has a maximum spin magnetization of 1 µB per moir´e unit cell. Our data reject this hypothesis, finding instead a maximum magnetization density of m in the range 2 − 4 µB per moir´e unit cell corresponding to an orbital magnetization of 1.8-3.6× 10−4µB/carbon atom. We conclude that the magnetic moment associated with the Chern magnet phase in tBLG is dominated by its orbital component.In an intrinsic orbital magnet in which all moments arise from conduction electrons, the magnetization depends strongly on the density. Additional density dependence arises from the fact that contributions to the orbital magnetization from both wave-packet angular momentum and Berry curvature need not be uniformly distributed within the Brillouin zone.

Transport observations of aquantum anomalous Hall effect measure only the total Berry curvature of a completely filled band. At partial band filling, however, extrinsic contributions from scattering complicate the relationship between transport and band properties. In contrast, measuring m provides direct information about the density-dependent occupation of the Bloch states in momentum space. Although crystalline defects on the atomic scale are unlikely in tBLG thanks to the high quality of the constituent graphene and hBN layers, the thermodynamic instability of magic angle twisted bilayer graphene makes it highly susceptible to inhomogeneity at scales larger than the moir´e period, as shown in prior spatially resolved studies. For example, the twist angle between the layers as well as their registry to the underlying hBN substrate may all vary spatially, providing potential pinning sites. Moir´e disorder may thus be analogous to crystalline disorder in conventional ferromagnets, which gives rise to Barkhausen noise as it was originally described. A subtler issue raised by our data is the density dependence of magnetic pinning; as shown in Fig. 5.3, Bc does not simply track 1/m across the entire density range, in particular failing to collapse with the rise in m in the Chern magnet gap. This suggests nontrivial dependence of either the pinning potential or the magnetocrystalline anisotropy energy on the realized many body state. Understanding the pinning dynamics is critical for stabilizing magnetism in tBLG and the growing class of related orbital magnets, which includes both moir´e systems as well as more traditional crystalline systems such as rhombohedral graphite. In order to understand the microscopic mechanism behind magnetic grain boundaries in the Chern magnet phase in tBLG/hBN, we used nanoSQUID magnetometry to map the local moir´e superlattice unit cell area, and thus the local twist angle, in this device, using techniques discussed in the literature.

This technique involves applying a large magnetic field to the tBLG/hBN device and then using the chiral edge state magnetization of the Landau levels produced by the gap between the moir´e band and the dispersive bands to extract the electron density at which full filling of the moir´e superlattice band occurs . The strength of this Landau level’s magnetization can be mapped in real space , and the density at which maximum magnetization occurs can be processed into a local twist angle as a function of position . It was noted in that the moir´e superlattice twist angle distribution in tBLG is characterized by slow long length scale variations interspersed with thin wrinkles, across which the local twist angle changes rapidly. These are also present in the sample imaged here . The magnetic grain boundaries we extracted by observing the domain dynamics of the Chern magnet appear to correspond to a subset of these moir´e superlattice wrinkles. It may thus be the case that these wrinkles serve a function in moir´e superlattice magnetism analogous to that of crystalline grain boundaries in more traditional transition metal magnets, pinning magnetic domain walls to structural disorder and producing Barkhausen noise in measurements of macroscopic properties. In tBLG, a set of moir´e subbands is created through rotational misalignment of a pair of identical graphene monolayers. In twisted monolayer-bilayer graphene a set of moir´e subbands is created through rotational misalignment of a graphene monolayer and a graphene bilayer. These systems both support Chern magnets. Both systems are also members of a class of moir´e superlattices known as homobilayers; in these systems, large plastic pots for plants the 2D crystals forming the moir´e superlattice share the same lattice constant, and the moir´e superlattice appears as a result of rotational misalignment, as illustrated in Fig. 5.17A. Homobilayers have many desirable properties; the most important one is that the twist angle can easily be used as a variational parameter for minimizing the bandwidth of the moir´e subbands, producing the so-called ‘magic angle’ tBLG and tMBG systems. Homobilayers do, however, have some undesirable properties. Although local variations in electron density are negligible in these devices, the local filling factor of the moir´e superlattice varies with the moir´e unit cell area, and thus with the relative twist angle. The tBLG moir´e superlattice is shown for two different twist angles in 5.17B-C across the magic angle regime; it is clear that the unit cell area couples strongly to twist angle in this regime, illustrating the sensitivity of these devices to twist angle disorder. The relative twist angle of the two crystals in moir´e superlattice devices is never uniform. Imaging studies have clearly shown that local twist angle variations provide the dominant source of disorder in tBLG . It is hard to exaggerate the significance of this problem to the study of moir´e superlattices. Orbital magnetism at B = 0 has only been realized in a handful of tBLG devices, and quantization of the anomalous Hall resistance has only been demonstrated in a single tBLG device, in spite of years of sustained effort by several research groups. A mixture of careful device design limiting the active area of devices and the use of local probes has allowed researchers to make many important discoveries while sidestepping the twist angle disorder issue- indeed, some exotic phases are known in tBLG only from a single device, or even from individual scanning probe experiments- but if the field is ever to realize sophisticated devices incorporating these exotic electronic ground states the problem needs to be addressed.

