The seasonal NO3–N concentrations in the domain varied from 0.01–7.03 mmol L 1 . Hutton et al. reported higher mobilization of nitrate at a shallower depth under drip irrigation of grapevine, and seasonal root zone nitrate concentrations ranging between 0 and 11.07 mmol L 1 in the Murrumbidgee Irrigation Areas in Australia. As the season continued and plant uptake was reduced, excess water further mobilised nitrate–nitrogen out of the root zone, as is evident from 27/04/07 and beyond . At the end of the crop season, little nitrogen remained in the soil system, and what did remain was well beyond the reach of the plants. This nitrogen is expected to continue leaching downwards over time and become a potential source of nitrate–nitrogen loading to the ground water. Additionally, peak NO3  –N concentrations in the soil profile L 1 and in drained water L 1 were significantly higher than the Australian environmental standard for protection of 80% NO3 –N L 1 and 95% of species NO3–N L 1. The NO3–N concentrations in the soil solution also occasionally exceeded the level of Australian drinking water quality standard for nitrate NO3–N L 1. High levels of nitrate–nitrogen below the crop root zone are undesirable, as some recharge to groundwater aquifers can occur, in addition to flow into downstream rivers, which are used for drinking water and irrigation. These findings are consistent with other studies , in which high nitrate concentrations in drainage water under drip and furrow fertigated irrigation systems have been reported.Estimated water balance components above and below the soil surface under a mandarin tree are presented in Table 4. It can be seen that in a highly precise drip irrigation system, a large amount of applied water drained out of the root zone, even though the amount of irrigation applied was based on estimated ETC. This drainage corresponded to 33.5% of applied water,hydroponics growing system and occurred because highly permeable light textured soils, such as those found in this study, are prone to deep drainage whenever the water application exceeds ETC.

The drainage amount in our study falls within the range of recharge fluxes to groundwater reported by Kurtzman et al. under citrus orchards in a semiarid Mediterranean climate. Mandarin root water uptake amounted to 307.3 mm, which constitutes about 49% of applied water. Root water uptake slightly increased when the model was run without considering solute stress , which is not a significant difference. It further substantiates the results obtained for seasonal ECsw in Fig. 6, where salinity remained below threshold over the season. Evaporation accounted for 17.7% of the total water applied through irrigation and rainfall. The modelling study overestimated the sink components of the water balance by 4.79 mm . There were major differences between water input and output from January 2007 onwards . During this period, irrigation and precipitation significantly exceeded tree water uptake , which eventually resulted in deep drainage from March 2007 onwards. Therefore, current irrigation scheduling requires adjustment during this period. This illustrates how simulations were helpful in evaluating the overall water dynamics in soil under the mandarin tree. The nitrogen balance is presented in Table 5. The nitrogen fertilizer was applied either in the form of NH4 + or NO3, but NH4 + transforms quickly to NO3  through the process of nitrification. Model simulations showed that nitrification of NH4 + was very rapid and most of the NH4 + –N converted to NO3  before it moved to a depth of 20 cm, and no traces of NH4 + were observed below this depth. It is apparent that the nitrification of NH4 + took place in the upper soil layer, which contains organic matter and moisture that supports microorganisms , facilitating the nitrification of NH4 + . Though NH4 + was initially nitrified to NO2 and consequently to NO3, NO2 was short-lived in the soil and decayed to NO3 quickly. Therefore, the simulated plant NH4 + –N uptake was only 0.71 kg ha 1 . Hence, the NO3–N form was responsible for most of the plant uptake, corresponding to about 85% of the applied nitrogen.

The monthly N applications were slightly higher than plant uptake during the flowering and fruit growth periods . However, the monthly uptake was slightly higher than the N application between these periods. High frequency of N applications in small doses resulted in similar nitrogen uptake efficiency in citrus as in other studies . Similarly, Scholberg et al. reported doubling of nitrogen use efficiency as a result of frequent application of N in a dilute solution. Slightly higher uptake was recorded when fertigation was applied in second last hour of an irrigation event , as compared to when it was applied early in the irrigation event . Hence, it can be concluded that timing of fertigation does not have a major impact in a normal fertigation schedule with small and frequent N doses within an irrigation event in light textured soils. Similar results were also obtained in our earlier study in a lysimeter planted with an orange tree , which revealed that timing of fertilizer N applications in small doses in an irrigation event with a low emitter rate had little impact on the nitrogen uptake efficiency. Nitrate–nitrogen leaching accounted for only 15% of the applied nitrogen . Monthly N balance revealed that most of the N leaching happened between March 2007 and August 2007, which was correlated with the extent of deep drainage occurring during this period. NO3 –N losses ranging from 2% to 15% were illustrated by Paramasivam et al. and Alva et al. , attributable in part to an improved management of N, which could be a contributor in the current estimation.In our study, it is evident that there were significant deep drainage and nitrate–nitrogen leaching losses , which could be reduced by appropriate management. Hence, different simulations involving the reduction of irrigation and fertigation applications during the whole or part of the crop season were conducted, to optimize water and nitrogen uptake and to reduce their losses from the soil .

Increasing the irrigation frequency with short irrigation events while maintaining the same irrigation volume, had no impact on deep drainage and N leaching . However, the seasonal salinity increased by 11% compared to the standard practice. This confirms that the current irrigation schedule followed with respect to the irrigation frequency seems to be optimal under the experimental conditions. In S2, Dr_W and Dr_N were reduced by 14.4% and 19%, respectively, but salinity increased by 11%. However, a sustained reduction in irrigation by 20% eventually reduced the Dr_W and Dr_N by 28.1 and 38.3%, respectively, at the expense of a 4.9% decline in plant water uptake, but with a 4% increase in N uptake. However, salinity increased by 25.8% compared to the normal practice, which would likely have a significant impact on plant growth. Scenarios S4 and S5 were based on decreasing the nitrogen application by 10% and 20%, resulting in a decrease in N leaching by 7.4% and 14.8%, respectively, along with a much higher reduction in plant N uptake , suggesting that the reduction in the fertilizer application alone is not a viable option to control N leaching under standard conditions. A combined reduction in irrigation and fertigation by 10% further reduced N leaching by 5.5%,flood table compared to reducing irrigation alone , but at the same time plant N uptake was reduced by 5% more than in S2. Similarly, reducing irrigation and N application by 20% produced a pronounced reduction in N leaching and water drainage , but it also resulted in a decrease in plant N uptake by 15.8% and water uptake by 4.8%, compared to normal practice. At the same time, salinity increased by 25.8%, which is similar to S3. The reduction in plant water and N uptake would have a major impact on plant growth and yield, and would adversely impact the sustainability of this expensive irrigation system. Hence, reducing fertilizer applications does not seem to be a good proposition under the current experimental conditions, as it results in an appreciable decline in plant N uptake. However, Kurtzman et al. reported that a 25% reduction in the application of N fertilizer is a suitable agro-hydrological strategy to lower the nitrate flux to groundwater by 50% under different environmental conditions. Rather, reducing irrigation alone seems to be a better option to control the deep drainage and N leaching losses under the conditions encounThered at the experimental site. Additionally, it is worth noting that in S3 and S7 the salinity during a period between October and December at a depth of 25 cm, and during December at a depth of 50 cm, increased considerably, and was higher than the threshold level , confirming that a sustained reduction in irrigation and fertigation is not a viable agro-hydrological option for controlling water and N leaching under the mandarin orchard. However, it seems unnecessary to reduce irrigation applications uniformly across the season as suggested by Lido9n et al. . Rather, irrigation could more profitably be reduced only during a particular time period when excess water was applied. The water and N balance data in our study revealed that an imbalance between water applications and uptake happened during the second half of the crop season, i.e., from January till August 2007, resulting in maximum drainage and N leaching , coinciding with the fruit maturation and harvesting stage. Hence, there is a need to reschedule irrigation within this period, rather than reducing water applications throughout the entire season.

Keeping this in mind, the following 5 scenarios were executed, in which irrigation was reduced during the second half of the crop season, i.e., between January and August, by 10%, 20%, 30%, 40%, and 50%, respectively. Scenarios S10, S11, and S12 showed an enormous potential for reducing water and N losses. In S10, Dr_W and Dr_N were reduced by 8% and 4% more than in S7, N uptake was increased by 6.9% , and salinity was also 4% less than in S7, which seems quite promising. On the other hand, in S11 and S12, the Dr_W and Dr_N were reduced to a greater extent than in S10, and soil salinity increased substantially , due to a considerable reduction in the leaching fraction. This is also shown in Fig. 12, which shows that monthly soil solution salinity in S11 and S12 at the 25 and 50 cm soil depths increased dramatically between January and August. Although ECsw remained below the threshold level, except at a 50 cm depth in S12 during March 2007, there is a significant likelihood of it increasing further in subsequent seasons, which would ultimately impact the growth and yield of mandarin trees. Hence, under current conditions, Scenario S10 represents the best option to control excessive water and N losses, and high salinity, and to increase the water and N efficiency for mandarin trees. Other permutations and combinations, involving fertilizer reductions along with S10, did not provide further improvements in controlling water and N leaching. It is concluded that simulations of irrigation and fertilizer applications, using HYDRUS, can be helpful in identifying strategies to improve the water and N efficiency for drip irrigation systems of perennial horticultural crops.Ex situ seed banks play an important role in accessing and propagating seeds for restoration of degraded and fragmented native habitats and for the conservation of threatened species. Accordingly, many botanic gardens and conservation agencies worldwide have established seed banks to address ongoing concerns about habitat destruction and in particular the loss of biodiversity. As well as providing seeds for use in conservation and restoration programs, seed banks provide unique opportunities for studies of the biology, ecology, and evolution of native species as well as opportunities for the commercialization of species with horticultural and agricultural potential. Given the value of these collections and in many cases the irreplaceable nature of the accessions held, it is imperative that seed banks are managed in ways that minimize age-related seed losses. Consequently, the maintenance of seed viability during long-term storage is a major focus of seed banks. However, the longevity of seed viability varies greatly among plant species and is significantly impacted by seed storage conditions. Therefore, the success of long-term ex situ seed conservation is dependent on regular monitoring using standardized and reproducible approaches.

DNA sequencing was performed using ABI BigDye Terminator according to the manufacturer’s protocol, except that 5-μl reactions were performed with 0.25 μl of BigDye on an ABI 3730xl DNA sequencing machine with 50 cm arrays. DNA sequences were analyzed with CodonCode Aligner v. 2.0.6 . We detected three types of polymorphism in our sequences – single feature polymorphism , insertions and deletions and variable number tandem repeats . Most of the SFPs that had been detected using the Affymetrix GeneChip were due to a single nucleotide polymorphism , but in five cases due to a single base indel. Since Haploview cannot handle missing values, missing bases were substituted prior to data analysis with an appropriate single nucleotide. Because all single-base indels could be tagged with SNPs from the same marker locus , we use the term SNP throughout the text. Both indels and VNTRs were excluded from data analysis, unless otherwise noted in the text.One hundred and ninety two RILs derived from a cross between an F1 of cv. Valmaine × cv. Salinas 88 and cv. Salinas were genotyped with EST-derived markers. Selection of markers for this first round of genotyping was based on the molecular linkage map developed from an interspecific cross between L. sativa cv. Salinas and Lactuca serriola accession UC96US23. Twenty markers were selected to evenly cover linkage group 2 in intervals of approximately 10 to 20 cM. After preliminary mapping of the resistance gene, the region containing Tvr1 was saturated with markers originating from a microarray-based study also carried out on the Salinas × UC96US23 population.

Marker polymorphism was tested with HRM analysis,25 liter pot plastic unless the difference between segregating alleles could be visually observed using gel electrophoresis. If polymorphism could not be observed with HRM analysis, PCR products from the two parental genotypes were sequenced and new primers were designed for HRM. Statistical analysis of the linkage between molecular markers and die back resistance was performed by MapManager QTX software. Dieback resistance for each RIL was considered as a bi-allelic qualitative trait and used for linkage analysis.Association mapping was performed on a set of 68 accessions from seven horticultural types of lettuce . In the first step, markers closely linked to the Tvr1 gene were amplified from each accession and sequenced. In the second step, the sequenced amplicons were analyzed for polymorphism with the CodonCode software and inputted into Haploview v. 4.2. Intra-locus SNPs were tagged in Haploview with the Tagger function at r2 = 1. Untagged SNPs from all markers and a representative SNP for each tag were then entered into TASSEL v. 2.0.1. TASSEL was subsequently used to test for association between individual SNPs and resistance to die back while accounting for the population structure. Both p-values for each SNP and percent of phenotypic variation explained by the model were calculated with TASSEL after 100,000 permutations. Prior to association analysis, the population structure in the set of 68 accessions was assessed with thirty EST-SSR markers distributed throughout the genome using the computer program STRUCTURE 2.2. Ten runs of STRUCTURE were done by setting the number of populations from 1 to 15. For each run, the number of iterations and burn-in period iterations were both set to 200,000. The ad hoc statistic was used to estimate the number of sub-populations. The optimum number of sub-populations was subsequently used to calculate the fraction of each individual’s genome that originates from each of the five sub-populations.

