Nanoparticle encapsulation to enhance seed treatment efficacy against Fusarium graminearum

Nanoparticle encapsulation to enhance seed treatment efficacy against Fusarium graminearum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nanoparticle encapsulation to enhance seed treatment efficacy against Fusarium graminearum Fernando Marcos, Balaji Narasimhan, Adam Mullis, Gary Munkvold This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4401757/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The importance of seed treatments has increased rapidly in the past decade, mainly due to their high efficacy controlling early-season pests and diseases, and their limited environmental impact. Chemical seed treatments require a smaller amount of pesticide use and reduce environmental spread compared to foliar or soil applications; similarly, selection pressure for the development of resistance in the pest population is reduced. However, the rapid dissipation of seed treatment active ingredients after planting is associated with unpredictable duration of control, limiting the performance of seed treatment technology. Polyanhydrides are synthetic biodegradable polymers that can be used to deliver active ingredients or pharmaceuticals in pathological systems. They can provide a steady and sustained release of active compounds, enhancing the treatment of diseases caused by pathogens. Our study consists of experiments using polyanhydride nanoparticle-encapsulated fludioxonil and thiabendazole (two fungicides commonly used against Fusarium graminearum ) at different rates on maize and soybean. We employed both rolled-towel assays (simulating a seedborne infection) and delayed emergence assays (simulating a soilborne infection). In the rolled-towel assay, nanoparticle-encapsulated fungicides performed similarly to standard formulations. However, when emergence was delayed for one week by low temperature, nanoparticle-encapsulated fungicides showed superior control over standard formulations. For longer emergence delay treatments, nanoparticle and conventional fungicide formulations showed similar levels of control. Polyanhydride encapsulated seed treatments showed the potential to prolong effectiveness of active ingredients when emergence is delayed due to cold temperatures, a very common situation in temperate maize production areas, such as the American Midwest. Biological sciences/Plant sciences Physical sciences/Nanoscience and technology Fusarium graminearum seed treatment nanoparticles seedling diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Seedling diseases can have a critical effect on crop productivity, causing damage that persists throughout the season. Early infections of plant roots are harmful to plant yield due to their high impact on plant population per area. Seedling diseases frequently are the result of the interaction of multiple organisms, including fungi, oomycetes, bacteria, and nematodes. In some cases, there is a synergistic effect between organisms in different kingdoms; for instance, wounds caused by nematode feeding predispose plants to fungal or bacterial colonization 1 . Areas with a high incidence of soilborne pathogens are common, resulting in failure to emerge, wilting, yellowing, chlorosis, root rot, and stunted growth. Seed rots, seedling blights, and root rots typically are caused by several genera of fungi and oomycetes, including Fusarium, Aspergillus, Pythium, Rhizoctonia, Penicillium , and Trichoderma . These genera are soil inhabitants and can harm seed germination and seedling development. Several of these genera, such as Fusarium , also can be seedborne. Cold soil conditions can promote seedling disease by delaying seed germination and emergence, although warmer soils favor some seedling pathogens. Soil temperatures under 12°C are favorable for fungal colonization by most seedling pathogens but not for seed germination, which increases the severity of seedling diseases in early plantings. These conditions are more common in no-till and reduced tillage systems 2 . Fusarium is a member of the Ascomycota and is one of the most heavily studied genera of plant pathogenic fungi due to its enormous relevance to agriculture. Species of this genus cause substantial economic losses registered over the past centuries on crops such as maize, soybean, banana, and wheat. F. graminearum sensu stricto is the most important species of the Fusarium graminearum species complex (FGSC) and is the primary pathogen responsible for Fusarium head blight (FHB) of cereals and Gibberella stalk and ear rot of maize, which have been reported in most parts of the world 5 , 6 . Its importance is reflected in its numerous hosts and its impact on grain yield and quality, and production of mycotoxins that are hazardous to animals and humans 7 . F. graminearum can produce a diverse array of mycotoxins, such as deoxynivalenol, zearalenone, fusarins, culmorins, and others 5 , 8 . The management of this pathogen requires the employment of several methods: options include planting resistant or tolerant cultivars, use of fungicides as seed treatment and as spray during flowering, and biological control. Seed treatment is being widely used to manage a range of diseases and pests; sharp increases in the value of the seed treatment market are due to many factors, including farm safety, reduced amounts of active ingredient, and reduced exposure of non-target organisms, compared to foliar or soil applications of crop protection chemicals. Seed treatment, therefore, has environmental advantages over foliar application for diseases and pests that can be further refined through seed-applied technologies. Fludioxonil and Thiabendazole are two fungicides known to have a broad spectrum of action against fungi and are very effective against Fusarium graminearum . They are two components of the commercial formulation Maxim Quattro®, commercialized by Syngenta Crop Science. Their use as seed-applied fungicides is a primary management strategy to control seedling diseases; however, it is constrained by several factors, including rapid dissipation of active ingredients, limiting uptake in plant tissues, and absorption by target organisms. Therefore, our research investigates improving the efficacy of seed-applied active ingredients through the use of nanoparticle delivery vehicles. Nanoparticles (NPs) are grouped into different classes based on their chemistry, size, and shape, such as metal, ceramic, and polymeric. The main feature of NPs is a unique set of characteristics, including a high surface to volume ratio and high reactivity. Due to their unique chemistry, they are promising candidates for application in many fields, from the delivery of vaccines to synthetic chemicals 13 , 14 . The use of NP-based approaches in pharmaceutical science has shown successful improvement over conventional methods of drug and vaccine delivery. 13 Recent advancements have shown that the small size and unique physicochemical properties of NPs can reduce toxicity, enhancing release, improving solubility, and bioavailability when employed for drug delivery 13 . Polymeric NPs have been employed in research settings for the delivery of drugs and vaccines. Their size ranges from 10 to 500 nm, and special features as biodegradability and biocompatibility offer advantages for the delivery of active compounds. Due to their controlled and sustained release patterns, stealth, and modified surface, they can be used for active and passive delivery of bioactives. Polyanhydride NPs have been extensively studied as delivery platforms for small molecule and biologics in biomedical applications due to their surface erosion kinetics, which provide tunable release timelines from days to years based on polymer composition and device geometry 13 , 15 . They are biodegradable and biocompatible, making them excellent candidates for agricultural controlled release 16 . Alteration of the proportion between monomer and co-monomer provides a range of release duration, ranging from weeks to months 15 , 17 , 18 . In this study, we report the use of polyanhydride NPs to encapsulate two fungicides that are active against a widespread pathogen, F. graminearum . Experiments were conducted to address three primary hypotheses: i) NP encapsulation can significantly improve the control of seed decay in vitro ; ii) NP encapsulation of seed treatment fungicides can prolong the release of active ingredients; and iii) prolonged release can enhance the control of Fusarium graminearum . Our main objectives were to improve the efficacy of seed treatments by slowing the dissipation of active ingredients, especially under conditions of colder temperatures and delayed emergence of seedlings and evaluate the storability of NPs simulating seed industry storage conditions. RESULTS Rolled towel assays This assay was conducted twice, and for the first run (Fig. 1 ), there were significant differences among all treatments. F -test p -values were highly significant ( p < 0.0001) for all the variables measured, i.e., severity, plant weight, root length, and shoot length. Thiabendazole (TBZ) and NP-encapsulated thiabendazole (N-TBZ) were not significantly different from one another for all variables. Each variable followed the same trend, where N-TBZ was slightly better, but not significantly so. However, conventional fludioxonil (FLD) and NP-encapsulated fludioxonil (N-FLD) were significantly different for all variables, with N-FLD demonstrating improved control of F. graminearum . In the second run, the trend of results was different from the first one. In pairwise comparisons, there were no significant differences between conventional formulations and NP-encapsulated fungicides. However, in orthogonal contrasts between the two conventional ingredients combined and the two combined NP-encapsulated treatments, there were significant differences for disease severity and shoot length, with p -values of 0.0450 and 0.0154, respectively, showing better results with the NPs for these measurements. (Fig. 2 ). However, the results showed no significant differences for root length ( p = 0.9556) and plant weight ( p = 0.2053). Additionally, three complementary treatments were tested: reduced rates (50%) of NP-encapsulated fungicides (HN-TBZ, HN-FLD) and an “empty” NP formulation (N-EMPTY). The reduced rates were as effective as full rates, and N-EMPTY showed no difference compared to the untreated control, indicating that the effect of the treatment comes from the release of the encapsulated fungicide without any antimicrobial effect from the polymer itself. Delayed emergence assays In these experiments, we evaluated the efficacy of the seed treatments against soilborne F. graminearum under conditions simulating delayed emergence in cold soil. The plants were assessed after 28 days, following treatments that differed in temperature sequence, to evaluate whether NP encapsulation could prolong the effects of the active ingredients under different environments. For the first trial (maize), there were similar trends of results through different seed treatments and environmental treatments. In the first environment, without delayed emergence (constant temperature of 24°C), all pairwise comparisons showed no significant differences among seed treatments. Generally, NP-encapsulated thiabendazole and conventional thiabendazole (N-TBZ and TBZ, respectively) provided slightly superior protection than conventional fludioxonil and nano-fludioxonil (FLD and N-FLD, respectively) but without statistical differences for all measurements. With a 1-week delay in emergence (7 days at 10°C followed by 21 days at 24°C), pairwise comparisons showed statistical differences among seed treatments only for plant weight (p = 0.0017), where N-TBZ had statistically higher plant weight compared to FLD (p = 0.0099). For all remaining measurements, NP-encapsulated treatments reached slightly higher values, however, no statistical differences were observed. In the third environment, with a 2-week delay, N-TBZ was significantly better than other treatments for shoot length, plant weight, and root length. For severity, TBZ was the best treatment, statistically different from N-FLD and FLD, and similar to N-TBZ. In the last environment when emergence was delayed for three weeks, there were no significant differences among seed treatments, except against the “Untreated” control. When employing orthogonal contrasts combining