Horticulture

Transcription factors act as master switches of transcriptional reprogramming, inducing diverse protective mechanisms in response to abiotic stresses.We identified a large number of TFs responsive to dehydration, and 287 TFs were responsive to rehydration from among the DTGs . Among these TFs, the bHLH, MYB, and WRKY families were the largest groups during both dehydration and rehydration . Members of these three TF families are implicated in stress responses in model plants. In Arabidopsis, bHLH-type genes AtMYC2 and AtAIB are involved in ABA signaling, and overexpression of AtMYC2 or AtAIB enhances the drought tolerance of transgenic plants.Multiple MYB genes have also been implicated in stress responses. For example, AtMYB2 is involved in the ABA-dependent drought tolerance pathway,and AtMYB108 is associated with both biotic and abiotic stress responses.WRKY TFs are well known for their involvement in the regulation of plant development and in response to abiotic stresses.Functional analyses of rice OsWRKY11 and soybean GmWRKY54 demonstrated that WRKYs are involved in drought signaling pathways.Our results revealed that 9 SBP and 5 TCP type TFs also showed a response to dehydration in M. flabellifolia . Further functional characterization of these TFs may shed light on their roles in desiccation tolerance. PKs are essential signaling regulators in the acquisition of desiccation tolerance. Among the DTGs,vertical grow system we identified strikingly large numbers that respond to dehydration and rehydration .

PKs may play a key regulatory role in drought stress adaptation in M. flabellifolia, as corroborated by comparison of our data with Arabidopsis and chrysanthemum drought stress expression profile data.In Arabidopsis, only 121 PKs were found to be responsive to osmotic stress.Xu et al. identified only 229 differentially expressed PKs during drought stress in chrysanthemum, a species with a large and complex polyploid genome and high heterozygosity.Protein family distribution analyses showed that three receptorlike kinase type families – leucine-rich repeat kinases, domain of unknown function DUF, and receptor like cytoplasmic kinases – were the largest groups of DTGs in both the dehydration and rehydration treatments . RLKs constitute the largest PK family in plants and have been implicated in the regulation of meristem proliferation, organ specification, reproduction, and hormone signal transduction.Drought transcript profiling in Arabidopsis has revealed that the transcript abundance of many RLK genes peaks 1 h after the start of drought treatment, indicating that RLKs may also be part of a rapid drought response.Several functional studies have also confirmed roles of RLKs in drought tolerance.In Arabidopsis, an LRR kinase, receptor protein kinase 1 , is induced by ABA. Repression of RPK1 decreases sensitivity to ABA, suggesting that RPK1 is involved in ABA perception.A DUF type kinase, CRK, and a receptor-like cytoplasmic type kinase, ARCK1, form a complex that negatively controls ABA and osmotic stress signal transduction.

In addition,DTGs encoding mitogen-activated protein kinases were identified during dehydration stress in M. flabellifolia . MAPK cascades function in transducing environmental and developmental cues to intracellular responses.In Arabidopsis, several MAPKs are involved in abiotic stress signaling pathways including MEKK1,MPK1,MPK3,MPK4,MPK6,MAP9,and MAP12.The transcript abundance of homologs of all these MAPKs, apart from MPK6 and MAP12, was altered in response to dehydration in M. flabellifolia . To identify early responsive regulatory genes, the expression pattern of differentially transcribed TFs and PKs was analyzed by hierarchical clustering . The transcript abundance of 53 TFs and 91 PKs peaked early in dehydration , indicating that broad regulatory networks are quickly employed to coordinate global transcriptional reprogramming during desiccation of M. flabellifolia. Among the up-regulated TFs during early dehydration, the strongest induction was observed for genes encoding putative DREB family members . The DREB TFs are well-known regulators of ABA-independent drought stress signaling pathways in other plant species.In addition, a total of nine genes encoding WRKY family members were included among the up-regulated TFs in early dehydration, making the WRKY family the biggest group of early dehydration up-regulated TFs . Furthermore, among the up-regulated PKs, the LRK10 L kinase family is one of the largest groups in during early dehydration , suggesting that LRK10 L kinases may be important upstream regulators of rapid responses to water deficit. For example, the transcript abundance of a DTG encoding an LRK10 L kinase increased nearly 120-fold in early dehydration .

LRK10 L genes have previously been reported to be associated with resistance to pathogen infection.Functional characterization of these LRK10 L kinases will be an important step toward elucidating their roles in desiccation tolerance.Various genetically encodable reporters have been developed to monitor gene expression, protein subcellular localization, protein stability, hormonal signaling, and impacts of environmental signals. The green fluorescent protein and its derivatives such as RFP, mCherry, and YFP have many applications as reporters for gene expression or as fusion proteins. Although GFP is easy to use, it needs light sources to visualize the fluorescence signals. The β-glucuronidase reporter has been widely used in plants for monitoring gene expression patterns and as a reporter for hormonal signaling. For example, DR5-GUS transgenic lines are commonly used to monitor auxin distribution and auxin signaling. Luciferase is another broadly used reporter in both animals and plants. Both GUS and luciferase require the addition of expensive substrates X-Gluc and luciferin, respectively. Whereas the traditional reporters have been very useful, they have limitations. Fluorescent proteins are often monitored under a microscope, rendering it less useful in analyzing plants in natural growing fields or analyzing large samples such as a tree. GUS staining is invasive and often requires sacrifice of the plants. Luciferase can be used non-invasively, but it requires a special camera and spraying the expensive substrate. It is also not very practical to use them in fields. GUS and luciferase may not be optimal for sterile conditions such as tissue culture because addition of substrates increases the chance for contamination of microbes. Therefore, there is a need to develop new reporter systems that can be widely used to monitor cellular activities noninvasively, continuously, and cost effectively. For the past few years,strawberry pots gene editing has been widely used in basic research and crop improvement. A visible marker for transgenes will greatly accelerate the isolation of edited plants that no longer harbor the gene editing machinery. Plants produce many colorful compounds that potentially can serve as reporters. For example, anthocyanins display bright red-blue colors and anthocyanin-producing rice plants have been used to generate interesting patternsin rice field. However, synthesis of anthocyanins requires multiple enzymes and varies greatly among different plants. It is difficult to use anthocyanin biosynthesis pathways as a universal visible reporter. Betalains are a class of plant natural products derived from the aminoacid tyrosine. The bright red color seen in beets, dragon fruit, Swiss chard, and other plants is resulted from accumulation of betalains. Biosynthesis of betalains has been well studied and only needs three enzymatic reactions to convert tyrosine into betalain. Tyrosine is first hydroxylated on the benzene ring, resulting in L-3,4-dihydroxyphenylalanine . The reaction is catalyzed by the P450 oxygenase CYP76AD1 . L-DOPA can be further oxidized into cyclo-DOPA by CYP76AD1 . Alternatively, LDOPA is catalyzed by L-DOPA 4,5-dioxygenase into betalamic acid, which is subsequently condensed with cyclo-DOPA into betanidin. The condensation reaction does not require an enzyme . Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the colorful betalain . Betalain has a very bright red color, which potentially can serve as a reporter to track gene expression or to visualize transgenic events. Because every cell contains the amino-acid tyrosine, exogenous application of tyrosine to tissues may not be required. We hypothesized that betalain would be a more convenient reporter than the aforementioned reporters. It is visible to naked eyes without any needs for special equipment. It does not require processing samples and it allows continuously monitoring events throughout the life cycle of an organism. Moreover, it is applicable to large plants grown under normal field conditions. Herein, we synthesize an artificial open reading frame named RUBY that when expressed can produce all of the enzymes required for betalain biosynthesis. We show that RUBY is a very effective marker for noninvasively selecting transformation events in both rice and Arabidopsis. Moreover, we show that RUBY can be used to visualize gene expression without any chemical treatments or special equipment, providing useful tools for visualizing gene expression in large plants under natural field growth conditions.Heterologous expression of CYP76AD1, DODA in tobacco, and other plants demonstrated that the betalain biosynthetic pathway can be re-constituted in plant cells. In order to use betalain as a visual reporter, we need to effectively co-express the entire pathway using a single promoter. We organized CYP76AD1, DODA, and Glucosyltransferase into a single open reading frame . The stop codons of CYP76AD1 and DODA were removed. The three genes were linked by sequences that encode 2A peptides. Upon transcription, the single transcript, which includes the coding regions of the three enzymes, produced the three separate enzymes through either 2A-mediated self-cleavage or ribosomal “skipping”.

The 2A system enables the expression of multiple proteins under the control of a single promoter. We name the 2A-linked unit of CYP76AD1, DODA, and Glucosyltransferase RUBY . RUBY can be expressed when a promoter is placed in front of it. The expression pattern and level of a particular gene may be inferred from the red color of betalain if the gene’s promoter is used to drive RUBY expression.We first placed RUBY under the control of Cauliflower Mosaic Virus 35S promoter, which is a widely used constitutively strong promoter. To test whether RUBY can produce functional enzymes for betalain synthesis, we infiltrated tobacco leaves with Agrobacteria that contain RUBY-expressing plasmid.Transient expression of RUBY led to the production of betalain in tobacco leaves, suggesting that the synthetic open reading frame RUBY can produce the functional enzymes for the synthesis of betalain. Moreover, we observed that betalain was not transported from the spots of Agrobacterium-infiltration spots to other leaves of the plant .We transformed the 35S:RUBY construct into Arabidopsis using Agrobacterium-mediated floral dipping. Two days after floral dipping, we noticed that the transformed plants displayed patches of red color , indicating that the RUBY cassette was functionally expressed and that RUBY may be used to monitor transient Arabidopsis transformation. Once the seeds from the Agrobacterium-dipped plants were harvested, transgenic seeds could be easily differentiated from non-transgenic seeds . The transformed seeds had a dark red color , demonstrating that RUBY can be used as a visual selection marker for transgenic events in Arabidopsis. We previously used mCherry as a very effective marker to select transgenic events , which requires a dissecting microscope with fluoresence capability. RUBY is a better option because it does not require special equipment. The 35S:RUBY plants produced sufficient amount of betalain to become visually evident . Consistent with previous reports that CaMV 35S promoter is constitutively active, we observed red color in all tissues throughout the plant life cycle . We also expressed RUBY reporter under the control of the Maize UBIQUITIN promoter, which has been widely used to over express genes in monocots. Similar to 35S:RUBY plants, UBQ:RUBY plants were also visibly red in leaves, stem, and flowers . These results clearly demonstrated that RUBY could be expressed in Arabidopsis and that our RUBY reporter was able to functionally re-constitute the betalain biosynthetic pathway. We expressed RUBY using the seed specific At2S3 promoter, which we previously used to drive mCherry expression in Arabidopsis to facilitate the selection of transgenes. As shown in Fig. 2c, the transgenic plants were indistinguishable from wild type plants. When we checked the seeds in a silique from an At2S3:RUBY T1 plant, RUBY-expressing seeds displayed strong red color, whereas the non-transgenic seeds were green . RUBY can be conveniently used to select single T-DNA insertion events by analyzing the ratio of red seeds to green seeds, which should be ~3:1 for single insertions. The At2S3:RUBY results demonstrate that RUBY could be an effective marker for Arabidopsis transformation. Furthermore, betalain was not widely transported from the sites of synthesis to other tissues as we did not see any red color in leaves . We also expressed RUBY under the control of the Arabidopsis YUC4 promoter . YUC4, which encodes a key enzyme in auxin biosynthesis, was shown to express in small regions of embryos, leaves, and flowers. GUS signals were observed in leaf tips and apical region of a gynoecium in YUC4 promoter:GUS transgenic plants. We observed similar patterns of betalain production in YUC4:RUBY lines .Unlike Arabidopsis, rice and many other plants are transformed through tissue culture and the formation of calli, which are often mosaic.

Environmental cues, especially temperature and light, have huge impacts on organ abscission. It has been reported in various plant species that high temperature accelerates reproductive organ abscission. In cotton, day temperatures above 40 °C can induce flower abscission. In soybean, flower abscission was found to increase with the elevated temperature treatment in three different soybean varieties, while no significant difference was found between control and cool temperature treatments. In addition, the light quality is also critical for organ abscission. Shading, as well as dark treatments, induced reproductive organ abscission in several plants. In pepper, shading treatment enhanced flower abscission in several cultivars. In apple, periods of darkness, shading, or cloudy weather have been showed to increase fruit abscission leading to early fruit drop. Nineteen days of shading treatment caused 98% of the fruit to abscise. In grape, five days of shading at bloom reduced the percentage of fruit set. However, the mechanisms of high temperature, or dark/low light-induced abscission and whether auxin is involved in these processes are still unknown. The DR5::GUS reporter system provides a visual indication of the activity of the auxin response in the auxin signal transduction pathway. We used tomatoes transformed with this reporter to investigate dynamics of the auxin response system during the different developmental stages and in response to environmental cues in pedicel AZ of tomato and to test the hypothesis that changes in the auxin response system are important in the regulation of abscission.