The qk values obtained from STRUCTURE were used as covariates in the statistical model given by TASSEL.The level of genetic variation at the nucleotide level was estimated as nucleotide polymorphism and nucleotide diversity . To test the neutrality of mutations, we employed Tajima’s D test, which is based on differences between π and θ. Analyses of genetic variation and estimates of haplotype diversity were carried out using DnaSP v. 5.00.04 software. Linkage disequilibrium between pairs of SNP loci in the genome was calculated with Haploview and the values were pooled over the entire data set. Decay of LD with distance was estimated using a logarithmic trend line that was fitted to the data. Distances between markers were calculated from their respective positions on the consensus molecular linkage map. The consensus map was created with JoinMap v. 2.0 from the Salinas × UC96US23 map and the × Salinas map . SNPs with frequency < 5% were excluded from the analysis.The resistance-SNP association observed in the set of 68 accessions was detected through sequencing of PCRamplicons from individual accessions. In order to accelerate and simplify the test of association, we developed a primer pair that allowed detection of polymorphism in the marker Cntg10192 through high-resolution melting analysis. These primers amplify a 185 bp product that contains all three SNPs detected in the marker Cntg10192 at the positions 54, 72, and 100. The first two SNPs match perfectly with the resistance allele, while the third SNP explains 40.9% of the trait variation. As with the first two SNPs, the third SNP has a C ⇔ T substitution. All susceptible genotypes carry the T allele, while resistant genotypes have either the T or C alleles at the third SNP. It appears that the T allele in the resistant material is associated with the resistance present in cv. Salinas and most of the other iceberg cultivars, whereas the C allele is associated with the resistance present in the three lines that originate from the romaine-like primitive accession PI491224.

Marker Cntg10192, therefore, not only allows for the detection of alleles associated with die back resistance, but also separates alleles of different origins. To further investigate polymorphism in this genomic region we sequenced two accessions from L. serriola, a wild species closely related to cultivated lettuce. One of the accessions is resistant to the disease, while the other one is susceptible.The resistant accession has a haplotype similar to cv. Salinas but instead of the T allele at position 54, it carries the C allele. The three SNPs at the marker Cntg10192 can thus distinguish four different haplotypes; three resistant and one associated with susceptibility . Haplotype R1 has the T-T-T allele combination at positions 54, 72, and 100. Haplotype R2 carries the T-T-C combination, while haplotype R3 carries the C-T-T alleles. Disease susceptibility was always associated with the S1 haplotype that carries the C-C-T combination. All four haplotypes can easily be separated through high-resolution melting analysis .Nucleotide polymorphism was observed in all nine markers that were sequenced from the region flanking the Tvr1gene. The rate of nucleotide substitutions in a set of 68 accessions translates into ~1 SNP per 149 bp between pairs of randomly selected sequences. This SNP frequency was somewhat lower when only coding regions were considered . These values are well within the range observed for other plant species. For example,25 litre plant pot the average SNP frequency is 60 bp in aspen, 87 bp in potato, 104 bp in maize, 130 bp in sugar beet, 232 bp in rice, 435 bp in sorghum, 585 bp in tomato, and 1030 bp in soybean. Both nucleotide polymorphism and nucleotide diversity of lettuce are similar to that observed in maize , potato , and sugar beet , but larger than in tomato , and soybean. If results from the analyzed region correspond to those for the whole genome, sequence variation in lettuce is relatively high for a selfing species. It was previously observed that selfing species have generally lower levels of sequence variation than outcrossing species because of smaller effective population sizes. Although polymorphism in lettuce appears to be considerably larger than in selfing soybean and tomato, it is similar to that observed in rice, which is also a self-pollinating species. The ratio of nucleotide diversity in coding and non-coding sequences was not analyzed in detail, since data from only four markers are available. However, the ratio haplotype frequencies, and calculated using the across these markers appears to be smaller than in DNAsp software.

This measure of diversity is analogous to Arabidopsis , soybean the heterozygosity at a single locus, and is at its maximum , maize , and potato . when haplotypes observed in the sample occur at equal This difference is likely due to a higher level of functional frequencies. Diversity based on haplotypes ranged constraint on the perigenic sequence of lettuce. Meas- from 0.593 in QGG19E03 to 0.809 in marker Cntg11275, ures of haplotype diversity were based on estimated with an average value of 0.732 ± 0.024. These values are higher than in rice , soybean , and human . It is possible that the high level of diversity is related to the way that selection of the 68 accessions was performed. We included die back resistant and susceptible material from all predominant horticultural types, thereby selecting haplotypes at similar frequencies. It would be interesting to observe how haplotype diversity changes in different genomic regions and/or for a different set of accessions. To test the neutrality of mutations, Tajima’s D was calculated for all surveyed markers. The average Dwas larger than in soybean , potato , and sorghum . A positive D value indicates a deficit of low-frequency alleles relative to what is expected. Since large D values can be caused by a population subdivision, it is possible that the presence of sub-populations in the analyzed set of lettuce accessions affects both haplotype diversity and the D values. When neutrality of mutations was tested in individual markers, three markers closely linked to the Tvr1 gene had Tajima’s D values significantly higher than expected . Again, the population structure or selection at the Tvr1 locus or the marker itself could have caused departures from neutrality.The decay of LD for the Trv1 region was relatively slow when measured both within individual markers and between markers flanking Tvr1. Estimated values of r2 were ~0.322 at 900 bp, and ~0.124 at 8 cM. A fitted logarithmic curve shows that the r2 value of 0.2 is reached somewhere between 0.5 cM to 1 cM. LD of SNP markers observed in some other selfing species was similar; LD in Arabidopsis was 250 kb or 1 cM and in soybean was ~50 kb. Intra-locus LD decayed very little in tomato, with the log trend showing r2 > 0.6 at 900 bp. However, it is problematic to compare decay of LD across species due to the large variability in LD quantification. LD depends on a combination of many factors, such as the origin of the population, selected set of accessions, analyzed genomic region, molecular marker system, and presence of unidentified sub-populations. Hyten compared four different soybean populations for levels of LD decline. While in the domesticated Asian G. max population LD did not decline along the 500 kb sequenced region, the wild Glycine soja population had a large LD decline within the LD block size averaging 12 kb. Comparable observations were not only made in the selfing Arabidopsis, but also in the out crossing maize and aspen. Our results show a large difference between estimates of LD when analyses were performed across all horticultural types or within each individual type. While the estimate of r2 at a distance of 8 cM was 0.124 for the whole set, it was 0.247 for romaine type and 0.345 for crisp head lettuce. Because only a relatively small part of the genome was analyzed in the present work, it is not possible to calculate LD at distances over 8 cM. However, the trend for the logarithmic curve suggests that LD could reach more than 15 cM in romaine and probably more than 25 cM in crisp head types before declining to the value of r2 < 0.2. When only iceberg types were included in the analysis, LD was still at its maximum at a distance of 8 cM . Although these observations come from a limited number of individuals, they are supported by the fact that the modern iceberg-type lettuce has an extremely limited genetic diversity that is frequently associated with extensive LD.We identified the genomic region carrying resistance against die back and nine markers closely linked with the Tvr1 gene through linkage analysis. We subsequently used this information to test the linked markers for association with the disease resistance on a set of 68 diverse accessions.

Both of these examples used sense transgenes, therefore this type of silencing effect commonly is referred to as co-suppression. Several other groups working with similar systems have reported analogous results . In contrast, antisense silencing was shown in tobacco to be not graft-transmissible regardless of whether the signal originated in the scion or root stock . In tomato grafting experiments with the ACC oxidase gene, antisense silencing of scion ACC oxidase was not seen early after graft establishment, however after several weeks a graft-transmissable silencing was observed . This time lag may account for why the earlier experiments concluded that there was no silencing in grafted antisense lines. A high level of expression of the target gene in the scion was necessary for the detection of silencing by Northern hybridization, as a result of expression of the antisense construct in the root stock, a situation similar to the nitrate reductase experiments discussed earlier . Thus, experimental time lines, the levels of target gene expression, and the model organisms used may be important determinants of the efficacy of antisense silencing in grafted systems. It has also been shown that even when target gene are not present in the recipient graft, transgenic siRNAs can accumulate from donor grafts . Arabidopsis containing a GFP inverted-repeat silencing construct as the donor was grafted withWT or GFP-expressing scions as recipients. The sRNAs identified in scion tissues included siRNAs generated as a result of the GFP construct and a substantial population of endogenous sRNAs from the root stock donor as well. Size classes ranging from 21 to 25 nt were most abundant, and the 24-nt class directed epigenetic modification of the GFP signal in the scion. The massively parallel deep sequencing methods used by this group showed that if a silencing target was not present in the recipient ,plastic planters bulk then siRNAs generated from hairpin-GFP in the root stock were still present in the scion, albeit at levels several of orders of magnitude lower.

This could be why previous experiments using less sensitive detection techniques, such as Northern blots, did not detect mobility of the signal. A recent report has shown that beyond the 24-nt siRNAs mentioned above, all size classes of siRNAs can trigger homologous sequence-specific methylation of targets at long-distances, at least in Arabidopsis . What facilitates the movement of sRNAs? sRNAs and associated RNPs are small enough to be translocated based on their size, since experiments have shown that a 27-kDa GFP is able to diffuse into the vascular system . Results of experiments where movement proteins are included indicate that spreading of the silencing signal is at least partially dependent on the size of the plasmodesmatal apertures . Alternatively, movement of the silencing signal might be selective, perhaps requiring protein– protein, or protein–nucleic acid interactions in order to obviate the apparent plasmodesmatal aperture size exclusion limit. This view is supported by experiments involving mutants deffective or deficient in the ability to move signals . Regardless of uncertainties related to the mechanism of sRNA movement, the evidence demonstrates that movement does indeed occur through the phloem component of the vascular system and is mediated by plasmodesmata, at least to some degree. Many experiments have been performed regarding the mobility of RNAs, both large and small, but whether the same pathways that are used for the movement of mRNA are used for miRNA or siRNA movement has not been determined. The emerging idea that sRNAs are involved in physiology, defense, and development, both cell autonomously and for long-distance signaling, is becoming more widely accepted . Given the variability in mobility detected across several studies, it seems that plasmodesmata-based transport of sRNAs is a regulated process. However, the molecular mechanisms that mediate sRNA mobility and whether they are cis or trans-acting are unknown.

Researchers have successfully employed strategies that utilize the expression of siRNAs in order to protect the plant root zone from pests and pathogens . For example, in soybean, resistance strategies that target soybean cyst nematode genes, including those associated with stimulating root growth in infected plants, sperm production, and female development have been tested . By grafting these plants to WT scions, systemic protection may be achieved in a manner similar to the virus resistance reported in tobacco and more recently in cassava in experiments demonstrating control of the devastating Cassava brown streak Uganda virus . Aside from pathogen resistance, down–regulation, and/or epigenetic modification of transcripts and genetic networks in the scion or the root stock also appear to be possible through the use of siRNAs and could influence scion-specific characteristics, such as flowering time, fruit production or quality, or root characteristics, such as tuberization in potatoes .In addition to RNAs, proteins may be transported over long distances in a regulated fashion. Certain motifs, reminiscent of nuclear localization signals, allow protein entry into CC and subsequently into the phloem for long-distance movement. Despite the evidence for selective and regulated processes for protein long-distance translocation, there is also evidence that shows nonspecific “leakage” of supposedly cell-autonomous proteins into sieve tubes and subsequently into sink tissues. Xylem vessels,which mainly transport water and low molecular weight inorganic and organic solutes, have been shown to contain proteins, although at lower concentrations than in phloem sap . Proteins targeted to the apoplast may inadvertently enter xylem or phloem vasculature and subsequently be transported to and unloaded in sink tissues. Examples of movement of proteins include exogenous viral movement proteins, endogenous transcription factors and xylem/phloem proteins . Some of the first studies of xylem protein transport involved viral movement proteins , but as knowledge has progressed, more researchers have been able to demonstrate mobility of endogenous plant proteins. For many years, proteins had been observed in the phloem, but the idea of a coordinated, selective, and regulated process of trafficking, influencing not only development, but plant responses to environmental cues is a more recent idea that has gained support .

Mobile proteins or non-cell-autonomous proteins may be encoded by as many as 20% of the genes in Arabidopsis . A comprehensive analysis of phloem sap proteins in pumpkin and cucumber using high resolution 2-D gel electrophoresis and partial sequencing by mass spectrometry identified several hundred proteins in the phloem, and the majority of these proteins may have roles in stress and defense reactions . Models of the mechanics underlying protein mobility in the vasculature include the structures associated with the vascular tissue. Within the phloem, SE, which lack a nucleus, ribosomes, and a vacuole, depend on neighboring CC for maintenance of their metabolic tasks . Because mature SE cells cannot synthesize proteins, the likely origins of proteins in the phloem are immature SE or CC. Structurally different from the plasmodesmata that connect mesophyll cells, specialized plasmodesmata between CC and SE are branched with all of the branches on the CC side funneling to a single opening on the SE membrane side. The requirements for specificity of transport between CC and SE are not completely known but accumulating evidence points to the importance of these branched plasmodesmata. Reviews from two research groups establish plasmodesmata as the “gatekeepers” of macromolecular transport into the SE . The specific mechanisms governing the regulation of plasmodesmatal apertures are still a mystery, but fluorescently labeled dextrans and GFP expression have been used to study plasmodesmatal size exclusion limits and their function under differing conditions. Through grafting,collection pot the vascular networks of both root stock and scion become connected and what is mobile in the root stock vascular networks is likely to become mobile in the vascular networks of the scion. In a thorough heterografting experiment involving 11 interspecific and intergeneric Cucurbit graft combinations, several structural P-proteins appeared in the recipient phloem exudate, as shown by SDS-PAGE and Coomassie staining. The results effectively demonstrated the direction of transmission was dependent on the combination of heterograft used, with some graft partners taking the role of donor or acceptor, and some able to perform both roles . This has clear implications for choosing of graft partners for GE-modifified root stocks. Fluorescence microscopy of graft junctions has shown sieve tube bridges connecting scion external bundle phloem to internal bundle root stock phloem when mobility was demonstrated. This observation identified physical continuity within the phloem as a prerequisite for mobility of proteins, but did not resolve the selective directionality observed .When two Cucurbit structural P-proteins, PP1 and PP2 were examined in intergeneric grafts, RT-PCR and Northern blots demonstrated that protein products rather than mRNA transcripts were translocated across the graft junctions. In addition to structural proteins, RNA-binding proteins appear to be abundant in the phloem translocation stream. Phloem sap collected and analyzed from four different sources all contained sRNAs of 18–25 nt sizes with various abundance profiles for each species. Fractionation of the phloem sap from pumpkin, cucumber, and lupine also identified a small∼27 kDa protein that bound strongly to 18–24 nt ssRNA. After cloning the pumpkin PSPR1 gene, microinjection studies demonstrated that PSPR1 specifically shuttled a high percentage of the ssRNAs across cell boundaries. In these studies, co-injection and subsequent movement of a 20-kDa fluorescent dextran showed that plasmodesmatal aperture was at least 20 kDa. Apparently, dilated plasmodesmata alone were not sufficient to allow the movement of ssRNAs between cells, since use of another protein shown to increase plasmodesmatal apertures was not sufficient to allow the movement of the ssRNAs .