conventional ingredients (TBZ and FLD), NP-encapsulated treatments (N-TBZ and N-FLD), and “Untreated”, clearer trends were evident (Fig. 3 ). For severity and root length, there were no significant differences. However, for plant weight (p = 0.0278) and shoot length (p = 0.0354), the NP-encapsulated treatments outperformed the conventional fungicide treatments for the 1-week emergence delay treatment only. The experiment was then repeated using soybean for a second run (Fig. 4 ). The results were similar to what was observed in the first run, where the level of protection provided by NP encapsulation or commercial treatments was comparable. However, when using contrasts once again, the same results were observed in the second environment. With a 1-week delay in emergence, NP formulations showed improved performance against F. graminearum in comparison with commercial treatments in two variables, root length (p = 0.0260) and plant weight (p = 0.0598). Storage assays In the storage assay, the conventional formulations showed superior control of the pathogen compared to the NP-encapsulated formulations for all sampling dates, according to the orthogonal contrasts (Fig. 5 ). Conventional formulations were consistent in maintaining low disease severity across sampling dates, without any loss of efficacy through the whole duration of the experiment. NP-encapsulated formulations showed a decrease in efficacy only between the first and second sampling dates ( p = 0.0002), and then consistent efficacy until the end. The water control showed a small increase in severity until the third month when it started to decrease. Severity of water control for the last sampling date was significantly different from the first ( p = 0.0068), second ( p < 0.0001) and third ( p < 0.0001) sampling dates. For root length, conventional formulations were very consistent until the fourth month in storage, with a loss in efficacy between the fourth sampling date and the last sampling date ( p = 0.0009). For shoot length, more variation was observed. Conventional formulations were not consistent during the whole experiment. The second, fourth, and fifth sampling dates had statistically inferior results compared to the first and third months. For plant weight, the analyses within treatments showed a steady effect of conventional formulations until the third month in storage, with statistical decreases in the fourth and fifth months. Fungicide Release Kinetics from Nanoparticles A fungicide release kinetics assay was performed to evaluate the maximal release rate of these payloads from nanoparticles in vitro in well-buffered, aqueous conditions with regular organic solvent washes to maintain infinite sink for payload release (Fig. 6 C-D). Release kinetics data were collected for the two different types of NPs used; F2–20:80 CPTEG: CPH NPs containing 20% fludioxonil, and F3–20:80 CPTEG: CPH NPs containing 15% thiabendazole. The particles containing fludioxonil exhibited a burst of fungicide release (~ 80% of total fludioxonil mass) within the first 24 hours. The remaining fludioxonil appeared to be released slowly through the end of the experiment. For thiabendazole loaded NPs, the trend was similar; however, the release occurred in a slower pattern. These NPs exhibited a burst of 60% after 24 hours, followed by a steady release of approximately 30% more until the end. There were no significant differences among the three different temperature treatments (i.e., 10°C, 15°C, and 24°C). We measured relatively high encapsulation efficiencies of 75.1 ± 1.3% for fludioxonil and 90.0 ± 1.8% for thiabendazole, yielding effective loadings of 15.0% and 13.5 ± 0.3% (w/w), respectively. DISCUSSION The results of this study showed that NP encapsulation of fungicides could provide some advantages for the control of seedborne and soilborne inoculum of F. graminearum . In both delayed emergence experiments, NP-encapsulated formulations resulted in better disease management than conventional formulations when emergence was delayed for 1-week by low temperature. However, in other environments, NP-encapsulated formulations and conventional formulations were statistically similar. These results showed a potential advantage to NP encapsulation when emergence was delayed for one week. This behavior could be related to the slow-release kinetics enabled by the specific polymer composition used. These results suggest that the NPs are prolonging the release of their payload(s) and hence improving the control of the F. graminearum pathogen during the first week of delayed emergence. The results may be enhanced using a mixture of different copolymer compositions and ratios together in the same slurry to target different windows of active ingredient release. Moreover, these experiments demonstrated the importance of seed-applied fungicides against seedling diseases. In both repetitions of the delayed emergence assay (first run using maize and second run with soybeans), the inoculated control had zero emergence when emergence was delayed for two and three weeks, while fungicide-based treatments were statistically superior. For the rolled-towel results, NP-encapsulated formulations were superior only in the first experiment, which is related to the poor control provided by FLD only in the first run. In contrast, TBZ and N-TBZ provided similar results. In the second experiment, when FLD was similar to N-FLD, the orthogonal contrasts showed no statistical differences between “nano” and “commercial”. Additionally, the severity of the inoculated and water-treated control of the second experiment was lower than in the first experiment. The lower disease pressure seen in the second run could explain the lack of significant differences in the second experiment. If the primary advantage of NP encapsulation is to prolong the efficacy of the seed treatment, this benefit would not be evident in the rolled-towel assays, which only assess control in the first week of germination and seedling growth. The rolled-towel assays demonstrated that NP-encapsulated formulations can work as well or better than conventional formulations, even with reduced rates. The half-rate-NP-encapsulated formulations provided similar results compared to the full rate of NP-encapsulated formulations and the standard rate of conventional formulations. Similarly, Washington et al. 19 observed equivalent levels of control using half rates. The results from the storage assay showed a relatively stable efficacy of NP-encapsulated formulations. For most of the variables measured, some efficacy was lost only after four or five months in storage, similar to the conventional formulations. Although maize seed is treated generally within one year before planting, this window of safe storage could be improved by more studies on copolymer composition. The release studies showed no difference in the time-release profile for both chemicals under different temperatures. These in vitro results indicated that NP encapsulation did not prolong active ingredient release over the desired time frame (1 to 3 weeks). However, the conditions of the in vitro release study likely do not adequately represent soil conditions. Additional polyanhydride copolymer formulations or combinations of the formulation(s) could be tested. Robust methods for measuring the time release of active ingredients in soil need to be developed. The results of our experiments in delayed-emergence environments might be related to interaction(s) between the pathogen and plant development. Lower temperatures can provide a competitive advantage to the fungus rather than the plant, and this interaction should be targeted by the employment of a seed treatment that can protect late-emerging seedlings. The results showing similar release profiles across temperatures can be advantageous to promote enhanced protection and prevent seed decay under various soil temperatures, including delayed emergence scenarios. This mechanism could be especially beneficial with a prolonged-release system to avoid the rapid dissipation of the active ingredient before germination. Prolonging the efficacy of seed treatments is a significant challenge that could greatly increase the value of this crop protection tactic. We hypothesized that the employment of NPs could address the limitation of rapid dissipation of active ingredients and promote a prolonged release, achieving enhanced protection against pests. Our results partially support this hypothesis; NP encapsulation displayed improved control for a specific environment (1-week delay) when tested with two different crop species. Further research should pursue the potential to enhance the prolonged effect to levels that could increase seedling protection for several weeks after planting. A better understanding of the chemical compatibility between the polymers and the active ingredients, as well studies with variable ratios of polyanhydride monomers, could bring additional flexibility (and benefits) to the pattern of release of a specific chemical. It is essential to understand the release patterns for different copolymer compositions and their behavior when applied in soil conditions. Additionally, other different types of chemicals should be used to understand which active ingredients are most compatible when using polyanhydride polymers. MATERIALS AND METHODS Seed preparation Before each trial, seeds were externally disinfested by a two-step process. Seeds were soaked in a solution of bleach and then rinsed using deionized sterile water. For maize, we used a solution of 10% bleach and a soaking period of five minutes. After the seeds were removed from the solution, seeds were rinsed at least five times until the bleach was removed completely. For soybean, the external disinfestation was performed with reduced bleach concentration (5%) and exposure period (1 minute), due to the high sensitivity of soybean seeds to physical and chemical damage. As bleach is commonly used to check the damage on soybean seed coats, we used this step to remove damaged seeds, such as swelled and cracked ones 20 . Finally, seeds were placed in labeled trays with paper towels inside a biosafety cabinet to dry before the next steps. Isolate and inoculum preparation The isolate of F. graminearum used for preliminary and primary experiments was FG27 (isolated in Iowa, USA). It was grown for each experiment from stored silica gel pellets, dated from December 2016. We used two forms of inoculation: a spore suspension for rolled-towel assays, simulating a seedborne infection, and infested millet for experiments in field soil, simulating soilborne inoculum. The spore suspension was prepared by reviving the fungus from silica pellets on potato dextrose agar (PDA) (BD, Franklin Lakes, New Jersey) for ten days at ambient temperature (24–26°C). To promote sporulation, mycelial plugs from PDA cultures were transferred to Spezieller Nährstoffarmer Agar (SNA) 21 plates. After approximately 15 days, spores from SNA plates were collected with sterile deionized water and filtered through sterile cheesecloth. The concentration was adjusted to reach 1 x 10 5 spores/mL using a hemocytometer. The inoculation was finally performed by soaking batches of 100 seeds in 30 ml of a spore suspension for five hours. All containers were placed on a shaker at ambient temperature at 92 r.p.m. For soybeans, the soaking period was reduced to one minute. After the inoculation, seeds were dried on paper towels inside a biosafety cabinet for twelve hours before treatment. Alternatively, to prepare inoculum for studies using field soil, we infested the soil using colonized millet. We pasteurized the millet by autoclaving it for 90 min at a temperature of 121°C. The millet remained at a temperature above 80°C for more than one hour after the autoclave cycle was finished. This process was done twice on two consecutive days. On the second day, after letting the bags containing the millet cool down, PDA plates containing 15-day-old F. graminearum were blended and poured into aerated bags containing autoclaved millet seeds in a one-plate-per-bag ratio. These bags were kept in a growth chamber for 15–20 days until the mycelial growth covered all millet kernels. For the non-inoculated control treatment, the colonized millet was autoclaved twice before mixing with soil. Millet inoculum and control millet samples were checked for viable F. graminearum by culturing on PDA. Millet was mixed