The foliar fertilizer product “CleanStart,” “Kick-Off,” and “GroZyme” were obtained from Ag Spectrum Company.0.1% Silwet L-77was added in each solution.The solutions were applied to the leaves of sunflower 8 h before darkness , and all plant Thissues except the sprayed leaf were covered to prevent inadvertent spray application.The petioles of all sprayed leaves were carefully protected by coating the leaf base petiole junction with lanolinand Teflon membranes.Four plants were treated as one replication, with three replications for each treatment.The foliar application of the fertilizers was replicated one time after 7 days, and then plants were harvested 7 days later.GroZyme is a microbial fermentation product derived from a proprietary mix of organic cereal grains inoculated with specific bacterial cultures and fermented.The fermentation process occurs under controlled environmental conditions until a specific metabolic profile is achieved at which time the live bacterium are liaised and the material is filtered to remove large particles.This concentrate is then extended and stabilized to make the final product.Soil applications of GroZyme have been reported to alter soil microbial activity and nitrogen transformations.The metabolic basis for the biological activity of foliar applied GroZyme is not known.However, field observations suggest that GroZyme functions to enhance plant growth by enhancing K metabolism and sugar transport.CleanStart is derived from ammonia, urea, orthophosphoric acid and potassium hydroxide,ebb flow tray and Kick-Off is a micro-nutrient mix of Fe, Mn, Cu, Zn predominantly derived from nitrate sources with additional surfactants and stabilizers.The elemental composition of all spray applications is provided in Table 1.Mid-sections of leaf petioles were cut from the leaves treated with different foliar treatments.Leaf cross-sections were cut with a cryotomeat a temperature of −20◦C.
Single sections of each treatment were selected under light microscopy for their ultra structural integrity and then freeze-dried under −20◦C for 3 days prior to μ-XRF analysis.Since μ-XRF analysis is time consuming and expensive only single samples from each treatment could be analyzed.Given that true replicate analyses could not be performed additional steps were taken to avoid the potential for experimental artifacts and to avoid any sample selection or analysis bias.All treatments were carefully controlled such that treatment conditions and experimental duration were identical; petioles were then taken from the four replicate plants and multiple sections from each petiole were prepared as described above.All sections were then assessed by light microscopy for ultra structural integrity and a single section was then selected and transported to the Stanford Synchrotron Radiation Laboratory for μ-XRF analysis.Samples selected in this fashion, therefore represent unbiased examples of treatment effects.To investigate the effects of different fertilizers on retranslocation of nutrients in the leaves of sunflower, μ-XRF mapping was performed.Cross sections of petioles were cut from the sun- flower plants at approximately 1.0 cm below the leaves and imaged under a light microscope prior to utilization for μ-XRF imaging.The cross section of petiole was composed of epidermis, parenchyma, vascular bundle containing xylem, phloem, and surrounding collenchyma.The microscope image in Figure 1 shows that the phloem within the vascular bundle exists as a discrete layer of cells on the abaxialside of the vascular bundle with xylem on the adaxial or upper side of the petiole.The entire vascular bundle is enclosed in Thissue that is likely collenchyma.Integrated intensity for Zn and other elements were calculated from the spectrum and normalized by the intensity of the Compton scattering peak.Elemental mapping for the measurement area was obtained from the normalized intensity for each element.The elemental distribution maps of Zn , Ca , and Kin the petioles collected from sunflowers plants with different treatments are presented in Figures 2A–F, together with corresponding photographs taken using an optical microscope.