Given that the ssRNAs were approximately 8 kDa, their lack of movement when KN1 was provided suggested a sequestration mechanism or a more complex ssRNA-binding protein interaction than is currently presumed. In an informative experiment, rice thioredoxin a major phloem sieve tube protein with basic antioxidant functions, was expressed in E. coli and fluorescently labeled with FITC . In tobacco, the labeled, heterologously expressed RPP13-1 protein was observed to migrate beyond the site of injection. However, the similarly purified and labeled E. coli homolog of RPP13-1 was not phloem-mobile under duplicate conditions, suggesting significant sequence or structure requirements for movement. Co-injection of rice RPP13-1 and FITC-labeled dextrans established that RPP13-1 increased the plasmodesmatal size exclusion limit to 9–20 kDa, from ∼1 kDa. Furthermore, two mutants of RPP13-1 that were deficient for mobility were identified and crystal structure prediction studies suggested that charged clusters of residues on the outer surface were responsible for binding and/or transport of RPP13-1 through the companion cell-plasmodesmata complex . Aoki et al. demonstrated the importance of protein structure for mobility using two heat shock proteins , CmHsc70-1 and CmHsc70-2, that had been isolated from pumpkin phloem sap. In microinjection experiments, CmHsc70-1 and CmHsc70-2, interacted with plasmodesmata, increasing the size exclusion limit and thereby, enhanced their own cell-to-cell transport. The C-terminal region of these HSPs potentiated their noncell-autonomous mobility through the plasmodesmata. A gainof-function experiment in which the C-terminal cucumber HSP motif was fused to a human Hsp70 protein established that the fusion protein, but not WT human Hsp70, could move from cellto-cell following micro-injection into pumpkin cotyledons, much like the movement of injected intact CmHsc70-1 and CmHsc70- 2. Interestingly, fusing the HSP C-terminal motif to GFP did notresult in cell-to-cell migration, suggesting that at least in this case, the targeting motif was only active in the context of highly conserved HSPs . Unlike nuclear localization signals or ER-targeting peptides, vascular system targeting peptides may have several different motifs, perhaps suggesting specialized interactions with different families of proteins, and/or selective import/export mechanisms. While targeting motifs appear to be important in regulating mobility, a non-regulated diffusion-based mechanism in the symplast from one cell to another is supported by the observation that protein size influences non-targeted movement of GFP but differences appear to be species- and developmental stage-dependent. Earlier studies indicated that non-regulated diffusion is limited to ∼50 kDa proteins in mature leaves and 60 kDa proteins in developing leaves . Unregulated diffusion-based movement across the sieve tube element–companion cell complex has been observed when CC specific promoters regulate 27 kDa GFP expression.While it was perhaps not surprising to detect the GFP in the vascular system due to the porous end plates of the SE, unloading of the GFP into the mesophyll sink cells was unexpected. Using the same promoter, GFP-fusions as large as 67 kDa subsequently were shown to traffic from CC to SE in root tips, although larger variants were restricted to a zone of cells adjacent to the mature protophloem.

A promising approach that can be used to guide “breeding for manipulation” is the use of plant and robot geometric models to co-design tree structures and machines to optimize manipulation reachability and throughput . Also, the use of large numbers of simpler, cheaper actuators that approach plants from different positions has shown promise in terms of reachability , and could be adopted to increase overall throughput.Agricultural robotics enable sensing and interacting with crops at fine spatial scales, even at the level of individual plants or plant parts. Thus, they enable high-throughput phenotyping for breeding improved crop cultivars, and ultra-precise farming, which is a key technology for increasing crop production in a sustainable manner. They can also generate crop-related data that can be used to increase food safety and traceability, and to optimize crop management. Furthermore, agricultural robots can reduce our dependence on unskilled farm labor, which is diminishing in many countries. Also, the emerging paradigm of replacing large conventional agricultural machines with teams of smaller autonomous vehicles could open up possibilities for dramatically changing the way we cultivate crops. Small machines reduce drastically soil compaction and are not necessarily restricted to crop rows; hence, they could be used to establish alternative, productive crop patterns that incorporate mixed cropping, which is known to reduce pest pressures and increase biodiversity. To accomplish their tasks, agricultural robotics face significant challenges. Their mechanical embodiments, electronics, and their sensing, perception and control software must operate with accuracy, repeatability, reliability and robustness under wide variations in environmental conditions; diversity in cropping systems; variation in crop physical and chemical characteristics and responses to environment and management,blueberry containers due to intraspecies biological variation; diversity and complexity of plant canopy structures. Essentially, agricultural robotics must combine the advanced perception and manipulation capabilities of robotic systems, with the throughput, efficiency and reliability of hard automation systems, in a cost-effective manner.

Sleep timing and duration in humans are determined in part by a master circadian clock entrained to local time by retinal inputs encoding environmental light-dark cycles. The clock is phase delayed by light in the evening and early night, and advanced by light in the late night and early morning. With industrialization and on-demand access to electric lighting, exposure to evening light has increased, while exposure to natural light during the day has decreased. The expected net effect is a delay in the phase at which the circadian clock aligns with local time, and thus a delay in the timing of the circadian sleep-wake cycle. Depending on an individual’s social schedule , this may result in a significant misalignment between biological time and social time, a state known as social jetlag. If sleep onset is delayed, but wake onset is fixed by the social schedule, then nocturnal sleep will be restricted. Epidemiological studies have uncovered associations between short sleep and population health, while experimental studies support a causal role for sleep restriction in metabolic and other health disorders currently described as epidemic. This leads to conjecture that a significant portion of the population in industrialized societies may sleep less than is physiologically optimal and that this may contribute to negative trends in population health. Although the logic supporting this conjecture is compelling, empirical support for the argument that industrialization has caused chronic sleep restriction is weak because information about sleep duration prior to widespread availability of electric lighting is anecdotal and based on self-report. Self-reports typically overestimate sleep, compared to objective measures such as actigraphy and polysomnography. Consequently, the degree to which sleep duration has declined with industrialization may be overestimated and is controversial. Trends over the past several decades within already industrialized societies are equivocal, with some studies showing increases, others showing decreases, and some showing no systematic change in sleep length.Another way to estimate the impacts of industrialization on sleep is to study sleep in indigenous communities living traditional lifestyles without electric lighting. Recently, several studies have used wrist-worn accelerometers and light sensors to examine sleep patterns in communities with little reliable access to electric lighting.

The findings generated from these studies have been variable. Daily sleep duration was observed to be unexpectedly short in traditional hunting and gathering societies in Africa and South America, an agrarian society in Madagasgar, and a pastoralist society in Namibia. By contrast, sleep duration was comparatively long in a traditional horticultural society of Papa New Guinea, and in an Argentinian society who were traditionally hunter-gatherers and showed a marked seasonal variation. In cases where societies are transitioning to electricity, groups with access to on-demand artificial lighting showed a delay in nocturnal sleep timing compared to groups without access . In some, but not all cases, delayed sleep was associated with reduced sleep duration. These results indicate that lifestyle may be an important determinant of habitual sleep duration and provide evidence for an effect of on-demand electric lighting on sleep timing and duration. To further examine the impact of lifestyle and electric lighting on sleep, we used actigraphy to measure sleep timing and duration in indigenous Ni-Vanuatu living traditional, small-scale subsistence lifestyles on Tanna Island, Vanuatu, in south pacific Melanesia. This study population provides some unique advantages, including homogeneity of ethnicity and lifestyle on the island, little seasonal variation in climate and daylength, and the availability of an electric grid in coastal but not inland villages, permitting a within-society comparison of sleep with and without access to on-demand electric lighting. Also, the latitude of Tanna Island, and thus the annual variation in photoperiod, is very close to the latitude of several hunter-gatherer societies previously shown to exhibit short sleep, thus permitting cross-cultural comparisons with a natural control for daylength.We recruited 91 adults living on Tanna Island to participate in our study. Forty-five of the participants lived in coastal villages with on-demand access to electricity, and 46 participants lived in villages that were up to 10 km inland and beyond the electric grid. Some data were lost due to equipment failure or were excluded due to non-compliance , leaving final sample sizes of 39 coastal participants and 43 inland participants .

Participants in both communities live similar lifestyles and rely primarily on small-scale farming for a livelihood.Data were collected from males, females, and females who were currently breastfeeding. It was expected that breastfeeding would lead to higher levels of sleep disruption due to mother-infant co-arousal, therefore breastfeeding females were maintained as a separate sample. Other females were not breastfeeding at the time of data collection and the ages of their children were over 2 years. Because fathers on Tanna Island typically take a less active role than the mother in infant rearing, it was deemed acceptable to collect males as one homogenous group irrespective of “father” status. Age and birthdays are not commonly tracked, so when documentation of age was unavailable, participant ages were estimated visually or relative to the birth of peers. Of those included in the final analysis, demographic variables and other sample characteristics are provided in Table 1. We obtained research permits from the Vanuatu Cultural Centre as well as permission from the elders and chiefs in the host communities. Participants were recruited by word of mouth. We explained the details of the study and obtained informed consent verbally from each participant, as outlined by the Office of Research Ethics at Simon Fraser University, Burnaby BC, Canada. Gifts equivalent to $5 CAD were given for participation. All procedures were performed in accordance with relevant guidelines and regulations and approved by Simon Fraser University.Sources of light at night were most often a singular incandescent light bulb inside dwellings powered by electrical grid ,best indoor plant pots and/or small solar powered LED lights . Solar torches were placed on the floor to facilitate household duties or were carried by hand when walking through the village. Light intensity provided by the torches did not exceed 2 lux, measured at 1-meter distance using the Actiwatch-2 light sensor . During the dates of this study, all participants from communities with access to electricity reported using artificial lights. Although seven participants living in electric communities did not have working electricity at the time of data collection, they each used solar torches, and reported exposure to electric light. Most of the participants from villages without electricity either owned or shared solar torches for use at night, and daily evening use was reported by 84% of participants in non-electric communities. Electronic devices were almost non-existent with the exception of some basic mobile phones, which were not a common source of light at night, especially in villages of the electric grid as there was no ready access to charging stations.Sleeping arrangements were variable, but most sleeping spaces consisted of blankets or foam mattresses on grass woven mats on the floor of the dwelling . Floors were commonly wood plank, or hard packed ground. Co-sleeping is typical on Tanna Island; all participants in this study shared sleeping quarters with multiple children or adults. Sleeping spaces were shared by immediate family , and sometimes with extended family as well . Sleeping arrangements are often flexible and can change readily. Traditional homes are made of local natural materials, carefully constructed to withstand cyclones Separate dwellings are used for sleeping and cooking.

A few dwellings incorporated cement or tin components . Little time is spent inside during the day. Temperature and humidity in typical sleeping spaces were measured with iButtons sampling at 20-minute intervals throughout the study interval. Te iButtons were placed in representative huts in one electric village and one non-electric village. Temperature in the non-electric village during this period averaged ~24.6 °C in the day and ~22.9 °C at night, with an average daily range of 3.9±1.8°C. Electric villages were slightly warmer and drier, averaging ~25.9 °C in the day and ~23.9 °C at night, with a daily range of 3.6±1.8 °C. Te daily temperature minimum occurred at ~06:20h, and the maximum between 13:20–14:00h, in both communities.Although coffee beans are exported from Tanna Island, cafeinated beverages are not commonly used or readily available in the villages studied. All men drink a beverage of kava root, which, although only a few of its constituents have been studied, has been found to have mild sedative, anxiolytic, and antinociceptive/analgesic properties. Sixty-five percent of male participants reported drinking kava on a daily basis. Women are not traditionally permitted to drink kava for leisure nor are they permitted to participate in Kava ceremonies. Food consists primarily of locally cultivated foods, such as root vegetables , seasonal fruit , and on occasion purchased rice, and chicken, beef, fish or pork for ceremonies/celebrations. Cooking takes place over a fire, or with hot stones in an earth oven cooking pit. Breakfast and dinner times were regimented but flexible, and men who attended the nakamals would often eat late. Lunch times were not regimented . Although both community types practice primarily subsistence horticultural lifestyles, 23% of the population of each community type report spending some time participating in wage labour. Farming is the primary daily activity for 93% of the non-electric community members compared to 41% of the electric community members, who report spending more time on other daily activities within the village . Most women report their primary evening activity to be caring for children and attendance at church groups, whereas men report spending time in the nakamal for kava drinking . Bislama is the national language in Vanuatu, but many distinct indigenous oral languages exist on Tanna, and can vary even between nearby villages. Although some terms exist to refer to times of the solar day, residents of Tanna Island do not quantitatively track time within a day , and there are no designated work and free days. Alarm clocks were not used by the participants of this study. Chickens and small pigs wander freely through the villages and often alert residents to sunrise. Individuals do report taking days of from agrarian responsibilities, but working in the garden on these days is replaced with obligations for religious worship . Formal education is not common, but is increasing in prevalence, especially for villages in close proximity to a school, which is the case for electric villages. Morning social obligations included early awakening by some women to prepare children for school , or to travel to Wednesday market to sell produce to make money for school fees. We did not collect information on the prevalence of school attendance but as noted, electric villages where closer to schools or to roads where vehicles travel.