To investigate the dynamics of the auxin response system in pedicel during flower development,vertical plant growing we collected pedicels two days before anthesis , at anthesis, and 5 and 10 days post anthesis/pollination . We examined the distribution of the auxin response activity using the DR5::GUS reporter system. GUS activity was concentrated in the vascular tissues, with the majority of activity, particularly in the young flowers and those at anthesis, on the distal side of the abscission zone , a clear disjunction or ‘step’ in the auxin response activity at the abscission zone. At anthesis the GUS activity seen in the proximal zone of the younger flowers had disappeared, increasing the difference in the auxin response across the AZ. GUS staining in pedicels at 5 and 10 DPA was considerably enhanced, particularly in the abscission zone and in the vascular tissues of the proximal portion of the pedicel . To examine the relationship between these auxin response changes on the control of abscission, we removed flowers 2 DBA, at anthesis, and 5 DPA. All of the pedicels whose flowers had been removed at anthesis had abscised 12 h after flower removal, but there was no abscission of flowers from young and older flowers .Following flower removal, pedicels were treated at the distal end, or at the junction between the pedicel and the peduncle with lanolin containing 1 mM auxin. Four hours after the start of the experiment there was little obvious change in distribution or intensity of the GUS staining , indicating that the response system was not rapidly responsive to changes in auxin concentration.Eight hours after flower removal pedicels began to separate and most had abscised by 12 h. . We tested the changes in the distribution of the auxin response after flower removal using the DR5::GUS reporter system.

Four hours after flower removal GUS staining in the pedicels was similar to that in the controls . RT-PCR visualization of GUS expression in the tissues confirmed that there was little change in expression in the early stages of the abscission process . There was a perceptible decrease in the sharp ‘step’ in GUS staining across the abscission zone 8 h after flower removal, and even more in pedicels that had not yet abscised 12 h after flower removal, suggesting a reduction in auxin response adjacent to the abscission zone in the later stages of abscission. GUS expression in pedicels 16 h after flower removal was confined to the distal portion of the pedicel .Treatment with the aubegan to abscise and most had abscised by 12 h . We tested the changes in the xin transport inhibitor mimicked the effect of flower removal on pedicel abscission. Eight hours after the treatment pedicels distribution of the auxin response after NPA treatment using the DR5:: GUS reporter system. Four hours after flower removal GUS staining in the pedicels was similar to that in the controls . There was a perceptible decrease in the sharp ‘step’ in GUS staining across the abscission zone 8 h after flower removal , suggesting a reduction in auxin response adjacent to the abscission zone in the later stages of abscission.Treatment with 10 ppm ethylene accelerated flower abscission following flower removal , while pretreatment for 24 h with 1-methylcyclopropene completely inhibited abscission . GUS activity in ethylene- and 1-MCP-treated pedicels 4 and 8 h after flower removal showed similar patterns to those seen in the controls . The sharp reduction in activity at the abscission zone showed little change 4 h after flower removal, even in ethylene-treated pedicels that had already abscised but was somewhat reduced after 8 h both in ethylene-treated and in 1-MCP-treated pedicels .Auxin is considered to be a key hormone in the initiation of abscission; the accepted model suggests that reduced transport of auxin through the AZ results in sensitization of the AZ to ethylene, which induces the chain of hydrolytic and other processes that lead to cell separation.

In a previous study, we demonstrated that a knotted homeobox transcription factor, KD1, plays a role in abscission, apparently by modulating transport of auxin through the AZ. Silencing KD1 increased auxin in the abscission zone, and microarray analysis suggested that this was associated with the down regulation of auxin efflux transporters, particularly PIN9. The study also suggested that the change in auxin distribution across the abscission zone resulting from KD1 activity was associated with a change in the activity of the auxin response pathway, and the experiments reported here were designed to test that hypothesis.High activity was seen in the distal portion of the pedicel during flower opening, with a marked disjunction or ‘step’ on the distal side of the AZ. Following pollination, response activity increased substantially, particularly in the young fruit, the AZ, and in the proximal region of the pedicel. Our data did not support the hypothesis that changes in distribution or activity of the auxin response system play an important role in the regulation of abscission. Removing the flowers at anthesis, which induces pedicel abscission within 8 h had little effect on the distribution or activity of auxin response , particularly in the early hours after excision,vertical farming when the abscission process is initiated. The visual results from GUS staining of the pedicels are supported by RT-PCR analysis of the expression of GUS transcripts , which shows a marked ‘step’ in transcript abundance across the abscission zone, and a change in expression pattern only at 12 h after excision, when most pedicels have already abscised.The conclusion that a change in distribution or activity of the auxin response system plays no regulatory role in pedicel abscission is supported by our additional data. None of the other manipulations that affected the occurrence or timing of abscission had a marked effect on GUS staining. Placement of auxin distal or proximal to the abscission zone, treatment with NPA, treatment with ethylene, or 1-MCP , placing inflorescences in the dark, or at high temperature, all had significant and varied effects on abscission, but the distribution of auxin response system as indicated by GUS activity was remarkably stable. In contrast, the data presented here demonstrated that the early stages of abscission were associated with marked changes in the distribution and activity of genes involved in auxin transport. This is in agreement with our earlier results. Application of the auxin transport inhibitor, NPA, resulted in a marked reduction of expression of genes encoding enzymes involved in auxin transport . Particularly striking decreases were seen in the expression of PIN1, PIN6, PIN9, and AUX/LAX2 in the abscission zone itself. This general pattern was also seen following flower removal, although the reduction in expression of the PIN genes appeared to be less tissue specific .

These changes are consistent with the observations of Shi et al, who also found a substantial reduction in SlPIN1 expression following flower removal, and suggested that it might play a role in modulating the auxin content of the AZ. Silencing of SlPIN1 expression accelerated pedicel abscission by simultaneously increasing auxin accumulation in the ovary and decreasing the auxin levels in the AZ, suggesting that auxin transport modulates auxin balance to influence pedicel abscission. Interestingly, PIN2/5/10 were not expressed in pedicels . This is different from other reports that down regulation of PIN5 in the flower pedicel reduces intracellular auxin accumulation in the endoplasmic reticulum , which is expected to control auxin availability for auxin signaling/response in the nuclei of AZ cells. The exact regulatory roles of these auxin transporters in the induction of abscission need further investigation in the future. Auxin and abiotic stress work together affecting plant growth and development. In Arabidopsis, the shoot ward auxin transport can be inhibited by the reduction of PIN1/ 3 transcripts under low temperature and increased by the upregulation of PIN2 under high temperature. In addition, high temperature induces hypocotyl elongation by regulating PIF4-mediated auxin biosynthesis. Our data showed that both high temperature and darkness can accelerate abscission . However, the intensity and distribution of the auxin response were almost little affected by these substantial changes in environmental conditions . It is still unknown if auxin transport can affect the pedicel abscission in tomato under these environmental conditions and would require further investigation. Our results are consistent with a model that places the primary control of abscission on the concentration of auxin in the abscission zone. Concurrent changes in the relative rates of influx and efflux might plausibly result in marked changes in auxin concentration, triggering the sensitivity to ethylene that results in the onset of the abscission process. Our data indicate that at 4 h after flower removal, expression of genes encoding auxin efflux enzymes fell while the expression of genes encoding influx enzymes increased. We can imagine a scenario where the activity of KD1 is controlled by auxin transported from the flower. When auxin flow falls, KD1 might modulate the expression of genes involved in auxin influx and efflux, amplifying the effect of small changes in auxin flow, and resulting in a marked fall in auxin content of the abscission zone, triggering the changes that result in separation. In this scenario, the auxin response system is an important factor, but it functions as a reporter of auxin content, and does not rapidly change activity or distribution in response to changes in auxin supply from the flower.Tomato inflorescences were harvested at 10 AM from plants grown in the greenhouse at the University of California Davis. inflorescences with at least two newly opened flowers at anthesis , two days before anthesis and five days post anthesis were cut on the proximal side of the AZ and placed in vials, and held in a chamber into which humid air was continuously flown through. For testing abscission triggered by auxin depletion via flower removal, flowers were removed with a sharp razor blade by cutting on the distal side of the AZ, and abscission of the remaining pedicel from the peduncle was monitored at intervals. For testing abscission triggered by auxin transporter inhibitor, N-1- naphthylphthalamic acid , the inflorescences were placed in vials containing 10 ml of 25 μM NPA solution. Flowers were not removed for NPA treatments. Control inflorescences were placed in a vial containing a solution of the equivalent concentration of dimethyl sulfoxide. For testing temperature-dependent abscission, the inflorescences were harvested at the anthesis stage. The pedicels with/out flowers were placed in the testing chambers in temperature-controlled rooms with indicated temperatures. For dark-induced pedicel abscission, the inflorescences were harvested at the anthesis stage with/ out flower removal and placed in the chambers under the dark conditions at 20 °C. For testing ethylene-triggered pedicel abscission, the inflorescences were harvested at the anthesis stage with flower removal and placed in the ethylene chambers. All the experiments were carried out with at least three biological replicates.

Benefit transfer approaches, SFA, and DEA yield results that can evaluate how well an NTS site is performing and how to improve their functioning, but have associated advantages and disadvantages . Benefit transfer approaches can provide an estimate for ecosystem services value, which can be used in cost-benefit analyses to determine net changes to well-being. In addition, benefit transfer can identify drivers of change through the application of regression models. Regardless of net costs or benefits, there exists a maximum level of ecosystem services provision that can be provided with given inputs. SFA and DEA can help determine whether systems are performing at this maximum level and where improvements can be made. Both methods apply somewhat similar regression models to benefit transfer approaches, yielding quantitative relationships among variables. Other options include a fixed effects model to estimate the best-practice frontier , and the multi-product distance function which allows for a stochastic error term and multiple outputs including negative externalities . In prioritizing ecosystem services,vertical hydroponic farming geographic location can dictate physical and social factors that can influence their value. For example, water issues have long been important in southern California and they are predicted to become more contentious due to climate change.

Further, new policies in California put an emphasis on climate-regulating services. The region is also a biodiversity hot spot at risk of alteration . Urbanization has reduced the amount of suitable habitat for organisms as natural ecosystems are replaced by human structures. Urban green spaces such as NTS can be important patches of habitat and act as corridors for organisms seeking refuge . One of the biggest challenges in characterizing and valuing ecosystem services associated with NTS is the lack of accessible data. There is no standard monitoring program for NTS, which makes it difficult to compare across time and across sites, and there is no monitoring of ecosystem services associated with NTS. Monitoring could help identify effective management strategies, e.g. timing of maintenance, and improve the above quantitative models for more accurate estimates of value and efficiency. In situ, mesocosm studies are also needed to evaluate NTS performance under actual environmental conditions . Other questions that still need to be addressed include how networks of NTS compare to single systems regarding both targeted and non-targeted ecosystem services, and how NTS operate over time. Prior to the widespread use of soil fumigants, Verticillium dahliae Kleb. was considered among the most important pathogens affecting commercial strawberry production in California . Plantations established in infested soils often suffered 50% or greater mortality , and wilt from this pathogen continues to be a major concern where strawberries are managed in perennial planting systems .

Breeding for resistance has been an important strategy for minimizing damage caused by V. dahliae in strawberry for several decades, and genetic variation for resistance to this pathogen has been demonstrated in many breeding populations . The University of California strawberry breeding program was initiated in 1930, and its goal since initiation has been to release cultivars specifically adapted to California production environments. In part due to concerns about the continued availability of effective soil fumigants, researchers in the UC strawberry breeding program began to develop a field-based resistance screening method in 1992 , and an ongoing resistance evaluation program has been conducted for this disease since 1994. Preliminary experimental results demonstrated substantial genetic variation for an overall low level of resistance to V. dahliae in the UC breeding population . Researchers also verified that resistance to the pathogen in this population has polygenic inheritance, is conditioned by both additive and dominance genetic effects, and that genotypes with high resistance were rare in the germplasm at that time. These results suggested that the infusion of non-California germplasm was not needed to obtain genetic progress, but that substantial time and effort would be needed to change the genetic composition of the breeding population and generate Verticilliumresistant strawberry cultivars with adequate frequency.Two broad strategies have been used to improve genetic resistance to soil disease in crop plants. One common approach is to first identify resistant germplasm and generate highly resistant lines, then back cross the resistance into elite cultivars.

