Author: naturehydro

The Berry phase not only has close connections to the optical selection rules that allow optical generation and detection of the valley-polarized carriers by circularly polarized photons, but also plays a central role in novel electron dynamics and transport phenomena in TMD and graphene layers, such as the valley Hall effect. In principle the Berry phase, together with other effects from inversion symmetry breaking, can have profound consequences for the wave function and energy spectrum of the excited states in two-dimensional materials. TMD monolayers are known to host strongly bound excitons with a remarkably large exciton binding energy due to enhanced Coulomb interactions in 2D. It was recently predicted that the Berry curvature of Bloch states can add an anomalous term to the group velocity of electrons and holes and creates an energy splitting between exciton states with opposite angular momentum Fig. 1a shows a simplified exciton energy spectrum illustrating the exciton fine structure based on our ab initio GW-Bethe-Salpeter equation calculations. The 2p+ and 2p− exciton states are split in energy with opposite order for the K and K’ valleys due to the opposite chirality in the two valleys. Such novel exciton fine structure, which embodies important wave function properties arising from the Bloch band geometry, can strongly modify the intraexcitonic light-matter interactions. Experimental observation of this predicted exciton spectrum, however, has been challenging, blueberry grow pot because it requires new spectroscopic probe that can distinguish both the momentum valley and the exciton angular momentum. Here, we report the first observation of the Berry-phase effect in the exciton spectrum of MoSe2 monolayer using intraexciton optical Stark spectroscopy.

We demonstrate that the degeneracy between the 2p±-exciton states is lifted by the Berry phase effect, and enabling a valley-dependent Autler-Townes doublet from strong intraexciton light-matter coupling. We coherently drive the intraexciton transitions using circularly-polarized infrared radiation, which couples the 1s exciton to the 2p+ or 2p− states selectively through the pump photon polarization . The pump-induced changes in the 1s exciton transition are detected by circularly polarized probes, which selectively measure the K or K’-valley excitons. Independent control of pump and probe photon polarization enables us to distinguish the exciton fine structures in the K and K’-valleys. We determine an energy splitting of 14 meV between the 2p+ and 2p− exciton states within a single valley, and this energy splitting changes sign between K and K’-valleys. We determine the 1s-2p transition dipole moment to be 55±6 Debye. This leads to an optical Stark shift that is almost 40 times larger than the interband counterpart under the same pump detuning and driving optical field strength. Such strong and valley-dependent intraexciton transitions open-up new pathways for the coherent manipulation of quantum states in 2D semiconducting materials using infrared radiation. To investigate the fine structure of the excitonic p-manifold, we fabricated a high quality MoSe2 monolayer that is encapsulated in hexagonal boron nitride layers using mechanical exfoliation and stacking following Ref. 21. The sandwiched hBN-MoSe2-hBN heterostructure was then transferred to an alumina-coated silver surface . The device was kept in vacuum at 77K for all optical measurements. This Aexciton peak arises from the optical transition between the ground state and the lowest energy 1s exciton state in MoSe2 monolayer, which is well-separated from the higher-lying exciton states due to strong Coulomb interactions in TMD monolayers.

It shows clearly that the 1s exciton transition exhibits avoided-crossing behavior in both valleys, which evolves gradually from energy blue shift to splitting and then to redshift as the pump photon energy is decreased. Due to the time-reversal symmetry between K and K’-valleys in MoSe2 monolayer, this observation also indicates that the 2p+ and 2p− exciton states are non-degenerate and has an energy difference of 14 meV in a single valley. We further plot the blue- and red-shifted 1s resonance as a function of the infrared pump photon energy in Fig. 3b. We find that the energy shifts induced by the intraexciton optical Stark effect are almost 40 times larger than its interband counterpart at the same pump intensity and resonance detuning21–23. To better understand the experimental results, we performed ab initio GW-BSE calculations using the BerkeleyGW26–28 package to determine the exciton energy levels and optical selection rules of exciton and intraexciton transitions in monolayer MoSe2. In these calculations, environmental screening effects from the hexagonal boron nitride encapsulation layers are included18 from first-principles . The simulation confirms the energy level diagram of the 1s, 2p+, and 2p− excitons and the optical selection rules in K and K’- valleys in Fig. 1a. Our calculations find that the energies of the 1s and 2p− exciton states are separated by 117 meV, with 2p+ exciton states further separated by 7 meV in K-valley. The energetic order of 2p+ , and 2p− excitons states is opposite in the K’-valley, as a result of time-reversal symmetry. Although the 2p± excitons are dark in linear optics, they are optically active when coupled to the 1s exciton with circularly-polarized light . For example, our calculations show that the 1s-2p+ intraexciton transition couple exclusively to the left-handed circularly polarized light with a transition dipole moment of 42 Debye. The 1s-2p− intraexciton transition, on the other hand, coupled exclusively to the right-handed circularly polarized light.

The experimentally observed intraexciton dipole moment and valley-dependent exciton fine structure match reasonably well with the ab initio GW-BSE calculations. The combination of 2p±-exciton splitting and extremely strong intraexcitonic light-matter interaction allow us to observe valley-dependent Autler-Townes doublets at higher pump intensity in MoSe2 monolayer. Towards this goal, we fabricated a hBN-encapsulated MoSe2 heterostructure on a zinc-sulphide substrate, where the local field factor on the sample for the infrared pump light is more favorable than that for MoSe2 on alumina coated silver substrate . Fig. 4c,d show the splitting energy in the Autler-Townes doublet at resonant excitation scales linearly with the excitation field strength, as expected from Eq. 129,30. At an effective driving intensity of 50±10 MW/cm2, which corresponds to a local optical field strength of 200±20 kV/cm, the Autler-Townes splitting can reach ~24 meV in both valleys. This Autler-Townes doublet leads to a valley dependent electromagnetically induced transparency in the 1s exciton transition, where the absorption at the 1s exciton resonance is reduced by more than 10-fold compared to the undriven exciton . Our findings offer a new and effective pathway to coherently manipulate the quantum states and excitonic excitations using infrared radiation coupled to the 1s-2p+ intraexciton transition.The dashed-lines indicate the peak position of unperturbed A-exciton. The dotted lines are guides to the eyes for the peak position at different driving energies. The spectra are offset for clarity and labelled according to the excitation energy . The spectra evolve from energy redshift to splitting and then to blue shift, as the driving energy is increased. The calculation is based on the Hamiltonian shown in Eq. 1. Exciton-photon coupling leads to avoided-crossing and the observed peak splitting at resonant coupling. This resonant coupling occurs at driving photon energy of 142 meV and 128 meV in the K and K’ valleys, respectively. The MoSe2 monolayer encapsulated in hBN flakes were prepared with a polyethylene terephthalate stamp by a dry transfer method21. Monolayer MoSe2 and hBN flakes were first exfoliated onto silicon substrate with a 90 nm oxide layer. We used PET stamp to pick-up the top hBN flake, monolayer MoSe2, and bottom hBN flake in sequence with accurate alignment based on an optical microscope. The hBN/MoSe2/hBN heterostructure was then stamped on a silver substrate coated with a 85 nm alumina layer or on a zinc sulphide substrate. Polymer and samples were heated to 60oC for the pickup and 130oC for the stamping process. Finally, hydroponic bucket the PET was dissolved in dichloromethane for 12 hours at room temperature. The sample temperature was kept at 77 K in a liquidnitrogen cooled cryostat equipped with BaF2 window during optical measurements. Pump-probe spectroscopy study is based on a regenerative amplifier seed by a mode-locked oscillator . The regenerative amplifier delivers femotosecond pulses at a repetition rate of 150 kHz and a pulse duration of 250 fs, which were split into two beams. One beam was used to pump an optical parametric amplifier and the other beam was focused onto a sapphire crystal to generate supercontinuum light for probe pulses. Femtosecond mid-infrared pump pulses with tunable photon energies were generated via difference frequency mixing of the idler pulses from the optical parametric amplifier and residual of fundamental output from regenerative amplifier in a 1 mm thick silver gallium sulphide crystal.

The mid-infrared pulse duration is ~350 fs. The pump-probe time delay was controlled by a motorized delay stage. The probe light was detected by high sensitivity CCD line camera operated at 1000 Hz. The helicity of pump and probe pulses was independently controlled using Fresnel rhomb and broadband quarter-wave plates, respectively. The experiment followed a reflection configuration with a normal incidence and collinear pump-probe geometry, where the absorption spectra are extracted from the reflectance contrast as described in the supporting information.This work was primarily supported by the Center for Computational Study of Excited State Phenomena in Energy Materials, which is funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division under Contract No. DE-AC02-05CH11231, as part of the Computational Materials Sciences Program which provided the experimental measurements and GW-BSE calculations. The sample fabrication and linear optical spectroscopy was supported by the US Army Research Office under MURI award W911NF-17-1-0312. The pump-probe setup was supported by the ARO MURI award W911NF- 15-1-0447. This research used resources of the National Energy Research Scientific Computing Center , a DOE Office of Science User Facility supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, and the Extreme Science and Engineering Discovery Environment , which is supported by National Science Foundation grant number ACI-1548562. S.T. acknowledges support from NSF DMR-1552220. K.W. and T.T. acknowledge support from the Elemental Strategy Initiative conducted by the MEXT, Japan and the CREST , JST. E.C.R acknowledges support from the Department of Defense through the National Defense Science & Engineering Graduate Fellowship Program. C.-K.Y. and C.S.O. acknowledges useful discussion with Prof. Ajit Srivastava. Viruses are obligate intracellular pathogens that require living host cells to replicate and spread in the infected plant. During compatible interactions, viruses overcome the plant immune system and hijack host cellular processes to establish active infections . Viruses disrupt the plant cell cycle, inhibit cell death pathways, restrict macromolecular trafficking, alter cell signaling, protein turnover, and transcriptional regulation, and suppress defense mechanisms. The interference with these processes in the host leads to a wide range of plant developmental and physiological defects . Cultivated grapevines are highly susceptible to a variety of viruses and viroids, which cause significant crop losses and shorten the productive life of vineyards. More than 65 different viral species classified in at least 15 families have been reported to infect grapevines, which represents the highest number of viruses so far detected in a single cultivated plant species . Although these viruses are generally transmitted by plant-feeding insects or soilborne nematodes, they can also be spread through infected propagation material . Grapevine red blotch is a viral disease discovered in northern California in 2008 that has become a major economic problem for the wine industry in the USA . This disease is caused by the Grapevine red blotch-associated virus , a circular ssDNA virus with resemblance to geminiviruses, which infects wine grape cultivars with significant detrimental effects on productivity . The incidence and severity of the red blotch symptoms vary depending on the grape cultivar, environmental conditions, and cultural practices . In red-skinned varieties, GRBaV infections result in the appearance of red patches on the leaf blades, veins, and petioles; in white-skinned varieties, they manifest as irregular chlorotic regions on the leaf blades. GRBaV also affects berry physiology, causing uneven ripening, higher titratable acidity, and lower sugar and anthocyanin content, among others . Consequently, must and wine produced from infected berries present altered flavor and aroma. To date, there is limited information on how GRBaV infections affect grape metabolism. Comprehensive analyses to study specific cellular processes that GRBaV exploits to promote infections in berries are still needed, in particular those that relate to changes in berry chemical composition during fruit development. Grape berry development exhibits a double sigmoid growth pattern with three distinct phases: early fruit development, lag phase, and berry ripening.

Using the Crop Sequence Boundaries dataset from USDA, I isolate farm plots that have been idle at least one year in growing years 2016-2023. The CSB produces estimates of field boundaries, crop acreage, and crop rotations using satellite data in combination with other publicly available data. This data is non-confidential, and not tied to or based off of specific producer information. The CSB provides the crop reported by the Cropland Data Layer for each area defined over an 8 year period. The field boundaries defined in this analysis are based off of cropping decisions for growing years 2016-2023 . I use the crop sequence boundary layer instead of yearly CDLs because the CSB aggregates land to field level, which reduces noise and any error that is inherent in the data used to construct the CDLs . The CSB also allows me to follow the cropping decisions for a single plot of land over multiple growing seasons. This allows me to have certainty when identifying the last crop grown on a plot of fallow land. Maps showing the crops last grown on fallow land are shown in figure 7. I use data from the California Department of Water Resources to construct the bounds of the analysis . The San Joaquin Valley is composed of the San Joaquin River and Tulare Lake hydrologic regions. I combine these two areas into a layer to use as the boundary for the SJV in the rest of the analysis. Hagerty constructs mean water requirement per acre and mean revenues per acre for 19 different crop categories using data from California 2007-2018. These data are shown in Appendix 1. I assign CDL crop code values found in the CSB data to these categories. I take the constructed mean revenue and mean water needs to use in equation , nft hydroponic system the farmer’s optimization problem.