into pasteurized field soil for all treatments, including controls. The percentage of inoculum in the soil (volume/volume) was 5%. Seed treatment application The traditional active ingredients chosen to be used in this research were fludioxonil and thiabendazole. While the dosage of fludioxonil is only 0.0065 mg/kernel (3.32% of the total slurry), thiabendazole has a much higher rate, 0.05 mg/kernel (26.5%). The recommended dosages of each active ingredient were used with the addition of a plantability polymer. The polymer used was Flo Rite® 1197 (BASF) following the manufacturer recommendation, approximately 0.44 ml per kernel. All compounds were then mixed with water (15 µL per seed) and vortexed before application on seeds. Similarly, for the NP formulations, we used the equivalent mass of the active ingredients. However, the total weight of active ingredient was adjusted to consider encapsulation efficiency and the total loading of the nanoparticles to ensure that the same active ingredient dosage would be delivered to each seed (Table 1 ). NP treatments were formulated with a surfactant, Span80, but without the additional plantability polymer. Before the application, treatments were sonicated using a VCX 130PB sonicator with a CV138 tip (Sonics and Materials, Newton, CT) at 30% amplitude to disperse the NPs into suspension. Table 1 Treatments employed in rolled-towel and delayed-emergence assays. All treatments included F. graminearum inoculation or infestation, except for CONTROL1. Treatment codes Treatment description Non-inoculated Water and plantability polymer without F. graminearum inoculation Untreated Water and plantability polymer with F. graminearum inoculation FLD Fludioxonil (0.0065 mg/kernel), water and plantability polymer N-FLD Polyanhydride nanoparticle encapsulated fludioxonil (0.0065 mg/kernel), water, and Span80 TBZ. Thiabendazole (0.05 mg/kernel), water and plantability polymer N-TBZ Polyanhydride nanoparticle encapsulated thiabendazole (0.05 mg/kernel), water, and Span80. Controls with no seed treatment with and without F. graminearum inoculation were added as well (Table 1 ). The treatments were applied by adding the slurry into plastic bags containing the specific number of seeds. After the application, the seeds were rubbed together to apply the solution evenly over the seeds. Seeds were dried again within a biosafety cabinet for 24 hours after treatment. Besides the treatments listed in Table 1 , additional treatments were included only in the second run of the rolled-towel assay. They consist of an empty NP formulation (N-EMPTY), and half rates of each fungicide, i.e., HN-TBZ (half dosage of N-TBZ) and HN-FLD (half dosage of N-FLD). Rolled towel assay To evaluate efficacy against seedborne inoculum using full and reduced rates of active ingredients, we conducted rolled-towel assays using rolled germination paper as described by Ellis et al . 22 , except that seeds were inoculated before “planting,” as already described. These assays were conducted using maize seeds under favorable conditions of germination (23–25°C) for seven days. For each replication, 15 seeds were placed on top of two layers of germination paper (Anchor Paper Co., St. Paul, MN) moistened with sterile water. A third sheet was placed on seeds, and they were rolled and placed inside a plastic bag containing sterile water. Each bag was placed inside buckets covered with a second bag to maintain moisture inside the bucket. They stayed inside a growth chamber at 24°C for one week, and then seven-day-old seedlings were analyzed for root length, shoot length, disease severity, and total fresh weight. Disease severity was measured on a one-to-five scale 23 , with five being the most severe disease, and one being completely healthy. Results of severity, root length, shoot length, and plant weight were analyzed for analysis of variance and mean separation using Tukey’s Honest Significant Difference test at p ≤ 0.05. Planned contrasts were employed as well to compare overall differences between nanoparticle encapsulation and standard formulations. Delayed Emergence assay In this assay, we assessed the efficacy of fungicides and their NP-encapsulated counterparts under conditions simulating an emergence delay due to cold temperatures in pathogen-infested soil. To study the prolonged-release effect, we conducted assays with a delayed-emergence scenario, in which non-inoculated seeds were planted in pasteurized soil infested with F. graminearum , and then subjected to a low temperature, unfavorable for germination, before a higher temperature, favorable for germination. These treatments simulated early planting in cold soils. As the rapid dissipation of active ingredients is one of the most important limitations of seed treatments, we employed this assay to evaluate if, in a delayed emergence situation, NP-encapsulated fungicides would prolong the release of active ingredients, and hence improve and extend protection from the pathogen. Treatments consisted of four different temperature combinations, varying in the number of weeks of emergence delay (weeks after planting at 10° C). Treatments ranged from no-emergence delay to a three-week emergence delay (Table 2 ). This assay was repeated twice: the first run was conducted using maize, and the second soybean. Table 2 Delayed emergence treatments for infested soil assay. Treatments Description No delay 24°C for four weeks 1-week delay 10°C for one week followed by three weeks at 24°C 2-week delay 10°C for two weeks followed by two weeks at 24°C 3-week delay 10°C for three weeks followed by one week at 24°C To assure correct temperatures for the duration of the experiments, data loggers (Spectrum Technologies, Aurora, IL) were placed in the growth chambers before the start to facilitate the temperature adjustment. Moreover, they remained through the entire experiment to monitor temperature fluctuations. Plants were uprooted after 28 days, and assessed for disease severity, root and shoot length, and plant weight, identically to measurements used for the rolled-towel assays. Data collected were analyzed by analysis of variance (ANOVA) in R version 3.6.1. Analysis of all main effects was conducted using all treatment combinations with Tukey’s pairwise adjustments. Mean separations were done using Tukey’s Honest Significant Difference test at p ≤ 0.05, and orthogonal contrasts, where we combined the means of traditional fungicides versus nanoparticle encapsulated versions using Tukey’s standard adjustment. Storage assay For the storage assay, maize and soybean seeds were inoculated, treated, and then stored in a cold room simulating the conditions employed in the industry (10°C and 50% RH 24 . Batches of seeds were inoculated using a spore suspension and then treated following the procedures mentioned above. Approximately 180 seeds per treatment, per sampling date, were placed inside envelopes (8 cm x 14 cm) and then arranged in larger envelopes (23 cm x 30 cm) and stored in the cold room. Seeds were sampled every month for five months in storage. At each sampling date, continued efficacy of treatments was assessed by rolled-towel assays already described. Seedlings were analyzed for root length, shoot length, disease severity, and total fresh weight. Disease severity was measured on a one-to-five scale, with five being the most severe disease, and one being completely healthy (Fig. 1 ). Results were analyzed for analysis of variance and mean separations using Tukey’s Honest Significant Difference test at p ≤ 0.05. Nanoparticle synthesis and characterization CPTEG and CPH monomers and 20:80 CPTEG:CPH copolymer were synthesized as described previously 25 , 26 , 27 , 28 . Fungicide-loaded nanoparticles were synthesized via flash nanoprecipitation by dissolving fungicide and copolymer in methylene chloride at 20 mg/mL, sonicating to homogenize, then rapidly pouring this solution into a -10°C pentane antisolvent bath at a solvent/antisolvent ratio of 1:250 (v/v) 29, 30 . The nanoparticles were collected by vacuum filtration and imaged via scanning electron microscopy (SEM, FEI Quanta 250, Hillsboro, OR). The resulting SEM micrographs were analyzed using the ParticleSizer plugin script in Fiji to calculate particle size distributions 31 . Additional experiments were conducted to characterize the release of active ingredients from NP encapsulation at three different temperatures and to assess the storability of NP-encapsulated seeds in typical storage conditions. To understand the release kinetics of the NP encapsulated formulations used in our experiments, we used HPLC (high-performance liquid chromatography) to estimate the mass of the fungicides released over time in an aqueous solution. In order to understand the release trends under different potential soil conditions, we performed this assay under three different temperatures, 10°C, 15°C, and 24°C. The process started by weighing 9–11 mg of each nanoparticle formulation in triplicate and suspending the particles in 0.5 mL of PBS (phosphate-buffered saline solution), pH 7.4. At each timepoint, the particles were centrifuged, the supernatant was collected, and particles were washed with acetonitrile to extract the remaining, non-dissolved fungicide. Fresh PBS was added, and the particles were re-dispersed by sonication. At the end of the aqueous (PBS) release, 40 mM NaOH was applied to rapidly degrade the remaining particles, allowing estimation of the remaining encapsulated fungicide. Released fludioxonil was quantified via RP-HPLC-UV/Vis (1200 series, Agilent Technologies, Santa Clara, CA, USA) in a constant solvent mix of 60% acetonitrile and 40% water, pH 2.27 (normalized with o-phosphoric acid) measuring absorbance at 280 nm, with expected elution time of 2 min. Similarly, thiabendazole was quantified via RP-HPLC-UV in constant solvent mixture of 40% acetonitrile 0.1% trifluoroacetic acid and 60% water 0.1% trifluoroacetic acid measuring absorbance at 254 nm, with expected elution time of 1.4 min. For each timepoint, the mass of drug released in PBS and acetonitrile samples were added to reflect maximal release rate. The encapsulation efficiency was calculated by dividing the total measured released drug mass by the nominal encapsulated mass. Declarations Acknowledgements We would like to thank the Iowa State University Presidential Interdisciplinary Research Seed Grant Program and the Iowa State University Seed Science Center for funding this research project and the staff of the Iowa State Seed Pathology Laboratory for their assisting in all of the steps of this research. The authors declare no conflict of interest. Author Contributions Fernando Marcos conducted and analyzed all efficacy experiments, made figures 1-5, and wrote the main manuscript text. Adam Mullis conducted and analyzed release kinetics experiments and made figure 6. Gary Munkvold and Balaji Narasimhan developed the original concept, contributed to design of the experiments, advised and consulted for all steps of the study, and revised the manuscript. Competing Interests The authors declare that they have no competing interests. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References da Silva, M.P., Tylka, G.L. & Munkvold, G.P. Seed treatment effects on maize seedlings coinfected with Fusarium spp. and Pratylenchus penetrans. Plant Dis. 100, 431–437 (2016). Robertson, A. & Munkvold, G.P. Check general root and mesocotyl health when assessing corn stands. Iowa State University Integrated Crop Management Newsletter. http://www.extension.iastate.edu/CropNews/2009/0519robertson.htm (2009) Munkvold, G.P. & O’Mara, J.K. Laboratory and growth chamber evaluation of fungicidal seed treatments for maize seedling blight caused by Fusarium species. Plant Dis. 86, 143–150 (2002). Arias, M. M. D., Leandro, L. F. & Munkvold, G. P. Aggressiveness of Fusarium species and impact of root infection on growth and yield of soybeans. Phytopathology. 103, 822–832. https://doi.org/10.1094/PHYTO-08-12-0207-R (2013). Munkvold, G.P. Fusarium species and their associated mycotoxins in Mycotoxigenic Fungi: Methods and Protocols, Methods in Molecular Biology (eds Moretti, A., Susca, A.) 51–106 (Humana Press, 2017). Agrios, G. N. Plant Pathology 534–538 (Elsevier Academic Press, 2005). Rubella, S. G. & Kistler, H.C. Heading for disaster: Fusarium graminearum on cereal. Mol. Plant Pathol. 5 (6), 515–525 (2004). Schmale III, D. G. & Bergstrom, G. C. Fusarium head blight in wheat. Plant Health Instr. 