Horticulture

Under baseline conditions, agricultural land use comprised 46% of the study area. Vineyard, pasture, and row crops make up approximately a quarter each of this agricultural area, followed by grains , while alfalfa, rice, and orchards accounted for less than 4% each. Under the enhanced agriculture scenario, the agricultural footprint increased to 68% of the study area, and is reduced slightly in the restoration and urban scenarios to 44% and 43%, respectively. The developed/urban class accounted for 9% of the study area under baseline, and increased in the urban scenario to 16% of the study area by 2050 .The total amount of carbon stored on the landscape under baseline conditions was ~784,000 Mg C ha-1 , with the majority stored in row crops , riparian forests , and grassland . Carbon storage in other agricultural classes was less than 4%. Carbon storage increased by 83% from baseline in the restoration scenario to ~1.4 million Mg C ha-1, largely associated with a 4-fold increase in carbon stored in riparian forests . Areas adjacent to the Cosumnes River and its tributaries became increasingly important for this ecosystem service . In the restored landscape, the carbon stored in riparian forests accounted for 63% of the total, with proportionally less harbored in row crops and grasslands compared to baseline. In the enhanced agriculture scenario, carbon storage increased by 12% from baseline to 879,000 Mg C ha-1 ,ebb and flow bench with parcels that harbor increased carbon storage scattered throughout the study area . Compared to baseline, the proportion of carbon stored in row crops increased slightly, from 47% to 53% of all carbon stored in the study area, while the proportion of carbon stored in vegetation classes such as riparian forest and grasslands decreased by ~4% each.

In the urban scenario, carbon storage decreased from baseline by 6% to ~740,000 Mg C ha-1 . The AUC score for the BRT model performance was 0.672 . Variables with the highest relative importance for predicting Swainson’s Hawk nest sites included riparian forest, row crops, pasture, grassland, vineyards, and urban/ developed areas. We found a positive relationship between nest sites and the proportion of riparian forest, row crops, pasture, and grassland within the surrounding 25-ha landscape, and a generally negative relationship with vineyards and urban/ developed areas, matching expectations based on field surveys of habitat use. Baseline conditions showed areas adjacent to the Cosumnes River had the highest suitability for Swainson’s Hawk . The predicted landscape suitability for Swainson’s Hawk changed substantially from baseline under each of the three management scenarios. It increased the most under the enhanced agriculture scenario , followed by the restoration scenario . Parcels close to the Cosumnes River became more suitable in the restoration scenario, and parcels throughout the study area became more suitable under the enhanced agriculture scenario . As might be expected, landscape suitability declined under the urban scenario . The average suitability of each land-use scenario as calculated for each of the 15 focal bird species showed subtle changes. Average suitability across all species increased under the restoration scenario by 5% and increased slightly under the enhanced agriculture scenario , but overall suitability declined by 1% in the urban scenario . In addition, using a 5% change threshold, the restoration land-use scenario resulted in a more suitable landscape for nine out of the 15 bird species. One species, the Yellow-Breasted Chat , experienced a 5% increase in suitability under the urban scenario, and no species experienced an increase or decrease in suitability exceeding 5% in the enhanced agriculture scenario.

The total amount of nitrous oxide emission associated with baseline agriculture in the study area is approximately 50,506kg N2O. Row crops had the highest N2O emissions, accounting for 60% of the baseline emissions. The next highest emissions were associated with grain and vineyards , while pasture, orchards, and rice were less than 3%. The total amount of nitrates leached from agricultural lands is approximately 2.1 million kg N. Patterns of nitrogen leaching were similar to nitrous oxide emissions, with row crops ranking highest , followed by grain and vineyards . Again, pasture, orchard, and rice comprised less than 2% of the total. Areas of high leaching and emissions under baseline were scattered throughout the agricultural lands of the study area . In the enhanced agriculture scenario, total N2O emissions and nitrate leaching in the study area increased by about 20%, concentrated in the northwest area . Nitrous oxide emissions increased to approximately 60,000 kg N2O, and nitrate leaching to 2.6 million kg N . Similar to baseline conditions, row crops accounted for the greatest proportion of emissions and leaching. In the restoration scenario where agricultural lands were replaced by natural vegetation, total N2O emissions and nitrate leaching decreased by 3% , mostly in close proximity to the Cosumnes River . There was also an approximate 5% decrease in these figures in the urban scenario as agricultural land is developed; N2O emissions decreased to around 48,000kg N2O and nitrate leaching to 2 million kg N, respectively . Patterns of emissions and leaching among types of agriculture are similar to baseline across both the restored and urbanized landscapes. The total agricultural commodity value of the study area under baseline was ~$184 million , with a handful of high-value parcels scattered throughout the study area . Row crops accounted for almost half of this value , followed by vineyards . Agricultural types favored by the Swainson’s Hawk such as alfalfa, grains, and pasture each accounted for less than 3%.

Under the enhanced agricultural scenario, revenue increased by 48% to ~$273 million , with revenues from row crops accounting for an even higher proportion of the total revenue . The restoration scenario resulted in a 4% decrease in agricultural revenue , while the urbanization scenario resulted in a 5% decrease .Our analysis examined a highly productive agricultural landscape and quantified how different land-management scenarios compared in terms of carbon storage, biodiversity values, ecosystem disservices, and agricultural returns at the parcel scale. More specifically, we looked at the trade offs under different land-use change scenarios, and evaluated whether positive benefits in financial agricultural returns were at the expense of biodiversity and other services. Underlain by the projected changes in land-cover types in 2050, our estimates indicated that the restoration scenario had multiple positive benefits from a conservation and environmental management perspective, similar to the conservation scenario generated by Nelson et al. . Restoration yielded substantial positive outcomes for carbon storage and habitat for Swanson’s Hawk, as well as 15 other focal bird species . Concurrently, ecosystem disservices decreased , and agricultural returns also decreased . Furthermore, the amount of agricultural land in the study area only decreased slightly under the restoration scenario. At the other extreme, urbanization had consistently negative effects on the landscape, and resulted in decreased carbon storage and landscape suitability for all bird species , along with a loss in financial agricultural returns . The only positive effect from a conservation and environmental management perspective was the reduction in nitrous oxide and nitrogen leaching . From a land-use planning viewpoint, however, these relatively negative effects of expanding the urban footprint would need to be examined in the context of alternatives for meeting the housing needs of local cities . The enhanced agriculture scenario was developed based on favoring the kinds of crops commonly protected or expanded as part of Swainson’s Hawk conservation and mitigation efforts. Although managing for a single species is not ideal, in some cases it is a necessity because mitigation for habitat loss or affected protected species demands it. In other cases, a single-species approach might be pursued by management if a keystone species is identified. Either way, evaluating the effect of this strategy on ecosystem services and disservices, agricultural returns,4x8ft rolling benches and potential multi-species benefits is valuable for management. By 2050, we estimated that the enhanced agricultural landscape had a highly positive effect on Swanson’s Hawk habitat value , higher even than the restoration scenario . Landscape suitability for the 15 focal bird species also increased marginally, i.e., favorable crop types for the Swanson’ hawk were also more suitable for some of these bird species. Agricultural returns increased by almost 50%. In contrast to other studies , we found carbon storage benefitted as well , owing to a replacement of grasslands with row crops, pastures, grains, and alfalfa with higher levels of carbon storage . However, these gains came at a cost, with increases in nitrous oxide emissions and nitrogen leaching by about one fifth . The approach and scenarios used in this study provide a framework which can be adaptively modified in the future to inform land utilization. Clearly, many different or complex management scenarios could be explored. Our intent was to evaluate a range of feasible options to demonstrate the effects of major restoration on the one hand to urban growth on the other, with the enhanced agriculture scenario in the middle. One future modification, for example, would be to optimize the configuration of native and adjacent agricultural land in the restoration scenario to increase connectivity across the landscape. Alternatively, future analyses could also account for carbon storage in urban green spaces, or the value of retaining mature trees to provide nesting habitat for a listed species in the urban scenario.

In addition, other services such as groundwater could be assessed, which is particularly relevant given the recently implemented Sustainable Groundwater Management Act, or recreation, given the high visitation rates of the study area. To improve estimates of ecosystem services associated with agricultural areas, better information is needed on crop rotations over time . The study area also harbors a few registered organic farms and some contracted organic production, such as rice cultivation. Future modifications of our approach could incorporate organic land management, given the association between organic practices and decreased ecosystem disservices. Although cultivation is largely conventional, compared to many parts of California it is highly diversified, with many different types of crops across small parcels. This spatial configuration makes farm scaping practices and buffer habitats a realistic option, which, in turn, can substantially affect carbon storage and biodiversity in agricultural lands . Estimates of the focal ecosystem services could also be improved by additional data, for example, including below-ground carbon storage estimates for the forest class, estimates of soil carbon, or nitrous oxide emissions for natural habitats. Our findings using the 15 focal bird species, although subtle, indicated that management actions for the Swainson’s Hawk yielded benefits for other bird species; however, it would be useful to assess how well this was reflected in other taxonomic groups. A number of studies from central California indicate that insects might respond similarly to the restoration scenario. For example, insectary hedgerows favor beneficial insects over pests by a ratio of three to one , with the highest numbers of insects correlated with the length of flowering period. Another study found that pollination by native bees depended on the proportion of natural habitat within 1 to 2.5km from the farm site . The authors compared rates of pollination of watermelon in Yolo County, California and found that farms with ≤1% natural habitat within 1 km experienced greatly reduced diversity and abundance of native bees compared to farms with ≥30% natural habitat within 1km, meaning that pollination services by native bees had to be supplemented by imported colonies of European honey bees. This study represents one of the few ecosystem services studies conducted at a spatial scale that is relevant to the on-the-ground decision-making of land managers, county planners, and conservation practitioners. Using parcels instead of pixels is useful because changes such as cropping patterns or fertilizer application occurs by these units. The identification of parcels that exhibit consistent, beneficial changes in carbon and biodiversity from baseline conditions across all scenarios may represent focal areas for targeted protection that constitute “no regrets” opportunities for conservation investment. In contrast, conflicted parcels that harbor both beneficial services and disservices might require land-owners and municipalities to work together to develop a management strategy that optimizes the value for beneficial ecosystem services. By restoring habitat, conservation biologists and restoration ecologists seek to promote the reassembly of diverse ecological communities, while also enhancing the ecosystem services these communities provide . Restoration of pollinator communities is of particular concern because pollinators play a critical role in plant reproduction in both natural and agricultural systems .

Mannuronate and guluronate blocks of the alginate have the capability for Mn2+ binding . However, at high alginate treatment the accumulation of Mn in roots was similar to the accumulation obtained with NPs alone. More experiments are needed to explain these results. In the case of Zn, the medium alginate treatment increased its accumulation in roots by 52% compared to control. The increase of elements in roots by CeO2 NPs may be due to the enhancement of soil cation exchange capacity produced by the CeO2 NPs. A small experiment was performed to determine if CeO2 NPs increased soil CEC. Results showed that the CEC in soil spiked with 400 mg/kg CeO2 NPs was 6× compared to control. Similar to soil natural colloids, e.g. kaolin and NOM, CeO2 NPs have a large surface area, with negative surface charge , that supplies many exchange sites for cation adsorption . to CeO The concentration of Na in roots of plants exposed 2 NPs alone decreased by 10.5% compared to control. In the CeO2 NP-alginate treatments, the reduction of Na in roots was higher . This could be associated with the increase in K absorption . As a C4 plant, corn needs Na to regenerate phosphorenolpyruvate,dutch bucket hydroponic the substrate for carboxylation . Thus, reduction of Na absorption could represent a toxicity pathway of CeO2 NPs to corn plants.Concentration of macro and micro-elements in corn shoots treated with CeO2 NPs and alginate are shown in Fig. 3.

As one can see in this figure, only the concentration of K was significantly affected by the NP treatments . All the CeO2 NP-containing treatments increased the concentration of K in corn shoots. However, there were no differences between the alginate and non-alginate treatments. An outward rectifying channel mediates potassium release into the xylem, which is controlled by the stress hormone abscisic acid . Perhaps the presence of NPs up regulates the production of abscisic acid, which in turn, increases the uptake of K. The fact that higher concentrations of Al, Fe, and Mn in roots, compared to control, were not observed in shoots could indicate that these elements were bound to NPs and stuck on the surface of the roots. Divalent cations have shown to bind with alginate.Chlorophyll and other leaf pigments are related to stress response.To elucidate if the CeO2 NPs coupled to alginate impacted the photosynthesis machinery in young corn plants , chlorophyll concentration in fresh leaves of one month-old plants was determined . As seen in this figure, none of the treatments affected Chl-b fluorescence. Chl-a was not affected by CeO2NPs alone; however, the combination of CeO2 NPs with alginate, at all concentrations, significantly reduced Chl-a fluorescence, compared to control. The CeO2 NPs plus alginate at low and medium concentration reduced Chl- a fluorescence by 16.5% and 18.4%, respectively.The soil was treated with CeO2 NPs with different concentration of alginate. Error bars stand for standard deviation. an indicative of stress . There exists the possibility that the CeO2 NP treatments affected the concentration of nitrogen or silicon in the corn plants, which are related to chlorophyll production or degradation.

