Tag: hydroponic

Posted on

Abiotic stress alters the susceptibility of plants to many pathogens

As sessile organisms, plants are presented with numerous biotic challenges such as herbivory and pathogen attack. Plants initiate responses to these challenges by harnessing tightly regulated phytohormone networks. Salicylic acid levels increase in plants following pathogen infection and SA is critical for the development of systemic acquired resistance . There are two enzymatic pathways for the generation of SA: one via phenylalanine ammonia lyase and the other via isochorismate synthase . In tomato , Arabidopsis and Nicotiana benthamiana, most pathogen-induced SA appears to be synthesized via the ICS pathway . Plants with compromised SA synthesis or signaling have greatly diminished defenses against pathogens, as is the case with SA-deficient transgenic plants expressing a bacterial salicylate hydroxylase or ICS mutants like sid2 , and mutants in downstream targets of SA such as npr1 . SAR induction by biotic agents coincides with increases in SA levels and a systemic transcriptional reprograming that primes the plant to respond rapidly to minimize the spread or severity of further infections . This transcriptional reprograming includes the expression of pathogenesis-related genes and deployment of peroxidases and other defense factors. In addition to induction by biotic agents, SAR responses are induced by exogenous application of SA to the foliage or roots . Plant activators are chemicals that have no direct antimicrobial activity but induce disease resistance . A number of synthetic compounds have been developed that induce SAR by increasing SA accumulation and/or by acting on downstream targets of SA . For example, the plant activator, probenazole, effective against bacterial, fungal, and oomycete diseases, stimulates SAR by increasing SA levels . 1,2,3-Benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester , sold under the trade name, Actigard,grow bucket stimulates SAR in many plant species without inducing SA accumulation . Tiadinil [TDL; N–4-methyl-1,2,3-thiadiazole-5-carboxamide] is a plant activator that was registered in Japan in 2003 under the trade name, V-GET. TDL was developed for disease management in rice where it is applied to nursery-grown seedlings for transplanting to production fields . TDL is very effective for control of rice blast disease caused by Magnaporthe oryzae and appears to induce resistance in a manner similar to BTH by acting on downstream targets of SA .

The TDL metabolite,4-methyl-1,2,3-thiadiazole-5-carboxylic acid, is responsible for the SAR activation .The effect of brief episodes of root stress such as salinity and water deficit at levels that commonly occur in agriculture is well documented in plant–oomycete interactions, wherein stress events predispose plants to levels of inoculum they would normally resist . The phytohormone abscisic acid accumulates rapidly in roots and shoots as an adaptive response to these abiotic stresses, but also contributes to the increased disease proneness of the plants . Antagonism between SA and ABA is well documented in relation to plant defense responses to pathogens . Previously, ABA was found to have an antagonistic effect on SAR which was induced by 1,2-benzisothiazol-3-one1,1-dioxide and BTH in Arabidopsis and tobacco . However, it is not known if plant activators that target SA signaling impact the ABA-mediated susceptibility to root pathogens that occurs following predisposing root stress in tomato. Because of the potential for unwanted trade offs and signaling conflicts in plants exposed to different stresses, as can occur in the field, we investigated how predisposing root stress impacts chemically induced resistance in tomato. The objective of this study was to determine the effect of pretreatment of tomato seedlings with TDL and BTH on salt-induced predisposition to the foliar bacterial pathogen Pseudomonas syringae pv. tomato and to the soil borne oomycete pathogen Phytophthora capsici. TDL is of particular interest in the context of soil borne pathogens such as Phytophthora capsici because it is often applied to plants as a root dip. We also determined the impact of SA, TDL and BTH on ABA accumulation during a predisposing episode of salt stress. The results show that TDL applied to roots strongly protects the leaves from disease caused by Pst in both non-stressed and salt-stressed plants. In contrast, neither TDL nor BTH protects roots from Phytophthora capsici.

The protection induced by plant activators against Pst does not result from reduced ABA accumulation and, although overall disease is less in both non-stressed and salt-stressed plants by chemically induced SAR, plant activators do not reverse the salt-induced increment in disease severity.To determine the effect of SA on ABA accumulation during salt stress, ABA levels were measured in WT plants pre-treated with SA, TDL, or BTH. Following salt stress treatment for 18 h, roots and shoots were collected and immediately frozen in liquid N2.The tissues were lyophilized and placed at −20◦C until extraction. The lyophilized tissue was ground in liquid N2 to a fine powder with a mortar and pestle, 50–100 mg samples were collected, and each sample transferred to a micro-fuge tube. Cold 80% methanol containing butylated hydroxytoluene at 10 μg ml−1 was added to each tube, which was then vortexed. The extracts were placed on ice and agitated occasionally for 30 min. The tubes were centrifuged for 5 min at 10,000 × g, and the supernatants collected. The pellet was extracted with 0.5 ml of 80% methanol and centrifuged to collect the supernatant. This step was repeated, all three supernatants were combined, and the methanol concentration of the extract adjusted to 70%. The extracts were applied to pre-wetted Sep-pak C18 columns and eluted with 5 ml of 70% methanol. The eluate containing ABA was concentrated to near dryness at 37◦C under vacuum and the volume adjusted to 300 μl with deionized water. The samples were analyzed by competitive immuno assay with an ABA immuno assay kit according to the manufacturer’s directions. Results are expressed as nanomoles of -ABA per gram dry weight of tissue. To determine the effect of the nahG transgene on ABA levels, roots and shoots from WT and NahG plants were processed using the same procedure as above.To determine if plant activators induce resistance to Pst under different stress regimes in our experimental format, roots of hydroponically grown seedlings of cv. “New Yorker” were treated with TDL and then either not salt-stressed or exposed to 0.2 M NaCl for 18 h prior to inoculation.

In preliminary experiments, several concentrations of TDL were evaluated for phytotoxicity and for efficacy against bacterial speck disease with 10 ppm TDL selected as this concentration provided an optimal response. Concentrations higher than 10 ppm of TDL caused a slight bronzing of the roots and depressed growth of the seedlings, suggesting a mild phytotoxicity of the chemical in our experimental format at these higher levels. Inoculated salt-stressed seedlings had more severe disease symptoms and a significantly higher titer of pathogen than non-stressed, inoculated plants. Pretreatment with TDL at 10 ppm significantly reduced Pst colonization and symptom severity in “New Yorker” plants in both non-stressed and salt-treated seedlings . However, TDL did not prevent the proportional increase in Pst colonization observed in salt-stressed plants relative to the non-stressed controls.Since TDL harnesses SA-mediated defenses, we treated SA deficient NahG plants to see if TDL induces resistance under the different stress regimes in this highly susceptible background. As expected, NahG plants were more susceptible to Pst and accumulated significantly less SA following Pst infection than the WT background “New Yorker.” However, TDL provided strong protection in the NahG plants and mitigated the predisposing effect of salt-stress on bacterial speck disease.In a previous study we showed that ABA-deficient tomato mutants displayed a much reduced predisposition phenotype to salt stress . To determine if the protective effect of TDL is altered within an ABA-deficient tomato mutant,dutch bucket for tomatoes seedlings of WT and an ABA-deficient mutant within this background, sitiens, were treated in the same format and stress regimes as above. TDL significantly reduced Pst symptoms and colonization in both non-stressed and salt-treated plants of “Rheinlands Ruhm.” However, 3.6- and 5.4-fold increases in pathogen titer as a result of salt-stress were observed in both the control and TDL-treated plants, respectively, indicating that TDL did not prevent the proportional increase in Pst colonization in salt-stressed plants, similar to the results with “New Yorker” and NahG plants. In contrast, the sitiens mutant was not predisposed to Pst by salt stress and had significantly reduced symptoms and colonization by the pathogen than the background “Rheinlands Ruhm” . Nonetheless, TDL pretreatment of sitiens provided further protection against Pst .To determine if plant activators protect tomato roots and crowns against the oomycete pathogen, Phytophthora capsici, and predisposing root stress, tomato seedlings were treated with TDL or BTH , not stressed or salt-stressed as above, and then inoculated. There was no protection provided by the plant activators against disease caused by Phytophthora capsici in either the control or salt-treated plants, as reflected in symptom severity and pathogen colonization .Because elevated levels of ABA in tomato can enhance susceptibility to Pst and Phytophthora capsici, the effect of SA, TDL, and BTH on ABA levels was determined in roots and shoots. ABA concentrations in either shoots or roots at the time selected for inoculation in our treatment sequence were not altered by SA . However, a trend of increasing ABA accumulation was observed in TDL- and BTH treated “New Yorker” plants relative to the corresponding control plants . Although the increase in ABA accumulation in the plants treated with these plant activators is not statistically significant at P ≤ 0.05, it can be said that SA, TDL, and BTH do not reduce ABA content relative to untreated plants . In addition, salt stress did not further increase the levels of ABA in plants that had been pretreated with TDL or BTH, which were similar to the salt stressed controls.In a previous study, we demonstrated the predisposing effect of salt stress and a role for ABA as a determinative factor in predisposition in the tomato–Phytophthora capsici interaction .

