Month: November 2023

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Magnesium is a key regulator of metabolic processes such as glycolysis and the Krebs cycle

Studies in yeasts and animal cells suggest that RPA-coating of single stranded DNA act as a signal to activate ATR dependent downstream phosphorylation, primarily through an associated protein called ATRIP . SUV2 is required for repair of UV induced damage, as is its namesake, and suv2 mutants are also sensitive to HU, MMC and CDDP. In sum, ATR, ALT2, SOG1 and SUV2 are all required for DNA damage response as a result of replication fork stalls. This requirement of ATR, ALT2, SOG1 and SUV2 for a plant’s survival in the response to specific genotoxins as seen with HU, MMC and CDDP heightens the curiosity as to what the true nature of Al’s impact on DNA is. As the sensitivity of atr,alt2, sog1 and suv2 mutant roots to defined genotoxins demonstrates, loss of these factors should lead to sensitivity to Al rather than tolerance if Al directly causes DNA damage. It seems counter intuitive that a plant gains Al tolerance by reducing the function of factors necessary for DNA damage response. If atr, alt2, sog1 and suv2 mutant roots can maintain root growth even in the presence of Al, what actual damage is detected by ATR, ALT2, SOG1 and SUV2 in the presence of Al? Identification of four factors that have clear roles in DNA damage responses suggests that a primary effect of Al toxicity is directly related to compromised genomic integrity, with Al possibly serving as a genotoxic agent, whether real or perceived. It is curious that loss of any one cell cycle checkpoint results in increased tolerance to Al rather than sensitivity as is observed with known genotoxic agents like HU, MMC and CDDP. This may suggest that these checkpoints are either so sensitive that even the limited amount of genomic stress that might directly or indirectly occur with Al could activate these factors yet in reality be relatively inconsequential to growth, or that Al is inappropriately perceived as a genotoxic agent by ATR, ALT2, SOG1 and SUV2. Based on the current findings on SOG1 and SUV2 in conjunction with the previous reports on ATR and ALT2,plastic nursery plant pot a DNA damage response is the primary cause of Al dependent root growth inhibition in Arabidopsis.

What kind of Al dependent DNA damage these factors are detecting is still unknown; however, in concurrence with research from the fields of Al toxicity and DNA damage responses, there could be a multitude of sources of this damage, including but not limited to: Reactive Oxygen Species, phosphate deficiency leading to dNTP depletion, competition with Mg2+ causing ATP depletion and enzymatic dysfunction, and topological strain affecting replication fork stalls or reduced transcriptional capabilities.Chronic high levels of Al exposure have been shown to result in peroxidation of lipids within the membranes of cells . Lipid peroxidation is likely a downstream result of Al damage, perhaps caused by Reactive Oxygen Species known to be generated by Al . ROS can cause DNA damage, where damage to individual bases may also be implicated, and could be tested for in a comet assay. In peripheral blood lymphocytes treated with Al, a high incidence of oxidized bases, particularly purines and apurinic/apyramidinic sites, were attributed to Al-generated ROS . In the human genome such base lesions are indeed repaired by ATR-mediated nucleotide excision repair . In Arabidopsis, over expression of a variety of factors in the antioxidant pathway have resulted in increased Al tolerance in Arabidopsis such as glutathione S-transferase and peroxidase . However, a loss-of-function mutant of At4g10500, an uncharacterized member of the 2-oxoglutarate and Fe-dependent oxygenase super family, tested for possible scavenging of ROS in response to Al exposure did not show any phenotypic changes in response to Al . Additionally, if Al-generated ROS were indeed primarily responsible for root growth inhibition, theloss-of-function mutants for DNA damage response factors would result in heightened sensitivity rather than tolerance to Al. Unrepaired damage caused by ROS leads to oxidative damage of lipids, amino acids, and DNA which can lead to cell death. Although ROS is likely a detrimental symptom of Al exposure, as Al toxicity is a complex and widely destructive biological assault, it is unlikely that ATR, ALT2, SOG1, and SUV2 are detecting damage caused by ROS.Al toxicity and phosphate deficiency typically coexist due to acid soil conditions that promote Al bio-availability while simultaneously reduce Pi uptake by the roots , and ALS3 has been identified as a required factor in a Pi starvation response in a sucrose-dependent manner . While Pi deficiency has many varied symptoms as phosphorous is required for photophosphorylation, genetic metabolism, transportation of nutrients, and phospholipid composition of cell membranes , it has dire effects on DNA replication as massive quantities of Pi are needed in the form of dNTP’s that are polymerized in order to form DNA as well as in the ATP consumed in the polymerization reaction. dNTP depletion caused by Pi deficiency would cause replication fork stalls, and lead to replication catastrophe, much like HU treatment.

A Pi starvation growth assay where seedlings of Col-0 wild type, als3-1, and atr- 4;als3-1 could be tested for Pi starvation responses. If atr-4 is capable of suppressing the als3-1 phosphate sensitivity, this could resolve whether or not Pi deficiency plays a predominant role in the Al-dependent damage response. However, damage caused by Pi deficiency would logically result in heightened Al sensitivity rather than tolerance as is observed for the loss-of-function mutants for ATR, ALT2, SOG1 and SUV2. Alternatively, with sucrose being a required factor for Pi sensitivity in als3-1, an Al growth assay where seedlings of Col-0 wild type and als3-1 are grown in the absence of sucrose with increasing amounts of AlCl3 could assess the ALS3-dependent PI starvation response. If Al toxicity is actually the result of Pi starvation, als3-1 should grow similarly to Col-0 wild type in the absence of sucrose, rather than a hypersensitive response to Al. It seems highly improbable given that Al exposure forces terminal differentiation through means of endore duplication, requiring rounds of DNA replication, as possible mechanism to inhibit root growth caused by Pi deficiency. Al3+ ions complete with magnesium ions for binding sites on the plasma membrane and decrease the uptake of magnesium into the root. Increasing concentrations of available magnesium in soil or nutrient media or over expressing magnesium transport genes enhance Al resistance as increased magnesium released into the rhizosphere competes with Al. Magnesium is the predominant ionic regulator of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell.It also acts as a second messenger for growth factors in regulation of protein synthesis and is required to maintain genomic stability. Besides its stabilizing effect on DNA and chromatin structure, magnesium is an essential cofactor in almost all enzymatic systems involved in DNA processing. Most obvious in DNA replication, its function is not only charge-related, but very specific with respect to the high fidelity of DNA synthesis . Furthermore, as an essential cofactor in nucleotide excision repair, base excision repair and mismatch repair, magnesium is required for the removal of DNA damage generated by environmental mutagens, endogenous processes, and DNA replication . More studies are warranted to study how Al interferes with the function of magnesium in plants under Al toxic conditions. Al growth responses in the presence of excess magnesium could be tested for atr-4;als3-1, alt2;als3-1, sog1-7;als3-1, and suv2;als3-1 in comparison with Col-0 wild type and als3-1 to determine if magnesium supplementation can alleviate the effects of Al toxicity.

This test would likely be indicative of Al resistance, showing magnesium outcompeting Al for entry into the cells of the root. If Al interferes with DNA replication machinery,seedling starter pot in vitro investigations such as PCR assays could be tested for amplicon lengths in the presence of Al to measure processivity, as such assays has been demonstrated in yeast . Such assays could be performed with commercially available DNA ploymerases, as Arabidopsis polymerase enzymes are not readily available.The effects of Al at the nuclear level are poorly understood. Al rapidly accumulates at high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . Al does not appear to have a base composition preference and it is likely that this lack of base discrimination is due Al binding to the phosphate backbone of DNA . This could result from an electrostatic attraction of Al3+ to the negative charges of the phosphodiester bonds. Such an association could increase the rigidity of euchromatin and relax supercoiled heterochromatin destabilizing genome topology through an ever-fluxing torsional tug-of-war. DNA gyrases and topoisomerases regulate topological strains such as supercoiled and relaxed DNA, especially caused by replisome progression, and are required to prevent replication fork stalls caused by supercoiled DNA in front of unwinding . Loss-of-function mutants of topI or topII could be tested for growth responses to Al. Perhaps in reality, overexpression mutants of a topoisomerase would be capable of counteracting the strain Al exerts on DNA and should be generated and tested. However, despite the effectiveness of topoisomerases to alleviate replication induced topological strains, their functions would not rid the nucleus of Al3+ and would likely be unable to ameliorate the affect of Al on the whole genome. Such a topological strain caused by binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide proper DNA separation as is necessary for DNA replication and mitotically relevant transcriptional events . Others have shown that Al causes DNA compaction, as well as compaction of chromatin, potentially through inhibition of unwinding of genomic DNA since Al3+ will raise the Tm of the double helix . Al has been shown to precipitate DNA out of solution in vitro, especially templates normally found in transcriptionally active euchromatin, extending the implication that Al compaction could lead to transcriptional repression. Importantly, many of these gene-silencing effects can be explained due to the extraordinary charge density of the Al ion, and perhaps this alone is reason enough to disrupt DNA processes that activate checkpoint responses. While gene silencing may indeed result from internalized Al, this is likely not a significant consequence of the effect of Al on DNA, as Al-inducible gene expression is a confirmed response to the internalization of Al . However, it is not unreasonable to predict that such conformational changes to the DNA could be perceived by ATR as being deleterious to replication fork progression. Such a topological strain on DNA caused by Al may cause a conformational change reminiscent of covalent crosslinkers, such as MMC and CDDP. Since Al3+ is expected to have high affinity for the negatively charged phosphodiester backbone of DNA and would presumably interact with this backbone differently than divalent cations and could cause an electrostatic interaction where Al acts as a non-covalent pseudo-crosslink. If Al acted as a pseudo-crosslinker that disrupts or restricts unwinding of DNA during processes such as DNA replication and RNA transcription, this may trigger ATR-dependent checkpoint activation, similar to a replication fork stall caused by a true DNA crosslink. As discussed, ATR, ALT2, SOG1 and SUV2 are all required to respond to DNA cross linking agents and all are linked to Al-dependent stoppage of root growth .Many uncertainties persist in regards to the nature of the unknown damage detected by ATR, ALT2, SOG1 and SUV2. It seems counter intuitive that a plant gains tolerance to an agent that causes DNA damage by reducing the function of a factor necessary for DNA-damage detection. Peculiarly, the genomic stability of Arabidopsis roots is secure enough to endore duplicate following Al exposure, indicating that unlike true DNA cross links, the effect of Al on the DNA is not severe enough to inhibit multiple rounds of DNA synthesis phases that would be required for the endore duplication cycle. Yet, visualization of micro-nuclei formation in cells of the root tip indicates that real breakage of chromosomes occurs following long term exposure to Al. Once more, it is perplexing that ATM is largely uninvolved with the Al response despite the consequent DNA breakage in the formation of micro-nuclei. Perhaps these factors force root growth inhibition in the presence of Al in order to prevent passage of minor damage to subsequent generations.

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Histochemical staining of Arabidopsis plants homozygous for full length ALS1 fused to GUS was performed

ALS1 encodes a half type ABC transporter that was localized to the vacuolar membrane of root tip cells, suggesting that it may be important for compartmentalization of internalized Al . Map based cloning revealed that the als1-1 mutation is a single amino acid substitution in this previously uncharacterized half type ABC transporter. It contains two distinct domains: the N-terminal domain contains at least four predicted transmembrane regions and appears to have a structure typical of the membrane spanning regions of ABC transporters and the C-terminal domain has all the required motifs associated with functional ATPases of ABC transporters . ALS1 also has significant homology to two distinct ABC transporters with different functions. ALS1 is homologous to mammalian TAP-type transporters associated with the endoplasmic reticulum to move short polypeptides for antigen processing. ALS1 is also homologous to yeast mitochondrial-localized subfamily B ABC transporter, Atm1p, and vacuolar localized Ycf1p. Atm1p is an essential component in the mitochondrial export of Fe/S clusters to the cytoplasm, indicating that it is essential for iron homeostasis in yeast. Ycf1p is required for movement of cadmium-glutathione into the yeast vacuole and loss of Ycf1p results in cadmium hypersensitivity. It is possible that ALS1 functions similarly to Atm1p and Ycf1p, thus transporting a metal complex in Arabidopsis . It was determined that ALS1 is primarily limited to vascular tissue in the roots, leaves, stems and flowers,bucket flower with high activity throughout the distal portion of the root tip. High GUS activity was also detected in the hydathodes and the area surrounding the water pore. ALS1 was found to be exclusively localized to the vacuolar membrane using ALS1 fused to GFP .