The physiological roles of mycorrhizal fungi, including arbuscular mycorrhizal fungi on seedlings and nut trees, are increasingly studied. Mycorrhizal fungi provide beneficial symbiosis in the roots of nut trees, accelerate plant growth and biomass production, nutrient acquisition, and increase potential tolerance to abiotic stress, e.g., drought and salt tolerance. Consequently, future outlooks in this field seem optimistic.In pistachio, several fungal and bacterial diseases can infest both the above-ground and under-ground tree parts. Among these, Phytophthora root and crown rot , Armillaria root rot , and Verticillium wilt are the three most serious soil borne fungal diseases of pistachio trees worldwide. The P. vera is the only pistachio species that produces edible nuts large enough for commercial use . Initial evaluations demonstrated that the P. vera seedling trees were susceptible to soilborne pathogens Phytophthora spp.; Verticillium dahlia, and nematodes.Therefore, other available Pistacia spp. were used as root stocks. Verticillium wilt has killed a majority of the trees in the late 1970s and caused growers the most severe economic losses ever experienced in California. A small number of P. integerrima seedlings from an Iranian seedling tree selected and planted at the USDA Plant Introduction Station in Chico were found to be tolerant to Verticillium wilt; the trees can be infested but exhibit few symptoms and no mortality. This P. integerrima seedling root stock was quickly commercialized as Pioneer Gold 1 . Verticillium-tolerant P. integerrima was then used to produce UCB1 which is moderately resistant to this disease; it exhibits mild symptoms when infested but, as with PGI, no mortality. The P. atlantica and P. terebinthus root stocks are susceptible to Verticillium wilt . Armillaria root rot occasionally affects pistachio and resistant root stocks would offer the best protection.

Field trials indicate that P. terebinthus and UCB1 are tolerant,blueberry plant pot whereas P. atlantica and P. integerrima are susceptible to this pathogen. Root and crown rot caused by Phytophthora spp. also can affect pistachio trees. According to Ferguson et al., UCB1 and P. atlantica are more tolerant to Phytophthora root and crown rot than P. integerima. Epstein et al. studied the resistance of four root stocks to Verticillium dahlia. Yield, growth, incidence of Verticillium symptoms, and mortality rates were studied for 10 consecutive years. UCB1 and P. integerrima showed the greatest tree vigor, and UCB1 had the fewest symptoms. UCB1 is also resistant to Phytophthora. Thus, UCB1 has become the major root stock in California. However, a stunted and difficult-to-graft phenotype has emerged in California in clonally reproduced UCB1 root stocks from multiple sources. This has been at times, since 2010, a serious production problem which has been variously attributed to either somaclonal mutation during in vitro propagation or to Rhodoccus sp. bacterial infection, Chang et al.. The syndrome was identified by its appearance as Pistachio Bushy Top Syndrome, PBTS. However, as this problem proved to be non-transmissible in the field, and nurseries can now identify it in young root stocks, it is no longer a problem. Nouri et al. reported a new pathogen, Macrophomina phaseolina in Kern County of California which is characterized by wilted foliage combined with crown rot of the root stock. UCB1 is highly susceptible to M. phaseolina and this pathogen is now an emerging threat to pistachio production in California. One important biotic stress in Persian walnut is black line disease caused by Cherry leaf roll virus. Persian walnut tolerates this virus and is generally symptomless. In contrast, J. hindsii or its hybrids are resistant to CLRV. Blackline symptoms occur when a hypersensitive root stock [Northern California Black walnut , other black walnuts, or hybrids of these with J. regia, are used as root stock for Persian walnut. The virus is transmitted through infected pollen and scions. The pollenborne virus enters through flowers during pollination and is systemically transported to the graft union. The resulting hypersensitive reaction of the root stock and death of tissue at the graft union blocks nutrient and water transport between the root stock and scion.

The hypersensitive response to this virus is controlled by a single dominant gene. To develop CLRV-resistant scion cultivars capable of blocking the virus at the pistillate flower and/or movement toward the graft union, a breeding program was initiated in 1984 the University of California-Davis to backcross resistance from Paradox into scion cultivars with commercially acceptable horticultural traits. This program is still ongoing. A DNA marker related to CLRV-resistance that maps to ~6.2 Mb onchromosome 14 has been developed in order to accelerate selection of CLRV-resistant offspring. In continuation of work started by E. Germain , a hybrid resistant to black line is in evaluation to be registered in France. In California, screening of a huge multi-species Juglans population, J. regia, J. microcarpa, J. major, J. cathayensis, and others and targeted interspecies hybridization between the selected superior genotypes to produce root stocks resistant to the soil borne pathogens, Agrobacterium tumefaciens, Phytophthora spp.; Pratylenchus vulnus, and Armillaria mellea, has been in progress for several years and is continuing. Crown gall is a major root stock issue in walnuts, particularly when using Paradox hybrid root stocks. This bacterial disease can significantly reduce production and increase management costs. The RNAi technology, RNA interference has been used experimentally to suppress genes involved in the plant response to the bacterium. Silencing of tryptophan monooxygenase and isopentenyl transferase genes blocks bacterial induction of de novo auxin and cytokinin and therefore prevents gall development. Using RNAi-mediated silencing technology, walnut researchers at UC-Davis were also able to develop apparent nematode resistance in Paradox microshoots evaluated in vitro but this work has not been confirmed in the greenhouse or field trials. Nematodes are another serious problem for nut growers. Three separate root-knot nematode resistance genes have been identified in Prunus species, Ma in the Myrobalan plum clones ‘P29800 and ‘P21750 , RMia in the peach root stock ‘Nemared’, and RMja in the bitter almond ‘Alnem’. Pyramiding of these three genes by interspecific crosses of almond × peach × Myrobalan is the main objective of the French root stock breeding program. 

To ensure the presence of the three genes in the same root stock, it has been necessary to develop effective molecular markers. The identification of intra-gene markers for nematode-resistance genes Ma and RMia has allowed the application of marker-assisted selection for these two genes. The RMja gene is located on linkage group 7 of the Prunus genome in the same region as the Ma gene. Chestnut cultivation has been threatened by chestnut blight and root rot diseases. The first pandemic disease for chestnut was root rot. Today two species of root rot are widelyspread in Europe and Asia Minor. The most effective method against root rot disease is using resistant root stocks. Due to the resistance to root rot, C. crenata and C. mollissima trees were imported into Europe at the beginning of the 19th century. The following years showed that their nut quality was low, and they were sensitive to spring frosts. Therefore, they were used as root stocks. However, graft incompatibility was observed. In France, two of these genotypes were registered as ‘Ipharra’ and ‘Marki’. Schad et al. planted some superior genotypes in orchards infected with Phytophthora spp. in 1946. As a result of this study, natural hybrids of C. crenata × C. sativa were obtained [260,261]. Amongst those, ‘Marsol’ and ‘Maraval’ have been used as resistant root stocks. ‘Marigoule’ has been used in forest areas due to its fast-growing characteristic. Now, ‘Marigoule’ is also used as root stocks in many countries due to its resistance to root rot and tolerance to the chestnut blight . However, seedlings of the ‘Marigoule’ are not tolerant or resistant to these diseases as a scion cultivar. Ten years of observation have demonstrated that ‘Marigoule’ seedling survival from root rot is only 10% greater than European chestnut seedlings. As a continuation of these studies, in 1980,plastic gardening pots a new breeding program was initiated in France. Early results showed that ‘Maridonne’ and ‘Marlhac’ root stocks could also be used against root rot . This breeding program is continuing. A similar program was also initiated in Spain by Gallasteguie in 1926 and continued by Urquijo. They imported some chestnut genotypes from Korea and Japan between 1917 and 1940. In this study, 263,000 genotypes were tested and 12,000 of these were found resistant to Phytophthora. As a result of this study, genotypes 111-1, 7521, and 1483 were selected for both resistance to root rot and better graft compatibility with chestnut cultivars. Genotypes ‘CHR-1510 , ‘CHR-1370 , ‘CHR-1680 , ‘CHR-1610 , ‘CHR-310 , ‘CHR-1490 , ‘CHR-1470 , ‘CHR-1670 , and ‘7760 also were found promising. Hybridization has been undertaken in several countries, including Portugal, Italy, Australia, and USA, to obtain root rot-resistant root stocks. A limited number of resulting hybrids were used commonly but most of them exhibited graft incompatibility problems. One example is ‘Menzies’ , commonly used as a seedling root stock source in Australia for its resistance to root rot. In Asia, chestnut production is from C. crenata and C. mollissima trees which are naturally resistant to chestnut blight and root rot but are sensitive to the Asian chestnut gall wasp . In Japan, seedlings of ‘Shibaguri’ have been used as scions for production but devastation from the gall wasp has reduced yield. In recent years, due to graft incompatibility problems, they have started using seedlings of the chosen scion cultivar as seedling root stocks. Scions and root stocks can interact at trans-graft-union movement at the molecular level in different ways. In some cases, mobile macromolecules and large signaling molecules can move through the graft union via the vascular system and regulate various physiological processes in scion including vigor, yield, water use efficiency, biotic and abiotic resistance, etc..

RNAs and proteins can be targeted to move up through the graft union and this process has been studied in various vegetable and fruit trees. In addition, some studies have focused on protein production in transgenic root stocks with targeted delivery to scions to control disease. Transgenic root stocks expressing a polygalacturonidase inhibitory protein , were able to protect wild type scion from both a bacterial disease caused by Xylella fastidiosa and a fungal disease caused by Botrytis cinereal, both pathogens use polygalacturonase as a virulence factor. Recently, the strategy of delivering therapeutic proteins from a root stock to a scion was validated in the field where transgenic root stocks were able to transgraft protected a sensitive wild type scion variety from succumbing to Pierces Disease.Transgrafting also holds great promise for the improvement of nut tree root stocks. Commercially accepted scion cultivars grafted onto transgenic root stocks could benefit from the root stock-mediated increase in productivity and/or disease resistance while avoiding potential consumer concerns regarding use of any transgenic scion. Rootstocks can also be improved with enhanced features while simultaneously designed to avoid transmission of macromolecules or products to the scion. As discussed in the biotic stress section, a crown gall resistant root stock was generated by silencing ipt and iaaM genes responsible for tumor formation. Examination for movement from the transgenic root stock to a standard untransformed scion showed that none of the genes or their products transfer through graft union. This method can produce root stocks with enhanced disease resistance or other features while avoiding concerns about changes in the scion or food product.The technique of producing trees on root stocks means two species are genetically joined and therefore can affect one another’s performance. The selection of root stock is an important aspect of orchard management. In nut tree crops, root stocks influence vigor, rooting ability, water and nutrient uptake, bud break timing, yield, nut quality, susceptibility to abiotic factors including temperatures, drought, waterlogging and salinity, and biotic factors, including crown gall, root rot, root-knot nematodes and soil borne fungal infections, harvest efficiency and post harvest nut quality. And now, producing sustainable orchards which can meet the challenges of climate change and economic production, producing better root stocks is even more important. Breeding tree nut crop root stocks began many years ago when local growers near the centers of a species origins started collecting and domesticating the best wild species tolerant to abiotic and biotic stresses and that also produced good nuts.

Advances in irrigation and soil management techniques, along with the application of chemical fertilizers and pesticides introduced by the Green Revolution in farming, resulted in a substantial increase in food production over the last 50 years. However, the potential of chemical fertilizers to disrupt soil health, the food chain, and ultimately human health has led to renewed interest in the consequences of their application, and has resulted in a substantial increase in the number of certified organic farms. Organic systems, defined by management practices lacking the application of synthetic fertilizers and pesticides, appear to reduce the burden of xenobiotics in the food chain, but there is still controversy regarding the nutritional advantages of organic versus conventionally produced food. Irrespective of the impacts on food quality, the potential for organic farming to impact soil health has been expounded as a significant benefit. However, only a few studies have explored these phenomena while taking into account all the confounding variables. Synthetic fertilizers can result in disrupted soil health, and may negatively impact plant growth as well as soil and plant microbial diversity and structure. Therefore, the cumulative use of such compounds could lead to the loss of productivity and economic revenue. Organic fertilizers are known to have several advantages to improve soil fertility, such as the ability to increase organic matter content in soil, improve the soil structure, enhance soil nitrogen content,planting blueberries in a pot enhance nutrient availability, and improve nutrient mobilization as well as increase root growth.