10.1094/PHI-I-2003-0612-01 (2003, updated 2010). Kebede, A. Z., Woldemariam, T., Reid, L.M. & Harris, L.J. Quantitative trait loci mapping for Gibberella ear rot resistance and associated agronomic traits using genotyping–by–sequencing in maize. Theor Appl Genet. 129,17–29 (2016). Munkvold, G. P. & White, D. G. Compendium of Corn Diseases, Fourth Edition. https://doi.org/10.1094/9780890544945.002 (The American Phytopathological Society, 2016) Munkvold, G. P. Seed pathology progress in academia and industry. Annu. Rev. Phytopathol. 47, 285–311(2009). Broders, K.D., Lipps, P.E., Paul, P.A. & Dorrance, A. E. Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling disease in Ohio. Plant Dis. 91, 1155–1160 (2007). Bhatia, S. Natural polymer drug delivery systems: Nanoparticles, plants, and algae in Natural Polymer Drug Delivery Systems: Nanoparticles, Plants, and Algae. https://doi.org/10.1007/978-3-319-41129-3 (Springer International Publishing, 2016). Khan, I., Saeed, K., & Khan, I. Nanoparticles: Properties, applications and toxicities. Arab. J. Chem. https://doi.org/10.1016/j.arabjc.2017.05.011 (2017). Phanse, Y. et al. Cellular internalization mechanisms of polyanhydride particles: Implications for rational design of drug delivery vehicles. J. Biomed. Nanotechnol. 12 (7), 1544–1552. https://doi.org/10.1166/jbn.2016.2259 (2016). Vela-Ramirez, J. E. et al. Safety and Biocompatibility of Carbohydrate-Functionalized Polyanhydride Nanoparticles. The AAPS Journal 17, 256–267 (2015). Berkland, C., Kipper, M. J., Narasimhan, B., Kim, K., & Pack, D. W. Microsphere size, precipitation kinetics and drug distribution control drug release from biodegradable polyanhydride microspheres. J. Control. Release. 94 (1), 129–141. https://doi.org/10.1016/j.jconrel.2003.09.011g (2004). Binnebose, A. M. et al. Polyanhydride nanoparticle delivery platform dramatically enhances killing of filarial worms. PLOS Negl. Trop. Dis. 9 (10). https://doi.org/10.1371/journal.pntd.0004173 (2015). Washington, L. A. Utilization of polyanhydride nanoparticle encapsulated fungicide seed treatments against seedborne and soilborne Fusarium graminearum on maize. Master’s Thesis, Iowa State Univeristy. https://lib.dr.iastate.edu/etd/15639 (2017). VanUtrecht, D., Bern, C. J., & Rukunudin, I. H. Soybean mechanical damage detection. Appl. Eng. Agric. 16 (2), 137–14. https://doi.org/10.13031/2013.5059 (2000). Leslie, J.F., & Summerell, B.A. The Fusarium Laboratory Manual (Blackwell Publishing, 2008). Ellis, M.L., Broders, K.D., Paul, P.A., Dorrance, A.E. Infection of soybean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions. Plant Dis. 95, 401–407 (2011). Cruz Jimenez, D. Soybean root rot caused by Fusarium oxysporum and Fusarium graminearum: interactions with biotic and abiotic factors. Doctoral dissertation, Iowa State University. https://lib.dr.iastate.edu/etd/15505 (2017). Harrington, J. F. Thumb rules of drying seeds. Crops & soils. 13 (1), 16–17 (1960). Shen E, Pizsczek R, Dziadul B, Narasimhan B. Microphase separation in bioerodible copolymers for drug delivery. Biomaterials. 22(3):201–10 (2001). Conix, A., Poly[l,3-Bis(p-carboxyphenoxyj-propane anhydride], Macromolecular Synthesis, Vol. 2, (Ed. J.R. Elliot) Wiley, NYC 1966. pp 95–99. Torres MP, Vogel BM, Narasimhan B, Mallapragada SK. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of Biomedical Materials Research - Part A. 2006;76(1):102–10. Mullis AS, Broderick SR, Binnebose AM, Peroutka-Bigus N, Bellaire BH, Rajan K, et al. Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release. Molecular Pharmaceutics. 2019;16(5):1917–28. Mullis AS, Broderick SR, Binnebose AM, Peroutka-Bigus N, Bellaire BH, Rajan K, et al. Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release. Molecular Pharmaceutics. 2019;16(5):1917–28. Mullis, A. S.; Broderick, S. R.; Phadke, K. S.; Peroutka-Bigus, N.; Bellaire, B. H.; Rajan, K.; Narasimhan, B. Data Analytics-Guided Rational Design of Antimicrobial Nanomedicines against Opportunistic, Resistant Pathogens. Nanomedicine: NBM 2023, 48, 102647. https://doi.org/10.1016/j.nano.2022.102647 . Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. Fiji: An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9, 676 – 682. Wagner T, Eglinger J. ParticleSizer. 2017. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4401757","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":304722210,"identity":"3ed3192c-8c89-42ab-a09e-7bbf053326ff","order_by":0,"name":"Fernando Marcos","email":"","orcid":"","institution":"Iowa State University","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"","lastName":"Marcos","suffix":""},{"id":304722212,"identity":"38cb1dec-5423-40af-8a27-cfa07a21f367","order_by":1,"name":"Balaji Narasimhan","email":"","orcid":"","institution":"Iowa State University","correspondingAuthor":false,"prefix":"","firstName":"Balaji","middleName":"","lastName":"Narasimhan","suffix":""},{"id":304722213,"identity":"7106f074-9267-4076-8bdd-6ed731bc8df0","order_by":2,"name":"Adam Mullis","email":"","orcid":"","institution":"Iowa State University","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Mullis","suffix":""},{"id":304722214,"identity":"4f76e3d3-133e-473c-b52b-1e65110957ed","order_by":3,"name":"Gary Munkvold","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYHACZiC2YOxnRogYEKNFgnFmM5A6AFFNpJYNB4jVojsj97AxT4WE7ObjzI8/f6j5I8fA3rxNAp8Wsxt5yck8ZySMtx1mM5M4cMzAmIHnWBkBLTnGB2e2SSRuO8xgxnCAzSCxQSLHjDgtm5vZP3848M+gvkH+DWEtCR+BWjYw8xhIHGwzSGCQ4CGg5cwbY4MPQL/MOMxTJnG2z9iwjSet2AKvluM5xhIJFTay/f3HN3+o+CYnz89+eOMNfFowARtpykfBKBgFo2AUYAMAYUJH1hTsTycAAAAASUVORK5CYII=","orcid":"","institution":"Iowa State University","correspondingAuthor":true,"prefix":"","firstName":"Gary","middleName":"","lastName":"Munkvold","suffix":""}],"badges":[],"createdAt":"2024-05-10 15:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4401757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4401757/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56934599,"identity":"88fd0e3d-8be6-40a6-93ba-4d950856834a","added_by":"auto","created_at":"2024-05-22 10:37:33","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":458443,"visible":true,"origin":"","legend":"\u003cp\u003eAverage of severity (A), plant weight (B), shoot length (C), and root length (D) for the first run of the rolled-towel assay of the four treatments and two controls (Untreated and Non-inoculated). Different treatments’ letters indicate significant differences by Tukey’s Honest Significant Difference test at 0.05. Error bars indicate standard error at 95% confidence intervals.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/cf3b5e613b05c1ce4bc0876a.jpeg"},{"id":56935019,"identity":"fa4ea507-21c9-40f4-b739-d683ddfa57eb","added_by":"auto","created_at":"2024-05-22 10:45:33","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345479,"visible":true,"origin":"","legend":"\u003cp\u003eAverage of severity (A), plant weight (B), shoot length (C), and root length (D) for the second run of the rolled-towel assay. Statistical analyses were conducted using orthogonal contrasts comparing nanoparticle-encapsulated formulations (N-TBZ and N-FLD), conventional formulations (TBZ and FLD), and a control (Untreated). Different treatments’ letters indicate significant difference using contrasts and standard Tukey’s adjustment at 0.05. \u003cem\u003eP-values \u003c/em\u003edisplayed are from the comparison between nanoparticle-encapsulated formulations versus\u003cem\u003e \u003c/em\u003econventional only. Error bars indicate standard error at 95% confidence intervals.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/6d2a9fea464bfae63f03aefb.jpeg"},{"id":56934602,"identity":"2b9fde93-0eb4-4f22-94c5-16a0cf0a514c","added_by":"auto","created_at":"2024-05-22 10:37:34","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":349708,"visible":true,"origin":"","legend":"\u003cp\u003eAverage plant weight (A) and shoot length (B) of the first run of the delayed-emergence assay. Statistical analyses were conducted using orthogonal contrasts comparing nanoparticle-encapsulated formulations (N-TBZ and N-FLD), conventional formulations (TBZ and FLD), and a control (Untreated). Different treatments’ letters indicate significant difference using contrasts and standard Tukey’s adjustment at 0.05. \u003cem\u003eP-values \u003c/em\u003edisplayed are from the comparison between nanoparticle-encapsulated formulations versus\u003cem\u003e \u003c/em\u003econventional only in the “1 week” environment. Error bars indicate standard error at 95% confidence intervals.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/90066ff72ffe3a576e924169.jpeg"},{"id":56934603,"identity":"2788ee98-82e5-448e-8c3f-d8083cdd0a96","added_by":"auto","created_at":"2024-05-22 10:37:34","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":352699,"visible":true,"origin":"","legend":"\u003cp\u003eAverage root length (A) and plant weight (B) of the second run of the delayed-emergence assay. Statistical analyses were conducted using orthogonal contrasts comparing nanoparticle-encapsulated formulations (N-TBZ and N-FLD), conventional formulations (TBZ and FLD), and a control (Untreated). Different treatments’ letters indicate significant difference using contrasts and standard Tukey’s adjustment at 0.05. \u003cem\u003eP-values \u003c/em\u003edisplayed are from the comparison between nanoparticle-encapsulated formulations versus\u003cem\u003e \u003c/em\u003econventional only in the “1 week” environment. Error bars indicate standard error at 95% confidence intervals.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/91d9a6359f1054559a610469.jpeg"},{"id":56934601,"identity":"587f43d0-3fb5-4936-b77d-4d75ae8bdd8b","added_by":"auto","created_at":"2024-05-22 10:37:34","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":446920,"visible":true,"origin":"","legend":"\u003cp\u003eAverage severity (A) and plant weight (B) across five sampling timings of storage. Statistical analyses were conducted using orthogonal contrasts comparing nanoparticle-encapsulated formulations (N-TBZ and N-FLD), conventional formulations (TBZ and FLD), and a control (Untreated). Different treatments’ letters indicate significant difference using contrasts and standard Tukey’s adjustment at 0.05. Error bars indicate standard error at 95% confidence intervals.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/733adefa2caece42cc55aaec.jpeg"},{"id":56935020,"identity":"9c23c79d-eca5-4a4d-bffd-b2785e388fe1","added_by":"auto","created_at":"2024-05-22 10:45:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":296384,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of fungicide-loaded nanoparticles. A-B) Scanning electron micrographs of 20:80 CPTEG:CPH 20% fludioxonil-loaded particles (A, count mean diameter 284 ± 134 nm) and 15% thiabendazole-loaded particles (B, count mean diameter 276 ± 82 nm). C-D) Release kinetics of fludioxonil-loaded (C) and thiabendazole-loaded (D) particles over 16 days at three different temperatures.