Use of non-intrusive laser exfoliation to improve substance uptake into citrus leaves

Background: Despite the presence of stomata in leaves, foliar application of agrochemicals can be extremely inefficient due to the low permeability of leaf cuticular surfaces to polar compounds. Methods: This study introduced a laser-based “wax exfoliation” method to facilitate the penetration of substances into the leaf and, together with enhancing their uptake into the phloem and subsequent transport across tissue. This investigation demonstrated the effectiveness and non-invasive properties of laser exfoliation to improve the penetration of foliar-applied substances into citrus leaves. Results: This work presents the use of laser energy to exfoliate the cuticle of a leaf, with the highest energy density of 0.76 J/ cm2 resulting in 85-90% exfoliation across the entire laser-spot area. The infrared wavelength of the erbium laser is specifically chosen to target the wax cuticle without causing damage to the underlying epidermal cells. This selective ablation allows for increased penetration of therapeutic compounds into the leaf and transportation throughout the plant's vasculature. This is demonstrated using a fluorescent glucose analog applied to the laser treated leaves, showing increased penetration and transport throughout the leaf. Conclusions: Our findings demonstrate that the use of laser technology for the foliar application of agrochemicals provides significant advantages, including improved foliage uptake of therapeutic compounds. The method of cuticle exfoliation presented in this study is highly effective and non-intrusive, limiting its effects to the cuticle only. Future work should focus on the development of prototypes for in-field applications, including testing at longer distances as the Er:YAG laser does not require a lens for this application.

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Introduction
One of the most prevalent methods used in modern-day agriculture to improve crop health, and hence yield, is the foliar application of agrochemicals. 1 However, several barriers cause retardation and interfere with the efficient penetration and utilization of these substances. For example, the leaf surface is coated by a waxy cuticle that serves as a barrier for the prevention of water loss and pathogenic entry into the plant body. Due to its water impermeable nature, the cuticle also prevents the entry of externally applied soluble compounds such as most agrochemicals. The movement of substances into the leaf occurs primarily through the stomata, 2 located mainly on the less-exposed abaxial side of plant leaves. 3 This property reduces the functional surface area leading to reduced agrochemical penetration through the foliar route. As consequence, considerable quantities of applied chemicals end up in the plant's natural environment and can generate undesirable ecological impacts.
An example of research focused on foliar application of substances into plant leaves is the study of agrochemicals to treat citrus Huanglongbing (HLB), 4,5 a bacterial disease of citrus that has spiraled the Florida citrus industry to the brink of disappearance and actively spreading across other regions of the world on a large scale. 6 In the case of HLB infections, therapies considered against the causative bacterium Candidatus liberibacter (CLas) 7 include treatment with antimicrobial agents as a promising alternative to enhance the crop's lifespan. 5 However, response to such treatments is diminished due to challenges associated with the application process and the ensuing slow penetration into the phloem, the conductive tissue where bacterial populations aggregate within infected citrus plants. 8 A novel alternative was recently identified to enhance penetration of agrochemicals into the leaf. This system involves the generation of leaf perforations of approximately 250 μm in diameter using a CO 2 laser directly focused upon the leaf surface. The perforations not only puncture the cuticle but also perforate the epidermis and few layers of underlying palisade parenchyma. 7 When applied on treated leaves, penetration of sample substances was increased over 2,000% over untreated leaves. 9 The use of laser micro-perforations as a plant pharmacodynamics-enhancing technique has several drawbacks. First, apart from the cuticle, laser-induced pores also affect the underlying leaf epidermis and palisade parenchyma due to the inevitable removal of material from both areas. 9,10 Second, this methodology requires highly focalized lasers to achieve maximum efficiency within field conditions, which is technologically complex due to the intrinsic irregularity of leaf surfaces, their random orientation, and depths within a tree.
The goal of this investigation was to devise a lesser invasive mechanism to enhance substance uptake into the leaves using specific laser beams. Our study describes a novel methodology for enhancing the penetration of agrochemicals into citrus leaves without the drastic requirement for physically perforating leaf tissues. This novel technique is based upon epidermal water content-dependent selective absorption of Erbium laser light. Partial separation of the waxy cuticle is successfully obtained across an area of several square centimeters through the application of a single laser shot. Since there is no damage afflicted to the leaf epidermal tissue, the cuticle rapidly regenerates within a brief period, thereby recovering its protective functions.