Organic practices rely upon crop rotations, crop residues, animal and/or green manure, off-farm organic wastes, mechanical cultivation, mineral bearing rocks, and aspects of biological pest control to maintain soil productivity and supply plant nutrients. It is generally assumed that greater soil microbial species’ richness promotes enhanced functional stability and thus soil health, but it is unknown whether short-term synthetic fertilizer application versus organic fertilizer will have a substantial impact on these ecological properties. Organic systems have previously been associated with either an increase in soil microbial richness or no significant change. Some of this variance may be explained by differences in the composition of the organic amendment, the time of harvesting, the studied time span, the rotation of crops planted, and so on. However, in general, studies suggest that short-term organic fertilizer amendment leads to a copiotrophic microbial community, while long-term application will result in a more stable community. Overall, Proteobacteria and Firmicutes dominate organic farming systems in long-term field experiments, with plant growth-promoting genera also enriched , while Actinobacteria and Acidobacteria predominate in conventionally managed lands. In the present study, soil type, land use, crop rotation, crop variety, and climate/weather were all standardized in a short-term field experiment conducted on adjacent plots that share the same land use history, same soil edaphic properties, and same environmental stressors . In each plot, a synthetic chemical fertilizer and a natural fertilizer were applied, followed by planting with Solanum lycopersicum and Beta vulgaris.Previously, this system was used to demonstrate that short-term organic fertilizer application did influence the accumulation of essential and non-essential elements within these crops, but overall, there was little or no clear effect of the fertilizer type on the elemental accumulation in the fruits, suggesting that the plant nutritional value was neither improved nor reduced in the short-term.

Here, we employed 16S rRNA amplicon sequencing to characterize how the soil microbial community responded to these treatments and to determine to what extent the community shifts are comparable to the changes occurring in the long term. We identified biomarkers of fertilization type and correlated bacterial shifts with the accumulation of chemical elements in soil and tomato and Swiss chard roots. The proportional differences in predicted bacterial functional genes in response to the fertilizer amendment were also evaluated. Extending the duration of the experiment would help to resolve whether the microbial shifts observed here are persistent over time and determine if the treatments have an impact on soil quality in the long term, which is ultimately required for evaluating the sustainability of land-use regimes. A field experiment was conducted on adjacent plots to study the prokaryotes community diversity and composition of soils under different fertilization treatments, conventional versus organic. In each of the plots, two plant species and B. vulgaris were planted in two subplots . Soils samples were collected over 3 months after fertilization and crop planting.While most studies agree that farming practices impact soil microbiota and the accumulation of elements within the plant, obtaining generalizable conclusions has been difficult, as results are dependent on the applied management, the composition of the organic amendments, the time of harvesting, the time span studied, the rotation of crops planted, and so on.In the present study, which standardizes for differences in soil properties, crop type, and climate conditions, changes in soil microbial richness were observed over the duration of the experiment associated with the crops’ developmental stage. Interestingly, microbial richness was greatest in July and lowest in August for both conventional and organic soils, suggesting that while nutrient supplies start to decrease, there is a decrease in richness.

In addition, the combined effect of ‘sampling date’ and ‘farming system’ significantly correlated with bacterial richness, with the values being higher in organic soils. While the higher richness in organic systems might be in part due to the introduction of microorganisms present in the manure into the soil , Bacteroidetes, and Chloroflflexi,previous data suggest that organic farming systems promote habitat diversification, favoring a more heterogeneous species distribution, or by stimulating the growth of copiotrophic microorganisms.In conventional soils, lower diversity might be expected because of the elimination or growth inhibition of particular bacteria in response to chemical compounds coming from pesticides/fungicides. Predicted functional diversity, however, did not change according to the farming system. pH is known to influence microbial composition as well as the mobility of heavy metals, influencing micronutrients’ uptake. In the present study, while pH was a determinant factor explaining the bacterial community structure found in the soil samples, it did not correlate with the alpha diversity estimate. Despite both plots starting at the same pH values, conventional fertilizer resulted in a reduced pH over time, consistent with the observed impact of chemical fertilizers in longer-term experiments. pH variation influences Proteobacteria, Actinobacteria, and Acidobacteria abundances. For example, Lauber et al. found that the relative abundance of Acidobacteria decreased with pH, while Actinobacteria and Bacteroidetes positively correlated with soil pH. In our study, Bacteroidetes positively correlated with pH,raspberries in pots while Actinobacteria was negatively correlated. The different trends observed in this study might result from the soil having only been fertilized once, which would select for copiotrophic taxa, compared with other studies where multiple recurrent fertilizations were applied over years. In addition, titanium abundance was positively associated with the community structure found in organic soils, and negatively associated with that of conventional soils. In contrast, the abundances of Ag, Mn, As, Fe, Co, Cd, and Ni were mainly associated with conventional soils. Titanium is considered to be a beneficial element for plant growth, improving crop performance through stimulating the activity of certain enzymes, enhancing chlorophyll content and photosynthesis, promoting nutrient uptake, strengthening stress tolerance, and improving crop yield and quality. In agreement, organic soils in the present study shifted towards the enrichment of taxa involved in nutrient cycling as well as in disease suppression. When studying the accumulation of chemical elements within the plants, Liñero et al. documented a differential accumulation according to the fertilization practice. Higher concentrations of Mn, Co, Na, Mg, Cd, and Tl were observed in conventionally grown tomato plants, while Mo, Cu, Zn, K, and Ba abundances were higher in the organically grown ones. Similarly, Swiss chards under organic amendment were more concentrated in Ba and some nutrients. Interestingly, the soil bacterial community of the present study was a good predictor of Mo and Cu accumulation in organic tomato and Swiss chard roots, thus suggesting that those elements’ absorption is favored, in part, by means of the soil microbial activity.

For instance, because of the spraying of copper sulfate on plant aerial parts, a higher accumulation of Cu might be expected in organic roots. Besides, its higher concentration in organic agricultural practices has been previously associated with a higher presence of arbuscular mycorrhizal fungi, and their synergistic interactions with several bacteria, such as species belonging to Rhizobiales and Methylococcales, are already well known. The soil’s bacterial community composition was significantly influenced by the management practice in the 3 months of the experiment, as observed in previous long-term studies. However, the experiment conducted should be extended over time to assess whether the microbial shifts associated with the farming system and the differential uptake of elements by the crops under study persist, in order to evaluate the farming system’s impact on the quality and health of soil, and hence the sustenance of the system. In any case, in the present study, similar to Lupatini et al., but in contrast to Wang et al., organic samples tended to have greater beta diversity compared with conventional samples, suggesting a greater heterogeneity in the microbial composition of organic soils. Hartmann et al. found that ~10% of bacterial and fungal OTUs were specific to the farming system and Lupatini et al. reported that Proteobacteria and Acidobacteria were highly sensitive to the farming practice. In the current study, the abundances of most common bacterial phyla were not statistically different between farming systems, but few phyla were significantly associated with each of the farming systems. For instance, Chloroflflexi, Thermi, Spirochaetes, and Euryarchaeota had greater mean relative abundance in organic soils, while a higher abundance of Firmicutes was observed in conventional soils. Within Chloroflflexi, members of the class Anaerolineae were enriched, which are known for their role in nitrogen cycling and have been previously identified as a highly represented bacterial group in manure. Several other denitrifiers were also augmented compared with conventional soils, including genera Azoarcus and Thauera, Parvibaculum, and Saccharomonospora, while nitrifiers were depleted. Ding et al. observed a similar result when studying microbial community changes in a long-term organic greenhouse farming, where the relative abundances of ammonia oxidizing microorganisms and anaerobic ammonium oxidation bacteria decreased in the organic soil. Furthermore, an increase in methylotrophic bacteria, for instance, those belonging to families Methylophilaceae and Methylococcaceae , as well as the archaea Methanosarcina, was observed, likely associated with their capability to metabolize methane and its derivative compounds that accumulated after the decomposition of the introduced organic matter in organic farming. Taxa belonging to Myxoccocales, Alteromonodaceae, and various Rhizobiales OTUs, known to contribute to general nutrient cycling , were also more abundant in organic soils, similar to previous reports. Members of Ignavibacteria order, suggested to be involved in the degradation of organic matter, were higher in the soil under the organic farming, similar to the results observed in longer-term field and greenhouse studies. However, other groups known to be capable of degrading various complex organic materials coming from manure or compost, such as several genera within Firmicutes phyla, did not respond in that direction. While it could be speculated that they might need recurrent organic amendments to respond, a 12-year greenhouse study suggested that Firmicutes were the least affected phyla by farming system. Interestingly, previous studies report that organic farming systems tend to increase the abundance of microbial taxa associated with plant health promotion. Several members of Firmicutes and Actinobacteria have been associated with disease suppression and have been reported to be augmented in organic farming. However, this was not the case in this study after a one-time manure application, where, for instance, higher abundances of Bacillus, Nocardia, Mycobacterium, and Rhodococcus were observed in the conventional soils. Besides, the plant growth-promoting Myxococcales was ~3.5 times more abundant in organic compared with conventional soils. They are considered to be micropredators that can produce secondary metabolites with antibacterial and anti-fungal activities, killing other microorganisms, and as such, have been suggested to likely play a key role as potential disease-suppressing bacteria in organic farming soils. Interestingly, Myxococcales, unlike the mentioned Firmicutes and Actinobacteria members, have been consistently found to be enriched in organic soils in both short- and long-term experiments, thus suggesting a rapid and lasting response of this bacteria to the organic amendment. Thus, these organisms’ population distribution and functional genes deserve further investigation.

The inoculum of the fungus is highly abundant and ubiquitous and usually comes from infected plant tissues . B. cinerea mainly enters the host via wounds or natural openings . Infections of non-senescing or unripe plant organs usually lead to limited damage and quiescent infections . Different types of quiescence have been described: delay of conidia germination or growth arrest after germination , endophytic symptomless growth in the apoplast , colonization of abscising flower organs followed by growth into ovaries or receptacles where growth arrests . Independent of the type of infection, the pathogen generally enters a short asymptomatic, biotrophic phase at the beginning of the disease cycle . An aggressive necrotrophic phase commonly succeeds the quiescent or asymptomatic phase once plant organs start to senesce or ripen, during which B. cinerea causes rapid decay of the infected tissues . B. cinerea’s infection mechanisms have been studied in model organisms and further characterized thanks to the availability of high-quality reference genome sequences . The fungus is known to actively promote plant susceptibility by employing a variety of virulence factors . In early stages, B. cinerea deploys sRNAs and effector proteins to suppress premature host cell death and immune responses, which enables the fungus to establish inside the host and accumulate biomass prior to the necrotrophic phase . It was demonstrated that B. cinerea Dicer-like proteins DCL1 and DCL2 produce sRNAs that are secreted from fungal hyphae and translocated to the plant cell where they interfere with the host RNAi mechanisms to silence host immune response genes in Arabidopsis and tomato leaves .

Some secreted virulence factors can lead to host cell death,blueberry pot like effector proteins, toxins and enzymes involved in reactive oxygen species production . B. cinerea can also secrete oxalic acid that lowers the pH of the host tissues and stimulates the production and activity of fungal enzymes like pectinases, laccases and proteases . Furthermore, oxalic acid accumulation leads to Ca2+ chelation, which in turn weakens the pectin structures of plant cell walls and inhibits the deposition of callose . Other virulence factors are cell wall degrading enzymes that enable B. cinerea to cause plant cell lysis and loosen walls to facilitate tissue penetration . The fungus is known to produce plant hormones or hormone analogues that may disturb the host’s cellular metabolism and immune responses. The relevance of these mechanisms for the capacity of B. cinerea to infect strawberry remains unknown.Grey mould in strawberries can result from B. cinerea infections of open flowers or by penetration of fruit receptacle tissues . In primary infections, B. cinerea infects flower organs during or right after flowering, allowing hyphae to grow into the receptacle . The sources of primary inoculum range from overwintering sclerotia to conidia or mycelium from infected neighbouring plants . Infected senescent petals, stamens and calyxes can facilitate primary infections in fruit . Histological studies have shown that even though styles are frequently infected, fungal growth appears to be strongly inhibited and never reaches the receptacle. In contrast, fungal growth in colonized stamens can reach the receptacle in some cultivars . Following infection of the unripe receptacle by B. cinerea, fungal growth is usually arrested and a symptomless quiescent phase occurs. The mechanisms that lead to quiescent infections are not yet fully understood. Proanthocyanins appear to induce B. cinerea quiescence in unripe fruit by restricting the activity of fungal enzymes like polygalacturonases that are necessary for aggressive infection of hosts .

Even though PA content in fruit remains constant during ripening, increasing polymerization of PAs leads to lower inhibitory activity in ripe fruit . Similarly, anthocyanins might delay B. cinerea infections or cause quiescence . For instance, strawberries illuminated with white fluorescent light showed increased anthocyanin content and delayed development of grey mould . Reduced fruit decay has also been observed in raspberries with high pigmentation and in transgenic tomatoes that accumulate anthocyanins . Other small phenolics, especially catechins, may have a role in quiescence. High levels of catechins inhibit fungal growth, and a decrease in catechins is correlated with a reduction of other anti-fungal compounds such as lipoxygenases . Interestingly, young and ripe fruit have low catechin concentration, suggesting that initial infections of young receptacles are possible because they do not yet accumulate enough catechins to stop colonization . B. cinerea quiescence is complex and involves additional factors besides the accumulation of phenolic compounds. It has been proposed that quiescence in unripe fruit is initiated by: lack of nutrients such as sugars from the host, presence of preformed anti-fungal compounds, unsuitable environment for fungal virulence factors . In unripe strawberries, factors from all three categories are present, including lack of available sugars , preformed anti-fungal compounds , and high activity of PG-inhibiting proteins . Induction of the necrotrophic phase in ripe strawberries could be triggered by changes in biochemical composition of the host tissues associated with the ripening process, such as increased sugar content, volatile production and alteration of plant deffences . These modifications promote not only fungal growth but also host susceptibility, e.g. via the release of oxalic acid and efflux of toxins .

