Ali Ozcan

Summary: The stem and bulb nematode, Ditylenchus dipsaci , is a plant-parasitic nematode that causes significant losses in crop production. Management practices against plant-parasitic nematodes are commonly implemented in the soil. Brown mustard, Brassica juncea , has significant potential as an Integrated Pest Management (IPM) product against plant-parasitic nematodes like D. dipsaci , but its efficacy depends on optimising cultivation and extraction parameters. This study investigated the influence of planting time, plant part and extraction method on the profile of B. juncea ’s bioactive compound. Additionally, the ability of B. juncea to immobilise D. dipsaci in vitro was examined. Seeds were cultivated at four different times (23 September and 11 October 2022, 1 March 2023 and 17 April 2023) and the plants were separated into parts (roots, stems, leaves, flowers, and whole plant), then processed using sonication (SAE) or microwave + sonication (MSAE) extraction. Phenolic compounds and glucosinolates of extracts were analysed. Furthermore, an in vitro nematode immobility test of the extracts was carried out against D. dipsaci . Ferulic acid was identified as the predominant phenolic compound in both extraction methods, particularly in the leaves and whole plant, followed by sinapic acid in the leaves. Sinigrin and glucoraphanin were the predominant glucosinolates. The highest total glucosinolate content was found in whole plant samples from the March planting by SAE. While the extraction method did not alter the yield of bioactive compounds, it significantly impacted nematode mobility. The highest nematode immobility (mean: 76.8%) was achieved with extracts from the stems, leaves and whole plant of the autumn plantings (September/October) using the SAE method. These results provide a framework for maximising the biopesticidal potential of brown mustard, contributing to the development of effective, plant-based crop protection products.

Summary: The stem and bulb nematode, a globally distributed pest, primarily affects onions and garlic, causing significant yield losses. Chemical treatments are a practical and effective method for controlling plant-parasitic nematodes to protect high-value crops. However, to minimise environmental damage, chemicals of natural origin need to be investigated. Brown mustard ( Brassica juncea ) plant is an annual plant in the Brassicaceae family, which is rich in bioactive compounds called glucosinolates. These bioactive compounds play a major role in the defence of Brassica plants against biotic stress factors in nature. Based on this point, efforts to develop plant protection products from the brown mustard plant are ongoing. In this study, the most suitable pre-extraction and extraction conditions were determined to ensure the maximum preservation of glucosinolate content and biological activity, to process brown mustard on an industrial scale. For this purpose, steam treatment was applied before extraction at 3 bar (=3 × 10 ⁵ Pa) pressure for 1 and 3 min. Four different temperatures (room temperature, 40, 55 and 70°C) and two different methanol concentrations (70% and 80%) were tested to optimise glucosinolates extraction from brown mustard. It was found that sinigrin was the most abundant glucosinolate, with extraction yields 1184.4 ± 14.4 mg kg ⁻¹ at 55°C for 3 min, and 2624.2 ± 98.5 mg kg ⁻¹ at 70°C/70% MeOH for 1 min. However, nematode immobility was higher at 55°C/70% MeOH for 3 min. It was determined that the application of hot steam for 3 min at 3 bar pressure as a pre-extraction process provided optimal conditions for methanolic extraction. The results may contribute significantly to the development of potential plant protection products with further studies.

Huanglongbing (HLB, aka citrus greening), one of the most devastating diseases of citrus, has wreaked havoc on the global citrus industry in recent decades. The culprit behind such a gloomy scenario is the phloem-limited bacteria “Candidatus Liberibacter asiaticus” (CLas), which are transmitted via psyllid. To date, there are no effective long-termcommercialized control measures for HLB, making it increasingly difficult to prevent the disease spread. To combat HLB effectively, introduction of multipronged management strategies towards controlling CLas population within the phloem system is deemed necessary. This article presents a comprehensive review of up-to-date scientific information about HLB, including currently available management practices and unprecedented challenges associated with the disease control. Additionally, a triangular disease management approach has been introduced targeting pathogen, host, and vector. Pathogen-targeting approaches include (i) inhibition of important proteins of CLas, (ii) use of the most efficient antimicrobial or immunity-inducing compounds to suppress the growth of CLas, and (iii) use of tools to suppress or kill the CLas. Approaches for targeting the host include (i) improvement of the host immune system, (ii) effective use of transgenic variety to build the host’s resistance against CLas, and (iii) induction of systemic acquired resistance. Strategies for targeting the vector include (i) chemical and biological control and (ii) eradication of HLB-affected trees. Finally, a hypothetical model for integrated disease management has been discussed to mitigate the HLB pandemic.