Methods
Plant material 'Valencia' orange leaves from greenhouse-reared plants of approximately one meter were selected. 'Valencia' orange is one of the main species affected by Huanglongbing (HLB) disease, and its leaves are covered by a thick cuticle layer that can impede the absorption of foliarly applied treatments, making it an ideal model organism. Laser treatments were applied to attached leaves and these remained on the tree for post-laser application in order to provide the live conditions. Treated leaves were consequently detached and transported to the laboratory for further analysis.

Energy levels
It is well-established that leaves have a high-water content including the epidermis. Since water has a strong absorption band in the 3000 nm wavelength region, an in-house Er:YAG laser (fundamental wavelength = 2940 nm; 200 μs pulse REVISED Amendments from Version 2 The first paragraph of the Results section was modified to include a clearer explanation of what is observed in Figure 1. The wording of the first sentence in the paragraph following Table 1 was improved. The explanation in the figure caption of Figure 3 was improved. The wording of the figure caption in Figure 5 was improved. Any further responses from the reviewers can be found at the end of the article duration) was used for foliage irradiation from a distance of approximately 30 cm. Each laser treatment consisted of a single shot performed without specific focusing of the laser on the leaf surface. In order to discern the varying effect of laser intensity on the leaf wax cuticle three laser energy levels were employed, with a spot area of 0.78 cm 2 . This spot area was determined by the diameter of the laser crystal used in the experiment (1cm), as no focusing element was employed. This allowed for a direct measurement of the effect of laser intensity on the leaf wax cuticle, without the added variable of a focusing element. No lens was used to focalize the laser and the beam divergence was 0.1 mrad, consequently disregarding the necessity for standardizing irradiation distance in such assays. In all cases, one single pulse sufficed to obtain the effect of wax cuticle exfoliation. The laser energies tested for the removal of wax across the investigated leaf foliage ranged from 0.3-0.76 J/cm 2 , depending upon the laser energy level.

Penetration of applied soluble fluorescent marker
To visualize the penetration of applied substances into the leaf, a fluorescent analog of glucose solution (NBDG: . The procedures and techniques described in this manuscript have been thoroughly described to allow for replication of the results. All relevant materials, steps, and parameters have been included to ensure full reproducibility of the experiment.

