Fly into the light: eliminating Drosophila melanogaster with chlorophyllin-based Photodynamic Inactivation.

Fruit flies spoil crops in agricultural settings. As conventional pesticides may generate negative off-target effects on humans or the environment, existing treatment methods need eco-friendly and safe alternatives. Photodynamic Inactivation (PDI) is based on the photosensitizer-mediated and light-induced overproduction of reactive oxygen species in targets. We here explore the potential of PDI for the control of fruit fly pests. Drosophila melanogaster serves as well-established model organism in this study. Two distinct experimental approaches are presented: the feed assay, in which fruit flies are provided with sodium magnesium chlorophyllin (Chl, approved as food additive E140) along with sucrose (3%) as their food, and the spray assay, where the photosensitizer is sprayed onto the insects. We show that PDI based on Chl can induce moribundity rates of Drosophila melanogaster of more than 99% with 5 mM Chl and LED illumination (395 nm, 8 h incubation in the dark, radiant exposure 78.9 J/cm2) with the feed assay. If the radiant exposure is doubled to 157.8 J/cm2, 88% of insects are killed by PDI based on 1 mM Chl. The photoactive compound is also effective if presented on strawberries without addition of sucrose with somewhat lower moribundity (71% at 5 mM Chl). Spraying Chl onto insects is less effective than feeding the photosensitizer: 5 mM Chl resulted in 79.5% moribundity (drug to light interval 8 h, radiant exposure 78.9 J/cm2), but if 5 h of sun light (532 J/cm2) and overnight (14 h) dark incubation is used for activation of Chl, more than 95% of insects are killed. As conclusion, Chl serves as effective photoinsecticide against Drosophila melanogaster if a drug to light interval of 8 h is maintained. Feeding the photoactive compound together with sucrose is more effective than spraying it onto insects and increasing the radiant exposure allows for lowering the photosensitizer concentration. Photodynamic Inactivation might therefore represent an eco-friendly addition to the farmers armamentarium against (semi-transparent) insects.

Photodynamic Inactivation in agriculture: combating fungal phytopathogens resistant to conventional treatment.

Botrytis cinerea is a severe threat in agriculture, as it can infect over 200 different crop species with gray mold affecting food yields and quality. The conventional treatment using fungicides lead to emerging resistance over the past decades. Here, we introduce Photodynamic Inactivation (PDI) as a strategy to combat B. cinerea infections, independent of fungicide resistance. PDI uses photoactive compounds, which upon illumination create reactive oxygen species toxic for killing target organisms. This study focuses on different formulations of sodium-magnesium-chlorophyllin (Chl, food additive E140) as photoactive compound in combination with EDTA disodium salt dihydrate (Na2EDTA) as cell-wall permeabilizer and a surfactant. In an in vitro experiment, three different photosensitizers (PS) with varying Chl and Na2EDTA concentrations were tested against five B. cinerea strains with different resistance mechanisms. We showed that all B. cinerea mycelial spheres of all tested strains were eradicated with concentrations as low as 224 µM Chl and 3.076 mM Na2EDTA (LED illumination with main wavelength of 395 nm, radiant exposure 106 J cm-2). To further test PDI as a Botrytis treatment strategy in agriculture a greenhouse trial was performed on B. cinerea infected bell pepper plants (Capsicum annum L). Two different rates (560 or 1120 g Ha-1) of PS formulation (0.204 M Chl and 1.279 M Na2EDTA) and a combination of PS formulation with 0.05% of the surfactant BRIJ L4 (560 g Ha-1) were applied weekly for 4 weeks by spray application. Foliar lesions, percentage of leaves affected, percentage of leaf area diseased and AUDPC were significantly reduced, while percentage of marketable plants were increased by all treatments compared to a water treated control, however, did not statistically differ from each other. No phytotoxicity was observed in any treatment. These results add to the proposition of employing PDI with the naturally sourced PS Chl in agricultural settings aimed at controlling B. cinerea disease. This approach seems to be effective regardless of the evolving resistance mechanisms observed in response to conventional antifungal treatments.

Alkylated EDTA potentiates antibacterial photodynamic activity of protoporphyrin.