It was proposed that ALS1 is potentially transporting a metal complex from the cytosol to the vacuole of the cell, similar to two yeast homologs Atm1p and Ycf1p. This would allow sequestration of Al into the vacuole and remove interaction of the Al cation within the cytosol. Due to this activity, it is possible that ALS3 loads and unloads Al from the phloem for movement of Al to less sensitive cells, while ALS1 transports intracellular Al from the cytosol to the vacuole for sequestration. Mutation of either of these two factors can lead to inappropriate accumulation of Al in sensitive tissues within the plant . ALS7/SLOWWALKER encodes a transcription factor that among other things is required for regulation of expression of genes whose products participated in the production of polyamines such as spermine . Since als7-1 has Al hypersensitive roots, it is expected that reduced production of polyamines lowers the protective effect of these multi-charged cations because of reduced capacity to compete with Al3+ for binding to anionic sites within the root tip. Anionic targets of Al3+ are expected to include negative charges in the plant cell wall as well as symplastic targets such as genomic DNA, which directly binds to polyamines such as spermine and spermidine . Aluminum resistantmutations have been identified by using EMS mutagenized Arabidopsis seedlings that had enhanced root growth on a phytotoxic level of AlCl3. From this screen, five lines were explored further in depth. Four of these lines were mapped to a similar location and all had increased organic acid release of malate and/or citrate. The fifth line did not have enhanced organic acidrelease, suggesting the resistance is conferred in a manner other than organic acid release, which has already been described previously. Unfortunately, fine scale mapping could not be accomplished due to the difficultly of identifying a population that had a clear Al resistant phenotype .

Since als3-1 has such a profound phenotype that is specific to Al stress, it presented a unique opportunity to use this mutation to help identify factors that confer increased Al tolerance and/or resistance using a suppressor mutagenesis approach. EMS chemical mutagenesis on als3-1 was performed and plants that masked als3-1 hypersensitivity on low levels of Al were isolated. The als3-1 suppressor lines represent mutations in factors that confer increased Al resistance and/or tolerance. By using this method, 12 strong suppressor mutants were identified, and 3 were initially chosen for further analysis. All three gave similar phenotypes and all mapped to the same region. Therefore only one of those lines was studied further, alt1-1. When the alt1- 1;als3-1 suppressor mutant was grown in the presence of increasing concentrations of AlCl3, its growth not only reversed AlCl3 hypersensitivity of als3-1 but it also had increased root growth compared to Col-0 wt in higher levels of AlCl3. It was found that alt1-1 is a mutation that does not exhibit any enhancement in Al exclusion, due to callose accumulation and Al content of alt1-1 being similar to wild type . Through map-based clonging, alt1-1was discovered to be a loss of function mutation in ATAXIA TELANGEICTASIA MUTATED AND RAD3-RELATED. In higher eukaryotes, ATR encodes a cell cyclecheckpoint that senses DNA damage as part of a signal transduction pathway capable of halting the cell cycle in order to repair damaged foci . In Arabidopsis, ATR is known to signal repair of persistence of single-stranded DNA, single-stranded DNA breaks, replication fork stalls, and cross links . Alt1-1 confers a single amino acid substitution of G1098E in the highly conserved yet uncharacterized UME domain, which is speculated to function in protein-protein interactions. A second alt allele, alt1-2 was also the results of a point mutation in ATR a L2553F substitution in the conserved phosphatidylinositol 3- and 4-kinase domain of ATR . Both of these dominant-negative mutant alleles were originally found because of their capability to suppress the hypersensitivity phenotype of als3-1 .

Further work has shown that a T- DNA insertion allele, atr-2, is also capable of fully suppressing als3- 1, thus supporting the argument that alt1-1and alt1-2 are mutations that reduce the function of ATR even though they are dominant . This is particularly evident when one considers the response of atr-2 and atr- 4 to other DNA-damage agents, which only adds to the conundrum of why Al activates this ATR-dependent pathway. While these mutants are Al tolerant, as demonstrated by their capacity to maintain root growth due to failure to arrest cell cycle progression and force QC differentiation, atr-2 and atr-4 roots exposed to different DNA-damage agents such as the replication fork poison hydroxyurea and the DNA cross linkers cisplatin and Mitomycin C exhibit extreme sensitivity . From these results, ATR isabsolutely necessary to repair stalled replication forks as well as DNA crosslinks yet loss of this repair factor confers Al tolerance. It is difficult to reconcile increased Al tolerance with loss of such a key DNA-damage response factor, although it could be argued that Al may bind to DNA in a manner similar to covalent crosslinkers but without the extreme detrimental effects of these cross linkers. This argument is based on the likelihood that Al would interact with DNA electrostatically in a reversible manner, with binding likely holding DNA in an unfavorable conformation that subsequently is perceived as a replication fork stall by ATR. Interestingly, even though treatment with Al has been shown to result in DSBs and micronuclei , a loss-of-function atm mutant was incapable of suppressing the Al hypersensitivity phenotype of als3-1suggesting that DSBs are not important to activating the DNA-damage checkpoint following Al treatment. The second als3-1 suppressor mutant identified is alt2-1, which is a loss-of function mutation in the cell cycle checkpoint TANMEI/ALT2. ALT2 encodes a WD- 40 motif containing protein homologous to an integral component of the mechanism required for response to DNA damage, AtCSA,cut flower bucket which is part of the DNA- damage binding machinery . WD-40 proteins are commonly found in many different biochemical pathways, serving as scaffolds for protein complexes. In some cases, such proteins participate in SCF ubiquitin ligase complexes in order to tag target proteins for degradation. Such a role for ALT2 in response to Al is consistent with the observation that CULLIN4, which is a key component of SCF ubiquitin ligases, interacts directly with DWD motif containing proteins . Such a function is distinctly different from what was previously described for human CSA, which functions in TCNER to monitor for conformational changes in DNA as assessed by blockage of transcription . Cooperation of a WD-40 motif containing protein with CULLIN4 is characteristic of Global Genome Nucleotide Excision repair and is independent of RNA polymerase II . At this point, it is unclear in which if either NER response pathway ALT2 may participate although the potential linkage of ALT2 to either is consistent with Al acting as a genotoxin. Like atr-2 and atr-4, alt2-1 falls into the same conundrum since it fails to halt the cell cycle and force differentiation of QC in the presence of a normally inhibitory concentration of Al yet it is highly sensitive to DNA cross linkers .

A double mutant representing the loss-of-function of both ATR and ALT2 showed no additive Al tolerance compared with the single mutants, suggesting that ATR and ALT2 act together to detect Al-dependent damage and actively halt root growth. Interestingly, alt2-1 does not exhibit hypersensitivity to the replication fork poison HU while it does show extreme sensitivity to CDDP and MMC, which is consistent with it being a key regulator of cell cycle progression following exposure to DNA-damage agents . Therefore, at this point the genotoxic nature of Al has yet to be defined. However, it is clear from the unbiased als3-1 suppressor screens, in which DNA damage response factors were identified as Al tolerance mutations, an ATR- and ALT2-dependent cell cycle checkpoint pathway is key to stoppage of root growth and promotion of terminal differentiation following Al treatment. Based on the results with ATR and ALT2, one could argue that Al-dependent DNA damage is a critical determinant of root growth inhibition, yet the effects of Al on the nucleus are just beginning to be elucidated. Al rapidly accumulates to high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . It is hypothesized that Al binds to the phosphate backbone of DNA , which would be expected to result from an electrostatic attraction of Al to the negative charges of the phosphodiester bonds. Such an association could increase the rigidity of euchromatin and relax supercoiled heterochromatin destabilizing genome topology through an ever-fluxing torsional tug-of-war. Binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide a viable template for replication, mitotically relevant transcriptional events, and even proper DNA separation . It is not unreasonable to predict that such conformational changes could be perceived by ATR as being deleterious to replication fork progression, thus activating this cell cycle checkpoint. It should be noted that chromosomal aberrations resulting in DNA breakage and intra-strand cross linking are a reported consequence of chronic exposure to Al that lead to micro-nuclei formation although it is not clear how relevant these are to stoppage of root growth since ATM does not appear to have a role in this process . Clearly, further studies are necessary to define if and how Al interacts with genomic DNA, especially since it is the Al3+ species that is predicted to bind to the negatively charged DNA backbone, yet intracellular pH is not expected to favor the formation of this species. In the end, utilizing Al toxicity as a real world model to study DNA damage in plant systems presents a novel system to study DNA damage response without the use of rare chemotherapy drugs or types of radiation not found in earthly environments. Ultimately, it seems counter intuitive that a plant gains tolerance to an agent that causes DNA damage by reducing the function of a factor necessary for DNA damage detection. This certainly begs the question—if atr and alt2 mutant roots can maintain root growth even in the presence of Al, what actual Al-dependent damage is detected by these cell cycle checkpoints? It cannot be ruled out that inappropriate activation of the cell cycle checkpoint machinery and the repair mechanisms that they regulate may actually be the cause of the damage such as DSBs and micro-nuclei. Consequently, one explanation could be that failure to activate this pathway prevents the program-dependent accumulation of the damage and results in roots that can grow in normally inhibitory levels of Al.

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Several QTL for chilling tolerance in rice have been high-resolution mapped and candidate genes identified

The heterozygous BC4 line 04GH0030 from Goodstal et al. containing a single copy of the S. habrochaites allele for fine-mapped QTL stm9 was used to generate a BC5 segregating for only the chromosome 9 region of interest from S. habrochaites. The BC5S1 generation was marker genotyped to identify recombinant individuals within the fine-mapped stm9 region flanked by markers T1670 and T0532 . Self-pollinated seed was collected from individuals chosen for their chromosome 9 introgressions, and these BC5S1 individuals were marker screened for further recombination events within the chromosome interval containing stm9. Self-seed from two heterozygous BC5S1 individuals and two heterozygous BC5S2 individuals were screened for recombinants via marker-assisted selection . Individuals that contained recombination events within the chromosome 9 fine-mapped stm9 region were selected, grown to maturity, and allowed to self-pollinate to produce seed of fully homozygous individual sub-NILs in the BC5S2 or BC5S3 generation . Recombinant homozygous individual sub-NILs were allowed to self-pollinate to generate ample seeds for replicated experiments. Phenotyping experiments were performed with one representative line from each recombinant class. All plant materials were grown in greenhouses at UC Davis. Seeds were planted in 73-cell flats containing soil media. Flats were watered daily, and plants were fertilized with a 10:30:20 NPK solution once a week. Greenhouses containing plants in flats, pots, and hydroponic tanks were maintained at ambient conditions of 25–37 °C with 55–80 % relative humidity during the day, and 18–25 °C with 20–55 % relative humidity at night. Plants from which seed was to be collected were transferred at the 4th to 5th true leaf stage to individual 8-L pots filled with soil media, and grown to maturity to obtain seed.After 2 weeks of growth in flats containing soil media ,procona London container the roots of two seedlings of each sub-NIL or control were carefully washed free of soil media in deionized water and transferred to a hydroponic growth tank set at 20 °C containing a modified Hoagland solution at 20 % of full strength .

Efforts were made to use vigorous seedlings of similar size. Plants were grown in the hydroponic tank for 1 week under ambient illumination in the greenhouse, with constant aeration and circulation of the nutrient solution. Subsequently, the plants were randomized and transferred to a separate refrigerated hydroponic tank containing fresh modified Hoagland solution at 20 % strength. Plants were acclimated overnight at a solution temperature of 20 °C. The following morning supplementary lighting was provided by one 1000 W metal halide lamp starting at 7:00 am and used throughout the experiment to maintain a light level above 1000 µmol m−2 s −2 PAR. The tank solution was maintained at 20 °C for 1 h after the supplementary lighting was turned on, and then the tank temperature was decreased to 6 °C. The tank was held at 6 °C for 2 h prior to phenotyping. Each experiment was conducted as a Randomized Complete Block Design and repeated in two seasons , with days as blocks and two replicate plants of each genotype per block. In addition to the two individual plants per recombinant sub-NIL, each replication of the experiment included controls: four plants of chilling-sensitive S. lycopersicum cv. T5, and two plants of a chilling-tolerant NIL, 03GH1322 that was also used as a tolerant control by Goodstal et al. . Six repetitions of the experiment that were conducted from May 9th to June 13th comprise the Spring data set, and four repetitions of the experiment that were carried out from October 7th to October 20th comprise the Fall data set. Plants were individually phenotyped for shoot turgor maintenance under root chilling according to the rating scale described in Goodstal et al. . Briefly, shoot turgor was scored for each plant on a scale of 0–3, with a stmscore of 0 denoting maintenance of shoot turgor, and a stmscore of 3 denoting severe loss of shoot turgor .The chromosomal location of stm9 in our study agrees with Goodstal et al. who fine-mapped stm9 to marker interval T1670–T1673 . We refined the location of stm9 to marker interval H358–T1673, a genetic distance of 0.32 cM. Our data suggests that the gene or polymorphisms controlling the tolerance phenotype are located close to marker H348 and within the marker interval H358–T1673. The chromosomal location of QTL stm9 detected in both data sets was coincident despite the significant Genotype × Season interaction in the ANOVA.