This method works well when the inheritance of resistance is conditioned by one or a few genes and the crop-specific breeding system permits back crossing and self-fertilization for rapid genetic transfer and stabilization. Intensive selection for resistance within the UC germplasm base has resulted in genotypes that exhibit few symptoms following inoculation with V. dahliae , and these genotypes have been useful in evaluating selection limits and the mechanisms of resistance within this genetic base . However, even asymptomatic plants suffer some yield loss under conditions of high disease pressure . Furthermore, due to the intensified focus on resistance in generating these genotypes, they all express substantial deficiencies for horticultural or productivity traits, and as a consequence this strategy has not been successful in generating cultivars that meet the commercial standards required of modern strawberry cultivars in California.An alternative breeding strategy is to obtain incremental increases in resistance through population improvement, by inclusion of V. dahliae–screening results in a multiple-trait selection system to choose the parents of elite breeding populations. This strategy is generally a superior alternative for developing resistance in cross-fertilized crops such as strawberry. This system is especially useful when inheritance of the resistance is polygenic, or determined by many genes, and back crossing systems are consequently less effective. Furthermore, with multiple-trait selection, Verticillium resistance is considered as one among the many characteristics required to render a cultivar useful to strawberry growers in commercial production. This selection system permits flexibility in the relative weighting of selection intensity for resistance and horticultural traits, and can result in cultivars of balanced commercial utility. Back crossing methods for resistance can be combined with multiple-trait selection for horticultural traits, but this strategy is generally most effective for inbred crops. UC has conducted strawberry breeding continuously since 1930 , and the improvement of traits important for commercial productivity has been substantial . The choice of parents for the population-improvement component of this program has depended in part on resistance to important pathogens,vertical gardening systems and the longest continuous screening effort has been dedicated to Verticillium dahliae Kleb. resistance. This study reports on progress in developing resistance to V. dahliae within the UC strawberry breeding program obtained through parent selection and population improvement since 1994.Between 1994 and 2008, 481 genotypes from the UC strawberry breeding program were screened for resistance to V. dahliae. This sample included 461 advanced selections, genotypes identified with superior characteristics and which might either serve as parents for future generations or eventually be released to growers as cultivars. These selections were obtained from controlled crosses conducted over 18 years , with 11 to 49 genotypes from each cross year. Twenty genotypes were tested from the original germplasm base present prior to 1988. Strawberry breeding proceeds with overlapping generations, but the yearly changes in resistance for the genotypes used as parents from 1988 to 2005 provided a reliable empirical index of genetic progress for this population. The advanced selections tested for resistance in each trial year included those genotypes considered most promising based on their performance in fruiting trials conducted at either the UC South Coast Research and Extension Center near Irvine, Calif. , or the Watsonville Strawberry Research Facility . The genotypes included from any cross year had not been evaluated for resistance prior to their selection for horticultural and productivity traits, and they provide a representative sample of the variation for resistance in that cohort. Changes in resistance parameters over time reflect the outcome of including resistance in the multitrait selection strategy for prior parent choice.

The selection intensities — which reflect the relative importance of the selected trait — applied to the improvement of Verticillium wilt resistance during the study period are impossible to quantify with precision, but the genetic progress reportedhere results from two sources. first, the most susceptible genotypes usually were eliminated entirely, and the participation of moderately susceptible genotypes as parents was limited to a few crosses. Moderately susceptible genotypes were included if they expressed outstanding horticultural characteristics, but they usually served as parents in crosses for just one year. Second, individual genotypic selections were made with knowledge of parental resistance scores, and fewer genotypes were retained from crosses expected to contain high frequencies of susceptible individuals.Runner plants from each tested genotype were immersed in an aqueous suspension of 1.0 x 106 V. dahliae spores per milliliter. All evaluations were conducted at the Wolf skill Experimental Orchard near Winters, Calif. . The inoculum included spores from one to three isolates originally obtained from symptomatic plants found in commercial production fields in California . Each genotype was represented by two plots of five inoculated runner plants per trial year; two noninoculated plants of the test genotype were placed adjacent to each plot, and disease ratings were made relative to these control plants. Plots were distributed between two replicates, and each year’s trial comprised a randomized complete block design. Inoculated plants and controls were established in the field from Oct. 5 to 18 each year. Many of the individual genotypes, 204 of the 481, were tested in more than one year, and the average number of test years per genotype was 1.7. In general, genotypes under consideration for release as commercial cultivars were tested with greater replication over years, and these superior genotypes were also heavily represented in crosses conducted to facilitate population improvement. Genotypes were evaluated in the spring following inoculation, by rating plants for symptoms of Verticillium wilt on a scale of 1 to 5, where 1 =severely diseased and 5 = no symptoms of disease . Individual plots were evaluated four to seven times in each trial year at approximately 3-week intervals beginning with the first symptoms on susceptible genotypes , and a combined score was obtained as the arithmetic mean of scores for all dates . An average resistance score was calculated for each genotype and year combination as the mean for the two replicates.Variation in resistance scores over trial years due to environmental factors — such as differences in ambient temperature, isolate source or inoculum quality — was confounded with genetic differences generated due to selection over years. Several of the genotypes were tested in most of the evaluation years, and the examination of these samples demonstrated relatively small yearly fluctuations in genotypic score. For example, on a resistance scale of 1 to 5, the relatively susceptible cultivar Camarosa had an average score of 2.52 and a standard deviation of 0.46 over all years, and the moderately resistant cultivar Camino Real had an average score of 4.25 and a standard deviation of 0.41 for trials conducted in 11 of the 15 years. This suggests that more than two-thirds of the samples will vary by no more than about 0.435 scoring units from year to year. The effect of trial year was not considered further, but is reflected as part of the error variation in our analyses. Changes in the Verticillium resistance scores due to selection were evaluated by first calculating a composite genotypic score as the average for each genotype over all trial years in which it was tested, then plotting and regressing these composite scores by cross year. The 20 genotypes from the original germplasm sample were considered representative of a base population present in 1987, and are included as such in plots and regression analyses. The average resistance scores were further resolved by calculating the percentage of genotypes in each cross-year population with a composite score greater than 3.0, and thus exhibiting at least moderate resistance. The percentages calculated for each cross year were plotted and treated by regression analysis as described for the resistance scores.The absence of reliable estimates for selection intensities and the presence of overlapping generations precludes precise determinations of the genetic response; however, the general trends for inclusion of Verticillium resistance in the population improvement program are evident from the steady increase in resistance scores over cross years . Regression of resistance scores for the 481 genotypes on their cross year demonstrated a highly significant increase over time, with b = 0.061 ± 0.007 . The predicted change in resistance score from this regression analysis over 18 cross years affected by selection is 1.10 resistance-score units, or a 46.3% improvement over the average resistance score for the original germplasm .

Assuming all of the roughly 16 000 smallholder farmers currently growing tobacco were to produce an average of 400kg of paprika, for example, this would result in a total yield of just 6 400 tons, equal to less than half of Zimbabwe’s record crop. Although it is not unreasonable to expect this could lead to lower international prices, smallholder farmers appear well positioned to cope. To the extent this provides a window of opportunity for Zimbabwe’s buyers to focus on lower-value smallholder paprika, there could be good potential for a gradual shift away from tobacco. Whether or not this potential can be realised, however, still depends on the development of crop extension services and input supply arrangements. As with all other enterprises, more comprehensive modelling is needed to assess the conditions under which such an investment would make financial and economic sense. Marigold flowers are a relatively new niche product in Zimbabwe and are grown almost exclusively by LSC farmers for their value as a colour extract. Marigold is a much lower-value commodity than either coffee or paprika,vertical plant rack but grows well on most soil types and is forgiving to management input.

As a bulky and relatively low-value commodity the returns to marigold are highly sensitive to transportation costs and most LSC farmers dry the crop in a cement-lined silage pit before delivery to Harare for processing. Dry flowers are converted to a compact pellet form for export or for domestic use either as an industrial food colour or ingredient in stock feed. Research is ongoing in Zimbabwe to determine an optimal pest control strategy and fertiliser recommendations for LSC marigold. Current production is around 700mt of dry flowers annually. Horticulture is Zimbabwe’s second most valuable agricultural export sector after tobacco and earned over USD 124.9 million in gross foreign income in the 1999/00 season, equal to approximately 1.5% of total GDP and 22% of the revenue generated by tobacco.An overview of sector performance by export season is given in Table 33 and shows that horticulture has grown rapidly in the past ten years with total value now five times greater than in 1990. But unstable political and macroeconomic conditions have slowed these growth rates and it is projected that total exports in the 2000/01 season could decline by 10% or more. Roses require a large expenditure on imported inputs, especially during establishment, for irrigation equipment, planting materials and greenhouse construction, and the total export values listed below would be considerably less if measured in net terms.

More than 80% of Zimbabwe’s horticultural exports are grown on LSC tobacco farms and were first developed using tobacco revenue. For most farmers, the main objective behind the introduction of roses and other horticultural exports was to lessen their dependence on tobacco. Not only can horticultural crops be extremely profitable, but like tobacco, they also earn foreign exchange and so have the potential to play a similar anchor role in a mixed farm system. Furthermore, although horticultural crops tend to be expensive because of high costs for pest and disease control, export vegetables and roses both provide a steady cash flow in their own right with weekly and even daily sales throughout the export season. From the social point of view, a further advantage of horticulture is that these crops are extremely labour intensive and create more jobs on a per hectare basis than any other farm enterprise including tobacco. These characteristics all make horticulture a very attractive sector, both for farmers and for Zimbabwe as a whole in terms of more diverse export earnings, employment creation and potential for growth linkages.Despite these advantages, the production and marketing of horticultural crops is a highly specialised business that cannot be entered into lightly. At the field level, success demands unparalleled attention to quality control and strict adherence to European standards governing the use of pesticides, fertilisers and other inputs.

It is not unusual for more than 30% of a vegetable crop to be rejected because of quality and this risk has discouraged many LSC farmers from continuing with production. Marketing, therefore, is one of the biggest obstacles to success. Although cut flowers can still be sold independently on the Dutch auction, most other produce must be sold as part of a forward contract negotiated by a central agent or export consortium with the capacity to supply the specific volumes and varieties of produce European buyers demand. 145. High freight costs and the availability of direct flights to Europe further add to the challenge of success with horticulture and are currently among the most pressing constraints facing Zimbabwe’s export sector. Over the past 12 months, direct flights from Harare to the UK, Europe and Australia have been reduced from 32 to 14 per week, resulting in a shortfall of approximately 150 tons of cargo space per week with a potential value USD 17 million over the 25-week peak export season.As a result, many flower exporters have been forced to drive their produce by road to Johannesburg nearly 1 200 kilometres away simply to find cargo space. Zimbabwe also has some of the highest costs for aviation fuel in the region leading to high air freight prices which now account for as much as 40% of total production costs for both vegetables and roses.For these and other reasons, more than 95% of vegetable exports and 100% of cut flowers are grown on individual LSC and large corporate farms, mainly within 200km of the International Airport in Harare. Smallholder farmers are only marginally involved in the export sector with fewer than 3 000 vegetable growers linked to established agents. This is in sharp contrast with the experience in Kenya where there are perhaps 60 000 to 75 000 smallholders involved in export-oriented vegetables, including production for canners and freezers. Despite more stringent marketing standards that apply today than when Kenyan horticulture first developed, there is still considerable scope for increased participation of Zimbabwean smallholders on an out grower basis in some of the high potential farm areas near Harare. Three indicative export vegetables are considered for this analysis including mangetout , baby carrots and baby corn. Other important export crops for LSC farmers include sugar snaps, fine beans,growing strawberries vertical system cherry tomatoes, courgettes and asparagus. Each vegetable crop is typically grown over an 8 to 9 week period with most LSC farmers producing three crops in rotation each season. Although some growers have tried to form local export associations with neighbouring producers, most vegetable crops are farmed on an out grower basis for a local contractor who collects, washes, grades and packs the produce as a shelf-ready product for delivery to a UK or other European supermarket chain. Individual farmers simply cannot guarantee the variety and continuity of supply needed to negotiate export contracts with supermarket buyers and this system is a vital link to the international market. To ensure a steady supply of good quality produce, most export agents also grow vegetable crops themselves, especially during the rainy season when many LSC farmers switch away from commodities that are difficult to grow.

