Specialized equipment used for harvesting distant food resources was left afield where needed . Equipment depots of this kind are discussed below. Of food caches, ceramic ollas hidden away in rock crevices and small shelters would be the most readily recoverable archaeologically and the most readily recognizable as actual caches. We would expect them to be uncommon occurrences at field camps or other locations far distant from residential sites. The woven platform granaries at residential sites would leave little recognizable evidence in the archaeological record. The term “forager” is applied to hunter-gatherers who moved about the landscape identifying exploitable food resources, mapping onto those resources, and seasonally moving the base camp to the place where the resources occurred. Residence lasted as long as the resources held out or untD other resources became available elsewhere. Foraging peoples probably were more keyed into the location and timing of food resources than any other hunter gatherers in prehistory. They knew where, when, and how to get food; had they not, such groups would have gone extinct, or at least gone hungry, with their first major error in subsistence planning. Ethnoarchaeological studies suggest that caching of foodstuffs by foragers was discouraged because of the constant need to move the base camp; it merely exacerbated the already uneven distribution of resources in time and space. Caching in such contexts ensured that the food would be where the people were not,vertical growing towers and someone would have to go back and get it later.

Caching of food would have been favored, however, if the foraging group intended to loop back and reoccupy the base camp for another purpose later in the season . In such cases, stored foods could augment those obtained on a subsequent visit. Concealment, such as in a rock-lined cache pit in a rock shelter, may have been important if the site was expected to be used by other groups during the interim. Such joint use of sites by two or more groups may have occurred in areas where water sources were few. Nomadic hunter-gatherers following a forager strategy occupied most of the California desert region in ethnographic times. These groups included the Panamint Shoshoni , Kawaiisu , and Southern Paiute-Chemehuevi , and they typified a lifeway common to almost the entire desert area for thousands of years. Little specific information is available on the caching behavior of California desert foragers. What information is available suggests that they adopted a collector strategy when resources were abundant and then regularly cached food. They cached autumn resources such as pinenuts collected to sustain the group over winter. At such times, the forager strategy definitely was abandoned. Steward reported that a single family might harvest at least 450 kg. of pine nuts in a good year. Storage on this scale meant that people often were compelled to spend the winter at or near the place of storage. If a decision was made to remain in the mountains where the nuts were gathered, the field camp became the winter village. Often, however, the winter village was located some distance away, preferably at a lower elevation that offered a more favorable climate and other amenities. In such cases, some of the nuts were transported to the village and the remainder were cached where collected. Descriptions of pine nut caches vary according to region and author, and are sketchy at best. Some accounts say the cones or nuts were buried in the ground and covered with grass and rocks; others suggest they were buried but not in a pit; still others suggest they were cached and covered with brush and earth within a circle of rocks .

AH agree the caching occurred outdoors in the pinyon groves, rather than in rock shelters. A thorough and systematic study of such caches has not been accomplished, but they should leave a conspicuous and decipherable record on the landscape. Field gear, such as pinyon hooks and poles, seemingly would have required little maintenance and special care, and would have been of little value for anything but harvesting pine nuts. Pinyon harvesting gear would have been left, usually in trees, where last used. Pointed or chiselended hardwood poles for extracting agave hearts would have been useful for little else, and likely would have been left near where agave was obtained. We have observed large block milling stones left in plain view as site furniture on the surface in patches of wild bunch grass at various locations in southern and western Nevada and eastern California. These milling stones mark the resource patch and also the field camp or work area from which the resource was harvested. Elsewhere in the same region we have seen similar milling stones around ephemeral ponds or lakes, perhaps left there as site furniture to be used when aquatic resources appeared at irregular intervals. Equipment of this nature likely would have been available for use by anyone in the group. The place it was left thus would not have been secret, and the equipment would have been used on subsequent visits to harvest the same resources. Such equipment would have been too heavy to carry away readily and almost indestructible anyway if someone else chanced upon it and used it. Because site furniture often tended to consist of large objects or facilities that remained at the site, anyone who used the place was likely to bring them into service. The situation can be described no more clearly than thus: “Upon arrival at a known site, one generally searches for the ‘furniture’ and pulls it ‘up’ out of its matrix for reuse. This means that large items of site furniture get continuously translated ‘upward’ if a deposit is forming” . A distinction must be made, however, between visible site furniture that is publicly accessible, and concealed site furnishings not intended for use by others.

Highly specialized and maintained personal equipment such as snare or trap bundles of the kind found at Ord Shelter, San Bernardino County, California , many similar examples reported by Janetski from across the American West, and a cache of dead fall triggers from Fortymile Canyon, Nye County, Nevada , are examples of equipment caches. Hidden equipment of this nature would have been retrieved from the cache and put to use only by the person that owned and cached it. The place of concealment would not have been generally known, and such gear would enter the archaeological record upon the death of its owner. A cache of basketry, ceramic, and metal containers was reported by King from Joshua Tree National Monument. This equipment cache was secreted in a small rock shelter. Whether it represents the activities of foragers, who left it for use on a subsequent trip to the same area, or whether it represents a field camp of collectors, cannot be determined on the basis of available information. Whatever the case,container vertical farming the equipment appears to be of too specialized and personal a nature to be considered site furniture. In few or no cases involving equipment of these kinds do we believe specialized caching or storage pits would have been used. Highly cared-for personal equipment would have been hidden away where it was safe; site furniture would have been given only the necessary care to ensure its presence the next time it was needed. Put another way, personal equipment was cached ; site furniture simply was abandoned . The models of hunter-gatherer organizational strategy discussed with reference to aboriginal life in the California deserts were derived largely from observations on remnant hunter gatherer populations in various parts of the world. In no case was the group being studied as residentially stable as those we have characterized as collectors in southeastern California, nor were they part-time horticulturalists. The models are idealized extremes that are of limited value to characterize the actual range of variation in aboriginal settlement and subsistence behavior in the California deserts. They tend to characterize extremes beyond the actual range of variation displayed by any group. Many ethnographic groups that were primarily foragers employed a combination of the two strategies, or switched from one strategy to the other and back again as circumstances changed, even in the course of a single year. Their organizational strategy therefore changed, and in some cases they may have occupied the same site more than once in a given year, each time with different agendas . The models are heuristic devices that we employ to force us to consider the nature of hunter gatherer organization and how it might help us characterize site function. WTien used in that way, they are useful; they simply cannot be taken too seriously or they lose their intended utility. Construction of better theoretical models is necessary for understanding the role and significance of food caches and the full range of food caching behavior in prehistory, and using such insights for anticipating other aspects of the archaeological record. Improved models will come from reanalysis of ethnographic accounts and from additional ethnoarchaeological observations, but the real contribution must come from carefully collected data from prehistoric contexts. AH hunter-gatherers probably manipulated their environments to one degree or another. Horticulture is merely one form of environmental manipulation, and it is an integral part of prehistoric cultural adaptations on parts of the Colorado Plateau, where during early Anasazi times extensive use was made of rock-lined cache pits that apparently were concealed in rock shelters.

Models that attempt to explain caching behavior should therefore not be limited strictly to non-horticultural hunter-gatherers, or they become even less useful for broad application to understanding prehistoric adaptations in the California deserts, the Southwest, and elsewhere. Indian Hill Rock shelter is a large overhang in Anza-Borrego Desert State Park of southeastern California . This region Ues m ethnographic Kamia territory on the eastern slope of the Peninsular Ranges and west of the Imperial Valley just north of the international border. Elevations in the desert range from below sea level to over 1,000 m. To the west, the Peninsular Range rises to nearly 2,000 m. Extensive excavations disclosed that aboriginal use of Indian Hill Rock shelter probably began as early as 5,000 years ago. Fairly continuous but non-intensive use of the site occurred into the historic period. Although ethnographic records for the region document irrigation of crops by the Kamia at nearby Jacumba, higher in the mountains to the southwest , no evidence of horticulture was found in the excavations. AH available evidence recovered from the shelter suggests a non-horticultural, hunter-gatherer adaptation, although it is recognized that the same group that used the shelter may have been involved in horticultural pursuits elsewhere in their seasonal round, at least in very recent times. The archaeological deposit is nearly two meters deep. The artifact assemblage includes abundant manos and metates, projectile points, hammer stones, and debitage. The assemblage suggests use of the shelter by groups engaged in hunting and plant gathering, but industries such as basketry and skin working are poorly represented. In short, the assemblage suggests the activities of task groups, not the residue of daily living. Excavations were conducted both within the shelter and in the exposed area to the front. Rock-lined cache pits or cists were encountered throughout the lower levels of the deposit, but only in the sheltered area behind the dripline. The upper levels ^ contain little evidence of cache pits, but instead contain ceramic sherds. Although radiocarbon analyses are still in progress, we anticipate that the levels containing abundant ceramics span no more than the last 1,000 years. Eleven rock-lined cache pits were exposed in the sandy deposits at Indian Hill Rock shelter. None of these was covered ; all had been opened and emptied of their contents in antiquity . Thus, what remained of many of the cache pits were the rock-lined or paved floors and some portions of the rock-lined walls. For this reason, no complete measurements for diameter, depth, or volume are meaningful. Several other features also may have been cache pits, but were lacking enough structural detail to be certain; these were recorded as “rock clusters.” No evidence of burning was found, so the features were not hearths. Three general methods of construction were noted during excavation: overlapping slabs; slabs placed in a mosaic fashion; and large, irregular rocks and/or milling stone fragments, sometimes chinked with smaller rocks.

The transitory increase in the cellular gallic acid concentration may regulate the expression/activity of UGT that convert gallic acid to the glucoside derivatives . Indeed, gallic acid 3-Oand 4-O-glucosides accumulated in the ugt84a23 ugt84a24 hairy roots . In addition, transcriptome and real-time qPCR analyses identified 11 UGTs with increased expression in ugt84a23 ugt84a24 and one of the candidate UGTs, PgUGT72BD1, exhibited regioselective glucosylation of gallic acid at the 4-OH position . However, none of the candidate UGTs produced gallic acid 3-O-glucoside, suggesting that the gallic acid 3-O-glucosylation activity may be regulated at a level other than transcription.Therefore, even though PgUGT72BD1 is expressed in the wild-type pomegranate roots and hairy roots, gallic acid is mainly used for the biosynthesis of β-glucogallin by PgUGT84A23 and PgUGT84A24 due to their much higher catalytic efficiencies than PgUGT72BD1. Indeed, our previous metabolite profiling analysis did not identify gallic acid 4-Oglucoside in any pomegranate tissues. These results also suggest that the primary role of PgUGT72BD1 in pomegranate roots could be glycosylating aglycones other than gallic acid. Intriguingly, HT production was not completely abolished in ugt84a23 ugt84a24 , suggesting that there could be additional UGT contributing to β-glucogallin formation in pomegranate. Of the 17 so far defined UGT phylogenetic groups in plants,vertical farming in shipping containers PgUGT72BD1 belongs to group E that contains UGT71, UGT72, and UGT88 gene families .

Regioselective glycosylation of hydroxybenzoic acids was previously reported for members of group E UGTs, including AtUGT71B1 that only glycosylates the 4-OH position and AtUGT71C1, AtUGT71C4, and AtUGT72B1 that specifically glycosylate the 3-OH position. Six amino acids are conserved in the hydroxybenzoic acid/ gallic acid 3-O or 4-O UGTs but distinct between the two groups of regioselective UGTs . The function of these amino acids in determining the regioselectivity of the corresponding UGTs can be explored by site-directed mutagenesis and enzyme assays. In addition, once the gallic acid 3-O UGT is cloned in pomegranate, the protein sequences and structural features of the gallic acid 3-O and 4-O UGTs can be compared to identify the key amino acid for regioselectivity. Furthermore, it was proposed that the regioselectivity for hydroxycoumarins was switched among the UGT71, UGT72, and UGT88 families during the evolution of group E UGTs. It will be interesting to understand whether regioselectivity switching event for gallic acid also occurred among these UGT gene families.Haplotype phasing and navigating between allelic and nonallelic variation are the major challenges in assembling genomes of out crossing species with high levels of heterozygosity such as found in members of the genus Juglans. Genome sequencing targeting out crossing plants employed inbred lines, haploids, and megagametophytes to avoid heterozygosity. Interspecific hybrids offer another strategy to avoid heterozygosity. Since the genome of an interspecific hybrid is usually comprised of haploid genomes of the parental species, interspecific hybrids have the same advantages for genome sequencing as haploids, but are usually easy to produce. Technical difficulties with allocating scaffolds to parental genomes have precluded the deployment of hybrids in genome sequencing.