Heat-shock proteins are general stress related proteins involved in the protection, restoration, and degradation of damaged cell components, especially proteins, during most abiotic stresses . Western-blot analytical technique was used to determine the content of HSP 70 in fresh leaves of one month-old corn plants. As seen in Fig. 5, HSP 70 was apparently over produced in the CeO2 NP treatments including medium and high amount of alginate. Khodakovskaya et al. discovered that multi-walled carbon nanotubes induce changes in gene expression in tomato leaves and roots, up-regulating the stress-related genes, such as heat shock protein 90. The present study showed that in corn plants, the HSP 70 increased expression in response to CeO2 NPs-alginate effect. However, the specific functions or structures protected by HSPs remain unknown. Heckathorn et al. reported that the chloroplast small HSP, which function is to protect photosynthesis during heavy metal stress in corn leaves , were trigged by heavy metal. Previous studies have shown that most of the CeO2 NPs taken by plants remain in the nanopariculate form; thus, it is possible that some of the NPs present in corn leaves reached the chloroplast affecting the HSP70 expression. More studies are needed in order to decipher these uncertainties. Land-based ecosystems in the northern hemisphere appear to remove, at least temporarily, a substantial portion of anthropogenic CO# from the atmosphere . The mechanisms behind this C sink are not well understood, even though knowledge of these processes is vital to predict and interpret the responses of ecosystems to global change .

Changes in plant productivity due to CO# enrichment , nitrogen deposition , land use change , and climatic effects have been investigated as potential components . However, the response of microbial communities to these perturbations, and their potential influence on C cycling, have received scarce attention. Mycorrhizal fungi in particular might play an important role in the sequestration of C in soil under elevated CO# and N deposition. This group, which symbiotically colonizes plant roots, forms associations with 80% of plant species and is found in nearly every habitat in the world . Plants allocate an estimated 10–20% of net photosynthate to mycorrhizal fungi, although this number can range from 5 to 85% among systems . A substantial amount of C allotted to mycorrhizal tissues could be long-lived in the soil. Chitin, which is not readily decomposed , can constitute up to 60% of fungal cell walls . Arbuscular mycorrhizal fungi are also the sole producers of glomalin, a potentially recalcitrant glycoprotein . AM hyphae in the absorptive hyphal network have lifespans of only 5–7 d , and with each cycle residual hyphal C should remain in the soil. Furthermore, some micro-arthropods prefer to graze on nonmycorrhizal fungi rather than on a variety of AM fungi , and therefore might not necessarily speed up tissue turnover significantly. As a result, glomalin alone can account for 30–60% of C in undisturbed soils , assuming that the protein is 30% C by weight M. C. Rillig, pers. comm. Likewise, portions of ectomycorrhizal biomass were responsible for approx. 15% of soil organic matter in two hardwood forests . Carbon derived from mycorrhizal tissue can account for a significantly sized pool within ecosystems and globally. Because mycorrhizal fungi acquire most or all their C directly from living plants, the nutrient status of foliage strongly affects mycorrhizal growth. As elevated CO# generally increases plant growth and root-to-shoot ratio , greater allocation of C to mycorrhizal structures might follow . Effects of elevated CO# on mycorrhizal growth have been reviewed by O’Neill , Diaz , Hodge , and Staddon & Fitter , with an emphasis on changes in percentage root length colonized and total root length colonized per plant. These reviews indicate that the percentage of roots with mycorrhizal structures might not necessarily change under elevated CO# . However,dutch buckets system as root biomass tends to rise, total mycorrhizal biomass per plant might do so as well. This response varies among systems and does not necessarily occur universally. By contrast, increases in N availability through deposition or fertilization tend to reduce root colonization and fruit body production by ECM fungi . Effects of CO# and N availability on the biomass or production of extraradical hyphae have been less intensively studied or summarized. In this review, we address the current state of knowledge regarding the potential for mycorrhizal tissue to form a sink or source of C in response to elevated CO# or N deposition. First, we present an overview of processes and pools involved in the cycling of mycorrhizal C and the relevance of various measures of mycorrhizal dynamics . Interand intraspecific variations in traits that could affect C dynamics are considered. Second, we discuss known effects of CO# concentration on hyphal biomass, turnover, tissue quality and community composition. Next, we focus on the influence of N availability on these same factors, and finally we address potential interactions between elevated CO# and N availability.Processes involved in the cycling of mycorrhizal C include production, survivorship and decomposition rates of tissue. As mycorrhizal tissue grows, C is transferred from the atmosphere via plants to the pool of live hyphae. Micro-arthropods might graze a fraction of live hyphae, but grazing on AM hyphae should be low, as in feeding trials mites and collembola appear to prefer nonmycorrhizal fungi.

When grazing of AM fungal hyphae does occur, animals often only clip the hyphae, severing connections to the root but not ingesting mycelial mass . Thus changes in micro-arthropod numbers might not have a major impact on C flux from AM hyphae to soil organic matter. Instead, death rates of live hyphae determine the flflux of C from the live to the dead hyphal pool. At this point, dead tissue is distributed between active and slow soil organic matter pools as a function of tissue quality . Active soil organic matter includes sugars and other metabolites that are processed relatively quickly by decomposers; slow soil organic matter consists of recalcitrant components such as chitin and glomalin, and might last from years to decades in the soil. In plant tissues, higher N content generally speeds decomposition rates . Finally, as soil organic matter decomposes, a portion of C remains in decomposer tissues, and the rest returns to the atmosphere. Each of these fluxes and pools might be affected directly by elevated CO# and N deposition, or indirectly through changes in the composition of the mycorrhizal community.Groups of mycorrhizal fungi differ in several factors, including growth rate, that could influence C cycling. For example, isolates of ECM fungi vary markedly in productivity, both within species and among species . In AM fungi, Sanders et al. observed significant differences in hyphal biomass among three Glomus species. In addition, after 16 wk growth, total hyphal lengths of Acaulospora denticulata and Scutellospora calospora were significantly greater than those of two Glomus species in a glasshouse experiment with Artemisia tridentata . If mycorrhizal communities are altered by climate change, then variation in growth and biomass among groups could affect the amount of atmospheric C that is initially drawn into the pool of live hyphae. Mycorrhizal groups also differ in tissue qualities that might affect the rate at which this mycorrhizal C is returned to the atmosphere. Wallander et al. found that five morphotypes of ECM fungi on field-grown Pinus sylvestris varied more than twofold in chitin concentration. Likewise, glomalin content in AM hyphae differed between Gigaspora and Glomus , and significantly between Glomus caledonium and Glomus intraradices . In addition, mean N concentrations in the hyphae of four isolates of Paxillus involutusranged from 5 to 9% when grown in culture.These results suggest that the identity, as well as the amount, of mycorrhizal fungi might be important in soil C dynamics.As most mycorrhizal structures are relatively delicate and often below ground, measurements of mycorrhizal biomass, growth rate or turnover present some challenges. Most mycorrhizal studies under elevated CO# or N deposition have quantified changes in mycorrhizal colonization . This measure might be an appropriate index for nutrient transfer to the host plant . However, because extraradical hyphae account for a large portion of fungal biomass , direct measures of hyphal length are a valuable indicator of the mycorrhizal C pool . Furthermore, root colonization does not necessarily increase linearly with hyphal biomass, and environmental changes might alter relationships between the two variables. For instance, the ratio of AM hyphal length : total root length colonized by AM varied nearly twofold among CO# and N treatments in Gutierrezia sarothrae , and was nearly three times greater under ambient versus elevated CO# in a serpentine grassland . In an additional study, Staddon et al. noted a decrease in this ratio with elevated CO# in Plantago lanceolata and Trifolium repens. For this reason we focus primarily on hyphal length or biomass in this review. We emphasize that hyphal length per unit soil area is a particularly meaningful variable in field studies, and might be used eventually to scale biomass to the ecosystem or regional level. The life stage of hyphae is also an important consideration when measuring fungal productivity. Few CO# or N studies have made the distinction between live and dead hyphae when determining hyphal length.

We also demonstrate the importance of resin selection and thorough chromatography operation optimization by evaluating the cost benefit of maximizing resin binding capacity to target product. Of course, further work is needed to verify whether the use of column chromatography is needed. Transient production of thaumatin in the edible crop Spinacia oleracea was also economically competitive and captures the benefits of obviating the need for an intensive DSP. According to this analysis, the cost to produce a kg of fresh weight of spinach is $0.10, as opposed to a cheaper price for tobacco . This is attributed to the higher cost of the seeds of spinach, the longer turnaround time assumed for spinach, and the higher plant density assumed for tobacco. It is evident that field operation is very labor intensive, due to the low recipe cycle time of 2 days, which is different than the traditional time frame of growing those crops. The potential for high intra-batch variations in product yield and quality due to meteorological factors is one of the concerns of using field grown plant material for this application. These variations in turns cause inconsistency in key facility performance parameters that should be quantified using a probabilistic approach and communicated to stakeholders and will be addressed in a follow-up communication. The cost of obtaining a more controlled supply of product is reflected in the indoor upstream facilities CAPEX and COGS. This should facilitate decision making when assessing the risk and reward of each scenario.

The large-scale recombinant production of thaumatin can address the growing market need for natural,nft hydroponic safe, non-caloric sweeteners. Like stevia, the advent of thaumatin as a sugar substitute is contingent on the feasibility of its large-scale manufacturing which was addressed in this work. However, there are also social, cultural, and behavioral factors impacting sugar consumption habits that were not considered. Consumer’s preference of such products will open the door for more plant-made bio-logics for food and beverage applications, which could drive the adoption of cost-effective solutions to rising challenges through environmentally friendly and sustainable processes.While the T-DNA approach is applicable to the model plants Arabidopsis and rice, where effective transformation methods are available, it may not be feasible in many other plant species whose transformation is inefficient. Transposon can be alternatively used for insertional mutagenesis in those plants, since the generation of new insertions occurs through crossing or propagation rather than through transformation. Supported by the United States National Science Foundation-Plant Genome Program . S.Q. was supported by the Research Start-up Grants of Zhejiang Academy of Agricultural Sciences , China. P.B.F.O. was supported by EU FP5 and FP6 projects Cereal Gene Tags and CEDROME . J.-S. J. was supported by the World Class University and the Crop Functional Genomics Center projects, Korean Ministry of Education, Science and Technology, Korea.The maize Ac-Ds transposable element has been shown to be active in the plant kingdom widely .

A number of important plant genes have been cloned using the Ac-Ds element . Ds insertion libraries have been generated in Arabidopsisand rice . However, the current strategies of transposon tagging are usually slow and labor intensive and have several drawbacks. For example, in the presence of Ac transposase , transposed Ds elements may continue secondary transpositions. Unstable Ds insertions and serial transposition events may cause untagged mutations because imprecise excision or a transposition footprint can result in a mutation that is no longer associated with the transposon . Another problem is that the Ac-Ds transposable elements are highly active in rice and can transpose early in newly transformed callus cells , which results in many sibling plants carrying the same Ds insertions and consequently decreasing gene tagging efficiency. In the present study, we constructed 12 Ac-Ds transposon tagging vectors based on three approaches: AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain chemical inducible expression system; deletion of AcTPase via Crelox site-specific recombination that was initially triggered by Ds excision; and suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element. We have tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful in functional genomics of rice and they will be useful for other crop plants as well.We constructed Ac-Ds transposon tagging vectors using a GVG-inducible expression system . The vectors pJJ86 and pDs-Ac-GVG carry an in cis two-element system that consists of Ds, 35S:GVG that expresses the chimeric GVG transcription activator, and AcTPase controlled by a GVG-inducible promoter.

The inducible promoter is transactivated through interaction between GVG and the 4xGAL4-upstream activating sequence . The transactivating activity of GVG is regulated by treatment with the steroid chemical dexamethasone . The Ds element in pJJ86 contains the 4x CaMV 35S enhancers for activation tagging , while the Ds in pDsAc-GVG does not. Excision of Ds from pJJ86 can be detected because in the resulting T-DNA fragment, the β-glucuronidase gene is driven by a CaMV 35S promoter. We also constructed a two-vector tagging system in which GVG-inducible AcTPase and Ds are in separate vectors . The strategy of the two-vector system is that transgenic plants carrying the GVG-inducible AcTPase and Ds are generated, respectively, and the AcTPase and Ds are combined in F1 by genetic crosses. In this case, Ds is mobilized in the presence of AcTPase in F1 plants, but stabilized after it is uncoupled from AcTPase in the subsequent generation. To test whether the inducible Ac-Ds system is functional in rice, we transformed rice cultivar Nipponbare with pJJ86. Independently transformed rice calli were cultured for 5 d on media with DEX to induce expression of AcTPase. Because Ds transposition can be detected by GUS activity, the DEX-treated calli and untreated controls were stained for GUS activity. DEX treatment of pJJ86-transformed calli exhibited stronger GUS staining than controls , indicating that the DEX inducible system in this vector is functional in rice. At the same time, there was low background of GUS activity in the untreated rice calli , suggesting that some background transposition occurred in the pJJ86 transformants.Because the Ac-Ds transposable elements are active in newly transformed callus cells and early transposition events lead to the same Ds insertions in sibling plants, we constructed a novel Ds element, designated HPT-Ds, and used the hygromycin resistance gene to suppress transposition. The pHPT-Ds1 vector carrying HPT-Ds and GVGinducible AcTPase in cis is shown in Figure 1E. The HPT gene in HPT-Ds has the same intron and triple splice acceptors as in the gene-trap Ds . Because HPT-Ds is immediately downstream of maize ubiquitin 1 promoter in T-DNA, the Ubi:HPT-Ds fusion confers hygromycin resistance, and transformed rice cells are thereby selected on hygromycin media. In case of transposition, HPT-Ds in the rice genome may not have a promoter nearby for transcription and the rice cells lose hygromycin resistance and can be counter-selected by hygromycin. To examine the efficacy of the HPT-Ds element,nft system we made a test construct containing Ubi:HPT-Ds and confirmed the function of the Ubi-driving HPT gene in a rice transformation experiment.