To examine the cause of the significant Genotype × Season interaction in more detail, we plotted recombinant sub-NIL stmscore means across the two seasons to create interaction plots . Inspection of the plots suggests that the changes in sub-NIL mean values across seasons primarily derived from greater chilling susceptibility of susceptible sub-NILs in the Spring than in the Fall . Magnitude differences would cause the size of the LOD peaks to differ among seasons, but not change the peak location, which is in agreement with our results . In addition to the increase in the magnitude of means for stmscore of the susceptible sub-NILs, two sub-NILs were classified as tolerant in the Spring dataset but as susceptible in the Fall dataset . Sub-NIL C4 had a mean of slightly over 1.0 in the Spring data set, and clearly grouped as susceptible in the Fall dataset . None of these lines contain the S. habrochaites introgression for high-resolution mapped stm9, but the introgressions do all flank stm9. These results suggest the possibility that there are environmentally sensitive genetic modifiers of the stmscore phenotype in this region of chromosome 9, and that the interaction of these modifiers with the environment could be causing the significant Genotype × Season interaction. Phenotypic plasticity in the presence of abiotic stress has been noted and reviewed previously . The rank changes seen within the tolerant group may be due to differences in the genomic content of S. habrochaites sequence in the flanking regions of QTL stm9, and not a direct effect of the environment on the gene or polymorphisms controlling the tolerant stm9 phenotype. Previous work in tomato has shown that the stomatal response of a plant when subjected to root chilling conditions differs between susceptible and tolerant phenotypes . Stomatal control is regulated by multiple environmental factors including light, temperature, day length, humidity, and CO2 levels . The Spring experiments were conducted under longer day lengths, higher air temperatures, and lower humidity than the Fall experiments . These seasonal differences affect the conditions in the greenhouse and may have contributed to the significant Genotype × Season interaction, as well as differences in relative response among the sub-NILs in the Spring versus Fall data sets. In the context of phenotypic plasticity, seasonal effects on sub-NIL performance would account for the more gradual separation of means in the Spring dataset compared to the Fall .Low marker density and small population sizes in initial genome-wide QTL mapping studies may bias upwards the estimation of QTL effects due to the inability to resolve closely linked, smaller effect QTL . Consequently,cut flower transport bucket single large effect QTL may resolve or fractionate into multiple, smaller effect QTL after fine- and high-resolution mapping . The original interspecific BC1 population used by Truco et al. to map QTL for shoot turgor maintenance under root chilling consisted of 196 individuals genotyped with 112 markers. Truco et al. mapped a major effect QTL to a 28-cM region on the short arm of chromosome 9 which accounted for 33 % of the phenotypic variation for shoot turgor maintenance under root chilling .

Despite the large initial genetic size of the QTL stm9 region detected by Truco et al. , subsequent fine-mapping by Goodstal et al. and high-resolution mapping in our present study do not provide any evidence of multiple QTL or QTL fractionation. The relatively small genetic size of high resolution mapped stm9 and the lack of QTL fractionation indicates that this level of resolution is suitable for the identification of candidate genes for stm9. There are numerous examples in the literature of environmentally stable, high resolution mapped QTL that have led to candidate gene identification and in some cases subsequent causal gene/ polymorphism determination.These QTL include qCTS12 , qCtss11 , and qCTB7 . Tomato-specific QTL examples include fw2.2, a fruit weight QTL, and se2.1, a stigma exsertion QTL, both identified in progeny derived from S. pennellii, another wild tomato relative . The causal gene underlying QTL fw2.2 was identified by Frary et al. , who proposed that changes in the regulation of ORFX , not changes in the sequence or structure of the expressed protein, are responsible for changes in fruit size. Chen and Tanksley determined the casual mutation underlying se2.1 is a mutation in the Style2.1 promoter that results in a down-regulation of Style2.1 expression during flower development. Collectively, the results from these studies suggest that candidate gene identification and functional testing for QTL stm9 should focus on mutations in regulatory and promoter regions of candidate genes in addition to mutations that may affect the sequence or structure of expressed proteins.Many genes have been identified as being involved directly or indirectly in plant tolerance or resistance to abiotic stresses , including chilling/cold tolerance . Plant responses to abiotic stresses can include multiple pathways that involve a variety of gene products such as receptors, signaling molecules, transporters, transcription regulators, and transcription factors . Many of the identified stress response pathways have been associated with tolerance to a range of abiotic stresses . The plant’s response to abiotic stress may result in both reversible and irreversible activation of stress response pathways . Because of the complex nature of the pathways involved, the specific genotype of the plant also has a large influence on abiotic stress response . Plant responses to abiotic stressors are dependent on the interplay of abiotic stress, environment, and genotype . Therefore, a particular abiotic stress applied in different environmental contexts may result in overlapping, but distinct responses from a single genotype . We analyzed the physical region in the cultivated tomato reference genome that is syntenic to the S. habrochaites QTL stm9 region because an assembled S. habrochaites whole genome sequence is not available. All of the protein products of the S. lycopersicum annotated genes located within 30 kb of the QTL stm9 peak marker have features that are shared with genes involved in responses to water stress and other abiotic stresses. In addition, the majority of the S. lycopersicum genes located within the syntenic high-resolution mapped stm9 region have been implicated in abiotic stress response pathways . It is possible that plant responses to root chilling stress may induce a more complex transcriptional response than other types of water stress such as those caused by salt or polyethylene-glycol , although overlap has been seen in the response to all three stresses . For example, in grape, under root chilling stress only transcripts for protein synthesis and the cell cycle were up-regulated to a lesser extent than under salt or PEG stress. The regulation of plant metabolism, protein metabolism, signal transduction, calcium signaling, stress hormone pathways, and transcription factors were all increased to a greater extent under root chilling in grape . These categories of genes account for the majority of genes located within the syntenic S. lycopersicum QTL stm9 region. While the total number of annotated genes within the S. lycopersicum reference genome region containing QTL stm9 is relatively small, there are no estimates available for S. habrochaites due to the unavailability of assembled whole genome sequence for this wild species. A comparison of the genetic and S. lycopersicum physical maps of the chromosome 9 region containing stm9 shows a variable rate of recombination across this region . The average kbp/cM for marker interval T1670–T1673 is 952 kbp/ cM, whereas for marker interval T1673–T0532, it is 385 kbp/cM. Recombination occurs more frequently in generich euchromatic regions, but can be suppressed due to lack of homology, heterochromatic regions, and/or the presence of repetitive elements . It is possible that this variable rate of recombination is due to the presence of repetitive elements or other local structural polymorphisms affecting the synteny and colinearity of the S. lycopersicum and S. habrochaites genome sequences in this region. In addition, our flow cytometry results indicated that the genome size of S. habrochaites is 1.5 × that of S. lycopersicum .

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Eight growers spanning the major rose-producing areas of California participated in the program

With the new bent-shoot method, plants are grown in raised containers in a modified hydroponics system. Most of the shoots are bent downward at the crown to intercept more light, creating a perennial lower canopy that exists for the 5 to 8 years of crop production. The upper canopy contains only stems that produce flowers, which take 45 to 52 days to develop. The bent-shoot method creates a spatial separation between the harvested flowers and perennial foliage that does not exist in standard roses. Pesticides to control western flower thrips and powdery mildew that are more compatible with mite predators have also recently become available. These developments, coupled with the difficulty that rose growers were facing in controlling spider mites, made us confident that we could develop a successful IPM program that rose growers would adopt. This project was initiated in 2000 with major funding from the Pest Management Alliance Program of the California Department of Pesticide Regulation and was later supplemented with additional funding. The goal of the Alliance project was to foster a team approach to the development and implementation of IPM programs in a given commodity and to document a reduction in traditional pesticide use. Our Alliance team included researchers, county-based advisors, growers, chemical and biological-control industry representatives,30 litre plant pots commodity associations and government officials. Our objective was to develop a cost-effective IPM program for the key pests of cut roses that included sampling, thresholds, biological control and directed sprays of reduced-risk pesticides.Each grower contributed an IPM and a conventional-practice greenhouse; all greenhouses were between 5,000 and 10,000 square feet in size.

All pest management decisions in the IPM greenhouses were based on the IPM program that we developed, while the grower made all pest management decisions in the conventional greenhouses. Data was collected and compared on a weekly basis by trained scouts using a comprehensive sampling plan that provided information about the density of insects, mites and diseases. The project included growers with several different rose varieties and both the bent-cane and hedgerow training techniques, but we kept these two variables standardized within a location. Implementation began in March 2000 and continued until January 2001. Fixed precision sampling plans that had been previously developed for two spotted spider mites and western flower thrips were used in our scouting program. This type of sampling plan was developed through intensive surveys of a crop to determine a pest’s spatial distribution. The degree of acceptable error was decided upon in advance, and the number of samples needed to obtain that precision was calculated using knowledge of the pest’s spatial distribution in the crop. We used a precision of 0.25, which is acceptable for pest management sampling . Generally, as spatial distribution becomes more aggregated , more samples are required to determine pest density with the desired precision. Although they take some effort to develop, these types of sampling plans are often more accurate and efficient than other sampling approaches. This study represents the first use of such plans in a floriculture IPM program. Sampling for all other pests was done during sampling and inspection for two spotted spider mites. Data was collated and summarized by the scouts and then discussed by members of the Alliance team. The scouts then met with the growers to discuss control strategies. Based on thresholds developed for each of the pests, no action was taken; cultural controls were used; biological control agents were released; or a pesticide application was made. Each greenhouse was a replicate, and ANOVA was used to determine whether there were differences between the conventional and IPM treatments.The first leaf above the bend on 38 randomly selected plants was sampled per 10,000 square feet of greenhouse area to estimate mite density at the desired precision.

Plants were classified as infested if the scout found more than five mobile mites on the sampled leaf, or not infested if there were five or fewer. These samples were also used to determine co-occurrence of two spotted spider mites with the predatory mite Phytoseiulus persimilis, and they were inspected for secondary pests and diseases. In addition to the fixed samples, the scouts took directed samples as they walked down each row and noticed damage by insects, mites or pathogens. These plants were flagged for potential spot treatments. In the IPM greenhouses, mite treatments were initiated according to the percentage of infested plants . Chemical controls included azadirachtin , bifenazate and insecticidal soap , all of which provide some level of compatibility with P. persimilis. Releases of predatory mites were based on the cooccurrence of two spotted spider mites and predators on the sampled leaf. Cooccurrence is the percentage of plants with two spotted spider mites on which P. persimilis also occurs. This idea has been discussed in the literature as a theoretical basis for natural enemy releases, but has never been tested in practice . We chose to include this method in our program because our natural enemy supplier recommended it to growers. Additional predatory mites were released when co-occurrence was less than 10%. All predator releases were made to leaves just below those on which two spotted spider mites were present. Predators were kept refrigerated and were released as soon as possible after arrival at the greenhouse, as per the supplier’s instructions.A fixed precision sampling plan for western flower thrips was also developed . This sampling plan used yellow sticky traps and a general threshold of 25 to 50 thrips per trap per week . Three 4-by-6-inch yellow sticky traps with both sides exposed were placed per 10,000 square feet . The traps were placed at flower level and were evenly distributed in the greenhouse . The lower threshold of 25 thrips per trap per week was used in more-susceptible varieties and in areas of heavy thrips pressure. The higher threshold of 50 thrips per trap per week was used in less-susceptible varieties . There is currently no cost-effective biological control agent for western flower thrips in cut roses, so control of this pest in the IPM greenhouses included both cultural and chemical methods.

Although the female thrips lays eggs in the flower or in foliage directly below the flower, the development time for eggs and larvae is longer than the 5 to 6 days between sepal split and flower harvest . Routine flower harvest removes immature thrips from the greenhouse and subsequently there is little thrips reproduction in the rose greenhouse unless open flowers are left on the rose plant. Teerling has measured significantly higher thrips populations in Canadian rose greenhouses when these flowers are not removed. Cultural control was the removal of open flowers, and chemical control was applications of spinosad or azadirachtin directed to the flowers when the thrips-per-trap-perweek threshold was reached. Research on the distribution of thrips in the rose range has revealed that most thrips are found near the developing flower . Based on these findings, we then conducted a trial to determine whether sprays directed toward the flowers would provide control equivalent to full-volume wet sprays. Such a study is critical to the implementation of IPM in the rose range, because a typical full-volume spray in roses may reach hundreds of gallons of water per acre. Such high volume thoroughly wets the foliage, but creates problems with runoff and affects biological control agents regardless of where they are on the plant. In separate rose greenhouses,25 liter pot plastic we initiated a replicated study where rose beds were divided into 20-foot sections and applications of registered pesticides were made using full-volume wet sprays at 275 gallons per acre versus the same material applied just to the upper canopy at 70 gallons per acre . Registered materials — acephate , methiocarb and spinosad — at label-recommended rates were used in the study. There were four replicated, 20-foot rows for each material per volume combination, and applications were made for 4 weeks. At the end of this time, 10 flowers were removed from each section and examined for the presence of thrips. Our effort to introduce IPM principles in the management of powdery mildew centered on an attempt to use a predictive model for powdery mildew of grapevines . The UC Davis powdery mildew risk-assessment model for grapevines is based on the effect of temperature on the reproductive rate of the pathogen following initial plant infection. As temperatures are recorded in vineyards, risk points are accumulated if temperatures are favorable or subtracted if temperatures are not favorable. When risk points reach a predetermined threshold, fungicide application is recommended. This model has been effective in determining if and when fungicide treatments need to be applied to grapevines, and has resulted in effective disease management with significantly reduced fungicide usage in California. In commercial rose greenhouses, growers spray regularly weekly during mildew season in Central California and all year long in Southern California. It is not unusual for half of all yearly pesticide sprays in a rose crop to be for mildew control, presenting a strong argument for matching applications to actual risks. Although the powdery mildew fungus attacking roses is a different species, its response to environmental conditions is similar to that of the species attacking grapevines. For this reason, we sought to determine whether the grapevine mildew model could be easily adapted to greenhouse grown roses. The greenhouses used in this effort were instrumented so that temperature, relative humidity and leaf wetness were measured at 30-minute intervals throughout the day and night. Temperature data was fed into the GMM to add or subtract risk points. In order to correlate actual disease development with the GMM risk points, a trained scout evaluated plants in the greenhouses weekly.