Using an interspecific hybrid between cultivated walnut and its wild relative J. microcarpa, we describe here a novel approach to sequencing hybrid genomes which results ina cost-effective high-quality genome assembly for both parents. The cultivated Persian/English walnut, Juglans regia, is native to Asia whereas J. microcarpa is native to North America, where it occurs in riparian areas in the southwestern USA. Both species are wind-pollinated, highly heterozygous, and intolerant of inbreeding. Their hybrids are infertile. Both have a genome size of about 600 MB with n = 16. English walnut is an important nut crop with 3.8 million tons harvested worldwide in 2017 . Walnut production has been steadily increasing in part due to health benefits derived from including walnuts in the human diet. In the USA, English walnut trees are grown commercially using rootstocks chosen for their ability to tolerate such soilborne pathogens as Phytophthora spp., lesion nematodes, and Agrobacterium tumefaciens, which are all serious pathogens of walnut. The commercial hybrid rootstock J. microcarpa × J. regia possessing tolerance to soil borne diseases is extensively used in walnut production in California. The development of genomic resources for walnut and its wild relatives, including reference-quality genome sequences, will accelerate genetic improvement of walnut scions and rootstocks. Juglans and its relatives in the family Juglandaceae are members of order Fagales, which includes many important forest trees. Reference-quality genome sequences will facilitate comparative genomics and will advance biology of this important group of woody perennials. Recent attempts to sequence the heterozygous genome of English walnut using traditional approaches resulted in assemblies with 4402 scaffolds with N50 = 640 kb and 25,670 scaffolds with N50 = 310 kb.

An attempt to sequence the heterozygous genome of J. microcarpa resulted in an even more fragmented assembly with 329,873 scaffolds with N50 = 136 kb.The novel sequencing approach described here exploits the synergy between long-read sequencing and optical genome mapping. The average length of reads produced with long read platforms exceeds the lengths of a vast majority of plant long-terminal-repeat retrotransposons , which results in a dramatically improved sequence assembly. Contigs or scaffolds assembled from long reads are sufficiently long to be aligned on genome-wide optical maps, which can be assembled with very high accuracy, even for large or polyploid plant genomes. Alignments of the optical maps of a hybrid onto the optical maps of its parents will assign contigs to parental genomes, while also serving as an assembly quality control. We used the assembled J. regia and J. microcarpa genome sequences in conjunction with the Juglandoid wholegenome-duplication, and the Juglandaceae fossil record to calibrate the molecular clock rate for woody perennials. We used the calibrated molecular clock to estimate the time of divergence of Juglans species and other woody perennials. Based on synteny within Juglans genomes, we allocated the 16 Juglans chromosomes produced by the Juglandoid WGD into eight homoeologous chromosome pairs and analyzed their evolution. Finally, we exploited the contiguity of the assemblies in the analyses of the structure and evolution of Juglans telomeres and centromeres and the distribution of disease resistance genes in the J. regia and J. microcarpa genomes.Next, we constructed two optical maps for the hybrid and one for each of its parents . The N50 of the optical contigs ranged from 1.31 to 2.90 Mb. The parental maps consisted of ‘haploid’ regions, in which the haplotypes were similar enough to collapse into a single contig, and ‘diploid’ regions, in which the haplotypes were dissimilar enough to be assembled into separate contigs . The haploid and diploid regions were identified by a map self-alignment . The mean length across the phased regions was 108 Mb in Serr and 33 Mb in 31.01 . Due to the inclusion of phased haplotypes in a map, the sum of the total lengths of the Serr and 31.01 optical maps was 13.4% longer than the length of the MS1-56 optical map . To ascertain whether the genome of our hybrid was complete, we compared the total length of its optical map to the sum of the lengths of the optical maps of the parents,vertical grow racks which we edited to disregard the redundant haplotypes from the phased regions . The total length of the edited maps of Serr and 31.01 differed from the length of the map of MS1-56 by only 1 Mb . We therefore concluded that the hybrid genome was a complete representation of the parental genomes. We aligned the sequence contigs on the optical map of the hybrid, stitched them into scaffolds , and allocated 40 of them comprising 99.85% of the hybrid genome assembly into the parental genomes with the aid of the optical maps of the parents . The remaining 224 scaffolds representing 0.15% of the hybrid genome sequence were too short to be aligned on an optical map and were aligned on Illuminasequence of the J. regia cv Chandler for assigning to parental genomes. Finally, we ordered and oriented scaffolds on high density genetic maps producing 16 pseudomolecules for each of the two genomes. They had only five gaps of unknown lengths ; the remaining gaps were estimated based on the optical map alignments. We then reduced gap lengths or closed them entirely with unassigned contigs or contigs produced with the 10X Genomics technology . We mapped 89X Illumina reads of the hybrid to the genome assemblies and detected 48,717 and 51,789 indels and 748 and 1,205 base substitutions in the Serr and 31.01 assemblies, respectively . We subsequently corrected these errors and produced the fifinal assemblies JrSerr_v1.0 and Jm31.01_v1.0 .Of the genes annotated on the Serr pseudomolecules, 26,403 were collinear with genes on the J. microcarpa pseudomolecules . The two genomes differed by 28 inversions involving > 3 collinear genes, 21 segmental duplications, 3 intra- and 14 inter-chromosomal interstitial translocations, but no terminal translocation . Only 90 of the JrSerr_v1.0 genes were not detected in the Jm31.01_v1.0 assembly.

We computed Ks divergence among the J. regia and J. microcarpa genes to analyze gene duplication and divergence. The Ks plot showed three peaks . The first peak mostly consisted of Ks values between orthologous genes in the two genomes and reflected their divergence. The second peak coincided with the major Ks peak in self-searches within the Serr and 31.01 genomes and reflected the divergence of paralogous genes, which originated by the Juglandoid WGD. The third peak coincided with the major peak in self-searches within the grape genome and reflected divergence between genes duplicated by the whole genome triplication first described in the grape genome. These inferences were confirmed by self-alignment plots , a plot of the JrSerr_v1.0 pseudomolecules against the grape pseudomolecules , and a plot against the Amborella trichopoda scaffolds.We analyzed gene collinearity along homoeologous chromosomes within the Serr genome and detected 38 paracentric inversions involving >3 collinear genes and 20 intra- and 16 inter-chromosomal interstitial translocations. The homoeologues did not differ by any terminal translocations and retained a 1:1 relationship. Using the grape genome as an outgroup, we assigned rearrangements to phylogenetic branches and computed the rates of their accumulation. The rates ranged from 0.4 to 1.4 major rearrangements per MY . The collinearity analysis showed that the 16 J. regia chromosomes can be built from 142 major synteny blocks making up the 19 grape chromosomes. Of them, 122 were shared by the J. regia homoeologous chromosomes, and were in the same order along the them . These rearrangements must have taken place prior to the Juglandoid WGD. We found 43.7% of the J. regia genes collinear with genes on the grape pseudomolecules .In each pair of homoeologous chromosomes within a Juglans genome, one chromosome contained more genes than the other . We denoted the homoeologues with more genes as “dominant” and those with fewer genes as “subdominant,” allocated the 16 Juglans chromosomes into eight homoeologous chromosome pairs, which we arranged in descending order based on the number of genes in the dominant chromosome , and renamed the chromosomes . There were 18,179 and 17,093 genes in the dominant subgenome and 13,107 and 12,304 genes in the subdominant subgenome in JrSerr_v1.0 and Jm31.01_v1.0, respectively . Both dominant and subdominant subgenomes had fewer genes than are in the A. trichopoda genome, which was not subject to a recent WGD . This comparison indicates that both dominant and subdominant Juglans subgenomes have lost genes since the WGD. The difference in gene loss between dominant and subdominant homoeologues was nearly constant among the homoeologous pairs , suggesting that the rate of gene loss was intrinsic to a subgenome. We subdivided homoeologous chromosomes into sections delineated by successive pairs of collinear genes and counted the numbers of singleton genes in the intervening intervals . In the JrSerr_v1.0 pseudomolecules, 13,791 singletons were on the dominant chromosomes but only 8,261 were on the subdominant chromosomes . The numbers of singletons per interval varied little along each chromosome, except for the proximal regions, indicating that gene loss was uniform along the chromosomes and occurred by many deletions involving one or few genes along a chromosome . In each chromosome, deletions were larger and more frequent in the proximal region than the rest of the chromosome, as the numbers of singletons per interval were greatly elevated in proximal regions. We also expressed the numbers of singletons per 2-Mb non-overlapping windows. Most of the windows in subdominant chromosomes contained fewer singletons than those in the dominant homoeologues . Since the divergence of the J. regia and J. microcarpa lineages 8 MYA, 28.7% of singletons were lost from the subdominant chromosomes in J. microcarpa but only 14.9% from the corresponding intervals in the dominant homoeologues . Thus, the factor that caused the asymmetry in fractionation has persisted in the subgenomes since their origin to the recent past. In 22 RNAseq datasets , genes on the dominant chromosomes were on average transcribed significantly more than their paralogues on the subdominant chromosomes .

Of the traditional field crops, cotton and groundnuts are the most labour intensive, but still demand considerably less labour than flue-cured tobacco. From the social perspective, an important advantage of these crops is that cotton and groundnuts both require casual workers to help at harvest time and so provide employment opportunities for workers beyond each farmer’s permanent labour force. Maize, soybeans and wheat, on the other hand, use considerably less labour with few jobs created for casual workers. For LSC farmers, this can be an important advantage since these crops avoid the trouble and expense of hiring outside workers. A comparison of total variable costs for tobacco and other traditional smallholder crops grown in Natural Region II is given in Table 10. As with the LSC sector, these data show that traditional field crops all cost much less to grow than tobacco on a per hectare basis. This is especially important to understanding the decisions smallholders make since only very few farmers have reliable access to seasonal credit or other sources of crop finance. Of the traditional field crops, cotton is generally the most expensive, but special input schemes help alleviate this pressure. It should also be kept in mind that smallholder farmers mostly cultivate less than a full hectare of most crops so that the actual costs and returns from each enterprise will less than the per hectare results shown below. At the national level,mobile vertical grow racks most maize in Zimbabwe is grown by communal and resettlement farmers for whom this is the principal food crop.

Although other food crops are of greater importance outside Natural Region II, virtually every smallholder farmer cultivates maize to some extent or another to meet at least some of their subsistence needs. Compared with LSC farmers, sales to the GMB are more common and most smallholder farmers also plan to earn at least some cash income from maize each year. Smallholder farmers normally account for 55% to 70% of total sales to the GMB. The financial indicators for smallholder maize are summarised in Table 15 for farmers who sell all of their output for cash. Management practices vary considerably in the smallholder sector and the low input level for communal and resettlement farmers is based on growers using recycled seed without fertiliser; all other production levels are for hybrid maize with more intensive use of fertiliser at each successive management level. Compared with LSC farmers, the results for smallholder maize are generally more favourable. Although crop profits are still very low compared with other smallholder enterprises, maize does at least return a positive gross and net profit at each management level. It should be noted, however, that for all models for hybrid maize the costs of production are greater than the estimated gross returns indicating that farmers must have some other source of cash income to sustain this activity. To the extent that tobacco is often grown in rotation with maize on smallholder farms, it is clearly important to include a similar high-value crop as part of the strategy for household and even national food security. Even for smallholders not growing tobacco, the crop can still help finance improved maize production through remittance income sent by family members or from casual employment on LSC tobacco farms. The imputed returns for small holder maize by marketing arrangement are summarised below.

In interpreting these data, it should be kept in mind that the optimal technological choice and marketing arrangement depends on each household’s own consumption requirements and availability of storage space. In these terms, production at the high and medium levels is much more profitable compared with low-input maize because of the additional grain harvested. Nevertheless, because the estimated profits are positive in all cases, the data show that some cash sales can still be justified at each production level to raise money for minor household expenses. Cotton is Zimbabwe’s third most valuable agricultural export after tobacco and horticulture and is a key economic sector in terms of contribution to GDP, employment and export earnings. More than 200 000 smallholder households derive their livelihood directly from cotton, which is well suited to production in more marginal agricultural areas. About 80% of the lint produced each year is exported with the balance used by a small but vibrant textile industry with numerous downstream jobs. Cottonseed is used for crushing and supplies a large portion of Zimbabwe’s vegetable oil and cake used in stock feed. Over the past five years, communal and resettlement farmers have accounted for 80% to 90% of total plantings by area and typically produce 60% to 80% of Zimbabwe’s total crop. The total annual export value for cotton normally ranges from USD 61 to 75 million depending on price and yield. The cotton industry was deregulated in 1995 with the commercialisation of the Cotton Marketing Board.Two other companies have since entered the industry and the resulting competition has benefited all cotton growers in the form of better market related prices, improved efficiencies and better service. Each buyer sets their own prices based on world market conditions and distinguish between four grades of smallholder cotton to encourage good crop management and post-harvest handling.