A total of 250 rice calli were transformed using a particle bombardment method and hygromycin-resistant cells were selected from 30 callus explants after 50 d of selection on hgromycin media. In constructing the pHPT-Ds1 vector, HPT-Ds was cloned between Ubi and GUS so that transposant cells canbe detected by GUS assay. The pHPT-Ds2 vector is similar to pHPT-Ds1 except that pHPT-Ds2 carries a Bar gene and transposition can be selected by herbicide resistance . pHPT-Ds1 was introduced into rice cultivar Nipponbare. A total of 26 stably transformed callus lines were obtained. In the condition without DEX treatment, five calli were randomly selected from each callus line and GUS-assayed for detection of transposant cells. Transposant cells were detected in 84.6% of callus lines but mosaic GUS patterns occurred at low frequency as compared with the GUS patterns of untreated pJJ86 calli . GUS assays were also carried out on 14 of pHPT-Ds1 transformed plantlets; 57.1% plantlets contained transposant cells that were rarely distributed in the tissue . The results of pJJ86 transformants and pHPT-Ds1 transformants indicated that there was background transposition activity in the rice calli and plantlets selected from hygromycinmedia, and that the growth of rice cells containing HPT-Ds transposition events were partially suppressed by hygromycin counter selection. To characterize the HPT-Ds excision events, rice genomic DNA of eight GUS-positive pHPT-Ds1 transformants was extracted and examined in nested polymerase chain reaction reactions using Ubi- and GUS-specific primers . Reconstructed Ubi:GUS sequence containing the HPT-Ds empty donor site was confirmed by sequencing the 657-bp PCR product . These results suggested that HPT-Ds elements in the pHPT-Ds1 transformants excised from the T-DNA. To get more information about the background transposition in the GVG-inducible AcPTase system, we constructed pHPT-Ds3 and pHPT-Ds4 by removing the 35S:GVG from pHPT-Ds1 and pHPT-Ds2, respectively. According to the GUS assay results of pHPT-Ds3 transformed callus lines, 57.1% of the callus lines showed somatic transposition. The mosaic GUS patterns of pHPT-Ds3 transformants were similar to those of pHPT-Ds1 transformants and the transposition frequency was a little lower than 84.6% of the pHPT-Ds1 calli. Our explanation for the results of pHPT-Ds1 and pHPT-Ds3 is that the background transposition in the GVG-inducible Ac-Ds system was primarily due to a low-level leaky expression of 4xUAS:AcTPase .The HPT-Ds element described in the present study is a novel Ds whose HPT gene has a dual function. During plant transformation and selection, HPT expression relies on the upstream Ubi promoter to confer resistance to hygromycin in selection media. In case of transposition, the HPT gene may be inactive because the 5 flanking sequence of HPT-Ds at a new genomic site may not be able to provide promoter activity. It is conceivable that most of the transposant cells become sensitive to hygromycin. Therefore, the counter-selection nature of the HPT gene in HPT-Ds can be used to diminish transposant cells in newly transformed rice calli on hygromycin media. In testing pHPT-Ds1 and pHPT-Ds3, it was observed that early transposition events in transformed calli and plantlets were suppressed by hygromycin. Few transposant cells in the calli and plantlets were able to grow under the hygromycin selection pressure, which might be due to escaping transposant cells or because of promoter activity of the 5 transposon flanking sequence. Because transposition requires transposase, an important theme in transposon tagging research is how to efficiently control transposase activity.

It was reported that AcTPase driven by strong promoters mediated high-frequency Ds excision in several dicot plants . Strong double enhancers of CaMV 35S promoter adjacent to wild type Ac element induced high-frequency Ac excision in rice transformation . In the present study, we have used the GVG-inducible promoter to control AcTPase expression and transposition was induced to high levels by DEX treatment of pJJ86 transformed callus. However, we also observed a leaky expression of AcTPase in the GVG-inducible Ac system in the transformants of pJJ86, pHPT-Ds1 and pHPT-Ds7 based on GUS assay results. Our explanation is that the transposition background was primarily from a low level of leaky expression of 4xUAS:AcTPase. Consistently, in the pHPT-Ds3 and pHPT-Ds5 vectors that do not have 35S:GVG, 57.1% of the pHPT-Ds3 transformants and 76.6% of the pHPT-Ds5 transformants still showed transposition in somatic cells. In spite of the wild type Ac element having a weak promoter that supports only 0.2% expression of the CaMV 35S promoter , the wild type Acitself can transpose in rice with a relatively low activity for three successive generations . This indicates that a weak expression of AcTPase can cause transposition events. In Southern blot analysis of genomic DNA of pHPT-Ds7 and pHPT-Ds8 transformants, the 5.4 kb hybridizing band represented the HPT-Ds at FDS in T-DNA. For the hybridizing bands larger or smaller than 5.4 kb, we explain that some of the bands might be from transposed HPT-Ds. The pHPTDs7 transformants showed transposition in somatic cells as suggested by GUS assay results. Because the rice genomic DNA for Southern hybridization was extracted from few leaves of a transformant, transposition in other leaves might not have been detected in the results. Also, since a rice transformant may have more than one T-DNA copy and may contain rearranged T-DNA, the hybridizing bands larger or smaller than 5.4 kb might possibly be from transgene rearrangement. Nevertheless, the efficacy of the HPT-Ds element when it was brought together with the GVG-inducible-AcTPase and the Cre-lox recombination system in pHPT-Ds7 and pHPT-Ds8 was confirmed by GUS assay and Southern blot analysis.

Since these technologies are unlikely to be mission critical during Artemis, their TRL can be increased and their risk factors studied through in-space evaluation. The Artemis missions also provide a test bed to evaluate the space-based evolution of microbes and alterations of seed stocks as a risk inherent to the biological component of the biomanufactory. This risk can be mitigated by incorporating backup seed and microbial freezer stocks to reset the system. However, ensuring that native and/or engineered traits remain robust over time is critical to avoid the resource penalties that are inherent to such a reset. Consequently, while optimal organisms and traits can be identified and engineered prior to a mission, testing their long-term performance on future NASA missions prior to inclusion in life support systems will help to assess whether engineered traits are robust to off-planet growth, whether microbial communities are stable across crop generations, and whether the in situ challenges that astronauts will face when attempting to reset the biomanufacturing system are surmountable. Quantifying these uncertainties during autonomous and crewed Artemis missions will inform trade off and optimization studies during the design of an enhanced life support system for Martian surface bio-operations.Crewed surface operations of ∼ 30 sols by four to six astronauts are projected to begin in 2031 ,growing tomatoes hydroponically with an additional mission similar in profile in 2033 . Given the short duration, a mission-critical biomanufactory as described herein is unlikely to be deployed.

However, these short-term, crewed missions RMA-S1, S2 provide opportunities to increase the TRL of biomanufactory elements for ∼ 500 sol surface missions RMA-L1 in ∼ 2040 and RMA-L2 in ∼ 2044. Building on the abiotic ISRU from early Artemis missions, we propose that RMA-S1 carry experimental systems for C-and-N-fixation processes such that a realized biomanufactory element can be properly scaled . Since RMA-S1, S2 will be crewed, regolith process testing becomes more feasible to be tested onsite on the surface of Mars, than during a complex sample return mission. Additionally, while relying on prepacked food for consumption, astronauts in RMA-S1 will be able to advance the TRL of platform combinations of agriculture hardware, crop cultivars, and operational procedures. An example is growing crops under various conditions to validate that a plant microbiome can provide a prolonged benefit in enclosed systems, and to determine resiliency in the event of pathogen invasion or a loss of microbiome function due to evolution. Additionally, the TRL for crop systems can be re-evaluated on account of partial gravity and/or microgravity. The RMA-S1 and RMA-S2 crews will be exposed for the first time to surface conditions after interplanetary travel, allowing for an initial assessment of health effects that can be contrasted to operations on the lunar surface , and that may be alleviated by potential biomanufactory pharmaceutical and functional food outputs . The RMA-S1 and RMA-S2 mission ISRU and FPS experiments will also provide insight into the input requirements for downstream biomanufactory processes. ISM technologies such as bioplastic synthesis and additive manufacture can be evaluated for sufficient TRL. Further, loop closure performance for several desired products can also be tested. This will help estimate the impact of waste stream characteristics changes on recycling .We have outlined the design and future deployment of a biomanufactory to support human surface operations during a 500 days manned Mars mission.

We extended previous stand-alone biological elements with space use potential into an integrated biomanufacturing system by bringing together the important systems of ISRU, synthesis, and recycling, to yield food, pharmaceuticals, and bio-materials. We also provided an envelope of future design, testing, and biomanufactory element deployment in a roadmap that spans Earth-based system development, testing on the ISS, integration with lunar missions, and initial construction during shorter-term initial human forays on Mars. The innovations necessary to meet the challenges of low-cost, energy and mass efficient, closed-loop, and regenerable biomanufacturing for space will undoubtedly yield important contributions to forwarding sustainable biomanufacturing on Earth. We anticipate that the path towards instantiating a biomanufactory will be replete with science, engineering, and ethical challenges. But that is the excitement—part-and-parcel—of the journey to Mars.Doubled haploid techniques have established themselves as useful tools in plant breeding and genetic research. As of 2005 doubled haploids had been produced in over 230 different crops . DH lines can be produced in several ways, by distant hybridization , by androgenesis or by gynogenesis . Each method has some advantages and disadvantages and not all are applicable or efficient in all crops. As discussed by Forster and Thomas 2005 the advantage of DH in breeding is in time savings between the initial cross and large-scale testing of developed lines.The advantage of DHs in genetic research is that perfectly homozygous lines can be produced relatively quickly and permit large scale replicated trials for detection of quantitative trait loci and their allocation to specific, sometimes very small, chromosome regions. In all cases, the critical issue in the use of double haploids is the efficiency of their production. This efficiency can be measured in several ways but it can always be brought down to the ultimate factor: how many useful lines can be produced in a given time with available labor and resources. For those involved in the DH development, the critical measure of efficiency is the number of green plants produced.

However, not all green plants need to be useful in breeding or research. Given that stability/uniformity is the main advantage of the DH lines, any deviation from it, including that caused by aneuploidy, is detrimental and in most cases will disqualify a line from any research or breeding. Triticale is a new cereal that in ca. 135 years since the first man-made wheat-rye hybrids were created has established itself as a competitive crop in certain environments. Unlike its progenitors, wheat and rye, triticale is generally amenable to anther culture and in most instances produces reasonable yields of haploids. First commercial cultivars of triticale developed with the aid of androgenesis have been released and the method is routinely used in breeding with thousands of DHs produced every year . The androgenic response in triticale appears to be under genetic control but in most cases, sufficiently large populations can be generated. However, despite a considerable progress in breeding for meiotic stability, triticale still has chromosome pairing issues and tends to show a fair proportion of aneuploids among sexually derived progenies, more so in hybrids than established lines . Perhaps for this reason,hydroponic growing supplies the issue of efficiency of the DH production and the quality of the recovered DH lines requires more attention in triticale than in most better established and more meiotically stable crops, such as wheat or barley. The wider context of this study was to generate sufficiently sized mapping populations of the DH triticale lines. Aneuploidy severely complicated this effort and appeared as an issue deserving to be analyzed with an additional effort. However, since from the start the emphasis was on the mapping populations and not the process of androgenesis itself, some data on population sizes and frequencies of specific events were never collected. Still, it appears that the issue of aneuploidy in triticale DH lines is important enough to be addressed.The measurements of the DNA contents were done on a population of spontaneously DHs of the Presto 9 Mungis combination. Ploidy levels of the regenerated plants were evaluated by fluorescence using a Partec II flflow cytometer . Samples were prepared according to Galbraith et al. with some modifications. Leaf fragments were chopped with a sharp razor blade in a Petri dish with 2 ml of the nuclei-isolation buffer Triton X-100 containing DAPI and the cell suspension was filtered through a 30-lm filter. The ploidy levels of regenerants were determined by comparing the G1 peaks of the analyzed samples to internal controls, here both parents, cv. Presto and Mungis. Plants with the peaks of the DNA contents distribution clearly separated from the controls were considered aneuploid.Root tips for karyotyping were collected either from seed germinated on wet filter paper in Petri dishes, or from plants grown in a hydroponic culture in an aerated full strength GroResearch Gromagnon 9-5-18 All-Purpose-Nutrient solution from American Hydroponics, Arcata, CA USA. For collections from germinating seedlings, seed were placed on wet filter paper in Petri dishes for 2–3 days at room temperature, refrigerated at ca.2℃ for 2–3 days, transferred to room temperature for 24 h and collected to ice water for ca. 27 h. Depending on the intended cytological protocol, roots were either fixed in 45% acetic acid overnight and squashed, or fixed in a Carnoy solution .