Results
The effect of increasing laser energy levels applied to the leaf surface was studied by treating 20 leaves from the same tree with three different energy levels. The results of these pulses on three representative leaves are presented in Figure 1. The yellow areas in Figure 1 represent the modified leaf cuticle according to the level of energy applied. The yellow color displayed in the irradiated area is due to the separation of the wax cuticle from the epidermis in that region, altering the reflection of the microscope's white light. In contrast, for the non-irradiated areas, the transparent wax cuticle remains in contact with the epidermis, which is green in color due to the chlorophyll. Exfoliation of the leaf cuticle increased proportionally with laser intensity level. For the highest energy density value at 0.76 J/cm 2 , the exfoliation effect was achieved across (85-90%) the entire laser-spot area ( Figure 1c). A close-up of a treated portion of a leaf using 0.76 J/cm 2 energy density is shown in Figure 2. The waxy material, which appears relatively smooth in untreated leaves (Figure 2a), aggregated and segregated after treatment, forming clean breaches into the epidermis (Figure 2b). Under these conditions, only the cuticle was affected as the green underlying epidermal layer of cells remained undisturbed by the energy applied. The image in Figure 2c (black-and-white) was generated using the color tool from Power Point ® (Microsoft™, USA) to facilitate the estimation of percent exposed areas. Regarding such estimations, the online tool 'Coolphptools' was employed, 11 to calculate the percentage area per color. The black-color areas, which corresponded to wax cuticle exfoliated regions, was estimated to cover as 0.61, indicating a 61% success rate in cuticle exfoliation of the total laser-irradiated area. Table 1 shows the average percentage of exfoliated area for each energy level.
The infrared emission wavelength of 2940 nm from the erbium laser coincides with the maximum of the absorption peak of water. Therefore, given the high-water content of the leaf, the absorption of the laser energy is very high.
This phenomenon provokes the rapid heating and exerting of pressure that 'pushes' the wax cuticle outwards. Since such a laser-energy/wavelength is not absorbed within the epidermal tissue or any other plant constituent, consequently, there is no irreversible physical damage imposed upon the leaf structural integrity, apart from cuticle exfoliation (temporary separation). The mechanism of selective ablation of plant parts/elements was previously utilized for the removal of cactus spines, where it is possible to pulverize and extract such spines through rapid heating of water content present within glochids. 12 Figure 3 presents a fluorescent view of a laser treated leaf using a green filter. In Figure 3a, the areas where the cuticular wax was "lifted" or exfoliated appear green in color. The fluorescent green color represents autofluorescence of undamaged epidermal cell walls. These open irregular-shaped areas range from several tens to hundreds of micrometers. The beige areas correspond to the exfoliated wax clumps. A cross section of a treated area in presented in Figure 3b.       detached) as a result of the pressure exerted by the laser-excited water (Figure 4b). Through such areas having detached and raised cuticles, therapeutic compounds can easily penetrate into the leaf epidermal layer and then follow its pathway to the plant transport system.
To demonstrate the effectiveness of the laser treatments in allowing externally applied hydrophobic substances to penetrate the leaf and travel throughout the plant vasculature, we applied a fluorescent NBDG to laser treated areas ( Figure 5). The images are viewed under green/red control untreated leaf is presented in Figure 5a. Conversely, Figure 5b depicts a treated leaf two hours after laser treatment and NBDG application. The externally applied fluorescent NBDG is visible throughout the leaf, especially in the veins containing the plant vascular tissue, clearly indicating successful solution penetration and distribution across the majority of leaf surface area.

Conclusion
The application of agrochemicals through the foliar route remains a "gold-standard" therapeutic administration route for enhancing crop productivity, treatment of diseases, and pathogen/parasite circumvention and prophylaxis. However, despite its wide application, penetration through leaves remains quite inefficient, causing dramatical environmental impact as > 90% of applied agrochemical doses by the foliar route are not absorbed by the plant and eventually lead to a detrimental impact on the immediate plant environment. 1 The use of laser technology for foliar application of agrochemicals contributes with a plethora of advantages. Aside from the improving foliage uptake of most therapeutic compounds/agrochemicals, it results in the reduction in agrochemical losses/wastage and eventual detrimental impact on the treated plant's immediate environment. The high efficacy provided by the cuticle exfoliation method presented in this communication eliminates perforation of live tissue by limiting its effect to the cuticle only, therefore, providing a less intrusive method for substance penetration. Future work requires the development of field application prototypes capable of operating at longer distances. We conducted experiments using an extremely simple optical arrangement that did not require a lens to focus the laser. Although the simplicity of the optics for long distances still needs to be verified, an estimate can be made based on simple calculations. For an Erbium laser with M2 < 2, we would have an angular divergence of 1.49 mrad. This means that for a distance of 5 meters from the aperture to the treatment area, we would have a beam diameter of approximately 1.48 cm and a spot area of 1.72 cm 2 . For this area, an energy density of 0.7 J/cm 2 could be achieved with pulses of 1.2 J of energy. A treatment distance of 5 meters would be sufficient for use under field conditions, in combination with substance sprayers currently used in the industry.