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/5f7e349140614206ee69caa8.png"},{"id":87212416,"identity":"f9ec1795-7f1e-4cd9-855a-88f1e2e03e2b","added_by":"auto","created_at":"2025-07-21 14:54:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2861521,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4401757/v1/3eea8d94-b5c5-481b-a432-0102df5a0d0e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanoparticle encapsulation to enhance seed treatment efficacy against Fusarium graminearum","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSeedling diseases can have a critical effect on crop productivity, causing damage that persists throughout the season. Early infections of plant roots are harmful to plant yield due to their high impact on plant population per area. Seedling diseases frequently are the result of the interaction of multiple organisms, including fungi, oomycetes, bacteria, and nematodes. In some cases, there is a synergistic effect between organisms in different kingdoms; for instance, wounds caused by nematode feeding predispose plants to fungal or bacterial colonization\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Areas with a high incidence of soilborne pathogens are common, resulting in failure to emerge, wilting, yellowing, chlorosis, root rot, and stunted growth. Seed rots, seedling blights, and root rots typically are caused by several genera of fungi and oomycetes, including \u003cem\u003eFusarium, Aspergillus, Pythium, Rhizoctonia, Penicillium\u003c/em\u003e, and \u003cem\u003eTrichoderma\u003c/em\u003e. These genera are soil inhabitants and can harm seed germination and seedling development. Several of these genera, such as \u003cem\u003eFusarium\u003c/em\u003e, also can be seedborne. Cold soil conditions can promote seedling disease by delaying seed germination and emergence, although warmer soils favor some seedling pathogens. Soil temperatures under 12\u0026deg;C are favorable for fungal colonization by most seedling pathogens but not for seed germination, which increases the severity of seedling diseases in early plantings. These conditions are more common in no-till and reduced tillage systems\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFusarium\u003c/em\u003e is a member of the Ascomycota and is one of the most heavily studied genera of plant pathogenic fungi due to its enormous relevance to agriculture. Species of this genus cause substantial economic losses registered over the past centuries on crops such as maize, soybean, banana, and wheat. \u003cem\u003eF. graminearum sensu stricto\u003c/em\u003e is the most important species of the \u003cem\u003eFusarium graminearum\u003c/em\u003e species complex (FGSC) and is the primary pathogen responsible for Fusarium head blight (FHB) of cereals and Gibberella stalk and ear rot of maize, which have been reported in most parts of the world\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Its importance is reflected in its numerous hosts and its impact on grain yield and quality, and production of mycotoxins that are hazardous to animals and humans\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eF. graminearum\u003c/em\u003e can produce a diverse array of mycotoxins, such as deoxynivalenol, zearalenone, fusarins, culmorins, and others\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe management of this pathogen requires the employment of several methods: options include planting resistant or tolerant cultivars, use of fungicides as seed treatment and as spray during flowering, and biological control. Seed treatment is being widely used to manage a range of diseases and pests; sharp increases in the value of the seed treatment market are due to many factors, including farm safety, reduced amounts of active ingredient, and reduced exposure of non-target organisms, compared to foliar or soil applications of crop protection chemicals. Seed treatment, therefore, has environmental advantages over foliar application for diseases and pests that can be further refined through seed-applied technologies.\u003c/p\u003e \u003cp\u003eFludioxonil and Thiabendazole are two fungicides known to have a broad spectrum of action against fungi and are very effective against \u003cem\u003eFusarium graminearum\u003c/em\u003e. They are two components of the commercial formulation Maxim Quattro\u0026reg;, commercialized by Syngenta Crop Science. Their use as seed-applied fungicides is a primary management strategy to control seedling diseases; however, it is constrained by several factors, including rapid dissipation of active ingredients, limiting uptake in plant tissues, and absorption by target organisms. Therefore, our research investigates improving the efficacy of seed-applied active ingredients through the use of nanoparticle delivery vehicles.\u003c/p\u003e \u003cp\u003eNanoparticles (NPs) are grouped into different classes based on their chemistry, size, and shape, such as metal, ceramic, and polymeric. The main feature of NPs is a unique set of characteristics, including a high surface to volume ratio and high reactivity. Due to their unique chemistry, they are promising candidates for application in many fields, from the delivery of vaccines to synthetic chemicals\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The use of NP-based approaches in pharmaceutical science has shown successful improvement over conventional methods of drug and vaccine delivery.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Recent advancements have shown that the small size and unique physicochemical properties of NPs can reduce toxicity, enhancing release, improving solubility, and bioavailability when employed for drug delivery\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePolymeric NPs have been employed in research settings for the delivery of drugs and vaccines. Their size ranges from 10 to 500 nm, and special features as biodegradability and biocompatibility offer advantages for the delivery of active compounds. Due to their controlled and sustained release patterns, stealth, and modified surface, they can be used for active and passive delivery of bioactives. Polyanhydride NPs have been extensively studied as delivery platforms for small molecule and biologics in biomedical applications due to their surface erosion kinetics, which provide tunable release timelines from days to years based on polymer composition and device geometry\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. They are biodegradable and biocompatible, making them excellent candidates for agricultural controlled release\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Alteration of the proportion between monomer and co-monomer provides a range of release duration, ranging from weeks to months\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we report the use of polyanhydride NPs to encapsulate two fungicides that are active against a widespread pathogen, \u003cem\u003eF. graminearum\u003c/em\u003e. Experiments were conducted to address three primary hypotheses: i) NP encapsulation can significantly improve the control of seed decay \u003cem\u003ein vitro\u003c/em\u003e; ii) NP encapsulation of seed treatment fungicides can prolong the release of active ingredients; and iii) prolonged release can enhance the control of \u003cem\u003eFusarium graminearum\u003c/em\u003e. Our main objectives were to improve the efficacy of seed treatments by slowing the dissipation of active ingredients, especially under conditions of colder temperatures and delayed emergence of seedlings and evaluate the storability of NPs simulating seed industry storage conditions.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRolled towel assays\u003c/h2\u003e \u003cp\u003eThis assay was conducted twice, and for the first run (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), there were significant differences among all treatments. \u003cem\u003eF\u003c/em\u003e-test \u003cem\u003ep\u003c/em\u003e-values were highly significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) for all the variables measured, i.e., severity, plant weight, root length, and shoot length. Thiabendazole (TBZ) and NP-encapsulated thiabendazole (N-TBZ) were not significantly different from one another for all variables. Each variable followed the same trend, where N-TBZ was slightly better, but not significantly so. However, conventional fludioxonil (FLD) and NP-encapsulated fludioxonil (N-FLD) were significantly different for all variables, with N-FLD demonstrating improved control of \u003cem\u003eF. graminearum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the second run, the trend of results was different from the first one. In pairwise comparisons, there were no significant differences between conventional formulations and NP-encapsulated fungicides. However, in orthogonal contrasts between the two conventional ingredients combined and the two combined NP-encapsulated treatments, there were significant differences for disease severity and shoot length, with \u003cem\u003ep\u003c/em\u003e-values of 0.0450 and 0.0154, respectively, showing better results with the NPs for these measurements. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the results showed no significant differences for root length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9556) and plant weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2053). Additionally, three complementary treatments were tested: reduced rates (50%) of NP-encapsulated fungicides (HN-TBZ, HN-FLD) and an \u0026ldquo;empty\u0026rdquo; NP formulation (N-EMPTY). The reduced rates were as effective as full rates, and N-EMPTY showed no difference compared to the untreated control, indicating that the effect of the treatment comes from the release of the encapsulated fungicide without any antimicrobial effect from the polymer itself.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDelayed emergence assays\u003c/h2\u003e \u003cp\u003eIn these experiments, we evaluated the efficacy of the seed treatments against soilborne \u003cem\u003eF. graminearum\u003c/em\u003e under conditions simulating delayed emergence in cold soil. The plants were assessed after 28 days, following treatments that differed in temperature sequence, to evaluate whether NP encapsulation could prolong the effects of the active ingredients under different environments. For the first trial (maize), there were similar trends of results through different seed treatments and environmental treatments. In the first environment, without delayed emergence (constant temperature of 24\u0026deg;C), all pairwise comparisons showed no significant differences among seed treatments. Generally, NP-encapsulated thiabendazole and conventional thiabendazole (N-TBZ and TBZ, respectively) provided slightly superior protection than conventional fludioxonil and nano-fludioxonil (FLD and N-FLD, respectively) but without statistical differences for all measurements. With a 1-week delay in emergence (7 days at 10\u0026deg;C followed by 21 days at 24\u0026deg;C), pairwise comparisons showed statistical differences among seed treatments only for plant weight (p\u0026thinsp;=\u0026thinsp;0.0017), where N-TBZ had statistically higher plant weight compared to FLD (p\u0026thinsp;=\u0026thinsp;0.0099). For all remaining measurements, NP-encapsulated treatments reached slightly higher values, however, no statistical differences were observed. In the third environment, with a 2-week delay, N-TBZ was significantly better than other treatments for shoot length, plant weight, and root length. For severity, TBZ was the best treatment, statistically different from N-FLD and FLD, and similar to N-TBZ. In the last environment when emergence was delayed for three weeks, there were no significant differences among seed treatments, except against the \u0026ldquo;Untreated\u0026rdquo; control. When employing orthogonal contrasts combining conventional ingredients (TBZ and FLD), NP-encapsulated treatments (N-TBZ and N-FLD), and \u0026ldquo;Untreated\u0026rdquo;, clearer trends were evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For severity and root length, there were no significant differences. However, for plant weight (p\u0026thinsp;=\u0026thinsp;0.0278) and shoot length (p\u0026thinsp;=\u0026thinsp;0.0354), the NP-encapsulated treatments outperformed the conventional fungicide treatments for the 1-week emergence delay treatment only.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe experiment was then repeated using soybean for a second run (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results were similar to what was observed in the first run, where the level of protection provided by NP encapsulation or commercial treatments was comparable. However, when using contrasts once again, the same results were observed in the second environment. With a 1-week delay in emergence, NP formulations showed improved performance against \u003cem\u003eF. graminearum\u003c/em\u003e in comparison with commercial treatments in two variables, root length (p\u0026thinsp;=\u0026thinsp;0.0260) and plant weight (p\u0026thinsp;=\u0026thinsp;0.0598).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStorage assays\u003c/h2\u003e \u003cp\u003eIn the storage assay, the conventional formulations showed superior control of the pathogen compared to the NP-encapsulated formulations for all sampling dates, according to the orthogonal contrasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConventional formulations were consistent in maintaining low disease severity across sampling dates, without any loss of efficacy through the whole duration of the experiment. NP-encapsulated formulations showed a decrease in efficacy only between the first and second sampling dates (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002), and then consistent efficacy until the end. The water control showed a small increase in severity until the third month when it started to decrease. Severity of water control for the last sampling date was significantly different from the first (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0068), second (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and third (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) sampling dates.