During secondary infections, the fungus initiates the necrotrophic phase without quiescence . The sources of conidia for secondary infections can also be diverse, from senescent leaves to infected fruit . Conidia from B. cinerea-infected flower parts are major sources of secondary inoculum . It has been estimated that more than 64% of the strawberry infections result from organic fragments that are in contact with the fruit, such as petals and stamens . Contrary to other fruit , senescent flower parts often adhere to strawberries long enough to retain water films for at least 8 h, which is the time needed for B. cinerea conidia germination . Secondary infections can also result from nesting, which corresponds to direct penetration of mycelia growing on neighbouring plant organs such as infected leaves and fruit . Generally, secondary infections proceed rapidly and B. cinerea can complete its germination and infection as fast as 16 h post-inoculation with a rapid increase in fungal biomass at 48 hpi . Early responses of strawberries to infection include higher expression of the defence genes FaPGIP and FaChi 2-1 , whereas lower expression of the reference gene DNA Binding Protein – FaDBP indicates extensive cell death induced by B. cinerea at late stages of infection .Fruit ripening influences the susceptibility of strawberry fruit to B. cinerea . Strawberries are mostly resistant to infection in their unripe stage, where they restrict fungal growth by causing quiescence. However, in the ripe stage, strawberries are highly susceptible and decay rapidly. Fruit susceptibility to fungal disease increases as ripening progresses; hence, B. cinerea appears to promote susceptibility in unripe fruit by activating specific ripening-related processes . In tomato fruit, master transcriptional regulators of ripening have been shown to have different roles in disease susceptibility. For example, the activity of the tomato transcription factor NON-RIPENING favours B. cinerea infection . Strawberries are non-climacteric fruit with a ripening programme different from that of climacteric tomatoes. Thus, a deeper understanding of strawberry ripening regulation and how B. cinerea may modulate particular ripening events are pivotal to characterize the dynamics of the strawberry-B. cinerea pathosystem. Recent transcriptomic studies of developing strawberries point out that ripening events start between the ‘large green’ and ‘white’ stages, and involve changes in cell wall composition, sugar metabolism, hormone biosynthesis and responses, pigmentation and antioxidant levels . Moreover, a general decrease of oxidative phosphorylation processes has been observed during strawberry ripening . Normal strawberry ripening involves a variety of biochemical and physiological processes,nursery pots some of which are discussed below in the context of B. cinerea interactions.Ripening is associated with the disassembly of the fruit cell walls, which leads to tissue softening.

Cell wall degradation benefits B. cinerea as it reduces mechanical barriers to infection and spread, increases the possibilities of bruising and provides the fungus with access to simple sugars as a carbon source . In strawberry, cell wall solubilization occurs early in fruit development when the walls start to swell . Cell wall solubilization correlates with an increase in fruit sugar content, resulting from polysaccharide breakdown. A decrease of acid-soluble pectins and the alcohol-insoluble fraction of cell walls occur during ripening, whereas the water-soluble content increases . The degree of pectin solubilization and depolymerization is highly related to strawberry fruit firmness . Silencing of an endogenous pectin lyase gene in strawberry resulted in fruit with higher external and internal firmness, mostly due to low pectin solubilization, stiffer cell walls, and increased cell to cell adhesion . Besides PL, other enzymes that may have affected strawberry firmness include PGs, β-galactosidases, endoglucanases, α-arabinofuranosidases and β-xylosidases . In addition to the fruit endogenous cell wall disassembly, B. cinerea secretes an extensive array of CWDEs that target most polysaccharides in the fruit cell walls, particularly pectins . These CWDEs include fungal PGs, such as Bcpg2, a gene that is mainly active in the early penetration stage . The expression of B. cinerea PGs is dependent on the host species, the plant tissue, temperature and the stage of infection .Another barrier for B. cinerea infection is the fruit cuticle. During fruit expansion and ripening the cuticle gets thinner, which makes strawberries more susceptible to initial penetration by germinating conidia. B. cinerea can penetrate the plant cuticle by secretion of cutinases . Additionally, cuticle properties can result in higher incidence of cracks and other damages through which B. cinerea can enter the fruit without the need of cutinases . Studies on strawberry cuticles are scarce and only exist for leaf tissues . In tomato fruit, thicker and stiffer cuticles lead to higher resistance to initial B. cinerea infections. Moreover, it is known that the chemical composition of the cuticle changes during tomato ripening, and this is likely to be the case in strawberry .During ripening, the content of sugar in strawberries increases and therefore can serve as nutrients for B. cinerea. In unripe strawberries, the main sugars are glucose and fructose with low concentrations of sucrose. Sucrose levels increase rapidly during de-greening and red colouring . In tomato, it has been shown that the Cnr mutant, which does not accumulate high levels of sugars is still highly susceptible to B. cinerea infection . This observation suggests that even though sugars may serve as a susceptibility factor, high sugar concentrations are not essential for B. cinerea infection. However, sugar content could still influence susceptibility to B. cinerea as specific sugars may serve as ripening initiation signals. For instance, sucrose regulates abscisic acid levels in strawberries, which are necessary for normal ripening and could influence fruit susceptibility as described below . Like other ripening-related events, B. cinerea can alter neutral sugar and sugar acid levels in the infected host tissues, mainly by degradation and depolymerization of cell walls. This was reported for infections in tobacco and Arabidopsis leaves, where the fungus degrades pectins to release the monosaccharide galacturonic acid .ABA biosynthesis during fruit ripening is triggered by a decrease in pH, turgor changes, sugar accumulation, and the switch of sugars from mainly glucose and fructose to sucrose . Effects of ABA on strawberry susceptibility to fungal disease have not been extensively studied, but down regulation of the ABA biosynthetic gene β-glucosidase FaBG3 has been reported to result in fruit with limited ripening and higher B. cinerea resistance . In tomato, ABA accumulation is related to higher pathogen susceptibility, probably via activation of senescence . During strawberry ripening, the increase of ABA is correlated with a decrease of auxin, which induces early fruit growth and expansion but is known to inhibit ripening processes . The role of auxin in fruit susceptibility seems to depend on the plant species, as indole acetic acid treatment in Arabidopsis leads to susceptibility, whereas IAA-treated tomato leaves and eggplant fruit show lower infection severity . Ethylene has a secondary organ-specific role in strawberry ripening, particularly in achenes and green and white receptacles . Ethylene increases the susceptibility of tomato to B. cinerea by inducing ripening; however, its functions during strawberry infections are yet to be fully characterized.

Seeds produced from these crosses were germinated at the University Lindcove Field Station in Tulare County and grown for approximately one year. In 1982, the seedlings were tested by isozyme procedures and the difficult-to-identify nucellars discarded. The hybrid seedlings are being field grown at the Lindcove Field Station and, when they come into fruiting will be tested by Dr. M. L. Roose of the Department of Botany and Plant Sciences for their disease resistance and ultimate horticultural characteristics as root stocks. New possibilities may also arise from recombinations of species, or genera, from graft hybrids or somatic hybrids. Grapefruit develops an excellent root system and although deeply rooted, spreads more widely than that of sweet or sour orange . It is apparently best adapted to growth in heavy or loamy soils and is poorly adapted to light sandy soils. Most Citrus varieties on grapefruit stocks show an overgrowth of the stock at the bud union similar to that of shaddock. In some cases, this is like a shoulder extending out from the bud union, in others it is smooth at the union and flares outward and downward as it approaches the ground line, sometimes referred to as an ‘elephant’s foot’ . The union is, however, cylindrical and shows no tendency for fluting or ridging. The bark is usually slightly thicker than the scion. With some selections, there is a slight bud union overgrowth with lemon scions . At the Citrus Research Center, Riverside, 1927 root stock plantings of trees on grapefruit root developed into normal-sized trees and, at 34 years of age,arandano cultivo were comparable in size to adjacent trees on sweet orange stock and larger than on sour stock with all scion varieties . In spite of their large size, such trees were characterized by surprisingly low yields, which in some cases was only 70 percent of their sweet orange counterpart.

The tendency for low yields has also been noted in Florida by Hume and Ziegler and Wolfe . Fruit size tended to be larger than on sweet orange stock and comparable to that on sour orange or Rough lemon stocks . The larger fruit size might possibly be associated with the fewer numbers of fruit per tree. Fruit quality was good, equal to that on sweet orange stock and frequently slightly superior . Grapefruit root stocks are probably slightly more resistance to Phytophthora than trees on sweet orange or Rough lemon, but are not as resistant as sour orange . That is one of the reasons California growers used it, although Wutscher indicates susceptibility to foot rot was one of the reasons it was discontinued in Florida. California lemon growers also used it because they felt it was more resistant to foot rot than sweet orange or Rough lemon, and that lemon trees budded on it were not as susceptible to shell bark as those on some orange and Rough lemon. Also, phloem necrosis of the lemon scion was less than on sour. They show no resistance to the citrus nematode or the burrowing nematode . Numerous cultivars tested for their resistance to tristeza were variable in their response, some varieties being almost as susceptible as the sour orange, others less, but none were tolerant . Under field conditions, trees declined from natural infection by tristeza at a much slower rate than those on sour orange, erroneously causing some growers in California to assume the combinations were tolerant to the disease. Under California conditions, the stock does not generally stem-pit from tristeza, or only mildly so. In experimental trials conducted by the Citrus Experiment Station, Riverside, the ‘Duncan’ variety did not perform very well. This was the original ‘Duncan’ variety introduced from Florida in the early 1900’s. Recently, all seedy varieties of grapefruit in Florida were lumped as ‘Duncans’. Unfortunately, the original ‘Duncan’ source in the Citrus Variety Collection at Riverside was eliminated and a seedling of it, of doubtful authenticity, substituted.

The original ‘Duncan’ may now be lost. Of all the varieties tested at Riverside, the C.E.S. No. 343 gave the best performance, but even it did not compare with the sweet orange or the sour orange . The C.E.S. No. 343 never gained commercial acceptance. In the eastern Los Angeles basin the ‘Jochimsen’ and the ‘Hall’ were grown. In Ventura County, the ‘Camulos’ was principally grown, and in Santa Barbara County, it was the ‘Stow’. Trees budded on grapefruit stock are less cold resistant in California than those on sour or sweet orange. Although both orange and lemon trees were budded commercially on grapefruit stock, but primarily lemons, results were not outstanding and the stock was never very popular. Because of tristeza, orange trees are no longer propagated on this stock in the U.S. Lemon trees on grapefruit stock can only be purchased through special orders to California nurserymen. Grapefruit cuttings, like shaddock marcots, have performed about equal to the budded trees.The root system, like those of the Valencia and the navel, was less penetrating than those of the seedlings, with a strong shallow lateral system. It is conceivable that in some areas commercial grapefruit orchards under the right conditions could be propagated from cuttings or grown as seedlings and eliminate any bud union problems. In areas where freezes are a major hazard, as in Texas, this could eliminate the practice of mounding to regenerate trees frozen to the bud union or groundline. Several hybrids of the grapefruit have been tried as root stocks and show some interesting results. Among these are the ‘Sampson’ tangelo, ‘Williams’ tangelo, ‘Siamelo’ and the citrumelo. The tangelo will be discussed with the mandarins, and citrumelos with the trifoliate hybrids. The Poorman’s orange is neither an orange nor a grapefruit, although it is used commercially as a grapefruit substitute. Sometimes it is referred to as ‘Morrison’s’ grapefruit or ‘New Zealand’ grapefruit . It is monoembryonic and passes along a genetic scaly bark to its progeny. Like the grapefruit, when budded to Valencia orange it was susceptible to tristeza . The Israeli Poorman is not a Poorman’s orange but something else, perhaps a seedling selection from Poorman .