This system also contributes to Zimbabwe’s very good reputation as a supplier of top quality lint. Compared with most neighbouring countries, Zimbabwe enjoys a very good ginning out turn of about 41% lint per unit of seed cotton. This is mostly because of a highly developed seed breeding and multiplication system that ensures all farmers start with good gene stock. Because lint is the most valuable product of cotton, this allows more favourable prices to be paid to farmers than in countries with a lower GOT. Compared with Zambia for example , producer prices in Zimbabwe are normally 20% higher in USD terms depending on world market conditions. About 98% of all cotton in Zimbabwe is handpicked which further contributes to the good quality and high prices on offer. Per hectare yields are highly dependent on adequate pest control and, like tobacco,vertical garden growing cotton makes intensive use of agro-chemicals and other purchased inputs. To help smallholder farmers afford these costs, each ginning company operates their own input scheme. Group lending, for example, has been used to good effect by The Cotton Company, which only extends credit through associations. Under this scheme, each association member must sign an agreement stating that the entire group will be ineligible for credit next year if just one individual defaults by selling outside the system. One of the new private companies, on the other hand, operates a voucher system in which farmers can choose to accept part of their payment in the form of a voucher exchangeable for inputs later in the year. Although this means that farmers are effectively put in the position of extending credit to the scheme operator, most growers have welcomed this system, both as a form of protection from inflation and as a way to avoid the problem of saving cash until later in the year to buy inputs. Compared with other traditional field crops, the results for LSC cotton are reasonably attractive. Of the traditional field crops, only wheat offers a potential for greater farmer profits. Although far less profitable than tobacco and other high-value crops, cotton is normally grown on large plots of 100 hectares or more and so can represent an important source of income when high yields are achieved. This is not necessarily true with low-input management and irrigated production is more profitable in every case despite higher production costs. As a handpicked crop, cotton is very labour intensive and requires an estimated 100 to 233 days of casual labour per hectare . This is slightly more than the estimated casual labour requirements for LSC tobacco.Compared with all other smallholder crops, the results for cotton are quite favourable and help explain why so many growers give high priority to this enterprise. Although somewhat expensive compared with other alternatives, the input schemes described above help cover many of these costs and cotton is not normally difficult to afford. On the other hand, input support packages do not always cover the costs of producing at the more intensive management levels and farmer profits are considerably greater with high and medium input use than with more typical low input management . To the extent that communal farmers are able to afford more intensive production as a result of remittances sent by family members working on LSC tobacco farms, a shift away from tobacco could impact the cotton sector.

The sensitivity indicators for cotton are among the best compared with all other smallholder crops and show that the good results for this enterprise are extremely robust. Cotton demands a large amount of labour, especially for good weed control and picking. Spraying is normally done with handheld knapsack sprayers. Smallholder farmers produce roughly 95% of Zimbabwe’s total groundnut crop, mainly on very small plots for home consumption. Groundnuts are one of the most important sources of protein in rural diets and are grown by nearly every smallholder farmer to some extent or another. Recently, total plantings by communal and resettlement farmers has varied between 100 000 to 140 000 hectares per annum. Only about 5% of these groundnuts are grown as a cash crop however and total deliveries to the GMB have been less 3 000 metric tons per year for the past three years. Smallholder groundnuts are generally not suitable for export due to problems with mixed and outdated varieties, and also because of problems with aflatoxin. To the extent that groundnuts can be promoted as a cash enterprise, the most urgent challenge is to develop a seed multiplication program to produce the varieties demanded by international buyers. Most LSC farmers grow long-season flamingo nuts as a dryland crop; only about one third of total production is irrigated. These pink skin confectionery nuts have many the characteristics sought by international buyers, but are not normally grown in sufficient quantities to attract European interest. Most trade therefore is with South Africa and Australia and any LSC groundnuts not exported are normally used domestically for the manufacture of peanut butter. An important advantage of groundnuts is that the crop performs well on the same sandy soils suited to tobacco and so makes an ideal rotation crop. Nevertheless, groundnuts are normally grown on small plots on most farms with only about one third of the area used for tobacco given to this activity. The total area planted to LSC groundnuts had fallen to just 1 000 hectares in recent years compared with more than 8 000 hectares annually in the early 1990s. The financial indicators LSC groundnuts are summarised in Table 19. Except with low input management, these data show that irrigated groundnuts are roughly twice as profitable compared with dryland management at each corresponding production level. Compared with other traditional LSC crops, on the other hand, cotton and wheat both offer higher producer profits. Although only about one third of total production is under irrigation, when high yields are achieved, the estimated gross profits from groundnuts equal about 22% of the pre -harvest cash costs for medium input dryland tobacco. In other words, the income from every 4.6 hectares of irrigated groundnuts, has the potential to finance roughly one hectare of flue-cured tobacco. In terms of labour requirements, groundnuts typically use some casual workers to help with harvesting and are more labour intensive compared with all other traditional field crops except cotton. The quantitative results for smallholder groundnuts are summarised in Table 20. These data show that groundnuts are relatively inexpensive for smallholders to grow, but that farmer profits are also very low. Because most households produce groundnuts mainly as a food crop, however, the imputed food security value should also be taken into account and returns would be greater than shown if the measured against the cost of buying groundnuts throughout the year. On the other hand, most farmers only cultivate a very small area of groundnuts and rarely grow the crop over an entire hectare.Importantly, these data show that groundnuts can be an attractive cash enterprise with the potential to generate similar net earnings to cotton. In order for this potential to be realised, however, farmers must have access to improved seed so as to produce a uniform crop with the characteristics international buyers demand.

The life cycle analysis could look at a future steady state after full application, but a more sophisticated analysis would consider expected rates of hardscape replacement and market penetration of new practices over the analysis period. The analysis could include use stage interactions with other systems, vehicles in particular. Initially, a greatly simplifying assumption would be that all hardscape would need to meet the same functional requirements with regard to vehicle interaction. Effects of hardscape on vehicles in cities include vehicle degradation and increased fuel use due to roughness, and increased fuel use due to energy consumption from deformation of the pavement itself . Very few cities manage their pavements based on roughness, and many do not want to manage roughness because of difficulties in handling roughness caused by manholes and other accommodations for under-pavement utilities. Deformation related energy consumption is primarily an issue for asphalt pavement carrying heavy, slow-moving trucks on hot days. Processes associated with those hardscape material flows, both inside and outside the system boundary would be modeled, as well as transportation of the materials, following LCA principles. The flows are the scaling factors for the processes at the urban area scale. Alternative scenarios for changes in the amount and types of materials, structures, changes in the amount of hardscape in different functional types , and changes in the amount of recycling within the urban boundary to reduce material transportation,vertical planters for vegetables and changes in the processes both within and outside the urban boundary to supply urban hardscape could be compared with current practices.

First-level analysis would look at the life cycle material flows, such as materials by type, massdistances of transportation and where applicable, land area needed within the urban area for stockpiling and processing. Second-level analysis would include calculation of important midpoint LCA indicators providing quantification of impacts of changes in flows. Hydrologic modeling of the most intense storm events would be performed to consider the hydrological impacts of changes in urban hardscape to make it more permeable, including partially and fully permeable pavement where they can both be used to also meet other functions. The effects of the changes in material flows and impacts from changing those flows caused by use of this type of hardscape would also be analyzed.In general, the quantification framework developed in this study could help identify where on the curve shown in Figure 5 we stand as of today. Implementation of better practice can cut down imports and flows resulting in lower costs and other environmental impacts.Hardscape covers large portions of urban surface areas, and has potentially large influence on air emissions, truck traffic and its associated problems, and the potential for flooding. Modeling the inflows of hardscape materials and the outflows of demolished hardscape and other rockbased products from buildings and other civil infrastructure is expected to provide a means to find solutions for reducing these flows and their impacts. Modeling of urban hydrology with respect to the effects of hardscape on surface and groundwater flows from precipitation is expected to provide a means to find solutions that will reduce the risk of flooding and improve groundwater recharge. The sustainability of urban areas can potentially be improved through changes in the built infrastructure of urban hardscapes. A conceptual framework does not exist, but is needed, that considers the entire urban area in order to evaluate high-level effects of changes in the design, construction and renovation of urban hardscape.

The original idea for combining UM and LCA to investigate urban hardscapes came from a plenary presentation at the Pavement Life Cycle Assessment Symposium in Davis, CA by Stephanie Pincetl from UCLA . Using Pincetl and her colleagues’ work as a starting point, a system’s approach that considers rock products and hydrological flows was selected for the modeling. An urban area can be considered as a system, with the city or developed urban area limits defining the system boundary as shown in Figure 6. From the standpoint of material and resource flows, this system can hypothetically be optimized if it maximizes reuse of existing resources within the system boundary; i.e., energy and material flows are generated, consumed, and restored within the system. Hardscape materials are primarily sand, gravel and crushed stone, collectively called aggregate, by mass, with small amounts of binding agents such as asphalt binder and Portland Cement, and even smaller amounts of other materials such as recycled tire rubber, other polymers, and chemical additives. A substantial part of most pavement structures is comprised of base and subbase layers consisting of compacted aggregates. For pavement surface materials, aggregates comprise 80 to 85 percent by volume of typical asphalt concrete and 62 to 68 percent by volume of concrete . On a mass basis, aggregate is about 95 percent of the mass of asphalt concrete and 85 percent of the mass of hydraulic cement concrete because the aggregate is much denser than the binding agents. The facts that aggregate makes up the large portion of hardscape materials, compacted aggregate is very dense , and aggregate must typically be hauled to cities from quarries has strong effects on the fuel use for its transportation and on the damage to the roads over which it is hauled.

The environmental impacts of imports of new materials and exports of waste materials into and out of the urban area can be assessed using LCA-type calculations. If material use is reduced through recycling and reuse within urban boundaries, these environmental benefits or impacts of such changes can be quantified by comparison to current conditions. Potential unintended negative consequences of materials reuse include increased land use and environmental problems from storage and processing of reused materials in the urban area, which can also be quantified by UM-LCA. Innovative approaches to hardscape systems that move towards a balance in demand and supply from reuse of material resources will require less material from outside the system boundaries and may require less transportation. Furthermore, the system may potentially produce less waste and pollution in the urban area and will export less waste and/or pollution out of the system. Opportunities to move closer to such a system can be analyzed based on the flows of import and export of resources, waste and emissions within and outside the system for environmental impacts in a life cycle perspective. Changes in hardscape extent and technology that improve permeability so as to capture, detain and infiltrate storm water can be analyzed with respect to flooding, storm water quality and groundwater recharge. Some of the means for increasing the permeability of hardscape include the use of permeable pavement, bio-swales, and catchments,vertical farming technology all of which increase the infiltration of storm water through the surface. All of these approaches also slow the rate of runoff, and where infiltration occurs, reduce total runoff. There are various types of permeable pavement available, all of which include a sub-surface porous layer made with gravel to retain or detain the water.The needs of different urban areas for improvements in flood control, water quality and recharge will differ greatly depending on current and predicted climate change effects on precipitation patterns, their topography, size, drainage systems and land use patterns. Together, the potential changes in hardscape systems to be more permeable and re-usable within an urban system can be analyzed together with the proposed framework.The UM model accounts for the energy and material flows within urban areas and between the urban area and its surroundings, helping researchers to study the interactions of nature and human systems, affecting the environmental impact of a city and effects on human and natural ecosystems. Kennedy et al. defined urban metabolism as “the sum total of the technical and socioeconomic processes that occur in cities resulting in growth, production of energy and elimination of waste” .