Mangetout and baby carrots, for example, are almost impossible to produce during the heavy rains, but baby corn is more forgiving and better suited to production throughout the year. In some cases, this means that export agents have even found it necessary to grow mangetout and other crops in plastic houses just to meet the conditions of regular supply demanded by European buyers. This can be justified for a few months each year, but is only feasible for a large supplier with effective economies of scale.Unlike roses, one important advantage of export vegetables is that these crops do not require a major investment in green houses and other specialised equipment by LSC farmers. Although most crops do best with drip line irrigation, it is perfectly acceptable to use existing overhead spray systems already available on most farms. For export agents, on the other hand, total investment costs can be very high and include the establishment of rural depots, grading and packing sheds, heat extraction units and cold rooms. Generators as well as insulated and refrigerated trucks are also required to ensure the cool chain to Europe is never broken.Large-scale commercial farmers. Per hectare results for each of the LSC vegetable crops covered here are summarised in Table 34. These calculations are based on delivery to a nearby depot where an agent collects the produce for final grading and export presentation. Importantly, LSC farmers are only paid for produce actually exported and yield assumptions are estimated on this basis. Unsurprisingly, this system has led to suspicions by LSC farmers that they are not always paid for all of the produce actually shipped abroad. Certainly, the grading system is not entirely transparent and this problem has caused some farmers to switch away from export vegetables in favour of other crops with more certain terms of payment. Because vegetable crops are all grown to a uniform standard, only one management level for each product is considered.Taken together, the results below are encouraging and show that, under the right conditions, export vegetables can be an important source of steady income for LSC farmers. Although per hectare profits are perhaps somewhat low compared with other enterprises on a single crop basis, export vegetables are normally triple cropped throughout the year and so easily rival flue-cured tobacco and other high-value enterprises in terms of total annual income. Assuming the three crops covered here were grown in rotation, for example, total annual income would be ZWD 102 503 per hectare, which is almost identical to the estimated profits for medium-input dryland flue-cured tobacco. Unlike tobacco, however, one the most important advantages of export vegetables is that these crops provide a steady cash flow in their own right. Not only are export vegetables planted in three to four cycles throughout the year, but most producers also aim to stagger their plantings on a weekly basis to produce a steady harvest throughout the season. This helps minimise the need for large cash expenditure at any one time and also provides a more regular income than almost any other farm enterprise. In terms of employment creation, the data show that an important advantage for Zimbabwe is that export vegetables are all very labour intensive and, on a per hectare basis, easily create more jobs than tobacco. Again assuming the three crops listed above were grown in rotation, a total of 760 days labour would be required per hectare, which is considerably more than for any other enterprise except roses. Although the precise situation will vary from farm to farm, casual workers normally fill more than 50% of the total labour requirement and vegetable exports can be an important source of employment and supplemental income for local residents. It should be stressed, however, that vegetable crops and other horticultural products could never substitute entirely for tobacco, which is grown on a much larger scale. Furthermore, crops with a large labour requirement are not always attractive from the farmer’s perspective because of potential management problems and incremental costs of housing and other benefits for permanent staff.Smallholder farmers. Vegetable crops have long been an important part of most smallholder farm systems and are grown both for household consumption and for sale in local markets. Crops including tomatoes, cabbage, onion, kale and okra all enjoy good demand in rural and urban markets and are an important part of many Zimbabwean diets.Domestic production, however, is entirely different from growing vegetables for the export market and there are currently fewer than 1 700 smallholder farmers within 100km of Harare producing baby corn, mangetout, butternut squash and sweet corn on an out grower basis for one leading export company. These farmers include established SSC producers also growing tobacco; irrigation scheme participants; and individual communal farmers with limited access to other sources of cash income. Depending on the time of year, these farmers produce from 50% to 90% of all baby corn and up to 5% of the mangetout shipped by the export company. Success with smallholder export horticulture depends heavily on technology transfer and the exporter provides extension agents to give advice and ensure that each crop is being grown to the required standards. A network of rural depots has been established to collect the produce and the out grower company itself does crop spraying with the cost deducted from each farmer’s final payment.

Zimbabwe exports most of her tobacco crop, however, so if global demand for tobacco were to fall in future , the impact on employment would depend on the extent to which commercial farmers were able to switch to other export crops such as roses, paprika, coffee and vegetables which are also highly labor intensive. Many other crops have similar per hectare labor requirements to tobacco, and some have further opportunities for downstream processing and growth linkages. Most of these crops are currently grown on a fairly limited scale, however, so the question would be how quickly and successfully farmers would be able to scale up exports and production of these crops, in the face of declining tobacco markets. This paper considers the financial costs and returns for tobacco and twelve other important crops grown by commercial and smallholder farmers in the intensive farming areas of northern and central Zimbabwe. Tobacco is of critical importance to the Zimbabwean economy and typically generates around USD 600 million in foreign revenue annually,vertical farming supplies equal to almost 10 percent of GDP and 30 percent of total exports.

Although global demand for tobacco is still strong, pressure from international health organisations, anti-smoking groups and new trade protocols all threaten the long-term prospects of this important crop. As Zimbabwe looks to the future, therefore, it is important to consider diversification options and growth strategies in which other crops could play an increasingly important role and eventually substitute for some of the foreign earnings and employment currently accounted for by tobacco.Towards this end, the primary aim of this analysis to provide an improved understanding of the trade-offs individual growers face in deciding which crops to grow. Agricultural production begins with the decisions farmers make and any successful diversification strategy must have the costs and returns for individual producers in mind. More specifically, at this critical juncture in the development of Zimbabwe, how do different crops and production technologies now compare in terms of total production costs, investment requirements, farmer profitability, employment creation, demand for credit and other matters of private and social importance? Are there attractive crop options that would justify farmers making a shift away from tobacco and what would be the implications for Zimbabwe’s trade balance and local employment opportunities? This paper has been prepared to help answer some of these questions and to provide an improved basis for discussion of recent development trends and future growth strategies. In geographic terms, it is first important to note that tobacco is grown almost exclusively in high-potential farm areas of Natural Region II in northeastern and central Zimbabwe.

This zone accounts for less than 15% of Zimbabwe’s total land area , but is ideally suited to intensive farming with a more or less reliable 750 to 1 000 mm of rainfall coming in the summer months from late-October until the end of March.By contrast, Natural Regions III, IV and V are much less suited to intensive farming with progressively arid conditions less suited to tobacco and other crops. Smallholder farmers occupy about 71% of the farmland in these lower-potential areas equal to about 18.7 million hectares in total. Natural Region I is mostly in the Eastern Highlands with a mild climate suited to specialised farming and forestry. Maps showing the location and rainfall expectations of each Natural Region and also the location of Zimbabwe’s 15 main flue-cured tobacco growing districts are given in Appendix 1. Further details on the breakdown of land classification by farm sector and area planted to different crops in each Natural Region are given in Appendix 2. In interpreting the discussion that follows, it is also important to bear in mind that large-scale commercial farmers dominate Zimbabwe’s tobacco sector. Although there are fewer than 2 000 commercial tobacco growers, these producers account for about 87% of area planted and 95% of the total crop equal to some 180 to 240 million kilos of flue-cured tobacco annually. Furthermore, most commercial farmers already have diversified sources of income. Although tobacco is still the backbone of commercial agriculture in most locations, other important crops for large-scale farmers include wheat, soybeans, maize, groundnuts and livestock, which are typically grown in rotation with tobacco.

Most commercial tobacco farmers practice a 5-year rotation and these crops are an important part of the overall land use system and help provide a steady cash flow. More recently, many commercial growers have also introduced other high-value crops including export roses, supermarket vegetables, paprika and coffee as part of their farm system specifically to lessen their dependence on tobacco. More than 80% of all horticultural exports, for example, are grown on tobacco farms and were first developed using tobacco revenue. Smallholder farmers, by comparison, are only marginally involved in the direct production of tobacco. Although there are roughly eight times as many smallholder tobacco growers compared with large commercial farmers, these account less than 1.5% of all smallholder households and just 7% of those in suitable agro-ecological areas. Certainly, the importance of tobacco as a high profit crop with fully developed market outlets cannot be overlooked for these producers, but it should also be noted that maize, cotton and groundnuts are all more important for smallholder farmers in most locations. In this respect, the greatest threat from shrinking tobacco markets for the smallholder sector is not so much the potential loss of direct farm enterprise income, but rather the loss of remittances sent by family members employed on large-scale commercial farms. Before proceeding, it should also be noted that this analysis has been prepared at a critical time in the development of Zimbabwe. Apart from the pressure on tobacco from international health organisations, new trade protocols and other sources, the overall future of the agriculture sector is now facing great uncertainty in terms of the controversial program of land acquisition and resettlement.

Although this paper has no interest to enter the political debate over land or to predict the outcome of current events, it is not possible to overlook the present situation and implications for development opportunities in agriculture. No matter how one interprets recent events, for example, it is clear that current conditions in Zimbabwe not only discourage new investment but also make it difficult to sustain existing farm production in terms of access to seasonal credit,vertical lettuce tower fuel shortages, high inflation, land occupations and foreign exchange controls that distort the cost of imported inputs. For large-scale commercial farmers, the strategy now is mainly one of survival and bracing for the worst rather than to invest in new technologies and infrastructure as part of a drive towards crop diversification. Smallholder farmers have likewise been affected by the poor investment climate and are perhaps even more dependent on improved conditions than commercial growers. One of the key findings from the analysis is that that the most profitable crops and management systems depend on specialised infrastructure and technical support services. Quite simply, until the economic and political situation improves, few entrepreneurs are willing to invest in these services, thereby limiting the opportunities for broad-based growth and diversification. Putting aside current political and economic constraints, the overall results of the analysis are encouraging and show that several crops apart from tobacco offer an opportunity for high producer profits and attractive rates of return. Other crops that rival and/or surpass tobacco in terms of potential net profit include roses, paprika, coffee and supermarket vegetables for large-scale commercial farmers and paprika, coffee and cotton for smallholder growers. These crops can all be grown in the same areas as tobacco and provide excellent rates of return to total production costs. On the other hand, these crops are also relatively expensive to produce and, with high-input management, are sometimes more costly than tobacco. This is especially true for high-value horticultural crops and long-season irrigated paprika on commercial farms. The analysis also shows that many traditional agricultural crops including wheat, soybeans, groundnuts and maize are now marginal activities for large-scale commercial farmers and, in some cases, even return a net loss and fail to cover the long-run depreciation cost of fixed assets. On the other hand these traditional crops do, in most cases, provide a positive gross income and are therefore important in terms of helping to finance other more profitable and expensive crops like tobacco, paprika and coffee. In turn, the very good financial returns from these high-value enterprises help offset the net losses from traditional field crops and the analysis shows these are still a fundamental ingredient to most successful farm strategies. In the case of paprika, for example, which is the most profitable smallholder crop apart from tobacco, yields are highly dependent on rainfall and can easily be wiped out by adverse growing conditions. Whereas large-scale farmers are able to protect themselves from some of these risks through irrigation, most smallholder farmers do not have access to this technology and paprika is typically considered a risky enterprise for smallholder growers.

Coffee is likewise an attractive possibility and can provide a net income that rivals many tobacco scenarios, but is a relatively new crop in tobacco areas and would require a substantial investment in farmer training and also pulping and processing facilities before it can be widely promoted in these locations. With respect to cotton, this enterprise generally provides a much lower income that tobacco , but does offer an excellent rate of return to cash and total production costs. At the national level, cotton is the most widely grown smallholder cash crop. Importantly, for all categories of farmer, the analysis shows that the most profitable crops and production technologies require specialised infrastructure, processing facilities and other support services. This is especially true with respect to roses and vegetable exports, which can be very profitable but are also extremely expensive to grow and extraordinarily skill intensive. These enterprises also require a large capital investment in processing and packing facilities, special irrigation equipment and other infrastructure including greenhouses and insulated trucks for roses. Some large vegetable exporters have been working with smallholder farmers near Harare to provide inputs and develop a network of collection points for baby corn and mangetout, but these programs are expensive to establish and are still relatively small with only limited farmer participation. In this respect, marketing constraints are one of the main obstacles to success with most high-value crops that could compete with tobacco in terms of profitability and potential export earnings. Again, this is most obvious in the case of roses and export vegetables, which must be grown to exacting European standards and delivered in fresh condition according to a tight time schedule. Paprika, on the other hand, is far more forgiving and can be sold to local export agents and processing companies on forward contract. Although this helps to minimise some production and marketing risks, relatively small world demand for paprika means that prices are highly sensitive to increased production and the crop could never substitute for tobacco on its own. In terms of labour requirements, the data show that tobacco generates more employment opportunities per hectare than nearly every other enterprise analysed. This is one factor often noted by proponents of tobacco to illustrate the overall importance of the crop to the national economy and the calculations here suggest that the total wage bill for tobacco grown on large-scale commercial farms could be as high as ZWD 2 600 million .To the extent that a share of this income is sent as remittance payments to family members in communal areas, tobacco can play a major role in helping to finance the inputs needed for improved management of major smallholder crops including maize and cotton. Although these benefits of tobacco cannot be ignored, the data show that many other crops are also very labour intensive and so offer similar opportunities for employment creation on a per hectare basis. Roses are perhaps the best example of this where just one hectare generates almost as many jobs as 25 hectares of flue-cured tobacco. Of the other field crops, paprika, coffee and export vegetables all demand a similar amount and sometimes more labour than tobacco with further opportunities for downstream processing and growth linkages. Because of limited market outlets and intensive management requirements, however, these crops are mostly grown on a much smaller-scale than tobacco and it is unlikely any single enterprise could ever substitute for a loss of tobacco employment without growth in other economic sectors including industry and tourism.