It is nucellar and does not show the flakey bark of Poorman. It has given fairly promising results in root stock trials in Israel , but its tristeza tolerance is no longer in doubt and it proved susceptible in trials at South Coast Field Station in l97__ . The Natsudaidai of Japan, or Japanese summer orange , is not an orange, or a grapefruit, although it too is commercially used as a grapefruit substitute in Japan where it is the second-most important variety. However, it is used to a very limited extent in Japan as a root stock for the Satsuma. The trees on it are vigorous and do not compare with the more productive trees on trifoliate orange,growing blueberries in containers but the fruit quality is inferior. In California at South Coast Field Station, orange trees budded upon it are tolerant to tristeza, show no stem pitting and are moderately vigorous. However, they are shy bearers . The pummelos or shaddocks, C. grandis [L.] Osbeck , are often confused with the grapefruit, particularly some of the smaller fruited varieties. The pummelos are referred to in some areas as pumplemousse, zabon, buntan, bankan, etc. They differ from most other potential root stocks in one major character, however, in that all varieties of pummelo are monoembryonic and produce no nucellar seedlings. In spite of this, most of their progeny are remarkably uniform in the nursery row and require a minimum of rogueing. When budded to major scion varieties, the scions are remarkably vigorous and uniform in size, even up to an age of 34 years , with surprising uniform performance at the Citrus Experiment Station, Riverside . Some of the better eating commercial varieties like the ‘Alemoen’, ‘Thong Dee’, ‘Kao Phuang’ and ‘Kao Panne’ have been among the most uniform. From the author’s experience, variability is not the major factor in their failure to obtain commercial root stock acceptance . Pummelo fruits, if not parthenocarpic, generally produce an abundance of large seeds, although under certain conditions they may be nearly seedless. Many are self-incompatible and require pollenizers to set seed . The seeds germinate readily and produce vigorous seedlings which are somewhat bushy in character, like the grapefruit, and require considerable training prior to budding. Numerous pummelo varieties have been successfully budded with most of the major commercial citrus varieties in California experiments and observed in various plantings for periods of from 13 to 34 years of age. The reported failure of Satsuma mandarin on pummelo cited by Webber was undoubtedly due to the presence of the tristeza virus in the Satsuma scion. All varieties of pummelos budded with orange scions and inoculated with tristeza by the author , were extremely susceptible to the disease. The reaction of pummelo to tristeza is very similar to sour orange in this respect with many of the young inoculated trees collapsing and dying within a few years. Trees affected with tristeza do not show stem pitting in the stock. This then , is the greatest drawback to the use of pummelo as a root stock, not its variability. In California, especially in root stock trials at the Citrus Experiment Station, oranges, grapefruit and lemons budded on pummelo stocks make large sized trees about equal to those on sweet orange stock. Like grapefruit stocks, yields tend to be somewhat low in proportion to the size of the trees. Like grapefruit, fruit sizes tend to be larger than on stocks like sweet orange and Cleopatra mandarin and more comparable to that on Rough lemon stock . Fruit quality is comparable to that on sweet orange or sour orange stock . The bud union of trees budded on pummelo is good, and like grapefruit stock, shows an overgrowth of the stock.

This may be either a slight shoulder effect or a tendency to slope outward and downward from the union in a slightly pyramidal shape . There is no distinctive character to the bark, except that it tends to be considerably thicker than the scion bark, much thicker than grapefruit. In some instances, the bark may be very thick and extremely rough, like that of an oak tree . Lemon bud union overgrowth does not appear to be a serious problem with the stock in California in the limited plantings observed. The root system of the pummelos is a strong, deeply penetrating tap root system with strong laterals. There are many fibrous roots, with the feeder roots tending to be coarser in texture than feeder roots of most other species . It shows no tolerance to the citrus nematode or the burrowing nematode . Pummelos, however, do have a high resistance to Phytophthora . No problems with gummosis were experienced in any of the author’s plantings. In Thailand, where pummelos are extensively grown, they are propagated principally as marcots and grown on soil beds a few feet above the water table and are relatively short-lived. It is probable that few other stocks would tolerate these conditions as long as the pummelo. In the Philippines, trees grafted on pummelo roots reportedly showed more mottle-leaf disease than on other root stocks observed . In California root stock trials, it was not observed that zinc deficiency was any more of a problem than on the other root stocks. In one lemon root stock trial in Ventura, California, where boron and salt were both somewhat of a problem, the Alemoen shaddock did not show the boron tolerance of C. macrophylla or C. pennivesiculata, but of 40 root stocks, gave essentially the best total performance after 25 years. In Thailand, it is grown under conditions where brackish water would be a problem for many other stocks, but in Thailand salinity, not boron, is the problem. Some varieties of pummelos are not very vigorous when grafted on other root stocks, nor as cuttings, at Riverside. Whether this is due to some unrecognized virus disease, or is of a genetic nature, is not known. Certain hybrids of the pummelo at Riverside have been extremely vigorous, as for example sibs of shaddock X St. Michael sweet orange. One of these hybrids provided fairly interesting root stock results, but proved to be very susceptible to tristeza . The pummelos therefore, have never gained commercial acceptance as root stocks and probably never will in a tristeza area.

The combined top growth of all six root stock seedlings was increased with the addition of VAN fungi and no phosphorus. However, the combined growth with VAN and phosphorus was slightly more. The root stock seedlings decreased in dependency on VAN in the following order: sour orange, Cleopatra mandarin, sweet orange, Rough lemon, Rangpur lime and Carrizo citrange. Root-shoot ratios indicate that root stock mycorrhizal dependency decreases as their capacity for root production increases. Emphasizing the importance of previous work, Timmer and Leyden state the interactions of copper and phosphorus in the fumigation-mycorrhizal syndrome are important. Copper deficiency has been frequently observed in citrus seedlings following the application of phosphate fertilizer. According to Timmer and Leyden, the application of phosphorus induces copper deficiency by stimulating growth of non-mycorrhizal seedlings until copper becomes limiting nutritionally. However, phosphorus-induced copper deficiency appears to be due to phosphorus inhibition of mycorrhizal development of seedlings inoculated with Glomus fasciculatus. Rhodes stresses the fact that mycorrhizae are recognized as being significantly beneficial to host-plant relationships, particularly where root systems are restricted and nutrient systems are low—although he makes no special reference to citrus. Mehraveran also discusses the mycorrhizal dependency of six citrus cultivars .Plants with mycorrhizae and a given phosphorus level are healthier than non-mycorrhizal plants with an equal phosphorus level. The type of mycorrhizae is important; the soil type, area of origin, effects on nematodes, Phytophthora, photosynthetic activity, and hydraulics are all factors to consider. Thus, Menge et al. found that in greenhouse experiments the addition of Glomus fasciculatus significantly increased the growth of Troyer citrange seedlings in 20 of 26 methyl bromide-fumigated soils from Southern California.

Of the six soils in which the mycorrhizal fungus provided no growth increase, two were greenhouse soils ,growing blueberries in pots three were nursery soils, and one was a field soil. Presence of the fungus increased foliar phosphorus, potassium, and copper and decreased foliar magnesium and sodium concentrations in the leaves of the Troyer citrange in the majority of the citrus soils. They present an interesting table on the mycorrhizal dependency of Troyer on Glomus fasciculatus on the 26 soils. While much of the work has been done with sandy soils, the whole soil-mycorrhizal complex is extremely important. Obviously there are different species of the mycorrhizal fungus and one wonders if they are equally effective and if their response varies in a different soil environment. To determine this, Graham, Linderman and Menge tested six VAN isolates. They found that isolates of Glomus fasciculatus was most efficient, and Glomus macrocarpum the least effective. Growth enhancement was significantly greater for Glomus isolates from California than from Florida. It is also stressed that growth enhancement for VAN fungi may vary with the soil type. Johnson reports on the effects of phosphorus nutrition on mycorrhizal colonization, photosynthesis, growth, and nutrient composition in sour orange seedlings. The sour orange seedlings were inoculated with the VAN fungus Glomus intraradices and fertilized with weekly applications of phosphorus. The photosynthetic rates correlated with a high phosphorus content in the leaf tissue of the central plants, but Johnson could find no correlation for the VAN-infected seedlings. He suggests that factors in addition to improved phosphorus nutrition influence the photosynthetic rate of VAN plants. The conclusions of Edriss, Davis and Burger were somewhat similar. Using sour orange seedlings and inoculating them with the mycorrhizal fungus Gigaspora heterogama, they found that the cytokinin production was greater than that of the check non-mycorrhizal plants despite the fact that there were similar dry weights and phosphorus concentrations in the leaves. The enhancement of the cytokinin production seemed to be associated with the mycorrhizal infection rather than increased phosphorus uptake.

The effect of VAN inoculations on nematode populations was first investigated by O’Bannon et al. in greenhouse studies in Florida. They found that when Rough lemon seedlings were inoculated with the citrus nematode, Tylenchulus semipenetrans and then transplanted into soil infected with VAN Glomus mosseae, that the presence of the fungus increased seedling growth. The seedling suppression by the citrus nematode alone was greater than the checks or the VAN inoculated. They made no studies on other nematodes such as the burrowing nematode Radopholus similis. Similarly, Hussey and Roncadori report that nematode suppression of vegetative growth or yield are partly offset by the presence of a VAN fungus. Using Rough lemon seedlings they found that the presence of the mycorrhizal fungus lessened the nematodes’ attraction to the citrus roots, hindered penetration, and the subsequent development and reproduction of the citrus nematode was suppressed. They do not specify why, except that a healthier plant is a more resistant plant. To some extent, this same hypothesis exists with the VAN fungus relationship with Phytophthora. Davis and Menge working with Pineapple sweet orange seedlings, present evidence that suggested that there was some tolerance to Phytophthora parasitica in seedlings infected with Glomus fasciculatus. They also felt this effect was caused by the ability of mycorrhizal roots to absorb more phosphorus and possibly other nutrients than non-mycorrhizal roots, as evidenced by root health and greater phosphorus uptake. Again, a healthier plant is a more resistant plant. They did not report on any studies with Phytophthora citrophthora. Davis and Menge again suggest that VAN fungi have a variable influence on the tolerance of seedlings of Pineapple sweet orange and Troyer citrange to Phytophthora parasitica. There are a number of papers on the effects of VAN fungi and water relationships of host citrus seedlings. The first report perhaps was that of Levy and Krikun . These two Israeli researchers working with Rough lemon seedlings studied recovery from water stress on similar-sized VAN infected seedlings and non-mycorrhizal seedlings. They found the VAN-infected seedlings affected stomatal conductance, photosynthesis and proline accumulation but not leaf water potential.

They suggest that most of the effect of mycorrhizal association is on stomatal regulation rather than on root resistance. Syvertsen studied the hydraulic conductivity of four commercial citrus root stocks. The hydraulic conductivity was estimated using a special pressure chamber technique. He found that Carrizo citrange and Rough lemon had the highest root conductivity, whereas Cleopatra mandarin and sour orange had the least. Further hydraulic studies were conducted by Graham and Syvertsen . They used seedlings of Carrizo citrange and sour orange grown in a low phosphorus sandy soil and either inoculated with Glomus intraradices or fertilized with phosphorus. The mycorrhizal-infected seedlings had sufficient levels of leaf phosphorus, but the non-mycorrhizal seedlings were phosphorus deficient. The root-shoot ratio of both root stocks was reduced by the mycorrhizal colonization, but root hydraulic conductivity per unit root length of mycorrhizal Carrizo and sour orange was more than twice that of non-mycorrhizal seedlings. The mycorrhizal plants had higher transpiration rates, apparently increased bythe conductivity of the roots. The authors felt the response was due to the mycorrhizal enhancement of phosphorus nutrition. In further studies, Graham and Syvertsen grew seedlings of five citrus root stocks in a low phosphorus sandy soil. The seedlings were incorporated into three treatments: inoculated with Glomus intraradices, non-inoculated, but fertilized with phosphorus, and non-inoculated and no phosphorus added. The order of the mycorrhizal dependency of the five root-stocks is as follows: sour orange = Cleopatra mandarin > Swingle citrumelo > Carrizo citrange > trifoliate orange. The less dependent root stocks, i.e., trifoliate orange and Carrizo citrange, had greater leaf phosphorus, finer roots, and slower growth rates than sour orange and Cleopatra mandarin. Rootstocks with a lower mycorrhizal dependency also generally had greater hydraulic conductivity of the roots,frambuesa cultivo greater transpiration and carbon dioxide assimilation rates. In additional but similar studies, Syvertsen and Graham amplify their work again with seedlings of Carrizo citrange, trifoliate orange, sour orange, Swingle citrumelo, and Cleopatra mandarin. Whole plant transpiration and maximum rates of net gas exchange or carbon dioxide and water vapor from single leaves were positively correlated with the hydraulic conductivity of the seedlings. Leaf nitrogen and phosphorus content and shoot-root ratio were also positively correlated with root conductivity. The differences in soil water depletion and plant water relations of trifoliate orange and Carrizo citrange during drought and recovery cycle is related to their root conductivity. They stress that the capability of root systems to conduct water and mineral elements is a very important factor in plant growth and physiological activity. The possibilities of seed inoculation or field inoculation was first mentioned by Newcomb . Hattingh and Gerdemann inoculated sour orange seed, successfully developing a special technique. They coated the seed with a mycorrhizal inoculum in a 1% solution of methyl cellulose. Menge, Lembright and Johnson indicated that commercial production of mycorrhizal inoculum for use in fumigated or sterilized soil was being attempted in several locations in the United States.

They felt the only current way to produce suitable quantities of mycorrhizal inoculum was on roots of susceptible host plants. Contamination by other pathogenic organisms can be a problem. Maybe better methods are available. The most common method for inoculating citrus in the field and in the greenhouse has been to mix the mycorrhizal inoculum with the soil prior to planting or transplanting. Menge et al. felt banding, layering and root inoculation were more efficient than seed inoculation. Root fragments can also be an important source of inoculation. Graham and Fardelmann found that root pieces stored up to one year under moist conditions did not lose the colonization potential with Glomus epigaeum. However, drying reduced this potential to nearly zero after nine months. Glomus intraradices was found sporulating in citrus roots found in orchard soil. They propose that dead root fragments account for a high percentage of the propagules in the citrus soil. The propagation of mycorrhizae cannot be too difficult. For a number of years a citrus nurseryman in California offered mycorrhizae for sale for inoculative purposes. This service is no longer available either because of propagation problems or little demand by the citrus industry. The knowledge of the necessity of the mycorrhizae in the seed bed or the transplant stage is the important thing. The mycorrhizae will gradually re-infect a fumigated soil. The nurseryman could leave the fumigated site fallow for a year or plant host cover crops of cereals and grasses or legumes prior to planting a seedbed or transplanting. Furthermore, field grown nursery trees are gradually diminishing. Container growing is so much more efficient and economical, and container trees can be grown in a shorter period of time. There is also less transplant shock. When the author visited South Africa in 1982, there were only three field grown nurseries left and they ceased with that planting. South Africa is essentially 100% in container growing. Spain and Australia are now heavily into container growing, with more citrus producing areas following suit. If the container planting mix is properly planned and prepared, there will no longer be this problem. For further information on this problem, a nice review of the total VAN potential benefits and interactions is presented by Graham . While the article is a review, it nicely presents the views and facts in pathology, horticulture, and physiology. A book on the subject is currently being written by J. A. Menge of the Department of Plant Pathology, Citrus Research Center, Riverside. Differences in cellular structure have infrequently but effectively been used to distinguish a limited number of stock species. Thus, Penzig found a striking difference in the cellular structure of the pith of twigs of trifoliate orange and sour orange, and this may probably also be true of the roots. Swingle suggested this method of distinguishing between these two species when used in Satsuma production in the U.S. Gulf states. Longitudinal sections of the pith of young stems of the trifoliate orange exhibit an irregular arrangement of thin-walled cells [labeled as] Fig. 66A, while similar sections of sour orange stems show only uniform thin-walled cells arranged in regular series and an entire absence of the crossplates of thick-walled cells . Wolf extended this method to distinguish Yuzu, which was found to have only thin-walled pith cells similar to those of the sour orange, but irregularly arranged . Thus, it differs from the sour orange, in which the cells are arranged in regular series or chains, and from the trifoliate, in which there are crossplates of thick-walled cells .