I leave A as a parameter and derive with respect to A. I solve equation for MCW to derive a ”choke price” of water per acre at which point a farmer would no longer want to plant their crop and instead will fallow their land. Crops are ordered from lowest value of MCW to highest. A low calculated value of MCW implies that the farmer cannot afford higher costs of water, and will likely make adaptations to reduce water costs. The land growing crops that have a low tolerance for rising water costs will be the first candidates for solar transition. The other ordering condition will be proximity to existing transmission lines. Transmission lines are hugely important in determining initial costs of bringing a solar farm online, and thus, planned solar projects in SJV are concentrated around existing infrastructure . Land parcels within 100 meters of high-voltage transmission lines will be preferred to those farther away.The calculated MCW values for crop categories are shown in the table below, listed from lowest value to highest value. These values represent the highest possible water cost per acre that a farmer growing a given crop would be willing to tolerate. These values are an upper bound estimates because the only costs considered in a farmer’s production function in this analysis are water costs. These values are calculated using Hagerty derived mean revenues and water needs per acre.6 In the map below, these values are represented with graduated colors representing the threshold water cost per acre values for the last crop grown in a non-fallow year on a land parcel. Farmers owning unirrigated grassland are unwilling to pay for water because they don’t use it on this kind of land. Should these lands be transitioned to solar energy generation,they would not provide any social benefit in the form of water savings, but would provide private benefit to the farmer by substantially increasing their revenues per acre. Other crops that have lower tolerances for water price shocks are safflower and alfalfa.

Safflower is not very water intensive, but does not provide much value per acre. Alfalfa, on the other hand, provides four times the revenue per acre of safflower , but has over twice the water needs per acre. Referring back to figures 2 and 3, safflower is preferred in drier years, and alfalfa grown on plots in wet years are commonly fallowed or swapped for vineyards, which have slightly lower water intensity. These kinds of farmers are the ideal targets for policy intervention to induce solar adoption. On the other end of the spectrum, truck crops like carrots and berries have extremely high tolerance for increasing water costs, as they are hugely valuable per acre, and aren’t hugely water intensive. These farmers will not likely be enticed into using their acreage for solar energy, and thus, the lands housing these crops should not be targeted by policy for land transition. Given the importance and expense of high-voltage transmission lines, I isolate plots of land that are within 100 meters of pre-existing high-voltage transmission infrastructure. These land parcels have been fallow in one or more growing season 2016-2023, and are symbolized based on the crop coverage in its last active season in figure 8. The number of single-cropped land parcels are displayed in parentheses. Such lands previously grew almonds and pistachios, grains, and tomatoes most commonly, followed by previously non-irrigated grassland. In total, there are over 30,000 acres of land identified in the SJV that have been fallow at least once out of the last eight growing seasons and are within 100m of existing high-transmission transmission lines. If the search criteria is expanded to include any farm plots within 400m of existing high-voltage transmission lines, an additional 60,000 acres qualify for solar transition. The maps of agricultural lands eligible for solar transition in the San Joaquin Valley are displayed in figure 9.

This analysis identifies over 90,000 acres of agricultural land that would benefit from solar transition. If all of the acreage identified were to produce solar energy instead of traditional crops, very little other agricultural land would need to be removed from irrigation to help the San Joaquin Valley achieve its groundwater conservation goals as set forth by SGMA. The land identified can provide 3-4x as much energy generating capacity as already exists in the San Joaquin Valley, given average generating capacity per acre in the area . The calculated threshold cost of water shows relative sensitivity to water pricing increases, as it is based on a ratio of water usage and crop value. The ordering of these values tells us which farmers are more likely to benefit privately from a solar land transition. As scarcity increases due to increased pressure from policymakers on cutting back agricultural water use, local governments may find it beneficial to target lowest-cost land transitions first, before taking more profitable agricultural land out of production. Using the calculated values of MCW , policymakers can better anticipate which farmers are more sensitive to water price shocks. These farmers growing crops that are especially sensitive may be targeted by policy interventions that incentivize investment in solar farming. Further, using existing transmission line infrastructure allows for lower input costs to bring solar farms online. Combining these two data can be a powerful way to guide land transition in the San Joaquin Valley. Future research can relax the assumption of equal water access across farmers, and allow for more nuanced production and cost functions for growers. These additions will better reflect the reality that farmers face with water, land, and other input costs. This ordering system provides a loose guide for which farmers may react to increased water prices induced by scarcity and regulation, and adding more flexibility will improve the ability to apply results to policy. This analysis aligns well with economic literature utilizing positive mathematical programming. These economic tools used in more comprehensive agricultural production models including Howitt , M´erel and Howitt , and Howitt et al. . Agriculture is a key human activity in terms of food production, hydroponic nft system economic importance and impact on the global carbon cycle. As the human population heads toward 9 billion or beyond by 2050, there is an acute need to balance agricultural output with its impact on the environment, especially in terms of greenhouse gas production. An evolving set of tools, approaches and metrics are being employed under the term “climate smart agriculture” to help—from small and industrial scale growers to local and national policy setters—develop techniques at all levels and find solutions that strike that production-environment balance and promote various ecosystem services. California epitomizes the agriculture-climate challenge, as well as its opportunities. As the United States’ largest agricultural producing state agriculture also accounted for approximately 8% of California’s greenhouse gas emissions statewide for the period 2000–2013. At the same time, California is at the forefront of innovative approaches to CSA . Given the state’s Mediterranean climate, part of an integrated CSA strategy will likely include perennial crops, such as winegrapes, that have a high market value and store C long term in woody biomass. Economically, wine production and retail represents an important contribution to California’s economy, generating $61.5 billion in annual economic impact. In terms of land use, 230,000 ha in California are managed for wine production, with 4.2 million tons of winegrapes harvested annually with an approximate $3.2 billion farm gate value.

This high level of production has come with some environmental costs, however, with degradation of native habitats, impacts to wildlife, and over abstraction of water resources. Although many economic and environmental impacts of wine production systems are actively being quantified, and while there is increasing scientific interest in the carbon footprint of vineyard management activities, efforts to quantify C capture and storage in annual and perennial biomass remain less well-examined. Studies from Mediterranean climates have focused mostly on C cycle processes in annual agroecosystems or natural systems. Related studies have investigated sources of GHGs, on-site energy balance, water use and potential impacts of climate change on productivity and the distribution of grape production. The perennial nature and extent of vineyard agroecosystems have brought increasing interest from growers and the public sector to reduce the GHG footprint associated with wine production. The ongoing development of carbon accounting protocols within the international wine industry reflects the increased attention that industry and consumers are putting on GHG emissions and offsets. In principle, an easy-to-use, wine industry specific, GHG protocol would measure the carbon footprints of winery and vineyard operations of all sizes. However, such footprint assessment protocols remain poorly parameterized, especially those requiring time-consuming empirical methods. Data collected from the field, such as vine biomass, cover crop biomass, and soil carbon storage capacity are difficult to obtain and remain sparse, and thus limit the further development of carbon accounting in the wine sector. Simple yet accurate methods are needed to allow vineyard managers to measure C stocks in situ and thereby better parameterize carbon accounting protocols. Not only would removing this data bottleneck encourage broader participation in such activities, it would also provide a reliable means to reward climate smart agriculture.Building on research that has used empirical data to compare soil and above ground C stocks in vineyards and adjacent oak woodlands in California, this study sought to estimate the C composition of a vine, including the relative contributions of its component parts . By identifying the allometric relationships among trunk diameter, plant height, and other vine dimensions, growers could utilize a reliable mechanism for translating vine architecture and biomass into C estimates. In both natural and agricultural ecosystems, several studies have been performed using allometric equations in order to estimate above ground biomass to assess potential for C sequestration. For example, functional relationships between the ground-measured Lorey’s height and above ground biomass were derived from allometric equations in forests throughout the tropics. Similarly, functional relationships have been found in tropical agriculture for above ground, below ground, and field margin biomass and C. In the vineyard setting, however, horticultural intervention and annual pruning constrain the size and shape of vines making existing allometric relationships less meaningful, though it is likely that simple physical measurements could readily estimate above ground biomass. To date, most studies on C sequestration in vineyards have been focused on soil C as sinks and some attempts to quantify biomass C stocks have been carried out in both agricultural and natural systems.

We identify TFP by estimating a Cobb-Douglas production function with inputs measured per hectare, implicitly imposing constant returns to scale on the production technology. In such a setting, the inclusion of a measure of farm size as an explanatory variable identifies any relationship between farm size and TFP . The Mexican Family Life Survey is a longitudinal survey of Mexican households, representative of the Mexican population at the national, urban, and rural levels. The MxFLS is a rich source of data for this analysis, as controlling for unobservable farm and community level characteristics using fixed effects is potentially important for determining the farm size – productivity relationship. Further, the decade long span of the surveys allows for a careful analysis of how the size-productivity relationship has evolved in the wake of NAFTA and contemporaneous reforms affecting the Mexican agricultural sector. The three survey rounds – 2002, 2005-06, and 2009-128 – tracked a broad range of individual, family, and community characteristics for the 8,437 initial households. The second and third waves of the survey successfully re-interviewed 90% and 94% of first wave households, respectively. Individuals from the first wave formed new households at annual rates of 3.6% and 4.7% between the first and second and the second and third waves, with 83% of newly formed households being re-interviewed in the third survey wave. While not representative of the Mexican agricultural sector per se, the MxFLS is representative of both rural and non-rural Mexican households. As such, the use of the dataset to study Mexican agriculture has the important caveat that it under represents the larger, stacking pots commercial agricultural operations to the degree that they are not family farms.

A comparison with the 2007 Agricultural Census reveals that both the census and MxFLS have less than 5% of farms that are greater than 50 ha. However, it is important to note that these “large” farms are not necessarily the same as those in the census because they are family-run farms and do not include corporate-run, commercial agricultural operations. In comparison to the 2007 census, the MxFLS over-represents farms less than 2 ha and under-represents farms between 20 ha and 50 ha. This is true for each survey wave, highlighting that while the MxFLS is not representative of the Mexican agricultural sector in its entirety, it is appropriate for studying household farms in Mexico. We employ a farm level analysis using all MxFLS households engaged in agricultural production. A plot-level analysis is not feasible because agricultural input data is recorded at the household level and is therefore not plot specific. However, as we are primarily concerned with documenting the farm size – productivity relationship in Mexico and how it has changed over time, and we are less concerned with fully explaining its determinants, a farm level analysis will suffice. Households in the MxFLS move in and out of agricultural production between survey waves. An unbalanced panel is constructed through two stages of restricting the MxFLS data: first, cross-sections of households with complete farm data are identified and cleaned to eliminate outliers, and second, the unbalanced panel is formed out of all households that appear in two or more MxFLS survey waves. Table 2.1 shows all households using plots for agricultural production in a given survey wave are referred to as agricultural households, whereas all households with plot size and output data for all non-fallow plots are referred to as complete farms.

The intermediate group, farms with farm size data, includes all farms with complete farm size data but not necessarily complete production data – this less restricted dataset increases the sample size at the expense of potentially introducing some measurement error, and is an alternative treatment of the data that is pursued below. Lastly, the number of farms in the panel includes the number of households with complete farm data in two or more of the survey years. These restrictions on the data leave us with a sample of 566 farms reappearing in two or more survey years. Table 2.2 describes these farms according to the combination of survey years in which they appear. Farms are classified into one of 7 farm size groups, as shown below in Table 2.3. The distribution of farms across these bins is roughly constant over time and across treatments of the data, although the share of farms between 0 and 0.5 ha is falling over time while the share of farms between 0.5 and 1 ha is increasing. Importantly, with the exception of the share of farms between 0.5 and 1 ha in 2002, the distribution does not change in any notable way as we restrict the cross section to form the panel, an indication that use of the panel has not introduced bias along this dimension. There is a considerable range in farm sizes in the sample, ranging from less than one hundredth of a hectare to 45,000 hectares. The median farm size in the panel is 2.5, 2.1, and 3.0 hectares in 2002, 2005, and 2009, respectively, with mean farm sizes of 101, 232, and 218 hectares. Around 75 percent of farms utilize only one plot for production in any given year. The preferred measure of agricultural output is a Fisher quantity index that includes all crop and livestock production for each farm in the MxFLS panel. Crop pricesfrom the Food and Agriculture Organization of the United Nations are used to aggregate crop output. Together with a measure of the value of livestock production, an output index is constructed as detailed in Appendix B.1. The MxFLS offers data on five agricultural inputs other than land: physical capital, draft animals, purchased intermediate inputs, family labor, and non-family labor.