This was accomplished by walking through the greenhouses in a predetermined pattern, stopping at regular intervals and evaluating one plant at each stop-point to assess disease incidence and severity. Disease incidence was determined by the presence or absence of mildew lesions on the leaves of harvestable stems. Disease severity was determined by counting the actual number of lesions on leaves attached to the harvestable stems. This data was used to calculate an overall disease rating for the crop that could be compared to risk predictions based on the GMM. Along with the disease incidence and severity data, we recorded the timing of all chemical fungicide and insecticide applications made by the growers in the IPM houses so that we could evaluate these effects on disease ratings. As a resistance management practice, growers typically varied the fungicide materials used throughout the season. A few fungicides were common across all locations, but growers did differ in some of the materials applied. For example, if powdery mildew became severe in a greenhouse, growers at all locations would typically apply piperalin because of its eradicative properties. Other materials used at the various locations included myclobutanil , chlorothalonil , benzeneacetic acid , azoxystrobin , insecticidal soap and potassium bicarbonate .Plants in both the IPM and conventional greenhouses were inspected for whiteflies, aphids, mealybugs, Botrytis, downy mildew and rust as part of the inspections for twospotted spider mites. The same traps that were used to monitor western flower thrips were also used to monitor white flies and winged aphids. We emphasized the use of materials that were compatible with the P. persimilis predator for control of these pests when necessary.Two spotted spider mites. Predatory mites were successfully used in all of the IPM greenhouses and almost eliminated the need for miticide applications in those houses. A comparison of two spotted spider mite levels under IPM and conventional control across all nurseries revealed that there were significantly more plants with no mites and significantly fewer plants with mites at the two levels measured in the IPM greenhouses . Similar results were observed at the individual nurseries. The cost of IPM during the first 8 weeks was higher than the cost of conventional control . Higher release rates were needed during this startup period for several reasons, including increased predator mortality as growers learned proper release techniques and the desire of some growers to begin biological control when twospotted spider mite densities were greater than the 25% infested threshold. After several releases had been made and predators became established, the release rate dropped and costs for the two control programs were comparable. Western flower thrips. 

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Sandwiched between the automobile bridges is a narrow pedestrian bridge

The day of my arrival in New Village, I ate lunch with many other construction workers in a private house marked with a sign saying paidang, a term referring to a low-budget restaurant for workers. The restaurant proprietor used his first-floor living room and garage for this purpose, setting up three square-shaped “eight sages” tables—each of which could accommodate eight customers. This type of table is a common piece of household furniture among farmers living in the lower Yangzi rice-growing region. I encountered several small restaurants in the New Village. Townhouse space was used for other informal businesses as well, including general stores, two majiang parlors, a computer IT repair store, and even a clothing factory. It was in one of the majiang parlors, run by an older couple in their two-story townhouse, that I first made the acquaintance of my host Mrs. Tang. During my stay, I accompanied her to this majiang parlor nearly every afternoon. The couple could make as much as USD $15 per day if all four majiang tables were in continual use. The computer repair shop, run by a young man in his 20s, was more difficult to find. Although situated in the house just behind the Tang’s, I did not notice it at first. The clothing factory, also a family-run business taking advantage of family household space, was comparatively more conspicuous. Although the majiang parlor and computer repair shop did not have signs, the name of the factory— “Jinhu Clothing Factory”—was mounted on a side wall along with an advertisement announcing that the factory was hiring experienced workers to operate the sewing machines. Even with all the above-mentioned survival strategies, many residents still cannot balance their family budgets. Thus, many young able-bodied men from Jinhu continue to migrate to distant urban centers for work. To be sure,maceta plastico cuadrada rural-to-urban migration is not a new strategy; it has been practiced by rural populations across China since the late 1980s.

As most rural young adults began to migrate to large cities for work, a great divide emerged between the productive space of cities, where able-bodied adults work in factories and in the service sectors, and the social reproduction space of villages, where grandparents take care of grandchildren, whose own parents in the factories do not have time to tend to their upbringing. In the villages, one generally encounters mostly women, children, and the elderly, often referred to as “left-behind elderly, children, and women” . The Jinhu development project was supposed to change this. Residents of the New Village were supposed to find employment at the nearby theme park. In fact, less than 100 people are employed there, with more than half of them hired from outside of the New Village. Moreover, one middle-aged woman complained to me that the developer hires his own relatives and friends for all of the higher paid positions. Residents of Jinhu are only hired as day laborers when seasonal jobs are needed, such as to cut weeds or plant trees and vegetables. As a result, to support themselves, residents have continued to find jobs far away. Mrs. Tang and her husband had both migrated to a large city in Zhejiang when I revisited Jinhu New Village in summer 2013. In all appearances, the “New Countryside” implemented at Jinhu merely reproduces the great divide between production space and social reproduction space that first emerged in the 1980s. In any case, Jinhu New Village is now, in many respects, merely a by-product of the major local development project, the Jinhu Rural World theme park. One of the most curious, even bizarre, aspects of the Jinhu site is the construction of a theme park. Not far from the New Village, a set of three bridges cross the artificial canal that separates the national highway from the theme park compound. Two of these bridges are restricted to automobile traffic, one serving as an entrance to the compound and one as an exit. Across the bridges one encounters the rectangular “Culture Plaza,” a vast space somewhat reminiscent of Tiananmen Square in appearance . Beyond the plaza is a large parking lot. Only after crossing this parking lot does one finally reach the ticket office, as well as the adjacent multi-story headquarters of the real estate development company responsible for the entire project—the Guoqiang Conglomerate.

All the elements of this entryway—not to mention the drive-in movie theater on the north side of the park—are tailored to car-owning clients, presumably arriving from large urban centers. Needless to say, the monumental entryway is also meant to impress. In order to obtain government funds and bank loans earmarked for agricultural development, the Rural World theme park is in principle designed to educate urbanites about agriculture in China. One particular focus is “natural disasters”—on the premise that agriculture is reliant on the conditions of the weather, the soil, and the natural environment. Thus, the rural theme park includes a small museum showing photographs and videos about floods, droughts, earthquakes, tornados, locust plagues, and tsunamis—all representing the various natural disasters afflicting the peasantry throughout China’s history. Associated with this theme of natural disaster—and billed as one of the highlights of visiting the park—is the“tsunami experience dome” . In contrast to the museum, which displays merely images and photographs of natural disasters, the water dome purportedly provides visitors with the “real” experience of a tsunami. Inside, visitors can surf six-meter high artificial waves generated by a wave machine. The Guoqiang Conglomerate is very proud of its innovative approach at combining entertainment with an educational experience. Indeed, Jinhu Rural World has been publicly acclaimed by both central government officials and the national media.Perhaps as part of its educational mission, the park is also a celebration of scientific management and high technology. As such, many of the park’s attractions provide insight into how agricultural development and modernization are imagined in contemporary China. By depicting natural disasters through historical photographs and videos contained within a museum, natural disasters are relegated to the realm of the historical past. By transforming tsunamis into entertainment, they become in essence domesticated by modern science. The theme of the human conquest of nature is ever present.

On either side of the vast empty plaza and parking lot is situated a large one-story green building, with signs identifying one as a “modern seedling center” and the other as an “agricultural high-tech center”. As one example of agricultural modernization, the agricultural high-tech center contains a section demonstrating hydroponic techniques for growing green vegetables, including lettuce and tomatoes. Water-drip irrigation and greenhouse farming are commonplace throughout the park. For example, the grape garden is covered with plastic,macetero de 7 litros producing a greenhouse-like environment that shelters the plants from uncontrollable nature. The grape vines are watered with a drip system and kept at constant temperature using a temperature control system. Elsewhere, delicate butterfly-shaped orchids are grown in glassware within laboratories in which staff members wear white medical coats and face masks . The park also features desert and tropical botanical gardens—containing plants like giant cacti and many exotic tropical plants—both housed within glass-roofed concrete buildings. Modern agriculture, as portrayed in the Rural World theme park, is not unlike urban modernity. Hygiene and cleanliness—represented by the face masks, lab coats, and hydroponics—are stressed. And modern agriculture is placed in a built environment almost entirely protected from the vagaries of the natural world. This modern, scientific, hygienic agriculture exists in curious contrast to another dominant theme of the park—the romanticization of the peasantry and rural life. The rural fantasy is evident in a variety of different “rural” activities in which tourists are invited to participate. It is this element of the park that is most evident when one searches for information on the internet. Numerous travel agencies based in Nanjing and Shanghai—two of the largest urban centers in Eastern China—describe nearly identical itineraries. Their websites highlight tsunami- and tornado-based water sports, as well as rural activities such as fruit and vegetable picking, watching “folk animal performances” , and growing crops in a four-by-four-meter rented vegetable plot in one of the greenhouses. Thus, during grape season, tourists can pick grapes from grape vines in the covered greenhouses, and then purchase what they have picked at a relatively high price— USD $5 to $6 per pound. The vegetable plots are rented for USD $215 per year; moreover, clients who do not wish to get their hands dirty can entrust the planting and maintenance of the crops to the theme park for an additional fee. Clients can also enjoy a variety of shows including cock fighting, goat fighting, and piglet racing. Also at an additional cost, one can go horseback riding around the park grounds. All of these fees are on top of the entry ticket of USD $25 per person. It is clear enough that what Jinhu Rural World represents is not a nostalgic recreation of traditional agricultural society; rather it is a playground for adventurous urbanites, featuring a clean, new, “modern” countryside. One key difference distinguishing Jinhu Rural World from traditional rural society is the apparent absence of peasants. In fact, at Jinhu, it is not the case that mechanization has replaced intensive farm work. Jinhu Rural World does hire local peasants to weed, prune grape vines, and grow vegetables for urbanites who rent the four-by-four plots. Yet peasants are absent from all publicity for the theme park. In the case of the vegetable plots, Jinhu’s innovative approach is to allow urbanites to monitor their plots over the internet—a form of e-commerce that Jinhu planners call “e-family farms” . Though there are peasants minding the plots, they rarely appear on the monitors; they become faceless and nearly invisible.

Peasants are not part of the new rural world. Instead, fabricated “folk animal performance,” agricultural machines, and new technologies are the key emphasis. It is as if the rural population itself is too backward to have a place in the modern new countryside. To build Jinhu Rural World theme park with no rural population in it, the Guoqiang Conglomerate embarked on a radical reorganization of village space. In 2007 and 2008, peasants had to move out of their original villages, which once dotted the landscape around the large lake now at the center of the theme park. Although the development company demolished most of the peasants’ houses immediately so that they could not be reoccupied, as of 2012, most of the fields were left fallow while awaiting the construction of the new theme park attractions. Village restructuring also involved a much more highly compartmentalized organization of space. To avoid confrontations with peasants seeking to exploit unused land, the company fenced off a large area, preventing access by all but authorized personnel. Formerly, villagers could walk anywhere, even across their neighbors’ fields. Their physical freedom of movement within villages was a longstanding part of peasant culture. Now things were different. When Mrs. Tang and her husband took me on a Honda motorcycle to see their old village, we were prevented by a guard from entering the compound, despite the husband’s angry insistence. The development of the Jinhu site involved a compartmentalizing of public and private space that led to previously unknown constraints on the free movement of villagers. Besides compartmentalization, development also entailed the labeling of space. All of the attractions in the Jinhu Rural World theme park were identified with signs. Jinhu New Village was also identified by means of the majestic gate placed at the entrance. Needless to say, local residents living in their natural villages had no need for such signs and labels. Labeling the landscape is done by outsiders and for outsiders. It is a tool used by the state to increase the legibility of the built environment. Living villagers were not the only ones to be moved. The village dead were also relocated—from graves once scattered throughout the peasants’ fields into a single cemetery just east of the new village. The Jinhu cemetery is graced with a brand-new gate just like the one for Jinhu New Village, albeit with characters written in white on black instead of in red .

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The effect size in each case represents the difference compared to the water control treatment group

MS-DIAL features were clustered by applying a Pearson correlation, with a minimum correlation of 0.8 and maximum p value of 0.05, retaining two features per cluster according to most intense and the most connected peak filters . Selected peaks were imported into MS-FINDER for annotation . Mass tolerance for MS1 and MS2 were set to 5 and 15 ppm respectively, the relative abundance cut off set at 1% and the formula finder was configured to use C, H, O, N, P, and S atoms. FooDB, PlantCyc, ChEBI, NPA, NANPDB, COCONUT, KNApSAcK, PubChem, and UNPD were used as local databases. During the final merge step in MS-CleanR, the best annotation for each peak was based on MS-FINDER scores. The normalized annotated peaks list produced by MS-CleanR was used for the final statistical analyses in R. A partial least squares supervised model of the complete log-transformed and Pareto-scaled dataset was done using the ropls package , with the three treatment groups as the response variables. Ellipses were drawn around treatments using stat_ellipsebased on a 95% confidence level. This distance type considers the correlation between variables and the ellipses are created around the centroid data point. Heatmaps were created using the log-transformed data within the ComplexHeatmap package in R , with hierarchical clustering according to the complete-linkage method and Euclidean distance measure across columns and rows, displayed as dendrograms.Pairwise multivariate analysis was performed across all time points between ANE and H2O, and between AA and H2O, using an orthogonal projections to latent structures discriminant analysis . OPLS-DA models were generated using the ropls package, macetas plastico cuadradas with the predictive components set to 1 and orthogonal components to 7. S-plots were generated following sample sum normalization and Pareto scaling via calculation of p1 and pcorr1 of the OPLS-DA scores using the muma package source code within R.