Roots from the hydroponic-grown plants were collected to ice water for ca. 27 h and fixed in the Carnoy solution. For the analyses of meiotic metaphase I chromosome pairing one anther from a spikelet of an ear was excised and live-squashed in a drop of 1% acetocarmine. If MI was present, the remaining two anthers were fixed in the Carnoy solution. All material in the Carnoy solution was fixed for 7 days at 37℃, stained in 1% acetocarmine for 2 h, re-fifixed and stored at -20℃ until used. All preparations of this type of material were made by squashing in a drop of 45% acetic acid using the two-coverslip technique. All C-banding on the 45% acetic acid- fixed root tips was according to Lukaszewski and Xu ; all C-bandning on the Carnoy-fixed material, whether root tips or anthers, was according to Giraldez et al. . In situ probing with labeled DNA was done according to the protocol of Dr. T. Endo, Kyoto University, Japan . Total genomic DNA of rye was labeled with digoxigenin and detected by the antidigoxigenin-fluorescein using standard kits and protocols from Roche Applied Science . The probe was mixed with blocking wheat DNA prepared according to MasoudiNejad et al. , usually in the probe to block ratio of ca. 1:100. Counter staining was with 1% propidium iodide. Sequential C-banding- in situ probing were done using the protocol provided by Dr. T. Endo, Kyoto University, Japan. For sequential C-banding- in situ probing, preparations were made in the same fashion as for standard in situ probing, they were C-banded, analyzed and photographed. The slides were then de-stained air dried, and probed using the Masoudi-Nejad et al. protocol. The cells photographed after C-banding were re-photographed after the in situ probing. All observations were made under a Zeiss Axioscope 20 equipped with epi-fluorescence, recorded with a SPOT RT Color digital camera , and processed using the SPOT Advanced and Adobe Photoshop CS software to improve contrast and resolution.The larger purpose of this study was to generate useful DH lines and no detailed records of the regeneration rates/ success were collected for every attempt or in every season. The total number of green androgenic plants transplanted to soil exceeded 3,500. Weak green plants that did not grow well in test tubes were not transplanted and no record exists of their numbers/proportions. Of the transplants, a certain proportion died before the colchicine treatment and the weakest of the survivors were not colchicine-treated. The estimated overall proportion of weak green plants rejected before colchicine treatment was about 8%, but in different cross combinations it ranged from ca. 2–3% toca. 70% in the group of late regenerants in Presto 9 NE422T. Mortality due to colchicine was low . Seed set per colchicine-treated plant ranged from a single seed to complete fertility. Complete fertility is probably indicative of spontaneous chromosome doubling in early microspore divisions rather than an effect of colchicine. In three cycles of androgenesis that included 644 colchicine-treated plants for which fairly detailed counts were made, 157 green plants were deemed to be aneuploid, based on their morphology at flowering. Most of these presumed aneuploids did not set seed. Those that did set seed and had their progeny karyotyped, all were aneuploid. Among plants that appeared normal in C0 and set seed, additional 16 were deemed aneuploid based on their morphology and seed set in C1, and all were found to be aneuploid upon karyotyping. Therefore, the average frequency of aneuploids among all regenerated green plants from all cross combinations must have exceeded 35%. In one of the most recalcitrant combinations, NE422T 9 Stan 1, among 72 plants that survived colchicine treatment, 50 were morphologically abnormal and were considered aneuploids.

The concentration of toluene in the building exhaust was 120 µg m-3, more than double the highest level measured indoors, suggesting a possible toluene source in the restrooms. The cleaning compound 2-butoxyethanol was slightly higher indoors, but at very low concentrations.The compounds listed in this category have many sources, including outdoor air. For the most part there was little difference across the building spaces for these compounds, and little difference from the ambient air measurement. The single exception to this observation is methylene chloride that appears to increase by about a factor of ten indoors. It is possible that this compound is in use as a cleaning solvent, or it may be present in computer equipment or other supplies. Methylene chloride is also used as a spot remover in dry cleaning processes and may be carried into the building on occupant clothing. The levels of this compound were low relative to health standards . Note that for compounds where measurements were below the LOQ the relative difference may be an artifact of imprecision of measurement rather than a reflection of real differences. These values should be interpreted carefully. 

In Table 4 the compounds which display relative reductions between two zones are shaded a tan color,growing tomatoes hydroponically while those with relative increase of factors of tens are shaded light green, and those with relative increases by factors of 100 are shaded red.  The HVAC inlet to the building which includes an air washer and filtration showed a reduction of all measured VOCs and aldehydes with the exception of octanal, 1,4‐ dichlorobenze, decamethylpentasiloxane, phenol, TXIB and diethylpthalate. The reductions were ranged from 4% to 100% for 2‐butoxyethanol, d‐ limonene, trichloroethylene, and a‐pinene. The plasticizer concentrations increased140% and 320% as the air moved from outside into the greenhouse. VOC concentrations all increased as the air was transmitted through the greenhouse. The concentration of the odorous compounds hexanal and nonanal increased by 68% and 110%, respectively in the greenhouse. Most other compounds showed modest increases on the order of 10s of percentage points, although styrene concentrations increased by almost a factor of 7 in the greenhouse.  With only a few exceptions , VOC and aldehyde concentrations increased as the ventilation air moved from the greenhouse into the occupied building spaces. Increases between the average indoor concentrations and the greenhouse air exit ranged from 6% for toluene to a factor of 120 for the cleaning solvent 2‐butoxyethanol. The cleaning solvent d‐limonene increased by a factor of 56, methylene chloride increased by a factor of 11, and a‐pinene by a factor of 64.Diethylpthalate increased by a factor of 21. 

Odorous compounds hexanal, nonanal, and octanal all increased by factors of two to four, as did the irritating and carcinogenic compound formaldehyde. The deodorant 1,4‐dichlorobenzene concentration increased by over a factor of six. The concentration of the VOCs and aldehydes in the HVAC exhaust stream was in only a single case lower than the incoming air , where it was zero in the exhaust. D‐limonene was 19 fold higher in the exhaust than the intake, 1,4‐ dichlorobenzen was 76 fold higher, and diethylpthalate increased 17 fold. Concentrations in the exhaust vs. greenhouse tended to be higher than the comparison to the ambient air – this is due the observed decrease at the intake relative to the greenhouse – possibly the benefit of the air washer. Overall, exhaust concentrations exceeded that supplied to the zones containing plants by small to large multiples. The concept that the building air is scrubbed clean of gaseous air contaminants by the plants is not supported by the data. This being said, it is not known what the building IAQ would be without the plants. Table 5 provides a comparison between the measured indoor VOC and aldehyde concentrations in the PBC and those measured in a survey of office buildings in the United States . From 1994 to 1998 the U.S. Environmental Protection Agency  conducted the Building Assessment and Survey Evaluation Study of 100 randomly selected office buildings in the continental U.S. . The data in Table 5 are summary statistics of measurements taken from the BASE study . Note that not all of the VOCs measured in the PBC were studied in the BASE study, and vise‐versa. The BASE study did not measure all of the listed compounds in all 100 buildings as can be seen from the Table. Compounds for which the PBC had more that a factor of two greater indoor concentrations than the BASE Study mean are 1,4‐dichlorobenzene, methylene chloride , and TXIB. 

The measured PBC formaldehyde and acetaldehyde levels were both above the mean BASE Study levels by more than one standard deviation. Compounds for which the PBC had one half or less of the concentration of the BASE Study indoor concentrations included 2‐butoxyethanol, acetone, d‐limonene, phenol, and styrene. Both 2‐butoxyethanol and d‐limonene are cleaning agents that may be used less frequently in the PBC than in the U.S. office buildings. The compound measured that is of greatest concern is formaldehyde, as discussed above, which has an 8‐hr average REL of 9 µg m‐3. The average across the measured spaces in Floors 5 and 3 was 28±1.4 µg m‐3. However, the observed formaldehyde concentrations fall within levels that have been recorded in U.S. office buildings . The observed levels of odorous compounds hexanal, nonanal, and octanal may detract from perceived air quality in the PBC, as the observed concentrations exceed document odor thresholds. However the levels may not be high enough to be irritating to occupants. The presence of 1,4‐dichlorobenzene in the ambient and greenhouse air suggests that some short circuiting of ventilation exhaust back into the building. It may also be possible that some leakage across the heat recovery wheel is slightly contaminating the fresh intake air. The removal of ambient particulate matter by the PBC rooftop air handler, the particle loss mechanisms in the air transit through greenhouse and ducting into the building, and the re‐circulating filtration of air by the floor level AHUs appears to be rather successful. Indoor PM2.5 was reduced to 26±8 µg m-3 7% of outdoor levels.  There are no indoor standards for PM2.5, but the World Health Organization has set the outdoor 24 hour standards at 25 µg/m3, and the annual PM2.5 standard at 10 µg/m3 . Considering the extremely high outdoor PM2.5 levels in New Delhi, the protective environment provided by the PBC is phenomenal. This one‐day study did not measure all possible air contaminants in the PBC or surrounding outdoor air. Ozone is an important air contaminant that was not measured, that may have a bearing on IAQ in the building. On January 1 the ozone level in the ambient air was likely around it’s lowest for the year as its formation is dependent on radiation from the sun, and it varies with the seasons. However, during other times of the year, ozone entrained into the building could have a significant impact on IAQ as it reacts with d‐limonene, a‐pinene, and other alkene compounds. Future work should include a study of ozone entry into the building. Rose production is currently the largest component of California’s $300 million cut-flower industry. In 2001, California growers produced 66% of the U.S. rose crop, with a wholesale value of $45 million . The key pests of cut roses are two spotted spider mites , western flower thrips and rose powdery mildew . The two spotted spider mite is a foliage feeder that extracts the cell contents from leaves. This feeding causes foliar stippling and can disrupt the plant’s photosynthetic and water balance mechanisms . The western flower thrips is both a foliage and flower feeder, although it feeds primarily on flowers in the cut-rose system . Powdery mildew is probably the most widespread and best-known disease of roses. The fungus produces a white, powdery-appearing growth of mycelium and conidia on leaves,hydroponic growing supplies which can cause distortion, discoloration and premature senescence. Although it causes some disruption of photosynthesis and transpiration control, the key impact of powdery mildew is reduced aesthetic value caused by the white, powdery spots and leaf distortion. Fresh cut roses are often harvested twice daily, so revised reentry intervals imposed by the U.S. Environmental Protection Agency after pesticide application limit the number of pesticides that are useful in this production system .

In addition, the typical number of pesticide sprays applied to roses grown for cut flowers has impeded the implementation of integrated pest management procedures, particularly the use of biological controls. The IPM approach to pest management incorporates all cost-effective control tactics appropriate for the crop, including biological, cultural and chemical controls. Pesticides that target hard-to-kill floriculture pests frequently kill natural enemies as well, which favors continued reliance on conventional pesticides while discouraging the adoption of biological control. Heavy pesticide use against key pests in the greenhouse has resulted in the widespread development of pesticide resistance in western flower thrips , mites , white flies , aphids and leaf miners . The heavy use of pesticides in cut roses is also a worker safety concern in global and local production. California rose growers reached a crisis point about 8 years ago, when pesticide resistance, costs and limited pesticide availability threatened the growers’ ability to effectively manage two spotted spider mites. At the same time, a new cut-rose production system that favors the success of IPM was gaining widespread acceptance. Roses were traditionally grown in soil with a hedgerow training system, where flowers are cut in a manner that gradually creates a 7-foot or taller hedge. The hedges are pruned back annually to about a 3-foot height and the process is begun again. With the new bent-shoot method, plants are grown in raised containers in a modified hydroponics system. Most of the shoots are bent downward at the crown to intercept more light, creating a perennial lower canopy that exists for the 5 to 8 years of crop production. The upper canopy contains only stems that produce flowers, which take 45 to 52 days to develop. The bent-shoot method creates a spatial separation between the harvested flowers and perennial foliage that does not exist in standard roses. Pesticides to control western flower thrips and powdery mildew that are more compatible with mite predators have also recently become available. These developments, coupled with the difficulty that rose growers were facing in controlling spider mites, made us confident that we could develop a successful IPM program that rose growers would adopt. This project was initiated in 2000 with major funding from the Pest Management Alliance Program of the California Department of Pesticide Regulation and was later supplemented with additional funding. The goal of the Alliance project was to foster a team approach to the development and implementation of IPM programs in a given commodity and to document a reduction in traditional pesticide use. Our Alliance team included researchers, county-based advisors, growers, chemical and biological-control industry representatives, commodity associations and government officials. Our objective was to develop a cost-effective IPM program for the key pests of cut roses that included sampling, thresholds, biological control and directed sprays of reduced-risk pesticides.Eight growers spanning the major rose-producing areas of California participated in the program. Each grower contributed an IPM and a conventional-practice greenhouse; all greenhouses were between 5,000 and 10,000 square feet in size. All pest management decisions in the IPM greenhouses were based on the IPM program that we developed, while the grower made all pest management decisions in the conventional greenhouses. Data was collected and compared on a weekly basis by trained scouts using a comprehensive sampling plan that provided information about the density of insects, mites and diseases. The project included growers with several different rose varieties and both the bent-cane and hedgerow training techniques, but we kept these two variables standardized within a location. Implementation began in March 2000 and continued until January 2001. Fixed precision sampling plans that had been previously developed for two spotted spider mites and western flower thrips were used in our scouting program. This type of sampling plan was developed through intensive surveys of a crop to determine a pest’s spatial distribution. The degree of acceptable error was decided upon in advance, and the number of samples needed to obtain that precision was calculated using knowledge of the pest’s spatial distribution in the crop.

Direct investigation of N2O production and fate in the soil profile has not advanced far, and is only beginning in the fertigation context. It is increasingly clear that much, if not most, soil N2O might be reduced before emission from surface. It is commonly assumed that the upper 20 cm play the central role in surface emissions ; in this study, production of emitted N2O was centered at the 10–15 cm depth. Calculations of net N2O production at different depths in the soil showed more overall N2O consumption than production at 20 cm and below, helping to explain lower emissions under NO3 treatments, where points of production were deeper. N2O production near surface has been difficult to measure, but is a missing link of great importance. The measurement and simulation of soil O2 availability, varying as it does at different points in soil aggregates, remains an obstacle that impedes comparison of field and laboratory soils. Basic questions also persist over fertilizer N availability in its different stages of transformation. And field trials have not verified whether fertilizer management can affect the completeness of N reduction in classical or nitrifier denitrification.In the past decade, nanoscale fertilizers and pesticides have been increasingly proposed and used in agriculture.In particular copper-containing nanopesticides are being introduced to the market due to their excellent antimicrobial and anti-fungal properties.The increasing use of nanopesticides in agriculture has motivated researchers to consider their environmental fate, bio-availability, and toxicity to edible plants.