The homolog of Arabidopsis TCP4, AqTCP4, was expressed at fairly consistent levels across all stages in the spurred taxa, but showed a substantial increase in expression in A. ecalcarata at DS3 and DS4, while AqTCP5 showed a gradual decrease in expression across all taxa, but a slightly higher level of expression in A. ecalcarata.Aquilegia has two BB homologs, one that is expressed at a low level in all taxa and shows a pattern of decreasing expression over development , and one that has fairly steady expression in the spurred taxa across phase I, but starts with a higher level of expression in A. ecalcarata .Variation in the shape of plant organs is determined by combinatorial differences in cell number and cell shape. The Aquilegia petal has evolved substantial morphological variation, particularly in several aspects of nectar spur shape, including length, width, and curvature. Our current understanding of the development of the Aquilegia nectar spur involves two phases, an early mitotic phase and a later cell expansion phase . Despite differences in adult morphology, detailed ontogenetic studies using SEM have shown that from petal initiation through the earliest stages of Phase I, petals of a spurred species, A. olympica, and those of the spurless species, A. ecalcarata, are quite similar in shape. In other species comparisons,vertical hydroponic garden differences in spur length at maturity have been largely attributed to differential cell elongation during phase II of development, however, the developmental basis of other aspects of spur shape have not been studied in detail.

Based on the sampling conducted here, discernible differences in several axes of shape, particularly width and curvature, are apparent quite early in Phase I of petal development, suggesting that cell division plays a role in determining their differences. By examining gene expression across early petal development in several species with variable morphologies, this study allows us to identify gene expression modules that appear to be conserved across petals with diverse morphologies as well as modules that differ in correlation with variation in spur morphology. Further, this comparative approach may provide insight into the developmental processes that underlie the morphologies.Over the course of petal development, GO enrichment analysis of genes commonly DE across all four taxa detected a pattern of declining expression of genes involved in mitotic activity. This pattern was also supported by correlations between WGCNA modules and developmental stages. Modules with eigengenes highly positively correlated with DS1 are enriched for GO terms related to mitosis. This finding of a decrease in mitotic activity throughout development is consistent both with petal developmental patterns in other model systems and with previous studies in Aquilegia. In contrast to this pattern, an enrichment of genes involved in oxidation reduction processes were found to be up-regulated at later developmental stages in all four taxa.

This is supported by both the DE analyses between DS1 and DS5 and by the WGCNA analyses where several modules with eigengenes highly positively correlated with DS5 are enriched for loci involved in oxidation-reduction processes. Exploring the types of genes that are differentially expressed between the spurred taxa and A. ecalcarata detected up-regulation of loci with GO categories related to mitosis in the spurred taxa, but only at DS5 . As Aquilegia petals develop, they begin to transition from cell division to expansion and differentiation, starting at the distal margin of the blade. In spurred taxa, this transition progresses from the petal margins toward the nascent nectary, with cell division persisting longest in the spur itself. Considering the pattern seen in the developmental comparisons and what is known about cellular processes during spur development, the increased expression of genes related to mitosis in the spurred taxa relative to A. ecalcarata suggests that the entire A. ecalcarata petal shifts into the differentiation phase at an earlier time point than in spurred taxa. Another pattern that supports this assertion is that there is an enrichment of loci involved with oxidation reduction processes expressed more highly late in development when considering all taxa, while when considering the loci DE between the spurred taxa and A. ecalcarata across development, there is an enrichment for loci with these processes at early stages in A. ecalcarata . A closer examination of genes with oxidation-reduction GO categorization in the developmental and spurred/spurlesss comparisons revealed a number of cytochrome P450 monooxygenases that at a molecular level function through heme/iron binding and oxidation. Although CYPs have similar molecular functions, they comprise the largest enzymatic gene family in plants and have evolved to play diverse roles in an array of cellular, developmental, and metabolic functions from hormone synthesis to pigment production. Many of these CYP functions appear to be important in differentiated cell types, rather than undifferentiated mitotically active cells. Thus, the upregulation of CYPs earlier in A. ecalcarata development may be consistent with our hypothesis that these petals are accelerated in their differentiation relative to those in spurred species.

Although the WGCNA identified a module that is highly correlated with the presence or absence of spurs , this module showed no GO enrichment. A curious result that emerged when examining the genes that are commonly up-regulated across development in only spurred taxa was the enrichment of genes involved in photosynthetic processes. This was also seen in WGCNA module 20, which contains genes expressed more highly in spurred taxa at DS5 and is enriched for GO terms related to photosynthesis. While this enrichment of photosynthetic genes seems perplexing given that petals are generally not considered photosynthetic organs, we hypothesize that this result is likely an indirect consequence of spur development, rather than a cause. During phase I of A. ecalcarata development, the entire petal is shaded from light by the enclosing sepals. In the spurred taxa, however, elongation of the petal spurs causes them to emerge from the bud and become exposed to direct light, likely inducing baseline expression of photosynthetic loci . Thus, this appears to be a background temporal component of spur development rather than a controlling factor.Although one might predict that more genes would be required for the development of the nectar spur, given its more complex three-dimensional structure compared to laminar petals and blades, a number of lines of evidence indicate that the number of loci required for early spur development may actually be relatively small. Considering the PCA conducted across the set of genes DE between DS1 and DS5 in any taxon , one might have expected that the presence or absence of spurs would be a major PC resulting in the grouping of samples from the spurred taxa apart from A. ecalcarata. However, none of the first 10 principle components examined capture such variation. The first principal component clustered samples by developmental stage,vertical home farming which is not surprising given how genes were selected for inclusion in the analysis. The second PC in this data set groups samples by geographic origin, with the Eurasian taxa clustering together and the North American taxa clustering together. This suggests that phylogenetic relatedness explains more shared developmental differences in gene expression in our data set than whether or not a nectar spur is produced. Several scenarios may contribute to this phenomenon. This pattern may indicate that relatively few genes are necessary to make a nectar spur and, thus, loci that consistently vary between the spurred taxa and A. ecalcarata do not explain a significant proportion of variation in this data set. Not mutually exclusive to this possibility, it may be that the genes important for spur production do not have variable expression levels across phase I of development and therefore were not captured in this set of analyses. Regardless, this result underscores the importance of sampling more than two species across divergent lineages for this type of study since, for instance, a pairwise comparison of A. ecalcarata and any single spurred species might have primarily identified loci that differ due to phylogenetic divergence rather than their morphological differences.

Given that crucial loci in nectar spur development may not be differentially expressed between DS1 and DS5, we also compared expression differences between the spurred taxa and A. ecalcarata at each of our developmental stages. These comparisons showed that during the earliest stages of spur development , there are fewer genes up-regulated in the spurred species than in A. ecalcarata. One possible explanation may be related to the proportion of differentiating cells in the petals of A. ecalcarata versus the spurred species. Given that the A. ecalcarata petal does not produce a spur and is entirely composed of blade tissue, a greater proportion of the A. ecalcarata petal may be in the differentiation phase relative to an equivalent sized petal that is producing a spur. Although a large set of genes are necessary for mitosis, the differentiation of cells into many specialized types may require the up-regulation of an even greater number of loci. In support of this hypothesis, several comparisons from the A. coerulea ‘Origami’ expression data set show a similar pattern. Comparing expression between the blade and the spur showed more loci are up-regulated in the blade and developmental comparisons between the 1mm and 3mm blade and spur cup tissue samples demonstrated that a greater number of genes are up-regulated in both tissues at the later developmental stage . Another consistent data point is seen in the expression of AqTCP4 . Previous studies have found that AqTCP4 is critical for the cell division to cell expansion/differentiation transition in Aquilegia and is expressed in a wave that starts at the blade margin and progresses towards the spur tip. However, in the comparison between the four sampled taxa, AqTCP4 is observed to peak during DS3-4 in A. ecalcarata while it is present at lower levels in the three spurred taxa. This is likely due to the absence of the prolonged proliferation needed for spur development, but may also reflect a developmental acceleration in A. ecalcarata relative to the spurred taxa, representing a heterochronic shift. Therefore, we infer that the greater up-regulation of genes in A. ecalcarata petals suggests that, across the organ, a larger proportion of cells have blade identity and have transitioned to expansion and differentiation. This pattern is likely to change over development as a greater proportion of the spurred petal begins to differentiate, including into cell types that are not present in the blade, such as trichomes and cells associated with the complex nectary. Along these lines, it was surprising to find that STY homologs were not strongly differentially expressed between A. ecalcarata and the spurred taxa, given the absence of nectaries in A. ecalcarata. This may reflect the fact that in situ expression studies have revealed early expression of STY homologs at the distal tip of developing petals, which is consistent with the more deeply conserved role for STY homologs in controlling auxin homeostasis in lateral organs. It is likely that this distal expression domain is also present in A. ecalcarata during the early stages sampled here, making the detection of differential expression in whole petals more difficult than in the previous study where dissected blade and spur tissues were compared at later stages. Even so, there is a discernible trend of increasing AqSTY expression across time in the spurred species relative to A. ecalcarata. While a relatively small number of genes were found to be consistently differentially expressed in entire early petals between the three spurred species and A. ecalcarata, an even smaller number of these genes were also found to be differentially expressed between the blade and the spur tissue of A. coerulea ‘Origami’. Only 35 genes showed consistent differential expression in what we term the ‘blade’ and ‘spur’ comparison classes between these studies, with 27 genes falling into the ‘blade’ class and 8 genes in the ‘spur’ class. Key loci in the development of nectar spurs are likely to act by prolonging mitosis in the spur cup, but none of these 35 loci are homologs of genes known to regulate the transition from cell division to differentiation in Arabidopsis by promoting or repressing mitosis. This list of 35 loci also does not contain several genes that are known to be necessary for proper spur formation. For example, the Aquilegia homolog of JAG has been shown to promote cell proliferation in petals as well as other organs. In the current dataset, AqJAG is expressed at somewhat lower levels in A. ecalcarata versus the spurred taxa, but the temporal expression dynamics across the five stages are quite similar .

As more sampling is completed, e.g. baseline surveys and during pilot mining programs, there will be an opportunity to build a database for genetic information that can be linked to specific species. Accomplishing this aim may require a combined approach.Benthic organisms can be classified by size: megafauna , macrofauna , meiofauna , and microbial communities. Megafauna are the only group that can be readily identified with imagery generated by photo or video methods, which can also contribute to our understanding of organism behavior and interaction . On the other end of the spectrum, microbial biodiversity is identified via molecular techniques such as sequencing . Macro- and meiofauna fall somewhere in the middle, amenable to both approaches but perhaps both are not always simultaneously appropriate. Macrofauna are often used as environmental indicators , and there is work to develop similar deep-sea measures using meiofauna where they dominate the biological community . It has been shown that MBT and metabarcoding can perform equally as well for biomonitoring of freshwater systems and assessment of marine aquaculture impacts .

In the deep sea,vertical aeroponic tower garden the limited taxonomic expertise on deep-sea meiofauna , may make the latter approach more appropriate for rapid assessment of biodiversity and environmental impacts.Molecular methods are better suited for identifying rare and cryptic species than MBT. Because of their low abundance and patchy distribution, missing rare species because of under sampling is often a problem, especially in the abyssal deep sea. Additionally, small, soft-bodied protists often disappear in fixed sediment and only show up in eDNA . Although rare species do not have significant biomass, they can contribute unique functions , and new species are discovered frequently with few representative individuals . Choosing clustering cutoffs for OTUs can affect results, losing rare species if too conservative and retaining technical artefacts if too lenient . Cryptic species usually cannot be identified via morphology and, therefore, require molecular methods to distinguish them. They have been documented in deep-sea taxa, such as polychaetes and gastropods , and can significantly impact estimates of species richness .MBT can provide demographic information and material that allow for examination of life histories, lifestyles, and functional attributes. On a practical level, sequencing technology cannot yet reveal these kinds of data. Whether demographic information is relevant to mining environmental requirements depends on the goals and objectives set by the ISA . Metrics such as density, dispersal, size structure, sex ratio, and physiological responses to environmental changes are often used to assess ecosystem health .

Metabarcoding is sufficient to estimate relative abundance, dispersal, and community changes , but additional techniques are required for the remaining metrics . However, capacity to interpret molecular data continues to increase, such as experimental calibration to calculate abundance data , or computational techniques to identify differentially abundant taxa and community shifts .In order to establish identification of deep-sea species and their ecological roles, it is likely MBT and molecular approaches are needed in combination. Metabarcoding yields sequences of genes that are clustered into OTUs which can serve as a proxy for species. However, in order to assign an OTU to a species, that species must have been morphologically identified and sequenced previously, or belong to a taxonomic clade well-supported in a robust phylogeny . Once described morphologically and genetically, metabarcoding alone can then be used to identify it. However, a molecular approach may greatly benefit from the improvement of MBT-based analyses, such as collating phylogenetic histories with environmental data . The former is sufficient if species diversity is the only interest. However, ISA draft exploitation regulation calls for the application of “an ecosystem approach” which implies information on functional diversity and ecological interactions are also necessary .There could be scenarios in which rapid results are required in order to respond appropriately to environmental impacts, such as when an environmental threshold is surpassed or a trigger is set off indicating serious harm to the marine environment . An ecological threshold is a “tipping point” at which the system undergoes rapid and irreversible change that triggers an action .