Delivering active ingredients with high antibacterial efficacy at infected sites in plants is essential to reach global goals for food security and sustainable agriculture productivity. Engineering nanomaterials is a suggested approach to attain systemic delivery of antibacterial active ingredients, thereby improving the treatment efficacy and minimizing harmful effects related to leaching in the environment. Herein, surface defect engineering of nanotherapeutics is used as a new form of active ingredient for systemic antimicrobial action in the phloem. The nanoparticle-based formulation, called Zinkicide®, features a spherical particle composed of a zinc oxide (ZnO) core and zinc peroxide (ZnO₂) shell with a total diameter below 5 nm. This formulation exhibits significant efficacy to manage citrus huanglongbing (HLB) disease as seen by the decrease in severity of symptoms and the increase from ∼7% to 19% of medium and large fruits in HLB infected citrus groves, during field trials. Further analysis of the bacterial responses to Zinkicide® in situ indicates high potency at a concentration as low as 9–18 μg mL⁻¹ and biofilm growth inhibition at a concentration of 50 μg mL⁻¹. Nanoscale infrared spectroscopy reveals morphology and secondary structure changes of the bacterial membrane upon treatment. The origin of the changes is considered, based on the optical signatures of the nanoparticles, indicative of surface defects. These inform a theoretical description of the participation of a ZnO₂/ZnO surface containing a pair of missing O atoms in the production of reactive oxygen species (ROS). The key participation of defects in the antibacterial action is confirmed experimentally by the slow decrease in antibacterial efficacy as nanoparticles age in media with passivation effects on the surface. This study reveals the importance of size of the nanoparticle and nature of surface defects in the design of nanotherapeutics for targeted delivery and antibacterial activity.

Nanotechnology may be used to design more performant copper agrochemicals. Assessing the benefits and risks of the novel nano-products requires a good understanding of their fate in soil relative to other forms of copper. This study compares nine forms of Cu including nano and conventional Cu-based formulations, as well as their salt or bulk equivalents. Readily available Cu was extracted with 0.01 M CaCl₂ over a period of 29 days in three soils with contrasting properties. For eight out of nine formulations, available Cu decreased over time indicating that complexation of Cu with soil phases was the process determining the availability of Cu. Freshly suspended CuO nanoparticles were the only form where available Cu increased over time, indicating that dissolution was limiting availability. The complexation/dissolution kinetics were mainly determined by the soil pH, whereas both soil pH and OC% determined the equilibrium concentration of Cu in the soil solution, in line with results from equilibrium modelling. Overall, the fraction of readily available Cu was very low in all cases, reaching <1% of the Cu added within a couple of weeks. Our results also show that the soil moisture content at the time of Cu amendment can greatly affect the measured available Cu. According to the Cu formulation, amending dry soil resulted in up to 100% more readily available Cu than amending wet soil (pre-incubated at 50% of its maximum water holding capacity for two weeks). Overall, we show that different Cu formulations and soil pre-incubation regime can significantly impact the short term availability of Cu, which is essential to consider when conducting impact or efficacy assessments of (nano)agrochemicals.