Extensive investigations of bio-accumulation and phytotoxicity of copper-based nano pesticides 2) on a variety of crop plants, for example, radish , ryegrass , cilantro , zucchini , bean , wheat , duck weeds , lettuce , alfalfa , have been conducted.However, most studies spiked NPs in soil or via water in hydroponic systems,hydroponic growing system whereas in commercial agriculture most copper related pesticides are applied through foliar spray.Compared with the soil-root transfer, the leaf-root transfer pathway and toxicity mechanism have rarely been investigated and are poorly understood.Terrestrial vascular plants have the ability to uptake metals, organic contaminants, even nanoparticles through the leaves employing different pathways.Ions are able to penetrate the leaf cuticle and enter the cytosol of epidermal cells or mesophyll cells.For fine particles , stomatal uptake is an important pathway.For example, Uzu et al. reported that lettuce leaves contained 335 ± 50 mg Pb kg−1 after exposure to Pb-enriched fine particles for 43 days.Hong et al. showed that CeO2 NPs were taken up through cucumber leaves and translocated to root tissues.Lettuce is a widely cultivated vegetable and usually used as a model plant in contaminant transfer studies.Furthermore, since the leaves are the edible part of lettuces, investigating their foliar uptake of copper based nanopesticides is of high interest for risk assessment of human and ecological health. Copper is a redox active transition metal and is involved in redox reaction in cells, generating O2 •− and ·OH via the Haber Weiss and Fenton reactions.Once copper particles/ions enter into plant tissues, no matter where they are localized, they may induce oxidative stress and affect several metabolic processes.Metabolomics is a powerful approach for gaining a comprehensive understanding of biological mechanisms, including toxicity, generally by monitoring low molecular weight metabolites.In recent years, various technologies have been employed for metabolic investigations of organism responses to environmental stressors.

Rather than target a limited number of metabolites or physiological parameters, nontargeted metabolomics can provide information on a large number of metabolites, which results in a deeper insight into the molecular mechanisms underlying the physiological and biochemical changes. In addition, metabolomics can be used to reveal the mechanism of plant defense and detoxification of contaminants.Pidatala et al.performed metabolomics studies and revealed detoxification and tolerance mechanism of Vetiver to Pb. Our recent study on cucumber plant root exudate metabolomics revealed that exposure to nano-Cu up-regulated a number of amino acids that bind with copper NPs and ions, likely to detoxify Cu from its nearby environment.The primary aim of this work was to determine the metabolic profile changes in plants exposed to Cu2 nanopesticides using GC-TOF-MS. The objective is to elucidate the toxicity and detoxification mechanisms underlying up- or down regulated metabolites. In addition, knowledge on the uptake and translocation of Cu2 nanopesticides and released Cu ions in lettuce leaves, through foliar application, is of high interest for risk assessment.The Cu2 nanopesticide used in this study were in the form of a commercial biocide . Detailed physicochemical properties of Kocide 3000 have been reported before.Specifically, the primary particle size is ∼50 to >1000 nm. The hydrodynamic diameter is 1532 ± 580 nm and the zeta potential is −47.6 ± 43 mV, measured via Dynamic Light Scattering , in NanoPure water at pH 7. Although Kocide 3000 particles are mainly micron-sized, these micronized particles are made up of nanosheets of Cu2 that are bound together by an organic composite and can potentially redissociate in water.For this reason the pesticide is considered “nano”. Copper content in Kocide 3000 is 26.5 ± 0.9%, while other elements including C, O, Na, Al, Si, S, Zn compose 73.5% of mass.Lettuce seeds were purchased from Seed Savers Exchange . The soil was collected from the Natural Reserve System of UC Santa Barbara , from the top 20 cm. The soil texture is sandy loamy grassland with sand/silt/clay percentage of 54.0%, 29.0% and 17.0%. Soil pH is 5.90 ± 0.04. Loss-onignition organic matter is 3.11 ± 0.07%. Cation exchange capacity is 25.8 ± 0.1 mequiv 100 g−1 .

More information regarding the soil composition was reported in a previous study.Lettuce seeds were planted in pots containing 250 g of soil. Each pot contained one seed. Plants were grown in a greenhouse, which was maintained at 28 °C by day and 20 °C by night. The daily light integral was 17.3 ± 3.6 mol m−2 d−1 . When plants were 24 days old, we began to spray them with Kocide 3000 suspended in NanoPure water at 105, 155, and 210 mg Kocide/100 mL. The doses were selected generally following the manufacturer’s recommendation . Before spraying, the suspension was sonicated for 30 min in cooled water. A hand-held spray bottle was used for spraying. The lettuces were sprayed a total of 8 times during 4 weeks; the amount sprayed each time was 8.75 , 12.9 and 17.5 mg/pot. Each spray was ∼1 mL. The seven treatments were: control; low, medium, and high NPs in uncovered soil; low, medium and high NPs in covered soil. Each treatment was replicated five times. In covered samples, the soil was covered with filter paper. In covered soil, Cu detected in root should be only from leaf translocation. In uncovered soil, Cu in root not only comes from translocation, but also from soil, because the soil was also exposed to some Cu2 nanopesticide during spraying. This allows us to determine whether the Cu NPs were translocated from leaf to root. At 52 days after planting, all plants were harvested.At harvest, the lettuce plants were gently removed from the soil, thoroughly rinsed with tap water for 5 min and then rinsed with NanoPure water three times. Leaf tissue was carefully separated from vascular and mesophyll tissues Figure S1. Mesophyll and root tissues were ground in liquid nitrogen and lyophilized for 5 days. Part of the freeze-dried mesophyll tissues were sent to UC Davis for metabolomics analysis, and another portion was ovendried at 70 °C for ICP-MS analysis. Since only a small amount of vascular tissue was available,vertical grow table it was only oven-dried for metal analysis.Dried plant tissues were digested with a mixture of 4 mL of H2O2 and 1 mL of plasma pure HNO3 using a microwave oven system at 165 °C for 1 h. The standard reference materials NIST 1547 and 1570a were also digested and analyzed as samples. The recoveries for all elements were between 90 and 99%. Cu and other six important mineral elements were analyzed using inductively coupled plasma mass spectrometry .The freeze-dried lettuce tissues samples were subjected to GC-TOF-MS analysis at the Genome Center Core Services, University of California Davis to identify the metabolites present in lettuce tissues. A description of sample pretreatment, analytical method and instrument has been described by Fiehn et al.Briefly, an Agilent 6890 gas chromatograph containing a Rtx-5Sil MS column with an additional 10 mm integrated guard column was used to run the samples, controlled using Leco ChromaTOF software version 2.32 . quantification was reported as peak height using the unique ion as default. Metabolites were unambiguously assigned by the BinBase identifier numbers using retention index and mass spectrum as the two most important identification criteria. More details regarding data acquisition, data processing and data reporting are provided in the SI.Partial least-squares discriminant analysis is a supervised clustering method, which uses a multiple linear regression technique to maximize the separation between groups and helps to understand which variables carry the class separating information.PLS-DA was run based on GC-TOFMS data using online resources.

Variable Importance in Projection is the weighted sum of the squares of the PLS-DA analysis, which indicates the importance of a variable to the entire model.A variable with a VIP above 1 is regarded as significant.Biological pathway analysis was performed based on all detected metabolites data using MetaboAnalyst 2.0.The impact value threshold calculated for pathway identification was set at 0.1.Cu in leaves increased in a dose-dependent manner in both covered and uncovered soil . Cu increased 82−140 times in vascular and 115−184 times in mesophyll tissues relative to the control, which indicates a high bio-accumulation of copper/nanoparticles in leaf tissues. Even though the leaves were thoroughly washed, copper ions and NPs remained on the surface or were incorporated into leaf tissues; it is likely that washing was not 100% efficient in removing them. Leaf exudates can form weak acids in the presence of water,which can accelerate dissolution of Cu2 nanopesticide, releasing cupric ions as long as the water remains on the leaf. This may result in a pathway for cupric ions to penetrate the epidermis cells and translocate to other tissues. In addition to ionized Cu, nanoparticles smaller than the stomatal diameter may enter past the guard cells. Stomatal diameters range from 8 to 12 μm for several species.Even though trichomes are not abundant on lettuce leaf surfaces, ESEM images taken after 24 h exposure to Cu2 nanopesticides show that many small particles were deposited on the lettuce leaf surface and stomatal cavities. The typical diameter observed for lettuce stomata is 13.1 μm , which is large enough to permit entry to Cu2 nanopesticides aggregates with an observed hydrodynamic diameter of 1530 ± 580 nm. Eichert demonstrated that 43 nm NPs entered stomata and migrated along the surface of stomatal pores.After passing the stomatal guard cells, the NPs may either attach to cell walls or move between cell walls. For example, Stamenkovic and Gustin showed the majority of foliar Hg was located in epidermal and stomatal cell walls and was rarely found in mesophyll or vascular tissue.However, Hong showed that Ce was present in cucumber root phloem after foliar application of CeO2 NPs.As seen in Table 1, the average [Cu] in control roots is 6.0 mg/kg, while in treated plants, [Cu] in root is 17.5−26.1 mg/kg in covered soil and 34.2−56.9 mg/kg in uncovered soil. Statistical analysis showed all the NP treated plants have significantly higher [Cu] in roots compared to controls, even though application was only foliar. In covered soil, where no direct root uptake could occur, Cu in the roots was translocated from the leaves via phloem loading. Liao et al. showed that some xylem-transported Cu was recirculated to roots via the phloem in chicory and tomato plants.Even though we observed evidence of Cu translocation to the roots, 97−99% of Cu mass was retained in the leaves. In addition, the translocation rate in NP-treated plants was 0.009 to 0.014, which is far lower than that in the control . This indicates plants sequestered most of the Cu in leaves.The threshold level for Cu to induce toxicity in plants is 20−30 mg/kg.However, high concentrations of Cu in lettuce leaves did not cause any visible toxic symptoms throughout the entire exposure period . On the contrary, the leaf biomass significantly increased at low and medium levels for uncovered treatment and medium level for covered treatment . Since a high amount of Cu was retained in leaf tissues but did not induce any toxic symptoms, lettuces likely employ a detoxification mechanism to build tolerance to excess Cu.

The [64Cu]-NP-uptake and accumulation amounts observed within lettuce seedlings were reasonable and comparable to others reports in the literature, reaching the same general conclusions that NP transport and accumulation in plants is species and size dependent.Smaller NPs have been demonstrated to have higher accumulation in plants than larger NPs. For example, Ni-NPs had very high NP uptake ranging from ∼13 200−38 983 μg/g in mesquite.The amount found in the leaves varied from 400 to 803 μg/g of mesquite with most the NPs remaining in the roots ranging from 12 835 to 38 183 μg/g.Another study using small CeO2-NPs exhibited NP accumulation ranging from 300 to 6000 μg/g of plant and indicated that NP accumulation was plant species dependent.NP sizes above active transport ranging from 14 to 40 nm had a large variation in uptake ranging from 0.25 to 3750 μg/g of plant, but typically had accumulation ranging from ∼1−1100 μg/g of plant again with the majority of the NPs contained within the root and with 0.5−183 μg/g in the leaves.NPs , had accumulation in mung bean of 8 μg/g and in wheat of 32 μg/ g.When comparing the accumulation of two similarly sized TiO2-NPs of different crystalline structure [22 nm and 25 nm ] in wheat different accumulation amounts were observed,vertical grow tables suggesting size was not the only limiting factor for transportation into a plant.In another study using radioactive NPs, Zhang et al.generated 141Ce by neutron bombardment of CeO2NPs synthesized via a precipitation method.

The fabrication of [ 141Ce]CeO2-NPs could make controlling the size distribution very difficult and the exact size of the radioactive [141Ce]CeO2- NPs was never determined. In addition, free radioactive 141Cemetal could dissolute and be transported into the plant, making it appear as if the intact-NPs were in the plant because possible leaching of radioactivity was not explored. We aimed to avoid complications of NP-fabrication in which the exact size distribution during the study could not be determined and to improve upon prior radio labeling methods, which gave low specific activity of 2.7 μCi/mg of NP.We were able to generate stable radioactive [ 64Cu]-NPs with high radio chemical purity and a specific activity of 2.2 mCi/mg of NP with a tight size distribution . Zhang et al.’s work also demonstrated a concentration and size dependence of the [141Ce]CeO2-NPs on plant accumulation in cucumber.These uptake differences may be attributed to the use of a different species and/or the NP solution administered had 2.4-times higher concentration . Autoradiography images showed [ 141Ce]CeO2-NPs movement to the leaves, implying that once NPs entered into the vascular cylinder, they move along with water flow. This was in good agreement with our study. In contrast, we saw no concentration dependence for either sized [ 64Cu]-NP using 48, 96, and 144 mg L−1 over a 2 h period with approximately the same accumulation amount at all concentrations . Similarly, another study using CuO-NPs administered two concentrations 10 mg L−1 and 100 mg L−1 for a period of 14 days in maize also observed no concentration dependence.