The present study is the first report of salt-induced predisposition to the bacterial speck pathogen, Pst, in tomato. Furthermore, the results with the ABA-deficient sitiens mutant are consistent with the salt-induced susceptibility to Pst being mediated by ABA . These results conform to studies in Arabidopsis where ABA has been reported to promote susceptibility to Pst .Because SA has been shown to protect tomato against salt stress, possibly by an ABA-dependent mechanism , plant activators that operate via the SA pathway were evaluated for effect on salt-induced predisposition. Protection of tomato against bacterial speck disease by BTH is well documented , and TDL has previously been shown to reduce the severity of bacterial and fungal infections without inducing SA accumulation . Here, TDL was shown to protect against Pst in both non-stressed and salt-stressed tomato plants. TDL pretreatment strongly reduced disease and colonization by Pst in both “New Yorker” and SA-deficient NahG plants. TDL, or more likely its biologically active metabolite, SV-03, presumably allows the NahG plants to mount an SAR response to Pst infection in the absence of SA accumulation . TDL provided protection in both non-stressed and salt-stressed plants, but did not reverse the predisposing effect of salt stress. An increase in Pst colonization was observed in the salt-stressed, TDL-pretreated plants of both genotypes, with comparable percentage increases relative to the corresponding non-stressed controls in “New Yorker” and NahG plants. This indicates that TDL does not reverse the salt-stress effect on disease, per se, and likely targets stress network signaling independently of an ABA-mediated process that conditions the salt-induced susceptibility observed in this system . “Rheinlands Ruhm” also displayed salt-induced predisposition to Pst. Pretreatment with TDL significantly reduced Pst colonization in both “Rheinlands Ruhm” and sitiens . Similarly, TDL provided protection in both non-stressed and saltstressed plants, but did not reverse the predisposing effect of salt stress in “Rheinlands Ruhm” plants. The salt-induced increment in colonization by the pathogen was comparable in both the untreated and TDL-treated plants . The ABA-deficient mutant, sitiens, is considerably less susceptible to Pst than its background “Rheinlands Ruhm,” and does not exhibit salt-induced predisposition .Protection by plant activators against foliar pathogens is well established . However, relatively few studies have examined these compounds against soilborne pathogens and so TDL and BTH were evaluated for protection against root infection by Phytophthora capsici. Neither TDL nor BTH induced resistance or impacted salt-induced predisposition to Phytophthora capsici . Phytophthora capsici is an aggressive root and crown pathogen with a hemibiotrophic parasitic habit that triggers both SA- and jasmonic acid-mediated responses during infection of tomato .

Posted on

Soil water extracts were prepared according to a published method with some modifications

The whole-plant N2 fixation potential was calculated by multiplying the total dry nodule biomass of each plant and the N2 fixation potential, which had been normalized to dry nodule biomass. To understand how plant effects were related to CNM concentration-dependent agglomeration in moist soils, the short- and long-term stabilities of CNMs were studied in soil water extracts. Briefly, control soil was weighed into separate 50 mL centrifuge tubes with 1:5 w/v Nanopure water . The centrifuge tubes were sealed securely and shaken horizontally on a shaker for 3 h . The extract was centrifuged to separate large solids, and the supernatant was decanted. The supernatant was vacuum filtered through a 0.22 μm membrane filter , and the filtrate was collected as the final soil extract and stored prior to use. A CNM stock solution was prepared by weighing dry CNM powder into the filtered soil extract, then mixing by brief sonication using a Branson 1510 bath sonicator . Aliquots of the dispersed CNM stock solution were further diluted by the filtered soil extract to yield a final lower concentration of 10 mg L−1. These two CNM concentrations were chosen for comparing the effect of lower versus higher CNM concentrations on CNM agglomeration in moist soil; both concentrations are relevant to the CNM doses used in the plant exposure experiment . The CNM suspensions were bathsonicated immediately before use in static agglomeration and sedimentation studies, hydroponic nft channel which were performed over a long time period . The changes of CNM hydrodynamic diameter and derived count rate with time were measured using dynamic light scattering in a Zetasizer NanoZS90 . DLS measurements were made every 15 s for the first 12 h, then daily from 1 to 7 d, and finally weekly until 56 d.

Meanwhile, dynamic CNM sedimentation in the soil extract was monitored by measuring the suspension absorbance at 600 nm using a UV-1800 spectrophotometer . Sedimentation patterns were inferred from the time course of normalized suspension absorbance at 600 nm . The UV-1800 spectrophotometer was zeroed using Nanopure water. The absorbance of the filtered soil extract alone was monitored over time as well, to confirm there was no interfering absorbance from the soil extract in the CNM suspensions. The zeta potential and electrophoretic mobility of the filtered soil extract and of 10 mg L−1 CNMs were also obtained using the Zetasizer NanoZS90. For either DLS, absorbance, ζ potential, or EPM, at least three replicate measurements were performed. Environmental scanning electron microscopy was performed to visualize the agglomerate morphologies of 10 and 300 mg L−1 CNMs in the soil extract, against a clean quartz sand substrate. Specimens were prepared by dispensing approximately 100 μL of the CNM suspensions onto clean quartz sand overlaying a 10 mm stainless steel conical-well Peltier stub. Imaging was by an FEI Co. XL30 field emission gun microscope , operated at 15 kV accelerating voltage, in a 3.5-torr chamber pressure with a gaseous secondary electron detector in environmental mode. Data are shown as the mean ± SE . For each CNM type, one-way analysis of variance with Tukey’s or Games-Howell post hoc multiple comparisons was used to determine significant differences between treatments . Homogeneity of variance was tested with Levene’s test. To explore dose–response relationships, correlations were performed between plant growth and end point metrics with soil CNM concentrations, using both two-tailed linear and power regression models. Correlation analyses were conducted both with and without the control data. Statistical analyses were performed using Microsoft Excel 2013, IBM SPSS Statistics 23, and SigmaPlot 12.3.P. vulgaris is characterized by a particular evolutionary history.

Recent analyses based on sequence data presented clear evidence of the Mesoamerican origin of common bean, which was most likely located in México . The expansion of this species to South America resulted in the development of two ecogeographic distinct genetic pools with partial reproductive isolation . After the formation of these genetic pools -between 500,000 and 100,000 years ago – domestication took place, independently in the Mesoamerican and the southern Andean regions of the American continent . Genome analysis of BAT93 and G19833 , P. vulgaris sequenced model genotypes, has initially revealed interesting differences, for example between their genome size and number of annotated genes . The common bean is the most important legume for human consumption. In less favored countries from Latin America and Africa, common bean are staple crops serving as the primary source of protein in the diet. Soil acidity in these tropical regions is a major constraint for crop productivity, usually resulting in a combination of nutrient deficiency and metal toxicity . In acidic soils, aluminum toxicity is the primary factor of growth restriction, resulting in the inhibition of root growth and function, as well as in the increased risk of plants to perish of drought and mineral deficiencies, thus decreasing crop production . High Al levels mainly affect roots causing an arrest of the growth of the principal and lateral roots . In Arabidopsis, the regulation of root growth is modulated by an ABC transporter‐like protein, annotated as ALUMINUM SENSITIVE PROTEIN 3 , which is localized in the tonoplast, suggesting a role in Al vacuolar sequestration . The LOW PHOSPHATE ROOT 1 ferroxidase, an ALS3– downstream protein of the phosphate-deficiency signaling pathway, is involved in root growth inhibition, by modulating iron homeostasis and ROS accumulation in root apical meristem and elongation zone . In root cells, AlT can affect multiple areas, as the plasma membrane, the cell wall and symplastic components .