As an example, a possible threshold to cessation of mining may be the loss of a specified amount of local biodiversity , implied by current mining guidelines for environmental management and monitoring plans , ISA, 2017; Annex VII , ISA, 2019. In a scenario using MBT, results can be obtained only after months of work, whereas metabarcoding results can be obtained within days after return to shore. This difference in response time may have significant impacts on the amount of environmental damage that ensues and, ultimately, the ability for an ecosystem to recover. The amount of time required to develop the expertise necessary for either approach differs greatly. Depending on taxon, it can take multiple years for a taxonomist to become accurate and efficient at species identification using MBT. In the deep sea, this expertise is especially lacking, particularly for small taxa, which can overwhelm experts’ workloads and increase turnaround time. Parataxonomy, which is the delegation of taks in an MBT workflow to non-experts, can alleviate some pressure on taxonomic experts. Although parataxonomy does not yield reproducible results, it can be useful when rapid biodiversity assessments are necessary . In contrast, popularity of molecular approaches has caused the proliferation of sequencing facilities, both academic and commercial, for turnaround time of approximately 2- 3 weeks from sample submission to data return. The more limited training required to process sequencing samples makes molecular approaches more accessible, and the automation that is actively being developed will further reduce processing times. Additionally, loss of taxonomic expertise over time can be mediated by genetic information which can be used as an immutable characteristic for taxonomic identification.One of the biggest challenges facing both MBT and molecular approaches is developing an adequate sampling design. There are still many gaps in our knowledge of deep-sea biodiversity , but it is unclear at what spatial and temporal scales scientists need to sample in order to accurately characterize it . Spatial heterogeneity is poorly defined and likely differs among habitat and location. In the CCZ, there can be unexpectedly high spatial heterogeneity . Additionally, adequate temporal resolution of sampling for accurate characterization of deep-sea habitats is unknown . MBT and metabarcoding often provide one snapshot in time and, given time constraints associated with MBT, a comprehensive monitoring program may incorporate both methodologies by employing metabarcoding more frequently and MBT at longer intervals. The study of deep-sea habitats is plagued by a lack of data, creating challenges that are exacerbated by the sheer vastness of the deep sea. The CCZ claim areas span almost 4500 km wide,vertical gardening in greenhouse larger than the continental U.S., which leads to high levels of uncertainty because of data limitation. A precautionary approach, wherein adequate measures are taken in order to manage risk, should be invoked . Therefore, collection of robust baseline data is critical for protection of the marine environment , as well as exhaustive monitoring, especially at the early stages of commercial exploitation . MBT and molecular approaches can be utilized together in these programs to facilitate future environmental assessment and monitoring.There are direct and indirect costs involved in collecting and processing sediment samples for MBT and metabarcoding. Here, they are separated into costs associated with consumables and labor . Other costs, such as laboratory equipment and bio-informatics pipelines, are held fixed. Choices within the decision networks are discussed in terms of how they affect the short-run economic cost. We assume that laboratories have basic amenities and access to people who are trained to do these tasks, i.e. identifying target taxa, preparing sediment samples for sequencing. Deep-sea samples, whether sediment, water, or individual organisms, are relatively expensive to collect because they often require research expeditions on global-class vessels equipped with specialized instruments.

One ship-day on a U.S. vessel can range from $42,000- 48,000 USD, which includes food, fuel, crew, and two technicians . The amount of time it would take to sail from the Scripps Institution of Oceanography pier to the eastern edge of the CCZ is approximately six days at full speed without stopping, resulting in over a quarter million dollars in transit time one-way. This cost estimate does not include the use of autonomous underwater vehicles , remotely-operated vehicles , or human-occupied vehicles , commonly used on deep-sea expeditions. In addition to collecting sediment samples for both MBT and sequencing, deep submergence vehicles can conduct visual surveys. The U.S. National Deep Submergence Facility operates their instruments at day-rates of $14,000 for AUV Sentry, $23,000 for ROV Jason, and $45,000 for HOV Alvin . Other sampling equipment includes box corers, multi-corers, epibenthic sleds, and CTD rosettes which can be deployed without special vehicles. Relevant results and sampling regimes from studies included in this paper are summarized in Table 3.2. Sediment samples were taken with multi-corers, box corers, or an epibenthic sled in one case, and the same sampling devices can be used for both approaches. The total number of samples used for analysis ranged from 5-41 and 15-42 per cruise for MBT and metabarcoding, respectively. Here, we assume number of samples equates to number of vertical fractions . Sampling costs are likely the biggest element but should not contribute significantly to cost differences between the two analytical approaches. However, the different methods used to process deep-sea sediment samples are a major factor.Consumables required for MBT are common to most labs and are readily available .Formalin is cheaper, easier to ship , and fixation results in little distortion of morphology. However, specimens fixed in ethanol can later be used for molecular studies which is advantageous and cannot be done with specimens fixed in formalin. The total cost of lab consumables for MBT ranges from $13.10-16.29 USD. Labor and expert time are the dominant costs of the MBT workflow . Based on a graduate student stipend in the U.S. of $29,500 USD annually, time alone ranges from $325-2,875 per sediment core slice. Additionally, the estimates shown here are only for the taxa listed for one vertical fraction of a sediment core although the top several centimeters are likely necessary for biodiversity characterization. The extensive time needed to manually sort and identify organisms, especially when using smaller sieve sizes that retain more sediment, is a large cost and a disadvantage of using MBT. Ludoxâ flotation, a method of concentrating animals in a liquid medium, can reduce picking and sorting times but adds additional consumable costs. The estimates we have provided are based on hand-sorting. There are many options when processing marine sediment for metabarcoding , influencing scientific outcomes and economic costs. A metabarcoding approach can also be used in parallel with other omics methods if relevant to objectives . The number of replicates in a study can refer to either sample replicates or PCR replicates. If there is enough sediment material in each sample, then pseudo-replicates can be added. PCR replicates are more often used and can help minimize errors due to PCR, such as unequal amplification and chimeras . Additional replicates yield more robust results and higher statistical power, but increase total cost . Sampling replicates and pseudoreplicates can also apply to MBT. Metabarcoding utilizes highly conserved fragments of DNA that all organisms have and within these fragments are markers, which are highly variable regions that allow for higher resolution taxonomic classification. Choosing markers, how many and which ones, can affect results and will depend on scientific objectives. 16S rRNA can be used to assess microbial diversity whereas eukaryote diversity is typically assessed with markers within the 18S rRNA or cytochrome oxidase I genes . More comprehensive diversity assessments may require multiple markers in order to completely capture the community. Advantages to working with widely-used markers include a more optimized protocol, the ability to compare to other studies, and a larger database. However, there is no truly universal marker that allows the retrieval of all taxa without bias. Additionally, a universal marker must be very conserved and therefore less useful for species-level taxonomic assignments. Development of markers targeting indicator taxa allows for better detection of them . These bioindicators can then be used for characterizing specific habitats, such as sediment plumes, and early detection of mining impacts .

Assessing the central nutritional role of indigenous agriculture alongside that of hunting and gathering might well suggest that malnutrition, rather than simply the spread of diseases through concentrated settlement, made communities less likely to recover from infectious epidemics. According to Snow, had “European expansion been less rapid, and had lethal epidemics not swept the landscape clear of Indian resistance as effectively as they did, the dynamics of historic cultural adaption” on the Great Plains and at the previous sites of European contact might have been different. But it is worth students and researchers asking a slightly different set of questions: Did the inability to reproduce horticultural and hunter-gathering methods actively contribute to Native American demographic decline following epidemics, rather than simply demonstrating another unfortunate result of the Biological Exchange? Is it possible to draw any stronger conclusions in regard to direct causation, rather than more general association, in assessing the dual disruption to hunter-gathering and horticultural practices? Did the change in Native American diets following European contact directly impact the attendant increase in mortality rates – as distinct from epidemics elsewhere in the world where demography was able to re-stabilize after around a century?

In order to consider such a possibility,plastic pots 30 liters students and researchers should turn to the literature on medical anthropology, paleo-archaeology, and modern experimental data on the link between health, immunity, and the consumption of vitamins and important minerals. Disrupted access to macro-nutrients and micro-nutrients – whether derived from hunted and gathered animals and plants, from indigenous agricultural practices, or a combination of both – might be defined as a co-factor alongside specifically pre-determined genetic loci and/or the biological exchange of diseases. Infectious diseases began to ravage Native American populations from the moment of first contact. But an inability to recover immunity and/or fertility, according to a working hypothesis, might have been exacerbated by declining access to ancestral sources of food. To assess these possibilities in greater detail, and how they might be understood by researchers and students, let us turn to more specific examples that relate to the hunting of animal products and the gathering and/or managed cultivation of vegetables, fruits, tubers, and seeds. They allow a potentially fruitful synthesis between early-contact history and modern research in evolutionary medicine and nutrition. Emphasizing the role of colonial human intervention rather than an amorphous biological exchange, modern historical scholarship provides further evidence of the problematic impact of European domestic agriculture on Native American health and fertility after contact.

Aside from its greater propensity to affect the spread of infectious diseases, we now know that the proliferation of small enclosures of cattle and grain physically disrupted hunting and gathering practices, as well as pre-contact forms of plant horticulture, and reduced their attendant nutritional gains. According to biological anthropologist Clark Spencer Larsen, the emphasis on disease in the biological exchange thesis “has overshadowed a host of other important consequences of contact such as population relocation, forced labor, dietary change, and other areas.”Meister similarly notes that “later population decline resulting from disease was made possible because Indians had been driven from their land and robbed of their other resources [including hunted animals and cultivated crops].”According to Anderson, “before long, the expansion of livestock-based agriculture ceased being a model for Indian improvement and instead served almost exclusively as a pretext for conquest, a very different expression of the cultural impact of distinct farming practices” among Europeans and Native Americans in eastern North America from the 1600s.As Kunitz has pointed out in a discussion of the paleoepidemiology of southwestern Native American communities, and their malnutrition following subsequent European colonization, “one does not need to invoke large-scale dramatic epidemics; prosaic entities like malnutrition… are more than sufficient to do the job [in demographic collapse].”Though it remains difficult to address the direct triggers for final mortality, Thornton has assessed much evidence on the history of Plains Native Americans in the two centuries after contact and concluded that their “mortality and fertility” was severely impacted “when the great herds of buffalo were destroyed” by European agricultural patterns, Native American over-hunting in response to curtailed nutritional sources elsewhere, and open warfare.

Other scholars concur that an association can be drawn between worsening health, the declining ability to hunt animals and/or cultivate plants, and a new reliance on European agricultural production as animals on the Great Plains came to be over-hunted.Before turning to cultivated plants, let us now consider how students and future researchers might understand the ways in which declining access to hunted animal products reduced the consumption of important minerals and fat-soluble vitamins, and affected overall health, immunity, and fertility. We would begin with a historical assessment of the role of hunted animals in Native American history. Particularly during the winter, many Native American communities relied on hunting and gathering fatty cuts of meat for optimal nutritional health, long after Neolithic-era Europeans had moved towards domesticated animal husbandry and grain production. Frison’s classic work on the pre-historic practices of the High Plains, Great Plains, and Rocky Mountain regions, for example, suggests that nutritious organs and fats from buffalo meat were favored over and above other portions.Having considered the importance of hunting in ancient populations, students might then turn to seventeenth-century and eighteenth-century European observations of indigenous communities in the northernmost parts of continental North America. Hunting and gathering patterns in those regions were less altered by colonization, and so provide a more accurate picture of their ancestral nutritional profile within their regional context. Their populations relied on fats from fish and land animals as a greater proportion of their diet due to their climate and ecology.Students could then move on to observational studies from pioneering ancestral health theorists such as Weston A. Price, as well as those from more recent anthropologists who have worked in regions such as British Columbia and sub-Arctic Canada. As Hearne noted elsewhere, community members tended to select only the fattiest parts of the animal, or nutrient-dense organ meats, throwing the rest away: “On the twenty-second of July we met several strangers,round plastic pots whom we joined in pursuit of the caribou, which were at this time so plentiful that we got everyday a sufficient number for our support, and indeed too frequently killed several merely for the tongues, marrow and fat.”Students might then examine oral histories, archival collections, archeological records, interviews, and participant observation of contemporary practices, using methods and materials from ethnographers such as Richard Daly, who has noted the historical preference for fish fats among Delgamuukw indigenous peoples in British Columbia, going back several centuries in communal memory. According to Daly, fat “rendered from salmon heads was prepared in summer, hung in bladder pouches in the rodent-resistant family meat caches, and saved for winter use.” Oils were “prepared from fatty fish and meat such as oolichan, salmon and beaver. Special processes were involved in preparing the heads- drying or boiling them for oil-as well as the eyes, bellies and eggs”. Moreover the “arrival of the oolichan. . . was traditionally announced with the cry, ‘Hlaa aat’ixshi halimootxw!’ or, ‘Our Saviour has just arrived!’” Ooligan grease was thus a prized gift in feasts and between neighbors.Surveying modern communities of Native Americans, Weston A. Price’s 1939 Nutrition and Physical Degeneration noted a similar preference for animal and fish fats, and organ meats, and suggested its provenance in ancestral food patterns that dated back centuries and even millennia.