Physical capital is measured as the value of tractors and other machines and equipment owned and draft animals is the value of horses, donkeys, and mules owned by each household in each survey year, deflated to 2002 values. Purchased intermediate inputs are measured using reported expenditures on each of nine agricultural inputs over the course of the previous year, again deflated to 2002 values. An index of family labor is constructed using household members’ time use and employment data in the MxFLS, and is an estimate of annual hours worked on the farm by all household members. In contrast, the non-family labor index is a measure of the number of non-household individuals that worked on each farm in each year, measured in workers and not labor hours. Appendix B.2 provides a detailed discussion of the source and construction of the family labor and non-family labor indices, including a set of alternative family labor indices. Table 2.4 shows the share of panel households using the different input categories in each year, with purchased intermediate inputs shown both collectively and further disaggregated into their nine components. For all of the inputs there exist at least some, if not a majority, of households that have zeros for that input category. This is expected, as farms in the sample are expected to span a range from low technology subsistence agriculture to more modern and input intensive operations. Furthermore, nft hydroponic many inputs may be substitutes for each other, and farms can access these inputs by owning them or by purchasing them in factor markets. Tractor services, for example, may be substituted for with draft animals. Households can either own some combination of these capital stocks or purchase their services from the market. We follow Battese to estimate production functions with observations having zero inputs. Of principle importance is any relationship between inputs per hectare and farm size, as systematic relationships between input intensity and farm size potentially drive a wedge between the farm size – land productivity and farm size – total factor productivity relationships . We calculate the correlation coefficients between logged input per hectare and logged farm size for those farms with non-zero values of usage of each input. These correlations are shown in Table 2.5. Conditional on using the input, the intensity of all inputs used declines with farm size, emphasizing the importance of moving from partial measures of productivity to a comprehensive measure such as TFP.The vast majority of plots are either privately owned property or are part of an ejido – a piece of communally held land where plots are farmed by designated households. It is commonly accepted that ejidos are less productive than privately held farms, although there is little empirical evidence comparing the TFP of these farms using micro data. At least 91% of privately held plots in the MxFLS have some form of formal documentation in any given year, while just 75-84% of MxFLS ejido properties do. Privately held plots primarily have a formal deed or title to the land as documentation, whereas ejido plots primarily have a certificate of ejido status or agricultural rights.

Formal documentation of property rights is important for accessing credit and is expected to be positively correlated with TFP. How property rights are formally documented matters, however, as a certificate of ejido status is often not acceptable to private financial institutions for use as collateral whereas formal deeds are. We control for both separately in the core empirical analysis. Because ejidos may function differently than privately owned parcels, we control for ejido status. Ejido farms make up 58% of the panel, and the ejido status of farms does not change for almost all farms in the panel. Panel farms are located in 92 distinct communities and are grouped into five regions in Mexico: the North, Center, Pacific, South, and Gulf. In the first survey wave, 26% of panel farms are in Northern states where agriculture is characterized by having larger commercial farms with greater importance of the commercial production of maize. In comparison, 50% of first wave farms are in Southern and Central states where agriculture is characterized by more traditional, smallholder maize producers and the commercial production of fruits and edible vegetables . In tests of heterogeneity, we introduce regional interactions with farm size in estimations of equation , allowing the farm size – TFP relationship to vary across agricultural regions. Additional household level controls are grouped into two broad categories: variables describing agricultural practices that are mostly endogenous, and demographic variables that are largely exogenous. Household demographic variables are based on predetermined characteristics of the household head. The panel farms predominantly have male, married, and Spanish speaking heads of household, with little differences across farm sizes or ejido status. Table B.3.5 in Appendix B.3 shows that farms larger than about 5 ha appear to be less likely to have an indigenous household head and more likely to have a literate household head than do smaller farms. Literacy is just one way to measure educational attainment of the household head, and it captures a rather low bar. We measure the education of household head by creating indicator variables for the highest level of formal schooling attended, from no formal education to elementary school, secondary school, high school, or college education. With little variation across survey years, Table B.3.6 in Appendix B.3 shows educational attainment by farm size for 2002 only, showing that a majority of farms have household heads with no more than an elementary school education, while almost one quarter of the panel’s household heads have no formal education at all. The following variables describing agricultural practices of farms are potentially endogenous, and for this reason are not included in the base specifications. They are introduced to shed light on potential channels affecting TFP and the farm size – TFP relationship. Any farm that does not bring any of its crop to market is classified as a subsistence farm, identifying farms that may behave differently than those who do. There is little difference in the prevalence of subsistence farming between ejido and non-ejido farms. As shown in Table B.3.1 in Appendix B.3, subsistence farming decreases with farm size, as expected. We calculate the share of each farm’s crop that is marketed – on average, those farms in the sample that do participate in the market sell around 75% of their production. This appears relatively constant across farm size bins. Alongside subsistence farming practices, Table B.3.1 in Appendix B.3 shows the share of farms engaged in monocropping.

One original model for this form of tribal litigation is depicted in University of Virginia professor Christian W. McMillen’s excellent study, Making Indian Law: The Hualapai Land Case and the Birth of Ethnohistory. Professor McMillen details the famous Indian land claim case United States v. Santa Fe Pacific Railroad Co., decided in 1941 by the Supreme Court, from its origins in a military order that recognized a Havasupai Nation boundary line that was about one-third of the nation’s traditional territory in 1881, confirmed by President Chester Arthur’s Executive Order on 4 January 1883. But, like many western reservations, railroad monopolies convinced Congress to open up the reservation boundaries to their interests. In early 1883, the Atchison, Topeka, and Santa Fe Pacific Railroad laid claim to the best water source on the reservation, Peach Springs, located on what became Route 66, leading to the conflict that consumed the Havasupai Indians for the next several decades. The Havasupai Reservation rests on lands that border a portion of the Grand Canyon’s southern edge in northern Arizona. Much of the land appears myth because it enables them to justify the appropriation of the land on the grounds that it is in need of management. In chapter 10, Madonna Moss describes Tlingit horticulture in Southeast Alaska, the northernmost portion of the Northwest Coast. Moss characterizes the Tlingits’ precontact management of indigenous plants as a system of selective harvesting. The exception was tobacco, which was grown prior to European contact using the horticultural management techniques of seeding, weeding, hydroponic nft and fertilizing. She proposes that it was their expertise with tobacco that enabled these people to raise the horticultural crops introduced in the eighteenth century successfully.

In the final case study, Douglas Deur describes the creation and maintenance of estuarine gardens by indigenous communities. Keeping it Living is a shining example of scientific reevaluation and concentrated inquiry of a long-held perspective, and it is as necessary as it is exemplary.Litigation involving Indian claims in the modern era often revolves around the complex and expensive reports prepared by ethnohistorians, historians, anthropologists, and other experts. Any claim involving the meaning of a treaty provision or whether a tribe qualifies for gaming on lands acquired after 1988 or even whether a tribe should be federally recognized will involve this battle of experts. Tribal victories in the Sioux Nation’s Black Hills land claim, Pacific Northwest and Great Lakes treaty fishing rights, and eastern land claims would have been unobtainable without careful expert testimony. One original model for this form of tribal litigation is depicted in University of Virginia professor Christian W. McMillen’s excellent study, Making Indian Law: The Hualapai Land Case and the Birth of Ethnohistory. Professor McMillen details the famous Indian land claim case United States v. Santa Fe Pacific Railroad Co., decided in 1941 by the Supreme Court, from its origins in a military order that recognized a Havasupai Nation boundary line that was about one-third of the nation’s traditional territory in 1881, confirmed by President Chester Arthur’s Executive Order on 4 January 1883. But, like many western reservations, railroad monopolies convinced Congress to open up the reservation boundaries to their interests. In early 1883, the Atchison, Topeka, and Santa Fe Pacific Railroad laid claim to the best water source on the reservation, Peach Springs, located on what became Route 66, leading to the conflict that consumed the Havasupai Indians for the next several decades.

Strawberry production in California accounts for more than 80% of total U.S. production, with an annual farm gate value of $1.10 billion , which is four times greater than all other states combined . In addition, California produces nearly one billion strawberry transplants each year in nurseries, and these transplants must meet strict phytosanitary standards for local production and export. Such a profitable industry in California has been made possible by the fumigation technology developed in the 1950s with methyl bromide and chloropicrin . Since then, preplant fumigation with methyl bromide and chloropicrin has become an integral part of the California strawberry production industry , and nearly all conventional strawberry production occurs in fumigated soils . Annual soil fumigation has contributed to the control of soilborne pathogens, nematodes, and weeds while also boosting the yields of strawberry plants. Historically, this also allowed breeding programs to focus on improving horticultural characteristics of strawberry cultivars in lieu of emphasizing disease resistance. Because of the negative effects of methyl bromide on stratospheric ozone, the fumigant was designated as a class I stratospheric ozone depleting substance by the Montreal Protocol and as a significant risk to human health . The continued availability of this efficient fumigant for agricultural soil fumigation beyond the 2005 phase-out date will be through critical-use exemptions. It has been estimated that annual losses in short term net farm income in California will be more than $162 million, with strawberry accounting for more than 60% of these losses . Over the past 10 years, research has focused on identifying alternative fumigants with efficacy comparable with methyl bromide . Alternative fumigants such as chloropicrin and Telone C35 have been identified, and improved application techniques have been developed to reduce emissions .

Although chloropicrin is as efficacious as methyl bromide + chloropicrin at high rates, these are not feasible for the growers due to regulatory limits placed on application rates. Regardless, chemical alternatives to methyl bromide will be subjected to increasing review and regulation and they may not be readily available over the longer term. It has been estimated that soilborne diseases caused by Pythium, Phytophthora, Cylindrocarpon, Macrophomina, Rhizoctonia, and Verticillium spp. result in 20 to 30% strawberry yield losses in the absence of fumigation . Therefore, longer-term research is required to develop nonchemical alternatives, and their adaptation will require effective integration with other methods of disease, pest, and crop management . In the post-methyl bromide era, Verticillium wilt is likely to reemerge as a major disease for conventional strawberry production. The disease already is a major problem in some organic production fields. In strawberry, symptoms begin to appear during early to mid-season, with outer leaves on infected plants turning yellow, drooping, and later turning brown and dry. Yield from these affected plants can be dramatically reduced and infected plants usually die before the end of the season . The fungus survives in the soil as microsclerotia for many years, and survives better in sandy loam soils typical of strawberry production fields in coastal California than in other types of soil . Large numbers of microsclerotia are formed in colonized tissue of susceptible crops, and a few are formed even on non-hosts . Whether microsclerotia are formed on infected strawberry plants is not known. Resistance to Verticillium wilt is unavailable in currently used commercial cultivars and tolerance in these cultivars is low. With the phase out of methyl bromide and possible future loss or restrictions on the use of alternative fumigants, hydroponic channel resistance to Verticillium wilt has now become a selection criterion in some breeding programs. As a result, resistance to Verticillium wilt in locally adapted strawberry cultivars may increase over time. The concept of rotating crops to manage plant diseases is perhaps one of the oldest cultural practices in agriculture . The utility of this practice in reducing Verticillium dahliae inoculum and subsequent disease intensity has been equivocal . Microsclerotia of V. dahliae survive in the soil up to 10 years, and the extensive host range and lack of host specificity reduce the usefulness of some crop rotations for Verticillium wilt management . However, recent work has shown that rotations with broccoli dramatically reduce microsclerotial numbers and Verticillium wilt incidence in susceptible crops. If rotations of broccoli are successful in strawberry, they will be equally applicable to both conventional and organic strawberry production systems. Although the benefits of rotations are numerous and quantification of these benefits in dollar terms is difficult, simple cost-benefit analysis of adapting rotations will inevitably lead to a better understanding of their composite benefits. Such information also may lead to increased adoption of crop rotations. The objectives of this study were to determine the effect of crop rotation on soil borne fungal inoculum density, disease severity, and strawberry growth and yield; to assess the effectiveness of crop rotation in soil with no detectable Verticillium spp. to improve strawberry growth and yield; and to obtain a cost-benefit analysis of this method of managing Verticillium wilt in strawberry. Strawberry plants grown in plots rotated with lettuce at both locations had a significantly smaller canopy diameter than other rotation treatments. In Watsonville, broccoli- and Brussels sprouts-rotated plots and fumigated control plots, plants had a greater canopy diameter than strawberry plants in lettuce rotation plots . In 2000, the canopy diameter of strawberry plants was higher in fumigated control and broccoli rotation plots than in the lettuce and Brussels sprouts rotation plots.