There is another way to make a moir´e superlattice. Two different 2D crystals with different lattice constants will form a moir´e superlattice without a relative twist angle; these systems are known as heterobilayers . These systems do not have ‘magic angles’ in the same sense that tBLG and tMBG do, and as a result there is no meaningful sense in which they are flat band systems, but interactions are so strong that they form interaction-driven phases at commensurate filling of the moir´e superlattice anyway. Indeed, many of the interaction-driven insulators these systems support survive to temperatures well above 100 K. The most important way in which heterobilayers differ from homobilayers, however, is in their insensitivity to twist angle disorder. In the small angle regime, the moir´e unit cell area of a heterobilayer is almost completely independent of twist angle, as illustrated in 5.17E-F. A new intrinsic Chern magnet was discovered in one of these systems, a heterobilayer moir´e superlattice formed through alignment of MoTe2 and WSe2 monolayers. The researchers who discovered this phase measured a well-quantized QAH effect in electronic transport in several devices, demonstrating much better repeatability than was observed in tBLG. The unit cell area as a function of twist angle is plotted for three moir´e superlattices that support Chern insulators in 5.17G, with the magic angle regime highlighted for the homobilayers, demonstrating greatly diminished sensitivity of unit cell area to local twist angle in the heterobilayer AB-MoTe2/WSe2. MoTe2/WSe2 does have its own sources of disorder, but it is now clear that the insensitivity of this system to twist angle disorder has solved the replication issue for Chern magnets in moir´e superlattices. Dozens of MoTe2/WSe2 devices showing well-quantized QAH effects have now been fabricated, and these devices are all considerably larger and more uniform than the singular tBLG device that was shown to support a QAH effect, and was discussed in the previous chapters. The existence of reliable, high-yield fabrication processes for repeatably realizing uniform intrinsic Chern magnets is an important development, and this has opened the door to a wide variety of devices and measurements that would not have been feasible in tBLG/hBN.The basic physics of this electronic phase differs markedly from the systems we have so far discussed, and we will start our discussion of MoTe2/WSe2 by comparing and contrasting it with graphene moir´e superlattices. In tBLG/hBN and its cousins, valley and spin degeneracy and the absence of significant spin-orbit coupling combine to make the moir´e subbands fourfold degenerate. When inversion symmetry is broken the resulting valley subbands can have finite Chern numbers, so that when the system forms a valley ferromagnet a Chern magnet naturally appears. Spin order may be present but is not necessary to realize the Chern magnet; it need not have any meaningful relationship with the valley order, since spin-orbit coupling is absent. MoTe2/WSe2 has strong spin-orbit coupling, and as a result, the spin order is locked to the valley degree of freedom. This manifests most obviously as a reduction of the degeneracy of the moir´e subbands; these are twofold degenerate in MoTe2/WSe2 and all other TMD-based moir´e superlattices. The closest imaginable analog of the tBLG/hBN Chern magnet in this system is one in which interactions favor the formation of a valley-polarized ferromagnet, at which point the finite Chern number of the valley subbands would produce a Chern magnet.

What they do provide is a convenient way for us to produce two dimensional monocrystalline devices with exceptionally low disorder for which electron density and band structure can be conveniently accessed as independent variables. That is valuable for furthering our understanding of condensed matter phenomena, independent of whether the fabrication procedures for making these material systems can ever be scaled up enough to be viable for use in technologies. The properties of crystals differ from the properties of atoms floating in free space because the atomic orbitals of the atoms in a crystal are close enough to those of adjacent atoms for electrons to hop between atoms. The resulting hybridization of atomic orbitals produces quantum states delocalized over the entire crystal with the capacity to carry momentum. This situation is shown in schematic form in Fig. 1.9A. For quantum states delocalized over the entire crystal, position ceases to be a useful basis. Instead, under these conditions we label electronic wave functions by their momenta, kx and ky. The atomic orbitals that prior to hybridization had discrete energy spectra now have energy spectra given by discrete functions of momentum, f. We call these functions electronic bands. Electrons loaded into the electronic bands of a two dimensional crystal will occupy the quantum states with the lowest available energies, so we can specify a maximum energy at which we expect to find electrons for any given electron density. We call that energy EF , the Fermi level. We can raise the Fermi level by adding additional electrons to the crystal, as shown in Fig. 1.9B. We have already discussed how two dimensional crystals naturally allow for manipulation of the electron density, square plant pots and thus the Fermi level. We have also already discussed how the application of an out-of-plane electric field to a two dimensional crystal will change the structure of the atomic orbitals supported by that crystal.

It naturally follows that atomic orbitals so modified will produce different electronic bands, as shown in Fig. 1.9C. It is relatively straightforward to compute how electronic bands will respond to the application of a displacement field. We will be using the momentum and energy basis for the rest of this document; this basis is known as momentum space. The simplest experiment we can perform to probe the electronic properties of a two dimensional crystal in this geometry is an electronic transport experiment, in which a voltage is applied to a region of the crystal with another region grounded, so that electrical current flows through the crystal. We can check if the crystal supports any electrical transport at all, and if it does we can measure the electrical resistance of the crystal this way, in close analogy to how this is done for three dimensional crystals. Crystals will only accept and thus conduct electrons if there are available quantum states at the Fermi level; we call these crystals metals , and they can be identified in band structure diagrams by the intersection of the Fermi level with an electronic band . Crystals without empty quantum states at the Fermi level will not accept and conduct electrons , and they can be identified in band structure diagrams with crystals for which the Fermi level does not intersect with an electronic band . There exists a variety of other experiments we can perform on two dimensional crystals in order to understand their properties. Two dimensional crystals can support electronic transport in the in-plane direction if they are metals, as shown in Fig. 1.10. Capacitors can also support electronic transport in the out-of-plane direction, as long as that electronic transport occurs at finite frequency. The same structure that we use to modify the electron density and ambient out-of-plane electric field of a two dimensional crystal can also be used as a capacitive AC conductor, as illustrated in Fig. 1.11A.

The conductance will depend only on the frequency at which an AC voltage is applied and the geometry of the parallel plate capacitor. However, if a two dimensional crystal is added in series, the capacitance of the top gate to the bottom gate may be substantially modified. If the two dimensional crystal is an insulator, electric fields will penetrate it and the capacitance between the two gates will not change. However, if the two dimensional crystal is a metal it will accept electrons and cancel the applied electric field, dramatically reducing the capacitance between the top and bottom gates and neutralizing the AC current through the capacitor. This technique can be used to measure the electronic properties of specifically the bulk of a two dimensional crystal; it is the property that was both calculated and measured in Fig. 1.2C and D. These two techniques are the bread and butter of the experimental study of two dimensional crystals, because they require only the ability to create stacks of two dimensional crystals and access to tools common to the study of all other microelectronic systems. We will discuss a considerable amount of electronic transport and capacitance data as well. However, the primary focus of this thesis will be on systems for which the nanoSQUID microscope can provide important information that is inaccessible to these techniques, and so we will discuss a few such systems next. Consider the following procedure: we obtain a pair of identical two dimensional atomic crystals. We slightly rotate one relative to the other, and then place the rotated crystal on top of the other . The resulting pattern brings the top layer atoms in alignment with the bottom layer atoms periodically, but with a lattice constant that is different from and in practice often much larger than the lattice constant of the original two atomic lattices. We call the resulting lattice a ‘moir´e superlattice.’ The idea to do this with two dimensional materials is relatively new, but the notion of a moir´e pattern is much older, and it applies to many situations outside of condensed matter physics. Pairs of incommensurate lattices will always produce moir´e patterns, and there are many situations in daily life in which we are exposed to pairs of incommensurate lattices, like when we look out a window through two slightly misaligned screens, or try to take pictures of televisions or computer screens with our camera phones. Of course these ‘crystals’ differ pretty significantly from the vast majority of crystals with which we have practical experience, so we’ll have to tread carefully while working to understand their properties. To start with, if we attempt to proceed as we normally would- by assigning atomicorbitals to all of the atoms in the unit cell, computing overlap integrals, and then diagonalizing the resulting matrix to extract the hybridized eigenstates of the system- we would immediately run into problems, because the unit cell has far too many atoms for this calculation to be feasible.