The use of NPs tagged with radioactivity and tracked by autoradiography and PET/CT has provided a noninvasive analytical tool to spatially visualize and quantify NP uptake and accumulation in plants. We investigated the fate of [64Cu]-NP transport into plants at the largely unexplored early time points, which would prevent dissolution events. Stability studies concluded that the [64Cu]-NPs were stable during the imaging and quantification time frame from 0.25 to 24 h resulting in intact NP-transport into lettuce seedlings. We further demonstrated that the transport of [64Cu]-NPs into lettuce was not concentration dependent but was size dependent with the 20 nm [64Cu]-NPs reaching a plateau with accumulation at ∼5.7 μg/g of lettuce and the smaller 10 nm NPs accumulation increasing linearly with the maximum amount at 24 h being ∼7.6 μg/g of lettuce.With the numerous factors that may dictate NP uptake and accumulation, further studies are warranted to fully understand the molecular mechanism of NP transport into plants.Legalization of cannabis production in 2017 has generated demands for state regulatory, research and extension agencies, including UC, to address the ecological, social and agricultural aspects of this crop, which has an estimated retail value of over $10 billion . Despite its enormous value and importance to California’s agricultural economy, remarkably little is known about how the crop is cultivated. While general information exists on cannabis cultivation, such as plant density, growing conditions, and nutrient, pest and disease management , only a few studies have attempted to measure or characterize some more specific aspects of cannabis production, such as yield per plant and regional changes in total production area . These data represent only a very small fraction of domestic or global activity and are likely skewed since they were largely derived not from field studies but indirectly from police seizure data or aerial imagery . In California, where approximately 66% of U.S. marijuana is grown , knowledge of the specific practices across the wide range of conditions under which it is produced is almost nonexistent.

Currently, 30 U.S. states have legalized cannabis production, sales and/or use, but strict regulations remain in place at the federal level, where it is classified as a Schedule I controlled substance. As a land-grant institution, UC receives federal support; were UC to engage in work that directly supports or enhances marijuana production or profitability, it would be inviolation of federal law and risk losing federal support. As a result, UC research on California cannabis production has been limited and focused on the geography of production and its environmental impacts . These studies have documented the negative effects of production on waterways, natural habitats and wildlife. While such effects are not unique to cannabis agriculture per se, they do present a significant threat to environmental quality and sensitive species in the watersheds where cannabis is grown . Science-based best management practices to mitigate or avoid impacts have not been developed for cannabis. Because information on cannabis production practices is so limited, it is currently not possible to identify key points of intervention to address the potential negative impacts of production. As a first step toward understanding cannabis production practices, we developed a statewide survey on cultivation techniques, pest and disease management, water use, labor and regulatory compliance. The objective was to provide a starting point from which UC scientists could build research and extension programs that promote best management practices — which are allowable as long as their intended purpose is not to improve yields, quality or profitability. Survey results also establish a baseline for documenting changes in cultivation practices over time as legal cannabis production evolves in California. To characterize key aspects of cannabis production in California,hydroponic growing systems we developed an anonymous online survey using Qualtrics survey software . A web-based survey that masked participants’ identity was determined to be the most suitable approach given that in-person interviews were limited by legal restrictions on UC researchers visiting cannabis farms, and mail or telephone surveys were constrained by the lack of any readily available mailing address or telephone contact information for most cannabis growers, who are understandably discrete with this information. An online survey was also the most cost-effective means of reaching a large number of cannabis growers. Survey questions focused on operational features , pest and water management, labor, farm revenue and grower demographics. Two draft surveys were reviewed by a subset of cannabis growers to improve the relevance of the questions and terminology. A consistent critique was that the survey was too long and asked for too much detail, taking up to 2 hours to complete, and that such a large time commitment would significantly reduce the response. We therefore made the survey more concise by eliminating or rephrasing many detailed questions across various aspects of cannabis production. The final survey included 37 questions: 12 openended and 25 structured . Structured questions presented either a list of answer choices or a text box to fill in with a number. Each list of answer choices included an “Other” option with a box for growers to enter text. Open-ended questions had a text entry box with no character limit. Condensing the survey to capture more respondents resulted in less detailed data, but the overall nature of the survey remained the same — a survey to broadly characterize multiple aspects of cannabis production in California. Data from the survey has supported and contextualized research by other scientists on specific aspects of cannabis production, such as water use , permitting , law enforcement , testing requirements , crop prices and perceptions of cannabis cultivation in the broader community . Recruitment of survey participants leveraged networks of California cannabis growers who had organized themselves for various economic and political purposes . These were a combination of county, regional and large statewide organizations, with many growers affiliating with multiple groups.

We identified the organizations through online searches and social media and sent recruitment emails to their membership list-serves. The emails contained an explanation of the survey goals, a link to the survey website and a message from the grower organization that endorsed the survey and encouraged members to participate. The emails were sent in July 2018 to approximately 17,500 email addresses, although not all members of these organizations necessarily cultivated cannabis, and the organizations noted that their mailing lists somewhat overlapped the lists of other groups that we contacted. For these reasons, the survey population was certainly less than 17,500 individual cannabis growers, but because we were not able to view mailing lists nor contact growers directly, and because there are no comprehensive surveys of the number of cannabis farms in California, we could not calculate a response rate or evaluate the representativeness of the sample. Respondents were given until Aug. 15, 2018, to complete the survey. All survey participants remained anonymous, and response data did not include any specific participant identifiers.In total, 101 surveys were either partially or fully completed. Responses to open-ended questions were coded before summary. Since incomplete surveys were included in this summary, the number of responses varied between questions. Each response was considered a unique grower and farm operation. As noted, survey response rate was difficult to quantify, and participants were self-selecting, which introduces bias. The survey data should be taken only as a starting point to guide more detailed evaluations of specific practices in the future, not as a basis for developing recommendations for production practices or policies.Survey respondents operated farms primarily in Humboldt , Mendocino and Nevada counties, but survey responses also came from Trinity , Santa Cruz , Sonoma , San Luis Obispo , Sacramento , Butte , Calaveras , Fresno , Los Angeles , San Diego , San Mateo and Siskiyou counties and Josephine County, Oregon . In line with California regulatory guidelines, small farms were defined as those of 10,000 sq ft or less, medium farms 10,001 to 22,000 sq ft and large farms 22,001 sq ft or more. Accordingly, 74% of farms were small, 16% were medium and 8% were large . For those growers who reported on their land use in 2013 , most farmed on land that was previously used entirely or in part for cannabis production . The other 22% indicated that the land was used in 2013 for agricultural crops, ranching, open space or “other” land uses.For this survey, we differentiated between outdoor , greenhouse and indoor farming . The most common ways to farm were all outdoors , combined outdoor and greenhouse and greenhouse only . This was followed by various combinations of greenhouse and indoor , greenhouse and other , outdoor and other , outdoor and indoor , all indoor and other . When measured by total plants, farms with combined outdoor and greenhouse facilities were responsible for 41% of crop production, followed by outdoor and other , greenhouse only , outdoor only , greenhouse and other , outdoor and indoor , greenhouse and indoor and other . A majority of survey respondents grew their cannabis crop in raised beds , native soil and/or grow bags , followed by hydroponic systems and plastic pots .Most growers reported groundwater as their primary water source for irrigation , with some growers reporting use of multiple water sources. Those using groundwater extracted 87% of annual volume between June and October. Of those storing water, most stored exclusively well or spring water, though some stored municipal water or rainwater .

The process of Velaglucerase alpha was design to modify the glycosylation profile of the protein toward oligomannose N-glycans to improve mannose-receptor mediated uptake of the drug into macrophages, the target cells. Here, we determined the optimal concentration of kifunensine and demonstrate that kifunensine addition at a concentration of 0.375 µM in the Agrobacterium infiltration solution of N. benthamiana plants during the vacuum infiltration process allows the production of exclusively high-mannose recombinant proteins. The anti-CD20 monoclonal antibody rituximab, approved for the treatment of non-Hodgkin’s lymphoma, was selected to evaluate the effectiveness of kifunensine for the production of an anti-cancer antibody with enhanced antibody-dependent cell mediated cytotoxicity . ADCC efficacy of rituximab is inversely correlated with the content of core fucose, suggesting that a rituximab variant with altered glycosylation would lower dosing requirements. More importantly, we demonstrate that afucosylated high-mannose decorated antibody, derived from the treated plants, exhibits increased ADCC effector function, as compared with rituximab derived from non-treated plants. The increased ADCC activity was verified using effector cells carrying both FcγRIIIa-V158 and FcγRIIIa-F158 allotypes. Several strategies could be implemented to modulate the plant-specific glycans: Protein containment in the Endoplasmic Reticulum using specific signal sequences, knockdown of fucosyltransferase and xylosyltransferase enzymesin N. benthamiana with RNA interference technology,hydroponic growing system knockout of fucosyltransferase and xylosyltransferase enzymes in N. benthamiana using gene editing, and replacement of plant glycans with human glycans through glyco-remodeling.

Gene editing using sequence-specific transcription-activator-like effector nucleases was only partially effective, while the use of transgenic knockdown lines at manufacturing require more exigent containment and cleaning procedures. Our approach eliminates the need for modification of the primary sequence or the use of transgenic, regulated material for manufacturing. Combined with the scalability and low manufacturing cost associated with the N. benthamiana transient expression system, this method represents an excellent alternative to the use of either glycoengineered or kifunensine-treated mammalian cell lines for the production of afucosylated anti-cancer antibody.We have hypothesized that a treatment with kifunensine would inhibit trimming of mannose residues in the endoplasmic reticulum , subsequently preventing the addition of α1,3-fucose and β1,2-xylose residues on the polysaccharide core . To do so, 60 plants per conditions were vacuum infiltrated in a solution of Agrobacteria with or without kifunensine, ranging from 0 to 5 µM . Visual observation of infiltrated plants from three to seven days post infiltration revealed no noticeable phenotypic or morphological differences between treated and untreated control plants . All leave and stems were collected from each infiltration and pooled for protein extraction and purification. Rituximab expression levels were quantified at 7 dpi and revealed a low to moderate increase in antibody expression between untreated and treated plants, with the average rituximab expression level ranging from 288 mg/kg to 385 mg/kg whole plant fresh weight in treated plants, compared to 287 mg/kg whole plant FW in untreated plants . These observations demonstrated that the kifunensine treatments were not detrimental to plant growth or protein expression. To evaluate the effect of kifunensine treatments on the integrity and assembly of rituximab, SDS-PAGE analysis was carried out under reduced and non-reduced conditions.

As illustrated in Figure 1D, rituximab derived from kifunensine treated and untreated conditions appeared intact and fully assembled. Non-reduced rituximab migrated at the expected molecular weight of ~145 kDa, while the reduced heavy and light chains migrated at the expected MW of ~50 kDa and ~25 kDa, respectively . Infiltration experiments were run in duplicates.The N-glycosylation profiles of purified rituximab expressed in N. benthamiana were evaluated by LC-MS/MS analysis . The major glycoforms were compared based on the relative intensity of the Asn297 glycopeptide masses identified by LC-MS/MS.As previously described in the literature, this plant-derived rituximab control exhibited primarily complex-type N-glycans, with the most abundant N-glycan structure being GlcNAc2Man3GlcNAc2 . On the other hand, there were significant differences in rituximab N-glycan profiles between untreated and treated samples. Complete conversion of plant complex glycans to oligomannose-type glycans was observed when N. benthamiana plants were infiltrated with ‘higher range’ and ‘medium range’ concentrations of kifunensine . The GlcNAc2Man9 and GlcNAc2Man8 were the major glycoforms observed with Man9 being the most abundant. In fact, the same oligomannose-type glycoform distribution was observed whether 0.375 µM or higher kifunensine concentration was used, indicating that 0.375 µM is sufficient to provide homogeneous rituximab with oligomannose-type glycans . When lower concentrations of kifunensine were used, a mixture of oligomannose, hybrid, and complex glycans was detected . For instance, the glycosylation profifile of rituximab from plants treated with 0.25 µM contained more than ~48% hybrid/complex glycan modififications and ~52% oligomannose glycosylation .

Importantly, no α1,3-fucose or β1,2-xylose residues were detected in rituximab derived from plants treated with ≥0.375 µM kifunensine.In this study, we have investigated the use of kifunensine in a plant expression platform that is established for large-scale manufacturing for recombinant protein. More specifically, we evaluated the concentration of kifunensine sufficient for the production of an afucosylated rituximab with enhanced biological activity. The vacuum infiltration of plants in a solution containing Agrobacterium culture supplemented with kifunensine at a concentration varying from 0.0625 µM to 5 µM did not affect the seven-day post-infiltration growth of the plants or the expression of rituximab. In fact, the expression of rituximab was slightly higher from kifunensine-treated plants. Similar tolerance to kifunensine treatment has also been described for the expression of antibodies in Chinese Hamster Ovary cells. However, it has been reported in one instance that kifunensine treatment of N. benthamiana plants via the growth medium led to a decrease in expression of a recombinant protein]. When applied during plant vacuum infiltration in the Agroinfiltration solution, kifunensine enters the interstitial spaces of the leaf tissue in contact with the host cells where recombinant protein expression occurs, rather than through uptake via the root system. The positive impact of infiltrated kifunensine on host cell tolerance and protein production may be due to its suppressing effects on the ER-associated degradation pathway where proteins with trimmed oligomannose glycans may be degraded if the polysaccharide chain is not further processed or proteins transported to the Golgi apparatus.Kifunensine treatment during the agroinfiltration ultimately results in protein afucosylation as it stops mannose trimming in the endoplasmic reticulum, yielding Man5-Man9 N-glycan structures. When delivered in this fashion, a minimum concentration of 0.375 µM kifunensine was sufficient to generate rituximab harboring only oligomannose glycan structures lacking fucose residues. In agreement with reports using mammalian cell cultures, the minimum required kifunensine concentration to generate antibody devoid of fucose residues in N. benthamiana falls somewhere between 0.25 and 0.375 µM . Kifunensine has the practical advantage of being active at 2-to-4-fold-lower concentrations than other inhibitors of the glycosylation pathway making it more cost-effective. Moreover, as described here and in mammalian cell cultures, treatments with kifunensine leads to a highly homogeneous product, with ultimately no formation of core-fucosylated hybrid structures.Proteins produced with glycan-engineering technologies not only lack potentially immunogenic plant-specific glycoforms,hydroponic growing but also provide enhanced effector function. The in vitro bioassay described in this study demonstrated enhanced ADCC activity from rituximab containing high-mannose glycoforms. It is expected that the reason for this increased ADCC activity lies in the absence of fucose residues on the glycosylation core rather than the high content of mannose residues, as many studies have reported the effect of afucosylation on the ADCC activity of anti-cancer antibodies including rituximab. In fact, similar ADCC results were obtained with antibodies derived from CHO cell cultures treated with kifunensine. It is important to note that ADCC activity was linearly proportional to the relative abundance of oligomannose glycoforms. This was particularly evident with ‘low-range’ concentrations of kifunensine applied, which generated a mixture of complex, hybrid, and oligomannose structures. With rituximab derived from a treatment of 0.25 µM kifunensine, generating a relatively small increase of oligomannose glycoforms, there was a significant but lower increase in ADCC activity . Importantly, plant-derived high-mannose rituximab glycoforms exhibited the same affinity for CD20 as Rituxan®, the commercial standard. Thus, the kifunensine treatment does not affect the paratope conformation of the plant-derived antibody.