Subsequently, however, the highest canopy diameter was observed in the fumigated control, followed by broccoli, Brussels sprouts, and lettuce rotation plots . At the Salinas site in 1998, strawberry plants in broccoli-rotated plots had the highest canopy diameter, and the differences between fumigated control and other rotations were variable. Conditions during fumigation in 1998 were not optimal; hence, strawberry plants did not show the typical robustness in these plots . In 2000, the response of strawberry canopy diameter to various rotation treatments was typical of what was observed at the Watsonville site, with the plants being more robust in the fumigated control followed by broccoli, cauliflower, and lettuce plants. There were significant differences between each of the treatments on both assessment dates . Repeated-measures ANOVA indicated that the rotation treatments significantly affected disease severity on strawberry for all observation dates at both locations. The highest wilt severity was observed in the lettuce rotation plots throughout the season in 1998 at the Watsonville site and the lowest was in the fumigated plots . Broccoli rotation plots had the lowest wilt severity among the three rotation treatments. Even though the differences in wilt severity between fumigated and broccoli rotation plots were significant through much of the season, final wilt severity was nearly identical between the two treatments . Wilt severity in the Brussels sprouts rotation plots was intermediate between broccoli and lettuce rotation plots throughout the season . In 2000, the onset of Verticillium wilt occurred 3 weeks later, and the severities were lower relative to 1998 . The response of different treatments, however, was nearly identical to 1998, with the least wilt severity recorded in fumigated plots followed by broccoli, Brussels sprouts, and lettuce . Final wilt severity in fumigated plots and plots that had broccoli residue incorporated was nearly identical and not statistically significant from each other . As in the Watsonville site, disease severity in the Salinas site was highest in the lettuce rotation treatment during both 1998 and 2000 . Because of the inefficient fumigation in 1997, disease severity on strawberry plants in fumigated plots was higher than in plots that had broccoli residue incorporated in 1998. However, there were no significant differences between these two treatments during the 2000 season . Strawberry plants in the broccoli-rotated plots showed a consistently lower disease severity than in the remaining vegetable rotation plots during all observed dates. Overall strawberry yield was higher in Salinas than in Watsonville . The fumigated control treatment produced the highest marketable and total yields at both locations until comparable dates of harvest . The experiment at Watsonville was terminated earlier but was continued at the Salinas site; hence, the final yields appear comparable between the two sites . Among the vegetable rotation treatments during both seasons at both sites, plots that had broccoli residue incorporated produced the highest strawberry yield. In 1998, total strawberry yield was about 22% less in plots with broccoli rotation relative to the fumigated plots in Watsonville. However, in 2000, fruit yield in fumigated plots was only 12% higher than in broccoli-rotated plots. Plots with lettuce rotation consistently had the lowest strawberry yield at both sites during both seasons. In Watsonville, Brussels sprouts plots had intermediate strawberry yield in 1998 but lowest yield in 2000 when it was similar to that in lettuce rotation plots. In 2000, the difference in yield between the lettuce and broccoli rotation plots was greater than in 1998 at the Salinas site .

The penicillin concentration for tissue decontamination ranges from 50 to 100 IU/mL, streptomycin ranges from 100 to 500 µg/mL, and amphotericin B from 0.25 to 2.5 µg/mL31. These antibiotics were placed into Leibovitz’s 15 medium, phosphate-buffered saline solution , or artificial seawater for washing the tissue samples. Other antibiotics such as ampicillin, gentamycin, and kanamycin have also been used. It is important to note that decontamination might vary depending on the source of the tissues, as some tissues have a higher initial microbial load. For instance, digestive glands, gills, and the mantle are prone to more contamination than the heart or adductor muscle because these organs are primarily involved in filtration. In addition, some marine microbes and parasites carry a symbiotic relationship with the animal, leading to more contamination and making it difficult to find optimal decontamination conditions. Aside from serum-free media needs, environmental factors such as oxygen, salt, pH, osmolarity, and temperature must be optimized. Fish cells are generally adapted to low oxygen environments with hypoxia-response genes. Some fish cells only grow in 5% carbon dioxide, while others utilize anoxic or standard oxygen tension. A comprehensive study on muscle lactate dehydrogenase in warm-water fish and mammalian cells reported significant differences in metabolic activity dependent on pH. Generally, seafood cells grow at lower temperatures than mammalian cells, blueberry packaging making them good candidates for producing cell-cultivated seafood with lower energy inputs. There are different fully defined basal media available for seafood cell culture including Eagle’s Medium, Modified Eagle’s Medium , Medium 199 and Leibowitz’s 15 .

While there have been significant advances in the development of serum-free culture media for mammalian cell lines, there has been limited progress for fish cells. Serum-free media has been achieved for a few fish cell lines in the past, however, these formulations were not well-defined or were proprietary within companies, resulting in significant challenges in broadening their utility for cell-cultivated seafood. Serum-free media containing lactalbumin hydrolysate, trypticase-soy broth, bacto-peptone, dextrose, yeast isolate, polyvinylpyrrolidone, and non-essential amino acids were studied with different fish cell lines , and cell growth and morphology of the cells was similar to those that were grown in serum-containing media. Bioprocessing was utilized to convert different feedstocks including whole oysters , whole mussels , whole lugworms , black soldier flies and crickets to protein hydrolysates for growing fish cells. These hydrolysates were cytotoxic for Zebrafish cells at high concentrations regardless of serum concentration, while, at lower concentrations , all of the hydrolysates supported cell growth. Black soldier fly hydrolysates could replace serum and provided a cost-effective source of peptides. The use of modeling tools also has the potential to foster more rapid identification of key media and related conditions for seafood cell growth and differentiation. For example, through the use of Design of Experiments and/or AI, the development of a serum-free medium can be pursued. Protein hydrolysates from marine byproducts could also provide inexpensive and high quality proteins and amino acids to develop serum-free media.There is limited knowledge on the in vitro differentiation and maturation of fish, crustacean and mollusk cells into fat or muscle tissues.

To screen for myogenesis in mackerel cells as an example, a variety of methods were utilized [e.g., serum starvation, reduced serum, reduced serum plus additives, reduced serum with insulin, 1-oleoyl lysophosphatidic acid and transferrin, reduced serum medium with insulin-like growth factor 1, reduced serum medium plus additives with IGF-1; medium with extracellular signal-regulated kinase inhibitor. Myogenic potential was assessed via RTqPCR using primers based on genome sequences from southern bluefin tuna , myogenin, along with immunohistochemistry. Differentiation via paired-box protein 7 and myosin heavy chain immunostaining was observed in a continuous muscle cell line developed from Atlantic mackerel. The cell line also exhibited an adipocyte-like phenotype, which was confirmed via Oil Red O staining and quantification of neutral lipids. MEF2A, Mrf-4, MyoD and Myf-5 expression was reported in a muscle cell line developed from a freshwater fish during differentiation of muscle cell culture. However, more detailed studies need to be carried out to facilitate selection of the right cell type for cultivated aquatic food development. Images of mackerel cells are provided in Fig. 3.While suspension culture-based approaches may be sufficient for unstructured seafood products like surimi, tissue-like products that replicate some of the complexity of muscle tissue, including texture/mechanics and mouthfeel after cooking and oral mastication, will require more sophisticated methods to impart structure to the final product. A variety of approaches are utilized that mainly rely on scaffolds to facilitate the transport of oxygen, nutrients, and waste products as tissues mature . Approaches to scaffolding and tissue engineering for cell-cultivated meat have been reviewed elsewhere.

The differences in requirements for scaffolds for seafood vs. terrestrial meat can be divided into two broad categories: those related to the cell requirements and those related to the effects of the scaffold on the organoleptic properties of the final product. Because scaffolds play a crucial role in delivering cues to the cells as they proliferate, differentiate, and mature, scaffolds that are appropriate for use with cells from one taxonomic group may not be optimal for another. Therefore, optimization of scaffold stiffness, topography, or surface functionalization may require significant differences between terrestrial animals, fish, and aquatic invertebrates. Scaffolds can also impact the acceptability of the final product due to texture, taste and flavor. For example, the melting temperature of fish collagen differs from that of collagen from terrestrial animals, with important impacts on cooking fish muscle, thus, the thermal properties of scaffolds for cell-cultivated seafood will need to be carefully considered. In addition, the 3D geometry of muscles from terrestrial animals, fish, crustaceans, and mollusks are different and need design considerations with scaffolds for whole cut cell-cultivated products. One of the earliest investigations into cell-cultivated meat or seafood was a NASA-funded study that demonstrated the in vitro expansion of goldfish muscle explants co-cultured with brown bullhead fibroblasts. While research into cell-cultivated seafood over the subsequent two decades has lagged behind that of cell-cultivated terrestrial meat, several recent studies have demonstrated progress. While this is an early example of a scaffold-free cell cultivated seafood prototype, there is precedent for the use of scaffold-free techniques in both academic and commercial efforts at producing cell-cultivated terrestrial meat, but less so for seafood-related goals. Recent advances with scaffold-free alternatives were reported for livestock-derived adipocyte cell cultures in 2D, that could also be applied to seafood cell cultures; the 2D systems were consolidated into 3D tissues via post cell growth aggregation using food grade cross-linking enzymes like transglutaminase or a gelling agent. The use of 3D bio-printing to produce cell-cultivated meat products has been a focus due to the level of control over structure, and this strategy was also applied for the formation of cell-cultivated large yellow croaker prototypes by printing with a bioink consisting of gelatin, alginate, and primary croaker satellite cells into a tissue-like structure. Microcarriers as scaffolds in suspension are also utilized towards cell production goals and scalability in cell-cultivated seafood production, providing large surface/volume ratios. These can have a temporary role or become part of the final product when developed from edible sources . Cells grown in the 2D environment inside or on the surface of the MCs can provide a smooth transition from flasks and bioreactors to finalized 3D tissue outcomes. Different types of marine polymers could be used for cell-cultivated seafood including hydrogels from algal sources, chitosan extracted from marine exoskeletons, blueberry packaging box and gelatin from underutilized species such as jellyfish, fish skin and seafood byproducts, which can also provide specific colors and flavors. In addition, extracellular matrix proteins and lipids can be integrated into the process via scaffolds and can have a significant role in the sensory and textural properties of fish meat. A recent study illustrated that some of the established lipid structure approaches, such as oleogels, could be integrated with cells cultured on microcarriers to form 3D structures simulating meat products. These approaches of combining structured fats with fibrous tissue scaffolds could enable the development of muscle-like fish products, however, there have been few studies reported in the literature to date. Cellular aggregates as self-scaffolding outcomes can also be pursued as a robust option for increasing biomass.Scaling-up using bioreactors for the 3D cell production environment is a major bottleneck for the cell-cultivated meat industry.

Most approaches being pursued are based on variations with stirred tank bioreactors derived from pharmaceutical industry designs, with a focus on cost reductions via simplified designs or those requiring lower energy impacts. These systems apply to cultivated meat and seafood alike. Other approaches generally being pursued in the field include hollow fiber-based bioreactors. In all cases, the costs of scaling are related to media, microcarriers, clean rooms, bioreactor hardware and labor. Innovative approaches will be required to reduce the cost of scaling up. For example, there are many unutilized nutrients and growth factors, which could be recovered and returned to the bioreactor after removing cell metabolites. This could be achieved using different approaches such as growing plants on the spent media to generate additional biomass for use in the production process, utilizing microbial communities for metabolic support to reduce inhibitor byproducts, along with more traditional selective membranes to isolate, recover and re-use key growth factors. Reductions in ammonia can be pursued using microorganisms and chemicals, which can help sustain cultures with reduced media changes or specific nutrient feeding. Glutamine substitutes including α-ketoglutarate , glutamate and pyruvate had a positive impact on cell proliferation and differentiation by reducing the rate of ammonia production. For instance, proliferation media containing αKG improved primary bovine fibro-adipogenic progenitor cell proliferation, while significantly reducing ammonia production rate due to the antioxidative and ammonia scavenging properties of αKG.In the US, both the Food and Drug Administration and the United States Department of Agriculture have established a joint agreement to address cell-cultivated meat and seafood safety and regulations. The FDA oversees cell collection, cell banks, cell growth and differentiation for all the seafood organisms, while the USDA/Food Safety and Inspection Service evaluates the products after harvest onwards for catfish. Codex Alimentarius also recently initiated programs on developing Hazard Analysis Critical Control Point and Good Manufacturing Practices for cell-cultivated meat and seafood. Complementary to regulatory organizations, the Food and Agriculture Organization , and the World Health Organization developed the first comprehensive food safety document that covers cell-cultivated seafood. This document outlines the food safety risks including zoonotic risks from cell lines and the production environment, biological contamination risks from initial cell sources to production, and risks from unwanted residues and novel inputs during production and processing of cell-cultivated meat products. These risk factors are combined with a food safety plan to address the challenges and regulatory requirements of both the FDA and the USDA along each step of cell-cultivated seafood production . Critical Control Points are biological, chemical, allergen and physical issues that need to be used for developing preventive controls. In the cell culture environment, bacteria can rapidly outgrow the animal cells, with additional hazards from other organisms including viruses, prions, fungi, protozoa and parasites. Escherichia coli, Listeria monocytogenes, Salmonella spp., Aeromonas hydrophyla, Vibrio spp., and Mycoplasma spp. are some of the most common bacterial contaminants in foods. Chemicals may be added intentionally or unintentionally to the production process and can pose food safety risks. These chemicals include antibiotics, drugs, sanitizers, cryoprotective agents, leachable chemicals , surfactants, and anti-foaming agents. There are approved chemicals listed by FDA that can be used for cell culture, but for new production processes these potential contaminants will need to be tracked. Physical hazards include objects, debris, plastics, and microplastics. A major issue with seafood is the allergens, with different types of proteins and allergens in fish and shellfish. For example, the major allergens in fish are parvalbumins, while in shellfish, tropomyosin, arginine kinase, and myosin light chain are the main allergens. Cellular aquaculture has the potential to reduce allergenicity in seafood by selectively growing specific cell types to avoid allergenic components. This can also be achieved through genetic modifications, such as using RNA Interference techniques to knock out causative genes. Additionally, the incorporation of food-grade additives like creatine or ethylenediamine tetra-acetic acid into the cell culture media may offer a route to address allergen-related issues by modulating the expression of parvalbumin, thereby reducing allergenicity.Cell-cultivated seafood industries need to comply with preventive controls rules established by the Food Safety Modernization Act .