There exist clever approximations that allow us to sidestep this issue, and these have been developed into very powerful tools over the past few years, but they are mostly beyond the scope of this document. I’d like to instead focus on conclusions we can draw about these systems using much simpler arguments. The physical arguments justifying the existence of electronic bands apply wherever and whenever an electron is exposed to an electric potential that is periodic, and thus has a set of discrete translation symmetries. For this reason, even though the moir´e superlattice is not an atomic crystal, we can always expect it to support electronic band structure for the same reason that we canal ways expect atomic crystals to support band structure. Two crystals with identical crystal symmetries will always produce moir´e superlattices with the same crystal symmetry, so we don’t need to worry about putting two triangular lattices together and ending up with something else.Another property we can immediately notice is that the electron density required to fill a moir´e superlattice band is not very large. This can be made clear by simply comparing the original atomic lattice to a moir´e superlattice in real space . Full depletion of a band in an atomic crystal requires removing an electron for every unit cell , plastic pots for planting and full filling of the band occurs when we have added an electron for every unit cell. We have already discussed how this is not possible for the vast majority of materials using only electrostatic gating, because the resulting charge densities are immense. Full depletion of the moir´e band, on the other hand, requires removing one electron per moir´e unit cell, and the moir´e unit cell contains many atoms . So the difference in charge density between full filling and full depletion of an electronic band in a moir´e superlattice is actually not so great , and indeed this is easily achievable with available technology. Before we go on, I want to make a few of the limitations of this argument clear. There are two things this argument does not necessarily imply: the moir´e bands we produce might not be near the Fermi level of the system at charge neutrality, and the bandwidth of the moir´e superlattice need not be small. In the first case, we won’t be apply to modify the electron density enough to reach the moir´e band, and in the latter, we won’t be able to fill the moir´e band’s highest energy levels using our electrostatic gate. We know of examples of real systems with moir´e superlattice bands that fail each of those criteria. But if these moir´e superlattice bands are near charge neutrality, and if their bandwidths are small, then we should be able to easily fill and deplete them with an electrostic gate.Finally, moir´e superlattice bands inherit any electronic degeneracies- like, for example, electron spin- that came with the original lattice. We haven’t discussed electronic degeneracies yet, and we will shortly. So if a moir´e superlattice satisfies all of these criteria, then it will provide a set of electronic bands that can be completely filled or depleted with an electronic gate. I’m sure this seems to the reader like a pretty niche system, and that’s more or less because it is. There aren’t too many material systems that need their atomic bonds aligned with a mechanical goniometer, and it’s hard to imagine ever integrating such a procedure into an industrial fabrication line. However, it’s tough to adequately express how hard it would be to replicate the properties of a moir´e superlattice band in an atomic crystal. I made an attempt to do so in the introduction to this thesis; suffice to say the control we have over the properties of these systems is more or less unprecedented within experimental condensed matter physics, and this means that we can perform experiments on electronic phases in these systems that would be difficult or impossible in atomic crystals. A variety of scanning probe microscopy techniques have been developed for examining condensed matter systems. It’s easy to justify why magnetic imaging might be interesting in gate-tuned two dimensional crystals, but magnetic properties of materials form only a small subset of the properties in which we are interested. Scanning tunneling microscopy is capable of probing the atomic-scale topography of a crystal as well as its local density of states, and a variety of scanning probe electrometry techniques exist as well, mostly based on single electron transistors. It’s worth pointing out that if you’re interested specifically in performing a scanning probe microscopy experiment on a dual-gated device, then these techniques both struggle, because the top gate both blocks tunnel current and screens out the electric fields to which a single electron transistor would be sensitive. Magnetic fields have an important advantage over electric fields: most materials have very low magnetic susceptibility, and thus magnetic fields pass unmodified through the vast majority of materials . This means that magnetic imaging is more than just one of many interesting things one can do with a dual-gated device; in these systems, magnetic imaging is a member of a very short list of usable scanning probe microscopy techniques. The simplest way in which we can use our nanoSQUID magnetometry microscope is as a DC magnetometer, probing the static magnetic field at a particular position in space .

Loss of integrity of the cuticle and epithelial cells of the integumentary and tracheal systems in T. molitor larvae due to densovirus infection may not only cause dehydration via excessive water loss, but also secondary infection by bacteria and fungi as a result of the loss of their primary defense barrier against infection. One limitation encountered in this study was the interpretation of a dark discoloration presented by densovirus-infected T. molitor larvae. Further investigation into the mechanism of this change is suggested as there remains uncertainty to whether this is in fact hemocyte granule prophenoloxidase-mediated melanization or some other possible explanation, such as the collection of debris over an unshed and desiccated cuticle. InI is the most important microscopical feature of the densovirus infection in T. molitor. InI observed on bright-field microscopy represents the DRAC observed on electron microscopy. The infection with TmDNV induces homogeneous basophilic- and eosinophilic-texturized InIs. Feulgen staining confirmed the DNA-dominant nature of the basophilic InIs, which correlates with DRACs accumulating maturing and mature virions on TEM as is shown in Figs. 6d and 7a. On the other hand, a great number of eosinophilic-texturized InIs presented with a fainter staining after the Feulgen reaction, which correlates with DRACs shown in TEM photomicrographs in Fig. 7c–f. Eosinophilic-texturized inclusions represented DRACs that ultrastructurally displayed an evolving mixture of heterogeneous viral matrix, maturing and mature virions, and ribosomes. Viral matrix observed in DARC was composed of electron-dense fibrillar small-aggregating-to-long-anastomosing streams, which evolved into a large compact mass that resembles the shape of a “rotary dial” . Similar structures were described in early studies of G. mellonella infected with densovirus. Later, H-1 parvovirus studies indicated that H-1 parvovirus–associated replication bodies, square plastic planter which are not uncommonly observed in DRAC in T. molitor , are composed of nucleolar fibrils and serve as the location of viral DNA replication. Differentiating maturing and mature virions from ribosomes in the infected nucleus on conventional plastic-embedded preparations posed a challenge.