Common bean is known to be highly sensitive to AlT but this sensitivity is genotype-dependent . In 2010, the evaluation of the root morphological traits related to AlT of 36 P. vulgaris genotypes revealed that Andean genotypes were more resistant to Al than Mesoamerican ones . Mendoza-Soto et al. reported that Mesoamerican common-bean plants subjected to high Al levels for short periods showed decreased root length as well as characteristic symptoms of AlT, such as ROS accumulation, callose deposition, lipoperoxidation and cell death in roots. Along other regulators, plant response to metal toxicity involves also microRNAs as part of the regulatory mechanisms. These molecules are a class of non-coding small RNAs of about 21 nucleotides in length, regulating gene expression at post-transcriptional level, guided by sequence complementarity, inducing cleavage or translational inhibition of the corresponding target transcript . The relevance of miRNA regulation in heavy metal tolerance is well documented; it has been demonstrated that heavy metal-responsive miRNAs show differential expression according to the toxicity level. Target genes of these miRNAs generally encode transcription factors that transcriptionally regulate networks relevant for the response to heavy metals. Additionally these encode transcripts for proteins that participate in metal absorption and transport, protein folding, antioxidant system, phytohormone signaling, or miRNA biogenesis and feedback regulation . High-throughput small RNA sequencing analyses have identified miRNAs that respond to AlT in roots of different plants species, however their function in response to AlT is largely unknown. Some of the target genes cleaved by AlT-responsive miRNAs encode disease resistance proteins, transcription factors or auxin signaling proteins . Our previous research indicated that P. vulgaris is no exception to this phenomenon. We identified common-bean miRNAs that respond to Al, these include conserved miRNAs that are Al-responsive in other plant species -i.e. miR319, miR390, miR393- and also miR1511 . miRNAs from the miR1511 family have been identified in non-legume plants like strawberry and poplar tree ,nft growing system although in the latter its nature as a miRNA has been discussed as it has been considered as part of a retrotransposon . Regarding legumes, miR1511 has been identified in Medicago truncatula and soybean . Also, miR1511 was identified in Mesoamerican common-bean cultivars, being more abundant in flowers and roots . However, this miRNA was not identified when analyzing the Andean G19833 reference genome . Genetic variation in MIR1511 has been reported in a comparative genotyping analysis of different Asian accession of domesticated soybean as well as its wild type progenitor Glycine soja. While sequences of mature miR1511 and miR1511* were found in G. max accessions, the sequences of annual wild G. soja showed insertion/deletion in the stem-loop region of MIR1511 that included complete or partial deletions of mature miR1511 sequence . Updated research indicates that the miR1511 target gene is not conserved in the different plants where it has been identified. In strawberry, the miR1511 targets an LTR retrotransposon gene .

Inconsistencies about the nature of miR1511 target gene also hold for legume species. For instance, different targets have been proposed for soybean ranging from genes coding for proteins involved in the regulation of nitrogen metabolism to proteins relevant in plant cell development . While in other species such as M. truncatula target genes have been searched but have not been identified. The SP1L1 transcript has been proposed as the common-bean miR1511 target , however despite several efforts from our and other groups this prediction could not be experimentally validated. These results suggested a species-specific selection of the corresponding target thus it was essential to experimentally validate the nature and possible function of the miR1511 target gene in common bean. Recent analyses led us to predict an ABC-2-type transporter-related gene, annotated as Aluminum Sensitive Protein 3 , as the target for miR1511. In this work we present its experimental validation. In addition, we genotyped MIR1511 in ecogeographically different common-bean cultivars and investigated the role of miR1511 and its corresponding target in the regulation of plant response to AlT. The comparison of MIR1511 sequence from BAT93 vs. G19833 P. vulgaris reference sequences showed a 58-bp deletion in the G19833 genotype. Such deletion comprised around 57% of pre-miR1511 sequence and included 7-bp and 10-bp of mature and star miR1511, respectively . To explore this phenomenon at a larger scale within the Phaseolus genus, we analyzed Genotyping-By-Sequencing data from 87 genotypes originated from a single genetic population , called non-admixed genotypes. These included genotypes from three Phaseolus species and different populations of wild P. vulgaris: three populations from the Mesoamerican , one from the Andean , and one from the Northern Peru–Ecuador gene pools . All the genotypes belonging to the Andean gene pool and part of the Mesoamerican genotypes displayed a truncated MIR1511, in contrast to the Northern Peru– Ecuador genotypes and the other Phaseolus species that presented a complete version of the MIR1511 in their genome. A population clustering of P. vulgaris genotypes confirmed these results and showed that in the three Mesoamerican populations only a part of the MW1 cluster presented the MIR1511 deletion . Predicted target genes for P. vulgaris miR1511 include SP1L1-like  and isopentyl-diphosphate delta-isomerase , previously reported , and a protein with unknown function and the Aluminum Sensitive Protein 3 , from our recent bio-informatic analysis. From these predicted targets, ALS3 is the only one possibly related to AlT, as reported for Arabidopsis , and showing an adequate binding-site penalty score , thus the 5’RLM-RACE assay was used to experimentally validate the ALS3 mRNA cleavage site. As shown in Figure 3a, a significant number independently cloned transcripts mapped to the predicted site of cleavage, between the nucleotides at positions 457 and 458 of the transcript, which corresponds to position 9 and 10 of the predicted miR1511 binding site, thus confirming a miR1511-induced degradation. The other two degradation events mapped to 7 nucleotides upstream and 17 nucleotides downstream of the miRNA-associated degradation site, suggesting random degradation. An additional action of miR1511 to induce translation inhibition of ALS3 mRNA in common bean, cannot be excluded. miR1511 target genes differ among plant species . In order to evaluate the specificity of the miR1511/ALS3 regulatory node in common bean, we analyzed the miR1511/ALS3 binding site sequence alignment from eight model plant species, including five legumes, which contain a precursor gene of miR1511 in their genome . Because of the deletion in MIR1511 from the G19833 genotype, we used the mature miR1511 and the corresponding ALS3 binding site sequences from the BAT93 Mesoamerican genotype, as representative of P. vulgaris. Among plant species analyzed, P. vulgaris was the only one that showed a binding-site penalty score lower than 5, corresponding to a score recommended to consider a small RNA-target binding as probably functional. For other species, the high penalty scores, ranging from 7.5 to 9, indicate a very low probability for the existence of a functional miR1511/ALS3 regulatory node .

Posted on

Hydroponic Agriculture: Cultivating the Future of Sustainable Farming

Interestingly, suppression of endodermal ABA signalling seems to contribute to the inactivation of aquaporin-mediated Lpr in a wild-type Scheduling low but frequent NO3 − applications, at-tuned to crop demand, allows the crop to take up most of the NO3 − before it passes through the low-salinity zone into the saline fringes. Figure 7 simulates continuous NO3 − application and a scenario which applies NO3 − only every 10 d, while the total amount of NO3 − applied is the same for both simulations. High-frequency applications of NO3 − using drip irrigation in-creased N uptake efficiency in some cases .Both Casparian strips and suberin lamellae, two extracellular hydrophobic barriers located in the wall of endodermal cells of the root, are thought to play important roles in restricting the free diffusion of solutes and water . Casparian strips act as apoplastic barriers not only to block solutes moving into the xylem through the free space between cells, but also to prevent their backfow from the stele to the apoplast of the cor-tex. Suberin lamellae, due to their deposition between the endodermal plasma membrane and secondary cell wall, do not block aploplastic transport but rather limit transcellular transport of nutrients and possibly water at the endodermis. Cross talk between the Casparian strip and suberin lamellae exists, with suberin being deposited in response to disruption of Casparian strips . Tese extracellular barriers are therefore at a cross-road between control of mineral nutrient and water uptake. However, the mechanisms that allow plants to integrate both these barrier functions to enable the simultaneous uptake of sufcient water and mineral nutrients remain under explored. Te dirigent-like protein Enhanced Suberin1 functions in the correct formation of Casparian strips by allowing the lignin, deposited at the Casparian Strip Domain through the action of Peroxidase64 and the Respiratory Burst Oxidase Homolog F ,blueberry packaging to form into a continuous ring. In the absence of this dirigent-like protein defective Casparian strips are formed along with enhanced and early deposition of suberin in the endodermis.

A similar pattern of Casparian strip disruption and response is also observed when the Casparian Strip Domain is disrupted through the loss of Casparian Strip Domain Proteins. Tese changes lead to systematic alterations in the profile of mineral nutrients and trace elements accumulating in leaves, and this phenotype provided the first tool for identification of genes involved in Casparian strip development. Detection of the diffusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 through interaction with the SCHENGEN3 receptor-like kinase is what drives this endodermal response to loss of Casparian strip integrity. Here, we report that detection of a loss of Casparian strip integrity at the root endodermis by the CIFs/SGN3 pathway leads to an integrated local and long-distance response. This response rebalances water and mineral nutrient uptake, compensating for breakage of the Casparian strip apoplastic seal between the stele and the cor-tex. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquapor-ins, and limitation of ion leakage through deposition of suberin in endodermal cell walls. This local root-based response is also coupled to a reduction in water demand in the shoot driven by ABA-mediated stomatal closure.Te dirigent-like protein Enhanced Suberin1 functions in the formation of Casparian strips by allowing the correct deposition of lignin at the Casparian strip domain. Te enhanced deposition of suberin in the esb1-1 mutant with disrupted Casparian strips can clearly be observed using the lipophilic stain Fluorol Yellow 088 close to the root tip , and this can be quantified by counting the number of endodermal cells afer the onset of cell expansion to the first appearance of yellow fuorescence . This early deposition of suberin is also verifed by the clear correspondence of FY 088 staining with enhanced promoter activity of known suberin biosynthetic genes, including GPAT5 monitored through both GUS staining and GFP fuorescence , and also others through GUS staining . This is further reinforced by the enhanced expression of known suberin biosynthetic genes in esb1-1 relative to wild-type . To better understand the causal link between Casparian strip integrity and enhanced deposition of suberin, we performed a reciprocal grafing experiment that revealed that the esb1-1 mutation is only required in the root to drive enhanced deposition of suberin at the endodermis, placing the function of ESB1 and the driver for increased suberin in the same tissue .