With less than 300 years of breeding, pedigrees for thousands of F. ananassa individuals have been recorded, albeit in disparate sources. To delve more deeply into the domestication history of cultivated strawberry, we assembled pedigree records from hundreds of sources and reconstructed the genealogy as deeply as possible. One of our initial motives for reconstructing the genealogy of cultivated strawberry was to identify historically important and genetically prominent ancestors of domesticated populations, in large part to guide the selection of individuals for whole-genome shotgun sequencing and DNA variant discovery, inform the development of single-nucleotide polymorphism genotyping platforms populated with octoploid genome-anchored subgenome-specific assays, and identify individuals for inclusion in genome-wide studies of biodiversity and population structure . The genetic relationships and genetic contributions of ancestors uncovered in the genealogy study described here guided the selection of individuals for downstream genomic studies that shed light on genetic variation and the genetic structure of domesticated populations worldwide . Our other early motive for reconstructing the genealogy of strawberry was to support the curation and stewardship of a historically and commercially important germplasm collection preserved at the University of California, Davis , plastic grow bag with accessions tracing to the early origins of the strawberry breeding program at the University of California, Berkeley , in the 1920s .

We sought to develop a complete picture of genetic relationships among living and extinct individuals in the California and worldwide populations, in part to assess how extinct individuals relate to living individuals preserved in public germplasm collections. Because 80% or more of the individuals we documented in the genealogy appear to be extinct, they could only be connected to living individuals through their pedigrees. One of the ways we explored ancestral interconnections between extinct and living individuals was through multivariate analyses of a combined pedigree–genomic relationship matrix estimated from genotyped and ungenotyped individuals . The holdings and history of the UCD Strawberry Germplasm Collection were shrouded in mystery when our study was initiated in 2015. The only individuals in the collection with pedigree records were publicly released and patented cultivars. The immediate challenge we faced in reconstructing the genealogy was the absence of pedigree records for 96% of the 1,287 accessions preserved in the collection, which is hereafter identified as the “California” population. To solve this problem, authenticate pedigrees, and fully reconstruct the genealogy of the California population, we applied an exclusion analysis in combination with high-density SNP genotyping . Here, we demonstrate the exceptional accuracy of diploid paternity analysis methods when applied to individuals in an allooctoploid organism genotyped with subgenome-specific SNPs on high-density arrays . Several thousand SNP markers common to the three arrays were integrated to develop a SNP profile database for the parentage analyses described here. SNPs on the 50-K and 850-K arrays are uniformly distributed across the octoploid genome and informative in octoploid populations worldwide . The 50-K SNP array harbors 1 SNP/16,200 bp, whereas the 850-K array harbors 1 SNP/953 bp, telomere-to-telomere across the 0.81-Gb octoploid genome.

The genealogies of domesticated plants, especially those with long-lived individuals, overlapping generations, and extensive migration and admixture, can be challenging to visualize and comprehend . We used Helium to visualize smaller targeted pedigrees; however, the strawberry pedigree networks we constructed and investigated were too large and mathematically complex to be effectively visualized and analyzed with Helium and other traditional hierarchical pedigree visualization approaches. Hierarchical methods often produce comprehensible insights and graphs when applied to pedigrees of individuals or small groups but yield exceedingly complex,labyrinthine graphs that are difficult to interpret when the genealogy contains a large number of individuals and lineages. We turned to social network analysis  to explore alternative approaches to search for patterns and extract information from the complex genealogy of strawberry. The pedigree networks of plants and animals share many of the features of social networks with nodes connected to one another through edges relationships. We used SNA methods, in combination with classic population genetic methods, to analyze the genealogy and develop deeper insights into the domestication history of strawberry . SNA approaches have been applied in diverse fields of study but have apparently not been applied to the problem of analyzing and characterizing pedigree networks . With SNA, narrative data are translated into relational data and summary statistics and visualized as sociograms . Here, we report insights gained from genealogical studies of domesticated strawberry populations worldwide. Our studies shed light on the complex wild ancestry of F. ananassa, the diversity of founders of domesticated populations of cultivated strawberry that have emerged over the past 300 years, and genetic relationships among extinct and extant ancestors in demographically unique domesticated populations tracing to the earliest ancestors and inter specific hybrids .

The genealogy does not account for lineages underlying what must have been millions of hybrid progeny screened in breeding programs worldwide; e.g., Johnson alone reported screening 600,000 progeny over 34 years at Driscoll’s . Cultivars are, nevertheless, an accurate barometer of global breeding activity and the only outward facing barometer of progress in strawberry breeding. When translated across the past 200 years of breeding, our selection cycle length estimates imply that the 2,656 cultivars in the genealogy of cultivated strawberry have emerged from the mathematical equivalent of only 12.9 cycles of selection . Even though offspring from 250 years of crosses have undoubtedly been screened worldwide since 1770, 15.5 years has elapsed on average between parents and offspring throughout the history of strawberry breeding . Because genetic gains are affected by selection cycle lengths, and faster generation times normally translate into greater genetic gains and an increase in the number of recombination events per unit of time , our analyses suggest that genetic gains can be broadly increased in strawberry by shortening selection cycle lengths. Genome-informed breeding and speed breeding are both geared towards that goal and have the potential to shorten selection cycle lengths and increase genetic gains . We reconstructed the genealogy of strawberry to inform the curation of a historically important germplasm collection, forensically identify the parents of individuals without pedigree records, authenticate the parents of individuals with pedigree records, shed light on the domestication history of strawberry, and retrospectively examine where we have been and how we got there. The reconstruction was greatly facilitated by the availability of outstanding SNP genotyping platforms , the development of an extensive DNA profile database to complement the pedigree database , and the application of robust and highly accurate diploid exclusion analysis methods for parent identification and pedigree authentication. We provided an open-source R code to support future parentage analyses in agricultural species. Our backward-facing genealogy study, in retrospect, pe grow bag yielded unexpected insights about the complex hybrid ancestry and breeding history of cultivated strawberry that should inspire future generations and guide where we should go from here. Our critical examination of historical selection cycle lengths was meant to be provocative and perhaps inspire the implementation of strategies for increasing breeding speed and accelerating the improvement of strawberry. We suspect that improvements can be achieved, at least in part, through changes in breeding schemes and the application of pedigree-informed predictive breeding methods. The open-source pedigree database we compiled should find broad utility in predictive breeding schemes and can be easily expanded and modified for specific breeding problems, other populations, and future analyses. Because of the depth and completeness of the pedigree records commonly available in strawberry, pedigree best linear unbiased prediction has the potential to increase genetic gains and enhance selection decisions, especially when combined with genomic prediction . The pedigree database we assembled will facilitate the application of pedigree-BLUP and identity-by-descent prediction of alleles and haplotypes , in addition to providing a solid foundation for expanding the genealogy over time.We are grateful to Clint Pumphrey, the manuscript curator of the special collections and archives of the Merrill-Cazier Library at Utah State University . Clint assisted the first author with acquiring and researching the laboratory notebooks and other records of Royce S. Bringhurst , a former faculty member and strawberry breeder at the UCD . The documents and photos associated with the collection yielded extensive pedigree records that were crucial for reconstructing the genealogy of the UCD Strawberry Breeding Program. We are equally grateful to Phillip Stewart, a strawberry breeder at Driscoll’s , for sharing copies of the UCB, pedigree records of Harold E.

Thomas , a former faculty member and strawberry breeder at UCB from 1927 to 1945. Those pedigree records greatly increased the completeness and depth of the database for the early years of the University of California Strawberry Breeding Program. The authors thank Thomas Sjulin, a former strawberry breeder at Driscoll’s , for sharing the public pedigree records he assembled over his career. Those nucleated the pedigree database we developed and were a catalyst for our study. SJK and GSC thank Robert Kerner for the computer forensic analyses that recovered several hundred pedigree records for UCD individuals from an obsolete electronic database, thus preventing the loss of those records for perpetuity. They were critical for integrating the UCD genealogy with the global genealogy for cultivated strawberry. SJK especially thanks Rachel Krevans, Matthew Chivvis, Jake Ewert, and Wesley Overson for their integrity, friendship, and steadfast support.Table grapes have become an important fresh commodity in Brazil for both internal market and exportation. Over the period of 2000–2016, Brazil presented an increase of ∼150% in table grape production, reaching around 970,000 MT in 2016 . The northern region of Paraná state is one of the main areas of table grape production. The mild winter and subtropical conditions of this region permit two crops of grapes per year, which allow Brazilian growers to time their production to coincide with market windows of other countries and compete for more advantageous prices. However, in these subtropical regions, berry ripening and harvest often occur during the rainy season, which is not ideal for Vitis vinifera cultivars because excess rain and moisture compromise the overall quality of the berries . Therefore, Brazilian table grape production is starting to incorporate American and/or hybrid grape cultivars that are better adapted to warm and rainy climates. Another disadvantage of growing table grapes in subtropical areas is that high temperatures during ripening can inhibit anthocyanin biosynthesis in the berries from V. labrusca and hybrid cultivars . This results in inadequate fruit color, and thereby a decrease in market acceptance and the potential economic value of the commodity . The seedless table grape Selection 21, a new hybrid of V. vinifera × V. labrusca recently developed by the Grape Genetic Breeding Program of Embrapa Grape and Wine, Brazil, obtained from the cross of [Arkansas 1976 × ] × “BRS Linda,” is a clear example of a cultivar that lacks red color development when grown in subtropical regions. The plant growth regulator ethephon, an ethylene-releasing agent, has long been known to improve berry color when applied at véraison . More recently, the application of –cis-abscisic acid has also been shown to stimulate anthocyanin accumulation and thereby improve berry color . S-ABA appears to be more effective than ethephon in enhancing grape color and it has other potential benefits compared to ethephon, including a shorter postharvest interval, and an exemption from tolerance in most countries. The introduction of S-ABA as an active ingredient in a commercial plant growth regulator prompted many studies on V. vinifera cultivars under temperate climate conditions. Such studies have shown that the efficacy of S-ABA varies with the cultivar , the S-ABA concentration , the time of application and the environmental conditions . Studies have shown that exogenous application of S-ABA can significantly increase the activity of a wide range of genes involved in anthocyanin biosynthesis . Most of these studies tested the effects of a single application of S-ABA before or during véraison. However, studies of the effects of S-ABA several applications at different concentrations and timings following véraison are still needed to optimize the use of this plant growth regulator in table grape cultivation . In grapes, the anthocyanin biosynthesis pathway involves multiple steps that are controlled by MYB transcription factors, such as VvMYBA1 and VvMYBA2 . In red varieties, the VvMYBA1 gene is only expressed after véraison. Both VvMYBA1 and VvMYBA2 regulate anthocyanin biosynthesis during ripening by strictly controlling the expression of the canonical UDP-glucose:flavonoid 3-Oglucosyltransferase gene .

Agricultural intensification simplifies ecosystems through management practices such as increases in agrochemical use, decreases in habitat complexity, and decreases in crop and vegetation diversity. Agricultural intensification alters functional biodiversity;in particular, reductions in habitat complexity impact the arthropod community composition, decrease arthropod diversity and reduce pest control services. Notably, biological pest control is likely the ecosystem service most affected by biodiversity loss at the local scale. In coffee agroecosystems, management intensification alters habitat complexity by impacting vegetation connectivity and structure. The management intensification gradient ranges in coffee systems from the least intensive traditional shaded “rustic system”, in which coffee grows under a diverse closed canopy of native forest, to the most intensive “sun monoculture”, which refers to rows of open unshaded coffee monoculture, that require high inputs of agrochemicals. On the shaded end of the intensification gradient, shade coffee habitats are naturally vegetatively complex, with diverse and dense shade canopies and vines and weeds that form connections between the shade trees and the coffee plants. This vegetation connectivity is an important aspect of habitat complexity that impacts species interactions at the local scale. However, while progressing along the management intensification gradient, reductions in habitat complexity, driven by decreases in shade trees, increases in herbicide use, and the clearing of vegetation between coffee plants, reduce vegetation connectivity and alter species interactions within ecological communities and the ecosystem services that they provide. Connectivity is one physical component of habitats that has a profound impact on arboreal insects and ant community structure. In the absence of connectivity, plastic square flower bucket trees are insular habitats with crown isolation that inhibits the movement of some taxa.