However, the large number of virus particles recovered on direct TEM supports the observation that large quantities of round vesicles/ particles of about 18 to 24 nm in diameter found within the DRAC were in fact maturing and mature densovirus virions rather than ribosomes. The DRAC displayed a very pleomorphic morphology, which varied from accumulation of maturing and mature virions to dense virus matrix that mimicked nucleolar structures and paracrystalline arrays. This heterogeneous appearance of the DRAC is an important feature that characterizes and facilitates TmDNV diagnosis at the ultrastructural level. Initial TEM studies on the G. mellonella densovirus demonstrated that the nucleoplasm is completely replaced by virions during the first 20 hours of infection. As in other autonomous parvoviruses that depend on the S phase of the infected cells, TmDNV replication and assembly is a unique and dynamic process that demands a deeper understanding. Nonetheless, crystallization of the DRAC forming “paracrystalline arrays” was interpreted as the final stage of nuclear infection with densovirus in T. molitor. Numerous infected cells presented cytoplasmic membrane–bound vesicles containing virus particles or paracrystalline arrays. Intracytoplasmic paracrystalline arrays have been considered a distinct feature of Densovirinae infections. The origin of the structures is not well understood, but it should be considered as a process of infected nuclear breakdown, heterophagia, or autophagia. Ultrastructural changes of the nuclei and cytoplasm in TmDNV–infected epithelial cells suggest apoptosis as the mechanism of cell death, although studies on viruses of the Parvoviridae family suggest that the mechanisms of Parvoviridae-induced cell death are unique and complex, warranting further investigation. Fruit, nut, and berry crops are commonly grouped into one of three categories: temperate, subtropical, and tropical. Temperate zone crops include almond, apple, apricot, peach, grape, blueberry, and strawberry . Avocado, citrus, and guava are considered to be subtropical, while banana, cashew, and pineapple are tropical . Generally, temperate and subtropical crops can be grown in San Mateo and San Francisco Counties , but tropical crops are rarely successful.

This publication focuses on temperate and subtropical crops. Temperate zone crops generally require a period of cold temperature during the winter months for successful flower and fruit development. This cold temperature period is measured in “chill hours” . Some crops require many chill hours, while others require few. This is called the crop’s “chill requirement.” When selecting temperate zone crops, it is important to choose only those crops that have a chill requirement that will be met at your location. Subtropical crops, such as citrus, loquat, and guava, require little or no chilling. Native to warm-climate regions, these crops can be injured by cold temperatures during winter and spring months, and they require heat during the growing season for fruit maturation and flavor.Selecting climate zones and meeting chill requirements are not the only factors necessary for good fruit production. Pollination, sunlight, heat accumulation, and wind are all important considerations. For fruit development to occur, flowers have to be pollinated; that is, pollen must move from the male organs to the female organs . Pollen transfer can be facilitated by bees, beetles, flies, butterflies, moths, birds, bats, wind, and water. The pollen can come from flowers on the same tree , or from flowers of other trees of the same species . In some cases, a crop may require another tree of a specific variety for pollination. For information on pollination requirements, see the “Crop and Variety Selection Table” at the end of this publication. For further details, see “Notes on Crops and Varieties” below. Note that even in self fertile crops, cross-pollination can increase fruit set. Also, poor pollination can occur as a result of insufficient pollinator activity, such as during cool and wet weather in the spring when bees may be less active . For most crops, plentiful sunlight and warm temperatures during the growing season are needed. Factors such as fog, wind, elevation, aspect , and distance from the ocean affect sunlight level and temperature.In recent years, a variety of novel two-dimensional van der Waals magnets have been discovered, founding the active field of 2D magnetism.

Among these prospective compounds, binary chromium tellurides Cr1–δTe are attractive owing to their rich magnetic properties, as well as inherent chemical and structural compatibility when forming heterostructures with other topological systems, such as tetradymite-type topological insulators or chalcogenide-based Dirac/ Weyl semimetals. Furthermore, the broken time-reversal symmetry and spin-orbit coupling offer unique opportunities for the interplay between spin configurations and reciprocal-space topology. In this regard, ferromagnetic Cr2Te3 with strong perpendicular magnetic anisotropy is an intriguing platform to host non-trivial topological physics, particularly for the high-quality thin films grown by molecular beam epitaxy. The intrinsic AHE is topological in nature and a hallmark of itinerant ferromagnets, which has also been observed in more exotic systems even without a net magnetization, such as spin liquids, antiferromagnets, and Weyl semimetals. When SOC coexists with long-range magnetic order, square plastic plant pot the Berry curvature can be significantly influenced near avoided band crossings, rendering the system an incredibly rich playground combining topology and magnetism. Here, we report the unique magnetotransport signatures of highquality quasi-2D Cr2Te3 MBE-grown thin films governed by non-trivial band topologies. Via synergetic structural, magnetic, and transport measurements, together with first-principles simulations, we have uncovered novel Berry-curvature-induced magnetism featuring an extraordinary sign reversal of the AHE as we modulate the temperature and the strain for the thin films containing 3–24 unit cells on Al2O3 or SrTiO3 substrates. Moreover, a hump-shaped Hall feature emerges, most likely due to the presence of multiple magnetic layers/domains under different levels of interfacial strain. This work identifies pristine ferromagnetic Cr2Te3 thin films as a fascinating platform for further engineering topological effects, given their nontrivial Berry curvature physics.The crystalline structure of Cr2Te3 thin films is described first, followed by the development of strain at the substrate/film interface by the epitaxy. Bulk Cr2Te3 crystalizes in a three-dimensional lattice with space group P31c ðD2 3d,No:163Þ, as shown in Fig. 1a–c, where each unit cell contains four vertically stacked hexagonal layers of Cr. There are three symmetrically unique sites for Cr, labeled Cr1, Cr2, and Cr3, respectively: The Cr1 atoms are sparsely arranged in a weakly antiferromagnetic sublattice, while the Cr2/Cr3 atoms form ferromagnetic layers similar to those in CrTe2. Since the Cr1 sites are often only partially filled , Cr2Te3 behaves essentially as a quasi-2D magnet. This quasi-2D nature of Cr2Te3 allows for high-quality, layer-by-layer epitaxial growth of c-oriented films on a variety of substrates. The hexagonal c axis is the easy magnetic axis, leading to PMA for the films. The sixfold in-plane symmetry is seen in the honeycombs visualized by atomic resolution scanning tunneling microscopy and scanning transmission electron microscopy high-angle annular dark-field imaging, as well as in the reflection high-energy electron diffraction and X-ray diffraction patterns.