Chemical class enrichment analysis was achieved using ChemRICH for each two-treatment comparison at each time point within R using the source code . A student’s t-test of the signal was conducted to generate p values and effect size. Previous transcriptomic work revealed a high level of congruency in differentially expressed genes in AA- and ANE-treated tomato seedlings compared to H2O-treated controls . To further this line of investigation, the metabolomic profiles of AA- and ANEroot-treated plants were compared to H2O after 24-, 48-, 72-, and 96-hours exposure to their respective treatments. Locally-treated roots and distal leaves were harvested, flash frozen, extracted for metabolites, and subsequently analyzed via LC-MS, which with underivatized samples primarily captures the nonvolatile metabolome . Partial least squares score plots of tomato root tissue revealed distinct clustering by treatment irrespective of time point . No overlap was observed in the 95% confidence ellipses for any treatment group. Likewise, heatmap visualization of the log10 signal of metabolites showed clear clustering of metabolomic profiles by treatment . Features displayed in the heat map were filtered from the total dataset with a p-value < 10−6 and absolute fold change > 5 in roots. Less defined clustering was observed in PLS score plots of distal leaf tissue across sampled timepoints . Ellipses representing the 95% confidence interval for both H2O and AA treatments both partially overlap with the ANE treatment group. Similarly, a heatmap depicting metabolite log10 signal showed more diffuse clustering by treatment . Visualized metabolites from leaves displayed in the heatmap used a p-value < 0.001 and an absolute fold-change > 2. These findings are refilective of distal tissue not directly treated with either elicitor. Changes in the distal leaves were not as robust as in the directly treated roots, likely due to diminution of systemic signals that effect metabolic changes throughout the plant. An assessment of the total annotated features across the metabolomic analysis revealed shared and unique annotated features between roots and leaves .

Roots and leaves share 44 features with leaves displaying the largest number of unique metabolic features . There were 330 unique identified features in leaves compared to 223 features unique to root tissue . A comparison of differential metabolic features for AA- and ANE-treated plants compared to the H2O control revealed robust overlap for both roots and leaves . AA- and ANE-treated roots shared 68 differential metabolic features, with AA and ANE treatments possessing 37 and 29 differential features unique to each, respectively . Less overlap was observed in leaves with 39 shared differential metabolites, with AA- and ANE-root treated plants displaying 34 and 19 uniquely differential metabolites, respectively .Chemical enrichment analyses were conducted to identify classes of metabolites whose accumulation was locally or systemically altered in AA- and ANE-root-treated tomato seedlings. Enrichment analyses of metabolites whose mean signal was significantly changed in AA- or ANE-treated plants compared to H2O identified numerous affected chemical classes . These changes were most robust in directly treated roots compared to distal leaves. Treatment of tomato seedlings with AA showed strong modulation of metabolomic features classified as triterpenoids and linoleic acid and derivatives in roots. AA-treated roots also showed increases in hydroxycinnamic acids and derivatives and fatty acyl glycosides of mono- and disaccharides. ANE-treated roots showed modulation in the accumulation of triterpenoids, steroidal glycosides, and hydroxycinnamic acids and derivatives. Similar to AA-treated plants, the roots of plants treated with ANE also showed increases in metabolites classified as fatty acyl glycosides of mono- and di-saccharides. Although less striking than the chemical enrichment analysis of roots, leaf tissue of root treated plants did reveal an altered metabolome . These changes in metabolite accumulation occurred most prominently at 96 hours, the latest tested time point. Increases in sesquiter penoids and steroidal saponins were seen in leaves of AA-treated plants at 96 hours.

A mix of accumulation and suppression of terpenoids and an increase of methoxyphenols was observed in the leaves of ANE-root-treated plants. Chemical enrichment analyses broadly revealed classes of metabolites that were induced or suppressed in AA- or ANE-treated plants. To examine which specific variables provide the strongest discriminatory power between the two treatment groups, a two-group comparative supervised multivariate analysis, orthogonal projections to latent structures discriminant analysis , was utilized. OPLS-DA score plots show strong between group variability discrimination between AA and ANE treatment groups compared to the H2O control across all tested time points with the x-axis describing the inter-treatment variability,macetas 30l and the y-axis showing the intra-treatment variability . S-plots derived from OPLS-DA were examined for both AA and ANE treatments in pairwise comparison with H2O control. S-plots of OPLS-DA revealed that treatment with AA or ANE induced shared changes in the levels of several defense-related metabolites in roots . Variables with the most negative and positive correlation and covariance values are the most influential in the model. These metabolites are located on either tail of the S-plot and contribute most greatly to the separation between treatment groups Bar charts depicting mean LC-MS signals for top OPLS-DA S-plot metabolites visualized across all time points illustrate that AA and ANE have similar effects on plant metabolic response . Treatment of tomato roots with AA and ANE resulted in a sharp increase in metabolic intermediates in ligno-suberin biosynthesis. This includes AA-induced accumulation of moupinamide and significant increases in coniferyl alcohol in the roots of ANE-treated plants across all tested time points. In roots,AA and ANE treatments also induced increased levels of N-ethyl phydroxycinnamide and N1-trans-feruloylagmatine compared to H2O treatment, refilecting strong upregulation of the shikimate pathway and phenolic compound synthesis. Reduced levels of tomatine and dehydrotomatine were observed in the roots of AA- and ANE-treated plants indicating suppression of steroid glycoalkaloid biosynthesis. Treated plants also showed lower levels of lyso-phosphatidyl ethanolamine that could refilect enhanced membrane lipid turnover. AA and ANE can induce disease resistance locally and systemically, alter the accumulation of key phytohormones, and change the transcriptional profile of tomato with a striking level of overlap between the two treatments . The current study examined and characterized the AA- and ANE-induced metabolomes of tomato. AA and ANE locally and systemically induce metabolome remodeling toward defense-associated metabolic features. Early studies investigating transcriptional and metabolic changes in potato revealed selective partitioning and shifting of terpenoid biosynthesis from steroidal glycoalkaloids to sesquiterpenes following treatment with AA or EPA or challenged with P. infestans . Similarly, our work here with AA- and ANE-treated tomato seedlings has shown a marked decrease in the levels of two abundant glycoalkaloids, tomatine and dehydrotomatine . Our data also show strong enrichment of sesquiterpenes in leaves of AA-treated plants at 96 hours post treatment, although the identity of these sesquiterpenes is unresolved .

This work further supports evidence for differential regulation and sub-functionalization of sterol/ glycoalkaloid and sesquiterpene biosynthetic pathways in solanaceous plants in different stress contexts . AA and EPA are strong elicitors that are abundant in structural and storage lipids of oomycete pathogens, but absent from higher plants. Although their initial perception by the plant is likely different from that of canonical MAMPs , there is some convergence in downstream defenses induced by these various MAMPs. Work to characterize the effect of canonical MAMP treatment on the metabolomes of various plant species has implicated common metabolic changes that prime for defense. Cells and leaf tissue of A. thaliana treated with lipopolysaccharide showed enrichment of phenylpropanoid pathway metabolites, including cinnamic acid derivatives and glycosides . In the same study, SA and JA were also positively correlated with LPS treatment, as we also observed in tomato following treatment with AA . Recent work in A. thaliana wild-type and receptor mutants treated with two chemotypes of LPS showed increases in hydroxycinnamic acid and derivatives and enrichment of the associated phenylpropanoid pathway . Work in tobacco similarly found treatment with LPS, chitosan, and flg22 all induced accumulation of hydroxycinnamic acid and derivatives, and that defense responses elicited by these MAMPs were modulated by both SA and JA . More recent work in the cells of Sorghum bicolor treated with LPS showed enrichment of hydroxycinnamic acids and other phenylpropanoids in coordination with accumulation of both SA and JA . Treatment of tomato with flg22 and flgII-28 also enriched hydroxycinnamic acids, and tomato treatment with cps22 revealed a metabolic shift toward the phenylpropanoid pathway with hydroxycinnamic acid, conjugates and derivatives as key biomarkers . Similar to traditional MAMPs, AA and the AA/EPA-containing complex mixture, ANE, both induce enrichment of cinnamic acid and derivatives in tomato seedlings . This supports the hypothesis that MAMPs broadly induce similar metabolic changes to enrich pools of specialized secondary metabolites that contribute to plant immunity. AA- and ANE-treated roots showed strong enrichment of metabolic features classified as fatty acyl glycosides of mono- and disaccharides . Fatty acyl glycosides have been studied in several plant families and are most extensively characterized in members of Solanaceae . Investigations into the function of fatty acyl glycosides in plants suggest they may act to protect against insect herbivory through various mechanisms and provide protection against fungal pathogens . A recent study isolated and identified fatty acyl glycosides from strawberry capable of inducing immune responses in A. thaliana, including ROS burst, callose deposition, increased expression of defense-related genes, and induced resistance to bacterial and fungal challenge . This same work also demonstrated that the strawberry-derived fatty acyl glycosides induced resistance in soybean and, due to their antimicrobial activity, also protected lemon fruits post harvest from fungal infection . AA- and ANE-root treatments locally elicit accumulation of the same class of defense associated metabolites that Grellet et al. illustrated to have direct antimicrobial activity and protect against disease . Cell wall fortification is an important plant defense often initiated upon pathogen infection. Cell wall lignification is a well-studied mechanism with localized accumulation of phenolic intermediates and lignin at attempted penetration sites . Lignification reinforces and rigidifies the cell wall to create an impervious barrier to microbial ingress . In our study, AA treatment of tomato roots induced accumulation of a phenylcoumaran intermediate in lignin biosynthesis, while ANE treatment induced accumulation of coniferyl alcohol, an important monomer unit of lignin. Interestingly, coniferyl alcohol has recently been shown to act in a signaling capacity in a regulatory feedback mechanism to intricately control lignin biosynthesis, an irreversible process that is energetically costly .

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Potential agricultural losses are exacerbated by a history of pesticide resistance development

Due to such unexpected effects, accurately predicting the consequences of specific CECs, even in model insects, is not yet possible. This problem is exacerbated by a lack of information regarding effects of pharmaceuticals and other CECs on the microbial communities of any terrestrial insects. Arthropods, such as insects and crustaceans, rely on hormones to grow, develop, mate, and produce pigmentation . However, many pharmaceuticals, especially mammalian sexhormones, are structurally similar to chemicals that these organisms rely on for growth and development. These pharmaceuticals then bind to receptors and either over express or suppress their counterparts’ natural function. This has been seen in birds, reptiles, and arthropods where endocrine disruption occurs, primary and secondary sexual characteristics are modified, and courtship behaviors are changed . Although most arthropod hormones do not closely match those of mammals, their molting hormone is very similar in structure to the mammalian female sex hormone 17β-estradiol. In crustaceans, mammalian hormones have been known to cause both increased molting events and inhibition of chitobiase, the enzyme responsible for digestion of the cuticle during insect molting . In insects, 17α-ethinylestradiol, a common synthetic birth control hormone, has been shown to alter molting and lead to deformities of C. riparius . In addition to these effects,macetas de plástico pharmaceuticals have been shown to have delayed cross-generational effects . The cabbage looper is a well-studied polyphagous insect native to North America and is found throughout much of the world . T. ni are yellow-green to green in color and can complete their life cycle in as little as 21 d depending on temperature . This species is a pest on many agricultural crops including crucifers and a variety of other vegetables in both field and greenhouse settings .

Currently, there is little to no information regarding pharmaceutical effects at the concentrations found in reclaimed water on the growth or microbial community composition of any terrestrial herbivore. Many herbivores can be exposed to these contaminants after the CECs enter surface waters, soil, and plants from wastewater reuse and unintended discharge. To investigate the function of the gut microbes in insects, several studies have used antibiotics applied at high doses . There is also no information regarding effects of CECs when translocated through plants to terrestrial insects. To test the hypothesis that common pharmaceuticals affect mortality, development, and microbial communities of T. ni, we conducted a series of bio-assays in artificial diet and on a key host plant utilizing surface water concentrations of common important pharmaceuticals. We used a culture-independent approach by performing a 16S rRNA gene survey on both diet and whole-body insects. Any effects would have potentially important implications from agricultural perspectives. Also, as there is currently no information on effects of CECs on terrestrial insects acquired through a plant matrix, our findings would have possible interest for integrated pest management research.In our study, CECs at concentrations found in reclaimed wastewater were shown to increase mortality of T. ni, especially on artificial diets contaminated with antibiotics, hormones, and a mixture of the chemicals. The mortality effect was also evident when T. ni were reared on plants grown in antibiotic-containing hydroponic growth media. Because plants grown in the hydroponic system contained quantifiable levels of ciprofloxacin in the leaf tissue , and the antibiotic treatments significantly changed the microbial community of the insect , we think this is possibly a cause of the mortality but we cannot exclude direct effects of the CECs on the insects or indirect effects through the plants. Ciprofloxacin is a quinolone topoisomerase IV and DNA gyrase inhibitor that acts by stabilizing the DNA-topoisomerase IV and DNA-girase so that it is no longer reversible . This blocks DNA replication and eventually causes cell death of bacteria.