To determine the cause and effect rela-tionship between damaged Casparian strips and enhanced suberin we carefully monitored the first appearance of both Casparian strips and enhanced suberin in esb1-1. Using lignin staining in the Casparian strip marker line pCASP1::CASP1::GFP, we are able to observe that damaged Casparian strips are visible 2.5 days afer sowing . This is at least 12hr before the first indication of enhanced suberin biosynthesis, which we monitor using promoter activity of suberin biosynthetic genes GPAT5, FAR4, FAR1 and FAR5 . This was also verified by the direct observation of suberin deposition with FY 088 . Te observation that treatment with the CIF2 peptide, normally leaked from the stele through loss of Casparian strip integrity, can enhance suberin deposition in wild-type plants supports our interpretation that enhanced suberin deposition is a response to loss of integrity of the Casparian strip-based apoplastic diffusion barrier. Furthermore, loss-of-function of the receptor-like kinase SGN3, required for sensing of CIFs, blocks the enhanced deposition of suberin in esb1-1 and casp1-1casp3-1 based on a chemical analysis of suberin in esb1-1 , and also on FY 088 staining. We conclude that Casparian strip defects sensed by the CIFs/SGN3 surveillance system lead to enhanced deposition of suberin in the endodermis.Te observation that enhanced suberin is deposited as a response to loss of integrity of the endodermal-based diffusion barrier between stele and cortex, raises the question, what is the function of this increased suberin deposition? Previously, the extent of endodermal suberin has been shown to be part of the response to nutrient status . We therefore tested the selectivity to solutes σNaCl, in roots varying in the extent of suberin deposition and the functionality of Casparian strips. For this, we measured solute leakage into xylem sap of pressurized roots at increasing sodium chloride concentrations in the solution bathing the roots. Taken individually, σNaCl of roots of esb1-1, sgn3-3 and wild-type were not significantly different from one another , which is surprising given the disruption of the Casparian strip-based apoplastic diffusion barrier in both mutants.

However, removal of suberin in esb1-1, by endodermal-specific ectopic expression of a cutinase ,blueberry packaging box caused a significant decrease in σNaCl compared to wild-type plants , and a similar tendency when compared to esb1-1 . This supports the notion that enhanced suberin deposition at the endodermis helps prevent passive solute leakage caused by defects in the Casparian strips of the esb1-1 mutant. We also observed a significant decrease in σNaCl in the double mutant esb1-1sgn3-3 compared to both wild-type and sgn3-3 . It is known that SGN3 is required for the enhanced deposition of suberin that occurs at the endodermis in esb1-1 . Our observation that removal of this enhanced suberin in esb1-1sgn3-3 decreases σNaCl further supports our conclusion that the role of this increased suberin deposition is to limit solute leakage where Casparian strip barriers are disrupted.It has also been suggested that endodermal suberin may impact water permeability, though how is still unclear. To further address the role of enhanced endodermal suberin, we investigated root hydraulic conductivity of esb1-1 and observed a significant reduction by 62% with respect to wild-type . Importantly, this difference in esb1-1 Lpr originates mainly from a reduction in an aquaporin-mediated water transport pathway . We also observed that the azide-resistant water transport pathway was lower in esb1-1 than in wild-type , yet to a lesser extent than the aquaporin mediated pathway. Te dra-matic reduction in aquaporin-mediated Lpr in esb1-1 we observe is an intriguing fnding, which led us to consider if this lack of aquaporin activity in esb1-1 roots is due to a direct output from the CIFs/SGN3 signalling pathway, or if it represents an efect downstream of enhanced suberin deposition. We found that removal of endodermal suberin in esb1-1 through expression of CDEF1 in the endodermis had no further efect on Lpr . This rules out a role for suberin in the reduced aquaporin-mediated Lpr of esb1-1. However, in the esb1-1sgn3-3 dou-ble mutant, as compared to esb1-1, we observed a full recovery of Lpr back to wild-type levels . Loss of Casparian strip integrity in esb1-1 therefore appears to be sensed by the CIFs/SGN3 signalling pathway, which leads to the inactivation of aquaporins, thereby reducing Lpr . To support this conclusion, we show that exogenous application of CIF2 to wild-type plants for 3h induces a reduction in Lpr, and only in the presence of a functional SGN3 . We have established the existence of two critical outputs of the CIFs/SGN3 diffusion-barrier surveillance system. Tese are enhanced deposition of endodermal suberin limiting solute leakage, and the inactivation of root aquaporin activity reducing Lpr. Do these two independent outputs of the CIFs/SGN3 diffusion barrier surveillance system work in parallel, or in series with one response leading to the other? Te fact that removal of endodermal suberin in esb1-1 does not compensate for its reduced Lpr suggests that enhanced suberin deposition is not the cause of the reduced aquaporin-mediated Lpr. However, reduced activity of aquaporins through loss-of-function of the two major aquaporins PIP2;1 and PIP2;2 in the pip2;1pip2;2 double mutant, does cause significant increases in endodermal suberin deposition . A similar increase in suberin is also observed afer treatment with the aquaporin inhibitor sodium azide through observation of the activity of the transcriptional reporter pGPAT5::mCITRINE-SYP122 for suberin biosynthesis. GPAT5 expression is observed to expand toward the root tip after 6 hours only of sodium azide treatment .

Based on this evidence, we propose the following sequence of events. Casparian strip defects are detected by the apoplastic leakage of CIFs from the stele, being sensed by SGN3. Once activated, SGN3 signals the inactivation of aqua-porins thereby reducing Lpr which in turn leads to the early and enhanced deposition of endodermal suberin. Insuch a model, SGN3 would inhibit aquaporin function, which may appear at variance with the usual activation of aquaporins through phosphorylation. Yet, such an inhibition was recently described in the case of FERONIA, a protein kinase inactivating PIP2; 1 function through an as yet unknown mechanism.Abscisic acid has been shown to be involved in regulating both aquaporin activity reviewed in and suberin deposition, making ABA an interesting can-didate worth exploring for a role in downstream CIFs/SGN3 signalling. To probe this possibility we expressed the dominant negative allele of the regulator of ABA signalling ABA-INSENSITIVE 1 in the endoder-mis of esb1-1 using pELTP::abi1. This abi1 construct specifically blocks ABA signalling at the endodermis and delays suberisation in a wild-type background as previously shown in . In esb1-1, we observed abi1 to have no effect on either the inactivation of aquaporins or the enhanced deposition suberin . We also observe that aquaporin inhibition with sodium azide in the pELTP::abi1-1 line still induces expression of the suberin biosynthesis gene GPAT5 toward the root tip in the pGPAT::mCITRINE-SYP122 line, as observed in wild-type . Based on this, activation of ABA signalling in the endodermis does not link perception of Casparian strip defects with the downstream responses of reduced aquaporin-mediated Lpr or suberin deposition. Suppbackground .The esb1-1 mutant is known to have reduced stomatal apertures and enhanced wilting resistance. This suggests that the CIFs/SGN3 sensing system not only initiates a local root response to Casparian strip integrity but is also involved in initiating long-distance responses in the shoot. We observe reduced stomatal apertures in esb1-1 , and an analysis of the expression of a set of known ABA signalling and response genes in leaves suggest that this stomatal closure is part of an ABA driven response. The aba1 mutation confers a strong ABA deficiency.By generating an esb1-1aba1 double mutant, we investigated the ABA-dependent component in the leaf response we observe in esb1-1. ABA-defciency in esb1-1aba1 suppressed both the reduced stomatal aperture and the activation of expression of ABA signalling and response genes that we observe in esb1-1 .