As the glycosylation profile of an anti-cancer antibody is correlated to its biological activity, it is therefore considered as a critical quality attribute that needs to be maintained during the manufacturing process. To that end, the glycosylation profile of kifunensine-treated antibodies is homogeneous, consistent, and easy to control at scale, which represent a significant advantage for this technology. There are reports that antibodies carrying high mannose glycans have a shorter serum half-life, as compared with other glycoforms. However, other pharmacokinetic studies with afucosylated high mannose antibodies indicated no impact on clearance. Thus, the pharmacokinetics property of any antibody will have to be evaluated based on its biological activity, the target indication, and dosage regimen. In conclusion, the application of kifunensine during transient agroinfiltration of the N. benthamiana host leverages the scalability and cost-effectiveness of the plant expression platform for the production of biobetter anti-cancer antibodies. First, the scale-up of rituximab expression was also demonstrated at the iBio CDMO facility using manufacturing procedures without affecting the expression or product quality. Second, using the process model described by Holtz et al. 2015, and the findings from this study , the cost of cGMP -grade kifunensine to produce high-mannose, afucosylated antibodies at manufacturing scale was estimated to be less than $0.80/g of antibody produced. Thus, kifunensine can be incorporated into already established manufacturing protocols without affecting production cost significantly. Further studies will focus on determining how long the inhibition effect of kifunensine lasts during and after the plant infiltration process. This attempt to increase anti-cancer efficacy of recombinant antibodies through in-process glycan engineering represents a promising alternative to meet unmet medical needs.The ADCC reporter assay was performed using Wil2-S cells as targets along with Jurkat-CD16 reporter cell lines. Two reporter cell lines stably expressing the FcγRIIIa receptor, V158 or F158 variants were used. Wil2-S cells were plated in a 96-well white bottom assay plate at 5000 cells per well. Serial dilutions of test antibodies were added to the plates containing the target cells and incubated at 4 ◦C for 15 min to allow opsonization. Jurkat-CD16 reporter cells were then added to assay plates already containing Wil2-S cells and antibodies. The fifinal concentration of antibodies ranged from 2 to 0.0003 µg/mL following several 3-fold dilutions. The effector:target cell ratio was 10:1. After a 6 h. incubation at 37 ◦C, One-Glo™ Luciferase Assay Reagent was added and luminescence was determined using a Gen5 microplate reader. Samples and controls were tested in triplicate, and the mean reporter signals of sample dilutions in Relative Luminescence Units were plotted against the antibody concentration. Antibody independent cellular cytotoxicity was measured in wells containing target and effector cells without antibodies. GraphPad prism software was used to plot normalized RLU versus Log10. The half maximal effective concentration values of plant-made rituximab and Fc variants were derived as dose responses obtained from non-linear regression curves. Fold of induction was calculated by taking the ratio of background subtracted induced RLU and background subtracted untreated control. Nitrogen is a crucial plant nutrient; to encourage large yields, farmers tend to apply excess nitrogen fertilizers to their crops. However, crop plants are generally inefficient at nitrogen uptake from the soil, with as much as 50 to 75% of applied N being unused by the plants . Crop plants compete for soil N against soil microbes involved with denitrification and nitrification, volatization to the atmosphere, as well as loss of N by leaching into waterways . It is important to breed and/or design nitrogen use efficient crop plants that can produce the same, or higher yields with less applied N fertilizer. Growth of NUE crop plants, coupled with implementation of best fertilizer management practices, would allow for a reduction of applied N fertilizer per hectare. This would both greatly reduce the N fertilizer expense for the farmer and greatly reduce the environmental pollution from excess N fertilizers. We have recently developed genetically engineered rice by introducing a barley alanine aminotransferase cDNA driven by a rice tissue specific promoter, OsAnt1 . This modification significantly increased biomass and grain yield in the transgenic plants compared to control plants when the plants were grown at a fixed, high amount of ammonium as the N source. As well, we analysed the transcriptomic profile of these transgenic plants grown at the fixed N concentration using Affymetrix Rice GeneChip microarrays to provide further insights into the nature of increased NUE of these transgenics . In this study, we compared various physiological and genetic data from alanine aminotransferase over-expressing transgenic plants to control plants grown at three different nitrogen levels and demonstrated significant changes between them.

These products pass through an elaborate biochemical cycle that eventually forms one molecule of a 6-carbon sugar and regenerates RuBP. The reaction of RuBP with oxygen oxidizes the RuBP, splits it into one molecule of a 3-carbon compound and one molecule of a 2-carbon compound , and subsequently releases carbon dioxide, hence the names C2 pathway or, more commonly, photo respiration. In total, photo respiration consumes biochemical energy, but does not result in any net production of sugar . Thus, photo respiration has been viewed as a wasteful process, a vestige of the high carbon dioxide atmospheres under which plants evolved . The balance between C3 carbon fixation and photo respiration depends on the relative amounts of carbon dioxide and oxygen entering the active site of rubisco and the affinity of the enzyme for each gas . At current atmospheric levels of carbon dioxide and oxygen , photo respiration in most crops dissipates over a quarter of the organic carbon produced during carbon dioxide assimilation . In contrast, C4 crops , which have a metabolic carbon dioxide pump that increases the concentration of this compound at the catalytic site of rubisco, minimize photo respiration at the expense of the additional energy required for pumping. Elevated levels of atmospheric carbon dioxide inhibit photo respiration in C3 plants,vertical greenhouse making photosynthesis more efficient. Initially, this accelerates both their photosynthetic carbon dioxide assimilation and their growth by about a third.

After a few days or weeks, however, carbon dioxide assimilation and growth both slow down until they are accelerated in the long term by only about 12% and 8%, respectively . Moreover, leaf nitrogen and protein concentrations ultimately decrease more than 12% under carbon dioxide enrichment . Such a loss of nitrogen and protein significantly diminishes the value of this plant material as food for animals and humans. Fig. 6. Differences in leaf carbon fixation capacity versus total nitrogen concentration between C3 plants grown at elevated and ambient carbon dioxide concentrations. Each symbol designates the mean ratio for a species. Shown are the regression line and 1:1 line . This data suggests that changes in photosynthesis from carbon dioxide enrichment derive from changes in plant nitrogen levels under carbon dioxide enrichment . Together these trends are known as carbon dioxide acclimation.According to this hypothesis, plants under carbon dioxide enrichment initially assimilate more carbon dioxide into carbohydrates than they can incorporate into their growing tissues. In response, they diminish carbon dioxide assimilation by decreasing their rubisco levels . This change in rubisco levels, however, is not necessarily selective; the decrease may instead just be part of the overall decline in protein and nitrogen concentrations .Another hypothesis for carbon dioxide acclimation is that shoots accumulate carbohydrates faster than roots can absorb nitrogen from soils, making leaf nitrogen concentrations decrease .

As these leaves senesce and drop to the ground, plant litter quality declines, microbial immobilization of soil nitrogen increases because of the high carbon-to-nitrogen ratios in the litter, soil nitrogen availability to plants further diminishes because more soil nitrogen is tied up in microorganisms, plants become even more nitrogen limited, plant protein levels decline and plant processes including photosynthesis slow down . This hypothesis, however, has difficulty in explaining the variation in carbon dioxide acclimation among sites and among methods of carbon dioxide enrichment .We have discovered another explanation for carbon dioxide acclimation: in C3 plants, shoot assimilation of nitrate into organic nitrogen compounds depends on photo respiration, so any condition that inhibits photo respiration also inhibits shoot nitrate assimilation . Thus, at elevated carbon dioxide concentrations, C3 plants that rely on nitrate as a nitrogen source suffer severe deprivation of organic nitrogen compounds such as proteins. The resulting decline in organic nitrogen compounds reduces the plants’ yield and biomass production. While high applications of nitrogen fertilizer may partially compensate for this, the plants’ nitrogen and protein concentrations still diminish . Ammonium and nitrate are the two main sources of nitrogen that are accessible to plants from the soil. Plants show a wide range of responses to carbon dioxide enrichment because the balance between nitrate and ammonium availability varies over seasons, years, locations and plant species. In an annual California grassland where nitrate was the predominant nitrogen source, net primary productivity diminished under carbon dioxide enrichment . This was presumably because elevated carbon dioxide inhibited plant nitrate assimilation , and the grasses became deprived of organic nitrogen.

In contrast, ammonium is the major form of nitrogen available to plants in marshes because wet, anaerobic soils promote denitrification and nitrate leaching . For example, the dominant C3 plant in the Chesapeake Bay marsh showed little carbon dioxide acclimation ; even after a decade of treatment, photosynthesis and growth remained about 35% greater under carbon dioxide enrichment , with little change in nitrogen concentrations . In wheat, another C3 plant, elevated carbon dioxide atmospheres stimulated less growth under nitrate than under ammonium nutrition .Several physiological mechanisms appear to be responsible for the dependency of nitrate assimilation on photo respiration. First, the initial biochemical step of nitrate assimilation is the conversion of nitrate to nitrite in leaves. This step is powered by the high-energy compound NADH , and photo respiration increases the availability of this compound . In contrast, C4 plants generate ample amounts of NADH in leaves via a different biochemical pathway. This explains why shoot nitrate assimilation is relatively independent of carbon dioxide concentrations in C4 plants . Second, the subsequent biochemical step of nitrate assimilation is the conversion of nitrite to ammonium in the chloroplasts of leaf cells, which requires the transport of nitrite into the chloroplast. Elevated carbon dioxide inhibits this transport . Third, this subsequent step also requires chemical energy from the oxidation of a different high-energy compound called ferredoxin. Several other processes — in particular, carbon dioxide assimilation — depend on the same energy source and seem to have priority in using it. Ferredoxin becomes involved in nitrate assimilation only when carbon dioxide availability limits C3 photosynthesis .Many crops in California depend on nitrate as their primary nitrogen source. As atmospheric carbon dioxide concentrations rise and nitrate assimilation diminishes, these crops will be depleted of organic nitrogen, including protein, and food quality will suffer . Wheat, rice and potato provide 21%, 14% and 2%, respectively, of protein in the human diet . At elevated carbon dioxide and standard fertilizer levels, wheat had 10% less grain protein . Similarly, grain protein in rice and tuber nitrogen in potato declined by about 10% at elevated carbon dioxide concentrations. Several approaches could mitigate these declines in food quality under carbon dioxide enrichment. Increased yields may compensate to some degree for total protein harvested . Several-fold increases in nitrogen fertilization could eliminate declines in food quality , but such fertilization rates would not be economically or environmentally feasible given the anticipated higher fertilizer prices and stricter regulations on nitrate leaching and nitrous oxide emissions. Greater reliance on ammonium fertilizers and inhibitors of nitrification might counteract food quality decreases. Nevertheless, the widespread adoption of such practices would require sophisticated management to avoid ammonium toxicity,vertical grow towers which occurs when plants absorb more of this compound than they can assimilate into amino acids and free ammonium then accumulates in their tissues . Several of these issues might be simultaneously addressed by fertigation, or frequent additions of small amounts of ammonium-based fertilizers in water delivered through micro-irrigation. These findings have broad implications for the future of plant distributions and food production. Enriched carbon dioxide atmospheres will not enhance the performance of C3 plants to the extent originally envisioned. A 10% decline in food protein content will further burden regions of the world already affected by hunger. With a better understanding of ammonium and nitrate use by crops and careful nitrogen management, we can turn these phenomena to our advantage.With its Mediterranean climate of moist, mild winters and dry moderate summers, a broad range of fruit and vegetable crops can be grown year round on the central coast of California. Monterey and Santa Cruz counties combined produced $912 million gross value of strawberries and over $2.7 billion worth of vegetables in 2011.