The sharp substrate/film interface is confirmed by the cross-sectional HAADF and the corresponding integrated differential phase contrast images. The intrinsic random distribution of Cr atoms on the Cr1 sites is resolved in the enlarged view of the atoms in Fig. 1k–m, shown overlaid with red circles, while the overall chemical composition of the thin film is uniform within the resolution of energy dispersive X-ray spectroscopy . Figure 1f illustrates the basic sample architecture, where the strain in the Cr2Te3 thin films is governed by the interface with the substrate.Upon reducing the thickness t, films grown on Al2O3 can develop an IP compressive strain up to −0.15%, as determined by XRD and summarized in Fig. 1d. A higher strain level can be sustained using SrTiO3 substrates. Such control of strain is well suited for exploring interface-sensitive properties in Cr2Te3 thin films.The magnetic properties of Cr2Te3 thin films with selected thicknesses were assessed using vibrating sample magnetometry . Figure 2a shows the temperature dependence of the magnetization M for a t = 24 u.c. film on Al2O3 substrate with an out-of-plane applied magnetic field μ0H = 0.1 T. Under the field-cool condition, M rises below the Curie temperature TC ~ 180 K, reaching M ~2.50 μB per Cr at 2 K in the 0.1 T field. The zero-FC scan, on the other hand, deviates from the FC curve below the blocking temperature Tb, signaling the freezing out of domains in a random direction in the absence of an aligning field. As illustrated in Fig. 2b, Cr2Te3 favors PMA with coercive field μ0Hc = 0.76 T and saturation magnetization Ms ~2.83 μB per Cr at 2 K for t = 24 u.c., whereas the IP measurements have weaker ferromagnetic hysteresis loops. The low-T zero-field kink feature in the OOP M becomes more prominent with reduced thickness . The multistep hysteresis attests to the presence of varied layer-dependent magnetic anisotropies, despite the overall chemical and phase homogeneity of the films34. This is consistent with the interfacial strain-driven magnetic profiles revealed by the depth-sensitive polarized neutron reflectometry as described below. The PNR experiments, responsive to the IP magnetization, were carried out at chosen T and H on samples with t = 24 and 6 u.c. to uncover the impact of interfacial strain and the details of the stepwise hysteresis loops due to the interplay between anisotropy and the Zeeman energies in an applied external magnetic field. The PNR spin asymmetry ratio SA = /, measured as a function of the wave vector transfer Q = 4πsin/λ with R+ and R− being the reflectivity for the neutron spin parallel or antiparallel to the external field, evidently confirms the magnetization . By simultaneously refining PNR and X-ray reflectivity data, the depth profiles of nuclear and magnetic scattering length densities at μ0H = 1 T, 0.8 T and 0.05 T for t = 24 u.c. were obtained and are shown in Fig. 2c. The uniform MSLD profile at the IP saturation field μ0H = 1 T attests to the high quality of the magnetic Cr2Te3 film with well-defined interfaces of 0.5 nm roughness. Remarkably, at reduced IP field μ0H = 0.8 T and 0.05 T, M develops a non-uniform depth-dependent profile with two distinct regions, possessing a lower IP magnetization value close to the substrate. Given that the NSLD depth profile of the Cr2Te3 layer is uniform and no changes are detected in the structure and chemical composition of the film, we attribute the reduced IP magnetization approaching the substrate to a canting of the magnetization vector towards the OOP direction .

Those differences could be explained by the diploid phasing of the Cabernet Sauvignon genome assembly and that multiple ISNT transcripts might correspond to a single gene locus. Nonetheless, similar relative amounts of Biological Process GO terms were found among the differentially expressed genes, confirming that the transcriptome obtained using Iso-Seq captured the transcriptional reprogramming underlying the main physiological and biochemical changes during grape berry development. In addition, gene expression analysis revealed that some private isoforms are significantly modulated during berry development, indicating that in addition to identifying the private gene space, the ISNT reference makes it possible to observe its expression. In conclusion, this study demonstrates that Iso-Seq data can be used to create and refine a comprehensive reference transcriptome that represents most genes expressed in a tissue undergoing extensive transcriptional reprogramming during development. In grapes, this approach can aid developing transcriptome references and is particularly valuable given diverse cultivars with private transcripts and accessions that are genetically distant from available genome references, like the non-vinifera Vitis species used as rootstocks or for breeding. The pipeline described here can be useful in efforts to reconstruct the gene space in plant species with large and complex genomes still unresolved.While San Joaquin Valley vineyards are currently fertilized with boron through the soil and foliage , some growers have expressed interest in applying boron via drip irrigation or “fertigation.” Fertigation is a relatively simple, cost-effective and efficient way to apply nutrients. However, irrigation water with more than 1 part per million boron can lead to vine toxicity, so the safety of boron fertigation is also a concern. Our research evaluates the safety and efficacy of boron fertigation in grapevines using drip irrigation. Boron is unique among the micronutrients due to the narrow range between deficiency and toxicity in soil and plant tissues. For grapevines, 25 liter plant pot this range is 0.15 ppm to 1 ppm in saturated soil extracts, and 30 ppm to 80 ppm in leaf tissue.