Posted on

TGB3 also appeared to function in redistribution of membrane vesicles throughout the cytosol

TGB1 and TGB2 also localized with membrane vesicles even though extensive membrane proliferation was not observed. However, ectopically expressed TGB3 elicited formation of a complex and well-defined ER network that is closely associated with, or houses, thick actin cables and TGB proteins.Virus movement and membrane reorganization, which is especially obvious in the perinuclear region, was disrupted by mutations in the central membranespanning domain of TGB3. Our findings thus suggest that host membrane associations are involved in several aspects of BSMV movement that merit future study. In addition to differences in requirements of the coat protein for cell-to-cell movement of hordeiviruses and potexviruses, a number of variations are evident in TGB1 protein structure, biochemical activities, interference with host gene silencing, and movement functions of the virgaviruses and the TGB-containing flexiviruses . A recent paper illuminating a TGB1 requirement for formation of X bodies associated with PVX highlights another major difference in functions of the TGB1 proteins of the hordeiviruses and the potexviruses that relates to BSMV actin remodeling. In contrast to BSMV, in which TGB3 expression has a major effect on actin architecture, PVX TGB1 is essential for X-body formation and functions in extensive actin and membrane remodeling . The multilayered membranous X-body is an important organelle that is required for normal levels of viral RNA replication and virion accumulation. Nevertheless, in plants and protoplasts infected with PVX mutants unable to express TGB1, morphogenesis of X bodies fails to occur, yet low levels of PVX replication can be detected and small amounts of virus particles accumulate . However, ectopic expression of PVX TGB1 results in massive remodeling of host actin and endomembranes, greenhouse ABS snap clamp and recruitment of these structures, as well as TGB2 and TGB3, to sites near the nucleus. Subsequently, X-bodies develop into complex multilayered membrane organelles adjacent to the nucleus, that selectively incorporate TGB proteins, ribosomes, viral RNA and virions to specific sites within the granular vesicular bodies .

These differences between the two viruses are further illustrated by conventional electron microscopic observations showing that structures corresponding to PVX X-bodies are not present in BSMV-infected cells, and that BSMV replicates in membrane vesicles formed from the chloroplast outer membrane . Our current results add to a growing list of major differences in the movement processes of TGB-encoding viruses . We previously reported that cytochalasin D treatment failed to affect TGB1 localization in BSMV infected protoplasts and as a result, postulated that cytoskeletal interactions of the protein were relatively minor. However, the experiments presented here reveal both actin remodeling and changes to ER structure as a consequence of BSMV infection and transient expression of TGB3 and TGB2/3. Our observations also provide evidence that the subcellular localization of the TGB proteins depends on actin cytoskeleton interactions. To investigate these interactions in more detail, we used LatB to inhibit actin polymerization in cells infiltrated with TGBreporter proteins. In contrast to cytochalasin D used in our earlier experiments , LatB can be up to 100-fold more potent than cytochalasins, and functions by shortening and thickening of actin filaments. After LatB treatment, the DsRed:Talin patterns in N. benthamiana infiltrated epidermal leaf cells exhibited a major shift from a filamentous actin network to thick cablelike structures.From these experiments, we conclude that actin cytoskeleton modifications are required for BSMV movement and that TGB3 has a critical role in cytoskeleton remodeling during movement. In contrast to the BSMV LatB experiments described above, experiments with the closely related PSLV TGB3 have resulted in different conclusions about mechanisms functioning in PD targeting . In the case of BSMV, actin cytoskeleton disruption by LatB interfered with CW localization of TGB3, and TGB1 when coexpressed with TGB2/3, whereas PSLV TGB3 CW localization was not dramatically affected by LatB treatment . These disparate results highlight fundamental differences in the mechanisms of subcellular transit of BSMV and PSLV. Such differences between related viruses may occur more often than previously realized, as illustrated by a previous report describing differences in the movement of two tobamoviruses .

In this direct comparison, movement of TMV is strongly inhibited by LatB treatment, whereas movement of the related TVCV is unaffected by LatB treatment. These results argue strongly that more than one mechanism may be operative in some closely related viruses, and our collective results suggest that BSMV and PSLV may fit within this category. Evidence for TGB3 associations with the Golgi membranes during coexpression of DsRed:TGB3 and the STGFP Golgi marker indicates that Golgi derived vesicles and DsRed:TGB3 co-localize with the CW after plasmolysis. BFA interference with Golgi stack integrity resulted in a major collapse of vesicles localized in close proximity to the CW, but BFA appears to have only limited effects on BSMV localization or PSLV TGB3 associations with “peripheral bodies” . Nevertheless, differences in the BSMV and PSLV LatB cytoskeleton disruption experiments suggest that different mechanisms may function in some TGB3 interactions culminating in PD targeting. Other than the preliminary experiments shown above, which suggest that BSMV infection does not result in obvious changes to microtubules, we have not extensively investigated possible direct interactions of BSMV TGB proteins with microtubules. However, in other experiments with the related Potato mop-top virus , colchicine treatments were used to disrupt tubulin polymerization and microtubule integrity . Colchicine can affect multiple metabolic and regulatory processes affecting a large number of functions that might interfere with TGB1 localization to the CW. However, the PD associations of PMTV mutants provided evidence for an association between microtubules and PMTV TGB1. Of particular interest, cells were observed for several days after transient expression of the three TGB proteins in ratios corresponding to those occurring during virus infection. During this period, a defined series of kinetic events were noted, beginning with PMTV TGB1 nucleolar interactions and proceeding through cytoplasmic granules to the CW. Thus, the effects of BSMV and PMTV on microtubule remodeling, seem to differ, and these experiments reinforce our suggestion that multiple pathways may operate in CW targeting during TGB1 expression of the virgaviruses. Unfortunately, individual events involved in viral movement from subcellular sites of replication to the PD and adjacent cells are difficult to dissect experimentally, and many of these problems have been discussed previously .

The infection front where important events are coordinated is a moving boundary consisting of a limited number of cells undergoing a series of asynchronous steps, so relatively few studies have probed events at this stage of infection. Variations in delivery protocols also contribute to experimental differences or artifacts that can lead to aberrant subcellular trafficking effects. In this regard,flower pot wholesale examples of the effects of over expression of PSLV TGB3 has been described recently in which anomalous cell death, membrane abnormalities and disrupted Golgi functions occur during transient infection . Third, pharmacological approaches can be quite variable in the hands of different researchers. Finally, a more diverse array of approaches, including infectivity studies applied to different hosts might provide interesting insights into alternative strategies employed by BSMV and other hordeiviruses. Although it would be preferable to investigate movement in the natural BSMV cereal hosts, these plants present technical difficulties that are difficult to circumvent. Fortunately, BSMV, unlike PSLV, is able to infect N. benthamiana, so we have been able to compare cytological and biochemical experiments with infectivity results in this host.Rust fungi are an order of >7000 species of highly specialized plant pathogens with a disproportionately large impact on agriculture, horticulture, forestry, and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. Asian soybean rust caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is a prime example of the damage that can be caused by rust fungi. It is a critical challenge for food security and one of the most damaging plant pathogens of this century. The disease is ubiquitously present in the soybean growing areas of Latin America, where 210 million metric tons of soybean are projected to be produced in 2022/23 , and on average representing a gross production value of U.S. $ 115 billion per season . A low incidence of this devastating disease can already affect yields and, if not managed properly, yield losses are reported of up to 80%. Chemical control in Brazil to manage the disease started in the 2002/03 growing season. In the following season, ~20 million hectares of soybeans were sprayed with fungicides to control this disease. The cost of managing P. pachyrhizi exceeds $2 billion USD per season in Brazil alone. The pathogen is highly adaptive and individually deployed resistance genes have been rapidly overcome when respective cultivars have been released. Similarly, the fungal tolerance to the main classes of site-specific fungicides is increasing, making chemical control less effective. Another remarkable feature for an obligate biotrophic pathogen is its wide host range, encompassing 153 species of legumes within 54 genera to date. Epidemiologically, this is relevant as it allows the pathogen to maintain itself in the absence of soybean on other legume hosts, such as overwintering on the invasive weed Kudzu in the United States. Despite the importance of the pathogen, not much was known about its genetic makeup as the large genome size , coupled to a high repeat content, high levels of heterozygosity and the dikaryotic nature of the infectious urediospores of the fungus have hampered whole genome assembly efforts. In this work, we provide reference quality assemblies and genome annotations of three P. pachyrhizi isolates. We uncover a genome with a total assembly size of up to 1.25 Gb.