As the interest in organic farming and the demand for organic produce has increased during the last decade, organic farming on the central coast has also greatly increased. There were over 9,300 certified organic hectares in Monterey and Santa Cruz Counties in 2011, five times the number recorded in 1998 . The total farm gate revenue from organic farming in these counties was over $197 million in 2011, representing a dramatic 12-fold increase in 13 years . This trend is also true for organic strawberry production. In 2000, 77 ha of organic strawberries were grown in central coastal California, but by 2012 this had increased to 509 ha, representing 8.3% of the total strawberry production in the area . Continued growth of organic strawberry production in this area, however, faces the challenge of managing soil-borne diseases without the use of synthetic fumigants and fungicides. Verticillium wilt is a soil-borne disease caused by Verticillium dahliae that can damage a wide range of important crops in California. Host crops include lettuce, tomatoes, potatoes, apples, cotton, artichokes, and strawberries . Due to its resilient overwintering structure , this pathogen can survive many years in soil even without host plants . In the premethyl bromide era, Verticillium wilt was a major limiting factor to strawberry production in California . Today, Verticillium wilt is one of several key soil-borne diseases facing California strawberry production and poses a long-term threat for organic strawberry production in the state.To avoid Verticillium wilt and other soil-borne diseases, as well as meet the requirements of the USDA National Organic Program , organic strawberry growers must implement crop rotation. Due to its high sensitivity to the disease, several years between strawberry plantings are necessary . For specialized strawberry growers in California, establishing a crop rotation system implies a major change in the design and management of the farming system. Due to the high costs of production and the high leasing fees of crop lands , specialized organic strawberry growers need to minimize the break time between strawberry crops as much as possible to stay in business. The following biological and cultural approaches to soil-borne disease management in strawberries have been tested: host resistance ; small cell transplants ; organic amendments such as compost ; high nitrogen organic fertilizers ; broccoli residues ; mustard residues , Sudan grass , and other cover crops ; microbial amendments including vesicular arbuscular mycorrhizal fungi ; plant growth promoting rhizobacteria ; crop rotations with broccoli, lettuce or Brussels sprouts; mustard seed meal ; soil-less trough production ; and anaerobic soil disinfestation . Further, a minimum of a three-year rotation is recommended for strawberries that do not use chemical fumigants in Europe , the Northeast and Midwest United States, and in eastern Canada . However, no research has yet integrated multiple biological and cultural practices for different rotation periods of organic strawberries in California. The objective of this project was to demonstrate the effects of strawberry planting frequency in organic strawberry/vegetable rotations and combined biological and cultural practices on fruit yield and disease level. We hypothesized that the use of non-host rotation crops for Verticillium wilt plus bio-fumigation with broccoli, mustard cover crop residues, relatively resistant strawberry cultivars, and compost application would suppress disease sufficiently to grow strawberries in rotation every two or three years. To test the above hypothesis, in 2001, we initiated a five year organic strawberry/vegetable rotation experiment in a commercial California field.The loss of soil productivity when crops are grown repeatedly on the same land resulting in poor plant growth and reduced yields is called “yield decline” , “soil sickness” , or “replant problem” . Such losses, called here “yield decline,” have been reported in many crops worldwide including strawberries . Biotic and abiotic factors can cause yield decline. Further, although one factor may possibly be responsible for yield decline, it is more likely that a combination of factors interact to cause the effect . This study also demonstrated the challenges researchers face when using a participatory process where farmer involvement is a key part of the experiment.

The sensitivity of XRD profiles to the amount, coordination, and position of high-Z interlayer scatterers, and to the number of vacant layer sites is illustrated next.Except for one sample obtained by metal sorption on poorly crystalline Mn oxides , the new Zn-rich phyllomanganate contains higher amounts of vacant layer sites and transition elements than any other natural and synthetic variety described so far . The constant Zn:Mn ratio of Mn-Zn precipitate suggests that Zn co-precipitated with Mn by a yet unknown mechanism to form a chemically well-defined phase as natural solids formed by metal sorption on preexisting mineral surfaces are chemically heterogeneous . Birnessite and vernadite minerals were given different names because the basal reflections of birnessite at 7.2-7.0 Å and 3.6-3.5 Å were not observed originally in the diffraction pattern of vernadite. However, recent studies have shown that natural vernadite and its biogenic and chemical analogs most often display a 001 reflection when their XRD pattern is recorded on modern diffractometers , thus confirming the view of Arrhenius et al. and Giovanoli that this mineral is a c-disordered variety of birnessite. Villalobos et al. showed that basal reflections are present when the diffracting crystallites have only 2-3 layers, on average. Here, this number is as low as 1.2 layers,vertical grow rack system meaning that Mn-Zn precipitate is essentially an assemblage of isolated layers.

Measurements of the Mn edge jumps on different Mn-Zn precipitates provide an inkling of how the constitutive nanoparticles are joined at particle or so-called grain boundaries. The Mn edge jump was typically between 0.2 and 0.3 for aggregates ~15-25 µm in diameter, which indicates that the phyllomanganate represents only a small fraction of the black precipitates, thus revealing a high micro-porosity. This porosity is possibly filled, at least partly, by organics that may help disrupt the parallelism of the layers, but also to tie them together.Since biological oxidation of Mn is generally faster than abiotic oxidation, most natural Mn oxides are considered to be biogenic. Manganese oxidation and the subsequent precipitation of Mn bioxides by microscopic fungi is also well documented . Here, we showed that Mn can be biomineralized also in higher living organisms, such as plants. Except for its atypical high Zn content and the structural consequences thereof, this new manganese biomineral is no exception to the intrinsic nanocrystalline nature of biogenic phyllomanganates. Although the mechanism of Mn to Mn oxidation is presently unknown, the constant Zn:Mn ratio of the new Mn biooxide suggests the existence of a well-defined bioactive process, likely in response to metal toxicity. The occurrence of Zn-Mn precipitate only in the root epidermis and the absence in the roots of any Zn-rich species from the soil matrix suggest that Mn oxidation did not occur in the rhizosphere, and thus does not result from bacterial activity or abiotic reaction.

Divalent manganese may have been complexed and transported to the roots by phytosiderophores , and then oxidized by the plant itself or by endomycorrhizal fungi, as shown for wheat and soybean .Knowing how to stimulate the formation of this new phase in biological systems, or how to synthesize it abiotically, would be a significant progress towards Zn immobilization in contaminated environments and their remediation. Formation of this new phase could in particular facilitate the growth of plants in highly contaminated environments in lowering the concentration of bio-available Zn in the rhizosphere. Rice is the primary staple food source for over half the world’s population. The crop is cultivated in at least 114 countries and is the primary source of income and employment for more than 100 million households in Asia and Africa. In recent years, especially in developing countries, rice production has not matched the food demands of an increasing population. To meet this growing demand, rice production has to be raised by at least 70% over the next three decades. The land area devoted to rice cultivation is limited and production cannot be increased by more acreage, so additional, applied research is needed to find other ways of increasing productivity. With limited land resources and increased demand for enhanced production attention is turning towards intensification through higher fertilizer inputs, which is predicted to result in higher yields. Despite a sound logic base supporting increased fertilizer inputs in some rice cropping systems, possible indirect adverse effects of increased fertilizer inputs were highlighted by Heong. In the last decade, plant hopper outbreaks in rice fields have intensified across Asia resulting in heavy yield losses. Over the past decade yield losses substantially increased due to a widespread outbreak of the brown plant hopper.

For example, the Central Plains of Thailand suffered from persistent Plant hopper outbreaks for 10 consecutive growing seasons from 2008 to 2012 and caused losses worth $52 million or equivalent to about 173,000 tons in 2010. The same pest was responsible for losses of around 1 million tons in Vietnam in 2007, and resulted in a government freeze on rice exports. Relationships among fertilizer applications and insect pest outbreaks are widely described in the scientific literature, especially in response to nitrogen fertilization. Specifically regarding rice-based cropping systems, there is a considerable body of research highlighting the indirect effects of fertilizer applications on crop susceptibility to pests. As an example, BPH prefer to feed and oviposit on rice plants supplied with nitrogen. BPH reared on plants with high nitrogen content had high feeding rates and honeydew excretion, less probing behavior, higher survival rates and population build-ups, higher fecundity and oöcyte production, and higher risk of economically important BPH outbreaks. Nitrogen fertilization has also significantly increased the populations of white-backed plant hoppers , green leaf hoppers, and small brown plant hoppers. Finally, Pandey reported higher incidence of rice leaf rollers damage at higher levels of nitrogen fertilization. Phosphorus fertilization has been reported to markedly increase population growth of BPH. Phosphorus alone or combinations with nitrogen and nitrogen-phosphorus-potassium treatments are reported to support moderate leaf hopper populations. Treatments with phosphorus alone and phosphorus in combination with nitrogen also increased populations of ear head bugs and associated grain damage. It has been suggested that phosphorus tends to increase abundance of yellow stem borers in rice, but to a lesser degree than nitrogen. High fertilization levels of both nitrogen and phosphorus caused higher levels of damage by blue beetles in rice crops. Regarding potassium fertilization, there are reports of negative associations between application rates and prevalence of insect pests in rice. As an example, Kulagold et al. reported that higher potassium fertilization of rice plants led to reduced densities of green leaf hoppers, yellow stem borers, blue beetles, rice leaf rollers and ear head bugs. The rate of rice stem borer infestation was greatest when there was no supply of potassium, but decreased in response to increased potassium concentration in rice plants. Silicon content in rice is reported several folds higher than N, P, and K, also promoting a beneficial effect in rice. Recently Guntzer et al. reported that Si increases resistance against insect pests,vertical farming companies pathogens and abiotic stresses including salinity, drought and storms. In this research, N*P*K interactions in a factorial experiments upon Si content of rice is one of the measured attributes. Ample evidence supports a general hypothesis that excessive crop fertilization regimes affects both risk of infestation and severity of economically insect-induced crop losses. However, there are few studies where the combined effects of nitrogen, phosphorous, and potassium fertilizers are studied in detail regarding BPH infestations and its life history characteristics on rice, and also as collectively altered by varying Si content of rice plant tissues. Our previous study showed that biochemical constituents of BPH varied with nutrient levels at different growth stages, and changes in relative water content of rice plants. Moreover, concentrations of N and P were found much higher in the BPH body than in its host rice plants, and this elemental mismatch is an inherent constraint on meeting nutritional requirements of BPH. In this study, rice plants were grown in pots under factorial combinations of fertilizer regimes and subsequently assessed as host plants for BPH. The following specific objectives were to explore direct effects of NPK fertilizer regimes on physiological characteristics of rice plants, and indirect effects of these regimes on fitness traits of BPH.The principal component analysis of fertilizer regimes, nutritional elements of rice plants, and BPH fitness trait responses showed that 78% of the total variance could be explained by the two principal axes, PCA1 and PCA2 . To improve visualization, four variables were not included in Fig. 1, but they were located within the space denoted “cluster 1”.

Due to the high level of variance explained in the two-dimensional space defined by the principal axes, we could make fairly strong inferences about the relative associations of the explanatory variables to show that: 1) there were very close associations between fertilizer regimes and the corresponding content of the same macro elements in rice plants; 2) all agronomic rice plant traits were positively associated with nitrogen fertilization ; 3) nitrogen fertilization was strongly associated with PCA1 explaining most of the variance in the data set; 4) the PCA2 axes was clearly associated with a negative relationship between free soluble sugar and potassium in rice plants.Nitrogen fertilization significantly increased BPH survival from egg to nymph or to adult and all other fitness traits were positively correlated, which indicated these traits improved significantly with the increase of nitrogen inputs . Fitness responses by BPH to phosphorus showed the same trend as nitrogen, but the level of association was more modest. There was a negative association between potassium and soluble sugar , and these two plant nutrition traits were orthogonal to the BPH fitness traits located along the principal axis, PCA1. This suggested that potassium and soluble sugar had only a minor influence on BPH fitness traits. Very interestingly, BPH nymphal development time was positively associated with silicon plant content and negatively correlated with all other principal BPH fitness traits.BPH is one of the most important insect pests of rice in Asia, and it has been an extremely severe pest of rice for decades. However, BPH was a relatively minor rice pest prior to the advent of high-input rice farming which became major after the development and widespread adoption of new high yielding varieties that required increased inputs of fertilizers and pesticides. These practices altered the crop microclimate and accentuated the pest problem such as shifting of the BPH from a minor to a major insect pest. Our data demonstrate that variation of fertilizer inputs to rice plants significantly affected fitness traits of BPH. Firstly, higher nitrogen input increased the survival rate from egg to nymph or adult stages. Secondly, nitrogen excess increased fresh body weight and enhanced the development of BPH. Thus, higher nitrogen input increased the all fitness traits of BPH. Phosphorus and potassium fertilizers had no significant effect BPH. But if phosphorus is supplied with higher nitrogen input , phosphorus significantly influenced several fitness traits including fecundity , fresh body weight and total number of adult BPH developed from one pair of BPH . Potassium has no significant effect on any fitness traits of BPH either alone or combined with higher nitrogen input , but may as shown by MANOVA and stylized 3D plots , exhibit subtle effects that merit further experiments. These results corroborate the first aim of this study by making it clear that altered fertilizer inputs to rice plants can trigger a bottom-up effect on the fitness traits of BPH. Thus, three questions remain to be answered. What are the physiological changes in the host plants that could explain the effects on the fitness traits of BPH? Do our findings support or refute the “Plant vigor hypothesis” which suggests that herbivorous insects prefer and perform better on rapidly-growing plants? Do our findings support or refute the “Plant stress hypothesis”and what can learn from this study to better manage rice production by fertilizer by pest relationships? Different fertilizer treatment combinations influenced rice plant physiology and in turn affected the fitness traits of BPH. When higher doses of nitrogen fertilizer were applied, plants accumulated higher amounts of nitrogen and soluble protein content in their plant tissue, which ultimately influenced herbivore growth and development . Egg hatch ability also increased with nitrogen content, resulting in more BPH produced with higher dry body weights.