The goal of boron fertilization of grapevines is to keep tissue levels within this narrow range, since both deficiency and toxicity can have serious negative effects on vine growth and production. Fertilization amounts must be precise to avoid toxicity while providing adequate boron to satisfy grapevine requirements . On the east side of the San Joaquin Valley, boron deficiency of grapevines occurs on soils formed from igneous rocks of the Sierra Nevada. This parent material is low in total boron, which is crystallized in borosilicate minerals that are highly resistant to weathering. Boron deficiency is often associated with sandy soils and vineyard areas with excessive leaching, such as in low spots or near leaky irrigation valves. Vine symptoms of boron deficiency are more widespread and pronounced following high rainfall years, when greater amounts of soluble boron are leached from the root zone. In addition, snow melt water has very low levels of boron, and vineyards irrigated primarily with this water have a greater risk of deficiency. Boron is required for the germination and growth of pollen during flowering, and vines that are deficient in this micronutrient will have clusters that set numerous shot berries, small berries with a distinctive pumpkin shape. When boron deficiency is severe, vines produce almost no crop. Foliar symptoms appear in the spring: shoots have shortened, swollen internodes and their tips sometimes die, and leaves have irregular, yellowish mottling between the veins. Grapevines are also sensitive to too much boron. Toxicity is common on the west side of the San Joaquin Valley, where most soils are derived from marine sedimentary and meta sedimentary parent material that is rich in easily weathered boron minerals. Symptoms of boron toxicity include leaves that are cupped downward in the spring and that develop brown spots adjacent to the leaf margin in middle or late summer, intensifying and leading to necrosis as boron accumulates. Yields are reduced, the result of diminished vine vigor and canopy development. When foliar boron sprays are applied in excess in the spring, juvenile leaves become cupped within 2 weeks; however, vines quickly recover and yields are usually unaffected. Toxicity also occurs when boron fertilizer is applied in excess, regardless of the soil type, and this can lead to yield loss. Over-fertilization is the sole reason for boron toxicity on the east side of the San Joaquin Valley, so it is critical to establish how much boron fertilizer can be applied safely and effectively.

Our research investigated the uptake of boron by grapevines when fertilizer was applied with a drip-irrigation system.Research was conducted from 1998 to 2001 in a mature ‘Thompson Seedless’ raisin vineyard near Woodlake in Tulare County. The vineyard was planted in Cajon sandy loam on a recent alluvial fan associated with the Kaweah River. This soil is derived from granitic parent materials, and the surface soil is highly micaceous with a slight to moderate amount of lime. The underlying soil has a coarse, sandy texture. At the onset of this study, the vineyard’s boron status was in the questionable range for deficiency. The vine’s leaf petioles and blades contained about 30 ppm boron. While the foliage had no symptoms of boron deficiency, in the past the grower had observed sticking caps and pumpkin-shaped shot berries, which are indicative of boron deficiency. During the course of the research, the vineyard was drip-irrigated from April through October. The vineyard canopy covered 60% of the land surface during summer months and about 20 inches of water was applied during the season. Boron treatments consisted of applying fertilizer in varying amounts 3 weeks prior to bloom on May 18, 1998, and then again 3 weeks prior to bloom the following year, on May 3, 1999. Growers who fertigate grapevines with a drip system generally inject material into the irrigation water over a 45-to- 60-minute period at the beginning of an irrigation set. We simulated fertigation by applying Solubor, a soluble boron product , to a shovel-sized hole beneath drippers during the first hour of the irrigation set. By doing this, precise amounts of boron could be applied to each plot and plot size could be reduced. This technique has been used successfully in previous research with other nutrients . The experiment was designed as a randomized complete block with five treatments, five blocks and five vine plots . To evaluate the rate of boron uptake and accumulation in tissue with consecutive years of fertilization, grape tissue samples were collected in 1998 and 1999 at bloom and then again about 6 weeks later during veraison. Veraison is the stage of development where berries begin to soften and/or color. To evaluate carryover, leaf tissue samples were also collected at this Tulare County site at both bloom and veraison in 2001, 2 years after the fertilization was discontinued. In each case, 100 petioles and 50 blades were sampled per plot from the center three vines. Petioles and blades were taken opposite inflorescences during bloom, and recently matured leaves were sampled at veraison. Samples were oven-dried, ground in a Wiley mill and sent to the UC Davis DANR Analytical Laboratory for analysis of total boron. Statistical analysis was by ANOVA using least significance difference to separate treatment means. A second experiment was conducted in 1998 in Fresno County near Selma, in a mature Thompson Seedless raisin vineyard planted on Pollaski sandy loam and drip-irrigated.