\u003c/p\u003e \u003cp\u003eFor root length, conventional formulations were very consistent until the fourth month in storage, with a loss in efficacy between the fourth sampling date and the last sampling date (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0009). For shoot length, more variation was observed. Conventional formulations were not consistent during the whole experiment. The second, fourth, and fifth sampling dates had statistically inferior results compared to the first and third months. For plant weight, the analyses within treatments showed a steady effect of conventional formulations until the third month in storage, with statistical decreases in the fourth and fifth months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFungicide Release Kinetics from Nanoparticles\u003c/h2\u003e \u003cp\u003eA fungicide release kinetics assay was performed to evaluate the maximal release rate of these payloads from nanoparticles \u003cem\u003ein vitro\u003c/em\u003e in well-buffered, aqueous conditions with regular organic solvent washes to maintain infinite sink for payload release (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Release kinetics data were collected for the two different types of NPs used; F2\u0026ndash;20:80 CPTEG: CPH NPs containing 20% fludioxonil, and F3\u0026ndash;20:80 CPTEG: CPH NPs containing 15% thiabendazole. The particles containing fludioxonil exhibited a burst of fungicide release (~\u0026thinsp;80% of total fludioxonil mass) within the first 24 hours. The remaining fludioxonil appeared to be released slowly through the end of the experiment. For thiabendazole loaded NPs, the trend was similar; however, the release occurred in a slower pattern. These NPs exhibited a burst of 60% after 24 hours, followed by a steady release of approximately 30% more until the end. There were no significant differences among the three different temperature treatments (i.e., 10\u0026deg;C, 15\u0026deg;C, and 24\u0026deg;C). We measured relatively high encapsulation efficiencies of 75.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3% for fludioxonil and 90.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% for thiabendazole, yielding effective loadings of 15.0% and 13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% (w/w), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe results of this study showed that NP encapsulation of fungicides could provide some advantages for the control of seedborne and soilborne inoculum of \u003cem\u003eF. graminearum\u003c/em\u003e. In both delayed emergence experiments, NP-encapsulated formulations resulted in better disease management than conventional formulations when emergence was delayed for 1-week by low temperature. However, in other environments, NP-encapsulated formulations and conventional formulations were statistically similar. These results showed a potential advantage to NP encapsulation when emergence was delayed for one week. This behavior could be related to the slow-release kinetics enabled by the specific polymer composition used. These results suggest that the NPs are prolonging the release of their payload(s) and hence improving the control of the \u003cem\u003eF. graminearum\u003c/em\u003e pathogen during the first week of delayed emergence. The results may be enhanced using a mixture of different copolymer compositions and ratios together in the same slurry to target different windows of active ingredient release. Moreover, these experiments demonstrated the importance of seed-applied fungicides against seedling diseases. In both repetitions of the delayed emergence assay (first run using maize and second run with soybeans), the inoculated control had zero emergence when emergence was delayed for two and three weeks, while fungicide-based treatments were statistically superior.\u003c/p\u003e \u003cp\u003eFor the rolled-towel results, NP-encapsulated formulations were superior only in the first experiment, which is related to the poor control provided by FLD only in the first run. In contrast, TBZ and N-TBZ provided similar results. In the second experiment, when FLD was similar to N-FLD, the orthogonal contrasts showed no statistical differences between \u0026ldquo;nano\u0026rdquo; and \u0026ldquo;commercial\u0026rdquo;. Additionally, the severity of the inoculated and water-treated control of the second experiment was lower than in the first experiment. The lower disease pressure seen in the second run could explain the lack of significant differences in the second experiment. If the primary advantage of NP encapsulation is to prolong the efficacy of the seed treatment, this benefit would not be evident in the rolled-towel assays, which only assess control in the first week of germination and seedling growth.\u003c/p\u003e \u003cp\u003eThe rolled-towel assays demonstrated that NP-encapsulated formulations can work as well or better than conventional formulations, even with reduced rates. The half-rate-NP-encapsulated formulations provided similar results compared to the full rate of NP-encapsulated formulations and the standard rate of conventional formulations. Similarly, Washington et al.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e observed equivalent levels of control using half rates.\u003c/p\u003e \u003cp\u003eThe results from the storage assay showed a relatively stable efficacy of NP-encapsulated formulations. For most of the variables measured, some efficacy was lost only after four or five months in storage, similar to the conventional formulations. Although maize seed is treated generally within one year before planting, this window of safe storage could be improved by more studies on copolymer composition.\u003c/p\u003e \u003cp\u003eThe release studies showed no difference in the time-release profile for both chemicals under different temperatures. These \u003cem\u003ein vitro\u003c/em\u003e results indicated that NP encapsulation did not prolong active ingredient release over the desired time frame (1 to 3 weeks). However, the conditions of the in vitro release study likely do not adequately represent soil conditions. Additional polyanhydride copolymer formulations or combinations of the formulation(s) could be tested. Robust methods for measuring the time release of active ingredients in soil need to be developed. The results of our experiments in delayed-emergence environments might be related to interaction(s) between the pathogen and plant development. Lower temperatures can provide a competitive advantage to the fungus rather than the plant, and this interaction should be targeted by the employment of a seed treatment that can protect late-emerging seedlings. The results showing similar release profiles across temperatures can be advantageous to promote enhanced protection and prevent seed decay under various soil temperatures, including delayed emergence scenarios. This mechanism could be especially beneficial with a prolonged-release system to avoid the rapid dissipation of the active ingredient before germination.\u003c/p\u003e \u003cp\u003eProlonging the efficacy of seed treatments is a significant challenge that could greatly increase the value of this crop protection tactic. We hypothesized that the employment of NPs could address the limitation of rapid dissipation of active ingredients and promote a prolonged release, achieving enhanced protection against pests. Our results partially support this hypothesis; NP encapsulation displayed improved control for a specific environment (1-week delay) when tested with two different crop species. Further research should pursue the potential to enhance the prolonged effect to levels that could increase seedling protection for several weeks after planting.\u003c/p\u003e \u003cp\u003eA better understanding of the chemical compatibility between the polymers and the active ingredients, as well studies with variable ratios of polyanhydride monomers, could bring additional flexibility (and benefits) to the pattern of release of a specific chemical. It is essential to understand the release patterns for different copolymer compositions and their behavior when applied in soil conditions. Additionally, other different types of chemicals should be used to understand which active ingredients are most compatible when using polyanhydride polymers.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSeed preparation\u003c/h2\u003e \u003cp\u003eBefore each trial, seeds were externally disinfested by a two-step process. Seeds were soaked in a solution of bleach and then rinsed using deionized sterile water. For maize, we used a solution of 10% bleach and a soaking period of five minutes. After the seeds were removed from the solution, seeds were rinsed at least five times until the bleach was removed completely. For soybean, the external disinfestation was performed with reduced bleach concentration (5%) and exposure period (1 minute), due to the high sensitivity of soybean seeds to physical and chemical damage. As bleach is commonly used to check the damage on soybean seed coats, we used this step to remove damaged seeds, such as swelled and cracked ones\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Finally, seeds were placed in labeled trays with paper towels inside a biosafety cabinet to dry before the next steps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIsolate and inoculum preparation\u003c/h2\u003e \u003cp\u003eThe isolate of \u003cem\u003eF. graminearum\u003c/em\u003e used for preliminary and primary experiments was FG27 (isolated in Iowa, USA). It was grown for each experiment from stored silica gel pellets, dated from December 2016. We used two forms of inoculation: a spore suspension for rolled-towel assays, simulating a seedborne infection, and infested millet for experiments in field soil, simulating soilborne inoculum. The spore suspension was prepared by reviving the fungus from silica pellets on potato dextrose agar (PDA) (BD, Franklin Lakes, New Jersey) for ten days at ambient temperature (24\u0026ndash;26\u0026deg;C). To promote sporulation, mycelial plugs from PDA cultures were transferred to Spezieller N\u0026auml;hrstoffarmer Agar (SNA)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e plates. After approximately 15 days, spores from SNA plates were collected with sterile deionized water and filtered through sterile cheesecloth. The concentration was adjusted to reach 1 x 10\u003csup\u003e5\u003c/sup\u003e spores/mL using a hemocytometer. The inoculation was finally performed by soaking batches of 100 seeds in 30 ml of a spore suspension for five hours. All containers were placed on a shaker at ambient temperature at 92 r.p.m. For soybeans, the soaking period was reduced to one minute. After the inoculation, seeds were dried on paper towels inside a biosafety cabinet for twelve hours before treatment.\u003c/p\u003e \u003cp\u003eAlternatively, to prepare inoculum for studies using field soil, we infested the soil using colonized millet. We pasteurized the millet by autoclaving it for 90 min at a temperature of 121\u0026deg;C. The millet remained at a temperature above 80\u0026deg;C for more than one hour after the autoclave cycle was finished. This process was done twice on two consecutive days. On the second day, after letting the bags containing the millet cool down, PDA plates containing 15-day-old \u003cem\u003eF. graminearum\u003c/em\u003e were blended and poured into aerated bags containing autoclaved millet seeds in a one-plate-per-bag ratio. These bags were kept in a growth chamber for 15\u0026ndash;20 days until the mycelial growth covered all millet kernels. For the non-inoculated control treatment, the colonized millet was autoclaved twice before mixing with soil. Millet inoculum and control millet samples were checked for viable \u003cem\u003eF. graminearum\u003c/em\u003e by culturing on PDA. Millet was mixed into pasteurized field soil for all treatments, including controls. The percentage of inoculum in the soil (volume/volume) was 5%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSeed treatment application\u003c/h2\u003e \u003cp\u003eThe traditional active ingredients chosen to be used in this research were fludioxonil and thiabendazole. While the dosage of fludioxonil is only 0.0065 mg/kernel (3.32% of the total slurry), thiabendazole has a much higher rate, 0.05 mg/kernel (26.5%). The recommended dosages of each active ingredient were used with the addition of a plantability polymer. The polymer used was Flo Rite\u0026reg; 1197 (BASF) following the manufacturer recommendation, approximately 0.44 ml per kernel. All compounds were then mixed with water (15 \u0026micro;L per seed) and vortexed before application on seeds.