Connectivity in the form of lianas and nylon ropes shape the local community structure of arboreal ants, with higher ant species richness often occurring in trees that are connected artificially or vegetatively as compared with trees without these physical connections, and higher ant species coexistence occurring in trees with higher levels of naturally occurring canopy connectivity. These results also reflect the nature of ants as highly efficient foragers, known to use branches and lianas as “opportunist walkways” that provide the quickest foraging routes by allowing for faster traveling speeds through avoiding obstacles on the ground, even if these routes are not necessarily the shortest distance. The variation in texture of natural walkways, characterized as “surface roughness”, further impacts both arboreal and ground ant running speeds and foraging efficiency. Physical connections between trees are thus important structures that facilitate not only arboreal ant mobility but also their foraging success, resource recruitment efficiency, and ant-provided ecosystem services, including pest removal. Ants play an important role in the control of the coffee berry borer , the most damaging insect pest of coffee. In particular, the aggressive arboreal ant Azteca sericeasur nests in shade trees, forages on coffee shrubs, and is a keystone predator that controls the CBB. Like many arboreal ants, A. sericeasur prefers walking on branches and vegetation to avoid traveling on the ground. Given the role of A. sericeasur as a biological control agent, understanding how connectivity at the local scale impacts these ants has potential implications for coffee agroecosystem management. In Chiapas, Mexico, Jiménez-Soto et al. found that artificially increasing connectivity between A. sericeasur nests and coffee plants by tying jute string between ant nest trees and coffee plants increased the capacity for A. sericeasur to remove the CBB by throwing them off the coffee plants.

These results suggest that naturally occurring vegetation connectivity might have a similar effect as that of artificial string connectivity on A. sericeasur activity and their associated pest removal services. Our study tests and expands on this hypothesis by examining the impact of both artificial connectivity and naturally occurring vegetation connectivity on A. sericeasur activity, its ability to recruit to resources, and its removal of the CBB with a manipulative experiment. Specifically, we tested the following hypotheses: We predicted that the coffee plants with vegetation or artificial connections to the ant nest tree have higher A. sericeasur activity than that of the isolated control plants; A. sericeasur ants recruit to resources more efficiently on coffee plants with vegetation or artificial connections to the nest tree; coffee plants with vegetation or artificial connections to the nest tree have greater CBB removal rates by A. sericeasur ants; and A. sericeasur activity, resource recruitment rates, and CBB removal rates decrease with increased distance from A. sericeasur nests. This study was conducted in the Soconusco region of Chiapas, Mexico at Finca Irlanda, a shaded, 300-hectare commercial polyculture coffee plantation. The plantation is located in the Sierra Madre de Chiapas Mountains at an elevation of 1100 m.a.s.l. The average canopy cover throughout the farm is 75 percent and the majority of the plantation shade trees are of the genus Inga. The climate is semi-tropical, with the rainy season occurring between May and October. Vegetation management at Finca Irlanda frequently includes “chaporreo”, in which farmworkers periodically use machetes to clear the weeds and epiphytes that grow between coffee plants. This management practice facilitates farmworker movement between coffee plants and reduces competition between weeds and coffee plants, but in the process inadvertently eliminates vegetation connections between the coffee plants and A. sericeasur nest trees.We collected data between June and August in the summer of 2022.

Within the 300 hectares of Finca Irlanda, we selected 17 trees with active A. sericeasur nests as study sites. Each site was located at least 10 m away from any other active A. sericeasur nests to prevent overlapping ant activity, following the methodology used by Jimenez-Soto et al.. We chose six coffee plants within a 5 m radius of each A. sericeasur nesting tree for a total of 102 coffee study plants. At each nesting tree site, we selected two coffee plants for the natural vegetation connectivity treatment, two for the artificial connectivity treatment, and two as isolated control plants . For the vegetation connectivity treatment, we selected two coffee plants with existing vegetation connections. The vegetation connections were either coffee branches directly touching the A. sericeasur nest tree or coffee branches touching a secondary plant, such as a vine or epiphyte that was touching the nest tree. We selected two coffee plants for the artificial connectivity treatment, in which we tied jute strings between the point of the nest tree trunk with the most active ant foraging trail and the central trunk of each coffee plant. We ensured that there were no existing vegetation connections on these plants and that the string was the only point of connection between each coffee plant and the nest tree. For the control treatment, we selected two isolated coffee plants with no connections between the coffee plants and the nest tree. We measured the distance between the central trunk of each study coffee plant and the ant nest tree. At each site, we quantified the ant activity on the coffee plants by counting the number of A. sericeasur that passed a central point on the central trunk of each coffee plant during 1 min . The observations took place between 7:30 AM and 2 PM before the afternoon rainy period. The observations were stopped if it began to rain, as rain significantly reduces ant activity. After setting up the strings, we returned to each site between 7 and 13 days after the initial setup and re-measured ant activity on the coffee plants. To assess the impact of artificial and vegetation connectivity on prey removal by A. sericeasur, plastic plant pot we placed five dead adult female CBB on white index cards on the centraltrunk of each coffee plant . We monitored A. sericeasur interactions on the cards for one hour, ensuring that only A. sericeasur were responsible for removing CBB, and counted the number of CBB removed. Because it has already been well-documented that A. sericeasur remove live CBB from coffee plants, we used dead prey to avoid the possibility of live CBB escaping during a longer observation period. The CBB were collected from infested coffee berries in the field, then frozen for up to 5 days before use. Recruitment is understood to be an integral component of trail-following in which ant workers follow chemical foraging trails to a food source, then re-apply chemical trails until that food source is exhausted. Tuna baiting is an effective and widely used method of assessing ant recruitment in coffee agroecosystems. To assess the impact of connectivity on ant resource recruitment efficiency, we placed 1 g of canned tuna on the central trunk of each coffee plant 1 m above ground and recorded the number of A. sericeasur that recruited to each tuna bait after 20 min. To test for statistical differences in ant activity, resource recruitment efficiency, and CBB removal between the control, string, and vegetation treatment coffee plants over the 5-week experiment, we fit our data with generalized linear mixed models using the lme4 package in R. For each response variable , we included the time , the treatment method , the distance between the coffee plant and the ant nest tree , the interaction between the treatment and the time, and the interaction between the time and the distance as fixed effects.

As random effects, we modeled the coffee plant identity nested within the site to control for site variation and spatial non-independence. To assess count data , we originally fit each model to a Poisson distribution. However, to correct for observed over-dispersion, we instead modified each model to a negative binomial distribution. We observed A. sericeasur using the artificial string connections at 12 of the 17 sites and on 20 of the 33 strings placed . A. sericeasur were the only ants observed using the strings. Out of 33 vegetation treatment plants , 20 plants included primary connections , and 13 plants were connected by secondary connections . We observed A. sericeasur utilizing vegetation connections on every vegetation treatment plant. Ant activity was higher on the vegetation treatment coffee plants than on both the control treatment and the string treatment . There was a significant effect of time on ant activity for the string treatment, indicating an increase in ant activity on the string plants after connecting the strings . There was no effect of time on ant activity for either the control or vegetation treatments, indicating that there was no significant change in ant activity for those treatments over the 5-week experiment . Treatment, time, and distance all impacted the ant recruitment to tuna baits. More ants recruited to the tuna baits on the vegetation treatment plants than on the strings, and more ants recruited to the bait on string plants as compared to the control plants . The overall number of ants that recruited to the baits decreased with time post string placement on both the control and vegetation plants, but there was no significant change in the number of ants recruiting to the baits on the string treatment . The number of ants recruiting to the baits on the control and string plants declined with distance from the nest tree, but was consistent over all distances for vegetation treatment plants .Treatment, time, and distance all impacted the number of CBB removed by ants. Ants removed more CBB from the natural vegetation treatment plants than from the string plants and removed more CBB on the string plants compared to the control plants . The overall number of CBB removed from the vegetation plants decreased with time post-string placement . The number of CBB removed on the control and string plants declined with distance from the nest tree, but was consistent over all distances for vegetation treatment plants .This study asked how connectivity, occurring naturally as vegetation or artificially as string connections, influences A. sericeasur activity, foraging efficiency, and pest removal services in coffee systems. Our research demonstrates that naturally occurring vegetation connectivity and, to a lesser extent, artificial connectivity between A. sericeasur nest trees and coffee plants increased both A. sericeasur mobility and CBB removal on coffee plants. Between the control, string, and vegetation connectivity treatments, all response variables were highest on coffee plants with naturally occurring vegetation bridges between the coffee plants and the ant nest tree.

The first three inoculated time points, 6, 12, and 24 hpi, had an average percentage of mapped reads between 85 and 88%. The last two time points, 36 and 48 hpi, where microscopic and macroscopic cell death was observed only had 52 and 64% of their reads mapped respectively. The complete results for total reads, percentage of reads mapped, and percentage of uniquely mapped reads are listed in Table 4.2.The total number of annotated proteins as well as DEGs are listed in Table 4.3. There were the highest number of both up-regulated and down-regulated DEGs at 24 hpi at 14,195 and 13,237 respectively. Using a cluster analysis of differentially expressed genes , the function of unknown genes can be recognized. In the hierarchical clustering, different areas with different colors, represent different groups of the cluster of genes up and down-regulated. This analysis shows two major clusters by treatment, one composed of all the mock uninoculated samples and inoculated samples corresponding to early time points . There were a large number of upregulated enriched genes associated with the ribosome and protein processing in the endoplasmic reticulum at 48-hpi, 321 and 189 respectively . There were also 72 enriched genes associated with the ribosome down-regulated at 12-hpi . Downregulated genes associated with the spliceosome were enriched at 12 and 36-hpi . There were many common differentially expressed plant defense genes that were upregulated in P. cinnamomi infected N. benthamiana leaves when compared to rootinoculated avocado, chestnut, and eucalyptus . Specifically, PAL, Thaumatin, Allene oxide synthase, F-box proteins, and cytochrome P450 were also significantly upregulated in avocado and L. longifolia roots. Genes encoding several members of the WRKY transcription factors were up-regulated in avocado, eucalyptus, chestnut, and L. longifolia roots. Glutathione S-transferase gene was up-regulated significantly in avocado and L. longifolia roots.

These and other defense related genes commonly expressed in avocado, round flower buckets other model systems, and the N. benthamiana pathosystem support the use of N. benthamiana to investigate defense gene response to P. cinnamomi.In this study we have elucidated the gene expression in response to P. cinnamomi infection in the N. benthamiana model system. By analyzing the transcriptome of N. benthamiana with RNAseq at five critical time points during the infection it was possible to identify important defense pathways using our model system. As early as 6-hpi there is a response by the infected host. We see a significant number of genes involved in the biosynthesis of secondary metabolites up-regulated. Specifically, known defense-related biosynthesis pathways such as flavonoid, terpenoid, and the phenilpropanoid pathways were enriched. Numerous plant-pathogen interaction genes were also enriched indicating the initiation of an active defense response to the pathogen. The highest number of enriched genes were involved in the biosynthesis of secondary metabolites at both 6 and 12-hpi. There was also a significant down-regulation of genes associated with the ribosome and plant hormone signal transduction especially auxin related genes. There is also a down-regulation in photosynthesis and metabolic pathways indicative of a decrease in resources allocated to growth and energy production in response to pathogen detection. The DEGs analysis confirms this early defense response with up-regulated hormone signaling, transcription factors, pathogen related genes, and resistance genes. At 24 hpi the KEGG enriched terms show a decrease in carbon fixation and metabolism which supports the allocation of resources towards plant defense. Salicylic acid binding is also down-regulated 5-fold which could be the result of pathogen effectors subverting the defense response. At the same time, we found transcription factors, PR genes, R genes, and anti-fungal genes that were up-regulated over 10-fold. Although P. cinnamomi is attempting to subvert the host defenses, the extensive colonization and intracellular growth at this stage of the infection has induced an extreme response in N. benthamiana.

At 36 hpi we found that besides significant up regulation in the plantpathogen interaction KEGG enrichment pathway there was up regulation of genes involved in endocytosis and phagosomes. Interestingly this is the time point where plant cell death becomes apparent in previously stained images of the infection process and is thought to be the stage where P. cinnamomi becomes necrotrophic in its infection strategy. There is also a 10-fold down regulation of SA binding and 5 to 10- fold up regulation of the JA pathway that further supports the necrotrophic infection occurring at this stage. At 48 hpi the KEGG pathway shows that 321 unigenes are involved in the up regulation of ribosome function. At the same time 770 unigenes are involved in the down regulation of metabolic pathways. Numerous defense genes are also being highly expressed at this time point including the continued up-regulation of the JA pathway. PAMP-triggered immunity is the plants first layer of defense against plant pathogens. Plants have developed pattern recognition receptors that initiate a defense response before the pathogen is able to infect the plant. This early PTI response is at a time in our model system when many of the encysted zoospores on the inoculated leaf surface haven’t germinated and there is no intracellular penetration . PTI response is linked to reactive oxygen species production, lignin and callose reinforcement, and the up-regulation of pathogenesis-related genes. Genes encoding Glutathione S-transferase, which serves to protect plant cells from ROS production was significantly up-regulated in our system as well as previously identified as being up-regulated during infection in Arabidopsis, Z. mays, and avocado . PR-genes up-regulated significantly in N. benthamiana in response to P. cinnamomi infection included; PR-1, PR-4 , PR-5 , and PR-9 . PR-5 was also up-regulated in infected avocado roots and PR-1 and PR-5 were up-regulated in eucalyptus roots . Hormone signaling plays an important role in a plants response to various pathogens and the pathway initiated varies depending on the type of pathogen. The jasmonic acid and ethylene signaling pathways are normally initiated in response to necrotrophic pathogens.