The quantification of the fluorescence yields was normalized by I0 and the dwell time.The normalized X-ray fluorescence intensities were scaled to different color brightness for individual elements, with the brightest spots corresponding to the highest elemental fluorescence.Each map indicates the relative distribution of the three elements, and the scale of fluorescence counts for individual elements is the same for each map.Very slight signals of Znwas noted in the petioles collected from the control and CleanStart treated plants of sunflower.At the resolution used in these experiments it is not possible to determine if the deposition of Zn in regions other than the phloem was resent in xylem or in collenchyma immediately adjacent to the xylem.A modest increase in the K signal in petioles was also observed in the Clean Start treatment.Foliar application of “Kick-Off,” a product containing Co, S, Fe, Cu, Mn, Mo, and Zn chelated with EDTAto sunflower leaves resulted in a marked increase in the concentration of Zn detected in the petioles with a notable deposition in a narrow band corresponding to the phloem Thissues of the petiole and a more diffuse band in the xylem/collenchyma region , while no such preferential localization to phloem Thissues was noted for control, CleanStart or ZnSO4 treatments and no phloem specific accumulation of other elements was observed.Application of ZnSO4 at the same Zn levels as used for all Zn treatments to the sunflower leaves also increased phloem Zn in the petiolesas compared with the controls , but the effect is much less pronounced than that of “Kick-Off.” The combined foliar application of “Kick-Off”with “CleanStart” resulted in a similar enhancement in Zn uptake and preferential distribution of Zn to phloem Thissues and xylem/collenchyma.The addition of the bio-stimulant product GroZyme resulted in a much more concentrated enrichment of Zn in the phloem and xylem/collenchyma region of the petiole vascular bundle.Low spatial resolution μ-XRF imaging provides only semiquantitative data.To further investigate the effects of “CleanStart” and “GroZyme” on phloem mobility of foliar applied Zn, microXRF scanning at higher resolution was performed, focusing on the vascular Thissues of the treated leaf veins.The Zn concentration in the petiole was also determined along a single scan line that transected the petiole and passed through the vascular system.Both “CleanStart” and “GroZyme,” which do not contain Zn, clearly increased the FIGURE 5 | Concentrations of Znin the petioles collected from sunflower plants treated with different foliar fertilizers.Full expanded leaves of sunflower were treated with control, CleanStart, Kick-Off, CleanStart + Kick-Off, CleanStart + Kick-Off + GroZyme, and ZnSO4, and the Zn concentration of leaf veins were analyzed by ICP-MS.
Compositions of the nutrients in different treatments were shown in Table 1.Data points and error bars represent means and SEs of three replicates.concentration of Zn following Kick-Off application.The patterns of Zn deposition in the “Kick-Off” + “CleanStart” and “KickOff” + “CleanStart” + “GroZyme” were far less diffuse and more intensely located in the phloem region and xylem/collenchyma than the pattern of Zn in petioles from leaves provided with “Kick-Off” or ZnSO4 alone.Intensity analysis across a single scan line through the vascular system of the petiole demonstrated that the peak of Zn densities in the phloem Thissues and xylem/collenchyma was markedly increased with addition of the biostimulant “GroZyme” to “Kick-Off”+“CleanStart” treatments.Total concentrations of Zn and other elements including Fe, K, Cu, Ca, and Mn etc.were determined by ICP-MS for the petioles collected from the sunflowers treated with different foliar fertilizers.The results showed that Zn concentrations in the leaf veins of sunflower ranged from 29.2 to 36.7 mg kg−1 DW.While the overall pattern of Zn concentration differences analyzed by ICPMS corresponded with the μ-XRF analysis results the total Zn concentration was not significantly different between treatments.Similarly, no difference in Fe, K, Cu, Ca, and Mn was observed among any treatments.The apparently greater sensitivity of μ-XRF is primarily a consequence of the ability of μ-XRF to analyze specifically within phloem and closely associated vascular organs while ICPMS provides analysis of the total petiole.Since vascular Thissue represents only a very small proportion of the petiole as a whole, and as the petile was fully mature at the time of treatment, changes in vascular Thissue element concentration may not be seen against the background of the bulk of petiole Thissue in which Zn was not increased.Efficacy of foliar applied nutrients depends not only on the absorption of the nutrients but also on the transport of these nutrients to other plant parts.It has been