However, unlike bacteria, when higher-level organisms evolved, the A and B subunits of the topoisomerases fused, creating homodimers that cannot be targets of ciprofloxacin , and thus damage to the ribosomes of insects is not a possible mechanism of toxicity. Interestingly, we did not see the increased time to adulthood in T. ni reared on plants compared with those reared on contaminated artificial diet. We postulate the discrepancy is possibly due to a number of factors such as dilution of CECs, as they were acquired from the water by the plants or there was bio-degradation of the chemicals occurring in the plant or by photo degradation. However, recent studies have shown pharmaceutical concentrations in surface waters, which appear to remain constant over the course of several years . More studies would be needed to determine how CECs at concentrations found in reclaimed water for agriculture would interact with current IPM strategies , and how soil matrices would affect the chemical acquisition and translocation by plants. Many insects rely on microbial communities and endosymbionts to grow and develop; however, it has been shown that Lepidoptera species do not have a vertically transmitted microbial community. In addition, because the effects of microbial communities on T. ni survival and development have not been documented, we present these data only to show that microbial communities change when exposed to CECs, and not as a proven factor influencing survival. We found significant shifts in the microbial community in the various life stages examined within the control treatments notably from third instar to subsequent life stages. A similar result has been reported for mosquitoes and other insects . However, there is one family, Lactobacillaceae, which appears in all treatments and life stages in high proportions, except for adults. They are fairly common in insects and can be responsible for at least 70% of the bacterial community .

Lactobacillaceae is responsible for ∼42% of the bacteria in all life stages, followed by Pseudomonadaceae, Alcaligenaceae, and Enterobacteriaceae. Lactobacillaceae have been shown to act as beneficial bacteria in Drosophila ; however, its function in T. ni is still unknown. Alcaligenaceae has been shown to be present in other moths , but Lepidopterans are not thought to have a functional microbiome . There are clear patterns regarding the changes in microbial community proportionality according to the heat map . In controls, third-instar microbial communities are relatively evenly spaced by family. The microbial community becomes predominately Lactobacillaceae for sixth instars and pupae. Once the insects reach the adult stage, their most predominant family is Pseudomonadaceae. This pattern holds in the acetaminophenand caffeine treatment groups as well. Interestingly, the other treatment groups do not share this pattern. For antibiotic- and hormone-treated T. ni, Lactobacillaceae is the predominant microbial family in the immature stages, but at the adult stage microbial community reverts to predominantly Pseudomonadaceae. We suspect that this is because,cultivo del frambueso once the larvae undergo metamorphosis and shed their gut contents in preparation for pupation, they are no longer exposed to the pressures exerted by the CECs on the microbial community. Fig. 3 provides a visual indication of the changes in the bacterial communities over time. The increase in β diversity after eclosion could be due to the larvae no longer being exposed to CECs or diet-borne bacteria after being moved to sterile containers. Also, when bacteria are lost as larvae digest their gut contents during pupation, the microbial β diversity could change. Interestingly, the hormone-treated T. ni follow a similar pattern to those exposed to antibiotics, but their ellipses are always much smaller, suggesting the entire insect population is showing a uniform response within their microbial communities. However, in the mixture-treated insects, larvae displayed a greater average diversity in their microbial community structure than either pupae or adults. This finding has not been shown in any single category of treatment, and we suspect the microbes exposed to mixtures could be experiencing potential interactive effects among chemicals . Such interactions should be the focus of future studies along with investigations of plant rhizosphere bacteria, particularly since we found a difference in the Bradyrhizobiaceae family for all treatments. These results show that a terrestrial insect pest of commercial crops can be affected by CECs found in reclaimed wastewater for agricultural use. Our results suggest that CECs found in wastewater can impact T. ni growth and development, survivorship, and alter their microbial communities. Because T. ni is a common agricultural pest found around the world, feeds on a wide variety of plants, and has a history of developing pesticide resistance, its ability to deal with toxins is likely higher than many other insects.

In addition, the responses we observed to CECs could have interesting implications for IPM practices on plants such as lowering the amount of pesticides needed or increasing susceptibility to insect pathogens, as has been shown in mosquitoes . These potential effects may be understated because some insects cannot detect the presence of the pharmaceuticals . However, we do not recommend purposefully exposing crops to CECs specifically for the control of insects because our study documented that these pharmaceuticals are translocated into crops and we do not yet know their possible effects on humans if consumed . We specifically want to note that ingestion of these compounds through uptake and translocation by a plant is not the only way T. ni or any other insect would be exposed to these compounds. Overhead sprinkler irrigation could cause contact absorption by the plants or insects, and simply drinking water on leaves at contaminated sites could expose insects to higher concentrations than were found in plant tissues. In fact, the ciprofloxacin concentration used was less than one-third of the highest rate . We urge caution in extrapolating to plants growing in soil, because variation in soil type and potential soil bacterial degradation could affect persistence [although soil bacteria are often negatively impacted by CECs ]. However, CEC exposures are considered pseudopersistent because they are reapplied with each irrigation. Thus, the effects reported here are likely to be conservative. Additional studies with other insects, particularly those with other feeding strategies, will be necessary before any patterns can be discerned.The majority of these associations are with arbuscular mycorrhiza fungi which penetrate into root cortex cells to form highly branched structures . The investment of photosynthetic carbon by plants to AMF is rewarded with increased nutrient availability made possible by the extended hyphal network in the soil. For instance, up to 90% of phosphorus uptake in plants can be contributed by symbiosis with AMF . AMF networks in the soil also influence water retention and soil aggregation further impacting plant growth . Moreover, next-generation sequencing technologies and advances in imaging techniques have greatly improved our knowledge on the taxonomical and functional properties of fungal communities in the rhizosphere . However, these methods are optimized for fine scale analysis and are not capable of assessing the foraging capabilities of hyphal networks which can span across centimeter to meter scales. Toward this end, several researchers have used compartment setups with physical barriers created by 20–37 µm nylon membranes which restrict movement of roots but not mycorrhizal fungi. This separation creates root-free and plantfree soil compartments connected only by mycorrhizal fungi to examine the transport of various compounds across these compartments. Using this set up, the importance of mycorrhizal fungi in the flow of different elements such as carbon , nitrogen and phosphorus between plants, soil and microbes over centimeter distances have been validated. Repeated disruption of the hyphal connections also led to a decreased resistance in plants to drought stress . The membranes can also be placed horizontally to create different depth gradients to investigate hyphal contributions to water uptake . In some studies, an additional 1.5–3 mm air gap is created between two membranes with a wire net to restrict solute movement between two chambers . A common feature of these set ups is the size-exclusion membranes which proved to be critical in distinguishing fungal hyphae processes in the rhizosphere soil. In addition to AMF interactions, a split root set up, which separates the roots of one plant into halves, can be introduced to investigate the systemic response of plants . In essence, the split-root system directs the growth of the roots to generally two different growth conditions and enables the investigation of whether a local stimuli have a local or global response which can be observed at the root or shoot level .

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The developmental time was rescued by reintroducing bacteria from the genus Asaia

Over expression of ecdysone during development could explain why BPA causes mouth deformities and increased pupation time in C. riparius 11. In mosquitoes , the vitellogenin gene is a target for ecdysteroid receptor, which can be modulated by the xenoestrogen BPA. This suggests that BPA and other xenoestrogens could have an effect on the production of vitellogenin, the egg yolk protein. This also would lead to altered viability of offspring, which we did not assess. Acetaminophen and the mixture of PPCPs significantly slowed developmental time of C. quinquefasciatus, but antibiotics alone did not. In contrast, Chouaia et al.described delayed larval development of Anopheles stephensi after treatment with the antibiotic rifampicin at 120 µg/ mL. In our study, there was relatively little difference in development of immature mosquitoes between the antibiotic treatments and the controls for the Acetobacteraceae, to which Asaia belongs. It is possible that the antibiotics chosen for this study are not effective in eliminating Asaia. Alternatively, the antibiotics in our study were used at doses much lower than the rifampicin tested by Chouaia et al., and might have achieved similar effects if applied at higher doses. With a combination of antibiotics, hormones and other constituents that occur in PPCP-contaminated reclaimed water, it is difficult to know exactly which chemical is affecting which bacterial family and which bacteria were responsible for the deleterious effects on developmental time. Notably, the antibiotic treatments had approximately 1/3 of the total number of bacteria relative to the control. Therefore even the approximately 8500 counts of Rickettsiaceaein the antibiotic treatments are lower in number than the Enterobacteriaceaefound in control treatments. Surprisingly,macetas de plastico the substantial loss of bacterial counts in the antibiotic only treatments did not slow development.

Although the antibiotics decreased overall bacterial counts in C. quinquefasciatus, W. pipientis appeared to be relatively unaffected. Wolbachia pipientis is susceptible to doxycycline and rifampin. Of the chemicals we tested, oxytetracycline should have had the most impact on Wolbachia. However, because the bacterial counts were relatively similar for W. pipientis in all treatments, we suspect either the oxytetracycline was too dilute to have an effect, it allowed a non-susceptible strain of W. pipientis to dominate, or it is simply ineffective against W. pipientis. However the vast majority of the eight bacterial families were greatly reduced in the antibiotic treatments. Of these families, Enterobacteriaceae and Microbacteriaceae were the most reduced in both the antibiotic and mixture treatments. The family Enterobacteriaceae is highly associated with insect endosymbionts, such as Buchnera in pea aphids. Buchnera bacteria are known to aid the aphid by supplying essential amino acids lacking from a nutritionally deficient diet. Without these endosymbionts the aphids do not develop properly, but the microorganisms apparently have no other direct biological effect on the aphid. The size of the Enterobacteriaceae populations probably had a minimal effect on larval developmental time, as thecounts in the acetaminophen treatment are relatively similar to the hormone-treated group. Additionally, the hormone treated group has a substantially reduced count of Enterobacteriaceae bacteria compared to the control, but showed no significant changes in developmental time. We suspect, therefore, that the acetaminophen is negatively impacting some other biological system in the larvae, but determination will require additional research. The use of reclaimed water for crop irrigation and the release of water from waste treatment plants and farm waste ponds into surface waters is occurring and likely to escalate as demand for fresh water increases. While, the research reported here suggests that PPCPs contaminating reclaimed water will have potentially useful effects for mosquito control, if the data can be extrapolated to other insect species, PPCPs will also have unintended negative effects on other aquatic insects. Very little is known regarding how these contaminants might bio-magnify or change chemically as they move through the food web. The eventual impact on populations is also unknown. Further, combinations of PPCPs may be more important for some insects than individual components.

Additional research is needed not only on aquatic insects living in surface waters, but also on uptake by plants and associated herbivores in terrestrial environments.Endosymbionts, bacterial species known to grow, develop, and vertically transmit in an organisms’ cells, usually for mutualistic symbiosis, are essential to the growth, development, and fecundity of many insect species. Many aphid species, such as Acyrthosiphon pisum, Megoura viciae, and Myzus persicae, rely heavily on endosymbionts to survive on the unbalanced diet of phloem sap. Their bacteriocyte endosymbiont, Buchnera spp, provides essential amino acids. Without Buchnera, aphids demonstrate reduced growth rates and development and produce few or no offspring. However, some endosymbionts in the genus Wolbachia are known to manipulate insects for their own benefit and can also lead to increased vector competency for transmission of human diseases such as West Nile Virus. Wolbachia species are common vertically transmitted endosymbionts in many mosquito species and typically infect reproductive tissue. There are many species of Wolbachia that influence a variety of insect behaviours and life-history traits. Some endosymbionts can also act defensively by killing parasitoid eggs after they are laid in the host . Because endosymbionts play major roles in insect development, they have been proposed for use in insect control . Due to the importance of endosymbionts for development in many insect species, some species vertically transfer endosymbionts . For example, Estes et al. describes the microbiome of dung beetles’ brood balls, which are used to nourish their offspring until they are adults. When the microbiota in the beetle offspring and their female parent were compared in sterilized dung and soil, they had proportionally identical 16S rRNA sequences from their microbiome. Interestingly, over their life stages, the proportions of the families of bacteria in the beetles’ microbiome changed. The predominant family of the first three instars varied by individual. However, from the pupal stage onwards, Enterobacteriaceae was the most common family in the dung beetle. Bees acquire important bacteria through social interaction and also from the environment . Without some of these bacteria, it is thought that honey bees could become more susceptible to outside diseases and increase incidents of colony collapse .