Antibiotic resistance has garnered significant attention due to the scarcity of new antibiotics in development. Protoporphyrin IX (PpIX)-mediated photodynamic therapy shows promise as a novel antibacterial strategy, serving as an alternative to antibiotics. However, the poor solubility of PpIX and its tendency to aggregate greatly hinder its photodynamic efficacy. In this study, we demonstrate that alkylated EDTA derivatives (aEDTA), particularly C14-EDTA, can enhance the solubility of PpIX by facilitating its dispersion in aqueous solutions. The combination of C14-EDTA and PpIX exhibits potent antibacterial activity against Staphylococcus aureus (S. aureus) when exposed to LED light irradiation. Furthermore, this combination effectively eradicates S. aureus biofilms, which are known to be strongly resistant to antibiotics, and demonstrates high therapeutic efficacy in an animal model of infected ulcers. Mechanistic studies reveal that C14-EDTA can disrupt PpIX crystallization, increase bacterial membrane permeability and sequester divalent cations, thereby improving the accumulation of PpIX in bacteria. This, in turn, enhances reactive oxygen species (ROS) production and the antibacterial photodynamic activity. Overall, this effective strategy holds great promise in combating antibiotic-resistant strains.

Low Molecular Weight Shuttle Molecules Enhance Polychloramide Antimicrobial Activity.

Investigated were the influences of succinimide (SI), 5,5-dimethylhydantoin (DMH), and 3-(hydroxymethyl)-5,5-dimethylhydantoin (HDMH) on the biocidal activity of chlorinated, water-soluble polyamide prepared by the reaction of isopropylamine with poly(styrene-alt-maleic anhydride). The resulting polymer was a negatively charged, water-soluble polymer bearing a carboxylic acid and an isopropylamide moiety on nearly every repeat unit. Subsequent treatment with NaOCl chlorinated the polymers to up to 4.4% Cl while inflicting some polymer chain scission. SI, DMH, or HDMH increased the biocidal activity of polychloramides toward planktonic Escherichia coli and Staphylococcus aureus. Independent solution studies confirmed that oxidative chlorine spontaneously transferred from aqueous polychloramides to small molecules. We concluded that SI, DMH, and HDMH acted as shuttles that extracted oxidative Cl from the polymer chloramides and transported oxidative Cl more efficiently to microbial surfaces. Succinimide was the most effective shuttle. These results warn that some low molecular weight soluble molecules in antimicrobial testing solutions may exaggerate the effectiveness of the polymer or immobilized antimicrobial agents.

Overview of methods and considerations for the photodynamic inactivation of microorganisms for agricultural applications.

Antimicrobial resistance in agriculture is a global concern and carries huge financial consequences. Despite that, practical solutions for growers that are sustainable, low cost and environmentally friendly have been sparse. This has created opportunities for the agrochemical industry to develop pesticides with novel modes of action. Recently the use of photodynamic inactivation (PDI), classically used in cancer treatments, has been explored in agriculture as an alternative to traditional chemistries, mainly as a promising new approach for the eradication of pesticide resistant strains. However, applications in the field pose unique challenges and call for new methods of evaluation to adequately address issues specific to PDI applications in plants and challenges faced in the field. The aim of this review is to summarize in vitro, ex vivo, and in vivo/in planta experimental strategies and methods used to test and evaluate photodynamic agents as photo-responsive pesticides for applications in agriculture. The review highlights some of the strategies that have been explored to overcome challenges in the field.

Impacts of non-microbial soils on polychloramide disinfectants.

The presence of some nonmicrobial chemicals and surfaces, herein called "soils", are known to degrade the performance of biocides, and biocidal assays often include mixtures of materials to mimic the effects of soils. We hypothesized that water-soluble anionic polychloramide biocides were less sensitive to soil interference than cationic polymeric biocides. The relationships between soil composition and antimicrobial polymer biocidal activity were compared for an anionic polychloramide, a cationic polychloramide, and a cationic poly(quaternary ammonium) biocide. The nanoscale soil models individually investigated were polyacrylic acid (PAA), cellulose nanocrystals (CNCs), and bovine serum albumin. The low molecular weight model soils were ammonium chloride, glycine, and succinimide. Three types of soil impacts were identified: 1) sequestration, whereby the soil physically inhibited transport of the biocide to microbes; 2) extraction, whereby the soil reduced or extracted oxidative chlorine, decreasing or eliminating the oxidative chlorine strength; and 3) extraction whereby the biocidal activity increases in the presence of a low molecular weight chemical that carries oxidative Cl from the polymer to the microbes. PAA and CNCs inhibit cationic biocides by sequestration but have little impact on anionic polychloramide. Glycine and BSA extract oxidative chlorine, lowering the biocidal activity of the anionic and cationic polychloramides while not impacting the poly(quaternary ammonium) biocide. Finally, the presence of succinimide increased bacteria deactivation of both anionic and cationic polychloramides. We propose that succinimide extracts oxidative chlorine from the polychloramides and transports it to the bacteria.