Approximately, 93% of the genome consists of TEs, of which two super families make up 80% of the TE content. The three P. pachyrhizi isolates collected from South America represent a single clonal lineage with high levels of heterozygosity. Studying the TEs in detail, we demonstrate that the expansion of TEs within the genome happened over the last 10 My and accelerated over the last 3 My, and did so in several bursts. Although TEs are tightly controlled during sporulation and appressoriaformation, we can see a clear relaxation of repression during the in planta life stages of the pathogen. Due to the nested TEs, it is not possible at present to correlate specific TEs to specific expanded gene families. However, we can see that the P. pachyrhizi genome is expanded in genes related to amino acid metabolism and energy production, which may represent key lifestyle adaptations. Overall, our data unveil that TEs that started their proliferation during the radiation of the Leguminosae play a prominent role in the P. pachyrhizi’s genome and may have a key impact on a variety of processes such as host range adaptation, stress responses and plasticity of the genome. The high-quality genome assembly and transcriptome data presented here are a key resource for the community. It represents a critical step for further in-depth studies of this pathogen to develop new methods of control and to better understand the molecular dialogue between P. pachyrhizi and its agriculturally relevant host, Soybean.The high repeat content and dikaryotic nature of the P. pachryrhizi genome poses challenges to genome assembly methods. Recent improvements in sequencing technology and assembly methods have provided contiguous genome assemblies for several rust fungi. Here, we have expanded the effort and provided reference-levelgenome assemblies of three P. pachyrhizi isolates using long-read sequencing technologies. All three isolates were collected from different regions of South America. We have used PacBio sequencing for the K8108 and MT2006 isolates and Oxford Nanopore for the UFV02 isolate to generate three high-quality genomes . Due to longer read lengths from Oxford nanopore, the UFV02 assembly is more contiguous compared to K8108 and MT2006 and is used as a reference in the current study . The total genome assembly size of up to 1.25 Gb comprising two haplotypes, makes the P. pachyrhizi genome one of the largest fungal genomes sequenced to date . Analysis of the TE content in the P. pachyrhizi genome indicates ~93% of the genome consist of repetitive elements, one of the highest TE contents reported for any organism to date . This high TE content may represent a key strategy to increase genetic variation in P. pachyrhizi. The largest class of TEs are class 1 retrotransposons, that account for 54.0% of the genome.

Posted on

Molecular diffusion was neglected as it was considered negligible relative to dispersion

High frequency irrigation systems involve fastidious planning and complex designs, so that timely and accurate additions of water and fertilizer can result in sustainable irrigation. At the same time these production systems are becoming more intensive, in an effort to optimise the return on expensive and scarce resources such as water and nutrients. Advanced fertigation systems combine drip irrigation and fertilizer application to deliver water and nutrients directly to the roots of crops, with the aim of synchronising the applications with crop demands , and maintaining the desired concentration and distribution of ions and water in the soil . Hence a clear understanding of water dynamics in the soil is important for the design, operation, and management of irrigation and fertigation under drip irrigation . However, there is a need to evaluate the performance of these systems, because considerable localised leaching canoccur near the drip lines, even under deficit irrigation conditions . The loss of nutrients, particularly nitrogen, from irrigation systems can be expensive and pose a serious threat to receiving water bodies . Citrus is one of the important horticultural crops being grown under advanced fertigation systems in Australia. Fertigation delivers nutrients in a soluble form with irrigation water directly into the root-zone, thus providing ideal conditions for rapid uptake of water and nutrients. Scholberg et al. demonstrated that more frequent applications of a dilute N solution to citrus seedlings doubled nitrogen uptake efficiency compared with less frequent applications of a more concentrated nutrient solution. Delivery of N through fertigation reduces N losses in the soil-plant system by ammonia volatilisation and nitrate leaching . However, poor irrigation management, i.e., an application of water in excess of crop requirements,hydroponic nft channel plus the storage capacity of the soil within the rooting depth, can contribute to leaching of water and nutrients below the rootzone.

Therefore, optimal irrigation scheduling is important to maximise the uptake efficiencies of water and nutrients . Most of the citrus production along the Murray River corridor is on sandy soils, which are highly vulnerable to rapid leaching of water and nutrients. Nitrogen is the key limiting nutrient and is therefore a main component of fertigation. An increasing use of nitrogenous fertilizers and their subsequent leaching as nitrate from the root zone of cropping systems is recognised as a potential source of groundwater contamination, because the harvested crop seldom takes up more than 25–70% of the total applied fertilizer . Several researchers have reported substantial leaching of applied N under citrus cultivation in field conditions . Similarly, in lysimeter experiments, Boaretto et al. showed 36% recovery of applied nitrogen by orange trees, while Jiang and Xia reported N leaching of 70% of the initial N value, and found denitrification and leaching to be the main processes for the loss of N. These studies suggest that knowledge of the nitrogen balance in cropping systems is essential for designing and managing drip irrigation systems and achieving high efficiency of N fertilizer use, thereby limiting the export of this nutrient as a pollutant to downstream water systems. Quantifying water and nitrogen losses below the root zone is highly challenging due to uncertainties associated with estimating drainage fluxes and solute concentrations in the leachate, even under well-controlled experimental conditions . Moreover, direct field measurements of simultaneous migration of water and nitrogen under drip irrigation is laborious, time-consuming and expensive . Hence simulation models have become valuable research tools for studying the complex and interactive processes of water and solute transport through the soil profile, as well as the effects of management practices on crop yields and on the environment .

In fact, models have proved to be particularly useful for describing and predicting transport processes, simulating conditions which are economically or technically impossible to carry out in field experiments . Several models have been developed to simulate flow and transport processes, nutrient uptake and biological transformations of nutrients in the soil . HYDRUS 2D/3D has been used extensively for evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequency and quality, timing of nutrient applications on wetting patterns and solute distribution because it has the capability to analyse water flow and nutrient transport in multiple spatial dimensions . In the absence of experimental data we can use multidimensional models solving water flow and nutrient transport equations to evaluate the multi-dimensional aspect of nitrate movement under fertigation . However, earlier simulation studies have reported contradictory results on nitrate distribution in soils. For example, Cote et al. reported that nitrate application at the beginning of an irrigation cycle reduced the risk of leaching compared to fertigation at the end of the irrigation cycle. On the other hand, Hanson et al. reported that fertigation at the end of an irrigation cycle resulted in a higher nitrogen use efficiency compared to fertigation at the beginning or middle of an irrigation cycle. These studies very well outlined the importance of numerical modelling in the design and management of irrigation and fertigation systems, especially when there is a lack of experimental data on nutrient transport in soils. However, there is still a need to verify the fate of nitrate in soils with horticultural crops and modern irrigation systems. Therefore, a lysimeter was established to observe water movement and drainage under drip irrigated navel orange, and to calibrate the HYDRUS 2D/3D model against collected experimental data. The model was then used, in the absence of experimental data on nitrate, to develop various modelling scenarios to assess the fate of nitrate for different irrigation and fertigation schemes.The study was conducted on a weighing lysimeter assembled and installed at the Loxton Research Centre of the South Australian Research and Development Institute. The lysimeter consisted of a PVC tank located on 1.2 m × 1.2 m pallet scales fifitted with 4 × 1 tonne load-cells, and connected to a computerised logging system which logged readings hourly.

A specially designed drainage system placed at the bottom of the lysimeter consisted of radially running drainage pipes,nft growing system which were connected to a pair of parallel pipes, which facilitated a rapid exit of drainage water from the lysimeter. These pipes were covered in a drainage sock and buried in a 25-cmlayer of coarse washed river sand at the base of the lysimeter, which ensured easy flushing of water through the drainage pipe. A layer of geo-textile material was placed over the top of the sand layer to prevent roots growing down into it, as this layer was intended to be only a drainage layer. A healthy young citrus tree was excavated from an orchard at the Loxton Research Centre and transplanted into the lysimeter. A soil profile approximately 85 cm deep was transferred to the tank with the tree and saturated to remove air pockets and to facilitate settling. The final soil surface was around 10 cm below the rim of the tank. Soil samples were collected from0 to 20, 20 to 40, 40 to 60, 60 to 85, and 85 to 110 cm depths to measure bulk density and to carry out particle size analysis. Two months after transplanting, the lysimeter was installed amongst existing trees in the orchard. Measurements were initiated after about six months, in order to enable the plant to adjust to the lysimeter conditions. The lysimeter was equipped with Sentek® EnviroSCAN® logging capacitance soil water sensors installed adjacent to the drip line at depths of 10, 20, 40, 60, and 80 cm to measure changes in the volumetric soil water content. Drainage water was directed through flexible piping into a large bin installed below ground level. The experimental site was approximately 240 m from an established weather station, which measured air temperature, relative humidity, wind speed , rainfall, and net radiation.Irrigation was applied using 3 pressure compensated emitters with a discharge rate of 4 L h−1. Emitters were located on a circle 25 cm away from the tree trunk at an equal distance from each other . The irrigation schedule was based on the average reference evapotranspiration during the last 10 years at the site, multiplied by the crop coefficient taken from Sluggett . The cumulative crop evapotranspiration during the 29 day experimental period was equal to 65.3 mm, and daily ETC varied from 1.68 to 3.39 mm. Irrigation was initiated on 16 August 2010 and terminated on 13 September, 2010. Irrigation and rainfall were recorded daily and drainage volume was measured 3 times per week throughout the trial period. Daily irrigation was applied in 5 short pulses using an automated irrigation controller, with 2 h breaks between irrigation pulses. The amount of irrigation water applied was slightly higher than ETC for the period. A total of 70 mm of rainfall fell during the experimental period, including a single event of 52 mm on 3 September 2010.The simulation domain was represented by a 110-cm deep and 100-cm wide cylindrical cross section. Drip irrigation was modelled as a circular line source 25 cm from the centre of the lysimeter with a uniform water flux along the drip line.