The indigenous communities Price encountered were seen to prize the fattiest parts of meat and fish, including organ meats, rather than muscle-cuts. As Fallon Morell and Enig have summarized, Price linked a diet high in fats from mammals and fish to “an almost complete absence of tooth decay and dental deformities among native Americans who lived as their ancestors did… [including among] the nomadic tribes living in the far northern territories of British Columbia and the Yukon, as well as the wary inhabitants of the Florida Everglades, who were finally coaxed into allowing him to take photographs… Skeletal remains of the Indians of Vancouver that Price studied were similar, showing a virtual absence of tooth decay, arthritis and any other kind of bone deformity…”Ironically, in order to move beyond conjecture when discussing declining health markers after European contact, paleo-anthropological and paleo-archaeological evidence for the precontact Native American consumption of maize provides students and researchers with a helpful framework. Evidence for diminishing health following the introduction of maize raises the hypothesis that consumption of the grain came at the expense of more nutrient-dense calorie sources from animals and other plant species. Familiarity with such a hypothesis should help students and researchers to examine a similar – albeit greatly increased – association between the intervention of colonial European agriculture and the failure of Native Americans to recover demographically from infectious diseases in the three centuries after contact . In eastern North America from A.D. 800 to 1100, a shift towards maize-centered agriculture took place in regions as far afield as the Southwest and the Eastern seaboard. Hunted and gathered meats, as well as nutrient-dense crops such as gourds, seeds, and tubers, declined as a proportion of overall calorific consumption. Across the South Atlantic and Gulf Coastal plains, the shift towards maize cultivation as a vital source of calories was associated with the development of “socially ranked societies” and “fortified civic ceremonial centers” placed near maize storage centers. Corn came to be central to the activities of Iroquois confederations stretching from the east coast to the Ohio River valley, as well as in the Mississippian chiefdoms that grew along the river-ways of the Southeast and Midwest.

On the one hand, the cost-to-yield ratio of pre-contact maize cultivation may have become seemingly more attractive because of an increased demographic pressure on both wild and domesticated resources. That is to say, increased mortality due to food scarcity from hunted and gathered sources might have been diminished by the calories provided by maize. Yet on the other hand, there is a growing scholarly consensus that health and immunity decreased following the indigenous production of maize among North American communities around 2000 years ago, particularly in the American southwest. It may well have provided a source of energy to keep Native American communities alive. But those communities, when compared to populations who had preceded the demographic pressure placed on hunter-gathering, seemed to register declining health markers. That which saved them from death through famine was not necessarily nutritionally optimal as a dominant calorie source. To be sure, we can find historical records from the contact and pre-contact era that suggest several indigenous Native American methods to increase the nutritional profile of corn, some of which may have been introduced in pre-historic communities as they shifted towards the grain. These include soaking it in lye made from wood ashes, in order to make it more easily ground. Doing do also likely made the protein and niacin in corn more bio-available, due to the alkalinizing effect of the wood ashes/slaked lime solution. Greater absorption of Niacin is associated with greater bio-availability of important minerals such as Calcium and Potassium. Nonetheless, the production of “hominy” from corn in this way may not always have been enough to counteract potential nutritional deficiencies associated with the shift toward the grain.In order to understand the distinction between famine-prevention and optimal nutritional health, students and researchers would be able to consider a number of different case studies. From the Great Lakes region to the southern plains, and from eastern coastal to Pacific populations, for example, the regular consumption of bison, deer, and fish likely prevented protein and iron deficiencies that later accompanied the reliance on maize for a greater proportion of the daily calorie output of Native American communities.Analysis of bones from prehistoric North America reveals that, long before European contact, potentially problematic indicators followed the move towards maize consumption. Several studies have focused on growth retardation and suggested its negative association with health markers more generally. Declining stature has been correlated with the onset of agricultural production of maize during the first centuries of the Common Era. Archaeological evidence from late prehistoric Dickson Mounds populations in west-central Illinois show that agricultural intensification led to a decline in skeletal weight and height. In the Lower Illinois Valley children under six in nascent maize producing societies have been found to have suffered growth retardation compared to hunter-gathering communities from nearby excavations.

Soil pathogen control with 1,3-D followed by metam sodium and 1,3-D with intermittent water seals was inconsistent between the two experiments, which suggests that specific micro- and macro-level differences in environmental and field conditions may contribute to greater treatment variability and risk to growers.When 1,3-D was sealed with HDPE and VIF, broad leaf weed density was reduced to less than 6 weeds per square meter, which was comparable to methyl bromide . These results are similar to a previous nursery study that indicated 1,3-D or 1,3-D plus chloropicrin sealed with HDPE or VIF resulted in weed seed viability and hand-weeding time comparable to methyl bromide . Generally, intermittent water seals after a 1,3-D application resulted in broad leaf weed density similar to the untreated control. Most weeds germinate near the soil surface,microgreen fodder system thus techniques such as intermittent water seals that limit upward fumigant movement into surface soils can adversely affect weed control.

The other surface treatments 1,3-D dual application and 1,3-D followed by metam sodium had intermediate broad leaf weed densities compared to untreated plots and methyl bromide. All fumigation treatments reduced grass weed populations compared to the control plots; however, the greatest reductions were observed in plots treated with methyl bromide, 1,3-D sealed with HDPE or VIF, and 1,3-D followed by metam sodium. It was clear in this study that effective surface treatments can greatly increase weed control with 1,3-D; however, even the best treatments will likely require supplemental weed control to meet grower expectations.Effects of surface seal treatments and 1,3-D soil fumigation on nursery stock vigor and performance in two nursery trials were evaluated in 2007 to 2010 . In the rose nursery trial, all treatments had similar root stock vigor and number of marketable plants except when 1,3-D was followed by metam sodium. During the 2008 growing season, roses grown in plots treated with 1,3-D followed by metam sodium had lower vigor than the other treatments; however, by harvest at the end of the second year, no differences in marketable plants were observed. In the tree nursery trial, tree root stock vigor was reduced in plots treated with 1,3-D followed by metam sodium and1,3-D with intermittent water seals compared with the other fumigation treatments, but root stock caliper at the end of the first growing season did not differ among treatments.

Compared with some other fumigation-dependent industries, perennial fruit and nut nursery stock production systems face a more difficult transition to methyl bromide alternatives . Despite several years of research, the following significant challenges to widespread adoption of alternatives in the perennial crop nursery industry remain: National and international market expectations for nematode-free nursery stock limit nursery stock producers to alternatives with very high nematode efficacy at significant depths in the soil. To meet California nursery certification requirements, producers are required to use approved fumigant treatments or conduct a post production inspection. A failed inspection may result in an essentially nonsalable crop. Most alternative treatment schedules are based on the use of 1,3-D , a fumigant that faces its own serious and evolving regulatory issues in California. No currently available alternative fumigant can be used in California to meet certification requirements in nurseries with fine-textured soil at registered rates. Methyl iodide, the alternative fumigant with performance most similar to methyl bromide, is not currently registered in the United States due to a voluntary withdrawal by the manufacturer. Concerns over control of weeds and fungal and bacterial pathogens in the short and long term may further limit adoption of alternatives with a narrower pest control spectrum. 

Containerized nursery stock production systems are being used in some parts of the industry, but the production costs, market acceptance and long-term viability of this system have not been addressed at the required scale.Adoption of methyl bromide alternatives, where they exist, in the perennial crop nursery industry will ultimately be driven by state and federal regulations and economics. Although it’s heavily regulated, 1,3-D is a viable alternative for growers with coarse-textured soil, but if 1,3-D becomes more difficult to use due to shortages or increasingly stringent regulations, it may be only a short-term solution. No viable fumigant alternatives exist for California nurseries with fine-textured soil, and some of them may be unable to produce certified nursery stock in the absence of methyl bromide. The cost of producing perennial nursery stock using more expensive, laborious or economically risky production methods will ultimately be passed on to customers and could have long-term impacts on the nursery, orchard, vineyard and ornamental industries.Citrus fruit is very important to the Indonesian farmer, since it can give him more income than other crops. According to some surveys , he can get at least five to six times as much income from a 5-6- year-old citrus planting as from groundnut and four to five times as much as from rice cultivated on equal acreages. Because of good yields and prices for certain mandarin varieties, more and more acreage is being planted. Although no exact statistics are available, it is estimated that in West Java alone, at least 10,000 ha are planted with citrus . Except in some places, i.e., the southern part of West Java and the northern part of West Borneo, mandarin trees are not planted as special citrus orchards, but are usually mixed together with other crops. Citrus plantings are scattered throughout the larger islands of Sumatra, Java, Madura, Borneo, Sulawesi, Bali, Lombok, etc. There are varieties that can be grown well at lower altitudes, like the Siam, but there are also good varieties that can grow and give good quality fruit at altitudes of 700 to 900 m like the well-known Garut variety. Therefore,barley fodder system the farmer has the opportunity to choose varieties suited for his situation. The gradual, but steady extension of citrus plantings carries with it some problems that are becoming more and more serious. Nearly every citrus grower knows that in Some places the original good quality mandarin trees which were many years old declined and were replaced by small, chlorotic citrus trees with marble-sized fruits. The farmer could not realize that introductions of new stock material from certain other areas could endanger the already established citrus trees, and instead, soil and climatic factors were blamed for the disease situation. The farmers tried to eliminate the symptoms by putting on more manure and fertilizers, and even by spraying the trees with pesticides. Presently, most of the citrus growers know that these efforts are useless. The magnitude of destruction is not small. In West Java alone, it was estimated that from 1960 onward, not less than 3 million trees were destroyed , and we know that this destruction is still taking place. Research on this problem was begun in 1954 and research workers of the Horticultural Research Institute, the Bogor Institute of Agriculture, and the Padjadjaran University have made contributions. Since deficiency symptoms of some macro- and microelements were found on leaves of declining trees nearly everywhere, much was done to correct these symptoms by applying fertilizers containing these elements to the soil or by foliar sprays. Reitsma et al.were optimistic that some carbamates such as Zn-dithiocarbamate, Fe-dithiocarbamate, and Mn-dithiocarbamate could remedy these respective deficiencies.