More studies are needed to fully understand the importance of microbiota in mosquitoes as they have been linked to increased transmission of pathogens from mosquitoes to humans. Mosquitoes are common disease vectors, which spend their juvenile stages in aquatic environments . Bacteria from the water, both symbiotic and free-living, have been shown to influence the microbiome of mosquitoes, suggesting that some of their possible endosymbionts are collected from the environment . Consequently, if the environment is altered, and some of these necessary bacteria are reduced/eliminated, there could be detrimental effects on the development of mosquito larvae. Such a removal effect may occur as the result of antibiotic runoff or other pollution,macetas rectangulares and/or environmental changes. For example, Rosi-Marshall et al. showed that common pharmaceuticals in streams will alter the respiration and diversity of biofilms. Pennington et al.reported differences in whole-body micro-biomes and increased developmental times for Culex quinquefasiatustreated with various pharmaceuticals and personal care products .Chemicals intended for human use often occur in aquatic environments and/or enter water supplies through water treatment plant overflow or from use of reclaimed water in water scarce areas. These chemicals can then affect bacterial communities in the water and the associated aquatic insect community. Presence of these contaminants can alter effectiveness of Bacillus thuringiensis subsp. israelensisa pesticide commonly applied for mosquito control . However, little is known about how such contaminants will influence the microbiome of such insects. Similarly, there is a lack of data determining if mosquito bacterial communities vary during the course of larval development; all available studies we are aware of examine only late instar larvae or mixed lower in stars and species, and information regarding mosquitoes’ endosymbionts and their function is very limited or non-existent outside of Wolbachia. Therefore, we describe the differences in the bacterial communities of the mosquito C. quinquefasciatus in multiple distinct larval stages, as well as when these mosquitoes are reared in environments contaminated with ecologically relevant concentrations of PPCPs that commonly occur in combinations in order to provide a baseline for more in-depth studies. In preparation for sequencing, three mosquitoes from each PPCP treatment were collected as second, third and fourth instars, as first instars were too small to collect without damage, and twice washed with 95% ethanol to remove any external microorganisms. Larvae were then transferred to individual sterile 2 mL microcentrifuge tubes with 95% ethanol and stored at -60 ± 3°C in an ultra cold freezer until DNA extraction. DNA was extracted using a Qiagen DNeasy® Blood and Tissue Kit following the manufacturers protocols amended as in Pennington et al.. In addition to mosquitoes, samples of water and water plus diet were also extracted using identical protocols, with the additional step of centrifugation at 2900 rpm in an IEC HN-SII tabletop centrifuge for 1 h to create a pellet. Upon extraction, nucleic acid concentration was quantified using a Nanodrop ND- 2000c Spectrophotometer , to confirm enough genetic material for sequencing. This process revealed no DNA in water or water and diet samples when no mosquitoes werepresent and thus they were not subjected to further analysis because any bacteria would have originated from the mosquitoes, their egg-rafts, or from the air after the water had been altered by the mosquitoes’ various biological processes. Roche 454 bacteria barcoded amplicon pyrosequencing was performed by a commercial sequencing facility .

The procedure used the forward primer 27Fmod and the reverse primer 519Rmodbio in a single-step 30 cycle PCR using HotStarTaq Plus Master Mix . PCR was performed using the following cycle conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s; 53°C for 40 s, 72°C for 1 min; and a final elongation at 72°C for 5 min. After PCR, all amplicons were mixed in equal concentrations and purified using Agencourt Ampure beads . Samples were then sequenced with Roche 454 FLX titanium instruments and reagents following the manufacturer’s guidelines.Operational taxonomic units were chosen by the default 97% identity threshold, which roughly correlates to species , via the UCLUST method as implemented in the pick_otus.py script . Representative OTUs were chosen using the pick_rep_set.py script and default settings. The Greengenes reference database clustered at 97% identity was used to assign taxonomy using the assign_taxonomy.py script. OTUs were counted and summarized using the make_otu_table.py and summarize_taxa.py scripts respectively.There were 658 distinct OTUs at the species level with 58 distinct families; OTUs failed to match any contained within the database and could not be assigned taxonomically. Fifteen families were chosen by their proportionality being greater than or equal to 1% in at least one sample. The cut-off was chosen at 1% as this was assumed to be the minimum to influence larval development. For alpha diversity, multiple rarefactions were performed using the multiple_rarefaction.py script with the lowest rarefaction depth of 2000, the highest rarefaction depth of 21000, a step size of 1000, and a replicate number of ten, which normalizes the data at each depth. Alpha diversity was calculated using the alpha_diversity.py script with the metrics observed species and Shannon Indices. Alpha diversity data was not averaged between replicate mosquitoes as they have been averaged by resampling-replicates and the complications and validity of this is still being considered. Metrics were summarized using the collate_alpha.py script. Statistical analyses were performed using R . Following processing through the QIIME pipeline, “Permutational MANOVA” in the Vegan package was used to compare the OTU data. Independent variables were instar , PPCP treatment and the interaction of the two, with three replicates of each instar in each PPCP treatment and control . PERMANOVA is analogous to MANOVA but is suited to address the non-normality that is commonly associated with count data in ecological community and genetic data . Microbial community data were further examined via principal component analysis performed in the FactoMineR package . PCA and PERMANOVA were conducted on each instar in the individual PPCP and control treatment groups. Following PCA, variables were examined for their contributions and correlation to each of the first two dimensions. Those variables that were ≥ 85% correlated were included in subsequent pairwise comparisons by instar in their respective treatment.

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The effect of the minerals on the activated carbon on NO adsorption efficiency was studied

Parametric studies on the production of activated carbon and the adsorption of NO by the carbon have been conducted. The optimal conditions and effectiveness of this procedure to regulate NO emissions have been determined.The activated carbons were characterized by the measurement of their average pore size and surface area. There are three types of pores which can develop in the carbon particles: micro-pores , mesopores and macropores . BET surface area of wheat straw char carbonized at temperatures between 300°C and 700o C is shown in Fig.3. The surface area passes through a maximum of 105.2 m2 /g at a pyrolysis and activation temperature of 600o C and 650o C respectively. The BET surface area of sweet potato stalk exhibits similar behaviors as that of wheat straw, being 88.1 m2 /g, 84.7 m2 /g, and 132 m2 /g at a pyrolysis temperature of 300°C, 400°C, and 600°C, respectively. The decrease in surface area beyond 600°C is caused by sample burning off. Using temperatures much lower than 600o C would compromise the maximum amount of effective adsorption surface area attainable . It is not only important to run reactions at temperatures low enough to prevent burn off and ash formation but also high enough to generate effective surface areas, which would be at 600o C for wheat straw. Fig.4 shows the BJH adsorption cumulative pore area of wheat straw activated carbon generated at different pyrolysis and activation times. It is evident from the plot that samples derived from longer pyrolysis and carbonization times exhibited a higher micro-pore count compared to shorter times. As with temperature generation optimum, however,greenhouse pot too long pyrolysis and activation reaction times cause an adverse increase in burn off percentage. Though wheat straw carbonized with a pyrolysis time of six hours and activation time of 2 hours still demonstrated a higher cumulative pore area than the shorter times, it also produces larger amounts of burn off. Since reaction temperatures were kept below 600o C, ash formation that would have diluted effective surface area was prevented.

Even though ash does not form, pyrolysis and activation times must still be chosen to create a balance between pore formation and burn off, one that would generate a high micro-pore count but at the same time, minimize material loss. The optimal pyrolysis and activation times for wheat straw are 2 hours and 1 hour respectively.Optimal pyrolysis and activation temperatures and times for carbon preparation were determined based on the amount of NOx that can be adsorbed by the activated carbon. The adsorption capacities of wheat straw activated carbons generated by different pyrolysis and activation temperatures are shown in Fig.5. A gas mixture containing 250ppm of NO, 5% O2, 10% CO2, with N2 as the balance was passed, at a flow rate of 250ml/min, through a turbular reactor containing 2g of activation carbon at 25o C. It is evident from the plots that the WS-2-600-1-650 activated carbon had the best adsorption efficiency. Samples carbonized above 600o C have higher ash concentrations than those carbonized below, while those carbonized below have lower percent micro-pore counts and surface area—both explaining why wheat straw generated at 600o C had the best adsorption efficiency. The NO adsorption efficiencies of wheat straw samples carbonized by differing pyrolysis and activation times are shown in Figure 6. It is evident from the plots that activated carbons carbonized by prolonged pyrolysis and activation times have better adsorption efficiencies than those carbonized by shorter times due to higher pore count and BET surface area. The micro-pore count and the surface area of activated carbon increases with an increase of the preparation time, which explains why the samples with the longest pyrolysis and activation times have the best adsorption efficiencies. However, prolong activation results in more burnoff and the production of ash. A balance must be reached when setting reaction parameters, one that will generate the largest surface area without a significant burnoff. We have found that the optimal pyrolysis and activation times for wheat straw are two and one hour, respectively. The hydroponic biomass possesses high mineral content. The activated carbon was first soaked in water to dissolve the soluble minerals and then dried to remove the moisture from the carbon particles. The adsorption experiments using the mineral-free activated carbon were performed and the results indicate that the NO adsorption efficiency was substantially improved .

A 100% NO removal efficiency was obtained for the entire 2 hr experiment, using a gas mixture containing 250 ppm NO and 10% O2 and at a W/F of 30g.min.L-1. The improved NO removal efficiency by the activated carbon with mineral removed is attributed to the increase of the carbon surface area, which was otherwise covered by the minerals. Because of this finding, the parametric study on the NO removal efficiency was performed mostly with the activated carbon having mineral content removed by water dissolution. The adsorbed NO can be desorbed from the activated carbon if temperature of the carbon bed is raised. Further increases of temperature results in the reduction of NO by activated carbon to produce N2. Simultaneously, the activated carbon is regenerated as a result of the reduction of NO to N2. Experiments on the reduction of the adsorbed NO by the activated carbon were performed by heating the NO saturated carbon under anaerobic conditions. In order to evaluate the behavior of the process over time, a purge gas flow of 1.0 L/min N2 was passed through the carbon bed and subsequently directed to the NOx analyzer. Desorption was conducted with a temperature ramp rate of 40°C/min from room temperature to 600o C. As the temperature of the carbon bed was increased, NO was desorbed from the surface of the activated carbon. Further increase of the temperature results in the reduction of NO by the activated carbon to N2. The fraction of the adsorbed NO that is reduced to N2 can be calculated by subtracting the NO coming out of the carbon bed from the total amount of NO adsorbed. The fraction of the adsorbed NO that is reduced depends on the temperature and the flow rate of N2 gas. Fig.13 shows the fraction of the desorbed NO integrated over the temperatures as the temperatures of the carbon bed was raised. As can be seen, the fraction of the total NO desorbed as NO was less than 100% of the total NO adsorbed, 38.5% and 9.1% for sweet potato and wheat straw activated carbons, respectively. The difference of which is attributed to the reaction of NO with the activated carbon to form N2. From the desorption curve as a function of temperature, the NO desorption mainly took place at temperature below 450°C, while the NO reduction by carbon occurred at temperature above 450°C,30 plant pot the higher the temperature the more effective the reduction is. Another set of experiments were performed to study the reduction of NO by activated carbon as a function of temperature and W/F, the ratio of the amount of carbon to flow rate of N2.