Gabriel Bilmes
Laser Ablation, Photophysics and 3D imaging Laboratory, Centro de Investigaciones Opticas (CONICETCIC-UNLP), La Plata, Buenos Aires, Argentina This paper describes a laser-based "wax exfoliation" method to facilitate the penetration of substances, as agrochemicals, into plants leaves. The authors applied the method to citrus leaves showing its effectiveness, the non-invasive properties of laser exfoliation and the advantages over other methods. The proposed method is original and novel. The methodology followed to test it and the experiments carried out are adequate, and the results obtained are conclusive. The conclusions are well founded. For all these reasons, I consider that the work should be indexed only with minor formal modifications, which are detailed below. ROW 6 of the introduction "…located mainly on the less-exposed abaxial side of citrus leaves". Consider modifying by: located mainly on the less-exposed abaxial side of plant leaves. ○ Last paragraph of the introduction: "…through the application of a single laser beam". Consider modifying by: through the application of a single laser shot. ○ In figure 1, both in the explanation and in the text, clarify how these images were taken, so that the treated part can be seen in yellow ○ Row 4 after table 1: "...The infrared wavelength inherent to the erbium laser, which is identical to the absorption peak of water, allows for considerable laser-energy absorption by such water content." Consider modifying by: The infrared emission wavelength of 2940 nm from the erbium laser coincides with the maximum of the absorption peak of water. Therefore, given the high-water content of the leaf, the absorption of the laser energy is very high.

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Row 4 after table 1: "...The infrared wavelength inherent to the erbium laser, which is identical to the absorption peak of water, allows for considerable laser-energy absorption by such water content." 1.
Consider modifying by: The infrared emission wavelength of 2940 nm from the erbium laser coincides with the maximum of the absorption peak of water. Therefore, given the high-water content of the leaf, the absorption of the laser energy is very high.

The paragraph was modified according to the reviewer's suggestion.
Consider reviewing the explanation of figure 3 in the text and the corresponding caption. It is not clearly written. Suggested wording: 1. The paragraph was modified according to the reviewer's suggestion.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Reviewer Expertise: Laser, optics, photothermal spectroscopy, digital holography microscopy I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 08 Apr 2023 luis ponce cabrera General comments: The reviewer is correct that it would have been interesting to see the results in the plant for a period after treatment, but this is beyond the scope and objectives of this work. We are presenting a method that allows the uptake of substances to be created and have demonstrated that there is an improvement. However, the consequences of the treatments can be very different depending on the substance applied. Each substance will require a specific study as the doses and timing will be different.
Regarding the productivity, it is also too early to tell as it will depend on how much substance is required and how many entry points will need to be opened in the plant. It may be sufficient to treat just one leaf in some cases, while in other cases it may be necessary to treat multiple leaves.
Answers to the referee's questions: Regarding the referee's suggestion to include a leaf before treatment in Figure 1, as can be seen in Figure 5, an image of the leaf where the treatment area is visible before (a) and after (b) treatment has already been included.

1.
As mentioned in the general comments, a statistical study on how the plant grows and whether this helps farmers is beyond the scope of this article. This method can be used to improve the capture of many different substances, each of which will require a specific study.

2.
Regarding the pulse duration, the available experimental setup does not allow for modification of that parameter, but we agree with the referee that it is worthwhile to conduct a study in the near future to optimize it. However, the penetration of the fluorescent glucose demonstrates that for the specific pulse duration we have used, the process significantly increases the capture of the substance, which was the main objective of this work.

3.
We did not include a discussion of efficiency and productivity because the development of prototypes applicable under field conditions is future work. However, we have modified the final part of the conclusions to include an estimate that gives an idea of the feasibility of this method. 4.
The answer to this question is related to the previous one. As mentioned earlier, the productivity for each specific substance will depend on many factors, and it is an analysis that is beyond the scope of this work.

5.
The cost of the device will be related to parameters such as power, pulse energy, scanning system, and others, as well as the size of the market. A rough estimate could be a few thousand dollars. The financial viability will depend on how effective the method is in saving on agrochemicals or improving plant treatments. It is still too early to make an accurate economic evaluation.

6.
Competing Interests: No competing interests were disclosed.
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