The soil was formed in place from the weathering of softly to moderately consolidated granitic sediments. The particle size distribution of the surface soil is 63% sand, 25% silt and 12% clay. At the onset of the experiment, boron tissue levels were in the adequate range, 40 ppm. In both experiments, drip irrigations during the season were based on a schedule using historical evapotranspiration and developed for raisin vineyards in the San Joaquin Valley . The irrigation source was high-quality pump water with a boron concentration less than 0.1 ppm. The experimental design and methods were identical in both vineyards, except that the 1/16-poundper-acre boron treatment was omitted in the second vineyard. The Fresno County trial was discontinued after tissue samples were taken at bloom and veraison in 1998.At both the Tulare and Fresno county sites, black plastic plant pots boron uptake was rapid when fertilizer was applied in the spring. In both vineyards, applying boron at 2/3 or 1 pound per acre increased the boron concentration in blades by bloom, 3 weeks after application. Boron increased further in blades by veraison . In the Tulare County vineyard, boron in bloom tissue increased from a questionable deficiency range to adequate; at the Fresno location, boron in bloom tissue increased from 40 ppm to 54 ppm, a dramatic increase considering boron fertilizer was applied just 3 weeks prior. This indicates that boron uptake is rapid. None of the fertigation treatments resulted in either symptoms of boron toxicity or deficiency. Applying boron at 1/3 pound per acre or less did not significantly increase boron tissue levels by bloom or veraison at either site the first year. Fertigation over consecutive years was evaluated at the Tulare County location. Boron in grapevine tissue continued to increase with consecutive years of application. At the higher fertilizer rate , boron levels in blades increased from 35 ppm in control vines to about 60 ppm. We speculate that continuing with annual applications of 1 pound boron per acre would result in toxicity within 4 to 5 years. The 1/3-pound-per-acre rate significantly elevated boron in blades by veraison of the second year to adequate levels . There were no visual signs of toxicity in any of the fertilizer treatments, even when boron was applied at 2 pounds per acre in a single application. Boron levels in tissue remained unchanged 2 years after fertilization was discontinued at the Tulare County location . This indicates substantial treatment longevity with fertigation of a drip-irrigated vineyard. Rainfall during this experiment was below normal, which helped minimize leaching. Also, well-managed drip irrigation minimizes leaching. Under drip irrigation, salts tend to accumulate near the soil surface and 2 to 3 feet away from the drip line, with minimal water and salt movement below the root zone when irrigations are accurately scheduled . Boron concentrated more in the blades than in the petioles in response to fertilization. At the onset of the Tulare County experiment, boron concentrations in petioles and blades were similar at 31 ppm and 34 ppm, respectively. Fertilizing with 1 pound boron per acre for 2 consecutive years resulted in a 25% increase of boron in petioles but a 76% increase in blades . All fertilizer treatments increased boron in blades more than in petioles, indicating that blades should be sampled when monitoring the vines’ boron status following fertilization. Potential boron toxicity values at the time of sampling during the bloom period are 80 ppm for petioles and 120 ppm for blades, and in mid- to late summer are 100 ppm for petioles and 300 ppm for blades.Annual boron fertigation at 1/3 pound per acre elevated grapevine tissue levels from questionable to the adequate range within 2 years . In addition, tissue boron levels remained unchanged 2 years after fertilization was discontinued. This is probably because leaching was reduced by two factors: below-normal rainfall and accurately scheduled drip irrigations. After fertilization, boron was concentrated more in blades than in petioles, indicating that blades are the best choice for monitoring toxicity. Blade samples should be monitored on a routine basis and fertilizer amounts should be adjusted accordingly to avoid boron toxicity or deficiency. The results of this research can be applied to other drip-irrigated vineyards in the San Joaquin Valley under similar conditions: rapidly drained soils, high quality irrigation water, and low boron content in soil, water and vine tissue. In other regions of the state where winter rainfall is much higher, there would presumably be more leaching of boron fertilizer during winter months and less carryover time after fertilization is discontinued. In contrast, less leaching and greater carryover of boron would be expected in areas of less rainfall or on soils with finer texture and higher water-holding capacity. These variables underscore the importance of monitoring boron in tissue when developing a long-term fertilization program.The study of crop domestication has long been used as a proxy for studying evolutionary processes, such as the genetic effects of bottlenecks and the detection of selection to identify agronomically important loci .

To further explore links between management and soil fertility, we used the results from the PCA to formalize a gradient in management across all farms, and then used this gradient as the basis for comparison between Field A and Field B across all indicators for soil fertility. Using the ggplot and tidy verse packages , we displayed the difference in values between Field A and Field B for each indicator for soil fertility sampled at each farm using bar plots. We also included error bars to show the range of uncertainty in these indicators for soil fertility. Lastly, we further compared Field A and Field B for each farm using radar plots. To generate the radar plots, we first scaled each soil indicator from 0 to 1. Using Jenks natural breaks optimization, we then grouped each farm based on low, medium, and high N-based fertilizer application, as this soil management metric was the strongest coefficient loading from the first principal component . Using the fmsb package in R , we used an averaging approach for each level of N-based fertilizer application to create three radar plots that each compared Field A and Field B across the eight indicators for soil fertility. Farmers provided an overview of their farm operation, including farm size , the total number of crops each farm planted per growing season at the whole farm level, the types of crops planted in their field during the initial field visit , the type and amount of nitrogen-based fertilizer they applied on farm, and key aspects of soil health in their own words . Farm sizes ranged from 15 to 800 acres, with about one third of farms in the 15 – 50-acre range, drainage pot another third in the 100 – 450-acre range, and roughly a final third in the 500 – 800 acre-range. Farmers grew primarily summer crops, including tomato, a variety of cucurbits, strawberry, herbs, nightshades, root vegetables, and sunflower/safflower for oil.

Farmers reported applying a range of external N-based organic fertilizers, including fish emulsion, Wiserg , pelleted chicken manure, and seabird guano, at varying rates . On the low end, farmers applied <1 kg-N/acre, and on the high end, farmers applied 90 – 180 kg-N/acre per season. About a third of farmers applied 2 – 25 kg-N/acre of N-based fertilizer. Farmer responses for describing key aspects of soil health were relatively similar and overlapped considerably in content and language . Specifically, farmers usually emphasized the importance of maintaining soil life and/or soil biology, promoting diversity, limiting soil compaction and minimizing disturbance to soil, and maintaining good soil structure and moisture. Several farmers also touched on the importance of using crops as indicators for monitoring soil health and the importance of limiting pests and disease. Discussion of the importance of promoting soil life, soil biology, and microbial and fungal activity had the highest count among farmers with ten mentions across the 13 farmers interviewed. Next to this topic, minimizing tillage and soil disturbance was the second most discussed with six of 13 farmers highlighting this key aspect of soil health. In addition to discussing soil health more broadly, farmers also provided in-depth responses to a series of questions related to soil fertility—such as key nutrients of interest on their farm, details about their fertility program, and the usefulness of soil tests in their farm operation— summarized in Table 2. When asked to elaborate on the extent to which they considered key nutrients, a handful of farmers readily listed several nutrients, including nitrogen, phosphorous, potassium , and other general macronutrients as well as one micronutrient .