Although others have studied the relationship of cities to their surroundings, Wolman in 1965 presented metabolism concept to address air and water quality in the U.S. and determined that increased urban population and industry growth created problems of water and air pollution. Furthermore, Wolman postulates that it is not the depletion of the water resource, but rather its mismanagement, that creates water problems. In subsequent years numerous cities worldwide including Brussels, Cape Town, Hamburg, Hong Kong, Lisbon, York, London, Singapore, Stockholm, Sydney, Tokyo, Toronto, and Vienna have been examined using the UM approach . Kennedy et al. studied data from urban metabolism studies since 1965 for eight metropolitan regions around the world and analyzed four fundamental cycles of energy, materials, water and nutrients. The authors found that water inputs per capita for six studies since the 1990s were higher than the four studies from the early 1970s. Due to the expansion of the cities, water table levels were found to be lower because of increased water demand for some cities such as Beijing and Mexico City while several other cities in the world water tables were found to be rising and water quality being highly affected mainly due to the discharge of wastewater into the ground. An increase in commercial and industrial waste was seen, however, the cities that had implemented recycling strategies at a large scale were producing less waste. The data also air pollution emissions over the same period. The study concluded that the urban metabolism results were different from city to city. Li et al. developed a general framework of an urban ecological infrastructure . Blue land , green land , grey land , exits and arteries build up the UEI system. All the sub-systems are tightly integrated and distributed amongst each other. Researchers have investigated transportation and street networks within the ‘grey land’ category have not been looked at in detail and almost no literature exists to guide roadway and other hardscape design in urban areas in light of responding to climate change effects on precipitation . Different methods such as the emergy or material flow analysis can be used in the UM approach. MFA is the most common and internationally accepted method that can be used to study the flows of resources into a city , followed by processing and consumption of resources, and flows of resources out of the city and emissions production . MFA approaches are typically focused entirely on the flows of materials or substances. However, researchers have also applied the UM approach under the umbrella of the three pillars of sustainability  and developed tools such as the integrated UM analysis tool . Others have suggested studying systems in a life cycle perspective and understanding urban sustainability by coupling UM and LCA, as is done in this white paper . Goldstein et al. applied the UM-LCA framework to five global cities and assessed the environmental impacts directly and indirectly caused by the mass and energy flows through the cities . The author concluded that the results of the study are first gross estimates due to inadequate data. Chester et al. presented evidence that better understanding of sustainable urban systems could be achieved by integrating UM and LCA frameworks, which would help in understanding physical flows and urban infrastructure system problems . A predecessor to this application of the UM-LCA framework to urban hardscape, Chester et al.used LCA to estimate the environmental impacts of building and operating parking spaces. Fraser and Chester used UM-LCA type analyses to estimate the materials and emissions investment in streets and roads in the Los Angeles area over time and the transition from heavy impacts from initial construction to increasing impacts from vehicle use and increasing demands for financial resources for maintenance and rehabilitation . This latter work indicates the importance of looking at urban impacts over a long enough analysis period, per LCA methods, to capture changes in impacts as the hardscape infrastructure both grows, or potentially shrinks if hardscape is converted to buildings or back to green space, and ages. Reyna and Chester evaluated the age and rate of demolition of building stock in Los Angeles . Recycling of concrete from building demotion recycling of all types of existing hardscape are the sources of raw aggregate based materials for new hardscape, the use of which is one of the cases proposed for exploration later in this document. LCA data and analysis approaches for use in an UM-LCA framework have greatly improved over the last ten years.

While the utility of these scores could be improved with faunal densities, they provide preliminary insight about what types of microhabitats and which environmental variables may be important to specific services at specific sites. Methane seeps have been recognized as essential fish habitat and this trait-based approach could provide additional evidence for spatial protections. The focus on ecosystem services provides a targeted effort that can help guide research and management priorities. In the case of the Del Mar seep, Grupe et al. found higher densities of commercially-valuable species at the active seep than in adjacent, background areas. Our results somewhat contrast because we found no significant differences in ecosystem services scores among the active seep and background area during the Del Mar dive. This suggests that the Del Mar area, in general, contributes more to fisheries and carbon services than our other study sites. However, background areas could potentially be influenced by the seep through movement of chemicals and animals from the seep to adjacent areas . One drawback to using deep-sea imagery for trait-based ecosystem services assessment is the need for visual evidence. The traits in Table 4.3 are not exhaustive of characteristics that can contribute to fisheries or carbon services, but they were ascertainable from our dive videos. While deep-sea imagery may not be able to confirm regulating services,vertical farming tower for sale like metatranscriptomics could , it does provide insight on animal behavior that can support ecosystem services. For example, mid-water fish would often be seen near the benthos and swimming into it , which has been previously observed .

This could potentially represent an important benthic-pelagic interaction that contributes to carbon export.Expensive and limited ship time can make it difficult to collect ideal video data. The need to juggle multiple scientific goals during each ROV dive makes it impractical to conduct quantitative visual transects, and to generate sufficient number of replicate transects. This can create downstream constraints for data analysis such as lack of quantitative measures of densities and difficulty standardizing observations. Best practices for collecting pictures and videos from deep-sea sampling instruments could be useful . Random and independent sampling could facilitate data analysis. Replicates could also be useful, in which cabled observatories may be appropriate . ROV and AUV transects should be conducted with consistent altitude, zoom, and speed, as well as with a scale for size . The resulting data can then be used to calculate faunal densities and other diversity metrics . A quantitative transect would also allow for comparison among locations and time periods . Accurate maps of the seafloor before dive operations can help ensure best use of time , but perhaps most important are the designation of detailed scientific goals and objectives prior to surveying.Environmental measurements should be made in association with imagery being taken. Physical and chemical properties, such as temperature, oxygen, and hydrogen sulfide at seeps, are important parameters that help shape the biological communities . Porewater chemistry influences the sediment community , which can contribute to fisheries services and carbon services . Scientific tools exist to assess water chemistry such as in situ mass spectrometers that can be mounted on ROVs and niskin bottles that can be used to sample water at discrete depths. These environmental properties can help explain differences in diversity and distribution, and provide insight on how communities may change with human impact such as climate change .

As imaging technology continues to advance, the resolution of pictures and videos becomes increasingly helpful for post-analysis . Imagery should be analyzed consistently, which may mean cross-referencing protocols and morphotypes if more than one person is conducting the analysis. Human bias is inherent to current image analysis but can be minimized with training . As more deep sea imagery is analyzed and libraries are produced, there are possibilities to incorporate machine learning algorithms in collaboration with computer science and programming . Machine learning techniques could facilitate mining of existing deep-sea imagery data. They often sit in labs untouched, representing an underutilized source of knowledge. These pictures and videos provide an opportunity to generate knowledge for habitats that are rarely visualized and are data-limited. Imagery is also routinely collected for non-biological purposes, such as instrument deployment, which could be an additional source of data.The approach developed in this study can serve as an environmental decision-making tool, such as in the designation of spatial protections, consideration of ecosystem service trade offs, and understanding of context-dependent roles of methane seeps. This analysis can identify areas of high ecosystem services provision, such as the Del Mar seep that had relatively high fisheries and carbon scores, which may be important for designating essential fish habitat or marine-protected areas . An ecosystem-services approach can also investigate trade offs that may need to be considered during the environmental decision-making process . For example, if methane seeps provide differential ecosystem services, one prioritization metric for spatial protections could be weighted ecosystem services scores . Lastly, results from this paper advance our understanding of ecosystem services associated with methane seeps. They highlight the context-dependent role of methane seeps in providing fisheries and carbon services along environmental gradients. For example, while the combination of seepage and low oxygen seemed to suppress ecosystem services scores at Point Dume, the Palos Verdes and Del Mar ecosystem services seemed to benefit from at least some seep activity.

Development and urbanization transform coastal landscapes by replacing vegetation with impermeable surfaces. Subsequent precipitation events can lead to decreased water infiltration, modified water flows, and introduction of contaminants into storm water runoff . As a result, flooding and property damage, increased safety and health risks, and environmental damage can occur . Urban planners and developers have traditionally addressed storm water runoff issues by building drainage systems that connect directly to large bodies of water or treatment plants. The former have been found to further alter hydrology and degrade water quality , while the latter can be energetically and chemically intensive . Natural storm water treatment systems are emerging as an alternative storm water management strategy . NTS are human-made systems that use natural processes to capture and treat storm water runoff. They come in different forms: bio-retention systems , infiltration basins and trenches, permeable pavements, dry wells and ponds, treatment wetlands, and combinations. Table 5.1 provides a summary,hydroponic vertical farm although there are differing opinions on what constitutes an NTS . Additionally, some of the listed systems can also treat wastewater, functioning in similar ways but obtaining water from different sources. This paper focuses primarily on bio-retention systems because they host biological communities as part of their design. Because NTS function as built ecosystems, they can support diverse ecosystem services, defined as direct and indirect benefits humans obtain from ecosystems . Ecosystem services associated with NTS have long been acknowledged , but they have largely been ignored by monitoring programs and economic valuation efforts, which have been limited to targeted water functions. Co-benefits have been described for other green spaces such as offsetting carbon emissions , cooling local temperatures , cultural services , and benefits to human health and well-being . The incorporation of ecosystem services and other co-benefits into environmental decision-making can present urban planners and developers with additional benefits, costs, and trade offs to consider in order to make optimized decisions . We use Los Angeles County as a case study because it experiences periodic water crises, hosts a dense human population near the coast, and has policy in place to encourage the use of NTS. There are spatial and temporal mismatches in the supply and demand for water in California, most precipitation occurring remotely from agricultural and metropolitan hubs that need consistent sources of water irrigated and imported . Additionally, California droughts and water shortages are predicted to increase in frequency and magnitude due to anthropogenic climate change .

As a state, California has passed several propositions to protect water supply and quality . In 2004, Los Angeles passed Proposition O which allowed the city to issue up to $500 million to fund projects that increase local water quality . In 2012, Los Angeles adopted its Low Impact Development Ordinance requiring development and redevelopment projects that alter impervious area to mitigate runoff by capturing precipitation and utilizing natural resources where possible. As a result of these environmental conditions and political momentum, green infrastructure and NTS have been broadly distributed throughout Los Angeles County and continue to be implemented. NTS are designed to capture storm water runoff for infiltration or reuse , and have been shown to be effective . Most systems are oriented vertically, using gravity to direct water flow through several layers that generally consist of a ponding area with vegetation, porous filter media, and a drainage zone . Infiltration rates of a system can vary widely depending on variables such as size , age , filter media , and other design factors . Vegetation also plays a role by intercepting precipitation and water flows , and preventing clogging of filter media to maintain infiltration capacity . Additionally, evapotranspiration by plants can account for 15- 20% of inflow . Urban areas with low permeable surface area can be prone to flooding and changes in groundwater recharge . Los Angeles County lacks infrastructure to handle large volumes of storm water , and flooding can occur when unexpectedly high precipitation occurs. This issue occurs in a state that experiences episodic drought , highlighting the need for proper storm water management and use. Targeted storm water infiltration by NTS can provide ecosystem services that help address these challenges.By altering landscapes and hydrology, urbanization can lead to increased flood risk caused by heavy precipitation and storm surge . NTS have been shown to significantly reduce runoff volume and magnitude of high-flow events by capturing and storing storm water runoff. Hatt et al. found reductions in runoff volume of 33% on average, as well as peak flow reductions of at least 80% in three bio-retention systems in Australia. In another field study, Davis reported mean peak flow reductions of 49% and 58% in two test bio-retention systems, as well as delays in flow peaks which can provide urban managers with time to put mitigation measures in place. Most Los Angeles NTS showed visual indicators of flood control services: permeable surfaces that allow for water infiltration, graded landscaping to help direct runoff, and ponding areas designed to temporarily store water. For example, Elmer Avenue Green Street was constructed specifically to address street flooding during precipitation events by incorporating bioswales, biofilters, permeable pavements, and rain barrels . However, additional information is needed in order to quantify these services. Measurements for flood hazard may be relevant , as well as those for flood vulnerability . Spatial models have also been developed, mapping urban surfaces to estimate infiltration capacity . The value of flood control has been extensively studied in the context of wetlands , generally by assessing differences in property damage along a spectrum of wetland area . Brander et al. conducted a meta-analysis to estimate the value of flood control by wetlands which resulted in a median of $20-30 1995 USD per hectare annually. Watson et al. estimated a net present value of less than $100 USD per hectare of wetland annually. NTS usually operate at smaller spatial scales than major wetland restoration projects so the main unknown here is whether NTS of different designs provide more localized flood control.Groundwater is used by more than half of the population in the U.S. and its recharge is an essential component of the water cycle . Major sources of recharge in urban environments include runoff infiltration, and leakages from the water supply and sewage systems . Urbanization can decrease groundwater recharge due to the installation of impermeable surfaces . However, the importation of large volumes of water to meet demand in highly-populated urban areas also leads to significant leakages and recharge . These changes can also degrade water quality, e.g. leakage from sewage systems, saltwater intrusion due to lowering of the water table . NTS can contribute to groundwater recharge by providing permeable surfaces and pore space in their filter media that allow storm water runoff to pass into the soil subsurface.

The historical record there and elsewhere thus suggests that during summer and early fall starches were consumed in far greater number than during the winter, when protein and fat consumption increased proportionally.By 1600, as another example, Western Apache communities had developed a “seasonal cycle” that veered between food gathering, horticulture, and winter hunts.In July they harvested saguaro fruit in the Gila Valley of modern day Arizona. In July they also began a month-long harvest of acorns. Fall and winter were dominated by hunting for animal meats. Similarly, on the Texas plains prior to European contact, Apache communities spent spring and summer in agricultural villages, moving towards hunting dominated nutrition in the winter.While surveying indigenous communities who had avoided the level of European contact suffered by their southern neighbors, Weston A. Price’s Nutrition and Physical Degeneration suggested that similar practices were maintained during the early twentieth century among communities living inside the Rocky Mountain Range in far northern Canada. Moving further towards speculation, or at least a working hypothesis, it is worth considering whether health declined as Native Americans were forced to consume maize and other starches during the winter months, having lost access to ancestral winter hunting grounds. After all, seasonal consumption of carbohydrates could have improved health by limiting the overall production of insulin in the bloodstream . In assessing such a hypothesis, students would be able to consult much recent research on the evolutionary dimension of obesity and metabolic syndrome. Firstly,stacking flower pot tower it has been suggested that the move towards all-year round abundance of food in many populations may have caused over-consumption at times when the human metabolism may otherwise have benefited from calorie reduction and/or greater insulin sensitivity.