\u003c/p\u003e \u003cp\u003eSimilarly, for the NP formulations, we used the equivalent mass of the active ingredients. However, the total weight of active ingredient was adjusted to consider encapsulation efficiency and the total loading of the nanoparticles to ensure that the same active ingredient dosage would be delivered to each seed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). NP treatments were formulated with a surfactant, Span80, but without the additional plantability polymer. Before the application, treatments were sonicated using a VCX 130PB sonicator with a CV138 tip (Sonics and Materials, Newton, CT) at 30% amplitude to disperse the NPs into suspension.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTreatments employed in rolled-towel and delayed-emergence assays. All treatments included F. graminearum inoculation or infestation, except for CONTROL1.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment codes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment description\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-inoculated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater and plantability polymer without \u003cem\u003eF. graminearum\u003c/em\u003e inoculation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUntreated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater and plantability polymer with \u003cem\u003eF. graminearum\u003c/em\u003e inoculation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFludioxonil (0.0065 mg/kernel), water and plantability polymer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-FLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolyanhydride nanoparticle encapsulated fludioxonil (0.0065 mg/kernel), water, and Span80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTBZ.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThiabendazole (0.05 mg/kernel), water and plantability polymer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-TBZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolyanhydride nanoparticle encapsulated thiabendazole (0.05 mg/kernel), water, and Span80.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eControls with no seed treatment with and without \u003cem\u003eF. graminearum\u003c/em\u003e inoculation were added as well (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The treatments were applied by adding the slurry into plastic bags containing the specific number of seeds. After the application, the seeds were rubbed together to apply the solution evenly over the seeds. Seeds were dried again within a biosafety cabinet for 24 hours after treatment.\u003c/p\u003e \u003cp\u003eBesides the treatments listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, additional treatments were included only in the second run of the rolled-towel assay. They consist of an empty NP formulation (N-EMPTY), and half rates of each fungicide, i.e., HN-TBZ (half dosage of N-TBZ) and HN-FLD (half dosage of N-FLD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRolled towel assay\u003c/h2\u003e \u003cp\u003eTo evaluate efficacy against seedborne inoculum using full and reduced rates of active ingredients, we conducted rolled-towel assays using rolled germination paper as described by Ellis \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, except that seeds were inoculated before \u0026ldquo;planting,\u0026rdquo; as already described. These assays were conducted using maize seeds under favorable conditions of germination (23\u0026ndash;25\u0026deg;C) for seven days.\u003c/p\u003e \u003cp\u003eFor each replication, 15 seeds were placed on top of two layers of germination paper (Anchor Paper Co., St. Paul, MN) moistened with sterile water. A third sheet was placed on seeds, and they were rolled and placed inside a plastic bag containing sterile water. Each bag was placed inside buckets covered with a second bag to maintain moisture inside the bucket. They stayed inside a growth chamber at 24\u0026deg;C for one week, and then seven-day-old seedlings were analyzed for root length, shoot length, disease severity, and total fresh weight. Disease severity was measured on a one-to-five scale\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, with five being the most severe disease, and one being completely healthy. Results of severity, root length, shoot length, and plant weight were analyzed for analysis of variance and mean separation using Tukey\u0026rsquo;s Honest Significant Difference test at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. Planned contrasts were employed as well to compare overall differences between nanoparticle encapsulation and standard formulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDelayed Emergence assay\u003c/h2\u003e \u003cp\u003eIn this assay, we assessed the efficacy of fungicides and their NP-encapsulated counterparts under conditions simulating an emergence delay due to cold temperatures in pathogen-infested soil. To study the prolonged-release effect, we conducted assays with a delayed-emergence scenario, in which non-inoculated seeds were planted in pasteurized soil infested with \u003cem\u003eF. graminearum\u003c/em\u003e, and then subjected to a low temperature, unfavorable for germination, before a higher temperature, favorable for germination. These treatments simulated early planting in cold soils. As the rapid dissipation of active ingredients is one of the most important limitations of seed treatments, we employed this assay to evaluate if, in a delayed emergence situation, NP-encapsulated fungicides would prolong the release of active ingredients, and hence improve and extend protection from the pathogen. Treatments consisted of four different temperature combinations, varying in the number of weeks of emergence delay (weeks after planting at 10\u0026deg; C). Treatments ranged from no-emergence delay to a three-week emergence delay (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This assay was repeated twice: the first run was conducted using maize, and the second soybean.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDelayed emergence treatments for infested soil assay.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u0026deg;C for four weeks\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1-week delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u0026deg;C for one week followed by three weeks at 24\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-week delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u0026deg;C for two weeks followed by two weeks at 24\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-week delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u0026deg;C for three weeks followed by one week at 24\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo assure correct temperatures for the duration of the experiments, data loggers (Spectrum Technologies, Aurora, IL) were placed in the growth chambers before the start to facilitate the temperature adjustment. Moreover, they remained through the entire experiment to monitor temperature fluctuations. Plants were uprooted after 28 days, and assessed for disease severity, root and shoot length, and plant weight, identically to measurements used for the rolled-towel assays.\u003c/p\u003e \u003cp\u003eData collected were analyzed by analysis of variance (ANOVA) in R version 3.6.1. Analysis of all main effects was conducted using all treatment combinations with Tukey\u0026rsquo;s pairwise adjustments. Mean separations were done using Tukey\u0026rsquo;s Honest Significant Difference test at p\u0026thinsp;\u0026le;\u0026thinsp;0.05, and orthogonal contrasts, where we combined the means of traditional fungicides \u003cem\u003eversus\u003c/em\u003e nanoparticle encapsulated versions using Tukey\u0026rsquo;s standard adjustment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStorage assay\u003c/h2\u003e \u003cp\u003eFor the storage assay, maize and soybean seeds were inoculated, treated, and then stored in a cold room simulating the conditions employed in the industry (10\u0026deg;C and 50% RH\u003csup\u003e24\u003c/sup\u003e. Batches of seeds were inoculated using a spore suspension and then treated following the procedures mentioned above. Approximately 180 seeds per treatment, per sampling date, were placed inside envelopes (8 cm x 14 cm) and then arranged in larger envelopes (23 cm x 30 cm) and stored in the cold room. Seeds were sampled every month for five months in storage. At each sampling date, continued efficacy of treatments was assessed by rolled-towel assays already described. Seedlings were analyzed for root length, shoot length, disease severity, and total fresh weight. Disease severity was measured on a one-to-five scale, with five being the most severe disease, and one being completely healthy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Results were analyzed for analysis of variance and mean separations using Tukey\u0026rsquo;s Honest Significant Difference test at p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle synthesis and characterization\u003c/h2\u003e \u003cp\u003eCPTEG and CPH monomers and 20:80 CPTEG:CPH copolymer were synthesized as described previously\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Fungicide-loaded nanoparticles were synthesized via flash nanoprecipitation by dissolving fungicide and copolymer in methylene chloride at 20 mg/mL, sonicating to homogenize, then rapidly pouring this solution into a -10\u0026deg;C pentane antisolvent bath at a solvent/antisolvent ratio of 1:250 (v/v)\u003csup\u003e29, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The nanoparticles were collected by vacuum filtration and imaged via scanning electron microscopy (SEM, FEI Quanta 250, Hillsboro, OR). The resulting SEM micrographs were analyzed using the ParticleSizer plugin script in Fiji to calculate particle size distributions\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditional experiments were conducted to characterize the release of active ingredients from NP encapsulation at three different temperatures and to assess the storability of NP-encapsulated seeds in typical storage conditions. To understand the release kinetics of the NP encapsulated formulations used in our experiments, we used HPLC (high-performance liquid chromatography) to estimate the mass of the fungicides released over time in an aqueous solution. In order to understand the release trends under different potential soil conditions, we performed this assay under three different temperatures, 10\u0026deg;C, 15\u0026deg;C, and 24\u0026deg;C. The process started by weighing 9\u0026ndash;11 mg of each nanoparticle formulation in triplicate and suspending the particles in 0.5 mL of PBS (phosphate-buffered saline solution), pH 7.4. At each timepoint, the particles were centrifuged, the supernatant was collected, and particles were washed with acetonitrile to extract the remaining, non-dissolved fungicide. Fresh PBS was added, and the particles were re-dispersed by sonication. At the end of the aqueous (PBS) release, 40 mM NaOH was applied to rapidly degrade the remaining particles, allowing estimation of the remaining encapsulated fungicide. Released fludioxonil was quantified via RP-HPLC-UV/Vis (1200 series, Agilent Technologies, Santa Clara, CA, USA) in a constant solvent mix of 60% acetonitrile and 40% water, pH 2.27 (normalized with o-phosphoric acid) measuring absorbance at 280 nm, with expected elution time of 2 min. Similarly, thiabendazole was quantified via RP-HPLC-UV in constant solvent mixture of 40% acetonitrile 0.1% trifluoroacetic acid and 60% water 0.1% trifluoroacetic acid measuring absorbance at 254 nm, with expected elution time of 1.4 min. For each timepoint, the mass of drug released in PBS and acetonitrile samples were added to reflect maximal release rate. The encapsulation efficiency was calculated by dividing the total measured released drug mass by the nominal encapsulated mass.