The cytochrome P450 super family is the largest enzymatic protein family in plants . CYP genes are involved in hormone signaling and associated with the JA pathway . CYP has been described as a JA-responsive gene which was up-regulated 37.75-fold in L. longifolia . Numerous genes in this family were up-regulated in all of the five time points we analyzed. Up-regulation of CYP has also been found in inoculated avocado . Some interesting and less well described anti-fungal genes in the CYP family, premnaspirodiene oxygenase and aristolochene synthase were identified that would be good candidates for further analysis. F-box proteins associated with the JA pathway were upregulated in 4 out of 5 of the time points in our system. The up-regulation of this gene in response to P. cinnamomi infection has also been discovered in avocado as well as numerous other model systems indicating a similar response among these different plants. Allene oxide synthase genes involved in JA biosynthesis , were also up-regulated in our system. JA response is traditionally associated with necrotrophic pathogen defense, plastic flower buckets wholesale but recent studies have shown that biotrophic pathogens such as Plasmopara viticola and hemi-biotrophs like P. infestans and P. cinnamomi can also trigger the activation of a JA triggered response. There were also some differentially expressed genes that are normally associated with the SA pathway discovered in the N. benthamiana system including up-regulated PAL genes and down-regulated SA binding genes. PAL is the key enzyme for the phenylpropanoid pathway which is involved in SA biosynthesis, lignin, and antimicrobial compounds such as flavonoids and phytoalexins . Auxin signaling has been shown to play and important role in the induction of resistance to P. cinnamomi . Plants using more than one defense pathway in response to P. cinnamomi infection has been seen previously in avocado and L. longifolia but was not found in Z. mays . Interestingly, genes encoding WRKY transcription factors were significantly upregulated at all of the time points in our system. In plants, WRKY transcription factors are encoded by a large family of genes, and they are involved in abiotic and biotic stress and are activated by pathogen perception . WRKY genes have been previously shown to be direct transcriptional targets of NPR1 in response to SA abundance . The significant up-regulation of WRKY transcription factors in our model system may indicate that there is some JA/ SA crosstalk in the infected samples. Up regulation of WRKY transcription factors in response to P. cinnamomi infection was also found in eucalyptus, chestnut, and L. longifolia roots. . WRKY51 transcription factor was selected as an ideal candidate for functional experiments because of its consistent up-regulated expression from 12 to 48 hpi shown in the RNAseq data and the uniform results during the qPCR validation. For the many reasons listed above WRKY51 transcription factor was chosen to functionally validate in our N. benthamiana model system. The transient over-expression of the WRKY51 construct was carefully timed through numerous experiments to discover at what point, if any, in comparison with the P. cinnamomi infection would the expression of this transcription factor have the greatest effect on the colonization of the pathogen. Transient expression of the WRKY51 protein 3 hours before inoculation with P. cinnamomi zoospores produced the clearest phenotype difference between the experimental and control groups. This early transient expression may induce an early defense response before the pathogen has a chance to colonize the host.

The RNAseq data in this study and others, and the functional assay using WRKY51 confirm that a close analysis of its expression in avocado is warranted. The RNAseq data as well as the functional work in this study provide a wealth of information concerning host defense response to P. cinnamomi infection. It is important however, to begin to make connections back to avocado which is the economically important crop we are interested in understanding more completely. By using the data obtained in this study and developing functional experiments that overcome the limitations associated with tree crops such as avocado we will be able to better address the long-term breeding concerns of avocado growers. Genes identified in our model system such as cytochrome P450 or identified and validated such as WRKY51 canbe used for marker assisted breeding. The avocado qPCR data for cytochrome P450 is an important first step in the goal to learn more about avocado defense gene response directly. The expression of cytochrome P450 in inoculated detached avocado leaves was similar to the expression in detached N. benthamiana leaves in both PS.54 and Dusa® rootstocks. Since cytochrome P450 has already been identified to be significantly up-regulated in P. cinnamomi infected avocado roots , it is reasonable to infer that this defense gene is similarly expressed in both roots and shoots. These universally expressed plant defense genes will provide vital information for resistance breeding projects in avocado. The next step is to find a functionally validated defense gene in our model system that is differentially expressed in the susceptible and tolerant avocado varieties and also transiently over express the avocado candidate genes homologs in N. benthamiana for functional validation. This would be an ideal candidate for marker assisted breeding. Using our model system, it is possible to identify such a candidate.Plant-parasitic nematodes infect a broad range of commercially important crop families such as the Solanaceae , Fabaceae , Malvaceae , Amaranthaceae , and Poaceae , causing an estimated annual loss of $80 billion USD . The most economically important group of PPNs are sedentary endoparasites, including root-knot nematodes and cyst nematodes . Sedentary endoparasites induce the formation of permanent feeding cells that provide specialized nutrient sources for nematodes . Infective second-stage juveniles of RKNs predominantly invade near the root tip and then migrate intercellularly toward the apical meristematic region without crossing the endodermis, making a U-turn to enter the vascular cylinder where they induce several giant cells as a feeding site by stimulating the redifferentiation of root cells into multi-nucleate giant cells by repeated nuclear divisions without cytoplasmic division. After maturation, adult RKN females lay eggs in a gelatinous egg mass on or below the surface of the root . In contrast, CNs move destructively through cells into the vascular cylinder, select a single cell, and form a syncytium as a feeding site by local dissolution of cell walls and protoplast fusion of neighboring cells. A CN female produces hundreds of eggs and its body forms a cyst that can protect the eggs for many years in the soil .

The need for additional replication may also have been reduced by using elevated amounts of genomic DNA and the use of end-labeling rather than BioPrime may have increased the reliability of calls. The protocol used for hybridization of lettuce genomic DNA was also subsequently highly effective for pepper and other Solanaceae. Furthermore, the use of genomic DNA is a desirable target because SFPs identified using cDNA may be a result of alternate splicing or gene expression differences. Rostoks et al. indicated that 40% of the SFPs they identified may have been falsely called and partially explained them as being mRNA structural variants. They also reported a high predicted false positive rate of 22% for SFPs detectedusing genomic DNA. We concluded that fragmented, end-labeled genomic DNA provided a suitable target for detection of polymorphisms while reducing false positive sequence polymorphism . The overlapping tile design increases the likelihood of detecting polymorphisms due to redundancy at individual positions, coverage along the contigs and optimal position of the SNP within a probe. Furthermore, the number of probes and hence the possible genome coverage was increased by substituting mismatch probes with AG probes for background correction and normalization of data. Because the peripheral 1 to 6 bases of a 25 bp oligonucleotide are less sensitive than the central bases, in terms of detecting sequence polymorphisms, procona system the tiling strategy reduces the loss of coverage due to probe position. The number and reliability of SPP calls in our experiments demonstrates that the overlapping tiling array design has improved coverage, sensitivity and specificity to detect polymorphisms.

SPP calls were validated using several approaches. The data from the two pair-wise comparisons yielded 20 to 41 thousand and 27 to 40 thousand SPPs respectively, depending on the criteria used for specificity and sensitivity. When SPPs from MSA and SFPdev were compared to the 51,552 SNPs detected between RNAseq reads of Salinas and US96UC23, 61.5% and 57.8% were found in or within at least 8 bp of the SPP range respectively, similar to that described by Gresham et al.. However, because of the high FDR associated with duplicated sequences, SPPs that were found to have a duplicated locus within the chip assembly, the gene space assembly or the genome assembly were removed from consideration; one third of the SPPs called that had duplicated loci did not contain a SNP in any of our validation tests. These identified SPPs likely were due to differences between paralogs rather than alleles at a single locus. Due to the increased redundancy provided by the mapping population of 213 RILs compared to the pair-wise comparison of the parents, SPPs in the SFPdev and MSA pair-wise comparisons that coincided with SPP mapped by Truco et al. but were absent of a SNP were considered real. Removal of duplicated loci and inclusion of mapped SPPs provided a balance between false positive and false negative rates and allowed us to optimize FDR while still discovering a high number of SPPs. Taking into consideration the lower observed FDR we concluded that the MSA method performed best as a pairwise comparison; however using multiple detection methods would yield a higher confidence in the subset of SPPs identified by both methods. The SPPs identified in the diversity panel that were polymorphic between L. sativa cv. Salinas and L. serriola acc. US96UC23 showed a low FDR. However, as a result of the filtering, sensitivity of this analysis was reduced compared to the two-genotype analyses by MSA and SFPdev. Specific analysis of the DP data for regions containing known SNPs showed that SFPdev values would have been significant in a pair-wise comparison, between SAL and SER but due to inclusion of data from all genotypes in the DP, the two were not called as polymorphic . The lack of some called SPPs in the DP may be due to larger genetic differences between L. perennis, L. virosa, or L. saligna relative to L. serriola and L. sativa. As a result of smaller hybridization differences between the more closely related genotypes, genotypes differing at a locus may have been grouped together reducing the number of SPPs called between the two genotypes.

Consequently, the DP analysis showed a lower false positive rate, but a higher false negative rate when comparing SAL and SER to sequence and mapping data. As part of our goal was to investigate the diversity and relationships of the genotypes in the DP, SPPs identified by the DP analysis were evaluated. Removal of SPPs in duplicated regions with inconsistent data or missing data was a reasonable method of removing unreliable data as these data may be from poorly performing probes in one or all replicates, heterozygous loci, paralogous genes or deleted genes. There was not a large difference in the observed FDRs for the three SPFdev cutoff values for the DP analysis; so in order to maximize the number of markers used in our phylogenetic analysis and principal component analysis, we used the least stringent cutoff value of 1.2. As the assumptions for analysis with the PHYLIP [21] package were not violated with the large number of markers, they were left as independent. To meet the constraints of the PC analysis software, markers were limited to those that were mapped. The markers discovered in our DP analysis were used to generate a phylogenetic tree showing species separation with 100% boot strap support. L. virosa and L. saligna are sexually incompatible species with L. sativa and appear to be more closely related to each other than to other species in the DP. Our data supports the conclusion by Kesseli et al., that these two species are not progenitors of L. sativa. By limiting markers to those polymorphic within cultivated lettuce we are able to separate most genotypes into classes representing each of the plant types. The butterhead type formed a distinct clade from the iceberg and cos types with 100% bootstrap support. However, the leafy type and the Batavia type both showed a wide distribution across the L. sativa clade. This is not unexpected and may reflect admixture between types during breeding programs. Alternatively, this distribution may indicate that these types are artificial polyphyletic groups based on loose morphological criteria. The leafy types are non-heading with a broad range of leaf morphology. Batavia types vary from heading to non-heading phenotypes. Batavia and iceberg cultivars are both considered crisphead types; however our phylogenetic and PC analyses showed that the two did not cluster together and are significantly different from each other . Rapid advancements in sequencing technology today are changing the methods for genetic analyses. Microarray technology presented in this paper yielded an in depth analysis of diversity for lettuce germplasm separating even closely related lines such as the crisp head class. It also has potentially several other uses including: detection of copy number variants, splice site identification, expression analysis or use with other species within the Compositae. TheSPPs identified in this study were highly reproducible and showed similar false positive results to current sequencing methods in the literature. This technology has also been used to create an ultra-dense, inter-specific genetic map between L. sativa cv. Salinas and L. serriola acc. US96UC23 to dissect phenotypic traits as well as validate and align genomic assemblies of lettuce into chromosomal linkage groups.Managing urban runoff and its associated pollutants is one of the most challenging environmental issues facing urban landscape management. The conversion of naturally pervious land surfaces to buildings, roads, parking lots, and other impervious surfaces results in a rapid surface runoff response for both time of concentration and peak flow. Impervious land surfaces adversely impact the quantity and quality of surface runoff because of their effects on surface water retention, infiltration, and contaminant fate and transport.