The latter has been linked to reduced carbohydrate and protein access in comparison to fat, often during winter. Secondly, a number of studies have shown a correlation between blood vitamin D levels and insulin sensitivity. Thus, from an evolutionary perspective, it may be postulated that some human populations may be adapted to consume more starch during those months when insulin sensitivity is higher due to raised blood vitamin D levels from available sunshine.What, then, might students make of the statements made by the Spanish colonizer Cabeza de Vaca, who lived with Coahuiltecan communities in what is now part of Texas, for eight years from 1538? During winter they hunted buffalo, deer, and javelin, while during the summer and fall they were sustained by fish, plants, and starches such as the mesquite bean. In what has been described as a “feast or famine economy”, animal-based diets in winter would thus give way to gorging thanks to “the ripening of fruit, or tuna, of the prickly pears [which] typically meant days of feasting until the fruit ran out.” During the winter period of hunting for animal proteins and fats, de Vaca noted the ability of Coahuiltecans to maintain aerobic activity for long periods of time: “The men could run after a deer for an entire day without resting and without apparent fatigue. . . one man near seven feet in stature. . . runs down a buffalo on foot and slays it with his knife or lance, as he runs by its side.”Students might even consider the above, and other similar original source testimonies, in light of modern scientific research on fat-adapted aerobic activity, including the use of ketones as an energy source. The metabolic state of ketosis is defined as the elevation of the ketone bodies D-beta-hydroxybutyrate and acetoacetate in the body in response to the consumption of a diet low in glucose and high in fats, or following long periods of fasting.

A number of recent studies have suggested that the metabolic use of ketones may benefit long periods of medium-intensity movement by reducing the need for regular consumption of carbohydrates. They have focused on the ability of endurance athletes to maintain or perhaps even increase performance whilst consuming a high fat diet .Students and researchers might consider the possibility that some indigenous communities prior to European contact would at least have cycled between periods of ketoadaptation and period of glucose-burning, depending on the season. It has certainly been suggested that communities in the northernmost parts of Canada and Alaska have historically utilized ketogenic or keto-adapted diets, burning fat obtained from meat and fish rather than glucose as a primary fuel for many months of the year .Though much more research is needed to determine the nature of fat burning during physical exercise, a useful hypothesis can be gleaned by noting that many Native Americans undertook long hunts over several hours at just the point in the season when they may have benefited from increased endurance due to their fat-adapted metabolic states and high fat consumption. Any European disruption to winter hunts, according to such a proposition, would also have disrupted ancestral metabolic patterns that incorporated seasonal fat-adaptation, or even seasonal ketosis. Fat-adapted metabolic diets have been found to be therapeutic for a number of medical disorders, particularly but not only neuropathies. Evidence of their potential health-promoting benefits, at least in periodic cycles, might also support the hypothesis that disruption to ancestral metabolic cycles could have prevented other benefits that are as yet unknown, pending further scientific research – thus potentially exacerbating Native American susceptibility to infectious diseases following European contact.

Of course, students would be encouraged to examine confounding evidence to such a hypothesis. In this case, for example, they might consider the historical record for Tarahumara communities in Northwestern Mexico, who ran great distances through the colonial era while eating comparatively few animal meat products. A similar association could be found with historic Apaches and Hopis, both of whom ran for long distances with a diet dominated by maize, squash, and beans. To be sure, it is ambiguous whether, historically, Tarahumaras relied solely on plant carbohydrates and proteins from beans, or whether such an account represents a teleological extrapolation from their diets which were examined during the second half of the twentieth century.But students would certainly be able to note the association between their high starch diet and their high-distance aerobic activities . Yet even here, other analytical indicators might become relevant. Several studies have shown low plasma cholesterol levels for modern Tarahumaras, at least in regard to HDL. But other studies have also shown relatively high levels of cardio-respiratory problems in rural populations who have maintained a high starch diet while decreasing their physical movement. That is to say, the problematic effects of high blood glucose might have been mitigated by intense exercise burning the substance for fuel rather than raising insulin to a sub-optimal level.The Tarahumara case study might even lead students to ask a further set of questions, which speak to recent research by O’Keefe and others on burning glucose during periods of aerobic endurance activity. Compared to long-distance aerobic activity in a fat-adapted state, students might ask, what are the long term consequences of burning glucose for fuel in a high state of oxidative stress, such as long distance running and hunting? Oxidative stress can be defined as the increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as glutathione, in association with intense consumption of oxygen during periods of activity . Even if exercise may have prevented glucose from being stored as fat among Tarahumaras, and lowered inflammatory markers,ebb and flow oxidative damage due to their metabolic state during exercise may have portended problematic cardiovascular health outcomes.Ongoing scientific discussions about optimal nutritional health – as defined in peer-reviewed studies – should help to inform our historical understanding of the contact period between Europeans and Native-Americans, from the sixteenth century through to the nineteenth century. Students should be in a position to question the perception of nomadic hunting as the only indigenous Native American nutritional and ecological practice in the three centuries following European contact. The regular or seasonal consumption of hunted wild animal products was not antithetical to horticultural cultivation. Both were often complementary during the pre-contact era, providing micro-nutrients and macro-nutrients that varied according to the season. Distrust of European agriculture – including its perceived association with the spread of diseases – did not require Native-Americans to conceive of themselves as a people who eschewed ecological cultivation altogether. If any generalizations are to be made, rather, students would be better off examining the ways in which Native American populations perceived colonial farming as a threat to their own land management of crops as well as to their continued hunter-gatherer activities outside indigenous cultivated settlements. We ought to examine the existence of both ecological systems in order to consider their distinction from those that followed the European encounter. During the era of contact, horticulture declined. But although hunting and gathering increased in response to colonial pressure on cultivated plants, its character and regional context changed in order to accompany new European technologies, often deployed in new lands. Those communities who migrated to the Great Plains – whether from the East, the West, or the Northern Great Lakes – eventually suffered from diminishing access to hunted animals, without the potential to mitigate that loss through renewed horticulture sustenance.

The same phenomenon also often occurred among Native American communities who remained at the site of first European contact. As is evident in the tragic history of Native American health and ecology in the three centuries after first contact, then, external interventions in the indigenous food system may well have contributed to heightened susceptibility to infectious diseases and near demographic collapse. The modern scientific literature on nutrition and health should inform our understanding of the negative health outcomes that were associated with diminished access to indigenously cultivated crops and/or hunter-gathered animals and plants. In evaluating declining health after European contact, students and researchers should assess the effects of curtailed hunting and gathering, the threat to pre-contact forms of horticulture, and the colonial misunderstanding of their symbiosis. Such an assessment could be informed by our understanding of the nutritional and ecological changes that accompanied the introduction of infectious diseases: a greater threat from zoonotically spread pathogens; diminished access to fats, fat soluble vitamins, proteins and essential minerals from animal and plant sources; an increasing inability to gather potentially beneficial indigenous starches and Resistant Starch sources; a growing threat to seasonal oscillations between winter higher fat diets and summer starches; and even a decline in cyclical ketosis in some regions. In light of current research on the relationship between nutritional density and immunity, and metabolic syndromes, diminished access to indigenous food sources can be related to the greater vulnerability to infectious diseases, above and beyond the differing historical immunities that distinguished Native Americans from Europeans. Questioning or modifying the Biological Exchange thesis should help students and researchers evaluate an important and ongoing question in public policy: historically, since European colonization efforts, why have top-down political interventions in nutrition so often accompanied a decline in ancestral health principles, health, and fertility? Indeed, such a correlation is suggested by events following the three centuries of European colonization, from the mid-nineteenth century to the present day. They might offer important insights as a concluding section or even a postscript to the proposed course, connecting the colonial period to the contemporary era. In 1867, for example, the Treaty of Medicine Lodge required Southern Plains Native Americans to give up land in return for government annuities. The federal government then began supplying them with food handouts, using the industrial and transportation systems developed to supply troops with grains during the Civil War. The containment of Native Americans in reserves severely limited the physical activity to which their communities had grown accustomed over previous centuries. It has been suggested that their decreased energy expenditure would have made them less likely to burn increasing volumes of glucose in their diet, contributing even further to the development of diabetes and other immune disorders through the twentieth century. Students and researchers would do well to examine the correlation between the Treaty of Medicine Lodge, other similar documents, and declining nutritional health among Native Americans during the post-colonial era. Aleš Hrdlička, a medic and an anthropologist, reported in his 1902 Physiological and Medical Observations among the Indians of Southwestern United States and Northern Mexico that obesity and associated “grave disease[s] of the liver” were “exclusively” found among Pima Indians on new reservations, rather than among those who relied on a more traditional system of hunting meats and gathering or cultivating fibrous seeds, chenopods, plants, and starchy tubers.

The cuticles of tomato and soybean leaves are much thinner, and therefore foliar absorption will be more rapid. Here, our findings indicate that a relatively high percentage of total Zn in the leaves from foliar applications would be bound to the cell wall rather than be present in a soluble, intercellular form. Zn mainly exists in a bound form in the cytoplasm, as well as in other cellular compartments, to avoid uncontrolled Zn2+ binding to nontarget sites. Various studies have also shown that the abundance of negatively charged sites in the cell wall limits the translocation of positively charged Zn2+ . For example, the major component of pectin in cell walls is polygalacturonic acid, which has a high binding capacity for Zn2+.Our previous research also shown that most of the Zn was deposited in the cell walls of apple leaves in coordination with different stages of development, with the nature of Zn binding being dependent on tissue age, as old and mature leaves exhibited higher proportions of Zn in the cell wall than young leaves. In general, there is low potential for remobilization of foliar-absorbed nutrients until the potential binding sites for that element within the leaf are saturated. Therefore, we hypothesized that there would be very limited utilization of Zn applied to the adaxial leaf surface because of the highly hydrophobic cuticular wax layer that would limit Zn penetration,dutch bucket wholesale as well as having a higher capability for cationic Zn fixation.

Previous studies have suggested that there is high diversity among trichome structure and function, which can hinder or promote water penetration. In the present study, we found that a relatively high trichome density resulted in a high degree of hydrophobicity , which was not consistent with the results of several studies that reported that trichomes may participate actively in the absorption of water and foliar-applied nutrient solutions. These differences may be related to the surface roughness provided by the high density, chemical composition, and structure of trichomes on apple leaves. The high density of nonglandular trichomes creates the hydrophobic characteristic of apple abaxial leaf surfaces to a certain degree, and repulsion of fertilizer drops by abaxial leaf trichomes could hinder the penetration of liquid Zn solutions. Our findings were similar to the results of Fernandez et al., who used Holm oak as a model to assess the capability of abaxial surfaces versus adaxial surfaces to absorb surface-deposited water drops. Another study by Li et al. also demonstrated that trichomes are not part of the primary pathway through which foliar-applied Zn moves across the leaf surface, even though Zn was found to accumulate around the base of trichomes. On the other hand, the possible contribution of stomata to the penetration of leaf-applied chemicals has been a matter of controversy for many decades, and it is still not fully understood whether stomata allow the penetration of foliar-applied solutes. Nonetheless, several studies show a clear effect of stomata in promoting foliar solute penetration, even in the absence of surfactants. However, the underlying mechanism is not understood, although it is hypothesized that penetration of solutes through stomata is restricted by their morphological and physical properties. Our study provides further evidence that supports the relevance of stomatal nutrient absorption, as indicated by the increased penetration rates from plant surfaces whose stomata were present and open.

Information about nutrient interactions will guide fertilization practices and optimize the efficiencies of fertilization strategies. The levels of two different elements for each pixel in an XRF data set can often provide important chemical information.Here, we analyzed the ionic changes that occurred in leaves in response to foliar Zn application to reveal the interactions between and homeostasis of Zn and other metals. Induction of element accumulation was observed after foliar Zn penetration for all the tested nutrients . One of the possible reasons for this promotional effect may be the important role of Zn in the constituents of enzymes involved in photosynthesis. The concentration of water-soluble Zn in leaves was found to be closely correlated with the levels of chlorophyll. The positive influence of foliar Zn fertilizer on photosynthesis and chlorophyll synthesis may help increase mineral nutrient absorption and accumulation in functional mature leaves. A reduction in this accumulation was observed for Mn and Fe along with a further increase in Zn. This might be due to the possible Zn toxic reaction in the treated leaves because locally toxic levels of Zn may occur at the point of fertilizer application. Excess Zn may cause uncontrolled displacement of essential cofactor metal cations such as Mn2+ and Fe2+. For example, it was reported that exposure to high Zn can subsequently inhibit PSII activity by replacing Mn in thylakoid membranes. The underlying mechanisms of the above effects remain uncertain, and further research is needed to uncover the complex interaction between foliar Zn status and plant nutrient responses. The increased spatial correlation of Zn versus P along with the penetration of foliar Zn fertilizer , together with the presence of Zn-phytic acid in the treated leaves , suggested that P may play an important role in the complexation of Zn, potentially in response to toxic concentrations of Zn.