Among these farmers that responded with a list of key nutrients, some talked about having their nutrients “lined up” as part of their fertility program. This approach involved keeping nutrients “in balance,” such as for example, monitoring pH to ensure magnesium levels did not impact calcium availability to plants. These farmers also emphasized that though nitrogen represented a key nutrient and was important to consider in their farm operation, tracking soil nitrogen levels was less important than other aspects of soil management, such as promoting soil biological processes, maintaining adequate soil moisture and aeration, or planting cover crops regularly. As one farmer put it, “if you add nutrients to the soil, and the biology is not right, the plants will not be able to absorb it.” Or, as another farmer emphasized, “It’s not about adding more [nitrogen]… I try to cover crop more too.” A third farmer emphasized, that “I don’t use any fertilizers because I honestly don’t believe in adding retroactively to fix a plant from the top down.” This same farmer relied on planting a cover crop once per year in each field, and discing that cover crop into the ground to ensure his crops were provided with adequate nitrogen for the following two seasons. While most farmers readily listed key nutrients, several farmers shifted conversation away from focusing on nutrients. These farmers generally found that this interview question missed the mark with regards to soil fertility. One farmer responded, “I’m not really a nutrient guy.” This same farmer added that he considered [soil fertility] a soil biology issue as much as a chemistry issue.” The general sentiment among these farmers emphasized that soil fertility was not about measuring and “lining up” nutrients, but about taking a more holistic approach. This approach focused on facilitating conditions in the soil and on-farm that promoted a soil-plant-microbe environment ideal for crop health and vigor. For example, the same farmer quoted above mentioned the importance of establishing and maintaining crop root systems, emphasizing that “if the root systems of a crop are not well established, that’s not something I can overcome just by dumping more nitrogen on the plants.” Another farmer similarly emphasized that they simply created the conditions for plants to “thrive,” and “have pretty much just stepped back and let our system do what it does; specifically, we feed our chickens whey-soaked wheat berries and then we rotate our chickens on the field prior to planting.

And we cover crop.” A third farmer also maintained that their base fertility program—a combination of planting a cover crop two seasons per year, an ICLS chicken rotation program, minimal liquid N-based fertilizer addition, and occasionally compost application—all worked together to “synergize with biology in the soil.” This synergy in the soil created by management practices—rather than focusing on nutrient levels—guided this farmer’s approach to building and assessing soil fertility on-farm. Another farmer called this approach “place-based” farming. This particular farmer elaborated on this concept, saying “I think the best style of farming is one where you come up with a routine [meaning like a fertility program] that uses resources you have: cover crops, waste materials beneficial to crops, animals” in order to build organic matter, which “seems to buffer some of the problems” that this farmer encountered on their farm. Similar to other farmers, this farmer asserted that adding more nitrogen-based fertilizer did not lead to better soil fertility or increase yields, drainage planter pot in their direct experience. Regardless of whether farmers listed key nutrients, a majority of farmers voiced that nitrogen was not a big concern for them on their farm. This sentiment was shared among most farmers in part because they felt the amount of nitrogen additions from fertilizers they added were insignificant compared to nitrogen additions by conventional farms. Farmers also emphasized that the amount of nitrogen they were adding was not enough to cause environmental harm; relatedly, a few farmers noted the absurdity and added economic burden of the recent nitrogen management plan requirements—specifically among organic farms with very low N-based fertilizer application. The majority of farmers also expressed that their use of cover crops and the small amount of N-based fertilizer additions as part of their soil fertility program ensured on-farm nitrogen demands were met for their crops. Across all farmers interviewed, cover cropping served as the baseline and heart of each fertility program, and was considered more effective than additional N-based fertilizers at maintaining and building soil fertility. Farmers used a range of cover crop species and often applied a mix of cover crops, including vetches and other legumes like red clover and cowpea , grains and cereals like oats . Farmers cited several reasons for the effectiveness of cover cropping, such as increased organic matter content, more established root systems, greater microbial activity, better aeration and crumble in their soils, greater number of earthworms and arthropods, improved drainage in their soils, and more bio-available N. Whereas farmers agreed that “more is not better” with regards to N-based fertilizers, farmers did agree that allocating more fields for planting cover crops over the course of the year was beneficial in terms of soil fertility. However, as one farmer pointed out, while cover crops provide the best basis for an effective soil fertility program, this approach is not always economically viable or physically possible. Several farmers expressed concern because they often must allocate more fields to cover crops than cash crops in any given season, which means that their farm operation requires more land to be able to produce the same amount of vegetables than if they had all their fields in cash crops.

Farmers also shared that in some circumstances, such as in early spring, they are not able to realize the full potential of a winter cover crop if they are forced to mow the cover crop early to plant cash crops and ensure the harvest timeline of a high-value summer vegetable crop. The cover crop approach to soil fertility takes “persistence,” as one farmer emphasized; another farmer similarly pointed out that the benefits of cover cropping “are not always realized in the crop year. You’re in it [organic agriculture] for the long haul, there is no quick fix.” Indeed, farmers who choose to regularly plant cover crops to build soil fertility, rather than just add N-based fertilizers, reported that they came up against issues of land tenure and access to land, market pressures, and long-term economic sustainability. To build on conversations about soil fertility, farmers also provided responses to interview questions that asked them to elaborate on the usefulness of available soil tests to gauge soil fertility more broadly—and then more specifically, the usefulness of soil tests in informing their soil fertility program and/or management approaches on-farm. Overall, only three of 13 farmers reported regularly using and relying on soil tests to inform their soil fertility program or aspects of their farm operation. These farmers offered very short responses and did not elaborate. For example, one farmer shared that they “test twice a year in general,” and that they “rely on the results of the soil tests to tweak [their] fertility program.” Another farmer said briefly, “We use soil tests… we utilize them to decide what to do to try to improve the soil.” A third farmer admitted that though he “used to do a soil test every year, literally used to spend hundreds of dollars per year on soil tests,” he found that the results of soil tests did not change year-to-year and were, as he put it, very “stable.” This particular farmer no longer regularly uses or relies on soil testing for their farm operation. The remaining ten farmers confirmed that they had previously submitted a soil test, usually once and most often to a local commercial lab in the region. These farmers expressed a range of sentiments when asked about the usefulness of soil tests, including disappointment, distrust, or both, particularly in the capacity of soil tests to inform soil fertility on their farm. Some farmers said directly, “I just don’t trust soil tests,” or “frankly, I don’t believe a lot in soil testing because it’s too standardized,” while other farmers initially stated they had used “limited” or “infrequent” soil tests, and then later admitted that they did not use or rely on soil tests on their farm operation. These farmers tended to focus on the limitations of soil tests that they encountered for their particular farm application. Limitations of soil tests discussed by farmers varied.