This simplification was made to enable HYDRUS to model this problem in a 2D axi-symmetrical mode , rather than in a full 3D mode, which would be computationally much more demanding. Additionally, since the surface wetted area and input flux densities under drippers were dynamic, an option that we would not be able to model with HYDRUS in a 3D mode, we assumed that the simplification of the problem to axi-symmetrical 2D was adequate. Moreover, the drainage system laid out in the lysimeter also supported the use of an axi-symmetrical domain as the drainage pipes run in a circular fashion to collect and flush drainage water out of the lysimeter. The transport domain was discretized into 3294 finite elements, with a very fine grid around the dripper and near the outflow , with gradually increasing element spacing farther from these two locations . Simulations were carried out over a period of 29 days.Since most soils on which citrus is grown in South Australia are coarse textured soils with good drainage, high oxygen levels, low organic matter, and low microbial populations, denitrification and mineralisation was assumed to be negligible in this study. Similarly, the soil adsorption of nitrate was also considered to be negligible since both nitrate and solid surfaces are negatively charged. Plant uptake of non-adsorbing nutrients like nitrate is controlled mainly by mass flow of water uptake . Therefore, it was assumed that nitrate was either passively taken up by the tree with root water uptake or moved downward with soil water. Spatial distribution of nitrate in the transport domain was thus simulated using the convection–dispersion equation for a nonreactive tracer. The longitudinal dispersivity was considered to be 5 cm, with the transverse dispersivity being one-tenth of this . Similar values of these parameters have been used in other studies .Citrus trees in this region are fertilised from early September till March, and in drip systems fertilizers are mostly applied with the second irrigation pulse for the day. All fertigation scenarios reported here are hypothetical. Fertigation was assumed to be supplied with the same quantity of water as in irrigations without fertigation and to conform to the 2D axi-symmetrical domain. For the initial scenario, fertigation pulses were applied from 30 August 2010 at the rate of one fertigation pulse each day. These were followed by 2 days without fertigation and then another daily fertigation pulses. The resultant dose of N for the period from August till September was calculated based on recommended fertilizer application practices for 5–6 year old orange tree. The seasonal recommended dose of nitrogen for an orange tree of this age is 139 g N applied from September to March . Hence for the seasonal simulation, nitrogen was assumed to be applied in equal monthly doses , in similar pulses as described for the experimental period. The simulation was run for 300 days in order to evaluate the fate of seasonally applied nitrogen fertilizer in citrus. Further scenarios examining the impact of timing of nitrogen application on the efficiency of nitrogen uptake simulated a fertilizer application either at the beginning , middle , or end of the daily irrigation scheme. Since the daily irrigation consisted of 5 pulses, fertigation was applied during the 2, 3 and 4 irrigation pulse in the PF1, PF2 and PF3 scenarios, respectively. It is a common practice that the initial and final irrigation pulses are fertilizer free to ensure a uniform fertilizer application and flushing of the drip lines. In addition to these simulations, two continuous fertigation scenarios were also performed to compare pulsed and continuous fertigation.

Posted on

Nft Hydroponic System Flow Rate

The flow rate of an NFT (Nutrient Film Technique) hydroponic system depends on various factors, including the size of the system, the number of plants, and the specific requirements of the plants being grown. However, I can provide you with a general guideline for the flow rate in an NFT system.

In an NFT system, a thin film of nutrient solution continuously flows over the roots of the plants, providing them with water and nutrients. The flow rate is typically measured in liters per hour (L/h) or gallons per hour (GPH).

A common recommendation for the flow rate in an NFT system is around 1-2 liters per minute per square meter of growing channel. This means that for every square meter of NFT channel, the system should provide a flow rate of 60-120 liters per hour (or approximately 15-30 gallons per hour). This guideline ensures that there is a sufficient supply of water and nutrients to the plants’ roots while also allowing for efficient drainage and oxygenation.

Keep in mind that this is a general guideline, and the flow rate can be adjusted based on the specific needs of your plants, the environmental conditions, and the stage of growth. It’s always a good idea to consult specific resources or seek advice from experienced hydroponic growers to determine the optimal flow rate for your particular setup.

Posted on

What Are The Different Ways To Grow Hydroponic Farming

Hydroponic farming offers various methods for growing plants without soil. Here are some of the different ways to practice hydroponic farming:

  1. Nutrient Film Technique (NFT): In this method, a thin film of nutrient-rich water continuously flows over the roots of the plants, allowing them to absorb nutrients while also receiving oxygen. The plants are usually placed in troughs or channels with a slight slope to facilitate the flow of the nutrient solution.
  2. Deep Water Culture (DWC): DWC involves suspending plant roots in a nutrient solution with the support of a floating platform or raft. The roots are submerged in the oxygenated solution, providing constant access to nutrients and oxygen.
  3. Drip System: Drip irrigation systems deliver nutrient-rich water directly to the plant’s root zone through small tubes or emitters. The solution is dripped or sprayed onto the medium or roots, allowing the plants to absorb the nutrients. Excess solution is collected and recirculated.
  4. Aeroponics: Aeroponics is a method where the plants’ roots are suspended in air, and a nutrient mist is sprayed directly onto the roots. This method ensures maximum oxygenation for the roots and efficient nutrient uptake.
  5. Wick System: The wick system is a passive hydroponic method where a wick, such as a cotton rope, transfers the nutrient solution from a reservoir to the plant’s growing medium. The wick draws up the solution, providing moisture and nutrients to the roots.
  6. Ebb and Flow (Flood and Drain): This method involves periodically flooding the growing tray or container with nutrient solution and then allowing it to drain away. The flooding and draining cycles provide oxygen to the roots while ensuring they receive the necessary nutrients.
  7. Vertical Farming: Vertical hydroponic systems utilize vertical space to grow plants in stacked layers or towers. This method maximizes the use of limited space and allows for high-density cultivation.
  8. Nutrient Film Technique (NFT): The NFT system uses a constant flow of nutrient-rich water in a shallow channel, allowing the plant roots to access the solution while being exposed to air. The nutrient film provides continuous nutrient supply and oxygenation.

These are just a few examples of the many hydroponic farming methods available. Each method has its own advantages and considerations, and the choice depends on factors such as plant type, available space, resources, and personal preference.

Posted on

How To Make A Nft Hydroponic System

To create an NFT (Nutrient Film Technique) hydroponic system, you will need the following materials and tools:

Materials:

  1. Growing channels or troughs (typically made of PVC or other suitable materials)
  2. Submersible water pump
  3. PVC pipes and connectors
  4. Growing media (such as Rockwool cubes or net pots)
  5. Nutrient solution
  6. Timer for controlling the water pump
  7. pH and EC meters for monitoring nutrient levels
  8. Light source appropriate for your plants
  9. Reservoir or tank for holding the nutrient solution
  10. Air pump and air stones (optional but recommended for oxygenation)

Tools:

  1. Saw or pipe cutter
  2. Drill with appropriate drill bits
  3. Measuring tape
  4. Screwdriver or wrench for tightening connectors

Here are the general steps to set up an NFT hydroponic system:

  1. Design and prepare the channels: Determine the size and layout of your growing channels. Cut the PVC pipes or troughs to the desired length and attach them to the frame or support structure.
  2. Set up the water pump: Install the submersible water pump in the reservoir or tank. Connect the pump to the PVC pipes or channels using suitable connectors and tubing. Ensure the pump is capable of providing a gentle flow of nutrient solution.
  3. Create a slope: Position the channels at a slight angle to allow the nutrient solution to flow from one end to the other. A slope of 1:30 to 1:40 is generally recommended.
  4. Install the growing media: Place the Rockwool cubes or net pots with your plant seedlings or clones into the channels, ensuring that the roots are exposed to the flowing nutrient solution.
  5. Connect the system: Connect the water pump to a timer, which will regulate the on/off cycles to maintain the desired nutrient flow. Set the timer to periodically pump the nutrient solution through the channels, allowing it to flow over the roots and then drain back into the reservoir.
  6. Mix and monitor the nutrient solution: Prepare the nutrient solution according to the instructions provided by the manufacturer. Regularly monitor the pH and electrical conductivity (EC) levels of the solution and adjust as necessary to maintain optimal plant health.
  7. Provide adequate lighting: Place suitable grow lights above the channels to provide the necessary light intensity and spectrum for plant growth. Adjust the height and duration of lighting based on the requirements of your plants.
  8. Oxygenate the solution (optional): If desired, connect an air pump and air stones to provide additional oxygenation to the nutrient solution. This helps ensure the roots receive sufficient oxygen for healthy growth.
  9. Monitor and maintain: Regularly check the system for proper flow, nutrient levels, and overall plant health. Keep an eye out for clogs, pests, or any other issues that may arise.