The formation of Zn-phytic acid in vegetative tissues was reported to occur under two circumstances: within some hyperaccumulator species or under conditions in which plants are exposed to high exogenous Zn concentrations, the binding of Zn to phytic acid helps plants limit Zn mobility and reduces toxicity. Our results were consistent with the previous findings showing an increased proportion of Zn-phytate in wheat leaves after Zn-EDTA and ZnSO4 applications. Agronomic strategies aim to deliver phyto available Zn via the application of Zn fertilizers to leaves. Knowledge of the ability and mechanism of Zn to penetrate the leaf surface from the site of application and the factors that are associated with penetration efficiency is critical for the development of strategies for Zn bio-fortification of crops. In the present study, the high spatial resolution and direct imaging capability of XRF was valuable for distinguishing differences in Zn penetration of epidermal cells,blueberry grow pot and these techniques provided a powerful strategy for investigating foliar microelement applications to plants with a high level of sensitivity. By the use of micro- and nano-XRF techniques, our results provide direct visual evidence for Zn penetration of the leaf surface. Furthermore, we provide new insights that can help in the development of Zn bio-fortification approaches in fruit crops. To further increase the Zn content in food crops, future studies need to focus on elucidating the pathway by which Zn penetrates plant leaves, the subcellular compart mentation of Zn, and the specific formulation of Zn foliar fertilizers.Educational theorists have recently begun to call for more immersion of trainee scientists and medical practitioners in the humanities, particular through the study of history as part of their educational program. In a widely circulated analysis that first appeared in an August 2014 Inside Higher Education supplement, Elizabeth H. Simmons suggests that “to fully prepare for careers in science, it is essential that students grasp how the impetus for scientific work arises from the world in which the scientist lives, often responds to problems the scientist has personally encountered, and ultimately impacts that society and those problems in its turn.” Every nascent scientist, according to Simmons, “should read, think, and write about how science and society have impacted one another across cultural and temporal context” because “ethical concepts absorbed” in such study will help them “hew more closely to the scientific ideal of seeking the truth.”Since C.P. Snow’s famous 1959 Rede Lecture lamented the gap between the “Two Cultures” of the sciences and humanities, academic initiatives such as Stanford University’s Science, Society, and Technology program have been founded to assert the wider societal impact of the natural sciences.Yet far fewer programs and courses have been designed to show how scientific endeavors might benefit from the study of the humanities, particularly history.

The newest version of the Medical School Admissions Test now encompasses questions on the psychological, social, and biological determinants of behavior to ensure that admitted medical students are “prepared to study the sociocultural and behavioral aspects of health.” But as Simmons notes, while “pre-medical and engineering students are being required to learn about issues linking science and culture, most students in science fields are still not pushed to learn about the human context of their major disciplines.”As Dean of the College of Liberal Arts at Michigan State University, Simmons may well know that her state’s own educational system incorporates a project with important implications for the nexus between the humanities and the sciences. The Decolonizing the Diet project at Northern Michigan University has begun to show how the study of early American history – particularly Native American history – can inform public policy and healthcare paradigms, while also impacting the agenda of cutting edge research in the biological, nutritional, and ecological sciences. In the project, students and local community members have learned how the move away from their ancestral nutritional principles has been detrimental to the health of Native Americans and the wider ecology of the Great Lakes region of North America. Aware of disproportionate instances of diabetes, heart-disease, depression, fertility problems, and chronic inflammatory conditions, project members have studied and then simulated pre-European contact Native American diets, using historical and anthropological methods. They have even begun to produce medically-relevant data on improvements in health and well-being after their nutritional intervention. A similar project, the American Indian Health and Diet Project , has been inspired by the work of Devon Abbott Mihesuah, a Choctaw historian and writer. Through her teaching and writing, Mihesuah has used the study of history, anthropology, and literature to inform contemporary health and nutritional practices – coming a little closer to the diets that many Native American communities consumed prior to European contact.The Decolonizing the Diet project is currently restricted to the Great Lakes region, where the authors of this article reside and work. As a professor of history and a researcher in biological science, we aim to show how a model course in the University of Minnesota system might offer a new pedagogical paradigm for the study of ancestral health principles. The course builds on the aims of the existing projects detailed above. But it also synthesizes their methods and objectives with the latest peer-reviewed research in evolutionary health and nutrition, bringing about new insights and research paradigms relating to the link between nutrition, metabolic health, and immunity to infectious disease. Some recently founded nutritional programs have aimed to educate public-sector workers in order to prevent a mismatch between their optimal nutrition, as understood from within an evolutionary health framework, and their current eating habits.This article demonstrates how the same public health agenda might be explored from within a large public research university system, led by a joint effort between professors in the humanities and teachers and researchers in the biological sciences. The 1862 Morrill Land-Grant Acts defined the American public research university’s ongoing mission “to promote the liberal and practical education of the industrial classes in the several pursuits and professions in life.”From the 1950s scientists used the land-grant system to ally their scientific and nutritional research with new policies that came to be promoted by the federal government. Within the University of Minnesota system, for example, Ancel Keys famously sought a new research agenda in order to question the health benefits of fats from animals, fish, and dairy. Contrary to those who highlighted the problematic medical implications of foods that were readily converted into glucose, as well as the potential inflammatory effects of polyunsaturated fats, Keys and others used their land-grant mandate to promote research that supported the government of the day in its stated desire to increase agricultural output in soy, wheat, corn, and seed oils.Scholars, scientists, public policy analysts, and journalists have begun to suggest that the resulting federally recommended food-pyramid has worsened certain aspects of American public health, particularly through the associated growth of metabolic disorders such as diabetes, and even in the prevalence of certain forms of heart disease .This article examines the present state of historical research in early American studies in order to assess whether similarly problematic associations and health markers appeared among Native American communities many decades – and even centuries – before the development of the problematic modern American food pyramid.

Gene Ontology enrichment was performed on the male and female differentially expressed genes showing at least twofold expression differences between sexes. Gene Ontology enrichment for the male-upregulated genes showed a significant over representation of 76 terms, including pollen and anther development, male gamete development, the terpenoid biosynthesis pathway, and cytokinin metabolism . The male-upregulated gene set showed an under representation for 71 terms, including terms relating to transcription and RNA regulation, splicing, and modification . Among the female-upregulated genes, there was an over representation of RNA transcription and metabolism and shoot and organ development , and an under representation of 47 terms, including cell metabolism and biosynthesis .WGCNA network analysis was performed to explore pathways that may be involved in floral sex dimorphism and development.In total, 17,953 genes were assigned to modules, accounting for 49.8% of all expressed genes and alternative transcripts. The remaining unassigned genes were placed in the “gray” module. Three modules accounted for the majority of assigned genes: the “purple”, “cyan”, and “brown” modules. The “purple” module was strongly correlated to the female sex and captured 30.2% of all female-expressed genes , this module likely represents the genes involved in primary and secondary female sex dimorphism. In the “purple” module, 17 GO terms were enriched,fodder system including RNA and nucleic acid metabolism and regulation, photosynthesis, and phenylpropanoid metabolism . This is consistent with the female differential expression of the majority of genes in this module and supports the contention that this module may be responsible for female sex-dimorphic traits.

The cyan module was the most male-correlated and included 45.5% of all male-upregulated genes likely representing the genes in primary and secondary male sex dimorphism pathways. The “cyan” module contained 230 significantly enriched GO terms, notably multiple terms related to pollen development . The “brown” module consisted mostly of genes not showing differential expression, and probably represents gene pathways involved in basic cell and biological processes.A total of 1,381,813 cis-eQTL and 811,499 trans-eQTL were identified after accounting forcovariates. Notably, there appears to be an eQTL “hotspot” on chr07, to which the expression levels of 550 genes are associated The exceptional number of genome-wide eQTL at this locus suggests that it may have a major role in regulating sex dimorphic gene expression in catkin tissue. Expression levels of 2127 genes were found to be associated with polymorphisms in the SDR, of which 1686 were trans . To identify top-level regulatory and intermediate pathway genes, 2127 genes with eQTL in the SDR were a subset for genes with involvement in secondary metabolism , hormone signaling , RNA splicing and regulation , or transcription . Of the 96 genome-wide transcription factors found to have SDR eQTL, 70 were differentially expressed in either males or females. Because these transcription factors include genes relating to floral development, phenylpropanoid production, and cytokinin signaling, they are candidates for top-level regulatory genes that may regulate further downstream expression. However, confirmation of such roles will require further investigation using methods such as ChIPSeq and DAP-Seq. Fourteen MADS-box and floral development genes, 15 phenylpropanoid pathway genes, and five terpenoid pathway genes were also found to have eQTL in the SDR , representing candidates for intermediate pathway genes directly responsible for dimorphisms in floral morphology, pigmentation, and volatile and secondary metabolite profiles.We identified eleven genes that are strong candidates as master regulators of sex using the following criteria: presence on chr15W and absence from chr15Z, a significant log2 M:F < − 1, presence in the femalecorrelated “purple” WGCNA module, and gene annotation either consistent with a possible floral sex dimorphism pathway or of unknown function.

Genes meeting all four of these criteria are expected to be present only in females and have expression levels and module membership that would implicate them in sex dimorphism. Four copies of ARR17, a truncated AGO4 gene, DRB1, GATA15, a CCHC zinc finger nuclease, and three genes coding hypothetical proteins met these criteria and were identified as candidate master regulator genes .Total RNA-Seq and small RNA-Seq captured the unique sex-specific transcriptomic profiles during catkin development, after floral meristem differentiation and prior to maturation of any stamens or pistils. Within a single maturing catkin, there are hundreds of individual flowers across a range of developmental stages, resulting in pooled expression data from across floral development time points as well as tissue types . In addition to the primary sex dimorphism genes responsible for anther and carpel development, this enables the identification of secondary sex dimorphisms, such as genes involved in pigmentation, volatile emission, and differences in catkin phenology, which can also inform differences in vegetative emergence and secondary metabolites. By using network analysis and incorporating genomic data through eQTL, we can hypothesize how the SDR may regulate differential gene expression in catkins. Nearly two-thirds of all expressed genes in the floral tissue exhibited differential expression between males and females. This number is due in part to the large sample size of 159 individuals, whereby there is enough statistical power to detect even slight differences in expression. Nevertheless, over 21% of the expressed genes showed at least twofold expression differences between sexes, providing evidence of global expression differences, which would require robust transcriptional regulation, ultimately leading back to the sex-determinant genes in the SDR. These genes provide important clues about the regulation of sex determination in this species and the molecular mechanism responsible for diecy and floral sex dimorphism, as described in more detail below.Four copies of ARR17, a type A cytokinin-response regulator, in the SDR, show high levels of expression in female S. purpurea: Sapur.15WG073500, Sapur.15WG073900, Sapur.15WG074000, and Sapur.15WG075200.