A multifunctional surface, subsurface and systemic therapeutic (MS3T) formulation comprised of two bactericides, both didecyldimethylammonium chloride (DDAC) and a zinc (Zn)-chelate, was developed as an alternative to copper pesticides for crop protection. Agricultural grade chemicals were used to prepare MS3T formulations. Minimal inhibitory concentration (MIC) was determined to be tested in vitro against Xanthomonas alfalfae subsp. citrumelonis (herein called Xa), Escherichia coli (E. coli), and Pseudomonas syringae (Ps). Assessment of the phytotoxic potential was carried out on tomato under greenhouse conditions. Moreover, field trials were conducted during three consecutive years on grapefruit (Chrysopelea paradise) groves to evaluate efficacy against citrus canker (Xanthomonas citri subsp. citri), scab (Elsinoe fawcetti), and melanose (Diaporthe citri). In addition to disease control, improvements to both fruit yield and quality were observed likely due to the nutritional activity of MS3T via the sustained release of plant nutrients (Zn and nitrogen). Zn residues of leaf tissues were analyzed via atomic absorption spectroscopy (AAS) at various time points before and after MS3T foliar applications throughout the duration of the 2018 field trial. Field trial results demonstrated MS3T to be an effective alternative to copper (Cu)-based formulations for the control of citrus canker.

The development of bacterial tolerance against pesticides poses a serious threat to the sustainability of food production. Widespread use of copper (Cu)-based products for plant disease management has led to the emergence of copper-tolerant pathogens such as Xanthomonas perforans (X. perforans) strains in Florida, which is very destructive to the tomato (Solanum lycopersicum) industry. In this study, we report a hybrid nanoparticle (NP)-based system, coined Locally Systemic Pesticide (LSP), which has been designed for improved efficacy compared to conventional Cu-based bactericides against Cu-tolerant X. perforans. The silica core–shell structure of LSP particles makes it possible to host ultra-small Cu NPs (in vitro antimicrobial properties were characterized for LSP NPs with core diameters from 50 to 600 nm. A concentration of 4 μg mL⁻¹ (Cu): 1 μg mL⁻¹ (Quat) was found to be sufficient to inhibit the growth of Cu-tolerant X. perforans compared to 100 μg mL⁻¹ (metallic Cu) required with standard Kocide 3000. Wetting properties of LSP exhibited contact angles below 60°, which constitutes a significant improvement from the 90° and 85° observed with water and Kocide 3000, respectively. The design was also found to provide slow Cu release to the leaves upon water washes, and to mitigate the phytotoxicity of water-soluble Cu and Quat agents. With Cu and Quat bound to the LSP silica core–shell structure, no sign of phytotoxicity was observed even at 1000 μg mL⁻¹ (Cu). In greenhouse and field experiments, LSP formulations significantly reduced the severity of bacterial spot disease compared to the water control. Overall, the study highlights the potential of using LSP particles as a candidate for managing tomato bacterial spot disease and beyond.

Zinkicide is a systemic bactericidal formulation containing protein-size fluorescent zinc oxide-based nanoparticles (nano-ZnO). Previous studies have shown that Zinkicide is effective in controlling citrus diseases. Its field performance as an antimicrobial agent has been linked to the bioavailability of zinc ions (Zn²⁺) at the target site. It is therefore important to monitor Zn²⁺ release from Zinkicide so that application rates and frequency can be estimated. In this study, we present a simplistic approach designed to monitor Zinkicide nanoparticle dissolution rates in water and acidic buffer solutions using traditional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The evolution of nano-ZnO in the polyacrylamide gel scaffolds was studied by exciting the sample with UV light and detecting the fluorescence of nano-ZnO. Fluorescence intensities measured with this assay allowed for quantitative analysis of molecular weight changes of nano-ZnO in citrate buffer, a surrogate of citrus juice. Our results demonstrated that citrate buffer induced the greatest degradation of Zinkicide. Fluorescence intensity fluctuations were observed over time, indicating interactions of citrate with the surface of nano-ZnO. These findings provide a new approach to quantify the dissolution of nanoparticles in simulated environments, even when other analytical methods lack sensitivity because of the small size of the system (≈4 nm).