Soil applications of NPK fertilization trials together with microelements were made by some other workers at the Bogor Institute of Agriculture. Some of these trials yielded a more-or-less positive result, but generally it was temporary. After the applications had ceased for some months, the deficiency symptoms reappeared as before. The idea that there was not only a shortage of some elements, but that there could be other factors causing the deficiency symptoms was suggested by some workers. Reitsma and Hadiwidjaja stated, “Though as a rule poor drainage and hardpan are the main fac- tors which lower the vitality of the trees, giving rise to injury of the rootlets by secondary parasites, malnutrition, on the other hand, resulting from insufficient or inadequate fertilization may indirectly intensify the severity of the disease.” Indeed. this statement was not totally incorrect, i.e., for trees not affected by the disease which will be discussed later. Tylenchulus semipenetrans. Cobb, a citrus nematode that causes some problems in other citrus regions of the world, was found by Thrower on citrus roots in several places in Java. Also he found Xiphinema sp. He applied Nemagon to correct the general vigor of the trees, but no positive reAsults were obtained. Inoculation trials with T. semipenetrans also failed to produce chlorotic symptoms on the inoculated seedlings, although the nematode was established on the roots . It was Terra who first expressed the opinion that the probable cause of the bad vigor of citrus trees in general, and the decline of some varieties on sour orange, in particular, was tristeza virus. Inoculation trials done by Thrower on lime seedlings indeed proved that tristeza virus was present in the declining mandarin trees tested. For the moment, it seemed certain that tristeza was to blame for the destruction of so many trees. However, if one considers the problem more closely, symptoms manifested on indicator plants such as vein clearing, vein flecking, and stem pitting merely indicate the presence of tristeza, and these symptoms are by no means generally found on declining mandarin or other citrus trees, except perhaps on limes. So these results did not yet prove that the decline was caused bv tristeza. Proof that the decline was caused by a virus or a complex of viruses required that certain symptoms generally present on declining trees could be reproduced constantly on inoculated seedlings of the same variety. Tirtawidjaja showed that certain symptoms could be reproduced consistently on graft-inoculated seedlings of several species and varieties, including the variety of mandarin declining in the field. At that time, there was no knowledge about mycoplasmas causing plant disease, so it was concluded that the cause of the constant and persistent symptoms was a virus or a complex of viruses . Owing to the absence of similarities with symptoms and other properties of known virus diseases at that time, the authors concluded that this was a new virus disease. It was named “Citrus vein phloem degeneration disease,” for the most specific symptom, the collapse of certain cells of the phloem in leaf veins. The phloem of the mature, yellow or chlorotic leaves is much thicker than normal . Sieve tubes and companion cells are collapsed, and form white bands extending from the sclerenchyma to the xylem. Ray cells remain intact, but are filled with abnormal quantities of starch granules.The external and the internal symptoms were used for diagnosis of CVPD,first in Java and later in other islands of western and central Indonesia. In 1964, the disease was found widespread in several places in west Java and was also found in central and southern parts of central Java and in certain areas of east Java . Further surveys in recent years revealed that CVPD is not confined to the island of Java, but is also present in several places in Sumatra. Nearly all provinces of the island have citrus areas with declining trees which show the characteristic symptoms. On the other hand, no indication of the disease was encountered in the citrus areas of west Borneo, south Borneo, Sulawesi , Madura, Lombok, and others, except on limited numbers of trees in some yards of Pontianak and Ujungpandang . So it is Sumatra and Java that suffer the most from CVPD. This is probably due to the fact that Pasarminggu was and still is functioning as a center of distribution for citrus nursery stock, and Pasarminggu is blamed for the distribution of CVPD in Java and Sumatra. A certain mandarin variety was once known only in Pasarminggu, but now it is widely grown throughout Java and Sumatra. The spread of CVPD was further enhanced by the acquired ability of nurserymen to produce budlings. In certain places, which are CVPDendemic areas, hundreds of thousands of mandarin budlings are being produced, sold and distributed to new and old citrus centers. The Garut area is the most important source of CVPD-affected nursery stock. With past experience that one could obtain more income from citrus than from other crops, but without the new knowledge that CVPD can wipe out whole areas of citrus, this nursery material is being used extensively, especially in Java and Sumatra.

Jasmonic acid and ET are known to be involved in defense responses against necrotrophs, such as mediating the host’s responses against them, but ET is also required for fruit ripening and senescence processes, which are conducive to disease susceptibility. Jasmonic acid can also mediate the disease resistance of fruit by increasing the fruit antioxidant capacity, but some fungi are able to hijack the JA signaling pathway to cause disease. Although the early steps of JA biosynthesis were highly induced upon M. laxa inoculation, downregulation of receptor genes was observed in mature fruit inoculated with M. laxa when compared to controls. These findings suggest that M. laxa could be somehow blocking the JA signaling pathway, although the mechanisms involved are unknown. Ethylene biosynthesis increases during ripening of climacteric fruit, such as nectarines. In our study, the control immature fruit produced basal ethylene levels, whereas ethylene production in control mature fruit increased through time after harvest. In inoculated immature fruit, there was a significant peak of ET production as compared to the control at 24 hpi. This discrete induction of ET can be part of the fruit defense responses against M. laxa.

Alternatively,dutch bucket for tomatoes the pathogen could be inducing fruit ethylene biosynthesis in immature fruit to accelerate ripening, in an attempt to promote fruit physicochemical changes that are conducive to disease. Along this line, ACS2 and ACO1, involved in system 2 ET production, were over expressed in inoculated immature tissues. Previous studies have reported on a similar modulation of ET biosynthesis by the pathogen. However, after 24 hpi, ethylene levels in inoculated immature fruit fell to control levels, and the fruit remained resistant. This may be in part due to the upregulation of the ethylene signaling inhibitors EBF1/2, which could mitigate the ethylene-induced ripening processes that contribute to susceptibility. In contrast, in inoculated mature fruit, ET production and signal transduction were lower at 6 hpi in inoculated fruit but grater from 24 hpi onward, following the autocatalytic system 2 ethylene biosynthesis. Overall, the results indicate the ability of M. laxa to differentially alter ET production to promote susceptibility and, in turn, the ability for immature fruit, but not mature fruit, to mitigate the consequences of this induction. The above observations indicate that although M. laxa was deploying some strategies to infect the immature tissues, it was not able to overcome either the surface or the active defense responses deployed by the immature fruit. Monilinia laxa remained on the immature tissue, increasing its biomass and multiplying on the surface, until 14 hpi when it ceased to grow.

It is known that Monilinia spp. can remain quiescent on fruit surfaces and that they can employ appressoria as resting structures on immature nectarines. After 14 hpi, M. laxa biomass and reads started to decrease, switching its transcriptional machinery by employing different sets of genes in order to deploy different strategies to survive on the fruit’s surface. Some results point out that M. laxa could either be starting a quiescence period or moving toward an autolysis process, breaking cells to feed on its remains. Another possibility is that the remaining M. laxa cells on immature fruit were being attacked by the host defenses. This is supported by the expression of M. laxa genes associated with response to oxidative stress at late time points, such as catalases, previously reported in detoxification during infection of tomato leaves by B. cinerea. Thus, it is likely that immature fruit was generating reactive oxygen species during the interaction through an oxidative burst to kill the pathogen. Monilinia laxa could also be producing ROS for its development and as a pathogenicity mechanism to damage the host tissue. Particularly, the NADPH oxidase complex is involved in both fungal ROS production and its use in sclerotia development and virulence. Some genes encoding the Nox regulator R  were found to be upregulated at 24 hpi in both mature and immature tissue. At later stages, a highly induced alcohol oxidase expressed in immature tissue at 48 hpi could be another ROS producer, previously described as an alternative ROS production system. Lin et al. demonstrated that AOX1 was involved in pathogenicity and oxygen stress responses in B. cinerea.

Concomitantly, nectarine counteracted the pathogen oxidative burst by expressing genes of antioxidant metabolism compounds such as glutathione and redox-related amino acids . Plant secondary metabolites such as terpenoids have been described to protect the fruit under biotic and abiotic stresses, although their role can be tissue dependent. Overall, the enrichment of genes involved in secondary metabolite biosynthesis was higher in resistant immature than susceptible mature tissue, which suggests that either the host was producing terpenoids in the resistant immature tissue to prevent the attack or that M. laxa was inhibiting its biosynthesis on mature tissue. Monilinia laxa could also be able to degrade and transform terpenoids as described for B. cinerea. The phenylpropanoid metabolism is also triggered in response to brown rot. In both immature and mature fruit, from 14 hpi to 48 hpi, phenylpropanoid-related pathways were highly induced. While on the immature tissue, these pathways could be involved in reinforcing the cell wall through lignin production, the role in the mature fruit could be more focused on the detoxification of fungal ROS production. Nevertheless, these hypotheses need to be further tested. On mature nectarines, M. laxa deployed other virulence factors in addition to ROS production and scavenging. The pathogen expressed upregulated DEGs related to proteolytic activity, containing domains such as the Pro −kumamolisin domain . The list of genes summarized in Table 1 could be putative pathogen target genes as they were expressed only when M. laxa infected the mature tissues, as none of the top five upregulated genes in mature tissue was found in the immature fruit. For instance, the highest expressed protease at in all time points is a homolog of a nonaspartyl protease found during pathogenesis in Sclerotinia sclerotiorum. Cell wall-degrading enzymes are commonly produced by necrotrophic fungi as virulence factors and their secretion by Monilinia spp. on culture media has been previously reported. A rhamnogalacturonan hydrolase ,blueberry grow pot which was highly expressed at both 24 and 48 hpi, was already described as a putative virulence factor in M. laxa infecting peaches. Current information regarding the strategies utilized by either Monilinia spp. or stone fruit or during their interaction is mainly focused on specific metabolic pathways or actions developed by one of the two players. As a novel feature of the present research, we demonstrated the synchronized responses from nectarine and M. laxa, by utilizing a resistant immature and susceptible mature fruit throughout a course of infection. Future research studies should be focused on delving into the host defense system for the ongoing development of nectarine cultivars with increased resistance to brown rot, as well as conducting in-depth fungal studies to alter the ability of M. laxa to cause disease.“Venus” nectarines Schneider were obtained from an organic orchard located in Raïmat . Fruit was bagged 6 weeks before the last harvest and then harvested at two different fruit developmental stages, “mature” and “immature” , and used immediately after harvest.

Injured or deformed fruit was discarded, and fruit for analysis was further homogenized by using a portable DA-Meter , based on the single index of absorbance difference . Other assessments of quality parameters were performed on 20 randomly selected fruit , according to the method of Baró-Montel et al..The M. laxa single-spore strain 8L was used for all experiments. Fungal conidial suspensions were maintained and prepared, as described by Baró-Montel et al..Each fruit was inoculated with the application of six 30- μL drops of a conidial suspension at a concentration of 106 conidia mL−1 on the fruit surface. Mock-inoculated fruits were equally treated with sterile water containing 0.01% Tween-80. Fruit were placed in closed containers with a relative humidity of 97 ± 3% at 20 ± 1 °C.Six cylinders of peel and pulp tissue encompassing the inoculation sites were sampled from each fruit and pooled for each replicate. Samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C until extraction. For symptom analysis, inoculated fruit was imaged at the set time points. Ethylene production of both mock and M. laxa inoculated immature and mature fruit was determined, as described by Baró-Montel.In the high input year round strawberry and vegetable cropping systems of coastal California, organic growers have to balance their desire to reduce environmental impacts with the multiple challenges of maintaining economically sustainable yields. These challenges include providing adequate plant nutrients while minimizing losses to protect water quality, maintaining low levels of disease, and building effective system-based pest management strategies. In comparison with conventionally managed systems, little research on organic systems has been available for growers to help meet these challenges and so in 2004 we formed a research-extension-grower network to develop and implement a collaborative organic research program . The California Collaborative Research and Extension network which we report on here grew out of this initial program. The first 4-5 years of research, known as the Organic Research Network Project, was funded by the USDA-Organic Agriculture Research and Extension Initiative program in 2004 , with the goals of building an organic research and extension network to support organic vegetable and strawberry producers in the region and to develop integrated fertility and pest management strategies to minimize negative impacts of agriculture on surrounding natural ecosystems and improve the economic viability of organic farming. Multiple field experiments were carried out including cover crop/fertility trials , a strawberry rotation trial , studies of the role of trap crops and hedgerows in arthropod biological control , and development of anaerobic soil disinfestation as a technique for controlling soil borne diseases in strawberry production . The current CAL-CORE program represents an extension and expansion of this earlier effort, bringing additional researchers, farmers, regions, and organizations into the network. Our goal is to provide a dynamic platform for network members to collaboratively research and evaluate integrated systems approaches to improve environmental sustainability and economic viability in a comprehensive manner. Using the information generated previously, a series of field experiments have been undertaken to examine the effect of crop rotations and specific management practices on productivity and economics as well as ecosystems services such as nutrient cycling; pest, disease, and weed suppression; soil carbon sequestration; and greenhouse gas emissions. A combination of direct measurement, modeling and Life Cycle Analysis are being used to describe the environmental imprint of the management systems being tested. The centerpiece of the project is a replicated rotation study being conducted at the University of California, Santa Cruz, Center for Agroecology and Sustainable Food Systems farm site which has been under organic management for 40 years. In addition a subset of the treatments in this study were chosen by six farmer collaborators to test on their fields. A number of additional satellite studies are also underway, but here we report on some of the preliminary findings from the main rotation study which is now in its 4th year and reflect on the functioning of the network as a mechanism for collaborative research and outreach. The goals of this study are to compare rotation length and crop composition on soil fertility and soil borne disease dynamics when used alone or in conjunction with soil amendments or anaerobic soil disinfestation. The CASFS field is an Elkhorn sandy loam , and the experiment is a split-split plot design with four replicates. It compares two and four year vegetable/strawberry rotations using combinations of crops believed to be either suppressive of a major soil borne disease , or more profitable but more conducive to disease. Superimposed on the rotations are fertility treatments : legume/cereal cover crop only, legume/cereal cover crop + compost + additional fertility amendments, cereal cover crop + mustard seed meal, or untreated control. In the two legume/cereal cover cropped treatments anaerobic soil disinfestation is used for disease management prior to planting strawberries. The study is in year 4 and all treatments were planted to strawberries in 2014. Winter cover crops in treatments 1a, 2a, 5a, 6a, 1b, 2b, 5b, and 6b were a mixture of bell bean 45%, purple vetch 45%, and cereal rye 10% and were planted at a rate of 367 kg ha-1 in November each fall and incorporated with a spader in April to May. In treatments 2a, 6a, 2b and 6b, compost and organic fertilizer were added pre-plant for each crop.