Remember to research and adjust the system parameters based on the specific requirements of your plants. Hydroponic gardening requires precise monitoring and adjustments to ensure optimal growth and yield.

Posted on

Strawberry Hydroponic Gutter System

A strawberry hydroponic gutter system is a method of growing strawberries using a hydroponic system that utilizes gutters as the growing medium. Hydroponics is a technique for cultivating plants without soil, where the plants receive all the necessary nutrients through a water-based solution.

In a hydroponic gutter system specifically designed for strawberries, a series of gutters are set up at an incline. The gutters typically have holes or channels where the strawberry plants are placed. The plants’ roots hang down into the flowing nutrient-rich water solution.

Here are the key components and considerations for a strawberry hydroponic gutter system:

  1. Gutters: The gutters used in the system can be made of various materials such as PVC, metal, or plastic. They should be designed to hold the plants securely while allowing the roots to access the nutrient solution.
  2. Nutrient Solution: The nutrient solution contains a balanced mix of essential nutrients that strawberries need for healthy growth. It typically includes macronutrients (such as nitrogen, phosphorus, and potassium) and micronutrients (such as iron, zinc, and calcium). The solution is regularly pumped or flowed through the gutters, ensuring the roots have access to the nutrients.
  3. Substrate: A substrate or growing medium may be used in the gutter system to support the strawberry plants. Common options include coconut coir, perlite, vermiculite, or rockwool. The substrate holds moisture and provides stability for the plants.
  4. Irrigation: An automated irrigation system is necessary to supply the nutrient solution to the gutters. This can be accomplished using drip irrigation, where small tubes or emitters deliver a regulated amount of nutrient solution to each plant or gutter.
  5. Lighting: Depending on the location and available natural light, supplemental artificial lighting may be needed to ensure the strawberries receive sufficient light for photosynthesis. LED grow lights are commonly used in hydroponic systems to provide the specific light spectrum needed for plant growth.
  6. Climate control: Controlling the temperature, humidity, and ventilation is crucial for optimal strawberry growth. Proper airflow and ventilation help prevent diseases and provide an environment suitable for plant development.
  7. Planting and maintenance: Strawberry plants are typically propagated from runners or small transplants and placed into the gutter system. Regular monitoring of pH levels, nutrient concentrations, and overall plant health is necessary. Pruning, removing dead leaves, and ensuring pollination (if not using self-pollinating varieties) are also important for productive plants.

A hydroponic gutter system offers several advantages for growing strawberries. It allows for high-density planting, efficient use of space, and easier access to the plants for maintenance and harvesting. Additionally, since hydroponics avoids soil, the risk of soil-borne diseases and pests is minimized.

However, it’s important to note that designing and managing a hydroponic gutter system requires some expertise in hydroponic gardening. It may be beneficial to consult resources, attend workshops, or seek guidance from experienced growers to ensure the best results.

Posted on

Our phylogenetic trees showed that P. turczaninovii always clustered with P. tenuiloba

Low resolution phylogenetic trees made using the chloroplast regions mentioned above have been reported for other taxa, including Curcuma and Sisyrinchium . The inadequate resolution may be due to the lower substitution rates and lack of variation found in single plastid regions. Thus, we do not recommend single plastid regions as DNA barcodes for this the genus. Among the candidate barcode genes, the Consortium for the Barcode of Life Plant Working Group suggested that rbcL, matK, and the rbcL+matK combination should be sufficient for a plant barcode, and that this combination should be supplemented with additional markers as required . In addition, Kress et al. and Chase et al. proposed that trnH-psbA can be used in two-locus or three-locus barcode systems to improve resolution. For instance, two of the three combinations of the three chloroplast loci tested in this study, rbcL+trnH-psbA and rbcL+matK+trnH-psbA exhibited higher discriminatory performance than any single marker. Consequently, this highlights the need to use chloroplast multi-locus barcodes to improve the resolution of species identification in Pulsatilla. The nuclear ITS region provided the highest inter-and intraspecific divergences and had a higher success rate for the correct identification of species in TAXONDNA . However, as for the treebuilding method,large pot with drainge the discriminatory performance of ITS is not satisfactory, as its highest resolution is 39.02% .

As evidenced by previous studies, the multi-locus barcode is one of the combinations that demonstrated the highest species resolution rate, e.g., Aceraceae , Lysimachia , Oberonia , Rhodiola and Schisandraceae . However, in this study, addition of ITS to different kinds of combinations of chloroplast markers did not increase the resolution rate obviously . The resolution rate based on tree-building analyses was 51.22% for BI and 58.54% for PWG. In addition, we found no distinct barcoding gap. This phenomenon may be due to the one or more of several reasons. first, incomplete lineage sorting and non-homogeneous concerted evolution are likely to occur at the ITS locus . Second, the three chloroplast regions cannot compensate for the drawbacks of ITS because they are sourced from a different genome. Although the nuclear genome is inherited biparentally, the chloroplast genome is inherited uniparentally. Thus, the chloroplast genome experiences more complete lineage sorting than the ITS locus does. Third, hybridizations may cause conflicts between ITS and chloroplast loci, as well as problematic results in ITS phylogeny due to the possibility of homogenization to paternal copies in some lineages and maternal copies in others. A combination of DNA markers from different genomes— which have different modes of inheritance and conflicting phylogenies—can hinder our understanding of species delimitation and the evolutionary processes of speciation. Because of its myriad variable sites that can reliably distinguish species, resulting from a high mutation rate and rapid concerted evolution, we recommend ITS as a good single barcode for the genus Pulsatilla.Phylogenetic identification and species recognition are foundationally important for biology . The results of the phylogenetic analyses performed in this study may shed some light on the identification and taxonomy of the genus Pulsatilla . Here, we found that Pulsatilla formed a monophyletic group with high support. Moreover, the three recognized subgenera — i.e. subg. Pulsatilla, subg. Kostyczewianae, and subg.

Preonanthus— were resolved as distinct monophyletic groups, which is consistent with the recent phylogenetic result . Within subgenus Pulsatilla, our analyses found that P. camanella and P. ambigua were resolved as sister to one another with high support. These two species share many common morphological characters, such as almost fully expanded leaves at anthesis,black plastic planting pots and dense, long trichomes. The flowers of both species nod before anthesis . However, during anthesis, the sepals of P. camanella can easily be distinguished from those of P. ambigua by color . At the same time, the micro-morphological characters of the leaves are also different . Actinocytic and anomocytic stomata exist in both species, but most stomata in P. camanella are actinocytic, whereas most are anomocytic in P. ambigua. Thus, molecular data as well as micromorphological characters can distinguish between these two species relatively well. Both types of evidence may be helpful to accurately identify specimens that are damaged or lack sufficient diagnostic characters. In addition to its use in identifying specimens, DNA barcoding is also useful for resolving taxonomic uncertainty .They did not have distinct barcodes. The micro-morphological characters were also found to be the same, since both plants showed polygonal epidermal cells with striation, a dense distribution of stomata, and glabrous or sparsely short trichomes. In addition, the geographical distribution of these two species overlaps in Inner Mongolia. Taken together, these distinct lines of evidence collectively suggest that P. turczaninovii and P. tenuiloba are the same species. The discovery of hybridization, introgression, and/or incomplete lineage sorting among species is another useful application of DNA barcoding . The chloroplast region is inherited maternally, but the nuclear genome, including the ITS region, is inherited biparentally . Thus, if there are different results in different phylogenetic analyses from chloroplast and nuclear data, we speculate that these differences may be caused by hybridization and/or introgression among species, which could result in a non-monophyletic clade. In subg. Pulsatilla, we found several complex groups. The samples of P. chinensis and P. cernua in cluster III, were indistinguishable. In the Bayesian inference tree based on ITS sequences , the samples of P. cernua clustered in a clade along with sample P. chinensis. However, in the Bayesian inference tree based on the combination of chloroplast sequences , sample P. chinensis clustered in a clade with all other samples of P. chinensis. P. chinensisis a widespread species and has a geographical range that covers that of P. cernua; in addition, sample P. chinensis was collected near populations of P. cernua in Jilin Province, China. Hybridization or introgression might have occurred during the speciation of P. chinensisis and P. cernua. A similar situation was also found for sample P. ambigua108 and cluster I , suggesting hybridization may have occurred between P. ambigua and P. tenuiloba/P. turczaninovii.