Efficacy of Oil and Photosensitizer against Frankliniella occidentalis in Greenhouse Sweet Pepper.

Many common insect pests have developed resistance against the pesticides currently available, to the point where pest and disease management has become extremely difficult and expensive, increasing pressure on agriculture and food production. There is an urgent need to explore and utilize alternatives. Due to their unique mode of action, photosensitizers may be able to control insect pests effectively, especially in combination with oil-based products, without the risk of resistance build-up. In this study, the efficacy of a mineral oil-based horticultural spray oil, PureSpray™ Green (PSG), and a sodium magnesium chlorophyllin photosensitizer formulation, SUN-D-06 PS, were evaluated and compared to a registered cyantraniliprole insecticide (as positive control) and a negative control against western flower thrips (WFT), Frankliniella occidentalis. In detached leaf ingestion assays, PSG at high concentration was more effective than low concentration, causing >70% WFT mortality, whilst SUN-D-06 PS + PSG caused higher mortality than cyantraniliprole after five days of feeding. The same combination was as effective as cyantraniliprole in the contact assay. In greenhouse pepper, the photosensitizer decreased the WFT more than mineral oil applied alone, whilst a combination treatment of SUN-D-06 PS + PSG was most effective, decreasing the WFT population to fewer than four WFT per plant. SUN-D-06 PS + PSG shows promise as a sustainable, economical way of controlling WFT, with the potential to be incorporated into existing integrated pest (and disease) management (IPM) programs with ease.

Fabrication, Characterization and In Planta Uptake of Engineered Surfactant Nanovesicles for the Delivery of the Biostimulant Sodium Copper Chlorophyllin.

Effective delivery of agrochemicals requires control over bioactive release kinetics coupled with effective penetration of the bioactive into plants. Herein, we demonstrate the fabrication of hybrid nanovesicles based on sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium bromide (CTAB) for enabling effective delivery of the biostimulant sodium copper chlorophyllin (Cu-chl) into plants. SDBS-CTAB nanovesicles exhibited a particle size of 107 nm with a well-defined spherical morphology, while modified formulations that included small fractions of the unsaturated dopant Span 80 yielded larger nanovesicles that were softer and more irregular in shape. All nanovesicles maintained high colloidal stability over >4 weeks and enabled sustained Cu-chl release, with the incorporation of Span 80 into the membranes enabling controllable acceleration of the release rate. Nanovesicle encapsulation improved the photostability of Cu-chl bioactive 3-4 × relative to that of free Cu-chl and enabled significant penetration of Cu-chl into the plant root without inducing any significant phytotoxicity.

Photosensitizer to the rescue: in planta and field application of photodynamic inactivation against plant pathogenic bacteria.

Control of plant pathogens using chemical and synthetic pesticides raises a major safety concern for humans and the environment. Despite the ongoing exploration of sustainable alternative methods, management practices for pathogens, especially bacteria, have remained almost unchanged over decades, whereby long-term uses of copper and antibiotics has led to widespread bacterial resistance in the field. Antimicrobial photodynamic inactivation (aPDI) of bacteria is emerging as an alternative strategy to combat resistant plant pathogens. aPDI utilizes light-sensitive molecules (photosensitizers) that upon illumination produce reactive oxygen species able to kill pathogens. Here we explore the potential of an anionic semisynthetic water-soluble derivative of chlorophyl (Sodium Magnesium Chlorophyllin: Mg-chl), as an antibacterial agent in planta, by simulating processes naturally occurring in the field. Mg-chl in combination with Na2EDTA (cell wall permeabilizing agent) was able to effectively inhibit Pseudomonas syringae pv. tomato DC3000 in vitro and in planta in both tomato and N. benthamiana. Notably, Mg-chl in combination with Na2EDTA and the common surfactant Morwet D-400 significantly reduced Xanthomonas hortorum pv. gardneri and Xanthomonas fragarie, respectively, in a commercial greenhouse trial against bacterial spot disease in tomato and in field experiments against angular leaf spot disease in strawberries.

Breaking the Rebellion: Photodynamic Inactivation against Erwinia amylovora Resistant to Streptomycin.