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Iowa State University Presidential Interdisciplinary Research Seed Grant Program and the Iowa State University Seed Science Center for funding this research project and the staff of the Iowa State Seed Pathology Laboratory for their assisting in all of the steps of this research. The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFernando Marcos conducted and analyzed all efficacy experiments, made figures 1-5, and wrote the main manuscript text. Adam Mullis conducted and analyzed release kinetics experiments and made figure 6. Gary Munkvold and Balaji Narasimhan developed the original concept, contributed to design of the experiments, advised and consulted for all steps of the study, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eda Silva, M.P., Tylka, G.L. \u0026amp; Munkvold, G.P. Seed treatment effects on maize seedlings coinfected with Fusarium spp. and Pratylenchus penetrans. Plant Dis. 100, 431\u0026ndash;437 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson, A. \u0026amp; Munkvold, G.P. Check general root and mesocotyl health when assessing corn stands. Iowa State University Integrated Crop Management Newsletter. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.extension.iastate.edu/CropNews/2009/0519robertson.htm\u003c/span\u003e\u003cspan address=\"http://www.extension.iastate.edu/CropNews/2009/0519robertson.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunkvold, G.P. \u0026amp; O\u0026rsquo;Mara, J.K. Laboratory and growth chamber evaluation of fungicidal seed treatments for maize seedling blight caused by Fusarium species. Plant Dis. 86, 143\u0026ndash;150 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArias, M. M. D., Leandro, L. F. \u0026amp; Munkvold, G. P. Aggressiveness of Fusarium species and impact of root infection on growth and yield of soybeans. Phytopathology. 103, 822\u0026ndash;832. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PHYTO-08-12-0207-R\u003c/span\u003e\u003cspan address=\"10.1094/PHYTO-08-12-0207-R\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunkvold, G.P. Fusarium species and their associated mycotoxins in Mycotoxigenic Fungi: Methods and Protocols, Methods in Molecular Biology (eds Moretti, A., Susca, A.) 51\u0026ndash;106 (Humana Press, 2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgrios, G. N. Plant Pathology 534\u0026ndash;538 (Elsevier Academic Press, 2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRubella, S. G. \u0026amp; Kistler, H.C. Heading for disaster: Fusarium graminearum on cereal. Mol. Plant Pathol. 5 (6), 515\u0026ndash;525 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmale III, D. G. \u0026amp; Bergstrom, G. C. Fusarium head blight in wheat. Plant Health Instr. 10.1094/PHI-I-2003-0612-01 (2003, updated 2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKebede, A. Z., Woldemariam, T., Reid, L.M. \u0026amp; Harris, L.J. Quantitative trait loci mapping for Gibberella ear rot resistance and associated agronomic traits using genotyping\u0026ndash;by\u0026ndash;sequencing in maize. Theor Appl Genet. 129,17\u0026ndash;29 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunkvold, G. P. \u0026amp; White, D. G. Compendium of Corn Diseases, Fourth Edition. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/9780890544945.002\u003c/span\u003e\u003cspan address=\"10.1094/9780890544945.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (The American Phytopathological Society, 2016)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunkvold, G. P. Seed pathology progress in academia and industry. Annu. Rev. Phytopathol. 47, 285\u0026ndash;311(2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroders, K.D., Lipps, P.E., Paul, P.A. \u0026amp; Dorrance, A. E. Evaluation of Fusarium graminearum associated with corn and soybean seed and seedling disease in Ohio. Plant Dis. 91, 1155\u0026ndash;1160 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatia, S. Natural polymer drug delivery systems: Nanoparticles, plants, and algae in Natural Polymer Drug Delivery Systems: Nanoparticles, Plants, and Algae. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-319-41129-3\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-41129-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Springer International Publishing, 2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan, I., Saeed, K., \u0026amp; Khan, I. Nanoparticles: Properties, applications and toxicities. Arab. J. Chem. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.arabjc.2017.05.011\u003c/span\u003e\u003cspan address=\"10.1016/j.arabjc.2017.05.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhanse, Y. et al. Cellular internalization mechanisms of polyanhydride particles: Implications for rational design of drug delivery vehicles. J. Biomed. Nanotechnol. 12 (7), 1544\u0026ndash;1552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1166/jbn.2016.2259\u003c/span\u003e\u003cspan address=\"10.1166/jbn.2016.2259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVela-Ramirez, J. E. et al. Safety and Biocompatibility of Carbohydrate-Functionalized Polyanhydride Nanoparticles. The AAPS Journal 17, 256\u0026ndash;267 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerkland, C., Kipper, M. J., Narasimhan, B., Kim, K., \u0026amp; Pack, D. W. Microsphere size, precipitation kinetics and drug distribution control drug release from biodegradable polyanhydride microspheres. J. Control. Release. 94 (1), 129\u0026ndash;141. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jconrel.2003.09.011g\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2003.09.011g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBinnebose, A. M. et al. Polyanhydride nanoparticle delivery platform dramatically enhances killing of filarial worms. PLOS Negl. Trop. Dis. 9 (10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pntd.0004173\u003c/span\u003e\u003cspan address=\"10.1371/journal.pntd.0004173\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWashington, L. A. Utilization of polyanhydride nanoparticle encapsulated fungicide seed treatments against seedborne and soilborne Fusarium graminearum on maize. Master\u0026rsquo;s Thesis, Iowa State Univeristy. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lib.dr.iastate.edu/etd/15639\u003c/span\u003e\u003cspan address=\"https://lib.dr.iastate.edu/etd/15639\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVanUtrecht, D., Bern, C. J., \u0026amp; Rukunudin, I. H. Soybean mechanical damage detection. Appl. Eng. Agric. 16 (2), 137\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13031/2013.5059\u003c/span\u003e\u003cspan address=\"10.13031/2013.5059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeslie, J.F., \u0026amp; Summerell, B.A. The Fusarium Laboratory Manual (Blackwell Publishing, 2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis, M.L., Broders, K.D., Paul, P.A., Dorrance, A.E. Infection of soybean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions. Plant Dis. 95, 401\u0026ndash;407 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCruz Jimenez, D. Soybean root rot caused by Fusarium oxysporum and Fusarium graminearum: interactions with biotic and abiotic factors. Doctoral dissertation, Iowa State University. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lib.dr.iastate.edu/etd/15505\u003c/span\u003e\u003cspan address=\"https://lib.dr.iastate.edu/etd/15505\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrington, J. F. Thumb rules of drying seeds. Crops \u0026amp; soils. 13 (1), 16\u0026ndash;17 (1960).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen E, Pizsczek R, Dziadul B, Narasimhan B. Microphase separation in bioerodible copolymers for drug delivery. Biomaterials. 22(3):201\u0026ndash;10 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConix, A., Poly[l,3-Bis(p-carboxyphenoxyj-propane anhydride], Macromolecular Synthesis, Vol. 2, (Ed. J.R. Elliot) Wiley, NYC 1966. pp 95\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorres MP, Vogel BM, Narasimhan B, Mallapragada SK. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. Journal of Biomedical Materials Research - Part A. 2006;76(1):102\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMullis AS, Broderick SR, Binnebose AM, Peroutka-Bigus N, Bellaire BH, Rajan K, et al. Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release. Molecular Pharmaceutics. 2019;16(5):1917\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMullis AS, Broderick SR, Binnebose AM, Peroutka-Bigus N, Bellaire BH, Rajan K, et al. Data Analytics Approach for Rational Design of Nanomedicines with Programmable Drug Release. Molecular Pharmaceutics. 2019;16(5):1917\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMullis, A. S.; Broderick, S. R.; Phadke, K. S.; Peroutka-Bigus, N.; Bellaire, B. H.; Rajan, K.; Narasimhan, B. Data Analytics-Guided Rational Design of Antimicrobial Nanomedicines against Opportunistic, Resistant Pathogens. Nanomedicine: NBM 2023, 48, 102647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.nano.2022.102647\u003c/span\u003e\u003cspan address=\"10.1016/j.nano.2022.102647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. Fiji: An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9, 676\u0026thinsp;\u0026ndash;\u0026thinsp;682. Wagner T, Eglinger J. ParticleSizer. 2017.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fusarium graminearum, seed treatment, nanoparticles, seedling diseases","lastPublishedDoi":"10.21203/rs.3.rs-4401757/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4401757/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe importance of seed treatments has increased rapidly in the past decade, mainly due to their high efficacy controlling early-season pests and diseases, and their limited environmental impact. Chemical seed treatments require a smaller amount of pesticide use and reduce environmental spread compared to foliar or soil applications; similarly, selection pressure for the development of resistance in the pest population is reduced. However, the rapid dissipation of seed treatment active ingredients after planting is associated with unpredictable duration of control, limiting the performance of seed treatment technology. Polyanhydrides are synthetic biodegradable polymers that can be used to deliver active ingredients or pharmaceuticals in pathological systems. They can provide a steady and sustained release of active compounds, enhancing the treatment of diseases caused by pathogens. Our study consists of experiments using polyanhydride nanoparticle-encapsulated fludioxonil and thiabendazole (two fungicides commonly used against \u003cem\u003eFusarium graminearum\u003c/em\u003e) at different rates on maize and soybean. We employed both rolled-towel assays (simulating a seedborne infection) and delayed emergence assays (simulating a soilborne infection). In the rolled-towel assay, nanoparticle-encapsulated fungicides performed similarly to standard formulations. However, when emergence was delayed for one week by low temperature, nanoparticle-encapsulated fungicides showed superior control over standard formulations. For longer emergence delay treatments, nanoparticle and conventional fungicide formulations showed similar levels of control. Polyanhydride encapsulated seed treatments showed the potential to prolong effectiveness of active ingredients when emergence is delayed due to cold temperatures, a very common situation in temperate maize production areas, such as the American Midwest.\u003c/p\u003e","manuscriptTitle":"Nanoparticle encapsulation to enhance seed treatment efficacy against Fusarium graminearum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 10:37:29","doi":"10.21203/rs.3.rs-4401757/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a0d5198-3571-406c-8738-2f73a0870064","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32171699,"name":"Biological sciences/Plant sciences"},{"id":32171700,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-07-21T14:53:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-22 10:37:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4401757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4401757","identity":"rs-4401757","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}