Large volumes of storm runoff from urbanized areas cause flooding, sewer system overflows, water pollution, groundwater recharge deficits, habitat destruction, beach closures, toxicity to aquatic organisms, procona valencia buckets and groundwater contamination. Traditional urban runoff management focuses on removing the surface runoff from urban areas as soon as possible to protect public safety. However, as excess surface water is quickly drained from urban areas, it is no longer available for recharging groundwater, irrigating urban landscapes, sustaining wildlife habitat and other uses. Green infrastructure uses natural or engineered systems that mimic natural processes to control storm water runoff. For example, traditional detention ponds have been widely used to treat storm runoff and permeable paving promotes infiltration of rain where it falls. Importantly, decentralized green infrastructure strategies control runoff and contaminants at their source. Vegetation is a green infrastructure strategy that can play an important role in surface runoff management . Large-scale tree planting programs have been established in many cities to mitigate the urban heat island effect, improve urban air quality, and reduce and treat urban runoff . There are municipal storm water credit programs in a growing number of cities that promote retaining existing tree canopy, as well as planting new trees . Although these programs encourage planning and management of urban forests to reduce runoff impacts , fertilizer is required to promote plant growth, and these added nutrients may contribute to contamination of surface runoff. Thus, reducing nutrients in storm runoff is a challenging task for landscape and water managers. Bioswales are shallow drainage courses that are filled with vegetation, compost, and/or riprap. As a part of the surface runoff flow path, they are designed to maximize the time water spends in the swale, which aids in the trapping and breakdown of certain pollutants. Bioswales have been widely recognized as an effective decentralized stormwater BMP to control urban runoff . Their effects are threefold; vegetation intercepts rainfall reducing net precipitation; plant uptake of water via transpiration reduces soil moisture, thereby increasing subsurface water storage capacity, and root channels improve infiltration . New bioswales are being developed for harvesting surface runoff and supporting urban tree growth. Bioswales that integrate engineered soil mixes and vegetation are being used to enhance treatment and storage of surface runoff . The composition of ESMs varies widely, from simple mixtures of stones and native soil to patented commercial products . Highly porous ESM mixes provide ample infiltration and pore space for temporary storage of surface runoff. Also, they support tree growth by providing more water and aeration to roots than compacted native soil alone. ESMs can reduce conflicts between surface roots and sidewalks by promoting deeper rooting systems. In California alone, over $70 million is spent annually to remediate damage by shallow tree roots to sidewalks, curbs and gutters, and street pavement . In Davis, California, a bioswale installed next to a parking lot reduced runoff from the parking lot by 88.8% and the total pollutant loading by 95.4% during the nearly two year monitoring period . Furthermore, a bioswale installed next to a turf grass patch at the University of California-Davis campus eliminated dry weather runoff from an irrigated urban landscape. The ESM used in these studies offered several advantages over other ESMs because the main structural element was locally quarried and relatively inexpensive lava rock . This ESM had a high porosity, high infiltration rate, and a high water storage capacity . The lava rock had many interstitial pores and a high surface area to volume ratio. It effectively fostered the growth of biofilms that retain nutrients and degrade organic pollutants. Because vegetated bioswale research is in its infancy, very few studies have monitored vegetation growth and its impacts on bioswale performance. Moreover, evaluation of system performance is generally conducted before vegetation is fully established . In contrast, this study evaluated the effectiveness of two bioswales on surface runoff reduction, pollutant reduction, and tree growth eight years after construction. The control bioswale contained native soil and the treatment contained an ESM. At the time of this study, the trees in the control and treatment bioswales were fully established and approaching mature size. Measurements recorded the differences in surface runoff dynamics and pollutant reduction rates, as well as tree and shrub growth. This study provides new information on the long-term effectiveness of engineered bioswales in a region with a Mediterranean climate. The water collection system was installed in 2007 to collect composited samples from natural runoff . In this study, surface runoff samples from the control site were collected at a high frequency using grab samples to better observe pollutant concentration dynamics for each experiment throughout a storm hydrograph.

The ant has several direct and indirect natural enemies, any one of which, or any combination thereof, could form the basis for the control that must occur to prevent the ant from taking over every shade tree in the plantation. One possibility we have suggested is that the phorid flies, known to reduce ant foraging activity, act as one suppressor. The reduction of foraging activity in the presence of phorids suggests that the latter could cause an ant colony to disappear, by either dying of starvation or being so harassed that the queen moves the nest to another site . A simple cellular automata model based on the natural history of the system captures the essential features of the clustering patterns of this ant . Furthermore, it has frequently been suggested that this sort of dynamic should lead to a power function distribution of the sizes of the clusters . As expected, the distribution of cluster sizes in our plot does follow a power function, as do the cluster sizes predicted by the CA model . It thus may be the case that the spatial pattern of the Azteca ants in coffee plantations forms by the same general rules that govern the formation of the spots of the jaguar or the stripes of the tiger, as suggested by the fundamental Turing process .As noted above, the relationship between A. sericeasur and the hemipteran C. viridis is a classic mutualism . While tending the scales, the ants also protect them from natural enemies, including parasitoids and at least two coccinellid predators, Azya orbigera and Diomus sp. . It is notable that these two coccinellid beetles appear to divide the habitat spatially, blueberry in pot with Diomus sp. able to feed on scales when they are separated in space from the local scale densities surrounding A. sericeasur nests while A. orbigera concentrate on those local densities near A. sericeasur nests.

Other ants are involved in tending the scales [especially the arboreal foraging but ground nesting Pheidole synanthropica , but including perhaps a dozen other species] but only on coffee plants that are not occupied by A. sericeasur . As is generally the case, coccinellid predators of hemipterans tended by ants face a dilemma; though adults can fly some distance to locate the isolated hemipterans that are not tended by ants, larvae are less mobile and need local concentrations of hemipterans to survive. However, the only place those high concentrations occur is where they are tended by ants. Consequently, the larvae of the coccinellid beetles, faced with the aggressive behavior of the protective ants, have evolved protective mechanisms against the ants, as noted above. In the network contained in the coffee system, Diomus larvae appear to engage in chemical mimicry that renders them invisible to the ants , and A. orbigera larvae are covered with waxy filaments, creating a barrier to ant attack . This arrangement provides a spatially explicit form of biological control in that the adult beetles range widely and consume hemipterans over a large area, but the larvae require the local concentrations of hemipterans that are provided only when under protection from ant mutualists, an ecosystem service provided by a simple level of ecological complexity . This also represents a spatially explicit community organization in that the ant P. synanthropica tends the same hemipterans, but never generates an extremely high density of the latter. The consequence is that when A. sericeasur searches for an alternative nesting site to escape its enemies, the residual concentrations of C. viridis supported by P. synanthropica provide them with an initial population of this key hemipteran mutualist. The alternative predator, Diomus sp., is spatially restricted to these less dense congregations of hemipterans but also participates in their overall control. Thus a spatially explicit organization of these interactions generates a unique community structure.As discussed in the section on trophic interactions, one of the main biological control agents of H. vastatrix is the white halo fungus, L. lecanii, which is commonly found in coffee plantations, especially associated with the hemipteran C. viridis when tended by Azteca ants . Because of this basic natural history , an obvious expectation is that the coffee rust disease incidence should be negatively correlated with the presence of ant nests because it is only under the protection of ants that C. viridis reaches densities high enough to attract the epizootics of the white halo fungus.

This is precisely what was found at three spatial scales. First, at a scale of 15 m, we reported a negative correlation between rust incidence and the distance to a coffee plant in which an epizootic of the white halo had killed all the hemipterans the previous year . Second, rust incidence data from plots approximately 50 m × 50 m show a negative correlation between rust incidence and coffee plants close to Azteca nests, although the R2 value was low, suggesting that many other factors affect the incidence of the disease . Finally, rust incidence data at a large scale similarly show a weak negative correlation between rust incidence and Azteca sites . It is notable that discerning the effect of L. lecanii first was facilitated by its relationship to the ant/hemipteran mutualism and thus its expected spatial pattern owing to the association of the latter with Azteca ants. At least eight other fungal pathogens are known through laboratory assessments to attack the causal agent of the coffee rust disease , yet finding spatial correlations that would indicate effectiveness in the field is not possible because of a lack of known spatial associations with other organisms. The essential ecological features of this disease are implicated in spatial ecology due to long distance dispersal by wind, local dispersal by touch and splash, mycoparasites, and other potential antagonists, and the need for a droplet of water for germination . The essential sociopolitical features include economic and political forces that cause coffee farming to either be undertaken or abandoned in a whole region, producing yet a larger spatial component of the system .Bunch grapes , notably European , are considered among the major fruit crops worldwide, producing roughly 70–80 million tons each year . Cultivars of V. vinifera L. are used for wine, juice, and table grape production. Grape berries are classified as nonclimacteric fruits, exhibiting a double-sigmoid developmental pattern with two rapid growth phases: the berry formation and the ripening phase , separated by an intermediate lag phase called the green plateau . The exponential increase in berry size characterizes both growth stages , but not the lag one .

During phases and , also known as immature stages, organic acids, mainly tartrate and malate, accumulate leading to induction of acidity levels . At the end of the lag phase, a step-change point takes place known as veraison, where acidity starts to decline while sugars, mostly glucose and fructose, as well as anthocyanins in colored varieties, increase. Of particular interest are phenolic compounds, which are major and ubiquitous plant secondary metabolites derived from the shikimate/phenylpropanoid and polyketide pathways, with three utmost categories: proanthocyanidins , also known as condensed tannins, the gallo- and ellagitannins , and the phlorotannins . Such diversity of polyphenols, with more than 8000 structural variants, bestows them a wide range of biological functions ranging from growth, development, and protection inside the plant to, to some extent, human-related issues . In grapevines, the accumulation pattern of phenolic compounds, plastic planters wholesale along with the aforementioned berry attributes, distinguishes each of the berry phases throughout berry development . Indeed, berry quality and sensory characteristics are notably defined by its polyphenol content . Remarkably, astringency is among the hardest sensory traits to depict and interpret as many intricate processes underpinning its perception . For instance, a sensory characterisation of the astringency of 11 varietals of Italian red wine revealed that neither total phenols nor PAs can predict how all astringency subtleties will be perceived . It is worth noting that the amounts, compositions, and proportions of polyphenols in a given species may vary widely depending on several factors, such as genotypic variations, developmental stages, and environmental circumstances . Scarlet Royal is a mid-season ripening table grape variety, producing seedless, red-skinned, oval-shaped, firm, and moderate to large berries with a sweet to neutral flavor . In the San Joaquin Valley, California, it typically ripens in mid to late August, filling the harvest window between Flame Seedless and Crimson Seedless, and has thus become a very popular red table grape variety in California. However, an undesirable astringent taste has been observed occasionally in some cases. In fact, the economic value of grapevines depends substantially on the environmental conditions, including climate, soil, cultural practices, cultivar, and rootstock. Hence, the term “terroir” is used in viticulture to describe the effect of such an interactive ecosystem on grapevine and wine quality . The current study aimed to understand the underlying mechanism of astringency development in Scarlet Royal berries at two contrasting vineyards . The first location produces well-colored, non-astringent berries; however, the second site yields astringent taste, poorly colored berries . The data showed a large variation in berry astringency within the same vineyard and from year to year. The data illustrated that the divergence in berry astringency stemmed from alterations in its polyphenol composition , most notably tannins. Additionally, the ripening stage was the most distinguishing platform for such variation between both vineyards. We were able to determine the tannins’ threshold level that causes the Scarlet Royal astringency taste to be ~ 400 mg/L. Given the changes in the levels of polyphenols during berry ripening, the question was raised: what is the mechanism governing the distinctive tannins accumulation pattern between V7-berries and V9-berries, and hence astringency diversity? To answer this question, RNA-seq data generated at one ripening timepoint was associated to the changes in polyphenolic levels using a systems biology approach, WGCNA .

The module-trait association analysis positively correlated the key flavonoid/PAs biosynthetic genes with the accumulation of tannins, catechin, and quercetin glycosides exclusively in V9-berries. The modulation of the berry’s transcriptomic profile is concomitant with its polyphenols’ composition, which finally disturbs berry quality, including astringency levels.Five-year-old V. vinifera cv. Scarlet Royal grafted on Freedom rootstock was chosen for its berry astringency diversity at two commercial vineyards located in Delano, San Joaquin Valley, California, USA. Vineyards were located at a close distance of 10 km, and the local weather conditions during the two seasons were collected from the Delano CIMIS weather station . Both vineyards were planted at the spacing of 2.44 and 3.66 m in an open gable trellis supporting system with East-West row orientation. Vines were pruned in a Quadrilateral cordon training with 7–8 spurs left on each cordon during the winter pruning. In addition, general UC guidelines practices were applied in both vineyard. Starting from veraison and until the end of the season, during two consecutive years . During the first year, sampling dates were July 8th , August 1st , August 10th , September 9th , September 15th , and October 19th ; and for the second year, sampling dates were: July 15th , August 10th , August 25th , September 10th , September 29th , and October 21st . Sampling dates varied from the first to the second year due to the vineyard’s accessibility. At each sampling point, two sets of fifty berries were collected periodically. The first set was used to measure the berry weight, and then these berries were macerated in an electric blender, filtered through a paper towel, and an aliquot of juice was used to determine soluble solids , pH, and titratable acidity . Soluble solids were determined using a tabletop Milwaukee MA871-BOX digital refractometer . The TA and pH were determined by titrating a 40 mL aliquot of juice with 0.1 N NaOH to a pH of 8.2 using an automatic titrator Excellence T5 . Another random 50 berries from each replicate were collected for color, tannins, and phenolic compounds and sent immediately in a cooler to EST laboratories. At harvest, which was during the month of September, an extra set of samples was collected and promptly frozen in liquid nitrogen and stored at −80°C for subsequent analysis, including RNA extraction and gene expression studies. Harvest time was determined by the growers, and the marketable clusters were picked based on the color, and yield was determined from the three harvest dates.At bloom, fifty leaves from each replicate were collected, resulting in a total of 200 leaves from each vineyard, for nutrient analysis.