Global crop production depends on strategies to counteract the ever-increasing spread of plant pathogens. Antibiotics are often used for large-scale treatments. As a result, Erwinia amylovora, causal agent of the contagious fire blight disease, has already evolved resistance to streptomycin (Sm). Photodynamic Inactivation (PDI) of microorganisms has been introduced as innovative method for plant protection. The aim of this study is to demonstrate that E. amylovora resistant to Sm (E. amylovoraSmR) can be killed by PDI. Two photosensitizers, the synthetic B17-0024, and the natural derived anionic sodium magnesium chlorophyllin (Chl) with cell-wall-permeabilizing agents are compared in terms of their photo-killing efficiency in liquid culture with or without 100 µg/mL Sm. In vitro experiments were performed at photosensitizer concentrations of 1, 10 or 100 µM and 5 or 30 min incubation in the dark, followed by illumination at 395 nm (radiant exposure 26.6 J/cm2). The highest inactivation of seven log steps was achieved at 100 µM B17-0024 after 30 min incubation. Shorter incubation (5 min), likely to represent field conditions, reduced the photo-killing to 5 log steps. Chlorophyllin at 100 µM in combination with 1.2% polyaspartic acid (PASA) reduced the number of bacteria by 6 log steps. While PASA itself caused some light independent toxicity, an antibacterial effect (3 log reduction) was achieved only in combination with Chl, even at concentrations as low as 10 µM. Addition of 100 µg/mL Sm to media did not significantly increase the efficacy of the photodynamic treatment. This study proves principle that PDI can be used to treat plant diseases even if causative bacteria are resistant to conventional treatment. Therefore, PDI based on natural photosensitizers might represent an eco-friendly treatment strategy especially in organic farming.

Water-soluble anionic polychloramide biocides based on maleic anhydride copolymers.

Our goal was to develop film-forming polymers to extend the antimicrobial lifetimes of cleaned and disinfected surfaces. Antimicrobial polymers were prepared by first reacting poly(ethylene-alt-maleic anhydride) with isopropylamine, partially consuming the anhydride groups, followed by hydrolysis to give water-soluble, highly anionic polyamide PC3. Chlorination with NaOCl gave PC3Cl with oxidative chlorine contents up to 9 wt%. Dried, 5 µm thick, PC3Cl films, gave log 4 reductions in the concentration of Escherichia coli or Staphylococcus aureus exposed to films. A unique feature of the maleic anhydride copolymer platform was the ability to form covalent grafts to surfaces via anhydride reactions. PC3 solution was impregnated into cellulosic filter paper, heated to form ester linkages with cellulose, followed by chlorination with sodium dichloroisocyanurate dihydrate giving grafted PC3Cl. The treated paper (0.3 wt% PC3Cl) gave a log 4 reduction of E. coli concentration in 30 min.

Photodynamic Inactivation of plant pathogens part II: fungi.

The constantly increasing demand for agricultural produce from organic and conventional farming calls for new, sustainable, and biocompatible solutions for crop protection. The overuse of fungicides leading to contamination of both produce and environment and the emergence of plant pathogenic fungi that are resistant to conventional treatments warrant the need for new methods to combat fungal infections in the field. We here deliver the follow-up study to our research on the Photodynamic Inactivation (PDI) of plant pathogenic bacteria (Glueck et al. in Photochem Photobiol Sci 18(7):1700-1708, 2019) by expanding the scope to fungal pathogens. Both fungal species employed in this study-Alternaria solani and Botrytis cinerea-cause substantial crop and economic losses. Sodium magnesium chlorophyllin (Chl, approved as food additive E140) in combination with Na2EDTA and the chlorin e6 derivative B17-0024 holding cationic moieties serve as eco-friendly photoactive compounds. Effectiveness of the antifungal PDI was measured by inhibition of growth of mycelial spheres (average diameter 2-3 mm) after incubation with the photosensitizer for 100 min and subsequent illumination using a LED array (395 nm, 106.6 J cm-2). One hundred micromolar Chl combined with 5 mM Na2EDTA was able to successfully photokill 94.1% of A. solani and 91.7% of B. cinerea samples. PDI based on B17-0024 can completely inactivate A. solani at 10 times lower concentration (10 µM); however, for B. cinerea, the concentration required for complete eradication was similar to that of Chl with Na2EDTA (100 µM). Using a plant compatibility assay based on Fragaria vesca, we further demonstrate that both photosensitizers neither affect host plant development nor cause significant leaf damage. The plants were sprayed with 300 µL of treatment solution used for PDI (one or three treatments on consecutive days) and plant growth was monitored for 21 days. Only minor leaf damage was observed in samples exposed to the chelators Na2EDTA and polyaspartic acid, but overall plant development was unaffected. In conclusion, our results suggest that sodium magnesium chlorophyllin in combination with EDTA and B17-0024 could serve as effective and safe photofungicides.

A Plant Based Modified Biostimulant (Copper Chlorophyllin), Mediates Defense Response in Arabidopsis thaliana under Salinity Stress.