Two additional copies of ARR17 are present on chr19 but are not differentially expressed. The cytokinin signaling pathway has been proposed as a common pathway for sex determination in angiosperms. Cytokinin-response regulators serve as feminizing factors in Actinidia, where they are master regulators, and Diospyros, where they act as top-level regulators downstream of the SDR. There is recent evidence implicating ARR17 as the master regulator of sex in the closely related genus Populus, where it may function as a feminizing factor whose expression is suppressed in males by small RNAs. The presence of two complete copies of ARR17 on S. purpurea chr19, expressed in both males and females, suggests that the dosage of ARR17 may play a role in sex determination in willow. Interestingly, these findings suggest a different mechanism for ARR17 than the leading model for sex determination in Populus proposed by Müller et al.. They proposed that functional ARR17 in P. alba is a feminizing factor, and in XY species, ARR17 is silenced by inverted repeats on the Y chromosome through the RNA-directed DNA methylation pathway, leading to a male phenotype. To confirm this, they silenced the ARR17 gene in an early-flowering female line and observed male flowers in tissue culture. We found no evidence of an ARR17 RNA interference mechanism in S. purpurea catkins. Salix purpurea has a similar truncated inverted repeat of ARR17 on chr15Z, but we did not observe small RNAs mapping to the ARR17 genes and their proximal regions, nor to the ARR17 homologs on Salix chr19. There were also no differential methylated regions in the putative promoter regions of any of the ARR17 genes, and S. purpurea males show expression of the ARR17 copies on chr19, whereas in P. trichocarpa males, there is no ARR17 expression. Furthermore, Carlson et al. did not find that ARR17 was differentially expressed in shoot tips containing floral primordia, indicating that this mechanism is not present at an earlier floral development stage either. Taken together, these results suggest that the RNAinterference mechanism of ARR17 may be absent in S. purpurea. The observation of ARR17 expression in both male and female S. purpurea, combined with a lack of small RNA loci in these same regions,fodder system for sale demonstrates that the Salix sex-determination mechanism is likely different from the model proposed by Müller et al.. Instead, our data suggest that if ARR17 is a master regulator in S. purpurea, it likely involves a unique mechanism, possibly through gene dosage, such that a threshold of ARR17 expression must be reached to activate a switch from male-to-female development. Alternatively, there may be another feature in the S. purpurea SDR that is suppressing this silencing mechanism, one such candidate is the adjacent AGO4 homolog described below.A single copy of an Arabidopsis AGO4 homolog, Sapur.15WG074400, is present within the ARR17-inverted repeat region of the chr15W SDR that exhibits a log2 M:F expression of −7.94, and has a cis-eQTL in the SDR. AGO4 is a component of the RNA-induced silencing complex in the RNA-dependent DNA methylation pathway, where it binds small RNAs and silences mRNA. In the bisulfite sequencing data, nearly three times as many regions showed increased methylation in males compared with females , supporting that methylation activity is down regulated in females and may have a role in mediating sex dimorphisms . The SDR AGO4 gene appears to be truncated to only 79 amino acids in length compared with five other catkin expressed AGO4 homologs in S. purpurea, which are 893–922 amino acids, and has multiple indels and substitutions when aligned .

The most similar AGO4 paralog to Sapur.15WG074400 by MUSCLE multiple-sequence alignment is Sapur.008G00580 which has a nearly seven fold greater expression in males . We speculate it is possible that the truncated version of AGO4 is interfering with expression of the full-length Sapur.008G00580 in males by a long noncoding RNA. This could have wide-ranging effects on sexually dimorphic gene expression and could explain the decreased genome-wide methylation observed in females. The findings from the bisulfite-sequencing data indicate that methylation is globally reduced in females. We hypothesize that the Sapur.15WG074400 could be competing for binding of siRNAs with a full-length AGO4 and sequestering male-specific RDDM in females. These global methylation differences could be responsible for sex determination, such as in Melandrium album where demethylation of male plants results in monoecy, with no effect on female plants. Such a mechanism could also explain ARR17 expression levels, and why no small RNAs were observed mapping to ARR17 in Salix, despite evidence for this mechanism in Populus.A female-expressed homolog of GATA15, Sapur.15WG 062800, is located in the W-specific region of the SDR and shows a cis-eQTL association with polymorphisms on Chr15. GATA15 is a transcriptional regulator that binds GAT or GATA motifs in gene promoters and is involved in cell differentiation, morphogenesis, and development. This is consistent with the GO enrichment analysis of female-expressed genes, which contains many significant terms related to morphogenesis and development. Furthermore, chr15 GATA15 was found by Carlson et al. to be differentially expressed in F1 S. purpurea shoot tips containing floral primordia, suggesting that this may be the earliest cue for floral sex differentiation, which would implicate it as a master regulator gene. While functional genomics data are required to elucidate its precise function, its expression in females both during floral differentiation and catkin emergence suggests that it could be directly involved in gynecium development.Four genes were identified that fit the criteria for candidate master regulator genes, but whose functions are not known or whose annotations are insuffificient for further analysis. These included Sapur.15WG068800, a CCHC-type zinc finger, and three hypothetical proteins: Sapur.15WG075300, Sapur.15WG074900, and Sapur.15 WG075700. While there is mounting evidence pointing toward ARR17 as the master regulator in Populus spp, the evidence for different expression profiles of ARR17 in Salix, as well as the presence of additional candidate genes, suggests that the mechanism may be more complicated or altogether different in Salix. Nevertheless, expression data from the ARR17 homologs in S. purpurea do support a possible role in sex determination, either as a single gene master regulator or part of a two-gene system in conjunction with another master regulator, and would provide further evidence to support cytokinin response as a common mechanism for diecy in angiosperms, assuggested by Montalvão et al. Further functional genomics studies will be necessary to elucidate the precise functions of candidate master regulators and their role in sex determination.Among the floral development genes with eQTL in the SDR were homologs of AGL11 and AGL32, AGL29 and AGL30, and AGL6, as well as TOC1, WOX1, RGA, and CONSTANS. While differential expression of MADS-box genes is expected in floral tissues, their association with the SDR through eQTL, even after accounting for sex as a covariate, suggests that the SDR may have a direct role in controlling expression of these genes and subsequent primary sex dimorphisms.

The interaction between K and Mg was found to be significant with the addition of Mg causing a significant increase in the N uptake at each level of K though the magnitude of increase diminished as levels of Mg increased. As the level of Mg application increased without K addition the N uptake was found to decrease. Addition of increased levels of K increased the uptake of phosphorus. The application of high rates of Mg decreases the favorable effect of K on P uptake and hence Mg is seen to have an antagonistic effect on P uptake. This reduction of P uptake in spite of increased P availability might be due to the interference of sulphate ions on the absorption of P by the plant. Increasing the rate of K application significantly increased the K uptake. As the level of Mg application increased the K uptake also showed a general increase, contrary to the reports that K and Mg are antagonistic. Thus Mg might have diminished only the solution concentration of K in soil and not the uptake. At higher levels of K, a significant increase in K uptake was noticed as Mg levels were increased. Thus a positive interaction existed between K and Mg on the uptake of K. This increase in K in the tissue with increasing Mg might be because of the decreased dry matter production associated with high Mg. Calcium uptake was significantly reduced with increasing K levels especially in the absence of Mg. Similar results were reported by Fageria who found an antagonistic effect of K application on Ca uptake.

When applied with high levels of Mg, though higher levels of K caused a reduction in the Ca uptake,hydroponic dutch buckets no significant decrease was generally obtained. Addition of increasing levels of Mg remarkably decreased the Ca uptake. The high Mg levels must have hindered the absorption of Ca by the plant due to the action of the Ca- Mg antagonistic effect. Such antagonism of Mg on the uptake of Ca was also reported by Kumar et al. . It is also seen that the antagonistic effect of increased levels of Mg occurred only in the absence of K. The decrease in tissue concentration and uptake of Ca with increasing concentration of Mg is presumably due to the replacement of Ca by Mg for the neutralization of negative charges within the vacuole and on the exchange sites in the apoplast of the plant cell . Though Mg uptake increases with its level of application, the presence of K especially at higher levels was found to decrease uptake by the crop. This may be because of an induced reduction in availability of Mg to the crop by the excess application of K, leading to less absorption of Mg by the plants. Thus, at higher Mg levels, by supplying additional excess levels of K, the antagonism of additional K is great enough to repress Mg absorption regardless of the Mg level. Thus at low soil K and sufficient exchangeable Mg levels, uptake of Mg is not hindered. The California strawberry industry produces about 85% of the strawberries grown in the United States, on 37,000 acres, with a value of $1.5 billion in 2008 . To control soil borne diseases and weeds, California strawberry fields have long been fumigated with methyl bromide plus chloropicrin . However, methyl bromide is being phased out as an ozone-depleting substance under the Montreal Protocol , an international treaty. Currently, some California strawberries can still be treated with methyl bromide under a critical-use exemption, subject to annual review by parties to the Montreal Protocol.

Alternative fumigants permitted for use in California strawberries are 1,3-dichloropropene , chloropicrin and, as of December 2010, methyl iodide. About 81% of California strawberries are grown in soils that were previously treated with chloropicrin , while 30% are also fumigated with 1,3-D and 43% with methyl bromide . Since soil treatments began in the 1960s, entire fields have been covered with polyethylene film to hold in the fumigant at concentrations needed to kill soil pests . More recently, a sizable portion of strawberry acreage has been treated with fumigants applied to beds via the drip irrigation system . The major alternatives to methyl bromide, 1,3-D and chloropicrin, are heavily regulated. The transition away from methyl bromide to alternatives has been complicated by regulations aimed at protecting workers and others from exposure to fumigants. In California, 1,3-D use per 36-square-mile township is limited to 90,250 pounds, called a “township cap,” which severely limits its availability in key strawberry production areas . The recent critical use nomination for strawberry indicates that “township caps currently limit the use of 1,3-D on 40% to 62% of total strawberry land” . In other words, methyl bromide use continues in California because restrictions on alternative fumigants leave few options. Among the reasons that fumigants are so heavily regulated in California is that they are classified as volatile organic compounds . Alternative fumigants such as 1,3-D are released into the air and, after reacting with nitrogen oxides, can convert to form ground-level ozone — a harmful air pollutant . Regulations have been developed to reduce the contribution of fumigants to ozone formation, which, for example, has seriously affected the use of fumigants in Ventura County, a key strawberry production area.impermeable film can greatly reduce fumigant emissions and enhance their distribution in soil, in comparison with conventional polyethylene films or uncovered soil .

VIF differs from traditional high-density polyethylene tarps in that it has additional gasimpermeable layers, such as nylon or polyaminides, between the polyethylene layers . Fumigant concentrations of 1,3-D and chloropicrin were higher under VIF than low-density polyethylene tarp, 1 to 4 days after drip fumigation . The improved retention of fumigants under VIF also provides more opportunity for them to degrade in the soil rather than be released into the atmosphere . A number of researchers have found that VIF as a tarp can reduce the amount of 1,3-D plus chloropicrin needed for effective soil disinfestations by 50% . Santos et al. found that reducing methyl bromide plus chloropicrin rates by one-half under VIF controlled nuts edge similarly to the full rate of 350 pounds per acre applied under standard films. A relatively new barrier, totally impermeable film , has been shown to apply easily and retain fumigant better than VIF . TIF is a five layer film with two thin ethylene vinyl alcohol layers embedded in three layers of standard polyethylene film . Our studies evaluated the compatibility of TIF and standard films with the two major fumigant application methods for strawberry, broadcast fumigation and chemigation. The primary objective was to compare fumigant retention under TIF and standard film. Secondary objectives were to measure the effects on strawberry fruit yield and weed control.We compared the retention of methyl bromide plus chloropicrin under TIF and standard films at a commercial farm near Salinas in 2007. The soil was a Chualar sandy loam. Methyl bromide 57% plus chloropicrin 43% and 1,3-D 61% plus chloropicrin 35% , both at 350 pounds per acre, were applied by a commercial applicator on Oct. 15, 2007. As the fumigant was applied,bato bucket it was immediately tarped by 13-foot-wide standard film  or 13-foot-wide TIF . The plots were 280 feet long and 33 feet wide to allow for three passes, each 11 feet wide, of the application tractor. The films were 13 feet wide overall with 1 foot on the leading edge used to anchor the film in the soil and 1 foot on the trailing edge used to glue to the leading edge of the previous pass, creating a 1-foot overlap. Hence, the applied film is like rows of overlapped roofing shingles. The proprietary glue used by the commercial applicator adhered to the TIF film and held it in place without incident. Each treatment was replicated two times and arranged in a randomized complete block design. Fumigant concentrations under the tarp were monitored with a MiniRae VOC meter at 3, 27, 51, 76, 97, 120 and 166 hours after application. The MiniRae VOC meter uses a photo ionization detector to measure the concentrations of volatile compounds such as fumigants. Fumigant samples were taken from airspace between the soil surface and the tarp at three random locations near the center of the plots. The film was cut and removed 192 hours after application. The field was then prepared for strawberry planting by the installation of 52-inch-wide raised beds with two drip irrigation lines per bed. ‘Albion’ strawberry was transplanted on Nov. 11, 2007. Strawberry fruit were harvested from two 40-plant sample stations per plot from April 18 to Sept. 1, 2008, and fruit were sorted into marketable and cull fruit by a trained crew. Weeds were sampled from two 125-square-foot sample areas on Feb. 15, April 28 and July 8, 2008.In 2008, we evaluated TIF at the USDA Agricultural Research Service farm on Spence Road near Salinas. The soil was a Chualar sandy loam. We injected 1,3-D 35% plus chloropicrin 60% plus an emulsifier 5%  through the drip irrigation system on Oct. 21, 2008, at 50, 100, 200, 300 and 400 pounds per acre, under both standard and TIF film . Briefly, the fumigants were injected in a closed system directly from nitrogen pressurized cylinders and metered into irrigation water with a flow meter . A static mixing device was installed at the point of injection to mix fumigants with irrigation water before distribution via the drip irrigation system.An emulsifiable formulation of methyl bromide 57% plus chloropicrin 43% was applied on Oct. 29, 2008, at 350 pounds per acre, also through the drip irrigation system. Each treatment was replicated four times, and the trial was arranged in a randomized complete block design.