In this study, temperatures were varied between 300 and 500°C and W/F between 10 and 40 g.min/L. Fig.14 shows that, in the case of the sweet potato activated carbon, 100% of NO was reduced to N2 at 500°C with a W/F above 10 g.min./L, while at 450°C it would require a W/F above 30 g.min./L. For wheat straw activated carbon, NO can be reduced to N2 at lower temperatures and smaller W/F ratios than those of sweet potato . All inlet NO was reduced to N2 at 450°C with a W/F above 15g.min.L-1. Higher temperatures or larger W/F ratios are favorable for NO reduction. The NO reduction efficiency also depends on the concentration of NO in the system. Fig. 16 shows NO reduction by sweet potato and wheat straw activated carbons at 450C with two inlet NO concentrations, 250 ppm and 1000 ppm. The results indicate that the higher the inlet NO concentrations, the smaller the fraction of the inlet NO is reduced at a given W/F. All of inlet NO was not reduced to N2 by the sweet potato activated carbon until the W/F reached 30 g.min/L and 40 g.min/L for 250 ppm and 1000ppm of inlet NO respectively. The activated carbon made from wheat straw can reduce NO more effectively than that from sweet potato. The entire inlet NO was reduced by the wheat straw activated carbon with a W/F of only 15 g.min/L and 30 g.min/L for 250ppm and 1000ppm of NO, respectively. The space maximum allowable concentration of NO in a human occupied cabin is 4.8ppm. Experiments were conducted to determine conditions for NO adsorption on SP-2-600-1-650 at room temperature such that the NO concentration exiting the carbon bed is less than 4.8ppm. The activated carbons before and after the removal of minerals with water dissolution were both examined. The parameters studied included inlet oxygen concentration , and carbon weight to flue gas flow rate ratio . The time that the carbon bed can hold before the NOx concentration exiting the bed exceeds the SMAC will be called SMAC time. Fig.17 and 18 show that the SMAC time for both activated carbons increase with the increase of W/F and oxygen concentrations. The removal of minerals from the activated carbon increases the surface areas and results in a much longer SMAC time. The SMAC time was 22 min., 30 min., 36 min., and 62 min. with a W/F of 45 g.min/L when flue gas contains 5%, 8%, 10%, and 15% O2, respectively using the activated carbon containing mineral. However, the SMAC time was substantially increased to 145 min, 215 min, 330 min, and 500 min at a W/F of 45 g.min/L with flue gas containing 5%, 8%, 10%, and 15% O2 using the activated carbon with mineral removed. Experiments were conducted to determine the effects of the regeneration on activated carbon in terms of NO removal efficiency, as assessed by the carbon’s SMAC time. The results indicate that regeneration improves the removal efficiency of NO. This phenomenon is attributed to the increase of surface area and micro-pores of the activated carbon. However, it was observed that additional carbon burns off occurs during regeneration, which causes the overall amount of activated carbon to decrease after each regeneration cycle. The loss of mass were determined to be about 0.99% for wheat straw and 0.49% for sweet potato stalk activated carbon per cycle of regeneration at 600°C. Lastly, due to the extent of the mission, the sufficiency of the amount of inedible biomass in providing the activated carbon for the life support systems should be assessed. With a six person crew, such as the mission planned for Mars, the average food consumption is about 0.25kg/d per capita . Six people would need 1.5kg of wheat per day. Wheat straw yield depends on the specific varieties harvested and is widely affected by agronomic and climatic factors. An average ratio of 1.3 kg of straw per kg of grain is found for most common varieties . Burnoff accounts for about 66.7% at 600o C in pyrolysis and 650o C in activation, while the loss of carbon mass from soaking and drying was determined to be about 14.4%. So as a result, 203kg of wheat straw activated carbon can be produced per year. By incinerating feces, inedible biomass and trash, between 3g and 30 g of NOx can be produced per day, depending mostly on the amount of nitrogen in the inedible biomass. The range in the amount of NOx produced covers 90% pre-treatment removal of nitrogen to no pretreatment . . About 10% of nitrogen in the waste forms NOx . Regeneration of the adsorbing activated carbon shall occur once a week via the reduction reaction. The loss of carbon mass was determined to be about 0.99% at 600o C per cycle of regeneration, After one year, all the activated carbon should be incinerated and replaced. Calculations based on the above assumptions yield 210g NO emissions per week, and the adsorption capacity of NO by the activated carbon is 5.46mg/g carbon, which will need at least 38.5kg of wheat straw activated carbon all year long. These can be distributed among one adsorption tank, just with a load of 64.6kg of wheat carbon in the beginning of the year. Every week, the tank should been regenerated.

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BER is characterized by water-soaking of the tissue and cell death in the blossom-end pericarp

Blossom-end rot in tomatoes and peppers causes significant losses in the vegetable industry each year. This is followed by blackening and sometimes drying of the affected tissue. BER occurs during fruit development prior to physiological maturity. While calcium deficiency in fruit tissue and abiotic stress can increase BER, the biological mechanism of cell death remains unknown and current treatments are not fully effective. Many previous studies on the biological drivers of BER development in tomatoes and peppers have relied on comparing measurements from BER affected fruit to healthy fruit. This approach has identified several possible causes of cell death. Comparing BER affected tomatoes grown in a low calcium hydroponic solution to healthy fruit grown with a higher calcium hydroponic solution, Mestre et al. found a reduction in glutathione content, glutathione reductase activity, and catalase activity. Increased hydrogen peroxide and lipid peroxidation were also associated with BER affected fruit. Aloni et al. found depleted apoplastic and symplastic ascorbic acid contents and increased apoplastic ascorbate oxidase activities in BER affected peppers compared to healthy peppers. An investigation of intracellular wash fuids by Turhan et al. found increased hydrogen peroxide concentration and peroxidase activity in BER affected fruit compared to healthy fruit. While these results strongly support the trend of increased oxidative stress during blossom-end rot development, the conclusions thus far are mainly correlative in nature. Trough over expression of a vacuolar calcium importer, de Freitas et al. found that increasing vacuolar calcium and decreasing apoplastic calcium increased BER development in tomatoes. These results suggest that decreased apoplastic calcium can lead to BER development,raspberry plant container though the hypothesis that adequate apoplastic calcium incurs a protective effect has not yet been tested.

Additionally, confounding factors at the whole plant, fruit, and cellular level make it difficult to determine the sequence of events leading to cell death during BER development. To eliminate these confounding factors and establish causative evidence regarding the biological cause of BER, direct manipulation of the apoplastic calcium concentration and antioxidant capacity is needed. Pericarp discs have been used previously to study ripening in tomatoes. Te fruit used in these experiments were at the mature green stage and the discs studied during these experiments followed ripening trends regarding physiology, visual appearance, and original tissue location. This paper describes a new method for studying BER development in immature tomatoes using the pericarp disc system.Immature green tomatoes showing no symptoms of BER were observed to develop BER symptoms during storage after harvest, if harvested just prior to the usual timing of BER development on the plant . BER development of the plant occurred within the first 3 days after harvest and the symptoms resembled BER regarding location and visual development. These results were replicated with 21-day old fruit of the HM variety and in the Rutgers variety.Pericarp discs were made from the stem-end, blossom-end, and columella tissue of immature HM tomatoes 21 days after pollination . During the first 12 h of storage, disc color lightened slightly and developed a “frosted” appearance as described previously. In discs made from stem-end pericarp, this appearance remained the same for 4 days. In blossom-end pericarp discs, symptoms developed resembling the progression of BER development on the plant. Water soaking of the tissue was first observed on day 2 of storage, usually followed by tissue darkening. While water soaking often started on day 2, initiation of water soaking also occurred on day 3 and 4 in some blossom-end discs. Water soaking and tissue blackening was initially localized to one area of the disc, and subsequently spread to the rest of the disc, as observed in Additional file 2: Video S1. An example of these symptoms is shown in . These results were replicated in Rutgers and Ailsa Craig tomatoes . Darkening in top discs was observed only when the discs were excessively handled for color or weight loss analysis, and was visually similar to symptom development in blossom-end discs. In columella discs, symptoms developed similarly to that of pericarp discs.

Discs taken from closer to the stem-end often maintained a green-white appearance, while discs taken from near the blossomend darkened over the 4-day storage period . In blossom-end pericarp discs made from fruit already showing BER symptoms, the blackening spread from the BER affected area to the non-affected area. HM and Ailsa Craig tomatoes produced the most consistent results . Rutgers tomatoes were more susceptible to symptom development at sites of damage from handling in stem-end discs .Color was measured in HM discs and whole fruit harvested with BER symptoms . L* and a* values were used to quantify darkening and loss of green color, respectively. Visual evaluations of symptom development in discs were also completed for comparison . Discs exhibiting symptoms and BER tissue in whole fruit harvested with BER symptoms had lower L* values and higher a* values than stem-end discs or the stem-end of whole fruit harvested with BER symptoms. Blossom-end discs that did not develop symptoms had similar values to those of the stem-end discs.HM disc weight loss over a 4-day storage period was not significantly different between stem-end discs and asymptomatic blossom-end discs . Discs with a visual symptom rating of 2 had significantly higher weight loss than asymptomatic blossom-end discs and stem-end discs. Weight loss in Rutgers discs was higher than HM discs. Weight loss for stem-end, side, and blossom-end Rutgers discs was slightly higher from day 2 to day 3.5 than day 0 to day 2 . Weight loss in columella discs was similar from day 2 to day 3.5 and day 0 to day 2.Enzyme analysis and color measurements were taken after 4 days of storage on 24 top and 24 bottom discs. A Pearson correlation analysis between enzyme activities and color measurements from HM discs showed that both pyrogallol peroxidase and ascorbate oxidase were significantly negatively correlated with L* values and positively correlated with a* values . Te mean ascorbate oxidase activity was 193.8 nmol min−1 g−1 . Te mean pyrogallol peroxidase activity was 11.7 μmol min−1 g−1 . Both ascorbate oxidase and pyrogallol peroxidase activity means were significantly higher in blossom-end discs compared to stem-end discs.

Treating discs with a 15 min soak in either 10 g/L calcium chloride or 500 mM ascorbic acid was effective in inhibiting the development of visual symptoms. Both visual evaluation and color data indicates that symptoms only developed in deionized water pH 5.58 and DI water pH 2.00 treated discs. Ascorbic acid treatment caused a mild loss of green color as seen in the slight increase in a* value compared to calcium treatments. Aside from this color change, ascorbic acid and calcium treated discs appeared healthy with no water soaking or darkening. Discs treated with DI water pH 2.00 showed water soaking and tissue degradation, and exhibited a gray color. Gray color development became apparent 3 days after harvest and disc formation, and became more noticeable on day 3.5, similar to the timing of tissue darkening observed in DI water-treated discs. In addition to 15 min soaking treatments, vacuum infiltration was tested as a method for inducing the uptake of treatment solutions. However, vacuum infiltration was found to induce a translucent and water-soaked appearance in all treatments, making water-soaked symptoms associated with blossom-end rot hard to identify . Te vacuum infiltration method was not pursued further.Symptom development in harvested whole fruit and disc systems closely resembled symptom development in tomato fruit on the plant. Harvested whole fruit developed symptoms only at the blossom-end of the fruit. Similarly,container raspberries only discs from the blossom-end of fruit developed symptoms in the pericarp disc system. Similar to trends in whole fruit, columella tissue taken from near the blossom-end of the fruit developed BER like symptoms while top columella tissue often did not develop symptoms. Visual BER symptom development in whole fruit on the plant begins with water soaking of the tissue at the blossom-end, usually in one or a small number of spots. Water-soaked tissue begins to darken until it has a blackened appearance. In discs, symptoms developed in a similar manner, starting with the development of a water soaked and somewhat translucent appearance. Water soaked areas then darkened. Drying of the affected tissue in discs was limited, likely due to the high relative humidity environment, though weight loss evidence suggests that increased water loss can occur during symptom development.

Drying of the BER affected area is also common in fruit that develop BER on the plant.Ascorbate oxidase activity was used as a measure of the overall trend in antioxidant depletion in BER affected fruit. Maintaining ascorbic acid concentrations in fruit has been suggested as key to BER resistance and increasing ascorbic acid concentrations in vulnerable fruit tissues is a possible treatment to inhibit BER incidence. However, conflicting ascorbate concentrations have been reported in BER fruit compared to healthy fruit. Tus, direct measurement of ascorbic acid is an unreliable indicator for the state of the antioxidant system and was deemed inappropriate for the purpose of comparing BER processes in whole fruit to symptom development in discs. Alternatively, ascorbate oxidase activity has been shown to be increased in BER affected peppers compared to healthy peppers. In tomato, ascorbate oxidase was found to be transcriptionally upregulated in fruit treated with the BER-inducing phytohormone gibberellin. Additional file 3: Figure S2 shows ascorbate oxidase activity in healthy top, healthy bottom, and BER affected pericarp tissue from BER affected fruit, and healthy top and healthy bottom pericarp tissue from BER unaffected fruit. This data indicates that the ascorbate oxidase activity from stem-end pericarp tissue of a BER-affected fruit is similar to the stem-end and blossom-end of healthy fruit. In our pericarp disc system, ascorbate oxidase activity was significantly correlated with objective measures of tissue darkening , suggesting that similar antioxidant depletion processes occur during symptom development in discs as in whole fruit.Peroxidase activity was chosen as a second indicator of enzymatic similarities between symptom development in discs and BER development on the plant. Peroxidase activity measured using an aromatic electron donor was shown to be greatly increased in BER fruit compared to healthy fruit. This trend has been linked to increased lignification, and peroxidase trends presented by Reitz and Mitcham are similar to those found for ascorbate oxidase activity in Additional file 3: Figure S2. In our disc system, peroxidase activity was significantly correlated to measures of tissue darkening , suggesting a similar trend to that of increased peroxidase activity in BER affected whole fruit on the plant as compared to healthy fruit.Regulation of respiration is vital to plant health, with increased respiration indicating an increase in energy consumption and possibly stress. Results presented here indicate symptom development is associated with an increase in respiration rate. Furthermore, the correlation between respiration on day 1 and color measurement on day 2 indicates this increase in respiration rate occurs prior to visual symptoms. Blossom-end disc respiration decreased to near the level of the stem-end discs on day 3, possibly due to cell death or exhaustion of the available chemical energy pool. This also indicates that processes associated with blossom-end rot are likely active prior to visible symptom development.Calcium deficiency in soil and fruit tissue has long been associated with BER incidence. However, abiotic stress can also increase BER incidence, bringing into question the direct relationship between BER occurrence and calcium deficiency. We applied calcium chloride and ascorbic acid to disc tissues to test the direct effect of increased calcium and antioxidant potential onBER development. Both treatments eliminated symptom development as evaluated visually and by colorimeter. Calcium localization within the cell is a key factor in BER incidence. De Freitas et al. directly studied the effect of calcium localization at the cellular level by increasing vacuolar calcium through increased expression of the sCAX1 gene. Increased vacuolar calcium was associated with decreased apoplastic calcium and increased BER incidence. Calcium applied to the pericarp disc systems would likely diffuse into the apoplast, testing the opposite localization state compared to that of De Freitas et al.. As expected, the opposite effect was observed when calcium was applied. Correlation of BER symptom development with increased ROS accumulation and depleted antioxidant activity has been well established in the literature.