To date, managing salinity stress in agriculture relies heavily on development of salt tolerant plant varieties, a time-consuming process particularly challenging for many crops. Plant based biostimulants (PBs) that enhance plant defenses under stress can potentially address this drawback, as they are not crop specific and are easy to apply in the field. Unfortunately, limited knowledge about their modes of action makes it harder to utilize them on a broader scale. Understanding how PBs enhance plant defenses at cellular and molecular levels, is a prerequisite for the development of sustainable management practices utilizing biostimulants to improve crop health. In this study we elucidated the protective mechanism of copper chlorophyllin (Cu-chl), a PB, under salinity stress. Our results indicate that Cu-chl exerts protective effects primarily by decreasing oxidative stress through modulating cellular H2O2 levels. Cu-chl treated plants increased tolerance to oxidative stress imposed by an herbicide, methyl viologen dichloride hydrate as well, suggesting a protective role against various sources of reactive oxygen species (ROS). RNA-Seq analysis of Cu-chl treated Arabidopsis thaliana seedlings subjected to salt stress identified genes involved in ROS detoxification, and cellular growth.

Preventing the release of copper chlorophyllin from crop spray deposits on hydrophobic surfaces.

The chlorophyll derivative copper chlorophyllin and related chlorins have promise as environmentally friendly agricultural chemicals, however, spray application is hindered by the propensity of dried spray deposits to wash off leaf surfaces during rain or irrigation. HYPOTHESIS: Polyelectrolyte complexes formed between anionic carboxymethyl cellulose and cationic polyamidoamine-epichlorohydrin can prevent the release of copper chlorophyllin from dried spray deposits on leaf surfaces when exposed to water. EXPERIMENTS: Sessile drops on parafilm and containing polyelectrolyte complex and copper chlorophyllin or Brilliant Sulfaflavine, an anionic water-soluble dye, were dried to form deposits that were physical models for crop spray drop deposits on hydrophobic leaf surfaces. Larger buffer drops were placed on the dried deposits and the release of copper chlorophyllin or the dye were measured. FINDINGS: Copper chlorophyllin showed some immediate (burst) release upon exposure to buffer whereas the remainder was immobilized on the parafilm. By contrast, Brilliant Sulfaflavine displayed rapid release following square root time dependence, typical of a diffusion-controlled process. The unusual behavior of copper chlorophyllin is attributed to the presence of CuChl nanoparticles when dispersed in water. The nanoparticles are encased in the polyelectrolyte complex that adheres to parafilm. The fraction of the added copper chlorophyllin lost in the burst release can be controlled by varying the polyelectrolyte complex composition and concentration.

Save the crop: Photodynamic Inactivation of plant pathogens I: bacteria.

The ever growing world-population poses challenges concerning the need for more food free of pesticide residues. The most common means to control plant pathogens is through the application of pesticides, which raises concerns over safety for humans and the environment. Recently, Photodynamic Inactivation (PDI) of microorganisms using natural photosensitizers has shown itself to be a powerful tool to combat bacteria and fungi. This study investigates the efficacy of PDI against the Gram(+) bacterial plant pathogen Rhodococcus fascians and Gram(-) Xanthomonas axonopodis and Erwinia amylovora using two chlorin e6 derivatives as photosensitizers: anionic sodium magnesium chlorophyllin (Chl, approved as food additive E140) in combination with cell wall permeabilizing agents (Na2EDTA or Polyaspartic acid sodium salt (PA)) and B17-0024, a mixture of chlorin e6 derivatives with cationic moieties at physiological pH. Both photosensitizers show excellent efficacy against R. fascians, whereby B17-0024 is phototoxic at a one order of magnitude lower concentration than Chl (10 µM B17-0024: relative inactivation (r.i.) >7.5 × 106, 100 µM Chl: r.i. 2.2 × 106, illumination with 26.6 J cm-2, 395 nm). The phototreatment of Gram(-) bacteria with Chl requires the obligatory use of cell wall permeabilizing agents like Na2EDTA (X. axonopodis) or PA (E. amylovora) to induce significant killing (more than 7 log units at 100 µM). On the other hand, B17-0024 proves to be a highly effective photosensitizer inducing bacterial inactivation at very low concentrations (10 µM for R. fascians and X. axonopodis, 100 µM for E. amylovora) without additives. In summary, PDI using both the natural photosensitizer Chl in combination with cell wall permeabilizing agents is effective and environmentally friendly. As an alternative, B17-0024 is highly photoactive against all model strains tested - even without cell wall permeabilizing agents. The photodynamic approach based on chlorin e6 derivatives should add to the growers' toolbox as a preferred alternative for the control of phytopathogens.