Plot sizes were a single 52-inch-wide by 75-foot-long bed. Fumigant concentrations under the tarp were sampled at one location near the plot center with a MiniRae VOC meter as described above, at 3, 8, 24, 48, 72, 96, 144, 192, 240 and 336 hours after application. The MiniRae meter was calibrated with known concentrations of 1,3-D and chloropicrin prior to each sampling. The plastic films were left on the beds for the length of the strawberry season. Before transplanting strawberries, planting holes were punched in the bed, and ‘Albion’ strawberry was transplanted by hand into all plots on Nov. 24, 2008. Visual crop injury was estimated on Jan. 6, 2009, using a scale of 0 = safe to 10 = dead. On March 10, 2009, diameters were measured on 20 plants per plot. Fruit were harvested from 50 sample stations in each plot once or twice weekly as needed from March 30 until Oct. 30, 2009. Fruit were graded as described in the 2007 trial. Weed densities were measured in 2007 and 2008. In 2008, nylon bags containing yellow nuts edge tubers and weed seeds were buried in each plot before the fumigant application, at a depth of 6 inches. These species were evaluated because they represent a range of susceptibility to fumigants from difficult , to intermediate , to easy . Little mallow and chickweed are common in strawberry. Weed seeds were retrieved 2 weeks after the methyl bromide plus chloropicrin application, and their viability was determined. The yellow nuts edge was planted in potting soil and placed in an illuminated growth chamber at 85°F for 4 weeks. Weed seed viability was determined using tetrazolium assays. Weed density ratings were measured in 125-square-foot sample areas on the bed tops, on Feb. 15, April 28 and July 8, 2008 , and Dec. 11, 2008, and Feb. 3 and March 17, 2009 .The data was subjected to analysis of variance in SAS v. 9.1 , and Duncan’s multiple range test was used for mean separation for all data at the 5% significance level. Weed seed and yellow nuts edge tuber survival data were analyzed to evaluate the effects of fumigant rate, film, and the interaction between rate and film. Linear contrasts were used to compare weed seed survival under the TIF and standard films using SAS PROC GLM. To determine if there was any correlation between strawberry fruit yield and fumigant concentrations, the 2008 data was tested using the SAS PROC CORR routine.

The first is based on the possibility that the bio-stimulant contains within it, previously unrecognized molecules that are the sole and discrete cause of the observed improvement in plant productivity. This concept emphasizes both the need for clear demonstration of plant productivity benefits and the unknown nature of the mode of action. Thus, a bio-stimulant could be defined as “a formulated product that improves plant productivity by a mechanism of action that is not the sole consequence of the presence of known essential plant nutrients, plant hormones, plant growth regulators or plant protective compounds.” By this definition, once the primary biological mechanism of bio-stimulant function has been identified it should henceforth, be subject to classification on the basis of that functional component. The majority of bio-stimulants in use today are complex mixtures of chemicals derived from a biological process or extraction of biological materials. The complexity of these mixtures is often considered to be essential to the performance of the bio-stimulant, and bio-stimulants may have properties of the whole, that cannot be fully elucidated by knowing the characteristics of the separate components or their combinations. This theory of complexity or “emergence” was described by Mayr ,planting gutter who argued that in many biological systems “the properties of the whole cannot be fully elucidated by knowing the characteristics of the separate components or their combinations.” “The term emergence describes the onset of novel properties that arise when a certain level of structural complexity is formed from components of lower complexity.

In the last few decades, emergence has been discussed in a number of different research fields, such as cybernetics, theory of complexity, artificial intelligence, non-linear dynamics, information theory, and social systems organization” . “Emergence” and “emergent properties” are thus closely related with the notion of the “systems biology” . Emergence was described by Johnson as “unexpected behaviors that stem from interaction between the components of an application and their environment,” “there is, however, considerable disagreement about the nature of ‘emergent properties.’ Others refer to emergent properties when an application exhibits behaviors that cannot be identified through functional decomposition. In other words, the system is more than the sum of its component parts” . Thus, a bio-stimulant could also be defined as “a formulated product of biological origin that improves plant productivity as a consequence of the emergent properties of its constituents.” To our knowledge, however, there have been no clear demonstrations that any bio-stimulant exhibits truly emergent properties. This is not however a unique challenge and all “biological systems are extremely complex and have emergent properties that cannot explained, or even predicted, by studying their individual parts” . Emergent properties have been demonstrated in the networks of biological signaling pathways ; in system-level study of traditional Chinese medicine , and in microbial communities . To adequately explain the biological complexity present in plants and their interactions with the environment, Lüttge and Bertolli et al. emphasize that classic reductionist biology/chemistry is indeed insufficient. While the two theoretical definitions provided in this section share a requirement that the mode of action is unknown, they differ in the core assumption that bio-stimulant function is a consequence of the discrete components in the bio-stimulant or as a consequence of the “emergent” properties of the bio-stimulant as a whole.

Each of these definitions is also incomplete in that it is certainly possible that a bio-stimulant may contain several molecules that act synergistically while not being truly “emergent,” and it is indeed possible and indeed likely, that even if a bio-stimulant is demonstrated to have emergent properties, that not all components of that bio-stimulant are required for that property to be expressed. We propose, therefore, a definition of a bio-stimulant that integrates these two concepts. Thus, a bio-stimulant is defined here as: “a formulated product of biological origin that improves plant productivity as a consequence of the novel, or emergent properties of the complex of constituents, and not as a sole consequence of the presence of known essential plant nutrients, plant growth regulators, or plant protective compounds.” Consistent with this definition, the ultimate identification of a novel molecule within a bio-stimulant that is found to be wholly responsible for the biological function of that bio-stimulant, would necessitate the classification of the bio-stimulant according to the discovered function. A review of the history of bio-stimulants and related products provides insight into the diversity of these products and the development of this field of study. The evolution of bio-stimulant classifications as described by various authors is presented in the Table 2. To the best of our knowledge, one of the first attempts to categorize bio-stimulants was provided by Filatov when 4 groupings of biogenic stimulants were suggested. Karnok compiled a list of 59 materials presenting in 15 bio-stimulants; Ikrina and Kolbin systematized patent literature and specified 9 categories of natural raw materials used to derive bio-stimulants; Basak suggested that bio-stimulants could be grouped on the basis of single or multi-component formulations and classified on the origin of the active ingredient, and the mode of action of the active ingredient. Du Jardin developed a scientific rationale of classification considering 8 categories of bio-stimulants and subsequently reduced this list to 7 categories .

Du Jardin was explicit in his exclusion of microorganisms from his categorization primarily to avoid conflict with existing categorization of microorganisms as bio-pesticides and sources of plant hormones. Later Bulgari et al. proposed a bio-stimulant classification on the basis of their mode of action rather than on their composition. Many bio-stimulant products have been classified into completely divergent groups and categories of function, use, and type of activity . For example, humate based products are often described as soil health amendments while plant growth promoting rhizobacteria could be categorized as bio-fertilizers, phytostimulators, and bio-pesticides . Du Jardin has proposed that bio-fertilisers are a subcategory of bio-stimulants. Seaweed extracts have been considered as bio-fertilizers and microorganisms have also been described as bio-fertilizers . Some inorganic elements or small molecules that are not known to be essential may also be classified as bio-stimulants if evidence of plant growth promotion is available . Thao and Yamakawa , for example, consider phosphites to be bio-stimulants since plant response to phosphites frequently cannot be explained as a consequence of the known anti-fungal function of these molecules. While the categorization of bio-stimulants by their origin does not, a priori, provide information on their mode of action this categorization may still be a useful tool to aid in the process of discovery and facilitate comparison between similar products. Registration of products used in agriculture is crucial to ensure their practical, safe and legitimate application. In the absence of a sound definition of bio-stimulants as a discrete group of products , the registration procedure and subsequent classification regime is untenable and this inevitably creates a barrier to trade and development. Various countries, states, and administrative regions have developed different categories for registration of potential bio-stimulants including terminology such as plant conditioners, “other fertilizers,” supplements, soil improvers, gutter berries plant strengtheners, fitofortificants, etc. . In many jurisdictions regulatory practices require an itemized description and identification of substances in all commercial product classifications while in others the registration of non-fully identified substances is allowed if those products are considered of complex composition. There is even a proposal for complex bio-stimulants to not specify the chemical name and note as “None” with the definition that “this product is a complex mixture of chemical substances” . If we accept the concept that a bio-stimulant is a product of clear benefit but unknown mode of action, then it can only be regulated by its safety and proof of efficacy. For example, in pharmacology it has been suggested that “the demand to demonstrate the mode of action of each single component in a phytopharmaceutical may not be obligatory any more” . The complex multi-component nature of many bio-stimulants clearly complicates discovery of their modes/mechanisms of action, production, registration and use. What is clearly needed however, is a regulatory mechanism to ensure that the products are “generally recognized as safe,” have “a positive benefit on crop productivity” and are discrete from exisiting categories of products. The task of identifying function and agronomic utility can then be pursued independently and will be driven by the marketplace imperative for product quality and consistency. Coordinating national legislation within this framework will become critical for the optimization of bio-stimulants and trade between different countries. The possible place of bio-stimulants in the regulatory system of pesticides and agrochemicals is illustrated in Figure 1.We have conducted an exhaustive analysis of the literature and categorized the majority of the reported bio-stimulants by origin . Microorganisms are widely used for the production of bio-stimulants and may be derived from bacteria, yeasts, and fungi. These preparations may include living and/or nonliving microorganisms and their metabolites. The concept of microorganism-based preparations as bio-stimulants is described by Xavier and Boyetchko , Sofo et al. , Colla et al. , Matyjaszczyk , and Ravensberg . different species of algae, mostly seaweeds, are also commonly used for producing bio-stimulants.

Seaweed-based preparations as bio-stimulants are described in reviews by Crouch and van Staden , Khan et al. , Craigie , Sharma et al. ; and experimental papers by Goatley and Schmidt , Jannin et al. , Billard et al. , Aremu et al. . Raw materials for bio-stimulants are also commonly based on higher plant parts including seeds, leaves, and roots and exudates from families Amaryllidaceae, Brassicacae, Ericaceae, Fabaceae, Fagaceae, Moringaceae, Plantaginaceae, Poaceae, Rosaceae, Solanaceae, Theaceae, Vitaceae, among others . bio-stimulants may also be based on protein hydrolysates and amino acids of animal origin including wastes and by-products , and insect derived chitin and chitosan derivatives . Humate-based raw materials are widely used to derive bio-stimulants and have been reviewed by Sanders et al. , Kelting et al. , Ertani et al. , and Jannin et al. . A final category of bio-stimulants includes those derived from extracts of food waste or industrial waste streams, composts and compost extracts, manures, vermicompost, aquaculture residues and waste streams, and sewage treatments among others. Because of the diversity of source materials and extraction technologies, the mode of action of these products is not easily determined. The technologies used in the production and preparation of bio-stimulants are highly diverse and include cultivation, extraction, fermentation, processing and purification, hydrolysis, and high-pressure cell rupture treatment . In some instances, a bio-stimulant product may also contain mixes of components derived from different sources and production methods. Frequently the rationale for utilizing extracts rather than raw biomass is a consequence of the need for a standardized manufacturing process to produce a uniform commercial product . For many products, the production processes are driven by process and marketing demands and are not the result of a targeted strategy to optimize the biological efficacy of the commercial product. While the ultimate composition and possible function of commercial bio-stimulant products may be partially determined by both the source of raw material and the process by which it is prepared , there may be manufacturing processes and product treatments utilized that result in compounds that are not present in the initial material. An example of this is the multitude of commercial seaweed extracts, often derived from the same species, that are rarely equivalent . Commercial bio-stimulant manufactured from similar sources are usually marketed as equivalent products, but may differ considerably in composition and thereby in efficiency . Many manufacturers do not reveal the technology of bio-stimulant production since that is a commercial secret .A diversity of substances contained in raw materials is used for the production of bio-stimulants. Whereas, primary metabolites are contained in most preparations de facto, the presence of secondary metabolites is more specific and depends to a large extent on the raw material used . Primary metabolites include amino acids, sugars, nucleotides, and lipids . Secondary metabolites are formed from different primary metabolic pathways, including glycolysis, the tricarboxylic acid cycle , aliphatic amino acids , the pentosephosphate and shikimate pathways which are primarily the source of aromatic AA and phenolic compounds , terpenoids/isoprenoids, nitrogen-containing compounds , sulfur-containing compounds. Frequently, bio-stimulants are shown to have a multicomponent composition and may include plant hormones or hormone-like substances, amino acids, betaines, peptides, proteins, sugars , aminopolysaccharides, lipids, vitamins, nucleotides or nucleosides, humic substances, beneficial elements, phenolic compounds, furostanol glycosides, sterols, etc. .