This antinociceptive response, termed stress-induced analgesia , is mediated, in part, by the release of opioid peptides. However, opioid-dependent and opioid-independent forms of SIA can be differentially activated based upon stressor parameters and duration. Recent research in our laboratories has demonstrated that an endocannabinoid signaling system mediates nonopioid SIA induced by continuous foot shock. A role for cannabinoid CB1 receptors in SIA was demonstrated by our observations that competitive CB1 antagonists, administered systemically or locally in the dorsolateral periaqueduc-tal gray , block nonopioid SIA. Furthermore, SIA is attenuated in rats rendered tolerant to cannabinoids, but not in rats rendered tolerant to morphine. In the midbrain PAG, a key structure implicated in the descending control of pain, stress triggers the rapid mobilization of two endocannabinoid lipids—2-arachidonoyl glycerol and anandamide. These compounds are hydrolyzed in vivo by distinct serine hydrolases. Anandamide is degraded by fatty-acid amide hydrolase, whereas 2-AG is hydrolyzed by monoacylglycerol lipase. Inhibition of either FAAH or MGL in the PAG also enhances SIA in a CB1-dependent manner, further supporting a role for endocannabinoids in regulating expression of SIA at the supraspinal level. The distribution of CB1 receptors in the brain suggests several anatomical regions where endocannabinoid actions could modulate SIA. One such region is the amygdala,best indoor vertical garden system an area of the limbic forebrain implicated in both fear conditioning and affective dimensions of pain. An ascending spino-pontoamygdaloid circuit has been postulated as an “affective” nociceptive pathway. CB1 immunore activity is dense in the basolateral nucleus of the amygdala, but is reportedly absent in the central nucleus of the amygdala. CB1 immunore activity is associated with a distinct sub-population of GABAergic interneurons in the BLA, corresponding to large cholecystokinin-positive cells.

The distribution of FAAH and MGL at this site also correlates well with the distribution of CB1 receptors. The anatomical localization of CB1 in the BLA is consistent with electrophysiological data demonstrating that activation of these receptors presynaptically modulates GABAergic transmission.For example, a form of short-term synaptic plasticity – depolarization-induced suppression of inhibition – in the BLA is blocked by CB1 antagonists. BLA efferents innervate the CeA, the main amygdaloid output nucleus, which sends projections to the PAG and other regions. Thus, an endocannabinoid-mediated reduction of GABA release would disinhibit principal neurons innervating the CeA, to control information processing in the amygdala. Unilateral micro-injection of cannabinoid agonists into the amygdala also induces antinociception in the tail- flick test, supporting a role for this structure in modulation of pain sensitivity. Furthermore, micro-injections of the GABA agonist muscimol in the CeA attenuates cannabinoid antinociception. Unilateral or bilateral lesions of the CeA also suppress the antinociceptive effects elicited by both systemic cannabinoids and diverse environmental challenges. Endocannabinoid signaling in the BLA also mediates extinction of aversive memories, suggesting that endocannabinoids modulate multiple responses to stress via actions in the amygdala. In the present study, we investigated the role of cannabinoid CB1 receptors in the BLA and CeA in nonopioid SIA in rats. First, the CB1-selective antagonist/inverse agonist rimonabant was micro-injected into the BLA and the CeA to examine the contribution of these sites to endocannabinoid-mediated SIA. Based upon the distributions of CB1 receptors in these sites, we hypothesized that pharmacological blockade of CB1 receptors in the BLA, but not the CeA, would suppress nonopioid SIA. To examine the contribution of endocannabinoids in the amygdala to SIA, we administered selective pharmacological inhibitors of FAAH and MGL locally in the BLA, at doses that enhanced nonopioid SIA following micro-injection into the midbrain PAG.

To this end, we used two pharmacological inhibitors that selectively target either FAAH or MGL. The FAAH inhibitor URB597 increases brain accumulation of anandamide but not 2- AG and enhances SIA in a CB1-dependent manner. On the other hand, the MGL inhibitor URB602 increases levels of 2-AG, but not anandamide, in the midbrain PAG and enhances CB1-mediated SIA when micro-injected into this structure. Sixty-three male Sprague–Dawley rats were used in these experiments. All procedures were approved by the University of Georgia Animal Care and Use Committee, and followed the guidelines of the International Association of the Study of Pain and the National Institutes of Health. Rimonabant was obtained from NIDA. URB597 was purchased from Cayman . URB602 was synthesized by reacting diimidazole- 1-ylmethanone with biphenyl-3-yl amine in acetonitrile in the presence of 4-dimethylaminopyradine and subsequently with cyclohexanol as described previously. Animals were anesthetized with a mixture of sodium pentobarbital and ketamine. Stainless steel guide cannulae were unilaterally implanted above either the BLA or CeA using zero points from bregma, the midline suture and the surface of the skull, respectively. Cannulae were fixed to the skull using skull screws and dental acrylic. Five to seven days after surgery, rats were habituated to restraining tubes prior to testing. The latency to remove the tail from a radiant heat source was measured using the tail-flick test. Drug or vehicle was micro-injected using a micro-infusion pump over 60 s into either the BLA, CeA or deliberately off-site. In experiment 1, rimonabant or vehicle was micro-injected into either the BLA or CeA 5 min prior to foot shock. In experiment 2, URB597 , URB602 , or vehicle was administered to the BLA 32 min prior to foot shock. Doses and delays were selected based upon previous studies demonstrating efficacy of the identical drug treatments following micro-injection into the midbrain PAG. SIA was induced by exposing rats to continuous foot shock using a Lafayette grid-shock apparatus and quantified behaviorally using the radiant heat tail-flick test. Removal of the tail from the heat source terminated application of thermal stimulation. Tail- flick latencies were monitored over 4 min immediately prior to exposure to the stressor to evaluate changes in basal nociceptive thresholds induced by pharmacological manipulations.

Tail withdrawal latencies were measured at 2-min intervals before and after foot shock. A ceiling tail-flick latency of 10 s was employed to prevent tissue damage. In all studies, the experimenter was blinded to the experimental condition. Following testing, rats were euthanized with sodium pentobarbital and perfused with saline followed by formalin. Brains were removed, cryoprotected overnight, cryostat-cut , and mounted onto gelatin-subbed slides. Sections were dried and stained with cresyl violet. Injection sites were confirmed histologically using a light microscope. micro-injection sites were confirmed for thirty-seven animals in the BLA and fifteen animals in the CeA . Eleven animals were used as off-site controls. Only animals with histologically confirmed micro-injection sites were included in data for analysis. Tail flick data were blocked for each subject by averaging every two adjacent tail-flick latencies into a single mean, as described previously. Means of two-trial blocks, calculated for each subject, were subjected to repeated measures analysis of variance and ANOVA, as appropriate. Post hoc comparisons were performed using the Fisher’s protected least squares difference , with P < 0.05 considered significant.In all studies, baseline tail-flick latencies did not differ between groups prior to administration of drug or vehicle. Moreover, latencies recorded just prior to foot shock, following injection of drug or vehicle, were similar between groups, indicating that the injection alone was not sufficient to induce antinociception. In all studies,plant pot with drainage foot shock increased tail-flick latencies .By contrast, intra-CeA micro-injection of rimonabant did not alter tail-flick latencies relative to vehicle . Neither the FAAH inhibitor URB597 nor the MGL inhibitor URB602 altered SIA , at doses that markedly enhanced SIA following micro-injection into the midbrain PAG. The present study demonstrates that pharmacological blockade of CB1 receptors in the BLA attenuates nonopioid SIA induced by continuous foot shock. micro-injection of rimonabant into the BLA, where CB1 receptors are dense, suppressed stress-induced antinociception relative to control conditions. By contrast, micro-injection of rimonabant into the CeA, where CB1 receptors are largely absent, failed to suppress nonopioid SIA. micro-injection of rimonabant into regions outside the amygdala also failed to suppress SIA, suggesting that the actions of the cannabinoid antagonist were not due to diffusion to distal sites. Our data are consistent with the observation that CB1 agonists depress monosynaptic evoked inhibitory post-synaptic potentials in the BLA but not in the CeA. Our results, therefore, suggest that CB1 receptors in the BLA modulate local inhibitory networks in the BLA to ultimately regulate expression of SIA. Nonetheless, neither the FAAH inhibitor URB597 nor the MGL inhibitor URB602 enhanced SIA following micro-injection into the BLA, at doses that markedly potentiated SIA following micro-injection into the midbrain dPAG. These differences likely reflect differential modulatory roles of distinct endocannabinoids in the ascending “affective” pain pathway compared to descending pain modulatory systems. Anatomical studies suggest that CB1 is preferentially associated with GABAergic, as opposed to glutamatergic, synapses in the BLA . Nonetheless, in the lateral amygdala, endocannabinoids mediate reductions in both local inhibitory inputs as well as excitatory transmission, whose actions could exert opposing effects. Low frequency stimulation of the lateral amygdala also mobilizes endocannabinoids from BLA neurons to presynaptically induce a long-term depression of inhibitory GABAergic transmission. Endocannabinoid LTDi in the BLA in turn, enhances excitatory synaptic transmission in the CeA. A specific role for anandamide, but not 2-AG, in this form of neuronal plasticity is suggested by two complementary observations. First, LTDi is enhanced in FAAH−/− mice, which are impaired in their ability to metabolize anandamide. Second, endocannabinoid mobilization in LTDi apparently requires the activation of the adenylyl-cyclase-protein kinase A pathway in the BLA, but not the phospholipase C-diacylglycerol lipase pathway that is implicated in 2-AG formation.This latter finding is in contrast to our observations that 2-AG appears to be the primary endocannabinoid implicated in nonopioid SIA; a strong temporal correspondence exists between cannabinoid SIA and the accumulation of 2-AG, but not anandamide, in the PAG.

Liquid chromatography mass spectrometric studies are required to determine how foot shock-induced endocannabinoid mobilization in the BLA differs from that observed previously in the PAG. It is also possible that changes in SIA could be more prominent when supraspinally-mediated measures of antinociception are employed that are more sensitive to the affective dimensions of pain . Our data do not preclude the possibility that the doses of FAAH and MGL inhibitors employed here were unable to surmount high levels of FAAH and MGL activity at this site relative to the PAG. It is also possible that enzymes other than FAAH or MGL may participate in endocannabinoid deactivation at these sites. We propose that CB1 receptor activation in the BLA removes inhibitory control over projection neurons innervating the CeA. The CeA thus receives multi-modal sensory information, and in turn, coordinates appropriate behavioral, hormonal, and autonomic responses to stress via efferent projections. The lack of effect of the FAAH inhibitor in our study is consistent with previous work demonstrating that FAAH−/− mice and mice treated with URB597, administered systemically, show similar amygdalar activation in response to restraint stress compared to control mice. In the BLA, restraint stress produced a low level of Fos induction, which was unaffected by cannabinoid treatment, whereas, the combination of restraint stress and CB1 agonist administration produced robust Fos induction within the CeA. These data support a synergistic interaction between environmental stress and CB1 receptor activation in the amygdala that could contribute to the behavioral phenotype observed here. CB1 as well as FAAH and MGL immunoreactivity are abundant in the BLA, suggesting that incoming stimuli may trigger the on-demand formation of endocannabinoids to activate CB1 receptors prior to undergoing enzymatic hydrolysis. Activation of CB1 receptors in the BLA, in turn, reduce the inhibitory tone exerted on principal neurons, eventually propagating this signal to brain regions implicated in antinociception, most notably the PAG. In the dPAG, foot shock stress stimulates mobilization of the endocannabinoids, 2-AG and anandamide, and micro-injection of CB1 antagonists into this site virtually abolishes nonopioid SIA. Our results suggest that endocannabinoids may be differentially modulated by stress in the amygdala. For example, in the amygdala, anandamide levels are decreased after the first exposure to restraint stress whereas 2-AG levels are unchanged. Our results collectively suggest that the facilitatory effects of endocannabinoids in specifically enhancing antinociceptive responses to foot shock stress occur downstream of receptor interactions in the BLA.In a variety of ways, Colorado remains a state in transition. As is the case with many states around the nation, the economy continues to improve from the recession. Demographically, the state is becoming increasingly diverse.