suggested that even a relatively small transport of foliar nutrients out of treated leaves and Thissues may have a short-term,flood and drain tray critical benefit to the plant.Knowledge of the ability of an element to be transported from the site of application is critical to provide insight into the longevity and potential nutritional impact of foliar application on non-sprayed Thissues.Analysis by μ-XRF in the present study shows clearly enhanced transport and localization of Zn in the vascular system of the sunflower petiole 7 days after application of “Kick-Off,” which contains Zn-EDTA , while Zn was not detectable in the petiole of the control plants and was very low in the petiole of ZnSO4 sprayed leaves.This demonstrates clearly that Zn is phloem mobile in sunflower and that the use of Zn-EDTA results in greater phloem Zn transport than ZnSO4 alone.While it has been demonstrated that Zn-EDTA is superior to ZnSO4 under some circumstances, it has not been demonstrated that the EDTA molecule can penetrate the leaf cuticle.It cannot be determined from this current research if the superiority of the EDTA containing Kick-Off material is a consequence of enhanced cuticular penetration or enhanced transport of the Zn once it enters the leaf.The ‘Kick-Off’ material also contains the micro-elements including Fe, Cu, Mn, and Mo and this may also enhance Zn uptake as has been observed previously.The addition of “CleanStart” derived from ammonia, urea, orthophosphoric acid and potassium hydroxide, significantly increased the phloem transport and xylem/collenchyma deposition of Zn when co-applied with ‘Kick-Off’.Addition of Urea to foliar Zn sprays, for example, is known to enhance Zn uptake and efficacy and the N status of cereals is known promote Zn retranslocation.While it is clear that addition of the multi-elements present in the Clean Start enhanced Zn retranslocation into sunflower petioles, the mechanism of this effect remains uncertain.
The chemical form in which a foliar nutrient is applied will influence plant nutrient uptake by altering the point of deliquescence of the applied foliar fertilizer thereby altering its solubility on the leaf surface, or by altering the charge on the ion of interest to facilitate its movement through the cuticle and cell wall.There is no direct evidence, however, to suggest that the formulation of a fertilizer spray can directly influence the transport of the absorbed nutrient from the site of application.The addition of the bio-stimulant “GroZyme” clearly enhanced Zn translocation when co-applied with “Kick-Off” and “CleanStart”.Grozyme is a non-living microbial fermentation product derived from a proprietary mix of organic cereal grains inoculated with specific bacterial cultures and fermented.The specific functional metabolite in GroZyme has not been identified.However, extensive field trials and research published in this issuehave demonstrated positive growth effects and enhanced translocation of K and other nutrient elements.Previous research utilizing soil applications of GroZyme has also shown that this product was able to alter microbial populations in a soil environment and improve N mobilization and uptake of soil nutrients especially organic nitrogen.The benefit of bacterial source metabolites on efficacy of foliar fertilizers has been demonstrated previously and it is plausible that the microbial extracts present in GroZyme have the capability to form metal complexes that enhance Zn uptake or mobility.Many putative bio-stimulants also contain plant growth hormones or plant signaling molecules that may alter plant metabolic processes and stimulate growth and indirectly influence the movement of substrates, including minerals, within the plant.In these experiment, the high spatial resolution and direct imaging capability of μ-XRF was useful in distinguishing differences in Zn transport through the vascular system of sunflower that could not be detected by ICP-MS.XRF provides a powerful strategy to trace foliar applied microelements within the plants with high sensitivity, a result that is consistent with our previous studies.This technique will be useful to facilitate the development of foliar fertilizers and application techniques that optimize transport of nutrients from site of application, which is one of the most important challenges to the foliar fertilizer industry.Heavy metal contamination, such as cadmiumand lead , in various environmental mediaposes a severe threat to ecological and human health as long as they are bio-available.Although there are natural sources of these elements, anthropogenic releases from activities such as metal mining and smelting, coal combustion, trace levels in fertilizers and even some wastewater sludge and biosolids, can increase concentrations to high levels in soils and sediment beds of lakes and rivers.